Self-assembling virus-like particles for delivery of nucleic acid programmable fusion proteins and methods of making and using same - Patents.com

Abstract

The present disclosure provides virus-like particles for delivering gene editing agents, such as nucleic acid programmable DNA binding proteins (napDNAbps) and base editor fusion proteins ("BE-VLPs" or "eVLPs"), and systems comprising such eVLPs. The present disclosure also provides polynucleotides encoding the eVLPs described herein, which may be useful for producing the eVLPs. Also provided herein is a method for editing the genome of a target cell by introducing the eVLPs described in the present invention into the target cell. The present disclosure also provides fusion proteins that constitute the components of the eVLPs described herein, as well as polynucleotides, vectors, cells, and kits.

Description

[Technical Field]

[0001] Related Applications This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. USSN 63 / 285,995, filed December 3, 2021, and U.S. Provisional Patent Application No. USSN 63 / 298,621, filed January 11, 2022, each of which is incorporated herein by reference.

[0002] Federally funded research This invention was made with government support under Grant Nos. UG3AI150551, U01AI142756, R35GM118062, RM1HG009490, R01EY009339, and T32GM095450 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] Background of the Invention Recently developed gene editing agents enable precise manipulation of genomic DNA within living organisms, raising the possibility of addressing the underlying causes of many genetic diseases (Anzalone et al., 2020; Doudna, 2020). Base editors (BEs) mediate targeted single-nucleotide changes without the need for double-stranded DNA breaks (DSBs), thereby minimizing undesirable consequences of editing, such as indels, large deletions (Kosicki et al., 2018; Song et al., 2020), translocations (Giannoukos et al., 2018; Stadtmauer et al., 2020; Webber et al., 2019), chromosomal aberrations (Leibowitz et al., 2021), or other chromosomal abnormalities. Cytosine base editors (CBEs) (Komor et al., 2016; Nishida et al., 2016) and adenine base editors (ABEs) (Gaudelli et al., 2017) can, in principle, together correct the majority of known disease-causing single nucleotide variants (Anzalone et al., 2020; Rees and Liu, 2018). Previously, BEs have been applied to correct pathogenic point mutations and rescue disease phenotypes in mice and non-human primates (Levy et al., 2020; Yeh et al., 2020), highlighting the potential of in vivo base editing as a therapeutic strategy.

[0004] Widespread therapeutic application of in vivo base editing requires safe and efficient methods for delivering BEs to multiple tissues and organs. To date, the most robust approaches for delivering BEs in vivo involve the use of viruses, such as adeno-associated viruses (AAV) or lentiviruses (LV), to deliver BE-encoding DNA to target tissues (Levy et al., 2020; Newby and Liu, 2021). However, viral delivery of DNA-encoding editing agents results in prolonged expression in transduced cells, which increases the frequency of off-target edits (Akcakaya et al., 2018; Davis et al., 2015; Wang et al., 2020; Yeh et al., 2018). Additionally, viral delivery of DNA increases the likelihood of viral vector integration into the genome of transduced cells, both of which can promote tumorigenesis or other adverse effects (Anzalone et al., 2020; Chandler et al., 2017). Furthermore, despite the constant evolution of transfection methods and the performance of viral delivery vectors (e.g., AAV or LV), the efficiency of these approaches can vary dramatically, especially in primary cells, which are highly sensitive to modifications of their environment and may change in response to transfection agents and / or vectors.

[0005] One alternative method for delivering gene editing agents (e.g., BEs) in vivo would be to directly deliver proteins (e.g., BEs) or ribonucleoproteins (RNPs) (e.g., BEs complexed with guide RNAs) instead of DNA. The short lifespan of RNPs within cells limits the opportunity for off-target editing, as demonstrated by previous reports that delivering BE RNPs instead of BE-encoding DNA or mRNA typically substantially reduces off-target editing without sacrificing on-target editing efficiency (Doman et al., 2020; Rees et al., 2017). Successful base editing has previously been reported in the mouse inner ear and retina after local administration of lipid-encapsulated BE RNPs (Yeh et al., 2018), while a generalizable strategy for delivering BE RNPs to multiple tissues and organs in vivo has not previously been reported. Therefore, there is a need for a system / method that effectively delivers BE ribonucleoproteins (RNPs) to cells, tissues, or organs of a subject in need thereof in a manner that improves overall safety by limiting and / or avoiding off-target editing without sacrificing targeted editing. Summary of the Invention

[0006] Summary of the Invention Virus-like particles (VLPs), assemblies of viral proteins capable of infecting cells but lacking viral genetic material, have emerged as potentially promising vehicles for delivering gene editing agents as ribonucleoproteins (RNPs) (Campbell et al., 2019; Choi et al., 2016; Gee et al., 2020; Hamilton et al., 2021; Indikova and Indik, 2020; Lyu et al., 2019; Lyu et al., 2021; Mangeot et al., 2019; Yao et al., 2021). VLPs delivering RNP cargo leverage the efficiency and tissue targeting advantages of viral delivery but avoid the risks associated with viral genome integration and long-term expression of editing agents. However, existing VLP-mediated strategies for delivering gene-editing RNPs have so far demonstrated low-to-moderate editing efficiencies in vivo or limited validation of their therapeutic efficacy (Campbell et al., 2019; Choi et al., 2016; Gee et al., 2020; Hamilton et al., 2021; Indikova and Indik, 2020; Lyu et al., 2019; Lyu et al., 2021; Mangeot et al., 2019; Yao et al., 2021). Indeed, therapeutic levels of postnatal in vivo gene editing using RNP-packaged VLPs have not previously been reported.

[0007] The present disclosure is based on the development and application of engineered virus-like particles (herein interchangeably referred to as either "VLPs" or "eVLPs") for packaging and delivering therapeutic RNPs, including Cas9 and base editors (or "BEs" as disclosed herein), in vitro and in vivo, which offer significant advantages of both viral and non-viral delivery strategies. In various embodiments, extensive VLP architecture engineering of an initial design based on previously reported VLPs (Mangeot et al., "Genome editing in primary cells and in vivo using viral-derived nanoblades loaded with Cas9-sgRNA ribonucleoproteins," Nature Communications, 2019) has yielded first-, second-, third-, and fourth-generation eVLPs capable of delivering ribonucleoproteins, such as Cas9 and BEs complexed with sgRNAs, to cells, tissues, or subjects. By iteratively engineering the VLP architecture to overcome bottlenecks in cargo packaging, release, and localization, optimized eVLPs were produced that mediated efficient on-target base editing in vitro across a variety of cell types and endogenous genomic loci, with minimal detected off-target editing and higher editing efficiency of eVLP-delivered BE cargo.

[0008] As described in various embodiments of the Examples, such eVLPs enable highly efficient base editing with minimal off-target editing in a variety of cell types, including multiple immortalized cell lines, primary human and mouse fibroblasts, and primary human T cells, and a 4.7-fold improvement in Cas9 nuclease-mediated indel formation compared to previously reported Cas9-VLPs. An exemplary application of the use of BE-VLPs described in the present invention is shown in the Examples, where a single in vivo injection of eVLPs into mice mediates efficient base editing of various target genes in multiple organs, strongly knocks down serum Pcsk9 levels, and partially restores visual function in a mouse model of genetic blindness. The present disclosure, including the Examples, establishes eVLPs as a useful platform for transiently delivering gene editing agents (e.g., Cas9 or BE ribonucleoprotein) in vivo and in vitro with therapeutically relevant efficiency and minimal risk of off-target editing or DNA integration, as well as improving the in vivo delivery of other proteins and RNPs.

[0009] In various embodiments, an eVLP (e.g., a BE-VLP) comprises a supramolecular assembly including: (a) (i) a lipid membrane (e.g., a monolayer or bilayer membrane) and (ii) an envelope comprising viral envelope glycoproteins; and (b) a multi-protein core region surrounded by the envelope, the multi-protein core region including: (i) a Gag protein, (ii) a Gag-Pro-Pol protein, and (iii) a Gag-cargo fusion protein comprising a Gag protein fused to a cargo protein (e.g., napDNAbp, Cas9, or BE) via a cleavable linker (e.g., a protease-cleavable linker, e.g., an MMLV protease-cleavable linker). In various embodiments, the cargo protein is napDNAbp (e.g., Cas9). In other embodiments, the cargo protein is a base editor. In various other embodiments, the multiprotein core region of the VLP further comprises one or more guide RNA molecules that are complexed with the napDNAbp or base editor to form a ribonucleoprotein (RNP). In various embodiments, the VLP is prepared in a production cell transiently transformed with plasmid DNA encoding the various protein and nucleic acid (sgRNA) components of the VLP. Without being bound by theory, the components self-assemble at the cell membrane and bud according to a naturally occurring budding mechanism (e.g., retroviral budding or other enveloped viral budding mechanisms) to release a fully mature VLP from the cell. Once formed, Gag-Pol-Pro cleaves the protease-sensitive linker of the gag-cargo (i.e., [Gag]-[cleavable linker]-[cargo], where the cargo can be BE-RNP or napDNAbp RNP), thereby releasing BE-RNP and / or napDNAbp RNA, as the case may be, within the VLP.Thus, in various embodiments, the present disclosure also provides VLPs in which the protease-sensitive linker has been cleaved (to produce two cleavage products, by way of example only, including (i) a fusion protein comprising a gag nucleocapsid protein and a nuclear export sequence, and (ii) a napDNAbp, which may be fused to one or more additional domains, such as an NLS and / or a deaminase (i.e., to form a base editor). For example, the present disclosure provides a VLP comprising a fusion protein comprising a gag nucleocapsid protein and a nuclear export sequence (NES), encapsulated by a group-specific antigen (gag) protease (pro) polyprotein, a nucleic acid programmable DNA binding protein (napDNAbp), and a lipid membrane and a viral envelope glycoprotein. In some embodiments, the present disclosure provides VLPs that contain a mixture of cleavage and non-cleavage products (i.e., some napDNAbp or BEs have been cleaved from the gag protein and are free, while some have not yet been cleaved from the gag protein). In some embodiments, more than 50%, more than 60%, more than 70%, more than 80%, or more than 90% of the napDNAbp or BEs are cleaved from the gag protein inside the VLP. When the VLP is administered to and taken up by a recipient cell, the contents of the VLP are released, for example, released BE RNPs and / or napDNAbp RNPs. Once inside the cell, the RNPs may translocate to the cell's nucleus (particularly where the NLS is contained as part of the RNP), where DNA editing, cleavage, or other modifications may occur at the target site(s) specified by the guide RNA. The present disclosure also provides polynucleotides and vectors encoding various components of the VLPs described herein.

[0010] In another aspect, the disclosure provides a composition (e.g., a pharmaceutical composition) comprising a virus-like particle (VLP) comprising a group-specific antigen (gag) protease (pro) polyprotein encapsulated by a viral envelope glycoprotein and a fusion protein, wherein the fusion protein comprises: (i) a gag nucleocapsid protein; (ii) a nucleic acid-programmable DNA-binding protein (napDNAbp); (iii) a cleavable linker; and (iv) a nuclear export sequence (NES). In some embodiments, the napDNAbp is fused to one or more additional domains, such as, for example, one or more NLSs and / or one or more deaminases (i.e., to form a base editor). In some embodiments, the pharmaceutical composition comprises a VLP comprising a group-specific antigen (gag) protease (pro) polyprotein, a nucleic acid-programmable DNA-binding protein (napDNAbp), and a fusion protein comprising a gag nucleocapsid protein and a nuclear export sequence (NES) encapsulated by a lipid membrane and a viral envelope glycoprotein (i.e., a VLP in which the cleavable linker has been cleaved by a protease). In some embodiments, the napDNAbp is fused to one or more additional domains, such as, for example, one or more NLSs and / or one or more deaminases (i.e., to form base editors). Each component of the pharmaceutical composition provided herein may comprise any of the options described above with reference to VLPs or any of the other options provided by the present disclosure. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0011] In another aspect, the present disclosure provides a method for editing a nucleic acid molecule in a target cell by base editing, comprising contacting the target cell with any of the compositions provided herein, thereby introducing one or more modifications to the nucleic acid molecule at the target site. In some embodiments, the cell is a mammalian cell (e.g., a human cell). In some embodiments, the cell is a cell from an animal related to veterinary or agricultural use. In some embodiments, the cell is in a subject. In certain embodiments, the subject is a human. In some embodiments, the one or more modifications to the nucleic acid molecule are associated with reducing, alleviating, or preventing symptoms of a disease or disorder.

[0012] In another aspect, the present disclosure provides a fusion protein comprising: (i) a group-specific antigen (gag) nucleocapsid protein; (ii) a nucleic acid-programmable DNA-binding protein (napDNAbp); (iii) a cleavable linker; and (iv) a nuclear export sequence (NES). Each component of the fusion proteins provided herein may comprise any of the options described herein with reference to BE-VLPs or any of the other options provided by the present disclosure. In other aspects, the present disclosure also provides polynucleotides encoding any of the eVLP components, including the fusion proteins provided herein, vectors comprising such polynucleotides, cells comprising any of the eVLP proteins, including the fusion proteins, polynucleotides, or vectors provided herein, and kits comprising any of a plurality of polynucleotides or eVLP proteins, including the fusion proteins, provided herein.

[0013] In another aspect, the present disclosure provides VLPs produced by transfecting, transducing, electroporating, or otherwise inserting any of the polynucleotides or vectors disclosed herein into a cell and expressing components of the VLP from the polynucleotide or vector, thereby allowing the virus-like particle to assemble spontaneously within the cell. In some embodiments, any of the compositions, methods, or cells provided herein may be used to produce the VLPs described herein.

[0014] In another aspect, the present disclosure provides compositions comprising any of the VLPs, polynucleotides, vectors, and fusion proteins provided herein.

[0015] In another aspect, the present disclosure provides methods of editing a nucleic acid molecule in a target cell using any of the VLPs, polynucleotides, compositions, and fusion proteins provided herein.

[0016] In another aspect, the disclosure provides a cell comprising any of the VLPs, polynucleotides, vectors, compositions, and fusion proteins described herein.

[0017] In another aspect, the disclosure provides kits comprising any of the VLPs, polynucleotides, vectors, compositions, and fusion proteins described herein.

[0018] It should be understood that the foregoing concepts, and additional concepts described below, may be arranged in any suitable combination, as the disclosure is not limited in this respect. Furthermore, other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments when considered in conjunction with the accompanying figures.

[0019] The following drawings form part of this specification and are included to further demonstrate certain aspects of the present disclosure which may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief explanation of the drawings]

[0020] [Figures 1A-1D]BE-VLP architecture and initial (v1) editing efficiency. Figure 1A: Schematic of BE-VLP. The base editor protein is fused to the C-terminus of the murine leukemia virus (MLV) gag polyprotein via a linker that is cleaved by MLV protease during particle maturation. BE = base editor. Figure 1B: Adenine base editing efficiency of v1 BE-VLP at two genomic loci in HEK293T cells. The protospacer position of the targeted adenine is indicated by a subscript (i.e., A5 = adenine at position 5), where the PAM is at positions 21-23. Data are shown as individual data points and mean ± standard error for n = 3 independent biological replicates. Figure 1C provides a generalized structure for a virus-like particle contemplated herein, which encompasses (a) a lipid membrane derived from the plasma membrane of the producer cell as a result of the retroviral budding process, (b) viral envelope glycoproteins (facilitating binding to and influencing tropism in recipient cells), and (c) a protein core or shell comprising a protein assembly including the retroviral Gag protein, where a portion of the Gag protein is fused to a cleavable protein cargo (by way of example, napDNAbp or BE) or Pro-Pol (containing protease activity). The cleavable protein cargo is joined to the Gag protein by a protease-cleavable linker and is cleaved by Pro-Pol at some point after assembly of the VLP. By way of background, Figure 1D provides a schematic diagram illustrating a typical retroviral budding process and the involvement of the Gag polyprotein, which includes the "MA" domain (matrix domain), the "CA" domain (capsid domain), and the "NC" domain (nucleocapsid domain). Without wishing to be bound by theory, it is believed that the Gag fusions, Gag-Pro-Pol fusions, and Gag-cargo fusions of the eVLPs described herein drive a similar budding process to form mature eVLPs that are released from the producer cells.

[0021] [Figures 2A-2G]Optimization of BE-VLPs (v2, v3, and v4 eVLPs were obtained by identifying and engineering solutions to bottlenecks limiting VLP efficacy). Figure 2A: More efficient linker cleavage results in improved cargo release after VLP maturation. Figure 2B: Adenine base editing efficiency of v1 and v2 BE-eVLPs at position A7 of the BCL11A enhancer site in HEK293T cells. Optimization of the protease-cleavable linker sequence is shown (see also Figure 8). Figure 2C: Demonstrates that improved localization of cargo in production cells results in more efficient incorporation into eVLPs. Figure 2D: Introducing a 3xNES motif upstream of the cleavable linker can promote cytoplasmic localization of gag-3xNES-cargo in production cells, but promotes nuclear localization of free ABE cargo in transduced cells. Figure 2E: Optimization of gag-ABE localization (see also Figures 9A-9B). Adenine base editing efficiency of v2.4 and v3 BE-eVLPs at position A7 of the BCL11A enhancer site in HEK293T cells. Figure 2F: Optimal gag-cargo:gag-pro-pol stoichiometry balances the amount of cargo protein per particle with the amount of MMLV protease required for efficient particle maturation. Figure 2G: Optimization of the gag-ABE:gag-pro-pol ratio. Adenine base editing efficiency of v3.4 eVLPs with different gag-ABE:gag-pro-pol stoichiometries at position A7 of the BCL11A enhancer site in HEK293T cells. The legend indicates the % gag-ABE plasmid of the total amount of gag-ABE and gag-pro-pol plasmids. Figure 2B, Figure 2E, and Figure 2G: Values ​​and error bars reflect the mean ± standard error of n = 3 independent biological replicates. Data were fitted to a four-parameter logistic curve using nonlinear regression.

[0022] [Figures 3A-3J]Characterization of BE-eVLPs. Figure 3A: Quantification of BE molecules per eVLP by anti-Cas9 and anti-MLV(p30) ELISA (see also Figures 10A-10C). Values ​​and error bars reflect the mean ± standard error of n = 3 independent replicates. Figure 3B: Quantification of relative sgRNA abundance by RT-qPCR using sgRNA-specific primers normalized to v1 sgRNA abundance. Values ​​and error bars reflect the mean ± standard error of n = 3 technical replicates. Figure 3C-3D: Comparing editing efficiency of v1, v2.4, v3.4, and v4 BE-eVLPs at the BCL11A enhancer site in HEK293T cells (Figure 3C) and the Dnmt1 site in NIH 3T3 cells (Figure 3D). Values ​​and error bars reflect the mean ± standard error of n = 3 independent biological replicates. Data were fitted to a four-parameter logistic curve using nonlinear regression. Figure 3E: Adenine base editing efficiency in HEK293T cells of a single BE-eVLP targeting either the HEK2 or BCL11A enhancer locus separately, or multiple v4 BE-eVLPs targeting both loci simultaneously. Data are shown as individual data points and means ± standard errors for n = 3 independent biological replicates. Figure 3F: Adenine base editing efficiency of FuG-B2-pseudotyped v4 BE-eVLPs in Neuro-2a cells or 3T3 fibroblasts. Data are shown as individual data points and means ± standard errors for n = 3 independent biological replicates. Figure 3G: Adenine base editing efficiency at three on-target genomic loci and their corresponding Cas-dependent off-target sites in HEK293T cells treated with v4 BE-eVLP or ABE8e plasmid. OT1 = off-target site 1, OT2 = off-target site 2, OT3 = off-target site 3. Figure 3H: Cas-independent off-target editing frequency at six off-target R-loops in HEK293T cells treated with v4 BE-eVLP or ABE8e plasmid. OTRL = off-target R-loop. (See also Figure 11A for experimental timeline and Figure 11B for on-target editing control.)Figure 3I: Molecules of BE-encoding DNA per v4 BE-eVLP detected by qPCR of lysed VLPs or lysis buffer only. Figure 3J: Amount of BE-encoding DNA detected by qPCR of lysates from cells either treated with BE-VLPs or transfected with a BE-encoding plasmid. Figures 3E-J: Data are shown as individual data points and mean ± standard error for n=3 independent biological replicates.

[0023] [Figures 4A-4C] Base editing in primary human and primary mouse cells using v4 BE-eVLP. Figure 4A: Correction efficiency of the COL7A1 (R185X) mutation in patient-derived primary human fibroblasts. Genomic DNA was harvested from cells 48 hours after transduction with v4 BE-VLP. Values ​​and error bars reflect the mean ± standard error of n = 3 independent biological replicates. Data were fitted to a four-parameter logistic curve using nonlinear regression. Figure 4B: Correction efficiency of the Idua (W392X) mutation in primary mouse fibroblasts. Genomic DNA was harvested from cells 48 hours after transduction with v4 BE-VLP. Values ​​and error bars reflect the mean ± standard error of n = 3 independent biological replicates. Data were fitted to a four-parameter logistic curve using nonlinear regression. Figure 4C: Adenine base editing efficiency at the B2M and CIITA loci in primary human T cells. T cells were transduced twice with v4 BE-VLPs, and genomic DNA was harvested from the cells 48 hours after the second transduction (see example). Data are presented as individual data points and means ± standard errors for n=3 independent biological replicates.

[0024] [Figure 5A-5B]In vivo base editing in the central nervous system using v4 BE-eVLPs. Figure 5A: Schematic of P0 ICV injection of v4 BE-eVLPs. Dnmt1-targeting v4 BE-eVLPs were co-injected with lentivirus encoding EGFP-KASH. Tissues were harvested 3 weeks post-injection, and the cortex and midbrain were isolated. Nuclei were dissociated for each tissue and analyzed by high-throughput sequencing as bulk unsorted (all nuclei) or GFP+ nuclei. Figure 5B: Adenine base editing efficiency at the Dnmt1 locus in bulk unsorted (all nuclei) and GFP+ populations. Data are shown as individual data points and mean ± standard error for n = 4 mice.

[0025] [Figures 6A-6E]In vivo knockdown of Pcsk9 from a single systemic injection of v4 BE-eVLPs. Figure 6A: Schematic of systemic injection of BE-eVLPs. Pcsk9-targeting BE-eVLPs were injected retroorbitally into 6-7 week-old C57BL / 6J mice. Organs were harvested 1 week after injection, and genomic DNA of unsorted cells was sequenced. Figure 6B: Adenine base editing efficiency at the Pcsk9 exon 1 splice donor in mouse liver after systemic injection of v1 BE-VLPs or v4 BE-eVLPs. Data are shown as individual data points and mean ± standard error for n = 3 mice (v1 BE-VLPs and v4 BE-eVLPs at 4 × 10 VLPs) or n = 4 mice (v4 BE-eVLPs at 7 × 10 eVLPs). Figure 6C: Adenine base editing efficiency at the Pcsk9 exon 1 splice donor in mouse heart, kidney, liver, lung, muscle, and spleen after systemic injection of 7 x 10 v4 BE-eVLPs. Data are shown as individual data points and mean ± standard error for n = 4 mice (treated) or n = 3 mice (untreated). Figure 6D: DNA sequencing reads containing A T-to-G C mutations within protospacer positions 4 to 10 for 14 CIRCLE-seq-designated off-target loci from the livers of v4 BE-eVLP-treated, AAV-treated, and untreated mice. Data are shown as individual data points and mean ± standard error for n = 4 mice (BE-eVLP), n = 5 mice (AAV), or n = 3 mice (untreated). vg = viral genome. Figure 6E: Serum Pcsk9 levels measured by ELISA. Data are presented as mean±standard error for individual data points and n=4 mice (treated) or n=3 mice (untreated).

[0026] [Figure 7A-7J]In vivo base editing by v4 BE-eVLP in a mouse model of genetic blindness. Figure 7A: Schematic diagram of Rpe65 exon 3 (shaded gray, PAM underlined) encompassing the R44X mutation (gray and italicized under the label "R44X"), which can be corrected by an A·T-to-G·C conversion at position A6 in the protospacer. The sequences shown are SEQ ID NO: 497 (top) and SEQ ID NO: 498 (bottom). Figure 7B: Schematic diagram of subretinal injection. Five weeks after injection, phenotypic rescue was assessed via electroretinogram (ERG), and tissues were subsequently harvested for sequencing. Figure 7C: Adenine base editing efficiency at positions A3, A6, and A8 of the protospacer in genomic DNA harvested from rd12 mice. Data are shown as individual data points and mean ± standard error for n = 6 mice (both treatment groups) or n = 4 mice (untreated). Figure 7D: Allele frequency distribution of genomic DNA collected from treated rd12 mice. Data are shown as mean ± standard error for n = 6 mice. 8e-LV = ABE8e-NG-LV, 8e-eVLP = v4 ABE8e-NG-eVLP. Figure 7E: Scotopic a-wave and b-wave amplitude measured by ERG after overnight dark adaptation. Data are shown as individual data points and mean ± standard error for n = 8 mice (wild-type), n = 6 mice (ABE8e-NG-LV and v4 ABE8e-NG-eVLP), or n = 4 mice (untreated). Figure 7F: Adenine base editing efficiency at positions A3, A6, and A8 of the protospacer in genomic DNA collected from rd12 mice. Data are shown as individual data points and mean ± standard error for n = 6 mice (v4 ABE7.10-NG-eVLP) or n = 4 mice (ABE7.10-NG-LV and untreated). P values ​​were calculated using a two-tailed t-test. Figure 7G: Allele frequency distribution of genomic DNA collected from treated rd12 mice. Data are shown as mean ± standard error for n = 6 mice (v4 ABE7.10-NG-eVLP) or n = 4 mice (ABE7.10-NG-LV and untreated).7.10-LV = ABE7.10-NG-LV, 7.10-eVLP = v4 ABE7.10-NG-eVLP. Figure 7H: Scotopic a- and b-wave amplitudes measured by ERG after overnight dark adaptation. Data are shown as individual data points and means ± standard error for n = 8 mice (wild-type), n = 7 mice (v4 ABE7.10-NG-eVLP), n = 5 mice (ABE7.10-NG-LV), or n = 4 mice (untreated). P values ​​were calculated using a two-tailed t-test. Figure 7I: Western blot of protein extracts from RPE tissue from wild-type, untreated, v4 ABE7.10-NG-eVLP-treated, and ABE7.10-NG-LV-treated mice. FIG. 7J: Representative ERG waveforms from wild-type, untreated, ABE7.10-NG-LV-treated, and v4 ABE7.10-NG-eVLP-treated mice.

[0027] [Figures 8A-8E]Engineering and characterization of v1 BE-VLPs and v2 BE-eVLPs. Figure 8A: Validation of VLP production. Immunoblot analysis of proteins from purified BE-VLPs using anti-Cas9, anti-p30, and anti-VSV-G antibodies. Figure 8B: Adenine base editing efficiency of v1 BE-VLPs at position A7 of the BCL11A enhancer site in HEK293T cells. Values ​​and error bars reflect the mean ± standard error of n = 3 independent biological replicates. Data were fitted to a four-parameter logistic curve using nonlinear regression. Figure 8C: Schematic of immature BE-VLPs with ABE8e fused to the gag structural protein. Various MMLV protease cleavage sites were inserted between gag and ABE8e to determine the optimal cleavable sequence that facilitates release of ABE8e from gag during proteolytic virion maturation. Arrows indicate the cleavage sites. The sequences shown are PRSSLY (SEQ ID NO: 499), PALTP (SEQ ID NO: 500), VQAL (SEQ ID NO: 501), VLTQ (SEQ ID NO: 502), PLQVL (SEQ ID NO: 503), TLNIERR (SEQ ID NO: 504), TSTLL (SEQ ID NO: 505), and MENSS (SEQ ID NO: 506). Figure 8D: Representative Western blot assessing cleaved ABE8e versus full-length gag-ABE8e in purified v2 BE-VLP variants. Figure 8E: Densitometry-based quantification of the cleaved ABE8e fraction from the Western blot. Data are shown as mean ± standard error for n=3 technical replicates.

[0028] [Figures 9A-9D]Improved gag-ABE localization in producer cells. Figure 9A: Schematic showing the localization of BE-RNP cargo in producer cells with (right) and without (left) a nuclear exclusion signal (NES). Figure 9B: v2.4 and v3 BE-eVLP constructs. Three HIV NESs were fused to either the C- or N-terminus of the gag-ABE fusion. A protease-cleavable linker was incorporated between the ABE and NES sequences such that the final BE cargo lacks the NES after proteolytic virion maturation. The protease cleavage sequences shown are TSTLL (SEQ ID NO: 505), MENSS (SEQ ID NO: 506), MSKLL (SEQ ID NO: 507), ATVVS (SEQ ID NO: 508), PLQVL (SEQ ID NO: 503), TLNIERR (SEQ ID NO: 504), IRKIL (SEQ ID NO: 509), and FLDG (SEQ ID NO: 510). Figure 9C: Representative immunofluorescence images of producer cells transfected with the v2.4 gag-ABE construct or the v3.4 gag-3 xNES-ABE construct. 48 hours after transfection, cells were fixed with paraformaldehyde and stained with anti-tubulin antibody to stain the cytoskeleton, DAPI to stain the nucleus, and anti-Cas9 antibody to visualize the gag-ABE fusion, as indicated in the legend. Scale bars represent 50 μm. Figure 9D: Automated image analysis-based quantification of the cytoplasmic localization of the v2.4 gag-ABE construct or the v3.4 gag-3 xNES-ABE construct. Data are shown as mean ± standard error for n = 3 technical replicates. P values ​​were calculated using a two-tailed t-test.

[0029] [Figures 10A-10G]Characterization of BE-eVLPs. Figure 10A: Representative negative-stain transmission electron micrograph (TEM) of v4 BE-eVLPs. Scale bar indicates 200 nm. Figures 10B-10C: Protein content of v1, v2.4, v3.4, and v4 BE-eVLPs was measured by anti-Cas9 or anti-MLV (p30) ELISA. Data are shown as individual data points and mean ± standard error for n=3 technical replicates. Figure 10D: Comparing particle number-normalized editing efficiency of v1, v2.4, v3.4, and v4 BE-VLPs at the BCL11A enhancer site in HEK293T cells. Data are shown as mean ± standard error for n=3 biological replicates. Figure 10E: Cell viability after v4 BE-eVLP treatment of HEK293T cells and NIH 3T3 fibroblasts. Data are shown as values ​​± standard error for n=3 biological replicates. Figure 10F: Indel frequencies produced by v1 Cas9-VLPs and v4 Cas9-eVLPs at the EMX1 locus in HEK293T cells. Data are shown as values ​​± standard error for n=3 biological replicates. Figure 10G: Adenine base editing efficiency of VSV-G-pseudotyped v4 BE-eVLPs in Neuro-2a cells or 3T3 fibroblasts. Data are shown as individual data points and mean ± standard error for n=3 biological replicates.

[0030] [Figures 11A-11D]Assessment of off-target editing by v4 BE-eVLP. Figure 11A: Experimental timeline for the orthogonal R-loop assay. Figure 11B: On-target editing control for the orthogonal R-loop experiment. Data are shown as individual data points and mean ± standard error for n=3 biological replicates. Figure 11C: Cell viability after v4 BE-VLP treatment of RDEB fibroblasts. Data are shown as mean ± standard error for n=3 biological replicates. Figure 11D: DNA sequencing reads containing A·T-to-G·C mutations within protospacer positions 4-10 for 10 previously identified off-target loci from genomic DNA of fibroblasts from a v4-BE-eVLP-treated RDEB patient. The gray dotted line represents the highest observed background mutation rate of 0.1%. Data are shown as individual data points and mean ± standard error for n=3 biological replicates.

[0031] [Figure 12] Editing efficiency of BE-VLPs in Neuro2a cells with Dnmt1.

[0032] [Figures 13A-13B] Flow cytometry analysis for nuclei sorting from mouse brain after P0 ICV injection. Figure 13A: Singlet nuclei were gated based on FSC / BSC ratio and DyeCycle Ruby signal. Row 1 demonstrates the gating strategy for GFP-negative samples. Bulk nuclei correspond to events that passed gate D for singlet nuclei. Figure 13B: Percentage of GFP-positive nuclei measured by flow cytometry after P0 ICV injection. Data are shown as mean ± standard error for n=3 biological replicates.

[0033] [Figures 14A-14C]Assessment of hepatotoxicity after systemic v4 BE-eVLP injection. Figure 14A: Plasma aspartate transaminase (AST) and alanine transaminase (ALT) levels 1 week after v4 BE-eVLP injection. Figures 14B-14C: Histopathological evaluation of livers by hematoxylin and eosin staining 1 week after injection from (Figure 14B) untreated mice and (Figure 14C) v4 BE-eVLP-treated mice. Representative examples of each are shown. Scale bars represent 50 μm.

[0034] [Figures 15A-15C] Sequencing analysis of RPE cDNA after v4 BE-eVLP or lentivirus treatment. Figure 15A: v4 BE-eVLP and lentivirus treatment resulted in 50-60% A T-to-G C conversion at the target adenine (A6). Data are shown as individual data points and mean ± standard error for n = 6 (ABE8e-NG-LV, ABE8e-NG-eVLP, and ABE7.10-NG-eVLP) or n = 4 (ABE7.10-NG-LV and untreated) replicates. Figures 15B-C: Off-target A-to-G RNA editing by v4 BE-eVLP and lentivirus as measured by high-throughput sequencing of Mcm3ap (Figure 15B) and Perp (Figure 15C) transcripts. Data are shown as mean ± standard error for n=6 (ABE8e-NG-LV, ABE8e-NG-eVLP, and ABE7.10-NG-eVLP) or n=4 (ABE7.10-NG-LV and naive) replicates.

[0035] [Figure 16]

[0049] An overview of one embodiment of the production of eVLPs, including BE RNPs (e.g., BE-VLPs), in producer cells using a set of expression plasmids encoding the various self-assembling components of the eVLP: (a) a plasmid encoding a Gag-BE fusion protein (e.g., a retroviral Gag, MMLV-Gag-BE fusion protein); (b) a plasmid encoding a Gag-Pro-Pol protein (e.g., a retroviral protein, such as the MMLV protease precursor); (c) a plasmid encoding a BE sgRNA; and (d) a plasmid encoding an envelope glycoprotein (e.g., the spike glycoprotein of vesicular stomatitis virus (VSV-G)). The plasmids are transiently co-transfected into producer cells, and the encoded proteins and sgRNA products are encoded. In some embodiments, such as the fourth-generation eVLPs described herein, the inventors have found an optimized stoichiometric ratio of gag-cargo fusion to Gag-Pro-Pol fusion protein that balances the amount of gag-cargo available for packaging into VLPs with the amount of retroviral protease (the "Pro" in the Gag-Pro-Pol fusion) required for VLP maturation. In one embodiment, the optimized ratio of Gag-cargo fusion to Gag-Pro-Pol fusion protein is achieved by transiently delivering appropriate ratios of plasmids encoding each component into production cells. In one embodiment, to adjust the stoichiometry of Gag-cargo fusion to Gag-Pro-Pol fusion, the ratio of Gag-cargo-encoding plasmid (e.g., Gag-3xNES-ABE8e) to wild-type MMLV gag-pro-pol plasmid transfected for VLP production was varied. We found that increasing the amount of gag-cargo plasmid beyond the original ratio used to produce v3.4 BE-eVLPs (38% gag-cargo plasmid and 62% gag-pro-pol plasmid) did not improve editing efficiency (Figure 2G). Decreasing the ratio of gag-cargo plasmid from 38% to 25% slightly improved editing efficiency (Figure 2G).However, further decreasing the proportion of gag-cargo plasmid below 25% reduced editing efficiency (Figure 2G). These results are consistent with a model in which the optimal gag-cargo:gag-pro-pol stoichiometry balances the amount of gag-cargo available for packaging into VLPs with the amount of MMLV protease (the "pro" in gag-pro-pol) required for VLP maturation. In one embodiment, the results of this final round of optimization revealed a fourth-generation (v4) BE-eVLP formulation (Figure 2G), which combined the optimal gag-BE:gag-pro-pol stoichiometry (gag-BE 25%) with the v3.4BE-eVLP architecture.

[0036] As illustrated in Figure 16, the present disclosure provides a plurality of polynucleotides encoding the eVLP (e.g., BE-VLP) self-assembling components described herein. In some embodiments, the present disclosure provides a plurality of polynucleotides comprising: (i) a first polynucleotide (e.g., a plasmid) comprising a nucleic acid sequence encoding a viral envelope glycoprotein; (ii) a second polynucleotide (e.g., a plasmid) comprising a nucleic acid sequence encoding a group-specific antigen (gag) protease (pro) polyprotein; (iii) a third polynucleotide (e.g., a plasmid) comprising a nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises: (a) a group-specific antigen (gag) nucleocapsid protein; (b) a nucleic acid-programmable DNA-binding protein (napDNAbp); (c) a cleavable linker; and (d) a nuclear export sequence (NES); and (iv) a fourth polynucleotide (e.g., a plasmid) comprising a nucleic acid sequence encoding a guide RNA (gRNA). In some embodiments, the gRNA binds to the napDNAbp of the fusion protein encoded by the third polynucleotide. In some embodiments, the ratio of the second polynucleotide to the third polynucleotide is approximately 10:1, approximately 9:1, approximately 8:1, approximately 7:1, approximately 6:1, approximately 5:1, approximately 4:1, approximately 3:1, approximately 2:1, approximately 1.5:1, approximately 1:1, or approximately 0.5:1. In certain embodiments, the ratio of the second polynucleotide to the third polynucleotide is approximately 3:1.

[0037] [Figures 17A-17B] v4 BE-eVLPs can efficiently edit primary human hematopoietic stem cells (HSCs). Figure 17A: Four-marker sorting for HSCs. Hematopoietic progenitor cells (HPCs): CD34+ / CD38+. HSCs: CD34+ / CD38- / CD90+ / CD45RA-. Figure 17B: Adenine base editing at the BCL11A enhancer locus.

[0038] [Figure 18] v4 BE-eVLPs minimally disrupt HSC cell viability.

[0039] [Figures 19A-19B] The v4 BE-eVLP enables efficient on-target editing with minimal off-target editing. Lower Cas-dependent off-target editing was observed compared to previous base editing approaches targeting the same site (see, for example, Zeng et al., Nat. Med. (2020)). DETAILED DESCRIPTION OF THE INVENTION

[0040] definition Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this invention belongs.The following references provide those skilled in the art with the general definitions of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The HarperCollins Dictionary of Biology (1991).As used herein, the following terms have the meanings ascribed to them unless otherwise specified.

[0041] Adenosine deaminase As used herein, the terms "adenosine deaminase" or "adenosine deaminase domain" refer to a protein or enzyme that catalyzes the deamination reaction of adenosine (or adenine). These terms are used interchangeably. In certain embodiments, the present disclosure provides a nucleobase editor fusion protein comprising one or more adenosine deaminase domains. Illustratively, the adenosine deaminase domain may comprise a heterodimer of a first adenosine deaminase domain and a second deaminase domain connected by a linker. The adenosine deaminase (by way of example, an engineered adenosine deaminase or an evolved adenosine deaminase) provided herein may be an enzyme that converts adenine (A) to inosine (I) in DNA or RNA. Such an adenosine deaminase can effect an A:T to G:C base pair conversion. In some embodiments, the deaminase is a variant of a naturally occurring deaminase from an organism. In some embodiments, the deaminase domain is not naturally occurring. For example, in some embodiments, the deaminase is 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 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally occurring deaminase.

[0042] In some embodiments, the adenosine deaminase is derived from a bacterium such as E. coli, S. aureus, S. typhi, S. putrefaciens, H. influenzae, or C. crescentus. In some embodiments, the adenosine deaminase is TadA deaminase. In some embodiments, the TadA deaminase is E. coli TadA deaminase (ecTadA). In some embodiments, the TadA deaminase is a truncated E. coli TadA deaminase. For example, the truncated ecTadA may lack one or more N-terminal amino acids compared to full-length ecTadA. In some embodiments, the truncated ecTadA may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues relative to full-length ecTadA. In some embodiments, the truncated ecTadA may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues relative to full-length ecTadA. In some embodiments, the ecTadA deaminase does not include an N-terminal methionine. In some embodiments, the adenosine deaminase comprises ecTadA(8e) (i.e., used in the base editor ABE8e), as further described herein. See U.S. Patent Application Publication No. 2018 / 0073012, published March 15, 2018, which is incorporated herein by reference.

[0043] BasesEdit "Base editing" refers to a genome editing technique that involves converting a specific nucleic acid base at a target genomic locus to another. In certain embodiments, this can be achieved without the need for a double-stranded DNA break (DSB) or a single-strand break (i.e., nicking). To date, other genome editing techniques, including CRISPR-based systems, begin with the introduction of a DSB at the target locus. Cellular DNA repair enzymes then repair the break, generally resulting in a random insertion or deletion of a base (indel) at the site of the DSB. However, if the desired goal is to introduce or correct a point mutation at a target locus rather than stochastically disrupting an entire gene, these genome editing techniques are inappropriate because of their low correction rate (e.g., typically 0.1% to 5%) and the primary genome editing product being an indel. To increase the efficiency of gene correction without simultaneously introducing random indels, the CRISPR / Cas9 system has been modified to directly convert one DNA base to another without DSB formation. See Komor, AC, et al., "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage," Nature 533, 420-424 (2016), the entire contents of which are incorporated herein by reference.

[0044] Base Editor The terms "base editor (BE)" and "nucleobase editor," used interchangeably herein, refer to an agent, including a polypeptide, capable of making modifications to bases (e.g., A, T, C, G, or U) in a nucleic acid sequence (e.g., DNA or RNA) that convert one base to another (e.g., A to G, A to C, A to T, C to T, C to G, C to A, G to A, G to C, G to T, T to A, T to C, or T to G). In some embodiments, the nucleobase editor is capable of deaminating a base in a nucleic acid, such as a base in a DNA molecule. In the case of an adenosine nucleobase editor, the nucleobase editor is capable of deaminating adenine (A) in DNA. Such nucleobase editors may include a nucleic acid-programmable DNA-binding protein (napDNAbp) fused to adenosine deaminase. Some nucleobase editors include CRISPR-mediated fusion proteins utilized in the base editing methods described herein. In some embodiments, the nucleobase editor comprises a nuclease-inactive Cas9 (dCas9) fused to a deaminase that binds to nucleic acids in a guide RNA-programmed manner via R-loop formation but does not cleave the nucleic acid. For example, the dCas9 domain of the fusion protein may include D10A and H840A mutations (which enable Cas9 to cleave only one strand of a nucleic acid duplex), as described in PCT Publication No. PCT / US2016 / 058344, published April 27, 2017 as International Publication No. WO 2017 / 070632, incorporated herein by reference. The DNA cleavage domain of S. pyogenes Cas9 includes two subdomains: an HNH nuclease subdomain and a RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA (the "target strand," or the strand on which editing or deamination occurs), while the RuvC1 subdomain cleaves the non-complementary strand containing the PAM sequence (the "non-edited strand").The RuvC1 mutation D10A produces a nick in the target strand, while the HNH mutation H840A produces a nick in the non-edited strand (see Jinek et al., Science, 337:816-821 (2012); Qi et al., Cell. 28;152(5):1173-83 (2013)).

[0045] In some embodiments, a nucleobase editor is a macromolecule or macromolecule complex that predominantly (by way of example, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 99%, greater than 99.9%, or 100%) effects the conversion of nucleobases in a polynucleotide sequence to another nucleobase (i.e., a transition or transversion) using a combination of (1) a nucleotide-, nucleoside-, or nucleobase-modifying enzyme and (2) a nucleic acid-binding protein that can be programmed to bind to a specific nucleic acid sequence.

[0046] In some embodiments, the nucleobase editor comprises a DNA-binding domain (e.g., a programmable DNA-binding domain such as dCas9 or nCas9) that directs it to a target sequence. In some embodiments, the nucleobase editor comprises a nucleobase-modifying domain fused to the programmable DNA-binding domain (e.g., dCas9 or nCas9). The terms "nucleobase-modifying enzyme" and "nucleobase-modifying domain," used interchangeably herein, refer to an enzyme that can modify a nucleobase and convert one nucleobase to another (e.g., a deaminase such as cytidine deaminase or adenosine deaminase). The nucleobase-modifying enzyme of the nucleobase editor may target a cytosine (C) base in a nucleic acid sequence and convert the C to a thymine (T) base. In some embodiments, editing C to T is performed by a deaminase, e.g., cytidine deaminase. In some embodiments, editing A to G is performed by a deaminase, e.g., adenosine deaminase. Nucleobase editors that can perform other types of base conversions (for example, C to G) are also contemplated.

[0047] A "split nucleobase editor" refers to a nucleobase editor that is provided as an N-terminal portion (also referred to as an N-terminal half) and a C-terminal portion (also referred to as a C-terminal half) encoded by two separate nucleic acids. Polypeptides corresponding to the N-terminal and C-terminal portions of a nucleobase editor may be combined to form a complete nucleobase editor. In some embodiments, for nucleobase editors comprising dCas9 or nCas9, "split" refers to the dCas9 or nCas9 domain being positioned as described herein in the split Cas9. Thus, in some embodiments, the N-terminal portion of the nucleobase editor contains the N-terminal portion of the split Cas9, and the C-terminal portion of the nucleobase editor contains the C-terminal portion of the split Cas9. Similarly, an intein-N or intein-C may be fused to the N-terminal or C-terminal portion of a nucleobase editor, respectively, to join the N-terminal and C-terminal portions of the nucleobase editor to form a complete nucleobase editor.

[0048] In some embodiments, the nucleobase editor converts C to T. In some embodiments, the nucleobase editor comprises a cytosine deaminase. "Cytosine deaminase" or "cytidine deaminase" refers to an enzyme that catalyzes the chemical reaction "cytosine + HO → uracil + NH" or "5-methyl-cytosine + HO → thymine + NH." As may be apparent from the reaction equation, such a chemical reaction results in a nucleobase change from C to U / T. In the context of a gene, such a nucleotide change or mutation may result in an amino acid change in the protein that may affect the function of the protein, for example, a loss-of-function or gain-of-function. In some embodiments, the C to T nucleobase editor comprises dCas9 or nCas9 fused to a cytidine deaminase. In some embodiments, the cytidine deaminase domain is fused to the N-terminus of dCas9 or nCas9. In some embodiments, the nucleobase editor further comprises a domain that inhibits uracil glycosylase and / or a nuclear localization signal.Such nucleobase editors have been described in the art, by way of example only, in Rees & Liu, Nat Rev Genet. 2018;19(12):770-788 and Koblan et al., Nat Biotechnol. 2018;36(9):843-846; and in U.S. Patent Publication No. 2018 / 0073012, published March 15, 2018 (issued October 30, 2018 as U.S. Patent No. 10,113,163); U.S. Patent Publication No. 2017 / 0121693, published May 4, 2017 (issued January 1, 2019 as U.S. Patent No. 10,167,457); PCT Publication No. WO2017 / 070633 published on June 27, 2015; U.S. Patent Publication No. 2015 / 0166980 published on June 18, 2015; U.S. Patent No. 9,840,699 published on December 12, 2017; U.S. Patent No. 10,077,453 published on September 18, 2018; PCT Publication No. WO2019 / 023680 published on January 31, 2019; PCT Publication No. WO PCT Application No. 2018 / 0176009, filed March 23, 2019; PCT Application No. PCT / US2019 / 033848, filed August 23, 2019; PCT Application No. PCT / US2019 / 47996, filed September 5, 2019; PCT Application No. PCT / US2019 / 049793, filed April 17, 2020; No. 028568; PCT Application No. PCT / US2019 / 61685, filed November 15, 2019; PCT Application No. PCT / US2019 / 57956, filed October 24, 2019; and PCT Application No. PCT / US2019 / 58678, filed October 29, 2019, the contents of each of which are incorporated herein by reference.

[0049] In some embodiments, the nucleobase editor converts A to G. In some embodiments, the nucleobase editor comprises an adenosine deaminase. "Adenosine deaminase" is an enzyme involved in purine metabolism. It is required for the breakdown of adenosine from food and the turnover of nucleic acids in tissues. Its primary function in humans is the development and maintenance of the immune system. Adenosine deaminase catalyzes the hydrolytic deamination of adenosine in the context of DNA (forming inosine, which base pairs as G). No naturally occurring adenosine deaminase is known to act on DNA. Instead, known adenosine deaminase enzymes act only on RNA (tRNA or mRNA). Evolved deoxyadenosine deaminase enzymes that accept a DNA substrate and deaminate dA to deoxyinosine are described, by way of example, in PCT Application No. PCT / US2017 / 045381, filed August 3, 2017 (published as International Publication No. WO2018 / 027078), PCT Application No. PCT / US2019 / 033848 (published as International Publication No. WO2019 / 226953), PCT Application No. PCT / US2019 / 033848 (filed May 23, 2019), and PCT Patent Application No. PCT / US2020 / 028568 (filed April 17, 2020), each of which is incorporated herein by reference.

[0050] Exemplary adenosine and cytidine nucleobase editors are also described in Rees & Liu, Base editing: precision chemistry on the genome and transcriptome of living cells, Nat. Rev. Genet. 2018;19(12):770-788; and U.S. Patent Publication No. 2018 / 0073012, published March 15, 2018 (issued October 30, 2018 as U.S. Patent No. 10,113,163); U.S. Patent Publication No. 2017 / 0121693, published May 4, 2017 (issued January 1, 2019 as U.S. Patent No. 10,167,457); PCT Publication No. WO No. 2017 / 070633 (published April 27, 2017); U.S. Patent Publication No. 2015 / 0166980 (published June 18, 2015); U.S. Patent No. 9,840,699 (issued December 12, 2017), and U.S. Patent No. 10,077,453 (issued September 18, 2018), the contents of each of which are incorporated herein by reference in their entirety.

[0051] cytosine deaminase As used herein, "cytosine deaminase" encoded by the CDA gene is an enzyme that catalyzes the removal of amine groups from cytidine (i.e., the base cytosine when attached to a ribose ring) to uridine (C to U) and from deoxycytidine to deoxyuridine (C to U). A non-limiting example of a cytosine deaminase is APOBEC1 ("apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1"). Another example is AID ("activation-induced cytosine deaminase"). Under standard Watson-Crick hydrogen bond pairing, the hydrogen of the cytosine base bonds to the guanine base. When cytidine is converted to uridine (or deoxycytidine is converted to deoxyuridine), the uridine (or the uracil base of uridine) hydrogen bonds with the base adenine. Thus, the conversion of "C" to uridine ("U") by cytosine deaminase results in the insertion of "A" instead of "G" during cellular repair and / or replication processes. Because adenine "A" pairs with thymine "T," cytosine deaminase, in concert with DNA replication, causes the conversion of C·G pairings to T·A pairings in double-stranded DNA molecules.

[0052] Cas9 The term "Cas9" or "Cas9 nuclease" refers to an RNA-guided nuclease comprising a Cas9 domain or a fragment thereof (e.g., a protein comprising an active or inactive DNA cleavage domain of Cas9 and / or a gRNA-binding domain of Cas9). A "Cas9 domain," as used herein, is a protein fragment comprising an active or inactive cleavage domain of Cas9 and / or a gRNA-binding domain of Cas9. A "Cas9 protein" is a full-length Cas9 protein. Cas9 nuclease is also sometimes referred to as casn1 nuclease or CRISPR (clustered regularly interspaced short palindromic repeats (CRISPR)). Clustered R egularly I nterspaced S hort P alindromic RCRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain a spacer, a sequence complementary to the preceding mobile element, and a targeting invading nucleic acid. The CRISPR cluster is transcribed and processed into CRISPR RNA (CRISPR RNA: crRNA). In type II CRISPR systems, corrective processing of the pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and a Cas9 domain. The tracrRNA acts as a guide for ribonuclease 3-assisted processing of the pre-crRNA. Subsequently, the Cas9 / crRNA / tracrRNA endonucleolytically cleaves linear or circular dsDNA targets complementary to the spacer. The target strand that is not complementary to the crRNA is first cut by an endonuclease, and then the 3'-5' is trimmed by an exonuclease. In nature, DNA binding and cleavage typically require both proteins and RNAs. However, a single guide RNA ("sgRNA," or simply "gRNA") can be engineered to incorporate both crRNA and tracrRNA aspects into a single RNA species. For example, see Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), the contents of which are incorporated herein by reference. Cas9 recognizes a short motif in the CRISPR repeat sequence (PAM or protospacer adjacent motif) to help distinguish self from non-self.Cas9 nuclease sequences and structures are well known to those skilled in the art (for example, “Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti et al., JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C., Sezate S., Suvorov AN, Kenton S., Lai HS, Lin SP, Qian Y.,Jia HG,Najar FZ,Ren Q.,Zhu H.,Song L.,White J.,Yuan X.,Clifton SW,Roe BA,McLaughlin RE,Proc.Natl.Acad.Sci.USA98:4658-4663(2001);“CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.”Deltcheva E., Chylinski K., Sharma See CM, Gonzales K., Chao Y., Pirzada ZA, Eckert MR, Vogel J., Charpentier E., Nature 471:602-607 (2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), the entire contents of each of which are incorporated herein by reference. Cas9 orthologs have been described in a variety of species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on the present disclosure.Such Cas9 nucleases and sequences also include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737, the entire contents of which are incorporated herein by reference. In some embodiments, the Cas9 nuclease comprises one or more mutations that partially impair or inactivate the DNA cleavage domain.

[0053] A Cas9 domain with inactivated nucleases may be interchangeably referred to as a "dCas9" protein (representing a nuclease-dead Cas9). Methods for producing a Cas9 domain (or a fragment thereof) with an inactive DNA cleavage domain are known (see, for example, Jinek et al., Science. 337:816-821 (2012); Qi et al., "Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression" (2013) Cell. 28;152(5):1173-83, the entire contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to contain two subdomains: an HNH nuclease subdomain and a RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, while the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al., Science. 337:816-821 (2012); Qi et al., Cell. 28; 152(5):1173-83 (2013)). In some embodiments, proteins comprising fragments of Cas9 are provided. For example, in some embodiments, the protein comprises one of two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or fragments thereof are referred to as "Cas9 variants." Cas9 variants share homology with Cas9 or fragments thereof.For example, a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, at least about 99.8% identical, or at least about 99.9% identical to a wild-type Cas9 (e.g., SpCas9 of SEQ ID NO: 13). In some embodiments, the Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more amino acid changes compared to wild-type Cas9 (e.g., SpCas9 of SEQ ID NO: 13). In some embodiments, the Cas9 variant comprises a fragment of Cas9 of SEQ ID NO: 13 (e.g., the gRNA binding domain or the DNA cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild-type Cas9 (e.g., SpCas9 of SEQ ID NO: 13). In some embodiments, the fragment is 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% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild-type Cas9 (e.g., SpCas9 of SEQ ID NO: 13).

[0054] CRISPR CRISPR is a family of DNA sequences (i.e., CRISPR clusters) in bacteria and archaea that represent snippets of preceding infection by viruses that have invaded prokaryotes. These snippets of DNA are used by prokaryotic cells to detect and destroy DNA from subsequent attacks by similar viruses, and together with an array of CRISPR-associated proteins (including Cas9 and its homologs) and CRISPR-associated RNAs, effectively comprise the prokaryotic immune defense system. In nature, CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In certain types of CRISPR systems (e.g., type II CRISPR systems), correct processing of the pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and the Cas9 protein. The tracrRNA acts as a guide for ribonuclease 3-assisted processing of the pre-crRNA. Cas9 / crRNA / tracrRNA then endonuclease-cleaves the linear or circular dsDNA target complementary to the RNA. Specifically, the target strand that is not complementary to the crRNA is first cut by an endonuclease, and then the 3'-5' is trimmed by an exonuclease. In nature, DNA binding and cleavage typically require a protein and both RNAs. However, a single guide RNA ("sgRNA" or simply "gRNA") can be engineered to incorporate aspects of both the crRNA and tracrRNA into the guide RNA of a single RNA species. For example, see Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Cas9 helps distinguish self from non-self by recognizing a short motif in the CRISPR repeat sequence (PAM or protospacer adjacent motif).CRISPR biology and Cas9 nuclease sequences and structures are well known to those skilled in the art (for example, “Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti et al., JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C., Sezate S., Suvorov AN, Kenton S., Lai HS,Lin SP,Qian Y.,Jia HG,Najar FZ,Ren Q.,Zhu H.,Song L.,White J.,Yuan X.,Clifton SW,Roe BA,McLaughlin RE,Proc.Natl.Acad.Sci.USA98:4658-4663(2001);“CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.”Deltcheva E.,Chylinski See K., Sharma CM, Gonzales K., Chao Y., Pirzada ZA, Eckert MR, Vogel J., Charpentier E., Nature 471:602-607 (2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), the entire contents of each of which are incorporated herein by reference. Cas9 orthologs have been described in a variety of species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on the present disclosure.Such Cas9 nucleases and sequences also include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737, the entire contents of which are incorporated herein by reference.

[0055] In certain types of CRISPR systems (e.g., type II CRISPR systems), correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and Cas9 protein. tracrRNA acts as a guide for ribonuclease 3-assisted processing of pre-crRNA. Cas9 / crRNA / tracrRNA then endonucleolytically cleaves linear or circular nucleic acid targets complementary to the RNA. Specifically, target strands not complementary to the crRNA are first cut by the endonuclease and then trimmed by a 3'-5 exonuclease. In nature, DNA binding and cleavage typically require both proteins and RNAs. However, single guide RNAs ("sgRNAs" or simply "gRNAs") can be engineered to incorporate aspects of both crRNA and tracrRNA into the guide RNA of a single RNA species.

[0056] In general, a "CRISPR system" collectively refers to the transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (transactivating CRISPR) sequences (e.g., tracrRNA or an active partial tracrRNA), tracr mate sequences (which, in the context of endogenous CRISPR systems, encompass "direct repeats" and partial direct repeats processed by the tracrRNA), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), or other sequences and transcripts from the CRISPR locus. The tracrRNA of the system is complementary (fully or partially) to the tracr mate sequence present on the guide RNA.

[0057] Deaminase The term "deaminase" or "deaminase domain" refers to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase is an adenosine (or adenine) deaminase, which catalyzes the hydrolytic deamination of adenine or adenosine. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine to inosine in deoxyribonucleic acid (DNA). In other embodiments, the deaminase is a cytidine (or cytosine) deaminase, which catalyzes the hydrolytic deamination of cytidine or cytosine.

[0058] The deaminase provided herein can be derived from any organism, such as bacteria.In some embodiments, deaminase or deaminase domain is the variant of the naturally occurring deaminase from an organism.In some embodiments, deaminase domain or deaminase domain does not occur in nature.For example, in some embodiments, deaminase or deaminase domain is 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 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to naturally occurring deaminase.

[0059] fusion proteins The term "fusion protein," as used herein, refers to a hybrid polypeptide containing protein domains derived from at least two different proteins. A protein may be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy-terminal (C-terminal) portion of the protein, thus forming an "amino-terminal fusion protein" or a "carboxy-terminal fusion protein," respectively. The protein may contain different domains, such as a nucleic acid-binding domain (e.g., the gRNA-binding domain of Cas9, which guides the protein to bind to a target site) and a nucleic acid-cleavage domain or catalytic domain of a nucleic acid-editing protein. Another example includes fusion of Cas9 or its equivalent to a deaminase. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced through recombinant protein expression and purification, which is particularly suitable for fusion proteins containing peptide linkers. Methods for recombinant protein expression and purification are well known and are described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4), the entire contents of which are incorporated herein by reference. thed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)).

[0060] group-specific antigen (gag) Without being limited by theory, in the context of a typical enveloped virus life cycle, Gag is the primary structural protein responsible for orchestrating most steps in virus assembly, including budding from a fully formed enveloped virion with (i) an envelope (comprising a lipid membrane formed from the plasma membrane during budding and one or more glycoproteins inserted therein) and (ii) an inner protein shell, the capsid. Most of these assembly steps occur through interactions with three Gag subdomains—the matrix (MA), capsid (CA), and nucleocapsid (NC; Figure 1). These three regions have a low level of sequence conservation among different retrovirus genera, which belies the observed high level of structural conservation. Outside of these three domains, the Gag protein can vary widely. For example, HIV-1 Gag further encodes a C-terminal p6 protein and two spacer proteins, SP1 and SP2, that define the CA-NC and NC-p6 junctions, but HTLV-1 does not contain additional sequences outside of MA, CA, and NC (Oroszlan and Copeland, 1985; Henderson et al., 1992).

[0061] Gag is also referred to as a "viral structural protein." As used herein, the term "viral structural protein" refers to a viral protein that contributes to the overall structure of the capsid protein or protein core of a virus. The term "viral structural protein" further encompasses functional fragments or derivatives of such viral proteins that contribute to the structure of the capsid protein or protein core of a virus. An example of a viral structural protein is MMLV Gag. Viral membrane fusion proteins are not considered viral structural proteins. Typically, the viral structural proteins are located inside the viral core.

[0062] Group-specific antigen (gag) nucleocapsid protein The term "group-specific antigen nucleocapsid protein" or "gag nucleocapsid protein" refers to a protein that constitutes a core structural component of the inner shell of many viruses, including retroviruses. The gag nucleocapsid protein used in the BE-VLPs of the present disclosure may be MMLV gag nucleocapsid protein, FMLV gag nucleocapsid protein, or a nucleocapsid protein from any other virus that produces such a protein.

[0063] Group-specific antigen (gag) protease ( pro ) Polyprotein "Group-specific antigen (gag) protease (pro) polyprotein" or "gag-pro polyprotein" refers to the gag nucleocapsid protein further comprising a viral protease linked thereto. The Gag-pro polyprotein mediates the proteolytic cleavage of the gag and gag-pol polyproteins or nucleocapsid proteins during or shortly after release of the virion from the plasma membrane. In the BE-VLPs described herein, the protease of the gag-pro polyprotein serves to cleave the cleavable linker in the fusion protein to release the base editor after delivery of the BE-VLP to a target cell. In some embodiments, the gag-pro polyprotein is an MMLV gag-pro polyprotein or an FMLV gag-pro polyprotein.

[0064] Guide RNA ("gRNA") As used herein, the term "guide RNA" generally refers to a specific type of guide nucleic acid that, in association with Cas9, directs the Cas9 protein to a specific sequence in a DNA molecule (including complementarity with the protospacer sequence of the guide RNA). However, the term also encompasses equivalent guide nucleic acid molecules associated with Cas9 equivalents, homologs, orthologs, or paralogs, whether naturally occurring or not (e.g., engineered or recombinant), that otherwise program the Cas9 equivalent to localize to a specific target nucleotide sequence. Cas9 equivalents may also include other napDNAbps from any type of CRISPR system (e.g., type II, type V, type VI), including Cpf1 (type V CRISPR-Cas system), C2c1 (type V CRISPR-Cas system), C2c2 (type VI CRISPR-Cas system), and C2c3 (type V CRISPR-Cas system). Further Cas equivalents are described in Makarova et al., "C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector," Science 2016;353(6299), the contents of which are incorporated herein by reference. Exemplary sequences and structures of guide RNAs are provided herein.

[0065] The term "guide RNA" is most commonly associated with the Cas protein of CRISPR-Cas9 and refers to a specific type of guide nucleic acid that associates with Cas9 and directs the Cas9 protein to a specific sequence in a DNA molecule (including complementarity to the protospacer sequence of the guide RNA). Functionally, the guide RNA associates with Cas9 and directs (or programs) the Cas9 protein to a specific sequence in a DNA molecule that includes a sequence complementary to the protospacer sequence of the guide RNA. The gRNA is a component of the CRISPR / Cas system. Typically, the guide RNA comprises a fusion of a CRISPR-targeting RNA (crRNA) and a trans-activating crRNA (tracrRNA), providing both targeting specificity and anchoring / binding ability for the Cas9 nuclease. The "crRNA" is a bacterial RNA that confers target specificity and is required for the tracrRNA to bind to Cas9. The "tracrRNA" links the crRNA to the Cas9 nuclease and is typically a bacterial RNA that can bind to any crRNA. The sequence specificity of the Cas DNA-binding protein is determined by the gRNA, which has nucleotide base-pairing complementarity to the target DNA sequence. Native gRNAs contain a 20-nucleotide (nt) specificity-determining sequence (SDS) or spacer that specifies the targeted DNA sequence, followed immediately by an 80-nt scaffold sequence that associates the gRNA with Cas9. In some embodiments, the SDS of the present disclosure has a length of 15-100 nucleotides or more. For example, the SDS may have a length of 15-90, 15-85, 15-80, 15-75, 15-70, 15-65, 15-60, 15-55, 15-50, 15-45, 15-40, 15-35, 15-30, or 15-20 nucleotides. In some embodiments, the SDS is 20 nucleotides long. For example, the SDS can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. At least a portion of the target DNA sequence is complementary to the SDS of the gRNA.For Cas9 to successfully bind to a DNA target sequence, a region of the target sequence must be complementary to the SDS of the gRNA sequence and immediately followed by the correct protospacer adjacent motif (PAM) sequence (e.g., NGG for Cas9 and TTN, TTTN, or YTN for Cpf1). In some embodiments, the SDS is 100% complementary to its target sequence. In some embodiments, the SDS sequence is less than 100% complementary to its target sequence and is therefore considered partially complementary to its target sequence. For example, a targeting sequence may be 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% complementary to its target sequence. In some embodiments, the SDS of the template DNA or target DNA may differ from the complementary region of the gRNA by 1, 2, 3, 4, or 5 nucleotides.

[0066] In some embodiments, the guide RNA is about 15-120 nucleotides in length and comprises a sequence of at least 10 contiguous nucleotides complementary to the target sequence. , 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 nucleotides in length. In some embodiments, the guide RNA comprises a sequence of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more consecutive nucleotides that are complementary to the target sequence. Sequence complementarity refers to the differential interactions between adenine and thymine (DNA) or uracil (RNA), and between guanine and cytosine.

[0067] Linker The term "linker," as used herein, refers to a molecule that connects two other molecules or moieties. A linker can be an amino acid sequence in the case of a linker that joins two fusion proteins. For example, Cas9 can be fused to a deaminase (such as adenosine deaminase or cytosine deaminase) by an amino acid linker sequence. A linker can also be a nucleotide sequence in the case of joining two nucleotide sequences together (such as in a gRNA). In other embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5 to 200 amino acids in length, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated.

[0068] A "cleavable linker" refers to a linker that can be split or cut by any means. The linker can be an amino acid sequence. In some embodiments, the linker between the NES and napDNAbp of the BE-VLPs provided herein comprises a cleavable linker. The cleavable linker may include a self-cleaving peptide (e.g., a 2A peptide such as EGRGSLLTCGDVEENPGP (SEQ ID NO: 9), ATNFSLLKQAGDVEENPGP (SEQ ID NO: 10), QCTNYALLKLAGDVESNPGP (SEQ ID NO: 11), or VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 12)). In some embodiments, the cleavable linker comprises a protease cleavage site that is cleaved after contact with a protease. For example, the present disclosure contemplates the use of a cleavable linker comprising a protease cleavage site for the amino acid sequence TSTLL MENSS (SEQ ID NO: 1), PRSSLYPALTP (SEQ ID NO: 2), VQALVLTQ (SEQ ID NO: 3), PLQVLTLNIERR (SEQ ID NO: 4), or an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 1-4. In certain embodiments, the cleavable linker comprises an MMLV protease cleavage site for an FMLV protease cleavage site.

[0069] napDNAbp As used herein, the term "nucleic acid programmable DNA binding protein" or "napDNAbp" (of which Cas9 is an example) refers to a protein that uses RNA:DNA hybridization to target and bind to specific sequences in a DNA molecule. Each napDNAbp is associated with at least one guide nucleic acid (e.g., a guide RNA), which localizes the napDNAbp to a DNA sequence containing a DNA strand (i.e., a target strand) complementary to the guide nucleic acid or a portion thereof (e.g., a protospacer of the guide RNA). In other words, the guide nucleic acid "programs" the napDNAbp (e.g., Cas9 or an equivalent) to localize and bind to the complementary sequence.

[0070] Without being bound by theory, the binding mechanism of the napDNAbp-guide RNA complex generally involves the napDNAbp forming an R-loop that induces the unwinding of the double-stranded DNA target, thereby separating the strands in the region bound by the napDNAbp. The guide RNA protospacer then hybridizes to the "target strand." This displaces the "non-target strand" that is complementary to the target strand, forming the single-stranded region of the R-loop. In some embodiments, the napDNAbp contains one or more nuclease activities that then cut the DNA to eliminate various types of lesions. For example, the napDNAbp may contain nuclease activities that cut the non-target strand at a first location and / or cut the target strand at a second location. Depending on the nuclease activity, the target DNA can be cut to form a "double-stranded break" in which both strands are cut. In other embodiments, the target DNA can be cut at only a single site, i.e., the DNA is "nicked" on one strand. Exemplary napDNAbps with different nuclease activities include "Cas9 nickase" ("nCas9") and inactivated Cas9, which has no nuclease activity ("dead Cas9" or "dCas9"). Exemplary sequences for these and other napDNAbps are provided herein.

[0071] Nickase As used herein, "nickase" refers to a napDNAbp (e.g., a Cas protein) that is capable of cleaving only one of the two complementary strands of a double-stranded target DNA sequence, thereby generating a nick in that strand. In some embodiments, the nickase cleaves the non-target strand of a double-stranded target DNA sequence. In some embodiments, the nickase comprises an amino acid sequence with one or more mutations in the catalytic domain of a canonical napDNAbp (e.g., a Cas protein), wherein the one or more mutations reduce or eliminate the nuclease activity of the catalytic domain. In some embodiments, the nickase is a Cas9 that comprises one or more mutations in the RuvC-like domain relative to the wild-type Cas9 sequence or relative to the equivalent amino acid position in another Cas9 variant or Cas9 equivalent. In some embodiments, the nickase is a Cas9 that comprises one or more mutations in the HNH-like domain relative to the wild-type Cas9 sequence or relative to the equivalent amino acid position in another Cas9 variant or Cas9 equivalent. In some embodiments, the nickase is a Cas9 containing an aspartate to alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 relative to the canonical Cas9 sequence or to the equivalent amino acid position in other Cas9 variants or Cas9 equivalents. In some embodiments, the nickase is a Cas9 containing an H840A, N854A, and / or N863A mutation relative to the canonical Cas9 sequence or to the equivalent amino acid position in other Cas9 variants or Cas9 equivalents. In some embodiments, the term "Cas9 nickase" refers to a Cas9 in which one of the two nuclease domains has been inactivated. This enzyme is capable of cleaving only one strand of target DNA. In some embodiments, the nickase is a Cas protein that is not a Cas9 nickase.

[0072] Nuclear export sequence (NES) The term "nuclear export sequence" or "NES" refers to an amino acid sequence that promotes the transport of proteins from the cell nucleus to the cytoplasm, for example, through the nuclear pore complex by nuclear transport. Nuclear export sequences are known in the art and will be clear to those skilled in the art. For example, NES sequences are described in Xu, D. et al. Sequence and structural analyses of nuclear export signals in the NESdb database. Mol Biol. Cell. 2012, 23(18)3677-3693, the contents of which are incorporated herein by reference.

[0073] Nuclear localization sequence (NLS) The term "nuclear localization sequence" or "NLS" refers to an amino acid sequence that facilitates the import of proteins into the cell nucleus, for example, by nuclear transport. Nuclear localization sequences are known in the art and will be apparent to those skilled in the art. For example, NLS sequences are described in International PCT Application PCT / EP2000 / 011690 to Plank et al., filed November 23, 2000, and published May 31, 2001 as WO / 2001 / 038547, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In some embodiments, the NLS comprises the amino acid sequence PKKKRKV (SEQ ID NO: 204).

[0074] nucleic acid molecule The term "nucleic acid" as used herein refers to a polymer of nucleotides. The polymer may be composed of natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C5 bromouridine, C5 fluorouridine, C5 iodouridine, C5 propynyluridine, C5 propynylcytidine, C5 methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)methylguanine, 4-acetylcytidine, The bases may include 5-(carboxyhydroxymethyl)uridine, dihydrouridine, methylpseudouridine, 1-methyladenosine, 1-methylguanosine, N6-methyladenosine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, 2'-O-methylcytidine, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioate and 5'-N phosphoramidite linkages).

[0075] Protease cleavage site The term "protease cleavage site," as used herein, refers to an amino acid sequence that is recognized and cleaved by a protease, i.e., an enzyme that catalyzes proteolysis and breaks down proteins into smaller polypeptides or single amino acids. In some embodiments, the protease cleavage site is included in a cleavable linker in a fusion protein as described herein. In certain embodiments, the protease cleavage site is cleaved by a protease of the gag-pro polyprotein. In some embodiments, the protease cleavage site comprises an MMLV protease cleavage site or an FMLV protease cleavage site. In certain embodiments, the protease cleavage site comprises one of the amino acid sequences TSTLLMEANSS (SEQ ID NO: 1), PRSSLYPALTP (SEQ ID NO: 2), VQALVLTQ (SEQ ID NO: 3), PLQVLTLNIERR (SEQ ID NO: 4), or an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 1-4. In some embodiments, the protease cleavage site comprises the amino acid sequence of any one of SEQ ID NOs: 1-8 or 499-510, or an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 1-8 or 499-510.

[0076] Proteins, peptides, and polypeptides The terms "protein," "peptide," and "polypeptide" are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to proteins, peptides, or polypeptides of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids in length. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified by the addition of a chemical entity, such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification. A protein, peptide, or polypeptide may also be a single molecule or a multimolecular complex. A protein, peptide, or polypeptide may be merely a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, synthetic, or any combination thereof. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced through recombinant protein expression and purification, which is particularly suitable for fusion proteins containing peptide linkers.Methods for recombinant protein expression and purification are well known and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)), the contents of which are incorporated herein by reference.

[0077] subject The term "subject," as used herein, refers to an individual organism, e.g., an individual mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, goat, cow, cat, or dog. In some embodiments, the subject is a vertebrate, amphibian, reptile, fish, insect, fly, or nematode. In some embodiments, the subject is a research animal. In some embodiments, the subject is genetically engineered, e.g., a genetically engineered non-human subject. The subject may be of either gender and at any stage of development.

[0078] treatment The terms "treatment," "treat," and "treating" refer to a clinical intervention intended to halt, alleviate, delay the onset of, or inhibit the progression of, a disease or disorder, as described herein, or one or more symptoms thereof. As used herein, the terms "treatment," "treat," and "treating" refer to a clinical intervention intended to halt, alleviate, delay the onset of, or inhibit the progression of, a disease or disorder, as described herein, or one or more symptoms thereof. In some embodiments, treatment may be administered after the onset of one or more symptoms and / or after a disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, e.g., to prevent or delay the onset of symptoms or inhibit the onset or progression of a disease. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence.

[0079] variant As used herein, the term "variant" should be interpreted as meaning something that exhibits qualities with a pattern that deviates from those found in nature. As an example, a variant Cas9 is a Cas9 that contains one or more changes in amino acid residues compared to the wild-type Cas9 amino acid sequence. The term "variant" encompasses homologous proteins that have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with a reference sequence and have the same or substantially the same functional activity(ies) as the reference sequence. The term also encompasses mutants, truncations, or domains of a reference sequence that exhibit the same or substantially the same functional activity(ies) as the reference sequence.

[0080] vector The term "vector," as used herein, refers to a nucleic acid that can be modified to encode a gene of interest, enter a host cell, mutate, or replicate within the host cell, and then transfer the replicative form of the vector to another host cell. Exemplary suitable vectors include viral vectors, such as retroviral vectors or bacteriophages and filamentous phages, and conjugative plasmids. Additional suitable vectors will be apparent to those skilled in the art based on the present disclosure. viral envelope glycoproteins The term "viral envelope glycoprotein" refers to a part of the viral envelope, i.e., an oligosaccharide-containing protein that forms the outermost layer of many types of viruses that protects viral genetic material as they travel between host cells. The glycoprotein may assist in identifying and binding to a receptor on the target cell membrane so that the viral envelope fuses with the membrane, allowing the contents of the viral particle (which may, by way of example, include a BE-VLP as described herein) to enter the host cell. This property may also be referred to as "tropism." The viral envelope glycoprotein used in the BE-VLPs (or, alternatively, eVLPs) of the present disclosure may include any glycoprotein from an enveloped virus. In some embodiments, the viral envelope glycoprotein is an adenovirus envelope glycoprotein, an adeno-associated virus envelope glycoprotein, a retrovirus envelope glycoprotein, or a lentivirus envelope glycoprotein. In certain embodiments, the viral envelope glycoprotein is vesicular stomatitis virus G protein (VSV-G), baboon retroviral envelope glycoprotein (BaEVRless), FuG-B2 envelope glycoprotein, HIV-1 envelope glycoprotein, or ecotropic murine leukemia virus (MLV) envelope glycoprotein.

[0081] Virus-like particles (VLPs) As used herein, a virus-like particle consists of (a) a supramolecular assembly comprising (i) a lipid membrane (e.g., a monolayer or bilayer) and (ii) an envelope comprising viral envelope glycoproteins; and (b) a multiprotein core region comprising (ii) a Gag protein, (ii) a first fusion protein comprising a Gag protein and Pro-Pol, and (iii) a second fusion protein comprising a Gag protein fused to a cargo protein via a protease-cleavable linker. In various embodiments, the cargo protein is a napDNAbp (e.g., Cas9). In other embodiments, the cargo protein is a base editor. In various other embodiments, the multiprotein core region of the VLP further comprises one or more guide RNA molecules complexed with the napDNAbp or base editor to form a ribonucleoprotein (RNP). In various embodiments, the VLP is prepared in a production cell transiently transformed with plasmid DNA encoding the various protein and nucleic acid (sgRNA) components of the VLP. The components self-assemble at the cell membrane and bud according to the natural mechanism of retroviral budding to release a fully mature VLP from the cell. Once formed, Pol-Pro cleaves the protease-sensitive linker joining the Gag-cargo linker (e.g., the linker joining Gag to the BE RNP or napDNAbp RNP), releasing the BE RNP and / or napDNAbp RNA, which may be present within the VLP. When the VLP is administered to a recipient cell and taken up by the cell, the contents of the VLP, including free BE RNP and / or napDNAbp RNA, are released. Once inside the cell, the RNP may translocate to nucleases of the cell (particularly where the NLS is included on the RNP), where DNA editing may occur at the target site specified by the guide RNA. Various embodiments include one or more improvements.

[0082] In one embodiment, the protease-cleavable linker is optimized to improve cleavage efficiency after VLP maturation, as demonstrated herein for v.2 VLPs (or "second generation" VLPs).

[0083] In another embodiment, the Gag-cargo fusion (for example, Gag::BE) further comprises one or more nuclear export signals at one or more locations along the length of the fusion polypeptide protein, which may be joined by a cleavable linker, such that during VLP assembly in the producer cell, the Gag-cargo fusion (due to the presence of a competing NLS signal) does not accumulate in the nucleus of the producer cell but is instead available in the cytoplasm to undergo the VLP assembly process at the cell membrane. Once inside the mature VLP after release from the producer cell, the NES may be cleaved by Pro-Pol, thereby separating the cargo (for example, napDNAbp or BE) from the NES. Thus, upon delivery to the recipient cell, the cargo (for example, napDNAbp or BE, typically flanked by one or more NLS elements) no longer contains the NES element, which would otherwise prevent the transport of the cargo to nucleases and disrupt gene editing activity. This is exemplified as the v.3 VLP (or "third generation" VLP) described herein.

[0084] In another embodiment, as demonstrated by the v.4 VLPs (or "fourth generation" VLPs) described herein, the inventors have found an optimized stoichiometric ratio of gag-cargo fusion to Gag-Pro-Pol fusion protein that balances the amount of gag-cargo available for packaging into VLPs with the amount of retroviral protease (the "Pro" in the Gag-Pro-Pol fusion) required for VLP maturation. In one embodiment, the optimized ratio of Gag-cargo fusion to Gag-Pro-Pol fusion protein is achieved by the appropriate ratio of plasmids encoding each component transiently delivered into production cells. In one embodiment, to adjust the stoichiometry of Gag-cargo fusion to Gag-Pro-Pol fusion, the ratio of Gag-cargo-encoding plasmid (e.g., Gag-3xNES-ABE8e) to wild-type MMLV gag-pro-pol plasmid transfected for VLP production was varied. We found that increasing the amount of gag-cargo plasmid beyond the original ratio (38% gag-cargo plasmid and 62% gag-pro-pol plasmid) used to produce v3.4 BE-eVLPs did not improve editing efficiency (Figure 2G). Reducing the gag-cargo plasmid ratio from 38% to 25% slightly improved editing efficiency (Figure 2G). However, further reducing the gag-cargo plasmid ratio below 25% reduced editing efficiency (Figure 2G). These results are consistent with a model in which the optimal gag-cargo:gag-pro-pol stoichiometry balances the amount of gag-cargo available for packaging into VLPs with the amount of MMLV protease (the "pro" in gag-pro-pol) required for VLP maturation. In one embodiment, the results of this final round of optimization revealed a fourth-generation (v4) BE-eVLP formulation (Figure 2G) that combined the optimal gag-BE:gag-pro-pol stoichiometry (gag-BE 25%) with the v3.4BE-eVLP architecture.

[0085] In some embodiments, the VLP comprises an additional agent for targeting the VLP for delivery to a specific cell type. For example, such an additional targeting agent may be incorporated into the outer lipid membrane encapsulation layer of the VLP. In some embodiments, the additional targeting agent is a protein. In certain embodiments, the additional targeting agent is an antibody.

[0086] Thus, as used herein, a virus-derived particle includes a virus-like particle formed by one or more virus-derived protein(s), which substantially lacks the viral genome such that the VLP is replication-incompetent when delivered to a recipient cell.

[0087] Wild type As used herein, the term "wild-type" is a term of art understood by those skilled in the art and means the typical form of an organism, strain, gene, or characteristic as occurring in nature, as distinguished from mutant or variant forms.

[0088] Detailed Description The present disclosure is based on the development and application of an engineered VLP (eVLP) platform for packaging and delivering ribonucleoprotein cargoes, such as napDNAbp-guide RNA cargoes or base editor guide RNA cargoes, in vitro and / or in vivo. In embodiments delivering base editor guide RNA ribonucleoprotein cargoes, the eVLP may be referred to as a base editor virus-like protein (BE-VLP). In various embodiments, optimized BE-VLPs enable highly efficient base editing with minimal off-target editing in a variety of cell types. In particular, the BE-VLPs described herein are based on the surprising discovery that both a nuclear export sequence (NES) and a nuclear localization sequence (NLS) may be incorporated into the same fusion protein to facilitate transport of the fusion protein to different parts of the cell during production and delivery. The BE-VLPs described in the present invention are produced in virus-producing cells and exported from the nucleus due to the presence of one or more NES sequences in the fusion protein inside the BE-VLP. After delivery to the target cell, when the BE is released from the VLP, the NES is cleaved from the fusion protein, allowing the BE (containing one or more NLS sequences) to enter the nucleus of the target cell and edit the genome. This disclosure also describes optimizing the protease cleavage site that separates the NES and VLP protein from the rest of the base editor to facilitate highly efficient cleavage and delivery of the BE. Finally, this disclosure also describes optimizing the ratios of the various components of the BE-VLP to ensure highly efficient production of the BE-VLP.

[0089] Thus, the present disclosure provides virus-like particles for delivering base editor fusion proteins (BE-VLPs), and systems comprising such BE-VLPs. The present disclosure also provides polynucleotides encoding the BE-VLPs described herein, which may be useful for producing the VLPs. Also provided herein are methods for editing the genome of a target cell by introducing the BE-VLPs described in the present invention into the target cell. The present disclosure also provides fusion proteins that constitute components of the BE-VLPs described herein, as well as polynucleotides, vectors, cells, and kits.

[0090] eVLP In various embodiments, an eVLP (e.g., a BE-VLP) comprises a supramolecular assembly including: (a) an envelope comprising (i) a lipid membrane (e.g., a monolayer or bilayer membrane) and (ii) a viral envelope glycoprotein; and (b) a multiprotein core region surrounded by the envelope, the multiprotein core region comprising: (i) a Gag protein, (ii) a Gag-Pro-Pol protein, and (iii) a Gag-cargo fusion protein comprising the Gag protein fused to a cargo protein (e.g., a napDNAbp or a BE) via a cleavable linker (e.g., a protease-cleavable linker). In various embodiments, the cargo protein is a napDNAbp (e.g., Cas9). In other embodiments, the cargo protein is a base editor. In various other embodiments, the multiprotein core region of the VLP further comprises one or more guide RNA molecules complexed with the napDNAbp or base editor to form a ribonucleoprotein (RNP). In various embodiments, VLPs are prepared in production cells transiently transformed with plasmid DNA encoding the various protein and nucleic acid (sgRNA) components of the VLP. Without being bound by theory, the components self-assemble at the cell membrane and bud according to a naturally occurring budding mechanism (e.g., retroviral budding or other enveloped viral budding mechanisms) to release a fully mature VLP from the cell. Once formed, Gag-Pol-Pro cleaves the protease-sensitive linker of the gag-cargo (i.e., [Gag]-[cleavable linker]-[cargo], where the cargo can be BE-RNP or napDNAbp RNP), thereby releasing BE-RNP and / or napDNAbp RNA, as the case may be, within the VLP. Thus, in various embodiments, the present disclosure also provides VLPs in which the napDNAbp or base editor has been cleaved from the gag protein and released within the VLP.For example, the present disclosure provides a VLP comprising a group-specific antigen (gag) protease (pro) polyprotein, a nucleic acid programmable DNA binding protein (napDNAbp), and a fusion protein comprising a gag nucleocapsid protein and a nuclear export sequence (NES), encapsulated by a lipid membrane and a viral envelope glycoprotein. In some embodiments, the present disclosure provides a VLP comprising a mixture of cleavage and uncleavage products (i.e., a mixture of napDNAbp that have been cleaved from the gag protein and that have not yet been cleaved from the gag protein). In some embodiments, the napDNAbp is fused to one or more additional domains, such as, for example, one or more NLSs and / or deaminases (e.g., to form a base editor).

[0091] When the VLP is administered to and taken up by a recipient cell, the contents of the VLP are released, for example, released BE RNPs and / or napDNAbp RNPs. Once inside the cell, the RNPs may translocate to nucleases of the cell (particularly where the NLS is contained on the RNP), where DNA editing may occur at the target site specified by the guide RNA. Various embodiments include one or more improvements.

[0092] In one embodiment, the protease-cleavable linker is optimized to improve cleavage efficiency after VLP maturation, as demonstrated herein for v.2 VLPs (or "second generation" VLPs).

[0093] In another embodiment, the Gag-cargo fusion (e.g., Gag::BE) further comprises one or more nuclear export signals at one or more locations along the length of the fusion polypeptide protein, which may be joined by a cleavable linker, such that during VLP assembly in the producer cell, the Gag-cargo fusion (due to the presence of a competing NLS signal) does not accumulate in the nucleus of the producer cell but is instead available in the cytoplasm to undergo the VLP assembly process at the cell membrane. Once inside the mature VLP after release from the producer cell, the NES may be cleaved by Gag-Pro-Pol, thereby separating the cargo (e.g., napDNAbp or BE) from the NES. Thus, upon delivery to the recipient cell, the cargo (e.g., napDNAbp or BE, typically flanked by one or more NLS elements) no longer contains the NES element, which would otherwise prevent the cargo from being transported to nucleases and disrupt gene editing activity. This is exemplified as the v.3 VLP (or "third generation" VLP) described herein.

[0094] In another embodiment, as demonstrated by the v.4 VLPs (or "fourth generation" VLPs) described herein, the inventors have found an optimized stoichiometric ratio of gag-cargo fusion to Gag-Pro-Pol fusion protein that balances the amount of gag-cargo available for packaging into VLPs with the amount of retroviral protease (the "Pro" in the Gag-Pro-Pol fusion) required for VLP maturation. In one embodiment, the optimized ratio of Gag-cargo fusion to Gag-Pro-Pol fusion protein is achieved by the appropriate ratio of plasmids encoding each component transiently delivered into production cells. In one embodiment, to adjust the stoichiometry of Gag-cargo fusion to Gag-Pro-Pol fusion, the ratio of Gag-cargo-encoding plasmid (e.g., Gag-3xNES-ABE8e) to wild-type MMLV gag-pro-pol plasmid transfected for VLP production was varied. We found that increasing the amount of gag-cargo plasmid beyond the original ratio (38% gag-cargo plasmid and 62% gag-pro-pol plasmid) used to produce v3.4 BE-eVLPs did not improve editing efficiency (Figure 2G). Reducing the gag-cargo plasmid ratio from 38% to 25% slightly improved editing efficiency (Figure 2G). However, further reducing the gag-cargo plasmid ratio below 25% reduced editing efficiency (Figure 2G). These results are consistent with a model in which the optimal gag-cargo:gag-pro-pol stoichiometry balances the amount of gag-cargo available for packaging into VLPs with the amount of MMLV protease (the "pro" in gag-pro-pol) required for VLP maturation. In one embodiment, the results of this final round of optimization revealed a fourth-generation (v4) BE-eVLP formulation (Figure 2G) that combined the optimal gag-BE:gag-pro-pol stoichiometry (gag-BE 25%) with the v3.4BE-eVLP architecture.In some embodiments, the ratio of gag-pro-polyprotein to gag-cargo is approximately 10:1, approximately 9:1, approximately 8:1, approximately 7:1, approximately 6:1, approximately 5:1, approximately 4:1, approximately 3:1, approximately 2:1, approximately 1.5:1, approximately 1:1, or approximately 0.5:1.

[0095] Thus, in one aspect, the present disclosure provides eVLPs comprising (a) an envelope and (b) a multiprotein core, wherein the envelope comprises a lipid membrane (e.g., a lipid monolayer or bilayer) and viral envelope glycoproteins, and the multiprotein core comprises a fusion protein comprising Gag (e.g., a retroviral Gag), a group-specific antigen (gag) protease (pro) polyprotein (i.e., "Gag-Pro-Pol"), and a Gag-cargo (e.g., Gag-napDNAbp or Gag-BE). In various embodiments, the Gag-cargo may comprise a ribonucleoprotein cargo, e.g., a napDNAbp or BE complexed with a guide RNA. In yet further embodiments, the Gag-cargo (e.g., Gag fused to a napDNAbp or BE) may comprise one or more NLS sequences and / or one or more NES sequences to regulate the cellular location of the cargo within the cell. The NLS sequence will facilitate the transport of the cargo to cellular nucleases to facilitate editing. The NES does the opposite, i.e., transports the cargo from the nucleus and / or prevents the transport of the cargo to the nucleus. In certain embodiments, the NES may be coupled to the fusion protein by a cleavable linker (e.g., a protease linker), so that during assembly in the production cell, the NES signal acts to retain the cargo in the cytoplasm and make it available for the packaging process. However, when the mature VLP is budded or released from the production cell in its mature form, the cleavable linker attached to the NES may be cleaved, thereby removing the association between the NES and the cargo. Thus, without the NES, the cargo translocates to the nuclease containing its NLS sequence, thereby facilitating editing. Various napDNAbps may be used in the system of the present disclosure. In some embodiments, the napDNAbp is a Cas9 protein (by way of example, a Cas9 nickase, a dead Cas9 (dCas9), or another Cas9 variant described herein). In some embodiments, the Cas9 protein is bound to a guide RNA (gRNA).The fusion protein may further comprise other protein domains, such as an effector domain. In some embodiments, the fusion protein further comprises a deaminase domain (by way of example, an adenosine deaminase domain or a cytosine deaminase domain). In certain embodiments, the fusion protein comprises a base editor such as ABE8e, or any of the other base editors described herein or known in the art.

[0096] In some embodiments, the fusion protein comprises two or more NESs (for example, two, three, four, five, six, seven, eight, nine, or ten or more NESs). In certain embodiments, the fusion protein further comprises a nuclear localization sequence (NLS), or two or more NLSs (for example, two, three, four, five, six, seven, eight, nine, or ten or more NLSs). In certain embodiments, the fusion protein may comprise at least one NES and one NLS.

[0097] The Gag-cargo fusion proteins described herein comprise one or more cleavable linkers. In one embodiment, the Gag-cargo fusion protein comprises a cleavable linker joining Gag to the cargo such that, once the Gag-cargo fusion is packaged into a mature VLP (which also contains Gag-Pro-Pol), protease activity can cleave the Gag-cargo cleavable linker, thereby releasing the cargo. In some embodiments, a cleavable linker may also be provided in a location such that, when the cleavable linker is cleaved (e.g., a Gag-Pro-Pol protein), the NES is separated from the cargo protein. This configuration of the fusion protein allows the fusion protein to be translocated from the nucleus of the producing cell during BE-VLP production, and the NES can be cleaved from the fusion protein after delivery to the target cell, or before delivery to the target cell but after packaging into the VLP, releasing the BE and allowing it to enter the nucleus of the target cell. In some embodiments, the cleavable linker comprises a protease cleavage site (e.g., a Moloney Murine Leukemia Virus (MMLV) protease cleavage site or a Friend Murine Leukemia Virus (FMLV) protease cleavage site). Various protease cleavage sites can be used in the fusion proteins of the present disclosure. In certain embodiments, the protease cleavage site comprises the amino acid sequence TSTLLMEANSS (SEQ ID NO: 1), PRSSLYPALTP (SEQ ID NO: 2), VQALVLTQ (SEQ ID NO: 3), PLQVLTLNIERR (SEQ ID NO: 4), or an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 1-4. In some embodiments, the protease cleavage site comprises the amino acid sequence of any one of SEQ ID NOs: 1-4, comprising one mutation, two mutations, three mutations, four mutations, five mutations, or more than five mutations compared to one of SEQ ID NOs: 1-4. In some embodiments, the cleavable linker of the fusion protein is cleaved by a protease of the gag-pro polyprotein.In certain embodiments, the cleavable linker of the fusion protein is not cleaved by the protease of the gag-pro polyprotein until the BE-VLP is assembled and delivered to a target cell. In some embodiments, the gag-pro polyprotein of a BE-VLP described herein comprises an MMLV gag-pro polyprotein or an FMLV gag-pro polyprotein. In some embodiments, the gag nucleocapsid protein of the fusion protein in a BE-VLP described herein comprises an MMLV gag nucleocapsid protein or an FMLV gag nucleocapsid protein.

[0098] In certain embodiments, the fusion protein comprises the following non-limiting structure: [gag nucleocapsid protein]-[1X-3X NES]-[cleavable linker]-[NLS]-[deaminase domain]-[napDNAbp]-[NLS], where ]-[ includes any linker (by way of example, an amino acid linker or any of the linkers provided herein); [1X-3X NES]-[gag nucleocapsid protein]-[cleavable linker]-[NLS]-[deaminase domain]-[napDNAbp]-[NLS], where ]-[ comprises any linker (by way of example, an amino acid linker or any of the linkers provided herein); or [gag nucleocapsid protein]-[1X-3X NES]-[cleavable linker]-[NLS]-[deaminase domain]-[napDNAbp]-[NLS]-[cleavable linker]-[1X-3X NES], where ]-[ includes any linker (by way of example, an amino acid linker or any of the linkers provided herein).

[0099] In embodiments in which the cleavable linker is cleaved by a protease within the VLP, the VLP may comprise a fusion protein comprising the structure [gag nucleocapsid protein]-[1X-3X NES] and a free napDNAbp or base editor. In certain embodiments, the base editor comprises the structure [NLS]-[deaminase domain]-[napDNAbp]-[NLS], where each instance of ]-[ comprises an optional linker (by way of example, an amino acid linker or any of the linkers provided herein).

[0100] In some embodiments, any of the above constructs comprises 3xNES.

[0101] The eVLPs provided by the present disclosure (e.g., BE-VLPs) comprise an outer encapsulation layer (or envelope layer) comprising a viral envelope glycoprotein. Any viral envelope glycoprotein described herein or known in the art may be used in the BE-VLPs of the present disclosure. In some embodiments, the viral envelope glycoprotein is an adenovirus envelope glycoprotein, an adeno-associated virus envelope glycoprotein, a retrovirus envelope glycoprotein, or a lentivirus envelope glycoprotein. In certain embodiments, the viral envelope glycoprotein is a retrovirus envelope glycoprotein. In some embodiments, the viral envelope glycoprotein is a vesicular stomatitis virus G protein (VSV-G), a baboon retrovirus envelope glycoprotein (BaEVRless), a FuG-B2 envelope glycoprotein, an HIV-1 envelope glycoprotein, or an ecotropic murine leukemia virus (MLV) envelope glycoprotein. In some embodiments, the viral envelope glycoprotein targets the system to a specific cell type (e.g., immune cells, neural cells, retinal pigment epithelial cells, etc.). For example, using different envelope glycoproteins in the eVLPs described herein may alter their cellular tropism, allowing the BE-VLPs to target specific cell types. In some embodiments, the viral envelope glycoprotein is the VSV-G protein, which targets the system to retinal pigment epithelium (RPE) cells. In some embodiments, the viral envelope glycoprotein is the HIV-1 envelope glycoprotein, which targets the system to CD4+ cells. In some embodiments, the viral envelope glycoprotein is the FuG-B2 envelope glycoprotein, which targets the system to neurons.

[0102] It will be appreciated that general methods for producing viral vector particles, which generally contain a coding nucleic acid of interest, are known in the art and may also be used to produce virus-derived particles according to the invention that do not contain a coding nucleic acid of interest but are instead designed to deliver a protein cargo (by way of example, a BE RNP).

[0103] Conventional viral vector particles encompass retrovirus, lentivirus, adenovirus and adeno-associated viral vector particles that are well known in the art.To outline the various viral vector particles that can be used, those skilled in the art can particularly refer to Kushnir et al. (2012, Vaccine, Vol.31:58-83), Zeltons (2013, Mol Biotechnol, Vol.53:92-107), Ludwig et al. (2007, Curr Opin Biotechnol, Vol.18(no 6):537-55) and Naskalaska et al. (2015, Vol.64(no 1):3-13). Furthermore, references to various methods of using virus-derived particles to deliver proteins to cells can be found by those skilled in the art in the articles by Maetzig et al. (2012, Current Gene Therapy, Vol. 12: 389-409) and Kaczmarczyk et al. (2011, Proc Natl Acad Sci USA, Vol. 108(no 41): 16998-17003).

[0104] Generally, the virus-like particles (which may also be referred to as "virus-derived particles") used in accordance with the present disclosure are formed by one or more virus-derived structural protein(s) and / or one more virus-derived envelope protein.

[0105] The virus-like particles used in accordance with the present invention are replication-incompetent in the host cells they invade.

[0106] In a preferred embodiment, the virus-like particle is formed by one or more retroviral-derived structural protein(s) and optionally one or more viral-derived envelope protein(s).

[0107] In a preferred embodiment, the viral structural protein is a retroviral Gag protein or a peptide fragment thereof. As known in the art, Gag and the Gag / pol precursor are expressed from full-length genomic RNA as polyproteins that require proteolytic cleavage mediated by retroviral protease (PR) to acquire a functional conformation. Furthermore, Gag, which is structurally conserved among retroviruses, is composed of at least three protein units: matrix protein (MA), capsid protein (CA), and nucleocapsid protein (NC), while Pol consists of retroviral protease (PR), reverse transcriptase (RT), and integrase (IN).

[0108] In some embodiments, the virus-derived particle comprises retroviral Gag proteins but does not comprise Pol proteins.

[0109] As known in the art, the host range of retroviral vectors, including lentiviral vectors, can be expanded or altered by a process known as pseudotyping. Pseudotyped lentiviral vectors consist of viral vector particles carrying glycoproteins derived from other enveloped viruses. Such pseudotyped viral vector particles retain the tropism of the virus from which the glycoproteins are derived.

[0110] In some embodiments, the virus-like particle is a pseudotyped virus-like particle, which comprises one or more viral structural proteins or viral envelope proteins that confer tropism to certain eukaryotic cells. The pseudotyped virus-like particle described herein may comprise, as the viral protein used for pseudotyping, a viral envelope protein selected from the group comprising VSV-G protein, measles virus HA 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, and foamy virus envelope protein, or a combination of two or more of these viral envelope proteins.

[0111] A well-known example of pseudotyping viral vector particles is the pseudotyping of viral vector particles using vesicular stomatitis virus glycoprotein (VSV-G). For the pseudotyping of viral vector particles, those skilled in the art can refer to, in particular, Yee et al. (1994, Proc Natl Acad Sci, USA, Vol. 91: 9564-9568) Cronin et al. (2005, Curr Gene Ther, Vol. 5 (no. 4): 387-398), which are incorporated herein by reference.

[0112] To produce virus-like particles, more precisely VSV-G pseudotyped virus-like particles, for delivering a protein(s) of interest to target cells, those skilled in the art may refer to Mangeot et al. (2011, Molecular Therapy, Vol. 19(no 9):1656-1666).

[0113] In some embodiments, the virus-like particle further comprises a viral envelope protein, wherein either (i) the viral envelope protein is derived from the same virus as the viral structural proteins, for example, the same virus as the viral Gag protein, or (ii) the viral envelope protein is derived from a different virus than the virus from which the viral structural proteins are derived, for example, the virus from which the viral Gag protein is derived.

[0114] As will be readily understood by one of skill in the art, virus-like particles for use in accordance with the present disclosure may be any of a variety of vector particles, including Moloney murine leukemia virus-derived vector particles, bovine immunodeficiency virus-derived vector particles, simian immunodeficiency virus-derived vector particles, feline immunodeficiency virus-derived vector particles, human immunodeficiency virus-derived vector particles, equine infection anemia virus-derived vector particles, caprine arthritis encephalitis virus-derived vector particles, and the like. particle), baboon endogenous virus-derived vector particles, rabies virus-derived vector particles, influenza virus-derived vector particles, norovirus virus-derived vector particles, respiratory syncytial virus virus-derived vector particles, hepatitis A virus-derived vector particles, hepatitis B virus-derived vector particles, hepatitis E virus-derived vector particles, Newcastle disease virus-derived vector particles, Norwalk virus-derived vector particles, parvovirus-derived vector particles, papillomavirus-derived vector particles, yeast retrotransposon-derived vector particles, measles virus-derived vector particles, and bacteriophage-derived vector particles.

[0115] In particular, the virus-like particles used in accordance with the present invention are derived from retroviruses, which may be selected from Moloney murine leukemia virus, bovine immunodeficiency virus, simian immunodeficiency virus, feline immunodeficiency virus, human immunodeficiency virus, equine infectious anemia virus, and caprine arthritis-encephalitis virus.

[0116] In another embodiment, the virus-like particle used in accordance with the present disclosure is a lentivirus-derived particle. Lentiviruses belong to the retrovirus family and have the unique ability to infect non-dividing cells.

[0117] Such lentivirus may be selected from among bovine immunodeficiency virus, simian immunodeficiency virus, feline immunodeficiency virus, human immunodeficiency virus, equine infectious anemia virus, and caprine arthritis-encephalitis virus.

[0118] To prepare Moloney murine leukemia virus-derived vector particles, those skilled in the art may refer to the methods disclosed by Sharma et al. (1997, Proc Natl Acad Sci USA, Vol. 94: 10803+-10808), Guibingua et al. (2002, Molecular Therapy, Vol. 5 (no. 5): 538-546), which are incorporated herein by reference. Moloney murine leukemia virus-derived (MLV-derived) vector particles may be selected from the group including MLV-A-derived vector particles and MLV-E-derived vector particles.

[0119] To prepare bovine immunodeficiency virus-derived vector particles, those skilled in the art may refer to the method disclosed by Rasmussen et al. (1990, Virology, Vol. 178(no 2):435-451), which is incorporated herein by reference.

[0120] To prepare simian immunodeficiency virus-derived vector particles, including VSV-G pseudotyped SIV virus-derived particles, those skilled in the art may refer to, in particular, the methods disclosed by Mangeot et al. (2000, Journal of Virology, Vol. 71 (no. 18): 8307-8315), Negre et al. (2000, Gene Therapy, Vol. 7: 1613-1623) and Mangeot et al. (2004, Nucleic Acids Research, Vol. 32 (no. 12), e102), which are incorporated herein by reference.

[0121] To prepare feline immunodeficiency virus-derived vector particles, those skilled in the art may refer in particular to the methods disclosed by Saenz et al. (2012, Cold Spring Harb Protoc, (1): 71-76; 2012, Cold Spring Harb Protoc, (1): 124-125; 2012, Cold Spring Harb Protoc, (1): 118-123), which are incorporated herein by reference.

[0122] To prepare human immunodeficiency virus-derived vector particles, those skilled in the art may refer, in particular, to the methods disclosed by Jalaguier et al. (2011, PlosOne, Vol. 6 (no 11), e28314), Cervera et al. (J Biotechnol, Vol. 166 (no 4): 152-165), and Tang et al. (2012, Journal of Virology, Vol. 86 (no 14): 7662-7676), which are incorporated herein by reference.

[0123] To prepare equine infectious anemia virus-derived vector particles, those skilled in the art may refer in particular to the methods disclosed by Olsen (1998, Gene Ther, Vol. 5 (no 11): 1481-1487), which are incorporated herein by reference.

[0124] To prepare vector particles derived from caprine arthritis-encephalitis virus, those skilled in the art may refer in particular to the method disclosed by Mselli-Lakhal et al. (2006, J Virol Methods, Vol. 136(no 1-2):177-184), which is incorporated herein by reference.

[0125] To prepare baboon endogenous virus-derived vector particles, those skilled in the art may refer in particular to the methods disclosed by Girard-Gagnepain et al. (2014, Blood, Vol. 124(no 8):1221-1231), which are incorporated herein by reference.

[0126] To prepare rabies virus-derived vector particles, those skilled in the art may refer in particular to the methods disclosed by Kang et al. (2015, Viruses, Vol. 7:1134-1152, doi:10.3390 / v7031134), Fontana et al. (2014, Vaccine, Vol. 32(no 24):2799-27804) or the PCT application published under No. WO2012 / 0618, which are incorporated herein by reference.

[0127] To prepare influenza virus-derived vector particles, those skilled in the art may refer, in particular, to the methods disclosed by Quan et al. (2012, Virology, Vol. 430:127-135) and Latham et al. (2001, Journal of Virology, Vol. 75(no 13):6154-6155), which are incorporated herein by reference.

[0128] To prepare norovirus-derived vector particles, those skilled in the art may refer, in particular, to the methods disclosed by Tome-Amat et al., (2014, Microbial Cell Factories, Vol. 13:134-142), which are incorporated herein by reference.

[0129] To prepare respiratory syncytial virus-derived vector particles, those skilled in the art may refer in particular to the methods disclosed by Walpita et al. (2015, PlosOne, DOI: 10.1371 / journal.pone.0130755), which are incorporated herein by reference.

[0130] To prepare hepatitis B virus-derived vector particles, those skilled in the art may refer in particular to the methods disclosed by Hong et al. (2013, Vol. 87(no 12):6615-6624), which are incorporated herein by reference.

[0131] To prepare hepatitis E virus-derived vector particles, those skilled in the art may refer in particular to the methods disclosed by Li et al. (1997, Journal of Virology, Vol. 71 (no 10): 7207-7213), which are incorporated herein by reference.

[0132] To prepare Newcastle disease virus-derived vector particles, those skilled in the art may refer in particular to the methods disclosed by Murawski et al. (2010, Journal of Virology, Vol. 84 (no 2): 1110-1123), which are incorporated herein by reference.

[0133] To prepare Norwalk virus-derived vector particles, those skilled in the art may refer in particular to the methods disclosed by Herbst-Kralovetz et al. (2010, Expert Rev Vaccines, Vol. 9 (no 3): 299-307), which are incorporated herein by reference.

[0134] To prepare parvovirus-derived vector particles, those skilled in the art may refer, in particular, to the methods disclosed by Ogasawara et al. (2006, In Vivo, Vol. 20:319-324), which are incorporated herein by reference.

[0135] To prepare papillomavirus-derived vector particles, those skilled in the art may refer in particular to the methods disclosed by Wang et al. (2013, Expert Rev Vaccines, Vol. 12 (no 2): doi:10.1586 / erv.12.151), which are incorporated herein by reference.

[0136] As used herein, a virus-like particle comprises a Gag protein, most preferably a Gag protein originating from a virus selected from the group consisting of Rous Sarcoma Virus (RSV), Feline Immunodeficiency Virus (FIV), Simian Immunodeficiency Virus (SIV), Moloney Leukemia Virus (MLV), and Human Immunodeficiency Virus (HIV-1 and HIV-2), in particular Human Immunodeficiency Virus Type 1 (HIV-1).

[0137] In some embodiments, the virus-like particle may also contain one or more viral envelope protein(s). The presence of one or more viral envelope protein(s) may confer more specific tropism to the virus-derived particle for targeted cells, as known in the art. The one or more viral envelope protein(s) may be selected from the group consisting of envelope proteins from retroviruses, envelope proteins from non-retroviral viruses, and chimeras of these viral envelope proteins with other peptides or proteins. An example of a non-lentiviral envelope glycoprotein of interest is the lymphocytic choriomeningitis virus (LCMV) strain WE54 envelope glycoprotein. These envelope glycoproteins increase the range of cells that can be transduced with retrovirus-derived vectors.

[0138] napDNAbp In various embodiments, the BE-VLPs disclosed herein, and the fusion proteins that make up the core components of the BE-VLPs described in the present invention, comprise a nucleic acid programmable DNA binding protein (napDNAbp).

[0139] In various embodiments, BE-VLPs and fusion proteins may comprise a napDNAbp domain with a wild-type Cas9 sequence, including, for example, the canonical Streptococcus pyogenes Cas9 sequence of SEQ ID NO: 13, as shown below: [Table A]

[0140] In other embodiments, BE-VLPs and fusion proteins may comprise a napDNAbp domain with a modified Cas9 sequence that includes the nickase variant of Streptococcus pyogenes Cas9 (of SEQ ID NO: 13) of SEQ ID NO: 14, which has an H840A substitution relative to wild-type SpCas9, for example, as shown below: [Table B]

[0141] The BE-VLPs and fusion proteins described herein may include any of the modified Cas9 sequences described above, or any variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto. In some embodiments, the base editor fusion proteins described herein include any of the following other wild-type SpCas9 sequences, optionally modified at the corresponding amino acid position by one or more mutations described herein: [Table C-1] [Table C-2] [Table C-3] [Table C-4] [Table C-5] [Table C-6]

[0142] The BE-VLPs and fusion proteins described herein may include any of the above SpCas9 sequences, or any variants thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto. In other embodiments, the Cas9 protein can be a wild-type Cas9 ortholog from another bacterial species different from the canonical Cas9 from S. pyogenes. For example, modified versions of the following Cas9 orthologs can be used in conjunction with the BE-VLPs and fusion proteins described herein by creating a mutation at the position corresponding to H840A or any other amino acid of interest in wild-type SpCas9. Additionally, any variant Cas9 orthologs having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any of the below orthologs can also be used with base editors. [Table D-1] [Table D-2] [Table D-3] [Table D-4] [Table D-5] [Table D-6]

[0143] The napDNAbp used in the BE-VLPs and fusion proteins described herein may include any suitable homolog and / or ortholog, or naturally occurring enzyme, such as Cas9. Cas9 homologs and / or orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. The Cas portion may be configured as a nickase (by way of example, mutagenized, recombinantly engineered, or otherwise naturally occurring), i.e., capable of cleaving only a single strand of the target double-stranded DNA. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure. Such Cas9 nucleases and sequences also include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737, the entire contents of which are incorporated herein by reference. In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain; i.e., the Cas9 is a nickase. In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of a Cas9 protein provided by any one of the Cas9 orthologs in the table above.

[0144] In some embodiments, the VLPs described herein can be used to deliver any Cas9 equivalent to a target cell. As used herein, the term "Cas9 equivalent" is a broad term that encompasses any napDNAbp protein that provides the same function as Cas9, even though its primary amino acid sequence and / or its three-dimensional structure may differ and / or it may be evolutionarily unrelated. Thus, while a Cas9 equivalent encompasses any evolutionarily related Cas9 ortholog, homolog, mutant, or variant described or encompassed herein, a Cas9 equivalent also encompasses proteins that may have evolved by convergent evolution processes to have the same or similar function as Cas9, but do not necessarily share any similarity in amino acid sequence and / or three-dimensional structure. Although a Cas9 equivalent may be based on a protein generated by convergent evolution, the VLPs described herein can be used to deliver any Cas9 equivalent that would provide the same or similar function as Cas9. By way of illustration, if Cas9 refers to the Type II enzyme of a CRISPR-Cas system, a Cas9 equivalent can refer to the Type V or Type VI enzyme of a CRISPR-Cas system.

[0145] For example, Cas12e (CasX) is a Cas9 equivalent that reportedly has the same function as Cas9 but evolved through convergent evolution. Thus, the Cas12e (CasX) protein described in Liu et al., "CasX enzymes comprise a distinct family of RNA-guided genome editors," Nature, 2019, Vol. 566:218-223, is contemplated for delivery using the VLPs described herein. Additionally, any variants or modifications of Cas12e (CasX) are envisioned and within the scope of the present disclosure.

[0146] Cas9 is a bacterial enzyme that has evolved in a wide variety of species, however, Cas9 equivalents contemplated herein may also be obtained from archaea, which constitute a distinct domain and kingdom of unicellular prokaryotic microorganisms from bacteria.

[0147] In some embodiments, Cas9 equivalents may refer to Cas12e (CasX) or Cas12d (CasY), as described, for example, in Burstein et al., "New CRISPR-Cas systems from uncultivated microbes." Cell Res. 2017 Feb 21. Doi:10.1038 / cr.2017.21, the entire contents of which are incorporated herein by reference. Using genome-resolution metagenomics, several CRISPR-Cas systems have been identified, including the first reported Cas9 in the archaeal domain of life. This diverse Cas9 protein was found as part of an active CRISPR-Cas system in the little-studied nanoarchaea. In bacteria, two previously unknown systems, CRISPR-Cas12e and CRISPR-Cas12d, have been discovered, representing the most compact systems discovered to date. In some embodiments, Cas9 refers to Cas12e or a variant of Cas12e. In some embodiments, Cas9 refers to Cas12d or a variant of Cas12d. It should be recognized that other RNA-guided DNA-binding proteins may be used as nucleic acid-programmable DNA-binding proteins (napDNAbp) and are within the scope of the present disclosure. See also Liu et al., "CasX enzymes comprise a distinct family of RNA-guided genome editors," Nature, 2019, Vol. 566:218-223. Any of these Cas9 equivalents are contemplated by the present disclosure.

[0148] In some embodiments, the Cas9 equivalent comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally occurring Cas12e (CasX) or Cas12d (CasY) protein. In some embodiments, the napDNAbp is a naturally occurring Cas12e (CasX) or Cas12d (CasY) protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a wild-type Cas moiety or any Cas moiety provided herein.

[0149] In various embodiments, nucleic acid programmable DNA binding proteins include, but are not limited to, Cas9 (e.g., dCas9 and nCas9), Cas12e (CasX), Cas12d (CasY), Cas12a (Cpf1), Cas12b1 (C2c1), Cas13a (C2c2), Cas12c (C2c3), Argonaute, and Cas12b1. An example of a nucleic acid programmable DNA binding protein with a PAM specificity different from Cas9 is clustered regularly interspaced short palindromic repeats 1 from Prevotella and Francisella (i.e., Cas12a (Cpf1)). Like Cas9, Cas12a (Cpf1) is also a class 2 CRISPR effector, but it is a type V subgroup of enzymes rather than a type II subgroup. Cas12a (Cpf1) has been shown to mediate robust DNA interference with characteristics distinct from Cas9. Cas12a (Cpf1) is a single RNA-guided endonuclease lacking tracrRNA and utilizing a T-rich protospacer adjacent motif (TTN, TTTN, or YTN). Furthermore, Cpf1 cleaves DNA via staggered DNA double-strand breaks. Of the 16 Cpf1 family proteins, two enzymes from Acidaminococcus and Lachnospiraceae have been shown to have efficient genome editing activity in human cells. Cpf1 proteins are known in the art and have previously been described, for example, in Yamano et al., "Crystal structure of Cpf1 in complex with guide RNA and target DNA," Cell (165) 2016, pp. 949-962, the entire contents of which are incorporated herein by reference.

[0150] In still other embodiments, the Cas protein is Cas12a, Cas12b1, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Csel, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6 , Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof, preferably containing a nickase mutation (e.g., a mutation corresponding to the D10A mutation in the wild-type Cas9 polypeptide of SEQ ID NO: 13).

[0151] In various other embodiments, the napDNAbp can be any of the following proteins: Cas9, Cas12a (Cpf1), Cas12e (CasX), Cas12d (CasY), Cas12b1 (C2c1), Cas13a (C2c2), Cas12c (C2c3), GeoCas9, CjCas9, Cas12g, Cas12h, Cas12i, Cas13b, Cas13c, Cas13d, Cas14, Csn2, xCas9, SpCas9-NG, circularly permuted Cas9, or an Argonaute (Ago) domain, or a variant thereof.

[0152] The VLPs described herein can also be used to deliver Cas12a(Cpf1)(dCpf1) variants, which can be used as programmable DNA-binding protein domains via guide nucleotide sequences. The Cas12a(Cpf1) protein has a RuvC-like endonuclease domain similar to the RuvC domain of Cas9, but does not have an HNH endonuclease domain, and the N-terminus of Cas12a(Cpf1) does not have the recognition lobe of the alpha-helix system of Cas9. Zetsche et al., Cell, 163, 759-771, 2015 (which is incorporated herein by reference) showed that the RuvC-like domain of Cas12a(Cpf1) is responsible for cleaving both DNA strands, and inactivating the RuvC-like domain inactivates Cas12a(Cpf1) nuclease activity.

[0153] In some embodiments, napDNAbp is a single effector of microbial CRISPR-Cas system.Single effectors of microbial CRISPR-Cas system include, but are not limited to, Cas9, Cas12a (Cpf1), Cas12b1 (C2c1), Cas13a (C2c2), and Cas12c (C2c3).Typically, microbial CRISPR-Cas systems are divided into class 1 and class 2 systems.Class 1 systems have multi-subunit effector complexes, while class 2 systems have a single protein effector.For example, Cas9 and Cas12a (Cpf1) are class 2 effectors. In addition to Cas9 and Cas12a (Cpf1), three distinct Class 2 CRISPR-Cas systems (Cas12b1, Cas13a, and Cas12c) have been described by Shmakov et al., "Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems", Mol. Cell, 2015 Nov 5;60(3):385-397, the contents of which are incorporated herein by reference.

[0154] The effectors of two of the systems, Cas12b1 and Cas12c, contain a RuvC-like endonuclease domain related to Cas12a. The third system, Cas13a, contains an effector with two predicted HEPN RNase domains. Unlike CRISPR RNA production by Cas12b1, mature CRISPR RNA production is tracrRNA-independent. Cas12b1 depends on both CRISPR RNA and tracrRNA for DNA cleavage. Bacterial Cas13a has been shown to possess an intrinsic RNase activity for CRISPR RNA maturation that is distinct from its RNA-activated single-stranded RNA degradation activity. These RNase functions are distinct from each other and from the CRISPR RNA processing behavior of Cas12a. See, for example, East-Seletsky, et al., "Two distinct RNase activities of CRISPR-Cas13a enable guide-RNA processing and RNA detection," Nature, 2016 Oct 13;538(7624):270-273, the entire contents of which are incorporated herein by reference. In vitro biochemical analysis of Cas13a in Leptotrichia shahii has shown that Cas13a can be programmed to cleave ssRNA targets carrying a complementary protospacer, guided by a single CRISPR RNA. Catalytic residues on two conserved HEPN domains mediate cleavage. Mutation of the catalytic residues produces a catalytically inactive RNA-binding protein. See, for example, Abudayyeh et al., "C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector," Science, 2016 Aug 5;353(6299), the entire contents of which are incorporated herein by reference.

[0155] The crystal structure of Alicyclobacillus acidoterrastris Cas12b1 (AacC2c1) has been reported in complex with a chimeric single-molecule guide RNA (sgRNA). See, e.g., Liu et al., "C2c1-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism," Mol. Cell, 2017 Jan 19;65(2):310-322, the entire contents of which are incorporated herein by reference. A crystal structure has also been reported for Alicyclobacillus acidoterrestris C2c1 bound to target DNA as a ternary complex. See, e.g., Yang et al., "PAM-dependent Target DNA Recognition and Cleavage by C2c1 CRISPR-Cas endonuclease," Cell, 2016 Dec 15;167(7):1814-1828, the entire contents of which are incorporated herein by reference. The catalytically competent conformation of AacC2c1 is independently captured by both the target and non-target DNA strands located within a single RuvC catalytic pocket, and C2c1-mediated cleavage results in a staggered seven-nucleotide break in the target DNA. Structural comparison between the C2c1 ternary complex and its previously identified Cas9 and Cpf1 counterparts demonstrates the diversity of the mechanisms employed by the CRISPR-Cas9 system.

[0156] In some embodiments, the napDNAbp may be a C2c1, C2c2, or C2c3 protein. In some embodiments, the napDNAbp is a C2c1 protein. In some embodiments, the napDNAbp is a Cas13a protein. In some embodiments, the napDNAbp is a Cas12c protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally occurring Cas12b1 (C2c1), Cas13a (C2c2), or Cas12c (C2c3) protein. In some embodiments, the napDNAbp is a naturally occurring Cas12b1 (C2c1), Cas13a (C2c2), or Cas12c (C2c3) protein.

[0157] Other programmable nucleases In various embodiments described herein, the VLPs disclosed herein are used to deliver napDNAbps, such as the Cas9 protein, either alone or as part of a fusion protein (e.g., a base editor). These proteins are "programmable" by becoming complexed with a guide RNA, which guides the Cas9 protein to a target site on DNA that possesses a sequence complementary to the spacer portion of the gRNA and also possesses the required PAM sequence. However, in certain embodiments contemplated herein, the napDNAbp may be replaced by a different type of programming protein, such as a zinc finger nuclease or a transcription activator-like effector nuclease (TALEN), which may be delivered to a target cell using the VLPs described herein.

[0158] In this manner, it is contemplated that nucleases suitable for delivery using the VLPs described herein need not necessarily be "programmed" with a nucleic acid targeting molecule (such as a guide RNA), but rather may be programmed by defining the specificity of a DNA-binding domain, such as a particular nuclease. Just as with the napDNAbp moiety, it may be preferable that such alternative programmable nucleases be modified such that only one strand of the target DNA is cut. In other words, the programmable nuclease may function as a nickase.

[0159] Suitable alternative programmable nucleases are well known in the art. TALENs are artificial restriction enzymes produced by fusing a TAL effector DNA binding domain to a DNA cleavage domain. These reagents enable efficient, programmable, and specific DNA cleavage, representing a powerful tool for in situ genome editing. Transcription activator-like effectors (TALEs) can be rapidly engineered to bind to virtually any DNA sequence. The term TALEN, as used herein, broadly encompasses monomeric TALENs that can cleave double-stranded DNA without the assistance of another TALEN. The term TALEN can also refer to one or both members of a pair of TALENs that are engineered to work together to cleave DNA at the same site. TALENs that work together can also be referred to as left- and right-handed TALENs, which refers to the handedness of DNA. See U.S. Ser. Nos. 12 / 965,590; 13 / 426,991 (U.S. Pat. No. 8,450,471); 13 / 427,040 (U.S. Pat. No. 8,440,431); 13 / 427,137 (U.S. Pat. No. 8,440,432); and 13 / 738,381, all of which are incorporated by reference herein in their entireties.In addition, TALENs are described in International Publication No. WO2015 / 027134, U.S. Patent No. 9,181,535, Boch et al., "Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors", Science, vol. 326, pp. 1509-1512 (2009), Bogdanove et al., TAL Effectors: Customizable Proteins for DNA Targeting, Science, vol. 333, pp. 1843-1846 (2011), Cade et al., "Highly efficient generation of heritable zebrafish gene mutations using homo- and heterodimeric TALENs", Nucleic Acids Research, vol. 40, pp. 8001-8010 (2012), and Cermak et al., "Efficient design and assembly of custom TALENs and other TAL effector-based constructs for DNA targeting". and "Targeting," Nucleic Acids Research, vol. 39, No. 17, e82 (2011), each of which is incorporated herein by reference.

[0160] Zinc finger nucleases can also be used as alternative programmable nucleases and delivered using the VLPs described herein. Like TALENs, ZFN proteins can be modified to function as nickases, i.e., engineer ZFNs to cleave only one strand of target DNA. ZFN proteins have been extensively described in the art, e.g., Carroll et al., "Genome Engineering with Zinc-Finger Nucleases," Genetics, Aug 2011, Vol. 188:773-782; Durai et al., "Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells," Nucleic Acids Res, 2005, Vol. 33:5978-90; and Gaj et al., "ZFN, TALEN, and CRISPR / Cas-based methods for genome engineering," Trends Biotechnol. 2013, Vol. 31:397-405, each of which is incorporated herein by reference in its entirety.

[0161] Deaminase domain In some embodiments, the BE-VLPs and fusion proteins described herein further comprise a deaminase domain (for example, when the base editor is encapsulated and delivered in the VLP). The deaminase domain may be a cytosine deaminase domain or an adenosine deaminase domain.

[0162] In some embodiments, the C to T conversion base editor comprises a cytosine deaminase. "Cytosine deaminase" refers to an enzyme that catalyzes the chemical reaction "cytosine + HO → uracil + NH" or "5-methyl-cytosine + HO → thymine + NH". As may be apparent from the reaction formula, such a chemical reaction results in a nucleobase change from C to U / T. In the context of a gene, such a nucleotide change or mutation may result in an amino acid change in the protein that may affect the function of the protein, for example, a loss-of-function or a gain-of-function. In some embodiments, the C to T base editor comprises dCas9 or nCas9 fused to a cytosine deaminase. In some embodiments, the cytosine deaminase domain is fused to the N-terminus of dCas9 or nCas9.

[0163] Non-limiting examples of suitable cytosine deaminase domains are provided below as SEQ ID NOs: 33-56.

[0164] Human AID MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGL (SEQ ID NO: 33)

[0165] Mouse AID MDSLLMKQKKFLYHFKNVRWAKGRHETYLCYVVKRRDSATSCSLDFGHLRNKSGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVAEFLRWNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIGIMTFKDYFYCWNTFVENRERTFKAWEGLHENSVRLTRQLRRILLPLYEVDDLRDAFRMLGF (SEQ ID NO: 34)

[0166] Inu AID MDSLLMKQRKFLYHFKNVRWAKGRHETYLCYVVKRRDSATSFSLDFGHLRNKSGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGYPNLSLRIFAARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENREKTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGL (SEQ ID NO: 35)

[0167] Cow AID MDSLLKKQRQFLYQFKNVRWAKGRHETYLCYVVKRRDSPTSFSLDFGHLRNKAGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGYPNLSLRIFTARLYFCDKERKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGL (SEQ ID NO: 36)

[0168] Mouse APOBEC-3 MGPFCLGCSHRKCYSPIRNLISQETFKFHFKNLGYAKGRKDTFLCYEVTRKDCDSPVSLHHGVFKNKDNIHAEICFLYWFHDKVLKVLSPREEFKITWYMSWSPCFECA EQIVRFLATHHNLSLDIFSSRLYNVQDPETQQNLCRLVQEGAQVAAMDLYEFKKCWKKFVDNGGRRFRPWKRLLTNFRYQDSKLQEILRPCYIPVPSSSSSTLSNICLT KGLPETRFCVEGRRMDPLSEEEFYSQFYNQRVKHLCYYHRMKPYLCYQLEQFNGQAPLKGCLLSEKGKQHAEILFLDKIRSMELSQVTITCYLTWSPCPNCAWQLAAFKRDRPDLILHIYTSRLYFHWKRPFQKGLCSLWQSGILVDVMDLPQFTDCWTNFVNPKRPFWPWKGLEIISRRTQRRLRRIKESWGLQDLVNDFGNLQLGPPMS (SEQ ID NO: 37)

[0169] Rat APOBEC-3 MGPFCLGCSHRKCYSPIRNLISQETFKFHFKNLRYAIDRKDTFLCYEVTRKDCDSPVSLHHGVFKNKDNIHAEICFLYWFHDKVLKVLSPREEFKITWYMSWSPCFECA EQVLRFLATHHNLSLDIFSSRLYNIRDPENQQNLCRLVQEGAQVAAMDLYEFKKCWKKFVDNGGRRFRPWKKLLTNFRYQDSKLQEILRPCYIPVPSSSSSTLSNICLT KGLPETRFCVERRRVHLLSEEEFYSQFYNQRVKHLCYYHGVKPYLCYQLEQFNGQAPLKGCLLSEKGKQHAEILFLDKIRSMELSQVIITCYLTWSPCPNCAWQLAAFKRDRPDLILHIYTSRLYFHWKRPFQKGLCSLWQSGILVDVMDLPQFTDCWTNFVNPKRPFWPWKGLEIISRRTQRRLHRIKESWGLQDLVNDFGNLQLGPPMS (SEQ ID NO: 38)

[0170] Rhesus APOBEC-3G MVEPMDPRTFVSNFNNRPILSGLNTVWLCCEVKTKDPSGPPLDAKIFQGKVYSKAKYHPEMRFLRWFHKWRQLHHDQEYKVTWYVSWSPCTRCANSVATFLAKDPKVTLTIFVARLYYFWKPDYQQALRILCQKRGGPHATMKIMNYNEFQDCWNKFVDGRGKPFKPRNNLPKHYTLLQATLGELLRHLMDPGTFTSNFNNKPWVSGQHETYLCYKVERLHNDTWVPLNQHRGFLRNQAPNIHGFPKGRHAELCFLDLIPFWKLDGQQYRVTCFTSWSPCFSCAQEMAKFISNNEHVSLCIFAARIYDDQGRYQEGLRALHRDGAKIAMMNYSEFEYCWDTFVDRQGRPFQPWDGLDEHSQALSGRLRAI (SEQ ID NO: 39)

[0171] Chimpanzee APOBEC-3G MKPHFRNPVERMYQDTFSDNFYNRPILSHRNTVWLCYEVKTKGPSRPPLDAKIFRGQVYSKLKYHPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKCTRDVATFLAEDPKVTLTIFVARLYYFWDPDYQEALRSLCQKRDGPRATMKIMNYDEFQHCWSKFVYSQRELFEPWNNLPKYYILLHIMLGEILRHSMDPPTFTSNFNNELWVRGRHETYLCYEVERLHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLHQDYRVTCFTSWSPCFSCAQEMAKFISNNKHVSLCIFAARIYDDQGRCQEGLRTLAKAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLEEHSQALSGRLRAILQNQGN (SEQ ID NO: 40)

[0172] Green monkey APOBEC-3G MNPQIRNMVEQMEPDIFVYYFNNRPILSGRNTVWLCYEVKTKDPSGPPLDANIFQGKLYPEAKDHPEMKFLHWFRKWRQLHRDQEYEVTWYVSWSPCTRCANSVATFLAEDPKVTLTIFVARLYYFWKPDYQQALRILCQERGGPHATMKIMNYNEFQHCWNEFVDGQGKPFKPRKNLPKHYTLLHATLGELLRHVMDPGTFTSNFNNKPWVSGQRETYLCYKVERSHNDTWVLLNQHRGFLRNQAPDRHGFPKGRHAELCFLDLIPFWKLDDQQYRVTCFTSWSPCFSCAQKMAKFISNNKHVSLCIFAARIYDDQGRCQEGLRTLHRDGAKIAVMNYSEFEYCWDTFVDRQGRPFQPWDGLDEHSQALSGRLRAI (SEQ ID NO: 41)

[0173] Human APOBEC-3G MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPPLDAKIFRGQVYSELKYHPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKCTRDMATFLAEDPKVTLTIFVARLYYFWDPDYQEALRSLCQKRDGPRATMKIMNYDEFQHCWSKFVYSQRELFEPWNNLPKYYILLHIMLGEILRHSMDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIYDDQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQNQEN (SEQ ID NO: 42)

[0174] Human APOBEC-3F MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPRLDAKIFRGQVYSQPEHHAEMCFLSWFCGNQLPAYKCFQITWFVSWTPCPDCVAKLAEFLAEHPNVTLTISAARLYYYWERDYRRALCRLSQAGARVKIMDDEEFAYCWENFVYSEGQPFMPWYKFDDNYAFLHRTLKEILRNPMEAMYPHIFYFHFKNLRKAYGRNESWLCFTMEVVKHHSPVSWKRGVFRNQVDPETHCHAERCFLSWFCDDILSPNTNYEVTWYTSWSPCPECAGEVAEFLARHSNVNLTIFTARLYYFWDTDYQEGLRSLSQEGASVEIMGYKDFKYCWENFVYNDDEPFKPWKGLKYNFLFLDSKLQEILE (SEQ ID NO: 43)

[0175] Human APOBEC-3B MNPQIRNPMERMYRDTFYDNFENEPILYGRSYTWLCYEVKIKRGRSNLLWDTGVFRGQVYFKPQYHAEMCFLSWFCGNQLPAYKCFQITWFVSWTPCPDCVAKLAEFLSEHPNVTLTISAARLYYYWERDYRRALCRLSQAGARVTIMDYEEFAYCWENFVYNEGQQFMPWYKFDENYAFLHRTLKEILRYLMDPDTFTFNFNNDPLVLRRRQTYLCYEVERLDNGTWVLMDQHMGFLCNEAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFEYCWDTFVYRQGCPFQPWDGLEEHSQALSGRLRAILQNQGN (SEQ ID NO: 44)

[0176] Human APOBEC-3C MNPQIRNPMKAMYPGTFYFQFKNLWEANDRNETWLCFTVEGIKRRSVVSWKTGVFRNQVDSETHCHAERCFLSWFCDDILSPNTKYQVTWYTSWSPCPDCAGEVAEFLARHSNVNLTIFTARLYYFQYPCYQEGLRSLSQEGVAVEIMDYEDFKYCWENFVYNDNEPFKPWKGLKTNFRLLKRRLRESLQ (SEQ ID NO: 45)

[0177] Human APOBEC-3A MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN (SEQ ID NO: 46)

[0178] Human APOBEC-3H MALLTAETFRLQFNNKRRLRRPYYPRKALLCYQLTPQNGSTPTRGYFENKKKCHAEICFINEIKSMGLDETQCYQVTCYLTWSPCSSCAWELVDFIKAHDHLNLGIFASRLYYHWCKPQQKGLRLLCGSQVPVEVMGFPKFADCWENFVDHEKPLSFNPYKMLEELDKNSRAIKRRLERIKIPGVRAQGRYMDILCDAEV (SEQ ID NO: 47)

[0179] Human APOBEC-3D MNPQIRNPMERMYRDTFYDNFENEPILYGRSYTWLCYEVKIKRGRSNLLWDTGVFRGPVLPKRQSNHRQEVYFRFENHAEMCFLSWFCGNRLPANRRFQITWFVSWNPCLPCVVKVTKFLAEHPNVTLTISAARLYYYRDRDWRWVLLRLHKAGARVKIMDYEDFAYCWENFVCNEGQPFMPWYKFDDNYASLHRTLKEILRNPMEAMYPHIFYFHFKNLLKACGRNESWLCFTMEVTKHHSAVFRKRGVFRNQVDPETHCHAERCFLSWFCDDILSPNTNYEVTWYTSWSPCPECAGEVAEFLARHSNVNLTIFTARLCYFWDTDYQEGLCSLSQEGASVKIMGYKDFVSCWKNFVYSDDEPFKPWKGLQTNFRLLKRRLREILQ (SEQ ID NO: 48)

[0180] Human APOBEC-1 MTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERDFHPSMSCSITWFLSWSPCWECSQAIREFLSRHPGVTLVIYVARLFWHMDQQNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLTFFRLHLQNCHYQTIPPHILLATGLIHPSVAWR (SEQ ID NO: 49)

[0181] Mouse APOBEC-1 MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSVWRHTSQNTSNHVEVNFLEKFTTERYFRPNTRCSITWFLSWSPCGECSRAITEFLSRHPYVTLFIYIARLYHHTDQRNRQGLRDLISSGVTIQIMTEQEYCYCWRNFVNYPPSNEAYWPRYPHLWVKLYVLELYCIILGLPPCLKILRRKQPQLTFFTITLQTCHYQRIPPHLLWATGLK (SEQ ID NO: 50)

[0182] Rat APOBEC-1 MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK (SEQ ID NO: 51)

[0183] Lamprey CDA1 (pmCDA1) MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKILHTTKSPAV (SEQ ID NO: 52)

[0184] Evolved pmCDA1 (evoCDA1) MTDAEYVRIHEKLDIYTFKKQFSNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIWVCKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMFQVKILHTTKSPAV (SEQ ID NO: 53)

[0185] Human APOBEC3G D316R_D317R MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPPLDAKIFRGQVYSELKYHPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKCTRDMATFLAEDPKVTLTIFVARLYYFWDPDYQEALRSLCQKRDGPRATMKIMNYDEFQHCWSKFVYSQRELFEPWNNLPKYYILLHIMLGEILRHSMDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIYRRQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQNQEN (SEQ ID NO: 54)

[0186] Human APOBEC3G chain A MDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIYDDQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQ (SEQ ID NO: 55)

[0187] Human APOBEC3G chain A D120R_D121R MDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIYRRQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQ (SEQ ID NO: 56)

[0188] In some embodiments, the base editor converts A to G. In some embodiments, the base editor comprises an adenosine deaminase. "Adenosine deaminase" is an enzyme involved in purine metabolism. It is required for the breakdown of adenosine from food and the turnover of nucleic acids in tissues. Its primary function in humans is the development and maintenance of the immune system. Adenosine deaminase catalyzes the hydrolytic deamination of adenosine in the context of DNA (forming inosine, which base pairs as G). No adenosine deaminase is known to act on DNA. Instead, known adenosine deaminase enzymes act only on RNA (tRNA or mRNA). Evolved deoxyadenosine deaminase enzymes that accept DNA substrates and deaminate dA to deoxyinosine and are used in the adenosine nucleobase editors herein are described, by way of example, in PCT Application No. PCT / US2017 / 045381, filed August 3, 2017 (published as International Publication No. WO2018 / 027078), PCT Application No. PCT / US2019 / 033848 (published as International Publication No. WO2019 / 226953), PCT Application No. PCT / US2019 / 033848 (filed May 23, 2019), and PCT Application No. PCT / US2020 / 028568 (filed April 17, 2020), each of which is incorporated herein by reference. Non-limiting examples of evolved adenosine deaminases that accept DNA as a substrate are provided below. In some embodiments, the adenosine deaminase comprises any of the following amino acid sequences, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 99.9% identical to any of the following amino acid sequences:

[0189] ecTadA SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 57)

[0190] ecTadA(D108N) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGAR N AKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 58)

[0191] ecTadA(D108G) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGAR G AKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 59)

[0192] ecTadA(D108V) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGAR V AKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 60)

[0193] ecTadA(H8Y,D108N,N127S) SEVEFS Y EYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGAR N AKTGAAGSLMDVLHHPGM S HRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 61)

[0194] ecTadA(H8Y,D108N,N127S,E155D) SEVEFS Y EYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGAR N AKTGAAGSLMDVLHHPGM S HRVEITEGILADECAALLSDFFRMRRQ D IKAQKKAQSSTD (SEQ ID NO: 62)

[0195] ecTadA(H8Y,D108N,N127S,E155G) SEVEFS Y EYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGAR N AKTGAAGSLMDVLHHPGM S HRVEITEGILADECAALLSDFFRMRRQ G IKAQKKAQSSTD (SEQ ID NO: 63)

[0196] ecTadA(H8Y,D108N,N127S,E155V) SEVEFS YEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGAR N AKTGAAGSLMDVLHHPGM S HRVEITEGILADECAALLSDFFRMRRQ V IKAQKKAQSSTD (SEQ ID NO: 64)

[0197] ecTadA(A106V,D108N,D147Y,and E155V) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFG V R N AKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLS Y FFRMRRQ V IKAQKKAQSSTD (SEQ ID NO: 65)

[0198] ecTadA(S2A,I49F,A106V,D108N,D147Y,E155V) A EVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRP F GRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFG V R N AKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLS Y FFRMRRQ V IKAQKKAQSSTD (SEQ ID NO: 66)

[0199] ecTadA(H8Y,A106T,D108N,N127S,K160S) SEVEFS YEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFG T R N AKTGAAGSLMDVLHHPGM S HRVEITEGILADECAALLSDFFRMRRQEIKAQ S KAQSSTD (SEQ ID NO: 67)

[0200] ecTadA(R26G,L84F,A106V,R107H,D108N,H123Y,A142N,A143D,D147Y,E155V,I156F) SEVEFSHEYWMRHALTLAKRAWDE G EVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVT F EPCVMCAGAMIHSRIGRVVFG VHN AKTGAAGSLMDVLH Y PGMNHRVEITEGILADEC ND LLS Y FFRMRRQ VF KAQKKAQSSTD (SEQ ID NO: 68)

[0201] ecTadA(E25G,R26G,L84F,A106V,R107H,D108N,H123Y,A142N,A143D,D147Y,E155V,I156F) SEVEFSHEYWMRHALTLAKRAWD GG EVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVT F EPCVMCAGAMIHSRIGRVVFG VHN AKTGAAGSLMDVLH Y PGMNHRVEITEGILADEC ND LLS Y FFRMRRQ VF KAQKKAQSSTD (SEQ ID NO: 69)

[0202] ecTadA(E25D,R26G,L84F,A106V,R107K,D108N,H123Y,A142N,A143G,D147Y,E155V,I156F SEVEFSHEYWMRHALTLAKRAWD DG EVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVT F EPCVMCAGAMIHSRIGRVVFG VKN AKTGAAGSLMDVLH Y PGMNHRVEITEGILADEC NG LLS Y FFRMRRQ VF KAQKKAQSSTD (SEQ ID NO: 70)

[0203] ecTadA(R26Q,L84F,A106V,D108N,H123Y,A142N,D147Y,E155V,I156F SEVEFSHEYWMRHALTLAKRAWDE Q EVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVT F EPCVMCAGAMIHSRIGRVVFG V R N AKTGAAGSLMDVLH Y PGMNHRVEITEGILADEC N ALLS Y FFRMRRQ VF KAQKKAQSSTD (SEQ ID NO: 71)

[0204] ecTadA(E25M,R26G,L84F,A106V,R107P,D108N,H123Y,A142N,A143D,D147Y,E155V,I156F SEVEFSHEYWMRHALTLAKRAWD MG EVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVT F EPCVMCAGAMIHSRIGRVVFG VPNAKTGAAGSLMDVLH Y PGMNHRVEITEGILADEC ND LLS Y FFRMRRQ VF KAQKKAQSSTD (SEQ ID NO: 72)

[0205] ecTadA(R26C,L84F,A106V,R107H,D108N,H123Y,A142N,D147Y,E155V,I156F) SEVEFSHEYWMRHALTLAKRAWDE C EVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVT F EPCVMCAGAMIHSRIGRVVFG VHN AKTGAAGSLMDVLH Y PGMNHRVEITEGILADEC N ALLS Y FFRMRRQ VF KAQKKAQSSTD (SEQ ID NO: 73)

[0206] ecTadA(L84F,A106V,D108N,H123Y,A142N,A143L,D147Y,E155V,I156F) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHEIMALRQGGLVMQNYRLIDATLYVT F EPCVMCAGAMIHSRIGRVVFG V R N AKTGAAGSLMDVLH Y PGMNHRVEITEGILADEC NL LLS Y FFRMRRQ VF KAQKKAQSSTD (SEQ ID NO: 74)

[0207] ecTadA(R26G,L84F,A106V,D108N,H123Y,A142N,D147Y,E155V,I156F) SEVEFSHEYWMRHALTLAKRAWDE GEVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVT F EPCVMCAGAMIHSRIGRVVFG V R N AKTGAAGSLMDVLH Y PGMNHRVEITEGILADEC N ALLS Y FFRMRRQ VF KAQKKAQSSTD (SEQ ID NO: 75)

[0208] ecTadA(R51H,L84F,A106V,D108N,H123Y,D147Y,E155V,I156F,K157N) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG H HDPTAHEIMALRQGGLVMQNYRLIDATLYVT F EPCVMCAGAMIHSRIGRVVFG V R N AKTGAAGSLMDVLH Y PGMNHRVEITEGILADECAALLS Y FFRMRRQ VFN AQKKAQSSTD (SEQ ID NO: 76)

[0209] ecTadA(E25A,R26G,L84F,A106V,R107N,D108N,H123Y,A142N,A143E,D147Y,E155V,I156F) SEVEFSHEYWMRHALTLAKRAWD AG EVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVT F EPCVMCAGAMIHSRIGRVVFG VNN AKTGAAGSLMDVLH Y PGMNHRVEITEGILADEC NE LLS Y FFRMRRQ VF KAQKKAQSSTD (SEQ ID NO: 77)

[0210] ecTadA(N37T,P48T,L84F,A106V,D108N,H123Y,D147Y,E155V,I156F) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVH T NRVIGEGWNR T IGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVT F EPCVMCAGAMIHSRIGRVVFG V R N AKTGAAGSLMDVLH Y PGMNHRVEITEGILADECAALLS Y FFRMRRQ VF KAQKKAQSSTD (SEQ ID NO: 78)

[0211] ecTadA(N37S,L84F,A106V,D108N,H123Y,D147Y,E155V,I156F) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVH S NRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVT F EPCVMCAGAMIHSRIGRVVFG V R N AKTGAAGSLMDVLH Y PGMNHRVEITEGILADECAALLS Y FFRMRRQ VF KAQKKAQSSTD (SEQ ID NO: 79)

[0212] ecTadA(H36L,L84F,A106V,D108N,H123Y,D147Y,E155V,I156F) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLV L NNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVT F EPCVMCAGAMIHSRIGRVVFG V R N AKTGAAGSLMDVLHY PGMNHRVEITEGILADECAALLS Y FFRMRRQ VF KAQKKAQSSTD (SEQ ID NO: 80)

[0213] ecTadA(H36L,P48L,L84F,A106V,D108N,H123Y,D147Y,E155V,I156F) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLV L NNRVIGEGWNR L IGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVT F EPCVMCAGAMIHSRIGRVVFG V R N AKTGAAGSLMDVLH Y PGMNHRVEITEGILADECAALLS Y FFRMRRQ VF KAQKKAQSSTD (SEQ ID NO: 81)

[0214] ecTadA(H36L,L84F,A106V,D108N,H123Y,D147Y,E155V,K57N,I156F) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLV L NNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVT F EPCVMCAGAMIHSRIGRVVFG V R N AKTGAAGSLMDVLH Y PGMNHRVEITEGILADECAALLS Y FFRMRRQ VFN AQKKAQSSTD (SEQ ID NO: 82)

[0215] ecTadA(H36L,L84F,A106V,D108N,H123Y,S146C,D147Y,E155V,I156F) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLV LNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVT F EPCVMCAGAMIHSRIGRVVFG V R N AKTGAAGSLMDVLH Y PGMNHRVEITEGILADECAALL CY FFRMRRQ VF KAQKKAQSSTD (SEQ ID NO: 83)

[0216] ecTadA(L84F,A106V,D108N,H123Y,S146R,D147Y,E155V,I156F) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHEIMALRQGGLVMQNYRLIDATLYVT F EPCVMCAGAMIHSRIGRVVFG V R N AKTGAAGSLMDVLH Y PGMNHRVEITEGILADECAALL RY FFRMRRQ VF KAQKKAQSSTD (SEQ ID NO: 84)

[0217] ecTadA(N37S,R51H,L84F,A106V,D108N,H123Y,D147Y,E155V,I156F SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVH S NRVIGEGWNRPIG H HDPTAHEIMALRQGGLVMQNYRLIDATLYVT F EPCVMCAGAMIHSRIGRVVFG V R N AKTGAAGSLMDVLH Y PGMNHRVEITEGILADECAALLS Y FFRMRRQ VF KAQKKAQSSTD (SEQ ID NO: 85)

[0218] ecTadA(R51L,L84F,A106V,D108N,H123Y,D147Y,E155V,I156F,K157N SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG L HDPTAHEIMALRQGGLVMQNYRLIDATLYVT F EPCVMCAGAMIHSRIGRVVFG V R N AKTGAAGSLMDVLH Y PGMNHRVEITEGILADECAALLS Y FFRMRRQ VFN AQKKAQSSTD (SEQ ID NO: 86)

[0219] saTadA(D108N) GSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLRETLQQPTAHAEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIPRVVYGAD N PKGGCSGSLMNLLQQSNFNHRAIVDKGVLKEACSTLLTTFFKNLRANKKSTN (SEQ ID NO: 87)

[0220] saTadA(D107A_D108N) GSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLRETLQQPTAHAEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIPRVVYGA AN PKGGCSGSLMNLLQQSNFNHRAIVDKGVLKEACSTLLTTFFKNLRANKKSTN (SEQ ID NO: 88)

[0221] saTadA(G26P_D107A_D108N) GSHMTNDIYFMTLAIEEAKKAAQL PEVPIGAIITKDDEVIARAHNLRETLQQPTAHAEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIPRVVYGA AN PKGGCSGSLMNLLQQSNFNHRAIVDKGVLKEACSTLLTTFFKNLRANKKSTN (SEQ ID NO: 89)

[0222] saTadA(G26P_D107A_D108N_S142A) GSHMTNDIYFMTLAIEEAKKAAQL P EVPIGAIITKDDEVIARAHNLRETLQQPTAHAEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIPRVVYGA AN PKGGCSGSLMNLLQQSNFNHRAIVDKGVLKEAC A TLLTTFFKNLRANKKSTN (SEQ ID NO: 90)

[0223] saTadA(D107A_D108N_S142A) GSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLRETLQQPTAHAEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIPRVVYGA AN PKGGCSGSLMNLLQQSNFNHRAIVDKGVLKEAC A TLLTTFFKNLRANKKSTN (SEQ ID NO: 91)

[0224] ecTadA(P48S) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNR S IGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 92)

[0225] ecTadA(P48T) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNR T IGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 93)

[0226] ecTadA(P48A) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNR A IGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 94)

[0227] ecTadA(A142N) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADEC N ALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 95)

[0228] ecTadA(W23R) SEVEFSHEYWMRHALTLAKRA R DEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 96)

[0229] ecTadA(W23L) SEVEFSHEYWMRHALTLAKRA L DEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 97)

[0230] ecTadA(R152P) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRM P RQEIKAQKKAQSSTD (SEQ ID NO: 98)

[0231] ecTadA(R152H) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRM H RQEIKAQKKAQSSTD (SEQ ID NO: 99)

[0232] ecTadA(L84F,A106V,D108N,H123Y,D147Y,E155V,I156F) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHEIMALRQGGLVMQNYRLIDATLYVT F EPCVMCAGAMIHSRIGRVVFG V R N AKTGAAGSLMDVLH YPGMNHRVEITEGILADECAALLS Y FFRMRRQ VF KAQKKAQSSTD (SEQ ID NO: 100)

[0233] ecTadA(H36L,R51L,L84F,A106V,D108N,H123Y,S146C,D147Y,E155V,I156F,K157N) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLV L NNRVIGEGWNRPIG L HDPTAHEIMALRQGGLVMQNYRLIDATLYVT F EPCVMCAGAMIHSRIGRVVFG V R N AKTGAAGSLMDVLH Y PGMNHRVEITEGILADECAALL CY FFRMRRQ VFN AQKKAQSSTD (SEQ ID NO: 101)

[0234] ecTadA(H36L,P48S,R51L,L84F,A106V,D108N,H123Y,S146C,D147Y,E155V,I156F,K157N) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLV L NNRVIGEGWNR S IG L HDPTAHEIMALRQGGLVMQNYRLIDATLYVT F EPCVMCAGAMIHSRIGRVVFG V R N AKTGAAGSLMDVLH Y PGMNHRVEITEGILADECAALL CY FFRMRRQ VFN AQKKAQSSTD (SEQ ID NO: 102)

[0235] ecTadA(H36L,P48A,R51L,L84F,A106V,D108N,H123Y,S146C,D147Y,E155V,I156F,K157N) SEVEFSHEYWMRHALTLAKROWDEREVPVGAVLV L NNRVIGEGWNR A IG L HDPTAHAEIMALRQGGLVMQNYRLIDATLIVT F EPCVMCAGAMIHSRIGRVVFG V R N ACTGAAGSLMDVLH Y PGMNHRVEITEGILADECAALL CY FFRMRRQ VFN AQKKAQSSTD(Database 103)

[0236] ecTadA(W23L,H36L,P48A,R51L,L84F,A106V,D108N,H123Y,S146C,D147Y,R152P,E155V,I156F,K157N) SEVEFSHEYWMRHALTLAKRA L THEREVPVGAVLV L NNRVIGEGWNR A IG L HDPTAHAEIMALRQGGLVMQNYRLIDATLIVT F EPCVMCAGAMIHSRIGRVVFG V R N ACTGAAGSLMDVLH Y PGMNHRVEITEGILADECAALL CY FFRM P RQ VFN AQKKAQSSTD(104)

[0237] ecTadA(W23R,H36L,P48A,R51L,L84F,A106V,D108N,H123Y,S146C,D147Y,R152P,E155V,I156F,K157N) SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD(SEQ ID NO: 113)

[0238] Staphylococcus aureus TadA: MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLRETLQQPTAHAEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIPRVVYGADDPKGGCSGSLMNLLQQSNFNHRAIVDKGVLKEACSTLLTTFFKNLRANKKSTN(SEQ ID NO: 105)

[0239] Bacillus subtilis TadA: MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEQRSIAHAEMLVIDEACKALGTWRLEGATLYVTLEPCPMCAGAVVLSRVEKVVFGAFDPKGGCSGTLMNLLQEERFNHQAEVVSGVLEEECGGMLSAFFRELRKKKKAARKNLSE(SEQ ID NO: 106)

[0240] Salmonella typhimurium (S. typhimurium) TadA: MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNHRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVLQNYRLLDTTLYVTLEPCVMCAGAMVHSRIGRVVFGARDAKTGAAGSLIDVLHHPGMNHRVEIIEGVLRDECATLLSDFFRMRRQEIKALKKADRAEGAGPAV(SEQ ID NO: 107)

[0241] Shewanella putrefaciens (S.putrefaciens) TadA: MDEYWMQVAMQMAEKAEAAGEVPVGAVLVKDGQQIATGYNLSISQHDPTAHAEILCLRSAGKKLENYRLLDATLYITLEPCAMCAGAMVHSRIARVVYGARDEKTGAAGTVVNLLQHPAFNHQVEVTSGVLAEACSAQLSRFFKRRRDEKKALKLAQRAQQGIE (SEQ ID NO: 108)

[0242] Haemophilus influenzae F3031 (H.influenzae) TadA: MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGWNLSIVQSDPTAHAEIIALRNGAKNIQNYRLLNSTLYVTLEPCTMCAGAILHSRIKRLVFGASDYKTGAIGSRFHFFDDYKMNHTLEITSGVLAEECSQKLSTFFQKRREEKKIEKALLKSLSDK (SEQ ID NO: 109)

[0243] Caulobacter crescentus (C.crescentus) TadA: MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIATAGNGPIAAHDPTAHAEIAAMRAAAAKLGNYRLTDLTLVVTLEPCAMCAGAISHARIGRVVFGADDPKGGAVVHGPKFFAQPTCHWRPEVTGGVLADESADLLRGFFRARRKAKI (SEQ ID NO: 110)

[0244] Geobacter sulfurreducens (G.sulfurreducens) TadA: MSSLKKTPIRDDAYWMGKAIREAAKAAARDEVPIGAVIVRDGAVIGRGHNLREGSNDPSAHAEMIAIRQAARRSANWRLTGATLYVTLEPCLMCMGAIILARLERVVFGCYDPKGGAAGSLYDLSADPRLNHQVRLSPGVCQEECGTMLSDFFRDLRRRKKAKATPALFIDERKVPPEP (SEQ ID NO: 111)

[0245] Streptococcus pyogenes (S.pyogenes)TadA MPYSLEEQTYFMQEALKEAEKSLQKAEIPIGCVIVKDGEIIGRGHNAREESNQAIMHAEIMAINEANAHEGNWRLLDTTLFVTIEPCVMCSGAIGLARIPHVIYGASNQKFGGADSLYQILTDERLNHRVQVERGLLAADCANIMQTFFRQGRERKKIAKHLIKEQSDPFD (SEQ ID NO: 112)

[0246] TadA 7.10: SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 113)

[0247] TadA 7.10(V106W)(E. coli) SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGWRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 114)

[0248] TadA-8e (E. coli) SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN (SEQ ID NO: 115)

[0249] TadA-8e(V106W)(E. coli) SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGWRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN (SEQ ID NO: 116)

[0250] Base Editor In some aspects, the present disclosure provides eVLPs and fusion proteins for delivering base editors. Base editors are known in the art, and the BE-VLPs described herein may be used to deliver any base editor already known or developed in the future. Base editors contemplated for delivery may comprise an amino acid sequence that is 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 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the base editor sequences provided herein.

[0251] In some aspects, the BE-VLP of the present disclosure comprises a cytidine base editor (CBE) comprising a napDNAbp domain and a cytosine deaminase domain, which enzymatically deaminates the cytosine nucleobase of a C:G nucleobase pair to uracil. The uracil can then be converted to thymine (T) by the cellular DNA repair and replication machinery. The mismatched guanine (G) on the opposite strand can then be converted to adenine (A) by the cellular DNA repair and replication machinery. In this manner, the target C:G nucleobase pair is ultimately converted to a T:A nucleobase pair.

[0252] In some aspects, the BE-VLPs of the present disclosure comprise the use of a cytidine base editor. Exemplary cytidine base editors include, but are not limited to, BE3, BE3.9max, BE4max, BE4-SaKKH, BE3.9-NG, BE3.9-NRRH, or BE4max-VRQR. Other cytidine base editors are known in the art, and one of skill in the art would recognize which cytidine base editors could be delivered using the BE-VLPs of the present disclosure.

[0253] The CBE in the BE-VLP described herein may further comprise one or more nuclear localization signals (NLS) and / or one or more uracil glycosylase inhibitor (UGI) domains. Thus, the base editor may comprise the structure: NH2-[first nuclear localization sequence]-[cytosine deaminase domain]-[napDNAbp domain]-[first UGI domain]-[second UGI domain]-[second nuclear localization sequence]-COOH, where each instance of "]-[" indicates the presence of an optional linker sequence. An exemplary CBE may have a structure comprising a "BE4max" architecture with an optimized nuclear localization signal, having the structure NH2-[NLS]-[cytosine deaminase]-[Cas9 nickase]-[UGI domain]-[UGI domain]-[NLS]-COOH, where the napDNAbp domain comprises a Cas9 nickase. This BE4max construct was reported to optimize codon usage for expression in human cells as reported in Koblan et al., Nat Biotechnol. 2018;36(9):843-846, which is incorporated herein by reference.

[0254] In other embodiments, a CBE may have a structure comprising a modified BE4max architecture containing a napDNAbp domain comprising a Cas9 variant other than a Cas9 nickase, such as SpCas9-NG, xCas9, or circularly permuted CP1028. Thus, an exemplary CBE may comprise the structure: NH2-[NLS]-[cytosine deaminase]-[xCas9]-[UGI domain]-[UGI domain]-[NLS]-COOH; or NH2-[NLS]-[cytosine deaminase]-[SpCas9-NG]-[UGI domain]-[UGI domain]-[NLS]-COOH, where each instance of "]-[" indicates the presence of an optional linker sequence.

[0255] The CBE in the BE-VLPs disclosed herein may comprise a modified (or evolved) cytosine deaminase domain, e.g., a deaminase domain that recognizes an extended PAM sequence, has improved efficiency in deaminating 5'-GC targets, and / or edits in a narrower target window, etc. In some embodiments, the disclosed cytidine base editor comprises an evolved nucleic acid programmable DNA binding protein (napDNAbp), such as an evolved Cas9.

[0256] Exemplary cytidine base editors are disclosed herein and may also include amino acid sequences that are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences disclosed herein. In certain embodiments, a cytidine base editor comprises an amino acid sequence that is at least 90% identical to any one of the CBE sequences disclosed herein. In certain embodiments, a disclosed cytidine nucleobase editor comprises the amino acid sequence of any one of the CBE sequences disclosed herein. Non-limiting examples of C to T nucleobase editors are provided below.

[0257] His6-rAPOBEC1-XTEN-dCas9 for expression in Escherichia coli

[0258] rAPOBEC1-XTEN-dCas9-NLS for mammalian expression

[0259] hAPOBEC1-XTEN-dCas9-NLS for mammalian expression

[0260] rAPOBEC1-XTEN-dCas9-UGI-NLS

[0261] rAPOBEC1-XTEN-SpCas9 Nickase-UGI-NLS(BE3)

[0262] pmCDA1-XTEN-dCas9-UGI (bacteria)

[0263] pmCDA1-XTEN-nCas9-UGI-NLS (mammalian construct)

[0264] huAPOBEC3G-XTEN-dCas9-UGI (bacteria)

[0265] huAPOBEC3G-XTEN-nCas9-UGI-NLS (mammalian construct)

[0266] huAPOBEC3G(D316R_D317R)-XTEN-nCas9-UGI-NLS (mammalian construct)

[0267] High-fidelity nucleobase editor

[0268] rAPOBEC1-XTEN-SaCas9n-UGI-NLS)(SaBE3 and SaBE3.9max) MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHH ADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPES KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEEASKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG SGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSPKKKRKV (SEQ ID NO: 128)

[0269] rAPOBEC1-XTEN-SaCas9n-UGI-NLS MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHH ADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPES KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEEASKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYKNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG SGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSPKKKRKV (SEQ ID NO: 129)

[0270] Nucleobase Editor 4-SSB

[0271] Nucleic acid base editor 4-(GGS)3

[0272] Nucleobase editor 4-XTEN

[0273] Nucleic acid base editor 4-32aa linker

[0274] Nucleic Acid Base Editor 4-2X UGI

[0275] Nucleobase Editor 4 (BE4)

[0276] BE4max (also AncBE4max)

[0277] AID-BE4max

[0278] AID-VRQR-BE4max

[0279] AncBE4max 689

[0280] YE1-BE4

[0281] YE2-BE4

[0282] YEE-BE4

[0283] EE-BE4

[0284] R33A-BE4

[0285] R33A+K34A-BE4

[0286] FERNY-BE4

[0287] AALN-BE4

[0288] BE4max ("BE4-NG") modified with SpCas9-NG

[0289] BE4max-SaKKH

[0290] BE4max-NRRH MKRTADGSEFESPKKKRKVSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYH HADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLKSGGSGGGSSGSETPGTSESATPESSGGSSGGS DKKYSIGLTIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMVKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGIIPHQIHLGELHAILRRQGDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRLRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKGNSDKLIARKKDWDPKKYGGFNSPTAAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIGFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLHKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGVPAAFKYFDTTIDKKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD SGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGS KRTADGSEFEPKKKRKV (SEQ ID NO: 150)

[0291] BE4max-VQR MKRTADGSEFESPKKKRKVSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYH HADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLKSGGSGGGSSGSETPGTSESATPESSGGSSGGS DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGS KRTADGSEFEPKKKRKV (SEQ ID NO: 151)

[0292] BE4max-VRQR MKRTADGSEFESPKKKRKV SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGL RDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLKSGGSSGGSSGSETPGTSESATPESSGGSSGGS DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD SGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGS KRTADGSEFEPKKKRKV (SEQ ID NO: 152)

[0293] In some aspects, the BE-VLP of the present disclosure comprises an adenine base editor. Exemplary adenine nucleobase editors include, but are not limited to, ABE7.10 (or ABEmax), ABE8e, ABE8e-SaKKH, ABE8e-NG, ABE-xCas9, ABE7.10-SaKKH, ABE7.10-NG, ABE7.10-VRQR, ABE7.10-VQR, ABE8e-NRTH, ABE8e-NRRH, ABE8e-VQR, or ABE8e-VRQR. In certain embodiments, the adenine base editor delivered by the BE-VLP is ABE8e or ABE7.10. ABE8e may be referred to herein as "ABE8" or "ABE8.0." ABE8e base editors and variants thereof may comprise an adenosine deaminase domain containing a TadA-8e adenosine deaminase monomer (monomeric form), or a TadA-8e adenosine deaminase homodimer or heterodimer (dimeric form). Other ABEs may be used to deaminate A nucleobases.

[0294] Some aspects of the present disclosure provide fusion proteins comprising a nucleic acid programmable DNA binding protein (napDNAbp) and at least two adenosine deaminase domains. Without wishing to be bound by any particular theory, dimerization of adenosine deaminase (e.g., in cis or trans) may improve the ability (e.g., efficiency) of the fusion protein to modify nucleic acid bases, for example, deaminating adenine. In some embodiments, any of the fusion proteins may contain two, three, four, or five adenosine deaminase domains. In some embodiments, any of the fusion proteins provided herein comprises two adenosine deaminases. In some embodiments, any of the fusion proteins provided herein contains only two adenosine deaminases. In some embodiments, the adenosine deaminases are the same. In some embodiments, the adenosine deaminases are any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminases are different.

[0295] In some embodiments, the general architecture of an exemplary fusion protein having a first adenosine deaminase, a second adenosine deaminase, and a napDNAbp comprises any one of the following structures, where the NLS is a nuclear localization sequence (by way of example, any NLS provided herein), NH2 is the N-terminus of the fusion protein, and COOH is the N-terminus of the fusion protein: NH2-[first adenosine deaminase]-[second adenosine deaminase]-[napDNAbp]-COOH;

[0296] The C-terminus of NH2-[first adenosine deaminase]-[napDNAbp]-[second adenosine deaminase]-COOH; NH2-[napDNAbp]-[first adenosine deaminase]-[second adenosine deaminase]-COOH; NH2-[second adenosine deaminase]-[first adenosine deaminase]-[napDNAbp]-COOH; NH2-[second adenosine deaminase]-[napDNAbp]-[first adenosine deaminase]-COOH; NH2-[napDNAbp]-[second adenosine deaminase]-[first adenosine deaminase]-COOH.

[0297] In some embodiments, the fusion proteins provided herein do not include a linker. In some embodiments, a linker is present between one or more domains or proteins (for example, a first adenosine deaminase, a second adenosine deaminase, and / or napDNAbp). In some embodiments, the "]-[" used in the general architecture above indicates the presence of an optional linker. Exemplary fusion proteins comprising a first adenosine deaminase, a second adenosine deaminase, napDNAbp, and an NLS are provided: NH2-[NLS]-[first adenosine deaminase]-[second adenosine deaminase]-[napDNAbp]-COOH; NH2-[first adenosine deaminase]-[NLS]-[second adenosine deaminase]-[napDNAbp]-COOH; NH2-[first adenosine deaminase]-[napDNAbp]-COOH; adenosine deaminase]-[second adenosine deaminase]-[NLS]-[napDNAbp]-COOH; NH2-[first adenosine deaminase]-[second adenosine deaminase]-[napDNAbp]-[NLS]-COOH; NH2-[NLS]-[first adenosine deaminase]-[napDNAbp]-[second adenosine deaminase]-COOH; NH2-[first adenosine deaminase]-[NLS] -[napDNAbp]-[second adenosine deaminase]-COOH;NH2-[first adenosine deaminase]-[napDNAbp]-[NLS]-[second adenosine deaminase]-COOH;NH2-[first adenosine deaminase]-[napDNAbp]-[second adenosine deaminase]-[NLS]-COOH;NH2-[NLS]-[napDNAbp]-[first adenosine deaminase]-[second adenosine deaminase] adenosine deaminase]-COOH;NH2-[napDNAbp]-[NLS]-[first adenosine deaminase]-[second adenosine deaminase]-COOH;NH2-[napDNAbp]-[first adenosine deaminase]-[NLS]-[second adenosine deaminase]-COOH;NH2-[napDNAbp]-[first adenosine deaminase]-[second adenosine deaminase]-[NLS]-COOH;NH2-[NLS]-[second adenosine deaminase]-[first adenosine deaminase]-[napDNAbp]-COOH;NH2-[second adenosine deaminase]-[NLS]-[first adenosine deaminase]-[napDNAbp]-COOH;NH2-[second adenosine deaminase]-[first adenosine deaminase]-[NLS]-[napDNAbp]-COOH; NH2-[second adenosine deaminase]-[first adenosine deaminase]-[napDNAbp]-[NLS]-COOH;NH2-[NLS]-[second adenosine deaminase]-[napDNAbp]-[first adenosine deaminase]-COOH;NH2-[second adenosine deaminase]-[NLS]-[napDNAbp]-[first adenosine deaminase]-COOH; NH2-[second adenosine deaminase]-[napDNAbp]-[NLS]-[first adenosine deaminase]-COOH;NH2-[second adenosine deaminase]-[napDNAbp]-[first adenosine deaminase]-[NLS]-COOH;NH2-[NLS]-[napDNAbp]-[second adenosine deaminase]-[first adenosine deaminase]-COOH; NH2-[napDNAbp]-[NLS]-[second adenosine deaminase]-[first adenosine deaminase]-COOH;NH2-[napDNAbp]-[second adenosine deaminase]-[NLS]-[first adenosine deaminase]-COOH;NH2-[napDNAbp]-[second adenosine deaminase]-[first adenosine deaminase]-[NLS]-COOH.

[0298] Exemplary ABEs include, but are not limited to, the following fusion proteins:

[0299] In some embodiments, the A to G base editor comprises the structure NH2-[second adenosine deaminase]-[first adenosine deaminase]-[dCas9]-COOH. In some embodiments, the second adenosine deaminase is wild-type ecTadA (SEQ ID NO: 153). In some embodiments, a linker is used between each domain. In some embodiments, the linker is 32 amino acids in length and comprises the amino acid sequence of SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 306). Exemplary adenine base editors comprise an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences of SEQ ID NOs: 153-203. In certain embodiments, the disclosed adenine base editors comprise an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 153-203. In certain embodiments, the disclosed adenine base editors comprise the amino acid sequence of any of SEQ ID NOs: 153-203.

[0300] Non-limiting examples of A-G base editors are provided below as SEQ ID NOs: 153-203.

[0301] ecTadA(wt)-XTEN-nCas9-NLS

[0302] ecTadA(D108N)-XTEN-nCas9-NLS: (Mammalian construct active on DNA)

[0303] ecTadA(D108G)-XTEN-nCas9-NLS: (Mammalian construct active on DNA, active on DNA, A to G editing)

[0304] ecTadA(D108V)-XTEN-nCas9-NLS: (Mammalian construct active on DNA, active on DNA, A to G editing)

[0305] ecTadA(D108N)-XTEN-nCas9-UGI-NLS (BE3 analog of A to G editor)

[0306] ecTadA(D108G)-XTEN-nCas9-UGI-NLS (BE3 analog of A to G editor)

[0307] ecTadA(D108V)-XTEN-nCas9-UGI-NLS (BE3 analog of A to G editor)

[0308] ecTadA(D108N)-XTEN-dCas9-UGI-NLS (BE2 ​​analogue for mammalian cells, A to G editor)

[0309] ecTadA(D108G)-XTEN-dCas9-UGI-NLS (BE2 ​​analogue of A to G editor for mammalian cells)

[0310] ecTadA(D108V)-XTEN-dCas9-UGI-NLS (BE2 ​​analogue of A to G editor for mammalian cells)

[0311] ecTadA(D108N)-XTEN-nCas9-AAG(E125Q)-NLS-cat. alkyladenosine glycosylase

[0312] ecTadA(D108G)-XTEN-nCas9-AAG(E125Q)-NLS-cat. alkyladenosine glycosylase

[0313] ecTadA(D108V)-XTEN-nCas9-AAG(E125Q)-NLS-cat. alkyladenosine glycosylase

[0314] ecTadA(D108N)-XTEN-nCas9-EndoV(D35A)-NLS: Contains cat. endonuclease V

[0315] ecTadA(D108G)-XTEN-nCas9-EndoV(D35A)-NLS: containing cat. endonuclease V

[0316] ecTadA(D108V)-XTEN-nCas9-EndoV(D35A)-NLS: Contains cat. endonuclease V

[0317] Variants arising from the first round of evolution (in bacteria) of ecTadA(H8Y_D108N_N127S)-XTEN-dCas9

[0318] Abundant variants from the second evolution (in bacteria) of ecTadA(H8Y_D108N_N127S_E155X)-XTEN-dCas9; X = D, G, or V

[0319] pNMG-160:ecTadA(D108N)-XTEN-nCas9-GGS-AAG*(E125Q)-GGS-NLS

[0320] pNMG-161:ecTadA(D108N)-XTEN-nCas9-GGS-EndoV*(D35A)-GGS-NLS

[0321] pNMG-371:ecTadA(L84F_A106V_D108N_H123Y_D147Y_E155V_I156F)-SGGS-SGGS-XTEN-SGGS-SGGS-ecTadA(L84F_A106V_D108N_H123Y_D147Y_E155V_I156F)-SGGS-SGGS-XTEN-SGGS-SGGS-nCas9-SGGS-NLS

[0322] pNMG-616 amino acid sequence: ecTadA (wild type)-(SGGS)2-XTEN-(SGGS)2-ecTadA(W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_SGGS_NLS

[0323] pNMG-624 amino acid sequence: ecTadA (wild type) - 32 aa linker - ecTadA (W23R_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N) - 24 aa linker _nCas9_SGGS_NLS

[0324] pNMG-476 amino acid sequence (Evolution #3 heterodimer, wild-type TadA + TadA Evolution #3 mutation): ecTadA (wild-type)-(SGGS)2-XTEN-(SGGS)2-ecTadA(L84F_A106V_D108N_H123Y_D147Y_E155V_I156F)-(SGGS)2-XTEN-(SGGS)2_nCas9_SGGS_NLS

[0325] pNMG-477 amino acid sequence: ecTadA (wild type)-(SGGS)2-XTEN-(SGGS)2-ecTadA(H36L_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_SGGS_NLS

[0326] pNMG-558 amino acid sequence: ecTadA (wild type) - 32 aa linker - ecTadA (H36L_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F _K157N) - 24 aa linker _nCas9_SGGS_NLS

[0327] pNMG-576 amino acid sequence: ecTadA (wild type)-(SGGS)2-XTEN-(SGGS)2-ecTadA(H36L_P48S_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F _K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_GGS_NLS

[0328] pNMG-577 amino acid sequence: ecTadA (wild type)-(SGGS)2-XTEN-(SGGS)2-ecTadA(H36L_P48S_R51L_L84F_A106V_D108N_H123Y_S146C_A142N_D147Y_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_GGS_NLS

[0329] pNMG-586 amino acid sequence: ecTadA (wild type)-(SGGS)2-XTEN-(SGGS)2-ecTadA(H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_GGS_NLS

[0330] pNMG-588 amino acid sequence: ecTadA (wild type)-(SGGS)2-XTEN-(SGGS)2-ecTadA(H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_A142N_D147Y_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_GGS_NLS

[0331] pNMG-620 amino acid sequence: ecTadA (wild type)-(SGGS)2-XTEN-(SGGS)2-ecTadA(W23R_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_GGS_NLS

[0332] pNMG-617 amino acid sequence: ecTadA (wild type)-(SGGS)2-XTEN-(SGGS)2-ecTadA(W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142A_S146C_D147Y_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_GGS_NLS

[0333] pNMG-618 amino acid sequence: ecTadA (wild type)-(SGGS)2-XTEN-(SGGS)2-ecTadA(W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142A_S146C_D147Y_R152P_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_GGS_NLS

[0334] pNMG-620 amino acid sequence: ecTadA (wild type)-(SGGS)2-XTEN-(SGGS)2-ecTadA(W23R_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_GGS_NLS

[0335] pNMG-621 amino acid sequence: ecTadA (wild type) - 32 aa linker - ecTadA (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N) - 24 aa linker _nCas9_GGS_NLS

[0336] pNMG-622 amino acid sequence: ecTadA (wild type) - 32 aa linker - ecTadA (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_R152P_E155V_I156F_K157N) - 24 aa linker _nCas9_GGS_NLS

[0337] pNMG-623 amino acid sequence: ecTadA (wild type) - 32 aa linker - ecTadA (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N) - 24 aa linker _nCas9_GGS_NLS

[0338] ABE6.3 ecTadA (wild type)-(SGGS)2-XTEN-(SGGS)2-ecTadA(H36L_P48S_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_SGGS_NLS

[0339] ABE7.8 ecTadA (wild type)-(SGGS)2-XTEN-(SGGS)2-ecTadA (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_SGGS_NLS

[0340] ABE7.9 ecTadA (wild type)-(SGGS)2-XTEN-(SGGS)2-ecTadA (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_R152P￢_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_SGGS_NLS

[0341] ABE7.10 ecTadA (wild type)-(SGGS)2-XTEN-(SGGS)2-ecTadA (W23R_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P￢_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_SGGS_NLS

[0342] ABE6.4:ecTadA(wild type)-(SGGS)2-XTEN-(SGGS)2-ecTadA(H36L_P48S_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_SGGS_NLS

[0343] ABEmax

[0344] ABE8e (monomer)

[0345] ABE8e (dimer)

[0346] SaABE8e

[0347] SpCas9NG-ABE8e(“ABE8e-NG”)

[0348] SaKKH-ABE8e(“ABE8e-KKH”)

[0349] ABE8-NRTH: NLS , TadA , linker, TadA, NRTH

[0350] ABE8-NRRH: NLS , TadA , linker, TadA, NRRH

[0351] xCas9(3.7)-ABE(7.10):(ecTadA(wt)-linker(32 aa)-ecTadA*(7.10)-linker(32 aa)-nxCas9(3.7)-NLS):

[0352] ABE8-VRQR: NLS , TadA , linker, TadA, SpCas9-VRQR

[0353] ABE8e(TadA-8e V106W)

[0354] Nuclear localization sequence (NLS) In various embodiments, the fusion proteins delivered by the BE-VLPs described herein may contain one or more nuclear localization sequences (NLSs) that help facilitate translocation of the protein to the cell nucleus. Such sequences are well known in the art and can include the following examples: [Table E]

[0355] The above NLS examples are not limiting. The fusion proteins delivered by the BE-VLPs described in the present invention may include any known NLS sequence, including any of those described in Cokol et al., "Finding nuclear localization signals," EMBO Rep., 2000, 1(5):411-415 and Freitas et al., "Mechanisms and Signals for the Nuclear Import of Proteins," Current Genomics, 2009, 10(8):550-7, each of which is incorporated herein by reference.

[0356] In various embodiments, the fusion proteins, constructs encoding the fusion proteins, and BE-VLPs disclosed herein further comprise one or more, preferably at least two, nuclear localization sequences. In certain embodiments, the fusion protein comprises at least two NLSs. In embodiments with at least two NLSs, the NLSs can be the same or they can be different NLSs. In some embodiments, one or more of the NLSs is a bipartite NLS ("bpNLS"). In certain embodiments, the fusion proteins of the present disclosure comprise two bipartite NLSs. In some embodiments, the fusion proteins of the present disclosure comprise more than two bipartite NLSs.

[0357] The location of the NLS fusion can be inserted at the N-terminus, C-terminus, or within the sequence of the fusion protein, for example, between the encoded napDNAbp component (e.g., Cas9) and the deaminase domain (e.g., adenosine or cytosine deaminase).

[0358] The NLS may be any known NLS sequence in the art. The NLS may also be any NLS discovered in the future for nuclear localization. The NLS may also be any naturally occurring NLS or any non-naturally occurring NLS (for example, an NLS with one or more desired mutations).

[0359] The term "nuclear localization sequence" or "NLS" refers to an amino acid sequence that facilitates the import of a protein into a cell nucleus, for example, by nuclear transport. Nuclear localization sequences are known in the art and will be apparent to those skilled in the art. For example, NLS sequences are described in International PCT Application PCT / EP2000 / 011690, filed November 23, 2000, by Plank et al., and published as WO / 2001 / 038547 on May 31, 2001, the contents of which are incorporated herein by reference. In some embodiments, the NLS comprises the amino acid sequence PKKKRKV (SEQ ID NO: 204), MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 206), KRTADGSEFESPKKKRKV (SEQ ID NO: 215), or KRTADGSEFEPKKKRKV (SEQ ID NO: 216). In other embodiments, the NLS comprises the amino acid sequence NLSKRPAAIKKAGQAKKKK (SEQ ID NO: 217), PAAKRVKLD (SEQ ID NO: 209), RQRRNELKRSF (SEQ ID NO: 218), or NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 219).

[0360] In one aspect of the present disclosure, a base editor or other fusion protein may be modified with one or more nuclear localization sequences (NLSs), preferably at least two NLSs. In certain embodiments, the fusion protein is modified with two or more NLSs. The present disclosure contemplates the use of any nuclear localization sequence known in the art at the time of this disclosure, or any nuclear localization sequence identified or otherwise made available by the state of the art at a later date as of the filing of this application. Exemplary nuclear localization sequences are peptide sequences that direct a protein to the nucleus of a cell in which the sequence is expressed. Nuclear localization signals are predominantly basic and can be located almost anywhere in the amino acid sequence of a protein; they generally comprise short sequences of 4 (Autieri & Agrawal, (1998) J. Biol. Chem. 273:14731-37, incorporated herein by reference) to 8 amino acids, and are typically rich in lysine and arginine residues (Magin et al., (2000) Virology 274:11-16, incorporated herein by reference). Nuclear localization sequences often contain proline residues. A variety of nuclear localization sequences have been identified and used to achieve the transport of biological molecules from the cytoplasm to the nucleus of a cell. See, for example, Tinland et al., (1992) Proc. Natl. Acad. Sci. USA 89:7442-46; Moede et al., (1999) FEBS Lett. 461:229-34, which are incorporated herein by reference. Translocation is currently thought to involve nuclear pore proteins.

[0361] Most NLSs can be classified into three general groups: (i) monokaryotic NLSs, exemplified by the SV40 large T antigen NLS (PKKKRKV (SEQ ID NO: 204)); (ii) bikaryotic motifs consisting of two basic domains separated by a variable number of spacer amino acids, exemplified by the Xenopus nucleoplasmin NLS (KRXXXXXXXXXXKKKL (SEQ ID NO: 220)); and (iii) non-canonical sequences, such as the M9 of the hnRNP A1 protein, the influenza virus nucleoprotein NLS, and the yeast Gal4 protein NLS (Dingwall and Laskey 1991), to name a few.

[0362] Nuclear localization sequences appear at various points in the amino acid sequence of a protein. NLSs have been identified at the N-terminus, C-terminus, and central region of a protein. Thus, the present disclosure provides fusion proteins that may be modified with one or more NLSs at the C-terminus and / or N-terminus, as well as at internal regions of the fusion protein. The residues of a longer sequence that do not function as component NLS residues should be selected so as not to interfere with the nuclear localization signal itself, for example, tonically or sterically. Therefore, although there is no strict limit to the composition of the sequence containing NLS, in practice, such a sequence can be functionally limited in length and composition.

[0363] The present disclosure contemplates any suitable means for modifying a fusion protein to include one or more NLSs. In one aspect, a fusion protein may be engineered to express a fusion protein translationally fused to one or more NLSs at its N-terminus or its C-terminus (or both), i.e., to form a base editor-NLS fusion construct. In other embodiments, a nucleotide sequence encoding a fusion protein may be genetically modified to incorporate a reading frame encoding one or more NLSs into an internal region of the encoded base editor. Additionally, the NLS may include various amino acid linker or spacer regions encoded between the base editor and an NLS amino acid sequence attached N-terminally, C-terminally, or internally, for example, to the central region of the protein. Thus, the present disclosure also provides nucleotide constructs, vectors, and host cells for expressing fusion proteins comprising a base editor and one or more NLSs, particularly between other components.

[0364] Fusion proteins delivered by the BE-VLPs described herein may also include nuclear localization sequences, which are linked to the base editor through one or more linkers, including, by way of example, polymeric, amino acid, nucleic acid, polysaccharide, chemical, or nucleic acid linker elements. Linkers within the contemplated scope of this disclosure are not intended to have any limitation and can be any suitable type of molecule (including, by way of example, a polymer, amino acid, polysaccharide, nucleic acid, lipid, or any synthetic chemical linker domain), and can be joined to the base editor by any suitable strategy that achieves the formation of a bond (including, by way of example, a covalent linkage, hydrogen bond) between the base editor and one or more NLSs.

[0365] Nuclear export sequence (NES) In various embodiments, the fusion proteins delivered by the BE-VLPs described herein may contain one or more nuclear export sequences (NESs), which help facilitate the translocation of proteins out of the cell nucleus. Nuclear export sequences (or nuclear export signals) have the opposite function to nuclear localization signals (NLSs). Such sequences are well known in the art (see, for example, Xu et al., "Sequence and structural analyses of nuclear export signals in the NESdb database," Mol. Biol. Cell, 2012, 23(18):3677-3693, the contents of which are incorporated herein by reference) and can include the following examples: [Table F-1] [Table F-2]

[0366] The above NES examples are not limiting. The fusion proteins delivered by the BE-VLPs described herein may include any known NES sequence, including those described in Xu, D. et al. Sequence and structural analyses of nuclear export signals in the NESdb database. Mol. Biol. Cell. 2012, 23(18), 3677-3693; Fung, H.Y. et al. Structural determinants of nuclear export signal orientation in binding to exportin CRM1. eLife. 2015, 4: e10034; and Kosugi, S. et al. Nuclear Export Signal Consensus Sequences Defined Using a Localization-based Yeast Selection System. Traffic. 2008, 9(12), 2053-2062, each of which is incorporated herein by reference.

[0367] In various embodiments, the fusion proteins, constructs encoding the fusion proteins, and BE-VLPs disclosed herein further comprise one or more, preferably at least three, nuclear export sequences. In certain embodiments, the fusion proteins comprise at least three NESs. In embodiments with at least three NESs, the NESs can be the same or different NESs. In certain other embodiments, the fusion proteins, constructs encoding the fusion proteins, and BE-VLPs may comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more NESs. Generally, the one or more NESs are each strong enough to drive the accumulation of a detectable amount of BE-VLP protein (e.g., Gag-cargo) in the cytoplasm of the producer cell.

[0368] The location of the NES fusion can be at the N-terminus, C-terminus, or within the sequence of the fusion protein (e.g., inserted between the encoded napDNAbp component (e.g., Cas9) and the gag nucleocapsid protein). In certain preferred embodiments, the NES (or multiple NESs, e.g., three NESs) is located between the napDNAbp and the gag nucleocapsid protein so that it can be cleaved from the napDNAbp upon delivery of the fusion protein to a target cell. The NES sequence may preferably be joined to the fusion protein via a cleavable linker, such as a protease-cleavable linker (e.g., Gag-Pro-Pol). In this way, as shown in the fourth generation eVLPs described herein, the NES may be removed from the cargo protein (e.g., BE or napDNAbp) after VLP maturation so that the BE and / or napDNAbp cargo may be freely translocated to the nucleus upon delivery to a recipient cell.

[0369] The NES may be any known NES sequence in the art. The NES may also be any future-discovered NES for nuclear transport. The NES may also be any naturally occurring NES or any non-naturally occurring NES (for example, an NES with one or more desired mutations).

[0370] The term "nuclear export sequence" or "NES" refers to an amino acid sequence that facilitates the export of a protein from the cell nucleus, e.g., by nuclear transport. Nuclear export sequences are known in the art and will be apparent to those skilled in the art.

[0371] In one aspect of the present disclosure, a base editor or other fusion protein may be modified with one or more nuclear export sequences (NESs), preferably at least three NESs. In certain embodiments, the fusion protein is modified with two or more, three or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more NESs. The present disclosure contemplates the use of any nuclear export sequence known in the art at the time of this disclosure or any nuclear export sequence identified or otherwise made available by the state of the art at a later date as of the filing of this application. Exemplary nuclear export sequences are peptide sequences that direct proteins out of the nucleus of a cell in which the sequence is expressed. NESs generally contain hydrophobic amino acid residues in the sequence LXXXLXXLXL, where L is a hydrophobic residue (often leucine) and X represents any amino acid. Nuclear export sequences often include leucine residues.

[0372] Fusion proteins delivered by the BE-VLPs described herein may also include nuclear export sequences, which are linked to the base editor through one or more linkers, for example, polymeric, amino acid, nucleic acid, polysaccharide, chemical, or nucleic acid linker elements. Linkers within the contemplated scope of this disclosure are not intended to have any limitation and can be any suitable type of molecule (for example, a polymer, amino acid, polysaccharide, nucleic acid, lipid, or any synthetic chemical linker domain) and can be joined to the base editor by any suitable strategy that achieves the formation of a bond (for example, a covalent linkage, hydrogen bond) between the base editor and one or more NESs. In some embodiments, the linker joining the one or more NESs to the base editor is a cleavable linker, as further described herein, such that the one or more NESs can be cleaved from the base editor upon delivery of the base editor to a target cell, for example.

[0373] In various embodiments, it may be useful to monitor the accumulation of BE-VLP proteins in the cytoplasm and / or nucleus, e.g., to confirm that the protein cargo (e.g., Gag-BE) is accumulating in the cytoplasm (and not the nucleus) during the process of VLP production in the producer cell. In other embodiments, it may be useful to monitor the accumulation of BE-VLP proteins in the nucleus and / or nucleases, e.g., to confirm that in recipient cells receiving eVLPs for BE delivery, the delivered BE is, in fact, ultimately transported to nucleases that may edit DNA. Detection of nuclear or cytoplasmic accumulation may be performed using any suitable technique, as the case may be. This can be achieved by any suitable technique. For example, a detectable marker may be fused to the BE so that its location within the cell may be visualized, such as in combination with a means for detecting the location of the nucleus (e.g., a nuclear-specific stain such as DAPI). Examples of detectable markers include fluorescent proteins (e.g., green fluorescent protein, or GFP; RFP; CFP) and epitope tags (e.g., HA tag, Flag tag, SNAP tag). Cell nuclei can also be isolated from cells, and their contents may then be analyzed by any suitable process for detecting proteins (e.g., immunohistochemistry, Western blot, or enzyme activity assay, etc.).

[0374] Linker The fusion proteins and BE-VLPs described herein may include one or more linkers. As defined above, the term "linker" as used herein refers to a chemical group or molecule that connects two molecules or moieties, for example, the binding domain and cleavage domain of a nuclease. In some embodiments, a linker connects the gRNA binding domain of an RNA-programmable nuclease with the catalytic domain of a deaminase (for example, cytosine deaminase or adenosine deaminase). In some embodiments, a linker connects dCas9 and a deaminase. Typically, a linker is located between or flanked by two groups, molecules, or other moieties, and is connected to each one via a covalent bond, thus connecting the two. In some embodiments, the linker is an amino acid or multiple amino acids (for example, a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5 to 100 amino acids in length, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated.

[0375] The linker may be as simple as a covalent bond, or it may be a polymeric linker many atoms in length. In certain embodiments, the linker is a polypeptide or amino acid-based. In other embodiments, the linker is not peptide-like. In certain embodiments, the linker is a covalent bond (by way of example, a carbon-carbon bond, a disulfide bond, a carbon-heteroatom bond, etc.). In certain embodiments, the linker is a carbon-nitrogen bond of an amide linkage. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched, aliphatic or heteroaliphatic linker. In certain embodiments, the linker is polymeric (by way of example, polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of an aminoalkanoic acid. In certain embodiments, the linker comprises an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutyric acid, 5-pentanoic acid, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx). In certain embodiments, the linker is based on a carbocyclic moiety (e.g., cyclopentane, cyclohexane). In other embodiments, the linker comprises a polyethylene glycol (PEG) moiety. In other embodiments, the linker comprises an amino acid. In certain embodiments, the linker comprises a peptide. In certain embodiments, the linker comprises an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring. The linker may include a functionalized moiety to facilitate attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker. Any electrophile may be used as part of the linker.Exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates.

[0376] In some other embodiments, the linker comprises the amino acid sequence (GGGGS) n (SEQ ID NO: 299), (G) n (SEQ ID NO: 300), (EAAAK) n (SEQ ID NO: 301), (GGS) n (SEQ ID NO: 302), (SGGS) n (SEQ ID NO: 303), (XP) n (SEQ ID NO: 304), or any combination thereof, wherein n is independently an integer from 1 to 30, and X is any amino acid. In some embodiments, the linker comprises the amino acid sequence (GGS) n (SEQ ID NO: 302), where n is 1, 3, or 7. In some embodiments, the linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 305). In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 306). In some embodiments, the linker comprises the amino acid sequence SGGSGGSGGS (SEQ ID NO: 307). In some embodiments, the linker comprises the amino acid sequence SGGS (SEQ ID NO: 303). In other embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESAGSYPYDVPDYAGSAAPAAKKKKLDGSGSGGSSGGS (SEQ ID NO: 308, 60AA). In some embodiments, the linker comprises the amino acid sequence GGS (SEQ ID NO: 302), GGSGGS (SEQ ID NO: 309), GGSGGSGGS (SEQ ID NO: 310), SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 311), SGSETPGTSESATPES (SEQ ID NO: 305), or SGGSSGGSSGSETPGTSESATPESAGSYPYDVPDYAGSAAPAAKKKKLDGSGSGGSSGG S (SEQ ID NO: 312).

[0377] In certain embodiments, linkers may be used to link any of the peptides or peptide domains or portions of the invention (such as, for example, a napDNAbp linked or fused to a deaminase domain, and / or a napDNAbp linked to one or more NESs). Any of the domains of the fusion proteins described herein may also be connected to each other through any of the linkers described in the invention.

[0378] In some embodiments, the linker is a cleavable linker (e.g., a linker that can be split or cut by any means). The cleavable linker may be an amino acid sequence. In some embodiments, the linker between one or more NESs and napDNAbp of the fusion proteins and BE-VLPs provided herein comprises a cleavable linker. The cleavable linker may include a self-cleaving peptide (e.g., a 2A peptide such as EGRGSLLTCGDVEENPGP (SEQ ID NO: 9), ATNFSLLKQAGDVEENPGP (SEQ ID NO: 10), QCTNYALLKLAGDVESNPGP (SEQ ID NO: 11), or VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 12)). In some embodiments, the cleavable linker comprises a protease cleavage site that is cleaved after contact with a protease. For example, the present disclosure contemplates the use of a cleavable linker comprising a protease cleavage site of the amino acid sequence TSTLLMEANS (SEQ ID NO: 1), PRSSLYPALTP (SEQ ID NO: 2), VQALVLTQ (SEQ ID NO: 3), PLQVLTLNIERR (SEQ ID NO: 4), or an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 1-4. In certain embodiments, the cleavable linker comprises an MMLV protease cleavage site of an FMLV protease cleavage site. In certain embodiments, the fusion proteins and BE-VLPs described herein comprise the cleavable linker TSTLLMEANS (SEQ ID NO: 1), which joins one or more NESs and the napDNAbp. In some embodiments, the linker is cleaved upon delivery of the BE-VLP / fusion protein to a target cell, releasing a free base editor that is capable of translocating into the nucleus of the target cell.

[0379] The protease cleavage site may be any sequence known in the art, or any sequence yet to be discovered, so long as the corresponding protease may be co-packaged into the eVLP to enable post-maturation cleavage within the mature eVLP particle. Such cleavage sites and their corresponding proteases include, but are not limited to, (a) Granzyme A, which recognizes and cleaves sequences containing ASPRAGGK (SEQ ID NO: 5), (b) Granzyme B, which recognizes and cleaves sequences containing YEADSLEE (SEQ ID NO: 6), (c) Granzyme K, which recognizes and cleaves sequences containing YQYRAL (SEQ ID NO: 7), and (d) Cathepsin D, which recognizes and cleaves sequences containing LGVLIV (SEQ ID NO: 8). Many other combinations of specific proteases and protease cleavage sites may be used in connection with the present disclosure by co-packaging specific proteases during the eVLP production process. Such proteases can include, but are not limited to, Arg-C proteinase, Asp-N endopeptidase, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 7, caspase 8, caspase 9, caspase 10, chymotrypsin, clostripain, enterokinase, factor Xa, glutamyl endopeptidase, granzyme B, neutrophil elastase, pepsin, prolyl-endopeptidase, proteinase K, staphylococcal peptidase I, thermolysin, thrombin, and trypsin. Any protease that pairs with its cognate recognition sequence may be used in the protease-sensitive linkers of the present disclosure, including any serine protease, cysteine ​​protease, aspartic acid protease, threonine protease, glutamic acid protease, metalloprotease, or aspartic acid peptide lyase (which constitute the major classes of known proteases). Specific protease cleavage sites for such enzymes are well known in the art and may be utilized in the linkers herein to provide protease-sensitive linkers.

[0380] Group-specific antigen (gag) proteins and viral envelope glycoproteins The BE-VLPs described herein include various viral envelope and capsid components used to encapsulate and deliver the base editor fusion proteins described herein. The use of viral envelope and capsid components for nucleic acid and protein delivery is known in the art, and those skilled in the art will readily understand the various options known in the art that can be used or substituted for these components in the BE-VLPs described herein. The use of such viral components for nucleic acid and / or protein delivery (e.g., delivery of Cas9) is described, for example, in Mangeot et al., Nat. Commun. 10, 45 (2019); Gutkin, et al., Nat. Biotechnol. (2021); and Hamilton, J.R. et al., Cell Reports 35(9), 109207 (2021), each of which is incorporated herein by reference.

[0381] In some embodiments, the BE-VLPs described herein comprise a viral envelope glycoprotein layer as the outermost layer of the BE-VLP. Viral envelope glycoproteins are part of the viral envelope, i.e., oligosaccharide-containing proteins that form the outermost layer of many types of viruses that protect viral genetic material as they travel between host cells. The glycoprotein may assist in identifying and binding to receptors on the target cell membrane so that the viral envelope fuses with the membrane, allowing the contents of the viral particle (which may, by way of example, include the fusion protein in a BE-VLP as described herein) to enter the host cell.

[0382] The viral envelope glycoprotein used in the BE-VLPs of the present disclosure may include any glycoprotein from an enveloped virus. In some embodiments, the viral envelope glycoprotein is an adenovirus envelope glycoprotein, an adeno-associated virus envelope glycoprotein, a retrovirus envelope glycoprotein, or a lentivirus envelope glycoprotein. In certain embodiments, the viral envelope glycoprotein is a vesicular stomatitis virus G protein (VSV-G), a baboon retrovirus envelope glycoprotein (BaEVRless), a FuG-B2 envelope glycoprotein, or an ecotropic murine leukemia virus (MLV) envelope glycoprotein.

[0383] Any known viral envelope glycoprotein can be used in the eVLPs of the present disclosure. Any viral envelope glycoprotein discovered or characterized in the future can also be used in the eVLPs of the present disclosure. Those skilled in the art will readily be able to identify additional viral envelope glycoproteins that could be used in the eVLPs described herein. For example, viral envelope glycoproteins are described in Banerjee, V. and Mukhopadhyay, S. Virus Disease (2016), 27(1), 1-11 and Li, Y. et al. Front. Immunol. (2021), 12, 1-12, each of which is incorporated herein by reference.

[0384] The viral envelope glycoproteins used in the VLPs described herein may also be capable of targeting the VLPs to specific cell types (e.g., immune cells, neural cells, retinal pigment epithelial cells, etc.). For example, using different envelope glycoproteins in the eVLPs described herein may alter their cellular tropism, allowing the eVLPs to be targeted to specific cell types. The process of producing viral vectors in combination with foreign viral envelope proteins is known as pseudotyping. Using pseudotyping, foreign viral envelope glycoproteins can be used to alter the cellular tropism of the VLP. The envelope glycoproteins incorporated into the VLPs allow them to easily enter different cell types that have the corresponding host receptors. Pseudotyping of viral vector systems is known in the art and is further described in, for example, Hamilton, J.R. et al. Targeted delivery of CRISPR-Cas9 and transgenes enables complex immune cell engineering. Cell Reports. 2021, 35, 109207; Kato, S. et al. Selective Neural Pathway Targeting Reveals Key Roles of Thalamostriatal Projection in the Control of Visual Discrimination. J. Neurosci. 2011, 31(47), 17169-17179; and Kato, S. et al. A lentiviral strategy for highly efficient retrograde gene transfer by pseudotyping with fusion envelope glycoprotein. Human Gene Ther. 2011, 22(2), 197-206, each of which is incorporated herein by reference.

[0385] Thus, the use of different glycoproteins in the VLPs described herein may be employed to alter their cellular tropism. Retroviral tropism may be easily adjusted by pseudotyping virions with different envelope glycoproteins, which may allow targeting of VLPs to specific cell types. In some embodiments, the viral envelope glycoprotein is the VSV-G protein, which targets VLPs to retinal pigment epithelial (RPE) cells. In some embodiments, the viral envelope glycoprotein is the HIV-1 envelope glycoprotein, which targets VLPs to CD4+ cells. In some embodiments, the viral envelope glycoprotein is the FuG-B2 envelope glycoprotein, which targets VLPs to neurons.

[0386] In some embodiments, exemplary viral envelope glycoproteins that may be used to target the VLPs described in the present invention to specific cell types include, but are not limited to, glycoproteins of the following amino acid sequences: [Table G]

[0387] In some embodiments, the eVLPs described herein further comprise an inner encapsulation layer comprising components from a viral capsid, including the gag-pro polyprotein (e.g., the gag nucleocapsid protein further comprising a viral protease linked thereto) and the gag nucleocapsid protein (e.g., the protein that constitutes the core structural component of the inner shell of many viruses, lacking the protease of the gag-pro polyprotein) as described herein.

[0388] The Gag-Pro polyprotein mediates the proteolytic cleavage of the Gag and Gag-Pol polyproteins or nucleocapsid proteins during or immediately after virion release from the plasma membrane. In the eVLPs described herein, the protease of the gag-pro polyprotein cleaves the cleavable linker in the fusion protein to release the base editor after delivery of the BE-VLP to a target cell. In some embodiments, the gag-pro polyprotein is an MMLV gag-pro polyprotein or an FMLV gag-pro polyprotein.

[0389] The gag nucleocapsid protein used in the eVLPs of the present disclosure may be an MMLV gag nucleocapsid protein, an FMLV gag nucleocapsid protein, or a nucleocapsid protein from any other virus that produces such a protein. In some embodiments, the gag nucleocapsid protein is fused to the napDNAbp (by way of example, as part of a base editor). In some embodiments, the fusion further comprises an NES described herein. In certain embodiments, the gag nucleocapsid protein and the NES are positioned on one side of a cleavable linker described herein, such that the napDNAbp or the base editor is positioned on the other side of the cleavable linker, thereby allowing the base editor to be released from the gag nucleocapsid protein upon cleavage of the cleavable linker by a protease of the gag-pro polyprotein after delivery of the BE-VLP to a target cell.

[0390] Both the gag-pro polyprotein and the gag nucleocapsid protein form the inner encapsulating layer of the eVLP described in the present invention, as shown in Figure 1. Any ratio of gag-pro polyprotein to gag nucleocapsid protein (i.e., as part of a fusion protein described herein) is contemplated in the eVLPs of the present disclosure. In some embodiments, the ratio of gag-pro polyprotein to fusion protein comprising gag nucleocapsid protein is approximately 10:1, approximately 9:1, approximately 8:1, approximately 7:1, approximately 6:1, approximately 5:1, approximately 4:1, approximately 3:1, approximately 2:1, approximately 1.5:1, approximately 1:1, or approximately 0.5:1. In certain embodiments, the ratio is approximately 3:1.

[0391] Methods for producing eVLPs In one aspect, the present disclosure relates to methods of producing the eVLPs described herein, as illustrated in Figure 16. In some embodiments, the methods of producing the eVLPs described in the present invention comprise transfecting, transducing, electroporating, or inserting one or more polynucleotides (by way of example, any of the polynucleotides described herein or any of the vectors described herein) that together encode all of the components of the eVLP into a production cell. In some embodiments, the polynucleotides transfected, transduced, electroporated, or otherwise inserted into the production cell comprise: (i) a first polynucleotide comprising a nucleic acid sequence encoding a viral envelope glycoprotein; (ii) a second polynucleotide comprising a nucleic acid sequence encoding a group-specific antigen (gag) protease (pro) polyprotein; (iii) a third polynucleotide comprising a nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises: (a) a group-specific antigen (gag) nucleocapsid protein; (b) a nucleic acid-programmable DNA-binding protein (napDNAbp); (c) a cleavable linker; and (d) a nuclear export sequence (NES); and (iv) a fourth polynucleotide comprising a nucleic acid sequence encoding a guide RNA (gRNA), wherein the gRNA binds to the napDNAbp of the fusion protein encoded by the third polynucleotide. In some embodiments, the present disclosure provides one or more vectors, which comprise one, two, three or all four of the polynucleotides provided herein.In certain embodiments, the first, second, third and fourth polynucleotides are each on separate vectors.In certain embodiments, one or more of the first polynucleotide, the second polynucleotide, the third polynucleotide and the fourth polynucleotide are on the same vector.

[0392] In some embodiments, when a producer cell expresses a polynucleotide, the various components of the eVLP spontaneously self-assemble within the producer cell. Assembly of the eVLP depends on the multimerization of the gag polyprotein encoded on the polynucleotide. The gag polyprotein (a portion of which is fused to a gene editing agent, such as a Cas9 protein or a base editor) multimerizes at the cell membrane of the producer cell and is then spontaneously released into the producer cell supernatant. Thus, BE-eVLPs can be produced by transient transfection of producer cells (e.g., Gesicle Producer 293T cells), as described in the examples herein. All of the polynucleotides required for eVLP production can be transfected into the producer cell simultaneously, or each required polynucleotide can be transfected one at a time. In some embodiments, a single polynucleotide encodes all of the components required to produce the eVLPs described herein. After transfection and incubation of the production cells (for example, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 15 hours, about 24 hours, about 36 hours, about 48 hours, or more than 48 hours), the production cell supernatant may be harvested and eVLPs may be purified therefrom.

[0393] Any cell capable of expressing an exogenous polynucleotide may be used to produce the eVLPs described herein. For example, the present disclosure contemplates the use of any of the cells listed in the kits and cells section herein for the production of eVLPs, or any other cell known in the art capable of expressing an exogenous polynucleotide.

[0394]

[0049] An overview of one embodiment of the production of eVLPs, including BE RNPs (e.g., BE-VLPs), in producer cells using a set of expression plasmids encoding the various self-assembling components of the eVLP: (a) a plasmid encoding a Gag-BE fusion protein (e.g., a retroviral Gag, MMLV-Gag-BE fusion protein); (b) a plasmid encoding a Gag-Pro-Pol protein (e.g., a retroviral protein, such as the MMLV protease precursor); (c) a plasmid encoding a BE sgRNA; and (d) a plasmid encoding an envelope glycoprotein (e.g., the spike glycoprotein of vesicular stomatitis virus (VSV-G)). The plasmids are transiently co-transfected into producer cells, and the encoded proteins and sgRNA products are encoded. In some embodiments, such as the fourth-generation eVLPs described herein, the inventors have found an optimized stoichiometric ratio of gag-cargo fusion to Gag-Pro-Pol fusion protein that balances the amount of gag-cargo available for packaging into VLPs with the amount of retroviral protease (the "Pro" in the Gag-Pro-Pol fusion) required for VLP maturation. In one embodiment, the optimized ratio of Gag-cargo fusion to Gag-Pro-Pol fusion protein is achieved by transiently delivering appropriate ratios of plasmids encoding each component into production cells. In one embodiment, to adjust the stoichiometry of Gag-cargo fusion to Gag-Pro-Pol fusion, the ratio of Gag-cargo-encoding plasmid (e.g., Gag-3xNES-ABE8e) to wild-type MMLV gag-pro-pol plasmid transfected for VLP production was varied. We found that increasing the amount of gag-cargo plasmid beyond the original ratio used to produce v3.4 BE-eVLPs (38% gag-cargo plasmid and 62% gag-pro-pol plasmid) did not improve editing efficiency (Figure 2G). Decreasing the ratio of gag-cargo plasmid from 38% to 25% slightly improved editing efficiency (Figure 2G).However, further decreasing the proportion of gag-cargo plasmid below 25% reduced editing efficiency (Figure 2G). These results are consistent with a model in which the optimal gag-cargo:gag-pro-pol stoichiometry balances the amount of gag-cargo available for packaging into VLPs with the amount of MMLV protease (the "pro" in gag-pro-pol) required for VLP maturation. In one embodiment, the results of this final round of optimization revealed a fourth-generation (v4) BE-eVLP formulation (Figure 2G), which combined the optimal gag-BE:gag-pro-pol stoichiometry (gag-BE 25%) with the v3.4BE-eVLP architecture.

[0395] As illustrated in Figure 16, the present disclosure provides a plurality of polynucleotides encoding the eVLP (e.g., BE-VLP) self-assembling components described herein. In some embodiments, the present disclosure provides a plurality of polynucleotides comprising: (i) a first polynucleotide (e.g., a plasmid) comprising a nucleic acid sequence encoding a viral envelope glycoprotein; (ii) a second polynucleotide (e.g., a plasmid) comprising a nucleic acid sequence encoding a group-specific antigen (gag) protease (pro) polyprotein; (iii) a third polynucleotide (e.g., a plasmid) comprising a nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises: (a) a group-specific antigen (gag) nucleocapsid protein; (b) a nucleic acid-programmable DNA-binding protein (napDNAbp); (c) a cleavable linker; and (d) a nuclear export sequence (NES); and (iv) a fourth polynucleotide (e.g., a plasmid) comprising a nucleic acid sequence encoding a guide RNA (gRNA). In some embodiments, the gRNA binds to the napDNAbp of the fusion protein encoded by the third polynucleotide. In some embodiments, the ratio of the second polynucleotide to the third polynucleotide is approximately 10:1, approximately 9:1, approximately 8:1, approximately 7:1, approximately 6:1, approximately 5:1, approximately 4:1, approximately 3:1, approximately 2:1, approximately 1.5:1, approximately 1:1, or approximately 0.5:1. In certain embodiments, the ratio of the second polynucleotide to the third polynucleotide is approximately 3:1.

[0396] Pharmaceutical Composition Another aspect of the present disclosure relates to pharmaceutical compositions comprising any of the eVLPs, fusion proteins, and polynucleotides / multiple polynucleotides or vectors described herein. The term "pharmaceutical composition," as used herein, refers to a composition formulated for pharmaceutical use. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition includes additional agents (e.g., for specific delivery, increased half-life, or other therapeutic compounds).

[0397] As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium, calcium talc, or zinc stearate or stearic acid), or solvent encapsulating material involved in carrying or transporting a compound from one location in the body (e.g., a delivery site) to another (e.g., an organ, tissue, or body part). A pharmaceutically acceptable carrier is "acceptable" in the sense of being compatible with the other ingredients of the formulation, but not toxic to the tissues of the subject (e.g., physiologically compatible, sterile, physiological pH, etc.).Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) (12) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG); (13) esters, such as ethyl oleate and ethyl laurate; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other compatible non-toxic substances employed in pharmaceutical formulations. Wetting agents, colorants, release agents, coating agents, sweeteners, flavorings, fragrances, preservatives, and antioxidants can also be present in the formulation. Terms such as "excipient," "carrier," "pharmaceutically acceptable carrier," or the like are used interchangeably herein.

[0398] In some embodiments, the pharmaceutical composition is formulated for delivery to a subject, for example, for gene editing.Suitable routes for administering the pharmaceutical composition described herein include but are not limited to: topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseous, periocular, intratumoral, intracerebral and intraventricular administration.

[0399] In some embodiments, the pharmaceutical compositions described herein are administered locally to the site of disease (for example, a tumor site). In some embodiments, the pharmaceutical compositions described herein are administered to a subject by injection, using a catheter, using a suppository, or using an implant (an implant of a porous material, a non-porous material, or a gelatin material (including membranes such as silastic membranes or fibers, etc.)).

[0400] In other embodiments, the pharmaceutical compositions described herein are delivered in a controlled release system.In one embodiment, a pump can be used (see, for example, Langer, 1990, Science 249:1527-1533; Sefton, 1989, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574).In another embodiment, polymeric materials can be used. (See, e.g., Medical Applications of Controlled Release (Langer and Wise eds., CRC Press, Boca Raton, Fla., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., Wiley, New York, 1984); Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61. See also, Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105.) Other controlled-release systems are discussed, for example, in Langer, supra.

[0401] In some embodiments, the pharmaceutical composition is formulated according to routine procedures as a composition suitable for intravenous or subcutaneous administration to a subject, for example, a human. In some embodiments, the pharmaceutical composition for administration by injection is a solution in a sterile, isotonic aqueous buffer. If necessary, the pharmaceutical composition can also include a solubilizing agent and a local anesthetic, such as lignocaine, to relieve pain at the injection site. Generally, the ingredients are supplied in unit dosage form, either individually or mixed together, for example, as a dry lyophilized powder or water-free concentrate in a sealed container, such as an ampoule or sachette, indicating the quantity of active agent. When the pharmaceutical composition is to be administered by injection, it can be dispensed with an infusion bottle containing sterile pharmaceutical-grade water or saline. When the pharmaceutical composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0402] Pharmaceutical compositions for systemic administration may be liquids, such as sterile saline, lactated Ringer's solution, or Hank's solution. In addition, pharmaceutical compositions may be in solid form and redissolved or suspended extemporaneously prior to use. Lyophilized forms are also contemplated.

[0403] Pharmaceutical compositions can be contained within lipid particles or vesicles, such as liposomes or microcrystals, which are also suitable for parenteral administration. The particles can be of any suitable structure, such as unilamellar or multilamellar, so long as the composition is contained therein. Compounds can be encapsulated in "stabilized plasmid-lipid particles" (SPLPs), which contain the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol%) of cationic lipids, and are stabilized by a polyethylene glycol (PEG) coating (Zhang Y. Pet et al., Gene Ther. 1999, 6:1438-47). Positively charged lipids, such as N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate, or "DOTAP," are particularly preferred for such particles and vesicles. The preparation of such lipid particles is well known. See, for example, U.S. Patent Nos. 4,880,635; 4,906,477; 4,911,928; 4,917,951; 4,920,016; and 4,921,757, each of which is incorporated herein by reference.

[0404] The pharmaceutical compositions described herein may be administered or packaged as, for example, unit doses. The term "unit dose", when used in relation to the pharmaceutical compositions of the present disclosure, refers to a physically discrete unit suitable for a subject as a unitary dosage, each unit containing a predetermined amount of active material, calculated to produce a desired therapeutic effect, together with the required diluent; i.e., carrier or vehicle.

[0405] Furthermore, the pharmaceutical composition can be provided as a pharmaceutical kit comprising (a) a container containing the compound of the invention in lyophilized form, and (b) a second container containing a pharmaceutically acceptable diluent for injection (e.g., sterile water). The pharmaceutically acceptable diluent can be used for reconstituting or diluting the lyophilized compound of the invention. Optionally, the notice associated with such container(s) can be in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, and the notice reflects approval by the agency of manufacture, use, or sale for human administration.

[0406] In another aspect, an article of manufacture containing materials useful for treating the above-mentioned diseases is included. In some embodiments, the article of manufacture includes a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The container may be formed from a variety of materials, such as glass or plastic. In some embodiments, the container holds a composition effective for treating a disease and may have a sterile access port. For example, the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic injection needle. The active agent in the composition is a compound of the present invention. In some embodiments, a label on or associated with the container indicates that the composition is used to treat a selected disease. The article of manufacture may further include a second container containing a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0407] Kits and Cells The fusion proteins, eVLPs, and compositions of the present disclosure may be assembled into kits. In some embodiments, the kits include polynucleotides for expression and assembly of the eVLPs described herein. In other embodiments, the kits further include a suitable guide nucleotide sequence, or a nucleic acid vector for expression of such a guide nucleotide sequence, to target the base editor Cas9 protein delivered by the eVLP to a desired target sequence.

[0408] The kits described herein may include one or more containers housing components for carrying out the methods described herein, and optionally instructions for use. Any of the kits described herein may further include components required for carrying out the base editing methods described herein. Each component of the kit may be provided in liquid form (e.g., in solution) or solid form (e.g., dry powder), if applicable. In certain cases, some of the components may be reconstituted or otherwise processable (e.g., into an active form), for example, by adding a suitable solvent or other species (e.g., water), which may or may not be provided with the kit.

[0409] In some embodiments, the kit may optionally include instructions and / or promotions for the use of the provided components. As used herein, "instructions" may define the instruction and / or promotion components and may typically involve written instructions on or in association with the packaging of the present disclosure. Instructions may also include any oral or electronic instructions provided in any format, such as audiovisual (e.g., videotape, DVD, etc.), internet, and / or web-based communications, etc., so that the user clearly recognizes that the instructions should be associated with the kit. The written instructions may be in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, and may also reflect agency approval of the manufacture, use, or sale for animal administration. As used herein, "promoted" includes all methods of business conduct related to the present disclosure, including educational methods, hospital and other clinical instructions, scientific research, drug discovery or development, academic research, pharmaceutical industry activities (including pharmaceutical sales), and any advertising or other promotional activities (including any form of written, oral, and electronic communication). Additionally, the kit may include other components depending on the particular use as described herein.

[0410] The kit may contain one or more of any of the components described herein in one or more containers. The components may be sterilely prepared, packaged in syringes, and shipped frozen. Alternatively, they may be contained in vials or other containers for storage. A second container may also contain other sterilely prepared components. Alternatively, the kit may include an active agent that is premixed and shipped in a vial, tube, or other container.

[0411] The kit may have various forms, such as a blister pouch, shrink-wrap pouch, vacuum-sealable pouch, sealable thermoformed tray, or similar pouch or tray form, with the accessories loosely packed in a pouch, one or more tubes, containers, boxes, or bags. The kit may be sterilized after the accessories are added, whereby the individual accessories in the container may be opened separately. The kit can be sterilized using any appropriate sterilization technique, such as radiation sterilization, heat sterilization, or other sterilization methods known in the art. The kit may also include other components, such as containers, cell culture media, salts, buffers, reagents, syringes, needles, fabrics such as gauze for applying or removing disinfectants, disposable gloves, supports for the drug before administration, etc., depending on the particular application. Some aspects of the present disclosure provide kits that include nucleic acid constructs that include nucleotide sequences encoding various components of the eVLPs described herein (including, by way of example only, napDNAbp, deaminase domain, gag protein, gRNA, and viral envelope glycoproteins). In some embodiments, the nucleotide sequence(s) include a heterologous promoter (or more than a single promoter) that drives expression of the BE-VLP system components.

[0412] Another aspect of the present disclosure provides kits that include one or more nucleic acid constructs encoding various components of the BE-VLP systems described herein, including, by way of example, nucleotide sequences that encode components of a BE-VLP system that are capable of delivering a base editor to a target cell. In some embodiments, the nucleotide sequences include a heterologous promoter that drives expression of the BE-VLP system component.

[0413] Cells that may contain any of the eVLPs, fusion proteins, and compositions described herein include prokaryotic and eukaryotic cells. In various aspects related to the production of eVLPs, the disclosure provides any suitable cells for use as VLP-producing cell lines, i.e., in various embodiments, cell lines that are transiently transformed with plasmids encoding the protein and nucleic acid components of the eVLP. In various other aspects related to the application of eVLPs, the disclosure provides any suitable target or recipient cells, for example, diseased cells or tissues of a subject in need of treatment with base editing delivered by a BE-VLP. The methods described herein may be used to deliver bases to eukaryotic cells (for example, mammalian cells, such as human cells). In some embodiments, the cells are in vitro (for example, cultured cells; in some embodiments, the cells are in vivo (for example, in a subject, such as a human subject); in some embodiments, the cells are ex vivo (for example, isolated from a subject and administered back to the same subject or to a different subject).

[0414] Typically, the eukaryotic cells are mammalian cells, such as human cells, chicken cells, or insect cells. Examples of suitable mammalian cells include, but are not limited to, HEK-293T cells, COS7 cells, Hela cells, and HEK-293 cells. Examples of suitable insect cells include, but are not limited to, High5 cells and Sf9 cells. In some embodiments, the insect cells lack undesirable human proteins, and their culture does not require animal serum.

[0415] Mammalian cells of the present disclosure include human cells, primate cells (e.g., Vero cells), rat cells (e.g., GH3 cells, OC23 cells), or mouse cells (e.g., MC3T3 cells). Various human cell lines include, but are not limited to, human embryonic kidney (HEK) cells, HeLa cells, cancer cells from the National Cancer Institute's 60 cancer cell lines (NCI60), DU145 (prostate cancer) cells, Lncap (prostate cancer) cells, MCF-7 (breast cancer) cells, MDA-MB-438 (breast cancer) cells, PC3 (prostate cancer) cells, T47D (breast cancer) cells, THP-1 (acute myeloid leukemia) cells, U87 (glioblastoma) cells, SHSY5Y human neuroblastoma cells (cloned from a myeloma), and Saos-2 (bone cancer) cells. In some embodiments, the eVLPs are delivered into human embryonic kidney (HEK) cells (e.g., HEK 293 or HEK 293T cells). In some embodiments, the eVLPs are delivered into stem cells (e.g., human stem cells), such as, for example, pluripotent stem cells (e.g., human pluripotent stem cells, including human induced pluripotent stem cells (hiPSCs)). Stem cells refer to cells that have the ability to divide indefinitely in culture and give rise to specialized cells. Pluripotent stem cells refer to a type of stem cell that can differentiate into all tissues of an organism, but is not capable of supporting the development of an entire organism by itself. Human induced pluripotent stem cells refer to somatic cells (e.g., mature or adult) that are reprogrammed into an embryonic stem cell-like state by forcing the expression of genes and factors important for maintaining the properties that define embryonic stem cells (see, e.g., Takahashi and Yamanaka, Cell 126(4):663-76, 2006, incorporated herein by reference). Human induced pluripotent stem cells express stem cell markers and are capable of producing cells characteristic of all three germ layers (ectoderm, endoderm, and mesoderm).

[0416] Additional non-limiting examples of cell lines that may be used in accordance with the present disclosure include 293-T, 293-T, 3T3, 4T1, 721, 9L, A-549, A172, A20, A253, A2780, A2780ADR, A2780cis, A431, ALC, B16, B35, BCP-1, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3, C2C12, C3H-10T1 / 2, C6, C6 / 36, Cal-27, CGR8, CHO, CML T1, CMT, COR-L23, COR-L23 / 5010, COR-L23 / CPR, COR-L23 / R23, COS-7, COV-434, CT26, D17, DH82, DU145, Du CaP, E14Tg2a, EL4, EM2, EM3, EMT6 / AR1, EMT6 / AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, Hepa1c1c7, High Five cells, HL-60, HMEC, HT-29, HUVEC, J558L cells, Jurkat, JY cells, K562 cells, KCL22, KG1, Ku812, KYO1, LNCap, Ma-Mel 1, 2, 3....48, MC-38, MCF-10A, MCF-7, MDA-MB-231, MDA-MB-435, MDA-MB-468, MDCK II, MG63, MONO-MAC 6, MOR / 0.2R, MRC5, MTD-1A, MyEnd, NALM-1, NCI-H69 / CPR, NCI-H69 / LX10, NCI-H69 / LX20, NCI-H69 / LX4, NIH-3T3, NW-145, OPCN / OPCT Peer, PNT-1A / PNT 2, PTK2, Raji, RBL cells, RenCa, RIN-5F, RMA / RMAS, S2, Saos-2 cells, Sf21, Sf9, SiHa, SKBR3, SKOV-3, T-47D, T2, T84, THP1, U373, U87, U937, VCaP, WM39, WT-49, X63, YAC-1, and YAR cells.

[0417] Some aspects of the present disclosure provide cells comprising any of the constructs disclosed herein. In some embodiments, host cells are transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, cells are transfected when naturally occurring in a subject. In some embodiments, the transfected cells are taken from a subject. In some embodiments, the cells are derived from cells taken from a subject, such as a cell line. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines are C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1, CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375 , ARH-77, Calu1, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB 55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelium, BALB / 3T3 mouse embryonic fibroblasts, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A 172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1 cell, BEAS-2B, bEnd.3, BHK-21, BR 293.BxPC3.C3H-10T1 / 2, C6 / 36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr- / -, COR-L23, COR-L23 / CPR, COR-L23 / 5010, COR-L23 / R23, COS-7, COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6 / AR1, EMT6 / AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepa1c1c7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812, KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MDCK 11, MOR / 0.2R, MONO-MAC 6, MTD-1A, MyEnd, NCI-H69 / CPR, NCI-H69 / LX10, NCI-H69 / LX20, NCI-H69 / LX4, NIH-3T3, NALM-1, NW-145, OPCN / OPCT cell lines, Peer, PNT-1A / PNT 2, RenCa, RIN-5F, RMA / RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell lines, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, and transgenic variants thereof.

[0418] Cell lines are available from a variety of sources known to those skilled in the art (see, for example, the American Type Culture Collection (ATCC) (Manassus, Va.)). In some embodiments, cells transfected with one or more vectors described herein are used to establish new cell lines comprising one or more vector-derived sequences. In some embodiments, cells transiently transfected with components of a CRISPR system as described herein (such as by transient transfection of one or more vectors or transfection with RNA) and modified through the activity of the CRISPR complex are used to establish new cell lines, including cells containing the modifications but lacking any other exogenous sequences. In some embodiments, cells transiently or non-transiently transfected with one or more vectors described herein, or cell lines derived from such cells, are used to evaluate one or more test compounds.

[0419] example Example 1. Therapeutic in vivo base editing with minimal off-target activity using engineered DNA-free virus-like particles (eVLPs) Base editors (BEs) enable the therapeutic correction of pathogenic point mutations in genomic DNA of living organisms. While various strategies have been used to deliver BEs in vivo, methods that deliver BE ribonucleoproteins (RNPs) to animal tissues would offer significant safety advantages over existing approaches for delivering DNA or mRNA. This paper reports the extensive engineering and application of engineered VLPs (eVLPs, also referred to herein as BE-VLPs), virus-like particles that efficiently package and deliver BEs or Cas9 RNPs without the potential for DNA delivery or unwanted DNA integration. By iteratively engineering the VLP architecture to overcome bottlenecks in cargo packaging, release, and localization, optimized fourth-generation eVLPs were produced that mediate efficient on-target base editing in vitro across various cell types and endogenous genomic loci, with minimal detected off-target editing and 4.7-fold higher editing after Cas9 nuclease delivery compared to first-generation VLPs. Using different glycoproteins in eVLPs alters their cellular tropism. The optimized eVLP also supported in vivo base editing in multiple organs after a single injection in mice, resulting in a 26-fold higher editing efficiency in the liver than previously described VLP architectures, a 78% knockdown of serum Pcsk9 levels, and partial restoration of visual function in a mouse model of genetic blindness. The frequency of off-target editing after treatment with eVLPs was substantially lower than base editing delivered by plasmid DNA or AAV, both in cultured cells and in vivo. eVLPs did not affect cell viability or induce detectable liver pathology. The cell-type tropism of eVLPs could be controlled by pseudotyping with different envelope glycoproteins. These results establish eVLPs as a promising method for in vivo therapeutic base editing that minimizes the risk of off-target editing or DNA integration.

[0420] Virus-like particles (VLPs), assemblies of viral proteins capable of infecting cells but lacking viral genetic material, have emerged as potentially promising vehicles for delivering gene editing agents as RNPs (Campbell et al., 2019; Choi et al., 2016; Gee et al., 2020; Hamilton et al., 2021; Indikova and Indik, 2020; Lyu et al., 2019; Lyu et al., 2021; Mangeot et al., 2019; Yao et al., 2021). VLPs delivering RNP cargo leverage the efficiency and tissue targeting advantages of viral delivery but avoid the risks associated with viral genome integration and long-term expression of editing agents. However, existing VLP-mediated strategies for delivering gene-editing RNPs have so far demonstrated low-to-moderate editing efficiencies in vivo or limited validation of their therapeutic efficacy (Campbell et al., 2019; Choi et al., 2016; Gee et al., 2020; Hamilton et al., 2021; Indikova and Indik, 2020; Lyu et al., 2019; Lyu et al., 2021; Mangeot et al., 2019; Yao et al., 2021). Indeed, therapeutic levels of postnatal in vivo gene editing using RNP-packaged VLPs have not previously been reported.

[0421] Described herein is the development and application of eVLPs, an engineered VLP platform for packaging and delivering therapeutic RNPs containing Cas9 nuclease and base editors in vitro and in vivo, which offers significant advantages over both viral and non-viral delivery strategies. Extensive VLP architecture engineering yielded fourth-generation eVLPs that package 16-fold more BE RNPs on average compared to previously reported initial VLP-based designs (Mangeot et al., 2019). These eVLPs enable highly efficient base editing with minimal off-target editing in a variety of cell types, including multiple immortalized cell lines, primary human and mouse fibroblasts, and primary human T cells, and a 4.7-fold improvement in Cas9 nuclease-mediated indel formation compared to previously reported Cas9-VLPs. A single in vivo injection of eVLPs into mice mediated efficient base editing of various target genes in multiple organs, robustly knocked down serum Pcsk9 levels, and partially restored visual function in a mouse model of genetic blindness. These results establish eVLPs as a useful platform for transiently delivering gene editing agents (as an example, BEs) in vivo with therapeutically relevant efficiency and minimal risk of off-target editing or DNA integration; the eVLPs described herein may similarly improve the in vivo delivery of other proteins and RNPs.

[0422] result Retroviral scaffolds support efficient base editor VLPs Retroviruses have been hypothesized to be attractive scaffolds for engineering base editor VLPs (BE-VLPs, also known as "eVLPs"). Retroviral capsids generally lack the rigid symmetry requirements of non-enveloped icosahedral viruses (Zhang et al., 2015), suggesting increased structural flexibility for incorporating non-native protein cargo. Additionally, retroviral tropism can be easily tuned by pseudotyping virions with different envelope glycoproteins, which can enable targeting of eVLPs to specific cell types (Cronin et al., 2005). Previous studies have demonstrated that fusing a desired protein cargo to the C-terminus of the retroviral gag polyprotein is sufficient to directly package the cargo protein into retroviral particles (Kaczmarczyk et al., 2011; Voelkel et al., 2010). More recently, similar strategies have been applied to package Cas9 RNPs within retroviral particles (Hamilton et al., 2021; Mangeot et al., 2019). Therefore, it was investigated whether retroviral scaffolds could support efficient BE-VLP formation in a manner that preserves BE activity.

[0423] For the initial (v1) BE-VLP design, ABE8e, a highly active adenine base editor (Richter et al., 2020), was fused to the C-terminus of the Friend murine leukemia virus (FMLV) gag polyprotein via a linker peptide cleaved by the FMLV protease during particle maturation (Figure 1A). FMLV-based VLPs have previously been successfully used to package and deliver Cas9 RNPs (Mangeot et al., 2019). eVLPs were produced by transfecting Gesicle 293T producer cells with plasmids expressing this FMLV gag-ABE8e fusion construct, wild-type FMLV gag-pro-pol polyprotein, VSV-G envelope glycoprotein, and sgRNAs targeting HEK293T cell genome site 2 or site 3 (hereafter referred to as HEK2 or HEK3).

[0424] After eVLP harvesting from the producer cell supernatant, HEK293T cells were transduced in vitro with the concentrated eVLPs. Encouragingly, v1 eVLPs robustly edited HEK2 and HEK3 genomic loci with over 97% efficiency at the highest dose in unsorted cells (Figure 1B). These eVLPs were confirmed via immunoblotting to contain Cas9, MLV capsid, and VSV-G protein (Figure 8A). These observations indicated that the FMLV retroviral scaffold supports eVLP formation and that v1 BE-VLPs can efficiently transduce and edit HEK293T cells in vitro.

[0425] Improved cargo release after VLP maturation While v1 eVLPs robustly edited the HEK2 and HEK3 loci in HEK293T cells, these commonly used test loci are not particularly amenable to gene editing and lack therapeutic relevance (Anzalone et al., 2020). To assess the therapeutic potential of eVLPs, their ability to introduce mutations into the BCL11A erythroid-specific enhancer, which upregulates fetal hemoglobin expression in red blood cells (Richter et al., 2020; Zeng et al., 2020), an established base editing strategy for treating β-hemoglobinopathies, was evaluated. v1 eVLPs achieved 73% editing efficiency at the BCL11A enhancer locus in HEK293T cells at high doses; however, editing levels declined sharply with decreasing doses (Figure 8B). These results indicated that v1 BE-VLP activity could be improved.

[0426] Cleavage of the gag-ABE8e linker by MLV protease after particle maturation is required to liberate free ABE8e RNPs. It was reasoned that linker cleavage efficiency may bottleneck BE-VLP editing (Figure 2A). To test this hypothesis, a series of second-generation (v2) engineered BE-eVLPs were constructed containing various protease-cleavable linker sequences between the MLV gag and ABE8e (Figure 8C). Initially, the retroviral scaffold was switched from Friend MLV to Moloney MLV (MMLV), a similar MLV strain whose protease substrate specificity has been extensively characterized (Feher et al., 2006). Four different linker sequences known to be cleaved with varying efficiencies by the MMLV protease were screened, and several novel gag-ABE8e linkers were identified that improved editing efficiency compared to v1 eVLPs (Figure 2B). Specifically, v2.4 BE-eVLPs exhibited 1.2- to 1.5-fold higher editing efficiency at all doses tested compared to v1 eVLPs (Figure 2B). To investigate the cleavage efficiency of the linker sequence in v2.1 to v2.4 BE-eVLPs, Western blots were performed to determine the fraction of cleaved ABE8e relative to full-length gag-ABE8e present in purified eVLPs. This analysis revealed that the v2.4 linker was cleaved more efficiently than the v2.1 and v2.2 linkers, but less efficiently than the v2.3 linker (Figures 8D-E).

[0427] These findings support a model in which the linker sequence in v2.4 BE-eVLP is cleaved at an optimal rate that supports efficient release of ABE8e RNP after VLP maturation but precludes premature release of ABE8e RNP before incorporation into VLPs. These findings demonstrate that the gag-cargo protein linker sequence is a critical parameter of VLP architecture, and that optimizing this sequence to balance linker cleavage kinetics between these two constraints can improve eVLP activity.

[0428] Improved cargo localization and loading into eVLPs Previously optimized BEs were fused to bipartite nuclear localization signals (NLSs) at their N- and C-termini, which facilitate nuclear import of BEs and enhance their access to genomic DNA (Koblan et al., 2018). However, gag-BE fusions must be localized to the cytoplasm and outer membrane of the producing cell in order to be incorporated into VLPs upon their formation (Figure 2C). The presence of two NLSs within the gag-BE fusion may interfere with localization of gag-BE to the outer membrane and prevent BE incorporation into VLPs.

[0429] To facilitate cytosolic gag-cargo localization in production cells, a third-generation (v3) eVLP architecture was designed containing a nuclear export signal (NES) in addition to the NLS. Previous studies have demonstrated that MLV-based VLPs can tolerate the addition of an NES at multiple locations within the gag protein (Wu and Roth, 2014). In the v3 design, an MLV protease-cleavable linker sequence was placed adjacent to the NES to ensure that the NES was cleaved from the cargo after VLP maturation (Figure 2D and Figure 9B), thereby liberating the NLS-flanked cargo protein, which could be efficiently imported into the nucleus of transduced cells.

[0430] All v3 BE-eVLP architectures contained the optimized gag-ABE8e linker sequence from v2.4 BE-eVLP. BE-eVLP v3.1, v3.2, and v3.3 retained the 3xNES motif fused to the C-terminus of ABE8e via an additional MMLV protease-cleavable linker and exhibited comparable or lower editing efficiency compared to v2.4 BE-eVLP (Figure 2E). However, v3.4 BE-eVLP, which contains the 3xNES motif at the C-terminus of MMLV gag immediately preceding the v2.4-optimized cleavable linker sequence, exhibited a 1.1- to 2.1-fold improvement in editing efficiency at the BCL11A enhancer locus compared to v2.4 BE-eVLP at all doses tested (Figure 2E). Notably, compared to the two separate cleavage events required for v3.1, v3.2, and v3.3 BE-eVLPs, v3.4 BE-eVLPs require only a single viral protease cleavage event, liberating an NES-free BE flanked by the NLS (Figures 2D and 9B). This may explain their superior efficiency. To further investigate the effect of NES addition on gag-ABE localization, immunofluorescence microscopy of producer cells transfected with the v3.4 gag-3xNES-ABE construct or the v2.4 gag-ABE construct was performed. This analysis revealed a 1.3-fold increase in the cytoplasmic localization of ABE protein detected in v3.4-transfected producer cells compared to v2.4-transfected producer cells (Figures 10C and 10D). These results demonstrate that BE-eVLP activity can be improved by promoting the extranuclear localization of gag-BE fusions in producer cells while maintaining the nuclear localization of BE released into transduced cells.

[0431] Finally, the gag-cargo:gag-pro-pol stoichiometry of v3.4 eVLPs was optimized. The optimal gag-cargo:gag-pro-pol stoichiometry was hypothesized to balance the amount of gag-cargo available for packaging into VLPs with the amount of MMLV protease (the "pro" in gag-pro-pol) required for VLP maturation (Figure 2F). To adjust this stoichiometry, the ratio of gag-3xNES-ABE 8e to wild-type MMLV gag-pro-pol plasmid transfected for VLP production was varied. Increasing the amount of gag-BE plasmid beyond the original ratio (38% gag-BE plasmid and 62% gag-pro-pol plasmid) used to produce v3.4 BE-eVLPs was found not to improve editing efficiency (Figure 2G). Reducing the gag-BE plasmid ratio from 38% to 25% slightly improved editing efficiency (Figure 2G). However, further decreasing the proportion of gag-BE plasmid below 25% reduced editing efficiency (Fig. 2G). These results are consistent with a model in which the optimal gag-BE:gag-pro-pol stoichiometry balances the amount of gag-BE available for packaging into VLPs with the amount of MMLV protease (the "pro" in gag-pro-pol) required for VLP maturation.

[0432] Results from this final round of optimization revealed a fourth-generation (v4) BE-eVLP formulation (Figure 2G) that combined the optimal gag-BE:gag-pro-pol stoichiometry (25% gag-BE) with the v3.4BE-eVLP architecture. The v4 BE-eVLPs were visualized by transmission electron microscopy, which confirmed their spherical morphology and approximate particle diameter of 100–150 nm (Figure 10A).

[0433] Next, the effect of this architecture engineering on the protein content of BE-eVLPs was determined. Anti-Cas9 and anti-MLV (p30) ELISA antibodies were performed to quantify the number of BE molecules and p30 (MLV capsid) molecules present in v1–v4 BE-eVLPs (Figure 10B–10C). These experiments revealed that v2.4, v3.4, and v4 BE-eVLPs contained 1.8-fold, 19.2-fold, and 11-fold more BE cargo protein molecules per particle, respectively, compared to v1 eVLPs (Figure 3A). This increase in BE protein content per particle correlates with an increase in the relative amount of sgRNA per particle, as measured by targeted RT-qPCR of lysed VLPs (Figure 3B). Interestingly, v4 BE-eVLPs contained fewer BE protein molecules per particle than v3.4 BE-eVLPs but the same amount of sgRNA molecules, suggesting that v3.4 and v4 BE-eVLPs may contain similar amounts of active BE RNP per particle. Additionally, v4 BE-eVLPs were produced at higher titers than v3.4 BE-eVLPs (Figure 10C).

[0434] These results support the model that increasing the number of active BE RNP molecules per particle can improve BE-eVLP editing efficiency. However, increasing the number of BE molecules per particle beyond a certain threshold can be detrimental, as these additional BE molecules do not appear to complex with the sgRNA, and there is a clear trade-off between the number of cargo molecules incorporated per VLP and overall VLP titer. Together, these results reveal additional important parameters that affect eVLP efficiency and demonstrate how these parameters can be improved by adjusting gag-cargo localization and gag-BE:gag-pro-pol stoichiometry.

[0435] v4 eVLPs support potent and highly efficient gene editing The above-described sequential VLP engineering efforts substantially improved the editing efficiency of v4 BE-eVLPs at the BCL11A enhancer locus in HEK293T cells to 95% at the highest dose (Figure 3C). v4 BE-eVLPs exhibited a 5.6-fold improvement in editing efficiency per unit volume compared to v1 eVLPs and a 2.2-fold improvement compared to v2.4 BE-eVLPs (Figure 3C). It was also observed that v4 BE-eVLPs exhibited an 8.5-fold improvement in base editing activity per viral particle in HEK293T cells (Figure 10D). To confirm that v4 VLP engineering supports general base editing improvements that are not restricted to one specific genomic locus or target cell line, v1, v2.4, v3.4, and v4 BE-eVLPs targeting the Dnmt1 locus in 3T3 mouse fibroblasts were tested. A very similar trend in editing efficiency was observed for the four eVLP architectures, with v4 eVLPs showing an 8.6-fold improvement in editing efficiency per unit volume compared to v1 eVLPs in 3T3 cells (Figure 3D). Furthermore, treatment with v4 eVLPs did not adversely affect the viability of HEK293T or 3T3 cells (Figure 10E). v4 BE-eVLPs also supported robust multiple editing of the BCL11A enhancer and HEK2 genomic loci in HEK293T cells (Figure 3E). These results demonstrate that v4 eVLPs mediate highly efficient base editing while minimizing perturbations to treated cells.

[0436] It was hypothesized that the engineered v4 eVLP architecture might similarly improve VLP-mediated delivery of other proteins in addition to base editors. To test this possibility, v1 and v4 VLPs were constructed that packaged Cas9 nuclease (Cas9-VLP) and an sgRNA targeting the EMX1 genomic locus. In HEK293T cells, a 4.7-fold improvement in indel frequency per unit volume produced by v4 Cas9-eVLPs compared to v1 Cas9-VLPs was observed (Figure 10F). This observation suggests that the optimized v4 eVLP architecture provides generalizable improvements to VLP-mediated delivery of proteins not limited to base editors.

[0437] An attractive feature of eVLPs is that their cellular tropism can, in principle, be tuned by producing them with different envelope glycoproteins. A similar strategy was previously used to tailor the tropism of Cas9-VLPs (Hamilton et al., 2021). To investigate whether eVLPs can be programmed to target specific cell types, we produced v4 eVLPs pseudotyped with the FuG-B2 envelope glycoprotein (Kato et al., 2011). FuG-B2 is an engineered envelope glycoprotein that contains the extracellular and transmembrane domains of the rabies virus envelope glycoprotein and the cytoplasmic domain of VSV-G, and can be used to pseudotype lentiviral vectors for neuron-specific transduction (Kato et al., 2011). Indeed, we observed that FuG-B2-pseudotyped v4 BE-eVLPs efficiently transduced and edited Neuro-2a cells (a mouse neuroblastoma cell line), but not mouse 3T3 fibroblasts (Figure 3F and Figure S10G). These results validate that the tissue specificity of eVLPs can be targeted by replacing them with other glycoproteins, such as those used in pseudotyped lentiviruses, to transduce specific cell populations.

[0438] Collectively, these findings identify factors that influence VLP activity and demonstrate that extensive manipulation of protease-cleavable linker sequences, gag-cargo localization, and gag-cargo:gag-pro-pol stoichiometry can overcome bottlenecks that limit VLP efficacy. These results also reveal novel insights into factors that influence VLP activity and establish v4 BE-eVLPs as a robust method for delivering BE RNPs to cultured cells.

[0439] v4 BE-eVLPs exhibit minimal off-target editing or DNA integration Given that v4 BE-eVLPs exhibit robust on-target base editing at several endogenous genomic loci in multiple cell types, their off-target editing profile was next assessed. BEs can mediate Cas-dependent off-target editing at a subset of Cas9 off-target binding sites, as well as low-level Cas-independent off-target editing throughout the genome (Anzalone et al., 2020). To assess Cas-dependent off-target editing by v4 BE-eVLPs compared to ABE8e plasmid transfection in HEK293T cells, targeted amplicon sequencing of known Cas9 off-target sites associated with three different sgRNAs targeting the HEK2, HEK3, and BCL11A enhancer loci was performed. Compared to plasmid transfection at these three genomic loci, v4 eVLP exhibited on-target editing efficiencies equal to or greater than those of v4 BE-eVLP; however, Cas-dependent off-target editing was observed to be 12-900-fold lower than that of v4 BE-eVLP (Figure 3G).

[0440] To assess Cas-independent off-target DNA editing, an orthogonal R-loop assay was performed, previously validated as a strategy to assess the ability of base editors to deaminate DNA in an unguided manner without the need for whole genome sequencing (Doman et al., 2020; Yu et al., 2020). Compared to transfection of a DNA plasmid encoding the same BE, v4 BE-eVLPs exhibited a >100-fold reduction in Cas-independent off-target editing, to virtually undetectable levels (Figure 3H, Figure 11B). These results confirm and extend previous findings that off-target editing by highly active BEs can be substantially minimized with RNP delivery ( Doman et al., 2020 ; Jang et al., 2021 ; Lyu et al., 2021 ; Newby et al., 2021 ; Rees and Liu, 2018 ; Richter et al., 2020 ; Yeh et al., 2018 ), and highlight the ability of eVLPs to support highly efficient on-target base editing with minimal off-target editing.

[0441] The DNA-free nature of eVLPs, in principle, avoids the possibility of DNA integration into the genome of transduced cells, a significant safety advantage over existing viral delivery modes (David and Doherty, 2017; Milone and O'Doherty, 2018). qPCR was used to verify that purified v4 BE-eVLPs contained less than 0.03 molecules of BE-encoding DNA per VLP (Figure 3I). Furthermore, while significant amounts (8.7 ng / μL) of BE-encoding DNA were detected in cell lysates from HEK293T cells transfected with the BE-encoding plasmid, no BE-encoding DNA was detected in cell lysates from v4 BE-eVLP-treated HEK293T cells above background levels (less than 0.02 ng / μL) in samples from untreated cells (Figure 3J). These results demonstrate that BE-eVLPs do not expose transduced cells to detectable levels of DNA encoding the base editor, thereby minimizing the potential for genomic integration of cargo DNA.

[0442] v4 BE-eVLPs efficiently edit primary human and mouse cells To further explore the utility of v4 BE-eVLPs, their ability to target and edit various primary human or mouse cells ex vivo was evaluated. ABE-mediated correction of a nonsense mutation in COL7A1, which causes recessive dystrophic epidermolysis bullosa (RDEB), has previously been demonstrated in primary human patient-derived fibroblasts (Osborn et al., 2020). After transduction of primary fibroblasts carrying a homozygous COL7A1 (R185X) mutation with v4 BE-eVLPs, over 95% editing was observed at the target adenine base, with no difference in cell viability between VLP-treated and untreated cells (Figure 4A and Figure S11C). Additionally, minimal Cas-dependent off-target editing was observed at 10 previously identified off-target sites (Osborn et al., 2020) (Figure S11D). The ability of v4 BE-eVLP to correct a nonsense mutation in primary fibroblasts derived from a mucopolysaccharidosis type IH mouse model (Wang et al., 2010) was also evaluated. Again, greater than 95% correction of the Idua (W392X) mutation was observed after v4 BE-eVLP transduction (Figure 4B). These results verify that BE-VLP activity is not restricted to immortalized cell lines and demonstrate that v4 BE-eVLP can achieve levels of base editing in primary human and mouse fibroblasts approaching 100%.

[0443] Next, BE-eVLP-mediated editing in primary human T cells was investigated. Gene editing strategies that reduce the expression of immunomodulatory proteins on the surface of T cells, including MHC class I and MHC class II, could advance T cell therapy by enabling "off-the-shelf" allogeneic chimeric antigen receptor (CAR) T cells. Previous reports have shown that disrupting the splice sites of the B2M and CIITA genes reduces MHC class I and MHC class II expression in primary human T cells (Gaudelli et al., 2020; LeibundGut-Landmann et al., 2004; Serreze et al., 1994). Treating primary human T cells with v4 BE-eVLP resulted in 45–60% disruption of the B2M and CIITA splice sites (Figure 4C). Collectively, these results confirm that eVLPs can efficiently edit clinically relevant primary human cell types ex vivo and lay the foundation for further optimization of BE-VLP editing efficiency in primary human T cells.

[0444] In vivo base editing in the CNS using eVLPs The robust activity of eVLPs ex vivo suggested that they may be a promising vehicle for delivering BE RNPs in vivo. To begin evaluating their in vivo efficacy, the ability of eVLPs to enable base editing within the mouse central nervous system (CNS) was first investigated. v4BE-eVLPs were produced to introduce silent mutations into mouse Dnmt1 at genomic loci known to be amenable to nuclease-mediated indel formation and adenine base editing in vivo (Levy et al., 2020; Swiech et al., 2015). To deliver BE-eVLPs to the CNS, neonatal intravenous (P0) intravenous (ICV) injection was performed, which bypasses the blood-brain barrier and is directly injected into the cerebrospinal fluid (CSF), similar to the intrathecal injection currently used to deliver nusinersen to patients with spinal muscular atrophy (Mercuri et al., 2018).

[0445] v4 BE-eVLPs were co-injected into each hemisphere with a VSV-G-pseudotyped lentivirus encoding EGFP fused to the nuclear membrane-localized Klarsicht / ANC-1 / Syne-1 homology (KASH) domain (Figure 5A). This strategy was reasoned to allow for the isolation of GFP-positive nuclei as a way to enrich for cells exposed to eVLPs. This approach is particularly useful for determining editing efficiency after injection into the brain, where many cells may be inaccessible. Three weeks after injection, bulk unsorted (all nuclei) and GFP-positive nuclei from cortical and midbrain tissue were analyzed, and base editing was assessed by high-throughput sequencing (Figure 5A).

[0446] The frequency of GFP-positive nuclei in both cortical and midbrain tissues was low (Figure S13B), consistent with previous reports that cells transduced by VSV-G pseudotyped lentivirus injected into mouse brains were localized near the injection site (Humbel et al., 2021; Parr-Brownlie et al., 2015). This is likely because the size of viral particles, with an average diameter approximately three times larger than the width of the brain extracellular space (Thorne and Nicholson, 2006), may hinder diffusion through bulk brain tissue. Encouragingly, we observed 53% and 55% editing in GFP-positive cortical and midbrain cells, respectively, corresponding to 6.1% and 4.4% editing in bulk cortex and midbrain (Figure S5B). These data establish BE-eVLP as a new non-viral delivery system for CNS base editing applications that delivers robust levels of active BE RNP per transduction event; however, improvements in transduction efficiency are required to achieve high levels of editing in bulk brain tissue.

[0447] In vivo liver base editing with eVLPs results in efficient knockdown of Pcsk9 To further explore the utility of BE-eVLPs in vivo, their ability to mediate therapeutic base editing in adult animals was investigated. First, proprotein convertase subtilisin / kexin type 9 (Pcsk9), a therapeutically relevant gene involved in cholesterol homeostasis (Abifadel et al., 2003; Fitzgerald et al., 2014), was targeted. Loss-of-function PCSK9 mutations occur naturally without apparent adverse health effects (Abifadel et al., 2003; Cohen et al., 2005; Cohen et al., 2006; Hooper et al., 2007; Rao et al., 2018). These individuals have lower levels of low-density lipoprotein (LDL) cholesterol in their blood, which reduces their risk of atherosclerotic cardiovascular disease, suggesting that disrupting the PCSK9 gene may be a promising strategy for treating familial hypercholesterolemia (Musunuru et al., 2021; Rothgangl et al., 2021). The optimized v4 BE-VLP architecture supported much more robust liver editing than the previously described VLP architecture (v1 BE-VLP), which mediated only 1.5% editing, 26-fold less than v4 eVLP at the same dose (Figure 6B).

[0448] A previously established base editing strategy for Pcsk9 knockdown in mouse liver, BE-eVLPs (Musunuru et al., 2021; Rothgangl et al., 2021), was designed and produced to target and disrupt the splice donor at the boundary between Pcsk9 exon 1 and intron 1. Systemic (retro-orbital) injections of eVLPs into 6-7 week-old adult C57BL / 6 mice were performed, and base editing in bulk liver was measured 1 week after injection (Figure 6A). At the maximum dose (7 × 10 11Following treatment with v4 BE-eVLP (Figure 6B), a 63% editing efficiency in bulk liver was observed, comparable to the editing efficiency typically achieved at this site with optimized state-of-the-art AAV-based delivery modalities and lipid nanoparticle (LNP)-based mRNA delivery systems (Musunuru et al., 2021; Rothgangl et al., 2021). The engineered v4 BE-eVLP architecture supported 26-fold higher editing levels in the liver than a VLP architecture based on a previously reported design (v1 BE-VLP) at the same dose (Figure 6B). These results establish efficient base editing by RNPs at therapeutically relevant loci in mouse liver.

[0449] In mice treated with the highest dose of v4 BE-eVLP, base editing efficiency was also evaluated in non-liver tissues, including the heart, skeletal muscle, lung, kidney, and spleen. 4.3% base editing was observed in the spleen, and no editing above background levels was observed in the lung, kidney, heart, and muscle. This tissue-wide editing pattern is consistent with the previously characterized tissue tropism of intravenously administered VSV-G-pseudotyped particles (Pan et al., 2002).

[0450] To assess whether treatment with BE-eVLPs results in Cas-dependent off-target editing in liver tissue, we performed CIRCLE-seq to designate potential off-target loci (Tsai et al., 2017). From the designated loci, 14 candidate off-target sites were selected and examined by targeted high-throughput sequencing based on homology near the PAM-proximal region of the protospacer. Detectable off-target editing above background levels was observed in 7x10 11No off-target editing was observed at either of these loci in genomic DNA isolated from the livers of mice treated with v4 BE-eVLPs (Figure 6D). In contrast, low but detectable (0.1–0.3%) levels of off-target editing were observed in the dual AAV8 vectors (1x10) encoding ABE8e and the same Pcsk9-targeting sgRNA. 11 This was observed at three of these loci in genomic DNA isolated from the livers of mice treated with v4 BE-eVLPs (Figure 6D). These results demonstrate that v4 BE-eVLPs can provide comparable on-target editing, but with minimal off-target editing in vivo, an improvement over existing virus-based delivery approaches.

[0451] Phenotypic analysis performed 1 week after injection showed a 7x10 increase in IL-1 expression compared to untreated mice. 11 We demonstrated a 78% reduction in serum Pcsk9 protein levels in mice treated with v4 BE-eVLPs (Figure 6E). To assess the potential toxicity of systemically administered eVLPs, 7x10 11 One week after injection of v4BE-eVLP, serum alanine aminotransferase (ALT) and aspartate transaminase (AST) levels, important biomarkers of hepatocellular injury (Meunier and Larrey, 2019), were assessed. All mice exhibited AST and ALT levels within the normal range, with no discernible differences between untreated and BE-eVLP-treated mice (Figure 14A). Furthermore, histological examination of the liver was performed on samples from eVLP-treated and untreated mice, revealing no obvious morphological differences attributable to BE-VLP treatment (Figures 14B-C). Collectively, these results demonstrate that v4 BE-eVLP can mediate efficient, therapeutically relevant base editing in mouse livers without detectable adverse effects or off-target editing.

[0452] v4 BE-eVLPs restore visual function in a mouse model of genetic blindness Finally, BE-eVLPs have been applied to correct disease-causing point mutations in adult mouse models of inherited retinal disorders. Loss-of-function mutations in multiple genes are associated with various forms of Leber congenital amaurosis (LCA), a family of monogenic retinal disorders that are responsible for retinal degeneration, early-onset visual impairment, and eventual blindness (Cideciyan, 2010; den Hollander et al., 2008). Gene editing approaches hold promise for treating and curing congenital blindness; an ongoing clinical trial (NCT03872479) uses AAV-delivered Cas9 nuclease to disrupt an aberrant splice site in CEP290 associated with the rare Leber congenital amaurosis 10 (LCA10). Loss-of-function mutations in other genes, including the retinoid isomerohydrolase RPE65, are also candidates for in vivo correction using precision gene editing agents ( Sodi et al., 2021 ; Suh et al., 2021 ).

[0453] We investigated whether v4 BE-eVLPs could restore visual function in a mouse model of LCA. rd12 mice carry a nonsense mutation in exon 3 of Rpe65 (c.130C>T; p.R44X), which causes near-complete loss of visual function (Pang et al., 2005; Suh et al., 2021). A homologous mutation responsible for LCA has recently been identified in humans (Zhong et al., 2019), highlighting the clinical relevance of the rd12 model.

[0454] A v4BE-eVLP (hereafter referred to as ABE8e-NG-eVLP) encapsulating the ABE8e-NG RNP and an sgRNA targeting the Rpe65 (R44X) mutation (Figure 7A) was designed and produced. The ABE8e-NG-eVLP was pseudotyped with VSV-G to enable efficient transduction of retinal pigment epithelial (RPE) cells (Puppo et al., 2014; Suh et al., 2021). The ABE8e-NG-eVLP was injected subretinally into 4-week-old rd12 mice. In a separate cohort, a replication-deficient lentivirus encoding the same ABE8e-NG and sgRNA construct (ABE8e-NG-LV) was also injected subretinally. Lentiviral delivery of ABE has previously been reported to successfully restore visual function in rd12 mice (Suh et al., 2021).

[0455] Five weeks after injection, RPE tissue was harvested and high-throughput sequencing of RPE genomic DNA was performed (Figure 7B). Encouragingly, sequencing analysis revealed that ABE8e-NG-VLP and ABE8e-NG-LV successfully mediated 21% and 11.5% correction of the R44X mutation at position A6 of the protospacer, respectively (Figure 7C). Notably, despite the transient nature of BE-VLP delivery, ABE8e-NG-VLP achieved 1.8-fold higher editing at the target base compared to ABE8e-NG-LV. These results demonstrate that eVLPs enable highly efficient correction of pathogenic mutations in mouse RPE.

[0456] While highly efficient correction of the targeted mutation was observed, both ABE8e-NG-eVLP and ABE8e-NG-LV induced significant levels of bystander editing (Figure 7C), due to the wide editing window of ABE8e-NG (Richter et al., 2020), such that the majority of edited alleles contained mutations at A3, A6, and / or A8, as opposed to A6 alone (Figure 7D). Bystander editing at positions A3 and A8 resulted in the Rpe65 missense mutations C45R and L43P, respectively. The L43P mutation has previously been shown to inactivate the Rpe65 enzyme (Suh et al., 2021). Indeed, after performing scotopic electroretinography (ERG) to assess retinal cell responses, minimal rescue of visual function was observed in both ABE8e-NG-eVLP- and ABE8e-NG-LV-injected eyes (Figure 7E). These results suggested that the wide base editing window of ABE8e-NG was not well suited to precisely correct the Rpe65(R44X) mutation.

[0457] To address this limitation, we designed and produced v4 BE-eVLPs encapsulating ABE7.10-NG, which exhibit a narrower editing window compared to ABE8e-NG (Huang et al., 2019; Richter et al., 2020). Subretinal injection of ABE7.10-NG-eVLPs into adult rd12 mice resulted in 12% correction of the R44X mutation in RPE genomic DNA with virtually no bystander editing (Figure 7F). Specifically, we observed that ABE7.10-NG-eVLP treatment resulted in 11% complete R44X correction without bystander editing, a 9-fold improvement in complete correction compared to ABE8e-NG-eVLP treatment (Figure 7G). Furthermore, treatment with ABE7.10-NG-eVLP resulted in a 1.4-fold improvement in bystander-free correction compared to treatment with ABE7.10-NG-LV, a lentivirus encoding the same ABE7.10-NG and sgRNA constructs, further demonstrating that transient delivery of v4 BE-eVLP can achieve comparable or higher editing efficiency compared to lentiviral BE delivery (Figure 7G).

[0458] Western blot analysis confirmed that ABE7.10-NG-eVLP treatment restored Rpe65 protein expression. Notably, ABE7.10-NG-LV-treated eyes expressed BE protein even 5 weeks after injection, whereas ABE7.10-NG-eVLP-treated eyes did not (Figure 7I), demonstrating transient exposure of cells to the base editor delivered using eVLP in vivo. Importantly, ABE7.10-NG-eVLP successfully rescued visual function to a similar level as ABE7.10-NG-LV, as measured by ERG in treated eyes (Figures 7H and 7J). This level of ERG rescue has previously been shown to correspond to other improvements in visual function, including restoration of visual chromophores and visual cortical responses (Suh et al., 2021). These results demonstrate that eVLPs can mediate efficient correction of pathogenic mutations in mouse RPE, along with amelioration of the disease phenotype.

[0459] To further analyze the editing outcomes, RNA was extracted from treated eyes, and targeted high-throughput sequencing of specific cDNAs was performed. As expected, in eVLP-treated eyes, up to 64% A·T-to-G·C conversion of the targeted adenine (A6) was observed in the on-target Rpe65 transcript (Figure 15A). The higher proportion of corrected Rpe65 transcripts compared to the Rpe65 genomic locus potentially reflects nonsense-mediated decay of uncorrected mRNAs.

[0460] BEs are known to exhibit low levels of transcriptome-wide Cas-independent off-target RNA editing (Anzalone et al., 2020). To investigate this possibility, off-target RNA editing by ABE-eVLPs and ABE-LVs was assessed by sequencing Mcm3ap and Perp transcripts from treated eyes, two transcripts previously identified as potential candidates for off-target RNA editing based on their sequence similarity to native TadA deaminase substrates (Jo et al., 2021). RNA off-target editing by ABE8e-NG-LVs in both transcripts, and low but detectable RNA off-target editing by ABE7.10-NG-LVs at a single adenine in Perp, was observed (Figures 15B-C). In contrast, no RNA off-target editing above background was detected in these two transcripts by ABE8e-NG-eVLPs or ABE7.10-NG-eVLPs (Figures 15B and 15C). Collectively, these findings highlight the therapeutic utility of eVLPs as a DNA-free method for transiently delivering BE RNPs in vivo with high on-target editing and minimal off-target editing.

[0461] Consideration Here, we present an efficiently engineered VLP platform capable of safely delivering RNPs for therapeutically relevant ex vivo and in vivo applications. By identifying and designing solutions to three distinct bottlenecks in VLP delivery efficiency, protein loading was improved by an average of 16-fold within v4 eVLPs compared to earlier designs based on VLP scaffolds. Base editing efficiency was improved by an average of 8-fold. These findings suggest that v4 eVLPs are highly versatile and suitable for a wide range of base editing a...

Claims

1. 1. A method for producing a virus-like particle (VLP), said method comprising: (a) introducing into a cell a polynucleotide comprising a nucleic acid sequence encoding a fusion protein, wherein the fusion protein is (i) group-specific antigen (gag) nucleocapsid proteins; (ii) nucleic acid programmable DNA binding protein (napDNAbp); (iii) a cleavable linker; (iv) a nuclear export sequence (NES); Including, (b) expressing the fusion protein; and (c) producing a VLP, wherein the VLP comprises (1) the fusion protein or a napDNAbp cleaved from the fusion protein; The method comprising:

2. (i) the VLP comprises napDNAbp cleaved from the fusion protein, and / or (ii) the VLP comprises a fusion protein; and / or (iii) The method of claim 1, wherein the VLP comprises a cleavage product comprising the gag nucleocapsid protein and the NES from the fusion protein, and the cleavage product lacks the nap DNAbp.

3. 3. The method of claim 1 or 2, further comprising introducing into the cell a polynucleotide comprising a nucleic acid sequence encoding a protein comprising a group-specific antigen (gag) and a protease (pro).

4. 4. The method of claim 3, wherein the protein comprises a Rous Sarcoma Virus (RSV) gag-pro polyprotein, a Feline Immunodeficiency Virus (FIV) gag-pro polyprotein, a Simian Immunodeficiency Virus (SIV) gag-pro polyprotein, a Human Immunodeficiency Virus Type 1 (HIV-1) gag-pro polyprotein, a Human Immunodeficiency Virus Type 2 (HIV-2) gag-pro polyprotein, a MMLV gag-pro polyprotein, or an FMLV gag-pro polyprotein.

5. 4. The method of claim 3, wherein the ratio of a polynucleotide comprising a nucleic acid sequence encoding a protein to a polynucleotide comprising a nucleic acid sequence encoding a fusion protein is approximately 10:1, approximately 9:1, approximately 8:1, approximately 7:1, approximately 6:1, approximately 5:1, approximately 4:1, approximately 3:1, approximately 2:1, approximately 1.5:1, approximately 1:1, or approximately 0.5:1, and optionally the ratio is approximately 3:

1.

6. 10. The method of claim 1, further comprising recovering the VLPs, and optionally further comprising purifying and / or concentrating the VLPs.

7. The method of claim 1, further comprising introducing into the cell a polynucleotide comprising a nucleic acid sequence encoding a viral envelope glycoprotein.

8. 8. The method of claim 7, wherein the viral envelope glycoprotein is an adenovirus envelope glycoprotein, an adeno-associated virus envelope glycoprotein, a retrovirus envelope glycoprotein, a lentivirus envelope glycoprotein, a vesicular stomatitis virus G protein (VSV-G), a baboon retroviral envelope glycoprotein (BaEVRless), a FuG-B2 envelope glycoprotein, an HIV-1 envelope glycoprotein, or an ecotropic murine leukemia virus (MLV) envelope glycoprotein.

9. 2. The method of claim 1, further comprising introducing into the cell a polynucleotide comprising a nucleic acid sequence encoding a guide RNA (gRNA), wherein the gRNA binds to the napDNAbp.

10. 2. The method of claim 1, wherein the napDNAbp is a Cas9 protein. Optionally, the Cas9 protein is a Cas9 nickase or a nuclease-inactive Cas9 (dCas9).

11. The fusion protein is (i) a deaminase domain, and / or (ii) at least three NESs; and / or (iii) at least one nuclear localization sequence (NLS); 2. The method of claim 1, further comprising: optionally, at least one NLS located at or near the N-terminus and / or C-terminus of the napDNAbp.

12. (i) a cleavable linker is positioned between the napDNAbp and the NES, and / or 2. The method of claim 1, wherein (ii) the cleavable linker comprises a protease cleavage site, optionally, the protease cleavage site is a Moloney murine leukemia virus (MMLV) protease cleavage site or a Friend murine leukemia virus (FMLV) protease cleavage site.

13. 2. The method of claim 1, wherein the gag nucleocapsid protein comprises an MMLV gag nucleocapsid protein or an FMLV gag nucleocapsid protein.

14. The fusion protein is, from the N-terminus to the C-terminus, NH 2 2. The method of claim 1, comprising -[gag nucleocapsid protein]-[1X-3X NES]-[cleavable linker]-[NLS]-[deaminase domain]-[nap DNAbp]-[NLS]-COOH, wherein each instance of ]-[ independently comprises an optional linker.

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