Virus-like particles for gene editing

Abstract

Provided are compositions and methods for preparing virus-like particles (VLPs) for delivering gene editors (e.g., base editors and prime editors) to a target cell to achieve highly efficient and accurate gene editing (e.g., base editing and prime editing). An example composition includes a first protein (Protein #1) comprising a Gag-Pol polyprotein, a second protein (Protein #2) comprising an envelope protein, a third protein (Protein #3) comprising a Cas protein without a Gag protein, a fourth protein (Protein #4) comprising a Gag protein fused to a first RNA binding domain (RBD#1), and a first RNA (RNA #1) comprising a guide RNA fused to a first RNA aptamer sequence (APT#1) that is recognizable by the RBD#1.

Description

Virus-Like Particles for Gene EditingCROSS REFERENCE TO RELATED APPLICATIONS [1] This application claims the benefit of PCT Application No. PCT / CN2024 / 079974, filed March 4, 2024, the content of which is hereby incorporated by reference in its entirety.Reference to an Electronic Sequence Listing[2] The content of the electronic sequence listing (49BD-382603.xml; Size: 20,749 bytes; and Date of Creation: March 3, 2025) is herein incorporated by reference in its entirety.BACKGROUND[3] CRISPR and its derived gene editing tools have the potential to treat many hereditary diseases. A base editor (BE) catalyzes precise base substitution without requiring double-stranded DNA breaks (DSBs) and thus largely reduces the adverse effects caused by DSBs, such as nucleotide insertions or deletions (indels), large genomic deletions, chromosomal breaks and other complex rearrangements.[4] The use of BE for the treatment of various genetic diseases in vivo requires its delivery to the target tissue or organ. Currently, the major approach for delivering gene editor in vivo is adeno-associated virus (AAV). Due to the cargo size limitation of AAT2A[5] V, however, BE has to be split and then packaged into separate AAVs, which may affect BE’s editing efficiency. Moreover, as AAV can remain in the delivered cells for a long time, the constant expression of BE proteins may also increase off-target effects of the BE. In contrast, the short life-time of mRNA or ribonucleoprotein (RNP) in delivered cells can significantly reduce the possibility of off-target effects of gene editing tools. Thus, efficient delivery of gene editing tools in the form of mRNA or RNP is a desirable and safer way, especially in the circumstances of therapeutic applications.[6] Virus-like particles (VLPs) are modified retroviral vectors that do not deliver DNA but can deliver mRNAs and proteins. There have been, however, limited reports for the use of VLP to deliver base editors or primer editors at high efficiency. SUMMARY[7] Virus-like particles (VLP) have been developed that package base editors and prime editors for delivery into a target cell for base editing or prime editing. To ensure that the Cas protein is encapsulated in the VLP but is later released from it, it is fused to the viral Gag protein through a protease cleavage site (PCS) that is cleaved during the VLP maturation process. It is discovered herein, however, that such a fusion can lead to frequent truncation and inactivation of the Cas protein. [8] Accordingly, various VLP preparation configurations have been designed tested to avoid such Cas truncation. As demonstrated in the accompanying experimental examples, such designs induced efficient and precise editing in different cells and in vivo. [9] Accordingly, one embodiment of the present disclosure provides a composition, comprising (a) a first protein (Protein #1) comprising a Gag-Pol polyprotein; (b) a second protein (Protein #2) comprising an envelope protein; (c) a third protein (Protein #3) comprising a Cas protein without a Gag protein; (d) a fourth protein (Protein #4) comprising a Gag protein fused to a first RNA binding domain (RBD#1); and (e) a first RNA (RNA #1) comprising a guide RNA fused to a first RNA aptamer (APT) sequence (APT#1) that is recognizable by the RBD#1.

[10] In some embodiments, in the Protein #4, the Gag protein is fused to the RBD#1 through a first protease cleavage site (PCS) cleavable by the Gag-Pol polyprotein upon proteolytic virion maturation.

[11] In some embodiments, the Protein #3 further comprises a nucleobase deaminase (ND) or a reverse-transcriptase (RT).

[12] In some embodiments, the composition further comprises a fifth protein (Protein #5) that comprises a nucleobase deaminase (ND) or a reverse-transcriptase (RT) fused to a second RNA binding domain (RBD#2), wherein the RNA #1 further comprises a second APT sequence (APT#2) that is recognizable by the RBD#2, wherein the RBD#1 and the RBD#2 have distinguishable RNA recognition properties.

[13] In some embodiments, the composition further comprises a fifth protein (Protein #5) that comprises a nucleobase deaminase (ND) or a reverse-transcriptase (RT) fused to a second RNA binding domain (RBD#2); and a second RNA (RNA #2) that comprises a helper guide RNA (hsgRNA) fused to a second copy of the APT#1 and a second APT sequence (APT#2) that is recognizable by the RBD#2, wherein the RBD#1 and the RBD#2 have distinguishable RNA recognition properties, and wherein the gRNA and the hsgRNA preferably recognize adjacent nucleotide sequences on a target genome.

[14] In some embodiments, the Protein #3 further comprises a nuclear export signal (NES) fused to the Cas protein, through a second PCS cleavable by the Gag-Pol polyprotein upon proteolytic virion maturation.

[15] In some embodiments, the Protein #4 further comprises a second NES that is separated from the RBD#1 upon cleavage of the first PCS.

[16] In some embodiments, the Protein #4 further comprises a first uracil glycosylase inhibitor (UGI) which stays fused to the RBD#1 upon cleavage of the first PCS.

[17] In some embodiments, the Protein #5 further comprises a second UGI.

[18] In some embodiments, the composition further comprises a sixth protein (Protein #6) that comprises a third UGI fused to a third RNA binding domain (RBD#3), wherein the RNA #1 further comprises a third APT sequence (APT#3) that is recognizable by the RBD#3, wherein the RBD#1, the RBD#2 and the RBD#3 each has distinguishable RNA recognition property.

[19] In some embodiments, the ND is fused to a nucleobase deaminase inhibitor through a protease recognition site.

[20] In some embodiments, the protease recognition site is recognized by Tev protease, TuMV protease, PPV protease, PVY protease, ZIKV protease or WNV protease.

[21] In some embodiments, the envelop protein is vesicular stomatitis virus glycoprotein (VSVG).

[22] Also provided, in one embodiment, is a virus-like particle (VLP) comprising: (a) a first protein (Protein #1) comprising a Gag-Pol polyprotein; (b) a second protein (Protein #2) comprising an envelope protein, which is preferably vesicular stomatitis virus glycoprotein (VSVG); (c) a third protein (Protein #3) comprising a Cas protein without a Gag protein; (d) a fourth protein (Protein #4) comprising a Gag protein; and (e) a first RNA (RNA #1) comprising a guide RNA fused to a first APT sequence (APT#1) that is recognizable by a first RNA binding domain (RBD#1).

[23] In some embodiments, the Protein #3 further comprises a nucleobase deaminase (ND) or a reverse-transcriptase (RT).

[24] In some embodiments, the VLP further comprises a fifth protein (Protein #5) that comprises a nucleobase deaminase (ND) or a reverse-transcriptase (RT) fused to a second RNA binding domain (RBD#2), wherein the RNA #1 further comprises a second APT sequence (APT#2) that is recognizable by the RBD#2, wherein the RBD#1 and the RBD#2 have distinguishable RNA recognition properties.

[25] In some embodiments, the VLP further comprises a fifth protein (Protein #5) that comprises a nucleobase deaminase (ND) or a reverse-transcriptase (RT) fused to a second RNA binding domain (RBD#2); and a second RNA (RNA #2) that comprises a helper guide RNA (hsgRNA) fused to a second copy of the APT#1 and a second APT sequence (APT#2) that is recognizable by the RBD#2, wherein the RBD#1 and the RBD#2 have distinguishable RNA recognition properties, and wherein the gRNA and the hsgRNA preferably recognize adjacent nucleotide sequences on a target genome.

[26] In some embodiments, the Protein #4 further comprises a second NES fused to the Gag protein.

[27] In some embodiments, the Protein #5 further comprises a first uracil glycosylase inhibitor (UGI).

[28] In some embodiments, the VLP further comprises a sixth protein (Protein #6) that comprises a second UGI fused to a third RNA binding domain (RBD#3), wherein the RNA #1 further comprises a third APT sequence (APT#3) that is recognizable by the RBD#3, wherein the RBD#1, the RBD#2 and the RBD#3 each has distinguishable RNA recognition property.

[29] In some embodiments, the VLP further comprises a cell targeting moiety which can be a standalone protein or is part of any other protein as discussed herein. In some embodiments, the cell targeting moiety is a CD117 ligand, such as an anti-CD117 antibody (e.g., briquilimab, 2G4, SR-1) or a fragment thereof.

[30] Another embodiment provides a method for preparing a virus-like particle (VLP), comprising culturing a cell engineered to express each of the proteins and RNA of the present disclosure in a medium, collecting supernatant from the medium, and precipitating VLP formed in the supernatant.

[31] Also provided is a method for delivering a Cas protein and associated guide RNA to a target cell, comprising transducing the target cell with the VLP of the disclosure.

[32] Still further provided is an isolated cell that expresses the proteins and RNA of the composition of the present disclosure, comprising, in addition to sequencing encoding the proteins and RNA, a construct encoding a first mRNA that encodes a Rev protein, wherein the first mRNA further comprises a first Rev response element (RRE).

[33] In some embodiments, the first mRNA further encodes the Protein #4.

[34] In some embodiments, within the first mRNA, the Rev-coding sequence and the RRE are separated from the Gag protein-coding sequence by a 2A self-cleavage peptide coding sequence.

[35] In some embodiments, the cell further comprises a second construct that can be transcribed into a second mRNA encoding the Protein #1, wherein the second mRNA further comprises a second RRE.

[36] Also provided is a method for preparing a composition of any one of claims 1-10, comprising culturing the isolated cell in a medium and collecting supernatant from the medium.

[37] Also provided, is a base editor, comprising (A) a first fusion protein that comprises a Cas protein and at least a UGI, (B) a second fusion protein that comprises a deaminase, a RNA binding domain (RBD) and at least a UGI, and (C1) a single guide RNA (sgRNA) fused to a RNA aptamer (APT), or (C2) a sgRNA and a helper sgRNA (hsgRNA) that is fused to the APT, wherein the RBD binds the ATP.

[38] In some embodiments, the first fusion protein comprises at least two, three, or four UGI. In some embodiments, the at least two or more UGI are disposed at the C-terminus of the Cas protein.

[39] In some embodiments, the second fusion protein comprises no more than one UGI. In some embodiments, the UGI is disposed at the N-terminus of the deaminase. In some embodiments, the deaminase is a cytidine deaminase.

[40] In some embodiments, the base editor includes at least 1, 2, or 3 UGI not included in the first or second fusion protein.BRIEF DESCRIPTION OF THE DRAWINGS

[41] FIG. 1: Version 0 of VLP system did not induce detectable editing at the on-target site. 1A: Schematic diagrams of the plasmids for the production of version 0 of VLP system (VLP-V0) containing BE3 or hA3A_BE3. 1B: The Sanger sequencing results of the PRR5L on-target site in the 293FT cells transfected with the plasmid expressing BE3, or transduced with the VLP_V0 containing BE3 or hA3A_BE3. The non-treated 293FT cells served as negative control. 1C: The base editing efficiencies at the C6 and C7 sites, which were quantified from the Sanger sequencing results in 1B. 1D: Western blot results showed the expression of BE3 and hA3A-BE3 in the 293FT cells transfected with the corresponding plasmids or transduced with the corresponding VLP_V0.

[42] FIG. 2: Comparison of different strategies for packing D10A in VLP. 2A: Schematic diagrams of the plasmids for the production of VLP-D10A, in which D10A was directly fused to Gag. 2B: Schematic diagrams of the plasmids for the production of VLP-COM / D10A, in which the COM protein was fused to Gag and D10A was recruited by the Com-containing hsgRNA and sgRNA. 2C: Western blot results showed the expression of D10A in the 293FT cells transfected with the D10A-expressing plasmid or transduced with VLP-D10A or VLP-COM / D10A.

[43] FIG. 3: Construction of VLP_V1-tBE_V1 and its editing efficiency. 3A: Schematic diagrams of the plasmids for the production of version 1 of VLP system containing tBE_V1 (VLP_V1-tBE_V1). 3B: The Sanger sequencing results of the PRR5L on-target site in the 293FT cells transduced with VLP_V1-tBE_V1. The non-treated 293FT cells served as negative control. 3C: The base editing efficiencies at the C6 and C7 sites, which were quantified from the Sanger sequencing results in 3B.

[44] FIG. 4: Construction of VLP_V2-tBE_V1 and its editing efficiency. 4A: Schematic diagrams of the plasmids for the production of version 2 of VLP system containing tBE_V1 (VLP_V2-tBE_V1). 4B: The Sanger sequencing results of the PRR5L on-target site in the 293FT cells transduced with VLP_V2-tBE_V1. The non-treated 293FT cells served as negative control. 4C: The base editing efficiencies at the C6 and C7 sites, which were quantified from the Sanger sequencing results in 4B.

[45] FIG. 5: Construction of VLP_V3-tBE_V1 and its editing efficiency. 5A: Schematic diagrams of the plasmids for the production of version 3 of VLP system containing tBE_V1 (VLP_V3-tBE_V1). 5B: The Sanger sequencing results of the PRR5L on-target site in the 293FT cells transduced with VLP_V3-tBE_V1. The non-treated 293FT cells served as negative control. 5C: The base editing efficiencies at the C6 and C7 sites, which were quantified from the Sanger sequencing results in 5B. 5D: The Sanger sequencing results of the HBG on-target site in the 293FT cells transduced with VLP_V3-tBE_V1. The non-treated 293FT cells served as negative control. 5E: The base editing efficiencies at the C6 and C7 sites, which were quantified from the Sanger sequencing results in 5D. 5F: The Sanger sequencing results of the PCSK9 on-target site in the N2A cells transduced with VLP_V3-tBE_V1. The non-treated N2A cells served as negative control. 5G: The base editing efficiencies at the C7 and C8 sites, which were quantified from the Sanger sequencing results in 5F.

[46] FIG. 6: Construction of VLP_V4-tBE_V1 and its editing efficiency. 6A: Schematic diagrams of the plasmids for the production of version 4 of VLP system containing tBE_V1 (VLP_V4-tBE_V1). 6B: The Sanger sequencing results of the PRR5L on-target site in the 293FT cells transduced with VLP_V4-tBE_V1. The non-treated 293FT cells served as negative control. 6C: The base editing efficiencies at the C6 and C7 sites, which were quantified from the Sanger sequencing results in 6B. 6D: The Sanger sequencing results of the HBG on-target site in the 293FT cells transduced with VLP_V4-tBE_V1. The non-treated 293FT cells served as negative control. 6E: The base editing efficiencies at the C6 and C7 sites, which were quantified from the Sanger sequencing results in 6D. 6F: The Sanger sequencing results of the PCSK9 on-target site in the N2A cells transduced with VLP_V4-tBE_V1. The non-treated N2A cells served as negative control. 6G: The base editing efficiencies at the C7 and C8 sites, which were quantified from the Sanger sequencing results in 6F.

[47] FIG. 7: Construction of VLP_V5-tBE_V1 and its editing efficiency. 7A: Schematic diagrams of the plasmids for the production of version 5 of VLP system containing tBE_V1 (VLP_V5-tBE_V1). 7B: The Sanger sequencing results of the PRR5L on-target site in the 293FT cells transduced with VLP_V5-tBE_V1. The non-treated 293FT cells served as negative control. 7C: The base editing efficiencies at the C6 and C7 sites, which were quantified from the Sanger sequencing results in 7B.

[48] FIG. 8: Construction of VLP_V3-SpCas9 and its editing efficiency. 8A: Schematic diagrams of the plasmids for the production of version 3 of VLP system containing SpCas9 (VLP_V3-SpCas9). 8B: The Sanger sequencing results of the PRR5L on-target site in the 293FT cells transduced with VLP_V3-SpCas9. The non-treated 293FT cells served as negative control. 8C: The DNA agarose gel for the PCR products amplified with the primers covering the PRR5L on-target site in the 293FT cells transduced with VLP_V3-SpCas9. The non-treated 293FT cells served as negative control. 8D: The indel frequencies at the on-target site, which were quantified from the DNA agarose gel in 8C.

[49] FIG. 9: Construction of VLP_V3-BE3 and its editing efficiency. 9A: Schematic diagrams of the plasmids for the production of version 3 of VLP system containing BE3 (VLP_V3-BE3). 9B: The Sanger sequencing results of the PRR5L on-target site in the 293FT cells transduced with VLP_V3-BE3. The non-treated 293FT cells served as negative control. 9C: The base editing efficiencies at the C6 and C7 sites, which were quantified from the Sanger sequencing results in 9B.

[50] FIG. 10: Construction of VLP_V5-BE3 and its editing efficiency. 10A: Schematic diagrams of the plasmids for the production of version 5 of VLP system containing BE3 (VLP_V5-BE3). 10B: The Sanger sequencing results of the PRR5L on-target site in the 293FT cells transduced with VLP_V5-BE3. The non-treated 293FT cells served as negative control. 10C: The base editing efficiencies at the C6 and C7 sites, which were quantified from the Sanger sequencing results in 10B.

[51] FIG. 11: Working mechanism of VLP_V5-tBE_V1. Schematic diagram of the working mechanism of VLP_V5-tBE_V1 to achieve efficient base editing.

[52] FIG.12: Construction of VLP_V6-tBE_V1 and its editing efficiency. 12A: Schematic diagrams of the plasmids for the production of version 6 of VLP system containing tBE_V1 (VLP_V6-tBE_V1). 12B: The Sanger sequencing results of the VEGFA on-target site in the 293FT cells transduced with VLP_V6-tBE_V1. The non-treated 293FT cells served as negative control. 12C: The base editing efficiencies at the C5 and C9 sites, which were quantified from the Sanger sequencing results in FIG.12B.

[53] FIG.13: Construction of VLP_V7-tBE_V1 and its editing efficiency. 13A: Schematic diagrams of the plasmids for the production of version 7 of VLP system containing tBE_V1 (VLP_V7-tBE_V1). 13B: The Sanger sequencing results of the VEGFA on-target site in the 293FT cells transduced with VLP_V7-tBE_V1. The non-treated 293FT cells served as negative control. 13C: The base editing efficiencies at the C5 and C9 sites, which were quantified from the Sanger sequencing results in FIG. 13B.

[54] FIG.14: Construction of VLP_V8-tBE_V1 and its editing efficiency. 14A: Schematic diagrams of the plasmids for the production of version 8 of VLP system containing tBE_V1 (VLP_V8-tBE_V1). 14B: The Sanger sequencing results of the VEGFA on-target site in the 293FT cells transduced with VLP_V8-tBE_V1. The non-treated 293FT cells served as negative control. 14C: The base editing efficiencies at the C5 and C9 sites, which were quantified from the Sanger sequencing results in FIG. 14B.

[55] FIG.15: Construction of Lenti-GFP, Lenti-GFP-VSVGmut, and Lenti-GFP-CD117 and its infection efficiency. 15A: Schematic diagrams of the plasmids for the production of Lenti-GFP, Lenti-GFP-VSVGmut, and Lenti-GFP-CD117. 15B: Flow cytometry results of Lenti-GFP, Lenti-GFP-VSVGmut, and Lenti-GFP-CD117 infected 293FT cells. 15C: Flow cytometry results of Lenti-GFP, Lenti-GFP-VSVGmut, and Lenti-GFP-CD117 infected 293FTCD117+ cells.

[56] FIG.16: Construction of VLP_V5.1-tBE_V1, VLP_V5.1-tBE_V1-VSVGmut, and VLP_V5.1-tBE_V1-CD117 and its editing efficiency. 16A: Schematic diagrams of the plasmids for the production of VLP_V5.1-tBE_V1, VLP_V5.1-tBE_V1-VSVGmut, and VLP_V5.1-tBE_V1-CD117. 16B: The Sanger sequencing results of the PRR5L on-target site in the 293FT cells transduced with VLP_V5.1-tBE_V1, VLP_V5.1-tBE_V1-VSVGmut, and VLP_V5.1-tBE_V1-CD117. The non-treated 293FT cells served as negative control. 16C: The Sanger sequencing results of the PRR5L on-target site in the 293FTCD117+ cells transduced with VLP_V5.1-tBE_V1, VLP_V5.1-tBE_V1-VSVGmut, and VLP_V5.1-tBE_V1-CD117. The non-treated 293FTCD117+ cells served as negative control.

[57] FIG.17: Construction of VLP_V6.1-tBE_V1, VLP_V6.2-tBE_V1, and VLP_V6.3-tBE_V1 and its editing efficiency. A: Schematic diagrams of the plasmids for the production of VLP_V6.1-tBE_V1, VLP_V6.2-tBE_V1, and VLP_V6.3-tBE_V1. B: The Sanger sequencing results of the VEGFA on-target site in the 293FT cells transduced with VLP_V6.1-tBE_V1, VLP_V6.2-tBE_V1, and VLP_V6.3-tBE_V1. The non-treated 293FT cells served as negative control. C: The base editing efficiencies at the C5 and C9 sites, which were quantified from the Sanger sequencing results in FIG. 17B. D: Total C-to-T editing efficiency of VLP_V6-tBE_V1, VLP_V6.1-tBE_V1, VLP_V6.2-tBE_V1, VLP_V6.3-tBE_V1, VLP_V7-tBE_V1, and VLP_V8-tBE_V1 at the VEGFA site in 293FT cell.DETAILED DESCRIPTIONDefinitions

[58] It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “an antibody,” is understood to represent one or more antibodies. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

[59] As used herein, the term “polypeptide” is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein”, “amino acid chain” or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of “polypeptide,” and the term “polypeptide” may be used instead of, or interchangeably with any of these terms. The term “polypeptide” is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modification by non- naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis.

[60] “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, though preferably less than 25% identity, with one of the sequences of the present disclosure.

[61] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 98 % or 99 %) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, default parameters are used for alignment.One alignment program is BLAST, using default parameters.

[62] The term “an equivalent nucleic acid or polynucleotide” refers to a nucleic acid having a nucleotide sequence having a certain degree of homology, or sequence identity, with the nucleotide sequence of the nucleic acid or complement thereof. A homolog of a double stranded nucleic acid is intended to include nucleic acids having a nucleotide sequence which has a certain degree of homology with or with the complement thereof. In one aspect, homologs of nucleic acids are capable of hybridizing to the nucleic acid or complement thereof. Likewise, “an equivalent polypeptide” refers to a polypeptide having a certain degree of homology, or sequence identity, with the amino acid sequence of a reference polypeptide. In some aspects, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some aspects, the equivalent polypeptide or polynucleotide has one, two, three, four or five addition, deletion, substitution and their combinations thereof as compared to the reference polypeptide or polynucleotide. In some aspects, the equivalent sequence retains the activity (e.g., epitope-binding) or structure (e.g., salt-bridge) of the reference sequence.

[63] The term “encode” as it is applied to polynucleotides refers to a polynucleotide which is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and / or translated to produce the mRNA for the polypeptide and / or a fragment thereof. The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.

[64] The “group-specific antigen” (Gag) is the primary structural protein responsible for viral assembly, including budding out of fully-formed enveloped virions having an (i) envelope (comprising a lipid membrane formed from cell membrane during budding out, and one or more glycoproteins inserted therein), and (ii) a capsid, which is the internal protein shell. Most of these assembly steps occur via interactions with three Gag subdomains - matrix (MA), capsid (CA), and nucleocapsid (NC). These three regions have a low level of sequence conservation among the different retroviral genera, which belies the observed high level of structural conservation. Outside of these three domains, Gag proteins can vary widely. For example, HIV-1 Gag additionally codes for a C-terminal p6 protein as well as two spacer proteins, SP1 and SP2, which demarcate the CA-NC and NC-p6 junctions, but HIV-1 contains no additional sequences outside of MA, CA, and NC (Oroszlan and Copeland, 1985; Henderson et al., 1992). \

[65] The term “group-specific antigen (gag) protease (pro) polyprotein” or “Gag-Pol polyprotein” refers to a gag nucleocapsid protein further comprising a viral protease linked thereto. Gag-pro polyproteins mediate proteolytic cleavage of gag and gag-pol polyproteins or nucleocapsid proteins during or shortly after the release of a virion from the plasma membrane. The protease of a gag-pro polyprotein can cleave a corresponding linker in the fusion protein. In some embodiments, a gag-pro polyprotein is an MMLV gag-pro polyprotein, an FMLV gag-pro polyprotein, or a HIV gag-pro polyprotein.

[66] “Guide RNAs” are non-coding short RNA sequences which bind to the complementary target DNA sequences. A guide RNA first binds to the Cas enzyme and the gRNA sequence guides the complex via pairing to a specific location on the DNA, where Cas performs its endonuclease activity by cutting the target DNA strand. A “single guide RNA,” frequently simply referred to as “guide RNA”, refers to synthetic or expressed single guide RNA (sgRNA) that consists of both the crRNA and tracrRNA as a single construct. The tracrRNA portion is responsible for Cas endonuclease activity and the crRNA portion binds to the target specific DNA region. Therefore, the trans activating RNA (tracrRNA, or scaffold region) and crRNA are two key components and are joined by tetraloop which results in formation of sgRNA.

[67] The term guide RNA also encompasses those used for prime editing, which can be more specifically referred to as pegRNA (prime editing guide RNA). In some embodiments, a pegRNA comprises one or more “extended regions” of nucleic acid sequence besides the conventional gRNA. The extended regions may comprise, but are not limited to, single- stranded RNA or DNA. In some embodiments, the extended region comprises a DNA synthesis template.

[68] A “nickase” refers to a Cas protein which 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 a non-target strand of a double stranded target DNA sequence. In some embodiments, the nickase is a Cas9 that comprises one or more mutations in a RuvC-like domain relative to a wild type Cas9 sequence or to an equivalent amino acid position in other Cas9 variants or Cas9 equivalents. In some embodiments, the nickase is a Cas9 that comprises one or more mutations in an HNH-like domain relative to a wild type Cas9 sequence or to an equivalent amino acid position in other Cas9 variants or Cas9 equivalents. In some embodiments, the nickase is a Cas9 that comprises an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 relative to a canonical Cas9 sequence or to an equivalent amino acid position in other Cas9 variants or Cas9 equivalents. In some embodiments, the nickase is a Cas9 that comprises an H840A, N854A, and / or N863A mutation relative to a canonical Cas9 sequence, or to an equivalent amino acid position in other Cas9 variants or Cas9 equivalents. In some embodiments, the term “Cas9 nickase” refers to a Cas9 with one of the two nuclease domains inactivated. This enzyme is capable of cleaving only one strand of a target DNA. In some embodiments, the nickase is a Cas protein that is not a Cas9 nickase.

[69] The term “nuclear export sequence” or “NES” refers to an amino acid sequence that promotes transport of a protein out of 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 would be apparent to the skilled artisan. An example NES sequence is provided in SEQ ID NO:2. Virus-Like Particles (VLPs) for Delivery of Base Editors and Prime Editors

[70] Virus-like particles (VLPs) are modified retroviral vectors that do not deliver DNA but can deliver mRNAs and proteins. Reports on the use of VLP to deliver CRISPR-related editors, however, are very limited and are associated with unsatisfactory efficiency.

[71] To assemble VLP, the key VLP proteins are needed, including the Gag-Pol polyprotein and an envelope protein (EP) (e.g., VSVG (vesicular stomatitis virus glycoprotein)). Other elements that are desired to be packaged into the VLP need to be coupled to one or more of these proteins. For instance, the conventional method of packaging a base editor, which includes a Cas protein and a nucleobase deaminase (ND), is to fuse it to the Gag protein. The sgRNA that is required by the base editor can bind to the base editor and thus is indirectly coupled to the Gag protein.

[72] In this context, the instant inventors made the unexpected discovery that, when a Cas9 protein, whether as part of a base editor (see, e.g.,FIG. 1A) or alone (see, e.g.,FIG. 2A), was fused to a Gag protein, it became truncated. By contrast, when the Cas9 protein was not fused to the Gag protein but was packaged into the VLP by other means (see, e.g.,FIG. 3A), no truncation occurred.

[73] Such a discovery, therefore, at least in part provides an explanation for why the use of VLP to deliver Cas-related editors has been by and large unsuccessful. Based on this unexpected discovery, the instant inventors have designed and tested a few new approaches, each of which exhibited greatly improved editing efficiency and accuracy following successful delivery.

[74] In one example approach, the Cas protein alone, or as part of a base editor or a prime editor is coupled, indirectly, to the Gag protein through a guide RNA (gRNA) that the Cas protein binds to. The binding between the Gag protein and the gRNA can be facilitated by an RNA binding domain (RBD) and a corresponding RNA aptamer (APT), without limitation.

[75] In a conventional base editor, the Cas protein (e.g., a catalytically impaired Cas9 endonuclease or a nickase) is fused to a nucleobase deaminase (ND), and the corresponding guide RNA is often referred to as a single guide RNA (sgRNA), which includes a tracrRNA responsible for Cas endonuclease activity and a crRNA that binds to the target specific DNA region. In a conventional prime editor, the Cas nickase is fused to a reverse-transcriptase (RT), and the corresponding guide RNA is often referred to as a prime editing guide RNA (pegRNA), which includes an sgRNA, a reverse transcription template and a primer binding site. A Cas protein can also work alone, without being part of a base editor or prime editor, with a gRNA to effect CRISPR-style gene editing.

[76] For base editing or prime editing, in some embodiments, the Cas protein is included in a fusion protein that further includes the ND or the RT. As further described later, however, the ND or the RT can be provided in sperate proteins which can be brought together to work with the Cas protein in a “split” configuration. A. Configuration #1 for CRISPR Gene Editing / Base Editing / Prime Editing

[77] An example configuration is illustrated in Table A, which lists protein and RNA molecules present in a packaging cell (also known as “producer cell”) for preparing a VLP. In this example, the packaging cell includes four proteins and an RNA molecule. Protein #1 includes a Gag-Pol polyprotein. Protein #2 includes an envelope protein (e.g., VSVG). Protein #3 includes a Cas protein, or a fusion with an ND (for base editing) or an RT (for prime editing). Protein #4 includes a Gag protein fused to an RNA binding domain (RBD) that recognizes a RNA aptamer (APT). The RNA molecule includes a gRNA (sgRNA or pegRNA) fused to the APT.

[78] Since the RBD can recognize and bind to the APT, RNA #1 can be coupled to Protein #4 by virtue of such binding. Meanwhile, Protein #3 is also brought to this complex since the Cas protein naturally binds the gRNA. Collectively, due to the RBD-APT and Cas-gRNA interactions, the Cas protein is brought to the Gag protein to be included as part of the VLP. This approach, without the need of a Gag-Cas fusion, avoids truncation of the Cas protein.

[79] When these protein / RNA molecules are produced in a packaging cell, they can assemble to various types of RNPs, and bud out to form VLPs. The Gag-Pol polyprotein and the envelope protein (EP) form a protein shell for VLP. The protein shell further includes Protein #4 (Gag + RBD), which is associated with the RNA #1 (gRNA_APT) and Protein #3 (Cas).

[80] In some embodiments, a protease cleavage site (PCS) is disposed between the Gag and RBD in Protein #4, which is cleavable during proteolytic virion maturation of the VLP, by the protease in the Gag-Pol polyprotein. Therefore, upon proteolytic virion maturation, the RBD is cleaved off from the Gag protein, hence the Protein #3 and RNA #1 are released from the protein shell which facilitates subsequent delivery of the Protein #3 and RNA #1 in the target cell for CRISPR gene / base / prime editing (Table A’). Table A. Example Configuration #1 in Packaging CellsMoleculeElement(s)NotesProtein #1Gag-Pol Protein #2EP (e.g., VSVG) Protein #3Cas (optionally fused with ND / RT) Protein #4Gag + (PCS) + RBDRBD recognizes APTRNA #1gRNA + APT Notes: EP – envelope protein; VSVG – vesicular stomatitis virus glycoprotein; RBD – RNA binding domain; PCS – protease cleavage site; ND – nucleobase deaminase; RT – reverse-transcriptase Table A’. Contents of Maturated VLP for Configuration Example #1 MoleculeElement(s)NotesProtein #1Gag-Pol Protein #2EP (e.g., VSVG) Protein #3Cas (optionally fused with ND / RT) Protein #4GagRBD is cleaved off from Gag upon VLP maturationRNA #1gRNA + APT OthersRBD  Table B. Example Configuration #2 in Packaging Cells (“split”)MoleculeElement(s)NotesProtein #1Gag-Pol Protein #2EP (e.g., VSVG) Protein #3Cas Protein #4Gag + (PCS) + RBD#1RBD#1 recognizes APT#1Protein #5ND / RT + RBD#2RBD#2 recognizes APT#2RNA #1gRNA + APT#1 + APT#2  Table B’. Contents of Maturated VLP for Configuration Example #2 MoleculeElement(s)NotesProtein #1Gag-Pol Protein #2EP (e.g., VSVG) Protein #3Cas Protein #4GagRBD#1 is cleaved off from Gag upon VLP maturationProtein #5ND / RT + RBD#2 RNA #1gRNA + APT#1 + APT#2 OthersRBD#1  Table C. Example Configuration #3 in Packaging Cells (“split with dual gRNA”)MoleculeElement(s)NotesProtein #1Gag-Pol Protein #2EP (e.g., VSVG) Protein #3Cas Protein #4Gag + (PCS) + RBD#1RBD#1 recognizes APT#1Protein #5ND / RT + RBD#2RBD#2 recognizes APT#2RNA #1gRNA + APT#1 RNA #2hsgRNA + APT#2 + APT#1  Table C’. Contents of Maturated VLP for Configuration Example #3MoleculeElement(s)NotesProtein #1Gag-Pol Protein #2EP (e.g., VSVG) Protein #3Cas Protein #4GagRBD#1 is cleaved off from Gag upon VLP maturationProtein #5ND / RT + RBD#2 RNA #1gRNA + APT#1 RNA #2hsgRNA + APT#2 + APT#1 OthersRBD#1  Table D. Example Configuration #4 in Packaging Cells (“Split with NES and free UGI”)MoleculeElement(s)NotesProtein #1Gag-Pol Protein #2EP (e.g., VSVG) Protein #3Cas + (PCS) + NES Protein #4Gag + NES + (PCS) + RBD#1RBD#1 recognizes APT#1Protein #5ND / RT+ UGI + RBD#2RBD#2 recognizes APT#2Protein #6UGI + RBD#3RBD#3 recognizes APT#3RNA #1gRNA + APT#1 + APT#3 RNA #2hsgRNA + APT#2 + APT#1 Note: NES – nuclear export signal; UGI – uracil glycosylase inhibitor Table D’. Contents of Maturated VLP for Configuration Example #4MoleculeElement(s)NotesProtein #1Gag-Pol Protein #2EP (e.g., VSVG) Protein #3CasNES is cleaved off from Cas upon VLP maturationProtein #4Gag + NESRBD#1 is cleaved off from Gag upon VLP maturationProtein #5ND / RT + UGI + RBD#2 Protein #6UGI + RBD#3 RNA #1gRNA + APT#1 + APT#3 RNA #2hsgRNA + APT#2 + APT#1 OthersNES,RBD#1  Table E. Example Configuration #5 in Packaging Cells (“free UGI cleaved from Gag”)MoleculeElement(s)NotesProtein #1Gag-Pol Protein #2EP (e.g., VSVG) Protein #3Cas + (PCS) + NES Protein #4Gag + NES + (PCS) + UGI + RBD#1RBD#1 recognizes APT#1Protein #5ND / RT + UGI + RBD#2RBD#2 recognizes APT#2RNA #1gRNA + APT#1 RNA #2hsgRNA + APT#2 + APT#1  Table E’. Contents of Maturated VLP for Configuration Example #5MoleculeElement(s)NotesProtein #1Gag-Pol Protein #2EP (e.g., VSVG) Protein #3CasNES is cleaved off from Cas upon VLP maturationProtein #4Gag + NESUGI + RBD#1 is cleaved off from Gag upon VLP maturationProtein #5ND / RT + UGI + RBD#2 RNA #1gRNA + APT#1 RNA #2hsgRNA + APT#2 + APT#1 OthersUGI + RBD#1,NESCleaved, standalone UGI improves editing efficiency  Table F. Example Construct Configuration #1MoleculeElement(s)Construct #1(Gag-Pol) + RREConstruct #2(EP) + (hsgRNA + APT#2 + APT#1) + (gRN + APT#1 + APT#3)Construct #3(Cas + (PCS) + NES) + 2A + (ND / RT + UGI + RBD#2)Construct #4(Gag + NES + (PCS) + RBD#1) + 2A + (Rev) + RREConstruct #5UGI + RBD#3Note: 2A – 2A self-cleaving peptide; RRE – Rev response element Table G. Example Construct Configuration #2MoleculeElement(s)Construct #1(Gag-Pol) + RREConstruct #2(EP) + (hsgRNA + APT#2 + APT#1) + (gRNA + APT#1)Construct #3(Cas + (PCS) + NES) + 2A + (ND / RT + UGI + RBD#2)Construct #4(Gag + NES + (PCS) + UGI + RBD#1) + 2A + (Rev) + RRE B. Configuration #2 for Split Base or Prime Editing

[81] As noted above, for a base editor or a prime editor, the ND or RT protein does not have to be fused to the Cas protein. Instead, in an example “split” configuration, the ND or RT protein is fused to an RNA binding domain (RBD) and gRNA is fused to a corresponding APT so that they can bind one another by virtue of the RBD-APT interaction. Meanwhile, since the Cas protein can bind to the gRNA naturally, all three components can be brought together to effect base editing or prime editing.

[82] For such a split base editing or prime editing setting, an example VLP configuration is illustrated in Table B. In this configuration, like in Table A, the packaging cell includes a Protein #1 (Gag-Pol polyprotein) and a Protein #2 (EP). In Protein #3, only the Cas protein (e.g., a catalytically impaired Cas9 endonuclease) is present (i.e., no ND or RT). In Protein #4, a Gag protein is fused, optionally through a PCS, to an RBD (RBD#1). In Protein #5, an ND or RT is fused to another RBD (RBD#2). In RNA#1, the gRNA (sgRNA or pegRNA) is fused to an RNA APT#1 corresponding to RBD#1 and a further RNA APT#2 corresponding to the RBD#2.

[83] Therefore, in this configuration, the gRNA can bind to the Protein #4 (Gag + (PCS) + RBD#1) by virtue of the RBD#1 / APT#1 binding, and can bind to the Protein #5 (ND / RT + RBD#2) through the RBD#2 / APT#2 binding. Meanwhile, the Cas protein in Protein #3 can naturally bind to the gRNA. As such, all of Proteins #3, Protein #4 and RNA#1 can associate with Protein #4 (Gag + (PCS) + RBD#1), and thus participate in VLP formation. Again, since this configuration avoids the use of a Gag-Cas fusion, it will avoid Cas truncation.

[84] Once the proteins and RNA are budded out from the packaging cell, VLPs can form. Like in the configuration of Table A, upon proteolytic virion maturation, the RBD#1 is cleaved off from the Gag protein in Protein #4. Consequently, the Protein #3, Protein #5 and RNA #1 are released from the protein shell, which facilitates subsequent delivery of the Protein #3, Protein #5 and RNA #1 in the target cell for base / prime editing (Table B’).C. Configuration #3 for Base or Prime Editing with Dual gRNA

[85] In some embodiments, the base editor or prime editor employs not just one gRNA, but two of them. They have similar structures and properties, and recognize two adjacent sites on the target nucleic acid to be edited. The use of dual gRNA enhances the accuracy of targeting and thus reduce off-target editing. One of them can be referred to as a gRNA (or sgRNA) while the other can be referred to as a helper single guide RNA (hsgRNA).

[86] In this context, a further example configuration is provided for preparing VLP with dual gRNAs, which is illustrated in Table C. In this configuration, like in Table B, the packaging cell includes a Protein #1 (Gag-Pol polyprotein), a Protein #2 (EP), a Protein #3 (Cas), a Protein #4 Gag + (PCS) + RBD#1), and a Protein #5 (ND or RT + RBD#2). Unlike the configuration in Table C which includes a single RNA molecule, this configuration includes two RNA molecules. RNA#1 includes a gRNA that is fused to APT#1 corresponding to RBD#1 fused to Gag, and RNA#2 includes a hsgRNA that is fused to both APT#1 and an APT#2 corresponding to RBD#2 which is fused to the ND or RT.

[87] Given the RBD#1 / APT1 and RBD#2 / APT2 binding relationship, the two gRNAs bind to Protein #4 (Gag + (PCS) + RBD#1) while RNA#2 further binds to Protein #5 (ND / RT + RBD#2). Also, because the Cas of Protein #3 can naturally bind to both gRNAs, all of Proteins #3 and #5 and RNA #1 and #2 are brought to Protein #4 for assembling into the VLP. Again, since this configuration avoids the use of a Gag-Cas fusion, it will avoid Cas truncation.

[88] Once the proteins and RNA are budded out from the packaging cell, VLPs can form. Like in the configuration of Tables A and B, upon proteolytic virion maturation, the RBD#1 is cleaved off from the Gag protein in Protein #4. Consequently, the Protein #3, Protein #5 and RNA #1 and #2 are released from the protein shell, which facilitates subsequent delivery of the Protein #3, Protein #5 and RNA #1 and #2 in the target cell for base / prime editing (Table C’).D. Further Inclusion and Timely Removal of NES

[89] In some embodiments, one or more additional elements can be added to the configuration to further improve their performance. One such example is a nuclear export signal (NES). Many NES sequences are known in the art. One such example is SEQ ID NO:2. In a preferred embodiment, three NES sequences are used in conjunction, and is noted as 3xNES (e.g., SEQ ID NO:3). An NES can improve the cytoplasmic localization of a protein, it is discovered. Therefore, inclusion of an NES can improve the cytoplasmic concentration of an associated protein and the budding out from the cell, for production of the VLP. On the other hand, however, such a property of an NES is not desired in the target cell for editing, as the genome editor is preferably localized in the nucleus to edit the chromosome.

[90] Based on such considerations, an NES can be included in one or more of the proteins in the packaging cell and is removed prior to VLP transduction. An example stage of such removal is during proteolytic virion maturation of the VLP. In some embodiments, therefore, an NES is fused to a protein, through a protease cleavage site (PCS) that is cleavable during the VLP maturation.

[91] For instance, in a Gag-PCS-RBD#1 fusion protein of any of the above configurations, an NES can be inserted between the Gag and the PCS (see, e.g.,FIG. 3A, pGag-NES-PCS-COM). The inclusion of the NES in this fusion protein enhances the cytoplasmic localization and thus budding out of this fusion protein. During VLP maturation, however, cleavage at the PCS releases the RBD#1 without the NES. Therefore, even if the RBD#1 is delivered to a target cell for editing, the NES will not accompany the RBD#1 which would inhibit the nuclear localization of the RBD#1 (or its associated gRNA / Cas).

[92] In another example, an NES is fused to the Cas protein, through a PCS (see, e.g.,FIG. 5A, pD10A-PCS-NES-T2A-MCP-UGI-mA3C). The inclusion of the NES in this fusion protein enhances the cytoplasmic localization and thus budding out of the Cas protein. During VLP maturation, however, cleavage at the PCS releases the Cas. Therefore, when the Cas protein is delivered to a target cell for editing, the NES will not accompany the Cas protein which would inhibit the nuclear localization of the Cas protein.E. Further Inclusion of Integrated and / or Free UGI

[93] It is discovered the further inclusion of a uracil glycosylase inhibitor (UGI) in the based editor or inclusion of a UGI as a standalone element can further improve the editing efficiency. A non-limiting example of UGI is found in Bacillus phage AR9 (YP_009283008.1). In some embodiments, the UGI comprises the amino acid sequence of SEQ ID NO:4 or has at least at least 90% sequence identity to SEQ ID NO:4 and retains the uracil glycosylase inhibition activity.

[94] In one example, a UGI is included in one of the proteins in any of the above configurations. For instance, the ND-RBD#2 fusion protein can further include a UGI (see, e.g.,FIG. 3A, pD10A-T2A-MCP-UGI-mA3C).

[95] In another example, a further protein is included in a configuration, such as the configuration of Table C. In this respect, in order to ensure that the UGI is also packaged into the VLP, it can be fused to a third RBD (RBD#3). Meanwhile, one of the gRNAs further includes a APT#3 that can be bound by the RBD#3. Such a configuration is illustrated in Table D. In the example, two of the proteins further include an NES, as discussed above.

[96] Once the proteins and RNA are budded out from the packaging cell, VLPs can form. Like in the configuration of Table C, upon proteolytic virion maturation, the RBD#1 is cleaved off from the Gap-NES protein in Protein #4; and the NES is also cleaved off from the Cas in Protein #3. Consequently, the Protein #3 (without the NES), Protein #5 (optionally including a further UGI), Protein #6 and RNA #1 and #2 are released from the protein shell, which facilitates subsequent delivery of the Protein #3 (without the NES), Protein #5, Protein #6 and RNA #1 and #2 in the target cell for base / prime editing (Table D’).

[97] In another example, the Gag-(PCS)-RBD#1 fusion protein further includes a UGI (see, e.g.,FIG. 7A, pGag-NES-PCS-UGI-COM-T2A-Rev-RRE). Note that in this example, the UGI should be placed at the same side as the RBD#1 from the PCS, such that, upon cleavage of the PCS, the UGI is still fused to the RBD#1. Relative to the core components of the base editor, however, this UGI is still standalone. As compared to the configuration of Table D, however, this configuration takes advantages of the existing RBD#1 / APT#1 and does not require a further pair of RBD / APT. This configuration is illustrated in Tables E / E’.

[98] In yet other examples, more UGI units can be added to the configurations. For instance, in FIG. 12, a total of three UGI units are included. Relative to FIG. 7A, FIG. 12 further includes a standalone UGI. In other words, the configuration of FIG. 12 combines the UGI of FIG. 6 and FIG. 7.

[99] In another example (illustrated in FIG. 13), a fourth UGI is fused to the C-terminus of the Cas protein. In yet another example (illustrated in FIG. 14), the standalone UGI protein includes two concatenated copies.F. Temporal Inhibition of Nucleobase Deaminase

[100] In some embodiments for base editing, it is preferred that the nucleobase deaminase (ND) is not active until the base editor has reached the target nucleic acid molecule to be edited. To this end, in one embodiment, an ND inhibitor is cleavably fused to the ND through a protease recognition site that is different from the PCS of the other proteins in the configurations.

[101] In some embodiments, the protease recognition site is recognized by a protease that is activated only in the target cell. In some embodiments, the protease recognition site is recognized by a protease that is activated only when the ND is bound to the target nucleic acid molecule to be edited. In some embodiments, the protease recognition site is a TEV protease recognition site. An example sequence of the TEV protease recognition site is SEQ ID NO:5. Other known examples of proteases include TuMV protease, PPV protease, PVY protease, ZIKV protease and WNV protease.

[102] The term “nucleobase deaminase” as used herein, refers to a group of enzymes that catalyze the hydrolytic deamination of nucleobases such as cytidine, deoxycytidine, adenosine and deoxyadenosine. Non-limiting examples of nucleobase deaminases include cytidine deaminases and adenosine deaminases.

[103] “Cytidine deaminase” refers to enzymes that catalyze the irreversible hydrolytic deamination of cytidine and deoxycytidine to uridine and deoxyuridine, respectively. Cytidine deaminases maintain the cellular pyrimidine pool. A family of cytidine deaminases is APOBEC (“apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like”). Members of this family are C-to-U editing enzymes. Some APOBEC family members have two domains, one domain of APOBEC like proteins is the catalytic domain, while the other domain is a pseudocatalytic domain. More specifically, the catalytic domain is a zinc dependent cytidine deaminase domain and is important for cytidine deamination. RNA editing by APOBEC-1 requires homodimerisation and this complex interacts with RNA binding proteins to form the editosome.

[104] Non-limiting examples of APOBEC proteins include APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, and activation-induced (cytidine) deaminase (AID).

[105] “Adenosine deaminase”, also known as adenosine aminohydrolase, or ADA, is an enzyme (EC 3.5.4.4) involved in purine metabolism. It is needed for the breakdown of adenosine from food and for the turnover of nucleic acids in tissues.

[106] Non-limiting examples of adenosine deaminases include tRNA-specific adenosine deaminase (TadA), adenosine deaminase tRNA specific 1 (ADAT1), adenosine deaminase tRNA specific 2 (ADAT2), adenosine deaminase tRNA specific 3 (ADAT3), adenosine deaminase RNA specific B1 (ADARB1), adenosine deaminase RNA specific B2 (ADARB2), adenosine monophosphate deaminase 1 (AMPD1), adenosine monophosphate deaminase 2 (AMPD2), adenosine monophosphate deaminase 3 (AMPD3), adenosine deaminase (ADA), adenosine deaminase 2 (ADA2), adenosine deaminase like (ADAL), adenosine deaminase domain containing 1 (ADAD1), adenosine deaminase domain containing 2 (ADAD2), adenosine deaminase RNA specific (ADAR) and adenosine deaminase RNA specific B1 (ADARB1).

[107] Some of the nucleobase deaminases have a catalytic domain (e.g., mA3-CDA1 and hA3B-CDA2) and an inhibitory domain (e.g., mA3-CDA2 and hA3B-CDA1). These inhibitory domains have general inhibitory activity against the catalytic domains of all nucleobase deaminases and thus can be used as generic inhibitors. Example sequences of such inhibitory domains are provided in SEQ ID NO:6 and 7. G. Expression Constructs and Optional Inclusion of RRE

[108] Expression constructs are also provided which can be used to generate the proteins / RNAs of each configuration. In some embodiments, one or more proteins and / or RNAs of the configurations can be combined, i.e., transcribed into a single mRNA or a single pre-protein before it is processed into individual molecules. In some embodiments, one or more proteins and / or RNAs of the configurations can be generated from a single construct under different promoters.

[109] It has been tested in the experimental examples that the inclusion of a Rev response element (RRE) (e.g., a HIV-1 RRE, SEQ ID NO:8) can improve the cytoplasmic localization of the corresponding mRNA, in the presence of a corresponding RRE Rev protein (SEQ ID NO:20). In some embodiments, therefore, the coding sequences of one or more of the proteins in a configuration includes an RRE, and at least one of the proteins includes a Rev.

[110] Example configurations of such constructs are illustrated in Tables F and G, with reference to FIG. 6 and 7, respectively. In Table F, a total of 5 constructs are prepared. Construct #1 expresses the single Gag-Pol polyprotein, but the mRNA further includes an RRE to enhance its cytoplasmic localization. Construct #2 encodes the envelop protein and two separate gRNA, each under the control of a different promoter.

[111] Construct #3 encodes a preprotein that is preprocessed by the 2A self-cleavage peptide to generate two separate proteins, Cas + (PCS) + NES and ND / RT + UGI + RBD#2. Construct #4 also includes an RRE in the mRNA, and its produced preprotein is processed (through the 2A self-cleavage) to generate a Rev protein that functions upon the RRE sequences for cytoplasmic localization. Construct #5 encodes a standalone UGI.

[112] The configuration of Table G is similar to that of Table F, but differs in that, in Table G, the standalone UGI is included in Construct #4 (cleavable from the Gag upon VLP maturation).

[113] “2A self-cleaving peptides” are 18-22 amino-acid-long viral oligopeptides that mediate “cleavage” of polypeptides during translation in eukaryotic cells. The designation “2A” refers to a specific region of the viral genome and different viral 2As have generally been named after the virus they were derived from. The first discovered 2A was F2A (foot-and-mouth disease virus), after which E2A (equine rhinitis A virus), P2A (porcine teschovirus-1 2A), and T2A (thosea asigna virus 2A) were also identified. A few non-limiting examples of 2A peptides are provided in SEQ ID NO:17-19.H. Optional Inclusion of a Cell-Targeting Moiety

[114] The expression constructs can optionally further encode a cell-targeting moiety which, when expressed, can facilitate targeting of the VLP to a particular cell type. In some embodiments, the target cells are hematopoietic stem cells (HSC) and a suitable targeting marker on the HSC is CD117.

[115] Accordingly, in some embodiments, one or more of the expression construct encodes a CD117 ligand. Examples of CD117 ligands include, without limitation, an anti-CD117 antibody or an antigen-binding fragment thereof (e.g., scFv) and human and animal stem cell factors (SCF). Example antibodies include briquilimab / AMG 191 (Kwon et al, Blood (2019) 133 (19): 2104–2108), 2G4 (Kim et al., Mol Cell Biochem 2023 Apr;478(4):861-873) and SR-1 (Pang et al., Blood 2019 May 9;133(19):2069-2078).I. Additional Definitions and Example Sequences

[116] The term “Cas protein” or “clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) protein” refers to RNA-guided DNA endonuclease enzymes associated with the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) adaptive immunity system in Streptococcus pyogenes, as well as other bacteria. Cas proteins include Cas9 proteins, Cas12a (Cpf1) proteins, Cas12b (formerly known as C2c1) proteins, Cas13 proteins and various engineered counterparts. Example Cas proteins include SpCas9, FnCas9, St1Cas9, St3Cas9, NmCas9, SaCas9, AsCpf1, LbCpf1, FnCpf1, VQR SpCas9, EQR SpCas9, VRER SpCas9, SpCas9-NG, xSpCas9, RHA FnCas9, KKH SaCas9, NmeCas9, StCas9, CjCas9, AsCpf1, FnCpf1, SsCpf1, PcCpf1, BpCpf1, CmtCpf1, LiCpf1, PmCpf1, Pb3310Cpf1, Pb4417Cpf1, BsCpf1, EeCpf1, BhCas12b, AkCas12b, EbCas12b, LsCas12b, RfCas13d, LwaCas13a, PspCas13b, PguCas13b, RanCas13b.

[117] In some embodiments, a Cas protein is fused to at least a UGI, preferably at the C-terminus of the Cas protein. In some embodiments, a Cas protein is fused to at least two UGI, preferably at the C-terminus of the Cas protein. In some embodiments, a Cas protein is fused to at least three UGI, preferably at the C-terminus of the Cas protein.

[118] Non-limiting examples of reverse-transcriptases (RT) that can be used for prime editing include human immunodeficiency virus (HIV) reverse-transcriptase, moloney murine leukemia virus (MMLV) reverse-transcriptase and avian myeloblastosis virus (AMV) reverse-transcriptase.

[119] There are many known RNA binding domain (RBD)-RNA aptamer (APT) pairs. In each pair, the RBD specifically recognize and bind the corresponding APT. In one example, the APT includes a MS2 sequence (SEQ ID NO:9) and the RNA binding domain includes a MS2 coat protein (MCP, SEQ ID NO:13). In another example, the APT includes a Com sequence (SEQ ID NO:10) and the RNA binding domain includes a COM protein (COM, SEQ ID NO:14). In another example, the APT includes a boxB sequence (SEQ ID NO:11) and the RNA binding domain includes a boxB coat protein (N22p, SEQ ID NO:15). In another example, the APT includes a PP7 sequence (SEQ ID NO:12) and the RNA binding domain includes a PP7 coat protein (PCP, SEQ ID NO: 16). These examples can be used as the APT#1 / RBD#1, APT#2 / RBD#2, and / or APT#3 / RBD#3 as illustrated above. Table H. Example SequencesNameSequenceSEQ ID NO:HIV protease cleavage site (PCS)SQNYPIVQ1nuclear export signal (NES)NINELALKFAGLDL23xNESNINELALKFAGLDLSGGSNINELALKFAGLDLSGGSNINELALKFAGLDL3uracil glycosylase inhibitor (UGI)TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML4TEV protease cleavage siteENLYFQS5Mouse APOBEC3 cytidine deaminase domain 2 (mA3-CDA2)MSSSTLSNICLTKGLPETRFWVEGRRMDPLSEEEFYSQFYNQRVKHLCYYHRMKPYLCYQLEQFNGQAPLKGCLLSEKGKQHAEILFLDKIRSMELSQVTITCYLTWSPCPNCAWQLAAFKRDRPDLILHIYTSRLYFHWKRPFQKGLCSLWQSGILVDVMDLPQFTDCWTNFVNPKRPFWPWKGLEIISRRTQRRLRRIKESWGLQDLVNDFGNLQLGPPMS6Human APOBEC3B cytidine deaminase domain 1 (hA3B-CDA1)MNPQIRNPMERMYRDTFYDNFENEPILYGRSYTWLCYEVKIKRGRSNLLWDTGVFRGQVYFKPQYHAEMCFLSWFCGNQLPAYKCFQITWFVSWTPCPDCVAKLAEFLSEHPNVTLTISAARLYYYWERDYRRALCRLSQAGARVKIMDYEEFAYCWENFVYNEGQ7HIV RREAGGAGCUUUGUUCCUUGGGUUCUUGGGAGCAGCAGGAAGCACUAUGGGCGCAGCGUCAAUGACGCUGACGGUACAGGCCAGACAAUUAUUGUCUGGUAUAGUGCAGCAGCAGAACAAUUUGCUGAGGGCUAUUGAGGCGCAACAGCAUCUGUUGCAACUCACAGUCUGGGGCAUCAAGCAGCUCCAGGCAAGAAUCCUGGCUGUGGAAAGAUACCUAAAGGAUCAACAGCUCCU8MS2ACAUGAGGAUCACCCAUGU9ComUGAAUGCCUGCGAGCAUC10boxBGCCCUGAAGAAGGGC11PP7GGAGCAGACGAUAUGGCGUCGCUCC12MS2 coat protein (MCP)MASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY13COMMGSMKSIRCKNCNKLLFKADSFDHIEIRCPRCKRHIIMLNACEHPTEKHCGKREKITHSDETVRYGSTSGHKLNGGGGGMDAKSLTAWS14boxB coat protein (N22p)MGNARTRRRERRAEKQAQWKAAN15PP7 coat protein (PCP)MGSKTIVLSVGEATRTLTEIQSTADRQIFEEKVGPLVGRLRLTASLRQNGAKTAYRVNLKLDQADVVDSGLPKVRYTQVWSHDVTIVANSTEASRKSLYDLTKSLVATSQVEDLVVNLVPLGR16P2AGSGATNFSLLKQAGDVEENPGP 17T2AGSGEGRGSLLTCGDVEENPGP 18E2AGSGQCTNYALLKLAGDVESNPGP 19RevMAGRSGDSDEDLLKAVRLIKFLYQSNPPPNPEGTRQARRNRRRRWRERQRQIHSISERILSTYLGRSAEPVPLQLPPLERLTLDCNEDCGTSGTQGVGSPQILVESPTILESGAKE20CD117 scFvMALPVTALLLPLALLLHAARPDYKDHDGDYKDHDIDYKDDDDKDIQMTQSPSSLSASVGDRVTITCRASQRGLRNVAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQWAVHSLITFGQGTKVEIKRTVGGGGSGGGGSGGGGSSSEVQLVESGGGLVQPGGSLRLSCAASGFNISVYMMHWVRQAPGKGLEWVASIYPYSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYVYHALDYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC21 J. Example Embodiments of the Present Disclosure

[120] In accordance with one embodiment of the present disclosure, provided is a composition useful for preparing a VLP. The composition may be present as a collected supernatant collected from a cell culture with cells producing various proteins / RNA as illustrated above.

[121] In one embodiment, provided is a composition that includes, among others, (a) a first protein (Protein #1) comprising a Gag-Pol polyprotein; (b) a second protein (Protein #2) comprising an envelope protein; (c) a third protein (Protein #3) comprising a Cas protein without a Gag protein; (d) a fourth protein (Protein #4) comprising a Gag protein fused to a first RNA binding domain (RBD#1); and (e) a first RNA (RNA #1) comprising a guide RNA fused to a first APT sequence (APT#1) that is recognizable by the RBD#1.

[122] In some embodiments, as illustrated in Table A, in the Protein #4, the Gag protein is fused to the RBD#1 through a first protease cleavage site (PCS) which is cleavable by the Gag-Pol polyprotein upon proteolytic virion maturation. In some embodiments, the Protein #3 further includes a nucleobase deaminase (ND) or a reverse-transcriptase (RT). The ND or RT can work with the Cas protein (e.g., a nickase) in concert, along with a suitable gRNA (e.g., sgRNA or pegRNA), to conduct base editing or prime editing.

[123] In some embodiments, the ND or RT is provided as a separate protein (Tables B and C). Accordingly, in one embodiment, the composition further includes a fifth protein (Protein #5) that comprises a nucleobase deaminase (ND) or a reverse-transcriptase (RT) fused to a second RNA binding domain (RBD#2). To this end, in some embodiments, the RNA #1 further includes a second APT sequence (APT#2) that is recognizable by the RBD#2. Preferably the RBD#1 and the RBD#2 have distinguishable RNA recognition properties. In other words, RBD#1 can bind to APT#1 but does not bind to APT#2, and vice versa.

[124] In some embodiments, as illustrated in Table C, the base editing or prime editing takes advantage of a dual-gRNA system, in which a helper gRNA is added that recognizes an adjacent sequence to that of the first gRNA. The use of two gRNA can further improve target specificity of the editors. Accordingly, in one embodiment, the composition further includes a fifth protein (Protein #5) that includes a nucleobase deaminase (ND) or a reverse-transcriptase (RT) fused to a second RNA binding domain (RBD#2) and a second RNA (RNA #2) that comprises a helper guide RNA (hsgRNA) fused to a second copy of the APT#1 and a second APT sequence (APT#2) that is recognizable by the RBD#2. In some embodiments, the RBD#1 and the RBD#2 have distinguishable RNA recognition properties, and wherein the gRNA and the hsgRNA preferably recognize adjacent nucleotide sequences on a target genome.

[125] In some embodiments, one or more nuclear export signal (NES) can be added to one or more proteins of the composition to facilitate their cytoplasmic localization. Preferably, the NES is not retained in any of the components required for gene editing in a target cell. Therefore, in some embodiments, the NES is cleaved during the VLP maturation process.

[126] In one embodiment, therefore, the Protein #3 further includes a nuclear export signal (NES) fused to the Cas protein, through a second PCS cleavable by the Gag-Pol polyprotein upon proteolytic virion maturation. In addition or alternatively, in some embodiments, the Protein #4 further includes a second NES that is separated from the RBD#1 upon cleavage of the first PCS.

[127] In some embodiments, one or more uracil glycosylase inhibitor (UGI) can be added as a standalone protein or as an additional element of any of the proteins of the composition. In one embodiment, the Protein #4 further comprises a first uracil glycosylase inhibitor (UGI) which stays fused to the RBD#1 upon cleavage of the first PCS. Upon the PCS cleavage, therefore, the UGI (fused to the RBD#1) serves as a standalone (relative to the base editing components) UGI.

[128] In addition or alternatively, to provide a standalone UGI, a sixth protein (Protein #6) is added to the composition that includes a third UGI fused to a third RNA binding domain (RBD#2), wherein the RNA #1 further comprises a third APT sequence (APT#3) that is recognizable by the RBD#3, wherein the RBD#1, the RBD#2 and the RBD#3 each has distinguishable RNA recognition property.

[129] In some embodiments, the Protein #5 further includes a UGI which stays fused to the ND during the preparation, transduction and editing processes.

[130] In some embodiments, the Protein #5 includes a nucleobase deaminase inhibitor that inhibits the activity of the ND, and can be cleaved off when the ND binds to the target DNA or RNA for editing. Therefore, in some embodiments, the ND inhibitor is fused to the ND through a protease recognition site, such as one for Tev protease, TuMV protease, PPV protease, PVY protease, ZIKV protease or WNV protease.

[131] Methods of preparing VLP with such compositions are also provided. In one embodiment, the method entails culturing a cell engineered to express each of the proteins and RNA of the above compositions in a medium, collecting supernatant from the medium, and precipitating VLP formed in the supernatant.

[132] Still further, provided are VLPs prepared with compositions and methods of the present disclosure. In one embodiment, as illustrated in Table A’, the VLP includes (a) a first protein (Protein #1) comprising a Gag-Pol polyprotein; (b) a second protein (Protein #2) comprising envelope protein; (c) a third protein (Protein #3) comprising a Cas protein without a Gag protein; (d) a fourth protein (Protein #4) comprising a Gag protein; and (e) a first RNA (RNA #1) comprising a guide RNA fused to a first APT sequence (APT#1) that is recognizable by a first RNA binding domain (RBD#1).

[133] In some embodiments, the Protein #3 further includes a nucleobase deaminase (ND) or a reverse-transcriptase (RT). In some embodiments, as illustrated in Table B’, the VLP further includes a fifth protein (Protein #5) that comprises a nucleobase deaminase (ND) or a reverse-transcriptase (RT) fused to a second RNA binding domain (RBD#2), wherein the RNA #1 further comprises a second APT sequence (APT#2) that is recognizable by the RBD#2, wherein the RBD#1 and the RBD#2 have distinguishable RNA recognition properties.

[134] In some embodiments, as illustrated in Table C’, the VLP further includes a fifth protein (Protein #5) that comprises a nucleobase deaminase (ND) or a reverse-transcriptase (RT) fused to a second RNA binding domain (RBD#2); and a second RNA (RNA #2) that comprises a helper guide RNA (hsgRNA) fused to a second copy of the APT#1 and a second APT sequence (APT#2) that is recognizable by the RBD#2. In some embodiments, the RBD#1 and the RBD#2 have distinguishable RNA recognition properties, and wherein the gRNA and the hsgRNA preferably recognize adjacent nucleotide sequences on a target genome.

[135] In some embodiments, as illustrated in Table D’, the Protein #4 further includes a second NES fused to the Gag protein. In some embodiments, as illustrated in Table E’, the Protein #5 further comprises a first uracil glycosylase inhibitor (UGI).

[136] In some embodiments, also as illustrated in Table D’, the VLP further includes a sixth protein (Protein #6) that comprises a second UGI fused to a third RNA binding domain (RBD#2), wherein the RNA #1 further comprises a third APT sequence (APT#3) that is recognizable by the RBD#3, wherein the RBD#1, the RBD#2 and the RBD#3 each has distinguishable RNA recognition property.

[137] Methods of using the VLP of the present disclosure to transduce a target cell is also provided, which can achieve delivery of components needed for gene editing. In one embodiment, provided is a method for delivering a Cas protein and associated guide RNA to a target cell, comprising transducing the target cell with a VLP of the present disclosure.

[138] Also provided, in some embodiments, are cells useful for producing the compositions as described above. In one embodiment, the isolated cell expresses the proteins and RNA of the composition includes, in addition to all other required coding sequences, a construct encoding a first mRNA that encodes a Rev protein, wherein the first mRNA further comprises a first Rev response element (RRE).

[139] In some embodiments, the first mRNA further encodes the Protein #4. In some embodiments, within the first mRNA, the Rev-coding sequence and the RRE are separated from the Gag protein-coding sequence by a 2A self-cleavage peptide coding sequence.

[140] In some embodiment, the cell further includes a second construct that can be transcribed into a second mRNA encoding the Protein #1, wherein the second mRNA further comprises a second RRE. Methods of Preparing VLPs

[141] The compositions of the present disclosure, in various configurations, can be used to prepare VLPs useful for delivering gene editing machinery to a target cell. In particular, a virus-like particle that is used herein is a retrovirus-derived particle. Such retrovirus may be selected among Moloney murine leukemia virus, Bovine immunodeficiency virus, Simian immunodeficiency virus, Feline immunodeficiency virus, Human immunodeficiency virus, Equine infection anemia virus, and Caprine arthritis encephalitis virus.

[142] In another embodiment, a virus-like particle that is used herein is a lentivirus-derived particle. Lentiviruses belong to the retrovirus family, and have the unique ability of being able to infect non-dividing cells. Such lentivirus may be selected among Bovine immunodeficiency virus, Simian immunodeficiency virus, Feline immunodeficiency virus, Human immunodeficiency virus, Equine infection anemia virus, and Caprine arthritis encephalitis virus.

[143] For preparing Moloney murine leukemia virus-derived vector particles, one skilled in the art may refer to the methods disclosed by Sharma et al. (1997, Proc Natl Acad Sci USA, Vol. 94: 1O8O3+- 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 in a group comprising MLV-A-derived vector particles and MLV-E-derived vector particles.

[144] For preparing Bovine Immunodeficiency virus-derived vector particles, one skilled in the art may refer to the methods disclosed by Rasmussen et al. (1990, Virology, Vol. 178(no 2): 435-451).

[145] For preparing Simian immunodeficiency virus-derived vector particles, including VSV-G pseudotyped SIV virus-derived particles, one skilled in the art may notably refer to 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), el02).

[146] For preparing Feline Immunodeficiency virus-derived vector particles, one skilled in the art may notably refer 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).

[147] For preparing Human immunodeficiency virus-derived vector particles, one skilled in the art may notably refer 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).

[148] For preparing Equine infection anemia virus-derived vector particles, one skilled in the art may notably refer to the methods disclosed by Olsen (1998, Gene Ther, Vol. 5(no 11): 1481-1487).

[149] For preparing Caprine arthritis encephalitis virus-derived vector particles, one skilled in the art may notably refer to the methods disclosed by Mselli-Lakhal et al. (2006, J Virol Methods, Vol. 136(no 1-2): 177-184).

[150] For preparing Baboon endogenous virus-derived vector particles, one skilled in the art may notably refer to the methods disclosed by Girard-Gagnepain et al. (2014, Blood, Vol. 124(no 8): 1221-1231).

[151] For preparing Rabies virus-derived vector particles, one skilled in the art may notably refer to the methods disclosed by Kang et al. (2015, Viruses, Vol. 7: 1134-1152, doi:10.3390 / v7031134) and Fontana et al. (2014, Vaccine, Vol. 32(no 24): 2799-27804), or to the PCT application published under no. WO 2012 / 0618. For preparing Influenza virus-derived vector particles, one skilled in the art may notably refer to the methods disclosed by Quan et al. (2012, Virology, Vol. 430: 127-135) and to Latham et al. (2001, Journal of Virology, Vol. 75(no 13): 6154-6155).

[152] For preparing Norovirus-derived vector particles, one skilled in the art may notably refer to the methods disclosed by Tome-Amat et al., (2014, Microbial Cell Factories, Vol. 13: 134-142). For preparing Respiratory syncytial virus-derived vector particles, one skilled in the art may notably refer to the methods disclosed by Walpita et al. (2015, PlosOne, DOI: 10.1371 / journal. pone.0130755).

[153] For preparing Hepatitis B virus-derived vector particles, one skilled in the art may notably refer to the methods disclosed by Hong et al. (2013, Journal of Virology, Vol. 87(no 12): 6615-6624). For preparing Hepatitis E virus-derived vector particles, one skilled in the art may notably refer to the methods disclosed by Li et al. (1997, Journal of Virology, Vol. 71(no 10): 7207-7213).

[154] For preparing Newcastle disease virus-derived vector particles, one skilled in the art may notably refer to the methods disclosed by Murawski et al. (2010, Journal of Virology, Vol. 84(no 2): 1110-1123). For preparing Norwalk virus-derived vector particles, one skilled in the art may notably refer to the methods disclosed by Herb st- Kralovetz et al. (2010, Expert Rev Vaccines, Vol. 9(no 3): 299-307).

[155] For preparing Parvovirus-derived vector particles, one skilled in the art may notably refer to the methods disclosed by Ogasawara et al. (2006, In Vivo, Vol. 20: 319-324). For preparing Papillomavirus -derived vector particles, one skilled in the art may notably refer to the methods disclosed by Wang et al. (2013, Expert Rev Vaccines, Vol. 12(no 2): doi:10.1586 / erv.l2.151).

[156] A virus-like particle includes a Gag protein, preferably a Gag protein originating from a virus selected from a group consisting of Rous Sarcoma Virus (RSV) Feline Immunodeficiency Virus (FIV), Simian Immunodeficiency Virus (SIV), Moloney Leukemia Virus (MLV), and Human Immunodeficiency Viruses (HIV-1 and HIV- 2), especially Human Immunodeficiency Virus of type 1 (HIV-1).

[157] In some embodiments, a virus-like particle may also include one or more viral envelope protein(s). The presence of one or more viral envelope protein(s) may impart to the virus-derived particle a more specific tropism for the cells which are targeted, as it is known in the art. The one or more viral envelope protein(s) may be selected from a 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 retroviral derived vectors.Compositions and Methods for Gene Editing

[158] The various compositions of the instant disclosure, such as the prepared VLPs, can be used for conducting gene editing in a target cell. To effect gene editing, in some embodiments, a target cell is placed in contact with a VLP of the instant disclosure to be transduced by the VLP. Upon transduction, the various proteins and guide RNA are delivered to the target cell. Editing is guided by the guide RNA and effected by the corresponding Cas protein or associated base / prime editor.

[159] Such transduction can be carried out in vitro, in particular in a cell culture. When the transduction is ex vivo, or in vivo, the VLP can have clinical / therapeutic significance.Split Base Editing with UGI’s

[160] The experimental examples demonstrated that addition of UGI to various locations in a base editor resulted in improved editing efficiency. For instance, the addition of a UGI (FIG. 12) or two concatenated ones (FIG. 14) increased the editing efficiency of design VLP_V6-tBE-V1 as compared to VLP_V5-tBE_V1 (FIG. 7). Then, an addition UGI placed at the C-terminus of the Cas protein (FIG. 13) further improved the editing efficiency.

[161] Interestingly, as shown in FIG. 17A-D, when a similar amount of UGI’s were used, it was more effective to place a greater number of UGI with the Cas protein instead of the deaminase (compare VLP_V6.2-tBE_V1 to VLP_V6.3-tBE_V1).

[162] In accordance with one embodiment of the present disclosure, therefore, provided is a split base editor that includes, on separate proteins, a Cas protein and a deaminase. In a preferred embodiment, the Cas protein is fused to at least 2 UGI, preferably at the C-terminus. In some embodiments, the Cas protein is fused to at least 3 UGI, preferably at the C-terminus of the Cas protein.

[163] In some embodiments, the deaminase is fused to at least a UGI. In some embodiments, the deaminase is fused to at least two UGI. In some embodiments, at least 1, 2 or more of the UGI are fused to the N-terminus of the deaminase.

[164] In some embodiments, the base editing system further includes 1, 2, 3, 4, 5, 6 or more standalone UGI. A standalone UGI is one that is directly fused to either the Cas protein or the deaminase, but can be fused to other proteins that becomes associated with the Cas protein or the deaminase through inter-molecule interaction.

[165] In some embodiments, the Cas protein and the deaminase are brought to the target DNA through interactions such as a RNA binding domain (RBD) that recognizes a corresponding RNA aptamer (APT).

[166] In a particular example, a base editing system of the present disclosure includes (A) a first fusion protein that includes a Cas protein and at least a UGI, (B) a second fusion protein that includes a deaminase, a RNA binding domain (RBD) and at least a UGI, and (C1) a single guide RNA (sgRNA) fused to a RNA aptamer (APT) that is recognized by the RBD, or (C2) a sgRNA and a helper sgRNA (hsgRNA) that is fused to the APT.

[167] In some embodiments, the first fusion includes at least 2 UGI.

[168] In some embodiments, the base editor further includes elements such as Gag, Pol, RRE, VSVG etc that facilitate packaging of the based editor into VLP. EXAMPLESExample 1. Fusion Base Editors with Reduced Off-Target Editing Activity

[169] This example tested various methods of delivering a cytosine base editor (CBE) to a target cell, through virus-like particle (VLP) packaging.

[170] To test whether a cytosine base editor (CBE) can be packaged in VLP, we first fused two commonly-used CBEs, i.e., BE3 and hA3A-BE3, to the C-terminus of the human immunodeficiency virus (HIV) Gag protein with three copies of nuclear export signals (3´NES) via an HIV protease cleavage site (PCS), i.e., the SQNYPIVQ (SEQ ID NO:1) motif (FIG.1A) and generated the plasmids pGag-NES-PCS-BE3 and pGag-NES-PCS-hA3A_BE3. Then, we produced the corresponding VLP_V0-BE3 (or VLP_V0-hA3A_BE3) by co-transfecting pGag-NES-PCS-BE3 (or pGag-NES-PCS-hA3A_BE3), psPAX2 (expressing the HIV Gag-Pol polyprotein and HIV Rev protein and containing an HIV Rev response element, RRE) and pMD2.G-sgRNA (expressing vesicular stomatitis virus glycoprotein, VSVG and single-guide RNA, sgRNA) (FIG.1A) into packaging cells.

[171] Next, we transduced 293FT cells with VLP_V0-BE3 (or VLP_V0-hA3A_BE3) and extracted the genomic DNA 48 hours after transduction. However, no obvious on-target base editing was detected (FIG.1B and 1C). In contrast, BE3 induced efficient base editing at the same target site in the plasmid-transfected 293FT cells (FIG.1B and 1C). These results suggested that CBEs were not successfully delivered into target cells. To explore why CBEs were not able to be delivered by fusing to HIV Gag protein, we performed immunoblots to determine the protein content of BE3 and hA3A-BE3 in VLP_V0-BE3 and VLP_V0-hA3A_BE3 with SpCas9-specific antibodies (FIG.1D). Compared to the BE3 and hA3A-BE3 expressed from plasmids, we surprisingly found that the protein contents of full-length BE3 and hA3A-BE3 (~200 kD) in VLPs were low but the contents of their truncated versions were high (FIG.1D), suggesting that BE3 and hA3A-BE3 were cleaved during the production or maturation of VLPs.

[172] Next, we attempted to package a split version of CBEs (tBE) into VLPs. As the nSpCas9 (D10A) is not fused with cytidine deaminase in the tBE system, we fused D10A to the C-terminus of Gag-NES with an HIV PCS (FIG.2A). However, we found that the D10A in VLPs was also cleaved (FIG.2C). Alternatively, we linked a COM protein to the C-terminus of Gag-NES protein via PCS and added a Com aptamer to the sgRNA and hsgRNA of tBE system (FIG.2B). Thus, the Gag-NES-PCS-COM fusion protein can recruit sgRNA-Com / D10A and hsgRNA-Com / D10A RNP complex into VLPs. We also examined the protein content of D10A in the produced VLPs and found that the unintended cleavage of D10A was significantly reduced when it was recruited by sgRNA-Com or hsgRNA-Com (FIG.2C).

[173] To produce functional VLPs that contain CBEs, e.g., tBE_V1, we co-transfected four plasmids into the packaging cells (FIG.3A). 1) pD10A-T2A-MCP-UGI-mA3C, which expresses D10A and the fusion protein of MCP (MS2-aptamer-binding protein), UGI (uracil DNA glycosylase inhibitor) and mA3C (mouse APOBEC3, catalytic domain) through a 2A self-cleaving peptide. 2) pGag-NES-PCS-COM, which expresses the fusion protein of Gag-NES, PCS and COM protein. 3) psPAX2, which expresses HIV Gag-Pol polyprotein and HIV Rev protein and also contains an RRE. 4) pMD2.G-hsgRNA_MS2_Com-sgRNA_Com, which expresses VSVG, hsgRNA_MS2_Com and sgRNA_Com. Theoretically, the COM protein fused to the C-terminus of Gag can be packaged into VLPs. Thus, the hsgRNA_MS2_Com / D10A and sgRNA_Com / D10A RNP complexes will be packaged into VLPs through the interaction of COM protein and Com aptamer. Meanwhile, the effector protein (MCP-UGI-mA3C) will also be packaged into VLPs through the interaction of MCP protein and MS2 aptamer of hsgRNA_MS2_Com. After the VLPs were produced from the packaging cells, we concentrated these tBE_V1-containing VLPs (VLP_V1-tBE_V1) and transduced 293FT cells with them. Forty-eight hours after transduction, we extracted the genomic DNA from the transduced cells and found C-to-T, C-to-A and C-to-G editing was all induced at the on-target site (FIG.3B). The total editing frequencies at two targeted cytosines (C6 and C7) were 57% (C-to-T), 38% (C-to-G) and 25% (C-to-A) respectively (FIG.3C). In contrast, no obvious editing was found in non-treated 293FT cells (FIG.3B and 3C).

[174] In order to improve the editing efficiency, we removed the Rev gene from psPAX2, resulting in the plasmid psPAX2-del-Rev (FIG.4A). Instead, we fused the Rev protein to the C-terminus of Gag-NES-PCS-COM protein via a 2a self-cleaving peptide and added the RRE to the 3’ end of the coding region of this fusion protein (FIG.4A, pGag-NES-PCS-COM-T2A-Rev-RRE). We produced the second version of tBE_V1-containing VLPs (VLP_V2-tBE_V1) by co-expressing pD10A-T2A-MCP-UGI-mA3C, pGag-NES-PCS-COM-T2A-Rev-RRE, psPAX2-del-Rev and pMD2.G-hsgRNA_MS2_COM-sgRNA_Com (FIG.4A) in packaging cells. After transducing 293FT cells with VLP_V2-tBE_V1, we found that the C-to-T (total 56%), C-to-G (total 45%) and C-to-A (total 31%) editing efficiencies at the two targeted cytosines (FIG.4B and 4C) were generally improved when compared to VLP_V1-tBE_V1 at the PRR5L target site (FIG.3B and 3C).

[175] To further improve the editing efficiency, we added HIV PCS and 3´NES to the C-terminal of D10A protein and constructed the plasmid pD10A-PCS-NES-T2A-MCP-UGI-mA3C (FIG.5A). As the NES can export protein from cell nucleus to cytoplasm, the level of D10A protein in the cytoplasm of packaging cells can increase, which should facilitate its packaging into VLP. During the maturation of VLPs, the HIV-protease-mediated cleavage at PCS can remove the NES from the D10A and therefore not compromise the importation of D10A into the nucleus of target cells. We produced the third version of tBE_V1-containing VLPs (VLP_V3-tBE_V1) by co-expressing pD10A-PCS-NES-T2A-MCP-UGI-mA3C, pGag-NES-PCS-COM-T2A-Rev-RRE, psPAX2-del-Rev and pMD2.G-hsgRNA_MS2_COM-sgRNA_Com (FIG.5A) in packaging cells. After transducing 293FT cells with VLP_V3-tBE_V1, we found that the C-to-T (total 69%), C-to-G (total 52%) and C-to-A (total 35%) editing efficiencies at the two targeted cytosines (FIG.5B and 5C) were higher when compared to VLP_V2-tBE_V1 at the PRR5L target site (FIG.4B and 4C). We also produced the VLP_V2-tBE_V1 targeting human HBG and mouse PCSK9 and transduced 293FT and N2A cells, respectively. Of note, obvious editing was detected at the HBG target site in 293FT cells and the PCSK9 target site in N2A cells (FIG.5D-5G)

[176] Though the editing efficiency induced by VLP_V2-tBE_V1 was improved, its product purity (the ratio of C-to-T editing to C-to-A / C-to-G editing) remained relatively low (FIG.5C). We found that the expression of UGI can improve both the editing efficiency and the product purity of CBE. Thus, we expressed the UGI fused with N22P, the protein that can specifically bind to the boxB aptamer of sgRNA-boxB-Com in the packaging cells and the N22P-UGI fusion should be packaged into VLPs. We produced the fourth version of tBE_V1-containing VLPs (VLP_V4-tBE_V1) by co-expressing pD10A-PCS-NES-T2A-MCP-UGI-mA3C, pGag-NES-PCS-COM-T2A-Rev-RRE, psPAX2-del-Rev, pMD2.G-hsgRNA_MS2_COM-sgRNA_boxB_Com and pN22P-UGI (FIG.6A) in packaging cells. After transducing 293FT cells with VLP_V4-tBE_V1, we found that the C-to-T editing efficiencies (total 137%) and the product purity at the two targeted cytosines (FIG.6B and 6C) were obviously improved when compared to VLP_V3-tBE_V1 at the PRR5L target site (FIG.5B and 5C). Similarly, the C-to-T editing efficiencies were also improved at the HBG target site in 293FT cells and the PCSK9 target site in N2A cells (FIG.6D-6G).

[177] As our study also found that the UGI expression level is positively correlated with the editing efficiency and product purity of CBE, we fused UGI to the N-terminus of COM protein and constructed the corresponding expression plasmid pGag-NES-PCS-UGI-COM-T2A-Rev-RRE (FIG.7A). Thus, the UGI-COM fusion protein can be recruited by the Com aptamers in both hsgRNA-MS2-Com and sgRNA-Com and the UGI level in VLPs can increase. We produced the fifth version of tBE_V1-containing VLPs (VLP_V5-tBE_V1) by co-expressing pD10A-PCS-NES-T2A-MCP-UGI-mA3C, pGag-NES-PCS-UGI-COM-T2A-Rev-RRE, psPAX2-del-Rev and pMD2.G-hsgRNA_MS2_Com-sgRNA_Com (FIG. 7A) in packaging cells. After transducing 293FT cells with VLP_V5-tBE_V1, we found that the C-to-T (total 158%) editing efficiencies and the product purity at the two targeted cytosines (FIG.7B and 7C) were obviously improved when compared to VLP_V4-tBE_V1 (FIG.6B and 6C).

[178] We then used our VLP_V3 system to package SpCas9 by co-expressing pGag-NES-PCS-COM-T2A-Rev-RRE, pSpCas9, psPAX2-del-Rev and pMD2.G-hsgRNA_MS2_Com-sgRNA_Com (FIG.8A) in packaging cells. After transducing 293FT cells with VLP_V3-SpCas9, we found that obvious deletion (~92%) were induced between the cutting sites directed by hsgRNA_MS2_Com and sgRNA_Com (FIG.8B-8D).

[179] We also used our VLP_V3 system to package BE3 by co-expressing pGag-NES-PCS-COM-T2A-Rev-RRE, pBE3, psPAX2-del-Rev and pMD2.G-hsgRNA_MS2_Com-sgRNA_Com (FIG.9A) in packaging cells. After transducing 293FT cells with VLP_V3-BE3, we found that detectable C-to-T editing efficiencies (total 24%) were induced (FIG.9B and 9C). We further used our VLP_V5 system to package BE3 by co-expressing pGag-NES-PCS-UGI-COM-T2A-Rev-RRE, pBE3, psPAX2-del-Rev and pMD2.G-hsgRNA_MS2_Com-sgRNA_Com (FIG.10A) in packaging cells. After transducing 293FT cells with VLP_V5-BE3, we found that the C-to-T (total 84%) editing efficiencies at the two targeted cytosines (FIG.10B and 10C) were obviously improved when compared to VLP_V3-BE3 (FIG.9B and 9C).

[180] As demonstrated above, we developed a new VLP system that can deliver CBEs into mammalian cells and induce efficient base editing at target sites (FIG.11). For the delivery of tBE-derived CBE system, Gag-Pol protein, Gag-UGI-COM protein, the hsgRNA and sgRNA that contain Com aptamer, D10A protein and the MCP-UGI-mA3c effector protein in VLP-packaging cells. The self-assembly of these proteins in the cytoplasm of packaging cells can form the cargoes for VLPs. The VSV-G protein on the surface of VLPs has a broad spectrum of transduction. In the transduced cells, the D10A / sgRNA protein complex and MCP-UGI-mA3c protein are released and efficient C-to-T base editing can be induced at the on-target editing sites. This new system has the advantage of delivering CBE into cells through VLPs and achieves efficient C-to-T base editing at on-target sites.

[181] Also, we expressed UGI fused to N22P in packaging cells based on VLP_V5-tBE_V1. We produced the sixth version of tBE_V1-containing VLPs (VLP_V6-tBE_V1) by co-expressing pD10A-PCS-NES-T2A-MCP-UGI-mA3C, pGag-NES-PCS-UGI-COM-T2A-Rev-RRE, psPAX2-del-Rev, pMD2.G-hsgRNA_MS2_Com-sgRNA_boxB_Com and pN22P-UGI (FIG.12A) in packaging cells. After transducing 293FT cells with VLP_V6-tBE_V1, we detected the C-to-T (total 99%) editing efficiencies and the product purity at the two targeted cytosines (FIG.12B and 12C).

[182] Furthermore, we fused UGI to the C-terminus of D10A and constructed the corresponding expression plasmid pD10A-UGI-PCS-NES-T2A-MCP-UGI-mA3C (FIG. 13A). We produced the seventh version of tBE_V1-containing VLPs (VLP_V7-tBE_V1) by co-expressing pD10A-UGI-PCS-NES-T2A-MCP-UGI-mA3C, pGag-NES-PCS-UGI-COM-T2A-Rev-RRE, psPAX2-del-Rev, pMD2.G-hsgRNA_MS2_Com-sgRNA_boxB_Com and pN22P-UGI (FIG. 9A) in packaging cells. After transducing 293FT cells with VLP_V7-tBE_V1, we found that the C-to-T (total 118%) editing efficiencies and the product purity at the two targeted cytosines (FIG.13B and 13C) were improved when compared to VLP_V6-tBE_V1 and VLP_V5-tBE_V1 (FIG.12B and 12C).

[183] Then we expressed 2×UGI fused to N22p in packaging cells based on VLP_V7-tBE_V1 and constructed the corresponding expression plasmid pN22p-2×UGI (FIG. 14A). We produced the eighth version of tBE_V1-containing VLPs (VLP_V8-tBE_V1) by co-expressing pD10A-UGI-PCS-NES-T2A-MCP-UGI-mA3C, pGag-NES-PCS-UGI-COM-T2A-Rev-RRE, psPAX2-del-Rev, pMD2.G-hsgRNA_MS2_Com-sgRNA_boxB_Com and pN22P-2×UGI (FIG.14A) in packaging cells. After transducing 293FT cells with VLP_V8-tBE_V1, we found that the C-to-T (total 135%) editing efficiencies and the product purity at the two targeted cytosines (FIG.14B and 14C) were improved when compared to VLP_V7-tBE_V1 (FIG.13B and 13C).

[184] VSVG, a widely used viral envelope protein, interacts with the low-density lipoprotein receptor (LDLR) on host cells. Some mutant VSVG pseudotyped viruses are defective in LDLR binding but retain the ability to fuse compared to wild-type VSVG pseudotyped viruses. Various CD117 ligands (such as anti-CD117 single-chain antibody fragment s(scFv), human stem cell factor and murine stem cell factor, etc.) are available that specifically recognize CD117. In this example, we co-expressed VSVGmut and CD117 ligands in packaging cells by constructing plasmids pVSVGmut and pCD117 ligands (FIG.15A). We produced Lenti-GFP, Lenti-GFP-VSVGmut, and Lenti-GFP-CD117 by co-expressing pLenti-GFP, psPAX2 and pMD2.G or pVSVGmut or pVSVGmut, pCD117 ligands (FIG.15A) in packaging cells. After transducing 293FT cells with Lenti-GFP, Lenti-GFP-VSVGmut, and Lenti-GFP-CD117, GFP+ cells were sorted by flow cytometry, which showed that Lenti-GFP-VSVGmut and Lenti-GFP-CD117 infection efficiency was significantly lower compared to Lenti-GFP (FIG.15B). After transducing 293FTCD117+ (293FT with high CD117 expression) cells with Lenti-GFP, Lenti-GFP-VSVGmut, and Lenti-GFP-CD117, GFP+ cells were sorted by flow cytometry, which showed that Lenti-GFP and Lenti-GFP-CD117 were able to be infected normally compared to Lenti-GFP-VSVGmut (FIG.15C).

[185] We then fused VSVGmut and CD117 ligands into VLP. We produced VLP_V5.1-tBE_V1, VLP_V5.1-tBE_V1-VSVGmut, and VLP_V5.1-tBE_V1-CD117 by co-expressing pD10A-PCS-NES-T2A-MCP-UGI-mA3C, pGag-NES-PCS-UGI-COM-T2A-Rev-RRE, psPAX2-del-Rev-hsgRNA_MS2_Com-sgRNA_boxB_Com and pMD2.G or pVSVGmut or pVSVGmut, pCD117 ligands (FIG.16A) in packaging cells. After transducing 293FT cells with VLP_V5.1-tBE_V1, VLP_V5.1-tBE_V1-VSVGmut, and VLP_V5.1-tBE_V1-CD117, we found that the C-to-T editing efficiencies of VLP_V5.1-tBE_V1-VSVGmut (total 30%) and VLP_V5.1-tBE_V1-CD117 (total 25%) at the two targeted cytosines (FIG.16B and FIG.16C) were reduced when compared to VLP_V5.1-tBE_V1 (total 110%). After transducing 293FTCD117+ cells with VLP_V5.1-tBE_V1, VLP_V5.1-tBE_V1-VSVGmut, and VLP_V5.1-tBE_V1-CD117, we found that the C-to-T editing efficiencies of VLP_V5.1-tBE_V1 (total 84%) and VLP_V5.1-tBE_V1-CD117 (total 123%) at the two targeted cytosines (FIG.16B and 16C) were improved when compared to VLP_V5.1-tBE_V1-VSVGmut (total 28%).

[186] Three additional designs (VLP_V6.1-tBE_V1, VLP_V6.2-tBE_V1, and VLP_V6.3-tBE_V1) were tested, each having UGI disposed at different locations (FIG. 17A). In VLP_V6.1-tBE_V1, two UGI’s were placed at the N-terminus of the mA3C; in VLP_V6.2-tBE_V1, one UGI was placed at the N-terminus of the mA3C and two UGI’s were placed at the C-terminus of the Cas protein; and in VLP_V6.3-tBE_V1, two UGI’s were placed at the N-terminus of the mA3C and one UGI was placed at the C-terminus of the Cas protein. These UGI’s were in addition to two UGI’s in other parts of the system.

[187] The comparison of these designs, along with VLP_V6-tBE_V1, VLP_V7-tBE_V1, and VLP_V8-tBE_V1, for editing the VEGFA site in 293FT cell is shown in FIG. 17D. As shown in the figure, among VLP_V6.1-tBE_V1, VLP_V6.2-tBE_V1, and VLP_V6.3-tBE_V1, VLP_V6.2-tBE_V1 has the highest editing efficiency. This suggests that more UGI’s (e.g., 2x) fused to the Cas protein was more effective than being fused to the deaminase.

[188] In this example, we have developed a VLP capable of delivering CBE systems with high editing efficiency at on-target sites. In addition, our VLP-CBE system induced efficient and precise editing in different cells. Therefore, the delivery method described herein can be used for highly precise and efficient base editing in various cells and various species.* * *

[189] The present disclosure is not to be limited in scope by the specific embodiments described which are intended as single illustrations of individual aspects of the disclosure, and any compositions or methods which are functionally equivalent are within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and compositions of the present disclosure without departing from the spirit or scope of the disclosure. Thus, it is intended that the present disclosure cover the modifications and variations of this disclosure provided they come within the scope of the appended claims and their equivalents.

[190] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. A composition, comprising:(a) a first protein (Protein #1) comprising a Gag-Pol polyprotein;(b) a second protein (Protein #2) comprising an envelope protein;(c) a third protein (Protein #3) comprising a Cas protein without a Gag protein;(d) a fourth protein (Protein #4) comprising a Gag protein fused to a first RNA binding domain (RBD#1); and(e) a first RNA (RNA #1) comprising a guide RNA fused to a first RNA aptamer (APT) sequence (APT#1) that is recognizable by the RBD#1.

2. The composition of claim 1, wherein, in the Protein #4, the Gag protein is fused to the RBD#1 through a first protease cleavage site (PCS) cleavable by the Gag-Pol polyprotein upon proteolytic virion maturation.

3. The composition of claim 1 or 2, wherein the Protein #3 further comprises a nucleobase deaminase (ND) or a reverse-transcriptase (RT).

4. The composition of claim 1 or 2, further comprising a fifth protein (Protein #5) that comprises a nucleobase deaminase (ND) or a reverse-transcriptase (RT) fused to a second RNA binding domain (RBD#2), wherein the RNA #1 further comprises a second APT sequence (APT#2) that is recognizable by the RBD#2, wherein the RBD#1 and the RBD#2 have distinguishable RNA recognition properties.

5. The composition of claim 1 or 2, further comprising:a fifth protein (Protein #5) that comprises a nucleobase deaminase (ND) or a reverse-transcriptase (RT) fused to a second RNA binding domain (RBD#2); and a second RNA (RNA #2) that comprises a helper guide RNA (hsgRNA) fused to a second copy of the APT#1 and a second APT sequence (APT#2) that is recognizable by the RBD#2, wherein the RBD#1 and the RBD#2 have distinguishable RNA recognition properties, and wherein the gRNA and the hsgRNA preferably recognize adjacent nucleotide sequences on a target genome.

6. The composition of any preceding claim, wherein the Protein #3 further comprises a nuclear export signal (NES) fused to the Cas protein, through a second PCS cleavable by the Gag-Pol polyprotein upon proteolytic virion maturation.

7. The composition of any one of claims 2-6, wherein the Protein #4 further comprises a second NES that is separated from the RBD#1 upon cleavage of the first PCS.

8. The composition of claim 3-7, wherein the Protein #4 further comprises a first uracil glycosylase inhibitor (UGI) which stays fused to the RBD#1 upon cleavage of the first PCS.

9. The composition of any one of claims 4-8, wherein the Protein #5 further comprises a second UGI.

10. The composition of any preceding claim, further comprising a sixth protein (Protein #6) that comprises a third UGI fused to a third RNA binding domain (RBD#3), wherein the RNA #1 further comprises a third APT sequence (APT#3) that is recognizable by the RBD#3, wherein the RBD#1, the RBD#2 and the RBD#3 each has distinguishable RNA recognition property.

11. The composition of any one of claims 3-10, wherein the ND is fused to a nucleobase deaminase inhibitor through a protease recognition site.

12. The composition of claim 11, wherein the protease recognition site is recognized by Tev protease, TuMV protease, PPV protease, PVY protease, ZIKV protease or WNV protease.

13. The composition of any one of claims 1-12, wherein the envelop protein is vesicular stomatitis virus glycoprotein (VSVG).

14. The composition of any one of claims 3-13, wherein the nucleobase deaminase (ND) is a cytidine deaminase, preferably APOBEC3C (A3C).

15. A virus-like particle (VLP) comprising:(a) a first protein (Protein #1) comprising a Gag-Pol polyprotein;(b) a second protein (Protein #2) comprising an envelope protein, which is preferably vesicular stomatitis virus glycoprotein (VSVG);(c) a third protein (Protein #3) comprising a Cas protein without a Gag protein;(d) a fourth protein (Protein #4) comprising a Gag protein; and(e) a first RNA (RNA #1) comprising a guide RNA fused to a first APT sequence (APT#1) that is recognizable by a first RNA binding domain (RBD#1).

16. The VLP of claim 15, wherein the Protein #3 further comprises a nucleobase deaminase (ND) or a reverse-transcriptase (RT).

17. The VLP of claim 15, further comprising a fifth protein (Protein #5) that comprises a nucleobase deaminase (ND) or a reverse-transcriptase (RT) fused to a second RNA binding domain (RBD#2), wherein the RNA #1 further comprises a second APT sequence (APT#2) that is recognizable by the RBD#2, wherein the RBD#1 and the RBD#2 have distinguishable RNA recognition properties.

18. The VLP of claim 15 or 16, further comprising:a fifth protein (Protein #5) that comprises a nucleobase deaminase (ND) or a reverse-transcriptase (RT) fused to a second RNA binding domain (RBD#2); and a second RNA (RNA #2) that comprises a helper guide RNA (hsgRNA) fused to a second copy of the APT#1 and a second APT sequence (APT#2) that is recognizable by the RBD#2, wherein the RBD#1 and the RBD#2 have distinguishable RNA recognition properties, and wherein the gRNA and the hsgRNA preferably recognize adjacent nucleotide sequences on a target genome.

19. The VLP of any one of claims 15-18, wherein the Protein #4 further comprises a second NES fused to the Gag protein.

20. The VLP of any one of claims 17-19, wherein the Protein #5 further comprises a first uracil glycosylase inhibitor (UGI).

21. The VLP of any one of claims 15-20, further comprising a sixth protein (Protein #6) that comprises a second UGI fused to a third RNA binding domain (RBD#3), wherein the RNA #1 further comprises a third APT sequence (APT#3) that is recognizable by the RBD#3, wherein the RBD#1, the RBD#2 and the RBD#3 each has distinguishable RNA recognition property.

22. The VLP of any one of claims 15-21, further comprising a cell targeting moiety, which is preferably a CD117 ligand, which is preferably an anti-CD117 antibody or antigen-binding fragment thereof.

23. A method for preparing a virus-like particle (VLP), comprising culturing a cell engineered to express each of the proteins and RNA of any one of claims 1-14 in a medium, collecting supernatant from the medium, and precipitating VLP formed in the supernatant.

24. A method for delivering a Cas protein and associated guide RNA to a target cell, comprising transducing the target cell with the VLP of any one of the claims 15-11.

25. An isolated cell that expresses the proteins and RNA of the composition of any one of claims 1-14, comprising, in addition to sequencing encoding the proteins and RNA, a construct encoding a first mRNA that encodes a Rev protein, wherein the first mRNA further comprises a first Rev response element (RRE).

26. The isolated cell of claim 25, wherein the first mRNA further encodes the Protein #4.

27. The isolated cell of claim 26, wherein, within the first mRNA, the Rev-coding sequence and the RRE are separated from the Gag protein-coding sequence by a 2A self-cleavage peptide coding sequence.

28. The isolated cell of any one of claims 24-27, further comprising a second construct that can be transcribed into a second mRNA encoding the Protein #1, wherein the second mRNA further comprises a second RRE.

29. A method for preparing a composition of any one of claims 1-14, comprising culturing the isolated cell of any one of claims 25-28 in a medium and collecting supernatant from the medium.

30. A base editor, comprising (A) a first fusion protein that comprises a Cas protein and at least a UGI, (B) a second fusion protein that comprises a deaminase, a RNA binding domain (RBD) and at least a UGI, and(C1) a single guide RNA (sgRNA) fused to a RNA aptamer (APT), or (C2) a sgRNA and a helper sgRNA (hsgRNA) that is fused to the APT,wherein the RBD binds the ATP.

31. The base editor of claim 30, wherein the first fusion protein comprises at least two UGI.

32. The base editor of claim 31, wherein the at least two UGI are disposed at the C-terminus of the Cas protein.

33. The base editor of any one of claims 30-32, wherein the second fusion protein comprises no more than one UGI.

34. The base editor of any one of claims 30-33, wherein the UGI is disposed at the N-terminus of the deaminase.

35. The base editor of any one of claims 30-34, wherein the deaminase is a cytidine deaminase.

36. The base editor of any one of claims 30-35, further comprising at least 1, 2, or 3 UGI not included in the first or second fusion protein.

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