WO2018071868A1 - Aav delivery of nucleobase editors - Google Patents

Aav delivery of nucleobase editors Download PDF

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Publication number
WO2018071868A1
WO2018071868A1 PCT/US2017/056671 US2017056671W WO2018071868A1 WO 2018071868 A1 WO2018071868 A1 WO 2018071868A1 US 2017056671 W US2017056671 W US 2017056671W WO 2018071868 A1 WO2018071868 A1 WO 2018071868A1
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WIPO (PCT)
Prior art keywords
sggs
seq
nucleotide sequence
cas9
composition
Prior art date
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PCT/US2017/056671
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English (en)
French (fr)
Inventor
David R. Liu
Jonathan Ma LEVY
Wei Hsi YEH
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Harvard University
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Harvard University
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Publication date
Priority to CN201780076474.6A priority Critical patent/CN110214180A/zh
Priority to CA3039928A priority patent/CA3039928A1/en
Priority to EP17794468.3A priority patent/EP3526320A1/en
Priority to JP2019520398A priority patent/JP7588390B2/ja
Priority to KR1020197013648A priority patent/KR102622411B1/ko
Priority to SG11201903089RA priority patent/SG11201903089RA/en
Priority to GB1906789.1A priority patent/GB2573062A/en
Priority to AU2017342543A priority patent/AU2017342543B2/en
Application filed by Harvard University filed Critical Harvard University
Priority to KR1020247000273A priority patent/KR20240007715A/ko
Publication of WO2018071868A1 publication Critical patent/WO2018071868A1/en
Priority to IL265900A priority patent/IL265900A/en
Anticipated expiration legal-status Critical
Priority to JP2022163015A priority patent/JP2023011629A/ja
Priority to JP2024169979A priority patent/JP2025038900A/ja
Ceased legal-status Critical Current

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Definitions

  • a Cas9 protein or a nucleobase editor is "split" into an N-terminal portion and a C- terminal portion.
  • the N-terminal portion or C-terminal portion of a Cas9 protein or a nucleobase editor may be fused to one member of the intein system, respectively.
  • the resulting fusion proteins when delivered on separate vectors (e.g.
  • rAAV vectors into one cell and co- expressed, may be joined to form a complete and functional Cas9 protein or nucleobase editor (e.g. , via intein-mediated protein splicing). Further provided herein are empirical testing of regulatory elements in the delivery vectors for high expression levels of the split Cas9 protein or the nucleobase editor.
  • compositions comprising: (i) a first nucleotide sequence encoding a N-terminal portion of a Cas9 protein fused at its C-terminus to an intein-N; and (ii) a second nucleotide sequence encoding an intein-C fused to the N-terminus of a C-terminal portion of the Cas9 protein, wherein the first nucleotide sequence or second nucleotide sequence is operably linked to a nucleotide sequence encoding at least one bipartite nuclear localization signal.
  • the N-terminal portion of the Cas9 protein comprises a portion of any one of SEQ ID NOs: 1-275 and 394-397 that corresponds to amino acids 1-573 or 1-637 of SEQ ID NO: 1.
  • the C-terminal portion of the Cas9 protein comprises a portion of any one of SEQ ID NOs: 1-275 and 394-397 that corresponds to amino acids 574- 1368 or 638-1368 of SEQ ID NO: 1.
  • the intein-N comprises the amino acid sequence as set forth in SEQ ID NO: 350-351 and 354-355.
  • the intein-C comprises the amino acid sequence as set forth in SEQ ID NO: 352-353 and 356-357.
  • the first nucleotide sequence or the second nucleotide sequence further comprises a nucleotide encoding a guide RNA (gRNA) operably linked to a promoter.
  • gRNA guide RNA
  • the first nucleotide sequence or the second nucleotide sequence further comprises a transcriptional terminator.
  • the transcriptional terminator is the transcriptional terminator from a bGH gene, hGH gene, or SV40 gene. In some embodiments, the transcriptional terminator is the transcriptional terminator from a bGH gene.
  • the first nucleotide sequence or the second nucleotide sequence further comprises a woodchuck hepatitis posttranscriptional regulatory element (WPRE) inserted 5' of the transcriptional terminator.
  • WPRE woodchuck hepatitis posttranscriptional regulatory element
  • the bipartite nuclear localization signal comprises an amino acid sequence selected from the group consisting of: KRPAATKKAGQAKKKK (SEQ ID NO: 344), KKTELQTTNAENKTKKL(SEQ ID NO: 345), KRGINDRNFWRGENGRKTR(SEQ ID NO: 346), and RKSGKIAAIVVKRPRK(SEQ ID NO: 347).
  • the bipartite nuclear localization signal comprises the amino acid sequence as set forth in SEQ ID NO: 344.
  • the Cas9 protein is a catalytically inactive Cas9 (dCas9) or a Cas9 nickase (nCas9), and wherein the first nucleotide sequence of (i) further comprises a nucleotide sequence encoding a nucleobase modifying enzyme fused to the N-terminus of the N-terminal portion of the Cas9 protein.
  • the Cas9 protein is a catalytically inactive Cas9 (dCas9) or a Cas9 nickase (nCas9), and wherein the second nucleotide sequence of (ii) further comprises a nucleotide sequence encoding a nucleobase modifying enzyme fused to the C-terminus of the C- terminal portion of the Cas9 protein.
  • the nucleobase modifying enzyme is a deaminase.
  • the deaminase is a cytosine deaminase.
  • the deaminase is an adenosine deaminase.
  • the second nucleotide sequence of (ii) further comprises a nucleotide sequence encoding a uracil glycosylase inhibitor (UGI) fused at the 3' end of the second nucleotide sequence.
  • the first nucleotide sequence of (i) further comprises a nucleotide sequence encoding a uracil glycosylase inhibitor (UGI) at the 5' end of the first nucleotide sequence.
  • UGI uracil glycosylase inhibitor
  • the UGI comprises the amino acids sequence of SEQ ID NOs: 299-302.
  • the first nucleotide sequence and the second nucleotide sequence are on different vectors.
  • the each of the different vectors is a genome of a recombinant adeno-associated virus (rAAV).
  • each vector is packaged in a rAAV particle.
  • compositions comprising: (i) a first recombinant adeno associated virus (rAAV) particle comprising a first nucleotide sequence encoding a N-terminal portion of a Cas9 protein fused at its C-terminus to an intein-N; and (ii) a second recombinant adeno associated virus (rAAV) particle comprising a second nucleotide sequence encoding an intein-C fused to the N-terminus of a C-terminal portion of the Cas9 protein, wherein the first nucleotide sequence or second nucleotide sequence is operably linked to a nucleotide sequence encoding at least one bipartite nuclear localization sign.
  • rAAV a first recombinant adeno associated virus
  • Cells comprising the compositions described herein are provided.
  • the N-terminal portion of the Cas9 protein and the C-terminal portion of the Cas9 protein are joined together to form the Cas9 protein.
  • the cell is a prokaryotic cell.
  • the cell is a bacterial cell.
  • the cell is a eukaryotic cell.
  • the cell is a yeast cell, a plant cell, or a mammalian cell.
  • the cell is a human cell.
  • kits comprising the any of the compositions described herein.
  • compositions comprising: (i) a first nucleotide sequence encoding a N-terminal portion of a nucleobase editor fused at its C-terminus to an intein-N; and (ii) a second nucleotide sequence encoding an intein-C fused to the N- terminus of a C-terminal portion of the nucleobase editor.
  • the intein-N comprises the amino acid sequence as set forth in SEQ ID NO: 350-351 and 354-355.
  • the intein-C comprises the amino acid sequence as set forth in SEQ ID NO: 352-353 and 356-357.
  • the first nucleotide sequence or the second nucleotide sequence further comprises a nucleotide encoding a guide RNA (gRNA) operably linked to a promoter.
  • gRNA guide RNA
  • the first nucleotide sequence or the second nucleotide sequence further comprises a transcriptional terminator.
  • the transcriptional terminator is a transcriptional terminator from a bGH gene, hGH gene, or SV40 gene.
  • the transcriptional terminal is from a bGH gene.
  • the first nucleotide sequence or the second nucleotide sequence further comprises a woodchuck hepatitis posttranscriptional regulatory element (WPRE) inserted 5' of the transcriptional terminator.
  • WPRE woodchuck hepatitis posttranscriptional regulatory element
  • the first nucleotide sequence or second nucleotide sequence are operably linked to a nucleotide sequence encoding at least one bipartite nuclear localization signal.
  • the bipartite nuclear localization signal comprises an amino acid sequence selected from the group consisting of: KRPAATKKAGQAKKKK (SEQ ID NO: 344), KKTELQTTNAENKTKKL(SEQ ID NO: 345), KRGINDRNFWRGENGRKTR(SEQ ID NO: 346), and RKSGKIAAIVVKRPRK(SEQ ID NO: 347).
  • the bipartite nuclear localization signal comprises the amino acid sequence as set forth in SEQ ID NO: 344.
  • the nucleobase editor comprises a cytosine deaminase fused to the N-terminus of a catalytically inactive Cas9 or a Cas9 nickase.
  • the cytosine deaminase is selected from the group consisting of: APOBEC1, APOBEC3, AID, and pmCDAl.
  • the nucleobase editor further comprises a uracil glycosylase inhibitor (UGI).
  • the UGI comprises the amino acids sequence of SEQ ID NOs: 299-302.
  • the first nucleotide sequence and the second nucleotide sequence are on different vectors.
  • each of the different vectors is a genome of a recombinant adeno-associated virus (rAAV).
  • the vector is packaged in a rAAV particle.
  • compositions comprising: (i) a first recombinant adeno associated virus (rAAV) particle comprising a first nucleotide sequence encoding a N-terminal portion of a nucleobase editor fused at its C-terminus to an intein-N; and (ii) a second recombinant adeno associated virus (rAAV) particle comprising a second nuclei acid encoding an intein-C fused to the N-terminus of a C-terminal portion of the nucleobase editor.
  • rAAV a first recombinant adeno associated virus
  • Cells comprising any of the compositions described herein are provided.
  • the N-terminal portion of the nucleobase editor and the C-terminal portion of the nucleobase editor are joined together to form the nucleobase editor.
  • the cell is a prokaryotic cell.
  • the cell is a bacterial cell.
  • the cell is an eukaryotic cell.
  • the cell is a yeast cell, a plant cell, or a mammalian cell.
  • the cell is a human cell.
  • kits comprising any of the compositions described herein.
  • Yet other aspects of the present disclosure provide methods comprising: contacting a cell with any of the compositions described herein, wherein the contacting results in the delivery of the first nucleotide sequence and the second nucleotide sequence into the cell, and wherein the N-terminal portion of the nucleobase editor and the C-terminal portion of the nucleobase editor are joined to form a nucleobase editor.
  • compositions described herein comprising: administering to a subject in need there of a therapeutically effective amount of any of the compositions described herein.
  • the subject has a disease or disorder.
  • the disease or disorder is selected from the group consisting of: cystic fibrosis, phenylketonuria, epidermolytic hyperkeratosis (EHK), chronic obstructive pulmonary disease (COPD), Charcot-Marie-Toot disease type 4J. , neuroblastoma (NB), von Willebrand disease (vWD), myotonia congenital, hereditary renal amyloidosis, dilated cardiomyopathy, hereditary lymphedema, familial Alzheimer' s disease, prion disease, chronic infantile neurologic cutaneous articular syndrome (CINCA), and desmin-related myopathy (DRM).
  • cystic fibrosis phenylketonuria
  • EHK epidermolytic hyperkeratosis
  • COPD chronic obstructive pulmonary disease
  • Charcot-Marie-Toot disease type 4J. neuroblastoma (NB), von Willebrand disease (vWD), myotonia congenital, hereditary renal amy
  • Figures 1A-1C are graphs showing a "split nucleobase editor” for delivery into cells using recombinant adeno associated virus (rAAV) vectors.
  • Figure 1A is a schematic
  • Figure IB shows that AAV-delivered split nucleobase editor can undergo protein splicing upon expression of the two halves in cells to form a complete nucleobase editor that has comparable activity to a nucleobase editor expressed as a whole.
  • Figure 1C shows the formation of a complete nucleobase editor from the two halves via protein splicing mediated by DnaE intein.
  • Figure 2 shows that U1118 cells were efficiently transfected by AAV2 containing nucleic acids encoding mCherry. Different viral titers were tested (2.5-10 ⁇ at 4.5 x 10 1 1 1 1 vg/ml * ) and all resulted in efficient transfection of U118 cells.
  • * vg/ml means viral genome-containing particles per microliter.
  • Figures 3A-3B are graphs showing high throughput sequence (HTS) results of nucleobase editing by r AAV-delivered split nucleobase editor in Ul 18 and HEK cells. Lipid-transfected nucleobase editor was used as a control. A sgRNA targeting R37 in the PRNP gene was used, and the PRNP gene locus was sequenced.
  • Figure 3A shows the HTS reads
  • Figure 3B summarizes the base editing results.
  • Figure 4 is a graph showing the optimization of the transcriptional terminator used in the AAV constructs encoding the split nucleobase editor. Transcriptional terminators of different sizes and origins were tested. bGH transcriptional terminator is relatively short and efficiently terminates transcription comparably to longer terminator sequences. It was therefore chosen to be used in the downstream experiments.
  • Figures 5A-5B are graphs showing the results of nucleobase editing with long term (up to 15 days) transduction of AAV encoding the split nucleobase editor in mouse astrocytes expressing human ApoE4 cDNA.
  • the target base is in the codon for arginine 112 and arginine 158 in ApoE4, which is converted to a cysteine upon base editing.
  • Figure 5A shows that the editing of arginine 158 increases overtime when the mouse astrocytes were transduced at 10 10 vg, while editing of arginine 112 remained minimal.
  • the nucleotide sequence 3 ' of the codon for arginine 158 sequence features a flanking NGG PAM allowing for high activity by SpCas9 (with guide sequence GAAGCGCCTGGCAGTGTACC, SEQ ID NO: 348), while the nucleotide sequence ⁇ of the codon for arginine 112 contains a flanking NAG PAM which does not allow for high activity (with guide sequence GACGTGCGCGGCCGCCTGGTG, SEQ ID NO: 349).
  • Figure 5B shows cells transduced with rAAV encoding mCherry at 10 10 vg (control).
  • Figure 6 is a schematic representation of the optimization of the nuclear localization signal in AAV constructs encoding the split nucleobase editor.
  • the nuclear localization signal controls nuclear import, which must occur for reconstituted nucleobase editor to associate with genomic DNA as a prerequisite for editing, and is a potential rate-limiting step in the process.
  • This schematic shows that the NLS (and NLS optimization) is critical for the nucleobase editor to be imported into the nucleus.
  • Figure 7 is a graph showing the results of base editing using different rAAV split nucleobase editor constructs containing different nuclear localization signals (NLS).
  • Figures 8A-8B are graphs showing the editing of DNMT1 gene in dissociated mouse cortical neurons using an AAV encoded split nucleobase editor.
  • Figure 9A-9B are graphs showing the editing of DNMT1 gene in mouse Neuro-2a cell line using either an AAV encoded split nucleobase editor, or a lipid transfected DNA encoded nucleobase editor.
  • Cas9 refers to an RNA- guided nuclease comprising a Cas9 protein (e.g., Cas9 nucleases from a variety of bacterial species), a fragment, a variant (e.g., a catalytically inactive Cas9 or a Cas9 nickase), or a fusion protein (e.g., a Cas9 fused to another protein domain) thereof.
  • a Cas9 protein e.g., Cas9 nucleases from a variety of bacterial species
  • a fragment e.g., a variant (e.g., a catalytically inactive Cas9 or a Cas9 nickase)
  • a fusion protein e.g., a Cas9 fused to another protein domain
  • a Cas9 nuclease is also referred to sometimes as a casnl nuclease or a CRISPR (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).
  • tracrRNA trans-encoded small RNA
  • rnc endogenous ribonuclease 3
  • Cas9 protein a trans-encoded small RNA
  • the tracrRNA serves as a guide for ribonuclease 3 -aided processing of pre-crRNA.
  • RNA molecules complementary to DNA -binding and cleavage typically requires protein and both RNAs.
  • single guide RNAs sgRNA, or simply “gNRA”
  • gRNA single guide RNAs
  • Non-limiting examples of Cas9 proteins and their respective amino acid sequence are provided in Example 1.
  • a nuclease-inactive Cas9 protein may interchangeably be referred to as a "dCas9" protein (for nuclease-"dead” Cas9).
  • Methods for generating a Cas9 protein (or a fragment thereof) having an inactive DNA cleavage domain are known (See, e.g., Jinek et al, Science. 337:816- 821(2012); Qi et al, (2013) Cell. 28;152(5): 1173-83, incorporated herein by reference).
  • the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvCl subdomain.
  • the HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvCl subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A and H840A completely inactivate the nuclease activity of S.
  • nuclease-inactive dCas9 domains include, but are not limited to, D10A/H840A, D10A/D839A/H840A, and D10A/D839A/H840A/N863A mutant domains (See, e.g., Prashant et al, Nature Biotechnology . 2013; 31(9):833-838, incorporated herein by reference).
  • a Cas9 nickase is used as part of the nucleobase editor.
  • a Cas9 nickase is able to cleave one strand of the double strand DNA.
  • a Cas9 nickase may be generated by introducing an inactivating mutation into either the HNH domain or the RuvCl domain.
  • an inactivating mutation D10A
  • D10A an inactivating mutation
  • the HNH domain remains active, i.e. , the residue at position 840 remains a histidine.
  • Such Cas9 variants are able to generate a single-strand DNA break (nick) at a specific location based on the gRNA-defined target sequence.
  • One skilled in the art is able to identify the catalytic residues in the RuvC l and HNH domains of any known Cas9 proteins and introduce inactivating mutations to generate a corresponding dCas9 or nCas9.
  • a "split Cas9 protein” or “split Cas9” refers to a Cas9 protein that is provided as an N- terminal portion (also referred to as an N-terminal half) and a C-terminal portion (also referred to as a C-terminal half) encoded by two separate nucleotide sequences.
  • the polypeptides corresponding to the N-terminal portion and the C-terminal portion of the Cas9 protein may be combined (joined) to form a complete Cas9 protein.
  • a Cas9 protein is known to consist of a bi- lobed structure linked by a disordered linker (e.g.
  • the "split" occurs between the two lobes, generating two portions of a Cas9 protein, each containing one lobe.
  • an "intein” is a segment of a protein that is able to excise itself and join the remaining portions (the exteins) with a peptide bond in a process known as protein splicing. Inteins are also referred to as “protein introns.” The process of an intein excising itself and joining the remaining portions of the protein is herein termed “protein splicing" or "intein-mediated protein splicing.”
  • an intein of a precursor protein an intein containing protein prior to intein-mediated protein splicing comes from two genes. Such intein is referred to herein as a split intein.
  • a split intein for example, in cyanobacteria, DnaE, the catalytic subunit a of DNA
  • polymerase III is encoded by two separate genes, dnaE-n and dnaE-c.
  • the intein encoded by the dnaE-n gene is herein referred as "intein-N.”
  • the intein encoded by the dnaE-c gene is herein referred as "intein-C.”
  • intein systems may also be used.
  • a synthetic intein based on the dnaE intein, the Cfa-N and Cfa-C intein pair has been described (e.g. , in Stevens et ah , J Am Chem Soc. 2016 Feb 24; 138(7):2162-5, incorporated herein by reference).
  • Non-limiting examples of intein pairs that may be used in accordance with the present disclosure include: Cfa DnaE intein, Ssp GyrB intein, Ssp DnaX intein, Ter DnaE3 intein, Ter ThyX intein, Rma DnaB intein and Cne Prp8 intein (e.g. , as described in US Patent 8,394,604, incorporated herein by reference.
  • nucleotide and amino acid sequences of inteins are provided.
  • Intein-N and intein-C may be fused to the N-terminal portion of the split Cas9 and the C- terminal portion of the split Cas9, respectively, for the joining of the N-terminal portion of the split Cas9 and the C-terminal portion of the split Cas9.
  • an intein-N is fused to the C-terminus of the N-terminal portion of the split Cas9, i.e.
  • an intein-C is fused to the N-terminus of the C-terminal portion of the split Cas9, i.e. , to form a structure of N-[intein-C]-[C-terminal portion of the split Cas9]-C.
  • the mechanism of intein- mediated protein splicing for joining the proteins the inteins are fused to is known in the art, e.g. , as described in Shah et ah , Chem Sci. 2014; 5(1):446-461, incorporated herein by reference.
  • nucleobase editor refers to a protein that edits a nucleotide base.
  • Edit refers to the conversion of one nucleobase to another (e.g. , A to G, A to C, A to T, C to T, C to G, C to A, G to A, G to C, G to T, T to A, T to C, T to G).
  • a nucleobase editor is a macromolecule or macromolecular complex that results primarily (e.g.
  • nucleobase in a polynucleic acid sequence into another nucleobase (i.e., a transition or transversion) using a combination of 1) a nucleotide-, nucleoside-, or nucleobase- modifying enzyme and 2) a nucleic acid binding protein that can be programmed to bind to a specific nucleic acid sequence.
  • 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 modifying enzyme fused to a programmable DNA binding domain (e.g. , a dCas9 or nCas9).
  • a "nucleobase modifying enzyme” is an enzyme that can modify a nucleobase and convert one nucleobase to another (e.g.
  • a deaminase such as a cytosine deaminase or a adenosine deaminase
  • the nucleobase editor may target cytosine (C) bases in a nucleic acid sequence and convert the C to thymine (T) base.
  • the C to T editing is carried out by a deaminase, e.g. , a cytosine deaminase.
  • Base editors that can carry out other types of base conversions (e.g. , adenosine (A) to guanine (G), C to G) are also contemplated.
  • Nucleobase editors that convert a C to T comprise a cytosine deaminase.
  • a "cytosine deaminase” refers to an enzyme that catalyzes the chemical reaction "cytosine + H 2 0 uracil + NH 3 " or "5-methyl-cytosine + H 2 0 thymine + NH 3 .” As it may be apparent from the reaction formula, such chemical reactions result in a C to U/T nucleobase change.
  • the C to T nucleobase 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.
  • the nucleobase editor further comprises a domain that inhibits uracil glycosylase, and/or a nuclear localization signal.
  • a nucleobase editor converts an A to G.
  • the nucleobase 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.
  • RNA RNA
  • tRNA or mRNA Evolved deoxyadenosine deaminase enzymes that accept DNA substrates and deaminate dA to deoxyinosine and here use in adenosine nucleobase editos have been described, e.g. , in US provisional application, U.S.S.N. 62/370,684, filed August 3, 2016; US provisional application, U.S.S.N. 62/370,684, filed February 3, 2017, US provisional application, U.S.S.N. 62/473,714, filed March 20, 2017, and PCT Application
  • the adenosine deaminase is E. coli TadA (SEQ ID NO: 314).
  • the possible mutations in ecTadA and constructs expressing nucleobase editors comprising the modified ecTadA are provided in Table 2.
  • the sequences of exemplary EcTadA mutants and nucleotibase editors comprising such mutants are provided in Example 1.
  • pNMG-284 a.a.)_ecTadA_XTEN_nCas9_SGGS_NLS (wild-type) + (A106V_D108N_D147Y_E155V) pCMV ecTadA LONGER LINKER (92 (A106V D108N D147Y E155V) + pNMG-285 a.a.)_ecTadA_XTEN_nCas9_SGGS_NLS (A106V_D108N_D147Y) pCMV ecTadA LONGER LINKER (92 (A106V D108N D147Y E155V) + pNMG-285b a.a.)_ecTadA_XTEN_nCas9_SGGS_NLS (A106V_D 108N_D 147 Y_E 155V) pNMG-286 pCMV_ecTadA_XTEN_nCas9
  • pCMV_ecTadA-92 a.a.-ecTadA-32 (L84F A106V D108N H123Y D147Y E155V 11 pNMG-514 a.a._nCas9_SGGS_NLS 56F)
  • pCMV_ecTadA-92 a.a.-ecTadA- 64 (L84F A106V D108N H123Y D147Y E155V 11 pNMG-515 a.a._nCas9_SGGS_NLS 56F)
  • pCMV_ecTadA-92 a.a.-ecTadA- 64 (L84F A106V D108N H123Y D147Y E155V 11 pNMG-516 a.a._nCas9_SGGS_NLS 56F)
  • pCMV_ecTadA- 32 a.a.-ecTadA- 64 (L84F A106V D108N H123Y D147Y E155V 11 pNMG-517 a.a._nCas9_SGGS_NLS 56F)
  • pCMV_ecTadA- 32 a.a.-ecTadA- 64 (L84F A106V D108N H123Y D147Y E155V 11 pNMG-518 a.a._nCas9_SGGS_NLS 56F)
  • pNMG-555 linker_nCas9_SGGS_NLS H36L R51 L L84F A106V D108N H123Y S146 C_D 147 Y_E 155V_1156 F _K157N
  • pCMV_ecTadA- 40 a.a. linker-ecTadA- 24 a.a. (H36L R51 L L84F A106V D108N H123Y S146 pNMG-560 linker_nCas9_SGGS_NLS C_D 147 Y_E 155V_1156 F _K157N)
  • pCMV_ecTadA- 40 a.a. linker-ecTadA- 32 a.a. (H36L R51 L L84F A106V D108N H123Y S146 pNMG-561 linker_nCas9_SGGS_NLS C_D 147 Y_E 155V_1156 F _K157N)
  • the A to G nucleobase editor comprises a dCas9 or nCas9 fused to an adenosine deaminase.
  • Such nucleobase editors are described in US provisional application, U.S.S.N. 62/370,684, filed August 3, 2016; US provisional application, U.S.S.N. 62/370,684, filed February 3, 2017, US provisional application, U.S.S.N. 62/473,714, filed March 20, 2017, and PCT Application PCT/US2017/045381, filed August 3, 2017; each of which is incorporated herein by reference.
  • an A to G nucleobase editor comprises the structure of NH2- [second adenosine deaminase] -[first adenosine deaminase] -[dCas9]-COOH.
  • the second adenosine deaminase is a wile-type ecTadA (SEQ ID NO: 314).
  • the a linker is used between each domain.
  • the linker is 32 amino acids long and comprises the amino acid sequence of
  • the adenosine deaminase comprises one or more of a W23X, H36X, N37X, P48X, I49X, R51X, N72X, L84X, S97X, A106X, D108X, H123X, G125X, A142X, S 146X, D147X, R152X, E155X, I156X, K157X, and/or K161X mutation in SEQ ID NO: 314, or one or more corresponding mutations in another adenosine deaminase, where the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase.
  • the adenosine deaminase comprises one or more of W23L, W23R, H36L, P48S, P48A, R51L, L84F, A106V, D108N, H123Y, A142N, S 146C, D147Y, R152P, E155V, I156F, and/or K157N mutation in SEQ ID NO: 314, or one or more corresponding mutations in another adenosine deaminase.
  • the adenosine deaminase comprises or consists of one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve mutations selected from H36X, P48X, R51X, L84X, A106X, D108X, H123X, S 146X, D147X, E155X, I156X, and K157X in SEQ ID NO: 314, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild- type adenosine deaminase.
  • the adenosine deaminase comprises or consists of one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve mutations selected from H36L, P48S, R51L, L84F, A106V, D108N, H123Y, S 146C, D147Y, E155V, I156F, and K157N in SEQ ID NO: 314, or a corresponding mutation or mutations in another adenosine deaminase.
  • the adenosine deaminse comprises or consists of a H36L, P48S, R51L, L84F, A106V, D108N, H123Y, S 146C, D147Y, E155V, I156F, and K157N mutation in SEQ ID NO: 314, or corresponding mutations in another adenosine deaminase.
  • the adenosine deaminase comprises or consists of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen mutations selected from H36X, P48X, R51X, L84X, A106X, D108X, H123X, A142X, S 146X, D147X, E155X, I156X, and K157X in SEQ ID NO: 314, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase.
  • the adenosine deaminase comprises or consists of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen mutations selected from H36L, P48S, R51L, L84F, A106V, D108N, H123Y, A142N, S 146C, D147Y, E155V, I156F, and K157N in SEQ ID NO: 314, or a corresponding mutation or mutations in another adenosine deaminase.
  • the adenosine deaminase comprises or consists of a H36L, P48S, R51L, L84F, A106V, D108N, H123Y, A142N, S 146C, D147Y, E155V, I156F, and K157N mutation in SEQ ID NO: 314, or corresponding mutations in another adenosine deaminase.
  • the adenosine deaminase comprises or consists of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen mutations selected from W23X, H36X, P48X, R51X, L84X, A106X, D108X, H123X, A142X, S 146X, D147X, E155X, I156X, and K157X in SEQ ID NO: 314, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase.
  • the adenosine deaminase comprises or consists of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen mutations selected from W23L, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, A142N, S 146C, D147Y, E155V, I156F, and K157N in SEQ ID NO: 314, or a corresponding mutation or mutations in another adenosine deaminase.
  • the adenosine deaminase comprises or consists of a W23L, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, A142N, S 146C, D147Y, E155V, I156F, and K157N mutation in SEQ ID NO: 314, or corresponding mutations in another adenosine deaminase.
  • the adenosine deaminase comprises or consists of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen mutations selected from W23X, H36X, P48X, R51X, L84X, A106X, D108X, H123X, A142X, S 146X, D147X, R152X, E155X, I156X, and K157X in SEQ ID NO: 314, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase.
  • the adenosine deaminase comprises or consists of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen mutations selected from W23L, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, A142N, S 146C, D147Y, R152P, E155V, I156F, and K157N in SEQ ID NO: 314, or a corresponding mutation or mutations in another adenosine deaminase.
  • the adenosine deaminase comprises or consists of a W23L, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, A142N, S 146C, D147Y, R152P, E155V, I156F, and K157N mutation in SEQ ID NO: 314, or corresponding mutations in another adenosine deaminase.
  • the adenosine deaminase comprises or consists of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen mutations selected from W23X, H36X, P48X, R51X, L84X, A106X, D108X, H123X, S 146X, D147X, R152X, E155X, I156X, and K157X in SEQ ID NO: 314, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase.
  • the adenosine deaminase comprises or consists of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen mutations selected from W23R, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, S 146C, D147Y, R152P, E155V, I156F, and K157N in SEQ ID NO: 314, or a corresponding mutation or mutations in another adenosine deaminase.
  • the adenosine deaminse comprises or consists of a W23R, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, S 146C, D147Y, R152P, E155V, I156F, and K157N mutation in SEQ ID NO: 314, or corresponding mutations in another adenosine deaminase.
  • a nucleobase editor converts a C to G.
  • Such nucleobase editors are described in US provisional application, U.S.S.N. 62/470, 175, filed March 10, 2017, US provisional application, U.S.S.N. 62/470, 175, filed March 10, 2017incorporated herein by reference.
  • nucleobase editor is a variant of the nucleobase editors described herein.
  • the nucleobase editor 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 nucleobase editor described herein (exemplary sequences are provided in Example 1).
  • the nucleobase editor comprises an amino acid sequence that is shorter or longer in length (e.g. , by no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, no more than 5%, no more than 1% longer or shorter) than any of the nucleobase editors provided herein.
  • the nucleobase editor comprises an amino acid sequence that is shorter or longer in length (e.g.
  • nucleobase editors by no more than 500 amino acids, no more than 450 amino acids, no more than 400 amino acids, no more than 350 amino acids, no more than 300 amino acids, no more than 250 amino acids, no more than 200 amino acids, no more than 200 amino acids, no more than 150 amino acids, no more than 100 amino acids, no more than 50 amino acids, no more than 10 amino acids, no more than 5 amino acids longer or shorter) than any of the nucleobase editors provided herein.
  • a “deaminase” refers to an enzyme that catalyzes the removal of an amine group from a molecule, or deamination, for example, through hydrolysis.
  • the deaminase is a cytidine deaminase, catalyzing the deamination of cytidine (C) to uridine (U), deoxycytidine (dC) to deoxyuridine (dU), or 5-methyl-cytidine to thymidine (T, 5-methyl-U), respectively.
  • C cytidine
  • U uridine
  • dC deoxycytidine
  • dU deoxyuridine
  • T 5-methyl-cytidine
  • the deaminase is a cytosine deaminase, catalyzing and promoting the conversion of cytosine to uracil ⁇ e.g., in RNA) or thymine ⁇ e.g., in DNA).
  • the deaminase is an adenosine deaminase that converts an A to G.
  • the deaminase is a naturally-occurring deaminase from an organism, such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase is a variant of a naturally-occurring deaminase from an organism, and the variants do 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 from an organism.
  • the deaminase comprises an amino acid sequence that is shorter or longer in length ⁇ e.g., by no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, no more than 5%, no more than 1% longer or shorter) than any of the deaminases provided herein.
  • the deaminase comprises an amino acid sequence that is shorter or longer in length ⁇ e.g., by no more than 100 amino acids, no more than 90 amino acids, no more than 80 amino acids, no more than 70 amino acids, no more than 60 amino acids, no more than 50 amino acids, no more than 40 amino acids, no more than 30 amino acids, no more than 20 amino acids, no more than 10 amino acids, no more than 5 amino acids, no more than 2 amino acids, longer or shorter) than any of the deaminases provided herein.
  • 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.
  • the N-terminal portion of the nucleobase editor contains the N-terminal portion of the split Cas9
  • the C-terminal portion of the nucleobase editor contains the C-terminal portion of the split Cas9.
  • intein-N or intein-C may be fused to the N-terminal portion or the C-terminal portion of the nucleobase editor, respectively, for the joining of the N- and C-terminal portions of the nucleobase editor to form a complete nucleobase editor.
  • linker refers to a chemical group or a molecule linking two molecules or moieties, e.g., two domains of a fusion protein, such as, for example, a nuclease-inactive Cas9 domain and a nucleic acid editing domain (e.g., a deaminase domain).
  • the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond, thus connecting the two.
  • the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein).
  • the linker is an organic molecule, group, polymer, or chemical moiety.
  • the linker is 5- 100 amino acids in length, for example, 1, 2, 3, 4, 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 linkeare also contemplated.
  • a "uracil glycosylase inhibitor (UGI)” refers to a protein that inhibits the activity of uracil-DNA glycosylase.
  • Suitable UGI proteins for use in accordance with the present disclosure include, for example, those published in Wang et al., J. Biol. Chem. 264: 1163-1171(1989); Lundquist et al, J. Biol. Chem. 272:21408-21419(1997); Ravishankar et al, Nucleic Acids Res. 26:4880-4887(1998); and Putnam et al, J. Mol. Biol. 287:331-346(1999), each of which is incorporated herein by reference.
  • Non-limiting, exemplary proteins that may be used as a UGI of the present disclosure and their respective sequences are provided in Example 1.
  • the UGI is a variant of a naturally-occurring deaminase from an organism, and the variants do not occur in nature.
  • the UGI 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 UGI from an organism or any UGIs provided herein (e.g. , in Example 1).
  • the UGI comprises an amino acid sequence that is shorter or longer in length (e.g. , by no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, no more than 5%, no more than 1% longer or shorter) than any of the UGIs provided herein.
  • the UGI comprises an amino acid sequence that is shorter or longer in length (e.g. , by no more than 20 amino acids, no more than 15 amino acids, no more than 10 amino acids, no more than 5 amino acids, no more than 2 amino acids longer or shorter) than any of the UGIs provided herein.
  • a gRNA is a component of the CRISPR/Cas system.
  • a "gRNA” guide ribonucleic acid herein refers to a fusion of a CRISPR-targeting RNA (crRNA) and a trans-activation crRNA (tracrRNA), providing both targeting specificity and scaffolding/binding ability for Cas9 nuclease.
  • crRNA CRISPR-targeting RNA
  • tracrRNA trans-activation crRNA
  • a "tracrRNA” is a bacterial RNA that links the crRNA to the Cas9 nuclease and typically can bind any crRNA.
  • the sequence specificity of a Cas DNA-binding protein is determined by gRNAs, which have nucleotide base-pairing complementarity to target DNA sequences.
  • the native gRNA comprises a 20 nucleotide (nt) Specificity Determining Sequence (SDS), which specifies the DNA sequence to be targeted, and is immediately followed by a 80 nt scaffold sequence, which associates the gRNA with Cas9.
  • nt nucleotide
  • SDS Specificity Determining Sequence
  • an SDS of the present disclosure has a length of 15 to 100 nucleotides, or more.
  • an SDS may have a length of 15 to 90, 15 to 85, 15 to 80, 15 to 75, 15 to 70, 15 to 65, 15 to 60, 15 to 55, 15 to 50, 15 to 45, 15 to 40, 15 to 35, 15 to 30, or 15 to 20 nucleotides.
  • the SDS is 20 nucleotides long.
  • the SDS may be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides long.
  • At least a portion of the target DNA sequence is complementary to the SDS of the gRNA.
  • a region of the target sequence is complementary to the SDS of the gRNA sequence and is immediately followed by the correct protospacer adjacent motif (PAM) sequence (e.g. , NGG for Cas9 and TTN, TTTN, or YTN for Cpfl).
  • PAM protospacer adjacent motif
  • an SDS is 100% complementary to its target sequence.
  • the SDS sequence is less than 100% complementary to its target sequence and is, thus, considered to be partially complementary to its target sequence.
  • a targeting sequence may be 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% complementary to its target sequence.
  • the SDS of template DNA or target DNA may differ from a complementary region of a gRNA by 1, 2, 3, 4 or 5 nucleotides.
  • the gRNA comprises a scaffold sequence (corresponding to the tracrRNA in the native CRISPR/Cas system) that is required for its association with Cas9 (referred to herein as the "gRNA handle").
  • the gRNA comprises a structure 5'-[SDS] -[gRNA handle]-3'.
  • the scaffold sequence comprises the nucleotide sequence of 5'-guuuuagagcuagaaauagcaaguuaaaauaaggcuaguc
  • gRNA handle sequences that may be used in accordance with the present disclosure are listed in Table 1.
  • thermophilus AGUUAAAAUAAGGCUUAGUCCGUACUCAACUUG
  • the guide RNA is about 15-120 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence.
  • the guide RNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 97, 98, 99, 100, 101, 102, 103, 104,
  • the guide RNA comprises a sequence of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more contiguous nucleotides that is complementary to a target sequence.
  • Sequence complementarity refers to distinct interactions between adenine and thymine (DNA) or uracil (RNA), and between guanine and cytosine.
  • a "protospacer adjacent motif (PAM) is typically a sequence of nucleotides located adjacent to (e.g., within 10, 9, 8, 7, 6, 5, 4, 3, 3, or 1 nucleotide(s) of a target sequence).
  • a PAM sequence is "immediately adjacent to" a target sequence if the PAM sequence is contiguous with the target sequence (that is, if there are no nucleotides located between the PAM sequence and the target sequence).
  • a PAM sequence is a wild-type PAM sequence. Examples of PAM sequences include, without limitation, NGG, NGR, NNGRR(TVN),
  • a PAM sequence is obtained from Streptococcus pyogenes (e.g., NGG or NGR). In some embodiments, a PAM sequence is obtained from Staphylococcus aureus (e.g., NNGRR(TVN)). In some embodiments, a PAM sequence is obtained from Neisseria meningitidis (e.g.,
  • a PAM sequence is obtained from Streptococcus thermophilus (e.g., NNAGAAW or NGGAG). In some embodiments, a PAM sequence is obtained from Treponema denticola (e.g., NAAAAC). In some embodiments, a PAM sequence is obtained from Escherichia coli (e.g., AWG). In some embodiments, a PAM sequence is obtained from Pseudomonas auruginosa (e.g., CC). Other PAM sequences are contemplated. A PAM sequence is typically located downstream (i.e., 3') from the target sequence, although in some embodiments a PAM sequence may be located upstream (i.e., 5') from the target sequence.
  • a "nuclear localization signal” or “NLS” refers to as an amino acid sequence that "tags" a protein for import into the cell nucleus by nuclear transport. Typically, this signal consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface.
  • One or more NLS may be added to the N- or C-terminus of a protein, or internally (e.g., between two protein domains). For example, one or more NLS may be added to the N- or C- terminus of a nucleobase editor, or between the Cas9 and the deaminase in a nucleobase editor. In some embodiments, 1, 2, 3, 4, 5, or more NLS may be added.
  • NLS sequences are known in the art and would be apparent to the skilled artisan. For example, NLS sequences are described in Plank et al, PCT/EP2000/011690, filed November 23, 2000, the contents of which are incorporated herein by reference for its disclosure of exemplary nuclear localization sequences.
  • a NLS comprises the amino acid sequence PKKKRKV (SEQ ID NO: 373) or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 374).
  • a linker is inserted between the Cas9 and the deaminase.
  • An NLS can be classified as monopartite or bipartite.
  • a non-limiting example of a monopartite NLS is the sequence PKKKRKV (SEQ ID NO: 373) in the SV40 Large T-antigen.
  • a bipartite NLS typically contains two clusters of basic amino acids, separated by a spacer of about 10 amino acids.
  • One non-limiting example of a bipartite NLS is the NLS of
  • the NLS used in accordance with the present disclosure is the NLS of
  • KRPAATKKAGQAKKKK nucleoplasmin comprising the amino acid sequence of KRPAATKKAGQAKKKK.
  • Other bipartite NLSs that may be used in accordance with the present disclosure include, without limitation: SV40 bipartite NLS (KRTADGSEFESPKKKRKV (SEQ ID NO: 375), e.g., as described in Hodel et ah, J Biol Chem. 2001 Jan 12;276(2): 1317-25, incorporated herein by reference); Kanadaptin bipartite NLS (KKTELQTTNAENKTKKL (SEQ ID NO: 345), e.g., as described in Hubner et ah, Biochem J.
  • influenza A nucleoprotein bipartite NLS KRGINDRNFWRGENGRKTR (SEQ ID NO: 346), e.g., as described in Ketha et ah, BMC Cell Biology. 2008;9:22, incorporated herein by reference
  • ZO-2 bipartite NLS RKS GKI A AIV VKRPRK (SEQ ID NO: 347), e.g., as described in Quiros et al, Nusrat A, ed. Molecular Biology of the Cell. 2013;24(16):2528-2543, incorporated herein by reference).
  • nucleotide sequence encoding an NLS is "operably linked" to the nucleotide sequence encoding a protein to which the NLS is fused (e.g., a Cas9 or a nucleobase editor) when two coding sequences are "in-frame with each other" and are translated as a single polypeptide fusing two sequences.
  • Nucleic acids of the present disclosure may include one or more genetic elements.
  • a "genetic element” refers to a particular nucleotide sequence that has a role in nucleic acid expression (e.g., promoter, enhancer, terminator) or encodes a discrete product of an engineered nucleic acid (e.g., a nucleotide sequence encoding a guide RNA, a protein and/or an RNA interference molecule).
  • a “promoter” refers to a control region of a nucleic acid sequence at which initiation and rate of transcription of the remainder of a nucleic acid sequence are controlled.
  • a promoter may also contain sub-regions at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors. Promoters may be constitutive, inducible, activatable, repressible, tissue-specific, or any combination thereof.
  • a promoter drives expression or drives transcription of the nucleic acid sequence that it regulates.
  • a promoter is considered to be “operably linked” when it is in a correct functional location and orientation in relation to a nucleic acid sequence it regulates to control (“drive”) transcriptional initiation and/or expression of that sequence.
  • a promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating the 5 ' non-coding sequences located upstream of the coding segment of a given gene or sequence. Such a promoter is referred to as an "endogenous promoter.” In some
  • a coding nucleic acid sequence may be positioned under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with the encoded sequence in its natural environment.
  • promoters may include promoters of other genes; promoters isolated from any other cell; and synthetic promoters or enhancers that are not "naturally occurring" such as, for example, those that contain different elements of different transcriptional regulatory regions and/or mutations that alter expression through methods of genetic engineering that are known in the art.
  • sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including polymerase chain reaction (PCR).
  • promoters used in accordance with the present disclosure are "inducible promoters," which are promoters that are characterized by regulating (e.g. , initiating or activating) transcriptional activity when in the presence of, influenced by or contacted by an inducer signal.
  • An inducer signal may be endogenous or a normally exogenous condition (e.g. , light), compound (e.g. , chemical or non-chemical compound) or protein that contacts an inducible promoter in such a way as to be active in regulating transcriptional activity from the inducible promoter.
  • a "signal that regulates transcription" of a nucleic acid refers to an inducer signal that acts on an inducible promoter.
  • a signal that regulates transcription may activate or inactivate transcription, depending on the regulatory system used. Activation of transcription may involve directly acting on a promoter to drive transcription or indirectly acting on a promoter by inactivation a repressor that is preventing the promoter from driving transcription. Conversely, deactivation of transcription may involve directly acting on a promoter to prevent transcription or indirectly acting on a promoter by activating a repressor that then acts on the promoter.
  • a "transcriptional terminator” is a nucleic acid sequence that causes transcription to stop. A transcriptional terminator may be unidirectional or bidirectional. It is comprised of a DNA sequence involved in specific termination of an RNA transcript by an RNA polymerase. A transcriptional terminator sequence prevents transcriptional activation of downstream nucleic acid sequences by upstream promoters. A transcriptional terminator may be necessary in vivo to achieve desirable expression levels or to avoid transcription of certain sequences.
  • transcriptional terminator is considered to be "operably linked to" a nucleotide sequence when it is able to terminate the transcription of the sequence it is linked to.
  • the most commonly used type of terminator is a forward terminator. When placed downstream of a nucleic acid sequence that is usually transcribed, a forward transcriptional terminator will cause transcription to abort.
  • bidirectional transcriptional terminators are provided, which usually cause transcription to terminate on both the forward and reverse strand.
  • reverse transcriptional terminators are provided, which usually terminate transcription on the reverse strand only.
  • Rho-independent terminators are generally composed of palindromic sequence that forms a stem loop rich in G-C base pairs followed by several T bases.
  • transcriptional termination is that the stem loop causes RNA polymerase to pause, and transcription of the poly-A tail causes the RNA:DNA duplex to unwind and dissociate from RNA polymerase.
  • the terminator region may comprise specific DNA sequences that permit site-specific cleavage of the new transcript so as to expose a polyadenylation site. This signals a specialized endogenous polymerase to add a stretch of about 200 A residues (polyA) to the 3' end of the transcript. RNA molecules modified with this polyA tail appear to more stable and are translated more efficiently.
  • a terminator may comprise a signal for the cleavage of the RNA.
  • the terminator signal promotes polyadenylation of the message.
  • Terminators for use in accordance with the present disclosure include any terminator of transcription described herein or known to one of ordinary skill in the art. Examples of terminators include, without limitation, the termination sequences of genes such as, for example, the bovine growth hormone terminator, and viral termination sequences such as, for example, the SV40 terminator, spy, yejM, secG-leuU, thrLABC, rrnB Tl, hisLGDCBHAFI, metZWV, rrnC, xapR, asp A and arc A terminator.
  • the termination signal may be a sequence that cannot be transcribed or translated, such as those resulting from a sequence truncation.
  • WPRE Woodchuck Hepatitis Virus
  • WPRE Posttranscriptional Regulatory Element
  • CTCCCTTTGGGCCGCCTCCCCGCATCGATACCG SEQ ID NO:376).
  • AAV adeno-associated virus
  • ssDNA positive- or negative-sensed.
  • the genome comprises two inverted terminal repeats (ITRs), one at each end of the DNA strand, and two open reading frames (ORFs): rep and cap between the ITRs.
  • the rep ORF comprises four overlapping genes encoding Rep proteins required for the AAV life cycle.
  • the cap ORF comprises overlapping genes encoding capsid proteins: VP1, VP2 and VP3, which interact together to form the viral capsid. VP1, VP2 and
  • VP3 are translated from one mRNA transcript, which can be spliced in two different manners: either a longer or shorter intron can be excised resulting in the formation of two isoforms of mRNAs: a -2.3 kb- and a -2.6 kb-long mRNA isoform.
  • the capsid forms a supramolecular assembly of approximately 60 individual capsid protein subunits into a non-enveloped, T-l icosahedral lattice capable of protecting the AAV genome.
  • the mature capsid is composed of VP1, VP2, and VP3 (molecular masses of approximately 87, 73, and 62 kDa respectively) in a ratio of about 1 : 1 : 10.
  • rAAV particles may comprise a nucleic acid vector (e.g. , a recombinant genome), which may comprise at a minimum: (a) one or more heterologous nucleic acid regions comprising a sequence encoding a protein or polypeptide of interest (e.g. , a split Cas9 or split nucleobase) or an RNA of interest (e.g. , a gRNA), or one or more nucleic acid regions comprising a sequence encoding a Rep protein; and (b) one or more regions comprising inverted terminal repeat (ITR) sequences (e.g. , wild-type ITR sequences or engineered ITR sequences) flanking the one or more nucleic acid regions (e.g.
  • ITR inverted terminal repeat
  • the nucleic acid vector is between 4 kb and 5 kb in size (e.g. , 4.2 to 4.7 kb in size). In some embodiments, the nucleic acid vector further comprises a region encoding a Rep protein. In some
  • the nucleic acid vector is circular. In some embodiments, the nucleic acid vector is single-stranded. In some embodiments, the nucleic acid vector is double-stranded. In some embodiments, a double-stranded nucleic acid vector may be, for example, a self-complimentary vector that contains a region of the nucleic acid vector that is complementary to another region of the nucleic acid vector, initiating the formation of the double-strandedness of the nucleic acid vector.
  • nucleic acid refers to a compound comprising a nucleobase and an acidic moiety, e.g. , a nucleotide, or a polymer of nucleotides.
  • polymeric nucleic acids e.g. , nucleic acid molecules comprising three or more nucleotides are linear molecules, in which adjacent nucleotides are linked to each other via a phosphodiester linkage.
  • nucleic acid refers to individual nucleic acid residues (e.g. nucleotides and/or nucleosides).
  • nucleic acid refers to an oligonucleotide chain comprising three or more individual nucleotide residues.
  • oligonucleotide and polynucleotide can be used interchangeably to refer to a polymer of nucleotides (e.g. , a string of at least three nucleotides).
  • nucleic acid encompasses RNA as well as single and/or double- stranded DNA.
  • Nucleic acids may be naturally occurring, for example, in the context of a genome, a transcript, an mRNA, tRNA, rRNA, siRNA, snRNA, a plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule.
  • a nucleic acid molecule may be a non- naturally occurring molecule, e.g. , a recombinant DNA or RNA, an artificial chromosome, an engineered genome (e.g., an engineered viral vector), an engineered vector, or fragment thereof, or a synthetic DNA, RNA, or DNA/RNA hybrid, optionally including non-naturally occurring nucleotides or nucleosides.
  • nucleic acid examples include nucleic acid analogs, e.g., analogs having other than a phosphodiester backbone.
  • Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g. , in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications. A nucleic acid sequence is presented in the 5' to 3 ' direction unless otherwise indicated.
  • a nucleic acid is or comprises natural nucleosides (e.g. adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and
  • deoxycytidine deoxycytidine
  • nucleoside analogs e.g. , 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo- pyrimidine, 3-methyl adenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5- fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g.
  • methylated bases methylated bases
  • intercalated bases modified sugars (e.g. , 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g. ,
  • 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.
  • One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc.
  • a protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex.
  • a protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide.
  • a protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof.
  • fusion protein refers to a hybrid polypeptide which comprises protein domains from at least two different proteins.
  • One protein may be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy-terminal (C-terminal) protein thus forming an "amino-terminal fusion protein” or a "carboxy-terminal fusion protein,” respectively.
  • a protein may comprise different domains, for example, a nucleic acid binding domain (e.g., the gRNA binding domain of Cas9 that directs the binding of the protein to a target site) and a nucleic acid cleavage domain or a catalytic domain of a nucleic-acid editing protein.
  • a protein is in a complex with, or is in association with, a nucleic acid, e.g., RNA or DNA.
  • a nucleic acid e.g., RNA or DNA.
  • 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)), which are incorporated herein by reference.
  • 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 (e.g., mouse, rat).
  • the subject is a domesticated animal.
  • the subject is a sheep, a goat, a cow, a cat, or a dog.
  • 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.
  • recombinant refers to proteins or nucleic acids that do not occur in nature, but are the product of human engineering.
  • a recombinant protein or nucleic acid molecule comprises an amino acid or nucleotide sequence that comprises at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations as compared to any naturally occurring sequence.
  • the fusion proteins (e.g., base editors) described herein are made recombinantly. Recombinant technology is familiar to those skilled in the art.
  • pharmaceutically-acceptable carrier means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g. , lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the compound from one site (e.g. , the delivery site) of the body, to another site (e.g. , organ, tissue or portion of the body).
  • a pharmaceutically acceptable carrier is "acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissue of the subject (e.g. , physiologically compatible, sterile, physiologic pH, etc.).
  • a therapeutically effective amount refers to the amount of each therapeutic agent (e.g. , nucleobase editor, rAAV) described in the present disclosure required to confer therapeutic effect on the subject, either alone or in combination with one or more other therapeutic agents. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual subject parameters including age, physical condition, size, gender, and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation.
  • a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a subject may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons. Empirical considerations, such as the half-life, generally will contribute to the determination of the dosage.
  • therapeutic agents that are compatible with the human immune system such as polypeptides comprising regions from humanized antibodies or fully human antibodies, may be used to prolong half-life of the polypeptide and to prevent the polypeptide being attacked by the host's immune system.
  • a subject in need thereof refers to an individual who has a disease, a sign and/or symptom of a disease, or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, the symptom of the disease, or the predisposition toward the disease.
  • the subject is a mammal.
  • the subject is a non-human primate.
  • the subject is human.
  • the mammal is a rodent.
  • the rodent is a mouse.
  • the rodent is a rat.
  • the mammal is a companion animal.
  • a "companion animal” refers to pets and other domestic animals.
  • companion animals include dogs and cats; livestock, such as horses, cattle, pigs, sheep, goats, and chickens; and other animals, such as mice, rats, guinea pigs, and hamsters.
  • treatment refers to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein.
  • treatment refers to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein.
  • treatment may be administered after one or more symptoms have developed and/or after a disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, e.g.
  • treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g. , in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence.
  • compositions e.g. , vectors, recombinant viruses
  • kits comprising nucleic acids encoding split Cas9 proteins or nucleobase editors, and methods of delivering a nucleobase editor or a Cas9 protein into a cell using such nucleic acids.
  • the N- terminal portion and C-terminal portion of a nucleobase editor or a Cas9 protein are encoded on separate nucleic acids and delivered into a cell, e.g. , a via recombinant adeno-associated virus (rAAV particles) delivery.
  • rAAV particles recombinant adeno-associated virus
  • polypeptides corresponding to the N-terminal portion and C- terminal portions of the nucleobase editor or Cas9 protein may be joined to form a complete nucleobase editor or Cas9 protein, e.g. , via intein-mediated protein splicing.
  • compositions comprising (i) a first nucleotide sequence encoding an N-terminal portion of a Cas9 protein fused at its C- terminus to an intein-N; and (ii) a second nucleotide sequence encoding an intein-C fused to the N-terminus of a C-terminal portion of the Cas9 protein, wherein the first nucleotide sequence or second nucleotide sequence is operably linked to a nucleotide sequence encoding at least one bipartite nuclear localization signal (NLS).
  • NLS bipartite nuclear localization signal
  • the Cas9 protein encoded by the first and second nucleotide sequence is herein referred as a "split Cas9.”
  • the Cas9 protein is known to have a N-terminal lobe and a C-terminal lobe linked by a disordered linker (e.g. , as described in Nishimasu et al. , Cell, Volume 156, Issue 5, pp. 935-949, 2014, incorporated herein by reference).
  • the N-terminal portion of the split Cas9 protein comprises the N-terminal lobe of a Cas9 protein.
  • the C-terminal portion of the split Cas9 comprises the C-terminal lobe of a Cas9 protein.
  • the N-terminal portion of the split Cas9 comprises a portion of any one of SEQ ID NO: 1-275 and 394-397 that corresponds to amino acids 1 -(550-650) in SEQ ID NO: 1.
  • "l-(550-650)” means starting from amino acid 1 and ending anywhere between amino acid 550-650 (inclusive).
  • the N-terminal portion of the split Cas9 may comprise a portion of any one of SEQ ID NOs: 1-275 and 394-397 that corresponds to amino acids 1-550, 1-551, 1-552, 1-553, 1-554, 1-555, 1-556, 1-557, 1-558, 1-559, 1-560, 1-561, 1-562, 1-563, 1- 564, 1-565, 1-566, 1-567, 1-568, 1-569, 1-570, 1-571, 1-572, 1-573, 1-574, 1-575, 1-576, 1-577, 1-578, 1-579, 1-580, 1-581, 1-582, 1-583, 1-584, 1-585, 1-586, 1-587, 1-588, 1-589, 1-590, 1- 591, 1-592, 1-593, 1-594, 1-595, 1-596, 1-597, 1-598, 1-599, 1-600, 1-601, 1-602, 1-603, 1-604, 1-605, 1-6
  • the C-terminal portion of the split Cas9 can be joined with the N-terminal portion of the split Cas9 to form a complete Cas9 protein.
  • the C-terminal portion of the Cas9 protein starts from where the N-terminal portion of the Cas9 protein ends.
  • the C-terminal portion of the split Cas9 comprises a portion of any one of SEQ ID NO: 1-275 and 394-397 that corresponds to amino acids (551-651)-1368 of SEQ ID NO: 1.
  • “(551-651)-1368" means starting at an amino acid between amino acids 551-651 (inclusive) and ending at amino acid 1368.
  • the C-terminal portion of the split Cas9 may comprise a portion of any one of SEQ ID NO: 1-275 and 394-397 that corresponds to amino acid 551-1368, 552-1368, 553-1368, 554-1368, 555-1368, 556-1368, 557-1368, 558-1368, 559-1368, 560-1368, 561-1368, 562-1368, 563-1368, 564-1368, 565-1368, 566-1368, 567-1368, 568-1368, 569-1368, 570-1368, 571-1368, 572-1368, 573-1368, 574-1368, 575-1368, 576-1368, 577-1368, 578-1368, 579-1368, 580-1368, 581-1368, 582-1368, 583-1368, 584-1368, 585-1368, 586-1368, 587-1368, 588-1368, 589-1368, 590-1368, 591-1368, 592-1368, 593
  • Cas9 variants may also be delivered to cells using the methods described herein.
  • a Cas9 variant may also be "split" as described herein.
  • a Cas9 variant 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 Cas9 sequences provided herein.
  • the Cas9 variant comprises an amino acid sequence that is shorter or longer in length (e.g., by no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, no more than 5%, no more than 1% longer or shorter) than any of the Cas9 proteins provided herein (e.g., in Example 1).
  • the UGI comprises an amino acid sequence that is shorter or longer in length (e.g., by no more than 200 amino acids, no more than 150 amino acids, no more than 100 amino acids, no more than 50 amino acids, no more than 10 amino acids, no more than 5 amino acids, or no more than 2 amino acids longer or shorter) than any of the Cas9 proteins provided herein.
  • the N-terminal portion of a split Cas9 comprises 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 the corresponding portion of any one of the Cas9 sequences provided herein (e.g. , in Example 1).
  • the N-terminal portion of the split Cas9 comprises an amino acid sequence that is shorter or longer in length (e.g.
  • the N-terminal portion of the split Cas9 comprises an amino acid sequence that is shorter or longer in length (e.g. , by no more than 200 amino acids, no more than 150 amino acids, no more than 100 amino acids, no more than 50 amino acids, no more than 10 amino acids, no more than 5 amino acids, or no more than 2 amino acids longer or shorter) than the corresponding portion of any of the Cas9 proteins provided herein.
  • the C-terminal portion of a split Cas9 comprises 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 the corresponding portion of any one of the Cas9 sequences provided herein (e.g. , in Example 1).
  • the C-terminal portion of the split Cas9 comprises an amino acid sequence that is shorter or longer in length (e.g.
  • the C-terminal portion of the split Cas9 comprises an amino acid sequence that is shorter or longer in length (e.g. , by no more than 200 amino acids, no more than 150 amino acids, no more than 100 amino acids, no more than 50 amino acids, no more than 10 amino acids, no more than 5 amino acids, or no more than 2 amino acids longer or shorter) than the corresponding portion of any of the Cas9 proteins provided herein.
  • the Cas9 variant is a dCas9 or nCas9.
  • the N-terminal portion of the split Cas9 comprises a mutation corresponding to a D10A mutation in SEQ ID NO: 1.
  • the N-terminal portion of the split Cas9 comprises a mutation corresponding to a D10A mutation in SEQ ID NO: 1 and the C-terminal portion of the split Cas9 comprises a mutation corresponding to a H840A mutation in SEQ ID NO: l .
  • the N-terminal portion of the split Cas9 comprises a mutation corresponding to a D10A mutation in SEQ ID NO: 1
  • the C-terminal portion of the split Cas9 comprises a histidine at the position corresponding to position 840 in SEQ ID NO: l .
  • an intein system may be used to join the N-terminal portion of the Cas9 protein and the C- terminal portion of the Cas9 protein.
  • the N- terminal portion of the Cas9 is fused to an intein-N.
  • the intein-N is fused to the C-terminus of the N-terminal portion of the Cas9 to form a structure of N3 ⁇ 4- [N-terminal portion of Cas9]-[intein-N]-COOH.
  • the intein-N is encoded by the dnaE- n gene.
  • the intein-N comprises the amino acid sequence of any one of SEQ ID NOs: 350-351 and 354-355.
  • the C-terminal portion of the Cas9 is fused to an intein-C, and the intein-C is fused to the N-terminus of the C-terminal portion of the Cas9 to form a structure of NH 2 -[intein-C]-[C-terminal portion of Cas9]-COOH.
  • the intein-C is encoded by the dnaE-c gene.
  • the intein-C comprises the amino acid sequence of any one of SEQ ID NOs: 352-353 and 356-357. Other split intein systems may also be used in the present disclosure and are known in the art.
  • Split nucleobase editors may be used in the present disclosure. Some aspects of the present disclosure relate to compositions comprising (i) a first nucleotide sequence encoding an N-terminal portion of a nucleobase editor fused at its C-terminus to an intein-N; and (ii) a second nucleotide sequence encoding an intein-C fused to the N-terminus of a C-terminal portion of the nucleobase editor.
  • Nucleobase editor variants are contemplated.
  • a nucleobase editor variant may also be "split" as described herein.
  • a nucleobase editor variant 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 nucleobase editor sequences (SEQ ID NOs: X-X) provided herein.
  • the N-terminal portion of a split nucleobase editor comprises 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 the corresponding portion of any one of the nucleobase editors provided herein (e.g. , in Example 1).
  • the N-terminal portion of the split nucleobase editor comprises an amino acid sequence that is shorter or longer in length (e.g.
  • the N-terminal portion of the split nucleobase editor comprises an amino acid sequence that is shorter or longer in length (e.g. , by no more than 200 amino acids, no more than 150 amino acids, no more than 100 amino acids, no more than 50 amino acids, no more than 10 amino acids, no more than 5 amino acids, or no more than 2 amino acids longer or shorter) than the corresponding portion of any of the nucleobase editors provided herein.
  • the C-terminal portion of a split nucleobase editor comprises 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 the corresponding portion of any one of the nucleobase editors provided herein (e.g. , in Example 1).
  • the C-terminal portion of the split nucleobase editor comprises an amino acid sequence that is shorter or longer in length (e.g.
  • the C-terminal portion of the split nucleobase editor comprises an amino acid sequence that is shorter or longer in length (e.g. , by no more than 200 amino acids, no more than 150 amino acids, no more than 100 amino acids, no more than 50 amino acids, no more than 10 amino acids, no more than 5 amino acids, or no more than 2 amino acids longer or shorter) than the corresponding portion of any of the nucleobase editors provided herein.
  • the N-terminal portion of a nucleobase editor comprises the N- terminal portion of a nuclease-inactive Cas9 protein (dCas9) or a Cas9 nickase (nCas9) .
  • the N-terminal portion of a nucleobase editor further comprises a nucleobase modifying enzyme (e.g.
  • nucleases nucleases, nickases, recombinases, deaminases, DNA repair enzymes, DNA damage enzymes, dismutases, alkylation enzymes, depurination enzymes, oxidation enzymes, pyrimidine dimer forming enzymes, integrases, transposases, polymerases, ligases, helicases, photolyases, glycosylases, epigenetic modifiers such as methylases, acetylases, methyltransferase, demethylase, etc.).
  • the nucleobase modifying enzyme is a deaminase (e.g.
  • the nucleobase modifying enzyme is fused to the N-terminus of the N-terminal portion of the split dCas9 or split nCas9.
  • the N-terminal portion of the nucleobase editor has of the structure: NH 2 - [nucleobase modifying enzyme]-[N- terminal portion of dCas9 or nCas9]-COOH.
  • the N-terminal portion of the nucleobase editor is fused to an intein N.
  • the intein-N is fused to the C-terminus of the N-terminal portion of the nucleobase editor.
  • the first nucleotide sequence encodes a polypeptide comprising the structure NH 2 - [nucleobase modifying enzyme] -[N-terminal portion of dCas9 or nCas9]- [intein-N]-COOH.
  • the C-terminal portion of the nucleobase editor comprises the C- terminal portion of a nuclease-inactive Cas9 protein (dCas9) or a Cas9 nickase (nCas9).
  • the nucleobase modifying enzyme is fused to the C-terminus of the C-terminal portion of the split dCas9 or split nCas9.
  • the C-terminal portion of the nucleobase editor is of the structure: NH 2 - [C-terminal portion of dCas9 or nCas9]-[nucleobase modifying enzyme]-COOH.
  • the C-terminal portion of the nucleobase editor comprises an intein-C fused to the C-terminal portion of the Cas9 protein.
  • the intein-C is fused to the N-terminus of the C-terminal portion of the nucleobase editor.
  • the second nucleotide sequence encodes a polypeptide of the structure: NH 2 -[intein-C]-[C-terminal portion of the Cas9 protein] -COOH.
  • the N-terminal portion of a split nucleobase editor further comprises an inhibitor of uracil glycosylase (UGI).
  • the first nucleotide sequence encodes a polypeptide of the structure: NH 2 - [UGI] -[nucleobase modifying enzyme]- [N-terminal portion of dCas9 or nCas9]-[intein-N].
  • the first nucleotide sequence encodes a polypeptide is of the structure: NH 2 - [nucleobase modifying enzyme] -[UGI] - [N-terminal portion of dCas9 or nCas9]-[intein-N].
  • the C-terminal portion of a split nucleobase editor further comprises an enzyme that inhibits the activity of uracil glycosylase (UGI).
  • UMI uracil glycosylase
  • the second nucleotide sequence encodes a polypeptide of the structure: NH 2 - [intein-C]- [C-terminal portion of dCas9 or nCas9]-[UGI]-COOH. In some embodiments, the second nucleotide sequence encodes a polypeptide of the structure: NH 2 - [intein-C] -[C-terminal portion of dCas9 or nCas9]-[nucleobase modifying enzyme]-[UGI]-COOH. In some embodiments, the second nucleotide sequence encodes a polypeptide of the structure: NH 2 - [intein-C] -[C-terminal portion of dCas9 or nCas9]-[nucleobase modifying enzyme]-[UGI]-COOH. In some
  • the second nucleotide sequence encodes a polypeptide of the structure: NH 2 - [intein-C]- [C-terminal portion of dCas9 or nCas9]-[UGI]-[nucleobase modifying enzyme]- COOH.
  • the split nucleobase editor may comprise any one of the following structures:
  • the first nucleotide sequence or the second nucleotide sequence (encoding either the split Cas9 protein or the split nucleobase editor) is operably linked to a nucleotide sequence encoding at least one bipartite nuclear localization signal (NLS).
  • NLS nuclear localization signal
  • the first nucleotide sequence may be operably linked to a nucleotide sequence encoding one or more (e.g. , 2, 3, 4, 5, or more) bipartite NLS.
  • the second nucleotide sequence may be operably linked to a nucleotide sequence encoding one or more (e.g. , 2, 3, 4, 5, or more) bipartite NLSs.
  • the split Cas9 or split nucleobase editor formed by joining the N-terminal portion and the C-terminal portion may comprise one or more bipartite NLSs.
  • the split Cas9 or split nucleobase editor may comprise any one of the following structures (bNLS means one or more bipartite nuclear localization signals):
  • linkers may be used to link any of the protein or protein domains described herein.
  • the linker may be as simple as a covalent bond, or it may be a polymeric linker many atoms in length.
  • the linker is a polypeptide or based on amino acids.
  • the linker is not peptide-like.
  • the linker is a covalent bond (e.g.
  • the linker is a carbon-nitrogen bond of an amide linkage.
  • the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linker.
  • the linker is polymeric (e.g. , polyethylene, polyethylene glycol, polyamide, polyester, etc.).
  • the linker comprises a monomer, dimer, or polymer of aminoalkanoic acid.
  • the linker comprises an aminoalkanoic acid (e.g.
  • the linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx).
  • the linker is based on a carbocyclic moiety (e.g. , cyclopentane, cyclohexane).
  • the linker comprises a polyethylene glycol moiety (PEG).
  • the linker comprises amino acids.
  • the linker comprises a peptide.
  • the linker comprises an aryl or heteroaryl moiety. In some embodiments, the linker is based on a phenyl ring.
  • the linker may include functionalized moieties to facilitate attachment of a nucleophile (e.g. , thiol, amino) from the peptide to the linker. Any electrophile may be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates.
  • the linker is an amino acid or a plurality of amino acids (e.g. , a peptide or protein).
  • the linker is a bond (e.g. , a covalent bond), an organic molecule, group, polymer, or chemical moiety.
  • the linker is 5- 100 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, 100-110, 110- 120, 120- 130, 130-140, 140- 150, or 150-200 amino acids in length.
  • a linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 377), which may also be referred to as the XTEN linker.
  • a linker comprises the amino acid sequence: SGGS (SEQ ID NO: 378).
  • a linker comprises the amino acid sequence: (SGGS) n (SEQ ID NO: 379), (GGGS) protest (SEQ ID NO: 380), (GGGGS) friendship (SEQ ID NO: 381), (G) context (SEQ ID NO: 390), (EAAAK) n (SEQ ID NO: 382), (GGS) thread, SGSETPGTSESATPES (SEQ ID NO: 377), or (XP)n motif, or a combination of any of these, wherein n is independently an integer between 1 and 30, inclusive, and wherein X is any amino acid.
  • n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.
  • the linker comprises the amino acid sequence: SGSETPGTSESATPES (SEQ ID NO: 377), and SGGS (SEQ ID NO: 378).
  • the linker comprises the amino acid sequence:
  • a linker comprises the amino acid sequence: SGGSSGGSSGS ETPGTS ES ATPES S GGS S GGS (SEQ ID NO: 384). In some embodiments, a linker comprises the amino acid sequence:
  • the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES (SEQ ID NO: 343). In some embodiments, the linker is 40 amino acids in length. In some embodiments, the linker comprises the amino acid sequence
  • the linker is 64 amino acids in length. In some embodiments, the linker comprises the amino acid sequence
  • the linker is 92 amino acids in length. In some embodiments, the linker comprises the amino acid sequence
  • the first and second nucleotide sequences are on the same nucleic acid vector. In some embodiments, the first and second nucleotide sequences are on different nucleic acid vectors. In some embodiments, the vector is a plasmid. In some embodiments, the nucleic acid vector is a recombinant genome of a adeno-associated virus (rAAV). In some embodiments, the nucleic acid vector is the genome of an adeno-associated virus packaged in a rAAV particle. In some embodiments, the first and/or the second nucleotide sequence is operably linked to a promoter.
  • rAAV adeno-associated virus
  • the nucleic acid vector further comprise a nucleotide sequence encoding one or more (e.g. , 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) gRNAs operably linked to a promoter.
  • the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter.
  • An inducible promoter of the present disclosure may be induced by (or repressed by) one or more physiological condition(s), such as changes in light, pH, temperature, radiation, osmotic pressure, saline gradients, cell surface binding, and the concentration of one or more extrinsic or intrinsic inducing agent(s).
  • An extrinsic inducer signal or inducing agent may comprise, without limitation, amino acids and amino acid analogs, saccharides and
  • polysaccharides polysaccharides, nucleic acids, protein transcriptional activators and repressors, cytokines, toxins, petroleum-based compounds, metal containing compounds, salts, ions, enzyme substrate analogs, hormones, or combinations thereof.
  • inducible promoters of the present disclosure include any inducible promoter described herein or known to one of ordinary skill in the art.
  • inducible promoters include, without limitation, chemically/biochemically-regulated and physically-regulated promoters such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g. , anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems, which include a tetracycline repressor protein (tetR), a tetracycline operator sequence (tetO) and a tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g.
  • promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid/retinoid/thyroid receptor superfamily include metal-regulated promoters (e.g. , promoters derived from metallothionein (proteins that bind and sequester metal ions) genes from yeast, mouse and human), pathogenesis-regulated promoters (e.g. , induced by salicylic acid, ethylene or benzothiadiazole (BTH)), temperature/heat-inducible promoters (e.g. , heat shock promoters), and light-regulated promoters (e.g. , light responsive promoters from plant cells).
  • metal-regulated promoters e.g. , promoters derived from metallothionein (proteins that bind and sequester metal ions) genes from yeast, mouse and human
  • pathogenesis-regulated promoters e.g. , induced by
  • inducible promoters of the present disclosure function in prokaryotic cells (e.g. , bacterial cells).
  • prokaryotic cells e.g. , bacterial cells.
  • inducible promoters for use prokaryotic cells include, without limitation, bacteriophage promoters (e.g. Pis Icon, T3, T7, SP6, PL) and bacterial promoters (e.g. , Pbad, PmgrB, Ptrc2, Plac/ara, Ptac, Pm), or hybrids thereof (e.g.
  • bacterial promoters for use in accordance with the present disclosure include, without limitation, positively regulated E. coli promoters, such as positively regulated ⁇ 70 promoters (e.g. , inducible pBad/araC promoter, Lux cassette right promoter, modified lamdba Prm promote, plac Or2-62 (positive), pBad/AraC with extra REN sites, pBad, P(Las) TetO, P(Las) CIO, P(Rhl), Pu, FecA, pRE, cadC, hns, pLas, pLux), oS promoters (e.g.
  • positively regulated E. coli promoters such as positively regulated ⁇ 70 promoters (e.g. , inducible pBad/araC promoter, Lux cassette right promoter, modified lamdba Prm promote, plac Or2-62 (positive), pBad/AraC with extra REN sites,
  • Pdps ⁇ 32 promoters (e.g. , heat shock), and ⁇ 54 promoters (e.g. , glnAp2); negatively regulated E. coli promoters such as negatively regulated ⁇ 70 promoters (e.g. , Promoter (PRM+), modified lamdba Prm promoter, TetR - TetR-4C P(Las) TetO, P(Las) CIO, P(Lac) IQ,
  • PRM+ Promoter
  • modified lamdba Prm promoter TetR - TetR-4C P(Las) TetO, P(Las) CIO, P(Lac) IQ
  • RecA_DlexO_DLac01 dapAp, FecA, Pspac-hy, pel, plux-cl, plux-lac, CinR, CinL, glucose controlled, modified Pr, modified Prm+, FecA, Pcya, rec A (SOS), Rec A (SOS),
  • Lutz-Bujard LacO with alternative sigma factor ⁇ 38 Lutz-Bujard LacO with alternative sigma factor ⁇ 38
  • ⁇ 32 promoters e.g. , Lutz-Bujard LacO with alternative sigma factor ⁇ 32
  • ⁇ 54 promoters e.g. , glnAp2
  • B. subtilis promoters such as repressible B. subtilis ⁇ promoters (e.g. , Gram-positive IPTG-inducible, Xyl, hyper-spank) and ⁇ promoters.
  • Other inducible microbial promoters may be used in accordance with the present disclosure.
  • inducible promoters of the present disclosure function in eukaryotic cells (e.g. , mammalian cells).
  • eukaryotic cells e.g. , mammalian cells.
  • inducible promoters for use eukaryotic cells include, without limitation, chemically-regulated promoters (e.g. , alcohol-regulated promoters, tetracycline-regulated promoters, steroid-regulated promoters, metal-regulated promoters, and pathogenesis-related (PR) promoters) and physically-regulated promoters (e.g. , temperature-regulated promoters and light-regulated promoters).
  • chemically-regulated promoters e.g. , alcohol-regulated promoters, tetracycline-regulated promoters, steroid-regulated promoters, metal-regulated promoters, and pathogenesis-related (PR) promoters
  • physically-regulated promoters e.g. , temperature-regulated promoters and light-regulated promoters
  • rAAV Recombinant Adeno-associated Virus
  • Some aspects of the present disclosure relate to using recombinant adeno-associated virus vectors for the delivery of a split Cas9 protein or a split nucleobase editor into a cell.
  • the N-terminal portion of the Cas9 protein or the nucleobase editor and the C-terminal portion of the Cas9 protein or the nucleobase editor are delivered by separate rAAV vectors or particles into the same cell, since the full-length Cas9 protein or nucleobase editors exceeds the packaging limit of rAAV (-4.9 kb).
  • a composition for delivering the split Cas9 protein or split nucleobase editor into a cell e.g.
  • the composition of the present disclosure comprises: (i) a first recombinant adeno- associated virus (rAAV) particle comprising a first nucleotide sequence encoding a N-terminal portion of a Cas9 protein or nucleobase editor fused at its C-terminus to an intein-N; and (ii) a second recombinant adeno-associated virus (rAAV) particle comprising a second nucleotide sequence encoding an intein-C fused to the N-terminus of a C-terminal portion of the Cas9 protein or nucleobase editor.
  • the rAAV particles of the present disclosure comprise a rAAV vector (i.e. , a recombinant genome of the rAAV) encapsidated in the viral capsid proteins.
  • the rAAV vector comprises: (1) a heterologous nucleic acid region comprising the first or second nucleotide sequence encoding the N-terminal portion or C- terminal portion of a split Cas9 protein or a split nucleobase editor in any form as described herein, (2) one or more nucleotide sequences comprising a sequence that facilitates expression of the heterologous nucleic acid region (e.g. , a promoter), and (3) one or more nucleic acid regions comprising a sequence that facilitate integration of the heterologous nucleic acid region
  • viral sequences that facilitate integration comprise Inverted Terminal Repeat (ITR) sequences.
  • ITR Inverted Terminal Repeat
  • the first or second nucleotide sequence encoding the N-terminal portion or C-terminal portion of a split Cas9 protein or a split nucleobase editor is flanked on each side by an ITR sequence.
  • the nucleic acid vector further comprises a region encoding an AAV Rep protein as described herein, either contained within the region flanked by ITRs or outside the region.
  • the ITR sequences can be derived from any AAV serotype (e.g. , 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) or can be derived from more than one serotype.
  • the ITR sequences are derived from AAV2 or AAV6.
  • ITR sequences and plasmids containing ITR sequences are known in the art and commercially available (see, e.g. , products and services available from Vector Biolabs,
  • Kessler PD Podsakoff GM, Chen X, McQuiston SA, Colosi PC, Matelis LA, Kurtzman GJ, Byrne BJ. Proc Natl Acad Sci USA. 1996 Nov 26;93(24): 14082-7; and Curtis A. Machida. Methods in Molecular MedicineTM. Viral Vectors for Gene Therapy Methods and Protocols. 10.1385/1-59259-304-6:201 ⁇ Humana Press Inc. 2003. Chapter 10. Targeted Integration by Adeno-Associated Virus.
  • the rAAV vector of the present disclosure comprises one or more regulatory elements to control the expression of the heterologous nucleic acid region (e.g. , promoters, transcriptional terminators, and/or other regulatory elements).
  • the first and/or second nucleotide sequence is operably linked to one or more (e.g. , 1, 2, 3, 4, 5, or more) transcriptional terminators.
  • transcriptional terminators include transcription terminators of the bovine growth hormone gene (bGH), human growth hormone gene (hGH), SV40, CW3, ⁇ , or combinations thereof.
  • the transcriptional terminator used in the present disclosure is a bGH transcriptional terminator.
  • the rAAV vector further comprises a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE).
  • WPRE Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element
  • the composition comprising the rAAV particle (in any form contemplated herein) further comprises a pharmaceutically acceptable carrier.
  • the composition is formulated in appropriate pharmaceutical vehicles for administration to human or animal subjects.
  • Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as
  • compositions described herein e.g. , compositions comprising nucleotide sequences encoding the split Cas9 or the split nucleobase editor or AAV particles containing nucleic acid vectors comprising such nucleotide sequences.
  • the contacting results in the delivery of such nucleotide sequences into a cell, wherein the N-terminal portion of the Cas9 protein or the nucleobase editor and the C-terminal portion of the Cas9 protein or the nucleobase editor are expressed in the cell and are joined to form a complete Cas9 protein or a complete nucleobase editor.
  • the split Cas9 protein or split nucleobase editor delivered using the methods described herein preferably have comparable activity compared to the original Cas9 protein or nucleobase editor (i.e. , unsplit protein delivered to a cell or expressed in a cell as a whole).
  • the split Cas9 protein or split nucleobase editor retains at least 50% (e.g. , at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) of the activity of the original Cas9 protein or nucleobase editor.
  • the split Cas9 protein or split nucleobase editor is more active (e.g. , 2-fold, 5-fold, 10-fold, 100-fold, 1000- fold, or more) than that of an original Cas9 protein or nucleobase editor.
  • compositions described herein may be administered to a subject in need thereof in a therapeutically effective amount to treat and/or prevent a disease or disorder the subject is suffering from. Any disease or disorder that maybe treated and/or prevented using
  • CRISPR/Cas9-based genome-editing technology may be treated by the split Cas9 protein or the split nucleobase editor described herein. It is to be understood that, if the nucleotide sequences encoding the split Cas9 protein or the nucleobase editor does not further encode a gRNA, a separate nucleic acid vector encoding the gRNA may be administered together with the compositions described herein.
  • Exemplary suitable diseases and disorders include, without limitation, [[The following diseases were included in the C to T editor application. Please indicate any that are still relevant and could be treated using an adenosine deaminase. ]]cystic fibrosis (see, e.g. , Schwank et ah , Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell stem cell. 2013; 13: 653-658; and Wu et. al., Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell stem cell.
  • phenylketonuria e.g., phenylalanine to serine mutation at position 835 (mouse) or 240 (human) or a homologous residue in
  • phenylalanine hydroxylase gene see, e.g., McDonald et al., Genomics. 1997; 39:402-405; Bernard-Soulier syndrome (BSS) - e.g., phenylalanine to serine mutation at position 55 or a homologous residue, or cysteine to arginine at residue 24 or a homologous residue in the platelet membrane glycoprotein IX (T>C mutation) - see, e.g., Noris et al. , British Journal of Haematology. 1997; 97: 312-320, and Ali et al, Hematol.
  • BSS Bernard-Soulier syndrome
  • EHK epidermolytic hyperkeratosis
  • accession number P04264 in the UNIPROT database at www[dot]uniprot[dot]org; chronic obstructive pulmonary disease (COPD) - e.g., leucine to proline mutation at position 54 or 55 (if counting the initiator methionine) or a homologous residue in the processed form of ai-antitrypsin or residue 78 in the unprocessed form or a homologous residue (T>C mutation) - see, e.g., Poller et al., Genomics.
  • COPD chronic obstructive pulmonary disease
  • von Willebrand disease e.g., cysteine to arginine mutation at position 509 or a homologous residue in the processed form of von Willebrand factor, or at position 1272 or a homologous residue in the unprocessed form of von Willebrand factor (T>C mutation) - see, e.g., Lavergne et al., Br. J. Haematol.
  • hereditary renal amyloidosis e.g., stop codon to arginine mutation at position 78 or a homologous residue in the processed form of apolipoprotein All or at position 101 or a homologous residue in the unprocessed form (T>C mutation) - see, e.g., Yazaki et al., Kidney Int. 2003; 64: 11-16; dilated cardiomyopathy (DCM) - e.g., tryptophan to Arginine mutation at position 148 or a homologous residue in the FOXD4 gene (T>C mutation), see, e.g., Minoretti et. al., Int. J.
  • DCM dilated cardiomyopathy
  • hereditary lymphedema - e.g., histidine to arginine mutation at position 1035 or a homologous residue in VEGFR3 tyrosine kinase (A>G mutation), see, e.g., Irrthum et ah, Am. J. Hum. Genet. 2000; 67: 295-301; familial Alzheimer's disease - e.g., isoleucine to valine mutation at position 143 or a homologous residue in presenilinl (A>G mutation), see, e.g., Gallo et. al., J. Alzheimer' s disease.
  • Prion disease e.g., methionine to valine mutation at position 129 or a homologous residue in prion protein (A>G mutation) - see, e.g., Lewis et. al., J. of General Virology. 2006; 87: 2443-2449; chronic infantile neurologic cutaneous articular syndrome (CINCA) - e.g., Tyrosine to Cysteine mutation at position 570 or a homologous residue in cryopyrin (A>G mutation) - see, e.g., Fujisawa et. al. Blood.
  • CINCA chronic infantile neurologic cutaneous articular syndrome
  • DRM desmin-related myopathy
  • Suitable routes of administrating the composition for pain suppression include, without limitation: topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseus, periocular, intratumoral, intracerebral, parenteral, and intracerebroventricular administration.
  • compositions of this disclosure may be administered or packaged as a unit dose, for example.
  • unit dose when used in reference to a pharmaceutical composition of the present disclosure refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent, i.e., a carrier or vehicle.
  • Treatment of a disease or disorder includes delaying the development or progression of the disease, or reducing disease severity. Treating the disease does not necessarily require curative results.
  • “delaying" the development of a disease means to defer, hinder, slow, retard, stabilize, and/or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individuals being treated.
  • a method that "delays" or alleviates the development of a disease, or delays the onset of the disease is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and/or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.
  • “Development” or “progression” of a disease means initial manifestations and/or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset.
  • onset or "occurrence” of a disease includes initial onset and/or recurrence.
  • Conventional methods known to those of ordinary skill in the art of medicine, can be used to administer the isolated polypeptide or pharmaceutical composition to the subject, depending upon the type of disease to be treated or the site of the disease.
  • kits comprising nucleic acid vectors for the expression of the nucleobase editors described herein.
  • the kit further comprises appropriate guide nucleotide sequences (e.g. , gRNAs) or nucleic acid vectors for the expression of such guide nucleotide sequences, to target the Cas9 protein or nucleobase editor to the desired target sequence.
  • guide nucleotide sequences e.g. , gRNAs
  • the kit described herein may include one or more containers housing components for performing the methods described herein and optionally instructions for use. Any of the kit described herein may further comprise components needed for performing the assay methods.
  • Each component of the kits where applicable, may be provided in liquid form (e.g. , in solution) or in solid form, (e.g. , a dry powder). In certain cases, some of the components may be reconstitutable or otherwise processible (e.g. , to an active form), for example, by the addition of a suitable solvent or other species (for example, water), which may or may not be provided with the kit.
  • the kits may optionally include instructions and/or promotion for use of the components provided.
  • instructions can define a component of instruction and/or promotion, and typically involve written instructions on or associated with packaging of the disclosure. Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g. , videotape, DVD, etc.), Internet, and/or web-based communications, etc.
  • the written instructions may be in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which can also reflect approval by the agency of manufacture, use or sale for animal administration.
  • kits includes all methods of doing business including methods of education, hospital and other clinical instruction, scientific inquiry, drug discovery or development, academic research, pharmaceutical industry activity including pharmaceutical sales, and any advertising or other promotional activity including written, oral and electronic communication of any form, associated with the disclosure. Additionally, the kits may include other components depending on the specific application, as described herein.
  • kits may contain any one or more of the components described herein in one or more containers.
  • the components may be prepared sterilely, packaged in a syringe and shipped refrigerated. Alternatively it may be housed in a vial or other container for storage. A second container may have other components prepared sterilely.
  • the kits may include the active agents premixed and shipped in a vial, tube, or other container.
  • kits may have a variety of forms, such as a blister pouch, a shrink wrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, or a similar pouch or tray form, with the accessories loosely packed within the pouch, one or more tubes, containers, a box or a bag.
  • the kits may be sterilized after the accessories are added, thereby allowing the individual accessories in the container to be otherwise unwrapped.
  • the kits can be sterilized using any appropriate sterilization techniques, such as radiation sterilization, heat sterilization, or other sterilization methods known in the art.
  • kits may also include other components, depending on the specific application, for example, containers, cell media, salts, buffers, reagents, syringes, needles, a fabric, such as gauze, for applying or removing a disinfecting agent, disposable gloves, a support for the agents prior to administration, etc.
  • a fabric such as gauze, for applying or removing a disinfecting agent, disposable gloves, a support for the agents prior to administration, etc.
  • Cells that may contain any of the compositions described herein include prokaryotic cells and eukaryotic cells.
  • the methods described herein are used to deliver a Cas9 protein or a nucleobase editor into a eukaryotic cell ⁇ e.g., a mammalian cell, such as a human cell).
  • the cell is in vitro ⁇ e.g., cultured cell.
  • the cell is in vivo ⁇ e.g., in a subject such as a human subject).
  • the cell is ex vivo ⁇ e.g., isolated from a subject and may be administered back to the same or a different subject).
  • Mammalian cells of the present disclosure include human cells, primate cells ⁇ e.g., vero cells), rat cells ⁇ e.g., GH3 cells, OC23 cells) or mouse cells ⁇ e.g., MC3T3 cells).
  • human cell lines including, without limitation, human embryonic kidney (HEK) cells, HeLa cells, cancer cells from the National Cancer Institute's 60 cancer cell lines (NCI60), DU145 (prostate cancer) cells, Lncap (prostate cancer) cells, MCF-7 (breast cancer) cells, MDA- MB-438 (breast cancer) cells, PC3 (prostate cancer) cells, T47D (breast cancer) cells, THP-1 (acute myeloid leukemia) cells, U87 (glioblastoma) cells, SHSY5Y human neuroblastoma cells (cloned from a myeloma) and Saos-2 (bone cancer) cells.
  • HEK human embryonic kidney
  • HeLa cells cancer cells from the National Cancer Institute's 60 cancer cell lines (NCI60)
  • DU145 (prostate cancer) cells Lncap (prostate cancer) cells
  • MCF-7 breast cancer
  • MDA- MB-438 breast cancer
  • PC3 prostate cancer
  • rAAV vectors are delivered into human embryonic kidney (HEK) cells ⁇ e.g., HEK 293 or HEK 293T cells).
  • rAAV vectors are delivered into stem cells ⁇ e.g., human stem cells) such as, for example, pluripotent stem cells ⁇ e.g., human pluripotent stem cells including human induced pluripotent stem cells (hiPSCs)).
  • stem cell refers to a cell with the ability to divide for indefinite periods in culture and to give rise to specialized cells.
  • a pluripotent stem cell refers to a type of stem cell that is capable of differentiating into all tissues of an organism, but not alone capable of sustaining full organismal development.
  • a human induced pluripotent stem cell refers to a somatic ⁇ e.g., mature or adult) cell that has been reprogrammed to an embryonic stem cell-like state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells ⁇ see, e.g., Takahashi and Yamanaka, Cell 126 (4): 663-76, 2006, incorporated by reference herein).
  • Human induced pluripotent stem cell cells express stem cell markers and are capable of generating cells characteristic of all three germ layers (ectoderm, endoderm, mesoderm).
  • Non-limiting examples of suitable Cas9 proteins and variants, and nucleobase editors and variants are provided.
  • the disclosure provides Cas9 variants, for example, Cas9 proteins from one or more organisms, which may comprise one or more mutations ⁇ e.g., to generate dCas9 or Cas9 nickase).
  • one or more of the amino acid residues, identified below by an asterisk, of a Cas9 protein may be mutated.
  • the D10 and/or H840 residues of the amino acid sequence provided in SEQ ID NO: 1, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 2-275 and 394-397 are mutated.
  • the D10 residue of the amino acid sequence provided in SEQ ID NO: 1, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 2-275 and 394-397 is mutated to any amino acid residue, except for D.
  • the D10 residue of the amino acid sequence provided in SEQ ID NO: 1, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 2- 275 and 394-397 is mutated to an A.
  • the H840 residue of the amino acid sequence provided in SEQ ID NO: 1, or a corresponding residue in any of the amino acid sequences provided in SEQ ID NOs: 2-275 and 394-397 is an H.
  • the H840 residue of the amino acid sequence provided in SEQ ID NO: 1, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 2-275 and 394-397 is mutated to any amino acid residue, except for H.
  • the H840 residue of the amino acid sequence provided in SEQ ID NO: 1, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 2-275 and 394-397 is mutated to an A.
  • the D10 residue of the amino acid sequence provided in SEQ ID NO: 1, or a corresponding residue in any of the amino acid sequences provided in SEQ ID NOs: 2-275 and 394-397 is a D.
  • a number of Cas9 sequences from various species were aligned to determine whether corresponding homologous amino acid residues of D10 and H840 of SEQ ID NO: 1 can be identified in other Cas9 proteins, allowing the generation of Cas9 variants with corresponding mutations of the homologous amino acid residues.
  • the alignment was carried out using the NCBI Constraint-based Multiple Alignment Tool (COBALT (accessible at st- va.ncbi.nlm.nih.gov/tools/cobalt)), with the following parameters. Alignment parameters: Gap penalties -11,-1; End-Gap penalties -5,-1.
  • CDD Parameters Use RPS BLAST on; Blast E-value 0.003; Find conserveed columns and Recompute on.
  • Query Clustering Parameters Use query clusters on; Word Size 4; Max cluster distance 0.8; Alphabet Regular.
  • VQR-nCas9 (D10A/D1135V/R1335Q/T1337R) S. pyogenes Cas9 Nickase
  • GLYETRIDLAKLGEG (SEQ ID NO: 9)
  • NMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG (SEQ ID NO: 10)
  • Francisella novicida Cpfl D917A (A917, E1006, and D1255 are bolded and underlined)
  • Francisella novicida Cpfl E1006A (D917, A1006, and D1255 are bolded and underlined)
  • Francisella novicida Cpfl D1255A (D917, E1006, and A1255 are bolded and underlined)
  • Francisella novicida Cpfl D917A/D1255A (A917, E1006, and A1255 are bolded and underlined)
  • Francisella novicida Cpfl E1006A/D1255A (D917, A1006, and A1255 are bolded and underlined)

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EP17794468.3A EP3526320A1 (en) 2016-10-14 2017-10-13 Aav delivery of nucleobase editors
JP2019520398A JP7588390B2 (ja) 2016-10-14 2017-10-13 核酸塩基エディターのａａｖ送達
KR1020197013648A KR102622411B1 (ko) 2016-10-14 2017-10-13 핵염기 에디터의 aav 전달
SG11201903089RA SG11201903089RA (en) 2016-10-14 2017-10-13 Aav delivery of nucleobase editors
AU2017342543A AU2017342543B2 (en) 2016-10-14 2017-10-13 AAV delivery of nucleobase editors
KR1020247000273A KR20240007715A (ko) 2016-10-14 2017-10-13 핵염기 에디터의 aav 전달
CN201780076474.6A CN110214180A (zh) 2016-10-14 2017-10-13 核碱基编辑器的aav递送
CA3039928A CA3039928A1 (en) 2016-10-14 2017-10-13 Aav delivery of nucleobase editors
IL265900A IL265900A (en) 2016-10-14 2019-04-08 Delivery of nuclear base editors using an adeno-associated virus
JP2022163015A JP2023011629A (ja) 2016-10-14 2022-10-11 核酸塩基エディターのａａｖ送達
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