Compositions and methods for targeted delivery of CRISPR-Cas effector polypeptides

Abstract

The present disclosure provides enveloped delivery vehicles (EDVs) comprising a nucleic acid-binding effector polypeptide or a nucleic acid encoding a nucleic acid-binding effector polypeptide, where the EDV comprises a fusion polypeptide comprising (i) a viral envelope protein and (ii) a targeting polypeptide that confers binding to a target cell. The present disclosure provides methods of using the EDVs of the present disclosure for delivery of, for example, a nucleic acid-binding effector polypeptide to a eukaryotic cell.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 400,166, filed August 23, 2022, and U.S. Provisional Patent Application No. 63 / 432,484, filed December 14, 2022, which applications are incorporated herein by reference in their entireties.

[0002] Incorporation by reference of a sequence listing provided as a sequence listing XML file The Sequence Listing is provided herewith as Sequence Listing XML "BERK-477WO_SEQ_LIST.xml", created on August 21, 2023, and having a size of 348,713 bytes. The contents of the Sequence Listing XML are incorporated herein by reference in their entirety.

[0003] Federal Funding Statement This invention was made with government support under Grant No. GM143461 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0004] Introduction The RNA-mediated adaptive immune system in bacteria and archaea relies on CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) genomic loci and CRISPR-associated (Cas) proteins, which function together to provide protection from invading viruses and plasmids. Genome editing can be performed using the CRISPR / Cas system, which includes CRISPR / Cas effector polypeptides and guide RNAs. The CRISPR / Cas system has revolutionized the fields of gene editing and genome engineering. Efficient methods for delivering CRISPR-Cas genome editing components to target cells are needed for both ex vivo and in vivo applications.

[0005] Current delivery strategies have drawbacks. For example, delivery of recombinant viruses encoding CRISPR-Cas effector polypeptides prolongs expression of the CRISPR-Cas effector polypeptides in target cells, thus increasing the likelihood of off-target gene editing events. Others use ribonucleoproteins (RNPs) containing CRISPR-Cas effector polypeptides and guide RNAs (gRNAs) to deliver genome editing components into cells.

[0006] There is a need in the art for additional strategies for delivering CRISPR-Cas effector polypeptides to target cells. Summary of the Invention

[0007] overview The present disclosure provides enveloped delivery vehicles (EDVs) (also referred to as virus-like particles (VLPs)) comprising a nucleic acid-binding effector polypeptide (e.g., a CRISPR Cas effector polypeptide) or a nucleic acid encoding a nucleic acid-binding effector polypeptide, wherein the EDV comprises a fusion polypeptide comprising (i) a viral envelope protein and (ii) a targeting polypeptide that confers binding to a target cell. The present disclosure provides methods of delivering a CRISPR-Cas effector polypeptide into a eukaryotic cell using the EDVs of the present disclosure. The present disclosure provides methods of in vivo gene editing using the EDVs of the present disclosure. [Brief explanation of the drawings]

[0008] [Figure 1A] Figures 1A-1C show genome editing of on-target versus bystander (non-target) cells using Gag-NES-2Xp53NLS-Cas9 and Cas9-EDV containing a guide RNA leading to knockout of beta-2 microglobulin (β2M) in 293T cells. [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 2] Genome editing of primary human cells (CD34+ hematopoietic stem and progenitor cells and T cells) using Cas9-EDV. [Figure 3] Genome editing of primary human T cells using Cas9-EDV containing guide RNA leading to knockout of TRAC is shown. EDVs contained envelope proteins including (i) a single-chain Fv (scFv) targeting CD3, (iii) a scFv targeting CD4, (iv) a scFv targeting CD19, (v) scFvs targeting CD3 and CD4, or (vi) no scFv. Cas9-EDV pseudotyped with HIV-1 and VSVG are presented as controls. HIV-1 Env-pseudotyped EDV specifically mediated genome editing in CD4+ T cells, while VSVG-pseudotyped EDV mediated genome editing in both CD4+ and CD8+ T cells. [Figure 4] 1 shows activation of primary human T cells by Cas9-EDV expressing anti-CD3 scFv and anti-CD28 scFv. [Figure 5] 1 shows a schematic of targeted lentiviral delivery in vivo. [Figure 6-1] Figures 6A-6F show targeted lentiviral delivery in vivo. Cas9-EDV containing envelope proteins containing anti-CD3 scFv and anti-CD28 scFv was used, along with a construct encoding a chimeric antigen receptor (CAR) containing anti-CD19 scFv. Data show the % of CAR-T cells (Figure 6D) and % of CD19 B cells (Figures 6E and 6F). [Figure 6-2] See description of Figure 6-1. [Figure 6-3] See description of Figure 6-1. [Figure 6-4] See description of Figure 6-1. [Figure 7] Schematic showing the evaluation of Cas9-EDV and lentivirus activity targeting T cells in vivo. [Figure 8] Figures 8A-8C show the in vivo generation of CAR-T cells. [Figure 9]

[0033] Figure 1 shows the in vivo generation of CAR-T cells. The Cas9-EDV contained a construct encoding the CAR and a guide RNA that resulted in knockout of TRAC. The control lentivirus contained a construct encoding the CAR but no guide RNA. [Figure 10] Figure 1 shows the in vivo generation of CAR-T cells, with the percentage of CD4+ T cells or CD8+ T cells shown. [Figure 11] 1 shows the in vivo generation of CD8+ CAR-T cells. [Figure 12] 12A-12B show genome editing in T cells in vivo. [Figure 13] 13A-13B show in vivo genome editing of CD8+ T cells. [Figure 14] Shows in vivo genome editing of mCherry+CAR-T cells. [Figure 15A] Figures 15A-15P provide the amino acid sequences of CRISPR-Cas effector polypeptides. [Figure 15B] See legend to Figure 15A. [Figure 15C] See legend to Figure 15A. [Figure 15D] See legend to Figure 15A. [Figure 15E] See legend to Figure 15A. [Figure 15F] See legend to Figure 15A. [Figure 15G] See legend to Figure 15A. [Figure 15H] See legend to Figure 15A. [Figure 15I] See legend to Figure 15A. [Figure 15J] See legend to Figure 15A. [Figure 15K] See legend to Figure 15A. [Figure 15L] See legend to Figure 15A. [Figure 15M] See legend to Figure 15A. [Figure 15N] See legend to Figure 15A. [Figure 15O] See legend to Figure 15A. [Figure 15P] See legend to Figure 15A. [Figure 16A] Figures 16A-16D provide the amino acid sequences of wild-type vesicular stomatitis virus (VSV) glycoprotein (VSVG) (Figure 16A), mutant VSVG (Figure 16B), wild-type measles virus hemagglutinin (HA) (Figure 16C), and mutant measles virus HA (Figure 16D). [Figure 16B] See legend to Figure 16A. [Figure 16C] See legend to Figure 16A. [Figure 16D] See legend to Figure 16A. [Figure 17] The amino acid sequence of the reverse transcriptase is provided. [Figure 18] A table is provided providing a description of the scFv polypeptides referred to in Example 2. [Figure 19A-1] Figures 19A-19F provide the nucleotide sequences (Figures 19A-19D and Figure 19F) encoding Gag-Cas9 (Figure 19A), Gag-Cas9 with 3xNES (Figure 19B), Gag-Cas9 with 2xp53 NLS (Figure 19C), Gag-Cas9 with 3xNES and 2xp53 NLS (Figure 19D), anti-CD19 scFv1 (Figure 19F), and the amino acid sequence of Gag-Cas9 with 3xNES and 2xp53 NLS (Figure 19E). [Figure 19A-2] See description of Figure 19A-1. [Figure 19B-1] See description of Figure 19A-1. [Figure 19B-2] See description of Figure 19A-1. [Figure 19C-1] See description of Figure 19A-1. [Figure 19C-2] See description of Figure 19A-1. [Figure 19D-1] See description of Figure 19A-1. [Figure 19D-2] See description of Figure 19A-1. [Figure 19D-3] See description of Figure 19A-1. [Figure 19E]See description of Figure 19A-1. [Figure 19F] See description of Figure 19A-1. [Figure 20A] Figures 20A-20E show cell-specific genome editing using antibody-targeted Cas9 virus-like particles (VLPs). [Figure 20B] See legend to Figure 20A. [Figure 20C] See legend to Figure 20A. [Figure 20D] See legend to Figure 20A. [Figure 20E] See legend to Figure 20A. [Figure 21A] Figures 21A-21C show the use of antibody-targeted VLPs for genome editing of specific cells. [Figure 21B] See legend to Figure 21A. [Figure 21C] See legend to Figure 21A. [Figure 22A]Figures 22A-22E. Cell-specific genome editing using antibody-targeted Cas9-EDV. (Figure 22A) Schematic of the scFv targeting molecule (blue) and VSVGmut (orange) on the outer surface of Cas9-EDV. Cas9-EDV packages a preformed Cas9-single guide RNA complex to avoid genetically encoding the genome editor within the viral genome. (Figure 22B) Experimental overview and schematic of the lentiviral vector used to engineer HEK293T EGFP cells expressing a heterologous ligand (e.g., CD19) on the plasma membrane. To facilitate cell engineering via a single lentiviral integration event, engineered cell mixtures were generated via a low multiplicity of infection to achieve less than 25% EGFP+ cells. The engineered cell mixtures were challenged with Cas9-EDV targeting B2M to test targeting molecule activity. (Figures 22C-22E) Evaluation of antibody-targeted Cas9-EDV activity. HEK293T and CD19 EGFP HEK293T cells were mixed at an approximate ratio of 3:1 and treated with B2M-targeting Cas9-EDVs exhibiting various targeting molecular pseudotypes. Cas9-EDV was concentrated 10-fold, and cells were treated with 50 μl of Cas9-EDV (Figures 22C, 22E) or a dilution curve (Figure 22D). Analysis was performed 7 days after treatment to assess B2M knockout in EGFP+ (on-target) and EGFP- (bystander) cells by flow cytometry (Figures 22C, 22D) and amplicon sequencing (Figure 22E). N = 3 technical replicates (N = 2) for all panels except for the 100 μl dose of CD19-scFv in (Figure 22D). Error bars represent the standard error of the mean. [Figure 22B] See legend to Figure 22A. [Figure 22C] See legend to Figure 22A. [Figure 22D] See legend to Figure 22A. [Figure 22E] See legend to Figure 22A. [Figure 23A]Figures 23A-23I. Optimization of Cas9-EDV for enhanced genome editing activity in primary human cells. (Figure 23A) Comparison of genome editing activity of CD19 antibody-targeted Cas9-EDV variants that package Cas9 ribonucleoproteins (RNPs) targeting B2M. B2M protein expression was assessed by flow cytometry in CD19-expressing target cells 7 days after treatment. (Figure 23B) Diagram of optimized Gag-Cas9 and Gag-pol Cas9-EDV production plasmids; updated features from Hamilton & Tsuchida et al., 2021, are highlighted in teal. (Figures 23C-23E) Genome editing activity of optimized VSVG-pseudotyped Cas9-EDV in primary human CD34+ cells (Figure 23C) and activated (Figure 23D) and resting primary human T cells (Figure 23E). B2M or TRAC genome editing was assessed by amplicon sequencing 7 days after treatment. (Figure 23F) Schematic of potential intraparticle Cas9-EDV configurations for packaged Cas9 RNP after proteolytic maturation. (Figure 23G) Schematic of compound GS-CA1, which inhibits either nuclear import and / or uncoating of HIV-1 capsids. (Figure 23H) mNeonGreen lentiviral vector was used to transduce HEK293T cells at the indicated MOI in the presence of GS-CA1 or DMSO. The percentage of mNeonGreen-positive cells was assessed by flow cytometry 3 days after treatment. TU = transduction unit. (Figure 23I) B2M-targeting Cas9-EDV, pre-titrated so that the highest treatment dose resulted in approximately 50% B2M- cells, was used to transduce HEK293T cells in the presence of GS-CA1 or DMSO. B2M expression was assessed by flow cytometry 3 days after treatment. Error bars represent standard deviation. N=3 technical replicates were used in all experiments. [Figure 23B] See legend to Figure 23A. [Figure 23C] See legend to Figure 23A. [Figure 23D] See legend to Figure 23A. [Figure 23E] See legend to Figure 23A. [Figure 23F] See legend to Figure 23A. [Figure 23G] See legend to Figure 23A. [Figure 23H] See legend to Figure 23A. [Figure 23I] See legend to Figure 23A. [Figure 24]Figures 24A-24E. Targeting and editing of primary human T cells with multiplexed antibodies. (Figures 24A-24B) Treatment of resting human T cells with Cas9-EDV co-presenting CD3 and CD28 scFvs results in cell activation (Figure 24A) and proliferation (Figure 24B), respectively, as measured by flow cytometric detection of CD25 and fold expansion over untreated T cell numbers 3 days after treatment. CD25 expression and cell proliferation were observed for CD3 / CD28 scFv Cas9-EDV regardless of whether the CD3 / CD28 scFv Cas9-EDV packaged a Cas9 RNP targeting PDCD1 or a non-targeting control. (Figure 24C) Genome editing 3 days after treatment, as detected by amplicon next-generation sequencing. In Figures A-C, Cas9-EDV was concentrated 62-fold, and 50 μl was used to treat 30k resting T cells. CD3 scFv-1 and CD28 scFv-2 were tested. (Figure 24D) Monodisplay screening of additional CD3 scFv targeting molecules with B2M-targeting Cas9-EDV on Jurkat T cell lines. B2M expression was assessed by flow cytometry 3 days after treatment. Cas9-EDV was concentrated 15-fold, and 50 μl was used to treat 30kJ Jurkat cells. (Figure 24E) Testing of a panel of T cell-targeted B2M-targeting Cas9-EDVs representing single or multiplexed scFv targeting molecules. Activated primary human T cells were treated with 1.38 x 10 Cas9-EDVs representing one or a combination of CD3 scFv-3, CD4 scFv-2, CD28 scFv-2, and control scFvs, and B2M expression in CD4+ and CD8+ T cells was assessed by flow cytometry 6 days after treatment. Error bars represent the standard error of the mean. N=3 technical replicates were used for all panels. [Figure 25-1]Figures 25A-25I. Programmable human cell delivery generates gene-edited CAR T cells in vivo. (Figure 25A) Summary of Cas9-EDV and lentivirus targeting T cells tested in PBMC-humanized mice. Both particles display multiplexed scFvs (CD3 scFv-3, CD4 scFv-1, and CD28 scFv-2). The Cas9-EDV vector co-packages the lentivirally encoded CAR-2A-mCherry transgene and the Cas9 RNP complex to disrupt the T cell receptor alpha constant (TRAC) gene, and the lentivirus encodes the CAR-2A-mCherry transgene. (Figure 25B) Experimental schematic for testing Cas9-EDV and lentivirus targeting T cells in PBMC-humanized mice via IV (intravenous) retroorbital injection. (Figure 25C) Representative flow cytometry plots demonstrating that CAR-expressing human T cells are present in the spleens of PBMC-humanized mice 10 days after administration of 1.5 x 10 Cas9-EDV (N = 2) or lentivirus (N = 3), as quantified in (Figure 25D). PBS = phosphate-buffered saline. (Figure 25E) Gene editing is observed in CAR-negative human T cells isolated from mice treated with Cas9-EDV targeted to T cells and is enriched in CAR-positive human T cells (Figure 25F). One CAR-positive lentivirus sample was excluded in (Figure 25F) due to sequencing failure. (Figure 25G) CAR-expressing human T cells are detectable in the spleens of PBMC-humanized mice 10 days after administration of 6.2 x 10 Cas9-EDV (N = 8) or lentivirus (N = 8). (Figure 25H) Genome editing is observed in CAR-negative human T cells isolated from mice treated with Cas9-EDV targeting T cells and is enriched in CAR-positive human T cells (Figure 25I). For all plots, the black line indicates the median value of the data set. LOD = limit of detection defined by the average modified reads from lentivirus-treated samples. [Figure 25-2] See description of Figure 25-1. [Figure 26]Figures 26A-26C. Functional kinetics of in vivo cell engineering. (Figure 26A) Depletion of CD19+ B cells is observed after administration of lentivirus targeting T cells (Experiment 2). Human CD45+ cells were isolated from PBMC-humanized spleens 10 days after systemic administration, and the percentage of CD19-expressing cells was assessed by flow cytometry. P=*** indicates p<0.001 (unpaired T-test). The black line indicates the median of the dataset. (Figure 26B) Model for in vivo generation of functional CAR-T cells with or without concomitant gene editing. Schematic generated in BioRender. (Figure 26C) TCR clonotypes of CAR-transduced T cells isolated from humanized mice treated with Cas9-EDV (mouse numbers 2, 4, and 6) or lentivirus (mouse numbers 13, 14, and 17) targeting T cells from Experiment 2. "n" indicates the unique number of clonotypes detected. [Figure 27A] Figures 27A-27C. Establishment of antibody-targeted Cas9-EDV delivery. (Figure 27A) Schematic of antibody-derived single-chain variable fragment (scFv) targeting molecules for Cas9-EDV display. The scFv is fused to the stalk and transmembrane domain (TMD) of human CD8α. (Figure 27B) Schematic of single-chain variable fragment (scFv) targeting molecules. The scFv targeting molecules were constructed in either a VH-VL or VL-VH orientation. Amino acid (AA) residues correspond to the CD8α protein sequence (UniProt P01732), linker (SEQ ID NO: 203). TMD = transmembrane domain. S = serine. (Figure 27C) Antibody-targeted Cas9-EDV mediates targeted genome editing regardless of target cell frequency. CD19 EGFP HEK293T cells were mixed with HEK293T cells to achieve a target cell frequency of approximately 2-92%. The heterogeneous cell mixture was challenged with antibody-targeted Cas9-EDV (100 μl, 2.5x concentration), and B2M knockout was assessed in EGFP+ (on-target) and EGFP- (bystander) cells by flow cytometry 7 days after treatment. N=3 technical replicates. Error bars represent the standard error of the mean. [Figure 27B] See legend to Figure 27A. [Figure 27C] See legend to Figure 27A. [Figure 28A] Figures 28A-28C. Antibody-targeted Cas9-EDV is a programmable strategy for mediating genome editing in specific cells. (Figure 28A) Verification of cell surface ligand expression in engineered cells. Engineered 293T cells transduced to express either CD19, CD20, CD4, or CD28 and EGFP were assayed for successful ligand expression. Cells were stained with monoclonal antibodies (CD19-PE, CD20-PE, CD4-PE-Cy7, CD28-PE) to confirm ligand expression only in EGFP+ cells. (Figure 28B) Various scFv targeting molecules were developed to target the ligands expressed on human immune cells: CD19, CD20, CD4, and CD28. The activity of cell-specific antibody-retargeted Cas9-EDV was assessed by flow cytometry 7 days after treatment for on-target, ligand+ cells (EGFP+) and off-target, bystander cells (EGFP-). (Figure 28C) Antibody-targeted Cas9-EDV-mediated genome editing is highly specific for cells expressing the scFv cognate ligand. scFv-Cas9-EDV was tested on engineered cells expressing cognate and non-cognate ligands, and genome editing activity was measured by flow cytometry 3 days after treatment. N=3 technical replicates. All error bars represent the standard error of the mean. [Figure 28B] See legend to Figure 28A. [Figure 28C] See legend to Figure 28A. [Figure 29-1]Figures 29A-29J. Optimization and study of Cas9-EDV activity. (Figure 29A) Diagram of the Gag-Cas9 plasmid showing the site of nuclear localization signal (NLS) variant addition to the N-terminus of Cas9. (Figure 29B) Evaluation of N-terminal Cas9 NLS addition on Cas9-EDV activity. Cas9-EDV was used to treat a mixture of CD19+ and CD19- 293T cells. Genome editing was assessed by flow cytometric detection of B2M expression in CD19+ target cells and CD19- bystander cells 7 days after treatment. BP = duplicates. (Figures 29C-29D) Direct comparison of genome editing in CD19+ on-target cells (Figure 29C) and CD19- bystander cells (Figure 29D) treated with Gag-[3xNES]-Cas9 EDV and Gag-Cas9 EDV. Genome editing was assessed by flow cytometry detection of B2M expression 7 days after treatment. (Figure 29E) Comparison of genome editing activity of CD19 antibody-targeted Cas9-EDV variants that package B2M-targeting Cas9 RNPs. B2M protein expression was assessed by flow cytometry in CD19-negative bystander cells 7 days after treatment. (Figure 29F) Direct comparison of genome editing in on-target and bystander cells with Gag-[3xNES]-[2xp53-NLS]-Cas9 EDV variants. The difference in genome editing between the two cell types is shown for Cas9-EDV treatment doses of 1.56 μl and 6.25 μl. (Figure 29G) Treatment of target cells with GS-CA1 does not alter genome editing by Cas9 RNP nucleofection. 293T cells were nucleofected with 50 pmol of B2M-targeting Cas9 RNP and cultured in the presence of either 25 nM GS-CA1, 0.05% DMSO, or Opti-MEM. Genome editing was assessed by flow cytometry detection of B2M expression 3 days after treatment. (Figure 29H) Diagram of the Gag-TCL-Cas9 and Gag-TCL-TEV plasmids for producing TEVp-Cas9-EDV, which releases Cas9 from Gag in a TEV protease-dependent manner. TCL = TEV protease-cleavable linker.(Figure 29I) Western blot analysis of the loss of Gag proteolytic processing in TEVp-Cas9-EDV, as indicated by the absence of p24 and other Gag proteolytic intermediates. CA = capsid, a component of the gag polypeptide. (Figure 29J) Genome editing activity of TEVp-Cas9-EDV packaged with Cas9 RNP, which activates tdTomato expression upon genome editing of a mouse neural progenitor cell line harboring a loxP-STOP-loxP-tdTomato reporter (51). Genome editing was assessed by flow cytometric detection of tdTomato reporter activation 7 days after treatment. N = 3 technical replicates were used in all experiments. [Figure 29-2] See description of Figure 29-1. [Figure 29-3] See description of Figure 29-1. [Figure 30A]Figures 30A-30M. Characterization and in vivo biodistribution of the optimized Cas9-EDV. (Figure 30A) Dynamic light scattering analysis of Cas9-EDV and lentiviral particles. Particles were pseudotyped with either VSVG or anti-CD19 scFv + VSVGmut, and the Z-average (Z-Ave) was measured in nanometers (d.nm). Commercially available 100 nm gold beads were used as a control. Dots indicate technical replicates. (Figure 30B) Representative transmission electron microscopy (TEM) image of anti-CD19 scFv + VSVGmut pseudotyped Cas9-EDV. (Figure 30C) Anti-myc immunogold TEM labeling of the anti-CD19 scFv 1 targeting molecule on Cas9-EDV (top). No staining is observed when the anti-CD19 scFv 1 myc tag is exchanged for a strep tag (bottom). (Figure 30D) Detection of B2M sgRNA in the produced Cas9-EDV and Cre-EDV. A custom qPCR primer / probe set was used for B2M sgRNA detection, and fold changes were calculated by normalizing to the Cre-EDV sample. Cre-EDV was generated by swapping the Cre recombinase in the Gag-Cas9 V2 plasmid with Cas9 (see Figure 2B). Cre-EDV (without sgRNA) was produced similarly, but with a plasmid lacking the U6-sgRNA expression cassette. ND = not detected. Dots indicate technical replicates. (Figure 30E) To assess unintended carryover of plasmid from producer cells to Cas9-EDV-treated cells, LentiX cells were transfected to produce Cas9-EDV in either the absence (top) or presence (bottom) of 1 μg of EGFP-expressing plasmid. Flow cytometry was performed on producer cells 3 days after transfection. (Figure 30F) Flow cytometry analysis of EGFP expression in Cas9-EDV-treated cells. 293T cells were treated with B2M Cas9-EDV produced either in the absence (top) or presence (bottom) of an EGFP-expressing plasmid. Flow cytometry was performed 6 days after Cas9-EDV treatment to assess EGFP and B2M expression.(Figure 30G) A standard curve quantifying B2M sgRNAs synthesized via a custom qPCR primer / probe set is used to interpolate the number of B2M sgRNAs associated with Cas9-EDV produced in one 10 cm dish (Figure 30H). Points represent biological replicates of Cas9-EDV produced in one 10 cm dish and concentrated 30-fold in 300 μl. (Figure 30I) The number of Cas9-EDV particles produced per 10 cm dish, as estimated by p24 ELISA. Points represent biological replicates (Figures 30J and 30K). B2M knockout in 293T cells after treatment with the indicated Cas9-EDV volumes (Cas9-EDV was concentrated 30-fold) (Figure 30J) or nucleofection with Cas9 RNP complexes (Figure 30K). Flow cytometry was used to detect B2M expression 6 days after treatment. Data were fitted using a four-parameter logistic curve. (Figure 30L) Genome editing comparison between Cas9-EDV treatment volume and Cas9 RNP nucleofection. Data from J and K were used to interpolate how Cas9-EDV treatment volume relates to Cas9 RNP pmol dose. B2M sgRNA molecule and particle numbers are scaled to Cas9-EDV treatment volume (Figures 30H and 30I). (Figure 30M) Biodistribution of VSVG and anti-CD19 scFv+VSVGmut lentivirus. 3E9 lentiviral copies, normalized by qPCR, were administered systemically to C57BL / 6 mice via retro-orbital injection. Five minutes after vector administration, the indicated tissues / organs were harvested, RNA was isolated, and qPCR was performed to detect the presence of lentiviral vector. Vector amount is plotted as fold change relative to the amount of vector in serum. Each point represents an individual mouse. Two mice received 100 μl of PBS as a vehicle control, and as expected, no lentiviral vector was detected in any organs (not shown). Floating points (D, H, I) represent the mean. All error bars represent the standard error of the mean. [Figure 30B] See legend to Figure 30A. [Figure 30C] See legend to Figure 30A. [Figure 30D]See legend to Figure 30A. [Figure 30E] See legend to Figure 30A. [Figure 30F] See legend to Figure 30A. [Figure 30G] See legend to Figure 30A. [Figure 30H] See legend to Figure 30A. [Figure 30I] See legend to Figure 30A. [Figure 30J] See legend to Figure 30A. [Figure 30K] See legend to Figure 30A. [Figure 30L] See legend to Figure 30A. [Figure 30M] See legend to Figure 30A. [Figure 31A]Figures 31A-31D. Evaluation of T cell-targeting molecules for Cas9-EDV delivery. (Figure 31A) Evaluation of molecules targeting human CD45. A panel of CD45-targeting Cas9-EDVs packaging B2M-targeting Cas9 RNPs was produced and concentrated 13.8-fold. 100 μl was used to treat 30kJ Jurkat cells, and B2M expression was assessed by flow cytometry on day 3. (Figure 31B) Evaluation of CD4- and CD45-targeting Cas9-EDVs in activated primary human T cells. Cas9-EDVs packaging TRAC-targeting Cas9 RNPs were produced, and 1.15 × 107 Cas9-EDVs were used to treat 15kJ activated T cells. T cell receptor (TCR) loss, indicative of TRAC genome editing, was assessed by flow cytometry 5 days after treatment. Representative plots are shown, and results are quantified in (Figure 31C). CD4 scFv-1 and CD45 scFv-5 targeting molecules were used. (Figure 31D) Evaluation of multiplexed targeting molecules for delivery to activated primary human T cells. 15k activated T cells were treated with 1.38 x 10 Cas9-EDVs packaged with Cas9 RNPs targeting B2M, and flow cytometry analysis of B2M expression was assessed on day 6. Representative flow cytometry plots are shown. CD3 scFv-3, CD4 scFv-2, and CD28 scFv-2 targeting molecules were used. Error bars represent the standard error of the mean. N = 3 technical replicates were evaluated (Figures 31A and 31C). [Figure 31B] See legend to Figure 31A. [Figure 31C] See legend to Figure 31A. [Figure 31D] See legend to Figure 31A. [Figure 32-1]Figures 32A-32G. Evaluation of in vivo CAR T generation using T cell-targeting vectors. (Figure 32A) Analysis of mCherry+ CAR T cells in spleens from humanized mouse experiment 1. Mice were treated with Cas9-EDV, lentivirus (LV), or PBS, and flow cytometry analysis was performed 10 days after treatment. Flow cytometry quantification of CD4+ (Figure 32B) and CD8+ CAR T cells (Figure 32C). (Figure 32D) Analysis of mCherry+ CAR T cells in spleens from humanized mouse experiment 2. Mice were treated with Cas9-EDV, lentivirus (LV), or PBS, and flow cytometry analysis was performed 10 days after treatment. The numbers in the upper left quadrant are mouse identifiers. Flow cytometry quantification of CD4+ (Figure 32E) and CD8+ CAR T cells (Figure 32F). (Figure 32G) Quantification of body weight change in humanized mice from experiment 2. The dotted line indicates 100%. Black lines indicate median values ​​(B-C, E-G). [Figure 32-2] See description of Figure 32-1. [Figure 32-3] See description of Figure 32-1. [Figure 33A] Figures 33A-33B. Analysis of delivery to off-target cells in vivo. (Figures 33A-33B) Representative confocal microscopy images of livers from PBMC-humanized mice 10 days after systemic administration of Cas9-EDV and lentivirus targeting T cells demonstrate that mCherry+ (red) cells colocalize with human CD3+ (green) T cells (Figure 33A) and F4 / 80+ (green) phagocytes (Figure 33B), but not with β-catenin+ (cyan) hepatocytes. Scale bar, 65 μm. [Figure 33B] See legend to Figure 33A. [Figure 34A]Figures 34A-34B. Assessment of CD19+ B cells and CAR T cell receptor clonality after in vivo cell manipulation. (Figure 34A) Flow cytometry analysis of CD19+ B cells in Cas9-EDV-treated or lentivirus-treated mice 10 days after administration. Human CD45+ cells were isolated from humanized mouse spleens prior to flow cytometry analysis. The upper left quadrant number is the mouse identifier. (Figure 34B) Correlation between the number of CAR T clonotypes generated in vivo and the number of sorted CAR T cells (Pearson's correlation coefficient, R=0.938). Dot markers indicate unique mice analyzed. [Figure 34B] See legend to Figure 34A. [Figure 35]

[0023] Figure 1 provides a table providing a description of the scFv polypeptides referred to in Example 3. Sequences: (GGGGSGGGGSGGGGS, SEQ ID NO: 194, TGGGGSGGGGSGGGGS, SEQ ID NO: 192, TGSTSGSGKPGSGEGSTKG, SEQ ID NO: 193, GGGGSGGGGSGGGGSS, SEQ ID NO: 203). DETAILED DESCRIPTION OF THE INVENTION

[0009] definition As used herein, "heterologous" refers to a nucleotide or polypeptide sequence that is not found in a naturally occurring nucleic acid or protein, respectively. For example, with respect to a CRISPR-Cas effector polypeptide, a heterologous polypeptide comprises an amino acid sequence derived from a protein other than the CRISPR-Cas effector polypeptide. As another example, a CRISPR-Cas effector protein (e.g., a dead CRISPR-Cas effector protein) can be fused to an activity domain from a non-CRISPR-Cas effector protein (e.g., a cytidine deaminase), and the sequence of the activity domain can be considered a heterologous polypeptide (it is heterologous to the CRISPR-Cas effector protein).

[0010] The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the terms include, but are not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The terms "polynucleotide" and "nucleic acid" should be understood to include single-stranded (such as sense or antisense) and double-stranded polynucleotides, as applicable to the described embodiments.

[0011] The terms "polypeptide," "peptide," and "protein," as used interchangeably herein, refer to polymeric forms of amino acids of any length and can include genetically and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. The terms encompass fusion proteins, including, but not limited to, fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences, with or without an N-terminal methionine residue, immunologically tagged proteins, and the like.

[0012] As used herein, the term "naturally-occurring" when applied to a nucleic acid, protein, cell, or organism refers to a nucleic acid, cell, protein, or organism that is found in nature.

[0013] As used herein, the term "isolated" is intended to describe a polynucleotide, polypeptide, or cell that is in an environment that is different from that in which the polynucleotide, polypeptide, or cell naturally occurs. An isolated genetically modified host cell may be present in a mixed population of genetically modified host cells.

[0014] As used herein, "heterologous" refers to a nucleotide or amino acid sequence that is not found in a naturally occurring nucleic acid or protein, respectively. For example, with respect to a Cas9 polypeptide, a heterologous polypeptide comprises an amino acid sequence derived from a protein other than the Cas9 polypeptide. Thus, for example, a polymerase polypeptide is heterologous to a Cas9 polypeptide.

[0015] As used herein, "recombinant" means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, and / or ligation steps that result in a construct having structural coding or non-coding sequences distinguishable from the endogenous nucleic acid found in natural systems. Generally, nucleotide sequences encoding structural coding sequences can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of synthetic oligonucleotides, to provide a synthetic nucleic acid that can be expressed from a recombinant transcription unit contained in an cellular or cell-free transcription and translation system. Such sequences can be provided in the form of an open reading frame uninterrupted by internal untranslated sequences, or introns, typically present in eukaryotic genes. Genomic DNA containing the relevant nucleotide sequence can also be used to form a recombinant gene or transcription unit. Sequences of untranslated DNA can be present 5' or 3' from the open reading frame; such sequences do not interfere with the manipulation or expression of the coding region and may, in fact, act to regulate the production of a desired product by various mechanisms (see "DNA Regulatory Sequences," below).

[0016] Thus, for example, the terms "recombinant" polynucleotide or "recombinant" nucleic acid refer to something that does not occur in nature, e.g., something that is created through human intervention by the artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished either by chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acid, e.g., genetic engineering techniques. Such artificial combinations can be performed to join nucleic acid segments of desired functions together to produce a desired combination of functions.

[0017] Similarly, the term "recombinant" polypeptide refers to a polypeptide that does not occur in nature, e.g., that is made through human intervention by the artificial combination of two otherwise separated segments of amino acid sequence. Thus, for example, a polypeptide that includes a heterologous amino acid sequence is recombinant.

[0018] "Construct" or "vector" means a recombinant nucleic acid, generally a recombinant DNA, generated for the purpose of expressing and / or propagating a particular nucleotide sequence or used in the construction of other recombinant nucleotide sequences.

[0019] The terms "DNA regulatory sequence," "control element," and "regulatory element," used interchangeably herein, refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, proteolysis signals, and the like, that provide and / or regulate the expression of a coding sequence and / or the production of an encoded polypeptide in a host cell.

[0020] The term "transformation" is used interchangeably with "genetic modification" herein and refers to a permanent or transient genetic change induced in a cell after the introduction of new nucleic acid (e.g., DNA exogenous to the cell) into the cell. The genetic change ("modification") can be achieved by integrating the new nucleic acid into the host cell's genome or by transient or stable maintenance of the new nucleic acid as an episomal element. If the cell is eukaryotic, a permanent genetic change can be achieved by introducing the new DNA into the cell's genome. In prokaryotic cells, the permanent change can be introduced into the chromosome or via extrachromosomal elements such as plasmids and expression vectors, which may contain one or more selectable markers to aid in their maintenance in the recombinant host cell. Suitable methods of genetic modification include viral infection, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, and the like. The choice of method generally depends on the type of cell being transformed and the context in which the transformation is being performed (i.e., in vitro, ex vivo, or in vivo). A general description of these methods can be found in Ausubel, et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995.

[0021] "Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. As used herein, the terms "heterologous promoter" and "heterologous control region" refer to promoters and other control regions that are not normally associated with a particular nucleic acid in nature. For example, a "transcriptional control region heterologous to a coding region" is a transcriptional control region that is not normally associated with the coding region in nature.

[0022] As used herein, a "host cell" refers to a eukaryotic cell, a prokaryotic cell, or a cell derived from a multicellular organism (e.g., a cell line) cultured in vivo or in vitro as a unicellular organism; a eukaryotic or prokaryotic cell can be or has been used as a recipient of a nucleic acid (e.g., an expression vector), including the progeny of the original cell that was genetically modified with the nucleic acid. It is understood that the progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent due to natural, accidental, or deliberate mutation. A "recombinant host cell" (also referred to as a "genetically modified host cell") is a host cell into which a heterologous nucleic acid, e.g., an expression vector, has been introduced. For example, a eukaryotic host cell is one that has been genetically modified by the introduction into a suitable eukaryotic host cell of a heterologous nucleic acid, e.g., an exogenous nucleic acid that is heterologous to the eukaryotic host cell, or a recombinant nucleic acid not normally found in the eukaryotic host cell.

[0023] The term "conservative amino acid substitution" refers to the interchangeability of amino acid residues in proteins that have similar side chains. For example, the group of amino acids with aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic hydroxyl side chains consists of serine and threonine; the group of amino acids with amide-containing side chains consists of asparagine and glutamine; the group of amino acids with aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains consists of lysine, arginine, and histidine; and the group of amino acids with sulfur-containing side chains consists of cysteine ​​and methionine. Exemplary conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[0024] A polynucleotide or polypeptide has a certain percentage of "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, a percentage of the bases or amino acids are the same and in the same relative positions when comparing the two sequences. Sequence similarity can be determined in several different ways. To determine sequence identity, sequences can be aligned using methods and computer programs, including BLAST, available on the World Wide Web at ncbi.nlm.nih.gov / BLAST. See, e.g., Altschul et al. (1990), J. Mol. Biol. 215:403-10. Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package from Oxford Molecular Group, Inc., a wholly owned subsidiary of Oxford Molecular Group, Inc., Madison, Wisconsin, USA. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, California, USA. Of particular interest are alignment programs that allow for gaps in sequences. Smith-Waterman is one type of algorithm that allows for gaps in sequence alignments. See Meth. Mol. Biol. 70:173-187 (1997). Sequences can also be aligned using the GAP program, which uses the Needleman and Wunsch alignment method. See J. Mol. Biol. 48:443-453 (1970).

[0025] The terms "chimeric antigen receptor" and "CAR," used interchangeably herein, generally refer to an artificial multimodular molecule capable of inducing or inhibiting immune cell activation, including, but not limited to, an extracellular domain (e.g., a ligand / antigen-binding domain), a transmembrane domain, and one or more intracellular signaling domains. The term CAR is not specifically limited to CAR molecules, but also includes CAR variants. CAR variants include split CARs, in which the extracellular portion (e.g., a ligand-binding portion) and the intracellular portion (e.g., an intracellular signaling portion) of the CAR are present on two separate molecules. CAR variants include, for example, split CARs, as well as on-switch CARs, which are conditionally activatable CARs in which the conditional heterodimerization of the two portions of the split CAR is pharmacologically controlled. CAR variants also include bispecific CARs containing a secondary CAR-binding domain that can amplify or inhibit the activity of a primary CAR. CAR variants also include, for example, inhibitory chimeric antigen receptors (iCARs), which can be used as components of bispecific CAR systems, in which binding of the secondary CAR-binding domain results in inhibition of primary CAR activation.CAR molecules and their derivatives (i.e., CAR variants) have been described, for example, in PCT Application No. US2014 / 016527, Fedorov et al. Sci Transl Med (2013); 5(215):215ra172, Glienke et al. Front Pharmacol (2015) 6:21, Kakarla & Gottschalk 52 Cancer J (2014) 20(2):151-5, Riddell et al. Cancer J (2014) 20(2):141-4, Pegram et al. Cancer J (2014) 20(2):127-33, Cheadle et al. Immunol Rev (2014) 257(1):91-106, Barrett et al. Annu Rev Med (2014) 65:333-47, Sadelain et al. Cancer J (2014) 20(2):127-33, Cheadle et al. Immunol Rev (2014) 257(1):91-106, Barrett et al. Annu Rev Med (2014) 65:333-47, Sadelain et al. Cancer Discov (2013) 3(4):388-98; Cartellieri et al., J Biomed Biotechnol (2010) 956304, the disclosures of which are incorporated herein by reference in their entireties.

[0026] The terms "antibody" and "immunoglobulin" include antibodies or immunoglobulins of any isotype, fragments of antibodies that retain specific binding to an antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies (scAbs), single-domain antibodies (dAbs), single-domain heavy chain antibodies, single-domain light chain antibodies, nanobodies, bispecific antibodies, multispecific antibodies, ebibodies, immunobodies, diabodies, and fusion proteins, including antigen-binding (also referred to herein as antigen binding) portions of antibodies and non-antibody proteins.

[0027] The term "nanobody" (Nb), as used herein, refers to the smallest antigen-binding fragment or single variable domain (V) derived from a naturally occurring heavy chain antibody. HH) and are known to those skilled in the art. They are derived from the heavy chain-only antibodies found in the Camelidae family. In the "Camelidae" family, immunoglobulins that lack polypeptide light chains are found. "Camelidae" includes Old World camelids (Bactrian camel (Camelus bactrianus) and dromedary (Camelus dromedarius)) and New World camelids (e.g., alpacas (Llama paccos), llamas (Llama glama), guanacos (Llama guanicoe), and vicuñas (Llama vicugna)). Single variable domain heavy chain antibodies are herein referred to as nanobodies or V HH They are called antibodies.

[0028] "Single-chain Fv" or "sFv" or "scFv" antibody fragments are fragments of the V of an antibody. H and V L In some embodiments, the Fv polypeptide comprises V domains, and these domains are present in a single polypeptide chain. H Domains and V L The sFv further comprises a polypeptide linker between the domains that enables the sFv to form the desired structure for antigen binding. For a review of sFvs, see Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0029] As used herein, the term "antibody mimetic" refers to a compound that can specifically and / or selectively bind to an antigen or other target in a manner similar to an antibody, but that is not structurally related to an antibody. Antibody mimetics are typically artificial peptides or proteins, although they are not limited to such embodiments. Typically, antibody mimetics are smaller than antibodies, with molar masses of approximately 3-20 kDa (whereas antibodies are generally approximately 150 kDa). Non-limiting examples of antibody mimetics include peptide aptamers, affimers, affilins, affibodies, affitins, alphabodies, anticalins, avimers, DARPins, fynomers, Kunitz domain peptides, nanoCLAMPs, affinity reagents, and scaffold proteins.

[0030] As used herein, the terms "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that a disease or its symptoms are completely or partially prevented, and / or therapeutic, in that a partial or complete cure is provided for the disease and / or adverse effects resulting from the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, e.g., a human, and includes (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed as having the disease, (b) inhibiting the disease, i.e., arresting its development, and (c) alleviating the disease, i.e., causing regression of the disease.

[0031] The terms "individual," "subject," "host," and "patient," used interchangeably herein, refer to individual organisms, e.g., mammals, including, but not limited to, mice, monkeys, non-human primates, humans, mammalian farm animals, mammalian sport animals, and mammalian pets.

[0032] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0033] Where a range of values ​​is provided, unless the context clearly indicates otherwise, it is understood that each intervening value is included, to the tenth of the unit of the lower limit, between the upper and lower limit of that range and any other stated or intervening value in that stated range. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and may also be encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0035] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a "guide RNA" includes a plurality of such guide RNAs, a reference to a "targeting polypeptide" includes a plurality of such polypeptides, a reference to a "CRISPR-Cas effector polypeptide" includes a reference to one or more CRISPR-Cas effector polypeptides and equivalents thereof known to those skilled in the art, and so forth. It should be further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a guide precedent for the use of exclusive terminology such as "solely," "solely," and the like or the use of "negative" limitations in connection with the recitation of claim elements.

[0036] In the context of describing the present disclosure (particularly in the context of the claims that follow), the use of the terms "a," "an," and "the" and similar referents should be construed to cover both the singular and the plural unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to") unless expressly stated otherwise. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated herein as if it were individually recited herein. For example, if a range of 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any and all examples provided herein, or the use of exemplary language (e.g., "etc."), are intended merely to better clarify embodiments of the present disclosure and do not impose limitations on the scope of the present disclosure unless otherwise stated.

[0037] As used herein, the term "about" when used in connection with a quantity indicates that the quantity may vary by up to 10% of the stated amount. For example, "about 100" means an amount of 90 to 110. When "about" is used in the context of a range, "about" when used in connection with a lower amount in a range means that the lower amount includes an amount 10% lower than the lower amount in the range, and "about" when used in connection with a higher amount in a range means that the higher amount includes an amount 10% higher than the higher amount in the range. For example, about 100 to about 1000 means that the range extends to 90 to 1100.

[0038] As used herein, the term "and / or" is intended to mean that phrases such as "A and / or B" include both A and B, A or B, A alone, and B alone. Similarly, as used herein, the term "and / or" is intended to mean that phrases such as "A, B, and / or C" include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone.

[0039] It is understood that aspects and embodiments of the present disclosure described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0040] It is understood that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments pertaining to the present invention are specifically embraced by the present invention and are disclosed herein as if each and every combination were individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein as if each and every such subcombination were individually and explicitly disclosed herein.

[0041] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0042] Detailed Description The terms enveloped delivery vehicle (EDV) and virus-like particle (VLP) are used interchangeably herein as synonyms. Thus, any reference to one (e.g., EDV) is considered a reference to the other (e.g., VLP). Accordingly, any disclosure / discussion herein of an EDV should be considered equivalent to a disclosure / discussion of a VLP, and vice versa.

[0043] The present disclosure provides an enveloped delivery vehicle (EDV) comprising a nucleic acid-binding effector polypeptide or a nucleic acid encoding a nucleic acid-binding effector polypeptide, wherein the EDV comprises a fusion polypeptide comprising (i) a viral envelope protein and (ii) a targeting polypeptide that confers binding to a target cell. The present disclosure provides a method for delivering the nucleic acid-binding effector polypeptide into a eukaryotic cell using the EDV of the present disclosure.

[0044] The present disclosure provides an EDV comprising a CRISPR-Cas effector polypeptide or a nucleic acid encoding a CRISPR-Cas effector polypeptide, wherein the EDV comprises a fusion polypeptide comprising (i) a viral envelope protein and (ii) a targeting polypeptide that confers binding to a target cell. The present disclosure provides a method of delivering a CRISPR-Cas effector polypeptide into a eukaryotic cell using the EDV of the present disclosure.

[0045] In some cases, the EDV comprises a nucleic acid comprising a nucleotide sequence encoding a therapeutic polypeptide, such as a chimeric antigen receptor (CAR). In some cases, the EDV comprises one or more CRISPR-Cas guide nucleic acids, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CRISPR-Cas guide nucleic acids, where the one or more CRISPR-Cas guide nucleic acids result in knockout of an endogenous nucleic acid. In some cases, the EDV comprises (i) a donor template nucleic acid and (ii) one or more CRISPR-Cas guide nucleic acids, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CRISPR-Cas guide nucleic acids, where contacting the target nucleic acid with the CRISPR-Cas effector polypeptide, the one or more CRISPR-Cas guide nucleic acids, and the donor template nucleic acid results in insertion of the donor template nucleic acid into the target nucleic acid. In some cases, the donor template nucleic acid comprises a nucleotide sequence encoding a therapeutic polypeptide.

[0046] Envelope-bearing delivery vehicles (EDVs) containing one or more targeting polypeptides The present disclosure provides enveloped delivery vehicles (EDVs) comprising a nucleic acid-binding effector polypeptide or a nucleic acid comprising a nucleotide sequence encoding a nucleic acid-binding effector polypeptide, the EDV comprising a fusion polypeptide comprising (i) a viral envelope protein (e.g., a viral envelope glycoprotein) and (ii) a polypeptide that confers binding to a target cell (a "targeting polypeptide").

[0047] The present disclosure provides an enveloped delivery vehicle (EDV) comprising a nucleic acid-binding effector polypeptide or a nucleic acid comprising a nucleotide sequence encoding the nucleic acid-binding effector polypeptide, where the EDV comprises a fusion polypeptide comprising (i) a viral envelope protein (e.g., a viral envelope glycoprotein) and (ii) one or more antibodies or antibody analogs that specifically bind to a target polypeptide on a target cell. In some cases, the EDV comprises a therapeutic polypeptide or a nucleic acid comprising a nucleotide sequence encoding a therapeutic polypeptide, encapsidated within the EDV. The EDV can be used in in vivo genome editing methods, which are also provided. In some cases, the EDV comprises a CRISPR-Cas effector polypeptide as the nucleic acid-binding effector polypeptide. In some cases, the EDV comprises a nucleic acid comprising a nucleotide sequence encoding the CRISPR-Cas effector polypeptide. In some cases, an EDV of the present disclosure comprises an RNP comprising (a) a CRISPR-Cas effector polypeptide and (b) a CRISPR-Cas guide nucleic acid, where the guide nucleic acid (e.g., guide RNA) comprises a nucleotide sequence comprising: (i) a protein-binding segment comprising a nucleotide sequence that binds to the CRISPR-Cas effector polypeptide, and a target-binding segment comprising a nucleotide sequence complementary to a target nucleotide sequence of target DNA in a cell (e.g., a eukaryotic cell, e.g., a eukaryotic cell present in an individual). In some cases, an EDV of the present disclosure comprises a nucleic acid (e.g., a recombinant expression vector) comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide and a nucleotide sequence encoding a CRISPR-Cas guide RNA. In some cases, an EDV of the present disclosure comprises a donor nucleic acid.

[0048] viral envelope proteins As described above, the EDV of the present disclosure comprises a fusion polypeptide comprising (i) a viral envelope protein (e.g., a viral envelope glycoprotein) and (ii) a polypeptide that confers binding to a target cell.

[0049] Suitable viral envelope proteins include, for example, vesicular stomatitis virus (VSV) glycoprotein (VSV-G protein), measles virus hemagglutinin (HA) protein and / or measles virus fusion glycoprotein, influenza virus neuraminidase (NA) protein, measles virus F protein, influenza virus HA protein, Moloney virus MLV-A protein, Moloney virus MLV-E protein, baboon endogenous retrovirus (BAEV) envelope protein, Ebola virus glycoprotein, foamy virus envelope protein, and combinations or two or more of the foregoing viral envelope proteins.

[0050] In some cases, the viral envelope protein is a VSV-G protein. In some cases, the viral envelope protein is a measles virus hemagglutinin protein. In some cases, the viral envelope protein is a measles virus F protein. In some cases, the viral envelope protein is an influenza virus hemagglutinin protein. In some cases, the viral envelope protein is a Moloney virus MLV-A protein. In some cases, the viral envelope protein is a Moloney virus MLV-E protein. In some cases, the viral envelope protein is a baboon endogenous retrovirus envelope protein. In some cases, the viral envelope protein is an Ebola virus glycoprotein. In some cases, the viral envelope protein is a foamy virus envelope protein.

[0051] In some cases, the viral envelope protein is a VSV-G protein. Suitable VSV-G proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000002.tif49159 (SEQ ID NO: 18).

[0052] In some cases, a suitable VSV-G protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000003.tif49159 (SEQ ID NO: 19).

[0053] In some cases, a suitable VSV-G protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in Figure 16A.

[0054] In some cases, the viral envelope protein is a BAEV-G protein. Suitable BAEV-G proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000004.tif56159 (SEQ ID NO: 49).

[0055] In some cases, the viral envelope protein is an influenza virus H1N1 hemagglutinin glycoprotein. Suitable influenza hemagglutinin proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000005.tif62157 (SEQ ID NO: 50; GenBank Accession No. ACP44189). Such glycoproteins may be useful for targeting the EDVs of the present disclosure to cells of the respiratory system (e.g., cells of the lung), including, for example, epithelial cells, goblet cells, Clara cells, type I pneumocytes, type II pneumocytes, monocytes, macrophages, dendritic cells, neutrophils, and natural killer (NK) cells.

[0056] In some cases, the viral envelope protein is an influenza virus H3N2 hemagglutinin glycoprotein. Suitable influenza hemagglutinin proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000006.tif62156 (SEQ ID NO: 51; GenBank Accession No: YP_308839). Such glycoproteins may be useful for targeting the EDVs of the present disclosure to cells of the respiratory system (e.g., cells of the lung), including, for example, epithelial cells, goblet cells, Clara cells, type I pneumocytes, type II pneumocytes, monocytes, macrophages, dendritic cells, neutrophils, and natural killer (NK) cells.

[0057] In some cases, the viral envelope protein is an influenza virus A H5N1 hemagglutinin glycoprotein. Suitable influenza hemagglutinin proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000007.tif62159 (SEQ ID NO: 52; GenBank Accession No: YP_308669). Such glycoproteins may be useful for targeting the EDVs of the present disclosure to cells of the respiratory system (e.g., cells of the lung), including, for example, epithelial cells, goblet cells, Clara cells, type I pneumocytes, type II pneumocytes, monocytes, macrophages, dendritic cells, neutrophils, and NK cells.

[0058] In some cases, the viral envelope protein is an influenza virus H7N9 hemagglutinin glycoprotein. Suitable influenza hemagglutinin proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000008.tif62155 (SEQ ID NO: 53; GenBank Accession No: YP_009118475). Such glycoproteins may be useful for targeting the EDVs of the present disclosure to cells of the respiratory system (e.g., cells of the lung), including, for example, epithelial cells, goblet cells, Clara cells, type I pneumocytes, type II pneumocytes, monocytes, macrophages, dendritic cells, neutrophils, and NK cells.

[0059] In some cases, the viral envelope protein is Hepatitis B virus (HBV) S glycoprotein. Suitable HBV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000009.tif23149 (SEQ ID NO: 54; GenBank Accession No.: ABV02793). Such heterologous glycoproteins may be useful for targeting the EDV of the present disclosure to hepatocytes.

[0060] In some cases, the viral envelope protein is the hepatitis B virus (HBV) middle S glycoprotein. Suitable HBV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000010.tif30152 (SEQ ID NO: 55; GenBank Accession No.: ACJ66136). Such heterologous glycoproteins may be useful for targeting the EDV of the present disclosure to hepatocytes.

[0061] In some cases, the viral envelope protein is the hepatitis B virus (HBV) large S glycoprotein. Suitable HBV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000011.tif43154 (SEQ ID NO: 56; GenBank Accession No.: AGR65633). Such heterologous glycoproteins may be useful for targeting the EDV of the present disclosure to hepatocytes.

[0062] In some cases, the viral envelope protein is the hepatitis B virus (HBV) small S glycoprotein. Suitable HBV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000012.tif23150 (SEQ ID NO: 57; GenBank Accession No.: AHC69850. Such heterologous glycoproteins may be useful for targeting the EDV of the present disclosure to hepatocytes.

[0063] In some cases, the viral envelope protein is Hepatitis B virus (HBV) pre-S glycoprotein. Suitable HBV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000013.tif43155 (SEQ ID NO: 58; GenBank Accession No.: CAA66700). Such heterologous glycoproteins may be useful for targeting the EDV of the present disclosure to hepatocytes.

[0064] In some cases, the viral envelope protein is Hepatitis B virus (HBV) pre-S2 glycoprotein. Suitable HBV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000014.tif30153 (SEQ ID NO: 59; GenBank Accession No.: AAO12662). Such heterologous glycoproteins may be useful for targeting the EDV of the present disclosure to hepatocytes.

[0065] In some cases, the viral envelope protein is a rabies virus. Suitable rabies virus proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000015.tif56159 (SEQ ID NO: 60; GenBank Accession No.: AWR88358). Such glycoproteins may be useful for targeting the EDVs of the present disclosure to neurons, astrocytes, oligodendrocytes, glia, and other cells of the central nervous system.

[0066] In some cases, the viral envelope protein is a Mokola virus glycoprotein. Suitable Mokola virus proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000016.tif56156 (SEQ ID NO: 61; GenBank Accession No.: AAB26292). Such glycoproteins may be useful for targeting the EDVs of the present disclosure to neurons, astrocytes, oligodendrocytes, glia, and other cells of the central nervous system.

[0067] In some cases, the viral envelope protein is a lymphocytic choriomeningitis virus (LCMV) glycoprotein. Suitable LCMV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000017.tif55158 (SEQ ID NO: 62; GenBank accession number: AIW66623).

[0068] In some cases, the viral envelope protein is lymphocytic choriomeningitis virus (LCMV) glycoprotein C. Suitable LCMV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000018.tif55157 (SEQ ID NO: 63; GenBank accession number: CAC01231).

[0069] In some cases, the viral envelope protein is a lymphocytic choriomeningitis virus (LCMV) glycoprotein. Suitable LCMV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000019.tif55158 (SEQ ID NO: 64; GenBank accession number: P09991).

[0070] In some cases, the viral envelope protein is lymphocytic choriomeningitis virus (LCMV) G1 glycoprotein. Suitable LCMV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000020.tif23158 (SEQ ID NO: 65; GenBank accession number: P09991).

[0071] In some cases, the viral envelope protein is lymphocytic choriomeningitis virus (LCMV) G2 glycoprotein. Suitable LCMV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000021.tif29156 (SEQ ID NO: 66; GenBank accession number: P09991).

[0072] In some cases, the viral envelope protein is Ross River virus E1 glycoprotein. Suitable Ross River virus proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000022.tif49155 (SEQ ID NO: 67; GenBank Accession No.: NP_740686). Such glycoproteins may be useful for targeting the EDVs of the present disclosure to cells that make up skeletal muscle, as well as joints, joint-associated connective tissue, bone, neurons, and lymphatic cells.

[0073] In some cases, the viral envelope protein is Ross River virus E2 glycoprotein. Suitable Ross River virus proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000023.tif49158 (SEQ ID NO: 68; GenBank Accession No.: NP_740684). Such glycoproteins may be useful for targeting the EDVs of the present disclosure to cells that make up skeletal muscle, as well as joints, joint-associated connective tissue, bone, neurons, and lymphatic cells.

[0074] In some cases, the viral envelope protein is the Semliki Forest virus E1 glycoprotein. Suitable Semliki Forest virus proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000024.tif49157 (SEQ ID NO: 69; GenBank Accession No: NP_819008). Such glycoproteins may be useful for targeting the EDVs of the present disclosure to muscle, pancreas, neurons, astrocytes, oligodendrocytes, glia, and other cells of the central nervous system.

[0075] In some cases, the viral envelope protein is the Semliki Forest virus E2 glycoprotein. Suitable Semliki Forest virus proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000025.tif49157 (SEQ ID NO: 48; GenBank Accession No: NP_819006). Such glycoproteins may be useful for targeting the EDVs of the present disclosure to muscle, pancreas, neurons, astrocytes, oligodendrocytes, glia, and other cells of the central nervous system.

[0076] In some cases, the viral envelope protein is Sindbis virus E1 glycoprotein. Suitable Sindbis virus proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000026.tif49156 (SEQ ID NO: 70; GenBank Accession No: NP_740677). Such glycoproteins may be useful for targeting the EDVs of the present disclosure to muscle, pancreas, neurons, astrocytes, oligodendrocytes, glia, and other cells of the central nervous system.

[0077] In some cases, the viral envelope protein is Sindbis virus E2 glycoprotein. Suitable Sindbis virus proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000027.tif49155 (SEQ ID NO: 71; GenBank Accession No.: NP_740675). Such glycoproteins may be useful for targeting the EDVs of the present disclosure to cells that make up skeletal muscle, as well as joints, joint-associated connective tissue, bone, neurons, and lymphatic cells.

[0078] In some cases, the viral envelope protein is an Ebola Zaire virus glycoprotein. Suitable Ebola Zaire virus proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000028.tif75158 (SEQ ID NO: 72; GenBank Accession No.: AAB81004). Such glycoproteins may be useful for targeting the EDVs of the present disclosure to hepatocytes, endothelial cells, dendritic cells, macrophages, and monocytes.

[0079] In some cases, the viral envelope protein is an Ebola Zaire virus glycoprotein. Suitable Ebola Zaire virus proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000029.tif69154 (SEQ ID NO: 73). Such glycoproteins may be useful for targeting the EDVs of the present disclosure to hepatocytes, endothelial cells, dendritic cells, macrophages, and monocytes.

[0080] In some cases, the viral envelope protein is an Ebola Reston virus glycoprotein. Suitable Ebola Reston virus proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000030.tif75155 (SEQ ID NO: 74; GenBank Accession No: NP_690583). Such glycoproteins may be useful for targeting the EDVs of the present disclosure to hepatocytes, endothelial cells, dendritic cells, macrophages, and monocytes.

[0081] In some cases, the viral envelope protein is a Marburg virus glycoprotein. Suitable Marburg virus proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000031.tif75158 (SEQ ID NO: 75); GenBank Accession No: CAA78117). Such glycoproteins may be useful for targeting the EDVs of the present disclosure to hepatocytes, endothelial cells, dendritic cells, macrophages, and monocytes.

[0082] In some cases, the viral envelope protein is a murine leukemia virus (MLV) glycoprotein. Suitable MLV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000032.tif75158 (SEQ ID NO: 76; GenBank accession number: AAA51037).

[0083] In some cases, the viral envelope protein is an MLV glycoprotein. Suitable MLV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000033.tif75158 (SEQ ID NO: 77; GenBank accession number: AID54959).

[0084] In some cases, the viral envelope protein is an MLV glycoprotein. Suitable MLV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000034.tif69158 (SEQ ID NO: 78; GenBank accession number: AAA46515).

[0085] In some cases, the viral envelope protein is an MLV glycoprotein. Suitable MLV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000035.tif69160 (SEQ ID NO: 79; GenBank accession number: AAA46514).

[0086] In some cases, the viral envelope protein is an MLV glycoprotein. Suitable MLV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000036.tif69157 (SEQ ID NO: 80; GenBank accession number: AAA46531).

[0087] In some cases, the viral envelope protein is a polytropic mink cell focus-forming virus glycoprotein. Suitable polytropic mink cell focus-forming virus proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000037.tif49158 (SEQ ID NO: 81; GenBank accession number: 2016415A).

[0088] In some cases, the viral envelope protein is a gibbon ape leukemia virus (GALV) glycoprotein. Suitable GALV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000038.tif75159 (SEQ ID NO: 82; GenBank accession number: P21415).

[0089] In some cases, the viral envelope protein is a GALV glycoprotein. Suitable GALV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000039.tif69158 (SEQ ID NO: 83).

[0090] In some cases, the viral envelope protein is a GALV glycoprotein. Suitable GALV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000040.tif69158 (SEQ ID NO: 83).

[0091] In some cases, the viral envelope protein is a GALV glycoprotein. Suitable GALV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000041.tif49158 (SEQ ID NO: 84).

[0092] In some cases, the viral envelope protein is a GALV glycoprotein. Suitable GALV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000042.tif17154 (SEQ ID NO: 85).

[0093] In some cases, the viral envelope protein is the RD114 retroviral glycoprotein. Suitable RD114 retroviral proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000043.tif63156 (SEQ ID NO: 86; GenBank accession number: YP_001497149).

[0094] In some cases, the viral envelope protein is a Sendai virus (SeV) glycoprotein. Suitable SeV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000044.tif62154 (SEQ ID NO: 87; GenBank accession number: P04855).

[0095] In some cases, the viral envelope protein is SeV F0 glycoprotein. Suitable SeV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000045.tif62154 (SEQ ID NO: 88; GenBank accession number: P04855).

[0096] In some cases, the viral envelope protein is SeV F2 glycoprotein. Suitable SeV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000046.tif10136 (SEQ ID NO: 89; GenBank accession number: P04855).

[0097] In some cases, the viral envelope protein is SeV F1 glycoprotein. Suitable SeV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000047.tif49159 (SEQ ID NO: 90; GenBank accession number: P04855).

[0098] In some cases, the viral envelope protein is the SeV hemagglutinin-neuraminidase glycoprotein. Suitable SeV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000048.tif62153 (SEQ ID NO: 91; GenBank accession number: BAA24391).

[0099] In some cases, the viral envelope protein is the Jaagsiekte ovine retrovirus (JSRV) glycoprotein. Suitable JSRV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000049.tif69154 (SEQ ID NO: 92; GenBank accession number: ABI50237).

[0100] In some cases, the viral envelope protein is a baculovirus gp64 glycoprotein. Suitable baculovirus proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000050.tif55157 (SEQ ID NO: 93; GenBank accession number: YP_009182316).

[0101] In some cases, the viral envelope protein is a baculovirus gp64 glycoprotein. Suitable baculovirus proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000051.tif55157 (SEQ ID NO: 94; GenBank accession number: YP_473216).

[0102] In some cases, the viral envelope protein is a Chandipura virus glycoprotein. Suitable Chandipura virus proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000052.tif55157 (SEQ ID NO: 95; GenBank accession number: YP_007641380).

[0103] In some cases, the viral envelope protein is a Venezuelan Equine Encephalitis virus glycoprotein. Suitable Venezuelan Equine Encephalitis virus proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000053.tif141160 (SEQ ID NO: 96; GenBank Accession No.: AAU89534). Such glycoproteins may be useful for the EDVs of the present disclosure to target dendritic cells, macrophages, and cells of the spleen, lymph nodes, thymus, pancreas, skeletal muscle, and central nervous system.

[0104] In some cases, the viral envelope protein is Venezuelan Equine Encephalitis virus E2 glycoprotein. Suitable Venezuelan Equine Encephalitis virus proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000054.tif49155 (SEQ ID NO: 97; GenBank Accession No.: AAU89534). Such glycoproteins may be useful for the EDVs of the present disclosure to target dendritic cells, macrophages, and cells of the spleen, lymph nodes, thymus, pancreas, skeletal muscle, and central nervous system.

[0105] In some cases, the viral envelope protein is Venezuelan Equine Encephalitis virus E1 glycoprotein. Suitable Venezuelan Equine Encephalitis virus proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000055.tif49159 (SEQ ID NO: 98; GenBank Accession No.: AAU89534). Such glycoproteins may be useful for the EDVs of the present disclosure to target dendritic cells, macrophages, and cells of the spleen, lymph nodes, thymus, pancreas, skeletal muscle, and central nervous system.

[0106] In some cases, the viral envelope protein is a Lassa virus glycoprotein. Suitable Lassa virus proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000056.tif56159 (SEQ ID NO: 99; GenBank accession number: ADY11070).

[0107] In some cases, the viral envelope protein is an avian leukosis virus glycoprotein. Suitable avian leukosis virus proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000057.tif69155 (SEQ ID NO: 100; GenBank accession number: ADO34853).

[0108] In some cases, the viral envelope protein is an avian leukosis virus glycoprotein. Suitable avian leukosis virus proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000058.tif69158 (SEQ ID NO: 101; GenBank accession number: AEF97639).

[0109] In some cases, the viral envelope protein is an avian leukosis virus glycoprotein. Suitable avian leukosis virus proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000059.tif62157 (SEQ ID NO: 102; GenBank accession number: AWM62167).

[0110] In some cases, the viral envelope protein is a human T-lymphotropic virus 1 (HTLV-1) glycoprotein. Suitable HTLV-1 proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000060.tif56156 (SEQ ID NO: 103; GenBank Accession No: AAU04884). Such glycoproteins may be useful for the EDV of the present disclosure to target CD4+ and CD8+ T cells.

[0111] In some cases, the viral envelope protein is a human foamy virus gp130 glycoprotein. Suitable human foamy virus proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000061.tif108159 (SEQ ID NO: 104; GenBank accession number: P14351).

[0112] In some cases, the viral envelope protein is a human foamy virus glycoprotein. Suitable human foamy virus proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000062.tif49159 (SEQ ID NO: 105).

[0113] In some cases, the viral envelope protein is a human foamy virus glycoprotein. Suitable human foamy virus proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000063.tif49157 (SEQ ID NO: 106).

[0114] In some cases, the viral envelope protein is the visna-maedi virus gp160 glycoprotein. Suitable visna-maedi virus proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000064.tif114159 (SEQ ID NO: 107; GenBank accession number: P35954).

[0115] In some cases, the viral envelope protein is a Visna-Maedi virus glycoprotein. Suitable Visna-Maedi virus proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000065.tif102159 (SEQ ID NO: 108).

[0116] In some cases, the viral envelope protein is a Visna-Maedi virus glycoprotein. Suitable Visna-Maedi virus proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000066.tif101159 (SEQ ID NO: 108).

[0117] In some cases, the viral envelope protein is a Visna-Maedi virus glycoprotein. Suitable Visna-Maedi virus proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000067.tif62157 (SEQ ID NO: 109).

[0118] In some cases, the viral envelope protein is a Visna-Maedi virus glycoprotein. Suitable Visna-Maedi virus proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000068.tif36159 (SEQ ID NO: 110).

[0119] In some cases, the viral envelope protein is the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein. Suitable SARS-CoV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000069.tif140158 (SEQ ID NO: 111; GenBank Accession No.: ABA02260). Such glycoproteins may be useful for targeting the EDVs of the present disclosure to cells of the respiratory system (e.g., cells of the lung), including, for example, epithelial cells, goblet cells, Clara cells, type I pneumocytes, type II pneumocytes, monocytes, macrophages, dendritic cells, neutrophils, and NK cells.

[0120] In some cases, the viral envelope protein is the SARS-CoV S2 glycoprotein. Suitable SARS-CoV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000070.tif68157 (SEQ ID NO: 112; GenBank Accession No.: ABD73002). Such glycoproteins may be useful for targeting the EDVs of the present disclosure to cells of the respiratory system (e.g., cells of the lung), including, for example, epithelial cells, goblet cells, Clara cells, type I pneumocytes, type II pneumocytes, monocytes, macrophages, dendritic cells, neutrophils, and NK cells.

[0121] In some cases, the viral envelope protein is a SARS-CoV spike receptor-binding domain glycoprotein. Suitable SARS-CoV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000071.tif29154 (SEQ ID NO: 113; GenBank Accession No.: ABD73002). Such glycoproteins may be useful for targeting the EDVs of the present disclosure to cells of the respiratory system (e.g., cells of the lung), including, for example, epithelial cells, goblet cells, Clara cells, type I pneumocytes, type II pneumocytes, monocytes, macrophages, dendritic cells, neutrophils, and NK cells.

[0122] In some cases, the viral envelope protein is respiratory syncytial virus (RSV) glycoprotein G. Suitable RSV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000072.tif36149 (SEQ ID NO: 114; UniProtKB: P03423-1). Such glycoproteins may be useful for targeting the EDVs of the present disclosure to cells of the respiratory system (e.g., cells of the lung), including, for example, epithelial cells, goblet cells, Clara cells, type I pneumocytes, type II pneumocytes, monocytes, macrophages, dendritic cells, neutrophils, and NK cells.

[0123] In some cases, the viral envelope protein is RSV glycoprotein F. Suitable RSV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000073.tif62155 (SEQ ID NO: 115; GenBank Accession No: P03420). Such glycoproteins may be useful for targeting the EDVs of the present disclosure to cells of the respiratory system (e.g., cells of the lung), including, for example, epithelial cells, goblet cells, Clara cells, type I pneumocytes, type II pneumocytes, monocytes, macrophages, dendritic cells, neutrophils, and NK cells.

[0124] In some cases, the viral envelope protein is a RSV glycoprotein. Suitable RSV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000074.tif62156 (SEQ ID NO: 116). Such glycoproteins may be useful for targeting the EDVs of the present disclosure to cells of the respiratory system (e.g., cells of the lung), including, for example, epithelial cells, goblet cells, Clara cells, type I pneumocytes, type II pneumocytes, monocytes, macrophages, dendritic cells, neutrophils, and NK cells.

[0125] In some cases, the viral envelope protein is the RSV F0 glycoprotein. Suitable RSV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000075.tif62156 (SEQ ID NO: 116; GenBank Accession No: P03420). Such glycoproteins may be useful for targeting the EDVs of the present disclosure to cells of the respiratory system (e.g., cells of the lung), including, for example, epithelial cells, goblet cells, Clara cells, type I pneumocytes, type II pneumocytes, monocytes, macrophages, dendritic cells, neutrophils, and NK cells.

[0126] In some cases, the viral envelope protein is the RSV F2 glycoprotein. Suitable RSV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000076.tif10147 (SEQ ID NO: 117; GenBank Accession No: P03420). Such glycoproteins may be useful for targeting the EDVs of the present disclosure to cells of the respiratory system (e.g., cells of the lung), including, for example, epithelial cells, goblet cells, Clara cells, type I pneumocytes, type II pneumocytes, monocytes, macrophages, dendritic cells, neutrophils, and NK cells.

[0127] In some cases, the viral envelope protein is the RSV F1 glycoprotein. Suitable RSV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000077.tif49155 (SEQ ID NO: 118; GenBank Accession No: P03420). Such glycoproteins may be useful for targeting the EDV of the present disclosure to lung / respiratory cells.

[0128] In some cases, the viral envelope protein is a RSV glycoprotein. Suitable RSV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000078.tif63156 (SEQ ID NO: 116). Such glycoproteins may be useful for targeting the EDV of the present disclosure to lung / respiratory cells.

[0129] In some cases, the viral envelope protein is a human parainfluenza virus type 3 hemagglutinin-neuraminidase glycoprotein. Suitable human parainfluenza virus type 3 proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000079.tif62154 (SEQ ID NO: 119; GenBank Accession No.: AAP35240). Such glycoproteins may be useful for targeting the EDVs of the present disclosure to cells of the respiratory system (e.g., cells of the lung), including, for example, epithelial cells, goblet cells, Clara cells, type I pneumocytes, type II pneumocytes, monocytes, macrophages, dendritic cells, neutrophils, and NK cells.

[0130] In some cases, the viral envelope protein is human parainfluenza virus type 3 glycoprotein F0. Suitable human parainfluenza virus type 3 proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000080.tif62152 (SEQ ID NO: 120; GenBank Accession No.: AXA52708). Such glycoproteins may be useful for targeting the EDVs of the present disclosure to cells of the respiratory system (e.g., cells of the lung), including, for example, epithelial cells, goblet cells, Clara cells, type I pneumocytes, type II pneumocytes, monocytes, macrophages, dendritic cells, neutrophils, and NK cells.

[0131] In some cases, the viral envelope protein is Hepatitis C virus (HCV) E1 glycoprotein. Suitable HCV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000081.tif23158 (SEQ ID NO: 121; GenBank Accession No: NP_751920). Such glycoproteins may be useful for targeting the EDV of the present disclosure to hepatocytes.

[0132] In some cases, the viral envelope protein is the HCV E2 glycoprotein. Suitable HCV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000082.tif42157 (SEQ ID NO: 122; GenBank Accession No: NP_751921). Such glycoproteins may be useful for targeting the EDV of the present disclosure to hepatocytes.

[0133] In some cases, the viral envelope protein is a fowl plague virus glycoprotein. Suitable fowl plague virus proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000083.tif62155 (SEQ ID NO: 123; GenBank accession number: 0601245A).

[0134] In some cases, the viral envelope protein is Autographa californica nuclear polyhedrosis virus (AcMNPV) major envelope glycoprotein gp64. Suitable AcMNPV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000084.tif56156 (SEQ ID NO: 158; UniProt accession number: P17501-1).

[0135] In some cases, the viral envelope protein is an AcMNPV glycoprotein. Suitable AcMNPV proteins include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000085.tif56159 (SEQ ID NO: 124).

[0136] In some cases, the viral envelope protein is a measles virus hemagglutinin (H) polypeptide. See, e.g., Levy et al. (2017) Blood Adv. 1:2088. Suitable measles virus H polypeptides include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000086.tif69156 (SEQ ID NO: 155). The EDVs of the present disclosure are capable of targeting T cells, B cells, monocytes, macrophages, dendritic cells, and hematopoietic stem cells (e.g., CD34 + Such glycoproteins may be useful for targeting specific target cells.

[0137] In some cases, the viral envelope protein is a measles virus fusion (F) polypeptide. Suitable measles virus F polypeptides include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000087.tif62153 (SEQ ID NO: 126). The EDVs of the present disclosure are capable of targeting T cells, B cells, monocytes, macrophages, dendritic cells, and hematopoietic stem cells (e.g., CD34 + Such glycoproteins may be useful for targeting specific target cells.

[0138] Variant viral envelope proteins In some cases, the EDV of the present disclosure comprises a fusion polypeptide comprising (i) a variant viral envelope protein and (ii) a targeting polypeptide that confers binding to a target cell, wherein the variant viral envelope protein comprises one or more amino acid substitutions compared to the wild-type viral envelope protein, and wherein the variant viral envelope protein exhibits reduced binding to its native receptor compared to the binding of the wild-type viral envelope protein to its native receptor. In some cases, the variant viral envelope protein retains endosomal fusion activity.

[0139] In some cases, the viral envelope protein is a variant VSV-G protein comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the VSV-G amino acid sequence shown in Figure 16A, wherein the variant VSV-G protein comprises one or more amino acid substitutions compared to the wild-type viral envelope protein (compared to the amino acid sequence shown in Figure 16A), and wherein the variant viral envelope protein exhibits reduced binding to its native receptor compared to the binding of a VSV-G polypeptide comprising the amino acid sequence shown in Figure 16A to its native receptor. The native receptor for VSV-G is the low-density lipoprotein receptor (LDLR).

[0140] In some cases, the VSV-G polypeptide contains one or more amino acid substitutions that reduce binding to a natural receptor for VSV-G while retaining the endosomal fusion function of the VSV-G polypeptide. In some cases, the viral envelope protein is a VSV-G protein comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the VSV-G amino acid sequence shown in Figure 16A, wherein amino acid 47 is other than Lys. In some cases, the viral envelope protein is a VSV-G protein comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the VSV-G amino acid sequence shown in Figure 16A, wherein amino acid 354 is other than Arg. In some cases, the viral envelope protein is a VSV-G protein comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the VSV-G amino acid sequence shown in Figure 16A, wherein amino acid 47 is other than Lys and amino acid 354 is other than Arg. In some cases, Lys at amino acid 47 is substituted with Ala. In some cases, Lys at amino acid 47 is substituted with Gln. In some cases, Arg at amino acid 354 is substituted with Ala. In some cases, Arg at amino acid 354 is substituted with Gln.

[0141] In some cases, the viral envelope protein is a VSV-G protein comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the VSV-G amino acid sequence shown in Figure 16B, wherein the VSV-G protein comprises a Gln at position 47 and an Ala at position 354.

[0142] In some cases, the viral envelope protein is a variant measles hemagglutinin (HA) protein comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the measles HA amino acid sequence shown in Figure 16C, wherein the variant measles HA protein comprises one or more amino acid substitutions compared to a wild-type viral envelope protein (compared to the amino acid sequence shown in Figure 16C), and the variant viral envelope protein exhibits reduced binding to its native receptor compared to the binding of a measles HA protein comprising the amino acid sequence shown in Figure 16C to its native receptor. In some cases, the variant measles HA protein comprises one or more substitutions of Y481, R533, S548, and F549, based on the amino acid numbering shown in Figure 16C. CD46 is the native receptor for measles virus HA.

[0143] In some cases, the viral envelope protein is a variant measles HA protein comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the measles HA amino acid sequence shown in Figure 16D, where amino acid 841 is other than Tyr, amino acid 533 is other than Arg, amino acid 548 is other than Ser, and amino acid 549 is other than Phe. In some cases, the viral envelope protein is a variant measles HA protein comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the measles HA amino acid sequence shown in Figure 16D, where amino acid 841 is Ala, amino acid 533 is Ala, amino acid 548 is Leu, and amino acid 549 is Ser.

[0144] Targeting Polypeptides The EDV of the present disclosure comprises a fusion polypeptide comprising (i) a viral glycoprotein and (ii) a targeting polypeptide (i.e., one or more targeting polypeptides) that confers binding to a target cell or target cell type. Targeting polypeptides include antibodies and antibody mimetics (also referred to as antibody analogs). Suitable antibody analogs include, for example, affibodies, affilins, affimers, affitins, alphabodies, anticalins, avimers, DARPins, finomers, Kunitz domain peptides, monobodies, repebodies, VLRs, and nanoCLAMPs. Suitable antibodies include single-chain Fv (scFv) polypeptides, diabodies, triabodies, and nanobodies. In some cases, the antibody is a single-chain Fv polypeptide. In some cases, the antibody is a nanobody. In some cases, the antibody is a bispecific antibody. In some cases, the EDV of the present disclosure comprises a fusion polypeptide comprising (i) a viral envelope protein and (ii) one or more antibodies or antibody analogs that specifically bind to a target polypeptide on a target cell. In some cases, the EDV of the present disclosure comprises a fusion polypeptide comprising (i) a viral envelope protein and (ii) two different antibodies (e.g., a first antibody and a second antibody), where the first antibody specifically binds to a first target polypeptide on a target cell and the second antibody specifically binds to a second target polypeptide on the same target cell.

[0145] In some examples, an EDV is provided that comprises two or more different targeting polypeptides. In some cases, an EDV is provided that comprises a bispecific targeting polypeptide, where the bispecific targeting polypeptide binds to two different targets on a targeted cell type. In some cases, the bispecific targeting polypeptide is a bispecific antibody or a derivative thereof.

[0146] In some cases, the targeting polypeptide provides selective binding to organs such as the kidney, liver, bone, pancreas, brain, lung, or heart. In some cases, the targeting polypeptide provides selective binding to a particular cell type. For example, in some cases, the targeting polypeptide provides selective binding to cells such as skeletal muscle cells, cardiomyocytes, adipocytes, epithelial cells, endothelial cells, macrophages, beta pancreatic islet cells, or immune cells (e.g., T cells, B cells, monocytes, natural killer cells, dendritic cells, etc.). In some cases, the targeting polypeptide provides selective binding to diseased cells compared to non-diseased cells of the same cell type. In some cases, the antibody provides selective binding to CAR-T cells, i.e., T cells modified to express a chimeric antigen receptor (CAR) on their surface.

[0147] Fusion Polypeptides In some cases, for example, when the antibody is an scFv or nanobody, the antibody itself is a fusion polypeptide comprising (i) the antibody and (ii) a heterologous polypeptide (a "fusion partner"). The fusion partner can be a polypeptide that improves the accessibility of the antibody to a target cell. Suitable fusion partners include, but are not limited to, a stalk portion of a polypeptide, a stalk and transmembrane domain of a polypeptide, an immunoglobulin hinge polypeptide, a linker polypeptide, and the like.

[0148] In some cases, the fusion partner is the stalk and transmembrane domain of a transmembrane protein. A "transmembrane domain," as used herein, is a portion of a transmembrane protein that includes a hydrophobic portion that can insert into or span a cell membrane. A transmembrane component or domain is thermodynamically stable within a cell membrane and generally has a three-dimensional structure that ranges in length from about 15 amino acids to about 30 amino acids. The structure of the transmembrane component or domain can include an alpha helix, a beta barrel, a beta sheet, a beta helix, or any combination thereof. In certain embodiments, the transmembrane component or domain includes or is derived from a known transmembrane protein (e.g., a CD4 transmembrane domain, a CD8 transmembrane domain, a CD27 transmembrane domain, a CD28 transmembrane domain, or any combination thereof).

[0149] In some cases, the fusion partner is the stalk and transmembrane domain of a CD8α polypeptide. In one example, the stalk and transmembrane domain has the following amino acid sequence: TIFF2025529869000088.tif10158 (SEQ ID NO: 20), and the stalk can comprise the amino acid sequence TIFF2025529869000089.tif4128 (SEQ ID NO: 21), and the TMD has the sequence TIFF2025529869000090.tif3128 (SEQ ID NO: 22). In another example, the fusion partner is the stalk domain of a CD8α polypeptide, e.g., the fusion partner has the amino acid sequence: Contains TIFF2025529869000091.tif4128 (SEQ ID NO: 21).

[0150] In some cases, the fusion partner is the stalk and transmembrane domain of the platelet-derived growth factor receptor (PDGFR).

[0151] In some cases, the fusion partner is a glycine-rich polypeptide having a length of 5 amino acids to about 50 amino acids, for example, the fusion partner comprises the sequence (GGGGS)n (SEQ ID NO: 38), where n is an integer between 1 and 10, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, for example, n is 3.

[0152] In some cases, the fusion partner is an immunoglobulin (Ig) hinge polypeptide. As used herein, "hinge polypeptide," "hinge region," or "hinge" refers to (a) an immunoglobulin hinge sequence (e.g., composed of the upper and core regions of an immunoglobulin hinge) or a functional fragment or variant thereof, (b) a type II C-lectin interdomain (stalk) region or a functional fragment or variant thereof, or (c) a cluster of differentiation (CD) molecule stalk region or a functional variant thereof. As used herein, "wild-type immunoglobulin hinge region" refers to the naturally occurring upper and middle hinge amino acid sequences found in the heavy chain of an antibody, inserted between and connected to the CH1 and CH2 domains (in the case of IgG, IgA, and IgD) or the CH1 and CH3 domains (in the case of IgE and IgM).

[0153] Antibodies targeting T cells In some cases, the targeting polypeptide is an antibody that targets (specifically binds to) an antigen expressed on the surface of a T cell, thereby targeting the EDV to the T cell. In some cases, the T cell is a CD4 + T cells. In some cases, the T cells are CD8 +The targeting polypeptide is a T cell. In some cases, the antibody is an scFv or nanobody that binds CD4. In some cases, the antibody is an scFv or nanobody that binds CD3. In some cases, the antibody is an scFv or nanobody that binds CD8. In some cases, the antibody is an scFv or nanobody that binds CD28. In some cases, the targeting polypeptide comprises one or more antibodies, for example, one or a combination of anti-CD3 (e.g., CD3 scFv-3), anti-CD4 (e.g., CD4 scFv-2), and anti-CD28 (e.g., CD28 scFv-2) (e.g., anti-CD3 and anti-CD4 antibodies, anti-CD3 and anti-CD28 antibodies, anti-CD3, anti-CD4 and anti-CD28 antibodies, etc.).

[0154] In some cases, the EDV comprises a fusion polypeptide comprising (i) a viral envelope protein, (ii) an anti-CD3 antibody, and (iii) an anti-CD8 antibody. In some cases, the EDV comprises a fusion polypeptide comprising (i) a viral envelope protein, (ii) an anti-CD3 antibody, and (iii) an anti-CD28 antibody. In some cases, the EDV comprises a fusion polypeptide comprising (i) a viral envelope protein, (ii) an anti-CD3 antibody, and (iii) an anti-CD4 antibody. In some cases, the EDV comprises a fusion polypeptide comprising (i) a viral envelope protein, (ii) an anti-CD3 antibody, and (iii) an anti-CD28 antibody. In some cases, the EDV comprises a fusion polypeptide comprising (i) a viral envelope protein, (ii) an anti-CD3 nanobody, and (iii) an anti-CD8 nanobody. In some cases, the EDV comprises a fusion polypeptide comprising (i) a viral envelope protein, (ii) an anti-CD3 nanobody, and (iii) an anti-CD28 nanobody. In some cases, the EDV comprises a fusion polypeptide comprising (i) a viral envelope protein, (ii) an anti-CD3 nanobody, and (iii) an anti-CD4 nanobody. In some cases, the EDV comprises a fusion polypeptide comprising (i) a viral envelope protein, (ii) an anti-CD3 nanobody, and (iii) an anti-CD28 nanobody. In some cases, the EDV comprises a fusion polypeptide comprising (i) a viral envelope protein, (ii) an anti-CD3 scFv, and (iii) an anti-CD8 scFv. In some cases, the EDV comprises a fusion polypeptide comprising (i) a viral envelope protein, (ii) an anti-CD3 scFv, and (iii) an anti-CD28 scFv. In some cases, the EDV comprises a fusion polypeptide comprising (i) a viral envelope protein, (ii) an anti-CD3 scFv, and (iii) an anti-CD4 scFv. In some cases, the EDV comprises a fusion polypeptide comprising (i) a viral envelope protein, (ii) an anti-CD3 scFv, and (iii) an anti-CD28 scFv.

[0155] Antibodies that target cancer cells In some cases, the targeting polypeptide is an antibody that targets a cancer antigen, thereby targeting the EDV to cancerous cells that display the cancer antigen on their cell surface.

[0156] Suitable antigens bound by antibodies present on the EDV of the present disclosure include, for example, CD3, epidermal growth factor receptor (EGFR), CA-125 (highly expressed on epithelial ovarian cancer cells), CD80, CD86, glycoprotein IIb / IIIa receptor, CD51, TNF-α, epithelial adhesion molecule EpcAM (CD326), vascular endothelial growth factor receptor-2 (VEGFR-2), CD52, mesothelin, activin receptor-like kinase 1 (ALK-1), phosphatidylserine, CD19, vascular endothelial growth factor A (VEGF-A), IL-6 receptor, CD11a, CD25, CD2, CD3 receptor, and the like.

[0157] Suitable antigens to which antibodies present in the EDV of the present disclosure bind include, for example, carbonic anhydrase IX, alpha-fetoprotein (AFP), α-actinin-4, A3, ART-4, B7, Ba733, BAGE, BrE3-antigen, CA125, CAMEL, CAP-1, CASP-8 / m, CCL19, CCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD80, CD83, CD95, CD126, CD132, CD133, CD138, CD1 47, CD154, CDC27, CDK-4 / m, CDKN2A, CTLA-4, CXCR4, CXCR7, CXCL12, HIF-1α, colon-specific antigen-p (CSAp ), CEACAM5, CEACAM6, c-Met, DAM, epidermal growth factor receptor (EGFR), EGFRvIII, EGP-1(TROP-2), EGP-2, ELF2-M, Ep-CAM, fibroblast growth factor (FGF), Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, gp100, GRO-β, HLA-DR, HM1.24, human chorionic gonadotropin (HCG) and its subunits, HER2 / neu, histone H2B, histone H3, histone H4, HMGB-1, hypoxia-inducible factor (HIF-1), HSP70-2M, HST-2, insulin-like growth factor-1 receptor (IGF-1R), IFN-γ IFN-α, IFN-β, IFN-λ, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, insulin-like growth factor-1 (IGF-1), KC4 antigen, KS-1 antigen, KS1-4, Le-Y, LDR / FUT, macrophage migration inhibitory factor (MIF), MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, These include MUC16, MUM-1 / 2, MUM-3, NCA66, NCA95, NCA90, PAM4 antigen, PD-1, PD-L1, PD-1 receptor, placental growth factor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, P1GF, ILGF, ILGF-1R, IL-6, IL-25, RS5, RANTES, T101, SAGE, 5100, survivin, survivin-2B, TAC, TAG-72, tenascin, TRAIL receptor, TNF-α, Tn antigen, tumor necrosis antigen, VEGFR, ED-B fibronectin, WT-1, 17-1A antigen, cofactors C3, C3a, C3b, C5a, and C5.

[0158] In some cases, the cancer-associated antigen is an antigen associated with a blood cancer. Examples of such antigens include, but are not limited to, BCMA, C5, CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD40, CD45, CD52, CD56, CD66, CD74, CD79a, CD79b, CD80, CD138, CTLA-4, CXCR4, DKK, EphA3, GM2, HLA-DR beta, integrin αVβ3, IGF-R1, IL6, KIR, PD-1, PD-L1, TRAILR1, TRAILR2, transferrin receptor, and VEGF. In some cases, the cancer-associated antigen is an antigen expressed by malignant B cells, such as CD19, CD20, CD22, CD25, CD38, CD40, CD45, CD74, CD80, CTLA-4, IGF-R1, IL6, PD-1, TRAILR2, or VEGF.

[0159] In some cases, the cancer-associated antigen is an antigen associated with a solid tumor. Examples of such antigens include CAIX, cadherin, CEA, c-MET, CTLA-4, EGFR family members, EpCAM, EphA3, FAP, folate binding protein, FR-alpha, gangliosides (such as GC2, GD3, and GM2), HER2, HER3, IGF-1R, integrin αVβ3, integrin α5β1, Le ガンマ , Liv1, mesothelin, mucin, NaPi2b, PD-1, PD-L1, PD-1 receptor, pgA33, PSMA, RANKL, ROR1, TAG-72, tenascin, TRAILR1, TRAILR2, VEGF, VEGFR, and others listed above.

[0160] In some cases, the cancer-associated antigen is an antigen associated with cancer stem cells. Examples of such antigens include, but are not limited to, SSEA3, SSEA4, TRA-1-60, TRA-1-81, CD133, CD90, CD326, Cripto-1, PODXL-1, ABCG2, CD24, CD49f, Notch2, CD146, CD10, CD117, and CD26 (Kim & Ryu (2017) BMB Rep 50(6):285-298).

[0161] Examples of suitable antibodies include, for example, abciximab (anti-glycoprotein IIb / IIIa), alemtuzumab (anti-CD52), bevacizumab (anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab (anti-CD20), panitumumab (anti-EGFR), rituximab (anti-CD20), tositumomab (anti-CD20), trastuzumab (anti-ErbB2), lambrolizumab (anti-PD-1 receptor), nivolumab (anti-PD-1), receptor), ipilimumab (anti-CTLA-4), abagovomab (anti-CA-125), adecatumumab (anti-EpCAM), atlizumab (anti-IL-6 receptor), benralizumab (anti-CD125), obinutuzumab (GA101, anti-CD20), CC49 (anti-TAG-72), tocilizumab (anti-IL-6 receptor), basiliximab (anti-CD25), daclizumab (anti-CD25), efalizumab (anti-CD11a), GA101 (anti-CD20; Glycar Roche), muromonab-CD3 (anti-CD3 receptor), and natalizumab (anti-alpha-4 integrin).

[0162] Non-limiting examples of antibodies targeting cancer-associated antigens that can be targeted include abituzumab (anti-CD51), LL1 (anti-CD74), LL2 or RFB4 (anti-CD22), veltuzumab (hA20, anti-CD20), rituxumab (anti-CD20), obinutuzumab (GA101, anti-CD20), ibalizumab (anti-CD4), daratumumab (anti-CD38), lambrolizumab ( anti-PD-1 receptor), nivolumab (anti-PD-1 receptor), ipilimumab (anti-CTLA-4), RS7 (anti-TROP-2), PAM4 or KC4 (both anti-mucins), MN-14 (anti-CEA), MN-15 or MN-3 (anti-CEACAM6), Mu-9 (anti-colon-specific antigen-p), Immu31 (anti-alpha-fetoprotein), R1 (anti-IGF-1R), A19 (anti-CD1 9), TAG-72 (e.g., CC49), Tn, J591 or HuJ591 (anti-PSMA), AB-PG1-XG1-026 (anti-PSMA dimer), D2 / B (anti-PSMA), G250 (anti-carbonic anhydrase IX), L243 (anti-HLA-DR), alemtuzumab (anti-CD52), oportuzumab (anti-EpCAM), bevacizumab (anti-VEGF), cetuximab (anti-EGFR), genomic DNA These include, but are not limited to, mutuzumab (anti-CD33), ibritumomab tiuxetan (anti-CD20), panitumumab (anti-EGFR), tositumomab (anti-CD20), PAM4 (clivatuzumab, also known as anti-mucin), trastuzumab (anti-HER2), pertuzumab (anti-HER2), polatuzumab (anti-CD79b), and anetuzumab (anti-mesothelin).

[0163] The VH and VL amino acid sequences of various cancer antigen-binding antibodies are known in the art, as are the light and heavy chain CDRs of such antibodies. See, for example, Ling et al. (2018) Frontiers Immunol. 9:469, WO2005 / 012493, US2019 / 0119375, and US2013 / 0066055. The following are non-limiting examples of antibodies that can be used as part of the targeting polypeptide of the subject EDVs. In some cases, the antibody comprises CDR sequences from the anti-CD19 scFv FMC63. In some cases, the antibody comprises CDR sequences from the anti-CD4 antibody ibalizumab (IMGT / mAb-DB ID 241). In some cases, the antibody comprises CDR sequences from the anti-CD20 antibody rituximab (IMGT / mAb-DB ID 161). In some cases, the antibody comprises CDR sequences from the anti-CD3 antibody acapatamab (IMGT / mAb-DB ID 1074). In some cases, the antibody comprises CDR sequences from the anti-CD3 antibody OKT3. In some cases, the antibody comprises CDR sequences from the anti-CD28 antibody PDB ID 1YJD. In some cases, the antibody comprises CDR sequences from the anti-CD19 antibody IMGT / mAb-DB ID 1232. In some cases, the antibody comprises CDR sequences from the anti-CD45 antibody Ab4122. In some cases, the antibody comprises CDR sequences from the anti-CD45 antibody Ab4129. In some cases, the antibody comprises CDR sequences from the anti-CD45 antibody apamistamab (IMGT / mAb-DB ID 633). See, e.g., Figures 18 and 35. The following are non-limiting examples of cancer antigen-binding antibodies.

[0164] anti-Her2 In some cases, the anti-Her2 antibody comprises (a) the amino acid sequence: and (b) a light chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025529869000092.tif23159 (SEQ ID NO: 159); and and a heavy chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025529869000093.tif50159 (SEQ ID NO: 160).

[0165] In some cases, the anti-Her2 antibody comprises a light chain variable region (VL) present in the light chain amino acid sequence provided above and a heavy chain variable region (VH) present in the heavy chain amino acid sequence provided above. For example, the anti-Her2 antibody may comprise (a) the amino acid sequence: and (b) a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025529869000094.tif10159 (SEQ ID NO: 161); and and a VH comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025529869000095.tif10158 (SEQ ID NO: 162). In some cases, the anti-Her2 antibody may comprise, in order from N-terminus to C-terminus, (a) the amino acid sequence: (b) a VH comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025529869000096.tif10158 (SEQ ID NO: 162); (c) an amino acid sequence: and a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025529869000097.tif10159 (SEQ ID NO: 161). Suitable linkers are described elsewhere herein and include, for example, (GGGGS)n (SEQ ID NO: 38), where n is an integer between 1 and 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0166] In some cases, the anti-Her2 antibody comprises a VL CDR1, a VL CDR2, and a VL CDR3 present in the light chain amino acid sequence provided above, and a VH CDR1, a CDR2, and a CDR3 present in the heavy chain amino acid sequence provided above.

[0167] For example, an anti-Her2 antibody may comprise a VL CDR1 having the amino acid sequence RASQDVNTAVA (SEQ ID NO: 164), a VL CDR2 having the amino acid sequence SASFLY (SEQ ID NO: 165), a VL CDR3 having the amino acid sequence QQHYTTPP (SEQ ID NO: 166), a VH CDR1 having the amino acid sequence GFNIKDTY (SEQ ID NO: 167), a VH CDR2 having the amino acid sequence IYPTNGYT (SEQ ID NO: 168), and a VH CDR3 having the amino acid sequence SRWGGDGFYAMDY (SEQ ID NO: 169).

[0168] In some cases, the anti-Her2 antibody is an scFv antibody. For example, the anti-Her2 scFv can comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000098.tif23159 (SEQ ID NO: 170).

[0169] As another example, in some cases, the anti-Her2 antibody has (a) the amino acid sequence: and (b) a light chain variable region (VL) comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025529869000099.tif23159 (SEQ ID NO: 171); and and a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025529869000100.tif49160 (SEQ ID NO: 172).

[0170] In some cases, the anti-Her2 antibody comprises a VL present in the light chain amino acid sequence provided above and a VH present in the heavy chain amino acid sequence provided above. For example, the anti-Her2 antibody comprises (a) the amino acid sequence: and (b) a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025529869000101.tif10158 (SEQ ID NO: 173); and and a VH comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025529869000102.tif10158 (SEQ ID NO: 174).

[0171] In some cases, an anti-Her2 antibody comprises a VL CDR1, a VL CDR2, and a VL CDR3 present in the light chain amino acid sequence provided above, and a VH CDR1, a CDR2, and a CDR3 present in the heavy chain amino acid sequence provided above. For example, an anti-HER2 antibody may comprise a VL CDR1 having the amino acid sequence KASQDVSIGVA (SEQ ID NO: 175), a VL CDR2 having the amino acid sequence SASYRY (SEQ ID NO: 176), a VL CDR3 having the amino acid sequence QQYYIYPY (SEQ ID NO: 177), a VH CDR1 having the amino acid sequence GFTFTDYTMD (SEQ ID NO: 178), a VH CDR2 having the amino acid sequence ADVNPNSGGSIYNQRFKG (SEQ ID NO: 179), and a VH CDR3 having the amino acid sequence ARNLGPSFYFDY (SEQ ID NO: 180).

[0172] In some cases, the anti-Her2 antibody is an scFv. For example, in some cases, the anti-Her2 scFv comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000103.tif23159 (SEQ ID NO: 170).

[0173] anti-CD19 Anti-CD19 antibodies are known in the art, and the VH and VL, or VH and VL CDRs, of any anti-CD19 antibody can be used. See, e.g., WO2005 / 012493.

[0174] In some cases, the anti-CD19 antibody comprises a VL CDR1 comprising the amino acid sequence KASQSVDYDGDSYLN (SEQ ID NO: 181), a VL CDR2 comprising the amino acid sequence DASNLVS (SEQ ID NO: 182), and a VL CDR3 comprising the amino acid sequence QQSTEDPWT (SEQ ID NO: 183). In some cases, the anti-CD19 antibody comprises a VH CDR1 comprising the amino acid sequence SYWMN (SEQ ID NO: 184), a VH CDR2 comprising the amino acid sequence QIWPGDGDTNYNGKFKG (SEQ ID NO: 185), and a VH CDR3 comprising the amino acid sequence RETTTVGRYYYAMDY (SEQ ID NO: 186). In some cases, the anti-CD19 antibody comprises a VL CDR1 comprising the amino acid sequence KASQSVDYDGDSYLN (SEQ ID NO: 181), a VL CDR2 comprising the amino acid sequence DASNLVS (SEQ ID NO: 182), a VL CDR3 comprising the amino acid sequence QQSTEDPWT (SEQ ID NO: 183), a VH CDR1 comprising the amino acid sequence SYWMN (SEQ ID NO: 184), a VH CDR2 comprising the amino acid sequence QIWPGDGDTNYNGKFKG (SEQ ID NO: 185), and a VH CDR3 comprising the amino acid sequence RETTTVGRYYYAMDY (SEQ ID NO: 186).

[0175] In some cases, the anti-CD19 antibody is an scFv. For example, in some cases, the anti-CD19 scFv comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000104.tif23159 (sequence number 187).

[0176] Antimesothelin Anti-mesothelin antibodies are known in the art, see, e.g., US2019 / 0000944, WO2009 / 045957, WO2014 / 031476, USPN8,460,660, US2013 / 0066055, and WO2009 / 068204.

[0177] In some cases, the anti-mesothelin antibody has (a) the amino acid sequence: a light chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025529869000105.tif23159 (SEQ ID NO: 215); (b) the amino acid sequence: and a heavy chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025529869000106.tif49160 (SEQ ID NO: 216).

[0178] In some cases, the anti-mesothelin antibody comprises a VL present in the light chain amino acid sequence provided above and a VH present in the heavy chain amino acid sequence provided above. For example, the anti-mesothelin antibody may comprise (a) the amino acid sequence: and (b) a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025529869000107.tif10157 (SEQ ID NO: 217); and and a VH comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025529869000108.tif10158 (SEQ ID NO: 218).

[0179] In some cases, the anti-mesothelin antibody comprises a VL CDR1, a VL CDR2, and a VL CDR3 present in the light chain amino acid sequence provided above, and a VH CDR1, a CDR2, and a CDR3 present in the heavy chain amino acid sequence provided above.

[0180] For example, an anti-mesothelin antibody may comprise a VL CDR1 having the amino acid sequence TGTSSDIGGYNSVS (SEQ ID NO: 219), a VL CDR2 having the amino acid sequence LMIYGVNNRPS (SEQ ID NO: 220), a VL CDR3 having the amino acid sequence SSYDIESATP (SEQ ID NO: 221), a VH CDR1 having the amino acid sequence GYSFTSYWIG (SEQ ID NO: 222), a VH CDR2 having the amino acid sequence WMGIIDPGDSRTRYSP (SEQ ID NO: 223), and a VH CDR3 having the amino acid sequence GQLYGGTYMDG (SEQ ID NO: 224).

[0181] The anti-mesothelin antibody can be an scFv. As one non-limiting example, an anti-mesothelin scFv has the following amino acid sequence: TIFF2025529869000109.tif23160 (SEQ ID NO: 225), where VH CDR1, CDR2, and CDR3 are underlined and VL CDR1, CDR2, and CDR3 are bold and underlined.

[0182] As one non-limiting example, an anti-mesothelin scFv may have the following amino acid sequence: TIFF2025529869000110.tif30160 (SEQ ID NO: 226), where VH CDR1, CDR2, and CDR3 are underlined and VL CDR1, CDR2, and CDR3 are bold and underlined.

[0183] anti-CD22 CD22 (also known as B lymphocyte cell adhesion molecule, sialic acid-binding Ig-like lectin 2, or SIGLEC2) is a sialic acid-binding adhesion molecule that is largely restricted to the B cell lineage and expressed in most B-lineage malignancies.

[0184] Anti-CD22 antibodies are known in the art, and the VH and VL or VH and VL CDRs of any anti-CD22 antibody can be used. See, for example, Xiao et al. (2009) Mabs 1:297 (depicting the fully human anti-CD22 m971 scFv) and U.S. Patent Publication No. 2020 / 0147134. Examples of anti-CD22 antibodies include epratuzumab and inotuzumab. See, for example, Lenoard et al. (2007) Oncogene 26:3704 and U.S. Patent No. 5,789,554 (depicting epratuzumab), and DiJoseph et al. (2007) Leukemia 21:2240 (depicting inotuzumab).

[0185] For example, an anti-CD22 antibody may comprise: (i) a heavy chain variable region (VH) CDR1 having the amino acid sequence: GDSVSSNSAA (SEQ ID NO: 227); (ii) a VH CDR2 having the amino acid sequence: TYYRSKWYN (SEQ ID NO: 228); (iii) a VH CDR3 having the amino acid sequence: AREVTGDLEDAFDI (SEQ ID NO: 229); (iv) a light chain variable region (VL) CDR1 having the amino acid sequence: QTIWSY (SEQ ID NO: 230); (v) a VL CDR2 having the amino acid sequence: AAS (Ala-Ala-Ser); and (vi) a VL CDR3 having the amino acid sequence: QQSYSIPQT (SEQ ID NO: 231).

[0186] anti-TROP-2 Trophoblast cell surface antigen 2 (Trop-2) (also known as epithelial glycoprotein-1, gastrointestinal tumor-associated antigen GA733-1, membrane component chromosome 1 surface marker-1, and tumor-associated calcium signal transducer-2) is a transmembrane glycoprotein that is upregulated in many cancer types and is the protein product of the TACSTD2 gene.

[0187] Anti-TROP-2 antibodies are known in the art, and the VH and VL, or VH and VL CDRs, of any anti-TROP-2 antibody can be used. (See, e.g., U.S. Patent No. 7,238,785.) In some cases, the anti-TROP-2 antibody comprises (i) the light chain CDR sequences CDR1 (KASQDVSIAVA; SEQ ID NO: 232), CDR2 (SASYRYT; SEQ ID NO: 233), and CDR3 (QQHYITPLT; SEQ ID NO: 234), and (ii) the heavy chain CDR sequences CDR1 (NYGMN; SEQ ID NO: 235), CDR2 (WINTYTGEPTYTDDFKG; SEQ ID NO: 236), and CDR3 (GGFGSSYWYFDV; SEQ ID NO: 237).

[0188] In some cases, the anti-TROP-2 antibody comprises (i) the heavy chain CDR sequences CDR1 (TAGMQ; SEQ ID NO: 238), CDR2 (WINTHSGVPKYAEDFKG (SEQ ID NO: 239), and CDR3 (SGFGSSYWYFDV; SEQ ID NO: 240), and (ii) the light chain CDR sequences CDR1 (KASQDVSTAVA; SEQ ID NO: 241), CDR2 (SASYRYT; SEQ ID NO: 233), and CDR3 (QQHYITPLT; SEQ ID NO: 234).

[0189] In some cases, the anti-TROP2 antibody has (a) the amino acid sequence: (b) a VL CDR1, a VL CDR2, and a VL CDR3 present in a light chain variable region (VL) comprising TIFF2025529869000111.tif10158 (SEQ ID NO: 244); and (b) the following amino acid sequence: and VH CDR1, CDR2, and CDR3 present in the heavy chain variable region (VH) comprising TIFF2025529869000112.tif10158 (SEQ ID NO: 245).

[0190] In some cases, the anti-TROP-2 antibody has (a) the amino acid sequence: and (b) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025529869000113.tif10158 (SEQ ID NO: 244); and and a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025529869000114.tif10158 (SEQ ID NO: 245).

[0191] anti-BCMA Anti-BCMA (B cell maturation antigen) antibodies are known in the art, and the VH and VL, or VH and VL CDRs, of any anti-BCMA antibody can be used. See, e.g., WO2014 / 089335, US2019 / 0153061, and WO2017 / 093942.

[0192] In some cases, the anti-BCMA antibody has (a) the amino acid sequence: a light chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025529869000115.tif23158 (SEQ ID NO: 248); (b) the amino acid sequence: and a heavy chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025529869000116.tif49159 (SEQ ID NO: 249).

[0193] In some cases, the anti-BCMA antibody comprises a VL present in the light chain amino acid sequence provided above and a VH present in the heavy chain amino acid sequence provided above. For example, the anti-BCMA antibody may comprise (a) the amino acid sequence: and (b) a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025529869000117.tif10158 (SEQ ID NO: 250); and and a VH comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025529869000118.tif16158 (SEQ ID NO: 251).

[0194] In some cases, the anti-BCMA antibody comprises a VL CDR1, a VL CDR2, and a VL CDR3 present in the light chain amino acid sequence provided above, and a VH CDR1, CDR2, and CDR3 present in the heavy chain amino acid sequence provided above.

[0195] For example, an anti-BCMA antibody may comprise a VL CDR1 having the amino acid sequence SSNIGSNT (SEQ ID NO: 252), a VL CDR2 having the amino acid sequence NYH, a VL CDR3 having the amino acid sequence AAWDDSLNGWV (SEQ ID NO: 253), a VH CDR1 having the amino acid sequence GFTFGDYA (SEQ ID NO: 254), a VH CDR2 having the amino acid sequence SRSKAYGGTT (SEQ ID NO: 255), and a VH CDR3 having the amino acid sequence ASSGYSSGWTPFDY (SEQ ID NO: 256).

[0196] The anti-BCMA antibody may be an scFv. As one non-limiting example, the anti-BCMA scFv may comprise the following amino acid sequence: TIFF2025529869000119.tif23160 (sequence number 257).

[0197] As another example, the anti-BCMA scFv may comprise the following amino acid sequence: TIFF2025529869000120.tif23160 (sequence number 258).

[0198] In some cases, the anti-BCMA antibody may comprise a VL CDR1 having the amino acid sequence SASQDISNYLN (SEQ ID NO: 259), a VL CDR2 having the amino acid sequence YTSNLHS (SEQ ID NO: 260), a VL CDR3 having the amino acid sequence QQYRKLPWT (SEQ ID NO: 261), a VH CDR1 having the amino acid sequence NYWMH (SEQ ID NO: 262), a VH CDR2 having the amino acid sequence ATYRGHSDTYYNQKFKG (SEQ ID NO: 263), and a VH CDR3 having the amino acid sequence GAIYNGYDVLDN (SEQ ID NO: 264).

[0199] In some cases, the anti-BCMA antibody has (a) the amino acid sequence: The invention also includes a light chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025529869000121.tif10157 (SEQ ID NO: 265).

[0200] In some cases, the anti-BCMA antibody has (a) the amino acid sequence: The invention further comprises a heavy chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025529869000122.tif10158 (SEQ ID NO: 266).

[0201] anti-MUC16 In some cases, the antibody is specific for MUC16 (also known as CA125). See, e.g., Yin et al. (2002) Int. J. Cancer 98:737. For example, the antibody can be specific for a MUC16 polypeptide present on cancer cells. See, e.g., US2018 / 0118848 and US2018 / 0112008. In some cases, the MUC16-specific antibody is an scFv. In some cases, the MUC16-specific antibody is a nanobody.

[0202] As an example, an anti-MUC16 antibody can comprise a VH CDR1 having the amino acid sequence GFTFSNYY (SEQ ID NO: 267), a VH CDR2 having the amino acid sequence ISGRGSTI (SEQ ID NO: 268), a VH CDR3 having the amino acid sequence VKDRGGYSPY (SEQ ID NO: 269), a VL CDR1 having the amino acid sequence QSISTY (SEQ ID NO: 270), a VL CDR2 having the amino acid sequence TAS, and a VL CDR3 having the amino acid sequence QQSYSTPPIT (SEQ ID NO: 271). See, e.g., US2018 / 0118848.

[0203] Anti-claudin-18.2 In some cases, the antibody is specific for claudin-18 isoform 2 ("claudin-18.2"). See, e.g., WO2013 / 167259. In some cases, the claudin-18.2-specific antibody is an scFv. In some cases, the claudin-18.2-specific antibody is a nanobody.

[0204] As an example, an anti-claudin-18.2 antibody may comprise a VH CDR1 having the amino acid sequence GYTFTDYS (SEQ ID NO: 272), a VH CDR2 having the amino acid sequence INTETGVP (SEQ ID NO: 273), a VH CDR3 having the amino acid sequence ARRTGFDY (SEQ ID NO: 274), a VL CDR1 having the amino acid sequence KNLLHSDGITY (SEQ ID NO: 275), a VL CDR2 having the amino acid sequence RVS, and a VL CDR3 having the amino acid sequence VQVLELPFT (SEQ ID NO: 276).

[0205] As another example, an anti-claudin-18.2 antibody may comprise a VH CDR1 having the amino acid sequence GFTFSSYA (SEQ ID NO: 277), a VH CDR2 having the amino acid sequence ISDGGSYS (SEQ ID NO: 278), a VH CDR3 having the amino acid sequence ARDSYYDNSYVRDY (SEQ ID NO: 279), a VL CDR1 having the amino acid sequence QDINTF (SEQ ID NO: 280), a VL CDR2 having the amino acid sequence RTN, and a VL CDR3 having the amino acid sequence LQYDEFPLT (SEQ ID NO: 281).

[0206] Examples of antibodies include, but are not limited to, natalizumab (Tysabri® Biogen Idec / Elan) which targets the α4 subunit of the α4β1 and α4β7 integrins (when used to treat MS and Crohn's disease); vedolizumab (MLN2; Millennium Pharmaceuticals / Takeda) which targets the α4β7 integrin (when used to treat UC and Crohn's disease); belimumab (Benlysta; Human Genome Research) which targets BAFF Sciences / GlaxoSmithKline) (when used to treat SLE); atacicept (TACI-Ig; Merck / Serono) which targets BAFF and APRIL (when used to treat SLE); alefacept (Amevive®; Astellas) which targets CD2 (when used to treat plaque psoriasis, GVHD); otelixizumab (TRX4; Tolerx / GlaxoSmithKline) which targets CD3 (when used to treat T1D); teplizumab (MGA031; MacroGenics / Eli Lilly) which targets CD3 (when used to treat T1D); rituximab (Rituxan® / Mabthera; Genentech / Roche / Biogen) which targets CD20 Idec) (when used to treat non-Hodgkin's lymphoma, RA (in patients with an inadequate response to TNF-blocking agents), and CLL); ofatumumab (Arzerra®; Genmab / GlaxoSmithKline) which targets CD20 (when used to treat CLL, RA); ocrelizumab (2H7; Genentech / Roche / Biogen Idec) which targets CD20 (when used to treat RA and SLE); epratuzumab (hLL2; Immunomedics / UCB) which targets CD22 (when used to treat SLE and non-Hodgkin's lymphoma); alemtuzumab (Campath® / MabCampath; Genzyme / Bayer) which targets CD52 (when used to treat CLL, MS); abatacept (Orencia®) which targets CD80 and CD86Bristol-Myers Squibb) (when used to treat RA and JIA, UC and Crohn's disease, and SLE); eculizumab (Soliris®; Alexion Pharmaceuticals) which targets the C5 complement protein (when used to treat paroxysmal nocturnal hemoglobinuria); omalizumab (Xolair®; Genentech / Roche / Novartis) which targets IgE (when used to treat moderate to severe persistent allergic asthma); canakinumab (Ilaris®; Novartis) which targets IL-1β (when used to treat cryopyrin-associated periodic syndrome, systemic JIA, neonatal-onset multisystem inflammatory disease, and acute gout); mepolizumab (Bosatria; GlaxoSmithKline) which targets IL-5 (when used to treat hypereosinophilic syndrome); and reslizumab (SCH55700; Ception) which targets IL-5. Therapeutics) (when used to treat eosinophilic esophagitis); tocilizumab (Actemra® / RoActemra®; Chugai / Roche) which targets IL-6R (when used to treat RA, JIA); ustekinumab (Stelara®; Centocor) which targets IL-12 and IL-23 (when used to treat plaque psoriasis, psoriatic arthritis, Crohn's disease); briakinumab (ABT-874; Abbott) which targets IL-12 and IL-23 (when used to treat psoriasis and plaque psoriasis); etanercept (Enbrel®) which targets TNF ; Amgen / Pfizer) (when used to treat RA, JIA, psoriatic arthritis, AS, and plaque psoriasis); TNF-targeted infliximab (Remicade®; Centocor / Merck) (when used to treat Crohn's disease, RA, psoriatic arthritis, UC, AS, and plaque psoriasis); TNF-targeted adalimumab (Humira® / Trudexa; Abbott) (when used to treat RA, JIA, psoriatic arthritis, Crohn's disease, AS, and plaque psoriasis); TNF-targeted certolizumab pegol (Cimzia®; UCB) (when used to treat Crohn's disease and RA);and golimumab (Simponi®; Centocor) which targets TNF (when used to treat RA, psoriatic arthritis, and AS). In some cases, the antibody whose production is induced by the intracellular domain of a synNotch polypeptide of the present disclosure is a therapeutic antibody for the treatment of cancer. Such antibodies include, for example, ipilimumab which targets CTLA-4 (when used to treat melanoma, prostate cancer, RCC); tremelimumab which targets CTLA-4 (when used to treat CRC, gastric cancer, melanoma, NSCLC); nivolumab which targets PD-1 (when used to treat melanoma, NSCLC, RCC); MK-3475 which targets PD-1 (when used to treat melanoma); and pidilizumab which targets PD-1 (when used to treat hematologic malignancies). BMS-936559, which targets PD-L1 (when used to treat melanoma, NSCLC, ovarian, and RCC); MEDI4736, which targets PD-L1; MPDL33280A, which targets PD-L1 (when used to treat melanoma); rituximab, which targets CD20 (when used to treat non-Hodgkin's lymphoma); ibritumomab tiuxetan and tositumomab (when used to treat lymphoma); brentuximab vedotin, which targets CD33 (when used to treat Hodgkin's lymphoma); gemtuzumab ozogaicin, which targets CD33 (when used to treat acute myeloid leukemia); alemtuzumab, which targets CD52 (when used to treat chronic lymphocytic leukemia); IGN101 and adecatumumab, which target EpCAM (when used to treat epithelial tumors (breast, colon, and lung)); and labetuzumab, which targets CEA (when used to treat breast, colon, and lung tumors). huA33, which targets gpA33 (when used to treat colorectal cancer); pemtumomab and oregovomab, which target mucin (when used to treat breast, colon, lung, and ovarian tumors); CC49 (miretumomab), which targets TAG-72 (when used to treat breast, colon, and lung tumors); cG250, which targets CAIX (when used to treat renal cell carcinoma); J591, which targets PSMA (when used to treat prostate cancer);MOv18 and MORAb-003 (faretuzumab), which target folate-binding proteins (when used to treat ovarian tumors); 3F8, ch14.18, and KW-2871 (e.g., GD2, GD3, and GM2), which target gangliosides (when used to treat neuroectodermal tumors and some epithelial tumors); hu3S193 and IgN311, which target Le y (when used to treat breast, colon, lung, and prostate tumors); bevacizumab, which targets VEGF (when used to treat tumor vasculature); IM-2C6 and CDP791, which target VEGFR (when used to treat solid tumors of epithelial origin); etaracizumab, which targets integrin V3 (when used to treat tumor vasculature); and volociximab, which targets integrin 51 (when used to treat tumor vasculature). cetuximab, panitumumab, nimotuzumab and 806 targeting EGFR (when used to treat glioma, lung, breast, colon, and head and neck tumors); trastuzumab and pertuzumab targeting ERBB2 (when used to treat breast, colon, lung, ovarian, and prostate tumors); MM-121 targeting ERBB3 (when used to treat breast, colon, lung, ovarian, and prostate tumors); AMG102, METMAB and SCH900105 targeting MET (when used to treat breast, AVE1642, IMC-A12, MK-0646, R1507, and CP751871, which target IGF1R (when used to treat glioma, lung, breast, head and neck, prostate, and thyroid cancers); KB004 and IIIA4, which target EPHA3 (when used to treat lung, kidney, and colon tumors, melanoma, glioma, and hematological malignancies); mapatumumab (HGS-ETR1), which targets TRAILR1 (when used to treat colon, lung, and pancreatic tumors and HGS-ETR2 and CS-1008 targeting TRAILR2; denosumab targeting RANKL (when used to treat prostate cancer and bone metastases); sibrotuzumab and F19 targeting FAP (when used to treat colon, breast, lung, pancreatic, and head and neck tumors); 81C6 targeting tenascin (when used to treat glioma, breast, and prostate tumors); blinatumomab (Blincyto;Amgen (when used to treat ALL); pembrolizumab, which targets PD-1 as used in cancer immunotherapy; and the 9E10 antibody, which targets c-Myc.

[0207] Examples of antibodies include, but are not limited to: abagovomab, abciximab, abituzumab, abrilumab, actoxumab, aducanumab, afelimomab, afutuzumab, alacizumab pegol, ALD518, alirocumab, altumomab pentetate, amatuximab, anatumomab mafenatox, anetumab ravtansine, anifrolumab, anrukinzumab, apolizumab, arcitumomab, asclinvacumab, acelizumab, atezolizumab, atinumab, atlizumab / tocilizumab, atorlimumab, baclofenib, Pineuzumab, basiliximab, bavituximab, bectumomab, begelomab, benralizumab, bertilimumab, besilesomab, bevacizumab / ranibizumab, bezlotoxumab, biciromab, bimagrumab, bimekizumab, bivatuzumab mertansine, brosozumab, bococizumab, brentuximab vedotin, brodalumab, brolucizumab, brontiximab, cantuzumab mertansine, cantuzumab ravtansine, caplacizumab, capromab pendetide, carlumab, catumaxomab, cBR96-doxorubicin immunoconjugate, cedelizumab Mabs, Ch.14.18, sitatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, codrituzumab, cortuximab ravtansine, conatumumab, concizumab, CR6261, crenezumab, dacetuzumab, daclizumab, darotuzumab, dapirolizumab pegol, daratumumab, dectrecumab, demcizumab, denintuzumab mafodotin, delrotuximab biotin, detumomab, dinutuximab, ziliadamumab, dorlimomab alitoxin, drozitumab, durigotumab, dupilumab , durvalumab, dusigitumab, ecloneximab, edovacomab, edrecolomab, efalizumab, efungumab, eldelumab, elgemtumab, elotuzumab, ersilimomab, emactuzumab, emibetuzumab, enavatuzumab, enfortumab vedotin, enlimomab pegol, enoblitzumab, enokizumab, enoticumab, ensituximab, epitumomab cituxetan, erlizumab, ertumaxomab, etrolizumab, evinacumab, evolocumab, exbivirumab, fanolesomab, faralimomab, faretuzumab,Fasinumab, FBTA05, felvizumab, fezakinumab, ficlatuzumab, figitumumab, filibumab, framvotumab, fretikumab, fontolizumab, foralumab, foravirumab, fresolimumab, furanumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gevokizumab, dilentuximab, glembatumumab vedotin, gomiliximab, guselkumab, ibalizumab, icrucumab, idarucizumab, igovomab, IMAB362, imalumab, imciromab, imgatuz Mab, inlacumab, indatuximab vedotin, indusatumab vedotin, inolimomab, inotuzumab ozogamicin, intetumumab, iratumumab, isatuximab, itolizumab, ixekizumab, keliximab, lambrolizumab, lampalizumab, lebrikizumab, remaresomab, lenzilumab, lerdelimumab, lexatumumab, ribivirumab, rifastuzumab vedotin, ligelizumab, rirotumab satetraxetan, lintuzumab, lirilumab, roderucizumab, roxivetomab, lorvostuzumab mertansine, lucatumab , lumiliximab pegol, lumiliximab, lumuletuzumab, margetuximab, maslimomab, matuzumab, mavrilimumab, metelimuab, milatuzumab, minletumomab, mirvetuximab soravtansine, mitumomab, mogamulizumab, morolimumab, morolimumab immunotherapy, motavizumab, moxetumomab pasudotox, muromonab-CD3, nacolomab butafenatox, namilumab, naptumomab estafenatox, narunatumab, nebacumab, necitumumab, nemolizumab, nerelimomab, nesvacumab, nofetumomab merpentane , obilutoxaximab, obinutuzumab, ocalatuzumab, ozlimomab, olaratumab, olokizumab, onartuzumab, ontuximab, opicinumab, oportuzumab monatox, olticumab, otreltuzumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, pancomab, panobacumab, palsatuzumab, pascolizumab, pasotuximab, pateclizumab, patritumab, perakizumab, pexelizumab, pinatuzumab vedotin, pintumomab, placumab, polatuzumab vedotin, ponezumab,Priliximab, plitoxaximab, pritumumab, PRO140, kiruizumab, racotumomab, radletumab, rafivirumab, ralpanzizumab, ramucirumab, ranibizumab, raxibacumab, refanezumab, regavirumab, rilotumumab, linucumab, lobatumumab, loredumab, romosozumab, lontalizumab, rovelizumab, ruplizumab, sacituzumab govitecan, samalizumab, sarilumab, satumomab pendetide, secukinumab, seribantumab, cetoxaximab, sevirumab, SGN-CD19A, SGN-CD33A, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, sofituzumab vedotin, solanezumab, solitomab, soneptizumab, sontuzumab, stamulumab, sulesomab, subizumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab , tanezumab, taplitumomab paptox, talexuzumab, tefibazumab, terimomab alitox, tenatumomab, teneliximab, teprotumumab, tesidolumab, tetulomab, TGN1412, ticilimumab / tremelimumab, tigatuzumab, tildrakizumab, TNX-650, toralizumab, tosatoxumab, tobetumab, tralokinumab, TRBS07, tregalizumab, treboglumab, tucotu Ibuprofen celmoleukin, tubilumab, ublituximab, urocuplumab, urelumab, urtoxazumab, bundeltuzumab vedotin, vanticizumab, vanucizumab, bapaliximab, varlilumab, batelizumab, veltuzumab, bepalimomab, besencumab, visilizumab, borsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, diralimumab, zolimomab alitox, etc.,

[0208] antibody mimics In some cases, the EDV of the present disclosure comprises an antibody mimic (also referred to as an "antibody analog"). Non-limiting examples of antibody mimics include peptide aptamers, affimers, affilins, affibodies, affitins, alphabodies, anticalins, avimers, DARPins, finomers, Kunitz domain peptides, nanoCLAMPs, affinity reagents, and scaffold proteins.

[0209] Nucleic Acid Binding Polypeptides As described above, the present disclosure provides an EDV comprising a nucleic acid-binding effector polypeptide or a nucleic acid encoding a nucleic acid-binding effector polypeptide, wherein the EDV comprises a fusion polypeptide comprising (i) a viral envelope protein and (ii) a targeting polypeptide that confers binding to a target cell. The present disclosure also provides a method for delivering the nucleic acid-binding effector polypeptide into a eukaryotic cell using the EDV of the present disclosure.

[0210] Suitable nucleic acid-binding effector polypeptides include nucleases. Suitable nucleases include, but are not limited to, homing nuclease polypeptides, FokI polypeptides, transcription activator-like effector nuclease (TALEN) polypeptides, MegaTAL polypeptides, meganuclease polypeptides, zinc finger nucleases (ZFNs), ARCUS nucleases, etc. Meganucleases can be engineered from LADLIDADG homing endonucleases (LHEs). MegaTAL polypeptides can comprise a TALE DNA-binding domain and an engineered meganuclease. For example, WO2004 / 067736 (homing endonucleases), Urnov et al. (2005) Nature 435:646 (ZFNs), Mussolino et al. (2011) Nucle. Acids Res. 39:9283 (TALE nucleases), Boissel et al. (2013) Nucl. Acids Res. 42:2591 (MegaTALs).

[0211] CRISPR-Cas effector polypeptides As described above, in some cases, the EDV of the present disclosure comprises a CRISPR-Cas effector polypeptide or a nucleic acid (e.g., a recombinant expression vector) comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide. The CRISPR-Cas effector polypeptide can be any of various CRISPR-Cas effector polypeptides. Suitable CRISPR-Cas effector polypeptides are described in detail below. For example, in some cases, the CRISPR-Cas effector polypeptide is a type II CRISPR-Cas effector polypeptide. In some cases, the type II CRISPR-Cas effector polypeptide is a Cas9 polypeptide. In some cases, the CRISPR-Cas effector polypeptide is a type V CRISPR-Cas effector polypeptide, such as a Cas12a, Cas12b, Cas12c, Cas12d, or Cas12e polypeptide. In some cases, the CRISPR-Cas effector polypeptide is a type VI CRISPR-Cas effector polypeptide, such as a Cas13a polypeptide, a Cas13b polypeptide, a Cas13c polypeptide, or a Cas13d polypeptide. In some cases, the CRISPR-Cas effector polypeptide is a Cas14 polypeptide. In some cases, the CRISPR-Cas effector polypeptide is a Cas14a polypeptide, a Cas14b polypeptide, or a Cas14c polypeptide. Also suitable for use are variant CRISPR-Cas effector polypeptides, where the variant CRISPR-Cas effector polypeptide has reduced nucleic acid cleavage activity. Also suitable for use are CRISPR-Cas effector fusion polypeptides, including (i) a variant CRISPR-Cas effector polypeptide with reduced nucleic acid cleavage activity and (ii) a heterologous fusion polypeptide. In some cases, the heterologous fusion polypeptide is a protein-modifying enzyme. In some cases, the heterologous fusion polypeptide is a nucleic acid-modifying enzyme. In some cases, the heterologous fusion polypeptide is a reverse transcriptase. In some cases, the heterologous fusion polypeptide is a cytidine deaminase.In some cases, the heterologous fusion polypeptide is an adenine deaminase. In some cases, the heterologous fusion polypeptide is a transcription factor. In some cases, the heterologous fusion polypeptide is a transcription activator. In some cases, the heterologous fusion polypeptide is a transcription repressor. Suitable protein-modifying enzymes and nucleic acid-modifying enzymes are described in detail below. For example, in some cases, the nucleic acid-modifying enzyme is a cytidine deaminase. In some cases, the nucleic acid-modifying enzyme is an adenosine deaminase. In some cases, the nucleic acid-modifying enzyme is a prime editor. As described in more detail below, in some cases, the CRISPR-Cas effector polypeptide comprises one or more nuclear localization signals.

[0212] An example of a CRISPR-Cas effector polypeptide is a CRISPR-Cas endonuclease (e.g., a Class 2 CRISPR-Cas effector polypeptide, such as a Type II, Type V, or Type VI CRISPR-Cas effector polypeptide). If a CRISPR-Cas effector polypeptide has endonuclease activity, the CRISPR-Cas effector polypeptide may also be referred to as a "CRISPR-Cas endonuclease." A CRISPR-Cas effector polypeptide may also have reduced or undetectable endonuclease activity. A CRISPR-Cas effector polypeptide may also be a fusion CRISPR-Cas effector polypeptide comprising a heterologous fusion partner. In some cases, a suitable CRISPR-Cas effector polypeptide is a Class 2 CRISPR-Cas effector polypeptide. In some cases, a suitable CRISPR-Cas effector polypeptide is a Class 2 Type II CRISPR-Cas effector polypeptide (e.g., a Cas9 protein). In some cases, a suitable CRISPR-Cas effector polypeptide is a Class 2 Type V CRISPR-Cas endonuclease (e.g., a Cpf1 protein, a C2c1 protein, or a C2c3 protein). In some cases, a suitable CRISPR-Cas effector polypeptide is a Class 2 Type VI CRISPR-Cas effector polypeptide (e.g., a C2c2 protein; also referred to as a "Cas13a" protein). Also suitable for use is a CasX protein. Also suitable for use is a CasY protein.

[0213] In some cases, a suitable CRISPR-Cas effector polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to an amino acid sequence shown in any one of Figures 15A-15P.

[0214] In some cases, the CRISPR-Cas effector polypeptide is a type II CRISPR-Cas effector polypeptide. In some cases, the CRISPR-Cas effector polypeptide is a Cas9 polypeptide. The Cas9 protein is directed to (e.g., stabilized at) a target site within a target nucleic acid sequence (e.g., a chromosomal or extrachromosomal sequence, such as an episomal sequence, a minicircle sequence, a mitochondrial sequence, a chloroplast sequence, etc.) by association with a protein-binding segment of the Cas9 guide RNA. In some cases, the Cas9 polypeptide comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or more than 99% amino acid sequence identity to the Streptococcus pyogenes Cas9 shown in Figure 15A.

[0215] In some cases, the Cas9 polypeptide is a Staphylococcus aureus (saCas9) polypeptide. In some cases, the saCas9 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the saCas9 amino acid sequence shown in Figure 15G.

[0216] In some cases, a suitable Cas9 polypeptide is a high-fidelity (HF) Cas9 polypeptide. Kleinstiver et al. (2016) Nature 529:490. For example, amino acids N497, R661, Q695, and Q926 of the amino acid sequence shown in Figure 15A are substituted, for example, with alanine. For example, an HF Cas9 polypeptide can include an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in Figure 15A, where amino acids N497, R661, Q695, and Q926 are substituted, for example, with alanine. In some cases, a suitable Cas9 polypeptide exhibits altered PAM specificity. See, for example, Kleinstiver et al. (2015) Nature 523:481.

[0217] In some cases, a suitable CRISPR-Cas effector polypeptide is a Type V CRISPR-Cas effector polypeptide. In some cases, the Type V CRISPR-Cas effector polypeptide is a Cpfl protein. In some cases, the Cpfl protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the Cpfl amino acid sequence shown in Figure 15H, Figure 15I, or Figure 15J.

[0218] In some cases, a suitable CRISPR-Cas effector polypeptide is a CasX or CasY polypeptide. CasX and CasY polypeptides are described in Burstein et al. (2017) Nature 542:237.

[0219] In some cases, a suitable CRISPR-Cas effector polypeptide is a fusion protein comprising a CRISPR-Cas effector polypeptide fused to a heterologous polypeptide (also referred to as a "fusion partner"). In some cases, the CRISPR-Cas effector polypeptide is fused to an amino acid sequence (the fusion partner) that provides subcellular localization, i.e., the fusion partner is a subcellular localization sequence (e.g., one or more nuclear localization signals (NLS) for targeting to the nucleus, two or more NLSs, three or more NLSs, etc.).

[0220] A nucleic acid that binds to a Class 2 CRISPR-Cas effector polypeptide (e.g., a Cas9 protein, a Type V or Type VI CRISPR-Cas protein, a Cpf1 protein, etc.) and targets the complex to a specific location within a target nucleic acid is referred to herein as a "guide RNA" or "CRISPR-Cas guide nucleic acid" or "CRISPR-Cas guide RNA." The guide RNA provides target specificity to the complex (RNP complex) by containing a targeting segment that includes a guide sequence (also referred to herein as a targeting sequence), which is a nucleotide sequence complementary to the sequence of the target nucleic acid.

[0221] In some cases, the guide RNA comprises two separate nucleic acid molecules, an "activator" and a "targeter," and is referred to herein as a "dual guide RNA," "dual-molecule guide RNA," "two-molecule guide RNA," or "dgRNA." In some cases, the guide RNA is a single molecule (e.g., for some Class 2 CRISPR-Cas proteins, the corresponding guide RNA is a single molecule; in some cases, the activator and targeter are covalently linked to each other, e.g., via an intervening nucleotide), and the guide RNA is referred to as a "single guide RNA," "single-molecule guide RNA," "one-molecule guide RNA," or simply "sgRNA."

[0222] In some cases, the EDV of the present disclosure comprises a CRISPR-Cas effector polypeptide, or both a CRISPR-Cas effector polypeptide and a guide RNA. In some cases, for example, when a target nucleic acid comprises a deleterious mutation in a defective allele (e.g., a deleterious mutation in a retinal cell target nucleic acid), the CRISPR-Cas effector polypeptide / guide RNA complex, along with a donor nucleic acid comprising a nucleotide sequence that corrects the deleterious mutation (e.g., a donor nucleic acid comprising a nucleotide sequence encoding a functional copy of a protein encoded by the defective allele), can be used to correct the deleterious mutation, for example, via homology-directed repair (HDR).

[0223] In some cases, an EDV of the present disclosure comprises (i) a CRISPR-Cas effector polypeptide and (ii) one guide RNA. In some cases, the guide RNA is a single-molecule (or "single guide") guide RNA ("sgRNA"). In some cases, the guide RNA is a double-molecule (or "dual guide") guide RNA ("dgRNA").

[0224] In some cases, the EDV of the present disclosure comprises (i) a CRISPR-Cas effector polypeptide and (ii) two or more gRNAs, wherein the two or more gRNAs result in multiplexed gene knockout, for example, each of the two or more guide RNAs targets a different gene. In some cases, the guide RNA is an sgRNA. In some cases, the guide RNA is a dgRNA.

[0225] In some cases, the EDV of the present disclosure comprises (i) a CRISPR-Cas effector polypeptide and (ii) two separate sgRNAs, where the two separate sgRNAs result in deletion ("knockout") of a target nucleic acid via non-homologous end joining (NHEJ). In some cases, the guide RNA is an sgRNA. In some cases, the guide RNA is a dgRNA.

[0226] Class 2 CRISPR-Cas effector polypeptidesIn Class 2 CRISPR systems, the function of the effector complex (e.g., cleaving target DNA) is carried out by a single endonuclease (see, e.g., Zetsche et al., Cell. 2015 Oct 22;163(3):759-71; Makarova et al., Nat Rev Microbiol. 2015 Nov;13(11):722-36; Shmakov et al., Mol Cell. 2015 Nov 5;60(3):385-97), and Shmakov et al. (2017) Nature Reviews Microbiology 15:169. Thus, the term "Class 2 CRISPR-Cas protein" is used herein to encompass CRISPR-Cas effector polypeptides (e.g., target nucleic acid cleavage proteins) from Class 2 CRISPR systems.Accordingly, as used herein, the term "Class 2 CRISPR-Cas effector polypeptide" includes type II CRISPR-Cas effector polypeptides (e.g., Cas9), type VA CRISPR-Cas effector polypeptides (e.g., Cpf1 (also referred to as "Cas12a")), type VB CRISPR-Cas effector polypeptides (e.g., C2c1 (also referred to as "Cas12b")), type VC CRISPR-Cas effector polypeptides (e.g., C2c3 (also referred to as "Cas12c")), type V-U1 CRISPR-Cas effector polypeptides (e.g., C2c4), type V-U2 CRISPR-Cas effector polypeptides (e.g., , C2c8), V-U5-type CRISPR-Cas effector polypeptides (e.g., C2c5), V-U4-type CRISPR-Cas proteins (e.g., C2c9), V-U3-type CRISPR-Cas effector polypeptides (e.g., C2c10), Type VI-A CRISPR-Cas effector polypeptides (e.g., C2c2 (also known as "Cas13a")), Type VI-B CRISPR-Cas effector polypeptides (e.g., Cas13b (also known as C2c4)), and Type VI-C CRISPR-Cas effector polypeptides (e.g., Cas13c (also known as C2c7)). To date, Class 2 CRISPR-Cas effector polypeptides encompass Type II, Type V, and Type VI CRISPR-Cas effector polypeptides, but the term is also meant to encompass any Class 2 CRISPR-Cas effector polypeptide suitable for binding to a corresponding guide RNA and forming an RNP complex.

[0227] In some cases, the CRISPR-Cas effector polypeptide is a fusion polypeptide comprising (i) a CRISPR-Cas effector polypeptide and (ii) one or more heterologous fusion partners (one or more heterologous fusion polypeptides). In some cases, the fusion CRISPR-Cas effector polypeptide comprises one or more localization signal peptides. In some cases, the fusion CRISPR-Cas effector polypeptide comprises one or more localization signal peptides. Suitable localization signals ("subcellular localization signals") include, for example, a nuclear localization signal (NLS) for targeting the nucleus, a sequence for retaining the fusion protein outside the nucleus, e.g., a nuclear export sequence (NES), a sequence for retaining the fusion protein in the cytoplasm, a mitochondrial localization signal for targeting the mitochondria, a chloroplast localization signal for targeting the chloroplast, an endoplasmic reticulum (ER) retention signal, and an ER export signal. In some cases, the fusion CRISPR-Cas effector polypeptide does not contain an NLS, such that the protein is not targeted to the nucleus (this can be advantageous, for example, when the target nucleic acid is RNA present in the cytosol). In some cases, the fusion CRISPR-Cas effector polypeptide contains both an NES and one or more NLSs. A suitable NES contains a hydrophobic amino acid residue, e.g., LXXXLXXLXL (SEQ ID NO: 207), where L is a hydrophobic amino acid residue (e.g., Leu) and X is any other amino acid. Suitable NESs are known in the art; see, e.g., Xu et al. (2012) Mol. Biol. Cell 23:3677. Non-limiting examples of suitable NESs include LPPLERLTL (SEQ ID NO: 188), LALKLAGLDL (SEQ ID NO: 189), LSQALASSFSV (SEQ ID NO: 190), and NELALKLAGLDI (SEQ ID NO: 191). In some cases, the NES comprises the amino acid sequence LPPLERLTL (SEQ ID NO: 188).

[0228] In some cases, the fusion CRISPR-Cas effector polypeptide includes (is fused to) a nuclear localization signal (NLS) (e.g., in some cases, two or more, three or more, four or more, or five or more NLSs). Thus, in some cases, the fusion polypeptide includes one or more NLSs (e.g., two or more, three or more, four or more, or five or more NLSs). In some cases, one or more NLSs (two or more, three or more, four or more, or five or more NLSs) are positioned at or near (e.g., within 50 amino acids) the N-terminus and / or C-terminus. In some cases, one or more NLSs (two or more, three or more, four or more, or five or more NLSs) are positioned at or near (e.g., within 50 amino acids) the N-terminus. In some cases, one or more NLSs (two or more, three or more, four or more, or five or more NLSs) are positioned at or near (e.g., within 50 amino acids) the C-terminus. In some cases, one or more NLSs (three or more, four or more, or five or more NLSs) are located at or near (e.g., within 50 amino acids of) both the N-terminus and the C-terminus. In some cases, an NLS is located at the N-terminus and an NLS is located at the C-terminus.

[0229] In some cases, the fusion CRISPR-Cas effector polypeptide comprises (i) a lentiviral Gag polypeptide, (ii) a nuclear export signal peptide, (iii) two copies of an NLS, and iv) a CRISPR-Cas effector polypeptide. Non-limiting examples of nucleotide sequences encoding Gag-Cas9 fusion polypeptides with NESs and / or NLSs are provided in Figures 19A-19D. Figure 19E provides an example of a Gag-Cas9 fusion polypeptide with three NESs and two NLSs.

[0230] Non-limiting examples of NLSs include the NLS of the SV40 virus large T antigen having the amino acid sequence PKKKRKV (SEQ ID NO: 1), an NLS derived from nucleoplasmin (e.g., the nucleoplasmin bisecting NLS having the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 2)), the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 3) or RQRRNELKRSP (SEQ ID NO: 4), the NLS of the SV40 virus large T antigen having the amino acid sequence PKKKRKV (SEQ ID NO: 1), the NLS of the nucleoplasmin bisecting NLS having the amino acid sequence KRPAATKKAGQAKKKK (SEQ ID NO: 2)), the NLS of the nucleoplasmin bisecting NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 3) or RQRRNELKRSP (S Sequence of hRNPA1 M9 NLS, IBB domain from importin-alpha, with TIFF2025529869000123.tif4128 (SEQ ID NO: 5) TIFF2025529869000124.tif4128 (SEQ ID NO: 6), the sequences VSRKRPRP (SEQ ID NO: 7) and PPKKARED (SEQ ID NO: 8) of the fibroid T protein, the sequence PQPKKKPL (SEQ ID NO: 9) of human p53, the sequence SALIKKKKKMAP (SEQ ID NO: 10) of mouse c-abl IV, the sequences DRLRR (SEQ ID NO: 11) and PKQKKRK (SEQ ID NO: 16) of influenza virus NS1, the sequence RKLKKKIKKL (SEQ ID NO: 12) of the hepatitis virus delta antigen, the sequence REKKKFLKRR (SEQ ID NO: 13) of the mouse Mx1 protein, the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 14) of human poly(ADP-ribose) polymerase, and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 15) of the steroid hormone receptor (human) glucocorticoid. In some cases, the NLS is TIFF2025529869000125.tif4128 (SEQ ID NO: 17). Generally, the NLS (or NLSs) is strong enough to promote the accumulation of a detectable amount of the fusion polypeptide in the nucleus of a eukaryotic cell. Detection of nuclear accumulation can be performed by any suitable technique. For example, a detectable marker can be fused to the fusion polypeptide so that its location within the cell can be visualized. Cell nuclei can also be isolated from the cells, and their contents can then be analyzed by any suitable process for detecting proteins, such as immunohistochemistry, Western blot, or enzyme activity assay. Nuclear accumulation can also be determined indirectly.

[0231] Fusion Polypeptides As described above, in some cases, the EDV of the present disclosure comprises a fusion polypeptide comprising (i) a CRISPR-Cas effector polypeptide and (ii) one or more heterologous polypeptides. The heterologous polypeptide is also referred to herein as a "fusion partner." In some cases, the CRISPR-Cas effector polypeptide is fused to one or more heterologous polypeptides that have / have a desired activity (e.g., a desired catalytic activity, a subcellular localization activity, etc.) to form a fusion protein.

[0232] In some cases, the fusion partner is capable of modulating transcription of the target DNA (e.g., inhibiting transcription, increasing transcription). For example, in some cases, the fusion partner is a protein (or a domain derived from a protein) that inhibits transcription (e.g., a transcription repressor, which is a protein that functions through recruiting a transcription inhibitory protein, modifying target DNA such as methylation, recruiting a DNA modifier, modulating histones associated with the target DNA, recruiting histone modifiers such as those that modify histone acetylation and / or methylation, etc.). In some cases, the fusion partner is a protein (or a domain derived from a protein) that increases transcription (e.g., a transcription activator, which is a protein that acts through recruiting a transcription activator protein, modifying target DNA such as demethylation, recruiting a DNA modifier, modulating histones associated with the target DNA, recruiting histone modifiers such as those that modify histone acetylation and / or methylation, etc.). In some cases, the fusion partner is a reverse transcriptase. In some cases, the fusion partner is a base editor. In some cases, the fusion partner is a deaminase.

[0233] In some cases, the CRISPR-Cas fusion polypeptide comprises a heterologous polypeptide having an enzymatic activity that modifies a target nucleic acid (e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, or glycosylase activity).

[0234] In some cases, the CRISPR-Cas fusion polypeptide comprises a heterologous polypeptide having an enzymatic activity (e.g., methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylating activity, deadenylating activity, sumoylating activity, desumoylating activity, ribosylation activity, deribosylation activity, myristoylating activity, or demyristoylating activity) that modifies a polypeptide (e.g., a histone) associated with a target nucleic acid.

[0235] Examples of proteins (or fragments thereof) that can be used to increase transcription include, but are not limited to, transcriptional activators such as VP16, VP64, VP48, VP160, p65 subdomains (e.g., from NFkB), and activation domains of EDLL and / or TAL activation domains (e.g., for activity in plants); histone lysine methyltransferases such as SET1A, SET1B, MLL1-5, ASH1, SYMD2, NSD1; histone lysine demethylases such as JHDM2a / b, UTX, JMJD3; histone acetyltransferases such as GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, SRC1, ACTR, P160, CLOCK; and DNA demethylases such as ten-eleven translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1.

[0236] Examples of proteins (or fragments thereof) that can be used to reduce transcription include transcription repressors such as Kruppel-associated box (KRAB or SKD); KOX1 repression domain; Mad mSIN3-interacting domain (SID); ERF repressor domain (ERD), SRDX repression domain (e.g., for repression in plants), etc.; histone lysine methyltransferases such as Pr-SET7 / 8, SUV4-20H1, and RIZ1; histone lysine demethylases such as JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARID1A / RBP2, JARID1B / PLU-1, JARID1C / SMCX, and JARID1D / SMCY; histone lysine deacetylases such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, and HDAC11; HhaI DNA These include, but are not limited to, DNA methylases such as m5c-methyltransferase (M.HhaI), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants); and peripheral recruitment elements such as lamin A and lamin B.

[0237] In some cases, the fusion partner has an enzymatic activity that modifies the target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA). Examples of enzymatic activities that can be provided by a fusion partner include nuclease activity, such as that provided by a restriction enzyme (e.g., FokI nuclease), methyltransferase activity (e.g., HhaI DNA cleavage enzyme), and the like. Methyltransferase activity, such as that provided by m5c-methyltransferase (M.HhaI), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants), etc.); demethylase activity, such as that provided by demethylases (e.g., ten-eleven translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1, etc.), DNA repair activity, DNA damage activity, and deaminases (e.g., cytosine deaminase enzymes, such as rat APOBEC1). These include, but are not limited to, deaminating activity, dismutase activity, alkylating activity, depurinating activity, oxidizing activity, pyrimidine dimer-forming activity, integrase activity such as that provided by integrases and / or resolvases (e.g., Gin invertase, Gin integrase such as a high activity mutant of GinH106Y; human immunodeficiency virus type 1 integrase (IN); Tn3 resolvase, etc.), transposase activity, recombinase activity such as that provided by recombinases (e.g., the catalytic domain of Gin recombinase), polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity.

[0238] In some cases, the fusion partner has an enzymatic activity that modifies a protein (e.g., histone, RNA-binding protein, DNA-binding protein, etc.) associated with a target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA). Examples of enzymatic activities (that modify a protein associated with a target nucleic acid) that can be provided by a fusion partner include histone methyltransferases (HMTs) (e.g., suppressor of diversity 3-9 homolog 1 (SUV39H1, also known as KMT1A), euchromatin histone lysine methyltransferase 2 (G9A, also known as KMT1C and EHMT2), SUV39H2, ESET / SETDB1, SET1A, SET1B, MLL1-5, ASH1, SYM methyltransferase activity, such as that provided by D2, NSD1, DOT1L, Pr-SET7 / 8, SUV4-20H1, EZH2, RIZ1), histone demethylases (e.g., lysine demethylase 1A (KDM1A, also known as LSD1), JHDM2a / b, JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARID1A / RBP2, JARID1B / PLU-1, JARID1C / SMCX, JARID1D / SMCY, demethylase activity such as that provided by histone acetyltransferases (e.g., catalytic cores / fragments of human acetyltransferases p300, GCN5, PCAF, CBP, TAF1, TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, HBO1 / MYST2, HMOF / MYST1, SRC1, ACTR, P160, CLOCK, etc.); histone deacetylases (e.g., These include, but are not limited to, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylating activity, deadenylating activity, sumoylating activity, desumoylating activity, ribosylation activity, deribosylation activity, myristoylating activity, and demyristoylating activity, such as those provided by HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, etc.

[0239] Additional examples of suitable fusion partners are dihydrofolate reductase (DHFR) destabilization domains (eg, to generate chemically controllable fusion polypeptides), and chloroplast transit peptides.

[0240] In some cases, the CRISPR-Cas fusion polypeptide comprises (a) a CRISPR-Cas effector polypeptide and (b) a chloroplast transit peptide. Thus, for example, a ribonucleoprotein (RNP) complex comprising a CRISPR-Cas effector polypeptide and a guide RNA of the present disclosure can target chloroplasts. In some cases, this targeting can be achieved by the presence of an N-terminal extension called a chloroplast transit peptide (CTP) or plastid transit peptide. If the expressed polypeptide is to be compartmentalized in plant plastids (e.g., chloroplasts), the chromosomal transgene from a bacterial source must have a sequence encoding a CTP sequence fused to the sequence encoding the expressed polypeptide. Therefore, localization of an exogenous polypeptide to chloroplasts is often achieved by operably linking a polynucleotide sequence encoding a CTP sequence to the 5' region of a polynucleotide encoding the exogenous polypeptide. The CTP is removed in a processing step during translocation into plastids. However, processing efficiency can be affected by the amino acid sequence of the CTP and sequences near the amino terminus of the peptide. Other options for targeting to chloroplasts that have been described are the maize cab-m7 signal sequence (U.S. Pat. No. 7,022,896, WO 97 / 41228), the pea glutathione reductase signal sequence (WO 97 / 41228), and the CTP described in US 2009 / 029861.

[0241] In some cases, the CRISPR-Cas fusion polypeptide comprises (a) a CRISPR-Cas effector polypeptide of the present disclosure and (b) an endosomal escape peptide. In some cases, the endosomal escape polypeptide comprises the amino acid sequence GLFXALLXLLXSLWXLLLXA (SEQ ID NO: 24), where each X is independently selected from lysine, histidine, and arginine. In some cases, the endosomal escape polypeptide comprises the amino acid sequence GLFHALLHLLHSLWHLLLHA (SEQ ID NO: 25).

[0242] Additional suitable heterologous polypeptides include, but are not limited to, polypeptides that directly and / or indirectly increase or decrease the transcription and / or translation of a target nucleic acid (e.g., transcriptional activators or fragments thereof, proteins or fragments thereof that recruit transcriptional activators, small molecule / drug-responsive transcriptional and / or translational regulators, translational regulatory proteins, etc.). Non-limiting examples of heterologous polypeptides for achieving increased or decreased transcription include transcriptional activator and transcriptional repressor domains. In some such cases, CRISPR-Cas fusion polypeptides are targeted to specific locations (i.e., sequences) within a target nucleic acid by a guide nucleic acid (guide RNA) to effect locus-specific regulation, such as blocking RNA polymerase binding to the promoter (selectively inhibiting transcriptional activator function) and / or modifying the local chromatin state (e.g., when using a fusion sequence that modifies the target nucleic acid or a polypeptide associated with the target nucleic acid). In some cases, the change is transient (e.g., transcriptional repression or activation). In some cases, the change is heritable (eg, when an epigenetic modification is made to the target nucleic acid or a protein associated with the target nucleic acid, such as a nucleosomal histone).

[0243] Non-limiting examples of heterologous polypeptides for use when targeting ssRNA target nucleic acids include (but are not limited to) splicing factors (e.g., RS domains); protein translation components (e.g., translation initiation, elongation, and / or release factors, e.g., eIF4G); RNA methylases; RNA editing enzymes (e.g., RNA deaminases, e.g., adenosine deaminases acting on RNA (ADARs), including enzymes that edit A to I and / or C to U); helicases; RNA binding proteins, and the like. It is understood that a heterologous polypeptide can comprise an entire protein, or in some cases, can comprise a fragment of a protein (e.g., a functional domain).

[0244] Heterologous polypeptides may be selected from a wide variety of proteins, whether transient or irreversible, direct or indirect, including endonucleases (e.g., RNase III from proteins such as SMG5 and SMG6, CRR22 DYW domain, Dicer, and PIN (PilT N-terminal domain); proteins and protein domains responsible for stimulating RNA cleavage (e.g., CPSF, CstF, CFIm, and CFIIm); exonucleases (e.g., XRN-1 or exonuclease T); deadenylases (e.g., HNT3); proteins and protein domains responsible for nonsense-mediated RNA decay (e.g., UPF1, UPF2, UPF3, UPF3b, RNP S1, Y14, DEK, REF2, and SRm160); proteins and protein domains responsible for stabilizing RNA (e.g., PABP); proteins and protein domains responsible for repressing translation (e.g., Ago2 and Ago4); proteins and protein domains responsible for stimulating translation (e.g., Staufen); proteins and protein domains responsible for (e.g., capable of) regulating translation (e.g., translation factors such as initiation factors, elongation factors, and release factors, e.g., eIF4G); proteins and protein domains responsible for polyadenylation of RNA (e.g., PAP1, GLD-2, and Star-PAP); proteins and protein domains responsible for polyuridinylation of RNA (e.g., CI D1 and terminal uridylate transferase); proteins and protein domains responsible for RNA localization (e.g., from IMP1, ZBP1, She2p, She3p, and Bicaudal-D); proteins and protein domains responsible for nuclear retention of RNA (e.g., Rrp6); proteins and protein domains responsible for nuclear export of RNA (e.g., TAP, NXF1, THO, TREX, REF, and Aly); proteins and protein domains responsible for suppression of RNA splicing (e.g., PTB, Sam68, and hnRNP A1); proteins and protein domains responsible for stimulation of RNA splicing (e.g., serine / arginine-rich (SR) domains); proteins and protein domains responsible for reducing transcription efficiency (e.g., FUS(TLS));and effector domains selected from the group including proteins and protein domains responsible for stimulating transcription (e.g., CDK7 and HIV Tat), and may be any domain capable of interacting with ssRNA (which for the purposes of this disclosure includes intra- and / or intermolecular secondary structures, e.g., double-stranded RNA duplexes such as hairpins, stem-loops, etc.). Alternatively, the effector domain may be selected from the group comprising endonucleases; proteins and protein domains capable of stimulating RNA cleavage; exonucleases; deadenylases; proteins and protein domains with nonsense-mediated RNA degradation activity; proteins and protein domains capable of stabilizing RNA; proteins and protein domains capable of repressing translation; proteins and protein domains capable of stimulating translation; proteins and protein domains capable of regulating translation (e.g., translation factors such as initiation factors, elongation factors, release factors, e.g., eIF4G); proteins and protein domains capable of polyadenylation of RNA; proteins and protein domains capable of polyuridylation of RNA; proteins and protein domains with RNA localization activity; proteins and protein domains capable of nuclear retention of RNA; proteins and protein domains with RNA nuclear export activity; proteins and protein domains capable of repressing RNA splicing; proteins and protein domains capable of stimulating RNA splicing; proteins and protein domains capable of reducing transcription efficiency; and proteins and protein domains capable of stimulating transcription. Another suitable heterologous polypeptide is a PUF RNA-binding domain, which is described in more detail in WO2012 / 068627, which is incorporated herein by reference in its entirety;

[0245] Some RNA splicing factors that can be used (in whole or as fragments thereof) as heterologous polypeptides for the fusion polypeptides of the present disclosure have a modular organization with distinct sequence-specific RNA-binding modules and splicing effector domains. For example, members of the serine / arginine-rich (SR) protein family contain an N-terminal RNA recognition motif (RRM) that binds to exon splicing enhancers (ESEs) within pre-mRNAs and a C-terminal RS domain that promotes exon inclusion. As another example, the hnRNP protein hnRNP A1 binds to exon splicing silencers (ESSs) via its RRM domain and inhibits exon inclusion via its C-terminal glycine-rich domain. Some splicing factors can regulate the alternative use of splice sites (SSs) by binding to regulatory sequences between the two alternative sites. For example, ASF / SF2 can recognize ESEs and promote the use of intron-proximal sites, whereas hnRNP A1 can bind to ESSs and shift splicing toward the use of intron-distal sites. One application of such factors is to generate ESFs that regulate the alternative splicing of endogenous genes, particularly disease-related genes. For example, Bcl-x pre-mRNA produces two splicing isoforms with two alternative 5' splice sites, encoding proteins with opposing functions. The long splicing isoform, Bcl-xL, is a potent apoptosis inhibitor expressed in long-lived, postmitotic cells and is upregulated in many cancer cells, protecting them from apoptotic signals. The short isoform, Bcl-xS, is a pro-apoptotic isoform and is expressed at high levels in cells with a high conversion rate (e.g., lymphocyte development). The ratio of the two Bcl-x splicing isoforms varies depending on the multiple splicing sites located either in the core exon region or in the exon extension region (i.e., between the two alternative 5' splice sites). Controlled by TIFF2025529869000126.tif3128. For further examples, see WO2010 / 075303, which is incorporated by reference in its entirety.

[0246] Additional suitable fusion partners include, but are not limited to, proteins (or fragments thereof) that are boundary elements (e.g., CTCF), proteins and fragments thereof that confer peripheral recruitment (e.g., lamin A, lamin B, etc.), protein docking elements (e.g., FKBP / FRB, Pil1 / Aby1, etc.).

[0247] nuclease In some cases, a CRISPR-Cas fusion polypeptide comprises (i) a CRISPR-Cas effector polypeptide and (ii) a heterologous polypeptide ("fusion partner"), where the heterologous polypeptide is a nuclease. In some cases, a CRISPR-Cas fusion polypeptide comprises a nucleic acid-binding effector polypeptide. Suitable nucleic acid-binding effector polypeptides can be nucleases, including, but not limited to, homing nuclease polypeptides, FokI polypeptides, transcription activator-like effector nuclease (TALEN) polypeptides, MegaTAL polypeptides, meganuclease polypeptides, zinc finger nucleases (ZFNs), ARCUS nucleases, and the like. Meganucleases can be engineered from LADLIDADG homing endonucleases (LHEs). MegaTAL polypeptides can comprise a TALE DNA-binding domain and an engineered meganuclease. For example, WO2004 / 067736 (homing endonucleases), Urnov et al. (2005) Nature 435:646 (ZFNs), Mussolino et al. (2011) Nucle. Acids Res. 39:9283 (TALE nucleases), Boissel et al. (2013) Nucl. Acids Res. 42:2591 (MegaTALs).

[0248] reverse transcriptase In some cases, a CRISPR-Cas fusion polypeptide comprises (i) a CRISPR-Cas effector polypeptide and (ii) a heterologous polypeptide ("fusion partner"), where the heterologous polypeptide is a reverse transcriptase polypeptide. Reverse transcriptases are known in the art, see, e.g., Cote and Roth (2008) Virus Res. 134:186. Suitable reverse transcriptases include, for example, murine leukemia virus reverse transcriptase, Rous sarcoma virus reverse transcriptase, human immunodeficiency virus type I reverse transcriptase, Moloney murine leukemia virus reverse transcriptase, transcriptional xenopolymerase (RTX), avian myeloblastosis virus reverse transcriptase (AMV-RT), Eubacterium rectale maturase reverse transcriptase (Marathon®), and the like. The reverse transcriptase fusion partner can include one or more mutations. For example, in some cases, the reverse transcriptase is an M-MLV reverse transcriptase polypeptide that includes one or more mutations selected from the group consisting of D200N, T306K, W313F, T330P, and L603W. In some cases, the reverse transcriptase is an M-MLV reverse transcriptase polypeptide that includes one or more mutations selected from the group consisting of D200N, T306K, W313F, T330P, and L603W. RT (e.g., containing the following substitutions: D200N / L603W / T330P / T306K / W313F) (where D200, L603, T330, T306, and W313 correspond to D199, L602, T329, T305, and W312 in the M-MLV RT amino acid sequence shown in Figure 17).

[0249] Base Editor In some cases, a CRISPR-Cas fusion polypeptide comprises (i) a CRISPR-Cas effector polypeptide and (ii) one or more heterologous polypeptides ("fusion partners"), wherein at least one of the one or more heterologous polypeptides is a deaminase. Suitable deaminases include, for example, adenosine deaminase, cytidine deaminase (e.g., activation-induced cytidine deaminase (AID)), APOBEC3G, etc.

[0250] A suitable adenosine deaminase is any enzyme capable of deaminating adenosine in DNA. In some cases, the deaminase is TadA deaminase.

[0251] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000127.tif17159 (SEQ ID NO: 26).

[0252] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000128.tif17157 (SEQ ID NO: 27).

[0253] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following Staphylococcus aureus TadA amino acid sequence: TIFF2025529869000129.tif16158 (SEQ ID NO: 28).

[0254] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following Bacillus subtilis TadA amino acid sequence: TIFF2025529869000130.tif16158 (SEQ ID NO: 29).

[0255] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following Salmonella typhimurium TadA: TIFF2025529869000131.tif17158 (SEQ ID NO: 30).

[0256] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following Shewanella putrefaciens TadA amino acid sequence: TIFF2025529869000132.tif17158 (SEQ ID NO: 31).

[0257] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following Haemophilus influenzae F3031 TadA amino acid sequence: TIFF2025529869000133.tif17159 (SEQ ID NO: 32).

[0258] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following Caulobacter crescentus TadA amino acid sequence: TIFF2025529869000134.tif17159 (SEQ ID NO: 33).

[0259] In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following Geobacter sulfurreducens TadA amino acid sequence: TIFF2025529869000135.tif17158 (SEQ ID NO: 34).

[0260] Cytidine deaminases suitable for inclusion in the CRISPR-Cas effector polypeptide fusion polypeptides of the disclosure include any enzyme capable of deaminating cytidine in DNA.

[0261] In some cases, the cytidine deaminase is a deaminase from the apolipoprotein B mRNA editing complex (APOBEC) family of deaminases. In some cases, the APOBEC family deaminase is selected from the group consisting of APOBEC1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase, and APOBEC3H deaminase. In some cases, the cytidine deaminase is activation-induced deaminase (AID).

[0262] In some cases, a suitable cytidine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000136.tif23159 (SEQ ID NO: 35).

[0263] In some cases, a suitable cytidine deaminase is AID and comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000137.tif23157 (SEQ ID NO: 36).

[0264] In some cases, a suitable cytidine deaminase is AID and comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000138.tif23157 (SEQ ID NO: 35).

[0265] transcription factors In some cases, the CRISPR-Cas fusion polypeptide comprises (i) a CRISPR-Cas effector polypeptide and (ii) a heterologous polypeptide ("fusion partner"), where the heterologous polypeptide is a transcription factor. The transcription factor can comprise (i) a DNA-binding domain and (ii) a transcriptional activator. The transcription factor can comprise (i) a DNA-binding domain and (ii) a transcriptional repressor. Suitable transcription factors include polypeptides comprising a transcriptional activator or transcriptional repressor domain (e.g., Kruppel-associated box (KRAB or SKD), Mad mSIN3-interacting domain (SID), ERF repressor domain (ERD), etc.), zinc finger-based artificial transcription factors (see, e.g., Sera (2009) Adv. Drug Deliv. 61:513), TALE-based artificial transcription factors (see, e.g., Liu et al. (2013) Nat. Rev. Genetics 14:781), and the like. In some cases, the transcription factor comprises a VP64 polypeptide (transcriptional activator). In some cases, the transcription factor comprises a Kruppel-associated box (KRAB) polypeptide (transcriptional repressor). In some cases, the transcription factor comprises a Mad mSIN3-interacting domain (SID) polypeptide (transcriptional repressor). In some cases, the transcription factor comprises an ERF repressor domain (ERD) polypeptide (transcriptional repressor). For example, in some cases, the transcription factor is a transcriptional activator, and the transcriptional activator is GAL4-VP16.

[0266] Recombinase In some cases, a CRISPR-Cas fusion polypeptide comprises (i) a CRISPR-Cas effector polypeptide and (ii) a heterologous polypeptide ("fusion partner"), where the heterologous polypeptide is a recombinase. Suitable recombinases include, for example, Cre recombinase, Hin recombinase, Tre recombinase, FLP recombinase, etc.

[0267] NLS In some cases, a CRISPR-Cas fusion polypeptide comprises (i) a CRISPR-Cas effector polypeptide and (ii) a heterologous polypeptide ("fusion partner"), where the heterologous polypeptide confers subcellular localization, i.e., the heterologous polypeptide comprises a subcellular localization sequence (e.g., a nuclear localization signal (NLS) for targeting to the nucleus, a sequence for retaining the fusion protein out of the nucleus, e.g., a nuclear export sequence (NES), a sequence for retaining the fusion protein in the cytoplasm, a mitochondrial localization signal for targeting to mitochondria, a chloroplast localization signal for targeting to chloroplasts, an endoplasmic reticulum (ER) retention signal, etc.). In some cases, the CRISPR-Cas fusion polypeptide does not comprise an NLS, such that the protein is not targeted to the nucleus (this can be advantageous, for example, when the target nucleic acid is RNA present in the cytosol). In some cases, the heterologous polypeptide can be provided with a tag (e.g., a fluorescent protein, e.g., green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), mCherry, tdTomato, etc.; a histidine tag, e.g., a 6XHis tag; a hemagglutinin (HA) tag; a FLAG tag; a Myc tag, etc.) to facilitate tracking and / or purification (i.e., the heterologous polypeptide is detectably labeled).

[0268] In some cases, a CRISPR-Cas fusion polypeptide comprises (a) a CRISPR-Cas effector polypeptide and (b) one or more nuclear localization signals (NLSs) (e.g., in some cases, two or more, three or more, four or more, or five or more NLSs). Thus, in some cases, a fusion polypeptide of the present disclosure comprises one or more NLSs (e.g., two or more, three or more, four or more, or five or more NLSs). In some cases, the one or more NLSs (two or more, three or more, four or more, or five or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the N-terminus and / or C-terminus. In some cases, the one or more NLSs (two or more, three or more, four or more, or five or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the N-terminus. In some cases, one or more NLSs (two or more, three or more, four or more, or five or more NLSs) are located at or near (e.g., within 50 amino acids of) the C-terminus. In some cases, one or more NLSs (three or more, four or more, or five or more NLSs) are located at or near (e.g., within 50 amino acids of) both the N-terminus and the C-terminus. In some cases, an NLS is located at the N-terminus and an NLS is located at the C-terminus.

[0269] In some cases, the CRISPR-Cas fusion polypeptide comprises a) a CRISPR-Cas effector polypeptide and b) 1 to 10 NLSs (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, or 2 to 5 NLSs). In some cases, the CRISPR-Cas fusion polypeptide comprises a) a CRISPR-Cas effector polypeptide and b) 2 to 5 NLSs (e.g., 2 to 4 NLSs, or 2 to 3 NLSs).

[0270] Non-limiting examples of NLSs include the NLS of the SV40 virus large T antigen having the amino acid sequence PKKKRKV (SEQ ID NO: 1), an NLS derived from nucleoplasmin (e.g., the nucleoplasmin bisecting NLS having the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 2)), the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 3) or RQRRNELKRSP (SEQ ID NO: 4), the NLS of the SV40 virus large T antigen having the amino acid sequence PKKKRKV (SEQ ID NO: 1), the NLS of the nucleoplasmin bisecting NLS having the amino acid sequence KRPAATKKAGQAKKKK (SEQ ID NO: 2)), the NLS of the nucleoplasmin bisecting NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 3) or RQRRNELKRSP (S Sequence of hRNPA1 M9 NLS, IBB domain from importin-alpha, with TIFF2025529869000139.tif4128 (SEQ ID NO: 5) TIFF2025529869000140.tif4128 (SEQ ID NO: 6), the sequences VSRKRPRP (SEQ ID NO: 7) and PPKKARED (SEQ ID NO: 8) of the fibroid T protein, the sequence PQPKKKPL (SEQ ID NO: 9) of human p53, the sequence SALIKKKKKMAP (SEQ ID NO: 10) of mouse c-abl IV, the sequences DRLRR (SEQ ID NO: 11) and PKQKKRK (SEQ ID NO: 16) of influenza virus NS1, the sequence RKLKKKIKKL (SEQ ID NO: 12) of the hepatitis virus delta antigen, the sequence REKKKFLKRR (SEQ ID NO: 13) of mouse Mx1 protein, the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 14) of human poly(ADP-ribose) polymerase, and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 15) of the steroid hormone receptor (human) glucocorticoid. Generally, the NLS (or NLSs) is strong enough to promote the accumulation of detectable amounts of CRISPR-Cas effector polypeptides in the nucleus of eukaryotic cells. Detection of nuclear accumulation can be carried out by any suitable technique. For example, a detectable marker can be fused to the CRISPR-Cas effector polypeptide so that its location within the cell can be visualized. Cell nuclei can also be isolated from cells, and their contents can then be analyzed by any suitable process for detecting proteins, such as immunohistochemistry, Western blot, or enzyme activity assay. Nuclear accumulation can also be determined indirectly.

[0271] PTD In some cases, a CRISPR-Cas fusion polypeptide contains a "protein transduction domain" or PTD (also known as a CPP—cell-penetrating peptide), which refers to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates crossing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule, which can range from small polar molecules to large macromolecules and / or nanoparticles, facilitates the molecule crossing the membrane, for example, moving from the extracellular space to the intracellular space, or from the cytosol into an organelle. In some cases, the PTD is covalently attached to the amino terminus of the CRISPR-Cas effector polypeptide. In some cases, the PTD is covalently attached to the carboxyl terminus of the CRISPR-Cas effector polypeptide. In some cases, the PTD is inserted internally into the CRISPR-Cas effector polypeptide (i.e., not at the N- or C-terminus of the CRISPR-Cas effector polypeptide) at a suitable insertion site. In some cases, a CRISPR-Cas fusion polypeptide comprises a) a CRISPR-Cas fusion polypeptide and b) one or more PTDs (e.g., two or more, three or more, four or more PTDs). In some cases, the PTD comprises a nuclear localization signal (NLS) (e.g., in some cases, two or more, three or more, four or more, or five or more NLSs). Thus, in some cases, a CRISPR-Cas fusion polypeptide comprises one or more NLSs (e.g., two or more, three or more, four or more, or five or more NLSs). In some cases, the PTD is covalently linked to a nucleic acid (e.g., a CRISPR-Cas guide nucleic acid, a polynucleotide encoding a CRISPR-Cas guide nucleic acid, a polynucleotide encoding a fusion polypeptide, a donor polynucleotide, etc.).Examples of PTDs include, but are not limited to, a minimal undecapeptide protein transduction domain (YGRKKRRQRRR; corresponding to residues 47-57 of HIV-1 TAT, comprising SEQ ID NO: 127); a polyarginine sequence containing a sufficient number of arginines to direct cell entry (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); a Drosophila antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52(7):1732-1737); a truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research 21:1248-1256); polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97:13003-13008); RRQRRTSKLMKR (SEQ ID NO: 128); Transportan. TIFF2025529869000141.tif3128 (SEQ ID NO: 129); TIFF2025529869000142.tif3128 (SEQ ID NO: 130); and RQIKIWFQNRRMKWKK (SEQ ID NO: 131). Exemplary PTDs include, but are not limited to, YGRKKRRQRRR (SEQ ID NO: 127), RKKRRQRRR (SEQ ID NO: 132); arginine homopolymers of from 3 to 50 arginine residues. Exemplary PTD domain amino acid sequences include, but are not limited to, any of the following: YGRKKRRQRRR (SEQ ID NO: 127), RKKRRQRR (SEQ ID NO: 133), YARAAARQARA (SEQ ID NO: 134), THRLPRRRRRR (SEQ ID NO: 135), and GGRRARRRRRR (SEQ ID NO: 136). In some cases, the PTD is an activatable CPP (ACPP) (see Aguilera et al. (2009) Integr Biol (Camb) June;1(5-6):371-381). ACPPs contain a polycationic CPP (e.g., Arg9 or "R9") connected to a matching polyanion (e.g., Glu9 or "E9") via a cleavable linker, which reduces the net charge to near zero, thereby inhibiting adhesion and uptake into cells. Upon cleavage of the linker, the polyanion is released, locally unmasking the polyarginine and its inherent adhesive properties, thus "activating" the ACPP to cross the membrane.

[0272] Linkers (e.g., for fusion partners) In some cases, the CRISPR-Cas polypeptide may be fused to the fusion partner via a linker polypeptide (e.g., one or more linker polypeptides). The linker polypeptide may have any of a variety of amino acid sequences. While other chemical bonds are not excluded, proteins can generally be linked by a spacer peptide that is flexible in nature. Suitable linkers include polypeptides ranging from 4 to 40 amino acids in length, or from 4 to 25 amino acids in length. These linkers can be produced by using oligonucleotides encoding a synthetic linker to couple the proteins, or they can be encoded by a nucleic acid sequence encoding the fusion protein. Peptide linkers with some degree of flexibility can be used. The linking peptide may have virtually any amino acid sequence, keeping in mind that preferred linkers generally have sequences that result in flexible peptides. The use of small amino acids such as glycine and alanine is used to create flexible peptides. Creating such sequences is routine for those of skill in the art. A variety of different linkers are commercially available and may be suitable for use.

[0273] Examples of linker polypeptides include glycine polymers (G) n (where n is at least one integer), glycine-serine polymers (e.g., (GS) n , (GSGGS) n (SEQ ID NO: 40), (GGSGGS) n (SEQ ID NO: 41), (GGGGS)n (SEQ ID NO: 38), and (GGGS) n(SEQ ID NO: 42) (where n is at least one integer; e.g., n is an integer between 1 and 10), glycine-alanine polymers, and alanine-serine polymers. Exemplary linkers can include amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 39), GGSGG (SEQ ID NO: 43), GSGSG (SEQ ID NO: 44), GSGGG (SEQ ID NO: 45), GGGSG (SEQ ID NO: 46), GSSSG (SEQ ID NO: 47), GGGGS (SEQ ID NO: 38), and the like. One of skill in the art will recognize that the design of a peptide conjugated to any desired element can include a linker that is fully or partially flexible, such that the linker can include a flexible linker and one or more moieties that confer a less flexible structure.

[0274] Guide nucleic acid As described above, the EDV of the present disclosure comprises a CRISPR-Cas effector polypeptide guide nucleic acid (e.g., RNA) or a nucleic acid comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide guide RNA.

[0275] A nucleic acid molecule that binds to a CRISPR-Cas effector polypeptide protein and targets the complex to a specific location within a target nucleic acid is referred to herein as a "CRISPR-Cas effector polypeptide guide RNA" or simply "guide RNA."

[0276] A guide RNA can be said to comprise two segments: a first segment (referred to herein as a "targeting segment") and a second segment (referred to herein as a "protein-binding segment"). A "segment" refers to a segment / section / region of a molecule, e.g., a contiguous stretch of nucleotides within a nucleic acid molecule. A segment can also refer to a region / section of a complex, such that a segment can comprise two or more molecular regions. The "targeting segment" is also referred to herein as the "variable region" of the guide RNA. The "protein-binding segment" is also referred to herein as the "constant region" of the guide RNA. In some cases, the guide RNA is a Cas9 guide RNA.

[0277] The first segment (targeting segment) of the guide RNA comprises a nucleotide sequence (guide sequence) that is complementary to (and therefore hybridizes with) a specific sequence (target site) within a target nucleic acid (e.g., a target DNA, e.g., ssDNA, dsDNA, or target RNA), such as the complementary strand of a double-stranded target DNA. The protein-binding segment (or "protein-binding sequence") interacts with (binds to) a CRISPR-Cas effector polypeptide. The protein-binding segment of the guide RNA comprises two stretches of complementary nucleotides that hybridize to each other to form a double-stranded RNA duplex (dsRNA duplex). Site-specific binding and / or cleavage of a target nucleic acid (e.g., genomic DNA) can occur at a location (e.g., a target sequence at a target locus) determined by base-pairing complementarity between the guide RNA (guide sequence of the guide RNA) and the target nucleic acid.

[0278] The guide RNA and CRISPR-Cas effector polypeptide form a complex (e.g., bound via non-covalent interactions). The guide RNA contains a targeting segment that includes a guide sequence (a nucleotide sequence complementary to the sequence of a target nucleic acid), thereby providing target specificity to the complex. The CRISPR-Cas effector polypeptide of the complex provides site-specific activity (e.g., cleavage activity or an activity provided by a CRISPR-Cas effector polypeptide when the CRISPR-Cas effector polypeptide is a CRISPR-Cas effector polypeptide fusion polypeptide, i.e., when it has a fusion partner). In other words, upon association with the guide RNA, the CRISPR-Cas effector polypeptide is guided to a target nucleic acid sequence (e.g., a chromosomal nucleic acid, e.g., a target sequence within a chromosome; a target sequence within an extrachromosomal nucleic acid, e.g., an episomal nucleic acid, a minicircle, ssRNA, ssDNA, etc.; a target sequence within a mitochondrial nucleic acid; a target sequence within a chloroplast nucleic acid; a target sequence within a plasmid; a target sequence within a viral nucleic acid, etc.).

[0279] The "guide sequence," also referred to as the "targeting sequence" of a guide RNA, can be modified to enable the guide RNA to target a CRISPR-Cas effector polypeptide to any desired sequence in any desired target nucleic acid, except that the guide RNA can take into account a protospacer adjacent motif (PAM) sequence. Thus, for example, a guide RNA can have a targeting segment having a sequence (guide sequence) that is complementary to (e.g., can hybridize with) a sequence in a nucleic acid within a eukaryotic cell, e.g., a viral nucleic acid, a eukaryotic nucleic acid (e.g., a eukaryotic chromosome, a chromosomal sequence, a eukaryotic RNA, etc.).

[0280] In some embodiments, the guide RNA comprises two separate nucleic acid molecules, an "activator" and a "targeter," and is referred to herein as a "dual guide RNA," "dual molecule guide RNA," or "dual molecule guide RNA," "dual guide RNA," or "dgRNA." In some embodiments, the activator and targeter are covalently linked to each other (e.g., via an intervening nucleotide), and the guide RNA is referred to as a "single guide RNA," "Cas9 single guide RNA," "single molecule Cas9 guide RNA," or "single molecule Cas9 guide RNA," or simply "sgRNA."

[0281] Guide RNAs include crRNA-like ("CRISPR RNA" / "targeting factor" / "crRNA" / "crRNA repeat") molecules and corresponding tracrRNA-like ("trans-acting CRISPR RNA" / "activator" / "tracrRNA") molecules. The crRNA-like molecule (targeting factor) contains both the targeting segment (single strand) of the guide RNA and a stretch of nucleotides ("duplex-forming segment") that forms one half of the dsRNA duplex of the protein-binding segment of the guide RNA. The corresponding tracrRNA-like molecule (activator / tracrRNA) contains a stretch of nucleotides (duplex-forming segment) that forms the other half of the dsRNA duplex of the protein-binding segment of the guide nucleic acid. In other words, the stretch of nucleotides of the crRNA-like molecule is complementary to and hybridizes with the stretch of nucleotides of the tracrRNA-like molecule to form the dsRNA duplex of the protein-binding domain of the guide RNA. Thus, each targeting agent molecule can be said to have a corresponding activator molecule (having a region that hybridizes with the targeting agent). The targeting agent molecule additionally provides a targeting segment. Thus, targeting agent and activator molecules (as a corresponding pair) hybridize to form a guide RNA. The exact sequence of a given crRNA or tracrRNA molecule is characteristic of the species in which the RNA molecule is found. A dual guide RNA can include any corresponding activator and targeting agent pair.

[0282] The terms "activator" or "activator RNA" are used herein to refer to a tracrRNA-like molecule (tracrRNA: "trans-acting CRISPR RNA") that is a dual-guide RNA (and thus a single-guide RNA when the "activator" and "targeting agent" are linked together, e.g., by an intervening nucleotide). Thus, for example, a guide RNA (dgRNA or sgRNA) contains an activator sequence (e.g., a tracrRNA sequence). A tracr molecule (tracrRNA) is a naturally occurring molecule that hybridizes with a CRISPR RNA molecule (crRNA) to form a dual-guide RNA. The term "activator" is used herein to encompass naturally occurring tracrRNAs, but also to encompass tracrRNAs with modifications (e.g., truncations, sequence mutations, base modifications, backbone modifications, linkage modifications, etc.) that retain at least one function of the tracrRNA (e.g., contributing to the dsRNA duplex to which the Cas9 protein binds). In some cases, the activator introduces one or more stem-loops that can interact with the Cas9 protein. An activator may be referred to as having a tracr sequence (tracrRNA sequence), and in some cases is a tracrRNA, although the term "activator" is not limited to naturally occurring tracrRNA.

[0283] The terms "targeting agent" or "targeting agent RNA" are used herein to refer to a crRNA-like molecule (crRNA: "CRISPR RNA") of a dual guide RNA (and thus of a single guide RNA when the "activator" and "targeting agent" are linked together, e.g., by an intervening nucleotide). Thus, for example, a guide RNA (dgRNA or sgRNA) comprises a targeting segment (which includes nucleotides that hybridize (are complementary to) the target nucleic acid) and a duplex-forming segment (e.g., a duplex-forming segment of a crRNA, which may also be referred to as a crRNA repeat). The sequence of the targeting segment (the segment that hybridizes with the target sequence of a target nucleic acid) of a targeting agent is modified by the user to hybridize with the desired target nucleic acid, and therefore the sequence of the targeting agent is often a non-naturally occurring sequence. However, the duplex-forming segment of a targeting agent (described in more detail below) that hybridizes with the duplex-forming segment of an activator can comprise a naturally occurring sequence (e.g., can comprise the sequence of a duplex-forming segment of a naturally occurring crRNA, which may also be referred to as a crRNA repeat). Thus, the term targeting agent is used herein to distinguish naturally occurring crRNAs, despite the fact that portions of the targeting agent (e.g., duplex-forming segments) often comprise naturally occurring sequences from crRNAs. However, the term "targeting agent" encompasses naturally occurring crRNAs.

[0284] A guide RNA can be said to comprise three portions: (i) a targeting sequence (a nucleotide sequence that hybridizes to a sequence of a target nucleic acid), (ii) an activator sequence (as described above) (sometimes referred to as a tracr sequence), and (iii) a sequence that hybridizes to at least a portion of the activator sequence to form a double-stranded duplex. The targeting agent comprises (i) and (iii), while the activator comprises (ii).

[0285] A guide RNA (e.g., a dual-guide RNA or a single-guide RNA) can be composed of any corresponding activator and targeting factor pair. In some cases, the duplex-forming segments can be exchanged between the activator and targeting factor. In other words, in some cases, the targeting factor contains a sequence of nucleotides from a duplex-forming segment of the tracrRNA (which sequence is normally part of the activator), while the activator contains a sequence of nucleotides from a duplex-forming segment of the crRNA (which sequence is normally part of the targeting factor).

[0286] As described above, a targeting agent comprises both a targeting segment (single strand) of the guide RNA and a stretch of nucleotides ("duplex-forming segment") that forms one half of the dsRNA duplex of the protein-binding segment of the guide RNA. The corresponding tracrRNA-like molecule (activator) comprises a stretch of nucleotides (duplex-forming segment) that forms the other half of the dsRNA duplex of the protein-binding segment of the guide RNA. In other words, the stretch of nucleotides of the targeting agent is complementary to the stretch of nucleotides of the activator and hybridizes with it to form the dsRNA duplex of the protein-binding segment of the guide RNA. Thus, each targeting agent can be said to have a corresponding activator (having a region that hybridizes with the targeting agent). The targeting agent molecule additionally provides a targeting segment. Thus, the targeting agent and activator (as a corresponding pair) hybridize to form the guide RNA. The specific sequence of a given naturally occurring crRNA or tracrRNA molecule is characteristic of the species in which the RNA molecule is found. Examples of suitable activators and targeting agents are known in the art.

[0287] knock out In some cases, as described above, the guide RNA present in an EDV of the present disclosure, or the guide RNA encoded by a guide RNA-encoding nucleic acid present in an EDV of the present disclosure, results in a deletion ("knockout") of the target nucleic acid.

[0288] For example, in some cases, an EDV of the present disclosure results in (i) delivery of a therapeutic protein and (ii) knockout of a target nucleic acid. As one non-limiting example, an EDV of the present disclosure can both (i) provide delivery of a therapeutic protein (such as a chimeric antigen receptor (CAR)) and (ii) knockout of an endogenous nucleic acid encoding a beta-2 microglobulin (β2M) polypeptide, where a guide RNA present in the EDV (or encoded by a nucleic acid present in the EDV) would comprise a nucleotide sequence that targets a nucleic acid encoding β2M in the target cell. Such EDVs may be useful for generating CAR-expressing T cells ("CAR-T cells") that do not express endogenous major histocompatibility complex (MHC) class I antigens on the cell surface, and thus may be useful for delivering allogeneic CAR-T cells.

[0289] As another example, in some cases, the EDV comprises a guide RNA or a nucleic acid comprising a nucleotide sequence encoding a guide RNA, which results in a knockout of the endogenous T-cell receptor alpha constant (TRAC) gene such that the TRAC polypeptide is not produced in the cell.

[0290] A TRAC polypeptide can include the following amino acid sequence: TIFF2025529869000143.tif16157 (sequence number 204).

[0291] As another example, in some cases, the EDV comprises a guide RNA or a nucleic acid comprising a nucleotide sequence encoding the guide RNA, where the guide RNA results in the knockout of an endogenous gene encoding an immune checkpoint, such as PD-1, PD-L1, CTLA4, and TIGIT.

[0292] Donor nucleic acid In some cases, the EDV of the present disclosure comprises a donor nucleic acid. "Donor nucleic acid" or "donor sequence" or "donor polynucleotide" or "donor template" refers to a nucleic acid sequence that is inserted into the site cleaved by a CRISPR-Cas effector protein (e.g., after dsDNA cleavage, after nicking the target DNA, after double-nicking the target DNA, etc.). The donor polynucleotide can contain sufficient homology to the genomic sequence at the target site, e.g., 70%, 80%, 85%, 90%, 95%, or 100% homology to the nucleotide sequence adjacent to the target site, e.g., within about 50 bases, e.g., within about 30 bases, within about 15 bases, within about 10 bases, or within about 5 bases of the target site, or to the nucleotide sequence immediately adjacent to the target site, to support homology-directed repair between the donor polynucleotide and the genomic sequence to which it has homology. Approximately 25, 50, 100, or 200 nucleotides, or more than 200 nucleotides (or any integer value between 10 and 200 nucleotides, or more) of sequence homology between the donor and genomic sequence can support homology-directed repair. The donor polynucleotide can be of any length, e.g., 10 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 500 nucleotides or more, 1000 nucleotides or more, 5000 nucleotides or more, etc.

[0293] The donor sequence is typically not identical to the genomic sequence it replaces. Rather, the donor sequence may contain at least one or more single base changes, insertions, deletions, inversions, or rearrangements relative to the genomic sequence, so long as there is sufficient homology to support homology-directed repair (e.g., for gene repair, e.g., to convert a disease-causing or non-disease-causing base pair). In some embodiments, the donor sequence contains non-homologous sequences flanking two homologous regions, so that homology-directed repair between the target DNA region and the two flanking sequences results in the insertion of the non-homologous sequence in the target region. The donor sequence may also contain a vector backbone that is not homologous to the DNA region of interest and contains sequences not intended for insertion into the DNA region of interest. Generally, the homologous regions of the donor sequence have at least 50% sequence identity to the genomic sequence with which recombination is desired. In certain embodiments, there is 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.9% sequence identity. Depending on the length of the donor polynucleotide, there can be any value between 1% and 100% sequence identity.

[0294] The donor sequence may contain certain sequence differences compared to the genomic sequence, such as restriction sites, nucleotide polymorphisms, selectable markers (e.g., drug resistance genes, fluorescent proteins, enzymes, etc.), which can be used to evaluate successful insertion of the donor sequence at the cleavage site or, in some cases, for other purposes (e.g., to indicate expression at the targeted genomic locus). In some cases, when located in a coding region, such nucleotide sequence differences do not change the amino acid sequence or result in silent amino acid changes (i.e., changes that do not affect the structure or function of the protein). Alternatively, these sequence differences may include flanking recombination sequences, such as FLP or loxP sequences, that can be subsequently activated to remove the marker sequence.

[0295] In some cases, the donor sequence is introduced into the cell as single-stranded DNA. In some cases, the donor sequence is introduced into the cell as double-stranded DNA. It can be introduced into the cell in linear or circular form. If introduced in linear form, the ends of the donor sequence can be protected (e.g., from exonucleolysis) by any convenient method, and such methods are known to those skilled in the art. For example, one or more dideoxynucleotide residues can be added to the 3' end of the linear molecule, and / or self-complementary oligonucleotides can be ligated to one or both ends. See, for example, Chang et al. (1987) Proc. Natl. Acad Sci USA 84:4959-4963; Nehls et al. (1996) Science 272:886-889. Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, the addition of terminal amino groups and the use of modified internucleotide linkages, such as phosphorothioates, phosphoramidates, and O-methylribose or deoxyribose residues. As an alternative to protecting the ends of linear donor sequences, additional lengths of sequence can be included outside the regions of homology that can be degraded without affecting recombination. Donor sequences can be introduced into cells as part of a vector molecule with additional sequences, such as an origin of replication, a promoter, and a gene encoding antibiotic resistance.

[0296] Therapeutic Proteins As noted above, in some cases, the EDV of the present disclosure comprises a nucleic acid comprising a nucleotide sequence encoding a therapeutic polypeptide.

[0297] Therapeutic polypeptides encoded by nucleic acids present in the EDV of the present disclosure can have a length of from about 250 amino acids to about 3000 amino acids. For example, in some cases, a therapeutic polypeptide encoded by a nucleic acid present in an EDV of the disclosure has a length of from about 250 amino acids to about 500 amino acids, from about 500 amino acids to about 1000 amino acids, from about 500 amino acids to about 750 amino acids, from about 750 amino acids to about 1500 amino acids, from about 750 amino acids to about 1000 amino acids, from about 1000 amino acids to about 1250 amino acids, from about 1000 amino acids to about 1500 amino acids, from about 1250 amino acids to about 1500 amino acids, from about 1250 amino acids to about 1750 amino acids, from about 1500 amino acids to about 1750 amino acids, from about 1500 amino acids to about 2000 amino acids, from about 1500 amino acids to about 2500 amino acids, from about 2000 amino acids to about 2500 amino acids, from about 2000 amino acids to about 3000 amino acids, or from about 2500 amino acids to about 3000 amino acids.

[0298] Suitable therapeutic proteins include, but are not limited to, chimeric antigen receptors (CARs), T cell receptors (TCRs), natural killer cell receptors (NKRs), synNotch polypeptides, antibodies, modular extracellular sensor architecture (MESA) receptors, etc. In some cases, the therapeutic protein is a functional version of a protein, e.g., cystic fibrosis transmembrane conductance (CFTR) protein, globin polypeptides (e.g., β-globin), etc.

[0299] antibody In some cases, the therapeutic protein is an antibody. Suitable antibodies include, for example, therapeutic antibodies. In some cases, the antibody is a single-chain Fv (scFv). In some cases, the antibody is a nanobody.

[0300] Suitable antibodies include, for example, natalizumab (Tysabri® Biogen Idec / Elan), which targets the α4 subunit of the α4β1 and α4β7 integrins (when used to treat MS and Crohn's disease); vedolizumab (MLN2; Millennium Pharmaceuticals / Takeda), which targets the α4β7 integrin (when used to treat UC and Crohn's disease); belimumab (Benlysta; Human Genome Research), which targets BAFF; Sciences / GlaxoSmithKline) (when used to treat SLE); atacicept (TACI-Ig; Merck / Serono) which targets BAFF and APRIL (when used to treat SLE); alefacept (Amevive®; Astellas) which targets CD2 (when used to treat plaque psoriasis, GVHD); otelixizumab (TRX4; Tolerx / GlaxoSmithKline) which targets CD3 (when used to treat T1D); teplizumab (MGA031; MacroGenics / Eli Lilly) which targets CD3 (when used to treat T1D); rituximab (Rituxan® / Mabthera; Genentech / Roche / Biogen) which targets CD20 Idec) (when used to treat non-Hodgkin's lymphoma, RA (in patients with an inadequate response to TNF-blocking agents), and CLL); ofatumumab (Arzerra®; Genmab / GlaxoSmithKline) which targets CD20 (when used to treat CLL, RA); ocrelizumab (2H7; Genentech / Roche / Biogen Idec) which targets CD20 (when used to treat RA and SLE); epratuzumab (hLL2; Immunomedics / UCB) which targets CD22 (when used to treat SLE and non-Hodgkin's lymphoma); alemtuzumab (Campath® / MabCampath; Genzyme / Bayer) which targets CD52 (when used to treat CLL, MS); abatacept (Orencia®) which targets CD80 and CD86Bristol-Myers Squibb) (when used to treat RA and JIA, UC and Crohn's disease, and SLE); eculizumab (Soliris®; Alexion Pharmaceuticals) which targets the C5 complement protein (when used to treat paroxysmal nocturnal hemoglobinuria); omalizumab (Xolair®; Genentech / Roche / Novartis) which targets IgE (when used to treat moderate to severe persistent allergic asthma); canakinumab (Ilaris®; Novartis) which targets IL-1β (when used to treat cryopyrin-associated periodic syndrome, systemic JIA, neonatal-onset multisystem inflammatory disease, and acute gout); mepolizumab (Bosatria; GlaxoSmithKline) which targets IL-5 (when used to treat hypereosinophilic syndrome); and reslizumab (SCH55700; Ception) which targets IL-5. Therapeutics) (when used to treat eosinophilic esophagitis); tocilizumab (Actemra® / RoActemra®; Chugai / Roche) which targets IL-6R (when used to treat RA, JIA); ustekinumab (Stelara®; Centocor) which targets IL-12 and IL-23 (when used to treat plaque psoriasis, psoriatic arthritis, Crohn's disease); briakinumab (ABT-874; Abbott) which targets IL-12 and IL-23 (when used to treat psoriasis and plaque psoriasis); etanercept (E nbrel®; Amgen / Pfizer) (when used to treat RA, JIA, psoriatic arthritis, AS, and plaque psoriasis); infliximab (Remicade®; Centocor / Merck) (when used to treat Crohn's disease, RA, psoriatic arthritis, UC, AS, and plaque psoriasis); adalimumab (Humira® / Trudexa®; Abbott) (when used to treat RA, JIA, psoriatic arthritis, Crohn's disease, AS, and plaque psoriasis); certolizumab pegol (Cimzia®;and golimumab (Simponi®; Centocor®), which targets TNF (used to treat RA, psoriatic arthritis, and AS). In some cases, antibodies whose production is induced by the intracellular domain of a synNotch polypeptide of the present disclosure are therapeutic antibodies for the treatment of cancer. Such antibodies include, for example, ipilimumab, which targets CTLA-4 (when used to treat melanoma, prostate cancer, and RCC); tremelimumab, which targets CTLA-4 (when used to treat CRC, gastric cancer, melanoma, and NSCLC); nivolumab, which targets PD-1 (when used to treat melanoma, NSCLC, and RCC); MK-3475, which targets PD-1 (when used to treat melanoma); pidilizumab, which targets PD-1 (when used to treat hematologic malignancies); BMS-936559, which targets PD-L1 (when used to treat melanoma, NSCLC, ovarian, and RCC); MEDI4736, which targets PD-L1; MPDL33280A, which targets PD-L1 (when used to treat melanoma); rituximab, which targets CD20 (when used to treat non-Hodgkin's lymphoma); ibritumomab tiuxetan and tositumomab (when used to treat lymphoma). brentuximab vedotin, which targets CD30 (when used to treat Hodgkin's lymphoma); gemtuzumab ozogaicin, which targets CD33 (when used to treat acute myeloid leukemia); alemtuzumab, which targets CD52 (when used to treat chronic lymphocytic leukemia); IGN101 and adecatumumab, which target EpCAM (when used to treat epithelial tumors (breast, colon, and lung)); labetuzumab, which targets CEA (when used to treat breast, colon, and lung tumors); huA33, which targets gpA33 (when used to treat colorectal cancer); pemtumomab and oregovomab, which target mucin (when used to treat breast, colon, lung, and ovarian tumors); CC49 (miretumomab), which targets TAG-72 (when used to treat breast, colon, and lung tumors); cG250, which targets CAIX (when used to treat renal cell carcinoma);J591, which targets PSMA (when used to treat prostate cancer); MOv18 and MORAb-003 (faretuzumab), which target folate-binding proteins (when used to treat ovarian tumors); 3F8, ch14.18, and KW-2871, which target gangliosides (e.g., GD2, GD3, and GM2), which are used to treat neuroectodermal tumors and some epithelial tumors; Le hu3S193 and IgN311 targeting γ (when used to treat breast, colon, lung, and prostate tumors); bevacizumab targeting VEGF (when used to treat tumor vasculature); IM-2C6 and CDP791 targeting VEGFR (when used to treat solid tumors of epithelial origin); etaracizumab targeting integrin V3 (when used to treat tumor vasculature); volociximab targeting integrin 51 (when used to treat tumor vasculature); cetuximab, panitumumab, nimotuzumab, and 806 targeting EGFR (when used to treat glioma, lung, breast, colon, and head and neck tumors); trastuzumab and pertuzumab targeting ERBB2 (when used to treat breast, colon, lung, ovarian, and prostate tumors); MM-121 targeting ERBB3 (when used to treat breast, colon, lung, ovarian, and prostate tumors); ME AMG102, METMAB, and SCH900105 targeting T (when used to treat breast, ovarian, and lung tumors); AVE1642, IMC-A12, MK-0646, R1507, and CP751871 targeting IGF1R (when used to treat glioma, lung, breast, head and neck, prostate, and thyroid cancers); and KB004 and IIIA4 targeting EPHA3 (when used to treat lung, kidney, and colon tumors, melanoma, glioma, and hematological malignancies). mapatumumab (HGS-ETR1), which targets TRAILR1 (when used to treat colon, lung, and pancreatic tumors and hematologic malignancies); HGS-ETR2 and CS-1008, which target TRAILR2; denosumab, which targets RANKL (when used to treat prostate cancer and bone metastases); sibrotuzumab and F19, which target FAP (when used to treat colon, breast, lung, pancreatic, and head and neck tumors);These include 81C6, which targets tenascin (used to treat glioma, breast cancer, and prostate tumors); blinatumomab (Blincyto; Amgen), which targets CD3 (used to treat ALL); pembrolizumab, which targets PD-1 as used in cancer immunotherapy; and 9E10, which targets c-Myc.

[0301] Suitable antibodies include, for example, abagovomab, abciximab, abituzumab, abrilumab, actoxumab, aducanumab, afelimomab, afutuzumab, alacizumab pegol, ALD518, alirocumab, altumomab pentetate, amatuximab, anatumomab mafenatox, anetumab ravtansine, anifrolumab, anrukinzumab, apolizumab, arcitumomab, asclinvacumab, acelizumab, atezolizumab, atinumab, atlizumab / tocilizumab, atorlimumab, bapineuzumab, basiliximab, bapineuzumab, basiliximab, bapineuzumab ... Bituximab, bectumomab, begelomab, benralizumab, bertilimumab, besilesomab, bevacizumab / ranibizumab, bezlotoxumab, biciromab, bimagrumab, bimekizumab, bivatuzumab mertansine, brosozumab, bococizumab, brentuximab vedotin, brodalumab, brolucizumab, brontiximab, cantuzumab mertansine, cantuzumab vedotin, caplacizumab, capromab pendetide, carlumab, catumaxomab, cBR96-doxorubicin immunoconjugate, cedelizumab, Ch.14.18, sitatuzumab Ibuprofen, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, codrituzumab, coltuximab ravtansine, conatumumab, concizumab, CR6261, crenezumab, dacetuzumab, daclizumab, darotuzumab, dapirolizumab pegol, daratumumab, dectrecumab, demcizumab, denintuzumab mafodotin, delrotuximab biotin, detumomab, dinutuximab, zilidabumab, dorlimomab alitoxin, drozitumab, durigotumab, dupilumab, durvalumab, dusigit Mab, ecromeximab, edovacomab, edrecolomab, efalizumab, efungumab, eldelumab, elgemtuzumab, elotuzumab, ersilimomab, emactuzumab, emibetuzumab, enavatuzumab, enfortumab vedotin, enlimomab pegol, enoblitzumab, enokizumab, enoticumab, encituximab, epitumomab cituxetan, erlizumab, ertumaxomab, etrolizumab, evinacumab, evolocumab, exbivirumab, fanolesomab, faralimomab, faretuzumab, fasinumab, FBTA05,Felvizumab, fezakinumab, ficlatuzumab, figitumumab, filibumab, framvotumab, fretikumab, fontolizumab, foralumab, foravirumab, fresolimumab, furanumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gevokizumab, dilentuximab, glembatumumab vedotin, gomiliximab, guselkumab, ibalizumab, icrucumab, idarucizumab, igovomab, IMAB362, imalumab, imciromab, imgatuzumab, inlacumab, indatu Ximab vedotin, indusatumab vedotin, inolimomab, inotuzumab ozogamicin, intetumumab, iratumumab, isatuximab, itolizumab, ixekizumab, keliximab, lambrolizumab, lampalizumab, lebrikizumab, remaresomab, lenzilumab, lerdelimumab, lexatumumab, ribivirumab, rifastuzumab vedotin, ligelizumab, rirotumab satetraxetan, lintuzumab, lirilumab, roderucizumab, roxivetomab, lorvotuzumab mertansine, lucatumumab, lulizumab pegol, lumiliximab Mab, lumletuzumab, margetuximab, maslimomab, matuzumab, mavrilimumab, metelimuab, milatuzumab, minletumomab, mirvetuximab soravtansine, mitumomab, mogamulizumab, morolimumab, morolimumab immunotherapy, motavizumab, moxetumomab pasudotox, muromonab-CD3, nacolomab butafenatox, namilumab, naptumomab estafenatox, narunatumab, nebacumab, necitumumab, nemolizumab, nerelimomab, nesvacumab, nofetumomab merpentane, obilutoxaximab, obinutuz Mab, ocaratuzumab, ozlimomab, olaratuzumab, olokizumab, onartuzumab, ontuxizumab, opicinumab, oportuzumab monatox, olticumab, otreltuzumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, pancomab, panobacumab, palsatuzumab, pascolizumab, pasotuximab, pateclizumab, patritumab, perakizumab, pexelizumab, pinatuzumab vedotin, pintumomab, placulumab, polatuzumab vedotin, ponezumab, priliximab, pritoxaximabPritumumab, PRO140, kiruzirumab, racotumomab, radletumab, rafivirumab, ralpanzirumab, ramucirumab, ranibizumab, raxibacumab, refanezumab, regavirumab, rilotumumab, linucumab, lobatumumab, loredumab, romosozumab, lontalizumab, rovelizumab, ruplizumab, sacituzumab govitecan, samalizumab, sarilumab, satumomab pendetide, secukinumab Mab, seribantumab, cetoxaximab, sevirumab, SGN-CD19A, SGN-CD33A, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, sofituzumab vedotin, solanezumab, solitomab, soneptizumab, sontuzumab, stamulumab, sulesomab, subizumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, tapritum Mabpaptox, talexuzumab, tefibazumab, terimomab-allitox, tenatumomab, teneliximab, teprotumumab, tesidolumab, tetulomab, TGN1412, ticilimumab / tremelimumab, tigatuzumab, tildrakizumab, TNX-650, toralizumab, tosatoxumab, tobetumab, tralokinumab, TRBS07, tregalizumab, trevoglumab, tucotuzumab-celmoloiqui These include tubilumab, ublituximab, urocupulumab, urelumab, urtoxazumab, bundeltuzumab vedotin, vanticizumab, vanucizumab, bapaliximab, varlilumab, batelizumab, veltuzumab, bepalimomab, besencumab, visilizumab, borsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, diralimumab, and zolimomab alitox.

[0302] Chimeric antigen receptor (CAR) A CAR generally comprises (a) an extracellular domain comprising an antigen-binding domain (antigen-binding polypeptide), (b) a transmembrane region, and (c) a cytoplasmic domain comprising an intracellular signaling domain (intracellular signaling polypeptide). In some cases, a CAR comprises (a) an extracellular domain comprising an antigen-binding domain, (b) a transmembrane region, and (c) a cytoplasmic domain comprising (i) one or more costimulatory polypeptides and (ii) an intracellular signaling domain. In some cases, a CAR comprises a hinge region between the extracellular antigen-binding domain and the transmembrane domain. Thus, in some cases, a CAR comprises (a) an extracellular domain comprising an antigen-binding domain, (b) a hinge region, (c) a transmembrane region, and (d) a cytoplasmic domain comprising an intracellular signaling domain. In some cases, a CAR comprises (a) an extracellular domain comprising an antigen-binding domain, (b) a hinge region, (c) a transmembrane region, and (d) a cytoplasmic domain comprising (i) one or more costimulatory polypeptides, and (ii) an intracellular signaling domain.

[0303] Exemplary CAR structures are known in the art (see, e.g., WO2009 / 091826, US2013 / 0287748, WO2015 / 142675, WO2014 / 055657, WO2015 / 090229, and U.S. Patent No. 9,587,020). In some cases, the CAR is a single polypeptide chain. In some cases, the CAR comprises two polypeptide chains. Generally, any CAR structure known to those of skill in the art can be used.

[0304] CARs specific for tumor antigens are already known in this field, for example, CD171-specific CAR (Park et al., Mol Ther (2007) 15(4):825-833), EGFRvIII-specific CAR (Morgan et al., Hum Gene Ther (2012) 23(10):1043-1053), EGF-R-specific CAR (Kobold et al., J. Natl Cancer Inst (2014) 107(1):364), carbonyl dehydratase IX-specific CAR (Lamers et al., Biochem Soc Trans (2016) 44(3):951-959), folate receptor-α (FR-α)-specific CAR (Kershaw et al., Clin Cancer Res (2006) 12 (20): 6106-6015), HER2-specific CAR (Ahmed et al., J Clin Oncol (2015) 33 (15) 1688-1696, Nakazawa et al., Mol Ther (2011) 19 (12): 2133-2143, Ahmed et al., Mol Ther(2009)17(10):1779-1787, Luo et al., Cell Res(2016)26(7):850-853, Morgan et al., Mol Ther(2010)18(4):843-851, Grada et al., Mol Ther Nucleic Acids(2013)9(2):32), CEA-specific CAR (Katz et al., Clin Cancer Res (2015) 21 (14): 3149-3159), IL-13Rα2-specific CAR (Brown et al., Clin Cancer Res (2015) 21 (18): 4062-4072), Gon GD2-specific CAR (Louis et al., Blood (2011) 118 (23): 6050-6056, Caruana et al., Nat Med (2015) 21 (5): 524-529, Yu et al. (2018) J. Hematol. Oncol. 11: 1), ErbB2-specific CAR (Wilkie et al., J Clin Immunol (2012) 32 (5): 1059-1070), VEGF-R-specific CAR (Chinnasamy et al., Cancer Res (2016) 22 (2): 436-447), FAP-specific CARs (Wang et al., Cancer Immunol Res (2014) 2 (2): 154-166), mesothelin (MSLN)-specific CARs (Moon et al., Clin Cancer Res (2011) 17 (14): 4719-30), NKG2D-specific CARs (VanSeggelen et al., Mol Ther (2015) 23 (10): 1600-1610), and CD19-specific CARs (axicabtagene ciloleucel (Yescarta™) and tisagenlecleucel (Kymriah™). Also, Li et al., J Hematol and Oncol (2018) 11: 22, reviewing clinical trials of tumor-specific CARs, Heyman and Yan (2019) Cancers 11:pii:E191, Baybutt et al. (2019) Clin. Pharmacol. Ther. 105:71.

[0305] As described above, a CAR comprises an extracellular domain comprising an antigen-binding domain. The antigen-binding domain present in a CAR can be any antigen-binding polypeptide, a wide variety of which are known in the art. In some cases, the antigen-binding domain is a single-chain Fv (scFv). Other antibody-based recognition domains are suitable, including cAb VHH (camelid antibody variable domains) and humanized versions, IgNAR VH (shark antibody variable domains) and humanized versions, sdAb VH (single-domain antibody variable domains), and "camelized" antibody variable domains. In some cases, the antigen-binding domain is a nanobody.

[0306] In some cases, the antigen bound by the antigen-binding domain of the CAR is a MUC1 polypeptide, an LMP2 polypeptide, an epidermal growth factor receptor (EGFR) vIII polypeptide, a HER-2 / neu polypeptide, a melanoma antigen family A, 3 (MAGE A3) polypeptide, a p53 polypeptide, a mutant p53 polypeptide, a NY-ESO-1 polypeptide, a folate hydrolase (prostate-specific membrane antigen;PSMA polypeptide, carcinoembryonic antigen (CEA) polypeptide, melanoma antigen recognized by T cells (melanA / MART1) polypeptide, Ras polypeptide, gp100 polypeptide, proteinase 3 (PR1) polypeptide, bcr-abl polypeptide, tyrosinase polypeptide, survivin polypeptide, prostate-specific antigen (PSA) polypeptide, hTERT polypeptide, sarcoma translocation breakpoint polypeptide, synovial sarcoma X (SSX) breakpoint polypeptide, EphA2 polypeptide, acid phosphatase, prostate (PAP) polypeptide, melanoma inhibitor of apoptosis (ML-IAP) polypeptide, epithelial cell adhesion molecule (EpCAM) polypeptide, ERG (TMPRSS2) polypeptide ETS fusion) polypeptide, NA17 polypeptide, paired box-3 (PAX3) polypeptide, anaplastic lymphoma kinase (ALK) polypeptide, androgen receptor polypeptide, cyclin B1 polypeptide, N-myc proto-oncogene (MYCN) polypeptide, Ras homolog gene family member C (RhoC) polypeptide, tyrosinase-related protein-2 (TRP-2) polypeptide, mesothelin polypeptide, prostate stem cell antigen (PSCA) polypeptide, melanoma-associated antigen-1 (MAGE A1) polypeptide, cytochrome P450 1B1 (CYP1B1) polypeptide, placenta-specific protein 1 (PLAC1) polypeptide, BORIS polypeptide (also known as CCCTC-binding factor or CTCF), ETV6-AML polypeptide, breast cancer antigen NY-BR-1 polypeptide (also known as ankyrin repeat domain-containing protein 30A), regulator of G protein signaling (RGS5) polypeptide, squamous cell carcinoma antigen recognized by T cells (SART3) polypeptide, carbonic anhydrase IX polypeptide, paired box-5 (PAX5) polypeptide, OY-TES1 (testis antigen;acrosin-binding protein) polypeptide, sperm protein 17 polypeptide, lymphoid cell-specific protein-tyrosine kinase (LCK) polypeptide, high-molecular-weight melanoma-associated antigen (HMW-MAA), A-kinase anchor protein-4 (AKAP-4), synovial sarcoma X breakpoint 2 (SSX2) polypeptide, X-antigen family member 1 (XAGE1) polypeptide, B7 homolog 3 (B7H3; also known as CD276) polypeptide, legman polypeptide (LGMN1; asparaginyl endopeptide) a tyrosine kinase with Ig and EGF homology domains-2 (Tie-2; also known as angiopoietin-1 receptor) polypeptide, a P antigen family member 4 (PAGE4) polypeptide, a vascular endothelial growth factor receptor 2 (VEGF2) polypeptide, a MAD-CT-1 polypeptide, a fibroblast activation protein (FAP) polypeptide, a platelet-derived growth factor receptor beta (PDGFβ) polypeptide, a MAD-CT-2 polypeptide, or a Fos-related antigen-1 (FOSL) polypeptide;

[0307] The antigen-binding polypeptide of the CAR can bind to any of a variety of cancer-associated antigens, including, for example, CD19, CD20, CD38, CD30, Her2 / neu, ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), B-cell maturation antigen (BCMA), high molecular weight melanoma-associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, and the like. Furthermore, examples of cancer-associated antigens include 4-1BB, 5T4, adenocarcinoma antigen, alpha-fetoprotein (AFP), BAFF, B lymphoma cells, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, and CD44. v6, CD51, CD52, CD56, CD74, CD80, CEA, CNTO888, CTLA-4, DRS, EGFR, EpCAM, CD3, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2 / neu, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgG1, L1-CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin α5β1, integrin Examples of mucins include mucin αvβ3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-Rα, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, ROR1, SCH900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF beta 2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, and vimentin.

[0308] The VH and VL amino acid sequences of various cancer-associated antigen-binding antibodies are known in the art, as are the light and heavy chain CDRs of such antibodies. See, for example, Ling et al. (2018) Frontiers Immunol. 9:469, WO2005 / 012493, US2019 / 0119375, and US2013 / 0066055. The following are non-limiting examples of antibodies that bind to cancer-associated antigens:

[0309] As one non-limiting example, in some cases, the CAR comprises an anti-CD19 antibody (e.g., an anti-CD19 scFv or an anti-CD19 nanobody). Anti-CD19 antibodies are known in the art, and the VH and VL, or VH and VL CDRs, of any anti-CD19 antibody can be included in the CAR. See, e.g., WO2005 / 012493.

[0310] In some cases, the anti-CD19 antibody comprises a VL CDR1 comprising the amino acid sequence KASQSVDYDGDSYLN (SEQ ID NO: 181), a VL CDR2 comprising the amino acid sequence DASNLVS (SEQ ID NO: 182), and a VL CDR3 comprising the amino acid sequence QQSTEDPWT (SEQ ID NO: 183). In some cases, the anti-CD19 antibody comprises a VH CDR1 comprising the amino acid sequence SYWMN (SEQ ID NO: 184), a VH CDR2 comprising the amino acid sequence QIWPGDGDTNYNGKFKG (SEQ ID NO: 185), and a VH CDR3 comprising the amino acid sequence RETTTVGRYYYAMDY (SEQ ID NO: 186). In some cases, the anti-CD19 antibody comprises a VL CDR1 comprising the amino acid sequence KASQSVDYDGDSYLN (SEQ ID NO: 181), a VL CDR2 comprising the amino acid sequence DASNLVS (SEQ ID NO: 182), a VL CDR3 comprising the amino acid sequence QQSTEDPWT (SEQ ID NO: 183), a VH CDR1 comprising the amino acid sequence SYWMN (SEQ ID NO: 184), a VH CDR2 comprising the amino acid sequence QIWPGDGDTNYNGKFKG (SEQ ID NO: 185), and a VH CDR3 comprising the amino acid sequence RETTTVGRYYYAMDY (SEQ ID NO: 186).

[0311] In some cases, the anti-CD19 antibody is an scFv. For example, in some cases, the anti-CD19 scFv comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025529869000144.tif23159 (sequence number 187).

[0312] Compositions containing EDV The present disclosure provides compositions, including pharmaceutical compositions, comprising the EDV of the present disclosure. The compositions may include pharmaceutically acceptable excipients, a variety of which are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients are described, for example, in "Remington: The Science and Practice of Pharmacy," 1999. th Ed. (1995), or the latest edition, Mack Publishing Co; A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy”, 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) HCAnsel et al., eds 7 th ed., Lippincott, Williams, & Wilkins, and Handbook of Pharmaceutical Excipients (2000) AHKibbe et al., eds., 3 rd It is well described in a variety of publications, including ed. Amer. Pharmaceutical Assoc.

[0313] The composition of the present disclosure can include (a) an EDV of the present disclosure, and (b) one or more of a buffer, surfactant, antioxidant, hydrophilic polymer, dextrin, chelating agent, suspending agent, solubilizing agent, thickening agent, stabilizer, bacteriostat, humectant, and preservative. Suitable buffers include N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (BIS-Tris), N-(2-hydroxyethyl)piperazine-N'-3-propanesulfonic acid (EPPS or HEPPS), glycylglycine, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 3-(N-morpholino)propanesulfonic acid (MOP), and the like. Suitable salts include, but are not limited to, N,N'-bis(2-ethanesulfonic acid) (PIPES), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), sodium bicarbonate, 3-(N-tris(hydroxymethyl)-methyl-amino)-2-hydroxy-propanesulfonic acid) TAPSO, (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), N-tris(hydroxymethyl)methyl-glycine (Tricine), tris(hydroxymethyl)-aminomethane (Tris), etc. Suitable salts include, for example, NaCl, MgCl, KCl, MgSO, etc.

[0314] In some cases, the composition is sterile. In some cases, the composition is suitable for administration to a human subject, e.g., if the composition is sterile and free of detectable pyrogens and / or other toxins.

[0315] In some cases, a composition of the disclosure includes (i) an EDV that does not include a donor template nucleic acid, and (ii) a donor template nucleic acid (provided separately from the EDV).

[0316] nucleic acid As described above, in some cases, the EDV of the present disclosure comprises a nucleic acid comprising a nucleotide sequence encoding a CRISPR-Cas effector polypeptide. In some cases, the EDV of the present disclosure comprises a nucleic acid comprising a nucleotide sequence encoding a guide RNA. In some cases, the EDV of the present disclosure comprises a nucleic acid comprising a nucleotide sequence encoding a therapeutic polypeptide.

[0317]

[0013] A coding sequence present in an EDV of the disclosure (e.g., a nucleotide sequence encoding a CRISPR-Cas effector polypeptide, a nucleotide sequence encoding a CRISPR-Cas guide RNA, a nucleotide sequence encoding a therapeutic protein) can be operably linked to a transcriptional control element (e.g., a promoter). The transcriptional control element can be a promoter. In some cases, the promoter is a constitutively active promoter. In some cases, the promoter is a regulatable promoter. In some cases, the promoter is an inducible promoter. In some cases, the promoter is a tissue-specific promoter. In some cases, the promoter is a cell-type-specific promoter. In some cases, the transcriptional control element (e.g., a promoter) functions in a targeted cell type or a targeted cell population. The promoter may be a constitutively active promoter (i.e., a promoter that is constitutively active / "ON" state), it may be an inducible promoter (i.e., a promoter whose state, active / "ON" or inactive / "OFF", is controlled by an external stimulus, e.g., a particular temperature, compound, or the presence of a protein), it may be a spatially restricted promoter (i.e., a transcriptional control element, enhancer, etc.) (e.g., a tissue-specific promoter, cell-type-specific promoter, etc.), or it may be a temporally restricted promoter (i.e., the promoter is in the "ON" or "OFF" state during a particular stage of embryonic development or a particular stage of a biological process, e.g., the hair follicle cycle in a mouse).

[0318] Suitable promoters may be derived from viruses and therefore may be referred to as viral promoters, or they may be derived from any organism, including prokaryotes or eukaryotes. Suitable promoters can be used to drive expression by any RNA polymerase (e.g., pol I, pol II, pol III). Exemplary promoters include, but are not limited to, the SV40 early promoter, the mouse mammary tumor virus long terminal repeat (LTR) promoter, the adenovirus major late promoter (Ad MLP), the herpes simplex virus (HSV) promoter, the cytomegalovirus (CMV) promoter, such as the CMV immediate early promoter region (CMVIE), the Rous sarcoma virus (RSV) promoter, the human U6 micronucleus promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), the enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep 1; 31 (17)), the human H1 promoter (H1), and the like.

[0319] In some cases, the coding nucleotide sequence is operably linked to (under the control of) a promoter operable in eukaryotic cells (e.g., a U6 promoter, an enhanced U6 promoter, an H1 promoter, etc.). As will be understood by those skilled in the art, when expressing an RNA (e.g., a guide RNA) from a nucleic acid (e.g., an expression vector) using a U6 promoter (e.g., in a eukaryotic cell) or another Pol III promoter, the RNA may need to be mutated if there are several consecutive Ts (encoding Us in the RNA). This is because a stretch of Ts (e.g., five Ts) in DNA can function as a terminator for polymerase III (Pol III). Therefore, to ensure transcription of the guide RNA in eukaryotic cells, it may be necessary to modify the sequence encoding the guide RNA to eliminate Ts. In some cases, the nucleotide sequence encoding the guide RNA is operably linked to a promoter operable in eukaryotic cells (e.g., a CMV promoter, an EF1α promoter, an estrogen receptor-regulated promoter, etc.).

[0320] Examples of inducible promoters include, but are not limited to, T7 RNA polymerase promoter, T3 RNA polymerase promoter, isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoter, lactose-inducible promoter, heat shock promoter, tetracycline-regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor-regulated promoter, etc. Thus, inducible promoters can be regulated by molecules including, but not limited to, doxycycline, estrogen and / or estrogen analogs, IPTG, etc.

[0321] Inducible promoters suitable for use include any inducible promoter described herein or known to those of skill in the art. Examples of inducible promoters include chemically / biochemically regulated and physically regulated promoters, such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems incorporating tetracycline repressor protein (tetR), tetracycline operator sequence (tetO), and tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., promoters based on rat glucocorticoid receptor, human estrogen receptor, moth ecdysone (moth These promoters include, but are not limited to, promoters derived from the ecdysone receptor and steroid / retinoid / thyroid receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (a protein that binds and sequesters metal ions) genes from yeast, mouse, and human), pathogenicity-regulated promoters (e.g., induced by salicylic acid, ethylene, or benzothiadiazole (BTH)), temperature / heat-inducible promoters (e.g., heat shock promoters), and light-regulated promoters (e.g., light-responsive promoters from plant cells).

[0322] In some cases, the promoter is a spatially restricted promoter (i.e., a cell-type specific promoter, a tissue-specific promoter, etc.) such that in a multicellular organism, the promoter is active (i.e., "ON") in a specific subset of cells. A spatially restricted promoter may also be referred to as an enhancer, a transcription control element, a regulatory sequence, etc. Any convenient spatially restricted promoter may be used, as long as the promoter is functional in the targeted host cell (e.g., eukaryotic cell, prokaryotic cell).

[0323] In some cases, the promoter is a reversible promoter.Suitable reversible promoters, including reversible inducible promoters, are known in the art.Such reversible promoters can be isolated and derived from many organisms, for example, eukaryotes and prokaryotes.Modification of a reversible promoter derived from a first organism for use in a second organism, for example, a first prokaryote and a second eukaryote, a first eukaryote and a second prokaryote, etc., is known in the art. Examples of such reversible promoters and systems based on such reversible promoters but which also contain additional regulatory proteins include, but are not limited to, alcohol-regulated promoters (such as the alcohol dehydrogenase I (alcA) gene promoter and promoters responsive to alcohol transactivator protein (AlcR)), tetracycline-regulated promoters (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid-regulated promoters (e.g., rat glucocorticoid receptor promoter system, human estrogen receptor promoter system, retinoid promoter system, thyroid promoter system, ecdysone promoter system, mifepristone promoter system, etc.), metal-regulated promoters (e.g., metallothionein promoter system, etc.), pathogenesis-related regulated promoters (e.g., salicylic acid-regulated promoters, ethylene-regulated promoters, benzothiadiazole-regulated promoters, etc.), temperature-regulated promoters (e.g., heat shock-inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light-regulated promoters, synthetic inducible promoters, and the like.

[0324] Methods for delivering nucleic acid-binding effector polypeptides The present disclosure provides methods for delivering a nucleic acid-binding effector polypeptide to a target eukaryotic cell. The methods generally involve contacting the cell with an EDV of the present disclosure or administering the EDV to an organism. In some cases, the target cell is in vitro. In some cases, the target cell is in vivo, and the method includes administering the EDV to an individual.

[0325] The present disclosure provides methods for delivering a CRISPR-Cas polypeptide to a target eukaryotic cell. The methods generally involve contacting the cell with an EDV of the present disclosure or administering the EDV to an organism. In some cases, the target cell is in vitro. In some cases, the target cell is in vivo, and the method includes administering the EDV to an individual.

[0326] When an EDV of the present disclosure includes a guide RNA, in some cases, the guide RNA results in knockout of the nucleic acid targeted by the guide RNA. Thus, in some cases, an EDV of the present disclosure results in (i) delivery of a therapeutic protein and (ii) knockout of the target nucleic acid. As one non-limiting example, an EDV of the present disclosure can both (i) provide delivery of a therapeutic protein (such as a chimeric antigen receptor (CAR)) and (ii) knockout of an endogenous nucleic acid encoding a beta-2 microglobulin (β2M) polypeptide, where the guide RNA present in the EDV (or encoded by a nucleic acid present in the EDV) would include a nucleotide sequence that targets the nucleic acid encoding β2M in the target cell. Such EDVs may be useful for generating CAR-expressing T cells ("CAR-T cells") that do not express endogenous major histocompatibility complex (MHC) class I antigens on the cell surface, and thus may be useful for delivering allogeneic CAR-T cells. As another non-limiting example, an EDV of the present disclosure can both (i) provide for the delivery of a therapeutic protein (e.g., an antibody, e.g., a cancer-specific antibody or other therapeutic antibody) and (ii) knock out endogenous nucleic acid encoding an antibody light chain (e.g., a kappa light chain) or an immunoglobulin (Ig) Fc polypeptide (e.g., an Ig Fc polypeptide of a particular isotype, such as IgG1). Such an EDV would be useful for generating B cells that produce therapeutic antibodies.

[0327] In some cases, the EDVs of the present disclosure effect homology-directed repair (HDR) of a defective target nucleic acid. In some cases, the EDVs of the present disclosure effect non-homologous end joining (NHEJ) of a target nucleic acid, for example, to result in knockout of the target nucleic acid.

[0328] Cells that function as recipients for the EDVs of the present disclosure can be any of a variety of eukaryotic cells, including, for example, in vitro cells, in vivo cells, ex vivo cells, primary cells, cancer cells, animal cells, plant cells, algae cells, fungal cells, etc. Cells that function as recipients for the EDVs of the present disclosure are referred to as "host cells" or "target cells."

[0329] In some cases, the cells are in vitro. In some cases, the cells are removed from an individual and contacted in vitro with an EDV of the present disclosure, such that the cells are modified to produce a therapeutic protein encoded by a nucleic acid present in the EDV, and the modified cells are returned to the individual from which the cells were obtained. In some cases, the cells are removed from an individual and contacted in vitro with an EDV of the present disclosure, such that the cells are modified to produce a therapeutic protein encoded by a nucleic acid present in the EDV, and the modified cells are administered to an individual other than the individual from which the cells were obtained.

[0330] Suitable cells include stem cells (e.g., embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, germ cells (e.g., oocytes, sperm, egg cells, sperm cells, etc.), somatic cells such as fibroblasts, oligodendrocytes, glial cells, hematopoietic cells, nerve cells, muscle cells, bone cells, liver cells, pancreatic cells, etc.

[0331] Suitable cells include human embryonic stem cells, fetal cardiomyocytes, myofibroblasts, mesenchymal stem cells, cardiomyocytes, adipocytes, totipotent cells, pluripotent cells, blood stem cells, myoblasts, adult stem cells, bone marrow cells, mesenchymal cells, embryonic stem cells, parenchymal cells, epithelial cells, endothelial cells, mesothelial cells, fibroblasts, osteoblasts, chondrocytes, exogenous cells, endogenous cells, stem cells, hematopoietic stem cells, bone marrow-derived progenitor cells, cardiomyocytes, skeletal cells, fetal cells, undifferentiated cells, multipotent progenitor cells, unipotent progenitor cells, monocytic cells, cardiac myoblasts, skeletal myoblasts, macrophages, capillary endothelial cells, xenogeneic cells, allogeneic cells, and postnatal stem cells.

[0332] In some cases, the cell is an immune cell, a neuron, an epithelial cell, an endothelial cell, or a stem cell. In some cases, the immune cell is a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, or a macrophage. In some cases, the immune cell is a cytotoxic T cell. In some cases, the immune cell is a helper T cell. In some cases, the immune cell is a regulatory T cell (Treg).

[0333] In some cases, the cell is a stem cell. Stem cells include adult stem cells. Adult stem cells are also called somatic stem cells. In some cases, the cell is a tissue-resident stem cell.

[0334] Adult stem cells reside in differentiated tissues but retain the properties of self-renewal and the ability to give rise to multiple cell types, usually those typical of the tissue they are found in. Numerous examples of somatic stem cells are known to those of skill in the art, including muscle stem cells, hematopoietic stem cells, epithelial stem cells, neural stem cells, mesenchymal stem cells, mammary stem cells, intestinal stem cells, mesodermal stem cells, endothelial stem cells, olfactory stem cells, neural crest stem cells, etc.

[0335] Stem cells of interest include mammalian stem cells, where the term "mammal" refers to any animal classified as a mammal, including humans, non-human primates, domestic and farm animals, and zoo, laboratory, sports, or pet animals such as dogs, horses, cats, cows, mice, rats, rabbits, etc. In some cases, the stem cells are human stem cells. In some cases, the stem cells are rodent (e.g., mouse, rat) stem cells. In some cases, the stem cells are non-human primate stem cells.

[0336] The stem cells can express one or more stem cell markers, such as SOX9, KRT19, KRT7, LGR5, CA9, FXYD2, CDH6, CLDN18, TSPAN8, BPIFB1, OLFM4, CDH17, and PPARGC1A.

[0337] In some cases, the stem cells are hematopoietic stem cells (HSCs). HSCs are mesodermally derived cells that can be isolated from bone marrow, blood, umbilical cord blood, fetal liver, and yolk sac. HSCs are characterized as CD34+ and CD3-. HSCs can repopulate erythroid, neutrophil-macrophage, megakaryocyte, and lymphoid hematopoietic cell lineages in vivo. In vitro, HSCs can be induced to undergo at least some self-renewal cell divisions and differentiate into the same lineages as those found in vivo. Thus, HSCs can be induced to differentiate into one or more of erythroid cells, megakaryocytes, neutrophils, macrophages, and lymphoid cells.

[0338] In another example, the stem cell is a neural stem cell (NSC). Neural stem cells (NSCs) can differentiate into neurons and glia (including oligodendrocytes and astrocytes). Neural stem cells are pluripotent stem cells that can divide multiple times and, under certain conditions, can produce daughter cells that are neural stem cells, or neural progenitor cells that can be neuroblasts or glioblasts, e.g., cells committed to becoming one or more types of neurons and glia, respectively. Methods for obtaining NSCs are known in the art.

[0339] In another example, the stem cell is a mesenchymal stem cell (MSC). MSCs derived from embryonic mesoderm and isolated from adult bone marrow can differentiate to form muscle, bone, cartilage, fat, bone marrow stroma, and tendon. Methods for isolating MSCs are known in the art, and any known method can be used to obtain MSCs. See, for example, U.S. Patent No. 5,736,396, which describes the isolation of human MSCs.

[0340] In some cases, the target cell is a lung cell. In some cases, the EDV includes a guide RNA or a nucleic acid including a nucleotide sequence encoding the guide RNA, where the guide RNA includes a targeting sequence that targets the CFTR (cystic fibrosis transmembrane conductance regulator) gene. For example, targeting the CFTR gene can treat cystic fibrosis. If the target gene contains a defect that leads to pathology, a donor nucleic acid including a non-defective nucleotide sequence can be included in the EDV so that the defect can be corrected.

[0341] In some cases, the target cells are CD34 + In some cases, the EDV includes a guide RNA or a nucleic acid including a nucleotide sequence encoding the guide RNA, where the guide RNA includes a targeting sequence that targets the HbF (fetal hemoglobin) gene. For example, targeting the HbF gene can treat sickle cell disease or beta-thalassemia. If the target gene contains a defect that leads to pathology, a donor nucleic acid including a non-defective nucleotide sequence can be included in the EDV so that the defect can be corrected.

[0342] In some cases, the target cells are CD8 +T cells. In some cases, the EDV comprises a guide RNA or a nucleic acid comprising a nucleotide sequence encoding the guide RNA, wherein the guide RNA comprises a targeting sequence that targets a gene selected from PD1 (programmed cell death 1), CTLA4 (cytotoxic T lymphocyte-associated protein 4), and TCR (T cell receptor). For example, targeting the PD-1 gene, the CTLA-4 gene, or the TCR gene can be used to generate chimeric antigen receptor (CAR)-T cells.

[0343] In some cases, the target cells are CD4 + T cells. In some cases, the EDV comprises a guide RNA or a nucleic acid comprising a nucleotide sequence encoding the guide RNA, wherein the guide RNA comprises a targeting sequence that targets the CCR5 gene or targets the integrated proviral HIV-1. Targeting the CCR5 gene can be used to enhance resistance to HIV. Targeting the integrated proviral HIV-1 can be used to reduce the pool of T cells that are a reservoir for latent HIV.

[0344] In some cases, the target cell is a skeletal muscle cell. In some cases, the EDV includes a guide RNA or a nucleic acid including a nucleotide sequence encoding the guide RNA, where the guide RNA includes a targeting sequence that targets the Duchenne muscular dystrophy (DMD) gene. Targeting the DMD gene can be used to treat Duchenne muscular dystrophy. If the target gene contains a defect that leads to pathology, a donor nucleic acid including a non-defective nucleotide sequence can be included in the EDV so that the defect is corrected.

[0345] In some cases, the target cell is an ocular cell (e.g., a retinal cell, a photoreceptor cell, etc.). In some cases, the EDV includes a guide RNA or a nucleic acid including a nucleotide sequence encoding the guide RNA, where the guide RNA includes a targeting sequence that targets the CEP290 (centrosomal protein 290) gene. Targeting the CEP290 gene can be used to treat Leber congenital amaurosis 10 (LCA10). If the target gene contains a defect that leads to pathology, a donor nucleic acid including a non-defective nucleotide sequence can be included in the EDV so that the defect is corrected.

[0346] In some cases, the target cell is an auditory cell (e.g., a hair cell, a cochlear cell, etc.). In some cases, the EDV includes a guide RNA or a nucleic acid including a nucleotide sequence encoding the guide RNA, where the guide RNA includes a targeting sequence that targets the USH2A (Usher Syndrome Type 2A) gene. Targeting the USH2A gene can be used to treat Usher Syndrome Type 2A. If the target gene contains a defect that leads to pathology, a donor nucleic acid including a non-defective nucleotide sequence can be included in the EDV so that the defect is corrected.

[0347] In some cases, the target cells are central nervous system cells (e.g., neurons (e.g., excitatory and inhibitory neurons); and glial cells (e.g., oligodendrocytes, astrocytes, and microglia)). In some cases, the EDV comprises a guide RNA or a nucleic acid comprising a nucleotide sequence encoding a guide RNA, wherein the guide RNA comprises a targeting sequence that targets a gene selected from Tau / MAPT-1, HTT (huntingtin), SOD1 (superoxide dismutase 1), SOCS3 (suppressors of cytokine signaling 3), USP8 (ubiquitin-specific peptidase 8), DOT1L (DOT1-like histone lysine methyltransferase), UFM1 (ufmylation; ubiquitin fold modifier 1), SOCS2 (suppressors of cytokine signaling 2), SOCS9 (suppressors of cytokine signaling 9), SOCS13 (suppressors of cytokine signaling 13), SOCS11 (suppressors of cytokine signaling 11), and SOCS5 (suppressors of cytokine signaling 5). For example, targeting the Tau gene can treat Alzheimer's disease. As another example, targeting the HTT gene can treat Huntington's disease. As another example, targeting the SOD1 gene can treat amyotrophic lateral sclerosis. As another example, targeting the Ufmylation, USP8, DOT1L, SOCS2, SOCS3, SOCS9, SOCS13, SOCS11, or SOCS5 gene can treat glioblastoma. If the target gene contains a defect that leads to pathology, a donor nucleic acid containing a non-defective nucleotide sequence can be included in the EDV so that the defect can be corrected.

[0348] In some cases, a single dose of a composition comprising an EDV of the present disclosure may be about 10 2 EDV ~ approx. 10 9 For example, a single dose of a composition comprising an EDV of the present disclosure may contain about 10 2 EDV ~ approx. 10 3 EDV, approx. 10 3 EDV ~ approx. 10 4EDV, approx. 10 4 EDV ~ approx. 10 5 EDV, approx. 10 5 EDV ~ approx. 10 6 EDV, approx. 10 6 EDV ~ approx. 10 7 EDV, approx. 10 7 EDV ~ approx. 10 8 EDV, approx. 10 8 EDV ~ approx. 10 9 EDV, or approximately 10 9 EDV ~ approx. 10 10 The EDV of the present disclosure may be administered via any of a variety of parenteral and oral administration routes. For example, the EDV of the present disclosure may be administered intravenously, intramuscularly, intratumorally, peritumorally, subcutaneously, intraperitoneally, etc. The EDV of the present disclosure may be administered via convection-enhanced delivery (CED) injection.

[0349] Methods for in vivo genome editing The present disclosure provides a method for modifying a target nucleic acid in a target eukaryotic cell in vivo, the method comprising administering to an individual in need thereof an effective amount of an EDV of the present disclosure, or a composition comprising an EDV of the present disclosure. The EDV enters the target eukaryotic cell in the individual and modifies the target nucleic acid in the target eukaryotic cell. In some cases, the target cell is a CD4 + In some cases, the target cells are CD8 + T cells.

[0350] In some cases, the target cell is an immune cell. In some cases, the immune cell is a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, or a macrophage. In some cases, the immune cell is a cytotoxic T cell. In some cases, the immune cell is a helper T cell. In some cases, the immune cell is a regulatory T cell (Treg). In some cases, the target cell is a CD4 + In some cases, the target cells are CD8 + T cells.

[0351] In some cases, EDV (a) insertion of a nucleic acid comprising a nucleotide sequence encoding a therapeutic polypeptide into the genome of a target cell; (b) deletion of one or more endogenous nucleic acids in the target cell; The nucleic acid sequence includes one or more guide RNAs (or one or more nucleic acids comprising a nucleotide sequence encoding one or more guide RNAs) that provide one or more of the following:

[0352] In some cases, the EDV comprises a nucleic acid comprising a nucleotide sequence encoding a CAR. In some cases, the EDV comprises (a) a nucleic acid comprising a nucleotide sequence encoding a CAR, and (b) a guide RNA that results in knockout of a nucleic acid encoding a TRAC in a target cell. In some cases, the EDV comprises a nucleic acid comprising a nucleotide sequence encoding a CAR. In some cases, the EDV comprises (a) a nucleic acid comprising a nucleotide sequence encoding a CAR, and (b) a guide RNA that results in knockout of an immune checkpoint in a target cell. In some of these embodiments, the target cell is a CD8 + T cells.

[0353] In some cases, a single dose of a composition comprising an EDV of the present disclosure comprises about 10 2 EDV ~ approx. 10 9 For example, a single dose of a composition comprising an EDV of the present disclosure may contain about 10 2 EDV ~ approx. 10 3 EDV, approx. 10 3 EDV ~ approx. 10 4 EDV, approx. 10 4 EDV ~ approx. 10 5 EDV, approx. 10 5 EDV ~ approx. 10 6 EDV, approx. 10 6 EDV ~ approx. 10 7 EDV, approx. 10 7 EDV ~ approx. 10 8 EDV, approx. 10 8 EDV ~ approx. 10 9 EDV, or approximately 10 9 EDV ~ approx. 10 10 Includes EDVs.

[0354] Compositions comprising the EDV of the present disclosure can be administered via any of a variety of parenteral and oral administration routes. For example, compositions comprising the EDV of the present disclosure can be administered intravenously, intramuscularly, intratumorally, peritumorally, subcutaneously, intraperitoneally, etc. The EDV of the present disclosure can be administered via convection-enhanced delivery (CED) injection.

[0355] Examples of Non-Limiting Aspects of the Disclosure Aspects, including embodiments of the present subject matter described above, may be useful alone or in combination with one or more other aspects or embodiments. Without limiting the foregoing, certain non-limiting aspects of the present disclosure are provided below (see, for example, Set A, Set B, and Set C). As will be apparent to one of skill in the art upon reading this disclosure, each individually numbered aspect may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects, and is not limited to the combinations of aspects explicitly provided below.

[0356] Set A Aspect 1. (a) a nucleic acid-binding effector polypeptide; (b)(i) a viral envelope protein, and (ii) a targeting polypeptide and one or more fusion polypeptides comprising Virus-like particles (VLPs) containing Embodiment 2. The VLP of embodiment 1, wherein the targeting polypeptide is one or more antibodies or one or more antibody analogs. Embodiment 3. The VLP of embodiment 2, wherein the one or more antibody analogs is an affibody, affilin, affimer, affitin, alphabody, anticalin, avimer, DARPin, finomer, Kunitz domain peptide, monobody, repebody, VLR, or nanoCLAMP. Embodiment 4. The VLP of embodiment 2, wherein the one or more antibodies are single-chain Fv (scFv) polypeptides, diabodies, diabodies, bispecific antibodies, triabodies, or nanobodies. Embodiment 5. The VLP of embodiment 1, wherein the targeting polypeptide is a fusion polypeptide comprising (i) an antibody or antibody analog and (ii) one or more heterologous polypeptides. Embodiment 6. The VLP of embodiment 5, wherein the antibody is a single-chain Fv (scFv) polypeptide, a diabody, a triabody, or a nanobody. Embodiment 7. The VLP of embodiment 5, wherein the antibody analog is an affibody, affilin, affimer, affitin, alphabody, anticalin, avimer, DARPin, finomer, Kunitz domain peptide, monobody, repebody, VLR, or nanoCLAMP. Embodiment 8. The VLP of any one of embodiments 5 to 7, wherein one of the number of heterologous polypeptides comprises a stalk portion of a transmembrane polypeptide. Embodiment 9. The VLP of any one of embodiments 5 to 7, wherein one of the number of heterologous polypeptides comprises a stalk portion and a transmembrane portion of a transmembrane polypeptide. Embodiment 10. The VLP according to embodiment 9, wherein the transmembrane polypeptide is a CD8 α-chain polypeptide or a platelet-derived growth factor polypeptide. Embodiment 11. The VLP of embodiment 8, wherein the one or more heterologous polypeptides comprise a stalk portion of a CD8 α chain polypeptide. Embodiment 12. The VLP of embodiment 8, wherein the one or more heterologous polypeptides comprise the stalk portion and transmembrane domain of a CD8 α chain polypeptide. 13. The stalk portion and the transmembrane domain have the amino acid sequence 13. The VLP of embodiment 12, comprising TIFF2025529869000145.tif10143 (SEQ ID NO: 20). Embodiment 14. The VLP according to any one of embodiments 1 to 13, wherein the viral envelope protein is selected from hepatitis B virus (HBV) glycoprotein, hepatitis C virus (HCV) glycoprotein, Marburg virus glycoprotein, Ebola virus glycoprotein, vesicular stomatitis virus (VSV) glycoprotein, influenza virus hemagglutinin, SARS-CoV glycoprotein, respiratory syncytial virus (RSV) glycoprotein, human parainfluenza virus glycoprotein, measles virus hemagglutinin and / or measles virus fusion glycoprotein, HTLV-1 glycoprotein, Ross River virus glycoprotein, rabies virus glycoprotein, Mokola virus glycoprotein, Semliki Forest virus glycoprotein, Sindbis virus glycoprotein, Venezuelan equine encephalitis virus glycoprotein. Embodiment 15. The VLP according to any one of embodiments 1 to 14, wherein the viral envelope protein is a variant viral envelope protein comprising one or more amino acid substitutions that reduce binding of the viral envelope protein to its receptor. Embodiment 16. The VLP according to embodiment 15, wherein the viral glycoprotein is a variant vesicular stomatitis virus glycoprotein (VSVG) comprising a substitution of K47 and / or R354, and wherein the amino acid numbering is based on the amino acid sequence shown in Figure 16A. Embodiment 17. The VLP of any one of embodiments 1 to 16, wherein the targeting polypeptide binds to a cancer cell, a hematopoietic stem cell, a lung cell, a neuron, an adipocyte, a liver cell, an endothelial cell, a muscle cell, a cardiac muscle cell, a retinal cell, or a T cell. Embodiment 18. The VLP according to any one of embodiments 1 to 16, wherein the targeting polypeptide binds to CD8+ T cells or CD4+ T cells. Embodiment 19. The VLP according to any one of embodiments 1 to 16, wherein the targeting polypeptide selectively binds to cancer cells. Embodiment 20. The VLP of any one of embodiments 1 to 19, wherein the nucleic acid-binding effector polypeptide is a CRISPR-Cas effector polypeptide, a zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease, wherein the CRISPR-Cas effector polypeptide is a type II CRISPR-Cas effector polypeptide, a type V CRISPR-Cas effector polypeptide, or a type VI CRISPR-Cas effector polypeptide. Embodiment 21. The VLP according to embodiment 20, wherein the CRISPR-Cas effector polypeptide is a type II CRISPR-Cas effector polypeptide, a type V CRISPR-Cas effector polypeptide, or a type VI CRISPR-Cas effector polypeptide. Embodiment 22. The VLP according to any one of embodiments 1 to 21, wherein the nucleic acid-binding effector polypeptide is a fusion polypeptide comprising (i) a CRISPR-Cas effector polypeptide and (ii) one or more heterologous polypeptides. Embodiment 23. The CRISPR-Cas effector polypeptide exhibits reduced catalytic activity compared to a wild-type CRISPR-Cas effector polypeptide; When the CRISPR-Cas effector polypeptide is complexed with a guide nucleic acid, the CRISPR-Cas effector polypeptide retains the ability to bind to the target nucleic acid. 23. The VLP of embodiment 22. Embodiment 24. The VLP according to embodiment 22 or 23, wherein at least one of the one or more heterologous polypeptides comprises a deaminase, a reverse transcriptase, a transcriptional regulator, or an epigenetic regulator. Embodiment 25. A VLP according to any one of embodiments 22 to 24, wherein the one or more heterologous polypeptides comprise one or more nuclear localization signals. Embodiment 26. The VLP of any one of embodiments 1 to 25, comprising a CRISPR-Cas guide RNA or a nucleic acid comprising a nucleotide sequence encoding a CRISPR-Cas guide RNA. Embodiment 27. The VLP of any one of embodiments 1 to 26, further comprising a donor template nucleic acid, or a nucleotide sequence encoding a donor template nucleic acid. Embodiment 28. The VLP according to any one of embodiments 1 to 27, further comprising a therapeutic polypeptide or a nucleic acid comprising a nucleotide sequence encoding a therapeutic polypeptide. Embodiment 29. A composition comprising the VLP of any one of embodiments 1 to 18. Embodiment 30. The composition of embodiment 29, comprising a pharmaceutically acceptable excipient. Embodiment 31. A method for delivering a nucleic acid-binding effector polypeptide to a eukaryotic cell, the method comprising contacting the cell with a VLP according to any one of embodiments 1 to 28, or with a composition according to embodiment 29 or embodiment 30. Embodiment 32 The method of embodiment 31, wherein the eukaryotic cell is in vivo. Embodiment 33 The method of embodiment 31, wherein the eukaryotic cell is in vitro. Embodiment 34. The method of any one of embodiments 31 to 33, wherein the eukaryotic cell is a cancer cell, a stem cell, a hematopoietic stem cell, a lung cell, a neuron, an adipocyte, a liver cell, an endothelial cell, a muscle cell, a cardiac muscle cell, a retinal cell, or a T cell. Embodiment 35. A method for modifying a target nucleic acid in a eukaryotic cell, the method comprising contacting the cell with a VLP according to any one of embodiments 1 to 28, or a composition according to embodiment 29 or embodiment 30, wherein said contacting results in delivery of a nucleic acid-binding effector polypeptide to the cell and modification of the target nucleic acid. Embodiment 36 The method of embodiment 35, wherein the eukaryotic cell is in vivo. Embodiment 37. The method of embodiment 35, wherein the eukaryotic cell is in vitro. Embodiment 38. The method of any one of embodiments 35 to 37, wherein the eukaryotic cell is a cancer cell, a stem cell, a hematopoietic stem cell, a lung cell, a neuron, an adipocyte, a liver cell, an endothelial cell, a muscle cell, a cardiac muscle cell, a retinal cell, or a T cell.

[0357] Set B Aspect 1. (a) a nucleic acid-binding effector polypeptide; (b)(i) a viral envelope protein, and (ii) one or more targeting polypeptides and one or more fusion polypeptides comprising: An enveloped delivery vehicle (EDV) comprising: Embodiment 2. The EDV of embodiment 1, wherein the one or more antibodies or antibody analogs are an affibody, affilin, affimer, affitin, alphabody, anticalin, avimer, DARPin, finomer, Kunitz domain peptide, monobody, repebody, VLR, or nanoCLAMP. Embodiment 3. The EDV of embodiment 2, wherein the one or more antibodies are single-chain Fv (scFv) polypeptides, diabodies, bispecific antibodies, triabodies, or nanobodies. Embodiment 4. The EDV of any one of embodiments 1 to 3, wherein the one or more antibodies or antibody analogs specifically bind to CD8+ T cells or CD4+ T cells. Embodiment 5. The EDV of embodiment 4, wherein the one or more antibodies comprise an anti-CD3 antibody and an anti-CD8 antibody. Embodiment 6. The EDV according to embodiment 5, wherein the anti-CD3 antibody and the anti-CD8 antibody are scFv polypeptides or nanobodies. Embodiment 7. The EDV according to any one of embodiments 1 to 6, wherein the viral envelope protein is selected from hepatitis B virus (HBV) glycoprotein, hepatitis C virus (HCV) glycoprotein, Marburg virus glycoprotein, Ebola virus glycoprotein, vesicular stomatitis virus (VSV) glycoprotein, influenza virus hemagglutinin, SARS-CoV glycoprotein, respiratory syncytial virus (RSV) glycoprotein, human parainfluenza virus glycoprotein, measles virus hemagglutinin and / or measles virus fusion glycoprotein, HTLV-1 glycoprotein, Ross River virus glycoprotein, rabies virus glycoprotein, Mokola virus glycoprotein, Semliki Forest virus glycoprotein, Sindbis virus glycoprotein, Venezuelan equine encephalitis virus glycoprotein. Embodiment 8. The EDV according to any one of embodiments 1 to 6, wherein the viral envelope protein is a variant viral envelope protein comprising one or more amino acid substitutions that reduce binding of the viral envelope protein to its receptor. Embodiment 9. The EDV according to embodiment 8, wherein the viral glycoprotein is a variant vesicular stomatitis virus glycoprotein (VSVG) comprising a substitution of K47 and / or R354, and wherein the amino acid numbering is based on the amino acid sequence depicted in Figure 16A. Embodiment 10. The EDV of any one of embodiments 1 to 9, wherein the nucleic acid-binding effector polypeptide is a CRISPR-Cas effector polypeptide, a zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease. The CRISPR-Cas effector polypeptide is a type II CRISPR-Cas effector polypeptide, a type V CRISPR-Cas effector polypeptide, or a type VI CRISPR-Cas effector polypeptide. Embodiment 11. The EDV of embodiment 10, wherein the CRISPR-Cas effector polypeptide is a type II CRISPR-Cas effector polypeptide, a type V CRISPR-Cas effector polypeptide, or a type VI CRISPR-Cas effector polypeptide. Embodiment 12. The EDV according to any one of embodiments 1 to 11, wherein the nucleic acid-binding effector polypeptide is a fusion polypeptide comprising (i) a CRISPR-Cas effector polypeptide and (ii) one or more heterologous polypeptides. Embodiment 13. The CRISPR-Cas effector polypeptide exhibits reduced catalytic activity compared to a wild-type CRISPR-Cas effector polypeptide; When the CRISPR-Cas effector polypeptide is complexed with a guide nucleic acid, the CRISPR-Cas effector polypeptide retains the ability to bind to the target nucleic acid. 13. The EDV of embodiment 12. Embodiment 14. The EDV according to embodiment 12 or 13, wherein at least one of the one or more heterologous polypeptides comprises a deaminase, a reverse transcriptase, a transcriptional regulator, or an epigenetic regulator. Embodiment 15. The EDV according to any one of embodiments 12 to 14, wherein at least one of the one or more heterologous polypeptides is a lentiviral Gag polypeptide. Embodiment 16. The EDV according to any one of embodiments 12 to 15, wherein the one or more heterologous polypeptides comprise one or more nuclear localization signals. Embodiment 17. The EDV according to any one of embodiments 12 to 16, wherein the one or more heterologous polypeptides comprise a nuclear export signal (NES) polypeptide. Embodiment 18. The EDV of any one of embodiments 1 to 17, comprising one or more CRISPR-Cas guide RNAs, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CRISPR-Cas guide RNAs. Embodiment 19. The EDV of any one of embodiments 1 to 18, further comprising a donor template nucleic acid, or a nucleotide sequence encoding a donor template nucleic acid. Embodiment 20. The EDV of any one of embodiments 1 to 19, further comprising a therapeutic polypeptide or a nucleic acid comprising a nucleotide sequence encoding a therapeutic polypeptide. Embodiment 21. The EDV according to embodiment 20, wherein the therapeutic polypeptide is a chimeric antigen receptor (CAR). Embodiment 22. The EDV according to embodiment 21, wherein the CAR comprises one or more scFvs or one or more nanobodies specific for a cancer-associated antigen. Aspect 23. (a) the cancer-associated antigen is a solid tumor-associated antigen selected from EGFR, HER2, EGFR806, mesothelin, PSCA, MUC1, claudin 18.2, EpCAM, GD2, VEGFR2, AFP, Nectin4 / FAP, CEA, LewisY, Glypican-3, EGFRIII, IL-13Rα2, CD171, MUC16, PSMA, AXL, CD20, CD80 / 86, c-MET, DLL-3, DR5, EpHA2, FR-α, gp100, MAGE-A1, MAGE-A3, MAGE-A4, and LMP1; or (b) The EDV of aspect 22, wherein the cancer-associated antigen is an antigen associated with a hematological cancer, and the cancer-associated antigen is selected from BCMA, C5, CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD40, CD45, CD52, CD56, CD66, CD74, CD79a, CD79b, CD80, CD138, CTLA-4, CXCR4, DKK, EphA3, GM2, HLA-DR beta, integrin αVβ3, IGF-R1, IL6, KIR, PD-1, PD-L1, TRAILR1, TRAILR2, transferrin receptor, and VEGF. Embodiment 24. The EDV according to any one of embodiments 18 to 23, wherein at least one of the one or more guide RNAs comprises a nucleotide sequence that hybridizes with a target nucleic acid and results in knockout of the target nucleic acid. Embodiment 25. The EDV according to embodiment 23, wherein the target nucleic acid to be knocked out encodes an immune checkpoint. Embodiment 26. The EDV according to embodiment 25, wherein the immune checkpoint is PD-1. Embodiment 27. The EDV according to embodiment 24, wherein the target nucleic acid to be knocked out encodes a T-cell receptor alpha constant (TRAC) polypeptide. Embodiment 28. A composition comprising the EDV according to any one of embodiments 1 to 27. Embodiment 29. The composition of embodiment 28, comprising a pharmaceutically acceptable excipient. Embodiment 30. A method for modifying a target nucleic acid in a target eukaryotic cell in vivo, the method comprising administering to an individual in need thereof an effective amount of an EDV of any one of embodiments 1 to 27, or a composition of embodiment 28 or embodiment 29, wherein the EDV enters the target eukaryotic cell in the individual and modifies the target nucleic acid in the target eukaryotic cell. Embod...

Claims

1. An enveloped delivery vehicle (EDV), comprising: (a) a nucleic acid-binding effector polypeptide; (b) (i) a viral envelope protein, and (ii) a targeting polypeptide that confers binding to the target cell; and one or more fusion polypeptides comprising An enveloped delivery vehicle (EDV) comprising:

2. The EDV of claim 1 , wherein the targeting polypeptide comprises one or more antibodies or antibody analogs.

3. 3. The EDV of claim 2, wherein the one or more antibody analogs are an affibody, affilin, affimer, affitin, alphabody, anticalin, avimer, DARPin, Fynomer, Kunitz domain peptide, monobody, repebody, VLR, or nanoCLAMP.

4. 3. The EDV of claim 2, wherein the one or more antibodies are single-chain Fv (scFv) polypeptides, diabodies, bispecific antibodies, triabodies, or nanobodies.

5. The EDV according to any one of claims 1 to 4, wherein the target cell is a cancer cell, a hematopoietic stem cell, a lung cell, a neuron, an adipocyte, a hepatocyte, an endothelial cell, a muscle cell, a cardiac muscle cell, a retinal cell, a tissue-resident stem cell, a monocyte, a macrophage, a B cell, or a T cell.

6. The EDV according to any one of claims 1 to 4, wherein the target cell is a cancer cell.

7. The target cells are CD8 ＋ T cells or CD4 ＋ The EDV according to any one of claims 1 to 4, which is a T cell.

8. The EDV of any one of claims 1 to 4, wherein the targeting polypeptide comprises an anti-CD19, anti-CD20, anti-CD4, anti-CD28, or anti-CD3 antibody.

9. The EDV of any one of claims 1 to 4, wherein the targeting polypeptide comprises (a) anti-CD3 and anti-CD4 antibodies, (b) anti-CD3 and anti-CD28 antibodies, or (c) anti-CD3, anti-CD4, and anti-CD28 antibodies.

10. the targeting polypeptide is (i) one or more antibodies or antibody analogs; (ii) one or more heterologous polypeptides; The EDV according to any one of claims 2 to 9, which is a fusion polypeptide comprising:

11. 11. The EDV of claim 10, wherein one of the multiple heterologous polypeptides comprises a stalk portion of a transmembrane polypeptide.

12. 11. The EDV of claim 10, wherein one of the multiple heterologous polypeptides comprises a stalk portion and a transmembrane portion of a transmembrane polypeptide.

13. The EDV according to claim 12, wherein the transmembrane polypeptide is a CD8 α-chain polypeptide or a platelet-derived growth factor polypeptide.

14. The EDV of claim 11 , wherein the one or more heterologous polypeptides comprise the stalk portion of a CD8 α-chain polypeptide.

15. The EDV of claim 11 , wherein the one or more heterologous polypeptides comprise the stalk portion and transmembrane domain of a CD8 α-chain polypeptide.

16. the stalk portion and the transmembrane domain having the amino acid sequence 16. The EDV of claim 15, comprising: (SEQ ID NO: 20).

17. 17. The EDV according to any one of claims 1 to 16, wherein the viral envelope protein is selected from hepatitis B virus (HBV) glycoprotein, hepatitis C virus (HCV) glycoprotein, Marburg virus glycoprotein, Ebola virus glycoprotein, vesicular stomatitis virus (VSV) glycoprotein, influenza virus hemagglutinin, SARS-CoV glycoprotein, respiratory syncytial virus (RSV) glycoprotein, human parainfluenza virus glycoprotein, measles virus hemagglutinin and / or measles virus fusion glycoprotein, HTLV-1 glycoprotein, Ross River virus glycoprotein, rabies virus glycoprotein, Mokola virus glycoprotein, Semliki Forest virus glycoprotein, Sindbis virus glycoprotein, Venezuelan equine encephalitis virus glycoprotein.

18. 17. The EDV according to any one of claims 1 to 16, wherein the viral envelope protein is a variant viral envelope protein comprising one or more amino acid substitutions that reduce binding of the viral envelope protein to its receptor.

19. 19. The EDV of claim 18, wherein the viral glycoprotein is a variant vesicular stomatitis virus glycoprotein (VSVG) comprising substitutions of K47 and / or R354, and the amino acid numbering is based on the amino acid sequence shown in Figure 16A.

20. The EDV according to any one of claims 1 to 19, wherein the nucleic acid-binding effector polypeptide is a CRISPR-Cas effector polypeptide, a zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease. The CRISPR-Cas effector polypeptide is a type II CRISPR-Cas effector polypeptide, a type V CRISPR-Cas effector polypeptide, or a type VI CRISPR-Cas effector polypeptide.

21. The EDV of claim 20, wherein the CRISPR-Cas effector polypeptide is a type II CRISPR-Cas effector polypeptide, a type V CRISPR-Cas effector polypeptide, or a type VI CRISPR-Cas effector polypeptide.

22. the nucleic acid binding effector polypeptide (i) a CRISPR-Cas effector polypeptide; and (ii) one or more heterologous polypeptides; The EDV according to any one of claims 1 to 21, which is a fusion polypeptide comprising:

23. the CRISPR-Cas effector polypeptide exhibits reduced catalytic activity compared to a wild-type CRISPR-Cas effector polypeptide; When the CRISPR-Cas effector polypeptide is complexed with a guide nucleic acid, the CRISPR-Cas effector polypeptide retains the ability to bind to a target nucleic acid.

23. The EDV of claim 22.

24. 24. The EDV of claim 22 or 23, wherein at least one of the one or more heterologous polypeptides comprises a deaminase, a reverse transcriptase, a transcriptional regulator, or an epigenetic regulator.

25. The EDV according to any one of claims 22 to 24, wherein at least one of the one or more heterologous polypeptides is a lentiviral Gag polypeptide.

26. The EDV according to any one of claims 22 to 25, wherein the one or more heterologous polypeptides comprise one or more nuclear localization signals.

27. The EDV according to any one of claims 22 to 26, wherein the one or more heterologous polypeptides comprise a nuclear export signal (NES) polypeptide.

28. one or more CRISPR-Cas guide RNAs, or one or more nucleic acids comprising a nucleotide sequence encoding said one or more CRISPR-Cas guide RNAs 28. The EDV according to any one of claims 1 to 27, comprising:

29. 29. The EDV of any one of claims 1 to 28, further comprising a donor template nucleic acid or a nucleotide sequence encoding said donor template nucleic acid.

30. a therapeutic polypeptide, or Nucleic acid comprising a nucleotide sequence encoding a therapeutic polypeptide 30. The EDV of any one of claims 1 to 29, further comprising:

31. 31. The EDV of claim 30, wherein the therapeutic polypeptide is a chimeric antigen receptor (CAR).

32. 32. The EDV of claim 31 , wherein the CAR comprises one or more scFvs or one or more nanobodies specific for cancer-associated antigens.

33. (a) the cancer-associated antigen is a solid tumor-associated antigen selected from EGFR, HER2, EGFR806, mesothelin, PSCA, MUC1, claudin 18.2, EpCAM, GD2, VEGFR2, AFP, Nectin4 / FAP, CEA, LewisY, Glypican-3, EGFRIII, IL-13Rα2, CD171, MUC16, PSMA, AXL, CD20, CD80 / 86, c-MET, DLL-3, DR5, EpHA2, FR-α, gp100, MAGE-A1, MAGE-A3, MAGE-A4, and LMP1; or (b) the cancer-associated antigen is an antigen associated with a blood cancer, and the cancer-associated antigen is selected from BCMA, C5, CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD40, CD45, CD52, CD56, CD66, CD74, CD79a, CD79b, CD80, CD138, CTLA-4, CXCR4, DKK, EphA3, GM2, HLA-DR beta, integrin αVβ3, IGF-R1, IL6, KIR, PD-1, PD-L1, TRAILR1, TRAILR2, transferrin receptor, and VEGF; 33. The EDV of claim 32.

34. At least one of the one or more guide RNAs A nucleotide sequence that hybridizes with a target nucleic acid and results in knockout of said target nucleic acid. The EDV according to any one of claims 28 to 33, comprising:

35. 35. The EDV of claim 34, wherein the target nucleic acid to be knocked out encodes an immune checkpoint.

36. The EDV of claim 35, wherein the immune checkpoint is PD-1.

37. 35. The EDV of claim 34, wherein the target nucleic acid to be knocked out encodes a T-cell receptor alpha constant (TRAC) polypeptide.

38. A composition comprising an EDV according to any one of claims 1 to 37.

39. 39. The composition of claim 38, comprising a pharmaceutically acceptable excipient.

40. 40. A method for delivering a nucleic acid-binding effector polypeptide to a eukaryotic cell, the method comprising contacting the eukaryotic cell with an EDV according to any one of claims 1 to 37, or a composition according to claim 38 or 39.

41. 41. The method of claim 40, wherein the eukaryotic cell is in vivo.

42. 41. The method of claim 40, wherein the eukaryotic cell is in vitro.

43. 43. The method of any one of claims 40 to 42, wherein the eukaryotic cell is a cancer cell, a stem cell, a hematopoietic stem cell, a lung cell, a neuron, an adipocyte, a hepatocyte, an endothelial cell, a muscle cell, a cardiomyocyte, a retinal cell, a tissue-resident stem cell, a monocyte, a macrophage, a B cell, or a T cell.

44. 1. A method for modifying a target nucleic acid in a eukaryotic cell, comprising: The method comprises contacting a eukaryotic cell with an EDV according to any one of claims 1 to 37 or a composition according to claim 38 or 39, said contacting results in delivery of said nucleic acid binding effector polypeptide to said cell and modification of a target nucleic acid within said cell. method.

45. 45. The method of claim 44, wherein the eukaryotic cell is in vivo.

46. 45. The method of claim 44, wherein the eukaryotic cell is in vitro.

47. 47. The method of any one of claims 44 to 46, wherein the eukaryotic cell is a cancer cell, a stem cell, a hematopoietic stem cell, a lung cell, a neuron, an adipocyte, a hepatocyte, an endothelial cell, a muscle cell, a cardiac muscle cell, a retinal cell, a tissue-resident stem cell, a monocyte, a macrophage, a B cell, or a T cell.

48. 1. A method for modifying a target nucleic acid in a target eukaryotic cell in vivo, comprising: The method comprises administering to an individual in need thereof an effective amount of an EDV according to any one of claims 1 to 37, or a composition according to claim 38 or 39, the EDV enters a target eukaryotic cell within the individual and modifies a target nucleic acid within the target eukaryotic cell; method.

49. The target eukaryotic cells are CD4 ＋ T cells or CD8 ＋ 49. The method of claim 48, wherein the cell is a T cell.

50. 50. The method of claim 49, wherein the targeting polypeptide comprises an anti-CD3 antibody and an anti-CD28 antibody.

51. The EDV is (a) a CRISPR-Cas effector polypeptide or a nucleic acid encoding the CRISPR-Cas effector polypeptide; (b) one or more CRISPR-Cas guide RNAs, or one or more nucleic acids encoding the one or more CRISPR-Cas guide RNAs; 51. The method of claim 50, comprising:

52. 49. The method of claim 48, wherein the targeting polypeptide is an antibody, antibody analog, single-chain Fv, diabody, triabody, nanobody, or bispecific antibody.

53. 53. The method of claim 52, wherein the targeting polypeptide binds to a surface antigen on a T cell.

54. 53. The method of claim 52, wherein the targeting polypeptide binds to CD19, CD20, CD4, CD28, or CD3.

55. 55. The method of any one of claims 48 to 54, wherein the EDV comprises a nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR).

56. 56. The method of claim 55, wherein the CAR comprises one or more scFvs or one or more nanobodies specific for a cancer-associated antigen.

57. (a) the cancer-associated antigen is a solid tumor-associated antigen selected from EGFR, HER2, EGFR806, mesothelin, PSCA, MUC1, claudin 18.2, EpCAM, GD2, VEGFR2, AFP, Nectin4 / FAP, CEA, LewisY, Glypican-3, EGFRIII, IL-13Rα2, CD171, MUC16, PSMA, AXL, CD20, CD80 / 86, c-MET, DLL-3, DR5, EpHA2, FR-α, gp100, MAGE-A1, MAGE-A3, MAGE-A4, and LMP1; or (b) the cancer-associated antigen is an antigen associated with a blood cancer, and the cancer-associated antigen is selected from BCMA, C5, CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD40, CD45, CD52, CD56, CD66, CD74, CD79a, CD79b, CD80, CD138, CTLA-4, CXCR4, DKK, EphA3, GM2, HLA-DR beta, integrin αVβ3, IGF-R1, IL6, KIR, PD-1, PD-L1, TRAILR1, TRAILR2, transferrin receptor, and VEGF; 57. The method of claim 56.

58. At least one of the one or more guide RNAs A nucleotide sequence that hybridizes with a target nucleic acid and results in knockout of said target nucleic acid.

58. The method of any one of claims 51 to 57, comprising:

59. 59. The method of Claim 58, wherein the target nucleic acid to be knocked out encodes an immune checkpoint.

60. 60. The method of claim 59, wherein the immune checkpoint is PD-1.

61. 59. The method of claim 58, wherein the target nucleic acid to be knocked out encodes a T-cell receptor alpha constant (TRAC) polypeptide.

62. 62. The method of any one of claims 57-61, wherein said administering treats cancer in said individual, said cancer comprising cells that express said cancer-associated antigen.

63. 63. The method of any one of claims 51-62, wherein said administering is by intravenous administration.

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