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Msg  177089 of 178609  at  12/5/2023 11:09:58 AM  by

JBWIN


Building IP: SGMO Patent Grant "Engineered Target Specific Base Editors"

Engineered Target Specific Base Editors

DOCUMENT ID

US 11834686 B2

DATE PUBLISHED

2023-12-05

INVENTOR INFORMATION

NAME

CITY

STATE

ZIP CODE

COUNTRY

Fauser; Friedrich
Richmond
CA
N/A
US
Miller; Jeffrey C.
Richmond
CA
N/A
US
Rebar; Edward
Richmond
CA
N/A
US

APPLICANT INFORMATION

NAME
Sangamo Therapeutics, Inc.
CITY
Richmond
STATE
CA
ZIP CODE
N/A
COUNTRY
US
AUTHORITY
N/A
TYPE
assignee

ASSIGNEE INFORMATION

NAME
Sangamo Therapeutics, Inc.
CITY
Brisbane
STATE
CA
ZIP CODE
N/A
COUNTRY
US
TYPE CODE
02

APPLICATION NO

16/545363

DATE FILED

2019-08-20

DOMESTIC PRIORITY (CONTINUITY DATA)

us-provisional-application US 62867565 20190627
us-provisional-application US 62817153 20190312
us-provisional-application US 62753696 20181031
us-provisional-application US 62721903 20180823

US CLASS CURRENT:

1/1

CPC CURRENT

TYPE

CPC

DATE

CPCI
2013-01-01
CPCI
2013-01-01
CPCI
2013-01-01
CPCI
2013-01-01
CPCI
2013-01-01
CPCI
2013-01-01
CPCA
2013-01-01
CPCA
2013-01-01
CPCA
2013-01-01
CPCA
2013-01-01
CPCA
2017-05-01

Abstract

Described herein are DNA-editing complexes, particularly DNA-editing complexes that specifically alter a single base pair in target DNA sequence as well as methods of making and using these DNA-editing complexes.

Background/Summary

CROSS-REFERENCE TO RELATED APPLICATIONS

(1) The present application claims the benefit of U.S. Provisional Application No. 62/721,903, filed Aug. 23, 2018; U.S. Provisional Application No. 62/753,696, filed Oct. 31, 2018; U.S. Provisional Application No. 62/817,153, filed Mar. 12, 2019; and U.S. Provisional Application No. 62/867,565, filed Jun. 27, 2019, the disclosures of which are hereby incorporated by reference in their entireties.

SEQUENCE LISTING

(1) The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 20, 2019, is named 8325-0180-S180-US1_SL.txt and is 225,511 bytes in size.

TECHNICAL FIELD

(2) The present disclosure is in the fields of polypeptide and genome engineering.

BACKGROUND

(3) Artificial nucleases, such as engineered zinc finger nucleases (ZFN), transcription-activator like effector nucleases (TALENs), the CRISPR/Cas system with an engineered crRNA/tracr RNA (‘single guide RNA’), also referred to as RNA guided nucleases, and/or nucleases based on the Argonaute system are revolutionizing the fields of medicine, biotechnology and agriculture. These molecular tools are allowing the genetic manipulation (e.g. editing) of genomes in organisms to a level never before possible. Artificial nucleases are capable of cleaving DNA such that following such cleavage, the cell is forced to ‘heal’ the break by either error-prone non-homologous end joining (NHEJ) or, in the presence of a substrate DNA with homology to the regions flanking the cut site, by insertion of the substrate DNA through homology-directed repair (HDR). Both of these processes start with a double strand break (DSB) in the DNA.

(4) In some instances, engineered nucleases could possibly result in unwanted consequences (e.g. translocations, inversions and deletions) that may occur due to the induction of multiple DSB in the chromosome of a genetically-edited cell. For example, some evidence of chromosomal rearrangements including translocations, inversions and deletions have been observed following nuclease treatment (Kosicki, et al. (2018) Nat Biotechnol 36:765 and Shin, et al. (2017) Nat Comm doi:10.1038/ncomms15646), and more recently, there has been concern about induction of the p53 pathway following cleavage in some cells leading to apoptosis using the CRISPR/Cas system (Ihry, et al. (2018) Nat Med 24:939-946 and Haapaniemi, et al. (2018) Nat Med 24:927-930). Also, HDR typically is very inefficient in most eukaryotes, making gene correction difficult (Eid, et al. (2018) Biochem J 475:1955-1964).

(5) In addition, Cas9 base editors such as AID-dCas9, APOBEC-dCas9 (e.g. APOBEC3G or APOBEC1), BE2, BE3 and BE4 (see, e.g., Komor, et al. (2016) Nature 533(7603):420-424; Komor, et al. (2017) Science Advances 3(8)_eaao4774; Kim, et al. (2017) Nat Biotechnol 35(4):371-376) can exhibit a lack of specificity (see Kim, et al. (2019) Nat Biotechnol 10.1038/s41587-019-0050-1; Zuo, et al. (2019) Science DOI:10.1126/science.aav9973), rendering them unsuitable for a variety of purposes, including in vivo and ex vivo therapeutic applications.

(6) Thus, there remains a need to accomplish genome (base) editing without inducing a double strand break and with high specificity.

SUMMARY

(7) The present disclosure provides methods and compositions to selectively edit DNA in a cell (for example, a base editor), including editing (e.g., of a single base) without making a double-stranded cut in the target DNA (e.g., the edited genome). Such base editors can be cytosine base editors (CBEs) which change a C:G to a T:A or adenine base editors (ABEs) which change A:T to G:C. Furthermore, because no double-stranded break is induced, there are no free DNA ends in the endogenous target and no translocations occur. Base editors as described herein can be used for gene knock out (e.g., changing a regular codon into a stop codon, for instance using a cytosine base editor and/or mutating a splice acceptor site using either cytosine or adenine base editors); introducing mutations (e.g., activating or repressing mutations) into a control element (e.g., promoter region) of a gene; correcting (reversing) disease-causing mutations (such as point mutations); and/or inducing mutations that that result in therapeutic benefits. The base editors as described herein may be provided (to a cell for in vitro or ex vivo uses or in vivo to a subject) for base editing in polypeptide and/or polynucleotide form. Among other advantages, the base editors of the invention can (1) increase specificity due to the additional DNA binding domain/length of the binding site an increased precision or targeting density due to reduced PAM requirements.; (2) expand (relax) PAM restrictions to allow targeting of sites not currently targetable; (3) increase editing efficiency at poorly performing PAM sites; and/or (4) improve efficiency at target sites targetable with non ZFP-anchored reagents and therefore supports a lower dose which then also results in lower off-target activity.

(8) Thus, described herein are base editing compositions comprising at least one functional domain (e.g., a DNA destabilizing molecule such as a nickase, a protein and/or a nucleotide) and at least one DNA-binding domain (e.g., a zinc finger protein). In certain embodiments, the base editing composition edits an adenine (A) or cytidine (C) base in DNA, wherein the composition comprises: (1) at least one zinc finger protein (ZFP) DNA-binding domain; (2) at least one DNA destabilizing molecule; and (3) at least one adenine or cytosine deaminase, wherein the composition does not make a double-stranded cut in the DNA.

(9) Any DNA destabilizing molecule may be used in the compositions described herein in any combination, including but not limited to a Cas9 nickase, a Cas9 protein (e.g., dCas) operably linked to a single guide RNA (sgRNA), any RNA programmable system, a zinc finger nuclease nickase (ZFN nickase), a TALEN nickase, one or more proteins such as those shown in Table A, and/or one more nucleotides (e.g., one or more peptide nucleic acids (PNAs), locked nucleic acids (LNAs) and/or bridged nucleic acids (BNAs)). In certain embodiments, the base editing composition comprises more than one DNA destabilizing molecule, for example one or more proteins (e.g., Table A, nickases, etc.) and/or one or more nucleotides. In certain embodiments, the composition comprises a ZFN nickase and one or more additional proteins and/or nucleotide DNA destabilizing molecules (e.g., one or more proteins of Table A and/or one or more nucleotides as described herein). In certain aspects, the base editing composition does not comprise a Cas9 protein, but may comprise other Cas protein (e.g, non-Cas9 RNA programmable systems). In certain embodiments, the DNA-destabilizing molecule comprises a zinc finger nuclease (ZFN) nickase.

(10) The at least one zinc finger protein (ZFP) DNA-binding domain of the base editing composition may be operably linked to one or more of the other components of the base editing composition, for example to one or more of the DNA destabilizing molecules (e.g., to Cas9 nickase, dCas9, etc.) and/or to the at least one adenine or cytosine deaminase. In certain embodiments, at least one ZFP DNA-binding domain is operably linked to the adenine or cytosine deaminase. In other embodiments, the base editing composition comprises first and second ZFP DNA-binding domains, wherein the first ZFP DNA-binding domain is operably linked to the Cas9 nickase. The ZFP DNA-binding domain may comprise 3, 4, 5, 6 or more fingers and may bind to a target site on either side (5′ or 3′) of the targeted base to be edited. In certain embodiments, the ZFP binds to a target site that is 1 to 100 (or any number therebetween) nucleotides on either side of the targeted base. In other embodiments, the ZFP binds to a target site that is 1 to 50 (or any number therebetween) nucleotides on either side of the targeted base.

(11) Any adenine or cytosine deaminase can be used in the compositions described herein, including wild-type and/or evolved domains. In certain embodiments, the adenine or cytosine deaminase is comprised of first and second inactive domains that dimerize to form an active adenine or cytosine deaminase. In certain embodiments, the first inactive domain of the adenine or cytosine deaminase is operably linked to the Cas9 nickase and the second inactive domain of the adenine or cytosine deaminase is operably linked to a ZFP DNA-binding domain. In still further embodiments, the adenine or cytosine deaminase and the ZFP DNA-binding domain are both operably linked to the Cas9 nickase. In other embodiments, the base editor comprises first and second ZFP DNA-domains, the first ZFP operably linked to the Cas9 nickase and the second ZFP DNA-binding domain operably linked to the adenine or cytosine deaminase.

(12) One or more polynucleotides encoding one or more base editing compositions as described herein are also provided. The polynucleotides may be carried on viral (e.g., AAV, Ad, etc.) and/or non-viral (e.g., plasmid, mRNA, etc.) vectors. Furthermore, a cell or population of cells comprising one or more compositions and/or the one or more polynucleotides as described herein are also provided, as well as descendants of such cells, wherein the cells comprise an edited base.

(13) Also provided are methods of editing a base in a target DNA (e.g., DNA double stranded endogenous gene or extrachromosomal (episomal) sequence) using one or more of the compositions and/or polynucleotides as described herein. In certain embodiments, the methods comprise: (i) editing a cytidine base (“C”) to a uracil base (“U”), optionally wherein the U is replaced with a thymidine base (“T”) during DNA replication; (ii) editing an adenine base (“A”) to an inosine (“I”), optionally wherein the I replaced with a guanine base (“G”) during replication; and/or (iii) editing a CA or AC dinucleotide to a UI or an IU. In other embodiments, the editing in the cell results in: (i) changing a C:G base pair to an T:A base pair; (ii) changing a C:G base pair to a G:C base pair; (iii) changing an A:T base pair to a G:C base pair; (iv) introduction of a stop codon; and/or (v) editing or creating a splicing sequence. The methods may be used to correct any disease mutation (e.g., point mutation), including in an exon or in an intron. wherein DNA in a chromosome or an extrachromosomal episome in the cell or the subject is edited. The method may be performed in vitro, ex vivo, or in vivo.

(14) In one aspect, described herein are compositions and systems comprising a DNA-editing composition (e.g., a base editing composition, also referred to herein as a base editing complex). The DNA-editing complex comprises at least one functional domain and a DNA-binding domain. In certain embodiments, the DNA-editing composition complex comprises a fusion molecule comprising a DNA-binding domain and, in addition, at least one DNA destabilizing molecule such as a nickase domain that makes a single-stranded cut in double-stranded DNA (e.g., a DNA-nickase). In other embodiments, the DNA-editing composition (complex) comprises multiple (two or more) fusion molecules, for example a first catalytically active fusion molecule comprising a nickase including a first DNA-binding domain and nickase domain and a second catalytically inactive fusion molecule comprising a second DNA-binding domain and optionally one or more additional fusion molecules, each comprising an additional DNA-binding domain and one or more functional domains as described herein. In certain embodiments, the base editor comprises a composition as shown in any of FIGS. 1A through 1D. In certain embodiments, binding of the first and second (and optionally additional DNA binding domains) results in base-editing, for example when the catalytically active and catalytically inactive fusion molecules dimerize. In some embodiments, the optional additional DNA binding domains bind to double stranded DNA, while in other embodiments, the DNA binding domains bind to single stranded DNA. In some embodiments, the DNA nickase is a ZFN nickase, a TALEN nickase or a CRISPR/Cas nickase, in which at least one functional (nickase) domain is operably linked to a DNA binding domain (e.g. a ZFP DNA binding domain, a TALE DNA binding domain and a sgRNA for use with a CRISPR/Cas system). In some embodiments, the DNA nickase (e.g., fusion molecule) comprises a linker sequence between the nickase domain and the DNA binding domain. The nickase domain(s) may be positioned on either side of the DNA-binding domain, including at the N- or C-terminal side of the fusion molecule (N- and/or C-terminal to DNA-binding domain). In some embodiments, the linkers are selected from a bacterial selection system from a large linker library (>10e8 members). In some embodiments, the linkers range from four to 22 amino acid residues. In some embodiments, the linkers allow for specific positioning of a functional domain (for example a nickase domain) relative to a DNA binding domain (for example, linkage of the nickase domain to the N- or C-terminal side of the DNA binding domain). In some examples, the linker is selected using the method disclosed in Paschon, et al. (2019) Nat Commun. 10:1133. One or more polynucleotides (e.g., constructs) encoding base editors (or components thereof) are also provided.

(15) The DNA-editing complexes as described herein comprise one or more functional domains, including, but not limited to, one or more adenine deaminase domains, one or more cytidine deaminases, and/or one or more uracil DNA glycosylase inhibitors. One or more functional domains may be included in the catalytically active and/or the catalytically inactive fusion molecule of the DNA-editing complexes described herein. In some embodiments, the cytidine deaminase is an apolipoprotein B mRNA-editing complex 1 (APOBEC1) domain. In some embodiments, the cytidine deaminase is an Activation Induced Deaminase (AID). In some embodiments, the deaminase is an adenine deaminase. In some embodiments, the adenine deaminase is a wild-type or mutated (evolved) TadA (tRNA adenine deaminase (see Gaudelli, et al. (2017) Nature 551:464-471). In some embodiments, the adenine deaminase is ABE 7.8, ABE 7.9 or ABE 7.10 (Gaudelli, ibid) or ABEmax (Koblan, et al. (2018) Nat Biotechnol. 36(9):843-846). In some embodiments, the deaminase (adenine or cytidine) functional domain is assembled from two polypeptides comprising operably linked zinc fingers (e.g., a split enzyme) or from one or more ZFPs operably linked to one part of the split enzyme and a Cas9 nickase operably linked to the other component of the split enzyme (see, e.g., FIG. 1B). In some embodiments, assembly of the deaminase is driven by the binding of the operably linked zinc fingers to DNA targets such that the polypeptides are positioned to allow assembly. In some embodiments, the base editor further comprises a uracil DNA glycosylase inhibitor (UGI).

(16) In one aspect, the base editor comprises a DNA-unwinding (also referred to as DNA-destabilizing) system derived from a CRISPR system, for instance a Cas9 (e.g., naturally occurring and/or engineered Cas9) protein (e.g., nickase) or a non-Cas9 protein. In certain embodiments, the base editor is a Cas9 base editor further comprises a zinc finger protein DNA-binding domain, which ZFP may be operably linked to any component of the Cas9 protein (e.g., wild-type or engineered nickase) in any orientation, for example a base editor comprising a ZFP (a ZFP anchor) operably linked to the Cas9 protein, the sgRNA of the Cas9 nickase or the deamimase (wild-type or engineered (evolved) ABE or CBE). In certain embodiments, the ZFP is operably linked to the Cas9 domain of the base editor. In certain embodiments, the base editor comprises the components as shown in the Cas9 base editors of FIG. 3.

(17) In another aspect, the base editor does not comprise a DNA-unwinding (DNA-destabilizing) element derived from a Cas9 protein (also referred to as “Cas9-free”). In certain embodiments, the Cas9-free base editors of the invention comprise a ZFP-deaminase fusion protein and a ZFN nickase, and optionally one or more DNA-destabilizing factors. In certain embodiments, the DNA-destabilizing factor is a protein (e.g., as shown in Table A) or an oligonucleotide (e.g., one or more PNAs, LNAs and/or BNAs). The one or more non Cas9 DNA-destabilizing (unwinding) factor(s) (e.g., proteins of Table A, LNAs, PNAs, BNAs, etc.) may be operably linked to any component of the base editor, for example either component of the ZFP-deaminase fusion protein and/or any of the components of the ZFN nickase. In some embodiments, the base editor comprises one or more protein and one or more nucleotide DNA-destabilizing (unwinding) factors. In still further embodiments, the Cas9-free base editors described herein comprise one or more proteins derived from a CRISPR system, which proteins are not Cas9 but have DNA-destabilizing (unwinding) properties.

(18) In certain embodiments, the base editor comprises one or more nucleotide sequences, for example one or more DNA oligonucleotides, RNA oligonucleotides, peptide nucleic acids (PNAs), locked nucleic acids (LNAs) and/or bridged nucleic acids (BNAs), which can be used to provide a single stranded DNA substrate for base editors at the target site. This can be facilitated by e.g. duplex invasion, triplex invasion or a tail-clamp (Quijano, et al. (2017) Yale J. Biol and Med. 90:583-598; Pellestor and Paulasova (2004) European J. Human Genetics 12:694-700; Schleifman, et al. (2011) Chem Biol. 18:1189-1198). The structure of the one or more nucleotide sequences of the base editor will vary in length; number and position of DNA and/or RNA and/or LNA and/or LNA and/or BNA bases; phosphorothioate bonds; other common modifications of these oligonucleotides depending on the target sequence composition.

(19) In certain embodiments, the base editor comprises one or more PNAs, for example, gamma PNAs containing miniPEG substitutions and the gamma position for enhanced binding, increased solubility and improved delivery (Bahal, et al. (2014) Current Gene Ther. 14(5):331-342. In certain embodiments, the PNAs comprise one or more O indicates 8-amino-2,6-dioxaoctanoic acid linkers and/or one or more cytosines (C) or pseudoisocytosine residues. Optionally, one or more lysine (Lys) residues are included in the PNA, for example on the N- and/or C-terminals of the PNA sequence. In certain embodiments, 1, 2, 3, 4, 5 or more Lys residues are included at one or both terminals of the PNA. In certain embodiments, two or more PNAs are used in the base editor, for example in the same or reverse orientation relative to each other. In certain embodiments, the PNA comprises one or more PNAs as shown in FIGS. 8B to 8E, including but not limited to one or more PNAs of the structure: N-Lys-Lys-Lys-NNNNNNNNNN-OOO-NNNNNNNNNN-Lys-Lys-Lys-C; N-Lys-Lys-Lys-NNNNNNNNNN-OOO-NNNNNNNNNNNNNNN-Lys-Lys-Lys-C; N-Lys-Lys-Lys-NNNNNNNNNN-OOO-NNNNNNNNNN-Lys-Lys-Lys-C; N-Lys-Lys-Lys-NNNNNNNNNN-OOO-NNNNNNNNNN-Lys-Lys-Lys-C; and/or N-Lys-Lys-Lys-NNNNNNNNNNNNNNN-Lys-Lys-Lys-C, where O indicates 8-amino-2,6-dioxaoctanoic acid linkers and C indicates cytosine. The Lys resides on the N- and/or C-terminals of the PNA sequence are optional and pseudoisocytosine be can substituted for cytosine.

(20) In other embodiments the base editor comprises one or more LNAs. LNAs can include a stacking linker and 2′-glycylamino-LNA for improved performance (Geny, et al. (2016) Nucleic Acids Res. 44(5):2007-2019). In certain embodiments, the LNA comprise one or more phosphorothioate bonds, optionally between one or more LNA residues and/or DNA residues. In other embodiments, the LNA comprises one or more Cholesterol-TEG, which may increase uptake into cells. In certain embodiments, the base editor comprises one or more LNAs as shown in FIG. 8F or 8G, including but not limited to one or more LNAs of the structure: 5′-NnNnNnNnNnNnNnNtctct nNnNnNnNnNnnNnnNnnNn-3′ (SEQ ID NO: 1); 5′-N*n*NnNnNnNnNnNnNtctctnNnNnNnNnNnNnNnNnnNnnNnnNnn*N*n-3′ (SEQ ID NO:69); and/or 5′-NnNnNnNnNnNnNnNtctct nNnNnNnNnNnnNnnNnnNn-Chol-TEG-3′ (SEQ ID NO:70), where LNA nucleotides are shown in uppercase; DNA nucleotides are in lower case; “*” indicates phosphorothioate bonds; and Chol-TEG indicates 3′ Cholesterol-TEG for increased uptake into cells.

(21) The components of the base-editing compositions described herein may be included in any combination (one or more nickase domains, one or more DNA-binding domains, one or more functional domains, etc.) and these components may be positioned in any order relative to each other. In some embodiments, the UGI, cytidine and/or adenine deaminase is(are) N-terminal of the DNA-binding domain of the catalytically inactive fusion molecule and/or N-terminal to the nickase domain of the catalytically active fusion molecule of the DNA-editing complex. In some embodiments, cytidine and/or adenine deaminase and/or UGI is(are) C-terminal of the DNA-binding domain of the catalytically inactive fusion molecule and/or C-terminal to the nickase domain of the catalytically active fusion molecule. In some embodiments, the one or more UGIs, cytidine and/or adenine deaminase(s) is(are) positioned between the DNA binding domain and the nickase domain(s) (in the catalytically active domain). In some embodiments, the fusion molecule comprises a cytidine deaminase and an adenine deaminase domain or a UGI, wherein the UGI, cytidine and adenine deaminases are positioned in any way with regard to the DNA-binding domain, each other and/or the nickase domain (e.g., both N-terminal to the DNA-binding domain of the catalytically inactive fusion molecule in any order, both C-terminal to the DNA-binding domain of the catalytically inactive fusion molecule in any order, one N-terminal to the DNA-binding domain of the catalytically inactive fusion molecule, one C-terminal to the DNA-binding domain of the catalytically inactive fusion molecule, N-terminal to the nickase domain and/or DNA-binding domain of the catalytically active fusion molecule, C-terminal to the nickase domain and/or DNA-binding domain of the catalytically active fusion molecule, one C-terminal to the nickase domain and/or DNA-binding domain of the catalytically active fusion molecule, one N-terminal to the nickase domain and/or DNA-binding domain of the catalytically active fusion molecule, between the nickase domain and the DNA-binding domain of the catalytically active fusion molecule, etc.). Non-limiting examples of configurations of one or more fusion molecules of the base-editing compositions are shown in the appended Figures and Examples. In some embodiments, the UGI, cytidine and/or adenine deaminase domains are linked to the other members of the DNA-editing complex using linkers known in the art. One or more polynucleotides encoding the base editors or components thereof are also provided.

(22) In still further aspects, the DNA-editing complex comprises one or more functional domains comprising at least one uracil DNA glycosylase inhibitor (e.g. UGI) domain. The, which UGI domain(s) is(are) incorporated into the DNA-editing complex in any way such that the DNA-editing complex is operable. In some embodiments, the base editing complex comprises a bacteriophage Gam protein. In some embodiments, the base editing complex comprises a deaminase, a nickase, a UGI and/or a GAM protein. In some embodiments, the components of the base editing complex are provided in one, two or more gene expression constructs encoding one, two or more fusion proteins. In some embodiments, one or more uracil DNA glycosylase inhibitor domain(s) is/are linked to the other members of the complex using the linkers described above and known in the art. In some embodiments, a linker is used to link the uracil DNA glycosylase inhibitor to other members of the complex wherein the linker is identified using the method disclosed in Paschon, et al. (2019) Nat Commun. 10:1133.

(23) In some embodiments, the DNA-editing (base editing) complex further comprises a molecule to assist in opening a double-strand DNA helix. In some embodiments, the molecule comprises an enzyme. In some embodiments, the enzyme is a helicase (for example, RecQ helicases (WRN, BLM, RecQL4 and RecQ5, (see Mo, et al. (2018) Cancer Lett. 413:1-10), DNA2 (Jia, et al. (2017) DNA Repair (Amst). 59:9-19) and any other eukaryotic helicases including for example, FANCJ, XPD, XPB, RTEL1, and PIF1 (Brosh (2013) Nat Rev Canc 13(8):542-558)). In some embodiments, the enzyme is a bacterial and/or a viral helicase. Exemplary viral helicases include those encoded by the Myoviridae family of viruses (for example gp41, Dda, UvsW, Gene α, and Ban); those encoded by the Podpviridae family of viruses (for example 4B); those encoded by the Siphoviridae, Baculoviridae, Herpesviridae, Polyomaviridae, Palillomaviridae and Poxviridae families (for example, G40P, p143, UL5, UL9, Tag, E1, NPH—I, NPH-II, A18R, and VETF), or any other viral helicase known in the art (see e.g. Frick and Lam (2006) Curr Pharm Des 12(11): 1315-1338). In some embodiments, the helicase enzyme is a bacterial enzyme. Exemplary bacterial helicases include the P. aeruginosa SF4 DnaB-like helicase, or the RecB and RecD helicases that are part of the bacterial RecBCD complex in bacteria such as E coli and H. pylori (Shadrick, et al. (2013) J. Biomol Screen 18(7):761-781). In some embodiments, engineered or evolved variants of multimeric helicases are used which result in monomeric helicase activity (see e.g. Brendza, et al. (2005) PNAS 102(29):10076-70081). In some embodiments, the molecule comprises a CRISPR/Cas complex. In some embodiments, the CRISPR/Cas complex comprises a guide RNA. In some embodiments, the complex comprises a Cas enzyme that is catalytically defective in its nuclease domains. In some embodiments, the complex comprises a Cas enzyme that is catalytically defective in one of its nuclease domains (for example a nickase). In some embodiments, the Cas enzyme is defective in its PAM recognition (Anders, et al. (2014) Nature 513(7519):569-573). In some embodiments, the Cas enzyme has relaxed (expanded) PAM requirements as compared to native PAM sequences (see for example Nishimasu, et al. (2018) Science 361:1259-1262). In certain embodiments, the Cas base editor as described herein exhibits relaxed (expanded) PAM requirements as compared to the NGG PAM sequence of SpCas9. In some embodiments, the molecule has helix-destabilizing properties. Exemplary helix-destabilizing molecules include ICP8 from herpes simplex virus type I (Boehmer and Lehman (1993) J Virol 67(2):711-715), Puralpha (Darbinian, et al. (2001) J Cell Biochem 80(4):589-95), and calf thymus DNA helix-destabilizing protein (Kohwi-Shigematsu, et al. (1978) Proc Natl Acad Sci USA 75(10):4689-93). In some embodiments, the molecule is involved in transcription and/or D loop formation/stabilization. Exemplary molecules of this class include Rad51, Rad52, RPA1, RPA2 and RPA3, Exol, BLM, and HMGB1 and HMGB2. Other proteins that can be utilized include Bovin ROA1 and E. coli RecA or E. coli rad51. Other protein domains that may act as DNA helix destabilizers include the RecI and Rec II domain from Cas9 or the RecII domain on its own, as well as any other helix destabilizing region from Cas9. Other non-limiting examples of suitable protein domains for use in the base editors described herein are shown in Table A.

(24) In some embodiments, the molecule is a nucleic acid, including but not limited to oligonucleotides, PNAs, LNAs, BNAs and the like. In some embodiments, the nucleic acid is a DNA with homology to the region near the targeted editing. In some embodiments, the nucleic acid is an RNA with homology to the region near the targeted editing. In some embodiments, the RNA is modified. In some embodiments, the fusion molecule comprises amino acid linker sequences between one or more domains of the fusion molecule. In some embodiments, the molecule(s) used to assist in opening a double-strand DNA helix is/are linked to the other members of the DNA-editing complex using the linkers described above. In some embodiments, the molecule(s) used to assist in opening a double strand DNA helix is linked to the other members of the DNA editing complex is identified using known methods.

(25) In certain embodiments, the nucleic acid comprises a PNA, for example a PNA comprising one or more O indicates 8-amino-2,6-dioxaoctanoic acid linkers and/or one or more cytosines (C) or pseudoisocytosine residues. Optionally, one or more lysine (Lys) residues are included in the PNA, for example on the N- and/or C-terminals of the PNA sequence. In certain embodiments, 1, 2, 3, 4, 5 or more Lys residues are included at one or both terminals of the PNA. In certain embodiments, two or more PNAs are used in the base editor, for example in the same or reverse orientation relative to each other. In certain embodiments, the one or more PNAs comprise: N-Lys-Lys-Lys-NNNNNNNNNN-OOO-NNNNNNNNNN-Lys-Lys-Lys-C; N-Lys-Lys-Lys-NNNNNNNNNN-OOO-NNNNNNNNNNNNNNN-Lys-Lys-Lys-C; N-Lys-Lys-Lys-NNNNNNNNNN-OOO-NNNNNNNNNN-Lys-Lys-Lys-C; N-Lys-Lys-Lys-NNNNNNNNNN-OOO-NNNNNNNNNN-Lys-Lys-Lys-C; and/or N-Lys-Lys-Lys-NNNNNNNNNNNNNNN-Lys-Lys-Lys-C(PNA #5), wherein O indicates 8-amino-2,6-dioxaoctanoic acid linkers and C indicates cytosine. The Lys resides on the N- and/or C-terminals of the PNA sequence are optional and pseudoisocytosine be can substituted for cytosine. See, also, FIGS. 8B to 8E.

(26) In other embodiments the base editor comprises one or more LNAs. LNAs can include a stacking linker and 2′-glycylamino-LNA for improved performance (Geny, et al. (2016) Nucleic Acids Res. 44(5):2007-2019. In certain embodiments, the LNA comprise one or more phosphorothioate bonds, optionally between one or more LNA residues and/or DNA residues. In other embodiments, the LNA comprises one or more Cholesterol-TEG, which may increase uptake into cells. In certain embodiments, the one or more LNAs comprise: 5′-NnNnNnNnNnNnNnNtctct nNnNnNnNnNnnNnnNnnNn-3′ (SEQ ID NO: 1); 5′-N*n*NnNnNnNnNnNnNtctctnNnNnNnNnNnNnNnNnnNnnNnnNnn*N*n-3′ (SEQ ID NO:69); and/or 5′-NnNnNnNnNnNnNnNtctct nNnNnNnNnNnnNnnNnnNn-Chol-TEG-3′ (SEQ ID NO:70), where LNA nucleotides are in uppercase; DNA nucleotides are in lower case; “*” indicates phosphorothioate bonds; and “Chol-TEG” indicates 3′ Cholesterol-TEG for increased uptake into cells. See, also, FIGS. 8F and 8G.

(27) These molecules may all be incorporated into the base editing system described herein, and may act to increase editing efficiency, decrease off target base editing, adjust the base editing window or alter the targeted type of nucleic acid base.

(28) In some embodiments, functional domains as described herein are included in single fusion molecule. Alternatively, DNA-editing complexes that include multiple functional domains may be separated into separate fusion molecules in any way. In some embodiments, one fusion molecule comprises a DNA binding domain, a cytidine and/or adenine deaminase and a UGI, while a second fusion molecule comprises a nickase or half-nickase domain. In some embodiments, one fusion molecule comprises a catalytically inactive (dead) FokI domain fused to a DNA binding domain fused to a deaminase domain, and the second fusion protein comprises a half FokI nickase protein, a DNA binding domain and a UGI domain. In some embodiments, one fusion protein comprises a catalytically inactive (dead) FokI domain fused to a deaminase domain fused to a UGI domain while a second fusion molecule comprises a functional nickase protein. In some embodiments, the one or more fusion proteins disclosed herein are fused in any order of domains within the fusion molecule that is operable. In some embodiments, the nickase domain is a Cas nickase domain, and in some embodiments, the nickase domain is a TALEN nickase domain. In some embodiments, one or more of the functional domains are linked to one or more other members of the complex using the linkers described above. In some embodiments, the one or more functional domains are linked to one or more other members of the complex using linkers identified using the methods disclosed in Paschon, et al. (2019) Nat Commun. 10:1133.

(29) The base editor(s) described herein may be encoded by one or more polynucleotides. The one or more polynucleotides may be carried on viral vectors (AAV, Ad, etc.), non-viral vectors (plasmid, mRNA, etc.) or combinations thereof. In certain embodiments, one polynucleotide includes all the components of the base editor while in other embodiments, the components of the base editor are carried by two or more polynucleotides (e.g., separate polynucleotides carrying split enzymes and/or ZFPs).

(30) In another aspect, described herein are methods of editing (e.g., gene editing) of a DNA molecule using one or more DNA-editing complexes as described herein. The methods described introducing one or more DNA-editing complexes into a cell such that the DNA molecule is edited. The cell may be isolated or may be in living subject (e.g., via intravenous or other administration to the subject). In some embodiments, the DNA molecule is a chromosome or an extrachromosomal episome in a cell. In some embodiments, the chromosome or extrachromosomal episome comprises a cytidine base (“C”) that is deaminated to a uracil base (“U”) by the fusion protein disclosed herein. In some embodiments, the U is replaced with a thymidine base (“T”) during DNA replication. In some embodiments, the chromosome or extrachromosomal episome comprises an adenine base (“A”) that is deaminated to an inosine (“I”) base by the fusion protein disclosed herein. In some embodiments, the I is replaced with a guanine base (“G”) during replication. In some embodiments, the chromosome or extrachromosomal episome comprises an adenine and a cytidine base that are deaminated by the deaminases disclosed herein such that a CA or AC dinucleotide is deaminated into a UI or an IU dinucleotide (FIG. 1 for exemplary systems).

(31) In some embodiments, the nickase domain is derived from a FokI DNA cleavage domain (see U.S. Pat. Nos. 5,436,150; 8,703,489; 9,200,266; and 9,631,186). In some embodiments, the FokI nickase comprises one or more mutations as compared to a parental FokI nickase. Mutations as described herein, include but are not limited to, mutations that change the charge of the cleavage domain, for example mutations of positively charged residues to non-positively charged residues (e.g., mutations of K and R residues (e.g., mutated to S); N residues (e.g., to D), and Q residues (e.g., to E); mutations to residues that are predicted to be close to the DNA backbone based on molecular modeling and that show variation in FokI homologs; and/or mutations at other residues (e.g., U.S. Pat. No. 8,623,618 and Guo, et al. (2010)J. Mol. Biol. 400(1):96-107). Nickases can be ZFN nickases, TALEN nickases and CRISPR/Cas systems such as Cas nickases.

(32) In some embodiments, the base editors comprise DNA-binding domains (e.g., engineered nickase domains) comprising cleavage domains that are derived from FokI or FokI homologues and comprise a mutation in one or more of amino acid residues 416, 418, 422, 447, 448, 476, 479, 481 and/or 525, numbered relative to the wild-type full length FokI as shown in SEQ ID NO:5, or corresponding residues in FokI homologues. In some embodiments, the cleavage half domains derived from FokI comprises a mutation in one or more of amino acid residues 414-426, 443-450, 467-488, 501-502, and/or 521-531, including one or more of 387, 393, 394, 398, 400, 416, 418, 422, 427, 434, 439, 441, 442, 444, 446, 448, 472, 473, 476, 478, 479, 480, 481, 487, 495, 497, 506, 516, 523, 525, 527, 529, 534, 559, 569, 570, and/or 571. The mutations may include mutations to residues found in natural restriction enzymes homologous to FokI at the corresponding positions. In some embodiments, the mutations are substitutions, for example substitution of the wild-type residue with any different amino acid, for example alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), histidine (H), phenylalanine (F), glycine (G), asparagine (N), serine (S) or threonine (T). In some embodiments, the FokI nuclease domain comprises a mutation at one or more of 416, 418, 422, 476, 447, 479, 481 and/or 525 (numbered relative to wild-type, SEQ ID NO:5). The nuclease domains may also comprise one or more mutations at positions 418, 432, 441, 448, 476, 481, 483, 486, 487, 490, 496, 499, 523, 527, 537, 538 and 559, including but not limited to ELD, KKR, ELE, KKS. See, e.g., U.S. Pat. No. 8,623,618. In some embodiments, the cleavage domain includes mutations at one or more of the residues 419, 420, 425, 446, 447, 470, 471, 472, 475, 478, 480, 492, 500, 502, 521, 523, 526, 530, 536, 540, 545, 573 and/or 574. In certain embodiments, the variant cleavage domains described herein include mutations to the residues involved in nuclease dimerization (dimerization domain mutations), and one or more additional mutations; for example to phosphate contact residues: e.g. dimerization mutants (such as ELD, KKR, ELE, KKS, etc.) in combination with one, two, three, four, five, six or more mutations at amino acid positions outside of the dimerization domain, for example in amino acid residues that may participate in phosphate contact. In some embodiments, the mutation at positions 416, 418, 422, 447, 448, 476, 479, 481 and/or 525 comprise replacement of a positively charged amino acid with an uncharged or a negatively charged amino acid. In other embodiments, mutations at positions 446, 472 and/or 478 (and optionally additional residues for example in the dimerization or catalytic domains) are made. In some embodiments, the mutations comprise I479Q and/or Q481A mutations.

(33) In some embodiments, the engineered cleavage half domain comprises mutations in the dimerization domain, for example, amino acid residues 490, 537, 538, 499, 496 and 486 in addition to the mutations described herein. In some embodiments, the invention provides fusion proteins wherein the engineered cleavage half-domain comprises a polypeptide in which the wild-type Gln (Q) residue at position 486 is replaced with a Glu (E) residue, the wild-type Ile (I) residue at position 499 is replaced with a Leu (L) residue and the wild-type Asn (N) residue at position 496 is replaced with an Asp (D) or a Glu (E) residue (“ELD” or “ELE”) in addition to one or more mutations described herein. In some embodiments, the engineered nickase half domains are derived from a wild-type FokI or FokI homologue cleavage half domain and comprise mutations in the amino acid residues 490, 538 and 537, numbered relative to wild-type FokI (SEQ ID NO:5) in addition to the one or more mutations at amino acid residues 416, 418, 422, 447, 448, 476, 479, 481 or 525. In some embodiments, the invention provides a fusion protein, wherein the engineered nickase half-domain comprises a polypeptide in which the wild-type Glu (E) residue at position 490 is replaced with a Lys (K) residue, the wild-type Ile (I) residue at position 538 is replaced with a Lys (K) residue, and the wild-type His (H) residue at position 537 is replaced with a Lys (K) residue or an Arg (R) residue (“KKK” or “KKR”) (see U.S. Pat. No. 8,962,281, incorporated by reference herein) in addition to one or more mutations described herein (see U.S. Patent Publication No. 2018/0087072).

(34) In some embodiments, fusion molecules comprising a DNA binding domain and an engineered FokI or homologue thereof cleavage half-domain as described herein that produce an artificial nuclease are provided. In some embodiments, the DNA-binding domain of the fusion molecule is a zinc finger binding domain (for example, an engineered zinc finger binding domain, ZFP). In some embodiments, the one or more of the zinc fingers are linked together using linkers identified using the methods disclosed in Paschon, et al., supra. In some embodiments, the DNA-binding domain is a TALE DNA-binding domain (TALE). In some embodiments, the DNA binding domain comprises a DNA binding molecule (e.g. guide RNA) and a catalytically inactive Cas or Cpf1 (also known as Cas12a) protein (for example dCas9 or dCpf1). In some embodiments, the DNA binding domain comprises a ZFP fused to a catalytically inactive Cas (dCas) protein. In some embodiments, the ZFP-dCas fusion protein comprises mutations to alter the PAM specificity. In some embodiments, the ZFP-dCas protein is not dependent on PAM recognition to specifically bind to a DNA sequence. In some embodiments, the DNA binding domain comprises a TALE fused to a dCas protein. In some embodiments, the TALE-dCas fusion protein comprises mutations to alter the PAM specificity. In some embodiments, the TALE-dCas protein is not dependent on PAM recognition to specifically bind to a DNA sequence. In any of the above embodiments, the linkers used to link the DNA binding domain (for example, ZFP, TALE or guide RNA and Cas system) to the engineered FokI or homologue thereof are identified using the methods known in the art. See, e.g., Paschon, et al. (2019) Nat Commun. 10:1133.

(35) In some embodiments, the DNA-editing complex edits specific DNA bases in a double stranded DNA. In some embodiments, the edits are made in a DNA molecule within a cell. In some embodiments, the DNA is in a chromosome in a cell. In some embodiments, the editing results in the change from a C:G base pair to a T:A base pair. In some embodiments, the editing results in a change from a C:G base pair to a G:C base pair. In some embodiments, the editing results in a change from a A:T base pair to a G:C base pair. In some embodiments, the editing is done in an exon. In some embodiments, the editing results in the introduction of a stop codon (for example TAA, TAG, TGA). In some embodiments, the base editing results in the knock-out of gene expression of a targeted gene. In some embodiments, the editing is done in a sequence encoding a splicing sequence (for example, a U2 splice sequence wherein a 5′ consensus sequence is G T A/G A/C/T G T/G/A/C A/G/T/C (T/C/G/A).sub.3 (SEQ ID NO:73) and the 3′ consensus sequence is (T/C).sub.10 T/C/A/G C/T A G (SEQ ID NO:74); and a U12 splice sequence wherein a 5′ consensus sequence is G/A T A T C T T/C and a 3′ consensus sequence is (T/G/A/G).sub.2 T/A/C/G (T/C/A/G).sub.2 C/T A G/C, see Turunen, et al. (2013) Wiley Interdiscip Rev RNA. 4(1):61-76). In some embodiments, a new splicing sequence is created. In some embodiments, a splicing sequence is altered such that it no longer functions as a splicing sequence. In some embodiments, alteration of a splicing sequence causes exon skipping. In some embodiments, a sequence is altered such that a rare codon in created. In some embodiments, base editing causes correction of a point mutation in a DNA sequence such that a gene associated with a disease is corrected. Non-limiting examples of base editing for treatment and/or prevention of disease include editing of JAK2 such that the V617F version is no longer expressed (thereby reducing activation of this gene which leads to uncontrolled blood cell production); base editing to knock out or repress other cancer genes such as BCR/ABL; base editing of A1AT; and the like. Exemplary diseases that may be treated include sickle cell disease, hemophilia, cystic fibrosis, phenylketonuria, Tay-Sachs, color blindness, Fabry disease, Friedreich's ataxia, prostate cancer, and many others.

(36) In some embodiments, the base editing complexes as disclosed herein act on RNA molecules. In some embodiments, the base editors utilize an RNA-specific deaminase such as ADAR2 (adenosine deaminase acting on RNA type 2) (see Cox, et al. (2017) Science 358(6366):1019-1027).

(37) Also disclosed herein are cells comprising any of the compositions (base-editing compositions and/or one or more polynucleotides encoding these compositions) as well as cells descended from these cells that have been modified by the methods and compositions disclosed herein. In some embodiments, the cell is a bacterial cell or a eukaryotic cell. In some embodiments, the cells comprise a base-editor complex and a base-editor complex induced DNA or RNA modification. The modified cells, and any cells derived from the modified cells do not necessarily comprise the base editor complex of the disclosure more than transiently, but the genomic modifications mediated by such base editor complexes remain.

(38) In yet another aspect, methods for targeted editing of cellular chromatin in a region of interest; methods of treating infection; and/or methods of treating disease are disclosed herein. These methods maybe practiced in vitro, ex vivo or in vivo or a combination thereof. The methods involve editing cellular chromatin at a predetermined region of interest in cells by expressing a base editing complex as described herein (for example fusion polypeptides and optionally any associated nucleic acids in which one or more fusion polypeptide(s) comprise the engineered nickases as disclosed herein). In certain embodiments, the targeted editing of the on-target site is found in 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the cells.

(39) The base editing complex as disclosed herein can be used in methods for targeted editing of cellular chromatin in a region of interest. Cells include cultured cells, cell lines, cells in an organism, cells that have been removed from an organism for treatment in cases where the cells and/or their descendants will be returned to the organism after treatment, and cells removed from an organism, modified using the fusion molecules of the invention, and then returned to the organism in a method of treatment (cell therapy). A region of interest in cellular chromatin can be, for example, a genomic sequence or portion thereof.

(40) A fusion molecule can be expressed in a cell, e.g., by delivering the fusion molecule to the cell as a polypeptide, or by delivering a polynucleotide encoding the fusion molecule to a cell, wherein the polynucleotide, if DNA, is transcribed and is translated, to generate the fusion molecule. Further, if the polynucleotide is an mRNA encoding the fusion molecule, following delivery of the mRNA to the cell, the mRNA is translated, thus generating the fusion molecule.

(41) In other aspects of the invention are provided methods and compositions for increasing base editing specificity. In some embodiments, methods are provided for increasing overall on-target editing specificity by decreasing off-target editing activity. In some embodiments, methods are provided for decreasing indel formation associated with base editing. In some embodiments, the engineered nickase components (nickase partners, for example a catalytically inactive ZFN partner and a catalytically active ZFN partner that form a ZFN nickase) of an engineered base editing complex are used to contact a cell, where each nickase partner of the complex is given in a ratio to the other partner other than one to one. In some embodiments, the ratio of the two partners is given at a 1:2, 1:3, 1:4, 1:5, 1:6, 1:8, 1:9, 1:10 or 1:20 ratio, or any value therebetween. In other embodiments, the ratio of the two partners is greater than 1:30. In some aspects, each partner is delivered to the cell as an mRNA or is delivered in a viral or non-viral vector where different quantities of mRNA or vector encoding each partner are delivered. In further embodiments, each partner of the nuclease complex may be comprised on a single viral or non-viral vector, but is deliberately expressed such that one partner is expressed at a higher or lower value that the other, ultimately delivering the cell a ratio of cleavage half domains that is other than one to one. In some embodiments, each cleavage half domain is expressed using different promoters with different expression efficiencies. In some embodiments, the two cleavage domains are delivered to the cell using a viral or non-viral vector where both are expressed from the same open reading frame, but the genes encoding the two partners are separated by a sequence (e.g. self-cleaving 2A sequence or IRES) that results in the 3′ partner being expressed at a lower rate, such that the ratios of the two partners are 1:2, 1:3, 1:4, 1:5, 1:6, 1:8, 1:9, 1:10 or 1:20 ratio, or any value therebetween. In other embodiments, the two partners are deployed at a ratio that is chosen to be different from 1:1.

(42) In another aspect, described herein is a population of cells produced using one or more base editors as described herein. In certain embodiments, more than 5%-20% (or any value therebetween), preferably more than 20%, even more preferably more than 50% and even more preferably between 80% and 100% of the cells include the modification to the targeted base (e.g., are base edited cells). In still further embodiments, the edited cells exhibit few or no off-target edits (unintended edits anywhere in the genome) and/or bystander (editing events in close proximity, for example 1-20 (or any value therebetween) nucleotides on either side of the intended target base, for example within the protospacer region of Cas9) mutations. Isolated populations of base edited cells as described herein can be used for ex vivo treatment of disease in a subject and/or can be further manipulated ex vivo (e.g., via further rounds of base editing as described herein) prior to use as an ex vivo treatment. In addition, base editing can be conducted in vivo such that the disease or condition is treated in the subject following correction of the disease-related mutations in vivo.

(43) In some embodiments, the nickase partners are fused to additional active domains. In some embodiments, the additional domains include one or more exemplary domains selected from one or more deaminases (for example A specific or C specific), a UGI domain, a helicase, and a GAM domain. In another aspect, described herein is a kit comprising a base editing complex as described herein or one or more polynucleotide(s) encoding one or more base editing complex proteins as described herein; ancillary reagents; and optionally instructions and suitable containers.

(44) These and other aspects will be readily apparent to the skilled artisan in light of disclosure as a whole.



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