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WO2024243460A1 - Vectors and methods for in vivo transduction - Google Patents

Vectors and methods for in vivo transduction Download PDF

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WO2024243460A1
WO2024243460A1 PCT/US2024/030878 US2024030878W WO2024243460A1 WO 2024243460 A1 WO2024243460 A1 WO 2024243460A1 US 2024030878 W US2024030878 W US 2024030878W WO 2024243460 A1 WO2024243460 A1 WO 2024243460A1
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sequence
viral
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vector
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Derek G. Ostertag
Traci OSTERTAG
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Abintus Bio Inc
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Abintus Bio Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/34—Vector systems having a special element relevant for transcription being a transcription initiation element

Definitions

  • CARs are engineered immune-receptors, which can redirect immune cells to selectively kill tumor cells or modulate an immune response at a site of inflammation.
  • the general premise for their use in cancer immunotherapy is to rapidly generate tumor- targeted immune cells.
  • the delivery vehicle is a nanoparticles, micelle, lipid nanoparticles, cationic polymetric nanoparticles, gold or other metallic-based nanoparticles, dendrimers, extracellular vesicles, DNA or RNA nanostructures, polypeptide structures, or silica-based Attorney Docket No.00156-005WO1 nanoparticles.
  • the recombinant polynucleotide comprises a sequence represented by: (CMV promoter)-(viral 5’R-U5)- (MMLV-psi)-(Viral gag)-(synthetic promoter)-(at least one transgene)-(WPRE domain)-(Viral 3’del-U3-R-U5).
  • the CMV promoter of comprises a sequence at least 85%- 100% identical to: CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAA TAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTA CGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAA TGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGT ACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGA TAGCGGTTTGACTCACGGGGATTTCCAAGTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCA CCAAAATCAATCAACGGGACTTTCCAAAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGAGTTTGTT
  • the R-U5 domain can comprise a sequence that is at least 80-100% identical to the sequence: GCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCA TCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGG TCTTTCATTT (SEQ ID NO:30).
  • the MMLV-psi sequence comprises a sequence that is at least 85%-100% identical to: AAGCTGGCCAGCAACTTATCTGTGTCTGTCCGATTGTCTAGTGTCTATGACTGATTTTATGCGCCTGC GTCGGTACTAGTTAGCTAACTAGCTCTGTATCTGGCGGACCCGTGGTGGAACTGACGAGTTCGGAACA CCCGGCCGCAACCCTGGGAGACGTCCCAGGGACTTCGGGGGCCGTTTTTGTGGCCCGACCTGAGTCCA AAAATCCCGATCGTTTTGGACTCTTTGGTGCACCCCCCTTAGAGGAGGGATATGTGGTTCTGGTAGGA GACGAGAACCTAAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGGACCGAAGCCGCGC CGCGCGTCTTGTCTGCTG (SEQ ID NO:31).
  • the viral gag domains comprise a sequence that is at least 80-100% identical to the sequence: GGCCAGACTGTTACCACTCCCTGAAGTTTGACCTTAGGTCACTGGAAAGATGTCGAGCGGATCGCTCA CAACCAGTCGGTAGATGTCAAGAAGAGACGTTGGGTTACCTTCTGCTCTGCAGAATGGCCAACCTTTA ACGTCGGATGGCCGCGAGACGGCACCTTTAACCGAGACCTCATCACCCAGGTTAAGATCAAGGTCTTT TCACCTGGCCCGCATGGACACCCAGACCAGGTCCCCTACATCGTGACCTGGGAAGCCTTGGCTTTTGA CCCCCCTCCCTGGGTCAAGCCCTTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCGT CTCTCCCCCTTGAACCTCCTCGTTCGACCCCGCCTCGATCCTCCCTTTATCCAGCCCTCACTCCTTCT CTAGGCGCC (SEQ ID NO:32).
  • the synthetic promoter comprises a sequence of SEQ ID NO:1, 12-25 or 26 Attorney Docket No.00156-005WO1 or sequences that are at least 80-99% identical thereto and can drive transcription of a downstream sequence.
  • the recombinant polynucleotide is used for in vivo transduction and the synthetic promoter comprises SEQ ID NO:4, 6-10 or 11 or sequence that are at least 80-99% identical thereto and which can drive transcription of a downstream sequence.
  • a transgene is operably linked and downstream of the synthetic promoter.
  • the transgene comprises a non-naturally occurring immune receptor construct.
  • the non-naturally occurring immune receptor is a chimeric antigen receptor (CAR), chimeric TCR (cTCR), or CAR-like construct.
  • the WPRE sequence can comprise a sequence that is at least 80%-100% identical to: AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTAC GCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCT CCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGC GTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCT TTCCGGGACTTTCGCTTTCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCT GCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCC
  • the viral 3’ delU3-R-U5 domain can comprise a sequence that is at least 80-100% identical to the sequence: GAGCTCAAAATAAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTAGGT TTGGCAAAATAAAAGAGCCCACAACCCCTCACTCGGCGCCAGTCCTCCGATTGACTGAGTCGCCCG GGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAG GGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTCTTTCATT (SEQ ID NO:34).
  • the viral miRNA target sequence domain can comprise a sequence that is at least 80-100% identical to the sequence: TGCTCAATAAATACCCGTTGAACGATTGCTCAATAAATACCCGTTGAAGCATTGCTCAATAAATACCC GTTGAACTACTGCTCAATAAATACCCGTTGAA (SEQ ID NO:35).
  • the viral miRNA target sequence domain can comprise a sequence that is at least 80-100% identical to the Attorney Docket No.00156-005WO1 sequence: CATGATTGCCACGTCTGCAGTACGATCATGATTGCCACGTCTGCAGTAGCATCATGATTGCCACGTCT GCAGTACTACCATGATTGCCACGTCTGCAGTA (SEQ ID NO:36).
  • the viral miRNA target sequence domain can comprise a sequence that is at least 80-100% identical to the sequence: TGATTGCCACTGTCTGCAGTACGATTGATTGCCACTGTCTGCAGTAGCATTGATTGCCACTGTCTGCA GTACTACTGATTGCCACTGTCTGCAGTA (SEQ ID NO:37).
  • the recombinant polynucleotide is a plasmid.
  • the disclosure also provides viral particle comprising an RNA sequence expressed from the foregoing plasmid.
  • the disclosure also provides a recombinant cell comprising the polynucleotide or plasmid of any of the foregoing.
  • the disclosure also provides a method or use of a selective synthetic promoter to prevent expression of transgenes and other encoded nucleotides operably linked to the selective synthetic promoter in non-intended cell types during in vivo therapy.
  • a therapeutic vector selected from the group consisting of gammaretrovirus-, lentivirus-, adenovirus-, adeno-associated virus-, herpesvirus-, human foamy virus-based products, transposons, plasmid, or artificial chromosomes comprise the selective synthetic promoter and operably linked transgene for in vivo therapy.
  • the disclosure also provides a recombinant vector comprising a sequence represented by: (CMV promoter)-(viral 5’R-U5)- (MMLV-psi)-(Viral gag)-(synthetic promoter)-(at least one transgene)-(WPRE domain)-(Viral 3’del-U3-R-U5) or (CMV promoter)- (viral 5’R-U5)-(MMLV-psi)-(Viral gag)-(WPRE domain)-(at least one transgene)-(synthetic promoter)-(Viral 3’del-U3-R-U5).
  • the CMV promoter of comprises a sequence at least 85%- 100% identical to: CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAA TAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTA CGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAA TGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGT ACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGA TAGCGGTTTGACTCACGGGGATTTCCAAGTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCA CCAAAATCAATCAACGGGACTTTCCAAAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGG
  • the R-U5 domain can comprise a sequence that is at least 80-100% identical to the sequence: GCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCA TCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGG TCTTTCATTT (SEQ ID NO:30).
  • the MMLV-psi sequence comprises a sequence that is at least 85%-100% identical to: AAGCTGGCCAGCAACTTATCTGTGTCTGTCCGATTGTCTAGTGTCTATGACTGATTTTATGCGCCTGC GTCGGTACTAGTTAGCTAACTAGCTCTGTATCTGGCGGACCCGTGGTGGAACTGACGAGTTCGGAACA CCCGGCCGCAACCCTGGGAGACGTCCCAGGGACTTCGGGGGCCGTTTTTGTGGCCCGACCTGAGTCCA AAAATCCCGATCGTTTTGGACTCTTTGGTGCACCCCCCTTAGAGGAGGGATATGTGGTTCTGGTAGGA GACGAGAACCTAAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGGACCGAAGCCGCGC CGCGCGTCTTGTCTGCTG (SEQ ID NO:31).
  • the viral gag domains comprise a sequence that is at least 80-100% identical to the sequence: GGCCAGACTGTTACCACTCCCTGAAGTTTGACCTTAGGTCACTGGAAAGATGTCGAGCGGATCGCTCA CAACCAGTCGGTAGATGTCAAGAAGAGACGTTGGGTTACCTTCTGCTCTGCAGAATGGCCAACCTTTA ACGTCGGATGGCCGCGAGACGGCACCTTTAACCGAGACCTCATCACCCAGGTTAAGATCAAGGTCTTT TCACCTGGCCCGCATGGACACCCAGACCAGGTCCCCTACATCGTGACCTGGGAAGCCTTGGCTTTTGA CCCCCCTCCCTGGGTCAAGCCCTTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCGT CTCTCCCCCTTGAACCTCCTCGTTCGACCCCGCCTCGATCCTCCCTTTATCCAGCCCTCACTCCTTCT CTAGGCGCC (SEQ ID NO:32).
  • the synthetic promoter comprises a sequence of SEQ ID NO:1, 12-25 or 26 or sequences that are at least 80-99% identical thereto and can drive transcription of a downstream sequence.
  • the recombinant polynucleotide is used for in vivo transduction and the synthetic promoter comprises SEQ ID NO:4, 6-10 or 11 or sequence that are at least 80-99% identical thereto and which can drive transcription of a downstream sequence.
  • a transgene is operably linked and downstream of the synthetic promoter.
  • the transgene comprises a non-naturally occurring immune receptor construct.
  • the non-naturally occurring immune receptor is a chimeric antigen receptor (CAR), chimeric TCR (cTCR), or CAR-like construct.
  • the WPRE sequence can comprise a sequence that is at least 80%-100% Attorney Docket No.00156-005WO1 identical to: AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTAC GCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCT CCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGC GTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGCATTGCCACCACCTGTCAGCTCCT TTCCGGGACTTTCGCTTTCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCT GCTGGACAGGGGGGGG
  • the viral 3’ delU3-R-U5 domain can comprise a sequence that is at least 80-100% identical to the sequence: GAGCTCAAAATAAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTAGGT TTGGCAAAATAAAAGAGCCCACAACCCCTCACTCGGCGCCAGTCCTCCGATTGACTGAGTCGCCCG GGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAG GGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTCTTTCATT (SEQ ID NO:34).
  • the viral miRNA target sequence domain can comprise a sequence that is at least 80-100% identical to the sequence: TGCTCAATAAATACCCGTTGAACGATTGCTCAATAAATACCCGTTGAAGCATTGCTCAATAAATACCC GTTGAACTACTGCTCAATAAATACCCGTTGAA (SEQ ID NO:35).
  • the viral miRNA target sequence domain can comprise a sequence that is at least 80-100% identical to the sequence: CATGATTGCCACGTCTGCAGTACGATCATGATTGCCACGTCTGCAGTAGCATCATGATTGCCACGTCT GCAGTACTACCATGATTGCCACGTCTGCAGTA (SEQ ID NO:36).
  • the viral miRNA target sequence domain can comprise a sequence that is at least 80-100% identical to the sequence: TGATTGCCACTGTCTGCAGTACGATTGATTGCCACTGTCTGCAGTAGCATTGATTGCCACTGTCTGCA GTACTACTGATTGCCACTGTCTGCAGTA (SEQ ID NO:37).
  • the recombinant vector comprises a plasmid.
  • the disclosure also provides a viral vector produced by expression of the plasmid as described above.
  • the host cell is a helper cell.
  • the viral vector is an RNA viral vector.
  • the viral Attorney Docket No.00156-005WO1 vector comprises an engineered retroviral vector.
  • the viral vector is engineered from a gammaretroviral vector.
  • the disclosure also provides a method of in vivo transduction or transfection, the method comprising administering the composition, or the viral vector of the disclosure to a subject, wherein the composition or viral vector transfects or transduces an immune cell in vivo.
  • BRIEF DESCRIPTION OF THE FIGURES [0012] Figure 1 shows that a YB-TATA element increases pSC1 activity 2-fold vs ADEp.
  • Figure 2 provides examples of promoter inactivity in 293T cells commonly used as a vector producing cell line after transfection with lentiviral vector encoding PSC1 immune cell selective promoter and mKATE2 fluorescent protein.
  • UBC is a universal promoter active in most cell types and acts as a positive control.
  • Figure 3 provides examples of promoter inactivity in HT1080 cells used as a vector producing cell line after transduction with retroviral vector encoding PSC1 immune cell selective promoter and GFP fluorescent protein.
  • EF1a is a universal promoter active in most cell types and acts as a positive control.
  • Figure 4 provides examples of promoter activity in primary CD4 T cells. Lentiviral vector encoding PSC1 immune cell selective promoter and mKate2 fluorescent protein.
  • Ubcp is a universal promoter active in most cell types and acts as a positive control.
  • Figure 5 provides examples of promoter inactivity in 293T cells used as a vector producing cell line after transduction with lentiviral vector encoding PSC1 immune cell selective promoter and mKate2 fluorescent protein.
  • Ubcp is a universal promoter active in most cell types and acts as a positive control.
  • DETAILED DESCRIPTION [0017] As used herein and in the appended claims, the singular forms "a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a subject” includes a plurality of such subjects and reference to Attorney Docket No.00156-005WO1 "the vector” includes reference to one or more vectors and equivalents thereof known to those skilled in the art, and so forth.
  • “about” can mean a range of plus or minus 20%, plus or minus 10%, plus or minus 5%, or plus or minus 1% of a given value.
  • the term can mean within an order of magnitude, within 5-fold, or within 2-fold, of a value.
  • variations can include an amount or concentration of 20%, 10%, 5%, 1 %, 0.5%, or even 0.1 % of the specified amount.
  • each intervening number there between with the same degree of precision is explicitly contemplated.
  • the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
  • the Retroviridae family of viruses may be used to create vectors that integrate into their host genome and provide long-term gene expression to the transduced cell and its descendants.
  • gammaretroviral vectors In general, gammaretroviral vectors, lentiviral vectors and foamy viral vectors are usable and useful to transduce cells including non- mobilized and mobilized stem cells.
  • the disclosure provides compositions and methods to transduce dividing or non-dividing cells (including hematopoietic stem cells) in vivo by direct administration of a vector to achieve therapeutic effects in many types of diseases including genetic diseases, cancer, infectious disease and autoimmune disease.
  • cells can be mobilized prior to in vivo infection.
  • Certain vector constructs of the disclosure can be considered “modular” with domains (sometimes referred to as cassettes) operably linked as described below.
  • a vector comprises repeats at the 5’ and 3’ ends, a transgene to be expressed (e.g., a CAR construct comprising a binding domain, an optional hinge or linker domain, a transmembrane domain, an intracellular domain; or a similar TCR construct) downstream (i.e., 3’) of a synthetic promoter, optionally one or more miRNA target domains, an optional kill switch domain and an Attorney Docket No.00156-005WO1 optional cell-activity-regulating domain.
  • a transgene to be expressed e.g., a CAR construct comprising a binding domain, an optional hinge or linker domain, a transmembrane domain, an intracellular domain; or a similar TCR construct
  • a synthetic promoter optionally one or more miRNA target domains, an optional kill switch
  • the binding domain, hinge/linker, transmembrane domain and intracellular domain generally comprise chimeric antigen receptors (CARs) including 1 generation, 2 generation, 3 generation and related constructs (e.g., TCR ⁇ and ⁇ chains comprising coding sequences for cleavable peptide linker between the TCR ⁇ and ⁇ , at least one of the TCR ⁇ and ⁇ chains linked to a binding domain to a peptide target).
  • CARs chimeric antigen receptors
  • 1 generation, 2 generation, 3 generation and related constructs e.g., TCR ⁇ and ⁇ chains comprising coding sequences for cleavable peptide linker between the TCR ⁇ and ⁇ , at least one of the TCR ⁇ and ⁇ chains linked to a binding domain to a peptide target.
  • antibody refers to a protein, or polypeptide sequence derived from an immunoglobulin molecule which specifically binds with an antigen.
  • Antibodies can be monoclonal, or polyclonal, multiple or single chain, or intact immunoglobulins, and may be derived from natural sources or from recombinant sources. Antibodies can be tetramers of immunoglobulin molecules. The antibody may be ‘humanized’, ‘chimeric’ or non-human. [0029]
  • antibody fragment refers to at least one portion of an antibody, that retains the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing/destabilizing, spatial distribution) an epitope of an antigen.
  • antibody fragments include, but are not limited to, Fab, Fab', Fv fragments, scFv antibody fragments, disulfide- linked Fvs, a Fd fragment consisting of the VH and CHl domains, linear antibodies, single domain antibodies (sdAb) such as either vL or vH, camelid vHH domains, multi-specific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody.
  • Fab fragment fragment fragment consisting of the VH and CHl domains
  • linear antibodies single domain antibodies (sdAb) such as either vL or vH, camelid vHH domains
  • sdAb single domain antibodies
  • multi-specific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an
  • An antigen binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005).
  • Antigen binding fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (Fn3) (see U.S. Patent No.: 6,703,199, which describes fibronectin polypeptide mini- bodies).
  • Fn3 fibronectin type III
  • antibody heavy chain refers to the larger of the two types of polypeptide chains present in antibody molecules in Attorney Docket No.00156-005WO1 their naturally occurring conformations, and which normally determines the class to which the antibody belongs.
  • antibody light chain refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa ( ⁇ ) and lambda ( ⁇ ) light chains refer to the two major antibody light chain isotypes.
  • Anticancer agent refers to agents that inhibit aberrant cellular division and growth, inhibit migration of neoplastic cells, inhibit invasiveness or prevent cancer growth and metastasis.
  • chemotherapeutic agents e.g., alkylating agents, anti-metabolites and the like
  • biological agents e.g., siRNA, viral vectors such as engineered MLV, lentivirus, foamy virus, adenoviruses, herpes virus that deliver cytotoxic genes
  • anticancer effect refers to a biological effect which can be manifested by various means including, but not limited to, a decrease in tumor volume, a decrease in the number of cancer cells, a decrease in the number of metastases, an increase in life expectancy, decrease in cancer cell proliferation, decrease in cancer cell survival, or amelioration of various physiological symptoms associated with the cancerous condition.
  • an “anticancer effect” can also be manifested by the ability of a CAR (or similar construct, e.g., TCR etc.) in prevention of the occurrence of cancer in the first place.
  • the term "antigen” or “Ag” refers to a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically- competent cells, or both.
  • antigens can be derived from recombinant or genomic DNA.
  • any DNA which comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein.
  • an antigen need not be encoded solely by a full length nucleotide sequence of a gene.
  • the disclosure includes, but is not limited to, the use of Attorney Docket No.00156-005WO1 partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response.
  • an antigen need not be encoded by a "gene” at all. It is readily apparent that an antigen can be synthesized or can be derived from a biological sample, or might be a macromolecule besides a polypeptide. Such a biological sample can include, but is not limited to, a tissue sample, a tumor sample, a cell or a fluid with other biological components. An antigen associated with a disease state (e.g., associated with an infection, or a cancer antigen) are targets of therapy.
  • Non-limiting examples of antigens that can be targeted include: CD5; CD19; CD20; CD22; CD24; CD30; CD33, CD34; CD38; CD69; CD72; CD97; CD123; CD171; CD269; CS1 (also referred to as CD2 subset 1, CRACC, MPL, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); epidermal growth factor receptor variant III (EGFRviii); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4 )bDGlcp(l-l)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAc ⁇ -Ser/Thr)); prostate-specific membrane antigen (PSMA); Receptor tyrosine kinase-like
  • an “antigen binding domain” refers to a polypeptide or peptide that due to its primary, secondary or tertiary sequence, Attorney Docket No.00156-005WO1 post-translational modifications, and/or charge binds to an antigen with a high degree of specificity.
  • the antigen binding domain may be derived from different sources, for example, an antibody (full length heavy chain, Fab fragments, single chain Fv (scFv) fragments, divalent single chain antibodies or diabodies), a non-immunoglobulin binding protein, a ligand or a receptor.
  • the antigen binding domain comprises T cell receptors (TCRs) or portions thereof.
  • anti-infection effect refers to a biological effect that can be manifested by various means including, but not limited to, decrease in the titer of the infectious agent, a decrease in colony counts of the infectious agent, amelioration of various physiological symptoms associated with the infectious condition.
  • anti-cancer effect refers to a biological effect which can be manifested by various means including, but not limited to, e.g., a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, inhibition of metastasis, or a decrease in tumor cell survival.
  • “beneficial results” may include, but are not limited to, lessening or alleviating the severity of the disease condition, preventing the disease condition from worsening, curing the disease condition, preventing the disease condition from developing, lowering the chances of a patient or subject developing the disease condition and prolonging a patient’s or subject’s life or life expectancy.
  • “beneficial results” may be alleviation of one or more symptom(s), diminishment of extent Attorney Docket No.00156-005WO1 of the deficit, stabilized (i.e., not worsening) state of cancer progression, delay or slowing of metastasis or invasiveness, and amelioration or palliation of symptoms associated with the cancer.
  • biological equivalent thereof is intended to be synonymous with “equivalent thereof” when referring to a reference protein, antibody or fragment thereof, polypeptide or nucleic acid, and refers to molecules having minimal homology to a reference molecule (e.g., protein, antibody etc.) while still maintaining desired structure and/or functionality.
  • an equivalent can comprise at least about 70% homology or identity, or at least 80% homology or identity and alternatively, or at least about 85%, or alternatively at least about 90%, or alternatively at least about 95%, or alternatively at least 98% percent homology or identity and exhibits substantially equivalent biological activity to the reference protein, polypeptide, antibody or fragment thereof or nucleic acid.
  • an equivalent thereof is a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complement and which has the same biological function (e.g., binds to a specific miRNA or encodes a protein or polypeptide having the same or similar biological effect to the polynucleotide to which it is being compared).
  • an equivalent thereof is an expressed polypeptide or protein from a polynucleotide that hybridizes under stringent conditions to the polynucleotide or its complement that encodes the reference polypeptide or protein.
  • cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • Examples of cancer include, but are not limited to, B-cell lymphomas (Hodgkin’s lymphomas and/or non- Hodgkin’s lymphomas), T cell lymphomas, myeloma, myelodysplastic syndrome, myeloproliferative disorders (e.g., polycythemia vera, myelofibrosis, essential thrombocythemia etc.), skin cancer, brain tumor, breast cancer, colon cancer, rectal cancer, esophageal cancer, anal cancer, cancer of unknown primary site, endocrine cancer, testicular cancer, lung cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, Attorney Docket No.00156-005WO1 liver cancer, bladder cancer, cancer of the urinary tract, cancer of reproductive organs thyroid cancer, renal cancer, carcinoma, melanoma, head and
  • tumor and cancer are used interchangeably herein, e.g., both terms encompass solid and liquid, e.g., diffuse or circulating, tumors.
  • cancer or tumor includes premalignant, as well as malignant cancers and tumors.
  • cancer is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.
  • cell-activity-regulating domain refers to any one or more of PDL1, PDL2, CD80, CD86, crmA, p35, NEMO-K277A (or derivative thereof), K13-opt, IKK2-SS/EE, IKK1-SS/EE, 41BBL, CD40L, vFLIP-K13, MC159, and the like and combination thereof that is expressed in an immune cell (e.g., T cell, e.g., CAR-T cell etc.) to decrease, regulate or modify the activity of the immune cell.
  • an immune cell e.g., T cell, e.g., CAR-T cell etc.
  • an accessory module is co-expressed with an immune receptor such as a CAR or engineered TCR to increase, decrease, regulate or modify the expression or activity of a CAR, engineered TCR, a CAR-expressing cell or engineered TCR-expressing cell.
  • the accessory module can be co-expressed with a CAR or engineered TCR using a single vector or using two or more different vectors.
  • “Chemotherapeutic agents” are compounds that are known to be of use in chemotherapy for cancer.
  • Non-limiting examples of chemotherapeutic agents can include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; a camptothecin (including the synthetic analogue topotecan); bryostatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics Attorney Docket No.00156-005WO1 such as the enediyne antibiotics (e.
  • dynemicin including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino- doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin),
  • CARs Chimeric antigen receptors
  • T cell immune cell
  • chimeric T-cell receptors or chimeric immunoreceptors.
  • CARs are constructed specifically to stimulate immune cell activation and proliferation in response to a specific antigen to which the CAR binds.
  • a CAR refers to a set of polypeptides, typically two in the simplest embodiments, which when expressed in an immune effector cell, provides the cell with specificity for a target antigen or cell, typically a cancer cell, and with intracellular signal generation.
  • a CAR comprises at least an extracellular antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to herein as "an intracellular signaling domain") comprising a functional signaling domain derived from a stimulatory molecule and/or costimulatory molecule.
  • the set of polypeptides are contiguous with each other.
  • the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of a CAR Attorney Docket No.00156-005WO1 fusion protein.
  • the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen binding domain, wherein the leader sequence is optionally cleaved from the antigen binding domain (e.g., a scFv) during cellular processing and localization of the CAR to the cellular membrane.
  • CARs are recombinant polypeptides comprising an antigen binding domain, a hinge region (HR), a transmembrane domain (TMD), an optional co-stimulatory domain (CSD) and an intracellular signaling domain (ISD).
  • the optional costimulatory domain is generally absent in 1 generation CAR constructs.
  • Second (2 ) generation CARs comprising antigen binding domains e.g., vL and vH fragments, vHH, ligands and receptors etc.
  • a costimulatory domain e.g., 4-1BB
  • CAR or “CARs” also encompasses newer approaches to conferring antigen specificity onto cells, such as Antibody-TCR chimeric molecules or Ab-TCR (WO2017/070608A1 incorporated herein by reference), TCR receptor fusion proteins or TFP (WO2016/187349A1 incorporated herein by reference), Tri- functional T cell antigen coupler (Tri-TAC or TAC) (see, WO2015/117229A1, incorporated herein by reference).
  • Tri-TAC or TAC Tri- functional T cell antigen coupler
  • the term “CAR-T cell” is used, to refer to T-cells that have been engineered to express a chimeric antigen receptor or TCR construct.
  • CAR-T lymphocytes bearing such CARs or cTCRs are generally referred to as CAR-T lymphocytes.
  • CARs can be also expressed in cells other than T cells, such as hematopoietic stem cells, induced pluripotent stem cells (iPSC), NK cells and macrophages.
  • iPSC induced pluripotent stem cells
  • NK cells NK cells and macrophages.
  • chimeric TCR or “cTCR” refers to a wild-type TCR nucleic acid coding sequence and the corresponding wild-type TCR protein linked to an antigen binding domain that is not derived from a TCR.
  • the antigen binding domain of a cTCR may comprise of a vL, vH or scFv fragments.
  • cTCR have been described in (Gross, Waks & Eshhar, Proc. Natl. Acad. Sci. USA, 1989). cTCRs are used in some embodiments.
  • the term “Ab-TCR” or “AbTCR” refers to a next generation CAR platform as described in WO 2017/070608 A1 which is incorporated herein by reference.
  • an Ab- TCR comprises an antibody moiety that specifically binds to a target antigen fused to a TCR module capable of recruiting at least one TCR Attorney Docket No.00156-005WO1 signaling module.
  • cTCRs and ab-TCRs can be used in the vectors of the disclosure.
  • Codon optimization or “controlling for species codon bias” refers to the preferred codon usage of a particular host cell.
  • the genetic code is redundant with 64 possible codons, but most organisms typically use a subset of these codons.
  • the codons that are utilized most often in a species are called optimal codons, and those not utilized very often are classified as rare or low-usage codons.
  • the coding sequences of a vector of the disclosure can be modified to reduce RNA destabilizing sequence elements, increase G/C content, limit ApoBec mediated mutations.
  • the vectors of the disclosure can be engineered to modify their stability and/or expression.
  • changes in expression can occur due to the frequency with which inactivating or attenuating mutations accumulate in the vector as it replicates in a cell. Investigation shows that one of the most frequent events is G to A mutations (corresponds to the C to T mutation) characteristic ApoBec mediated mutations in the negative strand of single stranded DNA from the first replicative step. This can cause changes in amino acid composition of vector-encoded proteins and a devastating change from TGG (Tryptophan) to stop codons (TAG or TGA).
  • this inactivating change is avoided by substitution with codons of other amino acids with similar chemical or structural properties such as phenylalanine or tyrosine at position of ApoBec modifications.
  • Such mutations can include modifications of one or more codons in the coding sequences of vector domains that change a tryptophan codon to a permissible codon that maintains the biological activity of the encoded protein.
  • the codon for tryptophan is UGG (TGG in DNA).
  • the “stop codon” is UAA, UAG or UGA (TAA, TAG or TGA in DNA).
  • a single point mutation in the tryptophan codon can cause an unnatural stop codon (e.g., UGG -> UAG or UGG -> UGA). It is also known that human APOBEC3GF (hA3G/F) inhibits retroviral Attorney Docket No.00156-005WO1 replication through G -> A hypermutations (Neogi et al., J. Int. AIDS Soc., 16(1):18472, Feb. 25, 2013). Thus, the disclosure contemplates modifications to the coding sequences of vectors of the disclosure to reduce ApoBec hypermutations by modifying tryptophan codons to permissible non-tryptophan codons.
  • a “conservative substitution” or “conservative sequence modifications” refers to amino acid modifications that do not significantly affect or alter the binding characteristics or function of the encoded protein.
  • “conservative sequence modifications” refers to amino acid modifications that do not significantly affect or alter the binding characteristics or function of a CAR construct of the disclosure (e.g., a conservative change in the constant chain, antibody, antibody fragment, or non- immunoglobulin binding domains).
  • conservative modifications include amino acid substitutions, additions and deletions.
  • Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g.
  • Co-stimulatory domain refers to a biological agent that enhances the proliferation, survival and/or development of T cells.
  • a co-stimulatory domain can comprise the costimulatory domain of any one or more of, for example, members of the TNFR superfamily, CD28, CD137 (4-1BB), CD134 (OX40), Dap10, Attorney Docket No.00156-005WO1 CD27, CD2, CD5, ICAM-1, LFA-1(CD11a/CD18), Lck, TNFR-I, TNFR-II, Fas, CD30, CD40 or combinations thereof.
  • Other co-stimulatory domains e.g., from other proteins will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the disclosure.
  • Cytokine Release Syndrome is a complication of cell therapies (e.g., CAR-T, bispecific T cell engaging antibodies etc.) that manifests itself with signs and symptoms such as fever, hypotension, shortness of breath, renal dysfunction, pulmonary dysfunction and/or capillary leak syndrome. CRS is usually due to excessive production of cytokines, such as IL6 and IL1.
  • a “delivery vehicle” or “polynucleotide delivery vehicle” refers to a composition that can be used in combination with a recombinant polynucleotide to deliver a recombinant polynucleotide to a cell in vivo.
  • Such delivery vehicles can be targeted delivery vehicles or non-targeted delivery vehicles.
  • delivery vehicles include micro- and nano-particles and micellular composition (e.g., liposomes).
  • a targeted delivery vehicles includes, for example, a liposome comprising an optional targeting moiety (e.g., an antibody) such that the liposome preferentially binds to a cell expression the cognate to the targeting moiety.
  • the delivery vehicle comprises a polynucleotide encoding a CAR or cTCR operably linked to a synthetic promoter.
  • the antigen binding domain retains sufficient antibody structure such that it has the required function, namely, the ability to bind to an antigen. It does not include any limitation to a particular process of producing the antibody.
  • “Domain” or “module” refers to a discrete section or part of a larger construct that can be replaced with a similar domain without affecting the function of other domains or module of the Attorney Docket No.00156-005WO1 construct.
  • the CAR in its simplest form, can be described as having a binding domain, a transmembrane domain and an intracellular domain.
  • Each “domain” of the CAR can be modified or changed without affecting the other domains of the CAR.
  • the binding domain can be one of a number of different binding domains as described herein.
  • the binding domain can be a polypeptide sequence that binds to a CD19 antigen. This CD19 binding domain can be replaced with a binding domain that binds to CD20 without affecting of having to change the transmembrane domain.
  • a retroviral vector of the disclosure contained in a viral capsid comprises a polynucleotide sense RNA strand having a number of domains including (from 5’ to 3’): a 5’Repeat(5’R)—U5—packaging sequence — a CAR sequence — (optional kill switch comprising an IRES domain linked to, e.g., thymidine kinase (TKO) coding sequence) — an miRNA targeting sequence(s) — a U3—3’Repeat(3’R).
  • TKO thymidine kinase
  • Each domain/module of the viral polynucleotide can be changed such that different CAR sequences can be provided, different kill switches (e.g., TKO or cytosine deaminase (CD)), different miRNA targeting sequences etc.
  • the constructs of the disclosure are modular in design.
  • Each domain/module of the construct, whether a polynucleotide construct or an encoded polypeptide construct can comprise minor variations in sequence so long as the variations do not destroy the biological activity of the domain.
  • a transmembrane domain can have 80-100% identity to a specific transmembrane sequence.
  • Genetically modified cells refer to cells that express, for example, a CAR or cTCR.
  • the genetically modified cells comprise vectors that encode a CAR or cTCR.
  • a “hinge region” (HR) as used herein refers to an optional hydrophilic region which is between the antigen binding domain and the transmembrane domain of a CAR.
  • the hinge region includes, but is not limited to, Fc fragments of antibodies or fragments or derivatives thereof, hinge regions of antibodies or fragments or derivatives thereof, CH2 regions of antibodies, CH3 Attorney Docket No.00156-005WO1 regions of antibodies, artificial spacer sequences or combinations thereof.
  • hinge regions include, but are not limited to, CD8a hinge, and artificial spacers made of polypeptides which may be as small as, for example, Gly or CH1 and CH3 domains of IgGs (such as human IgG4).
  • the hinge region is any one or more of (i) a hinge, CH2 and CH3 regions of IgG4, (ii) a hinge region of IgG4, (iii) a hinge and CH2 of IgG4, (iv) a hinge region of CD8a, (v) a hinge, CH2 and CH3 regions of IgG1, (vi) a hinge region of IgG1 or (vi) a hinge and CH2 region of IgG1.
  • Other hinge regions will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the disclosure. It should be noted that in some embodiments, a hinge region is not required and the antigen binding domain can be operably linked to the transmembrane domain directly or via a suitable linker.
  • Immuno cell refers to the cells of the mammalian immune system including, but not limited to, antigen presenting cells, B-cells, basophils, cytotoxic T-cells, dendritic cells, eosinophils, granulocytes, helper T-cells, leukocytes, lymphocytes, macrophages, mast cells, memory cells, monocytes, natural killer cells (e.g., NK and NKT cells), neutrophils, phagocytes, plasma cells and T-cells.
  • In vivo Immune cells refers to immune cells present in the body of a subject that have not been isolated or removed from the subject.
  • Immuno effector cell refers to a cell that is involved in an immune response, e.g., in the promotion of an immune effector response.
  • immune effector cells include T cells, e.g., alpha/beta T cells and gamma/delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.
  • T cells e.g., alpha/beta T cells and gamma/delta T cells
  • B cells natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.
  • NK natural killer
  • NKT natural killer T
  • Routes of in vivo delivery include intravenous injection, intramuscular injection, intranodal injection, intralesional, or intranasal/intra- lung inhalation.
  • Other versions of in vivo delivery e.g., direct delivery of therapeutic agent to cells via extracorporeal/machine- based mechanisms
  • In vivo delivery mechanisms can include particle injection, physiochemical, electroporation, ballistic injection, magnetic-based, photoporation, heat-based, pressure-based (osmotic or sound) or hydrodynamic delivery of selective synthetic promoters encoding therapeutic nucleic acids.
  • in vivo transduction refers to the process of a eukaryotic cell uptake of heterologous nucleic acids in situ.
  • in vivo transduction refers to the uptake by an immune effector cell of a vector of the disclosure within the human or animal body. Contrast this, with ex vivo transduction which described the removal of immune effector cells from the body and transducing them outside the body and then delivering the transduced cells back into the body.
  • in vivo transfection refers to the uptake by eukaryotic cells of a recombinant polynucleotide of the disclosure within (i.e., in situ) the body.
  • Intracellular signaling domain refers to an intracellular signaling portion of a molecule.
  • the intracellular signaling domain generates a signal that promotes an immune effector function of the cell. Examples of immune effector function include cytolytic activity and helper activity, including the secretion of cytokines.
  • domains that transduce the effector function signal include, but are not limited to, the z chain of the T-cell receptor complex or any of its homologs (e.g., h chain, FceR1g and b chains, MB1 (Iga) chain, B29 (Igb) chain, etc.), human CD3 zeta chain, CD3 polypeptides (D, d and e), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.) and other molecules involved in T-cell transduction, such as CD2, CD5 and CD28.
  • the z chain of the T-cell receptor complex or any of its homologs e.g., h chain, FceR1g and b chains, MB1 (Iga) chain, B29 (Igb) chain, etc.
  • human CD3 zeta chain CD3 polypeptides
  • the intracellular signaling domain can comprise a primary intracellular signaling domain.
  • Exemplary primary intracellular signaling domains include those derived from Attorney Docket No.00156-005WO1 the molecules responsible for primary stimulation, or antigen dependent simulation.
  • the intracellular signaling domain can comprise a costimulatory intracellular domain.
  • Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals, or antigen independent stimulation.
  • a primary intracellular signaling domain can comprise a cytoplasmic sequence of CD3z or CD3z1xx (Feucht et al., Nat.
  • a costimulatory intracellular signaling domain can comprise cytoplasmic sequence from co-receptor or costimulatory molecule, such as CD28 or 4-1BB.
  • a primary intracellular signaling domain can comprise a signaling motif which is known as an immunoreceptor tyrosine-based activation motif or ITAM.
  • ITAM containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta (CD3 ⁇ ), common FeR gamma (FCER1G), Fe gamma RIIa, FeR beta (Fe Epsilon R1b), CD3 gamma (CD3 ⁇ ), CD3 delta (CD3 ⁇ ), CD3 epsilon, CD79a, CD79b, DAPlO, and DAP12.
  • CD3 zeta CD3 ⁇
  • FCER1G common FeR gamma
  • FeR beta Fe Epsilon R1b
  • CD3 gamma CD3 ⁇
  • CD3 delta CD3 ⁇
  • CD3 epsilon CD79a, CD79b, DAPlO, and DAP12.
  • isolated refers to molecules or biologics or cellular materials being substantially free from other materials.
  • the term “isolated” refers to nucleic acid, such as DNA or RNA; protein or polypeptide; cell or cellular organelle(s); or tissue, separated from other DNAs or RNAs, or proteins or polypeptides, or cells or cellular organelles, or tissues or organs, respectively, which are present in the natural source.
  • isolated also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • an “isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
  • isolated is also used herein to refer to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides.
  • isolated is also used herein to refer to cells or tissues that are isolated from other cells or Attorney Docket No.00156-005WO1 tissues and is meant to encompass both, cultured and engineered cells or tissues.
  • linker refers to an oligonucleotide (operably connecting two polynucleotide domains) or a peptide (operably connecting two polypeptide domains) that joins together two or more domains or regions of a CAR polynucleotide or polypeptide, respectively.
  • the linker can be anywhere from 1 to 500 amino acids in length or 3 to 1500 nucleotides in length.
  • the “linker” is cleavable or non-cleavable. Unless specified otherwise, the term “linker” used herein means a non-cleavable linker.
  • Said non-cleavable linkers may be composed of flexible amino acid residues which allow freedom of motion of adjacent protein domains relative to one another.
  • residues include glycine and serine (e.g., glycine repeats or glycine-serine repeats in any order).
  • linkers include non-flexible amino acid residues.
  • cleavable linkers include 2A linkers (for example T2A), 2A-like linkers or functional equivalents thereof and combinations thereof.
  • the linkers include the picornaviral 2A-like linker, CHYSEL sequences of porcine teschovirus (P2A), Thosea asigna virus (T2A) or combinations, variants and functional equivalents thereof (e.g., GSG modified variants).
  • the linker sequences may comprise a motif that results in cleavage between the 2A glycine and the 2B proline.
  • Other cleavable linkers that may be used herein are readily appreciated by those of skill in the art.
  • flexible polypeptide linker refers to a peptide linker that consists of amino acids such as glycine and/or serine residues used alone or in combination, to link polypeptide chains together (e.g., variable heavy and variable light chain regions together).
  • the flexible polypeptide linker is a Gly/Ser linker and comprises the amino acid sequence (Gly-Ser), where n is a positive integer equal to or greater than 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 etc.).
  • “Mammal” as used herein refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees and other apes and monkey species; farm Attorney Docket No.00156-005WO1 animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like.
  • the term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included within the scope of this term.
  • off-target transduced cells refers to cells that are infected by a delivery vehicle or viral vector of the disclosure, but where expression of the vehicle’s or vector’s encoded genes/polynucleotides are unwanted or undesirable in the unintended cell types or to prevent expression of therapeutic transgenes on the surface of viral vector particles during production of retrovirus- or lentivirus-based products to reduce immunogenicity of a viral particle.
  • viral vectors can be “targeted” through incorporation of targeting proteins on the viral envelope.
  • expression of a viral polynucleotide or encoded gene can be controlled through the use of tissue specific promoters or other synthetic promoters.
  • the expression of the vector polynucleotide, gene/construct can be controlled through the use of cellular machinery that exists to control innate gene expression control.
  • RNAi target sequences can be used, whereby binding of innate miRNA to a target sequence(s) can be used to control expression in an off-target cell type.
  • operably linked refers to functional linkage or association between a first component and a second component such that each component can be functional.
  • operably linked includes the association between a regulatory sequence (e.g., a promoter) and a heterologous nucleic acid sequence resulting in expression of the latter.
  • a first nucleic acid sequence is operably linked with a Attorney Docket No.00156-005WO1 second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a first polypeptide functions in the manner it would independent of any linkage and a second polypeptide functions as it would absent a linkage between the two.
  • two polypeptides can be considered operably linked wherein the combined expression results in a desired or predicted activity that is lacking when either is expressed alone.
  • Percent identity in the context of two or more nucleic acids or polypeptide sequences, refers to two or more sequences that are related by percent sequence identity. Two sequences are "substantially identical" if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60% identity, optionally 70%, 71%. 72%.
  • the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more typically over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.
  • sequence comparison algorithm For sequence comparison, generally one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • the sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm Attorney Docket No.00156-005WO1 of Smith and Waterman, (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Bioi. 48:443, by the search for similarity method of Pearson and Lipman, (1988) Proc. Nat'l. Acad. Sci.
  • the percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, (1988) Comput. Appl. Biosci. 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (1970) J. Mol. Bioi.
  • promoter refers to a domain comprising nucleotide sequences that control or regulate the expression of an operably linked polynucleotide. Promoters are domains that drive the expression of a downstream polynucleotide.
  • regulatory sequence refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, enhancers and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a Attorney Docket No.00156-005WO1 recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell.
  • regulatory nucleic acid sequence from public databases and materials.
  • one skilled in the art can identify a regulatory sequence that is applicable for the intended use, for example, in vivo, ex vivo, or in vitro.
  • a “core promoter” refers to a minimal promoter comprising about 50-100 bp and lacks enhancer elements.
  • core promoters include, but are not limited to, super core promoter 1 (SCP1; see, Juven-Gershon et al., Nature, 3:917-922, 2006), AdML and CMV core promoters. More particularly, where a core-promoter cassette is present a second cassette (e.g., a second mini-promoter cassette, a polIII promoter cassette or IRES cassette) will be present.
  • a vector comprising a cassette with a core promoter specifically excludes the use of SCP1, AdML and CMV core promoters, but rather utilize designer core promoters as described further herein and below.
  • Core promoters include certain viral promoters.
  • Viral promoters are promoters that have a core sequence, but also usually include some further accessory elements.
  • the early promoter for SV40 contains three types of elements: a TATA box, an initiation site and a GC repeat (Barrera- Saldana et al., EMBO J, 4:3839-3849, 1985; Yaniv, Virology, 384:369– 374, 2009).
  • the TATA box is located approximately 20 base-pairs upstream from the transcriptional start site.
  • the GC repeat regions is a 21 base-pair repeat containing six GC boxes and is the site that determines the direction of transcription. This core promoter sequence is around 100 bp. Adding an additional 72 base-pair repeats, thus making it a “mini-promoter,” is useful as a transcriptional enhancer that increases the functionality of a promoter by a factor of about 10.
  • the SP1 protein interacts with the 21 bp repeats it binds either the first or the last three GC boxes. Binding of the first three initiates early expression, and binding of the last three initiates late expression.
  • the function of the 72 bp repeats is to enhance the amount of stable RNA and increase the rate of synthesis. This is done by binding Attorney Docket No.00156-005WO1 (dimerization) with the AP1 (activator protein 1) to give a primary transcript that is 3' polyadenylated and 5' capped.
  • Other viral promoters such as the Rous Sarcoma Virus (RSV), the HBV X gene promoter, and the Herpes Thymidine kinase core promoter can also be used as the basis for selection desired function.
  • RSV Rous Sarcoma Virus
  • HBV X gene promoter the Herpes Thymidine kinase core promoter
  • a core promoter typically encompasses ⁇ 40 to +40 relative to the +1 transcription start site (Juven-Gershon and Kadonaga, Dev. Biol.
  • RNA polymerase II interacts with a number of transcription factors that bind to DNA motifs in the promoter. These factors are commonly known as “general” or “basal” transcriptions factors and include, but are not limited to, TFIIA (transcription factor for RNA polymerase IIA), TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. These factors act in a “general” manner with all core promoters; hence they are often referred to as the “basal” transcription factors. [0076] Juven-Gershon et al., 2006 (supra), describe elements of core promoters.
  • the pRC/CMV core promoter consists of a TATA box and is 81 bp in length;
  • the CMV core promoter consists of a TATA box and an initiator site;
  • the SCP synthetic core promoters (SCP1 and SCP2) consist of a TATA box, an Inr (initiator), an MTE site (Motif Ten Element), and a DPE site (Downstream promoter element) and is about 81 bp in length.
  • the SCP synthetic promoter has improved expression compared to the simple pRC/CMV core promoter.
  • mini-promoter refers to a regulatory domain that promotes transcription of an operably linked gene or coding nucleic acid sequence.
  • the mini- promoter includes the minimal amount of elements necessary for effective transcription and/or translation of an operably linked coding sequence and can include other enhancing elements.
  • a mini-promoter can comprise a “core promoter” in combination with additional regulatory elements or a “modified core promoter”.
  • the mini-promoter or modified core promoter will be about 100-600 bp in length while a core promoter is typically less than about 100bp (e.g., about 70-80 bp).
  • a cassette comprising Attorney Docket No.00156-005WO1 a polynucleotide to be expressed will typically comprise an enhancer element or another element either upstream or downstream of the core promoter sequence that facilitates expression of an operably linked coding sequence above the expression levels of the core promoter alone.
  • mini-promoters e.g., modified core promoters
  • core promoter elements ⁇ 100, ⁇ 200, ⁇ 400 or ⁇ 600 bp
  • mini-promoters comprising core promoters plus minimal enhancer sequences and/or Kozak sequences to allow better gene expression compared to a core- promoter lacking such sequences that are still under 200, 400 or 600bp.
  • mini-promoters include modified core promoters and naturally occurring tissue specific promoters such as the elastin promoter (specific for pancreatic acinar cells, (204 bp; Hammer et al., Mol Cell Biol., 7:2956-2967, 1987) and the promoter from the cell cycle dependent ASK gene from mouse and man (63-380 bp; Yamada et al., J. Biol. Chem., 277: 27668–27681, 2002).
  • tissue specific promoters such as the elastin promoter (specific for pancreatic acinar cells, (204 bp; Hammer et al., Mol Cell Biol., 7:2956-2967, 1987) and the promoter from the cell cycle dependent ASK gene from mouse and man (63-380 bp; Yamada et al., J. Biol. Chem., 277: 27668–27681, 2002).
  • Ubiquitously expressed small promoters also include viral promoters such as the SV40 early and late promoters (about 340 bp), the RSV LTR promoter (about 270 bp) and the HBV X gene promoter (about 180 bp) (e.g., R Anish et al., PLoS One, 4: 5103, 2009) that has no canonical “TATTAA box” and has a 13 bp core sequence of 5’-CCCCGTTGCCCGG-3’ (SEQ ID NO:42).
  • viral promoters such as the SV40 early and late promoters (about 340 bp), the RSV LTR promoter (about 270 bp) and the HBV X gene promoter (about 180 bp) (e.g., R Anish et al., PLoS One, 4: 5103, 2009) that has no canonical “TATTAA box” and has a 13 bp core sequence of 5’-CCCCGTTGC
  • a cassette comprising at least one mini-promoter cassette will have expression levels that exceed, are about equal to, or about 1 fold to 2.5 fold less than the expression levels of an IRES cassette present in a replication competent retroviral vector (RRV).
  • RRV replication competent retroviral vector
  • Dispersed transcription is the most common mode of transcription in vertebrates. For instance, dispersed transcription is observed in about two-thirds of human genes. In vertebrates, focused transcription tends to be associated with regulated promoters, whereas dispersed transcription is typically observed in constitutive promoters in CpG islands.
  • a “synthetic promoter” is a promoter that is not "naturally occurring.” The synthetic promoters of the disclosure may be produced synthetically (e.g., via chemical synthesis), or using recombinant cloning and/or nucleic acid amplification technology, including polymerase chain reaction (PCR).
  • a synthetic promoter has characteristics of mini- or core-promoters and have enhanced cell-state specificity (see, e.g., Wu et al., Nature Communications volume 10, Article number: 2880 (2019)). [0081] In some embodiments, a synthetic promoter may be 10-300 nucleotides long.
  • the length of a synthetic promoter may be 10-300, 10-290, 10-280, 10-270, 10-260, 10-250, 10-240, 10- 230, 10-220, 10-210, 10-210, 10-200, 10-190, 10-180, 10-170, 10-160, 10-150, 10-140, 10-130, 10-120, 10-110, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-300, 20-290, 20-280, 20-270, 20-260, 20-250, 20-240, 20-230, 20-220, 20-210, 20-210, 20-200, 20- 190, 20-180, 20-170, 20-160, 20-150, 20-140, 20-130, 20-120, 20-110, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 30-300, 30- 290, 30-280, 30-270, 30-260, 30-250, 30-240, 30-230, 30-220, 30-210, 30-210, 30-210,
  • Promoters may be longer than 300 nucleotides, in some embodiments.
  • a synthetic promoter may be longer than 300 nucleotide (e.g., 300, 350, 400, 450, or 500 long or longer). [0082] In some the length of a synthetic promoter is 200 nucleotides or shorter.
  • a synthetic promoter may be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,
  • a synthetic promoters has differential activities in different cell lines or different cellular states. "Having differential activities” means the activity of a synthetic promoter is higher or lower in one type of cell or at a cellular state, compared to in a different type of cell or at a different cellular state, respectfully.
  • the activity of a synthetic promoter in one cell type or a cellular state is different from (higher or lower) the activity of the synthetic promoter in another cell type or another cellular state by at least 10% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8 -fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70- fold, 80-fold, 90-fold, 100-fold, 500-fold, or 1000-fold).
  • at least 10% e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8 -fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70- fold, 80-fold, 90-fold, 100-fold, 500-fold, or
  • the activity of a synthetic promoter in one cell type Attorney Docket No.00156-005WO1 or a cellular state is different from (higher or lower) than the activity of the synthetic promoter in another cell type or another cellular state by 10-100%.
  • the activity of a synthetic promoter in one cell type or a cellular state may be different from (higher or lower) than the activity of the synthetic promoter in another cell type or another cellular state by 10%-100%, 10%-90%, 10%-80%, 10%-70%, 10%-60%, 10%-50%, 10%-40%, 10%-30%, 10%-20%, 20%- 100%, 20%-90%, 20%-80%, 20%-70%, 20%-60%, 20%-50%, 20%-40%, 20%-30%, 30%-100%, 30%-90%, 30%-80%, 30%-70%, 30%-60%, 30%-50%, 30%-40%, 40%- 100%, 40%-90%, 40%-80%, 40%-70%, 40%-60%, 40%-50%, 50%-100%, 50%- 90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, or 90%-100%.
  • the activity of a synthetic promoter in one cell type or a cellular state is different from (higher or lower) the activity of the synthetic promoter in another cell type or another cellular state by 1-1000 fold.
  • the activity of a synthetic promoter in one cell type or a cellular state may be different from (higher or lower than) the activity of the synthetic promoter in another cell type or another cellular state by 1-1000, 1-900, 1-800, 1-700, 1-600, 1-500, 1-400, 1-300, 1-200, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1- 3, 1-2, 5-1000, 5-900, 5-800, 5-700, 5-600, 5-500, 5-400, 5-300, 5- 200, 5-100, 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-30, 5-20, 5-10, 5- 9, 5-8, 5-7, 5-6, 10-1000, 10-900
  • the activity of a synthetic promoter in one cell type or a cellular state may be different from (higher or lower than) the activity of the synthetic promoter in another cell type or another cellular state by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8 -fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50- fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 500-fold, or 1000-fold.
  • a synthetic promoter may be inactive in one cell type and active in another.
  • a synthetic promoter may be inactive in one cellular state and active in another.
  • Methods of measuring the activities of a promoter are known to those skilled in the art, e.g., as described in Jeyaseelan et al., Nucleic Acids Research. 29 (12), 2001; Allard et al., Cell Notes (21), 2008; and Zaslaver et al, Nature Methods. 3 (8): 623-628, 2006, each of which is incorporated herein by reference.
  • a synthetic promoter has differential activity (e.g., higher or lower) in one cellular state relative to another cellular state.
  • Non-limiting examples of different cell types that may transition between different cellular states include: embryonic stem cells, adult stem cells, induced pluripotent stem cells, neurons, cardiomyocytes, skin cells, liver cells, immune cells, epithelial cells, eye cells, astrocytes, and microglia.
  • the regulatory sequence e.g., a synthetic promoter
  • the regulatory element e.g., synthetic promoter
  • the regulatory element may have less sequence complexity than a natural promoter and its expression is Attorney Docket No.00156-005WO1 specific for a particular cell type to be transduced (e.g., an immune effector cell) with less expression (leakiness) in off target cell types and suitable for in vivo applications.
  • the disclosure provides a synthetic promoter comprising a core promoter (ital) linked to an adenoviral derived minipromoter (bold) (underlined & Ital denotes a linker): TGCTTTCCAGGAAATCTGCTTTCCAGGAAGCTTGCTTTCCAGGAATGATGCTTTCCAGGAACAGTGCT TTCCAGGAAATGTGCTTTCCAGGAACGTTGCTTTCCAGGAATACTGCTTTCCAGGAAGGCGCCAGA CGCTAGCGGGGGGCTATAAAAGGGGGTGGGGGGGCGTTCGTCCTCACTCT (SEQ ID NO:1); wherein the core promoter has the sequence: TGCTTTCCAGGAAATCTGCTTTCCAGGAAGCTTGCTTTCCAGGAATGATGCTTTCCAGGAACAGTGCT TTCCAGGAAATGTGCTTTCCAGGAACGTTGCTTTCCAGGAATACTGCTTTCCAGGAA (SEQ ID NO:2) and an adenoviral derived minimal promoter has the sequence:
  • adenoviral derived minipromoter in SEQ ID NO:1 is replaced with a YB TATA minipromoter to enhance expression in immune cells (see, e.g., data in Figure 1).
  • the synthetic promoter comprises SEQ ID NO:4 (Ital is core promoter (SEQ ID NO:2); YB TATA minipromoter is bold; underlined & Ital denotes a linker): TGCTTTCCAGGAAATCTGCTTTCCAGGAAGCTTGCTTTCCAGGAATGATGCTTTCCAGGAACAGTGCT TTCCAGGAAATGTGCTTTCCAGGAACGTTGCTTTCCAGGAATACTGCTTTCCAGGAAACGCGTTCTAG AGGGTATATAATGGGGGCCA (SEQ ID NO: 4) comprising of SEQ ID NO:2 and YB TATA minimal promoter TCTAGAGGGTATATAATGGGGGCCA (SEQ ID NO:5).
  • Table 1 Identifier Immune cell selective promoter sequence (bold is SEQ ID NO:3) G T C G T G C Attorney Docket No.00156-005WO1 TGGTGATTTCCAGGAAAGGGGGGCGCGCCAGACGCTAGCGGGGGGCTATAAAAGG GGGTGGGGGCGTTCGTCCTCACTCT G T G G T T T G A A T G T T T C T C G G G Attorney Docket No.00156-005WO1 AATGTGGCGCGCCAGACGCTAGCGGGGGGCTATAAAAGGGGGTGGGGGCGTTCGT CCTCACTCTAGATCTGCGATCTAAGTAAGCTTGTGTCCCTTGGGCTTGTCGTACTGTTG A G A T C G C C G A C C , comprise gene regulatory elements and minipromoters derived from specific cell types that subsequently confers cell specific selectivity in initiating transcription of
  • the disclosure provides a synthetic promoter comprising of gene regulatory and promoter elements derived from the CD2 gene that confers transcriptional selectivity to T cells: tttgtgtgagaattaaaatgcagcaagaaacacacactcataaacacatctgctttggcaaaggagc acatcagaagggctggcttgtgcgcgctcttgctctctgtgtatgtgtattatgtttttactgt aaaagatgtaaagagaggcacgtggttaagctctcggggtgtggactccaccagtctcacttcagttc ctttgcatgaagagctcagaatcaaaagaggaaccaacccctaag (SEQ ID NO: 12).
  • Table 2 T selective promoter examples Attorney Docket No.00156-005WO1 Identifier T cell selective promoter sequence g g a a c c c a a ct c a g c c ct c a a g g t a g a tt t [0091]
  • Table 3 NK selective promoter examples Identifier NK cell selective promoter sequence Attorney Docket No.00156-005WO1 SEQ ID gaggacattctgtccttttatcttctagttattctttcctattatgaattagcaaatacaaatcttttgtt NO:19 ccccagggtaaagaaaagaatttttaagaagtggt
  • a modular RNV-SIN plasmid of the disclosure can comprise: (CMV promoter)-(viral 5’R-U5)-(MMLV-psi)- (Viral gag)-(synthetic promoter)-(at least one transgene)-(WPRE domain)-(Viral 3’del-U3-R-U5).
  • the transgene can comprise any desired sequence to be expressed such as a 1 , 2 , 3 or other CAR construct, cTCR, ab-TCR, RNAi sequences and the like.
  • the RNV-SIN plasmid comprises a sequence (coded): CMV promoter (bold) Viral 5’ R-U5 (ital) MMLV ⁇ (ital & bold) Viral gag (underlined) Synthetic promoter pSC1 (bold/underlined/ital; Any of SEQ ID NOs:1, 4, 6-26 can be present in this domain) EmeraldGFP (emGFP) Transgene (double underlined) WPRE (dashed underlined) Viral 3’ delU3-R-U5 (dashed underlined & ital) CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAA TAATGACGTATGTTCCCATAGTAACGCCAATA
  • the RNAi target sequence is a sequence essentially complementary to at least one RNAi inducing molecule (interfering RNA).
  • the RNAi target sequence is a miRNA target sequence or a siRNA target sequence.
  • the RNAi target sequence is a miRNA target sequence.
  • the disclosure provides polynucleotide constructs containing a coding sequence for a CAR, one or more RNAi targeting sequences and an optional kill switch coding sequence.
  • single chain variable region refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived.
  • a synthetic linker e.g., a short flexible polypeptide linker
  • an scFv may have the vL and vH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise vL-linker-vH or may comprise vH-linker-vL. Alternatively, a scFv is also described as (vL+vH) or (vH+vL).
  • signal domain refers to the functional region of a protein which transmits information within the cell to regulate cellular activity via defined signaling pathways by generating second messengers or functioning as effectors by responding to such messengers.
  • subject is intended to include living organisms in which an immune response can be elicited (e.g., any domesticated mammals or a human).
  • subject or subject or “individual” or “animal” or “patient” are used interchangeably herein to refer to any subject, particularly a mammalian subject, for whom administration of a composition, vector, recombinant polynucleotide or pharmaceutical composition of the disclosure is desired.
  • Mammalian subjects include humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and the like, with humans being preferred.
  • T-cell and “T-lymphocyte” are interchangeable and used synonymously herein. Examples include, but are not limited to, na ⁇ ve T cells (“lymphocyte progenitors”), central memory T cells, effector memory T cells, stem memory T cells (T ), iPSC- derived T cells, synthetic T cells or combinations thereof.
  • lymphocyte progenitors na ⁇ ve T cells
  • central memory T cells effector memory T cells
  • stem memory T cells (T ) stem memory T cells (T )
  • T stem memory T cells
  • iPSC- derived T cells synthetic T cells or combinations thereof.
  • therapeutic effect refers to a biological effect which can be manifested by various means, including but not limited to, e.g., decrease in tumor volume, a decrease in the number of cancer cells, a decrease in the number of metastases, an increase in life expectancy, decrease in cancer cell proliferation, decrease in cancer cell survival, decrease in the titer of the infectious agent, a decrease in colony counts of the infectious agent, amelioration of various physiological symptoms associated with a disease condition.
  • a “therapeutic effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies in prevention of the occurrence of disease in the first place or in the prevention of relapse of the disease.
  • terapéuticaally effective amount refers to the amount of a pharmaceutical composition comprising vector or in vivo genetically engineered cells, to decrease at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect.
  • therapeutically effective amount means a sufficient amount of the composition to treat a disorder, at a reasonable benefit/risk ratio applicable to any medical treatment.
  • a therapeutically or prophylactically significant reduction in a symptom is, e.g., at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150% or more in a measured parameter as compared to a control or non-treated subject or the state of the subject prior to administering a vector as described herein.
  • Measured or measurable parameters include clinically detectable markers of disease, for example, elevated or depressed levels of a biological marker, as well as parameters related to a clinically accepted scale of symptoms or markers for cancer.
  • Transmembrane domain refers to the region of a CAR or TCR which crosses the plasma membrane.
  • the transmembrane domain of a CAR of the disclosure is the transmembrane region of a transmembrane protein (for example Type I transmembrane proteins), an artificial hydrophobic sequence or a combination thereof.
  • the TMD is selected from the transmembrane domain of an alpha, beta or zeta chain of a T-cell receptor, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CDl la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFl), CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6,
  • Vector refers to the vehicle by which a polynucleotide sequence (e.g. a foreign gene or coding sequence) can be introduced into a host cell, so as to transform the host cell and promote expression (e.g. transcription and translation) of the introduced sequence.
  • Vectors include plasmids, phages, viruses, etc.
  • viral vector refers to a vector obtained or derived from a virus. Typically the virus is a retrovirus including, but not limited to, lentiviruses and gammaretroviruses.
  • the viral vector of the disclosure may be a retroviral vector, such as a gammaretroviral vector.
  • the viral vector of the disclosure may be a lentiviral vector.
  • the vector may be based on a non-primate lentivirus such as equine infectious anemia virus (EIAV).
  • the viral vector of the disclosure may comprise a mitogenic T-cell activating Attorney Docket No.00156-005WO1 transmembrane protein and/or a cytokine-based T-cell activating transmembrane protein in the viral envelope.
  • the mitogenic T-cell activating transmembrane protein and/or cytokine-based T-cell activating transmembrane protein is/are derived from the host cell membrane.
  • virus like particle or “VLP” refers to a viral particle lacking a viral genome.
  • the VLP lacks an env protein. As with complete viral particles they contain an outer viral envelope made of the host cell lipid-bilayer (membrane), and hence contain host cell transmembrane proteins.
  • a VLP can be used in the methods and compositions of the disclosure as a vector for delivering a nucleic acid payload to a cell in vivo.
  • the disclosure provides a recombinant viral vector comprising a plurality of copies of one or more miRNA target sequences inserted into the vector to control expression of a coding sequence contained in the vector (e.g., CAR coding sequences) in off-target transduced cells.
  • a recombinant viral vector may comprise miRNA target sequence(s) inserted into an encapsidated viral polynucleotide.
  • miRNAs expressed in off-target cells can bind to such miRNA target sequence(s) in the viral polynucleotide and suppress expression of the viral polynucleotide containing the miRNA target sequence, thereby limiting viral replication and/or expression of vector- containing coding sequences (e.g., CARs) in the off-target transduced cells.
  • Such recombinant viral vectors can be referred to herein as "miR-attenuated”, “expression-restricted vectors” or “replication-restricted vectors” as they demonstrate reduced or attenuated replication and/or expression of vector-containing coding sequences in cells that express one or more miRNAs capable of binding to the incorporated miR target sequence(s) compared to cells that do not express, or have reduced expression of, the miR.
  • the one or more miRNA target sequence(s) are incorporated into the 3' untranslated region (UTR) and/or 3' UTR downstream of a transgene coding sequence.
  • the mRNA transcripts of a transgene coding sequence comprises an miR-target sequence (TS) comprising one or more miRNA target sequences (e.g., a miRNA target sequence cassette).
  • TS miR-target sequence
  • the miR-TS cassettes described herein comprise at least one miRNA target sequence.
  • the miR-TS cassettes described herein comprise a plurality of miRNA target sequences.
  • the miR-TS cassettes described herein comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more miRNA target sequences.
  • the miR-TS cassettes comprise two or more miRNA target sequences
  • the two or more target sequences can be the same or different.
  • the miR-TS cassettes comprise a plurality miRNA target sequences, wherein each miRNA target sequence of the plurality is a target sequence for the same miRNA.
  • the miR-TS cassettes may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of the same miR target sequence immediately contiguous or separated by a nucleotide spacer (e.g., 1-10 nucleotides).
  • the miR-TS cassettes comprise between 2 to 6 copies of the same miR target sequence.
  • the miR-TS cassettes comprise 3 copies of the same miR target sequence. In some embodiments, the miR-TS cassettes comprise 4 copies of the same miR target sequence. In some embodiments, the miR-TS cassettes comprise 5 copies of the same miR target sequence. In some embodiments, the miR-TS cassettes comprise 6 copies of the same miR target sequence. In some embodiments, the miR-TS cassettes comprise 7 copies of the same miR target sequence. In some embodiments, the miR-TS cassettes comprise 8 copies of the same miR target sequence. In some embodiments, the miR-TS cassettes comprise 9 copies of the same miR target sequence. In some embodiments, the miR-TS cassettes comprise 10 copies of the same miR target sequence.
  • the miR-TS cassettes described herein comprise a plurality of miRNA target sequences, wherein the plurality comprises at least two different miRNA target sequences.
  • the miR-TS cassettes described herein comprise 2, 3, 4, 5, 6, 7, 8, 9, or 10 different miRNA target sequences.
  • the miR-TS cassette may comprise one or more copies of a first miRNA target sequence and one or more copies of a second miRNA target sequence.
  • the miR-TS cassette comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies of a first miR target sequence and at least 2, 3, 4, 5, Attorney Docket No.00156-005WO1 6, 7, 8, 9, 10, or more copies of a second miR target sequence.
  • the miR-TS cassette comprises 3 or 4 copies of a first miR target sequence and 3 or 4 copies of a second miR target sequence.
  • the plurality of miRNA target sequences comprises at least 3 different miRNA target sequences.
  • the miR-TS cassette comprises one or more copies of a first miR target sequence, one or more copies of a second miR target sequence, and one or more copies of a third miR target sequence.
  • the miR-TS cassette comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies of a first miR target sequence, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies of a second miR target sequence, and at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies of a third miR target sequence.
  • the miR-TS cassette comprises 3 or 4 copies of a first miR target sequence, 3 or 4 copies of a second miR target sequence, and 3 or 4 copies of a third miR target sequence.
  • the plurality of miRNA target sequences comprises at least 4 different miRNA target sequences.
  • the miR-TS cassette comprises one or more copies of a first miR target sequence, one or more copies of a second miR target sequence, one or more copies of a third miR target sequence, and one or more copies of a fourth miR target sequence.
  • the miR-TS cassette comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies of a first miR target sequence, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies of a second miR target sequence, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies of a third miR target sequence, and at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies of a fourth miR target sequence.
  • the miR-TS cassette comprises 3 or 4 copies of a first miR target sequence, 3 or 4 copies of a second miR target sequence, 3 or 4 copies of a third miR target sequence, and 3 or 4 copies of a fourth miR target sequence.
  • the miR-TS cassette(s) comprise a plurality of miRNA target sequences, the plurality of miRNA target sequences may be arranged in tandem, without any intervening nucleic acid sequences.
  • the plurality of miRNA target sequences may be separated by a linker sequence.
  • the linker sequence comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or more nucleotides.
  • the linker sequence comprises about 4 to about 20 nucleotides. In further embodiments, the linker sequence comprises about 4 to about 16 nucleotides.
  • a miR-TS cassette may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the following subunits: a first miRNA target sequence-linker-a second miRNA target sequence, wherein adjacent subunits are separated by an additional linker sequence.
  • the first and the second miRNA target sequence are targets of the same miRNA. In some embodiments, the first and the second miRNA target sequence are targets of different miRNAs.
  • the miR target sequence is a target sequence for miR-155-5p, miR-3609, miR-25-3p, miR-93-5p, miR-4485- 3p, miR-221-3p, miR-181b-5p, mir-146a, mir-342, mir-4485, mir-21, miR-484, mir-320a, mir-92a-1, mir-30d, mir-155, mir-423, miR-1301- 3p, miR-3653-5p, miR-92b-3p, miR-122-5p, miR-1291, mir-222, miR- 1248.
  • the vector comprises a CAR or cTCR coding sequence whereby the above miRNA TS is used to prevent expression in Treg immune cells.
  • the miR target sequence is a target sequence for miR-1251-5p, miR-219a-5p, miR-219a-2-3p, miR-124-3p, miR-448, miR-138-2-3p, miR-490-5p, miR-129-1-3p, miR-1264, miR-3943, miR-490-3p, miR-383-5p, miR-133b, miR-129-2-3p, miR-128-2-5p, miR- 133a-3p, miR-129-5p, miR-1-3p, miR-885-3p, miR-124-5p, miR-759, miR- 7158-3p, miR-770-5p, miR-135a-5p, miR-885-5p, let-7g-5p, miR-100, miR-101, miR-106a
  • the vector comprises a CAR or cTCR coding sequence used to treat brain cancer.
  • the miR target sequence is a target sequence for miR-10b-5p, miR-126-3p, miR-145-3p, miR-451a, miR-199b- 5p, miR-5683, miR-3195, miR-3182, miR-1271-5p, miR-204-5p, miR-409- 5p, miR-136-5p, miR-514a-5p, miR-559, miR-483-3p, miR-1-3p, miR- 6080, miR-144-3p, miR-10b-3p, miR-6130, miR-6089, miR-203b-5p, miR- 4266, miR-4327, miR-5694, miR-193b, let-7a, let-7a-1, let-7a-2, let- 7a-3, let-7b, let-7c, let-7d, let-7e, let-7f-1
  • the vector comprises a CAR or cTCR coding sequence used to treat breast cancer.
  • the miR target sequence is a target sequence for miR-143, miR-145, miR-17-5p, miR-203, miR-214, miR-218, miR-335, miR-342-3p, miR-372, miR-424, miR-491-5p, miR-497, miR-7, miR-99a, miR-99b, miR-100, miR-101, miR-15a, miR-16, miR-34a, miR- 886-5p, miR-106a, miR-124, miR-148a, miR-29a, and/or miR-375.
  • the vector comprises a CAR or cTCR coding sequence used to treat cervical cancer.
  • the miR target sequence is a target sequence for miR-133a-5p, miR-490-5p, miR-124-3p, miR-137, miR-655- 3p, miR-376c-3p, miR-369-5p, miR-490-3p, miR-432-5p, miR-487b-3p, miR-342-3p, miR-223-3p, miR-136-3p, miR-136-3p, miR-143-5p, miR-1- Attorney Docket No.00156-005WO1 3p, miR-214-3p, miR-143-3p, miR-199a-3p, miR-199b-3p, miR-451a, miR- 127-3p, miR-133a-3p, miR-145-5p, miR-145-3p, miR-199a-5p, let-7a-1, let-7a-2, let-7
  • the vector comprises a CAR or cTCR coding sequence used to treat colon or colorectal cancer.
  • the miR target sequence is a target sequence for miR-101, miR-130a, miR-130b, miR-134, miR-143, miR-145, miR-152, miR-205, miR-223, miR-301a, miR-301b, miR-30c, miR-34a, miR-34c, miR-424, miR-449a, miR-543, and/or miR-34b.
  • the vector comprises a CAR or cTCR coding sequence used to treat endometrial cancer.
  • the miR target sequence is a target sequence for miR-125b, miR-138, miR-15a, miR-15b, miR-16, miR-16-1, miR-16-1-3p, miR-16-2, miR-181a, miR-181b, miR-195, miR-223, miR- 29b, miR-34b, miR-34c, miR-424, miR-10a, miR-146a, miR-150, miR-151, miR-155, miR-2278, miR-26a, miR-30e, miR-31, miR-326, miR-564, miR- 27a, let-7b, miR-124a, miR-142-3p, let-7c, miR-17, miR-20a, miR-29a, miR-30c, miR-720, miR-107, miR-342, miR-34a, miR-202, miR-142-5p, miR-29c, miR-145, miR-10a, miR-146
  • the vector comprises a CAR or cTCR coding sequence used to treat hematologic cancer.
  • the miR target sequence is a target sequence for miR-1, miR-145, miR-1826, miR-199a, miR-199a-3p, miR- 203, miR-205, miR-497, miR-508-3p, miR-509-3p, let-7a, let-7d, miR- 106a*, miR-126, miR-1285, miR-129-3p, miR-1291, miR-133a, miR-135a, miR-138, miR-141, miR-143, miR-182-5p, miR-200a, miR-218, miR-28-5p, miR-30a, miR-30c, miR-30d, miR-34a, miR-378, miR-429, miR-509-5p, miR-646, miR-133
  • the vector comprises a CAR or cTCR coding sequence used to treat kidney cancer.
  • the miR target sequence is a target sequence for let-7a-1, let-7a-2, let-7a-3, let-7b, let-7c, let-7d, let-7e, let-7f, let-7f-1, let-7f-2, let-7g, let-7i, miR-1, miR-100, miR-101, miR-105, miR-122, miR-122a, miR-1236, miR-124, miR-125b, miR-126, miR-127, miR-1271, miR-128-3p, miR-129-5p, miR-130a, miR- 130b, miR-133a, miR-134, miR-137, miR-138, miR-139, miR-139-5p, miR- 140-5p, miR-141, miR-142-3p, miR-143, miR-144, miR-145, miR-141, miR-142-3p, mi
  • the vector comprises a CAR or cTCR coding sequence used to treat liver cancer.
  • the liver cancer is hepatocellular carcinoma.
  • the miR target sequence is a target sequence for miR-143-3p, miR-126-3p, miR-126-5p, miR-1266-3p, miR- 6130, miR-6080, miR-511-5p, miR-143-5p, miR-223-5p, miR-199b-5p, miR-199a-3p, miR-199b-3p, miR-451a, miR-142-5p, miR-144, miR-150-5p, miR-142-3p, miR-214-3p, miR-214-5p, miR-199a-5p, miR-145-3p, miR- Attorney Docket No.00156-005WO1 145-5p, miR-1297, miR-141, miR-145, miR-16, miR-200a, miR-200b, miR- Attorney Docket No.00156-005WO1 145
  • the vector comprises a CAR or cTCR coding sequence used to treat lung cancer.
  • the miR target sequence is a target sequence for let-7b, miR-101, miR-125b, miR-1280, miR-143, miR-146a, miR-146b, miR-155, miR-17, miR-184, miR-185, miR-18b, miR-193b, miR- 200c, miR-203, miR-204, miR-205, miR-206, miR-20a, miR-211, miR-218, miR-26a, miR-31, miR-33a, miR-34a, miR-34c, miR-376a, miR-376c, miR- 573, miR-7-5p, miR-9, and/or miR-98.
  • the vector comprises a CAR or cTCR coding sequence used to treat melanoma.
  • the miR target sequence is a target sequence for let-7d, miR-218, miR-34a, miR-375, miR-494, miR-100, miR-124, miR-1250, miR-125b, miR-126, miR-1271, miR-136, miR-138, miR-145, miR-147, miR-148a, miR-181a, miR-206, miR-220a, miR-26a, miR-26b, miR-29a, miR-32, miR-323-5p, miR-329, miR-338, miR-370, miR-410, miR-429, miR-433, miR-499a-5p, miR-503, miR-506, miR-632, miR-646, miR-668, miR-877, and/or miR-9.
  • the vector comprises a CAR or cTCR coding sequence used to treat oral cancer.
  • the miR target sequence is a target sequence for let-7i, miR-100, miR-124, miR-125b, miR-129-5p, miR- 130b, miR-133a, miR-137, miR-138, miR-141, miR-145, miR-148a, miR- 152, miR-153, miR-155, miR-199a, miR-200a, miR-200b, miR-200c, miR- 212, miR-335, miR-34a, miR-34b, miR-34c, miR-409-3p, miR-411, miR- 429, miR-432, miR-449a, miR-494, miR-497, miR-498, miR-519d, miR- 655, miR-9, miR-
  • the vector comprises a CAR or cTCR coding sequence used to treat ovarian cancer.
  • the miR target sequence is a target sequence for miR-216a-5p, miR-802, miR-217, miR-145-3p, miR-143-3p, miR-451a, miR-375, miR-214-3p, miR-216b-3p, miR-432-5p, miR-216a-3p, miR-199b-5p, miR-199a-5p, miR-136-3p, miR-216b-5p, miR-136-5p, miR- 145-5p, miR-127-3p, miR-199a-3p, miR-199b-3p, miR-559, miR-129-2-3p, miR-4507, miR-1-3p, miR-148a-3p, miR-101, miR-1181, miR-124, miR- 1247, miR-133a, miR-141,
  • the vector comprises a CAR or cTCR coding sequence used to treat pancreatic cancer.
  • the miR target sequence is a target sequence for let-7a-3p, let-7c, miR-100, miR-101, miR-105, miR-124, miR-128, miR-1296, miR-130b, miR-133a-1, miR-133a-2, miR-133b, miR- 135a, miR-143, miR-145, miR-146a, miR-154, miR-15a, miR-187, miR- 188-5p, miR-199b, miR-200b, miR-203, miR-205, miR-212, miR-218, miR- 221, miR-224, miR-23a, miR-23b, miR-25, miR-26a, miR-26b, miR-29b, miR-302a, miR-30a, miR-30b, miR-30c
  • the vector comprises a CAR or cTCR coding sequence used to treat prostate cancer.
  • the miR target sequence is a target sequence for miR-101, miR-183, miR-204, miR-34a, miR-365b-3p, miR- 486-3p, and/or miR-532-5p.
  • the vector comprises a CAR or cTCR coding sequence used to treat retinoblastoma.
  • the miR target sequence is a target sequence for miR-143-3p, miR-133b, miR-1264, miR-448, miR-1298-5p, miR-490-5p, miR-138-2-3p, miR-144-3p, miR-144-5p, miR-150-5p, miR- 129-1-3p, miR-559, miR-1-3-p, miR-143-5p, miR-223-3p, miR-3943, miR- 338-3p, miR-124-3p, miR-219a-5p, miR-219a-2-3p, miR-451a, miR-142- 5p, miR-133a-3p, miR-145-5p, and/or miR-145-3p.
  • the vector comprises a CAR or cTCR coding sequence used to treat glioblastoma.
  • the miR target sequence is a target sequence for miR-143-3p, miR-223-3p, miR-6080, miR-208b-3p, miR-206, miR-133a-5p, miR-133b, miR-199a-5p, miR-199b-5p, miR-145-3p, miR- 145-5p, miR-150-5p, miR-142-3p, miR-144-3p, miR-144-5p, miR-338-3p, miR-214-3p, miR-559, miR-133a-3p, miR-1-3p, miR-126-3p, miR-142-5p, miR-451a, miR-199a-3p, and/or miR-199b-3p.
  • the vector comprises a CAR or cTCR coding sequence used to treat head and neck cancer.
  • a CAR or cTCR coding sequence used to treat head and neck cancer.
  • binding domains targeting any number of the “Targets” listing in Table 4 can be used to treat diseases associated with the target: [00131]
  • FSHR Prostate cancer ovarian cancer or breast cancer Attorney Docket No.00156-005WO1 TARGET EXEMPLARY DISEASE TARGETED BY CARs (i.e., conventional CARs and next generation CARs.
  • TCRB2 T cell leukemias and l m homas autoimmune disorders Attorney Docket No.00156-005WO1 TARGET EXEMPLARY DISEASE TARGETED BY CARs (i.e., conventional CARs and next generation CARs.
  • the method includes administering a viral construct comprising a viral capsid and envelope containing a polynucleotide derived from a viral genome.
  • the polynucleotide comprises RNA.
  • the polynucleotide is derived from a gammaretrovirus. In another embodiment, the polynucleotide is derived from a lentivirus. In another embodiment, the viral vector contains in the capsid or envelope a recombinant polynucleotide. In another embodiment, a delivery vehicle comprises or contains a recombinant polynucleotide. In still another embodiment, the polynucleotide comprises long terminal repeats at the 5’ and 3’ end. In yet another embodiment, the polynucleotide comprises a coding sequence for a CAR or cTCR. In yet another embodiment, the polynucleotide comprises one or more miRNA target sequence.
  • the miRNA target sequence are targets for miRNA present in off-target cells.
  • the polynucleotide comprises a sequence encoding a polypeptide that converts a prodrug to a toxic drug.
  • the disclosure provides methods of generating nanoparticles that deliver synthetic promoters, nucleic acids, proteins, or combinations thereof to immune cells or immune cell precursors in vivo (i.e., wherein the cells have not been isolated outside the body). Nanoparticles can comprise of micelles, lipid nanoparticles, cationic polymetric nanoparticles, gold or other metallic-based nanoparticles, dendrimers, extracellular vesicles, DNA or RNA nanostructures (e.g.
  • the disclosure provides a plasmid comprising a sequence that produces a polynucleotide that is encapsulated into a viral capsid.
  • the polynucleotide comprises RNA.
  • the polynucleotide is derived from a gammaretrovirus.
  • the polynucleotide comprises long terminal repeats at the 5’ and 3’ end.
  • the polynucleotide comprises a coding sequence for a CAR. In yet another embodiment, the polynucleotide comprises one or more miRNA target sequence. In still another embodiment, the miRNA target sequence are targets for miRNA present in off-target cells, but not in target cells, nor in cells used to make infectious vectors. In still another embodiment, the polynucleotide comprises a sequence encoding a polypeptide that converts a prodrug to a toxic drug.
  • the disclosure provide a plasmid polynucleotide construct comprising from 5’ to 3’, a CMV promoter, an “R-U5” domain from a gammaretrovirus operably linked to a viral gag coding sequence, which is upstream of a synthetic promoter, wherein the synthetic promoter is upstream of a coding sequence for a binding domain operably linked to an optional hinge/linker coding sequence operably linked to a transmembrane domain coding sequence operably linked to a signaling domain coding sequence followed by a “U3-R” domain from a gammaretrovirus.
  • the viral RNA can include a coding sequence for a kill switch operably linked to an IRES.
  • the IRES-Kill switch can be upstream or downstream (5’ or 3’) to an miRNA cassette.
  • the polynucleotide sequence can be schematically presented as (CMV promoter)-(viral 5’R-U5)-(MMLV-psi)- (Viral gag)-(synthetic promoter)-(transgene)-(WPRE domain)-(Viral 3’del-U3-R-U5).
  • the CMV promoter of the plasmid comprises a sequence at least 85%-100% identical to: CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAA TAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTA CGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAA TGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGT ACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGA Attorney Docket No.00156-005WO1 TAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCA CCAAAATCAATCAATCAACGGGACTTTCCAAAAAAAATC
  • the 5’ R-U5 domain can comprise a sequence that is at least 80-100% identical to the sequence: GCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCA GTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACC CGTCAGCGGGGGTCTTTCATTT (SEQ ID NO:30).
  • the MMLV-psi sequence comprises a sequence that is at least 85%-100% identical to: AAGCTGGCCAGCAACTTATCTGTGTCTGTCCGATTGTCTAGTGTCTATGACTGATTTTATGCGCCTGC GTCGGTACTAGTTAGCTAACTAGCTCTGTATCTGGCGGACCCGTGGTGGAACTGACGAGTTCGGAACA CCCGGCCGCAACCCTGGGAGACGTCCCAGGGACTTCGGGGGCCGTTTTTGTGGCCCGACCTGAGTCCA AAAATCCCGATCGTTTTGGACTCTTTGGTGCACCCCCCTTAGAGGAGGGATATGTGGTTCTGGTAGGA GACGAGAACCTAAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGGACCGAAGCCGCGC CGCGCGTCTTGTCTGCTG (SEQ ID NO:31).
  • the viral gag domains can comprise a sequence that is at least 80-100% identical to the sequence: GGCCAGACTGTTACCACTCCCTGAAGTTTGACCTTAGGTCACTGGAAAGATGTCGAGCGGATCGCTCA CAACCAGTCGGTAGATGTCAAGAAGAGACGTTGGGTTACCTTCTGCTCTGCAGAATGGCCAACCTTTA ACGTCGGATGGCCGCGAGACGGCACCTTTAACCGAGACCTCATCACCCAGGTTAAGATCAAGGTCTTT TCACCTGGCCCGCATGGACACCCAGACCAGGTCCCCTACATCGTGACCTGGGAAGCCTTGGCTTTTGA CCCCCCTCCCTGGGTCAAGCCCTTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCGT CTCTCCCCCTTGAACCTCCTCGTTCGACCCCGCCTCGATCCTCCCTTTATCCAGCCCTCACTCCTTCT CTAGGCGCC (SEQ ID NO:32).
  • the synthetic promoter comprises a sequence of SEQ ID NO:1, 4, or anyone of SEQ ID NO:6-26 or 38-47 (or sequence that are at least 80% identical thereto).
  • the WPRE sequence can comprise a sequence that is at least 80%-100% identical to: AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTAC GCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCT CCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGC GTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGCATTGCCACCACCTGTCAGCTCCT TTCCGGGACTTTCGCTTTCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCT GCTGGACAGGGGGGGG
  • the disclosure also provides for a viral capsid containing the expressed RNA polynucleotide from the plasmid construct.
  • RNA expressed from a DNA plasmid of the disclosure will have “T” replaced with “U”.
  • a retroviral polynucleotide of the disclosure will comprise “U” in place of “T” for any of the sequences provided herein, which sequences are expressly contemplated and one of skill in the art can readily envision.
  • the binding domain of the “CAR” can be any sequence that encodes a polypeptide that binds to a desired target antigen.
  • the binding domain can be an antibody fragment such as an scFv directed to a desired target antigen (see, e.g., Table 4). Sequences encoding various binding domains to the targets set forth in Table 4 are known in the art and published in numerous applications (see, e.g., WO 2018/102795, the disclosure of which is incorporated herein by reference including all sequences).
  • the CARs of the disclosure are modular in nature and thus different “binding domains” can be attached depending upon the desired target.
  • a ‘hinge’ or linker coding sequence can be operably linked to the binding domain of the CAR.
  • the ‘hinge’ is optional and the binding domain can be directly linked to the transmembrane domain coding sequence.
  • the binding domain and transmembrane domain are separated by a minimal peptide coding sequence or spacer.
  • Various hinge domains and spacers are known in the art and described herein.
  • the miR targeting sequence or cassette will typically comprise a target for an miRNA molecules that would inhibit expression of a polynucleotide of the viral construct.
  • the miR target sequence will be typically bind an miRNA that is expressed in a tissue or cell where expression of, e.g., a CAR is undesirable or unwanted, but the miRNA is not expressed in target cells nor in vector producer cells where expression from the viral construct is desired.
  • sequences or miRNA are not already known, they can be readily identified and characterized by making total RNA and performing deep bulk sequencing on such samples, from several examples of target tissues for which expression is not wanted (e.g., a tumor) and from several examples of cells where expression is desirable or needed (e.g., T cells) then using bioinformatic techniques known to those skilled in the art, candidate miRNAs and corresponding targets for further testing are identified.
  • a vector construct of the disclosure will include a kill switch as a further safety mechanism, such that expression of the vector construct will result in expression of, e.g., a suicide gene (e.g., a polypeptide having thymidine kinase (TK) or cytosine deaminase (CD) activity; see, e.g., WO 2010/045002, which is incorporated herein by reference).
  • a suicide gene e.g., a polypeptide having thymidine kinase (TK) or cytosine deaminase (CD) activity; see, e.g., WO 2010/045002, which is incorporated herein by reference).
  • the subject, tissue or cell is contacted with a pro-drug (e.g., 5- fluorocytosine; see, e.g., WO2010/002937, which is incorporated herein by reference) such that the cells expressing, e.g., a polypeptide having cytosine deaminase activity are contacted by the 5-FC wherein the 5-FC is converted to cytotoxic 5-FU at the site of the kill-switch’s expression thereby killing the vector-infected cell.
  • a pro-drug e.g., 5- fluorocytosine; see, e.g., WO2010/002937, which is incorporated herein by reference
  • compositions of the disclosure may comprise a viral vector, as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; Attorney Docket No.00156-005WO1 carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
  • Compositions of the disclosure can be formulated for intravenous administration.
  • compositions of the disclosure may be administered in a manner appropriate to the disease to be treated (or prevented).
  • the quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease.
  • an immunologically effective amount “an anti-tumor effective amount,” “a tumor-inhibiting effective amount,” or “therapeutic amount” or “anti-infective” is indicated
  • the amount of the compositions of the disclosure to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject) as the case may be.
  • a pharmaceutical composition is dosed at an amount sufficient to cause the transduction of immune effector cells (e.g., T cells, NK cells) sufficient to treat the disease or disorder.
  • a pharmaceutical composition of the disclosure comprises vector at 10 to 10 transforming units/dose (e.g., 10, 10, 10, 10, 10, 10, 10, 10 , 10 or any value between any of the foregoing two values).
  • the dose may be administered one time to several times per day and may be administered for consecutive days, weeks or months as necessary to induce immune effector cells in vivo.
  • the pharmaceutical composition comprising vectors of the disclosure can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med.

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Abstract

The disclosure provides compositions and methods for inducing an immunity via cellular expression of an antigen receptor binding construct in vivo.

Description

Attorney Docket No.00156-005WO1 VECTORS AND METHODS FOR IN VIVO TRANSDUCTION CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority under 35 U.S.C. §119 from U.S. Provisional Application Serial No. 63/468,497, filed May 23, 2023, and U.S. Provisional Application Serial No. 63/573,240, filed April 2, 2024, the disclosures of which are incorporated herein by reference in their entirety for all purposes. TECHNICAL FIELD [0002] The disclosure provides compositions and methods for cellular therapies of cancer, infection, allergic, degenerative and immune disorders. INCORPORATION BY REFERENCE OF SEQUENCE LISTING [0003] Accompanying this filing is a Sequence Listing entitled, “00156-005WO1.xml” created on May 23, 2024 and having 61,013 bytes of data, machine formatted on IBM-PC, MS-Windows operating system. The sequence listing is hereby incorporated by reference in its entirety for all purposes. BACKGROUND [0004] Adoptive immunotherapy has risen to the forefront of treatment approaches for cancer. Immune cells can be engineered to express artificial receptor (e.g., chimeric antigen receptors (CARs); TCRs; and 2 , 3 generation CARs) that recognize tumor associated antigens. CARs are engineered immune-receptors, which can redirect immune cells to selectively kill tumor cells or modulate an immune response at a site of inflammation. The general premise for their use in cancer immunotherapy is to rapidly generate tumor- targeted immune cells. SUMMARY [0005] The disclosure provides a composition comprising a delivery vehicle or a recombinant polynucleotide comprising or containing a polynucleotide having from 5’ to 3’ a synthetic promoter followed by at least one transgene. In one embodiment, the delivery vehicle is a nanoparticles, micelle, lipid nanoparticles, cationic polymetric nanoparticles, gold or other metallic-based nanoparticles, dendrimers, extracellular vesicles, DNA or RNA nanostructures, polypeptide structures, or silica-based Attorney Docket No.00156-005WO1 nanoparticles. In another embodiment, the recombinant polynucleotide comprises a sequence represented by: (CMV promoter)-(viral 5’R-U5)- (MMLV-psi)-(Viral gag)-(synthetic promoter)-(at least one transgene)-(WPRE domain)-(Viral 3’del-U3-R-U5). In yet a further embodiment, the CMV promoter of comprises a sequence at least 85%- 100% identical to: CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAA TAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTA CGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAA TGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGT ACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGA TAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCA CCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGC GTGTACGGTGGGAGGTCTATATAAGCAGAGCT (SEQ ID NO:28). In still another or further embodiment, the R-U5 domain can comprise a sequence that is at least 80-100% identical to the sequence: GCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCA TCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGG TCTTTCATTT (SEQ ID NO:30). In yet still another or further embodiment, the MMLV-psi sequence comprises a sequence that is at least 85%-100% identical to: AAGCTGGCCAGCAACTTATCTGTGTCTGTCCGATTGTCTAGTGTCTATGACTGATTTTATGCGCCTGC GTCGGTACTAGTTAGCTAACTAGCTCTGTATCTGGCGGACCCGTGGTGGAACTGACGAGTTCGGAACA CCCGGCCGCAACCCTGGGAGACGTCCCAGGGACTTCGGGGGCCGTTTTTGTGGCCCGACCTGAGTCCA AAAATCCCGATCGTTTTGGACTCTTTGGTGCACCCCCCTTAGAGGAGGGATATGTGGTTCTGGTAGGA GACGAGAACCTAAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGGACCGAAGCCGCGC CGCGCGTCTTGTCTGCTG (SEQ ID NO:31). In yet another or further embodiment, the viral gag domains comprise a sequence that is at least 80-100% identical to the sequence: GGCCAGACTGTTACCACTCCCTGAAGTTTGACCTTAGGTCACTGGAAAGATGTCGAGCGGATCGCTCA CAACCAGTCGGTAGATGTCAAGAAGAGACGTTGGGTTACCTTCTGCTCTGCAGAATGGCCAACCTTTA ACGTCGGATGGCCGCGAGACGGCACCTTTAACCGAGACCTCATCACCCAGGTTAAGATCAAGGTCTTT TCACCTGGCCCGCATGGACACCCAGACCAGGTCCCCTACATCGTGACCTGGGAAGCCTTGGCTTTTGA CCCCCCTCCCTGGGTCAAGCCCTTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCCCGT CTCTCCCCCTTGAACCTCCTCGTTCGACCCCGCCTCGATCCTCCCTTTATCCAGCCCTCACTCCTTCT CTAGGCGCC (SEQ ID NO:32). In another or further embodiment, the synthetic promoter comprises a sequence of SEQ ID NO:1, 12-25 or 26 Attorney Docket No.00156-005WO1 or sequences that are at least 80-99% identical thereto and can drive transcription of a downstream sequence. In yet another or further embodiment, the recombinant polynucleotide is used for in vivo transduction and the synthetic promoter comprises SEQ ID NO:4, 6-10 or 11 or sequence that are at least 80-99% identical thereto and which can drive transcription of a downstream sequence. In another or further embodiment, a transgene is operably linked and downstream of the synthetic promoter. In a further embodiment, the transgene comprises a non-naturally occurring immune receptor construct. In a further embodiment, the non-naturally occurring immune receptor is a chimeric antigen receptor (CAR), chimeric TCR (cTCR), or CAR-like construct. In still another or further embodiment, the WPRE sequence can comprise a sequence that is at least 80%-100% identical to: AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTAC GCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCT CCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGC GTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCT TTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCT GCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTT CCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGC CCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCC TTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGC (SEQ ID NO:33). In still another or further embodiment, the viral 3’ delU3-R-U5 domain can comprise a sequence that is at least 80-100% identical to the sequence: GAGCTCAAAATAAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTAGGT TTGGCAAAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCAGTCCTCCGATTGACTGAGTCGCCCG GGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAG GGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTCTTTCATT (SEQ ID NO:34). In yet another or further embodiment, the viral miRNA target sequence domain can comprise a sequence that is at least 80-100% identical to the sequence: TGCTCAATAAATACCCGTTGAACGATTGCTCAATAAATACCCGTTGAAGCATTGCTCAATAAATACCC GTTGAACTACTGCTCAATAAATACCCGTTGAA (SEQ ID NO:35). In another or further embodiment, the viral miRNA target sequence domain can comprise a sequence that is at least 80-100% identical to the Attorney Docket No.00156-005WO1 sequence: CATGATTGCCACGTCTGCAGTACGATCATGATTGCCACGTCTGCAGTAGCATCATGATTGCCACGTCT GCAGTACTACCATGATTGCCACGTCTGCAGTA (SEQ ID NO:36). In yet another or further embodiment, the viral miRNA target sequence domain can comprise a sequence that is at least 80-100% identical to the sequence: TGATTGCCACTGTCTGCAGTACGATTGATTGCCACTGTCTGCAGTAGCATTGATTGCCACTGTCTGCA GTACTACTGATTGCCACTGTCTGCAGTA (SEQ ID NO:37). In another or further embodiment, the recombinant polynucleotide is a plasmid. [0006] The disclosure also provides viral particle comprising an RNA sequence expressed from the foregoing plasmid. [0007] The disclosure also provides a recombinant cell comprising the polynucleotide or plasmid of any of the foregoing. [0008] The disclosure also provides a method or use of a selective synthetic promoter to prevent expression of transgenes and other encoded nucleotides operably linked to the selective synthetic promoter in non-intended cell types during in vivo therapy. In one embodiment, a therapeutic vector selected from the group consisting of gammaretrovirus-, lentivirus-, adenovirus-, adeno-associated virus-, herpesvirus-, human foamy virus-based products, transposons, plasmid, or artificial chromosomes comprise the selective synthetic promoter and operably linked transgene for in vivo therapy. [0009] The disclosure also provides a recombinant vector comprising a sequence represented by: (CMV promoter)-(viral 5’R-U5)- (MMLV-psi)-(Viral gag)-(synthetic promoter)-(at least one transgene)-(WPRE domain)-(Viral 3’del-U3-R-U5) or (CMV promoter)- (viral 5’R-U5)-(MMLV-psi)-(Viral gag)-(WPRE domain)-(at least one transgene)-(synthetic promoter)-(Viral 3’del-U3-R-U5). In one embodiment, the CMV promoter of comprises a sequence at least 85%- 100% identical to: CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAA TAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTA CGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAA TGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGT ACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGA TAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCA CCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGC Attorney Docket No.00156-005WO1 GTGTACGGTGGGAGGTCTATATAAGCAGAGCT (SEQ ID NO:28). In another or further embodiment, the R-U5 domain can comprise a sequence that is at least 80-100% identical to the sequence: GCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCA TCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGG TCTTTCATTT (SEQ ID NO:30). In still another or further embodiment, the MMLV-psi sequence comprises a sequence that is at least 85%-100% identical to: AAGCTGGCCAGCAACTTATCTGTGTCTGTCCGATTGTCTAGTGTCTATGACTGATTTTATGCGCCTGC GTCGGTACTAGTTAGCTAACTAGCTCTGTATCTGGCGGACCCGTGGTGGAACTGACGAGTTCGGAACA CCCGGCCGCAACCCTGGGAGACGTCCCAGGGACTTCGGGGGCCGTTTTTGTGGCCCGACCTGAGTCCA AAAATCCCGATCGTTTTGGACTCTTTGGTGCACCCCCCTTAGAGGAGGGATATGTGGTTCTGGTAGGA GACGAGAACCTAAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGGACCGAAGCCGCGC CGCGCGTCTTGTCTGCTG (SEQ ID NO:31). In yet another or further embodiment, the viral gag domains comprise a sequence that is at least 80-100% identical to the sequence: GGCCAGACTGTTACCACTCCCTGAAGTTTGACCTTAGGTCACTGGAAAGATGTCGAGCGGATCGCTCA CAACCAGTCGGTAGATGTCAAGAAGAGACGTTGGGTTACCTTCTGCTCTGCAGAATGGCCAACCTTTA ACGTCGGATGGCCGCGAGACGGCACCTTTAACCGAGACCTCATCACCCAGGTTAAGATCAAGGTCTTT TCACCTGGCCCGCATGGACACCCAGACCAGGTCCCCTACATCGTGACCTGGGAAGCCTTGGCTTTTGA CCCCCCTCCCTGGGTCAAGCCCTTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCCCGT CTCTCCCCCTTGAACCTCCTCGTTCGACCCCGCCTCGATCCTCCCTTTATCCAGCCCTCACTCCTTCT CTAGGCGCC (SEQ ID NO:32). In still another or further embodiment, the synthetic promoter comprises a sequence of SEQ ID NO:1, 12-25 or 26 or sequences that are at least 80-99% identical thereto and can drive transcription of a downstream sequence. In another or further embodiment, the recombinant polynucleotide is used for in vivo transduction and the synthetic promoter comprises SEQ ID NO:4, 6-10 or 11 or sequence that are at least 80-99% identical thereto and which can drive transcription of a downstream sequence. In another or further embodiment, a transgene is operably linked and downstream of the synthetic promoter. In a further embodiment, the transgene comprises a non-naturally occurring immune receptor construct. In yet a further embodiment, the non-naturally occurring immune receptor is a chimeric antigen receptor (CAR), chimeric TCR (cTCR), or CAR-like construct. In still another or further embodiment, the WPRE sequence can comprise a sequence that is at least 80%-100% Attorney Docket No.00156-005WO1 identical to: AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTAC GCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCT CCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGC GTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCT TTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCT GCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTT CCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGC CCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCC TTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGC (SEQ ID NO:33). In another or further embodiment, the viral 3’ delU3-R-U5 domain can comprise a sequence that is at least 80-100% identical to the sequence: GAGCTCAAAATAAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTAGGT TTGGCAAAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCAGTCCTCCGATTGACTGAGTCGCCCG GGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAG GGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTCTTTCATT (SEQ ID NO:34). In still another or further embodiment, the viral miRNA target sequence domain can comprise a sequence that is at least 80-100% identical to the sequence: TGCTCAATAAATACCCGTTGAACGATTGCTCAATAAATACCCGTTGAAGCATTGCTCAATAAATACCC GTTGAACTACTGCTCAATAAATACCCGTTGAA (SEQ ID NO:35). In yet another or further embodiment, the viral miRNA target sequence domain can comprise a sequence that is at least 80-100% identical to the sequence: CATGATTGCCACGTCTGCAGTACGATCATGATTGCCACGTCTGCAGTAGCATCATGATTGCCACGTCT GCAGTACTACCATGATTGCCACGTCTGCAGTA (SEQ ID NO:36). In another or further embodiment, the viral miRNA target sequence domain can comprise a sequence that is at least 80-100% identical to the sequence: TGATTGCCACTGTCTGCAGTACGATTGATTGCCACTGTCTGCAGTAGCATTGATTGCCACTGTCTGCA GTACTACTGATTGCCACTGTCTGCAGTA (SEQ ID NO:37). In another or further embodiment, the recombinant vector comprises a plasmid. [0010] The disclosure also provides a viral vector produced by expression of the plasmid as described above. In another embodiment, the host cell is a helper cell. In yet another embodiment, the viral vector is an RNA viral vector. In a further embodiment, the viral Attorney Docket No.00156-005WO1 vector comprises an engineered retroviral vector. In still a further embodiment, the viral vector is engineered from a gammaretroviral vector. [0011] The disclosure also provides a method of in vivo transduction or transfection, the method comprising administering the composition, or the viral vector of the disclosure to a subject, wherein the composition or viral vector transfects or transduces an immune cell in vivo. BRIEF DESCRIPTION OF THE FIGURES [0012] Figure 1 shows that a YB-TATA element increases pSC1 activity 2-fold vs ADEp. [0013] Figure 2 provides examples of promoter inactivity in 293T cells commonly used as a vector producing cell line after transfection with lentiviral vector encoding PSC1 immune cell selective promoter and mKATE2 fluorescent protein. UBC is a universal promoter active in most cell types and acts as a positive control. [0014] Figure 3 provides examples of promoter inactivity in HT1080 cells used as a vector producing cell line after transduction with retroviral vector encoding PSC1 immune cell selective promoter and GFP fluorescent protein. EF1a is a universal promoter active in most cell types and acts as a positive control. [0015] Figure 4 provides examples of promoter activity in primary CD4 T cells. Lentiviral vector encoding PSC1 immune cell selective promoter and mKate2 fluorescent protein. Ubcp is a universal promoter active in most cell types and acts as a positive control. [0016] Figure 5 provides examples of promoter inactivity in 293T cells used as a vector producing cell line after transduction with lentiviral vector encoding PSC1 immune cell selective promoter and mKate2 fluorescent protein. Ubcp is a universal promoter active in most cell types and acts as a positive control. DETAILED DESCRIPTION [0017] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a subject" includes a plurality of such subjects and reference to Attorney Docket No.00156-005WO1 "the vector" includes reference to one or more vectors and equivalents thereof known to those skilled in the art, and so forth. [0018] Also, the use of “or” means “and/or” unless stated otherwise. Similarly, “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting. [0019] It is to be further understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of.” [0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Allen et al., Remington: The Science and Practice of Pharmacy 22 ed., Pharmaceutical Press (September 15, 2012); Hornyak et al., Introduction to Nanoscience and Nanotechnology, CRC Press (2008); Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology 3 ed., revised ed., J. Wiley & Sons (New York, NY 2006); Smith, March’s Advanced Organic Chemistry Reactions, Mechanisms and Structure 7 ed., J. Wiley & Sons (New York, NY 2013); Singleton, Dictionary of DNA and Genome Technology 3 ed., Wiley-Blackwell (November 28, 2012); and Green and Sambrook, Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provide one skilled in the art with a general guide to many of the terms used in the present application. For references on how to prepare antibodies, see Greenfield, Antibodies A Laboratory Manual 2 ed., Cold Spring Harbor Press (Cold Spring Harbor NY, 2013); Köhler and Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur. J. Immunol. 1976 Jul, 6(7):511-9; Queen and Selick, Humanized immunoglobulins, U. S. Patent No. 5,585,089 (1996 Dec); and Riechmann et al., Reshaping human antibodies for therapy, Nature 1988 Mar 24, 332(6162):323-7 All headings and subheading provided herein are solely for ease of reading and should not be construed to limit the invention. Although methods and materials similar or equivalent to those described herein can be Attorney Docket No.00156-005WO1 used in the practice or testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and specific examples are illustrative only and not intended to be limiting. [0021] All publications mentioned herein are incorporated herein by reference in full for the purpose of describing and disclosing the methodologies, which might be used in connection with the description herein. Moreover, with respect to any term that is presented in one or more publications that is similar to, or identical with, a term that has been expressly defined in this disclosure, the definition of the term as expressly provided in this disclosure will control in all respects. [0022] It should be understood that this disclosure is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments or aspects only and is not intended to limit the scope of the present disclosure. [0023] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about." The term "about" when used to described the present invention, in connection with percentages means ±1%. The term “about,” as used herein can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which can depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. Alternatively, “about” can mean a range of plus or minus 20%, plus or minus 10%, plus or minus 5%, or plus or minus 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5-fold, or within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an Attorney Docket No.00156-005WO1 acceptable error range for the particular value can be assumed. Also, where ranges and/or subranges of values are provided, the ranges and/or subranges can include the endpoints of the ranges and/or subranges. In some cases, variations can include an amount or concentration of 20%, 10%, 5%, 1 %, 0.5%, or even 0.1 % of the specified amount. [0024] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated. [0025] The Retroviridae family of viruses may be used to create vectors that integrate into their host genome and provide long-term gene expression to the transduced cell and its descendants. In general, gammaretroviral vectors, lentiviral vectors and foamy viral vectors are usable and useful to transduce cells including non- mobilized and mobilized stem cells. The examples below focus on gammaretroviral vectors, but one skilled in the art will quickly realize that the invention can use all types of integrating vectors including viral and non-viral vectors such as adenoviral-retroviral hybrids, piggy-bac and sleeping beauty transposons etc. [0026] The disclosure provides compositions and methods to transduce dividing or non-dividing cells (including hematopoietic stem cells) in vivo by direct administration of a vector to achieve therapeutic effects in many types of diseases including genetic diseases, cancer, infectious disease and autoimmune disease. In some embodiments, cells can be mobilized prior to in vivo infection. [0027] Certain vector constructs of the disclosure can be considered “modular” with domains (sometimes referred to as cassettes) operably linked as described below. For example, in one embodiment, a vector comprises repeats at the 5’ and 3’ ends, a transgene to be expressed (e.g., a CAR construct comprising a binding domain, an optional hinge or linker domain, a transmembrane domain, an intracellular domain; or a similar TCR construct) downstream (i.e., 3’) of a synthetic promoter, optionally one or more miRNA target domains, an optional kill switch domain and an Attorney Docket No.00156-005WO1 optional cell-activity-regulating domain. When the transgene is a CAR, the binding domain, hinge/linker, transmembrane domain and intracellular domain generally comprise chimeric antigen receptors (CARs) including 1 generation, 2 generation, 3 generation and related constructs (e.g., TCRα and β chains comprising coding sequences for cleavable peptide linker between the TCRα and β, at least one of the TCRα and β chains linked to a binding domain to a peptide target). [0028] The term "antibody," as used herein, refers to a protein, or polypeptide sequence derived from an immunoglobulin molecule which specifically binds with an antigen. Antibodies can be monoclonal, or polyclonal, multiple or single chain, or intact immunoglobulins, and may be derived from natural sources or from recombinant sources. Antibodies can be tetramers of immunoglobulin molecules. The antibody may be ‘humanized’, ‘chimeric’ or non-human. [0029] The term "antibody fragment" refers to at least one portion of an antibody, that retains the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing/destabilizing, spatial distribution) an epitope of an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', Fv fragments, scFv antibody fragments, disulfide- linked Fvs, a Fd fragment consisting of the VH and CHl domains, linear antibodies, single domain antibodies (sdAb) such as either vL or vH, camelid vHH domains, multi-specific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody. An antigen binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen binding fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (Fn3) (see U.S. Patent No.: 6,703,199, which describes fibronectin polypeptide mini- bodies). [0030] The term "antibody heavy chain," refers to the larger of the two types of polypeptide chains present in antibody molecules in Attorney Docket No.00156-005WO1 their naturally occurring conformations, and which normally determines the class to which the antibody belongs. [0031] The term "antibody light chain," refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes. [0032] “Anticancer agent” refers to agents that inhibit aberrant cellular division and growth, inhibit migration of neoplastic cells, inhibit invasiveness or prevent cancer growth and metastasis. The term includes chemotherapeutic agents (e.g., alkylating agents, anti-metabolites and the like), biological agents (e.g., siRNA, viral vectors such as engineered MLV, lentivirus, foamy virus, adenoviruses, herpes virus that deliver cytotoxic genes), antibodies and the like. [0033] The term "anticancer effect" refers to a biological effect which can be manifested by various means including, but not limited to, a decrease in tumor volume, a decrease in the number of cancer cells, a decrease in the number of metastases, an increase in life expectancy, decrease in cancer cell proliferation, decrease in cancer cell survival, or amelioration of various physiological symptoms associated with the cancerous condition. An "anticancer effect" can also be manifested by the ability of a CAR (or similar construct, e.g., TCR etc.) in prevention of the occurrence of cancer in the first place. [0034] The term "antigen" or "Ag" refers to a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically- competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full length nucleotide sequence of a gene. The disclosure includes, but is not limited to, the use of Attorney Docket No.00156-005WO1 partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be synthesized or can be derived from a biological sample, or might be a macromolecule besides a polypeptide. Such a biological sample can include, but is not limited to, a tissue sample, a tumor sample, a cell or a fluid with other biological components. An antigen associated with a disease state (e.g., associated with an infection, or a cancer antigen) are targets of therapy. [0035] Non-limiting examples of antigens that can be targeted include: CD5; CD19; CD20; CD22; CD24; CD30; CD33, CD34; CD38; CD69; CD72; CD97; CD123; CD171; CD269; CS1 (also referred to as CD2 subset 1, CRACC, MPL, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); epidermal growth factor receptor variant III (EGFRviii); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4 )bDGlcp(l-l)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAcα-Ser/Thr)); prostate-specific membrane antigen (PSMA); Receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms Like Tyrosine Kinase 3 (FLT3); Tumor-associated glycoprotein 72 (TAG72); CD44v6; a glycosylated CD43 epitope; Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); Interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); Mesothelin; Interleukin 11 receptor alpha (IL-llRa); prostate stem cell antigen (PSCA); Protease Serine 21 (Testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis(Y) antigen; Platelet-derived growth factor receptor beta (PDGFR-beta); Stage-specific embryonic antigen-4 (SSEA-4); Folate receptor alpha (FRa or FR1); Folate receptor beta (FRb); Receptor tyrosine-protein kinase ERBB2 (Her2/neu); Mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); Prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); Ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor); carbonic anhydrase IX (CAlX); Proteasome Attorney Docket No.00156-005WO1 (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2); glycoprotein 100 (gpl00); oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type-A receptor 2 (EphA2); sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2- 3)bDClalp(l- 4)bDGlcp(l-1)Cer); transglutaminase 5 (TGS5); high molecular weight-melanoma associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); tumor endothelial marker 1 (TEM1/CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); G protein coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD179a; anaplastic lymphoma kinase (ALK); Polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion of globoH glycoceramide (GloboH); mammary gland differentiation antigen (NY- BR-1); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K 9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumor protein (WT1); Cancer/testis antigen 1 (NY-ESO-1); Cancer/testis antigen 2 (LAGE- 1a); Melanoma-associated antigen 1 (MAGE-A1); ETS translocation- variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X Antigen Family, Member lA (XAGEl); angiopoietin- binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; survivin; telomerase; prostate carcinoma tumor antigen-1 (PCT A-1 or Galectin 8); melanoma antigen recognized by T cells 1 (MelanA or MARTI); Rat sarcoma (Ras) mutant; human Telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetyl glucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin Bl; v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family Member C (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 lB 1 (CYPlB 1); CCCTC-Binding Factor (Zinc Finger Protein)-Like Attorney Docket No.00156-005WO1 (BORIS or Brother of the Regulator of Imprinted Sites), Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3); Paired box protein Pax-5 (PAX5); proacrosin binding protein sp32 (OY-TESl); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); Receptor for Advanced Glycation End products (RAGE-1); renal ubiquitous 1 (RUl); renal ubiquitous 2 (RU2); legumain; human papilloma virus E6 (HPV E6); human papilloma virus E7 (HPV E7); intestinal carboxyl esterase; heat shock protein 70-2 mutated (mut hsp70-2); CD79a; CD79b; Leukocyte-associated immunoglobulin-like receptor 1 (LAIRl); Fc fragment of IgA receptor (FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); immunoglobulin lambda-like polypeptide 1 (IGLLl); MPL; Biotin; c-MYC epitope Tag; LAMP1 TROP2; GFRalpha4; CDH17; CDH6; NYBR1; CDH19; CD200R; Slea (CA19.9; Sialyl Lewis Antigen); Fucosyl-GM1; PTK7; gpNMB; CDH1-CD324; DLL3; CD276/B7H3; IL11Ra; IL13Ra2; CD179b-IGLl1; TCRgamma-delta; NKG2D; CD32 (FCGR2A) ; Tn ag; Tim1-/HVCR1; CSF2RA (GM-CSFR-alpha); TGFbetaR2; Lews Ag; TCR-beta1 chain; TCR-beta2 chain; TCR-gamma chain; TCR-delta chain; FITC; Leutenizing hormone receptor (LHR); Follicle stimulating hormone receptor (FSHR) ; Gonadotropin Hormone receptor (CGHR or GR) ; CCR4; GD3; SLAMF6; SLAMF4; HIV1 envelope glycoprotein; HTLV1-Tax; CMV pp65; EBV-EBNA3c; KSHV K8.1; KSHV-gH; influenza A hemagglutinin (HA); GAD; PDL1; Guanylyl cyclase C (GCC); auto antibody to desmoglein 3 (Dsg3); auto antibody to desmoglein 1 (Dsg1); HLA; HLA-A; HLA-A2; HLA-B; HLA-C; HLA-DP; HLA-DM; HLA-DOA; HLA-DOB; HLA-DQ; HLA-DR; HLA-G; IgE; CD99; Ras G12V; Tissue Factor 1 (TF1); AFP; GPRC5D; Claudin18.2 (CLD18A2 or CLDN18A.2); P- glycoprotein; STEAP1; Liv1; Nectin-4; Cripto; gpA33; BST1/CD157; low conductance chloride channel; and the antigen recognized by TNT antibody. [0036] An “antigen binding domain” refers to a polypeptide or peptide that due to its primary, secondary or tertiary sequence, Attorney Docket No.00156-005WO1 post-translational modifications, and/or charge binds to an antigen with a high degree of specificity. The antigen binding domain may be derived from different sources, for example, an antibody (full length heavy chain, Fab fragments, single chain Fv (scFv) fragments, divalent single chain antibodies or diabodies), a non-immunoglobulin binding protein, a ligand or a receptor. There are, however, numerous alternatives, such as linked cytokines (which leads to recognition of cells bearing the cytokine receptor), affibodies, ligand binding domains from naturally occurring receptors, soluble protein/peptide ligand for a receptor (for example on a tumor cell), peptides, and vaccines to prompt an immune response, which may each be used in various embodiments of the disclosure. In some embodiments, almost any molecule that binds a given cognate or antigen with high affinity can be used as an antigen binding domain, as will be appreciated by those of skill in the art. In some embodiments, the antigen binding domain comprises T cell receptors (TCRs) or portions thereof. [0037] The term "anti-infection effect" refers to a biological effect that can be manifested by various means including, but not limited to, decrease in the titer of the infectious agent, a decrease in colony counts of the infectious agent, amelioration of various physiological symptoms associated with the infectious condition. [0038] The term "antitumor effect" or “anti-cancer effect” refers to a biological effect which can be manifested by various means including, but not limited to, e.g., a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, inhibition of metastasis, or a decrease in tumor cell survival. [0039] As used herein “beneficial results” may include, but are not limited to, lessening or alleviating the severity of the disease condition, preventing the disease condition from worsening, curing the disease condition, preventing the disease condition from developing, lowering the chances of a patient or subject developing the disease condition and prolonging a patient’s or subject’s life or life expectancy. As non-limiting examples, “beneficial results” may be alleviation of one or more symptom(s), diminishment of extent Attorney Docket No.00156-005WO1 of the deficit, stabilized (i.e., not worsening) state of cancer progression, delay or slowing of metastasis or invasiveness, and amelioration or palliation of symptoms associated with the cancer. [0040] As used herein, the term “biological equivalent thereof” is intended to be synonymous with “equivalent thereof” when referring to a reference protein, antibody or fragment thereof, polypeptide or nucleic acid, and refers to molecules having minimal homology to a reference molecule (e.g., protein, antibody etc.) while still maintaining desired structure and/or functionality. For example, an equivalent can comprise at least about 70% homology or identity, or at least 80% homology or identity and alternatively, or at least about 85%, or alternatively at least about 90%, or alternatively at least about 95%, or alternatively at least 98% percent homology or identity and exhibits substantially equivalent biological activity to the reference protein, polypeptide, antibody or fragment thereof or nucleic acid. Alternatively, when referring to polynucleotides, an equivalent thereof is a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complement and which has the same biological function (e.g., binds to a specific miRNA or encodes a protein or polypeptide having the same or similar biological effect to the polynucleotide to which it is being compared). Alternatively, when referring to polypeptides or proteins, an equivalent thereof is an expressed polypeptide or protein from a polynucleotide that hybridizes under stringent conditions to the polynucleotide or its complement that encodes the reference polypeptide or protein. [0041] “Cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, B-cell lymphomas (Hodgkin’s lymphomas and/or non- Hodgkin’s lymphomas), T cell lymphomas, myeloma, myelodysplastic syndrome, myeloproliferative disorders (e.g., polycythemia vera, myelofibrosis, essential thrombocythemia etc.), skin cancer, brain tumor, breast cancer, colon cancer, rectal cancer, esophageal cancer, anal cancer, cancer of unknown primary site, endocrine cancer, testicular cancer, lung cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, Attorney Docket No.00156-005WO1 liver cancer, bladder cancer, cancer of the urinary tract, cancer of reproductive organs thyroid cancer, renal cancer, carcinoma, melanoma, head and neck cancer, brain cancer (e.g., glioblastoma multiforme), prostate cancer (including but not limited to androgen- dependent prostate cancer and androgen-independent prostate cancer), and leukemia. Other cancer and cell proliferative disorders will be readily recognized in the art. The terms "tumor" and "cancer" are used interchangeably herein, e.g., both terms encompass solid and liquid, e.g., diffuse or circulating, tumors. As used herein, the term "cancer" or "tumor" includes premalignant, as well as malignant cancers and tumors. The term "cancer" is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. [0042] The term “cell-activity-regulating domain” refers to any one or more of PDL1, PDL2, CD80, CD86, crmA, p35, NEMO-K277A (or derivative thereof), K13-opt, IKK2-SS/EE, IKK1-SS/EE, 41BBL, CD40L, vFLIP-K13, MC159, and the like and combination thereof that is expressed in an immune cell (e.g., T cell, e.g., CAR-T cell etc.) to decrease, regulate or modify the activity of the immune cell. In some embodiments, an accessory module is co-expressed with an immune receptor such as a CAR or engineered TCR to increase, decrease, regulate or modify the expression or activity of a CAR, engineered TCR, a CAR-expressing cell or engineered TCR-expressing cell. The accessory module can be co-expressed with a CAR or engineered TCR using a single vector or using two or more different vectors. [0043] “Chemotherapeutic agents” are compounds that are known to be of use in chemotherapy for cancer. Non-limiting examples of chemotherapeutic agents can include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; a camptothecin (including the synthetic analogue topotecan); bryostatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics Attorney Docket No.00156-005WO1 such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino- doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; Attorney Docket No.00156-005WO1 gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE® Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR® gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, lapatinib (Tykerb); inhibitors of PKC- alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above or combinations thereof. [0044] “Chimeric antigen receptors” (CARs) are artificial (non- naturally occurring) immune cell (e.g., T cell) receptors contemplated for use as a therapy for cancer, autoimmune diseases and infectious diseases, using a technique called adoptive cell transfer. CARs are also known as artificial T-cell receptors, chimeric T-cell receptors or chimeric immunoreceptors. CARs are constructed specifically to stimulate immune cell activation and proliferation in response to a specific antigen to which the CAR binds. Generally, a CAR refers to a set of polypeptides, typically two in the simplest embodiments, which when expressed in an immune effector cell, provides the cell with specificity for a target antigen or cell, typically a cancer cell, and with intracellular signal generation. In some embodiments, a CAR comprises at least an extracellular antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to herein as "an intracellular signaling domain") comprising a functional signaling domain derived from a stimulatory molecule and/or costimulatory molecule. In some embodiments, the set of polypeptides are contiguous with each other. In one embodiment, the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of a CAR Attorney Docket No.00156-005WO1 fusion protein. In one embodiment, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen binding domain, wherein the leader sequence is optionally cleaved from the antigen binding domain (e.g., a scFv) during cellular processing and localization of the CAR to the cellular membrane. In various embodiments, CARs are recombinant polypeptides comprising an antigen binding domain, a hinge region (HR), a transmembrane domain (TMD), an optional co-stimulatory domain (CSD) and an intracellular signaling domain (ISD). The optional costimulatory domain is generally absent in 1 generation CAR constructs. Second (2 ) generation CARs comprising antigen binding domains (e.g., vL and vH fragments, vHH, ligands and receptors etc.) typically incorporate a costimulatory domain (e.g., 4-1BB). Unless specified otherwise, as used herein, the term “CAR” or “CARs” also encompasses newer approaches to conferring antigen specificity onto cells, such as Antibody-TCR chimeric molecules or Ab-TCR (WO2017/070608A1 incorporated herein by reference), TCR receptor fusion proteins or TFP (WO2016/187349A1 incorporated herein by reference), Tri- functional T cell antigen coupler (Tri-TAC or TAC) (see, WO2015/117229A1, incorporated herein by reference). Typically, the term “CAR-T cell” is used, to refer to T-cells that have been engineered to express a chimeric antigen receptor or TCR construct. Thus, T lymphocytes bearing such CARs or cTCRs are generally referred to as CAR-T lymphocytes. CARs can be also expressed in cells other than T cells, such as hematopoietic stem cells, induced pluripotent stem cells (iPSC), NK cells and macrophages. [0045] The term “chimeric TCR,” or “cTCR” refers to a wild-type TCR nucleic acid coding sequence and the corresponding wild-type TCR protein linked to an antigen binding domain that is not derived from a TCR. In exemplary embodiment, the antigen binding domain of a cTCR may comprise of a vL, vH or scFv fragments. cTCR have been described in (Gross, Waks & Eshhar, Proc. Natl. Acad. Sci. USA, 1989). cTCRs are used in some embodiments. The term “Ab-TCR” or “AbTCR” refers to a next generation CAR platform as described in WO 2017/070608 A1 which is incorporated herein by reference. In an embodiment, an Ab- TCR comprises an antibody moiety that specifically binds to a target antigen fused to a TCR module capable of recruiting at least one TCR Attorney Docket No.00156-005WO1 signaling module. cTCRs and ab-TCRs can be used in the vectors of the disclosure. [0046] “Codon optimization” or “controlling for species codon bias” refers to the preferred codon usage of a particular host cell. As will be understood by those of skill in the art, it can be advantageous to modify a coding sequence to enhance RNA message stability and its expression in a particular host. The genetic code is redundant with 64 possible codons, but most organisms typically use a subset of these codons. The codons that are utilized most often in a species are called optimal codons, and those not utilized very often are classified as rare or low-usage codons. As part of a codon optimization, the coding sequences of a vector of the disclosure can be modified to reduce RNA destabilizing sequence elements, increase G/C content, limit ApoBec mediated mutations. In one embodiment, the vectors of the disclosure can be engineered to modify their stability and/or expression. For example, changes in expression can occur due to the frequency with which inactivating or attenuating mutations accumulate in the vector as it replicates in a cell. Investigation shows that one of the most frequent events is G to A mutations (corresponds to the C to T mutation) characteristic ApoBec mediated mutations in the negative strand of single stranded DNA from the first replicative step. This can cause changes in amino acid composition of vector-encoded proteins and a devastating change from TGG (Tryptophan) to stop codons (TAG or TGA). In one embodiment this inactivating change is avoided by substitution with codons of other amino acids with similar chemical or structural properties such as phenylalanine or tyrosine at position of ApoBec modifications. [0047] Such mutations can include modifications of one or more codons in the coding sequences of vector domains that change a tryptophan codon to a permissible codon that maintains the biological activity of the encoded protein. It is known in the art that the codon for tryptophan is UGG (TGG in DNA). Moreover, it is known in the art that the “stop codon” is UAA, UAG or UGA (TAA, TAG or TGA in DNA). A single point mutation in the tryptophan codon can cause an unnatural stop codon (e.g., UGG -> UAG or UGG -> UGA). It is also known that human APOBEC3GF (hA3G/F) inhibits retroviral Attorney Docket No.00156-005WO1 replication through G -> A hypermutations (Neogi et al., J. Int. AIDS Soc., 16(1):18472, Feb. 25, 2013). Thus, the disclosure contemplates modifications to the coding sequences of vectors of the disclosure to reduce ApoBec hypermutations by modifying tryptophan codons to permissible non-tryptophan codons. [0048] A “conservative substitution” or "conservative sequence modifications" refers to amino acid modifications that do not significantly affect or alter the binding characteristics or function of the encoded protein. For example, "conservative sequence modifications" refers to amino acid modifications that do not significantly affect or alter the binding characteristics or function of a CAR construct of the disclosure (e.g., a conservative change in the constant chain, antibody, antibody fragment, or non- immunoglobulin binding domains). Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a CAR of the disclosure can be replaced with other amino acid residues from the same side chain family and the altered CAR can be tested using the binding and/or functional assays described herein. [0049] “Co-stimulatory domain” as used herein refers to a biological agent that enhances the proliferation, survival and/or development of T cells. A co-stimulatory domain can comprise the costimulatory domain of any one or more of, for example, members of the TNFR superfamily, CD28, CD137 (4-1BB), CD134 (OX40), Dap10, Attorney Docket No.00156-005WO1 CD27, CD2, CD5, ICAM-1, LFA-1(CD11a/CD18), Lck, TNFR-I, TNFR-II, Fas, CD30, CD40 or combinations thereof. Other co-stimulatory domains (e.g., from other proteins) will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the disclosure. [0050] Cytokine Release Syndrome (CRS) is a complication of cell therapies (e.g., CAR-T, bispecific T cell engaging antibodies etc.) that manifests itself with signs and symptoms such as fever, hypotension, shortness of breath, renal dysfunction, pulmonary dysfunction and/or capillary leak syndrome. CRS is usually due to excessive production of cytokines, such as IL6 and IL1. [0051] A “delivery vehicle” or “polynucleotide delivery vehicle” refers to a composition that can be used in combination with a recombinant polynucleotide to deliver a recombinant polynucleotide to a cell in vivo. Such delivery vehicles can be targeted delivery vehicles or non-targeted delivery vehicles. Examples of delivery vehicles include micro- and nano-particles and micellular composition (e.g., liposomes). A targeted delivery vehicles includes, for example, a liposome comprising an optional targeting moiety (e.g., an antibody) such that the liposome preferentially binds to a cell expression the cognate to the targeting moiety. In some embodiments, the delivery vehicle comprises a polynucleotide encoding a CAR or cTCR operably linked to a synthetic promoter. [0052] "Derived from" as that term is used herein, indicates a relationship between a first and a second molecule. It generally refers to structural similarity between the first molecule and a second molecule and does not include a process or source limitation on a first molecule that is derived from a second molecule. For example, in the case of an antigen binding domain that is derived from an antibody molecule, the antigen binding domain retains sufficient antibody structure such that it has the required function, namely, the ability to bind to an antigen. It does not include any limitation to a particular process of producing the antibody. [0053] “Domain” or “module” refers to a discrete section or part of a larger construct that can be replaced with a similar domain without affecting the function of other domains or module of the Attorney Docket No.00156-005WO1 construct. For example, in a chimeric antigen receptor (CAR) polypeptide or coding nucleic acid sequence, the CAR, in its simplest form, can be described as having a binding domain, a transmembrane domain and an intracellular domain. Each “domain” of the CAR can be modified or changed without affecting the other domains of the CAR. For example, the binding domain can be one of a number of different binding domains as described herein. The binding domain can be a polypeptide sequence that binds to a CD19 antigen. This CD19 binding domain can be replaced with a binding domain that binds to CD20 without affecting of having to change the transmembrane domain. Similarly, a retroviral vector of the disclosure contained in a viral capsid comprises a polynucleotide sense RNA strand having a number of domains including (from 5’ to 3’): a 5’Repeat(5’R)—U5—packaging sequence — a CAR sequence — (optional kill switch comprising an IRES domain linked to, e.g., thymidine kinase (TKO) coding sequence) — an miRNA targeting sequence(s) — a U3—3’Repeat(3’R). Each domain/module of the viral polynucleotide can be changed such that different CAR sequences can be provided, different kill switches (e.g., TKO or cytosine deaminase (CD)), different miRNA targeting sequences etc. The constructs of the disclosure are modular in design. Each domain/module of the construct, whether a polynucleotide construct or an encoded polypeptide construct can comprise minor variations in sequence so long as the variations do not destroy the biological activity of the domain. Thus, for example, a transmembrane domain can have 80-100% identity to a specific transmembrane sequence. [0054] “Genetically modified cells”, “redirected cells”, “genetically engineered cells” or “modified cells” as used herein refer to cells that express, for example, a CAR or cTCR. In some embodiments, the genetically modified cells comprise vectors that encode a CAR or cTCR. [0055] A “hinge region” (HR) as used herein refers to an optional hydrophilic region which is between the antigen binding domain and the transmembrane domain of a CAR. The hinge region includes, but is not limited to, Fc fragments of antibodies or fragments or derivatives thereof, hinge regions of antibodies or fragments or derivatives thereof, CH2 regions of antibodies, CH3 Attorney Docket No.00156-005WO1 regions of antibodies, artificial spacer sequences or combinations thereof. Examples of hinge regions include, but are not limited to, CD8a hinge, and artificial spacers made of polypeptides which may be as small as, for example, Gly or CH1 and CH3 domains of IgGs (such as human IgG4). In some embodiments, the hinge region is any one or more of (i) a hinge, CH2 and CH3 regions of IgG4, (ii) a hinge region of IgG4, (iii) a hinge and CH2 of IgG4, (iv) a hinge region of CD8a, (v) a hinge, CH2 and CH3 regions of IgG1, (vi) a hinge region of IgG1 or (vi) a hinge and CH2 region of IgG1. Other hinge regions will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the disclosure. It should be noted that in some embodiments, a hinge region is not required and the antigen binding domain can be operably linked to the transmembrane domain directly or via a suitable linker. [0056] “Immune cell” as used herein refers to the cells of the mammalian immune system including, but not limited to, antigen presenting cells, B-cells, basophils, cytotoxic T-cells, dendritic cells, eosinophils, granulocytes, helper T-cells, leukocytes, lymphocytes, macrophages, mast cells, memory cells, monocytes, natural killer cells (e.g., NK and NKT cells), neutrophils, phagocytes, plasma cells and T-cells. “In vivo Immune cells” refers to immune cells present in the body of a subject that have not been isolated or removed from the subject. [0057] "Immune effector cell," as that term is used herein, refers to a cell that is involved in an immune response, e.g., in the promotion of an immune effector response. Examples of immune effector cells include T cells, e.g., alpha/beta T cells and gamma/delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes. [0058] The term “In vivo delivery” as used herein refers to direct delivery of therapeutic agents without ex vivo manipulation to cells (e.g., without isolating the target cells). Routes of in vivo delivery include intravenous injection, intramuscular injection, intranodal injection, intralesional, or intranasal/intra- lung inhalation. Other versions of in vivo delivery (e.g., direct delivery of therapeutic agent to cells via extracorporeal/machine- based mechanisms) will be apparent to those of skill in the art and Attorney Docket No.00156-005WO1 may be used in connection with alternate embodiments of the disclosure. In vivo delivery mechanisms can include particle injection, physiochemical, electroporation, ballistic injection, magnetic-based, photoporation, heat-based, pressure-based (osmotic or sound) or hydrodynamic delivery of selective synthetic promoters encoding therapeutic nucleic acids. [0059] “In vivo transduction” or “In vivo transfection” refers to the process of a eukaryotic cell uptake of heterologous nucleic acids in situ. For example, in vivo transduction refers to the uptake by an immune effector cell of a vector of the disclosure within the human or animal body. Contrast this, with ex vivo transduction which described the removal of immune effector cells from the body and transducing them outside the body and then delivering the transduced cells back into the body. Similarly, in vivo transfection refers to the uptake by eukaryotic cells of a recombinant polynucleotide of the disclosure within (i.e., in situ) the body. Transduction is often associated with viral delivery, while transfection is often associated with naked DNA or RNA uptake. [0060] An "intracellular signaling domain," (ISD) or “cytoplasmic domain” refers to an intracellular signaling portion of a molecule. The intracellular signaling domain generates a signal that promotes an immune effector function of the cell. Examples of immune effector function include cytolytic activity and helper activity, including the secretion of cytokines. Examples of domains that transduce the effector function signal include, but are not limited to, the z chain of the T-cell receptor complex or any of its homologs (e.g., h chain, FceR1g and b chains, MB1 (Iga) chain, B29 (Igb) chain, etc.), human CD3 zeta chain, CD3 polypeptides (D, d and e), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.) and other molecules involved in T-cell transduction, such as CD2, CD5 and CD28. Other intracellular signaling domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the disclosure. [0061] In another embodiment, the intracellular signaling domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from Attorney Docket No.00156-005WO1 the molecules responsible for primary stimulation, or antigen dependent simulation. In another embodiment, the intracellular signaling domain can comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals, or antigen independent stimulation. For example, a primary intracellular signaling domain can comprise a cytoplasmic sequence of CD3z or CD3z1xx (Feucht et al., Nat. Med., 25:82-88, 2019), and a costimulatory intracellular signaling domain can comprise cytoplasmic sequence from co-receptor or costimulatory molecule, such as CD28 or 4-1BB. [0062] A primary intracellular signaling domain can comprise a signaling motif which is known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAM containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta (CD3ζ), common FeR gamma (FCER1G), Fe gamma RIIa, FeR beta (Fe Epsilon R1b), CD3 gamma (CD3ɣ), CD3 delta (CD3δ), CD3 epsilon, CD79a, CD79b, DAPlO, and DAP12. [0063] The term “isolated” as used herein refers to molecules or biologics or cellular materials being substantially free from other materials. In one embodiment, the term “isolated” refers to nucleic acid, such as DNA or RNA; protein or polypeptide; cell or cellular organelle(s); or tissue, separated from other DNAs or RNAs, or proteins or polypeptides, or cells or cellular organelles, or tissues or organs, respectively, which are present in the natural source. The term “isolated” also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Moreover, an “isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term “isolated” is also used herein to refer to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides. The term “isolated” is also used herein to refer to cells or tissues that are isolated from other cells or Attorney Docket No.00156-005WO1 tissues and is meant to encompass both, cultured and engineered cells or tissues. [0064] As used herein, the term “linker” (also “linker domain” or “linker region”) refers to an oligonucleotide (operably connecting two polynucleotide domains) or a peptide (operably connecting two polypeptide domains) that joins together two or more domains or regions of a CAR polynucleotide or polypeptide, respectively. The linker can be anywhere from 1 to 500 amino acids in length or 3 to 1500 nucleotides in length. In some embodiments the “linker” is cleavable or non-cleavable. Unless specified otherwise, the term “linker” used herein means a non-cleavable linker. Said non-cleavable linkers may be composed of flexible amino acid residues which allow freedom of motion of adjacent protein domains relative to one another. Non-limiting examples of such residues include glycine and serine (e.g., glycine repeats or glycine-serine repeats in any order). In some embodiments, linkers include non-flexible amino acid residues. Examples of cleavable linkers include 2A linkers (for example T2A), 2A-like linkers or functional equivalents thereof and combinations thereof. In some embodiments, the linkers include the picornaviral 2A-like linker, CHYSEL sequences of porcine teschovirus (P2A), Thosea asigna virus (T2A) or combinations, variants and functional equivalents thereof (e.g., GSG modified variants). In some embodiments, the linker sequences may comprise a motif that results in cleavage between the 2A glycine and the 2B proline. Other cleavable linkers that may be used herein are readily appreciated by those of skill in the art. [0065] The term "flexible polypeptide linker" as used herein refers to a peptide linker that consists of amino acids such as glycine and/or serine residues used alone or in combination, to link polypeptide chains together (e.g., variable heavy and variable light chain regions together). In one embodiment, the flexible polypeptide linker is a Gly/Ser linker and comprises the amino acid sequence (Gly-Ser), where n is a positive integer equal to or greater than 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 etc.). [0066] “Mammal” as used herein refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees and other apes and monkey species; farm Attorney Docket No.00156-005WO1 animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included within the scope of this term. One of skill in the art will recognize that CAR constructs and related sequences are optimized for use in a particular mammalian species (e.g., the encoded protein/polypeptide is derived from the mammalian species being treated or optimized to be expressed by codon optimization in a particular species). [0067] As used herein, “off-target transduced cells” refers to cells that are infected by a delivery vehicle or viral vector of the disclosure, but where expression of the vehicle’s or vector’s encoded genes/polynucleotides are unwanted or undesirable in the unintended cell types or to prevent expression of therapeutic transgenes on the surface of viral vector particles during production of retrovirus- or lentivirus-based products to reduce immunogenicity of a viral particle. It will be recognized in the art that viral vectors can be “targeted” through incorporation of targeting proteins on the viral envelope. In addition, or alternatively, the expression of a viral polynucleotide or encoded gene can be controlled through the use of tissue specific promoters or other synthetic promoters. In still other or further embodiments, the expression of the vector polynucleotide, gene/construct can be controlled through the use of cellular machinery that exists to control innate gene expression control. In this instance RNAi target sequences can be used, whereby binding of innate miRNA to a target sequence(s) can be used to control expression in an off-target cell type. [0068] The term "operably linked" or “functionally linked” refers to functional linkage or association between a first component and a second component such that each component can be functional. For example, operably linked includes the association between a regulatory sequence (e.g., a promoter) and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a Attorney Docket No.00156-005WO1 second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. In the context of two polypeptides that are operably linked, a first polypeptide functions in the manner it would independent of any linkage and a second polypeptide functions as it would absent a linkage between the two. Alternatively, two polypeptides can be considered operably linked wherein the combined expression results in a desired or predicted activity that is lacking when either is expressed alone. [0069] “Percent identity” in the context of two or more nucleic acids or polypeptide sequences, refers to two or more sequences that are related by percent sequence identity. Two sequences are "substantially identical" if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60% identity, optionally 70%, 71%. 72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more typically over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length. [0070] For sequence comparison, generally one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Methods of alignment of sequences for comparison are well known in the art and are publicly available. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm Attorney Docket No.00156-005WO1 of Smith and Waterman, (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Bioi. 48:443, by the search for similarity method of Pearson and Lipman, (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Brent et al., (2003) Current Protocols in Molecular Biology). [0071] Two examples of algorithms that can be used for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al., (1990) J. Mol. Bioi. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI). [0072] The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, (1988) Comput. Appl. Biosci. 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (1970) J. Mol. Bioi. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossom 62 matrix or a P AM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. [0073] The term “regulatory sequence” or “promoter” refers to a domain comprising nucleotide sequences that control or regulate the expression of an operably linked polynucleotide. Promoters are domains that drive the expression of a downstream polynucleotide. The term "regulatory sequence" refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, enhancers and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a Attorney Docket No.00156-005WO1 recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell. One skilled in the art can readily identify regulatory nucleic acid sequence from public databases and materials. Furthermore, one skilled in the art can identify a regulatory sequence that is applicable for the intended use, for example, in vivo, ex vivo, or in vitro. As used herein, a “core promoter” refers to a minimal promoter comprising about 50-100 bp and lacks enhancer elements. Such core promoters include, but are not limited to, super core promoter 1 (SCP1; see, Juven-Gershon et al., Nature, 3:917-922, 2006), AdML and CMV core promoters. More particularly, where a core-promoter cassette is present a second cassette (e.g., a second mini-promoter cassette, a polIII promoter cassette or IRES cassette) will be present. In some embodiments, a vector comprising a cassette with a core promoter specifically excludes the use of SCP1, AdML and CMV core promoters, but rather utilize designer core promoters as described further herein and below. [0074] Core promoters include certain viral promoters. Viral promoters, as used herein, are promoters that have a core sequence, but also usually include some further accessory elements. For example, the early promoter for SV40 contains three types of elements: a TATA box, an initiation site and a GC repeat (Barrera- Saldana et al., EMBO J, 4:3839-3849, 1985; Yaniv, Virology, 384:369– 374, 2009). The TATA box is located approximately 20 base-pairs upstream from the transcriptional start site. The GC repeat regions is a 21 base-pair repeat containing six GC boxes and is the site that determines the direction of transcription. This core promoter sequence is around 100 bp. Adding an additional 72 base-pair repeats, thus making it a “mini-promoter,” is useful as a transcriptional enhancer that increases the functionality of a promoter by a factor of about 10. When the SP1 protein interacts with the 21 bp repeats it binds either the first or the last three GC boxes. Binding of the first three initiates early expression, and binding of the last three initiates late expression. The function of the 72 bp repeats is to enhance the amount of stable RNA and increase the rate of synthesis. This is done by binding Attorney Docket No.00156-005WO1 (dimerization) with the AP1 (activator protein 1) to give a primary transcript that is 3' polyadenylated and 5' capped. Other viral promoters, such as the Rous Sarcoma Virus (RSV), the HBV X gene promoter, and the Herpes Thymidine kinase core promoter can also be used as the basis for selection desired function. [0075] A core promoter typically encompasses −40 to +40 relative to the +1 transcription start site (Juven-Gershon and Kadonaga, Dev. Biol. 339:225–229, 2010), which defines the location at which the RNA polymerase II machinery initiates transcription. Typically, RNA polymerase II interacts with a number of transcription factors that bind to DNA motifs in the promoter. These factors are commonly known as “general” or “basal” transcriptions factors and include, but are not limited to, TFIIA (transcription factor for RNA polymerase IIA), TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. These factors act in a “general” manner with all core promoters; hence they are often referred to as the “basal” transcription factors. [0076] Juven-Gershon et al., 2006 (supra), describe elements of core promoters. For example, the pRC/CMV core promoter consists of a TATA box and is 81 bp in length; the CMV core promoter consists of a TATA box and an initiator site; while the SCP synthetic core promoters (SCP1 and SCP2) consist of a TATA box, an Inr (initiator), an MTE site (Motif Ten Element), and a DPE site (Downstream promoter element) and is about 81 bp in length. The SCP synthetic promoter has improved expression compared to the simple pRC/CMV core promoter. [0077] As used herein a “mini-promoter” or “small promoter” refers to a regulatory domain that promotes transcription of an operably linked gene or coding nucleic acid sequence. The mini- promoter, as the name implies, includes the minimal amount of elements necessary for effective transcription and/or translation of an operably linked coding sequence and can include other enhancing elements. A mini-promoter can comprise a “core promoter” in combination with additional regulatory elements or a “modified core promoter”. Typically, the mini-promoter or modified core promoter will be about 100-600 bp in length while a core promoter is typically less than about 100bp (e.g., about 70-80 bp). In other embodiments, where a core promoter is present, a cassette comprising Attorney Docket No.00156-005WO1 a polynucleotide to be expressed will typically comprise an enhancer element or another element either upstream or downstream of the core promoter sequence that facilitates expression of an operably linked coding sequence above the expression levels of the core promoter alone. [0078] Accordingly, the disclosure provides mini-promoters (e.g., modified core promoters) derived from cellular elements as determined for “core promoter” elements (<100, <200, <400 or <600 bp) that allow ubiquitous expression at significant levels in target cells and are useful for stable incorporation into vectors, in general, and non-replicating retroviral vectors in particular, to allow efficient expression of transgenes or polynucleotide operably linked to the core promoter. Also provided are mini-promoters comprising core promoters plus minimal enhancer sequences and/or Kozak sequences to allow better gene expression compared to a core- promoter lacking such sequences that are still under 200, 400 or 600bp. Such mini-promoters include modified core promoters and naturally occurring tissue specific promoters such as the elastin promoter (specific for pancreatic acinar cells, (204 bp; Hammer et al., Mol Cell Biol., 7:2956-2967, 1987) and the promoter from the cell cycle dependent ASK gene from mouse and man (63-380 bp; Yamada et al., J. Biol. Chem., 277: 27668–27681, 2002). Ubiquitously expressed small promoters also include viral promoters such as the SV40 early and late promoters (about 340 bp), the RSV LTR promoter (about 270 bp) and the HBV X gene promoter (about 180 bp) (e.g., R Anish et al., PLoS One, 4: 5103, 2009) that has no canonical “TATTAA box” and has a 13 bp core sequence of 5’-CCCCGTTGCCCGG-3’ (SEQ ID NO:42). In yet other embodiments, a cassette comprising at least one mini-promoter cassette will have expression levels that exceed, are about equal to, or about 1 fold to 2.5 fold less than the expression levels of an IRES cassette present in a replication competent retroviral vector (RRV). [0079] Transcription from a core- or mini-promoter occurs through the interaction of various elements. In focused transcription, for example, there is either a single major transcription start site or several start sites within a narrow region of several nucleotides. Focused transcription is the Attorney Docket No.00156-005WO1 predominant mode of transcription in simpler organisms. In dispersed transcription, there are several weak transcription start sites over a broad region of about 50 to 100 nucleotides. Dispersed transcription is the most common mode of transcription in vertebrates. For instance, dispersed transcription is observed in about two-thirds of human genes. In vertebrates, focused transcription tends to be associated with regulated promoters, whereas dispersed transcription is typically observed in constitutive promoters in CpG islands. [0080] As used herein a “synthetic promoter” is a promoter that is not "naturally occurring." The synthetic promoters of the disclosure may be produced synthetically (e.g., via chemical synthesis), or using recombinant cloning and/or nucleic acid amplification technology, including polymerase chain reaction (PCR). In some embodiments, a synthetic promoter has characteristics of mini- or core-promoters and have enhanced cell-state specificity (see, e.g., Wu et al., Nature Communications volume 10, Article number: 2880 (2019)). [0081] In some embodiments, a synthetic promoter may be 10-300 nucleotides long. For example, the length of a synthetic promoter may be 10-300, 10-290, 10-280, 10-270, 10-260, 10-250, 10-240, 10- 230, 10-220, 10-210, 10-210, 10-200, 10-190, 10-180, 10-170, 10-160, 10-150, 10-140, 10-130, 10-120, 10-110, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-300, 20-290, 20-280, 20-270, 20-260, 20-250, 20-240, 20-230, 20-220, 20-210, 20-210, 20-200, 20- 190, 20-180, 20-170, 20-160, 20-150, 20-140, 20-130, 20-120, 20-110, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 30-300, 30- 290, 30-280, 30-270, 30-260, 30-250, 30-240, 30-230, 30-220, 30-210, 30-210, 30-200, 30-190, 30-180, 30-170, 30-160, 30-150, 30-140, 30- 130, 30-120, 30-110, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30- 40, 40-300, 40-290, 40-280, 40-270, 40-260, 40-250, 40-240, 40-230, 40-220, 40-210, 40-210, 40-200, 40-190, 40-180, 40-170, 40-160, 40- 150, 40-140, 40-130, 40-120, 40-110, 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, 50-300, 50-290, 50-280, 50-270, 50-260, 50-250, 50- 240, 50-230, 50-220, 50-210, 50-210, 50-200, 50-190, 50-180, 50-170, 50-160, 50-150, 50-140, 50-130, 50-120, 50-110, SO-100, 50-90, 50- 80, 50-70, 50-60, 60-300, 60-290, 60-280, 60-270, 60-260, 60-250, Attorney Docket No.00156-005WO1 -240, 60-230, 60-220, 60-210, 60-210, 60-200, 60-190, 60-180, 60-0, 60-160, 60-150, 60-140, 60-130, 60-120, 60-110, 60-100, 60-90,-80, 60-70, 70-300, 70-290, 70-280, 70-270, 70-260, 70-250, 70-0, 70-230, 70-220, 70-210, 70-210, 70-200, 70-190, 70-180, 70-170,-160, 70-150, 70-140, 70-130, 70-120, 70-110, 70-100, 70-90, 70-, 80-300, 80-290, 80-280, 80-270, 80-260, 80-250, 80-240, 80-230,-220, 80-210, 80-210, 80-200, 80-190, 80-180, 80-170, 80-160, 80-0, 80-140, 80-130, 80-120, 80-110, 80-100, 80-90, 90-300, 90-290,-280, 90-270, 90-260, 90-250, 90-240, 90-230, 90-220, 90-210, 90-0, 90-200, 90-190, 90-180, 90-170, 90-160, 90-150, 90-140, 90-130,-120, 90-110, 90-100, 100-300, 100-290, 100-280, 100-270, 100-260,0-250, 100-240, 100-230, 100-220, 100-210, 100-210, 100-200, 100-0, 100-180, 100-170, 100-160, 100-150, 100-140, 100-130, 100-120,0-110, 110-300, 110-290, 110-280, 110-270, 110-260, 110-250, 110-0, 110-230, 110-220, 110-210, 110-210, 110-200, 110-190, 110-180,0-170, 110-160, 110-150, 110-140, 110-130, 110-120, 120-300, 120-0, 120-280, 120-270, 120-260, 120-250, 120-240, 120-230, 120-220,0-210, 120-210, 120-200, 120-190, 120-180, 120-170, 120-160, 120-0, 120-140, 120-130, 130-300, 130-290, 130-280, 130-270, 130-260,0-250, 130-240, 130-230, 130-220, 130-210, 130-210, 130-200, 130-0, 130-180, 130-170, 130-160, 130-150, 130-140, 140-300, 140-290,0-280, 140-270, 140-260, 140-250, 140-240, 140-230, 140-220, 140-0, 140-210, 140-200, 140-190, 140-180, 140-170, 140-160, 140-150,0-300, 150-290, 150-280, 150-270, 150-260, 150-250, 150-240, 150-0, 150-220, 150-210, 150-210, 150-200, 150-190, 150-180, 150-170,0-160, 160-300, 160-290, 160-280, 160-270, 160-260, 160-250, 160-0, 160-230, 160-220, 160-210, 160-210, 160-200, 160-190, 160-180,0-170, 170-300, 170-290, 170-280, 170-270, 170-260, 170-250, 170-0, 170-230, 170-220, 170-210, 170-210, 170-200, 170-190, 170-180,0-300, 180-290, 180-280, 180-270, 180-260, 180-250, 180-240, 180-0, 180-220, 180-210, 180-210, 180-200, 180-190, 190-300, 190-290,0-280, 190-270, 190-260, 190-250, 190-240, 190-230, 190-220, 190-0, 190-210, 190-200, 200-300, 200-290, 200-280, 200-270, 200-260,0-250, 200-240, 200-230, 200-220, 200-210, 200-210, 210-300, 210-0, 210-280, 210-270, 210-260, 210-250, 210-240, 210-230, 210-220,0-300, 220-290, 220-280, 220-270, 220-260, 220-250, 220-240, 220-0, 230-300, 230-290, 230-280, 230-270, 230-260, 230-250, 230-240, Attorney Docket No.00156-005WO1 240-300, 240-290, 240-280, 240-270, 240-260, 240-250, 250-300, 250- 290, 250-280, 250-270, 250-260, 260-300, 260-290, 260-280, 260-270, 270-300, 270-290, 270-280, 280-300, 280-290, or 290-300 nucleotides in length. Promoters may be longer than 300 nucleotides, in some embodiments. In some embodiments, a synthetic promoter may be longer than 300 nucleotide (e.g., 300, 350, 400, 450, or 500
Figure imgf000039_0001
long or longer). [0082] In some
Figure imgf000039_0002
the length of a synthetic promoter is 200 nucleotides or shorter. In some embodiments, a synthetic promoter may be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,
Figure imgf000039_0003
119, 120, 121, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 165, 166, 167, 168, 169, 170,
Figure imgf000039_0004
171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 nucleotides long. [0083] In some embodiments, a synthetic promoters has differential activities in different cell lines or different cellular states. "Having differential activities" means the activity of a synthetic promoter is higher or lower in one type of cell or at a cellular state, compared to in a different type of cell or at a different cellular state, respectfully. In some embodiments, the activity of a synthetic promoter in one cell type or a cellular state is different from (higher or lower) the activity of the synthetic promoter in another cell type or another cellular state by at least 10% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8 -fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70- fold, 80-fold, 90-fold, 100-fold, 500-fold, or 1000-fold). In some embodiments, the activity of a synthetic promoter in one cell type Attorney Docket No.00156-005WO1 or a cellular state is different from (higher or lower) than the activity of the synthetic promoter in another cell type or another cellular state by 10-100%. For example, the activity of a synthetic promoter in one cell type or a cellular state may be different from (higher or lower) than the activity of the synthetic promoter in another cell type or another cellular state by 10%-100%, 10%-90%, 10%-80%, 10%-70%, 10%-60%, 10%-50%, 10%-40%, 10%-30%, 10%-20%, 20%- 100%, 20%-90%, 20%-80%, 20%-70%, 20%-60%, 20%-50%, 20%-40%, 20%-30%, 30%-100%, 30%-90%, 30%-80%, 30%-70%, 30%-60%, 30%-50%, 30%-40%, 40%- 100%, 40%-90%, 40%-80%, 40%-70%, 40%-60%, 40%-50%, 50%-100%, 50%- 90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, or 90%-100%. In some embodiments, the activity of a synthetic promoter in one cell type or a cellular state is different from (higher or lower) the activity of the synthetic promoter in another cell type or another cellular state by 1-1000 fold. For example, the activity of a synthetic promoter in one cell type or a cellular state may be different from (higher or lower than) the activity of the synthetic promoter in another cell type or another cellular state by 1-1000, 1-900, 1-800, 1-700, 1-600, 1-500, 1-400, 1-300, 1-200, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1- 3, 1-2, 5-1000, 5-900, 5-800, 5-700, 5-600, 5-500, 5-400, 5-300, 5- 200, 5-100, 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-30, 5-20, 5-10, 5- 9, 5-8, 5-7, 5-6, 10-1000, 10-900, 10-800, 10-700, 10-600, 10-500, 10-400, 10-300, 10-200, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-1000, 20-900, 20-800, 20-700, 20-600, 20- 500, 20-400, 20-300, 20-200, 20-100, 20-90, 20-80, 20-70, 20-60, 20- 50, 20-40, 20-30, 30-1000, 30-900, 30-800, 30-700, 30-600, 30-500, 30-400, 30-300, 30-200, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40, 40-1000, 40-900, 40-800, 40-700, 40-600, 40-500, 40-400, 40- 300, 40-200, 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, 50-1000, 50- 900, 50-800, 50-700, 50-600, 50-500, 50-400, 50-300, 50-200, 50-100, 50-90, 50-80, 50-70, 50-60, 60-1000, 60-900, 60-800, 60-700, 60-600, 60-500, 60-400, 60-300, 60-200, 60-100, 60-90, 60-80, 60-70, 70- 1000, 70-900, 70-800, 70-700, 70-600, 70-500, 70-400, 70-300, 70- 200, 70-100, 70-90, 70-80, 80-1000, 80-900, 80-800, 80-700, 80-600, 80-500, 80-400, 80-300, 80-200, 80-100, 80-90, 90-1000, 90-900, 90- Attorney Docket No.00156-005WO1 800, 90-700, 90-600, 90-500, 90-400, 90-300, 90-200, 90-100, 100- 1000, 100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300, 100-200, 200-1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200- 400, 200-300, 300-1000, 300-900, 300-800, 300-700, 300-600, 300-500, 300-400, 400-1000, 400-900, 400-800, 400-700, 400-600, 400-500, 500- 1000, 500-900, 500-800, 500-700, 500-600, 600-1000, 600-900, 600- 800, 600-700, 700-1000, 700-900, 700-800, 800-1000, 800-900, or 900- 1000 fold. In some embodiments, the activity of a synthetic promoter in one cell type or a cellular state may be different from (higher or lower than) the activity of the synthetic promoter in another cell type or another cellular state by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8 -fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50- fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 500-fold, or 1000-fold. In some embodiments, a synthetic promoter may be inactive in one cell type and active in another. In some embodiments, a synthetic promoter may be inactive in one cellular state and active in another. Methods of measuring the activities of a promoter (e.g., a synthetic promoter) are known to those skilled in the art, e.g., as described in Jeyaseelan et al., Nucleic Acids Research. 29 (12), 2001; Allard et al., Cell Notes (21), 2008; and Zaslaver et al, Nature Methods. 3 (8): 623-628, 2006, each of which is incorporated herein by reference. [0084] In some embodiments, a synthetic promoter has differential activity (e.g., higher or lower) in one cellular state relative to another cellular state. Non-limiting examples of different cell types that may transition between different cellular states include: embryonic stem cells, adult stem cells, induced pluripotent stem cells, neurons, cardiomyocytes, skin cells, liver cells, immune cells, epithelial cells, eye cells, astrocytes, and microglia. [0085] In one embodiment, the regulatory sequence (e.g., a synthetic promoter) comprises a minimal nucleic acid sequence sufficient for initiating transcription of a downstream gene or polynucleotide in in vivo applications. In still other embodiments, the regulatory element (e.g., synthetic promoter) may have less sequence complexity than a natural promoter and its expression is Attorney Docket No.00156-005WO1 specific for a particular cell type to be transduced (e.g., an immune effector cell) with less expression (leakiness) in off target cell types and suitable for in vivo applications. For example, the disclosure provides a synthetic promoter comprising a core promoter (ital) linked to an adenoviral derived minipromoter (bold) (underlined & Ital denotes a linker): TGCTTTCCAGGAAATCTGCTTTCCAGGAAGCTTGCTTTCCAGGAATGATGCTTTCCAGGAACAGTGCT TTCCAGGAAATGTGCTTTCCAGGAACGTTGCTTTCCAGGAATACTGCTTTCCAGGAAGGCGCGCCAGA CGCTAGCGGGGGGCTATAAAAGGGGGTGGGGGCGTTCGTCCTCACTCT (SEQ ID NO:1); wherein the core promoter has the sequence: TGCTTTCCAGGAAATCTGCTTTCCAGGAAGCTTGCTTTCCAGGAATGATGCTTTCCAGGAACAGTGCT TTCCAGGAAATGTGCTTTCCAGGAACGTTGCTTTCCAGGAATACTGCTTTCCAGGAA (SEQ ID NO:2) and an adenoviral derived minimal promoter has the sequence: GCTAGCGGGGGGCTATAAAAGGGGGTGGGGGCGTTCGTCCTCACTCT (SEQ ID NO:3). In another example, adenoviral derived minipromoter in SEQ ID NO:1 is replaced with a YB TATA minipromoter to enhance expression in immune cells (see, e.g., data in Figure 1). In this example, the synthetic promoter comprises SEQ ID NO:4 (Ital is core promoter (SEQ ID NO:2); YB TATA minipromoter is bold; underlined & Ital denotes a linker): TGCTTTCCAGGAAATCTGCTTTCCAGGAAGCTTGCTTTCCAGGAATGATGCTTTCCAGGAACAGTGCT TTCCAGGAAATGTGCTTTCCAGGAACGTTGCTTTCCAGGAATACTGCTTTCCAGGAAACGCGTTCTAG AGGGTATATAATGGGGGCCA (SEQ ID NO: 4) comprising of SEQ ID NO:2 and YB TATA minimal promoter TCTAGAGGGTATATAATGGGGGCCA (SEQ ID NO:5). [0086] Other examples of synthetic promoters based on synthetic core and minimal promoters that can be used in vivo are provided in Table 1: [0087] Table 1: Identifier Immune cell selective promoter sequence (bold is SEQ ID NO:3) G T C G T G C
Figure imgf000042_0001
Attorney Docket No.00156-005WO1 TGGTGATTTCCAGGAAAGGGGGGCGCGCCAGACGCTAGCGGGGGGCTATAAAAGG GGGTGGGGGCGTTCGTCCTCACTCT G T G G T T T G A A T G T T T C T C G G G
Figure imgf000043_0001
Attorney Docket No.00156-005WO1 AATGTGGCGCGCCAGACGCTAGCGGGGGGCTATAAAAGGGGGTGGGGGCGTTCGT CCTCACTCTAGATCTGCGATCTAAGTAAGCTTGTGTCCCTTGGGCTTGTCGTACTGTTG A G A T C G C C G A C C
Figure imgf000044_0001
, comprise gene regulatory elements and minipromoters derived from specific cell types that subsequently confers cell specific selectivity in initiating transcription of a downstream gene or polynucleotide. For example, in this embodiment, the disclosure provides a synthetic promoter comprising of gene regulatory and promoter elements derived from the CD2 gene that confers transcriptional selectivity to T cells: tttgtgtgagaattaaaatgcagcaagaaaacacacactcataaacacatctgctttggcaaaggagc acatcagaagggctggcttgtgcgcgctcttgctctctgtgtatgtgtattatgttttatgttactgt aaaagatgtaaagagaggcacgtggttaagctctcggggtgtggactccaccagtctcacttcagttc cttttgcatgaagagctcagaatcaaaagaggaaaccaacccctaag (SEQ ID NO: 12). [0089] Additional embodiments of synthetic promoters derived from specific cells types to confer selectivity in T cells or NK cells are summarized in Tables 2 and 3 respectively: [0090] Table 2: T selective promoter examples Attorney Docket No.00156-005WO1 Identifier T cell selective promoter sequence g g a a c c c a a ct c a g c c ct c a a g g t a g a tt t
Figure imgf000045_0001
[0091] Table 3: NK selective promoter examples Identifier NK cell selective promoter sequence
Figure imgf000045_0002
Attorney Docket No.00156-005WO1 SEQ ID gaggacattctgtccttttatcttctagttattctatcatcagtcctttccttattatgaattagcaaatacaaaatctttgtt NO:19 ccccagggtaaagaaaagaattttaagaagtggtttttcttagccccctcagcaccctagtctatttctaacaccaaaa a g tt ct t c t a t t c t g a t g ct g a c g c tt a g g a c t
Figure imgf000046_0001
[0092] In one embodiment of the disclosure a cell-specific synthetic promoter is inserted into an RNV-SIN(pro) plasmid Attorney Docket No.00156-005WO1 downstream of the viral gag sequence and upstream of the transgene sequence (e.g. Emerald GFP as presented below; however in place of the Emerald GFP one could insert a CAR or cTCR coding sequence), typically with a short (<50bp) non-coding sequence in between the gag coding domain and the synthetic promoter and in between the synthetic promoter and the transgene. [0093] In one embodiment, a modular RNV-SIN plasmid of the disclosure can comprise: (CMV promoter)-(viral 5’R-U5)-(MMLV-psi)- (Viral gag)-(synthetic promoter)-(at least one transgene)-(WPRE domain)-(Viral 3’del-U3-R-U5). As mentioned above, the transgene can comprise any desired sequence to be expressed such as a 1 , 2 , 3 or other CAR construct, cTCR, ab-TCR, RNAi sequences and the like. In a particular embodiment, the RNV-SIN plasmid comprises a sequence (coded): CMV promoter (bold) Viral 5’ R-U5 (ital) MMLV Ψ (ital & bold) Viral gag (underlined) Synthetic promoter pSC1 (bold/underlined/ital; Any of SEQ ID NOs:1, 4, 6-26 can be present in this domain) EmeraldGFP (emGFP) Transgene (double underlined) WPRE (dashed underlined) Viral 3’ delU3-R-U5 (dashed underlined & ital) CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAA TAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTA CGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAA TGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGT ACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGA TAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCA CCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGC GTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGGCGCCAGTCCTCCGATTGACTG AGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTT CCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTCTTTCATTTGGGGGCTCGTCC GGGATCGGGAGACCCCTGCCCAGGGACCACCGACCCACCACCGGGAGGTAAGCTGGCCAGCAACTTAT CTGTGTCTGTCCGATTGTCTAGTGTCTATGACTGATTTTATGCGCCTGCGTCGGTACTAGTTAGCTAA CTAGCTCTGTATCTGGCGGACCCGTGGTGGAACTGACGAGTTCGGAACACCCGGCCGCAACCCTGGGA GACGTCCCAGGGACTTCGGGGGCCGTTTTTGTGGCCCGACCTGAGTCCAAAAATCCCGATCGTTTTGG ACTCTTTGGTGCACCCCCCTTAGAGGAGGGATATGTGGTTCTGGTAGGAGACGAGAACCTAAAACAGT TCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGGACCGAAGCCGCGCCGCGCGTCTTGTCTGCTGC AGCATCGTTCTGTGTTGTCTCTGTCTGACTGTGTTTCTGTATTTGTCTGAGAATTAAGGCCAGACTGT TACCACTCCCTGAAGTTTGACCTTAGGTCACTGGAAAGATGTCGAGCGGATCGCTCACAACCAGTCGG TAGATGTCAAGAAGAGACGTTGGGTTACCTTCTGCTCTGCAGAATGGCCAACCTTTAACGTCGGATGG CCGCGAGACGGCACCTTTAACCGAGACCTCATCACCCAGGTTAAGATCAAGGTCTTTTCACCTGGCCC GCATGGACACCCAGACCAGGTCCCCTACATCGTGACCTGGGAAGCCTTGGCTTTTGACCCCCCTCCCT GGGTCAAGCCCTTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCCCGTCTCTCCCCCTT GAACCTCCTCGTTCGACCCCGCCTCGATCCTCCCTTTATCCAGCCCTCACTCCTTCTCTAGGCGCCGG AATTAATTCTCGAGTGCTTTCCAGGAAATCTGCTTTCCAGGAAGCTTGCTTTCCAGGAATGATGCTTT CCAGGAACAGTGCTTTCCAGGAAATGTGCTTTCCAGGAACGTTGCTTTCCAGGAATACTGCTTTCCAG Attorney Docket No.00156-005WO1 GAAGGCGCGCCAGACGCTAGCGGGGGGCTATAAAAGGGGGTGGGGGCGTTCGTCCTCACTCTGCGGCC GCTTCGAGATGGCCAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGAC GGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCT GACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTGA CCTACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCC ATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGC CGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGG ACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAAGGTCTATATCACCGCCGAC AAGCAGAAGAACGGCATCAAGGTGAACTTCAAGACCCGCCACAACATCGAGGACGGCAGCGTGCAGCT CGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACC TGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTC GTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTGATCGCGAGTCGACAAGCTTGG ATCCATCGATCAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTT GCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGC TTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCA
Figure imgf000048_0001
[0094] In another embodiment, the vector can be a lentiviral SIN backbone having a sequence (coded as follows): RSV promoter (bold) Viral 5’ R-U5 (ital) PBS (underlined) HIV-1 Ψ (double underlined) RRE cPPT/CTS Synthetic promoter pSC1(Any of SEQ ID NOs:1, 4, 6-26 can be present in this domain) (Bold/Ital/underlined) EmeraldGFP (emGFP) WPRE PPT Viral 3’ delU3-R-U5 (ital and dashed underlined) AATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACATGCCTTAC AAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGA AGGCAACAGACGGGTCTGACATGGATTGGACGAACCACTGAATTGCCGCATTGCAGAGATATTGTATT TAAGTGCCTAGCTCGATACATAAACGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTG GCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCC CGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAG CAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCG GCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTA GCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGA TGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAG CAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATAC TGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCA ACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGA AGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGGCCGCTGATCTTCAGACCTGGAGGAGG Attorney Docket No.00156-005WO1 AGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAG TAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTG TTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGC CAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGC ATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATAC CTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCC TTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGG ACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAA AAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAA TTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTG CTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCA ACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATC CATTCGATTAGTGAACGGATCTCGACGGTATCGGTTAACCTTTAAAAGAAAAGGGGGGATTGGGGGGT ACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAA ATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCAGTTTATCGATAATTGCT TTCCAGGAAATCTGCTTTCCAGGAAGCTTGCTTTCCAGGAATGATGCTTTCCAGGAACAGTGCTTTCC AGGAAATGTGCTTTCCAGGAACGTTGCTTTCCAGGAATACTGCTTTCCAGGAAGGCGCGCCAGACGCT AGCGGGGGGCTATAAAAGGGGGTGGGGGCGTTCGTCCTCACTCTGCGGCCGCTTCGAGATGGCCAGCA AGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCAC AAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTG CACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTGACCTACGGCGTGCAGTGCT TCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTC CAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGG CGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGC ACAAGCTGGAGTACAACTACAACAGCCACAAGGTCTATATCACCGCCGACAAGCAGAAGAACGGCATC AAGGTGAACTTCAAGACCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCA GAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCC TGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATC ACTCTCGGCATGGACGAGCTGTACAAGTGAACGCGTCTGGAACAATCAACCTCTGGATTACAAAATTT GTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATG CCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCT GTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACG CAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTC CCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGG CACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCA CCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCC CGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTC CCTTTGGGCCGCCTCCCCGCCTGAATACGAGCTCGGTACCTTTAAGACCAATGACTTACAAGGCAGCT GTAGATCTTAGCCACTTTTTAAAAGAAAAGAGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACA AGATCTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCT AACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGT CTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCA (SEQ ID NO:28) [0095] The term "RNAi target sequence", or “miR target sequence” or “miR target cassette” as used herein, relates to a nucleic acid sequence specifically hybridizing to a dsRNA inducing RNA interference (interfering RNA). Thus, the RNAi target sequence is a sequence essentially complementary to at least one RNAi inducing molecule (interfering RNA). The RNAi target sequence is a miRNA target sequence or a siRNA target sequence. Typically the RNAi target sequence is a miRNA target sequence. As described more fully Attorney Docket No.00156-005WO1 below the disclosure provides polynucleotide constructs containing a coding sequence for a CAR, one or more RNAi targeting sequences and an optional kill switch coding sequence. [0096] The term “single chain variable region” or "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein an scFv may have the vL and vH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise vL-linker-vH or may comprise vH-linker-vL. Alternatively, a scFv is also described as (vL+vH) or (vH+vL). [0097] The term "signaling domain" refers to the functional region of a protein which transmits information within the cell to regulate cellular activity via defined signaling pathways by generating second messengers or functioning as effectors by responding to such messengers. [0098] The term "subject" is intended to include living organisms in which an immune response can be elicited (e.g., any domesticated mammals or a human). The terms "subject" or "individual" or "animal" or "patient" are used interchangeably herein to refer to any subject, particularly a mammalian subject, for whom administration of a composition, vector, recombinant polynucleotide or pharmaceutical composition of the disclosure is desired. Mammalian subjects include humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and the like, with humans being preferred. [0099] The terms “T-cell” and “T-lymphocyte” are interchangeable and used synonymously herein. Examples include, but are not limited to, naïve T cells (“lymphocyte progenitors”), central memory T cells, effector memory T cells, stem memory T cells (T ), iPSC- derived T cells, synthetic T cells or combinations thereof. Attorney Docket No.00156-005WO1 [00100] The term "therapeutic effect" refers to a biological effect which can be manifested by various means, including but not limited to, e.g., decrease in tumor volume, a decrease in the number of cancer cells, a decrease in the number of metastases, an increase in life expectancy, decrease in cancer cell proliferation, decrease in cancer cell survival, decrease in the titer of the infectious agent, a decrease in colony counts of the infectious agent, amelioration of various physiological symptoms associated with a disease condition. A “therapeutic effect" can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies in prevention of the occurrence of disease in the first place or in the prevention of relapse of the disease. [00101] The term “therapeutically effective amount” as used herein refers to the amount of a pharmaceutical composition comprising vector or in vivo genetically engineered cells, to decrease at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect. The phrase "therapeutically effective amount" as used herein means a sufficient amount of the composition to treat a disorder, at a reasonable benefit/risk ratio applicable to any medical treatment. [00102] A therapeutically or prophylactically significant reduction in a symptom is, e.g., at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150% or more in a measured parameter as compared to a control or non-treated subject or the state of the subject prior to administering a vector as described herein. Measured or measurable parameters include clinically detectable markers of disease, for example, elevated or depressed levels of a biological marker, as well as parameters related to a clinically accepted scale of symptoms or markers for cancer. It will be understood, however, that the total daily usage of the compositions and formulations as disclosed herein will be decided by the attending physician within the scope of sound medical judgment. The exact amount required will Attorney Docket No.00156-005WO1 vary depending on factors such as the type of disease being treated, gender, age, and weight of the subject. [00103] “Transmembrane domain” (TMD) as used herein refers to the region of a CAR or TCR which crosses the plasma membrane. The transmembrane domain of a CAR of the disclosure is the transmembrane region of a transmembrane protein (for example Type I transmembrane proteins), an artificial hydrophobic sequence or a combination thereof. Other transmembrane domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the disclosure. In some embodiments, the TMD is selected from the transmembrane domain of an alpha, beta or zeta chain of a T-cell receptor, CD3γ, CD3ε, CD3δ, CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CDl la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFl), CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, and/or NKG2C. [00104] “Vector”, “cloning vector” and “expression vector” as used herein refer to the vehicle by which a polynucleotide sequence (e.g. a foreign gene or coding sequence) can be introduced into a host cell, so as to transform the host cell and promote expression (e.g. transcription and translation) of the introduced sequence. Vectors include plasmids, phages, viruses, etc. [00105] The term “viral vector” refers to a vector obtained or derived from a virus. Typically the virus is a retrovirus including, but not limited to, lentiviruses and gammaretroviruses. The viral vector of the disclosure may be a retroviral vector, such as a gammaretroviral vector. The viral vector of the disclosure may be a lentiviral vector. The vector may be based on a non-primate lentivirus such as equine infectious anemia virus (EIAV). The viral vector of the disclosure may comprise a mitogenic T-cell activating Attorney Docket No.00156-005WO1 transmembrane protein and/or a cytokine-based T-cell activating transmembrane protein in the viral envelope. The mitogenic T-cell activating transmembrane protein and/or cytokine-based T-cell activating transmembrane protein is/are derived from the host cell membrane. [00106] As used herein “virus like particle” or “VLP” refers to a viral particle lacking a viral genome. In some cases, the VLP lacks an env protein. As with complete viral particles they contain an outer viral envelope made of the host cell lipid-bilayer (membrane), and hence contain host cell transmembrane proteins. A VLP can be used in the methods and compositions of the disclosure as a vector for delivering a nucleic acid payload to a cell in vivo. [00107] In another embodiment, the disclosure provides a recombinant viral vector comprising a plurality of copies of one or more miRNA target sequences inserted into the vector to control expression of a coding sequence contained in the vector (e.g., CAR coding sequences) in off-target transduced cells. In certain embodiments, a recombinant viral vector may comprise miRNA target sequence(s) inserted into an encapsidated viral polynucleotide. miRNAs expressed in off-target cells can bind to such miRNA target sequence(s) in the viral polynucleotide and suppress expression of the viral polynucleotide containing the miRNA target sequence, thereby limiting viral replication and/or expression of vector- containing coding sequences (e.g., CARs) in the off-target transduced cells. Such recombinant viral vectors can be referred to herein as "miR-attenuated", “expression-restricted vectors” or "replication-restricted vectors" as they demonstrate reduced or attenuated replication and/or expression of vector-containing coding sequences in cells that express one or more miRNAs capable of binding to the incorporated miR target sequence(s) compared to cells that do not express, or have reduced expression of, the miR. [00108] In certain embodiments, the one or more miRNA target sequence(s) are incorporated into the 3' untranslated region (UTR) and/or 3' UTR downstream of a transgene coding sequence. When transcribed, the mRNA transcripts of a transgene coding sequence comprises an miR-target sequence (TS) comprising one or more miRNA target sequences (e.g., a miRNA target sequence cassette). In some Attorney Docket No.00156-005WO1 embodiments, the miR-TS cassettes described herein comprise at least one miRNA target sequence. In some embodiments, the miR-TS cassettes described herein comprise a plurality of miRNA target sequences. For example, in some embodiments, the miR-TS cassettes described herein comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more miRNA target sequences. In such embodiments, wherein the miR-TS cassettes comprise two or more miRNA target sequences, the two or more target sequences can be the same or different. [00109] In some embodiments, the miR-TS cassettes comprise a plurality miRNA target sequences, wherein each miRNA target sequence of the plurality is a target sequence for the same miRNA. For example, the miR-TS cassettes may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of the same miR target sequence immediately contiguous or separated by a nucleotide spacer (e.g., 1-10 nucleotides). In some embodiments, the miR-TS cassettes comprise between 2 to 6 copies of the same miR target sequence. In some embodiments, the miR-TS cassettes comprise 3 copies of the same miR target sequence. In some embodiments, the miR-TS cassettes comprise 4 copies of the same miR target sequence. In some embodiments, the miR-TS cassettes comprise 5 copies of the same miR target sequence. In some embodiments, the miR-TS cassettes comprise 6 copies of the same miR target sequence. In some embodiments, the miR-TS cassettes comprise 7 copies of the same miR target sequence. In some embodiments, the miR-TS cassettes comprise 8 copies of the same miR target sequence. In some embodiments, the miR-TS cassettes comprise 9 copies of the same miR target sequence. In some embodiments, the miR-TS cassettes comprise 10 copies of the same miR target sequence. [00110] In some embodiments, the miR-TS cassettes described herein comprise a plurality of miRNA target sequences, wherein the plurality comprises at least two different miRNA target sequences. In some embodiments, the miR-TS cassettes described herein comprise 2, 3, 4, 5, 6, 7, 8, 9, or 10 different miRNA target sequences. For example, in some embodiments, the miR-TS cassette may comprise one or more copies of a first miRNA target sequence and one or more copies of a second miRNA target sequence. In some embodiments, the miR-TS cassette comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies of a first miR target sequence and at least 2, 3, 4, 5, Attorney Docket No.00156-005WO1 6, 7, 8, 9, 10, or more copies of a second miR target sequence. In some embodiments, the miR-TS cassette comprises 3 or 4 copies of a first miR target sequence and 3 or 4 copies of a second miR target sequence. In some embodiments, the plurality of miRNA target sequences comprises at least 3 different miRNA target sequences. For example, in some embodiments, the miR-TS cassette comprises one or more copies of a first miR target sequence, one or more copies of a second miR target sequence, and one or more copies of a third miR target sequence. In some embodiments, the miR-TS cassette comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies of a first miR target sequence, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies of a second miR target sequence, and at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies of a third miR target sequence. In some embodiments, the miR-TS cassette comprises 3 or 4 copies of a first miR target sequence, 3 or 4 copies of a second miR target sequence, and 3 or 4 copies of a third miR target sequence. In some embodiments, the plurality of miRNA target sequences comprises at least 4 different miRNA target sequences. For example, in some embodiments, the miR-TS cassette comprises one or more copies of a first miR target sequence, one or more copies of a second miR target sequence, one or more copies of a third miR target sequence, and one or more copies of a fourth miR target sequence. In some embodiments, the miR-TS cassette comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies of a first miR target sequence, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies of a second miR target sequence, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies of a third miR target sequence, and at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies of a fourth miR target sequence. In some embodiments, the miR-TS cassette comprises 3 or 4 copies of a first miR target sequence, 3 or 4 copies of a second miR target sequence, 3 or 4 copies of a third miR target sequence, and 3 or 4 copies of a fourth miR target sequence. [00111] In some embodiments, the miR-TS cassette(s) comprise a plurality of miRNA target sequences, the plurality of miRNA target sequences may be arranged in tandem, without any intervening nucleic acid sequences. In some embodiments, the plurality of miRNA target sequences may be separated by a linker sequence. In some Attorney Docket No.00156-005WO1 embodiments, the linker sequence comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or more nucleotides. In some embodiments, the linker sequence comprises about 4 to about 20 nucleotides. In further embodiments, the linker sequence comprises about 4 to about 16 nucleotides. As an illustrative embodiment, a miR-TS cassette may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the following subunits: a first miRNA target sequence-linker-a second miRNA target sequence, wherein adjacent subunits are separated by an additional linker sequence. In some embodiments, the first and the second miRNA target sequence are targets of the same miRNA. In some embodiments, the first and the second miRNA target sequence are targets of different miRNAs. [00112] In some embodiments, the miR target sequence is a target sequence for miR-155-5p, miR-3609, miR-25-3p, miR-93-5p, miR-4485- 3p, miR-221-3p, miR-181b-5p, mir-146a, mir-342, mir-4485, mir-21, miR-484, mir-320a, mir-92a-1, mir-30d, mir-155, mir-423, miR-1301- 3p, miR-3653-5p, miR-92b-3p, miR-122-5p, miR-1291, mir-222, miR- 1248. In this embodiment, the vector comprises a CAR or cTCR coding sequence whereby the above miRNA TS is used to prevent expression in Treg immune cells. [00113] In some embodiments, the miR target sequence is a target sequence for miR-1251-5p, miR-219a-5p, miR-219a-2-3p, miR-124-3p, miR-448, miR-138-2-3p, miR-490-5p, miR-129-1-3p, miR-1264, miR-3943, miR-490-3p, miR-383-5p, miR-133b, miR-129-2-3p, miR-128-2-5p, miR- 133a-3p, miR-129-5p, miR-1-3p, miR-885-3p, miR-124-5p, miR-759, miR- 7158-3p, miR-770-5p, miR-135a-5p, miR-885-5p, let-7g-5p, miR-100, miR-101, miR-106a, miR-124, miR-124a, miR-125a, miR-125a-5p, miR- 125b, miR-127-3p, miR-128, miR-129, miR-136, miR-137, miR-139-5p, miR-142-3p, miR-143, miR-145, miR-146b-5p, miR-149, miR-152, miR- 153, miR-195, miR-21, miR-212-3p, miR-219-5p, miR-222, miR-29b, miR- 31, miR-3189-3p, miR-320, miR-320a, miR-326, miR-330, miR-331-3p, miR-340, miR-342, miR-34a, miR-376a, miR-449a, miR-483-5p, miR-503, miR-577, miR-663, miR-7, miR-7-5p, miR-873, let-7a, let-7f, miR-107, miR-122, miR-124-5p, miR-139, miR-146a, miR-146b, miR-15b, miR-16, miR-181a, miR-181a-1, miR-181a-2, miR-181b, miR-181b-1, miR-181b-2, miR-181c, miR-181d, miR-184, miR-185, miR-199a-3p, miR-200a, miR- 200b, miR-203, miR-204, miR-205, miR-218, miR-23b, miR-26b, miR-27a, Attorney Docket No.00156-005WO1 miR-29c, miR-328, miR-34c-3p, miR-34c-5p, miR-375, miR-383, miR-451, miR-452, miR-495, miR-584, miR-622, miR-656, miR-98, miR-124-3p, miR-181b-5p, miR-200b, and/or miR-3189-3p. In a further embodiment, the vector comprises a CAR or cTCR coding sequence used to treat brain cancer. [00114] In some embodiments, the miR target sequence is a target sequence for miR-10b-5p, miR-126-3p, miR-145-3p, miR-451a, miR-199b- 5p, miR-5683, miR-3195, miR-3182, miR-1271-5p, miR-204-5p, miR-409- 5p, miR-136-5p, miR-514a-5p, miR-559, miR-483-3p, miR-1-3p, miR- 6080, miR-144-3p, miR-10b-3p, miR-6130, miR-6089, miR-203b-5p, miR- 4266, miR-4327, miR-5694, miR-193b, let-7a, let-7a-1, let-7a-2, let- 7a-3, let-7b, let-7c, let-7d, let-7e, let-7f-1, let-7f-2, let-7g, let-7i, miR-100, miR-107, miR-10a, miR-10b, miR-122, miR-124, miR- 1258, miR-125a-5p, miR-125b, miR-126, miR-127, miR-129, miR-130a, miR-132, miR-133a, miR-143, miR-145, miR-146a, miR-146b, miR-147, miR-148a, miR-149, miR-152, miR-153, miR-15a, miR-16, miR-17-5p, miR-181a, miR-1826, miR-183, miR-185, miR-191, miR-193a-3p, miR-195, miR-199b-5p, miR-19a-3p, miR-200a, miR-200b, miR-200c, miR-205, miR- 206, miR-211, miR-216b, miR-218, miR-22, miR-26a, miR-26b, miR-300, miR-30a, miR-31, miR-335, miR-339-5p, miR-33b, miR-34a, miR-34b, miR-34c, miR-374a, miR-379, miR-381, miR-383, miR-425, miR-429, miR- 450b-3p, miR-494, miR-495, miR-497, miR-502-5p, miR-517a, miR-574- 3p, miR-638, miR-7, miR-720, miR-873, miR-874, miR-92a, miR-98, miR- 99a, mmu-miR-290-3p, and/or mmu-miR-290-5p. In a further embodiment, the vector comprises a CAR or cTCR coding sequence used to treat breast cancer. [00115] In some embodiments, the miR target sequence is a target sequence for miR-143, miR-145, miR-17-5p, miR-203, miR-214, miR-218, miR-335, miR-342-3p, miR-372, miR-424, miR-491-5p, miR-497, miR-7, miR-99a, miR-99b, miR-100, miR-101, miR-15a, miR-16, miR-34a, miR- 886-5p, miR-106a, miR-124, miR-148a, miR-29a, and/or miR-375. In a further embodiment, the vector comprises a CAR or cTCR coding sequence used to treat cervical cancer. [00116] In some embodiments, the miR target sequence is a target sequence for miR-133a-5p, miR-490-5p, miR-124-3p, miR-137, miR-655- 3p, miR-376c-3p, miR-369-5p, miR-490-3p, miR-432-5p, miR-487b-3p, miR-342-3p, miR-223-3p, miR-136-3p, miR-136-3p, miR-143-5p, miR-1- Attorney Docket No.00156-005WO1 3p, miR-214-3p, miR-143-3p, miR-199a-3p, miR-199b-3p, miR-451a, miR- 127-3p, miR-133a-3p, miR-145-5p, miR-145-3p, miR-199a-5p, let-7a-1, let-7a-2, let-7a-3, let-7b, let-7c, let-7d, let-7e, let-7f-1, let- 7f-2, let-7g, let-7i, miR-100, miR-101, miR-126, miR-142-3p, miR- 143, miR-145, miR-192, miR-200c, miR-21, miR-214, miR-215, miR-22, miR-25, miR-302a, miR-320, miR-320a, miR-34a, miR-34c, miR-365, miR- 373, miR-424, miR-429, miR-455, miR-484, miR-502, miR-503, miR-93, miR-98, miR-186, miR-30a-5p, miR-627, let-7a, miR-1, miR-124, miR- 125a, miR-129, miR-1295b-3p, miR-1307, miR-130b, miR-132, miR-133a, miR-133b, miR-137, miR-138, miR-139, miR-139-5p, miR-140-5p, miR- 148a, miR-148b, miR-149, miR-150-5p, miR-154, miR-15a, miR-15b, miR- 16, miR-18a, miR-191, miR-193a-5p, miR-194, miR-195, miR-196a, miR- 198, miR-199a-5p, miR-203, miR-204-5p, miR-206, miR-212, miR-218, miR-224, miR-24-3p, miR-26b, miR-27a, miR-28-3p, miR-28-5p, miR-29b, miR-30a-3p, miR-30b, miR-328, miR-338-3p, miR-342, miR-345, miR-34a- 5p, miR-361-5p, miR-375, miR-378, miR-378a-3p, miR-378a-5p, miR-409- 3p, miR-422a, miR-4487, miR-483, miR-497, miR-498, miR-518a-3p, miR- 551a, miR-574-5p, miR-625, miR-638, miR-7, miR-96-5p, miR-202-3p, miR-30a, and/or miR-451. In a further embodiment, the vector comprises a CAR or cTCR coding sequence used to treat colon or colorectal cancer. [00117] In some embodiments, the miR target sequence is a target sequence for miR-101, miR-130a, miR-130b, miR-134, miR-143, miR-145, miR-152, miR-205, miR-223, miR-301a, miR-301b, miR-30c, miR-34a, miR-34c, miR-424, miR-449a, miR-543, and/or miR-34b. In a further embodiment, the vector comprises a CAR or cTCR coding sequence used to treat endometrial cancer. [00118] In some embodiments, the miR target sequence is a target sequence for miR-125b, miR-138, miR-15a, miR-15b, miR-16, miR-16-1, miR-16-1-3p, miR-16-2, miR-181a, miR-181b, miR-195, miR-223, miR- 29b, miR-34b, miR-34c, miR-424, miR-10a, miR-146a, miR-150, miR-151, miR-155, miR-2278, miR-26a, miR-30e, miR-31, miR-326, miR-564, miR- 27a, let-7b, miR-124a, miR-142-3p, let-7c, miR-17, miR-20a, miR-29a, miR-30c, miR-720, miR-107, miR-342, miR-34a, miR-202, miR-142-5p, miR-29c, miR-145, miR-193b, miR-199a, miR-214, miR-22, miR-137, and/or miR-197. In a further embodiment, the vector comprises a CAR or cTCR coding sequence used to treat hematologic cancer. Attorney Docket No.00156-005WO1 [00119] In some embodiments, the miR target sequence is a target sequence for miR-1, miR-145, miR-1826, miR-199a, miR-199a-3p, miR- 203, miR-205, miR-497, miR-508-3p, miR-509-3p, let-7a, let-7d, miR- 106a*, miR-126, miR-1285, miR-129-3p, miR-1291, miR-133a, miR-135a, miR-138, miR-141, miR-143, miR-182-5p, miR-200a, miR-218, miR-28-5p, miR-30a, miR-30c, miR-30d, miR-34a, miR-378, miR-429, miR-509-5p, miR-646, miR-133b, let-7b, let-7c, miR-200c, miR-204, miR-335, miR- 377, and/or miR-506. In a further embodiment, the vector comprises a CAR or cTCR coding sequence used to treat kidney cancer. [00120] In some embodiments, the miR target sequence is a target sequence for let-7a-1, let-7a-2, let-7a-3, let-7b, let-7c, let-7d, let-7e, let-7f, let-7f-1, let-7f-2, let-7g, let-7i, miR-1, miR-100, miR-101, miR-105, miR-122, miR-122a, miR-1236, miR-124, miR-125b, miR-126, miR-127, miR-1271, miR-128-3p, miR-129-5p, miR-130a, miR- 130b, miR-133a, miR-134, miR-137, miR-138, miR-139, miR-139-5p, miR- 140-5p, miR-141, miR-142-3p, miR-143, miR-144, miR-145, miR-146a, miR-148a, miR-148b, miR-150-5p, miR-15b, miR-16, miR-181a-5p, miR- 185, miR-188-5p, miR-193b, miR-195, miR-195-5p, miR-197, miR-198, miR-199a, miR-199a-5p, miR-199b, miR-199b-5p, miR-200a, miR-200b, miR-200c, miR-202, miR-203, miR-204-3p, miR-205, miR-206, miR-20a, miR-21, miR-21-3p, miR-211, miR-212, miR-214, miR-217, miR-218, miR- 219-5p, miR-22, miR-223, miR-26a, miR-26b, miR-29a, miR-29b-1, miR- 29b-2, miR-29c, miR-302b, miR-302c, miR-30a, miR-30a-3p, miR-335, miR-338-3p, miR-33a, miR-34a, miR-34b, miR-365, miR-370, miR-372, miR-375, miR-376a, miR-377, miR-422a, miR-424, miR-424-5p, miR-433, miR-4458, miR-448, miR-450a, miR-451, miR-485-5p, miR-486-5p, miR- 497, miR-503, miR-506, miR-519d, miR-520a, miR-520b, miR-520c-3p, miR-582-5p, miR-590-5p, miR-610, miR-612, miR-625, miR-637, miR-675, miR-7, miR-877, miR-940, miR-941, miR-98, miR-99a, miR-132, and/or miR-31. In a further embodiment, the vector comprises a CAR or cTCR coding sequence used to treat liver cancer. In further embodiments, the liver cancer is hepatocellular carcinoma. [00121] In some embodiments, the miR target sequence is a target sequence for miR-143-3p, miR-126-3p, miR-126-5p, miR-1266-3p, miR- 6130, miR-6080, miR-511-5p, miR-143-5p, miR-223-5p, miR-199b-5p, miR-199a-3p, miR-199b-3p, miR-451a, miR-142-5p, miR-144, miR-150-5p, miR-142-3p, miR-214-3p, miR-214-5p, miR-199a-5p, miR-145-3p, miR- Attorney Docket No.00156-005WO1 145-5p, miR-1297, miR-141, miR-145, miR-16, miR-200a, miR-200b, miR- 200c, miR-29b, miR-381, miR-409-3p, miR-429, miR-451, miR-511, miR- 99a, let-7a-1, let-7a-2, let-7a-3, let-7b, let-7c, let-7d, let-7e, let-7f-1, let-7f-2, let-7g, let-7i, miR-1, miR-101, miR-133b, miR- 138, miR-142-5p, miR-144, miR-1469, miR-146a, miR-153, miR-15a, miR- 15b, miR-16-1, miR-16-2, miR-182, miR-192, miR-193a-3p, miR-194, miR-195, miR-198, miR-203, miR-217, miR-218, miR-22, miR-223, miR- 26a, miR-26b, miR-29c, miR-33a, miR-34a, miR-34b, miR-34c, miR-365, miR-449a, miR-449b, miR-486-5p, miR-545, miR-610, miR-614, miR-630, miR-660, miR-7515, miR-9500, miR-98, miR-99b, miR-133a, let-7a, miR- 100, miR-106a, miR-107, miR-124, miR-125a-3p, miR-125a-5p, miR-126, miR-126*, miR-129, miR-137, miR-140, miR-143, miR-146b, miR-148a, miR-148b, miR-149, miR-152, miR-154, miR-155, miR-17-5p, miR-181a-1, miR-181a-2, miR-181b, miR-181b-1, miR-181b-2, miR-181c, miR-181d, miR-184, miR-186, miR-193b, miR-199a, miR-204, miR-212, miR-221, miR-224, miR-27a, miR-27b, miR-29a, miR-30a, miR-30b, miR-30c, miR- 30d, miR-30d-5p, miR-30e-5p, miR-32, miR-335, miR-338-3p, miR-340, miR-342-3p, miR-361-3p, miR-373, miR-375, miR-4500, miR-4782-3p, miR-497, miR-503, miR-512-3p, miR-520a-3p, miR-526b, miR-625*, and/or miR-96. In a further embodiment, the vector comprises a CAR or cTCR coding sequence used to treat lung cancer. [00122] In some embodiments, the miR target sequence is a target sequence for let-7b, miR-101, miR-125b, miR-1280, miR-143, miR-146a, miR-146b, miR-155, miR-17, miR-184, miR-185, miR-18b, miR-193b, miR- 200c, miR-203, miR-204, miR-205, miR-206, miR-20a, miR-211, miR-218, miR-26a, miR-31, miR-33a, miR-34a, miR-34c, miR-376a, miR-376c, miR- 573, miR-7-5p, miR-9, and/or miR-98. In a further embodiment, the vector comprises a CAR or cTCR coding sequence used to treat melanoma. [00123] In some embodiments, the miR target sequence is a target sequence for let-7d, miR-218, miR-34a, miR-375, miR-494, miR-100, miR-124, miR-1250, miR-125b, miR-126, miR-1271, miR-136, miR-138, miR-145, miR-147, miR-148a, miR-181a, miR-206, miR-220a, miR-26a, miR-26b, miR-29a, miR-32, miR-323-5p, miR-329, miR-338, miR-370, miR-410, miR-429, miR-433, miR-499a-5p, miR-503, miR-506, miR-632, miR-646, miR-668, miR-877, and/or miR-9. In a further embodiment, Attorney Docket No.00156-005WO1 the vector comprises a CAR or cTCR coding sequence used to treat oral cancer. [00124] In some embodiments, the miR target sequence is a target sequence for let-7i, miR-100, miR-124, miR-125b, miR-129-5p, miR- 130b, miR-133a, miR-137, miR-138, miR-141, miR-145, miR-148a, miR- 152, miR-153, miR-155, miR-199a, miR-200a, miR-200b, miR-200c, miR- 212, miR-335, miR-34a, miR-34b, miR-34c, miR-409-3p, miR-411, miR- 429, miR-432, miR-449a, miR-494, miR-497, miR-498, miR-519d, miR- 655, miR-9, miR-98, miR-101, miR-532-5p, miR-124a, miR-192, miR- 193a, and/or miR-7. In a further embodiment, the vector comprises a CAR or cTCR coding sequence used to treat ovarian cancer. [00125] In some embodiments, the miR target sequence is a target sequence for miR-216a-5p, miR-802, miR-217, miR-145-3p, miR-143-3p, miR-451a, miR-375, miR-214-3p, miR-216b-3p, miR-432-5p, miR-216a-3p, miR-199b-5p, miR-199a-5p, miR-136-3p, miR-216b-5p, miR-136-5p, miR- 145-5p, miR-127-3p, miR-199a-3p, miR-199b-3p, miR-559, miR-129-2-3p, miR-4507, miR-1-3p, miR-148a-3p, miR-101, miR-1181, miR-124, miR- 1247, miR-133a, miR-141, miR-145, miR-146a, miR-148a, miR-148b, miR- 150*, miR-150-5p, miR-152, miR-15a, miR-198, miR-203, miR-214, miR- 216a, miR-29c, miR-335, miR-34a, miR-34b, miR-34c, miR-373, miR-375, miR-410, miR-497, miR-615-5p, miR-630, miR-96, miR-132, let-7a, let- 7a-1, let-7a-2, let-7a-3, let-7b, let-7c, let-7d, let-7e, let-7f-1, let-7f-2, let-7g, let-7i, miR-126, miR-135a, miR-143, miR-144, miR- 150, miR-16, miR-200a, miR-200b, miR-200c, miR-217, miR-218, miR- 337, miR-494, and/or miR-98 inserted into the 5' UTR or 3' UTR of one or more viral genes required for viral replication. In a further embodiment, the vector comprises a CAR or cTCR coding sequence used to treat pancreatic cancer. [00126] In some embodiments, the miR target sequence is a target sequence for let-7a-3p, let-7c, miR-100, miR-101, miR-105, miR-124, miR-128, miR-1296, miR-130b, miR-133a-1, miR-133a-2, miR-133b, miR- 135a, miR-143, miR-145, miR-146a, miR-154, miR-15a, miR-187, miR- 188-5p, miR-199b, miR-200b, miR-203, miR-205, miR-212, miR-218, miR- 221, miR-224, miR-23a, miR-23b, miR-25, miR-26a, miR-26b, miR-29b, miR-302a, miR-30a, miR-30b, miR-30c-1, miR-30c-2, miR-30d, miR-30e, miR-31, miR-330, miR-331-3p, miR-34a, miR-34b, miR-34c, miR-374b, miR-449a, miR-4723-5p, miR-497, miR-628-5p, miR-642a-5p, miR-765, Attorney Docket No.00156-005WO1 and/or miR-940. In a further embodiment, the vector comprises a CAR or cTCR coding sequence used to treat prostate cancer. [00127] In some embodiments, the miR target sequence is a target sequence for miR-101, miR-183, miR-204, miR-34a, miR-365b-3p, miR- 486-3p, and/or miR-532-5p. In a further embodiment, the vector comprises a CAR or cTCR coding sequence used to treat retinoblastoma. [00128] In some embodiments, the miR target sequence is a target sequence for miR-143-3p, miR-133b, miR-1264, miR-448, miR-1298-5p, miR-490-5p, miR-138-2-3p, miR-144-3p, miR-144-5p, miR-150-5p, miR- 129-1-3p, miR-559, miR-1-3-p, miR-143-5p, miR-223-3p, miR-3943, miR- 338-3p, miR-124-3p, miR-219a-5p, miR-219a-2-3p, miR-451a, miR-142- 5p, miR-133a-3p, miR-145-5p, and/or miR-145-3p. In a further embodiment, the vector comprises a CAR or cTCR coding sequence used to treat glioblastoma. [00129] In some embodiments, the miR target sequence is a target sequence for miR-143-3p, miR-223-3p, miR-6080, miR-208b-3p, miR-206, miR-133a-5p, miR-133b, miR-199a-5p, miR-199b-5p, miR-145-3p, miR- 145-5p, miR-150-5p, miR-142-3p, miR-144-3p, miR-144-5p, miR-338-3p, miR-214-3p, miR-559, miR-133a-3p, miR-1-3p, miR-126-3p, miR-142-5p, miR-451a, miR-199a-3p, and/or miR-199b-3p. In a further embodiment, the vector comprises a CAR or cTCR coding sequence used to treat head and neck cancer. [00130] Depending upon the designed CAR or cTCR and the binding domain engineered into/onto the CAR or cTCR, the methods and compositions can be used to treat a number of disease and disorders. For example, binding domains targeting any number of the “Targets” listing in Table 4 can be used to treat diseases associated with the target: [00131] Table 4: TARGET EXEMPLARY DISEASE TARGETED BY CARs (i.e., conventional CAR d i CAR E Ab TCR d TFP
Figure imgf000062_0001
Attorney Docket No.00156-005WO1 TARGET EXEMPLARY DISEASE TARGETED BY CARs (i.e., conventional CARs and next generation CARs. E.g., Ab-TCR, and TFP) BCMA M eloma PEL lasma cell leukemia Waldenstrom’s macro lobinemia ors
Figure imgf000063_0001
Attorney Docket No.00156-005WO1 TARGET EXEMPLARY DISEASE TARGETED BY CARs (i.e., conventional CARs and next generation CARs. E.g., Ab-TCR, and TFP) FSHR Prostate cancer ovarian cancer or breast cancer
Figure imgf000064_0001
Attorney Docket No.00156-005WO1 TARGET EXEMPLARY DISEASE TARGETED BY CARs (i.e., conventional CARs and next generation CARs. E.g., Ab-TCR, and TFP) TCRB2 T cell leukemias and l m homas autoimmune disorders
Figure imgf000065_0001
Attorney Docket No.00156-005WO1 TARGET EXEMPLARY DISEASE TARGETED BY CARs (i.e., conventional CARs and next generation CARs. E.g., Ab-TCR, and TFP) HIV1-env HIVI/AIDS and related conditions
Figure imgf000066_0001
immunity to a subject in need thereof comprising administering a vector or delivery vehicle of the disclosure encoding a chimeric antigen receptor (CAR) or chimeric TCR (cTCR) to the subject such that the CAR or cTCR is selectively expressed in a desired immune cell type or immune cell stem cell (e.g., hematopoietic stem cell). In one embodiment, the method includes administering a viral construct comprising a viral capsid and envelope containing a polynucleotide derived from a viral genome. In one embodiment, the polynucleotide comprises RNA. In another embodiment, the polynucleotide is derived from a gammaretrovirus. In another embodiment, the polynucleotide is derived from a lentivirus. In another embodiment, the viral vector contains in the capsid or envelope a recombinant polynucleotide. In another embodiment, a delivery vehicle comprises or contains a recombinant polynucleotide. In still another embodiment, the polynucleotide comprises long terminal repeats at the 5’ and 3’ end. In yet another embodiment, the polynucleotide comprises a coding sequence for a CAR or cTCR. In yet another embodiment, the polynucleotide comprises one or more miRNA target sequence. In still another embodiment, the miRNA target sequence are targets for miRNA present in off-target cells. In still another embodiment, the polynucleotide comprises a sequence encoding a polypeptide that converts a prodrug to a toxic drug. [00133] The disclosure provides methods of generating nanoparticles that deliver synthetic promoters, nucleic acids, proteins, or combinations thereof to immune cells or immune cell precursors in vivo (i.e., wherein the cells have not been isolated outside the body). Nanoparticles can comprise of micelles, lipid nanoparticles, cationic polymetric nanoparticles, gold or other metallic-based nanoparticles, dendrimers, extracellular vesicles, DNA or RNA nanostructures (e.g. aptamers), polypeptide structures, or silica-based nanoparticles. Other versions of nanoparticles Attorney Docket No.00156-005WO1 (e.g., from other encapsulating mechanisms) will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the disclosure. [00134] The disclosure provides a plasmid comprising a sequence that produces a polynucleotide that is encapsulated into a viral capsid. In one embodiment, the polynucleotide comprises RNA. In another embodiment, the polynucleotide is derived from a gammaretrovirus. In still another embodiment, the polynucleotide comprises long terminal repeats at the 5’ and 3’ end. In yet another embodiment, the polynucleotide comprises a coding sequence for a CAR. In yet another embodiment, the polynucleotide comprises one or more miRNA target sequence. In still another embodiment, the miRNA target sequence are targets for miRNA present in off-target cells, but not in target cells, nor in cells used to make infectious vectors. In still another embodiment, the polynucleotide comprises a sequence encoding a polypeptide that converts a prodrug to a toxic drug. [00135] The disclosure provide a plasmid polynucleotide construct comprising from 5’ to 3’, a CMV promoter, an “R-U5” domain from a gammaretrovirus operably linked to a viral gag coding sequence, which is upstream of a synthetic promoter, wherein the synthetic promoter is upstream of a coding sequence for a binding domain operably linked to an optional hinge/linker coding sequence operably linked to a transmembrane domain coding sequence operably linked to a signaling domain coding sequence followed by a “U3-R” domain from a gammaretrovirus. In some embodiments, the viral RNA can include a coding sequence for a kill switch operably linked to an IRES. In some embodiments, the IRES-Kill switch can be upstream or downstream (5’ or 3’) to an miRNA cassette. The polynucleotide sequence can be schematically presented as (CMV promoter)-(viral 5’R-U5)-(MMLV-psi)- (Viral gag)-(synthetic promoter)-(transgene)-(WPRE domain)-(Viral 3’del-U3-R-U5). [00136] In one embodiment, the CMV promoter of the plasmid comprises a sequence at least 85%-100% identical to: CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAA TAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTA CGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAA TGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGT ACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGA Attorney Docket No.00156-005WO1 TAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCA CCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGC GTGTACGGTGGGAGGTCTATATAAGCAGAGCT (SEQ ID NO:29). [00137] In a further embodiment, the 5’ R-U5 domain can comprise a sequence that is at least 80-100% identical to the sequence: GCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCA GTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACC CGTCAGCGGGGGTCTTTCATTT (SEQ ID NO:30). [00138] In another embodiment or further embodiment, the MMLV-psi sequence comprises a sequence that is at least 85%-100% identical to: AAGCTGGCCAGCAACTTATCTGTGTCTGTCCGATTGTCTAGTGTCTATGACTGATTTTATGCGCCTGC GTCGGTACTAGTTAGCTAACTAGCTCTGTATCTGGCGGACCCGTGGTGGAACTGACGAGTTCGGAACA CCCGGCCGCAACCCTGGGAGACGTCCCAGGGACTTCGGGGGCCGTTTTTGTGGCCCGACCTGAGTCCA AAAATCCCGATCGTTTTGGACTCTTTGGTGCACCCCCCTTAGAGGAGGGATATGTGGTTCTGGTAGGA GACGAGAACCTAAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGGACCGAAGCCGCGC CGCGCGTCTTGTCTGCTG (SEQ ID NO:31). [00139] In one embodiment, the viral gag domains can comprise a sequence that is at least 80-100% identical to the sequence: GGCCAGACTGTTACCACTCCCTGAAGTTTGACCTTAGGTCACTGGAAAGATGTCGAGCGGATCGCTCA CAACCAGTCGGTAGATGTCAAGAAGAGACGTTGGGTTACCTTCTGCTCTGCAGAATGGCCAACCTTTA ACGTCGGATGGCCGCGAGACGGCACCTTTAACCGAGACCTCATCACCCAGGTTAAGATCAAGGTCTTT TCACCTGGCCCGCATGGACACCCAGACCAGGTCCCCTACATCGTGACCTGGGAAGCCTTGGCTTTTGA CCCCCCTCCCTGGGTCAAGCCCTTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCCCGT CTCTCCCCCTTGAACCTCCTCGTTCGACCCCGCCTCGATCCTCCCTTTATCCAGCCCTCACTCCTTCT CTAGGCGCC (SEQ ID NO:32). [00140] In another or further embodiment, the synthetic promoter comprises a sequence of SEQ ID NO:1, 4, or anyone of SEQ ID NO:6-26 or 38-47 (or sequence that are at least 80% identical thereto). [00141] In another or further embodiment, the WPRE sequence can comprise a sequence that is at least 80%-100% identical to: AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTAC GCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCT CCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGC GTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCT TTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCT GCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTT CCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGC CCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCC TTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGC (SEQ ID NO:33) [00142] In one embodiment, the viral 3’ delU3-R-U5 domain can comprise a sequence that is at least 80-100% identical to the sequence: Attorney Docket No.00156-005WO1 GAGCTCAAAATAAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTAGGT TTGGCAAAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCAGTCCTCCGATTGACTGAGTCGCCCG GGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAG GGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTCTTTCATT (SEQ ID NO:34) [00143] As mentioned above and elsewhere, the transgene downstream of the synthetic promoter selected from SEQ ID NO:1, 4, 6-26, 38-46 or 47) can be any desire coding sequence (e.g., a CAR or subsequently improved generations of a CAR or cTCR). [00144] The disclosure also provides for a viral capsid containing the expressed RNA polynucleotide from the plasmid construct. [00145] It will be recognized by one of skill in the art that RNA expressed from a DNA plasmid of the disclosure will have “T” replaced with “U”. In will be readily apparent that a retroviral polynucleotide of the disclosure, will comprise “U” in place of “T” for any of the sequences provided herein, which sequences are expressly contemplated and one of skill in the art can readily envision. [00146] The binding domain of the “CAR” can be any sequence that encodes a polypeptide that binds to a desired target antigen. For example, the binding domain can be an antibody fragment such as an scFv directed to a desired target antigen (see, e.g., Table 4). Sequences encoding various binding domains to the targets set forth in Table 4 are known in the art and published in numerous applications (see, e.g., WO 2018/102795, the disclosure of which is incorporated herein by reference including all sequences). The CARs of the disclosure are modular in nature and thus different “binding domains” can be attached depending upon the desired target. [00147] As described above, a ‘hinge’ or linker coding sequence can be operably linked to the binding domain of the CAR. In some instances the ‘hinge’ is optional and the binding domain can be directly linked to the transmembrane domain coding sequence. In another embodiment, the binding domain and transmembrane domain are separated by a minimal peptide coding sequence or spacer. Various hinge domains and spacers are known in the art and described herein. [00148] The miR targeting sequence or cassette will typically comprise a target for an miRNA molecules that would inhibit expression of a polynucleotide of the viral construct. For example, Attorney Docket No.00156-005WO1 the miR target sequence will be typically bind an miRNA that is expressed in a tissue or cell where expression of, e.g., a CAR is undesirable or unwanted, but the miRNA is not expressed in target cells nor in vector producer cells where expression from the viral construct is desired. When such sequences or miRNA are not already known, they can be readily identified and characterized by making total RNA and performing deep bulk sequencing on such samples, from several examples of target tissues for which expression is not wanted (e.g., a tumor) and from several examples of cells where expression is desirable or needed (e.g., T cells) then using bioinformatic techniques known to those skilled in the art, candidate miRNAs and corresponding targets for further testing are identified. Additionally, using the disclosure one of skill in the art can readily identify a binding domain for treating a particular cancer or disease, as well as an miRNA targeting sequence that would prevent expression of the CAR in undesirable tissues and/or cells and suitable ‘hinge’, ‘transmembrane domain’ and intracellular domains. [00149] In some embodiments, a vector construct of the disclosure will include a kill switch as a further safety mechanism, such that expression of the vector construct will result in expression of, e.g., a suicide gene (e.g., a polypeptide having thymidine kinase (TK) or cytosine deaminase (CD) activity; see, e.g., WO 2010/045002, which is incorporated herein by reference). In instances where a subject, cells or tissues have developed unwanted vector expression, the subject, tissue or cell is contacted with a pro-drug (e.g., 5- fluorocytosine; see, e.g., WO2010/002937, which is incorporated herein by reference) such that the cells expressing, e.g., a polypeptide having cytosine deaminase activity are contacted by the 5-FC wherein the 5-FC is converted to cytotoxic 5-FU at the site of the kill-switch’s expression thereby killing the vector-infected cell. [00150] Pharmaceutical compositions of the disclosure may comprise a viral vector, as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; Attorney Docket No.00156-005WO1 carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the disclosure can be formulated for intravenous administration. The composition may further comprise a secondary active agent (e.g., an anticancer, antiviral or antibiotic agent). [00151] Pharmaceutical compositions of the disclosure may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease. When "an immunologically effective amount," "an anti-tumor effective amount," "a tumor-inhibiting effective amount," or "therapeutic amount" or “anti-infective” is indicated, the amount of the compositions of the disclosure to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject) as the case may be. It can generally be stated that a pharmaceutical composition is dosed at an amount sufficient to cause the transduction of immune effector cells (e.g., T cells, NK cells) sufficient to treat the disease or disorder. In one embodiment, a pharmaceutical composition of the disclosure comprises vector at 10 to 10 transforming units/dose (e.g., 10, 10, 10, 10, 10, 10, 10, 10 , 10 or any value between any of the foregoing two values). The dose may be administered one time to several times per day and may be administered for consecutive days, weeks or months as necessary to induce immune effector cells in vivo. The pharmaceutical composition comprising vectors of the disclosure can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). [00152] Use of selective synthetic promoters in therapeutic vectors to prevent expression of transgenes and other encoded nucleotides during the manufacturing of therapeutic vectors like gammaretrovirus- or lentivirus-based products for in vivo use. The Attorney Docket No.00156-005WO1 synthetic promoter is, for example, not active in HEK 293T or human sarcoma HT1080 cell lines, but is active in primary immune cells. [00153] This selectivity would prevent expression of any encoding nucleotides downstream of the synthetic promoter during vector production and generation of producer cell lines. This enables several advantages over current vector production methods: 1. Higher titers resulting from lack of competition between transcriptional machinery making vector genome and transcriptional machinery transcribing transgene(s) off the synthetic promoter. 2. Reduced immunogenicity of vector particles as transgenes are not expressed and therefore not packaged or expressed as part of vector particles or exosomes from producer cell lines. 3. Potential for increased titer, stability, and particle safety profile by preventing transgene signaling in producer cell lines. Activity of transgenes may negatively affect production cell activity and metabolism, may result in production of dangerous signaling molecules from producer cells, alter what cellular proteins are packaged into vector particles, and change efficiency of vector packaging and production. [00154] A number of embodiments have been set forth above to illustrate the disclosure. The following claims further set forth what the Applicants regard as their invention.

Claims

Attorney Docket No.00156-005WO1 What is claimed is: 1. A composition comprising (i) a delivery vehicle comprising a recombinant polynucleotide or (ii) a viral vector comprising a recombinant polynucleotide, wherein the recombinant polynucleotide comprises from 5’ to 3’ a synthetic promoter followed by at least one transgene. 2. The composition of claim 1, wherein the delivery vehicle is a nanoparticles, micelle, lipid nanoparticles, cationic polymetric nanoparticles, gold or other metallic-based nanoparticles, dendrimers, extracellular vesicles, DNA or RNA nanostructures, polypeptide structures, or silica-based nanoparticles. 3. The composition of claim 1, wherein the recombinant polynucleotide comprises a sequence represented by: (viral 5’R-U5)- (MMLV-psi)-(Viral gag)-(synthetic promoter)-(at least one transgene)-(WPRE domain)-(Viral 3’del-U3-R-U5). 4. The composition of claim 3, wherein the R-U5 domain can comprise a sequence that is at least 80-100% identical to the sequence: GCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCA GTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACC CGTCAGCGGGGGTCTTTCATTT (SEQ ID NO:30). 5. The composition of claim 3, wherein the MMLV-psi sequence comprises a sequence that is at least 85%-100% identical to: AAGCTGGCCAGCAACTTATCTGTGTCTGTCCGATTGTCTAGTGTCTATGACTGATTTTATGCGCCTGC GTCGGTACTAGTTAGCTAACTAGCTCTGTATCTGGCGGACCCGTGGTGGAACTGACGAGTTCGGAACA CCCGGCCGCAACCCTGGGAGACGTCCCAGGGACTTCGGGGGCCGTTTTTGTGGCCCGACCTGAGTCCA AAAATCCCGATCGTTTTGGACTCTTTGGTGCACCCCCCTTAGAGGAGGGATATGTGGTTCTGGTAGGA GACGAGAACCTAAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGGACCGAAGCCGCGC CGCGCGTCTTGTCTGCTG (SEQ ID NO:31) 6. The composition of claim 3, wherein the viral gag domains comprise a sequence that is at least 80-100% identical to the sequence: GGCCAGACTGTTACCACTCCCTGAAGTTTGACCTTAGGTCACTGGAAAGATGTCGAGCGGATCGCTCA CAACCAGTCGGTAGATGTCAAGAAGAGACGTTGGGTTACCTTCTGCTCTGCAGAATGGCCAACCTTTA ACGTCGGATGGCCGCGAGACGGCACCTTTAACCGAGACCTCATCACCCAGGTTAAGATCAAGGTCTTT TCACCTGGCCCGCATGGACACCCAGACCAGGTCCCCTACATCGTGACCTGGGAAGCCTTGGCTTTTGA CCCCCCTCCCTGGGTCAAGCCCTTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCCCGT Attorney Docket No.00156-005WO1 CTCTCCCCCTTGAACCTCCTCGTTCGACCCCGCCTCGATCCTCCCTTTATCCAGCCCTCACTCCTTCT CTAGGCGCC (SEQ ID NO:32). 7. The composition of claim 3, wherein the synthetic promoter comprises a sequence of SEQ ID NO:1, 12-25 or 26 or sequences that are at least 80-99% identical thereto and can drive transcription of a downstream sequence. 8. The composition of claim 3, wherein the recombinant polynucleotide is used for in vivo transduction and the synthetic promoter comprises SEQ ID NO:4, 6-10 or 11 or sequence that are at least 80-99% identical thereto and which can drive transcription of a downstream sequence. 9. The composition of claim 7 or 8, wherein a transgene is operably linked and downstream of the synthetic promoter. 10. The composition of claim 9, wherein the transgene comprises a non-naturally occurring immune receptor construct. 11. The composition of claim 10, wherein the non-naturally occurring immune receptor is a chimeric antigen receptor (CAR), chimeric TCR (cTCR), or CAR-like construct. 12. The composition of claim 3, wherein the WPRE sequence can comprise a sequence that is at least 80%-100% identical to: AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTAC GCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCT CCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGC GTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCT TTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCT GCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTT CCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGC CCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCC TTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGC (SEQ ID NO:33). 13. The composition of claim 3, wherein the viral 3’ delU3-R-U5 domain can comprise a sequence that is at least 80-100% identical to the sequence: GAGCTCAAAATAAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTAGGT TTGGCAAAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCAGTCCTCCGATTGACTGAGTCGCCCG GGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAG GGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTCTTTCATT (SEQ ID NO:34). Attorney Docket No.00156-005WO1 14. The composition of claim 3, wherein the viral miRNA target sequence domain can comprise a sequence that is at least 80-100% identical to the sequence: TGCTCAATAAATACCCGTTGAACGATTGCTCAATAAATACCCGTTGAAGCATTGCTCAATAAATACCC GTTGAACTACTGCTCAATAAATACCCGTTGAA (SEQ ID NO:35). 15. The composition of claim 3, wherein the viral miRNA target sequence domain can comprise a sequence that is at least 80-100% identical to the sequence: CATGATTGCCACGTCTGCAGTACGATCATGATTGCCACGTCTGCAGTAGCATCATGATTGCCACGTCT GCAGTACTACCATGATTGCCACGTCTGCAGTA (SEQ ID NO:36). 16. The composition of claim 3, wherein the viral miRNA target sequence domain can comprise a sequence that is at least 80-100% identical to the sequence: TGATTGCCACTGTCTGCAGTACGATTGATTGCCACTGTCTGCAGTAGCATTGATTGCCACTGTCTGCA GTACTACTGATTGCCACTGTCTGCAGTA (SEQ ID NO:37). 17. The composition of any one of claims 3-16, wherein in any sequence “T” is “U”. 18. A recombinant DNA plasmid or vector comprising a sequence represented by: (CMV promoter)-(viral 5’R-U5)-(MMLV-psi)-(Viral gag)-(synthetic promoter)-(at least one transgene)-(WPRE domain)- (Viral 3’del-U3-R-U5). 19. A viral particle comprising an RNA sequence expressed from the plasmid of claim 18. 20. A recombinant cell comprising the polynucleotide of any one of claim 1-17 or the plasmid of claim 18. 21. A method or use of a selective synthetic promoter to prevent expression of transgenes and other encoded nucleotides operably linked to the selective synthetic promoter in non-intended cell types during in vivo therapy. 22. The method or use of claim 21, wherein a therapeutic vector selected from the group consisting of gammaretrovirus-, lentivirus-, adenovirus-, adeno-associated virus-, herpesvirus-, human foamy Attorney Docket No.00156-005WO1 virus-based products, transposons, plasmid, or artificial chromosomes comprise the selective synthetic promoter and operably linked transgene. 23. A recombinant vector comprising a sequence represented by: (CMV promoter)-(viral 5’R-U5)-(MMLV-psi)-(Viral gag)-(synthetic promoter)-(at least one transgene)-(WPRE domain)-(Viral 3’del-U3-R- U5) or (CMV promoter)-(viral 5’R-U5)-(MMLV-psi)-(Viral gag)-(WPRE domain)-(at least one transgene)-(synthetic promoter)-(Viral 3’del- U3-R-U5). 24. The recombinant vector of claim 23, wherein the CMV promoter of comprises a sequence at least 85%-100% identical to: CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAA TAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTA CGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAA TGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGT ACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGA TAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCA CCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGC GTGTACGGTGGGAGGTCTATATAAGCAGAGCT (SEQ ID NO:28). 25. The recombinant vector of claim 23, wherein the R-U5 domain can comprise a sequence that is at least 80-100% identical to the sequence: GCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCA GTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACC CGTCAGCGGGGGTCTTTCATTT (SEQ ID NO:30) or the foregoing SEQ ID NO:30 wherein “T” can be “U”. 26. The recombinant vector of claim 23, wherein the MMLV-psi sequence comprises a sequence that is at least 85%-100% identical to: AAGCTGGCCAGCAACTTATCTGTGTCTGTCCGATTGTCTAGTGTCTATGACTGATTTTATGCGCCTGC GTCGGTACTAGTTAGCTAACTAGCTCTGTATCTGGCGGACCCGTGGTGGAACTGACGAGTTCGGAACA CCCGGCCGCAACCCTGGGAGACGTCCCAGGGACTTCGGGGGCCGTTTTTGTGGCCCGACCTGAGTCCA AAAATCCCGATCGTTTTGGACTCTTTGGTGCACCCCCCTTAGAGGAGGGATATGTGGTTCTGGTAGGA GACGAGAACCTAAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGGACCGAAGCCGCGC CGCGCGTCTTGTCTGCTG (SEQ ID NO:31) or the foregoing SEQ ID NO:31, wherein “T” can be “U”, 27. The recombinant vector of claim 23, wherein the viral gag domains comprise a sequence that is at least 80-100% identical to the sequence: Attorney Docket No.00156-005WO1 GGCCAGACTGTTACCACTCCCTGAAGTTTGACCTTAGGTCACTGGAAAGATGTCGAGCGGATCGCTCA CAACCAGTCGGTAGATGTCAAGAAGAGACGTTGGGTTACCTTCTGCTCTGCAGAATGGCCAACCTTTA ACGTCGGATGGCCGCGAGACGGCACCTTTAACCGAGACCTCATCACCCAGGTTAAGATCAAGGTCTTT TCACCTGGCCCGCATGGACACCCAGACCAGGTCCCCTACATCGTGACCTGGGAAGCCTTGGCTTTTGA CCCCCCTCCCTGGGTCAAGCCCTTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCCCGT CTCTCCCCCTTGAACCTCCTCGTTCGACCCCGCCTCGATCCTCCCTTTATCCAGCCCTCACTCCTTCT CTAGGCGCC (SEQ ID NO:32) or the foregoing SEQ ID NO:32, wherein “T” can be “U”. 28. The recombinant vector of claim 23, wherein the synthetic promoter comprises a sequence of SEQ ID NO:1, 12-25 or 26 or sequences that are at least 80-99% identical thereto or an RNA sequence thereof and can drive transcription of a downstream sequence. 29. The recombinant vector of claim 23, wherein the recombinant polynucleotide is used for in vivo transduction and the synthetic promoter comprises SEQ ID NO:4, 6-10 or 11 or sequence that are at least 80-99% identical thereto or an RNA sequence thereof and which can drive transcription of a downstream sequence. 30. The recombinant vector of claim 28 or 29, wherein a transgene is operably linked and downstream of the synthetic promoter. 31. The recombinant vector of claim 30, wherein the transgene comprises a non-naturally occurring immune receptor construct. 32. The recombinant vector of claim 31, wherein the non-naturally occurring immune receptor is a chimeric antigen receptor (CAR), chimeric TCR (cTCR), or CAR-like construct. 33. The recombinant vector of claim 23, wherein the WPRE sequence can comprise a sequence that is at least 80%-100% identical to: AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTAC GCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCT CCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGC GTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCT TTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCT GCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTT CCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGC CCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCC TTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGC (SEQ ID NO:33) or the foregoing SEQ ID NO:33, wherein “T” can be “U”. Attorney Docket No.00156-005WO1 34. The recombinant vector of claim 23, wherein the viral 3’ delU3-R-U5 domain can comprise a sequence that is at least 80-100% identical to the sequence: GAGCTCAAAATAAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTAGGT TTGGCAAAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCAGTCCTCCGATTGACTGAGTCGCCCG GGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAG GGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTCTTTCATT (SEQ ID NO:34) or the foregoing SEQ ID NO:34, wherein “T” can be “U”. 35. The recombinant vector of claim 23, wherein the viral miRNA target sequence domain can comprise a sequence that is at least 80- 100% identical to the sequence: TGCTCAATAAATACCCGTTGAACGATTGCTCAATAAATACCCGTTGAAGCATTGCTCAATAAATACCC GTTGAACTACTGCTCAATAAATACCCGTTGAA (SEQ ID NO:35) or the foregoing SEQ ID NO:35, wherein “T” can be “U”. 36. The recombinant vector of claim 23, wherein the viral miRNA target sequence domain can comprise a sequence that is at least 80- 100% identical to the sequence: CATGATTGCCACGTCTGCAGTACGATCATGATTGCCACGTCTGCAGTAGCATCATGATTGCCACGTCT GCAGTACTACCATGATTGCCACGTCTGCAGTA (SEQ ID NO:36) or the foregoing SEQ ID NO:36, wherein “T” can be “U”. 37. The recombinant vector of claim 23, wherein the viral miRNA target sequence domain can comprise a sequence that is at least 80- 100% identical to the sequence: TGATTGCCACTGTCTGCAGTACGATTGATTGCCACTGTCTGCAGTAGCATTGATTGCCACTGTCTGCA GTACTACTGATTGCCACTGTCTGCAGTA (SEQ ID NO:37) or the foregoing SEQ ID NO:37 wherein “T” can be “U”. 38. The recombinant vector of claim 23, wherein the recombinant vector comprises a plasmid. 39. A viral vector produced by expression of the plasmid of claim 38 in a host cell. 40. The viral vector of claim 39, wherein the host cell is a helper cell. Attorney Docket No.00156-005WO1 41. The viral vector of claim 39 or 40, wherein the viral vector is an RNA viral vector. 42. The viral vector of claim 41, wherein the viral vector comprises an engineered retroviral vector. 43. The viral vector of claim 42, wherein the viral vector is engineered from a gammaretroviral vector. 44. A method of in vivo transduction or transfection, the method comprising administering the composition of any one of claim 1-18, or the viral vector of any one of claim 39-43 to a subject, wherein the composition or viral vector transfects or transduces an immune cell in vivo.
PCT/US2024/030878 2023-05-23 2024-05-23 Vectors and methods for in vivo transduction Pending WO2024243460A1 (en)

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US20210171977A1 (en) * 2017-03-13 2021-06-10 Massachusetts Institute Of Technology Synthetic promoters
US20200095573A1 (en) * 2017-05-17 2020-03-26 Seattle Children's Hospital (dba Seattle Children's Research Institute) Generating mammalian t cell activation inducible synthetic promoters (syn+pro) to improve t cell therapy
CN116059405A (en) * 2022-09-26 2023-05-05 华中科技大学同济医学院附属协和医院 A synthetic gene network system with specificity and sensitivity for the treatment of malignant tumors

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