阅读说明：本技术 线粒体基因组的遗传修饰 (Genetic modification of mitochondrial genome ) 是由 米查尔·明丘克 张雷 帕亚姆·A·伽玛吉 于 2019-03-21 设计创作，主要内容包括：本公开文本涉及基因组工程化,尤其是线粒体DNA(mtDNA)的靶向遗传修饰的领域。(The present disclosure relates to the field of genome engineering, in particular targeted genetic modification of mitochondrial dna (mtdna).)

1. Use of a zinc finger nuclease comprising a first and a second Zinc Finger Nuclease (ZFN), wherein the first ZFN comprises a cleavage domain and a Zinc Finger Protein (ZFP) that binds to a target site in a wild-type mitochondrial dna (mtDNA), and the second ZFN comprises a cleavage domain and a ZFP that binds to a target site in a mutant mtDNA, such that mutant mtDNA in the subject is reduced or eliminated, for treating a mitochondrial disorder in a subject in need thereof.

2. The use of claim 1, wherein the first ZFN is a left ZFN and the second ZFN is a right ZFN.

3. The use of claim 1, wherein the zinc finger nuclease is encoded by one or more polynucleotides.

4. The use of claim 1 or claim 2 or claim 3, wherein the polynucleotide is carried by one or more AAV vectors.

5. The use of any one of claims 1 to 4, wherein the subject is a human subject.

6. The use of any one of claims 1 to 5, wherein said mtDNA is in the heart, brain, lung and/or muscle of said subject.

7. The use of any one of claims 1 to 6, wherein the mutant mtDNA comprises the following mutations: m.5024c > T, 1555G, 1624T, 3243G, 3460A, 3271C, 4300G, 5545T, 7445G, 7472 random insertions, 8344G, 8356C 8993G, 9176G/C, 10158C, 10191C, 10197A, 11777A, 11778A, 13513a, 14459A, 14484C, 14487C, and/or 14709C.

8. The use of any one of claims 1 to 7, wherein the mutant mtDNA comprises the 5024C > T mutation and the left ZFP binds to the target site within SEQ ID NO 33 and the right ZFP binds to the target site within SEQ ID NO 34.

9. The use according to claim 8, wherein the zinc finger nuclease according to claim 2, wherein the left ZFN comprises a ZFP named WTM1/48960 and the right ZFN comprises a ZFP named MTM62/48962, MTM24/51024, MTM25/51025, MTM26/51026, MTM27/51027, MTM28/51028, MTM29/51029, MTM30/51030, MTM32/51032, MTM33/51033, MTM36/51036, MTM37/51037, MTM39/51039, MTM42/51042, MTM43/51043 or MTM 45/51045.

10. A method of treating a mitochondrial disease in a subject, the method comprising expressing the ZFN of any of claims 1 to 9 in a subject in need thereof such that mutant mtDNA in the subject is reduced or eliminated.

11. A zinc finger nuclease comprising a left zinc finger nuclease and a right Zinc Finger Nuclease (ZFN), wherein the left ZFN comprises a cleavage domain and a Zinc Finger Protein (ZFP) that binds to a target site in wild-type mitochondrial DNA within SEQ ID NO 33, and the right ZFN comprises a cleavage domain and a ZFP that binds to a target site in mutant mitochondrial DNA within SEQ ID NO 34 or SEQ ID NO 35.

12. One or more polynucleotides encoding the nuclease according to claim 11, optionally carried by one or more AAV vectors.

13. A cell comprising the nuclease according to claim 10 or one or more polynucleotides according to claim 12, optionally a heart cell, brain cell, lung cell and/or muscle cell.

14. The cell of claim 13, wherein mutant mtDNA at position 5024 in the cell is reduced or eliminated.

15. A cell or cell line produced or evolved from the cell of claim 13 or claim 14.

16. A pharmaceutical composition comprising a zinc finger nuclease according to claim 11, a polynucleotide according to claim 12 and/or a cell according to any of claims 13-15.

17. A kit comprising a nuclease according to claim 11, a polynucleotide according to claim 12, a cell according to any one of claims 13 to 15 and/or a pharmaceutical composition according to claim 16.

Technical Field

The present disclosure relates to the field of genome engineering, in particular targeted modification of mitochondrial genomes (mtDNA).

Background

Mitochondrial diseases are a genetically diverse class of inherited multisystem disorders (often affecting organs that require the greatest amount of energy, such as the heart, brain, muscles and lungs) that are mostly propagated by mutations in mitochondrial dna (mtdna), affecting adults around 1/5,000. See, for example, Gorman et al (2015) Ann neuron 77: 753-. At least 250 pathogenic mtDNA mutations have been characterized to date (Tuppen et al (2010) Biochem Biophys Acta 1797: 113-. Human mtDNA is a small double-stranded multicopy genome, present at about 100 and 10,000 copies/cell. In disease states, mutant mtDNA often co-exists with wild-type mtDNA in a phenomenon known as "heterogeneity" (resulting from multiple mitochondria being inherited through the maternal line of the egg cell). Because mutant mtDNA is generally functionally recessive, the presence of mutant mtDNA is promoted by the wild-type genome, and disease severity of the condition caused by heterogeneous mtDNA mutations correlates with mutation burden. See, e.g., Gorman et al (2016) Nat Rev Dis Primers 2: 16080. Threshold effects (where the mutant mtDNA burden must exceed > 60% before symptoms manifest) are a clear feature of heterogeneous mtDNA disease, and attempts to shift the heterogeneity ratio below this threshold have driven many studies on treating these incurable and essentially untreatable disorders.

One such method relies on targeted nucleolysis of mtDNA using mitochondrially targeted zinc finger nucleases (mtzfns) and other genome engineering tools. See, e.g., Srivastava et al (2001) Hum Mol Genet 10:3093-3099 (2001); bacman et al (2013) Nat Med 19: 1111-; gamma et al (2014) EMBO Mol Med 6: 458-466; reddy et al (2015) Cell 161: 459-469; gamma et al (2016) Nucleic Acids Res 44: 7804-7816; gamma et al (2018) Trends Gene 34 (2: 101). Because mammalian mitochondria lack an efficient DNA Double Strand Break (DSB) repair pathway, selective introduction of DSBs into mutant mtDNA results in rapid degradation of these molecules by incompletely characterized mechanisms. Because mtDNA copy number is maintained at a cell type specific steady state level, selective elimination of mutant mtDNA stimulates replication of the remaining mtDNA pool, causing a shift in the heterogeneity ratio. Methods for delivering ZFNs to mitochondria in cultured cells have been shown to be capable of generating large heterogeneity excursions that lead to phenotypic rescue of patient-derived cell cultures. See, e.g., Minczuk et al (2006) Proc Natl Acad Sci U S A103: 19689-19694 (2006); minczuk et al (2010) Nat Protoc 5: 342-356; minczuk et al, (2008) Nucleic Acids Res 36: 3926-; gaude et al (2018) Mol Cell 69: 581-593; U.S. patent No. 9,139,628.

Although mutations in mtDNA associated with human disease that occurred later in the 80's of the 20 th century were initially described (see, e.g., Holt et al (1988) Nature 331: 717-719; Wallace et al (1988) Science 242: 1427-1430; Wallace et al (1988) Cell 55:601-610), effective treatment of heterogeneous mitochondrial disease has not occurred for decades in between. Prevention of the transmission of mtDNA mutations by mitochondrial replacement therapy has received attention (see, e.g., Craven et al (2010) Nature 465: 82-85; Tachibana et al (2013) Nature 493: 627-631; Hyslop et al (2016) Nature 534: 383-386; Kang et al (2016) Nature 540:270-275), but mitochondrial replacement may have only limited utility in view of the Nature of the mtDNA bottleneck (Floros et al (2018) Nat Cell Biol 20:144-151), heterogeneous mitochondrial disease presentation (vafaii et al (2012) Nature 491: 383), and subsequent deletion of family history of most new cases of mitochondrial disease in 374. In addition, molecular approaches for treating mitochondrial diseases do not provide clinically relevant therapies for heterogeneous mitochondrial diseases. See, e.g., Viscomi et al (2015) Biochim Biophy Acta 1847: 544-557; pfeffer et al (2013) Nat Rev Neurol 9: 474-481.

Thus, there remains a need for additional methods and compositions for mtDNA gene modification, particularly heterogeneity excursions of mtDNA, to provide a universal therapeutic for treating mitochondrial diseases of various genetic origins by reducing the amount of mutant mitochondrial sequences.

Disclosure of Invention

The present invention describes compositions and methods for gene therapy and genome engineering. In particular, the methods and compositions relate to nuclease-mediated modification (e.g., one or more insertions and/or deletions) of the endogenous mitochondrial genome (mutant or wild-type). The mitochondrial genome can be targeted for correction of pathogenic mutations, including by altering: in a subject having a mitochondrial disorder, a nuclease-mediated shift in the ratio of mutant to wild-type mtDNA in one or more specific tissues and/or organs (e.g., in cardiac tissue) is included that results in reversal of the phenotype of the targeted tissue to wild-type (e.g., molecular and biochemical phenotypes). This reversal occurs through a heterogeneity shift in which the ratio of mutant to wild-type mtDNA is altered by: cleaving the mutant sequence such that the mutant mtDNA associated with the disease degrades following selective cleavage by the targeted nuclease in the absence of an effective DNA repair mechanism (such as in mitochondria).

Thus, the one or more genomic modifications (e.g., heterogeneity shifts) may comprise cleavage followed by degradation of the cleaved mtDNA sequences, and these genetic modifications and/or cells comprising these modifications may be used in ex vivo or in vivo methods.

Thus, described herein is the use of a zinc finger nuclease comprising a left zinc finger nuclease and a right Zinc Finger Nuclease (ZFN) (or a pharmaceutical composition comprising the zinc finger nuclease) for treating a mitochondrial disorder in a subject in need thereof, wherein one ZFN partner comprises a cleavage domain and a Zinc Finger Protein (ZFP) that binds to a target site in a mutant mitochondrial DNA (mutant mtDNA), and the other ZFN partner comprises a cleavage domain and a Zinc Finger Protein (ZFP) that binds to a target site in a wild-type mitochondrial DNA (mtDNA) or a mutant mtDNA (mutant mtDNA), such that the mutant mtDNA in the subject is reduced or eliminated (e.g., the heterogeneity ratio of wild-type to mutant mtDNA is shifted). In some embodiments, both the right and left ZFPs bind to a target in the mutant mtDNA, while in other embodiments one ZFN partner binds to the wild-type mtDNA and the other ZFN partner binds to the mutant mtDNA. In other embodiments, the ZFN that binds to the wild-type mtDNA is the left ZFN and the right ZFN binds to the mutant mtDNA, or the right ZFN binds to the wild-type mtDNA and the left ZFN binds to the mutant mtDNA. Also described are methods of treating a mitochondrial disorder in a subject in need thereof by expressing a ZFN described herein. In any of the uses or methods described herein, the mutant mtDNA comprises one or more of the following mutations: 5024C > T, 1555G, 1624T, 3243G, 3460A, 3271C, 4300G, 5545T, 7445G, 7472 random insertion, 8344G, 8356C 8993G, 9176G/C, 10158C, 10191C, 10197A, 11777A, 11778A, 13513a, 14459A, 14484C, 14487C, and/or 14709C. In certain embodiments, the zinc finger nuclease is encoded by one or more polynucleotides (e.g., separate polynucleotides encoding the left and right ZFNs or the same polynucleotide encoding both the left and right ZFNs), including but not limited to one or more polynucleotides carried by one or more AAV vectors. The subject may be a human subject, and the mtDNA may be in any tissue of the subject. In some embodiments, the mtDNA may be in the brain, lung, and/or muscle of the subject. The ZFNs and/or polynucleotides may be administered by any suitable means, including intravenous injection. In embodiments where the mutant mtDNA comprises a 5024C > T mutation, the left ZFP may bind to a target site within SEQ ID NO:33 and the right ZFP may bind to a target site within SEQ ID NO:34, including but not limited to ZFNs wherein the left ZFP comprises a ZFP designated WTM1/48960 and the right ZFN comprises a ZFP designated MTM62/48962, MTM24/51024, MTM25/51025, MTM26/51026, MTM27/51027, MTM28/51028, MTM29/51029, MTM30/51030, MTM32/51032, MTM33/51033, MTM36/51036, MTM 37/37, MTM39/51039, MTM42/51042, MTM43/51043, or MTM 45/51045.

Also described is a zinc finger nuclease comprising a left zinc finger nuclease and a right Zinc Finger Nuclease (ZFN), wherein the left ZFN comprises a cleavage domain and a Zinc Finger Protein (ZFP) that binds to a target site in wild-type mitochondrial DNA within SEQ ID NO:33, and the right ZFN comprises a cleavage domain and a ZFP that binds to a target site in mutant mitochondrial DNA within SEQ ID NO:34 or SEQ ID NO: 35. In certain embodiments, the ZFNs are encoded by one or more polynucleotides (e.g., carried by an AAV vector). Also described are cells (e.g., heart, brain, lung, and/or muscle cells) comprising nucleases and/or polynucleotides as set forth herein, including cells in which mutant mtDNA at position 5024 is reduced or eliminated, as well as cells, cell lines, and partially or fully differentiated cells (which may not comprise a ZFN or polynucleotide encoding a ZFN) evolved from these cells. Also provided are methods comprising one or more zinc finger nucleases; a pharmaceutical composition of one or more polynucleotides and/or a cell as described herein.

In one aspect, disclosed herein are methods and compositions for targeted modification of mtDNA genes using one or more nucleases. Nucleases (e.g., engineered meganucleases, Zinc Finger Nucleases (ZFNs) (the term "ZFNs" includes ZFN pairs), TALE-nucleases (TALENs comprising fusions of TALE effector domains with nuclease domains from restriction endonucleases and/or from meganucleases such as megatales and compact TALENs) (the term "TALENs" includes TALEN pairs)), Ttago systems, and/or CRISPR/Cas nuclease systems are used to cut DNA at a mitochondrial genome, typically a mutant mitochondrial genome, such that heterogeneity (such as that between wild-type and mutant mitochondrial genomes) is offset and the amount of mutant mtDNA is reduced. The target (e.g., mutant mtDNA) can be inactivated after cleavage because the double repair pathway in mtDNA is inefficient, and thus selective cleavage of mutant mtDNA (where wild-type mtDNA is not cleaved) results in rapid degradation of the mutant mtDNA and a corresponding shift in the heterogeneity ratio of wild-type to mutant mtDNA. The nucleases described herein can induce Double Strand Breaks (DSBs) or single strand breaks (nicks) in the target DNA. In some embodiments, the DSB is generated by introducing two nicks using two nicking enzymes. In some cases, the nickase is a ZFN, while in other cases, the nickase is a TALEN or CRISPR/Cas nickase. Any of the nucleases (e.g., ZFNs, TALENs, CRISPR/Cas, etc.) described herein can specifically target a target mutant mtDNA, including, for example, the target sequences shown in table 2, including, for example, target sites comprising 9 to 20 or more (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) contiguous or non-contiguous nucleotides of the wild-type or mutant sequence. Any mutant may be targeted by the DNA binding domain, including but not limited to m.5024c > T. For example, a non-limiting group of human diseases associated with mtDNA mutations include Kearns-Sayre syndrome (KSS; progressive myopathy, ophthalmoplegia, cardiomyopathy); CPEO: chronic progressive external ophthalmoplegia; and Pearson Syndrome (pancytopenia, lactic acidosis), in which all three diseases are associated with a single large deletion (approximately 5kb) in the mitochondrial genome. Other human diseases associated with mutant mitochondria are MELAS (myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) which are associated with 3243A > G mutations (also referred to herein as a3243G or 3243G) and/or 3271T > C mutations (also referred to herein as T3271C or 3271C) in the TRNL1 gene or sporadic mutations in ND1 and ND 5; MERRF (myoclonic epilepsy with broken red fibers, myopathy) associated with 8344A > G and/or 8356T > C mutations (referred to herein as a8344G or 8344G/T8356C or 8356C) in the TRNK gene; NARP (neuropathy, ataxia, retinitis pigmentosa) which is associated with the 8993T > G mutation in the ATP6 gene (referred to herein as T8993G or 8993G); MILS (progressive brainstem disease, also known as maternally inherited Leigh syndrome), which is also associated with 8993T > G/C mutations (referred to herein as T8993G, T8993C, 8993G, 8993C) and/or 9176T > G/C mutations (referred to herein as T9176G, T9176C, 9176G, 9176C) in ATP 6; MIDD (diabetes, deafness), which is associated with a 3243A > G mutation (referred to herein as a3243G or 3243G) in the TRNL1 gene; LHON (optic neuropathy) associated with a 3460G > a mutation in ND1 (referred to herein as G3460A or 3460A), a 11778G > a mutation in ND4 (referred to herein as G11778A or 11778A), and/or a 14484T > C mutation in ND6 gene (referred to herein as T14484C or 14484C); myopathies and diabetes, which are associated with 14709T > C mutations (referred to herein as T14709C or 14709C) in the TRNE gene; sensorineural deafness and deafness, which are associated with 1555A > G mutations in the RNR1 gene (referred to herein as a1555G or 1555G) and sporadic mutations in the TRNS1 gene; exercise intolerance, which is associated with sporadic mutations in the CYB gene; and the lethal infantile encephalopathy Leigh/Leigh-like syndrome associated with the 10158T > C mutation (referred to as T10158C or 10158C) and/or the 10191T > C mutation (referred to as T10191C or 10191C) and/or the 10197G > a mutation (referred to as G10197A or 10197A) in the ND3 gene. Other mutations in mtDNA include 14709T > C mutation in ND6 gene (referred to as T14709C or 14709C); 14459G > a and/or 14487T > C mutation in ND6 gene (referred to herein as G14459A or 14459A and T14487C or 14487C) and/or 11777C > a mutation in ND4 gene (referred to as C11777A or 11777a) and/or 1624C > T mutation associated with Leigh syndrome (referred to as C1624T or 1624T); the 13513G > a mutation in ND5 gene (designated G13513A or 13513 a); 7445A > G mutation (referred to as A7445G or 7445G) and/or insertion at 7472 associated with deafness and myopathy; 5545C > T mutations associated with multiple system disorders (referred to as C5545T or 5545T) and 4300A > G mutations associated with cardiomyopathy (referred to as a4300G or 4300G). See, e.g., great and Taylor (2006) IUBMB Life 58(3): 143-); taylor and Turnbull (2005) Nat Rev Genet 6(5): 389-402; and Tuppen et al (2010) supra). All mutations are numbered relative to the wild-type sequence.

In one aspect, described herein are non-naturally occurring Zinc Finger Proteins (ZFPs) that bind to target sites in an mtDNA genome, wherein the ZFPs comprise one or more engineered zinc finger binding domains. In one embodiment, the ZFP is a Zinc Finger Nuclease (ZFN) that cleaves a target genomic region of interest, wherein the ZFN comprises one or more engineered zinc finger binding domains and a nuclease cleavage domain or cleavage half-domain. The cleavage domains and cleavage half-domains can be obtained, for example, from various restriction endonucleases and/or homing endonucleases and can be wild-type or engineered (mutant). In one embodiment, the cleavage half-domain is derived from a type IIS restriction endonuclease (e.g., fokl). In certain embodiments, the zinc finger domain comprises a zinc finger protein having recognition helix domains ordered as shown in a single row of table 1. Nucleases comprising these zinc finger proteins can include any linker sequence (e.g., linked to a cleavage domain) and any cleavage domain (e.g., dimerization mutants, such as ELD mutants; fokl domains with mutations at one or more of 416, 422, 447, 448 and/or 525; and/or catalytic domain mutants that result in nickase function). See, for example, U.S. patent nos. 8,703,489; 9,200,266, respectively; 8,623,618, respectively; and 7,914,796; and U.S. patent publication No. 20180087072. In certain embodiments, the ZFPs of the ZFNs bind to target sites of 9 to 18 or more nucleotides within the sequences shown in table 2. In certain embodiments, the ZFN selectively binds to mutant mtDNA (as compared to wild-type mtDNA) such that the ZFN selectively cleaves mutant mtDNA (as compared to cleavage of wild-type mtDNA). In other embodiments, the ZFN selectively binds to a target site in the mutant mtDNA comprising one or more of the following mutations: 1555G, 1624T, 3243G, 3460A, 3271C, 4300G, 5545T, 7445G, 7472 random insertions, 8344G, 8356C 8993G, 9176G/C, 10158C, 10191C, 10197A, 11777A, 11778A, 13513A, 14459A, 14484C, 14487C, or 14709C numbered relative to the wild type sequence, wherein the nucleotides after said positions indicate a mutant sequence. Any ZFN described herein can comprise a ZFN pair (e.g., left and right), wherein one member of the pair binds to the mutant mtDNA sequence and the other member of the pair binds to the wild-type mtDNA. Alternatively, the ZFNs described herein may comprise pairs of ZFNs (left and right), wherein both ZFNs bind to wild-type mtDNA, or both ZFNs bind to mutant mtDNA.

In another aspect, described herein are transcriptional activator-like effector (TALE) proteins that bind to a target site (e.g., a target site in mtDNA comprising at least 9 or 12 (e.g., 9 to 20 or more) nucleotides of a target sequence set forth in table 2), wherein the TALE comprises one or more engineered TALE binding domains. In one embodiment, the TALE is a nuclease (TALEN) that cleaves a target genomic region of interest, wherein the TALEN comprises one or more engineered TALE DNA binding domains and a nuclease cleavage domain or cleavage half-domain. The cleavage domains and cleavage half-domains can be obtained, for example, from various restriction endonucleases and/or homing endonucleases (meganucleases). In one embodiment, the cleavage half-domain is derived from a type IIS restriction endonuclease (e.g., fokl). In other embodiments, the cleavage domain is derived from a meganuclease, which can also exhibit DNA binding function. In certain embodiments, the TALEN selectively binds to mutant mtDNA (as compared to wild-type mtDNA) such that the TALEN selectively cleaves mutant mtDNA (as compared to cleavage of wild-type mtDNA). In other embodiments, the TALEN selectively binds to a target site comprising the following mutations: 1555G, 1624T, 3243G, 3460A, 3271C, 4300G, 5545T, 7445G, 7472 random insertions, 8344G, 8356C 8993G, 9176G/C, 10158C, 10191C, 10197A, 11777A, 11778A, 13513A, 14459A, 14484C, 14487C, or 14709C numbered relative to the wild type sequence, wherein the nucleotides after said positions indicate a mutant sequence. Any TALEN described herein can comprise a TALEN pair (e.g., left and right), wherein one member of the pair binds to the mutant mtDNA sequence and the other member of the pair binds to the wild-type mtDNA. Alternatively, TALENs as described herein can include pairs of TALENs (left and right) where both TALENs bind to wild-type mtDNA or both TALENs bind to mutant mtDNA.

In another aspect, described herein is a CRISPR/Cas system that binds to a target site in mtDNA, wherein the CRISPR/Cas system comprises one or more engineered single guide RNAs or functional equivalents and a Cas9 nuclease. In certain embodiments, the single guide rna (sgrna) binds to a sequence of 9, 12, or more contiguous nucleotides comprising a target site as set forth in table 2. In certain embodiments, the sgRNA selectively binds to mutant mtDNA (as compared to wild-type mtDNA) such that the CRISPR/Cas nuclease selectively cleaves mutant mtDNA (as compared to cleavage of wild-type mtDNA). In other embodiments, the CRISPR/Cas system selectively binds to a target site comprising the following mutations: 1555G, 1624T, 3243G, 3460A, 3271C, 4300G, 5545T, 7445G, 7472 random insertions, 8344G, 8356C 8993G, 9176G/C, 10158C, 10191C, 10197A, 11777A, 11778A, 13513A, 14459A, 14484C, 14487C, or 14709C numbered relative to the wild type sequence, wherein the nucleotides after said positions indicate a mutant sequence. Any of the sgrnas described herein can selectively bind to a mutant or alternatively a wild-type mtDNA. In the case of using sgRNA pairs, one or both members can bind to the wild-type or mutant mtDNA.

Nucleases (e.g., ZFNs, CRISPR/Cas systems, Ttago, and/or TALENs) as described herein can bind to and/or cleave a region of interest (e.g., like a leader sequence, trailer sequence, or intron) in a coding or non-coding region of mtDNA or within a non-transcribed region upstream or downstream of the coding region. The target site may be 9-18 or more nucleotides in length, including target sites as shown in table 2 or target sites encompassing 1555G, 1624T, 3243G, 3460A, 3271C, 4300G, 5545T, 7445G, 7472 random insertions, 8344G, 8356C 8993G, 9176G/C, 10158C, 10191C, 10197A, 11777A, 11778A, 13513a, 14459A, 14484C, 14487C, or 14709C in mtDNA. In certain embodiments, the DNA binding domain of the nuclease (ZFP, TALE, sgRNA, etc.) selectively binds to the mutant mtDNA (as compared to cleavage of the wild-type mtDNA). In some embodiments, the DNA binding domain of the one or more nucleases binds to a selected position in the TRNL1, ND1, ND5, TRNK, ATP6, ND4, ND6, TRNE, RNR1, TRNS, CYB, CYTb, 12SrRNA, and/or ND3 mitochondrial gene.

In another aspect, described herein are one or more polynucleotides encoding one or more nucleases (e.g., ZFNs, CRISPR/Cas systems, Ttago, and/or TALENs described herein). In certain embodiments, the same polynucleotide encodes one nuclease (e.g., both the left and right monomers of a paired nuclease or all components of the CRISPR/Cas system), while in other embodiments separate polynucleotides are used for components of the nuclease (e.g., a first polynucleotide encodes one member of a paired nuclease (e.g., left member/monomer) and a second polynucleotide encodes the other member of a paired nuclease (e.g., right monomer/member)). The polynucleotides may be formulated in viral or non-viral vectors, including but not limited to AAV, Ad, retroviral vectors, and the like, as well as in mRNA, plasmids, minicircle DNA, and the like. In certain embodiments, the vector is targeted to a specific tissue or organ, e.g., the AAV vector is targeted to the heart (cardiac tissue). In certain embodiments, the nuclease is a ZFN comprising a left ZFN and a right ZFN, separately formulated as an AAV vector composition and administered simultaneously (e.g., formulated as a single pharmaceutical composition comprising two AAV vectors).

In another aspect, described herein is a ZFN, CRISPR/Cas system, Ttago, and/or TALEN expression vector comprising a polynucleotide operably linked to a promoter encoding one or more nucleases (e.g., ZFNs, CRISPR/Cas systems, Ttago, and/or TALENs) as described herein. In one embodiment, the expression vector is a viral vector (e.g., an AAV vector). In one aspect, the viral vector exhibits tissue-specific tropism.

In another aspect, described herein is a host cell comprising one or more nuclease (e.g., ZFNs, CRISPR/Cas systems, Ttago, and/or TALENs) expression vectors.

In another aspect, a pharmaceutical composition comprising an expression vector as described herein (e.g., comprising one or more components of one or more nucleases) is provided. In some embodiments, the pharmaceutical composition may comprise more than one expression vector. In some embodiments, the pharmaceutical composition comprises a first expression vector having a first polynucleotide and a second expression vector having a second polynucleotide. In some embodiments, the first polynucleotide and the second polynucleotide are different. In some embodiments, the first polynucleotide and the second polynucleotide are substantially identical. In certain embodiments, the pharmaceutical composition comprises a first AAV vector encoding a left monomer of a ZFN pair and/or a second AAV vector encoding a right monomer of the ZFN pair. In certain embodiments, the pharmaceutical composition (e.g., a drug comprising a polynucleotide, such as an AAV vector comprising one or two monomers) is at a concentration of 1x10¹⁰To 1x10¹⁴(or any value therebetween) between vector genomes (vg)/cell or subject. In some embodiments, the concentration of the pharmaceutical composition is 1x10¹²、5x10¹²Or 1x10¹³Individual vg per cell or subject (e.g., by tail vein injection). The pharmaceutical composition is suitable for delivery to a subject, including but not limited to systemic, intraperitoneal, intravenous, intramuscular, mucosal, or topical delivery methods, or combinations thereof. The pharmaceutical composition may also comprise a donor sequence (e.g., a transgene encoding a protein that is absent or deficient in a disease or disorder, such as a mitochondrial disorder). In some embodiments, the donor is a human, aThe sequence is associated with an expression vector.

In some embodiments, fusion proteins are provided that comprise a DNA binding domain (e.g., a zinc finger protein or TALE or sgRNA or meganuclease) and a wild-type or engineered cleavage domain or cleavage half-domain.

In another aspect, described herein are compositions comprising one or more nucleases (e.g., ZFNs, TALENs, TtAgo, and/or CRISPR/Cas systems) described herein, including nucleases comprising a DNA binding molecule (e.g., ZFP, TALE, sgRNA, etc.) and a nuclease (cleavage) domain. In certain embodiments, the composition comprises one or more nucleases in combination with a pharmaceutically acceptable excipient. In some embodiments, the composition comprises two or more sets of (pairs of) nucleases, each set having a different specificity. In other aspects, the compositions comprise different types of nucleases. In some embodiments, the composition comprises a polynucleotide encoding an mtDNA-specific nuclease, while in other embodiments, the composition comprises an mtDNA-specific nuclease protein. In certain embodiments, the composition is suitable for delivery to a subject, including via systemic delivery.

In another aspect, described herein is a polynucleotide encoding one or more nucleases or nuclease components described herein (e.g., a nuclease domain of a ZFN, TALEN, TtAgo, or CRISPR/Cas system). The polynucleotide may be, for example, mRNA or DNA. In some aspects, the mRNA can be chemically modified (see, e.g., Kormann et al, (2011) Nature Biotechnology 29(2): 154-. In other aspects, the mRNA can comprise an ARCA cap (see U.S. Pat. Nos. 7,074,596; and 8,153,773). In further embodiments, the mRNA may comprise a mixture of unmodified and modified nucleotides (see U.S. patent publication No. 2012/0195936). In another aspect, described herein is a nuclease expression vector comprising a polynucleotide operably linked to a promoter encoding one or more of the ZFNs, TALENs, TtAgo, or CRISPR/Cas systems described herein. In one embodiment, the expression vector is a viral vector, such as an AAV vector.

In another aspect, described herein is a host cell comprising one or more nucleases, one or more nuclease expression vectors as described herein. In certain embodiments, the host cell comprises wherein the amount of mutant mtDNA is reduced or eliminated, thereby shifting the heterogeneity ratio of mtDNA in the cell (as compared to a wild-type cell). In certain embodiments, the heterogeneity ratio shifts towards wild-type (non-mutant mtDNA) by at least 5% or more, preferably at least 10% or more, and even more preferably at least 20% or more. The host cell may be stably transformed or transiently transfected with one or more nuclease expression vectors or any combination thereof. In other embodiments, the one or more nuclease expression vectors express one or more nucleases in the host cell. In another embodiment, the host cell may further comprise an exogenous polynucleotide donor sequence. In any of the embodiments described herein, the host cell can include an embryonic cell, such as one or more mouse, rat, rabbit, or other mammalian cell embryos (e.g., non-human primates). In some embodiments, the host cell comprises a tissue. Also described are cells or cell lines produced or evolved from the cells described herein, including pluripotent, totipotent, multipotent or differentiated cells comprising a modification of mtDNA (e.g., a heterogeneous ratio of mtDNA). In certain embodiments, described herein are differentiated cells as described herein comprising modifications as described herein, the differentiated cells evolved from stem cells as described herein. In certain embodiments, the host cell is a cardiac cell or a stem cell, such as a hematopoietic stem cell or an inducible pluripotent stem cell.

In another aspect, described herein is a method for cleaving an mtDNA gene in a cell, the method comprising: (a) introducing one or more polynucleotides encoding one or more nucleases into the cell under conditions such that the one or more nucleases target mtDNA are expressed and cleave the mtDNA. In certain embodiments, the mutant mtDNA is selectively cleaved compared to the wild-type mtDNA. This results in a shift in the heterogeneity ratio of mutant mtDNA to wild-type mtDNA. Optionally, the method further comprises administering to the cell a donor (e.g., a therapeutic protein) that can be integrated into the genome of the cell or mtDNA. Integration of one or more donor nucleases occurs via Homology Directed Repair (HDR) or through non-homologous end joining (NHEJ) related repair. In addition, the one or more nuclease-encoding polynucleotides and/or donors can be introduced into the cell using any one or combination of delivery systems (e.g., a non-viral vector, LNP, or viral vector). In certain embodiments, vectors specific for certain cell, tissue and/or organ types are used, e.g., AAV vectors specific for heart tissue, brain tissue, lung tissue, muscle tissue, and the like. In certain embodiments, cleavage of the mutant mtDNA shifts heterogeneity towards wild-type (e.g., including partial or complete reversion to wild-type sequences) sequences, thereby treating and/or preventing mitochondrial disease in a subject in need thereof. In certain embodiments, the mutant mtDNA that is cleaved and restored to wild type comprises a point mutation (e.g., 5024C > T).

In any of the compositions or methods described herein, the one or more polynucleotides may be provided and/or delivered at any concentration (dose) that provides the desired effect. In a preferred embodiment, the one or more polynucleotides are derived from adeno-associated virus (AAV) vectors at 10,000-1x10¹⁴One or more vector genomes/cells or subjects (or any value therebetween). In certain embodiments, the one or more polynucleotides are delivered at an MOI of between 250 and 1,000 (or any value therebetween) using a lentiviral vector. In other embodiments, the one or more polynucleotides are delivered using a plasmid vector at 150 and 1,500ng/100,000 cells (or any value therebetween). In other embodiments, the one or more polynucleotides are delivered as mRNA at 150- > 1,500ng/100,000 cells (or any value therebetween). Where two or more polynucleotides are delivered, the vectors may be the same or different vectors, and the same vectors may be in any ratio, including but not limited toLimited to 1:1 ratio delivery. In certain embodiments, two AAV vectors are used at any concentration per monomer, including but not limited to 1x10¹⁰To 1x10¹⁴(or any value therebetween), optionally at 5x10¹²Each vg/monomer delivers a component of a paired nuclease (e.g., ZFN comprising MTM25 monomer and WTM1 monomer). In certain embodiments, the dose of the individual monomers, or alternatively the total dose (of both monomers), is 1x10¹²、5x10¹²Or 1x10¹³Individual vg per cell or subject (e.g., by tail vein injection). In some embodiments, the ZFNs are administered at doses of: a total AAV dose of 5e12 vg/kg (e.g., 2.5e12 vg/kg per AAV-ZFN monomer); a total AAV dose of 1e13 vg/kg (e.g., 0.5e13 vg/kg per AAV-ZFN monomer); a total AAV dose of 5e13 vg/kg (e.g., 2.5e13 vg/kg per AAV-ZFN monomer); a total AAV dose of 1e14 vg/kg (e.g., 0.5e14 vg/kg per AAV-ZFN monomer); a total AAV dose of 5e14 vg/kg (e.g., 2.5e14 vg/kg per AAV-ZFN monomer); or a total AAV dose of 1e15 vg/kg (e.g., 0.5e15 vg/kg per AAV-ZFN monomer). In certain embodiments, the AAV is administered by intravenous injection.

In yet another aspect, provided herein is a cell comprising a genetically modified mtDNA, e.g., a cell that alters the heterogeneity ratio of wild type to mutant mtDNA by reducing and/or eliminating mutant mtDNA in the cell. In certain embodiments, the cellular heterogeneity ratio is reduced compared to cells from a subject having a mitochondrial disorder. The mutant mtDNA is reduced and/or eliminated from a mutant mtDNA by degradation following cleavage of the mutant mtDNA by a nuclease specific for a mutant form of mtDNA (e.g., a nuclease that targets a sequence of 9-20 or more base pairs as shown in table 2 or encompassing one or more of 1555G, 1624T, 3243G, 3460A, 3271C, 4300G, 5545T, 7445G, 7472 random insertion, 8344G, 8356C 8993G, 9176G/C, 10158C, 10191C, 10197A, 11777A, 11778A, 13513a, 14459A, 14484C, 14487C, or 14709C). Cleavage that contributes to degradation may be within the one or more target sites and/or one or more cleavage sites and/or within 1-50 base pairs of the edge of a target site of 9-18 or more base pairs of the target sequence. The modified cell as described herein can be isolated or can be within a subject, e.g., a subject having a mitochondrial disorder.

In any of the methods and compositions described herein, the cell can be any eukaryotic cell. In certain embodiments, the cell is a differentiated cell, such as a heart cell, brain cell, liver cell, kidney cell, muscle cell, nerve cell, intestinal cell, eye cell, and/or ear cell, and the like. In other embodiments, the cell is a stem cell. In other embodiments, the cells are patient-derived, such as autologous CD34+ (hematopoietic) stem cells (e.g., mobilized from the bone marrow into the peripheral blood in a patient via Granulocyte Colony Stimulating Factor (GCSF) administration). CD34+ cells may be harvested, purified, cultured, and the nuclease introduced into the cells by any suitable method.

In another aspect, the methods and compositions of the invention provide for the use of a composition (nuclease, pharmaceutical composition, polynucleotide, expression vector, cell line and/or animal such as a transgenic animal) as described herein, for example, for the treatment and/or prevention of a mitochondrial disease. In certain embodiments, these compositions are used to screen drug libraries and/or other therapeutic compositions (i.e., antibodies, structural RNAs, etc.) for the treatment of mitochondrial disorders. Such screening can begin at the cellular level with manipulated cell lines or primary cells, and can be performed to the therapeutic level of the entire animal (e.g., veterinary or human therapy). Thus, in certain aspects, described herein is a method of treating and/or preventing a mitochondrial disease in a subject in need thereof, the method comprising administering to the subject one or more nucleases, polynucleotides and/or cells as described herein. The method may be ex vivo or in vivo. In certain embodiments, the cells as described herein are administered to the subject. In any of the methods described herein, the cell can be a stem cell (patient-derived stem cell) derived from the subject.

In any of the compositions and methods described herein, the nuclease is introduced in mRNA form and/or using one or more non-viral, LNP, or viral vectors. In certain embodiments, the one or more nucleases are introduced in the form of mRNA. In other embodiments, the one or more nucleases are introduced using a viral vector, such as an adeno-associated vector (AAV) (including AAV1, AAV3, AAV4, AAV5, AAV6, AAV8, AAV8.2, AAV9, AAV rh10, AAV2/8, AAV2/5, and AAV2/6) or via a lentivirus or integration-defective lentivirus vector.

Once delivered to the cell, the one or more nucleases are transcribed and/or translated, and the nuclease protein is taken up by the mitochondria. Thus, in some embodiments, the one or more nucleases comprise a mitochondrial targeting peptide (see, e.g., U.S. Pat. No. 9,139,628; Omuta (1998) J. biochem 123(6): 1010-6). In certain embodiments, tissue or cell specific vectors are used, for example vectors specific for the heart (cardiac tissue).

Any cell can be modified using the compositions and methods of the invention, including but not limited to prokaryotic or eukaryotic cells, such as cells, insect, yeast, fish, mammalian (including non-human mammalian) and plant cells. In certain embodiments, the cell is a heart cell, brain cell, liver cell, spleen cell, intestinal cell, or immune cell, such as a T cell (e.g., CD4+, CD3+, CD8+, etc.), dendritic cell, B cell, or the like. In other embodiments, the cell is a pluripotent, totipotent or multipotent cell, such as an Induced Pluripotent Stem Cell (iPSC), a hematopoietic stem cell (e.g., CD34+), an embryonic stem cell, or the like. Specific stem cell types that may be used in the methods and compositions of the invention include Embryonic Stem Cells (ESCs), Induced Pluripotent Stem Cells (iPSCs), and hematopoietic stem cells (e.g., CD34+ cells). The ipscs can be derived from patient samples and/or normal controls, wherein patient-derived ipscs can be mutated to a normal or wild-type gene sequence at a gene of interest, or normal cells can be altered to a known disease allele at a gene of interest. Similarly, hematopoietic stem cells can be isolated from a patient or from a donor.

Thus, described herein are methods and compositions for altering an mtDNA genome, including but not limited to selectively cleaving mutant mtDNA to alter the heterogeneity ratio of mutant to wild-type mtDNA in a cell, organ and/or tissue (e.g., a cell, organ and/or tissue of a subject in need thereof), thereby treating and/or preventing mitochondrial disease. The compositions and methods can be used in vitro, in vivo, or ex vivo and include administering an artificial transcription factor or nuclease comprising a DNA binding domain targeted to mtDNA.

Kits comprising the nucleic acids, nucleases, and/or cells of the invention are also provided. The kit can comprise a nucleic acid encoding the nuclease (e.g., a gene encoding an RNA molecule or ZFN, TALEN, TtAgo, or CRISPR/Cas system contained in a suitable expression vector), or an aliquot of the nuclease protein, a donor molecule, a suitable sternness modulator, a cell, instructions for performing the methods of the invention, and the like.

These and other aspects will be readily apparent to those skilled in the art in view of this disclosure as a whole.

Drawings

Fig. 1A to 1G depict the design of nucleases targeting mitochondrial DNA and mtDNA heterogeneity modification in vivo. FIG. 1A is a schematic diagram showing monomer "WTM 1" binding to the sequence upstream of m.5024 (SEQ ID NO:1) in the wild type and mutant genomes and mutant specific monomer "MTM 25" binding preferentially to the mutation site (SEQ ID NO:2) due to C > T mutation (indicated by x) in the target site. Forced heterodimer fokl domain dimerization creates a DNA double strand break, resulting in specific deletion of mutant mtDAN. Figure 1B depicts a schematic of the library (left panel) and screening assay (right panel) used to screen for nucleases targeted to mouse mtDNA. For screening, the mtm (n) mtZFN library (labeled "mtm (n)") was cloned into a backbone containing the ribosome stuttering T2A site ("2A"), WTM1 mtZFN ("WTM 1"), and hammerhead ribozyme ("HHR"), such that the backbone also co-expressed mCherry from a separate promoter (SV 40). These constructs were transfected into Mouse Embryonic Fibroblasts (MEFs) with m.5024c > T and transfectants were sorted by Fluorescence Activated Cell Sorting (FACS) at 24 hours; heterogeneity shifts in extracted DNA and transfected fibroblasts were determined by pyrosequencing. FIG. 1C shows the results of pyrosequencing analysis of m.5024C > T heterogeneity from MEFs transfected with control or MTM25/WTM1 facilitated by tetracycline-sensitive HHR 7 at different concentrations. The variation in the m.5024c > T heterogeneity ("Δ m.5024c > T (%)") was plotted according to different test conditions. "utZFN" is mtZFN that does not have a target site in mouse mtDNA 7. n is 4-8. Error bars indicate SD. Statistical analysis performed: two-tailed student t-test × p < 0.01. Fig. 1D is a schematic diagram depicting an in vivo experiment. MTM25 and WTM1 were encoded in separate AAV genomes, which were packaged in AAV9.45 and then administered systemically (tail vein) simultaneously. Animals were sacrificed 65 days post injection. Figure 1E shows western blot analysis of total cardiac protein from animals injected with MTM25 and/or WTM 1. Both proteins include an HA tag and are distinguished by molecular weight. FIG. 1E shows pyrosequencing analysis of m.5024C > T heterogeneity of total DNA from ear and heart. The m.5024c > T change (Δ) between these was plotted. n is 4-20 (table S1). Error bars indicate SEM. Statistical analysis performed: two-tailed student t-test. P < 0.001. Figure 1F shows the analysis of mtDAN copy number by qPCR. Each square indicates an animal. n is 4 to 8 (table S1). Error bars indicate SEM. Statistical analysis performed: two-tailed student t-test × p < 0.01.

FIGS. 2A-2E depict m.5024C>The reduction in T mtDNA heterogeneity results in phenotypic rescue in living subjects. FIG. 2A is a graph formed by m.5024C>Exemplary of the mt-tRNA encoded by the T mutation is ALA. Circle indicates due to 5024C>Position of mutant "a" where T mutation was inserted. Given the nature and location of this mutation, transcribed tRNA molecules containing a mutation mismatch are less likely to fold or aminoacylate correctly, resulting in m.5024C at high levels>Steady state levels of mt-tRNA ALA decrease under T heterogeneity. Figure 2B shows quantification of northern blot analysis of total cardiac RNA extracts. mt-tRNA abundance was normalized to 5S rRNA. n is 4-6. Error bars indicate SEM. Statistical analysis performed: two-tailed student's t-test。***p<0.001. The data indicate an increase in the presence of mt tRNA: ALA as normalized to mt tRNA: CYS in cells treated with WTM1/MTM25ZFN pairs compared to untreated cells. FIG. 2C depicts a schematic representation of the evaluation of mt-tRNA^ALAMedian dose of physiological effects of molecular phenotype rescue (5e12 vg/animal) AAV-treated mice and age/initial heterogeneity-matched (vehicle-treated) controls for Principal Component Analysis (PCA) plots of metabolomics data. Each square indicates one animal (see example 2). Figure 2D shows measurements of total metabolite abundance (phosphoenolpyruvate in the left panel; pyruvate in the middle panel; lactate in the right panel) from mouse heart tissue from medium dose AAV treated mice (right panel "+ AAV") and age/initial heterogeneity-matched controls (left panel "VEH") by LC/MS. The chemical structure of the final glycolytic metabolites and the reactions linking these metabolites are depicted in the upper panel. Error bars indicate SEM. Statistical analysis performed: and (5) carrying out t test on a single student. P<0.05. Figure 2E shows the chemical structure (top panel) and in vivo abundance of the initial reactants and products of the glycolytic pathway from mouse heart tissue. The elevated glucose levels in the hearts of treated animals (left panel) in combination with reduced downstream metabolite abundance (glucose-6-phosphate shown in the middle panel and fructose-6-phosphate shown in the right panel) resulted in the spectrum of restored mitochondrial metabolism and enhanced aerobic glycolysis observed in treated animals (right panel "+ AAV") compared to controls (left panel "VEH").

Detailed Description

Disclosed herein are compositions and methods for targeted modification of mtDNA (including selective cleavage of mutant mtDNA) to offset heterogeneity of mtDNA and effect reversion of molecular and biochemical phenotypes to wild-type.

The present invention contemplates genetic modifications to mtDNA, including but not limited to selective cleavage of mtDNA for the treatment and/or prevention of any genetically-derived mitochondrial disease in a subject in need thereof. Any mutant mtDNA can be targeted by a DNA binding domain including, but not limited to, m.5024c > T, 1555G, 1624T, 3243G, 3460A, 3271C, 4300G, 5545T, 7445G, 7472 random insertions, 8344G, 8356C 8993G, 9176G/C, 10158C, 10191C, 10197A, 11777A, 11778A, 13513a, 14459A, 14484C, 14487C, and/or 14709C.

SUMMARY

The practice of the methods and preparation and use of the compositions disclosed herein employ, unless otherwise indicated, conventional techniques of molecular biology, biochemistry, chromatin structure and analysis, computational chemistry, cell culture, recombinant DNA and related arts, which are well known in the art. These techniques are explained fully in the literature. See, e.g., Sambrook et al, Molecular CLONING, A Laboratory Manual, second edition, Cold Spring Harbor LABORATORY Press,1989, and third edition, 2001; ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; book of books, METHODS IN enzymolyy, Academic Press, san diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, third edition, Academic Press, san Diego, 1998; METHODS IN ENZYMOLOGY, volume 304, "Chromatin" (edited by p.m. wassarman and a.p. wolffe), Academic Press, san diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol 119, "chromatography Protocols" (edited by P.B. Becker) Humana Press, Totorwa, 1999.

Definition of

The terms "nucleic acid", "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymer of deoxyribonucleotides or ribonucleotides in either a linear or circular conformation and in either single-or double-stranded form. For the purposes of this disclosure, these terms should not be construed as limiting in terms of the length of the polymer. These terms may encompass known analogs of natural nucleotides, as well as nucleotides that are modified in the base, sugar, and/or phosphate moieties (e.g., phosphorothioate backbones). Typically, analogs of a particular nucleotide have the same base-pairing specificity; that is, the analog of A will base pair with T.

The terms "polypeptide", "peptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acids are chemical analogs or modified derivatives of the corresponding naturally occurring amino acid.

"binding" refers to a sequence-specific, non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). Not all components of a binding interaction need be sequence specific (e.g., in contact with a phosphate residue in the DNA backbone), so long as the interaction as a whole is sequence specific. Such interactions are typically characterized by a dissociation constant (K)_d) Is 10^-6M^-1Or lower. "affinity" refers to the strength of binding: enhanced binding affinity with lower K_dAnd (4) correlating.

A "binding domain" is a molecule that is capable of non-covalent binding to another molecule. The binding molecule may bind to, for example, a DNA molecule (a DNA binding protein, such as a zinc finger protein or TAL-effector domain protein or a single guide RNA), an RNA molecule (an RNA binding protein), and/or a protein molecule (a protein binding protein). In the case of a protein-binding molecule, it may bind to itself (to form homodimers, homotrimers, etc.), and/or it may bind to one or more molecules of one or more different proteins. The binding molecule may have more than one type of binding activity. For example, zinc finger proteins have DNA binding, RNA binding, and protein binding activities. Thus, DNA binding molecules (including DNA binding components of artificial nucleases and transcription factors) include, but are not limited to, ZFPs, TALEs, and sgrnas.

A "zinc finger DNA binding protein" (or binding domain) is a domain within a protein or larger protein that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain that are structurally stabilized by coordination of zinc ions. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP. Artificial nucleases and transcription factors can comprise a ZFP DNA-binding domain and a functional domain (the nuclease domain of ZFN or the transcriptional regulatory domain of ZFP-TF). The term "zinc finger nuclease" includes one ZFN and pairs of ZFNs (including a first ZFN and a second ZFN, also referred to as left ZFNs and right ZFNs) that dimerize to cleave a target gene.

A "TALE DNA binding domain" or "TALE" is a polypeptide comprising one or more TALE repeat domains/units. The repeat domain is involved in binding of the TALE to its cognate target DNA sequence. A single "repeat unit" (also referred to as a "repeat") is typically 33-35 amino acids in length and exhibits at least some sequence homology to other TALE repeat sequences in naturally occurring TALE proteins. See, for example, U.S. patent No. 8,586,526. The artificial nucleases and transcription factors can comprise a TALE DNA binding domain and a functional domain (nuclease domain of TALEN or transcription regulatory domain of TALEN-TF). The term "TALEN" includes one TALEN as well as pairs of TALENs (including a first TALEN and a second TALEN, also known as left TALEN and right TALEN) that dimerize to cleave a target gene.

Zinc fingers and TALE binding domains can be "engineered" to bind to a predetermined nucleotide sequence, for example, by engineering (changing one or more amino acids) the recognition helix region of a naturally occurring zinc finger or TALE protein. Thus, an engineered DNA binding protein (zinc finger or TALE) is a non-naturally occurring protein. A non-limiting example of a method of engineering a DNA binding protein is design and selection. The designed DNA binding proteins are proteins that do not occur in nature, and their design/composition is derived primarily from sound criteria. Reasonable criteria for design include the application of replacement rules and computerized algorithms for processing information in a database storing information of existing ZFP and/or TALE design and binding data. See, for example, U.S. patent nos. 6,140,081; 6,453,242; 6,534,261; and 8,585,526; see also international patent publication nos. WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536; and WO 03/016496.

A "selected" zinc finger protein or TALE is a protein not found in nature, which results primarily from empirical processes such as phage display, interaction traps, or hybrid selection. See, for example, U.S. patent nos. 5,789,538; 5,925,523, respectively; 6,007,988, respectively; 6,013,453, respectively; 6,200,759, respectively; 8,586,526, respectively; and international patent publication No. WO 95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970; WO 01/88197; and WO 02/099084.

"TtAgo" is a prokaryotic Argonaute protein, believed to be involved in gene silencing. TtAgo is derived from the bacterium Thermus thermophilus (Thermus thermophilus). See, e.g., Swarts et al, supra, g.sheng et al, (2013) proc.natl.acad.sci.u.s.a.111, 652). The "TtAgo system" is all components required, including, for example, guide DNA for cleavage by TtAgo enzyme.

"recombination" refers to the process of exchanging genetic information between two polynucleotides, including but not limited to donor capture by non-homologous end joining (NHEJ) and homologous recombination. For the purposes of this disclosure, "Homologous Recombination (HR)" refers to a specialized form of this exchange that occurs, for example, during repair of a double-stranded break in a cell by a homology-directed repair mechanism. This process requires nucleotide sequence homology, repairs the "target" molecule (i.e., the molecule that undergoes a double strand break) using a "donor" molecular template, and is variously referred to as "non-crossover type gene conversion" or "short strand gene conversion" because it results in the transfer of genetic information from the donor to the target. Without wishing to be bound by any particular theory, such transfer may involve mismatch correction of heteroduplex DNA formed between the fragmented target and donor, and/or "synthesis-dependent strand annealing" (where genetic information is used for weight synthesis that will be part of the target), and/or related processes. This specialized HR typically results in a change in the sequence of the target molecule such that part or all of the sequence of the donor polynucleotide is incorporated into the target polynucleotide.

In the methods of the present disclosure, one or more targeted nucleases as described herein generate a Double Strand Break (DSB) in a target sequence (e.g., cellular chromatin) at a predetermined site. DSBs may be deleted and/or inserted by homology directed repair or by non-homology directed repair mechanisms. Deletions may include any number of base pairs. Similarly, insertions can include any number of base pairs, including, for example, the incorporation of a "donor" polynucleotide that optionally shares homology with the nucleotide sequence in the break region. The donor sequence may be physically integrated, or alternatively, the donor polynucleotide serves as a template for repair of the break by homologous recombination, resulting in the introduction of all or part of the nucleotide sequence in the donor into cellular chromatin. Thus, a first sequence in cellular chromatin can be altered, and in certain embodiments, the first sequence can be converted to a sequence present in a donor polynucleotide. Thus, use of the term "replace" or "replacement" can be understood to mean the replacement of one nucleotide sequence by another nucleotide sequence (i.e., a sequence replacement in the informational sense), and does not necessarily require the physical or chemical replacement of one polynucleotide by another.

In any of the methods described herein, additional zinc finger protein pairs, TALENs, TtAgo, or CRISPR/Cas systems may be used for additional double-strand cleavage of additional target sites within the cell.

Any of the methods described herein can be used to insert donors of any size and/or partially or completely inactivate one or more target sequences in a cell by targeted integration of donor sequences that disrupt the expression of one or more genes of interest. Cell lines having partially or fully inactivated genes are also provided.

In any of the methods described herein, the exogenous nucleotide sequence (the "donor sequence" or "transgene") can comprise a sequence that is homologous but not identical to a genomic sequence in the region of interest, thereby stimulating homologous recombination to insert a different sequence in the region of interest. Thus, in certain embodiments, the portion of the donor sequence that is homologous to a sequence in the region of interest exhibits about 80% to 99% (or any integer therebetween) sequence identity relative to the genomic sequence being replaced. In other embodiments, the homology between the donor and genomic sequence is greater than 99%, for example, if more than 100 consecutive base pairs of donor and genomic sequence differ by only 1 nucleotide. In some cases, a non-homologous portion of the donor sequence can comprise a sequence that is not present in the region of interest, thereby introducing a new sequence into the region of interest. In these cases, the non-homologous sequence is typically flanked by 50-1,000 base pairs (or any integer value therebetween) or any number of base pairs greater than 1,000 that are homologous or identical to the sequence in the region of interest. In other embodiments, the donor sequence is non-homologous to the first sequence and is inserted into the genome by a non-homologous recombination mechanism.

"cleavage" refers to the breaking of the covalent backbone of a DNA molecule. Cleavage can be performed by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of phosphodiester bonds. Both single-stranded and double-stranded cleavage are possible, and double-stranded cleavage can occur as a result of two different single-stranded cleavage events. DNA cleavage can be such that blunt ends or staggered ends are produced. In certain embodiments, the fusion polypeptide is used to target double-stranded DNA cleavage.

A "cleavage half-domain" is a polypeptide sequence that binds a second polypeptide (the same or different) to form a complex with cleavage activity, preferably double-stranded cleavage activity. The terms "first and second cleavage half-domains", "+ and-cleavage half-domains" and "left and right cleavage half-domains" are used interchangeably to refer to a dimerized cleavage half-domain pair.

An "engineered cleavage half-domain" is a cleavage half-domain that has been modified so as to form an obligate heterodimer with another cleavage half-domain (e.g., another engineered cleavage half-domain). See also, U.S. patent nos. 8,623,618; 7,888,121; 7,914,796, respectively; and 8,034,598, which are incorporated herein by reference in their entirety.

The term "sequence" refers to a nucleotide sequence of any length, which may be DNA or RNA; may be linear, circular or branched, and may be single-stranded or double-stranded. The term "donor sequence" refers to a nucleotide sequence that is inserted into a genome. The donor sequence may be of any length, for example between 2 and 100,000,000 nucleotides in length (or any integer value therebetween or above), preferably between about 100 and 100,000 nucleotides in length (or any integer therebetween), more preferably between about 2000 and 20,000 nucleotides in length (or any value therebetween) and even more preferably between about 5 and 15kb (or any value therebetween).

"chromatin" is a nucleoprotein structure comprising the genome of a cell. Cellular chromatin comprises nucleic acids (primarily DNA) and proteins (including histone and non-histone chromosomal proteins). Most eukaryotic chromatin exists in the form of nucleosomes in which the nucleosome core comprises approximately 150 base pairs of DNA associated with an octamer comprising two each of histones H2A, H2B, H3 and H4, and linker DNA (of variable length, depending on the organism) extends between the nucleosome cores. The histone H1 molecule is typically associated with a linker DNA. For the purposes of this disclosure, the term "chromatin" is meant to encompass all types of nuclear proteins, both prokaryotic and eukaryotic. Cellular chromatin comprises chromosomes and episomal chromatin.

A "chromosome" is a chromatin complex that comprises all or a portion of a cell genome. The genome of a cell is generally characterized by its karyotype, which is the collection of all chromosomes that comprise the genome of the cell. The genome of the cell may comprise one or more chromosomes.

An "episome" is a replicative nucleic acid, a nucleoprotein complex or other structure comprising a nucleic acid that is not part of the chromosomal karyotype of a cell. Examples of episomes include plasmids and certain viral genomes.

An "accessible region" is a site in cellular chromatin where a target site present in a nucleic acid can be bound by an exogenous molecule that recognizes the target site. Without wishing to be bound by any particular theory, it is believed that the accessible regions are regions that are not packaged into the nucleosome structure. The different structures of the accessible regions can often be detected by their sensitivity to chemical and enzymatic probes (e.g., nuclease).

A "target site" or "target sequence" is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind, provided that sufficient conditions for binding are present. The target site may be of any length, for example 9 to 20 or more nucleotides, and the length and bound nucleotides may be contiguous or non-contiguous.

An "exogenous" molecule is a molecule that is not normally present in a cell but can be introduced into a cell by one or more genetic, biochemical, or other methods. "normally present in a cell" is determined with respect to a particular developmental stage and environmental conditions of the cell. Thus, for example, a molecule that is only present during embryonic development of muscle is an exogenous molecule with respect to adult muscle cells. Similarly, the molecule introduced by heat shock is an exogenous molecule with respect to a non-heat shocked cell. The exogenous molecule can comprise, for example, a functional form of a malfunctioning endogenous molecule or a malfunctioning form of a normally functioning endogenous molecule.

The foreign molecule may in particular be a small molecule, such as produced by a combinatorial chemical process, or a macromolecule, such as a protein, a nucleic acid, a carbohydrate, a lipid, a glycoprotein, a lipoprotein, a polysaccharide, any modified derivative of the above, or any complex comprising one or more of the above. Nucleic acids include DNA and RNA, and may be single-stranded or double-stranded; may be linear, branched or cyclic; and may be any length. Nucleic acids include those capable of forming a double helix, as well as nucleic acids that form a triple helix. See, for example, U.S. Pat. nos. 5,176,996 and 5,422,251. Proteins include, but are not limited to, DNA binding proteins, transcription factors, chromatin remodeling factors, methylated DNA binding proteins, polymerases, methylases, demethylases, acetyl esterases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, gyrases, and helicases.

The exogenous molecule may be the same type of molecule as the endogenous molecule, e.g., an exogenous protein or nucleic acid. For example, the exogenous nucleic acid may comprise an infectious viral genome, plasmid or episome introduced into the cell, or a chromosome that is not normally present in the cell. Methods for introducing exogenous molecules into cells are known to those of skill in the art and include, but are not limited to, lipid-mediated transfer (i.e., liposomes, including neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer, and viral vector-mediated transfer. The exogenous molecule may also be a molecule of the same type as the endogenous molecule, but derived from a different species than the cell source. For example, the human nucleic acid sequence may be introduced into a cell line originally derived from a mouse or hamster.

In contrast, an "endogenous" molecule is a molecule that normally exists in a particular cell at a particular developmental stage under particular environmental conditions. For example, an endogenous nucleic acid can comprise the genome of a chromosome, mitochondria, chloroplast or other organelle, or a naturally occurring episomal nucleic acid. Other endogenous molecules may include proteins, such as transcription factors and enzymes.

As used herein, the term "product of an exogenous nucleic acid" includes polynucleotide and polypeptide products, such as transcription products (polynucleotides such as RNA) and translation products (polypeptides).

A "fusion" molecule is a molecule in which two or more subunit molecules are linked (preferably covalently linked). The subunit molecules may be molecules of the same chemical type, or may be molecules of different chemical types. Examples of the first type of fusion molecule include, but are not limited to, fusion proteins (e.g., fusions between ZFPs or TALE DNA binding domains and one or more activation domains) and fusion nucleic acids (e.g., nucleic acids encoding the fusion proteins described above). Examples of the second type of fusion molecule include, but are not limited to, fusions between triple helix forming nucleic acids and polypeptides, and fusions between minor groove binders and nucleic acids.

Expression of the fusion protein in the cell can be derived from delivering the fusion protein to the cell or obtained by delivering a polynucleotide encoding the fusion protein to the cell, wherein the polynucleotide is transcribed and the transcript is translated to produce the fusion protein. Trans-splicing, polypeptide cleavage, and polypeptide ligation may also be involved in the expression of proteins in cells. Methods of delivering polynucleotides and polypeptides to cells are presented elsewhere in this disclosure.

For purposes of this disclosure, "gene" includes a region of DNA encoding a gene product (see below), as well as all regions of DNA that regulate the production of a gene product, whether or not such regulatory sequences are contiguous with the coding sequence and/or the transcribed sequence. Thus, genes include, but are not necessarily limited to, promoter sequences, terminators, translational regulatory sequences (such as ribosome binding sites and internal ribosome entry sites), enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus control regions.

"Gene expression" refers to the conversion of information contained in a gene into a gene product. The gene product can be a direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other type of RNA) or a protein resulting from translation of an mRNA. Gene products also include RNA modified by methods such as capping, polyadenylation, methylation, and editing, as well as proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP ribosylation, myristoylation, and glycosylation.

"modulation" of gene expression refers to a change in gene activity. Modulation of expression may include, but is not limited to, gene activation and gene repression. Genome editing (e.g., cleavage, alteration, inactivation, random mutation) can be used to modulate expression. Gene inactivation refers to any reduction in gene expression as compared to a cell that does not include a ZFP, TALE, TtAgo, or CRISPR/Cas system as described herein. Thus, gene inactivation may be partial inactivation or complete inactivation.

A "region of interest" is any region of cellular chromatin, such as, for example, a gene or a non-coding sequence within or adjacent to a gene, where it may be desired to bind a foreign molecule. Binding may be used for the purpose of targeted DNA cleavage and/or targeted recombination. For example, the region of interest can be present in a chromosome, episome, organelle genome (e.g., mitochondria, chloroplasts), or infectious viral genome. The region of interest may be located within the coding region of the gene, within a transcribed non-coding region (e.g., a leader sequence, trailer sequence or intron), or within a non-transcribed region upstream or downstream of the coding region. The length of the region of interest can be as small as a single nucleotide pair or as many as 2,000 nucleotide pairs, or any integer value of nucleotide pairs.

"eukaryotic" cells include, but are not limited to, fungal cells (such as yeast), plant cells, animal cells, mammalian cells, and human cells (e.g., T cells), including stem cells (pluripotent and multipotent).

The terms "operably linked" and "operably linked" (or "operably linked") are used interchangeably with respect to the juxtaposition of two or more components (such as sequential elements) wherein the components are arranged such that the two components function normally and allow for the possibility of: at least one of the components may mediate a function exerted on at least one other component. By way of illustration, a transcriptional regulatory sequence (such as a promoter) is operably linked to a coding sequence if the transcriptional regulatory sequence controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. Transcriptional regulatory sequences are typically operably linked in cis to the coding sequence, but need not be directly adjacent thereto. For example, an enhancer is a transcriptional regulatory sequence operably linked to a coding sequence, even if it is not contiguous.

With respect to functional polypeptides, the term "operably linked" may refer to the fact that: each component functions identically when it is linked to another component as it does not. For example, with respect to a fusion polypeptide in which a ZFP, TALE, TtAgo, or Cas DNA binding domain is fused to an activation domain, the ZFP, TALE, TtAgo, or Cas DNA binding domain is in operative linkage with the activation domain if, in the functional polypeptide, the ZFP, TALE, TtAgo, or Cas DNA binding domain portion is capable of binding its target site and/or its binding site while the activation domain is capable of upregulating gene expression. When the ZFP, TALE, TtAgo, or Cas DNA-binding domain in the fusion polypeptide is fused to the cleavage domain, the ZFP, TALE, TtAgo, or Cas DNA-binding domain and the cleavage domain are in operative linkage if, in the fusion polypeptide, the ZFP, TALE, TtAgo, or Cas DNA-binding domain portion is capable of binding to its target site and/or its binding site while the cleavage domain is capable of cleaving DNA in the vicinity of the target site.

A "functional fragment" of a protein, polypeptide, or nucleic acid is a protein, polypeptide, or nucleic acid that differs in sequence from a full-length protein, polypeptide, or nucleic acid, but retains the same function as the full-length protein, polypeptide, or nucleic acid. Functional fragments may have more, fewer, or the same number of residues as the corresponding native molecule, and/or may contain one or more amino acid or nucleotide substitutions. Methods for determining the function of a nucleic acid (e.g., encoding function, ability to hybridize to another nucleic acid) are well known in the art. Similarly, methods for determining the function of proteins are well known. For example, the DNA binding function of a polypeptide can be determined, for example, by filter binding, electrophoretic mobility shift, or immunoprecipitation assays. DNA cleavage can be determined by gel electrophoresis. See Ausubel et al, supra. The ability of a protein to interact with another protein can be determined, for example, by co-immunoprecipitation, two-hybrid assays, or complementation (both genetic and biochemical). See, e.g., Fields et al (1989) Nature 340: 245-246; U.S. patent No. 5,585,245 and international patent publication No. WO 98/44350.

A "vector" is capable of transferring a gene sequence to a target cell. Typically, "vector construct", "expression vector" and "gene transfer vector" mean any nucleic acid construct capable of directing the expression of a gene of interest and capable of transferring the gene sequence to a target cell. Thus, the term includes cloning and expression vehicles, as well as integrating vectors.

The terms "subject" and "patient" are used interchangeably and refer to mammals such as human patients and non-human primates, as well as laboratory animals such as rabbits, dogs, cats, rats, mice, and other animals. Thus, the term "subject" or "patient" as used herein means any mammalian patient or subject to which the nuclease, donor and/or genetically modified cell of the invention can be administered. The subject of the present invention includes subjects suffering from an obstacle.

"sternness" refers to the relative ability of any cell to function in a manner similar to a stem cell, i.e., totipotency, pluripotency, or oligopotency, and the degree of expansion or unlimited self-renewal that any particular stem cell may have.

An "ACTR" is an antibody-coupled T cell receptor, i.e., an engineered T cell component capable of binding to an exogenously supplied antibody. Binding of the antibody to the ACTR component enables the T cell to interact with the antigen recognized by the antibody, and upon encountering this antigen, the T cell-containing ACTR is triggered to interact with the antigen (see U.S. patent publication No. 2015/0139943).

Fusion molecules

Described herein are compositions, e.g., nucleases, useful for cleaving a selected target gene of mtDNA in a cell.

Recombinant transcription factors comprising DNA-binding domains from zinc finger proteins ("ZFPs") or TAL effector domains ("TALEs") and engineered nucleases comprising zinc finger nucleases ("ZFNs"), TALENs, CRISPR/Cas nuclease systems, and homing endonucleases all designed to bind to a target DNA site have the ability to regulate gene expression of endogenous genes and are useful for genome engineering, gene therapy, and treatment of mitochondrial disorders. See, for example, U.S. patent nos. 9,394,545; 9,150,847, respectively; 9,206,404, respectively; 9,045,763, respectively; 9,005,973, respectively; 8,956,828; 8,936,936, respectively; 8,945,868, respectively; 8,871,905, respectively; 8,586,526, respectively; 8,563,314, respectively; 8,329,986, respectively; 8,399,218, respectively; 6,534,261; 6,599,692, respectively; 6,503,717, respectively; 6,689,558, respectively; 7,067,317, respectively; 7,262,054, respectively; 7,888,121; 7,972,854, respectively; 7,914,796, respectively; 7,951,925, respectively; 8,110,379, respectively; 8,409,861; U.S. patent publication numbers 2003/0232410; 2005/0208489, respectively; 2005/0026157, respectively; 2005/0064474, respectively; 2006/0063231, respectively; 2008/0159996, respectively; 2010/0218264, respectively; 2012/0017290, respectively; 2011/0265198, respectively; 2013/0137104, respectively; 2013/0122591, respectively; 2013/0177983, respectively; 2013/0177960, respectively; and 2015/0056705, the disclosure of which is incorporated by reference in its entirety for all purposes. In addition, targeted nucleases based on the Argonaute system (e.g., from Thermus thermophilus, referred to as "TtAgo", see Swarts et al (2014) Nature 507(7491): 258-.

Nuclease-mediated gene therapy can be used to genetically engineer a cell to have one or more inactivated genes and/or cause the cell to express products not previously produced in the cell (e.g., via transgene insertion and/or via correction of endogenous sequences). Examples of uses for transgene insertion include insertion of one or more genes encoding one or more novel therapeutic proteins, insertion of coding sequences encoding proteins that are absent in a cell or in an individual, insertion of a wild-type gene in a cell containing a mutated gene sequence, and/or insertion of sequences encoding a structural nucleic acid (e.g., shRNA or siRNA). Examples of useful applications for "correction" of endogenous gene sequences include alterations in disease-related genetic mutations, shifts in heterogeneity, alterations in sequences encoding splice sites, alterations in regulatory sequences, and targeted alterations in sequences encoding structural features of proteins. The transgene construct can be inserted by Homology Directed Repair (HDR) or by end capture during non-homologous end joining (NHEJ) driven processes. See, for example, U.S. patent nos. 9,045,763; 9,005,973, respectively; 7,888,121; and 8,703,489.

Clinical trials using these engineered transcription factors and nucleases have shown that these molecules are capable of treating various conditions, including cancer, HIV, and/or blood disorders (such as hemoglobinopathy and/or hemophilia). See, e.g., Yu et al, (2006) FASEB J.20: 479-481; tebas et al, (2014) New Eng J Med 370(10): 901. Thus, these methods may be used to treat diseases.

In certain embodiments, one or more components of the fusion molecule (e.g., nuclease) are naturally occurring. In other embodiments, one or more components of the fusion molecule (e.g., nuclease) are non-naturally occurring, i.e., engineered in the DNA binding molecule and/or one or more cleavage domains. For example, the DNA-binding portion of a naturally occurring nuclease can be altered to bind to a selected target site (e.g., a CRISPR/Cas system or a single guide RNA of a meganuclease that has been engineered to bind to a site different from the homologous binding site). In other embodiments, the nuclease comprises a heterologous DNA binding domain and a cleavage domain (e.g., a zinc finger nuclease; TAL effector domain DNA binding protein; meganuclease DNA binding domain with a heterologous cleavage domain). Thus, any nuclease can be used to practice the invention, including but not limited to at least one ZFN, TALEN, meganuclease, CRISPR/Cas nuclease, and the like that cleaves the target gene resulting in a genomic modification of the target gene (e.g., an insertion and/or deletion in the cleaved gene).

Also described herein is the increase in specificity of cleavage activity by independent titration of engineered cleavage half-domain partners of nuclease complexes. In some embodiments, the ratio of the two partners (half-cleavage domains) is given in 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 other embodiments, the two partners are disposed in a ratio selected to be different from 1: 1. The methods and compositions of the present invention provide unexpected and unexpected increases in targeting specificity via reduction of off-target cleavage activity, when used alone or in combination. Nucleases used in these embodiments can include ZFNs, TALENs, CRISPR/Cas, CRISPR/dCas, and TtAgo, or any combination thereof.

DNA binding molecules

The fusion molecules described herein can include any DNA binding molecule (also referred to as a DNA binding domain), including protein domains and/or polynucleotide DNA binding domains. In certain embodiments, the DNA binding domain binds to a target site of 9-18 or more nucleotides, wherein the target site comprises one or more mutant mtDNA sequences. The mutation may be a point mutation, for example a target site comprising an m.5024c > T mutation.

In certain embodiments, the compositions and methods described herein employ a meganuclease (homing endonuclease) DNA-binding domain for binding to a donor molecule and/or to a region of interest in the genome of a cell. Naturally occurring meganucleases recognize cleavage sites of 15-40 base pairs and are generally divided into four families: the LAGLIDADG family, the GIY-YIG family, the His-Cyst box family and the HNH family. Exemplary homing endonucleases include 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. The recognition sequences of the exemplary homing endonucleases described above are known. See also U.S. patent nos. 5,420,032 and 6,833,252; belfort et al, (1997) Nucleic Acids Res.25: 3379-3388; dujon et al, (1989) Gene 82: 115-118; perler et al, (1994) Nucleic Acids Res.22, 1125-1127; jasin (1996) Trends Genet.12: 224-228; gimble et al, (1996) J.mol.biol.263: 163-; argast et al, (1998) J.mol.biol.280:345-353 and New England Biolabs catalog. In addition, the DNA binding specificity of homing endonucleases and meganucleases can be engineered to bind to non-native target sites. See, e.g., Chevalier et al, (2002) Molec. cell 10: 895-905; epinat et al (2003) Nucleic Acids Res.31: 2952-2962; ashworth et al, (2006) Nature 441: 656-; paques et al, (2007) Current Gene Therapy 7: 49-66; and U.S. patent publication No. 2007/0117128. The DNA binding domains of the homing endonucleases and meganucleases can be altered as a whole in the context of the nucleases (i.e., such that the nucleases comprise homologous cleavage domains) or can be fused to heterologous cleavage domains.

In other embodiments, the DNA-binding domain of one or more nucleases used in the methods and compositions described herein comprises a naturally occurring or engineered (non-naturally occurring) TAL effector DNA-binding domain. See, for example, U.S. patent No. 8,586,526, which is incorporated by reference herein in its entirety. Phytopathogenic bacteria of the genus Xanthomonas (Xanthomonas) are known to cause a wide variety of diseases in important crop plants. The pathogenicity of xanthomonas depends on a conserved type III secretion (T3S) system, which injects more than 25 different effector proteins into plant cells. Among these proteins injected are transcription activator-like (TAL) effectors that mimic plant transcription activators and manipulate plant transcriptomes (see Kay et al, (2007) Science 318: 648-651). These proteins contain a DNA binding domain and a transcription activation domain. One of the most well characterized TAL effectors is the AvrBs3 from Xanthomonas campestris var vesiculosis pv. Vesicoria (see Bonas et al, (1989) Mol Gen Genet 218: 127-. TAL effectors contain a centralized domain of tandem repeats, each containing about 34 amino acids, which are critical to the DNA binding specificity of these proteins. In addition, it contains a nuclear localization sequence and an acidic transcription activation domain (for a review see Schornack et al (2006) J Plant Physiol 163(3): 256-272). In addition, in the plant pathogenic bacterium Ralstonia solanacearum, two genes named brg11 and hpx17 have been found to be homologous to the AvrBs3 family of the genus Xanthomonas in the Ralstonia solanacearum biovariant 1 strain GMI1000 and the biovariant 4 strain RS1000 (see Heuer et al (2007) apple and Envir Micro 73(13): 4379-. The nucleotide sequences of these genes are 98.9% identical to each other, but differ by a deletion of 1,575bp in the repeat domain of hpx 17. However, both gene products have less than 40% sequence identity to the AvrBs3 family protein of xanthomonas. See, for example, U.S. patent No. 8,586,526, which is incorporated by reference herein in its entirety.

The specificity of these TAL effectors depends on the sequence found in the tandem repeat. The repeat sequences comprise approximately 102bp, and the repeat sequences are typically 91% -100% homologous to each other (Bonas et al, supra). Polymorphisms in the repeat sequence are typically located at positions 12 and 13, and a one-to-one correspondence appears to exist between the identity of the hypervariable diresidues (RVDs) at positions 12 and 13 and the identity of consecutive nucleotides in the target sequence of the TAL effector (see Moscou and Bogdanove, (2009) Science 326:1501 and Boch et al, (2009) Science 326: 1509-. In experiments, it has been determined that the DNA of these TAL effectors recognizes the native code such that the HD sequences at positions 12 and 13 result in binding to cytosine (C), NG to T, NI to A, C, G or T, NN to a or G, and ING to T. These DNA binding repeats have been assembled into proteins with new combinations and numbers of repeats to make artificial transcription factors that interact with the new sequences and activate the expression of non-endogenous reporters in plant cells (Boch et al, supra). Engineered TAL proteins have been linked to fokl cleavage half-domains to generate TAL effector domain nuclease fusions (TALENs). See, for example, U.S. patent nos. 8,586,526; christian et al (2010) Genetics epub 10.1534/genetics.110.120717). In certain embodiments, the TALE domain comprises an N-cap and/or a C-cap, as described in U.S. patent No. 8,586,526.

In certain embodiments, the DNA-binding domain of one or more nucleases for use in vivo cleavage and/or targeted cleavage of the genome of a cell comprises a zinc finger protein. Preferably, the zinc finger protein is non-naturally occurring and is engineered to bind to a target site of choice. See, for example, Beerli et al (2002) Nature Biotechnol.20: 135-141; pabo et al (2001) Ann. Rev. biochem.70: 313-340; isalan et al (2001) Nature Biotechnol.19: 656-660; segal et al, (2001) Curr, Opin, Biotechnol.12: 632-637; choo et al (2000) curr. Opin. struct. biol.10: 411-416; U.S. Pat. nos. 6,453,242; 6,534,261; 6,599,692, respectively; 6,503,717, respectively; 6,689,558, respectively; 7,030,215, respectively; 6,794,136, respectively; 7,067,317, respectively; 7,262,054, respectively; 7,070,934, respectively; 7,361,635, respectively; 7,253,273, respectively; and U.S. patent publication numbers 2005/0064474; 2007/0218528, respectively; 2005/0267061, all incorporated herein by reference in their entirety.

The engineered zinc finger binding domains may 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 a database comprising triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, wherein each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of a zinc finger that binds to a particular triplet or quadruplet sequence. See, for example, commonly owned U.S. Pat. nos. 6,453,242 and 6,534,261, which are incorporated herein by reference in their entireties.

Exemplary selection methods, including phage display and two-hybrid systems, are disclosed in U.S. Pat. nos. 5,789,538; 5,925,523, respectively; 6,007,988, respectively; 6,013,453, respectively; 6,410,248, respectively; 6,140,466, respectively; 6,200,759, respectively; and 6,242,568; and international patent publication No. WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197; and GB patent No. 2,338,237. In addition, enhancement of binding specificity for zinc finger binding domains has been described, for example, in commonly owned international patent publication No. WO 02/077227.

In addition, as disclosed in these and other references, the zinc finger domains and/or the multi-finger zinc finger proteins may be joined together using any suitable linker sequence (including, for example, linkers of 5 or more amino acids in length). For exemplary linker sequences of 6 or more amino acids in length, see also U.S. patent nos. 6,479,626; 6,903,185, respectively; and 7,153,949. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein.

ZFPs can be operably associated (linked) with one or more nuclease (cleavage) domains to form ZFNs. The term "ZFN" encompasses pairs of ZFNs that dimerize to cleave a target gene. The methods and compositions can also be used to increase the specificity of ZFNs (including nuclease pairs) for their intended target relative to other unintended cleavage sites (referred to as off-target sites) (see U.S. patent publication No. 20180087072). Thus, a nuclease described herein can comprise a mutation in one or more of its DNA-binding domain backbone regions and/or one or more mutations in its nuclease cleavage domain. These nucleases can include mutations to amino acids within the ZFP DNA-binding domain ("ZFP backbone") that can interact non-specifically with phosphates on the DNA backbone, but which do not include changes in the DNA recognition helix. Thus, the invention includes mutations in the ZFP backbone of cationic amino acid residues that are not required for nucleotide target specificity. In some embodiments, these mutations in the ZFP backbone comprise mutating cationic amino acid residues to central or anionic amino acid residues. In some embodiments, the mutations in the ZFP backbone comprise mutating polar amino acid residues to neutral or non-polar amino acid residues. In a preferred embodiment, the mutation is at position (-5), (-9) and/or position (-14) relative to the DNA binding helix. In some embodiments, the zinc finger may comprise one or more mutations at (-5), (-9), and/or (-14). In further embodiments, one or more zinc fingers of a multi-fingered zinc finger protein may comprise a mutation in (-5), (-9), and/or (-14). In some embodiments, amino acids (e.g., arginine (R) or lysine (K)) at (-5), (-9), and/or (-14) are mutated to alanine (A), leucine (L), Ser (S), Asp (N), Glu (E), Tyr (Y), and/or glutamine (Q).

In some aspects, the DNA binding domain (e.g., ZFP, TALE, sgRNA, etc.) preferentially targets mutant mtDNA as compared to wild-type. In paired nucleases, one DNA binding domain may target a wild-type sequence and the other DNA binding domain may target a mutant sequence. Alternatively, both DNA binding domains may be targeted to wild-type or mutant sequences. In certain embodiments, the DNA binding domain targets a site (9 to 18 nucleotides or more) in the mutant mtDNA (e.g., m.5024c > T) as shown in table 2. In other embodiments, the DNA binding domain targets a sequence in the mutant mtDNA comprising one or more of the following mutations: 1555G, 1624T, 3243G, 3460A, 3271C, 4300G, 5545T, 7445G, 7472 random insertion, 8344G, 8356C 8993G, 9176G/C, 10158C, 10191C, 10197A, 11777A, 11778A, 13513A, 14459A, 14484C, 14487C, or 14709C.

Selecting a target site; ZFPs and methods for designing and constructing fusion proteins (and polynucleotides encoding the fusion proteins) are known to those skilled in the art and are described in detail in the following documents: U.S. patent nos. 6,140,081; 5,789,538, respectively; 6,453,242; 6,534,261; 5,925,523, respectively; 6,007,988, respectively; 6,013,453, respectively; 6,200,759, respectively; and international patent publication No. WO 95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970; WO 01/88197; WO 02/099084; WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536; and WO 03/016496.

In addition, as disclosed in these and other references, the zinc finger domains and/or the multi-finger zinc finger proteins may be joined together using any suitable linker sequence (including, for example, linkers of 5 or more amino acids in length). For exemplary linker sequences of 6 or more amino acids in length, see also U.S. patent nos. 6,479,626; 6,903,185, respectively; and 7,153,949. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein.

In certain embodiments, the DNA-binding molecule is part of a CRISPR/Cas nuclease system. See, for example, U.S. patent No. 8,697,359 and U.S. patent publication No. 2015/0056705. The CRISPR (clustered regularly interspaced short palindromic repeats) locus (which encodes the RNA component of the system) and the Cas (CRISPR-associated) locus (which encodes the protein) (Jansen et al (2002) mol. Microbiol.43: 1565-. CRISPR loci in microbial hosts contain a combination of CRISPR-associated (Cas) genes and non-coding RNA elements capable of specifically programming CRISPR-mediated nucleic acid cleavage.

Type II CRISPRs are one of the most well characterized systems and perform targeted DNA double strand breaks in four consecutive steps. First, two non-coding RNAs, namely the pre-crRNA array and the tracrRNA, are transcribed from the CRISPR locus. In the second step, the tracrRNA hybridizes to the repeat region of the pre-crRNA and mediates the processing of the pre-crRNA into mature crRNA comprising a separate spacer sequence. tracrRNA complex directs Cas9 to target DNA through Watson-Crick base pairing between a spacer on the crRNA and a protospacer on the target DNA, which is an additional requirement for target recognition near the Protospacer Adjacent Motif (PAM). Finally, Cas9 mediates cleavage of the target DNA to create a double strand break within the protospacer. The activity of the CRISPR/Cas system comprises three steps: (i) insertion of foreign DNA sequences into CRISPR arrays to prevent subsequent attack in a process called "adaptation" (ii) expression of the relevant proteins, and expression and processing of the arrays, followed by (iii) RNA-mediated interference with foreign nucleic acids. Thus, in bacterial cells, several so-called "Cas" proteins are involved in the natural function of the CRISPR/Cas system and play a role in functions such as insertion of foreign DNA.

In certain embodiments, the Cas protein may be a "functional derivative" of a naturally occurring Cas protein. "functional derivatives" of native sequence polypeptides are compounds that have qualitative biological properties in common with native sequence polypeptides. "functional derivatives" include, but are not limited to, fragments of the native sequence and derivatives of the native sequence polypeptide and fragments thereof, provided that they have the common biological activity of the corresponding native sequence polypeptide. The biological activity contemplated herein is the ability of the functional derivative to hydrolyze a DNA substrate into fragments. The term "derivative" encompasses amino acid sequence variants, covalent modifications, and fusions thereof of a polypeptide. Suitable derivatives of Cas polypeptides or fragments thereof include, but are not limited to, mutants, fusions, covalent modifications of Cas proteins or fragments thereof. Cas proteins, including Cas proteins or fragments thereof, and derivatives of Cas proteins or fragments thereof, may be obtained from cells or chemically synthesized or obtained by a combination of both procedures. The cell may be one that naturally produces a Cas protein, or one that naturally produces a Cas protein and is genetically engineered to produce an endogenous Cas protein at a higher expression level or to produce a Cas protein from an exogenous introduced nucleic acid encoding the same or a different Cas than the endogenous Cas. In some cases, the cell does not naturally produce the Cas protein, and is genetically engineered to produce the Cas protein. In some embodiments, the Cas protein is a small Cas9 ortholog, delivered via AAV vectors (Ran, et al (2015) Nature 510, page 186).

In some embodiments, the DNA binding molecule is part of a TtAgo system (see Swarts et al, supra; Sheng et al, supra). In eukaryotes, gene silencing is mediated by the argonaute (ago) family of proteins. In this paradigm, Ago binds to small (19-31nt) RNA. This protein-RNA silencing complex recognizes the target RNA by Watson-Crick base pairing between the small RNA and the target, and endonucleolytically cleaves the target RNA (Vogel (2014) Science 344: 972-973). In contrast, prokaryotic Ago proteins bind to small single-stranded DNA fragments and may act to detect and remove foreign (often viral) DNA (Yuan et al (2005) mol.cell 19,405; Olovnikov et al (2013) mol.cell 51,594; Swarts et al, supra). Exemplary prokaryotic Ago proteins include those from Aquifex aeolicus, Rhodobacter sphaeroides, and Thermus thermophilus.

One of the most well characterized prokaryotic Ago proteins is that from Thermus thermophilus (T. thermophilus) (Ttago; Swarts et al, supra). TtAgo associates with 15nt or 13-25nt single stranded DNA fragments having a 5' phosphate group. This "guide DNA" to TtAgo binding is used to guide the protein-DNA complex to bind the Watson-Crick complementary DNA sequence in a third party DNA molecule. Once the sequence information in these guide DNAs allows identification of the target DNA, the TtAgo-guide DNA complex cleaves the target DNA. This mechanism is also supported by the structure of the TtAgo-directed DNA complex that binds to its target DNA (g.sheng et al, supra). Ago from rhodobacter sphaeroides (RsAgo) has similar properties (Olovnikov et al, supra).

Exogenous guide DNA of any DNA sequence can be loaded onto the TtAgo protein (Swarts et al, supra). Because the specificity of TtAgo cleavage is guided by the guide DNA, TtAgo-DNA complexes formed from exogenous, investigator-specified guide DNA direct TtAgo target DNA cleavage to complementary, investigator-specified target DNA. In this way, a targeted double-strand break can be created in the DNA. The use of a TtAgo-guided DNA system (or an orthologous Ago-guided DNA system from other organisms) allows targeted cleavage of genomic DNA within cells. This cleavage may be single-stranded or double-stranded. For cleaving mammalian genomic DNA, a form of TtAgo codon optimized for expression in mammalian cells is preferably used. Furthermore, it may be preferred to treat the cells with a TtAgo-DNA complex formed in vitro, wherein the TtAgo protein is fused to a cell penetrating peptide. In addition, it may be preferred to use a form of the TtAgo protein that has been altered by mutagenesis to have improved activity at 37 ℃. Using standard techniques in the art that utilize DNA fragmentation, Ago-RNA mediated DNA cleavage can be used to affect a panoramic picture of the results (including gene knockout, targeted gene addition, gene correction, targeted gene deletion).

Thus, nucleases comprise DNA binding molecules that specifically bind to a target site in any gene into which it is desired to insert a donor (transgene).

B. Cleavage domain

Any suitable cleavage domain may be operably linked to the DNA-binding domain to form a nuclease. For example, ZFP DNA-binding domains have been fused with nuclease domains to form ZFNs, which are functional entities that are capable of recognizing their intended nucleic acid targets through their engineered (ZFP) DNA-binding domains and causing DNA to be cleaved near the ZFP binding site by nuclease activity, including for genomic modification in various organisms. See, for example, U.S. patent nos. 7,888,121; 8,623,618, respectively; 7,888,121; 7,914,796 and 8,034,598; and U.S. patent publication No. 2011/0201055. Likewise, the TALE DNA binding domain has been fused to a nuclease domain to produce a TALEN. See, for example, U.S. patent No. 8,586,526.

As described above, the cleavage domain may be heterologous to the DNA binding domain, such as a zinc finger DNA binding domain and cleavage domain from a nuclease or TALEN DNA binding domain, or a meganuclease DNA binding domain and cleavage domain from a different nuclease. The heterologous cleavage domain may be obtained from any endonuclease or exonuclease. Exemplary endonucleases from which the cleavage domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. Additional enzymes that cleave DNA are known (e.g., S1 nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease). One or more of these enzymes (or functional fragments thereof) may be used as a source of cleavage domains and cleavage half-domains.

Similarly, the cleavage half-domain may be derived from any of the nucleases listed above, or parts thereof, which require dimerization to produce cleavage activity. Typically, if the fusion protein comprises a cleavage half-domain, then two fusion proteins are required for cleavage. Alternatively, a single protein comprising two cleavage half-domains may be used. The two cleavage half-domains may be derived from the same endonuclease (or functional fragments thereof), or each cleavage half-domain may be derived from a different endonuclease (or functional fragments thereof). In addition, the target sites of the two fusion proteins are preferably arranged with respect to each other such that binding of the two fusion proteins to their respective target sites places the cleavage half-domains in a spatial orientation with respect to each other, which allows the cleavage half-domains to form a functional cleavage domain, e.g., by dimerization. Thus, in certain embodiments, the proximal edges of the target sites are separated by 5-8 nucleotides or 15-18 nucleotides. However, any integer number of nucleotides or nucleotide pairs (e.g., 2 to 50 nucleotide pairs or more) may be inserted between the two target sites. Typically, the cleavage site is located between the target sites.

Restriction endonucleases (restriction enzymes) are present in many species and are capable of sequence-specific binding to DNA (at a recognition site) and of cleaving DNA at or near the binding site. Certain restriction enzymes (e.g., type IIS) cleave DNA at sites remote from the recognition site and have separable binding and cleavage domains. For example, type IIS FokI enzymes catalyze double-stranded cleavage of DNA, cleaving at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other strand. See, for example, U.S. Pat. nos. 5,356,802; 5,436,150, respectively; and 5,487,994; and Li et al (1992) Proc. Natl.Acad.Sci.USA 89: 4275-; li et al (1993) Proc. Natl.Acad.Sci.USA 90: 2764-; kim et al (1994a) Proc.Natl.Acad.Sci.USA 91: 883-887; kim et al (1994b) J.biol.chem.269:31,978-31, 982. Thus, in one embodiment, the fusion protein comprises a cleavage domain (or cleavage half-domain) from at least one type IIS restriction enzyme and one or more zinc finger binding domains (which may or may not be engineered).

An exemplary type IIS restriction enzyme whose cleavage domain can be separated from the binding domain is FokI. This particular enzyme is active as a dimer. Bitinaite et al, (1998) Proc. Natl. Acad. Sci. USA 95:10,570-10, 575. Thus, for the purposes of this disclosure, the portion of the FokI enzyme used in the disclosed fusion proteins is considered to be the cleavage half-domain. Thus, for targeted double-stranded cleavage and/or targeting of alternative cell sequences using zinc finger-fokl fusions, two fusion proteins each comprising a fokl cleavage half-domain can be used to reconstitute a catalytically active cleavage domain. Alternatively, a single polypeptide molecule comprising a zinc finger binding domain and two fokl cleavage half-domains may also be used. Parameters for targeted cleavage and targeted sequence changes using zinc finger-fokl fusions are provided elsewhere in the disclosure.

The cleavage domain or cleavage half-domain may be any portion of a protein that retains cleavage activity, or retains the ability to multimerize (e.g., dimerize) to form a functional cleavage domain.

Exemplary type IIS restriction enzymes are described in International publication WO 07/014275, which is incorporated herein in its entirety. Additional restriction enzymes also comprise separable binding and cleavage domains, and this disclosure contemplates these. See, e.g., Roberts et al, (2003) Nucleic Acids Res.31: 418-420.

In certain embodiments, the cleavage domain comprises one or more engineered cleavage half-domains (also referred to as dimerization domain mutants) that minimize or prevent homodimerization, e.g., as described in U.S. patent nos. 8,623,618; 7,888,121; 7,914,796, respectively; and 8,034,598; and U.S. patent publication No. 2011/0201055, the entire disclosure of which is incorporated herein by reference in its entirety. Amino acid residues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 of fokl are all targets for affecting dimerization of the fokl cleavage half-domains.

In certain embodiments, the engineered cleavage half-domain is derived from fokl and comprises one or more mutations in one or more of amino acid residues 416, 422, 447, 448, and/or 525 (see, e.g., U.S. patent publication No. 20180087072) numbered relative to the wild-type fokl cleavage half-domain (residues 394-579 of full-length fokl) as shown below:

wild type FokI cleavage half-domain (SEQ ID NO:1)

QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF

These mutations reduce non-specific interactions between the FokI domain and the DNA molecule. In other embodiments, the cleavage half-domain derived from FokI comprises a mutation at one or more of amino acid residues 414-426, 443-450, 467-488, 501-502 and/or 521-531. The mutations may include mutations of residues present in natural restriction enzymes homologous to FokI. In certain embodiments, the mutation is a substitution, e.g., a substitution of the wild-type residue with a different amino acid, e.g., serine (S), e.g., R416S or K525S. In a preferred embodiment, the mutation at position 416, 422, 447, 448 and/or 525 comprises the substitution of a positively charged amino acid with an uncharged or negatively charged amino acid. In another embodiment, the engineered cleavage half-domain comprises mutations in amino acid residues 499, 496, and 486 in addition to mutations in one or more of amino acid residues 416, 422, 447, 448, or 525. In a preferred embodiment, the invention provides a fusion protein wherein the engineered cleavage half-domain comprises a polypeptide wherein 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 glu (e) residue ("ELD" or "ELE") in addition to the one or more mutations at positions 416, 422, 447, 448 or 525.

Cleavage domains with more than one mutation, e.g., at positions 490(E → K) and 538(I → K) in one cleavage half-domain, can be used to generate an engineered cleavage half-domain designated "E490K: I538K" and by mutating positions 486(Q → E) and 499(I → L) in the other cleavage half-domain to generate an engineered cleavage half-domain designated "Q486E: I499L"; a mutation that replaces the wild-type gln (q) residue at position 486 with a glu (e) residue, the wild-type iso (i) residue at position 499 with a leu (l) residue, and the wild-type asn (n) residue at residue 496 with an asp (d) or a glu (e) residue (also referred to as "ELD" and "ELE" domains, respectively); engineered cleavage half-domains comprise mutations at positions 490, 538 and 537 (numbered relative to wild-type fokl), for example mutations that replace the wild-type glu (e) residue at position 490 with a lys (k) residue, the wild-type iso (i) residue at position 538 with a lys (k) residue and the wild-type his (h) residue at position 537 with a lys (k) residue or a arg (r) residue (also referred to as "KKK" and "KKR" domains, respectively); and/or the engineered cleavage half-domain comprises mutations at positions 490 and 537 (numbered relative to wild-type fokl), for example mutations that replace the wild-type glu (e) residue at position 490 with a lys (k) residue and the wild-type his (h) residue at position 537 with a lys (k) residue or a arg (r) residue (also referred to as "KIK" and "KIR" domains, respectively). See, for example, U.S. patent nos. 7,914,796; 8,034,598, respectively; and 8,623,618; the disclosure of which is incorporated by reference in its entirety for all purposes. In other embodiments, the engineered cleavage half-domain comprises a "Sharkey" and/or "Sharkey" mutation (see Guo et al, (2010) J.mol.biol.400(1): 96-107).

Alternatively, nucleases can be assembled at nucleic acid target sites in vivo using so-called "split-enzyme" technology (see, e.g., U.S. patent publication No. 2009/0068164). Components of such a cleavage enzyme may be expressed on separate expression constructs or may be linked in one open reading frame, with the individual components separated by, for example, a self-cleaving 2A peptide or IRES sequence. The components may be individual zinc finger binding domains or domains of meganuclease nucleic acid binding domains.

Nucleases can be screened for activity prior to use, for example in a yeast-based staining system, as described in U.S. patent No. 8,563,314.

Cas 9-associated CRISPR/Cas system comprises two RNA non-coding components: tracrRNA and pre-crRNA arrays containing nuclease guide sequences (spacers) separated by the same Direct Repeat (DR). In order to achieve genome engineering using the CRISPR/Cas system, two functions of these RNAs must exist (see Cong et al (2013) science xpress 1/10.1126/science 1231143). In some embodiments, the tracrRNA and pre-crRNA are supplied via separate expression constructs or as separate RNAs. In other embodiments, chimeric RNAs are constructed in which an engineered mature crRNA (conferring target specificity) is fused to a tracrRNA (supplying interaction with Cas 9) to produce a chimeric cr-RNA-tracrRNA hybrid (also referred to as a single guide RNA). (see Jinek et al (2012) Science 337: 816-821; Jinek et al (2013) eLife 2: e00471.DOI:10.7554/eLife.00471 and Cong, supra).

In some embodiments, the CRISPR-Cpf1 system is used. The CRISPR-Cpf1 system identified in Francisella spp is a class 2 CRISPR-Cas system that mediates robust DNA interference in human cells. Although Cpf1 and Cas9 are functionally conserved, they differ in many ways, including in the guide RNA and substrate specificity of Cpf1 and Cas9 (see Fagerlund et al (2015) Genom Bio 16: 251). The main difference between Cas9 and Cpf1 proteins is that Cpf1 does not utilize tracrRNA, and therefore only crRNA is required. FnCpf1 crRNA is 42-44 nucleotides in length (19-nucleotide repeats and 23-25-nucleotide spacers) and contains a single stem-loop that can tolerate sequence changes that preserve secondary structure. Furthermore, Cpf1 crRNA is significantly shorter than the engineered sgRNA of about 100 nucleotides required for Cas9, and the PAM requirement for FnCpfl is 5'-TTN-3' and 5'-CTA-3' on the replacement strand. Although both Cas9 and Cpf1 produce double-strand breaks in the target DNA, Cas9 uses its RuvC and HNH-like domains to make blunt-end cuts within the seed sequence of the guide RNA, while Cpf1 uses RuvC-like domains to make staggered cuts outside the seed sequence. Since Cpf1 is staggered from the critical seed region, NHEJ does not disrupt the target site, thus ensuring that Cpf1 can continue to cleave the same site until the desired HDR recombination event occurs. Thus, in the methods and compositions described herein, the term "Cas" is understood to include both Cas9 and Cpf1 proteins. Thus, as used herein, "CRISPR/Cas system" refers to CRISPR/Cas and/or CRISPR/Cpf1 systems, including both nuclease and/or transcription factor systems.

Target site

As described above, the DNA binding domain may be engineered to bind to any sequence of choice. The engineered DNA binding domain may have novel binding specificity compared to naturally occurring DNA binding domains.

In certain embodiments, the one or more nucleases can target any wild-type or mutant mtDNA sequence that selectively targets mutant mtDNA, for example encompassing target sites of 9-25 or more nucleotides (contiguous or non-contiguous) of a mutant mtDNA sequence such as: 1555G, 1624T, 3243G, 3460A, 3271C, 4300G, 5545T, 7445G, 7472 random insertions, 8344G, 8356C 8993G, 9176G/C, 10158C, 10191C, 10197A, 11777A, 11778A, 13513A, 14459A, 14484C, 14487C, or 14709C or target sites as shown in Table 2.

Following the teachings of the present specification, construction of such expression cassettes utilizes methods well known in the art of molecular biology (see, e.g., Ausubel or manitis). Prior to use of the expression cassette to generate transgenic animals, the responsiveness of the expression cassette to a stress inducer that associates with the selected control element can be tested by introducing the expression cassette into a suitable cell line (e.g., a primary cell, transformed cell, or immortalized cell line).

Furthermore, although not necessary for expression, the exogenous sequence may also be a transcriptional or translational regulatory sequence, such as a promoter, enhancer, insulator, internal ribosome entry site, sequence encoding a 2A peptide, hammerhead ribozyme, targeting peptide, and/or polyadenylation signal. In addition, the control elements of the gene of interest can be operably linked to a reporter gene to produce a chimeric gene (e.g., a reporter expression cassette).

Targeted insertion of non-coding nucleic acid sequences may also be achieved. Sequences encoding antisense RNA, RNAi, shRNA, and microrna (mirna) may also be used for targeted insertion.

In further embodiments, the donor nucleic acid may comprise non-coding sequences that are specific target sites designed for additional nucleases. Subsequently, additional nucleases can be expressed in the cell, allowing cleavage and modification of the original donor molecule by insertion of another donor molecule of interest. In this way, repeated integration of donor molecules can be generated, allowing for a characteristic stacking (trail stacking) at a specific locus of interest or at a safe harbor locus.

Cells

Thus, provided herein are genetically modified cells comprising a genetically modified mtDNA gene. In certain embodiments, the modification comprises cleaving the mutant mtDNA such that a heterogeneity ratio of the mtDNA is altered. In certain embodiments, the mutant mtDNA is cleaved in a patient having one or more mitochondrial disorders such that the disorder or symptoms associated therewith are treated and/or prevented. The nuclease can bind and cleave any mutant mtDNA differentially, including but not limited to binding at a point mutation (such as m.5024c > T).

Unlike random cleavage, targeted cleavage ensures that mutant forms of mtDNA are preferentially cleaved compared to wild-type, e.g., when nucleases are designed such that the DNA binding domain binds to the mutated sequence and exhibits specificity for the mutant form.

Any cell type, including but not limited to cells and cell lines, can be genetically modified as described herein. Other non-limiting examples of cells containing modified mtDNA include heart cells, brain cells, lung cells, liver cells, T cells (e.g., CD4+, CD3+, CD8+, etc.); a dendritic cell; b cells; autologous (e.g., patient-derived) or allogeneic pluripotent, totipotent or multipotent stem cells (e.g., CD34+ cells, including pluripotent stem cells (ipscs), embryonic stem cells, etc.). In certain embodiments, the cell as described herein is a CD34+ cell derived from a patient.

The cells as described herein are useful for treating and/or preventing mitochondrial disease in a subject suffering from the disorder, e.g., by in vivo or ex vivo therapy. For ex vivo therapy, nuclease-modified cells can be amplified using standard techniques and then reintroduced into the patient. See, e.g., Tebas et al, (2014) New Eng J Med 370(10): 901. In the case of stem cells, these precursors differentiate into mtDNA-expressing cells at an altered heterogeneity rate compared to wild-type (diseased) cells after infusion into a subject. Also provided are pharmaceutical compositions comprising cells as described herein. In addition, the cells may be cryopreserved prior to administration to a patient.

The cells and ex vivo methods as described herein provide for the treatment and/or prevention of disorders (e.g., mitochondrial disorders) in a subject (e.g., a mammalian subject) and eliminate the need for continuous prophylactic administration or risky procedures, such as allogeneic bone marrow transplantation or gamma retrovirus delivery. Thus, the invention described herein provides a safer, cost-effective and time-saving way of treating and/or preventing mitochondrial disorders.

Delivery of

The nucleases, polynucleotides encoding such nucleases, donor polynucleotides, and compositions comprising the proteins and/or polynucleotides described herein can be delivered by any suitable means. In certain embodiments, the nuclease and/or donor is delivered in vivo. In other embodiments, nucleases and/or donors are delivered to isolated cells (e.g., autologous or heterologous stem cells) to provide modified cells that can be used for ex vivo delivery to a patient.

Methods of delivering nucleases as described herein are described, for example, in the following documents: U.S. Pat. nos. 6,453,242; 6,503,717, respectively; 6,534,261; 6,599,692, respectively; 6,607,882, respectively; 6,689,558, respectively; 6,824,978, respectively; 6,933,113, respectively; 6,979,539, respectively; 7,013,219, respectively; and 7,163,824, the entire disclosure of which is incorporated herein by reference in its entirety.

Nucleases and/or donor constructs as described herein can also be delivered using any nucleic acid delivery mechanism, including naked DNA and/or RNA (e.g., mRNA) and vectors containing sequences encoding one or more components. Any vector system may be used, including but not limited to plasmid vectors, DNA micro-loops, retroviral vectors, lentiviral vectors, adenoviral vectors, poxvirus vectors; herpes virus vectors and adeno-associated virus vectors, and the like, and combinations thereof. See also U.S. Pat. nos. 6,534,261; 6,607,882, respectively; 6,824,978, respectively; 6,933,113, respectively; 6,979,539, respectively; 7,013,219, respectively; and 7,163,824; and U.S. patent publication No. 2014/0335063, which is incorporated by reference herein in its entirety. Further, it is clear that any of these systems may contain one or more sequences required for processing. Thus, when one or more nucleases and donor constructs are introduced into a cell, the nucleases and/or donor polynucleotides may be carried on the same delivery system or on different delivery mechanisms. When multiple systems are used, each delivery mechanism may comprise a sequence encoding one or more nucleases and/or a donor construct (e.g., mRNA encoding one or more nucleases and/or mRNA or AAV carrying one or more donor constructs).

Introduction into cells (e.g., mammalian cells) and target tissues can be accomplished using conventional viral and non-viral based gene transfer methodsNucleic acids encoding nucleases and donor constructs are included. Non-viral vector delivery systems include DNA plasmids, DNA minicircles, naked nucleic acids, and nucleic acids complexed with a delivery vehicle, such as a liposome or poloxamer. Viral vector delivery systems include DNA and RNA viruses that have an episomal or integrated genome after delivery to a cell. For a review of gene therapy programs, see Anderson, Science 256: 808-; nabel and Felgner, TIBTECH 11:211-217 (1993); mitani and Caskey, TIBTECH 11:162-166 (1993); dillon, TIBTECH 11: 167-; miller, Nature 357:455-460 (1992); van Brunt, Biotechnology 6(10):1149-1154 (1988); vigne, reactive Neurology and Neuroscience 8:35-36 (1995); kremer and Perricaudet, British Medical Bulletin 51(1):31-44 (1995); haddada et al, Current Topics in Microbiology and Immunology Doerfler and(1995); and Yu et al, Gene Therapy 1:13-26 (1994).

Non-viral delivery methods of nucleic acids include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycations or lipids nucleic acid conjugates, Lipid Nanoparticles (LNPs), naked DNA, naked RNA, capped RNA, artificial viral particles, and agent-enhanced DNA uptake. Nucleic acids can also be delivered using sonoporation using, for example, the Sonitron 2000 system (Rich-Mar).

Other exemplary nucleic acid delivery systems include those provided by: amaxa Biosystems (colongene, germany), Maxcyte, inc. (Rockville, maryland), BTX molecular delivery system (Holliston, MA), and copernius Therapeutics Inc (see, e.g., U.S. patent No. 6,008,336). Lipofection is described, for example, in U.S. patent nos. 5,049,386; 4,946,787, respectively; and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam)^TMAnd Lipofectin^TM). Suitable cationic and neutral lipids for efficient receptor recognition lipofection of polynucleotides include those of Felgner, international patent publication nos. WO 91/17424, WO 91/16024. In some aspectsIn one aspect, the nuclease is delivered as mRNA, and the transgene is delivered via other forms (such as viral vectors, minicircle DNA, plasmid DNA, single-stranded DNA, linear DNA, liposomes, nanoparticles, and the like).

Preparation of nucleic acid complexes (including targeted liposomes such as immunoliposome) is well known to those skilled in the art (see, e.g., Crystal, Science 270:404- & 410 (1995); Blaese et al, Cancer Gene Ther.2:291- & 297 (1995); Behr et al, Bioconjugate chem.5:382- & 389 (1994); Remy et al, Bioconjugate chem.5:647- & 654 (1994); Gao et al, Gene Therapy 2:710- & 722 (1995); Ahmad et al, Cancer Res.52:4817- & 4820 (1992); U.S. Pat. Nos. 4,186,183; 4,217,344; 4,235,871; 4,261,975; 4,485,054; 4,501,728; 4,774,085; 4,837,028; and 4,946,787).

Other delivery methods include the use of packaging of the nucleic acid to be delivered into an EnGeneIC Delivery Vehicle (EDV). These EDVs are specifically delivered into a target tissue using a bispecific antibody, where one arm of the antibody is specific for the target tissue and the other arm is specific for the EDV. The antibody loads the EDV to the target cell surface, after which the EDV is introduced into the cell by endocytosis. After entering the cell, the contents are released (see MacDiarmid et al, (2009) Nature Biotechnology 27(7): 643).

Delivery of nucleic acids encoding engineered CRISPR/Cas systems using RNA or DNA virus based systems utilizes highly evolved methods to target the virus to specific cells in vivo and transport the virus payload into the nucleus. The viral vector may be administered directly to the subject (in vivo), or it may be used to treat cells in vitro, as well as to administer the modified cells to the subject (ex vivo). Conventional virus-based systems for delivering CRISPR/Cas systems include, but are not limited to, retroviral, lentiviral, adenoviral, adeno-associated viral, vaccinia viral and herpes simplex viral vectors for gene transfer. Integration into the host genome is possible using retroviral, lentiviral, and adeno-associated viral gene transfer methods, often resulting in long-term expression of the inserted transgene. In addition, high transduction efficiencies are observed in many different cell types and target tissues.

The tropism of retroviruses can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and typically producing high viral titers. The choice of retroviral gene transfer system depends on the target tissue. Retroviral vectors include cis-acting long terminal repeats with packaging capability for up to 6-10kb of foreign sequences. The minimal cis-acting LTR is sufficient for replication and packaging of the vector, which is then used to integrate the therapeutic gene into the target cell to provide durable transgene expression. Widely used retroviral vectors include those based on: murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immunodeficiency Virus (SIV), Human Immunodeficiency Virus (HIV) and combinations thereof (see, e.g., Buchscher et al (1992) J.Virol.66: 2731-.

In applications where transient expression is preferred, an adenovirus-based system may be used. Adenovirus-based vectors are capable of extremely high transduction efficiency in many cell types and do not require cell division. Using such vectors, high titers and high expression levels have been obtained. Such a carrier can be produced in large quantities in a relatively simple system. Adeno-associated virus ("AAV") vectors are also useful for transducing cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo Gene Therapy programs (see, e.g., West et al, Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; International patent publication No. WO 93/24641; Kotin (1994) Human Gene Therapy 5: 793. 801; Muzyczka, J.Clin.invest.94:1351 (1994)) the construction of recombinant AAV vectors is described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al, mol.cell.biol.5: 3251-42 60 (1985); Tratschin et al, mol.cell.biol.4:2072-2081 (1984); and Muzycza serotype, PNAS.81: 3266 (Satsu et al., AAV 3262; and AAV strains, such as AAV 3978, 1989, 6778; AAV strains; see, AAV strains, such as ATCC No. 3, 1985, 1984; and AAV strains; see, AAV strains, e.3, 1989, 2, 1984; and AAV strains, 2, AAV strains, such as AAV strains, 2, AAV2/8, AAV2/5 and AAV 2/6.

At least six viral vector approaches are currently available for gene transfer in clinical trials using a pathway involving complementation of defective vectors by genes inserted into helper cell lines to produce transducints.

pLASN and MFG-S are examples of retroviral vectors that have been used in clinical trials (Dunbar et al, Blood 85: 3048-. PA317/pLASN was the first therapeutic vector to be used in gene therapy trials. (Blaese et al, Science 270: 475-. Transduction efficiency of MFG-S packaged vectors has been observed to be 50% or higher. (Ellem et al, Immunol Immunother.44(1):10-20 (1997); Dranoff et al, hum. Gene ther.1:111-2 (1997)).

Recombinant adeno-associated viral vectors (rAAV) are promising alternative gene delivery systems based on defective and non-pathogenic parvoviral adeno-associated type 2 viruses. All vectors were derived from plasmids that only retained AAV 145 base pair (bp) inverted terminal repeats flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery due to integration into the genome of the transduced cell are key features of this vector system. (Wagner et al, Lancet 351: 91171702-3 (1998)), Kearns et al, Gene ther.9:748-55 (1996)). Other AAV serotypes may also be used in accordance with the present invention, including AAV1, AAV3, AAV4, AAV5, AAV6, AAV8, AAV8.2, AAV9, AAV9.45, and AAVrh10, as well as pseudotyped AAV such as AAV2/8, AAV2/5, and AAV 2/6. In some embodiments, AAV serotypes that target the heart, lung, brain, and/or muscle are used, including but not limited to use AAV serotypes that are capable of crossing the blood brain barrier.

Replication-defective recombinant adenovirus vectors (Ad) can be produced at high titers and are susceptible to infection of a variety of different cell types. Most adenoviral vectors are engineered such that the transgene replaces the Ad E1a, E1b, and/or E3 genes; the replication-defective vector is then propagated in human 293 cells which supply the deleted gene function in trans. Ad vectors can transduce various types of tissues in vivo, including non-dividing differentiated cells such as those found in the liver, kidney, and muscle. Conventional Ad vectors have greater carrying capacity. An example of the use of Ad vectors in clinical trials involves polynucleotide therapy for anti-tumor immunity using intramuscular injection (Sterman et al, hum. Gene Ther.7:1083-9 (1998)). Additional examples of the use of adenoviral vectors for gene transfer in clinical trials include Rosenecker et al, Infectin 24: 15-10 (1996); sterman et al, hum. Gene Ther.9:71083-1089 (1998); welsh et al, hum.Gene ther.2:205-18 (1995); alvarez et al, hum. Gene ther.5:597-613 (1997); topf et al, Gene ther.5:507-513 (1998); sterman et al, hum. Gene ther.7:1083-1089 (1998).

The packaging cells are used to form viral particles that infect the host cell. Such cells include 293 cells packaging adenovirus and ψ 2 cells or PA317 cells packaging retrovirus. Viral vectors for use in gene therapy are typically produced by a production cell line that packages the nucleic acid vector into a virion. The vector typically contains the minimal viral sequences required for packaging and subsequent integration into the host (if applicable), with the other viral sequences being replaced by an expression cassette encoding the protein to be expressed. The lost viral function is supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically have only Inverted Terminal Repeat (ITR) sequences from the AAV genome that are required for packaging and integration into the host genome. Viral DNA is packaged in cell lines containing helper plasmids encoding other AAV genes (i.e., rep and cap) but lacking ITR sequences. The cell line is also infected with adenovirus as a helper. Helper viruses promote replication of AAV vectors and expression of AAV genes from helper plasmids. Helper plasmids are not packaged in large quantities due to the lack of ITR sequences. Contamination with adenovirus can be reduced by, for example, heat treatment to which adenovirus is more sensitive than AAV.

In many gene therapy applications, it may be desirable to deliver gene therapy vectors to specific tissue types with a high degree of specificity. Thus, a viral vector can be modified to have specificity for a given cell type by expressing the ligand as a fusion protein with the viral coat protein on the outer surface of the virus. The ligand is selected to have affinity for a receptor known to be present on the cell type of interest. For example, Han et al, Proc. Natl. Acad. Sci. USA 92:9747-9751(1995) reported that Moloney (Moloney) murine leukemia virus can be modified to express human nerve growth factor fused to gp70, and that the recombinant virus infects certain human breast cancer cells expressing human epidermal growth factor receptors. This principle can be extended to other virus-target cell pairs, where the target cell expresses a receptor and the virus expresses a fusion protein comprising a ligand for a cell surface receptor. For example, filamentous phage can be engineered to display antibody fragments (e.g., FAB or Fv) with specific binding affinity to virtually any selected cellular receptor. Although the above description applies primarily to viral vectors, the same principles may apply to non-viral vectors. Such vectors can be engineered to contain specific uptake sequences that facilitate uptake by specific target cells.

Gene therapy vectors can be delivered to an individual subject, typically by topical application via systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, sublingual, or intracranial infusion) as described below, or in vivo administration via pulmonary inhalation. Alternatively, the vector may be delivered to the cells ex vivo, such as cells explanted from individual patients (e.g., lymphocytes, bone marrow aspirate, tissue biopsy) or fully donor hematopoietic stem cells, before the cells are reimplanted into the patient, typically after selection of cells into which the vector has been incorporated.

Vectors containing nucleases and/or donor constructs (e.g., retroviruses, adenoviruses, liposomes, etc.) can also be administered directly to an organism to transduce cells in vivo. Alternatively, naked DNA may be administered. Administration is by any route commonly used to introduce molecules for eventual contact with blood or tissue cells, including but not limited to injection, infusion, topical application, inhalation, and electroporation. Suitable methods of using such nucleic acids are available and well known to those skilled in the art, and while more than one route may be used to administer a particular composition, a particular route may generally provide a more direct and more efficient reaction than another route.

Vectors suitable for introducing the polynucleotides described herein include non-integrating lentiviral vectors (IDLV). See, e.g., Ory et al (1996) Proc. Natl. Acad. Sci. USA 93: 11382. 11388; dull et al (1998) J.Virol.72: 8463-8471; zuffery et al (1998) J.Virol.72: 9873-; follenzi et al (2000) Nature Genetics 25: 217-222; U.S. patent No. 8,936,936.

Pharmaceutically acceptable carriers depend, in part, on the particular composition being administered, as well as on the particular method used to administer the composition. Thus, there are a wide variety of suitable formulations of Pharmaceutical compositions that can be used, as described below (see, e.g., Remington's Pharmaceutical Sciences, 17 th edition, 1989).

Clearly, the nuclease encoding sequence and donor construct can be delivered using the same or different systems. For example, the donor polynucleotide can be carried by AAV, while the one or more nucleases can be carried by mRNA. In addition, different systems may be administered by the same or different routes (intramuscular, tail vein, other intravenous, intraperitoneal, and/or intramuscular). Multiple vectors may be delivered simultaneously or in any order.

Formulations for ex vivo and in vivo administration include suspensions in liquids or emulsified liquids. The active ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, the compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizing agents or other agents that enhance the effectiveness of the pharmaceutical compositions.

The effective amount administered for modifying mtDNA, including for treating and/or preventing mitochondrial disorders, will vary between subjects and depending on the mode of administration and the site of administration. Thus, the effective amount is optimally determined by the person administering the composition and is common in the artThe skilled person can easily determine the appropriate dosage. In certain embodiments, after allowing sufficient time for expression (e.g., typically 2-15 days or more), analysis of mtDNA modified serum or other tissue levels and comparison to prior administration will determine whether the amount administered is too low, within an appropriate range, or too high. Suitable regimens for initial and subsequent administration are also variable, but are represented by an initial administration followed by a subsequent administration, if desired. Subsequent administrations may be given at variable intervals ranging from daily to yearly to every few years. In certain embodiments, where a viral vector such as AAV is used, the total dose or component dose administered may be at 1x10¹⁰And 5x10¹⁵Between vg/ml (or any value therebetween), even more preferably at 1x10¹¹And 1x10¹⁴Between vg/ml (or any value therebetween), even more preferably at 1x10¹²And 1x10¹³Between vg/ml (or any value therebetween). In some embodiments, the total dose may be administered intravenously and may be between 5e12 vg/kg and 1e15 vg/kg (or any value therebetween), even more preferably between 5e13 vg/kg and 5e14 vg/kg (or any value therebetween), even more preferably between 5e13 vg/kg and 1e14 vg/kg (or any value therebetween).

Applications of

The methods and compositions disclosed herein are useful for providing therapy to mitochondrial diseases or disorders, e.g., by altering the heterogeneous ratio of mutant mtDNA to wild-type DNA such that the disease or disorder is treated and/or prevented. The cells may be modified in vivo or may be modified ex vivo and subsequently administered to a subject. Thus, the methods and compositions provide for the treatment and/or prevention of mitochondrial disorders.

Non-limiting examples of mitochondrial disorders that can be treated and/or prevented using the methods and compositions described herein include: LHON (Leber's hereditary optic neuropathy), MM (mitochondrial myopathy), AD (Alzheimer's disease), LIMM (lethal infant mitochondrial myopathy), ADPD (Alzheimer's disease and Parkinson's disease), MMC (maternal myopathy and cardiomyopathy), NARP (neurogenic muscular weakness, ataxia and retinitis pigmentosa; alternating phenotype at this locus reported as Leigh disease), FICP (lethal infant cardiomyopathy plus MELAS-associated cardiomyopathy), MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like attacks), LDYT (Leber's hereditary optic neuropathy and dystonia), MERRF (myoclonic epilepsy and fragmented red muscle fibers), CM (maternal hereditary hypertrophic disease), CPEO (chronic progressive extraocular paralysis), KSS (Sararn syndrome), DM (diabetes), DMDF (diabetes + deafness), O (chronic intestinal paralysis with obstruction of the eye), and pseudoparalysis of the eye, DEAF (maternally inherited deafness or aminoglycoside induced deafness), PEM (progressive encephalopathy), SNHL (sensorineural deafness), aging, encephalomyopathy, FBSN (familial bilateral striatal necrosis), PEO and SNE (subacute necrotizing encephalopathy).

Nuclease-mediated cleavage can be used to correct mtDNA sequences associated with disease (e.g., point mutations, substitution mutations, etc.). Correction can be via, for example, degradation of mtDNA sequences that are cleaved in the absence of effective DNA repair mechanisms, as is typically the case in mitochondria. Specific mutant human mtdnas that can be targeted include 1555G, 1624T, 3243G, 3460A, 3271C, 4300G, 5545T, 7445G, 7472 insertions, 8344G, 8356C 8993G, 9176G/C, 10158C, 10191C, 10197A, 11777A, 11778A, 13513a, 14459A, 14484C, 14487C, and 14709C.

By way of non-limiting example, the methods and compositions described herein may be used to treat and/or prevent mitochondrial disorders including, but not limited to, mitochondrial myopathy; diabetes and deafness (DAD); leber's disease; leber Hereditary Optic Neuropathy (LHON), characterized by progressive loss of central vision due to degeneration of the optic nerve and retina, affecting 1 in 50,000 in finland; leigh syndrome; maternally inherited Leigh syndrome; leigh-like syndrome; neuropathy, ataxia, retinitis pigmentosa, and ptosis (NARP); myoneurogenic gastrointestinal encephalopathy (MNGIE); myoclonic Epilepsy (MERRF) with ragged red fibers; mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like episodes (MELAS); mtDNA-deficient mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), cardiomyopathy, deafness, other disorders.

The following examples relate to exemplary embodiments of the disclosure, wherein the nuclease comprises a Zinc Finger Nuclease (ZFN). It is understood that this is for exemplary purposes only and that other nucleases can be used, such as TALENs, TtAgo and CRISPR/Cas systems, homing endonucleases (meganucleases) with engineered DNA-binding domains and/or fusions of naturally occurring engineered homing endonucleases (meganucleases) DNA-binding domains with heterologous cleavage domains and/or fusions of meganucleases with TALE proteins. For example, additional nucleases can be designed to bind to sequences comprising 9 to 12 contiguous nucleotides of the sequences disclosed herein (e.g., table 2). In addition, the following examples relate to nucleases in which a DNA binding domain (ZFP, TAL effector domain, sgRNA, etc.) selectively binds to mtDNA having a 5024C > T mutation (as shown in table 2). It will be clear that this is for exemplary purposes only and that nucleases binding to other mutant mtDNA sequences are envisaged, including but not limited to one or more mutations at one or more of the following positions: 1555G, 1624T, 3243G, 3460A, 3271C, 4300G, 5545T, 7445G, 7472 random insertions, 8344G, 8356C 8993G, 9176G/C, 10158C, 10191C, 10197A, 11777A, 11778A, 13513A, 14459A, 14484C, 14487C, and/or 14709C.

Examples

Example 1: mtDNA nuclease

Zinc finger proteins targeted to mtDNA were designed and incorporated into mRNA, plasmid, AAV or adenoviral vectors essentially as described in the following literature: gamma et al (2014) EMBO Mol Med.6: 458-466; gamma et al (2016) Methods in mol. biol.1351: 145-162; U.S. Pat. nos. 6,534,261 and 9,139,628.

Specifically, ZFP pairs with single nucleotide binding specificity for mutant mtDNA (m5024C > T) were generated. See fig. 1. Since this site in mouse mtDNA is challenging for ZFPs, the choice of targeting strategies with different numbers of zinc finger motifs, spacer lengths and additional linkers was employed. Assembly of candidate ZFPs results in 24 unique ZFPs that target the m.5024c > T site, called mutant-specific monomers (MTM); and a library of single partner ZFPs targeting adjacent sequences on opposite strands, called wild type specific monomer 1(WTM1) (fig. 1A).

The MTM (n) T2A _ WTM1 m.5024C > T candidate library was cloned by inserting the MTM ZFP domain upstream of FokI (+) between the 5 'EcoRI and 3' BamHI restriction sites. This product was then PCR amplified to include the 5' ApaI site and remove the 3' stop codon, while also incorporating the T2A sequence and the 3' XhoI site. This fragment was then cloned into pcmCherry (addge 62803) using the ApaI/XhoI sites. The WTM1 ZFP was cloned separately upstream of fokl (-) in the pcmCherry _3k19 vector (addge 104499) incorporating the 3' hammerhead ribozyme (HHR) using 5' EcoRI and 3' BamHI sites, and the resulting product was PCR amplified to include 5' XhoI and 3' AflII sites, allowing for cloning downstream of the mtm (n) variant.

MTM25(+) and WTM1(-) monomers were also cloned into separate pcmchery and pTracer vectors as previously described in Gamma et al (2016) Methods in mol.biol.1351: 145-. Vector construction of mtzfns intended for AAV production was achieved by PCR amplification of MTM25(+) _ HHR and WTM1(-) _ HHR transgenes incorporating 5 'EagI and 3' BglII sites.

These products were then cloned into rAAV2-CMV between the 5 'EagI and 3' BamHI sites. The FLAG epitope of WTM1(-) was replaced by a Hemagglutinin (HA) tag by PCR. The resulting plasmids were used to generate recombinant AAV2/9.45-CMV-MTM25 and AAV2/9.45-CMV-WTM1 virus particles in the Vector Core laboratory (UNC Gene Therapy Center, Vector Core Facility, Church mountain, N.C.). The 3K19 hammerhead ribozyme (HHR) sequence (Beilstein et al (2015) ACS Synth Biol 4: 526-.

Table 1 shows recognition helices within the DNA binding domains of exemplary mtDNA ZFP DNA binding domains and the target sites for these ZFPs (DNA target sites are indicated in uppercase letters; non-contacting nucleotides are indicated in lowercase letters). Nucleotides in target sites of helix-contact recognized by ZFPs are indicated in uppercase letters; non-contact nucleotides are indicated by lower case letters. TALENs and/or sgrnas are also designed into the sequences shown in table 2 (e.g., target sites comprising 9 to 20 or more (including 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more) nucleotides (contiguous or non-contiguous nucleotides) of the target sites shown in table 2) following methods known in the art. See, e.g., U.S. patent No. 8,586,526 (classical or non-classical RVD using TALENs) and U.S. patent publication No. 2015/0056705.

Table 1: mtDNA zinc finger protein recognition helix design

Table 2: target sites for zinc finger proteins

MTM#	SBS#	Target site
			WTM1	48960	aaGTTAAACTTGTGTGTtttcttagggc(SEQ ID NO:33)
MTM62	48962	tgATAAGGATTGTAaGACTTCatcctac(SEQ ID NO:34)
			MTM24	51024	tgATAAGGATTGTAaGACTTCatcctac(SEQ ID NO:34)
MTM25	51025	tgATAAGGATTGTAaGACTTCatcctac(SEQ ID NO:34)
			MTM26	51026	tgATAAGGATTGTAaGACTTCatcctac(SEQ ID NO:34)
MTM27	51027	tgATAAGGATTGTAaGACTTCatcctac(SEQ ID NO:34)
			MTM28	51028	tgATAAGGATTGTAaGACTTCatcctac(SEQ ID NO:34)
MTM29	51029	tgATAAGGATTGTAaGACTTCatcctac(SEQ ID NO:34)
			MTM30	51030	tgATAAGGATTGTAaGACTTCatcctac(SEQ ID NO:34)
MTM32	51032	tgATAAGGATTGTAaGACTTCatcctac(SEQ ID NO:34)
			MTM33	51033	tgATAAGGATTGTAaGACTTCatcctac(SEQ ID NO:34)
MTM36	51036	tgATAAGGATTGTAaGACTTCatcctac(SEQ ID NO:34)
			MTM37	51037	tgATAAGGATTGTAaGACTTCatcctac(SEQ ID NO:34)
MTM39	51039	tgATAAGGATTGTAaGACTTCatcctac(SEQ ID NO:34)
			MTM42	51042	tgATAAGGATTGTAaGACTTCatcctac(SEQ ID NO:34)
MTM43	51043	tgATAAGGATTGTAaGACttcatcctac(SEQ ID NO:34)
			MTM45	51045	tgATAAGGATTGTAAGActtcatcctac(SEQ ID NO:34)
MTM65	48965	gaTAAGGATTGTAAGACttcatcctaca(SEQ ID NO:35)
			MTM66	48966	gaTAAGGATTGTAAGACttcatcctaca(SEQ ID NO:35)
MTM48	51048	gaTAAGGATTGTAAGACttcatcctaca(SEQ ID NO:35)
			MTM49	51049	gaTAAGGATTGTAAGACttcatcctaca(SEQ ID NO:35)
MTM50	51050	gaTAAGGATTGTAAGACttcatcctaca(SEQ ID NO:35)
			MTM52	51052	gaTAAGGATTGTAAgacttcatcctaca(SEQ ID NO:35)
MTM55	51055	gaTAAGGATTGTAAGACttcatcctaca(SEQ ID NO:35)
			MTM56	51056	gaTAAGGATTGTAAGACttcatcctaca(SEQ ID NO:35)

All ZFN pairwise combinations were tested for cleavage activity. Wild type and m.5024C > T Mouse Embryo Fibroblast (MEF) cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 2mM L-glutamine, 110mg/L sodium pyruvate (Life Technologies) and 10% FCS (PAA laboratories). Cells were transfected by electroporation using MEF1 kit and T20 program using Nucleofector II device (Lonza). Fluorescence Activated Cell Sorting (FACS) was performed as described in Gamma et al (2016) Methods Mol Biol 1352: 145-162. Control of mtZFN expression is achieved by titration of tetracycline into the medium, controlling the rate of HHR autocatalysis, as previously described in gamma et al (2016) Nucleic Acids Res 44:7804, which also describes how total cellular protein extraction is performed. Detection of proteins by western blot was achieved by resolving 20-100 μ g of extracted proteins on SDS-PAGE 4% -12% bis-tris Bolt gels. These were transferred to nitrocellulose using an iBlot 2 transfer tank (Life Technologies). Antibodies used for western blotting in this work: rat anti-HA (Roche, 1:500), goat anti-rat HRP (Santa Cruz, 1: 1000). Gels were stained with coomassie brilliant blue (Life Technologies) for loading. All pairs were found to be active.

Constructs were also subjected to several rounds of screening in Mouse Embryonic Fibroblasts (MEFs) carrying approximately 65% m.5024c > T to assess heterogeneous shift activity. As shown in figure 1, these screens identified consistent specific activity against MTM25/WTM1, which produced approximately 20% shift from 65% to 45% m.5024c > T in MEF cell lines as determined by pyrosequencing. Briefly, the assessment of m.5024c > T mtDNA heterogeneity was performed by pyrosequencing. A40 cycle PCR reaction for pyrosequencing was prepared using KOD DNA polymerase (Takara) using 100ng of template DNA with the following primers:

m.4,962-4,986 Forward direction

5’ATACTAGTCCGCGAGCCTTCAAAG 3’(SEQ ID NO:36)

m.5,360-m.5,383 in the reverse direction

5’[Btn]GAGGGTTCCGATATCTTTGTGATT 3’(SEQ ID NO:37)

m.5003-m.5022 sequencing primer

5’AAGTTTAACTTCTGATAAGG 3’(SEQ ID NO:38)

In addition, mitochondrial localization was confirmed by immunofluorescence in fixed MEF cells as described in Minczuk et al (2010) Methods Mol Biol 649: 257-270. MTM25 and WTM1 were uniquely localized in mitochondria and this pair was selected for further in vivo experiments.

It will be clear that these designs may include any linker between any of the finger molecules and/or between the ZFP and the cleavage domain, including but not limited to classical and/or non-classical linkers (between the fingers) and/or linkers between the ZFP and the cleavage domain as described in us patent No. 9,394,531. See also, U.S. patent No. 8,772,453 and U.S. patent publication No. 2015/0064789.

In addition, nucleases other than ZFNs (including CRISPR/Cas nucleases, TALENs, etc.) can be designed to target sites of 9-18 or more nucleotides as shown above. Any of the nucleases (ZFNs, CRISPR/Cas systems, and TALENs) can comprise an engineered cleavage domain, such as the heterodimers disclosed in U.S. patent No. 8,623,618 (e.g., ELD and KKR engineered cleavage domains), and/or a cleavage domain having one or more mutations in positions 416, 422, 447, 448, and/or 525 as described in U.S. patent publication No. 20180087072. These mutants are used in conjunction with the exemplary ZFP DNA binding domains described herein.

Example 2: in vivo nuclease activity

Nuclease activity was also tested in mice. The C57BL/6j-t RNA used in this study^ALAMice were housed in temperature-controlled (21 ℃) rooms with one to four cages each, with a 12h light-dark cycle and a relative humidity of 60%.

MTM25 and WTM1 mtZFN monomers were encoded in separate viral genomes and packaged in heart-targeted engineered AAV9.45 serotypes (fig. 1D). See Pulicheria et al (2011) Mol Ther 19: 1070-. Detection of proteins by western blot was achieved by resolving 20-100 μ g of extracted proteins on SDS-PAGE 4% -12% bis-tris Bolt gels. These were transferred to nitrocellulose using an iBlot 2 transfer tank (Life Technologies). Antibodies used for western blotting in this work: rat anti-HA (Roche, 11867431001, 1:500), goat anti-rat HRP (Santa Cruz, SC2065, 1: 1000). Gels were stained with coomassie brilliant blue (Life Technologies) for loading.

As shown in fig. 1E, 5x10 was administered systemically (tail vein)¹²Robust expression of MTM25 and WTM1 in total mouse heart tissue was detected by western blot after individual viral genomes (vg)/monomer/mouse.

Further in vivo experiments were performed as follows. Female mice 2 to 8 months old with 44% -81% m.5024c > T heterogeneity (20 vehicles, 7 single monomers, 4/mtZFN-AAV9.45 dose) were treated in groups as shown below:

treatment with vehicle (1 XPBS, 350mM NaCl, 5% w/v D-sorbitol) and AAV was given systemically by tail vein injection.

For mouse heart tissue, 50mg of it was homogenized in RIPA buffer (150mM NaCl, 50mM Tris (pH 8), 1% (v/v) Triton X-100, 0.5% (v/v) deoxycholate, 0.1% (v/v) SDS) using a GentleMesACS dissociator (Miltenyi). The resulting homogenate was centrifuged at 10,000x g at 4 ℃ for 10 minutes, and then the supernatant was recovered and centrifuged at 10,000x g at 4 ℃ for 10 minutes. The concentration of cell and tissue protein extracts was determined by BCA assay (Pierce).

An assessment of mtDNA heterogeneity by pyrosequencing was performed and expressed as the change (Δ) between auricular hole genotype and postmortem heart genotype determined at two weeks of age (prior to experimental intervention). Briefly, mitochondrial DNA copy number of mouse heart samples was determined by qPCR using PowerUp SYBR Green master mix according to the manufacturer's protocol (Applied Biosystems). Samples were analyzed using a 7900HT fast real-time PCR system (Thermo Fisher). The following primers were used:

MT-COI forward direction

5’TGCTAGCCGCAGGCATTACT 3’(SEQ ID NO:39)

MT-COI reversal

5’CGGGATCAAAGAAAGTTGTGTTT 3’(SEQ ID NO:40)

RNaseP forward direction

5’GCCTACACTGGAGTCCGTGCTACT 3’(SEQ ID NO:41)

RNaseP reverse direction

5’CTGACCACACACGAGCTGGTAGAA3’(SEQ ID NO:42)

All primers for pyrosequencing and qPCR were designed against the C57BL/6j mouse nuclear and mitochondrial genomes, respectively, using NCBI reference sequences grcm38.p6 and NC _ 005089.1.

As shown in fig. 1F and fig. 1G, animals injected 65 days after injection revealed m.5024c in mtZFN-treated mice>Elimination of T mutant mtDNA, but not in vehicle or single monomer injected controls. The degree of change in heterogeneity by mtZFN treatment followed a biphasic AAV dose-dependent trend with intermediate doses (5x 10)¹²V g) in elimination of m.5024C>The T mutant mtDNA is most effective. Lowest (1x 10)¹²Individual vg) doses did not result in heterogeneity shifts, possibly due to insufficient concentrations of mtzfns and/or AAV resulting in mosaic transduction of targeted tissues. Highest dose (1X 10)¹³Vg) exhibited a comparable intermediate dose (5x 10)¹²Vg) compared to reduced heterogeneous shift activity, probably due to off-target effects resulting in partial mtDNA copy number deletions, which was not observed when lower doses were administered (fig. 1G). The latter result is consistent with our past observations, underscoring the importance of fine-tuning mtZFN levels in mitochondria for effective mtDNA heterogeneity modification.

Having defined conditions for achieving robust excursions of the m.5024C > T heterogeneity in vivo, we next addressed disease-associated phenotypic correction in animal models of mitochondrial disease (see Kauppila et al (2016) Cell Rep 16: 2980;. 2990. the common feature of mt-tRNA molecules in mitochondrial disease that is reproduced in a tRNAALA mouse model is instability of mt-tRNA molecules proportional to mutant loading. see FIG. 2A; Yarham et al (2010) Wiley Indressip Rev RNA 1: 304-.

mtZ for cross-dose range to evaluate mtZFN treatmentEffect of mt-tRNAALA stability in the Heart of FN treated animals northern blots were performed essentially as described in Pearce et al (2017) Elife 6, doi: 10-7554/eLife.27596. Briefly, total RNA was extracted from 25mg of mouse heart tissue using trizol (ambion) by homogenization using a GentlemACS dissociator (Miltenyi). Specifically, 5ug of total RNA was resolved on a 10% polyacrylamide gel containing 8M urea. The gel was blotted dry onto a positively charged nylon membrane (Hybond-N +) where the resulting membrane was exposed to 254nm UV light at 120mJ/cm²And (4) carrying out crosslinking. For the tRNA probe, the cross-linked membrane was hybridized with a radioactively labeled RNA probe T7 transcribed from a PCR fragment corresponding to the appropriate region of mouse mtDNA. Using complementary alpha 32P]End-labeled DNA oligo probe 5 SrRNA. The film was exposed to a storage phosphor screen and scanned using a Typhoon phosphorescence imaging system (GE Healthcare). The signal was quantified using Fiji software. The sequence of the oligomer is as follows:

MT-TA Forward

5’

TAATACGACTCACTATAGGGAGACTAAGGACTGTAAGACTTCATC 3’(SEQ ID NO:43)

MT-TA reverse (SEQ ID NO:44)

5’GAGGTCTTAGCTTAATTAAAG 3’

MT-TC Forward (SEQ ID NO:45)

5’TAATACGACTCACTATAGGGAGACAAGTCTTAGTAGAGATTTCTC3’

MT-TC reverse (SEQ ID NO:46)

5’GGTCTTAAGGTGATATTCATG 3

5S rRNA oligomer:

5’

AAGCCTACAGCACCCGGTATTCCCAGGCGGTCTCCCATCCAAGTACTAACCA 3’(SEQ ID NO:47)

all primers for northern blot and qPCR were designed for the C57BL/6j mouse nuclear and mitochondrial genomes, respectively, using NCBI reference sequences grcm38.p6 and NC _ 005089.1.

As shown in fig. 2B, there was a significant increase in the steady state level of mt-tRNAALA, which is proportional to the heterogeneity shift detected in these mice (fig. 1F). The deletion of mtDNA copy number associated with administration of high viral dose (fig. 1G) did not appear to affect the recovery of mt-tRNAALA steady-state levels after a heterogeneity excursion, consistent with previously published data, i.e. even severe mtDNA deletions did not show a proportional change in mitochondrial RNA steady-state levels. See Jazayeri et al (2003) J.biol.chem 278: 9823-.

Further experiments were conducted to assess the physiological role of the mt-tRNAALA molecular phenotypic rescue. In particular, titers from the used intermediate viruses were assessed (5X 10)¹²Individual vg) steady state metabolite abundance in cardiac tissue of treated mice. Briefly, frozen tissue samples were cut and weighed into precell tubes pre-filled with ceramic beads (Stretton Scientific ltd., debarkshire, uk). The correct volume of extraction solution (30% acetonitrile, 50% methanol and 20% water) was added to obtain 40mg of sample per mL of extraction solution. Tissue samples were lysed using a Precellys 24 homogenizer (Stretton Scientific ltd., debarkshire, uk). The suspensions were mixed and incubated in a thermal mixer (Eppendorf, Germany) for 15 minutes at 4 ℃ followed by centrifugation (16,000g, 15 minutes at 4 ℃). The supernatant was collected and transferred to an autosampler glass vial, which was stored at-80 ℃ until further analysis. The samples were randomized in order to avoid bias due to machine settling and were treated blindly. LC-MS analysis was performed using a QOxctive Orbitrap Mass spectrometer in combination with a Dionex U3000 UHPLC system (Thermo). The liquid chromatography system was equipped with a ZIC-pHILIC column (150 mm. times.2.1 mm) and a guard column (20 mm. times.2.1 mm) from Merck Millipore (Germany), and the temperature was maintained at 40 ℃. The mobile phase consisted of 20mM ammonium carbonate and 0.1% ammonium hydroxide in water (solvent a) and acetonitrile (solvent B). The flow rate was set at 200. mu.L/min, with the gradient as previously described in Mackay et al (2015) Methods Enzymol 561: 171-. The mass spectrometer was operated in both full MS and polarity switched modes. The spectra obtained were analyzed using XCalibur Qual Browser and XCalibur Quan Browser software (Thermo Scientific).

As shown in fig. 2C-2E, this analysis revealed altered metabolic characteristics in mtZFN-treated mice (fig. 2C), demonstrating increased phosphoenolpyruvate and pyruvate levels and lower lactate levels compared to controls (fig. 2D). In addition, the treated animals exhibited higher glucose levels, but lower glucose-6-phosphate and fructose-6-phosphate levels (fig. 2E).

Thus, the recovery of mitochondrial function after m.5024c > T heterogeneity migration using nuclease was achieved.

Taken together, the data demonstrate that nucleases targeting mutant mitochondrial DNA sequences can be used in vitro and in vivo to manipulate heterogeneous mutations in mouse mtDNA, thereby generating molecular and physiological rescue of disease phenotypes in heart tissue.

All patents, patent applications, and publications mentioned herein are hereby incorporated by reference in their entirety.

Although the disclosure has been provided in detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit or scope of the disclosure. Accordingly, the foregoing description and examples should not be considered as limiting.