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  • C12Y304/21061—Kexin (3.4.21.61), i.e. proprotein convertase subtilisin/kexin type 9

Definitions

• Cytosine base editors (CBEs) (Komor et al., 2016; Nishida et al., 2016) and adenine base editors (ABEs) (Gaudelli et al., 2017) in principle can together correct the majority of known disease-causing single-nucleotide variants (Anzalone et al., 2020; Rees and Liu, 2018).
• BEs have been applied to correct pathogenic point mutations and rescue disease phenotypes in mice and non-human primates (Levy et al., 2020; Yeh et al., 2020), highlighting the potential of in vivo base editing as a therapeutic strategy.
• eVLPs as a useful platform for transiently delivering gene editing agents (e.g., Cas9 or BE ribonucleoproteins) in vitro and in vivo with therapeutically relevant efficiencies and with minimized risk of off- target editing or DNA integration and similarly improves the in vivo delivery of other proteins and RNPs.
• gene editing agents e.g., Cas9 or BE ribonucleoproteins
• the present disclosure provides VLPs comprising a group- specific antigen (gag) protease (pro) polyprotein, a nucleic acid programmable DNA binding protein (napDNAbp), and a fusion protein comprising a gag nucleocapsid protein and a nuclear export sequence (NES), encapsulated by a lipid membrane and a viral envelope glycoprotein.
• the present disclosure provides VLPs comprising a mixture of cleaved and uncleaved products (i.e., some of the napDNAbps or BEs have been cleaved from the gag proteins and are free, while some have not yet been cleaved from the gag proteins).
• the napDNAbp is fused to one or more additional domains such as one or more NLS and/or one or more deaminase (z.e., to form a base editor).
• additional domains such as one or more NLS and/or one or more deaminase (z.e., to form a base editor).
• Each component of the pharmaceutical compositions provided herein may comprise any of the options described above in reference to the VLPs, or any of the other options provided by the present disclosure.
• a pharmaceutical composition further comprises a pharmaceutically acceptable excipient.
• FIGS. 2A-2G Optimization of BE-VLPs (identifying and engineering solutions to bottlenecks that limit VLP potency results in v2, v3, and v4 eVLPs).
• FIG. 2A More efficient linker cleavage leads to improved cargo release after VLP maturation.
• FIG. 2B Adenine base editing efficiencies of vl and v2 BE-eVLPs at position A7 of the BCL11A enhancer site in HEK293T cells. Optimization of protease-cleavable linker sequence is shown (see also FIG. 8).
• FIG 2C Improved localization of cargo in producer cells leads to more efficient incorporation into eVLPs.
• FIG. 1 Improved localization of cargo in producer cells leads to more efficient incorporation into eVLPs.
• FIG. 31 Molecules of BE-encoding DNA per v4 BE-eVLP detected by qPCR of lysed VLPs or lysis buffer only.
• FIG. 3J Amount of BE-encoding DNA detected by qPCR of lysate from cells that were either treated with BE- VLPs or transfected with BE-encoding plasmids.
• 8e- LV ABE8e-NG-LV
• 8e-eVLP v4 ABE8e-NG-eVLP.
• FIG. 7E Scotopic a-wave and b- wave amplitudes measured by ERG following overnight dark adaptation.
• FIG. 10D Comparison of editing efficiencies with particle
• the present disclosure provides pluralities of polynucleotides encoding the eVLP (e.g., BE- VLP) self-assembling component as described herein.
• the present disclosure provides pluralities of polynucleotides comprising: (i) a first polynucleotide (e.g., a plasmid) comprising a nucleic acid sequence encoding a viral envelope glycoprotein; (ii) a second polynucleotide (e.g., a plasmid) comprising a nucleic acid sequence encoding a group- specific antigen (gag) protease (pro) polyprotein; (iii) a third polynucleotide (e.g., a plasmid) comprising a nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises: (a) a group-specific antigen (gag) nucleocap
• FIGs. 19A-19B v4 BE-eVLPs enable efficient on-target editing with minimal off- target editing. Lower Cas-dependent off-target editing was observed compared to previous base editing approaches targeting the same site (e.g., Zeng et al., Nat. Med. (2020)).
• the ecTadA deaminase does not comprise an N-terminal methionine.
• the adenosine deaminase comprises ecTadA(8e) (z.e., as used in the base editor ABE8e) as described further herein. Reference is made to U.S. Patent Publication No. 2018/0073012, published March 15, 2018, which is incorporated herein by reference.
• Base editing refers to genome editing technology that involves the conversion of a specific nucleic acid base into another at a targeted genomic locus. In certain embodiments, this can be achieved without requiring double- stranded DNA breaks (DSB), or single stranded breaks (z.e., nicking).
• DSB double- stranded DNA breaks
• z.e., nicking single stranded breaks
• CRIS PR-based systems begin with the introduction of a DSB at a locus of interest. Subsequently, cellular DNA repair enzymes mend the break, commonly resulting in random insertions or deletions (indels) of bases at the site of the DSB.
• nucleobase editor is capable of deaminating an adenine (A) in DNA.
• nucleobase editors may include a nucleic acid programmable DNA binding protein (napDNAbp) fused to an adenosine deaminase.
• napDNAbp nucleic acid programmable DNA binding protein
• Some nucleobase editors include CRISPR-mediated fusion proteins that are utilized in the base editing methods described herein.
• the nucleobase editor comprises a DNA binding domain (e.g., a programmable DNA binding domain such as a dCas9 or nCas9) that directs it to a target sequence.
• the nucleobase editor comprises a nucleobase modification domain fused to a programmable DNA binding domain (e.g., dCas9 or nCas9).
• a to G editing is carried out by a deaminase, e.g., an adenosine deaminase.
• Nucleobase editors that can carry out other types of base conversions (e.g., C to G) are also contemplated.
• a “split nucleobase editor” refers to a nucleobase editor that is provided as an N- terminal portion (also referred to as a N-terminal half) and a C-terminal portion (also referred to as a C-terminal half) encoded by two separate nucleic acids.
• the polypeptides corresponding to the N-terminal portion and the C-terminal portion of the nucleobase editor may be combined to form a complete nucleobase editor.
• the “split” is located in the dCas9 or nCas9 domain, at positions as described herein in the split Cas9.
• cytosine deaminase refers to an enzyme that catalyzes the chemical reaction “cytosine + H2O uracil + NH3” or “5-methyl-cytosine + H2O thymine + NH3.”
• cytosine deaminase refers to an enzyme that catalyzes the chemical reaction “cytosine + H2O uracil + NH3” or “5-methyl-cytosine + H2O thymine + NH3.”
• cytosine deaminase or “cytidine deaminase” refers to an enzyme that catalyzes the chemical reaction “cytosine + H2O uracil + NH3” or “5-methyl-cytosine + H2O thymine + NH3.”
• a Cas9 nuclease is also referred to sometimes as a casnl nuclease or a CRTS PR (Clustered Regularly Interspaced Short Palindromic Repeat)-associated nuclease.
• CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids).
• CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids.
• CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA).
• CRISPR biology as well as Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g., “Complete genome sequence of an Ml strain of Streptococcus pyogenes.” Ferretti et al., J.
• the deaminases provided herein may be from any organism, such as a bacterium.
• the deaminase or deaminase domain is a variant of a naturally-occurring deaminase from an organism.
• the deaminase or deaminase domain does not occur in nature.
• the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring deaminase.
• Another example includes fusion of a Cas9 or equivalent thereof to a deaminase.
• Any of the proteins provided herein may be produced by any method known in the art.
• the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker.
• Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4 th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which is incorporated herein by reference.
• Gag proteins can vary widely.
• 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 HTLV-1 contains no additional sequences outside of MA, CA, and NC (Oroszlan and Copeland, 1985; Henderson et al., 1992).
• the linker is 5-200 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45- 50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated.
• a “cleavable linker” refers to a linker that can be split or cut by any means.
• the linker can be an amino acid sequence.
• the linker between the NES and the napDNAbp of the BE-VLPs provided herein comprises a cleavable linker.
• a cleavable linker may comprise a self-cleaving peptide (e.g., a 2A peptide such as EGRGSLLTCGDVEENPGP (SEQ ID NO: 9), ATNFSLLKQAGDVEENPGP (SEQ ID NO: 10), QCTNYALLKLAGDVESNPGP (SEQ ID NO: 11), or VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 12)).
• a self-cleaving peptide e.g., a 2A peptide such as EGRGSLLTCGDVEENPGP (SEQ ID NO: 9), ATNFSLLKQAGDVEENPGP (SEQ ID NO: 10), QCT
• the binding mechanism of a napDNAbp - guide RNA complex includes the step of forming an R-loop whereby the napDNAbp induces the unwinding of a double-strand DNA target, thereby separating the strands in the region bound by the napDNAbp.
• the guide RNA protospacer then hybridizes to the “target strand.” This displaces a “non-target strand” that is complementary to the target strand, which forms the single strand region of the R-loop.
• the napDNAbp includes one or more nuclease activities, which then cut the DNA, leaving various types of lesions.
• a "nickase” refers to a napDNAbp (e.g., 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.
• the nickase cleaves a non-target strand of a double stranded target DNA sequence.
• the nickase comprises an amino acid sequence with one or more mutations in a catalytic domain of a canonical napDNAbp (e.g., a Cas protein), wherein the one or more mutations reduces or abolishes nuclease activity of the catalytic domain.
• nuclear export sequence 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.
• NES sequences are described in Xu, D. et al. Sequence and structural analyses of nuclear export signals in the NESdb database. Mol Biol. Cell. 2012, 23(18) 3677-3693, the contents of which are incorporated herein by reference.
• a protease cleavage site comprises an MMLV protease cleavage site or an FMLV protease cleavage site.
• a protease cleavage site comprises one of the amino acid sequences TSTLLMENSS (SEQ ID NO: 1 ), PRSSLYPALTP (SEQ ID NO: 2), VQALVLTQ (SEQ ID NO: 3), PLQVLTLNIERR (SEQ ID NO: 4), or an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 1-4.
• protein refers to a polymer of amino acid residues linked together by peptide (amide) bonds.
• the terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long.
• a protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins.
• the term “subject,” as used herein, refers to an individual organism, for example, an individual mammal.
• the subject is a human.
• the subject is a non-human mammal.
• the subject is a non-human primate.
• the subject is a rodent.
• the subject is a sheep, a goat, a cattle, a cat, or a dog.
• the subject is a vertebrate, an amphibian, a reptile, a fish, an insect, a fly, or a nematode.
• the subject is a research animal.
• the subject is genetically engineered, e.g., a genetically engineered non-human subject. The subject may be of either sex and at any stage of development.
• vector refers to a nucleic acid that can be modified to encode a gene of interest and that is able to enter a host cell, mutate, and replicate within the host cell, and then transfer a replicated form of the vector into another host cell.
• exemplary suitable vectors include viral vectors, such as retroviral vectors or bacteriophages and filamentous phage, and conjugative plasmids. Additional suitable vectors will be apparent to those of skill in the art based on the instant disclosure.
• a viral envelope glycoprotein is an adenoviral envelope glycoprotein, an adeno-associated viral envelope glycoprotein, a retroviral envelope glycoprotein, or a lentiviral envelope glycoprotein.
• a viral envelope glycoprotein is a vesicular stomatitis virus G protein (VSV-G), a baboon retroviral envelope glycoprotein (BaEVRless), a FuG-B2 envelope glycoprotein, an HIV-1 envelope glycoprotein, or an ecotropic murine leukemia virus (MLV) envelope glycoprotein.
• VSV-G vesicular stomatitis virus G protein
• BaEVRless baboon retroviral envelope glycoprotein
• FuG-B2 envelope glycoprotein an HIV-1 envelope glycoprotein
• MMV ecotropic murine leukemia virus
• the cargo protein is a base editor.
• the multi-protein core region of the VLPs further comprises one or more guide RNA molecules which are complexed with the napDNAbp or the base editor to form a ribonucleoprotein (RNP).
• RNP ribonucleoprotein
• the VLPs are prepared in a producer cell that is transiently transformed with plasmid DNA that encodes that various protein and nucleic acid (sgRNA) components of the VLPs. The components self-assemble at the cell membrane and bud out in accordance with the naturally occurring mechanism of retroviral budding in order to release from the cell fully-matured VLPs.
• the Gag-cargo fusion (e.g., Gag::BE) further comprises one or more nuclear export signals at one or more locations along the length of the fusion polypeptide protein which may be joined by a cleavable linker such that during VLP assembly in the producer cell, the Gag-cargo fusions (due to presence of competing NLS signals) do not accumulate in the nucleus of the producer cells but instead are available in the cytoplasm to undergo the VLP assembly process at the cell membrane.
• the NES may be cleaved by Pro-Pol thereby separating the cargo (e.g., napDNAbp or a BE) from the NES.
• the cargo e.g., napDNAbp or BE, typically flanked with one or more NLS elements
• the cargo will not comprise an NES element, which may otherwise prohibit the transport of the carbo into the nuclease and hinder gene editing activity.
• This is exemplified as v.3 VLPs described herein (or “third generation” VLPs).
• the inventors found an optimized stoichiometry ratio of Gag-cargo fusion to Gag-Pro-Pol fusion protein which balances the amount of Gag-cargo available to be packaged into VLPs with the amount of retrovirus protease (the “Pro” in the Gag-Pro-Pol fusion) required for VLP maturation.
• the optimized ratio of Gag-cargo fusion to Gag-Pro-Pol fusion protein is achieved by the appropriate ratio of plasmids encoding each component which are transiently delivered to the producer cells.
• wild type is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms.
• the present disclosure also describes the optimization of a protease cleavage site which separates the NES and VLP proteins from the rest of the base editor to promote highly efficient cleavage and delivery of the BE. Finally, the present disclosure also describes the optimization of the ratios of various components of the BE-VLPs, ensuring high efficiency of BE- VLP production.
• the eVLPs comprise a supra-molecular assembly comprising (a) an envelope comprising (i) a lipid membrane (e.g., single-layer or bi-layer membrane) and a (ii) viral envelope glycoprotein and (b) a multi-protein core region enclosed by the envelope and comprising (i) a Gag protein, (ii) a Gag-Pro-Pol protein, and (iii) a Gag-cargo fusion protein comprising a Gag protein fused to a cargo protein (e.g., a napDNAbp or BE) via a cleavable linker (e.g., a protease-cleavable linker).
• a cleavable linker e.g., a protease-cleavable linker
• the VLP is administered to a recipient cell and taken up by said recipient cell, the contents of the VLP are released, e.g., released BE RNP and/or napDNAbp RNP.
• the RNPs may translocate to the nuclease of the cell (in particular, where NLSs are included on the RNPs), where DNA editing may occur at target sites specified by the guide RNA.
• Various embodiments comprise one or more improvements.
• the Gag-cargo fusion (e.g., Gag-BE) further comprises one or more nuclear export signals at one or more locations along the length of the fusion polypeptide protein which may be joined by a cleavable linker such that during VLP assembly in the producer cell, the Gag-cargo fusions (due to presence of competing NLS signals) do not accumulate in the nucleus of the producer cells but instead are available in the cytoplasm to undergo the VLP assembly process at the cell membrane.
• the NES may be cleaved by Gag- Pro-Pol thereby separating the cargo (e.g., napDNAbp or a BE) from the NES.
• the present disclosure provides an eVLP comprising an (a) envelope, and (b) a multi-protein core, wherein the envelope comprises a lipid membrane (e.g., a lipid mono- or bi-layer membrane) and a viral envelope glycoprotein, and wherein the multi-protein core comprises a Gag (e.g., a retroviral Gag), a group- specific antigen (gag) protease (pro) polyprotein (z.e., “Gag-Pro-Pol”), and a fusion
• the Gag-cargo fusion proteins described herein comprise one or more cleavable linkers.
• the Gag-cargo fusion proteins comprise a cleavable linker joining the Gag to the cargo, such that once the Gag-cargo fusion has been packaged in mature VLPs (which will also contain the Gag-Pro-Pol, the protease activity can cleave the Gag-cargo cleavable linker, thereby releasing the cargo.
• a cleavable linker may also be provided in such a location such that when the cleavable linker is cleaved (e.g., by the Gag-Pro-Pol protein), the NES is separated away from the cargo protein.
• the gag-pro polyprotein of the BE-VLPs described herein comprises an MMLV gag-pro polyprotein or an FMLV gag-pro polyprotein.
• the gag nucleocapsid protein of the fusion protein in the BE-VLPs described herein comprises an MMLV gag nucleocapsid protein or an FMLV gag nucleocapsid protein.
• the fusion protein comprises the following non-limiting structures:
• viral vector particles which generally contain coding nucleic acids of interest
• virus-derived particles which do not contain coding nucleic acids of interest but instead are designed to deliver a protein cargo (e.g., a BE RNP).
• a protein cargo e.g., a BE RNP
• viral vector particles encompass retroviral, lentiviral, adenoviral, and adeno-associated viral vector particles that are well known in the art.
• the one skilled in the art may notably refer to Kushnir et al. (2012, Vaccine, Vol. 31: 58-83), Zeltons (2013, Mol Biotechnol, Vol. 53: 92-107), Ludwig et al. (2007, Curr Opin Biotechnol, Vol. 18(no 6): 537-55) and Naskalaska et al. (2015, Vol. 64 (no 1): 3-13).
• retroviral vector including lentiviral vectors
• the host range of retroviral vector may be expanded or altered by a process known as pseudotyping.
• Pseudotyped lentiviral vectors consist of viral vector particles bearing glycoproteins derived from other enveloped viruses. Such pseudotyped viral vector particles possess the tropism of the virus from which the glycoprotein is derived.
• a virus-like particle further comprises a viral envelope protein, wherein either (i) the said viral envelope protein originates from the same virus as the viral structural protein, e.g., originates from the same virus as the viral Gag protein, or (ii) the said viral envelope protein originates from a virus distinct from the virus from which originates the viral structural protein, e.g., originates from a virus distinct from the virus from which originates the viral Gag protein.
• Influenza virus-derived vector particles 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 Eatham et al. (2001, Journal of Virology, Vol. 75(no 13): 6154-6155), which is incorporated herein by reference.
• Hepatitis B virus-derived vector particles 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, Vol. 87(no 12): 6615-6624), which is incorporated herein by reference.
• Hepatitis E virus-derived vector particles 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), which is incorporated herein by reference.
• Parvovirus-derived vector particles 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), which is incorporated herein by reference.
• the BE-VLPs disclosed herein, as well as the fusion proteins that make up the core component of the presently described BE-VLPs comprise a nucleic acid programmable DNA binding protein (napDNAbp).
• napDNAbp nucleic acid programmable DNA binding protein
• the BE-VLPs and fusion proteins may include a napDNAbp domain having a wild type Cas9 sequence, including, for example the canonical Streptococcus pyogenes Cas9 sequence of SEQ ID NO: 13 , shown as follows:
• the BE-VLPs and fusion proteins described herein may include any of the above SpCas9 sequences, or any variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.
• the Cas9 protein can be a wild type Cas9 ortholog from another bacterial species different from the canonical Cas9 from S. pyogenes.
• modified versions of the following Cas9 orthologs can be used in connection with the BE-VLPs and fusion proteins described in this specification by making mutations at positions corresponding to H840A or any other amino acids of interest in wild type SpCas9.
• any variant Cas9 orthologs having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any of the below orthologs may also be used with the base editors.
• Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference.
• a Cas9 nuclease has an inactive (e.g., an inactivated) DNA cleavage domain; that is, the Cas9 is a nickase.
• the Cas9 protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of a Cas9 protein as provided by any one of the Cas9 orthologs in the above tables.
• the VLPs described herein can be used for delivery of any Cas9 equivalent to a target cell.
• Cas9 equivalent is a broad term that encompasses any napDNAbp protein that serves the same function as Cas9 despite that its amino acid primary sequence and/or its three-dimensional structure may be different and/or unrelated from an evolutionary standpoint.
• Cas9 is a bacterial enzyme that evolved in a wide variety of species. However, the Cas9 equivalents contemplated herein may also be obtained from archaea, which constitute a domain and kingdom of single-celled prokaryotic microbes different from bacteria.
• Cas9 equivalents may refer to Casl2e (CasX) or Casl2d (CasY), which have been described in, for example, Burstein et al., “New CRISPR-Cas systems from uncultivated microbes.” Cell Res. 2017 Feb 21. Doi: 10.1038/cr.2017.21, the entire contents of which is hereby incorporated by reference.
• CasX Casl2e
• CasY Casl2d
• Cas9 refers to Casl2e, or a variant of Casl2e. In some embodiments, Cas9 refers to a Casl2d, or a variant of Casl2d. It should be appreciated that other RNA-guided DNA binding proteins may be used as a nucleic acid programmable DNA binding protein (napDNAbp) and are within the scope of this disclosure. Also see Liu et al., “CasX enzymes comprises a distinct family of RNA-guided genome editors,” Nature, 2019, Vol.566: 218-223. Any of these Cas9 equivalents are contemplated by the present disclosure.
• the Cas9 equivalent comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring Casl2e (CasX) or Casl2d (CasY) protein.
• the napDNAbp is a naturally-occurring Casl2e (CasX) or Casl2d (CasY) protein.
• the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a wild-type Cas moiety or any Cas moiety provided herein.
• Casl2a Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 (i.e., Casl2a (Cpfl)). Similar to Cas9, Casl2a (Cpfl) is also a Class 2 CRISPR effector, but it is a member of type V subgroup of enzymes, rather than the type II subgroup. It has been shown that Casl2a (Cpfl) mediates robust DNA interference with features distinct from Cas9.
• Casl2a is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich proto spacer- adjacent motif (TTN, TTTN, or YTN). Moreover, Cpfl cleaves DNA via a staggered DNA double- stranded break.
• TTN T-rich proto spacer- adjacent motif
• TTTN TTTN
• YTN T-rich proto spacer- adjacent motif
• Cpfl cleaves DNA via a staggered DNA double- stranded break.
• Cpfl proteins are known in the art and have been described previously, for example, in Yamano et al., “Crystal structure of Cpfl in complex with guide RNA and target DNA.” Cell (165) 2016, p. 949-962; the entire contents of which is hereby incorporated by reference.
• the Cas protein may include any CRISPR associated protein, including but not limited to, Casl2a, Casl2bl, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions
• the napDNAbp can be any of the following proteins: a Cas9, a Casl2a (Cpfl), a Casl2e (CasX), a Casl2d (CasY), a Casl2bl (C2cl), a Casl3a (C2c2), a Casl2c (C2c3), a GeoCas9, a CjCas9, a Casl2g, a Casl2h, a Casl2i, a Casl3b, a Casl3c, a Casl3d, a Casl4, a Csn2, an xCas9, an SpCas9-NG, a circularly permuted Cas9, or an Argonaute (Ago) domain, or a variant thereof.
• a Cas9 a Casl2a (Cpfl), a Casl2e (CasX), a Ca
• the napDNAbp is a single effector of a microbial CRISPR-Cas system.
• Single effectors of microbial CRISPR-Cas systems include, without limitation, Cas9, Casl2a (Cpfl), Casl2bl (C2cl), Casl3a (C2c2), and Casl2c (C2c3).
• microbial CRISPR-Cas systems are divided into Class 1 and Class 2 systems. Class 1 systems have multi-subunit effector complexes, while Class 2 systems have a single protein effector.
• Cas9 and Cas 12a (Cpfl) are Class 2 effectors.
• Catalytic residues in the two conserved HEPN domains mediate cleavage. Mutations in the catalytic residues generate catalytically inactive RNA-binding proteins. See e.g., Abudayyeh et al., “C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector”, Science, 2016 Aug 5;
• TALENS are described in WO 2015/027134, U.S. 9,181,535, Boch et al., “Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors”, Science, vol. 326, pp. 1509-1512 (2009), Bogdanove et al., TAL Effectors: Customizable Proteins for DNA Targeting, Science, vol. 333, pp. 1843-1846 (2011), Cade et al., “Highly efficient generation of heritable zebrafish gene mutations using homo- and heterodimeric TALENs”, Nucleic Acids Research, vol. 40, pp.
• the BE-VLPs and fusion proteins described herein further comprise a deaminase domain (e.g., when a base editor is being encapsulated and delivered in the VLP).
• a deaminase domain may be a cytosine deaminase domain or an adenosine deaminase domain.
• Base editors that convert a C to T in some embodiments, comprise a cytosine deaminase.
• the C to T base editor comprises a dCas9 or nCas9 fused to a cytosine deaminase.
• the cytosine deaminase domain is fused to the N-terminus of the dCas9 or nCas9.
• a base editor converts an A to G.
• the base editor comprises an adenosine deaminase.
• An “adenosine deaminase” is an enzyme involved in purine metabolism. It is needed for the breakdown of adenosine from food and for the turnover of nucleic acids in tissues. Its primary function in humans is the development and maintenance of the immune system.
• An adenosine deaminase catalyzes hydrolytic deamination of adenosine (forming inosine, which base pairs as G) in the context of DNA. There are no known adenosine deaminases that act on DNA.
• an adenosine deaminase comprises any of the following amino acid sequences, or an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 99.9% identical to any of the following amino acid sequences:
• ecTadA (D108N) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTA
• ecTadA E25M, R26G, L84F, A106V, R107P, D108N, H123Y, A142N, A143D,
• ecTadA (R26C, L84F, A106V, R107H, D108N, H123Y, A142N , D147Y, E155V,
• ecTadA N37T, P48T, L84F, A106V, D108N, H123Y, D147Y, E155V, I156F
• ecTadA H36L, L84F, A106V, D108N, H123Y, D147Y, E155V, I156F
• ecTadA H36L, P48L, L84F, A106V, D108N, H123Y, D147Y, E155V, I156F
• ecTadA H36L, L84F, A106V, D108N, H123Y, S146C, D147Y, E155V, I156F
• Shewanella putrefaciens S. putrefaciens
• TadA Shewanella putrefaciens
• TadA-8e(V106W) E. coli
• SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTA HAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGWRNSKR GAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN SEQ ID NO: 116
• the present disclosure provides eVLPs and fusion proteins for delivering base editors.
• Base editors are known in the art, and the presently described BE- VLPs may be used to deliver any base editor that is already known, or that is developed in the future.
• the base editors contemplated for delivery may comprise an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the base editor sequences provided herein.
• the BE-VLPs of the present disclosure comprise cytidine base editors (CBEs) comprising a napDNAbp domain and a cytosine deaminase domain that enzymatically deaminates a cytosine nucleobase of a C:G nucleobase pair to a uracil.
• CBEs cytidine base editors
• the uracil may be subsequently converted to a thymine (T) by the cell’s DNA repair and replication machinery.
• T thymine
• the mismatched guanine (G) on the opposite strand may subsequently be converted to an adenine (A) by the cell’s DNA repair and replication machinery.
• a target C:G nucleobase pair is ultimately converted to a T:A nucleobase pair.
• the CBEs in the BE-VLPs described herein may further comprise one or more nuclear localization signals (NLSs) and/or one or more uracil glycosylase inhibitor (UGI) domains.
• the base editors may comprise the structure: NH2-[first nuclear localization sequence]-[cytosine deaminase domain] -[napDNAbp domain] -[first UGI domain] -[second UGI domain] -[second nuclear localization sequence] -COOH, wherein each instance of “]-[” indicates the presence of an optional linker sequence.
• the CBEs in the presently disclosed BE-VLPs may comprise modified (or evolved) cytosine deaminase domains, such as deaminase domains that recognize an expanded PAM sequence, have improved efficiency of deaminating 5'-GC targets, and/or make edits in a narrower target window.
• the disclosed cytidine base editors comprise evolved nucleic acid programmable DNA binding proteins (napDNAbp), such as an evolved nucleic acid programmable DNA binding proteins (napDNAbp), such as an evolved
• Exemplary cytidine base editors are disclosed herein and may also comprise amino acid sequences that are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences disclosed herein.
• the cytidine base editors comprise an amino acid sequence that is at least 90% identical to any one of the CBE sequences disclosed herein.
• the disclosed cytidine nucleobase editors comprise the amino acid sequence of any one of the
• V (SEQ ID NO: 125)

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