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WO2025229499A1 - Compositions and methods for modulating t cells - Google Patents

Compositions and methods for modulating t cells

Info

Publication number
WO2025229499A1
WO2025229499A1 PCT/IB2025/054401 IB2025054401W WO2025229499A1 WO 2025229499 A1 WO2025229499 A1 WO 2025229499A1 IB 2025054401 W IB2025054401 W IB 2025054401W WO 2025229499 A1 WO2025229499 A1 WO 2025229499A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
rte
nucleic acid
polypeptide
driver
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/IB2025/054401
Other languages
French (fr)
Inventor
David BASSAN
Ruben Alvarez Rodriguez
Devin Trudeau
Maya RON
Yaron BEN SHUSHAN-GOLECZKI
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Averna Therapeutics Ltd
Original Assignee
Averna Therapeutics Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Averna Therapeutics Ltd filed Critical Averna Therapeutics Ltd
Publication of WO2025229499A1 publication Critical patent/WO2025229499A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex

Definitions

  • T cells for example T cells, redirected with engineered immune receptors to kill target cells have shown success in treating patients with hematological malignancies and autoimmune disorders.
  • T cells are isolated from the blood of a patient by apheresis, ex vivo engineered with a viral vector to express an engineered immune receptor, expanded, and infused to the patient.
  • the engineered T cells After the engineered T cells are administered to a patient, they recognize and bind to specific proteins on the surface of target cells, and once bound, the engineered T cells activate an immune response, leading to the destruction of the target cells.
  • this ex vivo engineering of immune cells is a lengthy and expensive procedure, in part because the therapeutic agent, the engineered immune cells, has to be custom-engineered for each patient from isolated autologous cells.
  • the engineered immune cells has to be custom-engineered for each patient from isolated autologous cells.
  • compositions and methods for delivering an engineered immune receptor to, e.g., immune cells comprising: (a) a nucleic acid sequence encoding a T cell receptor fused antigen modifier (TRAM) comprising an antigen binding domain and a TCR subunit, and (b) a retrotransposable element untranslated region (RTE-UTR).
  • TCR subunit is selected from the group consisting of TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ .
  • the RTE-UTR comprises a 3’ RTE-UTR and/or a 5’ RTE-UTR.
  • the antigen binding domain is selected from the group consisting of an scFv, a VHH, a TCR-like antibody, a natural ligand, an FcR-binding receptor (e.g. CD16), and an NK killing receptor.
  • a retrotransposable-element (RTE) integration system comprising a driver nucleic acid and a template nucleic acid, wherein: (a) the driver nucleic acid comprises a nucleic acid sequence encoding an RTE polypeptide, wherein the RTE polypeptide is a site-specific RTE polypeptide; and (b) the template nucleic acid comprises: a nucleic acid sequence encoding an engineered immune receptor, and an RTE-UTR capable of being bound by the RTE polypeptide.
  • the engineered immune receptor comprises an antigen binding domain.
  • the engineered immune receptor is selected from the group consisting of a T cell receptor (TCR), a TCR fused antigen modifier (TRAM), a TRuC and a chimeric antigen receptor (CAR).
  • TCR T cell receptor
  • TRAM TCR fused antigen modifier
  • CAR chimeric antigen receptor
  • a retrotransposable-element (RTE) integration system comprising a driver nucleic acid and a template nucleic acid, wherein: (a) the driver nucleic acid comprises a nucleic acid sequence encoding an RTE polypeptide; and (b) the template nucleic acid comprises a nucleic acid sequence encoding a T cell receptor fused antigen 2 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 modifier (TRAM) comprising an antigen binding domain and a TCR subunit, and an RTE-UTR capable of being bound by the RTE polypeptide [0016]
  • claim the LNP is covalently linked to an antibody or any fragment thereof that recognizes a T cell antigen.
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a nucleic acid of the disclosure, a RTE integration system of the disclosure, or a LNP of the disclosure.
  • a method of treating a disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a nucleic acid of the disclosure, a RTE integration system of the disclosure, a LNP of the disclosure, or a pharmaceutical composition of the disclosure.
  • the subject is pre-treated with a T cell activator or concomitantly treated with a T cell activator.
  • the T cell activator is a CD3 agonist or a TCR agonist.
  • subject is further administered prior, concurrently, or subsequent to said administering with at least one ⁇ -chain receptor agonist.
  • a method of in vivo genome manipulation comprising pre-administering a T cell activator and administering a pharmaceutical composition comprising a reprograming agent.
  • the reprogramming agent is a genome manipulation system comprising one or more nucleic acids.
  • the one or more nucleic acids comprise at least one nucleic acid sequence comprised in or derived from a retrotransposon.
  • the reprograming agent meditates durable expression by integrating one or more nucleic acids into a genome.
  • the T cell activator is an CD3 or TCR agonist, or an immune cell engager, e.g., Bispecific T cell engager (BiTe).
  • a method of in vivo genomic manipulation in an immune cell comprising contacting the immune cell with a nucleic acid of the disclosure, a RTE integration system of the disclosure, a LNP of the disclosure, or a pharmaceutical composition of the disclosure, wherein the immune cell natively expresses a TCR-CD3 complex.
  • 3 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 BRIEF DESCRIPTION OF DRAWINGS
  • the present disclosure including the drawings, include reference to the term TCRF (T Cell Receptor Fusion Protein) which may be used interchangeably with TRAM (T Cell Receptor fused Antigen Modifier).
  • FIG.1A and FIG.1B illustrate non-limiting examples of nucleic acids useful for gene delivery, using an RTE polypeptide to promote insertion of a transgene (e.g., a heterologous nucleic acid comprising a gene of interest (“GOI”)) into a genome.
  • a transgene e.g., a heterologous nucleic acid comprising a gene of interest (“GOI”)
  • FIG.1A demonstrates a cis configuration where one polynucleotide (e.g. mRNA) encodes both a protein that is capable of promoting retrotransposition (an RTE polypeptide, represented as ORF2) and an antisense transgene flanked by terminal regions.
  • FIG.1B demonstrates an exemplary trans-configuration with two polynucleotides (e.g.
  • the first polynucleotide encodes ORF2 in an mRNA format
  • the second polynucleotide comprises a GOI flanked by terminal regions.
  • the GOI e.g. CAR or TRAM
  • the gene of interest may be inserted in a forward (i.e., sense) configuration relative to the 5’UTR and or polyA tail.
  • the template nucleic acid is not an mRNA (i.e. lacks a 5’ cap structure, or a 3’ polyA tail, or both).
  • FIG.2 illustrates examples of TCR, canonical CAR, and TRAM components.
  • FIG.3 illustrates exemplary CAR and TRAM designs. Designs shown for anti-CD19 CAR and TRAM.
  • FIG.4A- FIG.4F illustrate expression of anti-CD19 CAR or TRAMs (with T2A-GFP) in several cell types following lentiviral transduction.
  • FIG.5A- FIG.5F illustrate anti-CD19 CAR or TRAM expression and CD19 masking in Nalm6 cells following lentiviral transduction gated on transduced (GFP+) cells.
  • FIG.6A- FIG.6F illustrate anti-CD19 CAR or TRAM expression and CD19 masking in Raji cells following lentiviral transduction gated on transduced (GFP+) cells.
  • FIG.7A- FIG.7F illustrate the Vingi1 RTE system for GFP and anti-CD19 CAR expression.
  • FIG.8A- FIG.8I illustrate CART-19 expression under MNDopt or EF1 ⁇ promotors in several donor T cells.
  • FIGS.8A-8F show results with Vingi1 driver.
  • FIGS.8G-8I show results 4 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 with lentivirus (LV).
  • FIGS.8A, 8D and 8G show dPCR results.
  • FIGS.8B, 8E and 8H show %CAR positive cells as determined by FACS.
  • FIGS.8C, 8F and 8I show quantification as receptors/cell.
  • FIG.9A- FIG.9C illustrate anti-CD19 CAR or TRAMs ( ⁇ ) with T2A-GFP using Vingi1 RTE.
  • FIG.10A- FIG.10C illustrate anti-CD19 CAR or TRAMs ( ⁇ (without T2A-GFP) expression using Vingi1 RTE.
  • FIG.11A- FIG.11D illustrate anti-CD19 CAR or TRAMs ( ⁇ ) (without T2A-GFP) killing in Nalm6 cells using Vingi1 RTE.
  • FIG.12A- FIG.12C illustrate anti-CD19 ⁇ TRAMs with EF1 ⁇ or MNDopt promoters using Vingi1 RTE.
  • FIG.13A- FIG.13B illustrate anti-CD20 CAR or ⁇ TRAMs with different spacers under EF1 ⁇ promoter using Vingi1 RTE.
  • FIG.14A- FIG.14E illustrate T cell specific promoters with GFP transgene using Vingi1 RTE.
  • FIG.15A- FIG.15C illustrate exemplary nucleic acid designs with microRNA (miR) binding sites.
  • FIG.16A- FIG.16B demonstrate GFP transgene integration and reduced expression in hepatocytes in the presence of liver-restricted miR-122 with GFP transgene under MNDopt promotor driven by a Vingi1 RTE.
  • FIG.17A- FIG.17F demonstrate results of Donor 10 T cells transiently transfected with targeted LNPs (tLNPs) comprising GFP mRNA with several binders driven by Vingi1.
  • tLNPs targeted LNPs
  • FIG.18A- FIG.18B demonstrate targeted LNPs (tLNPs) with several binders containing mRNA for Vingi1 driver and MNDopt-GFP RTEs reporter, in trans.
  • FIG.19A- FIG.19D demonstrate expression of GFP effected by LNPs (non-targeted), containing RNA for Vingi1 or R2-1_TG driver and MNDopt-GFP RTE reporters, transfected in the presence of antibodies.
  • FIG.20A- FIG.20D demonstrate the kinetics of LDL-R expression following activation.
  • FIG.21A- FIG.21H demonstrate the comparison of activation and transient GFP RNA- LNP transfection following antibody activation on day 0 or day 2. 5 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0049]
  • FIG.22A- FIG.22D demonstrate the comparison of activation and Vingi1 GFP RTE- LNP transfection following antibody activation on day 0 or day 2.
  • FIG.23A- FIG.23B demonstrate LDL-R expression following antibody activation on day 0.
  • FIG.24A- FIG.24F demonstrate expression (FIGS.24A-24C) and integration (FIGS.
  • FIG.25A- FIG.25B demonstrate integration and expression of LNP (non-targeted) containing mRNA for R2-1_TG engineered driver with different MNDopt-GFP R2 reporters at different ratios 2 days post TransActTM activation.
  • FIG.26A -FIG.26B demonstrate integration of TRAMs mediated by tLNP and measured by FACS and dPCR.
  • FIG.27A-FIG.27D show activation with FDA-approved anti-CD3 antibodies. Anti- CD3 or BiTes enable R2 driver- and Vingi1-driver GFP transgene (in cis) integration with R2 driver and Vingi1 driver as measured by FACS and dPCR.
  • FIG.28A-FIG.28E demonstrate expression data and functional activity (killing tumor cells) with several anti-CD19 scFvs TRAMs integrated into PBMCs with Vingi1.
  • FIG.29A-FIG.29D demonstrate expression data with CARs and TRAMs comprising anti-CD20 scFvs integrated into PBMCs with a Vingi1 driver.
  • FIG.30A and FIG.30B demonstrate proliferation of TRAM+ PBMCs produced via driver-based transgene integration in co-culture
  • FIG.31A and FIG.31B present integration and expression data for ten R2 drivers -Vpx (FIG.31A) or +VPX (FIG.31B) with GFP reporter.
  • FIG.32A and FIG.32B are bar graphs demonstrating CAR and TRAM expression (FIG.32A) and integration (FIG.32B) by an R2 driver having amino acid sequence set forth in SEQ ID NO: 642 driving CMV anti-CD19 CD28zeta CAR (nucleic acid sequence set forth in SEQ ID NO: 3312) or TRAM in donor PBMCs over 12 days.
  • FIG.33A - FIG.33C are bar graphs demonstrating CAR and TRAM expression (FIG.
  • FIG.34A demonstrate killing efficacy (FIG.34A) and interferon gamma secretion (FIG.34B) of an R2 driver having amino acid sequence set forth in SEQ ID NO: 1690 driving CMV or CMVg TRAMs.
  • FIG.35 demonstrates the in vivo tumor killing activity using ex vivo generated Vingi1 TRAM.
  • Retrotransposable element (RTE) Integration Systems [0067] Retrotransposable elements (RTEs) have naturally evolved to facilitate the incorporation of DNA copies of RNA into genomes.
  • the RTE integration systems of the disclosure take advantage of RTE function, and offer a novel method for introducing engineered immune receptors into T cells by guiding insertional synthesis of a DNA sequence from an RNA template into host cell DNA.
  • RTE integration systems comprising a driver nucleic acid and a template nucleic acid, wherein (a) the driver nucleic acid comprises a nucleic acid sequence encoding an RTE polypeptide (e.g. a site specific RTE polypeptide); and 7 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 (b) the template nucleic acid comprises: (i) a nucleic acid sequence encoding an engineered immune receptor; and (ii) an RTE untranslated region (RTE-UTR) capable of being bound by the RTE polypeptide.
  • RTE-UTR RTE untranslated region
  • the RTE polypeptide is capable of mediating integration, into a host cell genome, of a DNA copy of the nucleic acid sequence encoding the engineered immune receptor.
  • a “DNA copy” refers to a DNA sequence that is produced based on the nucleic acid sequence encoding the engineered immune receptor being a template for transcription and/or reverse transcription.
  • the DNA copy may be the DNA product of reverse transcription of the nucleic acid sequence encoding the engineered immune receptor by the RTE polypeptide.
  • the DNA copy may be the DNA product of reverse transcription, by the RTE polypeptide, of an RNA transcript of the nucleic acid sequence encoding the engineered immune receptor.
  • the template nucleic acid comprises a promoter sequence capable of driving transcription of the nucleic acid sequence encoding the engineered immune receptor after insertion into the host cell DNA.
  • the template nucleic acid comprises 5’ UTR between the start of transcription and the beginning of the nucleic acid sequence encoding the engineered immune receptor.
  • the template nucleic acid comprises a 3’ UTR between the nucleic acid sequence encoding the engineered immune receptor and the end of transcription, with the 3’UTR optionally comprising a polyadenylation sequence.
  • the engineered immune receptor may be a T cell receptor (TCR), a TCR fused antigen modifier (TRAM), a T cell receptor fusion construct (TRuC), a chimeric antigen receptor (CAR), and a costimulatory receptor fusion, as described further herein below.
  • the host cell is an immune cell.
  • the immune cell may be a T cell, a B cell, a NK (natural killer) cell, or an NK-T cell.
  • the immune cell may be an immune cell that natively expresses a TCR-CD3 (T cell receptor-cluster of differentiation 3) complex, e.g., a T cell or a NK-T cell.
  • a “template nucleic acid” as used herein refers to a nucleic acid, e.g., DNA or RNA, and is used interchangeably with “template construct” or simply “template”.
  • a template that is an RNA may be referred to herein as an “RNA template”, “RNA template nucleic acid”, or “RNA template construct”.
  • a “driver nucleic acid” as used herein refers to a nucleic acid, e.g., DNA or RNA, encoding a driver protein and is used interchangeably with “driver nucleic acid”.
  • RNA driver construct A driver nucleic acid that is an RNA may be referred to herein as an “RNA driver construct” or “RNA driver nucleic acid”).
  • the transgene comprised in the template nucleic acid may be in a sense orientation or an anti-sense orientation with respect to the template nucleic acid.
  • the template nucleic acid and the driver nucleic acid are separate nucleic acids, i.e., in trans.
  • the template nucleic acid and the driver nucleic acid are present on a single nucleic acid, i.e., in cis.
  • the template nucleic acid includes a transgene which may be flanked 5’ and 3’, independently, with one or more regulatory elements.
  • the template nucleic acid includes a driver nucleic acid and may further include one or more regulatory elements at the termini of the nucleic acid.
  • the template nucleic acid includes an adjacent driver nucleic acid and further includes one or more regulatory elements at the termini of the nucleic acid.
  • the driver encoded by the driver nucleic acid may be any one of the engineered drivers described elsewhere herein.
  • the driver nucleic acid may be DNA or RNA (e.g. mRNA).
  • the template nucleic acid may be DNA or RNA (e.g. mRNA).
  • the driver nucleic acid and template are provided in trans, on separate nucleic acids (e.g. on separate RNAs).
  • the driver nucleic acid and template are in a cis configuration and provided in a single nucleic acid.
  • they are provided in cis as DNA.
  • they are provided in cis, as RNA, e.g. mRNA.
  • they are provided in trans as two separate DNA strands.
  • they are provided in trans, as RNA, as two separate RNA strands.
  • a driver nucleic acid encodes a “driver” or “driver protein” (interchangeably used herein) that comprises or consists of an RTE polypeptide (as discussed in further detail below). 9 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0075]
  • the driver nucleic acid includes untranslated regions (UTRs) that stabilize a transcript.
  • the driver nucleic acid comprises a nucleic acid sequence encoding an RTE that is flanked by a 5’UTR (driver 5’UTR) and/or a 3’UTR (driver 3’UTR).
  • the driver 5’UTR comprises a nucleic acid sequence of SEQ ID NO: 29, SEQ ID NO: 80 or SEQ ID NO: 96.
  • the driver 3’ UTR comprises a nucleic acid sequence of SEQ ID NO: 83.
  • the driver nucleic acid is an RNA, optionally mRNA, and comprises one or more chemical and/or sequence modifications.
  • the modification is an RNA CAP, a modified polyA length (e.g., relative to a natural polyA), a chemical modification (e.g., a pseudouridine and/or a methylpseudouridine), a 5’ UTR modification, a 3’ UTR modification, a modified Kozak sequence, a modified (e.g., truncated) stem loop, an RNA stabilization motif, a 5-methoxyuridine (5-MO-U) modification, a 5- methylcytidine (5mC) modification, or one or more additional and/or modified microsatellites.
  • a modified polyA length e.g., relative to a natural polyA
  • a chemical modification e.g., a pseudouridine and/or a methylpseudouridine
  • a 5’ UTR modification e.g., a 3’ UTR modification
  • a modified Kozak sequence e.g., truncated
  • a nucleic acid sequence encoding an engineered protein and/or a transgene is codon optimized (e.g., codon optimized for expression in human cells).
  • codon optimization is for RNA optimization.
  • RNA optimization comprises reducing the Uracil (U) load of an RNA molecule.
  • Engineered drivers [0077] Retrotransposable elements, which are also referred to herein interchangeably as “retroelements” or “RTEs”, or “retrotransposons” are nucleotide sequences that have naturally evolved to facilitate the proliferation of their own nucleotide sequences throughout the genome of a cell.
  • RTE typically encodes a protein, which may be referred to herein as an “RTE protein” or “driver”, that includes, among other domains, a reverse transcriptase (RT) domain and an endonuclease (EN) domain.
  • RTE protein or “driver”
  • RT reverse transcriptase
  • EN endonuclease
  • the RNA template is generally the RNA that encoded the RTE protein (which may be referred to herein as a “RTE transcript”), such that the reverse transcription results in replicative insertion of the original RTE in cDNA form.
  • the RNA copy of the RTE comprises one or more untranslated regions (RTE-UTRs) that, as RNA, form structures capable of being bound by the driver, e.g., by a compatible RNA binding domain (RBD) within the driver, to create a ribonucleoprotein complex that traverses back into the nucleus to initiate and ultimately 10 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 complete the reverse transcription of the RTE transcript by the RTE protein.
  • RTD RNA binding domain
  • the driver and one or more RTE-UTRs of a given RTE have co-evolved to have sufficient binding affinity for each other to allow them to bind to each other in the environment of a cell interior, e.g., in the cytoplasm.
  • the EN domain initiates the reverse transcription of the RTE transcript by creating a nick at a target site within genomic DNA of a cell.
  • new DNA is synthesized using the RTE transcript as a template.
  • the RTE protein switches to a second nick and exposed 3’ hydroxyl to begin synthesis of the second DNA strand.
  • the two nicks are sealed by the action of one or more host cell DNA repair mechanisms.
  • a “driver protein” refers to an RTE protein, or an engineered variant thereof comprising an RTE protein or a portion thereof.
  • the RTE protein is modified with amino acid substitutions, truncation, domain fusions (as described in further detail hereinbelow).
  • the driver comprises one or more heterologous polypeptides, e.g., fused directly or indirectly to an RTE protein or a portion thereof (as described in further detail hereinbelow).
  • an “engineered driver protein” refers to a driver that is an engineered variant of a naturally occurring, wild type (WT) RTE protein (which may be referred to herein as a “WT driver”).
  • Engineered drivers of the disclosure may retain the same or substantially the same function as a WT driver, e.g., at least an RT activity and an EN activity.
  • a “polypeptide” of a protein refers to either a full-length version of a protein or to a portion thereof.
  • a polypeptide of a protein may also comprise one or more amino acid substitutions with respect to the protein.
  • an “RTE polypeptide” refers to a full-length RTE protein or to a portion thereof.
  • the portion may be, for example, a functional domain of the protein.
  • the portion may be what remains of an RTE protein after deletion of one or more amino acids, segments, functional domains, binding domains, motifs, or combinations thereof.
  • An “RTE polypeptide” as used herein may also include a full-length RTE protein or a portion thereof that has been subjected to one or more amino acid substitutions.
  • RTE polypeptide For example, what remains of an RTE protein after deletion of the first 100 amino acids from the N-terminus, along with substitutions of certain amino acids in the RT domain and EN domain, may be referred to herein 11 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 as an RTE polypeptide.
  • RTE polypeptide For example, what remains of an RTE protein after deletion of the first 100 amino acids from the N-terminus, as well as the RT domain, may be referred to herein as an RTE polypeptide.
  • engineered drivers comprising an RTE protein or portion thereof (e.g., an RTE polypeptide), and optionally comprising one or more heterologous polypeptides, that can genomically incorporate template nucleotides with an improvement in at least one of efficiency, specificity, accuracy, fidelity or processivity compared to a reference driver, e.g., a naturally occurring, wild type (WT) RTE protein.
  • a reference driver e.g., a naturally occurring, wild type (WT) RTE protein.
  • WT wild type
  • the present disclosure also provides nucleic acids (which may be interchangeably referred to herein as “polynucleotides”, and may be DNA, RNA or a DNA/RNA hybrid) that encode the engineered drivers.
  • Engineered drivers of the disclosure may comprise an RTE polypeptide of an RTE protein encoded in one of various RTEs, including those known in the art, as well as new RTEs described herein.
  • the RTE is a non-LTR retroelement.
  • the non-LTR retroelement is a long-interspersed element polypeptide (LINE) or a short-interspersed element (SINE).
  • LINEs Long INterspersed Elements
  • SINEs Short INterspersed Elements
  • Wild-type LINEs typically encode a protein with reverse transcriptase and endonuclease activities. SINEs do not encode reverse transcriptase or endonuclease and depend on reverse transcriptase and endonuclease encoded by partner LINEs.
  • the LINE is a LINE-1, a LINE-2, or a LINE-3.
  • the RTE polypeptide is a non-site specific RTE polypeptide. In some embodiments, the RTE polypeptide is a site specific RTE polypeptide.
  • the RTE polypeptide is derived from a Class I transposable element that utilizes an intermediate RNA during integration by a copy-and-paste mechanism, with or without Long Terminal Repeats (LTR) or Inverted Terminal Repeats (ITR) in their structure, and that can be derived from a superclade selected from the group consisting of retrons (include clades AbiA, AbiK, AbiP2, CRISPR, CRISPR-like, DCRs, G2L4, G2L, G2Lb, G2Lc, GII, Retrons, UG1, 12 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 UG2, UG3, UG4, UG5, UG6, UG7, UG8, UG9, UG10, UG11, UG12, UG13, UG14, UG15, UG16, UG17, UG18, UG19, UG20, UG21, UG22, UG
  • the RTE polypeptide is derived from an RTE selected from the group consisting of CRE, R2 group (including R4 clade and R2 clade), Hero, NeSL, RandI, Proto1, L1, Tx1, RTEPT, Proto2, RTEX, RTE, Outcast, Ingi (which includes sub-clade Vingi), I, Nimb, Tad1, Loa, R1, Jockey, Rex1, CR1, L2, L2A, L2B, Daphne, and Crack.
  • the RTE polypeptide is a Vingi1-Acar RTE protein or portion thereof.
  • the Vingi1-Acar polypeptide has an amino acid sequence set forth in SEQ ID NO: 31 (WT Vingi1-Acar RTE protein), or a sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • WT Vingi1-Acar RTE protein WT Vingi1-Acar RTE protein
  • R2 group RTEs in nature are non-long terminal repeat (non-LTR) retroelements that specifically insert DNA sequences into ribosomal RNA (rRNA) gene loci in the genomic DNA of a host cell.
  • R2 group RTEs which include members of the R2 clade and R4 clade, typically include a single open reading frame (ORF) that encodes a single protein.
  • RTEs of the R2 clade as well as the R4 clade may be referred to herein as “R2 RTEs”. 13 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0088]
  • Non-R2 RTEs including LTR RTEs and non-LTR RTEs enumerated above that are not in the R2 group, also typically encode an RTE protein that comprises an RT domain and an EN domain, and are referred to herein as “non-R2 RTE proteins”.
  • These non-R2 RTEs may have, depending on their classification, one or more additional ORFs that encode chaperone proteins that have certain RNA- or DNA-binding domains.
  • an engineered driver comprises an R2 polypeptide, that is, a polypeptide of an R2 protein.
  • the R2 polypeptide may be fused to one or more heterologous polypeptides, of which various embodiments are described herein.
  • R2 RTEs are widely distributed across various animal species, with a presence in invertebrates such as insects and mollusks, as well as in vertebrates such as birds, reptiles, and amphibians.
  • R2 RTEs are typically integrated into the 28S ribosomal RNA (rRNA) genes and are maintained in a stable, lineage-specific manner across generations.
  • R2 RTE proteins from different animal species show sequence variation, reflecting the adaptation of R2 elements to different host genomes and their co-evolution.
  • a particular species of animal may have multiple versions of R2 group RTEs, each with different nucleotide sequences (as well as different amino acid sequences for their respective RTE proteins), integrated within their genome.
  • Each unique RTE from a given animal species may be described herein with the following nomenclature: A clade identifier (e.g. R2) followed by the name of the organisms from which the RTE originated, or a two- to four-letter abbreviation thereof.
  • a clade identifier e.g. R2
  • an RTE from clade R2 of Taeniopygia guttata may be referred to herein as a “R2-TG”.
  • the clade identifier and the species abbreviation may be interspersed with a counter numeral.
  • a first RTE from clade R2 of Chamaetylas choloensis may be referred to as R2-1-ChCh (or R2_1-ChCh, or R2-1_ChCh)
  • a second RTE from clade R2 of Chamaetylas choloensis may be referred to as R2-2-ChCh (or R2_2-ChCh, or R2-2_ChCh).
  • RTE proteins and polypeptides, and nucleic acids encoding them may be referred to in a similar manner.
  • a protein encoded in an R2 RTE may be referred to herein as an R2 protein
  • an R2 14 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 protein encoded in the R2-1-ChCh RTE may be referred to herein as an R2-1-ChCh protein
  • an R2 polypeptide of an R2 protein encoded in the R2-1-ChCh RTE may be referred to herein as an R2-1-ChCh polypeptide.
  • an engineered driver comprising a given RTE polypeptide may be referred to by the particular RTE polypeptide comprised in the driver, e.g., an engineered driver comprising an R2 polypeptide may be referred to herein as a “R2 driver”, and an engineered driver comprising an R2-TG polypeptide may be referred to herein as a “R2-TG driver”.
  • an R2 polypeptide of an R2 protein may be the wildtype (WT) version of the corresponding R2 protein.
  • WT wildtype version of the corresponding R2 protein.
  • an R2 polypeptide of an R2 protein may be a portion of the WT version of the corresponding R2 protein.
  • the R2 polypeptide of an R2 protein may comprise one or more amino acid substitutions compared to the WT version of the corresponding R2 protein.
  • a WT version of an R2 protein may be referred to as a “WT R2 protein”.
  • a WT R2 protein typically comprises the following domains: a DNA binding domain cluster, which may comprise, for example, one or more zinc-finger (ZF) domains and one or more Myb domains; an RNA binding domain, a reverse transcriptase (RT) domain, and an endonuclease (EN) domain.
  • ZF zinc-finger
  • RT reverse transcriptase
  • EN endonuclease
  • the DNA binding domain cluster is located approximately at residues 216- 319.
  • the DNA binding domain cluster comprises a first ZF domain located approximately at residues 216-281, a second ZF domain located approximately at residues 297-329, and a Myb domain located approximately at residues 337-391.
  • the RT domain is located approximately at residues 591-1029, and the EN domain is located approximately at residues 1154-1390.
  • segments of polypeptides between domains may be designated as “linkers” that link adjacent domains. It will be appreciated that the boundaries of the domains are approximate and largely based on homology with existing protein structures.
  • the location of any given domain may be offset by, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 residues.
  • the R2 polypeptide may have least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the WT version of the corresponding R2 protein.
  • % Sequence identity may be calculated in one of a number of ways. % Sequence identity may be calculated in one of a number of ways. In certain embodiments, the % sequence identity may be a % subject identity, which is the percentage of identical matches between a query sequence and a subject sequence, relative to the length of the query sequence.
  • the % sequence identity may be a % query identity, which is the percentage of identical matches between a query sequence and a subject sequence, relative to the length of the subject sequence.
  • the % sequence identity may be a % alignment identity, which restricts the calculation of the % identity to the region(s) of alignment, so that large deletions or added domains are discounted.
  • an engineered driver of the disclosure may comprise an RTE polypeptide of one of the following R2 proteins, as listed in Table 1: Table 1: R2 RTE protein sequences (amino acid and DNA sequence) of representative RTEs RTE name Organism species Amino acid sequence Nucleotide of RTE protein sequence encoding SEQ ID NO RTE protein SEQ ID NO 1 R2-1-TG* Taeniopygia guttata 75 2379 2 R2-ZA Zonotrichia albicollis 373 3 R4-AC Anolis carolinensis 374 2380 4 R4-1-PH Parhyale hawaiensis 795 29 * R2-1-TG is also referred to herein as “R2-1_TG” or “R2-TG”, which are all to be understood to refer to the same RTE, with the WT RTE protein having the amino acid sequence set forth in SEQ ID NO: 75, and encoded by the DNA sequence set forth in SEQ ID NO: 2379 (or a RNA version thereof
  • an engineered driver of the disclosure may comprise an RTE polypeptide of one of the following R2 proteins, as listed in Table 2: 16 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 Table 2: R2 RTE protein sequences (amino acid and DNA sequence) of R2 RTEs RTE name Abbre Organism Amino acid Nucleotide viation species sequence of sequence of RTE protein RTE protein SEQ ID NO SEQ ID NO 1 R2-1_Acridotheres tristis R2-AT Acridotheres tristis 582 1714 2 R2-1_Acrocephalus R2- Acrocephalus _scirpaceus _scirpaceus ASS scirpaceus scirpaceus 583 1715
  • an engineered driver of the disclosure may comprise an R2 polypeptide of one of the following R2 proteins: R2-XC protein (SEQ ID NO: 624), R2-LS protein (SEQ ID NO: 607), R2-MC protein (SEQ ID NO: 608), R2-GP protein (SEQ ID NO: 603), or R2-PTT protein (SEQ ID NO: 621).
  • an engineered driver comprises a non-R2 RTE polypeptide.
  • the non-R2 RTE polypeptide may be fused to one or more heterologous polypeptides, of which various embodiments are described herein.
  • Engineered drivers comprising an RTE protein or portion thereof, and which may be optionally fused to one or more heterologous polypeptides.
  • the engineered drivers of the disclosure can genomically incorporate template nucleotides with an improvement in at least one of efficiency, specificity, accuracy, fidelity or processivity compared to a reference driver, e.g., a naturally occurring, wild type (WT) RTE protein.
  • WT wild type
  • Efficiency of genomic integration can be measured, for example, by amplicon sequencing and comparing the number of insertions to non- insertions at a target site and characterizing as a percentage, or by genomically integrating a transgene encoding a fluorescent protein (e.g., GFP), and measuring a percentage of fluorescent protein-positive cells.
  • a fluorescent protein e.g., GFP
  • efficiency and activity are used interchangeably when used with respect to genomic integration of transgenes by engineered drivers of the disclosure.
  • engineered drivers of the disclosure may exhibit improvement in efficiency of genomic integration by at least 1.5x fold, at least 2x fold, at least 2.5x fold, at least 3.5x fold, at least 4x fold, at least 4.5x fold, at least 5x fold, at least 10x fold, at least 15x fold, at least 20x fold, at least 25x fold, at least 30x fold, at least 40x fold, at least 50x fold, at least 60x fold, at least 70x fold, at least 80x fold, at least 90x fold, at least about 100x fold, at least about 150x fold, at least about 200x fold, at least about 300x fold, at least about 400x fold, at least about 500x fold, at least about 750x fold, or about 1000x fold compared to a reference driver.
  • the term “specificity” with respect to gene insertion as used herein refers to the proportion of insertions at a specific target site versus another site. An engineered protein with high specificity would exhibit few or no off-target insertions compared to insertions into a safe harbor site. Specificity may be measured by, for example, amplicon sequencing of known or predicted off-target sites. Engineered drivers with greater specificity are desired. [0106] The term “accuracy” with respect to gene insertions as used herein refers to the percentage of full length insertions at the target site and can be measured by, for example, sequencing the target site and comparing correct insertions to total insertions at the site of interest. Engineered drivers with greater accuracy are desired.
  • fidelity with respect to gene insertion as used herein refers to the nucleotide misincorporation rate as measured, for example, on a per nucleotide basis of the DNA sequence compared to the RNA template sequence. Engineered drivers with greater fidelity are desired.
  • processing with respect to gene insertion as used herein is a measure of the proportional presence of some sequence distal to the RT initiation site versus some sequence proximal to the RT initiation site. Engineered drivers with higher processivity are desired. In some embodiments, large insertions may be desired. A large insertion may be a nucleic acid sequence of about 20 to about 10,000 bases or more.
  • heterologous polypeptides For example, about 20 bases, about 50 bases, about100 bases, about 200 bases, about 300 bases, about 400 bases, about 500 bases, about 200 bases, about 300 bases, about 400 bases, about 500 bases, about 750 bases, about 1000 bases, about 1250 bases, about 1500 bases, about 2,000 bases, about 3,000 bases, about 21 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 4,000 bases, about 5,000 bases, about 6,000 bases, about 7,000 bases, about 8,000 bases, about 9,000 bases, about 10,000 bases or more.
  • engineered drivers that comprises an RTE polypeptide fused to one or more heterologous polypeptides (e.g.
  • any disclosures herein regarding embodiments of engineered drivers, RTE polypeptides, and heterologous polypeptides fused thereto, should be understood as also disclosing nucleic acids that encode the embodiments of engineered drivers, RTE polypeptides, RTE polypeptide fragments and heterologous polypeptides fused thereto.
  • a “heterologous” polypeptide of the engineered driver refers to a polypeptide that is not a portion of the natural RTE protein from which the RTE polypeptide is derived, and is separate and independent from the heterologous template nucleic acid of the disclosure (which are described further herein below).
  • some of the polypeptides disclosed herein may be known to have more than one function and hence be present in one or more functional category.
  • Rad 51 and RAD17 are listed as both RNA/DNA processing polypeptides and RNA/DNA repair polypeptides.
  • the driver comprises an RTE polypeptide fused to at least one (e.g., 1, 2, 3, 4, etc.) heterologous polypeptide at the N-terminus of the RTE polypeptide. In certain embodiments, the driver comprises an RTE polypeptide fused to at least one (e.g., 1, 2, 3, 4, etc.) heterologous polypeptide at the C-terminus of the RTE polypeptide. In certain embodiments, the driver comprises at least one (e.g., 1, 2, 3, 4, etc.) heterologous polypeptide inserted within an RTE polypeptide.
  • the driver comprises an RTE polypeptide fused to at least two heterologous polypeptides, at least one at the N-terminus of the RTE polypeptide and at least one at the C-terminus of the RTE polypeptide. In certain embodiments, the driver comprises an RTE polypeptide fused to at least two heterologous polypeptides, at least one at the N-terminus of the RTE polypeptide and at least one inserted within the RTE polypeptide.
  • the driver comprises an RTE polypeptide fused to at least two heterologous polypeptides, at least one at the C-terminus of the RTE polypeptide and at least one inserted within the RTE 22 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 polypeptide.
  • the driver comprises an RTE polypeptide fused to at least two heterologous polypeptides, fused at the N-terminus of the RTE polypeptide [0113]
  • the at least one heterologous polypeptide comprises a heterologous nucleosome binding polypeptide, a heterologous nucleic acid binding polypeptide, a heterologous RNA/DNA repair polypeptide, a heterologous RNA/DNA processing polypeptide, heterologous nuclear localization signal, a heterologous reverse transcriptase, a heterologous endonuclease, a heterologous intrinsically disordered polypeptide, or any combination thereof.
  • pairs of adjacent heterologous polypeptides may be connected directly, or via a linker.
  • a heterologous polypeptide and an RTE polypeptide situated adjacent to each other may be directly connected, or connected via a linker.
  • the linker is a rigid linker.
  • the linker is a flexible linker. Flexible linkers are generally made up of small, non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. Alternating Gly and Ser residues provides flexibility.
  • the linker is a glycine-serine based linker or a XTEN peptide linker.
  • the linker is a cleavable linker. Solubility of the linker and associated sequences may be enhanced by the inclusion of charged residues, e.g., two positively charged residues (e.g., Lys) and one negatively charged residue (e.g., Glu).
  • the linker may be 2-35 amino acids long.
  • the linker is selected from any one of SEQ ID NOs: 427, 428 or 429.
  • the at least one heterologous polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 76, 383-400, 402-422, 539, 544, 585, 591, 592, 598, 599, 691, 696-700, 706, 707, 712, 713, 718, 719, 720, 725, 726, 736, 737, 742, 747, 748, 749, 754, 755, 759- 763, 768- 771, 776- 779, 784-787, 792-794, 835- 838, 1214-or an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or 99% identical, to a
  • Non-limiting examples of engineered drivers that can be used (e.g., directly or encoded on an RNA and/or DNA molecule) to promote insertion of a heterologous gene into a target nucleic acid in a host cell include protein fusions comprising an RTE polypeptide, optionally fused to at least one heterologous polypeptide that redirects and/or enhances insertion of the heterologous gene.
  • each of the at least one heterologous polypeptide comprises one or more of, e.g., an RNA/DNA processing polypeptide, an RNA/DNA repair polypeptide, a nucleic acid binding polypeptide, or a nucleosome binding polypeptide.
  • the at least one heterologous polypeptide can be fused to the N-terminus and/or C-terminus of an RTE polypeptide, and/or internally within an RTE polypeptide (e.g., between two domains of the RTE polypeptide).
  • Nucleic acids encoding one or more engineered drivers can be used to promote insertion of a DNA copy of a template nucleic acid (e.g., a template nucleic acid comprising a gene of interest) into a target nucleic acid.
  • a template nucleic acid e.g., a template nucleic acid comprising a gene of interest
  • a second nucleic acid a driver nucleic acid that encodes an engineered driver, which together can be delivered or administered to a cell or subject.
  • the nucleic acid that encodes the template and the driver nucleic acid are provided (e.g., administered to a subject) as separate nucleic acid molecules (i.e., in trans), or on a single nucleic acid molecule (i.e., in cis).
  • One or more nucleic acids (e.g., RNA and/or DNA) encoding at least one of these engineered drivers can be provided, in trans or in cis, to target cells (e.g., ex vivo or in vivo) along with one or more nucleic acids (e.g., RNA and/or DNA) encoding a transgene of the present disclosure to promote integration of the transgene into a nucleic acid (e.g., a genomic nucleic acid) of the target cells.
  • an RTE polypeptide is modified to remove the natural EN domain.
  • a heterologous endonuclease domain is fused to the RT domain (e.g., to replace the natural EN domain).
  • hEN heterologous endonuclease domain
  • Various embodiments of heterologous polypeptides that may be fused to an RTE polypeptide in an engineered driver of the disclosure are described in more detail hereinbelow.
  • Heterologous nucleosome binding polypeptide Nucleosome binding polypeptides are characterized by being capable of binding a nucleosome, a section of genomic DNA that is wrapped around a core of histone proteins, which 24 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 forms a unit of DNA packaging in eukaryotes.
  • nucleosome binding polypeptides have various functions that affect access to genomic DNA, including modulating chromatin structure and chromatin accessibility, and altering the activity of genomic editing proteins [0121]
  • the at least one heterologous polypeptide fused to an RTE polypeptide comprises a nucleosome binding polypeptide.
  • the nucleosome binding polypeptide may be a HMGN1 (high mobility group nucleosome-binding domain-containing protein 1) polypeptide, an HMGB1 (high mobility group protein B1) polypeptide, or a StkC (sticky-C) DNA binding domain polypeptide.
  • the HMGN1 polypeptide may comprise an amino acid sequence set forth in SEQ ID NO: 77 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the HMGN1 polypeptide comprises at least one amino acid substitution, wherein the at least one amino acid substitution is at the D100 residue, e.g. D100E and/or D100G relative to SEQ ID NO: 77.
  • the HMGB1 polypeptide may comprise an amino acid sequence set forth in SEQ ID NO: 404 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the StkC binding domain polypeptide may comprise an amino acid sequence set forth in SEQ ID NO: 420 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • an engineered driver of the disclosure may comprise one or more, for example 1, 2, 3, 4, or 5, heterologous nucleosome binding polypeptides, optionally in combination with one or more other heterologous polypeptides as described herein.
  • the engineered driver protein comprises an R2 RTE polypeptide fused to one or more heterologous nucleosome binding polypeptides, e.g., an HMGN1 polypeptide, an HMGB1 polypeptide, an StkC DNA binding domain polypeptide, or combinations thereof.
  • the engineered driver protein comprises an R2 RTE polypeptide fused to an HMGN1 polypeptide.
  • the HMGN1 polypeptide may be situated internally in the R2 RTE protein, on the N-terminal end of the R2 RTE polypeptide or the C-terminal end of the R2 25 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 RTE polypeptide.
  • SEQ ID NOs: 82, 1232, 1233 or 642 set forth amino acid sequences of exemplary driver proteins comprising a HMGN1 polypeptide.
  • Heterologous nucleic acid binding polypeptide [0127]
  • the at least one heterologous polypeptide fused to an RTE polypeptide comprises a nucleic acid binding polypeptide that binds to RNA and/or DNA.
  • a nucleic acid binding polypeptide binds to RNA. In certain embodiments, a nucleic acid binding polypeptide binds to DNA. [0128] In certain embodiments, the nucleic acid binding polypeptide may be a sequence non-specific DNA binding polypeptide. In certain embodiments, the sequence non-specific DNA binding domain may be a Sto7d DNA binding domain polypeptide or a Sso7d DNA binding domain polypeptide. Sso7D is from thermophilic archaea Sulfolobus solfataricus, and the Sto7d protein, is a Sso7D counterpart from Sulfolobus tokodaii.
  • sequence non-specific DNA binding domain may be a UL42 DNA binding domain polypeptide or a Guinea pig cytomegalovirus (CMV) DNA binding domain.
  • CMV Guinea pig cytomegalovirus
  • sequence non-specific DNA binding domains e.g., from the Sso7d homology group can improve processivity of DNA polymerases and reverse transcriptases, and can be used to promote integration of a transgene by an engineered driver.
  • the Sto7d DNA binding domain polypeptide comprises an amino acid sequence set forth in SEQ ID NO 405, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the Sso7d DNA binding domain polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 385, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the UL42 DNA binding domain polypeptide (binding domain from herpes simplex virus 1 (HSV1)) comprises an amino acid sequence set forth in SEQ ID NO: 598, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the Guinea pig CMV DNA binding domain polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 835, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the Rat CMV DNA binding domain polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 836, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the sequence non-specific DNA binding domain may be a single-stranded (ss) DNA binding polypeptide.
  • the ss DNA binding polypeptide may be a RPA3 DNA binding domain polypeptide, which may comprise an amino acid sequence set forth in SEQ ID NO: 388, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the ss DNA binding polypeptide may be a RecT polypeptide, which may comprise an amino acid sequence set forth in SEQ ID NOs: 400 (csRecT) or SEQ ID NO: 402 (paRecT), or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • csRecT amino acid sequence set forth in SEQ ID NOs: 400
  • paRecT SEQ ID NO: 402
  • the ss DNA binding polypeptide may be a single stranded binding (SSB) polypeptide, which may comprise an amino acid sequence set forth in SEQ ID NOs: 421, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the nucleic acid binding polypeptide may be a sequence specific DNA binding polypeptide.
  • site-specific DNA binding domains may improve site-specific integration of a transgene of the disclosure into the genome (e.g., by improving local interaction of the retroelement protein at the genomic site of interest) and can be used to introduce site-specificity into otherwise non-site-specific drivers and/or improve site- specificity for site-specific drivers.
  • the sequence specific DNA binding polypeptide may be a Cas protein.
  • Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, 27 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologues thereof, or modified versions thereof.
  • the Cas protein may be a nucleolytically inactive Cas protein(e.g., a dead SpCas9 having D10A and/or H840A amino acid substitutions).
  • the sequence specific DNA binding polypeptide may be a Zinc finger DNA binding domain (e.g., a Zinc finger DNA binding domain targeting AAVS1, a transcription activator-like effector (TALE) DNA binding domain, or a dead SpuFz1 (e.g., dSpuFz1, a SpuFz1 with a D606A amino acid substitution).
  • TALE transcription activator-like effector
  • the nucleic acid binding polypeptide provides sequence specificity, and may comprise a sequence set forth in any one of SEQ ID NOs: 406-417, or 422- 424, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • a sequence-specific endonuclease may be used, e.g., to enhance or replace a native EN domain of an RTE polypeptide. Such domains may retarget integration to a site of interest.
  • Non-limiting examples of an EN fusion/replacement include a site-specific homing endonuclease targeting the AAVS1 gene fused to RTE polypeptide deficient in endonuclease activity, either through an inactivating mutation in the endonuclease domain (e.g., by a D237A, H238 substitution, and/or D216A in an L2-2 domain or a corresponding mutation in an alternative domain), or through a deletion of the EN domain.
  • an engineered driver of the disclosure may comprise one or more, for example 1, 2, 3, 4, or 5, heterologous nucleic acid binding polypeptides, optionally in combination with one or more other heterologous polypeptides as described herein.
  • the engineered driver protein comprises an R2 RTE polypeptide fused to one or more heterologous nucleic acid binding polypeptides.
  • the engineered driver protein comprises an R2 RTE polypeptide fused to a Sto7d DNA binding domain polypeptide or the UL42 DNA binding domain polypeptide.
  • the fused polypeptide may be situated internally in the R2 RTE protein, on the N-terminal end of the R2 RTE polypeptide or the C-terminal end of the R2 RTE polypeptide.
  • SEQ ID NOs: 500, 509, 631, 1232, 688 or 832 set forth amino acid sequences of exemplary driver proteins comprising a Sto7d DNA binding domain polypeptide or a UL42 DNA binding domain polypeptide.
  • 28 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 Heterologous RNA/DNA repair polypeptide [0143]
  • the at least one heterologous polypeptide fused to an RTE polypeptide comprises an RNA/DNA repair polypeptide, a protein that is or interacts with a host repair protein that acts on RNA and/or DNA.
  • the DNA repair polypeptide is selected from a group consisting of a non-homologous end joining (NHEJ) pathway protein, a mismatch repair (MMR) protein, a microhomology-mediated end-joining (MMEJ) protein, a homology directed repair (HDR) pathway protein, and a DNA damage response protein.
  • NHEJ non-homologous end joining
  • MMR mismatch repair
  • MMEJ microhomology-mediated end-joining
  • HDR homology directed repair
  • Activation or inhibition of certain host cell proteins via a heterologous polypeptide that is fused to an RTE polypeptide e.g., a reverse transcriptase and/or endonuclease domain
  • the precise class of host cell proteins will depend on the mechanism of integration (e.g., depending on the RTE polypeptide that is used.
  • the RNA/DNA repair polypeptide may be a RAD51 polypeptide, a RAD17 polypeptide, a BRCA2 polypeptide, a ANKRD28 polypeptide, an HSV-1 alkaline nuclease polypeptide (e.g., UL12), a proliferating cell nuclear antigen (PCNA) polypeptide, a polypeptide containing a PCNA interacting protein (PIP) motif, a MDC1 polypeptide, a MSH4 polypeptide, a SCML1 polypeptide, a CDKN2A polypeptide, a p53 inhibitor peptide, or a CTIP (CtBP-interacting protein, also known as DNA endonuclease RBBP8 or Retinoblastoma-binding protein 8) polypeptide.
  • PCNA proliferating cell nuclear antigen
  • PIP PCNA interacting protein
  • the RNA/DNA repair polypeptide is a heterologous CTIP polypeptide.
  • the RAD17 polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 396, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the ANKRD28 polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 393, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the HSV-1 alkaline nuclease polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 384, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the BRCA2 polypeptide comprises an amino acid sequence that set forth in SEQ ID NO: 418, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the PCNA polypeptide comprises an amino acid sequence that is set forth in SEQ ID NO: 398 or SEQ ID NO: 399, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the MDC1 polypeptide comprises an amino acid sequence that set forth in SEQ ID NO: 394, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the MSH4 polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 395, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the SCML1 polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 397, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the CDKN2A polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 398, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the p53 inhibitor is a MDM2 polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 390, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the p53 inhibitor is a peptide 14-derived peptide comprising an amino acid sequence set forth in SEQ ID NO: 419, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the CTIP polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 76, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the CTIP polypeptide comprises at least one amino acid substitution, wherein the at least one amino acid substitution is at one or more of (e.g.
  • the CTIP polypeptide comprises the amino acid substitutions of T15I, R123K, P181L, M202L, M202F, M202T, A237L, A237I, A237N, A237K, N288D, D18F, I49C, S17D, D18C, Q46T, I2A, C198S, E155S, L281G, I186S, and Q46K, relative to SEQ ID NO: 76.
  • RNA/DNA repair polypeptide may contain a proliferating cell nuclear antigen (PCNA) interaction (PIP) domain.
  • PCNA proliferating cell nuclear antigen
  • an RTE polypeptide has a modification that inactivates its PIP.
  • an RTE polypeptide has a modification that activates a PIP domain). The PIP domain is believed to recruit the cellular PCNA protein which may play a role in DNA replication.
  • the native RTE PIP domain is replaced with a PIP domain from another protein (such as p21, FEN1 or CHAF1A) which may improve PCNA recruitment.
  • the modification comprises deletion.
  • the modification comprises one or more mutations (e.g., point mutations).
  • the modification is in the EN domain.
  • the modification is in the RT domain.
  • the modification is in the RNA binding domain.
  • PIP polypeptides may be added as a heterologous polypeptide in the C-terminal or N-terminal of the RTE protein, or internally in the RTE protein.
  • the PIP polypeptide is a p21 polypeptide comprising an amino acid sequence that set forth in SEQ ID NO: 419, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the PIP polypeptide is a FEN1 polypeptide comprising an amino acid sequence that set forth in SEQ ID NO: 386, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the PIP polypeptide is a CHAF1A polypeptide comprising an amino acid sequence that set forth in SEQ ID NO: 399, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • an engineered driver of the disclosure may comprise one or more, for example 1, 2, 3, 4, or 5, heterologous RNA/DNA repair polypeptides, optionally in combination with one or more other heterologous polypeptides as described herein.
  • the engineered driver protein comprises an R2 RTE polypeptide fused to one or more heterologous RNA/DNA binding polypeptides.
  • the engineered driver protein comprises an R2 RTE polypeptide fused to a CTIP binding domain polypeptide.
  • the fused CTIP polypeptide may be situated internally in the R2 RTE protein, on the N-terminal end of the R2 RTE polypeptide or the C-terminal end of the R2 RTE polypeptide.
  • SEQ ID NOs: 82, or 652-680 set forth amino acid sequences of exemplary driver proteins comprising a CTIP polypeptide.
  • RNA/DNA processing polypeptide is an enzyme that causes chemical changes to RNA and/or DNA, for example by promoting or retarding RNA degradation.
  • proteins that directly process and/or repair RNA/DNA intermediates involved in transgene integration also may, without being bound by theory, improve transgene integration efficiency.
  • an RNA/DNA processing polypeptide may improve transgene integration efficiency and/or redirect transgene integration to a different target location (e.g., within the genome of a cell).
  • the at least one heterologous polypeptide fused to an RTE polypeptide comprises an RNA/DNA processing polypeptide.
  • the at least one heterologous polypeptide comprises an RNA/DNA processing polypeptide selected 32 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 from an RNA helicase polypeptide, an RNA helicase recruitment motif polypeptide, a RAD51 polypeptide, a RAD17 polypeptide, or a RAD6 polypeptide.
  • RNA helicases are enzymes that unwind RNA structures, and play a role in various cellular processes, including but limited to transcription, RNA splicing, and translation, as well as in the regulation of gene expression.
  • a RNA helicase recruitment motif is a specific sequence or structural feature in proteins that facilitates the binding and recruitment of RNA helicases to their target sites.
  • the at least one heterologous RNA/DNA processing polypeptide comprises an RNA helicase polypeptide.
  • the RNA helicase polypeptide is an EIF4A (Eukaryotic initiation factor 4A) polypeptide.
  • the EIF4A polypeptide comprises an amino acid set forth in SEQ ID NO: 592, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the RNA helicase polypeptide is a Dengue virus RNA helicase polypeptide.
  • the Dengue virus RNA helicase polypeptide comprises an amino acid set forth in SEQ ID NO: 1654, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the at least one heterologous RNA/DNA processing polypeptide comprises a RNA helicase recruitment motif polypeptide.
  • the RNA helicase recruitment motif polypeptide is a TUDOR domain polypeptide from TDRD3 which may recruit RNA helicase DHX9 (DExH box helicase 9).
  • the TUDOR domain polypeptide comprises an amino acid set forth in SEQ ID NO: 591, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the RNA helicase recruitment peptide is derived from EIF4G which recruits EIF4A RNA helicase, as set forth in SEQ ID NO: 837, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the RNA helicase recruitment motif polypeptide is a canine hepacivirus core protein motif polypeptide.
  • the canine hepacivirus core protein motif polypeptide (which recruits cellular DDX3) comprises an amino acid set forth in 33 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 SEQ ID NO: 838, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the canine hepacivirus core protein motif polypeptide comprises an amino acid set forth in SEQ ID NO: 599, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the canine hepacivirus core protein motif polypeptide comprises an amino acid set forth in SEQ ID NO: 838, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • fusing known RNA helicase domains to an RTE polypeptide, or fusing peptides that can recruit cellular RNA helicases to an RTE polypeptide may improve reverse transcription in genome editing systems by removing secondary structure in the RNA template that may block reverse transcription thus reducing activity and processivity.
  • RAD51 is a protein involved in the homology directed repair (HDR) pathway, and a RAD51 domain directly binds to single-stranded DNA and searches for matching DNA in the genome as an early stem in homologous recombination.
  • HDR homology directed repair
  • RAD51 fusion to an RTE polypeptide may improve integration at genomic sites that are homologous to sequences that are included on a template nucleic acid (for example sequences that flank the transgene and are homologous to one or more genomic sequences in the vicinity of the target gene).
  • the RAD51 polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 383, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the RAD17 polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 396, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the RAD6 polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 389, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, 34 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • an engineered driver of the disclosure may comprise one or more, for example 1, 2, 3, 4, or 5, heterologous RNA/DNA processing polypeptides, optionally in combination with one or more other heterologous polypeptides as described herein.
  • the engineered driver protein comprises an R2 RTE polypeptide fused to one or more heterologous RNA/DNA processing polypeptides.
  • the engineered driver protein comprises an R2 RTE polypeptide fused to a RNA helicase polypeptide (e.g., a EIF4A polypeptide or a Dengue virus RNA helicase polypeptide) or a RNA helicase recruitment motif polypeptide (e.g., a TUDOR domain polypeptide).
  • the fused polypeptide may be internal to the R2 RTE protein, on the N-terminal end of the R2 RTE polypeptide or the C- terminal end of the R2 RTE polypeptide.
  • SEQ ID NOs: 592 or 1654 set forth amino acid sequences of exemplary driver proteins comprising a RNA helicase polypeptide.
  • SEQ ID NOs: 591 or 838 set forth amino acid sequences of exemplary driver proteins comprising a RNA helicase recruitment motif polypeptide.
  • the engineered driver protein comprises a non-R2 RTE polypeptide fused to an RNA helicase polypeptide.
  • the non-R2 RTE may be, e.g., Vingi RTE, R2NS RTE,_L4_ACar RTE, a CR1_10_Ami RTE, a ZFL2-2 RTE, or any other non-R2 RTE disclosed elsewhere herein.
  • Heterologous localization signal [0177]
  • the engineered drivers of the disclosure may comprise heterologous localization sequences.
  • a nuclear localization sequence is a polypeptide that promotes import of a protein into a nucleus of a cell.
  • a nucleolar localization sequence is a polypeptide that promotes import of a protein into a nucleolus of a cell.
  • the at least one heterologous polypeptide fused to an RTE polypeptide comprises a localization polypeptide.
  • the localization polypeptide may be a nuclear localization signal (NLS).
  • the localization polypeptide may be a nucleolar localization signal (NoLS).
  • the NLS comprises an amino acid sequence set forth in any one of SEQ ID NOs: 430, 431, 391, 435 or 1677.
  • an NLS comprises a SV40 sequence (e.g., SEQ ID NO: 430), a nucleoplasmin sequence (e.g., SEQ ID NO: 431), a TOPBN1 sequence (e.g., SEQ ID NO: 391), or a bipartite SV40 (bp-SV40) sequence (e.g., SEQ ID NO: 435 or SEQ ID NO: 1677).
  • the NoLS comprises an amino acid sequence set forth in any one of SEQ ID NOs: 432, 433 or 434.
  • a NoLS comprises a PNRC sequence (e.g., SEQ ID NO: 432), a poly R sequence (e.g., SEQ ID NO: 433), or a H2B sequence (e.g., SEQ ID NO: 434).
  • an engineered driver of the disclosure may comprise one or more, for example 1, 2, 3, 4, or 5, heterologous localization sequences, optionally in combination with one or more other heterologous polypeptides as described herein.
  • the engineered driver protein comprises an R2 RTE polypeptide fused to one or more heterologous localization sequences.
  • the engineered driver protein comprises an R2 RTE polypeptide or fragment thereof fused to a NLS or a NoLS.
  • the fused polypeptide may be situated at the N-terminal end of the R2 RTE polypeptide or the C-terminal end of the R2 RTE polypeptide, or internal to the R2 RTE polypeptide.
  • SEQ ID NOs: 82, 751, 752 or 753 set forth amino acid sequences of exemplary driver proteins comprising a NLS or NoLS.
  • Intrinsically disordered regions are regions of proteins that lack a fixed three- dimensional (3D) structure. These IDRs, which lack an amino acid sequence that form, for example, alpha helices, beta sheets, and other canonical 3D protein structures, are thought to provide flexibility, allowing the protein to have ordered interactions with multiple molecular partners within the intracellular environment.
  • IDRs also demonstrate a function of forming and maintaining condensates. Condensates can form and maintain organization through a range of different processes, the most well-known of which is phase separation of proteins and RNA. There are many different examples of intrinsically disordered proteins forming condensates.
  • Condensation is also called phase- separation, and may provide beneficial properties.
  • condensed proteins might require or benefit from phase-separated environment with different 36 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 and optimized chemical-physiological conditions or help with energetic limitations by reducing the entropy and increasing the enthalpy of the system.
  • phase separation can also prevent degradation, and help with nuclear import or to improve protein-RNA intermolecular interactions. For example, it has been shown that for LINE-1 elements, ORF1 tends to condensate.
  • R2-TG has an intrinsically disordered region at its N- terminal (Residues 1-161, 1-184 or 1-218).
  • the at least one heterologous polypeptide fused to an RTE polypeptide comprises a heterologous IDR polypeptide.
  • the heterologous IDR polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOS: 691, 696-700, 706-707, 712-713, 718-720, 725-726, 736-737, 742, 747-749, 754-755, 759-763, 768-771, 776-779, 784-787, 792-794 and 1214-1216, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the heterologous IDR polypeptide may be a condensation polypeptide comprising any one (or a combination of two or more) of the following: the N- terminal 161 residues of R2-TG protein (SEQ ID NO: 1214); the N-terminal 184 residues of R2- TG RTE protein (SEQ ID NO: 1215); the N-terminal 218 residues of R2-TG RTE protein (SEQ ID NO: 1216), a condensation peptide from the first IDR of UBQ2 polypeptide (SEQ ID NO: 691), a condensation peptide from the second IDR of UBQ2 polypeptide (SEQ ID NO: 696), a condensation peptide from the third IDR of UBQ2 polypeptide (SEQ ID NO: 697), a condensation peptide from the second IDR of RBM14 polypeptide (SEQ ID NO: 698), a condensation peptide from the fourth IDR of RBM14 polypeptide (SEQ ID NO: 699),
  • condensation peptide included in an engineered driver is capable of directly binding to the template RNA or to other condensation fused peptides.
  • condensation peptide fusion to an RTE polypeptide may improve integration at genomic sites that are homologous to sequences that are included on a template nucleic acid (for example sequences that flank the transgene and are homologous to one or more genomic sequences in the vicinity of the target gene, e.g., homology arms).
  • the transgene may be in sense or anti-sense orientation.
  • Many RTE proteins have native IDRs.
  • an R2 RTE proteins typically have a native IDR at their N-terminal region.
  • the RTE polypeptide to which the heterologous IDR polypeptide is fused is an RTE polypeptide having a deletion of its native IDR or portion thereof.
  • an engineered driver of the disclosure may comprise one or more, for example 1, 2, 3, 4, or 5, heterologous IDR polypeptides, optionally in combination with one or more other heterologous polypeptides as described herein.
  • the engineered driver protein comprises an R2 RTE polypeptide fused to one or more heterologous IDR polypeptides. In certain embodiments, the engineered driver protein comprises an R2 RTE polypeptide fused to a heterologous IDR polypeptide.
  • the fused heterologous IDR polypeptide may be situated internal to the R2 RTE protein, at the N- terminal end of the R2 RTE polypeptide or the C-terminal end of the R2 RTE polypeptide.
  • SEQ ID NOs: 694-695, 701-705, 708-711, 714-717, 721- 724, 727-735, 738-741, 743-746 or 825-831 set forth amino acid sequences of exemplary driver proteins comprising a heterologous IDR polypeptide.
  • the engineered driver protein comprises a non-R2 RTE polypeptide fused to one or more heterologous IDR polypeptides.
  • the non-R2 RTE may be, e.g., Vingi RTE, R2NS RTE,_L4_ACar RTE, a CR1_10_Ami RTE, a ZFL2-2 RTE, or any other non-R2 RTE disclosed elsewhere herein.
  • Heterologous EN – Chimeric drivers [0192]
  • the at least one heterologous polypeptide fused to an RTE polypeptide comprises a heterologous EN domain polypeptide.
  • an RTE protein typically comprises an EN domain and an RT domain.
  • a heterologous EN domain polypeptide in the context of engineered drivers as described herein refers to an EN domain polypeptide of a protein that is not the RTE protein from which the RTE polypeptide (which may comprise the RT domain) of the engineered driver is derived.
  • the 39 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 heterologous EN domain may be a EN domain polypeptide of a different RTE protein, or a EN domain polypeptide of a non-RTE protein.
  • the heterologous EN domain may be a EN domain polypeptide of a different R2 protein (e.g., an R2-2-AcOr RTE protein or a R4-AC RTE protein).
  • the engineered driver would be a chimeric driver protein comprising EN and RT domains derived from different R2 proteins.
  • the heterologous EN domain polypeptide may be a EN domain polypeptide of a non-R2 RTE protein.
  • the engineered driver would be a chimeric driver protein comprising EN and RT domains derived from different RTE proteins.
  • the heterologous EN domain may be the EN domain of a non- RTE protein.
  • the engineered driver would be a chimeric driver protein comprising an EN domain from a non-RTE source.
  • non-RTE proteins comprising a EN domain include but are not limited to Cas nucleases (e.g., SpCas9 nuclease), a homing endonuclease, or a FokI nuclease.
  • Fanzor nuclease e.g., SpuFz1
  • OMEGA RNA-guided system
  • the heterologous EN domain includes but is not limited to a Fanzor1 or Fanzor2 nuclease.
  • Non- limiting examples of drivers utilizing a Fanzor1 or Fanzor2 nuclease have amino acid sequence set forth in any one of SEQ ID NOs: 473-484.
  • the heterologous EN domain polypeptide may functionally replace the native EN activity of the RTE polypeptide of the engineered driver.
  • the RTE polypeptide of the engineered driver may be lacking a functional EN domain, for example through deletion of the EN domain or portion thereof, and/or through one or more amino acid substitutions that reduce or eliminate enzymatic activity of the native EN domain of the RTE polypeptide.
  • Examples of chimeric drivers with a heterologous EN domain polypeptide provided herein include at least the following: SEQ ID NO: 448 sets forth the amino acid sequence of a chimeric driver of different R2 proteins (R2-ZA and R2-TG), comprising an R2-TG RTE polypeptide and an R2-ZA EN domain (which replaced the R2-TG EN domain).
  • SEQ ID NO: 40 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 473 sets forth the amino acid sequence of a SpuFz1 nuclease R2-TG fusion. The SpuFz1 portion is fused at its N-terminus via a linker to a C-terminal R2-TG protein.
  • SEQ ID NO: 474 sets forth the amino acid sequence of a SpuFz1 nuclease R2-TG fusion.
  • the SpuFz1 portion is fused at its N-terminus via a linker to a truncated C-terminal R2-TG protein, lacking first 209 amino acid residues.
  • SEQ ID NO: 475 sets forth the amino acid sequence of a SpuFz1 nuclease R2-TG fusion.
  • the SpuFz1 portion is fused at its N-terminus via a linker to a truncated C-terminal R2- TG protein, with native endonuclease domain deleted.
  • SEQ ID NO: 476 sets forth the amino acid sequence of a SpuFz1 nuclease R2-TG fusion.
  • the SpuFz1 portion is fused at its C- terminus via a linker to a truncated N-terminal R2-TG protein, with native endonuclease domain deleted.
  • SEQ ID NO: 477 sets forth the amino acid sequence of a SpuFz1 nuclease R2-TG fusion.
  • the SpuFz1 portion is fused at its C-terminus via a linker to an N-terminal R2-TG protein, carrying a K1307A mutation.
  • SEQ ID NO: 478 sets forth the amino acid sequence of a dead SpuFz1 nuclease R2-TG fusion.
  • the SpuFz1 carries a D606A mutation and is fused at the N-terminus fused via a linker to a C-terminal R2-TG protein.
  • SEQ ID NO: 479 sets forth the amino acid sequence of a dead SpuFz1 nuclease R2-TG fusion.
  • the SpuFz1 carries a D606A mutation and is fused at its N-terminus via a linker to a C-terminal R2-TG protein, lacking first 209 amino acid residues.
  • SEQ ID NO: 480 sets forth the amino acid sequence of a SpuFz1 nuclease R2-TG fusion.
  • the SpuFz1 is fused at its N-terminus via a linker to a C-terminal R2- TG protein (lacking 209 N-terminal amino acids.
  • SEQ ID NO: 481 sets forth the amino acid sequence of a SpuFz1 nuclease R2-TG fusion.
  • the SpuFz1 is fused at its N-terminus via a linker to a C-terminal R2-TG protein without the native DNA binding domain, and carrying K1307A mutation.
  • SEQ ID NO: 482 sets forth the amino acid sequence of a SpuFz1 nuclease R2-TG fusion.
  • the SpuFz1 is fused at its N-terminus via a linker to a C-terminal R2-TG protein carrying a K1307A mutation.
  • SEQ ID NO: 483 sets forth the amino acid sequence of a SpuFz1 nuclease R2-TG fusion.
  • the SpuFz1 is fused at the C-terminus via a linker to an N-terminal R2- TG protein (lacking 209 N-terminal amino acid residues).
  • SEQ ID NO: 484 sets forth the amino acid sequence of a SpuFz1 nuclease R2-TG fusion. In each of the above, the SpuFz1 is fused at the C-terminus via a linker to a truncated N-terminal R2-TG protein.
  • the at least one heterologous polypeptide fused to an RTE polypeptide comprises a heterologous RT domain polypeptide.
  • 41 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032
  • an RTE protein typically comprises an EN domain and an RT domain.
  • a heterologous RT domain polypeptide in the context of engineered drivers as described herein refers to an RT domain polypeptide of a protein that is not the RTE protein from which the engineered driver is derived.
  • the heterologous RT domain polypeptide may be an RT domain polypeptide of a different RTE protein, or an RT domain polypeptide of a non-RTE protein.
  • a given engineered driver comprises an RTE polypeptide of an R2-1-TG RTE protein
  • the heterologous RT domain polypeptide may be an RT domain polypeptide of a different R2 protein (e.g., an R2-2-AcOr RTE protein or a R4-AC RTE protein).
  • the engineered driver would be a chimeric driver protein comprising EN and RT domains derived from different R2 proteins.
  • the RT domain polypeptide of a different R2 RTE protein may be derived from an R2-Toc RT domain, optionally comprising an amino acid sequence set forth in SEQ ID NO: 539, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto.
  • the heterologous RT domain polypeptide in an engineered driver may be an RT domain polypeptide of a non-R2 RTE protein.
  • the engineered driver would be a chimeric driver protein comprising EN and RT domains derived from different RTE proteins.
  • the RT domain polypeptide of a non-R2 RTE protein may be from an LTR retroelement or an ERV.
  • the RT domain polypeptide of a non-R2 RTE protein may be from a non-LTR retroelement.
  • the RT domain polypeptide of a non-R2 RTE protein may be from a Vingi RTE.
  • the RT domain polypeptide of a non-R2 RTE protein may be from an R2-NS RTE.
  • the RT domain polypeptide of a non-R2 RTE protein may be from a L2_L4_ACar RTE. In certain embodiments, the RT domain polypeptide of a non-R2 RTE protein may be from a CR1_10_Ami RTE. In certain embodiments, the RT domain polypeptide of a non-R2 RTE protein may be from a ZFL2-2 RTE. [0203] In certain embodiments, if a given engineered driver comprises an RTE polypeptide of an RTE (e.g. R2 RTE) protein, then the heterologous RT domain polypeptide may be the RT domain polypeptide of a non-RTE protein.
  • RTE e.g. R2 RTE
  • the engineered driver would be a chimeric driver protein comprising an RT domain from a non-RTE source.
  • the RT domain comprises a sequence may be from a MMLV RT. Marathon RT, a TGIRT_III RT, or a SSIV RT.
  • the heterologous RT domain polypeptide may functionally replace the native RT activity of the RTE polypeptide of the engineered driver.
  • the RTE polypeptide of the engineered driver may be lacking a functional RT domain, for example through deletion of the RT domain or portion thereof, and/or through one or more amino acid substitutions that damage or eliminate enzymatic activity of the native RT domain of the RTE polypeptide.
  • SEQ ID NO: 534 sets forth an amino acid sequence of a Vingi RT.
  • SEQ ID NO: 535 sets forth amino acid sequence of an MMLV RT.
  • SEQ ID NO: 536 sets forth amino acid sequence of an R2-NS RT.
  • SEQ ID NO: 537 sets forth amino acid sequence of a L2_L4_ACar RT.
  • SEQ ID NO: 538 sets forth amino acid sequence of a CR1-10_Ami RT.
  • SEQ ID NO: 539 sets forth amino acid sequence of an R2 Toc RT.
  • SEQ ID NO: 540 sets forth amino acid sequence of a ZFL2-2 RT.
  • SEQ ID NO: 425 sets forth amino acid sequence of a Marathon RT.
  • SEQ ID NO: 541 sets forth amino acid sequence of a TGIRT_III RT.
  • SEQ ID NO: 542 sets forth amino acid sequence of an MMLV derived RT peptide.
  • SEQ ID NO: 543 sets forth amino acid sequence of a SSIV RT.
  • Examples of chimeric drivers with a heterologous RT domain polypeptide provided herein include at least the following: R2-TG chimera with Vingi RT (SEQ ID NO: 517), R2-TG chimera with MMLV RT (SEQ ID NO: 518), R2-TG chimera with R2NS RT (SEQ ID NO: 519), R2-TG chimera with L2_24_ACar-RT (SEQ ID NO: 520), R2-TG chimera with CR1- 10_Ami-RT (SEQ ID NO: 521), R2-TG chimera with R2Toc-RT (SEQ ID NO: 522), R2-TG chimera with ZFl2-2 RT (SEQ ID NO: 523), R2-TG chimera with Marathon RT (SEQ ID NO: 524), R2-TG chimera with TGIRT_III RT (SEQ ID NO: 525), R2-TG mutated RT with YADD RT motif (SEQ ID NO: 526),
  • SEQ ID NO: 376 sets forth the amino acid sequence of a chimeric driver between different R2 proteins (R2-ZA 43 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 and R2-TG), comprising an R2-TG RTE polypeptide and an R2-ZA RT domain (which replaces the R2-TG RT domain).
  • R2-ZA 43 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 and R2-TG
  • R2-ZA RT domain which replaces the R2-TG RT domain.
  • an engineered driver fused to a CTIP polypeptide and an HMGN1 polypeptide as well as an engineered driver, e.g., a driver comprising an R2 polypeptide, fused to a Sto7d polypeptide and an HMGN1 polypeptide.
  • the engineered driver comprises an R2 polypeptide fused to a CTIP polypeptide and an HMGN1 polypeptide.
  • the engineered driver comprises, in an N-terminal to C-terminal order, the CTIP polypeptide, the HMGN1 polypeptide, and the R2 polypeptide.
  • the R2 polypeptide may be lacking a native N-terminal intrinsically disordered region.
  • the engineered driver comprises an R2 polypeptide fused to a CTIP polypeptide, an HMGN1 polypeptide, and a heterologous intrinsic disordered region.
  • the engineered driver comprises, in an N-terminal to C-terminal order, a heterologous intrinsic disordered region, a CTIP polypeptide, a HMGN1 polypeptide, and an R2 polypeptide.
  • the R2 polypeptide may be lacking a native N-terminal intrinsically disordered region.
  • a driver polypeptide having the above-noted arrangement of components may comprise an amino acid sequence set forth in any one of SEQ ID NOs: 694, 695, 701, 702, 703, 704, 705, 708, 709, 710, 711, 714, 715, 716, 717, 721, 722, 723, 724, 727, 728, 729, 730, 731, 732, 733, 734, 735, 738, 739, 740, 741, 743, 744, 745 or 746 [0210]
  • the engineered driver comprises an R2 polypeptide fused to a CTIP polypeptide, an HMGN1 polypeptide, and a RNA helicase polypeptide.
  • the engineered driver comprises, in an N-terminal to C-terminal order, a RNA helicase polypeptide, a CTIP polypeptide, an HMGN1 polypeptide, and an R2 polypeptide.
  • the RNA helicase polypeptide may be, e.g., an EI4A polypeptide or a Dengue virus RNA helicase polypeptide.
  • the R2 polypeptide may comprises a deletion within its native N-terminal intrinsically disordered region.
  • a driver polypeptide 44 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 having the above-noted arrangement of components may comprise an amino acid sequence set forth in SEQ ID NO: 833.
  • the engineered driver comprises an R2 polypeptide fused to a Sto7d polypeptide and an HMGN1 polypeptide.
  • the engineered driver comprises, in an N-terminal to C-terminal order, the Sto7d polypeptide, the HMGN1 polypeptide, and an R2 polypeptide.
  • the R2 polypeptide may be lacking a native N-terminal intrinsically disordered region.
  • the engineered driver comprises an R2 polypeptide fused to a Sto7d polypeptide, an HMGN1 polypeptide, and a RNA helicase recruitment motif polypeptide.
  • the engineered driver comprises, in an N-terminal to C-terminal order, the RNA helicase recruitment motif polypeptide, the Sto7d polypeptide, the HMGN1 polypeptide, and an R2 polypeptide.
  • the RNA helicase recruitment motif polypeptide may be, e.g., a TUDOR domain polypeptide.
  • the R2 polypeptide may comprise a deletion within its native N-terminal intrinsically disordered region.
  • an engineered driver having the above-noted arrangement of components may comprise an amino acid sequence set forth in SEQ ID NO: 1232, which may be encoded by a nucleotide sequence set forth in SEQ ID NO: 3330 (exemplary IVT sequence set forth in SEQ ID NO: 3329).
  • the engineered driver having the amino acid set forth in SEQ ID NO:1232 comprises: an amino acid sequence of R2-1_TG Orf2 polypeptide with N-terminal deletion of residues 1-184; N-terminal fusion of the following: SV40-NLS polypeptide SEQ ID NO:430, TUDOR polypeptide SEQ ID NO:3269, sto7D polypeptide SEQ ID NO:405, and HMGN polypeptide SEQ ID NO:77; and amino acid substitutions P280K, D555K, T1011S, I1219N, A1282G.
  • IVT nucleotide sequence SEQ ID NO:3329 which encodes the SEQ ID NO:1232 amino acid sequence, includes the following: synthetic 5' UTR SEQ ID NO:80; mouse alpha globin 3'UTR SEQ ID NO:83; and A29N10A70 polyA signal SEQ ID NO:3201. The orientation of the sequences between external UTRs is in antisense.
  • the engineered driver comprises an R2 polypeptide fused to a Sto7d polypeptide and an HMGN1 polypeptide.
  • SEQ ID NO:1690 sets forth the amino acid sequence of an engineered driver comprising: an R2-1_TG Orf2 polypeptide with N-terminal deletion of residues 1-184; N-terminal fusion of the following: SV40-NLS polypeptide SEQ ID NO:430, Sto7D polypeptide SEQ ID NO:405, and HMGN polypeptide SEQ ID NO:77; and 45 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 amino acid substitutions P280K, D555K, D923K, T1011S, I1219N, A1282G.
  • SEQ ID NO:3009 sets forth a nucleotide sequence encoding the SEQ ID NO:1690 protein, and includes synthetic 5' UTR SEQ ID NO:80; mouse alpha globin 3'UTR SEQ ID NO:83; and A29N10A70 polyA signal SEQ ID NO:3201. The orientation of the sequences between external UTRs is in antisense.
  • SEQ ID NO: 642 is the amino acid sequence of an exemplary R2 driver comprising: R2- 1_TG polypeptide with deletion of N-terminal residues 1-184; N-terminal fusion of the following: SV40-NLS polypeptide SEQ ID NO:3199, sto7D polypeptide SEQ ID NO:405, HMGN polypeptide SEQ ID NO:77; and amino acid substitutions P280K, D555K, T1011S, I1219N, A1282G.
  • SEQ ID NO:3299 sets forth a nucleotide sequence encoding the SEQ ID NO:642 protein, and includes synthetic 5' UTR SEQ ID NO:80 ; mouse alpha globin 3'UTR SEQ ID NO:83; and A29N10A70 polyA signal SEQ ID NO:3201. The orientation of the sequences between external UTRs is in antisense.
  • Modifications made to RTE polypeptides [0216]
  • an RTE polypeptide comprises at least one amino acid substitution, at least one amino acid deletion, at least one amino acid addition, or a combination thereof, when compared to a wildtype (WT) RTE protein.
  • the RTE polypeptide may have least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the WT RTE protein.
  • an RTE polypeptide comprises at least one amino acid substitution, relative to the corresponding WT RTE protein.
  • an amino acid substitution e.g., in an RT domain or an EN domain
  • the at least one amino acid substitution is relative to SEQ ID NO: 75.
  • the at least one amino acid substitution corresponds to at least 46 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 one substitution (e.g., 1, 2, 3, 4, 5, etc.
  • the at least one amino acid substitution corresponds to at least one substitution (e.g., 1, 2, 3, 4, 5, etc.
  • the at least one amino acid substitution is a substitution corresponds to at least one of positions F148, K700, F667, C145, P280, D555, T1011 relative to SEQ ID NO: 75.
  • the at least one amino acid substitution may be a substitution corresponding to position F148 relative to SEQ ID NO: 75. In certain embodiments, the at least one amino acid substitution may be a substitution corresponding to position K700 relative to SEQ ID NO: 75. In certain embodiments, the at least one amino acid substitution may be a substitution corresponding to position F667 relative to SEQ ID NO: 75. In certain embodiments, the at least one amino acid substitution may be a substitution corresponding to position P280 relative to SEQ ID NO: 75. In certain embodiments, the at least one amino acid substitution may be a substitution corresponding to position D555 relative to SEQ ID NO: 75.
  • the at least one amino acid substitution may be a substitution corresponding to position T1011 relative to SEQ ID NO: 75. In certain embodiments, the at least one amino acid substitution may be a substitution corresponding to position K700 relative to SEQ ID NO: 75.
  • the retroelement polypeptide is an R2-TG polypeptide, which may, e.g., have an amino acid sequence set forth in any one of SEQ ID NOs: 490, 491, 492, 493, 494, 495, 496, 497 or 498. [0221]
  • an RT domain of the RTE polypeptide is a variant RT domain, relative to a natively occurring unsubstituted RT domain.
  • the at least one (in some embodiments 1, 2, 3, etc.) amino acid substitution may be selected from: L18P, E19L, S26N, V27R, A29T, I30A, R32G, N35I, S36R, L37K, A38R, L49F, G209R, K289R, L316H, E399M, E472G, Y477H, T514R, D537Y, T538K, H544Q, D555K, N562D, W762R, R822K, S895K, Q909N, R964K, G966N, T989Y, I1003M, T1011S, A1012G, L1013R, T1016L, Q1019R, E1027D, P1033R, C1079K, K1085R, L1089R, H1090Y, I1219N, A1282G, T989I, K579R, K
  • the at least one (in some embodiments 1, 2, 3, etc.) amino acid substitution may be selected from E399M, Y477H, D555K, W762R, R822K, H1090Y, F956I, P78V, W677C, K314R, K385M, P437M, T448Y, C518Y, T305R, P280K, N269R, L213K, E214K, Q215K, T1011R, R435K, L438A, R1197K, G1331A, T989L, Q955R, L952I, E947S, Q642R, G647T, N941K, V727L, M874L, D227W, D227F, C344V, R669V, V600T, T989V, M449E, T679L, Y478L, M671S, F956V, M686
  • the at least one (in some embodiments 1, 2, 3, etc.) amino acid substitution may be selected from E399M, Y477H, D555K, W762R, R822K, H1090Y, F956I, P78V, W677C, K314R, K385M, P437M, T448Y, C518Y, T305R, P280K, N269R, L213K, E214K, Q215K, E214R, T1011R, K547R, C145G, I1219N, G257M, K700D, F148K, F667G, and F667Y, or combinations thereof, relative to SEQ ID NO: 75.
  • the at least one amino acid substitution comprises a substitution corresponding to at least one ((in some embodiments 1, 2, 3, etc.) of positions F148, K700, F667, C145, P280, D555, T1011, I1219, and A1282 relative to SEQ ID NO: 75.
  • the at least one amino acid substitution comprises a substitution corresponding to a substitution in position P280 relative to SEQ ID NO: 75.
  • the substitution may be selected from, for example, P280K, P280R, P280Q, P280N, and P280H.
  • the at least one amino acid substitution comprises a substitution corresponding to a substitution in position D555 relative to SEQ ID NO: 75.
  • the substitution may be selected from, for example, D555K, D555R, D555Q, D555A, D555H, D555S, D555C, D555Y, D555W, D555F, D555L, D555V, D555M, and D555I relative to SEQ ID NO: 75.
  • the at least one amino acid substitution comprises the substitution of D555K relative to SEQ ID NO: 75.
  • SEQ ID NO: 490 sets forth the mutated amino acid sequence of an exemplary R2-TG driver with the D555K amino acid substitution.
  • the at least one amino acid substitution comprises a substitution corresponding to a substitution in position T1011.
  • the substitution may be selected from, for example, to T1011R, T1011A, T1011K, T1011S, T1011F, T1011Y, T1011W, and T1011H relative to SEQ ID NO: 75.
  • the amino acid substitution is in position F148.
  • the substituted amino acid may be selected from, for example, F148R, F148A, F148K, F148S, F148F, F148Y, F148W, F148H.
  • the amino acid substitution is in position K700.
  • the substituted amino acid may be selected from, for example, K700D, K700E, K700S, K700Q, K700T, K700R, K700H.
  • the at least one amino acid substitution comprises at least the following substitutions: P280K, D555K, T1011S, I1219N, and A1282G relative to SEQ ID NO: 75.
  • Examples of engineered drivers comprising the P280K, D555K, T1011S, I1219N, and A1282G substitutions relative to SEQ ID NO: 75 are provided in SEQ ID NOs: 82, 642, 1232, 833 or 1690.
  • the at least one (e.g.1, 2, 3, 4, 5, etc.) amino acid substitution comprises one or more of the following substitutions: G257R, P280K, N363H, E399M, V539G, H423Q, A442P, D537Y, K547R, D555K, R669L, N889G, F973E, T1011A, Q1019R, D1133P, K1085R, R1177G, Y1242K, and A1282G, or combinations thereof, relative to SEQ ID NO: 75.
  • the at least one amino acid substitution comprises at least the following substitutions: G257R, P280K, N363H, E399M, V539G, H423Q, A442P, D537Y, K547R, D555K, R669L, N889G, F973E, T1011A, Q1019R, D1133P, K1085R, R1177G, and Y1242K, A1282G relative to SEQ ID NO: 75.
  • An example of an engineered driver comprising the substitutions of G257R, P280K, N363H, E399M, V539G, H423Q, A442P, D537Y, K547R, D555K, R669L, N889G, F973E, T1011A, Q1019R, D1133P, K1085R, R1177G, Y1242K, and A1282G, relative to SEQ ID NO: 75 is provided in SEQ ID NO: 681.
  • Examples of engineered drivers comprising at least one amino acid substitution relative to SEQ ID NO: 75 are provided in SEQ ID NOs: 490, 491, 492, 493, 494, 495, 496, 497, 498, 681 or 1301. [0232] In certain embodiments, the at least one amino acid substitution is relative to SEQ ID NO: 373.
  • the at least one (in some embodiments 1, 2, 3, etc.) amino acid substitution may be selected from G1057D, S931R, A289P, V996S, L1100F, M287A, C482S, M685L, S547P, V916I, A1256E, M1259A, N332G, M358L, Y919P, T950A, M376P, P151K, D584L, Y1162Q, M640S, C314L, R1131G, A965S, Y1044L, D1236G, A970L, Q1039R, C474Y, and T404, or combinations thereof, relative to SEQ ID NO: 373.
  • the at least one amino acid substitution comprises a substitution corresponding to at least one of positions S931, Q1039 or M287 relative to SEQ ID NO: 373.
  • Amino acid deletion in RTE protein [0235] In certain embodiments, the RTE polypeptide comprises at least one amino acid deletion.
  • the deletion can be a deletion of individual amino acids, or a longer segment, including deletion of functional domains, binding domains, motifs, protein regions, or portions thereof. Segments of an RTE protein that may be deleted in an RTE polypeptide may be, an RT domain, EN domain, a DNA binding domain, a RNA binding domain, a PIP domain, an intrinsically disordered region (IDR), or combinations thereof.
  • N-terminal intrinsically disordered region [0236]
  • the N-terminal region of some RTE proteins e.g., R2 proteins encoded in Group 2 RTE, contain intrinsically disordered regions (IDRs), which appear to lack a fixed three-dimensional (3D) structure. These IDRs are thought to provide flexibility, allowing the protein to have ordered interactions with multiple molecular partners within the intracellular environment.
  • IDRs intrinsically disordered regions
  • an RTE polypeptide comprised in an engineered driver protein may comprise a deletion of a N-terminal intrinsically disordered region (IDR) or a portion thereof with respect to the WT RTE protein (e.g., a WT R2 RTE protein).
  • IDR intrinsically disordered region
  • the deletion is a deletion of between about 10 amino acids and about 300 amino acids, between about 20 amino acids and about 300 amino acids, between about 40 amino acids and about 300 amino acids, between about 50 amino acids and about 300 amino acids, between about 60 amino acids and about 300 amino acids, between about 80 amino acids and about 300 amino acids, between about 90 amino acids and about 300 amino acids, between about 100 amino acids and about 300 amino acids, between about 120 amino acids and about 300 amino acids, between about 140 amino acids and about 300 amino acids, between about 150 amino acids and about 300 amino acids, between about 160 amino acids and about 300 amino acids, between about 180 amino acids and about 300 amino acids, between about 200 amino acids and about 300 amino acids, between about 220 amino acids and about 300 amino acids, between about 250 amino acids and about 300 amino acids, between about 10 amino acids and about 250 amino acids, between about 20 amino acids and about 250 amino acids, between about 40 amino acids and about 250 amino acids, between about 50 amino acids and about 250 amino acids, between about 60 amino acids and about 250 amino acids, between about 80 amino acids and about
  • the deletion is truncation from the N-terminus of the RTE protein.
  • the RTE protein is an R2 protein.
  • the deletion of the N-terminal IDR is relative to a WT R2-TG protein having an amino acid sequence set forth in SEQ ID NO: 75.
  • the engineered driver protein comprises an R2 RTE polypeptide comprising a deletion of a N-terminal intrinsically disordered region (IDR) or a portion thereof of the corresponding WT RTE protein.
  • SEQ ID NOs: 694, 695, 701- 705, 708- 711, 714- 717, 721-724, 727- 735, 738- 741, 743- 746, 825- 831 set forth amino acid sequences of exemplary driver proteins comprising an R2 RTE polypeptide comprising a deletion of a N-terminal intrinsically disordered region (IDR) or a portion thereof.
  • exemplary engineered drivers [0239]
  • an engineered driver comprises one or more domains provided in the Examples.
  • an engineered driver comprises or has an amino acid sequence of any one of SEQ ID NOs: 75–86, 169–230, 277–384, 418–533, 539–555, 587–621, 631–834, or 838–1179, or an amino acid sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% 53 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical) thereto.
  • SEQ ID NOs: 75–86, 169–230, 277–384, 418–533, 539–555, 587–621, 631–834, or 838–1179 or an amino acid sequence that is at least 70% identical (e.g., at least
  • an engineered driver is encoded by a DNA sequence (or a corresponding RNA version thereof) set forth in any one of SEQ ID NOs: 24–37, 76–128, 144–372, 559–630, 1692–1929, 1989–2342, 2381–2578, or 2580–3011, or, a DNA sequence (or a corresponding RNA version thereof) that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical) thereto.
  • a DNA sequence or a corresponding RNA version thereof set forth in any one of SEQ ID NOs: 24–37, 76–128, 144–372, 559–630, 1692–1929, 1989–2342, 2381–2578, or
  • the engineered driver having the sequence set forth in SEQ ID NO: 436 is encoded by a DNA having the sequence set forth in SEQ ID Ns: 2381 (or by a corresponding RNA version thereof).
  • the engineered driver having the sequence set forth in SEQ ID NO: 1682 is encoded by a DNA having the sequence set forth in SEQ ID NO: 3003 (or by a corresponding RNA version thereof).
  • an engineered driver has an amino acid sequence of SEQ ID NO: 82 (encoded by, e.g., a nucleic acid sequence set forth in SEQ ID NO: 2506 or an IVT nucleic acid sequence set forth in SEQ ID NO: 3328). In certain embodiments, an engineered driver has an amino acid sequence of SEQ ID NO: 631 (encoded by, e.g., a nucleic acid sequence set forth in SEQ ID NO: 1799).
  • an engineered driver has an amino acid sequence of SEQ ID NO: 642 (encoded by, e.g., a nucleic acid sequence set forth in SEQ ID NO: 2496, or an IVT nucleic acid sequence set forth in SEQ ID NO: 3203). In certain embodiments, an engineered driver has an amino acid sequence of SEQ ID NO: 642 (encoded by, e.g., a nucleic acid sequence set forth in SEQ ID NO: 2603 or an IVT nucleic acid sequence set forth in SEQ ID NO: 3299, codon optimized).
  • an engineered driver has an amino acid sequence of SEQ ID NO: 1232 (encoded by, e.g., a nucleic acid sequence set forth in SEQ ID NO: 3330, or an IVT nucleic acid sequence set forth in SEQ ID NO: 3329). In certain embodiments, an engineered driver has an amino acid sequence of SEQ ID NO: 681 (encoded by, e.g., a nucleic acid sequence set forth in SEQ ID NO: 1791 or an IVT nucleic acid sequence set forth in SEQ ID NO: 3265).
  • an engineered driver has an amino acid sequence of SEQ ID NO: 833 (encoded by, e.g., a nucleic acid sequence set forth in 54 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 SEQ ID NO: 2576).
  • an engineered driver has an amino acid sequence of SEQ ID NO: 1690 (encoded by, e.g., an IVT nucleic acid sequence set forth in SEQ ID NO: 3009).
  • an engineered driver is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical) to any one of the aforementioned amino acid or DNA sequences.
  • the nucleic acids and polypeptides disclosed herein may be produced by methods known in the art.
  • the DNA or RNA molecules disclosed herein may be prepared synthetically via methods known in the art, or in the case of RNA, via in vitro transcription (IVT) methods known in the art.
  • the engineered driver and components thereof may be produced via recombinant protein expression and purification, which is well suited for, e.g., fusion proteins.
  • Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.
  • polynucleotide refers to a polymer of nucleotides.
  • the polynucleotide comprises one or more chemical and/or sequence modifications.
  • the polymer may include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine and deoxycytidine), and nucleoside analogs having modified bases, modified sugars (e.g., 2′-fluororibose, 2′-methoxy), or modified phosphate groups (e.g., phosphorothioates, 2’-5’ linkage).
  • a nucleic acid comprises one or more chemical and/or sequence modifications.
  • the modification is an RNA cap, a modified polyA (e.g., relative to a natural polyA), a chemically modified nucleotide, a 5’ UTR (untranslated region) modification, a 3’ UTR modification, a modified Sirloin (SINE- derived nuclear RNA localization) sequence, a modified (e.g., truncated) stem loop, an RNA stabilization motif (which may be a WPRE motif), a 5-methoxyuridine (5-MO-U) modification, a 5-methylcytidine (5mC) modification, or one or more additional and/or modified microsatellites.
  • RNA cap e.g., relative to a natural polyA
  • a chemically modified nucleotide e.g., relative to a natural polyA
  • a chemically modified nucleotide e.g., relative to a natural polyA
  • a chemically modified nucleotide e.g., relative to a natural polyA
  • a nucleic acid is sequence optimized (e.g., codon optimized) to enhance, for example, expression, reverse transcription, or transgene function.
  • RNA sequence optimization comprises one or more of the following modifications compared to the starting RNA molecule: reducing the uracil (U) load of an RNA molecule; reducing the GC% content of an RNA molecule; reducing the length and/or number of intron sequences of an RNA molecule; reducing RNA binding motifs or sites within an RNA molecule; lowering ⁇ G (free energy) of an RNA molecule; reducing the nucleotide repeats found in a sequence of an RNA molecule; adjusting the frequency of usage of particular codons in an RNA molecule; reducing the number of palindromic sequences in an RNA molecule; maximizing pairing of bases of an in an RNA molecule; removing splicing site
  • an RNA driver nucleic acid encoding an engineered driver may include an RNA pseudoknot at or near the 5’ and/or 3’ end of the nucleic acid.
  • An RNA pseudoknot is a structural RNA motif where bases in a loop pair with a sequence outside the loop, forming intertwined stem-loops.
  • the driver nucleic acid comprises an RNA pseudoknot.
  • the template nucleic acid comprises an RNA pseudoknot.
  • the pseudoknot is a EvopreQ1 pseudoknot (e.g., SEQ ID NO: 1926). In certain embodiments, the pseudoknot is a Mpknot1 pseudoknot(e.g., SEQ ID NO: 1927). In certain embodiments, the pseudoknot is a MALAT1 pseudoknot (e.g., SEQ ID NO: 1928).
  • T cell Specific Elements As provided herein, the driver nucleic acids of the disclosure are provided to mediate the integration of a DNA copy of a template into the genome of a T cell, whether provided in cis or in trans. Accordingly, the disclosure provides elements and methods to enhance T cell specificity.
  • driver nucleic acids with binding sites for differentially expressed microRNAs e.g. miRs that can prevent integration or expression in undesired cell types and can increase integration in T cells.
  • miRs differentially expressed microRNAs
  • altering T cells to express transgenes using RTEs with use of miRs enables safe and potent immunotherapy for cancer, autoimmunity, and other conditions.
  • the T cell specific element is one or more miR binding site(s) that do not express in T cells but do express in other cells in which expression is not 56 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 desired.
  • the binding of miRs to an mRNA leads to degradation and is used to knock-down transgene expression.
  • the differentially expressed miR is miR-122.
  • the differentially expressed miR is derived from Ludwig et al. Nucleic Acids Research, 44(8): 3865– 3877 (2016), the distribution of miRNAs in human tissues and is presented in, and downloadable from the Human miRNA tissue atlas each of which is by reference the present disclosure.
  • a “template nucleic acid” (or “template”) comprises a sequence of interest destined for integration into a genome, and one or more sequence elements (e.g., RTE-UTRs described in more detail herein below) that interact with a driver, e.g., an engineered driver of the present disclosure.
  • the sequence of interest may be referred to as a transgene or a gene of interest (“GOI”), that encodes an RNA or protein (e.g., a therapeutic RNA or protein).
  • GOI gene of interest
  • the template nucleic acid may be referred to herein as a “reporter”, “reporter nucleic acid” a “reporter construct”, a “gene delivery construct”, or a “template construct”.
  • the driver nucleic acid may be DNA, RNA (e.g. mRNA), or a hybrid RNA/DNA molecule.
  • the template nucleic acid may be DNA or RNA (e.g. mRNA), or a hybrid RNA/DNA molecule.
  • the driver nucleic acid and template nucleic acid are provided in trans, as separate RNA molecules, DNA molecules, or separate RNA/DNA molecules.
  • the driver nucleic acid and template nucleic acid are in a cis configuration and provided in a single nucleic acid. In certain embodiments, they are provided in cis as DNA. In certain embodiments, they are provided in cis as RNA. In certain embodiments, they are provided in cis as chimeric RNA/DNA. [0250]
  • the template in a trans configuration, includes the transgene that may be flanked, on each side independently, with a terminal region comprising one or more functional elements (e.g. RTE-UTRs, homology arms, etc.).
  • the template nucleic acid portion includes an adjacent driver nucleic acid.
  • the combined nucleic acid in cis configuration may be flanked, on each side independently, with a terminal region comprising one or more functional elements (e.g. RTE-UTRs, homology arms, etc.).
  • 57 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032
  • the driver nucleic acid and the template can be provided as DNA, as RNA, or as a mixture of both. It is contemplated in other embodiments that the driver nucleic acid and the template can be provided as DNA, as RNA (e.g. as mRNA), or as a mixture of both encapsulated in a carrier, e.g. in a lipid nanoparticle (LNP).
  • RNA e.g. as mRNA
  • LNP lipid nanoparticle
  • an RTE transcript of a natural RTE comprises one or more retrotransposable element untranslated regions (RTE-UTRs) that, as RNA, forms a structure that is capable of being bound by the RTE protein (e.g., by a compatible RNA binding domain comprised in the RTE protein), as a pre-requisite to achieve the reverse transcription of the RTE transcript by the RTE protein.
  • RTE-UTRs retrotransposable element untranslated regions
  • the template 5’ terminal region and the 3’ terminal region may comprise, independently, at least one RTE-UTR that is capable (e.g., as RNA) of binding an RTE polypeptide comprised in a driver encoded by the driver nucleic acid.
  • the RTE protein and the one or more RTE-UTRs of a given RTE typically have co-evolved to have sufficient binding affinity for each other.
  • the one or more RTE-UTRs comprised the template may be derived from the same RTE as the RTE polypeptide comprised in the driver.
  • RTE-UTRs and RTE polypeptides where the RTE-UTRs and the RTE polypeptides are derived from different RTEs (e.g., different species of R2 RTEs) have sufficient affinity to enable productive interaction of the template and the driver.
  • Some heterologous pairings may be even more effective in facilitating transgene integration that homologous pairing.
  • a template nucleic acid comprising a 5’ RTE-UTR (SEQ ID NOs: 150) and a 3’ RTE-UTR (SEQ ID NOs: 126) from R2-1-Phylloscopus trochilus trochilus (PTT)
  • R2-1-Phylloscopus trochilus trochilus was more effective in facilitating transgene integration than another template nucleic acid comprising an RTE-UTR derived from R2-1-TG.
  • the one or more RTE-UTRs comprising the template may be derived from a different RTE as the RTE polypeptide comprised in the driver.
  • RTE-UTR may be referred to by the species of RTE from which it is derived.
  • R2-1-TG RTE- UTR an RTE-UTR derived from R2-1-TG
  • a template may be referred to herein by the RTE from which the one or more RTE-UTRs comprised therein are derived.
  • a template comprising one or more RTE- UTRs derived from the R2-1-TG RTE may be referred to herein as a “R2-1-TG template” or “R2-1-TG reporter”.
  • RTE-UTR comprised in a 5’ terminal region of the template (when in trans configuration) or of the combined nucleic acid (when in cis configuration) may be referred to herein as a 5’ RTE-UTR.
  • AN RTE-UTR comprised in a 3’ terminal region of the template (when in trans configuration) or of the combined nucleic acid (when in cis configuration) may be referred to herein as a 3’ RTE-UTR.
  • RTE-UTRs comprised in the template (when in trans configuration) or the combined nucleic acid (when in cis configuration) may be RTE-UTRs derived from natural RTEs (e.g.
  • the RTE-UTRs may be modified RTE-UTRs based on RTE-UTRs from one or more natural RTEs.
  • the 3 ⁇ RTE-UTR e.g., of an R2-TG, may comprise a conserved Stem Loop (SL) region and a variable number of a microsatellite repeats (e.g., a minimal 3 ⁇ UTR required for efficient transgene integration).
  • the one or more RTE-UTR(s) are derived from a non-LTR RTE.
  • the one or more RTE-UTR(s) are derived from a Class I transposable element that utilizes an intermediate RNA during integration by a copy-and-paste mechanism (PMIDs: 32955944 and 18261821) and that lacks Long Terminal Repeats (LTR) or Inverted Terminal Repeats (ITR) in their structure.
  • a copy-and-paste mechanism PMIDs: 32955944 and 18261821
  • LTR Long Terminal Repeats
  • ITR Inverted Terminal Repeats
  • the one or more RTE-UTR(s) are derived from a superclade selected from the group consisting of Retrons (include clades AbiA, AbiK, AbiP2, CRISPR, CRISPR-like, DCRs, G2L4, G2L, G2Lb, G2Lc, GII, Retrons, UG1, UG2, UG3, UG4, UG5, UG6, UG7, UG8, UG9, UG10, UG11, UG12, UG13, UG14, UG15, UG16, UG17, UG18, UG19, UG20, UG21, UG22, UG23, UG24, UG25, UG26, UG27, UG28 and UG28b), Group II introns (include clades E, ML, B, CL1A, CL1B, CL2A, CL2B, F, D and C), Penelope-like retroelements (in
  • a template of the disclosure may comprise an RTE-UTR (a 3’ RTE-UTR and/or a 5’ RTE-UTR) derived from an R2 RTE.
  • RTE-UTRs from R2 retroelements found in a variety of organisms are set forth in SEQ ID NOs: 33, 34, 85, 86, 99, 100, 125-128, 144-257, 259- 292, 294- 356, 358-372 or 486-489.
  • 5’RTE-UTRs are set forth in SEQ ID NOs: 33, 85, 99, 125, 127, any one of 144-249, any one of 359-367 374, 486, or 488.3’RTE-UTRs are set forth in SEQ ID NOs: 34, 86, 100, 126, 128, , 250-358368-372, 487, 489.
  • Other non-limiting examples of 5’ RTE-UTRs are set forth in any one of SEQ ID NOs: 359-367.
  • Other non-limiting examples of 3’RTE-UTRs are set forth in any one SEQ ID NOs: 357- 372.
  • the RTE-UTR sequences in the present application are provided as DNA sequences.
  • a template of the disclosure may comprise an RTE-UTR (a 3’ RTE-UTR and/or a 5’ RTE-UTR) derived from one of the following R2 RTEs, as listed in Table 3: Table 3: RTE-UTRs of representative R2 RTEs RTE name Organism species SEQ ID NO for SEQ ID NO for 5' RTE-UTR 3' RTE-UTR 1 R2-1-TG Taeniopygia guttata 85 86 2 R2-ZA Zonotrichia albicollis 486 487 3 R4-AC Anolis carolinensis 488 489 4 R4-1-PH Parhyale hawaiensis 99 100 [0261] Table 3 provides the DNA sequence of the 5’ RTE-UTR and the 3’ RTE-UTR from each of the listed RTE species.
  • an RTE integration system of the disclosure may utilize an RTE-UTR (a 3’ RTE-UTR and/or a 5’ RTE-UTR) derived from one of the following R2 RTEs, as listed in Table 4: 60 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 Table 4: RTE-UTRs of R2 RTEs RTE name Abbreviation Organism SEQ ID SEQ ID NO species NO for 5' for 3' RTE- RTE-UTR UTR 1 R2-1_Acridotheres_tristis R2-AT Acridotheres tristis 171 279 2 R2-1_Acrocephalus R2-ASS Acrocephalus _scirpaceus _scirpaceus scirpaceus scirpaceus
  • RTE-UTR sequences in Table 4 are provided as DNA sequences. For each RTE-UTR sequence provided, it will be understood that the present disclosure also includes a respective RNA version thereof.
  • an R2 RTE-UTR comprised in a template may an identical sequence with the RTE-UTR sequence comprised in a natural R2 RTE, for example as listed in 63 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 the tables above.
  • the R2 RTE-UTR used in a template may have least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the RTE-UTR sequence comprised in a natural R2 RTE, for example as listed in the tables above.
  • an “ancestral reconstruction process” refers to the method of extrapolating back in time and to model the ancient parents of the identified sequences, (see for example Campitelli et al., Reconstruction of full-length LINE-1 progenitors from ancestral genomes. Genetics.2022 Jul; 221(3): iyac074.).
  • an R2 RTE-UTR used in a template may be an “ancestral RTE-UTR” based on an ancestral reconstruction based on a plurality of R2 RTE species.
  • a template of the disclosure comprises an ancestral RTE-UTR (a 3’ RTE-UTR and/or a 5’ RTE-UTR) derived from one of the following combinations of R2 RTEs, as listed in Table 5.
  • Table 5 Ancestral RTE-UTRs RTEs included in ancestral reconstruction SEQ ID NO for SEQ ID NO for 3' process 5' RTE-UTR RTE-UTR 1 3utr-AR1-4 (SEQ ID NO: 3077, 290 or 289) 85 357 2 3utr-AR2-5(SEQ ID NO: 128, 292, 273 or 282) 85 2378 3 3utr-AR3-7(SEQ ID NO: 280, 256 or 283) 85 551 4 5utr-AR1-4(SEQ ID NO: 1337, 1338 or 1339) 359 86 5 5utr-AR2-5(SEQ ID NO: 280, 256 or 283) 360 86 6 5utr-AR3-4 (SEQ ID NO: 148, 187 or 2275) 361 86 7 5utr-AR4-6 (SEQ ID NO: 145 or 85) 362 86 8 5utr-AR5-4 SEQ ID NO: 180, 173 or 188) 363 86 64 318294
  • the modified RTE-UTRs which included modified 5’ RTE-UTRs and/or 3’ RTE-UTRs were based on a plurality of R2 RTEs. Different sets of R2 RTEs, as described herein below, were used to generate different modified RTE-UTRs based on ancestral reconstruction (see, e.g., Campitelli et al., Reconstruction of full-length LINE-1 progenitors from ancestral genomes, Genetics, Volume 221, Issue 3, July 2022, and Diallo AB et al., Ancestors 1.0: a web server for ancestral sequence reconstruction. Bioinformatics. 2010;26(1):130–131, whose ancestral reconstruction procedures are incorporated herewith).
  • the RTE-UTR sequences in Table 5 are provided as DNA sequences. For each RTE- UTR sequence provided, it will be understood that the present disclosure also includes a respective RNA version thereof.
  • Homology arms [0270]
  • the 5’ terminal region of the template, the 3’ terminal region of the template, or both, may comprise, independently, a homology arm with nucleic acid sequences having homology to nucleic acid sequences in the human genome.
  • each homology arm is independently selected and is from about 4 to about 200 nucleotides in length or more, for example about 4, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200 or more nucleotides long.
  • the homology 65 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 arm corresponds to a sequence in the 28S rDNA locus in the human genome.
  • the homology arm corresponds to a sequence in the AAVS1 locus in the human genome .
  • the nucleic acid sequence of the homology arm is in a reading frame that is different than the open reading frame of the transgene comprised in the template.
  • the nucleic acid sequence of the homology arm is in the same reading frame as the transgene.
  • a template comprises (e.g. in a 5’ terminal region and/or a 3’ terminal region) one or more sequences (e.g. homology arms) that are homologous to a target sequence (e.g., a target sequence in a host genome).
  • target sequences include safe harbor genomic targets.
  • a template may comprise one or more RNA nuclear localization sequences (e.g., a SAFB motif) and/or one or more stabilization motifs (e.g., a WPRE motif).
  • the template also may comprise flanking regions homologous to a target sequence in a genome.
  • the template nucleic acid comprises one or more chemical or sequence modifications.
  • the modification is a RNA cap, a modified polyA (e.g., relative to a natural polyA), a chemical modification (e.g., a pseudouridine and/or a methylpseudouridine), a 5’ end modification, a 3’ end modification, a modified Kozak sequence, a modified (e.g., truncated) stem loop, a RNA stabilization motif (which may be a WPRE motif), a 5-methoxyuridine (5-moU) modification, a 5-methylcytidine (5-mC) modification, or one or more additional and/or modified microsatellites.
  • a modified polyA e.g., relative to a natural polyA
  • a chemical modification e.g., a pseudouridine and/or a methylpseudouridine
  • a 5’ end modification e.g., a 3’ end modification
  • a modified Kozak sequence e.g., truncated stem
  • a nucleic acid sequence encoding an engineered driver and/or a template is sequence optimized (e.g., codon optimized) to enhance expression, reverse transcription or transgene function.
  • RNA sequence optimization comprises one or more of the following modifications compared to the starting RNA molecule: reducing the uracil (U) load of an RNA molecule; reducing the GC% content of an RNA molecule; reducing the length and/or number of intron sequences of an RNA molecule; reducing RNA binding motifs or sites within an RNA molecule; lowering ⁇ G (free energy) of an RNA molecule; reducing the nucleotide repeats found in a sequence of an RNA molecule; adjusting the frequency of usage of particular codons 66 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 in an RNA molecule; reducing the number of palindromic sequences in an RNA molecule; maximizing pairing of bases of an in an RNA
  • a template of the disclosure comprises a sequence of interest, destined for integration into a genome, which may be referred to as a transgene or a gene of interest (“GOI”), and which encodes a RNA or protein (e.g., a therapeutic RNA or protein).
  • a template may comprise a plurality of transgenes.
  • the transgene comprises a promoter and specifies an RNA, which may or may not encode a protein.
  • the transgene comprises 5’ and 3’ UTRs such that the RNA will function as an mRNA.
  • the transgene includes a polyadenylation (“polyA”) signal.
  • the transgene includes one or more introns.
  • the transgene may be in sense or anti-sense orientation with respect to the template.
  • the promoter may be an inducible promoter. In some embodiments, the promoter is a tissue-specific promoter.
  • the promoter may be, e.g., a MNDopt promoter, a MNDu promoter, a EF1a promoter, a CMV promoter, a A1AT promoter, a Albumin promoter, or a ApoE promoter.
  • a template may comprise a segment encoding a detection polypeptide, e.g. useful to detect integration of the transgene by a given driver.
  • a detection polypeptide is a human influenza hemagglutinin (HA) Flag (DYKDDDDK; SEQ ID NOs: 392), green fluorescent protein (GFP or variants such as EGFP, e.g.
  • the transgene comprises a nucleic acid that encodes an immune cell engager, including, for example, a T cell engager (e.g., BiTE, DART), an NK cell engager (e.g., BiKE, TriKE), a macrophage engager (e.g., BiME), and an innate cell engager (e.g., ICE).
  • a T cell engager e.g., BiTE, DART
  • an NK cell engager e.g., BiKE, TriKE
  • a macrophage engager e.g., BiME
  • an innate cell engager e.g., ICE
  • the template of the disclosure comprises a nucleic acid that encodes an engineered protein.
  • the engineered protein is an engineered immune receptor.
  • an engineered immune receptor comprises an antigen binding 67 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 domain and is designed to redirect the specificity and function of immune cells (e.g., T cells) towards particular target cells (e.g., based on the antigen binding domain) and are useful for immunotherapies.
  • the engineered immune receptor is selected from the group consisting of a T cell receptor (TCR), TCR fused antigen modifier (TRAM), a T cell receptor fusion construct (TRuC) (Baeuerle, Patrick A., et al.
  • TCR-CD3 complex comprises six (6) unique polypeptides: TCR ⁇ and TCR ⁇ polypeptides, and CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ polypeptides (see FIG.2).
  • the TCR ⁇ and TCR ⁇ polypeptides are responsible for recognition of antigens presented on the Major Histocompatibility Complex (MHC) and have a very short intracellular domain with no signaling moieties.
  • the CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ polypeptides each contain a single immunoreceptor tyrosine-based activation motif (ITAM), and CD3 ⁇ contains 3 ITAM motifs.
  • ITAM immunoreceptor tyrosine-based activation motif
  • the full TCR-CD3 complex has eight polypeptide: 1 TCR ⁇ , 1 TCR ⁇ , 1 CD3 ⁇ , 1 CD3 ⁇ , 2 CD3 ⁇ , and 2 CD3 ⁇ chains. Without being held to theory or mechanism, engagement of a TCR initiates T cell activation and is referred to as “signal 1”, which is necessary for T cell activation and effector functions.
  • a T Cell Receptor fused Antigen Modifier is a single polypeptide that contains an antigen binding domain fused to one of the standard polypeptides of the TCR-CD3 complex (FIG.2).
  • a TRAM is incorporated into the TCR-CD3 complex in lieu of the cognate standard polypeptide, such that binding of the antigen recognized by the TRAM initiates T cell signaling in a similar manner to natural TCR signaling following recognition of a specific 68 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 peptide-MHC complex.
  • FMC63 mouse anti-hCD19 antibody
  • VH Variable Heavy Chain
  • VL Variable Light Chain
  • SP signal peptide
  • TM Transmembrane
  • IC Intracellular
  • C ⁇ Constant Alpha Chain
  • C ⁇ Constant Beta Chain
  • hTCR human TCR
  • mTCR murine TCR
  • co-stim co-stimulatory domain.
  • a conventional CAR contains an antigen binding domain, hinge, transmembrane domain, and signaling domains (FIG.2).
  • the antigen binding domain is typically derived from the variable fragments of antibodies in the form of a single chain variable fragment (scFv) where the variable light (VL) and variable heavy (VH) chains are fused using a flexible linker.
  • the transmembrane and signaling domains are typically derived from various immune receptors.
  • First generation CARs contain one signaling domain, typically the intracellular portion of CD3 ⁇ .
  • Second generation CARs have the addition of a second signaling domain from a co-stimulatory receptor such as 4-1BB (CD137) or CD28.
  • Third generation CARs have an additional co-stimulatory signaling domain.
  • the canonical 2nd generation anti-CD19 CAR used here “CART-19” has been used extensively in the clinic (Kymriah®) and consists of the CD8 ⁇ signal peptide (“SP”, SEQ ID NO: 1), FMC63 VL (SEQ ID NO: 2), 3xG4S linker (SEQ ID NO: 3), FMC63 VH (SEQ ID NO: 4), CD8 ⁇ Hinge (SEQ ID NO: 5), CD8 ⁇ Transmembrane (“TM”, SEQ ID NO: 6), 4-1BB co-stimulatory domain (SEQ ID NO: 7), and CD3 ⁇ signaling domain (SEQ ID NO: 8).
  • SP CD8 ⁇ signal peptide
  • FMC63 VL SEQ ID NO: 2
  • 3xG4S linker SEQ ID NO: 3
  • FMC63 VH SEQ ID NO: 4
  • CD8 ⁇ Hinge SEQ ID NO: 5
  • TM CD8 ⁇ Transmembrane
  • SEQ ID NO: 7 4-1BB co-
  • a CAR contains a signal that is capable of expressing on the cell surface and providing signal 1 independently of the TCR-CD3 complex to the extent that the T cell is activated and can perform effector functions.
  • a TRAM requires the full TCR-CD3 complex to express on the cell surface and provide signal 1 (the single ITAM of CD3 ⁇ , CD3 ⁇ , or CD3 ⁇ without the scaffolding and/or additional ITAMs from CD3 ⁇ is typically not sufficient to activate T cells to perform effector functions).
  • CARs can contain a co-stimulatory signaling domain that provides signal 2 (e.g. in 2 nd generation and beyond CARs).
  • the engineered immune receptor is a TRAM.
  • the TRAM comprises an antigen binding domain fused to any one or more TCR subunits (TCR ⁇ , TCR ⁇ ), CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ ), or a functional fragment thereof.
  • TCR ⁇ , TCR ⁇ TCR subunits
  • CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ TCR subunits
  • the present disclosure provides TRAMs which may be used as compositions 69 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 with engineered retrotransposon systems (that may be delivered with RNA-LNP), have T cell specific expression, and/or prevent antigen masking.
  • antigen masking refers to the blocking of the epitope of an antigen, which in turn prevents the recognition of the antigen by a T cell.
  • Antigen masking can be mediated by an engineered immune receptor that is capable of binding the antigen in cis when expressed in the target cell itself, or in trans when expressed in a different cell in proximity to the target cell.
  • the engineered immune receptor-encoding transgene is integrated into a host cell using an RTE integration system as described herein.
  • the cell is an immune cell, which may be an immune cell that natively expresses a TCR-CD3 complex, e.g., a T cell.
  • the transgene integration into the cell is performed in vivo.
  • the transgene integration into the cell is performed in vitro.
  • the transgene integration into the cell is performed ex vivo.
  • the engineered immune receptor comprises one or more of the following elements: an antigen binding domain that binds to a target antigen, a transmembrane domain, a signal peptide, a co-stimulatory domain, an immunoreceptor tyrosine-based activation motifs (ITAMs), and/or a T cell-specific moiety.
  • Transmembrane (TM) domains [0289]
  • the engineered immune receptors of the disclosure comprise a transmembrane domain.
  • the transmembrane domain is selected from the group consisting of TCR ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ , CD28, CD45, CD4, CD5, CD7, CD8, CD8 alpha, CD8beta, CD9, CD11a, CD11b, CD11c, CD11d, CD16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD134, CD137, TNFSFR25, CD154, 4-1BB/CD137, activating NK cell receptors, an Immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD276 (B7-H3), CD29, CD30, CD40, CD49a, CD49D, CD49f, CD69, CD84, CD96 (Tactile),
  • the engineered immune receptor is a CAR comprising a transmembrane domain selected from the group consisting of CD4, CD8, CD16, CD28, CD34, CD137, IgG1, IgG4, IgD, NGFR, LIR1, and PD1.
  • the engineered immune receptor is a TRAM comprising a TCR subunit selected from the group consisting of TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ .
  • the TRAM comprises a transmembrane domain selected from the group consisting of TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and a variant thereof.
  • the engineered immune receptors of the disclosure comprise an antigen binding domain that binds to a target antigen.
  • the antigen binding domain contains one or more single chain variable fragments (scFvs) comprising a variable light chain (VL) and variable heavy chain (VH), the variable domain of the heavy chain of a heavy-chain-only antibody (VHH), variable heavy chain only (VH), TCR-like antibody, single chain TCR V ⁇ V ⁇ , natural ligand, D-domain, FcR-binding receptor (e.g., CD16), or an NK killing receptor (e.g., NKG2D, NKp30, NKp44, NKp46).
  • the antigen binding domain comprises a VH and VL fused by a linker.
  • the linker is a flexible linker.
  • the flexible linker comprises the amino acid sequence of 3xG4S (SEQ ID NO: 3) or Whitlow linker (SEQ ID NO: 9).
  • the antigen binding domain is linked to the N terminus of the TCR subunit by a linker and/or a hinge.
  • the hinge and/or linker is, is from, or is derived from (e.g., comprises all or a fragment of) CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8.
  • CD8.beta. CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SEAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c 71 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CEEC2), CD79A (B cell antigen receptor complex-associated alpha chain), CD79B (B cell antigen receptor complex- associated beta chain), CD84 (SEAMF5), CD96 (Tactile), CD100 (SEMA4D
  • the linker and/or hinge is selected from the group consisting of the following amino acid sequences (or fragments thereof): GGGGS (SEQ ID NO: 129), EAAAK (SEQ ID NO: 130), PAPAP (SEQ ID NO: 131), ALEA (SEQ ID NO: 132), GGGGGG (SEQ ID NO: 133), AAA (SEQ ID NO: 10), Whitlow (SEQ ID NO: 9), SESATPES (XTEN linker, SEQ ID NO: 78), AEQQRQQQEAAQKAQ (SEQ ID NO: 117), CD8a hinge (SEQ ID NO: 5), CD28 hinge (SEQ ID NO:28), CD4 hinge (SEQ ID NO: 134), CD7 hinge (SEQ ID NO: 135), CD34 hinge (SEQ ID NO: 136), CD137 hinge (SEQ ID NO: 137), IgG1 hinge (SEQ ID NO: 138), IgG4 hinge (SEQ ID NO: 139), I
  • the target antigen is selected from the group consisting of 2B4 (CD244), 4-1BB, 5T4, A33 antigen, adenocarcinoma antigen, adrenoceptor beta 3 (ADRB3), A kinase anchor protein 4 (AKAP-4), alpha-fetoprotein (AFP), anaplastic lymphoma kinase (ALK), Androgen receptor, B7H3 (CD276), p2-integrins, BAFF, B-lymphoma cell, B cell maturation antigen (BCMA), bcr-abl (oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl), BhCG, bone marrow stromal cell antigen 2 (BST2), CCCTC-Binding Factor (Zinc Finger Protein)-Like 72 318294750 Attorney Docket No.:AVRT-010/02WO 351047
  • the antigen binding domain is specific for one or more of any of the target antigens of the disclosure. In some embodiments, the antigen binding domain is specific for GPCR5D (G-protein coupled receptor class 5D), BCMA, CD22, CD19 and/or CD20.
  • GPCR5D G-protein coupled receptor class 5D
  • BCMA CD22, CD19 and/or CD20.
  • the CD19 antigen binding domain is derived from an antibody selected from the group consisting of FMC63, murine 4G7 (VL SEQ ID NO: 109, VH SEQ ID NO: 110), human Hu19 (VL SEQ ID NO: 111, VH SEQ ID NO: 112), Hu1E7, huB4, hBU12, humanized FMC63, humanized 4G7, GR37, GR41, SRB-37, SRB-85, and modified versions of any of the preceding.
  • an antibody selected from the group consisting of FMC63, murine 4G7 (VL SEQ ID NO: 109, VH SEQ ID NO: 110), human Hu19 (VL SEQ ID NO: 111, VH SEQ ID NO: 112), Hu1E7, huB4, hBU12, humanized FMC63, humanized 4G7, GR37, GR41, SRB-37, SRB-85, and modified versions of any of the preceding.
  • the CD20 antigen binding domain is derived from an antibody selected from the group consisting of Leu16, rituximab, ofatumumab (VH SEQ ID NO: 115, VL SEQ ID NO: 116), ocrelizumab, obinutuzumab (VH SEQ ID NO: 113, VL SEQ ID NO 114), ibritumomab tiuxetan, AME-133v, IMMU-106, TRU-015, tositumomab, and modified versions of any of the preceding.
  • an antibody selected from the group consisting of Leu16, rituximab, ofatumumab (VH SEQ ID NO: 115, VL SEQ ID NO: 116), ocrelizumab, obinutuzumab (VH SEQ ID NO: 113, VL SEQ ID NO 114), ibritumomab tiuxetan, AME-133v,
  • the BCMA antigen binding domain is derived from an antibody selected from the group consisting of erlanatamab, alnuctamab, linvoseltamab, teclistamab, 75 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 belantamab, pavurutamab, icatolimab, and human anti-TNFRSF17 clone hSG16.17 clone hSG16.45, or modified versions of any of the preceding.
  • the antigen binding domain is bispecific. In some embodiments, the antigen binding domain is bispecific for CD19 and CD20.
  • the bispecific antigen binding domain comprises multiple scFvs.
  • the multiple scFvs are linked by a linker or hinge selected from the group consisting of flexible 3xG4S linker (SEQ ID NO: 3), rigid 3xEAAAK (SEQ ID NO: 41), machine-learning assisted rigid AEQQRQQQEAAQKAQ (SEQ ID NO: 117), semi-flexible GGGSEAAAKGGGS (SEQ ID NO: 42), semi-rigid EAAAKGGGSEAAAK (SEQ ID NO: 43), and rigid 3xPAPAP (SEQ ID NO: 118).
  • the linker or hinge is from, or is derived from (e.g., comprises all or a fragment of) CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8. alpha., CD8.beta., CDl la (ITGAL), CDl lb (ITGAM), CDl lc (ITGAX), CDl ld (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SEAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CEEC2), CD79A (B cell antigen receptor
  • the engineered immune receptor further comprises a signal peptide.
  • Signal peptides are nucleic acids that direct proteins to the secretory pathway.
  • the signal peptide is selected from the group consisting of CD8 ⁇ signal peptide, a TCR ⁇ signal peptide, and a TCR ⁇ signal peptide.
  • the signal peptide is a CD8 ⁇ SP (SEQ ID NO: 1), mTCR ⁇ SP (SEQ ID NO: 12), or hTCR ⁇ SP (SEQ ID NO: 16).
  • the signal peptide is a CD8 ⁇ signal peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the signal peptide is a TCR ⁇ signal peptide comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the signal peptide is a TCR ⁇ signal peptide comprising the amino acid sequence of SEQ ID NO: 16.
  • Co-stimulatory domains [0303] In some embodiments, the engineered immune receptor further comprises one or more co-stimulatory domains. Co-stimulatory domains generally play a functional role in the activation of an immune cell.
  • the co-stimulatory domain is selected from the group consisting of CD2, CD5, CD27, CD28, DAP10, DAP12, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), OX40, and 4-1BB (CD137).
  • the co- stimulatory domain is a signaling region of a protein selected from the group consisting of DAP- 10, CD28, OX-40, 4-IBB (CD137), CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD- 1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), tumor necrosis factor superfamily member 14, TNFSF14, LIGHT), NKG2C, Ig alpha (CD79a), Fc gamma receptor, MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, CDS, GITR, BAFFR,
  • the engineered immune receptor further comprises one or more ITAMs.
  • ITAMs generally play a functional role in the transmission of signals from immune receptors.
  • the ITAMs are selected from the group consisting of TCR ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , Fc epsilon receptor 1 chain, Fc ⁇ R2, Fc ⁇ R1, Fc ⁇ R2a, Fc ⁇ R2b1, Fc ⁇ R2b2, Fc ⁇ R3a, Fc ⁇ R3b Fc ⁇ R1, DAP10, DAP12, CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, and CD66d.
  • RTE integration systems of the present disclosure can be delivered in vivo in an LNP format.
  • engineered immune receptors in vivo delivery to, and stable expression in, cells other than T cells could lead to antigen masking, where access to the target antigen by the T cell is blocked.
  • Certain engineered immune receptors e.g., TRAMs, are designed to reduce this effect by being unstable in cells other than TCR-expressing (i.e., T) cells.
  • TRAMs are designed to reduce this effect by being unstable in cells other than TCR-expressing (i.e., T) cells.
  • the disclosure also describes additional methods to enhance T cell specificity before the stage of surface expression, for example, According to some embodiments of the present disclosure, driver or template RNAs with binding sites for differentially expressed miRs can be prevented from integrating or expressing in certain cell types.
  • the transgene comprises a promoter.
  • the promoter is a naturally occurring promoter.
  • the promoter is a recombinant promoter.
  • the promoter is a constitutive, inducible, and/or tissue or cell specific promoter.
  • the cell specific promoter is a T cell specific promoter.
  • the T cell specific promoter comprises one or more of hCCL5 (SEQ ID NO: 46), mCCL5 (SEQ ID NO: 47), hCD2 (SEQ ID NO: 48), mCD2 (SEQ ID NO: 49), hCD3 ⁇ (SEQ ID NO: 50) , hCD3 ⁇ (SEQ ID NO: 51), mCD3 ⁇ (SEQ ID NO: 52), hCD3 ⁇ (SEQ ID NO: 53), mCD3 ⁇ , minimal mCD3 ⁇ (SEQ ID NO: 55), and any fragment thereof.
  • the promotor is an inducible promoter containing binding sites for T cell transcription factors.
  • the T cell transcription factor binding sites are selected from the group consisting of NR4A (SEQ ID NO: 57), AP1 (SEQ ID NO: 58), NFAT (SEQ ID NO: 59), NF ⁇ B (SEQ ID NO: 60), and any combination thereof.
  • the template further comprises a T cell specific enhancer.
  • the T cell specific enhancer comprises chr16-445 (SEQ ID NO: 56).
  • the template further comprises one or more binding sites for a differentially expressed miR.
  • the one or more miRs are located in the 5’ RTE-UTR or RTE3’UTR in either the sense orientation or anti-sense orientation.
  • the miR is miR-122.
  • the differentially expressed miRs are derived from Ludwig et al.
  • Sample TRAM designs [0310] The following are non-limiting examples of TRAM engineered immune receptor designs.
  • FIG.3 also depicts exemplary TRAM designs.
  • CD19 scFv for the following TRAMs comprise FMC63 VL (SEQ ID NO: 2), Whitlow linker (SEQ ID NO: 9), FMC63 VH (SEQ ID NO: 4) and are fused to CD3 component via 3xA (SEQ ID NO: 10) and G4Sx3 linker (SEQ ID NO: 3).
  • This scFv with spacer is referred to herein as “FMC63 LH scFv- G4S” (SEQ ID NO: 11).
  • mC ⁇ + ⁇ TRAM comprises mTCR ⁇ SP (SEQ ID NO: 12), FMC63 VH (SEQ ID NO: 4), 3xA (SEQ ID NO: 10), mTCR-C ⁇ (SEQ ID NO: 13), T2A (SEQ ID NO: 14), FMC63 VL (SEQ ID NO: 2), 3xA (SEQ ID NO: 10), and mTCR-C ⁇ (SEQ ID NO: 15).
  • mC ⁇ TRAM comprises mTCR ⁇ SP (SEQ ID NO: 12), mTCR-C ⁇ (SEQ ID NO: 13), T2A (SEQ ID NO: 14), FMC63 LH scFv-G4S (SEQ ID NO:11), and mTCR-C ⁇ (SEQ ID NO: 15).
  • hC ⁇ TRAM comprises hTCR ⁇ SP (SEQ ID NO: 16), FMC63 LH scFv-G4S (SEQ ID NO: 11), and hTCR- C ⁇ (SEQ ID NO: 17).
  • ⁇ TRAM comprises of CD8 ⁇ SP (SEQ ID NO: 1), FMC63 LH scFv-G4S (SEQ ID NO: 11), and CD3 ⁇ (SEQ ID NO: 18).
  • ⁇ TRAM comprises of CD8 ⁇ SP (SEQ ID NO: 1), FMC63 LH scFv-G4S (SEQ ID NO: 11), and CD3 ⁇ (SEQ ID NO: 19).
  • ⁇ TRAM comprises CD8 ⁇ SP (SEQ ID NO: 1), FMC63 LH scFv-G4S (SEQ ID NO: 11), and CD3 ⁇ (SEQ ID NO: 20). comprises of CD8 ⁇ SP (SEQ ID NO: 1), FMC63 LH scFv-G4S (SEQ ID NO: 11), CD28 hinge (SEQ ID NO: 21) since CD3 ⁇ has only 8 amino acids extracellularly, CD3 ⁇ HTM (SEQ ID NO: 22), and CD3 ⁇ signaling domain (SEQ ID NO: 8).
  • Delivery Systems [0318] The present disclosure also describes delivery systems which deliver the RTE integration systems (also referred to herein as “RTE system”) of the disclosure to a host cell.
  • the host cell is a T cell.
  • the delivery systems described in the disclosure are useful for delivering one or more templates to a host cell and promoting integration of a DNA copy of the template nucleic acid into the host nucleic acid (e.g., a genomic nucleic acid of a host cell), for example for therapeutic purposes (e.g., to provide or supplement expression of an RNA and/or polypeptide that provides a therapeutic benefit to a subject, for example a human subject having a disease or disorder associated with a loss of normal gene function).
  • the delivery systems of the disclosure are useful for high efficiency delivery of driver and template nucleic acid to specific cells or populations of cells.
  • LNPs Lipid nanoparticles
  • the RTE system is delivered using LNPs.
  • LNPs are particles composed of lipids, typically spherical in shape and a micron or less in diameter, that are used as delivery vehicles for genetic material, such as DNA or RNA, into cells. Without being limited by theory, LNPs protect the genetic material from degradation and facilitate its entry into target cells.
  • LNPs comprise an ionizable lipid, a phospholipid, cholesterol, a PEGylated lipid or any combination thereof.
  • the ionizable lipid is an ionizable 80 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 cationic lipid.
  • the LNP of the disclosure comprises: an ionizable cationic lipid, a phospholipid, a cholesterol, and a PEGylated lipid.
  • the average size of the LNP is between about 10nm and about 1000nm in diameter, between about 100nm and about 1000nm in diameter, between about 100nm and about 1000nm in diameter, between about 500nm and about 1000nm in diameter, between about 10nm and about 100nm in diameter, between about 10nm and about 200nm in diameter, between about 10nm and about 500nm in diameter, or between about 100nm and about 500nm in diameter.
  • Any technique known in the art may be used to determine the size of the LNP.
  • LNP size could be measured using dynamic light scattering (DLS).
  • the composition comprises one or more nucleic acids packaged into an LNP.
  • the driver nucleic acid and template nucleic acid of the disclosure are packaged into the LNP.
  • the driver nucleic acid and template nucleic acid are provided as RNAs and packaged into the LNP.
  • a driver nucleic acid and template nucleic acid are packaged independently into LNPs, which may be comprised of the same or different components.
  • the driver may be delivered as an amino acid sequence.
  • Targeted LNPs [0325] In some embodiments, the RTE system is delivered using a targeted LNP (also referred to herein as “tLNP”).
  • the tLNP is covalently modified with an attached antibody (also referred to herein as a “targeting moiety”) or antigen binding domain that recognizes a target antigen present on the target cell of interest, which can also increase specificity.
  • the target antigen is a T cell specific antigen.
  • the targeting moiety is specific to a single target (e.g., CD3 ⁇ ). In some embodiments, the targeting moiety is bispecific (e.g., CD3 ⁇ and CD20).
  • targeting moieties of the disclosure are fused to tLNPs, bind and/or activate T cells, and enhance delivery of the nucleic acids packaged in the tLNPs.
  • the tLNP is comprised of 1,2-dioleoyl-sn-glycero-3-phosphate (18PA) which can be used to target LNPs using the Selective organ-targeting (SORT) LNP method.
  • the LNP is covalently linked to an antibody that recognizes a T cell antigen.
  • the covalently linked antibody comprises a silent Fc domain. 81 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032
  • the antibody is specific for one or more of CD2, CD5, CD7, TCR, CD8, and CD3.
  • the antibody is selected from the group consisting of OKT3 (VH SEQ ID NO: 63, VL SEQ ID NO: 64), BMA031 (VH SEQ ID NO: 65, VL SEQ ID NO: 66), H65 (VH SEQ ID NO: 67, VL SEQ ID NO: 68), 3A1E (VH SEQ ID NO: 69, VL SEQ ID NO: 70), OKT8 (VH SEQ ID NO: 71, VL SEQ ID NO: 72), Visilizumab (VH SEQ ID NO: 73, VL SEQ ID NO: 74), Teplizumab (VH SEQ ID NO: 88, VL SEQ ID NO: 89), Siplizumab (VH SEQ ID NO: 92, VL SEQ ID NO: 93), Urelumab (VH SEQ ID NO: 94, VL SEQ ID NO: 95), Otelixizumab (VH SEQ ID NO: 101, VL SEQ ID NO:
  • the tLNP is covalently linked to an antibody fragment that recognizes a T cell antigen.
  • the antibody fragment may be a scFv, an antigen-binding fragment (Fab), a (Fab)2, a diabody, a single-domain antibody (sdAb) or a variable domain of the heavy chain of a heavy-chain-only antibody (VHH).
  • a non-limiting selection of antigens (with corresponding antibodies or fragments thereof that specifically said antigen) useful for tLNPs targeting a T cell includes, Anti-human CD2 (VIP VIIIC8 and Siplizumab), CD3 ⁇ (OKT3, Visilizumab, Teplizumab, Otexlizumab, Foralumab, CD3_F2B), CD5 (H65, 5G7, and UHCT2), CD7 (3A1E), CD8 (OKT8), and 4-1BB (Urelumab).
  • Immune cell conditioning [0331] Also provided herein are methods for conditioning immune cells (e.g., T cells) to be more responsive to in vivo or ex vivo transfection.
  • adjuvant conditioning is utilized to improve the efficacy of treatment.
  • the adjuvant conditioning acts via the recruitment of other elements of the immune system.
  • the immune cell conditioning comprises activation of T cells.
  • T cell activator encompasses any agent that elicits a response from T cells, such as but not limited to proliferation, differentiation and engagement in immune- mediated functions.
  • a T cell activator such as an activating antibody, is administered to T cells concurrently with, or prior to, transfection.
  • a T 82 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 cell activator such as an activating antibody
  • the activating antibody or T cell activator is selected from the group consisting of Anti-human CD3 clones: OKT3, UCHT1, SK7, HIT3a YTH 12.5, Teplizumab (HzOKT3 ⁇ 1), Otelixumab, Foralumab, CD3_F2B, and CD3_F1F. and anti-TCR clone BMA031, or any variant thereof.
  • the activating antibody or T cell activator is an immune cell engager such as a bispecific T cell engager (“BiTe”) which binds an activating moiety on the T cells (typically CD3) as well as a tumor antigen.
  • a bispecific T cell engager such as a bispecific T cell engager (“BiTe”) which binds an activating moiety on the T cells (typically CD3) as well as a tumor antigen.
  • the BiTe is selected from the group consisting of Mosunetuzumab (Heavy chain 1 SEQ ID NO: 121, Light chain 1 SEQ ID NO: 122, Heavy chain 2 SEQ ID NO: 123, Light chain 2 SEQ ID NO: 124), Blinatumomab, MGD006, XmAb14045, AMG 330, AMG 420, AMG 564, AMG 701, BiTe 197, REGN1979, RG6026, Mosunetuzumab, AFM11, MGD006, tebentafusp, Catuxomab, Pasotuxizumab, Acapatamab, AMV564, Glofitamab, Solitomab, Talquetamab, Tarlatamab, Tebentafusp, Odronextamab, Epcoritamab, and modified versions of any of the preceding.
  • Mosunetuzumab Heavy chain 1 SEQ ID NO: 121, Light chain 1 SEQ ID NO:
  • T cells are activated in a subject with the administration by intravenous or subcutaneous infusion or injection.
  • the subject is treated or conditioned with a ⁇ -chain receptor agonist.
  • the ⁇ -chain receptor agonist it administered by intravenous or subcutaneous infusion or injection.
  • the ⁇ -chain receptor agonist comprises IL-15, IL-2, IL-7, or IL-21.
  • T cells are activated in a subject with the administration of one or more immune checkpoint inhibitors (inhibitors or the checkpoint associated with CTLA-4, PD-1, PD-L1, Tim-3 LAG-3, OX40, GITR, CD40, CD122, CD137, CD122, CD40, ICOS, TIGIT, Siglec-15, or B7H3).
  • Enhancing Moieties [0339] Also provided herein are enhancing moieties that can be included into the RTE integration systems of the disclosure.
  • SAM and HD domain containing protein 1 is a tetrameric enzyme with dNTP triphosphate hydrolase (dNTPase) activity.
  • SAMHD1 has been shown to limit retroviral infectivity (e.g. HIV infectivity) by depleting the intracellular dNTP pools, which is required for 83 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 efficient reverse transcription.
  • SAMHD1 inhibitors may be provided as an enhancing moiety to be used in conjunction with RTE integration systems of the disclosure, to improve the efficiency of transgene genomic insertion.
  • the SAMHD1 inhibitor reduces SAMHD1 ⁇ mediated dNTP degradation.
  • Non-limiting SAMHD1 inhibitors are provided herein.
  • the SAMHD1 inhibitor is a viral inhibitor of SAMHD1, or is derived from such a viral inhibitor.
  • the SAMHD1 inhibitor is from a human immunodeficiency virus (HIV), which may be HIV-1 or HIV-2. In other embodiments the SAMHD1 inhibitor is from SIV.
  • the SAMH1 inhibitor comprises a Vpx polypeptide or a functional variant thereof (i.e. retaining the functional properties of Vpx).
  • Vpx includes wild-type naturally occurring Vpx (from any source), as well as modifications and variants thereof which retain the functional properties of Vpx.
  • amino acid sequence of Vpx comprises the amino acid sequence of SEQ ID NO: 1568 or comprises an amino acid sequence comprising at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the nucleic acid sequence of Vpx is set forth in SEQ ID NO: 3399 or comprises a nucleic acid sequence comprising at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • polypeptide-based SAMHD1 inhibitors may be provided to facilitate transgene integration in the polypeptide form itself, or as a nucleic acid (DNA or RNA, e.g. mRNA) encoding said polypeptide-based SAMHD1 inhibitor.
  • the SAMHD1 inhibitors described herein may be used in conjunction with RTE integration systems of the disclosure, in order to effectuate the noted enhanced transgene integration.
  • the RTE integration systems further comprise at least one 84 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 SAMHD1 inhibitor.
  • the at least one SAMHD1 inhibitor is provided in combination with an RTE integration system of the disclosure.
  • the SAMHD1 inhibitor is genetically encoded (e.g. provided as a nucleic acid sequence encoding a SAMHD1 inhibitor polypeptide).
  • the driver nucleic acid and template nucleic acid are provided in cis (i.e. on a single nucleic acid), and a genetically encoded SAMHD1 inhibitor is provided in trans, and is provided as a separate nucleic acid sequence encoding the SAMHD1 inhibitor polypeptide.
  • the driver nucleic acid and template nucleic acid are provided in cis (i.e.
  • the driver nucleic acid and template nucleic acid are provided in trans (i.e. on separate nucleic acids), and a genetically encoded SAMHD1 inhibitor is provided for in cis with the template.
  • the driver nucleic acid and template nucleic acid are provided in trans (i.e. on separate nucleic acids), and a genetically encoded SAMHD1 inhibitor is provided further in trans, and is provided as yet another separate nucleic acid encoding the SAMHD1 inhibitor polypeptide.
  • the driver nucleic acid and template nucleic acid are provided in trans (i.e. on separate nucleic acids), and a genetically encoded SAMHD1 inhibitor is provided for in cis with the driver nucleic acid.
  • the driver nucleic acid comprising the nucleic acid sequence encoding the RTE polypeptide and the nucleic acid sequence encoding the SAMHD1 inhibitor polypeptide is bicistronic.
  • the driver nucleic acid comprising the nucleic acid sequence encoding the RTE polypeptide and the nucleic acid sequence encoding the SAMHD1 inhibitor polypeptide further comprises a IRES sequence.
  • the driver nucleic acid comprising the nucleic acid sequence encoding the RTE polypeptide and the nucleic acid sequence encoding the SAMHD1 inhibitor polypeptide, further comprises a nucleic acid sequence encoding a self-cleaving 2A peptide, optionally a T2A peptide, a P2A peptide, E2A peptide, or a F2A peptide.
  • the RTE integration systems of the disclosure comprise at least one SAMHD1 inhibitor.
  • the SAMHD1 inhibitor is not genetically encoded and is provided in the form of a protein.
  • compositions comprising any of the nucleic acids, RTE integration systems, and/or delivery systems of the disclosure, and optionally a pharmaceutically acceptable carrier.
  • Methods of Treatment Provided herein are methods of treatment using the nucleic acids, systems, LNPs, or pharmaceutical compositions of the disclosure.
  • the nucleic acids, systems, LNPs, or pharmaceutical compositions of the disclosure are used in a method of treatment of treating a disease or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the nucleic acids, systems, LNPs, or pharmaceutical compositions of the disclosure.
  • the disease or condition is cancer.
  • the cancer is a lymphoma or leukemia.
  • the disease or condition is an autoimmune disease.
  • the disease or condition is an inborn error of metabolism.
  • one or more nucleic acid(s) and/or proteins are provided, e.g., in a pharmaceutical composition of the disclosure, for delivery to a cell, or to a subject.
  • the subject is a mammal.
  • the subject is a human.
  • the route of administration includes, but is not limited to intravenous, intraperitoneal, intramuscular, subcutaneous, intrathecal, and intradermal administration.
  • administration is via injection or intravenous infusion.
  • the injection is intramuscular, intraperitoneal, intravascular, or subcutaneous.
  • two or more of the nucleic acids, systems, LNPs, or pharmaceutical compositions of the disclosure can be administered together or simultaneously.
  • two or more two or more of the nucleic acids, systems, LNPs, or pharmaceutical compositions of the disclosure can be administered separately (e.g., sequentially).
  • a subject is pre-treated with an activating antibody or T cell activator.
  • the T cell activator is an antibody.
  • the T cell activator is an CD3 or TCR agonist.
  • the CD3 agonist is an antibody 86 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 selected from the group consisting of OKT3, Teplizumab, Otelixizumab, Foralumab, Vislizumab, CD3_F1F, CD3_F2B, and modified versions of any of the preceding.
  • the TCR agonist is BMA031 or a modified version thereof.
  • the T cell activator is an immune cell engager such as a Bispecific T cell engager (BiTe).
  • the BiTe is selected from the group consisting of Blinatumomab, MGD006, XmAb14045, AMG 330, AMG 420, AMG 564, AMG 701, BiTe 197, REGN1979, RG6026, Mosunetuzumab, AFM11, MGD006, tebentafusp, Catuxomab, Pasotuxizumab, Acapatamab, AMV564, Glofitamab, Solitomab, Talquetamab, Tarlatamab, Tebentafusp, Odronextamab, Epcoritamab, and modified versions of any of the preceding.
  • the subject is administered prior, concurrently, or after administration of an activating agent with at least one ⁇ -chain receptor agonist.
  • the at least one ⁇ -chain receptor agonist is selected from the group consisting of IL-2, IL-7, IL-15, IL-21, and modified versions of any of the preceding.
  • the immune cell may be a T cell, a B cell, a NK (natural killer) cell, or an NK-T cell.
  • the immune cell may be an immune cell that natively expresses a TCR-CD3 (T cell receptor-cluster of differentiation 3) complex, e.g., a T cell or a NK-T cell.
  • the method of in vivo genomic manipulation comprises priming (pre-activating) T cells.
  • the T cells are primed by pre-administering a T cell activator, or concomitantly administering the T cell activator with any of the nucleic acids, systems, LNPs, or pharmaceutical compositions of the disclosure.
  • the T cell activator is an antibody.
  • the T cell activator is a CD3 or TCR agonist.
  • the CD3 agonist is an antibody selected from the group consisting of OKT3, Teplizumab, Otelixizumab, Foralumab, Vislizumab, CD3_F1F, CD3_F2B, and modified versions of any of the preceding.
  • the TCR agonist is BMA031 or a 87 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 modified version thereof.
  • the T cell activator is an immune cell engager such as a BiTe.
  • the BiTe is selected from the group consisting of Blinatumomab, MGD006, XmAb14045, AMG 330, AMG 420, AMG 564, AMG 701, BiTe 197, REGN1979, RG6026, Mosunetuzumab, AFM11, MGD006, tebentafusp, Catuxomab, Pasotuxizumab, Acapatamab, AMV564, Glofitamab, Solitomab, Talquetamab, Tarlatamab, Tebentafusp, Odronextamab, Epcoritamab, and modified versions of any of the preceding.
  • the method of in vivo manipulation further comprises administering a reprograming agent.
  • the reprogramming agent is an RTE integration system comprising one or more nucleic acids.
  • the reprograming agent meditates durable expression by integrating one or more nucleic acids into a genome.
  • the reprograming agent mediates transient expression of one or more nucleic acids.
  • the one or more nucleic acids comprise at least one nucleic acid sequence comprised in or derived from a retrotransposon, a transposon, a Cas protein, a guide RNA, or a combination thereof.
  • the one or more nucleic acids is packaged in a viral vector or an LNP.
  • the viral vector is selected from the group consisting of a lentivirus, a retrovirus, an Adeno-associated viral vector (AAV), herpes simplex viral vector, and an adenovirus.
  • the RTE integration system comprises the driver nucleic acids, templates, delivery systems, and/or pharmaceutical compositions of the disclosure. In some embodiments, the RTE integration system comprises one or more nucleic acids.
  • the one or more nucleic acids comprise at least one nucleic acid sequence comprised in or derived from a retrotransposon, a transposon, a Cas protein, a guide RNA, or a combination thereof.
  • ENUMERATED EMBODIMENTS [0360] Provided herein are non-limited exemplary embodiments of the disclosure. Set I [0361] Embodiment I-1.
  • TAM T cell receptor fused antigen modifier
  • Embodiment I-4 The nucleic acid construct of any one of I-1 to I-3, wherein the RTE- UTR comprises a 3' RTE-UTR and/or a 5' RTE-UTR.
  • Embodiment I-5 The nucleic acid construct of any one of I-1 to I-4, wherein the RTE- UTR is derived from a non-LTR RE.
  • Embodiment I-6 Embodiment I-6.
  • RTE-UTR is derived from an retrotransposable element (RTE) selected from the group consisting of CRE, R4, Hero, NeSL, R2, RandI, Proto1, L1, Tx1, RTEPT, Proto2, RTEX, RTE, Outcast, Ingi (which includes sub-clade Vingi), I, Nimb, Tad1, Loa, R1, Jockey, Rex1, CR1, L2, L2A, L2B, Daphne, and Crack.
  • RTE retrotransposable element
  • nucleic acid construct of any one of I-4 to I-6, wherein the 5’ RTE- UTR comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 85, SEQ ID NO: 99, SEQ ID NO: 125, SEQ ID NO: 127, any one of SEQ ID NO: 144-249, and any one of SEQ ID NO: 359-367, or a corresponding RNA version thereof.
  • Embodiment I-9 The nucleic acid construct of any one of I-1 to I-8, wherein the antigen binding domain is linked to the N terminus of the TCR subunit by a linker and/or hinge.
  • Embodiment I-10 Embodiment I-10.
  • Embodiment I-11 The nucleic acid construct of any one of I-1 to I-10, wherein the antigen binding domain is specific for BCMA.
  • Embodiment I-12 The nucleic acid construct of any one of I-1 to I-10, wherein the antigen binding domain is specific for CD22. 89 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0373] Embodiment I-13.
  • Embodiment I-14 The nucleic acid construct of I-14, wherein the CD19 antigen binding domain is derived from an antibody selected from the group consisting of FMC63, murine 4G7, human Hu19, Hu1E7, huB4, hBU12, humanized FMC63, humanized 4G7, GR37, GR41, SRB- 37, SRB-85, and a modified version of any of the preceding.
  • Embodiment I-15 The nucleic acid construct of any one of I-1 to I-10, wherein the antigen binding domain is specific for CD20.
  • Embodiment I-16 The nucleic acid construct of I-16, wherein the CD20 antigen binding domain is derived from an antibody selected from the group consisting of Leu16, rituximab, ofatumumab, ocrelizumab, obinutuzumab, Ibritumomab tiuxetan, AME-133v, IMMU-106, TRU-015, tositumomab, and a modified version of any of the preceding.
  • Embodiment I-17 The nucleic acid construct of any one of I-1 to I-10, wherein the antigen binding domain is bispecific.
  • Embodiment I-18 Embodiment I-18.
  • nucleic acid construct of any one of I-1 to I-18, wherein the nucleic acid comprises DNA.
  • Embodiment I-19 The nucleic acid construct of any one of I-1 to I-18, wherein the nucleic acid comprises RNA.
  • Embodiment I-20 The nucleic acid construct of I-20, wherein the RNA is mRNA.
  • Embodiment I-21 The nucleic acid construct of I-21, wherein the RNA is mRNA.
  • a retrotransposable element (RTE) transgene integration system comprising a driver construct and a template, wherein (a) the driver nucleic acid comprises a nucleic acid encoding a site specific RTE polypeptide; and (b) the template comprises (i) a nucleic acid encoding an engineered immune receptor; (ii) a retroelement (RTE)-UTR, wherein the RTE-UTR is capable of binding the RTE polypeptide; and wherein the site specific RTE polypeptide mediates integration of the nucleic acid encoding the engineered immune receptor into a T cell genome.
  • the driver nucleic acid comprises a nucleic acid encoding a site specific RTE polypeptide
  • the template comprises (i) a nucleic acid encoding an engineered immune receptor; (ii) a retroelement (RTE)-UTR, wherein the RTE-UTR is capable of binding the RTE polypeptide; and wherein the site specific RTE polypeptide mediates integration of
  • TCR T cell receptor
  • TCR fused antigen modifier TAM
  • CAR chimeric antigen receptor
  • a retrotransposable element (RTE) transgene integration system comprising a driver construct and a template, wherein (a) the driver construct comprises a nucleic acid encoding an RTE polypeptide; and (b) the template comprises (i) a nucleic acid encoding a T cell receptor fused antigen modifier (TRAM); and (ii) a retroelement (RE)-UTR, wherein the RTE-UTR is capable of binding the RTE polypeptide; and wherein the RTE polypeptide mediates integration of the nucleic acid encoding the TRAM into a genome.
  • the driver construct comprises a nucleic acid encoding an RTE polypeptide
  • the template comprises (i) a nucleic acid encoding a T cell receptor fused antigen modifier (TRAM); and (ii) a retroelement (RE)-UTR, wherein the RTE-UTR is capable of binding the RTE polypeptide; and wherein the RTE polypeptide mediates integration of the nucleic
  • Embodiment I-25 The system of any one of I-22 to I-25, wherein the driver construct and the template are in a trans configuration and provided as separate nucleic acids.
  • Embodiment I-25 The system of any one of I-22 to I-25, wherein the driver construct and the template are in a cis configuration and provided in the same nucleic acid.
  • Embodiment I-26 The system of any one of I-22 to I-29, wherein the RTE-UTR comprises a 3' RTE-UTR and/or a 5' RTE-UTR .
  • Embodiment I-27 Embodiment I-27.
  • RTE-UTR comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 85, SEQ ID NO: 99, SEQ ID NO: 125, SEQ ID NO: 127, any one of SEQ ID NO: 144-249, and any one of SEQ ID NO: 359-367, or a corresponding RNA version thereof.
  • Embodiment I-30 The system of I-30 or I-31, wherein the RTE3’ UTR comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 86, SEQ ID NO: 100, SED ID NO: 126, SEQ ID NO: 128, any one of SEQ ID NO: 250-358, and any one of SEQ ID NO: 368-372, or a corresponding RNA version thereof.
  • Embodiment I-29 The system of any one of I-22 to I-32, wherein the TRAM comprises an antigen binding domain, and a TCR subunit selected from the group consisting of TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ .
  • Embodiment I-31 The system of any one of I-22 to I-33, wherein the antigen binding domain is selected from the group consisting of an scFv, VHH, TCR-like antibody, natural ligand, FcR-binding receptor (e.g. CD16), and an NK killing receptor.
  • Embodiment I-31 The system of I-34, wherein the antigen binding domain is specific for CD19. 91 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0392] Embodiment I-32.
  • the system of I-35 wherein the CD19 antigen binding domain is selected from the group consisting of FMC63, murine 4G7, human Hu19, Hu1E7, huB4, hBU12, humanized FMC63, humanized 4G7, GR37, GR41, SRB-37, SRB-85, and modified versions of any of the preceding.
  • Embodiment I-33 The system of I-34, wherein the antigen binding domain is specific for CD20.
  • Embodiment I-34 wherein the antigen binding domain is specific for CD20.
  • Embodiment I-38 wherein the CD20 antigen binding domain is selected from the group consisting of Leu16, rituximab, ofatumumab, ocrelizumab, obinutuzumab, Ibritumomab tiuxetan, AME-133v, IMMU-106, TRU-015, tositumomab, and modified versions of any of the preceding.
  • Embodiment I-35 The system of I-34, wherein the antigen binding domain is bispecific for CD19 and CD20.
  • Embodiment I-36 The system of any one of I-22 to I-40, wherein the RTE polypeptide is a non-LTR RTE.
  • Embodiment I-37 The system of any one of I-22 to I-42, wherein the RTE polypeptide is derived from an RTE selected from the group consisting of CRE, R4, Hero, NeSL, R2, RandI, Proto1, L1, Tx1, RTEPT, Proto2, RTEX, RTE, Outcast, Ingi (which includes sub-clade Vingi), I, Nimb, Tad1, Loa, R1, Jockey, Rex1, CR1, L2, L2A, L2B, Daphne, and Crack.
  • Embodiment I-38 The system of any one of I-22 to I-43, wherein RTE polypeptide is a naturally occurring RTE polypeptide.
  • Embodiment I-39 The system of any one of I-22 to I-43, wherein RTE polypeptide is an engineered RTE polypeptide.
  • Embodiment I-40 The system of any one of I-22 to I-45, wherein the template comprises a binding site for a differentially expressed microRNA (miR).
  • Embodiment I-41 The system of I-46, wherein the binding site for the differentially expressed miR is located within the RTE3’ UTR region.
  • Embodiment I-42 The system of I-46 or I-47, wherein the binding site for the differentially expressed miR is specific for miR-122.
  • Embodiment I-43 The system of any one of I-22 to I-43, wherein RTE polypeptide is an engineered RTE polypeptide.
  • Embodiment I-40 The system of any one of I-22 to I-45, wherein the template comprises a binding site for a differentially expressed microRNA (miR).
  • Embodiment I-41 The system of
  • Embodiment I-44 The system of I-49, wherein the binding site for the differentially expressed miR is specific for miR-122. 92 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0405] Embodiment I-45.
  • the system of any one of I-22 to I-50, wherein the driver construct comprises DNA.
  • Embodiment I-46 The system of any one of I-22 to I-50, wherein the driver construct comprises RNA. [0407] Embodiment I-47.
  • Embodiment I-48 The system of any one of I-22 to I-54, wherein the template comprises DNA.
  • Embodiment I-50 The system of I-56, wherein the RNA is mRNA.
  • Embodiment I-51 A lipid nanoparticle (LNP) comprising the system of any one of I-22 to I-58.
  • Embodiment I-52 The LNP of I-59, wherein the LNP is covalently linked to an antibody that recognizes an T cell antigen.
  • Embodiment I-53 The LNP of I-60, wherein the covalently linked antibody comprises a silent Fc domain.
  • Embodiment I-54 The LNP of I-60 or I-61, wherein the antibody is specific for one or more of CD2, CD5, CD7, TCR, CD8, and CD3.
  • Embodiment I-55 Embodiment I-55.
  • Embodiment I-56 A pharmaceutical composition comprising: (a) the nucleic acid construct of any one of I-1 to I-21; (b) the RTE transgene integration system of any one of I-22 to I-58; or (c) the LNP of any one of I-59 to I-63, and a pharmaceutically acceptable carrier. 93 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0417]
  • Embodiment I-57 A method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of I-64. [0418] Embodiment I-58.
  • Embodiment I-65 wherein the subject is pre-treated with a T cell activator.
  • Embodiment I-59 The method of I-66, wherein the T cell activator is a CD3 or TCR agonist.
  • Embodiment I-60 The method of I-67, wherein the CD3 agonist is an antibody selected from the group consisting of OKT3, Teplizumab, Otelixizumab, Foralumab, Vislizumab, CD3_F1F, CD3_F2B, and modified versions of any of the preceding.
  • Embodiment I-61 The method of I-67, wherein the TCR agonist is BMA031 or a modified version thereof.
  • Embodiment I-62 The method of I-66, wherein the T cell activator is an immune cell engager such as a Bispecific T cell engager (BiTe).
  • Embodiment I-63 The method of I-70, wherein the BiTe is selected from the group consisting of Blinatumomab, MGD006, XmAb14045, AMG 330, AMG 420, AMG 564, AMG 701, BiTe 197, REGN1979, RG6026, Mosunetuzumab, AFM11, MGD006, tebentafusp, Catuxomab, Pasotuxizumab, Acapatamab, AMV564, Glofitamab, Solitomab, Talquetamab, Tarlatamab, Tebentafusp, Odronextamab, Epcoritamab, and modified versions of any of the preceding.
  • Embodiment I-64 The method of any one of I-65 to I-72, wherein the subject is further administered prior, concurrently, or after said administering with at least one ⁇ -chain receptor agonist.
  • Embodiment I-65 The method of I-73, wherein the at least one ⁇ -chain receptor agonist is selected from the group consisting of IL-2, IL-7, IL-15, IL-21, and modified versions of any of the preceding.
  • Embodiment I-66 The method of any one of I-65 to I-74, wherein the disease is cancer or an autoimmune disease.
  • Embodiment I-67 Embodiment I-67.
  • Embodiment I-68 A method of in vivo gene editing comprising pre-administering a T cell activator and administering a pharmaceutical composition comprising a reprograming agent.
  • Embodiment I-69 The method of I-77, wherein the reprogramming agent is a gene editing system comprising one or more nucleic acids.
  • Embodiment I-71 The method of I-77, wherein the reprograming agent mediates transient expression of one or more nucleic acids.
  • Embodiment I-72 The method of any one of I-77 to I-80, wherein the one or more nucleic acids comprise at least one nucleic acid sequence comprised in or derived from a retrotransposon, a transposon, a Cas protein, a guide RNA, or a combination thereof.
  • Embodiment I-73 Embodiment I-73.
  • Embodiment I-74 The method of I-83, wherein the viral vector is selected from the group consisting of a lentivirus, a retrovirus, and an adenovirus.
  • Embodiment I-75 The method of any one of I-77 to I-84, wherein the T cell activator is a CD3 or TCR agonist.
  • Embodiment I-76 The method of any one of I-77 to I-81, wherein the one or more nucleic acids is packaged in a viral vector or a lipid nanoparticle (LNP).
  • Embodiment I-74 The method of I-83, wherein the viral vector is selected from the group consisting of a lentivirus, a retrovirus, and an adenovirus.
  • Embodiment I-75 The method of any one of I-77 to I-84, wherein the T cell activator is a CD3 or TCR agonist.
  • Embodiment I-76 Embodiment I-76.
  • Embodiment I-85 wherein the CD3 agonist is an antibody selected from the group consisting of OKT3, Teplizumab, Otelixizumab, Foralumab, Vislizumab, CD3_F1F, CD3_F2B, and modified versions of any of the preceding.
  • Embodiment I-77 The method of I-85, wherein the TCR agonist is BMA031 or a modified version thereof.
  • Embodiment I-78 The method of I-77 to I-84, wherein the T cell activator is an immune cell engager such as a BiTe.
  • Embodiment I-79 Embodiment I-79.
  • the BiTe is selected from the group consisting of Blinatumomab, MGD006, XmAb14045, AMG 330, AMG 420, AMG 564, AMG 701, BiTe 197, REGN1979, RG6026, Mosunetuzumab, AFM11, MGD006, tebentafusp, Catuxomab, Pasotuxizumab, Acapatamab, AMV564, Glofitamab, Solitomab, Talquetamab, Tarlatamab, Tebentafusp, Odronextamab, Epcoritamab, and modified versions of any of the preceding.
  • Embodiment II-1 A nucleic acid encoding a T cell receptor fused antigen modifier (TRAM), the nucleic acid comprising: (a) a nucleic acid sequence encoding an antigen binding domain; (b) a nucleic acid sequence encoding a TCR subunit; and (c) a retroelement (RTE)-UTR.
  • TAM T cell receptor fused antigen modifier
  • Embodiment II-2 The nucleic acid of II-1, wherein the nucleic acid construct comprises more than one construct.
  • Embodiment II-3 Embodiment II-3.
  • Embodiment II-4 The nucleic acid of any one of II-1 to II-3, wherein the RTE-UTR comprises a 3’ RTE-UTR and/or a 5’ RTE-UTR.
  • Embodiment II-5 The nucleic acid of any one of II-1-4, wherein the RTE-UTR is derived from a non-LTR RE.
  • Embodiment II-6 The nucleic acid of any one of II-1-4, wherein the RTE-UTR is derived from a non-LTR RE.
  • RTE-UTR is derived from an retrotransposable element (RTE) selected from the group consisting of CRE, R4, Hero, NeSL, R2, RandI, Proto1, L1, Tx1, RTEPT, Proto2, RTEX, RTE, Outcast, Ingi (which includes sub- clade Vingi), I, Nimb, Tad1, Loa, R1, Jockey, Rex1, CR1, L2, L2A, L2B, Daphne, and Crack.
  • RTE retrotransposable element
  • nucleic acid of any one of II-4-6, wherein the 5’ RTE-UTR comprises a nucleic acid sequence selected from the group consisting of SEQ ID SEQ ID NO: 33, SEQ ID NO: 85, SEQ ID NO: 99, SEQ ID NO: 125, SEQ ID NO: 127, any one of SEQ ID NO: 144-249, and any one of SEQ ID NO: 359-367, or a corresponding RNA version thereof.
  • the nucleic acid of any one of II-4-7, wherein the 3’ RTE UTR comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 86, SEQ ID NO: 100, SED ID NO: 126, SEQ ID NO: 128, any one of SEQ ID NO: 250- 358, and any one of SEQ ID NO: 368-372, or a corresponding RNA version thereof.
  • Embodiment II-9 The nucleic acid of any one of II-1-8, wherein the antigen binding domain is linked to the N terminus of the TCR subunit by a linker and/or hinge.
  • 96 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032
  • Embodiment II-11 The nucleic acid of any one of II-1-10, wherein the antigen binding domain is specific for GPCR5D.
  • Embodiment II-12 The nucleic acid of any one of II-1-10, wherein the antigen binding domain is specific for BCMA.
  • Embodiment II-13 The nucleic acid of any one of II-1-10, wherein the antigen binding domain is specific for BCMA.
  • Embodiment II-14 The nucleic acid of any one of II-1-10, wherein the antigen binding domain is specific for CD19.
  • Embodiment II-15 The nucleic acid of II-14, wherein the CD19 antigen binding domain is derived from an antibody selected from the group consisting of FMC63, murine 4G7, human Hu19, Hu1E7, huB4, hBU12, humanized FMC63, humanized 4G7, GR37, GR41, SRB-37,SRB- 85, and a modified version of any of the preceding.
  • Embodiment II-16 Embodiment II-16.
  • Embodiment II-17 The nucleic acid of claim16, wherein the CD20 antigen binding domain is derived from an antibody selected from the group consisting of Leu16, rituximab, ofatumumab, ocrelizumab, obinutuzumab, Ibritumomab tiuxetan, AME-133v, IMMU-106, TRU-015, tositumomab, and a modified version of any of the preceding.
  • Embodiment II-18 Embodiment II-18.
  • Embodiment II-19 The nucleic acid of any one of II-1 to II-18, wherein the nucleic acid construct comprises DNA.
  • Embodiment II-20 The nucleic acid of any one of II-1 to II-18, wherein the nucleic acid construct comprises RNA.
  • Embodiment II-21 The nucleic acid of II-20, wherein the RNA is mRNA.
  • Embodiment II-22 Embodiment II-22.
  • a retrotransposable-element (RTE) transgene integration system comprising a driver nucleic acid and a template, wherein (a) the driver nucleic acid comprises a nucleic acid encoding a site specific RTE polypeptide; and (b) the template comprises (i) a nucleic acid encoding an engineered immune receptor; 97 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 (ii) a retroelement (RE)-UTR, wherein the RTE-UTR is capable of being bound by the RTE polypeptide; and wherein the site specific RTE polypeptide mediates integration of a DNA copy of the template into a T cell genome.
  • the driver nucleic acid comprises a nucleic acid encoding a site specific RTE polypeptide
  • the template comprises (i) a nucleic acid encoding an engineered immune receptor; 97 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 (
  • TCR T cell receptor
  • TCR fused antigen modifier TAM
  • TRuC chimeric antigen receptor
  • CAR chimeric antigen receptor
  • a retrotransposable-element (RTE) transgene integration system comprising a driver nucleic acid and a template, wherein a) the driver nucleic acid comprises a nucleic acid encoding an RTE polypeptide; and (b) the template comprises (i) a nucleic acid encoding a T cell receptor fused antigen modifier (TRAM); and (ii) a retroelement (RTE)-UTR, wherein the RTE-UTR is capable of being bound by the RTE polypeptide; and wherein the RTE polypeptide mediates a DNA copy of the template into a T cell genome.
  • the driver nucleic acid comprises a nucleic acid encoding an RTE polypeptide
  • the template comprises (i) a nucleic acid encoding a T cell receptor fused antigen modifier (TRAM); and (ii) a retroelement (RTE)-UTR, wherein the RTE-UTR is capable of being bound by the RTE polypeptide; and wherein the
  • Embodiment II-26 The system of any one of II-22 to II-24, wherein the driver nucleic acid and the template are in a trans configuration and provided as separate nucleic acid constructs.
  • Embodiment II-26 The system of any one of II-22 to II-24, wherein the driver nucleic acid and the template are in a cis configuration and provided in the same nucleic acid construct.
  • Embodiment II-27 The system of any one of II-22 to II-26, wherein the RTE-UTR comprises a 3’ RTE-UTR and/or a 5’ RTE-UTR.
  • Embodiment II-28 Embodiment II-28.
  • RTE-UTR comprises a nucleic acid sequence selected from the group consisting of SEQ ID SEQ ID NO: 33, SEQ ID NO: 85, SEQ ID NO: 99, SEQ ID NO: 125, SEQ ID NO: 127, any one of SEQ ID NO: 144-249, and any one of SEQ ID NO: 359-367, or a corresponding RNA version thereof.
  • SEQ ID SEQ ID NO: 33 SEQ ID NO: 85, SEQ ID NO: 99, SEQ ID NO: 125, SEQ ID NO: 127, any one of SEQ ID NO: 144-249, and any one of SEQ ID NO: 359-367, or a corresponding RNA version thereof.
  • the system of II-27 or II-28, wherein the 3’ RTE UTR comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 86, SEQ ID NO: 100, SED ID NO: 126, SEQ ID NO: 128, any one of SEQ ID NO: 250-358, and any one of SEQ ID NO: 368-372, or a corresponding RNA version thereof.
  • 98 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0469] Embodiment II-30.
  • Embodiment II-31 The system of any one of II-22-29, wherein the TRAM comprises an antigen binding domain, and a TCR subunit selected from the group consisting of TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ .
  • Embodiment II-31 The system of any one of II-22-30, wherein the antigen binding domain is selected from the group consisting of an scFv, VHH, TCR-like antibody, natural ligand, FcR-binding receptor (e.g. CD16), and an NK killing receptor.
  • Embodiment II-32 The system of II-31, wherein the antigen binding domain is specific for CD19.
  • Embodiment II-33 The system of II-31, wherein the antigen binding domain is specific for CD19.
  • Embodiment II-34 The system of II-31, wherein the antigen binding domain is specific for CD20.
  • Embodiment II-35 The system of II-31, wherein the antigen binding domain is specific for CD20.
  • Embodiment II-34 wherein the CD20 antigen binding domain is selected from the group consisting of Leu16, rituximab, ofatumumab, ocrelizumab, obinutuzumab, Ibritumomab tiuxetan, AME-133v, IMMU-106, TRU-015, tositumomab, and modified versions of any of the preceding.
  • Embodiment II-36 The system of II-31, wherein the antigen binding domain is bispecific for CD19 and CD20.
  • Embodiment II-37 The system of any one of II-22 to II-36, wherein the RTE polypeptide is a non-LTR RTE.
  • Embodiment II-38 The system of any one of II-22 to II-37, wherein the RTE polypeptide is derived from an RTE selected from the group consisting of CRE, R4, Hero, NeSL, R2, RandI, Proto1, L1, Tx1, RTEPT, Proto2, RTEX, RTE, Outcast, Ingi (which includes sub-clade Vingi), I, Nimb, Tad1, Loa, R1, Jockey, Rex1, CR1, L2, L2A, L2B, Daphne, and Crack.
  • Embodiment II-39 The system of any one of II-22 to II-38, wherein RTE polypeptide is a naturally occurring RTE polypeptide.
  • Embodiment II-40 The system of any one of II-22 to II-38, wherein RTE polypeptide is an engineered RTE polypeptide. 99 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032
  • Embodiment II-41 The system of any one of II-22 to II-40, wherein the template comprises a binding site for a differentially expressed microRNA (miR).
  • Embodiment II-42 The system of II-41, wherein the binding site for the differentially expressed miR is located within the 3’ RTE UTR region.
  • Embodiment II-43 Embodiment II-43.
  • Embodiment II-44 The system of any one of II-22 to II-43, wherein the driver nucleic acid comprises a binding site for a differentially expressed miR.
  • Embodiment II-45 The system of II-44, wherein the binding site for the differentially expressed miR is specific for miR-122.
  • Embodiment II-46 The system of any one of II-22 to II-45, wherein the driver nucleic acid comprises DNA.
  • Embodiment II-47 The system of any one of II-22 to II-45, wherein the driver nucleic acid comprises RNA.
  • Embodiment II-48 The system of II-47, wherein the RNA is mRNA.
  • Embodiment II-49 The system of any one of II-22 to II-48, wherein the template comprises DNA.
  • Embodiment II-50 The system of any one of II-22 to II-48, wherein the template comprises RNA.
  • Embodiment II-51 The system of II-50, wherein the template comprises a promotor and a polyA signal.
  • Embodiment II-52 A lipid nanoparticle (LNP) comprising the nucleic acid of any one of II-1 to II-21 or the system of any one of II-22to II-51.
  • Embodiment II-53 A lipid nanoparticle (LNP) comprising the nucleic acid of any one of II-1 to II-21 or the system of any one of II-22to II-51.
  • Embodiment II-52 wherein the LNP is covalently linked to an antibody or any fragment thereof that recognizes an T cell antigen.
  • Embodiment II-54 The LNP of II-53, wherein the covalently linked antibody comprises a silent Fc domain.
  • Embodiment II-55 The LNP of II-53 or II-54, wherein the antibody is specific for one or more of CD2, CD5, CD7, TCR, CD8, and CD3.
  • Embodiment II-57 A pharmaceutical composition comprising: (a) the nucleic acid of any one of II-1 to II-21; (b) the RTE transgene integration system of any one of II-22 to II-51; or (c) the LNP of any one of II-52 to II-56. [0497] and a pharmaceutically acceptable carrier. [0498] Embodiment II-58.
  • a method of treating a disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of (a) the nucleic acid of any one of II-1 to II-21; (b) the RTE transgene integration system of any one of II-22 to II-51; or (c) the LNP of any one of II-52 to II-56, (d) the pharmaceutical composition of II-57.
  • Embodiment II-59 The method of II-58, wherein the subject is pre-treated with a T cell activator.
  • Embodiment II-60 The method of II-59, wherein the T cell activator is an CD3 or TCR agonist.
  • Embodiment II-60 wherein the CD3 agonist is an antibody selected from the group consisting of OKT3, Teplizumab, Otelixizumab, Foralumab, Vislizumab, CD3_F1F, CD3_F2B, and modified versions of any of the preceding.
  • Embodiment II-62 The method of II-60, wherein the TCR agonist is BMA031 or a modified version thereof.
  • Embodiment II-63 The method of II-59, wherein the T cell activator is an immune cell engager such as a Bispecific T cell engager (BiTe).
  • Embodiment II-64 Embodiment II-64.
  • BiTe is selected from the group consisting of Blinatumomab, MGD006, XmAb14045, AMG 330, AMG 420, AMG 564, AMG 701, BiTe 197, REGN1979, RG6026, Mosunetuzumab, AFM11, MGD006, tebentafusp, Catuxomab, Pasotuxizumab, Acapatamab, AMV564, Glofitamab, Solitomab, Talquetamab, 101 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 Tarlatamab, Tebentafusp, Odronextamab, Epcoritamab, and modified versions of any of the preceding.
  • Embodiment II-65 The method of any one of II-58-64, wherein the subject is further administered prior, concurrently, or after said administering with at least one ⁇ -chain receptor agonist.
  • Embodiment II-66 The method of II-65, wherein the at least one ⁇ -chain receptor agonist is selected from the group consisting of IL-2, IL-7, IL-15, IL-21, and modified versions of any of the preceding.
  • Embodiment II-67 The method of any one of II-58 to II-66, wherein the disease is cancer or an autoimmune disease.
  • Embodiment II-68 The method of any one of II-58 to II-66, wherein the disease is cancer or an autoimmune disease.
  • Embodiment II-69 A method of in vivo gene editing comprising pre-administering a T cell activator and administering a pharmaceutical composition comprising a reprograming agent.
  • Embodiment II-70 The method of II-69, wherein the reprogramming agent is a gene editing system comprising one or more nucleic acids.
  • Embodiment II-72 The method of II-69, wherein the reprograming agent mediates transient expression of one or more nucleic acids.
  • Embodiment II-73 The method of any one of II-69 to II-72, wherein the one or more nucleic acids comprise at least one nucleic acid sequence comprised in or derived from a retrotransposon, a transposon, a Cas protein, a guide RNA, or a combination thereof.
  • Embodiment II-74 Embodiment II-74.
  • Embodiment II-75 The method of II-74, wherein the viral vector is selected from the group consisting of a lentivirus, a retrovirus, and an adenovirus.
  • Embodiment II-76 The method of any one of II-69 to II-75, wherein the T cell activator is an CD3 or TCR agonist. 102 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0517] Embodiment II-77.
  • Embodiment II-76 wherein the CD3 agonist is an antibody selected from the group consisting of OKT3, Teplizumab, Otelixizumab, Foralumab, Vislizumab, CD3_F1F, CD3_F2B, and modified versions of any of the preceding.
  • Embodiment II-78 The method of II-76, wherein the TCR agonist is BMA031 or a modified version thereof.
  • Embodiment II-79 The method of II-69 to II-75, wherein the T cell activator is an immune cell engager such as a BiTe.
  • Embodiment II-80 Embodiment II-80.
  • a method of in vivo genomic manipulation in an immune cell comprising contacting the immune cell with: (a) the nucleic acid of any one of II-1 to II-21; (b) the system of any one of II-22 to II-51; (c) the LNP of any one of II-52 to II-56; (d) the pharmaceutical composition of II-57; or (e) a combination thereof.
  • Set III [0522] Embodiment III-1.
  • a nucleic acid comprising: (a) a nucleic acid sequence encoding a T cell receptor fused antigen modifier (TRAM) comprising: (i) an antigen binding domain; and (ii) a TCR subunit, and (b) a retrotransposable element untranslated region (RTE-UTR).
  • TAM T cell receptor fused antigen modifier
  • RTE-UTR retrotransposable element untranslated region
  • Embodiment III-2 The nucleic acid of III-1, wherein the nucleic acid comprises more than one separate nucleic acids. 103 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0524] Embodiment III-3. The nucleic acid of III-1 or Embodiment III-2, wherein the TCR subunit is selected from the group consisting of TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ . [0525] Embodiment III-4. The nucleic acid of any one of III-1 to III-3, wherein the RTE-UTR comprises a 3’ RTE-UTR and/or a 5’ RTE-UTR.
  • Embodiment III-5 The nucleic acid of any one of III-1 to III-4, wherein the RTE-UTR is derived from a non-LTR RTE.
  • Embodiment III-6 The nucleic acid of III-5, wherein the non-LTR RTE is selected from the group consisting of CRE, R4, Hero, NeSL, R2, RandI, Proto1, L1, Tx1, RTEPT, Proto2, RTEX, RTE, Outcast, Ingi, I, Nimb, Tad1, Loa, R1, Jockey, Rex1, CR1, L2, L2A, L2B, Daphne, and Crack.
  • Embodiment III-7 Embodiment III-7.
  • nucleic acid of any one of III-4 to III-6, wherein the 5’ RTE-UTR comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 85, SEQ ID NO: 99, SEQ ID NO: 125, SEQ ID NO: 127, any one of SEQ ID NO: 144- 249, and any one of SEQ ID NO: 359-367, or modified versions of any of the preceding sequences having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, or a corresponding RNA version thereof.
  • Embodiment III-8 Embodiment III-8.
  • nucleic acid of any one of III-4 to III-7, wherein the 3’ RTE UTR comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 86, SEQ ID NO: 100, SED ID NO: 126, SEQ ID NO: 128, any one of SEQ ID NO: 250- 358, and any one of SEQ ID NO: 368-372, or modified versions of any of the preceding sequences having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, or a corresponding RNA version thereof.
  • Embodiment III-9 Embodiment III-9.
  • Embodiment III-10 The nucleic acid of any one of III-1to III-9, wherein the antigen binding domain is selected from the group consisting of an scFv, a VHH, a TCR-like antibody, a natural ligand, an FcR-binding receptor (e.g. CD16), and an NK killing receptor.
  • Embodiment III-11 The nucleic acid of any one of III-1 to III-10, wherein the antigen binding domain is specific for GPCR5D.
  • Embodiment III-12 Embodiment III-12.
  • the nucleic acid of III-14 wherein the CD19 antigen binding domain is derived from an antibody selected from the group consisting of FMC63, murine 4G7, human Hu19, Hu1E7, huB4, hBU12, humanized FMC63, humanized 4G7, GR37, GR41, SRB- 37, and SRB-85, or a modified version of any of the preceding.
  • Embodiment III-16 The nucleic acid of any one of III-1 to III-10, wherein the encoded antigen binding domain is specific for CD20.
  • Embodiment III-17 Embodiment III-17.
  • the nucleic acid of III- wherein the CD20 antigen binding domain is derived from an antibody selected from the group consisting of Leu16, rituximab, ofatumumab, ocrelizumab, obinutuzumab, Ibritumomab tiuxetan, AME-133v, IMMU-106, TRU-015, and tositumomab, or a modified version of any of the preceding.
  • Embodiment III-18 The nucleic acid of any one of III-1 to III-17, wherein the antigen binding domain is a bispecific antigen binding domain.
  • nucleic acid of any one of III-1 to III-18, wherein the nucleic acid comprises DNA.
  • Embodiment III-20 The nucleic acid of any one of claims 1-18 wherein the nucleic acid comprises RNA.
  • Embodiment III-21 The nucleic acid of any one of III-1 to III-18, wherein the nucleic acid is a RNA/DNA hybrid.
  • Embodiment III-22 The nucleic acid of any one of III-1 to III-18, wherein the nucleic acid is a RNA/DNA hybrid.
  • a retrotransposable-element (RTE) integration system comprising a driver nucleic acid and a template nucleic acid, wherein: (a) the driver nucleic acid comprises a nucleic acid sequence encoding an RTE polypeptide, wherein the RTE polypeptide is a site-specific RTE polypeptide; and (b) the template nucleic acid comprises: (i) a nucleic acid sequence encoding an engineered immune receptor; and (ii) an RTE-UTR capable of being bound by the RTE polypeptide.
  • Embodiment III-23 The system of III-22, wherein the engineered immune receptor comprises an antigen binding domain.
  • Embodiment III-24 The system of III-22 or III-23, wherein the engineered immune receptor is selected from the group consisting of a T cell receptor (TCR), a TCR fused antigen modifier (TRAM), a TRuC and a chimeric antigen receptor (CAR).
  • TCR T cell receptor
  • TRM TCR fused antigen modifier
  • TRuC TRuC
  • CAR chimeric antigen receptor
  • a retrotransposable-element (RTE) integration system comprising a driver nucleic acid and a template nucleic acid, wherein: (a) the driver nucleic acid comprises a nucleic acid sequence encoding an RTE polypeptide; and (b) the template nucleic acid comprises (i) a nucleic acid sequence encoding a T cell receptor fused antigen modifier (TRAM) comprising an antigen binding domain and a TCR subunit; and (ii) an RTE-UTR capable of being bound by the RTE polypeptide.
  • T cell receptor fused antigen modifier T cell receptor fused antigen modifier
  • Embodiment III-27 The system of III-26, wherein the immune cell that natively expresses a TCR-CD3 complex is a T-cell.
  • Embodiment III-28 The system of any one of III-22 to III-27, wherein the driver nucleic acid and the template nucleic acid are provided as separate nucleic acids.
  • Embodiment III-29 The system of any one of III-22 to III-25, wherein the RTE polypeptide mediates integration of a DNA copy of the nucleic acid sequence encoding an engineered immune receptor into a genome of an immune cell that natively expresses a TCR- CD3 complex.
  • Embodiment III-30 The system of any one of III-22 to III-29, wherein the RTE-UTR comprises a 3’ RTE-UTR and/or a 5’ RTE-UTR.
  • Embodiment III-31 Embodiment III-31.
  • RTE-UTR comprises a nucleic acid sequence selected from the group consisting of SEQ ID SEQ ID NO: 33, SEQ ID NO: 85, SEQ ID NO: 99, SEQ ID NO: 125, SEQ ID NO: 127, any one of SEQ ID NO: 144-249, and any one of SEQ ID NO: 359-367, or modified versions of any of the preceding sequences having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, or a corresponding RNA version thereof.
  • the system of III-30 or III-31, wherein the 3’ RTE UTR comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 86, SEQ ID NO: 100, SED ID NO: 126, SEQ ID NO: 128, any one of SEQ ID NO: 250- 106 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 358, and any one of SEQ ID NO: 368-372, or modified versions of any of the preceding sequences having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, or a corresponding RNA version thereof.
  • Embodiment III-33 Embodiment III-33.
  • Embodiment III-34 The system of any one of III-23 to III-33, wherein the antigen binding domain is selected from the group consisting of an scFv, a VHH, a TCR-like antibody, a natural ligand, an FcR-binding receptor (e.g. CD16), and an NK killing receptor.
  • Embodiment III-35 The system of any one of III-25 to III-32, wherein the TCR subunit is selected from the group consisting of TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ .
  • Embodiment III-36 The system of III-35, wherein the antigen binding domain is specific for CD19.
  • Embodiment III-37 The system of III-35, wherein the CD19 antigen binding domain is selected from the group consisting of FMC63, murine 4G7, human Hu19, Hu1E7, huB4, hBU12, humanized FMC63, humanized 4G7, GR37, GR41, SRB-37, SRB-85, and modified versions of any of the preceding.
  • Embodiment III-38 Embodiment III-38.
  • Embodiment III-39 The system of III-38, wherein the CD20 antigen binding domain is selected from the group consisting of Leu16, rituximab, ofatumumab, ocrelizumab, obinutuzumab, Ibritumomab tiuxetan, AME-133v, IMMU-106, TRU-015, tositumomab, and modified versions of any of the preceding.
  • Embodiment III-40 The system of any one of III-23 to III-36, wherein the antigen binding domain is bispecific.
  • Embodiment III-41 The system of any one of III-23 to III-36, wherein the antigen binding domain is bispecific.
  • Embodiment III-42 The system of any one of III-22 to III-41, wherein the RTE polypeptide is a non-LTR RTE.
  • Embodiment III-43 The system of III-42, wherein the non-LTR RTE is selected from the group consisting of CRE, R4, Hero, NeSL, R2, RandI, Proto1, L1, Tx1, RTEPT, Proto2, RTEX, RTE, Outcast, Ingi, I, Nimb, Tad1, Loa, R1, Jockey, Rex1, CR1, L2, L2A, L2B, Daphne, and Crack.
  • Embodiment III-44 The system of any one of III-22 to III-43, wherein the RTE polypeptide is a naturally occurring RTE polypeptide.
  • Embodiment III-45 The system of any one of III-22 to III-43, wherein the RTE polypeptide is an engineered RTE polypeptide.
  • Embodiment III-46 The system of any one of III-22 to III-45, wherein the template nucleic acid comprises a binding site for a differentially expressed microRNA (miR).
  • Embodiment III-47 Embodiment III-47.
  • Embodiment III-48 The system of III-46 or III-47, wherein the binding site for the differentially expressed miR is specific for miR-122.
  • Embodiment III-49 The system of any one of III-22 to III-48, wherein the driver nucleic acid comprises a binding site for a differentially expressed miR.
  • Embodiment III-50 The system of III-49, wherein the binding site for the differentially expressed miR is specific for miR-122.
  • Embodiment III-51 Embodiment III-51.
  • Embodiment III-52 The system of any one of III-22 to III-50, wherein the driver nucleic acid comprises DNA.
  • Embodiment III-52 The system of any one of III-22 to III-50, wherein the driver nucleic acid comprises RNA.
  • Embodiment III-53 The system of any one of III-22 to III-50, wherein the driver nucleic acid comprises a DNA/RNA hybrid.
  • Embodiment III-54 The system of III-52, wherein the RNA is mRNA.
  • Embodiment III-55 The system of any one of III-22 to III-54, wherein the template nucleic acid comprises DNA.
  • Embodiment III-56 The system of any one of III-22 to III-50, wherein the driver nucleic acid comprises DNA.
  • Embodiment III-57 The system of any one of claims III-22 to III-54, wherein the template nucleic acid comprises a DNA/RNA hybrid.
  • Embodiment III-58 The system of III-56, wherein the template nucleic acid comprises a promotor and a polyA signal.
  • Embodiment III-59 A lipid nanoparticle (LNP) comprising the nucleic acid of any one of III-1 to III-21 or the system of any one of III-22 to III-58.
  • LNP lipid nanoparticle
  • Embodiment III-60 The LNP of III-59, wherein the LNP is covalently linked to an antibody or any fragment thereof that recognizes a T cell antigen.
  • Embodiment III-61 The LNP of III-60, wherein the covalently linked antibody comprises a silent Fc domain.
  • Embodiment III-62 The LNP of III-60 or III-61, wherein the antibody is specific for one or more of CD2, CD5, CD7, TCR, CD8, and CD3.
  • Embodiment III-63 Embodiment III-63.
  • Embodiment III-64 A pharmaceutical composition comprising a pharmaceutically acceptable carrier and: (a) the nucleic acid of any one of III-1 to III-21; (b) the RTE integration system of any one of III-22 to 58; (c) the LNP of any one of III-59 to III-63; or (d) a combination thereof. [0586] Embodiment III-65.
  • a method of treating a disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of (a) the nucleic acid of any one of III-1 to III-21; (b) the RTE integration system of any one of III-22 to III-58; (c) the LNP of any one of III-59 to III-63, (d) the pharmaceutical composition of III-64, or (e) a combination thereof.
  • Embodiment III-66 The method of III-65, wherein the subject is pre-treated with a T cell activator or concomitantly treated with a T cell activator. 109 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0588] Embodiment III-67.
  • Embodiment III-68 The method of III-67, wherein the CD3 agonist is an antibody selected from the group consisting of OKT3, Teplizumab, Otelixizumab, Foralumab, Vislizumab, CD3_F1F, and CD3_F2B, or modified versions of any of the preceding.
  • Embodiment III-69 The method of III-67, wherein the TCR agonist is BMA031 or a modified version thereof.
  • Embodiment III-70 The method of III-66, wherein the T cell activator is an immune cell engager.
  • Embodiment III-71 The method of III-70, wherein the immune cell engager is a Bispecific T cell engager (BiTe).
  • Embodiment III-72 The method of III-71, wherein the BiTe is selected from the group consisting of Blinatumomab, MGD006, XmAb14045, AMG 330, AMG 420, AMG 564, AMG 701, BiTe 197, REGN1979, RG6026, Mosunetuzumab, AFM11, MGD006, tebentafusp, Catuxomab, Pasotuxizumab, Acapatamab, AMV564, Glofitamab, Solitomab, Talquetamab, Tarlatamab, Tebentafusp, Odronextamab, and Epcoritamab, or modified versions of any of the preceding.
  • Embodiment III-73 The method of any one of III-65 to III-72, wherein the subject is further administered prior, concurrently, or subsequent to said administering with at least one ⁇ - chain receptor agonist.
  • Embodiment III-74 The method of III-73, wherein the at least one ⁇ -chain receptor agonist is selected from the group consisting of IL-2, IL-7, IL-15, IL-21, and modified versions of any of the preceding.
  • Embodiment III-75 The method of any one of III-65 to III-74, wherein the disease is cancer or an autoimmune disease.
  • Embodiment III-76 Embodiment III-76.
  • Embodiment III-77 A method of in vivo genome manipulation comprising pre- administering a T cell activator and administering a pharmaceutical composition comprising a reprograming agent. 110 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0599] Embodiment III-78. The method of III-77, wherein the reprogramming agent is a genome manipulation system comprising one or more nucleic acids. [0600] Embodiment III-79.
  • Embodiment III-80 The method of III-77, wherein the reprograming agent mediates transient expression of one or more nucleic acids.
  • Embodiment III-81 The method of any one of III-77 to III-80, wherein the one or more nucleic acids comprise at least one nucleic acid sequence comprised in or derived from a retrotransposon, a transposon, a Cas protein, a guide RNA, or a combination thereof.
  • Embodiment III-82 The method of any one of III-77 to III-80, wherein the one or more nucleic acids comprise at least one nucleic acid sequence comprised in or derived from a retrotransposon, a transposon, a Cas protein, a guide RNA, or a combination thereof.
  • the method of III-79, wherein the one or more nucleic acids comprise at least one nucleic acid sequence comprised in or derived from a retrotransposon.
  • 83 The method of any one of III-77 to III-81, wherein the one or more nucleic acids is packaged in a viral vector or a lipid nanoparticle (LNP).
  • Embodiment III-84 The method of III-83, wherein the viral vector is selected from the group consisting of a lentivirus, a retrovirus, and an adenovirus.
  • Embodiment III-85 The method of any one of III-77 to III-84, wherein the T cell activator is an CD3 or TCR agonist.
  • Embodiment III-86 The method of III-85, wherein the CD3 agonist is an antibody selected from the group consisting of OKT3, Teplizumab, Otelixizumab, Foralumab, Vislizumab, CD3_F1F, CD3_F2B, and modified versions of any of the preceding.
  • Embodiment III-87 The method of III-85, wherein the TCR agonist is BMA031 or a modified version thereof.
  • Embodiment III-88 The method of III-77 to III-84, wherein the T cell activator is an immune cell engager.
  • Embodiment III-89 Embodiment III-89.
  • the method of III-88, wherein the immune cell engager is a Bispecific T cell engager (BiTe).
  • BiTe Bispecific T cell engager
  • the method of III-88, wherein the BiTe is selected from the group consisting of Blinatumomab, MGD006, XmAb14045, AMG 330, AMG 420, AMG 564, AMG 701, BiTe 197, REGN1979, RG6026, Mosunetuzumab, AFM11, MGD006, tebentafusp, Catuxomab, Pasotuxizumab, Acapatamab, AMV564, Glofitamab, Solitomab, Talquetamab, Tarlatamab, Tebentafusp, Odronextamab, Epcoritamab, and modified versions of any of the preceding.
  • Embodiment III-91 A method of in vivo genomic manipulation in an immune cell, the method comprising contacting the immune cell with: (a) the nucleic acid of any one of III-1 to III-21; (b) the RTE integration system of any one of III-22 to 58; or (c) the LNP of any one of III-59 to III-63, (d) the pharmaceutical composition of III-64; or (e) a combination thereof. [0613] wherein the immune cell natively expresses a TCR-CD3 complex.
  • transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative embodiments, as “consisting of” and “consisting essentially of” the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B”, the disclosure also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B”.
  • Example 1 Materials and Methods [0617] Unless stated otherwise, material and methods for the subsequent Examples were performed as described herein below.
  • Cell lines and PBMCs [0618] Nalm6-Luc, Raji-Luc, Jurkat, and THP1 cells were cultured in complete RPMI (“cRPMI”) containing RPMI (Capricorn) supplemented with 10% fetal-calf serum (“FCS”, Gibco), 2 mM L-Glutamine (Bio-West), and 1x Penicillin–Streptomycin (“Pen/strep,” Bio- West).
  • cRPMI complete RPMI
  • FCS fetal-calf serum
  • FCS fetal-calf serum
  • Bio-West 2 mM L-Glutamine
  • Pen/strep Penicillin–Streptomycin
  • Huh7, U2-OS, and HepG2 cells were cultured in complete DMEM (“cDMEM) containing DMEM (Capricorn) supplemented with 10% FCS, 2mM L-Glutamine, and 1x Pen/strep.
  • cDMEM complete DMEM
  • FCS FCS
  • 2mM L-Glutamine 2mM L-Glutamine
  • 1x Pen/strep 1x Pen/strep
  • PHL Primary human hepatocytes
  • Primary human hepatocytes were cultured in Human Hepatocyte Medium (Primacyt) using the Hepatocyte Plating and Thawing Kit (Primacyt)
  • PBMCs peripheral blood mononuclear cells
  • T-cells a mixture of cell types including, e.g., T-cells, B-cells, and NK cells, from healthy donors (Cell Generation)
  • T cell medium containing ImmunoCultTM-XF T cell Expansion Medium (Stem Cell), 10% FCS, 1x Pen/strep, and 100 U/ml IL-2 (Miltenyi) thereby resulting unless otherwise noted.
  • PBMCs were activated for 2 days after thawing in T cell medium 113 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 with the addition of TransActTM (Miltenyi) at a 1:100 dilution, driving expansion of T cells.
  • TransActTM TransActTM
  • IL-2 was replaced with 10ng/ml IL-7 (Miltenyi) and 10ng/ml IL-15 (Miltenyi).
  • the indicated antibodies at the indicated doses were added to the culture medium (with IL-7 and IL-15). All cells were grown in incubators with 5% CO2 at 37 o C.
  • Donor PBMCs are referred to herein by donor number, For example, PBMCs from donor 13 are referred to as “DP13”.
  • Plasmids [0621] All plasmids were synthesized by GenScript. The CAR or TRAM transgenes were codon optimized using an in-house algorithm. They contain the T7 promoter and a unique Type IIS restriction site at the 3 ⁇ end. In-vitro transcription (IVT) [0622] Plasmids were linearized using restriction enzyme according to manufacturer’s protocol, purified with AMPure XP beads, placed on magnetic tube rack, washed three times with 70% ethanol while on magnet, dried, and resuspended in water.
  • RNA from in vitro translation (IVT) 60 ⁇ g total was loaded from one syringe and lipid mix (SM-102, DSPC, Cholesterol, and DMG- PEG2000 unless otherwise noted) from a second syringe and mixed using the Ignite machine.
  • LNPs were then washed with 40ml PBS, added to a tube with centrifugal filter (Amicon) and centrifuged at 2000g at 4C until volume was reduced to approximately 500 ⁇ l.
  • Encapsulated RNA concentration was determined using a RibogreeenTM assay before and after lysing LNPs, where the encapsulated RNA is the total mRNA (following lysis) minus the free RNA (before lysis).
  • the Stunner machine was used, which analyzes UV/Vis concentration, dynamic light scattering (DLS), and static light scattering (SLS). LNPs were stored at 4C, typically for up to one week.
  • LNP Targeted LNP
  • the lipid mix also contained DPSE-PEG2000-Maleimide.
  • LNPs with Maleimide (“LNP-Mal”) were produced using the above method for LNP production and mixed with antibodies that were reduced using Tris (2-carboxyethyl) phosphine (TCEP).
  • TEP Tris (2-carboxyethyl) phosphine
  • Targeted LNPs were purified from unconjugated antibodies using a Size Exclusion Column (SEC).
  • RNA concentration Fractions containing tLNPs were combined and analyzed using the same methods.
  • Antibodies for cell culture or tLNPs [0625] Anti-human CD2 (VIP VIIIC8 and Siplizumab), CD3 ⁇ (OKT3, Visilizumab, Teplizumab, Otexlizumab, Foralumab, CD3_F2B), CD5 (H65, 5G7, and UHCT2), CD7 (3A1E), CD8 (OKT8), and 4-1BB (Urelumab) antibody VH and VL portions were incorporated into human IgG1 with Fc silencing mutations or tested as they are used clinically, where relevant. Selected antibodies were also tested in additional formats such as Fab, (Fab)2, or diabodies.
  • Isotype control for binders acquired from Absolute Antibodies was anti-FITC (4-4-20) or a standard Isotype control from all other vendors (MiMabs, Selleckhem, FJ Bio, etc.).
  • Anti-mouse CD3 ⁇ (145-2C11) VH and VL portions were incorporated into mouse IgG1 with or without Fc silencing.
  • T cells Two days post thaw, T cells were centrifuged at 300g for 5 min at RT, suspended to 1x10 6 cells/ml in T cell medium supplemented with 2 ⁇ g/ml Apolipoprotein E (ApoE; Peptrotech), and seeded in 96-well TC plates, 1x10 5 cells per well. LNPs were added at dose of 400 ng mRNA per well in duplicates unless otherwise noted and incubated for 24. Cells were then centrifuged, suspended in T cell medium, 200 ⁇ l per well, and incubated for 24 h.
  • Apolipoprotein E Apolipoprotein E
  • Cells were then expanded, for GFP studies in TC plates (Thermo-Fisher) or for CAR/TRAM in gas- permeable 24-well G-Rex plates (Wilson Wolf) according to the manufacturer’s protocol.
  • 115 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 Lentiviral transduction [0627]
  • Cells were transduced with VSVG-pseudotyped lentiviruses (LV) (VectorBuilder) at a multiplicity of infection (MOI) of 20.
  • Primary human T cells were transduced 2 days post thaw in the same conditions as LNP transfection but without ApoE and expanded in the same manner.
  • the gating strategy was removal of debris based on FSC-W and FSC-A, removal of dead cells based on DAPI (or L/D EF506), selection of cells based on SSC-A and FSC-A, and selection of singlets based FSC-H and FSC-A.
  • Quantification of CAR or TRAM receptors [0629] Quantum APC MESF beads (Bang Laboratories) were run with the same APC voltage as the T cells stained with either anti-FMC63-APC or anti-Flag-APC.
  • MESF Equivalent Soluble Fluorochrome
  • dPCR Digital PCR
  • Insertion site was testing using Lumi-Seq. Frozen pellets of cells were thawed, lysed, DNA extracted, and sheared in using Adaptive Focused Acoustics (AFA) technology (Covaris). All purification steps were performed with AMPure XP beads. DNA was blunted using NEBNext End Repair Module and purified. Then dA tailed was performed using NEBNext dA- Tailing Module and purified. Uniquely labeled adapters were ligated with T4 DNA ligase and purified.
  • AFA Adaptive Focused Acoustics
  • DNA cassette was enriched using a PCR with P5 and target specific primers, purified, a second PCR with P5 and nested primers, flanked by Illumina adapter sequence, purified and size selected, followed by and a third PCR with P5 primers and unique index adapters.
  • Library QC was done using Qubit and TapeStation, and then run on the Iseq100 high throughput sequencer.
  • Luciferase killing assay [0632] Firefly luciferase (Luc)-labelled target cells (Nalm6-Luc or Raji-Luc) were seeded in wells of white 96-well TC plates (Greiner) in cRPMI, 1x10 5 cells/well in 70 ⁇ l.
  • Effector cells were added in 50 ⁇ l cRPMI at indicated Effector:Target (E:T) ratios between 20:1 and 0.01:1 calculated based on CAR+ or TRAM+ cells.
  • Non-treated (NT) T cells were added to have the same total cell number as the average of the CAR+ or TRAM+ cells.
  • Co-culture was incubated for 20 h, and BioGloTM Luciferase Assay System reagent (Promega) was added, 20 ⁇ l 117 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 per well, and incubated for 10 mins.
  • Luciferase signal was measured with the Infinite 200 Pro plate reader (Tecan) with an exposure time of 500 ms. The higher the luciferase signal the more viable target cells per well. Relative viability was calculated as relative units (RLU) of a given well divided by the maximum RLU for the given effector.
  • EC50 curves were generated with Prism 10 (GraphPad) by fitting a non-linear sigmodial curve, 4PL, X is concentration. The bottom of the curve constrained to equal 0 to ensure uniformity between curves and at very high E:Ts which may be larger than 20:1 no target cells would be viable. The lower the EC50 the more potent the killing.
  • Cytokine secretion assay For cytokine secretion analysis an additional 80 ⁇ l of medium was added to the wells in the above describe Luc killing assay and harvested after 20 h of co-culture from in-vitro cultures or from serum of mice and analyzed using the human IFN ⁇ ELISA Kit (Ray Biotech) or Th1/Th2 cytokine bead array “CBA” kit (BD) according to manufacturer’s protocols.
  • In vivo studies in mice [0634] For activation or LNP studies for human T cells, immunodeficient mice were injected I.V or I.P as indicated with the indicated quantity of human PBMCs at indicated time before treatment.
  • mice For activation and LNP studies with immunocompetent mice C57Bl/6 mice were used unless otherwise indicated. For studies with mRNA-LNPs for transient expression mice were sacrificed and organs were harvested 24h post injection unless otherwise noted. For LNP studies with integrating mRNA-LNPs blood was sampled at indicated time points and mice were sacrificed at indicated time point. For anti-tumor response studies, Nalm6-Luc or Raji-Luc cells were injected IV and after tumor establishment CAR-T or TRAM-T cells or LNPs were injected I.V. at the indicated doses. Tumor burden was measured by IVIS at indicated time points and blood was sampled for FACS and/or dPCR analysis at indicated time points. All mouse studies were approved by IACUC ethics committee.
  • FIG.3 also depicts exemplary TRAM designs.
  • CD19 scFv for the following TRAMs comprise FMC63 VL (SEQ ID NO: 2), Whitlow linker (SEQ ID NO: 9), FMC63 VH 118 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 (SEQ ID NO: 4) and are fused to CD3 component via 3xA (SEQ ID NO: 10) and G4Sx3 linker (SEQ ID NO: 3).
  • FMC63 LH scFv-G4S This scFv with spacer is referred to herein as “FMC63 LH scFv-G4S” (SEQ ID NO: 11).
  • “mC ⁇ + ⁇ TRAM” comprises mTCR ⁇ SP (SEQ ID NO: 12), FMC63 VH (SEQ ID NO: 4), 3xA (SEQ ID NO: 10), mTCR-C ⁇ (SEQ ID NO: 13), T2A (SEQ ID NO: 14), FMC63 VL (SEQ ID NO: 2), 3xA (SEQ ID NO: 10), and mTCR-C ⁇ (SEQ ID NO: 15).
  • mC ⁇ TRAM comprises mTCR ⁇ SP (SEQ ID NO: 12), mTCR-C ⁇ (SEQ ID NO: 13), T2A (SEQ ID NO: 14), FMC63 LH scFv-G4S (SEQ ID NO:11), and mTCR-C ⁇ (SEQ ID NO: 15).
  • hC ⁇ TRAM comprises hTCR ⁇ SP (SEQ ID NO: 16), FMC63 LH scFv-G4S (SEQ ID NO: 11), and hTCR- C ⁇ (SEQ ID NO: 17).
  • ⁇ TRAM comprises of CD8 ⁇ SP (SEQ ID NO: 1), FMC63 LH scFv-G4S (SEQ ID NO: 11), and CD3 ⁇ (SEQ ID NO: 18).
  • ⁇ TRAM comprises of CD8 ⁇ SP (SEQ ID NO: 1), FMC63 LH scFv-G4S (SEQ ID NO: 11), and CD3 ⁇ (SEQ ID NO: 19).
  • ⁇ TRAM comprises CD8 ⁇ SP (SEQ ID NO: 1), FMC63 LH scFv-G4S (SEQ ID NO: 11), and CD3 ⁇ (SEQ ID NO: 20).
  • Example 2 Lentiviral transduction of several cell types with Anti-CD19 CAR or TRAMs [0643] Whereas a canonical CAR can express stably on the surface of any cell, a TRAM should only express on T cells, which are the only cells that express all 6 TCR-CD3 chains.
  • lentiviral vectors were generated with an anti-CD19 CAR (nucleic acid sequence set forth SEQ ID NO: 3336, amino acid sequence set forth in SEQ ID NO: 23) or TRAM according to the designs depicted in FIG.3 and described in Example 1, followed by a T2A self-cleaving peptide (SEQ ID NO: 14) and GFP (amino acid sequence set forth in SEQ ID NO: 24, nucleic acid sequence set forth in SEQ ID NO: 3331).
  • MNDopt (SEQ ID NO: 25) is a truncated version of the MND promoter (SEQ ID NO: 26) which is referred to herein to as “MNDu.”
  • Lentiviral vectors were used due to high transduction efficiency in a wide variety of cell types, as RTEs expression can be low in certain cell types. Inclusion of the T2A-GFP allows 119 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 for observation of cells that underwent transduction, regardless of the stability of the CAR or TRAM.
  • FIG.4C Expression 6 days post transduction in liver cell line HUH7.
  • FIG.4D Expression 6 days post transduction in B cell line Nalm6-Luc.
  • FIG.4E Expression 6 days post transduction in B cell line Raji-Luc.
  • FIG.4F Median fluorescence intensity (MFI) of CAR or TRAM expression on HUH7 cells gated on GFP+ (transduced) cells. Most TRAMs expressed similarly to and slightly lower than CART-19. For mC ⁇ + ⁇ and hC ⁇ TRAMs, expression was lower and not all GFP+ cells were stained with the anti-FMC63 antibody.
  • FIG.5-6 For Raji and Nalm6, CD19+ malignant B cell lines, it is advantageous to test whether expression of CARs or TRAMs lead to CD19 antigen masking.
  • CAR/TRAM and CD19 surface expression was analyzed within the transduced (GFP+) cells (FIGS.5-6).
  • FIG.5 cells were transduced with lentiviruses at an MOI of 20. Further analysis of cells shown in FIG.4D.
  • NT non-treated cells.
  • FIG.5A %GFP+ gated on singlets.
  • FIGGS.5B-5F Analysis gated on transduced (GFP+) cells.
  • FIG.5B Detectable %CAR+ or %TRAM+ cells, indicating cell surface expression.
  • FIG.5C Detectable %CD19+ indicative of cells without full 120 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 CD19 masking.
  • FIGS.5D- 5F Median fluorescence intensity (MFI) of indicated marker.
  • FIG. 5D GFP MFI.
  • FIG.5E Surface CAR or TRAM MFI.
  • FIG.5F Detectable (unmasked) CD19 MFI. As expected, CART-19 highly expressed on the cell surface and completely masked CD19 in both cell lines.
  • FIG.6A %GFP+ gated on singlets.
  • FIGS.6B- 6F Analysis gated on transduced (GFP+) cells.
  • FIG.6B Detectable %CAR+ or %TRAM+ cells, indicating cell surface expression.
  • FIG.6C Detectable %CD19+ indicative of cells without full CD19 masking.
  • FIGS.6D-6F Median fluorescence intensity (MFI) of indicated marker.
  • FIG. 6D GFP MFI.
  • FIG.6E Surface CAR or TRAM MFI.
  • FIG.6F Detectable (unmasked) CD19 MFI. GFP expression was seen in 30-60% of cells, as shown in FIG.6A. The amount of the GFP positive cells mC ⁇ , hC ⁇ , and ⁇ TRAMs was detected on the surface of 12-24% of cells, whereas mC ⁇ + ⁇ , ⁇ , ⁇ , and ⁇ TRAMs were not detectable, as shown in FIG.6B. With regard to CD19 masking, CART-19 completely masked CD19 and it was not detectable, and for the TRAMs there were different degrees of masking, though CD19 was still detectable in most or all of the cells ( as shown in FIG.6C and FIG.6F).
  • the ⁇ TRAM masked to the highest degree and led to a 445 fold reduction in MFI.
  • the mC ⁇ and hC ⁇ TRAMS also masked to a large degree and reduced MFI by 40 fold (FIG.6F).
  • ⁇ TRAM led to a 20 fold reduction in MFI, consistent with high degree of masking by this TRAM in Nalm6 cells, though staining was all above background.
  • the mC ⁇ + ⁇ , ⁇ and ⁇ TRAMs did not mask CD19 much, reducing MFI only 2-5 fold and cells staining strongly positive.
  • Vingi1-Acar a non-LTR and non-site specific RTE derived from Anolis carolinensis (green Anole), which was found to mediate highly efficient gene transfer of GFP or CART-19 to primary T cells (FIGS. 7A-7F).
  • FIGS.7A-7C GFP transgene under indicated promoters.
  • FIGS.7D-7F CART-19 transgene under indicated promoters.
  • FIG.7A, FIG.7D Genomic integrations according to dPCR with the indicated probes.
  • FIG.7B % GFP positive according to FACS.
  • FIG.7E % CAR positive according to FACS.
  • FIG.7C GFP expression level median fluorescence intensity (MFI).
  • FIG.7F Quantification of CAR expression levels was calculated using FACS and Quantum beads for samples with >500 CAR+ (Day 12 only).
  • Vingi1 driver full transcript (SEQ ID NO: 27) consists of the T7 Clean Cap (SEQ ID NO: 28), Vingi1 driver 5’ UTR (SEQ ID NO: 29), Vingi1 ORF2 (DNA SEQ ID NO: 30, protein SEQ ID NO: 31), and Vingi1 driver 3’UTR and polyA (SEQ ID NO: 32).
  • the Vingi1 reporter consists of the T7 Clean Cap (SEQ ID NO: 28), Vingi1 reporter 5’ UTR (SEQ ID NO: 33), the anti-sense of the cargo (promoter, transgene, polyA signal) of interest in the inverse orientation, 122 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 and Vingi1 reporter 3’UTR and polyA (SEQ ID NO: 34).
  • the cargo was placed in the reverse orientation such that only following integration can the gene be transcribed (sense strand) and translated. All cargo consisted of a promoter, Kozak sequence (SEQ ID NO: 35), transgene on interest, and was followed by a synthetic polyA signal (SEQ ID NO: 36).
  • T cells were transfected with LNPs containing only the reporter, which will not integrate into the cells without the presence of the driver.
  • the ideal promoter for GFP (SEQ ID NO: 24) and CART-19 (amino acid sequence set forth in SEQ ID NO: 23, nucleic acid sequence set forth in SEQ ID NO: 3336) expression was different.
  • the promoters tested were MNDopt (542bp, SEQ ID NO: 25), MNDu (635bp, SEQ ID NO: 26), and the Elongation factor 1 ⁇ (EF1 ⁇ ) promoter (1179 bp, SEQ ID NO: 37).
  • the MNDopt promoter showed >5 fold higher rate of integrations detected by digital PCR (dPCR) with a GFP transgene probe (as shown in FIG.7A), and >4 fold higher %GFP+ detected by flow cytometry than with the EF1 ⁇ and MNDu promotors (as shown in FIG.7B).
  • the ranking was MNDu > EF1 ⁇ > MNDopt (as shown in FIG.7C).
  • the results with GFP for EF1 ⁇ and MNDopt are as expected, since the shorter MNDopt promoter is expected to integrate more efficiently due to processivity of the RT whereas EF1 ⁇ is longer but known to lead to very high levels of expression.
  • MNDopt exhibited highest rate of genomic integrations detected by dPCR (as shown in FIG.7D), however in terms of %CAR+ detectable on the cell surface by FACS, the EF1 ⁇ promotor exhibited the highest expression (as shown in FIG.7E). CAR receptors per cell were quantified using Quantum beads for day 12 samples and the ranking was EF1 ⁇ > MNDu > MNDopt (as shown in FIG.7F). It is worth noting that although the mechanism is unknown surface CAR expression at 5 days post transfection (pt) for Vingi1 RTE was very low and increased substantially by 12 days.
  • FIGS.8A-8I T cells were transfected with indicated LV (FIGS.8G-8I) or Vingi1 RNA-LNPs with CART-19 transgene (FIGS.8A-8F) and expression analyzed at indicated days post transfection. CAR surface expression detected by FACS with an anti-FMC63-APC antibody. Error bars show SD of duplicates.
  • FIGS.8A-8C Vingi1 with MNDopt promoter.
  • FIGS.8D-8F Vingi1 with EF1 ⁇ promoter.
  • FIG.8G-8I LV with EF1 ⁇ promoter.
  • FIGS.8A, 8D, and 8G Genomic integrations according to dPCR with a 123 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 CAR probe.
  • FIGS.8B, 8E, and 8H %CAR detected by FACS.
  • FIGS.8C, 8F, and 8I Quantification of CAR expression levels was calculated using FACS and Quantum beads for samples with >500 CAR+.
  • Example 4 Anti-CD19 CAR or TRAMs with T2A-GFP using Vingi1 driver [0652]
  • reporters with the CART-19 or human CD19 TRAMs were tested followed by T2A-GFP under the MNDopt promoter (FIGS.9A-9C), the same format tested in several cell types with lentiviruses.
  • FIG.9B CAR-T or TRAM-T effector cells were co-cultured with Nalm6-Luc target cells at indicated E:T (effector:target) ratios between 20:1-0.2:1for 20h and luciferase signal read. Relative viability calculated as relative units (RLU) of luminescence relative to maximum RLU of a given effector.
  • FIG.9C Killing EC50s for E:T where half of the targets are killed. CART-19 without the T2A-GFP under the MNDopt or EF1 ⁇ promoters were included as controls.
  • FIG.9A The ⁇ , ⁇ , ⁇ , and ⁇ TRAMs all expressed well whereas hC ⁇ TRAM and CART- 19 with T2A-GFP showed no expression. CART-19 without T2A-GFP was detectable but low under the MNDopt promoter and highly under the EF1 ⁇ promoter. A Nalm6-Luc killing assay was performed using all TRAMs and CARs-T with >7% expression.
  • the E:T (effector:target ratio) EC50 (effective concentration 50%) is the E:T ratio at which 50% of the target cells are alive. A lower EC50 indicates higher sensitivity and potency, as it achieves the same killing with fewer T cells.
  • the ⁇ , ⁇ , ⁇ , and ⁇ TRAMs (with T2A-GFP and under MNDopt) killed similarly to each other (as shown in FIGS.9B and 9C). They all kill better than EF1 ⁇ -CART-19 (without T2A-GFP). It is unclear why the MNDopt TRAM expression is much better than CART-19 using a Vingi1 driver and slightly lower than CART-19 in lentiviruses. In fact, considering how much lower CART-19 expression is with the Vingi1 driver compared to lentiviruses (as shown in FIGS.8A-8I), it would be expected that TRAMs may not achieve detectable expression with RTE drivers.
  • TRAMs are particularly suitable for RTEs due to the surprisingly improved expression compared to a canonical CAR.
  • Example 5 Anti-CD19 CAR or TRAMs under MNDopt using Vingi1 driver (without T2A- GFP) 124 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0653]
  • Vingi1 driver without T2A- GFP
  • 124 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032
  • TRAMs were compared to CART-19 with the same MNDopt (“Mo”) promoter or EF1 ⁇ promoter.
  • T cells were transfected with indicated Vingi1 RNA-LNPs and analyzed by dPCR and FACS at 5- and 12-days post transfection. Results are shown in FIGS.10A-10C.
  • NT non-treated cells.
  • FIG. 10A Genomic integrations according to dPCR to the synthetic poly A probes.
  • FIG.10B %positive according detected by FACS following staining with an anti-FMC63-APC.
  • FIG. 10C Quantification of CAR expression levels was calculated using FACS and Quantum beads. Again, the TRAMs showed superior integration and expression than CART-19.
  • the ranking was ⁇ and ⁇ TRAMs > ⁇ TRAM > Mo-CART- 19 > EF1 ⁇ -CART-19 (FIG.10A).
  • percent CAR or TRAM positive analyzed by flow cytometry the ranking was ⁇ and ⁇ TRAMs > ⁇ TRAM > EF1 ⁇ -CART-19 > Mo-CART-19 (FIG. 10B). It should be noted that the TRAMs were already >23% positive 5 days post transfection, when the Mo-CART-19 was undetectable and EF1a CAR was 2% or 8% CAR depending on donor.
  • FIGS.11A11B Donor 10.
  • FIGS. 11C-11D Donor 13.
  • FIGS.11B and 11D EC50s for E:T where half of the targets are killed. In donor 10 ⁇ TRAM killing lower than the others, yet in donor 13 ⁇ TRAM did kill like the others.
  • ⁇ TRAMs have the highest expression and consistently potent killing, but ⁇ TRAM also expresses and kills well. Since ⁇ 125 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 TRAM doesn’t prevent masking, attention was focused on ⁇ TRAM as the lead design with ⁇ TRAM a good backup.
  • Example 6 Anti-CD19 ⁇ TRAM with MNDopt or EF1 ⁇ promoters using Vingi1 RTE [0654] Since EF1 ⁇ was superior to MNDopt for CART-19 expression, the ⁇ TRAM with these two promoters was then tested (FIGS.12A-12C).
  • FIG.12A Genomic integrations according to dPCR to the synthetic poly A probes.
  • FIG.12B %positive according detected by FACS following staining with an anti-FMC63-APC.
  • FIG.12C Quantification of CAR expression levels were calculated using FACS and Quantum beads. Integrations were again highest for MNDopt (FIG.12A).
  • TRAMs can be used to other target antigens, for example CD20. CD20 is also expressed on many B cell malignancies.
  • CAR or ⁇ TRAMs with a scFv derived from Leu16 VL (SEQ ID NO: 38), Whitlow linker (SEQ ID NO: 9), and Leu16 VH (SEQ ID NO: 39) were tested.
  • a Flag tag SEQ ID NO: 40 was added at the N’ terminus (between the signal peptide and VL). Incorporating some elements such as 41BBzeta or CD3 ⁇ or other subunit denotes canonical CAR T or TRAM. Initially these were tested under the EF1 ⁇ promoter (FIGS.13A-13B).
  • FIG.13A Genomic integrations according to dPCR to the synthetic poly A probes. Error bars show SD of duplicates.
  • FIG.13B %positive 126 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 according detected by FACS following staining with an anti-Flag-APC.
  • the ⁇ TRAMs were compared to a conventional CAR consisting of the same Leu16 scFv followed by the CD28 hinge (SEQ ID NO: 21), CD28 transmembrane (SEQ ID NO: 45), 4-1BB costimulatory domain (SEQ ID NO: 7), and CD3 ⁇ signaling domain (SEQ ID NO: 8).
  • Expression of the Leu16 CARs or TRAMs under the EF1a promoter was lower than CART-19, with the conventional CAR detected in 2.6% and TRAMs 0.8-1.5% of cells 12 days post transfection (FIG.13B).
  • TRAMs can prevent surface expression in non-T cells even after transcription, but an approach to prevent transcription is to use promoters that express specifically in T cells.
  • Vingi1 driver with a GFP transgene template
  • several natural and synthetic promoters compared to MNDopt were tested (FIGS.14A-14E).
  • Donor 10 T cells were transfected with indicated Vingi1 RNA-LNPs.
  • a single reporter only (R only) is shown and all other R only controls showed the same results. Error bars show SD of duplicates.
  • NT non-treated cells.
  • FIGS.14A-14B Natural T cell promoters analyzed 5 days post LNP transfection.
  • FIGS.14C- 14E Synthetic T cell promoters analyzed 6 days post LNP transfection.
  • FIGS.14A and 14C Genomic integrations according to dPCR with a GFP probe.
  • FIGS.14B and 14D %GFP+ detected by FACS.
  • FIG.14E GFP MFI (median) of GFP+ cells where there were >500 events.
  • TRAMs to achieve T cell specific surface expression allows for use of strong and non-specific promoters like MNDopt to drive expression. If T cell promoters do generate sufficiently high protection, TRAMs would still be beneficial as an additional fail-safe mechanism in case the promoter is leaky in other cell types.
  • Example 9 Using microRNAs to attain cell specificity [0657] An additional approach to increase cell specificity is by adding microRNA (miR) sites that do not express in T cells but do express in other cells in which expression is not desired. Binding of miRs to mRNA which leads to degradation, may be used to knock-down transgene expression (typically up to 90% reduction).
  • miR microRNA
  • FIGS.15A-C and 16A-16B show a non-limiting example of sketches of nucleic acids with miR binding sites. Three binding sites were added were added to nucleic acids in several configurations.
  • FIG.15A Binding sites added to the sense strand of the transgene in the 3’ UTR are expected to lead to degradation following transcription.
  • FIG.15B Binding sites added to reporter RNA on the anti-sense strand (the orientation of the IVT) upstream to the inverted transgene which to degradation prior to integration.
  • FIG.15C Binding sites added to the 3’ UTR of driver RNA.
  • One is in the 3’ UTR of the transcribed transgene (“miR122 R”) which is expected to knock-down expression similar to the typical use of miRs to regulate transgenes (FIG.15A).
  • FIG.16A miR122 R led to 8 fold decrease in GFP expression without decreases integrations, as expected.
  • FIG.16A primary human hepatocytes (PHH) were transfected with LNPs 1 day post thaw and analyzed by dPCR to GFP probe and FACS 3 days post transfection.
  • miR122 R AS almost completely prevented integration instead of only knocking down integration.
  • miR122 D led to a 4-fold decrease in integration and GFP expression, as expected.
  • FIG.16B In primary T cells (FIG.16B) there would be an expectation that there would be no effect as miR-122 is not expressed in T cells. However, in fact it had substantial and unexpected effects though the mechanism is unknown.
  • miR122 R led to a ⁇ 2-fold decrease in integration and expression
  • miR122 R AS led to a >2 fold increase in integration and expression
  • miR122 D led to a 1.2 fold increase in integration and expression.
  • the combination of miR122 D + miR122 R AS increased %GFP+ from 24% with the WT to 60%. Inclusion of miR sites on the reporter in anti-sense and on the driver would both completely prevent integration in undesired cell types that express the miR, and potentially increase integration in T cells.
  • finding miRs that differentiate between T cells and all other cell types of concern is challenging, and this approach may not be enough to achieve the required level of specificity.
  • miR-122 is well characterized as liver specific, many others are not as well defined and may express to some degree in T cells. It is particularly difficult to find miRs that are not expressed at all in T cells yet express highly in B cells, the off-target cells of highest concern, as they are closely related. Therefore, there may be some benefit to using TRAMs to ensure cell specific surface expression when transcribed, which can be combined with other approaches that enhance specificity at the stage of integration or transcription.
  • TRAMs Targeted LNPs
  • binders Targeted LNPs (tLNPs) with several binders
  • LNPs in the blood become coated with Apolipoprotein E (ApoE) and enter the cell via the low-density lipoprotein receptor (LDL-R).
  • LDL-R surface expression on resting T cells is very low, and most T cells in circulation are in the resting state.
  • One method of 129 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 mediating efficient delivery to resting T cells is to coat the LNPs with an antibody (“binder”) that recognizes an antigen on T cells leading to internalization.
  • Several such antibodies were tested, including some that mediate T cell activation.
  • the targets tested were CD2, CD5, CD7, TCR, and CD3 and all antibodies were human IgG1 Fc silent.
  • T cells were cultured with cytokines IL-7 and IL-15 without the addition of an activating agent such as TransActTM.
  • tLNPs containing mRNA for transient GFP expression (“EX1494” SEQ ID NO: 62) were tested to assess activation and transfection efficiency (FIGS.17A-17F).
  • Donor 10 T cells were transfected transiently with GFP mRNA-tLNPs to the indicated antigen target with clone indicated in parenthesis. All antibodies were human IgG1 Fc silent. Isotype antibody was an anti-FITC, which does not express on human cells. FACS was performed 24h post LNP transfection. Error bars show SD of duplicates.
  • FIG.17A Expression of CD69 activation marker.
  • FIG.17B Expression of 41BB activation marker.
  • FIG.17C %GFP+ following transfection without ApoE.
  • FIG.17D GFP geometric mean fluorescence intensity (gMFI) of the GFP+ cells following transfection without ApoE. Background gMFI of singlets in NT cells was subtracted from the gMFI of each sample.
  • FIG.17E %GFP+ following transfection in the presence of ApoE.
  • FIG.17F GFP gMFI of the GFP+ cells following transfection in the presence of ApoE. Background gMFI of singlets in NT cells was subtracted from the gMFI of each sample.
  • Anti-CD3 (clone OKT3, VH SEQ ID NO: 63, VL SEQ ID NO: 64) and Anti-TCR (clone BMA031, VH SEQ ID NO: 65, VL SEQ ID NO: 66) coated tLNPs mediated activation as indicated by an increase in CD69 and 41BB, whereas anti-CD2 (clone VIP VIIIC8), CD5 (clone H65, VH SEQ ID NO: 67, VL SEQ ID NO: 68), CD7 (clone 3A1E, VH SEQ ID NO: 69, VL SEQ ID NO: 70), CD8 (clone OKT8, VH SEQ ID NO: 71, VL SEQ ID NO: 72), and CD3 (clone Visilizumab, VH SEQ ID NO: 73, VL SEQ ID NO: 74) did not (FIGS.17A-17B).
  • the expression level of GFP (MFI of GFP+ cells) ranking was CD3 (OKT3), TCR > CD7, CD8, CD5 > CD2 > CD3 (Visilizumab) > Isotype (FIG.17D).
  • ApoE where the tLNPs can enter either via the target antigen or LDL-R, the trends 130 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 were similar except that the isotype and CD8 were closer in %GFP+ to the other tLNPs (FIG.
  • Donor 10 T cells were transfected with Vingi1 GFP mRNA-tLNPs to the indicated antigen target with clone indicated in parenthesis. All antibodies were human IgG1 Fc silent and isotype antibody was an anti-FITC, which does not express on human cells. FACS was performed 5 days post LNP transfection. Error bars show SD of duplicates.
  • FIG.18A %GFP+ following transfection without ApoE.
  • FIG.18B %GFP+ following transfection in the presence of ApoE. Without ApoE only the activating tLNPs showed integration above 0.05% GFP+ which increased between the tested doses of 200ng, 400ng, and 1200ng RNA (FIG.18A).
  • CD3 (OKT3) mediated more efficient integration than TCR (0.11 vs 0.05%, 0.21 vs 0.04% and 0.54% vs 0.09% at doses of 200, 400, and 1200ng respectively).
  • the activating tLNPs were again better than the others, though there were some GFP+ cells in some of the other targets and integration for activating antibodies decreased at the 1200ng dose (FIG.18B).
  • CD3 (OKT3) mediated more efficient integration than TCR (0.5 vs 0.36% at the 200ng dose).
  • Donor 10 or donor 12 T cells were transfected with RNA-LNPs with the indicated conditions: Vingi1 driver and reporter in FIG.19A, Vingi1 driver only in FIG.19B, R2-1_TG driver and reporter in FIG.19A, R2-1_TG driver only in FIG.19B.
  • Positive control for strong activation using 131 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 TransActTM 1:100 was added either on day 0 (day of thawing PBMCs, 2 days prior to LNPs; “TransAct day 0”) and together with LNPs (“TransAct day 2”). “No stimulation” was cultured in only IL-7 and IL-15.
  • Test samples had 250ng/ml of antibody to the indicated antigen target with clone indicated in parenthesis added together with the LNPs. All antibodies were human IgG1 Fc silent and isotype antibody was an anti-FITC. LNP transfection was in the presence of ApoE. FACS was performed 5 days post LNP transfection. Error bars show SD of duplicates. FIG. 19A: %GFP+ following transfection without ApoE. FIG.19B: %GFP+ following transfection in the presence of ApoE.
  • Antibodies initially tested were activating antibodies against TCR (BMA031) or CD3 (OKT3), and non-activating antibodies against FITC (“Isotype hIgG1”) CD5 (H65) or CD3 (Vislizumab).
  • Stimulation controls were TransActTM (“TA”) activation on day of thaw (day 0, 2 days before LNPs) or in parallel to LNP transfection like the other antibodies (day 2) and no stimulation (cultured in only IL-7 and IL-15). As shown in FIG.19A, integration was highest for Vingi1 TA day 0, with 24-28% GFP+.
  • Activation mediated by TA, TCR (BMA031) or CD3 (OKT3) concurrent with LNPs showed between 3-14% GFP+, and non-activating conditions had ⁇ 0.8% GFP+.
  • a reference driver on a site-specific RTE for the ribosomal 28S loci, R2-1_TG, derived from the Australian zebra finch (Taeniopygia guttata) in the same conditions was also tested (FIGS.19C-19D).
  • the R2-1_TG driver used had undergone several rounds of engineering from the natural driver (SEQ ID NO: 75).
  • the first 160 amino acids were removed and the carboxy- terminal binding protein interacting protein (“CtIP”, SEQ ID NO: 76) followed by the high mobility group nucleosome binding domain 1 (“HMGN1”, SEQ ID NO: 77) were fused to the 5’ UTR with the XTEN linker (SEQ ID NO: 78) between each added domain. Additionally, several point mutations were introduced (P280K, D555K, T1011S, I1219N, A1282G).
  • An exemplary engineered R2-1_TG driver nucleic acid (SEQ ID NO: 82 encoded by SEQ ID NO: 3328) comprises a T7 Clean Cap (SEQ ID NO: 28), an R2-1_TG driver 5’ UTR (SEQ ID NO: 80), a nucleotide segment encoding an engineered R2-1_TG polypeptide (SEQ ID NO: 2506), a R2- 1_TG driver 3' UTR (SEQ ID NO: 83), and a polyA signal.
  • the R2-1_TG reporter consists of the T7 Clean Cap (SEQ ID NO: 28), an R25’ homology arm (SEQ ID NO: 84), an R2-1_TG 5’ RTE-UTR (SEQ ID NO: 85), the anti-sense of the cargo (e.g., comprising a promoter, a transgene, and optionally a polyA signal) of interest in the inverse orientation, an R2-1_TG 3’ 132 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 RTE-UTR (SEQ ID NO: 86), an R23’ homology arm (SEQ ID NO: 87), and a polyA signal.
  • the cargo e.g., comprising a promoter, a transgene, and optionally a polyA signal
  • the cargo tested was GFP (SEQ ID NO: 24) with the MNDopt promoter (SEQ ID NO: 25).
  • SEQ ID NO: 24 MNDopt promoter
  • TA day 0 showed GFP of 0.16-0.27% (FIG.19C) that was discernable from the background 0.01-0.08% GFP+) seen with the reporter only (FIG.19D).
  • %GFP for the site-specific RTE is lower than Vingi1, the potential safety advantages of a site-specific RTE are immense, especially considering reports of T cell cancers from CAR-T cells. These cancers were likely caused by integration of the retroviral or lentiviral vector into genes involved in preventing cancer.
  • LDL-R expression following activating antibody addition is a marker for T cell activation and mediates entry of LNPs. As shown in FIGS.20A-20D, the kinetics of LDL-R expression was tested to investigate why TransActTM activation 48h prior to LNP addition was superior to contemporaneous LNP addition and if applicable to antibody mediated activation.
  • Donor 10 PBMCs were thawed and seeded in IL-7 or IL-15 without TransActTM for all samples except for TransActTM day 0 (TA Day0) where TransActTM 1:100 and IL-2 were added at thaw. After 2 days, indicated antibodies were added at indicated concentrations or positive activation control TransActTM 1:100 (TA Day2) or negative control with no added reagents. All antibodies were human IgG1 Fc silent and Isotype antibody was an anti-FITC. Cells were stained with Allophycocyanin-tagged antibodies against -LDL-R (LDL-R-APC antibodies) at indicated time points after the time of antibody addition on day 2. Error bars show SD of duplicates.
  • FIG.20A shows a bar graph showing percentages of LDL-R positive cells (%LDL- R+) at all antibody doses, from 10 ng/ml to 1000 ng/ml.
  • FIG.20B shows an X-Y plot showing %LDL-R+ for antibody dose of 1000ng/ml and controls.
  • FIG.20C shows LDL-R median fluorescence intensity (MFI) of LDL-R+ cells bar graph at all antibody doses.
  • FIG.20D shows LDL-R MFI (median) of LDL-R+ cells XY graph for antibody dose of 1000ng/ml and controls.
  • MFI median fluorescence intensity
  • Activating antibodies against TCR BMA031) or CD3 (OKT3), or non-activating antibodies against Isotype or CD7 (3A1E) were added to T cells 2 days post thaw (concurrently with LNPs).
  • Control conditions include no stimulation, or TransActTM activation added at the same 133 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 time (TA day2) or upon thaw (TA day0).
  • LDL-R expression kinetics were measured over 48h from antibody addition.
  • Example 13 Addition of activating antibodies to T cells 2 days prior to LNP transfection improves transfection and integration more than coadministration with LNPs [0669] Based on the results described in Example 12, supra, transfection and integration were tested when adding the activating anti-TCR (BMA031) antibody either when thawing PBMCs 2 days prior LNPs (on day 0) or together with LNPs (on day 2), with controls of no-stimulation (cultured in only IL-7+IL-15) or strongly activating TransActTM+IL2 (TA+IL2).
  • BMA031 activating anti-TCR
  • transient GFP LNPs were added and FACS was performed 24h post LNP transfection (FIGS.21A-21H).
  • Donor 12 or donor 15 PBMCs were thawed and either cultured only in IL-7 and IL-15 (“no stim”), IL-7 and IL-15 with the addition of 250ng/ml anti-TCR (BMA031) activating antibody (“TCR d0”), or IL-2 with the addition of TransActTM1:100 “TA+IL2.”
  • TCR d0 250ng/ml anti-TCR
  • TCR d0 250ng/ml anti-TCR
  • IL-2 with the addition of TransActTM1:100 “TA+IL2.”
  • tLNPs coated with anti-CD5 (H65) antibody and containing mRNA for transient GFP expression were added in the presence of ApoE.
  • FIG. 21A shows percent of GFP positive cells (%GFP+).
  • FIGS.21B-21D shows percent of cells expressing activation markers: FIG.21B shows percent of cells positive for activation marker LDL-R (%LDL-R+), FIG.21C shows cells percent of positive for activation marker 4-1BB (CD137) (%4-1BB+), FIG.21D shows percent of cells positive for activation marker CD69 (%CD69+).
  • FIGS.21E-21H shows the expression level of the respective markers as measured by MFI gated on positive cells: FIG.21E shows the MFI of GFP, FIG.21F shows the MFI of 134 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 LDL-R-APC, FIG.21G shows the MFI of 4-1BB-PE, FIG.21H shows the MFI of CD69-PE- Cy7. Both %GFP+ and LDL-R expression were much higher when cells were activated with TCR antibody or TransActTM on day 0 (2 days prior to LNPs) compared to TCR antibody on day 2 or without stimulation (FIGS.21A, 21B, 21E, and 21F).
  • Donor 12 or donor 15 PBMCs were thawed and either cultured only in IL-7 and IL-15 “no stim”, IL-7 and IL-15 with the addition of 250ng/ml anti-TCR (BMA031) “TCR d0”, or IL-2 with the addition of TransActTM 1:100 “TA+IL2.” Two days later LNPs (non-targeted) containing indicated mRNAs were added. For the “TCR d2” treatment, 250ng/ml anti-TCR (BMA031) was added to the cells cultured in only IL-7 and IL-15.
  • FIG.22A and FIG.22B shows the genomic integration of GFP transgene with a Vingi1 driver and a MNDopt-GFP reporter, as measured by FACS (FIG.22A) and digital PCR (FIG.22B).
  • FIG.22C and FIG.22D shows a negative control experiment with Vingi1 MNDopt-GFP reporter only (no driver nucleic acid).
  • Teplizumab VH SEQ ID NO: 88, VL SEQ ID NO: 89, full heavy chain SEQ ID NO: 90, full light chain SEQ ID NO: 91
  • an anti-CD3 ⁇ humanized antibody OKT3 with mutations in the human IgG1 to reduce binding to Fc receptors (HzOKT3, Ala-Ala)
  • HzOKT3, Ala-Ala an anti-CD3 ⁇ humanized antibody with mutations in the human IgG1 to reduce binding to Fc receptors
  • This stands in contrast with the mouse OKT3 (without Fc silencing) that induced considerable side effects.
  • the activation potential based on LDL-R expression 48h post addition of antibodies or TransActTM that were added when thawing PBMCs were compared (FIGS.23A-23B).
  • Donor 15 PBMCs were thawed and seeded with IL-7 or IL-15 for all samples except for “TA (IL2)” which was seeded with IL-2 and TransActTM 1:100.
  • Antibodies to indicated antigen target with clone indicated in parenthesis were added at the indicated concentrations (0.08 ng/ml, 0.40 ng/ml, 2 ng/ml, etc. as provided in the x-axis).
  • Isotype IgG (anti-FITC), OKT3, and Teplizumab are human IgG1 Fc silent, Siplizumab is human IgG1, Urelumab is human IgG4.
  • FIG.23A shows the percentage of cells positive for activation marker LDL-R (%LDL-R+) as measure by FACS.
  • FIG.23B shows the MFI of of LDL-R+ cells.
  • Culture conditions tested were no stimulation (IL-7 and IL-15 only), isotype IgG, non-activating anti-CD2 (clone Siplizumab, VH SEQ ID NO: 92, VL SEQ ID NO: 93), co-stimulatory anti-41BB (clone Urelumab, VH SEQ ID NO: 94, VL SEQ ID NO: 95), Teplizumab + anti-41BB, TransActTM 1:100 with IL-7 and IL-15 (like the antibodies) or TransActTM 1:100 in IL2.
  • the Isotype, CD2, and 41BB antibodies alone did not upregulate LDL-R compared to no stimulation.
  • Teplizumab was extremely potent and is nearly saturated even with a very low dose of 0.08ng/ml and similar to TransActTM, whereas there was no indication of activation in the presence of anti-41BB antibody.
  • the mouse OKT3 activated cells to a lower extent, and this activation was substantially reduced below 2ng/ml and was similar to no stimulation at 0.08ng/ml. However,it should be noted that it was from a different vendor (OKT3 from Absolute Antibodies, Teplizumab from Selleckchem).
  • FIGS.24A-24F Integration of GFP under the MNDopt promoter was tested from several drivers 48h post thaw and activation with Teplizumab 250ng/ml, OKT3 250ng/ml, or TransActTM and control with no stimulation (FIG.24).
  • Donor 12 T cells were transfected with the indicated RNA-LNPs 2 days following the indicated stimulation.
  • the LNPs tested were not targeted LNPs.
  • FACS and digital PCR with MNDopt or GFP probes was performed 5 days post LNP transfection.
  • FIGS.24A-24C show the percentage of GFP positive cells (%GFP+) detected by FACS among PBMCs transfected with a Vingi1 driver (amino acid sequence set forth in SEQ ID NO: 31) and reporter (FIG.24A), PBMCs transfected with a R4-1_PH driver and reporter (FIG.24B), and PBMCs transfected with an R2-1_TG driver and reporter (FIG.24C).
  • FIGS.24D-24F shows the genomic integrations detected by dPCR with the MNP (promoter) probe or the GFP (transgene) probe as indicated in each figure, among PBMCs transfected with a Vingi1 driver and reporter(FIG.24D), PBMCs transfected with a R4-1_PH driver and reporter(FIG.24E), and PBMCs transfected with an R2-1_TG driver and reporter (FIG.24F).
  • MNP promoter
  • GFP transgene
  • the R4-1_PH driver full transcript comprises the T7 Clean Cap (SEQ ID NO: 28), a R4-1_PH driver 5’ UTR (SEQ ID NO: 96), a R4-1_PH ORF2 encoding an RTE polypeptide (DNA SEQ ID NO: 97, protein SEQ ID NO: 98), optionally a R4-1_PH driver 3’UTR, and optionally polyA .
  • the R4-1_PH reporter comprises the T7 Clean Cap (SEQ ID NO: 28), a R4-1_PH 5’ RTE-UTR (SEQ ID NO: 99), an anti-sense of the cargo (comprising, e.g., a promoter, a transgene, and a polyA signal) of interest in the inverse orientation, and a R4-1_PH 3’ RTE-UTR (SEQ ID NO: 100) and polyA.
  • %GFP+ was 0.26% without stimulation, 12.3% with Teplizumab, 6.4% with OKT3, and 18.2% with TransActTM (FIG.24A).
  • %GFP+ was 0.03% without stimulation, 0.47% with Teplizumab, 0.54% with OKT3, and 1.26% with TransActTM (FIG.24B).
  • %GFP+ was 0.06% without stimulation, 0.22% with Teplizumab, 0.03% with OKT3, and 0.21% with TransActTM (FIG.24C).
  • FIGS.24D-24F showed similar trends, except that in general R4-1_PH driver integrations were lower than with 137 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 R2-1_TG driver (FIGS.24E-24F) and that R2-1_TG driver integrations were about 2-fold higher with TransActTM activation than with Teplizumab or OKT3 (FIG.24F).
  • R2-1_TG driver integrations were about 2-fold higher with TransActTM activation than with Teplizumab or OKT3 (FIG.24F).
  • Teplizumab activation on day 0 mediates efficient integration of several different RTEs more than OKT3 and less than TransActTM.
  • Example 15 R2-1_TG engineered driver with reporters from different species [0677]
  • the R2-TG driver nucleic acid encoding the highly engineered R2-TG driver (SEQ ID NO: 82) was tested with R2 template comprising a MNDopt- driven GFP transgene with RTE-UTRs from R2 retroelements from several different species.
  • Donor 12 PBMC were activated with TransActTM, then transfected with the indicated RNA- LNPs for 2 days, and FACS and digital PCR with MNDopt or GFP probes was performed 5 days post LNP transfection. These experiments were run with TransActTM activation to see a high signal.
  • RTE-UTRs of R2 RTEs from the following species were tested: Taeniopygia guttata (TG), Phylloscopus trochilus trochilus (PTT), Limnodromus scolopaceus (LS), and Melierax canorus (MC).
  • TG Taeniopygia guttata
  • PTT Phylloscopus trochilus trochilus
  • LS Limnodromus scolopaceus
  • MC Melierax canorus
  • TG Taeniopygia guttata
  • PTT Phylloscopus trochilus trochilus
  • LS Limnodromus scolopaceus
  • MC Melierax canorus.
  • R2_TG D + TG R means that the cells were transfected with an R2-TG driver nucleic acid (SEQ ID NO: 82) and an R2 reporter comprising RTE-UTRs from the same species that the driver polypeptide was derived from, namely R2-TG from Taeniopygia guttata.
  • R2_TG D + PTT R means that the cells were transfected with an R2-1 TG driver nucleic acid (IVT sequence set forth in SEQ ID NO:3328) 138 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 and an R2 reporter comprising RTE-UTRs from a different species, namely R2-PTT. It was found, as shown in FIG.25A, that the R2-1_PTT reporter was better than R2-1_TG reporter when used with the R2-TG driver while R2-1_LS and R2-1_MC were worse than R2-1_TG (FIG.25A).
  • the PTT reporter comprises a 5’ UTR (SEQ ID NO: 125) comprising a 5’ RTE- UTR (SEQ ID NO: 126) from R2-PTT, an R2 retroelement found in Phylloscopus trochilus trochilus, and a 3’ UTR comprising a 3’ RTE-UTR from the R2-PTT and a PolyA signal.
  • the combination of the R2-1 TG driver (SEQ ID NO: 82) and the R2-1_PTT reporter were tested at several driver nucleic acid : reporter ratios (by weight of encapsulated RNA, with a constant total of 60ug per LNP), with a driver nucleic acid : reporter ratio of 1:1 being the control condition.
  • the R2-1_PTT reporter was more effective in driving the GFP transgene expression than the T2-1_TG reporter. Expression levels were mostly the same across the different ratios. That said, the 2:1 driver:reporter nucleic acid ratio was somewhat higher, achieving 43 integrations of the MND probe and 25 integrations of the GFP probe as assessed by dPCR and 0.41% GFP+ by FACS.
  • Example 16 Integration of TRAM with Vingi1 driver and Targeted LNPs
  • tLNPs coated with one of the following antibodies were tested: non-activating anti-CD7 (3A1E), activating anti-TCR (BMA031), or activating anti-CD3 (OKT3) were tested.
  • non-activating anti-CD7 3A1E
  • activating anti-TCR BMA031
  • activating anti-CD3 OKT3
  • the test Vingi1 driver had an amino acid sequence set forth in SEQ ID NO: 27; and the template having a nucleotide sequence set forth in SEQ ID NO:3354, was driven by an MNDopt promotor and comprised an anti-CD19 FMC63 VL-VH ⁇ TRAM.
  • the Vingi1 template nucleotide sequence includes GOI anti-CD19 FMC63 VL-VH ⁇ with the following features: Signal peptide SEQ ID NO: 3320; scFv chain 1 SEQ ID NO: 3322; intra scFv linker SEQ ID NO: 3211; scFv chain 2 SEQ ID NO:3316; Linkers after scFv (GCCGCCGCT); Hinge SEQ ID NO: 3321; transmembrane (tm) SEQ ID NO: 3317; Co stimulatory domain SEQ ID NO:3314; Signaling SEQ ID NO:3318 SEQ ID NO:3313. TRAM is in antisense orientation. Vpx enhancement was not used.
  • Non-treated (NT), cysteine and isotype hIgG1 are negative controls.
  • 139 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032
  • TRAM transgene integration was measured by FACS (detecting anti-TRAM antibody- based fluorescence) and by dPCR using a polyA signal probe (indicating full integration of the TRAM transgene) 11 days post transfection of PBMCs from donor 13 (DP13).
  • FIGS.26A-26B demonstrate that tLNPs are capable of mediating transfection and integration of TRAMs as measured by FACS (FIG.26A) and dPCR (polyA probe, FIG.26B).
  • Test arms LNP transfection in PBMCs from donor 10 (DP10). Test arms: No stimulus, 10pM or 1nM of Teplizumab (humanized anti-CD3 monoclonal antibody), Mosunetuzumab, Epcoritamab and Glofitamab, or TransActTM each with NT (no treatment), R2 driver only, R2 driver+R2 GFP template+Vpx, or Vingi-GFP template (in cis) The R2 driver has an amino acid sequence set forth in SEQ ID NO: 642.
  • SEQ ID NO:3203 sets forth the IVT nucleotide sequence encoding SEQ ID NO:642 protein.
  • the nucleotide sequence includes Synthetic 5' UTR SEQ ID NO:80 ; mouse alpha globin 3'UTR SEQ ID NO:83 ; A29N10A70 polyA signal SEQ ID NO:3201. The orientation of the sequences between external UTRs is in antisense.
  • the R2 GFP template has a nucleotide sequence set forth in SEQ ID NO: 2236.
  • the Vingi1 driver-GFP reporter (in cis) has an IVT nucleotide sequence set forth in SEQ ID NO: 3332 and has the following features, in 5’ to 3’ order: T7 Clean Cap (SEQ ID NO: 28), Vingi1 driver 5’ UTR (SEQ ID NO: 29), Vingi1 ORF2 (DNA SEQ ID NO: 30, protein SEQ ID NO: 31), synthetic PolyA signal (SEQ ID NO: 36), inverted GFP (amino acid SEQ ID: 24), MNDopt promoter (SEQ ID NO: 25), Vingi1 reporter 3’UTR and polyA (SEQ ID NO: 34).
  • FIGS.27A-27 B show GFP expression as determined by FACS; FIGS.27C-27D show integration as copies per 100 genomes, assessed by dPCR.
  • No integration is seen for Vingi 140 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 or R2 driver + R2 GFP template +Vpx in absence of an activating antibody or BiTe.
  • Both anti- CD3/CD20 BiTes Epcoritamab and Glofitamab mediate transgene integration with R2 driver as well as TransActTM when applied at a concentration of 1000 pM (1 nM). That said, integration with R2 driver was lower than integration with Vingi1 driver.
  • Teplizumab also enabled integration, albeit lower than integration with the BiTes.
  • Additional anti-CD3 activating antibodies that may be useful for pre-activation or as a targeting moiety on LNPs include: Otelixizumab (VH SEQ ID NO: 101, VL SEQ ID NO: 102, full heavy chain SEQ ID NO: 103, full light chain SEQ ID NO: 104), an anti-CD3 ⁇ humanized YTH12.5 IgG2 antibody; and Foralumab (VH SEQ ID NO: 105, VL SEQ ID NO: 106, full heavy chain SEQ ID NO: 107, full light chain SEQ ID NO: 108), an fully human IgG1 antibody with Fc silencing mutations.
  • Example 18 Vingi Anti-CD-19 scFv TRAM Expression and in vitro Killing [0687]
  • Several anti-CD19 scFvs having different orientations of Light and Heavy chains were tested for activity in PBMC donor 13 activated with TransAct + IL2. All scFvs were tested as a ⁇ TRAM and contained a “Flag” tag on the N-terminus of the mature peptide following cleavage of the signal peptide.
  • Anti-CD19 scFvs FMC63, 4G7, and Hu19 were tested in both VH-VL (variable heavy chain-variable light chain) and the reverse VL-VH orientations fused to CD3gamma.
  • the Vingi 1 driver had an amino acid sequence set forth in SEQ ID NO: 31, encoded by a nucleic acid sequence set forth in SEQ ID NO:3326.
  • the templates used are shown in Table 6. LH refers to a light chain -heavy chain orientation, while HL refers to a heavy chain-light chain orientation.
  • Table 6 Templates used for testing with Vingi1 driver Description SEQ ID NO: pV1-FMC63-LH 3210 pV1-FMC63-HL 3234 pV1-4G7-LH 3237 pV1-4G7-LH 3247 141 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 pV1-Hu19-LH 3249 pV1-Hu19-LH 3255 [0689] FIGS.28A-C showed that all TRAMs expressed on the surface and showed genomic integrations at all time points, highest at 12 days post LNPs (17-28% TRAM+ and 34-54 copies per 100 genomes).
  • the receptors/TRAM+ cells ranged between 6300 and 24000 (FIG.28D). All TRAMs mediated killing of Nalm-6 Luc target cells above the background killing of non-treated (NT) cells in a luciferase killing assay (FIG.28E). The data generally show that the system works well with a variety of scFv. [0690] FMC63 (mouse) TRAM exhibited high levels of integration (FIG.28A-28C), expression (FIG.28D) and killing of Nalm6 cells (FIG.28E).
  • TRAM expression is initially low post-transfection (e.g., day 6 as shown in FIG.28A, and day 9 as shown in FIG.28B), but is similar to FMC63 TRAM by day 12 (FIG.28C).
  • the order of the heavy chain (H) and light chain (L) may affect killing capacity.
  • the LH solid square
  • the HL open square
  • the HL exhibited more effective killing than the LH.
  • Example 19 Identification of anti-CD20 scFv binders [0692] Template nucleic acids encoding CARs or TRAMs comprising one of several anti-CD20 scFv TRAMs in different orientations, in combination with Vingi 1 driver nucleic acid (amino acid sequence set forth in SEQ ID NO: 31, encoded by a nucleic acid sequence set forth in SEQ ID NO:3326), were tested for genomic integration of the scFv transgene in Donor 13 PBMC.
  • Vingi 1 driver nucleic acid amino acid sequence set forth in SEQ ID NO: 31, encoded by a nucleic acid sequence set forth in SEQ ID NO:3326
  • FACS %TRAM+ cells
  • FACS % CAR or % TRAM
  • quantification of receptor per cell of PBMCs transfected (using LNPs) with the Vingi1 driver and a CAR- or TRAM-encoding template nucleic acid were performed at 6-, 9-, and 12-days post transfection.
  • NT Non-treated and R only (template alone) are negative controls.
  • Template nucleic acids were prepared.
  • Each template nucleic acid comprised a transgene driven by an MNDopt promoter and encoded a CAR or TRAM comprising an anti-CD20 scFv, Leu16, Obinutuzumab (Obi), or Ofatumumab (Ofa), either in LH (light chain-heavy chain) 142 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 orientation or HL (heavy chain-light chain) orientation fused to CD3 ⁇ , and further comprising a Flag tab.
  • FIGS.29C-29D show the results for the Leu16 TRAM, which expresses well with both the flexible and rigid linkers.
  • Example 20 Expansion of TRAM+ Cells [0698] A functional analysis shows that PBMCs transfected with a Vingi1-TRAM system comprising a Vingi1 driver (amino acid sequence set forth in SEQ ID NO: 31, encoded by a 143 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 nucleic acid sequence set forth in SEQ ID NO: 3326), and the Vingi1 template with anti-CD19 FMC63 VL-VH ⁇ as GOI as described in Example 16) are able to expand.
  • Vingi1 driver amino acid sequence set forth in SEQ ID NO: 31, encoded by a 143 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 nucleic acid sequence set forth in SEQ ID NO: 3326
  • the Vingi1 template with anti-CD19 FMC63 VL-VH ⁇ as GOI as described in Example 16 are able to expand.
  • FIG.30A shows integration and expression of TRAM at 12-days post transfection with LNP.
  • FIG.30B shows integration and expression of TRAM at 4 day and 7 day post co- culture with Nalm6 cells (CD19 +).
  • Example 21 Mouse studies for LNP delivery and RTE driven CAR or TRAM integration in vivo with or without pre-treatment with activating antibodies [0699] The efficiency and biodistribution of LNP delivery in vivo in mice with or without pre- treatment of activating antibodies is tested. For immunodeficient mice with human PBMCs, an anti-human CD3 ⁇ antibody (e.g., Teplizumab) will be used. For immunocompetent mice an anti- mouse CD3 ⁇ antibody (e.g., 145-2C11) will be used.
  • an anti-human CD3 ⁇ antibody e.g., Teplizumab
  • an anti- mouse CD3 ⁇ antibody e.g., 145-2C11
  • RNA-LNPs For biodistribution and transfection efficiency transient RNA-LNPs will be used, and for integration RTE RNA-LNPs will be used.
  • Example 22 Anti-tumor response mediated by CAR-T or TRAM-T cells engineered with RTEs [0700] The anti-tumor response to a lymphoma model (e.g. Nalm6 or Raji) is tested in immunodeficient mice mediated by CAR or TRAM-expressing T cells engineered with driver- based transgene integration.
  • a lymphoma model e.g. Nalm6 or Raji
  • Example 23 Testing engineered R2-TG drivers in Immune cells
  • Various R2-TG drivers having amino acid and nucleic acid sequences shown in Table 8 were tested in PBMCs from Donor 13, with a GFP reporter template having nucleic acid sequence set forth in SEQ ID NO: 2362.
  • 4ug Vpx mRNA (SEQ ID NO: 3399 encoding amino acid sequence set forth in SEQ ID NO:1568) was co-encapsulated with 30ug driver and 30ug reporter into SM102 LNPs.
  • 144 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0702] PBMC from Donor 13 were activated by TransActTM +IL2.
  • SEQ ID NO: 82 sets forth the amino acid sequence of an exemplary R2-1_TG driver comprising: an R2-1_TG polypeptide having, relative to SEQ ID NO: 75, deletion of 161 N- terminal amino acid residues; N-terminal fusion of the following: SV40-NLS polypeptide SEQ ID NO:430, CtIP polypeptide SEQ ID NO:76, HMGN poly
  • SEQ ID NO: 3327 sets forth a corresponding DNA sequence encoding the above R2-1_TG driver.
  • SEQ ID NO: 681 sets forth the amino acid sequence of an exemplary R2-1_TG driver comprising: an R2-1_TG polypeptide having, relative to SEQ ID NO: 75 deletion of 161 N-terminal amino acid residues; N-terminal fusion of the following: SV40-NLS polypeptide SEQ ID NO:430, EIF4a-2 polypeptide SEQ ID NO:592, CtIP polypeptide SEQ ID NO:76, HMGN polypeptide SEQ ID NO:77; and amino acid substitutions G257R, P280K, N363H, E399M, H423Q, A442P, D537Y, V539G, K547R, D555K, R669L, N889G, F973E, T1011A, 145 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 Q1019R
  • SEQ ID NO: 3266 sets forth a corresponding DNA sequence encoding the above R2-1_TG driver.
  • SEQ ID NO: 642 sets forth the amino acid sequence of an exemplary R2-1_TG driver comprising: an R2-1_TG polypeptide having, relative to SEQ ID NO: 75 having a deletion of 184 N-terminal amino acid residues; N-terminal fusion of the following: SV40-NLS polypeptide SEQ ID NO:430, sto7D polypeptide SEQ ID NO:405, HMGN polypeptide SEQ ID NO:77; and amino acid substitutions P280K, D555K, T1011S, I1219N, A1282G.
  • SEQ ID NO: 3204 sets forth a corresponding DNA sequence encoding the above R2-1_TG driver.
  • SEQ ID NO: 658 sets forth the amino acid sequence of an exemplary R2-1_TG driver comprising: an R2-1_TG polypeptide having, relative to SEQ ID NO: 75 having a deletion of 161 N-terminal amino acid residues; N-terminal fusion of the following: SV40-NLS polypeptide SEQ ID NO:430, CtIP polypeptide SEQ ID NO:76, HMGN polypeptide SEQ ID NO:77; and amino acid substitutions P280K, D555K, D923K, Q955R, T1011S, I1219N, A1282G.
  • SEQ ID NO: 3284 sets forth a corresponding DNA sequence encoding the above R2- 1_TG driver.
  • SEQ ID NO: 695 sets forth the amino acid sequence of an exemplary R2-1_TG driver comprising: an R2-1_TG polypeptide having, relative to SEQ ID NO: 75 having a deletion of 161 N-terminal amino acid residues; N-terminal fusion of the following: hnRPA1-2 polypeptide SEQ ID NO:707, SV40-NLS polypeptide SEQ ID NO:430, CtIP polypeptide SEQ ID NO:76, HMGN polypeptide SEQ ID NO:77; and amino acid substitutions P280K, D555K, D923K, T1011S, I1219N, A1282G.
  • SEQ ID NO: 3294 sets forth a corresponding DNA sequence encoding the above R2-1_TG driver.
  • SEQ ID NO: 1208 sets forth the amino acid sequence of an exemplary R2-1_TG driver comprising: an R2-1_TG polypeptide having, relative to SEQ ID NO: 75 having a deletion of 161 N-terminal amino acid residues; N-terminal fusion of the following: SV40-NLS polypeptide SEQ ID NO:430, CtIP polypeptide SEQ ID NO:76, HMGN polypeptide SEQ ID NO:77; and amino acid substitutions P280K, D555K, E888A, N891A, T892A, S895A, E898A, T899A, N902A, T1011S, I1219N, A1282G.
  • SEQ ID NO: 3261 sets forth a corresponding DNA sequence encoding the above R2-1_TG driver.
  • SEQ ID NO: 1240 sets forth the amino acid sequence of an exemplary R2-1_TG driver comprising: an R2-1_TG polypeptide having, relative to SEQ ID NO: 75 having a deletion of 161 N-terminal amino acid residues; an N-terminal fusion of the following: bp-NLS (engineered 146 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 bipartite-NLS) polypeptide SEQ ID NO:3280, SV40-NLS polypeptide SEQ ID NO:3275, CHV polypeptide SEQ ID NO:599, UL42 polypeptide SEQ ID NO:598, CtIP polypeptide SEQ ID NO:76, HMGN polypeptide SEQ ID NO:77; and amino acid substitutions P280K, D555K, D923K, T1011S, I1219N, A1282G.
  • SEQ ID NO: 3281 sets forth a corresponding DNA sequence encoding the above R2-1_TG driver.
  • SEQ ID NO: 675 sets forth the amino acid sequence of an exemplary R2-1_TG driver comprising: an R2-1_TG polypeptide having, relative to SEQ ID NO: 75 having a deletion of 161 N-terminal amino acid residues; an N-terminal fusion of the following: SV40-NLS polypeptide SEQ ID NO:430, CtIP-I2A polypeptide SEQ ID NO:3291, HMGN polypeptide SEQ ID NO:77; and amino acid substitutions P280K, D555K, T1011S, I1219N, A1282G.
  • SEQ ID NO: 3289 sets forth a corresponding DNA sequence encoding the above R2-1_TG driver.
  • SEQ ID NO: 693 sets forth the amino acid sequence of an exemplary R2-1_TG driver comprising: an R2-1_TG polypeptide having, relative to SEQ ID NO: 75 having a deletion of 161 N-terminal amino acid residues; an N-terminal fusion of the following: SV40-NLS polypeptide SEQ ID NO:430, TUDOR polypeptide SEQ ID NO:3269, CtIP polypeptide SEQ ID NO:76, HMGN polypeptide SEQ ID NO:77; and amino acid substitutions P280K, D555K, T1011S, I1219N, A1282G.
  • SEQ ID NO: 3272 sets forth a corresponding DNA sequence encoding the above R2-1_TG driver.
  • SEQ ID NO: 753 sets forth the amino acid sequence of an exemplary R2-1_TG driver comprising: an R2-1_TG polypeptide having, relative to SEQ ID NO: 75 having a deletion of 161 N-terminal amino acid residues; an N-terminal fusion of the following: SV40-NLS polypeptide SEQ ID NO:430, CtIP polypeptide SEQ ID NO:76, HMGN polypeptide SEQ ID NO:77; and amino acid substitutions P280K, D555K, D923K, T1011S, I1219N, A1282G.
  • FIGS.31A and 31B show FACS and dPCR results. For each group of three columns, the column represents polyA dPCR. GFP expression as assessed by FACS is shown as a solid circle adjacent and to and to the right of the third column.
  • FIG.31A in the absence of Vpx, the drivers having amino acid sequence set forth in SEQ ID NO: 642, SEQ ID NO: 695 and SEQ ID NO: 1208 exhibited highest GFP expression above driver having amino acid sequence set forth in SEQ ID NO: 82. These results were repeated in the presence of Vpx, as shown in FIG 31B.
  • Engineered R2-TG driver drives integration and expression of TRAMs and CD19 CAR (CD19 CD28zeta)
  • Engineered R2-TG driver having amino acid sequence set forth in SEQ ID NO: 642 was tested in PBMC from Donor 13, with a CD3gamma TRAM template (SEQ ID NO: 3308) or CD19 CD28zeta CAR template (nucleic acid sequence set forth in SEQ ID NO: 3312, amino acid sequence set forth in SEQ ID NO:3311).
  • Vpx mRNA (nucleic acid sequence set forth in SEQ ID NO: 3399 encoding AA sequence set forth in SEQ ID NO: 1568) was co-encapsulated with 30ug driver and 30ug reporter into SM102 LNPs.
  • CD19 CD28zeta CAR template nucleic acid comprises a transgene encoding an anti- CD19 CD28zeta CAR, which comprises an anti-CD19 FMC63 scFv fused to a CD28 hinge, TM and co-stimulating regions and a CD3zeta receptor, driven by a CMV promotor.
  • CD3gamma TRAM template nucleic acid utilizes FMC63 scFv fused to a CD gamma TCR, driven by a CMV promotor.
  • the template further comprises 5’ TEV UTR and 3’ HAG UTR.
  • 'HAG' refers to mouse hemoglobin alpha
  • '3’UTR HAG' refers to the mouse hemoglobin alpha 3'UTR (SEQ ID NO: 83)
  • '5’UTR HAG' mouse hemoglobin alpha 5'UTR SEQ ID NO: 80
  • TEV refers to the Tobacco Etch Virus 5’ UTR (SEQ ID NO:3307).
  • PBMC from Donor 13 were activated by TransActTM +IL2.
  • NT refers to non-treated cells.
  • SEQ ID NO: 642 is the amino acid sequence of an exemplary R2 driver comprising R2- 1_TG polypeptide with deletion of 184 N-terminal residues; and N-terminal fusion of the following: SV40-NLS polypeptide SEQ ID NO:3199, sto7D polypeptide SEQ ID NO:3202, HMGN-2 polypeptide SEQ ID NO:3298; and amino acid substitutions P280K, D555K, T1011S, I1219N, A1282G.
  • SEQ ID NO:3312 sets forth the nucleotide sequence encoding SEQ ID NO:3311.
  • the template nucleotide sequence includes as the GOI, a conventional anti-CD19 CAR with FMC63 scFv, CD28 hinge-transmembrane, CD28 co-stimulatory region, and CD3zeta signaling and has the following features: Signal peptide SEQ ID NO:3320 ; Scfv chain 1 SEQ ID NO:3322 ; intra scFv linker SEQ ID NO:3211 ; scFv chain 2 SEQ ID NO:3316 ; Linkers after scFv (GCCGCCGCT) ; Hinge SEQ ID NO:3321 ; transmembrane SEQ ID NO:3317 ; Co stim SEQ 148 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 ID NO:3314 ; Signaling SEQ ID NO:3318.
  • SEQ ID NO:3315 sets forth the template IVT nucleotide sequence comprising SEQ ID NO:3312.
  • SEQ ID NO:3301 sets forth the amino acid sequence encoded by SEQ ID NO:3302.
  • the amino acid sequence includes as the GOI a TRAM comprising FMC63 LH-CD3G, and has the following features: Signal peptide SEQ ID NO:1 ; scFv chain 1 SEQ ID NO:2 ; intra scFv linker SEQ ID NO:9 ; scFv chain 2 SEQ ID NO:4 ; Linkers after scFv (AAA) and SEQ ID NO:3 ; Cd3 chain hinge transmembrane signaling SEQ ID NO:18 SEQ ID NO:3301.
  • FIG.32A shows percent CAR or percent TRAM as measured by FACS with an anti- FMC63 antibody.
  • the anti-CD19 CD28zeta CAR exhibited highest expression (0.54%) at 12 days, while the TRAM exhibited 0.73% expression at 9 days.
  • Full integrations by the R2-TG driver are shown in Fig.32B, whereby integration of the anti-CD19 CD28zeta CAR increases over 16 days while expression of TRAM appears to peak at 12-16 days.
  • Example 25 Engineered R2-TG drivers drive TRAM expression
  • Engineered R2-TG driver having amino acid sequence set forth in SEQ ID NO: 1690 was tested in PBMCs from Donor 13, with a TRAM template driven by CMV promotor (SEQ ID NO: 3303) or by CMVg promotor (SEQ ID NO: 3309).
  • Vpx mRNA (SEQ ID NO: 3399) was co-encapsulated with 30ug driver and 30ug reporter into SM102 LNPs.
  • Cells were analyzed by FACS (anti-FMC63) and dPCR (CMV probe) at 6-, 9- and 12-days and number of receptors per cell at 9- and 12-days post transfection (as described in Example 1, supra).
  • NT refers to non- treated cells
  • R only refers to transfection of reporter (template) only.
  • SEQ ID NO:1690 sets forth the amino acid sequence of R2-1_TG Orf2 polypeptide; with deletion of 184 N-terminal residues; and N-terminal fusion of the following: SV40-NLS polypeptide SEQ ID NO:430, Sto7D polypeptide SEQ ID NO:405, HMGN polypeptide SEQ ID NO:77; and mutations P280K, D555K, D923K, T1011S, I1219N, A1282G.
  • SEQ ID NO:3009 sets forth the nucleotide sequence encoding SEQ ID NO:1690 protein.
  • the nucleotide sequence includes utr5 external:Bancov SEQ ID NO:80; utr3 external:HAG-3UTR SEQ ID NO:83; A29N10A70 polyA signal SEQ ID NO:3201.
  • the orientation of the sequences between external UTRs is in antisense.
  • 149 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0725]
  • CMV promotor having a nucleic acid sequence set forth in SEQ ID NO: 3305 is the immediate/early promoter enhancer of cytomegalovirus (CMV) and is commonly used as an in vitro and in vivo promoter for driving the expression of transgenes in mammalian cells.
  • CMV immediate/early promoter enhancer of cytomegalovirus
  • CMVg promotor having a nucleic acid sequence set forth in SEQ ID NO: 3310 is the CMV promotor with a nucleotide substitution (C>G at position 408) believed to minimize silencing and increase stability.
  • SEQ ID NO:3303 sets forth the nucleotide sequence encoding SEQ ID NO:3301 protein.
  • the nucleotide sequence includes R220bp upstream homology arm SEQ ID NO:84 ; R2-1_PTT external 5'UTR SEQ ID NO:125 ; mir:miR-122 SEQ ID NO:3306 ; mir:miR-122 SEQ ID NO:3306 ; mir:miR-122 SEQ ID NO:3306 ; CMV promoter SEQ ID NO:3305 ; internal 5'UTR: TEV-pMRNAxp (minusG) SEQ ID NO:3307; reporter goi:TRuC_FMC63 LH-CD3G-sets forth the amino acid sequence of CART gene with the following features: Signal peptide SEQ ID NO:3214 ; scFv chain 1 SEQ ID NO:3212; intra scFv linker SEQ ID NO:3211; scFv chain 2 SEQ ID NO:3208; Linkers after scFv (GCTGCAGCT) and SEQ ID NO:3215; CD3 chain hinge -
  • SEQ ID NO:3309 sets forth the nucleotide sequence encoding SEQ ID NO:3301 protein.
  • the nucleotide sequence includes R220bp upstream homology arm SEQ ID NO:84 ; R2-1_PTT external 5'UTR SEQ ID NO:125; mir:miR-122 SEQ ID NO:3306 ; mir:miR-122 SEQ ID NO:3306; mir:miR-122 SEQ ID NO:3306 ; promoter:CMVg-The sequence harbors eCMV+pCMVg and additional 3' and 5' SEQ ID NO:3310; internal 5'UTR: TEV- pMRNAxp(minusG) SEQ ID NO:3307; reporter GOI: FMC63 LH-CD3G-sets forth the amino acid sequence of CAR gene with the following features: Signal peptide SEQ ID NO:3214; scFv chain 1 SEQ ID NO:
  • FIG.33A-FIG33C show FACS, dPCR and quantification of receptors/cell, respectively.
  • FIG.33A shows that the R2-TG driver and template having a CMV or CMVg promotor expressed TRAM. At 12-days post transfection, the template with the CMVg promotor exhibits about twice the number of TRAM+ than the template with the CMV promotor.
  • FIG.33B shows that the two templates result in approximately the same number of integrations (copies per 100 genomes) at all time points.
  • FIG.33C shows that the R2-TG driver and template driven by CMV or CMVg express TRAM with template having the CMV or CMVg promotor but with higher expression of receptor with the CMVG promotor.
  • Example 26 T cells with 28S integrated CAR/TRAM exhibit tumor cytotoxicity and cytokine production
  • a Nalm6-Luc killing assay was performed using R2-TG driver and CMV-driven TRAM or CMVg-driven TRAM as shown in FIG.3, at 12 days post LNP transfection.
  • Engineered R2- TG driver having amino acid sequence set forth in SEQ ID NO: 1690 was tested in PBMCs from Donor 13, with a CMV-driven TRAM template (SEQ ID NO: 3303) or with CMVg-driven TRAM template (SEQ ID NO: 3309).
  • a Nalm6-Luc killing assay was performed with PBMCs transfected via lentivirus (LV) to integrate a EF1a-CART-19.
  • LV lentivirus
  • CD19-41BBzeta CAR has an amino acid sequence set forth in SEQ ID NO: 23 (encoded by a nucleic acid sequence set forth in SEQ ID NO: 3336), and consists of the CD8 ⁇ signal peptide (“SP”, SEQ ID NO: 1), FMC63 VL (SEQ ID NO: 2), 3xG4S linker (SEQ ID NO: 3), FMC63 VH (SEQ ID NO: 4), CD8 ⁇ Hinge (SEQ ID NO: 5), CD8 ⁇ Transmembrane (“TM”, SEQ ID NO: 6), 4-1BB co-stimulatory domain (SEQ ID NO: 7), and CD3 ⁇ signaling domain (SEQ ID NO: 8).
  • SP CD8 ⁇ signal peptide
  • FMC63 VL SEQ ID NO: 2
  • 3xG4S linker SEQ ID NO: 3
  • FMC63 VH SEQ ID NO: 4
  • CD8 ⁇ Hinge SEQ ID NO: 5
  • TM CD8 ⁇ Transmembrane
  • FIG 34A shows the results of a Nalm6-luciferase killing assay, with R only as negative control and CD1941BBzeta CAR as positive control.
  • the engineered R2-TG driver driving the TRAM templates exhibited effective tumor killing.
  • FIG.34B shows levels of interferon gamma (IFNg) secretion by the Nalm6 cells as assessed by ELISA.
  • IFNg interferon gamma
  • Example 27 Reduction in Tumor Burden with Vingi1 CD3G TRAM T cells in a peripheralized NALM-6-Luc B cell leukemia model [0736]
  • the primary objective of this study was to test the effects in an in vivo cancer model of T cells integrated with a TRAM transgene generated using Vingi1 driver RTE platform. Tumor suppression was tested using the NALM-6 tumor model for 28 days.
  • the example provides ex vivo produced cells for an in vivo animal model of tumor growth.
  • the Nalm6 cells express luciferase and the IVIS measures photons released by luciferase.
  • test driver nucleic acid was Vingi1 driven by MNDopt promotor (having amino acid sequence set forth in SEQ ID NO: 31; and the Vingi1 template comprised a transgene encoding a TRAM (template nucleic acid described in Example 16, supra and having a nucleic acid sequence set forth in SEQ ID NO:3354).2x10e5 Nalm6 luciferase cells were injected into groups of NSG (NOD scid gamma) immunodeficient mouse. The two negative control groups were A) PBS treated (5 mice) and B) untreated T cells treated (NT, 1 mouse).
  • the positive control C) was a lentivirus (LV) EF1a CART-19 (7 mice) and test was D) Vingi1 TRAM as described herein (3 mice). 2.58 x 10e6 CAR or TRAM positive cells in a total of 15x 10e6 T cells were injected on day 4 to group C and D, respectively. IVIS was measured on Days 8, 12, 15, 19, 21, 26 and 28 post Nalm6 injection into mice. [0738] FIG.35 shows tumor burden as measured by IVIS (total flux (photons/sec)). Groups A and B show steady tumor growth over time, while groups C and D show no tumor growth. In this experiment, the Vingi1 TRAM system killed tumors as well as the commercial LV EF1a CART-19. 152 318294750

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Abstract

The present disclosure provides compositions and methods for delivering an engineered immune receptor to immune cells, e.g., T cells, in vivo, ex vivo, or in vitro. Provided are recombinant nucleic acids and templates that are useful for immune cell reprogramming with high specificity and efficiency.

Description

Attorney Docket No.:AVRT-010/02WO 351047-2032 COMPOSITIONS AND METHODS FOR MODULATING T CELLS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Application No.63/640,193 filed on April 29, 2024 and U.S. Provisional Application No.63/786,249 filed on April 9, 2025. These applications are hereby incorporated by reference in their entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING [0002] The contents of the electronic sequence listing (AVRT_010_02WO_SeqList_ST26.xml; Size: 11,885,551 bytes; and Date of Creation: April 24, 2025) are herein incorporated by reference in its entirety. BACKGROUND [0003] Immune cells, for example T cells, redirected with engineered immune receptors to kill target cells have shown success in treating patients with hematological malignancies and autoimmune disorders. Typically, T cells are isolated from the blood of a patient by apheresis, ex vivo engineered with a viral vector to express an engineered immune receptor, expanded, and infused to the patient. After the engineered T cells are administered to a patient, they recognize and bind to specific proteins on the surface of target cells, and once bound, the engineered T cells activate an immune response, leading to the destruction of the target cells. [0004] However, this ex vivo engineering of immune cells, as with many other ex vivo cell therapy approaches, is a lengthy and expensive procedure, in part because the therapeutic agent, the engineered immune cells, has to be custom-engineered for each patient from isolated autologous cells. Thus, there is a need for technologies and compositions to enable safe, efficient, scalable, and cost-effective reprogramming of immune cells. [0005] Additionally, the generation of immune cells expressing engineered immune receptors in vivo runs the risk of inadvertent expression in non-T cells, which can lead to antigen binding in cis or trans, masking the antigen, and preventing recognition by engineered T cells. Thus, there is also a need for methods of generating T cells expressing engineered immune receptors, to reduce the risk of antigen masking on target cells. 1 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0006] This disclosure provides compositions and methods for in vivo, in vitro and ex vivo engineering of immune cells in a safe, and cost-effective manner to satisfy these urgent needs in the field. SUMMARY [0007] Aspects of the disclosure provide compositions and methods for delivering an engineered immune receptor to, e.g., immune cells. [0008] In one aspect, provided herein is a nucleic acid comprising: (a) a nucleic acid sequence encoding a T cell receptor fused antigen modifier (TRAM) comprising an antigen binding domain and a TCR subunit, and (b) a retrotransposable element untranslated region (RTE-UTR). [0009] In some embodiments, the TCR subunit is selected from the group consisting of TCRα, TCRβ, CD3δ, CD3γ, and CD3ε. [0010] In some embodiments, the RTE-UTR comprises a 3’ RTE-UTR and/or a 5’ RTE-UTR. [0011] In some embodiments, the antigen binding domain is selected from the group consisting of an scFv, a VHH, a TCR-like antibody, a natural ligand, an FcR-binding receptor (e.g. CD16), and an NK killing receptor. [0012] In another aspect, provided herein is a retrotransposable-element (RTE) integration system comprising a driver nucleic acid and a template nucleic acid, wherein: (a) the driver nucleic acid comprises a nucleic acid sequence encoding an RTE polypeptide, wherein the RTE polypeptide is a site-specific RTE polypeptide; and (b) the template nucleic acid comprises: a nucleic acid sequence encoding an engineered immune receptor, and an RTE-UTR capable of being bound by the RTE polypeptide. [0013] In some embodiments, the engineered immune receptor comprises an antigen binding domain. [0014] In some embodiments, the engineered immune receptor is selected from the group consisting of a T cell receptor (TCR), a TCR fused antigen modifier (TRAM), a TRuC and a chimeric antigen receptor (CAR). [0015] In another aspect, provided herein is a retrotransposable-element (RTE) integration system comprising a driver nucleic acid and a template nucleic acid, wherein: (a) the driver nucleic acid comprises a nucleic acid sequence encoding an RTE polypeptide; and (b) the template nucleic acid comprises a nucleic acid sequence encoding a T cell receptor fused antigen 2 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 modifier (TRAM) comprising an antigen binding domain and a TCR subunit, and an RTE-UTR capable of being bound by the RTE polypeptide [0016] In another aspect, provided herein is a lipid nanoparticle (LNP) comprising a nucleic acid of the disclosure or a RTE integration system of the disclosure. In some embodiments, claim the LNP is covalently linked to an antibody or any fragment thereof that recognizes a T cell antigen. [0017] In another aspect, provided herein is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a nucleic acid of the disclosure, a RTE integration system of the disclosure, or a LNP of the disclosure. [0018] In another aspect, provided herein is a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a nucleic acid of the disclosure, a RTE integration system of the disclosure, a LNP of the disclosure, or a pharmaceutical composition of the disclosure. [0019] In some embodiments, the subject is pre-treated with a T cell activator or concomitantly treated with a T cell activator. [0020] In some embodiments, the T cell activator is a CD3 agonist or a TCR agonist. [0021] In some embodiments, subject is further administered prior, concurrently, or subsequent to said administering with at least one γ-chain receptor agonist. [0022] In another aspect, provided herein is a method of in vivo genome manipulation comprising pre-administering a T cell activator and administering a pharmaceutical composition comprising a reprograming agent. [0023] In some embodiments, the reprogramming agent is a genome manipulation system comprising one or more nucleic acids. Optionally, the one or more nucleic acids comprise at least one nucleic acid sequence comprised in or derived from a retrotransposon. [0024] In some embodiments, the reprograming agent meditates durable expression by integrating one or more nucleic acids into a genome. [0025] In some embodiments, the T cell activator is an CD3 or TCR agonist, or an immune cell engager, e.g., Bispecific T cell engager (BiTe). [0026] In another aspect, provided herein is a method of in vivo genomic manipulation in an immune cell, the method comprising contacting the immune cell with a nucleic acid of the disclosure, a RTE integration system of the disclosure, a LNP of the disclosure, or a pharmaceutical composition of the disclosure, wherein the immune cell natively expresses a TCR-CD3 complex. 3 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 BRIEF DESCRIPTION OF DRAWINGS [0027] The present disclosure, including the drawings, include reference to the term TCRF (T Cell Receptor Fusion Protein) which may be used interchangeably with TRAM (T Cell Receptor fused Antigen Modifier). [0028] FIG.1A and FIG.1B illustrate non-limiting examples of nucleic acids useful for gene delivery, using an RTE polypeptide to promote insertion of a transgene (e.g., a heterologous nucleic acid comprising a gene of interest (“GOI”)) into a genome. FIG.1A demonstrates a cis configuration where one polynucleotide (e.g. mRNA) encodes both a protein that is capable of promoting retrotransposition (an RTE polypeptide, represented as ORF2) and an antisense transgene flanked by terminal regions. FIG.1B demonstrates an exemplary trans-configuration with two polynucleotides (e.g. which could be RNAs or mRNAs), wherein the first polynucleotide (the “driver nucleic acid”) encodes ORF2 in an mRNA format and the second polynucleotide (the “template nucleic acid”) comprises a GOI flanked by terminal regions. In some embodiments, as shown in the figure, the GOI, e.g. CAR or TRAM, may be encoded in an inverted (i.e., antisense) configuration relative to the 5’UTR and or polyA tail. In some embodiments, the gene of interest (GOI, e.g. CAR or TRAM) may be inserted in a forward (i.e., sense) configuration relative to the 5’UTR and or polyA tail. In other embodiments, the template nucleic acid is not an mRNA (i.e. lacks a 5’ cap structure, or a 3’ polyA tail, or both). [0029] FIG.2 illustrates examples of TCR, canonical CAR, and TRAM components. [0030] FIG.3 illustrates exemplary CAR and TRAM designs. Designs shown for anti-CD19 CAR and TRAM. [0031] FIG.4A- FIG.4F illustrate expression of anti-CD19 CAR or TRAMs (with T2A-GFP) in several cell types following lentiviral transduction. [0032] FIG.5A- FIG.5F illustrate anti-CD19 CAR or TRAM expression and CD19 masking in Nalm6 cells following lentiviral transduction gated on transduced (GFP+) cells. [0033] FIG.6A- FIG.6F illustrate anti-CD19 CAR or TRAM expression and CD19 masking in Raji cells following lentiviral transduction gated on transduced (GFP+) cells. [0034] FIG.7A- FIG.7F illustrate the Vingi1 RTE system for GFP and anti-CD19 CAR expression. [0035] FIG.8A- FIG.8I illustrate CART-19 expression under MNDopt or EF1α promotors in several donor T cells. FIGS.8A-8F show results with Vingi1 driver. FIGS.8G-8I show results 4 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 with lentivirus (LV). FIGS.8A, 8D and 8G show dPCR results. FIGS.8B, 8E and 8H show %CAR positive cells as determined by FACS. FIGS.8C, 8F and 8I show quantification as receptors/cell. [0036] FIG.9A- FIG.9C illustrate anti-CD19 CAR or TRAMs (^^^^^^^^^^) with T2A-GFP using Vingi1 RTE. [0037] FIG.10A- FIG.10C illustrate anti-CD19 CAR or TRAMs (^^^^^^^^ (without T2A-GFP) expression using Vingi1 RTE. [0038] FIG.11A- FIG.11D illustrate anti-CD19 CAR or TRAMs (^^^^^^^) (without T2A-GFP) killing in Nalm6 cells using Vingi1 RTE. [0039] FIG.12A- FIG.12C illustrate anti-CD19 γ TRAMs with EF1α or MNDopt promoters using Vingi1 RTE. [0040] FIG.13A- FIG.13B illustrate anti-CD20 CAR or γ TRAMs with different spacers under EF1α promoter using Vingi1 RTE. [0041] FIG.14A- FIG.14E illustrate T cell specific promoters with GFP transgene using Vingi1 RTE. [0042] FIG.15A- FIG.15C illustrate exemplary nucleic acid designs with microRNA (miR) binding sites. [0043] FIG.16A- FIG.16B demonstrate GFP transgene integration and reduced expression in hepatocytes in the presence of liver-restricted miR-122 with GFP transgene under MNDopt promotor driven by a Vingi1 RTE. [0044] FIG.17A- FIG.17F demonstrate results of Donor 10 T cells transiently transfected with targeted LNPs (tLNPs) comprising GFP mRNA with several binders driven by Vingi1. [0045] FIG.18A- FIG.18B demonstrate targeted LNPs (tLNPs) with several binders containing mRNA for Vingi1 driver and MNDopt-GFP RTEs reporter, in trans. [0046] FIG.19A- FIG.19D demonstrate expression of GFP effected by LNPs (non-targeted), containing RNA for Vingi1 or R2-1_TG driver and MNDopt-GFP RTE reporters, transfected in the presence of antibodies. [0047] FIG.20A- FIG.20D demonstrate the kinetics of LDL-R expression following activation. [0048] FIG.21A- FIG.21H demonstrate the comparison of activation and transient GFP RNA- LNP transfection following antibody activation on day 0 or day 2. 5 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0049] FIG.22A- FIG.22D demonstrate the comparison of activation and Vingi1 GFP RTE- LNP transfection following antibody activation on day 0 or day 2. [0050] FIG.23A- FIG.23B demonstrate LDL-R expression following antibody activation on day 0. [0051] FIG.24A- FIG.24F demonstrate expression (FIGS.24A-24C) and integration (FIGS. 24D—24F) of GFP effected by LNP (non-targeted) containing RNA for Vingi1, R4-1_PH, or R2-1_TG drivers and MNDopt-GFP RTE reporters 2 days post antibody or TransAct™ activation. [0052] FIG.25A- FIG.25B demonstrate integration and expression of LNP (non-targeted) containing mRNA for R2-1_TG engineered driver with different MNDopt-GFP R2 reporters at different ratios 2 days post TransAct™ activation. [0053] FIG.26A -FIG.26B demonstrate integration of TRAMs mediated by tLNP and measured by FACS and dPCR. [0054] FIG.27A-FIG.27D show activation with FDA-approved anti-CD3 antibodies. Anti- CD3 or BiTes enable R2 driver- and Vingi1-driver GFP transgene (in cis) integration with R2 driver and Vingi1 driver as measured by FACS and dPCR. [0055] FIG.28A-FIG.28E demonstrate expression data and functional activity (killing tumor cells) with several anti-CD19 scFvs TRAMs integrated into PBMCs with Vingi1. [0056] FIG.29A-FIG.29D demonstrate expression data with CARs and TRAMs comprising anti-CD20 scFvs integrated into PBMCs with a Vingi1 driver. [0057] FIG.30A and FIG.30B demonstrate proliferation of TRAM+ PBMCs produced via driver-based transgene integration in co-culture [0058] FIG.31A and FIG.31B present integration and expression data for ten R2 drivers -Vpx (FIG.31A) or +VPX (FIG.31B) with GFP reporter. [0059] FIG.32A and FIG.32B are bar graphs demonstrating CAR and TRAM expression (FIG.32A) and integration (FIG.32B) by an R2 driver having amino acid sequence set forth in SEQ ID NO: 642 driving CMV anti-CD19 CD28zeta CAR (nucleic acid sequence set forth in SEQ ID NO: 3312) or TRAM in donor PBMCs over 12 days. [0060] FIG.33A - FIG.33C are bar graphs demonstrating CAR and TRAM expression (FIG. 33A), integration (FIG.33B) and receptor quantification (FIG.33C) by an R2 driver having amino acid sequence set forth in SEQ ID NO: 1690 driving CMV or CMVg TRAMs in donor PBMCs over 12 days. 6 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0061] FIG.34A and FIG.34B demonstrate killing efficacy (FIG.34A) and interferon gamma secretion (FIG.34B) of an R2 driver having amino acid sequence set forth in SEQ ID NO: 1690 driving CMV or CMVg TRAMs. [0062] FIG.35 demonstrates the in vivo tumor killing activity using ex vivo generated Vingi1 TRAM. DETAILED DESCRIPTION Definitions [0063] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms. [0064] The indefinite articles “a” and “an,” as used herein, unless clearly indicated to the contrary, should be understood to mean “at least one.” [0065] The term “about,” as used herein, is intended to qualify the numerical values which it modifies, denoting such a value as variable within a margin of ±10%. [0066] The disclosure provides methods and compositions useful to promoting the integration of a transgene encoding a T cell receptor, fusion or fragment thereof, into a genome of a T cell. Without being bound by theory or mechanism, the inventors show that by using polynucleotide compositions that encode an engineered driver protein and a transgene nucleic acid (e.g., GFP, gene of interest (GOI)), the transgene can be integrated into the genome of T cells. Retrotransposable element (RTE) Integration Systems [0067] Retrotransposable elements (RTEs) have naturally evolved to facilitate the incorporation of DNA copies of RNA into genomes. The RTE integration systems of the disclosure take advantage of RTE function, and offer a novel method for introducing engineered immune receptors into T cells by guiding insertional synthesis of a DNA sequence from an RNA template into host cell DNA. [0068] Generally, provided herein are RTE integration systems comprising a driver nucleic acid and a template nucleic acid, wherein (a) the driver nucleic acid comprises a nucleic acid sequence encoding an RTE polypeptide (e.g. a site specific RTE polypeptide); and 7 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 (b) the template nucleic acid comprises: (i) a nucleic acid sequence encoding an engineered immune receptor; and (ii) an RTE untranslated region (RTE-UTR) capable of being bound by the RTE polypeptide. In some embodiments, the RTE polypeptide is capable of mediating integration, into a host cell genome, of a DNA copy of the nucleic acid sequence encoding the engineered immune receptor. As used herein, a “DNA copy” refers to a DNA sequence that is produced based on the nucleic acid sequence encoding the engineered immune receptor being a template for transcription and/or reverse transcription. By way of example, where the template nucleic acid is RNA, the DNA copy may be the DNA product of reverse transcription of the nucleic acid sequence encoding the engineered immune receptor by the RTE polypeptide. By way of another example, where the template nucleic acid is DNA, the DNA copy may be the DNA product of reverse transcription, by the RTE polypeptide, of an RNA transcript of the nucleic acid sequence encoding the engineered immune receptor. [0069] In some embodiments, the template nucleic acid comprises a promoter sequence capable of driving transcription of the nucleic acid sequence encoding the engineered immune receptor after insertion into the host cell DNA. In some embodiments, the template nucleic acid comprises 5’ UTR between the start of transcription and the beginning of the nucleic acid sequence encoding the engineered immune receptor. In some embodiments, the template nucleic acid comprises a 3’ UTR between the nucleic acid sequence encoding the engineered immune receptor and the end of transcription, with the 3’UTR optionally comprising a polyadenylation sequence. In some embodiments, the engineered immune receptor may be a T cell receptor (TCR), a TCR fused antigen modifier (TRAM), a T cell receptor fusion construct (TRuC), a chimeric antigen receptor (CAR), and a costimulatory receptor fusion, as described further herein below. [0070] In some embodiments, the host cell is an immune cell. In some embodiments, where a template encodes an engineered immune receptor that is not a TRAM (e.g., where the engineered immune response is a CAR), the immune cell may be a T cell, a B cell, a NK (natural killer) cell, or an NK-T cell. In some embodiments, the immune cell may be an immune cell that natively expresses a TCR-CD3 (T cell receptor-cluster of differentiation 3) complex, e.g., a T cell or a NK-T cell. 8 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0071] A “template nucleic acid” as used herein refers to a nucleic acid, e.g., DNA or RNA, and is used interchangeably with “template construct” or simply “template”. A template that is an RNA may be referred to herein as an “RNA template”, “RNA template nucleic acid”, or “RNA template construct”. A “driver nucleic acid” as used herein refers to a nucleic acid, e.g., DNA or RNA, encoding a driver protein and is used interchangeably with “driver nucleic acid”. A driver nucleic acid that is an RNA may be referred to herein as an “RNA driver construct” or “RNA driver nucleic acid”). [0072] In some embodiments, the transgene comprised in the template nucleic acid may be in a sense orientation or an anti-sense orientation with respect to the template nucleic acid. In some embodiments, the template nucleic acid and the driver nucleic acid are separate nucleic acids, i.e., in trans. In some embodiments, the template nucleic acid and the driver nucleic acid are present on a single nucleic acid, i.e., in cis. For clarity, in a trans configuration, the template nucleic acid includes a transgene which may be flanked 5’ and 3’, independently, with one or more regulatory elements. In a cis configuration, the template nucleic acid includes a driver nucleic acid and may further include one or more regulatory elements at the termini of the nucleic acid. In a cis configuration, the template nucleic acid includes an adjacent driver nucleic acid and further includes one or more regulatory elements at the termini of the nucleic acid. In certain embodiments, the driver encoded by the driver nucleic acid may be any one of the engineered drivers described elsewhere herein. [0073] It is noted that the driver nucleic acid may be DNA or RNA (e.g. mRNA). Likewise, the template nucleic acid may be DNA or RNA (e.g. mRNA). In exemplary embodiments, the driver nucleic acid and template are provided in trans, on separate nucleic acids (e.g. on separate RNAs). In some embodiments, the driver nucleic acid and template are in a cis configuration and provided in a single nucleic acid. In some embodiments, they are provided in cis as DNA. In some embodiments, they are provided in cis, as RNA, e.g. mRNA. In some embodiments, they are provided in trans as two separate DNA strands. In some embodiments, they are provided in trans, as RNA, as two separate RNA strands. Driver nucleic acids [0074] A driver nucleic acid encodes a “driver” or “driver protein” (interchangeably used herein) that comprises or consists of an RTE polypeptide (as discussed in further detail below). 9 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0075] In some embodiments, the driver nucleic acid includes untranslated regions (UTRs) that stabilize a transcript. In some embodiments, the driver nucleic acid comprises a nucleic acid sequence encoding an RTE that is flanked by a 5’UTR (driver 5’UTR) and/or a 3’UTR (driver 3’UTR). In some embodiments, the driver 5’UTR comprises a nucleic acid sequence of SEQ ID NO: 29, SEQ ID NO: 80 or SEQ ID NO: 96. In some embodiments, the driver 3’ UTR comprises a nucleic acid sequence of SEQ ID NO: 83. [0076] In some embodiments, the driver nucleic acid is an RNA, optionally mRNA, and comprises one or more chemical and/or sequence modifications. In some embodiments, the modification is an RNA CAP, a modified polyA length (e.g., relative to a natural polyA), a chemical modification (e.g., a pseudouridine and/or a methylpseudouridine), a 5’ UTR modification, a 3’ UTR modification, a modified Kozak sequence, a modified (e.g., truncated) stem loop, an RNA stabilization motif, a 5-methoxyuridine (5-MO-U) modification, a 5- methylcytidine (5mC) modification, or one or more additional and/or modified microsatellites. In some embodiments, a nucleic acid sequence encoding an engineered protein and/or a transgene (e.g., encoding a therapeutic RNA or protein) is codon optimized (e.g., codon optimized for expression in human cells). In some embodiments, codon optimization is for RNA optimization. In some embodiments, RNA optimization comprises reducing the Uracil (U) load of an RNA molecule. Engineered drivers [0077] Retrotransposable elements, which are also referred to herein interchangeably as “retroelements” or “RTEs”, or “retrotransposons” are nucleotide sequences that have naturally evolved to facilitate the proliferation of their own nucleotide sequences throughout the genome of a cell. An RTE typically encodes a protein, which may be referred to herein as an “RTE protein” or “driver”, that includes, among other domains, a reverse transcriptase (RT) domain and an endonuclease (EN) domain. In nature, the RNA template is generally the RNA that encoded the RTE protein (which may be referred to herein as a “RTE transcript”), such that the reverse transcription results in replicative insertion of the original RTE in cDNA form. Typically (but without being held to theory or mechanism), the RNA copy of the RTE comprises one or more untranslated regions (RTE-UTRs) that, as RNA, form structures capable of being bound by the driver, e.g., by a compatible RNA binding domain (RBD) within the driver, to create a ribonucleoprotein complex that traverses back into the nucleus to initiate and ultimately 10 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 complete the reverse transcription of the RTE transcript by the RTE protein. Typically, it is thought that the driver and one or more RTE-UTRs of a given RTE have co-evolved to have sufficient binding affinity for each other to allow them to bind to each other in the environment of a cell interior, e.g., in the cytoplasm. Without being held to theory or mechanism, the EN domain initiates the reverse transcription of the RTE transcript by creating a nick at a target site within genomic DNA of a cell. At the 3’ hydroxyl exposed by the nick, new DNA is synthesized using the RTE transcript as a template. Upon completion, the RTE protein switches to a second nick and exposed 3’ hydroxyl to begin synthesis of the second DNA strand. The two nicks are sealed by the action of one or more host cell DNA repair mechanisms. However, these RT and EN mechanisms can be used to incorporate other templates with nucleotide sequences encoding a gene of interest (“GOI”), i.e., a transgene. [0078] As used herein, a “driver protein” (or simply a ”driver”) refers to an RTE protein, or an engineered variant thereof comprising an RTE protein or a portion thereof. In some embodiments, the RTE protein is modified with amino acid substitutions, truncation, domain fusions (as described in further detail hereinbelow). In some embodiments, the driver comprises one or more heterologous polypeptides, e.g., fused directly or indirectly to an RTE protein or a portion thereof (as described in further detail hereinbelow). For clarity of presentation, an “engineered driver protein” (or “engineered driver”) as used herein refers to a driver that is an engineered variant of a naturally occurring, wild type (WT) RTE protein (which may be referred to herein as a “WT driver”). Engineered drivers of the disclosure may retain the same or substantially the same function as a WT driver, e.g., at least an RT activity and an EN activity. [0079] As used herein, a “polypeptide” of a protein refers to either a full-length version of a protein or to a portion thereof. A polypeptide of a protein may also comprise one or more amino acid substitutions with respect to the protein. [0080] As used herein, an “RTE polypeptide” refers to a full-length RTE protein or to a portion thereof. The portion may be, for example, a functional domain of the protein. Alternatively, the portion may be what remains of an RTE protein after deletion of one or more amino acids, segments, functional domains, binding domains, motifs, or combinations thereof. An “RTE polypeptide” as used herein may also include a full-length RTE protein or a portion thereof that has been subjected to one or more amino acid substitutions. For example, what remains of an RTE protein after deletion of the first 100 amino acids from the N-terminus, along with substitutions of certain amino acids in the RT domain and EN domain, may be referred to herein 11 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 as an RTE polypeptide. In another example, what remains of an RTE protein after deletion of the first 100 amino acids from the N-terminus, as well as the RT domain, may be referred to herein as an RTE polypeptide. [0081] As will be described in further detail herein below, the present disclosure provides embodiments of engineered drivers, comprising an RTE protein or portion thereof (e.g., an RTE polypeptide), and optionally comprising one or more heterologous polypeptides, that can genomically incorporate template nucleotides with an improvement in at least one of efficiency, specificity, accuracy, fidelity or processivity compared to a reference driver, e.g., a naturally occurring, wild type (WT) RTE protein. The present disclosure also provides nucleic acids (which may be interchangeably referred to herein as “polynucleotides”, and may be DNA, RNA or a DNA/RNA hybrid) that encode the engineered drivers. RTEs [0082] Engineered drivers of the disclosure may comprise an RTE polypeptide of an RTE protein encoded in one of various RTEs, including those known in the art, as well as new RTEs described herein. [0083] In certain embodiments, the RTE is a non-LTR retroelement. In certain embodiments, the non-LTR retroelement is a long-interspersed element polypeptide (LINE) or a short-interspersed element (SINE). LINEs (Long INterspersed Elements) and SINEs (Short INterspersed Elements) are non-LTR retroelements that are found in almost all eukaryotes and are among the most common retroelements in the human genome. Wild-type LINEs typically encode a protein with reverse transcriptase and endonuclease activities. SINEs do not encode reverse transcriptase or endonuclease and depend on reverse transcriptase and endonuclease encoded by partner LINEs. In certain embodiments, the LINE is a LINE-1, a LINE-2, or a LINE-3. [0084] In some embodiments, the RTE polypeptide is a non-site specific RTE polypeptide. In some embodiments, the RTE polypeptide is a site specific RTE polypeptide. In certain embodiments, the RTE polypeptide is derived from a Class I transposable element that utilizes an intermediate RNA during integration by a copy-and-paste mechanism, with or without Long Terminal Repeats (LTR) or Inverted Terminal Repeats (ITR) in their structure, and that can be derived from a superclade selected from the group consisting of retrons (include clades AbiA, AbiK, AbiP2, CRISPR, CRISPR-like, DCRs, G2L4, G2L, G2Lb, G2Lc, GII, Retrons, UG1, 12 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 UG2, UG3, UG4, UG5, UG6, UG7, UG8, UG9, UG10, UG11, UG12, UG13, UG14, UG15, UG16, UG17, UG18, UG19, UG20, UG21, UG22, UG23, UG24, UG25, UG26, UG27, UG28 and UG28b), Group II introns (including clades E, ML, B, CL1A, CL1B, CL2A, CL2B, F, D and C), non-LTR retroelements (including clades CRE, R2 group (including R4 clade and R2 clade), Hero, NeSL, RandI, Proto1, L1, Tx1, RTEPT, Proto2, RTEX, RTE, Outcast, Ingi (which includes sub-clade Vingi), I, Nimb, Tad1, Loa, R1, Jockey, Rex1, CR1, L2, L2A, L2B, Daphne, and Crack), LTR-retroelements or Endogenous Retroviruses (ERV) (including clades Ty1/Copia, Hepadnaviridae, BEL/Pao, Retroviridae, Caulimoviridae, and Ty3/Gypsy), Penelope-like retroelements (including clades Pen1, Pen1a, Pen2, Pen3, Pen3a, Pen3b, Pen4, Pen5, Pen5a and Pen6), Tyrosine recombinase retroelements (including clades CryptonA, CryptonF, CryptonS and CryptonI) and DIRS retroelements (including clades DIRS and Ngaro). [0085] In some embodiments, the RTE polypeptide is derived from an RTE selected from the group consisting of CRE, R2 group (including R4 clade and R2 clade), Hero, NeSL, RandI, Proto1, L1, Tx1, RTEPT, Proto2, RTEX, RTE, Outcast, Ingi (which includes sub-clade Vingi), I, Nimb, Tad1, Loa, R1, Jockey, Rex1, CR1, L2, L2A, L2B, Daphne, and Crack. [0086] In some embodiments, the RTE polypeptide is a Vingi1-Acar RTE protein or portion thereof. In some embodiments, the Vingi1-Acar polypeptide has an amino acid sequence set forth in SEQ ID NO: 31 (WT Vingi1-Acar RTE protein), or a sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. The amino acid sequence of SEQ ID NO: 31 may be encoded by the nucleic acid sequence of SEQ ID NO: 27. Ving1-ACAR may be referred to herein as “Vingi1”. R2 group RTEs [0087] R2 group RTEs in nature are non-long terminal repeat (non-LTR) retroelements that specifically insert DNA sequences into ribosomal RNA (rRNA) gene loci in the genomic DNA of a host cell. R2 group RTEs, which include members of the R2 clade and R4 clade, typically include a single open reading frame (ORF) that encodes a single protein. As used herein, RTEs of the R2 clade as well as the R4 clade may be referred to herein as “R2 RTEs”. 13 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0088] Non-R2 RTEs, including LTR RTEs and non-LTR RTEs enumerated above that are not in the R2 group, also typically encode an RTE protein that comprises an RT domain and an EN domain, and are referred to herein as “non-R2 RTE proteins”. These non-R2 RTEs may have, depending on their classification, one or more additional ORFs that encode chaperone proteins that have certain RNA- or DNA-binding domains. Such additional proteins are referred to herein as “non-R2 chaperone proteins”. [0089] An RTE protein encoded in R2 RTE’s, including R2 clade and R4 clade RTEs, may be referred to herein as an “R2 RTE protein”, or an “R2 protein”. An R2 RTE protein or a portion thereof may be referred to herein as an “R2 RTE polypeptide” or an “R2 polypeptide”. [0090] In certain embodiments, an engineered driver comprises an R2 polypeptide, that is, a polypeptide of an R2 protein. In certain embodiments, the R2 polypeptide may be fused to one or more heterologous polypeptides, of which various embodiments are described herein. [0091] R2 RTEs are widely distributed across various animal species, with a presence in invertebrates such as insects and mollusks, as well as in vertebrates such as birds, reptiles, and amphibians. R2 RTEs are typically integrated into the 28S ribosomal RNA (rRNA) genes and are maintained in a stable, lineage-specific manner across generations. R2 RTE proteins from different animal species show sequence variation, reflecting the adaptation of R2 elements to different host genomes and their co-evolution. Also note that a particular species of animal may have multiple versions of R2 group RTEs, each with different nucleotide sequences (as well as different amino acid sequences for their respective RTE proteins), integrated within their genome. [0092] Each unique RTE from a given animal species may be described herein with the following nomenclature: A clade identifier (e.g. R2) followed by the name of the organisms from which the RTE originated, or a two- to four-letter abbreviation thereof. As such, for example, an RTE from clade R2 of Taeniopygia guttata (zebra finch), may be referred to herein as a “R2-TG”.In instances where multiple RTEs have been identified in a given organism species, the clade identifier and the species abbreviation may be interspersed with a counter numeral. As such, a first RTE from clade R2 of Chamaetylas choloensis may be referred to as R2-1-ChCh (or R2_1-ChCh, or R2-1_ChCh), and a second RTE from clade R2 of Chamaetylas choloensis may be referred to as R2-2-ChCh (or R2_2-ChCh, or R2-2_ChCh). RTE proteins and polypeptides, and nucleic acids encoding them, may be referred to in a similar manner. As such, for example, a protein encoded in an R2 RTE may be referred to herein as an R2 protein, an R2 14 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 protein encoded in the R2-1-ChCh RTE may be referred to herein as an R2-1-ChCh protein, and an R2 polypeptide of an R2 protein encoded in the R2-1-ChCh RTE may be referred to herein as an R2-1-ChCh polypeptide. An engineered driver comprising a given RTE polypeptide maybe referred to by the particular RTE polypeptide comprised in the driver, e.g., an engineered driver comprising an R2 polypeptide may be referred to herein as a “R2 driver”, and an engineered driver comprising an R2-TG polypeptide may be referred to herein as a “R2-TG driver”. [0093] In certain embodiments, an R2 polypeptide of an R2 protein may be the wildtype (WT) version of the corresponding R2 protein. In certain embodiments, an R2 polypeptide of an R2 protein may be a portion of the WT version of the corresponding R2 protein. In certain embodiments, the R2 polypeptide of an R2 protein may comprise one or more amino acid substitutions compared to the WT version of the corresponding R2 protein. As used herein, a WT version of an R2 protein may be referred to as a “WT R2 protein”. [0094] A WT R2 protein typically comprises the following domains: a DNA binding domain cluster, which may comprise, for example, one or more zinc-finger (ZF) domains and one or more Myb domains; an RNA binding domain, a reverse transcriptase (RT) domain, and an endonuclease (EN) domain. In a WT R2-TG protein, having an amino acid sequence as set forth in SEQ ID NO: 75, the DNA binding domain cluster is located approximately at residues 216- 319. The DNA binding domain cluster comprises a first ZF domain located approximately at residues 216-281, a second ZF domain located approximately at residues 297-329, and a Myb domain located approximately at residues 337-391. The RT domain is located approximately at residues 591-1029, and the EN domain is located approximately at residues 1154-1390. In addition, segments of polypeptides between domains may be designated as “linkers” that link adjacent domains. It will be appreciated that the boundaries of the domains are approximate and largely based on homology with existing protein structures. As such, depending how the domains are determined, the location of any given domain, including those listed above, may be offset by, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 residues. [0095] In certain embodiments, the R2 polypeptide may have least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the WT version of the corresponding R2 protein. 15 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0096] The percent identity between two such polypeptides (as recited herein above and elsewhere throughout to present disclosure) can be determined by manual inspection of the two optimally aligned polypeptide sequences or by using software programs or algorithms (e.g., CLUSTAL, MUSCLE, MAFFT) using standard parameters, familiar to one with skill in the art. % Sequence identity may be calculated in one of a number of ways. % Sequence identity may be calculated in one of a number of ways. In certain embodiments, the % sequence identity may be a % subject identity, which is the percentage of identical matches between a query sequence and a subject sequence, relative to the length of the query sequence. In certain embodiments, the % sequence identity may be a % query identity, which is the percentage of identical matches between a query sequence and a subject sequence, relative to the length of the subject sequence. In certain embodiments, the % sequence identity may be a % alignment identity, which restricts the calculation of the % identity to the region(s) of alignment, so that large deletions or added domains are discounted. [0097] In certain embodiments, an engineered driver of the disclosure may comprise an RTE polypeptide of one of the following R2 proteins, as listed in Table 1: Table 1: R2 RTE protein sequences (amino acid and DNA sequence) of representative RTEs RTE name Organism species Amino acid sequence Nucleotide of RTE protein sequence encoding SEQ ID NO RTE protein SEQ ID NO 1 R2-1-TG* Taeniopygia guttata 75 2379 2 R2-ZA Zonotrichia albicollis 373 3 R4-AC Anolis carolinensis 374 2380 4 R4-1-PH Parhyale hawaiensis 795 29 * R2-1-TG is also referred to herein as “R2-1_TG” or “R2-TG”, which are all to be understood to refer to the same RTE, with the WT RTE protein having the amino acid sequence set forth in SEQ ID NO: 75, and encoded by the DNA sequence set forth in SEQ ID NO: 2379 (or a RNA version thereof). [0098] Table 1 provides the amino acid and DNA sequences of an R2 protein from each of the listed RTE species. For each DNA sequence provided, it will be understood that the present disclosure also includes a respective RNA version thereof. [0099] In certain embodiments, an engineered driver of the disclosure may comprise an RTE polypeptide of one of the following R2 proteins, as listed in Table 2: 16 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 Table 2: R2 RTE protein sequences (amino acid and DNA sequence) of R2 RTEs RTE name Abbre Organism Amino acid Nucleotide viation species sequence of sequence of RTE protein RTE protein SEQ ID NO SEQ ID NO 1 R2-1_Acridotheres tristis R2-AT Acridotheres tristis 582 1714 2 R2-1_Acrocephalus R2- Acrocephalus _scirpaceus _scirpaceus ASS scirpaceus scirpaceus 583 1715 3 R2-1_Agelaius_tricolor R2- Agelaius tricolor AgT 584 1716 4 R2-1_Ammodramus R2- Ammodramus _caudacutus AC caudacutus 586 1717 5 R2-1_Ammospiza_nelsoni R2- Ammospiza AN nelsoni 587 1718 6 R2-1_Buteo_platypterus R2-BP Buteo platypterus 589 1719 7 R2-1_Chamaetylas_ R2-CC Chamaetylas choloensis choloensis 590 1720 8 R2-1_Chamaetylas R2-CF Chamaetylas _fuelleborni fuelleborni 593 1721 9 R2-1_Cichladusa_arquata R2- Cichladusa CA arquata 594 1722 10 R2-1_Ciconia_episcopus R2-CE Ciconia episcopus 595 1723 11 R2-1_Colaptes_auratus R2- Colaptes auratus CoAu 597 1724 12 R2-1_Cossypha_archeri R2- Cossypha archeri CoAr 600 1725 13 R2-1_Cossypha_dichroa R2- Cossypha CD dichroa 601 1726 14 R2-1_Falco_fasciinucha R2-FF Falco fasciinucha 602 1727 15 R2- R2-GP Gallinago 1_Gallinago_paraguaiae paraguaiae 603 1728 16 R2-1_Garrulus_glandarius R2- Garrulus GG glandarius 604 1729 17 R2-1_Geranoaetus R2- Geranoaetus _albicaudatus GA albicaudatus 605 1730 18 R2- R2- Harpyopsis 1_Harpyopsis_novaeguine HN novaeguineae ae 606 1731 17 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 RTE name Abbre Organism Amino acid Nucleotide viation species sequence of sequence of RTE protein RTE protein SEQ ID NO SEQ ID NO 19 R2- R2-LS Limnodromus 1_Limnodromus_scolopac scolopaceus eus 607 1732 20 R2-1_Melierax_canorus R2- Melierax canorus MC 608 1733 21 R2- R2- Myiozetetes 1_Myiozetetes_cayanensis MyC cayanensis 609 1734 22 R2-1_Otis_tarda R2-OT Otis tarda 610 1735 23 R2-1_Prinia_subflava R2-PS Prinia subflava 611 1736 24 R2-1_Serinus_canaria R2-SC Serinus canaria 613 1737 25 R2- R2- Aphelocoma 1_Aphelocoma_californica ApC-1 californica 616 1738 26 R2- R2- Oenanthe 1_Oenanthe_melanoleuca OM melanoleuca 620 1739 27 R2- R2- Phylloscopus 1_Phylloscopus_trochilus PTT trochilus _trochilus trochilus 621 1740 28 R2-1_Geothlypis_trichas R2-GT Geothlypis trichas 623 1741 29 R2-1_Xenus_cinereus R2- Xenus cinereus XC 624 1742 30 R2- R2-2- Aphelocoma 2_Aphelocoma_californica ApC californica 625 1743 31 R2-2_Cichladusa_arquata R2- Cichladusa CiA arquata 626 1744 32 R2-1_Scolopax_minor R2- Scolopax minor SM 630 1745 33 R2-1_Tyto_alba R2-Ta Tyto alba 559 1746 34 R2-2_Acrocera_orbiculus R2- Acrocera AcC orbiculus 567 1747 35 R2- R2- Drosophila 1_Drosophila_sulfurigaster DSS sulfurigaster _sulfurigaster sulfurigaster 575 1748 36 R2-1_Rhopalosiphum_padi R2-RP Rhopalosiphum padi 576 1749 37 R2-1_Nemotelus_nigrinus R2- Nemotelus NN nigrinus 578 1750 38 R2-1_Chlorops_oryzae R2- Chlorops oryzae CO 580 1713 18 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 RTE name Abbre Organism Amino acid Nucleotide viation species sequence of sequence of RTE protein RTE protein SEQ ID NO SEQ ID NO 39 R2-2_Andrena_bucephala R2- Andrena AB bucephala 581 1751 40 R2-3_Ascidia_mentula R2- Ascidia mentula AM 1692 1752 41 R2- R2- Crotalus 1_Crotalus_oreganus_helle COH oreganus helleri ri 1693 1753 42 R2- R2-GF Gopherus 1_Gopherus_flavomarginat flavomarginatus us 1694 1754 43 R2-1_Malaclemys_terrapin R2- Malaclemys _pileata MTP terrapin pileata 1695 1756 44 R2- R2- Eublepharis 1_Eublepharis_macularius EM macularius 1696 1757 45 R2-1_Emys_orbicularis R2-EO Emys orbicularis 1697 1758 46 R2-1_Caretta_caretta R2-CC Caretta caretta 1698 1759 47 R2-1_Pleurodeles_waltl R2- Pleurodeles waltl PW 1699 1755 48 R2-1_Aspidoscelis_tigris R2-1- Aspidoscelis _stejnegeri ATS tigris stejnegeri 1700 1760 49 R2-1_Aspidoscelis_tigris R2-2- Aspidoscelis _stejnegeri ATS tigris stejnegeri 1701 1761 50 R2-1_Mauremys_mutica R2-1- Mauremys MM mutica 1702 1762 51 R2-3_Mauremys_mutica R2-1- Mauremys MM mutica 1703 1763 52 R2-1_Xiphophorus_hellerii R2- Xiphophorus XH hellerii 1704 1764 53 R2-1_Gambusia_affinis R2- Gambusia affinis GamA 1705 1765 54 R2-1_Schizopygopsis R2- Schizopygopsis _malacanthus SMa malacanthus 1706 1766 55 R2- R2-1- Schizopygopsis 1_Schizopygopsis_pylzovi SP pylzovi 1707 1767 56 R2- R2-2- Schizopygopsis 2_Schizopygopsis_pylzovi SP pylzovi 1708 1768 57 R2-1_Gila_orcuttii R2- Gila orcuttii GO 1709 1769 58 R2-1_Ahaetulla_prasina R2-AP Ahaetulla prasina 1710 1770 19 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 RTE name Abbre Organism Amino acid Nucleotide viation species sequence of sequence of RTE protein RTE protein SEQ ID NO SEQ ID NO 59 R2- R2- Aldabrachelys 1_Aldabrachelys_gigantea AG gigantea 1711 1771 60 R2-1_Ascidia_mentula R2- Ascidia mentula AsMe 1712 1772 [0100] Table 2 provides the amino acid and DNA sequences of an R2 protein from each of the listed RTE species. For each DNA sequence provided, it will be understood that the present disclosure also includes a respective RNA version thereof. [0101] In certain embodiments, an engineered driver of the disclosure may comprise an R2 polypeptide of one of the following R2 proteins: R2-XC protein (SEQ ID NO: 624), R2-LS protein (SEQ ID NO: 607), R2-MC protein (SEQ ID NO: 608), R2-GP protein (SEQ ID NO: 603), or R2-PTT protein (SEQ ID NO: 621). [0102] In certain embodiments, an engineered driver comprises a non-R2 RTE polypeptide. In certain embodiments, the non-R2 RTE polypeptide may be fused to one or more heterologous polypeptides, of which various embodiments are described herein. Engineered drivers [0103] As noted above, the present disclosure provides engineered drivers, comprising an RTE protein or portion thereof, and which may be optionally fused to one or more heterologous polypeptides. The engineered drivers of the disclosure can genomically incorporate template nucleotides with an improvement in at least one of efficiency, specificity, accuracy, fidelity or processivity compared to a reference driver, e.g., a naturally occurring, wild type (WT) RTE protein. [0104] The term “efficiency” with respect to gene insertion as used herein refers to the percent of transgene integrations at a target site in the genome. Efficiency of genomic integration can be measured, for example, by amplicon sequencing and comparing the number of insertions to non- insertions at a target site and characterizing as a percentage, or by genomically integrating a transgene encoding a fluorescent protein (e.g., GFP), and measuring a percentage of fluorescent protein-positive cells. As used herein, “efficiency” and “activity” are used interchangeably when used with respect to genomic integration of transgenes by engineered drivers of the disclosure. 20 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 For example, in certain working Examples provided below, embodiments of the engineered drivers of the disclosure demonstrated their “enhanced” or “improved” activity through improved efficiency in the genomic integration of transgenes encoded in templates. Engineered drivers with greater efficiency are desired. In certain embodiments, engineered drivers of the disclosure may exhibit improvement in efficiency of genomic integration by at least 1.5x fold, at least 2x fold, at least 2.5x fold, at least 3.5x fold, at least 4x fold, at least 4.5x fold, at least 5x fold, at least 10x fold, at least 15x fold, at least 20x fold, at least 25x fold, at least 30x fold, at least 40x fold, at least 50x fold, at least 60x fold, at least 70x fold, at least 80x fold, at least 90x fold, at least about 100x fold, at least about 150x fold, at least about 200x fold, at least about 300x fold, at least about 400x fold, at least about 500x fold, at least about 750x fold, or about 1000x fold compared to a reference driver. [0105] The term “specificity” with respect to gene insertion as used herein refers to the proportion of insertions at a specific target site versus another site. An engineered protein with high specificity would exhibit few or no off-target insertions compared to insertions into a safe harbor site. Specificity may be measured by, for example, amplicon sequencing of known or predicted off-target sites. Engineered drivers with greater specificity are desired. [0106] The term “accuracy” with respect to gene insertions as used herein refers to the percentage of full length insertions at the target site and can be measured by, for example, sequencing the target site and comparing correct insertions to total insertions at the site of interest. Engineered drivers with greater accuracy are desired. [0107] The term “fidelity” with respect to gene insertion as used herein refers to the nucleotide misincorporation rate as measured, for example, on a per nucleotide basis of the DNA sequence compared to the RNA template sequence. Engineered drivers with greater fidelity are desired. [0108] The term “processivity” with respect to gene insertion as used herein is a measure of the proportional presence of some sequence distal to the RT initiation site versus some sequence proximal to the RT initiation site. Engineered drivers with higher processivity are desired. In some embodiments, large insertions may be desired. A large insertion may be a nucleic acid sequence of about 20 to about 10,000 bases or more. For example, about 20 bases, about 50 bases, about100 bases, about 200 bases, about 300 bases, about 400 bases, about 500 bases, about 200 bases, about 300 bases, about 400 bases, about 500 bases, about 750 bases, about 1000 bases, about 1250 bases, about 1500 bases, about 2,000 bases, about 3,000 bases, about 21 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 4,000 bases, about 5,000 bases, about 6,000 bases, about 7,000 bases, about 8,000 bases, about 9,000 bases, about 10,000 bases or more. Heterologous polypeptides [0109] The present disclosure provides embodiments of engineered drivers that comprises an RTE polypeptide fused to one or more heterologous polypeptides (e.g. fused to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more heterologous polypeptides), and also provides as nucleic acids that encode said engineered drivers fused to said heterologous polypeptides. As such, any disclosures herein regarding embodiments of engineered drivers, RTE polypeptides, and heterologous polypeptides fused thereto, should be understood as also disclosing nucleic acids that encode the embodiments of engineered drivers, RTE polypeptides, RTE polypeptide fragments and heterologous polypeptides fused thereto. [0110] It is to be noted that a “heterologous” polypeptide of the engineered driver refers to a polypeptide that is not a portion of the natural RTE protein from which the RTE polypeptide is derived, and is separate and independent from the heterologous template nucleic acid of the disclosure (which are described further herein below). Furthermore, some of the polypeptides disclosed herein may be known to have more than one function and hence be present in one or more functional category. For example, Rad 51 and RAD17 are listed as both RNA/DNA processing polypeptides and RNA/DNA repair polypeptides. [0111] In certain embodiments, the driver comprises an RTE polypeptide fused to at least one (e.g., 1, 2, 3, 4, etc.) heterologous polypeptide at the N-terminus of the RTE polypeptide. In certain embodiments, the driver comprises an RTE polypeptide fused to at least one (e.g., 1, 2, 3, 4, etc.) heterologous polypeptide at the C-terminus of the RTE polypeptide. In certain embodiments, the driver comprises at least one (e.g., 1, 2, 3, 4, etc.) heterologous polypeptide inserted within an RTE polypeptide. [0112] In certain embodiments, the driver comprises an RTE polypeptide fused to at least two heterologous polypeptides, at least one at the N-terminus of the RTE polypeptide and at least one at the C-terminus of the RTE polypeptide. In certain embodiments, the driver comprises an RTE polypeptide fused to at least two heterologous polypeptides, at least one at the N-terminus of the RTE polypeptide and at least one inserted within the RTE polypeptide. In certain embodiments, the driver comprises an RTE polypeptide fused to at least two heterologous polypeptides, at least one at the C-terminus of the RTE polypeptide and at least one inserted within the RTE 22 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 polypeptide. In certain embodiments, the driver comprises an RTE polypeptide fused to at least two heterologous polypeptides, fused at the N-terminus of the RTE polypeptide [0113] In certain embodiments, the at least one heterologous polypeptide comprises a heterologous nucleosome binding polypeptide, a heterologous nucleic acid binding polypeptide, a heterologous RNA/DNA repair polypeptide, a heterologous RNA/DNA processing polypeptide, heterologous nuclear localization signal, a heterologous reverse transcriptase, a heterologous endonuclease, a heterologous intrinsically disordered polypeptide, or any combination thereof. [0114] In certain embodiments, in an engineered driver comprising a plurality of heterologous polypeptides in sequence, pairs of adjacent heterologous polypeptides may be connected directly, or via a linker. In certain embodiments, a heterologous polypeptide and an RTE polypeptide situated adjacent to each other may be directly connected, or connected via a linker. In certain embodiments, the linker is a rigid linker. In other embodiments, the linker is a flexible linker. Flexible linkers are generally made up of small, non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. Alternating Gly and Ser residues provides flexibility. In certain embodiments, the linker is a glycine-serine based linker or a XTEN peptide linker. In certain embodiments, the linker is a cleavable linker. Solubility of the linker and associated sequences may be enhanced by the inclusion of charged residues, e.g., two positively charged residues (e.g., Lys) and one negatively charged residue (e.g., Glu). The linker may be 2-35 amino acids long. In certain embodiments, the linker is selected from any one of SEQ ID NOs: 427, 428 or 429. [0115] In certain embodiments, the at least one heterologous polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 76, 383-400, 402-422, 539, 544, 585, 591, 592, 598, 599, 691, 696-700, 706, 707, 712, 713, 718, 719, 720, 725, 726, 736, 737, 742, 747, 748, 749, 754, 755, 759- 763, 768- 771, 776- 779, 784-787, 792-794, 835- 838, 1214-or an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or 99% identical, to a sequence of any of SEQ ID NOs: 76, 383-400, 402-422, 539, 544, 585, 591, 592, 598, 599, 691, 696-700, 706, 707, 712, 713, 718, 719, 720, 725, 726, 736, 737, 742, 747, 748, 749, 754, 755, 759- 763, 768- 771, 776- 779, 784-787, 792-794, 835- 838, 1214. 23 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 Arrangement of heterologous polypeptides fused to an RTE polypeptide [0116] Non-limiting examples of engineered drivers that can be used (e.g., directly or encoded on an RNA and/or DNA molecule) to promote insertion of a heterologous gene into a target nucleic acid in a host cell include protein fusions comprising an RTE polypeptide, optionally fused to at least one heterologous polypeptide that redirects and/or enhances insertion of the heterologous gene. In some embodiments, each of the at least one heterologous polypeptide comprises one or more of, e.g., an RNA/DNA processing polypeptide, an RNA/DNA repair polypeptide, a nucleic acid binding polypeptide, or a nucleosome binding polypeptide. The at least one heterologous polypeptide can be fused to the N-terminus and/or C-terminus of an RTE polypeptide, and/or internally within an RTE polypeptide (e.g., between two domains of the RTE polypeptide). Nucleic acids (e.g., RNA and/or DNA) encoding one or more engineered drivers can be used to promote insertion of a DNA copy of a template nucleic acid (e.g., a template nucleic acid comprising a gene of interest) into a target nucleic acid. Provided in some embodiments is a first nucleic acid (a template nucleic acid) comprising a gene of interest, with a second nucleic acid (a driver nucleic acid) that encodes an engineered driver, which together can be delivered or administered to a cell or subject. In some embodiments, the nucleic acid that encodes the template and the driver nucleic acid are provided (e.g., administered to a subject) as separate nucleic acid molecules (i.e., in trans), or on a single nucleic acid molecule (i.e., in cis). [0117] One or more nucleic acids (e.g., RNA and/or DNA) encoding at least one of these engineered drivers can be provided, in trans or in cis, to target cells (e.g., ex vivo or in vivo) along with one or more nucleic acids (e.g., RNA and/or DNA) encoding a transgene of the present disclosure to promote integration of the transgene into a nucleic acid (e.g., a genomic nucleic acid) of the target cells. [0118] In some embodiments, an RTE polypeptide is modified to remove the natural EN domain. In some embodiments, a heterologous endonuclease domain (hEN) is fused to the RT domain (e.g., to replace the natural EN domain). [0119] Various embodiments of heterologous polypeptides that may be fused to an RTE polypeptide in an engineered driver of the disclosure are described in more detail hereinbelow. Heterologous nucleosome binding polypeptide [0120] Nucleosome binding polypeptides are characterized by being capable of binding a nucleosome, a section of genomic DNA that is wrapped around a core of histone proteins, which 24 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 forms a unit of DNA packaging in eukaryotes. Without being bound by theory, nucleosome binding polypeptides have various functions that affect access to genomic DNA, including modulating chromatin structure and chromatin accessibility, and altering the activity of genomic editing proteins [0121] In certain embodiments, the at least one heterologous polypeptide fused to an RTE polypeptide comprises a nucleosome binding polypeptide. In certain embodiments, the nucleosome binding polypeptide may be a HMGN1 (high mobility group nucleosome-binding domain-containing protein 1) polypeptide, an HMGB1 (high mobility group protein B1) polypeptide, or a StkC (sticky-C) DNA binding domain polypeptide. [0122] In certain embodiments, the HMGN1 polypeptide may comprise an amino acid sequence set forth in SEQ ID NO: 77 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. In certain embodiments, the HMGN1 polypeptide comprises at least one amino acid substitution, wherein the at least one amino acid substitution is at the D100 residue, e.g. D100E and/or D100G relative to SEQ ID NO: 77. [0123] In certain embodiments, the HMGB1 polypeptide may comprise an amino acid sequence set forth in SEQ ID NO: 404 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. The StkC binding domain polypeptide may comprise an amino acid sequence set forth in SEQ ID NO: 420 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. [0124] It will be appreciated that an engineered driver of the disclosure may comprise one or more, for example 1, 2, 3, 4, or 5, heterologous nucleosome binding polypeptides, optionally in combination with one or more other heterologous polypeptides as described herein. [0125] In certain embodiments, the engineered driver protein comprises an R2 RTE polypeptide fused to one or more heterologous nucleosome binding polypeptides, e.g., an HMGN1 polypeptide, an HMGB1 polypeptide, an StkC DNA binding domain polypeptide, or combinations thereof. [0126] In certain embodiments, the engineered driver protein comprises an R2 RTE polypeptide fused to an HMGN1 polypeptide. The HMGN1 polypeptide may be situated internally in the R2 RTE protein, on the N-terminal end of the R2 RTE polypeptide or the C-terminal end of the R2 25 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 RTE polypeptide. SEQ ID NOs: 82, 1232, 1233 or 642 set forth amino acid sequences of exemplary driver proteins comprising a HMGN1 polypeptide. Heterologous nucleic acid binding polypeptide [0127] In certain embodiments, the at least one heterologous polypeptide fused to an RTE polypeptide comprises a nucleic acid binding polypeptide that binds to RNA and/or DNA. In certain embodiments, a nucleic acid binding polypeptide binds to RNA. In certain embodiments, a nucleic acid binding polypeptide binds to DNA. [0128] In certain embodiments, the nucleic acid binding polypeptide may be a sequence non- specific DNA binding polypeptide. In certain embodiments, the sequence non-specific DNA binding domain may be a Sto7d DNA binding domain polypeptide or a Sso7d DNA binding domain polypeptide. Sso7D is from thermophilic archaea Sulfolobus solfataricus, and the Sto7d protein, is a Sso7D counterpart from Sulfolobus tokodaii. In certain embodiments, the sequence non-specific DNA binding domain may be a UL42 DNA binding domain polypeptide or a Guinea pig cytomegalovirus (CMV) DNA binding domain. Without being limited by theory, sequence non-specific DNA binding domains, e.g., from the Sso7d homology group can improve processivity of DNA polymerases and reverse transcriptases, and can be used to promote integration of a transgene by an engineered driver. [0129] In certain embodiments, the Sto7d DNA binding domain polypeptide comprises an amino acid sequence set forth in SEQ ID NO 405, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. [0130] In certain embodiments, the Sso7d DNA binding domain polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 385, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. [0131] In certain embodiments, the UL42 DNA binding domain polypeptide (binding domain from herpes simplex virus 1 (HSV1)) comprises an amino acid sequence set forth in SEQ ID NO: 598, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. 26 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0132] In certain embodiments, the Guinea pig CMV DNA binding domain polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 835, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. [0133] In certain embodiments, the Rat CMV DNA binding domain polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 836, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. [0134] In certain embodiments, the sequence non-specific DNA binding domain may be a single-stranded (ss) DNA binding polypeptide. In certain embodiments, the ss DNA binding polypeptide may be a RPA3 DNA binding domain polypeptide, which may comprise an amino acid sequence set forth in SEQ ID NO: 388, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. [0135] In certain embodiments, the ss DNA binding polypeptide may be a RecT polypeptide, which may comprise an amino acid sequence set forth in SEQ ID NOs: 400 (csRecT) or SEQ ID NO: 402 (paRecT), or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. [0136] In certain embodiments, the ss DNA binding polypeptide may be a single stranded binding (SSB) polypeptide, which may comprise an amino acid sequence set forth in SEQ ID NOs: 421, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. [0137] In certain embodiments, the nucleic acid binding polypeptide may be a sequence specific DNA binding polypeptide. Without being bound by theory, site-specific DNA binding domains may improve site-specific integration of a transgene of the disclosure into the genome (e.g., by improving local interaction of the retroelement protein at the genomic site of interest) and can be used to introduce site-specificity into otherwise non-site-specific drivers and/or improve site- specificity for site-specific drivers. [0138] In certain embodiments, the sequence specific DNA binding polypeptide may be a Cas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, 27 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologues thereof, or modified versions thereof. The Cas protein may be a nucleolytically inactive Cas protein(e.g., a dead SpCas9 having D10A and/or H840A amino acid substitutions). In certain embodiments, the sequence specific DNA binding polypeptide may be a Zinc finger DNA binding domain (e.g., a Zinc finger DNA binding domain targeting AAVS1, a transcription activator-like effector (TALE) DNA binding domain, or a dead SpuFz1 (e.g., dSpuFz1, a SpuFz1 with a D606A amino acid substitution). [0139] In certain embodiments, the nucleic acid binding polypeptide provides sequence specificity, and may comprise a sequence set forth in any one of SEQ ID NOs: 406-417, or 422- 424, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. [0140] In certain embodiments, a sequence-specific endonuclease may be used, e.g., to enhance or replace a native EN domain of an RTE polypeptide. Such domains may retarget integration to a site of interest. Non-limiting examples of an EN fusion/replacement include a site-specific homing endonuclease targeting the AAVS1 gene fused to RTE polypeptide deficient in endonuclease activity, either through an inactivating mutation in the endonuclease domain (e.g., by a D237A, H238 substitution, and/or D216A in an L2-2 domain or a corresponding mutation in an alternative domain), or through a deletion of the EN domain. [0141] It will be appreciated that an engineered driver of the disclosure may comprise one or more, for example 1, 2, 3, 4, or 5, heterologous nucleic acid binding polypeptides, optionally in combination with one or more other heterologous polypeptides as described herein. [0142] In certain embodiments, the engineered driver protein comprises an R2 RTE polypeptide fused to one or more heterologous nucleic acid binding polypeptides. In certain embodiments, the engineered driver protein comprises an R2 RTE polypeptide fused to a Sto7d DNA binding domain polypeptide or the UL42 DNA binding domain polypeptide. The fused polypeptide may be situated internally in the R2 RTE protein, on the N-terminal end of the R2 RTE polypeptide or the C-terminal end of the R2 RTE polypeptide. SEQ ID NOs: 500, 509, 631, 1232, 688 or 832 set forth amino acid sequences of exemplary driver proteins comprising a Sto7d DNA binding domain polypeptide or a UL42 DNA binding domain polypeptide. 28 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 Heterologous RNA/DNA repair polypeptide [0143] In certain embodiments, the at least one heterologous polypeptide fused to an RTE polypeptide comprises an RNA/DNA repair polypeptide, a protein that is or interacts with a host repair protein that acts on RNA and/or DNA. In some embodiments, the DNA repair polypeptide is selected from a group consisting of a non-homologous end joining (NHEJ) pathway protein, a mismatch repair (MMR) protein, a microhomology-mediated end-joining (MMEJ) protein, a homology directed repair (HDR) pathway protein, and a DNA damage response protein. [0144] Activation or inhibition of certain host cell proteins via a heterologous polypeptide that is fused to an RTE polypeptide (e.g., a reverse transcriptase and/or endonuclease domain) may improve transgene insertion efficiency. The precise class of host cell proteins will depend on the mechanism of integration (e.g., depending on the RTE polypeptide that is used. [0145] In certain embodiments, the RNA/DNA repair polypeptide may be a RAD51 polypeptide, a RAD17 polypeptide, a BRCA2 polypeptide, a ANKRD28 polypeptide, an HSV-1 alkaline nuclease polypeptide (e.g., UL12), a proliferating cell nuclear antigen (PCNA) polypeptide, a polypeptide containing a PCNA interacting protein (PIP) motif, a MDC1 polypeptide, a MSH4 polypeptide, a SCML1 polypeptide, a CDKN2A polypeptide, a p53 inhibitor peptide, or a CTIP (CtBP-interacting protein, also known as DNA endonuclease RBBP8 or Retinoblastoma-binding protein 8) polypeptide. In certain embodiments, the RNA/DNA repair polypeptide is a heterologous CTIP polypeptide. [0146] In certain embodiments, the RAD17 polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 396, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. [0147] In certain embodiments, the ANKRD28 polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 393, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. [0148] In certain embodiments, the HSV-1 alkaline nuclease polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 384, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. 29 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0149] In certain embodiments, the BRCA2 polypeptide comprises an amino acid sequence that set forth in SEQ ID NO: 418, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. [0150] In certain embodiments, the PCNA polypeptide comprises an amino acid sequence that is set forth in SEQ ID NO: 398 or SEQ ID NO: 399, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. [0151] In certain embodiments, the MDC1 polypeptide comprises an amino acid sequence that set forth in SEQ ID NO: 394, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. [0152] In certain embodiments, the MSH4 polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 395, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. [0153] In certain embodiments, the SCML1 polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 397, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. [0154] In certain embodiments, the CDKN2A polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 398, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. [0155] In certain embodiments, the p53 inhibitor is a MDM2 polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 390, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. [0156] In certain embodiments, the p53 inhibitor is a peptide 14-derived peptide comprising an amino acid sequence set forth in SEQ ID NO: 419, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. 30 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0157] In certain embodiments, the CTIP polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 76, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. In certain embodiments, the CTIP polypeptide comprises at least one amino acid substitution, wherein the at least one amino acid substitution is at one or more of (e.g. at least 1,2,3,4,5,etc.) T15I, R123K, P181L, M202L, M202F, M202T, A237L, A237I, A237N, A237K, N288D, D18F, I49C, S17D, D18C, Q46T, I2A, C198S, E155S, L281G, I186S, Q46K, or a combination thereof relative to SEQ ID NO: 76. In exemplary embodiments, the CTIP polypeptide comprises the amino acid substitutions of T15I, R123K, P181L, M202L, M202F, M202T, A237L, A237I, A237N, A237K, N288D, D18F, I49C, S17D, D18C, Q46T, I2A, C198S, E155S, L281G, I186S, and Q46K, relative to SEQ ID NO: 76. [0158] Exemplary embodiments of engineered drivers with a mutated CTIP polypeptide with one or more of the above-noted amino acid substitutions (relative to SEQ ID NO: 76) are set forth in SEQ ID NOs: 789-791, 795-800 or 1106-1116. [0159] In certain embodiments, the RNA/DNA repair polypeptide may contain a proliferating cell nuclear antigen (PCNA) interaction (PIP) domain. In certain embodiments, an RTE polypeptide has a modification that inactivates its PIP. In certain embodiments, an RTE polypeptide has a modification that activates a PIP domain). The PIP domain is believed to recruit the cellular PCNA protein which may play a role in DNA replication. In certain embodiments, the native RTE PIP domain is replaced with a PIP domain from another protein (such as p21, FEN1 or CHAF1A) which may improve PCNA recruitment. In certain embodiments, the modification comprises deletion. In certain embodiments, the modification comprises one or more mutations (e.g., point mutations). In certain embodiments, the modification is in the EN domain. In certain embodiments, the modification is in the RT domain. In certain embodiments, the modification is in the RNA binding domain. In certain embodiments, PIP polypeptides (such as a p21 polypeptide, a FEN1 polypeptide or a CHAF1A polypeptide) may be added as a heterologous polypeptide in the C-terminal or N-terminal of the RTE protein, or internally in the RTE protein. [0160] In certain embodiments, the PIP polypeptide is a p21 polypeptide comprising an amino acid sequence that set forth in SEQ ID NO: 419, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. 31 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0161] In certain embodiments, the PIP polypeptide is a FEN1 polypeptide comprising an amino acid sequence that set forth in SEQ ID NO: 386, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. [0162] In certain embodiments, the PIP polypeptide is a CHAF1A polypeptide comprising an amino acid sequence that set forth in SEQ ID NO: 399, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. [0163] It will be appreciated that an engineered driver of the disclosure may comprise one or more, for example 1, 2, 3, 4, or 5, heterologous RNA/DNA repair polypeptides, optionally in combination with one or more other heterologous polypeptides as described herein. [0164] In certain embodiments, the engineered driver protein comprises an R2 RTE polypeptide fused to one or more heterologous RNA/DNA binding polypeptides. In certain embodiments, the engineered driver protein comprises an R2 RTE polypeptide fused to a CTIP binding domain polypeptide. The fused CTIP polypeptide may be situated internally in the R2 RTE protein, on the N-terminal end of the R2 RTE polypeptide or the C-terminal end of the R2 RTE polypeptide. SEQ ID NOs: 82, or 652-680, set forth amino acid sequences of exemplary driver proteins comprising a CTIP polypeptide. Heterologous RNA/DNA processing polypeptide [0165] An RNA/DNA processing polypeptide is an enzyme that causes chemical changes to RNA and/or DNA, for example by promoting or retarding RNA degradation. In addition to interacting with host cell repair and DNA-damage response proteins, proteins that directly process and/or repair RNA/DNA intermediates involved in transgene integration also may, without being bound by theory, improve transgene integration efficiency. Further without being bound by theory, an RNA/DNA processing polypeptide may improve transgene integration efficiency and/or redirect transgene integration to a different target location (e.g., within the genome of a cell). [0166] In certain embodiments, the at least one heterologous polypeptide fused to an RTE polypeptide comprises an RNA/DNA processing polypeptide. In certain embodiments, the at least one heterologous polypeptide comprises an RNA/DNA processing polypeptide selected 32 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 from an RNA helicase polypeptide, an RNA helicase recruitment motif polypeptide, a RAD51 polypeptide, a RAD17 polypeptide, or a RAD6 polypeptide. [0167] RNA helicases are enzymes that unwind RNA structures, and play a role in various cellular processes, including but limited to transcription, RNA splicing, and translation, as well as in the regulation of gene expression. A RNA helicase recruitment motif is a specific sequence or structural feature in proteins that facilitates the binding and recruitment of RNA helicases to their target sites. [0168] In certain embodiments, the at least one heterologous RNA/DNA processing polypeptide comprises an RNA helicase polypeptide. In certain embodiments, the RNA helicase polypeptide is an EIF4A (Eukaryotic initiation factor 4A) polypeptide. In certain embodiments, the EIF4A polypeptide comprises an amino acid set forth in SEQ ID NO: 592, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. In certain embodiments, the RNA helicase polypeptide is a Dengue virus RNA helicase polypeptide. In certain embodiments, the Dengue virus RNA helicase polypeptide comprises an amino acid set forth in SEQ ID NO: 1654, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. [0169] In certain embodiments, the at least one heterologous RNA/DNA processing polypeptide comprises a RNA helicase recruitment motif polypeptide. In certain embodiments, the RNA helicase recruitment motif polypeptide is a TUDOR domain polypeptide from TDRD3 which may recruit RNA helicase DHX9 (DExH box helicase 9). In certain embodiments, the TUDOR domain polypeptide comprises an amino acid set forth in SEQ ID NO: 591, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. In certain embodiments, the RNA helicase recruitment peptide is derived from EIF4G which recruits EIF4A RNA helicase, as set forth in SEQ ID NO: 837, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. [0170] In certain embodiments, the RNA helicase recruitment motif polypeptide is a canine hepacivirus core protein motif polypeptide. In certain embodiments, the canine hepacivirus core protein motif polypeptide (which recruits cellular DDX3) comprises an amino acid set forth in 33 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 SEQ ID NO: 838, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. In certain embodiments, the canine hepacivirus core protein motif polypeptide comprises an amino acid set forth in SEQ ID NO: 599, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. In certain embodiments, the canine hepacivirus core protein motif polypeptide comprises an amino acid set forth in SEQ ID NO: 838, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. [0171] Without wishing to be bound by theory, fusing known RNA helicase domains to an RTE polypeptide, or fusing peptides that can recruit cellular RNA helicases to an RTE polypeptide, may improve reverse transcription in genome editing systems by removing secondary structure in the RNA template that may block reverse transcription thus reducing activity and processivity. [0172] RAD51 is a protein involved in the homology directed repair (HDR) pathway, and a RAD51 domain directly binds to single-stranded DNA and searches for matching DNA in the genome as an early stem in homologous recombination. Without being bound by theory, in site- specific transgene integration, e.g., transgene integration by R2 RTE proteins or certain embodiments of drivers comprising R2 RTE polypeptides, RAD51 fusion to an RTE polypeptide may improve integration at genomic sites that are homologous to sequences that are included on a template nucleic acid (for example sequences that flank the transgene and are homologous to one or more genomic sequences in the vicinity of the target gene). [0173] In certain embodiments, the RAD51 polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 383, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. In certain embodiments, the RAD17 polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 396, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. In certain embodiments, the RAD6 polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 389, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, 34 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. [0174] It will be appreciated that an engineered driver of the disclosure may comprise one or more, for example 1, 2, 3, 4, or 5, heterologous RNA/DNA processing polypeptides, optionally in combination with one or more other heterologous polypeptides as described herein. [0175] In certain embodiments, the engineered driver protein comprises an R2 RTE polypeptide fused to one or more heterologous RNA/DNA processing polypeptides. In certain embodiments, the engineered driver protein comprises an R2 RTE polypeptide fused to a RNA helicase polypeptide (e.g., a EIF4A polypeptide or a Dengue virus RNA helicase polypeptide) or a RNA helicase recruitment motif polypeptide (e.g., a TUDOR domain polypeptide). The fused polypeptide (RNA helicase polypeptide or RNA helicase recruitment motif polypeptide) may be internal to the R2 RTE protein, on the N-terminal end of the R2 RTE polypeptide or the C- terminal end of the R2 RTE polypeptide. SEQ ID NOs: 592 or 1654 set forth amino acid sequences of exemplary driver proteins comprising a RNA helicase polypeptide. SEQ ID NOs: 591 or 838 set forth amino acid sequences of exemplary driver proteins comprising a RNA helicase recruitment motif polypeptide. [0176] In certain embodiments, the engineered driver protein comprises a non-R2 RTE polypeptide fused to an RNA helicase polypeptide. In certain embodiments, the non-R2 RTE may be, e.g., Vingi RTE, R2NS RTE,_L4_ACar RTE, a CR1_10_Ami RTE, a ZFL2-2 RTE, or any other non-R2 RTE disclosed elsewhere herein. Heterologous localization signal [0177] The engineered drivers of the disclosure may comprise heterologous localization sequences. For example, a nuclear localization sequence (NLS) is a polypeptide that promotes import of a protein into a nucleus of a cell. A nucleolar localization sequence (NoLS) is a polypeptide that promotes import of a protein into a nucleolus of a cell. NLS’s and NoLS’s, and methods for assessing their ability to direct a polypeptide to the nucleus or the nucleolus, respectively, are known to those with skill in the art. [0178] In certain embodiments, the at least one heterologous polypeptide fused to an RTE polypeptide comprises a localization polypeptide. In certain embodiments, the localization polypeptide may be a nuclear localization signal (NLS). In certain embodiments, the localization polypeptide may be a nucleolar localization signal (NoLS). 35 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0179] In certain embodiments, the NLS comprises an amino acid sequence set forth in any one of SEQ ID NOs: 430, 431, 391, 435 or 1677. In certain embodiments, an NLS comprises a SV40 sequence (e.g., SEQ ID NO: 430), a nucleoplasmin sequence (e.g., SEQ ID NO: 431), a TOPBN1 sequence (e.g., SEQ ID NO: 391), or a bipartite SV40 (bp-SV40) sequence (e.g., SEQ ID NO: 435 or SEQ ID NO: 1677). [0180] In non-limiting embodiments, the NoLS comprises an amino acid sequence set forth in any one of SEQ ID NOs: 432, 433 or 434. In certain embodiments, a NoLS comprises a PNRC sequence (e.g., SEQ ID NO: 432), a poly R sequence (e.g., SEQ ID NO: 433), or a H2B sequence (e.g., SEQ ID NO: 434). [0181] It will be appreciated that an engineered driver of the disclosure may comprise one or more, for example 1, 2, 3, 4, or 5, heterologous localization sequences, optionally in combination with one or more other heterologous polypeptides as described herein. [0182] In certain embodiments, the engineered driver protein comprises an R2 RTE polypeptide fused to one or more heterologous localization sequences. In certain embodiments, the engineered driver protein comprises an R2 RTE polypeptide or fragment thereof fused to a NLS or a NoLS. The fused polypeptide (NLS or NoLS) may be situated at the N-terminal end of the R2 RTE polypeptide or the C-terminal end of the R2 RTE polypeptide, or internal to the R2 RTE polypeptide. SEQ ID NOs: 82, 751, 752 or 753 set forth amino acid sequences of exemplary driver proteins comprising a NLS or NoLS. Heterologous intrinsically disordered polypeptide [0183] Intrinsically disordered regions (IDRs) are regions of proteins that lack a fixed three- dimensional (3D) structure. These IDRs, which lack an amino acid sequence that form, for example, alpha helices, beta sheets, and other canonical 3D protein structures, are thought to provide flexibility, allowing the protein to have ordered interactions with multiple molecular partners within the intracellular environment. [0184] IDRs also demonstrate a function of forming and maintaining condensates. Condensates can form and maintain organization through a range of different processes, the most well-known of which is phase separation of proteins and RNA. There are many different examples of intrinsically disordered proteins forming condensates. Condensation is also called phase- separation, and may provide beneficial properties. Without wishing to be bound by theory, condensed proteins might require or benefit from phase-separated environment with different 36 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 and optimized chemical-physiological conditions or help with energetic limitations by reducing the entropy and increasing the enthalpy of the system. Without wishing to be bound by theory, phase separation can also prevent degradation, and help with nuclear import or to improve protein-RNA intermolecular interactions. For example, it has been shown that for LINE-1 elements, ORF1 tends to condensate. R2-TG has an intrinsically disordered region at its N- terminal (Residues 1-161, 1-184 or 1-218). Without wishing to be bound by theory, if a N- terminal intrinsically disordered region is responsible for/promotes condensation of R2 proteins, then other condensation peptides may be substituted for the disordered domains of RTE proteins and improve function. [0185] In certain embodiments, the at least one heterologous polypeptide fused to an RTE polypeptide comprises a heterologous IDR polypeptide. In certain embodiments, the heterologous IDR polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOS: 691, 696-700, 706-707, 712-713, 718-720, 725-726, 736-737, 742, 747-749, 754-755, 759-763, 768-771, 776-779, 784-787, 792-794 and 1214-1216, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. [0186] In some embodiments, the heterologous IDR polypeptide may be a condensation polypeptide comprising any one (or a combination of two or more) of the following: the N- terminal 161 residues of R2-TG protein (SEQ ID NO: 1214); the N-terminal 184 residues of R2- TG RTE protein (SEQ ID NO: 1215); the N-terminal 218 residues of R2-TG RTE protein (SEQ ID NO: 1216), a condensation peptide from the first IDR of UBQ2 polypeptide (SEQ ID NO: 691), a condensation peptide from the second IDR of UBQ2 polypeptide (SEQ ID NO: 696), a condensation peptide from the third IDR of UBQ2 polypeptide (SEQ ID NO: 697), a condensation peptide from the second IDR of RBM14 polypeptide (SEQ ID NO: 698), a condensation peptide from the fourth IDR of RBM14 polypeptide (SEQ ID NO: 699), a condensation peptide from the first IDR of HSPB8 polypeptide (SEQ ID NO: 700), a condensation peptide from the first IDR of hnRNPA1 polypeptide (SEQ ID NO: 706), a condensation peptide from the second IDR of hnRNPA1 polypeptide (SEQ ID NO: 707), a condensation peptide from the third IDR of TAF15 polypeptide (SEQ ID NO: 712), a condensation peptide from the fourth IDR of TAF15 polypeptide (SEQ ID NO: 713), a condensation peptide from the first IDR of TDP43 polypeptide (SEQ ID NO: 718), a condensation peptide from the second IDR of TDP43 polypeptide (SEQ ID NO: 719), a 37 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 condensation peptide from the first IDR of DDX4 polypeptide (SEQ ID NO: 720), a condensation peptide from the first IDR of FUS polypeptide (SEQ ID NO: 725), a condensation peptide from the second IDR of FUS polypeptide (SEQ ID NO: 726), a condensation peptide from the third IDR of FUS polypeptide (SEQ ID NO: 736), a condensation peptide from the first IDR of DYRK3 polypeptide (SEQ ID NO: 737), a condensation peptide from the first IDR of CWC27 polypeptide (SEQ ID NO: 742), a condensation peptide from the second IDR of CWC27 polypeptide (SEQ ID NO: 747), a condensation peptide from the first IDR of SPAG7 polypeptide (SEQ ID NO: 748), a condensation peptide from the second IDR of SPAG7 polypeptide (SEQ ID NO: 749), a condensation peptide from the first IDR of RS10L polypeptide (SEQ ID NO: 754), a condensation peptide from the first IDR of RS4Y2 polypeptide (SEQ ID NO: 755), a condensation peptide from the first IDR of RBMY1D polypeptide (SEQ ID NO: 759), a condensation peptide from the second IDR of RBMY1D polypeptide (SEQ ID NO: 760), a condensation peptide from the first IDR of KHDC4 polypeptide (SEQ ID NO: 761), a condensation peptide from the second IDR of KHDC4 polypeptide (SEQ ID NO: 762), a condensation peptide from the third IDR of KHDC4 polypeptide (SEQ ID NO: 763), a condensation peptide from the first IDR of RP9 polypeptide (SEQ ID NO: 768), a condensation peptide from the second IDR of RP9 polypeptide (SEQ ID NO: 769), a condensation peptide from the first IDR of PHP14 polypeptide (SEQ ID NO: 770), a condensation peptide from the first IDR of LMOD1a polypeptide (SEQ ID NO: 771), a condensation peptide from the second IDR of LMOD1a polypeptide (SEQ ID NO: 776), a condensation peptide from the first IDR of H2A1H polypeptide (SEQ ID NO: 777), a condensation peptide from the first IDR of CWC25 polypeptide (SEQ ID NO: 778), a condensation peptide from the first IDR of RAD51AP1 polypeptide (SEQ ID NO: 779), a condensation peptide from the first IDR of SPA24 polypeptide (SEQ ID NO: 784), a condensation peptide from the first IDR of HSB1L polypeptide (SEQ ID NO: 785), a condensation peptide from the first IDR of H1T polypeptide (SEQ ID NO: 786), a condensation peptide from the first IDR of RAMAC polypeptide (SEQ ID NO: 787), a condensation peptide from the first IDR of AIMP1 polypeptide (SEQ ID NO: 792), a condensation peptide from the first IDR of MRPL1 polypeptide (SEQ ID NO: 793), a condensation peptide from the second IDR of RAD51AP1 polypeptide (SEQ ID NO: 794). For clarity, the sequence identifiers provided above are the predicted IDRs of the polypeptides. [0187] In some embodiments, a condensation peptide included in an engineered driver is capable of directly binding to the template RNA or to other condensation fused peptides. In 38 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 some embodiments, in site-specific transgene integration, condensation peptide fusion to an RTE polypeptide may improve integration at genomic sites that are homologous to sequences that are included on a template nucleic acid (for example sequences that flank the transgene and are homologous to one or more genomic sequences in the vicinity of the target gene, e.g., homology arms). In general, the transgene may be in sense or anti-sense orientation. [0188] Many RTE proteins have native IDRs. For example, as noted above, an R2 RTE proteins typically have a native IDR at their N-terminal region. In certain embodiments, the RTE polypeptide to which the heterologous IDR polypeptide is fused is an RTE polypeptide having a deletion of its native IDR or portion thereof. [0189] It will be appreciated that an engineered driver of the disclosure may comprise one or more, for example 1, 2, 3, 4, or 5, heterologous IDR polypeptides, optionally in combination with one or more other heterologous polypeptides as described herein. [0190] In certain embodiments, the engineered driver protein comprises an R2 RTE polypeptide fused to one or more heterologous IDR polypeptides. In certain embodiments, the engineered driver protein comprises an R2 RTE polypeptide fused to a heterologous IDR polypeptide. The fused heterologous IDR polypeptide may be situated internal to the R2 RTE protein, at the N- terminal end of the R2 RTE polypeptide or the C-terminal end of the R2 RTE polypeptide. SEQ ID NOs: 694-695, 701-705, 708-711, 714-717, 721- 724, 727-735, 738-741, 743-746 or 825-831 set forth amino acid sequences of exemplary driver proteins comprising a heterologous IDR polypeptide. [0191] In certain embodiments, the engineered driver protein comprises a non-R2 RTE polypeptide fused to one or more heterologous IDR polypeptides. In certain embodiments, the non-R2 RTE may be, e.g., Vingi RTE, R2NS RTE,_L4_ACar RTE, a CR1_10_Ami RTE, a ZFL2-2 RTE, or any other non-R2 RTE disclosed elsewhere herein. Heterologous EN – Chimeric drivers [0192] In certain embodiments, the at least one heterologous polypeptide fused to an RTE polypeptide comprises a heterologous EN domain polypeptide. As described herein, an RTE protein typically comprises an EN domain and an RT domain. As such, a heterologous EN domain polypeptide in the context of engineered drivers as described herein refers to an EN domain polypeptide of a protein that is not the RTE protein from which the RTE polypeptide (which may comprise the RT domain) of the engineered driver is derived. As such, the 39 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 heterologous EN domain may be a EN domain polypeptide of a different RTE protein, or a EN domain polypeptide of a non-RTE protein. [0193] For example, if a given engineered driver comprises an RTE polypeptide of an R2-1-TG RTE protein, then the heterologous EN domain may be a EN domain polypeptide of a different R2 protein (e.g., an R2-2-AcOr RTE protein or a R4-AC RTE protein). In such a case, the engineered driver would be a chimeric driver protein comprising EN and RT domains derived from different R2 proteins. [0194] In certain embodiments, if a given engineered driver comprises an RTE polypeptide of an R2 protein, then the heterologous EN domain polypeptide may be a EN domain polypeptide of a non-R2 RTE protein. In such a case, the engineered driver would be a chimeric driver protein comprising EN and RT domains derived from different RTE proteins. [0195] In certain embodiments, if a given engineered driver comprises an RTE polypeptide of an RTE (e.g. R2 RTE) protein, then the heterologous EN domain may be the EN domain of a non- RTE protein. In such a case, the engineered driver would be a chimeric driver protein comprising an EN domain from a non-RTE source. Examples of non-RTE proteins comprising a EN domain include but are not limited to Cas nucleases (e.g., SpCas9 nuclease), a homing endonuclease, or a FokI nuclease. Fanzor nuclease (e.g., SpuFz1) is a RNA-guided system (aka OMEGA). Like the Cas systems, it uses a guide RNA molecule to guide the binding and cleavage of specific and non-specific dsDNA sequences. In certain embodiments, the heterologous EN domain includes but is not limited to a Fanzor1 or Fanzor2 nuclease. Non- limiting examples of drivers utilizing a Fanzor1 or Fanzor2 nuclease have amino acid sequence set forth in any one of SEQ ID NOs: 473-484. [0196] In certain embodiments, the heterologous EN domain polypeptide may functionally replace the native EN activity of the RTE polypeptide of the engineered driver. In certain embodiments, the RTE polypeptide of the engineered driver may be lacking a functional EN domain, for example through deletion of the EN domain or portion thereof, and/or through one or more amino acid substitutions that reduce or eliminate enzymatic activity of the native EN domain of the RTE polypeptide. [0197] Examples of chimeric drivers with a heterologous EN domain polypeptide provided herein include at least the following: SEQ ID NO: 448 sets forth the amino acid sequence of a chimeric driver of different R2 proteins (R2-ZA and R2-TG), comprising an R2-TG RTE polypeptide and an R2-ZA EN domain (which replaced the R2-TG EN domain). SEQ ID NO: 40 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 473 sets forth the amino acid sequence of a SpuFz1 nuclease R2-TG fusion. The SpuFz1 portion is fused at its N-terminus via a linker to a C-terminal R2-TG protein. SEQ ID NO: 474 sets forth the amino acid sequence of a SpuFz1 nuclease R2-TG fusion. The SpuFz1 portion is fused at its N-terminus via a linker to a truncated C-terminal R2-TG protein, lacking first 209 amino acid residues. SEQ ID NO: 475 sets forth the amino acid sequence of a SpuFz1 nuclease R2-TG fusion. The SpuFz1 portion is fused at its N-terminus via a linker to a truncated C-terminal R2- TG protein, with native endonuclease domain deleted. SEQ ID NO: 476 sets forth the amino acid sequence of a SpuFz1 nuclease R2-TG fusion. The SpuFz1 portion is fused at its C- terminus via a linker to a truncated N-terminal R2-TG protein, with native endonuclease domain deleted. SEQ ID NO: 477 sets forth the amino acid sequence of a SpuFz1 nuclease R2-TG fusion. The SpuFz1 portion is fused at its C-terminus via a linker to an N-terminal R2-TG protein, carrying a K1307A mutation. SEQ ID NO: 478 sets forth the amino acid sequence of a dead SpuFz1 nuclease R2-TG fusion. The SpuFz1 carries a D606A mutation and is fused at the N-terminus fused via a linker to a C-terminal R2-TG protein. SEQ ID NO: 479 sets forth the amino acid sequence of a dead SpuFz1 nuclease R2-TG fusion. The SpuFz1 carries a D606A mutation and is fused at its N-terminus via a linker to a C-terminal R2-TG protein, lacking first 209 amino acid residues. SEQ ID NO: 480 sets forth the amino acid sequence of a SpuFz1 nuclease R2-TG fusion. The SpuFz1 is fused at its N-terminus via a linker to a C-terminal R2- TG protein (lacking 209 N-terminal amino acids. SEQ ID NO: 481 sets forth the amino acid sequence of a SpuFz1 nuclease R2-TG fusion. The SpuFz1 is fused at its N-terminus via a linker to a C-terminal R2-TG protein without the native DNA binding domain, and carrying K1307A mutation. SEQ ID NO: 482 sets forth the amino acid sequence of a SpuFz1 nuclease R2-TG fusion. The SpuFz1 is fused at its N-terminus via a linker to a C-terminal R2-TG protein carrying a K1307A mutation. SEQ ID NO: 483 sets forth the amino acid sequence of a SpuFz1 nuclease R2-TG fusion. The SpuFz1 is fused at the C-terminus via a linker to an N-terminal R2- TG protein (lacking 209 N-terminal amino acid residues). SEQ ID NO: 484 sets forth the amino acid sequence of a SpuFz1 nuclease R2-TG fusion. In each of the above, the SpuFz1 is fused at the C-terminus via a linker to a truncated N-terminal R2-TG protein. Heterologous RT domain [0198] In certain embodiments, the at least one heterologous polypeptide fused to an RTE polypeptide comprises a heterologous RT domain polypeptide. 41 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0199] As described herein, an RTE protein typically comprises an EN domain and an RT domain. As such, a heterologous RT domain polypeptide in the context of engineered drivers as described herein refers to an RT domain polypeptide of a protein that is not the RTE protein from which the engineered driver is derived. As such, the heterologous RT domain polypeptide may be an RT domain polypeptide of a different RTE protein, or an RT domain polypeptide of a non-RTE protein. [0200] For example, if a given engineered driver comprises an RTE polypeptide of an R2-1-TG RTE protein, then the heterologous RT domain polypeptide may be an RT domain polypeptide of a different R2 protein (e.g., an R2-2-AcOr RTE protein or a R4-AC RTE protein). In such a case, the engineered driver would be a chimeric driver protein comprising EN and RT domains derived from different R2 proteins. In certain embodiments, the RT domain polypeptide of a different R2 RTE protein may be derived from an R2-Toc RT domain, optionally comprising an amino acid sequence set forth in SEQ ID NO: 539, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, or at least 99% identical thereto. [0201] In certain embodiments, the heterologous RT domain polypeptide in an engineered driver may be an RT domain polypeptide of a non-R2 RTE protein. In such a case, the engineered driver would be a chimeric driver protein comprising EN and RT domains derived from different RTE proteins. [0202] In certain embodiments, the RT domain polypeptide of a non-R2 RTE protein may be from an LTR retroelement or an ERV. In certain embodiments, the RT domain polypeptide of a non-R2 RTE protein may be from a non-LTR retroelement. In certain embodiments, the RT domain polypeptide of a non-R2 RTE protein may be from a Vingi RTE. In certain embodiments, the RT domain polypeptide of a non-R2 RTE protein may be from an R2-NS RTE. In certain embodiments, the RT domain polypeptide of a non-R2 RTE protein may be from a L2_L4_ACar RTE. In certain embodiments, the RT domain polypeptide of a non-R2 RTE protein may be from a CR1_10_Ami RTE. In certain embodiments, the RT domain polypeptide of a non-R2 RTE protein may be from a ZFL2-2 RTE. [0203] In certain embodiments, if a given engineered driver comprises an RTE polypeptide of an RTE (e.g. R2 RTE) protein, then the heterologous RT domain polypeptide may be the RT domain polypeptide of a non-RTE protein. In such a case, the engineered driver would be a chimeric driver protein comprising an RT domain from a non-RTE source. In certain 42 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 embodiments, the RT domain comprises a sequence may be from a MMLV RT. Marathon RT, a TGIRT_III RT, or a SSIV RT. [0204] In certain embodiments, the heterologous RT domain polypeptide may functionally replace the native RT activity of the RTE polypeptide of the engineered driver. In certain embodiments, the RTE polypeptide of the engineered driver may be lacking a functional RT domain, for example through deletion of the RT domain or portion thereof, and/or through one or more amino acid substitutions that damage or eliminate enzymatic activity of the native RT domain of the RTE polypeptide. [0205] SEQ ID NO: 534 sets forth an amino acid sequence of a Vingi RT. SEQ ID NO: 535 sets forth amino acid sequence of an MMLV RT. SEQ ID NO: 536 sets forth amino acid sequence of an R2-NS RT. SEQ ID NO: 537 sets forth amino acid sequence of a L2_L4_ACar RT. SEQ ID NO: 538 sets forth amino acid sequence of a CR1-10_Ami RT. SEQ ID NO: 539 sets forth amino acid sequence of an R2 Toc RT. SEQ ID NO: 540 sets forth amino acid sequence of a ZFL2-2 RT. SEQ ID NO: 425 sets forth amino acid sequence of a Marathon RT. SEQ ID NO: 541 sets forth amino acid sequence of a TGIRT_III RT. SEQ ID NO: 542 sets forth amino acid sequence of an MMLV derived RT peptide. SEQ ID NO: 543 sets forth amino acid sequence of a SSIV RT. [0206] Examples of chimeric drivers with a heterologous RT domain polypeptide provided herein include at least the following: R2-TG chimera with Vingi RT (SEQ ID NO: 517), R2-TG chimera with MMLV RT (SEQ ID NO: 518), R2-TG chimera with R2NS RT (SEQ ID NO: 519), R2-TG chimera with L2_24_ACar-RT (SEQ ID NO: 520), R2-TG chimera with CR1- 10_Ami-RT (SEQ ID NO: 521), R2-TG chimera with R2Toc-RT (SEQ ID NO: 522), R2-TG chimera with ZFl2-2 RT (SEQ ID NO: 523), R2-TG chimera with Marathon RT (SEQ ID NO: 524), R2-TG chimera with TGIRT_III RT (SEQ ID NO: 525), R2-TG mutated RT with YADD RT motif (SEQ ID NO: 526), R2-TG with RT deletion (SEQ ID NO: 527), R2-TG internal fusion with MMLV RT derived peptide in the RT domain (SEQ ID NO: 528), R2-TG chimera with mutated R2-TG and MMLV RT sequence from SEQ ID NO: 528(SEQ ID NO: 529), R2- TG chimera with SSIV RT (SEQ ID NO: 530), mutated R2-TG fusion with SSIV RT (SEQ ID NO: 531), R2-TG internal fusion with N-terminal fused RNA binding domain (RBD) motif (SEQ ID NO: 532), R2-TG internal fusion of RBD motif (SEQ ID NO: 533). SEQ ID NO: 376 sets forth the amino acid sequence of a chimeric driver between different R2 proteins (R2-ZA 43 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 and R2-TG), comprising an R2-TG RTE polypeptide and an R2-ZA RT domain (which replaces the R2-TG RT domain). Specific combinations of heterologous polypeptides [0207] Certain combinations of heterologous polypeptides were found to provide unexpectedly advantageous properties in the resulting engineered drivers, for example as demonstrated in the Examples below. These advantageous combinations include, but are not limited to, an engineered driver fused to a CTIP polypeptide and an HMGN1 polypeptide, as well as an engineered driver, e.g., a driver comprising an R2 polypeptide, fused to a Sto7d polypeptide and an HMGN1 polypeptide. [0208] In certain embodiments, the engineered driver comprises an R2 polypeptide fused to a CTIP polypeptide and an HMGN1 polypeptide. In certain embodiments, the engineered driver comprises, in an N-terminal to C-terminal order, the CTIP polypeptide, the HMGN1 polypeptide, and the R2 polypeptide. In certain embodiments, the R2 polypeptide may be lacking a native N-terminal intrinsically disordered region. [0209] In certain embodiments, the engineered driver comprises an R2 polypeptide fused to a CTIP polypeptide, an HMGN1 polypeptide, and a heterologous intrinsic disordered region. In certain embodiments, the engineered driver comprises, in an N-terminal to C-terminal order, a heterologous intrinsic disordered region, a CTIP polypeptide, a HMGN1 polypeptide, and an R2 polypeptide. In certain embodiments, the R2 polypeptide may be lacking a native N-terminal intrinsically disordered region. By way of example, a driver polypeptide having the above-noted arrangement of components may comprise an amino acid sequence set forth in any one of SEQ ID NOs: 694, 695, 701, 702, 703, 704, 705, 708, 709, 710, 711, 714, 715, 716, 717, 721, 722, 723, 724, 727, 728, 729, 730, 731, 732, 733, 734, 735, 738, 739, 740, 741, 743, 744, 745 or 746 [0210] In certain embodiments, the engineered driver comprises an R2 polypeptide fused to a CTIP polypeptide, an HMGN1 polypeptide, and a RNA helicase polypeptide. In certain embodiments, the engineered driver comprises, in an N-terminal to C-terminal order, a RNA helicase polypeptide, a CTIP polypeptide, an HMGN1 polypeptide, and an R2 polypeptide. The RNA helicase polypeptide may be, e.g., an EI4A polypeptide or a Dengue virus RNA helicase polypeptide. In certain embodiments, the R2 polypeptide may comprises a deletion within its native N-terminal intrinsically disordered region. By way of example, a driver polypeptide 44 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 having the above-noted arrangement of components may comprise an amino acid sequence set forth in SEQ ID NO: 833. [0211] In certain embodiments, the engineered driver comprises an R2 polypeptide fused to a Sto7d polypeptide and an HMGN1 polypeptide. In certain embodiments, the engineered driver comprises, in an N-terminal to C-terminal order, the Sto7d polypeptide, the HMGN1 polypeptide, and an R2 polypeptide. In certain embodiments, the R2 polypeptide may be lacking a native N-terminal intrinsically disordered region. [0212] [0213] In certain embodiments, the engineered driver comprises an R2 polypeptide fused to a Sto7d polypeptide, an HMGN1 polypeptide, and a RNA helicase recruitment motif polypeptide. In certain embodiments, the engineered driver comprises, in an N-terminal to C-terminal order, the RNA helicase recruitment motif polypeptide, the Sto7d polypeptide, the HMGN1 polypeptide, and an R2 polypeptide. The RNA helicase recruitment motif polypeptide may be, e.g., a TUDOR domain polypeptide. In certain embodiments, the R2 polypeptide may comprise a deletion within its native N-terminal intrinsically disordered region. By way of example, an engineered driver having the above-noted arrangement of components may comprise an amino acid sequence set forth in SEQ ID NO: 1232, which may be encoded by a nucleotide sequence set forth in SEQ ID NO: 3330 (exemplary IVT sequence set forth in SEQ ID NO: 3329). The engineered driver having the amino acid set forth in SEQ ID NO:1232 comprises: an amino acid sequence of R2-1_TG Orf2 polypeptide with N-terminal deletion of residues 1-184; N-terminal fusion of the following: SV40-NLS polypeptide SEQ ID NO:430, TUDOR polypeptide SEQ ID NO:3269, sto7D polypeptide SEQ ID NO:405, and HMGN polypeptide SEQ ID NO:77; and amino acid substitutions P280K, D555K, T1011S, I1219N, A1282G. IVT nucleotide sequence SEQ ID NO:3329, which encodes the SEQ ID NO:1232 amino acid sequence, includes the following: synthetic 5' UTR SEQ ID NO:80; mouse alpha globin 3'UTR SEQ ID NO:83; and A29N10A70 polyA signal SEQ ID NO:3201. The orientation of the sequences between external UTRs is in antisense. [0214] In some embodiments, the engineered driver comprises an R2 polypeptide fused to a Sto7d polypeptide and an HMGN1 polypeptide. SEQ ID NO:1690 sets forth the amino acid sequence of an engineered driver comprising: an R2-1_TG Orf2 polypeptide with N-terminal deletion of residues 1-184; N-terminal fusion of the following: SV40-NLS polypeptide SEQ ID NO:430, Sto7D polypeptide SEQ ID NO:405, and HMGN polypeptide SEQ ID NO:77; and 45 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 amino acid substitutions P280K, D555K, D923K, T1011S, I1219N, A1282G. SEQ ID NO:3009 sets forth a nucleotide sequence encoding the SEQ ID NO:1690 protein, and includes synthetic 5' UTR SEQ ID NO:80; mouse alpha globin 3'UTR SEQ ID NO:83; and A29N10A70 polyA signal SEQ ID NO:3201. The orientation of the sequences between external UTRs is in antisense. [0215] SEQ ID NO: 642 is the amino acid sequence of an exemplary R2 driver comprising: R2- 1_TG polypeptide with deletion of N-terminal residues 1-184; N-terminal fusion of the following: SV40-NLS polypeptide SEQ ID NO:3199, sto7D polypeptide SEQ ID NO:405, HMGN polypeptide SEQ ID NO:77; and amino acid substitutions P280K, D555K, T1011S, I1219N, A1282G. SEQ ID NO:3299 sets forth a nucleotide sequence encoding the SEQ ID NO:642 protein, and includes synthetic 5' UTR SEQ ID NO:80 ; mouse alpha globin 3'UTR SEQ ID NO:83; and A29N10A70 polyA signal SEQ ID NO:3201. The orientation of the sequences between external UTRs is in antisense. Modifications made to RTE polypeptides [0216] In certain embodiments, an RTE polypeptide comprises at least one amino acid substitution, at least one amino acid deletion, at least one amino acid addition, or a combination thereof, when compared to a wildtype (WT) RTE protein. [0217] In certain embodiments, the RTE polypeptide may have least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the WT RTE protein. Amino acid substitution [0218] In certain embodiments, an RTE polypeptide comprises at least one amino acid substitution, relative to the corresponding WT RTE protein. [0219] In certain embodiments, an amino acid substitution (e.g., in an RT domain or an EN domain) is a stabilizing amino acid substitution. [0220] In certain embodiments, the at least one amino acid substitution is relative to SEQ ID NO: 75. In certain embodiments, the at least one amino acid substitution corresponds to at least 46 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 one substitution (e.g., 1, 2, 3, 4, 5, etc. substitutions) selected from E399M, Y477H, D555K, W762R, R822K, H1090Y, F956I, P78V, W677C, K314R, K385M, P437M, T448Y, C518Y, T305R, P280K, N269R, L213K, E214K, Q215K, E214R, T1011R, K547R, C145G, I1219N, G257M, K700D, F148K, F667G, and F667Y relative to SEQ ID NO: 75. In various embodiments, the at least one amino acid substitution corresponds to at least one substitution (e.g., 1, 2, 3, 4, 5, etc. substitutions) selected from E399M, Y477H, D555K, W762R, R822K, H1090Y, F956I, P78V, W677C, K314R, K385M, P437M, T448Y, C518Y, T305R, P280K, N269R, L213K, E214K, Q215K, E214R, and T1011R relative to SEQ ID NO: 75. In certain embodiments, the at least one amino acid substitution is a substitution corresponds to at least one of positions F148, K700, F667, C145, P280, D555, T1011 relative to SEQ ID NO: 75. In certain embodiments, the at least one amino acid substitution may be a substitution corresponding to position F148 relative to SEQ ID NO: 75. In certain embodiments, the at least one amino acid substitution may be a substitution corresponding to position K700 relative to SEQ ID NO: 75. In certain embodiments, the at least one amino acid substitution may be a substitution corresponding to position F667 relative to SEQ ID NO: 75. In certain embodiments, the at least one amino acid substitution may be a substitution corresponding to position P280 relative to SEQ ID NO: 75. In certain embodiments, the at least one amino acid substitution may be a substitution corresponding to position D555 relative to SEQ ID NO: 75. In certain embodiments, the at least one amino acid substitution may be a substitution corresponding to position T1011 relative to SEQ ID NO: 75. In certain embodiments, the at least one amino acid substitution may be a substitution corresponding to position K700 relative to SEQ ID NO: 75. In certain embodiments, the retroelement polypeptide is an R2-TG polypeptide, which may, e.g., have an amino acid sequence set forth in any one of SEQ ID NOs: 490, 491, 492, 493, 494, 495, 496, 497 or 498. [0221] In certain embodiments, an RT domain of the RTE polypeptide is a variant RT domain, relative to a natively occurring unsubstituted RT domain. [0222] In certain embodiments, the at least one (in some embodiments 1, 2, 3, etc.) amino acid substitution may be selected from: L18P, E19L, S26N, V27R, A29T, I30A, R32G, N35I, S36R, L37K, A38R, L49F, G209R, K289R, L316H, E399M, E472G, Y477H, T514R, D537Y, T538K, H544Q, D555K, N562D, W762R, R822K, S895K, Q909N, R964K, G966N, T989Y, I1003M, T1011S, A1012G, L1013R, T1016L, Q1019R, E1027D, P1033R, C1079K, K1085R, L1089R, H1090Y, I1219N, A1282G, T989I, K579R, K547R, V660I, T608P, F956I, I1003L, T1016S, 47 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 T680S, L594P, P52R, P418S, P78G, L51G, P78V, P180S, V539G, P180K, P437R, V539R, N640A, W677C, V687W, R866I, A747G, W762E, T908I, W959K, D1037N, Y1194L, R1292P, K1210R, K993R, K755R, C518R, K314R, K1182R, K385M, P437M, T448Y, C518Y, E1008R, T305R, P280K, A1012R, N269R, L213K, E214K, Q215K, E214R, T1011R, E1015R, L213R, G257M, G209F, W762D, W959K, T908I, Y579R, T1011A, Y571H, E630D, D1075E, L1089M, A442P, H423Q, E173D, P188L, C145G, N301G, R355K, G257R, V251G, T603A, N363H, P1033S, F667Y, F1365L, V501L, G257R+C145G, W762R+C145G, G257R+E173D, N363H+E173D, A442P+E173D, W762R+E173D, G257R+P188L, W762R+P188L, G257R+V251G, S895K+V251G, N363H+G257R, R568S, E542K, P985R, G561R, Y563R, L1184K, H1030R, A557R, I558R, Q567R, N271W, E350R, L316R, F304K, D923K, A317W, E1010R, Q1021R, E419R, T1011F, E769R, W345R, V110R, R568G, F135G, N104Y, V142Y, P115K, A111Y, L129R, V98R, D126R, E144R, Q112R, Y136R, F148K, T1016K, K700D, E188D, E188A, R435K, L438A, A1191L, P1195G, P1195G, R1197K, L1200I, G1331A, G1335A, P593G, S595V, E596S, S599V, P593A, S595A, E596A, S599A, K612D, D616K, K612A, D616A, K856D, D6860K, K856A, D6860A, N891S, T892V, S895V, N891A, T892A, S895A, E888S, E898S, T899V, N902S, E888A, E898A, T899A, N902A, T934V, G937P, P939G, T934A, G937A, K1252D, D1255K, W1256G, K1252A, D1255A, W1256A, A877V, A877I, A877M, F876Y, A877H, Q955R, L952I, T989L, E947S, G658R, Q642R, F973R, G647T, D990V, D990R, N941K, V727L, M874L, D227W, D227F, C344V, R669V, V600T, R866L, T989V, M449E, T679L, Y478L, M671S, F956V, M686S, W1126P, T1367R, C404S, E1363K, F973Q, M420T, R669F, G689E, N1219L, K480R, K508Q, E812K, V1368R, R1157M, G217P, H1320E, N891K, K1269G, K1269G, D1255G, T670L, T670L, H1180Q, K907R, T996A, I778F, R574G, K1151H, E194G, R574S, D1288A, F186G, T969E, G432Q, D591Q, M553R, T174K, W1028K, D534Y, D591W, D972R, W1028Y, D972Q, P169R, L975R, C587K, I978K, D591R, D182R, D972M, A1024K, C170K, P985K, G172R, S178R, G172K, S177R, A166R, S177W, P548R, S204R, H544R, G575H, F1135T, N1123K, P1116D, P998H, S645Q, P998Q, D879A, D879M, K576M, D879W, E643R, T994W, N1140R, D1133R, D583R, M644R, G575R, A1128R, A649R, E1125R, F1135Y, C750K, P1129R, F1135R, L1057R, H1006K, L582K, D573R, T994R, D878R, A578K, D839R, G311S, T532K, T728K, P1116K, L243K, E247K, L249F, K255R, V258I, F260Y, Y272H, E286K, P291A, D303S, I308R, I327L, K333R, G339R, I370L, I374L, K377R, S383K, L474I, T481I, L482I, F498L, M502L, Q516R, F519I, G520K, C521L, S526K, D534K, L572K, S589R, F611Y, P545K, F549R, L564R, 48 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 L569R, F570Y, D573N, K377R, L582I, I585K, I597V, F601Y, F628L, L631P, V641L, A649S, P650A, V661K, E666K, E672R, F674Y, W677I, V687L, C690S, N738H, Q741R, L754I, A764S, H768R, L771I, V776I, F846Y, L848I, I896L, W933F, Y995F, A1038G, A1063L, I778F, V785I, L794M, E798G, K821G, K821R, V835I, L844I, L854I, C855R, H865K, C901L, D929N, A932P, M942L, K950R, Y951H, D957N, S965P, T969E, P998S, Y1002H, L1014I, T1023K, E1027K, Y1041H, S1043R, K1053R, V1061R, Q1062R, T1130K, Q1173R, Q1233K, F1278L, V1287I, V1291I, E1317Q, V1318I, M1361L, E1363K, R1064K, K1078R, F1080L, E1082K, K1092R, G1098R, E1127K, E1127Q, K1132R, Q1155H, F1162Y, W1171F, L1184I, Q1188K, C1215Y, N1228Q, E1249K, V1257T, H1313P, F1345N, K1355R, K1386R, V727M, T759G, Q795R, I814C, T819E, H825K, Q868G, Q868D, A931P, D944G, F973S, L175S, A1012S, I683L, S699G, I707P, W710Y, T714A, I718T, I726V, N813G, V881T, L906M, S648K, S699K, S717P, A748R, D800P, V239M, E334A, T532G, K622Q, T1367R, H1378R, F1380Y, Y817R, Q911K, Q911S, L657Y, L720Y, A779S, K780N, D199K, A779K, D783G, D851N, E949Y, E949Y, V299E, E949Y, G520A, D591G, S599D, T1122L, L1285I, N1103D, P739R, E638L, N889G, Y1242K, E400M, D1133P, S645K, V639T, I745R, G772A, A747S, A748P, I778F, M841L, M850L, E949L, F983E, R669L, L213K, L564Q, F973E, R669I, F667G, and F973E, or combinations thereof, relative to SEQ ID NO: 75. [0223] In certain embodiments, the at least one (in some embodiments 1, 2, 3, etc.) amino acid substitution may be selected from E399M, Y477H, D555K, W762R, R822K, H1090Y, F956I, P78V, W677C, K314R, K385M, P437M, T448Y, C518Y, T305R, P280K, N269R, L213K, E214K, Q215K, T1011R, R435K, L438A, R1197K, G1331A, T989L, Q955R, L952I, E947S, Q642R, G647T, N941K, V727L, M874L, D227W, D227F, C344V, R669V, V600T, T989V, M449E, T679L, Y478L, M671S, F956V, M686S, W1126P, T1367R, C404S, E1363K, K508Q, E812K, V1368R, R1157M, N1123K, P1116D, P998H, S645Q, P998Q, N1123K, P1116D, P998H, S645Q, P998Q, D879A, D879M, K576M, G311S, T532K, L572K, S589R, F611Y, F846Y, L848I, I896L, W933F, Y995F, A1038G, A1063L, T1130K, Q1173R, Q1233K, F1278L, V1287I, V1291I, E1317Q, V1318I, M1361L, E1363K, K1386R, V727M, T759G, Q795R, I814C, T819E, H825K, Q868G, Q868D, A931P, D944G, F973S, L175S, A1012S, I683L, S699G, I707P, W710Y, T714A, I718T, I726V, N813G, V881T, L906M, S648K, S699K, S717P, A748R, D800P, V239M, E334A, T532G, K622Q, D923K, P739R, E638L, N889G, Y1242K, E400M, T1011S, T1011A, I1219N, A1282G, L213R, A1012G, E542K, G257R, C145G, L316R, D537Y, D1133P, and K1085R, or combinations thereof, relative to SEQ ID NO: 75. 49 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0224] In certain embodiments, the at least one (in some embodiments 1, 2, 3, etc.) amino acid substitution may be selected from E399M, Y477H, D555K, W762R, R822K, H1090Y, F956I, P78V, W677C, K314R, K385M, P437M, T448Y, C518Y, T305R, P280K, N269R, L213K, E214K, Q215K, E214R, T1011R, K547R, C145G, I1219N, G257M, K700D, F148K, F667G, and F667Y, or combinations thereof, relative to SEQ ID NO: 75. [0225] In certain embodiments, the at least one amino acid substitution comprises a substitution corresponding to at least one ((in some embodiments 1, 2, 3, etc.) of positions F148, K700, F667, C145, P280, D555, T1011, I1219, and A1282 relative to SEQ ID NO: 75. [0226] In certain embodiments, the at least one amino acid substitution comprises a substitution corresponding to a substitution in position P280 relative to SEQ ID NO: 75. The substitution may be selected from, for example, P280K, P280R, P280Q, P280N, and P280H. [0227] In certain embodiments, the at least one amino acid substitution comprises a substitution corresponding to a substitution in position D555 relative to SEQ ID NO: 75. The substitution may be selected from, for example, D555K, D555R, D555Q, D555A, D555H, D555S, D555C, D555Y, D555W, D555F, D555L, D555V, D555M, and D555I relative to SEQ ID NO: 75. In certain embodiments, the at least one amino acid substitution comprises the substitution of D555K relative to SEQ ID NO: 75. SEQ ID NO: 490 sets forth the mutated amino acid sequence of an exemplary R2-TG driver with the D555K amino acid substitution. [0228] In certain embodiments, the at least one amino acid substitution comprises a substitution corresponding to a substitution in position T1011. The substitution may be selected from, for example, to T1011R, T1011A, T1011K, T1011S, T1011F, T1011Y, T1011W, and T1011H relative to SEQ ID NO: 75. In certain embodiments the amino acid substitution is in position F148. The substituted amino acid may be selected from, for example, F148R, F148A, F148K, F148S, F148F, F148Y, F148W, F148H. In certain embodiments the amino acid substitution is in position K700. The substituted amino acid may be selected from, for example, K700D, K700E, K700S, K700Q, K700T, K700R, K700H. [0229] In certain embodiments, the at least one amino acid substitution comprises at least the following substitutions: P280K, D555K, T1011S, I1219N, and A1282G relative to SEQ ID NO: 75. Examples of engineered drivers comprising the P280K, D555K, T1011S, I1219N, and A1282G substitutions relative to SEQ ID NO: 75 are provided in SEQ ID NOs: 82, 642, 1232, 833 or 1690. 50 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0230] In certain embodiments, the at least one (e.g.1, 2, 3, 4, 5, etc.) amino acid substitution comprises one or more of the following substitutions: G257R, P280K, N363H, E399M, V539G, H423Q, A442P, D537Y, K547R, D555K, R669L, N889G, F973E, T1011A, Q1019R, D1133P, K1085R, R1177G, Y1242K, and A1282G, or combinations thereof, relative to SEQ ID NO: 75. In certain embodiments, the at least one amino acid substitution comprises at least the following substitutions: G257R, P280K, N363H, E399M, V539G, H423Q, A442P, D537Y, K547R, D555K, R669L, N889G, F973E, T1011A, Q1019R, D1133P, K1085R, R1177G, and Y1242K, A1282G relative to SEQ ID NO: 75. An example of an engineered driver comprising the substitutions of G257R, P280K, N363H, E399M, V539G, H423Q, A442P, D537Y, K547R, D555K, R669L, N889G, F973E, T1011A, Q1019R, D1133P, K1085R, R1177G, Y1242K, and A1282G, relative to SEQ ID NO: 75 is provided in SEQ ID NO: 681. [0231] Examples of engineered drivers comprising at least one amino acid substitution relative to SEQ ID NO: 75 are provided in SEQ ID NOs: 490, 491, 492, 493, 494, 495, 496, 497, 498, 681 or 1301. [0232] In certain embodiments, the at least one amino acid substitution is relative to SEQ ID NO: 373. [0233] In certain embodiments, the at least one (in some embodiments 1, 2, 3, etc.) amino acid substitution may be selected from G1057D, S931R, A289P, V996S, L1100F, M287A, C482S, M685L, S547P, V916I, A1256E, M1259A, N332G, M358L, Y919P, T950A, M376P, P151K, D584L, Y1162Q, M640S, C314L, R1131G, A965S, Y1044L, D1236G, A970L, Q1039R, C474Y, and T404, or combinations thereof, relative to SEQ ID NO: 373. [0234] In certain embodiments, the at least one amino acid substitution comprises a substitution corresponding to at least one of positions S931, Q1039 or M287 relative to SEQ ID NO: 373. Amino acid deletion in RTE protein [0235] In certain embodiments, the RTE polypeptide comprises at least one amino acid deletion. The deletion can be a deletion of individual amino acids, or a longer segment, including deletion of functional domains, binding domains, motifs, protein regions, or portions thereof. Segments of an RTE protein that may be deleted in an RTE polypeptide may be, an RT domain, EN domain, a DNA binding domain, a RNA binding domain, a PIP domain, an intrinsically disordered region (IDR), or combinations thereof. 51 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 Deletion of N-terminal intrinsically disordered region [0236] The N-terminal region of some RTE proteins, e.g., R2 proteins encoded in Group 2 RTE, contain intrinsically disordered regions (IDRs), which appear to lack a fixed three-dimensional (3D) structure. These IDRs are thought to provide flexibility, allowing the protein to have ordered interactions with multiple molecular partners within the intracellular environment. In certain embodiments, an RTE polypeptide comprised in an engineered driver protein may comprise a deletion of a N-terminal intrinsically disordered region (IDR) or a portion thereof with respect to the WT RTE protein (e.g., a WT R2 RTE protein). [0237] In certain embodiments, the deletion is a deletion of between about 10 amino acids and about 300 amino acids, between about 20 amino acids and about 300 amino acids, between about 40 amino acids and about 300 amino acids, between about 50 amino acids and about 300 amino acids, between about 60 amino acids and about 300 amino acids, between about 80 amino acids and about 300 amino acids, between about 90 amino acids and about 300 amino acids, between about 100 amino acids and about 300 amino acids, between about 120 amino acids and about 300 amino acids, between about 140 amino acids and about 300 amino acids, between about 150 amino acids and about 300 amino acids, between about 160 amino acids and about 300 amino acids, between about 180 amino acids and about 300 amino acids, between about 200 amino acids and about 300 amino acids, between about 220 amino acids and about 300 amino acids, between about 250 amino acids and about 300 amino acids, between about 10 amino acids and about 250 amino acids, between about 20 amino acids and about 250 amino acids, between about 40 amino acids and about 250 amino acids, between about 50 amino acids and about 250 amino acids, between about 60 amino acids and about 250 amino acids, between about 80 amino acids and about 250 amino acids, between about 90 amino acids and about 250 amino acids, between about 100 amino acids and about 250 amino acids, between about 120 amino acids and about 250 amino acids, between about 140 amino acids and about 250 amino acids, between about 150 amino acids and about 250 amino acids, between about 160 amino acids and about 250 amino acids, between about 180 amino acids and about 250 amino acids, between about 200 amino acids and about 250 amino acids, between about 220 amino acids and about 250 amino acids, between about 10 amino acids and about 200 amino acids, between about 20 amino acids and about 200 amino acids, between about 40 amino acids and about 200 amino acids, between about 50 amino acids and about 200 amino acids, between about 60 amino acids and about 200 amino acids, between about 80 amino acids and about 200 amino acids, between about 90 52 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 amino acids and about 200 amino acids, between about 100 amino acids and about 200 amino acids, between about 120 amino acids and about 200 amino acids, between about 140 amino acids and about 200 amino acids, between about 150 amino acids and about 200 amino acids, between about 160 amino acids and about 200 amino acids, between about 180 amino acids and about 250 amino acids, between about 10 amino acids and about 150 amino acids, between about 20 amino acids and about 150 amino acids, between about 40 amino acids and about 150 amino acids, between about 50 amino acids and about 150 amino acids, between about 60 amino acids and about 150 amino acids, between about 80 amino acids and about 150 amino acids, between about 90 amino acids and about 150 amino acids, between about 100 amino acids and about 150 amino acids, between about 120 amino acids and about 150 amino acids, between about 140 amino acids and about 150 amino acids, between about 150 amino acids and about 150 amino acids, between about 160 amino acids and about 150 amino acids, between about 180 amino acids and about 250 amino acids, between about 160 amino acids and about 240 amino acids, about 161 amino acids, about 184 amino acids, and about 184 amino acids, within the N-terminal intrinsically disordered region of the RTE protein, or at the N-terminus of the RTE protein. In certain embodiments, the deletion is truncation from the N-terminus of the RTE protein. In certain embodiments, the RTE protein is an R2 protein. In certain embodiments, the deletion of the N-terminal IDR is relative to a WT R2-TG protein having an amino acid sequence set forth in SEQ ID NO: 75. [0238] In certain embodiments, the engineered driver protein comprises an R2 RTE polypeptide comprising a deletion of a N-terminal intrinsically disordered region (IDR) or a portion thereof of the corresponding WT RTE protein. SEQ ID NOs: 694, 695, 701- 705, 708- 711, 714- 717, 721-724, 727- 735, 738- 741, 743- 746, 825- 831 set forth amino acid sequences of exemplary driver proteins comprising an R2 RTE polypeptide comprising a deletion of a N-terminal intrinsically disordered region (IDR) or a portion thereof. Exemplary engineered drivers [0239] In certain embodiments, an engineered driver comprises one or more domains provided in the Examples. In certain embodiments, an engineered driver comprises or has an amino acid sequence of any one of SEQ ID NOs: 75–86, 169–230, 277–384, 418–533, 539–555, 587–621, 631–834, or 838–1179, or an amino acid sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% 53 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical) thereto. In certain embodiments, an engineered driver is encoded by a DNA sequence (or a corresponding RNA version thereof) set forth in any one of SEQ ID NOs: 24–37, 76–128, 144–372, 559–630, 1692–1929, 1989–2342, 2381–2578, or 2580–3011, or, a DNA sequence (or a corresponding RNA version thereof) that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical) thereto. [0240] The above-noted amino acid and nucleotide sequences correspond to each other as follows: the engineered driver having the sequence set forth in SEQ ID NO: 436 is encoded by a DNA having the sequence set forth in SEQ ID Ns: 2381 (or by a corresponding RNA version thereof). In another example, the engineered driver having the sequence set forth in SEQ ID NO: 1682 is encoded by a DNA having the sequence set forth in SEQ ID NO: 3003 (or by a corresponding RNA version thereof). [0241] In certain embodiments, an engineered driver has an amino acid sequence of SEQ ID NO: 82 (encoded by, e.g., a nucleic acid sequence set forth in SEQ ID NO: 2506 or an IVT nucleic acid sequence set forth in SEQ ID NO: 3328). In certain embodiments, an engineered driver has an amino acid sequence of SEQ ID NO: 631 (encoded by, e.g., a nucleic acid sequence set forth in SEQ ID NO: 1799). In certain embodiments, an engineered driver has an amino acid sequence of SEQ ID NO: 642 (encoded by, e.g., a nucleic acid sequence set forth in SEQ ID NO: 2496, or an IVT nucleic acid sequence set forth in SEQ ID NO: 3203). In certain embodiments, an engineered driver has an amino acid sequence of SEQ ID NO: 642 (encoded by, e.g., a nucleic acid sequence set forth in SEQ ID NO: 2603 or an IVT nucleic acid sequence set forth in SEQ ID NO: 3299, codon optimized). In certain embodiments, an engineered driver has an amino acid sequence of SEQ ID NO: 1232 (encoded by, e.g., a nucleic acid sequence set forth in SEQ ID NO: 3330, or an IVT nucleic acid sequence set forth in SEQ ID NO: 3329). In certain embodiments, an engineered driver has an amino acid sequence of SEQ ID NO: 681 (encoded by, e.g., a nucleic acid sequence set forth in SEQ ID NO: 1791 or an IVT nucleic acid sequence set forth in SEQ ID NO: 3265). In certain embodiments, an engineered driver has an amino acid sequence of SEQ ID NO: 833 (encoded by, e.g., a nucleic acid sequence set forth in 54 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 SEQ ID NO: 2576). In certain embodiments, an engineered driver has an amino acid sequence of SEQ ID NO: 1690 (encoded by, e.g., an IVT nucleic acid sequence set forth in SEQ ID NO: 3009). In certain embodiments, an engineered driver is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical) to any one of the aforementioned amino acid or DNA sequences. [0242] The nucleic acids and polypeptides disclosed herein may be produced by methods known in the art. For example, the DNA or RNA molecules disclosed herein may be prepared synthetically via methods known in the art, or in the case of RNA, via in vitro transcription (IVT) methods known in the art. Likewise, the engineered driver and components thereof, e.g. RTE polypeptides, heterologous polypeptides, and other polypeptides and proteins described herein, may be produced via recombinant protein expression and purification, which is well suited for, e.g., fusion proteins. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference. [0243] The terms “polynucleotide,” “nucleic acid” and “NA” may be used interchangeably and refer to a polymer of nucleotides. In certain embodiments, the polynucleotide comprises one or more chemical and/or sequence modifications. The polymer may include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine and deoxycytidine), and nucleoside analogs having modified bases, modified sugars (e.g., 2′-fluororibose, 2′-methoxy), or modified phosphate groups (e.g., phosphorothioates, 2’-5’ linkage). In certain embodiments, a nucleic acid comprises one or more chemical and/or sequence modifications. In certain embodiments, the modification is an RNA cap, a modified polyA (e.g., relative to a natural polyA), a chemically modified nucleotide, a 5’ UTR (untranslated region) modification, a 3’ UTR modification, a modified Sirloin (SINE- derived nuclear RNA localization) sequence, a modified (e.g., truncated) stem loop, an RNA stabilization motif (which may be a WPRE motif), a 5-methoxyuridine (5-MO-U) modification, a 5-methylcytidine (5mC) modification, or one or more additional and/or modified microsatellites. In certain embodiments, a nucleic acid is sequence optimized (e.g., codon optimized) to enhance, for example, expression, reverse transcription, or transgene function. In 55 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 certain embodiments, RNA sequence optimization comprises one or more of the following modifications compared to the starting RNA molecule: reducing the uracil (U) load of an RNA molecule; reducing the GC% content of an RNA molecule; reducing the length and/or number of intron sequences of an RNA molecule; reducing RNA binding motifs or sites within an RNA molecule; lowering ΔG (free energy) of an RNA molecule; reducing the nucleotide repeats found in a sequence of an RNA molecule; adjusting the frequency of usage of particular codons in an RNA molecule; reducing the number of palindromic sequences in an RNA molecule; maximizing pairing of bases of an in an RNA molecule; removing splicing site sequences from an RNA molecule; removing rare codons or other slowly translated codons. A person with skill in the art will be able to generate a polynucleotide encoding any one of the polypeptides disclosed herein, based on the polypeptide sequence, as provided herein. [0244] In certain embodiments, an RNA driver nucleic acid encoding an engineered driver may include an RNA pseudoknot at or near the 5’ and/or 3’ end of the nucleic acid. An RNA pseudoknot is a structural RNA motif where bases in a loop pair with a sequence outside the loop, forming intertwined stem-loops. In certain embodiments, the driver nucleic acid comprises an RNA pseudoknot. In certain embodiments, the template nucleic acid comprises an RNA pseudoknot. In certain embodiments, the pseudoknot is a EvopreQ1 pseudoknot (e.g., SEQ ID NO: 1926). In certain embodiments, the pseudoknot is a Mpknot1 pseudoknot(e.g., SEQ ID NO: 1927). In certain embodiments, the pseudoknot is a MALAT1 pseudoknot (e.g., SEQ ID NO: 1928). T cell Specific Elements [0245] As provided herein, the driver nucleic acids of the disclosure are provided to mediate the integration of a DNA copy of a template into the genome of a T cell, whether provided in cis or in trans. Accordingly, the disclosure provides elements and methods to enhance T cell specificity. For example, provided herein are driver nucleic acids with binding sites for differentially expressed microRNAs (miRs), e.g. miRs that can prevent integration or expression in undesired cell types and can increase integration in T cells. [0246] Therefore, without being limited by theory, altering T cells to express transgenes using RTEs with use of miRs enables safe and potent immunotherapy for cancer, autoimmunity, and other conditions. In some embodiments, the T cell specific element is one or more miR binding site(s) that do not express in T cells but do express in other cells in which expression is not 56 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 desired. In some embodiments, the binding of miRs to an mRNA leads to degradation and is used to knock-down transgene expression. [0247] In some embodiments, the differentially expressed miR is miR-122. In some embodiments the differentially expressed miR is derived from Ludwig et al. Nucleic Acids Research, 44(8): 3865– 3877 (2016), the distribution of miRNAs in human tissues and is presented in, and downloadable from the Human miRNA tissue atlas each of which is by reference the present disclosure. Template nucleic acids [0248] A “template nucleic acid” (or “template”) comprises a sequence of interest destined for integration into a genome, and one or more sequence elements (e.g., RTE-UTRs described in more detail herein below) that interact with a driver, e.g., an engineered driver of the present disclosure. The sequence of interest may be referred to as a transgene or a gene of interest (“GOI”), that encodes an RNA or protein (e.g., a therapeutic RNA or protein). In some instances, the template nucleic acid may be referred to herein as a “reporter”, “reporter nucleic acid” a “reporter construct”, a “gene delivery construct”, or a “template construct”. [0249] It is noted that the driver nucleic acid may be DNA, RNA (e.g. mRNA), or a hybrid RNA/DNA molecule. Likewise, the template nucleic acid may be DNA or RNA (e.g. mRNA), or a hybrid RNA/DNA molecule. In exemplary embodiments, the driver nucleic acid and template nucleic acid are provided in trans, as separate RNA molecules, DNA molecules, or separate RNA/DNA molecules. In other certain embodiments, the driver nucleic acid and template nucleic acid are in a cis configuration and provided in a single nucleic acid. In certain embodiments, they are provided in cis as DNA. In certain embodiments, they are provided in cis as RNA. In certain embodiments, they are provided in cis as chimeric RNA/DNA. [0250] For clarity, in a trans configuration, the template includes the transgene that may be flanked, on each side independently, with a terminal region comprising one or more functional elements (e.g. RTE-UTRs, homology arms, etc.). In a cis configuration, the template nucleic acid portion includes an adjacent driver nucleic acid. The combined nucleic acid in cis configuration may be flanked, on each side independently, with a terminal region comprising one or more functional elements (e.g. RTE-UTRs, homology arms, etc.). 57 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0251] In certain embodiments, the driver nucleic acid and the template can be provided as DNA, as RNA, or as a mixture of both. It is contemplated in other embodiments that the driver nucleic acid and the template can be provided as DNA, as RNA (e.g. as mRNA), or as a mixture of both encapsulated in a carrier, e.g. in a lipid nanoparticle (LNP). In yet other embodiments, the driver nucleic acid and the template can be provided in a delivery vector, e.g. a viral vector. These delivery formats are discussed in greater detail herein. RTE-UTRs [0252] As described elsewhere herein, an RTE transcript of a natural RTE comprises one or more retrotransposable element untranslated regions (RTE-UTRs) that, as RNA, forms a structure that is capable of being bound by the RTE protein (e.g., by a compatible RNA binding domain comprised in the RTE protein), as a pre-requisite to achieve the reverse transcription of the RTE transcript by the RTE protein. Whether in cis or trans configurations, the template 5’ terminal region and the 3’ terminal region may comprise, independently, at least one RTE-UTR that is capable (e.g., as RNA) of binding an RTE polypeptide comprised in a driver encoded by the driver nucleic acid. [0253] As noted elsewhere, the RTE protein and the one or more RTE-UTRs of a given RTE typically have co-evolved to have sufficient binding affinity for each other. As such, in certain embodiments, the one or more RTE-UTRs comprised the template may be derived from the same RTE as the RTE polypeptide comprised in the driver. However, it has also been found that certain heterologous pairs RTE-UTRs and RTE polypeptides, where the RTE-UTRs and the RTE polypeptides are derived from different RTEs (e.g., different species of R2 RTEs) have sufficient affinity to enable productive interaction of the template and the driver. Some heterologous pairings may be even more effective in facilitating transgene integration that homologous pairing. For example, in a system comprising an R2-1_TG driver nucleic acid encoding an engineered driver comprising an R2-1_TG RTE polypeptide, a template nucleic acid (an R2-1-PTT reporter) comprising a 5’ RTE-UTR (SEQ ID NOs: 150) and a 3’ RTE-UTR (SEQ ID NOs: 126) from R2-1-Phylloscopus trochilus trochilus (PTT), was more effective in facilitating transgene integration than another template nucleic acid comprising an RTE-UTR derived from R2-1-TG. As such, in certain embodiments, the one or more RTE-UTRs comprising the template may be derived from a different RTE as the RTE polypeptide comprised in the driver. 58 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0254] An RTE-UTR may be referred to by the species of RTE from which it is derived. For example, an RTE-UTR derived from R2-1-TG may be referred to herein as a “R2-1-TG RTE- UTR”. Similarly, a template may be referred to herein by the RTE from which the one or more RTE-UTRs comprised therein are derived. As such, a template comprising one or more RTE- UTRs derived from the R2-1-TG RTE may be referred to herein as a “R2-1-TG template” or “R2-1-TG reporter”. [0255] An RTE-UTR comprised in a 5’ terminal region of the template (when in trans configuration) or of the combined nucleic acid (when in cis configuration) may be referred to herein as a 5’ RTE-UTR. AN RTE-UTR comprised in a 3’ terminal region of the template (when in trans configuration) or of the combined nucleic acid (when in cis configuration) may be referred to herein as a 3’ RTE-UTR. In certain embodiments, RTE-UTRs comprised in the template (when in trans configuration) or the combined nucleic acid (when in cis configuration) may be RTE-UTRs derived from natural RTEs (e.g. from any one of the RTEs, including R2 RTEs, described herein). In certain embodiments, the RTE-UTRs may be modified RTE-UTRs based on RTE-UTRs from one or more natural RTEs. [0256] In some embodiments, the 3´ RTE-UTR, e.g., of an R2-TG, may comprise a conserved Stem Loop (SL) region and a variable number of a microsatellite repeats (e.g., a minimal 3´ UTR required for efficient transgene integration). [0257] In certain embodiments, the one or more RTE-UTR(s) are derived from a non-LTR RTE. In certain embodiments, the one or more RTE-UTR(s) are derived from a Class I transposable element that utilizes an intermediate RNA during integration by a copy-and-paste mechanism (PMIDs: 32955944 and 18261821) and that lacks Long Terminal Repeats (LTR) or Inverted Terminal Repeats (ITR) in their structure. In some embodiments, the one or more RTE-UTR(s) are derived from a superclade selected from the group consisting of Retrons (include clades AbiA, AbiK, AbiP2, CRISPR, CRISPR-like, DCRs, G2L4, G2L, G2Lb, G2Lc, GII, Retrons, UG1, UG2, UG3, UG4, UG5, UG6, UG7, UG8, UG9, UG10, UG11, UG12, UG13, UG14, UG15, UG16, UG17, UG18, UG19, UG20, UG21, UG22, UG23, UG24, UG25, UG26, UG27, UG28 and UG28b), Group II introns (include clades E, ML, B, CL1A, CL1B, CL2A, CL2B, F, D and C), Penelope-like retroelements (include clades Pen1, Pen1a, Pen2, Pen3, Pen3a, Pen3b, Pen4, Pen5, Pen5a and Pen6), and non-LTR retroelements (include clades CRE, R4, Hero, NeSL, R2, RandI, Proto1, L1, Tx1, RTEPT, Proto2, RTEX, RTE, Outcast, Ingi (which includes 59 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 sub-clade Vingi), I, Nimb, Tad1, Loa, R1, Jockey, Rex1, CR1, L2, L2A, L2B, Daphne, and Crack). [0258] In certain embodiments, a template of the disclosure may comprise an RTE-UTR (a 3’ RTE-UTR and/or a 5’ RTE-UTR) derived from an R2 RTE. [0259] Non limiting examples of RTE-UTRs from R2 retroelements found in a variety of organisms are set forth in SEQ ID NOs: 33, 34, 85, 86, 99, 100, 125-128, 144-257, 259- 292, 294- 356, 358-372 or 486-489. Of these, 5’RTE-UTRs are set forth in SEQ ID NOs: 33, 85, 99, 125, 127, any one of 144-249, any one of 359-367 374, 486, or 488.3’RTE-UTRs are set forth in SEQ ID NOs: 34, 86, 100, 126, 128, , 250-358368-372, 487, 489. Other non-limiting examples of 5’ RTE-UTRs are set forth in any one of SEQ ID NOs: 359-367. Other non-limiting examples of 3’RTE-UTRs are set forth in any one SEQ ID NOs: 357- 372. The RTE-UTR sequences in the present application are provided as DNA sequences. For each RTE-UTR sequence provided, it will be understood that the present disclosure also includes a respective RNA version thereof. [0260] In certain embodiments, a template of the disclosure may comprise an RTE-UTR (a 3’ RTE-UTR and/or a 5’ RTE-UTR) derived from one of the following R2 RTEs, as listed in Table 3: Table 3: RTE-UTRs of representative R2 RTEs RTE name Organism species SEQ ID NO for SEQ ID NO for 5' RTE-UTR 3' RTE-UTR 1 R2-1-TG Taeniopygia guttata 85 86 2 R2-ZA Zonotrichia albicollis 486 487 3 R4-AC Anolis carolinensis 488 489 4 R4-1-PH Parhyale hawaiensis 99 100 [0261] Table 3 provides the DNA sequence of the 5’ RTE-UTR and the 3’ RTE-UTR from each of the listed RTE species. The RTE-UTR sequences in Table 3 are provided as DNA sequences. For each RTE-UTR sequence provided, it will be understood that the present disclosure also includes a respective RNA version thereof. [0262] In certain embodiments, an RTE integration system of the disclosure may utilize an RTE-UTR (a 3’ RTE-UTR and/or a 5’ RTE-UTR) derived from one of the following R2 RTEs, as listed in Table 4: 60 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 Table 4: RTE-UTRs of R2 RTEs RTE name Abbreviation Organism SEQ ID SEQ ID NO species NO for 5' for 3' RTE- RTE-UTR UTR 1 R2-1_Acridotheres_tristis R2-AT Acridotheres tristis 171 279 2 R2-1_Acrocephalus R2-ASS Acrocephalus _scirpaceus _scirpaceus scirpaceus scirpaceus 187 295 3 R2-1_Agelaius_tricolor R2-AgT Agelaius tricolor 145 251 4 R2- R2-AC Ammodramus 1_Ammodramus_caudacutus caudacutus 146 252 5 R2-1_Ammospiza_nelsoni R2-AN Ammospiza nelsoni 146 254 6 R2-1_Buteo_platypterus R2-BP Buteo platypterus 190 298 7 R2- R2-CC Chamaetylas 1_Chamaetylas_choloensis choloensis 175 283 8 R2- R2-CF Chamaetylas 1_Chamaetylas_fuelleborni fuelleborni 188 296 9 R2-1_Cichladusa_arquata R2-CA Cichladusa arquata 176 284 10 R2-1_Ciconia_episcopus R2-CE Ciconia episcopus 163 271 11 R2-1_Colaptes_auratus R2-CoAu Colaptes auratus 192 300 12 R2-1_Cossypha_archeri R2-CoAr Cossypha archeri 179 287 13 R2-1_Cossypha_dichroa R2-CD Cossypha dichroa 180 288 14 R2-1_Falco_fasciinucha R2-FF Falco fasciinucha 168 276 15 R2-1_Gallinago_paraguaiae R2-GP Gallinago paraguaiae 165 273 16 R2-1_Garrulus_glandarius R2-GG Garrulus glandarius 166 274 17 R2- R2-GA Geranoaetus 1_Geranoaetus_albicaudatus albicaudatus 181 289 18 R2- R2-HN Harpyopsis 1_Harpyopsis_novaeguineae novaeguineae 182 290 19 R2- R2-LS Limnodromus 1_Limnodromus_scolopaceus scolopaceus 127 128 20 R2-1_Melierax_canorus R2-MC Melierax canorus 183 291 61 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 RTE name Abbreviation Organism SEQ ID SEQ ID NO species NO for 5' for 3' RTE- RTE-UTR UTR 21 R2- R2-MyC Myiozetetes 1_Myiozetetes_cayanensis cayanensis 177 285 22 R2-1_Otis_tarda R2-OT Otis tarda 169 277 23 R2-1_Prinia_subflava R2-PS Prinia subflava 178 286 24 R2-1_Serinus_canaria R2-SC Serinus canaria 149 257 25 R2- R2-ApC-1 Aphelocoma 1_Aphelocoma_californica californica 144 250 26 R2-1_Oenanthe_melanoleuca R2-OM Oenanthe melanoleuca 148 256 27 R2-1_Phylloscopus_trochilus R2-PTT Phylloscopus _trochilus trochilus trochilus 125 126 28 R2-1_Geothlypis_trichas R2-GT Geothlypis trichas 153 261 29 R2-1_Xenus_cinereus R2-XC Xenus cinereus 174 282 30 R2- R2-2-ApC Aphelocoma 2_Aphelocoma_californica californica 172 280 31 R2-2_Cichladusa_arquata R2-CiA Cichladusa arquata 173 281 32 R2-1_Scolopax_minor R2-SM Scolopax minor 184 292 33 R2-1_Tyto_alba R2-Ta Tyto alba 189 297 34 R2-2_Acrocera_orbiculus R2-AcC Acrocera orbiculus 201 309 35 R2- R2-DSS Drosophila 1_Drosophila_sulfurigaster sulfurigaster _sulfurigaster sulfurigaster 210 318 36 R2-1_Rhopalosiphum_padi R2-RP Rhopalosiphum padi 211 319 37 R2-1_Nemotelus_nigrinus R2-NN Nemotelus nigrinus 212 320 38 R2-1_Chlorops_oryzae R2-CO Chlorops oryzae 214 322 39 R2-2_Andrena_bucephala R2-AB Andrena bucephala 215 323 40 R2-3_Ascidia_mentula R2-AM Ascidia mentula 161 269 41 R2- R2-COH Crotalus 1_Crotalus_oreganus_helleri oreganus helleri 157 265 42 R2- R2-GF Gopherus 1_Gopherus_flavomarginatus flavomarginatus 158 266 43 R2-1_Malaclemys_terrapin R2-MTP Malaclemys _pileata terrapin pileata 159 267 62 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 RTE name Abbreviation Organism SEQ ID SEQ ID NO species NO for 5' for 3' RTE- RTE-UTR UTR 44 R2- R2-EM Eublepharis 1_Eublepharis_macularius macularius 241 348 45 R2-1_Emys_orbicularis R2-EO Emys orbicularis 193 301 46 R2-1_Caretta_caretta R2-CC Caretta caretta 195 303 47 R2-1_Pleurodeles_waltl R2-PW Pleurodeles waltl 196 304 48 R2-1_Aspidoscelis_tigris R2-1-ATS Aspidoscelis _stejnegeri tigris stejnegeri 217 325 49 R2-1_Aspidoscelis_tigris R2-2-ATS Aspidoscelis _stejnegeri tigris stejnegeri 218 326 50 R2-1_Mauremys_mutica R2-1-MM Mauremys mutica 243 350 51 R2-3_Mauremys_mutica R2-1-MM Mauremys mutica 219 327 52 R2-1_Xiphophorus_hellerii R2-XH Xiphophorus hellerii 237 345 53 R2-1_Gambusia_affinis R2-GamA Gambusia affinis 239 346 54 R2-1_Schizopygopsis R2-SMa Schizopygopsis _malacanthus malacanthus 235 342 55 R2- R2-1-SP Schizopygopsis 1_Schizopygopsis_pylzovi pylzovi 235 344 56 R2- R2-2-SP Schizopygopsis 2_Schizopygopsis_pylzovi pylzovi 240 347 57 R2-1_Gila_orcuttii R2-GO Gila orcuttii 242 349 58 R2-1_Ahaetulla_prasina R2-AP Ahaetulla prasina 244 351 59 R2- R2-AG Aldabrachelys 1_Aldabrachelys_gigantea gigantea 248 355 60 R2-1_Ascidia_mentula R2-AsMe Ascidia mentula 156 264 [0263] Table 4 provides the DNA sequence of the 5’ RTE-UTR and the 3’ RTE-UTR from each of the listed RTE species. The RTE-UTR sequences in Table 4 are provided as DNA sequences. For each RTE-UTR sequence provided, it will be understood that the present disclosure also includes a respective RNA version thereof. [0264] In certain embodiments, an R2 RTE-UTR comprised in a template may an identical sequence with the RTE-UTR sequence comprised in a natural R2 RTE, for example as listed in 63 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 the tables above. In certain embodiments, the R2 RTE-UTR used in a template may have least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the RTE-UTR sequence comprised in a natural R2 RTE, for example as listed in the tables above. [0265] An “ancestral reconstruction process” refers to the method of extrapolating back in time and to model the ancient parents of the identified sequences, (see for example Campitelli et al., Reconstruction of full-length LINE-1 progenitors from ancestral genomes. Genetics.2022 Jul; 221(3): iyac074.). In certain embodiments, an R2 RTE-UTR used in a template may be an “ancestral RTE-UTR” based on an ancestral reconstruction based on a plurality of R2 RTE species. [0266] In certain embodiments, a template of the disclosure comprises an ancestral RTE-UTR (a 3’ RTE-UTR and/or a 5’ RTE-UTR) derived from one of the following combinations of R2 RTEs, as listed in Table 5. Table 5: Ancestral RTE-UTRs RTEs included in ancestral reconstruction SEQ ID NO for SEQ ID NO for 3' process 5' RTE-UTR RTE-UTR 1 3utr-AR1-4 (SEQ ID NO: 3077, 290 or 289) 85 357 2 3utr-AR2-5(SEQ ID NO: 128, 292, 273 or 282) 85 2378 3 3utr-AR3-7(SEQ ID NO: 280, 256 or 283) 85 551 4 5utr-AR1-4(SEQ ID NO: 1337, 1338 or 1339) 359 86 5 5utr-AR2-5(SEQ ID NO: 280, 256 or 283) 360 86 6 5utr-AR3-4 (SEQ ID NO: 148, 187 or 2275) 361 86 7 5utr-AR4-6 (SEQ ID NO: 145 or 85) 362 86 8 5utr-AR5-4 SEQ ID NO: 180, 173 or 188) 363 86 64 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 RTEs included in ancestral reconstruction SEQ ID NO for SEQ ID NO for 3' process 5' RTE-UTR RTE-UTR 9 5utr-AR7-5 (SEQ ID NO: 149, 486 or 146) 364 86 10 5utr-AR8m-10 (SEQ ID NO: 187 or 2275) 365 86 11 5utr-AR8-11(SEQ ID NO: 148, 145 or 85) 363 86 12 5utr-AR8f-10 (SEQ ID NO: 187 or 125) 364 86 [0267] Modified RTE-UTR sequences were created through ancestral reconstruction of multiple R2 RTEs and improved R2 transgene integration [0268] Modified R2 RTE-UTR elements were created through ancestral reconstruction using an ancestral genome reconstruction program Ancestors 1.1, based on RTE-UTR elements from a plurality of R2 RTEs. The modified RTE-UTRs, which included modified 5’ RTE-UTRs and/or 3’ RTE-UTRs were based on a plurality of R2 RTEs. Different sets of R2 RTEs, as described herein below, were used to generate different modified RTE-UTRs based on ancestral reconstruction (see, e.g., Campitelli et al., Reconstruction of full-length LINE-1 progenitors from ancestral genomes, Genetics, Volume 221, Issue 3, July 2022, and Diallo AB et al., Ancestors 1.0: a web server for ancestral sequence reconstruction. Bioinformatics. 2010;26(1):130–131, whose ancestral reconstruction procedures are incorporated herewith). [0269] The RTE-UTR sequences in Table 5 are provided as DNA sequences. For each RTE- UTR sequence provided, it will be understood that the present disclosure also includes a respective RNA version thereof. Homology arms [0270] In certain embodiments, the 5’ terminal region of the template, the 3’ terminal region of the template, or both, may comprise, independently, a homology arm with nucleic acid sequences having homology to nucleic acid sequences in the human genome. In some embodiments, each homology arm is independently selected and is from about 4 to about 200 nucleotides in length or more, for example about 4, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200 or more nucleotides long. In some embodiments, the homology 65 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 arm corresponds to a sequence in the 28S rDNA locus in the human genome. In some embodiments, the homology arm corresponds to a sequence in the AAVS1 locus in the human genome .In some embodiments, the nucleic acid sequence of the homology arm is in a reading frame that is different than the open reading frame of the transgene comprised in the template. In some embodiments, the nucleic acid sequence of the homology arm is in the same reading frame as the transgene. [0271] In some embodiments, a template comprises (e.g. in a 5’ terminal region and/or a 3’ terminal region) one or more sequences (e.g. homology arms) that are homologous to a target sequence (e.g., a target sequence in a host genome). Non-limiting examples of target sequences include safe harbor genomic targets. In some non-limiting embodiments, safe harbor genomic targets are located in the genes AAVS1, 28SrDNA, hROSA26, CCR5, SHS231, and PCSK9. Other optional template elements [0272] In some embodiments, a template may comprise one or more RNA nuclear localization sequences (e.g., a SAFB motif) and/or one or more stabilization motifs (e.g., a WPRE motif). In some embodiments, the template also may comprise flanking regions homologous to a target sequence in a genome. [0273] In certain embodiments, the template nucleic acid comprises one or more chemical or sequence modifications. In certain embodiments, the modification is a RNA cap, a modified polyA (e.g., relative to a natural polyA), a chemical modification (e.g., a pseudouridine and/or a methylpseudouridine), a 5’ end modification, a 3’ end modification, a modified Kozak sequence, a modified (e.g., truncated) stem loop, a RNA stabilization motif (which may be a WPRE motif), a 5-methoxyuridine (5-moU) modification, a 5-methylcytidine (5-mC) modification, or one or more additional and/or modified microsatellites. In certain embodiments, a nucleic acid sequence encoding an engineered driver and/or a template is sequence optimized (e.g., codon optimized) to enhance expression, reverse transcription or transgene function. In certain embodiments, RNA sequence optimization comprises one or more of the following modifications compared to the starting RNA molecule: reducing the uracil (U) load of an RNA molecule; reducing the GC% content of an RNA molecule; reducing the length and/or number of intron sequences of an RNA molecule; reducing RNA binding motifs or sites within an RNA molecule; lowering ΔG (free energy) of an RNA molecule; reducing the nucleotide repeats found in a sequence of an RNA molecule; adjusting the frequency of usage of particular codons 66 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 in an RNA molecule; reducing the number of palindromic sequences in an RNA molecule; maximizing pairing of bases of an in an RNA molecule; removing splicing site sequences from an RNA molecule; removing rare codons or other slowly translated codons. A person with skill in the art will be able to generate a polynucleotide encoding any one of the polypeptides disclosed herein, based on the polypeptide sequence, as provided herein. Transgenes [0274] As noted above, a template of the disclosure comprises a sequence of interest, destined for integration into a genome, which may be referred to as a transgene or a gene of interest (“GOI”), and which encodes a RNA or protein (e.g., a therapeutic RNA or protein). In certain embodiments, a template may comprise a plurality of transgenes. [0275] In certain embodiments, the transgene comprises a promoter and specifies an RNA, which may or may not encode a protein. In certain embodiments, the transgene comprises 5’ and 3’ UTRs such that the RNA will function as an mRNA. In certain embodiments, the transgene includes a polyadenylation (“polyA”) signal. In certain embodiments, the transgene includes one or more introns. In some embodiments, the transgene may be in sense or anti-sense orientation with respect to the template. [0276] In some embodiments, the promoter may be an inducible promoter. In some embodiments, the promoter is a tissue-specific promoter. In certain embodiments, the promoter may be, e.g., a MNDopt promoter, a MNDu promoter, a EF1a promoter, a CMV promoter, a A1AT promoter, a Albumin promoter, or a ApoE promoter. [0277] In certain embodiments, a template may comprise a segment encoding a detection polypeptide, e.g. useful to detect integration of the transgene by a given driver. In certain embodiments, a detection polypeptide is a human influenza hemagglutinin (HA) Flag (DYKDDDDK; SEQ ID NOs: 392), green fluorescent protein (GFP or variants such as EGFP, e.g. encoded by a nucleic acid sequence set forth in SEQ ID NO: 3331), or mCherry. [0278] In some embodiments, the transgene comprises a nucleic acid that encodes an immune cell engager, including, for example, a T cell engager (e.g., BiTE, DART), an NK cell engager (e.g., BiKE, TriKE), a macrophage engager (e.g., BiME), and an innate cell engager (e.g., ICE). [0279] In some embodiments, the template of the disclosure comprises a nucleic acid that encodes an engineered protein. In some embodiments, the engineered protein is an engineered immune receptor. Generally, an engineered immune receptor comprises an antigen binding 67 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 domain and is designed to redirect the specificity and function of immune cells (e.g., T cells) towards particular target cells (e.g., based on the antigen binding domain) and are useful for immunotherapies. [0280] In some embodiments, the engineered immune receptor is selected from the group consisting of a T cell receptor (TCR), TCR fused antigen modifier (TRAM), a T cell receptor fusion construct (TRuC) (Baeuerle, Patrick A., et al. "Synthetic TRuC receptors engaging the complete T cell receptor for potent anti-tumor response." Nature communications 10.1 (2019): 2087), and chimeric antigen receptor (CAR), and a costimulatory receptor fusion. One of skill in the art would recognize that the invention encompasses sequence variations of TRAMs, CARs, and costimulatory receptor fusions. TCRF (T Cell Receptor Fusion Protein) may be used interchangeably with TRAM. [0281] Generally, a naturally occurring TCR-CD3 complex comprises six (6) unique polypeptides: TCRα and TCRβ polypeptides, and CD3δ, CD3γ, CD3ε, and CD3ζ polypeptides (see FIG.2). The TCRα and TCRβ polypeptides are responsible for recognition of antigens presented on the Major Histocompatibility Complex (MHC) and have a very short intracellular domain with no signaling moieties. The CD3γ, CD3δ, and CD3ε polypeptides each contain a single immunoreceptor tyrosine-based activation motif (ITAM), and CD3ζ contains 3 ITAM motifs. The full TCR-CD3 complex has eight polypeptide: 1 TCRα, 1 TCRβ, 1 CD3γ, 1 CD3δ, 2 CD3ε, and 2 CD3ζ chains. Without being held to theory or mechanism, engagement of a TCR initiates T cell activation and is referred to as “signal 1”, which is necessary for T cell activation and effector functions. Engagement of co-stimulatory receptors then provides “signal 2” which augment the response and in some cases is necessary to avoid induction of anergy or tolerance where the T cell loses functionality. Cytokines can provide “signal 3” which can influence many pathways including those connected to proliferation, metabolism, and potency. A difference between the different signals is that without signal 1 there is no T cell activation, whereas the initiation of signal 1 without signals 2 or 3 does activate the cell but can exhibit diminished activity. [0282] A T Cell Receptor fused Antigen Modifier (TRAM) is a single polypeptide that contains an antigen binding domain fused to one of the standard polypeptides of the TCR-CD3 complex (FIG.2). When present, a TRAM is incorporated into the TCR-CD3 complex in lieu of the cognate standard polypeptide, such that binding of the antigen recognized by the TRAM initiates T cell signaling in a similar manner to natural TCR signaling following recognition of a specific 68 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 peptide-MHC complex. Examples of TRAM designs are shown in FIG.2 and FIG.3. Legend to FIG.2: scFv = Single chain variable fragment, TM = transmembrane, co-stim = co-stimulatory TRAM = T cell receptor fusion Antigen Modifier. Legend FIG.3: FMC63 = mouse anti-hCD19 antibody FMC63, VH = Variable Heavy Chain, VL = Variable Light Chain, SP = signal peptide, TM = Transmembrane, IC = Intracellular, Cα = Constant Alpha Chain, Cβ = Constant Beta Chain, hTCR = human TCR, mTCR = murine TCR, co-stim = co-stimulatory domain. [0283] Generally, a conventional CAR contains an antigen binding domain, hinge, transmembrane domain, and signaling domains (FIG.2). The antigen binding domain is typically derived from the variable fragments of antibodies in the form of a single chain variable fragment (scFv) where the variable light (VL) and variable heavy (VH) chains are fused using a flexible linker. The transmembrane and signaling domains are typically derived from various immune receptors. First generation CARs contain one signaling domain, typically the intracellular portion of CD3ζ. Second generation CARs have the addition of a second signaling domain from a co-stimulatory receptor such as 4-1BB (CD137) or CD28. Third generation CARs have an additional co-stimulatory signaling domain. For example, the canonical 2nd generation anti-CD19 CAR used here “CART-19” (FIG.3) has been used extensively in the clinic (Kymriah®) and consists of the CD8α signal peptide (“SP”, SEQ ID NO: 1), FMC63 VL (SEQ ID NO: 2), 3xG4S linker (SEQ ID NO: 3), FMC63 VH (SEQ ID NO: 4), CD8α Hinge (SEQ ID NO: 5), CD8α Transmembrane (“TM”, SEQ ID NO: 6), 4-1BB co-stimulatory domain (SEQ ID NO: 7), and CD3ζ signaling domain (SEQ ID NO: 8). [0284] A TRAM differs from a CAR in several aspects. A CAR contains a signal that is capable of expressing on the cell surface and providing signal 1 independently of the TCR-CD3 complex to the extent that the T cell is activated and can perform effector functions. Conversely, a TRAM requires the full TCR-CD3 complex to express on the cell surface and provide signal 1 (the single ITAM of CD3γ, CD3δ, or CD3ε without the scaffolding and/or additional ITAMs from CD3ζ is typically not sufficient to activate T cells to perform effector functions). Additionally, CARs can contain a co-stimulatory signaling domain that provides signal 2 (e.g. in 2nd generation and beyond CARs). [0285] In exemplary embodiments, the engineered immune receptor is a TRAM. In some embodiments, the TRAM, as provide herein, comprises an antigen binding domain fused to any one or more TCR subunits (TCRα, TCRβ), CD3δ, CD3γ, CD3ε, and CD3ζ), or a functional fragment thereof. The present disclosure provides TRAMs which may be used as compositions 69 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 with engineered retrotransposon systems (that may be delivered with RNA-LNP), have T cell specific expression, and/or prevent antigen masking. [0286] The term “antigen masking” refers to the blocking of the epitope of an antigen, which in turn prevents the recognition of the antigen by a T cell. Antigen masking can be mediated by an engineered immune receptor that is capable of binding the antigen in cis when expressed in the target cell itself, or in trans when expressed in a different cell in proximity to the target cell. [0287] In some embodiments, the engineered immune receptor-encoding transgene is integrated into a host cell using an RTE integration system as described herein. In some embodiments, the cell is an immune cell, which may be an immune cell that natively expresses a TCR-CD3 complex, e.g., a T cell. In some embodiments, the transgene integration into the cell is performed in vivo. In some embodiments, the transgene integration into the cell is performed in vitro. In some embodiments, the transgene integration into the cell is performed ex vivo. [0288] In some embodiments, the engineered immune receptor comprises one or more of the following elements: an antigen binding domain that binds to a target antigen, a transmembrane domain, a signal peptide, a co-stimulatory domain, an immunoreceptor tyrosine-based activation motifs (ITAMs), and/or a T cell-specific moiety. Transmembrane (TM) domains [0289] The engineered immune receptors of the disclosure comprise a transmembrane domain. In some embodiments, the transmembrane domain is selected from the group consisting of TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ, CD3ε, CD28, CD45, CD4, CD5, CD7, CD8, CD8 alpha, CD8beta, CD9, CD11a, CD11b, CD11c, CD11d, CD16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD134, CD137, TNFSFR25, CD154, 4-1BB/CD137, activating NK cell receptors, an Immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD276 (B7-H3), CD29, CD30, CD40, CD49a, CD49D, CD49f, CD69, CD84, CD96 (Tactile), CD5, CEACAM1, CRT AM, cytokine receptor, DAP- 10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL- 7R alpha, inducible T cell costimulator (ICOS), integrins, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB 1, KIRDS2, LAT, LFA-1, LFA-1, a ligand that binds with CD83, LIGHT, LIGHT, LTBR, Ly9 (CD229), lymphocyte function- associated antigen-1 (LFA-1; CDl-la/CD18), MHC class 1 molecule, NKG2C, NKG2D, NKp30, 70 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG/Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), Signaling Lymphocytic Activation Molecules (SLAM proteins), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; LylO8), SLAMF7, SLP-76, TNF receptor proteins, TNFR2, TNFSF14, TNFRSF19, a Toll ligand receptor, TRANCE/RANKL, VLA1, or VLA-6, or a fragment, truncation, or a combination thereof. [0290] In some embodiments, the engineered immune receptor is a CAR comprising a transmembrane domain selected from the group consisting of CD4, CD8, CD16, CD28, CD34, CD137, IgG1, IgG4, IgD, NGFR, LIR1, and PD1. [0291] In exemplary embodiments, the engineered immune receptor is a TRAM comprising a TCR subunit selected from the group consisting of TCRα, TCRβ, CD3δ, CD3γ, CD3ε, and CD3ζ. In some embodiments, the TRAM comprises a transmembrane domain selected from the group consisting of TCRα, TCRβ, CD3δ, CD3γ, CD3ε, and a variant thereof. Antigen binding domains [0292] The engineered immune receptors of the disclosure comprise an antigen binding domain that binds to a target antigen. In some embodiments, the antigen binding domain contains one or more single chain variable fragments (scFvs) comprising a variable light chain (VL) and variable heavy chain (VH), the variable domain of the heavy chain of a heavy-chain-only antibody (VHH), variable heavy chain only (VH), TCR-like antibody, single chain TCR VαVβ, natural ligand, D-domain, FcR-binding receptor (e.g., CD16), or an NK killing receptor (e.g., NKG2D, NKp30, NKp44, NKp46). [0293] In some embodiments, the antigen binding domain comprises a VH and VL fused by a linker. In some embodiments the linker is a flexible linker. In some embodiments, the flexible linker comprises the amino acid sequence of 3xG4S (SEQ ID NO: 3) or Whitlow linker (SEQ ID NO: 9). [0294] In some embodiments the antigen binding domain is linked to the N terminus of the TCR subunit by a linker and/or a hinge. In some embodiments, the hinge and/or linker is, is from, or is derived from (e.g., comprises all or a fragment of) CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8. alpha., CD8.beta., CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SEAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c 71 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CEEC2), CD79A (B cell antigen receptor complex-associated alpha chain), CD79B (B cell antigen receptor complex- associated beta chain), CD84 (SEAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (0X40), CD137 (4-1BB), CD150 (SEAMF1), CD158A (KIR2DL1), CD158B 1 (KIR2DE2), CD158B2 (KIR2DE3), CD158C (KIR3DP1), CD158D (KIRDE4), CD158F1 (KIR2DE5A), CD158F2 (KIR2DE5B), CD158K (KIR3DE2), CD160 (BY55), CD162 (SEEPEG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 (CD3-zeta), CD258 (LIGHT), CD268 (BAFFR), CD270 (TNFSF14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-1), CD314 (NKG2D), CD319 (SLAMF7), CD335 (NK-p46), CD336 (NK-p44), CD337 (NK-p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRTAM), CD357 (TNFRSF18), inducible T cell co-stimulator (ICOS), LFA-1 (CDl la/CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL- 2R beta, IL-2R gamma, IL-7R alpha, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1/CBP, a CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, a TNF receptor protein, an immunoglobulin protein, a cytokine receptor, an integrin, activating NK cell receptors, or Toll ligand receptor, or which is a fragment or combination thereof. [0295] In some embodiments, the linker and/or hinge is selected from the group consisting of the following amino acid sequences (or fragments thereof): GGGGS (SEQ ID NO: 129), EAAAK (SEQ ID NO: 130), PAPAP (SEQ ID NO: 131), ALEA (SEQ ID NO: 132), GGGGGG (SEQ ID NO: 133), AAA (SEQ ID NO: 10), Whitlow (SEQ ID NO: 9), SESATPES (XTEN linker, SEQ ID NO: 78), AEQQRQQQEAAQKAQ (SEQ ID NO: 117), CD8a hinge (SEQ ID NO: 5), CD28 hinge (SEQ ID NO:28), CD4 hinge (SEQ ID NO: 134), CD7 hinge (SEQ ID NO: 135), CD34 hinge (SEQ ID NO: 136), CD137 hinge (SEQ ID NO: 137), IgG1 hinge (SEQ ID NO: 138), IgG4 hinge (SEQ ID NO: 139), IgD hinge (SEQ ID NO: 140), LIR1 hinge (SEQ ID NO: 141), NGFR hinge (SEQ ID NO: 142), PD1 hinge (SEQ ID NO: 143). [0296] In some embodiments, the target antigen is selected from the group consisting of 2B4 (CD244), 4-1BB, 5T4, A33 antigen, adenocarcinoma antigen, adrenoceptor beta 3 (ADRB3), A kinase anchor protein 4 (AKAP-4), alpha-fetoprotein (AFP), anaplastic lymphoma kinase (ALK), Androgen receptor, B7H3 (CD276), p2-integrins, BAFF, B-lymphoma cell, B cell maturation antigen (BCMA), bcr-abl (oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl), BhCG, bone marrow stromal cell antigen 2 (BST2), CCCTC-Binding Factor (Zinc Finger Protein)-Like 72 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 (BORIS or Brother of the Regulator of Imprinted Sites), BST2, C242 antigen, 9-0-acetyl- CA19- 9 marker, CA-125, CAEX, calreticulin, carbonic anhydrase 9 (CAIX), C-MET, CCR4, CCR5, CCR8, CD2, CD3, CD4, CD5, CD8, CD7, CD10, CD16, CD19, CD20, CD22, CD23 (IgE receptor), CD24, CD25, CD27, CD28, CD30 (TNFRSF8), CD33, CD34, CD38, CD40, CD40L, CD41, CD44, CD44V6, CD49f, CD51, CD52, CD56, CD63, CD70, CD72, CD74, CD79a, CD79b, CD80, CD84, CD96, CD97, CD100, CD123, CD125, CD133, CD137, CD138, CD150, CD152 (CTLA-4), CD160, CD171, CD174, CD179a, CD200, CD221, CD229, CD244, CD272 (BTLA), CD274 (PDL-1, B7H1), CD279 (PD-1), CD352, CD358, CD300 molecule-like family member f (CD300LF), Carcinoembryonic antigen (CEA), claudin 6 (CLDN6), CLDN9, CLDN18.2, C-type lectin-like molecule-1 (CLL-1 or CLECL1), C-type lectin domain family 12 member A (CLEC12A), a cytomegalovirus (CMV) infected cell antigen, CNTO888, CRTAM (CD355), CS-1 (also referred to as CD2 subset 1, CRACC, CD319, and 19A24), CSPG4, CTLA-4, Cyclin B 1, chromosome X open reading frame 61 (CXORF61), Cytochrome P450 IB 1 (CYP1B 1), DNAM-1 (CD226), desmoglein 4, DR3, DR5, E-cadherin neoepitope, EDB-E, epidermal growth factor receptor (EGFR), EGF1R, epidermal growth factor receptor variant III (EGFRvIII), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), EGF- like module-containing mucin-like hormone receptor-like 2 (EMR2), elongation factor 2 mutated (ELF2M), endosialin, Epithelial cell adhesion molecule (EPCAM), ephrin type-A receptor 2 (EphA2), Ephrin B2, receptor tyrosine-protein kinases erb-B2,3,4 (erb-B2,3,4), ERBB, ERBB2 (Her2/neu), ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), ETA, ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML), Fc fragment of IgA receptor (FCAR or CD89), fibroblast activation protein alpha (FAP), FBP, Fc receptor- like 5 (FCRL5), fetal acetylcholine receptor (AChR), fibronectin extra domain-B, Fms-Like Tyrosine Kinase 3 (FLT3), folate-binding protein (FBP), folate receptor 1, Folate receptor alpha, Folate receptor beta, Fos-related antigen 1, Fucosyl, Fucosyl GM1; GM2, ganglioside G2 (GD2), ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(l-l)Cer),o- acetyl-GD2 ganglioside (0AcGD2), GITR (TNFRSF 18), GM1, ganglioside GM3 (aNeu5Ac(2-3)bDGalp(l- 4)bDGlcp(l-l)Cer), GP 100, hexasaccharide portion of globoH glycoceramide (GloboH), glycoprotein 75, Glypican-3 (GPC3), glycoprotein 100 (gp100), GPNMB, G protein-coupled receptor 20 (GPR20), G protein-coupled receptor class C group 5, member D (GPRC5D), Hepatitis A virus cellular receptor 1 (HAVCR1), human Epidermal Growth Factor Receptor 2 (HER-2), HER2/neu, HER3, HER4, HGF, HIV-1 gp1, HIV-1 ph120, high molecular weight- 73 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 melanoma-associated antigen (HMWMAA), human papilloma virus E6 (HPV E6), human papilloma virus E7 (HPV E7), heat shock protein 70-2 mutated (mut hsp70-2), human scatter factor receptor kinase, human Telomerase reverse transcriptase (hTERT), HVEM, ICOS, insulin-like growth factor receptor 1 (IGF-1 receptor), IGF-I, IgGl, immunoglobulin lambdalike polypeptide 1 (IGLL1), IL-6, Interleukin 11 receptor alpha (IL-11Ra), IL-13, Interleukin- 13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2), insulin-like growth factor I receptor (IGF1- R), integrin a5pi, integrin avP3, intestinal carboxyl esterase, K-light chain, KCS1, kinase insert domain receptor (KDR), KIR, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL2, KIR- L, KG2D ligands, KIT (CD117), KLRGI, LAGE-la, LAG3, lymphocyte- specific protein tyrosine kinase (LCK), Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), lambda light chain, legumain, Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Lewis(Y) antigen, LeY, LG, L11 cell adhesion molecule (L1-CAM), LIGHT, LMP2, lymphocyte antigen 6 complex, LTBR, locus K 9 (LY6K), Ly-6, lymphocyte antigen 75 (LY75), melanoma cancer testis antigen- 1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT- 2), MAGE, Melanoma- associated antigen 1 (MAGE-A1), MAGE-A3 melanoma antigen recognized by T cells 1 (MelanA or MARTI), MelanA/MARTl, Mesothelin, MAGE A3, MAGE-A6, MAGE-A12, melanoma inhibitor of apoptosis (ML-IAP), melanoma- specific chondroitin- sulfate proteoglycan (MCSCP), MORAb-009, MS4A1, Mucin 1 (MUC1), MUC2, MUC3, MUC4, MUC5AC, MUC5b, MUC7, MUC16, mucin CanAg, Mullerian inhibitory substance (MIS) receptor type II, v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN), N-glycolylneuraminic acid, N-Acetyl glucosaminyl-transferase V (NA17), neural cell adhesion molecule (NCAM), NKG2A, NKG2C, NKG2D, NKG2E ligands, NKR-P IA,NPC-1C, NTB-A, mammary gland differentiation antigen (NY-BR-1), NY-ESO-1, oncofetal antigen (h5T4), Olfactory receptor 51E2 (OR51E2), 0X40, plasma cell antigen, poly SA, proacrosin binding protein sp32 (OY-TES 1), p53, p53 mutant, pannexin 3 (PANX3), prostatic acid phosphatase (PAP), paired box protein Pax-3 (PAX3), Paired box protein Pax-5 (PAX5), prostate carcinoma tumor antigen- 1 (PCTA-1 or Galectin 8), PD-1H, Platelet-derived growth factor receptor alpha (PDGFR- alpha), PDGFR-beta, PDL192, PEN- 5, phosphatidylserine, placenta- specific 1 (PLAC1), Polysialic acid, Prostase, prostatic carcinoma cells, prostein, Protease Serine 21 (Testisin or PRSS21), Proteinase3 (PR1), PSA, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), Proteasome (Prosome, Macropain) Subunit, Beta Type, Receptor for Advanced Glycation Endproducts (RAGE-1), 74 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 RANKL, Ras mutant, Ras Homolog Family Member C (RhoC), RON, Receptor tyrosine kinase- like orphan receptor 1 (R0R1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), sarcoma translocation breakpoints, Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3), SAS, SDC1, SLAMF7, sialyl Lewis adhesion molecule (sLe), Siglec-3, Siglec-7, Siglec-9, sonic hedgehog (SHH), sperm protein 17 (SPA17), Stage- specific embryonic antigen- 4 (SSEA-4), STEAP, sTn antigen, synovial sarcoma, X breakpoint 2 (SSX2), Survivin, Tumor- associated glycoprotein 72 (TAG72), TCR5y, TCRa, TCRB, TCR Gamma Alternate Reading Frame Protein (TARP), telomerase, TIGIT TNF-a precursor, tumor endothelial marker 1 (TEM1/CD248), tumor endothelial marker 7-related (TEM7R), tenascin C, TGF beta 2, TGF-P, transglutaminase 5 (TGS5), angiopoietin-binding cell surface receptor 2 (Tie 2), TIM1, TIM2, TIM3, Tn Ag, TRAIL-R1, TRAIL-R2, Tyrosinase-related protein 2 (TRP-2), thyroid stimulating hormone receptor (TSHR), tumor antigen CTAA16.88, Tyrosinase, TYRP-1, R0R1, TAG- 72, uroplakin 2 (UPK2), VEGF-A, VEGFR-1, vascular endothelial growth factor receptor 2 (VEGFR2), vimentin, Wilms tumor protein (WT1), and X Antigen Family, Member 1A (XAGE1). [0297] In some embodiments, the antigen binding domain is specific for one or more of any of the target antigens of the disclosure. In some embodiments, the antigen binding domain is specific for GPCR5D (G-protein coupled receptor class 5D), BCMA, CD22, CD19 and/or CD20. [0298] In some embodiments, the CD19 antigen binding domain is derived from an antibody selected from the group consisting of FMC63, murine 4G7 (VL SEQ ID NO: 109, VH SEQ ID NO: 110), human Hu19 (VL SEQ ID NO: 111, VH SEQ ID NO: 112), Hu1E7, huB4, hBU12, humanized FMC63, humanized 4G7, GR37, GR41, SRB-37, SRB-85, and modified versions of any of the preceding. [0299] In some embodiments, the CD20 antigen binding domain is derived from an antibody selected from the group consisting of Leu16, rituximab, ofatumumab (VH SEQ ID NO: 115, VL SEQ ID NO: 116), ocrelizumab, obinutuzumab (VH SEQ ID NO: 113, VL SEQ ID NO 114), ibritumomab tiuxetan, AME-133v, IMMU-106, TRU-015, tositumomab, and modified versions of any of the preceding. [0300] In some embodiments, the BCMA antigen binding domain is derived from an antibody selected from the group consisting of erlanatamab, alnuctamab, linvoseltamab, teclistamab, 75 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 belantamab, pavurutamab, icatolimab, and human anti-TNFRSF17 clone hSG16.17 clone hSG16.45, or modified versions of any of the preceding. [0301] In some embodiments, the antigen binding domain is bispecific. In some embodiments, the antigen binding domain is bispecific for CD19 and CD20. In some embodiments, the bispecific antigen binding domain comprises multiple scFvs. In some embodiments, the multiple scFvs are linked by a linker or hinge selected from the group consisting of flexible 3xG4S linker (SEQ ID NO: 3), rigid 3xEAAAK (SEQ ID NO: 41), machine-learning assisted rigid AEQQRQQQEAAQKAQ (SEQ ID NO: 117), semi-flexible GGGSEAAAKGGGS (SEQ ID NO: 42), semi-rigid EAAAKGGGSEAAAK (SEQ ID NO: 43), and rigid 3xPAPAP (SEQ ID NO: 118). In some embodiments, the linker or hinge is from, or is derived from (e.g., comprises all or a fragment of) CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8. alpha., CD8.beta., CDl la (ITGAL), CDl lb (ITGAM), CDl lc (ITGAX), CDl ld (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SEAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CEEC2), CD79A (B cell antigen receptor complex-associated alpha chain), CD79B (B cell antigen receptor complex-associated beta chain), CD84 (SEAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (0X40), CD137 (4-1BB), CD150 (SEAMF1), CD158A (KIR2DL1), CD158B 1 (KIR2DE2), CD158B2 (KIR2DE3), CD158C (KIR3DP1), CD158D (KIRDE4), CD158F1 (KIR2DE5A), CD158F2 (KIR2DE5B), CD158K (KIR3DE2), CD160 (BY55), CD162 (SEEPEG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 (CD3-zeta), CD258 (LIGHT), CD268 (BAFFR), CD270 (TNFSF14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-1), CD314 (NKG2D), CD319 (SLAMF7), CD335 (NK-p46), CD336 (NK-p44), CD337 (NK-p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRTAM), CD357 (TNFRSF18), inducible T cell costimulator (ICOS), LFA-1 (CDl la/CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL- 2R beta, IL-2R gamma, IL-7R alpha, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1/CBP, a CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, a TNF receptor protein, an immunoglobulin protein, a cytokine receptor, an integrin, activating NK cell receptors, and Toll ligand receptor, or which is a fragment or combination thereof. 76 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 Signal peptides [0302] As previously mentioned, in some embodiments, the engineered immune receptor further comprises a signal peptide. Signal peptides (SPs) are nucleic acids that direct proteins to the secretory pathway. In some embodiments, the signal peptide is selected from the group consisting of CD8α signal peptide, a TCRα signal peptide, and a TCRβ signal peptide. In some embodiments, the signal peptide is a CD8α SP (SEQ ID NO: 1), mTCRα SP (SEQ ID NO: 12), or hTCRβ SP (SEQ ID NO: 16). In some embodiments, the signal peptide is a CD8α signal peptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the signal peptide is a TCRα signal peptide comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the signal peptide is a TCRβ signal peptide comprising the amino acid sequence of SEQ ID NO: 16. Co-stimulatory domains [0303] In some embodiments, the engineered immune receptor further comprises one or more co-stimulatory domains. Co-stimulatory domains generally play a functional role in the activation of an immune cell. In some embodiments, the co-stimulatory domain is selected from the group consisting of CD2, CD5, CD27, CD28, DAP10, DAP12, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), OX40, and 4-1BB (CD137). In some embodiments, the co- stimulatory domain is a signaling region of a protein selected from the group consisting of DAP- 10, CD28, OX-40, 4-IBB (CD137), CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD- 1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), tumor necrosis factor superfamily member 14, TNFSF14, LIGHT), NKG2C, Ig alpha (CD79a), Fc gamma receptor, MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, CDS, GITR, BAFFR, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD (CDl ld), ITGAE (CD103), ITGAL (CDl la), ITGAM (CDl lb), ITGAX (CDl lc), ITGB1, CD29, ITGB2, CD 18, ITGB7, NKG2D, TNFR2, TRANCE (RANKL), DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), 77 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG (Cbp), CD 19a, a ligand that specifically binds with CD83, and combinations thereof. Immunoreceptor tyrosine-based activation motifs (ITAMs) [0304] In some embodiments, the engineered immune receptor further comprises one or more ITAMs. ITAMs generally play a functional role in the transmission of signals from immune receptors. In some embodiments, the ITAMs are selected from the group consisting of TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ, CD3ε, CD3ζ, Fc epsilon receptor 1 chain, FcεR2, FcγR1, FcγR2a, FcγR2b1, FcγR2b2, FcγR3a, FcγR3b FcβR1, DAP10, DAP12, CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, and CD66d. T cell specific elements [0305] RTE integration systems of the present disclosure can be delivered in vivo in an LNP format. For engineered immune receptors, in vivo delivery to, and stable expression in, cells other than T cells could lead to antigen masking, where access to the target antigen by the T cell is blocked. Certain engineered immune receptors, e.g., TRAMs, are designed to reduce this effect by being unstable in cells other than TCR-expressing (i.e., T) cells. The disclosure also describes additional methods to enhance T cell specificity before the stage of surface expression, for example, According to some embodiments of the present disclosure, driver or template RNAs with binding sites for differentially expressed miRs can be prevented from integrating or expressing in certain cell types. Templates comprising transgenes encoding T cell-specific promoters or enhancers can also bias expression specifically in T cells. [0306] In some embodiments, the transgene comprises a promoter. In some embodiments, the promoter is a naturally occurring promoter. In some embodiments, the promoter is a recombinant promoter. In some embodiments, the promoter is a constitutive, inducible, and/or tissue or cell specific promoter. In some embodiments, the cell specific promoter is a T cell specific promoter. In some embodiments, the T cell specific promoter comprises one or more of hCCL5 (SEQ ID NO: 46), mCCL5 (SEQ ID NO: 47), hCD2 (SEQ ID NO: 48), mCD2 (SEQ ID NO: 49), hCD3γ (SEQ ID NO: 50) , hCD3δ (SEQ ID NO: 51), mCD3δ (SEQ ID NO: 52), hCD3ε (SEQ ID NO: 53), mCD3ε, minimal mCD3δ (SEQ ID NO: 55), and any fragment thereof. 78 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0307] In some embodiments, the promotor is an inducible promoter containing binding sites for T cell transcription factors. In some embodiments the T cell transcription factor binding sites are selected from the group consisting of NR4A (SEQ ID NO: 57), AP1 (SEQ ID NO: 58), NFAT (SEQ ID NO: 59), NFκB (SEQ ID NO: 60), and any combination thereof. [0308] In some embodiments, the template further comprises a T cell specific enhancer. In some embodiments, the T cell specific enhancer comprises chr16-445 (SEQ ID NO: 56). [0309] In some embodiments, the template further comprises one or more binding sites for a differentially expressed miR. In some embodiments, the one or more miRs are located in the 5’ RTE-UTR or RTE3’UTR in either the sense orientation or anti-sense orientation. In some embodiments, the miR is miR-122. In some embodiments the differentially expressed miRs are derived from Ludwig et al. Nucleic Acids Research, 44(8): 3865– 3877 (2016), the distribution of miRNAs in human tissues and is presented in, and downloadable from the Human miRNA tissue atlas at https://ccb-compute2.cs.uni-saarland.de/mirnatissueatlas_2025 each of which is incorporated by reference in its entirety to the extent that is not inconsistent with the present disclosure. Sample TRAM designs [0310] The following are non-limiting examples of TRAM engineered immune receptor designs. FIG.3 also depicts exemplary TRAM designs. Unless otherwise noted the CD19 scFv for the following TRAMs comprise FMC63 VL (SEQ ID NO: 2), Whitlow linker (SEQ ID NO: 9), FMC63 VH (SEQ ID NO: 4) and are fused to CD3 component via 3xA (SEQ ID NO: 10) and G4Sx3 linker (SEQ ID NO: 3). This scFv with spacer is referred to herein as “FMC63 LH scFv- G4S” (SEQ ID NO: 11). [0311] “mCα+β TRAM” comprises mTCRα SP (SEQ ID NO: 12), FMC63 VH (SEQ ID NO: 4), 3xA (SEQ ID NO: 10), mTCR-Cα (SEQ ID NO: 13), T2A (SEQ ID NO: 14), FMC63 VL (SEQ ID NO: 2), 3xA (SEQ ID NO: 10), and mTCR-Cβ (SEQ ID NO: 15). [0312] “mCβ TRAM” comprises mTCRα SP (SEQ ID NO: 12), mTCR-Cα (SEQ ID NO: 13), T2A (SEQ ID NO: 14), FMC63 LH scFv-G4S (SEQ ID NO:11), and mTCR-Cβ (SEQ ID NO: 15). [0313] “hCβ TRAM” comprises hTCRβ SP (SEQ ID NO: 16), FMC63 LH scFv-G4S (SEQ ID NO: 11), and hTCR- Cβ (SEQ ID NO: 17). 79 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0314] “γ TRAM” comprises of CD8α SP (SEQ ID NO: 1), FMC63 LH scFv-G4S (SEQ ID NO: 11), and CD3γ (SEQ ID NO: 18). [0315] “δ TRAM” comprises of CD8α SP (SEQ ID NO: 1), FMC63 LH scFv-G4S (SEQ ID NO: 11), and CD3δ (SEQ ID NO: 19). [0316] “ε TRAM” comprises CD8α SP (SEQ ID NO: 1), FMC63 LH scFv-G4S (SEQ ID NO: 11), and CD3ε (SEQ ID NO: 20). comprises of CD8α SP (SEQ ID NO: 1), FMC63 LH scFv-G4S (SEQ ID NO: 11), CD28 hinge (SEQ ID NO: 21) since CD3ζ has only 8 amino acids extracellularly, CD3ζ HTM (SEQ ID NO: 22), and CD3ζ signaling domain (SEQ ID NO: 8). Delivery Systems [0318] The present disclosure also describes delivery systems which deliver the RTE integration systems (also referred to herein as “RTE system”) of the disclosure to a host cell. In some embodiments, the host cell is a T cell. [0319] In some embodiments, the delivery systems described in the disclosure are useful for delivering one or more templates to a host cell and promoting integration of a DNA copy of the template nucleic acid into the host nucleic acid (e.g., a genomic nucleic acid of a host cell), for example for therapeutic purposes (e.g., to provide or supplement expression of an RNA and/or polypeptide that provides a therapeutic benefit to a subject, for example a human subject having a disease or disorder associated with a loss of normal gene function). [0320] In some embodiments, the delivery systems of the disclosure are useful for high efficiency delivery of driver and template nucleic acid to specific cells or populations of cells. Lipid nanoparticles (LNPs) [0321] In some embodiments, the RTE system is delivered using LNPs. LNPs are particles composed of lipids, typically spherical in shape and a micron or less in diameter, that are used as delivery vehicles for genetic material, such as DNA or RNA, into cells. Without being limited by theory, LNPs protect the genetic material from degradation and facilitate its entry into target cells. [0322] Generally, LNPs comprise an ionizable lipid, a phospholipid, cholesterol, a PEGylated lipid or any combination thereof. In some embodiments, the ionizable lipid is an ionizable 80 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 cationic lipid. In certain embodiments, the LNP of the disclosure comprises: an ionizable cationic lipid, a phospholipid, a cholesterol, and a PEGylated lipid. [0323] In certain embodiments, the average size of the LNP is between about 10nm and about 1000nm in diameter, between about 100nm and about 1000nm in diameter, between about 100nm and about 1000nm in diameter, between about 500nm and about 1000nm in diameter, between about 10nm and about 100nm in diameter, between about 10nm and about 200nm in diameter, between about 10nm and about 500nm in diameter, or between about 100nm and about 500nm in diameter. Any technique known in the art may be used to determine the size of the LNP. For example, LNP size could be measured using dynamic light scattering (DLS). [0324] In some embodiments, the composition comprises one or more nucleic acids packaged into an LNP. In some embodiments, the driver nucleic acid and template nucleic acid of the disclosure are packaged into the LNP. In certain embodiments, the driver nucleic acid and template nucleic acid are provided as RNAs and packaged into the LNP. In certain embodiments, a driver nucleic acid and template nucleic acid are packaged independently into LNPs, which may be comprised of the same or different components. In certain embodiments, the driver may be delivered as an amino acid sequence.. Targeted LNPs [0325] In some embodiments, the RTE system is delivered using a targeted LNP (also referred to herein as “tLNP”). In some embodiments, the tLNP is covalently modified with an attached antibody (also referred to herein as a “targeting moiety”) or antigen binding domain that recognizes a target antigen present on the target cell of interest, which can also increase specificity. In some embodiments, the target antigen is a T cell specific antigen. [0326] In some embodiments, the targeting moiety is specific to a single target (e.g., CD3ε). In some embodiments, the targeting moiety is bispecific (e.g., CD3ε and CD20). In some embodiments, targeting moieties of the disclosure are fused to tLNPs, bind and/or activate T cells, and enhance delivery of the nucleic acids packaged in the tLNPs. [0327] In some embodiments, the tLNP is comprised of 1,2-dioleoyl-sn-glycero-3-phosphate (18PA) which can be used to target LNPs using the Selective organ-targeting (SORT) LNP method. [0328] In some embodiments, the LNP is covalently linked to an antibody that recognizes a T cell antigen. In some embodiments, the covalently linked antibody comprises a silent Fc domain. 81 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 In some embodiments, the antibody is specific for one or more of CD2, CD5, CD7, TCR, CD8, and CD3. In some embodiments, the antibody is selected from the group consisting of OKT3 (VH SEQ ID NO: 63, VL SEQ ID NO: 64), BMA031 (VH SEQ ID NO: 65, VL SEQ ID NO: 66), H65 (VH SEQ ID NO: 67, VL SEQ ID NO: 68), 3A1E (VH SEQ ID NO: 69, VL SEQ ID NO: 70), OKT8 (VH SEQ ID NO: 71, VL SEQ ID NO: 72), Visilizumab (VH SEQ ID NO: 73, VL SEQ ID NO: 74), Teplizumab (VH SEQ ID NO: 88, VL SEQ ID NO: 89), Siplizumab (VH SEQ ID NO: 92, VL SEQ ID NO: 93), Urelumab (VH SEQ ID NO: 94, VL SEQ ID NO: 95), Otelixizumab (VH SEQ ID NO: 101, VL SEQ ID NO: 102), Foralumab (VH SEQ ID NO: 105, VL SEQ ID NO: 106), and modified versions of any of the preceding, e.g., having at least 80%, 85%, 90%, 95%, or 99% sequence identity of each aforementioned VH or VL. [0329] In some embodiments, the tLNP is covalently linked to an antibody fragment that recognizes a T cell antigen. In some embodiments, the antibody fragment may be a scFv, an antigen-binding fragment (Fab), a (Fab)2, a diabody, a single-domain antibody (sdAb) or a variable domain of the heavy chain of a heavy-chain-only antibody (VHH). [0330] A non-limiting selection of antigens (with corresponding antibodies or fragments thereof that specifically said antigen) useful for tLNPs targeting a T cell includes, Anti-human CD2 (VIP VIIIC8 and Siplizumab), CD3ε (OKT3, Visilizumab, Teplizumab, Otexlizumab, Foralumab, CD3_F2B), CD5 (H65, 5G7, and UHCT2), CD7 (3A1E), CD8 (OKT8), and 4-1BB (Urelumab). Immune cell conditioning [0331] Also provided herein are methods for conditioning immune cells (e.g., T cells) to be more responsive to in vivo or ex vivo transfection. In some embodiments, adjuvant conditioning is utilized to improve the efficacy of treatment. [0332] Without wishing to be bound to theory, the adjuvant conditioning acts via the recruitment of other elements of the immune system. In some embodiments, the immune cell conditioning comprises activation of T cells. [0333] As used herein the term “T cell activator” encompasses any agent that elicits a response from T cells, such as but not limited to proliferation, differentiation and engagement in immune- mediated functions. In some embodiments, a T cell activator, such as an activating antibody, is administered to T cells concurrently with, or prior to, transfection. In some embodiments, a T 82 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 cell activator, such as an activating antibody, is administered to a patient concurrently with, or prior to, in vivo delivery (e.g., by LNP administration). [0334] In some embodiments, the activating antibody or T cell activator is selected from the group consisting of Anti-human CD3 clones: OKT3, UCHT1, SK7, HIT3a YTH 12.5, Teplizumab (HzOKT3γ1), Otelixumab, Foralumab, CD3_F2B, and CD3_F1F. and anti-TCR clone BMA031, or any variant thereof. [0335] In some embodiments, the activating antibody or T cell activator is an immune cell engager such as a bispecific T cell engager (“BiTe”) which binds an activating moiety on the T cells (typically CD3) as well as a tumor antigen. In some embodiments, the BiTe is selected from the group consisting of Mosunetuzumab (Heavy chain 1 SEQ ID NO: 121, Light chain 1 SEQ ID NO: 122, Heavy chain 2 SEQ ID NO: 123, Light chain 2 SEQ ID NO: 124), Blinatumomab, MGD006, XmAb14045, AMG 330, AMG 420, AMG 564, AMG 701, BiTe 197, REGN1979, RG6026, Mosunetuzumab, AFM11, MGD006, tebentafusp, Catuxomab, Pasotuxizumab, Acapatamab, AMV564, Glofitamab, Solitomab, Talquetamab, Tarlatamab, Tebentafusp, Odronextamab, Epcoritamab, and modified versions of any of the preceding. [0336] In some embodiments, T cells are activated in a subject with the administration by intravenous or subcutaneous infusion or injection. [0337] In some embodiments, the subject is treated or conditioned with a γ-chain receptor agonist. In some embodiments, the γ-chain receptor agonist it administered by intravenous or subcutaneous infusion or injection. In some embodiments the γ-chain receptor agonist comprises IL-15, IL-2, IL-7, or IL-21. [0338] In some embodiments, T cells are activated in a subject with the administration of one or more immune checkpoint inhibitors (inhibitors or the checkpoint associated with CTLA-4, PD-1, PD-L1, Tim-3 LAG-3, OX40, GITR, CD40, CD122, CD137, CD122, CD40, ICOS, TIGIT, Siglec-15, or B7H3). Enhancing Moieties [0339] Also provided herein are enhancing moieties that can be included into the RTE integration systems of the disclosure. [0340] The SAM and HD domain containing protein 1 (SAMHD1) is a tetrameric enzyme with dNTP triphosphate hydrolase (dNTPase) activity. SAMHD1 has been shown to limit retroviral infectivity (e.g. HIV infectivity) by depleting the intracellular dNTP pools, which is required for 83 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 efficient reverse transcription. In this disclosure, SAMHD1 inhibitors may be provided as an enhancing moiety to be used in conjunction with RTE integration systems of the disclosure, to improve the efficiency of transgene genomic insertion. In some embodiments, the SAMHD1 inhibitor reduces SAMHD1‐mediated dNTP degradation. Non-limiting SAMHD1 inhibitors are provided herein. [0341] In some embodiments, the SAMHD1 inhibitor is a viral inhibitor of SAMHD1, or is derived from such a viral inhibitor. In some embodiments, the SAMHD1 inhibitor is from a human immunodeficiency virus (HIV), which may be HIV-1 or HIV-2. In other embodiments the SAMHD1 inhibitor is from SIV. In some embodiments, the SAMH1 inhibitor comprises a Vpx polypeptide or a functional variant thereof (i.e. retaining the functional properties of Vpx). As used herein, the term “Vpx” includes wild-type naturally occurring Vpx (from any source), as well as modifications and variants thereof which retain the functional properties of Vpx. In some embodiments, amino acid sequence of Vpx comprises the amino acid sequence of SEQ ID NO: 1568 or comprises an amino acid sequence comprising at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the nucleic acid sequence of Vpx is set forth in SEQ ID NO: 3399 or comprises a nucleic acid sequence comprising at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. [0342] As contemplated herein, polypeptide-based SAMHD1 inhibitors (e.g. Vpx, Vpx functional variants thereof, defective SAMHD1 variants) may be provided to facilitate transgene integration in the polypeptide form itself, or as a nucleic acid (DNA or RNA, e.g. mRNA) encoding said polypeptide-based SAMHD1 inhibitor. [0343] The SAMHD1 inhibitors described herein may be used in conjunction with RTE integration systems of the disclosure, in order to effectuate the noted enhanced transgene integration. In some embodiments, the RTE integration systems further comprise at least one 84 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 SAMHD1 inhibitor. In other embodiments, the at least one SAMHD1 inhibitor is provided in combination with an RTE integration system of the disclosure. In some embodiments, the SAMHD1 inhibitor is genetically encoded (e.g. provided as a nucleic acid sequence encoding a SAMHD1 inhibitor polypeptide). [0344] Accordingly, in some embodiments, the driver nucleic acid and template nucleic acid are provided in cis (i.e. on a single nucleic acid), and a genetically encoded SAMHD1 inhibitor is provided in trans, and is provided as a separate nucleic acid sequence encoding the SAMHD1 inhibitor polypeptide. In some embodiments, the driver nucleic acid and template nucleic acid are provided in cis (i.e. on a single nucleic acid), and a genetically encoded SAMHD1 inhibitor is further provided on the same nucleic acid. [0345] Alternatively in other embodiments, the driver nucleic acid and template nucleic acid are provided in trans (i.e. on separate nucleic acids), and a genetically encoded SAMHD1 inhibitor is provided for in cis with the template. In some embodiments, the driver nucleic acid and template nucleic acid are provided in trans (i.e. on separate nucleic acids), and a genetically encoded SAMHD1 inhibitor is provided further in trans, and is provided as yet another separate nucleic acid encoding the SAMHD1 inhibitor polypeptide. In some embodiments, the driver nucleic acid and template nucleic acid are provided in trans (i.e. on separate nucleic acids), and a genetically encoded SAMHD1 inhibitor is provided for in cis with the driver nucleic acid. [0346] In some embodiments, the driver nucleic acid comprising the nucleic acid sequence encoding the RTE polypeptide and the nucleic acid sequence encoding the SAMHD1 inhibitor polypeptide is bicistronic. In some embodiments, the driver nucleic acid comprising the nucleic acid sequence encoding the RTE polypeptide and the nucleic acid sequence encoding the SAMHD1 inhibitor polypeptide, further comprises a IRES sequence. In some embodiments, the driver nucleic acid comprising the nucleic acid sequence encoding the RTE polypeptide and the nucleic acid sequence encoding the SAMHD1 inhibitor polypeptide, further comprises a nucleic acid sequence encoding a self-cleaving 2A peptide, optionally a T2A peptide, a P2A peptide, E2A peptide, or a F2A peptide. [0347] As noted above, the RTE integration systems of the disclosure comprise at least one SAMHD1 inhibitor. In some embodiments, the SAMHD1 inhibitor is not genetically encoded and is provided in the form of a protein. 85 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 Pharmaceutical Compositions [0348] Also provided herein are pharmaceutical compositions comprising any of the nucleic acids, RTE integration systems, and/or delivery systems of the disclosure, and optionally a pharmaceutically acceptable carrier. Methods of Treatment [0349] Provided herein are methods of treatment using the nucleic acids, systems, LNPs, or pharmaceutical compositions of the disclosure. In some embodiments, the nucleic acids, systems, LNPs, or pharmaceutical compositions of the disclosure are used in a method of treatment of treating a disease or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the nucleic acids, systems, LNPs, or pharmaceutical compositions of the disclosure. In some embodiments, the disease or condition is cancer. In some embodiments, the cancer is a lymphoma or leukemia. In some embodiments, the disease or condition is an autoimmune disease. In some embodiments, the disease or condition is an inborn error of metabolism. [0350] In some embodiments, one or more nucleic acid(s) and/or proteins are provided, e.g., in a pharmaceutical composition of the disclosure, for delivery to a cell, or to a subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. [0351] In some embodiments, the route of administration includes, but is not limited to intravenous, intraperitoneal, intramuscular, subcutaneous, intrathecal, and intradermal administration. In some embodiments, administration is via injection or intravenous infusion. In some embodiments, the injection is intramuscular, intraperitoneal, intravascular, or subcutaneous. [0352] In some embodiments, two or more of the nucleic acids, systems, LNPs, or pharmaceutical compositions of the disclosure (e.g., different compositions, for example comprising different nucleic acids) can be administered together or simultaneously. In some embodiments, two or more two or more of the nucleic acids, systems, LNPs, or pharmaceutical compositions of the disclosure (e.g., different compositions, comprising different nucleic acids) can be administered separately (e.g., sequentially). [0353] In some embodiments, a subject is pre-treated with an activating antibody or T cell activator. In some embodiments, the T cell activator is an antibody. In some embodiments, the T cell activator is an CD3 or TCR agonist. In some embodiments, the CD3 agonist is an antibody 86 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 selected from the group consisting of OKT3, Teplizumab, Otelixizumab, Foralumab, Vislizumab, CD3_F1F, CD3_F2B, and modified versions of any of the preceding. In some embodiments, the TCR agonist is BMA031 or a modified version thereof. In some embodiments, the T cell activator is an immune cell engager such as a Bispecific T cell engager (BiTe). In some embodiments, the BiTe is selected from the group consisting of Blinatumomab, MGD006, XmAb14045, AMG 330, AMG 420, AMG 564, AMG 701, BiTe 197, REGN1979, RG6026, Mosunetuzumab, AFM11, MGD006, tebentafusp, Catuxomab, Pasotuxizumab, Acapatamab, AMV564, Glofitamab, Solitomab, Talquetamab, Tarlatamab, Tebentafusp, Odronextamab, Epcoritamab, and modified versions of any of the preceding. [0354] In some embodiments, the subject is administered prior, concurrently, or after administration of an activating agent with at least one γ-chain receptor agonist. In some embodiments, the at least one γ-chain receptor agonist is selected from the group consisting of IL-2, IL-7, IL-15, IL-21, and modified versions of any of the preceding. Methods of In Vivo Genomic Manipulation of an immune cell [0355] Also provided herein are methods of in vivo genomic manipulation of an immune cell. In some embodiments, the method of in vivo genomic manipulation comprises contacting the immune cell with any of the nucleic acids, systems, LNPs, or pharmaceutical compositions of the disclosure. In some embodiments, where a template encodes an engineered immune receptor that is not a TRAM (e.g., where the engineered immune response is a CAR), the immune cell may be a T cell, a B cell, a NK (natural killer) cell, or an NK-T cell. In some embodiments, the immune cell may be an immune cell that natively expresses a TCR-CD3 (T cell receptor-cluster of differentiation 3) complex, e.g., a T cell or a NK-T cell. [0356] In some embodiments, the method of in vivo genomic manipulation comprises priming (pre-activating) T cells. In some embodiments, the T cells are primed by pre-administering a T cell activator, or concomitantly administering the T cell activator with any of the nucleic acids, systems, LNPs, or pharmaceutical compositions of the disclosure. In some embodiments, the T cell activator is an antibody. [0357] In some embodiments, the T cell activator is a CD3 or TCR agonist. In some embodiments, the CD3 agonist is an antibody selected from the group consisting of OKT3, Teplizumab, Otelixizumab, Foralumab, Vislizumab, CD3_F1F, CD3_F2B, and modified versions of any of the preceding. In some embodiments, the TCR agonist is BMA031 or a 87 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 modified version thereof. In some embodiments, the T cell activator is an immune cell engager such as a BiTe. In some embodiments, the BiTe is selected from the group consisting of Blinatumomab, MGD006, XmAb14045, AMG 330, AMG 420, AMG 564, AMG 701, BiTe 197, REGN1979, RG6026, Mosunetuzumab, AFM11, MGD006, tebentafusp, Catuxomab, Pasotuxizumab, Acapatamab, AMV564, Glofitamab, Solitomab, Talquetamab, Tarlatamab, Tebentafusp, Odronextamab, Epcoritamab, and modified versions of any of the preceding. [0358] In some embodiments, the method of in vivo manipulation further comprises administering a reprograming agent. In some embodiments, the reprogramming agent is an RTE integration system comprising one or more nucleic acids. In some embodiments, the reprograming agent meditates durable expression by integrating one or more nucleic acids into a genome. In some embodiments, the reprograming agent mediates transient expression of one or more nucleic acids. In some embodiments, the one or more nucleic acids comprise at least one nucleic acid sequence comprised in or derived from a retrotransposon, a transposon, a Cas protein, a guide RNA, or a combination thereof. In some embodiments, the one or more nucleic acids is packaged in a viral vector or an LNP. In some embodiments, the viral vector is selected from the group consisting of a lentivirus, a retrovirus, an Adeno-associated viral vector (AAV), herpes simplex viral vector, and an adenovirus. [0359] In some embodiments, the RTE integration system comprises the driver nucleic acids, templates, delivery systems, and/or pharmaceutical compositions of the disclosure. In some embodiments, the RTE integration system comprises one or more nucleic acids. In some embodiments, the one or more nucleic acids comprise at least one nucleic acid sequence comprised in or derived from a retrotransposon, a transposon, a Cas protein, a guide RNA, or a combination thereof. ENUMERATED EMBODIMENTS [0360] Provided herein are non-limited exemplary embodiments of the disclosure. Set I [0361] Embodiment I-1. A nucleic acid construct encoding a T cell receptor fused antigen modifier (TRAM), the nucleic acid construct comprising: (a) a nucleic acid sequence encoding an antigen binding domain; (b) a nucleic acid sequence encoding a TCR subunit; and 88 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 (c) a retroelement (RTE)-UTR. [0362] Embodiment I-2. The nucleic acid construct of I-1, wherein the nucleic acid construct comprises more than one construct. [0363] Embodiment I-3. The nucleic acid construct of I-1 or I-2, wherein the TCR subunit is selected from the group consisting of TCRα, TCRβ, CD3δ, CD3γ, and CD3ε. [0364] Embodiment I-4.The nucleic acid construct of any one of I-1 to I-3, wherein the RTE- UTR comprises a 3' RTE-UTR and/or a 5' RTE-UTR. [0365] Embodiment I-5. The nucleic acid construct of any one of I-1 to I-4, wherein the RTE- UTR is derived from a non-LTR RE. [0366] Embodiment I-6. The nucleic acid construct of I-5, wherein the RTE-UTR is derived from an retrotransposable element (RTE) selected from the group consisting of CRE, R4, Hero, NeSL, R2, RandI, Proto1, L1, Tx1, RTEPT, Proto2, RTEX, RTE, Outcast, Ingi (which includes sub-clade Vingi), I, Nimb, Tad1, Loa, R1, Jockey, Rex1, CR1, L2, L2A, L2B, Daphne, and Crack. [0367] Embodiment I-7. The nucleic acid construct of any one of I-4 to I-6, wherein the 5’ RTE- UTR comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 85, SEQ ID NO: 99, SEQ ID NO: 125, SEQ ID NO: 127, any one of SEQ ID NO: 144-249, and any one of SEQ ID NO: 359-367, or a corresponding RNA version thereof. [0368] Embodiment I-8. The nucleic acid construct of any one of I-4 to I-7, wherein the 3’ RTE UTR comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 86, SEQ ID NO: 100, SED ID NO: 126, SEQ ID NO: 128, any one of SEQ ID NO: 250-358, and any one of SEQ ID NO: 368-372, or a corresponding RNA version thereof. [0369] Embodiment I-9. The nucleic acid construct of any one of I-1 to I-8, wherein the antigen binding domain is linked to the N terminus of the TCR subunit by a linker and/or hinge. [0370] Embodiment I-10. The nucleic acid construct of any one of I-1 to I-9, wherein the antigen binding domain contains an scFv, VHH, TCR-like antibody, natural ligand, FcR- binding receptor (e.g. CD16), and an NK killing receptor. [0371] Embodiment I-11. The nucleic acid construct of any one of I-1 to I-10, wherein the antigen binding domain is specific for BCMA. [0372] Embodiment I-12. The nucleic acid construct of any one of I-1 to I-10, wherein the antigen binding domain is specific for CD22. 89 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0373] Embodiment I-13. The nucleic acid construct of any one of I-1 to I-10, wherein the antigen binding domain is specific for CD19. [0374] Embodiment I-14. The nucleic acid construct of I-14, wherein the CD19 antigen binding domain is derived from an antibody selected from the group consisting of FMC63, murine 4G7, human Hu19, Hu1E7, huB4, hBU12, humanized FMC63, humanized 4G7, GR37, GR41, SRB- 37, SRB-85, and a modified version of any of the preceding. [0375] Embodiment I-15. The nucleic acid construct of any one of I-1 to I-10, wherein the antigen binding domain is specific for CD20. [0376] Embodiment I-16. The nucleic acid construct of I-16, wherein the CD20 antigen binding domain is derived from an antibody selected from the group consisting of Leu16, rituximab, ofatumumab, ocrelizumab, obinutuzumab, Ibritumomab tiuxetan, AME-133v, IMMU-106, TRU-015, tositumomab, and a modified version of any of the preceding. [0377] Embodiment I-17. The nucleic acid construct of any one of I-1 to I-10, wherein the antigen binding domain is bispecific. [0378] Embodiment I-18. The nucleic acid construct of any one of I-1 to I-18, wherein the nucleic acid comprises DNA. [0379] Embodiment I-19. The nucleic acid construct of any one of I-1 to I-18, wherein the nucleic acid comprises RNA. [0380] Embodiment I-20. The nucleic acid construct of I-20, wherein the RNA is mRNA. [0381] Embodiment I-21. A retrotransposable element (RTE) transgene integration system comprising a driver construct and a template, wherein (a) the driver nucleic acid comprises a nucleic acid encoding a site specific RTE polypeptide; and (b) the template comprises (i) a nucleic acid encoding an engineered immune receptor; (ii) a retroelement (RTE)-UTR, wherein the RTE-UTR is capable of binding the RTE polypeptide; and wherein the site specific RTE polypeptide mediates integration of the nucleic acid encoding the engineered immune receptor into a T cell genome. [0382] Embodiment I-22. The system of I-22, wherein the engineered immune receptor is selected from the group consisting of a T cell receptor (TCR), TCR fused antigen modifier (TRAM), and chimeric antigen receptor (CAR). 90 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0383] Embodiment I-23. A retrotransposable element (RTE) transgene integration system comprising a driver construct and a template, wherein (a) the driver construct comprises a nucleic acid encoding an RTE polypeptide; and (b) the template comprises (i) a nucleic acid encoding a T cell receptor fused antigen modifier (TRAM); and (ii) a retroelement (RE)-UTR, wherein the RTE-UTR is capable of binding the RTE polypeptide; and wherein the RTE polypeptide mediates integration of the nucleic acid encoding the TRAM into a genome. [0384] Embodiment I-24. The system of any one of I-22 to I-25, wherein the driver construct and the template are in a trans configuration and provided as separate nucleic acids. [0385] Embodiment I-25. The system of any one of I-22 to I-25, wherein the driver construct and the template are in a cis configuration and provided in the same nucleic acid. [0386] Embodiment I-26. The system of any one of I-22 to I-29, wherein the RTE-UTR comprises a 3' RTE-UTR and/or a 5' RTE-UTR . [0387] Embodiment I-27. The system of I-30, wherein the 5’ RTE-UTR comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 85, SEQ ID NO: 99, SEQ ID NO: 125, SEQ ID NO: 127, any one of SEQ ID NO: 144-249, and any one of SEQ ID NO: 359-367, or a corresponding RNA version thereof. [0388] Embodiment I-28. The system of I-30 or I-31, wherein the RTE3’ UTR comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 86, SEQ ID NO: 100, SED ID NO: 126, SEQ ID NO: 128, any one of SEQ ID NO: 250-358, and any one of SEQ ID NO: 368-372, or a corresponding RNA version thereof. [0389] Embodiment I-29. The system of any one of I-22 to I-32, wherein the TRAM comprises an antigen binding domain, and a TCR subunit selected from the group consisting of TCRα, TCRβ, CD3δ, CD3γ, and CD3ε. [0390] Embodiment I-30. The system of any one of I-22 to I-33, wherein the antigen binding domain is selected from the group consisting of an scFv, VHH, TCR-like antibody, natural ligand, FcR-binding receptor (e.g. CD16), and an NK killing receptor. [0391] Embodiment I-31. The system of I-34, wherein the antigen binding domain is specific for CD19. 91 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0392] Embodiment I-32. The system of I-35, wherein the CD19 antigen binding domain is selected from the group consisting of FMC63, murine 4G7, human Hu19, Hu1E7, huB4, hBU12, humanized FMC63, humanized 4G7, GR37, GR41, SRB-37, SRB-85, and modified versions of any of the preceding. [0393] Embodiment I-33. The system of I-34, wherein the antigen binding domain is specific for CD20. [0394] Embodiment I-34. The system of I-38, wherein the CD20 antigen binding domain is selected from the group consisting of Leu16, rituximab, ofatumumab, ocrelizumab, obinutuzumab, Ibritumomab tiuxetan, AME-133v, IMMU-106, TRU-015, tositumomab, and modified versions of any of the preceding. [0395] Embodiment I-35. The system of I-34, wherein the antigen binding domain is bispecific for CD19 and CD20. [0396] Embodiment I-36. The system of any one of I-22 to I-40, wherein the RTE polypeptide is a non-LTR RTE. [0397] Embodiment I-37.The system of any one of I-22 to I-42, wherein the RTE polypeptide is derived from an RTE selected from the group consisting of CRE, R4, Hero, NeSL, R2, RandI, Proto1, L1, Tx1, RTEPT, Proto2, RTEX, RTE, Outcast, Ingi (which includes sub-clade Vingi), I, Nimb, Tad1, Loa, R1, Jockey, Rex1, CR1, L2, L2A, L2B, Daphne, and Crack. [0398] Embodiment I-38. The system of any one of I-22 to I-43, wherein RTE polypeptide is a naturally occurring RTE polypeptide. [0399] Embodiment I-39. The system of any one of I-22 to I-43, wherein RTE polypeptide is an engineered RTE polypeptide. [0400] Embodiment I-40. The system of any one of I-22 to I-45, wherein the template comprises a binding site for a differentially expressed microRNA (miR). [0401] Embodiment I-41. The system of I-46, wherein the binding site for the differentially expressed miR is located within the RTE3’ UTR region. [0402] Embodiment I-42. The system of I-46 or I-47, wherein the binding site for the differentially expressed miR is specific for miR-122. [0403] Embodiment I-43. The system of any one of I-22 to I-48, wherein the driver nucleic acid comprises a binding site for a differentially expressed miR. [0404] Embodiment I-44. The system of I-49, wherein the binding site for the differentially expressed miR is specific for miR-122. 92 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0405] Embodiment I-45. The system of any one of I-22 to I-50, wherein the driver construct comprises DNA. [0406] Embodiment I-46. The system of any one of I-22 to I-50, wherein the driver construct comprises RNA. [0407] Embodiment I-47. The system of I-52, wherein the RNA is mRNA. [0408] Embodiment I-48. The system of any one of I-22 to I-54, wherein the template comprises DNA. [0409] Embodiment I-49. The system of any one of I-22 to I-54, wherein the template comprises RNA. [0410] Embodiment I-50. The system of I-56, wherein the RNA is mRNA. [0411] Embodiment I-51. A lipid nanoparticle (LNP) comprising the system of any one of I-22 to I-58. [0412] Embodiment I-52. The LNP of I-59, wherein the LNP is covalently linked to an antibody that recognizes an T cell antigen. [0413] Embodiment I-53. The LNP of I-60, wherein the covalently linked antibody comprises a silent Fc domain. [0414] Embodiment I-54. The LNP of I-60 or I-61, wherein the antibody is specific for one or more of CD2, CD5, CD7, TCR, CD8, and CD3. [0415] Embodiment I-55. The LNP of any one of I-60 to I-62, wherein the antibody is selected from the group consisting of OKT3 (VH SEQ ID NO: 63, VL SEQ ID NO: 64), BMA031 (VH SEQ ID NO: 65, VL SEQ ID NO: 66), H65 (VH SEQ ID NO: 67, VL SEQ ID NO: 68), 3A1E (VH SEQ ID NO: 69, VL SEQ ID NO: 70), OKT8 (VH SEQ ID NO: 71, VL SEQ ID NO: 72), Visilizumab (VH SEQ ID NO: 73, VL SEQ ID NO: 74), Teplizumab (VH SEQ ID NO: 88, VL SEQ ID NO: 89), Siplizumab (VH SEQ ID NO: 92, VL SEQ ID NO: 93), Urelumab (VH SEQ ID NO: 94, VL SEQ ID NO: 95), Otelixizumab (VH SEQ ID NO: 101, VL SEQ ID NO: 102), Foralumab (VH SEQ ID NO: 105, VL SEQ ID NO: 106), and modified versions of any of the preceding. [0416] Embodiment I-56. A pharmaceutical composition comprising: (a) the nucleic acid construct of any one of I-1 to I-21; (b) the RTE transgene integration system of any one of I-22 to I-58; or (c) the LNP of any one of I-59 to I-63, and a pharmaceutically acceptable carrier. 93 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0417] Embodiment I-57. A method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of I-64. [0418] Embodiment I-58. The method of I-65, wherein the subject is pre-treated with a T cell activator. [0419] Embodiment I-59. The method of I-66, wherein the T cell activator is a CD3 or TCR agonist. [0420] Embodiment I-60. The method of I-67, wherein the CD3 agonist is an antibody selected from the group consisting of OKT3, Teplizumab, Otelixizumab, Foralumab, Vislizumab, CD3_F1F, CD3_F2B, and modified versions of any of the preceding. [0421] Embodiment I-61. The method of I-67, wherein the TCR agonist is BMA031 or a modified version thereof. [0422] Embodiment I-62. The method of I-66, wherein the T cell activator is an immune cell engager such as a Bispecific T cell engager (BiTe). [0423] Embodiment I-63. The method of I-70, wherein the BiTe is selected from the group consisting of Blinatumomab, MGD006, XmAb14045, AMG 330, AMG 420, AMG 564, AMG 701, BiTe 197, REGN1979, RG6026, Mosunetuzumab, AFM11, MGD006, tebentafusp, Catuxomab, Pasotuxizumab, Acapatamab, AMV564, Glofitamab, Solitomab, Talquetamab, Tarlatamab, Tebentafusp, Odronextamab, Epcoritamab, and modified versions of any of the preceding. [0424] Embodiment I-64. The method of any one of I-65 to I-72, wherein the subject is further administered prior, concurrently, or after said administering with at least one γ-chain receptor agonist. [0425] Embodiment I-65. The method of I-73, wherein the at least one γ-chain receptor agonist is selected from the group consisting of IL-2, IL-7, IL-15, IL-21, and modified versions of any of the preceding. [0426] Embodiment I-66. The method of any one of I-65 to I-74, wherein the disease is cancer or an autoimmune disease. [0427] Embodiment I-67. The method of any one of I-65 to I-75, wherein the route of administration is selected from the group consisting of intravenous, intraperitoneal, intramuscular, subcutaneous, intrathecal, and intradermal administration. 94 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0428] Embodiment I-68. A method of in vivo gene editing comprising pre-administering a T cell activator and administering a pharmaceutical composition comprising a reprograming agent. [0429] Embodiment I-69. The method of I-77, wherein the reprogramming agent is a gene editing system comprising one or more nucleic acids. [0430] Embodiment I-70. The method of I-77, wherein the reprograming agent meditates durable expression by integrating one or more nucleic acids into a genome. [0431] Embodiment I-71. The method of I-77, wherein the reprograming agent mediates transient expression of one or more nucleic acids. [0432] Embodiment I-72. The method of any one of I-77 to I-80, wherein the one or more nucleic acids comprise at least one nucleic acid sequence comprised in or derived from a retrotransposon, a transposon, a Cas protein, a guide RNA, or a combination thereof. [0433] Embodiment I-73. The method of any one of I-77 to I-81, wherein the one or more nucleic acids is packaged in a viral vector or a lipid nanoparticle (LNP). [0434] Embodiment I-74. The method of I-83, wherein the viral vector is selected from the group consisting of a lentivirus, a retrovirus, and an adenovirus. [0435] Embodiment I-75. The method of any one of I-77 to I-84, wherein the T cell activator is a CD3 or TCR agonist. [0436] Embodiment I-76. The method of I-85, wherein the CD3 agonist is an antibody selected from the group consisting of OKT3, Teplizumab, Otelixizumab, Foralumab, Vislizumab, CD3_F1F, CD3_F2B, and modified versions of any of the preceding. [0437] Embodiment I-77. The method of I-85, wherein the TCR agonist is BMA031 or a modified version thereof. [0438] Embodiment I-78. The method of I-77 to I-84, wherein the T cell activator is an immune cell engager such as a BiTe. [0439] Embodiment I-79. The method of I-78, wherein the BiTe is selected from the group consisting of Blinatumomab, MGD006, XmAb14045, AMG 330, AMG 420, AMG 564, AMG 701, BiTe 197, REGN1979, RG6026, Mosunetuzumab, AFM11, MGD006, tebentafusp, Catuxomab, Pasotuxizumab, Acapatamab, AMV564, Glofitamab, Solitomab, Talquetamab, Tarlatamab, Tebentafusp, Odronextamab, Epcoritamab, and modified versions of any of the preceding. 95 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 Set II [0440] Embodiment II-1. A nucleic acid encoding a T cell receptor fused antigen modifier (TRAM), the nucleic acid comprising: (a) a nucleic acid sequence encoding an antigen binding domain; (b) a nucleic acid sequence encoding a TCR subunit; and (c) a retroelement (RTE)-UTR. [0441] Embodiment II-2. The nucleic acid of II-1, wherein the nucleic acid construct comprises more than one construct. [0442] Embodiment II-3. The nucleic acid of II-1 or II-2, wherein the TCR subunit is selected from the group consisting of TCRα, TCRβ, CD3δ, CD3γ, and CD3ε. [0443] Embodiment II-4. The nucleic acid of any one of II-1 to II-3, wherein the RTE-UTR comprises a 3’ RTE-UTR and/or a 5’ RTE-UTR. [0444] Embodiment II-5. The nucleic acid of any one of II-1-4, wherein the RTE-UTR is derived from a non-LTR RE. [0445] Embodiment II-6. The nucleic acid of II-5, wherein the RTE-UTR is derived from an retrotransposable element (RTE) selected from the group consisting of CRE, R4, Hero, NeSL, R2, RandI, Proto1, L1, Tx1, RTEPT, Proto2, RTEX, RTE, Outcast, Ingi (which includes sub- clade Vingi), I, Nimb, Tad1, Loa, R1, Jockey, Rex1, CR1, L2, L2A, L2B, Daphne, and Crack. [0446] Embodiment II-7. The nucleic acid of any one of II-4-6, wherein the 5’ RTE-UTR comprises a nucleic acid sequence selected from the group consisting of SEQ ID SEQ ID NO: 33, SEQ ID NO: 85, SEQ ID NO: 99, SEQ ID NO: 125, SEQ ID NO: 127, any one of SEQ ID NO: 144-249, and any one of SEQ ID NO: 359-367, or a corresponding RNA version thereof. [0447] Embodiment II-8. The nucleic acid of any one of II-4-7, wherein the 3’ RTE UTR comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 86, SEQ ID NO: 100, SED ID NO: 126, SEQ ID NO: 128, any one of SEQ ID NO: 250- 358, and any one of SEQ ID NO: 368-372, or a corresponding RNA version thereof. [0448] Embodiment II-9. The nucleic acid of any one of II-1-8, wherein the antigen binding domain is linked to the N terminus of the TCR subunit by a linker and/or hinge. [0449] Embodiment II-10. The nucleic acid of any one of II-1-9, wherein the antigen binding domain is selected from the group consisting of an scFv, VHH, TCR-like antibody, natural ligand, FcR-binding receptor (e.g. CD16), and an NK killing receptor. 96 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0450] Embodiment II-11. The nucleic acid of any one of II-1-10, wherein the antigen binding domain is specific for GPCR5D. [0451] Embodiment II-12. The nucleic acid of any one of II-1-10, wherein the antigen binding domain is specific for BCMA. [0452] Embodiment II-13. The nucleic acid of any one of II-1-10, wherein the antigen binding domain is specific for CD22. [0453] Embodiment II-14. The nucleic acid of any one of II-1-10, wherein the antigen binding domain is specific for CD19. [0454] Embodiment II-15. The nucleic acid of II-14, wherein the CD19 antigen binding domain is derived from an antibody selected from the group consisting of FMC63, murine 4G7, human Hu19, Hu1E7, huB4, hBU12, humanized FMC63, humanized 4G7, GR37, GR41, SRB-37,SRB- 85, and a modified version of any of the preceding. [0455] Embodiment II-16. The nucleic acid of any one of II-1-10, wherein the encoded antigen binding domain is specific for CD20. [0456] Embodiment II-17. The nucleic acid of claim16, wherein the CD20 antigen binding domain is derived from an antibody selected from the group consisting of Leu16, rituximab, ofatumumab, ocrelizumab, obinutuzumab, Ibritumomab tiuxetan, AME-133v, IMMU-106, TRU-015, tositumomab, and a modified version of any of the preceding. [0457] Embodiment II-18. The nucleic acid of any one of II-1-10, wherein the encoded antigen binding domain is bispecific. [0458] Embodiment II-19. The nucleic acid of any one of II-1 to II-18, wherein the nucleic acid construct comprises DNA. [0459] Embodiment II-20. The nucleic acid of any one of II-1 to II-18, wherein the nucleic acid construct comprises RNA. [0460] Embodiment II-21. The nucleic acid of II-20, wherein the RNA is mRNA. [0461] Embodiment II-22. A retrotransposable-element (RTE) transgene integration system comprising a driver nucleic acid and a template, wherein (a) the driver nucleic acid comprises a nucleic acid encoding a site specific RTE polypeptide; and (b) the template comprises (i) a nucleic acid encoding an engineered immune receptor; 97 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 (ii) a retroelement (RE)-UTR, wherein the RTE-UTR is capable of being bound by the RTE polypeptide; and wherein the site specific RTE polypeptide mediates integration of a DNA copy of the template into a T cell genome. [0462] Embodiment II-23. The system of II-22, wherein the engineered immune receptor is selected from the group consisting of a T cell receptor (TCR), TCR fused antigen modifier (TRAM), TRuC and chimeric antigen receptor (CAR). [0463] Embodiment II-24. A retrotransposable-element (RTE) transgene integration system comprising a driver nucleic acid and a template, wherein a) the driver nucleic acid comprises a nucleic acid encoding an RTE polypeptide; and (b) the template comprises (i) a nucleic acid encoding a T cell receptor fused antigen modifier (TRAM); and (ii) a retroelement (RTE)-UTR, wherein the RTE-UTR is capable of being bound by the RTE polypeptide; and wherein the RTE polypeptide mediates a DNA copy of the template into a T cell genome. [0464] Embodiment II-25. The system of any one of II-22 to II-24, wherein the driver nucleic acid and the template are in a trans configuration and provided as separate nucleic acid constructs. [0465] Embodiment II-26. The system of any one of II-22 to II-24, wherein the driver nucleic acid and the template are in a cis configuration and provided in the same nucleic acid construct. [0466] Embodiment II-27. The system of any one of II-22 to II-26, wherein the RTE-UTR comprises a 3’ RTE-UTR and/or a 5’ RTE-UTR. [0467] Embodiment II-28. The system of II-27, wherein the 5’ RTE-UTR comprises a nucleic acid sequence selected from the group consisting of SEQ ID SEQ ID NO: 33, SEQ ID NO: 85, SEQ ID NO: 99, SEQ ID NO: 125, SEQ ID NO: 127, any one of SEQ ID NO: 144-249, and any one of SEQ ID NO: 359-367, or a corresponding RNA version thereof. [0468] Embodiment II-29. The system of II-27 or II-28, wherein the 3’ RTE UTR comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 86, SEQ ID NO: 100, SED ID NO: 126, SEQ ID NO: 128, any one of SEQ ID NO: 250-358, and any one of SEQ ID NO: 368-372, or a corresponding RNA version thereof. 98 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0469] Embodiment II-30. The system of any one of II-22-29, wherein the TRAM comprises an antigen binding domain, and a TCR subunit selected from the group consisting of TCRα, TCRβ, CD3δ, CD3γ, and CD3ε. [0470] Embodiment II-31. The system of any one of II-22-30, wherein the antigen binding domain is selected from the group consisting of an scFv, VHH, TCR-like antibody, natural ligand, FcR-binding receptor (e.g. CD16), and an NK killing receptor. [0471] Embodiment II-32. The system of II-31, wherein the antigen binding domain is specific for CD19. [0472] Embodiment II-33. The system of II-32, wherein the CD19 antigen binding domain is selected from the group consisting of FMC63, murine 4G7, human Hu19, Hu1E7, huB4, hBU12, humanized FMC63, humanized 4G7, GR37, GR41, SRB-37, SRB-85, and modified versions of any of the preceding. [0473] Embodiment II-34. The system of II-31, wherein the antigen binding domain is specific for CD20. [0474] Embodiment II-35. The system of II-34, wherein the CD20 antigen binding domain is selected from the group consisting of Leu16, rituximab, ofatumumab, ocrelizumab, obinutuzumab, Ibritumomab tiuxetan, AME-133v, IMMU-106, TRU-015, tositumomab, and modified versions of any of the preceding. [0475] Embodiment II-36. The system of II-31, wherein the antigen binding domain is bispecific for CD19 and CD20. [0476] Embodiment II-37. The system of any one of II-22 to II-36, wherein the RTE polypeptide is a non-LTR RTE. [0477] Embodiment II-38. The system of any one of II-22 to II-37, wherein the RTE polypeptide is derived from an RTE selected from the group consisting of CRE, R4, Hero, NeSL, R2, RandI, Proto1, L1, Tx1, RTEPT, Proto2, RTEX, RTE, Outcast, Ingi (which includes sub-clade Vingi), I, Nimb, Tad1, Loa, R1, Jockey, Rex1, CR1, L2, L2A, L2B, Daphne, and Crack. [0478] Embodiment II-39. The system of any one of II-22 to II-38, wherein RTE polypeptide is a naturally occurring RTE polypeptide. [0479] Embodiment II-40. The system of any one of II-22 to II-38, wherein RTE polypeptide is an engineered RTE polypeptide. 99 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0480] Embodiment II-41. The system of any one of II-22 to II-40, wherein the template comprises a binding site for a differentially expressed microRNA (miR). [0481] Embodiment II-42. The system of II-41, wherein the binding site for the differentially expressed miR is located within the 3’ RTE UTR region. [0482] Embodiment II-43. The system of II-41 or II-42, wherein the binding site for the differentially expressed miR is specific for miR-122. [0483] Embodiment II-44. The system of any one of II-22 to II-43, wherein the driver nucleic acid comprises a binding site for a differentially expressed miR. [0484] Embodiment II-45. The system of II-44, wherein the binding site for the differentially expressed miR is specific for miR-122. [0485] Embodiment II-46. The system of any one of II-22 to II-45, wherein the driver nucleic acid comprises DNA. [0486] Embodiment II-47. The system of any one of II-22 to II-45, wherein the driver nucleic acid comprises RNA. [0487] Embodiment II-48. The system of II-47, wherein the RNA is mRNA. [0488] Embodiment II-49. The system of any one of II-22 to II-48, wherein the template comprises DNA. [0489] Embodiment II-50. The system of any one of II-22 to II-48, wherein the template comprises RNA. [0490] Embodiment II-51. The system of II-50, wherein the template comprises a promotor and a polyA signal. [0491] Embodiment II-52. A lipid nanoparticle (LNP) comprising the nucleic acid of any one of II-1 to II-21 or the system of any one of II-22to II-51. [0492] Embodiment II-53. The LNP of II-52, wherein the LNP is covalently linked to an antibody or any fragment thereof that recognizes an T cell antigen. [0493] Embodiment II-54. The LNP of II-53, wherein the covalently linked antibody comprises a silent Fc domain. [0494] Embodiment II-55. The LNP of II-53 or II-54, wherein the antibody is specific for one or more of CD2, CD5, CD7, TCR, CD8, and CD3. [0495] Embodiment II-56. The LNP of any one of II-53 to II-55, wherein the antibody is selected from the group consisting of OKT3 (VH SEQ ID NO: 63, VL SEQ ID NO: 64), BMA031 (VH SEQ ID NO: 65, VL SEQ ID NO: 66), H65 (VH SEQ ID NO: 67, VL SEQ ID 100 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 NO: 68), 3A1E (VH SEQ ID NO: 69, VL SEQ ID NO: 70), OKT8 (VH SEQ ID NO: 71, VL SEQ ID NO: 72), Visilizumab (VH SEQ ID NO: 73, VL SEQ ID NO: 74), Teplizumab (VH SEQ ID NO: 88, VL SEQ ID NO: 89), Siplizumab (VH SEQ ID NO: 92, VL SEQ ID NO: 93), Urelumab (VH SEQ ID NO: 94, VL SEQ ID NO: 95), Otelixizumab (VH SEQ ID NO: 101, VL SEQ ID NO: 102), Foralumab (VH SEQ ID NO: 105, VL SEQ ID NO: 106), and modified versions of any of the preceding. [0496] Embodiment II-57. A pharmaceutical composition comprising: (a) the nucleic acid of any one of II-1 to II-21; (b) the RTE transgene integration system of any one of II-22 to II-51; or (c) the LNP of any one of II-52 to II-56. [0497] and a pharmaceutically acceptable carrier. [0498] Embodiment II-58. A method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of (a) the nucleic acid of any one of II-1 to II-21; (b) the RTE transgene integration system of any one of II-22 to II-51; or (c) the LNP of any one of II-52 to II-56, (d) the pharmaceutical composition of II-57. [0499] Embodiment II-59. The method of II-58, wherein the subject is pre-treated with a T cell activator. [0500] Embodiment II-60 The method of II-59, wherein the T cell activator is an CD3 or TCR agonist. [0501] Embodiment II-61. The method of II-60, wherein the CD3 agonist is an antibody selected from the group consisting of OKT3, Teplizumab, Otelixizumab, Foralumab, Vislizumab, CD3_F1F, CD3_F2B, and modified versions of any of the preceding. [0502] Embodiment II-62. The method of II-60, wherein the TCR agonist is BMA031 or a modified version thereof. [0503] Embodiment II-63. The method of II-59, wherein the T cell activator is an immune cell engager such as a Bispecific T cell engager (BiTe). [0504] Embodiment II-64. The method of II-63, wherein the BiTe is selected from the group consisting of Blinatumomab, MGD006, XmAb14045, AMG 330, AMG 420, AMG 564, AMG 701, BiTe 197, REGN1979, RG6026, Mosunetuzumab, AFM11, MGD006, tebentafusp, Catuxomab, Pasotuxizumab, Acapatamab, AMV564, Glofitamab, Solitomab, Talquetamab, 101 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 Tarlatamab, Tebentafusp, Odronextamab, Epcoritamab, and modified versions of any of the preceding. [0505] Embodiment II-65. The method of any one of II-58-64, wherein the subject is further administered prior, concurrently, or after said administering with at least one γ-chain receptor agonist. [0506] Embodiment II-66. The method of II-65, wherein the at least one γ-chain receptor agonist is selected from the group consisting of IL-2, IL-7, IL-15, IL-21, and modified versions of any of the preceding. [0507] Embodiment II-67. The method of any one of II-58 to II-66, wherein the disease is cancer or an autoimmune disease. [0508] Embodiment II-68. The method of any one of II-58 to II-67, wherein the route of administration is selected from the group consisting of intravenous, intraperitoneal, intramuscular, subcutaneous, intrathecal, and intradermal administration. [0509] Embodiment II-69. A method of in vivo gene editing comprising pre-administering a T cell activator and administering a pharmaceutical composition comprising a reprograming agent. [0510] Embodiment II-70. The method of II-69, wherein the reprogramming agent is a gene editing system comprising one or more nucleic acids. [0511] Embodiment II-71. The method of II-69, wherein the reprograming agent meditates durable expression by integrating one or more nucleic acids into a genome. [0512] Embodiment II-72. The method of II-69, wherein the reprograming agent mediates transient expression of one or more nucleic acids. [0513] Embodiment II-73. The method of any one of II-69 to II-72, wherein the one or more nucleic acids comprise at least one nucleic acid sequence comprised in or derived from a retrotransposon, a transposon, a Cas protein, a guide RNA, or a combination thereof. [0514] Embodiment II-74. The method of any one of II-69 to II-73, wherein the one or more nucleic acids is packaged in a viral vector or a lipid nanoparticle (LNP). [0515] Embodiment II-75. The method of II-74, wherein the viral vector is selected from the group consisting of a lentivirus, a retrovirus, and an adenovirus. [0516] Embodiment II-76. The method of any one of II-69 to II-75, wherein the T cell activator is an CD3 or TCR agonist. 102 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0517] Embodiment II-77. The method of II-76, wherein the CD3 agonist is an antibody selected from the group consisting of OKT3, Teplizumab, Otelixizumab, Foralumab, Vislizumab, CD3_F1F, CD3_F2B, and modified versions of any of the preceding. [0518] Embodiment II-78. The method of II-76, wherein the TCR agonist is BMA031 or a modified version thereof. [0519] Embodiment II-79. The method of II-69 to II-75, wherein the T cell activator is an immune cell engager such as a BiTe. [0520] Embodiment II-80. The method of II-79, wherein the BiTe is selected from the group consisting of Blinatumomab, MGD006, XmAb14045, AMG 330, AMG 420, AMG 564, AMG 701, BiTe 197, REGN1979, RG6026, Mosunetuzumab, AFM11, MGD006, tebentafusp, Catuxomab, Pasotuxizumab, Acapatamab, AMV564, Glofitamab, Solitomab, Talquetamab, Tarlatamab, Tebentafusp, Odronextamab, Epcoritamab, and modified versions of any of the preceding. [0521] Embodiment II-81. A method of in vivo genomic manipulation in an immune cell, the method comprising contacting the immune cell with: (a) the nucleic acid of any one of II-1 to II-21; (b) the system of any one of II-22 to II-51; (c) the LNP of any one of II-52 to II-56; (d) the pharmaceutical composition of II-57; or (e) a combination thereof. Set III [0522] Embodiment III-1. A nucleic acid comprising: (a) a nucleic acid sequence encoding a T cell receptor fused antigen modifier (TRAM) comprising: (i) an antigen binding domain; and (ii) a TCR subunit, and (b) a retrotransposable element untranslated region (RTE-UTR). [0523] Embodiment III-2. The nucleic acid of III-1, wherein the nucleic acid comprises more than one separate nucleic acids. 103 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0524] Embodiment III-3. The nucleic acid of III-1 or Embodiment III-2, wherein the TCR subunit is selected from the group consisting of TCRα, TCRβ, CD3δ, CD3γ, and CD3ε. [0525] Embodiment III-4. The nucleic acid of any one of III-1 to III-3, wherein the RTE-UTR comprises a 3’ RTE-UTR and/or a 5’ RTE-UTR. [0526] Embodiment III-5. The nucleic acid of any one of III-1 to III-4, wherein the RTE-UTR is derived from a non-LTR RTE. [0527] Embodiment III-6. The nucleic acid of III-5, wherein the non-LTR RTE is selected from the group consisting of CRE, R4, Hero, NeSL, R2, RandI, Proto1, L1, Tx1, RTEPT, Proto2, RTEX, RTE, Outcast, Ingi, I, Nimb, Tad1, Loa, R1, Jockey, Rex1, CR1, L2, L2A, L2B, Daphne, and Crack. [0528] Embodiment III-7. The nucleic acid of any one of III-4 to III-6, wherein the 5’ RTE-UTR comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 85, SEQ ID NO: 99, SEQ ID NO: 125, SEQ ID NO: 127, any one of SEQ ID NO: 144- 249, and any one of SEQ ID NO: 359-367, or modified versions of any of the preceding sequences having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, or a corresponding RNA version thereof. [0529] Embodiment III-8. The nucleic acid of any one of III-4 to III-7, wherein the 3’ RTE UTR comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 86, SEQ ID NO: 100, SED ID NO: 126, SEQ ID NO: 128, any one of SEQ ID NO: 250- 358, and any one of SEQ ID NO: 368-372, or modified versions of any of the preceding sequences having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, or a corresponding RNA version thereof. [0530] Embodiment III-9. The nucleic acid of any one of III-1 to III-8, wherein the antigen binding domain is linked to the N terminus of the TCR subunit, optionally by a linker and/or hinge. [0531] Embodiment III-10. The nucleic acid of any one of III-1to III-9, wherein the antigen binding domain is selected from the group consisting of an scFv, a VHH, a TCR-like antibody, a natural ligand, an FcR-binding receptor (e.g. CD16), and an NK killing receptor. [0532] Embodiment III-11. The nucleic acid of any one of III-1 to III-10, wherein the antigen binding domain is specific for GPCR5D. [0533] Embodiment III-12. The nucleic acid of any one of III-1 to III-10, wherein the antigen binding domain is specific for BCMA. 104 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0534] Embodiment III-13. The nucleic acid of any one of III-1 to III-10, wherein the antigen binding domain is specific for CD22. [0535] Embodiment III-14. The nucleic acid of any one of III-1 to III-10, wherein the antigen binding domain is specific for CD19. [0536] Embodiment III-15. The nucleic acid of III-14, wherein the CD19 antigen binding domain is derived from an antibody selected from the group consisting of FMC63, murine 4G7, human Hu19, Hu1E7, huB4, hBU12, humanized FMC63, humanized 4G7, GR37, GR41, SRB- 37, and SRB-85, or a modified version of any of the preceding. [0537] Embodiment III-16. The nucleic acid of any one of III-1 to III-10, wherein the encoded antigen binding domain is specific for CD20. [0538] Embodiment III-17. The nucleic acid of III-, wherein the CD20 antigen binding domain is derived from an antibody selected from the group consisting of Leu16, rituximab, ofatumumab, ocrelizumab, obinutuzumab, Ibritumomab tiuxetan, AME-133v, IMMU-106, TRU-015, and tositumomab, or a modified version of any of the preceding. [0539] Embodiment III-18. The nucleic acid of any one of III-1 to III-17, wherein the antigen binding domain is a bispecific antigen binding domain. [0540] Embodiment III-19. The nucleic acid of any one of III-1 to III-18, wherein the nucleic acid comprises DNA. [0541] Embodiment III-20. The nucleic acid of any one of claims 1-18 wherein the nucleic acid comprises RNA. [0542] Embodiment III-21. The nucleic acid of any one of III-1 to III-18, wherein the nucleic acid is a RNA/DNA hybrid. [0543] Embodiment III-22. A retrotransposable-element (RTE) integration system comprising a driver nucleic acid and a template nucleic acid, wherein: (a) the driver nucleic acid comprises a nucleic acid sequence encoding an RTE polypeptide, wherein the RTE polypeptide is a site-specific RTE polypeptide; and (b) the template nucleic acid comprises: (i) a nucleic acid sequence encoding an engineered immune receptor; and (ii) an RTE-UTR capable of being bound by the RTE polypeptide. [0544] Embodiment III-23. The system of III-22, wherein the engineered immune receptor comprises an antigen binding domain. 105 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0545] Embodiment III-24. The system of III-22 or III-23, wherein the engineered immune receptor is selected from the group consisting of a T cell receptor (TCR), a TCR fused antigen modifier (TRAM), a TRuC and a chimeric antigen receptor (CAR). [0546] Embodiment III-25. A retrotransposable-element (RTE) integration system comprising a driver nucleic acid and a template nucleic acid, wherein: (a) the driver nucleic acid comprises a nucleic acid sequence encoding an RTE polypeptide; and (b) the template nucleic acid comprises (i) a nucleic acid sequence encoding a T cell receptor fused antigen modifier (TRAM) comprising an antigen binding domain and a TCR subunit; and (ii) an RTE-UTR capable of being bound by the RTE polypeptide. [0547] Embodiment III-26. The system of any one of III-22 to III-25, wherein the RTE polypeptide mediates integration of a DNA copy of the nucleic acid sequence encoding an engineered immune receptor into a genome of an immune cell that natively expresses a TCR- CD3 complex. [0548] Embodiment III-27. The system of III-26, wherein the immune cell that natively expresses a TCR-CD3 complex is a T-cell. [0549] Embodiment III-28. The system of any one of III-22 to III-27, wherein the driver nucleic acid and the template nucleic acid are provided as separate nucleic acids. [0550] Embodiment III-29. The system of any one of III-22 to III-27, wherein the driver nucleic acid and the template are provided in a same nucleic acid. [0551] Embodiment III-30. The system of any one of III-22 to III-29, wherein the RTE-UTR comprises a 3’ RTE-UTR and/or a 5’ RTE-UTR. [0552] Embodiment III-31. The system of III-30, wherein the 5’ RTE-UTR comprises a nucleic acid sequence selected from the group consisting of SEQ ID SEQ ID NO: 33, SEQ ID NO: 85, SEQ ID NO: 99, SEQ ID NO: 125, SEQ ID NO: 127, any one of SEQ ID NO: 144-249, and any one of SEQ ID NO: 359-367, or modified versions of any of the preceding sequences having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, or a corresponding RNA version thereof. [0553] Embodiment III-32. The system of III-30 or III-31, wherein the 3’ RTE UTR comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 86, SEQ ID NO: 100, SED ID NO: 126, SEQ ID NO: 128, any one of SEQ ID NO: 250- 106 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 358, and any one of SEQ ID NO: 368-372, or modified versions of any of the preceding sequences having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, or a corresponding RNA version thereof. [0554] Embodiment III-33. The system of any one of III-25 to III-32, wherein the TCR subunit is selected from the group consisting of TCRα, TCRβ, CD3δ, CD3γ, and CD3ε. [0555] Embodiment III-34. The system of any one of III-23 to III-33, wherein the antigen binding domain is selected from the group consisting of an scFv, a VHH, a TCR-like antibody, a natural ligand, an FcR-binding receptor (e.g. CD16), and an NK killing receptor. [0556] Embodiment III-35. The system of any one of III-23 to III-34, wherein the antigen binding domain is specific for GPCR5D, BCMA, CD22, CD19 or CD20. [0557] Embodiment III-36. The system of III-35, wherein the antigen binding domain is specific for CD19. [0558] Embodiment III-37. The system of III-35, wherein the CD19 antigen binding domain is selected from the group consisting of FMC63, murine 4G7, human Hu19, Hu1E7, huB4, hBU12, humanized FMC63, humanized 4G7, GR37, GR41, SRB-37, SRB-85, and modified versions of any of the preceding. [0559] Embodiment III-38. The system of III-35, wherein the antigen binding domain is specific for CD20. [0560] Embodiment III-39. The system of III-38, wherein the CD20 antigen binding domain is selected from the group consisting of Leu16, rituximab, ofatumumab, ocrelizumab, obinutuzumab, Ibritumomab tiuxetan, AME-133v, IMMU-106, TRU-015, tositumomab, and modified versions of any of the preceding. [0561] Embodiment III-40. The system of any one of III-23 to III-36, wherein the antigen binding domain is bispecific. [0562] Embodiment III-41. The system of III-40, wherein the antigen binding domain is bispecific for CD19 and CD20. [0563] Embodiment III-42. The system of any one of III-22 to III-41, wherein the RTE polypeptide is a non-LTR RTE. [0564] Embodiment III-43. The system of III-42, wherein the non-LTR RTE is selected from the group consisting of CRE, R4, Hero, NeSL, R2, RandI, Proto1, L1, Tx1, RTEPT, Proto2, RTEX, RTE, Outcast, Ingi, I, Nimb, Tad1, Loa, R1, Jockey, Rex1, CR1, L2, L2A, L2B, Daphne, and Crack. 107 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0565] Embodiment III-44. The system of any one of III-22 to III-43, wherein the RTE polypeptide is a naturally occurring RTE polypeptide. [0566] Embodiment III-45. The system of any one of III-22 to III-43, wherein the RTE polypeptide is an engineered RTE polypeptide. [0567] Embodiment III-46. The system of any one of III-22 to III-45, wherein the template nucleic acid comprises a binding site for a differentially expressed microRNA (miR). [0568] Embodiment III-47. The system of III-46, wherein the binding site for the differentially expressed miR is located within the 3’ RTE UTR region. [0569] Embodiment III-48. The system of III-46 or III-47, wherein the binding site for the differentially expressed miR is specific for miR-122. [0570] Embodiment III-49. The system of any one of III-22 to III-48, wherein the driver nucleic acid comprises a binding site for a differentially expressed miR. [0571] Embodiment III-50. The system of III-49, wherein the binding site for the differentially expressed miR is specific for miR-122. [0572] Embodiment III-51. The system of any one of III-22 to III-50, wherein the driver nucleic acid comprises DNA. [0573] Embodiment III-52. The system of any one of III-22 to III-50, wherein the driver nucleic acid comprises RNA. [0574] Embodiment III-53. The system of any one of III-22 to III-50, wherein the driver nucleic acid comprises a DNA/RNA hybrid. [0575] Embodiment III-54. The system of III-52, wherein the RNA is mRNA. [0576] Embodiment III-55. The system of any one of III-22 to III-54, wherein the template nucleic acid comprises DNA. [0577] Embodiment III-56. The system of any one of III-22 to III-54, wherein the template nucleic acid comprises RNA. [0578] Embodiment III-57. The system of any one of claims III-22 to III-54, wherein the template nucleic acid comprises a DNA/RNA hybrid. [0579] Embodiment III-58. The system of III-56, wherein the template nucleic acid comprises a promotor and a polyA signal. [0580] Embodiment III-59. A lipid nanoparticle (LNP) comprising the nucleic acid of any one of III-1 to III-21 or the system of any one of III-22 to III-58. 108 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0581] Embodiment III-60. The LNP of III-59, wherein the LNP is covalently linked to an antibody or any fragment thereof that recognizes a T cell antigen. [0582] Embodiment III-61. The LNP of III-60, wherein the covalently linked antibody comprises a silent Fc domain. [0583] Embodiment III-62. The LNP of III-60 or III-61, wherein the antibody is specific for one or more of CD2, CD5, CD7, TCR, CD8, and CD3. [0584] Embodiment III-63. The LNP of any one of III-60 to III-62, wherein the antibody is selected from the group consisting of OKT3 (VH SEQ ID NO: 63, VL SEQ ID NO: 64), BMA031 (VH SEQ ID NO: 65, VL SEQ ID NO: 66), H65 (VH SEQ ID NO: 67, VL SEQ ID NO: 68), 3A1E (VH SEQ ID NO: 69, VL SEQ ID NO: 70), OKT8 (VH SEQ ID NO: 71, VL SEQ ID NO: 72), Visilizumab (VH SEQ ID NO: 73, VL SEQ ID NO: 74), Teplizumab (VH SEQ ID NO: 88, VL SEQ ID NO: 89), Siplizumab (VH SEQ ID NO: 92, VL SEQ ID NO: 93), Urelumab (VH SEQ ID NO: 94, VL SEQ ID NO: 95), Otelixizumab (VH SEQ ID NO: 101, VL SEQ ID NO: 102), and Foralumab (VH SEQ ID NO: 105, VL SEQ ID NO: 106), or modified versions of any of the preceding having at least 80%, 85%, 90%, 95%, or 99% sequence identity of each aforementioned VH or VL. [0585] Embodiment III-64. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and: (a) the nucleic acid of any one of III-1 to III-21; (b) the RTE integration system of any one of III-22 to 58; (c) the LNP of any one of III-59 to III-63; or (d) a combination thereof. [0586] Embodiment III-65. A method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of (a) the nucleic acid of any one of III-1 to III-21; (b) the RTE integration system of any one of III-22 to III-58; (c) the LNP of any one of III-59 to III-63, (d) the pharmaceutical composition of III-64, or (e) a combination thereof. [0587] Embodiment III-66. The method of III-65, wherein the subject is pre-treated with a T cell activator or concomitantly treated with a T cell activator. 109 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0588] Embodiment III-67. The method of III-66, wherein the T cell activator is a CD3 agonist or a TCR agonist. [0589] Embodiment III-68. The method of III-67, wherein the CD3 agonist is an antibody selected from the group consisting of OKT3, Teplizumab, Otelixizumab, Foralumab, Vislizumab, CD3_F1F, and CD3_F2B, or modified versions of any of the preceding. [0590] Embodiment III-69. The method of III-67, wherein the TCR agonist is BMA031 or a modified version thereof. [0591] Embodiment III-70. The method of III-66, wherein the T cell activator is an immune cell engager. [0592] Embodiment III-71. The method of III-70, wherein the immune cell engager is a Bispecific T cell engager (BiTe). [0593] Embodiment III-72. The method of III-71, wherein the BiTe is selected from the group consisting of Blinatumomab, MGD006, XmAb14045, AMG 330, AMG 420, AMG 564, AMG 701, BiTe 197, REGN1979, RG6026, Mosunetuzumab, AFM11, MGD006, tebentafusp, Catuxomab, Pasotuxizumab, Acapatamab, AMV564, Glofitamab, Solitomab, Talquetamab, Tarlatamab, Tebentafusp, Odronextamab, and Epcoritamab, or modified versions of any of the preceding. [0594] Embodiment III-73. The method of any one of III-65 to III-72, wherein the subject is further administered prior, concurrently, or subsequent to said administering with at least one γ- chain receptor agonist. [0595] Embodiment III-74. The method of III-73, wherein the at least one γ-chain receptor agonist is selected from the group consisting of IL-2, IL-7, IL-15, IL-21, and modified versions of any of the preceding. [0596] Embodiment III-75. The method of any one of III-65 to III-74, wherein the disease is cancer or an autoimmune disease. [0597] Embodiment III-76. The method of any one of III-65 to III-75, wherein the route of administration is selected from the group consisting of intravenous, intraperitoneal, intramuscular, subcutaneous, intrathecal, and intradermal administration. [0598] Embodiment III-77. A method of in vivo genome manipulation comprising pre- administering a T cell activator and administering a pharmaceutical composition comprising a reprograming agent. 110 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0599] Embodiment III-78. The method of III-77, wherein the reprogramming agent is a genome manipulation system comprising one or more nucleic acids. [0600] Embodiment III-79. The method of III-77, wherein the reprograming agent meditates durable expression by integrating one or more nucleic acids into a genome. [0601] Embodiment III-80. The method of III-77, wherein the reprograming agent mediates transient expression of one or more nucleic acids. [0602] Embodiment III-81. The method of any one of III-77 to III-80, wherein the one or more nucleic acids comprise at least one nucleic acid sequence comprised in or derived from a retrotransposon, a transposon, a Cas protein, a guide RNA, or a combination thereof. [0603] Embodiment III-82. The method of III-79, wherein the one or more nucleic acids comprise at least one nucleic acid sequence comprised in or derived from a retrotransposon. [0604] 83. The method of any one of III-77 to III-81, wherein the one or more nucleic acids is packaged in a viral vector or a lipid nanoparticle (LNP). [0605] Embodiment III-84. The method of III-83, wherein the viral vector is selected from the group consisting of a lentivirus, a retrovirus, and an adenovirus. [0606] Embodiment III-85. The method of any one of III-77 to III-84, wherein the T cell activator is an CD3 or TCR agonist. [0607] Embodiment III-86. The method of III-85, wherein the CD3 agonist is an antibody selected from the group consisting of OKT3, Teplizumab, Otelixizumab, Foralumab, Vislizumab, CD3_F1F, CD3_F2B, and modified versions of any of the preceding. [0608] Embodiment III-87. The method of III-85, wherein the TCR agonist is BMA031 or a modified version thereof. [0609] Embodiment III-88. The method of III-77 to III-84, wherein the T cell activator is an immune cell engager. [0610] Embodiment III-89. The method of III-88, wherein the immune cell engager is a Bispecific T cell engager (BiTe). [0611] Embodiment III-90. The method of III-88, wherein the BiTe is selected from the group consisting of Blinatumomab, MGD006, XmAb14045, AMG 330, AMG 420, AMG 564, AMG 701, BiTe 197, REGN1979, RG6026, Mosunetuzumab, AFM11, MGD006, tebentafusp, Catuxomab, Pasotuxizumab, Acapatamab, AMV564, Glofitamab, Solitomab, Talquetamab, Tarlatamab, Tebentafusp, Odronextamab, Epcoritamab, and modified versions of any of the preceding. 111 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0612] Embodiment III-91. A method of in vivo genomic manipulation in an immune cell, the method comprising contacting the immune cell with: (a) the nucleic acid of any one of III-1 to III-21; (b) the RTE integration system of any one of III-22 to 58; or (c) the LNP of any one of III-59 to III-63, (d) the pharmaceutical composition of III-64; or (e) a combination thereof. [0613] wherein the immune cell natively expresses a TCR-CD3 complex. [0614] While several inventive embodiments are described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described here. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure. [0615] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document. [0616] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited. 112 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 In the claims, as well as in the specification, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative embodiments, as “consisting of” and “consisting essentially of” the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B”, the disclosure also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B”. EXAMPLES Example 1: Materials and Methods [0617] Unless stated otherwise, material and methods for the subsequent Examples were performed as described herein below. Cell lines and PBMCs [0618] Nalm6-Luc, Raji-Luc, Jurkat, and THP1 cells were cultured in complete RPMI (“cRPMI”) containing RPMI (Capricorn) supplemented with 10% fetal-calf serum (“FCS”, Gibco), 2 mM L-Glutamine (Bio-West), and 1x Penicillin–Streptomycin (“Pen/strep,” Bio- West). Huh7, U2-OS, and HepG2 cells were cultured in complete DMEM (“cDMEM) containing DMEM (Capricorn) supplemented with 10% FCS, 2mM L-Glutamine, and 1x Pen/strep. [0619] Primary human hepatocytes (PHH) were cultured in Human Hepatocyte Medium (Primacyt) using the Hepatocyte Plating and Thawing Kit (Primacyt) [0620] Cryopreserved human peripheral blood mononuclear cells (PBMCs), a mixture of cell types including, e.g., T-cells, B-cells, and NK cells, from healthy donors (Cell Generation) were cultured in “T cell medium” containing ImmunoCult™-XF T cell Expansion Medium (Stem Cell), 10% FCS, 1x Pen/strep, and 100 U/ml IL-2 (Miltenyi) thereby resulting unless otherwise noted. Unless otherwise noted, PBMCs were activated for 2 days after thawing in T cell medium 113 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 with the addition of TransAct™ (Miltenyi) at a 1:100 dilution, driving expansion of T cells. For non-activating conditions, IL-2 was replaced with 10ng/ml IL-7 (Miltenyi) and 10ng/ml IL-15 (Miltenyi). For antibody activation, the indicated antibodies at the indicated doses were added to the culture medium (with IL-7 and IL-15). All cells were grown in incubators with 5% CO2 at 37oC. Donor PBMCs are referred to herein by donor number, For example, PBMCs from donor 13 are referred to as “DP13”. Plasmids [0621] All plasmids were synthesized by GenScript. The CAR or TRAM transgenes were codon optimized using an in-house algorithm. They contain the T7 promoter and a unique Type IIS restriction site at the 3´end. In-vitro transcription (IVT) [0622] Plasmids were linearized using restriction enzyme according to manufacturer’s protocol, purified with AMPure XP beads, placed on magnetic tube rack, washed three times with 70% ethanol while on magnet, dried, and resuspended in water. IVT was performed using NEB HiScribe™ T7 High Yield RNA Synthesis Kit. Then for the DNase phase, the TURBO™ DNase kit AM2238 was utilized. The RNA transcript was then purified using Monarch RNA cleanup Kit T2050. Quantification and quality were determined by nanodrop, Agilent TapeStation, and Controller software. LNP production [0623] To encapsulate RNA (e.g., mRNA) in LNPs, RNA from in vitro translation (IVT) (60 μg total) was loaded from one syringe and lipid mix (SM-102, DSPC, Cholesterol, and DMG- PEG2000 unless otherwise noted) from a second syringe and mixed using the Ignite machine. LNPs were then washed with 40ml PBS, added to a tube with centrifugal filter (Amicon) and centrifuged at 2000g at 4C until volume was reduced to approximately 500 μl. Encapsulated RNA concentration was determined using a Ribogreeen™ assay before and after lysing LNPs, where the encapsulated RNA is the total mRNA (following lysis) minus the free RNA (before lysis). To analyze LNP average size and variance the Stunner machine was used, which analyzes UV/Vis concentration, dynamic light scattering (DLS), and static light scattering (SLS). LNPs were stored at 4C, typically for up to one week. 114 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 Targeted LNP (tLNP) production [0624] For targeted LNP production, the lipid mix also contained DPSE-PEG2000-Maleimide. LNPs with Maleimide (“LNP-Mal”) were produced using the above method for LNP production and mixed with antibodies that were reduced using Tris (2-carboxyethyl) phosphine (TCEP). Targeted LNPs were purified from unconjugated antibodies using a Size Exclusion Column (SEC). Each fraction was analyzed by for RNA concentration (Ribogreen™), DLS and SLS (Stunner), DLS and particle count (Malvern), and protein concentration (BCA or Nano orange assay). Fractions containing tLNPs were combined and analyzed using the same methods. Antibodies for cell culture or tLNPs [0625] Anti-human CD2 (VIP VIIIC8 and Siplizumab), CD3ε (OKT3, Visilizumab, Teplizumab, Otexlizumab, Foralumab, CD3_F2B), CD5 (H65, 5G7, and UHCT2), CD7 (3A1E), CD8 (OKT8), and 4-1BB (Urelumab) antibody VH and VL portions were incorporated into human IgG1 with Fc silencing mutations or tested as they are used clinically, where relevant. Selected antibodies were also tested in additional formats such as Fab, (Fab)2, or diabodies. Isotype control for binders acquired from Absolute Antibodies was anti-FITC (4-4-20) or a standard Isotype control from all other vendors (MiMabs, Selleckhem, FJ Bio, etc.). Anti-mouse CD3ε (145-2C11) VH and VL portions were incorporated into mouse IgG1 with or without Fc silencing. Primary human T cell LNP transfection [0626] Two days post thaw, T cells were centrifuged at 300g for 5 min at RT, suspended to 1x106 cells/ml in T cell medium supplemented with 2 μg/ml Apolipoprotein E (ApoE; Peptrotech), and seeded in 96-well TC plates, 1x105 cells per well. LNPs were added at dose of 400 ng mRNA per well in duplicates unless otherwise noted and incubated for 24. Cells were then centrifuged, suspended in T cell medium, 200 μl per well, and incubated for 24 h. Cells were then expanded, for GFP studies in TC plates (Thermo-Fisher) or for CAR/TRAM in gas- permeable 24-well G-Rex plates (Wilson Wolf) according to the manufacturer’s protocol. 115 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 Lentiviral transduction [0627] Cells were transduced with VSVG-pseudotyped lentiviruses (LV) (VectorBuilder) at a multiplicity of infection (MOI) of 20. Primary human T cells were transduced 2 days post thaw in the same conditions as LNP transfection but without ApoE and expanded in the same manner. All cell lines were transduced in the presence of 8 μg/ml polybrene and washed 24 h post transduction. Nalm6-Luc, Raji-Luc, and THP1 cells were transfected in 96-well plates, 5x104 cells/well. HUH7 (liver derived) cells were transfected in 24-well plates, 1x105 cells/well. Flow cytometry (also referred to as “FACS” (Fluorescence-Activated Cell Sorting)): [0628] Cells were analyzed at indicated time point post transfection. Cells were stained with antibodies for 15 min at RT in the dark in Cell Staining Buffer (Biolegend) at the recommended dilution unless otherwise noted. Cells were then washed, suspended in PBS with DAPI 0.5 μg/ml added to stain dead cells unless otherwise noted, and analyzed with the CytoFLEX S V4- B2-Y4-R3 Flow Cytometer (Beckman Coulter) followed by CytExpert 2.5 (Beckman Coulter). Antibodies used were anti-FMC63- Allophycocyanin (APC) (Miltenyi), Flag-APC (Miltenyi), HA tag-APC (GG8-1F3.3.1, Miltenyi), hCD3-PerCP (REA613, Miltenyi), hCD4-BV421 (REA623, Miltenyi), hCD8-APC-Vio770 (REA734, Miltenyi), hCD69-PE-Vio770 (REA824, Miltenyi), hCD45RA-BV650 (HI100, Biolegend), hCD62L-PE (DREG-56, Biolegend), hCD45RO-PE-Cy7 (UCHL1, Biolegend), aCD223 (LAG3)-PE (11C3C65, Biolegend), hCD127 (4-1BB)-PE (4B41, Biolegend), hCD279 (PD1)-PE-Cy7 (EH12.2H7, Biolegend), Fixable viability dye eFluor506 (“L/D EF506”, Thermo-Fisher). Unless otherwise noted the gating strategy was removal of debris based on FSC-W and FSC-A, removal of dead cells based on DAPI (or L/D EF506), selection of cells based on SSC-A and FSC-A, and selection of singlets based FSC-H and FSC-A. Quantification of CAR or TRAM receptors: [0629] Quantum APC MESF beads (Bang Laboratories) were run with the same APC voltage as the T cells stained with either anti-FMC63-APC or anti-Flag-APC. Molecules of Equivalent Soluble Fluorochrome (MESF) units were calculated from median fluorescence intensity (MFI) using QuickCal® 3.0 (Bangs Laboratories) of CAR+ or TRAM+ cells if there were >500 events. MESF units were converted to antigen binding capacity (ABC) units, representing the number of 116 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 antibodies, by building a Fluorescence/Protein (F/P) curve for each antibody lot. F/P curves determine the ratio of MESF to ABC, essentially fluorophores per antibody. F/P curve generated by staining human QSC beads (Bangs laboratories) with the antibody which generates ABC values run in parallel to MESF beads. Using QuickCal®, MESF were converted to ABC to determine the number of antibodies bound to the cell which was assumed to be roughly equal to the receptors/cell. Digital PCR (dPCR) [0630] Cells were analyzed for absolute quantification of the number of genomic integrations using dPCR with probes targeted to the transgene (either promoter, middle of transgene, or synthetic polyA after the transgene as indicated), and compared to a probe targeting a known reference gene. Frozen pellets of cells were thawed, lysed, DNA extracted, and analyzed with the QIAcuity dPCR machine (Qiagen). Ligation-mediated Unique Molecular Identifier Sequencing (Lumi-Seq) insertion site analysis [0631] Insertion site was testing using Lumi-Seq. Frozen pellets of cells were thawed, lysed, DNA extracted, and sheared in using Adaptive Focused Acoustics (AFA) technology (Covaris). All purification steps were performed with AMPure XP beads. DNA was blunted using NEBNext End Repair Module and purified. Then dA tailed was performed using NEBNext dA- Tailing Module and purified. Uniquely labeled adapters were ligated with T4 DNA ligase and purified. DNA cassette was enriched using a PCR with P5 and target specific primers, purified, a second PCR with P5 and nested primers, flanked by Illumina adapter sequence, purified and size selected, followed by and a third PCR with P5 primers and unique index adapters. Library QC was done using Qubit and TapeStation, and then run on the Iseq100 high throughput sequencer. Luciferase killing assay [0632] Firefly luciferase (Luc)-labelled target cells (Nalm6-Luc or Raji-Luc) were seeded in wells of white 96-well TC plates (Greiner) in cRPMI, 1x105 cells/well in 70 μl. Effector cells (T cells) were added in 50μl cRPMI at indicated Effector:Target (E:T) ratios between 20:1 and 0.01:1 calculated based on CAR+ or TRAM+ cells. Non-treated (NT) T cells were added to have the same total cell number as the average of the CAR+ or TRAM+ cells. Co-culture was incubated for 20 h, and BioGlo™ Luciferase Assay System reagent (Promega) was added, 20 μl 117 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 per well, and incubated for 10 mins. Luciferase signal was measured with the Infinite 200 Pro plate reader (Tecan) with an exposure time of 500 ms. The higher the luciferase signal the more viable target cells per well. Relative viability was calculated as relative units (RLU) of a given well divided by the maximum RLU for the given effector. EC50 curves were generated with Prism 10 (GraphPad) by fitting a non-linear sigmodial curve, 4PL, X is concentration. The bottom of the curve constrained to equal 0 to ensure uniformity between curves and at very high E:Ts which may be larger than 20:1 no target cells would be viable. The lower the EC50 the more potent the killing. Cytokine secretion assay [0633] For cytokine secretion analysis an additional 80 μl of medium was added to the wells in the above describe Luc killing assay and harvested after 20 h of co-culture from in-vitro cultures or from serum of mice and analyzed using the human IFNγ ELISA Kit (Ray Biotech) or Th1/Th2 cytokine bead array “CBA” kit (BD) according to manufacturer’s protocols. In vivo studies in mice: [0634] For activation or LNP studies for human T cells, immunodeficient mice were injected I.V or I.P as indicated with the indicated quantity of human PBMCs at indicated time before treatment. For activation and LNP studies with immunocompetent mice C57Bl/6 mice were used unless otherwise indicated. For studies with mRNA-LNPs for transient expression mice were sacrificed and organs were harvested 24h post injection unless otherwise noted. For LNP studies with integrating mRNA-LNPs blood was sampled at indicated time points and mice were sacrificed at indicated time point. For anti-tumor response studies, Nalm6-Luc or Raji-Luc cells were injected IV and after tumor establishment CAR-T or TRAM-T cells or LNPs were injected I.V. at the indicated doses. Tumor burden was measured by IVIS at indicated time points and blood was sampled for FACS and/or dPCR analysis at indicated time points. All mouse studies were approved by IACUC ethics committee. Sample TRAM designs [0635] The following are non-limiting examples of TRAM transgene designs. FIG.3 also depicts exemplary TRAM designs. Unless otherwise noted the CD19 scFv for the following TRAMs comprise FMC63 VL (SEQ ID NO: 2), Whitlow linker (SEQ ID NO: 9), FMC63 VH 118 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 (SEQ ID NO: 4) and are fused to CD3 component via 3xA (SEQ ID NO: 10) and G4Sx3 linker (SEQ ID NO: 3). This scFv with spacer is referred to herein as “FMC63 LH scFv-G4S” (SEQ ID NO: 11). [0636] “mCα+β TRAM” comprises mTCRα SP (SEQ ID NO: 12), FMC63 VH (SEQ ID NO: 4), 3xA (SEQ ID NO: 10), mTCR-Cα (SEQ ID NO: 13), T2A (SEQ ID NO: 14), FMC63 VL (SEQ ID NO: 2), 3xA (SEQ ID NO: 10), and mTCR-Cβ (SEQ ID NO: 15). [0637] “mCβ TRAM” comprises mTCRα SP (SEQ ID NO: 12), mTCR-Cα (SEQ ID NO: 13), T2A (SEQ ID NO: 14), FMC63 LH scFv-G4S (SEQ ID NO:11), and mTCR-Cβ (SEQ ID NO: 15). [0638] “hCβ TRAM” comprises hTCRβ SP (SEQ ID NO: 16), FMC63 LH scFv-G4S (SEQ ID NO: 11), and hTCR- Cβ (SEQ ID NO: 17). [0639] “γ TRAM” comprises of CD8α SP (SEQ ID NO: 1), FMC63 LH scFv-G4S (SEQ ID NO: 11), and CD3γ (SEQ ID NO: 18). [0640] “δ TRAM” comprises of CD8α SP (SEQ ID NO: 1), FMC63 LH scFv-G4S (SEQ ID NO: 11), and CD3δ (SEQ ID NO: 19). [0641] “ε TRAM” comprises CD8α SP (SEQ ID NO: 1), FMC63 LH scFv-G4S (SEQ ID NO: 11), and CD3ε (SEQ ID NO: 20). comprises of CD8α SP (SEQ ID NO: 1), FMC63 LH scFv-G4S (SEQ ID NO: 11), CD28 hinge (SEQ ID NO: 21) since CD3ζ has only 8 amino acids extracellularly, CD3ζ HTM (SEQ ID NO: 22), and CD3ζ signaling domain (SEQ ID NO: 8). Example 2: Lentiviral transduction of several cell types with Anti-CD19 CAR or TRAMs [0643] Whereas a canonical CAR can express stably on the surface of any cell, a TRAM should only express on T cells, which are the only cells that express all 6 TCR-CD3 chains. To confirm this, lentiviral vectors were generated with an anti-CD19 CAR (nucleic acid sequence set forth SEQ ID NO: 3336, amino acid sequence set forth in SEQ ID NO: 23) or TRAM according to the designs depicted in FIG.3 and described in Example 1, followed by a T2A self-cleaving peptide (SEQ ID NO: 14) and GFP (amino acid sequence set forth in SEQ ID NO: 24, nucleic acid sequence set forth in SEQ ID NO: 3331). The promoter used, “MNDopt” (SEQ ID NO: 25) is a truncated version of the MND promoter (SEQ ID NO: 26) which is referred to herein to as “MNDu.” Lentiviral vectors were used due to high transduction efficiency in a wide variety of cell types, as RTEs expression can be low in certain cell types. Inclusion of the T2A-GFP allows 119 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 for observation of cells that underwent transduction, regardless of the stability of the CAR or TRAM. The cell types evaluated included primary T cells derived from PBMCs from healthy donors, Nalm6 (B-ALL cell line), Raji (NHL B cell line), and HUH7 (liver cell line). In T cells, all TRAMs and CARs were expressed on the cell surface (FIG.4A and 4B). Indicated cells were transduced with lentiviruses at an MOI of 20. CAR or TRAM surface expression detected by FACS with an anti-FMC63-APC antibody. Results gated on live single cells unless otherwise noted. NT = non-treated cells. (FIG.4A and FIG.4B) Expression 12 days post transduction in primary human T cells from PBMCs. (FIG.4C) Expression 6 days post transduction in liver cell line HUH7. (FIG.4D) Expression 6 days post transduction in B cell line Nalm6-Luc. (FIG.4E) Expression 6 days post transduction in B cell line Raji-Luc. (FIG.4F) Median fluorescence intensity (MFI) of CAR or TRAM expression on HUH7 cells gated on GFP+ (transduced) cells. Most TRAMs expressed similarly to and slightly lower than CART-19. For mCα+β and hCβ TRAMs, expression was lower and not all GFP+ cells were stained with the anti-FMC63 antibody. For mCα+β TRAM this may be due to non-functional folding of the split VH and VL chimeras or mispairing with endogenous TCR chains. For hCβ TRAM this may be due to competition with endogenous TCRβ. [0644] In all other cell types, CART-19 CAR expressed as expected and γ TRAM did not express on the cell surface as expected (FIG.4C-F). Surprisingly, all of the other TRAMs did express on the cell surface of other tested cell types despite these cells not expressing the other TCR-CD3 chains (FIG.4C-4E). The mCβ, hCβ, and ζ TRAMs expressed on all other cell types, and in the case of HUH7 cells the ζ TRAM appears to have killed the cells. The mCα+β and ε TRAMs expressed moderately in HUH7 cells, in about 5% of Nalm6 cells, but not in Raji cells. The δ TRAM expressed moderately is HUH7 cells, but not on Nalm6 or Raji cells. It should be noted that the expression levels of the TRAMs that did express on HUH7 cells were lower than CART-19 (FIG.4F). [0645] For Raji and Nalm6, CD19+ malignant B cell lines, it is advantageous to test whether expression of CARs or TRAMs lead to CD19 antigen masking. For this purpose, CAR/TRAM and CD19 surface expression was analyzed within the transduced (GFP+) cells (FIGS.5-6). In FIG.5, cells were transduced with lentiviruses at an MOI of 20. Further analysis of cells shown in FIG.4D. NT = non-treated cells. (FIG.5A) %GFP+ gated on singlets. (FIGS.5B-5F) Analysis gated on transduced (GFP+) cells. (FIG.5B) Detectable %CAR+ or %TRAM+ cells, indicating cell surface expression. (FIG.5C) Detectable %CD19+ indicative of cells without full 120 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 CD19 masking. (FIGS.5D- 5F) Median fluorescence intensity (MFI) of indicated marker. (FIG. 5D) GFP MFI. (FIG.5E) Surface CAR or TRAM MFI. (FIG.5F) Detectable (unmasked) CD19 MFI. As expected, CART-19 highly expressed on the cell surface and completely masked CD19 in both cell lines. In Nalm6 cells, γ and δ TRAMs were not detected on the cell surface, mCα+β and ε TRAMs were detected on 5-7% of cells, and surprisingly mCβ, hCβ, and ζ TRAMs were detected in nearly all GFP+ cells (FIG.5B). Accordingly, CD19 staining was positive γ and δ TRAMs and negative due to masking for mCβ, hCβ, and ζ TRAMs (FIG.5C). It should be noted the level of CD19 detected (according to Median Fluorescence Intensity, “MFI”) was 6-11 fold lower for γ or δ TRAMs than non-treated (NT) cells the staining was well separated from background and should allow for recognition and killing by TRAM-T cells. Interestingly, mCα+β and ε TRAMs masked to a higher degree (in 38% and 76% of cells, respectively, FIG. 5C) than what would be expected based on TRAM surface expression (in 7% and 5% of cells, respectively, FIG.5B). [0646] In Raji cells, the trends are essentially the same, but overall transduction was lower and starting point of CD19 expression is higher (as shown in FIGS.6A-6F). In the data shown in FIGS.6A-6F, cells were transduced with lentiviruses at an MOI of 20. Further analysis of cells shown in FIG.6D. NT = non-treated cells. FIG.6A: %GFP+ gated on singlets. FIGS.6B- 6F: Analysis gated on transduced (GFP+) cells. FIG.6B: Detectable %CAR+ or %TRAM+ cells, indicating cell surface expression. FIG.6C: Detectable %CD19+ indicative of cells without full CD19 masking. FIGS.6D-6F: Median fluorescence intensity (MFI) of indicated marker. FIG. 6D: GFP MFI. FIG.6E: Surface CAR or TRAM MFI. FIG.6F: Detectable (unmasked) CD19 MFI. GFP expression was seen in 30-60% of cells, as shown in FIG.6A. The amount of the GFP positive cells mCβ, hCβ, and ζ TRAMs was detected on the surface of 12-24% of cells, whereas mCα+β, γ, δ, and ε TRAMs were not detectable, as shown in FIG.6B. With regard to CD19 masking, CART-19 completely masked CD19 and it was not detectable, and for the TRAMs there were different degrees of masking, though CD19 was still detectable in most or all of the cells ( as shown in FIG.6C and FIG.6F). The ζ TRAM masked to the highest degree and led to a 445 fold reduction in MFI. The mCβ and hCβ TRAMS also masked to a large degree and reduced MFI by 40 fold (FIG.6F). Despite not expressing on the surface, ε TRAM led to a 20 fold reduction in MFI, consistent with high degree of masking by this TRAM in Nalm6 cells, though staining was all above background. The mCα+β, γ and δ TRAMs did not mask CD19 much, reducing MFI only 2-5 fold and cells staining strongly positive. 121 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0647] The results of surface expression in non-T cells and CD19 masking highlight the importance of T cell specific expression. Cancer cells with masked CD19 expression may not be completely killed by CAR T cells and may pose a safety risk. The surface expression of all TRAMs except for γ TRAM in non-T cells, though the mechanism unclear, was unexpected. Additionally, masking of CD19 by the ε TRAM in B cells despite the lack of surface expression further may indicate that not all TRAMs, including the most commonly used ε TRAM, solve the masking concern. Although only anti-CD19 CAR and TRAMs were tested, it is reasonable to assume that the same patterns would be seen with other scFvs as the main cause for stability on the surface is likely the other components. Further work was mainly focused on γ, δ, and ε TRAMs which were at least not expressed on the surface of B cell lines, but γ TRAM would be the most attractive due the exquisitely specific expression and lowest CD19. Example 3: Vingi1 RTE system for GFP and Anti-CD19 CAR expression [0648] After characterizing the specificity of expression using lentiviruses, testing of CARs and TRAMs using RTEs followed. The RTE used for initial studies was Vingi1-Acar (“Vingi1”) a non-LTR and non-site specific RTE derived from Anolis carolinensis (green Anole), which was found to mediate highly efficient gene transfer of GFP or CART-19 to primary T cells (FIGS. 7A-7F). Donor 10 T cells were transfected with indicated Vingi1 RNA-LNPs, where reporter only (R only) is a negative control that should not integrate whereas and driver + reporter should integrate and express. Cells were analyzed by dPCR and FACS at 5- and 12-days post transfection. Error bars show SD of duplicates. NT = non-treated cells. FIGS.7A-7C: GFP transgene under indicated promoters. FIGS.7D-7F: CART-19 transgene under indicated promoters. FIG.7A, FIG.7D: Genomic integrations according to dPCR with the indicated probes. FIG.7B: % GFP positive according to FACS. FIG.7E: % CAR positive according to FACS. FIG.7C: GFP expression level median fluorescence intensity (MFI). FIG.7F: Quantification of CAR expression levels was calculated using FACS and Quantum beads for samples with >500 CAR+ (Day 12 only). [0649] The Vingi1 driver full transcript (SEQ ID NO: 27) consists of the T7 Clean Cap (SEQ ID NO: 28), Vingi1 driver 5’ UTR (SEQ ID NO: 29), Vingi1 ORF2 (DNA SEQ ID NO: 30, protein SEQ ID NO: 31), and Vingi1 driver 3’UTR and polyA (SEQ ID NO: 32). The Vingi1 reporter consists of the T7 Clean Cap (SEQ ID NO: 28), Vingi1 reporter 5’ UTR (SEQ ID NO: 33), the anti-sense of the cargo (promoter, transgene, polyA signal) of interest in the inverse orientation, 122 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 and Vingi1 reporter 3’UTR and polyA (SEQ ID NO: 34). The cargo was placed in the reverse orientation such that only following integration can the gene be transcribed (sense strand) and translated. All cargo consisted of a promoter, Kozak sequence (SEQ ID NO: 35), transgene on interest, and was followed by a synthetic polyA signal (SEQ ID NO: 36). As a negative control, T cells were transfected with LNPs containing only the reporter, which will not integrate into the cells without the presence of the driver. Interestingly, the ideal promoter for GFP (SEQ ID NO: 24) and CART-19 (amino acid sequence set forth in SEQ ID NO: 23, nucleic acid sequence set forth in SEQ ID NO: 3336) expression was different. The promoters tested were MNDopt (542bp, SEQ ID NO: 25), MNDu (635bp, SEQ ID NO: 26), and the Elongation factor 1α (EF1α) promoter (1179 bp, SEQ ID NO: 37). [0650] For GFP, the MNDopt promoter showed >5 fold higher rate of integrations detected by digital PCR (dPCR) with a GFP transgene probe (as shown in FIG.7A), and >4 fold higher %GFP+ detected by flow cytometry than with the EF1α and MNDu promotors (as shown in FIG.7B). In terms of the level of expression, the ranking was MNDu > EF1α > MNDopt (as shown in FIG.7C). The results with GFP for EF1α and MNDopt are as expected, since the shorter MNDopt promoter is expected to integrate more efficiently due to processivity of the RT whereas EF1α is longer but known to lead to very high levels of expression. [0651] For CART-19, MNDopt exhibited highest rate of genomic integrations detected by dPCR (as shown in FIG.7D), however in terms of %CAR+ detectable on the cell surface by FACS, the EF1α promotor exhibited the highest expression (as shown in FIG.7E). CAR receptors per cell were quantified using Quantum beads for day 12 samples and the ranking was EF1α > MNDu > MNDopt (as shown in FIG.7F). It is worth noting that although the mechanism is unknown surface CAR expression at 5 days post transfection (pt) for Vingi1 RTE was very low and increased substantially by 12 days. This trend was true for several PBMC donors with Vingi1 CART-19 under the MNDopt (FIG.8A-C) and EF1α promoters (as shown in FIGS.8D-8F), unlike lentiviruses with EF1α-CART-19 where expression was already high (>58%) at 5 days post transduction (FIGS.8G-8I). In FIGS.8A-8I, T cells were transfected with indicated LV (FIGS.8G-8I) or Vingi1 RNA-LNPs with CART-19 transgene (FIGS.8A-8F) and expression analyzed at indicated days post transfection. CAR surface expression detected by FACS with an anti-FMC63-APC antibody. Error bars show SD of duplicates. FIGS.8A-8C: Vingi1 with MNDopt promoter. FIGS.8D-8F: Vingi1 with EF1α promoter. FIG.8G-8I: LV with EF1α promoter. FIGS.8A, 8D, and 8G: Genomic integrations according to dPCR with a 123 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 CAR probe. FIGS.8B, 8E, and 8H: %CAR detected by FACS. FIGS.8C, 8F, and 8I: Quantification of CAR expression levels was calculated using FACS and Quantum beads for samples with >500 CAR+. Example 4: Anti-CD19 CAR or TRAMs with T2A-GFP using Vingi1 driver [0652] To test the TRAMs using Vingi1 driver, reporters with the CART-19 or human CD19 TRAMs were tested followed by T2A-GFP under the MNDopt promoter (FIGS.9A-9C), the same format tested in several cell types with lentiviruses. T cells were transfected with indicated Vingi1 RNA-LNPs and expression analyzed at indicated days post transfection. NT = non- treated cells. FIG.9B: CAR-T or TRAM-T effector cells were co-cultured with Nalm6-Luc target cells at indicated E:T (effector:target) ratios between 20:1-0.2:1for 20h and luciferase signal read. Relative viability calculated as relative units (RLU) of luminescence relative to maximum RLU of a given effector. FIG.9C: Killing EC50s for E:T where half of the targets are killed. CART-19 without the T2A-GFP under the MNDopt or EF1α promoters were included as controls. FIG.9A: The γ, δ, ε, and ζ TRAMs all expressed well whereas hCβ TRAM and CART- 19 with T2A-GFP showed no expression. CART-19 without T2A-GFP was detectable but low under the MNDopt promoter and highly under the EF1α promoter. A Nalm6-Luc killing assay was performed using all TRAMs and CARs-T with >7% expression. The E:T (effector:target ratio) EC50 (effective concentration 50%) is the E:T ratio at which 50% of the target cells are alive. A lower EC50 indicates higher sensitivity and potency, as it achieves the same killing with fewer T cells. The γ, δ, ε, and ζ TRAMs (with T2A-GFP and under MNDopt) killed similarly to each other (as shown in FIGS.9B and 9C). They all kill better than EF1α-CART-19 (without T2A-GFP). It is unclear why the MNDopt TRAM expression is much better than CART-19 using a Vingi1 driver and slightly lower than CART-19 in lentiviruses. In fact, considering how much lower CART-19 expression is with the Vingi1 driver compared to lentiviruses (as shown in FIGS.8A-8I), it would be expected that TRAMs may not achieve detectable expression with RTE drivers. However, the opposite was true, and it seems that TRAMs are particularly suitable for RTEs due to the surprisingly improved expression compared to a canonical CAR. Example 5: Anti-CD19 CAR or TRAMs under MNDopt using Vingi1 driver (without T2A- GFP) 124 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0653] Next, inducing genomic integration of CD19 TRAM with Vingi1 drivers expression was tested under the MNDopt promoter without the T2A-GFP, which is not desired in the final product and reduces insert size by 795bp. The γ and δ TRAMs were tested because they prevent CD19 masking and ε TRAM was also tested since it is most commonly used. These TRAMs were compared to CART-19 with the same MNDopt (“Mo”) promoter or EF1α promoter. T cells were transfected with indicated Vingi1 RNA-LNPs and analyzed by dPCR and FACS at 5- and 12-days post transfection. Results are shown in FIGS.10A-10C. NT = non-treated cells. FIG. 10A: Genomic integrations according to dPCR to the synthetic poly A probes. FIG.10B: %positive according detected by FACS following staining with an anti-FMC63-APC. (FIG. 10C) Quantification of CAR expression levels was calculated using FACS and Quantum beads. Again, the TRAMs showed superior integration and expression than CART-19. In terms of genomic integrations analyzed using dPCR with a probe to the synthetic polyA signal sequence found immediately after the transgene the ranking was γ and ε TRAMs > δ TRAM > Mo-CART- 19 > EF1α-CART-19 (FIG.10A). In terms of percent CAR or TRAM positive analyzed by flow cytometry the ranking was γ and ε TRAMs > δ TRAM > EF1α-CART-19 > Mo-CART-19 (FIG. 10B). It should be noted that the TRAMs were already >23% positive 5 days post transfection, when the Mo-CART-19 was undetectable and EF1a CAR was 2% or 8% CAR depending on donor. In terms of receptor levels detected by flow cytometry the ranking was ε TRAM > γ TRAM > EF1α-CART-19 > δ TRAM > Mo-CART-19 (FIG.10C). It is expected that ε TRAM would have the highest expression levels among TRAMs as each TCR-CD3 subunit has one γ, one δ, and two ε chains. Overall, the γ, δ, and ε TRAMs integrated and expressed much better than CART-19 in RTEs with the same MNDopt promoter, opposite to the trend in lentiviruses. A Nalm6-Luc killing assay with γ, δ, and ε TRAMs and CART-19 under MNDopt showed overall similar killing (FIG.11). CAR-T or TRAM-T effector cells were co-cultured with Nalm6-Luc target cells at indicated E:T ratios between 20:1-0.2:1 for 20h and luciferase signal read. Error bars show SD of triplicates. NT = non-treated cells. FIGS.11A11B: Donor 10. FIGS. 11C-11D: Donor 13. FIGS.11A and 11C) Kill curves. Relative viability calculated as RLU to maximum RLU of a given effector. FIGS.11B and 11D: EC50s for E:T where half of the targets are killed. In donor 10 δ TRAM killing lower than the others, yet in donor 13 δ TRAM did kill like the others. Considering both expression and killing data γ and ε TRAMs have the highest expression and consistently potent killing, but δ TRAM also expresses and kills well. Since ε 125 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 TRAM doesn’t prevent masking, attention was focused on γ TRAM as the lead design with δ TRAM a good backup. Example 6: Anti-CD19 γ TRAM with MNDopt or EF1α promoters using Vingi1 RTE [0654] Since EF1α was superior to MNDopt for CART-19 expression, the γ TRAM with these two promoters was then tested (FIGS.12A-12C). Donor 13 T cells were transfected with indicated Vingi1 RNA-LNPs and analyzed by dPCR and FACS at 6-, 9-, and 12-days post transfection. Error bars show SD of duplicates. NT = non-treated cells. FIG.12A: Genomic integrations according to dPCR to the synthetic poly A probes. FIG.12B: %positive according detected by FACS following staining with an anti-FMC63-APC. FIG.12C: Quantification of CAR expression levels were calculated using FACS and Quantum beads. Integrations were again highest for MNDopt (FIG.12A). Unlike CART-19, surface expression of γ TRAM was higher in both %TRAM+ and receptor levels for MNDopt compared to EF1α (FIGS.12B-12C). The higher surface expression of γ TRAM with MNDopt promoter is in line with the genomic integrations and results with GFP. Expression under EF1α promoter was similar for γ TRAM and CART-19. These results highlight that that specific optimization for CAR vs. TRAM payloads may be needed. Example 7: Additional scFvs in TRAMs [0655] TRAMs can be used to other target antigens, for example CD20. CD20 is also expressed on many B cell malignancies. CAR or γ TRAMs with a scFv derived from Leu16 VL (SEQ ID NO: 38), Whitlow linker (SEQ ID NO: 9), and Leu16 VH (SEQ ID NO: 39) were tested. To simplify detection, a Flag tag (SEQ ID NO: 40) was added at the N’ terminus (between the signal peptide and VL). Incorporating some elements such as 41BBzeta or CD3γ or other subunit denotes canonical CAR T or TRAM. Initially these were tested under the EF1α promoter (FIGS.13A-13B). T cells were transfected with indicated Vingi1 RNA-LNPs and analyzed by dPCR and FACS at 6-, 9-, and 12-days post transfection. All contain an Anti-CD20 Leu16 VL- VH scFv antigen binding domain with Flag tag on the N’ terminus. Several spacers between the scFv and CD3γ were tested with different levels of rigidity. Flexible 3xG4S, Rigid 3xEAAAK, SR = semi-rigid EAAAKGGGSEAAAK, SF = semi-flexible GGGSEAAAKGGGS, IgG4 short hinge = ESKYGPPCPPCPM. NT = non-treated cells. FIG.13A: Genomic integrations according to dPCR to the synthetic poly A probes. Error bars show SD of duplicates. FIG.13B: %positive 126 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 according detected by FACS following staining with an anti-Flag-APC. Several different spacers were compared between the scFv and CD3γ: flexible 3xG4S linker (SEQ ID NO: 3), rigid 3xEAAAK (SEQ ID NO:41), semi-flexible GGGSEAAAKGGGS (SEQ ID NO:42), semi-rigid EAAAKGGGSEAAAK (SEQ ID NO: 43), or IgG4 short 14aa hinge (SEQ ID NO: 44). The γ TRAMs were compared to a conventional CAR consisting of the same Leu16 scFv followed by the CD28 hinge (SEQ ID NO: 21), CD28 transmembrane (SEQ ID NO: 45), 4-1BB costimulatory domain (SEQ ID NO: 7), and CD3ζ signaling domain (SEQ ID NO: 8). Expression of the Leu16 CARs or TRAMs under the EF1a promoter was lower than CART-19, with the conventional CAR detected in 2.6% and TRAMs 0.8-1.5% of cells 12 days post transfection (FIG.13B). Example 8: T cell-specific promoters [0656] TRAMs can prevent surface expression in non-T cells even after transcription, but an approach to prevent transcription is to use promoters that express specifically in T cells. Using the Vingi1 driver with a GFP transgene template, several natural and synthetic promoters compared to MNDopt were tested (FIGS.14A-14E). Donor 10 T cells were transfected with indicated Vingi1 RNA-LNPs. A single reporter only (R only) is shown and all other R only controls showed the same results. Error bars show SD of duplicates. NT = non-treated cells. FIGS.14A-14B: Natural T cell promoters analyzed 5 days post LNP transfection. FIGS.14C- 14E: Synthetic T cell promoters analyzed 6 days post LNP transfection. FIGS.14A and 14C: Genomic integrations according to dPCR with a GFP probe. FIGS.14B and 14D: %GFP+ detected by FACS. FIG.14E: GFP MFI (median) of GFP+ cells where there were >500 events. The natural human (h) and mouse (m) T cell promoters hCCL5 (SEQ ID NO: 46), mCCL5 (SEQ ID NO: 47), hCD2 (SEQ ID NO: 48), mCD2 (SEQ ID NO: 49), hCD3γ (SEQ ID NO: 50) , hCD3δ (SEQ ID NO: 51), mCD3δ (SEQ ID NO: 52), hCD3ε (SEQ ID NO: 53), and mCD3ε (SEQ ID NO: 54) resulted in <0.5% GFP+ (FIG.14A) with integrations detected by dPCR somewhat higher for mCCL5 followed by mCD3δ but still much lower than MNDopt (FIG. 14B). All of these promoters are >1260bp so the low integration is not surprising and naturally have lower expression than constitutive promoters typically used for transgene expression like MND and EF1α. Next, several modified or synthetic promoters were tested (FIGS.14C-E). A minimalized mCD3δ promoter (SEQ ID NO: 55) increased %GFP+ to approximately 3.3% and a synthetic T cell promoter with enhancer from chr16-445 and miniCMV (SEQ ID NO: 56) 127 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 mediated 2.3% GFP+ (FIG.14C). Several activation dependent synthetic promoters using binding several sites for NR4A (SEQ ID NO: 57), AP1 (SEQ ID NO: 58), NFAT (SEQ ID NO: 59), and NFκB (SEQ ID NO: 60) were tested which can be used to further enhance specific expression following activation. Only NFκBx4 showed good expression of up to 24% GFP (FIG. 14C). However, the efficiency of GFP expression compared to integrations, and the MFI of GFP+ cells was lower for all T cell promoters compared to MNDopt (FIGS.14D and 14E). It may be possible to combine several of these promoters to improve expression. The approach of using TRAMs to achieve T cell specific surface expression allows for use of strong and non- specific promoters like MNDopt to drive expression. If T cell promoters do generate sufficiently high protection, TRAMs would still be beneficial as an additional fail-safe mechanism in case the promoter is leaky in other cell types. Example 9: Using microRNAs to attain cell specificity [0657] An additional approach to increase cell specificity is by adding microRNA (miR) sites that do not express in T cells but do express in other cells in which expression is not desired. Binding of miRs to mRNA which leads to degradation, may be used to knock-down transgene expression (typically up to 90% reduction). For PoC, several configurations of 3 binding sites for the liver restricted miR-122 (SEQ ID NO: 61) on a Vingi1 driver with aGFP transgene under the MNDopt promoter were tested (as shown in FIGS.15A-C and 16A-16B). FIGS.15A-15C shows a non-limiting example of sketches of nucleic acids with miR binding sites. Three binding sites were added were added to nucleic acids in several configurations.(FIG.15A: Binding sites added to the sense strand of the transgene in the 3’ UTR are expected to lead to degradation following transcription. FIG.15B: Binding sites added to reporter RNA on the anti-sense strand (the orientation of the IVT) upstream to the inverted transgene which to degradation prior to integration. FIG.15C: Binding sites added to the 3’ UTR of driver RNA. One is in the 3’ UTR of the transcribed transgene (“miR122 R”) which is expected to knock-down expression similar to the typical use of miRs to regulate transgenes (FIG.15A). Another is on the anti-sense strand of the template in the orientation that is transfected to the cell (“miR122 R AS”) upstream to the flipped transgene template, which is expected to degrade the template RNA prior to integration (FIG.15B). Another is on the 3’ UTR of the driver RNA (“miR122 D”), which is expected to degrade the driver mRNA prior to translation of the ORF1 machinery required to integrate the 128 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 transgene (FIG.15C). The miR sites can be designed in other areas or configurations to achieve the same goal. [0658] In primary human hepatocytes FIG.16A: miR122 R led to 8 fold decrease in GFP expression without decreases integrations, as expected. In FIG.16A, primary human hepatocytes (PHH) were transfected with LNPs 1 day post thaw and analyzed by dPCR to GFP probe and FACS 3 days post transfection. In FIG.16B, Donor 12 T cells were transfected with indicated Vingi1 RNA-LNPs and analyzed by dPCR and FACS at 5 days post transfection. Error bars show SD of duplicates. NT = non-treated cells. Surprisingly, miR122 R AS almost completely prevented integration instead of only knocking down integration. miR122 D led to a 4-fold decrease in integration and GFP expression, as expected. The combination of miR122 D + miR122 R AS led to 0 integrations and expression. [0659] In primary T cells (FIG.16B) there would be an expectation that there would be no effect as miR-122 is not expressed in T cells. However, in fact it had substantial and unexpected effects though the mechanism is unknown. The miR122 R led to a <2-fold decrease in integration and expression, miR122 R AS led to a >2 fold increase in integration and expression, and miR122 D led to a 1.2 fold increase in integration and expression. The combination of miR122 D + miR122 R AS increased %GFP+ from 24% with the WT to 60%. Inclusion of miR sites on the reporter in anti-sense and on the driver would both completely prevent integration in undesired cell types that express the miR, and potentially increase integration in T cells. However, finding miRs that differentiate between T cells and all other cell types of concern is challenging, and this approach may not be enough to achieve the required level of specificity. Although miR-122 is well characterized as liver specific, many others are not as well defined and may express to some degree in T cells. It is particularly difficult to find miRs that are not expressed at all in T cells yet express highly in B cells, the off-target cells of highest concern, as they are closely related. Therefore, there may be some benefit to using TRAMs to ensure cell specific surface expression when transcribed, which can be combined with other approaches that enhance specificity at the stage of integration or transcription. Example 10: Targeted LNPs (tLNPs) with several binders [0660] Typically, LNPs in the blood become coated with Apolipoprotein E (ApoE) and enter the cell via the low-density lipoprotein receptor (LDL-R). LDL-R surface expression on resting T cells is very low, and most T cells in circulation are in the resting state. One method of 129 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 mediating efficient delivery to resting T cells is to coat the LNPs with an antibody (“binder”) that recognizes an antigen on T cells leading to internalization. Several such antibodies were tested, including some that mediate T cell activation. The targets tested were CD2, CD5, CD7, TCR, and CD3 and all antibodies were human IgG1 Fc silent. To work with T cells closer to a resting state, T cells were cultured with cytokines IL-7 and IL-15 without the addition of an activating agent such as TransAct™. [0661] First, tLNPs containing mRNA for transient GFP expression (“EX1494” SEQ ID NO: 62) were tested to assess activation and transfection efficiency (FIGS.17A-17F). Donor 10 T cells were transfected transiently with GFP mRNA-tLNPs to the indicated antigen target with clone indicated in parenthesis. All antibodies were human IgG1 Fc silent. Isotype antibody was an anti-FITC, which does not express on human cells. FACS was performed 24h post LNP transfection. Error bars show SD of duplicates. FIG.17A: Expression of CD69 activation marker. FIG.17B: Expression of 41BB activation marker. FIG.17C: %GFP+ following transfection without ApoE. FIG.17D: GFP geometric mean fluorescence intensity (gMFI) of the GFP+ cells following transfection without ApoE. Background gMFI of singlets in NT cells was subtracted from the gMFI of each sample. FIG.17E: %GFP+ following transfection in the presence of ApoE. FIG.17F: GFP gMFI of the GFP+ cells following transfection in the presence of ApoE. Background gMFI of singlets in NT cells was subtracted from the gMFI of each sample. Anti-CD3 (clone OKT3, VH SEQ ID NO: 63, VL SEQ ID NO: 64) and Anti-TCR (clone BMA031, VH SEQ ID NO: 65, VL SEQ ID NO: 66) coated tLNPs mediated activation as indicated by an increase in CD69 and 41BB, whereas anti-CD2 (clone VIP VIIIC8), CD5 (clone H65, VH SEQ ID NO: 67, VL SEQ ID NO: 68), CD7 (clone 3A1E, VH SEQ ID NO: 69, VL SEQ ID NO: 70), CD8 (clone OKT8, VH SEQ ID NO: 71, VL SEQ ID NO: 72), and CD3 (clone Visilizumab, VH SEQ ID NO: 73, VL SEQ ID NO: 74) did not (FIGS.17A-17B). Without the presence of ApoE, where tLNP entry is mediated target antigen alone. Without ApoE at all doses (200, 400, 1200ng RNA) the isotype tLNPs transfected <10% of cells, most tLNPs (CD2, CD5, CD7, TCR, CD3 OKT3) transfected all T cells (~85% of cells), CD8 tLNPs only transfected CD8+ cells (~20% of cells) (FIG.17C). CD3 Visilizumab tLNPs transfected some of the T cells in a dose dependent manner (44% at 200ng, 51% at 400ng, and 69% at 1200ng) (FIG.17C). The expression level of GFP (MFI of GFP+ cells) ranking was CD3 (OKT3), TCR > CD7, CD8, CD5 > CD2 > CD3 (Visilizumab) > Isotype (FIG.17D). In the presence of ApoE, where the tLNPs can enter either via the target antigen or LDL-R, the trends 130 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 were similar except that the isotype and CD8 were closer in %GFP+ to the other tLNPs (FIG. 17E), the MFI overall was higher, and the difference in MFI between the activating TCR and CD3 (OKT) tLNPs and all other tLNPs was greater (FIG.17F). Taken together, these results show that tLNPs can mediate efficient uptake even in the absence of ApoE, and that activating tLNPs mediate superior transfection/expression especially in the presence of ApoE since they upregulate LDL-R. [0662] Next, tLNPs containing Vingi1 driver mRNAs with GFP cargo were tested (FIGS.18A- 18B). Donor 10 T cells were transfected with Vingi1 GFP mRNA-tLNPs to the indicated antigen target with clone indicated in parenthesis. All antibodies were human IgG1 Fc silent and isotype antibody was an anti-FITC, which does not express on human cells. FACS was performed 5 days post LNP transfection. Error bars show SD of duplicates. FIG.18A: %GFP+ following transfection without ApoE. FIG.18B: %GFP+ following transfection in the presence of ApoE. Without ApoE only the activating tLNPs showed integration above 0.05% GFP+ which increased between the tested doses of 200ng, 400ng, and 1200ng RNA (FIG.18A). CD3 (OKT3) mediated more efficient integration than TCR (0.11 vs 0.05%, 0.21 vs 0.04% and 0.54% vs 0.09% at doses of 200, 400, and 1200ng respectively). In the presence of ApoE the activating tLNPs were again better than the others, though there were some GFP+ cells in some of the other targets and integration for activating antibodies decreased at the 1200ng dose (FIG.18B). Again CD3 (OKT3) mediated more efficient integration than TCR (0.5 vs 0.36% at the 200ng dose). It should be noted that the integration upon transfection of resting T cells, even with the activating tLNPs (maximum 0.5% GFP+), is much lower than the 20-40% GFP+ typically observed for cells activated with TransAct™ 2 days prior non-targeted LNP addition with the same Vingi1 GFP RTE. Example 11: Addition of activating antibodies to T cells added with LNPs improves integration [0663] To begin to further understand the influence of activation, particularly mediated by activating antibodies, on transfection and integration, several different stimulations added in parallel to non-targeted LNPs in the presence of ApoE were tested (FIGS.19A-19D). Donor 10 or donor 12 T cells were transfected with RNA-LNPs with the indicated conditions: Vingi1 driver and reporter in FIG.19A, Vingi1 driver only in FIG.19B, R2-1_TG driver and reporter in FIG.19A, R2-1_TG driver only in FIG.19B. Positive control for strong activation using 131 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 TransAct™ 1:100 was added either on day 0 (day of thawing PBMCs, 2 days prior to LNPs; “TransAct day 0”) and together with LNPs (“TransAct day 2”). “No stimulation” was cultured in only IL-7 and IL-15. Test samples had 250ng/ml of antibody to the indicated antigen target with clone indicated in parenthesis added together with the LNPs. All antibodies were human IgG1 Fc silent and isotype antibody was an anti-FITC. LNP transfection was in the presence of ApoE. FACS was performed 5 days post LNP transfection. Error bars show SD of duplicates. FIG. 19A: %GFP+ following transfection without ApoE. FIG.19B: %GFP+ following transfection in the presence of ApoE. [0664] Antibodies initially tested were activating antibodies against TCR (BMA031) or CD3 (OKT3), and non-activating antibodies against FITC (“Isotype hIgG1”) CD5 (H65) or CD3 (Vislizumab). Stimulation controls were TransAct™ (“TA”) activation on day of thaw (day 0, 2 days before LNPs) or in parallel to LNP transfection like the other antibodies (day 2) and no stimulation (cultured in only IL-7 and IL-15). As shown in FIG.19A, integration was highest for Vingi1 TA day 0, with 24-28% GFP+. Activation mediated by TA, TCR (BMA031) or CD3 (OKT3) concurrent with LNPs showed between 3-14% GFP+, and non-activating conditions had <0.8% GFP+. The Vingi1 reporter only negative control, as shown in FIG.19B, showed <0.1% GFP+ for all conditions, well below background for the activating conditions. [0665] A reference driver on a site-specific RTE for the ribosomal 28S loci, R2-1_TG, derived from the Australian zebra finch (Taeniopygia guttata) in the same conditions was also tested (FIGS.19C-19D). The R2-1_TG driver used had undergone several rounds of engineering from the natural driver (SEQ ID NO: 75). The first 160 amino acids were removed and the carboxy- terminal binding protein interacting protein (“CtIP”, SEQ ID NO: 76) followed by the high mobility group nucleosome binding domain 1 (“HMGN1”, SEQ ID NO: 77) were fused to the 5’ UTR with the XTEN linker (SEQ ID NO: 78) between each added domain. Additionally, several point mutations were introduced (P280K, D555K, T1011S, I1219N, A1282G). An exemplary engineered R2-1_TG driver nucleic acid (SEQ ID NO: 82 encoded by SEQ ID NO: 3328) comprises a T7 Clean Cap (SEQ ID NO: 28), an R2-1_TG driver 5’ UTR (SEQ ID NO: 80), a nucleotide segment encoding an engineered R2-1_TG polypeptide (SEQ ID NO: 2506), a R2- 1_TG driver 3' UTR (SEQ ID NO: 83), and a polyA signal. The R2-1_TG reporter consists of the T7 Clean Cap (SEQ ID NO: 28), an R25’ homology arm (SEQ ID NO: 84), an R2-1_TG 5’ RTE-UTR (SEQ ID NO: 85), the anti-sense of the cargo (e.g., comprising a promoter, a transgene, and optionally a polyA signal) of interest in the inverse orientation, an R2-1_TG 3’ 132 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 RTE-UTR (SEQ ID NO: 86), an R23’ homology arm (SEQ ID NO: 87), and a polyA signal. The cargo tested was GFP (SEQ ID NO: 24) with the MNDopt promoter (SEQ ID NO: 25). Using this system, only the TA day 0 showed GFP of 0.16-0.27% (FIG.19C) that was discernable from the background 0.01-0.08% GFP+) seen with the reporter only (FIG.19D). [0666] Although the %GFP for the site-specific RTE is lower than Vingi1, the potential safety advantages of a site-specific RTE are immense, especially considering reports of T cell cancers from CAR-T cells. These cancers were likely caused by integration of the retroviral or lentiviral vector into genes involved in preventing cancer. The 28S ribosomal site has many copies in the genome, and therefore integration of an RTE into one of the 28S sites is likely to be safe. Taken together it seems that activation substantially improves transfection and integration, and this improvement is much greater if the cells are activated 2 days before LNP transfection rather than concurrent with LNP transfection. Example 12: Kinetics of LDL-R expression following activation [0667] LDL-R expression following activating antibody addition is a marker for T cell activation and mediates entry of LNPs. As shown in FIGS.20A-20D, the kinetics of LDL-R expression was tested to investigate why TransAct™ activation 48h prior to LNP addition was superior to contemporaneous LNP addition and if applicable to antibody mediated activation. [0668] Donor 10 PBMCs were thawed and seeded in IL-7 or IL-15 without TransAct™ for all samples except for TransAct™ day 0 (TA Day0) where TransAct™ 1:100 and IL-2 were added at thaw. After 2 days, indicated antibodies were added at indicated concentrations or positive activation control TransAct™ 1:100 (TA Day2) or negative control with no added reagents. All antibodies were human IgG1 Fc silent and Isotype antibody was an anti-FITC. Cells were stained with Allophycocyanin-tagged antibodies against -LDL-R (LDL-R-APC antibodies) at indicated time points after the time of antibody addition on day 2. Error bars show SD of duplicates. FIG.20A shows a bar graph showing percentages of LDL-R positive cells (%LDL- R+) at all antibody doses, from 10 ng/ml to 1000 ng/ml. FIG.20B shows an X-Y plot showing %LDL-R+ for antibody dose of 1000ng/ml and controls. FIG.20C shows LDL-R median fluorescence intensity (MFI) of LDL-R+ cells bar graph at all antibody doses. FIG.20D shows LDL-R MFI (median) of LDL-R+ cells XY graph for antibody dose of 1000ng/ml and controls. Activating antibodies against TCR (BMA031) or CD3 (OKT3), or non-activating antibodies against Isotype or CD7 (3A1E) were added to T cells 2 days post thaw (concurrently with LNPs). Control conditions include no stimulation, or TransAct™ activation added at the same 133 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 time (TA day2) or upon thaw (TA day0). LDL-R expression kinetics were measured over 48h from antibody addition. There was a modest increase within the first 30h of activation (up to 1.7 fold) and marked increase by 48h post-activation (up to 3.5 fold in %LDL-R+ and 10 fold in LDL-R MFI of LDL-R+ cells). TCR (BMA031) and CD3 (OKT3) performed similarly and were slightly lower than TransAct™ day 2 and much higher at 48h than non-activating conditions. These data indicate that it would be beneficial to effect LNP delivery 48h post activation rather than activating at the same time as LNP addition, either through activating tLNPs (LNPs conjugated with an activating antibody) or addition of soluble activating antibodies. Additionally, activation of cells before LNP addition gives time for the T cell metabolism and proliferation to be higher during the LNP treatment, which also should improve integration. Example 13: Addition of activating antibodies to T cells 2 days prior to LNP transfection improves transfection and integration more than coadministration with LNPs [0669] Based on the results described in Example 12, supra, transfection and integration were tested when adding the activating anti-TCR (BMA031) antibody either when thawing PBMCs 2 days prior LNPs (on day 0) or together with LNPs (on day 2), with controls of no-stimulation (cultured in only IL-7+IL-15) or strongly activating TransAct™+IL2 (TA+IL2). To analyze activation markers and transfection, transient GFP LNPs were added and FACS was performed 24h post LNP transfection (FIGS.21A-21H). Donor 12 or donor 15 PBMCs were thawed and either cultured only in IL-7 and IL-15 (“no stim”), IL-7 and IL-15 with the addition of 250ng/ml anti-TCR (BMA031) activating antibody (“TCR d0”), or IL-2 with the addition of TransAct™1:100 “TA+IL2.” Two days later, tLNPs coated with anti-CD5 (H65) antibody and containing mRNA for transient GFP expression were added in the presence of ApoE. For the “TCR d2” treatment, 250ng/ml anti-TCR activating antibody (BMA031) was added to the cells cultured in only IL-7 and IL-15 concurrently with the transient GFP LNPs. FACS was performed 24h post LNP transfection. NT = Non-treated (cultured in only IL-7 and IL-15). FIG. 21A shows percent of GFP positive cells (%GFP+). FIGS.21B-21D shows percent of cells expressing activation markers: FIG.21B shows percent of cells positive for activation marker LDL-R (%LDL-R+), FIG.21C shows cells percent of positive for activation marker 4-1BB (CD137) (%4-1BB+), FIG.21D shows percent of cells positive for activation marker CD69 (%CD69+). FIGS.21E-21H shows the expression level of the respective markers as measured by MFI gated on positive cells: FIG.21E shows the MFI of GFP, FIG.21F shows the MFI of 134 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 LDL-R-APC, FIG.21G shows the MFI of 4-1BB-PE, FIG.21H shows the MFI of CD69-PE- Cy7. Both %GFP+ and LDL-R expression were much higher when cells were activated with TCR antibody or TransAct™ on day 0 (2 days prior to LNPs) compared to TCR antibody on day 2 or without stimulation (FIGS.21A, 21B, 21E, and 21F). Within the day 0 activation, activation with TransAct™ showed about 2-3 fold higher GFP MFI than activation with the TCR antibody. Additional activation markers of 4-1BB and CD69 were also highest in %positive for day 0 activations, though TCR day 2 was closer to the day 0 activations than it was for GFP and LDL- R in particular for donor 12 (FIG.21C+21D). Consistent with the known kinetics of 4-1BB and CD69, the MFI was higher for TCR day 0 for 41BB and TCR day 2 for CD69, at least for donor 12 (FIGS 21G and 21H). [0670] Next, a similar study was conduct with Vingi1 driver-based GFP transgene integration. Donor 12 or donor 15 PBMCs were thawed and either cultured only in IL-7 and IL-15 “no stim”, IL-7 and IL-15 with the addition of 250ng/ml anti-TCR (BMA031) “TCR d0”, or IL-2 with the addition of TransAct™ 1:100 “TA+IL2.” Two days later LNPs (non-targeted) containing indicated mRNAs were added. For the “TCR d2” treatment, 250ng/ml anti-TCR (BMA031) was added to the cells cultured in only IL-7 and IL-15. FACS (based on GFP fluorescence) and digital PCR with MNDopt (promoter) or GFP (transgene) probes were performed 5 days post LNP transfection. “NT” indicates Non-treated (cultured in only Il-7 and IL-15). Error bars show SD of duplicates. FIG.22A and FIG.22B shows the genomic integration of GFP transgene with a Vingi1 driver and a MNDopt-GFP reporter, as measured by FACS (FIG.22A) and digital PCR (FIG.22B). FIG.22C and FIG.22D shows a negative control experiment with Vingi1 MNDopt-GFP reporter only (no driver nucleic acid). Regarding integration of GFP transgene with the Vingi1 driver (FIG.22A). TCR activation on day 0 was better than day 2 (11.7 vs 3.1% for donor 12 and 5.8 vs 1.3% for donor 15), yet 3-4 fold lower than TransAct™ day 0 (29.1% and 24.2% in donors 12 and 15, respectively). Trends were similar for genomic integrations according to dPCR (FIG.22B). The reporter-only negative control showed, as expected, no GFP expression or integrations (FIGS.22C-22D). Taken together these results imply that activation of cells 2 days prior to LNP treatment improves integration more than antibody activation together with LNPs. These data may also be applicable to activating tLNPs. 135 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 Example 14: Humanized OKT3 (Teplituzumab) added 2 days prior to LNPs mediates potent activation and efficient integration [0671] The approach of activating T cells prior to LNP transfection may be applied to in vivo CAR-T therapy. In this case it may be preferable to select an activating antibody that is humanized or fully human, available for in vivo administration, and shown to be tolerable in clinical trials. One such activating antibody is Teplizumab (VH SEQ ID NO: 88, VL SEQ ID NO: 89, full heavy chain SEQ ID NO: 90, full light chain SEQ ID NO: 91), an anti-CD3ε humanized antibody (OKT3) with mutations in the human IgG1 to reduce binding to Fc receptors (HzOKT3, Ala-Ala), which has been approved for treatment of type 1 diabetes with manageable side effects. This stands in contrast with the mouse OKT3 (without Fc silencing) that induced considerable side effects. First, the activation potential based on LDL-R expression 48h post addition of antibodies or TransAct™ that were added when thawing PBMCs were compared (FIGS.23A-23B). [0672] Donor 15 PBMCs were thawed and seeded with IL-7 or IL-15 for all samples except for “TA (IL2)” which was seeded with IL-2 and TransAct™ 1:100. Antibodies to indicated antigen target with clone indicated in parenthesis were added at the indicated concentrations (0.08 ng/ml, 0.40 ng/ml, 2 ng/ml, etc. as provided in the x-axis). Isotype IgG (anti-FITC), OKT3, and Teplizumab are human IgG1 Fc silent, Siplizumab is human IgG1, Urelumab is human IgG4. “CD3 (Tep) + 41BB” contained indicated concentration each of Teplizumab and Urelumab. Cells were stained with anti-LDL-R-APC 48h post treatment. FIG.23A shows the percentage of cells positive for activation marker LDL-R (%LDL-R+) as measure by FACS. FIG.23B shows the MFI of of LDL-R+ cells. Culture conditions tested were no stimulation (IL-7 and IL-15 only), isotype IgG, non-activating anti-CD2 (clone Siplizumab, VH SEQ ID NO: 92, VL SEQ ID NO: 93), co-stimulatory anti-41BB (clone Urelumab, VH SEQ ID NO: 94, VL SEQ ID NO: 95), Teplizumab + anti-41BB, TransAct™ 1:100 with IL-7 and IL-15 (like the antibodies) or TransAct™ 1:100 in IL2. As expected, the Isotype, CD2, and 41BB antibodies alone did not upregulate LDL-R compared to no stimulation. Teplizumab was extremely potent and is nearly saturated even with a very low dose of 0.08ng/ml and similar to TransAct™, whereas there was no indication of activation in the presence of anti-41BB antibody. The mouse OKT3 activated cells to a lower extent, and this activation was substantially reduced below 2ng/ml and was similar to no stimulation at 0.08ng/ml. However,it should be noted that it was from a different vendor (OKT3 from Absolute Antibodies, Teplizumab from Selleckchem). 136 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0673] Next, as shown in FIGS.24A-24F, integration of GFP under the MNDopt promoter was tested from several drivers 48h post thaw and activation with Teplizumab 250ng/ml, OKT3 250ng/ml, or TransAct™ and control with no stimulation (FIG.24). Donor 12 T cells were transfected with the indicated RNA-LNPs 2 days following the indicated stimulation. The LNPs tested were not targeted LNPs. FACS and digital PCR with MNDopt or GFP probes was performed 5 days post LNP transfection. NT = Non-treated, R only negative control, D+R = driver nucleic acid + reporter. Error bars show SD of duplicates. FIGS.24A-24C show the percentage of GFP positive cells (%GFP+) detected by FACS among PBMCs transfected with a Vingi1 driver (amino acid sequence set forth in SEQ ID NO: 31) and reporter (FIG.24A), PBMCs transfected with a R4-1_PH driver and reporter (FIG.24B), and PBMCs transfected with an R2-1_TG driver and reporter (FIG.24C). FIGS.24D-24F shows the genomic integrations detected by dPCR with the MNP (promoter) probe or the GFP (transgene) probe as indicated in each figure, among PBMCs transfected with a Vingi1 driver and reporter(FIG.24D), PBMCs transfected with a R4-1_PH driver and reporter(FIG.24E), and PBMCs transfected with an R2-1_TG driver and reporter (FIG.24F). [0674] As shown in FIGS.24A-24F, in addition to Vingi1 (SEQ ID NO: 31, encoded by IVT nucleic acid set forth in SEQ ID NO: 3332) and R2-1_TG (SEQ ID NO: 82), drivers, a driver based on R4-1_PH, a non-LTR retrotransposon that is predicted to be site specific for 28S rDNA derived from Parhyale hawaiensis was also tested. The R4-1_PH driver full transcript comprises the T7 Clean Cap (SEQ ID NO: 28), a R4-1_PH driver 5’ UTR (SEQ ID NO: 96), a R4-1_PH ORF2 encoding an RTE polypeptide (DNA SEQ ID NO: 97, protein SEQ ID NO: 98), optionally a R4-1_PH driver 3’UTR, and optionally polyA . The R4-1_PH reporter comprises the T7 Clean Cap (SEQ ID NO: 28), a R4-1_PH 5’ RTE-UTR (SEQ ID NO: 99), an anti-sense of the cargo (comprising, e.g., a promoter, a transgene, and a polyA signal) of interest in the inverse orientation, and a R4-1_PH 3’ RTE-UTR (SEQ ID NO: 100) and polyA. [0675] For Vingi1 driver-based genomic integration, %GFP+ was 0.26% without stimulation, 12.3% with Teplizumab, 6.4% with OKT3, and 18.2% with TransAct™ (FIG.24A). For R4- 1_PH driver-based genomic integration, %GFP+ was 0.03% without stimulation, 0.47% with Teplizumab, 0.54% with OKT3, and 1.26% with TransAct™ (FIG.24B). For R2-1_TG driver- based genomic integration, %GFP+ was 0.06% without stimulation, 0.22% with Teplizumab, 0.03% with OKT3, and 0.21% with TransAct™ (FIG.24C). The dPCR results FIGS.24D-24F showed similar trends, except that in general R4-1_PH driver integrations were lower than with 137 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 R2-1_TG driver (FIGS.24E-24F) and that R2-1_TG driver integrations were about 2-fold higher with TransAct™ activation than with Teplizumab or OKT3 (FIG.24F). Taken together, Teplizumab activation on day 0 mediates efficient integration of several different RTEs more than OKT3 and less than TransAct™. [0676] These results highlight the potential to enhance LNP uptake and RTE integration with pre-treatment with an activating antibody. There are several advantages of such an approach to activation mediated by activating targeted LNPs. Firstly, it gives enough time for the cells to reach an activation state and increase LDL-R expression for better uptake and integration. Secondly, the tolerability of activating antibodies tested in the clinic is well understood, whereas the tolerability of activating tLNPs is less understood and established. It should be noted that for Teplizumab to achieve the clinical benefit for diabetes, it is administered daily for 14 days, whereas for activation for LNP transfection and RTE integration, fewer doses may be required. Example 15: R2-1_TG engineered driver with reporters from different species [0677] To potentially increase R2 integration, the R2-TG driver nucleic acid encoding the highly engineered R2-TG driver (SEQ ID NO: 82) was tested with R2 template comprising a MNDopt- driven GFP transgene with RTE-UTRs from R2 retroelements from several different species. Donor 12 PBMC were activated with TransAct™, then transfected with the indicated RNA- LNPs for 2 days, and FACS and digital PCR with MNDopt or GFP probes was performed 5 days post LNP transfection. These experiments were run with TransAct™ activation to see a high signal. RTE-UTRs of R2 RTEs from the following species were tested: Taeniopygia guttata (TG), Phylloscopus trochilus trochilus (PTT), Limnodromus scolopaceus (LS), and Melierax canorus (MC). [0678] As shown in FIG.25A, the experimental conditions shown in the X-axis are to be interpreted as follows: NT = Non-treated, R only negative control, and D+R = driver nucleic acid + reporter. Error bars show SE of duplicates. Also as shown in FIG.25A, TG = Taeniopygia guttata, PTT = Phylloscopus trochilus trochilus, LS = Limnodromus scolopaceus, MC = Melierax canorus. As such, for example, “R2_TG D + TG R” means that the cells were transfected with an R2-TG driver nucleic acid (SEQ ID NO: 82) and an R2 reporter comprising RTE-UTRs from the same species that the driver polypeptide was derived from, namely R2-TG from Taeniopygia guttata. In another example, , “R2_TG D + PTT R” means that the cells were transfected with an R2-1 TG driver nucleic acid (IVT sequence set forth in SEQ ID NO:3328) 138 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 and an R2 reporter comprising RTE-UTRs from a different species, namely R2-PTT. It was found, as shown in FIG.25A, that the R2-1_PTT reporter was better than R2-1_TG reporter when used with the R2-TG driver while R2-1_LS and R2-1_MC were worse than R2-1_TG (FIG.25A). The PTT reporter comprises a 5’ UTR (SEQ ID NO: 125) comprising a 5’ RTE- UTR (SEQ ID NO: 126) from R2-PTT, an R2 retroelement found in Phylloscopus trochilus trochilus, and a 3’ UTR comprising a 3’ RTE-UTR from the R2-PTT and a PolyA signal. [0679] As shown in FIG.25B, the combination of the R2-1 TG driver (SEQ ID NO: 82) and the R2-1_PTT reporter were tested at several driver nucleic acid : reporter ratios (by weight of encapsulated RNA, with a constant total of 60ug per LNP), with a driver nucleic acid : reporter ratio of 1:1 being the control condition. [0680] The R2-1_PTT reporter was more effective in driving the GFP transgene expression than the T2-1_TG reporter. Expression levels were mostly the same across the different ratios. That said, the 2:1 driver:reporter nucleic acid ratio was somewhat higher, achieving 43 integrations of the MND probe and 25 integrations of the GFP probe as assessed by dPCR and 0.41% GFP+ by FACS. Example 16: Integration of TRAM with Vingi1 driver and Targeted LNPs [0681] Transfection of targeted LNPs (tLNPs) was tested in PBMCs from donor 13 (DP13) cultured in IL7+IL15 without an activating agent, using Vingi1 driven integration of a TRAM. tLNPs coated with one of the following antibodies were tested: non-activating anti-CD7 (3A1E), activating anti-TCR (BMA031), or activating anti-CD3 (OKT3) were tested. At doses of 200 ng and 400 ng. The test Vingi1 driver had an amino acid sequence set forth in SEQ ID NO: 27; and the template having a nucleotide sequence set forth in SEQ ID NO:3354, was driven by an MNDopt promotor and comprised an anti-CD19 FMC63 VL-VH γ TRAM. The Vingi1 template nucleotide sequence includes GOI anti-CD19 FMC63 VL-VH γ with the following features: Signal peptide SEQ ID NO: 3320; scFv chain 1 SEQ ID NO: 3322; intra scFv linker SEQ ID NO: 3211; scFv chain 2 SEQ ID NO:3316; Linkers after scFv (GCCGCCGCT); Hinge SEQ ID NO: 3321; transmembrane (tm) SEQ ID NO: 3317; Co stimulatory domain SEQ ID NO:3314; Signaling SEQ ID NO:3318 SEQ ID NO:3313. TRAM is in antisense orientation. Vpx enhancement was not used. Non-treated (NT), cysteine and isotype hIgG1 (anti-FITC) are negative controls. 139 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0682] TRAM transgene integration was measured by FACS (detecting anti-TRAM antibody- based fluorescence) and by dPCR using a polyA signal probe (indicating full integration of the TRAM transgene) 11 days post transfection of PBMCs from donor 13 (DP13). FIGS.26A-26B demonstrate that tLNPs are capable of mediating transfection and integration of TRAMs as measured by FACS (FIG.26A) and dPCR (polyA probe, FIG.26B). Data as shown in both figures indicate that using tLNPs coated with activating antibodies anti-CD3 OKT3 and anti- TCR BMA031 results in substantially higher genomic integration than controls, (FIG.26B). Using LNPs coated with non-activating anti-CD7 antibody only weakly improved genomic integration over controls. Example 17: Influence of clinically approved antibodies and BiTes on integration of a GOI [0683] FDA-approved antibodies may be effective activators and various antibodies were tested for their effect on driver-based genomic integration. Bispecific T cell engagers (BiTEs) are bispecific antibodies that redirect T cells to target antigen-expressing tumors. Exemplary clinically approved BiTes that target both CD20 and CD3 are Mosunetuzumab, Epcoritamab and Glofitamab. [0684] Test arms: LNP transfection in PBMCs from donor 10 (DP10). Test arms: No stimulus, 10pM or 1nM of Teplizumab (humanized anti-CD3 monoclonal antibody), Mosunetuzumab, Epcoritamab and Glofitamab, or TransAct™ each with NT (no treatment), R2 driver only, R2 driver+R2 GFP template+Vpx, or Vingi-GFP template (in cis) The R2 driver has an amino acid sequence set forth in SEQ ID NO: 642. SEQ ID NO:3203 sets forth the IVT nucleotide sequence encoding SEQ ID NO:642 protein. The nucleotide sequence includes Synthetic 5' UTR SEQ ID NO:80 ; mouse alpha globin 3'UTR SEQ ID NO:83 ; A29N10A70 polyA signal SEQ ID NO:3201. The orientation of the sequences between external UTRs is in antisense. The R2 GFP template has a nucleotide sequence set forth in SEQ ID NO: 2236. The Vingi1 driver-GFP reporter (in cis) has an IVT nucleotide sequence set forth in SEQ ID NO: 3332 and has the following features, in 5’ to 3’ order: T7 Clean Cap (SEQ ID NO: 28), Vingi1 driver 5’ UTR (SEQ ID NO: 29), Vingi1 ORF2 (DNA SEQ ID NO: 30, protein SEQ ID NO: 31), synthetic PolyA signal (SEQ ID NO: 36), inverted GFP (amino acid SEQ ID: 24), MNDopt promoter (SEQ ID NO: 25), Vingi1 reporter 3’UTR and polyA (SEQ ID NO: 34). [0685] Results: FIGS.27A-27 B show GFP expression as determined by FACS; FIGS.27C-27D show integration as copies per 100 genomes, assessed by dPCR. No integration is seen for Vingi 140 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 or R2 driver + R2 GFP template +Vpx in absence of an activating antibody or BiTe. Both anti- CD3/CD20 BiTes Epcoritamab and Glofitamab mediate transgene integration with R2 driver as well as TransAct™ when applied at a concentration of 1000 pM (1 nM). That said, integration with R2 driver was lower than integration with Vingi1 driver. Teplizumab also enabled integration, albeit lower than integration with the BiTes. [0686] Additional anti-CD3 activating antibodies that may be useful for pre-activation or as a targeting moiety on LNPs include: Otelixizumab (VH SEQ ID NO: 101, VL SEQ ID NO: 102, full heavy chain SEQ ID NO: 103, full light chain SEQ ID NO: 104), an anti-CD3ε humanized YTH12.5 IgG2 antibody; and Foralumab (VH SEQ ID NO: 105, VL SEQ ID NO: 106, full heavy chain SEQ ID NO: 107, full light chain SEQ ID NO: 108), an fully human IgG1 antibody with Fc silencing mutations. Example 18: Vingi Anti-CD-19 scFv TRAM Expression and in vitro Killing [0687] Several anti-CD19 scFvs having different orientations of Light and Heavy chains were tested for activity in PBMC donor 13 activated with TransAct + IL2. All scFvs were tested as a γ TRAM and contained a “Flag” tag on the N-terminus of the mature peptide following cleavage of the signal peptide. Anti-CD19 scFvs FMC63, 4G7, and Hu19 were tested in both VH-VL (variable heavy chain-variable light chain) and the reverse VL-VH orientations fused to CD3gamma. Analysis was performed with FACS following staining with an APC-conjugated anti-Flag antibody, and digital PCR for full integrations (R2-PTT 5’ UTR probe). Data was collected at 6, 9, and 12 days following LNP treatment. The killing potential of the cells was further tested in Nalm6 cells. [0688] The Vingi 1 driver had an amino acid sequence set forth in SEQ ID NO: 31, encoded by a nucleic acid sequence set forth in SEQ ID NO:3326. The templates used are shown in Table 6. LH refers to a light chain -heavy chain orientation, while HL refers to a heavy chain-light chain orientation. Table 6: Templates used for testing with Vingi1 driver Description SEQ ID NO: pV1-FMC63-LH 3210 pV1-FMC63-HL 3234 pV1-4G7-LH 3237 pV1-4G7-LH 3247 141 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 pV1-Hu19-LH 3249 pV1-Hu19-LH 3255 [0689] FIGS.28A-C showed that all TRAMs expressed on the surface and showed genomic integrations at all time points, highest at 12 days post LNPs (17-28% TRAM+ and 34-54 copies per 100 genomes). At 12 days post transfection, the receptors/TRAM+ cells ranged between 6300 and 24000 (FIG.28D). All TRAMs mediated killing of Nalm-6 Luc target cells above the background killing of non-treated (NT) cells in a luciferase killing assay (FIG.28E). The data generally show that the system works well with a variety of scFv. [0690] FMC63 (mouse) TRAM exhibited high levels of integration (FIG.28A-28C), expression (FIG.28D) and killing of Nalm6 cells (FIG.28E). For Hu19 (fully human) TRAM, TRAM expression is initially low post-transfection (e.g., day 6 as shown in FIG.28A, and day 9 as shown in FIG.28B), but is similar to FMC63 TRAM by day 12 (FIG.28C). [0691] The order of the heavy chain (H) and light chain (L) may affect killing capacity. For example, as shown in FIG.28E, in cells expressing FMC63 TRAM, the LH (solid square) exhibited more effective killing than the HL (open square). However, in cells expressing 4G7 TRAM or Hu19 TRAM, the HL exhibited more effective killing than the LH. Example 19: Identification of anti-CD20 scFv binders [0692] Template nucleic acids encoding CARs or TRAMs comprising one of several anti-CD20 scFv TRAMs in different orientations, in combination with Vingi 1 driver nucleic acid (amino acid sequence set forth in SEQ ID NO: 31, encoded by a nucleic acid sequence set forth in SEQ ID NO:3326), were tested for genomic integration of the scFv transgene in Donor 13 PBMC. FACS (%TRAM+ cells) and quantification of receptor per cell were tested [0693] FACS ( % CAR or % TRAM) and quantification of receptor per cell of PBMCs transfected (using LNPs) with the Vingi1 driver and a CAR- or TRAM-encoding template nucleic acid were performed at 6-, 9-, and 12-days post transfection. NT = Non-treated and R only (template alone) are negative controls. [0694] Template nucleic acids were prepared. Each template nucleic acid comprised a transgene driven by an MNDopt promoter and encoded a CAR or TRAM comprising an anti-CD20 scFv, Leu16, Obinutuzumab (Obi), or Ofatumumab (Ofa), either in LH (light chain-heavy chain) 142 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 orientation or HL (heavy chain-light chain) orientation fused to CD3^, and further comprising a Flag tab. Templates used are shown in Table 7: Table 7 Description scFv Hinge- TM Co-stim- Type SEQ ID Signaling NO: Leu16_HL_28 Leu16_H CD28 41BB-CD3ζ CAR 3085 Leu16_LH_28 Leu16_L CD28 41BB-CD3ζ CAR 3101 Leu16_HL_8 Leu16_H CD8 41BB-CD3ζ CAR 3119 Leu16_LH_8 Leu16_L CD8 41BB-CD3ζ CAR 3125 Obi_HL_28 Obi_HL CD28 41BB-CD3ζ CAR 3133 Obi_LH_28 Obi_LH CD28 41BB-CD3ζ CAR 3141 Obi_HL_8 Obi_HL CD8 41BB-CD3ζ CAR 3151 Obi_LH_8 Obi_LH CD8 41BB-CD3ζ CAR 3162 Ofa_HL_28 Ofa_HL CD28 41BB-CD3ζ CAR 3167 Ofa_LH_28 Ofa_LH CD28 41BB-CD3ζ CAR 3176 Ofa_HL_8 Ofa_HL CD8 41BB-CD3ζ CAR 3185 Ofa_LH_8 Ofa_LH CD8 41BB-CD3ζ CAR 3194 Leu16 LH-G4S- Leu16_L CD3γ (without co-stim) TRAM 3217 CD3G (flexible linker) Leu16 LH- Leu16_L CD3γ (without co-stim) TRAM 3227 EAAAK-CD3G (rigid linker) [0695] FIGS.29A-29B are graphs showing % CAR and quantification of receptors/cell 6-, 9- or 12-days following transfection with Vingi1 driver and CAR templates disclosed in Table 7. [0696] There appears to be some difference in expression for the H and L chain orientations for Obinutuzumab (Obi), Ofatumumab (Ofa), which translates to differences in quantification of receptor/cell, with the LH orientation showing better expression than the HL orientation. [0697] FIGS.29C-29D show the results for the Leu16 TRAM, which expresses well with both the flexible and rigid linkers. Example 20: Expansion of TRAM+ Cells [0698] A functional analysis shows that PBMCs transfected with a Vingi1-TRAM system comprising a Vingi1 driver (amino acid sequence set forth in SEQ ID NO: 31, encoded by a 143 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 nucleic acid sequence set forth in SEQ ID NO: 3326), and the Vingi1 template with anti-CD19 FMC63 VL-VH γ as GOI as described in Example 16) are able to expand. Specific expansion of TRAM+ cells following LNP-RTE transfection, with no activation (cultured in IL7, IL15) was found to take place when the TRAM+ cells were co-cultured with a CD19+ B cell line (Nalm6). Such a co-culturing may more accurately model cell expansion in vivo where TRAM+ cells would be expected to be activated and proliferate following an encounter with CD19+ cells (e.g., tumor cells).FIG.30A shows integration and expression of TRAM at 12-days post transfection with LNP. FIG.30B shows integration and expression of TRAM at 4 day and 7 day post co- culture with Nalm6 cells (CD19 +). Data show enhanced expression and integration of TRAM indicating that the transfected T cells have been activated and are proliferating. Example 21: Mouse studies for LNP delivery and RTE driven CAR or TRAM integration in vivo with or without pre-treatment with activating antibodies [0699] The efficiency and biodistribution of LNP delivery in vivo in mice with or without pre- treatment of activating antibodies is tested. For immunodeficient mice with human PBMCs, an anti-human CD3ε antibody (e.g., Teplizumab) will be used. For immunocompetent mice an anti- mouse CD3ε antibody (e.g., 145-2C11) will be used. For biodistribution and transfection efficiency transient RNA-LNPs will be used, and for integration RTE RNA-LNPs will be used. Example 22: Anti-tumor response mediated by CAR-T or TRAM-T cells engineered with RTEs [0700] The anti-tumor response to a lymphoma model (e.g. Nalm6 or Raji) is tested in immunodeficient mice mediated by CAR or TRAM-expressing T cells engineered with driver- based transgene integration. Example 23: Testing engineered R2-TG drivers in Immune cells [0701] Various R2-TG drivers having amino acid and nucleic acid sequences shown in Table 8 were tested in PBMCs from Donor 13, with a GFP reporter template having nucleic acid sequence set forth in SEQ ID NO: 2362. In half of the samples, 4ug Vpx mRNA (SEQ ID NO: 3399 encoding amino acid sequence set forth in SEQ ID NO:1568) was co-encapsulated with 30ug driver and 30ug reporter into SM102 LNPs. 144 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0702] PBMC from Donor 13 were activated by TransAct™ +IL2. Transfection (1200ng LNP per well) and expansion were performed using ImmunoCult™-XF T cell Expansion Medium as described in Example 1, supra. [0703] FACS and digital PCR with MNDopt or GFP probes were performed 5 days post LNP transfection. NT = Non-treated, R only negative control. Error bars show SD of duplicates. Table 8 Type AA SEQ ID NO: NA SEQ ID NO: IVT SEQ ID NO 1 Engineered R2-TG 82 3327 3328 2 Engineered R2-TG 681 3266 3265 3 Engineered R2-TG 642 3204 3203 4 Engineered R2-TG 658 3284 3285 5 Engineered R2-TG 695 3294 3297 6 Engineered R2-TG 1208 3261 3262 7 Engineered R2-TG 1240 3281 3278 8 Engineered R2-TG 675 3289 3290 9 Engineered R2-TG 693 3272 3271 10 Engineered R2-TG 753 3286 3287 [0704] SEQ ID NO: 82 sets forth the amino acid sequence of an exemplary R2-1_TG driver comprising: an R2-1_TG polypeptide having, relative to SEQ ID NO: 75, deletion of 161 N- terminal amino acid residues; N-terminal fusion of the following: SV40-NLS polypeptide SEQ ID NO:430, CtIP polypeptide SEQ ID NO:76, HMGN polypeptide SEQ ID NO:77; and amino acid substitutions P280K, D555K, T1011S, I1219N, A1282G. SEQ ID NO: 3327 sets forth a corresponding DNA sequence encoding the above R2-1_TG driver. [0705] SEQ ID NO: 681 sets forth the amino acid sequence of an exemplary R2-1_TG driver comprising: an R2-1_TG polypeptide having, relative to SEQ ID NO: 75 deletion of 161 N-terminal amino acid residues; N-terminal fusion of the following: SV40-NLS polypeptide SEQ ID NO:430, EIF4a-2 polypeptide SEQ ID NO:592, CtIP polypeptide SEQ ID NO:76, HMGN polypeptide SEQ ID NO:77; and amino acid substitutions G257R, P280K, N363H, E399M, H423Q, A442P, D537Y, V539G, K547R, D555K, R669L, N889G, F973E, T1011A, 145 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 Q1019R, K1085R, D1133P, R1177G, Y1242K, A1282G. SEQ ID NO: 3266 sets forth a corresponding DNA sequence encoding the above R2-1_TG driver. [0706] SEQ ID NO: 642 sets forth the amino acid sequence of an exemplary R2-1_TG driver comprising: an R2-1_TG polypeptide having, relative to SEQ ID NO: 75 having a deletion of 184 N-terminal amino acid residues; N-terminal fusion of the following: SV40-NLS polypeptide SEQ ID NO:430, sto7D polypeptide SEQ ID NO:405, HMGN polypeptide SEQ ID NO:77; and amino acid substitutions P280K, D555K, T1011S, I1219N, A1282G. SEQ ID NO: 3204 sets forth a corresponding DNA sequence encoding the above R2-1_TG driver. [0707] SEQ ID NO: 658 sets forth the amino acid sequence of an exemplary R2-1_TG driver comprising: an R2-1_TG polypeptide having, relative to SEQ ID NO: 75 having a deletion of 161 N-terminal amino acid residues; N-terminal fusion of the following: SV40-NLS polypeptide SEQ ID NO:430, CtIP polypeptide SEQ ID NO:76, HMGN polypeptide SEQ ID NO:77; and amino acid substitutions P280K, D555K, D923K, Q955R, T1011S, I1219N, A1282G. SEQ ID NO: 3284 sets forth a corresponding DNA sequence encoding the above R2- 1_TG driver. [0708] SEQ ID NO: 695 sets forth the amino acid sequence of an exemplary R2-1_TG driver comprising: an R2-1_TG polypeptide having, relative to SEQ ID NO: 75 having a deletion of 161 N-terminal amino acid residues; N-terminal fusion of the following: hnRPA1-2 polypeptide SEQ ID NO:707, SV40-NLS polypeptide SEQ ID NO:430, CtIP polypeptide SEQ ID NO:76, HMGN polypeptide SEQ ID NO:77; and amino acid substitutions P280K, D555K, D923K, T1011S, I1219N, A1282G. SEQ ID NO: 3294 sets forth a corresponding DNA sequence encoding the above R2-1_TG driver. [0709] SEQ ID NO: 1208 sets forth the amino acid sequence of an exemplary R2-1_TG driver comprising: an R2-1_TG polypeptide having, relative to SEQ ID NO: 75 having a deletion of 161 N-terminal amino acid residues; N-terminal fusion of the following: SV40-NLS polypeptide SEQ ID NO:430, CtIP polypeptide SEQ ID NO:76, HMGN polypeptide SEQ ID NO:77; and amino acid substitutions P280K, D555K, E888A, N891A, T892A, S895A, E898A, T899A, N902A, T1011S, I1219N, A1282G. SEQ ID NO: 3261 sets forth a corresponding DNA sequence encoding the above R2-1_TG driver. [0710] SEQ ID NO: 1240 sets forth the amino acid sequence of an exemplary R2-1_TG driver comprising: an R2-1_TG polypeptide having, relative to SEQ ID NO: 75 having a deletion of 161 N-terminal amino acid residues; an N-terminal fusion of the following: bp-NLS (engineered 146 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 bipartite-NLS) polypeptide SEQ ID NO:3280, SV40-NLS polypeptide SEQ ID NO:3275, CHV polypeptide SEQ ID NO:599, UL42 polypeptide SEQ ID NO:598, CtIP polypeptide SEQ ID NO:76, HMGN polypeptide SEQ ID NO:77; and amino acid substitutions P280K, D555K, D923K, T1011S, I1219N, A1282G. SEQ ID NO: 3281 sets forth a corresponding DNA sequence encoding the above R2-1_TG driver. [0711] SEQ ID NO: 675 sets forth the amino acid sequence of an exemplary R2-1_TG driver comprising: an R2-1_TG polypeptide having, relative to SEQ ID NO: 75 having a deletion of 161 N-terminal amino acid residues; an N-terminal fusion of the following: SV40-NLS polypeptide SEQ ID NO:430, CtIP-I2A polypeptide SEQ ID NO:3291, HMGN polypeptide SEQ ID NO:77; and amino acid substitutions P280K, D555K, T1011S, I1219N, A1282G. SEQ ID NO: 3289 sets forth a corresponding DNA sequence encoding the above R2-1_TG driver. [0712] SEQ ID NO: 693 sets forth the amino acid sequence of an exemplary R2-1_TG driver comprising: an R2-1_TG polypeptide having, relative to SEQ ID NO: 75 having a deletion of 161 N-terminal amino acid residues; an N-terminal fusion of the following: SV40-NLS polypeptide SEQ ID NO:430, TUDOR polypeptide SEQ ID NO:3269, CtIP polypeptide SEQ ID NO:76, HMGN polypeptide SEQ ID NO:77; and amino acid substitutions P280K, D555K, T1011S, I1219N, A1282G. SEQ ID NO: 3272 sets forth a corresponding DNA sequence encoding the above R2-1_TG driver. [0713] SEQ ID NO: 753 sets forth the amino acid sequence of an exemplary R2-1_TG driver comprising: an R2-1_TG polypeptide having, relative to SEQ ID NO: 75 having a deletion of 161 N-terminal amino acid residues; an N-terminal fusion of the following: SV40-NLS polypeptide SEQ ID NO:430, CtIP polypeptide SEQ ID NO:76, HMGN polypeptide SEQ ID NO:77; and amino acid substitutions P280K, D555K, D923K, T1011S, I1219N, A1282G. SEQ ID NO: 3286 sets forth a corresponding DNA sequence encoding the above R2-1_TG driver. [0714] FIGS.31A and 31B show FACS and dPCR results. For each group of three columns, the column represents polyA dPCR. GFP expression as assessed by FACS is shown as a solid circle adjacent and to and to the right of the third column. In FIG.31A, in the absence of Vpx, the drivers having amino acid sequence set forth in SEQ ID NO: 642, SEQ ID NO: 695 and SEQ ID NO: 1208 exhibited highest GFP expression above driver having amino acid sequence set forth in SEQ ID NO: 82. These results were repeated in the presence of Vpx, as shown in FIG 31B. 147 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 Example 24: Engineered R2-TG driver drives integration and expression of TRAMs and CD19 CAR (CD19 CD28zeta) [0715] Engineered R2-TG driver having amino acid sequence set forth in SEQ ID NO: 642 was tested in PBMC from Donor 13, with a CD3gamma TRAM template (SEQ ID NO: 3308) or CD19 CD28zeta CAR template (nucleic acid sequence set forth in SEQ ID NO: 3312, amino acid sequence set forth in SEQ ID NO:3311). Vpx mRNA (nucleic acid sequence set forth in SEQ ID NO: 3399 encoding AA sequence set forth in SEQ ID NO: 1568) was co-encapsulated with 30ug driver and 30ug reporter into SM102 LNPs. [0716] CD19 CD28zeta CAR template nucleic acid comprises a transgene encoding an anti- CD19 CD28zeta CAR, which comprises an anti-CD19 FMC63 scFv fused to a CD28 hinge, TM and co-stimulating regions and a CD3zeta receptor, driven by a CMV promotor. [0717] CD3gamma TRAM template nucleic acid utilizes FMC63 scFv fused to a CD gamma TCR, driven by a CMV promotor. The template further comprises 5’ TEV UTR and 3’ HAG UTR. Where utilized, 'HAG' refers to mouse hemoglobin alpha, '3’UTR HAG' refers to the mouse hemoglobin alpha 3'UTR (SEQ ID NO: 83), '5’UTR HAG' mouse hemoglobin alpha 5'UTR (SEQ ID NO: 80). TEV refers to the Tobacco Etch Virus 5’ UTR (SEQ ID NO:3307). [0718] PBMC from Donor 13 were activated by TransAct™ +IL2. Transfection (1200ng LNP per well) and expansion were performed using Immunol™-XF T cell Expansion Medium as described in Example 1, supra. Cells were analyzed by FACS (based on anti-FMC63 immunofluorescence) and dPCR (CMV probe) at 6-, 9-, 12- and 16-days post transfection. NT refers to non-treated cells. [0719] SEQ ID NO: 642 is the amino acid sequence of an exemplary R2 driver comprising R2- 1_TG polypeptide with deletion of 184 N-terminal residues; and N-terminal fusion of the following: SV40-NLS polypeptide SEQ ID NO:3199, sto7D polypeptide SEQ ID NO:3202, HMGN-2 polypeptide SEQ ID NO:3298; and amino acid substitutions P280K, D555K, T1011S, I1219N, A1282G. [0720] SEQ ID NO:3312 sets forth the nucleotide sequence encoding SEQ ID NO:3311.The template nucleotide sequence includes as the GOI, a conventional anti-CD19 CAR with FMC63 scFv, CD28 hinge-transmembrane, CD28 co-stimulatory region, and CD3zeta signaling and has the following features: Signal peptide SEQ ID NO:3320 ; Scfv chain 1 SEQ ID NO:3322 ; intra scFv linker SEQ ID NO:3211 ; scFv chain 2 SEQ ID NO:3316 ; Linkers after scFv (GCCGCCGCT) ; Hinge SEQ ID NO:3321 ; transmembrane SEQ ID NO:3317 ; Co stim SEQ 148 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 ID NO:3314 ; Signaling SEQ ID NO:3318. The orientation of the CAR is in sense orientation. SEQ ID NO:3315 sets forth the template IVT nucleotide sequence comprising SEQ ID NO:3312. [0721] SEQ ID NO:3301 sets forth the amino acid sequence encoded by SEQ ID NO:3302.The amino acid sequence includes as the GOI a TRAM comprising FMC63 LH-CD3G, and has the following features: Signal peptide SEQ ID NO:1 ; scFv chain 1 SEQ ID NO:2 ; intra scFv linker SEQ ID NO:9 ; scFv chain 2 SEQ ID NO:4 ; Linkers after scFv (AAA) and SEQ ID NO:3 ; Cd3 chain hinge transmembrane signaling SEQ ID NO:18 SEQ ID NO:3301. The orientation of the TRAM from signal peptide to CD3 chain is in sense orientation. SEQ ID NO:3309 sets forth the template IVT nucleotide sequence comprising SEQ ID NO:3301. [0722] FIG.32A shows percent CAR or percent TRAM as measured by FACS with an anti- FMC63 antibody. The anti-CD19 CD28zeta CAR exhibited highest expression (0.54%) at 12 days, while the TRAM exhibited 0.73% expression at 9 days. Full integrations by the R2-TG driver are shown in Fig.32B, whereby integration of the anti-CD19 CD28zeta CAR increases over 16 days while expression of TRAM appears to peak at 12-16 days. Example 25: Engineered R2-TG drivers drive TRAM expression [0723] Engineered R2-TG driver having amino acid sequence set forth in SEQ ID NO: 1690 was tested in PBMCs from Donor 13, with a TRAM template driven by CMV promotor (SEQ ID NO: 3303) or by CMVg promotor (SEQ ID NO: 3309). Vpx mRNA (SEQ ID NO: 3399) was co-encapsulated with 30ug driver and 30ug reporter into SM102 LNPs. Cells were analyzed by FACS (anti-FMC63) and dPCR (CMV probe) at 6-, 9- and 12-days and number of receptors per cell at 9- and 12-days post transfection (as described in Example 1, supra). NT refers to non- treated cells, R only refers to transfection of reporter (template) only. [0724] SEQ ID NO:1690 sets forth the amino acid sequence of R2-1_TG Orf2 polypeptide; with deletion of 184 N-terminal residues; and N-terminal fusion of the following: SV40-NLS polypeptide SEQ ID NO:430, Sto7D polypeptide SEQ ID NO:405, HMGN polypeptide SEQ ID NO:77; and mutations P280K, D555K, D923K, T1011S, I1219N, A1282G. SEQ ID NO:3009 sets forth the nucleotide sequence encoding SEQ ID NO:1690 protein. The nucleotide sequence includes utr5 external:Bancov SEQ ID NO:80; utr3 external:HAG-3UTR SEQ ID NO:83; A29N10A70 polyA signal SEQ ID NO:3201. The orientation of the sequences between external UTRs is in antisense. 149 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0725] CMV promotor having a nucleic acid sequence set forth in SEQ ID NO: 3305 is the immediate/early promoter enhancer of cytomegalovirus (CMV) and is commonly used as an in vitro and in vivo promoter for driving the expression of transgenes in mammalian cells. CMVg promotor having a nucleic acid sequence set forth in SEQ ID NO: 3310 is the CMV promotor with a nucleotide substitution (C>G at position 408) believed to minimize silencing and increase stability. [0726] SEQ ID NO:3303 sets forth the nucleotide sequence encoding SEQ ID NO:3301 protein. The nucleotide sequence includes R220bp upstream homology arm SEQ ID NO:84 ; R2-1_PTT external 5'UTR SEQ ID NO:125 ; mir:miR-122 SEQ ID NO:3306 ; mir:miR-122 SEQ ID NO:3306 ; mir:miR-122 SEQ ID NO:3306 ; CMV promoter SEQ ID NO:3305 ; internal 5'UTR: TEV-pMRNAxp (minusG) SEQ ID NO:3307; reporter goi:TRuC_FMC63 LH-CD3G-sets forth the amino acid sequence of CART gene with the following features: Signal peptide SEQ ID NO:3214 ; scFv chain 1 SEQ ID NO:3212; intra scFv linker SEQ ID NO:3211; scFv chain 2 SEQ ID NO:3208; Linkers after scFv (GCTGCAGCT) and SEQ ID NO:3215; CD3 chain hinge -transmembrane-signaling (htm signaling) SEQ ID NO:3304 SEQ ID NO:3308; mouse alpha globin 3'UTR SEQ ID NO:83; synthetic polyadenylation signal SEQ ID NO:1641; R2-1_PTT external 3'UTR SEQ ID NO:126; R220bp downstream homology arm SEQ ID NO:87; A29N10A70 polyA SEQ ID NO:3201. The orientation of the sequences between external UTRs is in antisense. [0727] SEQ ID NO:3309 sets forth the nucleotide sequence encoding SEQ ID NO:3301 protein. The nucleotide sequence includes R220bp upstream homology arm SEQ ID NO:84 ; R2-1_PTT external 5'UTR SEQ ID NO:125; mir:miR-122 SEQ ID NO:3306 ; mir:miR-122 SEQ ID NO:3306; mir:miR-122 SEQ ID NO:3306 ; promoter:CMVg-The sequence harbors eCMV+pCMVg and additional 3' and 5' SEQ ID NO:3310; internal 5'UTR: TEV- pMRNAxp(minusG) SEQ ID NO:3307; reporter GOI: FMC63 LH-CD3G-sets forth the amino acid sequence of CAR gene with the following features: Signal peptide SEQ ID NO:3214; scFv chain 1 SEQ ID NO:3212 ; intra scFv linker SEQ ID NO:3211 ; scFv chain 2 SEQ ID NO:3208 ; Linkers after scFv (GCTGCAGCT) and SEQ ID NO:3215 ; CD3 chain htm signaling SEQ ID NO:3304 SEQ ID NO:3308 ; mouse alpha globin 3'UTR SEQ ID NO:83 ; synthetic polyadenylation signal SEQ ID NO:1641; R2-1_PTT external 3'UTR SEQ ID NO:126 ; R220bp downstream homology arm SEQ ID NO:87 ; A29N10A70 polyA signal SEQ ID NO:3201. The orientation of the sequences between external UTRs is in antisense orientation. 150 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 [0728] FIG.33A-FIG33C show FACS, dPCR and quantification of receptors/cell, respectively. [0729] FIG.33A shows that the R2-TG driver and template having a CMV or CMVg promotor expressed TRAM. At 12-days post transfection, the template with the CMVg promotor exhibits about twice the number of TRAM+ than the template with the CMV promotor. [0730] FIG.33B shows that the two templates result in approximately the same number of integrations (copies per 100 genomes) at all time points. [0731] FIG.33C shows that the R2-TG driver and template driven by CMV or CMVg express TRAM with template having the CMV or CMVg promotor but with higher expression of receptor with the CMVG promotor. Example 26: T cells with 28S integrated CAR/TRAM exhibit tumor cytotoxicity and cytokine production [0732] A Nalm6-Luc killing assay was performed using R2-TG driver and CMV-driven TRAM or CMVg-driven TRAM as shown in FIG.3, at 12 days post LNP transfection. Engineered R2- TG driver having amino acid sequence set forth in SEQ ID NO: 1690 was tested in PBMCs from Donor 13, with a CMV-driven TRAM template (SEQ ID NO: 3303) or with CMVg-driven TRAM template (SEQ ID NO: 3309). A Nalm6-Luc killing assay was performed with PBMCs transfected via lentivirus (LV) to integrate a EF1a-CART-19. [0733] Cells transfected via LNP with driver and template nucleic acids were cocultured with Nalm6 luciferase for 20 hours. [0734] Commercially available CD19-41BBzeta CAR has an amino acid sequence set forth in SEQ ID NO: 23 (encoded by a nucleic acid sequence set forth in SEQ ID NO: 3336), and consists of the CD8α signal peptide (“SP”, SEQ ID NO: 1), FMC63 VL (SEQ ID NO: 2), 3xG4S linker (SEQ ID NO: 3), FMC63 VH (SEQ ID NO: 4), CD8α Hinge (SEQ ID NO: 5), CD8α Transmembrane (“TM”, SEQ ID NO: 6), 4-1BB co-stimulatory domain (SEQ ID NO: 7), and CD3ζ signaling domain (SEQ ID NO: 8). [0735] FIG 34A shows the results of a Nalm6-luciferase killing assay, with R only as negative control and CD1941BBzeta CAR as positive control. The engineered R2-TG driver driving the TRAM templates exhibited effective tumor killing. FIG.34B shows levels of interferon gamma (IFNg) secretion by the Nalm6 cells as assessed by ELISA. The CMVg TRAM secreted more than twice the IFNg than CMV-TRAM. 151 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 Example 27: Reduction in Tumor Burden with Vingi1 CD3G TRAM T cells in a peripheralized NALM-6-Luc B cell leukemia model [0736] The primary objective of this study was to test the effects in an in vivo cancer model of T cells integrated with a TRAM transgene generated using Vingi1 driver RTE platform. Tumor suppression was tested using the NALM-6 tumor model for 28 days. The example provides ex vivo produced cells for an in vivo animal model of tumor growth. The Nalm6 cells express luciferase and the IVIS measures photons released by luciferase. [0737] The test driver nucleic acid was Vingi1 driven by MNDopt promotor (having amino acid sequence set forth in SEQ ID NO: 31; and the Vingi1 template comprised a transgene encoding a TRAM (template nucleic acid described in Example 16, supra and having a nucleic acid sequence set forth in SEQ ID NO:3354).2x10e5 Nalm6 luciferase cells were injected into groups of NSG (NOD scid gamma) immunodeficient mouse. The two negative control groups were A) PBS treated (5 mice) and B) untreated T cells treated (NT, 1 mouse). The positive control C) was a lentivirus (LV) EF1a CART-19 (7 mice) and test was D) Vingi1 TRAM as described herein (3 mice). 2.58 x 10e6 CAR or TRAM positive cells in a total of 15x 10e6 T cells were injected on day 4 to group C and D, respectively. IVIS was measured on Days 8, 12, 15, 19, 21, 26 and 28 post Nalm6 injection into mice. [0738] FIG.35 shows tumor burden as measured by IVIS (total flux (photons/sec)). Groups A and B show steady tumor growth over time, while groups C and D show no tumor growth. In this experiment, the Vingi1 TRAM system killed tumors as well as the commercial LV EF1a CART-19. 152 318294750

Claims

Attorney Docket No.:AVRT-010/02WO 351047-2032 CLAIMS What is claimed is: 1. A nucleic acid comprising: (a) a nucleic acid sequence encoding a T cell receptor fused antigen modifier (TRAM) comprising: (i) an antigen binding domain; and (ii) a TCR subunit, and (b) a retrotransposable element untranslated region (RTE-UTR). 2. The nucleic acid of claim 1, wherein the nucleic acid comprises more than one separate nucleic acids. 3. The nucleic acid of claim 1 or 2, wherein the TCR subunit is selected from the group consisting of TCRα, TCRβ, CD3δ, CD3γ, and CD3ε. 4. The nucleic acid of any one of claims 1-3, wherein the RTE-UTR comprises a 3’ RTE-UTR and/or a 5’ RTE-UTR. 5. The nucleic acid of any one of claims 1-4, wherein the RTE-UTR is derived from a non-LTR RTE. 6. The nucleic acid of claim 5, wherein the non-LTR RTE is selected from the group consisting of CRE, R4, Hero, NeSL, R2, RandI, Proto1, L1, Tx1, RTEPT, Proto2, RTEX, RTE, Outcast, Ingi, I, Nimb, Tad1, Loa, R1, Jockey, Rex1, CR1, L2, L2A, L2B, Daphne, and Crack. 7. The nucleic acid of any one of claims 4-6, wherein the 5’ RTE-UTR comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 85, SEQ ID NO: 99, SEQ ID NO: 125, SEQ ID NO: 127, any one of SEQ ID NO: 144-249, and any one of SEQ ID NO: 359-367, or modified versions of any of the preceding sequences having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, or a corresponding RNA version thereof. 8. The nucleic acid of any one of claims 4-7, wherein the 3’ RTE UTR comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 86, SEQ ID NO: 100, SED ID NO: 126, SEQ ID NO: 128, any one of SEQ ID NO: 250-358, and any one of SEQ ID NO: 368-372, or modified versions of any of the preceding sequences having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, or a corresponding RNA version thereof. 153 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 9. The nucleic acid of any one of claims 1-8, wherein the antigen binding domain is linked to the N terminus of the TCR subunit, optionally by a linker and/or hinge. 10. The nucleic acid of any one of claims 1-9, wherein the antigen binding domain is selected from the group consisting of an scFv, a VHH, a TCR-like antibody, a natural ligand, an FcR- binding receptor (e.g. CD16), and an NK killing receptor. 11. The nucleic acid of any one of claims 1-10, wherein the antigen binding domain is specific for GPCR5D. 12. The nucleic acid of any one of claims 1-10, wherein the antigen binding domain is specific for BCMA. 13. The nucleic acid of any one of claims 1-10, wherein the antigen binding domain is specific for CD22. 14. The nucleic acid of any one of claims 1-10, wherein the antigen binding domain is specific for CD19. 15. The nucleic acid of claim 14, wherein the CD19 antigen binding domain is derived from an antibody selected from the group consisting of FMC63, murine 4G7, human Hu19, Hu1E7, huB4, hBU12, humanized FMC63, humanized 4G7, GR37, GR41, SRB-37, and SRB-85, or a modified version of any of the preceding. 16. The nucleic acid of any one of claims 1-10, wherein the encoded antigen binding domain is specific for CD20. 17. The nucleic acid of claim16, wherein the CD20 antigen binding domain is derived from an antibody selected from the group consisting of Leu16, rituximab, ofatumumab, ocrelizumab, obinutuzumab, Ibritumomab tiuxetan, AME-133v, IMMU-106, TRU-015, and tositumomab, or a modified version of any of the preceding. 18. The nucleic acid of any one of claims 1-17, wherein the antigen binding domain is a bispecific antigen binding domain. 19. The nucleic acid of any one of claims 1-18, wherein the nucleic acid comprises DNA. 20. The nucleic acid of any one of claims 1-18 wherein the nucleic acid comprises RNA. 21. The nucleic acid of any one of claims 1-18, wherein the nucleic acid is a RNA/DNA hybrid. 22. A retrotransposable-element (RTE) integration system comprising a driver nucleic acid and a template nucleic acid, wherein: (a) the driver nucleic acid comprises a nucleic acid sequence encoding an RTE polypeptide, wherein the RTE polypeptide is a site-specific RTE polypeptide; and 154 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 (b) the template nucleic acid comprises: (i) a nucleic acid sequence encoding an engineered immune receptor; and (ii) an RTE-UTR capable of being bound by the RTE polypeptide. 23. The system of claim 22, wherein the engineered immune receptor comprises an antigen binding domain. 24. The system of claim 22 or claim 23, wherein the engineered immune receptor is selected from the group consisting of a T cell receptor (TCR), a TCR fused antigen modifier (TRAM), a TRuC and a chimeric antigen receptor (CAR). 25. A retrotransposable-element (RTE) integration system comprising a driver nucleic acid and a template nucleic acid, wherein: (a) the driver nucleic acid comprises a nucleic acid sequence encoding an RTE polypeptide; and (b) the template nucleic acid comprises (i) a nucleic acid sequence encoding a T cell receptor fused antigen modifier (TRAM) comprising an antigen binding domain and a TCR subunit; and (ii) an RTE-UTR capable of being bound by the RTE polypeptide. 26. The system of any one of claims 22-25, wherein the RTE polypeptide mediates integration of a DNA copy of the nucleic acid sequence encoding an engineered immune receptor into a genome of an immune cell that natively expresses a TCR-CD3 complex. 27. The system of claim 26, wherein the immune cell that natively expresses a TCR-CD3 complex is a T-cell. 28. The system of any one of claims 22-27, wherein the driver nucleic acid and the template nucleic acid are provided as separate nucleic acids. 29. The system of any one of claims 22-27, wherein the driver nucleic acid and the template are provided in a same nucleic acid. 30. The system of any one of claims 22-29, wherein the RTE-UTR comprises a 3’ RTE-UTR and/or a 5’ RTE-UTR. 31. The system of claim 30, wherein the 5’ RTE-UTR comprises a nucleic acid sequence selected from the group consisting of SEQ ID SEQ ID NO: 33, SEQ ID NO: 85, SEQ ID NO: 99, SEQ ID NO: 125, SEQ ID NO: 127, any one of SEQ ID NO: 144-249, and any one of SEQ ID NO: 359-367, or modified versions of any of the preceding sequences having at 155 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, or a corresponding RNA version thereof. 32. The system of claim 30 or 31, wherein the 3’ RTE UTR comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 86, SEQ ID NO: 100, SED ID NO: 126, SEQ ID NO: 128, any one of SEQ ID NO: 250-358, and any one of SEQ ID NO: 368-372, or modified versions of any of the preceding sequences having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto, or a corresponding RNA version thereof. 33. The system of any one of claims 25-32, wherein the TCR subunit is selected from the group consisting of TCRα, TCRβ, CD3δ, CD3γ, and CD3ε. 34. The system of any one of claims 23-33, wherein the antigen binding domain is selected from the group consisting of an scFv, a VHH, a TCR-like antibody, a natural ligand, an FcR- binding receptor (e.g. CD16), and an NK killing receptor. 35. The system of any one of claims 23-34, wherein the antigen binding domain is specific for GPCR5D, BCMA, CD22, CD19 or CD20. 36. The system of claim 35, wherein the antigen binding domain is specific for CD19. 37. The system of claim 35, wherein the CD19 antigen binding domain is selected from the group consisting of FMC63, murine 4G7, human Hu19, Hu1E7, huB4, hBU12, humanized FMC63, humanized 4G7, GR37, GR41, SRB-37, SRB-85, and modified versions of any of the preceding. 38. The system of claim 35, wherein the antigen binding domain is specific for CD20. 39. The system of claim 38, wherein the CD20 antigen binding domain is selected from the group consisting of Leu16, rituximab, ofatumumab, ocrelizumab, obinutuzumab, Ibritumomab tiuxetan, AME-133v, IMMU-106, TRU-015, tositumomab, and modified versions of any of the preceding. 40. The system of any one of claims 23-36, wherein the antigen binding domain is bispecific. 41. The system of claim 40, wherein the antigen binding domain is bispecific for CD19 and CD20. 42. The system of any one of claims 22-41, wherein the RTE polypeptide is a non-LTR RTE. 43. The system of claim 42, wherein the non-LTR RTE is selected from the group consisting of CRE, R4, Hero, NeSL, R2, RandI, Proto1, L1, Tx1, RTEPT, Proto2, RTEX, RTE, Outcast, Ingi, I, Nimb, Tad1, Loa, R1, Jockey, Rex1, CR1, L2, L2A, L2B, Daphne, and Crack. 156 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 44. The system of any one of claims 22-43, wherein the RTE polypeptide is a naturally occurring RTE polypeptide. 45. The system of any one of claims 22-43, wherein the RTE polypeptide is an engineered RTE polypeptide. 46. The system of any one of claims 22-45, wherein the template nucleic acid comprises a binding site for a differentially expressed microRNA (miR). 47. The system of claim 46, wherein the binding site for the differentially expressed miR is located within the 3’ RTE UTR region. 48. The system of claim 46 or 47, wherein the binding site for the differentially expressed miR is specific for miR-122. 49. The system of any one of claims 22-48, wherein the driver nucleic acid comprises a binding site for a differentially expressed miR. 50. The system of claim 49, wherein the binding site for the differentially expressed miR is specific for miR-122. 51. The system of any one of claims 22-50, wherein the driver nucleic acid comprises DNA. 52. The system of any one of claims 22-50, wherein the driver nucleic acid comprises RNA. 53. The system of any one of claims 22-50, wherein the driver nucleic acid comprises a DNA/RNA hybrid. 54. The system of claim 52, wherein the RNA is mRNA. 55. The system of any one of claims 22-54, wherein the template nucleic acid comprises DNA. 56. The system of any one of claims 22-54, wherein the template nucleic acid comprises RNA. 57. The system of any one of claims 22-54, wherein the template nucleic acid comprises a DNA/RNA hybrid. 58. The system of claim 56, wherein the template nucleic acid comprises a promotor and a polyA signal. 59. A lipid nanoparticle (LNP) comprising the nucleic acid of any one of claims 1-21 or the system of any one of claims 22-58. 60. The LNP of claim 59, wherein the LNP is covalently linked to an antibody or any fragment thereof that recognizes a T cell antigen. 61. The LNP of claim 60, wherein the covalently linked antibody comprises a silent Fc domain. 62. The LNP of claim 60 or 61, wherein the antibody is specific for one or more of CD2, CD5, CD7, TCR, CD8, and CD3. 157 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 63. The LNP of any one of claims 60-62, wherein the antibody is selected from the group consisting of OKT3 (VH SEQ ID NO: 63, VL SEQ ID NO: 64), BMA031 (VH SEQ ID NO: 65, VL SEQ ID NO: 66), H65 (VH SEQ ID NO: 67, VL SEQ ID NO: 68), 3A1E (VH SEQ ID NO: 69, VL SEQ ID NO: 70), OKT8 (VH SEQ ID NO: 71, VL SEQ ID NO: 72), Visilizumab (VH SEQ ID NO: 73, VL SEQ ID NO: 74), Teplizumab (VH SEQ ID NO: 88, VL SEQ ID NO: 89), Siplizumab (VH SEQ ID NO: 92, VL SEQ ID NO: 93), Urelumab (VH SEQ ID NO: 94, VL SEQ ID NO: 95), Otelixizumab (VH SEQ ID NO: 101, VL SEQ ID NO: 102), and Foralumab (VH SEQ ID NO: 105, VL SEQ ID NO: 106), or modified versions of any of the preceding having at least 80%, 85%, 90%, 95%, or 99% sequence identity of each aforementioned VH or VL. 64. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and: (a) the nucleic acid of any one of claims 1-21; (b) the RTE integration system of any one of claims 22-58; (c) the LNP of any one of claims 59-63; or (d) a combination thereof. 65. A method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of (a) the nucleic acid of any one of claims 1-21; (b) the RTE integration system of any one of claims 22-58; (c) the LNP of any one of claims 59-63; (d) the pharmaceutical composition of claim 64; or (e) a combination thereof. 66. The method of claim 65, wherein the subject is pre-treated with a T cell activator or concomitantly treated with a T cell activator. 67. The method of claim 66, wherein the T cell activator is a CD3 agonist or a TCR agonist. 68. The method of claim 67, wherein the CD3 agonist is an antibody selected from the group consisting of OKT3, Teplizumab, Otelixizumab, Foralumab, Vislizumab, CD3_F1F, and CD3_F2B, or modified versions of any of the preceding. 69. The method of claim 67, wherein the TCR agonist is BMA031 or a modified version thereof. 70. The method of claim 66, wherein the T cell activator is an immune cell engager. 71. The method of claim 70, wherein the immune cell engager is a Bispecific T cell engager (BiTe). 158 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 72. The method of claim 71, wherein the BiTe is selected from the group consisting of Blinatumomab, MGD006, XmAb14045, AMG 330, AMG 420, AMG 564, AMG 701, BiTe 197, REGN1979, RG6026, Mosunetuzumab, AFM11, MGD006, tebentafusp, Catuxomab, Pasotuxizumab, Acapatamab, AMV564, Glofitamab, Solitomab, Talquetamab, Tarlatamab, Tebentafusp, Odronextamab, and Epcoritamab, or modified versions of any of the preceding. 73. The method of any one of claims 65-72, wherein the subject is further administered prior, concurrently, or subsequent to said administering with at least one γ-chain receptor agonist. 74. The method of claim 73, wherein the at least one γ-chain receptor agonist is selected from the group consisting of IL-2, IL-7, IL-15, IL-21, and modified versions of any of the preceding. 75. The method of any one of claims 65-74, wherein the disease is cancer or an autoimmune disease. 76. The method of any one of claims 65-75, wherein the route of administration is selected from the group consisting of intravenous, intraperitoneal, intramuscular, subcutaneous, intrathecal, and intradermal administration. 77. A method of in vivo genome manipulation comprising pre-administering a T cell activator and administering a pharmaceutical composition comprising a reprograming agent. 78. The method of claim 77, wherein the reprogramming agent is a genome manipulation system comprising one or more nucleic acids. 79. The method of claim 77, wherein the reprograming agent meditates durable expression by integrating one or more nucleic acids into a genome. 80. The method of claim 77, wherein the reprograming agent mediates transient expression of one or more nucleic acids. 81. The method of any one of claims 77-80, wherein the one or more nucleic acids comprise at least one nucleic acid sequence comprised in or derived from a retrotransposon, a transposon, a Cas protein, a guide RNA, or a combination thereof. 82. The method of claim 79, wherein the one or more nucleic acids comprise at least one nucleic acid sequence comprised in or derived from a retrotransposon. 83. The method of any one of claims 77-81, wherein the one or more nucleic acids is packaged in a viral vector or a lipid nanoparticle (LNP). 84. The method of claim 83, wherein the viral vector is selected from the group consisting of a lentivirus, a retrovirus, and an adenovirus. 159 318294750 Attorney Docket No.:AVRT-010/02WO 351047-2032 85. The method of any one of claims 77-84, wherein the T cell activator is an CD3 or TCR agonist. 86. The method of claim 85, wherein the CD3 agonist is an antibody selected from the group consisting of OKT3, Teplizumab, Otelixizumab, Foralumab, Vislizumab, CD3_F1F, CD3_F2B, and modified versions of any of the preceding. 87. The method of claim 85, wherein the TCR agonist is BMA031 or a modified version thereof. 88. The method of claim 77-84, wherein the T cell activator is an immune cell engager. 89. The method of claim 88, wherein the immune cell engager is a Bispecific T cell engager (BiTe). 90. The method of claim 88, wherein the BiTe is selected from the group consisting of Blinatumomab, MGD006, XmAb14045, AMG 330, AMG 420, AMG 564, AMG 701, BiTe 197, REGN1979, RG6026, Mosunetuzumab, AFM11, MGD006, tebentafusp, Catuxomab, Pasotuxizumab, Acapatamab, AMV564, Glofitamab, Solitomab, Talquetamab, Tarlatamab, Tebentafusp, Odronextamab, Epcoritamab, and modified versions of any of the preceding. 91. A method of in vivo genomic manipulation in an immune cell, the method comprising contacting the immune cell with: (a) the nucleic acid of any one of claims 1-21; (b) the RTE integration system of any one of claims 22-58; (c) the LNP of any one of claims 59-63; (d) the pharmaceutical composition of claim 64; or (e) a combination thereof, wherein the immune cell natively expresses a TCR-CD3 complex. 160 318294750
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210324332A1 (en) * 2020-03-27 2021-10-21 Dcprime B.V. Ex vivo use of modified cells of leukemic origin for enhancing the efficacy of adoptive cell therapy
US20220184230A1 (en) * 2019-09-03 2022-06-16 Myeloid Therapeutics, Inc. Methods and compositions for genomic integration
US20220193137A1 (en) * 2019-04-04 2022-06-23 Umc Utrecht Holding B.V. Modified immune receptor constructs
US20230141052A1 (en) * 2021-05-11 2023-05-11 Myeloid Therapeutics, Inc. Methods and compositions for genomic integration

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220193137A1 (en) * 2019-04-04 2022-06-23 Umc Utrecht Holding B.V. Modified immune receptor constructs
US20220184230A1 (en) * 2019-09-03 2022-06-16 Myeloid Therapeutics, Inc. Methods and compositions for genomic integration
US20210324332A1 (en) * 2020-03-27 2021-10-21 Dcprime B.V. Ex vivo use of modified cells of leukemic origin for enhancing the efficacy of adoptive cell therapy
US20230141052A1 (en) * 2021-05-11 2023-05-11 Myeloid Therapeutics, Inc. Methods and compositions for genomic integration

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
"United States Patent Office Manual of Patent Examining Procedures"
BAEUERLE, PATRICK A. ET AL.: "Synthetic TRuC receptors engaging the complete T cell receptor for potent anti-tumor response.", NATURE COMMUNICATIONS, vol. 10, no. 1, 2019, pages 2087, XP055620180, DOI: 10.1038/s41467-019-10097-0
CAMPITELLI ET AL.: "Reconstruction of full-length LINE-1 progenitors from ancestral genomes", GENETICS, vol. 221, no. 3, July 2022 (2022-07-01), pages iyac074
DIALLO AB ET AL.: "Ancestors 1.0: a web server for ancestral sequence reconstruction", BIOINFORMATICS, vol. 26, no. 1, 2010, pages 130 - 131
HAN JEFFREY S: "Non-long terminal repeat (non-LTR) retrotransposons: mechanisms, recent developments, and unanswered questions", MOBILE DNA, BIOMED CENTRAL LTD, LONDON, UK, vol. 1, no. 1, 12 May 2010 (2010-05-12), pages 15, XP021084898, ISSN: 1759-8753, DOI: 10.1186/1759-8753-1-15 *
LUDWIG ET AL., NUCLEIC ACIDS RESEARCH, vol. 44, no. 8, 2016, pages 3865 - 3877

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