Novel zinc finger fusion proteins for nucleobase editing

Abstract

Provided herein are base editor systems comprising fusion proteins comprising zinc finger proteins and cytidine deaminase domains, and methods of using the base editor systems, which can be used to specifically modify single base pairs in a target DNA sequence.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 292,817, filed December 22, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a sequence listing that has been submitted electronically in XML format. The sequence listing is incorporated herein by reference in its entirety. The XML copy created on November 13, 2022 is named 025297WO041.xml and is 165,097 bytes in size. [Background technology]

[0003] Single-base high-precision DNA editing has a variety of applications in the treatment and understanding of disorders, such as genetic diseases. For example, knockout of one or more genes can be achieved by converting normal codons to stop codons, or by mutating splice acceptor sites to introduce exon skipping and / or frameshift mutations. Furthermore, DNA point mutations are associated with a wide range of disorders. Single-base editing can be used to correct harmful mutations or to introduce beneficial genetic modifications.

[0004] Cytidine deaminases convert the nucleobase cytosine to thymine (or the nucleoside deoxycytidine to thymidine). These enzymes function in the pyrimidine salvage pathway, acting primarily on single-stranded DNA to convert cytosine to uracil, which is subsequently replaced by thymine bases during DNA replication or repair. A cytidine deaminase identified in the bacterium Burkholderia cenocepacia, DddA, can catalyze the deamination of cytosine to uracil in double-stranded DNA. Thus, DddA avoids the need to unwind dsDNA to ssDNA (Mok et al., Nature (2020) 583: 631-7). Mok's study reports C to T base editing at the human CCR5 locus using a DddA-derived cytosine base editor fused to a transcription activator-like effector (TALE) protein, but it is unclear how broadly applicable this approach is. Furthermore, the new deaminases acting on double-stranded DNA may have improved or modified base editing activity compared to DddA.

[0005] Thus, there is a continuing need to develop high-precision base editing systems for the prevention and treatment of many diseases. Summary of the Invention

[0006] The present disclosure provides a zinc finger protein (ZFP)-based nucleobase editing system and uses thereof. In one aspect, the present disclosure provides a system for changing a cytosine to a thymine in the genome of a cell (e.g., a eukaryotic or prokaryotic cell, where the eukaryotic cell can be a mammalian cell, such as a human cell, or a plant cell), comprising a first fusion protein and a second fusion protein, or a first and a second expression construct for expressing the first and second fusion proteins, respectively, wherein a) the first fusion protein comprises i) a first zinc finger protein (ZFP) domain that binds to a first sequence in a target genomic region in the cell, and ii) a first portion of a cytidine deaminase polypeptide (e.g., the cytidine deaminase is represented by SEQ ID NO:1). a) a toxin-derived deaminase (TDD) comprising an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 13-24; b) a second fusion protein comprising i) a second ZFP domain that binds to a second sequence in the target genomic region, and ii) a second portion of a cytidine deaminase polypeptide; c) upon binding of the first fusion protein and the second fusion protein to the target genomic region, the first and second portions dimerize, and the dimerized portions form an active cytidine deaminase capable of changing a cytosine to a uracil in the target genomic region. In some embodiments, the first and second portions lack cytidine deaminase activity by themselves. In some embodiments, the first and second portions form an active cytidine deaminase comprising an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 13-24. In some embodiments, the first and second portions form an active cytidine deaminase comprising any one of the amino acid sequences of SEQ ID NOs: 13-24. In some embodiments, the target genomic region can be specific to a particular allele of a gene in a cell. In some embodiments, the targeted cytosine can be between the proximal end of the first sequence and the proximal end of the second sequence in the target genomic region, optionally with the proximal ends spaced no more than 100 bp apart.

[0007] Also provided are multiplex versions of the base editor system comprising two or more pairs of first and second fusion proteins, each pair of fusion proteins binding to a different target genomic region, and optionally the first and second cytidine deaminase moieties of one pair of fusion proteins differ from the first and second moieties of another pair of fusion proteins.

[0008] In some embodiments, the base editor system further comprises a nickase that induces a single-stranded DNA break in the unedited or edited strand, the DNA break being about 500 bp or less, optionally 200 bp or less, optionally about 10-50 bp from the cytosine to be edited. The nickase may be, for example, a ZFP-based nickase, a TALE-based nickase, or a CRISPR-based nickase. In some embodiments, the nickase is a ZFP-based nickase formed by dimerization of a first nickase domain and a second nickase domain, each fused to two ZFP domains that bind to the target genomic region, one of the nickase domains comprising an inactivating mutation. In certain embodiments, one of the nickase domains is fused to a first or second ZFP-cytidine deaminase fusion protein, and the other nickase domain is fused to a third ZFP domain that binds to a third sequence in the target genomic region. Alternatively, the two nickase domains may be fused to a third ZFP domain that binds to a third sequence in the target genomic region and a fourth ZFP domain that binds to a fourth sequence in the target genomic region, respectively. In certain embodiments, the first and second nickase domains are derived from FokI (e.g., FokI-ELD and FokI-KKR, optionally with an inactivating mutation of D450N).

[0009] In some embodiments, the base editor system further comprises an inhibitory component of a cytidine deaminase, e.g., a toxin-derived deaminase inhibitor (TDDI), where the cytidine deaminase is a TDD. In certain embodiments, the system comprises a third fusion protein or a third expression construct for expressing a third fusion protein in a cell, the third fusion protein comprising i) a ZFP domain that binds to a third sequence in the target genomic region, and ii) an inhibitory domain of a cytidine deaminase, wherein upon binding of the third fusion protein to the target genomic region, the inhibitory domain interacts with the dimerized cytidine deaminase moiety, thereby inhibiting the cytidine deaminase activity of the dimerized cytidine deaminase moiety.

[0010] In some embodiments of a base editor system containing an inhibitory domain, the system comprises a third fusion protein or a third expression construct for expressing the third fusion protein in a cell, and a fourth fusion protein or a fourth expression construct for expressing the fourth fusion protein in a cell, where the third fusion protein comprises i) a ZFP domain that binds to a third sequence in the target genomic region, and ii) a first dimerization domain; the fourth fusion protein comprises i) an inhibitory domain of a cytidine deaminase, and ii) a second dimerization domain capable of pairing with the first dimerization domain in the presence of a dimerization-inducing agent; when the third fusion protein binds to the target genomic region and the third and fourth fusion proteins dimerize, the inhibitory domain binds to the dimerized cytidine deaminase moiety, thereby inhibiting cytidine deaminase activity of the dimerized cytidine deaminase moiety.

[0011] In some embodiments of a base editor system containing an inhibitory domain, the system comprises a third fusion protein or a third expression construct for expressing the third fusion protein in a cell, and a fourth fusion protein or a fourth expression construct for expressing the fourth fusion protein in a cell, where the third fusion protein comprises i) a ZFP domain that binds to a third sequence in the target genomic region, and ii) a first dimerization domain; the fourth fusion protein comprises i) an inhibitory domain of a cytidine deaminase, and ii) a second dimerization domain capable of pairing with the first dimerization domain in the absence of a dimerization inhibitor; when the third fusion protein binds to the target genomic region and the third and fourth fusion proteins dimerize, the inhibitory domain binds to the dimerized cytidine deaminase moiety, thereby inhibiting cytidine deaminase activity of the dimerized cytidine deaminase moiety.

[0012] In certain embodiments, the base editor systems described herein comprise both a nickase component and an inhibitory domain component described herein.

[0013] Any ZFP domain used in the fusion proteins described herein can independently have 2, 3, 4, 5, 6, 7, or 8 zinc fingers.

[0014] In some embodiments, the protein components of the base editor system are provided to the cell by expression cassettes or constructs. Such cassettes or constructs may be provided to the cell on the same or separate expression vectors, such as viral vectors. The viral vector may be, for example, an adeno-associated viral (AAV) vector, an adenoviral vector, or a lentiviral vector.

[0015] In some embodiments of the base editor system described herein, the cytidine deaminase is a TDD. In certain embodiments, the TDD comprises an amino acid sequence at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1-12, or a toxic domain of a TDD comprising said sequence. In certain embodiments, the TDD comprises an amino acid sequence at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 13-24. In a particular embodiment, the TDD comprises the amino acid sequence of any one of SEQ ID NOs: 13-24.

[0016] In some embodiments, the first and second cytidine deaminase portions comprise SEQ ID NOs:25 and 26, SEQ ID NOs:27 and 28, SEQ ID NOs:29 and 30, SEQ ID NOs:31 and 32, SEQ ID NOs:33 and 34, SEQ ID NOs:35 and 36, SEQ ID NOs:37 and 38, SEQ ID NOs:39 and 40, SEQ ID NOs:41 and 42, SEQ ID NOs:43 and 44, SEQ ID NOs:45 and 46, SEQ ID NOs:47 and 48, SEQ ID NOs:49 and 50, SEQ ID NOs:51 and 52, SEQ ID NOs:53 and 54, SEQ ID NOs:55 and 56, SEQ ID NOs:57 and 58, SEQ ID NOs:59 and 60, SEQ ID NOs:61 and 62, SEQ ID NOs:63 and 64, SEQ ID NOs:65 and 66, SEQ ID NOs:67 and 68, SEQ ID NOs:69 and 70, SEQ ID NOs:71 and 72, SEQ ID NOs:73 and 74, SEQ ID NOs:75 and 76, SEQ ID NOs:77 and 78, SEQ ID NOs:79 and 80, SEQ ID NOs:81 and 82, or SEQ ID NOs:83 and 84, respectively, or vice versa.

[0017] In a related aspect, the disclosure also provides a fusion protein comprising: i) a zinc finger protein (ZFP) domain that binds to a gene (which may be a eukaryotic gene, e.g., a human gene); and ii) a cytidine deaminase polypeptide or fragment thereof (e.g., a half-domain), e.g., the cytidine deaminase is a TDD comprising an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 13-24, and optionally, the ZFP domain and the cytidine deaminase or fragment thereof are linked by a peptide linker (e.g., comprising an amino acid sequence of any one of SEQ ID NOs: 85-95). In some embodiments, the TDD comprises the amino acid sequence of any one of SEQ ID NOs: 13-24.

[0018] In a related aspect, the disclosure provides a fusion protein comprising i) a zinc finger protein (ZFP) domain that binds to a gene (which may be a eukaryotic gene, e.g., a human gene), and ii) a cytidine deaminase inhibitory domain, e.g., the cytidine deaminase is a TDD comprising an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 13-24, and optionally the ZFP domain and the inhibitory domain are linked by a peptide linker (e.g., comprising an amino acid sequence of any one of SEQ ID NOs: 85-95). In some embodiments, when the cytidine deaminase is a TDD, the cytidine deaminase inhibitory domain is a TDDI. In some embodiments, the TDD comprises an amino acid sequence of any one of SEQ ID NOs: 13-24.

[0019] In a related aspect, the disclosure provides a fusion protein comprising: i) a zinc finger protein (ZFP) domain that binds to a gene (which may be a eukaryotic gene, e.g., a human gene); and ii) a nickase (e.g., a nickase domain described herein) or a fragment thereof, optionally wherein the ZFP domain and the nickase or fragment thereof are linked by a peptide linker (e.g., comprising the amino acid sequence of any one of SEQ ID NOs: 85-95).

[0020] In one aspect, the disclosure provides a pair of fusion proteins comprising: a) a first fusion protein comprising i) a zinc finger protein (ZFP) domain that binds to a gene (which may be a eukaryotic gene, e.g., a human gene), and a first dimerization domain; and b) a second fusion protein comprising i) a cytidine deaminase inhibitory domain, e.g., a cytidine deaminase is a TDD comprising an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 13-24, and ii) a second dimerization domain, wherein the first and second dimerization domains are capable of dimerizing in the presence of a dimerization inducer. In some embodiments, when the cytidine deaminase is a TDD, the cytidine deaminase inhibitory domain is a TDDI. In certain embodiments, the TDD comprises the amino acid sequence of any one of SEQ ID NOs: 13-24.

[0021] In another aspect, the disclosure provides a pair of fusion proteins comprising: a) a first fusion protein comprising i) a zinc finger protein (ZFP) domain that binds to a gene (which may be a eukaryotic gene, e.g., a human gene), and a first dimerization domain; and b) a second fusion protein comprising i) a cytidine deaminase inhibitory domain, e.g., a cytidine deaminase is a TDD comprising an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 13-24, and ii) a second dimerization domain, wherein the first and second dimerization domains are capable of dimerizing in the absence of a dimerization inhibitor. In some embodiments, when the cytidine deaminase is a TDD, the cytidine deaminase inhibitory domain is a TDDI. In certain embodiments, the TDD comprises the amino acid sequence of any one of SEQ ID NOs: 13-24.

[0022] In one aspect, the present disclosure provides one or more nucleic acid molecules encoding the fusion protein(s) described herein, as well as expression constructs comprising the nucleic acid molecule(s), and viral vectors comprising the expression construct(s), optionally the viral vector can be an adeno-associated viral vector, an adenoviral vector, or a lentiviral vector. Also provided is a cell (which can be a eukaryotic cell, e.g., a mammalian cell or a plant cell) comprising the base editor system described herein, the fusion protein(s) described herein, the isolated nucleic acid molecule(s) described herein, the expression construct(s) described herein, or the viral vector(s) described herein. In some embodiments, the mammalian cell is a human cell, e.g., a human embryonic stem cell or a human induced pluripotent stem cell.

[0023] In some aspects, the present disclosure provides a method of changing a cytosine to a thymine in a target genomic region in a cell (which may be a eukaryotic cell, e.g., a mammalian or plant cell), comprising delivering a base editor system described herein to the cell. In some embodiments, changing a cytosine to a thymine results in a stop codon in the target genomic region. A multiplex format of the system may target two or more genomic regions (e.g., 2, 3, 4, or 5 genomic regions). Editing may be performed in vivo, ex vivo, or in vitro.

[0024] Also provided are genetically engineered cells (which may be eukaryotic cells, e.g., mammalian cells such as human iPSCs or plant cells) obtained by the editing methods.

[0025] The engineered cells (e.g., engineered human cells) described herein (including pharmaceutical compositions comprising the cells and a pharma- ceutically acceptable carrier) may be used to treat a patient (e.g., a human patient in need thereof) or may be used in the manufacture of a medicament for treating a patient in need thereof. In some embodiments, the patient has cancer, an autoimmune disorder, an autosomal dominant disease, or a mitochondrial disorder. In some embodiments, the patient has sickle cell disease, hemophilia, cystic fibrosis, phenylketonuria, Tay-Sachs disease, prion disease, color blindness, a lysosomal storage disease, Friedreich's ataxia, or prostate cancer. Kits and articles of manufacture comprising the cells are also contemplated.

[0026] Other features, objects, and advantages of the present invention will be apparent in the following detailed description. It should be understood, however, that the detailed description, while indicating embodiments and aspects of the present invention, is given by way of example only, not by way of limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the detailed description. [Brief description of the drawings]

[0027] [Figure 1]Schematic diagram showing a pair of ZFP-TDD fusion proteins for C to T base editing. The rectangle represents the DNA-binding zinc finger in the ZFP domain of the fusion protein. The arrow shape above the underlined C nucleotide represents the dimerized TDD domain of the fusion protein. The black line between the zinc finger domain and the TDD domain represents a peptide linker. [Diagram 2] Schematic diagram showing ZFP design for ZFP-TDD fusion protein pair targeting CIITA. G2, G5, C6, C8, G10, G11, G14, C15, and C16 are target nucleotides for base editing. Top strand (left to right): SEQ ID NO: 114. Bottom strand (right to left): SEQ ID NO: 115. [Diagram 3] FIG. 1 is a schematic showing an example of the combined use of a ZFP-TDD base editing system with a CRISPR / Cas-based nickase system. [Figure 4] FIG. 1 is a schematic showing an example of a trimeric ZFP-TDD+FokI nickase base editing system. [Diagram 5] Schematic diagram showing ZFP designs for use in combination with ZFP-nickases in ZFP-TDD fusion proteins targeting CIITA. Top nucleotide strand (left to right): SEQ ID NO: 116. Bottom nucleotide strand (right to left): SEQ ID NO: 117. Middle amino acid strand (left to right): SEQ ID NO: 118. [Figure 6] FIG. 1 is a table showing heat map results of C to T base editing at the human CIITA locus ("site 2") by a series of ZFP-TDD fusion protein pairs. The degree of editing activity corresponds to the intensity of the shading in the cell. Each s2_left_6_ZFP construct is fused to FokI-ELD-D450N. "Yes" in the column titled "Nickase" indicates co-delivery of FokI-KKR. [Figure 7] FIG. 1 is a schematic showing the design for inhibition of TDD using targeted ZFP-TDDI. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] The present disclosure provides systems and methods for base editing, e.g., from cytosine (C) to thymine (T), in cellular DNA, such as genomic DNA. The systems involve the use of ZFP-toxin derived deaminase (TDD) fusion proteins (ZFP-TDD). By providing high-precision gene editing in a cellular context, the systems and methods can be used to prevent and / or treat many diseases. These systems and methods are believed to be particularly useful for cell-based therapies that require the simultaneous knockout of multiple human genes.

[0029] The systems and methods can convert a targeted C:G base pair to a T:A base pair. In some embodiments, the base editing system may also include a protein (e.g., UGI) that increases the stability of the conversion and / or an endonuclease that nicks DNA near the targeted base to stimulate DNA repair in the edited region and promote the correction of the G nucleotide on the opposite strand to an A to form the edited T:A base pair.

[0030] The present system and method is advantageous in part due to the compact size of the ZFP domain in the fusion protein. In comparison, the large physical size of TALEs and the long C-terminal TALE linker can limit how small the base editing window can be, as well as the design density. In addition, the size and highly repetitive nature of engineered TALEs make it difficult to deliver TALE-based base editors to human cells using common viral vectors. The present ZFP-derived base editing system avoids these issues. For example, the compactness of these ZFP-derived systems can allow for packaging within a single AAV vector, as opposed to TALE base editor systems (e.g., TALE-TDD) or CRISPR / Cas base editor systems. In addition, the small size of the fusion proteins herein allows the editing system to include a nickase to generate a DNA nick near the edited base, thereby facilitating DNA repair mechanisms to change the base opposite the edited C from G to the corresponding A, allowing the correct T:A base pair to be formed. The inclusion of a nickase can significantly increase the base editing efficiency.

[0031] I. Zinc Finger Fusion Proteins Provided are fusion proteins that contain a DNA-binding zinc finger protein (ZFP) domain, a cytidine deaminase inhibitor (e.g., TDDI) domain, and / or a nickase domain (e.g., FokI domain) fused to a base editor domain (e.g., a cytidine deaminase domain, which can be a TDD such as those described herein) or a fragment thereof. As used herein, "fusion protein" refers to a polypeptide in which heterologous functional domains (i.e., functional domains that do not naturally occur in the same protein in nature) are covalently linked (e.g., via a peptidyl bond). These fusion proteins, which can be recombinantly produced, are components of the present base editor system. In some embodiments, the ZFP fusion proteins herein comprise a cytidine deaminase domain (e.g., from a TDD described herein) and additionally a nickase domain and / or a UGI domain.

[0032] Other formats of the system are also contemplated herein. For example, instead of peptidyl linkage, the two functional domains may be linked together by non-covalent bonds. In some embodiments, the two functional domains (e.g., ZFP domain and cytidine deaminase inhibitor domain; or ZFP domain and nickase domain) are each fused to a dimerization partner (e.g., leucine zipper and further described herein), whereby the two functional domains are linked together through the interaction of the dimerization partners. In certain embodiments, the dimerization of these domains can be controlled by the presence or absence of a specific agent (e.g., a small molecule or peptide). It is believed that such formats can be substituted for fusion proteins in any aspect of the invention.

[0033] Each component of the present base editor system is described in further detail below.

[0034] A. Base Editor The ZFP-cytidine deaminase fusion protein of the present disclosure comprises a cytidine deaminase domain or a fragment thereof in addition to the ZFP domain. The term "deaminase" or "deaminase domain" as used herein refers to a protein that catalyzes the deamination reaction of a nucleoside (e.g., cytidine, adenosine, deoxycytidine, or deoxyadenosine) in the context of free base, RNA, or DNA. For example, the cytidine deaminase domain can catalyze the deamination of cytosine to uracil, which is replaced by a thymine base during DNA replication or repair. The deaminase domain can be naturally occurring or engineered. In some embodiments, the cytidine deaminase of the present disclosure acts on double-stranded DNA.

[0035] In some embodiments, cytidine deaminase is derived from a toxin, which may be derived from, for example, prokaryotic or eukaryotic organisms. In certain embodiments, the organism may be bacteria or fungi. Such cytidine deaminase is referred to herein as toxin-derived deaminase (TDD). As used herein, cytidine deaminase "derived from" toxin may refer to the cytidine deaminase that is the same as the naturally occurring toxin, or to the cytidine deaminase that is a modified version of the toxin that retains deaminase activity.

[0036] In certain embodiments, the TDD may comprise an amino acid sequence selected from, for example, SEQ ID NO:1 ("TDD20"), SEQ ID NO:2 ("TDD21"), SEQ ID NO:3 ("TDD22"), SEQ ID NO:4 ("TDD23"), SEQ ID NO:5 ("TDD24"), SEQ ID NO:6 ("TDD25"), SEQ ID NO:7 ("TDD26"), SEQ ID NO:8 ("TDD27"), SEQ ID NO:9 ("TDD28"), SEQ ID NO:10 ("TDD29"), SEQ ID NO:11 ("TDD30"), and SEQ ID NO:12 ("TDD31"), or a portion of the foregoing amino acid sequences capable of cytidine deaminase activity (e.g., a "toxic domain"). These amino acid sequences are shown below: TDD20 (NCBI accession number WP_212528010.1) TDD21 (NCBI accession number WP_166233360.1) MAAPGACTRTRLVTRSNSPCPPRAQASAGRGLAAPGTGAVASQSRGRRPVMDQTPPEARYVAAGHIEGKAALQVRAAASSGGTIHHNNPFGTCIYCQQQVPTLLPDGARLTAEPPAQARPRSAWWSPPGPVTMVGNVADPASHPVATAQ (SEQ ID NO: 2) TDD22 (NCBI accession number MBK7217240.1) MKHGRDSWYPIASDADLLVKTLLFASVADASFDKASDGEEYLGHLPTRTRTRDKTHGLLVLDDEPPIPVVSGQNGPAATIPRGSRGFDAITQTHVEGHVAALMVNHGWTRATLYINNVPCPSCDRLLPRMLPPGARLNVIGPNGFQKLYVGGRW (SEQ ID NO: 3) TDD23 (NCBI accession number RTL35118.1) MPAEVTEKSQPAPPAQAESGMSQAAQSAWAPGSNYFRLGAVCNDTFNQSLSTQYDGLPQCAIAGIGEGLKKLSAIGEVGVGVAADVASVVVADLQIGYEAITYPSLAEGNSDRLAHKLRELQTKYEPLTQSVKQGYEGVQHDLNDIGAQSNNDSAAYARLVTDPAQSIVKAIDKFTGQTPAEQVKDIFDQGTQIAVAAALGKAAALASGSCRLEEIAAKFDALFSKMEETKQAFGTQLEHCIASGKKVQSSDYIRLSKAGDQDGLSRPADDKISESCNSVDRSYFERLPQRMRDSDKTAGILVADGQELDVLSGRKGPAQSMPAQSRGFDLITKTHAEGHAIAHMIQHGIKEAKIYINNNEICSSCEKLIPRMLPPGSKLEVHMPSGAVRNFVGEPR (SEQ ID NO: 4) TDD24 (NCBI accession number WP_216577045.1) RGSVRAVTDQTGTVTDRYSYSLYGERTHDTGDGDHIFGYCGRDGVITERDGLLYMRARYYSPELKRFISADSVIGNLSSTPSLNRYAYVNGNPVTQIDPFGLSAEPGTKKAKKNINWREAVHDALDIAGSITGYGDICDAVNAVLYLTEGDYINAGLSALSIIPGADAFTKTGKASKVLGNGIVESGLK HLDNVADGIKKLVKKADDAIDLEKHLPKKLLTEEAFAKKADKVADDVVEDAAKAIKGGSSSINLPEYDGKTTHGVLVLDDGTQVPFSSGNANPNYKNYIPASHVEGKSAIYMRENGINNGTVFHNNTDGTCPYCDKMLPTLLEEGSTLTVVPPANANAPKPSWVDTVKTYIGNDKIPKKPK (SEQ ID NO: 5) TDD25 (NCBI accession no. CP070896.1; bp 1412377-1412931) FFYLKTAQRRAIIFLIEYENSPFVAYLSTPPEGSGQKNNGNTTTPDFYVSPDGHPIPRAAYNLPEYDGSTTHGVLITSDGSEISFTSGGGDPRYTNYANNGHVEQKSSLYMRDNNISSGVVYHNNTEGTCGFCNTMTSTFLPEGATLTVVPPSNAVATNSKAVDVPKTYTGNSKDPKISSRYTGN (SEQ ID NO: 6) TDD26 (NCBI accession number MBV9870847.1) TDD27 MQGFVNAGELAIGDELLDSSGNILFVEKFDVELTEKPTKVYNFQVEDFHTYYVGENGVWVHNASSEYGEVPELPEFNGKKTEGVFRTADGKEIKFESGGSTEYKNPSASHAEGKAAIYMRENGIKEGTVFHNNPNGTCNYCDKGLATLLPEGARLTVVPPIGAVAPNKYWVDVPKTYTGNGNLPSMK (SEQ ID NO: 8) TDD28 MKKRLISFMLLITVISCVGFNTDYFKNLLTLISQAAKEVIYEETESYDEETSDSAVDRLEQELEKLDVRNFFEDESETESGSSSINLPEYDGKTTHGVLVLDDGTQVPFSSGNANPNYKNYIPASHVEGKSAIYMRENGINNGTVFHNNTDGTCPYCDKMLPTLLDEGSTLTVVPPTNASAPKPSWVDTVKTYIGNDKIPKKPK (SEQ ID NO: 9) TDD29 MRKKILKFILIQLMLVSFIFTNCGSMLKEMCTFAEQISSNEENTDKASIISCGDFNSDGKINVFDLMRSKQHVLNKEEQPSGLADINADGAFNILDSLILQDYILLRDTKIKDKVNVPPSDNIPDVSSDDSYRYVIFSEDKETPIQINSQLTTTNGKIATNNNFEGNRSINGDGKIIENANISMVYMDDKIVNTYFSNPEEYYEFVIENDDEANKIIDTPTISYDNILIEKSVTINSCMMAMNDIDIKSNTDNTGNTVIYSKYGDINIDCSNINLVGLIYAPFGTVKLSAQNVQIDGIIIAKNVIIDSEQKINLNPNNSIADFIGVDTEKMVIPYRDWCYIDKPDDAVLPEIVEEQIYEDPLSVDDAWLEDKYGSLIVDEYSAQMKKELVECLYICISERISNLSELIELFSFAAQGNAKDYVKLKTSLKNGKKDVSLTIGDYDISVEKEKSDKVIYGPMPYNPFVFRYYLTPNKTSDDSDNTDEPQEEPKKFKINDIGLFFELAAAGVSTAVVNVATTAGTAGTAVIGAGTAVIGAGTAAAATAINTVTTTVTKITTKAEQRIEQAVQNGKDSIVNIKNEVETSIKDTLSDIADVDLFGMQYVPLTNGFGYNIGSVRLNFIKEVGSWTSQTILNGEPINIDKIKTVVDLDTLNNINSISPQIYKDIASNIMNDKDLEKTYKIIDCINEKTDEFKLAYEQLKDIEQAYEYANIDKDKIDIPDFDGKITWGILVLEDGTCITFSSGNANPMFNHYIPASHAEGKAAIYMRQKGIKHGVIFHNNTDGTCPYCNTMLPTLLEENSTLIVVPPINAVAKKRGWIDKIKIYTGNNKIPKTN(SEQ ID NO: 10) TDD30 (NCBI Accession No. MBR1615955.1) MGNYESDKSRLTPYLFIVEVDGIQTMRFQKCEGLEAETEVFEYQEGGGTVHNFKGRSRFPNIVLEKGIVDSDDLWKWYNKTAEGNVERQSGSVVLCNLQGEEIKRWNFFRAFPCRWVGPALNAMDRRTFAVERIEIAHEWLEVDGGDEEEENLTSNNEKKKNANKQKIELPPYDGKTTYGVLILDDGKQYSFNSGKPAPIYRNYIPASHVEGKAAIYMRENKIQSGTVYHNNTDGTCPYCDKMLPTLLEKDSTLKVVPPQNATSSKKGWITNEKIYIGNDKIPKTAR (SEQ ID NO: 11) TDD31 DMEAGLGESGRAHIALPEYDGGTTHGVLVLDDGTQIPLSSGKGDPPGTNYAASGHVEGKAALIMRENGITSATLYINNPNGTCGYCDSQLPTLLPEGATLTVVPPANAVAPKSRWIDYPKTYIGNSNDPKPWPRYKGN (SEQ ID NO: 12) In some embodiments, the sequence, if present, does not include a signal sequence.

[0037] In some embodiments, the cytidine deaminase may include a toxic domain of TDD. Examples of toxic domains for TDD20-31 are, for example, SEQ ID NO:13 (TDD20), SEQ ID NO:14 (TDD21), SEQ ID NO:15 (TDD22), SEQ ID NO:16 (TDD23), SEQ ID NO:17 (TDD24), SEQ ID NO:18 (TDD25), SEQ ID NO:19 (TDD26), SEQ ID NO:20 (TDD27), SEQ ID NO:21 (TDD28), SEQ ID NO:22 (TDD29), SEQ ID NO:23 (TDD30), and SEQ ID NO:24 (TDD31), as shown in Table 3. As used herein, unless otherwise specified, the term "TDD" refers to the TDD toxic domain.

[0038] In certain embodiments, the cytidine deaminase domain (e.g., from a TDD described herein) is a "split enzyme" consisting of a first and a second "half domain" or "split entity" that lacks cytidine deaminase activity alone but dimerizes to form an active cytidine deaminase. As used herein, an "inactive" or "lacking cytidine deaminase activity" half-domain can be a half-domain that i) lacks any cytidine deaminase activity (e.g., any detectable cytidine deaminase activity), ii) lacks specific cytidine deaminase activity, or iii) lacks significant cytidine deaminase activity (i.e., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or more of on-target base editing activity, which in certain embodiments can be 10% or more). For example, assembly of an active cytidine deaminase can be driven by binding zinc finger proteins with linked half-domains to a DNA target in close proximity to each other, positioning the half-domains in a location that allows assembly of a functional cytidine deaminase.

[0039] It is understood that a "half-domain" pair as described herein may refer to any pair of cytidine deaminase polypeptide sequences that, separately, do not have cytidine deaminase activity, but together form a functional cytidine deaminase domain (either wild-type or a variant as discussed herein). In some embodiments, the toxic domains of TDD20-TDD31 are divided into half-domains at the residues shown in Table 3. In certain embodiments, the half-domain pairs of the TDD may comprise the amino acid sequences of SEQ ID NOs:25 and 26, SEQ ID NOs:27 and 28, SEQ ID NOs:29 and 30, SEQ ID NOs:31 and 32, SEQ ID NOs:33 and 34, SEQ ID NOs:35 and 36, SEQ ID NOs:37 and 38, SEQ ID NOs:39 and 40, SEQ ID NOs:41 and 42, SEQ ID NOs:43 and 44, SEQ ID NOs:45 and 46, SEQ ID NOs:47 and 48, SEQ ID NOs:49 and 50, SEQ ID NOs:51 and 52, SEQ ID NOs:53 and 54, SEQ ID NOs:55 and 56, SEQ ID NOs:57 and 58, SEQ ID NOs:59 and 60, SEQ ID NOs:61 and 62, SEQ ID NOs:63 and 64, SEQ ID NOs:65 and 66, SEQ ID NOs:67 and 68, SEQ ID NOs:69 and 70, SEQ ID NOs:71 and 72, SEQ ID NOs:73 and 74, SEQ ID NOs:75 and 76, SEQ ID NOs:77 and 78, SEQ ID NOs:79 and 80, SEQ ID NOs:81 and 82, or SEQ ID NOs:83 and 84.

[0040] When the present disclosure refers to a cytidine deaminase (e.g., a TDD as described herein), it is contemplated that other cytidine deaminases can be used in the fusion proteins and cell editing systems described herein. The cytidine deaminase can include wild-type or evolved domains. In certain embodiments, the cytidine deaminase can be, for example, an apolipoprotein B mRNA editing complex 1 (APOBEC1) domain or an activation-induced deaminase (AID).

[0041] The present disclosure also provides other possible cytidine deaminases. Such cytidine deaminases can be used, for example, in the fusion proteins and cell editing systems described herein. In some embodiments, the cytidine deaminase is a functional analog of the TDD described herein. A functional analog of a TDD is a molecule that has the same or substantially the same biological function (i.e., cytidine deaminase function) as the TDD. For example, a functional analog may be an isoform or variant of a TDD, such as an isoform or variant that contains a portion of the TDD with or without additional amino acid residues and / or contains a mutation relative to the TDD (e.g., a variant having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the TDD (e.g., a TDD comprising any one of the amino acid sequences of SEQ ID NOs: 1-12) or a toxic domain thereof (e.g., a toxic domain comprising any one of the amino acid sequences of SEQ ID NOs: 13-24). In certain embodiments, the functional analog is an orthologue of a TDD described herein. In certain embodiments, the orthologue of a TDD may comprise an amino acid sequence that is at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the TDD (e.g., a TDD comprising any one of the amino acid sequences of SEQ ID NOs: 1-12). In certain embodiments, an ortholog of a TDD may comprise a toxic domain having an amino acid sequence that is at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of a toxic domain of a TDD described herein (e.g., a toxic domain comprising the amino acid sequence of any one of SEQ ID NOs: 13-24).

[0042] In certain embodiments, the cytidine deaminase described herein may target cytidine in AC sequence, TC sequence, GC sequence, CC sequence, AAC sequence, TAC sequence, GAC sequence, CAC sequence, ATC sequence, TTC sequence, GTC sequence, CTC sequence, AGC sequence, TGC sequence, GGC sequence, CGC sequence, ACC sequence, TCC sequence, GCC sequence, CCC sequence, or any combination thereof.In certain embodiments, the cytidine deaminase described herein has increased efficiency and / or activity compared to DddA.In some embodiments, the increased efficiency or activity may be, for example, in any one or combination of the above target sequences.

[0043] The term "percent identity" for amino acid or nucleotide sequences refers to the percentage of residues in two sequences that are the same when aligned for maximum correspondence. The percentage identity of two amino acid sequences (or two nucleic acid sequences) may be obtained, for example, by BLAST (registered trademark) (available at the National Center for Biotechnology Information website of the National Library of Medicine) using default parameters. In some embodiments, the length of the reference sequence that is aligned for comparison purposes is at least 30% (e.g., at least 40, 50, 60, 70, 80, or 90%, or 100%) of the reference sequence.

[0044] It is also contemplated that an adenine deaminase (e.g., TadA) can be used in the fusion proteins and cell editing systems described herein to convert A:T base pairs to G:C base pairs. In certain embodiments, the TDD may be mutated at residues that form the nucleotide pocket to enable the enzyme to act as an adenine deaminase and / or reduce TC sequence bias within the base editing window.

[0045] B. Zinc finger protein domains The fusion proteins described herein (such as ZFP-cytidine deaminase (e.g., ZFP-TDD), ZFP-cytidine deaminase inhibitor (e.g., ZFP-TDDI), or ZFP-nickase fusion proteins) contain a zinc finger protein (ZFP) domain. "Zinc finger protein" or "ZFP" refers to a protein having a DNA binding domain stabilized by zinc. ZFPs bind to DNA in a sequence-specific manner. Each DNA binding domain is called a "finger." ZFPs have at least one finger, and each finger binds 2-4 base pairs of nucleotides, typically 3 or 4 base pairs of DNA (contiguous or non-contiguous). Each zinc finger typically contains approximately 30 amino acids and chelates zinc. Engineered ZFPs can have novel binding specificities compared to naturally occurring zinc finger proteins. Engineering methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, the use of databases that contain triplet (or tetrad) nucleotide sequences and individual zinc finger amino acid sequences, where each triplet or tetrad nucleotide sequence is associated with one or more amino acid sequences of zinc fingers that bind to that particular triplet or tetrad sequence (see, e.g., U.S. Patent Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,140,081; 6,200,759; 6,453,242; 6,534,261; 6,979,539; and 8,586,526; and International Patent Publication Nos. WO 95 / 19431; WO 96 / 06166; See the ZFP design methods detailed in WO98 / 53057; WO98 / 53058; WO98 / 53059; WO98 / 53060; WO98 / 54311; WO00 / 27878; WO01 / 60970; WO01 / 88197; WO02 / 016536; WO02 / 099084; and WO03 / 016496.

[0046] The ZFP domain of the present ZFP fusion proteins may comprise at least three (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or more) zinc fingers. Individual zinc fingers are typically spaced three base pairs apart when binding to DNA, unless connected by an engineered linker that allows for skipping one or more bases (see, e.g., Paschon et al., Nat Commun. (2019) 10:1133, and U.S. Pat. Nos. 8,772,453; 9,163,245; 9,394,531, and 9,982,245). ZFP domains with three fingers typically recognize target sites that contain 9 or 12 nucleotides. ZFP domains with four fingers typically recognize target sites that contain 12-15 nucleotides. ZFP domains with five fingers typically recognize target sites that contain 15-18 nucleotides. A ZFP domain with six fingers can recognize target sites containing 18 to 21 nucleotides.

[0047] The target specificity of the ZFP domain may be improved by mutations to the ZFP backbone, for example, as described in U.S. Patent Publication No. 2018 / 0087072. Mutations include those made to residues in the ZFP backbone that can nonspecifically interact with phosphates on the DNA backbone but are not involved in nucleotide target specificity. In some embodiments, the mutations include mutating cationic amino acid residues to neutral or anionic amino acid residues. In some embodiments, the mutations include mutating polar amino acid residues to neutral or nonpolar amino acid residues. In further embodiments, the mutations are made at positions (-4), (-5), (-9), and / or (-14) relative to the DNA binding helix. In some embodiments, the zinc fingers may include one or more mutations at positions (-4), (-5), (-9), and / or (-14). In further embodiments, one or more zinc fingers in a multi-finger ZFP domain may include a mutation at positions (-4), (-5), (-9), and / or (-14). In some embodiments, the amino acid at position (-4), (-5), (-9), and / or (-14) (e.g., arginine (R) or lysine (K)) is mutated to alanine (A), leucine (L), Ser (S), Asp (N), Glu (E), Tyr (Y), and / or glutamine (Q). In some embodiments, the R residue at position (-4) is mutated to Q.

[0048] Alternatively, the DNA binding domain may be derived from a nuclease, for example, the recognition sequences of homing endonucleases and meganucleases, such as I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII, and I-TevIII, are known. See also U.S. Patent Nos. 5,420,032 and 6,833,252; Belfort et al., Nucleic Acids Res. (1997) 25:3379-88; Dujon et al., Gene (1989) 82:115-8; Perler et al., Nucleic Acids Res. (1994) 22:1125-7; Jasin, Trends Genet. (1996) 12:224-8; Gimble et al., J Mol Biol. (1996) 263:163-80; Argast et al., J Mol Biol. (1998) 280:345-53; and the New England Biolabs catalog. In addition, the DNA binding specificity of homing endonucleases and meganucleases can be engineered to bind to non-natural target sites. See, e.g., Chevalier et al., Mol Cell (2002) 10:895-905; Epinat et al., Nucleic Acids Res. (2003) 31:2952-62; Ashworth et al., Nature (2006) 441:656-59; Paques et al., Current Gene Therapy (2007) 7:49-66; and U.S. Patent Publication No. 2007 / 0117128.

[0049] In some embodiments, the ZFP fusion protein comprises one or more zinc finger domains. The domains may be linked together via an extensible flexible linker, for example, one domain may comprise one or more (e.g., 3, 4, 5, or 6) zinc fingers and another domain may comprise an additional one or more (e.g., 3, 4, 5, or 6) zinc fingers. In some embodiments, the linker is a standard inter-finger linker, such that the finger array comprises one DNA binding domain comprising 8, 9, 10, 11, or 12 or more fingers. In other embodiments, the linker is an atypical linker, such as a flexible linker. For example, two ZFP domains may be linked (N- to C-terminally) to a cytidine deaminase, inhibitor, or nickase domain ("domain") such as those described herein in a ZFP-ZFP-domain, domain-ZFP-ZFP, ZFP-domain-ZFP, or ZFP-domain-ZFP-domain (two ZFP-domain fusion proteins fused together via a linker) arrangement.

[0050] In some embodiments, the ZFP fusion proteins are "two-handed", i.e., they contain two zinc finger clusters (two ZFP domains) separated by an intervening amino acid such that the two ZFP domains bind to two discontinuous target sites. An example of a two-handed zinc finger binding protein is SIP1, where a cluster of four zinc fingers is located at the amino terminus of the protein and a cluster of three fingers is located at the carboxyl terminus (see Remacle et al., EMBO J. (1999) 18(18):5073-84). Each cluster of zinc fingers in these proteins is capable of binding to a unique target sequence, and the spacing between the two target sequences can include many nucleotides.

[0051] The DNA-binding ZFP domain of the ZFP fusion protein described herein targets the protein to a DNA target region. In some embodiments, the DNA target region is at least 8 bp in length. For example, the target region may be 8 bp to 40 bp in length, for example, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 bp in length.

[0052] In certain embodiments, the ZFP binds to a target site that is 1-100 nucleotides (or any number in between) on either side of the targeted base. In other embodiments, the ZFP binds to a target site that is 1-50 nucleotides (or any number in between) on either side of the targeted base.

[0053] In certain embodiments, a ZFP domain or ZFP fusion protein of the disclosure may be as described in this Example.

[0054] C. Base Editor Inhibitors In some embodiments, the base editor systems described herein may include an inhibitor of the editor to better regulate the base editing activity of the system in time and space. For example, if the cytidine deaminase is a TDD described herein, the inhibitor may be TDDI, which inhibits said TDD. An example of a TDD / TDDI pair is the cytidine deaminase DddA and its inhibitor DddI.

[0055] Thus, in some embodiments, the base editor system comprises a TDDI component in addition to the ZFP-TDD fusion protein, which can be brought into proximity with the TDD complex via a DNA binding domain that is covalently fused to it, or via dimerization with a DNA binding domain that is not covalently linked to it.

[0056] In some embodiments, the base editing system includes a ZFP-inhibitor fusion protein that includes a ZFP domain and an inhibitor domain, where the ZFP domain binds to a sequence in a DNA target region that is close (e.g., within 50-100 nt) to the binding site of the ZFP-cytidine deaminase fusion protein. When the ZFP-inhibitor fusion protein is introduced into a cell, the inhibitor domain becomes proximal to the cytidine deaminase complex and inhibits the base editing activity of cytidine deaminase at the locus by binding to the complex. The presence of the sequence bound by the ZFP domain of the ZFP-inhibitor determines the inhibitory activity of the inhibitor.

[0057] In some embodiments, the binding of the inhibitor domain to the cytidine deaminase complex may be modulated by an agent (e.g., a small molecule or peptide). For example, the inhibitor domain may be fused to a dimerization domain, and its dimerization partner may be fused to a ZFP domain that binds to a sequence in the DNA target region close (e.g., within 50-100 nt) to the binding site of the ZFP-cytidine deaminase fusion protein. The inhibitor and ZFP dimerization domains may dimerize in the presence of a dimerization inducer (e.g., a small molecule or peptide). In the presence of the agent, the inhibitor domain will come into proximity to the DNA target region via dimerization, thereby binding to and inactivating the cytidine deaminase complex. When the agent is removed, the inhibitor domain will no longer be captured in the vicinity of the DNA target region and will detach from the cytidine deaminase complex, allowing the base editing process to proceed. Examples of such agents and dimerization domains are shown in Table 1 below: [Table 1]

[0058] Conversely, dimerization of the domain fused to the ZFP and inhibitor domain can be inhibited, rather than promoted, by a dimerization inhibitor (e.g., a small molecule or peptide), such that the presence of the agent allows activity of the cytidine deaminase complex, and if the agent is removed, the inhibitor domain is allowed to bind to the cytidine deaminase complex and the base editing process is inhibited.

[0059] D. Uracil DNA Glycosylase Inhibitors The term "uracil glycosylase inhibitor" or "UGI" as used herein refers to a protein that can inhibit the uracil-DNA glycosylase base excision repair enzyme. Upon detection of a G:U mismatch, cells respond via base excision repair initiated by removal of the mismatched uracil by uracil N-glycosylase (UNG). In some embodiments, the base editor system described herein further comprises one or more UGIs to protect the edited G:U intermediate from removal by UNG. In certain embodiments, the ZFP-cytidine deaminase (e.g., ZFP-TDD) fusion proteins described herein may comprise one or more UGI domains, e.g., linked by a linker described herein. In some embodiments, the linker is a SGGS linker (SEQ ID NO: 95). The UGI domain(s) may be located at the N-terminus, C-terminus, or any combination thereof of the fusion protein (e.g., one UGI domain at the C-terminus, one UGI domain at the N-terminus, two UGI domains at the C-terminus, two UGI domains at the N-terminus, or any combination thereof). Additionally or alternatively, the one or more UGI domains may be on another ZFP fusion protein ("ZFP-UGI"). In certain embodiments, the UGI domain comprises the amino acid sequence of SEQ ID NO:99.

[0060] E. Nickase In some embodiments, the base editor system described herein further comprises a nickase that induces a single-stranded DNA break near the edited DNA target region (e.g., within 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nt from the edited base). Once the nick is created, DNA repair mechanisms are triggered so that the region downstream of the nick is removed and replaced to create a completely edited double-stranded DNA target region. The nick can be, for example, 5' or 3' of the edited base, on the same or opposite strand. In certain embodiments, the nickase is part of a ZFP fusion protein.

[0061] In some embodiments, a nickase is composed of a first and a second monomer (also referred to herein as the "half-domain" of the nickase) that dimerize to form an active nickase. One monomer may contain a mutation that renders it catalytically inactive (whereby only one DNA strand is nicked by the active monomer). In some embodiments, the monomers may be derived from FokI (e.g., FokI-ELD and FokI-KKR), and one of the monomers may have an inactivating mutation, such as D450N. In certain embodiments, a pair of monomers that form a complete active nickase may include a FokI-ELD-D450N monomer (e.g., having the amino acid sequence of SEQ ID NO: 109) and a FokI-KKR monomer (e.g., having the amino acid sequence of SEQ ID NO: 110).

[0062] In some embodiments, the base editor system described herein has a trimeric structure to include a nickase function. For example, one half-domain of a dimeric nickase can be fused to a ZFP-cytidine deaminase (e.g., a ZFP-TDD described herein), and the other half-domain can be fused to an independent ZFP, such that both ZFP domains generate an active nickase capable of generating a single-strand break when bound to their DNA target region. See, for example, FIG. 4.

[0063] In some embodiments, the base editor systems described herein have a tetrameric structure to include a nickase function. In addition to two ZFP-cytidine deaminase (e.g., a ZFP-TDD described herein) fusion proteins, such systems also include two ZFP-nickases, where one half-domain of the dimeric nickase is fused to a first ZFP domain and the other half-domain is fused to a second ZFP domain, such that an active nickase capable of generating a single-strand break is generated when both ZFP domains bind to their DNA target region.

[0064] In some embodiments, the nickase may be, for example, a ZFN nickase, a TALEN nickase, or a CRISPR / Cas nickase. In certain embodiments, the nickase is derived from the DNA cleavage domain of FokI. In some embodiments, the FokI nickase comprises one or more mutations compared to the parent FokI nickase, such as a mutation that changes the charge of the cleavage domain; a mutation to a residue that is predicted to be close to the DNA backbone based on molecular modeling and shows variation in FokI homologs, and / or a mutation at another residue (see, for example, U.S. Patent No. 8,623,618, and Guo et al., J Mol Biol. (2010) 400(1):96-107). An exemplary pair of half-domains of FokI may be, for example, SEQ ID NOs: 109 and 110.

[0065] In the ZFP fusion proteins described herein, the nickase domain(s) can be located on either side of the DNA-binding ZFP domain, including on the N- or C-terminal side of the fusion molecule (N-terminal and / or C-terminal to the ZFP domain). In some embodiments, a ZFP-cytidine deaminase (e.g., a ZFP-TDD described herein) fusion protein described herein comprises a cytidine deaminase domain at the N- or C-terminus and a nickase domain at the opposite end.

[0066] F. Peptide Linker In the fusion proteins described herein, the ZFP, cytidine deaminase (e.g., a TDD described herein), inhibitor (e.g., TDDI), nickase, and / or UGI domains may be arranged in any order relative to each other. In some embodiments, the domains may be linked to each other by a direct peptidyl bond, a peptide linker, or any combination thereof. In some embodiments, two or more of the domains may be linked to each other by dimerization (e.g., via a leucine zipper, a STAT protein N-terminal domain, or an FK506 binding protein).

[0067] In some embodiments, the ZFP, cytidine deaminase (e.g., a TDD described herein), inhibitor (e.g., TDDI), UGI, and / or nickase domains, and / or zinc fingers within the ZFP domains may be linked via a peptide linker, e.g., a non-cleavable peptide linker of about 5-200 amino acids (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 or more amino acids). Preferred linkers are flexible amino acid subsequences that are typically synthesized as recombinant fusion proteins. See, e.g., U.S. Patent Nos. 6,479,626; 6,903,185; 7,153,949; 8,772,453; and 9,163,245; and PCT Patent Publication No. WO2011 / 139349. The proteins described herein may include any combination of suitable linkers.

[0068] In some embodiments, the peptide linker is 3-30 amino acid residues in length and is rich in G and / or S. Non-limiting examples of such linkers are the SGGS linker (SEQ ID NO: 95), as well as G4S-type linkers, i.e., linkers containing one or more (e.g., 2, 3, or 4) GGGGS (SEQ ID NO: 119) motifs, or variants of that motif (such as those having insertions, deletions, and / or substitutions of 1, 2, or 3 amino acids from that motif).

[0069] In certain embodiments, the peptide linker used in the fusion proteins described herein may be L26 (SEQ ID NO:85), L21 (SEQ ID NO:86), L18 (SEQ ID NO:87), L13 (SEQ ID NO:88), L11 (SEQ ID NO:89), L9 (SEQ ID NO:90), L7A (SEQ ID NO:91), L6 (SEQ ID NO:92), L4 (SEQ ID NO:93), L0 (SEQ ID NO:94), SGGS (SEQ ID NO:95), or N6A (SEQ ID NO:96), as shown in Table 4.

[0070] II. Base Editor Systems The present disclosure provides a base editor system comprising the ZFP fusion protein described herein. The base editor system can be used to edit the cytosine base in a DNA target region to a uracil base, and the uracil is replaced by a thymine base during DNA replication or repair. In certain embodiments, this edit changes the targeted C:G base pair to a T:A base pair. Figure 1 shows the base editing system of the present disclosure.

[0071] The base editor system described herein can be used to knock out genes (e.g., by changing normal codons to stop codons and / or by mutating splice acceptor sites to introduce exon skipping and / or frameshift mutations); introduce mutations in regulatory elements of genes (e.g., promoter or enhancer regions) to increase or decrease expression; correct disease-causing mutations (e.g., point mutations); and / or induce mutations that provide therapeutic benefit. The target DNA can be within a chromosome in a cell or in an extrachromosomal sequence (e.g., mitochondrial DNA). The base editing can be performed in vitro, ex vivo, or in vivo.

[0072] In some embodiments, the base editor systems described herein perform one or more codon conversions, e.g., CAA to TAA; CAG to TAG; CGA to TGA; or TGG to TAG, TGA, or TAA; or any combination thereof, thereby introducing a stop codon(s).

[0073] The base editor systems of the disclosure may include, in addition to a ZFP-cytidine deaminase (e.g., a ZFP-TDD described herein) fusion protein, components such as an inhibitor domain (e.g., TDDI), UGI, and nickase, or any combination thereof, described herein, that may serve to regulate or enhance the editing activity of the system. In certain embodiments, the systems may be packaged within a single viral vector (e.g., an AAV vector).

[0074] In some embodiments, a base editor system of the disclosure comprises a pair of ZFP-cytidine deaminase (e.g., a ZFP-TDD described herein) fusion proteins, each comprising a cytidine deaminase half-domain that lacks cytidine deaminase activity by itself, such that binding of the ZFP to its respective nucleotide target results in an active cytidine deaminase molecule that is capable of editing a targeted C base to a T (e.g., by replacing the C with a U, which in turn is replaced by a T during DNA replication or repair).

[0075] For example, in some embodiments, a base editor system may include a) a first fusion protein (ZFP-TDD left) comprising i) a first ZFP domain that binds to a nucleotide of a double-stranded DNA target region on one side of the base targeted for editing; and ii) an N-half domain of a TDD; and b) a second fusion protein (ZFP-TDD right) comprising i) a second ZFP domain that binds to a nucleotide of a double-stranded DNA target region on the other side of the base targeted for editing; and ii) a C-half domain of a TDD; binding of the ZFP-TDD left and ZFP-TDD right to their respective nucleotides results in an active TDD molecule capable of editing a DNA target region by changing a C base to a T. The ZFP-TDD and / or the DNA target region may be, for example, as described herein.

[0076] In some embodiments, the base editor system described herein has a trimeric structure to include a nickase function. For example, the base editor system may include the first and second ZFP-TDD fusion proteins described above, where the ZFP-TDD left and ZFP-TDD right bind to their respective nucleotides to generate an active TDD molecule capable of editing a DNA target region by changing a C base to a T. One half-domain of the dimeric nickase may be fused to the first or second ZFP-TDD fusion protein, and the other half-domain may be fused to an independent ZFP, such that both ZFP domains bind to their DNA target region to generate an active nickase capable of generating a single-strand break. See, e.g., FIG. 4. The ZFP-TDD, DNA target region, and nickase domain may be, for example, as described herein.

[0077] In some embodiments, the base editor system described herein has a tetrameric structure to include a nickase function. For example, the base editor system may include the first and second ZFP-TDD fusion proteins described above, where the ZFP-TDD left and ZFP-TDD right bind to their respective nucleotides to generate an active TDD molecule capable of editing a DNA target region by changing a C base to a T. In addition to the two ZFP-TDD fusion proteins, such a system may also include two ZFP-nickase fusion proteins, where one half-domain of the dimeric nickase is fused to a first ZFP domain and the other half-domain is fused to a second ZFP domain, such that both ZFP domains bind to their DNA target region to generate an active nickase capable of generating a single-strand break. The ZFP-TDD, DNA target region, and nickase domain may be, for example, as described herein.

[0078] In some embodiments, a base editor system may comprise a) a first fusion protein (ZFP-TDDI) that binds to a nucleotide in the first DNA target region, comprising i) a zinc finger protein (ZFP) domain that binds to a nucleotide in the first DNA target region; and ii) a TDDI domain; b) a second fusion protein (ZFP-TDD left) that comprises i) a ZFP domain that binds to a nucleotide in the second DNA target region on one side of the base targeted for editing; and ii) an N-half domain of a TDD; and c) a third fusion protein (ZFP-TDD right) that comprises i) a ZFP domain that binds to a nucleotide in the second DNA target region on the other side of the base targeted for editing; and ii) a C-half domain of a TDD; binding of the ZFP-TDD left and ZFP-TDD right to their respective nucleotides results in an active TDD molecule capable of editing the second DNA editing region by changing a C base to a T; binding of the ZFP-TDDI to the first DNA target region prevents editing of the second DNA target region by the TDD. The ZFP-TDD, ZFP-TDDI, and DNA target region can be, for example, as described herein.

[0079] In some embodiments, the base editor system comprises: a) a first fusion protein comprising i) a zinc finger protein (ZFP) domain that binds to a nucleotide in a first DNA target region; and ii) a dimerization domain; b) a second fusion protein comprising i) a TDDI domain; and ii) a dimerization domain that pairs with the dimerization domain of a); c) a third fusion protein comprising i) a ZFP domain that binds to a nucleotide in the second DNA target region on one side of the base targeted for editing; and ii) an N-half domain of a TDD (ZFP-TDD-left); and d) a second fusion protein comprising i) a ZFP domain that binds to a nucleotide in the second DNA target region on one side of the base targeted for editing; and ii) an N-half domain of a TDD (ZFP-TDD-left); a) a ZFP domain that binds to a nucleotide of the second DNA target region on the other side of the targeted base; and ii) a fourth fusion protein (ZFP-TDD right) that includes a C-half domain of a TDD; binding of the ZFP-TDD left and ZFP-TDD right to their respective nucleotides results in an active TDD molecule that is capable of editing the second DNA editing region by changing the C base to a T; the fusion proteins of a) and b) dimerize to form a ZFP-TDDI, and binding of the ZFP of a) to the first DNA target region prevents editing of the second DNA target region by the TDD. The ZFP-TDD, ZFP-TDDI, and / or DNA target region may be, for example, as described herein.

[0080] In some embodiments, the dimerization domains of the fusion proteins of a) and b) pair in the presence of a dimerization-inducing agent to form a ZFP-TDDI and inhibit the activity of the TDD.

[0081] In some embodiments, the dimerization domains of the fusion proteins of a) and b) are inhibited from pairing to form ZFP-TDDI in the presence of a dimerization inhibitor, allowing activity of the TDD.

[0082] In some embodiments, the ZFP-TDDI is specific to the sequence that is to be protected from the base editing activity of the TDD. For example, the ZFP domain may bind to the allele that is to be preserved in its non-edited form (e.g., when another allele, such as a mutant allele, is targeted for editing), or to a known site of off-target editing. In some embodiments, the base editing of the TDD may convert a normal codon to a stop codon in the non-protected allele.

[0083] In some embodiments, expression of the ZFP-TDDI (or a component thereof) may be under the control of an inducible promoter. In certain embodiments, such a system may be used as a "kill switch," where the ZFP-TDDI protects essential genes in a cell from being edited, and reducing or eliminating expression of the ZFP-TDDI causes the cell to die.

[0084] When the assembly of ZFP-TDDI is under the control of a dimerization inducer or dimerization inhibitor, base editing can be conditional on the presence or absence of the agent. Such conditional systems can also be used for "kill switches", for example, ZFP-TDDI protects essential genes in cells from being edited in the presence of a dimerization inducer or in the absence of a dimerization inhibitor, and the removal or administration of the agent, respectively, kills the cell.

[0085] In certain embodiments, the base editor system of the present disclosure may be a multiplex system that includes two or more pairs of ZFP-TDD left and ZFP-TDD right, and such a system may be capable of editing two or more DNA target regions at once. In certain embodiments, to increase the specificity of editing, the multiplex system includes ZFP-TDD pairs, and the N-half domain and C-half domain of the TDD are split at different positions (e.g., positions described herein) within the TDD sequence in each pair. In certain embodiments, the DNA target regions edited by the ZFP-TDD pairs of the multiplex system may be in different genes. In certain embodiments, the DNA target regions may be in the same gene.

[0086] In any of the above embodiments, the TDD and TDDI may be any of those described herein. It is also contemplated that other cytidine deaminases and inhibitors may be used in place of the TDD and TDDI. In certain embodiments, the multiplex system described herein may include a first ZFP-cytidine deaminase pair and a second ZFP-cytidine deaminase pair, where the first and second pairs utilize different cytidine deaminases (e.g., selected from those described herein).

[0087] In some embodiments, the systems and methods described herein result in targeted editing of DNA target regions in at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of cells. In some embodiments, the edited cells exhibit little or no off-target indels (e.g., less than 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% off-target indels). In some embodiments, the edited cells exhibit little or no off-target base editing (e.g., less than 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% off-target base editing). However, higher percentages may be possible, as base edits at off-target sites may be less likely to result in translocations or other genomic arrangements.

[0088] The present disclosure also provides nucleic acid molecules encoding the ZFP fusion proteins described herein, which can be part of a viral or non-viral vector. Additionally, the present disclosure provides cells or populations of cells comprising the base editor systems described herein, as well as progeny of such cells, which comprise one or more edited bases.

[0089] III. Delivery of ZFP Fusion Proteins The ZFP fusion proteins of the present disclosure can be introduced into target cells as proteins through various methods (e.g., electroporation, fusion with a receptor ligand for the protein, lipid nanoparticles, cationic or anionic liposomes, or nuclear localization signals (e.g., in combination with liposomes)). In other embodiments, the fusion protein is introduced into target cells via a nucleic acid molecule encoding it, e.g., a DNA plasmid or mRNA. The nucleic acid molecule can be in a nucleic acid expression vector, which can include expression control sequences, e.g., promoters, enhancers, transcription signal sequences, and transcription termination sequences, that allow expression of the coding sequence of the ZFP fusion protein. "Delivery of the system," as described herein, can refer to either delivery of the fusion protein system described herein, or delivery of a nucleic acid molecule encoding the fusion protein system or a vector or expression construct that includes the nucleic acid molecule.

[0090] In some embodiments, the promoter on the vector for inducing expression of the ZFP fusion protein is a constitutively active promoter or an inducible promoter. Suitable promoters include, but are not limited to, the Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), the CMV immediate early promoter, the simian virus 40 (SV40) promoter, the dihydrofolate reductase (DHFR) promoter, the β-actin promoter, the phosphoglycerate kinase (PGK) promoter, the EFlα promoter, the Moloney murine leukemia virus (MoMLV) LT Examples of promoters include R, creatine kinase-based (CK6) promoter, transthyretin promoter (TTR), thymidine kinase (TK) promoter, tetracycline-responsive promoter (TRE), hepatitis B virus (HBV) promoter, human alpha 1-antitrypsin (hAAT) promoter, chimeric liver-specific promoter (LSP), E2 factor (E2F) promoter, human telomerase reverse transcriptase (hTERT) promoter, CMV enhancer / chicken beta-actin / rabbit beta-globin promoter (CAG promoter; Niwa et al., Gene (1991) 108(2):193-9), and RU-486-responsive promoter. In addition, the promoter may contain one or more autoregulatory elements, whereby a ZFP fusion protein can bind and suppress its own expression level to a preset threshold. See U.S. Patent No. 9,624,498.

[0091] Any method of introducing the nucleotide sequence into the cell may be used, including but not limited to electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, liposomes combined with nuclear localization signals, naturally occurring liposomes (e.g., exosomes), or viral transduction. In certain embodiments, the nucleotide sequence is in the form of mRNA and is delivered to the cell via electroporation.

[0092] Viral transduction may be used for in vivo delivery of expression vectors. A person skilled in the art may adapt various viral vectors known in the art for use in the present disclosure, such as vaccinia vectors, adenoviral vectors, lentiviral vectors, poxvirus vectors, adeno-associated viral (AAV) vectors, retroviral vectors, and hybrid viral vectors. In some embodiments, the viral vector used in the present disclosure is a recombinant AAV (rAAV) vector. Any suitable AAV serotype may be used. For example, the AAV may be AAV1, AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV8.2, AAV9, AAV.PHP.B, AAV.PHP.eB, or AAVrhlO, or a novel serotype or pseudotype, such as AAV2 / 8, AAV2 / 5, AAV2 / 6, AAV2 / 9, or AAV2 / 6 / 9. In some embodiments, the expression vector is an AAV viral vector, which is introduced into target human cells by recombinant AAV virions. The genome of the recombinant AAV virions comprises a construct with AAV inverted repeat (ITR) sequences at both ends to allow production of AAV virions in production systems such as insect cell / baculovirus production systems or mammalian cell production systems. AAV may be engineered so that its capsid protein has low immunogenicity or high transduction capacity in humans. The viral vectors described herein may be produced using methods known in the art. Any suitable permissive or packaging cell type may be used to produce viral particles. For example, mammalian (e.g., 293) cells or insect (e.g., sf9) cells may be used as packaging cell lines.

[0093] Any type of cell can be targeted by the base editing methods described herein. For example, the cell can be eukaryotic or prokaryotic. In some embodiments, the cell is a mammalian (e.g., human) cell or a plant cell. Human cells can include, for example, T cells, natural killer (NK) cells, NK T cells, alpha-beta T cells, gamma-delta T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, B cells, human embryonic stem cells, tumor-infiltrating lymphocytes (TILs), or pluripotent stem cells (e.g., induced pluripotent stem cells (iPSCs)) that can differentiate into lymphoid cells. In some embodiments, the system can be used to modify pluripotent stem cells before they differentiate into multiple cell types. For example, lymphoid cell precursors can be modified before differentiation into lymphoid types, such as regulatory T cells, effector T cells, and natural killer cells. The multiplex base editor system of the present disclosure (comprising two or more ZFP-cytidine deaminase (e.g., ZFP-TDD) pairs) can be used to prepare cells with multiple base edits at once, including, inter alia, pluripotent cells. In some embodiments, the multiplex system may be used to prepare, for example, allogeneic T cells. As described herein, when the system includes a ZFP-cytidine deaminase inhibitor (e.g., ZFP-TDDI) that can be induced to assemble in the presence or absence of a dimerization modulating agent, it is believed that the edited cells can be placed under the control of a "kill switch" that is activated upon administration of the agent.

[0094] For agricultural applications, any method for introducing proteins or nucleic acid molecules into plant cells is contemplated, such as Agrobacterium tumefaciens-mediated T-DNA delivery.

[0095] IV.Medicinal Uses The present disclosure provides a method of editing cytosines to thymine bases in cellular DNA, comprising delivering a base editor system described herein to a cell (e.g., from a patient), thereby replacing the targeted C base with a T base. The cell may be in the patient (in vivo treatment), or the method described herein may be performed on cells removed from the patient, and the edited cells may then be delivered to the patient (ex vivo treatment). In some embodiments, the cells are further manipulated ex vivo before being used as a treatment. The term "treatment" encompasses alleviating symptoms, preventing the onset of symptoms, slowing disease progression, improving quality of life, and increasing survival. In some embodiments, the patient treated with the methods described herein is a mammal, e.g., a human.

[0096] In some embodiments, the method of the present disclosure is used to edit genes or regulatory sequences associated with disease. For example, in certain embodiments, base editing can correct point mutations in DNA sequences to restore normal gene expression or activity. In certain embodiments, base editing can introduce stop codons in harmful genes (e.g., cancer genes). In certain embodiments, base editing can introduce mutations that provide therapeutic benefits.

[0097] In some embodiments, the patient has cancer. In certain embodiments, cells from the patient are further modified before or after base editing to provide resistance to chemotherapeutic agents. The patient can then be treated with a chemotherapeutic agent, which in some embodiments can result in a higher survival rate for edited cells than for non-edited cells.

[0098] In some embodiments, the patient has an autoimmune disorder.

[0099] In some embodiments, the patient has an autosomal dominant disease, such as autosomal dominant polycystic kidney disease.

[0100] In some embodiments, the patient has a mitochondrial disorder.

[0101] In some embodiments, the patient has sickle cell disease, hemophilia (e.g., hemophilia A, B, or C), cystic fibrosis, phenylketonuria, Tay-Sachs disease, prion disease, color blindness, lysosomal storage disease (e.g., Fabry disease), Friedreich's ataxia, or prostate cancer.

[0102] In some embodiments, the method of the present disclosure can target a specific allele of a gene, for example, a wild type or mutant allele, for base editing. In certain embodiments, the allele can be associated with cancer. For example, the method can target the V617F mutant allele of JAK2, which leads to constitutive tyrosine phosphorylation activity and plays a crucial role in the expansion of myeloproliferative neoplasms. For example, knocking out the expression of the allele with the V617F mutation by introducing a stop codon can promote successful treatment of JAK2 V617F disorders.

[0103] The disclosure further provides pharmaceutical compositions comprising elements of the base editing systems described herein, e.g., a ZFP-cytidine deaminase (e.g., a ZFP-TDD) pair, and optionally a nickase component (e.g., a ZFP-nickase component for a trimer or tetramer system described herein) and / or a cytidine deaminase inhibitor (e.g., a TDDI) component (e.g., a ZFP-cytidine deaminase inhibitor component), or a nucleotide sequence encoding said elements (e.g., in a viral or non-viral vector described herein). The pharmaceutical compositions may further comprise a pharma- ceutically acceptable carrier, e.g., water, saline (e.g., phosphate buffered saline), dextrose, glycerol, sucrose, lactose, gelatin, dextran, albumin, or pectin. In addition, the compositions may contain auxiliary substances, e.g., wetting or emulsifying agents, pH buffers, stabilizers, or other agents that enhance the effectiveness of the pharmaceutical composition. The pharmaceutical compositions may contain delivery vehicles, such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, and vesicles.

[0104] In some embodiments, the base editor systems described herein include 2B4 (CD244), 4-1BB (CD137), A2aR, AAVS1, ACTB, AID, ALB, B2M, B7.1, B7.2, B7-H2, B7-H3, B7-H4, B7-H6, BAFFR, BCL11A, BLAME (SLAMF8), BTLA, butyrophilin, CIITA, CCR5, CD100 (SEMA4D), CD103, CD3 zeta, CD4, CD5, CD7, CD11a, CD11b, CD11c, CD11d, CD150, IPO-3), CD1 60, CD160(BY55), CD18, CD19, CD2, CD27, CD28, CD29, CD30, CD4, CD40, CD47, CD48, CD49a, CD49D, CD49f, CD52, CD69, CD7, CD83, CD84, CD8 alpha, CD8 beta, CD96(Tactile), CDS, CEACAM1, CISH, CRTAM, CTLA4, CXCR4, DCK, DGK, DGKA, DGKB, DGKD, DGKE, DGKG, DGKI, DGKK, DGKQ, DGKZ, DHFR, DNAM1(CD226), EP2 / 4 Receptors, Adenosine receptors including A2AR, FAS, FASLG, GADS, GITR, GM-CSF, gp49B, HHLA2, HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HIV-LTR (long terminal repeat), HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-I, HVEM, HVEM, IA4, ICAM-1, ICOS, ICOS, ICOS (CD278), IFN-alpha / beta / gamma, IL-1 beta, IL-12, IL-15, IL-18, IL-23, IL2R beta, IL2R gamma, IL2RA, IL-6, IL7R alpha, ILT-2, ILT-4, immunoglobulin heavy chain locus, immunoglobulin light chain locus, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIR family receptors, KLRG1, Lag-3, LAIR-1, LAT, LIGHT, LTBR, ​​Ly9 (CD229), MNK1 / 2, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX2R,The genomic loci can be engineered to target a genomic locus selected from OX40, PAG / Cbp, PD-1, PD-L1, PD-L2, PGE2 receptor, PIR-B, PPP1R12C, PRNP1, PSGL1, PTPN2, RANCE / RANKL, RFX5, ROSA26, SELPLG (CD162), SIRP alpha (CD47), SLAM (SLAMF1, SLAMF4 (CD244, 2B4), SLAMF5, SLAMF6 (NTB-A, Ly108), SLAMF7, SLP-76, SOCS1, SOCS3, tetherin, TGFBR2, TIGIT, TIM-1, TIM-3, TIM-4, TMIGD2, TRA, TRAC, TRB, TRD, TRG, TNF, TNF-alpha, TNFR2, TRIM5, TUBA1, VISTA, VLA1, and VLA-6.

[0105] It is understood that the ZFP fusion proteins and base editor systems described herein may be used in the therapeutic methods described herein, may be for use in the treatments described herein, or may be used in the manufacture of a medicament for the treatments described herein.

[0106] V. Agricultural use The described systems and methods for editing cytosines to thymine bases in cellular DNA may also be used in agricultural applications. For example, in certain embodiments, base editing can correct one or more point mutations in a DNA sequence to restore normal gene expression or activity. In certain embodiments, base editing can introduce a stop codon in one or more deleterious genes. In certain embodiments, base editing can introduce one or more beneficial mutations. In certain embodiments, the systems and methods described herein are used to edit crop plants.

[0107] Unless otherwise defined herein, all technical and scientific terms used in connection with this disclosure shall have the meanings commonly understood by those skilled in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure. In case of conflict, the present specification, including definitions, will control. In general, the nomenclature used in connection with cardiology, medicine, medicinal and pharmaceutical chemistry, and cell biology described herein, and the techniques thereof, are well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly performed in the art, or as described herein.

[0108] Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Throughout this specification and the embodiments, the word "have" or "comprises" or variations such as "has," "having," "comprises," or "comprising" are understood to imply the inclusion of the described element or group of elements but not the exclusion of any other element or group of elements. It is also noted that the term "or" is generally used in the sense of including "and / or" unless the content clearly dictates otherwise. As used herein, the term "about" refers to a numerical range that is plus or minus 10%, 5%, or 1% from the stated numerical value within the context of the particular use. Moreover, the headings provided herein are merely for convenience and do not indicate the scope or meaning of the claimed embodiments.

[0109] All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents form part of the common general knowledge in the art.

[0110] In order that this invention may be better understood, the following examples are set forth. These examples are for illustrative purposes only and are not to be construed as limiting the scope of the invention in any way. EXAMPLES

[0111] Example 1: Base editing activity of TDD in cells We identified novel cytidine deaminases, TDD20 to TDD31 (Table 2), which were tested for base editing activity in K562 cells. [Table 2] To prepare the ZFP-TDD fusion protein pairs, the TDD toxic domain was split into two halves, each lacking cytidine deaminase activity (see Table 3). [Table 3] TIFF2025504606000004.tif226159TIFF2025504606000005.tif230159TIFF2025504606000006.tif228159TIFF2025504606000007.tif77159

[0112] ZFP-TDD design To evaluate editing at the human CIITA site 2 locus, a left and right ZFP pair was designed to target one half of the TDD to a site at the locus ("site 2" shown in Figure 2) such that the halves dimerize at the target site and restore catalytic activity of the TDD. Figure 2 shows a left ZFP and two right ZFPs that can be used to form an exemplary left and right pair (e.g., CIITA_site_2_left_6+CIITA_site2_right1 or CIITA_site_2_left_6+CIITA_site2_right5). In this experiment, the pair CIITA_site_2_left_6+CIITA_site2_right5 was used.

[0113] The N-terminal half of each split TDD pair was fused to the C-terminus of the right ZFP and the C-terminal half was fused to the C-terminus of the left ZFP using the L26 linker (SEQ ID NO: 85). A UGI (uracil DNA glycosylase inhibitor) domain was fused to the C-terminus of each N- and C-terminal half using the SGGS linker (SEQ ID NO: 95). All ZFP-TDD fusion constructs further contained a 3xFLAG tag (e.g., SEQ ID NO: 97 or 98, or the above sequence without the start codon), as well as an SV40 nuclear localization signal fused to the N-terminus of the ZFP.

[0114] Exemplary component sequences, such as those contained in the ZFP-TDDs described above, are shown in Table 4 below. [Table 4]

[0115] TDD base editing activity at the CIITA locus The base editing frequency of TDD20-TDD31 was tested at nucleotides G2, G5, C6, C8, G10, G11, G14, C15, and C16 of the target sequence CIITA site 2 (Figure 2). In some cases, the base editing system included a ZFP-FokI nickase to improve base editing activity. The efficiency of the base editor can be increased by nicking the unmodified DNA strand with a nickase. The unmodified DNA strand is then recognized by the cell as newly synthesized, and the natural DNA repair machinery repairs the nicked DNA strand using the modified strand as a template. The unmodified strand can be nicked using a FokI-derived ZFN or TALEN or CRISPR / Cas-derived nickase. However, all three approaches require the delivery of two additional constructs (two peptides for ZFN or TALEN nickase, one peptide and one sgRNA for CRISPR / Cas nickase; Figure 3).

[0116] A trimeric ZFP-TDD base editor structure can be used to overcome this limitation, facilitating delivery and increasing the editing efficiency by making base editing and DNA nicking more likely to occur simultaneously. In such a trimeric structure, one half-domain of a dimeric nickase (e.g., FokI, in this case the halves can be, for example, SEQ ID NOs: 109 and 110) can be fused to the N-terminus of the left or right ZFP-TDD, and the corresponding other half-domain of the nickase can be targeted to the site of interest via an independent ZFP-nickase peptide (Figure 4). The trimeric ZFP-TDD-nickase system was tested in K562 cells using FokI nickase according to the protocol described above.

[0117] The trimeric ZFP design at the CIITA locus is shown in Figure 5. Three fusion proteins were constructed as follows: 1.CIITA_site_2_right_5ZFP-TDD half domain#1; 2. Nickase half-domain #1-CIITA_site_2_left_6ZFP-TDD half-domain #2, and 3. CIITA_site_2_nickase ZFP-nickase half-domain #2.

[0118] The fusion protein was constructed as follows: Right ZFP#5-TDD construct: [3xFlag+NLS]-[CIITA_site_2_right_5ZFP]-[L26 linker]-[TDD half-domain#1]-[SGGS]-[UGI]-*; in particular, [SEQ ID NO:97]-[SEQ ID NO:103]-[SEQ ID NO:85]-[TDD half domain #1]-[SEQ ID NO:95]-[SEQ ID NO:99]-* Nickase-left ZFP#6-TDD construct: [3xFLAG-NLS]-[Nickase half-domain #1(FokI-ELD_D450N)]-[N6A]-[CIITA_site_2_left_6ZFP]-[L26 linker]-[TDD half-domain #2]-[SGGS]-[UGI]-*; in particular, [SEQ ID NO: 112 without the start codon] - [SEQ ID NO: 96] - [SEQ ID NO: 101] - [SEQ ID NO: 85] - [TDD half domain #2] - [SEQ ID NO: 95] - [SEQ ID NO: 99] - * ZFP-Nickase construct: [3xFlag+NLS]-[CIITA_site_2_Nickase ZFP]-[L0 linker]-[Nickase half-domain#2(FokI-KKR)]-[Scaffold]-*; Specifically, sequence number 113. Table 6 below includes certain sequences of these fusion proteins. [Table 5]

[0119] To assay base editing in cells using the constructs prepared as described above, K562 (ATCC, CCL243) cells were obtained from ATCC and maintained at 37°C, 5% CO2 in RPMI1640 with 10% FBS and 1x penicillin-streptomycin-glutamine (PSG) (Gibco, 10378-016). 400ng of pDNA encoding the paired ZFP-TDD was electroporated into K562 cells using the SF Cell Line 96-well Nucleofector Kit (Lonza, V4SC-2960) according to the manufacturer's instructions. Briefly, cells were washed twice with 1x PBS (without divalent cations) and 2x10 6 DNA fragments were electroporated per 15μL of supplemented SF Cell Line 96-well Nucleofector solution. 5The cells were resuspended in 150 μL of pDNA. For each transfection, 15 μL of cell suspension was mixed with 5 μL of pDNA, transferred to a Lonza Nucleocuvette plate, and then electroporated in an Amaxa Nucleofector 96-well Shuttle System (Lonza) using the protocol for K562 cells (Nucleofector program 96-FF-120). The electroporated cells were incubated at room temperature for 10 minutes and then transferred to 150 μL of pre-warmed complete medium in a 96-well tissue culture plate. The cells were incubated for 72 hours and then harvested for quantification of base editing.

[0120] PCR primers for the CIITA site 2 locus were designed using Primer3 with the following optimization conditions: amplicon size of 200 nucleotides; melting temperature of 60° C.; primer length of 20 nucleotides; and GC content of 50%. The primer and amplicon sequences are shown in Table 5 below. [Table 6]

[0121] Adapters were added for the second PCR reaction to append the Illumina library sequences (forward primer: ACACGACGCTCTTCCGATCT (SEQ ID NO: 107); reverse primer: GACGTGTGCTCTTCCGAT (SEQ ID NO: 108)). The CIITA site 2 locus was amplified in 25 μL using AccuPrime HiFi (Invitrogen) with 100 ng of genomic DNA. Primers were used at a final concentration of 0.1 μM with the following thermocycling conditions: initial melt at 95° C. for 5 min; 35 cycles of 95° C. for 30 s, 55° C. for 30 s, 68° C. for 40 s; and a final extension at 68° C. for 10 min. The PCR product was diluted 1:20 in water. 2 μL of the diluted PCR product was used in a 20 μL PCR reaction to append the Illumina library sequences using Phusion High-Fidelity PCR MasterMix with HF Buffer (NEB). Primers were used at a final concentration of 0.5 μM with the following conditions: initial melt at 98° C. for 30 seconds; 12 cycles of 98° C. for 10 seconds, 60° C. for 30 seconds, 72° C. for 40 seconds, and a final extension at 72° C. for 10 minutes. A second PCR reaction was then performed to add sample-specific sequence barcodes. PCR libraries were purified using a QIAquick PCR Purification Kit (Qiagen). Samples were quantified with a Qubit dsDNA HS Assay Kit (Invitrogen) and diluted to 2 nM. Libraries were then run on either an Illumina MiSeq using a standard 300 cycle kit or an Illumina NextSeq 500 using a medium-power 300 cycle kit according to the manufacturer's instructions.

[0122] result As shown in Figure 6, at least 17 TDDs showed detectable base editing activity (>0.25% base editing) at the CIITA locus, especially at the CIITA site 2 target. In some cases, nicking appeared to improve editing activity.

[0123] Example 2: Effect of different linkers on TDD base editing activity at the CIITA locus To assess whether different linkers between the deaminase and ZFP domains affect base editing activity, the editing frequency at the CIITA locus of the TDDs described herein can be assessed using different linkers, e.g., L26, L21, L18, L13, L11, L9, L6, and L4. Different linker lengths could alter the base editing profile within the base editing window.

[0124] Example 3: Targeting TDD inhibitors (TDDIs) to TDD The TDDs described herein are believed to be inactivated by TDD inhibitors (TDDIs). TDDIs linked to ZFPs or TALEs can target potential TDD-derived cytosine base editor sites and prevent them from being edited (Figure 7). TDDIs can be linked to ZFPs using dimerization domains whose effects are enhanced by small molecules, thereby placing editing activity under small molecule control.

[0125] By designing the targeting TDDI construct to be allele specific, editing can be selectively targeted to a particular allele, for example, by editing the stop codon only when the mutation is present, a deleterious mutation can be knocked out. For example, JAK2 V617F can be knocked out by editing the stop codon only when the V617F mutation is present.

[0126] This TDDI approach may also be used to reduce editing at off-target sites, especially when editing cannot be eliminated by other means.

[0127] It is further contemplated that other cytidine deaminases and their inhibitors may be used in place of TDD and TDDI.

Claims

1. 1. A system for changing a cytosine to a thymine in the genome of a cell, comprising a first fusion protein and a second fusion protein, or first and second expression constructs for expressing said first and second fusion proteins, respectively; a) the first fusion protein comprises: i) a first zinc finger protein (ZFP) domain that binds to a first sequence in a target genomic region in the cell; and ii) a first portion of a cytidine deaminase polypeptide, wherein the cytidine deaminase is a toxin-derived deaminase (TDD) comprising an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 13-24; b) the second fusion protein comprises: i) a second ZFP domain that binds to a second sequence in the target genomic region; and ii) a second portion of the cytidine deaminase polypeptide; c) the first portion and the second portion, by themselves, lack cytidine deaminase activity; d) when the first fusion protein and the second fusion protein bind to the target genomic region, the first and second portions dimerize, and the dimerized portions form an active cytidine deaminase capable of converting cytosines in the target genomic region to thymines; Optionally, the cell is a eukaryotic cell, Optionally, the eukaryotic cell is a mammalian cell or a plant cell; Further optionally, the mammalian cell is a human cell. The system.

2. The system of claim 1 , wherein the target genomic region is specific to a particular allele of a gene in the cell.

3. 3. The system of claim 1 or 2, wherein the cytosine is between a proximal end of the first sequence and a proximal end of the second sequence in the target genomic region, and optionally the proximal ends are spaced apart by 100 bp or less.

4. The system according to any one of claims 1 to 3, comprising two or more pairs of the first and second fusion proteins, each pair of the fusion proteins binding to a different target genomic region.

5. 5. The system of claim 4, wherein the first and second cytidine deaminase portions of one pair of fusion proteins are different from the first and second portions of another pair of fusion proteins.

6. 6. The system of any one of claims 1 to 5, further comprising a nickase that induces a single-stranded DNA break in the non-edited or edited strand, wherein the DNA break is about 500 bp or less, optionally 200 bp or less, optionally about 10-50 bp from the cytosine to be edited.

7. The system of claim 6, wherein the nickase is a ZFP-based nickase, a TALE-based nickase, or a CRISPR-based nickase.

8. 8. The system of claim 7, wherein the nickase is a ZFP-based nickase formed by dimerization of a first nickase domain and a second nickase domain each fused to two ZFP domains that bind to the target genomic region, one of the nickase domains comprising an inactivating mutation.

9. one of the nickase domains is fused to the first or second fusion protein; The other nickase domain is fused to a third ZFP domain that binds to a third sequence in the target genomic region. The system of claim 8.

10. The two nickase domains are i) a third ZFP domain that binds to a third sequence in the target genomic region; and ii) a fourth ZFP domain that binds to a fourth sequence in the target genomic region. are fused into The system of claim 8.

11. The system according to any one of claims 8 to 10, wherein the first and second nickase domains are derived from FokI.

12. a third fusion protein or a third expression construct for expressing said third fusion protein in said cell; e) the third fusion protein comprises: i) a ZFP domain that binds to a third sequence in the target genomic region; and ii) comprises an inhibitory domain of said cytidine deaminase; f) when the third fusion protein binds to the target genomic region, the inhibitory domain binds to the dimerized cytidine deaminase moiety, thereby inhibiting the cytidine deaminase activity of the dimerized cytidine deaminase moiety. A system according to any one of claims 1 to 7.

13. a third fusion protein or a third expression construct for expressing the third fusion protein in the cell, and a fourth fusion protein or a fourth expression construct for expressing the fourth fusion protein in the cell; e) the third fusion protein comprises: i) a ZFP domain that binds to a third sequence in the target genomic region; and ii) comprises a first dimerization domain; f) the fourth fusion protein comprises: i) an inhibitory domain of said cytidine deaminase, and ii) a second dimerization domain capable of pairing with the first dimerization domain in the presence of a dimerization-inducing agent; g) when the third fusion protein binds to the target genomic region and the first and second dimerization domains dimerize, the inhibitory domain binds to the dimerized cytidine deaminase moiety, thereby inhibiting cytidine deaminase activity of the dimerized cytidine deaminase moiety. A system according to any one of claims 1 to 7.

14. a third fusion protein or a third expression construct for expressing the third fusion protein in the cell, and a fourth fusion protein or a fourth expression construct for expressing the fourth fusion protein in the cell; e) the third fusion protein comprises: i) a ZFP domain that binds to a third sequence in the target genomic region; and ii) comprises a first dimerization domain; f) the fourth fusion protein comprises: i) an inhibitory domain of said cytidine deaminase, and ii) a second dimerization domain capable of pairing with the first dimerization domain in the absence of a dimerization inhibitor; g) when the third fusion protein binds to the target genomic region and the first and second dimerization domains dimerize, the inhibitory domain binds to the dimerized cytidine deaminase moiety, thereby inhibiting cytidine deaminase activity of the dimerized cytidine deaminase moiety. A system according to any one of claims 1 to 7.

15. The system of any one of the preceding claims, wherein the ZFP domains independently have 2, 3, 4, 5, 6, 7, or 8 zinc fingers.

16. 2. The system of any one of the preceding claims, wherein the expression constructs are on the same or separate viral vectors.

17. The system of claim 16, wherein the viral vector is an adeno-associated viral (AAV) vector, an adenoviral vector, or a lentiviral vector.

18. The system according to any one of claims 1 to 17, wherein the TDD comprises an amino acid sequence of any one of SEQ ID NOs: 1 to 12.

19. The system of any one of claims 1 to 17, wherein the TDD comprises a toxic domain of the TDD comprising an amino acid sequence of any one of SEQ ID NOs: 1 to 12.

20. The system according to any one of claims 1 to 17, wherein the cytidine deaminase is a TDD comprising an amino acid sequence at least 95% identical to any one of the amino acid sequences of SEQ ID NOs: 13 to 24.

21. The system according to any one of claims 1 to 17, wherein the TDD comprises an amino acid sequence of any one of SEQ ID NOs: 13 to 24.

22. wherein the first and second cytidine deaminase portions comprise SEQ ID NOs:25 and 26, SEQ ID NOs:27 and 28, SEQ ID NOs:29 and 30, SEQ ID NOs:31 and 32, SEQ ID NOs:33 and 34, SEQ ID NOs:35 and 36, SEQ ID NOs:37 and 38, SEQ ID NOs:39 and 40, SEQ ID NOs:41 and 42, SEQ ID NOs:43 and 44, SEQ ID NOs:45 and 46, SEQ ID NOs:47 and 48, SEQ ID NOs:49 and 50, SEQ ID NOs:51 and 52, SEQ ID NOs:53 and 54, SEQ ID NOs:55 and 56, SEQ ID NOs:57 and 58, SEQ ID NOs:59 and 60, SEQ ID NOs:61 and 62, SEQ ID NOs:63 and 64, SEQ ID NOs:65 and 66, SEQ ID NOs:67 and 68, SEQ ID NOs:69 and 70, SEQ ID NOs:71 and 72, SEQ ID NOs:73 and 74, SEQ ID NOs:75 and 76, SEQ ID NOs:77 and 78, SEQ ID NOs:79 and 80, SEQ ID NOs:81 and 82, or SEQ ID NOs:83 and 84, respectively, or vice versa. A system according to any one of claims 1 to 17.

23. 1. A fusion protein comprising: i) a zinc finger protein (ZFP) domain that binds to a gene; and ii) a fragment of a cytidine deaminase polypeptide, wherein the cytidine deaminase is a toxin-derived deaminase (TDD) comprising an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 13-24, optionally wherein the ZFP domain and the cytidine deaminase fragment are linked by a peptide linker, optionally wherein the gene is a eukaryotic gene, and optionally wherein the eukaryotic gene is a human gene.

24. 1. A fusion protein comprising: i) a zinc finger protein (ZFP) domain that binds to a gene; and ii) a cytidine deaminase inhibitory domain, wherein the cytidine deaminase is a toxin-derived deaminase (TDD) comprising an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 13-24, optionally wherein the ZFP domain and the inhibitory domain are linked by a peptide linker, optionally wherein the gene is a eukaryotic gene, and optionally wherein the eukaryotic gene is a human gene.

25. The fusion protein of claim 23 or 24, wherein the TDD comprises an amino acid sequence of any one of SEQ ID NOs: 13 to 24.

26. The fusion protein according to any one of claims 23 to 25, wherein the linker comprises any one of SEQ ID NOs: 85 to 95.

27. a) a first fusion protein comprising i) a zinc finger protein (ZFP) domain that binds to a gene, and ii) a first dimerization domain; and b) a second fusion protein comprising: i) a cytidine deaminase inhibitory domain, wherein the cytidine deaminase is a toxin-derived deaminase (TDD) comprising an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 13-24; and ii) a second dimerization domain. A pair of fusion proteins comprising: the first and second dimerization domains are capable of dimerizing in the presence of a dimerization-inducing agent; Optionally, said gene is a eukaryotic gene, and optionally, said eukaryotic gene is a human gene. The pair of fusion proteins.

28. a) a first fusion protein comprising i) a zinc finger protein (ZFP) domain that binds to a gene, and ii) a first dimerization domain; and b) a second fusion protein comprising: i) a cytidine deaminase inhibitory domain, wherein the cytidine deaminase is a toxin-derived deaminase (TDD) comprising an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 13-24; and ii) a second dimerization domain. wherein the first and second dimerization domains are capable of dimerizing in the absence of a dimerization inhibitor; Optionally, said gene is a eukaryotic gene, and optionally, said eukaryotic gene is a human gene. The pair of fusion proteins.

29. 29. A pair of fusion proteins according to claim 27 or 28, wherein the TDD comprises the amino acid sequence of any one of SEQ ID NOs: 13 to 24.

30. 30. One or more isolated nucleic acid molecules encoding the fusion protein(s) of any one of claims 23 to 29.

31. 31. One or more expression constructs comprising the nucleic acid molecule(s) of claim 30.

32. 32. One or more viral vectors comprising the expression construct(s) of claim 31 , optionally wherein the viral vector is an adeno-associated viral vector, an adenoviral vector, or a lentiviral vector.

33. 32. A cell comprising the system of any one of claims 1 to 22, the fusion protein(s) of any one of claims 23 to 29, the isolated nucleic acid molecule(s) of claim 30, the expression construct(s) of claim 31, or the viral vector(s) of claim 32, said cell optionally being a eukaryotic cell.

34. 34. The cell of claim 33, wherein the cell is a mammalian cell, optionally a human cell, further optionally a human embryonic stem cell or a human pluripotent stem cell.

35. 23. A method for changing a cytosine to a thymine in a target genomic region in a cell, comprising delivering to the cell a system according to any one of claims 1 to 22, optionally wherein the cell is a eukaryotic cell.

36. 36. The method of claim 35, wherein changing the cytosine to the thymine results in a stop codon in the target genomic region.

37. 37. The method of claim 35 or 36, wherein the system targets two or more genomic regions.

38. 38. The method of any one of claims 35 to 37, comprising delivering a system according to any one of claims 13 and 15 to 22 and the dimerization-inducing agent, wherein the agent induces dimerization of the first and second dimerization domains, thereby activating binding of the inhibitory domain to the dimerized cytidine deaminase portion.

39. 38. The method of any one of claims 35 to 37, comprising delivering the system of any one of claims 14 to 22 and the dimerization inhibitor, wherein the agent inhibits dimerization of the first and second dimerization domains, thereby preventing binding of the inhibitory domain to the dimerized cytidine deaminase portion.

40. The method of any one of claims 35 to 39, wherein the cell is an in vivo human cell.

41. The method of any one of claims 35 to 39, wherein the cells are ex vivo human cells.

42. 42. A genetically engineered cell obtainable by the method of claim 41, said genetically engineered cell being optionally a eukaryotic cell, optionally a human cell.

43. 43. A method of treating a patient in need of treatment, comprising delivering to said patient a genetically engineered cell of claim 42, optionally wherein said cell and said patient are human.

44. 43. The genetically engineered cell of claim 42 for use in treating a patient in need of treatment.

45. 43. Use of the genetically engineered cell of claim 42 for the manufacture of a medicament for treating a patient in need thereof.

46. 46. ​​The method, cell or use of any one of claims 43 to 45, wherein the patient has cancer, an autoimmune disorder, an autosomal dominant disease, or a mitochondrial disorder.

47. 46. ​​The method, cell or use of any one of claims 43 to 45, wherein the patient has sickle cell disease, hemophilia, cystic fibrosis, phenylketonuria, Tay-Sachs disease, prion disease, color blindness, lysosomal storage disease, Friedreich's ataxia, or prostate cancer.

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