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WO2024129976A2 - Particle delivery systems - Google Patents

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WO2024129976A2
WO2024129976A2 PCT/US2023/084036 US2023084036W WO2024129976A2 WO 2024129976 A2 WO2024129976 A2 WO 2024129976A2 US 2023084036 W US2023084036 W US 2023084036W WO 2024129976 A2 WO2024129976 A2 WO 2024129976A2
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sequence
protein
seq
xdp
ligand
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WO2024129976A3 (en
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Gayathri VIJAYAKUMAR
Trent GOMBERG
Anthony Mauriello
Isabel COLIN
Sean Higgins
Hannah SPINNER
Brett T. STAAHL
Maroof ADIL
Benjamin OAKES
Jason Fernandes
Emeric Jean Marius CHARLES
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Scribe Therapeutics Inc
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Scribe Therapeutics Inc
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • 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
    • A61K48/0075—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 delivery route, e.g. oral, subcutaneous
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • C—CHEMISTRY; METALLURGY
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/22—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a Strep-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • DNA oligos with the sequence TGAAGCTGACAGCATTCGGGCCGAGATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGC GCT (non-target strand, NTS (SEQ ID NO: 968)) and AGCGCGAGCACAGCTAAGGCCACGGAGCGAGACATCTCGGCCCGAATGCTGTCAGC TTCA (target strand, TS (SEQ ID NO: 969)) were purchased with 5’ fluorescent labels (LI- COR TM IRDye® 700 and 800, respectively).
  • Cleavage-competent fractions were also determined using the same protocol for CasX2 protein in combination with guides 2, 32, 64 and 174 and targeting sequence 7.37 144 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 (CasX2.2.7.37, CasX2.32.7.37, CasX2.64.7.37), and CasX2.174.7.37 to be 16 ⁇ 3%, 13 ⁇ 3%, 5 ⁇ 2%, and 22 ⁇ 5%, as shown in FIG.2 and Table 10.
  • CasX RNPs were reconstituted with the indicated CasX protein (see FIG.4) at a final concentration of 1 ⁇ M with 1.5-fold excess of the indicated guide in 1 ⁇ cleavage buffer (20 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM TCEP, 5% glycerol, 10 mM MgCl2) at 37° C for 10 min before being moved to ice until ready to use.
  • Cleavage reactions were set up with a final RNP concentration of 200 nM and a final target concentration of 10 nM.
  • Reactions were carried out at 37° C except where otherwise noted and initiated by the addition of the target DNA. Aliquots were taken at 0.25, 0.5, 1, 2, 5, and 10 minutes and quenched by adding to 95% formamide, 20 mM EDTA. Samples were denatured by heating at 95° C for 10 minutes and run on a 10% urea- PAGE gel. The gels were imaged with a LI-COR Odyssey CLx and quantified using the LI- COR Image Studio software or imaged with a Cytiva Typhoon and quantified using the Cytiva IQTL software.
  • CasX variants had improved cleavage rates relative to the wild-type CasX2.
  • CasX 457 cleaved more slowly than 119, despite having a higher competent fraction as determined above.
  • CasX488 and CasX491 had the highest cleavage rates by a large margin; as the target was almost entirely cleaved in the first timepoint, the true cleavage rate exceeds the resolution of this assay, and the reported kcleave should be taken as a lower bound.
  • the data indicate that the CasX variants have a higher level of activity, with k cleave rates reaching at least 30-fold higher compared to wild-type CasX2. 5.
  • V 0 for CasX2 with guides 2, 32, 64, and 174 were 20.4 ⁇ 1.4 nM/min, 18.4 ⁇ 2.4 nM/min, 7.8 ⁇ 1.8 nM/min, and 49.3 ⁇ 1.4 nM/min (see Table 10 and FIGS.5and FIG.6).
  • Guide 174 showed substantial improvement in the cleavage rate of the resulting RNP ( ⁇ 2.5-fold relative to 2, see FIG.6), while guides 32 and 64 performed similar to or worse than guide 2.
  • guide 64 supports a cleavage rate lower than that of guide 2 but performs much better in vivo (data not shown).
  • both 515.174 and 526.174 cut essentially all of the target DNA by the first timepoint, exceeding the resolution of the assay and resulting in estimated cleavage rates of 17.10 and 19.87 min -1 , respectively (FIG.9, Table 10).
  • An RNP of 2.2 by contrast, cut on average less than 60% of the target DNA by the final 10-minute timepoint and has an estimated k cleave nearly two orders of magnitude lower than the engineered RNPs.
  • the modifications made to the protein and guide have resulted in RNPs that are more stable, more likely to form active particles, and cut DNA much more efficiently on a per-particle basis as well.
  • plasmid pXDP1 was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments containing CasX variant, HIV-1, or MS2 CP components were amplified and cloned using In-Fusion® primers with 15-20 base pair overlaps and KAPA HiFi DNA polymerase according to the manufacturer’s protocols.
  • the fragments were purified by gel extraction and cloned into plasmid backbones using In-Fusion® HD Cloning Kit from Takara (Cat# 639650) according to the manufacturer’s protocols. Assembled products were transformed into chemically-competent Turbo Competent E. coli bacterial cells, plated on LB-Agar plates containing ampicillin and incubated at 37 ⁇ C. Individual colonies were picked and miniprepped using QIAprep® Spin Miniprep Kit following the manufacturer’s The resultant plasmids were sequenced 148 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 using Sanger sequencing to ensure correct assembly.
  • the amino acid sequence of the MS2 CP is provided in SEQ ID NO: 4140, and the amino acid sequence of the Gag polyprotein fused to the MS2 CP is provided in SEQ ID NO: 4141.
  • Guide plasmid cloning [0440] The tdTomato targeting guide plasmids used in these experiments were pSG50 (guide scaffold 188; FIG.12) and pSG54 (guide scaffold 228; FIG.13), which were cloned from pSG33 and pSG34, respectively.
  • the mammalian expression backbone contained a cPPT, ampicillin resistance, and a colEI replication site and was amplified using primers with appropriate overlaps to accept the U6 promoter and guide RNA scaffold cassette.
  • the backbone, pSG3, was digested using NdeI and XbaI. Synthetic DNA fragments corresponding to scaffold variants were amplified and cloned as described, above.
  • the resultant plasmids, pSG33 and pSG34 were sequenced using Sanger sequencing to ensure correct assembly (Table 12).
  • Cloning tdTomato spacer 12.7 into pSG3 and pSG14 [0441] To clone the targeting pSG50 and pSG54 plasmids from the non-targeting pSG33 and pSG34, the spacer 12.7 was cloned using the following protocol.
  • the targeting spacer sequence DNA for the tdTomato targeting spacer 12.7 was ordered as single-stranded DNA (ssDNA) oligos (Integrated DNA Technologies) consisting of the targeting sequence (CTGCATTCTAGTTGTGGTTT, SEQ ID NO: 1018) and the reverse complement of this sequence. These two oligos were annealed together and cloned into a pSG plasmid with an alternate scaffold by Golden Gate assembly using T4 DNA Ligase (New England BioLabs Cat# M0202L) and Esp3I restriction enzyme from New England BioLabs (NEB Cat# R0734L). Golden Gate products were transformed into chemically competent NEB® Turbo competent E.
  • ssDNA single-stranded DNA
  • pGP2 Glycoprotein plasmid cloning [0442] Sequences encoding the VSV-G glycoprotein and the cytomegalovirus (CMV) promoter were amplified from pMD2.G and cloned as described for the structural plasmids, above. The backbone was taken from a kanamycin resistant plasmid and amplified and cloned using the same methods.
  • Plasmid ratios in Table 11 were used in all version 206 XDPs used in this assay, based on prior experimental data from other XDP versions.
  • Table 11 Plasmids and ratios used in XDP constructs XDP version 206 Structural plasmid plasmids ratios Gag-(-1)-PR* 10% Gag-MS2* 45% CasX* 45% *transcript contains RRE and produces REV Collection and concentration [0444] Media was aspirated from the plates 24 hours post-transfection and replaced with Opti- MEMTM (Thermo Fisher). XDP-containing media was collected 72 hours post-transfection and filtered through a 0.45 ⁇ m PES filter. The supernatant was concentrated and purified via centrifugation.
  • Pellets were either resuspended in Storage Buffer (PBS + 113 mM NaCl, 15% Trehalose dihydrate, pH 8 or an appropriate media by gentle trituration and vortexing.
  • XDPs were resuspended in 300 ⁇ L of DMEM/ F12 supplemented with GlutaMAXTM, HEPES, non-essential amino acids, Pen/Strep, 2-mercaptoethanol, B-27 without vitamin A, and N2.
  • Resuspension and transduction [0446] tdTomato neural progenitor cells (NPCs) were resuspended and transduced with XDPs.
  • tdTomato NPCs were grown in DMEM/F12 supplemented with GlutaMAXTM, HEPES, NEAA, Pen/Strep, 2-mercaptoethanol, B-27 without vitamin A, and N2.
  • Cells were harvested using StemPro Accutase Cell Dissociation Reagent and seeded on PLF-coated 96-well plates.48 hours later, cells were transduced with XDPs containing a tdTomato targeting spacer. Cells were then centrifuged for 15 minutes at 1000 x g.
  • Transduced NPCs were grown for 96 hours before analyzing tdTomato fluorescence by flow cytometry as a marker of editing at the tdTomato locus, with the EC50 determined as the number of XDP particles needed to achieve editing in 50% of the cells, as determined by flow cytometry.
  • tdTomato fluorescence was measured using flow cytometry. The assays were run 2-3 times for each sample, with similar results. Results: [0447]
  • the MS2 bacteriophage relies on the non-covalent affinity between its genomic RNA and the MS2 coat protein for the packaging of its genome in an icosahedral viral shell.
  • the high- affinity element in the RNA genome is termed the MS2 hairpin, which binds to the coat protein with a Kd of approximately 3e -9 .
  • two high affinity variants of the MS2 hairpin were incorporated into the extended stem of the guide scaffold 174, thereby introducing into the CasX:guide RNP an affinity for the MS2 coat protein.
  • the resulting guide scaffolds 188 and 228 were tested in XDP version 168; a version that relies on a Gag-CasX fusion configuration and lacks the MS2 coat protein, while version 206 (FIG.11) has the incorporated MS2 coat protein fused to Gag.
  • MS2 hairpin variant sequences of these scaffolds are ACATGAGGATCACCCATGT (SEQ ID NO: 1131) and CGTACACCATCAGGGTACG (SEQ ID NO: 1132), respectively.
  • MS2-based recruitment of these variant scaffolds was tested in XDP version 206. This version is composed of the Gag-(-1)-PR, Gag-MS2, and CasX architectures. This version relies 151 295377244 Attorney Docket No.
  • SCRB-050/01WO 333322-2386 on orthogonal recruitment of CasX via the MS2 coat protein and MS2 hairpin system of the guide rather than a direct fusion between CasX and a recruiting protein. This is demonstrated in FIG.15, where constructs with both guide scaffold 188 and 228 edit well in the tdTomato assay, in contrast to constructs with guide scaffold 174, which lacks the MS2 hairpin and edits poorly. Additionally, XDP version 206 with scaffold 188 edits better at the same dosage over XDP version 168 with scaffold 174 (see FIGS.14 and 15). At 0.6 ⁇ L of XDPs delivered, editing was ⁇ 70% with XDP version 206 with guide scaffold 188.
  • ⁇ 20% editing was achieved at the same treatment volume for XDP version 168 (a Gag-CasX fusion) with guide scaffold 174 and version 206 with guide scaffold 228.
  • XDP version 206 with guide scaffold 188 is 2-3x more potent than version 168 with guide scaffold 174.
  • This increase in editing from version 168 to version 206 could be attributed to the lack of a direct fusion of Gag to CasX, causing less steric hindrance in particle formation.
  • the similarity between guide scaffolds 188 and 228 in editing in version 168 suggests that the difference in potency in XDP version 206 is due to the MS2 hairpin’s affinity for the coat protein linked to Gag.
  • the CasX protein and guide scaffold RNA form the apoenzyme RNP in the cytoplasm of the producer cell that then binds the Gag-MS2 protein by interactions of the MS2 hairpin in the guide extended stem and the MS2 coat protein.
  • the second possible mechanism is that the guide scaffold RNA hairpin first binds the MS2 coat protein and then forms the apoenzyme with the CasX protein.
  • MS2 coat protein variants have several point mutations that alter their affinity to its hairpin RNA. Usage of these variants in version 206 could result in higher potency variants. Fusing multiple coat proteins to the HIV Gag protein could further increase potency as well.
  • NCR non-covalent recruitment
  • RNA combinations from humans and retroviruses include the Iron Responsive Element (IRE)-Iron Binding element, U1 hairpin II, retrovirus Tat-trans-activation response (TAR) system, Csy4, Pardaxin, tRNA or Psi-Nucleocapsid. 152 295377244 Attorney Docket No.
  • IRE Iron Responsive Element
  • TAR retrovirus Tat-trans-activation response
  • Csy4 Pardaxin
  • tRNA or Psi-Nucleocapsid 152 295377244 Attorney Docket No.
  • the MS2 packaging system consists of two major components: the phage coat protein and its cognate binding partner, which is a short hairpin stem loop structure.
  • the short hairpin stem loop structure is engineered into the sgRNA incorporated into the XDP.
  • the encoding sequence for the phage coat protein is fused to either the encoding sequence for the Gag polyprotein (derived from any retroviruses) or to any other protein domains derived from the Gag polyprotein of any retroviral origin.
  • RNA fold structures were generated with RNAfold web server and Varna java-based software.
  • pXDP17, pXDP161, pXDP164 and pXDP166 was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments containing CasX variant, HIV-1, or MS2 CP components were amplified and cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
  • MS2 was placed either on the N- or the C-terminus of the Capsid (Version 263- pXDP276, Version 264-pXDP277, Version 265-pXDP278 and Version 266-pXDP279), with and without cleavage sites. MS2 was placed either on the N- or the C-terminal of the Nucleocapsid (Version 267-pXDP280, Version 268-pXDP281, Version 269-pXDP282 and Version 270-pXDP283), with and without cleavage sites. The sequences for these constructs are provided in Table 16.
  • the designed constructs were synthesized as transgenes and purchased pre-cloned into pTWIST expression plasmids from Twist Bioscience®. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
  • Guide plasmid cloning [0455] The guide plasmids used in these experiments were pSG50 and pSG17, encoding guide scaffold 188. Spacer 12.7 was cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation.
  • the guide plasmid used in all MS2 constructs is pSG50.
  • the guide plasmid used in control construct (V168) is pSG17.
  • pGP2 Glycoprotein plasmid cloning Sequences encoding the VSV-G glycoprotein and the CMV promoter were amplified from pMD2.G and cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Cell culture and transfection; collection and concentration; resuspension and transduction [0457] HEK293T Lenti-XTM cell culture was performed as described in Example 2, above. For transfection, the XDP structural plasmids of Table 16 were used in amounts ranging from 13 to 80.0 ⁇ g. Each transfection also received 13 ⁇ g of pSG50 or pSG17 and 0.25 ⁇ g of pGP2.
  • Table 15 presents the results of percent editing of the dtTomato target sequence when 16.6 ⁇ l of the concentrated XDP prep was used to treat NPCs.
  • the results show that it is feasible to fuse MS2 with or without a cleavage sequence to either the capsid or the nucleocapsid.
  • the results indicate that fusing MS2 to the C-terminal of the capsid results in more potent XDP as compared to a fusion to the N-terminal.
  • introduction of a cleavage site in between MS2 and CA on the C-terminal does improve potency as shown in FIG.52.
  • FIG.16 depicts the fold improvement in EC50 over the base control V168 (CasX fused to full length HIV Gag- polyprotein) and it shows that V265, V269 and V270 show about 5 to 8-fold improvement in potency.
  • FIG.17 depicts the fold improvement in EC50 over the base control V206 (MS2 fused to full length HIV Gag-polyprotein and the results demonstrate that V265, V269 and V270 show about 6- to 9-fold improvement in terms of overall editing potency.
  • Table 15 Percent editing at the second dilution (16.6 ⁇ l) XDP version Plasmid number Encoded Configuration** % Editing 168 pXDP17 MA*-CA*-NC*-P1*-P6*-CasX 92.6 206 pXDP164 MA*-CA*-NC*-P1*-P6-MS2 92.3 263 pXDP276 MA*-CA-MS2*-NC*-P1*-P6 85.5 264 pXDP277 MA*-MS2-CA*-NC*-P1*-P6 9.8 265 pXDP278 MA*-CA* -MS2*-NC*-P1*-P6 75.6 266 pXDP279 MA*-MS
  • this enhanced architecture can be translated to proteins derived from the Gag polyproteins of Alpharetroviral, Betaretroviral, Gammaretroviral, Deltaretroviral, Epsilonretroviral, Lentiviral and Spumaretroviral origin, serving as an orthogonal recruitment mechanism for CasX or any other payload that can be coupled with a cognate short hairpin RNA element in an XDP or other particle-delivery system.
  • Table 16 Plasmid sequences XDP version Plasmid number SEQ ID NO of Encoding Sequence pGP2 979 pSG50 1143 pXDP161 1139 168 pXDP17 1144 pXDP166 1142 206 pXDP164 1140 301 pXPD276 1145 302 pXPD277 1146 303 pXPD278 1147 304 pXPD279 1148 305 pXPD280 1149 306 pXPD281 1150 307 pXPD282 1151 308 pXPD283 1152 pSG17 1153
  • Example 4 Non-covalent recruitment with RNA binding - Retro-MS2 [0460] The purpose of these experiments was to demonstrate the utility of a recruitment method for the incorporation of RNP into XDP an MS2-based system and Gag 157 295377244 Attorney Docket No.
  • the guide RNA spacer used in all of these experiments was 12.7 targeting the tdTomato locus.
  • the guide scaffold used in all the MS2 constructs was 188, along with spacer 12.7.
  • RNA fold structures were generated with RNAfold web server and Varna java- based software.
  • Structural plasmid cloning MS2 was fused to the Gag-protease, Gag or partial Gag polyproteins derived from Alpharetroviruses (Versions 271, 272, 273), Betaretroviruses (Versions 277, 279), Gammaretroviruses (Versions 276, 278), Deltaretroviruses (Versions 274, 275) and Lentiviruses (Versions 280, 281, 282) with their respective species-specific cleavage sites. The sequences for these constructs are provided in Table 18.
  • the designed constructs were synthesized as transgenes and purchased pre-cloned into pTWIST expression plasmids from Twist Biosciences. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Guide plasmid cloning [0463] The guide plasmid used in these experiments was pSG50. To clone the targeting pSG50 spacer 12.7 was cloned in as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation.
  • pGP2 Glycoprotein plasmid cloning [0464] Sequences encoding the VSV-G glycoprotein and the CMV promoter were cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
  • Cell culture and transfection [0465] HEK293T Lenti-XTM cell culture was performed as described in Example 2, above. For transfection, the XDP structural plasmids of Table 18 were used in amounts ranging from 13 to 80.0 ⁇ g. Each transfection also received 13 ⁇ g of p42.174.12.7 and 0.25 ⁇ g of pGP2. Polyethylenimine (PEI MAX® from Polyplus) was then added as described in Example 2.
  • Table 17 represents the percent editing of the dtTomato target sequence when 16.6 ⁇ l of the concentrated XDP prep was used to treat NPCs.
  • V275 derived from HTLV1 (Deltaretroviruses)
  • V279 derived from MPMV (Betaretroviruses)
  • V281 derived from EIAV (Lentivirus) showed successful editing ranging from 76.5, 61.6, to 48.7% at the tdT locus, respectively.
  • V273 derived from RSV, Alpharetroviruses
  • V274 derived from BLV, Deltaretroviruses
  • V276 derived from FLV, Gammaretroviruses
  • V277 derived from MMTV, Betretroviruses
  • V278 derived from MMLV, Gammaretroviruses
  • V280 derived from EIAV, Lentivirus
  • V282 derived from SIV, Lentivirus
  • V280 has MS2 fused to Gag-pro polyprotein
  • V281 has MS2 fused to the MA-CA polyprotein.
  • Table 17 Percent editing at the second dilution (16.6 ⁇ l) XDP version Plasmid Genus/or Virus with n umber der Virus c onfiguration % Editing 206 pXDP164 Lentivirus HIV HIV Gag-MS2 95.1 271 pXDP354 Alpharetrovirus ALV ALV Gag-pro-MS2 88.7 159 295377244 Attorney Docket No.
  • Table 18 Plasmid sequences XDP Plasmid version number SEQ ID NO of DNA Encoding Sequence pGP2 979 pSG50 1143 pXDP166 1142 206 pXDP161 1139 206 pXDP164 1140 271 pXDP354 1155 272 pXDP355 1156 160 295377244 Attorney Docket No.
  • All plasmids encoding CasX proteins utilized the CasX 491 variant protein. All XDPs contained sgRNAs with scaffold 188 (see FIG.12) and spacer 12.7. Structural plasmid cloning [0472] In order to generate the structural plasmids, listed below, pXDP1was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments encoding CasX variant, HIV-1, or MS2 CP components were amplified and cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
  • the tdTomato targeting guide plasmid used in these experiments was pSG50 (guide scaffold 188), which was cloned from pSG33.
  • the mammalian expression backbone contained a cPPT, ampicillin resistance, and a colEI replication site and was amplified using primers with appropriate overlaps to accept the U6 promoter and guide RNA scaffold cassette.
  • the backbone, pSG3, was digested using NdeI and XbaI. Synthetic DNA fragments corresponding to novel 161 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 scaffolds were amplified and cloned as described in Example 2, above.
  • the resultant plasmid, pSG33 was sequenced using Sanger sequencing to ensure correct assembly.
  • Cloning tdTomato spacer 12.7 [0474] To clone the targeting pSG50 plasmid from the non-targeting pSG33, spacer 12.7 was cloned as described in Example 2, above. The resultant plasmid was sequenced using Sanger sequencing to ensure correct ligation (see Table 20).
  • pGP2 Glycoprotein plasmid cloning [0475] Sequences encoding the VSV-G glycoprotein and the CMV promoter were amplified from pMD2.G and cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
  • Table 23 lists the plasmid structural and glycoprotein plasmid components.
  • Cell culture and transfection [0476] HEK293T Lenti-XTM cell culture was performed as described in Example 2, above.
  • the XDP structural plasmids (also encoding the CasX variants) of Table 21 were used in amounts ranging from 13 to 80.0 ⁇ g. Each transfection also received 13 ⁇ g of pSG50 and 0.25 ⁇ g of pGP2.
  • Polyethylenimine (PEI MAX® from Polyplus) was then added as described in Example 2. Plasmid ratios in Table 19 were used in all Version 206 XDPs used in this assay and are based on prior data from other XDP versions.
  • Table 19 Construct plasmids and ratios of plasmids used XDP version 206 plasmids Structural plasmid ratios Gag-(-1)-PR* 10% Gag-MS2* 45% CasX* 45% *transcript contains RRE and produces REV Collection and concentration; resuspension and transduction [0477] XDPs were collected and concentrated as described in Example 2, above. tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above, and tdTomato fluorescence was measured using flow cytometry. The assays were run 2-3 times for each sample, with similar results. 162 295377244 Attorney Docket No.
  • Table 22 MS2 variants Plasmid M utation i Inverse EC50 n umber n MS2 Affinity by volume pXDP164 WT WT 1.6 pXDP321 V29I K d : 4e-10 M 1.5 pXDP335 K43R K d : 1e-9 M 2.7 pXDP336 K66R K d : 3e-9 M 4.7 pXDP337 N55R unknown 2.2 pXDP338 N87S K d : 6.3e-8 M 0.3 pXDP339 T59A K d : 1.2e-7 M 0.4 pXDP340 V68_V80 dInc Dimerization incompetent 2.2 Table 23: XDP Version and pseudotyping descriptions X DP version Architectures and g lycoproteins Plasmid numbers Rev expression 206 Gag-(-1)-PR pXDP161 Yes Gag-MS2 pXDP164 CasX pXDP166 VSV-G p
  • pXDP1 was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments containing CasX, HIV-1, retrovirus Tat, IRP1, IRP2, truncated U1A, U1A, phage Q ⁇ coat protein, phage GA coat protein, phage ⁇ N coat protein, or truncated phage ⁇ N coat protein components were amplified using In Fusion primers with 15-20 base pair overlaps and Kapa HiFi DNA polymerase according to the manufacturer’s protocols.
  • fragments were purified by gel extraction from a 1% agarose gel using Zymoclean Gel DNA Recovery Kit according to the manufacturer’s protocol.
  • fragments containing dual boxB hairpin, retrovirus transactivation response (TAR) element, Iron Responsive Element (IRE), U1A hairpin, phage Q ⁇ hairpin, phage GA hairpin, phage ⁇ N hairpin, or phage PP7 hairpins were amplified and cloned in guide scaffolds based on guide scaffold 174 or guide scaffold 235. Sequences of guide RNA scaffolds with dual hairpins are provided in Table 26, below. Scaffolds 188 and 251 were used as controls.
  • SCRB-050/01WO 333322-2386 cPPT ampicillin resistance
  • a colEI replication site was amplified using primers with appropriate overlaps to accept the U6 promoter and guide RNA scaffold cassette.
  • Fragments containing the retrovirus TAR, Iron Responsive Element (IRE), U1A hairpin II, phage Q ⁇ hairpin, phage GA hairpin, phage ⁇ N hairpin (also referred to herein as a boxB hairpin or boxB element), or phage PP7 hairpin were amplified and cloned as described in Example 2, above.
  • the targeting spacer sequence DNA for the tdTomato targeting spacer 12.7 was cloned into pSG33 and pSG34 as described in Example 2, above.
  • the resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation.
  • pGP2 Glycoprotein plasmid cloning [0486] Sequences encoding the VSV-G glycoprotein and the CMV promoter were amplified and cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
  • Cell culture and transfection [0487] HEK293T Lenti-XTM cell culture was performed as described in Example 2, above.
  • the XDP structural plasmids (also encoding the CasX variants) of Table 24 were used in amounts ranging from 13 to 80.0 ⁇ g. Each transfection will also receive 13 ⁇ g of a pSG plasmid and 0.25 ⁇ g of pGP2.
  • Polyethylenimine PEI MAX® from Polyplus
  • Collection and concentration; resuspension and transduction; titering [0488] XDPs were collected and concentrated as described in Example 2, above.
  • tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above, and tdTomato fluorescence was measured using flow cytometry.
  • the assays were run 2-3 times for each sample with similar results. Titers were quantified for each version of XDPs using the NanoSight NS300. Results: [0489]
  • the CasX guide scaffold extended stem region is highly modifiable. The extended stem loop protrudes out from the RNP, and so additions to this region have little effect on RNP formation and editing potency, as seen in other experiments described herein. This feature was used to add on one or two of several different RNA hairpins to the extended stem loop to engineer the CasX gRNA to bind their corresponding RNA-binding proteins.
  • Table 24 shows the sequences of the Gag-NCR protein plasmids and their complementary sgRNAs with non- targeting spacers that were employed to create the versions.
  • Table 25 shows the amino acid and 166 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 RNA sequences of the Gag-NCR proteins and their complementary sgRNAs, respectively.
  • the amino acid sequences of the NCR proteins (not fused to the Gag polyprotein) are provided in SEQ ID NOs: 4130-4139. [0490] It was expected that inclusion of these NCR proteins into the constructs would likely yield more potent XDP configurations as it has previously been demonstrated that different K d s of NCR proteins, such as MS2, can modify the potency of XDPs. There is a large variety of K d s and sizes across these NCR proteins.
  • XDPs with the MS2, PP7, Tat, or U1A NCR systems produced the highest levels of editing in the mouse tdTomato NPCs. Indeed, XDPs with the PP7, Tat or U1A NCR systems produced higher levels of editing than XDPs with the MS2 NCR system. Both Tat and U1A NCR systems are monomeric in nature. Therefore, that both Tat and U1A NCR systems produced higher levels of editing suggests that MS2 dimerization has a detrimental effect on XDP architecture formation. It is anticipated that the relatively small size of the Tat protein could make it amenable to stacking (i.e., adding multiple Tat binding sites), which could enable better recruitment and packaging of the CasX RNP.
  • RNA hairpin and the NCR protein have a higher binding affinity (Kd of 1 nM) compared to that of the MS2 system (of Kd of ⁇ 2.6 nM). This may explain the higher level of editing observed with the PP7 system compared to the MS2 system (FIGS.20 and 21).
  • Titers were quantified for each version of XDP particles produced using the NanoSight NS300, and the number of transduced mouse NPCs was counted.
  • the bar chart in FIG.22 shows the number of XDPs containing the indicated NCR systems per edited mouse NPC
  • the bar chart in FIG.23 shows the average number of XDPs containing the indicated NCR systems per mouse NPC.
  • Table 26 shows the sequences of guide scaffolds based on guide scaffold 174 or scaffold 235, with two copies of each of the indicated hairpins.
  • the guide scaffolds in Table 26 were tested in combination with the NCR proteins provided in Table 25.
  • Table 28 Construct plasmids and ratios of plasmids used XDP version 206 XDP version 309 Structural plasmid plasmids plasmids ratios Gag-(-1)-PR* Gag-(-1)-PR* pXDP161 pXDP161 10% Gag-MS2* Gag-MS2-MS2* pXDP164 pXDP288 45% CasX* CasX* pXDP166 pXDP166 45% *transcript contains RRE and produces REV Table 29: XDP plasmids for evaluation NLS effects X DP version Architectures and g lycoproteins Plasmid numbers Gag-(-1)-PR pXDP161 V206 Gag-MS2 pXDP164 CasX pXDP166 VSV-G pGP2 Gag-(-1)-PR pXDP161 V206 NLS 240 Gag-MS2 pXDP164 CasX with NLS 240
  • tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above, and tdTomato fluorescence was measured using flow cytometry. tdTomato fluorescence was measured using flow cytometry. The assays were run 2-3 times for each sample, with similar results. Results: [0513] XDP version 309 is identical to version 206 except there is an additional MS2 CP fused to the first MS2 in this system, so pXDP164 (which encodes Gag-MS2) is replaced with pXDP288, which encodes Gag-MS2-MS2.
  • Example 8 Evaluation of non-covalent recruitment (NCR) systems with dual MS2 hairpins for MS2 coat protein binding [0514]
  • the ur ose of these ex eriments was to determine if the incor oration of two MS2 Table 31: sgRNA encoding sequences Plasmid Scaffold Spacer Encoding gui SEQ ID Encoding hairpin SEQ ID n umbers de sequence NO sequence NO ACTGGCGCTTTTATCTGATTACTT CAGCGTCAATGACG pSG67 250 NT TGAGAGCCATCACCAGCGACTATG 1200 CTGACGGTACAGGC 1204 CACATGAGGATCAC 184 295377244 Attorney Docket No.
  • pGP2 Glycoprotein plasmid cloning [0519] Sequences encoding the VSV-G glycoprotein and the CMV promoter were amplified from pMD2.G and cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
  • Cell culture and transfection [0520] HEK293T Lenti-XTM cell culture was performed as described in Example 2, above. For transfection, the XDP structural plasmids (also encoding the CasX variants) of Table 33 were used in amounts ranging from 13 to 80.0 ⁇ g.
  • Table 32 Plasmids and ratios used XDP version 206 plasmids Structural plasmid ratios Gag-(-1)-PR 10% Gag-MS2 45% CasX 45%
  • Table 33 Plasmid architecture and glycoprotein sequences Plasmid n umbers Architecture SEQ ID NO of Encoding sequence pGP2 VSV-G 979 pXDP161 Gag-(-1)-PR 1139 pXDP164 Gag-MS2 1140 pXDP166 SV40NLS-CasX491-SV40 NLS 1142 AAV122_Cmyc_NLS- pXDP344 BPSV40_NLS_(GGGS)2_PG-CasX- 1206 SV40 pXDP350 AAV119-CasX-AAV129 1207
  • Table 34 Version and pseudotyping descriptions XDP version Architectures and glycoproteins Plasmid numbers Gag-(-1)-PR pXDP161 206 Gag-MS2 pX
  • SCRB-050/01WO 333322-2386 1. 194 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 lentiviral and Alpharetroviral constructs bearing the glycoprotein variants. 195 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 [0549]
  • pXDP1 was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments containing CasX ALV and HIV-1 components were amplified and cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
  • Plasmids and their sequences are listed in Table 42.
  • Table 42 Plasmid sequences for structural plasmids and glycoproteins X DP version number/ Viral source Plasmid n umber SEQ ID NO 168 pXDP161 1139 168 pXDP17 1144 - pSG17 1153 - pSG005 1253 44 pXDP40 1254 102 pXDP145 1255 pGP2 979 H5N1 pGP80 1256 H7N9 pGP81 1257 Eastern equine encephalitis virus (EEEV) pGP65 1258 196 295377244 Attorney Docket No.
  • SCRB-050/01WO 333322-2386 Guide plasmid cloning [0550]
  • the guide plasmids used in these experiments were either pSG005 or pSG17.
  • pSG17 has both the spacer 12.7 targeting tdTomato as well as the guide scaffold 226 that has the RRE/RBE element that has been described in previous examples.
  • pSG005 has guide scaffold 174 along with the spacer 12.7 targeting tdTomato.
  • spacer 12.7 was cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation.
  • pGP Glycoprotein plasmid cloning Encoding sequences for glycoproteins derived from Togaviridae, Paramyxoviridae, Rhabdoviridae, Orthomyxoviridae, Retroviridae and Flaviviridae are provided in Table 42. The designed constructs were synthesized as transgenes and purchased pre-cloned into pTWIST expression plasmids from Twist Bioscience. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly (see Table 42). Cell culture and transfection [0552] HEK293T Lenti-XTM cell culture was performed as described in Example 2, above.
  • XDPs derived from HIV lentiviral-based architecture were pseudotyped with GPs from Togaviridae (pGP65, 66, 67, 68, 69 and 70), Rhabdoviridae (pGP29.7, 30) and Moloney Murine leukemia virus (pGP10).
  • XDPs derived from two different alpha retroviral-based architectures (ALV V44 and ALV V102) were pseudotyped with GPs from Rhabdoviridae (pGP29.7).
  • the XDP structural plasmids (configurations are listed in Table 42) were used in amounts ranging from 13 to 80.0 ⁇ g.
  • Each transfection also received 13 ⁇ g of either pSG005 or pSG17 and 2.5 ⁇ g of pGP2 or any other GPs.
  • Polyethylenimine PEI MAX® from Polyplus
  • PEI MAX® Polyethylenimine
  • XDPs were collected and concentrated as described in Example 2, above.
  • tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above, and tdTomato fluorescence was measured using flow cytometry. The assays were run 2-3 times for each sample with similar results.
  • V168 XDPs pseudotyped with the different GPs in terms of number of particles added to the tdTomato NPCs, are shown in FIG.41.
  • V168 pseudotyped with pGP2 served as the base control XDP for comparisons.
  • the results show that GPs derived from Togaviridae (in particular Semliki, WEEV, EEEV, VEEV) and Rhabdoviridae (Mokola and Rabies), as well as MoMLV are potent in NPCs, suggesting properties of neural tropism.
  • GPs derived from Togaviridae such as pGP68, pGP68, pGP66 and pGP65 seemed particularly potent (in that order) ranging in editing efficiencies from 74% to 36% when 50 ⁇ l of concentrated XDPs were used to treat NPCs. They also show that both architectural versions of ALV derived XDPs (V44 and V102) can be pseudotyped with GPs derived from Rhabdoviridae (pGP 29.7), ranging in editing efficacies from 7% to 27% when 50 ⁇ l of concentrated XDPs were used to treat NPCs, in addition to VSV-G, where they show efficacies ranging from 39% to 30% as shown in FIG.40.
  • V168 XDPs Titers for the V168 XDPs were determined by P24 ELISA, as shown in FIG.42, and they demonstrate that XDPs can be produced that are pseudotyped with the different glycoproteins without affecting overall titer.
  • the difference in potency that is seen in tdT NPCs is most likely due to inherent differences in cellular and tissue tropism between these glycoproteins.
  • the difference in editing profiles of ALV V44 and ALV102 pseudotyped with Rabies (pGP29.7) also highlights the possibility of the XDP internal architecture having an independent effect on the packaging of the targeting moiety on the surface of these particles.
  • GPs such as pGP70 and pGP69 as compared to other Togaviridae GPs might be due to incompatibility with the internal architecture, in addition to inherent differences in tropism. Therefore, these GPs might show potency with other architectural variants of HIV based XDPs, in addition to XDPs derived from other architectural variants of Alpharetroviral, Betaretroviral, Gammaretroviral, Deltaretroviral, Epsilonretroviral, Lentiviral and Spumaretroviral origin.
  • XDPs derived from HIV lentiviral-based architecture were pseudotyped with GPs from different rabies variants from the Rhabdoviridae family (pGP29, 29.2, 29.3, 29.4, 29.5, 29.6, 29.8). V168 pseudotyped with pGP2 served as the base control XDP for comparisons.
  • Several rabies variants showed potency in mouse NPCs, with pGP29 and pGP29.4 showing particular promise with editing efficiencies at the tdTomato locus ranging from 70% to 25% when 16.6 ⁇ l of the concentrated XDPs were used to treat NPCs, as shown in FIG.43 and 200 295377244 Attorney Docket No.
  • FIG.44 V168 pseudotyped with pGP2 demonstrated the most efficacy at 85%.
  • the rabies variants pGP29 and pGP29.4 would allow specific targeting of cells of neuronal origin, suggesting a better safety profile in vivo for neural indications, thereby making up for their lower editing potencies relative to VSV-G (pGP2).
  • XDPs derived from HIV lentiviral-based architecture were pseudotyped with GPs from Paramyxoviridae (pGP35.1, 35.2, 34.1, 34.2), Orthomyxoviridae (pGP80, 81, 82) and Flaviviridae (pGP25, 26, 27, 28, 75) families. Almost all the GPs showed activity at the 50 ⁇ l dose, as shown in FIG.45.
  • XDPs pseudotyped with Orthomyxoviridae (pGP80, 82) and Paramyxoviridae (pGP35.1, 35.2, 34.1, 34.2) demonstrated about 35%, 11% and 10% editing, respectively, as shown in FIG.46.
  • Titers for the V168 XDPs were determined by P24 ELISA as shown in FIG.47 and demonstrate that pseudotyping XPDs with the different glycoproteins didn’t affect production titers.
  • V168 XDPs have been successfully pseudotyped with these diverse glycoproteins
  • Example 12 Enhancing RNA export mechanisms for the formation of XDP using a Rev/RRE system - Scaffold 174 vs 226
  • the purpose of these experiments was to evaluate the effects of incorporation of a portion of an HIV-1 Rev response element (RRE) sequence into the guide RNA scaffold to determine whether RNA export, recruitment of the guide into XDP, and resultant potency of the XDP was enhanced, with and without a direct Gag-CasX fusion.
  • the HIV-1 RRE is a ⁇ 350 nucleotide RNA element in the HIV-1 genome that is recognized by the HIV-1 Rev protein and 201 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 is essential for HIV-1 replication.
  • XDPs were pseudotyped with 10% VSV-G (percentage of plasmid relative to the other plasmids utilized for the XDP construct).
  • RNA fold structures were generated with RNAfold web server and Varna java-based software.
  • Structural plasmid cloning [0561] In order to generate the structural plasmids used to make the XDP, pXDP1 was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments containing CasX or HIV-1 Gag components were amplified and cloned as described in Example 2, above. The sequence for Rev was incorporated into the backbone of the Gag plasmid. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
  • the tdTomato and PTBP-1 targeting guide plasmids used in these experiments were pSG5, pSG17, pSG47, and pSG48 cloned from pSG3 for the first and pSG14 for the latter 3 plasmids.
  • the mammalian expression backbone contained a cPPT, ampicillin resistance, and a colEI replication site and was amplified using primers with appropriate overlaps to accept the U6 promoter and guide RNA scaffold cassette.
  • the backbone was digested using NdeI and XbaI. Synthetic DNA fragments corresponding to novel scaffolds were amplified and cloned as described in Example 2, above.
  • the targeting spacer sequence DNA for the tdTomato targeting spacer 12.7 was ordered as single-stranded DNA (ssDNA) oligos (Integrated DNA Technologies) consisting of the targeting sequence (CTGCATTCTAGTTGTGGTTT, SEQ ID NO: 1018) and the reverse complement of this sequence.
  • the targeting spacer sequence DNA for the tdTomato targeting spacer 12.2 was ordered as single-stranded DNA (ssDNA) oligos (Integrated DNA Technologies) consisting of the targeting sequence (TATAGCATACATTATACGAA, SEQ ID NO: 1541) and the reverse complement of this sequence.
  • the targeting spacer sequence DNA for the PTBP-1 targeting spacer 28.10 was ordered as single-stranded DNA (ssDNA) oligos (Integrated DNA Technologies) consisting of the targeting sequence (CAGCGGGGATCCGACGAGCT, SEQ ID 203 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 NO: 1542) and the reverse complement of this sequence.
  • ssDNA single-stranded DNA
  • pGP2 Glycoprotein plasmid cloning [0564] Sequences encoding the VSV-G glycoprotein and the CMV promoter were cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
  • Cell culture and transfection [0565] HEK293T Lenti-XTM cell culture was performed as described in Example 2, above. For transfection, the XDP structural plasmids (also encoding the CasX variants) of Table 44 were used in amounts ranging from 13 to 80.0 ⁇ g. Each transfection also received 13 ⁇ g of p42.174.12.7 and 0.25 ⁇ g of pGP2.
  • tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above. Editing of tdTomato was assessed by measuring fluorescence or by Next Generation Sequencing to assess rate of edits. The assays were run 2-3 times for each sample with similar results. Results: [0568] The RRE binds strongest to Rev at Stem II (circled in FIG.48), therefore, this region was incorporated into scaffold 174 (FIG.49), resulting in scaffold 226, depicted in FIG.50.
  • Guide scaffold 226 was evaluated using three different spacer sequences; 12.7 (CTGCATTCTAGTTGTGGTTT, SEQ ID NO: 1018), 12.2 (TATAGCATACATTATACGAA, SEQ ID NO: 1541), targeting tdTomato, and 28.10 (CAGCGGGGATCCGACGAGCT, SEQ ID NO: 1542) targeting PTBP-1. Editing using spacers 12.7 and 12.2 were read out using the tdTomato system and 28.10 was analyzed using NGS of the PTBP-1 locus. In each case, XDP incorporating scaffold 226 resulted in 3- to 5-fold greater editing per XDP than XDP incorporating scaffold 174 (Table 46; results presented as the ratio of the EC50 for scaffold 174 to 226).
  • Table 46 EC50 results from editing assays Spacer EC50174/EC50226 12.7 3.06 205 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Spacer EC50174/EC50226 12.2 5.31 28.10 3.64 [0569] To further interrogate the mechanism of the increases in potency using the RRE/Rev system, three assays were performed. First, it was demonstrated that the increase in potency is Rev-dependent by testing the 226 guide scaffold in the XDP V1 and V7 architectures (see Table 45 above). Plasmids in the V1 architecture encode the Rev protein whereas the Rev protein is absent in the V7 architecture.
  • FIG.56 demonstrates that editing with XDP incorporating scaffold 174 or scaffold 226 is very similar in the V7 architecture; scaffold 226 does not increase editing in the Rev-independent V7 construct but does in V1, a Rev-containing architecture.
  • efficiency of scaffold 226 in the absence of an additional recruitment system e.g., Gag-CasX fusion, Gag-MS2, tVSVG-Stx
  • XDP version 207 lacks any architectural recruitment mechanism for CasX to be incorporated into the XDP.
  • XDPs with guide scaffold 174 were unable to edit NPCs in this construct whereas XDPs with scaffold 226 were able to achieve >20% editing (FIG.57).
  • the editing data with XDP incorporating guide scaffold 226 demonstrate a consistent pattern of increased potency over XDP incorporating guide scaffold 174.
  • the data show that without changing the nuclease function, the potency of XDPs can be increased by designing constructs that incorporate an RNA nuclear export pathway such as the Rev/RRE system. These enhanced effects were seen across different gene targets and multiple spacers.
  • the data demonstrate the utility of incorporating retroviral RNA transport elements into the RNP scaffold to increase potency of XDP particles. 206 295377244 Attorney Docket No.
  • pGP2 Glycoprotein plasmid cloning [0579] Sequences encoding the VSV-G glycoprotein and the CMV promoter were cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly (see Table 50).
  • Cell culture and transfection [0580] HEK293T Lenti-XTM cell culture was performed as described in Example 2, above. For transfection, the XDP structural plasmids (also encoding the CasX-NLS variants of Table 49) of Table 48 were used in amounts ranging from 13 to 80.0 ⁇ g. Each transfection also received 13 ⁇ g of sgRNA plasmid and 0.25 ⁇ g of pGP2.
  • SCRB-050/01WO 333322-2386 . . 211 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 212 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 213 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 t e gycoproten was p . or transecton, te structura pasm s ste aove an n 214 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 215 295377244 Attorney Docket No.
  • SCRB-050/01WO 333322-2386 8 protein (RBP). Binding of the RNA hairpin to MS2 RBP facilitates enhanced recruitment of 216 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 [0599] gRNAs incorporating RNA hairpin variants with varying affinities for the MS2 RBP were evaluated using a high-throughput, in vitro biochemical assay to assess equilibrium binding and dissociation kinetics (Buenrostro et al., Quantitative analysis of RNA-protein interactions on a massively parallel array reveals biophysical and evolutionary landscapes. Nat Biotechnol. 32(6):562 (2014)).
  • gRNA hairpin variants and their associated K d (dissociation constant) values are listed in Table 56, sequences of the guide plasmids encoding the different MS2 RNA hairpin variants are provided in Table 57 and the sequences of the MS2 hairpins are provided in Table 58.
  • Table 56 gRNA scaffolds containing MS2 hairpin variants with varying affinities and their dissociation constant values (Kd). Specific positions for the indicated nucleotide mutations refer to the positions of the base MS2 hairpin (scaffold 188) depicted in FIG.62 Positions of indicated – ⁇ log(K K OF )/ Scaffold No.
  • XDPs were pseudotyped with 10% VSV-G (percentage of VSV-G plasmid relative to other XDP structural plasmids).
  • RNA fold structures were generated with RNAfold web server and VARNA software. The methods to produce XDPs are described herein, as well as in WO2021113772A1, incorporated by reference in its entirety.
  • Structural plasmid cloning [0601] Briefly, to generate the XDP structural plasmids, the Gag-pol sequence was removed from pXDP1, and amplified and purified fragments encoding CasX 491, HIV-1, or MS2 CP components were cloned as described in Example 2, above.
  • HEK293T Lenti-XTM cells were seeded in 15 cm dishes at 20 x 10 6 cells per dish 24 hours before transfection to reach 70-90% confluency. The next day, Lenti-XTM cells were transfected with the following plasmids PEI MAX® (Polypus): XDP structural 220 295377244 Attorney Docket No.
  • SCRB-050/01WO 333322-2386 plasmids also encoding the CasX variants; Table 57), pSG50 (or other guide plasmid variants listed in Table 57), and pGP2 for XDP pseudotyping.24 hours post-transfection, media was replaced with Opti-MEM (Thermo Fisher). XDP-containing media was collected 72 hours post- transfection and filtered through a 0.45 ⁇ m PES filter. The supernatant was concentrated and purified via centrifugation.
  • XDPs were resuspended in 500 ⁇ L of DMEM/F12 supplemented with GlutaMAXTM, HEPES, NEAA, Pen/Strep, 2-mercaptoethanol, B-27 without vitamin A, and N2.
  • NPCs tdTomato neural progenitor cells
  • FIG.63 shows the percent editing at the tdTomato locus as measured by tdTomato fluorescence using flow cytometry when 0.007 ⁇ L of concentrated XDP preps were used to transduce NPCs.
  • high- affinity scaffold variants 296 and 298 demonstrated enhanced potency with both MS2 WT and MS2 353, with K d values ranging from 1.8 to 2.1 nM.
  • FIG.64 illustrates EC50 results across the different gRNA scaffolds incorporating the MS2 WT and MS 353 configurations. Scaffold variants 296, 297, and 305 exhibited a slightly higher potency compared to scaffold 188, an advantage that was more evident with the MS2353 configuration.
  • FIG.65 shows a clear correlation between the affinity (Kd) of the gRNA MS2 hairpin and resulting XDP potency (EC50), with an R 2 value of 0.81 (p ⁇ 0.001).
  • XDP comprising MS2 having an affinity of ⁇ 35nM resulted in efficient recruitment and packaging of the CasX RNP into XDPs.
  • affinity Kd
  • gRNA MS2 hairpin a cytokine therapeutic payload
  • GM-CSF cytokine granulocyte-macrophage colony-stimulating factor
  • SCRB-050/01WO 333322-2386 Methods: Structural plasmid cloning [0608] In order to generate the structural plasmids used to make the XDPs, mouse or human GMCSF was directly fused to a Gag structural protein, as described in Table 59, below. Cloning was performed as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
  • Table 59 Configurations of XDPs for carrying GM-CSF XDP description Plasmid Encoded Components** 1 MA-p2A-p2B-p10-CA-NC- Pro ⁇ -GMCSF ALV GM-CSF 2 VSVG 1 MA*-CA*-NC*-p1*-p6*-GMCSF Version 168-GM-CSF 2 MA*-CA*-NC*-p1*-p6*-Pro ⁇ 3 VSVG 1 MA-GMCSF VSV M-GM-CSF 2 VSVG * indicates cleavage sequence between adjacent components ** 5' to 3' orientation ⁇ indicates a -1 frame-shift in the encoded construct (Gag-TFR-PR polyprotein) Cell culture and transfection [0609] HEK293T Lenti-XTM cell culture was performed as described in Example 2, above.
  • XDP structural plasmids of Table 70 were used in amounts ranging from 13 to 80.0 ⁇ g. Collection and concentration [0610] XDPs were collected and concentrated as described in Example 2, above. Enzyme-linked immunosorbent assays (ELISAs) [0611] ELISAs were performed to measure the amount of GM-CSF per XDP. Specifically, XDPs were lysed with the lysis reagent and the number of GM-CSF molecules packaged per XDP was quantified using the Mouse GM-CSF Quantikine® ELISA kit (R&D, Cat no. MGM00) and Human GM-CSF Quantikine® ELISA kit (R&D, Cat no.
  • XDPs were engineered to carry human or mouse GM-CSF via the direct fusion of GM- CSF to the protein scaffold, and the amount of GM-CSF per XDP was measured via ELISA. As shown in Table 60, below, the XDPs contained GM-CSF, with between 40-527 molecules of GM-CSF per XDP. The results demonstrate that XDP constructs can be created to incorporate heterologous payloads, and in different configurations.
  • Table 60 Number and concentration of GM-CSF molecules in XDPs GM-CSF species XDP description Molecules of GM-CSF/XDP ALV GM-CSF 46 Mouse V168-GM-CSF 35 VSV M-mGM-CSF 149 ALV GM-CSF 101 Human V168-GM-CSF 527 VSV M-mGM-CSF 40
  • Example 17 Engineering of XDPs for incorporating catalytically-dead CasX repressor (dXR) system [0613] Experiments were performed to demonstrate that XDPs can be used to incorporate a catalytically-dead CasX repressor (dXR) system as the therapeutic payload.
  • dXR catalytically-dead CasX repressor
  • XDPs were generated using the version 168 or version 206 configuration.
  • Cloning was performed as described in Example 2, above. The constructs were designed with sequences coding for catalytically-dead CasX protein 491 (dCasX491; SEQ ID NO: 878) linked to the ZNF10 KRAB domain or the ZIM3 KRAB domain (dXR, see FIG.86 for a diagram), along with guide RNA scaffold variant 226 or 251, and spacer sequence 7.37 targeted to human B2M 7.37 (GGCCGAGATGTCTCGCTCCG, SEQ ID NO: 1017) or a non- targeting spacer (CGAGACGTAATTACGTCTCG; SEQ ID NO: 1019).
  • XDPs were collected and concentrated as described in Example 2, above. Results: [0617] XDPs were engineered to carry a dXR system targeting the B2M locus for repression. The XDPs were administered to human NPCs, and the level of B2M repression was measured. As shown in FIG.67, both the version 168 and version 206 XDPs were able to induce repression of B2M. The version 206 XDP with dCasX491 linked to the Zim3 KRAB domain produced the highest level of repression. [0618] The results of the experiments support that XDPs can be generated carrying functional dXR systems that result in targeted gene repression.
  • Example 18 Quantification of CasX ribonucleoproteins (RNPs) in XDPs [0619] Experiments were performed to measure the amount of CasX RNPs incorporated into XDPs. Methods: [0620] XDPs were generated using the version 168 configuration with guide scaffold 226, or the version 206 configuration with guide scaffold 251 (see FIG.26) or guide scaffold 188 (see FIG.12). [0621] Cloning was performed as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Cell culture and transfection [0622] HEK293T Lenti-XTM cell culture was performed as described in Example 2, above.
  • XDPs were collected and concentrated as described in Example 2, above.
  • 224 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Quantification of CasX RNPs via Western blot analysis
  • a semi-quantitative Western blot analysis was performed using XDP version 206 with guide scaffold 251, XDP version 168 with guide scaffold 226, and version XDP version 206 with guide scaffold 188 (FIG.68).
  • the protein amount in XPD particles was measured using a Pierce 660 assay.
  • XPD particles were lysed in Laemmli sample buffer and resolved by SDS-PAGE followed by Western blotting using a polyclonal antibody against the CasX protein.
  • the gel also contained a range of purified CasX to establish a standard curve, shown in FIG.69.
  • the resulting immunoblot was imaged using a ChemiDoc Touch, and the CasX protein levels were quantified by densitometry using Image Lab software from BioRad. Quantification of the CasX molecules in each XDP particle sample was determined using the standard curve.
  • Results of the Western blot analysis demonstrated that XDP version 168 with guide scaffold 226 contained approximately 227-239 CasX molecules/XDP particle (FIG.70) and, by inference, RNP.
  • the XDP version 206 with guide scaffold 188 contained approximately 240- 257 CasX molecules/XDP particle
  • XDP version 206 with guide scaffold 251 contained approximately 966-1112 CasX molecules/XDP particle, showing the superiority of scaffold 251 for facilitating incorporation of RNP into the XDP particles.
  • the fold differences relative to XDP version 168 with guide scaffold 226 are shown in FIG.71.
  • Example 19 Evaluation of orthogonal recruitment system with MS2 linked to Gag plus a nuclear export signal (NES) linked to CasX
  • the purpose of these experiments was to evaluate whether linking cleavable nuclear export signals (NESs) to CasX in an XDP construct could prevent the sequestration of CasX in the nucleus in packaging cells and promote the packaging of CasX RNPs into XDPs.
  • a potential concern during XDP production is the sequestration of the CasX RNP in the nucleus of the producer cell line as a result of the strong nuclear localization signals on the CasX protein. This possible nuclear sequestration might affect RNP packaging into XDPs and, therefore, XDP editing potency.
  • cleavable nuclear export signals (NESs) linked to CasX in an XDP construct so as to prevent the sequestration of CasX in the nucleus in packaging cells and promote the packaging of CasX RNPs into XDPs was evaluated.
  • NESs cleavable nuclear export signals
  • 225 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Methods: [0627] Cleavable NESes were added to the XDP version 206 system (plasmid configurations are shown in Table 62. The NESes were linked to the C-terminus of CasX 676 via an HIV cleavage sequence and a rigid linker.
  • Table 62 Configurations of version 206 XDPs with or without NESes XDP description Plasmid Encoded Components** 1 MA*-CA*-NC*-p1*-p6-MS2 2 MA*-CA*-NC*-p1*-p6*-Pro ⁇ Version 206 3 NLS-CasX-NLS 4 sgRNA (scaffold 251) 1 MA*-CA*-NC*-p1*-p6-MS2 Version 206 with 2 MA*-CA*-NC*-p1*-p6*-Pro ⁇ NES 3 NLS-CasX-NLS*-NES 4 sgRNA (scaffold 251) * indicates cleavage sequence between adjacent components ** 5' to 3' orientation ⁇ indicates a -1 frame-shift in the encoded construct (Gag-TFR-PR polyprotein) [0628] CRM1 (chromosomal maintenance 1) plays a major role in the export of proteins with leucine-rich nuclear export signals.
  • Nuclear export signals that utilize the CRM1 nuclear export pathway with a range of affinities were selected and attached to the C-terminus of CasX in cleavable manner, such that during the maturation process post-XDP budding, the HIV protease would cleave the NES such that the CasX RNP would not have an attached NES when delivered into the target cell.
  • 15 different NESs that use the CRM1 pathway with different Rc/n and K d values were selected (see Fu, S. et al., Mol Biol Cell.2018 Aug 15;29(17):2037- 2044), and six additional NESs were selected from NESdb, a database of NES-containing CRM1 cargoes (see Xu, D., et al.
  • SCRB-050/01WO 333322-2386 S ource of NES Amino acid sequence of NES SEQ ID NO HPV 11 E1 ISPRLDAIKL 1058 Menin DLVLLSLVL 1059 mRNA export factor P VSKITF 1060 EB2 VTL Nucleoprotein - 1061 Porcine epidemic LAPNVAALLFGGNVAVRELADSYEITYNYKMTVPKSDPNV diarrhea virus Nuclear export 1062 protein - Influenza ILMRMSKMQL A virus NS-2 of MVM DEMTKKFGTLTIHDTEKYASQPELCNN 1063 P axillin QRVTSTQQQTRISASSATRELDELMASLSDFKFMAQGKTGSSS P PGGPPKPGSQLDSMLGSLQSDLNKLGV 1064 Phosphoprotein of I I 1065 hPIV-2 ELLKGLDL HCMV Protein CILCQL 1066 UL94 LLLY VEEV Capsid T DPFLAMQVQELTRSMANLTFKQRR
  • Example 20 Screen of XDPs with diverse incorporated viral glycoproteins to evaluate tropism and editing capabilities
  • the glycoprotein belonging to VSV Indiana species within the Vesiculovirus genus is usually the most widely used glycoprotein for pseudotyping purposes. The purpose of these experiments was to explore the transduction capabilities of glycoproteins belonging to other species, and test whether the cellular tropism of XDPs could be altered by pseudotyping XDPs with various glycoproteins as targeting moieties in various cell types.
  • Methods [0632] The screen of glycoproteins was conducted in the XDP version 206 construct configuration.
  • the version 206 XDPs pseudotyped with glycoproteins of Table 64 were transduced into mouse tdTomato neural progenitor cells (NPCs), in which editing of the tdTomato locus was measured, or human Jurkat T cells, K562 lymphoblasts, ARPE-19 retinal pigment epithelial (RPE) cells, Y79 retinoblastoma cells, induced neurons, human NPCs, or astrocytes, in which editing of the B2M locus was measured.
  • NPCs mouse tdTomato neural progenitor cells
  • RPE retinal pigment epithelial
  • Table 64 Description of glycoproteins tested l ycoprotein SEQ ID NO ( Amino acid) VSVG 573 V SAV 654 A BVV 655 C ARV 656 C HPV 657 C OCV 658 V SIV 659 I SFV 660 J URV 661 M SPV 662 M ARV 663 M ORV 664 V SNJV 665 P ERV 666 P IRYV 667 R ADV 668 Y BV 669 VSV CEN AM - 670 94GUB 229 295377244 Attorney Docket No.
  • SCRB-050/01WO 333322-2386 l ycoprotein SEQ ID NO ( Amino acid) VSV South America 671 85CLB E EEV 634 V EEV 635 y man e al ng e ted to . 230 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 in ES, Cells ated DPs 10 re re yzed Cs d, well were well - d es at ys ated. . re ed 231 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 tes at y.
  • SCRB-050/01WO 333322-2386 acer s sX S in a th a X NPs ato- 3) th a or l- and 235 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 iency an ent ids id o R) or R In ed in DP 236 295377244 Attorney Docket No.
  • the fusion proteins of the dXR constructs were made up of, from N- to C-terminus, a catalytically-dead CasX 491, and a ZNF10 or ZIM3 KRAB domain (see FIG.86; “RD1” is “Repressor Domain 1” and denotes the KRAB domain of interest).
  • the fusion proteins of the ELXR configuration #1 constructs were made up of, from N- to C-terminus, a catalytic domain from DNMT3A, an interaction domain from DNMT3L, a catalytically-dead CasX 491, and a ZNF10 or ZIM3 KRAB domain (see ELXR configuration #1 in FIG.87), along with amino acid linkers and NLS sequences.
  • Catalytically-active CasX 491 herein termed “CasX”; SEQ ID NO: 189) was also included as a control.
  • CasX Catalytically-active CasX 491
  • the ELXR constructs also contained a 2x FLAG tag.
  • Table 67 DNA and protein sequences of components of dXR and ELXR Key DNA SEQ ID Protein SEQ component sequence NO sequence ID NO ATGGATGCTAAGTCACTAACTGCCTGG MDAKSLTAWSRTLVTFKDVFV ZNF10 TCCCGGACACTGGTGACCTTCAAGGAT DFTREEWKLLDTAQQIVYRNV GTATTTGTGGACTTCACCAGGGAGGAG 24 MLENYKNLVSLGYQLTKPDVI KRAB 82 TGGAAGCTGCTGGACACTGCTCAGCAG LRLEKGEEP 2494 domain ATCGTGTACAGAAATGTGATGCTGGAG AACTATAAGAACCTGGTTTCCTTGGGT 237 295377244 Attorney Docket No.
  • RNA sequences of the guide scaffolds are provided in Table 6. Sequences of spacers 7.37 targeted to human B2M and a non- targeting spacer are provided in Table 68. All resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. 241 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Table 68: Sequences of spacers used in constructs 0.0 Non-target N/A CGAGACGUAAUUACGUCUCG 2449 [0664] Cell culture and transfection, collection and concentration of XDPs, and resuspension and transduction of XDPs is performed as described in Example 2 above analysis of cytometry. XDPs.
  • XDPs were able to carry either the dXR or ELXR configuration #1 systems as therapeutic payloads and achieve transcriptional repression of a target locus.
  • Example 24 Engineering of XDPs for carrying ELXR systems
  • XDPs are generated with ELXR configuration #1, #4, or #5 molecules as the payload (see FIGS.87 and 88 for diagrams of the configurations).
  • Materials and Methods Description of XDPs and CasX constructs tested 242 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386
  • XDP configuration versions 168 and 206 are generated with various ELXR molecules.
  • Table 66 summarizes the plasmids used to encode the components of the version 168 and 206 XDP systems.
  • ELXR molecules in configurations #1, #4, and #5 which contain a catalytically- inactive CasX 491, are tested, as diagrammed in FIG.88.
  • Table 69 provides amino acid sequences of configurations #1, #4, and #5 ELXR molecules, showing the sequences of the components of the proteins from N- to C-terminus in the table.
  • the repressor domain 1 shown in Table 69 (“RD1”in FIG.88) may be a repressor domain from the species Columba livia, Rattus norvegicus, Cebus imitator, chimpanzee, Chlorocebus sabaeus, Ophiophagus hannah, Ailuropoda melanoleuca, Peromyscus maniculatus bairdii, or Phyllostomus discolor, in place of the human ZNF10 or ZIM3 KRAB domains that were tested in Example 23.
  • Other catalytically- inactive CasX variants can be used in place of catalytically-inactive CasX 491; these variants are listed in Table 4.
  • RNA sequences of the guide scaffolds are provided in Table 6. Sequences of spacers 7.37 targeted to human B2M and a non-targeting spacer control are provided in Table 68, above. All resultant plasmids are sequenced using Sanger sequencing to ensure correct assembly. [0673] Cell culture and transfection, collection and concentration of XDPs, and resuspension and transduction of XDPs is performed as in Example 2, above. Repression of the 248 295377244 Attorney Docket No.
  • SCRB-050/01WO 333322-2386 B2M locus is assessed at 7 days and 14 days, when cells are harvested for analysis of HLA immunostaining as detected using flow cytometry, to demonstrate the ability of the constructs to deliver the therapeutic payload and repress expression of B2M.
  • Example 25 Evaluation of non-covalent recruitment (NCR) systems with protein-ligand pairs attached to Gag and protein cargo [0674] Experiments were performed to evaluate a protein-based NCR recruitment system for packaging cargo in XDPs.
  • XDPs were generated in which an NCR protein was fused to the Gag polyprotein, and a ligand for the NCR protein was fused to the cargo of the XDP, i.e., to the N-terminus of CasX.
  • Table 70 summarizes the plasmids used to encode the components of these XDP systems.
  • Table 70 Summary of version XDPs with protein recruitment of CasX Plasmid Encoded Components, 5' to 3' 1 MA*-CA*-NC*-p1*-p6-NCR protein 2 MA*-CA*-NC*-p1*-p6*-PR ⁇ 3
  • Ligand-CasX 4 VSV-G 5 sgRNA * indicates cleavage sequence between adjacent components ⁇ indicates a -1 frame-shift in the encoded construct (Gag-TFR-PR polyprotein) [0676]
  • the protein-ligand pairs that were tested are provided in Table 71 (each row is a protein-ligand pair).
  • Some of the listed protein-ligand pairs were split fluorescent proteins that were anticipated to emit fluorescence when the two portions of the split protein are bound, such as mNeon-Green and sfCherry.
  • Table 72 provides the amino acid sequences of the NCR proteins
  • Table 73 provides the amino acid sequences of the ligands.
  • Table 71 Protein-ligand pairs for protein-based recruitment to XDPs Protein attached to Gag Ligand attached to CasX Protein A Fc 249 295377244 Attorney Docket No.
  • XDPs with each of the protein-ligand pairs tested produced editing of the tdTomato locus. Indeed, for many of the XDPs with protein- ligand pairs, as well as for version 206 XDPs, editing levels were near 100%. This was true even at the lowest volumes of XDPs administered to the cells. Accordingly, editing levels were likely saturated in this assay. [0680] NPCs transduced with split fluorescent protein NCR systems were imaged for red fluorescence (indicating editing of the tdTomato locus) and green fluorescence (indicating binding of the two portions of the split fluorescent protein).
  • the three split mNeon-Green NCR systems (mNG2 1-10 +mNG11, mNG3A 1-10 +mNG3 11 , and mNG3K 1-10 +mNG3 11 ) and the split CloGFP system (CloGFP1-10+CloGFP11) each produced cells with red and green fluorescence, indicating both editing and the presence of reconstituted split fluorescent proteins (data not shown).
  • the green fluorescence was often found in puncta, which is believed to indicate nuclear 253 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 localization of the CasX:gRNA RNP along with the bound fluorescent protein.
  • the SpyCatcherOO3 protein comprises a sequence of SEQ ID NO: 2465, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the SpyTag003, and the SpyTag003 comprises a sequence of SEQ ID NO: 2478, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the SpyCatcher003 protein;
  • the Strep-Tactin protein comprises a sequence of SEQ ID NO: 2466, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the Twin Strep tag II
  • the Twin Strep tag II comprises a sequence of SEQ ID NO: 2479, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Strep-Tactin protein
  • the Strep-Tactin protein comprises a sequence of SEQ ID NO: 2466, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the Strep tag II
  • the Strep tag II comprises a sequence of SEQ ID NO: 2480, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Strep-Tactin protein
  • the Avidin protein comprises a sequence of SEQ ID NO: 2467, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the Avi tag
  • the Avi tag comprises a sequence of SEQ ID NO: 2481, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Avidin protein;
  • the mNG2i-io protein comprises a sequence of SEQ ID NO: 2453, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the mNGn ligand
  • the mNGn ligand comprises a sequence of SEQ ID NO: 2470, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mNG2i-io protein;
  • the sfCherry2i-io protein comprises a sequence of SEQ ID NO: 2454, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the sfCherry2n ligand, and the sfCherry2n ligand comprises a sequence of SEQ ID NO: 2471, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the sfCherry2i-io protein;
  • the mNG3 Ai-io protein comprises a sequence of SEQ ID NO: 2455, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the mNGn ligand
  • the mNGn ligand comprises a sequence of SEQ ID NO: 2470, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mNG3Ai-io protein;
  • the mNG3ki-io protein comprises a sequence of SEQ ID NO: 2456, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the mNGn ligand
  • the mNGn ligand comprises a sequence of SEQ ID NO: 2470, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mNG3ki-io protein;
  • the sfGFP i-io protein comprises a sequence of SEQ ID NO: 2457, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the GFPn ligand
  • the GFPn ligand comprises a sequence of SEQ ID NO: 2474, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the sfGFPi-io protein;
  • the mClover3 i-io protein comprises a sequence of SEQ ID NO: 2458, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the mClover3n ligand, and the mClover3n ligand comprises a sequence of SEQ ID NO: 2473, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mClover3i-io protein;
  • the CloGFPO.2i-io protein comprises a sequence of SEQ ID NO: 2459, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the GFPn ligand
  • the GFPn ligand comprises a sequence of SEQ ID NO: 2474, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the CloGFPO.2i-io protein; or
  • the CloGFP i-io protein comprises a sequence of SEQ ID NO: 2460, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the GFPn ligand
  • the GFPn ligand comprises a sequence of SEQ ID NO: 2474, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the CloGFPi-io protein.
  • RNA binding-protein comprises:
  • a truncated AN protein and the ligand is a AN hairpin
  • a Tat protein and the ligand is a transactivation response (TAR) element
  • an iron-responsive binding element protein (IRE-BP) and the ligand is an iron response element (IRE);
  • the MS2 coat protein comprises a sequence of SEQ ID NO: 4140, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the MS2 hairpin;
  • the PP7 coat protein comprises a sequence of SEQ ID NO: 4132, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the PP7 hairpin;
  • the QP coat protein comprises a sequence of SEQ ID NO: 4138, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the QP hairpin;
  • the AN protein comprises a sequence of SEQ ID NO: 4131, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the AN hairpin;
  • the truncated AN protein comprises a sequence of SEQ ID NO: 4130; , or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the AN hairpin;
  • the Tat protein comprises a sequence of SEQ ID NO: 4133, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the TAR element;
  • the phage GA coat protein comprises a sequence of SEQ ID NO: 4139, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the phage GA hairpin;
  • the IRE-BP comprises a sequence of SEQ ID NO: 4134 or 4135, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the IRE;
  • the U1A signal recognition particle comprises a sequence of SEQ ID NO: 4137, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the UI hairpin II; or
  • the truncated U1A signal recognition particle comprises a sequence of SEQ ID NO: 4136, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the UI hairpin II.
  • the MS2 hairpin comprises a sequence of SEQ ID NO: 910;
  • the PP7 hairpin comprises a sequence of SEQ ID NO: 914;
  • the QP hairpin comprises a sequence of SEQ ID NO: 911;
  • the AN hairpin comprises a sequence of SEQ ID NO: 954;
  • the TAR comprises a sequence of SEQ ID NO: 951;
  • the phage GA hairpin comprises a sequence of SEQ ID NO: 953;
  • the IRE comprises a sequence of SEQ ID NO: 952;
  • the UI hairpin II comprises a sequence of SEQ ID NO: 912.
  • the XDP system of embodiment 7 or embodiment 8 wherein the MS2 hairpin comprises a sequence selected from the group consisting of ACAUGAGGAUCACCCAUGU (SEQ ID NO: 910), ACCUGAGGAUCACCCAGGU (SEQ ID NO: 1847), GCAUGAGGAUCACCCAUGC (SEQ ID NO: 1848), GCCUGAGGAUCACCCAGGC (SEQ ID NO: 1849), GCCUGAGCAUCAGCCAGGC (SEQ ID NO: 1850), ACAUGAGCAUCAGCCAUGU (SEQ ID NO: 1851), ACUUGAGGAUCACCCAUGU (SEQ ID NO: 1852), ACAUUAGGAUCACCAAUGU (SEQ ID NO: 1853), and ACAUGAGGACCACCCAUGU (SEQ ID NO: 1854).
  • ACAUGAGGAUCACCCAUGU SEQ ID NO: 910
  • ACCUGAGGAUCACCCAGGU SEQ ID NO: 1847
  • GCAUGAGGAUCACCCAUGC SEQ ID NO
  • RNA-binding protein comprises MS2 coat protein and the ligand comprises the MS2 hairpin
  • therapeutic payload comprising the MS2 hairpin exhibits a dissociation constant (KD) to the MS2 coat protein of less than 100 nM, less than 50 nM, less than 35 nM, less than 10 nM, less than 3 nM, or less than 2 nM in an in vitro assay.
  • KD dissociation constant
  • the encoded therapeutic payload comprises a nucleic acid selected from the group consisting of a single-stranded antisense oligonucleotide (ASO), a double-stranded RNA interference (RNAi) molecule, a DNA aptamer, an RNA aptamer , a first CRISPR guide ribonucleic acid (gRNA), a first and a second gRNA, or any combination thereof.
  • ASO single-stranded antisense oligonucleotide
  • RNAi double-stranded RNA interference
  • the encoded therapeutic payload comprises a first, and optionally a second gRNA selected from the group consisting of a Class 2 Type II, a Class 2 Type V, and a Class 2 Type VI CRISPR system gRNA.
  • the first, and optionally the second gRNA is a single-molecule guide RNA (sgRNA) comprising a scaffold sequence and a targeting sequence, wherein the targeting sequence comprises between 15 and 20 nucleotides and is complementary to a target nucleic acid sequence.
  • sgRNA single-molecule guide RNA
  • RNP ribonucleoprotein
  • the first, and optionally the second gRNA scaffold sequence comprises a sequence selected from the group consisting of the sequences of SEQ ID NOS: 2249, 2308, 2312, 2314-2317, 2319, 2380, and 2417-2429, or a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto, wherein the gRNA is capable of complexing with a CRISPR protein to form a ribonucleoprotein (RNP).
  • RNP ribonucleoprotein
  • RRE Stem IIB of Rev response element
  • XDP system of any one of embodiments 16 -28 comprising a second gRNA scaffold sequence identical to the first gRNA scaffold sequence, and a targeting sequence complementary to a different region of the target nucleic acid, wherein the second gRNA is capable of complexing with a CRISPR protein to form a ribonucleoprotein (RNP).
  • RNP ribonucleoprotein
  • the encoded therapeutic payload comprises a protein payload selected from the group consisting of a cytokine, an interleukin, an enzyme, a receptor, a microprotein, a hormone, erythropoietin, a ribonuclease (RNase), a deoxyribonuclease (DNase), a blood clotting factor, an anticoagulant, a bone morphogenetic protein, an engineered protein scaffold, a thrombolytic protein, a CRISPR protein, granulocyte-macrophage colony-stimulating factor (GMCSF), a transcription factor, a repressor domain, a transposon, a reverse transcriptase, a viral interferon antagonist, a tick protein, and an anti-cancer biologic.
  • a protein payload selected from the group consisting of a cytokine, an interleukin, an enzyme, a receptor, a microprotein, a hormone, erythropoietin,
  • CRISPR protein is a Class 2 CRISPR protein selected from the group consisting of a Class 2 Type II, a Class 2 Type V, or a Class 2 Type VI CRISPR protein.
  • the XDP system of embodiment 34 wherein the CasX is a CasX variant comprising a sequence selected from the group consisting of SEQ ID NOS: 135-169, 181-320, 322-366, 368-457, 797-804, 806-829, 831, 832, 834-842, 937, 938, 940, or 942, or a sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto, wherein the CasX variant retains the ability to form an RNP with a gRNA and retains nuclease activity.
  • the CasX variant comprises a sequence selected from the group consisting of SEQ ID NOS: 189, 196, 354, and 813, or a sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto, wherein the CasX variant retains the ability to form an RNP with a gRNA and retains nuclease activity.
  • dCasX catalytically - dead CasX variant
  • the CRISPR protein is a catalytically - dead CasX variant (dCasX) comprising a sequence selected from the group consisting of SEQ ID NOS: 877-896 and 4112-4117, or a sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto, wherein the dCasX retains the ability to form an RNP with a gRNA.
  • dCasX catalytically - dead CasX variant
  • dCasX is linked to a first repressor domain (RD1) as a fusion protein (dXR), wherein the fusion protein is capable of reducing expression of the target nucleic acid by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%.
  • RD1 first repressor domain
  • dXR fusion protein
  • RD1 comprises an amino acid sequence motif selected from the group consisting of: a) PX1X2X3X4X5X6EX7, wherein i) Xi is A, D, E, or N, ii) X2isLorV, iii) X3 is I or V, iv) X4isS, T, orF, v) X 5 is H, K, L, Q, R or W, vi) XeisLorM, and vii) X7 is G, K, Q, or R; b) X1X2X3X4GX5X6X7X8X9, wherein i) Xi is L or V, ii) X 2 is A, G, L, TorV, iii) X3 is A, F, or S, iv) X4isLorV, v) X 5 is C, F, H, I
  • the first amino acid sequence motif comprises the sequence LYX1X2VMX3EX4X5X6X7X8X9X10 (SEQ ID NO: 4109), wherein i) Xi is K or R, ii) X2 is D or E, iii) X3 is L, Q, or R, iv) Xiis N or T, v) X5 is F or Y, vi) Xe is A, E, G, Q, R, or S, vii) X?is H, L, or N, viii) Xs is L or V, ix) X9 is A, G, I, L, T, or V, and x) X10 is A, F, or S; and
  • the second amino acid sequence motif comprises the sequence FX1DVX2X3X4FX5X6X7EWX8 (SEQ ID NO: 4110), wherein i) Xi is A, E, G, K, or R, ii) X2 is A, S, or T, iii) X3 is I or V, iv) X 4 is D, E, N, or Y, v) X5 is S or T, vi) Xe is E, L, P, Q, R, or W, vii)X?is D or E, and viii) Xs is A, E, G, Q, or R.
  • RD1 comprises a sequence selected from the group consisting of SEQ ID NOS: 2509-2603, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.
  • RD1 comprises a sequence selected from the group consisting of SEQ ID NOS: 2509-2517, or sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.
  • DNMT3A CD comprises a sequence of SEQ ID NO: 2504, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.
  • DNMT3L ID comprises a sequence of SEQ ID NO: 2497, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.
  • 57 The XDP system of any one of embodiments 53-56, wherein the fourth repressor is an ATRX-DNMT3-DNMT3L (ADD) domain of DNMT3A.
  • ADD domain comprises the sequence of SEQ ID NO: 2503, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.
  • NLS-Linker-DNMT3 A CD-Linker- DNMT3L ID-Linker-Linker-dCasX- Linker3-RD1-NLS;
  • NLS-DNMT3 A CD-Linker-DNMT3L ID-Linker-RD 1 -Linker-dCasX-Linker- NLS.
  • NLS-RD1 -Linker- ADD-DNMT3 A CD-Linker2-DNMT3L ID-Linkerl-dCasX- Linker-NLS; or
  • Linker-NLS [0343] 62.
  • glycoprotein has a sequence selected from the group consisting of SEQ ID NOS: 573-613, 615-682, 684-692, 694-698, 700, 702-706, 708-727, 729-730, 732, 734, 738, 740-746, 749-796, or a sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.
  • the therapeutic payload comprises the CRISPR protein and the first gRNA complexed as a first ribonucleoprotein complex (RNP), and the CRISPR protein and a second gRNA complexed as a second RNP.
  • RNP ribonucleoprotein complex
  • MA matrix polypeptide
  • CA capsid polypeptide
  • NC nucleocapsid polypeptide
  • pl pl protein
  • p6 protein p6 protein
  • PCS protease cleavage sequence
  • the Gag-TFR-PR polyprotein comprises, from N-terminus to C-terminus, MA-PCS-CA-PCS-NC-PCS-pl-PCS- p6-PCS-protease, wherein the protease is capable of cleaving the PCS of the Gag and the Gag- TRF-PR.
  • a eukaryotic cell comprising the XDP system of any one of embodiments 1-86.
  • eukaryotic cell of embodiment 87 or embodiment 88 wherein the eukaryotic cell is selected from the group consisting of a Baby Hamster Kidney fibroblast (BHK) cell, a human embryonic kidney 293 (HEK293) cell, a human embryonic kidney 293T (HEK293T) cell, a NS0 cell, a SP2/0 cell, a YO myeloma cell, a P3X63 mouse myeloma cell, a PER cell, a PER.C6 cell, a hybridoma cell, an NIH3T3 cell, a CV-1 (simian) in Origin with SV40 genetic material (COS) cell, a HeLa cell, a Chinese hamster ovary (CHO) cell, and an HT1080 cell.
  • BHK Baby Hamster Kidney fibroblast
  • HEK293 human embryonic kidney 293
  • HEK293T human embryonic kidney 293T
  • NS0 cell a SP
  • 91 The eukaryotic cell of any one of embodiment 87-90, wherein the eukaryotic cell is modified to reduce expression of a cell surface marker.
  • 93 The eukaryotic cell of any one of embodiment 87-92, wherein the eukaryotic cell is modified to express one or more cell surface markers selected from CD46, CD47, CD55, and CD59, wherein the incorporation of the cell surface marker on the surface of the XDP released from the eukaryotic cell is increased compared to XDP released from a eukaryotic cell that has not be modified.
  • a method of making an XDP comprising a therapeutic payload comprising:
  • the XDP of embodiment 100 comprising a therapeutic payload of RNPs of a CRISPR protein and a gRNA.
  • the XDP of embodiment 101 comprising a therapeutic payload of RNPs of a CasX variant and a guide RNA variant.
  • 105 The XDP of embodiment 104, wherein incorporation of the ligand and NCR protein into the XDP system results in at least a 2-fold, at a least 3 -fold, or at least a 4-fold increase repressing potency of the XDP compared to an equivalent XDP without the incorporated ligand and NCR protein, when assayed in vitro under comparable conditions.
  • 106 A delivery particle (XDP) comprising cleavage products of a retroviral Gag polyprotein, a therapeutic payload, and a tropism factor, wherein:
  • the cleavage products of the Gag polyprotein comprise MA, CA, NC, pl, and p6, wherein p6 is fused to a non-covalent recruitment (NCR) protein,
  • the therapeutic payload is fused to a ligand and encapsidated within the XDP, wherein the NCR protein has an affinity for the ligand.
  • a NbALFA protein and the ligand comprises an ALFA tag
  • a SpyCatcher protein and the ligand comprises a SpyTag
  • a SpyCatcher002 protein and the ligand comprises a SpyTag002;
  • a SpyCatcher003 protein and the ligand comprises a SpyTag003;
  • a Strep-Tactin protein and the ligand comprises a Strep tag II
  • a sfCherry2i-io protein and the ligand comprises a sfCherry2n ligand
  • a mNG3ki-io protein and the ligand comprises a mNGn ligand
  • a sfGFPi-io protein and the ligand comprises a GFPn ligand
  • a mClover3i-io protein and the ligand comprises a mClover3n ligand
  • a CloGFPO.2i-io protein and the ligand comprises a GFPn ligand
  • the Protein A comprises a sequence of SEQ ID NO: 2450, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Fc region
  • the Fc region comprises a sequence of SEQ ID NO: 2468, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Protein A;
  • the truncated Protein A comprises a sequence of SEQ ID NO: 2451, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Fc region
  • the Fc region comprises a sequence of SEQ ID NO: 2468, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the truncated protein A;
  • the CL7 protein comprises a sequence of SEQ ID NO: 2452, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the IM7 ligand
  • the IM7 ligand comprises a sequence of SEQ ID NO: 2469, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the CL7 protein;
  • the NbALFA protein comprises a sequence of SEQ ID NO: 2462, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the ALFA tag
  • the ALFA tag comprises a sequence of SEQ ID NO: 2476, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the NbALFA tag;
  • the SpyCatcher protein comprises a sequence of SEQ ID NO: 2463, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the SpyTag
  • the SpyTag comprises a sequence of SEQ ID NO: 2477, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the SpyCatcher protein
  • the SpyCatcher002 protein comprises a sequence of SEQ ID NO: 2461 or 2464, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the SpyTag002, and the SpyTag002 comprises a sequence of SEQ ID NO: 2475, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the SpyCatcher002 protein;
  • the SpyCatcher003 protein comprises a sequence of SEQ ID NO: 2465, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the SpyTag003, and the SpyTag003 comprises a sequence of SEQ ID NO: 2478, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the SpyCatcher003 protein;
  • the Strep-Tactin protein comprises a sequence of SEQ ID NO: 2466, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Twin Strep tag II
  • the Twin Strep tag II comprises a sequence of SEQ ID NO: 2479, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Srep-Tactin protein
  • the Strep-Tactin protein comprises a sequence of SEQ ID NO: 2466, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the to the Strep tag II
  • a Strep tag II comprises the sequence of SEQ ID NO: 2480, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Strep-Tactin protein
  • the Avidin protein comprises a sequence of SEQ ID NO: 2467, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Avi tag
  • the Avi tag comprises a sequence of SEQ ID NO: 2481, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Avidin protein;
  • the mNG2i-io protein comprises a sequence of SEQ ID NO: 2453, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mNGn ligand
  • the mNGn ligand comprises a sequence of SEQ ID NO: 2470, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mNG2i-io protein
  • the sfCherry2i-io protein comprises a sequence of SEQ ID NO: 2454, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the sfCherry2n ligand, and the sfCherry2n ligand comprises a sequence of SEQ ID NO: 2471, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the sfCherry2i-io protein;
  • the mNG3 Ai-io protein comprises a sequence of SEQ ID NO: 2455, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mNGn ligand
  • the mNGn ligand comprises a sequence of SEQ ID NO: 2470, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mNG3 Ai-io protein
  • the mNG3ki-io protein comprises a sequence of SEQ ID NO: 2456, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence
  • the sfGFP i-io protein comprises a sequence of SEQ ID NO: 2457, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the GFPn ligand
  • the GFP11 ligand comprises a sequence of SEQ ID NO: 2474, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the sfGFPi-io protein;
  • the mClover3i-io protein comprises a sequence of SEQ ID NO: 2458, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mClover3n ligand
  • the mClover3n ligand comprises a sequence of SEQ ID NO: 2473, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mClover3 i-io protein;
  • the CloGFPO.2i-io protein comprises a sequence of SEQ ID NO: 2459, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the GFPn ligand
  • the GFPn ligand comprises a sequence of SEQ ID NO: 2474, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the CloGFPO.2i-io protein; or
  • the CloGFP i-io protein comprises a sequence of SEQ ID NO: 2460, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the GFPn ligand
  • the GFPn ligand comprises a sequence of SEQ ID NO: 2474, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the CloGFP0.2i-io protein.
  • an MS2 coat protein or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the MS2 hairpin, and the ligand is an MS2 hairpin;
  • a PP7 coat protein or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the PP7 hairpin, and the ligand is a PP7 hairpin;
  • a QP coat protein or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the QP hairpin and the ligand is a QP hairpin;
  • a AN protein or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the AN hairpin, and the ligand is a AN hairpin;
  • a truncated AN protein or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the AN hairpin, and the ligand is a AN hairpin;
  • a Tat protein or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the TAR element, and the ligand is a transactivation response (TAR) element;
  • a phage GA coat protein or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the phage GA hairpin, and the ligand is a phage GA hairpin;
  • an iron-responsive binding element protein (h) an iron-responsive binding element protein (IRE-BP) , or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the IRE, and the ligand is an iron response element (IRE);
  • a U1A signal recognition particle or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the U1 hairpin II, and the ligand is a U1 hairpin II; or
  • the MS2 hairpin comprises the sequence of SEQ ID NO: 910;
  • the PP7 hairpin comprises the sequence of SEQ ID NO: 914;
  • the QP hairpin comprises the sequence of SEQ ID NO: 911;
  • the AN hairpin comprises the sequence of SEQ ID NO: 954;
  • the TAR comprises the sequence of SEQ ID NO: 951;
  • the phage GA hairpin comprises the sequence of SEQ ID NO: 953;
  • the IRE comprises the sequence of SEQ ID NO: 952;
  • the U1 hairpin II comprises the sequence of SEQ ID NO: 912.
  • the XDP of embodiment 112, wherein the MS2 hairpin comprises a sequence selected from the group consisting of ACAUGAGGAUCACCCAUGU (SEQ ID NO: 910), ACCUGAGGAUCACCCAGGU (SEQ ID NO: 1847), GCAUGAGGAUCACCCAUGC (SEQ ID NO: 1848), GCCUGAGGAUCACCCAGGC (SEQ ID NO: 1849), GCCUGAGCAUCAGCCAGGC (SEQ ID NO: 1850), ACAUGAGCAUCAGCCAUGU (SEQ ID NO: 1851), ACUUGAGGAUCACCCAUGU (SEQ ID NO: 1852), ACAUUAGGAUCACCAAUGU (SEQ ID NO: 1853), and ACAUGAGGACCACCCAUGU (SEQ ID NO: 1854).
  • ACAUGAGGAUCACCCAUGU SEQ ID NO: 910
  • ACCUGAGGAUCACCCAGGU SEQ ID NO: 1847
  • GCAUGAGGAUCACCCAUGC SEQ ID NO: 1848
  • RNA- binding protein comprises MS2 and the ligand comprises the MS2 hairpin
  • therapeutic payload comprising the MS2 hairpin exhibits a dissociation constant (KD) to the MS2 coat protein of less than 100 nM, less than 50 nM, less than 35 nM, less than 10 nM, less than 3 nM, or less than 2 nM in an in vitro assay.
  • KD dissociation constant
  • the therapeutic payload comprises a nucleic acid selected from the group consisting of a single-stranded antisense oligonucleotide (ASO), a double-stranded RNA interference (RNAi) molecule, a DNA aptamer, an RNA aptamer, a first CRISPR guide ribonucleic acid (gRNA), a first and a second gRNA, or any combination thereof.
  • ASO single-stranded antisense oligonucleotide
  • RNAi double-stranded RNA interference
  • the therapeutic payload comprises a protein payload selected from the group consisting of a cytokine, an interleukin, an enzyme, a receptor, a microprotein, a hormone, erythropoietin, a ribonuclease (RNase), a deoxyribonuclease (DNase), a blood clotting factor, an anticoagulant, a bone morphogenetic protein, an engineered protein scaffold, a thrombolytic protein, a CRISPR protein, granulocytemacrophage colony-stimulating factor (GMCSF), a transcription factor, a transposon, a reverse transcriptase, a viral interferon antagonist, a tick protein, and an anti-cancer modality.
  • a protein payload selected from the group consisting of a cytokine, an interleukin, an enzyme, a receptor, a microprotein, a hormone, erythropoietin, a ribonuclease (
  • 118 The XDP of any one of embodiments 106-117, wherein the therapeutic payload comprises of RNPs of a CRISPR protein and a gRNA, RNPs of a CasX variant and a guide RNA variant, or RNPs of a dCasX and linked repressor domain(s) and a guide RNA variant.
  • a method of modifying a target nucleic acid sequence in a population of cells comprising contacting the cells with the XDP of any one of embodiments 100-120, wherein the XDP comprises a therapeutic payload of RNPs of a CasX variant and a gRNA variant, and wherein said contacting comprises introducing the into the cells the RNP, wherein the target nucleic acid targeted by the gRNA variant is modified by the CasX variant.
  • a method of repressing a target nucleic acid sequence in a population of cells, wherein the XDP comprises a therapeutic payload of RNPs of a dXR and a guide RNA wherein the method comprises contacting the cells with the XDP of embodiment 104 or embodiment 105, wherein said contacting comprises introducing the into the cells the RNP, wherein a target nucleic acid targeted by the guide RNA is repressed by the dXR.
  • composition comprising the XDP of any one of embodiments 100-120 for use as a medicament for the treatment of a subject having a disease.
  • RNP of CasX and single guide RNA were either prepared immediately before experiments or prepared and snap-frozen in liquid nitrogen and stored at -80°C for later use.
  • the CasX protein was incubated with sgRNA at 1 : 1.2 molar ratio. Briefly, sgRNA was added to Buffer# 1 (25 mM NaPi, 150 mM NaCl, 200 mM trehalose, 1 mM MgC12), then the CasX was added to the sgRNA solution, slowly with swirling, and incubated at 37°C for 10 min to form RNP complexes.
  • Buffer# 1 25 mM NaPi, 150 mM NaCl, 200 mM trehalose, 1 mM MgC12
  • RNP complexes were filtered before use through a 0.22 pm Costar® 8160 filters that were pre-wet with 200 pl Buffer#l. If needed, the RNP sample was concentrated with a 0.5 ml Ultra 100-Kd cutoff filter, (MilliporeTM part #UFC510096), until the desired volume was obtained. Formation of competent RNP was assessed as described below.
  • the ability of CasX variants to form active RNP compared to reference CasX was determined using an in vitro cleavage assay.
  • the beta-2 microglobulin (B2M) 7.37 target for the cleavage assay was created as follows.
  • DNA oligos with the sequence TGAAGCTGACAGCATTCGGGCCGAGATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGC GCT (non-target strand, NTS (SEQ ID NO: 968)) and AGCGCGAGCACAGCTAAGGCCACGGAGCGAGACATCTCGGCCCGAATGCTGTCAGC TTCA (target strand, TS (SEQ ID NO: 969)) were purchased with 5’ fluorescent labels (LI- CORTM IRDye® 700 and 800, respectively).
  • dsDNA targets were formed by mixing the oligos in a 1 : 1 ratio in lx cleavage buffer (20 mM Tris HC1 pH 7.5, 150 mM NaCl, 1 mM TCEP, 5% glycerol, 10 mM MgCh), heating to 95° C for 10 minutes, and allowing the solution to cool to room temperature.
  • CasX RNPs were reconstituted with the indicated CasX and guides (see graphs) at a final concentration of 1 pM with 1.5-fold excess of the indicated guide unless otherwise specified in 1 * cleavage buffer (20 mM Tris HC1 pH 7.5, 150 mM NaCl, 1 mM TCEP, 5% glycerol, 10 mM MgCh) at 37° C for 10 min before being moved to ice until ready to use.
  • the 7.37 target was used, along with sgRNAs having spacers complementary to the 7.37 target.
  • Cleavage reactions were prepared with final RNP concentrations of 100 nM and a final target concentration of 100 nM.
  • CasX acts essentially as a single-turnover enzyme under the assayed conditions, as indicated by the observation that sub-stoichiometric amounts of enzyme fail to cleave a greater-than-stoichiometric amount of target even under extended time-scales and instead approach a plateau that scales with the amount of enzyme present.
  • the fraction of target cleaved over long time-scales by an equimolar amount of RNP is indicative of what fraction of the RNP is properly formed and active for cleavage.
  • the cleavage traces were fit with a biphasic rate model, as the cleavage reaction clearly deviates from monophasic under this concentration regime, and the plateau was determined for each of three independent replicates. The mean and standard deviation were calculated to determine the active fraction (Table 10).
  • Apparent active (competent) fractions were determined for RNPs formed for reference CasX2 + guide 174 + 7.37 spacer, CasX 119 + guide 174 + 7.37 spacer, CasX 457 + guide 174 +7.37 spacer, CasX 488 + guide 174 + 7.37 spacer, and CasX 491 + guide 174 + 7.37 spacer, as shown in FIG. 1.
  • the determined active fractions are shown in Table 10. All CasX variants had higher active fractions than the wild-type CasX2, indicating that the engineered CasX variants form significantly more active and stable RNP with the identical guide under tested conditions compared to wild-type CasX.
  • Cleavage-competent fractions were also determined using the same protocol for CasX2 protein in combination with guides 2, 32, 64 and 174 and targeting sequence 7.37 (CasX2.2.7.37, CasX2.32.7.37, CasX2.64.7.37), and CasX2.174.7.37 to be 16 ⁇ 3%, 13 ⁇ 3%, 5 ⁇ 2%, and 22 ⁇ 5%, as shown in FIG. 2 and Table 10.
  • CasX RNPs were reconstituted with the indicated CasX protein (see FIG. 4) at a final concentration of 1 pM with 1.5-fold excess of the indicated guide in l x cleavage buffer (20 mM Tris HC1 pH 7.5, 150 mM NaCl, 1 mM TCEP, 5% glycerol, 10 mM MgCh) at 37° C for 10 min before being moved to ice until ready to use.
  • Cleavage reactions were set up with a final RNP concentration of 200 nM and a final target concentration of 10 nM. Reactions were carried out at 37° C except where otherwise noted and initiated by the addition of the target DNA.
  • CasX488 and CasX491 had the highest cleavage rates by a large margin; as the target was almost entirely cleaved in the first timepoint, the true cleavage rate exceeds the resolution of this assay, and the reported kcieave should be taken as a lower bound.
  • Cleavage assays were also performed with wild-type reference CasX2 and reference guide 2 compared to gRNA variants 32, 64, and 174 to determine whether the variants improved cleavage.
  • the experiments were performed as described above. As many of the resulting RNPs did not approach full cleavage of the target in the time tested, initial reaction velocities (Vo) were determined rather than first-order rate constants. The first two timepoints (15 and 30 seconds) were fitted with a line for each CasX:sgRNA combination and replicate. The mean and standard deviation of the slope for three replicates were determined.
  • the Vo for CasX2 with guides 2, 32, 64, and 174 were 20.4 ⁇ 1.4 nM/min, 18.4 ⁇ 2.4 nM/min, 7.8 ⁇ 1.8 nM/min, and 49.3 ⁇ 1.4 nM/min (see Table 10 and FIGS. 5and FIG. 6).
  • Guide 174 showed substantial improvement in the cleavage rate of the resulting RNP ( ⁇ 2.5-fold relative to 2, see FIG. 6), while guides 32 and 64 performed similar to or worse than guide 2.
  • guide 64 supports a cleavage rate lower than that of guide 2 but performs much better in vivo (data not shown).
  • sequence alterations to generate guide 64 likely improve in vivo transcription at the cost of a nucleotide involved in triplex formation. Improved expression of guide 64 likely explains its improved activity in vivo, while its reduced stability may lead to improper folding in vitro.
  • RNPs of 515.174 and 526.174 had competent fractions of 76% and 91%, respectively, as compared to 16% for 2.2 (FIG. 8, Table 10).
  • both 515.174 and 526.174 cut essentially all of the target DNA by the first timepoint, exceeding the resolution of the assay and resulting in estimated cleavage rates of 17.10 and 19.87 min' 1 , respectively (FIG. 9, Table 10).
  • An RNP of 2.2 by contrast, cut on average less than 60% of the target DNA by the final 10-minute timepoint and has an estimated kcieave nearly two orders of magnitude lower than the engineered RNPs.
  • the modifications made to the protein and guide have resulted in RNPs that are more stable, more likely to form active particles, and cut DNA much more efficiently on a per-particle basis as well.
  • RNA fold structures were generated with RNAfold web server and Varna java-based software.
  • plasmid pXDPl was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments containing CasX variant, HIV-1, or MS2 CP components were amplified and cloned using In-Fusion® primers with 15-20 base pair overlaps and KAPA HiFi DNA polymerase according to the manufacturer’s protocols. The fragments were purified by gel extraction and cloned into plasmid backbones using In-Fusion® HD Cloning Kit from Takara (Cat# 639650) according to the manufacturer’s protocols. Assembled products were transformed into chemically-competent Turbo Competent E.
  • the amino acid sequence of the MS2 CP is provided in SEQ ID NO: 4140, and the amino acid sequence of the Gag polyprotein fused to the MS2 CP is provided in SEQ ID NO: 4141.
  • the tdTomato targeting guide plasmids used in these experiments were pSG50 (guide scaffold 188; FIG. 12) and pSG54 (guide scaffold 228; FIG. 13), which were cloned from pSG33 and pSG34, respectively.
  • the mammalian expression backbone contained a cPPT, ampicillin resistance, and a colEI replication site and was amplified using primers with appropriate overlaps to accept the U6 promoter and guide RNA scaffold cassette.
  • the backbone, pSG3 was digested using Ndel and Xbal. Synthetic DNA fragments corresponding to scaffold variants were amplified and cloned as described, above.
  • the resultant plasmids, pSG33 and pSG34 were sequenced using Sanger sequencing to ensure correct assembly (Table 12). Cloning tdTomato spacer 12. 7 into pSG3 and pSG14
  • the spacer 12.7 was cloned using the following protocol.
  • the targeting spacer sequence DNA for the tdTomato targeting spacer 12.7 was ordered as single-stranded DNA (ssDNA) oligos (Integrated DNA Technologies) consisting of the targeting sequence (CTGCATTCTAGTTGTGGTTT, SEQ ID NO: 1018) and the reverse complement of this sequence.
  • HEK293T Lenti-XTM cells were maintained in 10% FBS supplemented DMEM with HEPES and GlutaMAXTM (Thermo Fisher®). Cells were seeded in 15 cm dishes at 20 x 10 6 cells per dish in 20 mL of media. Cells were allowed to settle and grow for 24 hours before transfection. At the time of transfection, cells were 70-90% confluent.
  • the XDP structural plasmids also encoding the CasX variants
  • Table 13 were used in amounts ranging from 13 to 80.0 pg. Each transfection also received 13 pg of p42.174.12.7 and 0.25 pg of pGP2.
  • Polyethylenimine (PEI MAX®, Polyplus) was then added to the plasmid mixture, mixed, and allowed to incubate at room temperature before being added to the cell culture. Plasmid ratios in Table 11 were used in all version 206 XDPs used in this assay, based on prior experimental data from other XDP versions.
  • XDPs were resuspended in 300 pL of DMEM/ F12 supplemented with GlutaMAXTM, HEPES, non-essential amino acids, Pen/Strep, 2-mercaptoethanol, B-27 without vitamin A, and N2.
  • tdTomato neural progenitor cells were resuspended and transduced with XDPs.
  • tdTomato NPCs were grown in DMEM/F12 supplemented with GlutaMAXTM, HEPES, NEAA, Pen/Strep, 2-mercaptoethanol, B-27 without vitamin A, and N2.
  • Cells were harvested using StemPro Accutase Cell Dissociation Reagent and seeded on PLF-coated 96-well plates. 48 hours later, cells were transduced with XDPs containing a tdTomato targeting spacer. Cells were then centrifuged for 15 minutes at 1000 x g.
  • Transduced NPCs were grown for 96 hours before analyzing tdTomato fluorescence by flow cytometry as a marker of editing at the tdTomato locus, with the EC50 determined as the number of XDP particles needed to achieve editing in 50% of the cells, as determined by flow cytometry.
  • tdTomato fluorescence was measured using flow cytometry. The assays were run 2-3 times for each sample, with similar results.
  • the MS2 bacteriophage relies on the non-covalent affinity between its genomic RNA and the MS2 coat protein for the packaging of its genome in an icosahedral viral shell.
  • the high- affinity element in the RNA genome is termed the MS2 hairpin, which binds to the coat protein with a Kd of approximately 3e' 9 .
  • two high affinity variants of the MS2 hairpin were incorporated into the extended stem of the guide scaffold 174, thereby introducing into the CasX:guide RNP an affinity for the MS2 coat protein.
  • the resulting guide scaffolds 188 and 228 were tested in XDP version 168; a version that relies on a Gag-CasX fusion configuration and lacks the MS2 coat protein, while version 206 (FIG. 11) has the incorporated MS2 coat protein fused to Gag.
  • Guides 188 and 228 performed similarly to guide scaffold 174 in total editing across all volumes tested, demonstrating that the insertion of the MS2 hairpin was benign to the function of the RNP.
  • the MS2 hairpin variant sequences of these scaffolds are ACATGAGGATCACCCATGT (SEQ ID NO: 1131) and CGTACACCATCAGGGTACG (SEQ ID NO: 1132), respectively.
  • XDP version 206 This version is composed of the Gag-(-l)-PR, Gag-MS2, and CasX architectures. This version relies on orthogonal recruitment of CasX via the MS2 coat protein and MS2 hairpin system of the guide rather than a direct fusion between CasX and a recruiting protein. This is demonstrated in FIG. 15, where constructs with both guide scaffold 188 and 228 edit well in the tdTomato assay, in contrast to constructs with guide scaffold 174, which lacks the MS2 hairpin and edits poorly. Additionally, XDP version 206 with scaffold 188 edits better at the same dosage over XDP version 168 with scaffold 174 (see FIGS. 14 and 15).
  • the CasX protein and guide scaffold RNA form the apoenzyme RNP in the cytoplasm of the producer cell that then binds the Gag-MS2 protein by interactions of the MS2 hairpin in the guide extended stem and the MS2 coat protein.
  • the second possible mechanism is that the guide scaffold RNA hairpin first binds the MS2 coat protein and then forms the apoenzyme with the CasX protein.
  • MS2 coat protein variants have several point mutations that alter their affinity to its hairpin RNA. Usage of these variants in version 206 could result in higher potency variants. Fusing multiple coat proteins to the HIV Gag protein could further increase potency as well.
  • NCR RNA hairpin - non-covalent recruitment
  • RNA combinations from humans and retroviruses include the Iron Responsive Element (IRE)-Iron Binding element, U1 hairpin II, retrovirus Tat-trans-activation response (TAR) system, Csy4, Pardaxin, tRNA or Psi-Nucleocapsid.
  • IRE Iron Responsive Element
  • TAR retrovirus Tat-trans-activation response
  • Csy4 Pardaxin
  • tRNA or Psi-Nucleocapsid Table 12: sgRNA encoding sequences
  • the MS2 packaging system consists of two major components: the phage coat protein and its cognate binding partner, which is a short hairpin stem loop structure.
  • the short hairpin stem loop structure is engineered into the sgRNA incorporated into the XDP.
  • the encoding sequence for the phage coat protein is fused to either the encoding sequence for the Gag polyprotein (derived from any retroviruses) or to any other protein domains derived from the Gag polyprotein of any retroviral origin.
  • RNA fold structures were generated with RNAfold web server and Varna java-based software.
  • pXDPl was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments containing CasX variant, HIV-1, or MS2 CP components were amplified and cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
  • MS2 was placed either on the N- or the C-terminus of the Capsid (Version 263- pXDP276, Version 264-pXDP277, Version 265-pXDP278 and Version 266-pXDP279), with and without cleavage sites.
  • MS2 was placed either on the N- or the C-terminal of the Nucleocapsid (Version 267-pXDP280, Version 268-pXDP281, Version 269-pXDP282 and Version 270-pXDP283), with and without cleavage sites.
  • the sequences for these constructs are provided in Table 16.
  • the designed constructs were synthesized as transgenes and purchased pre-cloned into pTWIST expression plasmids from Twist Bioscience®. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
  • the guide plasmids used in these experiments were pSG50 and pSG17, encoding guide scaffold 188. Spacer 12.7 was cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation.
  • the guide plasmid used in all MS2 constructs is pSG50.
  • the guide plasmid used in control construct (VI 68) is pSG17.
  • HEK293T Lenti-XTM cell culture was performed as described in Example 2, above.
  • the XDP structural plasmids of Table 16 were used in amounts ranging from 13 to 80.0 pg. Each transfection also received 13 pg of pSG50 or pSG17 and 0.25 pg of pGP2.
  • Example 2 Polyethylenimine (PEI MAX® from Polyplus) was then added as described in Example 2. XDPs were collected and concentrated as described in Example 2, above. tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above. tdTomato fluorescence was measured using flow cytometry. The assays were run 2-3 times for each sample, with similar results.
  • PEI MAX® Polyethylenimine
  • FIG. 52 Fusing MS2 to the N- or C-terminal of nucleocapsid with and without a cleavage site may be superior to a capsid fusion, with a fusion to the N-terminal of NC being marginally better in terms of editing as shown in FIG. 53.
  • the EC50 for the different constructs were calculated and plotted as shown in FIG. 54 and recapitulates the differences in potency described above.
  • FIG. 16 depicts the fold improvement in EC50 over the base control VI 68 (CasX fused to full length HIV Gagpolyprotein) and it shows that V265, V269 and V270 show about 5 to 8-fold improvement in potency.
  • FIG. 17 depicts the fold improvement in EC50 over the base control V206 (MS2 fused to full length HIV Gag-polyprotein and the results demonstrate that V265, V269 and V270 show about 6- to 9-fold improvement in terms of overall editing potency.
  • this enhanced architecture can be translated to proteins derived from the Gag polyproteins of Alpharetroviral, Betaretroviral, Gammaretroviral, Deltaretroviral, Epsilonretroviral, Lentiviral and Spumaretroviral origin, serving as an orthogonal recruitment mechanism for CasX or any other payload that can be coupled with a cognate short hairpin RNA element in an XDP or other particle-delivery system.
  • MS2 was fused to the Gag-protease, Gag or partial Gag polyproteins derived from Alpharetroviruses (Versions 271, 272, 273), Betaretroviruses (Versions 277, 279), Gammaretroviruses (Versions 276, 278), Deltaretroviruses (Versions 274, 275) and Lentiviruses (Versions 280, 281, 282) with their respective species-specific cleavage sites.
  • the sequences for these constructs are provided in Table 18.
  • the designed constructs were synthesized as transgenes and purchased pre-cloned into pTWIST expression plasmids from Twist Biosciences. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
  • the guide plasmid used in these experiments was pSG50. To clone the targeting pSG50 spacer 12.7 was cloned in as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation. pGP2 Glycoprotein plasmid cloning
  • HEK293T Lenti-XTM cell culture was performed as described in Example 2, above.
  • the XDP structural plasmids of Table 18 were used in amounts ranging from 13 to 80.0 pg. Each transfection also received 13 pg of p42.174.12.7 and 0.25 pg of pGP2.
  • Polyethylenimine (PEI MAX® from Polyplus) was then added as described in Example 2. Collection and concentration; resuspension and transduction
  • tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above, and tdTomato fluorescence was measured using flow cytometry. The assays were run 2-3 times for each sample with similar results.
  • V275 derived from HTLV1 (Deltaretroviruses)
  • V279 derived from MPMV (Betaretroviruses)
  • V281 derived from EIAV (Lentivirus) showed successful editing ranging from 76.5, 61.6, to 48.7% at the tdT locus, respectively.
  • V273 derived from RSV, Alpharetroviruses
  • V274 derived from BLV, Deltaretroviruses
  • V276 derived from FLV, Gammaretroviruses
  • NIH derived from MMTV, Betretroviruses
  • V278 derived from MMLV, Gammaretroviruses
  • V280 derived from EIAV, Lentivirus
  • V282 derived from SIV, Lentivirus
  • V280 has MS2 fused to Gag-pro polyprotein
  • V281 has MS2 fused to the MA-CA polyprotein.
  • All plasmids encoding CasX proteins utilized the CasX 491 variant protein.
  • All XDPs contained sgRNAs with scaffold 188 (see FIG. 12) and spacer 12.7.
  • pXDPl was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments encoding CasX variant, HIV-1, or MS2 CP components were amplified and cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
  • the tdTomato targeting guide plasmid used in these experiments was pSG50 (guide scaffold 188), which was cloned from pSG33.
  • the mammalian expression backbone contained a cPPT, ampicillin resistance, and a colEI replication site and was amplified using primers with appropriate overlaps to accept the U6 promoter and guide RNA scaffold cassette.
  • the backbone, pSG3, was digested using Ndel and Xbal. Synthetic DNA fragments corresponding to novel scaffolds were amplified and cloned as described in Example 2, above.
  • the resultant plasmid, pSG33 was sequenced using Sanger sequencing to ensure correct assembly.
  • HEK293T Lenti-XTM cell culture was performed as described in Example 2, above.
  • the XDP structural plasmids (also encoding the CasX variants) of Table 21 were used in amounts ranging from 13 to 80.0 pg. Each transfection also received 13 pg of pSG50 and 0.25 pg of pGP2.
  • Polyethylenimine (PEI MAX® from Polyplus) was then added as described in Example 2. Plasmid ratios in Table 19 were used in all Version 206 XDPs used in this assay and are based on prior data from other XDP versions.
  • XDPs were collected and concentrated as described in Example 2, above.
  • tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above, and tdTomato fluorescence was measured using flow cytometry. The assays were run 2-3 times for each sample, with similar results.
  • Table 20 sgRNA and hairpin encoding sequences (DNA)
  • MS2 variants had affinity KdS ranging from 1.2e-7 M to 4e-10 M, with the wild-type version being 3e-9 M (a lower Kd value indicates greater affinity between the MS2 hairpin and coat protein).
  • Results of the assays showed that the XDP with MS2 having lower Kd variants tended to perform with better editing than higher Kd variants (see Table 22) with a gRNA having a single MS2 hairpin (gRNA 188).
  • the data were analyzed with a correlation analysis between the Kd of the MS2 coat protein and the inverse of the EC50 (by volume of XDP introduced into assay); a measure of potency that increases with more potent XDP constructs.
  • NCR non-covalent recruitment
  • RNA fold structures were generated with RNAfold web server and Varna java-based software.
  • pXDPl was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments containing CasX, HIV-1, retrovirus Tat, IRP1, IRP2, truncated U1A, U1A, phage QP coat protein, phage GA coat protein, phage kN coat protein, or truncated phage kN coat protein components were amplified using In Fusion primers with 15-20 base pair overlaps and Kapa HiFi DNA polymerase according to the manufacturer’s protocols. The fragments were purified by gel extraction from a 1% agarose gel using Zymoclean Gel DNA Recovery Kit according to the manufacturer’s protocol.
  • fragments containing dual boxB hairpin, retrovirus transactivation response (TAR) element, Iron Responsive Element (IRE), U1A hairpin, phage QP hairpin, phage GA hairpin, phage kN hairpin, or phage PP7 hairpins were amplified and cloned in guide scaffolds based on guide scaffold 174 or guide scaffold 235. Sequences of guide RNA scaffolds with dual hairpins are provided in Table 26, below. Scaffolds 188 and 251 were used as controls.
  • the guide plasmids modified in these experiments were pSG50, encoding guide scaffold 188 (see FIG. 12).
  • the non-targeting guide plasmids used in these experiments were pSG82 to pSG88, encoding guide scaffold 188.
  • the mammalian expression backbone had a cPPT, ampicillin resistance, and a colEI replication site and was amplified using primers with appropriate overlaps to accept the U6 promoter and guide RNA scaffold cassette.
  • HEK293T Lenti-XTM cell culture was performed as described in Example 2, above.
  • the XDP structural plasmids (also encoding the CasX variants) of Table 24 were used in amounts ranging from 13 to 80.0 pg. Each transfection will also receive 13 pg of a pSG plasmid and 0.25 pg of pGP2.
  • Polyethylenimine (PEI MAX® from Polyplus) was then added as described in Example 2.
  • XDPs were collected and concentrated as described in Example 2, above.
  • tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above, and tdTomato fluorescence was measured using flow cytometry. The assays were run 2-3 times for each sample with similar results. Titers were quantified for each version of XDPs using the NanoSight NS300.
  • the CasX guide scaffold extended stem region is highly modifiable.
  • the extended stem loop protrudes out from the RNP, and so additions to this region have little effect on RNP formation and editing potency, as seen in other experiments described herein.
  • This feature was used to add on one or two of several different RNA hairpins to the extended stem loop to engineer the CasX gRNA to bind their corresponding RNA-binding proteins.
  • Table 24 shows the sequences of the Gag-NCR protein plasmids and their complementary sgRNAs with nontargeting spacers that were employed to create the versions.
  • Table 25 shows the amino acid and RNA sequences of the Gag-NCR proteins and their complementary sgRNAs, respectively.
  • the amino acid sequences of the NCR proteins are provided in SEQ ID NOs: 4130-4139.
  • XDPs with the MS2, PP7, Tat, or U1 A NCR systems produced the highest levels of editing in the mouse tdTomato NPCs. Indeed, XDPs with the PP7, Tat or U1 A NCR systems produced higher levels of editing than XDPs with the MS2 NCR system. Both Tat and U1 A NCR systems are monomeric in nature. Therefore, that both Tat and U1 A NCR systems produced higher levels of editing suggests that MS2 dimerization has a detrimental effect on XDP architecture formation. It is anticipated that the relatively small size of the Tat protein could make it amenable to stacking (i.e., adding multiple Tat binding sites), which could enable better recruitment and packaging of the CasX RNP.
  • RNA hairpin and the NCR protein have a higher binding affinity (Ka of 1 nM) compared to that of the MS2 system (of Kd of - 2.6 nM). This may explain the higher level of editing observed with the PP7 system compared to the MS2 system (FIGS. 20 and 21).
  • Titers were quantified for each version of XDP particles produced using the NanoSight NS300, and the number of transduced mouse NPCs was counted.
  • the bar chart in FIG. 22 shows the number of XDPs containing the indicated NCR systems per edited mouse NPC
  • the bar chart in FIG. 23 shows the average number of XDPs containing the indicated NCR systems per mouse NPC.
  • use of the Gag-UIA, Gag-Tat, or Gag-PP7 NCR systems required the lowest average number of XDPs to edit a single mouse NPC (FIGS. 22 and 23), which is consistent with the high editing levels seen in FIGS. 20-21.
  • Table 25 Amino acid sequences of Gag-NCR proteins and RNA sequences of guide scaffolds based on guide scaffold 174
  • Table 26 shows the sequences of guide scaffolds based on guide scaffold 174 or scaffold 235, with two copies of each of the indicated hairpins.
  • the guide scaffolds in Table 26 were tested in combination with the NCR proteins provided in Table 25.
  • FIGS. 91-99 The results of the editing assays testing guide scaffolds with dual RNA hairpins are provided in FIGS. 91-99.
  • FIG. 92 use of guide scaffold 188, which has a single MS2 hairpin, produced a slightly lower editing potency than use of guide scaffold 251, which has two copies of the MS2 hairpin (“dual hairpin”). This is consistent with the results provided below in Example 9.
  • Example 7 Evaluation of non-covalent recruitment (NCR) systems with dual MS2 coat protein for RNA binding
  • pXDPl was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments containing CasX variant, HIV-1, or MS2 CP components were amplified and cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
  • the tdTomato targeting guide plasmid used in these experiments were pSG50 (scaffold 188; see FIG. 12) and pSG5 (scaffold 174), which were cloned from pSG33 and pSG3 respectively.
  • the mammalian expression backbone contained a cPPT, ampicillin resistance, and a colEI replication site and was amplified using primers with appropriate overlaps to accept the U6 promoter and guide RNA scaffold cassette.
  • the backbone was digested using Ndel and Xbal. Synthetic DNA fragments corresponding to guide scaffolds incorporating the MS2 hairpin were amplified and cloned as described in Example 2, above.
  • the resultant plasmids, pSG3 and pSG33 were sequenced using Sanger sequencing to ensure correct assembly.
  • HEK293T Lenti-XTM cell culture was performed as described in Example 2, above.
  • the XDP structural plasmids (also encoding the CasX variants) of Table 30 were used in amounts ranging from 13 to 80.0 pg. Each transfection also received 13 pg of pSG50 or pSG5 and 0.25 pg of pGP2.
  • the descriptions of the plasmids used to evaluate the NLS are listed in Table 29.
  • Polyethylenimine (PEI MAX® from Polyplus) was then added as described in Example 2. Plasmid ratios in Table 28 were used in all version 206 XDPs used in this assay and are based on prior data from other XDP versions.
  • XDPs were collected and concentrated as described in Example 2, above.
  • tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above, and tdTomato fluorescence was measured using flow cytometry.
  • tdTomato fluorescence was measured using flow cytometry. The assays were run 2-3 times for each sample, with similar results.
  • XDP version 309 is identical to version 206 except there is an additional MS2 CP fused to the first MS2 in this system, so pXDP164 (which encodes Gag-MS2) is replaced with pXDP288, which encodes Gag-MS2-MS2. While the hypothesis was that inclusion of the additional MS2 would increase the avidity of the RNP with MS2 hairpin in the scaffolds for these coat proteins, thereby increasing the incorporation of RNP into the budding XDP, it was observed that there was a significant decrease in editing with the constructs incorporating the second MS2 coat protein (see FIG. 24).
  • Example 8 Evaluation of non-covalent recruitment (NCR) systems with dual MS2 hairpins for MS2 coat protein binding
  • pXDPl was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments containing CasX variant, HIV-1, or MS2 CP components were amplified and cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
  • the tdTomato targeting guide plasmid used in these experiments were pSG72 (scaffold 250; see FIG. 25) and pSG68 (scaffold 251; see FIG. 26) which were cloned from pSG67 and pSG68 respectively.
  • the mammalian expression backbone contained a cPPT, ampicillin resistance, and a colEI replication site and was amplified using primers with appropriate overlaps to accept the U6 promoter and guide RNA scaffold cassette.
  • the backbone was digested using Ndel and Xbal. Synthetic DNA fragments corresponding to novel scaffolds were cloned as described in Example 2, above.
  • the resultant plasmids, pSG72 and pSG73, were sequenced using Sanger sequencing to ensure correct assembly (see Table 31).
  • HEK293T Lenti-XTM cell culture was performed as described in Example 2, above.
  • the XDP structural plasmids (also encoding the CasX variants) of Table 33 were used in amounts ranging from 13 to 80.0 pg. Each transfection also received 13 pg of pSG50 or pSG5 and 0.25 pg of pGP2.
  • Polyethylenimine (PEI MAX® from Polyplus) was then added as described in Example 2. Plasmid ratios in Table 32 were used in all version 206 XDPs used in this assay, based on prior data. Plasmid sequences are listed in Table 33. XDP version and components incorporated are listed in Table 34.
  • tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above, and tdTomato fluorescence was measured using flow cytometry. tdTomato fluorescence was measured using flow cytometry. The assays were run 2-3 times for each sample, with similar results.
  • This approach is applicable to not just MS2 hairpins but may apply to any RNA hairpin that can be used in CasX recruitment in XDPs such as TAR, Iron Responsive Element, U1 A RNA, phage QP hairpin, phage GA hairpin, phage AN hairpin, Cys4 RNA stem loop, or other element with an RNA that binds protein in a sequence specific interaction with high affinity.
  • the purpose of the experiments was to evaluate the utility of the MS2 and RRE systems in constructs to assess their ability to enhance the creation and potency of XDP.
  • the generation of XDPs is described in which CasX is recruited into the XDPs by fusing MS2 coat protein to different proteins within the HIV Gag polyprotein and the guide scaffold has one or two MS2 hairpins and portions of the HIV-1 Rev Response Element (e.g., the Rev Binding Element, or RBE).
  • All plasmids encoding CasX proteins utilized the CasX 491 variant protein. All XDPs were pseudotyped with 10% VSV-G. RNA fold structures were generated with RNAfold web server and Varna java-based software.
  • pXDPl was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments containing CasX, HIV-1, or MS2 CP components were amplified using In Fusion primers with 15-20 base pair overlaps and Kapa HiFi DNA polymerase according to the manufacturer’s protocols. The fragments were purified by gel extraction from a 1% agarose gel using ZymocleanTM Gel DNA Recovery Kit according to the manufacturer’s protocol. These fragments were cloned into plasmid backbones using In-Fusion HD Cloning Kit from Takara according to the manufacturer’s protocols. Assembled products were transformed into chemically-competent Turbo Competent E.
  • the tdTomato targeting guide plasmids used in these experiments were pSG17, pSG72 to pSG76 cloned from non-targeting plasmids pSG14 and pSG67 to pSG71, respectively.
  • the configurations and the sequences of these plasmids and the inserted elements are provided in Tables 36 and 37, respectively (see the listed Figures within the tables showing the sequence and configurations).
  • the mammalian expression backbone contained a cPPT, ampicillin resistance, and a colEI replication site and was amplified using primers with appropriate overlaps to accept the U6 promoter and guide RNA scaffold cassette.
  • the backbone was digested using Ndel and Xbal.
  • Synthetic DNA fragments corresponding to novel scaffolds were amplified and cloned as described in Example 2, above.
  • the resultant plasmids, pSG3 and pSG5, were sequenced using Sanger sequencing to ensure correct assembly (see Table 37 for description of construct).
  • the targeting spacer sequence DNA for the tdTomato targeting spacer 12.7 was cloned into pSG3, pSG14, pSG13, and pSG67 to pSG71 as described in Example 2, above.
  • the resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation (see Table 38).
  • HEK293T Lenti-XTM cell culture was performed as described in Example 2, above.
  • the XDP structural plasmids (also encoding the CasX variants) of Table 39 were used in amounts ranging from 13 to 80.0 pg.
  • Each transfection also received 13 pg of a pSG plasmid and 0.25 pg of pGP2.
  • Polyethylenimine (PEI MAX® from Polyplus) was then added as described in Example 2.
  • XDPs were collected and concentrated as described in Example 2, above.
  • tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above, and tdTomato fluorescence was measured using flow cytometry. The assays were run 2-3 times for each sample with similar results (see Table 40).
  • VSV-G Vesicular stomatitis virus envelope glycoprotein
  • XDPs VI 68 with scaffold 226 targeting tdTomato
  • RNA fold structures were generated with RNAfold web server and Varna javabased software.
  • pXDPl was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments containing CasX and HIV-1 components were amplified and cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. The structural plasmids and their sequences are listed in Table 41.
  • the guide plasmid used in these experiments was pSG17, which encodes the spacer 12.7 targeting tdTomato incorporated into the guide scaffold 226 that also has the RBE element described in previous examples.
  • spacer 12.7 was cloned as described in Example 2, above.
  • the resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation.
  • Encoding sequences for glycoproteins from different species within the Vesiculovirus genus were derived and are provided in Table 41.
  • the designed constructs were synthesized as transgenes and purchased pre-cloned into pTWIST expression plasmids from Twist Biosciences. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
  • pGP2 (which serves as the control GP) was cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Cell culture and transfection
  • HEK293T Lenti-XTM cell culture was performed as described in Example 2, above.
  • the XDP structural plasmids (sequences listed in Table 41) were used in amounts ranging from 13 to 80.0 pg. Each transfection also received 13 pg of p42.174.12.7 and 2.5 pg of pGP2.
  • Polyethylenimine (PEI MAX® from Polyplus) was then added as described in Example 2. Collection and concentration; resuspension and transduction
  • tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above, and tdTomato fluorescence was measured using flow cytometry. The assays were run 2-3 times for each sample, with similar results.
  • FIG. 36 shows that the XDPs pseudotyped with different glycoproteins produce to comparable levels with equivalent titers relative to the control construct.
  • Example 11 Enhancing tropism and editing potency with glycoprotein variants for XDP based on lentiviral and Alpharetrovirus constructs
  • VSV-G Vesicular stomatitis virus envelope glycoprotein
  • RNA fold structures were generated with RNAfold web server and Varna java-based software Structural plasmid cloning
  • pXDPl was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments containing CasX ALV and HIV-1 components were amplified and cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Plasmids and their sequences are listed in Table 42.
  • Table 42 Plasmid sequences for structural plasmids and glycoproteins Guide plasmid cloning
  • the guide plasmids used in these experiments were either pSG005 or pSG17.
  • pSG17 has both the spacer 12.7 targeting tdTomato as well as the guide scaffold 226 that has the RRE/RBE element that has been described in previous examples.
  • pSG005 has guide scaffold 174 along with the spacer 12.7 targeting tdTomato.
  • spacer 12.7 was cloned as described in Example 2, above.
  • the resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation.
  • Encoding sequences for glycoproteins derived from Togaviridae, Paramyxoviridae, Rhahdoviridae, Orthomyxoviridae, Retroviridae and Flaviviridae are provided in Table 42.
  • the designed constructs were synthesized as transgenes and purchased pre-cloned into pTWIST expression plasmids from Twist Bioscience. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly (see Table 42).
  • HEK293T Lenti-XTM cell culture was performed as described in Example 2, above.
  • XDPs derived from HIV lentiviral -based architecture (VI 68) were pseudotyped with GPs from Togaviridae (pGP65, 66, 67, 68, 69 and 70), Rhahdoviridae (pGP29.7, 30) and Moloney Murine leukemia virus (pGPIO).
  • XDPs derived from two different alpha retroviral -based architectures (ALV V44 and ALV V102) were pseudotyped with GPs from Rhahdoviridae (pGP29.7).
  • the XDP structural plasmids (configurations are listed in Table 42) were used in amounts ranging from 13 to 80.0 pg. Each transfection also received 13 pg of either pSG005 or pSG17 and 2.5 pg of pGP2 or any other GPs. Polyethylenimine (PEI MAX® from Polyplus) was then added as described in Example 2.
  • tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above, and tdTomato fluorescence was measured using flow cytometry. The assays were run 2-3 times for each sample with similar results.
  • Percent editing of the tdTomato target sequence in tdT NPCs are shown for all XDP constructs derived from HIV (VI 68) as well as XDP constructs derived from ALV (V44 and VI 02) in FIG. 38, in terms of volume of XDPs used to treat the cells. This is broken up further with the percent editing in tdT NPCs elicited when 50 pl and 16 pl of the concentrated XDP preps were used to treat NPCs, as shown in FIG. 39 and FIG. 40, respectively. Percent editing for the VI 68 XDPs pseudotyped with the different GPs, in terms of number of particles added to the tdTomato NPCs, are shown in FIG. 41.
  • VI 68 pseudotyped with pGP2 served as the base control XDP for comparisons.
  • the results show that GPs derived from Togaviridae (in particular Semliki, WEEV, EEEV, VEEV) and Rhabdoviridae (Mokola and Rabies), as well as MoMLV are potent in NPCs, suggesting properties of neural tropism.
  • GPs derived from Togaviridae such as pGP68, pGP68, pGP66 and pGP65 seemed particularly potent (in that order) ranging in editing efficiencies from 74% to 36% when 50 pl of concentrated XDPs were used to treat NPCs.
  • these GPs might show potency with other architectural variants of HIV based XDPs, in addition to XDPs derived from other architectural variants of Alpharetroviral, Betaretroviral, Gammaretroviral, Deltaretroviral, Epsilonretroviral, Lentiviral and Spumaretroviral origin.
  • XDPs derived from HIV lentiviral -based architecture were pseudotyped with GPs from different rabies variants from the Rhabdoviridae family (pGP29, 29.2, 29.3, 29.4, 29.5, 29.6, 29.8).
  • VI 68 pseudotyped with pGP2 served as the base control XDP for comparisons.
  • Several rabies variants showed potency in mouse NPCs, with pGP29 and pGP29.4 showing particular promise with editing efficiencies at the tdTomato locus ranging from 70% to 25% when 16.6 pl of the concentrated XDPs were used to treat NPCs, as shown in FIG. 43 and FIG. 44.
  • VI 68 pseudotyped with pGP2 demonstrated the most efficacy at 85%.
  • the rabies variants pGP29 and pGP29.4 would allow specific targeting of cells of neuronal origin, suggesting a better safety profile in vivo for neural indications, thereby making up for their lower editing potencies relative to VSV-G (pGP2).
  • XDPs derived from HIV lentiviral -based architecture were pseudotyped with GPs from Paramyxoviridae (pGP35.1, 35.2, 34.1, 34.2), Orthomyxoviridae (pGP80, 81, 82) and Flaviviridae (pGP25, 26, 27, 28, 75) families. Almost all the GPs showed activity at the 50 pl dose, as shown in FIG. 45.
  • XDPs pseudotyped with Orthomyxoviridae (pGP80, 82) and Paramyxoviridae (pGP35.1, 35.2, 34.1, 34.2) demonstrated about 35%, 11% and 10% editing, respectively, as shown in FIG. 46.
  • Titers for the VI 68 XDPs were determined by P24 ELISA as shown in FIG. 47 and demonstrate that pseudotyping XPDs with the different glycoproteins didn’t affect production titers.
  • V168 XDPs have been successfully pseudotyped with these diverse glycoproteins
  • Example 12 Enhancing RNA export mechanisms for the formation of XDP using a Rev/RRE system - Scaffold 174 vs 226
  • the purpose of these experiments was to evaluate the effects of incorporation of a portion of an HIV-1 Rev response element (RRE) sequence into the guide RNA scaffold to determine whether RNA export, recruitment of the guide into XDP, and resultant potency of the XDP was enhanced, with and without a direct Gag-CasX fusion.
  • the HIV-1 RRE is a -350 nucleotide RNA element in the HIV-1 genome that is recognized by the HIV-1 Rev protein and is essential for HIV-1 replication.
  • REV shuttles the HIV-1 RNA genome out of the nucleus into the cytoplasm by binding to the RRE, RanGTP, and Crml.
  • portions of the RRE element were incorporated into the extended stem region of the CasX scaffold 174 To enhance nuclear export of the sgRNAs into the cytoplasm of the XDP -producing LentiX cells.
  • the proposed recruitment mechanism using RRE elements in enhancing nuclear export of the gRNA is depicted in FIG. 51.
  • RNA fold structures were generated with RNAfold web server and Varna java-based software.
  • pXDPl was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments containing CasX or HIV-1 Gag components were amplified and cloned as described in Example 2, above. The sequence for Rev was incorporated into the backbone of the Gag plasmid. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
  • the tdTomato and PTBP-1 targeting guide plasmids used in these experiments were pSG5, pSG17, pSG47, and pSG48 cloned from pSG3 for the first and pSG14 for the latter 3 plasmids.
  • the mammalian expression backbone contained a cPPT, ampicillin resistance, and a colEI replication site and was amplified using primers with appropriate overlaps to accept the U6 promoter and guide RNA scaffold cassette.
  • the backbone was digested using Ndel and Xbal. Synthetic DNA fragments corresponding to novel scaffolds were amplified and cloned as described in Example 2, above.
  • the resultant plasmids, pSG3 and pSG5, were sequenced using Sanger sequencing to ensure correct assembly (see Table 43).
  • the targeting spacer sequence DNA for the tdTomato targeting spacer 12.7 was ordered as single-stranded DNA (ssDNA) oligos (Integrated DNA Technologies) consisting of the targeting sequence (CTGCATTCTAGTTGTGGTTT, SEQ ID NO: 1018) and the reverse complement of this sequence.
  • ssDNA single-stranded DNA
  • the targeting spacer sequence DNA for the tdTomato targeting spacer 12.2 was ordered as single-stranded DNA (ssDNA) oligos (Integrated DNA Technologies) consisting of the targeting sequence (TATAGCATACATTATACGAA, SEQ ID NO: 1541) and the reverse complement of this sequence.
  • the targeting spacer sequence DNA for the PTBP-1 targeting spacer 28.10 was ordered as single-stranded DNA (ssDNA) oligos (Integrated DNA Technologies) consisting of the targeting sequence (CAGCGGGGATCCGACGAGCT, SEQ ID NO: 1542) and the reverse complement of this sequence.
  • HEK293T Lenti-XTM cell culture was performed as described in Example 2, above.
  • the XDP structural plasmids (also encoding the CasX variants) of Table 44 were used in amounts ranging from 13 to 80.0 pg. Each transfection also received 13 pg of p42.174.12.7 and 0.25 pg of pGP2.
  • Polyethylenimine (PEI MAX® from Polyplus) was then added as described in Example 2.
  • the XDP versions, architectures and plasmids utilized in the transfection are listed in Table 45.
  • tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above. Editing of tdTomato was assessed by measuring fluorescence or by Next Generation Sequencing to assess rate of edits. The assays were run 2-3 times for each sample with similar results.
  • XDP version 207 lacks any architectural recruitment mechanism for CasX to be incorporated into the XDP.
  • XDPs with guide scaffold 174 were unable to edit NPCs in this construct whereas XDPs with scaffold 226 were able to achieve >20% editing (FIG. 57).
  • the editing data with XDP incorporating guide scaffold 226 demonstrate a consistent pattern of increased potency over XDP incorporating guide scaffold 174.
  • the data show that without changing the nuclease function, the potency of XDPs can be increased by designing constructs that incorporate an RNA nuclear export pathway such as the Rev/RRE system. These enhanced effects were seen across different gene targets and multiple spacers.
  • All plasmids encoding CasX proteins utilized the CasX 491 variant protein. All XDPs were pseudotyped with 10% VSV-G (percentage of plasmid relative to the other plasmids utilized for the XDP construct). RNA fold structures were generated with RNAfold web server and Varna java-based software.
  • pXDPl was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments containing CasX, HIV-1, or MS2 CP components were amplified and cloned as described in Example 2, above. CasX was tested with various combinations of NLS sequences, as summarized in Table 49. Specifically, each CasX-NLS combination had N- and C-terminal NLS made up of various NLS sequences (e.g., c-Myc, SV40, and nucleoplasmid NLS sequences) with various linkers. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
  • NLS sequences e.g., c-Myc, SV40, and nucleoplasmid NLS sequences
  • the guide plasmids used in these experiments were pSG50, pSG72, pSG73, and pSG76 which were cloned from non-targeting plasmids pSG33, pSG67, pSG68, and pSG71.
  • the mammalian expression backbone contained a cPPT, ampicillin resistance, and a colEI replication site and was amplified using primers with appropriate overlaps to accept the U6 promoter and guide RNA scaffold cassette. These fragments were amplified and cloned as described in Example 2, above.
  • the resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly (see Table 47). Table 47: Guide scaffold sequences
  • the targeting spacer sequence DNA for the tdTomato targeting spacer 12.7 was ordered as single-stranded DNA (ssDNA) oligos consisting of the targeting sequence (CTGCATTCTAGTTGTGGTTT, SEQ ID NO: 1018) and the reverse complement of this sequence. These two oligos were annealed together and cloned into pSG33, pSG67, pSG68, and pSG71 plasmids done by Golden Gate assembly, as described in Example 2, above.
  • the nontargeting (NT) spacer 0.0 encoded by the sequence CGAGACGTAATTACGTCTCG, SEQ ID NO: 1019) was used as a control and was cloned in a similar manner. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation.
  • HEK293T Lenti-XTM cell culture was performed as described in Example 2, above.
  • the XDP structural plasmids also encoding the CasX-NLS variants of Table 49
  • Table 48 were used in amounts ranging from 13 to 80.0 pg.
  • Each transfection also received 13 pg of sgRNA plasmid and 0.25 pg of pGP2.
  • Polyethylenimine was then added as described in Example 2, above.
  • Table 49 CasX-NLS plasmids, NLS descriptions, and NLS sequences for each tested NLS are bolded and italicized (PKKKRKV; SEQ ID NO: 35), and a nucleoplasmin NLS is underlined and italicized (KRPAATKKAGQAKKKK; SEQ ID NO: 36).
  • tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above, and tdTomato fluorescence was measured using flow cytometry.
  • the base V206 contains a CasX protein with flanking SV40 NLSs on the N- and C- terminal domains to increase potency by localizing CasX to the nucleus in the target cell.
  • NLS 115, 240, 247, 248, 251, 252, 255, 256, and 269 nine alternate NLS sequence combinations were tested, termed NLS 115, 240, 247, 248, 251, 252, 255, 256, and 269 (see Table 49).
  • Six of the nine tested NLS combinations performed better than the base SV40 NLS, with the top three variants being NLS 240, 248, and 251. These performed 11-, 10-, and 14-fold better by inverse EC50 by volume than the base V206 SV40 NLS (Table 51).
  • NLS 240 and 255 Two NLS variants, NLS 240 and 255, were selected to be tested with guide scaffolds that were engineered to contain a portion of the RRE, termed “RBE”.
  • Scaffolds 250, 251, and 254 each contain one or two RBEs and one or two MS2 hairpins (see Table 53). These scaffolds were compared to scaffold 188 (FIG. 12) which contains one MS2 hairpin and no RBEs.
  • the results of the assay show that scaffolds containing an RBE performed 2- to 6-fold better with NLS 240 and 10- to 23 -fold better with NLS 255 (see Table 52).
  • scaffold 251 performed best with a 6-fold increase with NLS 240 and a 23-fold increase with NLS 255. There was a slight decrease in potency with RBE containing scaffolds in base V206 with the least potent scaffold, scaffold 254, being 40% as potent as scaffold 188 in the base V206 XDP construct.
  • Table 52 Potency of RRE scaffolds
  • Table 53 Features of MS2 and RBE containing scaffolds
  • NLS variants can be designed that can increase the potency of the XDP, and that potency can be further enhanced with the use of guide scaffolds with incorporated RBE.
  • Example 14 Enhancing export mechanisms - NLS Variants +/- RRE evaluated in vivo [0586] The purpose of these experiments was to evaluate the effects on in vivo editing potency of the addition of NLS sequences to the N- and/or C-terminal end of CasX and RRE into guide RNA sequences that are incorporated into XDP constructs.
  • RNA fold structures were generated with RNAfold web server and Varna java-based software.
  • the guide plasmids used in these experiments were either pSG005 or pSG17.
  • pSG17 has both the spacer 12.7 targeting tdTomato as well as the scaffold 226 that has the RRE/RBE element that has been described in previous examples.
  • pSG005 has the scaffold 174 along with the spacer 12.7 targeting tdTomato.
  • spacer 12.7 was cloned as described in Example 2, above.
  • the resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation.
  • HEK293T Lenti-XTM cell culture was performed as described in Example 2, above.
  • Structural plasmid 1 for XDP Version 1 (pXDP17), Version 310 (pXDP240) and Version 311 (pXDP255) all encode for CasX 491 with different NLS.
  • the amino acid sequence of CasX with NLS for pXDP240 is provided in SEQ ID NO: 1631
  • the amino acid sequence of CasX with NLS for pXDP255 is provided in SEQ ID NO: 1646.
  • Structural plasmid 2 for all the versions is pXDPOOl.
  • the guide plasmids used in these experiments were either pSG005 or pSG17.
  • pSG17 has both the spacer 12.7 targeting tdTomato as well as the scaffold 226.
  • the plasmid encoding the glycoprotein was pGP2.
  • the XDP structural plasmids listed above and in Table 54 were used in amounts ranging from 13 to 80.0 pg. Each transfection also received 13 pg of pSG17 (gRNA) and 0.25 pg of pGP2. Polyethylenimine (PEI MAX® from Polyplus) was then added as described in Example 2.
  • Table 55 Plasmid sequences for different Gag-CasX NLS constructs as well XDP structural plasmids
  • the XDP filtered supernatant was divided evenly into an appropriate number of centrifuge tubes or bottles and l/5th of the supernatant volume of sucrose buffer (50mM Tris- HCL, lOOmM NaCl, 10% sucrose, pH 7.4) was underlaid using serological pipettes.
  • sucrose buffer 50mM Tris- HCL, lOOmM NaCl, 10% sucrose, pH 7.4
  • the samples were centrifuged at 10,000 x g, 4°C, in a swinging-bucket rotor for 4 hours with no brake.
  • the supernatant was carefully removed and the pellet briefly dried by inverting the centrifuge vessels.
  • Pellets were resuspended in Storage Buffer (PBS + 113 mM NaCl, 15% trehalose dihydrate, pH8) by gentle trituration and vortexing.
  • mice were group housed and experiments were conducted in conformance with approved IACUC protocols. Prior to infection, mice were anesthetized with isoflurane. The anesthetized mouse was aligned on an Angle two stereotactic frame (Leica, Germany) and craniotomies were performed by stereotaxic surgery to target the substantia nigra (SN). Mice received a unilateral XDP injection with 8.15xl0 8 particles of one of the three XDP test articles. Mice were sacrificed 3 weeks post-injection, brains harvested and fixed with 4% PFA and cryosectioned (10 pm thick sections) and mounted on microscope slides. TH+ dopaminergic neurons in the SN were labeled with TH antibody and cell nuclei labeled with DAPI.
  • the XDPs differed in the composition and arrangement of the nuclear localization signals (NLS) on the CasX protein. 8.15xl0 8 XDPs of each preparation were delivered as determined by the Nanosight physical titering method to the substantia nigra (SN). Tyrosine hydroxylase (TH) antibody staining marks SN dopaminergic neurons.
  • XDP version 1 showed sparse editing activity in astrocytes surrounding the TH+ neurons. Significantly more editing activity was observed (approx. 10 to 100-fold), as determined by the amount of tdTomato+ cells, with XDP versions 310 (pXDP240) and 311 (pXDP255) compared to version 1 (FIG. 60).
  • editing potency of XDPs was improved using a recruitment strategy in which the gRNA of the CasX:gRNA RNP complex contained a functionalized RNA extended stem region with an MS2 hairpin having high affinity for a Gag-MS2 RNA-binding protein (RBP). Binding of the RNA hairpin to the MS2 RBP facilitates enhanced recruitment of the CasX RNP cargo to the budding XDP particle. Upon delivery of the XDP to the target cell for editing, this RNA hairpin-MS2 RBP is expected to dissociate, allowing CasX to translocate to the nucleus.
  • RBP Gag-MS2 RNA-binding protein
  • gRNAs incorporating RNA hairpin variants with varying affinities for the MS2 RBP were evaluated using a high-throughput, in vitro biochemical assay to assess equilibrium binding and dissociation kinetics (Buenrostro et al., Quantitative analysis of RNA-protein interactions on a massively parallel array reveals biophysical and evolutionary landscapes. Nat Biotechnol. 32(6):562 (2014)).
  • gRNA hairpin variants and their associated Kd (dissociation constant) values are listed in Table 56, sequences of the guide plasmids encoding the different MS2 RNA hairpin variants are provided in Table 57 and the sequences of the MS2 hairpins are provided in Table 58.
  • Table 56 gRNA scaffolds containing MS2 hairpin variants with varying affinities and their dissociation constant values (Kd). Specific positions for the indicated nucleotide mutations refer to the positions of the base MS2 hairpin (scaffold 188) depicted in FIG. 62
  • All plasmids encoding CasX proteins utilized CasX variant 491. All XDPs were pseudotyped with 10% VSV-G (percentage of VSV-G plasmid relative to other XDP structural plasmids). RNA fold structures were generated with RNAfold web server and VARNA software. The methods to produce XDPs are described herein, as well as in WO2021113772A1, incorporated by reference in its entirety.
  • the Gag-pol sequence was removed from pXDPl, and amplified and purified fragments encoding CasX 491, HIV-1, or MS2 CP components were cloned as described in Example 2, above. Individual colonies were picked, miniprepped, and Sanger-sequenced for assembly verification. Plasmid sequences are listed in Table 57.
  • HEK293T Lenti-XTM cells were seeded in 15 cm dishes at 20 x 10 6 cells per dish 24 hours before transfection to reach 70-90% confluency. The next day, Lenti-XTM cells were transfected with the following plasmids using PEI MAX® (Polypus): XDP structural plasmids (also encoding the CasX variants; Table 57), pSG50 (or other guide plasmid variants listed in Table 57), and pGP2 for XDP pseudotyping. 24 hours post-transfection, media was replaced with Opti-MEM (Thermo Fisher).
  • XDP-containing media was collected 72 hours posttransfection and filtered through a 0.45 pm PES filter. The supernatant was concentrated and purified via centrifugation. XDPs were resuspended in 500 pL of DMEM/F12 supplemented with GlutaMAXTM, HEPES, NEAA, Pen/Strep, 2-mercaptoethanol, B-27 without vitamin A, and N2.
  • NPCs neural progenitor cells
  • XDPs composed of Gag-MS2, Gag-pro, CasX, gRNA scaffold variants, and VSV-G were produced as version 206 either with the original MS2 (MS2 WT) or an MS2 high-affinity variant (MS2 353).
  • Produced XDPs were subsequently assessed for their editing efficiency at the tdTomato locus in NPCs.
  • FIG. 63 shows the percent editing at the tdTomato locus as measured by tdTomato fluorescence using flow cytometry when 0.007 pL of concentrated XDP preps were used to transduce NPCs.
  • high- affinity scaffold variants 296 and 298 demonstrated enhanced potency with both MS2 WT and MS2 353, with Kd values ranging from 1.8 to 2.1 nM.
  • medium-affinity scaffold variants 303, 304, 305, 307, 310 and 313, with Kd values ranging from 9.2 to 36.9 nM resulted in promising editing efficiencies.
  • FIG. 64 illustrates EC50 results across the different gRNA scaffolds incorporating the MS2 WT and MS 353 configurations. Scaffold variants 296, 297, and 305 exhibited a slightly higher potency compared to scaffold 188, an advantage that was more evident with the MS2 353 configuration.
  • FIG. 64 illustrates EC50 results across the different gRNA scaffolds incorporating the MS2 WT and MS 353 configurations. Scaffold variants 296, 297, and 305 exhibited a slightly higher potency compared to scaffold 188, an advantage that was more evident with the MS2 353 configuration.
  • Example 16 Engineering of XDPs with a cytokine therapeutic payload
  • XDPs can be used to carry the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) as the therapeutic protein payload.
  • mouse or human GMCSF was directly fused to a Gag structural protein, as described in Table 59, below. Cloning was performed as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
  • ** 5' to 3' orientation f indicates a -1 frame-shift in the encoded construct (Gag-TFR-PR polyprotein)
  • HEK293T Lenti-XTM cell culture was performed as described in Example 2, above.
  • the XDP structural plasmids of Table 70 were used in amounts ranging from 13 to 80.0 pg.
  • ELISAs were performed to measure the amount of GM-CSF per XDP. Specifically, XDPs were lysed with the lysis reagent and the number of GM-CSF molecules packaged per XDP was quantified using the Mouse GM-CSF Quantikine® ELISA kit (R&D, Cat no. MGM00) and Human GM-CSF Quantikine® ELISA kit (R&D, Cat no. DGM00) per the manufacturer’s instruction. Results:
  • XDPs were engineered to carry human or mouse GM-CSF via the direct fusion of GM- CSF to the protein scaffold, and the amount of GM-CSF per XDP was measured via ELISA. As shown in Table 60, below, the XDPs contained GM-CSF, with between 40-527 molecules of GM-CSF per XDP. The results demonstrate that XDP constructs can be created to incorporate heterologous payloads, and in different configurations.
  • Example 17 Engineering of XDPs for incorporating catalytically-dead CasX repressor (dXR) system
  • XDPs were generated using the version 168 or version 206 configuration.
  • HEK293T Lenti-XTM cell culture was performed as described in Example 2, above.
  • XDPs were collected and concentrated as described in Example 2, above.
  • XDPs were engineered to carry a dXR system targeting the B2M locus for repression.
  • the XDPs were administered to human NPCs, and the level of B2M repression was measured.
  • both the version 168 and version 206 XDPs were able to induce repression of B2M.
  • the version 206 XDP with dCasX491 linked to the Zim3 KRAB domain produced the highest level of repression.
  • XDPs were generated using the version 168 configuration with guide scaffold 226, or the version 206 configuration with guide scaffold 251 (see FIG. 26) or guide scaffold 188 (see FIG. 12).
  • HEK293T Lenti-XTM cell culture was performed as described in Example 2, above. Collection and concentration
  • the resulting immunoblot was imaged using a ChemiDoc Touch, and the CasX protein levels were quantified by densitometry using Image Lab software from BioRad. Quantification of the CasX molecules in each XDP particle sample was determined using the standard curve.
  • XDP version 168 with guide scaffold 226 contained approximately 227-239 CasX molecules/XDP particle (FIG. 70) and, by inference, RNP.
  • the XDP version 206 with guide scaffold 188 contained approximately 240- 257 CasX molecules/XDP particle
  • XDP version 206 with guide scaffold 251 contained approximately 966-1112 CasX molecules/XDP particle, showing the superiority of scaffold 251 for facilitating incorporation of RNP into the XDP particles.
  • the fold differences relative to XDP version 168 with guide scaffold 226 are shown in FIG. 71.
  • Example 19 Evaluation of orthogonal recruitment system with MS2 linked to Gag plus a nuclear export signal (NES) linked to CasX
  • Cleavable NESes were added to the XDP version 206 system (plasmid configurations are shown in Table 62.
  • the NESes were linked to the C-terminus of CasX 676 via an HIV cleavage sequence and a rigid linker.
  • ** 5' to 3' orientation f indicates a -1 frame-shift in the encoded construct (Gag-TFR-PR polyprotein)
  • CRM1 chromosomal maintenance 1 plays a major role in the export of proteins with leucine-rich nuclear export signals.
  • Nuclear export signals that utilize the CRM1 nuclear export pathway with a range of affinities were selected and attached to the C-terminus of CasX in cleavable manner, such that during the maturation process post-XDP budding, the HIV protease would cleave the NES such that the CasX RNP would not have an attached NES when delivered into the target cell.
  • 15 different NESs that use the CRM1 pathway with different Rc/n and Kd values were selected (see Fu, S. et al., Mol Biol Cell.
  • the XDPs were transduced into human Jurkat T cells or neural progenitor cells (NPCs), and editing of the B2M locus was measured.
  • Example 20 Screen of XDPs with diverse incorporated viral glycoproteins to evaluate tropism and editing capabilities
  • glycoprotein belonging to VSV Indiana species within the Vesiculovirus genus is usually the most widely used glycoprotein for pseudotyping purposes.
  • the purpose of these experiments was to explore the transduction capabilities of glycoproteins belonging to other species, and test whether the cellular tropism of XDPs could be altered by pseudotyping XDPs with various glycoproteins as targeting moieties in various cell types.
  • the screen of glycoproteins was conducted in the XDP version 206 construct configuration.
  • the version 206 XDPs pseudotyped with glycoproteins of Table 64 were transduced into mouse tdTomato neural progenitor cells (NPCs), in which editing of the tdTomato locus was measured, or human Jurkat T cells, K562 lymphoblasts, ARPE-19 retinal pigment epithelial (RPE) cells, Y79 retinoblastoma cells, induced neurons, human NPCs, or astrocytes, in which editing of the B2M locus was measured.
  • NPCs mouse tdTomato neural progenitor cells
  • RPE retinal pigment epithelial
  • the XDPs were designed to contain ribonucleoproteins (RNP) of CasX 676 complexed with single guide RNA variant 251 having spacer sequence 12.7 targeted to tdTomato (encoded by CTGCATTCTAGTTGTGGTTT, SEQ ID NO: 1018) or spacer sequence 7.37 targeted to human B2M (SEQ ID NO: 2448).
  • RNP ribonucleoproteins
  • the XDPs were produced by transient transfection of LentiX HEK293T cells (Takara Bio) with two structural plasmids encoding components of the Gag-pol HIV-1 system, a plasmid encoding a pseudotyping glycoprotein, and a plasmid encoding the guide RNA.
  • the pStx42 plasmid was created with a human U6 promoter upstream of the guide RNA cassette A plasmid encoding a glycoprotein for pseudotyping the XDP was also used. All plasmids contained either an ampicillin or kanamycin resistance gene, were generated using standard molecular biology techniques, and were sequenced using Sanger sequencing to ensure correct assembly.
  • HEK293T Lenti-XTM cell culture was performed as described in Example 2, above. Collection and concentration
  • XDPs were transduced into tdTomato mouse NPCs, human Jurkat T cells, K562 lymphoblasts, ARPE-19 retinal pigment epithelial (RPE) cells, retinoblastoma Y79 cells, induced neurons, human NPCs, or human astrocytes.
  • tdTomato NPCs were resuspended and transduced with XDPs packaged with 12.7 spacer targeting the tdTomato locus as described in Example 2, above.
  • Human NPCs were grown in DMEM/ F12 supplemented with GlutaMAXTM, HEPES, non-essential amino acids, Pen/Strep, 2-mercaptoethanol, B-27 without vitamin A, and N2. Cells were harvested using StemProTM Accutase Cell Dissociation Reagent and seeded on PLF coated 96-well plates. Cells were allowed to grow for 24 hours before being treated for targeting XDPs (having a spacer for B2M) starting with a neatly resuspended virus and proceeding through 10 half-log dilutions. Cells were then centrifuged for 15 minutes at 1000 x g. Human NPCs were grown for 96 hours before analysis of B2M editing by flow cytometry.
  • Human astrocytes were similarly treated, where two independent biological replicates were also performed and analyzed (data are shown in FIGS. 77 and 85, where similar findings are observed).
  • Human induced neurons were grown in N2B27-based media. Briefly, to induce neuronal differentiation, iPSCs were plated in neuronal plating media (N2B27 base media with doxycycline, L-ascorbic acid, dibutyryl cAMP sodium salt, CultureOneTM, BDNF, and GDNF).
  • induced neurons were seeded on a 96-well plate at -30,000-50,000 cells per well and were cultured for at least one week in plating media and thereafter, half-media changes were performed once every week using feeding media (N2B27 base media with L-ascorbic acid, dibutyryl cAMP sodium salt, BDNF, and GDNF).
  • feeding media N2B27 base media with L-ascorbic acid, dibutyryl cAMP sodium salt, BDNF, and GDNF.
  • 30,000 induced neurons were seeded per well of a 96-well plate; 24 hours later, cells were transduced with targeting XDPs (having a B2M- targeting spacer) as described earlier.
  • Jurkat cells were grown in RPMI supplemented with FBS. 20,000 cells were transduced with the targeting XDPs (having a spacer for B2M) starting with neat-resuspended virus and proceeding through 10 half-log dilutions. Cells were then centrifuged for 15 minutes at 1000 x g. Jurkats were grown for 96 hours before analysis of B2M editing by flow. The assays were run 2 times for each sample with similar results. K562 lymphoblasts were similarly treated. [0639] ARPE-19 cells were grown in DMEM-F12 supplemented with FBS and Pen/Strep. Y79 cells were grown in RPMI supplemented with FBS.
  • 20,000 cells were transduced with the targeting XDPs (having a spacer for 2M) starting with neat-resuspended virus and proceeding through 10 half-log dilutions. Cells were then centrifuged for 15 minutes at 1000 x g. Jurkats were grown for 96 hours before analysis of B2M editing by flow cytometry.
  • tdTomato fluorescence and editing of the B2M locus was measured using flow cytometry. The assays were run 2-3 times for each sample, with similar results.
  • VSV-G-mediated cell entry occurs by binding to the low-density lipoprotein receptor (LDL-R), which is a ubiquitous receptor found on most cell types. Accordingly, the tropism of XDPs pseudotyped with VSV-G is broad. To alter the tropism of XDPs relative to XDPs pseudotyped with VSV-G, XDPs were generated with diverse viral glycoproteins as targeting moieties.
  • LDL-R low-density lipoprotein receptor
  • a comparison of the mouse and human NPC editing data revealed that the XDPs did not edit mouse and human NPCs at the same levels. Specifically, almost all XDPs with vesiculoviral glycoproteins showed a higher level of editing in mouse NPCs (FIG. 75) than they did in human NPCs (FIG. 76). XDPs with alphaviral glycoproteins showed a higher level of editing in human NPCs than in mouse NPCs. Interestingly, XDPs with rabies glycoprotein showed a higher level of editing in mouse NPCs than in human NPCs. Conversely, XDPs with Mokola glycoprotein showed a higher level of editing in human NPCs than in mouse NPCs.
  • XDPs with certain glycoproteins belonging to the vesiculoviral family showed substantially higher levels of editing in human astrocytes (FIGS. 77 and 85) than in either human NPCs (FIG. 76) or human induced neurons (FIG. 83). This finding may be particularly useful to skew XDP tropism towards glial cells rather than neurons, which would be beneficial for glial cell targets.
  • Example 21 Generation of exemplary version 206 XDPs
  • Plasmids encoding CasX proteins are generated to encode the CasX 491 variant, the CasX 515 variant, the CasX 676 variant, or the CasX 812 variant using methods described in previous examples.
  • Plasmids encoding guide scaffold 188 or guide scaffold 251, each with a targeting sequence are generated using methods described in previous examples.
  • Structural plasmids encoding retroviral components and NCR, guide plasmids, and pGP2 glycoprotein plasmids are cloned as described in Example 2, above. Exemplary DNA sequences of version 206 components are provided in Table 65.
  • HEK293T Lenti-XTM cell culture is performed as described in Example 2, above, using the five plasmids of Table 65, selecting either CasX 491, 515, 676, or 812 and guide 188 or 251. Collection and concentration
  • This process is expected to generate version 206 XDPs with either CasX 491, CasX 515, CasX 676, or CasX 812 and guide scaffold 188 or guide scaffold 251, complexed as RNP as the therapeutic payload, which are then evaluated for editing of target nucleic acid.
  • Example 22 Demonstration of dual-editing at two different genomic loci using two types of CasX RNPs packaged and delivered via a single XDP particle in vitro
  • XDP particles were generated to contain a CasX protein with gRNAs targeting the PTBP1 locus and the tdTomato STOP cassette and used to transduce mouse tdTomato neural progenitor cells (NPCs) to demonstrate editing at the two genomic loci in vitro.
  • NPCs mouse tdTomato neural progenitor cells
  • XDP configurations were used to generate XDPs in these experiments. Specifically, VI 68 XDPs were produced incorporating guide scaffold 226, while V206 XDPs were produced incorporating guide scaffold 251. XDPs were engineered to package two types of RNPs within a single XDP: CasX variant 491 complexed with a PTBP1 -targeting gRNA and CasX variant 491 complexed with a tt/Zomato-targeting gRNA. All XDP particles were pseudotyped with the VSV-G glycoprotein.
  • XDP structural plasmid cloning was performed as described in Example 2.
  • XDP production using HEK293T Lenti-XTM cells was performed as described in Example 2. Briefly, adherent Lenti-XTM cells were seeded in 15cm plates at 2E7 cells per plate in 20mL of media.
  • XDP structural plasmids encoding the HIV-1 Gag-pol structural components (as well as CasX 491 for VI 68), a plasmid encoding for CasX 491 (relevant for V206), a plasmid encoding a single gRNA with either scaffold 226 (for VI 68 XDPs) or scaffold 251 (for V206 XDPs) and PTSTV-targeting spacer 28.10 (CAGCGGGGAUCCGACGAGCU; SEQ ID NO: 982), a plasmid encoding a single gRNA with either scaffold 226 or 251 and tt/Zomato-targeting spacer 12.7 (CUGCAUUCUAGUUGUGGUUU; SEQ ID NO: 1855), and a plasmid encoding the VSV-G glycoprotein.
  • PEI MAX® Polypus
  • XDP-containing media was collected and filtered through a 0.45 pm PES filter. The supernatant was concentrated and purified via centrifugation. XDPs were resuspended in a freezing buffer.
  • XDPs containing dual-CasX RNPs using spacer 28.10 with a non-targeting (NT) spacer or dual-CasX RNPs using spacer 12.7 with an NT spacer were also produced and assessed for editing.
  • NT non-targeting
  • XDP transduction of tdTomato NPCs was performed as described in Example 2. Editing at the tdTomato locus was assessed by analyzing tdTomato fluorescence detected by flow cytometry, while editing at the PTBP1 locus was assessed as indel rate detected by NGS using methods as described in Example 2.
  • VI 68 XDPs were produced to achieve packaging of two types of CasX RNPs within a single XDP.
  • V168 XDPs contained either 1) RNPs of CasX 491 complexed with a ⁇ /Tomato-targeting gRNA and CasX 491 complexed with a / J 7 -targeting gRNA (VI 68 12.7-28.10), or 2) RNPs of CasX 491 complexed with a /t/Zb/iia/o-targeting gRNA and CasX 491 complexed with a non-targeting gRNA (V168 12.7-NT).
  • V168 XDPs were subsequently assessed for their editing efficiency at the tdTomato locus or PTBP1 locus in mNPCs, and the results are illustrated in FIG. 79.
  • the data demonstrate that VI 68 XDPs containing two types of CasX RNPs were able to achieve dose-dependent editing at both the tdTomato and PTBP1 loci when delivered into mNPCs.
  • the data further suggest that similar levels of editing were achieved at both loci at the indicated volumes of XDP application.
  • V206 XDPs were produced to achieve dual-CasX RNP packaging: 1) RNPs of CasX 491 complexed with a /t/Z /iia/o-targeting gRNA and CasX 491 complexed with a 7 J 777/ J 7 -targeting gRNA (V206 12.7-28.10); 2) RNPs of CasX 491 complexed with a tdTomato- targeting gRNA and CasX 491 complexed with a non-targeting gRNA (V206 12.7-NT); or 3) RNPs of CasX 491 complexed with a 7 J 77>7 J 7 -targeting gRNA and CasX 491 complexed with a non-targeting gRNA (V206 28.10-NT).
  • an MS2-based non-covalent recruitment (NCR) system is used in which an MS2 coat protein is fused to the HIV Gag polyprotein and an MS2 hairpin is incorporated into the guide RNA, which is used to non-covalently recruit the RNP into the XDP during its formation.
  • Table 66 Summary of version 168 and 206 XDPs with dXR, ELXR, or CasX
  • the fusion proteins of the dXR constructs were made up of, from N- to C-terminus, a catalytically-dead CasX 491, and a ZNF10 or ZIM3 KRAB domain (see FIG. 86; “RD1” is “Repressor Domain 1” and denotes the KRAB domain of interest).
  • the fusion proteins of the ELXR configuration #1 constructs were made up of, from N- to C-terminus, a catalytic domain from DNMT3A, an interaction domain from DNMT3L, a catalytically-dead CasX 491, and a ZNF10 or ZIM3 KRAB domain (see ELXR configuration #1 in FIG. 87), along with amino acid linkers and NLS sequences.
  • Catalytically-active CasX 491 herein termed “CasX”; SEQ ID NO: 189) was also included as a control.
  • the DNA and protein sequences of the components of the dXR and ELXR configuration #1 constructs are provided in Table 67, below.
  • the ELXR constructs also contained a 2x FLAG tag.
  • Guide RNA scaffold variant 226 was used with the version 168 XDPs, and guide scaffold variant 251 was used with the version 206 XDPs.
  • the RNA sequences of the guide scaffolds are provided in Table 6. Sequences of spacers 7.37 targeted to human B2M and a nontargeting spacer are provided in Table 68. All resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Table 68: Sequences of spacers used in constructs
  • FIGS. 89 and 90 show repression of the B2M locus in cells treated with 50 pL of XDPs.
  • Version 168 or 206 XDPs were engineered to carry the dXR molecule with the single repressor domain or the ELXR molecule having configuration #1 for targeting the B2M locus for repression. As shown in FIG. 89, six days following XDP administration, only version 168 XDPs carrying the dXR system with a ZNF10 KRAB domain repressed B2M. Meanwhile, version 206 XDPs carrying the dXR with a ZNF10 KRAB domain, dXR with a ZIM3 KRAB domain, and ELXR having configuration #1 with a ZIM3 KRAB domain all repressed the B2M locus.
  • XDPs were able to carry either the dXR or ELXR configuration #1 systems as therapeutic payloads and achieve transcriptional repression of a target locus.
  • Example 24 Engineering of XDPs for carrying ELXR systems
  • XDPs are generated with ELXR configuration #1, #4, or #5 molecules as the payload (see FIGS. 87 and 88 for diagrams of the configurations).
  • XDP configuration versions 168 and 206 are generated with various ELXR molecules.
  • Table 66 summarizes the plasmids used to encode the components of the version 168 and 206 XDP systems.
  • ELXR molecules in configurations #1, #4, and #5, which contain a catalytically- inactive CasX 491, are tested, as diagrammed in FIG. 88.
  • Table 69 provides amino acid sequences of configurations #1, #4, and #5 ELXR molecules, showing the sequences of the components of the proteins from N- to C-terminus in the table.
  • 88 may be a repressor domain from the species Columba livia, Rattus norvegicus, Cebus imitator, chimpanzee, Chlorocebus sabaeus, Ophiophagus hannah, Ailuropoda melanoleuca, Peromyscus maniculatus bairdii, or Phyllostomus discolor, in place of the human ZNF10 or ZIM3 KRAB domains that were tested in Example 23.
  • Other catalytically- inactive CasX variants can be used in place of catalytically-inactive CasX 491; these variants are listed in Table 4.
  • Guide RNA scaffold variant 226 is used with the version 168 XDPs, and guide scaffold variant 251 is used with the version 206 XDPs.
  • the RNA sequences of the guide scaffolds are provided in Table 6. Sequences of spacers 7.37 targeted to human B2M and a non-targeting spacer control are provided in Table 68, above. All resultant plasmids are sequenced using Sanger sequencing to ensure correct assembly.
  • Example 25 Evaluation of non-covalent recruitment (NCR) systems with protein-ligand pairs attached to Gag and protein cargo
  • XDPs were generated in which an NCR protein was fused to the Gag polyprotein, and a ligand for the NCR protein was fused to the cargo of the XDP, i.e., to the N-terminus of CasX.
  • Table 70 summarizes the plasmids used to encode the components of these XDP systems.
  • Table 70 Summary of version XDPs with protein recruitment of CasX
  • the protein-ligand pairs that were tested are provided in Table 71 (each row is a protein-ligand pair). Some of the listed protein-ligand pairs were split fluorescent proteins that were anticipated to emit fluorescence when the two portions of the split protein are bound, such as mNeon-Green and sfCherry.
  • Table 72 provides the amino acid sequences of the NCR proteins, and Table 73 provides the amino acid sequences of the ligands.
  • Guide scaffold 226 was used (SEQ ID NO: 2380), with the 12.7 spacer for targeting the tdTomato locus (CUGCAUUCUAGUUGUGGUUU, SEQ ID NO: 1855).
  • XDPs were generated using an NCR system based on the binding of a protein-ligand pair in which the protein was fused to the C-terminus of the Gag polyprotein and the ligand was fused to the N-terminus of CasX. As shown in FIG. 100, XDPs with each of the protein-ligand pairs tested produced editing of the tdTomato locus. Indeed, for many of the XDPs with proteinligand pairs, as well as for version 206 XDPs, editing levels were near 100%. This was true even at the lowest volumes of XDPs administered to the cells. Accordingly, editing levels were likely saturated in this assay.
  • NPCs transduced with split fluorescent protein NCR systems were imaged for red fluorescence (indicating editing of the tdTomato locus) and green fluorescence (indicating binding of the two portions of the split fluorescent protein).
  • the three split mNeon-Green NCR systems mNG2i-io+mNGl 1, mNG3Ai-io+mNG3n, and mNG3Ki-io+mNG3n
  • the split CloGFP system (CloGFPi-io+CloGFPn) each produced cells with red and green fluorescence, indicating both editing and the presence of reconstituted split fluorescent proteins (data not shown).
  • the green fluorescence was often found in puncta, which is believed to indicate nuclear localization of the CasX:gRNA RNP along with the bound fluorescent protein.
  • cells transduced with version 206 XDPs, and CL7+IM7 and sfCherry2i-io+sfCherry2n protein NCR systems were also examined and were found to show red fluorescence (indicating editing of the tdTomato locus) but not green fluorescence.

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Abstract

Provided herein are delivery particle systems (XDP) useful for the delivery of payloads of any type. In some embodiments, a XDP particle system with tropism for target cells of interest is used to deliver CRISPR/Cas polypeptides (e.g., CasX proteins) and guide nucleic acids (gNA), for the modification of nucleic acids in target cells. Also provided are methods of making and using such XDP to modify the nucleic acids in such cells.

Description

Attorney Docket No. SCRB-050/01WO 333322-2386 EXAMPLES Example 1: CasX:gRNA In Vitro Cleavage Assays 1. Assembly of RNP [0418] Purified RNP of CasX and single guide RNA (sgRNA) were either prepared immediately before experiments or prepared and snap-frozen in liquid nitrogen and stored at −80oC for later use. To prepare the RNP complexes, the CasX protein was incubated with sgRNA at 1:1.2 molar ratio. Briefly, sgRNA was added to Buffer#1 (25 mM NaPi, 150 mM NaCl, 200 mM trehalose, 1 mM MgCl2), then the CasX was added to the sgRNA solution, slowly with swirling, and incubated at 37°C for 10 min to form RNP complexes. RNP complexes were filtered before use through a 0.22 μm Costar® 8160 filters that were pre-wet with 200 μl Buffer#1. If needed, the RNP sample was concentrated with a 0.5 ml Ultra 100-Kd cutoff filter, (MilliporeTM part #UFC510096), until the desired volume was obtained. Formation of competent RNP was assessed as described below. 2. In vitro cleavage assays: Determining cleavage-competent fractions for protein variants compared to wild-type reference CasX [0419] The ability of CasX variants to form active RNP compared to reference CasX was determined using an in vitro cleavage assay. The beta-2 microglobulin (B2M) 7.37 target for the cleavage assay was created as follows. DNA oligos with the sequence TGAAGCTGACAGCATTCGGGCCGAGATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGC GCT (non-target strand, NTS (SEQ ID NO: 968)) and AGCGCGAGCACAGCTAAGGCCACGGAGCGAGACATCTCGGCCCGAATGCTGTCAGC TTCA (target strand, TS (SEQ ID NO: 969)) were purchased with 5’ fluorescent labels (LI- CORTM IRDye® 700 and 800, respectively). dsDNA targets were formed by mixing the oligos in a 1:1 ratio in 1x cleavage buffer (20 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM TCEP, 5% glycerol, 10 mM MgCl2), heating to 95° C for 10 minutes, and allowing the solution to cool to room temperature. [0420] CasX RNPs were reconstituted with the indicated CasX and guides (see graphs) at a final concentration of 1 µM with 1.5-fold excess of the indicated guide unless otherwise specified in 1× cleavage buffer (20 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM TCEP, 5% glycerol, 10 mM MgCl2) at 37° C for 10 min before being moved to ice until ready to use. The 7.37 target was used, along with sgRNAs having spacers complementary to the 7.37 target. 143 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 [0421] Cleavage reactions were prepared with final RNP concentrations of 100 nM and a final target concentration of 100 nM. Reactions were carried out at 37° C and initiated by the addition of the 7.37 target DNA. Aliquots were taken at 5, 10, 30, 60, and 120 minutes and quenched by adding to 95% formamide, 20 mM EDTA. Samples were denatured by heating at 95° C for 10 minutes and run on a 10% urea-PAGE gel. The gels were either imaged with a LI-COR Odyssey® CLx and quantified using the LI-COR Image Studio software or imaged with a Cytiva TyphoonTM and quantified using the Cytiva IQTL software. The resulting data were plotted and analyzed using Prism. It was assumed that CasX acts essentially as a single-turnover enzyme under the assayed conditions, as indicated by the observation that sub-stoichiometric amounts of enzyme fail to cleave a greater-than-stoichiometric amount of target even under extended time-scales and instead approach a plateau that scales with the amount of enzyme present. Thus, the fraction of target cleaved over long time-scales by an equimolar amount of RNP is indicative of what fraction of the RNP is properly formed and active for cleavage. The cleavage traces were fit with a biphasic rate model, as the cleavage reaction clearly deviates from monophasic under this concentration regime, and the plateau was determined for each of three independent replicates. The mean and standard deviation were calculated to determine the active fraction (Table 10). [0422] Apparent active (competent) fractions were determined for RNPs formed for reference CasX2 + guide 174 + 7.37 spacer, CasX 119 + guide 174 + 7.37 spacer, CasX 457 + guide 174 +7.37 spacer, CasX 488 + guide 174 + 7.37 spacer, and CasX 491 + guide 174 + 7.37 spacer, as shown in FIG.1. The determined active fractions are shown in Table 10. All CasX variants had higher active fractions than the wild-type CasX2, indicating that the engineered CasX variants form significantly more active and stable RNP with the identical guide under tested conditions compared to wild-type CasX. This may be due to an increased affinity for the sgRNA, increased stability or solubility in the presence of sgRNA, or greater stability of a cleavage-competent conformation of the engineered CasX:sgRNA complex. An increase in solubility of the RNP was indicated by a notable decrease in the observed precipitate formed when CasX 457, CasX 488, or CasX 491 was added to the sgRNA compared to CasX2. 3. In vitro cleavage assays – Determining cleavage-competent fractions for single guide variants relative to reference single guides [0423] Cleavage-competent fractions were also determined using the same protocol for CasX2 protein in combination with guides 2, 32, 64 and 174 and targeting sequence 7.37 144 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 (CasX2.2.7.37, CasX2.32.7.37, CasX2.64.7.37), and CasX2.174.7.37 to be 16 ± 3%, 13 ± 3%, 5 ± 2%, and 22 ± 5%, as shown in FIG.2 and Table 10. [0424] A second set of guides were tested under different conditions to better isolate the contribution of the guide to RNP formation. Guides 174, 175, 185, 186, 196, 214, and 215 with 7.37 spacer were mixed with CasX 491 at final concentrations of 1 µM for the guide and 1.5 µM for the protein, rather than with excess guide as before. Results are shown in FIG.3 and Table 10. Many of these guides exhibited additional improvement over 174, with 185 and 196 achieving 91 ± 4% and 91 ± 1% competent fractions, respectively, compared with 80 ± 9% for 174 under these guide-limiting conditions. [0425] The data indicate that both CasX variants and sgRNA variants are able to form a higher degree of active RNP with guide RNA compared to wild-type CasX and wild-type sgRNA. [0426] The apparent cleavage rates of CasX variants 119, 457, 488, and 491 compared to wild-type reference CasX were determined using an in vitro fluorescent assay for cleavage of the target 7.37. 4. In vitro cleavage assays – Determining kcleave for CasX variants compared to wild-type reference CasX [0427] CasX RNPs were reconstituted with the indicated CasX protein (see FIG.4) at a final concentration of 1 µM with 1.5-fold excess of the indicated guide in 1× cleavage buffer (20 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM TCEP, 5% glycerol, 10 mM MgCl2) at 37° C for 10 min before being moved to ice until ready to use. Cleavage reactions were set up with a final RNP concentration of 200 nM and a final target concentration of 10 nM. Reactions were carried out at 37° C except where otherwise noted and initiated by the addition of the target DNA. Aliquots were taken at 0.25, 0.5, 1, 2, 5, and 10 minutes and quenched by adding to 95% formamide, 20 mM EDTA. Samples were denatured by heating at 95° C for 10 minutes and run on a 10% urea- PAGE gel. The gels were imaged with a LI-COR Odyssey CLx and quantified using the LI- COR Image Studio software or imaged with a Cytiva Typhoon and quantified using the Cytiva IQTL software. The resulting data were plotted and analyzed using Prism, and the apparent first- order rate constant of non-target strand cleavage (kcleave) was determined for each CasX:sgRNA combination replicate individually. The mean and standard deviation of three replicates with independent fits are presented in Table 10, and the quantification of competent fractions of RNP of CasX variants traces are shown in FIG 8. 145 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 [0428] Apparent cleavage rate constants were determined for wild-type CasX2, and CasX variants 119, 457, 488, and 491 with guide 174 and spacer 7.37 utilized in each assay (see Table 10 and FIG.4). All CasX variants had improved cleavage rates relative to the wild-type CasX2. CasX 457 cleaved more slowly than 119, despite having a higher competent fraction as determined above. CasX488 and CasX491 had the highest cleavage rates by a large margin; as the target was almost entirely cleaved in the first timepoint, the true cleavage rate exceeds the resolution of this assay, and the reported kcleave should be taken as a lower bound. [0429] The data indicate that the CasX variants have a higher level of activity, with kcleave rates reaching at least 30-fold higher compared to wild-type CasX2. 5. In vitro cleavage assays: Determination of cleavage rates for guide variants compared to reference single guides [0430] Cleavage assays were also performed with wild-type reference CasX2 and reference guide 2 compared to gRNA variants 32, 64, and 174 to determine whether the variants improved cleavage. The experiments were performed as described above. As many of the resulting RNPs did not approach full cleavage of the target in the time tested, initial reaction velocities (V0) were determined rather than first-order rate constants. The first two timepoints (15 and 30 seconds) were fitted with a line for each CasX:sgRNA combination and replicate. The mean and standard deviation of the slope for three replicates were determined. [0431] Under the assayed conditions, the V0 for CasX2 with guides 2, 32, 64, and 174 were 20.4 ± 1.4 nM/min, 18.4 ± 2.4 nM/min, 7.8 ± 1.8 nM/min, and 49.3 ± 1.4 nM/min (see Table 10 and FIGS.5and FIG.6). Guide 174 showed substantial improvement in the cleavage rate of the resulting RNP (~2.5-fold relative to 2, see FIG.6), while guides 32 and 64 performed similar to or worse than guide 2. Notably, guide 64 supports a cleavage rate lower than that of guide 2 but performs much better in vivo (data not shown). Some of the sequence alterations to generate guide 64 likely improve in vivo transcription at the cost of a nucleotide involved in triplex formation. Improved expression of guide 64 likely explains its improved activity in vivo, while its reduced stability may lead to improper folding in vitro. [0432] Additional experiments were carried out with guides 174, 175, 185, 186, 196, 214, and 215 with spacer 7.37 and CasX 491 to determine relative cleavage rates. To reduce cleavage kinetics to a range measurable with our assay, the cleavage reactions were incubated at 10° C. Results are in FIG.7 and Table 10. Under these conditions, 215 was the only guide that supported a faster cleavage rate than 174.196, which exhibited the highest active fraction of 146 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 RNP under guide-limiting conditions, had kinetics essentially the same as 174, again highlighting that different variants result in improvements of distinct characteristics. [0433] The data support that use of the majority of the guide variants with CasX results in RNP with a higher level of activity than one with the wild-type guide, with improvements in initial cleavage velocity ranging from ~2-fold to >6-fold. Numbers in Table 10 indicate, from left to right, CasX variant, sgRNA scaffold, and spacer sequence of the RNP construct. In the RNP construct names in the table below, CasX protein variant, guide scaffold and spacer are indicated from left to right. 6. In vitro cleavage assays: Comparing cleavage rate and competent fraction of 515.174 and 526.174 against reference 2.2 [0434] We wished to compare engineered protein CasX variants 515 and 526 in complex with engineered single-guide variant 174 against the reference wild-type protein 2 (SEQ ID NO: 2) and minimally-engineered guide variant 2 (SEQ ID NO: 5). RNP complexes were assembled as described above, with 1.5-fold excess guide. Cleavage assays to determine kcleave and competent fraction were performed as described above, with both performed at 37°C, and with different timepoints used to determine the competent fraction for the wild-type vs engineered RNPs due to the significantly different times needed for the reactions to near completion. [0435] The resulting data clearly demonstrate the dramatic improvements made to RNP activity by engineering both protein and guide. RNPs of 515.174 and 526.174 had competent fractions of 76% and 91%, respectively, as compared to 16% for 2.2 (FIG.8, Table 10). In the kinetic assay, both 515.174 and 526.174 cut essentially all of the target DNA by the first timepoint, exceeding the resolution of the assay and resulting in estimated cleavage rates of 17.10 and 19.87 min-1, respectively (FIG.9, Table 10). An RNP of 2.2, by contrast, cut on average less than 60% of the target DNA by the final 10-minute timepoint and has an estimated kcleave nearly two orders of magnitude lower than the engineered RNPs. The modifications made to the protein and guide have resulted in RNPs that are more stable, more likely to form active particles, and cut DNA much more efficiently on a per-particle basis as well. Table 10: Results of cleavage and RNP formation assays RNP Construct kcleave* Initial velocity* Competent fraction 2.2.7.37 - 20.4 ± 1.4 nM/min 16 ± 3% 2.32.7.37 - 18.4 ± 2.4 nM/min 13 ± 3% 2.64.7.37 - 7.8 ± 1.8 nM/min 5 ± 2% 2.174.7.37 0.51 ± 0.01 min-1 49.3 ± 1.4 nM/min 22 ± 5% 147 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 RNP Construct kcleave* Initial velocity* Competent fraction 119.174.7.37 6.29 ± 2.11 min-1 - 35 ± 6% 457.174.7.37 3.01 ± 0.90 min-1 - 53 ± 7% 488.174.7.37 15.19 min-1 - 67% 491.174.7.37 16.59 min-1 / 0.293 83% / 17% (guide- min -1 (10° C) - limited) 491.175.7.37 0.089 min-1 (10° C) - 5% (guide-limited) 491.185.7.37 0.227 min-1 (10° C) - 44% (guide-limited) 491.186.7.37 0.099 min-1 (10° C) - 11% (guide-limited) 491.196.7.37 0.292 min-1 (10° C) - 46% (guide-limited) 491.214.7.37 0.284 min-1 (10° C) - 30% (guide-limited) 491.215.7.37 0.398 min-1 (10° C) - 38% (guide-limited) 515.174.7.37 17.10 min-1** - 76% 526.174.7.37 19.87 min-1** - 91% *Mean and standard deviation **Rate exceeds resolution of assay Example 2: Non-covalent recruitment with RNA binding - Gag-MS2 [0436] These experiments evaluated the ability of an MS2-based non-covalent recruitment (NCR) system to improve the generation of XDP in packaging host cells where the CasX RNP is recruited into the XDPs by fusing MS2 coat proteins (CPs) to the HIV Gag polyprotein and an MS2 hairpin is incorporated into the guide RNA. Methods: [0437] All plasmids encoding CasX proteins utilized the CasX 491 variant protein. [0438] RNA fold structures were generated with RNAfold web server and Varna java-based software. Structural plasmid cloning [0439] In order to generate the structural plasmids used below, plasmid pXDP1 was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments containing CasX variant, HIV-1, or MS2 CP components were amplified and cloned using In-Fusion® primers with 15-20 base pair overlaps and KAPA HiFi DNA polymerase according to the manufacturer’s protocols. The fragments were purified by gel extraction and cloned into plasmid backbones using In-Fusion® HD Cloning Kit from Takara (Cat# 639650) according to the manufacturer’s protocols. Assembled products were transformed into chemically-competent Turbo Competent E. coli bacterial cells, plated on LB-Agar plates containing ampicillin and incubated at 37^C. Individual colonies were picked and miniprepped using QIAprep® Spin Miniprep Kit following the manufacturer’s The resultant plasmids were sequenced 148 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 using Sanger sequencing to ensure correct assembly. The amino acid sequence of the MS2 CP is provided in SEQ ID NO: 4140, and the amino acid sequence of the Gag polyprotein fused to the MS2 CP is provided in SEQ ID NO: 4141. Guide plasmid cloning [0440] The tdTomato targeting guide plasmids used in these experiments were pSG50 (guide scaffold 188; FIG.12) and pSG54 (guide scaffold 228; FIG.13), which were cloned from pSG33 and pSG34, respectively. The mammalian expression backbone contained a cPPT, ampicillin resistance, and a colEI replication site and was amplified using primers with appropriate overlaps to accept the U6 promoter and guide RNA scaffold cassette. The backbone, pSG3, was digested using NdeI and XbaI. Synthetic DNA fragments corresponding to scaffold variants were amplified and cloned as described, above. The resultant plasmids, pSG33 and pSG34, were sequenced using Sanger sequencing to ensure correct assembly (Table 12). Cloning tdTomato spacer 12.7 into pSG3 and pSG14 [0441] To clone the targeting pSG50 and pSG54 plasmids from the non-targeting pSG33 and pSG34, the spacer 12.7 was cloned using the following protocol. The targeting spacer sequence DNA for the tdTomato targeting spacer 12.7 was ordered as single-stranded DNA (ssDNA) oligos (Integrated DNA Technologies) consisting of the targeting sequence (CTGCATTCTAGTTGTGGTTT, SEQ ID NO: 1018) and the reverse complement of this sequence. These two oligos were annealed together and cloned into a pSG plasmid with an alternate scaffold by Golden Gate assembly using T4 DNA Ligase (New England BioLabs Cat# M0202L) and Esp3I restriction enzyme from New England BioLabs (NEB Cat# R0734L). Golden Gate products were transformed into chemically competent NEB® Turbo competent E. coli (NEB Cat #C2984I), plated on LB-Agar plates containing carbenicillin and incubated at 37oC. Individual colonies were picked and miniprepped as described above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation. pGP2 Glycoprotein plasmid cloning [0442] Sequences encoding the VSV-G glycoprotein and the cytomegalovirus (CMV) promoter were amplified from pMD2.G and cloned as described for the structural plasmids, above. The backbone was taken from a kanamycin resistant plasmid and amplified and cloned using the same methods. Assembled products were transformed into chemically-competent Turbo Competent E. coli bacterial cells, plated on LB-Agar plates containing kanamycin and 149 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 incubated at 37^C. The resultant plasmids in this five-plasmid system (Gag-(-1)-PR, Gag-MS2, CasX, gRNA, and GP) were sequenced using Sanger sequencing to ensure correct assembly. Cell culture and transfection [0443] HEK293T Lenti-X™ cells were maintained in 10% FBS supplemented DMEM with HEPES and GlutaMAX™ (Thermo Fisher®). Cells were seeded in 15 cm dishes at 20 x 106 cells per dish in 20 mL of media. Cells were allowed to settle and grow for 24 hours before transfection. At the time of transfection, cells were 70-90% confluent. For transfection, the XDP structural plasmids (also encoding the CasX variants) of Table 13 were used in amounts ranging from 13 to 80.0 µg. Each transfection also received 13 µg of p42.174.12.7 and 0.25 µg of pGP2. Polyethylenimine (PEI MAX®, Polyplus) was then added to the plasmid mixture, mixed, and allowed to incubate at room temperature before being added to the cell culture. Plasmid ratios in Table 11 were used in all version 206 XDPs used in this assay, based on prior experimental data from other XDP versions. Table 11: Plasmids and ratios used in XDP constructs XDP version 206 Structural plasmid plasmids ratios Gag-(-1)-PR* 10% Gag-MS2* 45% CasX* 45% *transcript contains RRE and produces REV Collection and concentration [0444] Media was aspirated from the plates 24 hours post-transfection and replaced with Opti- MEM™ (Thermo Fisher). XDP-containing media was collected 72 hours post-transfection and filtered through a 0.45 μm PES filter. The supernatant was concentrated and purified via centrifugation. [0445] Filtered supernatant was divided evenly into an appropriate number of centrifuge tubes or bottles and 1/5th of the supernatant volume of Sucrose Buffer (50mM Tris-HCL, 100mM NaCl, 10% Sucrose, pH 7.4) was underlaid using serological pipettes. The samples were centrifuged at 10,000xg, 4 ̊C, in a swinging- rotor for 4 hours with no brake. The 150 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 supernatant was carefully removed and the pellet briefly dried by inverting the centrifuge vessels. Pellets were either resuspended in Storage Buffer (PBS + 113 mM NaCl, 15% Trehalose dihydrate, pH 8 or an appropriate media by gentle trituration and vortexing. XDPs were resuspended in 300 μL of DMEM/ F12 supplemented with GlutaMAX™, HEPES, non-essential amino acids, Pen/Strep, 2-mercaptoethanol, B-27 without vitamin A, and N2. Resuspension and transduction [0446] tdTomato neural progenitor cells (NPCs) were resuspended and transduced with XDPs. In brief, tdTomato NPCs were grown in DMEM/F12 supplemented with GlutaMAX™, HEPES, NEAA, Pen/Strep, 2-mercaptoethanol, B-27 without vitamin A, and N2. Cells were harvested using StemPro Accutase Cell Dissociation Reagent and seeded on PLF-coated 96-well plates.48 hours later, cells were transduced with XDPs containing a tdTomato targeting spacer. Cells were then centrifuged for 15 minutes at 1000 x g. Transduced NPCs were grown for 96 hours before analyzing tdTomato fluorescence by flow cytometry as a marker of editing at the tdTomato locus, with the EC50 determined as the number of XDP particles needed to achieve editing in 50% of the cells, as determined by flow cytometry. tdTomato fluorescence was measured using flow cytometry. The assays were run 2-3 times for each sample, with similar results. Results: [0447] The MS2 bacteriophage relies on the non-covalent affinity between its genomic RNA and the MS2 coat protein for the packaging of its genome in an icosahedral viral shell. The high- affinity element in the RNA genome is termed the MS2 hairpin, which binds to the coat protein with a Kd of approximately 3e-9. Here, two high affinity variants of the MS2 hairpin were incorporated into the extended stem of the guide scaffold 174, thereby introducing into the CasX:guide RNP an affinity for the MS2 coat protein. The resulting guide scaffolds 188 and 228 were tested in XDP version 168; a version that relies on a Gag-CasX fusion configuration and lacks the MS2 coat protein, while version 206 (FIG.11) has the incorporated MS2 coat protein fused to Gag. Guides 188 and 228 performed similarly to guide scaffold 174 in total editing across all volumes tested, demonstrating that the insertion of the MS2 hairpin was benign to the function of the RNP. The MS2 hairpin variant sequences of these scaffolds are ACATGAGGATCACCCATGT (SEQ ID NO: 1131) and CGTACACCATCAGGGTACG (SEQ ID NO: 1132), respectively. [0448] MS2-based recruitment of these variant scaffolds was tested in XDP version 206. This version is composed of the Gag-(-1)-PR, Gag-MS2, and CasX architectures. This version relies 151 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 on orthogonal recruitment of CasX via the MS2 coat protein and MS2 hairpin system of the guide rather than a direct fusion between CasX and a recruiting protein. This is demonstrated in FIG.15, where constructs with both guide scaffold 188 and 228 edit well in the tdTomato assay, in contrast to constructs with guide scaffold 174, which lacks the MS2 hairpin and edits poorly. Additionally, XDP version 206 with scaffold 188 edits better at the same dosage over XDP version 168 with scaffold 174 (see FIGS.14 and 15). At 0.6 µL of XDPs delivered, editing was ~70% with XDP version 206 with guide scaffold 188. In the same assay, ~20% editing was achieved at the same treatment volume for XDP version 168 (a Gag-CasX fusion) with guide scaffold 174 and version 206 with guide scaffold 228. These data suggested that XDP version 206 with guide scaffold 188 is 2-3x more potent than version 168 with guide scaffold 174. This increase in editing from version 168 to version 206 could be attributed to the lack of a direct fusion of Gag to CasX, causing less steric hindrance in particle formation. Furthermore, the similarity between guide scaffolds 188 and 228 in editing in version 168 suggests that the difference in potency in XDP version 206 is due to the MS2 hairpin’s affinity for the coat protein linked to Gag. [0449] The results suggest two possible mechanisms of recruitment of the CasX RNP to XDP particles in version 206. First, the CasX protein and guide scaffold RNA form the apoenzyme RNP in the cytoplasm of the producer cell that then binds the Gag-MS2 protein by interactions of the MS2 hairpin in the guide extended stem and the MS2 coat protein. The second possible mechanism is that the guide scaffold RNA hairpin first binds the MS2 coat protein and then forms the apoenzyme with the CasX protein. Collectively, the results demonstrate the utility of the incorporation of the MS2 system for the formation of more potent XDP particles with increased numbers of RNP and higher editing capabilities. Additionally, the MS2 coat protein variants have several point mutations that alter their affinity to its hairpin RNA. Usage of these variants in version 206 could result in higher potency variants. Fusing multiple coat proteins to the HIV Gag protein could further increase potency as well. Alternatively, there are also several RNA hairpin – non-covalent recruitment (NCR) protein combinations, such as Qβ phage, GA phage, PP7 phage, or λN, that could be used to replace MS2. Other protein RNA combinations from humans and retroviruses include the Iron Responsive Element (IRE)-Iron Binding element, U1 hairpin II, retrovirus Tat-trans-activation response (TAR) system, Csy4, Pardaxin, tRNA or Psi-Nucleocapsid. 152 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Table 12: sgRNA encoding sequences Plasmid Enco Scaffold Spac SEQ ID ding Hairpin SEQ number er Full Encoding Sequence NO sequence ID NO ACTGGCGCTTTTATCTGATTACTTT GAGAGCCATCACCAGCGACTATGTC ACATGAGG pSG0033 188 NT GTAGTGGGTAAAGCTCACATGAGGA 1135 ATCACCCA 1131 TCACCCATGTGAGCATCAAAGCGAG TGT ACGTAATTACGTCTCG ACTGGCGCTTTTATCTGATTACTTT GAGAGCCATCACCAGCGACTATGTC CGTACACC pSG0034 228 NT GTAGTGGGTAAAGCTCCCCGTACAC 1134 ATCAGGGT 1132 CATCAGGGTACGGGGAGCATCAAAG ACG CGAGACGTAATTACGTCTCG ACTGGCGCTTTTATCTGATTACTTT GAGAGCCATCACCAGCGACTATGTC CGTACACC pSG0035 229 NT GTAGTGGGTAAAGCTCCCCGTACAC 1136 ATTAGGGT 955 CATTAGGGTACGGGGAGCATCAAAG ACG CGAGACGTAATTACGTCTCG ACTGGCGCTTTTATCTGATTACTTT GAGAGCCATCACCAGCGACTATGTC ACATGAGG pSG50 188 12.7 GTAGTGGGTAAAGCTCACATGAGGA 1825 ATCACCCA 1131 TCACCCATGTGAGCATCAAAGCTGC TGT ATTCTAGTTGTGGTTT ACTGGCGCTTTTATCTGATTACTTT GAGAGCCATCACCAGCGACTATGTC CGTACACC pSG54 228 12.7 GTAGTGGGTAAAGCTCCCCGTACAC 4146 ATCAGGGT 1132 CATCAGGGTACGGGGAGCATCAAAG ACG CTGCATTCTAGTTGTGGTTT Table 13: Architecture and glycoprotein sequences Plasmid number Architecture DNA Sequence (SEQ ID NO) pXDP17 Gag-CasX491-Hatag 1138 pXDP161* Gag-(-1)-PR 1139 pXDP164* Gag-MS2 1140 pXDP165* Gag-MS2-p1*-p6-(-1)-PR 1141 pXDP166* SV40NLS-CasX491-SV40 NLS 1142 *backbone of plasmid expresses Rev 153 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Table 14: Version and pseudotyping descriptions XDP version Architectures and glycoproteins Plasmid numbers Rev expression Gag-(-1)-PR pXDP161 168 Gag-CasX pXDP17 Yes VSV-G pGP2 Gag-(-1)-PR pXDP161 206 Gag-MS2 pXDP164 CasX pXDP166 Yes VSV-G pGP2 Example 3: Non-covalent recruitment with RNA binding - Partial Gag-MS2 [0450] The purpose of these experiments was to demonstrate the utility of a non-covalent recruitment (NCR) method for the incorporation of RNP into XDP using an MS2-based system where the RNPs are recruited into the XDPs by fusing the MS2 coat protein (CP) to different proteins within an HIV Gag polyprotein in the XDP construct. [0451] The MS2 packaging system consists of two major components: the phage coat protein and its cognate binding partner, which is a short hairpin stem loop structure. In this orthogonal phage RNA-based recruitment system, the short hairpin stem loop structure is engineered into the sgRNA incorporated into the XDP. The encoding sequence for the phage coat protein is fused to either the encoding sequence for the Gag polyprotein (derived from any retroviruses) or to any other protein domains derived from the Gag polyprotein of any retroviral origin. This would enable the recruitment of the expressed CasX RNP into the XDP particle by the targeted interaction between the short hairpin stem loop structure engineered into the sgRNA, which is complexed with the CasX as an RNP, and the phage coat protein fused to the Gag polyprotein or any proteins derived from the Gag polyprotein. Here, XDPs in which the RNP is recruited into the XDPs by fusing the MS2 coat protein (CP) to different proteins within an HIV Gag polyprotein in the XDP construct are described. Methods: [0452] All plasmids containing CasX proteins encoded the CasX 491 variant protein. All XDPs were pseudotyped with 10% VSV-G (percentage of plasmid relative to the other plasmids utilized for the XDP construct). The guide scaffold used in all the MS2 constructs was 188 along with spacer 12.7 targeting the tdTomato locus. The guide scaffold used in the control construct (V168) was 226, also with spacer 12.7. This has the RRE/RBE element described in 154 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 other examples herein. RNA fold structures were generated with RNAfold web server and Varna java-based software. Structural plasmid cloning [0453] In order to generate the structural plasmids (pXDP17, pXDP161, pXDP164 and pXDP166), pXDP1 was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments containing CasX variant, HIV-1, or MS2 CP components were amplified and cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. [0454] MS2 was placed either on the N- or the C-terminus of the Capsid (Version 263- pXDP276, Version 264-pXDP277, Version 265-pXDP278 and Version 266-pXDP279), with and without cleavage sites. MS2 was placed either on the N- or the C-terminal of the Nucleocapsid (Version 267-pXDP280, Version 268-pXDP281, Version 269-pXDP282 and Version 270-pXDP283), with and without cleavage sites. The sequences for these constructs are provided in Table 16. The designed constructs were synthesized as transgenes and purchased pre-cloned into pTWIST expression plasmids from Twist Bioscience®. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Guide plasmid cloning [0455] The guide plasmids used in these experiments were pSG50 and pSG17, encoding guide scaffold 188. Spacer 12.7 was cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation. The guide plasmid used in all MS2 constructs is pSG50. The guide plasmid used in control construct (V168) is pSG17. pGP2 Glycoprotein plasmid cloning [0456] Sequences encoding the VSV-G glycoprotein and the CMV promoter were amplified from pMD2.G and cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Cell culture and transfection; collection and concentration; resuspension and transduction [0457] HEK293T Lenti-X™ cell culture was performed as described in Example 2, above. For transfection, the XDP structural plasmids of Table 16 were used in amounts ranging from 13 to 80.0 µg. Each transfection also received 13 µg of pSG50 or pSG17 and 0.25 µg of pGP2. Polyethylenimine (PEI MAX® from Polyplus) was then added as described in Example 2. XDPs were collected and concentrated as described in Example 2, above. tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above. tdTomato fluorescence 155 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 was measured using flow cytometry. The assays were run 2-3 times for each sample, with similar results. Results: [0458] Percent editing of the tdTomato target sequence in tdT NPCs are shown for all the constructs in FIG.52 in terms of number of particles added and the volume of XDPs added (FIG.53). Table 15 presents the results of percent editing of the dtTomato target sequence when 16.6 μl of the concentrated XDP prep was used to treat NPCs. The results show that it is feasible to fuse MS2 with or without a cleavage sequence to either the capsid or the nucleocapsid. The results indicate that fusing MS2 to the C-terminal of the capsid results in more potent XDP as compared to a fusion to the N-terminal. In addition, introduction of a cleavage site in between MS2 and CA on the C-terminal does improve potency as shown in FIG.52. Fusing MS2 to the N- or C-terminal of nucleocapsid with and without a cleavage site may be superior to a capsid fusion, with a fusion to the N-terminal of NC being marginally better in terms of editing as shown in FIG.53. The EC50 for the different constructs were calculated and plotted as shown in FIG.54 and recapitulates the differences in potency described above. FIG.16 depicts the fold improvement in EC50 over the base control V168 (CasX fused to full length HIV Gag- polyprotein) and it shows that V265, V269 and V270 show about 5 to 8-fold improvement in potency. FIG.17 depicts the fold improvement in EC50 over the base control V206 (MS2 fused to full length HIV Gag-polyprotein and the results demonstrate that V265, V269 and V270 show about 6- to 9-fold improvement in terms of overall editing potency. Table 15: Percent editing at the second dilution (16.6μl) XDP version Plasmid number Encoded Configuration** % Editing 168 pXDP17 MA*-CA*-NC*-P1*-P6*-CasX 92.6 206 pXDP164 MA*-CA*-NC*-P1*-P6-MS2 92.3 263 pXDP276 MA*-CA-MS2*-NC*-P1*-P6 85.5 264 pXDP277 MA*-MS2-CA*-NC*-P1*-P6 9.8 265 pXDP278 MA*-CA* -MS2*-NC*-P1*-P6 75.6 266 pXDP279 MA*-MS2*- CA*-NC*-P1*-P6 45 267 pXDP280 MA*-CA*-NC-MS2*-P1*-P6 90.1 268 pXDP281 MA*-CA*-NC*- MS2*-P1*-P6 91.4 269 pXDP282 MA*-CA*-MS2-NC*-P1*-P6 91.6 270 pXDP283 MA*-CA*-MS2*- NC*-P1*-P6 82.1 * indicates cleavage sequence between adjacent components ** 5' to 3' orientation 156 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 [0459] These results show that it is functionally feasible to fuse MS2 with or without a cleavage sequence to the capsid or the nucleocapsid derived from the HIV Gag polyprotein to create XDP that results in enhanced editing of the target nucleic acid. These results also show that it is possible to improve potency depending on the location within the Gag polyprotein (or its components) where the MS2 is fused. It is also believed that this enhanced architecture can be translated to proteins derived from the Gag polyproteins of Alpharetroviral, Betaretroviral, Gammaretroviral, Deltaretroviral, Epsilonretroviral, Lentiviral and Spumaretroviral origin, serving as an orthogonal recruitment mechanism for CasX or any other payload that can be coupled with a cognate short hairpin RNA element in an XDP or other particle-delivery system. Table 16: Plasmid sequences XDP version Plasmid number SEQ ID NO of Encoding Sequence pGP2 979 pSG50 1143 pXDP161 1139 168 pXDP17 1144 pXDP166 1142 206 pXDP164 1140 301 pXPD276 1145 302 pXPD277 1146 303 pXPD278 1147 304 pXPD279 1148 305 pXPD280 1149 306 pXPD281 1150 307 pXPD282 1151 308 pXPD283 1152 pSG17 1153 Example 4: Non-covalent recruitment with RNA binding - Retro-MS2 [0460] The purpose of these experiments was to demonstrate the utility of a recruitment method for the incorporation of RNP into XDP an MS2-based system and Gag 157 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 polyproteins or components of Gag polyproteins derived from five genera of retroviruses, including Alpharetroviruses, Betaretroviruses, Gammaretroviruses, Deltaretroviruses and Lentiviruses. Methods: [0461] All plasmids containing CasX proteins encoded the CasX 491 protein. All XDPs were pseudotyped with 10% VSV-G (percentage of plasmid relative to the other plasmids utilized for the XDP construct). The guide RNA spacer used in all of these experiments was 12.7 targeting the tdTomato locus. The guide scaffold used in all the MS2 constructs was 188, along with spacer 12.7. RNA fold structures were generated with RNAfold web server and Varna java- based software. Structural plasmid cloning [0462] MS2 was fused to the Gag-protease, Gag or partial Gag polyproteins derived from Alpharetroviruses (Versions 271, 272, 273), Betaretroviruses (Versions 277, 279), Gammaretroviruses (Versions 276, 278), Deltaretroviruses (Versions 274, 275) and Lentiviruses (Versions 280, 281, 282) with their respective species-specific cleavage sites. The sequences for these constructs are provided in Table 18. The designed constructs were synthesized as transgenes and purchased pre-cloned into pTWIST expression plasmids from Twist Biosciences. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Guide plasmid cloning [0463] The guide plasmid used in these experiments was pSG50. To clone the targeting pSG50 spacer 12.7 was cloned in as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation. pGP2 Glycoprotein plasmid cloning [0464] Sequences encoding the VSV-G glycoprotein and the CMV promoter were cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Cell culture and transfection [0465] HEK293T Lenti-X™ cell culture was performed as described in Example 2, above. For transfection, the XDP structural plasmids of Table 18 were used in amounts ranging from 13 to 80.0 µg. Each transfection also received 13 µg of p42.174.12.7 and 0.25 µg of pGP2. Polyethylenimine (PEI MAX® from Polyplus) was then added as described in Example 2. 158 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Collection and concentration; resuspension and transduction [0466] XDPs were collected and concentrated as described in Example 2, above. [0467] tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above, and tdTomato fluorescence was measured using flow cytometry. The assays were run 2-3 times for each sample with similar results. Results: [0468] Percent editing of the dtTomato target sequence in tdT NPCs are shown for all the constructs in FIG.55 across the dilution curve for the volume of XDPs added. Table 17 represents the percent editing of the dtTomato target sequence when 16.6 µl of the concentrated XDP prep was used to treat NPCs. These results show that, as compared to V206, which was the control XDP in this experiment (derived from HIV, lentivirus) and which edited at 95% efficacy at the tdTomato locus when 16.6 µl of the concentrated XDP was used, V271 and V272, which are different architectural variants derived from ALV (Alpharetroviruses) showed editing efficacies ranging from 79 to 88%. V275 derived from HTLV1 (Deltaretroviruses), V279 derived from MPMV (Betaretroviruses) as well as V281 derived from EIAV (Lentivirus) showed successful editing ranging from 76.5, 61.6, to 48.7% at the tdT locus, respectively. Other XDPs such as V273 (derived from RSV, Alpharetroviruses), V274 (derived from BLV, Deltaretroviruses), V276 (derived from FLV, Gammaretroviruses), V277 (derived from MMTV, Betretroviruses), V278 (derived from MMLV, Gammaretroviruses), V280 (derived from EIAV, Lentivirus), V282 (derived from SIV, Lentivirus) showed above background editing at the tdT locus ranging from 10.6 to 4.03%. The variation in editing efficiencies observed between the different constructs may be due to the architectural differences between the retroviral families used. Editing differences between V280 (editing at 10.6%) as compared to V281 (editing at 48.7%) is an example of this as both versions are derived from EIAV (Lentivirus) but differ in the architectural sequence. V280 has MS2 fused to Gag-pro polyprotein, whereas V281 has MS2 fused to the MA-CA polyprotein. Table 17: Percent editing at the second dilution (16.6 µl) XDP version Plasmid Genus/or Virus with number der Virus configuration % Editing 206 pXDP164 Lentivirus HIV HIV Gag-MS2 95.1 271 pXDP354 Alpharetrovirus ALV ALV Gag-pro-MS2 88.7 159 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 XDP version Plasmid Genus/o Virus with number rder Virus configuration % Editing 272 pXDP355 Alpharetrovirus ALV ALV Gag-MS2 79.1 273 pXDP356 Alpharetrovirus RSV RSV Gag-pro-MS2 5.05 274 pXDP357 Deltaretrovirus BLV BLV Gag-pro-MS2 6.63 275 pXDP358 Deltaretrovirus HTLV1 HTLV1 Nat Gag-pro- 76.5 MS2 276 pXDP359 Gammaretrovirus FLV FLV Gag-pro-MS2 5.3 277 pXDP360 Betaretrovirus MMTV MMTV Gag-pro-MS2 4.03 278 pXDP361 Gammaretrovirus MMLV MMLV Gag-pro-MS2 5.03 279 pXDP362 Betaretrovirus MPMV MPMV Gag-pro-MS2 61.6 280 pXDP363 Lentivirus EIAV EIAV Gag-pro-MS2 10.6 281 pXDP364 Lentivirus EIAV EIAV MA-CA-MS2 48.7 282 pXDP365 Lentivirus SIV SIV Gag-pro-MS2 9.05 [0469] Overall, these results show that fusing MS2 with the Gag-protease, Gag or partial Gag polyproteins of diverse retroviral origin that include Alpharetroviruses, Betaretroviruses, Gammaretroviruses, Deltaretroviruses and Lentiviruses creates XDPs that result in editing of the target nucleic acid. It is believed that supplementing these versions with another plasmid that encodes for the respective Gag-protease or Gag polyprotein could further augment editing functions. Table 18: Plasmid sequences XDP Plasmid version number SEQ ID NO of DNA Encoding Sequence pGP2 979 pSG50 1143 pXDP166 1142 206 pXDP161 1139 206 pXDP164 1140 271 pXDP354 1155 272 pXDP355 1156 160 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 XDP Plasmid version number SEQ ID NO of DNA Encoding Sequence 273 pXDP356 1157 274 pXDP357 1158 275 pXDP358 1159 276 pXDP359 1160 277 pXDP360 1161 278 pXDP361 1162 279 pXDP362 1163 280 pXDP363 1164 281 pXDP364 1165 282 pXDP365 1166 Example 5: Non-covalent recruitment with MS2 variants [0470] Experiments were conducted to evaluate the ability of an MS2-based recruitment system using MS2 variants having altered affinities to the MS2 hairpin in order to improve the generation of XDP in packaging host cells. Methods: [0471] All plasmids encoding CasX proteins utilized the CasX 491 variant protein. All XDPs contained sgRNAs with scaffold 188 (see FIG.12) and spacer 12.7. Structural plasmid cloning [0472] In order to generate the structural plasmids, listed below, pXDP1was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments encoding CasX variant, HIV-1, or MS2 CP components were amplified and cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Guide plasmid cloning [0473] The tdTomato targeting guide plasmid used in these experiments was pSG50 (guide scaffold 188), which was cloned from pSG33. The mammalian expression backbone contained a cPPT, ampicillin resistance, and a colEI replication site and was amplified using primers with appropriate overlaps to accept the U6 promoter and guide RNA scaffold cassette. The backbone, pSG3, was digested using NdeI and XbaI. Synthetic DNA fragments corresponding to novel 161 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 scaffolds were amplified and cloned as described in Example 2, above. The resultant plasmid, pSG33, was sequenced using Sanger sequencing to ensure correct assembly. Cloning tdTomato spacer 12.7 [0474] To clone the targeting pSG50 plasmid from the non-targeting pSG33, spacer 12.7 was cloned as described in Example 2, above. The resultant plasmid was sequenced using Sanger sequencing to ensure correct ligation (see Table 20). pGP2 Glycoprotein plasmid cloning [0475] Sequences encoding the VSV-G glycoprotein and the CMV promoter were amplified from pMD2.G and cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Table 23 lists the plasmid structural and glycoprotein plasmid components. Cell culture and transfection [0476] HEK293T Lenti-X™ cell culture was performed as described in Example 2, above. For transfection, the XDP structural plasmids (also encoding the CasX variants) of Table 21 were used in amounts ranging from 13 to 80.0 µg. Each transfection also received 13 µg of pSG50 and 0.25 µg of pGP2. Polyethylenimine (PEI MAX® from Polyplus) was then added as described in Example 2. Plasmid ratios in Table 19 were used in all Version 206 XDPs used in this assay and are based on prior data from other XDP versions. Table 19: Construct plasmids and ratios of plasmids used XDP version 206 plasmids Structural plasmid ratios Gag-(-1)-PR* 10% Gag-MS2* 45% CasX* 45% *transcript contains RRE and produces REV Collection and concentration; resuspension and transduction [0477] XDPs were collected and concentrated as described in Example 2, above. tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above, and tdTomato fluorescence was measured using flow cytometry. The assays were run 2-3 times for each sample, with similar results. 162 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Table 20: sgRNA and hairpin encoding sequences (DNA) Plasmid Guide SEQ Hairpin Encoding SEQ number scaffold Spacer DNA Sequence ID NO Sequence ID NO ACTGGCGCTTTTATCTGATTACTTT GAGAGCCATCACCAGCGACTATGTC pSG0033 188 NT GTAGTGGGTAAAGCTCACATGAGGA 1135 ACATGAGGATCA CCCATG 1131 TCACCCATGTGAGCATCAAAGCGAG T ACGTAATTACGTCTCG ACTGGCGCTTTTATCTGATTACTTT GAGAGCCATCACCAGCGACTATGTC pSG50 188 12.7 GTAGTGGGTAAAGCTCACATGAGGA 1825 ACATGAGGATCA CCATGTGAGCATCAAAGCTGC C 1131 TCAC CCATGT ATTCTAGTTGTGGTTT Table 21: XDP component architecture and glycoprotein sequences Plasmid number Architecture SEQ ID NO of DNA Sequence pGP2 VSV-G 979 pXDP161 Gag-(-1)-PR 1139 pXDP164 Gag-MS2 1140 pXDP165 Gag-MS2-p1*-p6-(-1)-PR 1141 pXDP166 SV40NLS-CasX491-SV40 NLS 1142 pXDP321 Gag-MS2 (V29I) 1167 pXDP335 Gag-MS2(K43R) 1168 pXDP336 Gag-MS2(K66R) 1169 pXDP337 Gag-MS2(N55R) 1170 pXDP338 Gag-MS2(N87S) 1171 pXDP339 Gag-MS2(T59A 1172 pXDP340 Gag-MS2(dInc) 1173 pXDP353 Gag-MS2(N55K) 1174 Results: [0478] In all, wild-type and 5 different MS2 variants were tested, as well as one dimerization- incompetent variant. These variants were tested in the same Gag-MS2 system as previous examples specified and this configuration is depicted in FIG.18. To test these variants, pXDP164, which encodes the wild-type Gag-MS2 in XDP version 206, was replaced with either pXDP321, pXDP335, pXDP336, pXDP337, pXDP338, pXDP339, or pXDP340. These MS2 163 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 variants had affinity KdS ranging from 1.2e-7 M to 4e-10 M, with the wild-type version being 3e-9 M (a lower Kd value indicates greater affinity between the MS2 hairpin and coat protein). [0479] Results of the assays showed that the XDP with MS2 having lower Kd variants tended to perform with better editing than higher Kd variants (see Table 22) with a gRNA having a single MS2 hairpin (gRNA 188). The data were analyzed with a correlation analysis between the Kd of the MS2 coat protein and the inverse of the EC50 (by volume of XDP introduced into assay); a measure of potency that increases with more potent XDP constructs. This resulted in an r value of -0.625 as seen in FIG.19, demonstrating that incorporation of MS2 with lower Kds correlated with resultant increased editing potency. The results support that by altering the binding affinity of the RNA hairpin and NCR protein, the potency of XDPs can be effectively modulated, thereby improving the XDP constructs. This approach may be similarly used with other RNA binding proteins, such as Qβ phage, GA phage, PP7 phage, or Λ N for engineering more potent XDPs. Table 22: MS2 variants Plasmid Mutation i Inverse EC50 number n MS2 Affinity by volume pXDP164 WT WT 1.6 pXDP321 V29I Kd: 4e-10 M 1.5 pXDP335 K43R Kd: 1e-9 M 2.7 pXDP336 K66R Kd: 3e-9 M 4.7 pXDP337 N55R unknown 2.2 pXDP338 N87S Kd: 6.3e-8 M 0.3 pXDP339 T59A Kd: 1.2e-7 M 0.4 pXDP340 V68_V80 dInc Dimerization incompetent 2.2 Table 23: XDP Version and pseudotyping descriptions XDP version Architectures and glycoproteins Plasmid numbers Rev expression 206 Gag-(-1)-PR pXDP161 Yes Gag-MS2 pXDP164 CasX pXDP166 VSV-G pGP2 164 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Example 6: Evaluation of non-covalent recruitment (NCR) systems with RNA binding proteins linked to Gag [0480] The purpose of these experiments was to evaluate the ability of various non-covalent recruitment (NCR) proteins linked to HIV Gag polyprotein and one or two copies of their cognate binding partner hairpin structures (“single hairpin” or “dual hairpins” respectively) integrated into the guide RNA scaffolds to improve the generation of XDP in packaging host cells. Methods: [0481] All XDPs were pseudotyped with 10% VSV-G (percentage of plasmid relative to the other plasmids utilized for the XDP construct). RNA fold structures were generated with RNAfold web server and Varna java-based software. Structural plasmid cloning [0482] In order to generate the structural plasmids used to make the XDP, pXDP1 was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments containing CasX, HIV-1, retrovirus Tat, IRP1, IRP2, truncated U1A, U1A, phage Qβ coat protein, phage GA coat protein, phage λN coat protein, or truncated phage λN coat protein components were amplified using In Fusion primers with 15-20 base pair overlaps and Kapa HiFi DNA polymerase according to the manufacturer’s protocols. The fragments were purified by gel extraction from a 1% agarose gel using Zymoclean Gel DNA Recovery Kit according to the manufacturer’s protocol. [0483] Further, fragments containing dual boxB hairpin, retrovirus transactivation response (TAR) element, Iron Responsive Element (IRE), U1A hairpin, phage Qβ hairpin, phage GA hairpin, phage λN hairpin, or phage PP7 hairpins were amplified and cloned in guide scaffolds based on guide scaffold 174 or guide scaffold 235. Sequences of guide RNA scaffolds with dual hairpins are provided in Table 26, below. Scaffolds 188 and 251 were used as controls. [0484] These fragments were cloned into plasmid backbones as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Guide plasmid cloning [0485] The guide plasmids modified in these experiments were pSG50, encoding guide scaffold 188 (see FIG.12). The non-targeting guide plasmids used in these experiments were pSG82 to pSG88, encoding guide scaffold 188. The mammalian expression backbone had a 165 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 cPPT, ampicillin resistance, and a colEI replication site and was amplified using primers with appropriate overlaps to accept the U6 promoter and guide RNA scaffold cassette. Fragments containing the retrovirus TAR, Iron Responsive Element (IRE), U1A hairpin II, phage Qβ hairpin, phage GA hairpin, phage λN hairpin (also referred to herein as a boxB hairpin or boxB element), or phage PP7 hairpin were amplified and cloned as described in Example 2, above. The targeting spacer sequence DNA for the tdTomato targeting spacer 12.7 was cloned into pSG33 and pSG34 as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation. pGP2 Glycoprotein plasmid cloning [0486] Sequences encoding the VSV-G glycoprotein and the CMV promoter were amplified and cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Cell culture and transfection [0487] HEK293T Lenti-X™ cell culture was performed as described in Example 2, above. For transfection, the XDP structural plasmids (also encoding the CasX variants) of Table 24 were used in amounts ranging from 13 to 80.0 µg. Each transfection will also receive 13 µg of a pSG plasmid and 0.25 µg of pGP2. Polyethylenimine (PEI MAX® from Polyplus) was then added as described in Example 2. Collection and concentration; resuspension and transduction; titering [0488] XDPs were collected and concentrated as described in Example 2, above. tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above, and tdTomato fluorescence was measured using flow cytometry. The assays were run 2-3 times for each sample with similar results. Titers were quantified for each version of XDPs using the NanoSight NS300. Results: [0489] The CasX guide scaffold extended stem region is highly modifiable. The extended stem loop protrudes out from the RNP, and so additions to this region have little effect on RNP formation and editing potency, as seen in other experiments described herein. This feature was used to add on one or two of several different RNA hairpins to the extended stem loop to engineer the CasX gRNA to bind their corresponding RNA-binding proteins. Table 24 shows the sequences of the Gag-NCR protein plasmids and their complementary sgRNAs with non- targeting spacers that were employed to create the versions. Table 25 shows the amino acid and 166 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 RNA sequences of the Gag-NCR proteins and their complementary sgRNAs, respectively. The amino acid sequences of the NCR proteins (not fused to the Gag polyprotein) are provided in SEQ ID NOs: 4130-4139. [0490] It was expected that inclusion of these NCR proteins into the constructs would likely yield more potent XDP configurations as it has previously been demonstrated that different Kds of NCR proteins, such as MS2, can modify the potency of XDPs. There is a large variety of Kds and sizes across these NCR proteins. [0491] As shown in FIGS.20 and 21, XDPs with the MS2, PP7, Tat, or U1A NCR systems produced the highest levels of editing in the mouse tdTomato NPCs. Indeed, XDPs with the PP7, Tat or U1A NCR systems produced higher levels of editing than XDPs with the MS2 NCR system. Both Tat and U1A NCR systems are monomeric in nature. Therefore, that both Tat and U1A NCR systems produced higher levels of editing suggests that MS2 dimerization has a detrimental effect on XDP architecture formation. It is anticipated that the relatively small size of the Tat protein could make it amenable to stacking (i.e., adding multiple Tat binding sites), which could enable better recruitment and packaging of the CasX RNP. Furthermore, while the PP7 NCR system also dimerizes, the RNA hairpin and the NCR protein have a higher binding affinity (Kd of 1 nM) compared to that of the MS2 system (of Kd of ~ 2.6 nM). This may explain the higher level of editing observed with the PP7 system compared to the MS2 system (FIGS.20 and 21). [0492] Titers were quantified for each version of XDP particles produced using the NanoSight NS300, and the number of transduced mouse NPCs was counted. The bar chart in FIG.22 shows the number of XDPs containing the indicated NCR systems per edited mouse NPC, and the bar chart in FIG.23 shows the average number of XDPs containing the indicated NCR systems per mouse NPC. Overall, use of the Gag-U1A, Gag-Tat, or Gag-PP7 NCR systems required the lowest average number of XDPs to edit a single mouse NPC (FIGS.22 and 23), which is consistent with the high editing levels seen in FIGS.20-21. [0493] In addition, it is anticipated that the location of the NCR protein in the Gag polyprotein or the viral protein used can both be modified, and enhanced guide RNA scaffolds could lead to further improvements in potency. 167 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Table 24: Sequences encoding Gag-NCR proteins and guide scaffolds based on guide scaffold 174 GAG- Protein Hairpin SEQ Scaffold 174 + SEQ NCR Plasmid protein Architecture number Encoding encoding ID Hairpin Encoding ID Sequence sequence NO Sequence NO SEQ ID NO λN - just RNA ACTGGCGCTTTTAT binding Gag-tλN pXDP366 1175 CTGATTACTTTGAG site GCCCTGAAGAA AGCCATCACCAGCG GGG 1185 ACTATGTCGTAGTG 1192 λN - full C GGTAAAGCTGCACG antitermina CCCTGAAGAAGGGC tion protein Gag-λN pXDP367 1176 GTGCAGCATCAAAG N ACTGGCGCTTTTAT CTGATTACTTTGAG AAGGAGTTTAT AGCCATCACCAGCG PP7 Gag-PP7 pXDP342 1177 ATGGAAACCCT 1186 ACTATGTCGTAGTG GG 1193 T TAAAGCTGCACT AAGGAGTTTATATG GAAACCCTTAGTGC AGCATCAAAG ACTGGCGCTTTTAT CTGATTACTTTGAG AGCCATCACCAGCG GGCTCGTGTAG ACTATGTCGTAGTG TAT/Tar Gag-Tat pXDP368 1178 CTCATTAGCTC 1187 GGTAAAGCTGCACG 1194 CGAGCC GCTCGTGTAGCTCA TTAGCTCCGAGCCG TGCAGCATCAAAG ACTGGCGCTTTTAT CTGATTACTTTGAG AGCCATCACCAGCG CCGTGTGCATC ACTATGTCGTAGTG Gag-IRP1 pXDP369 1179 CGCAGTGTCGG 1188 GGTAAAGCTGCACC 1195 ATCCACGG CGTGTGCATCCGCA GTGTCGGATCCACG GGTGCAGCATCAAA G IRP ACTGGCGCTTTTAT CTGATTACTTTGAG AGCCATCACCAGCG CCGTGTGCATC ACTATGTCGTAGTG Gag-IRP2 pXDP370 1180 CGCAGTGTCGG 1188 GGTAAAGCTGCACC 1195 ATCCACGG CGTGTGCATCCGCA GTGTCGGATCCACG GGTGCAGCATCAAA G 168 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 GAG- NCR Plas Protein Hairpin SEQ Scaffold 174 + SEQ protein Architecture mid Encoding encoding ID Hairpin Encoding ID number Sequence sequence NO Sequence NO SEQ ID NO ACTGGCGCTTTTAT CTGATTACTTTGAG GGAATCCATT U1A- AGCCATCACCAGCG Truncated Gag-tU1A pXDP371 1181 GCACTCCGGA 1189 ACTATGTCGTAGTG 1197 TTTCACTAG GGTAAAGCTGCACG GAATCCATTGCACT CCGGATTTCACTAG GTGCAGCATCAAAG ACTGGCGCTTTTAT CTGATTACTTTGAG GGAATCCATT AGCCATCACCAGCG U1A-Full Gag-U1A pXDP372 1182 GCACTCCGGA 1189 ACTATGTCGTAGTG 1197 TTTCACTAG GGTAAAGCTGCACG GAATCCATTGCACT CCGGATTTCACTAG GTGCAGCATCAAAG ACTGGCGCTTTTAT CTGATTACTTTGAG AGCCATCACCAGCG Qβ Gag-Qβ pXDP373 1183 ATGCATGTCT AAGACAGCAT 1190 ACTATGTCGTAGTG GGTAAAGCTGCACA 1198 TGCATGTCTAAGAC AGCATGTGCAGCAT CAAAG ACTGGCGCTTTTAT CTGATTACTTTGAG AAAACATAAG AGCCATCACCAGCG ACTATGTCGTAGTG GA Gag-GA pXDP362 1184 GAAAACCTAT 1191 GGTAAAGCTGCACA 1199 GTT AAACATAAGGAAAA CCTATGTTGTGCAG CATCAAAG Table 25: Amino acid sequences of Gag-NCR proteins and RNA sequences of guide scaffolds based on guide scaffold 174 NCR Archite SEQ SEQ Scaffold 174 + SEQ protein cture GAG-Protein AA Sequence ID Hairpin RNA ID Hairpin Sequence ID NO sequence NO (RNA) NO ATMGARASVLSGGELDRWEKIR ACUGGCGCUUUUA LRPGGKKKYKLKHIVWASRELE UCUGAUUACUUUG λN - just RFAVNPGLLETSEGCRQILGQL GCCCUGAAG AGAGCCAUCACCA RNA- Gag-tλN QPSLQTGSEELRSLYNTVATLY 2435 A binding site Q AGGGC 954 GCGACUAUGUCGU 2314 CVHQRIEIKDTKEALDKIEEE AGUGGGUAAAGCU NKSKKKAQQAAADTGHSNQVSQ GCACGCCCUGAAG AAGGGCGUGCAGC 169 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 NCR Archite SEQ SEQ Scaffold 174 + SEQ protein cture GAG-Protein AA Sequence ID Hairpin RNA sequen ID Hairpin Sequence ID NO ce NO (RNA) NO NAWVKVVEEKAFSPEVIPMFSA AUCAAAG LSEGATPQDLNTMLNTVGGHQA AMQMLKETINEEAAEWDRVHPV HAGPIAPGQMREPRGSDIAGTT STLQEQIGWMTHNPPIPVGEIY KRWIILGLNKIVRMYSPTSILD IRQGPKEPFRDYVDRFYKTLRA EQASQEVKNWMTETLLVQNANP DCKTILKALGPGATLEEMMTAC QGVGGPGHKARVLAEAMSQVTN PATIMIQKGNFRNQRKTVKCFN CGKEGHIAKNCRAPRKKGCWKC GKEGHQMKDCTERQANFLGKIW PSHKGRPGNFLQSRPEPTAPPE ESFRFGEETTTPSQKQEPIDKE LYPLASLRSLFGSDPSSQMDAQ TRRRERRAEKQAQWKAANGGSL ATMGARASVLSGGELDRWEKIR LRPGGKKKYKLKHIVWASRELE RFAVNPGLLETSEGCRQILGQL QPSLQTGSEELRSLYNTVATLY CVHQRIEIKDTKEALDKIEEEQ NKSKKKAQQAAADTGHSNQVSQ NYPIVQNIQGQMVHQAISPRTL NAWVKVVEEKAFSPEVIPMFSA LSEGATPQDLNTMLNTVGGHQA AMQMLKETINEEAAEWDRVHPV HAGPIAPGQMREPRGSDIAGTT λN - full STLQEQIGWMTHNPPIPVGEIY antiterminat KRWIILGLNKIVRMYSPTSILD ion protein Gag-λN IRQGPKEPFRDYVDRFYKTLRA 2436 N EQASQEVKNWMTETLLVQNANP DCKTILKALGPGATLEEMMTAC QGVGGPGHKARVLAEAMSQVTN PATIMIQKGNFRNQRKTVKCFN CGKEGHIAKNCRAPRKKGCWKC GKEGHQMKDCTERQANFLGKIW PSHKGRPGNFLQSRPEPTAPPE ESFRFGEETTTPSQKQEPIDKE LYPLASLRSLFGSDPSSQMDAQ TRRRERRAEKQAQWKAANPLLV GVSAKPVNRPILSLNRKPKSRV ESALNPIDLTVLL ATMGARASVLSGGELDRWEKIR LRPGGKKKYKLKHIVWASRELE RFAVNPGLLETSEGCRQILGQL ACUGGCGCUUUUA QPSLQTGSEELRSLYNTVATLY UCUGAUUACUUUG CVHQRIEIKDTKEALDKIEEEQ AAGGAGUUUA AGAGCCAUCACCA PP7 Gag-PP7 NKSKKKAQQAAADTGHSNQVSQ 2437 UAUGGAAACC 914 GCGACUAUGUCGU 2312 NYPIVQNIQGQMVHQAISPRTL CUU AGUGGGUAAAGCU NAWVKVVEEKAFSPEVIPMFSA GCACUAAGGAGUU LSEGATPQDLNTMLNTVGGHQA UAUAUGGAAACCC AMQMLKETINEEAAEWDRVHPV UUAGUGCAGCAUC AAAG 170 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 NCR Archite SEQ Hair SEQ Scaffold 174 + SEQ protein cture GAG-Protein AA Sequence ID pin RNA ID Hairpin Sequence ID NO sequence NO (RNA) NO STLQEQIGWMTHNPPIPVGEIY KRWIILGLNKIVRMYSPTSILD IRQGPKEPFRDYVDRFYKTLRA EQASQEVKNWMTETLLVQNANP DCKTILKALGPGATLEEMMTAC QGVGGPGHKARVLAEAMSQVTN PATIMIQKGNFRNQRKTVKCFN CGKEGHIAKNCRAPRKKGCWKC GKEGHQMKDCTERQANFLGKIW PSHKGRPGNFLQSRPEPTAPPE ESFRFGEETTTPSQKQEPIDKE LYPLASLRSLFGSDPSSQSKTI VLAVGEATRTLTEIQSTADRQI FEEKVGPLVGRLRLTASLRQNG AKTAYRVNLKLDQADVVDASTS VAGELPKVRYTQVWSHDVTIVA NSTEASRKSLYDLTKSLVATSQ VEDLVVNLVPLGRL ATMGARASVLSGGELDRWEKIR LRPGGKKKYKLKHIVWASRELE RFAVNPGLLETSEGCRQILGQL QPSLQTGSEELRSLYNTVATLY CVHQRIEIKDTKEALDKIEEEQ NKSKKKAQQAAADTGHSNQVSQ NYPIVQNIQGQMVHQAISPRTL NAWVKVVEEKAFSPEVIPMFSA ACUGGCGCUUUUA LSEGATPQDLNTMLNTVGGHQA AMQMLKETINEEAAEWDRVHPV UCUGAUUACUUUG HAGPIAPGQMREPRGSDIAGTT AGAGCCAUCACCA GGCUCGUGUA GCGACUAUGUCGU STLQEQIGWMTHNPPIPVGEIY TAT/Tar Gag-Tat GCUCAUUAGC AGUGGGUAAAGCU KRWIILGLNKIVRMYSPTSILD 2438 951 2315 UCCGAGCC GCACGGCUCGUGU IRQGPKEPFRDYVDRFYKTLRA EQASQEVKNWMTETLLVQNANP AGCUCAUUAGCUC DCKTILKALGPGATLEEMMTAC CGAGCCGUGCAGC AUCAAAG QGVGGPGHKARVLAEAMSQVTN PATIMIQKGNFRNQRKTVKCFN CGKEGHIAKNCRAPRKKGCWKC GKEGHQMKDCTERQANFLGKIW PSHKGRPGNFLQSRPEPTAPPE ESFRFGEETTTPSQKQEPIDKE LYPLASLRSLFGSDPSSQSGPR PRGTRGKGRRIRRL ATMGARASVLSGGELDRWEKIR LRPGGKKKYKLKHIVWASRELE RFAVNPGLLETSEGCRQILGQL ACUGGCGCUUUUA UCUGAUUACUUUG QPSLQTGSEELRSLYNTVATLY AGAGCCAUCACCA CVHQRIEIKDTKEALDKIEEEQ IRP Gag- CCGUGUGCAU GCGACUAUGUCGU NKSKKKAQQAAADTGHSNQVSQ CCGCAGUGUC AGUGG IRP1 NYPIVQNIQGQMVHQAISPRTL 2439 952 GUAAAGCU 2316 NAWVKVVEEKAFSPEVIPMFSA GGAUCCACGG GCACCCGUGUGCA UCCGCAGUGUCGG LSEGATPQDLNTMLNTVGGHQA AUCCACGGGUGCA AMQMLKETINEEAAEWDRVHPV GCAUCAAAG HAGPIAPGQMREPRGSDIAGTT 171 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 NCR Archite SEQ SEQ Scaffold 174 + SEQ protein cture GAG-Protein AA Sequence ID Hairpin RNA ID Hairpin Sequence ID NO sequence NO (RNA) NO KRWIILGLNKIVRMYSPTSILD IRQGPKEPFRDYVDRFYKTLRA EQASQEVKNWMTETLLVQNANP DCKTILKALGPGATLEEMMTAC QGVGGPGHKARVLAEAMSQVTN PATIMIQKGNFRNQRKTVKCFN CGKEGHIAKNCRAPRKKGCWKC GKEGHQMKDCTERQANFLGKIW PSHKGRPGNFLQSRPEPTAPPE ESFRFGEETTTPSQKQEPIDKE LYPLASLRSLFGSDPSSQMSNP FAHLAEPLDPVQPGKKFFNLNK LEDSRYGRLPFSIRVLLEAAIR NCDEFLVKKQDIENILHWNVTQ HKNIEVPFKPARVILQDFTGVP AVVDFAAMRDAVKKLGGDPEKI NPVCPADLVIDHSIQVDFNRRA DSLQKNQDLEFERNRERFEFLK WGSQAFHNMRIIPPGSGIIHQV NLEYLARVVFDQDGYYYPDSLV GTDSHTTMIDGLGILGWGVGGI EAEAVMLGQPISMVLPQVIGYR LMGKPHPLVTSTDIVLTITKHL RQVGVVGKFVEFFGPGVAQLSI ADRATIANMCPEYGATAAFFPV DEVSITYLVQTGRDEEKLKYIK KYLQAVGMFRDFNDPSQDPDFT QVVELDLKTVVPCCSGPKRPQD KVAVSDMKKDFESCLGAKQGFK GFQVAPEHHNDHKTFIYDNTEF TLAHGSVVIAAITSCTNTSNPS VMLGAGLLAKKAVDAGLNVMPY IKTSLSPGSGVVTYYLQESGVM PYLSQLGFDVVGYGCMTCIGNS GPLPEPVVEAITQGDLVAVGVL SGNRNFEGRVHPNTRANYLASP PLVIAYAIAGTIRIDFEKEPLG VNAKGQQVFLKDIWPTRDEIQA VERQYVIPGMFKEVYQKIETVN ESWNALATPSDKLFFWNSKSTY IKSPPFFENLTLDLQPPKSIVD AYVLLNLGDSVTTDHISPAGNI ARNSPAARYLTNRGLTPREFNS YGSRRGNDAVMARGTFANIRLL NRFLNKQAPQTIHLPSGEILDV FDAAERYQQAGLPLIVLAGKEY GAGSSRDWAAKGPFLLGIKAVL AESYERIHRSNLVGMGVIPLEY LPGENADALGLTGQERYTIIIP ENLKPQMKVQVKLDTGKTFQAV MRFDTDVELTYFLNGGILNYMI RKMAKL Gag- ATMGARASVLSGGELDRWEKIR CCGUGUGCAU A IRP2 SRELE 2440 CCGCAGUGUC 95 CUGGCGCUUUUA LRPGGKKKYKLKHIVWA 2 UCUGAUUACUUUG 2316 172 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 NCR Archite SEQ SEQ Scaffold 174 + SEQ protein cture GAG-Protein AA Sequence ID Hairpin RNA ID Hairpin Sequence ID NO sequence NO (RNA) NO RFAVNPGLLETSEGCRQILGQL GGAUCCACGG AGAGCCAUCACCA QPSLQTGSEELRSLYNTVATLY GCGACUAUGUCGU CVHQRIEIKDTKEALDKIEEEQ AGUGGGUAAAGCU NKSKKKAQQAAADTGHSNQVSQ GCACCCGUGUGCA NYPIVQNIQGQMVHQAISPRTL UCCGCAGUGUCGG NAWVKVVEEKAFSPEVIPMFSA AUCCACGGGUGCA LSEGATPQDLNTMLNTVGGHQA GCAUCAAAG AMQMLKETINEEAAEWDRVHPV HAGPIAPGQMREPRGSDIAGTT STLQEQIGWMTHNPPIPVGEIY KRWIILGLNKIVRMYSPTSILD IRQGPKEPFRDYVDRFYKTLRA EQASQEVKNWMTETLLVQNANP DCKTILKALGPGATLEEMMTAC QGVGGPGHKARVLAEAMSQVTN PATIMIQKGNFRNQRKTVKCFN CGKEGHIAKNCRAPRKKGCWKC GKEGHQMKDCTERQANFLGKIW PSHKGRPGNFLQSRPEPTAPPE ESFRFGEETTTPSQKQEPIDKE LYPLASLRSLFGSDPSSQMDAP KAGYAFEYLIETLNDSSHKKFF DVSKLGTKYDVLPYSIRVLLEA AVRNCDGFLMKKEDVMNILDWK TKQSNVEVPFFPARVLLQDFTG IPAMVDFAAMREAVKTLGGDPE KVHPACPTDLTVDHSLQIDFSK CAIQNAPNPGGGDLQKAGKLSP LKVQPKKLPCRGQTTCRGSCDS GELGRNSGTFSSQIENTPILCP FHLQPVPEPETVLKNQEVEFGR NRERLQFFKWSSRVFKNVAVIP PGTGMAHQINLEYLSRVVFEEK DLLFPDSVVGTDSHITMVNGLG ILGWGVGGIETEAVMLGLPVSL TLPEVVGCELTGSSNPFVTSID VVLGITKHLRQVGVAGKFVEFF GSGVSQLSIVDRTTIANMCPEY GAILSFFPVDNVTLKHLEHTGF SKAKLESMETYLKAVKLFRNDQ NSSGEPEYSQVIQINLNSIVPS VSGPKRPQDRVAVTDMKSDFQA CLNEKVGFKGFQIAAEKQKDIV SIHYEGSEYKLSHGSVVIAAVI SCTNNCNPSVMLAAGLLAKKAV EAGLRVKPYIRTSLSPGSGMVT HYLSSSGVLPYLSKLGFEIVGY GCSICVGNTAPLSDAVLNAVKQ GDLVTCGILSGNKNFEGRLCDC VRANYLASPPLVVAYAIAGTVN IDFQTEPLGTDPTGKNIYLHDI WPSREEVHRVEEEHVILSMFKA LKDKIEMGNKRWNSLEAPDSVL FPWDLKSTYIRCPSFFDKLTKE 173 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 NCR Archite SEQ SEQ Scaffold 174 + SEQ protein cture GAG-Protein AA Sequence ID Hairpin RNA ID Hairpin Sequence ID NO sequence NO (RNA) NO DHISPAGSIARNSAAAKYLTNR GLTPREFNSYGARRGNDAVMTR GTFANIKLFNKFIGKPAPKTIH FPSGQTLDVFEAAELYQKEGIP LIILAGKKYGSGNSRDWAAKGP YLLGVKAVLAESYEKIHKDHLI GIGIAPLQFLPGENADSLGLSG RETFSLTFPEELSPGITLNIQT STGKVFSVIASFEDDVEITLYK HGGLLNFVARKFSL ATMGARASVLSGGELDRWEKIR LRPGGKKKYKLKHIVWASRELE RFAVNPGLLETSEGCRQILGQL
Figure imgf000034_0001
NAWVKVVEEKAFSPEVIPMFSA LSEGATPQDLNTMLNTVGGHQA AMQMLKETINEEAAEWDRVHPV HAGPIAPGQMREPRGSDIAGTT ACUGGCGCUUUUA STLQEQIGWMTHNPPIPVGEIY KRWIILGLNKIVRMYSPTSILD UCUGAUUACUUUG IRQGPKEPFRDYVDRFYKT GGAAUCCAUU AGAGCCAUCACCA U1A- Gag- LRA Truncated tU1A QEVKNWMTETLLVQNANP 24 GCACUCCGGA GCGACUAUGUCGU EQAS 41 912 2317 UUUCACUAG AGUGGGUAAAGCU DCKTILKALGPGATLEEMMTAC GCACGGAAUCCAU QGVGGPGHKARVLAEAMSQVTN PATIMIQKGNFRNQRKTVKCFN UGCACUCCGGAUU CGKEGHIAKNCRAPRKKGCWKC UCACUAGGUGCAG CAUCAAAG GKEGHQMKDCTERQANFLGKIW PSHKGRPGNFLQSRPEPTAPPE ESFRFGEETTTPSQKQEPIDKE LYPLASLRSLFGSDPSSQMAVP ETRPNHTIYINNLNEKIKKDEL KKSLYAIFSQFGQILDILVSRS LKMRGQAFVIFKEVSSATNALR SMQGFPFYDKPMRIQYAKTDSD IIAKMKGTFL ATMGARASVLSGGELDRWEKIR LRPGGKKKYKLKHIVWASRELE RFAVNPGLLETSEGCRQILGQL QPSLQTGSEELRSLYNTVATLY ACUGGCGCUUUUA CVHQRIEIKDTKEALDKIEEEQ NKSKKKAQQAAADTGHSNQVSQ UCUGAUUACUUUG NYPIVQNIQGQMVHQAISPRTL AGAGCCAUCACCA GGAAUCCAUU GCGACUAUG U1A-Full Gag- UCGU NAWVKVVEEKAFSPEVIPMFSA GCACUCCGGA AGUGGGUAAAGCU U1A LSEGATPQDLNTMLNTVGGHQA 2442 912 2446 UUUCACUAG GCACAGCUAUCCA AMQMLKETINEEAAEWDRVHPV HAGPIAPGQMREPRGSDIAGTT UUGCACUCCGGAU STLQEQIGWMTHNPPIPVGEIY AGCUGUGCAGCAU CAAAG KRWIILGLNKIVRMYSPTSILD IRQGPKEPFRDYVDRFYKTLRA EQASQEVKNWMTETLLVQNANP 174 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 NCR Archite SEQ H SEQ Scaffold 174 + SEQ protein cture GAG-Protein AA Sequence ID airpin RNA ID Hairpin Sequence ID NO sequence NO (RNA) NO QGVGGPGHKARVLAEAMSQVTN PATIMIQKGNFRNQRKTVKCFN CGKEGHIAKNCRAPRKKGCWKC GKEGHQMKDCTERQANFLGKIW PSHKGRPGNFLQSRPEPTAPPE ESFRFGEETTTPSQKQEPIDKE LYPLASLRSLFGSDPSSQMAVP ETRPNHTIYINNLNEKIKKDEL KKSLYAIFSQFGQILDILVSRS LKMRGQAFVIFKEVSSATNALR SMQGFPFYDKPMRIQYAKTDSD IIAKMKGTFVERDRKREKRKPK SQETPATKKAVQGGGATPVVGA VQGPVPGMPPMTQAPRIMHHMP GQPPYMPPPGMIPPPGLAPGQI PPGAMPPQQLMPGQMPPAQPLS ENPPNHILFLTNLPEETNELML SMLFNQFPGFKEVRLVPGRHDI AFVEFDNEVQAGAARDALQGFK ITQNNAMKISFAKKL ATMGARASVLSGGELDRWEKIR LRPGGKKKYKLKHIVWASRELE RFAVNPGLLETSEGCRQILGQL QPSLQTGSEELRSLYNTVATLY CVHQRIEIKDTKEALDKIEEEQ NKSKKKAQQAAADTGHSNQVSQ NYPIVQNIQGQMVHQAISPRTL NAWVKVVEEKAFSPEVIPMFSA LSEGATPQDLNTMLNTVGGHQA AMQMLKETINEEAAEWDRVHPV HAGPIAPGQMREPRGSDIAGTT ACUGGCGCUUUUA STLQEQIGWMTHNPPIPVGEIY KRWIILGLNKIVRMYSPTSILD UCUGAUUACUUUG IRQGPKEPFRDYVDRFYKTLRA AGAGCCAUCACCA Gag-Qβ 2443 AUGCAUGUC GCGACUAUGUCGU Qβ EQASQEVKNWMTETLLVQNANP U AAGACAGCAU 911 AGUGGGUAAAGCU 2318 DCKTILKALGPGATLEEMMTAC GCACAUGCAUGUC QGVGGPGHKARVLAEAMSQVTN PATIMIQKGNFRNQRKTVKCFN UAAGACAGCAUGU CGKEGHIAKNCRAPRKKGCWKC GCAGCAUCAAAG GKEGHQMKDCTERQANFLGKIW PSHKGRPGNFLQSRPEPTAPPE ESFRFGEETTTPSQKQEPIDKE LYPLASLRSLFGSDPSSQMAKL ETVTLGNIGKDGKQTLVLNPRG VNPTNGVASLSQAGAVPALEKR VTVSVSQPSRNRKNYKVQVKIQ NPTACTANGSCDPSVTRQAYAD VTFSFTQYSTDEERAFVRTELA ALLASPLLIDAIDQLNPAYL ATMGARASVLSGGELDRWEKIR ACUGGCGCUUUUA LRPGGKKKYKLKHIVWASRELE AAAACAUAAG UCUGAUUACUUUG GA Gag-GA RFAVNPGLLETSEGCRQILGQL 2444 GAAAACCUAU 953 AGAGCCAUCACCA 2319 QPSLQTGSEELRSLYNTVATLY GUU GCGACUAUGUCGU AGUGGGUAAAGCU 175 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 NCR Archite SEQ Hairpin R SEQ Scaffold 174 + SEQ protein cture GAG-Protein AA Sequence ID NA ID Hairpin Sequence ID NO sequence NO (RNA) NO NKSKKKAQQAAADTGHSNQVSQ GCACAAAACAUAA NYPIVQNIQGQMVHQAISPRTL GGAAAACCUAUGU NAWVKVVEEKAFSPEVIPMFSA UGUGCAGCAUCAA LSEGATPQDLNTMLNTVGGHQA AG AMQMLKETINEEAAEWDRVHPV HAGPIAPGQMREPRGSDIAGTT STLQEQIGWMTHNPPIPVGEIY KRWIILGLNKIVRMYSPTSILD IRQGPKEPFRDYVDRFYKTLRA EQASQEVKNWMTETLLVQNANP DCKTILKALGPGATLEEMMTAC QGVGGPGHKARVLAEAMSQVTN PATIMIQKGNFRNQRKTVKCFN CGKEGHIAKNCRAPRKKGCWKC GKEGHQMKDCTERQANFLGKIW PSHKGRPGNFLQSRPEPTAPPE ESFRFGEETTTPSQKQEPIDKE LYPLASLRSLFGSDPSSQMATL RSFVLVDNGGTGNVTVVPVSNA NGVAEWLSNNSRSQAYRVTASY RASGADKRKYAIKLEVPKIVTQ VVNGVELPGSAWKAYASIDLTI PIFAATDDVTVISKSLAGLFKV GNPIAEAISSQSGFYAL [0494] Further experiments were conducted using sgRNAs with two hairpins for binding by NCR proteins. Table 26, below, shows the sequences of guide scaffolds based on guide scaffold 174 or scaffold 235, with two copies of each of the indicated hairpins. The guide scaffolds in Table 26 were tested in combination with the NCR proteins provided in Table 25. Table 26: Guide scaffold sequences with dual hairpins Enc DNA RNA Scaffold Architecture oding Sequence (5’- 3’) SEQ ID sgRNA (5’-3’) SEQ NO ID NO ACTGGCGCTTTTATCTGATT ACTTTGAGAGCCATCACCAG ACUGGCGCUUUUAUCUGAUUAC CGACTATGTCGTAGTGGGTA UUUGAGAGCCAUCACCAGCGAC PP7 Dual HP UAUGUCGUAGUGGGUAAAGCUG AAGCTGCACTATGGGCGCAG CACUAUGGGCGCAGCAAGGAGU 362 - 174 Scaffold CAAGGAGTTTATATGGAAAC 1312 UUAUAUGGAAACCCUUGCUGAC 2417 based CCTTGCTGACGGTACAGGCC AAGGAGTTTATATGGAAACC GGUACAGGCCAAGGAGUUUAUA CTTGGTATAGTGCAGCATCA UGGAAACCCUUGGUAUAGUGCA GCAUCAAAG AAG λN Dual HP - ACTGGCGCTTTTATCTGATT ACUGGCGCUUUUAUCUGAUUAC ACTTTGAGAGCCATCACCAG UUUGAGAGCCAUCACCAGCGAC 363 174 Scaffold CGACTATGTCGTAGTGGGTA 1313 UAUGUCGUAGUGGGUAAAGCUG 2418 based AAGCTGCACTATGGGCGCAG CACUAUGGGCGCAGCGCCCUGA 176 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 old Architecture Encodin DNA RNA Scaff g Sequence (5’- 3’) SEQ ID sgRNA (5’-3’) SEQ NO ID NO CGCCCTGAAGAAGGGCGCTG AGAAGGGCGCUGACGGUACAGG ACGGTACAGGCCGCCCTGAA CCGCCCUGAAGAAGGGCGGUAU GAAGGGCGGTATAGTGCAGC AGUGCAGCAUCAAAG ATCAAAG ACTGGCGCTTTTATCTGATT ACUGGCGCUUUUAUCUGAUUAC ACTTTGAGAGCCATCACCAG CGACTATGTCGTAGTGGGTA UUUGAGAGCCAUCACCAGCGAC Tar Dual HP - AAGCTGCACTATGGGCGCAG UAUGUCGUAGUGGGUAAAGCUG CACUAUGGGCGCAGCGGCUCGU 364 174 Scaffold CGGCTCGTGTAGCTCATTAG 1314 GUAGCUCAUUAGCUCCGAGCCG 2419 based CTCCGAGCCGCTGACGGTAC CUGACGGUACAGGCCGGCUCGU AGGCCGGCTCGTGTAGCTCA TTAGCTCCGAGCCGGTATAG GUAGCUCAUUAGCUCCGAGCCG TGCAGCATCAAAG GUAUAGUGCAGCAUCAAAG ACTGGCGCTTTTATCTGATT ACUGGCGCUUUUAUCUGAUUAC ACTTTGAGAGCCATCACCAG UUUGAGAGCCAUCACCAGCGAC CGACTATGTCGTAGTGGGTA UAUGUCGUAGUGGGUAAAGCUG IRE Dual HP AAGCTGCACTATGGGCGCAG CACUAUGGGCGCAGCCCGUGUG 365 - 174 Scaffold CCCGTGTGCATCCGCAGTGT 1315 CAUCCGCAGUGUCGGAUCCACG 2420 based CGGATCCACGGGCTGACGGT GGCUGACGGUACAGGCCCCGUG ACAGGCCCCGTGTGCATCCG UGCAUCCGCAGUGUCGGAUCCA CAGTGTCGGATCCACGGGGT CGGGGUAUAGUGCAGCAUCAAA ATAGTGCAGCATCAAAG G ACTGGCGCTTTTATCTGATT ACUGGCGCUUUUAUCUGAUUAC ACTTTGAGAGCCATCACCAG UUUGAGAGCCAUCACCAGCGAC CGACTATGTCGTAGTGGGTA U1A Dual HP AAGCTGCACTATGGGCGCAG UAUGUCGUAGUGGGUAAAGCUG CATCC CACUAUGGGCGCAGCAUCCAUU 366 - 174 Scaffold ATTGCACTCCGGATA 1316 GCACUCCGGAUAGCUGCUGACG 2421 based GCTGCTGACGGTACAGGCCA GUACAGGCCAUCCAUUGCACUC TCCATTGCACTCCGGATAGC CGGAUAGCUGGUAUAGUGCAGC TGGTATAGTGCAGCATCAAA G AUCAAAG ACTGGCGCTTTTATCTGATT ACUGGCGCUUUUAUCUGAUUAC ACTTTGAGAGCCATCACCAG UUUGAGAGCCAUCACCAGCGAC Qβ Dual HP - CGACTATGTCGTAGTGGGTA UAUGUCGUAGUGGGUAAAGCUG AAGCTGCACTATGGGCGCAG CACUAUGGGCGCAGCAUGCAUG 367 174 Scaffold CATGCATGTCTAAGACAGCA 1317 UCUAAGACAGCAUGCUGACGGU 2422 based TGCTGACGGTACAGGCCATG ACAGGCCAUGCAUGUCUAAGAC CATGTCTAAGACAGCATGGT AGCAUGGUAUAGUGCAGCAUCA ATAGTGCAGCATCAAAG AAG ACTGGCGCTTTTATCTGATT ACTTTGAGAGCCATCACCAG ACUGGCGCUUUUAUCUGAUUAC UUUGAGAGCCAUCACCAGCGAC CGACTATGTCGTAGTGGGTA GA Dual HP UAUGUCGUAGUGGGUAAAGCUG AAGCTGCACTATGGGCGCAG CACUAUGGGCGCAGCAAAACAU 368 - 174 Scaffold CAAAACATAAGGAAAACCTA 1318 AAGGAAAACCUAUGUU 2423 TGTTCTGACGGT CUGACG based ACAGGCCA AAACATAAGGAAAACCTATG GUACAGGCCAAAACAUAAGGAA AACCUAUGUUGGUAUAGUGCAG TTGGTATAGTGCAGCATCAA CAUCAAAG AG ACTGGCGCTTCTATCTGATT ACUGGCGCUUCUAUCUGAUUAC PP7 Dual HP ACTCTGAGCGCCATCACCAG UCUGAGCGCCAUCACCAGCGAC 369 - 235 Scaffold CGACTATGTCGTAGTGGGTA 1319 UAUGUCGUAGUGGGUAAAGCCG 2424 based AAGCCGCTTACGGACTATGG CUUACGGACUAUGGGCGCAGCA AGGAGUUUAUAUGGAAACCCUU 177 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 ld Architecture Encodin DNA RNA Scaffo g Sequence (5’- 3’) SEQ ID sgRNA (5’-3’) SEQ NO ID NO GGAAACCCTTGCTGACGGTA GCUGACGGUACAGGCCAAGGAG CAGGCCAAGGAGTTTATATG UUUAUAUGGAAACCCUUGGUAU GAAACCCTTGGTATAGTCCG AGUCCGUAAGAGGCAUCAGAG TAAGAGGCATCAGAG ACTGGCGCTTCTATCTGATT ACUGGCGCUUCUAUCUGAUUAC ACTCTGAGCGCCATCACCAG UCUGAGCGCCAUCACCAGCGAC λN Dual HP - CGACTATGTCGTAGTGGGTA UAUGUCGUAGUGGGUAAAGCCG AAGCCGCTTACGGACTATGG CUUACGGACUAUGGGCGCAGCG 370 235 Scaffold GCGCAGCGCCCTGAAGAAGG 1320 CCCUGAAGAAGGGCGCUGACGG 2425 based GCGCTGACGGTACAGGCCGC UACAGGCCGCCCUGAAGAAGGG CCTGAAGAAGGGCGGTATAG CGGUAUAGUCCGUAAGAGGCAU TCCGTAAGAGGCATCAGAG CAGAG ACTGGCGCTTCTATCTGATT ACTCTGAGCGCCATCACCAG ACUGGCGCUUCUAUCUGAUUAC CGACTATGTCGTAGTGGGTA UCUGAGCGCCAUCACCAGCGAC UAUGUCGUAGUGGGUAAAGCCG TAR Dual HP AAGCCGCTTACGGACTATGG CUUACGGACUAUGGGCGCAGCG GCGCAGCGGCTCGTGTAGCT 371 - 235 Scaffold CATTAGCTCCGAGCCGCTGA 1243 GCUCGUGUAGCUCAUUAGCUCC 2426 based CGGTACAGGCCGGCTCGTGT GAGCCGCUGACGGUACAGGCCG AGCTCATTAGCTCCGAGCCG GCUCGUGUAGCUCAUUAGCUCC GAGCCGGUAUAGUCCGUAAGAG GTATAGTCCGTAAGAGGCAT GCAUCAGAG CAGAG ACTGGCGCTTCTATCTGATT ACUGGCGCUUCUAUCUGAUUAC ACTCTGAGCGCCATCACCAG UCUGAGCGCCAUCACCAGCGAC CGACTATGTCGTAGTGGGTA P AAGCCGCTT UAUGUCGUAGUGGGUAAAGCCG IRE Dual H ACGGACTATGG GCGCAGCCCGTGTGCATCCG CUUACGGACUAUGGGCGCAGCC 372 - 235 Scaffold CGUGUGCAUCCGCAGUGUCGGA CAGTGTCGGATCCACGGGCT 1322 2427 based UCCACGGGCUGACGGUACAGGC GACGGTACAGGCCCCGTGTG CCCGUGUGCAUCCGCAGUGUCG CATCCGCAGTGTCGGATCCA CGGGGTATAGTCCGTAAGAG GAUCCACGGGGUAUAGUCCGUA GCATCAGAG AGAGGCAUCAGAG ACTGGCGCTTCTATCTGATT ACTCTGAGCGCCATCACCAG ACUGGCGCUUCUAUCUGAUUAC UCUGAGCGCCAUCACCAGCGAC CGACTATGTCGTAGTGGGTA U1A Dual HP UAUGUCGUAGUGGGUAAAGCCG AAGCCGCTTACGGACTATGG CUUACGGACUAUGGGCGCAGCA 373 - 235 Scaffold GCGCAGCATCCATTGCACTC 1323 UCCAUUGCACUC 2428 CGGATAGCT CGGAUAGCUG based GCTGACGGTAC AGGCCATCCATTGCACTCCG CUGACGGUACAGGCCAUCCAUU GCACUCCGGAUAGCUGGUAUAG GATAGCTGGTATAGTCCGTA UCCGUAAGAGGCAUCAGAG AGAGGCATCAGAG ACTGGCGCTTCTATCTGATT ACUGGCGCUUCUAUCUGAUUAC ACTCTGAGCGCCATCACCAG UCUGAGCGCCAUCACCAGCGAC CGACTATGTCGTAGTGGGTA Qβ Dual HP - AAGCCGCTTACGGACTATGG UAUGUCGUAGUGGGUAAAGCCG CUUACG 74 235 Scaffold GC GACUAUGGGCGCAGCA 3 GCAGCATGCATGTCTAAG 1324 UGCAUGUCUAAGACAGCAUGCU 2429 based ACAGCATGCTGACGGTACAG GACGGUACAGGCCAUGCAUGUC GCCATGCATGTCTAAGACAG UAAGACAGCAUGGUAUAGUCCG CATGGTATAGTCCGTAAGAG GCATCAGAG UAAGAGGCAUCAGAG GA Dual HP ACTGGCGCTTCTATCTGATT ACUGGCGCUUCUAUCUGAUUAC 375 - 235 Scaffold ACTCTGAGCGCCATCACCAG 1325 UCUGAGCGCCAUCACCAGCGAC 2430 based UAUGUCGUAGUGGGUAAAGCCG 178 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 rchitecture Encoding Se DNA RNA Scaffold A quence (5’- 3’) SEQ ID sgRNA (5’-3’) SEQ NO ID NO AAGCCGCTTACGGACTATGG CUUACGGACUAUGGGCGCAGCA GCGCAGCAAAACATAAGGAA AAACAUAAGGAAAACCUAUGUU AACCTATGTTGCTGACGGTA GCUGACGGUACAGGCCAAAACA CAGGCCAAAACATAAGGAAA UAAGGAAAACCUAUGUUGGUAU ACCTATGTTGGTATAGTCCG AGUCCGUAAGAGGCAUCAGAG TAAGAGGCATCAGAG [0495] The results of the editing assays testing guide scaffolds with dual RNA hairpins are provided in FIGS.91-99. As shown in FIG.92, use of guide scaffold 188, which has a single MS2 hairpin, produced a slightly lower editing potency than use of guide scaffold 251, which has two copies of the MS2 hairpin (“dual hairpin”). This is consistent with the results provided below in Example 9. [0496] As shown as FIG.91, use of guide scaffolds with one or two copies of the PP7 hairpin produced similarly high levels of editing. This was true for the guide scaffolds based on either guide scaffold 174 and 235. [0497] In the λN NCR system, using a truncated λN protein made up of the RNA-binding site as the NCR protein (“tλN”), use of the dual boxB hairpin guide scaffold produced the highest level of editing in the guide scaffold 174 background, followed by the dual hairpin guide scaffold in the scaffold 235 background (FIG.93). Therefore, adding two copies of the boxB hairpin improved editing levels when paired with the tλN NCR protein.
Figure imgf000039_0001
179 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386
Figure imgf000040_0001
188; see FIG.12) and pSG5 (scaffold 174), were cloned from pSG33 and pSG3 180 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386
Figure imgf000041_0001
GGGCGCAGCTCATGAGGATCACCC GGATCACCCATGTGG ATGAGCTGACGGTACAGGCCACAT TATAGTGC GAGGATCACCCATGTGGTATAGTG CAGCATCAAAGCTGCATTCTAGTT GTGGTTT 181 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 pGP2 Glycoprotein plasmid cloning [0510] Sequences encoding the VSV-G glycoprotein and the CMV promoter were cloned as described in Example 2. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly (see Table 30). Cell culture and transfection [0511] HEK293T Lenti-X™ cell culture was performed as described in Example 2, above. For transfection, the XDP structural plasmids (also encoding the CasX variants) of Table 30 were used in amounts ranging from 13 to 80.0 µg. Each transfection also received 13 µg of pSG50 or pSG5 and 0.25 µg of pGP2. The descriptions of the plasmids used to evaluate the NLS are listed in Table 29. Polyethylenimine (PEI MAX® from Polyplus) was then added as described in Example 2. Plasmid ratios in Table 28 were used in all version 206 XDPs used in this assay and are based on prior data from other XDP versions. Table 28: Construct plasmids and ratios of plasmids used XDP version 206 XDP version 309 Structural plasmid plasmids plasmids ratios Gag-(-1)-PR* Gag-(-1)-PR* pXDP161 pXDP161 10% Gag-MS2* Gag-MS2-MS2* pXDP164 pXDP288 45% CasX* CasX* pXDP166 pXDP166 45% *transcript contains RRE and produces REV Table 29: XDP plasmids for evaluation NLS effects XDP version Architectures and glycoproteins Plasmid numbers Gag-(-1)-PR pXDP161 V206 Gag-MS2 pXDP164 CasX pXDP166 VSV-G pGP2 Gag-(-1)-PR pXDP161 V206 NLS 240 Gag-MS2 pXDP164 CasX with NLS 240 pXDP344 182 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 XDP version Architectures and glycoproteins Plasmid numbers VSV-G pGP2 Gag-(-1)-PR pXDP161 V206 NLS 255 Gag-MS2 pXDP164 CasX with NLS 255 pXDP350 VSV-G pGP2 Table 30: Plasmid architecture and glycoprotein sequences Plasmid numbers Architecture SEQ ID NO of Encoding sequence pGP2 VSV-G 979 pXDP161 Gag-(-1)-PR 1139 pXDP164 Gag-MS2 1140 pXDP166 SV40NLS-CasX491-SV40 NLS 1142 pXDP344 AAV122_Cmyc_NLS- BPSV40_NLS_(GGGS)2_PG-CasX-SV40 1206 pXDP350 AAV119-CasX-AAV129 1207 Collection and concentration; resuspension and transduction [0512] XDPs were collected and concentrated as described in Example 2, above. tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above, and tdTomato fluorescence was measured using flow cytometry. tdTomato fluorescence was measured using flow cytometry. The assays were run 2-3 times for each sample, with similar results. Results: [0513] XDP version 309 is identical to version 206 except there is an additional MS2 CP fused to the first MS2 in this system, so pXDP164 (which encodes Gag-MS2) is replaced with pXDP288, which encodes Gag-MS2-MS2. While the hypothesis was that inclusion of the additional MS2 would increase the avidity of the RNP with MS2 hairpin in the scaffolds for these coat proteins, thereby increasing the incorporation of RNP into the budding XDP, it was observed that there was a significant decrease with the constructs incorporating the 183 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 second MS2 coat protein (see FIG.24). The inverse of the EC50 by volume was 1.6 µL-1 for V206 (single MS2) and 0.075 µL-1 for V309 (double MS2). While V309 was still more potent than the negative control V206 without an MS2 hairpin containing scaffold (scaffold 174), which had an inverse EC50 of 0.012 µL-1, the results nevertheless underscore the utility of incorporating the MS2 system in the XDP constructs. Example 8: Evaluation of non-covalent recruitment (NCR) systems with dual MS2 hairpins for MS2 coat protein binding [0514] The ur ose of these ex eriments was to determine if the incor oration of two MS2
Figure imgf000044_0001
Table 31: sgRNA encoding sequences Plasmid Scaffold Spacer Encoding gui SEQ ID Encoding hairpin SEQ ID numbers de sequence NO sequence NO ACTGGCGCTTTTATCTGATTACTT CAGCGTCAATGACG pSG67 250 NT TGAGAGCCATCACCAGCGACTATG 1200 CTGACGGTACAGGC 1204 CACATGAGGATCAC 184 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Plasmid Scaffold Spacer E SEQ ID Encoding hairpin SEQ ID numbers ncoding guide sequence NO sequence NO GGGCGCAGCGTCAATGACGCTGAC CCATGTGGTATAGT GGTACAGGCCACATGAGGATCACC GC CATGTGGTATAGTGCAGCATCAAA GCGAGACGTAATTACGTCTCG ACTGGCGCTTTTATCTGATTACTT TGAGAGCCATCACCAGCGACTATG TCGTAGTGGGTAAAGCTGCACTAT CAGCTCATGAGGAT CACCCATGAGCTGA GGGCGCAGCTCATGAGGATCACCC pSG68 251 NT CGGTACAGGCCACA ATGAGCTGACGGTACAGGCCACAT 1201 1205 TGAGGATCACCCAT GAGGATCACCCATGTGGTATAGTG CAGCATCAAAGCGAGACGTAATTA GTGGTATAGTGC CGTCTCG ACTGGCGCTTTTATCTGATTACTT TGAGAGCCATCACCAGCGACTATG CAGCGTCAATGACG TCGTAGTGGGTAAAGCTGCACTAT CTGACGGTACAGGC pSG72 250 12.7 GGGCGCAGCGTCAATGACGCTGAC 1202 CACATGAGGATCAC 1204 GGTACAGGCCACATGAGGATCACC CCATGTGGTATAGT CATGTGGTATAGTGCAGCATCAAA GC GCTGCATTCTAGTTGTGGTTT ACTGGCGCTTTTATCTGATTACTT TGAGAGCCATCACCAGCGACTATG CAGCTCATGAGGAT TCGTAGTGGGTAAAGCTGCACTAT GGGCGCAGCTCATGAGGATCACCC CACCCATGAGCTGA pSG73 251 12.7 ATGAGCTGACGGTACAGGCCACAT 1203 CGGTACAGGCCACA 1205 TGAGGATCACCCAT GAGGATCACCCATGTGGTATAGTG GTGGTATAGTGC CAGCATCAAAGCTGCATTCTAGTT GTGGTTT Cloning tdTomato spacer 12.7 into pSG67 and pSG68 [0518] To clone the targeting pSG72 and 73 plasmids from the non-targeting pSG67 and pSG68, spacer 12.7 was cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation. pGP2 Glycoprotein plasmid cloning [0519] Sequences encoding the VSV-G glycoprotein and the CMV promoter were amplified from pMD2.G and cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Cell culture and transfection [0520] HEK293T Lenti-X™ cell culture was performed as described in Example 2, above. For transfection, the XDP structural plasmids (also encoding the CasX variants) of Table 33 were used in amounts ranging from 13 to 80.0 µg. Each transfection also received 13 µg of pSG50 or pSG5 and 0.25 µg of pGP2. Polyethylenimine (PEI MAX® from Polyplus) was then added as described in Example 2. Plasmid ratios in Table 32 were used in all version 206 XDPs used in 185 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 this assay, based on prior data. Plasmid sequences are listed in Table 33. XDP version and components incorporated are listed in Table 34. Table 32: Plasmids and ratios used XDP version 206 plasmids Structural plasmid ratios Gag-(-1)-PR 10% Gag-MS2 45% CasX 45% Table 33: Plasmid architecture and glycoprotein sequences Plasmid numbers Architecture SEQ ID NO of Encoding sequence pGP2 VSV-G 979 pXDP161 Gag-(-1)-PR 1139 pXDP164 Gag-MS2 1140 pXDP166 SV40NLS-CasX491-SV40 NLS 1142 AAV122_Cmyc_NLS- pXDP344 BPSV40_NLS_(GGGS)2_PG-CasX- 1206 SV40 pXDP350 AAV119-CasX-AAV129 1207 Table 34: Version and pseudotyping descriptions XDP version Architectures and glycoproteins Plasmid numbers Gag-(-1)-PR pXDP161 206 Gag-MS2 pXDP164 CasX pXDP166 VSV-G pGP2 Gag-(-1)-PR pXDP161 Gag-MS2 pXDP164 206 NLS 240 CasX w/NLS 240 pXDP344 VSV-G pGP2 186 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 XDP version Architectures and glycoproteins Plasmid numbers Gag-(-1)-PR pXDP161 206 NLS 255 Gag-MS2 pXDP164 CasX w/ NLS 255 pXDP350 VSV-G pGP2 Collection and concentration; resuspension and transduction [0521] XDPs were collected and concentrated as described in Example 2, above. [0522] tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above, and tdTomato fluorescence was measured using flow cytometry. tdTomato fluorescence was measured using flow cytometry. The assays were run 2-3 times for each sample, with similar results. Results: [0523] Two guide scaffolds, scaffold 250 (FIG.25) and 251 (FIG.26), were assayed in the version 206 system. Scaffold 250 had one MS2 hairpin and one RRE, and scaffold 251 had two MS2 hairpins and one RRE. These versions were tested in three different compositions. First was V206 which contains SV40 NLSs on either side of the protein. Second was V206 with NLS 240, which is a stronger NLS than the SV40 in V206. Third was V206 with NLS 255 which had an NLS comparable to NLS 240. The results showed that with the NLS variants, the dual MS2 scaffold 251 performed better than 250, and the opposite was true for V206 with the normal SV40 NLS (FIG.27). However, as seen in Table 35 and FIG.27, these scaffolds were able to edit very similarly across all conditions. The potency was measured by inverse EC50, and with
Figure imgf000047_0001
187 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386
Figure imgf000048_0001
fragments were cloned into plasmid backbones using In-Fusion HD Cloning Kit from Takara according to the manufacturer’s protocols. products were transformed into 188 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386
Figure imgf000049_0001
252 Dual RBE - RBE - 189 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386
Figure imgf000050_0001
190 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386
Figure imgf000051_0001
191 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386
Figure imgf000052_0001
envelope glycoproteins derived from other species within the Vesiculovirus genus to produce potent particles that can successfully edit This would offer several advantages: 1) 192 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386
Figure imgf000053_0001
JURV pGP87 1240 193 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386
Figure imgf000054_0001
2. 194 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386
Figure imgf000055_0001
lentiviral and Alpharetroviral constructs bearing the glycoprotein variants. 195 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386
Figure imgf000056_0001
[0549] In order to generate the structural plasmids used to make the XDP, pXDP1 was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments containing CasX ALV and HIV-1 components were amplified and cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Plasmids and their sequences are listed in Table 42. Table 42: Plasmid sequences for structural plasmids and glycoproteins XDP version number/ Viral source Plasmid number SEQ ID NO 168 pXDP161 1139 168 pXDP17 1144 - pSG17 1153 - pSG005 1253 44 pXDP40 1254 102 pXDP145 1255 pGP2 979 H5N1 pGP80 1256 H7N9 pGP81 1257 Eastern equine encephalitis virus (EEEV) pGP65 1258 196 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 XDP version number/ Viral source Plasmid number SEQ ID NO Venezuelan equine encephalitis viruses (VEEV) pGP66 1259 Western equine encephalitis virus (WEEV) pGP67 1260 Semliki Forest virus pGP68 1261 Sindbis virus pGP69 1262 Chikungunya virus (CHIKV) pGP70 1263 Bornavirus BoDV-1 pGP58 1264 Tick-borne encephalitis virus (TBEV) pGP71 1265 Rabies virus (strain Nishigahara RCEH) (RABV) pGP29.3 1266 Rabies virus (strain India) (RABV) pGP29.4 1267 Rabies virus (strain CVS-11) (RABV) pGP29.5 1268 Rabies virus (strain ERA) (RABV) pGP29.6 1269 Rabies virus (strain SAD B19) (RABV) pGP29.7 1270 Rabies virus (strain Vnukovo-32) (RABV) pGP29.8 1271 Rabies virus (strain Pasteur vaccins / PV) (RABV) pGP29.9 1272 Rabies virus (strain PM1503/AVO1) (RABV) pGP29.1 1273 Rabies virus (strain China/DRV) (RABV) pGP29.11 1274 Rabies virus (strain China/MRV) (RABV) pGP29.12 1275 Rabies virus (isolate Human/Algeria/1991) (RABV) pGP29.13 1276 Rabies virus (strain HEP-Flury) (RABV) pGP29.14 1277 Rabies virus (strain silver-haired bat- associated) (RABV) (SHBRV) pGP29.15 1278 Codon optimized rabies virus pGP29.2 1279 Rabies Virus pGP29 1280 Mokola Virus pGP30 1281 Measles Virus pGP32.1 1282 Measles Virus pGP32.2 1283 Mouse mammary tumor virus pGP6 1284 197 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 XDP version number/ Viral source Plasmid number SEQ ID NO Human T-lymphotropic virus 1 pGP7 1284 RD114 Endogenous Feline Retrovirus pGP8 1286 Gibbon ape leukemia virus pGP9 1287 Moloney Murine leukemia virus pGP10 1288 Baboon Endogenous Virus pGP11 1289 Human Foamy Virus pGP12 1290 Ebola Zaire Virus pGP41 1291 Dengue pGP25 1292 Zika virus pGP26 1293 West Nile Virus pGP27 1294 Japanese Encephalitis Virus pGP28 1295 Mumps Virus F pGP31.1 1296 Mumps Virus HN pGP31.2 1297 Sendai Virus F pGP33.1 1298 Sendai Virus HN pGP33.2 1299 AcMNPV gp64 pGP59 1300 Ross River Virus pGP54 1301 N1 Neuraminidase pGP82 1302 Dengue virus 2 pGP75 1303 Dengue virus 3 pGP76 1304 Dengue virus 4 pGP77 1305 Nipah Virus pGP34.1 1306 Nipah Virus pGP34.2 1307 Hendra Virus pGP35.1 1308 Hendra Virus pGP35.2 1309 Newcastle disease virus pGP37.1 1310 Newcastle disease virus pGP37.2 1311 198 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Guide plasmid cloning [0550] The guide plasmids used in these experiments were either pSG005 or pSG17. pSG17 has both the spacer 12.7 targeting tdTomato as well as the guide scaffold 226 that has the RRE/RBE element that has been described in previous examples. pSG005 has guide scaffold 174 along with the spacer 12.7 targeting tdTomato. To clone the targeting pSG005 and pSG17 guide plasmids, spacer 12.7 was cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation. pGP Glycoprotein plasmid cloning [0551] Encoding sequences for glycoproteins derived from Togaviridae, Paramyxoviridae, Rhabdoviridae, Orthomyxoviridae, Retroviridae and Flaviviridae are provided in Table 42. The designed constructs were synthesized as transgenes and purchased pre-cloned into pTWIST expression plasmids from Twist Bioscience. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly (see Table 42). Cell culture and transfection [0552] HEK293T Lenti-X™ cell culture was performed as described in Example 2, above. XDPs derived from HIV lentiviral-based architecture (V168) were pseudotyped with GPs from Togaviridae (pGP65, 66, 67, 68, 69 and 70), Rhabdoviridae (pGP29.7, 30) and Moloney Murine leukemia virus (pGP10). XDPs derived from two different alpha retroviral-based architectures (ALV V44 and ALV V102) were pseudotyped with GPs from Rhabdoviridae (pGP29.7). For transfection, the XDP structural plasmids (configurations are listed in Table 42) were used in amounts ranging from 13 to 80.0 µg. Each transfection also received 13 µg of either pSG005 or pSG17 and 2.5 µg of pGP2 or any other GPs. Polyethylenimine (PEI MAX® from Polyplus) was then added as described in Example 2. Collection and concentration; resuspension and transduction [0553] XDPs were collected and concentrated as described in Example 2, above. [0554] tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above, and tdTomato fluorescence was measured using flow cytometry. The assays were run 2-3 times for each sample with similar results. Results: [0555] Percent editing of the tdTomato target sequence in tdT NPCs are shown for all XDP constructs derived from HIV (V168) as well as XDP constructs derived from ALV (V44 and V102) in FIG.38, in terms of volume of XDPs used to treat the cells. This is broken up further 199 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 with the percent editing in tdT NPCs elicited when 50 µl and 16 µl of the concentrated XDP preps were used to treat NPCs, as shown in FIG.39 and FIG.40, respectively. Percent editing for the V168 XDPs pseudotyped with the different GPs, in terms of number of particles added to the tdTomato NPCs, are shown in FIG.41. V168 pseudotyped with pGP2 served as the base control XDP for comparisons. The results show that GPs derived from Togaviridae (in particular Semliki, WEEV, EEEV, VEEV) and Rhabdoviridae (Mokola and Rabies), as well as MoMLV are potent in NPCs, suggesting properties of neural tropism. GPs derived from Togaviridae such as pGP68, pGP68, pGP66 and pGP65 seemed particularly potent (in that order) ranging in editing efficiencies from 74% to 36% when 50 μl of concentrated XDPs were used to treat NPCs. They also show that both architectural versions of ALV derived XDPs (V44 and V102) can be pseudotyped with GPs derived from Rhabdoviridae (pGP 29.7), ranging in editing efficacies from 7% to 27% when 50 μl of concentrated XDPs were used to treat NPCs, in addition to VSV-G, where they show efficacies ranging from 39% to 30% as shown in FIG.40. Titers for the V168 XDPs were determined by P24 ELISA, as shown in FIG.42, and they demonstrate that XDPs can be produced that are pseudotyped with the different glycoproteins without affecting overall titer. The difference in potency that is seen in tdT NPCs is most likely due to inherent differences in cellular and tissue tropism between these glycoproteins. The difference in editing profiles of ALV V44 and ALV102 pseudotyped with Rabies (pGP29.7) also highlights the possibility of the XDP internal architecture having an independent effect on the packaging of the targeting moiety on the surface of these particles. The lack of potencies with particular GPs such as pGP70 and pGP69 as compared to other Togaviridae GPs might be due to incompatibility with the internal architecture, in addition to inherent differences in tropism. Therefore, these GPs might show potency with other architectural variants of HIV based XDPs, in addition to XDPs derived from other architectural variants of Alpharetroviral, Betaretroviral, Gammaretroviral, Deltaretroviral, Epsilonretroviral, Lentiviral and Spumaretroviral origin. [0556] XDPs derived from HIV lentiviral-based architecture (V168) were pseudotyped with GPs from different rabies variants from the Rhabdoviridae family (pGP29, 29.2, 29.3, 29.4, 29.5, 29.6, 29.8). V168 pseudotyped with pGP2 served as the base control XDP for comparisons. Several rabies variants showed potency in mouse NPCs, with pGP29 and pGP29.4 showing particular promise with editing efficiencies at the tdTomato locus ranging from 70% to 25% when 16.6 μl of the concentrated XDPs were used to treat NPCs, as shown in FIG.43 and 200 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 FIG.44. V168 pseudotyped with pGP2 demonstrated the most efficacy at 85%. However, as compared to pGP2, the rabies variants (pGP29 and pGP29.4) would allow specific targeting of cells of neuronal origin, suggesting a better safety profile in vivo for neural indications, thereby making up for their lower editing potencies relative to VSV-G (pGP2). [0557] XDPs derived from HIV lentiviral-based architecture (V168) were pseudotyped with GPs from Paramyxoviridae (pGP35.1, 35.2, 34.1, 34.2), Orthomyxoviridae (pGP80, 81, 82) and Flaviviridae (pGP25, 26, 27, 28, 75) families. Almost all the GPs showed activity at the 50 µl dose, as shown in FIG.45. At the second dilution (when 16.6 μl of the concentrated XDPs were used to treat NPCs), XDPs pseudotyped with Orthomyxoviridae (pGP80, 82) and Paramyxoviridae (pGP35.1, 35.2, 34.1, 34.2) demonstrated about 35%, 11% and 10% editing, respectively, as shown in FIG.46. Titers for the V168 XDPs were determined by P24 ELISA as shown in FIG.47 and demonstrate that pseudotyping XPDs with the different glycoproteins didn’t affect production titers. [0558] These data support the conclusion that XDPs can be effectively pseudotyped with different glycoproteins derived from diverse viral genera. The differences in potency that were seen in tdT NPCs suggests inherent differences in cellular and tissue tropism properties that exist amongst these glycoproteins. The observed selectively can be harnessed with XDPs designed to safely and selectively deliver the payload to therapeutically-relevant cells. Overall, these results show that XDPs can be engineered to possess selective cell tropism by effectively pseudotyping them with envelope glycoproteins derived from different viral families that retain good editing potency. Given that V168 XDPs have been successfully pseudotyped with these diverse glycoproteins, it should be possible to use these glycoproteins to pseudotype other versions of XDPs derived from any architectural variants of Alpharetroviral, Betaretroviral, Gammaretroviral, Deltaretroviral, Epsilonretroviral, Lentiviral and Spumaretroviral origin. Example 12: Enhancing RNA export mechanisms for the formation of XDP using a Rev/RRE system - Scaffold 174 vs 226 [0559] The purpose of these experiments was to evaluate the effects of incorporation of a portion of an HIV-1 Rev response element (RRE) sequence into the guide RNA scaffold to determine whether RNA export, recruitment of the guide into XDP, and resultant potency of the XDP was enhanced, with and without a direct Gag-CasX fusion. The HIV-1 RRE is a ~350 nucleotide RNA element in the HIV-1 genome that is recognized by the HIV-1 Rev protein and 201 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 is essential for HIV-1 replication. Early in the HIV-1 replication cycle, REV shuttles the HIV-1 RNA genome out of the nucleus into the cytoplasm by binding to the RRE, RanGTP, and Crm1. As described herein, portions of the RRE element were incorporated into the extended stem region of the CasX scaffold 174 To enhance nuclear export of the sgRNAs into the cytoplasm of the XDP-producing LentiX cells. The proposed recruitment mechanism using RRE elements in enhancing nuclear export of the gRNA is depicted in FIG.51. Methods: [0560] All plasmids containing CasX proteins had the CasX variant 491 protein. All XDPs were pseudotyped with 10% VSV-G (percentage of plasmid relative to the other plasmids utilized for the XDP construct). RNA fold structures were generated with RNAfold web server and Varna java-based software. Structural plasmid cloning [0561] In order to generate the structural plasmids used to make the XDP, pXDP1 was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments containing CasX or HIV-1 Gag components were amplified and cloned as described in Example 2, above. The sequence for Rev was incorporated into the backbone of the Gag plasmid. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Guide plasmid cloning [0562] The tdTomato and PTBP-1 targeting guide plasmids used in these experiments were pSG5, pSG17, pSG47, and pSG48 cloned from pSG3 for the first and pSG14 for the latter 3 plasmids. The mammalian expression backbone contained a cPPT, ampicillin resistance, and a colEI replication site and was amplified using primers with appropriate overlaps to accept the U6 promoter and guide RNA scaffold cassette. The backbone was digested using NdeI and XbaI. Synthetic DNA fragments corresponding to novel scaffolds were amplified and cloned as described in Example 2, above. The resultant plasmids, pSG3 and pSG5, were sequenced using Sanger sequencing to ensure correct assembly (see Table 43). Table 43: Guide plasmids and sequences Plasmid Scaffold Target Encoding Guide scaffold se SEQ ID number number quence NO ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACC pSG3 174 NT AGCGACTATGTCGTAGTGGGTAAAGCTCCCTCTTCGGA 1021 GGGAGCATCAAAGCGAGACGTAATTACGTCTCG 202 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Plasmid Scaffold Ta SEQ ID number number rget Encoding Guide scaffold sequence NO ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACC pSG4 174 12.2 AGCGACTATGTCGTAGTGGGTAAAGCTCCCTCTTCGGA 1536 GGGAGCATCAAAGTATAGCATACATTATACGAA ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACC pSG5 174 12.7 AGCGACTATGTCGTAGTGGGTAAAGCTCCCTCTTCGGA 1021 GGGAGCATCAAAGCGAGACGTAATTACGTCTCG ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACC pSG30 174 28.10 AGCGACTATGTCGTAGTGGGTAAAGCTCCCTCTTCGGA 1537 GGGAGCATCAAAGCAGCGGGGATCCGACGAGCT ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACC AGCGACTATGTCGTAGTGGGTAAAGCTGCACTATGGGC pSG14 226 0.0 GCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATT 1538 ATTGTCTGGTATAGTGCAGCATCAAAGCGAGACGTAAT TACGTCTCG ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACC AGCGACTATGTCGTAGTGGGTAAAGCTGCACTATGGGC pSG17 226 12.7 GCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATT 1539 ATTGTCTGGTATAGTGCAGCATCAAAGCTGCATTCTAG TTGTGGTTT ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACC AGCGACTATGTCGTAGTGGGTAAAGCTGCACTATGGGC pSG0047 226 12.2 GCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATT 1540 ATTGTCTGGTATAGTGCAGCATCAAAGTATAGCATACA TTATACGAA ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACC AGCGACTATGTCGTAGTGGGTAAAGCTGCACTATGGGC pSG0048 226 28.10 GCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATT 4148 ATTGTCTGGTATAGTGCAGCATCAAAGCAGCGGGGATC CGACGAGCT Cloning tdTomato spacer 12.7 into pSG3 and pSG14 [0563] To clone the targeting plasmids from their respective non-targeting plasmids spacers 12.7, 12.2, and 28.10 were cloned using the following protocol. The targeting spacer sequence DNA for the tdTomato targeting spacer 12.7 was ordered as single-stranded DNA (ssDNA) oligos (Integrated DNA Technologies) consisting of the targeting sequence (CTGCATTCTAGTTGTGGTTT, SEQ ID NO: 1018) and the reverse complement of this sequence. The targeting spacer sequence DNA for the tdTomato targeting spacer 12.2 was ordered as single-stranded DNA (ssDNA) oligos (Integrated DNA Technologies) consisting of the targeting sequence (TATAGCATACATTATACGAA, SEQ ID NO: 1541) and the reverse complement of this sequence. The targeting spacer sequence DNA for the PTBP-1 targeting spacer 28.10 was ordered as single-stranded DNA (ssDNA) oligos (Integrated DNA Technologies) consisting of the targeting sequence (CAGCGGGGATCCGACGAGCT, SEQ ID 203 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 NO: 1542) and the reverse complement of this sequence. For each spacer the two oligos were annealed together and cloned into pSG3 or pSG14 by Golden Gate assembly, as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation. pGP2 Glycoprotein plasmid cloning [0564] Sequences encoding the VSV-G glycoprotein and the CMV promoter were cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Cell culture and transfection [0565] HEK293T Lenti-X™ cell culture was performed as described in Example 2, above. For transfection, the XDP structural plasmids (also encoding the CasX variants) of Table 44 were used in amounts ranging from 13 to 80.0 µg. Each transfection also received 13 µg of p42.174.12.7 and 0.25 µg of pGP2. Polyethylenimine (PEI MAX® from Polyplus) was then added as described in Example 2. The XDP versions, architectures and plasmids utilized in the transfection are listed in Table 45. Table 44: Architecture and pseudotyping plasmid sequences Plasmid number Architecture SEQ ID NO of DNA sequence pGP2 VSV-G 979 pXDP161* Gag-(-1)-PR 1139 pXDP164* Gag-MS2 1140 pXDP166* SV40NLS-CasX491- SV40 NLS 1142 pXDP17* Gag-CasX491-HAtag 1210 * Backbone of plasmid expressed Rev Table 45: XDP version and pseudotyping descriptions XDP version Architectures and glycoprotein Plasmid numbers Rev expression Gag-(-1)-PR-RT-Int pXDP1 1 Gag-CasX pXDP17 Yes VSV-G pGP2 7 Gag-CasX pXDP17 VSV-G pGP2 No 204 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 XDP version Architectures and glycoprotein Plasmid numbers Rev expression Gag-(-1)-PR pXDP161 168 Gag-CasX pXDP17 Yes VSV-G pGP2 Gag-MS2 pXDP164 206 Gag-(-1)-PR pXDP161 CasX pXDP166 Yes VSV-G pGP2 Gag-(-1)-PR pXDP161 207 CasX pXDP166 Yes VSV-G pGP2 Collection and concentration; resuspension and transduction [0566] XDPs were collected and concentrated as described in Example 2, above. [0567] tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above. Editing of tdTomato was assessed by measuring fluorescence or by Next Generation Sequencing to assess rate of edits. The assays were run 2-3 times for each sample with similar results. Results: [0568] The RRE binds strongest to Rev at Stem II (circled in FIG.48), therefore, this region was incorporated into scaffold 174 (FIG.49), resulting in scaffold 226, depicted in FIG.50. Guide scaffold 226 was evaluated using three different spacer sequences; 12.7 (CTGCATTCTAGTTGTGGTTT, SEQ ID NO: 1018), 12.2 (TATAGCATACATTATACGAA, SEQ ID NO: 1541), targeting tdTomato, and 28.10 (CAGCGGGGATCCGACGAGCT, SEQ ID NO: 1542) targeting PTBP-1. Editing using spacers 12.7 and 12.2 were read out using the tdTomato system and 28.10 was analyzed using NGS of the PTBP-1 locus. In each case, XDP incorporating scaffold 226 resulted in 3- to 5-fold greater editing per XDP than XDP incorporating scaffold 174 (Table 46; results presented as the ratio of the EC50 for scaffold 174 to 226). Table 46: EC50 results from editing assays Spacer EC50174/EC50226 12.7 3.06 205 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Spacer EC50174/EC50226 12.2 5.31 28.10 3.64 [0569] To further interrogate the mechanism of the increases in potency using the RRE/Rev system, three assays were performed. First, it was demonstrated that the increase in potency is Rev-dependent by testing the 226 guide scaffold in the XDP V1 and V7 architectures (see Table 45 above). Plasmids in the V1 architecture encode the Rev protein whereas the Rev protein is absent in the V7 architecture. FIG.56 demonstrates that editing with XDP incorporating scaffold 174 or scaffold 226 is very similar in the V7 architecture; scaffold 226 does not increase editing in the Rev-independent V7 construct but does in V1, a Rev-containing architecture. [0570] Next, efficiency of scaffold 226 in the absence of an additional recruitment system (e.g., Gag-CasX fusion, Gag-MS2, tVSVG-Stx) was assessed. XDP version 207 lacks any architectural recruitment mechanism for CasX to be incorporated into the XDP. XDPs with guide scaffold 174 were unable to edit NPCs in this construct whereas XDPs with scaffold 226 were able to achieve >20% editing (FIG.57). These data suggested that there may be an orthogonal mechanism of recruitment affected by guide scaffold 226, especially since the increase in editing is greater than was seen in the V168 and V1 XDPs. [0571] Lastly, the edits made by XDP with guide scaffold 174 and 226 were assessed to ensure that the nature of edits caused by the RNP was preserved across these two scaffolds. NGS data from samples from the constructs evaluated in FIG.58 were run through CRISPResso to assess the indel profile. Insertions and deletions were graphed by their frequency on the total read population. Analysis of the data showed that the proportion of insertions and deletions remained similar across the two scaffolds. FIG.59 shows the data for the calculated EC50 values for the editing experiments. [0572] The editing data with XDP incorporating guide scaffold 226 demonstrate a consistent pattern of increased potency over XDP incorporating guide scaffold 174. The data show that without changing the nuclease function, the potency of XDPs can be increased by designing constructs that incorporate an RNA nuclear export pathway such as the Rev/RRE system. These enhanced effects were seen across different gene targets and multiple spacers. [0573] The data demonstrate the utility of incorporating retroviral RNA transport elements into the RNP scaffold to increase potency of XDP particles. 206 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Example 13: Evaluation of nuclear import and export systems - NLS Variants +/- RRE (XDP Version 206) [0574] The purpose of these experiments was to demonstrate the effects of incorporating a variety of nuclear localization signals (NLS) linked to the CasX molecule in the MS2-based recruitment system of XDP version 206. Additionally, experiments were performed to determine if the inclusion of a portion of the HIV-1 rev response element (RRE) or modified portions of the RRE in the sgRNA would increase the potency of these NLS-enhanced constructs in order to determine whether the nuclear export ability of the RRE-Rev system would counteract the effects of the NLSs in the producer cell. Methods: [0575] All plasmids encoding CasX proteins utilized the CasX 491 variant protein. All XDPs were pseudotyped with 10% VSV-G (percentage of plasmid relative to the other plasmids utilized for the XDP construct). RNA fold structures were generated with RNAfold web server and Varna java-based software. Structural plasmid cloning [0576] In order to generate the structural plasmids used below, pXDP1 was digested using
Figure imgf000067_0001
207 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Table 47: Guide scaffold sequences Plasmid Scaffold Target G SEQ number number uide encoding sequence ID NO ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACCAGCG pSG0033 188 0.0 ACTATGTCGTAGTGGGTAAAGCTCACATGAGGATCACCCATG 1135 TGAGCATCAAAGCGAGACGTAATTACGTCTCG ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACCAGCG pSG50 188 12.7 ACTATGTCGTAGTGGGTAAAGCTCACATGAGGATCACCCATG 1825 TGAGCATCAAAGCTGCATTCTAGTTGTGGTTT ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACCAGCG ACTATGTCGTAGTGGGTAAAGCTGCACTATGGGCGCAGCGTC pSG67 250 0.0 AATGACGCTGACGGTACAGGCCACATGAGGATCACCCATGTG 1200 GTATAGTGCAGCATCAAAGCGAGACGTAATTACGTCTCG ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACCAGCG ACTATGTCGTAGTGGGTAAAGCTGCACTATGGGCGCAGCTCA pSG68 251 0.0 TGAGGATCACCCATGAGCTGACGGTACAGGCCACATGAGGAT 1201 CACCCATGTGGTATAGTGCAGCATCAAAGCGAGACGTAATTA CGTCTCG ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACCAGCG ACTATGTCGTAGTGGGTAAAGCTGCACTATGGGCGCAGCGTC pSG69 252 0.0 AATGACGCTGACGGTACAGGCCACATGGCAGTCGTAACGACG 1227 CGGGTGGTATAGTGCAGCATCAAAGCGAGACGTAATTACGTC TCG ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACCAGCG ACTATGTCGTAGTGGGTAAAGCTGCACTATGGGCGCAGACAT pSG71 254 0.0 GGCAGTCGTAACGACGCGGGTCTGACGGTACAGGCCACATGA 1228 GGATCACCCATGTGGTATAGTGCAGCATCAAAGCGAGACGTA ATTACGTCTCG ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACCAGCG ACTATGTCGTAGTGGGTAAAGCTGCACTATGGGCGCAGCGTC pSG72 250 12.7 AATGACGCTGACGGTACAGGCCACATGAGGATCACCCATGTG 1202 GTATAGTGCAGCATCAAAGCTGCATTCTAGTTGTGGTTT ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACCAGCG ACTATGTCGTAGTGGGTAAAGCTGCACTATGGGCGCAGCTCA pSG73 251 12.7 TGAGGATCACCCATGAGCTGACGGTACAGGCCACATGAGGAT 1203 CACCCATGTGGTATAGTGCAGCATCAAAGCTGCATTCTAGTT GTGGTTT ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACCAGCG ACTATGTCGTAGTGGGTAAAGCTGCACTATGGGCGCAGACAT pSG76 254 12.7 GGCAGTCGTAACGACGCGGGTCTGACGGTACAGGCCACATGA 1230 GGATCACCCATGTGGTATAGTGCAGCATCAAAGCTGCATTCT AGTTGTGGTTT Cloning tdTomato spacer 12.7 into pSG33, pSG67, pSG68, and 71 [0578] The targeting spacer sequence DNA for the tdTomato targeting spacer 12.7 was ordered as single-stranded DNA (ssDNA) oligos consisting of the targeting sequence (CTGCATTCTAGTTGTGGTTT, SEQ ID NO: 1018) and the reverse complement of this sequence. These two oligos were annealed together and cloned into pSG33, pSG67, pSG68, and 208 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 pSG71 plasmids done by Golden Gate assembly, as described in Example 2, above. The non- targeting (NT) spacer 0.0 (encoded by the sequence CGAGACGTAATTACGTCTCG, SEQ ID NO: 1019) was used as a control and was cloned in a similar manner. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation. pGP2 Glycoprotein plasmid cloning [0579] Sequences encoding the VSV-G glycoprotein and the CMV promoter were cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly (see Table 50). Cell culture and transfection [0580] HEK293T Lenti-X™ cell culture was performed as described in Example 2, above. For transfection, the XDP structural plasmids (also encoding the CasX-NLS variants of Table 49) of Table 48 were used in amounts ranging from 13 to 80.0 µg. Each transfection also received 13 µg of sgRNA plasmid and 0.25 µg of pGP2. Polyethylenimine was then added as described in Example 2, above. Table 48: Plasmid ratios of V206 XDP version 206 plasmids Structural plasmid ratios Gag-(-1)-PR* 10% Gag-MS2* 45% CasX* 45% *transcript contains RRE and produces REV Table 49: CasX-NLS plasmids, NLS descriptions, and NLS sequences for each tested NLS Plasmid NLS number number N-terminal NLS sequence* C-terminal NLS sequence* PAAKRVKLDGGSPAAKRVKLDGGSPAAKR VKLDGGSPAAKRVKLDGGSPAAKRVKLDG TSPKKKRKVALEYPYDVPDYA pXDP343 NLS 115 GSPAAKRVKLDSR (SEQ ID NO: (SEQ ID NO: 499) 4142) PAAKRVKLDGGKRTADGSEFESPKKKRKV TLEGGSPKKKRKV pXDP344 NLS 240 GGGSGGGSPGSRDISR (SEQ ID NO: 4128) (SEQ ID NO: 1740) 5 NLS 245 PAAKRVKLDP TLEGGSPKKKRKV pXDP34 PPPKKKRKVPGSRDISR (SEQ ID NO: 4143) (SEQ ID NO:1740) 209 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Plasmid NLS number number N-terminal NLS sequence* C-terminal NLS sequence* TLEVGPKRTADSQHSTPPKTKRKVEFEP pXDP346 NLS 247 PKKKRKVSRDISR (SEQ ID NO: KKKRKV 4144) (SEQ ID NO:1750) TLEVGGGSGGGSKRTADSQHSTPPKTKR pXDP347 NLS 248 PKKKRKVSRDISR (SEQ ID NO: KVEFEPKKKRKV 4144) (SEQ ID NO:1751) TLEVGPAEAAAKEAAAKEAAAKAPAAKR pXDP348 NLS 251 PKKKRKVSRDISR (SEQ ID NO: VKLD 4144) (SEQ ID NO:1754) PKKKRKVSR TLEVGPGGGSGGGSGGGSPAAKRVKLD pXDP349 NLS 252 DISR (SEQ ID NO: 4144) (SEQ ID NO: 1755) TLEVGPKRTADSQHSTPPKTKRKVEFEP pXDP350 NLS 255 PAAKRVKLDGGKRTADGSEFESPKKKRKV KKKRKV GGSSRDISR (SEQ ID NO: 4129) (SEQ ID NO: 1750) PAAKRVKLDGGKRTADGSEFESPKKKRKV TLEVGGGSGGGSKRTADSQHSTPPKTKR GGGSGGGSPGSRDISR (SEQ I KVEFEPKKKRKV pXDP351 NLS 256 D NO: 4128) (SEQ ID NO: 1751) PAAKRVKLDGGSPAAKRVKLDGGSPAAKR GSKRPAATKKAGQAKKKK pXDP352 NLS 269 VKLDGGSPAAKRVKLDSR (SEQ ID NO: 4145) (SEQ ID NO: 1760) *c-Myc NLS sequences are bolded (PAAKRVKLD; SEQ ID NO: 37) SV40 NLS sequences
Figure imgf000070_0001
210 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386
Figure imgf000071_0001
. . 211 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386
Figure imgf000072_0001
212 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386
Figure imgf000073_0001
213 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 t
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e gycoproten was p . or transecton, te structura pasm s ste aove an n 214 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386
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215 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 8
Figure imgf000076_0001
protein (RBP). Binding of the RNA hairpin to MS2 RBP facilitates enhanced recruitment of 216 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386
Figure imgf000077_0001
[0599] gRNAs incorporating RNA hairpin variants with varying affinities for the MS2 RBP were evaluated using a high-throughput, in vitro biochemical assay to assess equilibrium binding and dissociation kinetics (Buenrostro et al., Quantitative analysis of RNA-protein interactions on a massively parallel array reveals biophysical and evolutionary landscapes. Nat Biotechnol. 32(6):562 (2014)). gRNA hairpin variants and their associated Kd (dissociation constant) values are listed in Table 56, sequences of the guide plasmids encoding the different MS2 RNA hairpin variants are provided in Table 57 and the sequences of the MS2 hairpins are provided in Table 58. Table 56: gRNA scaffolds containing MS2 hairpin variants with varying affinities and their dissociation constant values (Kd). Specific positions for the indicated nucleotide mutations refer to the positions of the base MS2 hairpin (scaffold 188) depicted in FIG.62 Positions of indicated –∆log(K K OF )/ Scaffold No. nucleotide changes within d (nM) KOFF (1/s) F -∆∆G MS2 hairpin (scaffold 188) High affinity MS2 hairpin variants 188 2.558 0.001 251 2.558 296 -13C, 1G 1.881 0.001 0.510 297 -15G, 3C 2.112 0.001 0.062 298 -15G, -13C, 1G, 3C 299 -15G, -13C, -8C, -3G, 1G, 3C 300 -8C, -3G 2.686 0.002 >1 Medium-high affinity MS2 hairpin variants 304 -13U 9.346 0.002 0.286 307 -11U, -1A 9.226 0.003 0.579 217 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Positions of indicated –∆log(K old No. nucleotide changes within K O )/ Scaff FF d (nM) KOFF (1/s) -∆∆G MS2 hairpin (scaffold 188) 313 -6C 9.274 0.002 0.400 Medium affinity MS2 hairpin variants 301 -8U, -3A 34.084 0.007 0.654 303 -15C, -13U 0.002 0.089 305 -13C 17.634 0.002 0.163 306 -1U 0.015 0.854 310 -5U 36.912 0.016 0.910 314 -14G, 3G 0.002 0.120 Medium-low affinity MS2 hairpin variants 308 -11U, -1G 77.562 0.018 0.754 Low affinity MS2 hairpin variants 309 -11U, -1U 453.563 N/A N/A 311 -5A 415.477 N/A N/A 302 -8A, -3G 1489.244 N/A N/A No affinity MS2 hairpin variants 312 -5G 12506.440 N/A N/A 315 -10G 18018.92728 N/A N/A Table 57: Sequences of XDP plasmids Plasmid number Description DNA Sequence pXDP161 Gag-(-1)-PR SEQ ID NO: 1139 pXDP164 Gag-MS2 SEQ ID NO: 1140 pXDP353 Gag-MS2 (N55K) SEQ ID NO: 1174 pXDP344 CasX 491 NLS 240 SEQ ID NO: 1206 ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACCAGCGACT pSG50 Scaffold 188 ATGTCGTAGTGGGTAAAGCTCACATGAGGATCACCCATGTGAGCA TCAAAGCTGCATTCTAGTTGTGGTTT (SEQ ID NO: 1825) ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACCAGCGACT ATGTCGTAGTGGGTAAAGCTGCACTATGGGCGCAGCTCATGAGGA pSG73 Scaffold 251 TCACCCATGAGCTGACGGTACAGGCCACATGAGGATCACCCATGT GGTATAGTGCAGCATCAAAGCTGCATTCTAGTTGTGGTTT (SEQ ID NO: 1203) ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACCAGCGACT pSG170 Scaffold 296 ATGTCGTAGTGGGTAAAGCTCACCTGAGGATCACCCAGGTGAGCA TCAAAGCTGCATTCTAGTTGTGGTTT (SEQ ID NO: 1826) ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACCAGCGACT pSG171 Scaffold 297 ATGTCGTAGTGGGTAAAGCTCGCATGAGGATCACCCATGCGAGCA TCAAAGCTGCATTCTAGTTGTGGTTT (SEQ ID NO: 1827) ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACCAGCGACT pSG172 Scaffold 298 ATGTCGTAGTGGGTAAAGCTCGCCTGAGGATCACCCAGGCGAGCA TCAAAGCTGCATTCTAGTTGTGGTTT (SEQ ID NO: 1828) ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACCAGCGACT pSG173 Scaffold 299 ATGTCGTAGTGGGTAAAGCTCGCCTGAGCATCAGCCAGGCGAGCA (SEQ ID NO: 1829) 218 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Plasmid number Description DNA Sequence ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACCAGCGACT pSG174 Scaffold 300 ATGTCGTAGTGGGTAAAGCTCACATGAGCATCAGCCATGTGAGCA TCAAAGCTGCATTCTAGTTGTGGTTT (SEQ ID NO: 1830) ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACCAGCGACT pSG175 Scaffold 301 ATGTCGTAGTGGGTAAAGCTCACATGAGTATCAACCATGTGAGCA TCAAAGCTGCATTCTAGTTGTGGTTT (SEQ ID NO: 1831) ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACCAGCGACT pSG176 Scaffold 302 ATGTCGTAGTGGGTAAAGCTCACATGAGAATCAGCCATGTGAGCA TCAAAGCTGCATTCTAGTTGTGGTTT (SEQ ID NO: 1832) ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACCAGCGACT pSG177 Scaffold 303 ATGTCGTAGTGGGTAAAGCTCCCTTGAGGATCACCCATGTGAGCA TCAAAGCTGCATTCTAGTTGTGGTTT (SEQ ID NO: 1833) ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACCAGCGACT pSG178 Scaffold 304 ATGTCGTAGTGGGTAAAGCTCACTTGAGGATCACCCATGTGAGCA TCAAAGCTGCATTCTAGTTGTGGTTT (SEQ ID NO: 1834) ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACCAGCGACT pSG179 Scaffold 305 ATGTCGTAGTGGGTAAAGCTCACCTGAGGATCACCCATGTGAGCA TCAAAGCTGCATTCTAGTTGTGGTTT (SEQ ID NO: 1835) ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACCAGCGACT pSG180 Scaffold 306 ATGTCGTAGTGGGTAAAGCTCACATGAGGATCACCTATGTGAGCA TCAAAGCTGCATTCTAGTTGTGGTTT (SEQ ID NO: 1836) ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACCAGCGACT pSG181 Scaffold 307 ATGTCGTAGTGGGTAAAGCTCACATTAGGATCACCAATGTGAGCA TCAAAGCTGCATTCTAGTTGTGGTTT (SEQ ID NO: 1837) ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACCAGCGACT pSG182 Scaffold 308 ATGTCGTAGTGGGTAAAGCTCACATTAGGATCACCGATGTGAGCA TCAAAGCTGCATTCTAGTTGTGGTTT (SEQ ID NO: 1838) ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACCAGCGACT pSG183 Scaffold 309 ATGTCGTAGTGGGTAAAGCTCACATTAGGATCACCTATGTGAGCA TCAAAGCTGCATTCTAGTTGTGGTTT (SEQ ID NO: 1839) ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACCAGCGACT pSG184 Scaffold 310 ATGTCGTAGTGGGTAAAGCTCACATGAGGATTACCCATGTGAGCA TCAAAGCTGCATTCTAGTTGTGGTTT (SEQ ID NO: 1840) ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACCAGCGACT pSG185 Scaffold 311 ATGTCGTAGTGGGTAAAGCTCACATGAGGATAACCCATGTGAGCA TCAAAGCTGCATTCTAGTTGTGGTTT (SEQ ID NO: 1841) ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACCAGCGACT pSG186 Scaffold 312 ATGTCGTAGTGGGTAAAGCTCACATGAGGATGACCCATGTGAGCA TCAAAGCTGCATTCTAGTTGTGGTTT (SEQ ID NO: 1842) ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACCAGCGACT pSG187 Scaffold 313 ATGTCGTAGTGGGTAAAGCTCACATGAGGACCACCCATGTGAGCA TCAAAGCTGCATTCTAGTTGTGGTTT (SEQ ID NO: 1843) ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACCAGCGACT pSG188 Scaffold 314 ATGTCGTAGTGGGTAAAGCTCAGATGAGGATCACCCATGGGAGCA TCAAAGCTGCATTCTAGTTGTGGTTT (SEQ ID NO: 1844) ACTGGCGCTTTTATCTGATTACTTTGAGAGCCATCACCAGCGACT pSG189 Scaffold 315 ATGTCGTAGTGGGTAAAGCTCACATGGGGATCACCCATGTGAGCA TCAAAGCTGCATTCTAGTTGTGGTTT (SEQ ID NO: 1845) Table 58: MS2 hairpin variant sequences Scaffold No. MS2 Sequences SEQ ID NO 188 ACAUGAGGAUCACCCAUGU 219 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Scaffold No. MS2 Sequences SEQ ID NO 251 910 296 ACCUGAGGAUCACCCAGGU 1847 297 GCAUGAGGAUCACCCAUGC 1848 298 GCCUGAGGAUCACCCAGGC 1849 299 GCCUGAGCAUCAGCCAGGC 1850 300 ACAUGAGCAUCAGCCAUGU 1851 304 ACUUGAGGAUCACCCAUGU 1852 307 ACAUUAGGAUCACCAAUGU 1853 313 ACAUGAGGACCACCCAUGU 1854 Materials and Methods: [0600] All plasmids encoding CasX proteins utilized CasX variant 491. All XDPs were pseudotyped with 10% VSV-G (percentage of VSV-G plasmid relative to other XDP structural plasmids). RNA fold structures were generated with RNAfold web server and VARNA software. The methods to produce XDPs are described herein, as well as in WO2021113772A1, incorporated by reference in its entirety. Structural plasmid cloning [0601] Briefly, to generate the XDP structural plasmids, the Gag-pol sequence was removed from pXDP1, and amplified and purified fragments encoding CasX 491, HIV-1, or MS2 CP components were cloned as described in Example 2, above. Individual colonies were picked, miniprepped, and Sanger-sequenced for assembly verification. Plasmid sequences are listed in Table 57. Guide plasmid cloning [0602] All guide plasmids containing MS2 RNA hairpin variants (Tables 57 and 58) incorporated the tdTomato targeting spacer 12.7 (CUGCAUUCUAGUUGUGGUUU; SEQ ID NO: 1855). pGP2 glycoprotein plasmid cloning [0603] Sequences encoding the VSV-G glycoprotein and CMV promoter were cloned as described in Example 2, above. XDP production [0604] Briefly, HEK293T Lenti-X™ cells were seeded in 15 cm dishes at 20 x 106 cells per dish 24 hours before transfection to reach 70-90% confluency. The next day, Lenti-X™ cells were transfected with the following plasmids PEI MAX® (Polypus): XDP structural 220 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 plasmids (also encoding the CasX variants; Table 57), pSG50 (or other guide plasmid variants listed in Table 57), and pGP2 for XDP pseudotyping.24 hours post-transfection, media was replaced with Opti-MEM (Thermo Fisher). XDP-containing media was collected 72 hours post- transfection and filtered through a 0.45 μm PES filter. The supernatant was concentrated and purified via centrifugation. XDPs were resuspended in 500 μL of DMEM/F12 supplemented with GlutaMAX™, HEPES, NEAA, Pen/Strep, 2-mercaptoethanol, B-27 without vitamin A, and N2. [0605] XDP transduction of tdTomato neural progenitor cells (NPCs) was conducted as described in Example 2. Results: [0606] XDPs composed of Gag-MS2, Gag-pro, CasX, gRNA scaffold variants, and VSV-G were produced as version 206 either with the original MS2 (MS2 WT) or an MS2 high-affinity variant (MS2353). Produced XDPs were subsequently assessed for their editing efficiency at the tdTomato locus in NPCs. FIG.63 shows the percent editing at the tdTomato locus as measured by tdTomato fluorescence using flow cytometry when 0.007 µL of concentrated XDP preps were used to transduce NPCs. In addition to the base control gRNA scaffolds 188 and 251, high- affinity scaffold variants 296 and 298 demonstrated enhanced potency with both MS2 WT and MS2 353, with Kd values ranging from 1.8 to 2.1 nM. Furthermore, medium-affinity scaffold variants 303, 304, 305, 307, 310 and 313, with Kd values ranging from 9.2 to 36.9 nM, resulted in promising editing efficiencies. FIG.64 illustrates EC50 results across the different gRNA scaffolds incorporating the MS2 WT and MS 353 configurations. Scaffold variants 296, 297, and 305 exhibited a slightly higher potency compared to scaffold 188, an advantage that was more evident with the MS2353 configuration. FIG.65 shows a clear correlation between the affinity (Kd) of the gRNA MS2 hairpin and resulting XDP potency (EC50), with an R2 value of 0.81 (p<0.001). XDP comprising MS2 having an affinity of <35nM resulted in efficient recruitment and packaging of the CasX RNP into XDPs. However, there was no correlation observed between the affinity (Kd) of the gRNA MS2 hairpin and resulting XDP titer (FIG.66). Example 16: Engineering of XDPs with a cytokine therapeutic payload [0607] Experiments were performed to demonstrate that XDPs can be used to carry the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) as the therapeutic protein payload. 221 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Methods: Structural plasmid cloning [0608] In order to generate the structural plasmids used to make the XDPs, mouse or human GMCSF was directly fused to a Gag structural protein, as described in Table 59, below. Cloning was performed as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Table 59: Configurations of XDPs for carrying GM-CSF XDP description Plasmid Encoded Components** 1 MA-p2A-p2B-p10-CA-NC- Pro†-GMCSF ALV GM-CSF 2 VSVG 1 MA*-CA*-NC*-p1*-p6*-GMCSF Version 168-GM-CSF 2 MA*-CA*-NC*-p1*-p6*-Pro† 3 VSVG 1 MA-GMCSF VSV M-GM-CSF 2 VSVG * indicates cleavage sequence between adjacent components ** 5' to 3' orientation † indicates a -1 frame-shift in the encoded construct (Gag-TFR-PR polyprotein) Cell culture and transfection [0609] HEK293T Lenti-X™ cell culture was performed as described in Example 2, above. For transfection, the XDP structural plasmids of Table 70 were used in amounts ranging from 13 to 80.0 µg. Collection and concentration [0610] XDPs were collected and concentrated as described in Example 2, above. Enzyme-linked immunosorbent assays (ELISAs) [0611] ELISAs were performed to measure the amount of GM-CSF per XDP. Specifically, XDPs were lysed with the lysis reagent and the number of GM-CSF molecules packaged per XDP was quantified using the Mouse GM-CSF Quantikine® ELISA kit (R&D, Cat no. MGM00) and Human GM-CSF Quantikine® ELISA kit (R&D, Cat no. DGM00) per the manufacturer’s instruction. 222 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Results: [0612] XDPs were engineered to carry human or mouse GM-CSF via the direct fusion of GM- CSF to the protein scaffold, and the amount of GM-CSF per XDP was measured via ELISA. As shown in Table 60, below, the XDPs contained GM-CSF, with between 40-527 molecules of GM-CSF per XDP. The results demonstrate that XDP constructs can be created to incorporate heterologous payloads, and in different configurations. Table 60: Number and concentration of GM-CSF molecules in XDPs GM-CSF species XDP description Molecules of GM-CSF/XDP ALV GM-CSF 46 Mouse V168-GM-CSF 35 VSV M-mGM-CSF 149 ALV GM-CSF 101 Human V168-GM-CSF 527 VSV M-mGM-CSF 40 Example 17: Engineering of XDPs for incorporating catalytically-dead CasX repressor (dXR) system [0613] Experiments were performed to demonstrate that XDPs can be used to incorporate a catalytically-dead CasX repressor (dXR) system as the therapeutic payload. Methods: Structural plasmid cloning [0614] XDPs were generated using the version 168 or version 206 configuration. [0615] Cloning was performed as described in Example 2, above. The constructs were designed with sequences coding for catalytically-dead CasX protein 491 (dCasX491; SEQ ID NO: 878) linked to the ZNF10 KRAB domain or the ZIM3 KRAB domain (dXR, see FIG.86 for a diagram), along with guide RNA scaffold variant 226 or 251, and spacer sequence 7.37 targeted to human B2M 7.37 (GGCCGAGATGTCTCGCTCCG, SEQ ID NO: 1017) or a non- targeting spacer (CGAGACGTAATTACGTCTCG; SEQ ID NO: 1019). The amino acid sequences of the dXR constructs are provided in Table 61, below. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. 223 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Table 61: Amino acid sequences of dXR constructs XDP Plasmid version number Description Amino acid SEQ ID NO 318 pXDP538 V168-XR.ZIM3 956 319 pXDP539 V168-XR.ZNF10 957 320 pXDP540 V206-XR.ZIM3 958 321 pXDP541 V206-XR.ZNF10 959 Cell culture and transfection; collection and concentration [0616] HEK293T Lenti-X™ cell culture was performed as described in Example 2, above. XDPs were collected and concentrated as described in Example 2, above. Results: [0617] XDPs were engineered to carry a dXR system targeting the B2M locus for repression. The XDPs were administered to human NPCs, and the level of B2M repression was measured. As shown in FIG.67, both the version 168 and version 206 XDPs were able to induce repression of B2M. The version 206 XDP with dCasX491 linked to the Zim3 KRAB domain produced the highest level of repression. [0618] The results of the experiments support that XDPs can be generated carrying functional dXR systems that result in targeted gene repression. Example 18: Quantification of CasX ribonucleoproteins (RNPs) in XDPs [0619] Experiments were performed to measure the amount of CasX RNPs incorporated into XDPs. Methods: [0620] XDPs were generated using the version 168 configuration with guide scaffold 226, or the version 206 configuration with guide scaffold 251 (see FIG.26) or guide scaffold 188 (see FIG.12). [0621] Cloning was performed as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Cell culture and transfection [0622] HEK293T Lenti-X™ cell culture was performed as described in Example 2, above. Collection and concentration [0623] XDPs were collected and concentrated as described in Example 2, above. 224 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Quantification of CasX RNPs via Western blot analysis [0624] To determine the number of CasX molecules per XPD particle, a semi-quantitative Western blot analysis was performed using XDP version 206 with guide scaffold 251, XDP version 168 with guide scaffold 226, and version XDP version 206 with guide scaffold 188 (FIG.68). The protein amount in XPD particles was measured using a Pierce 660 assay. XPD particles were lysed in Laemmli sample buffer and resolved by SDS-PAGE followed by Western blotting using a polyclonal antibody against the CasX protein. The gel also contained a range of purified CasX to establish a standard curve, shown in FIG.69. The resulting immunoblot was imaged using a ChemiDoc Touch, and the CasX protein levels were quantified by densitometry using Image Lab software from BioRad. Quantification of the CasX molecules in each XDP particle sample was determined using the standard curve. Results: [0625] Results of the Western blot analysis demonstrated that XDP version 168 with guide scaffold 226 contained approximately 227-239 CasX molecules/XDP particle (FIG.70) and, by inference, RNP. The XDP version 206 with guide scaffold 188 contained approximately 240- 257 CasX molecules/XDP particle, and XDP version 206 with guide scaffold 251 contained approximately 966-1112 CasX molecules/XDP particle, showing the superiority of scaffold 251 for facilitating incorporation of RNP into the XDP particles. The fold differences relative to XDP version 168 with guide scaffold 226 are shown in FIG.71. Example 19: Evaluation of orthogonal recruitment system with MS2 linked to Gag plus a nuclear export signal (NES) linked to CasX [0626] The purpose of these experiments was to evaluate whether linking cleavable nuclear export signals (NESs) to CasX in an XDP construct could prevent the sequestration of CasX in the nucleus in packaging cells and promote the packaging of CasX RNPs into XDPs. A potential concern during XDP production is the sequestration of the CasX RNP in the nucleus of the producer cell line as a result of the strong nuclear localization signals on the CasX protein. This possible nuclear sequestration might affect RNP packaging into XDPs and, therefore, XDP editing potency. Therefore, the use of adding cleavable nuclear export signals (NESs) linked to CasX in an XDP construct so as to prevent the sequestration of CasX in the nucleus in packaging cells and promote the packaging of CasX RNPs into XDPs was evaluated. 225 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Methods: [0627] Cleavable NESes were added to the XDP version 206 system (plasmid configurations are shown in Table 62. The NESes were linked to the C-terminus of CasX 676 via an HIV cleavage sequence and a rigid linker. Table 62: Configurations of version 206 XDPs with or without NESes XDP description Plasmid Encoded Components** 1 MA*-CA*-NC*-p1*-p6-MS2 2 MA*-CA*-NC*-p1*-p6*-Pro† Version 206 3 NLS-CasX-NLS 4 sgRNA (scaffold 251) 1 MA*-CA*-NC*-p1*-p6-MS2 Version 206 with 2 MA*-CA*-NC*-p1*-p6*-Pro† NES 3 NLS-CasX-NLS*-NES 4 sgRNA (scaffold 251) * indicates cleavage sequence between adjacent components ** 5' to 3' orientation † indicates a -1 frame-shift in the encoded construct (Gag-TFR-PR polyprotein) [0628] CRM1 (chromosomal maintenance 1) plays a major role in the export of proteins with leucine-rich nuclear export signals. Nuclear export signals that utilize the CRM1 nuclear export pathway with a range of affinities were selected and attached to the C-terminus of CasX in cleavable manner, such that during the maturation process post-XDP budding, the HIV protease would cleave the NES such that the CasX RNP would not have an attached NES when delivered into the target cell. Specifically, 15 different NESs that use the CRM1 pathway with different Rc/n and Kd values were selected (see Fu, S. et al., Mol Biol Cell.2018 Aug 15;29(17):2037- 2044), and six additional NESs were selected from NESdb, a database of NES-containing CRM1 cargoes (see Xu, D., et al. Mol Biol Cell.2012 Sep;23(18):3673-6). The amino acid sequences of the nuclear export signals are listed in Table 63, below. Further nuclear export signals have been identified for future testing, and are also listed in Table 63. 226 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Table 63: Sequences of nuclear export signals (NESs) Source of NES Amino acid sequence of NES SEQ ID NO PKI NSNELALKLAGLDINK 1022 CPEB4 RTFDMHSLESSLIDIMR 1023 MEK1 TNLEALQKKLEELELDE 1024 ADAR1 RGVDCLSSHFQELSIYQ 1025 FMRP RSFEMTEFNQALEEIKG 1026 hRio2 LKEVDQLRLERLQID 1027 Super PKI NLNELALKLAGLDINK 1028 X11L2 SSLQELVQQFEALPGDLV 1029 SMAD4 ERVVSPGIDLSGLTLQ 1030 HDAC5 EAETVSAMALLSVG 1031 SNUPN MEELSQALASSFSVSQDLNS 1032 REV LQLPPLERLTLDC 1033 MVM NS2 STVDEMTKKFGTLTIHD 1034 HPV E7 HVDIRTLEDLLMGTLGIVC 1035 Pax RELDELMASLSDFKFMA 1036 P53 EMFRELNEALELKD 1037 NMD3 RERENMDTDDERQYQDFLEDLEEDEAIRKNVNIYRDSAIPVES DTDDEGAPRISLAEMLEDLHISQDATGEEGASMLT 1038 Rex ALSAQLYSSLSLDS 1039 IκBα MFQAAERPQEWAMEGPRDGLKKERLLDDRHDSGLDSMKDEEYE QMVKELQEIRLE 1040 NFE2L2 FLNAFEDSFSSILS 1041 MLXIP IDASLTKLFECMTLAY 1042 Influenza NP MIDGIGRFYI 1043 NPM mutant E DLWQSLAQVSLRK 1044 Rabies P EVDNLPEDMKRLHLDD 1045 IRF3 QEDILDELLGNMVLA 1046 NS2 LVSLIRLKSKL 1047 Tax-1 YKRIEELLYKISLTT 1048 Nucleocapsid N 104 otein of PRRSV CTLSDSGRISYTVEFSL 9 pr PTHHTVRLIRVTASPSA ICP27 LEELCAARRLSL 1050 Adenoviral E1A VSQIFPDSVMLAVQEGIDLL 1051 BIRC2 PNCPFLENSLETLRFSISNLSMQ 1052 CALM LDSSLANLVGNLGIGNGT 1053 BVP-1 E1 protein ELITFINALKL 1054 X protein of BDV LRLTLLELVRRL 1055 HBZ MVNFVSVGLFRCLPVPCPEDLLVEELVDGLLSL 1056 Influenza M1 LFGDTIAYLLSL 1057 227 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Source of NES Amino acid sequence of NES SEQ ID NO HPV 11 E1 ISPRLDAIKL 1058 Menin DLVLLSLVL 1059 mRNA export factor PVSKITF 1060 EB2 VTL Nucleoprotein - 1061 Porcine epidemic LAPNVAALLFGGNVAVRELADSYEITYNYKMTVPKSDPNV diarrhea virus Nuclear export 1062 protein - Influenza ILMRMSKMQL A virus NS-2 of MVM DEMTKKFGTLTIHDTEKYASQPELCNN 1063 Paxillin QRVTSTQQQTRISASSATRELDELMASLSDFKFMAQGKTGSSS PPGGPPKPGSQLDSMLGSLQSDLNKLGV 1064 Phosphoprotein of II 1065 hPIV-2 ELLKGLDL HCMV Protein CILCQL 1066 UL94 LLLY VEEV Capsid TDPFLAMQVQELTRSMANLTFKQRRDAPPEG 1067 protein PSAKKPKK VP1 of CAV ELDTNFFTLYVAQG 1068 triplex capsid 1069 protein VP19C - Human herpesvirus LERLFGRLRI 1 (strain F) (HHV-1) (Human herpes simplex virus 1) cGAS EQCERA 1070 cGAS LEKLKL 1071 [0629] The XDPs were transduced into human Jurkat T cells or neural progenitor cells (NPCs), and editing of the B2M locus was measured. Results [0630] Overall, of the 21 nuclear export signals tested, about 10 showed improvements in editing, which suggests that they improved packaging of CasX RNPs into the XDPs. Specifically, the nuclear export signals that worked the best in Jurkat and/or NPCs were hRIO2, iKbA, MEK1, P53, Pax, PK1, Rex, Smad4, CPEB4, ADAR1, FMRP and SNUPN (FIGS.72- 74). 228 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Example 20: Screen of XDPs with diverse incorporated viral glycoproteins to evaluate tropism and editing capabilities [0631] The glycoprotein belonging to VSV Indiana species within the Vesiculovirus genus is usually the most widely used glycoprotein for pseudotyping purposes. The purpose of these experiments was to explore the transduction capabilities of glycoproteins belonging to other species, and test whether the cellular tropism of XDPs could be altered by pseudotyping XDPs with various glycoproteins as targeting moieties in various cell types. Methods: [0632] The screen of glycoproteins was conducted in the XDP version 206 construct configuration. The version 206 XDPs pseudotyped with glycoproteins of Table 64 were transduced into mouse tdTomato neural progenitor cells (NPCs), in which editing of the tdTomato locus was measured, or human Jurkat T cells, K562 lymphoblasts, ARPE-19 retinal pigment epithelial (RPE) cells, Y79 retinoblastoma cells, induced neurons, human NPCs, or astrocytes, in which editing of the B2M locus was measured. [0633] The amino acid sequences of the glycoproteins tested are provided in Table 64, below. Table 64: Description of glycoproteins tested lycoprotein SEQ ID NO (Amino acid) VSVG 573 VSAV 654 ABVV 655 CARV 656 CHPV 657 COCV 658 VSIV 659 ISFV 660 JURV 661 MSPV 662 MARV 663 MORV 664 VSNJV 665 PERV 666 PIRYV 667 RADV 668 YBV 669 VSV CEN AM - 670 94GUB 229 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 lycoprotein SEQ ID NO (Amino acid) VSV South America 671 85CLB EEEV 634 VEEV 635 y man e al ng e ted to .
Figure imgf000090_0001
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ed 231 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 tes at y. tor of g id they ith y gher ) or pism ARV, is n T ine.
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** 5' to 3' orientation 233 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 sing 251. X NP es of iver itro. s es of d fly, a.24 X d
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234 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 acer s sX S in a th a X NPs ato- 3) th a or l-
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and 235 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 iency an ent ids id o R) or R In ed in DP
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236 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386
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4 VSV-G 5 sgRNA * indicates cleavage sequence between adjacent components † indicates a -1 frame-shift in the encoded construct (Gag-TFR-PR polyprotein) [0661] The fusion proteins of the dXR constructs were made up of, from N- to C-terminus, a catalytically-dead CasX 491, and a ZNF10 or ZIM3 KRAB domain (see FIG.86; “RD1” is “Repressor Domain 1” and denotes the KRAB domain of interest). The fusion proteins of the ELXR configuration #1 constructs were made up of, from N- to C-terminus, a catalytic domain from DNMT3A, an interaction domain from DNMT3L, a catalytically-dead CasX 491, and a ZNF10 or ZIM3 KRAB domain (see ELXR configuration #1 in FIG.87), along with amino acid linkers and NLS sequences. Catalytically-active CasX 491 (herein termed “CasX”; SEQ ID NO: 189) was also included as a control. [0662] The DNA and protein sequences of the components of the dXR and ELXR configuration #1 constructs are provided in Table 67, below. The ELXR constructs also contained a 2x FLAG tag. Table 67: DNA and protein sequences of components of dXR and ELXR Key DNA SEQ ID Protein SEQ component sequence NO sequence ID NO ATGGATGCTAAGTCACTAACTGCCTGG MDAKSLTAWSRTLVTFKDVFV ZNF10 TCCCGGACACTGGTGACCTTCAAGGAT DFTREEWKLLDTAQQIVYRNV GTATTTGTGGACTTCACCAGGGAGGAG 24 MLENYKNLVSLGYQLTKPDVI KRAB 82 TGGAAGCTGCTGGACACTGCTCAGCAG LRLEKGEEP 2494 domain ATCGTGTACAGAAATGTGATGCTGGAG AACTATAAGAACCTGGTTTCCTTGGGT 237 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Key DNA SEQ ID Protein SEQ component sequence NO sequence ID NO TATCAGCTTACTAAGCCAGATGTGATC CTCCGGTTGGAGAAGGGAGAAGAGCCC ATGAACAATTCCCAGGGAAGAGTGACC MNNSQGRVTFEDVTVNFTQGE TTCGAGGATGTCACTGTGAACTTCACC WQRLNPEQRNLYRDVMLENYS CAGGGGGAGTGGCAGCGGCTGAATCCC NLVSVGQGETTKPDVILRLEQ GAACAGAGAAACTTGTACAGGGATGTG GKEPWLEEEEVLGSGRAEKNG ZIM3 ATGCTGGAGAATTACAGCAACCTTGTC DIGGQIWKPKDVKESL TCTGTGGGACAAGGGGAAACCACCAAA KRAB 2483 CCCGATGTGATCTTGAGGTTGGAACAA 2495 domain GGAAAGGAGCCATGGTTGGAGGAAGAG GAAGTGCTGGGAAGTGGCCGTGCAGAA AAAAATGGGGACATTGGAGGGCAGATT TGGAAGCCAAAGGATGTGAAAGAGAGT CTC ATGAACCATGACCAGGAATTTGACCCC MNHDQEFDPPKVYPPVPAEKR CCAAAGGTTTACCCACCTGTGCCAGCT KPIRVLSLFDGIATGLLVLKD GAGAAGAGGAAGCCCATCCGCGTGCTG LGIQVDRYIASEVCEDSITVG TCTCTCTTTGATGGGATTGCTACAGGG MVRHQGKIMYVGDVRSVTQKH CTCCTGGTGCTGAAGGACCTGGGCATC IQEWGPFDLVIGGSPCNDLSI CAAGTGGACCGCTACATTGCCTCCGAG VNPARKGLYEGTGRLFFEFYR GTGTGTGAGGACTCCATCACGGTGGGC LLHDARPKEGDDRPFFWLFEN ATGGTGCGGCACCAGGGAAAGATCATG VVAMGVSDKRDISRFLESNPV TACGTCGGGGACGTCCGCAGCGTCACA MIDAKEVSAAHRARYFWGNLP CAGAAGCATATCCAGGAGTGGGGCCCA GMNRPLASTVNDKLELQECLE TTCGACCTGGTGATTGGAGGCAGTCCC HGRIAKFSKVRTITTRSNSIK TGCAATGACCTCTCCATTGTCAACCCT QGKDQHFPVFMNEKEDILWCT GCCCGCAAGGGACTTTATGAGGGTACT EMERVFGFPVHYTDVSNMSRL GGCCGCCTCTTCTTTGAGTTCTACCGC ARQRLLGRSWSVPVIRHLFAP CTCCTGCATGATGCGCGGCCCAAGGAG LKEYFACV DNMT3A GGAGATGATCGCCCCTTCTTCTGGCTC TTTGAGAATGTGGTGGCCATGGGCGTT 2484 catalytic AGTGACAAGAGGGACATCTCGCGATTT 2496 domain CTTGAGTCTAACCCCGTGATGATTGAC GCCAAAGAAGTGTCTGCTGCACACAGG GCCCGTTACTTCTGGGGTAACCTTCCT GGCATGAACAGGCCTTTGGCATCCACT GTGAATGATAAGCTGGAGCTGCAAGAG TGTCTGGAGCACGGCAGAATAGCCAAG TTCAGCAAAGTGAGGACCATTACCACC AGGTCAAACTCTATAAAGCAGGGCAAA GACCAGCATTTCCCCGTCTTCATGAAC GAGAAGGAGGACATCCTGTGGTGCACT GAAATGGAAAGGGTGTTTGGCTTCCCC GTCCACTACACAGACGTCTCCAACATG AGCCGCTTGGCGAGGCAGAGACTGCTG GGCCGATCGTGGAGCGTGCCGGTCATC CGCCACCTCTTCGCTCCGCTGAAGGAA TATTTTGCTTGTGTG DNMT3L ATGGGCCCTATGGAGATATACAAGACA MGPMEIYKTVSAWKRQPVRVL GTGTCTGCATGGAAGAGACAGCCAGTG nteraction 2 SLFRNIDKVLKSLGFLESGSG i 485 CGGGTACTGAGCCTCTTCAGAAACATC SGGGTLKYVEDVTNVVRRDVE 2497 domain GACAAGGTACTAAAGAGTTTGGGCTTC KWGPFDLVYGSTQPLGSSCDR 238 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Key DNA SEQ ID Protein SEQ component sequence NO sequence ID NO TTGGAAAGCGGTTCTGGTTCTGGGGGA CPGWYMFQFHRILQYALPRQE GGAACGCTGAAGTACGTGGAAGATGTC SQRPFFWIFMDNLLLTEDDQE ACAAATGTCGTGAGGAGAGACGTGGAG TTTRFLQTEAVTLQDVRGRDY AAATGGGGCCCCTTTGACCTGGTGTAC QNAMRVWSNIPGLKSKHAPLT GGCTCGACGCAGCCCCTAGGCAGCTCT PKEEEYLQAQVRSRSKLDAPK TGTGATCGCTGTCCCGGCTGGTACATG VDLLVKNCLLPLREYFKYFSQ TTCCAGTTCCACCGGATCCTGCAGTAT NSLPL GCGCTGCCTCGCCAGGAGAGTCAGCGG CCCTTCTTCTGGATATTCATGGACAAT CTGCTGCTGACTGAGGATGACCAAGAG ACAACTACCCGCTTCCTTCAGACAGAG GCTGTGACCCTCCAGGATGTCCGTGGC AGAGACTACCAGAATGCTATGCGGGTG TGGAGCAACATTCCAGGGCTGAAGAGC AAGCATGCGCCCCTGACCCCAAAGGAA GAAGAGTATCTGCAAGCCCAAGTCAGA AGCAGGAGCAAGCTGGACGCCCCGAAA GTTGACCTCCTGGTGAAGAACTGCCTT CTCCCGCTGAGAGAGTACTTCAAGTAT TTTTCTCAAAACTCACTTCCTCTT CAAGAGATCAAGAGAATCAACAAGATC QEIKRINKIRRRLVKDSNTKK 878 AGAAGGAGACTGGTCAAGGACAGCAAC AGKTGPMKTLLVRVMTPDLRE ACAAAGAAGGCCGGCAAGACAGGCCCC RLENLRKKPENIPQPISNTSR ATGAAAACCCTGCTCGTCAGAGTGATG ANLNKLLTDYTEMKKAILHVY ACCCCTGACCTGAGAGAGCGGCTGGAA WEEFQKDPVGLMSRVAQPASK AACCTGAGAAAGAAGCCCGAGAACATC KIDQNKLKPEMDEKGNLTTAG CCTCAGCCTATCAGCAACACCAGCAGG FACSQCGQPLFVYKLEQVSEK GCCAACCTGAACAAGCTGCTGACCGAC GKAYTNYFGRCNVAEHEKLIL TACACCGAGATGAAGAAAGCCATCCTG LAQLKPEKDSDEAVTYSLGKF CACGTGTACTGGGAAGAGTTCCAGAAA GQRALDFYSIHVTKESTHPVK GACCCCGTGGGCCTGATGAGCAGAGTT PLAQIAGNRYASGPVGKALSD GCTCAGCCTGCCAGCAAGAAGATCGAC ACMGTIASFLSKYQDIIIEHQ CAGAACAAGCTGAAGCCCGAGATGGAC KVVKGNQKRLESLRELAGKEN GAGAAGGGCAATCTGACCACAGCCGGC LEYPSVTLPPQPHTKEGVDAY TTTGCCTGCTCTCAGTGTGGCCAGCCT NEVIARVRMWVNLNLWQKLKL CTGTTCGTGTACAAGCTGGAACAGGTG SRDDAKPLLRLKGFPSFPLVE TCCGAGAAAGGCAAGGCCTACACCAAC RQANEVDWWDMVCNVKKLINE dCasX491 TACTTCGGCAGATGTAACGTGGCCGAG 2486 KKEDGKVFWQNLAGYKRQEAL CACGAGAAGCTGATTCTGCTGGCCCAG RPYLSSEEDRKKGKKFARYQL CTGAAACCTGAGAAGGACTCTGATGAG GDLLLHLEKKHGEDWGKVYDE GCCGTGACCTACAGCCTGGGCAAGTTT AWERIDKKVEGLSKHIKLEEE GGACAGAGAGCCCTGGACTTCTACAGC RRSEDAQSKAALTDWLRAKAS ATCCACGTGACCAAAGAAAGCACACAC FVIEGLKEADKDEFCRCELKL CCCGTGAAGCCCCTGGCTCAGATCGCC QKWYGDLRGKPFAIEAENSIL GGCAATAGATACGCCTCTGGACCTGTG DISGFSKQYNCAFIWQKDGVK GGCAAAGCCCTGTCCGATGCCTGCATG KLNLYLIINYFKGGKLRFKKI GGAACAATCGCCAGCTTCCTGAGCAAG KPEAFEANRFYTVINKKSGEI TACCAGGACATCATCATCGAGCACCAG VPMEVNFNFDDPNLIILPLAF AAGGTGGTCAAGGGCAACCAGAAGAGA GKRQGREFIWNDLLSLETGSL CTGGAAAGCCTGAGGGAGCTGGCCGGC KLANGRVIEKTLYNRRTRQDE AAAGAGAACCTGGAATACCCCAGCGTG PALFVALTFERREVLDSSNIK ACCCTGCCTCCTCAGCCTCACACAAAA PMNLIGVARGENIPAVIALTD GAAGGCGTGGACGCCTACAACGAAGTG PEGCPLSRFKDSLGNPTHILR IGESYKEKQRTIQAKKEVEQR 239 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Key DNA SEQ ID Protein SEQ component sequence NO sequence ID NO CTGAACCTGTGGCAGAAGCTGAAACTG RAGGYSRKYASKAKNLADDMV TCCAGGGACGACGCCAAGCCTCTGCTG RNTARDLLYYAVTQDAMLIFA AGACTGAAGGGCTTCCCTAGCTTCCCT NLSRGFGRQGKRTFMAERQYT CTGGTGGAAAGACAGGCCAATGAAGTG RMEDWLTAKLAYEGLSKTYLS GATTGGTGGGACATGGTCTGCAACGTG KTLAQYTSKTCSNCGFTITSA AAGAAGCTGATCAACGAGAAGAAAGAG DYDRVLEKLKKTATGWMTTIN GATGGCAAGGTTTTCTGGCAGAACCTG GKELKVEGQITYYNRYKRQNV GCCGGCTACAAGAGACAAGAAGCCCTG VKDLSVELDRLSEESVNNDIS AGGCCTTACCTGAGCAGCGAAGAGGAC SWTKGRSGEALSLLKKRFSHR CGGAAGAAGGGCAAGAAGTTCGCCAGA PVQEKFVCLNCGFETHAAEQA TACCAGCTGGGCGACCTGCTGCTGCAC ALNIARSWLFLRSQEYKKYQT CTGGAAAAGAAGCACGGCGAGGACTGG NKTTGNTDKRAFVETWQSFYR GGCAAAGTGTACGATGAGGCCTGGGAG KKLKEVWKPAV AGAATCGACAAGAAGGTGGAAGGCCTG AGCAAGCACATTAAGCTGGAAGAGGAA AGAAGGAGCGAGGACGCCCAATCTAAA GCCGCTCTGACCGATTGGCTGAGAGCC AAGGCCAGCTTTGTGATCGAGGGCCTG AAAGAGGCCGACAAGGACGAGTTCTGC AGATGCGAGCTGAAGCTGCAGAAGTGG TACGGCGATCTGAGAGGCAAGCCCTTC GCCATTGAGGCCGAGAACAGCATCCTG GACATCAGCGGCTTCAGCAAGCAGTAC AACTGCGCCTTCATTTGGCAGAAAGAC GGCGTCAAGAAACTGAACCTGTACCTG ATCATCAATTACTTCAAAGGCGGCAAG CTGCGGTTCAAGAAGATCAAACCCGAG GCCTTCGAGGCTAACAGATTCTACACC GTGATCAACAAAAAGTCCGGCGAGATC GTGCCCATGGAAGTGAACTTCAACTTC GACGACCCCAACCTGATTATCCTGCCT CTGGCCTTCGGCAAGAGACAGGGCAGA GAGTTCATCTGGAACGATCTGCTGAGC CTGGAAACCGGCTCTCTGAAGCTGGCC AATGGCAGAGTGATCGAGAAAACCCTG TACAACAGGAGAACCAGACAGGACGAG CCTGCTCTGTTTGTGGCCCTGACCTTC GAGAGAAGAGAGGTGCTGGACAGCAGC AACATCAAGCCCATGAACCTGATCGGC GTGGCCCGGGGCGAGAATATCCCTGCT GTGATCGCCCTGACAGACCCTGAAGGA TGCCCACTGAGCAGATTCAAGGACTCC CTGGGCAACCCTACACACATCCTGAGA ATCGGCGAGAGCTACAAAGAGAAGCAG AGGACAATCCAGGCCAAGAAAGAGGTG GAACAGAGAAGAGCCGGCGGATACTCT AGGAAGTACGCCAGCAAGGCCAAGAAT CTGGCCGACGACATGGTCCGAAACACC GCCAGAGATCTGCTGTACTACGCCGTG ACACAGGACGCCATGCTGATCTTCGCG AATCTGAGCAGAGGCTTCGGCCGGCAG GGCAAGAGAACCTTTATGGCCGAGAGG CAGTACACCAGAATGGAAGATTGGCTC ACAGCTAAACTGGCCTACGAGGGACTG 240 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Key DNA SEQ ID Protein SEQ component sequence NO sequence ID NO GCCCAGTATACCTCCAAGACCTGCAGC AATTGCGGCTTCACCATCACCAGCGCC GACTACGACAGAGTGCTGGAAAAGCTC AAGAAAACCGCCACCGGCTGGATGACC ACCATCAACGGCAAAGAGCTGAAGGTT GAGGGCCAGATCACCTACTACAACAGG TACAAGAGGCAGAACGTCGTGAAGGAT CTGAGCGTGGAACTGGACAGACTGAGC GAAGAGAGCGTGAACAACGACATCAGC AGCTGGACAAAGGGCAGATCAGGCGAG GCTCTGAGCCTGCTGAAGAAGAGGTTT AGCCACAGACCTGTGCAAGAGAAGTTC GTGTGCCTGAACTGCGGCTTCGAGACA CACGCCGCTGAACAGGCTGCCCTGAAC ATTGCCAGAAGCTGGCTGTTCCTGAGA AGCCAAGAGTACAAGAAGTACCAGACC AACAAGACCACCGGCAACACCGACAAG AGGGCCTTTGTGGAAACCTGGCAGAGC TTCTACAGAAAAAAGCTGAAAGAAGTC TGGAAGCCCGCCGTG GGAGGGCCGAGCTCTGGCGCACCCCCA GGPSSGAPPPSGGSPAGSPTS CCAAGTGGAGGGTCTCCTGCCGGGTCC TEEGTSESATPESGPGTSTEP CCAACATCTACTGAAGAAGGCACCAGC SEGSAPGSPAGSPTSTEEGTS Linker 1 GAATCCGCAACGCCCGAGTCAGGCCCT TEPSEGSAPGTSTEPSE GGTACCTCCACAGAACCATCTGAAGGT 2487 2498 AGTGCGCCTGGTTCCCCAGCTGGAAGC CCTACTTCCACCGAAGAAGGCACGTCA ACCGAACCAAGTGAAGGATCTGCCCCT GGGACCAGCACTGAACCATCTGAG TCTAGCGGCAATAGTAACGCTAACAGC SSGNSNANSRGPSFSSGLVPL Linker 2 CGCGGGCCGAGCTTCAGCAGCGGCCTG 2488 SLRGSH 2499 GTGCCGTTAAGCTTGCGCGGCAGCCAT Linker 3A GGCGGTTCCGGCGGAGGAAGC 2489 GGSGGGS 2500 Linker 3B GGCGGTTCCGGCGGAGGTTCC 2490 Linker 4 GGATCAGGCTCTGGAGGTGGA 2491 GSGSGGG 2501 NLS A CCAAAGAAGAAGCGGAAGGTC 2492 PKKKRKV 35 NLS B CCAAAAAAGAAGAGAAAGGTA 2493 [0663] Guide RNA scaffold variant 226 was used with the version 168 XDPs, and guide scaffold variant 251 was used with the version 206 XDPs. The RNA sequences of the guide scaffolds are provided in Table 6. Sequences of spacers 7.37 targeted to human B2M and a non- targeting spacer are provided in Table 68. All resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. 241 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Table 68: Sequences of spacers used in constructs
Figure imgf000102_0001
0.0 Non-target N/A CGAGACGUAAUUACGUCUCG 2449 [0664] Cell culture and transfection, collection and concentration of XDPs, and resuspension and transduction of XDPs is performed as described in Example 2 above analysis of cytometry. XDPs. with the single
Figure imgf000102_0002
repressor oma n or e mo ecu e av ng con gura on or arge ng he B2M locus for repression. As shown in FIG.89, six days following XDP administration, only version 168 XDPs carrying the dXR system with a ZNF10 KRAB domain repressed B2M. Meanwhile, version 206 XDPs carrying the dXR with a ZNF10 KRAB domain, dXR with a ZIM3 KRAB domain, and ELXR having configuration #1 with a ZIM3 KRAB domain all repressed the B2M locus. [0667] As shown in FIG.90, 14 days following XDP administration, most of the dXR and ELXR configuration #1 systems had lost B2M repression. This was in contrast to the XDPs with catalytically-active CasX 491, which achieved long-term repression of the B2M locus via editing of the locus. Notably, the version 206 XDPs with an ELXR configuration #1 system with a ZIM3 KRAB domain showed a two-fold reduction at day 14 as compared to day six (FIG.89) and retained the most repression activity as compared to the other dXRs and ELXRs tested. [0668] Accordingly, XDPs were able to carry either the dXR or ELXR configuration #1 systems as therapeutic payloads and achieve transcriptional repression of a target locus. Example 24: Engineering of XDPs for carrying ELXR systems [0669] XDPs are generated with ELXR configuration #1, #4, or #5 molecules as the payload (see FIGS.87 and 88 for diagrams of the configurations). Materials and Methods: Description of XDPs and CasX constructs tested 242 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 [0670] XDP configuration versions 168 and 206 are generated with various ELXR molecules. Table 66, above, summarizes the plasmids used to encode the components of the version 168 and 206 XDP systems. [0671] ELXR molecules in configurations #1, #4, and #5, which contain a catalytically- inactive CasX 491, are tested, as diagrammed in FIG.88. Table 69 provides amino acid sequences of configurations #1, #4, and #5 ELXR molecules, showing the sequences of the components of the proteins from N- to C-terminus in the table. The repressor domain 1 shown in Table 69 (“RD1”in FIG.88) may be a repressor domain from the species Columba livia, Rattus norvegicus, Cebus imitator, chimpanzee, Chlorocebus sabaeus, Ophiophagus hannah, Ailuropoda melanoleuca, Peromyscus maniculatus bairdii, or Phyllostomus discolor, in place of the human ZNF10 or ZIM3 KRAB domains that were tested in Example 23. Other catalytically- inactive CasX variants can be used in place of catalytically-inactive CasX 491; these variants are listed in Table 4. Table 69: Amino acid sequences of ELXR configuration #1, #4, and #5 molecules ELXR Compone SEQ # nts Domains AA sequence ID NO START MAPKKKRKVSR 2502 codon + NLS + linker START MERLVYEVRQKCRNIEDICISCGSLNVTLEHPLFIGGMCQ 2503 codon + NCKNCFLECAYQYDDDGYQSYCTICCGGREVLMCGNNNCC DNMT3A RCFCVECVDLLVGPGAAQAAIKEDPWNCYMCGHKGTYGLL ADD RRREDWPSRLQMFFAN domain DNMT3A NHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLK 2504 catalytic DLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVT ELXR1 domain QKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLF with FEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISR FLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVND ADD KLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVF domain MNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGR SWSVPVIRHLFAPLKEYFACV Linker SSGNSNANSRGPSFSSGLVPLSLRGSH 2499 DNMT3L MGPMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSLGFLESG 2497 interaction SGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGS domain SCDRCPGWYMFQFHRILQYALPRQESQRPFFWIFMDNLLL TEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGL KSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLP LREYFKYFSQNSLPL Linker GGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTST 2498 EPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE Linker GGSGGGSA 2505 243 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 ELXR Compone SEQ # nts Domains AA sequence ID NO dCasX491 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDL 878 RERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAI LHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKG NLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNV AEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIH VTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASF LSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVT LPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDA KPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKE DGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLG DLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEE ERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCE LKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQ KDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVI NKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWND LLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFE RREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSR FKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYS RKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSR GFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKT LAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTIN GKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNND ISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHA AEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVET WQSFYRKKLKEVWKPAV Buffer + RSGGSGGGSTS 2506 linker Repressor Human ZIM3 MNNSQGRVTFEDVTVNFTQGEWQRLNPEQRNLYRDVMLEN 2495 domain 1 YSNLVSVGQGETTKPDVILRLEQGKEPWLEEEEVLGSGRA EKNGDIGGQIWKPKDVKESL Human ZNF10 MDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYR 2494 NVMLENYKNLVSLGYQLTKPDVILRLEKGEEP Columba livia QDVVTFKDVAIYFSPEEWVRLSAGQRELYQEVMLDNYELV 2509 repressor TSLDRESKLLYKMDPEEESCEGVPYSSADSGAPDSSSTSA domain C Rattus ALVTFEDVAVRFTQEEWALLDPSQKILYRDVMRETYRNLT 2510 norvegicus SVGINWECWDLEACFRSLGRNLRVQVVKRKCELTNSGPCA repressor E domain Cebus imitator SKAPITFGDLAIYFSQEEWEWLSPIQKDLYEDVMLENYRN 2511 repressor LVSLGLSFRRPNVITLLEKGKAPWMVEPARRRRGPDSGSK domain V Chimpanzee EMGLLTFRDIAIEFSLEEWQCLDCAQRNLYRDVMLENYRN 2512 repressor LVSLGIAVSKPDLITCLEQNKESQNIKRNKMVAKHPVMHS domain H Chlorocebus SQESVAFEDVAVYFTTKEWAIMVPAERALYRDVMLENYEA 2513 sabaeus VAFVAVPPTSKPALVSHLEQGKESCFIRPPGVLSRSDWRA repressor G domain Ophiophagus STPVTFEDVVVYFTAAEWVHLTNWQRDFYQAVMMETYELV 2514 hannah ASVAGDGVPMAEDEEGGVERPVWQYIPRGKRRRKTPQPRA repressor D domain 244 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 ELXR Compone SEQ # nts Domains AA sequence ID NO Ailuropoda LLTFEDVAVSFSEEEWELLDPPQKTLYNDVMQENYETVIS 2515 melanoleuca LGLKLKNDTGNDQPISISALEMQASGSKVLRKARMKVAQK repressor T domain Peromyscus VTYNDVHVDITQEEWALMDPSQRNLYKDVMVETYMNLTAI 2516 maniculatus GYNWENLEVEEPCQNPLKHGRHERPHTGEKPYEYNQCGKA bairdii FAQP repressor domain Phyllostomus VAFRDVIVDFTQEEWQQLKPAQKDLYRDVMMEIYWNLVSL 2517 discolor DLETEGDMNEPDPEKDSWEDRTSIVVVEGLMRNGAQGYAC repressor EKAGIQGCRV domain Buffer + TSPKKKRKV 2507 NLS START MAPKKKRKVSR 2502 codon + NLS + linker Repressor Human ZIM3 MNNSQGRVTFEDVTVNFTQGEWQRLNPEQRNLYRDVMLEN 2495 domain 1 YSNLVSVGQGETTKPDVILRLEQGKEPWLEEEEVLGSGRA EKNGDIGGQIWKPKDVKESL Human ZNF10 MDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYR 2494 NVMLENYKNLVSLGYQLTKPDVILRLEKGEEP Columba livia QDVVTFKDVAIYFSPEEWVRLSAGQRELYQEVMLDNYELV 2509 repressor TSLDRESKLLYKMDPEEESCEGVPYSSADSGAPDSSSTSA domain C Rattus ALVTFEDVAVRFTQEEWALLDPSQKILYRDVMRETYRNLT 2510 norvegicus SVGINWECWDLEACFRSLGRNLRVQVVKRKCELTNSGPCA repressor E domain Cebus imitator SKAPITFGDLAIYFSQEEWEWLSPIQKDLYEDVMLENYRN 2511 repr LVSLGLSFRRPNVITLLEKGKAPWMVEPARRRRGPDSGSK ELXR4 essor do V with main Chimp EMGLLTFRDIAIEFSLEEWQCLDCAQRNLYRDVMLENYRN ADD anzee 2512 LVSLGIAVSKPDLITCLEQNKESQNIKR n r NKMVAKHPVMHS domai epressor domain H Chlorocebus SQESVAFEDVAVYFTTKEWAIMVPAERALYRDVMLENYEA 2513 sabaeus VAFVAVPPTSKPALVSHLEQGKESCFIRPPGVLSRSDWRA repressor G domain Ophiophagus STPVTFEDVVVYFTAAEWVHLTNWQRDFYQAVMMETYELV 2514 hannah ASVAGDGVPMAEDEEGGVERPVWQYIPRGKRRRKTPQPRA repressor D domain Ailuropoda LLTFEDVAVSFSEEEWELLDPPQKTLYNDVMQENYETVIS 2515 melanoleuca LGLKLKNDTGNDQPISISALEMQASGSKVLRKARMKVAQK repressor T domain Peromyscus VTYNDVHVDITQEEWALMDPSQRNLYKDVMVETYMNLTAI 2516 maniculatus GYNWENLEVEEPCQNPLKHGRHERPHTGEKPYEYNQCGKA bairdii FAQP repressor domain 245 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 ELXR Compone SEQ # nts Domains AA sequence ID NO Phyllostomus VAFRDVIVDFTQEEWQQLKPAQKDLYRDVMMEIYWNLVSL 2517 discolor DLETEGDMNEPDPEKDSWEDRTSIVVVEGLMRNGAQGYAC repressor EKAGIQGCRV domain Linker GGSGGGSA 2505 START MERLVYEVRQKCRNIEDICISCGSLNVTLEHPLFIGGMCQ 2503 codon + NCKNCFLECAYQYDDDGYQSYCTICCGGREVLMCGNNNCC DNMT3A RCFCVECVDLLVGPGAAQAAIKEDPWNCYMCGHKGTYGLL ADD RRREDWPSRLQMFFAN domain DNMT3A NHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLK 2504 catalytic DLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVT domain QKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLF FEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISR FLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVND KLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVF MNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGR SWSVPVIRHLFAPLKEYFACV Linker SSGNSNANSRGPSFSSGLVPLSLRGSH 2499 DNMT3L MGPMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSLGFLESG 2497 interaction SGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGS domain SCDRCPGWYMFQFHRILQYALPRQESQRPFFWIFMDNLLL TEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGL KSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLP LREYFKYFSQNSLPL Linker GGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTST 2498 EPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE dCasX491 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDL 878 RERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAI LHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKG NLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNV AEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIH VTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASF LSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVT LPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDA KPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKE DGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLG DLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEE ERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCE LKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQ KDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVI NKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWND LLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFE RREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSR FKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYS RKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSR GFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKT LAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTIN GKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNND ISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHA AEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVET WQSFYRKKLKEVWKPAV Buffer + RSGGSGGGSTS 2506 linker NLS PKKKRKV 35 246 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 ELXR Compone SEQ # nts Domains AA sequence ID NO START MAPKKKRKVSR 2502 codon + NLS + linker START MERLVYEVRQKCRNIEDICISCGSLNVTLEHPLFIGGMCQ 2503 codon + NCKNCFLECAYQYDDDGYQSYCTICCGGREVLMCGNNNCC DNMT3A RCFCVECVDLLVGPGAAQAAIKEDPWNCYMCGHKGTYGLL ADD RRREDWPSRLQMFFAN domain DNMT3A NHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLK 2504 catalytic DLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVT domain QKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLF FEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISR FLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVND KLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVF MNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGR SWSVPVIRHLFAPLKEYFACV Linker SSGNSNANSRGPSFSSGLVPLSLRGSH 2499 DNMT3L MGPMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSLGFLESG 2497 interaction SGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGS domain SCDRCPGWYMFQFHRILQYALPRQESQRPFFWIFMDNLLL TEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGL KSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLP LREYFKYFSQNSLPL Linker GGSGGG 2508 ELXR5 Repressor Human ZIM3 MNNSQGRVTFEDVTVNFTQGEWQRLNPEQRNLYRDVMLEN 2495 with domain 1 YSNLVSVGQGETTKPDVILRLEQGKEPWLEEEEVLGSGRA ADD EKNGDIGGQIWKPKDVKESL domain Human ZNF10 MDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYR 2494 NVMLENYKNLVSLGYQLTKPDVILRLEKGEEP Columba livia QDVVTFKDVAIYFSPEEWVRLSAGQRELYQEVMLDNYELV 2509 repressor TSLDRESKLLYKMDPEEESCEGVPYSSADSGAPDSSSTSA domain C Rattus ALVTFEDVAVRFTQEEWALLDPSQKILYRDVMRETYRNLT 2510 norvegicus SVGINWECWDLEACFRSLGRNLRVQVVKRKCELTNSGPCA repressor E domain Cebus imitator SKAPITFGDLAIYFSQEEWEWLSPIQKDLYEDVMLENYRN 2511 repressor LVSLGLSFRRPNVITLLEKGKAPWMVEPARRRRGPDSGSK domain V Chimpanzee EMGLLTFRDIAIEFSLEEWQCLDCAQRNLYRDVMLENYRN 2512 repressor LVSLGIAVSKPDLITCLEQNKESQNIKRNKMVAKHPVMHS domain H Chlorocebus SQESVAFEDVAVYFTTKEWAIMVPAERALYRDVMLENYEA 2513 sabaeus VAFVAVPPTSKPALVSHLEQGKESCFIRPPGVLSRSDWRA repressor G domain Ophiophagus STPVTFEDVVVYFTAAEWVHLTNWQRDFYQAVMMETYELV 2514 hannah ASVAGDGVPMAEDEEGGVERPVWQYIPRGKRRRKTPQPRA repressor D domain Ailuropoda LLTFEDVAVSFSEEEWELLDPPQKTLYNDVMQENYETVIS 2515 melanoleuca LGLKLKNDTGNDQPISISALEMQASGSKVLRKARMKVAQK T 247 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 ELXR Compone SEQ # nts Domains AA sequence ID NO repressor domain Peromyscus VTYNDVHVDITQEEWALMDPSQRNLYKDVMVETYMNLTAI 2516 maniculatus GYNWENLEVEEPCQNPLKHGRHERPHTGEKPYEYNQCGKA bairdii FAQP repressor domain Phyllostomus VAFRDVIVDFTQEEWQQLKPAQKDLYRDVMMEIYWNLVSL 2517 discolor DLETEGDMNEPDPEKDSWEDRTSIVVVEGLMRNGAQGYAC repressor EKAGIQGCRV domain Linker GGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTST 2498 EPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE dCasX491 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDL 878 RERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAI LHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKG NLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNV AEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIH VTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASF LSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVT LPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDA KPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKE DGKVFWQNLAGYKRQEALRPYLSSEEDRKKGKKFARYQLG DLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEE ERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCE LKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQ KDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVI NKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWND LLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFE RREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSR FKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYS RKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSR GFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKT LAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTIN GKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNND ISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHA AEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVET WQSFYRKKLKEVWKPAV Buffer + RSGGSGGGSTS 2506 linker NLS PKKKRKV 35 [0672] Guide RNA scaffold variant 226 is used with the version 168 XDPs, and guide scaffold variant 251 is used with the version 206 XDPs. The RNA sequences of the guide scaffolds are provided in Table 6. Sequences of spacers 7.37 targeted to human B2M and a non-targeting spacer control are provided in Table 68, above. All resultant plasmids are sequenced using Sanger sequencing to ensure correct assembly. [0673] Cell culture and transfection, collection and concentration of XDPs, and resuspension and transduction of XDPs is performed as in Example 2, above. Repression of the 248 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 B2M locus is assessed at 7 days and 14 days, when cells are harvested for analysis of HLA immunostaining as detected using flow cytometry, to demonstrate the ability of the constructs to deliver the therapeutic payload and repress expression of B2M. Example 25: Evaluation of non-covalent recruitment (NCR) systems with protein-ligand pairs attached to Gag and protein cargo [0674] Experiments were performed to evaluate a protein-based NCR recruitment system for packaging cargo in XDPs. Materials and Methods: [0675] XDPs were generated in which an NCR protein was fused to the Gag polyprotein, and a ligand for the NCR protein was fused to the cargo of the XDP, i.e., to the N-terminus of CasX. Table 70, below, summarizes the plasmids used to encode the components of these XDP systems. Table 70: Summary of version XDPs with protein recruitment of CasX Plasmid Encoded Components, 5' to 3' 1 MA*-CA*-NC*-p1*-p6-NCR protein 2 MA*-CA*-NC*-p1*-p6*-PR† 3 Ligand-CasX 4 VSV-G 5 sgRNA * indicates cleavage sequence between adjacent components † indicates a -1 frame-shift in the encoded construct (Gag-TFR-PR polyprotein) [0676] The protein-ligand pairs that were tested are provided in Table 71 (each row is a protein-ligand pair). Some of the listed protein-ligand pairs were split fluorescent proteins that were anticipated to emit fluorescence when the two portions of the split protein are bound, such as mNeon-Green and sfCherry. Table 72 provides the amino acid sequences of the NCR proteins, and Table 73 provides the amino acid sequences of the ligands. Table 71: Protein-ligand pairs for protein-based recruitment to XDPs Protein attached to Gag Ligand attached to CasX Protein A Fc 249 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Protein attached to Gag Ligand attached to CasX Protein A truncated Fc CL7 Im7 mNeon-Green (mNG)21-10 mNG11 sfCherry21-10 sfCherry211 mNG3A1-10 mNG11 mNG3k1-10 mNG11 sfGFP1-10 GFP11 mClover31-10 mClover311 CloGFP0.21-10 GFP11 CloGFP1-10 GFP11 SpyCatcher 002 (variant 1) SpyTag 002 NbALFA ALFA tag SpyCatcher SpyTag SpyCatcher 002 (variant 2) SpyTag002 SpyCatcher 003 SpyTag003 Strep-Tactin® Twin Strep tag II Strep-Tactin® Strep tag II Avidin Avi tag Table 72: Amino acid sequences of proteins fused to Gag Protein attached Amino acid se SEQ ID to Gag quence of protein attached to Gag NO MKKKNIYSIRKLGVGIASVTLGTLLISGGVTPAANAAQHDEAQQNAFYQVL NMPNLNADQRNGFIQSLKDDPSQSANVLGEAQKLNDSQAPKADAQQNNFNK DQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEAKKLNESQAPK ADNNFNKEQQNAFYEILNMPNLNEEQRNGFIQSLKDDPSQSANLLSEAKKL NESQAPKADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANL Protein A LAEAKKLNDAQAPKADNKFNKEQQNAFYEILHLPNLTEEQRNGFIQSLKDD 2450 PSVSKEILAEAKKLNDAQAPKEEDNNKPGKEDNNKPGKEDNNKPGKEDNNK PGKEDNNKPGKEDGNKPGKEDNKKPGKEDGNKPGKEDNKKPGKEDGNKPGK EDGNKPGKEDGNGVHVVKPGDTVNDIAKANGTTADKIAADNKLADKNMIKP GQELVVDKKQPANHADANKAQALPETGEENPFIGTTVFGGLSLALGAALLA GRRREL 250 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Protein attached Amino a SEQ ID to Gag cid sequence of protein attached to Gag NO Truncated Protein MADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKK A LNDAQAPKA 2451 SKSNEPGKATGEGKPNKWLNNAGKDLGSPVPDRIANKLRDKEFESFDDFRE CL7 TFWEEVSKDPELSKQFSRNNNDRMKVGKAPKTRTQDVSGKRTSFELNHQKP 2452 IEQNGGVYDMDNISWTPKRNIDIEG MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLKS TKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQF mNG21-10 EDGASLTVNYRYTYEGSHIKGEAQVMGTGFPADGPVMTNTLTAADWCMSKK 2453 TYPNDKTIISTFKWSYTTVNGKRYRSTARTTYTFAKPMAANYLKNQPMYVF RKTELKHSM MEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGHPYEGTQTAKLKVTK GGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFTWERVMNFED sfCherry21-10 GGVVTVTQDSSLQDGQFIYKVKLLGINFPSDGPVMQKKTMGWEASTERMYP 2454 EDGALKGEINQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVDIKLDITS HNED MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLKS TKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQF mNG3A1-10 EDGASLTVNYRYTYEGSHIKGEAQVMGTGFPADGPVMTNTLTAADLCVSKM 2455 TYPNDKTIISTFKWSYTTVNGKRYRSTARTTYTFAKPMAAKYLKNQPMYVL RKTELKHSM MVSKGEEDNMASLPATHELHIFGSINDVDFDMVGQGTGNPNEGYEELNLKS TKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQF mNG3k1-10 EDGASLTVNYRYTYEGSHIKGEAQVIGTGFPADGPVMTNTLTAADWCMSKM 2456 TYPNDKTIISTFKWSYITVNGKRYRSTARTTYTFAKPMAANYLKNQPMYVF RKTELKHSM MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTG KLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKD sfGFP1-10 DGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYIT 2457 ADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQ SVLSKDPNEK MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTT GKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFK mClover31-10 DDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHYVYI 2458 TADKQKNCIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSH QSKLSKDPNEK MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTTG KLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKD CloGFP0.21-10 DGKYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHYVYIT 2459 ADKQKNCIKANFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQ TKLSKDPNEK MSKGEELFTGVVPILVELDGDVNGHKFSVRGQGEGDATIGKLTLKLICTTG KLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFRD CloGFP1-10 DGKYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHYVYIT 2460 ADKQKNCIKANFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLLHQ TKLSKDPNEK 251 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Protein attached Amino acid sequence of pr SEQ ID to Gag otein attached to Gag NO GAMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRD SpyCatcher 002 SSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQ (variant 1) 2461 VTVNGEATKGDAHT EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGERRVMV NbALFA AAVSERGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLE 2462 DRVDSFHDYWGQGTQVTVSS VDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSG SpyCatcher KTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTV 2463 NGKATKGDAHI SpyCatcher002 VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSG KTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTV (variant 2) 2464 NGEATKGDAHT VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSG SpyCatcher003 KTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTV 2465 DGEATEGDAHT MAEAGITGTWYNQLGSTFIVTAGADGALTGTYVTARGNAESRYVLTGRYDS Strep-Tactin® APATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSG 2466 TTEANAWKSTLVGHDTFTKVKPSAAS MVHATSPLLLLLLLSLALVAPGLSARKCSLTGKWTNDLGSNMTIGAVNSRG Avidin EFTGTYITAVTATSNEIKESPLHGTQNTINKRTQPTFGFTVNWKFSESTTV 2467 FTGQCFIDRNGKEVLKTMWLLRSSVNDIGDDWKATRVGINIFTRLRTQKE Table 73: Amino acid sequences of ligands fused to CasX Ligand attached Amino aci SEQ ID to CasX d sequence of ligand attached to CasX NO EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK Fc EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL 2468 VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK MGKIEEGKQETWNNGDKGYNGRAEDGGCGGAIEELEEEVRRHQMRLMALQL EEQLMGGCGDVGDMEFRNSISDYTEEEFVRLLRGIERENVAATDDRLDWML IM7 EHFVEITEHPDGTDLIYYPSDNRDDSPEGIVEEIREWREANGRPGFKQGGS 2469 TDGGDVGEERNRRCEELNEEIEEHRERLRQLEETREECRT mNG11 TELNFKEWQKAFTDMM 2470 sfCherry211 YTIVEQYERAEARHST 2471 mClover311 RDHMVLLEFVTAAGITHGMDELYK 2473 GFP11 RDHMVLHEYVNAAGIT 2474 252 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 Ligand attached Amino acid sequence of ligand at SEQ ID to CasX tached to CasX NO SpyTag002 VPTIVMVDAYKRYK 2475 ALFA tag PSRLEEELRRRLTEP 2476 SpyTag AHIVMVDAYKPTK 2477 SpyTag003 RGVPHIVMVDAYKRYK 2478 Twin Strep tag II WSHPQFEKGSAGSAAGSGAGWSHPQFEK 2479 Strep tag II WSHPQFEK 2480 Avi tag GLNDIFEAQKIEWHE 2481 [0677] Guide scaffold 226 was used (SEQ ID NO: 2380), with the 12.7 spacer for targeting the tdTomato locus (CUGCAUUCUAGUUGUGGUUU, SEQ ID NO: 1855). [0678] Cell culture and transfection, collection and concentration of XDPs, and resuspension and transduction of XDPs was performed as described in Example 2, above. tdTomato fluorescence was measured using flow cytometry. NPCs transduced with split fluorescent protein NCR systems were imaged by fluorescence microscopy 24 hours following transduction. Version 206 XDPs using RNA recruitment and targeting tdTomato were included as a control. Results: [0679] XDPs were generated using an NCR system based on the binding of a protein-ligand pair in which the protein was fused to the C-terminus of the Gag polyprotein and the ligand was fused to the N-terminus of CasX. As shown in FIG.100, XDPs with each of the protein-ligand pairs tested produced editing of the tdTomato locus. Indeed, for many of the XDPs with protein- ligand pairs, as well as for version 206 XDPs, editing levels were near 100%. This was true even at the lowest volumes of XDPs administered to the cells. Accordingly, editing levels were likely saturated in this assay. [0680] NPCs transduced with split fluorescent protein NCR systems were imaged for red fluorescence (indicating editing of the tdTomato locus) and green fluorescence (indicating binding of the two portions of the split fluorescent protein). The three split mNeon-Green NCR systems (mNG21-10+mNG11, mNG3A1-10+mNG311, and mNG3K1-10+mNG311) and the split CloGFP system (CloGFP1-10+CloGFP11) each produced cells with red and green fluorescence, indicating both editing and the presence of reconstituted split fluorescent proteins (data not shown). The green fluorescence was often found in puncta, which is believed to indicate nuclear 253 295377244 Attorney Docket No. SCRB-050/01WO 333322-2386 localization of the CasX:gRNA RNP along with the bound fluorescent protein. As controls, cells transduced with version 206 XDPs, and CL7+IM7 and sfCherry21-10+sfCherry211 protein NCR systems were also examined and were found to show red fluorescence (indicating editing of the tdTomato locus) but not green fluorescence. [0681] The results described herein demonstrate that the XDPs with protein-ligand pairs facilitate recruitment of CasX (and, hence, the complexed RNP) into the XDP particles, and are able to deliver RNPs to cells and produce genome editing of the target nucleic acid. 254 295377244 (g) the SpyCatcherOO3 protein comprises a sequence of SEQ ID NO: 2465, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the SpyTag003, and the SpyTag003 comprises a sequence of SEQ ID NO: 2478, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the SpyCatcher003 protein;
(h) the Strep-Tactin protein comprises a sequence of SEQ ID NO: 2466, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the Twin Strep tag II, and the Twin Strep tag II comprises a sequence of SEQ ID NO: 2479, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Strep-Tactin protein;
(i) the Strep-Tactin protein comprises a sequence of SEQ ID NO: 2466, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the Strep tag II, and the Strep tag II comprises a sequence of SEQ ID NO: 2480, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Strep-Tactin protein;
(j) the Avidin protein comprises a sequence of SEQ ID NO: 2467, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the Avi tag, and the Avi tag comprises a sequence of SEQ ID NO: 2481, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Avidin protein;
(k) the mNG2i-io protein comprises a sequence of SEQ ID NO: 2453, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the mNGn ligand, and the mNGn ligand comprises a sequence of SEQ ID NO: 2470, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mNG2i-io protein;
(l) the sfCherry2i-io protein comprises a sequence of SEQ ID NO: 2454, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the sfCherry2n ligand, and the sfCherry2n ligand comprises a sequence of SEQ ID NO: 2471, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the sfCherry2i-io protein;
(m) the mNG3 Ai-io protein comprises a sequence of SEQ ID NO: 2455, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the mNGn ligand, and the mNGn ligand comprises a sequence of SEQ ID NO: 2470, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mNG3Ai-io protein;
(n) the mNG3ki-io protein comprises a sequence of SEQ ID NO: 2456, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the mNGn ligand, and the mNGn ligand comprises a sequence of SEQ ID NO: 2470, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mNG3ki-io protein;
(o) the sfGFP i-io protein comprises a sequence of SEQ ID NO: 2457, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the GFPn ligand, and the GFPn ligand comprises a sequence of SEQ ID NO: 2474, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the sfGFPi-io protein;
(p) the mClover3 i-io protein comprises a sequence of SEQ ID NO: 2458, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the mClover3n ligand, and the mClover3n ligand comprises a sequence of SEQ ID NO: 2473, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mClover3i-io protein;
(q) the CloGFPO.2i-io protein comprises a sequence of SEQ ID NO: 2459, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the GFPn ligand, and the GFPn ligand comprises a sequence of SEQ ID NO: 2474, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the CloGFPO.2i-io protein; or
(r) the CloGFP i-io protein comprises a sequence of SEQ ID NO: 2460, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the GFPn ligand, and the GFPn ligand comprises a sequence of SEQ ID NO: 2474, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the CloGFPi-io protein.
[0287] 6. The XDP system of embodiment 1 or embodiment 2, wherein the NCR protein is an RNA-binding protein and the ligand is an RNA.
[0288] 7. The XDP system of embodiment 6, wherein the RNA binding-protein comprises:
(a) an MS2 coat protein and the ligand is an MS2 hairpin;
(b) a PP7 coat protein and the ligand is a PP7 hairpin;
(c) a QP coat protein and the ligand is a Q|3 hairpin;
(d) a AN protein and the ligand is a AN hairpin;
(e) a truncated AN protein and the ligand is a AN hairpin; (f) a Tat protein and the ligand is a transactivation response (TAR) element;
(g) a phage GA coat protein and the ligand is a phage GA hairpin;
(h) an iron-responsive binding element protein (IRE-BP) and the ligand is an iron response element (IRE);
(i) a U1A signal recognition particle and the ligand is a U1 hairpin II; or
(j) a truncated U1 A signal recognition particle and the ligand is a U1 hairpin II. [0289] 8. The XDP system of embodiment 7, wherein
(a) the MS2 coat protein comprises a sequence of SEQ ID NO: 4140, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the MS2 hairpin;
(b) the PP7 coat protein comprises a sequence of SEQ ID NO: 4132, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the PP7 hairpin;
(c) the QP coat protein comprises a sequence of SEQ ID NO: 4138, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the QP hairpin;
(d) the AN protein comprises a sequence of SEQ ID NO: 4131, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the AN hairpin;
(e) the truncated AN protein comprises a sequence of SEQ ID NO: 4130; , or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the AN hairpin;
(f) the Tat protein comprises a sequence of SEQ ID NO: 4133, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the TAR element; (g) the phage GA coat protein comprises a sequence of SEQ ID NO: 4139, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the phage GA hairpin;
(h) the IRE-BP comprises a sequence of SEQ ID NO: 4134 or 4135, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the IRE;
(i) the U1A signal recognition particle comprises a sequence of SEQ ID NO: 4137, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the UI hairpin II; or
(j) the truncated U1A signal recognition particle comprises a sequence of SEQ ID NO: 4136, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the UI hairpin II.
[0290] 9. The XDP system of embodiment 7 or embodiment 8, wherein
(a) the MS2 hairpin comprises a sequence of SEQ ID NO: 910;
(b) the PP7 hairpin comprises a sequence of SEQ ID NO: 914;
(c) the QP hairpin comprises a sequence of SEQ ID NO: 911;
(d) the AN hairpin comprises a sequence of SEQ ID NO: 954;
(e) the TAR comprises a sequence of SEQ ID NO: 951;
(f) the phage GA hairpin comprises a sequence of SEQ ID NO: 953;
(g) the IRE comprises a sequence of SEQ ID NO: 952; or
(h) the UI hairpin II comprises a sequence of SEQ ID NO: 912.
[0291] 10. The XDP system of embodiment 7 or embodiment 8 wherein the MS2 hairpin comprises a sequence selected from the group consisting of ACAUGAGGAUCACCCAUGU (SEQ ID NO: 910), ACCUGAGGAUCACCCAGGU (SEQ ID NO: 1847), GCAUGAGGAUCACCCAUGC (SEQ ID NO: 1848), GCCUGAGGAUCACCCAGGC (SEQ ID NO: 1849), GCCUGAGCAUCAGCCAGGC (SEQ ID NO: 1850), ACAUGAGCAUCAGCCAUGU (SEQ ID NO: 1851), ACUUGAGGAUCACCCAUGU (SEQ ID NO: 1852), ACAUUAGGAUCACCAAUGU (SEQ ID NO: 1853), and ACAUGAGGACCACCCAUGU (SEQ ID NO: 1854).
[0292] 11. The XDP system of any one of embodiments 7-10, wherein the RNA-binding protein comprises MS2 coat protein and the ligand comprises the MS2 hairpin, and wherein the therapeutic payload comprising the MS2 hairpin exhibits a dissociation constant (KD) to the MS2 coat protein of less than 100 nM, less than 50 nM, less than 35 nM, less than 10 nM, less than 3 nM, or less than 2 nM in an in vitro assay.
[0293] 12. The XDP system of any one of embodiment 6-11, wherein the encoded therapeutic payload comprises two RNA hairpins.
[0294] 13. The XDP system of embodiment 12, wherein the two RNA hairpins are identical.
[0295] 14. The XDP system of any one of embodiments 1-13, wherein the encoded therapeutic payload comprises a protein, a nucleic acid, or both a protein and a nucleic acid.
[0296] 15. The XDP system of any one of embodiments 1-14, wherein the encoded therapeutic payload comprises a nucleic acid selected from the group consisting of a single-stranded antisense oligonucleotide (ASO), a double-stranded RNA interference (RNAi) molecule, a DNA aptamer, an RNA aptamer , a first CRISPR guide ribonucleic acid (gRNA), a first and a second gRNA, or any combination thereof.
[0297] 16. The XDP system of embodiment 15, wherein the encoded therapeutic payload comprises a first, and optionally a second gRNA selected from the group consisting of a Class 2 Type II, a Class 2 Type V, and a Class 2 Type VI CRISPR system gRNA.
[0298] 17. The XDP system of embodiment 16, wherein the first, and optionally the second gRNA is a Class 2 Type II CRISPR system gRNA.
[0299] 18. The XDP system of embodiment 16, wherein the first, and optionally the second gRNA is a Class 2 Type V CRISPR system gRNA.
[0300] 19. The XDP system of any one of embodiments 16-18, wherein the first, and optionally the second gRNA is a single-molecule guide RNA (sgRNA) comprising a scaffold sequence and a targeting sequence, wherein the targeting sequence comprises between 15 and 20 nucleotides and is complementary to a target nucleic acid sequence.
[0301] 20. The XDP system of embodiment 19, wherein the targeting sequence has 18, 19, or 20 nucleotides. [0302] 21. The XDP system of embodiment 19 or embodiment 20, wherein the first, and optionally the second gRNA scaffold sequence comprises a sequence selected from the group consisting of the sequences of SEQ ID NOS: 2101-2430, and 4106, or a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto, wherein the gRNA is capable of complexing with a CRISPR protein to form a ribonucleoprotein (RNP).
[0303] 22. The XDP system of any one of embodiments 19-21, wherein the first, and optionally the second gRNA scaffold sequence comprises a sequence selected from the group consisting of the sequences of SEQ ID NOS: 2101-2430, and 4106.
[0304] 23. The XDP system of embodiment 22, wherein the first, and optionally the second gRNA scaffold sequence comprises a sequence selected from the group consisting of the sequences of SEQ ID NOS: 2238, 2292, 2380, and 4106.
[0305] 24. The XDP system of any one of embodiments 16 -23, wherein scaffold of the first, and optionally the second gRNA comprises an extended stem loop comprising the RNA hairpin. [0306] 25. The XDP system of any one of embodiments 16 -24, wherein the first, and optionally the second gRNA is a CasX gRNA comprising an extended stem loop comprising the RNA hairpin.
[0307] 26. The XDP system of embodiment 25, wherein the first, and optionally the second gRNA scaffold sequence comprises a sequence selected from the group consisting of the sequences of SEQ ID NOS: 2249, 2308, 2312, 2314-2317, 2319, 2380, and 2417-2429, or a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto, wherein the gRNA is capable of complexing with a CRISPR protein to form a ribonucleoprotein (RNP).
[0308] 27. The XDP system of embodiment 26, wherein the first, and optionally the second gRNA scaffold sequence comprises a sequence selected from the group consisting of the sequences of SEQ ID NOS: 2249, 2308, 2312, 2314-2317, 2319, 2380, and 2417-2429. [0309] 28. The XDP system of any one of embodiments 16-27, wherein the first, and optionally the second gRNA comprises a scaffold comprising one or more binding partner elements selected from the group consisting of: i) a Stem IIB of Rev response element (RRE; SEQ ID NO: 569), ii) a Stem II-V of RRE (SEQ ID NO: 571), iii) a Stem II of RRE (SEQ ID NO: 570), iv) a Rev-binding element (RBE) of Stem IIB (SEQ ID NO: 565), and v) and a full-length RRE (SEQ ID NO: 572), wherein the one or more binding partner elements are capable of binding Rev protein.
[0310] 29. The XDP system of any one of embodiments 16 -28, comprising a second gRNA scaffold sequence identical to the first gRNA scaffold sequence, and a targeting sequence complementary to a different region of the target nucleic acid, wherein the second gRNA is capable of complexing with a CRISPR protein to form a ribonucleoprotein (RNP).
[0311] 30. The XDP system of any one of embodiments 1-29, wherein the encoded therapeutic payload comprises a protein payload selected from the group consisting of a cytokine, an interleukin, an enzyme, a receptor, a microprotein, a hormone, erythropoietin, a ribonuclease (RNase), a deoxyribonuclease (DNase), a blood clotting factor, an anticoagulant, a bone morphogenetic protein, an engineered protein scaffold, a thrombolytic protein, a CRISPR protein, granulocyte-macrophage colony-stimulating factor (GMCSF), a transcription factor, a repressor domain, a transposon, a reverse transcriptase, a viral interferon antagonist, a tick protein, and an anti-cancer biologic.
[0312] 31. The XDP system of any one of embodiments 1-30, wherein the encoded therapeutic payload comprises a CRISPR protein.
[0313] 32. The XDP system of embodiment 31, wherein the CRISPR protein is a Class 2 CRISPR protein selected from the group consisting of a Class 2 Type II, a Class 2 Type V, or a Class 2 Type VI CRISPR protein.
[0314] 33. The XDP system of embodiment 32, wherein the Class 2 Type II CRISPR protein is a Cas9 protein.
[0315] 34. The XDP system of embodiment 32, wherein the Class 2 Type V CRISPR protein selected from the group consisting of Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f, Cast 2g, Casl2h, Casl2i, Casl2j, Cast 2k, Cast 4, and Cas . [0316] 35. The XDP system of embodiment 34, wherein the CasX is a CasX variant comprising a sequence selected from the group consisting of SEQ ID NOS: 135-169, 181-320, 322-366, 368-457, 797-804, 806-829, 831, 832, 834-842, 937, 938, 940, or 942, or a sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto, wherein the CasX variant retains the ability to form an RNP with a gRNA and retains nuclease activity.
[0317] 36. The XDP system of embodiment 35, wherein the CasX variant comprises a sequence selected from the group consisting of SEQ ID NOS: 189, 196, 354, and 813, or a sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto, wherein the CasX variant retains the ability to form an RNP with a gRNA and retains nuclease activity.
[0318] 37. The XDP system of embodiment 35, wherein the CasX variant comprises a sequence selected from the group consisting of SEQ ID NOS: 189, 196, 354, and 813.
[0319] 38. The XDP system of any one of embodiments 31-37, wherein the CRISPR protein comprises a nuclear localization signal (NLS).
[0320] 39. The XDP system of embodiment 38, wherein the NLS is a c-myc NLS or an SV40 NLS.
[0321] 40. The XDP system of embodiment 39, wherein the NLS comprises a sequence selected from the group consisting of SEQ ID NOs: 35, 37, 1740, 1750, 4128, and 4129.
[0322] 41. The XDP system of embodiment 31, wherein the CRISPR protein is a catalytically - dead CasX variant (dCasX) comprising a sequence selected from the group consisting of SEQ ID NOS: 877-896 and 4112-4117, or a sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto, wherein the dCasX retains the ability to form an RNP with a gRNA.
[0323] 42. The XDP system of embodiment 41, wherein the dCasX comprises a sequence selected from the group consisting of SEQ ID NOS: 877-896 and 4112-4117, and wherein the dCasX retains the ability to form an RNP with a gRNA. [0324] 43. The XDP system of embodiment 42, wherein the dCasX comprises the sequence of SEQ ID NO: 878.
[0325] 44. The XDP system of embodiment 42 or embodiment 43, wherein the dCasX is linked to a first repressor domain (RD1) as a fusion protein (dXR), wherein the fusion protein is capable of reducing expression of the target nucleic acid by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%.
[0326] 45. The XDP system of embodiment 44, wherein the RD1 comprises an amino acid sequence motif selected from the group consisting of: a) PX1X2X3X4X5X6EX7, wherein i) Xi is A, D, E, or N, ii) X2isLorV, iii) X3 is I or V, iv) X4isS, T, orF, v) X5 is H, K, L, Q, R or W, vi) XeisLorM, and vii) X7 is G, K, Q, or R; b) X1X2X3X4GX5X6X7X8X9, wherein i) Xi is L or V, ii) X2is A, G, L, TorV, iii) X3 is A, F, or S, iv) X4isLorV, v) X5 is C, F, H, I, L or Y, vi) Xe is A, C, P, Q, or S, vii) X7 is A, F, G, I, S, or V, viii) Xs is A, P, S, or T, and ix) XAsKorR; c) QX1X2LYRX3VMX4 (SEQ ID NO: 4107), wherein i) Xi is K or R, ii) X2is A, D, E, G, N, S, or T, iii) X3 is D, E, or S, and iv) X4isLorR; d) X1X2X3FX4DVX5X6X7FX8X9X10X11 (SEQ ID NO: 4108), wherein i) Xi is A, L, P, or S, ii) X2isLorV, iii) X3 is S or T, iv) X4 is A, E, G, K, or R, v) X5 is A or T, vi) XeisIorV, vii) X7 is D, E, N, or Y, viii) Xs is S or T, ix) X9is E, P, Q, R, or W, x) Xiois E or N, and xi) XnisEorQ; e) X1X2X3PX4X5X6X7X8X9X10, wherein i) Xi is E, G, or R, ii) X2isEorK, iii) X3 is A, D, or E, iv) X4isCorW, v) X5 is I, K, L, M, T, or V, vi) X6isI,L,P, orV, vii) X7is D, E, K, or V, viii) Xs is E, G, K, P, or R, ix) X9 is A, D, R, G, K, Q, or V, and x) X10 is D, E, G, I, L, R, S, or V; f) L YX1X2VMX3EX4X5X6X7X8X9X10 (SEQ ID NO : 4109), wherein i) Xi is K or R, ii) X2isDorE, iii) X3 is L, Q, or R, iv) X4isNorT, v) X5 is F or Y, vi) Xe is A, E, G, Q, R, or S, vii) X?isH, L, orN, viii) Xs is Lor V, ix) X9 is A, G, I, L, T, or V, and x) Xiois A, F, or S, g) FX1DVX2X3X4FX5X6X7EWX8 (SEQ ID NO: 4110), wherein i) Xi is A, E, G, K, or R, ii) X2isA, S, orT, iii) X3 is I or V, iv) X4isD, E,N, or Y, v) X5 is S or T, vi) X6is E, L, P, Q, R, or W, vii) X?isDorE, and viii) Xs is A, E, G, Q, or R; h) X1PX2X3X4X5 X6LEX7X8X9X10X11X12, wherein i) Xi is K or R, ii) X2 is A, D, E, or N, iii) X3 is I, L, M, or V, iv) X4isIorV, v) X5 is F, S, or T, vi) X6is H, K, L, Q, R, or W, vii) X?isK, Q, orR, viii) XsisE, G, orR, ix) XgisD, E, orK, x) Xiois A, D, or E, xi) XnisL orP, and xii) XnisCorW; i) X1LX2X3X4QX5X6, wherein i) Xi is C, H, L, Q, or W, ii) X2isD, G,N,R, or S, iii) X3 is L, P, S, or T, iv) X4isA, S, orT, v) X5 is K or R, and vi) Xe is A, D, E, K, N, S, or T.
[0327] 46. The XDP system of embodiment 44, wherein the RD1 comprises a first and a second amino acid sequence motif wherein:
(a) the first amino acid sequence motif comprises the sequence LYX1X2VMX3EX4X5X6X7X8X9X10 (SEQ ID NO: 4109), wherein i) Xi is K or R, ii) X2 is D or E, iii) X3 is L, Q, or R, iv) Xiis N or T, v) X5 is F or Y, vi) Xe is A, E, G, Q, R, or S, vii) X?is H, L, or N, viii) Xs is L or V, ix) X9 is A, G, I, L, T, or V, and x) X10 is A, F, or S; and
(b) the second amino acid sequence motif comprises the sequence FX1DVX2X3X4FX5X6X7EWX8 (SEQ ID NO: 4110), wherein i) Xi is A, E, G, K, or R, ii) X2 is A, S, or T, iii) X3 is I or V, iv) X4is D, E, N, or Y, v) X5 is S or T, vi) Xe is E, L, P, Q, R, or W, vii)X?is D or E, and viii) Xs is A, E, G, Q, or R.
[0328] 47. The XDP system of any one of embodiments 44-46, wherein the RD1 comprises a sequence selected from the group consisting of SEQ ID NOS: 2509-4105, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. [0329] 48. The XDP system of any one of embodiments 44-47, wherein the RD1 comprises a sequence selected from the group consisting of SEQ ID NOS: 2509-2603, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.
[0330] 49. The XDP system of any one of embodiments 44-48, wherein the RD1 comprises a sequence selected from the group consisting of SEQ ID NOS: 2509-2517, or sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.
[0331] 50. The XDP system of any one of embodiments 44-49, wherein the dXR comprises a second, a third, and a fourth repressor domain.
[0332] 51. The XDP system of embodiment 50, wherein the second, the third, and the fourth repressor domains are each a DNA methyltransferase (DNMT) domain.
[0333] 52. The XDP system of embodiment 51, wherein the second repressor domain is a DNMT3 A domain or a subdomain thereof.
[0334] 53. The XDP system of embodiment 52, wherein the second repressor domain is a catalytic domain of DNMT3A (DNMT3A CD).
[0335] 54. The XDP system of embodiment 53, wherein the DNMT3A CD comprises a sequence of SEQ ID NO: 2504, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.
[0336] 55. The XDP system of any one of embodiments 50-54, wherein the third transcription repressor domain is a DNMT3L interaction domain (DNMT3L ID).
[0337] 56. The XDP system of embodiment 55, wherein the DNMT3L ID comprises a sequence of SEQ ID NO: 2497, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.
[0338] 57. The XDP system of any one of embodiments 53-56, wherein the fourth repressor is an ATRX-DNMT3-DNMT3L (ADD) domain of DNMT3A. [0339] 58. The XDP system of embodiment 57, wherein the ADD domain comprises the sequence of SEQ ID NO: 2503, or a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.
[0340] 59. The XDP system of any one of embodiments 44-58, wherein the dXR comprises one or more linker peptide sequences and/or nuclear localization signals (NLS).
[0341] 60. The XDP system of embodiment 59, wherein the dXR is configured, from N- terminus to C-terminus:
(a) NLS-Linker-DNMT3 A CD-Linker- DNMT3L ID-Linker-Linker-dCasX- Linker3-RD1-NLS;
(b) NLS-Linker-dCasX-Linker3-RDl-NLS-Linker-DNMT3A CD-Linker-DNMT3L ID;
(c) NLS-Linker-dCasX-Linker-DNMT3A CD-Linker-DNMT3L ID-Linker-RD 1- NLS;
(d) NLS-RD1-Linker-DNMT3A CD-Linker-DNMT3L ID-Linkerl-dCasX-Linker- NLS, or
(e) NLS-DNMT3 A CD-Linker-DNMT3L ID-Linker-RD 1 -Linker-dCasX-Linker- NLS.
[0342] 61. The XDP system of embodiment 59, wherein the dXR is configured, from N- terminus to C-terminus:
(a) NLS-ADD-DNMT3A CD-Linker-DNMT3L ID-Linker-Linker-dCasX-Linker- RD1-NLS;
(b) NLS-Linker-dCasX-Linker-RDl-NLS-Linker-ADD-DNMT3A CD-Linker- DNMT3L ID;
(c) NLS-Linker-dCasX-Linker-ADD-DNMT3 A CD-Linker-DNMT3L ID-Linker- RD 1 -NLS;
(d) NLS-RD1 -Linker- ADD-DNMT3 A CD-Linker2-DNMT3L ID-Linkerl-dCasX- Linker-NLS; or
(e) NLS-ADD-DNMT3A CD-Linker-DNMT3L ID-Linker-RD 1-Linker-dCasX-
Linker-NLS. [0343] 62. The XDP system of any one of embodiment 1-61, wherein the encoded tropism factor is a glycoprotein.
[0344] 63. The XDP system of embodiment 62, wherein the glycoprotein has binding affinity for a cell surface marker of a target cell and facilitates entry of the XDP into the target cell.
[0345] 64. The XDP system of embodiment 62 or embodiment 63, wherein the glycoprotein has a sequence selected from the group consisting of SEQ ID NOS: 573-613, 615-682, 684-692, 694-698, 700, 702-706, 708-727, 729-730, 732, 734, 738, 740-746, 749-796, or a sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.
[0346] 65. The XDP system of embodiment 64, wherein the glycoprotein has a sequence selected from the group consisting of the sequences of SEQ ID NOS: 573-613, 615-682, 684- 692, 694-698, 700, 702-706, 708-727, 729-730, 732, 734, 738, 740-746, 749-796.
[0347] 66. The XDP system of any one of embodiments 1-65, wherein the one or more retroviral components, therapeutic payload, and tropism factor are encoded on three, four, or five nucleic acids.
[0348] 67. The XDP system of embodiment 66, wherein separate vectors comprise each nucleic acid, and wherein each vector comprises a promoter operably linked to the nucleic acid. [0349] 68. The XDP system of embodiment 67, wherein the promoter comprises a Pol II or a Pol III promoter.
[0350] 69. The XDP system of embodiment 67 or embodiment 68, wherein the vector comprising the nucleic acid encoding the Gag polyprotein fused to the NCR protein or functional domain thereof comprises a sequence encoding a Rev protein.
[0351] 70. The XDP system of any one of embodiment 67-69, wherein the XDPs are capable of self-assembly when the separate vectors are introduced into eukaryotic packaging cells and the cells are cultured under conditions allowing expression of the encoded components.
[0352] 71. The XDP system of embodiment 70, wherein the encoded therapeutic payload is encapsidated within the XDPs upon self-assembly in the eukaryotic packaging cell.
[0353] 72. The XDP of embodiment 70 or embodiment 71, wherein the encoded tropism factor is incorporated on the surface of the XDP upon self-assembly and release from the eukaryotic packaging cell. [0354] 73. The XDP system of embodiment 71 or embodiment 72, wherein the therapeutic payload comprises a CRISPR protein and a first gRNA complexed as a ribonucleoprotein complex (RNP).
[0355] 74. The XDP system of embodiment 73, wherein the therapeutic payload comprises the CRISPR protein and the first gRNA complexed as a first ribonucleoprotein complex (RNP), and the CRISPR protein and a second gRNA complexed as a second RNP.
[0356] 75. The XDP system of embodiment 73 or embodiment 74, wherein inclusion of the sequences encoding NCR protein and its corresponding ligand in the nucleic acids encoding the Gag polyprotein and the gRNA, respectively, results in enhanced incorporation of the numbers of RNP into the XDPs during self-assembly when compared to an equivalent XDP system not comprising the NCR protein and its corresponding ligand.
[0357] 76. The XDP system of embodiment 75, wherein at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, or at least about 1000 RNP particles are incorporated into the XDP.
[0358] 77. The XDP system of embodiment 76, wherein at least about 100 to about 1000 RNP, at least about 200 to about 800 RNP, or at least about 300 to about 600 RNP are incorporated into the XDP.
[0359] 78. The XDP system of any one of embodiments 1-77, wherein the encoded retroviral components are derived from a Lentivirus.
[0360] 79. The XDP system of embodiment 78, wherein the Lentivirus is a human immunodeficiency type 1 (HIV-1) virus or human immunodeficiency type 2 (HIV-2) virus.
[0361] 80. The XDP system of embodiment 78 or embodiment 79, wherein the Gag polyprotein fused to the NCR protein comprises one or more components selected from the group consisting of a matrix polypeptide (MA), a capsid polypeptide (CA), a nucleocapsid polypeptide (NC), a pl protein (pl), a p6 protein (p6), and a protease cleavage sequence (PCS). [0362] 81. The XDP system of embodiment 80, wherein the Gag polyprotein comprises, from N-terminus to C-terminus, MA-PCS-CA-PCS-NC-PCS-pl-PCS-p6.
[0363] 82. The XDP system of any one of embodiments 1-81, wherein the therapeutic payload is encoded on a separate nucleic acid from the Gag polyprotein. [0364] 83. The XDP system of any one of embodiments 1-82, wherein the Gag-TFR-PR polyprotein comprises, from N-terminus to C-terminus, MA-PCS-CA-PCS-NC-PCS-pl-PCS- p6-PCS-protease, wherein the protease is capable of cleaving the PCS of the Gag and the Gag- TRF-PR.
[0365] 84. The XDP system of any one of embodiments 1-83, wherein the nucleic acid sequences encoding the components are arranged, from 5' to 3', to encode proteins according to a configuration of:
(i) MA-PCS-CA-PCS-NC-PCS-pl-PCS-p6-PCS-CasX;
(ii) MA-PCS -CA-PCS-NC-PCS-pl-PCS-p6-MS2;
(iii) MA-PCS-CA-MS2-PCS-NC-PCS-pl-PCS -p6;
(iv) MA-PCS-MS2-CA-PCS-NC-PCS-pl-PCS -p6;
(v) MA-PCS-CA-PCS-MS2-PCS-NC-PCS-pl-PCS-p6;
(vi) MA-PCS-MS2-PCS-CA-PCS-NC-PCS-pl-PCS-p6;
(vii) MA-PCS-CA-PCS-NC-MS2-PCS-pl-PCS-p6;
(viii) MA-PCS-CA-PCS-NC-PCS-MS2-PCS-pl-PCS-p6;
(ix) MA-PCS-CA-PCS-MS2-NC-PCS-pl-PCS-p6;
(x) MA-PCS-CA-PCS-MS2-PCS-NC-PCS-pl-PCS-p6;
(xi) MA-PCS-CA-PCS-NC-PCS-pl-PCS-p6-NCR protein;
(xii) MA-PCS -CA-PCS -NC-PCS-pl-PCS-p6-PCS-Pro;
(xiii) MA-p2A-p2B-plO-CA-NC- Pro-GMCSF;
(xiv) MA-PCS-CA-PCS-NC-PCS-pl-PCS-p6-PCS-GMCSF;
(xv) MA-PCS-CA-PCS-NC-PCS-pl-PCS-p6-PCS-Pro;
(xvi) MA-PCS-CA-PCS-NC-PCS-pl-PCS-p6-NCR protein; or
(xvii) MA-PCS-CA-PCS-NC-PCS-pl-PCS-p6-PCS-Pro.
[0366] 85. The XDP system of any one of embodiments 1-84, wherein the one or more of the components are encoded by nucleic acids selected from the group consisting of the sequences of SEQ ID NOS: 19-31, 196, 813, 848, 975-977, 979, 1021, 1134-1136, 1138-1153, 1155-1195, 1127-1199, 1200-1207, 1227-1230, 1233-1250, 1253-1284, 1286-1320, 1322-1325, 1536-1540, 1572, 1587, 1781, 1783-1787, 1789, 1790, 1845, 2249, 2308, 2482-2493, and 4146-4148, or sequences having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.
[0367] 86. The XDP system of any one of embodiments 1-85, wherein the one or more of the retroviral components are encoded by a nucleic acid selected from the group consisting of the sequences of SEQ ID NOS: 19-31, 196, 813, 975-979, 2249, and 2308.
[0368] 87. A eukaryotic cell comprising the XDP system of any one of embodiments 1-86. [0369] 88. The eukaryotic cell of embodiment 87, wherein the cell is a packaging cell.
[0370] 89. The eukaryotic cell of embodiment 87 or embodiment 88, wherein the eukaryotic cell is selected from the group consisting of a Baby Hamster Kidney fibroblast (BHK) cell, a human embryonic kidney 293 (HEK293) cell, a human embryonic kidney 293T (HEK293T) cell, a NS0 cell, a SP2/0 cell, a YO myeloma cell, a P3X63 mouse myeloma cell, a PER cell, a PER.C6 cell, a hybridoma cell, an NIH3T3 cell, a CV-1 (simian) in Origin with SV40 genetic material (COS) cell, a HeLa cell, a Chinese hamster ovary (CHO) cell, and an HT1080 cell. [0371] 90. The eukaryotic cell of embodiment 89, wherein the eukaryotic cell is a HEK293 cell.
[0372] 91. The eukaryotic cell of any one of embodiment 87-90, wherein the eukaryotic cell is modified to reduce expression of a cell surface marker.
[0373] 92. The eukaryotic cell of embodiment 91, wherein the cell surface marker is selected from the group consisting of B2M, CD47 and HLA-E KI, wherein the incorporation of the cell surface marker on the surface of the XDP released from the eukaryotic cell is reduced compared to XDP released from a eukaryotic cell that has not be modified.
[0374] 93. The eukaryotic cell of any one of embodiment 87-92, wherein the eukaryotic cell is modified to express one or more cell surface markers selected from CD46, CD47, CD55, and CD59, wherein the incorporation of the cell surface marker on the surface of the XDP released from the eukaryotic cell is increased compared to XDP released from a eukaryotic cell that has not be modified.
[0375] 94. A method of making an XDP comprising a therapeutic payload, the method comprising:
(a) propagating the eukaryotic cell of any one of embodiment 87-93 under conditions such that an XDP is produced; and
(b) harvesting the XDP produced by the eukaryotic cell. [0376] 95. The method of embodiment 94, wherein the eukaryotic cell is a HEK293 cell.
[0377] 96. The method of embodiment 94 or embodiment 95, wherein the therapeutic payload comprises RNP of i) the CasX variant or the dXR, and ii) the first and, optionally the second guide RNA.
[0378] 97. The method of embodiment 96, wherein expression of the sequences encoding the NCR protein and its corresponding ligand results in enhanced incorporation of the numbers of RNP into the XDP during self-assembly compared to an equivalent method not comprising the NCR protein and its corresponding ligand.
[0379] 98. The method of embodiment 97, wherein at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, or at least about 1000 RNP particles are incorporated into the XDP.
[0380] 99. The method of embodiment 97, wherein at least about 100 to about 1000 RNP, at least about 200 to about 800 RNP, or at least about 300 to about 600 RNP particles are incorporated into the XDP.
[0381] 100. An XDP produced by the method of any one of embodiments 94-99.
[0382] 101. The XDP of embodiment 100, comprising a therapeutic payload of RNPs of a CRISPR protein and a gRNA.
[0383] 102. The XDP of embodiment 101, comprising a therapeutic payload of RNPs of a CasX variant and a guide RNA variant.
[0384] 103. The XDP of embodiment 101 or embodiment 102, wherein incorporation of the ligand and NCR protein into the XDP system results in at least a 2-fold, at a least 3 -fold, or at least a 4-fold increase in editing potency of the XDP compared to an equivalent XDP without the incorporated ligand and NCR protein, when assayed in vitro under comparable conditions.
[0385] 104. The XDP of embodiment 101 or embodiment 102, comprising a therapeutic payload of RNPs of a dXR and a guide RNA variant.
[0386] 105. The XDP of embodiment 104, wherein incorporation of the ligand and NCR protein into the XDP system results in at least a 2-fold, at a least 3 -fold, or at least a 4-fold increase repressing potency of the XDP compared to an equivalent XDP without the incorporated ligand and NCR protein, when assayed in vitro under comparable conditions. [0387] 106. A delivery particle (XDP) comprising cleavage products of a retroviral Gag polyprotein, a therapeutic payload, and a tropism factor, wherein:
(a) the cleavage products of the Gag polyprotein comprise MA, CA, NC, pl, and p6, wherein p6 is fused to a non-covalent recruitment (NCR) protein,
(b) the tropism factor is incorporated on the surface of the XDP; and
(c) the therapeutic payload is fused to a ligand and encapsidated within the XDP, wherein the NCR protein has an affinity for the ligand.
[0388] 107. The XDP of embodiment 106, wherein the NCR protein is fused to the C-terminus of p6.
[0389] 108. The XDP of embodiment 106 or embodiment 107, wherein the NCR protein is a protein-binding protein and the ligand is a protein ligand, wherein the NCR has binding affinity for the ligand.
[0390] 109. The XDP of embodiment 108, wherein the NCR protein comprises:
(a) a Protein A and the ligand is an Fc region;
(b) a truncated Protein A and the ligand comprises an Fc region;
(c) a CL7 protein and the ligand comprises an IM7 ligand;
(d) a NbALFA protein and the ligand comprises an ALFA tag;
(e) a SpyCatcher protein and the ligand comprises a SpyTag;
(f) a SpyCatcher002 protein and the ligand comprises a SpyTag002;
(g) a SpyCatcher003 protein and the ligand comprises a SpyTag003;
(h) a Strep-Tactin protein and the ligand comprises a Twin Strep tag II;
(i) a Strep-Tactin protein and the ligand comprises a Strep tag II;
(j) an Avidin protein and the ligand comprises an Avi tag;
(k) a mNG2i-io protein and the ligand comprises a mNGn ligand;
(l) a sfCherry2i-io protein and the ligand comprises a sfCherry2n ligand;
(m) a mNG3 Ai-io protein and the ligand comprises a mNGn ligand;
(n) a mNG3ki-io protein and the ligand comprises a mNGn ligand;
(o) a sfGFPi-io protein and the ligand comprises a GFPn ligand;
(p) a mClover3i-io protein and the ligand comprises a mClover3n ligand;
(q) a CloGFPO.2i-io protein and the ligand comprises a GFPn ligand; or
(r) a CloGFPi-io protein and the ligand comprises a GFPn ligand. [0391] 110. The XDP of embodiment 109, wherein:
(a) the Protein A comprises a sequence of SEQ ID NO: 2450, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Fc region, and the Fc region comprises a sequence of SEQ ID NO: 2468, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Protein A;
(b) the truncated Protein A comprises a sequence of SEQ ID NO: 2451, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Fc region, and the Fc region comprises a sequence of SEQ ID NO: 2468, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the truncated protein A;
(c) the CL7 protein comprises a sequence of SEQ ID NO: 2452, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the IM7 ligand, and the IM7 ligand comprises a sequence of SEQ ID NO: 2469, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the CL7 protein;
(d) the NbALFA protein comprises a sequence of SEQ ID NO: 2462, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the ALFA tag, and the ALFA tag comprises a sequence of SEQ ID NO: 2476, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the NbALFA tag;
(e) the SpyCatcher protein comprises a sequence of SEQ ID NO: 2463, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the SpyTag, and the SpyTag comprises a sequence of SEQ ID NO: 2477, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the SpyCatcher protein;
(f) the SpyCatcher002 protein comprises a sequence of SEQ ID NO: 2461 or 2464, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the SpyTag002, and the SpyTag002 comprises a sequence of SEQ ID NO: 2475, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the SpyCatcher002 protein;
(g) the SpyCatcher003 protein comprises a sequence of SEQ ID NO: 2465, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the SpyTag003, and the SpyTag003 comprises a sequence of SEQ ID NO: 2478, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the SpyCatcher003 protein;
(h) the Strep-Tactin protein comprises a sequence of SEQ ID NO: 2466, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Twin Strep tag II, and the Twin Strep tag II comprises a sequence of SEQ ID NO: 2479, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Srep-Tactin protein;
(i) the Strep-Tactin protein comprises a sequence of SEQ ID NO: 2466, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the to the Strep tag II, and a Strep tag II comprises the sequence of SEQ ID NO: 2480, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Strep-Tactin protein;
(j) the Avidin protein comprises a sequence of SEQ ID NO: 2467, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Avi tag, and the Avi tag comprises a sequence of SEQ ID NO: 2481, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Avidin protein;
(k) the mNG2i-io protein comprises a sequence of SEQ ID NO: 2453, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mNGn ligand, and the mNGn ligand comprises a sequence of SEQ ID NO: 2470, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mNG2i-io protein;
(l) the sfCherry2i-io protein comprises a sequence of SEQ ID NO: 2454, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the sfCherry2n ligand, and the sfCherry2n ligand comprises a sequence of SEQ ID NO: 2471, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the sfCherry2i-io protein;
(m) the mNG3 Ai-io protein comprises a sequence of SEQ ID NO: 2455, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mNGn ligand, and the mNGn ligand comprises a sequence of SEQ ID NO: 2470, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mNG3 Ai-io protein; (n) the mNG3ki-io protein comprises a sequence of SEQ ID NO: 2456, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mNGn ligand, and the mNGn ligand comprises a sequence of SEQ ID NO: 2470, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mNG3ki-io protein;
(o) the sfGFP i-io protein comprises a sequence of SEQ ID NO: 2457, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the GFPn ligand, and the GFP11 ligand comprises a sequence of SEQ ID NO: 2474, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the sfGFPi-io protein;
(p) the mClover3i-io protein comprises a sequence of SEQ ID NO: 2458, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mClover3n ligand, and the mClover3n ligand comprises a sequence of SEQ ID NO: 2473, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mClover3 i-io protein;
(q) the CloGFPO.2i-io protein comprises a sequence of SEQ ID NO: 2459, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the GFPn ligand, and the GFPn ligand comprises a sequence of SEQ ID NO: 2474, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the CloGFPO.2i-io protein; or
(r) the CloGFP i-io protein comprises a sequence of SEQ ID NO: 2460, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the GFPn ligand, and the GFPn ligand comprises a sequence of SEQ ID NO: 2474, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the CloGFP0.2i-io protein.
[0392] 111. The XDP of embodiment 106 or embodiment 107, wherein the NCR protein is an RNA-binding protein and the ligand is an RNA.
[0393] 112. The XDP of embodiment 111, wherein the NCR protein comprises:
(a) an MS2 coat protein, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the MS2 hairpin, and the ligand is an MS2 hairpin;
(b) a PP7 coat protein, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the PP7 hairpin, and the ligand is a PP7 hairpin;
(c) a QP coat protein, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the QP hairpin and the ligand is a QP hairpin;
(d) a AN protein, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the AN hairpin, and the ligand is a AN hairpin;
(e) a truncated AN protein, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the AN hairpin, and the ligand is a AN hairpin;
(f) a Tat protein, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the TAR element, and the ligand is a transactivation response (TAR) element; (g) a phage GA coat protein, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the phage GA hairpin, and the ligand is a phage GA hairpin;
(h) an iron-responsive binding element protein (IRE-BP) , or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the IRE, and the ligand is an iron response element (IRE);
(i) a U1A signal recognition particle, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the U1 hairpin II, and the ligand is a U1 hairpin II; or
(j) a truncated U1 A signal recognition particle, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the U1 hairpin II, and the ligand is a U1 hairpin II.
[0394] 113. The XDP of embodiment 112, wherein
(a) the MS2 hairpin comprises the sequence of SEQ ID NO: 910;
(b) the PP7 hairpin comprises the sequence of SEQ ID NO: 914;
(c) the QP hairpin comprises the sequence of SEQ ID NO: 911;
(d) the AN hairpin comprises the sequence of SEQ ID NO: 954;
(e) the TAR comprises the sequence of SEQ ID NO: 951;
(f) the phage GA hairpin comprises the sequence of SEQ ID NO: 953;
(g) the IRE comprises the sequence of SEQ ID NO: 952; or
(h) the U1 hairpin II comprises the sequence of SEQ ID NO: 912.
[0395] 114. The XDP of embodiment 112, wherein the MS2 hairpin comprises a sequence selected from the group consisting of ACAUGAGGAUCACCCAUGU (SEQ ID NO: 910), ACCUGAGGAUCACCCAGGU (SEQ ID NO: 1847), GCAUGAGGAUCACCCAUGC (SEQ ID NO: 1848), GCCUGAGGAUCACCCAGGC (SEQ ID NO: 1849), GCCUGAGCAUCAGCCAGGC (SEQ ID NO: 1850), ACAUGAGCAUCAGCCAUGU (SEQ ID NO: 1851), ACUUGAGGAUCACCCAUGU (SEQ ID NO: 1852), ACAUUAGGAUCACCAAUGU (SEQ ID NO: 1853), and ACAUGAGGACCACCCAUGU (SEQ ID NO: 1854).
[0396] 115. The XDP of any one of embodiments embodiment 112-114, wherein the RNA- binding protein comprises MS2 and the ligand comprises the MS2 hairpin, and wherein the therapeutic payload comprising the MS2 hairpin exhibits a dissociation constant (KD) to the MS2 coat protein of less than 100 nM, less than 50 nM, less than 35 nM, less than 10 nM, less than 3 nM, or less than 2 nM in an in vitro assay.
[0397] 116. The XDP of any one of embodiments 106-115, wherein the therapeutic payload comprises a nucleic acid selected from the group consisting of a single-stranded antisense oligonucleotide (ASO), a double-stranded RNA interference (RNAi) molecule, a DNA aptamer, an RNA aptamer, a first CRISPR guide ribonucleic acid (gRNA), a first and a second gRNA, or any combination thereof.
[0398] 117. The XDP of any one of embodiments 106-116, wherein the therapeutic payload comprises a protein payload selected from the group consisting of a cytokine, an interleukin, an enzyme, a receptor, a microprotein, a hormone, erythropoietin, a ribonuclease (RNase), a deoxyribonuclease (DNase), a blood clotting factor, an anticoagulant, a bone morphogenetic protein, an engineered protein scaffold, a thrombolytic protein, a CRISPR protein, granulocytemacrophage colony-stimulating factor (GMCSF), a transcription factor, a transposon, a reverse transcriptase, a viral interferon antagonist, a tick protein, and an anti-cancer modality.
[0399] 118. The XDP of any one of embodiments 106-117, wherein the therapeutic payload comprises of RNPs of a CRISPR protein and a gRNA, RNPs of a CasX variant and a guide RNA variant, or RNPs of a dCasX and linked repressor domain(s) and a guide RNA variant.
[0400] 119. The XDP of any one of embodiments 106-118, wherein the cleavage products of the Gag polyprotein are derived from a Lentivirus.
[0401] 120. The XDP of embodiment 119, wherein the Lentivirus is an HIV-1 virus or an HIV-2 virus.
[0402] 121. A method of modifying a target nucleic acid sequence in a population of cells, comprising contacting the cells with the XDP of any one of embodiments 100-120, wherein the XDP comprises a therapeutic payload of RNPs of a CasX variant and a gRNA variant, and wherein said contacting comprises introducing the into the cells the RNP, wherein the target nucleic acid targeted by the gRNA variant is modified by the CasX variant. [0403] 122. The method of embodiment 121, wherein the RNP of the CasX variant and the guide RNA exhibits at least a 2-fold improvement in cleavage velocity of a target nucleic acid compared to an RNP of a reference CasX and a reference guide RNA, when assayed in vitro under comparable conditions.
[0404] 123. The method of embodiment 121 or embodiment 122, wherein incorporation of the binding partner element and the NCR protein in the XDP results in at least a 2-fold, at a least 3- fold, at least a 4-fold, at least a 5-fold increase in editing potency of the XDP for a target nucleic acid compared to an equivalent XDP without the one or more binding partner elements and the NCR protein, when assayed in vitro under comparable conditions.
[0405] 124. The method of any one of embodiments 121-123, wherein the modification comprises introducing one or more single-stranded breaks in the target nucleic acid sequence. [0406] 125. The method of any one of embodiments 121-123, wherein the modification comprises introducing one or more double-stranded breaks in the target nucleic acid sequence. [0407] 126. The method of any one of embodiments 121-125, wherein the modification comprises introducing an insertion, deletion, substitution, duplication, or inversion of one or more nucleotides in the target nucleic acid sequence.
[0408] 127. The method of any one of embodiments 121-125, wherein the modification comprises introducing an in-frame mutation, a frame-shifting mutation, or a premature stop codon in the target nucleic acid coding sequence.
[0409] 128. The method of any one of embodiments 121-127, wherein the cells are modified in vitro or ex vivo.
[0410] 129. The method of any one of embodiments 121-127, wherein the cells are modified in vivo.
[0411] 130. A method of repressing a target nucleic acid sequence in a population of cells, wherein the XDP comprises a therapeutic payload of RNPs of a dXR and a guide RNA, wherein the method comprises contacting the cells with the XDP of embodiment 104 or embodiment 105, wherein said contacting comprises introducing the into the cells the RNP, wherein a target nucleic acid targeted by the guide RNA is repressed by the dXR.
[0412] 131. The method of embodiment 130, wherein the cells are repressed in vivo.
[0413] 132. The method of any one of embodiments 121-131, wherein the XDP is administered to a subject using a therapeutically effective dose. [0414] 133. The method of embodiment 132, wherein the subject is the subject is selected from the group consisting of a mouse, a rat, a pig, or a non-human primate.
[0415] 134. The method of embodiment 132, wherein the subject is a human.
[0416] 135. The method of embodiment 131 or embodiment 132, wherein the XDP is administered by a route of administration selected from the group consisting of subcutaneous, intradermal, intraneural, intranodal, intramedullary, intramuscular, intravenous, intracerebroventricular, intracisternal, intrathecal, intracranial, intralumbar, intratracheal, intraosseous, intraportal, inhalatory, intracontralateral striatum, intraocular, intravitreal, intralymphatical, intraperitoneal and sub-retinal routes, wherein the administering method is injection, transfusion, or implantation.
[0417] 136. A composition comprising the XDP of any one of embodiments 100-120 for use as a medicament for the treatment of a subject having a disease.
EXAMPLES
Example 1: CasX:gRNA In Vitro Cleavage Assays
1. Assembly of RNP
[0418] Purified RNP of CasX and single guide RNA (sgRNA) were either prepared immediately before experiments or prepared and snap-frozen in liquid nitrogen and stored at -80°C for later use. To prepare the RNP complexes, the CasX protein was incubated with sgRNA at 1 : 1.2 molar ratio. Briefly, sgRNA was added to Buffer# 1 (25 mM NaPi, 150 mM NaCl, 200 mM trehalose, 1 mM MgC12), then the CasX was added to the sgRNA solution, slowly with swirling, and incubated at 37°C for 10 min to form RNP complexes. RNP complexes were filtered before use through a 0.22 pm Costar® 8160 filters that were pre-wet with 200 pl Buffer#l. If needed, the RNP sample was concentrated with a 0.5 ml Ultra 100-Kd cutoff filter, (Millipore™ part #UFC510096), until the desired volume was obtained. Formation of competent RNP was assessed as described below.
2. In vitro cleavage assays: Determining cleavage-competent fractions for protein variants compared to wild-type reference CasX
[0419] The ability of CasX variants to form active RNP compared to reference CasX was determined using an in vitro cleavage assay. The beta-2 microglobulin (B2M) 7.37 target for the cleavage assay was created as follows. DNA oligos with the sequence TGAAGCTGACAGCATTCGGGCCGAGATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGC GCT (non-target strand, NTS (SEQ ID NO: 968)) and AGCGCGAGCACAGCTAAGGCCACGGAGCGAGACATCTCGGCCCGAATGCTGTCAGC TTCA (target strand, TS (SEQ ID NO: 969)) were purchased with 5’ fluorescent labels (LI- COR™ IRDye® 700 and 800, respectively). dsDNA targets were formed by mixing the oligos in a 1 : 1 ratio in lx cleavage buffer (20 mM Tris HC1 pH 7.5, 150 mM NaCl, 1 mM TCEP, 5% glycerol, 10 mM MgCh), heating to 95° C for 10 minutes, and allowing the solution to cool to room temperature.
[0420] CasX RNPs were reconstituted with the indicated CasX and guides (see graphs) at a final concentration of 1 pM with 1.5-fold excess of the indicated guide unless otherwise specified in 1 * cleavage buffer (20 mM Tris HC1 pH 7.5, 150 mM NaCl, 1 mM TCEP, 5% glycerol, 10 mM MgCh) at 37° C for 10 min before being moved to ice until ready to use. The 7.37 target was used, along with sgRNAs having spacers complementary to the 7.37 target. [0421] Cleavage reactions were prepared with final RNP concentrations of 100 nM and a final target concentration of 100 nM. Reactions were carried out at 37° C and initiated by the addition of the 7.37 target DNA. Aliquots were taken at 5, 10, 30, 60, and 120 minutes and quenched by adding to 95% formamide, 20 mM EDTA. Samples were denatured by heating at 95° C for 10 minutes and run on a 10% urea-PAGE gel. The gels were either imaged with a LI-COR Odyssey® CLx and quantified using the LI-COR Image Studio software or imaged with a Cytiva Typhoon™ and quantified using the Cytiva IQTL software. The resulting data were plotted and analyzed using Prism. It was assumed that CasX acts essentially as a single-turnover enzyme under the assayed conditions, as indicated by the observation that sub-stoichiometric amounts of enzyme fail to cleave a greater-than-stoichiometric amount of target even under extended time-scales and instead approach a plateau that scales with the amount of enzyme present. Thus, the fraction of target cleaved over long time-scales by an equimolar amount of RNP is indicative of what fraction of the RNP is properly formed and active for cleavage. The cleavage traces were fit with a biphasic rate model, as the cleavage reaction clearly deviates from monophasic under this concentration regime, and the plateau was determined for each of three independent replicates. The mean and standard deviation were calculated to determine the active fraction (Table 10).
[0422] Apparent active (competent) fractions were determined for RNPs formed for reference CasX2 + guide 174 + 7.37 spacer, CasX 119 + guide 174 + 7.37 spacer, CasX 457 + guide 174 +7.37 spacer, CasX 488 + guide 174 + 7.37 spacer, and CasX 491 + guide 174 + 7.37 spacer, as shown in FIG. 1. The determined active fractions are shown in Table 10. All CasX variants had higher active fractions than the wild-type CasX2, indicating that the engineered CasX variants form significantly more active and stable RNP with the identical guide under tested conditions compared to wild-type CasX. This may be due to an increased affinity for the sgRNA, increased stability or solubility in the presence of sgRNA, or greater stability of a cleavage-competent conformation of the engineered CasX: sgRNA complex. An increase in solubility of the RNP was indicated by a notable decrease in the observed precipitate formed when CasX 457, CasX 488, or CasX 491 was added to the sgRNA compared to CasX2.
3. In vitro cleavage assays - Determining cleavage-competent fractions for single guide variants relative to reference single guides
[0423] Cleavage-competent fractions were also determined using the same protocol for CasX2 protein in combination with guides 2, 32, 64 and 174 and targeting sequence 7.37 (CasX2.2.7.37, CasX2.32.7.37, CasX2.64.7.37), and CasX2.174.7.37 to be 16 ± 3%, 13 ± 3%, 5 ± 2%, and 22 ± 5%, as shown in FIG. 2 and Table 10.
[0424] A second set of guides were tested under different conditions to better isolate the contribution of the guide to RNP formation. Guides 174, 175, 185, 186, 196, 214, and 215 with 7.37 spacer were mixed with CasX 491 at final concentrations of 1 pM for the guide and 1.5 pM for the protein, rather than with excess guide as before. Results are shown in FIG. 3 and Table 10. Many of these guides exhibited additional improvement over 174, with 185 and 196 achieving 91 ± 4% and 91 ± 1% competent fractions, respectively, compared with 80 ± 9% for 174 under these guide-limiting conditions.
[0425] The data indicate that both CasX variants and sgRNA variants are able to form a higher degree of active RNP with guide RNA compared to wild-type CasX and wild-type sgRNA. [0426] The apparent cleavage rates of CasX variants 119, 457, 488, and 491 compared to wild-type reference CasX were determined using an in vitro fluorescent assay for cleavage of the target 7.37.
4. In vitro cleavage assays - Determining kcieave for CasX variants compared to wild-type reference CasX
[0427] CasX RNPs were reconstituted with the indicated CasX protein (see FIG. 4) at a final concentration of 1 pM with 1.5-fold excess of the indicated guide in l x cleavage buffer (20 mM Tris HC1 pH 7.5, 150 mM NaCl, 1 mM TCEP, 5% glycerol, 10 mM MgCh) at 37° C for 10 min before being moved to ice until ready to use. Cleavage reactions were set up with a final RNP concentration of 200 nM and a final target concentration of 10 nM. Reactions were carried out at 37° C except where otherwise noted and initiated by the addition of the target DNA. Aliquots were taken at 0.25, 0.5, 1, 2, 5, and 10 minutes and quenched by adding to 95% formamide, 20 mM EDTA. Samples were denatured by heating at 95° C for 10 minutes and run on a 10% urea- PAGE gel. The gels were imaged with a LI-COR Odyssey CLx and quantified using the LI- COR Image Studio software or imaged with a Cytiva Typhoon and quantified using the Cytiva IQTL software. The resulting data were plotted and analyzed using Prism, and the apparent first- order rate constant of non -target strand cleavage (kcieave) was determined for each CasX: sgRNA combination replicate individually. The mean and standard deviation of three replicates with independent fits are presented in Table 10, and the quantification of competent fractions of RNP of CasX variants traces are shown in FIG 8. [0428] Apparent cleavage rate constants were determined for wild-type CasX2, and CasX variants 119, 457, 488, and 491 with guide 174 and spacer 7.37 utilized in each assay (see Table 10 and FIG. 4). All CasX variants had improved cleavage rates relative to the wild-type CasX2. CasX 457 cleaved more slowly than 119, despite having a higher competent fraction as determined above. CasX488 and CasX491 had the highest cleavage rates by a large margin; as the target was almost entirely cleaved in the first timepoint, the true cleavage rate exceeds the resolution of this assay, and the reported kcieave should be taken as a lower bound.
[0429] The data indicate that the CasX variants have a higher level of activity, with kcieave rates reaching at least 30-fold higher compared to wild-type CasX2.
5. In vitro cleavage assays: Determination of cleavage rates for guide variants compared to reference single guides
[0430] Cleavage assays were also performed with wild-type reference CasX2 and reference guide 2 compared to gRNA variants 32, 64, and 174 to determine whether the variants improved cleavage. The experiments were performed as described above. As many of the resulting RNPs did not approach full cleavage of the target in the time tested, initial reaction velocities (Vo) were determined rather than first-order rate constants. The first two timepoints (15 and 30 seconds) were fitted with a line for each CasX:sgRNA combination and replicate. The mean and standard deviation of the slope for three replicates were determined.
[0431] Under the assayed conditions, the Vo for CasX2 with guides 2, 32, 64, and 174 were 20.4 ± 1.4 nM/min, 18.4 ± 2.4 nM/min, 7.8 ± 1.8 nM/min, and 49.3 ± 1.4 nM/min (see Table 10 and FIGS. 5and FIG. 6). Guide 174 showed substantial improvement in the cleavage rate of the resulting RNP (~2.5-fold relative to 2, see FIG. 6), while guides 32 and 64 performed similar to or worse than guide 2. Notably, guide 64 supports a cleavage rate lower than that of guide 2 but performs much better in vivo (data not shown). Some of the sequence alterations to generate guide 64 likely improve in vivo transcription at the cost of a nucleotide involved in triplex formation. Improved expression of guide 64 likely explains its improved activity in vivo, while its reduced stability may lead to improper folding in vitro.
[0432] Additional experiments were carried out with guides 174, 175, 185, 186, 196, 214, and 215 with spacer 7.37 and CasX 491 to determine relative cleavage rates. To reduce cleavage kinetics to a range measurable with our assay, the cleavage reactions were incubated at 10° C. Results are in FIG. 7 and Table 10. Under these conditions, 215 was the only guide that supported a faster cleavage rate than 174. 196, which exhibited the highest active fraction of RNP under guide-limiting conditions, had kinetics essentially the same as 174, again highlighting that different variants result in improvements of distinct characteristics. [0433] The data support that use of the majority of the guide variants with CasX results in RNP with a higher level of activity than one with the wild-type guide, with improvements in initial cleavage velocity ranging from ~2-fold to >6-fold. Numbers in Table 10 indicate, from left to right, CasX variant, sgRNA scaffold, and spacer sequence of the RNP construct. In the RNP construct names in the table below, CasX protein variant, guide scaffold and spacer are indicated from left to right.
6. In vitro cleavage assays: Comparing cleavage rate and competent fraction of 515.174 and 526.174 against reference 2.2
[0434] We wished to compare engineered protein CasX variants 515 and 526 in complex with engineered single-guide variant 174 against the reference wild-type protein 2 (SEQ ID NO: 2) and minimally-engineered guide variant 2 (SEQ ID NO: 5). RNP complexes were assembled as described above, with 1.5-fold excess guide. Cleavage assays to determine kcieave and competent fraction were performed as described above, with both performed at 37°C, and with different timepoints used to determine the competent fraction for the wild-type vs engineered RNPs due to the significantly different times needed for the reactions to near completion.
[0435] The resulting data clearly demonstrate the dramatic improvements made to RNP activity by engineering both protein and guide. RNPs of 515.174 and 526.174 had competent fractions of 76% and 91%, respectively, as compared to 16% for 2.2 (FIG. 8, Table 10). In the kinetic assay, both 515.174 and 526.174 cut essentially all of the target DNA by the first timepoint, exceeding the resolution of the assay and resulting in estimated cleavage rates of 17.10 and 19.87 min'1, respectively (FIG. 9, Table 10). An RNP of 2.2, by contrast, cut on average less than 60% of the target DNA by the final 10-minute timepoint and has an estimated kcieave nearly two orders of magnitude lower than the engineered RNPs. The modifications made to the protein and guide have resulted in RNPs that are more stable, more likely to form active particles, and cut DNA much more efficiently on a per-particle basis as well.
Table 10: Results of cleavage and RNP formation assays
Figure imgf000149_0001
Figure imgf000150_0001
*Mean and standard deviation
**Rate exceeds resolution of assay
Example 2: Non-covalent recruitment with RNA binding - Gag-MS2
[0436] These experiments evaluated the ability of an MS2-based non-covalent recruitment (NCR) system to improve the generation of XDP in packaging host cells where the CasX RNP is recruited into the XDPs by fusing MS2 coat proteins (CPs) to the HIV Gag polyprotein and an MS2 hairpin is incorporated into the guide RNA.
Methods:
[0437] All plasmids encoding CasX proteins utilized the CasX 491 variant protein.
[0438] RNA fold structures were generated with RNAfold web server and Varna java-based software.
Structural plasmid cloning
[0439] In order to generate the structural plasmids used below, plasmid pXDPl was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments containing CasX variant, HIV-1, or MS2 CP components were amplified and cloned using In-Fusion® primers with 15-20 base pair overlaps and KAPA HiFi DNA polymerase according to the manufacturer’s protocols. The fragments were purified by gel extraction and cloned into plasmid backbones using In-Fusion® HD Cloning Kit from Takara (Cat# 639650) according to the manufacturer’s protocols. Assembled products were transformed into chemically-competent Turbo Competent E. coli bacterial cells, plated on LB-Agar plates containing ampicillin and incubated at 37°C. Individual colonies were picked and miniprepped using QIAprep® Spin Miniprep Kit following the manufacturer’s protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. The amino acid sequence of the MS2 CP is provided in SEQ ID NO: 4140, and the amino acid sequence of the Gag polyprotein fused to the MS2 CP is provided in SEQ ID NO: 4141.
Guide plasmid cloning
[0440] The tdTomato targeting guide plasmids used in these experiments were pSG50 (guide scaffold 188; FIG. 12) and pSG54 (guide scaffold 228; FIG. 13), which were cloned from pSG33 and pSG34, respectively. The mammalian expression backbone contained a cPPT, ampicillin resistance, and a colEI replication site and was amplified using primers with appropriate overlaps to accept the U6 promoter and guide RNA scaffold cassette. The backbone, pSG3, was digested using Ndel and Xbal. Synthetic DNA fragments corresponding to scaffold variants were amplified and cloned as described, above. The resultant plasmids, pSG33 and pSG34, were sequenced using Sanger sequencing to ensure correct assembly (Table 12). Cloning tdTomato spacer 12. 7 into pSG3 and pSG14
[0441] To clone the targeting pSG50 and pSG54 plasmids from the non-targeting pSG33 and pSG34, the spacer 12.7 was cloned using the following protocol. The targeting spacer sequence DNA for the tdTomato targeting spacer 12.7 was ordered as single-stranded DNA (ssDNA) oligos (Integrated DNA Technologies) consisting of the targeting sequence (CTGCATTCTAGTTGTGGTTT, SEQ ID NO: 1018) and the reverse complement of this sequence. These two oligos were annealed together and cloned into a pSG plasmid with an alternate scaffold by Golden Gate assembly using T4 DNA Ligase (New England BioLabs Cat# M0202L) and Esp3I restriction enzyme from New England BioLabs (NEB Cat# R0734L). Golden Gate products were transformed into chemically competent NEB® Turbo competent E. coli (NEB Cat #C2984I), plated on LB-Agar plates containing carbenicillin and incubated at 37°C. Individual colonies were picked and miniprepped as described above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation. pGP2 Glycoprotein plasmid cloning
[0442] Sequences encoding the VSV-G glycoprotein and the cytomegalovirus (CMV) promoter were amplified from pMD2.G and cloned as described for the structural plasmids, above. The backbone was taken from a kanamycin resistant plasmid and amplified and cloned using the same methods. Assembled products were transformed into chemically-competent Turbo Competent A. coli bacterial cells, plated on LB-Agar plates containing kanamycin and incubated at 37°C. The resultant plasmids in this five-plasmid system (Gag-(-l)-PR, Gag-MS2, CasX, gRNA, and GP) were sequenced using Sanger sequencing to ensure correct assembly. Cell culture and transfection
[0443] HEK293T Lenti-X™ cells were maintained in 10% FBS supplemented DMEM with HEPES and GlutaMAX™ (Thermo Fisher®). Cells were seeded in 15 cm dishes at 20 x 106 cells per dish in 20 mL of media. Cells were allowed to settle and grow for 24 hours before transfection. At the time of transfection, cells were 70-90% confluent. For transfection, the XDP structural plasmids (also encoding the CasX variants) of Table 13 were used in amounts ranging from 13 to 80.0 pg. Each transfection also received 13 pg of p42.174.12.7 and 0.25 pg of pGP2. Polyethylenimine (PEI MAX®, Polyplus) was then added to the plasmid mixture, mixed, and allowed to incubate at room temperature before being added to the cell culture. Plasmid ratios in Table 11 were used in all version 206 XDPs used in this assay, based on prior experimental data from other XDP versions.
Table 11: Plasmids and ratios used in XDP constructs
Figure imgf000152_0001
*transcript contains RRE and produces REV
Collection and concentration
[0444] Media was aspirated from the plates 24 hours post-transfection and replaced with Opti- MEM™ (Thermo Fisher). XDP-containing media was collected 72 hours post-transfection and filtered through a 0.45 pm PES filter. The supernatant was concentrated and purified via centrifugation.
[0445] Filtered supernatant was divided evenly into an appropriate number of centrifuge tubes or bottles and l/5th of the supernatant volume of Sucrose Buffer (50mM Tris-HCL, lOOmM NaCl, 10% Sucrose, pH 7.4) was underlaid using serological pipettes. The samples were centrifuged at 10,000xg, 4 °C, in a swinging-bucket rotor for 4 hours with no brake. The supernatant was carefully removed and the pellet briefly dried by inverting the centrifuge vessels. Pellets were either resuspended in Storage Buffer (PBS + 113 mM NaCl, 15% Trehalose dihydrate, pH 8 or an appropriate media by gentle trituration and vortexing. XDPs were resuspended in 300 pL of DMEM/ F12 supplemented with GlutaMAX™, HEPES, non-essential amino acids, Pen/Strep, 2-mercaptoethanol, B-27 without vitamin A, and N2.
Resuspension and transduction
[0446] tdTomato neural progenitor cells (NPCs) were resuspended and transduced with XDPs. In brief, tdTomato NPCs were grown in DMEM/F12 supplemented with GlutaMAX™, HEPES, NEAA, Pen/Strep, 2-mercaptoethanol, B-27 without vitamin A, and N2. Cells were harvested using StemPro Accutase Cell Dissociation Reagent and seeded on PLF-coated 96-well plates. 48 hours later, cells were transduced with XDPs containing a tdTomato targeting spacer. Cells were then centrifuged for 15 minutes at 1000 x g. Transduced NPCs were grown for 96 hours before analyzing tdTomato fluorescence by flow cytometry as a marker of editing at the tdTomato locus, with the EC50 determined as the number of XDP particles needed to achieve editing in 50% of the cells, as determined by flow cytometry. tdTomato fluorescence was measured using flow cytometry. The assays were run 2-3 times for each sample, with similar results.
Results:
[0447] The MS2 bacteriophage relies on the non-covalent affinity between its genomic RNA and the MS2 coat protein for the packaging of its genome in an icosahedral viral shell. The high- affinity element in the RNA genome is termed the MS2 hairpin, which binds to the coat protein with a Kd of approximately 3e'9. Here, two high affinity variants of the MS2 hairpin were incorporated into the extended stem of the guide scaffold 174, thereby introducing into the CasX:guide RNP an affinity for the MS2 coat protein. The resulting guide scaffolds 188 and 228 were tested in XDP version 168; a version that relies on a Gag-CasX fusion configuration and lacks the MS2 coat protein, while version 206 (FIG. 11) has the incorporated MS2 coat protein fused to Gag. Guides 188 and 228 performed similarly to guide scaffold 174 in total editing across all volumes tested, demonstrating that the insertion of the MS2 hairpin was benign to the function of the RNP. The MS2 hairpin variant sequences of these scaffolds are ACATGAGGATCACCCATGT (SEQ ID NO: 1131) and CGTACACCATCAGGGTACG (SEQ ID NO: 1132), respectively.
[0448] MS2-based recruitment of these variant scaffolds was tested in XDP version 206. This version is composed of the Gag-(-l)-PR, Gag-MS2, and CasX architectures. This version relies on orthogonal recruitment of CasX via the MS2 coat protein and MS2 hairpin system of the guide rather than a direct fusion between CasX and a recruiting protein. This is demonstrated in FIG. 15, where constructs with both guide scaffold 188 and 228 edit well in the tdTomato assay, in contrast to constructs with guide scaffold 174, which lacks the MS2 hairpin and edits poorly. Additionally, XDP version 206 with scaffold 188 edits better at the same dosage over XDP version 168 with scaffold 174 (see FIGS. 14 and 15). At 0.6 L of XDPs delivered, editing was -70% with XDP version 206 with guide scaffold 188. In the same assay, -20% editing was achieved at the same treatment volume for XDP version 168 (a Gag-CasX fusion) with guide scaffold 174 and version 206 with guide scaffold 228. These data suggested that XDP version 206 with guide scaffold 188 is 2-3x more potent than version 168 with guide scaffold 174. This increase in editing from version 168 to version 206 could be attributed to the lack of a direct fusion of Gag to CasX, causing less steric hindrance in particle formation. Furthermore, the similarity between guide scaffolds 188 and 228 in editing in version 168 suggests that the difference in potency in XDP version 206 is due to the MS2 hairpin’s affinity for the coat protein linked to Gag.
[0449] The results suggest two possible mechanisms of recruitment of the CasX RNP to XDP particles in version 206. First, the CasX protein and guide scaffold RNA form the apoenzyme RNP in the cytoplasm of the producer cell that then binds the Gag-MS2 protein by interactions of the MS2 hairpin in the guide extended stem and the MS2 coat protein. The second possible mechanism is that the guide scaffold RNA hairpin first binds the MS2 coat protein and then forms the apoenzyme with the CasX protein. Collectively, the results demonstrate the utility of the incorporation of the MS2 system for the formation of more potent XDP particles with increased numbers of RNP and higher editing capabilities. Additionally, the MS2 coat protein variants have several point mutations that alter their affinity to its hairpin RNA. Usage of these variants in version 206 could result in higher potency variants. Fusing multiple coat proteins to the HIV Gag protein could further increase potency as well. Alternatively, there are also several RNA hairpin - non-covalent recruitment (NCR) protein combinations, such as QP phage, GA phage, PP7 phage, or kN, that could be used to replace MS2. Other protein RNA combinations from humans and retroviruses include the Iron Responsive Element (IRE)-Iron Binding element, U1 hairpin II, retrovirus Tat-trans-activation response (TAR) system, Csy4, Pardaxin, tRNA or Psi-Nucleocapsid. Table 12: sgRNA encoding sequences
Figure imgf000155_0001
Table 13: Architecture and glycoprotein sequences
Figure imgf000155_0002
*backbone of plasmid expresses Rev Table 14: Version and pseudotyping descriptions
Figure imgf000156_0001
Example 3: Non-covalent recruitment with RNA binding - Partial Gag-MS2
[0450] The purpose of these experiments was to demonstrate the utility of a non-covalent recruitment (NCR) method for the incorporation of RNP into XDP using an MS2-based system where the RNPs are recruited into the XDPs by fusing the MS2 coat protein (CP) to different proteins within an HIV Gag polyprotein in the XDP construct.
[0451] The MS2 packaging system consists of two major components: the phage coat protein and its cognate binding partner, which is a short hairpin stem loop structure. In this orthogonal phage RNA-based recruitment system, the short hairpin stem loop structure is engineered into the sgRNA incorporated into the XDP. The encoding sequence for the phage coat protein is fused to either the encoding sequence for the Gag polyprotein (derived from any retroviruses) or to any other protein domains derived from the Gag polyprotein of any retroviral origin. This would enable the recruitment of the expressed CasX RNP into the XDP particle by the targeted interaction between the short hairpin stem loop structure engineered into the sgRNA, which is complexed with the CasX as an RNP, and the phage coat protein fused to the Gag polyprotein or any proteins derived from the Gag polyprotein. Here, XDPs in which the RNP is recruited into the XDPs by fusing the MS2 coat protein (CP) to different proteins within an HIV Gag polyprotein in the XDP construct are described.
Methods:
[0452] All plasmids containing CasX proteins encoded the CasX 491 variant protein. All XDPs were pseudotyped with 10% VSV-G (percentage of plasmid relative to the other plasmids utilized for the XDP construct). The guide scaffold used in all the MS2 constructs was 188 along with spacer 12.7 targeting the tdTomato locus. The guide scaffold used in the control construct (V168) was 226, also with spacer 12.7. This scaffold has the RRE/RBE element described in other examples herein. RNA fold structures were generated with RNAfold web server and Varna java-based software.
Structural plasmid cloning
[0453] In order to generate the structural plasmids (pXDP17, pXDP161, pXDP164 and pXDP166), pXDPl was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments containing CasX variant, HIV-1, or MS2 CP components were amplified and cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
[0454] MS2 was placed either on the N- or the C-terminus of the Capsid (Version 263- pXDP276, Version 264-pXDP277, Version 265-pXDP278 and Version 266-pXDP279), with and without cleavage sites. MS2 was placed either on the N- or the C-terminal of the Nucleocapsid (Version 267-pXDP280, Version 268-pXDP281, Version 269-pXDP282 and Version 270-pXDP283), with and without cleavage sites. The sequences for these constructs are provided in Table 16. The designed constructs were synthesized as transgenes and purchased pre-cloned into pTWIST expression plasmids from Twist Bioscience®. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
Guide plasmid cloning
[0455] The guide plasmids used in these experiments were pSG50 and pSG17, encoding guide scaffold 188. Spacer 12.7 was cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation. The guide plasmid used in all MS2 constructs is pSG50. The guide plasmid used in control construct (VI 68) is pSG17. pGP2 Glycoprotein plasmid cloning
[0456] Sequences encoding the VSV-G glycoprotein and the CMV promoter were amplified from pMD2.G and cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
Cell culture and transfection; collection and concentration; resuspension and transduction [0457] HEK293T Lenti-X™ cell culture was performed as described in Example 2, above. For transfection, the XDP structural plasmids of Table 16 were used in amounts ranging from 13 to 80.0 pg. Each transfection also received 13 pg of pSG50 or pSG17 and 0.25 pg of pGP2.
Polyethylenimine (PEI MAX® from Polyplus) was then added as described in Example 2. XDPs were collected and concentrated as described in Example 2, above. tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above. tdTomato fluorescence was measured using flow cytometry. The assays were run 2-3 times for each sample, with similar results.
Results:
[0458] Percent editing of the tdTomato target sequence in tdT NPCs are shown for all the constructs in FIG. 52 in terms of number of particles added and the volume of XDPs added (FIG. 53). Table 15 presents the results of percent editing of the dtTomato target sequence when 16.6 pl of the concentrated XDP prep was used to treat NPCs. The results show that it is feasible to fuse MS2 with or without a cleavage sequence to either the capsid or the nucleocapsid. The results indicate that fusing MS2 to the C-terminal of the capsid results in more potent XDP as compared to a fusion to the N-terminal. In addition, introduction of a cleavage site in between MS2 and CA on the C-terminal does improve potency as shown in FIG. 52. Fusing MS2 to the N- or C-terminal of nucleocapsid with and without a cleavage site may be superior to a capsid fusion, with a fusion to the N-terminal of NC being marginally better in terms of editing as shown in FIG. 53. The EC50 for the different constructs were calculated and plotted as shown in FIG. 54 and recapitulates the differences in potency described above. FIG. 16 depicts the fold improvement in EC50 over the base control VI 68 (CasX fused to full length HIV Gagpolyprotein) and it shows that V265, V269 and V270 show about 5 to 8-fold improvement in potency. FIG. 17 depicts the fold improvement in EC50 over the base control V206 (MS2 fused to full length HIV Gag-polyprotein and the results demonstrate that V265, V269 and V270 show about 6- to 9-fold improvement in terms of overall editing potency.
Table 15: Percent editing at the second dilution (16.6pl)
Figure imgf000158_0001
* indicates cleavage sequence between adjacent components
** 5' to 3' orientation [0459] These results show that it is functionally feasible to fuse MS2 with or without a cleavage sequence to the capsid or the nucleocapsid derived from the HIV Gag polyprotein to create XDP that results in enhanced editing of the target nucleic acid. These results also show that it is possible to improve potency depending on the location within the Gag polyprotein (or its components) where the MS2 is fused. It is also believed that this enhanced architecture can be translated to proteins derived from the Gag polyproteins of Alpharetroviral, Betaretroviral, Gammaretroviral, Deltaretroviral, Epsilonretroviral, Lentiviral and Spumaretroviral origin, serving as an orthogonal recruitment mechanism for CasX or any other payload that can be coupled with a cognate short hairpin RNA element in an XDP or other particle-delivery system.
Table 16: Plasmid sequences
Figure imgf000159_0001
Example 4: Non-covalent recruitment with RNA binding - Retro-MS2
[0460] The purpose of these experiments was to demonstrate the utility of a recruitment method for the incorporation of RNP into XDP using an MS2-based system and Gag polyproteins or components of Gag polyproteins derived from five genera of retroviruses, including Alpharetroviruses, Betaretroviruses, Gammaretroviruses, Deltaretroviruses and Lentiviruses.
Methods:
[0461] All plasmids containing CasX proteins encoded the CasX 491 protein. All XDPs were pseudotyped with 10% VSV-G (percentage of plasmid relative to the other plasmids utilized for the XDP construct). The guide RNA spacer used in all of these experiments was 12.7 targeting the tdTomato locus. The guide scaffold used in all the MS2 constructs was 188, along with spacer 12.7. RNA fold structures were generated with RNAfold web server and Varna javabased software.
Structural plasmid cloning
[0462] MS2 was fused to the Gag-protease, Gag or partial Gag polyproteins derived from Alpharetroviruses (Versions 271, 272, 273), Betaretroviruses (Versions 277, 279), Gammaretroviruses (Versions 276, 278), Deltaretroviruses (Versions 274, 275) and Lentiviruses (Versions 280, 281, 282) with their respective species-specific cleavage sites. The sequences for these constructs are provided in Table 18. The designed constructs were synthesized as transgenes and purchased pre-cloned into pTWIST expression plasmids from Twist Biosciences. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
Guide plasmid cloning
[0463] The guide plasmid used in these experiments was pSG50. To clone the targeting pSG50 spacer 12.7 was cloned in as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation. pGP2 Glycoprotein plasmid cloning
[0464] Sequences encoding the VSV-G glycoprotein and the CMV promoter were cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
Cell culture and transfection
[0465] HEK293T Lenti-X™ cell culture was performed as described in Example 2, above. For transfection, the XDP structural plasmids of Table 18 were used in amounts ranging from 13 to 80.0 pg. Each transfection also received 13 pg of p42.174.12.7 and 0.25 pg of pGP2.
Polyethylenimine (PEI MAX® from Polyplus) was then added as described in Example 2. Collection and concentration; resuspension and transduction
[0466] XDPs were collected and concentrated as described in Example 2, above.
[0467] tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above, and tdTomato fluorescence was measured using flow cytometry. The assays were run 2-3 times for each sample with similar results.
Results:
[0468] Percent editing of the dtTomato target sequence in tdT NPCs are shown for all the constructs in FIG. 55 across the dilution curve for the volume of XDPs added. Table 17 represents the percent editing of the dtTomato target sequence when 16.6 pl of the concentrated XDP prep was used to treat NPCs. These results show that, as compared to V206, which was the control XDP in this experiment (derived from HIV, lentivirus) and which edited at 95% efficacy at the tdTomato locus when 16.6 pl of the concentrated XDP was used, V271 and V272, which are different architectural variants derived from ALV (Alpharetroviruses) showed editing efficacies ranging from 79 to 88%. V275 derived from HTLV1 (Deltaretroviruses), V279 derived from MPMV (Betaretroviruses) as well as V281 derived from EIAV (Lentivirus) showed successful editing ranging from 76.5, 61.6, to 48.7% at the tdT locus, respectively. Other XDPs such as V273 (derived from RSV, Alpharetroviruses), V274 (derived from BLV, Deltaretroviruses), V276 (derived from FLV, Gammaretroviruses), NIH (derived from MMTV, Betretroviruses), V278 (derived from MMLV, Gammaretroviruses), V280 (derived from EIAV, Lentivirus), V282 (derived from SIV, Lentivirus) showed above background editing at the tdT locus ranging from 10.6 to 4.03%. The variation in editing efficiencies observed between the different constructs may be due to the architectural differences between the retroviral families used. Editing differences between V280 (editing at 10.6%) as compared to V281 (editing at 48.7%) is an example of this as both versions are derived from EIAV (Lentivirus) but differ in the architectural sequence. V280 has MS2 fused to Gag-pro polyprotein, whereas V281 has MS2 fused to the MA-CA polyprotein.
Table 17: Percent editing at the second dilution (16.6 pl)
Figure imgf000161_0001
Figure imgf000162_0001
[0469] Overall, these results show that fusing MS2 with the Gag-protease, Gag or partial Gag polyproteins of diverse retroviral origin that include Alpharetroviruses, Betaretroviruses, Gammaretroviruses, Deltaretroviruses and Lentiviruses creates XDPs that result in editing of the target nucleic acid. It is believed that supplementing these versions with another plasmid that encodes for the respective Gag-protease or Gag polyprotein could further augment editing functions.
Table 18: Plasmid sequences
Figure imgf000162_0002
Figure imgf000163_0001
Example 5: Non-covalent recruitment with MS2 variants
[0470] Experiments were conducted to evaluate the ability of an MS2-based recruitment system using MS2 variants having altered affinities to the MS2 hairpin in order to improve the generation of XDP in packaging host cells.
Methods:
[0471] All plasmids encoding CasX proteins utilized the CasX 491 variant protein. All XDPs contained sgRNAs with scaffold 188 (see FIG. 12) and spacer 12.7.
Structural plasmid cloning
[0472] In order to generate the structural plasmids, listed below, pXDPlwas digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments encoding CasX variant, HIV-1, or MS2 CP components were amplified and cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
Guide plasmid cloning
[0473] The tdTomato targeting guide plasmid used in these experiments was pSG50 (guide scaffold 188), which was cloned from pSG33. The mammalian expression backbone contained a cPPT, ampicillin resistance, and a colEI replication site and was amplified using primers with appropriate overlaps to accept the U6 promoter and guide RNA scaffold cassette. The backbone, pSG3, was digested using Ndel and Xbal. Synthetic DNA fragments corresponding to novel scaffolds were amplified and cloned as described in Example 2, above. The resultant plasmid, pSG33, was sequenced using Sanger sequencing to ensure correct assembly.
Cloning tdTomato spacer 12. 7
[0474] To clone the targeting pSG50 plasmid from the non-targeting pSG33, spacer 12.7 was cloned as described in Example 2, above. The resultant plasmid was sequenced using Sanger sequencing to ensure correct ligation (see Table 20). pGP2 Glycoprotein plasmid cloning
[0475] Sequences encoding the VSV-G glycoprotein and the CMV promoter were amplified from pMD2.G and cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Table 23 lists the plasmid structural and glycoprotein plasmid components.
Cell culture and transfection
[0476] HEK293T Lenti-X™ cell culture was performed as described in Example 2, above. For transfection, the XDP structural plasmids (also encoding the CasX variants) of Table 21 were used in amounts ranging from 13 to 80.0 pg. Each transfection also received 13 pg of pSG50 and 0.25 pg of pGP2. Polyethylenimine (PEI MAX® from Polyplus) was then added as described in Example 2. Plasmid ratios in Table 19 were used in all Version 206 XDPs used in this assay and are based on prior data from other XDP versions.
Table 19: Construct plasmids and ratios of plasmids used
Figure imgf000164_0001
*transcript contains RRE and produces REV
Collection and concentration; resuspension and transduction
[0477] XDPs were collected and concentrated as described in Example 2, above. tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above, and tdTomato fluorescence was measured using flow cytometry. The assays were run 2-3 times for each sample, with similar results. Table 20: sgRNA and hairpin encoding sequences (DNA)
Figure imgf000165_0001
Table 21: XDP component architecture and glycoprotein sequences
Figure imgf000165_0002
Results:
[0478] In all, wild-type and 5 different MS2 variants were tested, as well as one dimerizationincompetent variant. These variants were tested in the same Gag-MS2 system as previous examples specified and this configuration is depicted in FIG. 18. To test these variants, pXDP164, which encodes the wild-type Gag-MS2 in XDP version 206, was replaced with either pXDP321, pXDP335, pXDP336, pXDP337, pXDP338, pXDP339, or pXDP340. These MS2 variants had affinity KdS ranging from 1.2e-7 M to 4e-10 M, with the wild-type version being 3e-9 M (a lower Kd value indicates greater affinity between the MS2 hairpin and coat protein). [0479] Results of the assays showed that the XDP with MS2 having lower Kd variants tended to perform with better editing than higher Kd variants (see Table 22) with a gRNA having a single MS2 hairpin (gRNA 188). The data were analyzed with a correlation analysis between the Kd of the MS2 coat protein and the inverse of the EC50 (by volume of XDP introduced into assay); a measure of potency that increases with more potent XDP constructs. This resulted in an r value of -0.625 as seen in FIG. 19, demonstrating that incorporation of MS2 with lower KdS correlated with resultant increased editing potency. The results support that by altering the binding affinity of the RNA hairpin and NCR protein, the potency of XDPs can be effectively modulated, thereby improving the XDP constructs. This approach may be similarly used with other RNA binding proteins, such as QP phage, GA phage, PP7 phage, or A N for engineering more potent XDPs.
Table 22: MS2 variants
Figure imgf000166_0001
Table 23: XDP Version and pseudotyping descriptions
Figure imgf000166_0002
Example 6: Evaluation of non-covalent recruitment (NCR) systems with RNA binding proteins linked to Gag
[0480] The purpose of these experiments was to evaluate the ability of various non-covalent recruitment (NCR) proteins linked to HIV Gag polyprotein and one or two copies of their cognate binding partner hairpin structures (“single hairpin” or “dual hairpins” respectively) integrated into the guide RNA scaffolds to improve the generation of XDP in packaging host cells.
Methods:
[0481] All XDPs were pseudotyped with 10% VSV-G (percentage of plasmid relative to the other plasmids utilized for the XDP construct). RNA fold structures were generated with RNAfold web server and Varna java-based software.
Structural plasmid cloning
[0482] In order to generate the structural plasmids used to make the XDP, pXDPl was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments containing CasX, HIV-1, retrovirus Tat, IRP1, IRP2, truncated U1A, U1A, phage QP coat protein, phage GA coat protein, phage kN coat protein, or truncated phage kN coat protein components were amplified using In Fusion primers with 15-20 base pair overlaps and Kapa HiFi DNA polymerase according to the manufacturer’s protocols. The fragments were purified by gel extraction from a 1% agarose gel using Zymoclean Gel DNA Recovery Kit according to the manufacturer’s protocol.
[0483] Further, fragments containing dual boxB hairpin, retrovirus transactivation response (TAR) element, Iron Responsive Element (IRE), U1A hairpin, phage QP hairpin, phage GA hairpin, phage kN hairpin, or phage PP7 hairpins were amplified and cloned in guide scaffolds based on guide scaffold 174 or guide scaffold 235. Sequences of guide RNA scaffolds with dual hairpins are provided in Table 26, below. Scaffolds 188 and 251 were used as controls.
[0484] These fragments were cloned into plasmid backbones as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
Guide plasmid cloning
[0485] The guide plasmids modified in these experiments were pSG50, encoding guide scaffold 188 (see FIG. 12). The non-targeting guide plasmids used in these experiments were pSG82 to pSG88, encoding guide scaffold 188. The mammalian expression backbone had a cPPT, ampicillin resistance, and a colEI replication site and was amplified using primers with appropriate overlaps to accept the U6 promoter and guide RNA scaffold cassette. Fragments containing the retrovirus TAR, Iron Responsive Element (IRE), U1A hairpin II, phage QP hairpin, phage GA hairpin, phage AN hairpin (also referred to herein as a boxB hairpin or boxB element), or phage PP7 hairpin were amplified and cloned as described in Example 2, above. The targeting spacer sequence DNA for the tdTomato targeting spacer 12.7 was cloned into pSG33 and pSG34 as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation. pGP2 Glycoprotein plasmid cloning
[0486] Sequences encoding the VSV-G glycoprotein and the CMV promoter were amplified and cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
Cell culture and transfection
[0487] HEK293T Lenti-X™ cell culture was performed as described in Example 2, above. For transfection, the XDP structural plasmids (also encoding the CasX variants) of Table 24 were used in amounts ranging from 13 to 80.0 pg. Each transfection will also receive 13 pg of a pSG plasmid and 0.25 pg of pGP2. Polyethylenimine (PEI MAX® from Polyplus) was then added as described in Example 2.
Collection and concentration; resuspension and transduction; titering
[0488] XDPs were collected and concentrated as described in Example 2, above. tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above, and tdTomato fluorescence was measured using flow cytometry. The assays were run 2-3 times for each sample with similar results. Titers were quantified for each version of XDPs using the NanoSight NS300.
Results:
[0489] The CasX guide scaffold extended stem region is highly modifiable. The extended stem loop protrudes out from the RNP, and so additions to this region have little effect on RNP formation and editing potency, as seen in other experiments described herein. This feature was used to add on one or two of several different RNA hairpins to the extended stem loop to engineer the CasX gRNA to bind their corresponding RNA-binding proteins. Table 24 shows the sequences of the Gag-NCR protein plasmids and their complementary sgRNAs with nontargeting spacers that were employed to create the versions. Table 25 shows the amino acid and RNA sequences of the Gag-NCR proteins and their complementary sgRNAs, respectively. The amino acid sequences of the NCR proteins (not fused to the Gag polyprotein) are provided in SEQ ID NOs: 4130-4139.
[0490] It was expected that inclusion of these NCR proteins into the constructs would likely yield more potent XDP configurations as it has previously been demonstrated that different Kas of NCR proteins, such as MS2, can modify the potency of XDPs. There is a large variety of Kas and sizes across these NCR proteins.
[0491] As shown in FIGS. 20 and 21, XDPs with the MS2, PP7, Tat, or U1 A NCR systems produced the highest levels of editing in the mouse tdTomato NPCs. Indeed, XDPs with the PP7, Tat or U1 A NCR systems produced higher levels of editing than XDPs with the MS2 NCR system. Both Tat and U1 A NCR systems are monomeric in nature. Therefore, that both Tat and U1 A NCR systems produced higher levels of editing suggests that MS2 dimerization has a detrimental effect on XDP architecture formation. It is anticipated that the relatively small size of the Tat protein could make it amenable to stacking (i.e., adding multiple Tat binding sites), which could enable better recruitment and packaging of the CasX RNP. Furthermore, while the PP7 NCR system also dimerizes, the RNA hairpin and the NCR protein have a higher binding affinity (Ka of 1 nM) compared to that of the MS2 system (of Kd of - 2.6 nM). This may explain the higher level of editing observed with the PP7 system compared to the MS2 system (FIGS. 20 and 21).
[0492] Titers were quantified for each version of XDP particles produced using the NanoSight NS300, and the number of transduced mouse NPCs was counted. The bar chart in FIG. 22 shows the number of XDPs containing the indicated NCR systems per edited mouse NPC, and the bar chart in FIG. 23 shows the average number of XDPs containing the indicated NCR systems per mouse NPC. Overall, use of the Gag-UIA, Gag-Tat, or Gag-PP7 NCR systems required the lowest average number of XDPs to edit a single mouse NPC (FIGS. 22 and 23), which is consistent with the high editing levels seen in FIGS. 20-21.
[0493] In addition, it is anticipated that the location of the NCR protein in the Gag polyprotein or the viral protein used can both be modified, and enhanced guide RNA scaffolds could lead to further improvements in potency. Table 24: Sequences encoding Gag-NCR proteins and guide scaffolds based on guide scaffold 174
Figure imgf000170_0001
Figure imgf000171_0001
Table 25: Amino acid sequences of Gag-NCR proteins and RNA sequences of guide scaffolds based on guide scaffold 174
Figure imgf000171_0002
Figure imgf000172_0001
Figure imgf000173_0001
Figure imgf000174_0001
Figure imgf000175_0001
Figure imgf000176_0001
Figure imgf000177_0001
Figure imgf000178_0001
[0494] Further experiments were conducted using sgRNAs with two hairpins for binding by NCR proteins. Table 26, below, shows the sequences of guide scaffolds based on guide scaffold 174 or scaffold 235, with two copies of each of the indicated hairpins. The guide scaffolds in Table 26 were tested in combination with the NCR proteins provided in Table 25.
Table 26: Guide scaffold sequences with dual hairpins
Figure imgf000178_0002
Figure imgf000179_0001
Figure imgf000180_0001
Figure imgf000181_0001
[0495] The results of the editing assays testing guide scaffolds with dual RNA hairpins are provided in FIGS. 91-99. As shown in FIG. 92, use of guide scaffold 188, which has a single MS2 hairpin, produced a slightly lower editing potency than use of guide scaffold 251, which has two copies of the MS2 hairpin (“dual hairpin”). This is consistent with the results provided below in Example 9.
[0496] As shown as FIG. 91, use of guide scaffolds with one or two copies of the PP7 hairpin produced similarly high levels of editing. This was true for the guide scaffolds based on either guide scaffold 174 and 235.
[0497] In the AN NCR system, using a truncated AN protein made up of the RNA-binding site as the NCR protein (“tAN”), use of the dual boxB hairpin guide scaffold produced the highest level of editing in the guide scaffold 174 background, followed by the dual hairpin guide scaffold in the scaffold 235 background (FIG. 93). Therefore, adding two copies of the boxB hairpin improved editing levels when paired with the tAN NCR protein.
[0498] In the AN NCR system using the full-length antitermination protein N as the NCR protein, use of the dual boxB hairpin guide scaffold also produced the highest level of editing in the guide scaffold 174 background (FIG. 94). Use of the guide scaffold with dual boxB hairpins in the guide scaffold 235 background and the guide scaffold with a single boxB hairpins produced lower levels of editing.
[0499] In the Tat/TARNCR system, as shown in FIG. 95, use of the guide scaffold with dual TAR elements in the scaffold 235 background produced the highest level of editing for the majority of the doses of XDPs tested, followed by the guide scaffolds with dual TAR elements in the guide scaffold 174 background.
[0500] Ula NCR systems using a truncated U1 a protein (“tUla”; see FIG. 96) or a full-length Ula protein (see FIG. 97) were tested only for the guide scaffold using a single hairpin or dual hairpins in the guide scaffold 235 background. As shown in FIG. 96 and FIG. 97, the scaffolds with dual hairpins in the guide scaffold 235 background produced a higher level of editing than the single hairpin for both Ula systems.
[0501] In the QP system, use of the guide scaffolds with dual QP hairpins resulted in editing at similarly high levels, and they were substantially more potent than the guide scaffold with a single QP hairpin (see FIG. 98).
[0502] Finally, in the phage GA NCR system, use of the guide scaffold with two phage GA hairpins in the guide scaffold 174 background produced the highest level of editing, followed by the guide scaffold with a single phage GA hairpin. The guide scaffold with two phage GA hairpins in the guide scaffold 235 background produced low levels of editing (see FIG. 99).
[0503] Notably, in this experiment, many of the NCR systems tested produced substantially higher levels of editing than either MS2 system shown in FIG. 92. For example, all the NCR systems with PP7 hairpins and NCR proteins (FIG. 91) resulted in higher levels of editing than either MS2 system (FIG. 92), when delivered at the same dose of XDPs.
[0504] Taken together, the results described herein show that various NCR systems can be used to recruit CasX-gRNA RNPs into XDPs.
Example 7: Evaluation of non-covalent recruitment (NCR) systems with dual MS2 coat protein for RNA binding
[0505] The purpose of these experiments was to evaluate whether inclusion of dual MS2 coat proteins (CP) linked to Gag and a single MS2 hairpin integrated into the guide RNA scaffold would enhance the potency of XDPs generated using this system, compared to constructs having a single copy of MS2 CP.
Methods:
[0506] All plasmids encoding CasX proteins utilized the CasX 491 variant protein.
Structural plasmid cloning
[0507] In order to generate the structural plasmids used to make the XDP, pXDPl was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments containing CasX variant, HIV-1, or MS2 CP components were amplified and cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
Guide plasmid cloning
[0508] The tdTomato targeting guide plasmid used in these experiments were pSG50 (scaffold 188; see FIG. 12) and pSG5 (scaffold 174), which were cloned from pSG33 and pSG3 respectively. The mammalian expression backbone contained a cPPT, ampicillin resistance, and a colEI replication site and was amplified using primers with appropriate overlaps to accept the U6 promoter and guide RNA scaffold cassette. The backbone was digested using Ndel and Xbal. Synthetic DNA fragments corresponding to guide scaffolds incorporating the MS2 hairpin were amplified and cloned as described in Example 2, above. The resultant plasmids, pSG3 and pSG33, were sequenced using Sanger sequencing to ensure correct assembly.
Cloning tdTomato spacer 12.7 into pSG3 and pSG33
[0509] To clone the targeting pSG50 and pSG5 plasmids from the non-targeting pSG33 and pSG3 spacer 12.7 was cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation (see Table 27).
Table 27: Encoded Guide and hairpin sequences
Figure imgf000183_0001
pGP2 Glycoprotein plasmid cloning
[0510] Sequences encoding the VSV-G glycoprotein and the CMV promoter were cloned as described in Example 2. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly (see Table 30).
Cell culture and transfection
[0511] HEK293T Lenti-X™ cell culture was performed as described in Example 2, above. For transfection, the XDP structural plasmids (also encoding the CasX variants) of Table 30 were used in amounts ranging from 13 to 80.0 pg. Each transfection also received 13 pg of pSG50 or pSG5 and 0.25 pg of pGP2. The descriptions of the plasmids used to evaluate the NLS are listed in Table 29. Polyethylenimine (PEI MAX® from Polyplus) was then added as described in Example 2. Plasmid ratios in Table 28 were used in all version 206 XDPs used in this assay and are based on prior data from other XDP versions.
Table 28: Construct plasmids and ratios of plasmids used
Figure imgf000184_0001
*transcript contains RRE and produces REV
Table 29: XDP plasmids for evaluation NLS effects
Figure imgf000184_0002
Figure imgf000185_0001
Table 30: Plasmid architecture and glycoprotein sequences
Figure imgf000185_0002
Collection and concentration; resuspension and transduction
[0512] XDPs were collected and concentrated as described in Example 2, above. tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above, and tdTomato fluorescence was measured using flow cytometry. tdTomato fluorescence was measured using flow cytometry. The assays were run 2-3 times for each sample, with similar results.
Results:
[0513] XDP version 309 is identical to version 206 except there is an additional MS2 CP fused to the first MS2 in this system, so pXDP164 (which encodes Gag-MS2) is replaced with pXDP288, which encodes Gag-MS2-MS2. While the hypothesis was that inclusion of the additional MS2 would increase the avidity of the RNP with MS2 hairpin in the scaffolds for these coat proteins, thereby increasing the incorporation of RNP into the budding XDP, it was observed that there was a significant decrease in editing with the constructs incorporating the second MS2 coat protein (see FIG. 24). The inverse of the EC50 by volume was 1.6 pL'1 for V206 (single MS2) and 0.075 pL'1 for V309 (double MS2). While V309 was still more potent than the negative control V206 without an MS2 hairpin containing scaffold (scaffold 174), which had an inverse EC50 of 0.012 pL'1, the results nevertheless underscore the utility of incorporating the MS2 system in the XDP constructs.
Example 8: Evaluation of non-covalent recruitment (NCR) systems with dual MS2 hairpins for MS2 coat protein binding
[0514] The purpose of these experiments was to determine if the incorporation of two MS2 hairpin RNA elements into the CasX sgRNA increased the potency of XDPs based on the MS2 coat protein hairpin recruitment system.
Methods:
[0515] All plasmids encoding CasX proteins utilized the CasX 491 variant protein.
Structural plasmid cloning
[0516] In order to generate the structural plasmids used to make the XDP, pXDPl was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments containing CasX variant, HIV-1, or MS2 CP components were amplified and cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
Guide plasmid cloning
[0517] The tdTomato targeting guide plasmid used in these experiments were pSG72 (scaffold 250; see FIG. 25) and pSG68 (scaffold 251; see FIG. 26) which were cloned from pSG67 and pSG68 respectively. The mammalian expression backbone contained a cPPT, ampicillin resistance, and a colEI replication site and was amplified using primers with appropriate overlaps to accept the U6 promoter and guide RNA scaffold cassette. The backbone was digested using Ndel and Xbal. Synthetic DNA fragments corresponding to novel scaffolds were cloned as described in Example 2, above. The resultant plasmids, pSG72 and pSG73, were sequenced using Sanger sequencing to ensure correct assembly (see Table 31).
Table 31: sgRNA encoding sequences
Figure imgf000186_0001
Figure imgf000187_0001
Cloning tdTomato spacer 12. 7 into pSG67 and pSG68
[0518] To clone the targeting pSG72 and 73 plasmids from the non-targeting pSG67 and pSG68, spacer 12.7 was cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation. pGP2 Glycoprotein plasmid cloning
[0519] Sequences encoding the VSV-G glycoprotein and the CMV promoter were amplified from pMD2.G and cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
Cell culture and transfection
[0520] HEK293T Lenti-X™ cell culture was performed as described in Example 2, above. For transfection, the XDP structural plasmids (also encoding the CasX variants) of Table 33 were used in amounts ranging from 13 to 80.0 pg. Each transfection also received 13 pg of pSG50 or pSG5 and 0.25 pg of pGP2. Polyethylenimine (PEI MAX® from Polyplus) was then added as described in Example 2. Plasmid ratios in Table 32 were used in all version 206 XDPs used in this assay, based on prior data. Plasmid sequences are listed in Table 33. XDP version and components incorporated are listed in Table 34.
Table 32: Plasmids and ratios used
Figure imgf000188_0001
Table 33: Plasmid architecture and glycoprotein sequences
Figure imgf000188_0002
Table 34: Version and pseudotyping descriptions
Figure imgf000188_0003
Figure imgf000189_0001
Collection and concentration; resuspension and transduction
[0521] XDPs were collected and concentrated as described in Example 2, above.
[0522] tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above, and tdTomato fluorescence was measured using flow cytometry. tdTomato fluorescence was measured using flow cytometry. The assays were run 2-3 times for each sample, with similar results.
Results:
[0523] Two guide scaffolds, scaffold 250 (FIG. 25) and 251 (FIG. 26), were assayed in the version 206 system. Scaffold 250 had one MS2 hairpin and one RRE, and scaffold 251 had two MS2 hairpins and one RRE. These versions were tested in three different compositions. First was V206 which contains SV40 NLSs on either side of the protein. Second was V206 with NLS 240, which is a stronger NLS than the SV40 in V206. Third was V206 with NLS 255 which had an NLS comparable to NLS 240. The results showed that with the NLS variants, the dual MS2 scaffold 251 performed better than 250, and the opposite was true for V206 with the normal SV40 NLS (FIG. 27). However, as seen in Table 35 and FIG. 27, these scaffolds were able to edit very similarly across all conditions. The potency was measured by inverse EC50, and with no NLS scaffold 250’s inverse EC50 was 1.45 pL'1 and 251’s was 1.01 pL'1. Versions with the NLS scaffold 251 were more potent than versions with the 250 scaffold. For version 206 NLS 240 scaffold 250, the inverse EC50 was 46.25 pL'1 and for scaffold 251 was more than two-fold higher, at 98.33 pL'1.
[0524] The results supported that guide scaffolds with two MS2 hairpins are capable of forming more potent XDP particles compared to guides with a single MS2 hairpin. The results also showed that in some cases, with CasX variants with alternate NLSs, the dual MS2 hairpin scaffolds can be beneficial to potency. This approach is applicable to not just MS2 hairpins but may apply to any RNA hairpin that can be used in CasX recruitment in XDPs such as TAR, Iron Responsive Element, U1 A RNA, phage QP hairpin, phage GA hairpin, phage AN hairpin, Cys4 RNA stem loop, or other element with an RNA that binds protein in a sequence specific interaction with high affinity.
Table 35: Summary of scaffolds and editing results
Figure imgf000190_0001
Example 9: Evaluation of RNA binding partners RRE and MS2
[0525] The purpose of the experiments was to evaluate the utility of the MS2 and RRE systems in constructs to assess their ability to enhance the creation and potency of XDP. Here the generation of XDPs is described in which CasX is recruited into the XDPs by fusing MS2 coat protein to different proteins within the HIV Gag polyprotein and the guide scaffold has one or two MS2 hairpins and portions of the HIV-1 Rev Response Element (e.g., the Rev Binding Element, or RBE).
Methods:
[0526] All plasmids encoding CasX proteins utilized the CasX 491 variant protein. All XDPs were pseudotyped with 10% VSV-G. RNA fold structures were generated with RNAfold web server and Varna java-based software.
Structural plasmid cloning
[0527] In order to generate the structural plasmids used to make the XDP, pXDPl was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments containing CasX, HIV-1, or MS2 CP components were amplified using In Fusion primers with 15-20 base pair overlaps and Kapa HiFi DNA polymerase according to the manufacturer’s protocols. The fragments were purified by gel extraction from a 1% agarose gel using Zymoclean™ Gel DNA Recovery Kit according to the manufacturer’s protocol. These fragments were cloned into plasmid backbones using In-Fusion HD Cloning Kit from Takara according to the manufacturer’s protocols. Assembled products were transformed into chemically-competent Turbo Competent E. coli bacterial cells, plated on LB-Agar plates containing ampicillin and incubated at 37°C. Individual colonies were picked and miniprepped using Qiagen spin Miniprep Kit following the manufacturer’s protocol. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly (see Table 36).
Table 36: Structural sequences
Figure imgf000191_0001
*Backbone of plasmid expresses Rev
Guide plasmid cloning
[0528] The tdTomato targeting guide plasmids used in these experiments were pSG17, pSG72 to pSG76 cloned from non-targeting plasmids pSG14 and pSG67 to pSG71, respectively. The configurations and the sequences of these plasmids and the inserted elements are provided in Tables 36 and 37, respectively (see the listed Figures within the tables showing the sequence and configurations). The mammalian expression backbone contained a cPPT, ampicillin resistance, and a colEI replication site and was amplified using primers with appropriate overlaps to accept the U6 promoter and guide RNA scaffold cassette. The backbone was digested using Ndel and Xbal. Synthetic DNA fragments corresponding to novel scaffolds were amplified and cloned as described in Example 2, above. The resultant plasmids, pSG3 and pSG5, were sequenced using Sanger sequencing to ensure correct assembly (see Table 37 for description of construct).
Table 37: Guide scaffold design
Figure imgf000191_0002
Figure imgf000192_0001
Table 38: Scaffold sequences
Figure imgf000192_0002
Figure imgf000193_0001
Cloning tdTomato spacer 12. 7 into pSG3, pSG14, pSG13, and pSG67 to pSG71
[0529] The targeting spacer sequence DNA for the tdTomato targeting spacer 12.7 was cloned into pSG3, pSG14, pSG13, and pSG67 to pSG71 as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation (see Table 38). pGP2 Glycoprotein plasmid cloning
[0530] Sequences encoding the VSV-G glycoprotein and the CMV promoter were amplified and cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
Cell culture and transfection
[0531] HEK293T Lenti-X™ cell culture was performed as described in Example 2, above. For transfection, the XDP structural plasmids (also encoding the CasX variants) of Table 39 were used in amounts ranging from 13 to 80.0 pg. Each transfection also received 13 pg of a pSG plasmid and 0.25 pg of pGP2. Polyethylenimine (PEI MAX® from Polyplus) was then added as described in Example 2.
Table 39: XDP plasmids
Figure imgf000193_0002
Collection and concentration; resuspension and transduction
[0532] XDPs were collected and concentrated as described in Example 2, above. tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above, and tdTomato fluorescence was measured using flow cytometry. The assays were run 2-3 times for each sample with similar results (see Table 40).
Results:
[0533] The results depicted in FIG. 29, FIG. 30, FIG. 31, and Table 40 demonstrate that the inclusion of RBEs in guide scaffolds 250, 251, and 254 did not significantly decrease the potency of V206 XDPs, so long as the guide scaffold also contains at least one MS2 hairpin. Scaffolds 250 and 254 had EC50s within 2-fold of scaffold 188 (MS2 hairpin only). The results presented in FIG. 30 demonstrate that guide scaffold 251, which has two MS2 hairpins, was only slightly less potent than guide scaffold 188, which could be due to the second MS2 hairpin in this scaffold. This is consistent with the comparison of scaffolds 188 and 251 provided in Example 6 (see FIG. 92).
Table 40: EC50 results of XDP configured with MS2 and RBE
Figure imgf000194_0001
Example 10: Enhancing tropism and editing potency with Vesiculovirus glycoprotein variants
[0534] The purpose of these experiments was to evaluate the ability of diverse glycoprotein variants to enhance tropism for target cells and improve overall editing of the XDP constructs bearing the glycoprotein variants compared to a standard control VSV-G glycoprotein.
[0535] Editing efficiency and specificity can be altered and enhanced with the method of CasX delivery that is employed. Vesicular stomatitis virus envelope glycoprotein (VSV-G) has been widely used to pseudotype viral vectors. However, VSV-G has been shown to be susceptible to human complement inactivation. Experiments were conducted to demonstrate that XDPs (VI 68 with scaffold 226 targeting tdTomato) can be effectively pseudotyped with envelope glycoproteins derived from other species within the Vesiculovirus genus to produce potent particles that can successfully edit target cells. This would offer several advantages: 1) some of these variant glycoproteins maybe relatively resistant to complement inactivation with human serum; 2) some of these variant glycoproteins may exhibit enhanced tropism; and 3) having XDPs pseudotyped with different glycoproteins that are distinct from each other may enable repeated dosing of the therapeutic modality (with different glycoproteins) to circumvent the humoral immune response that could be induced to the previous glycoprotein.
Methods:
[0536] All plasmids containing CasX proteins encoded the CasX 491 protein. Furthermore, the guide RNA scaffold 226 and spacer 12.7, which targets the tdTomato locus, were used in these experiments. RNA fold structures were generated with RNAfold web server and Varna javabased software.
Structural plasmid cloning
[0537] To generate the structural plasmids used to make the XDP, pXDPl was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments containing CasX and HIV-1 components were amplified and cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. The structural plasmids and their sequences are listed in Table 41.
Table 41: Plasmid sequences for structural plasmids and glycoproteins
Figure imgf000195_0001
Figure imgf000196_0001
Guide plasmid cloning
[0538] The guide plasmid used in these experiments was pSG17, which encodes the spacer 12.7 targeting tdTomato incorporated into the guide scaffold 226 that also has the RBE element described in previous examples. To clone the targeting pSG17, spacer 12.7 was cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation. pGP Glycoprotein plasmid cloning
[0539] Encoding sequences for glycoproteins from different species within the Vesiculovirus genus were derived and are provided in Table 41. The designed constructs were synthesized as transgenes and purchased pre-cloned into pTWIST expression plasmids from Twist Biosciences. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
[0540] pGP2 (which serves as the control GP) was cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Cell culture and transfection
[0541] HEK293T Lenti-X™ cell culture was performed as described in Example 2, above. For transfection, the XDP structural plasmids (sequences listed in Table 41) were used in amounts ranging from 13 to 80.0 pg. Each transfection also received 13 pg of p42.174.12.7 and 2.5 pg of pGP2. Polyethylenimine (PEI MAX® from Polyplus) was then added as described in Example 2. Collection and concentration; resuspension and transduction
[0542] XDPs were collected and concentrated as described in Example 2, above.
[0543] tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above, and tdTomato fluorescence was measured using flow cytometry. The assays were run 2-3 times for each sample, with similar results.
Results:
[0544] Percent editing of the dtTomato target sequence in tdT NPCs are shown for all the constructs in FIG. 32 in terms of number of XDP particles used to treat the cells and, in FIG. 33 in terms of the volume of XDPs used to treat the cells. This is broken down further, with the percent editing in tdT NPCs elicited when 0.2 pl and 0.06 pl of the concentrated XDP prep were used to treat NPCs shown in FIG. 34. The EC50 values for the different constructs were calculated and plotted as shown in FIG. 35 and fold-improvements in EC50 over the base control GP (pGP2) are shown in FIG. 37. The data demonstrate that incorporation of 8 different GPs (pGPlOl, pGPlOO, 99, 98, 95, 93, 91 and 88) resulted in between a 2 to 7-fold improvement over the base control GP (pGP2). FIG. 36 shows that the XDPs pseudotyped with different glycoproteins produce to comparable levels with equivalent titers relative to the control construct. These results show that XDPs can be effectively pseudotyped with envelope glycoproteins derived from other species within the Vesiculovirus genus to produce potent particles that can successfully edit the target cell (tdT NPCs). In particular, several glycoproteins, including pGPlOl, pGPlOO, 99, 98, 95, 93, 91 and 88, showed promise for enhanced tropism and editing by the resulting XDP.
[0545] Given that XDPs based on an HIV architecture have been successfully pseudotyped with these variant glycoproteins, it should be possible to use these glycoproteins to pseudotype other versions of XDPs derived from any architectural variants based on components from Alpharetroviral, Betaretroviral, Gammaretroviral, Deltaretroviral, Epsilonretroviral, Lentiviral and Spumaretroviral origin.
Example 11: Enhancing tropism and editing potency with glycoprotein variants for XDP based on lentiviral and Alpharetrovirus constructs
[0546] The purpose of these experiments was to evaluate the ability of diverse glycoprotein variants to enhance tropism for target cells and improve overall editing of XDP based on lentiviral and Alpharetroviral constructs bearing the glycoprotein variants. [0547] Editing efficiency and specificity can be altered and enhanced with the method of CasX delivery that is employed. Vesicular stomatitis virus envelope glycoprotein (VSV-G) has been widely used to pseudotyped viral vectors. However, VSV-G has been shown to be susceptible to human complement inactivation. Experiments were conducted to demonstrate that XDPs derived from lentiviral-based HIV (V168 with scaffold 226 targeting TdTomato) as well as other retroviruses such as ALV (V44 and V102 with scaffold 174 targeting TdTomato) can be effectively pseudotyped with envelope glycoproteins derived from other viral families, including, but not limited to Togaviridae , Paramyxoviridae, Rhabdoviridae, Orthomyxoviridae, Retroviridae and Flaviviridae to produce potent particles that can successfully edit target cells. Methods:
[0548] All plasmids containing CasX proteins encoded the CasX 491 variant protein. RNA fold structures were generated with RNAfold web server and Varna java-based software Structural plasmid cloning
[0549] In order to generate the structural plasmids used to make the XDP, pXDPl was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments containing CasX ALV and HIV-1 components were amplified and cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Plasmids and their sequences are listed in Table 42.
Table 42: Plasmid sequences for structural plasmids and glycoproteins
Figure imgf000198_0001
Figure imgf000199_0001
Figure imgf000200_0001
Guide plasmid cloning
[0550] The guide plasmids used in these experiments were either pSG005 or pSG17. pSG17 has both the spacer 12.7 targeting tdTomato as well as the guide scaffold 226 that has the RRE/RBE element that has been described in previous examples. pSG005 has guide scaffold 174 along with the spacer 12.7 targeting tdTomato. To clone the targeting pSG005 and pSG17 guide plasmids, spacer 12.7 was cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation. pGP Glycoprotein plasmid cloning
[0551] Encoding sequences for glycoproteins derived from Togaviridae, Paramyxoviridae, Rhahdoviridae, Orthomyxoviridae, Retroviridae and Flaviviridae are provided in Table 42. The designed constructs were synthesized as transgenes and purchased pre-cloned into pTWIST expression plasmids from Twist Bioscience. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly (see Table 42).
Cell culture and transfection
[0552] HEK293T Lenti-X™ cell culture was performed as described in Example 2, above. XDPs derived from HIV lentiviral -based architecture (VI 68) were pseudotyped with GPs from Togaviridae (pGP65, 66, 67, 68, 69 and 70), Rhahdoviridae (pGP29.7, 30) and Moloney Murine leukemia virus (pGPIO). XDPs derived from two different alpha retroviral -based architectures (ALV V44 and ALV V102) were pseudotyped with GPs from Rhahdoviridae (pGP29.7). For transfection, the XDP structural plasmids (configurations are listed in Table 42) were used in amounts ranging from 13 to 80.0 pg. Each transfection also received 13 pg of either pSG005 or pSG17 and 2.5 pg of pGP2 or any other GPs. Polyethylenimine (PEI MAX® from Polyplus) was then added as described in Example 2.
Collection and concentration; resuspension and transduction
[0553] XDPs were collected and concentrated as described in Example 2, above.
[0554] tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above, and tdTomato fluorescence was measured using flow cytometry. The assays were run 2-3 times for each sample with similar results.
Results:
[0555] Percent editing of the tdTomato target sequence in tdT NPCs are shown for all XDP constructs derived from HIV (VI 68) as well as XDP constructs derived from ALV (V44 and VI 02) in FIG. 38, in terms of volume of XDPs used to treat the cells. This is broken up further with the percent editing in tdT NPCs elicited when 50 pl and 16 pl of the concentrated XDP preps were used to treat NPCs, as shown in FIG. 39 and FIG. 40, respectively. Percent editing for the VI 68 XDPs pseudotyped with the different GPs, in terms of number of particles added to the tdTomato NPCs, are shown in FIG. 41. VI 68 pseudotyped with pGP2 served as the base control XDP for comparisons. The results show that GPs derived from Togaviridae (in particular Semliki, WEEV, EEEV, VEEV) and Rhabdoviridae (Mokola and Rabies), as well as MoMLV are potent in NPCs, suggesting properties of neural tropism. GPs derived from Togaviridae such as pGP68, pGP68, pGP66 and pGP65 seemed particularly potent (in that order) ranging in editing efficiencies from 74% to 36% when 50 pl of concentrated XDPs were used to treat NPCs. They also show that both architectural versions of ALV derived XDPs (V44 and VI 02) can be pseudotyped with GPs derived from Rhabdoviridae (pGP 29.7), ranging in editing efficacies from 7% to 27% when 50 pl of concentrated XDPs were used to treat NPCs, in addition to VSV-G, where they show efficacies ranging from 39% to 30% as shown in FIG. 40. Titers for the VI 68 XDPs were determined by P24 ELISA, as shown in FIG. 42, and they demonstrate that XDPs can be produced that are pseudotyped with the different glycoproteins without affecting overall titer. The difference in potency that is seen in tdT NPCs is most likely due to inherent differences in cellular and tissue tropism between these glycoproteins. The difference in editing profiles of ALV V44 and ALV102 pseudotyped with Rabies (pGP29.7) also highlights the possibility of the XDP internal architecture having an independent effect on the packaging of the targeting moiety on the surface of these particles. The lack of potencies with particular GPs such as pGP70 and pGP69 as compared to other Togaviridae GPs might be due to incompatibility with the internal architecture, in addition to inherent differences in tropism. Therefore, these GPs might show potency with other architectural variants of HIV based XDPs, in addition to XDPs derived from other architectural variants of Alpharetroviral, Betaretroviral, Gammaretroviral, Deltaretroviral, Epsilonretroviral, Lentiviral and Spumaretroviral origin.
[0556] XDPs derived from HIV lentiviral -based architecture (VI 68) were pseudotyped with GPs from different rabies variants from the Rhabdoviridae family (pGP29, 29.2, 29.3, 29.4, 29.5, 29.6, 29.8). VI 68 pseudotyped with pGP2 served as the base control XDP for comparisons. Several rabies variants showed potency in mouse NPCs, with pGP29 and pGP29.4 showing particular promise with editing efficiencies at the tdTomato locus ranging from 70% to 25% when 16.6 pl of the concentrated XDPs were used to treat NPCs, as shown in FIG. 43 and FIG. 44. VI 68 pseudotyped with pGP2 demonstrated the most efficacy at 85%. However, as compared to pGP2, the rabies variants (pGP29 and pGP29.4) would allow specific targeting of cells of neuronal origin, suggesting a better safety profile in vivo for neural indications, thereby making up for their lower editing potencies relative to VSV-G (pGP2).
[0557] XDPs derived from HIV lentiviral -based architecture (VI 68) were pseudotyped with GPs from Paramyxoviridae (pGP35.1, 35.2, 34.1, 34.2), Orthomyxoviridae (pGP80, 81, 82) and Flaviviridae (pGP25, 26, 27, 28, 75) families. Almost all the GPs showed activity at the 50 pl dose, as shown in FIG. 45. At the second dilution (when 16.6 pl of the concentrated XDPs were used to treat NPCs), XDPs pseudotyped with Orthomyxoviridae (pGP80, 82) and Paramyxoviridae (pGP35.1, 35.2, 34.1, 34.2) demonstrated about 35%, 11% and 10% editing, respectively, as shown in FIG. 46. Titers for the VI 68 XDPs were determined by P24 ELISA as shown in FIG. 47 and demonstrate that pseudotyping XPDs with the different glycoproteins didn’t affect production titers.
[0558] These data support the conclusion that XDPs can be effectively pseudotyped with different glycoproteins derived from diverse viral genera. The differences in potency that were seen in tdT NPCs suggests inherent differences in cellular and tissue tropism properties that exist amongst these glycoproteins. The observed selectively can be harnessed with XDPs designed to safely and selectively deliver the payload to therapeutically-relevant cells. Overall, these results show that XDPs can be engineered to possess selective cell tropism by effectively pseudotyping them with envelope glycoproteins derived from different viral families that retain good editing potency. Given that V168 XDPs have been successfully pseudotyped with these diverse glycoproteins, it should be possible to use these glycoproteins to pseudotype other versions of XDPs derived from any architectural variants of Alpharetroviral, Betaretroviral, Gammaretroviral, Deltaretroviral, Epsilonretroviral, Lentiviral and Spumaretroviral origin.
Example 12: Enhancing RNA export mechanisms for the formation of XDP using a Rev/RRE system - Scaffold 174 vs 226
[0559] The purpose of these experiments was to evaluate the effects of incorporation of a portion of an HIV-1 Rev response element (RRE) sequence into the guide RNA scaffold to determine whether RNA export, recruitment of the guide into XDP, and resultant potency of the XDP was enhanced, with and without a direct Gag-CasX fusion. The HIV-1 RRE is a -350 nucleotide RNA element in the HIV-1 genome that is recognized by the HIV-1 Rev protein and is essential for HIV-1 replication. Early in the HIV-1 replication cycle, REV shuttles the HIV-1 RNA genome out of the nucleus into the cytoplasm by binding to the RRE, RanGTP, and Crml. As described herein, portions of the RRE element were incorporated into the extended stem region of the CasX scaffold 174 To enhance nuclear export of the sgRNAs into the cytoplasm of the XDP -producing LentiX cells. The proposed recruitment mechanism using RRE elements in enhancing nuclear export of the gRNA is depicted in FIG. 51.
Methods:
[0560] All plasmids containing CasX proteins had the CasX variant 491 protein. All XDPs were pseudotyped with 10% VSV-G (percentage of plasmid relative to the other plasmids utilized for the XDP construct). RNA fold structures were generated with RNAfold web server and Varna java-based software.
Structural plasmid cloning
[0561] In order to generate the structural plasmids used to make the XDP, pXDPl was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments containing CasX or HIV-1 Gag components were amplified and cloned as described in Example 2, above. The sequence for Rev was incorporated into the backbone of the Gag plasmid. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
Guide plasmid cloning
[0562] The tdTomato and PTBP-1 targeting guide plasmids used in these experiments were pSG5, pSG17, pSG47, and pSG48 cloned from pSG3 for the first and pSG14 for the latter 3 plasmids. The mammalian expression backbone contained a cPPT, ampicillin resistance, and a colEI replication site and was amplified using primers with appropriate overlaps to accept the U6 promoter and guide RNA scaffold cassette. The backbone was digested using Ndel and Xbal. Synthetic DNA fragments corresponding to novel scaffolds were amplified and cloned as described in Example 2, above. The resultant plasmids, pSG3 and pSG5, were sequenced using Sanger sequencing to ensure correct assembly (see Table 43).
Table 43: Guide plasmids and sequences
Figure imgf000204_0001
Figure imgf000205_0001
Cloning tdTomato spacer 12. 7 into pSG3 and pSG14
[0563] To clone the targeting plasmids from their respective non-targeting plasmids spacers 12.7, 12.2, and 28.10 were cloned using the following protocol. The targeting spacer sequence DNA for the tdTomato targeting spacer 12.7 was ordered as single-stranded DNA (ssDNA) oligos (Integrated DNA Technologies) consisting of the targeting sequence (CTGCATTCTAGTTGTGGTTT, SEQ ID NO: 1018) and the reverse complement of this sequence. The targeting spacer sequence DNA for the tdTomato targeting spacer 12.2 was ordered as single-stranded DNA (ssDNA) oligos (Integrated DNA Technologies) consisting of the targeting sequence (TATAGCATACATTATACGAA, SEQ ID NO: 1541) and the reverse complement of this sequence. The targeting spacer sequence DNA for the PTBP-1 targeting spacer 28.10 was ordered as single-stranded DNA (ssDNA) oligos (Integrated DNA Technologies) consisting of the targeting sequence (CAGCGGGGATCCGACGAGCT, SEQ ID NO: 1542) and the reverse complement of this sequence. For each spacer the two oligos were annealed together and cloned into pSG3 or pSG14 by Golden Gate assembly, as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation. pGP2 Glycoprotein plasmid cloning
[0564] Sequences encoding the VSV-G glycoprotein and the CMV promoter were cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
Cell culture and transfection
[0565] HEK293T Lenti-X™ cell culture was performed as described in Example 2, above. For transfection, the XDP structural plasmids (also encoding the CasX variants) of Table 44 were used in amounts ranging from 13 to 80.0 pg. Each transfection also received 13 pg of p42.174.12.7 and 0.25 pg of pGP2. Polyethylenimine (PEI MAX® from Polyplus) was then added as described in Example 2. The XDP versions, architectures and plasmids utilized in the transfection are listed in Table 45.
Table 44: Architecture and pseudotyping plasmid sequences
Figure imgf000206_0001
* Backbone of plasmid expressed Rev
Table 45: XDP version and pseudotyping descriptions
Figure imgf000206_0002
Figure imgf000207_0001
Collection and concentration; resuspension and transduction
[0566] XDPs were collected and concentrated as described in Example 2, above.
[0567] tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above. Editing of tdTomato was assessed by measuring fluorescence or by Next Generation Sequencing to assess rate of edits. The assays were run 2-3 times for each sample with similar results.
Results:
[0568] The RRE binds strongest to Rev at Stem II (circled in FIG. 48), therefore, this region was incorporated into scaffold 174 (FIG. 49), resulting in scaffold 226, depicted in FIG. 50. Guide scaffold 226 was evaluated using three different spacer sequences; 12.7 (CTGCATTCTAGTTGTGGTTT, SEQ ID NO: 1018), 12.2 (TATAGCATACATTATACGAA, SEQ ID NO: 1541), targeting tdTomato, and 28.10 (CAGCGGGGATCCGACGAGCT, SEQ ID NO: 1542) targeting PTBP-1. Editing using spacers 12.7 and 12.2 were read out using the tdTomato system and 28.10 was analyzed using NGS of the PTBP-1 locus. In each case, XDP incorporating scaffold 226 resulted in 3- to 5-fold greater editing per XDP than XDP incorporating scaffold 174 (Table 46; results presented as the ratio of the EC50 for scaffold 174 to 226).
Table 46: EC50 results from editing assays
Figure imgf000207_0002
Figure imgf000208_0001
[0569] To further interrogate the mechanism of the increases in potency using the RRE/Rev system, three assays were performed. First, it was demonstrated that the increase in potency is Rev-dependent by testing the 226 guide scaffold in the XDP VI and V7 architectures (see Table 45 above). Plasmids in the VI architecture encode the Rev protein whereas the Rev protein is absent in the V7 architecture. FIG. 56 demonstrates that editing with XDP incorporating scaffold 174 or scaffold 226 is very similar in the V7 architecture; scaffold 226 does not increase editing in the Rev-independent V7 construct but does in VI, a Rev-containing architecture.
[0570] Next, efficiency of scaffold 226 in the absence of an additional recruitment system (e.g., Gag-CasX fusion, Gag-MS2, tVSVG-Stx) was assessed. XDP version 207 lacks any architectural recruitment mechanism for CasX to be incorporated into the XDP. XDPs with guide scaffold 174 were unable to edit NPCs in this construct whereas XDPs with scaffold 226 were able to achieve >20% editing (FIG. 57). These data suggested that there may be an orthogonal mechanism of recruitment affected by guide scaffold 226, especially since the increase in editing is greater than was seen in the VI 68 and VI XDPs.
[0571] Lastly, the edits made by XDP with guide scaffold 174 and 226 were assessed to ensure that the nature of edits caused by the RNP was preserved across these two scaffolds. NGS data from samples from the constructs evaluated in FIG. 58 were run through CRISPResso to assess the indel profile. Insertions and deletions were graphed by their frequency on the total read population. Analysis of the data showed that the proportion of insertions and deletions remained similar across the two scaffolds. FIG. 59 shows the data for the calculated EC50 values for the editing experiments.
[0572] The editing data with XDP incorporating guide scaffold 226 demonstrate a consistent pattern of increased potency over XDP incorporating guide scaffold 174. The data show that without changing the nuclease function, the potency of XDPs can be increased by designing constructs that incorporate an RNA nuclear export pathway such as the Rev/RRE system. These enhanced effects were seen across different gene targets and multiple spacers.
[0573] The data demonstrate the utility of incorporating retroviral RNA transport elements into the RNP scaffold to increase potency of XDP particles. Example 13: Evaluation of nuclear import and export systems - NLS Variants +/- RRE (XDP Version 206)
[0574] The purpose of these experiments was to demonstrate the effects of incorporating a variety of nuclear localization signals (NLS) linked to the CasX molecule in the MS2-based recruitment system of XDP version 206. Additionally, experiments were performed to determine if the inclusion of a portion of the HIV-1 rev response element (RRE) or modified portions of the RRE in the sgRNA would increase the potency of these NLS-enhanced constructs in order to determine whether the nuclear export ability of the RRE-Rev system would counteract the effects of the NLSs in the producer cell.
Methods:
[0575] All plasmids encoding CasX proteins utilized the CasX 491 variant protein. All XDPs were pseudotyped with 10% VSV-G (percentage of plasmid relative to the other plasmids utilized for the XDP construct). RNA fold structures were generated with RNAfold web server and Varna java-based software.
Structural plasmid cloning
[0576] In order to generate the structural plasmids used below, pXDPl was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments containing CasX, HIV-1, or MS2 CP components were amplified and cloned as described in Example 2, above. CasX was tested with various combinations of NLS sequences, as summarized in Table 49. Specifically, each CasX-NLS combination had N- and C-terminal NLS made up of various NLS sequences (e.g., c-Myc, SV40, and nucleoplasmid NLS sequences) with various linkers. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
Guide plasmid cloning
[0577] The guide plasmids used in these experiments were pSG50, pSG72, pSG73, and pSG76 which were cloned from non-targeting plasmids pSG33, pSG67, pSG68, and pSG71. The mammalian expression backbone contained a cPPT, ampicillin resistance, and a colEI replication site and was amplified using primers with appropriate overlaps to accept the U6 promoter and guide RNA scaffold cassette. These fragments were amplified and cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly (see Table 47). Table 47: Guide scaffold sequences
Figure imgf000210_0001
Cloning tdTomato spacer 12. 7 into pSG33, pSG67, pSG68, and 71
[0578] The targeting spacer sequence DNA for the tdTomato targeting spacer 12.7 was ordered as single-stranded DNA (ssDNA) oligos consisting of the targeting sequence (CTGCATTCTAGTTGTGGTTT, SEQ ID NO: 1018) and the reverse complement of this sequence. These two oligos were annealed together and cloned into pSG33, pSG67, pSG68, and pSG71 plasmids done by Golden Gate assembly, as described in Example 2, above. The nontargeting (NT) spacer 0.0 (encoded by the sequence CGAGACGTAATTACGTCTCG, SEQ ID NO: 1019) was used as a control and was cloned in a similar manner. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation. pGP2 Glycoprotein plasmid cloning
[0579] Sequences encoding the VSV-G glycoprotein and the CMV promoter were cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly (see Table 50).
Cell culture and transfection
[0580] HEK293T Lenti-X™ cell culture was performed as described in Example 2, above. For transfection, the XDP structural plasmids (also encoding the CasX-NLS variants of Table 49) of Table 48 were used in amounts ranging from 13 to 80.0 pg. Each transfection also received 13 pg of sgRNA plasmid and 0.25 pg of pGP2. Polyethylenimine was then added as described in Example 2, above.
Table 48: Plasmid ratios of V206
Figure imgf000211_0001
*transcript contains RRE and produces REV
Table 49: CasX-NLS plasmids, NLS descriptions, and NLS sequences for each tested NLS
Figure imgf000211_0002
Figure imgf000212_0001
are bolded and italicized (PKKKRKV; SEQ ID NO: 35), and a nucleoplasmin NLS is underlined and italicized (KRPAATKKAGQAKKKK; SEQ ID NO: 36).
Table 50: Architecture and glycoprotein sequences
Figure imgf000212_0002
Figure imgf000213_0001
*backbone of plasmid expresses rev
Collection and concentration; Resuspension and transduction
[0581] XDPs were collected and concentrated as described in Example 2, above.
[0582] tdTomato neural progenitor cells were resuspended and transduced as described in Example 2, above, and tdTomato fluorescence was measured using flow cytometry.
Results:
[0583] The base V206 contains a CasX protein with flanking SV40 NLSs on the N- and C- terminal domains to increase potency by localizing CasX to the nucleus in the target cell. In these assays, nine alternate NLS sequence combinations were tested, termed NLS 115, 240, 247, 248, 251, 252, 255, 256, and 269 (see Table 49). Six of the nine tested NLS combinations performed better than the base SV40 NLS, with the top three variants being NLS 240, 248, and 251. These performed 11-, 10-, and 14-fold better by inverse EC50 by volume than the base V206 SV40 NLS (Table 51).
Table 51: Potency of NLS variants
Figure imgf000213_0002
Figure imgf000214_0001
[0584] Two NLS variants, NLS 240 and 255, were selected to be tested with guide scaffolds that were engineered to contain a portion of the RRE, termed “RBE”. Scaffolds 250, 251, and 254 (FIGS. 25, 26, 28, respectively) each contain one or two RBEs and one or two MS2 hairpins (see Table 53). These scaffolds were compared to scaffold 188 (FIG. 12) which contains one MS2 hairpin and no RBEs. The results of the assay show that scaffolds containing an RBE performed 2- to 6-fold better with NLS 240 and 10- to 23 -fold better with NLS 255 (see Table 52). In both NLS 240 and NLS 255, scaffold 251 performed best with a 6-fold increase with NLS 240 and a 23-fold increase with NLS 255. There was a slight decrease in potency with RBE containing scaffolds in base V206 with the least potent scaffold, scaffold 254, being 40% as potent as scaffold 188 in the base V206 XDP construct.
Table 52: Potency of RRE scaffolds
Figure imgf000214_0002
Table 53: Features of MS2 and RBE containing scaffolds
Figure imgf000215_0001
[0585] These data show that NLS variants can be designed that can increase the potency of the XDP, and that potency can be further enhanced with the use of guide scaffolds with incorporated RBE. These findings support additional efforts to expand the types and combinations of NLS variants and HIV-1 interacting scaffolds in order to further increase the potency of XDP.
Example 14: Enhancing export mechanisms - NLS Variants +/- RRE evaluated in vivo [0586] The purpose of these experiments was to evaluate the effects on in vivo editing potency of the addition of NLS sequences to the N- and/or C-terminal end of CasX and RRE into guide RNA sequences that are incorporated into XDP constructs.
Methods:
[0587] All plasmids containing CasX proteins encoded the CasX 491 variant protein. All XDPs were pseudotyped with 10% VSV-G (percentage of plasmid relative to the other plasmids utilized for the XDP construct). RNA fold structures were generated with RNAfold web server and Varna java-based software.
Structural plasmid cloning
[0588] In order to generate the structural plasmids used below, pXDPl was digested using EcoRI to remove the Gag-pol sequence. Between one and three fragments containing the CasX 491 variant protein with the different NLS constructs as shown in Table 54 and HIV-1 components were amplified and cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. The plasmids with the sequences and corresponding version numbers are listed in Table 55. Table 54: Amino acid sequences of the N- and C-terminal NLS of the encoded CasX 491
Figure imgf000216_0001
Guide plasmid cloning
[0589] The guide plasmids used in these experiments were either pSG005 or pSG17. pSG17 has both the spacer 12.7 targeting tdTomato as well as the scaffold 226 that has the RRE/RBE element that has been described in previous examples. pSG005 has the scaffold 174 along with the spacer 12.7 targeting tdTomato. To clone the targeting pSG005 and pSG17 guide plasmids, spacer 12.7 was cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct ligation. pGP2 Glycoprotein plasmid cloning
[0590] Sequences encoding the VSV-G glycoprotein and the CMV promoter cloned as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
Cell culture and transfection
[0591] HEK293T Lenti-X™ cell culture was performed as described in Example 2, above. Structural plasmid 1 for XDP Version 1 (pXDP17), Version 310 (pXDP240) and Version 311 (pXDP255) all encode for CasX 491 with different NLS. The amino acid sequence of CasX with NLS for pXDP240 is provided in SEQ ID NO: 1631, and the amino acid sequence of CasX with NLS for pXDP255 is provided in SEQ ID NO: 1646. Structural plasmid 2 for all the versions is pXDPOOl. The guide plasmids used in these experiments were either pSG005 or pSG17. pSG17 has both the spacer 12.7 targeting tdTomato as well as the scaffold 226. The plasmid encoding the glycoprotein was pGP2. For transfection, the XDP structural plasmids listed above and in Table 54 were used in amounts ranging from 13 to 80.0 pg. Each transfection also received 13 pg of pSG17 (gRNA) and 0.25 pg of pGP2. Polyethylenimine (PEI MAX® from Polyplus) was then added as described in Example 2.
Table 55: Plasmid sequences for different Gag-CasX NLS constructs as well XDP structural plasmids
Figure imgf000217_0001
Collection and concentration
[0592] XDPs were collected and concentrated as described in Example 2, above. Resuspension and transduction
[0593] The XDP filtered supernatant was divided evenly into an appropriate number of centrifuge tubes or bottles and l/5th of the supernatant volume of sucrose buffer (50mM Tris- HCL, lOOmM NaCl, 10% sucrose, pH 7.4) was underlaid using serological pipettes. The samples were centrifuged at 10,000 x g, 4°C, in a swinging-bucket rotor for 4 hours with no brake. The supernatant was carefully removed and the pellet briefly dried by inverting the centrifuge vessels. Pellets were resuspended in Storage Buffer (PBS + 113 mM NaCl, 15% trehalose dihydrate, pH8) by gentle trituration and vortexing.
Stereotaxic infusion of CasX RNPs in mice and processing of brain tissues
[0594] Adult tdTOM/tdTOM mice were group housed and experiments were conducted in conformance with approved IACUC protocols. Prior to infection, mice were anesthetized with isoflurane. The anesthetized mouse was aligned on an Angle two stereotactic frame (Leica, Germany) and craniotomies were performed by stereotaxic surgery to target the substantia nigra (SN). Mice received a unilateral XDP injection with 8.15xl08 particles of one of the three XDP test articles. Mice were sacrificed 3 weeks post-injection, brains harvested and fixed with 4% PFA and cryosectioned (10 pm thick sections) and mounted on microscope slides. TH+ dopaminergic neurons in the SN were labeled with TH antibody and cell nuclei labeled with DAPI.
Results:
[0595] Here, in vivo gene-editing activity of three XDPs delivered by stereotaxic injection into the mouse brain was measured. CasX RNPs packaged in the XDPs were programmed to edit a STOP cassette between a promoter and tdTomato Red Fluorescent Protein gene that when deleted causes expression of tdTomato protein only in edited cells. Therefore, the presence of tdTomato+ signal visually reports gene editing. TdTomato protein can be visualized using standard fluorescent microscopes without additional signal amplification.
[0596] The XDPs differed in the composition and arrangement of the nuclear localization signals (NLS) on the CasX protein. 8.15xl08 XDPs of each preparation were delivered as determined by the Nanosight physical titering method to the substantia nigra (SN). Tyrosine hydroxylase (TH) antibody staining marks SN dopaminergic neurons. XDP version 1 showed sparse editing activity in astrocytes surrounding the TH+ neurons. Significantly more editing activity was observed (approx. 10 to 100-fold), as determined by the amount of tdTomato+ cells, with XDP versions 310 (pXDP240) and 311 (pXDP255) compared to version 1 (FIG. 60).
Further, there was a dose-dependent increase in editing observed when different doses of XDP version 311 were administered (FIG. 61).
[0597] These data show how engineering the composition and organization of the nuclear localization sequences appended to the N- or C-terminus of CasX protein results in increased in vivo potency of the XDPs. The creation of potent XDPs confers the benefits of lowering the required therapeutic XDP dose to achieve therapeutically-relevant levels of cell editing, increased patient safety, and smaller scale manufacturing processes; factors important for the use of XDPs as therapeutic biologies for gene editing applications in vivo.
Example 15: Improving MS2 hairpin binding affinity enhances XDP editing potency
[0598] In previous examples, editing potency of XDPs was improved using a recruitment strategy in which the gRNA of the CasX:gRNA RNP complex contained a functionalized RNA extended stem region with an MS2 hairpin having high affinity for a Gag-MS2 RNA-binding protein (RBP). Binding of the RNA hairpin to the MS2 RBP facilitates enhanced recruitment of the CasX RNP cargo to the budding XDP particle. Upon delivery of the XDP to the target cell for editing, this RNA hairpin-MS2 RBP is expected to dissociate, allowing CasX to translocate to the nucleus. Thus, increasing the stability of the MS2 protein-RNA complex supports XDP formation, which may be achieved by changing the MS2 RNA-binding protein or RNA hairpin sequences to increase the binding affinity between these components. Accordingly, experiments were conducted to investigate whether gRNAs containing MS2 hairpin variants with improved binding affinity would enhance XDP formation or editing potency. To explore this principle further, multiple MS2 hairpin variants with varying equilibrium binding affinities were assessed for their effects on XDP potency and titer. Several non-binding variants were also included in these experiments.
[0599] gRNAs incorporating RNA hairpin variants with varying affinities for the MS2 RBP were evaluated using a high-throughput, in vitro biochemical assay to assess equilibrium binding and dissociation kinetics (Buenrostro et al., Quantitative analysis of RNA-protein interactions on a massively parallel array reveals biophysical and evolutionary landscapes. Nat Biotechnol. 32(6):562 (2014)). gRNA hairpin variants and their associated Kd (dissociation constant) values are listed in Table 56, sequences of the guide plasmids encoding the different MS2 RNA hairpin variants are provided in Table 57 and the sequences of the MS2 hairpins are provided in Table 58.
Table 56: gRNA scaffolds containing MS2 hairpin variants with varying affinities and their dissociation constant values (Kd). Specific positions for the indicated nucleotide mutations refer to the positions of the base MS2 hairpin (scaffold 188) depicted in FIG. 62
Figure imgf000219_0001
Figure imgf000220_0001
Table 57: Sequences of XDP plasmids
Figure imgf000220_0002
Figure imgf000221_0001
Table 58: MS2 hairpin variant sequences
Figure imgf000221_0002
Figure imgf000222_0001
Materials and Methods:
[0600] All plasmids encoding CasX proteins utilized CasX variant 491. All XDPs were pseudotyped with 10% VSV-G (percentage of VSV-G plasmid relative to other XDP structural plasmids). RNA fold structures were generated with RNAfold web server and VARNA software. The methods to produce XDPs are described herein, as well as in WO2021113772A1, incorporated by reference in its entirety.
Structural plasmid cloning
[0601] Briefly, to generate the XDP structural plasmids, the Gag-pol sequence was removed from pXDPl, and amplified and purified fragments encoding CasX 491, HIV-1, or MS2 CP components were cloned as described in Example 2, above. Individual colonies were picked, miniprepped, and Sanger-sequenced for assembly verification. Plasmid sequences are listed in Table 57.
Guide plasmid cloning
[0602] All guide plasmids containing MS2 RNA hairpin variants (Tables 57 and 58) incorporated the tdTomato targeting spacer 12.7 (CUGCAUUCUAGUUGUGGUUU; SEQ ID NO: 1855). pGP2 glycoprotein plasmid cloning
[0603] Sequences encoding the VSV-G glycoprotein and CMV promoter were cloned as described in Example 2, above.
XDP production
[0604] Briefly, HEK293T Lenti-X™ cells were seeded in 15 cm dishes at 20 x 106 cells per dish 24 hours before transfection to reach 70-90% confluency. The next day, Lenti-X™ cells were transfected with the following plasmids using PEI MAX® (Polypus): XDP structural plasmids (also encoding the CasX variants; Table 57), pSG50 (or other guide plasmid variants listed in Table 57), and pGP2 for XDP pseudotyping. 24 hours post-transfection, media was replaced with Opti-MEM (Thermo Fisher). XDP-containing media was collected 72 hours posttransfection and filtered through a 0.45 pm PES filter. The supernatant was concentrated and purified via centrifugation. XDPs were resuspended in 500 pL of DMEM/F12 supplemented with GlutaMAX™, HEPES, NEAA, Pen/Strep, 2-mercaptoethanol, B-27 without vitamin A, and N2.
[0605] XDP transduction of tdTomato neural progenitor cells (NPCs) was conducted as described in Example 2.
Results:
[0606] XDPs composed of Gag-MS2, Gag-pro, CasX, gRNA scaffold variants, and VSV-G were produced as version 206 either with the original MS2 (MS2 WT) or an MS2 high-affinity variant (MS2 353). Produced XDPs were subsequently assessed for their editing efficiency at the tdTomato locus in NPCs. FIG. 63 shows the percent editing at the tdTomato locus as measured by tdTomato fluorescence using flow cytometry when 0.007 pL of concentrated XDP preps were used to transduce NPCs. In addition to the base control gRNA scaffolds 188 and 251, high- affinity scaffold variants 296 and 298 demonstrated enhanced potency with both MS2 WT and MS2 353, with Kd values ranging from 1.8 to 2.1 nM. Furthermore, medium-affinity scaffold variants 303, 304, 305, 307, 310 and 313, with Kd values ranging from 9.2 to 36.9 nM, resulted in promising editing efficiencies. FIG. 64 illustrates EC50 results across the different gRNA scaffolds incorporating the MS2 WT and MS 353 configurations. Scaffold variants 296, 297, and 305 exhibited a slightly higher potency compared to scaffold 188, an advantage that was more evident with the MS2 353 configuration. FIG. 65 shows a clear correlation between the affinity (Kd) of the gRNA MS2 hairpin and resulting XDP potency (EC50), with an R2 value of 0.81 (p<0.001). XDP comprising MS2 having an affinity of <35nM resulted in efficient recruitment and packaging of the CasX RNP into XDPs. However, there was no correlation observed between the affinity (Kd) of the gRNA MS2 hairpin and resulting XDP titer (FIG. 66).
Example 16: Engineering of XDPs with a cytokine therapeutic payload
[0607] Experiments were performed to demonstrate that XDPs can be used to carry the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) as the therapeutic protein payload. Methods:
Structural plasmid cloning
[0608] In order to generate the structural plasmids used to make the XDPs, mouse or human GMCSF was directly fused to a Gag structural protein, as described in Table 59, below. Cloning was performed as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
Table 59: Configurations of XDPs for carrying GM-CSF
Figure imgf000224_0001
* indicates cleavage sequence between adjacent components
** 5' to 3' orientation f indicates a -1 frame-shift in the encoded construct (Gag-TFR-PR polyprotein)
Cell culture and transfection
[0609] HEK293T Lenti-X™ cell culture was performed as described in Example 2, above. For transfection, the XDP structural plasmids of Table 70 were used in amounts ranging from 13 to 80.0 pg.
Collection and concentration
[0610] XDPs were collected and concentrated as described in Example 2, above. Enzyme-linked immunosorbent assays (ELISAs)
[0611] ELISAs were performed to measure the amount of GM-CSF per XDP. Specifically, XDPs were lysed with the lysis reagent and the number of GM-CSF molecules packaged per XDP was quantified using the Mouse GM-CSF Quantikine® ELISA kit (R&D, Cat no. MGM00) and Human GM-CSF Quantikine® ELISA kit (R&D, Cat no. DGM00) per the manufacturer’s instruction. Results:
[0612] XDPs were engineered to carry human or mouse GM-CSF via the direct fusion of GM- CSF to the protein scaffold, and the amount of GM-CSF per XDP was measured via ELISA. As shown in Table 60, below, the XDPs contained GM-CSF, with between 40-527 molecules of GM-CSF per XDP. The results demonstrate that XDP constructs can be created to incorporate heterologous payloads, and in different configurations.
Table 60: Number and concentration of GM-CSF molecules in XDPs
Figure imgf000225_0001
Example 17: Engineering of XDPs for incorporating catalytically-dead CasX repressor (dXR) system
[0613] Experiments were performed to demonstrate that XDPs can be used to incorporate a catalytically-dead CasX repressor (dXR) system as the therapeutic payload.
Methods:
Structural plasmid cloning
[0614] XDPs were generated using the version 168 or version 206 configuration.
[0615] Cloning was performed as described in Example 2, above. The constructs were designed with sequences coding for catalytically-dead CasX protein 491 (dCasX491; SEQ ID NO: 878) linked to the ZNF10 KRAB domain or the ZIM3 KRAB domain (dXR, see FIG. 86 for a diagram), along with guide RNA scaffold variant 226 or 251, and spacer sequence 7.37 targeted to human B2M 7.37 (GGCCGAGATGTCTCGCTCCG, SEQ ID NO: 1017) or a nontargeting spacer (CGAGACGTAATTACGTCTCG; SEQ ID NO: 1019). The amino acid sequences of the dXR constructs are provided in Table 61, below. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Table 61: Amino acid sequences of dXR constructs
Figure imgf000226_0001
Cell culture and transfection; collection and concentration
[0616] HEK293T Lenti-X™ cell culture was performed as described in Example 2, above. XDPs were collected and concentrated as described in Example 2, above.
Results:
[0617] XDPs were engineered to carry a dXR system targeting the B2M locus for repression. The XDPs were administered to human NPCs, and the level of B2M repression was measured. As shown in FIG. 67, both the version 168 and version 206 XDPs were able to induce repression of B2M. The version 206 XDP with dCasX491 linked to the Zim3 KRAB domain produced the highest level of repression.
[0618] The results of the experiments support that XDPs can be generated carrying functional dXR systems that result in targeted gene repression.
Example 18: Quantification of CasX ribonucleoproteins (RNPs) in XDPs
[0619] Experiments were performed to measure the amount of CasX RNPs incorporated into XDPs.
Methods:
[0620] XDPs were generated using the version 168 configuration with guide scaffold 226, or the version 206 configuration with guide scaffold 251 (see FIG. 26) or guide scaffold 188 (see FIG. 12).
[0621] Cloning was performed as described in Example 2, above. The resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly.
Cell culture and transfection
[0622] HEK293T Lenti-X™ cell culture was performed as described in Example 2, above. Collection and concentration
[0623] XDPs were collected and concentrated as described in Example 2, above. Quantification of CasX RNPs via Western blot analysis
[0624] To determine the number of CasX molecules per XPD particle, a semi-quantitative Western blot analysis was performed using XDP version 206 with guide scaffold 251, XDP version 168 with guide scaffold 226, and version XDP version 206 with guide scaffold 188 (FIG. 68). The protein amount in XPD particles was measured using a Pierce 660 assay. XPD particles were lysed in Laemmli sample buffer and resolved by SDS-PAGE followed by Western blotting using a polyclonal antibody against the CasX protein. The gel also contained a range of purified CasX to establish a standard curve, shown in FIG. 69. The resulting immunoblot was imaged using a ChemiDoc Touch, and the CasX protein levels were quantified by densitometry using Image Lab software from BioRad. Quantification of the CasX molecules in each XDP particle sample was determined using the standard curve.
Results:
[0625] Results of the Western blot analysis demonstrated that XDP version 168 with guide scaffold 226 contained approximately 227-239 CasX molecules/XDP particle (FIG. 70) and, by inference, RNP. The XDP version 206 with guide scaffold 188 contained approximately 240- 257 CasX molecules/XDP particle, and XDP version 206 with guide scaffold 251 contained approximately 966-1112 CasX molecules/XDP particle, showing the superiority of scaffold 251 for facilitating incorporation of RNP into the XDP particles. The fold differences relative to XDP version 168 with guide scaffold 226 are shown in FIG. 71.
Example 19: Evaluation of orthogonal recruitment system with MS2 linked to Gag plus a nuclear export signal (NES) linked to CasX
[0626] The purpose of these experiments was to evaluate whether linking cleavable nuclear export signals (NESs) to CasX in an XDP construct could prevent the sequestration of CasX in the nucleus in packaging cells and promote the packaging of CasX RNPs into XDPs. A potential concern during XDP production is the sequestration of the CasX RNP in the nucleus of the producer cell line as a result of the strong nuclear localization signals on the CasX protein. This possible nuclear sequestration might affect RNP packaging into XDPs and, therefore, XDP editing potency. Therefore, the use of adding cleavable nuclear export signals (NESs) linked to CasX in an XDP construct so as to prevent the sequestration of CasX in the nucleus in packaging cells and promote the packaging of CasX RNPs into XDPs was evaluated. Methods:
[0627] Cleavable NESes were added to the XDP version 206 system (plasmid configurations are shown in Table 62. The NESes were linked to the C-terminus of CasX 676 via an HIV cleavage sequence and a rigid linker.
Table 62: Configurations of version 206 XDPs with or without NESes
Figure imgf000228_0001
* indicates cleavage sequence between adjacent components
** 5' to 3' orientation f indicates a -1 frame-shift in the encoded construct (Gag-TFR-PR polyprotein)
[0628] CRM1 (chromosomal maintenance 1) plays a major role in the export of proteins with leucine-rich nuclear export signals. Nuclear export signals that utilize the CRM1 nuclear export pathway with a range of affinities were selected and attached to the C-terminus of CasX in cleavable manner, such that during the maturation process post-XDP budding, the HIV protease would cleave the NES such that the CasX RNP would not have an attached NES when delivered into the target cell. Specifically, 15 different NESs that use the CRM1 pathway with different Rc/n and Kd values were selected (see Fu, S. et al., Mol Biol Cell. 2018 Aug 15 ;29(17):2037- 2044), and six additional NESs were selected from NESdb, a database of NES-containing CRM1 cargoes (see Xu, D., et al. Mol Biol Cell. 2012 Sep;23(18):3673-6). The amino acid sequences of the nuclear export signals are listed in Table 63, below. Further nuclear export signals have been identified for future testing, and are also listed in Table 63. Table 63: Sequences of nuclear export signals (NESs)
Figure imgf000229_0001
Figure imgf000230_0001
[0629] The XDPs were transduced into human Jurkat T cells or neural progenitor cells (NPCs), and editing of the B2M locus was measured.
Results
[0630] Overall, of the 21 nuclear export signals tested, about 10 showed improvements in editing, which suggests that they improved packaging of CasX RNPs into the XDPs.
Specifically, the nuclear export signals that worked the best in Jurkat and/or NPCs were hRIO2, iKbA, MEK1, P53, Pax, PK1, Rex, Smad4, CPEB4, AD ARI, FMRP and SNUPN (FIGS. 72- 74). Example 20: Screen of XDPs with diverse incorporated viral glycoproteins to evaluate tropism and editing capabilities
[0631] The glycoprotein belonging to VSV Indiana species within the Vesiculovirus genus is usually the most widely used glycoprotein for pseudotyping purposes. The purpose of these experiments was to explore the transduction capabilities of glycoproteins belonging to other species, and test whether the cellular tropism of XDPs could be altered by pseudotyping XDPs with various glycoproteins as targeting moieties in various cell types.
Methods:
[0632] The screen of glycoproteins was conducted in the XDP version 206 construct configuration. The version 206 XDPs pseudotyped with glycoproteins of Table 64 were transduced into mouse tdTomato neural progenitor cells (NPCs), in which editing of the tdTomato locus was measured, or human Jurkat T cells, K562 lymphoblasts, ARPE-19 retinal pigment epithelial (RPE) cells, Y79 retinoblastoma cells, induced neurons, human NPCs, or astrocytes, in which editing of the B2M locus was measured.
[0633] The amino acid sequences of the glycoproteins tested are provided in Table 64, below.
Table 64: Description of glycoproteins tested
Figure imgf000231_0001
Figure imgf000232_0001
The XDPs were designed to contain ribonucleoproteins (RNP) of CasX 676 complexed with single guide RNA variant 251 having spacer sequence 12.7 targeted to tdTomato (encoded by CTGCATTCTAGTTGTGGTTT, SEQ ID NO: 1018) or spacer sequence 7.37 targeted to human B2M (SEQ ID NO: 2448). Utilizing methods described in the sections below, the XDPs were produced by transient transfection of LentiX HEK293T cells (Takara Bio) with two structural plasmids encoding components of the Gag-pol HIV-1 system, a plasmid encoding a pseudotyping glycoprotein, and a plasmid encoding the guide RNA. For the plasmid encoding the guide RNAs, the pStx42 plasmid was created with a human U6 promoter upstream of the guide RNA cassette A plasmid encoding a glycoprotein for pseudotyping the XDP was also used. All plasmids contained either an ampicillin or kanamycin resistance gene, were generated using standard molecular biology techniques, and were sequenced using Sanger sequencing to ensure correct assembly.
Cell culture and transfection
[0634] HEK293T Lenti-X™ cell culture was performed as described in Example 2, above. Collection and concentration
[0635] XDPs were collected and concentrated as described in Example 2, above. Resuspension and transduction
[0636] XDPs were transduced into tdTomato mouse NPCs, human Jurkat T cells, K562 lymphoblasts, ARPE-19 retinal pigment epithelial (RPE) cells, retinoblastoma Y79 cells, induced neurons, human NPCs, or human astrocytes. tdTomato NPCs were resuspended and transduced with XDPs packaged with 12.7 spacer targeting the tdTomato locus as described in Example 2, above.
[0637] Human NPCs were grown in DMEM/ F12 supplemented with GlutaMAX™, HEPES, non-essential amino acids, Pen/Strep, 2-mercaptoethanol, B-27 without vitamin A, and N2. Cells were harvested using StemPro™ Accutase Cell Dissociation Reagent and seeded on PLF coated 96-well plates. Cells were allowed to grow for 24 hours before being treated for targeting XDPs (having a spacer for B2M) starting with a neatly resuspended virus and proceeding through 10 half-log dilutions. Cells were then centrifuged for 15 minutes at 1000 x g. Human NPCs were grown for 96 hours before analysis of B2M editing by flow cytometry. Human astrocytes were similarly treated, where two independent biological replicates were also performed and analyzed (data are shown in FIGS. 77 and 85, where similar findings are observed). Human induced neurons were grown in N2B27-based media. Briefly, to induce neuronal differentiation, iPSCs were plated in neuronal plating media (N2B27 base media with doxycycline, L-ascorbic acid, dibutyryl cAMP sodium salt, CultureOne™, BDNF, and GDNF). After three days of differentiation, induced neurons were seeded on a 96-well plate at -30,000-50,000 cells per well and were cultured for at least one week in plating media and thereafter, half-media changes were performed once every week using feeding media (N2B27 base media with L-ascorbic acid, dibutyryl cAMP sodium salt, BDNF, and GDNF). 30,000 induced neurons were seeded per well of a 96-well plate; 24 hours later, cells were transduced with targeting XDPs (having a B2M- targeting spacer) as described earlier.
[0638] Jurkat cells were grown in RPMI supplemented with FBS. 20,000 cells were transduced with the targeting XDPs (having a spacer for B2M) starting with neat-resuspended virus and proceeding through 10 half-log dilutions. Cells were then centrifuged for 15 minutes at 1000 x g. Jurkats were grown for 96 hours before analysis of B2M editing by flow. The assays were run 2 times for each sample with similar results. K562 lymphoblasts were similarly treated. [0639] ARPE-19 cells were grown in DMEM-F12 supplemented with FBS and Pen/Strep. Y79 cells were grown in RPMI supplemented with FBS. For each cell type, 20,000 cells were transduced with the targeting XDPs (having a spacer for 2M) starting with neat-resuspended virus and proceeding through 10 half-log dilutions. Cells were then centrifuged for 15 minutes at 1000 x g. Jurkats were grown for 96 hours before analysis of B2M editing by flow cytometry.
[0640] tdTomato fluorescence and editing of the B2M locus was measured using flow cytometry. The assays were run 2-3 times for each sample, with similar results.
Results:
[0641] VSV-G-mediated cell entry occurs by binding to the low-density lipoprotein receptor (LDL-R), which is a ubiquitous receptor found on most cell types. Accordingly, the tropism of XDPs pseudotyped with VSV-G is broad. To alter the tropism of XDPs relative to XDPs pseudotyped with VSV-G, XDPs were generated with diverse viral glycoproteins as targeting moieties.
[0642] A comparison of the mouse and human NPC editing data revealed that the XDPs did not edit mouse and human NPCs at the same levels. Specifically, almost all XDPs with vesiculoviral glycoproteins showed a higher level of editing in mouse NPCs (FIG. 75) than they did in human NPCs (FIG. 76). XDPs with alphaviral glycoproteins showed a higher level of editing in human NPCs than in mouse NPCs. Interestingly, XDPs with rabies glycoprotein showed a higher level of editing in mouse NPCs than in human NPCs. Conversely, XDPs with Mokola glycoprotein showed a higher level of editing in human NPCs than in mouse NPCs.
[0643] Additionally, XDPs with certain glycoproteins belonging to the vesiculoviral family (including PERV, YBV, JURV, ISFV, PIRYV, RADV and CHIPV) showed substantially higher levels of editing in human astrocytes (FIGS. 77 and 85) than in either human NPCs (FIG. 76) or human induced neurons (FIG. 83). This finding may be particularly useful to skew XDP tropism towards glial cells rather than neurons, which would be beneficial for glial cell targets.
[0644] Furthermore, ARPE-19 RPE cells and retinoblastoma Y79 cells were treated with XDPs pseudotyped with various glycoproteins, and the editing levels at the B2M locus were assessed. As illustrated in the bar plots shown in FIG. 81 (Y79 cells) and FIG. 84 (ARPE-19 cells), use of nearly the same glycoproteins within the vesiculoviral family (e.g., VSVG, MARV, COCV, VSIV, VSVCENAM, VSVSAM, CARV, ABVV, and MORV) resulted in relatively higher levels of editing compared to the levels resulting from use of other glycoproteins. This finding shows that these glycoproteins exhibit tropism towards retina-derived cells.
[0645] Finally, the level of editing of the B2M locus was measured in Jurkat cells, a human T lymphocyte cell line, as well as in K562 cells, an immortalized myelogenous leukemia cell line. Only XDPs with certain glycoproteins belonging to the vesiculoviral family showed high levels of editing in Jurkat cells (FIG. 78), with minimal editing in Jurkat cells exhibited by XDPs with glycoproteins belonging to lyssaviruses, alphaviruses, paramyxoviruses and retroviruses. In comparison of the effects achieved in XDP -treated Jurkat cells with the editing levels achieved in XDP -treated K562 cells (FIG. 82), use of the VS AV glycoprotein resulted in editing at the B2M locus in Jurkat cells than in K562 cells (compare FIG. 78 with FIG. 82), suggesting that use of VSAV for pseudotyping XDPs confers T lymphocyte tropism.
[0646] The results of the experiments support that viral glycoproteins can be selectively utilized to preferentially confer tropism on cells intended for gene editing.
Example 21: Generation of exemplary version 206 XDPs
[0647] Experiments are conducted to generate exemplary version 206 XDPs having different CasX nucleases and gRNA.
Methods:
Plasmid cloning
[0648] Plasmids encoding CasX proteins are generated to encode the CasX 491 variant, the CasX 515 variant, the CasX 676 variant, or the CasX 812 variant using methods described in previous examples. Plasmids encoding guide scaffold 188 or guide scaffold 251, each with a targeting sequence, are generated using methods described in previous examples. Structural plasmids encoding retroviral components and NCR, guide plasmids, and pGP2 glycoprotein plasmids are cloned as described in Example 2, above. Exemplary DNA sequences of version 206 components are provided in Table 65.
Table 65: DNA sequences of components of version 206 XDPs
Figure imgf000235_0001
f indicates a -1 frame-shift in the encoded construct (Gag-TFR-PR polyprotein)
Cell culture and transfection
[0649] HEK293T Lenti-X™ cell culture is performed as described in Example 2, above, using the five plasmids of Table 65, selecting either CasX 491, 515, 676, or 812 and guide 188 or 251. Collection and concentration
[0650] XDPs are collected and concentrated as described in Example 2, above.
[0651] This process is expected to generate version 206 XDPs with either CasX 491, CasX 515, CasX 676, or CasX 812 and guide scaffold 188 or guide scaffold 251, complexed as RNP as the therapeutic payload, which are then evaluated for editing of target nucleic acid.
Example 22: Demonstration of dual-editing at two different genomic loci using two types of CasX RNPs packaged and delivered via a single XDP particle in vitro
[0652] Experiments were performed to demonstrate the ability to encode, package and deliver two types of CasX RNPs within a single XDP particle for targeted editing at two different genomic loci. Here, XDP particles were generated to contain a CasX protein with gRNAs targeting the PTBP1 locus and the tdTomato STOP cassette and used to transduce mouse tdTomato neural progenitor cells (NPCs) to demonstrate editing at the two genomic loci in vitro. Materials and Methods:
XDP construct cloning
[0653] Two XDP configurations were used to generate XDPs in these experiments. Specifically, VI 68 XDPs were produced incorporating guide scaffold 226, while V206 XDPs were produced incorporating guide scaffold 251. XDPs were engineered to package two types of RNPs within a single XDP: CasX variant 491 complexed with a PTBP1 -targeting gRNA and CasX variant 491 complexed with a tt/Zomato-targeting gRNA. All XDP particles were pseudotyped with the VSV-G glycoprotein.
[0654] XDP structural plasmid cloning was performed as described in Example 2. XDP production using HEK293T Lenti-X™ cells was performed as described in Example 2. Briefly, adherent Lenti-X™ cells were seeded in 15cm plates at 2E7 cells per plate in 20mL of media. 24 hours later, cells were transfected with the following plasmids using PEI MAX® (Polypus): XDP structural plasmids encoding the HIV-1 Gag-pol structural components (as well as CasX 491 for VI 68), a plasmid encoding for CasX 491 (relevant for V206), a plasmid encoding a single gRNA with either scaffold 226 (for VI 68 XDPs) or scaffold 251 (for V206 XDPs) and PTSTV-targeting spacer 28.10 (CAGCGGGGAUCCGACGAGCU; SEQ ID NO: 982), a plasmid encoding a single gRNA with either scaffold 226 or 251 and tt/Zomato-targeting spacer 12.7 (CUGCAUUCUAGUUGUGGUUU; SEQ ID NO: 1855), and a plasmid encoding the VSV-G glycoprotein. 72 hours post-transfection, XDP-containing media was collected and filtered through a 0.45 pm PES filter. The supernatant was concentrated and purified via centrifugation. XDPs were resuspended in a freezing buffer. As experimental controls, XDPs containing dual-CasX RNPs using spacer 28.10 with a non-targeting (NT) spacer or dual-CasX RNPs using spacer 12.7 with an NT spacer were also produced and assessed for editing. [0655] XDP transduction of tdTomato NPCs was performed as described in Example 2. Editing at the tdTomato locus was assessed by analyzing tdTomato fluorescence detected by flow cytometry, while editing at the PTBP1 locus was assessed as indel rate detected by NGS using methods as described in Example 2.
Results:
[0656] VI 68 XDPs were produced to achieve packaging of two types of CasX RNPs within a single XDP. Specifically, V168 XDPs contained either 1) RNPs of CasX 491 complexed with a ^/Tomato-targeting gRNA and CasX 491 complexed with a /J 7
Figure imgf000237_0001
-targeting gRNA (VI 68 12.7-28.10), or 2) RNPs of CasX 491 complexed with a /t/Zb/iia/o-targeting gRNA and CasX 491 complexed with a non-targeting gRNA (V168 12.7-NT). Produced V168 XDPs were subsequently assessed for their editing efficiency at the tdTomato locus or PTBP1 locus in mNPCs, and the results are illustrated in FIG. 79. The data demonstrate that VI 68 XDPs containing two types of CasX RNPs were able to achieve dose-dependent editing at both the tdTomato and PTBP1 loci when delivered into mNPCs. The data further suggest that similar levels of editing were achieved at both loci at the indicated volumes of XDP application.
[0657] Similarly, V206 XDPs were produced to achieve dual-CasX RNP packaging: 1) RNPs of CasX 491 complexed with a /t/Z /iia/o-targeting gRNA and CasX 491 complexed with a 7J 777/J 7 -targeting gRNA (V206 12.7-28.10); 2) RNPs of CasX 491 complexed with a tdTomato- targeting gRNA and CasX 491 complexed with a non-targeting gRNA (V206 12.7-NT); or 3) RNPs of CasX 491 complexed with a 7J 77>7J 7 -targeting gRNA and CasX 491 complexed with a non-targeting gRNA (V206 28.10-NT). Produced V206 XDPs were subsequently assessed for their editing efficiency at the tdTomato locus or PTBP1 locus in mNPCs, and the results are illustrated in FIG. 80. The data demonstrate that V206 XDPs packaging CasX RNPs for dualtargeting were able to induce similar levels of dose-dependent editing at both the tdTomato and PTBP1 loci when delivered into mNPCs. In addition, the data demonstrate that editing efficiency achieved by V206 XDPs containing CasX RNPs with either spacer 28.10 or 12.7 and a nontargeting (NT) spacer did not affect editing levels at either of the two targeted loci.
[0658] The results from these experiments show that XDPs with different configurations can be engineered to package two types of CasX RNPs (i.e., CasX is complexed with two different gRNAs targeting different sequences) within a single XDP particle to achieve editing at two different genomic loci. Furthermore, while the experiments here utilized two separate plasmids to express the two different gRNAs for targeting, future experiments will use a single plasmid for dual-gRNA expression. These findings also justify additional studies to investigate in vivo editing after delivering XDPs containing CasX RNPs for dual-targeting of different genes. Demonstrating the potential to use XDPs to induce editing at multiple genomic loci offers a therapeutic opportunity to address polygenic diseases.
Example 23: Engineering of XDPs for carrying catalytically-dead CasX repressor (dXR) or epigenetic long-term CasX repressor (ELXR) systems
[0659] Experiments were performed to test whether XDPs can be used to incorporate catalytically-dead CasX repressor systems as the therapeutic payload.
Materials and Methods:
Description of XDPs and CasX constructs tested
[0660] XDP configuration versions 168 and 206 were generated with various CasX and repressor protein constructs. Table 66, below, summarizes the plasmids used to encode the components of the version 168 and 206 XDP systems. In version 168 XDP systems, the dXR with a single repressor domain or with an ELXR protein (see FIGS. 87-88 for diagrams of ELXR proteins) containing multiple repressor domains is fused to the HIV Gag polyprotein. In contrast, in version 206 XDPs, an MS2-based non-covalent recruitment (NCR) system is used in which an MS2 coat protein is fused to the HIV Gag polyprotein and an MS2 hairpin is incorporated into the guide RNA, which is used to non-covalently recruit the RNP into the XDP during its formation.
Table 66: Summary of version 168 and 206 XDPs with dXR, ELXR, or CasX
Figure imgf000238_0001
Figure imgf000239_0001
* indicates cleavage sequence between adjacent components f indicates a -1 frame-shift in the encoded construct (Gag-TFR-PR polyprotein)
[0661] The fusion proteins of the dXR constructs were made up of, from N- to C-terminus, a catalytically-dead CasX 491, and a ZNF10 or ZIM3 KRAB domain (see FIG. 86; “RD1” is “Repressor Domain 1” and denotes the KRAB domain of interest). The fusion proteins of the ELXR configuration #1 constructs were made up of, from N- to C-terminus, a catalytic domain from DNMT3A, an interaction domain from DNMT3L, a catalytically-dead CasX 491, and a ZNF10 or ZIM3 KRAB domain (see ELXR configuration #1 in FIG. 87), along with amino acid linkers and NLS sequences. Catalytically-active CasX 491 (herein termed “CasX”; SEQ ID NO: 189) was also included as a control.
[0662] The DNA and protein sequences of the components of the dXR and ELXR configuration #1 constructs are provided in Table 67, below. The ELXR constructs also contained a 2x FLAG tag.
Table 67: DNA and protein sequences of components of dXR and ELXR
Figure imgf000239_0002
Figure imgf000240_0001
Figure imgf000241_0001
Figure imgf000242_0001
Figure imgf000243_0001
[0663] Guide RNA scaffold variant 226 was used with the version 168 XDPs, and guide scaffold variant 251 was used with the version 206 XDPs. The RNA sequences of the guide scaffolds are provided in Table 6. Sequences of spacers 7.37 targeted to human B2M and a nontargeting spacer are provided in Table 68. All resultant plasmids were sequenced using Sanger sequencing to ensure correct assembly. Table 68: Sequences of spacers used in constructs
Figure imgf000244_0001
[0664] Cell culture and transfection, collection and concentration of XDPs, and resuspension and transduction of XDPs is performed as described in Example 2, above.
[0665] Cells were grown for six (see FIG. 89) or 14 (see FIG. 90) days before analysis of fluorescence as a marker of repression of the B2M locus, as measured using flow cytometry. FIGS. 89 and 90 show repression of the B2M locus in cells treated with 50 pL of XDPs.
Results:
[0666] Version 168 or 206 XDPs were engineered to carry the dXR molecule with the single repressor domain or the ELXR molecule having configuration #1 for targeting the B2M locus for repression. As shown in FIG. 89, six days following XDP administration, only version 168 XDPs carrying the dXR system with a ZNF10 KRAB domain repressed B2M. Meanwhile, version 206 XDPs carrying the dXR with a ZNF10 KRAB domain, dXR with a ZIM3 KRAB domain, and ELXR having configuration #1 with a ZIM3 KRAB domain all repressed the B2M locus.
[0667] As shown in FIG. 90, 14 days following XDP administration, most of the dXR and ELXR configuration #1 systems had lost B2M repression. This was in contrast to the XDPs with catalytically-active CasX 491, which achieved long-term repression of the B2M locus via editing of the locus. Notably, the version 206 XDPs with an ELXR configuration #1 system with a ZIM3 KRAB domain showed a two-fold reduction at day 14 as compared to day six (FIG. 89) and retained the most repression activity as compared to the other dXRs and ELXRs tested.
[0668] Accordingly, XDPs were able to carry either the dXR or ELXR configuration #1 systems as therapeutic payloads and achieve transcriptional repression of a target locus.
Example 24: Engineering of XDPs for carrying ELXR systems
[0669] XDPs are generated with ELXR configuration #1, #4, or #5 molecules as the payload (see FIGS. 87 and 88 for diagrams of the configurations).
Materials and Methods:
Description of XDPs and CasX constructs tested [0670] XDP configuration versions 168 and 206 are generated with various ELXR molecules. Table 66, above, summarizes the plasmids used to encode the components of the version 168 and 206 XDP systems.
[0671] ELXR molecules in configurations #1, #4, and #5, which contain a catalytically- inactive CasX 491, are tested, as diagrammed in FIG. 88. Table 69 provides amino acid sequences of configurations #1, #4, and #5 ELXR molecules, showing the sequences of the components of the proteins from N- to C-terminus in the table. The repressor domain 1 shown in Table 69 (“RDl”in FIG. 88) may be a repressor domain from the species Columba livia, Rattus norvegicus, Cebus imitator, chimpanzee, Chlorocebus sabaeus, Ophiophagus hannah, Ailuropoda melanoleuca, Peromyscus maniculatus bairdii, or Phyllostomus discolor, in place of the human ZNF10 or ZIM3 KRAB domains that were tested in Example 23. Other catalytically- inactive CasX variants can be used in place of catalytically-inactive CasX 491; these variants are listed in Table 4.
Table 69: Amino acid sequences of ELXR configuration #1, #4, and #5 molecules
Figure imgf000245_0001
Figure imgf000246_0001
Figure imgf000247_0001
Figure imgf000248_0001
Figure imgf000249_0001
Figure imgf000250_0001
[0672] Guide RNA scaffold variant 226 is used with the version 168 XDPs, and guide scaffold variant 251 is used with the version 206 XDPs. The RNA sequences of the guide scaffolds are provided in Table 6. Sequences of spacers 7.37 targeted to human B2M and a non-targeting spacer control are provided in Table 68, above. All resultant plasmids are sequenced using Sanger sequencing to ensure correct assembly.
[0673] Cell culture and transfection, collection and concentration of XDPs, and resuspension and transduction of XDPs is performed as described in Example 2, above. Repression of the B2Mlocus is assessed at 7 days and 14 days, when cells are harvested for analysis of HLA immunostaining as detected using flow cytometry, to demonstrate the ability of the constructs to deliver the therapeutic payload and repress expression of B2M.
Example 25: Evaluation of non-covalent recruitment (NCR) systems with protein-ligand pairs attached to Gag and protein cargo
[0674] Experiments were performed to evaluate a protein-based NCR recruitment system for packaging cargo in XDPs.
Materials and Methods:
[0675] XDPs were generated in which an NCR protein was fused to the Gag polyprotein, and a ligand for the NCR protein was fused to the cargo of the XDP, i.e., to the N-terminus of CasX. Table 70, below, summarizes the plasmids used to encode the components of these XDP systems.
Table 70: Summary of version XDPs with protein recruitment of CasX
Figure imgf000251_0001
* indicates cleavage sequence between adjacent components f indicates a -1 frame-shift in the encoded construct (Gag-TFR-PR polyprotein)
[0676] The protein-ligand pairs that were tested are provided in Table 71 (each row is a protein-ligand pair). Some of the listed protein-ligand pairs were split fluorescent proteins that were anticipated to emit fluorescence when the two portions of the split protein are bound, such as mNeon-Green and sfCherry. Table 72 provides the amino acid sequences of the NCR proteins, and Table 73 provides the amino acid sequences of the ligands.
Table 71: Protein-ligand pairs for protein-based recruitment to XDPs
Figure imgf000251_0002
Figure imgf000252_0001
Table 72: Amino acid sequences of proteins fused to Gag
Figure imgf000252_0002
Figure imgf000253_0001
Figure imgf000254_0001
Table 73: Amino acid sequences of ligands fused to CasX
Figure imgf000254_0002
Figure imgf000255_0001
[0677] Guide scaffold 226 was used (SEQ ID NO: 2380), with the 12.7 spacer for targeting the tdTomato locus (CUGCAUUCUAGUUGUGGUUU, SEQ ID NO: 1855).
[0678] Cell culture and transfection, collection and concentration of XDPs, and resuspension and transduction of XDPs was performed as described in Example 2, above. tdTomato fluorescence was measured using flow cytometry. NPCs transduced with split fluorescent protein NCR systems were imaged by fluorescence microscopy 24 hours following transduction. Version 206 XDPs using RNA recruitment and targeting tdTomato were included as a control. Results:
[0679] XDPs were generated using an NCR system based on the binding of a protein-ligand pair in which the protein was fused to the C-terminus of the Gag polyprotein and the ligand was fused to the N-terminus of CasX. As shown in FIG. 100, XDPs with each of the protein-ligand pairs tested produced editing of the tdTomato locus. Indeed, for many of the XDPs with proteinligand pairs, as well as for version 206 XDPs, editing levels were near 100%. This was true even at the lowest volumes of XDPs administered to the cells. Accordingly, editing levels were likely saturated in this assay.
[0680] NPCs transduced with split fluorescent protein NCR systems were imaged for red fluorescence (indicating editing of the tdTomato locus) and green fluorescence (indicating binding of the two portions of the split fluorescent protein). The three split mNeon-Green NCR systems (mNG2i-io+mNGl 1, mNG3Ai-io+mNG3n, and mNG3Ki-io+mNG3n) and the split CloGFP system (CloGFPi-io+CloGFPn) each produced cells with red and green fluorescence, indicating both editing and the presence of reconstituted split fluorescent proteins (data not shown). The green fluorescence was often found in puncta, which is believed to indicate nuclear localization of the CasX:gRNA RNP along with the bound fluorescent protein. As controls, cells transduced with version 206 XDPs, and CL7+IM7 and sfCherry2i-io+sfCherry2n protein NCR systems were also examined and were found to show red fluorescence (indicating editing of the tdTomato locus) but not green fluorescence.
[0681] The results described herein demonstrate that the XDPs with protein-ligand pairs facilitate recruitment of CasX (and, hence, the complexed RNP) into the XDP particles, and are able to deliver RNPs to cells and produce genome editing of the target nucleic acid.

Claims

Attorney Docket No. SCRB-050/01WO 333322-2386 CLAIMS What is claimed is: 1. A delivery particle (XDP) system comprising one or more nucleic acids encoding components comprising: (a) a retroviral Gag polyprotein fused to a non-covalent recruitment (NCR) protein or a functional domain thereof, wherein the NCR protein is a protein-binding protein and the ligand is a protein ligand, wherein the NCR has binding affinity for the ligand; (b) a retroviral Gag-transframe region protease polyprotein (Gag-TFR-PR); (c) a therapeutic payload comprising a ligand capable of binding the NCR protein; and (d) a tropism factor. 2. The XDP system of claim 1, wherein the NCR protein is fused to the C-terminus of the Gag polyprotein. 3. The XDP system of claim 1, wherein the protein-binding protein comprises: (a) a Protein A and the ligand comprises an Fc region; (b) a truncated Protein A and the ligand comprises an Fc region; (c) a CL7 protein and the ligand comprises an IM7 ligand; (d) a NbALFA protein and the ligand comprises an ALFA tag; (e) a SpyCatcher protein and the ligand comprises a SpyTag; (f) a SpyCatcher002 protein and the ligand comprises a SpyTag002; (g) a SpyCatcher003 protein and the ligand comprises a SpyTag003; (h) a Strep-Tactin protein and the ligand comprises a Twin Strep tag II; (i) a Strep-Tactin protein and the ligand comprises a Strep tag II; (j) an Avidin protein and the ligand comprises an Avi tag; (k) a mNG21-10 protein and the ligand comprises a mNG11 ligand; (l) a sfCherry21-10 protein and the ligand comprises a sfCherry211 ligand; (m) a mNG3A1-10 protein and the ligand comprises a mNG11 ligand; (n) a mNG3k1-10 protein and the ligand comprises a mNG11 ligand; (o) a sfGFP1-10 protein and the ligand comprises a GFP11 ligand; (p) a mClover31-10 protein and the ligand comprises a mClover311 ligand; (q) a CloGFP0.21-10 protein and the ligand comprises a GFP11 ligand; or 255 295377236 Attorney Docket No. SCRB-050/01WO 333322-2386 (r) a CloGFP1-10 protein and the ligand comprises a GFP11 ligand. 4. The XDP system of claim 3, wherein: (a) the Protein A comprises a sequence of SEQ ID NO: 2450, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Fc region, and the Fc region comprises a sequence of SEQ ID NO: 2468, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Protein A; (b) the truncated Protein A comprises a sequence of SEQ ID NO: 2451, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the Fc region, and the Fc region comprises a sequence of SEQ ID NO: 2468, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the truncated Protein A; (c) the CL7 protein comprises a sequence of SEQ ID NO: 2452, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the IM7 ligand, and the IM7 ligand comprises a sequence of SEQ ID NO: 2469, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the CL7 protein; (d) the NbALFA protein comprises a sequence of SEQ ID NO: 2462, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the ALFA tag, and the ALFA tag comprises a sequence of SEQ ID NO: 2476, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 295377236 Attorney Docket No. SCRB-050/01WO 333322-2386 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the NbALFA protein; (e) the SpyCatcher protein comprises a sequence of SEQ ID NO: 2463, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the SpyTag, and the SpyTag comprises a sequence of SEQ ID NO: 2477, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the SpyCatcher protein; (f) the SpyCatcher002 protein comprises a sequence of SEQ ID NO: 2461 or 2464, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the SpyTag002, and the SpyTag002 comprises a sequence of SEQ ID NO: 2475, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the SpyCatcher002 protein; (g) the SpyCatcher003 protein comprises a sequence of SEQ ID NO: 2465, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the SpyTag003, and the SpyTag003 comprises a sequence of SEQ ID NO: 2478, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the SpyCatcher003 protein; (h) the Strep-Tactin protein comprises a sequence of SEQ ID NO: 2466, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the Twin Strep tag II, and the Twin Strep tag II comprises a sequence of SEQ ID NO: 2479, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 295377236 Attorney Docket No. SCRB-050/01WO 333322-2386 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Strep-Tactin protein; (i) the Strep-Tactin protein comprises a sequence of SEQ ID NO: 2466, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the Strep tag II, and the Strep tag II comprises a sequence of SEQ ID NO: 2480, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Strep-Tactin protein; (j) the Avidin protein comprises a sequence of SEQ ID NO: 2467, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the Avi tag, and the Avi tag comprises a sequence of SEQ ID NO: 2481, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Avidin protein; (k) the mNG21-10 protein comprises a sequence of SEQ ID NO: 2453, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the mNG11 ligand, and the mNG11 ligand comprises a sequence of SEQ ID NO: 2470, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mNG21-10 protein; (l) the sfCherry21-10 protein comprises a sequence of SEQ ID NO: 2454, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the sfCherry211 ligand, and the sfCherry211 ligand comprises a sequence of SEQ ID NO: 2471, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 295377236 Attorney Docket No. SCRB-050/01WO 333322-2386 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the sfCherry21-10 protein; (m) the mNG3A1-10 protein comprises a sequence of SEQ ID NO: 2455, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the mNG11 ligand, and the mNG11 ligand comprises a sequence of SEQ ID NO: 2470, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mNG3A1-10 protein; (n) the mNG3k1-10 protein comprises a sequence of SEQ ID NO: 2456, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the mNG11 ligand, and the mNG11 ligand comprises a sequence of SEQ ID NO: 2470, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mNG3k1-10 protein; (o) the sfGFP1-10 protein comprises a sequence of SEQ ID NO: 2457, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the GFP11 ligand, and the GFP11 ligand comprises a sequence of SEQ ID NO: 2474, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the sfGFP1-10 protein; (p) the mClover31-10 protein comprises a sequence of SEQ ID NO: 2458, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the mClover311 ligand, and the mClover311 ligand comprises a sequence of SEQ ID NO: 2473, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 295377236 Attorney Docket No. SCRB-050/01WO 333322-2386 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mClover31-10 protein; (q) the CloGFP0.21-10 protein comprises a sequence of SEQ ID NO: 2459, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the GFP11 ligand, and the GFP11 ligand comprises a sequence of SEQ ID NO: 2474, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the CloGFP0.21-10 protein; or (r) the CloGFP1-10 protein comprises a sequence of SEQ ID NO: 2460, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding the GFP11 ligand, and the GFP11 ligand comprises a sequence of SEQ ID NO: 2474, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the CloGFP1-10 protein. 5. The XDP system of claim 1, wherein the encoded therapeutic payload comprises a protein, a nucleic acid, or both a protein and a nucleic acid. 6. The XDP system of claim 5, wherein the nucleic acid comprises a first, and optionally a second gRNA selected from the group consisting of a Class 2 Type II, a Class 2 Type V, and a Class 2 Type VI CRISPR system gRNA. 7. The XDP system of claim 6, wherein the first, and optionally the second gRNA is a Class 2 Type II CRISPR system gRNA or a Class 2 Type V CRISPR system gRNA. 8. The XDP system of claim 6, wherein the first, and optionally the second gRNA is a single-molecule guide RNA (sgRNA) comprising a scaffold sequence and a targeting sequence, wherein the targeting sequence comprises between 15 and 20 nucleotides and is complementary to a target nucleic acid sequence. 9. The XDP system of claim 8, wherein the first, and optionally the second gRNA scaffold sequence comprises a sequence selected from the group consisting of the sequences of SEQ ID NOS: 2101-2430, and 4106, or a sequence having at least about 70%, at least about 75%, at least 260 295377236 Attorney Docket No. SCRB-050/01WO 333322-2386 about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto, wherein the gRNA is capable of complexing with a CRISPR protein to form a ribonucleoprotein (RNP). 10. The XDP system of claim 8, wherein the first, and optionally the second gRNA scaffold sequence comprises a sequence selected from the group consisting of the sequences of SEQ ID NOS: 2101-2430, and 4106. 11. The XDP system of claim 10, wherein the first, and optionally the second gRNA scaffold sequence comprises a sequence selected from the group consisting of the sequences of SEQ ID NOS: 2238, 2292, 2380, and 4106. 12. The XDP system of claim 8, wherein the first, and optionally the second gRNA is a CasX gRNA comprising an extended stem loop comprising the RNA hairpin. 13. The XDP system of claim 12, wherein the first, and optionally the second gRNA scaffold sequence comprises a sequence selected from the group consisting of the sequences of SEQ ID NOS: 2249, 2308, 2312, 2314-2317, 2319, 2380, and 2417-2429, or a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto, wherein the gRNA is capable of complexing with a CRISPR protein to form a ribonucleoprotein (RNP). 14. The XDP system of claim 8, wherein the first, and optionally the second gRNA comprises a scaffold comprising one or more binding partner elements selected from the group consisting of: i) a Stem IIB of Rev response element (RRE; SEQ ID NO: 569), ii) a Stem II-V of RRE (SEQ ID NO: 571), iii) a Stem II of RRE (SEQ ID NO: 570), iv) a Rev-binding element (RBE) of Stem IIB (SEQ ID NO: 565), and v) and a full-length RRE (SEQ ID NO: 572), wherein the one or more binding partner elements are capable of binding Rev protein. 15. The XDP system of claim 8, comprising a second gRNA scaffold sequence identical to the first gRNA scaffold sequence, and a targeting sequence complementary to a different region 295377236 Attorney Docket No. SCRB-050/01WO 333322-2386 of the target nucleic acid, wherein the second gRNA is capable of complexing with a CRISPR protein to form a ribonucleoprotein (RNP). 16. The XDP system of claim 1, wherein the encoded therapeutic payload comprises a protein payload selected from the group consisting of a cytokine, an interleukin, an enzyme, a receptor, a microprotein, a hormone, erythropoietin, a ribonuclease (RNase), a deoxyribonuclease (DNase), a blood clotting factor, an anticoagulant, a bone morphogenetic protein, an engineered protein scaffold, a thrombolytic protein, a CRISPR protein, granulocyte- macrophage colony-stimulating factor (GMCSF), a transcription factor, a repressor domain, a transposon, a reverse transcriptase, a viral interferon antagonist, a tick protein, and an anti- cancer biologic. 17. The XDP system of claim 1, wherein the encoded therapeutic payload comprises a CRISPR protein. 18. The XDP system of claim 17, wherein the CRISPR protein is a Class 2 CRISPR protein selected from the group consisting of a Class 2 Type II, a Class 2 Type V, or a Class 2 Type VI CRISPR protein. 19. The XDP system of claim 18, wherein the Class 2 Type II CRISPR protein is a Cas9 protein. 20. The XDP system of claim 18, wherein the Class 2 Type V CRISPR protein selected from the group consisting of Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12f, Cas12g, Cas12h, Cas12i, Cas12j, Cas12k, Cas14, and CasΦ. 21. The XDP system of claim 20, wherein the CasX is a CasX variant comprising a sequence selected from the group consisting of SEQ ID NOS: 135-169, 181-320, 322-366, 368-457, 797- 804, 806-829, 831, 832, 834-842, 937, 938, 940, or 942, or a sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto, wherein the CasX variant retains the ability to form an RNP with a gRNA and retains nuclease activity. 22. The XDP system of claim 21, wherein the CasX variant comprises a sequence selected from the group consisting of SEQ ID NOS: 189, 196, 354, and 813, or a sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, 295377236 Attorney Docket No. SCRB-050/01WO 333322-2386 or at least about 99% sequence identity thereto, wherein the CasX variant retains the ability to form an RNP with a gRNA and retains nuclease activity. 23. The XDP system of claim 21, wherein the CasX variant comprises a sequence selected from the group consisting of SEQ ID NOS: 189, 196, 354, and 813. 24. The XDP system of any one of claims 17-23, wherein the CRISPR protein comprises a nuclear localization signal (NLS). 25. The XDP system of claim 24, wherein the NLS comprises a sequence selected from the group consisting of SEQ ID NOs: 35, 37, 1740, 1750, 4128, and 4129. 26. The XDP system of claim 17, wherein the CRISPR protein is a catalytically-dead CasX variant (dCasX) comprising a sequence selected from the group consisting of SEQ ID NOS: 877-896 and 4112-4117, or a sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto, wherein the dCasX retains the ability to form an RNP with a gRNA. 27. The XDP system of claim 26, wherein the dCasX comprises a sequence selected from the group consisting of SEQ ID NOS: 877-896 and 4112-4117, and wherein the dCasX retains the ability to form an RNP with a gRNA. 28. The XDP system of claim 27, wherein the dCasX comprises the sequence of SEQ ID NO: 878. 29. The XDP system of claim 27 or claim 28, wherein the dCasX is linked to at least a first repressor domain (RD1) as a fusion protein, wherein the fusion protein is capable of reducing expression of the target nucleic acid by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. 30. The XDP system of claim 1, wherein the encoded tropism factor is a glycoprotein. 31. The XDP system of claim 30, wherein the glycoprotein has binding affinity for a cell surface marker of a target cell and facilitates entry of the XDP into the target cell. 32. The XDP system of claim 30, wherein the glycoprotein has a sequence selected from the group consisting of SEQ ID NOS: 573-613, 615-682, 684-692, 694-698, 700, 702-706, 708-727, 729-730, 732, 734, 738, 740-746, and 749-796, or a sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. 295377236 Attorney Docket No. SCRB-050/01WO 333322-2386 33. The XDP system of any one of claims 1-32, wherein the one or more retroviral components, therapeutic payload, and tropism factor are encoded on three, four, or five nucleic acids. 34. The XDP system of claim 33, wherein separate vectors comprise each nucleic acid, and wherein each vector comprises a promoter operably linked to the nucleic acid. 35. The XDP system of claim 34, wherein the vector comprising the nucleic acid encoding the Gag polyprotein fused to the NCR protein or functional domain thereof comprises a sequence encoding a Rev protein. 36. The XDP system of claim 34 or 35, wherein the XDPs are capable of self-assembly when the separate vectors are introduced into eukaryotic packaging cells and the cells are cultured under conditions allowing expression of the encoded components. 37. The XDP system of claim 36, wherein the XDP comprises a CRISPR protein and a first gRNA complexed as a ribonucleoprotein complex (RNP). 38. The XDP system of claim 36, wherein the XDP comprises the CRISPR protein and the first gRNA complexed as a first ribonucleoprotein complex (RNP), and the CRISPR protein and a second gRNA complexed as a second RNP. 39. The XDP system of claim 37 or claim 38, wherein inclusion of the sequences encoding NCR protein and its corresponding ligand in the nucleic acids encoding the Gag polyprotein and the gRNA, respectively, results in enhanced incorporation of the numbers of RNP into the XDPs during self-assembly when compared to an equivalent XDP system not comprising the NCR protein and its corresponding ligand. 40. The XDP system of claim 39, wherein at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, or at least about 1000 RNP particles are incorporated into the XDP. 41. The XDP system of claim 40, wherein at least about 100 to about 1000 RNP, at least about 200 to about 800 RNP, or at least about 300 to about 600 RNP are incorporated into the XDP. 42. The XDP system of any one of claims 1-41, wherein the encoded retroviral components are derived from a Lentivirus. 43. The XDP system of claim 42, wherein the Gag polyprotein fused to the NCR protein comprises one or more components selected from the group consisting of a matrix polypeptide 295377236 Attorney Docket No. SCRB-050/01WO 333322-2386 (MA), a capsid polypeptide (CA), a nucleocapsid polypeptide (NC), a p1 protein (p1), a p6 protein (p6), and a protease cleavage sequence (PCS). 44. The XDP system of claim 43, wherein the Gag polyprotein comprises, from N-terminus to C-terminus, MA-PCS-CA-PCS-NC-PCS-p1-PCS-p6. 45. The XDP system of any one of claims 1-44, wherein the therapeutic payload is encoded on a separate nucleic acid from the Gag polyprotein. 46. The XDP system of any one of claims 1-45, wherein the Gag-TFR-PR polyprotein comprises, from N-terminus to C-terminus, MA-PCS-CA-PCS-NC-PCS-p1-PCS-p6-PCS- protease, wherein the protease is capable of cleaving the PCS of the Gag and the Gag-TRF-PR. 47. The XDP system of any one of claims 1-46, wherein the one or more of the components are encoded by nucleic acids selected from the group consisting of the sequences of SEQ ID NOS: 19-31, 196, 813, 848, 975-977, 979, 1021, 1134-1136, , 1138-1153, 1155-1195, 1127- 1199, 1200-1207, 1227-1230, 1233-1250, 1253-1284, 1286-1320, 1322-1325, 1536-1540, 1572, 1587, 1781, 1783-1787, 1789, 1790, 1845, 2249, 2308, 2482-2493, and 4146-4148, or sequences having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. 48. The XDP system of any one of claims 1-46, wherein the one or more of the retroviral components are encoded by a nucleic acid selected from the group consisting of the sequences of SEQ ID NOS: 19-31, 196, 813, 848, 975-977, 979, 2249, and 2308. 49. A eukaryotic cell comprising the XDP system of any one of claims 1-48. 50. The eukaryotic cell of claim 49, wherein the eukaryotic cell is modified to reduce expression of a cell surface marker. 51. The eukaryotic cell of claim 50, wherein the cell surface marker is selected from the group consisting of B2M, CD47 and HLA-E KI, wherein the incorporation of the cell surface marker on the surface of the XDP released from the eukaryotic cell is reduced compared to XDP released from a eukaryotic cell that has not be modified. 52. The eukaryotic cell of any one of claim 49-51, wherein the eukaryotic cell is modified to express one or more cell surface markers selected from CD46, CD47, CD55, and CD59, wherein the incorporation of the cell surface marker on the surface of the XDP released from the 295377236 Attorney Docket No. SCRB-050/01WO 333322-2386 eukaryotic cell is increased compared to XDP released from a eukaryotic cell that has not be modified. 53. A method of making an XDP comprising a therapeutic payload, the method comprising: (a) propagating the eukaryotic cell of any one of claim 49-52 under conditions such that an XDP is produced; and (b) harvesting the XDP produced by the eukaryotic cell. 54. The method of claim 53, wherein expression of the sequences encoding the NCR protein and its corresponding ligand results in enhanced incorporation of the numbers of RNP into the XDP during self-assembly compared to an equivalent method not comprising the NCR protein and its corresponding ligand. 55. An XDP produced by the method of claim 53 or 54. 56. A delivery particle (XDP) comprising cleavage products of a retroviral Gag polyprotein, a therapeutic payload, and a tropism factor, wherein: (a) the cleavage products of the Gag polyprotein comprise MA, CA, NC, p1, and p6, wherein p6 is fused to a non-covalent recruitment (NCR) protein, wherein the NCR protein is a protein-binding protein and the ligand is a protein ligand, wherein the NCR has binding affinity for the ligand, (b) the tropism factor is incorporated on the surface of the XDP; and (c) the therapeutic payload is fused to a ligand and encapsidated within the XDP, wherein the NCR protein has an affinity for the ligand. 57. The XDP of claim 56, wherein the NCR protein is fused to the C-terminus of p6. 58. The XDP of claim 56 or 57, wherein the NCR protein comprises: (a) a Protein A and the ligand is an Fc region; (b) a truncated Protein A and the ligand comprises an Fc region; (c) a CL7 protein and the ligand comprises an IM7 ligand; (d) a NbALFA protein and the ligand comprises an ALFA tag; (e) a SpyCatcher protein and the ligand comprises a SpyTag; (f) a SpyCatcher002 protein and the ligand comprises a SpyTag002; (g) a SpyCatcher003 protein and the ligand comprises a SpyTag003; (h) a Strep-Tactin protein and the ligand comprises a Twin Strep tag II; (i) a Strep-Tactin protein and the ligand comprises a Strep tag II; (j) an Avidin protein and the ligand comprises an Avi tag; 266 295377236 Attorney Docket No. SCRB-050/01WO 333322-2386 (k) a mNG21-10 protein and the ligand comprises a mNG11 ligand; (l) a sfCherry21-10 protein and the ligand comprises a sfCherry211 ligand; (m) a mNG3A1-10 protein and the ligand comprises a mNG11 ligand; (n) a mNG3k1-10 protein and the ligand comprises a mNG11 ligand; (o) a sfGFP1-10 protein and the ligand comprises a GFP11 ligand; (p) a mClover31-10 protein and the ligand comprises a mClover311 ligand; (q) a CloGFP0.21-10 protein and the ligand comprises a GFP11 ligand; or (r) a CloGFP1-10 protein and the ligand comprises a GFP11 ligand. 59. The XDP of claim 58, wherein: (a) the Protein A comprises a sequence of SEQ ID NO: 2450, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Fc region, and the Fc region comprises a sequence of SEQ ID NO: 2468, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Protein A; (b) the truncated Protein A comprises a sequence of SEQ ID NO: 2451, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Fc region, and the Fc region comprises a sequence of SEQ ID NO: 2468, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the truncated protein A; (c) the CL7 protein comprises a sequence of SEQ ID NO: 2452, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the IM7 ligant, and the IM7 ligand comprises a sequence of SEQ ID NO: 2469, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 295377236 Attorney Docket No. SCRB-050/01WO 333322-2386 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the CL7 protein; (d) the NbALFA protein comprises a sequence of SEQ ID NO: 2462, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the ALFA tag, and the ALFA tag comprises a sequence of SEQ ID NO: 2476, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the NbALFA tag; (e) the SpyCatcher protein comprises a sequence of SEQ ID NO: 2463, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the SpyTag, and the SpyTag comprises a sequence of SEQ ID NO: 2477, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the SpyCatcher protein; (f) the SpyCatcher002 protein comprises a sequence of SEQ ID NO: 2461 or 2464, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the SpyTag002, and the SpyTag002 comprises a sequence of SEQ ID NO: 2475, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the SpyCatcher002 protein; (g) the SpyCatcher003 protein comprises a sequence of SEQ ID NO: 2465, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the SpyTag003, and the SpyTag003 comprises a sequence of SEQ ID NO: 2478, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at 268 295377236 Attorney Docket No. SCRB-050/01WO 333322-2386 least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the SpyCatcher003 protein; (h) the Strep-Tactin protein comprises a sequence of SEQ ID NO: 2466, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Twin Strep tag II, and the Twin Strep tag II comprises a sequence of SEQ ID NO: 2479, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Srep-Tactin protein; (i) the Strep-Tactin protein comprises a sequence of SEQ ID NO: 2466, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the to the Strep tag II, and a Strep tag II comprises the sequence of SEQ ID NO: 2480, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Strep-Tactin protein; (j) the Avidin protein comprises a sequence of SEQ ID NO: 2467, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Avi tag, and the Avi tag comprises a sequence of SEQ ID NO: 2481, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the Avidin protein; (k) the mNG21-10 protein comprises a sequence of SEQ ID NO: 2453, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mNG11 ligand, and the mNG11 ligand comprises a sequence of SEQ ID NO: 2470, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least 295377236 Attorney Docket No. SCRB-050/01WO 333322-2386 about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mNG21-10 protein; (l) the sfCherry21-10 protein comprises a sequence of SEQ ID NO: 2454, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the sfCherry211 ligand, and the sfCherry211 ligand comprises a sequence of SEQ ID NO: 2471, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the sfCherry21-10 protein; (m) the mNG3A1-10 protein comprises a sequence of SEQ ID NO: 2455, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mNG11 ligand, and the mNG11 ligand comprises a sequence of SEQ ID NO: 2470, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mNG3A1-10 protein; (n) the mNG3k1-10 protein comprises a sequence of SEQ ID NO: 2456, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mNG11 ligand, and the mNG11 ligand comprises a sequence of SEQ ID NO: 2470, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mNG3k1-10 protein; (o) the sfGFP1-10 protein comprises a sequence of SEQ ID NO: 2457, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the GFP11 ligand, and the GFP11 ligand comprises a sequence of SEQ ID NO: 2474, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least 295377236 Attorney Docket No. SCRB-050/01WO 333322-2386 about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the sfGFP1-10 protein; (p) the mClover31-10 protein comprises a sequence of SEQ ID NO: 2458, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mClover311 ligand, and the mClover311 ligand comprises a sequence of SEQ ID NO: 2473, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the mClover31-10 protein; (q) the CloGFP0.21-10 protein comprises a sequence of SEQ ID NO: 2459, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the GFP11 ligand, and the GFP11 ligand comprises a sequence of SEQ ID NO: 2474, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the CloGFP0.21-10 protein; or (r) (r)the CloGFP1-10 protein comprises a sequence of SEQ ID NO: 2460, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the GFP11 ligand, and the GFP11 ligand comprises a sequence of SEQ ID NO: 2474, or a sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto that is capable of binding to the CloGFP0.21-10 protein. 60. The XDP of any one of claims 56-59, wherein the therapeutic payload comprises a protein payload selected from the group consisting of a cytokine, an interleukin, an enzyme, a receptor, a microprotein, a hormone, erythropoietin, a ribonuclease (RNase), a deoxyribonuclease (DNase), a blood clotting factor, an anticoagulant, a bone morphogenetic protein, an engineered protein scaffold, a thrombolytic protein, a CRISPR protein, granulocyte- 295377236 Attorney Docket No. SCRB-050/01WO 333322-2386 macrophage colony-stimulating factor (GMCSF), a transcription factor, a transposon, a reverse transcriptase, a viral interferon antagonist, a tick protein, and an anti-cancer modality. 61. The XDP of any one of claims 56-60, wherein the therapeutic payload comprises of RNPs of a CRISPR protein and a gRNA, RNPs of a CasX variant and a guide RNA variant, or RNPs of a dCasX and linked repressor domain(s) and a guide RNA variant. 62. The XDP of any one of claims 56-61, wherein the cleavage products of the Gag polyprotein are derived from a Lentivirus. 63. A method of modifying a target nucleic acid sequence in a population of cells, comprising contacting the cells with the XDP of any one of claims 55-62, wherein the XDP comprises a therapeutic payload of RNPs of a CasX variant and a gRNA variant, wherein said contacting comprises introducing the into the cells the RNP, and wherein the target nucleic acid targeted by the gRNA variant is modified by the CasX variant. 64. A method of repressing a target nucleic acid sequence in a population of cells, comprising contacting the cells with the XDP of any one of claims 55-62, wherein the XDP comprises a therapeutic payload of RNPs of a dCasX linked to at least a first repressor domain (RD1) as a fusion protein and a guide RNA, and wherein said contacting comprises introducing the into the cells the RNP, wherein a target nucleic acid targeted by the guide RNA is repressed by the dCsaX linked to the at least a first repressor domain. 65. A composition comprising the XDP of any one of claims 55-62 for use as a medicament for the treatment of a subject having a disease. 66. A composition comprising the XDP of any one of claims 55-62 for use in treating a disease in a subject in need thereof. 295377236
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