WO2017197141A2 - Targeted treatment of androgenic alopecia - Google Patents

Abstract

Disclosed herein are methods and compositions for inactivating genes associated with androgenic alopecia, using engineered nucleases comprising a DNA binding domain and a cleavage domain or cleavage half-domain in conditions able to preserve cell viability. Polynucleotides encoding nucleases, vectors comprising polynucleotides encoding nucleases, and cells comprising polynucleotides encoding nucleases and/or cells comprising nucleases are also provided.

Description

TARGETED TREATMENT OF ANDROGENIC ALOPECIA

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. Provisional

Application No. 62/335,952, filed May 13, 2016, the disclosure of which is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

[0002] The present disclosure is in the field of genome modification of human cells, including skin cells and structures found in the skin.

BACKGROUND

[0003] Gene therapy holds enormous potential for a new era of human therapeutics. These methodologies will allow treatment for conditions that have not been addressable by standard medical practice. Gene therapy can include the many variations of genome editing techniques such as disruption or correction of a gene locus, and insertion of an expressible transgene that can be controlled either by a specific exogenous promoter fused to the transgene, or by the endogenous promoter found at the site of insertion into the genome.

[0004] Recombinant transcription factors comprising the DNA binding domains from zinc finger proteins ("ZFPs"), TAL-effector domains ("TALEs") and CRISPR/Cas transcription factor systems have the ability to regulate gene expression of endogenous genes (see, e.g., U.S. Patent Nos. 8,586,526; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,067,317; 7,262,054; Perez-Pinera et al. (2013) Nature Methods 10:973 -976; Platek et al. (2014) Plant Biotechnology J. doi :

10.1111/pbi.12284). Clinical trials using these engineered transcription factors containing zinc finger proteins have shown that these novel transcription factors are capable of treating various conditions, (see, e.g., Yu et al. (2006) FASEB J. 20:479- 481).

[0005] In addition, artificial nucleases comprising the DNA binding domains from zinc finger proteins ("ZFPs"), TAL-effector domains ("TALEs"), Ttago and CRISPR/Cas nuclease systems (including Cas and/or Cfpl) have the ability to modify gene expression of endogenous genes via nuclease-mediated modification of the gene, including either homology directed repair (HDR), following non-homologous end joining ( HEJ) and/or by end capture during non-homologous end joining ( HEJ) driven processes. See, e.g., U.S. Patent Nos. 9,394,531; 9,255,250; 9,200,266;

9,045,763; 9,005,973; 9,150,847; 8,956,828; 8,945,868; 8,703,489; 8,586,526;

6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,067,317; 7,262,054; 7,888, 121;

7,972,854; 7,914,796; 7,951,925; 8, 110,379; 8,409,861; U.S. Patent Publications 2003/0232410; 2005/0208489; 2005/0026157; 2005/0064474; 2006/0063231;

2008/0159996; 2010/00218264; 2012/0017290; 2011/0265198; 2013/0137104;

2013/0122591; 2013/0177983; 2013/0196373; 2015/0056705 and 2015/0335708. Further, targeted nucleases are being developed based on the Argonaute system (e.g., from T. thermophilics, known as 'TtAgo', see Swarts et al (2014) Nature 507(7491): 258-261), which also may have the potential for uses in genome editing and gene therapy. This nuclease-mediated approach to transgene integration offers the prospect of improved transgene expression, increased safety and expressional durability, as compared to classic integration approaches, since it allows exact transgene

positioning for a minimal risk of gene silencing or activation of nearby oncogenes.

[0006] Androgenetic alopecia (AGA) is the most common form of hair loss in humans, occurring both in men and women. In men, this condition is commonly known as male pattern baldness (MPB), as hair is lost in a well-defined pattern, beginning above both temples. Over time, the hairline recedes and hair also thins at the crown (near the top of the head), often progressing to partial or complete baldness. In Caucasians, MPB is noticeable in about 20% of men aged 20, and increases steadily with age, so that a male in his 90s has a 90% chance of having some degree of MPB. In women, onset of androgenetic alopecia tends to occur later and in a milder form. The pattern of hair loss in women also differs from male-pattern baldness. In women, the hair becomes thinner all over the head, and the hairline does not recede. Androgenetic alopecia in women rarely leads to total baldness. A familial tendency to MPB and racial variation in the prevalence is well recognized, with heredity accounting for approximately 80% of predisposition. Normal levels of androgens are sufficient to cause hair loss in genetically susceptible individuals.

[0007] The key pathophysiological features of AGA are alteration in hair cycle development, follicular miniaturization and inflammation. In general, hair growth occurs in a cycle that can last from a few months (e.g. for shorter terminal length hairs such as those in the eyebrow) to several years. Hair is produced in the anagen or growth phase where cell division takes place in the matrix of the hair bulb outside the dermal papilla. Keratinocytes then move up into the thinner part of the hair follicle, differentiating into the layers of the hair and its surrounding sheath. Melanocytes in the bulb also transfer pigment to the hair keratinocytes to give the hair color. Anagen is followed by a short regressive phase called catagen which is a transitional phase that lasts 2-3 weeks. The final stage is called telogen which is a resting phase that can last about 2-3 months. At the end of telogen, the hair is shed and the cycle starts over (Blume-Peytave et al (2008) in Hair Growth and Disorders (Blume-Peytave, Tosti Whiting, Trueb Eds.). Regulation of the growth cycle in humans is very complex and is dependent of such factors as race, hair location, age, sex, etc. and even may display seasonal variations, and there are many growth factors that are thought to play a part. For example, signaling through WNT, Hedgehog and Wingless plays a role as well as other well-known growth factors such as F-kB, TGFp, TGFp2, TGFpR-II, βΐ-integrin, NCAM, FGF1, FGF2, FGF4, FGFr2, IGF-1, growth hormone and many others (Blume-Peytave et al, ibid).

[0008] Androgens also play significant roles in hair growth. Interestingly, their relative roles differ depending on the type of hair. For example, androgens have almost no impact on the growth of eyelashes, while having significant impact on beard growth. In many types of cells, androgen (testosterone) diffuses from the blood through the plasma membrane. Inside the cell, like other steroid hormones, testosterone may bind to specific androgen receptors. This type of activity occurs in skeletal muscle cells, and some hair follicles such as those that produce pubic and axillary hair. In certain tissues however, particularly the secondary sexual organs such as the prostate, or in beard and balding hair follicles, testosterone is metabolized by one of the 5a-reductases into the more potent androgen 5a-dihydrotestosterone which has been demonstrated to have more affinity for the androgen receptor than testosterone.

[0009] In AGA, the anagen phase decreases with each cycle, while the length of telogen remains constant or is prolonged. Ultimately, anagen duration becomes so short that the growing hair fails to achieve sufficient length to reach the surface of the skin, leaving an empty follicular pore. Hair follicle miniaturization is the histological hallmark of androgenetic alopecia. Once the arrector pili muscle, which attaches circumferentially around the primary follicle, has detached from all secondary follicles and primary follicles have undergone miniaturization and detachment, hair loss is likely irreversible (see Cranwell W, Sinclair R. Male Androgenetic Alopecia. [Updated 2016 Feb 29]. In: De Groot LJ, Beck-Peccoz P, Chrousos G, et al., editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000-.).

[0010] Treatment of AGA currently involve the use of topical minoxidil and oral finasteride which are approved by the Food and Drug Administration (USA) for the treatment of AGA. Both medications prevent further hair loss, but only partially reverse baldness, and require continuous use to maintain the effect. Topical minoxidil is well tolerated as a 5% solution and acts as a potassium channel agonist and vasodilator, potentially increasing blood supply to the hair follicle. Finasteride is a potent and selective antagonist of type II 5a-reductase, thus preventing the conversion of testosterone into 5a-dihydrotestosterone (DHT). Both treatments are not completely effective however. For example, Minoxidil is most effective on small areas of hair loss, and in patients under 40 years of age who have only recently started losing hair, but loss returns when Monoxidil use is stopped. Finasteride (Propecia) is only available for men, and can cause birth defects if a pregnant woman is exposed to it during pregnancy. It is also associated with an increased risk of prostate cancer and impotence, even after discontinuing use (Traish et al (2014) Korean J Urol 55 (6): 367- 379).

[0011] To date, genome-wide association studies (GWAS) have identified 12 potential regions of interest, with the strongest association found regions on the long arm of the X chromosome (Heilmann-Heimbach et al (2016) Exp Derm 25: 251).

Along with other genetic loci, the gene encoding the androgen receptor (AR) is found in this location. The eleven other regions are found in autosomal regions, which may in part explain why some baldness patterns are similar between fathers and their sons. Specific genes located in these autosomal regions include the histone deactylases HDAC4 and/or histone deacetylase HDAC9, both of which are expressed in the hair follicle and both may play roles in the activity of AR; 5-alpha-reductase2 (SRD5A2), responsible for the enzymatic production of DHT from testosterone (see prostaglandin D2 synthase (PTGDS); GPR44 receptor (also known as PTGDR2 or DP2)); the TWIST 1 and TWIST2 transcription factors where TWIST 1 has been shown to play a role in the transition between anagen and catagen phase and TWIST2 has been shown to play a role in the establishment of mesenchymal cell lineages; genes involved in WNT signaling which may regulate AR signaling and cell cycle in the follicle such as WntlOA which is expressed in early hair morphogenesis and is expressed in the follicle, WNT3, also expressed in the follicle and may serve as a receptor for IP3, and ITPR2, another potential IP3 receptor, both of which are involved in WNT signaling; and then a number of other genes with no known specific tie to hair growth including TARDBP, SUC R1, MB L1, EBF1, AUTS2, IMP5, and SSETBP1. In addition, elevated levels of prostaglandin D2 synthase (PTGDS) has been shown to lead to follicular miniaturization (Garza et al (2012) Sci Transl Med 4(126): 126ra34).

PTGDS catalyzes the production of prostaglandin D2 (PGD2) which then binds to the GPR44 receptor (also known as PD-2), which has been shown to be necessary for the hair growth decrease caused by PGD2 (Nieves and Garza (2014) Exp Dermatol 23(4):224-22. Other genes that may play a role in AGA include AR-co-activators such as Hic-5/ARA55 which acts as a AR transcription potentiator by being a scaffold protein which stabilizes or recruits chromatin structure allowing access for transcription (Inui and Itami (2011) J of Derm Sci 61 : 1-6); TGFpi and 2 which have been identified as an androgen-inducible growth suppressor found in dermal papilla cells and DKK1, also upregulated in alopecia, and also which inhibits growth and induces apoptosis in outer root sheath cell (Inui ibid). Another GWAS study (Prodi et al (2008) J Invest Derm 128:2268) identified a strong correlation with the

ectodysplasin A2 receptor (EDA2R), also found on the X-chromosome in the vicinity of the AR. EDA2R is capable of activating the FkB pathway and may also be involved in c-Jun activation.

[0012] Thus, there remains a need for methods and compositions that can be used to prevent or treat androgenic alopecia through treatments that rely on specific gene targeting.

SUMMARY

[0013] Disclosed herein are compositions and methods for partial or complete inactivation or disruption of a gene involved in androgenic alopecia. In certain embodiments, the compositions are delivered to cells such that the gene is knocked out and the androgenic alopecia is treated.

[0014] Thus, in one aspect, described herein are cells in which the expression of at least one gene involved in androgenic alopecia (an AGA-related gene) is modulated. In some embodiments, cells are described that comprise an engineered nuclease to cause a knockout of a gene such that the alopecia is treated. In other embodiments, cells are described that comprise an engineered transcription factor (TF) such that the expression of a gene related to AGA is modulated. In preferred embodiments, the gene to be targeted is the androgen receptor (AR), the ectodysplasin A2 receptor (EDA2R), histone deactylases HDAC4 and/or histone deacetylase HDAC9, prostaglandin D2 synthase (PTGDS), GPR44 receptor (PTGDR2, or DP2), the TWIST 1 and TWIST2 transcription factors, WntlOA, WNT3, ITPR2, TARDBP, SUC R1, MBNL1, EBF 1, AUTS2, IMP5, and SSETBP1. Further preferred genes include Hic5/ARA55, TGFpl and 2, DKK1, and SRD5A2. In preferred

embodiments, the modification of gene results in increased production of hair. In some embodiments, the cells are dermal or hair follicle cells.

[0015] Described herein is a DNA-binding domain (e.g., zinc-finger protein (ZFP), TALE effector domain, or single guide RNA of a CRISPR/Cas system) that binds to target site in a AGA-related gene in a genome. In certain embodiments, the target site recognized by the DNA-binding domain is in PTGDS gene. In other embodiments, the target site recognized by the DNA-binding domain is in a PTGDR2 gene. In yet other embodiments, the target site recognize by the DNA-binding domain is in an AR gene. The target site may be in an intron or exon of the targeted gene. In certain embodiments, the target site comprises a sequence of 12-25

(including target sites of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) or more nucleotides as shown in the target sites of Tables 1, 2, 3 or 7 or a target sequence in the same gene from a related species, including target sites from other species (e.g., human) that exhibit 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more homology or hybridize under stringent conditions (as known in the art) to the target sites shown in Tables 1, 2, 3 or 7. In certain embodiments, the target site is in a human AGA-related gene, such as PTGDS, PTGDR2 or AR. In certain embodiments, the DNA-binding domain comprises a zinc finger protein with the recognition helix regions as shown in a single row of Tables 1, 2 or 3 (recognition helix regions of F l- F5 or F 1-F6). In other embodiments, the DNA-binding domain comprises a sgRNA as shown in in a single row of Table 7.

[0016] In one embodiment, the DNA-binding domain is in association (e.g., as a fusion protein, or interacts with, in the case of a single guide RNA) with a functional domain to form an artificial transcription factor (e.g., where the functional domain is a transcriptional regulatory domain or is an inactive (e.g. 'dead') nuclease domain) or an artificial nuclease (e.g., where the functional domain is a cleavage domain). The transcriptional regulatory domain may be an activation domain or a repression domain. [0017] In other embodiments, the DNA-binding domain is in association with at least one cleavage domain (or cleavage half-domain) to form an artificial nuclease. Cleavage domains and cleavage half domains can be obtained, for example, from various restriction endonucleases and/or homing endonucleases. In one embodiment, the cleavage half-domains are derived from an endonuclease, for example a Type IIS restriction endonuclease (e.g., Fok I) and/or a Cas endonuclease. In certain embodiments, the DNA-binding domain recognizes a target site in an AGA-related {e.g., PTGDS, PTGDR2 or AR) gene {e.g., a target site comprising 12 or more nucleotides of a target site as shown in Tables 1, 2, 3 or 7, including for example, a zinc finger protein comprising the recognition helix regions as shown in a single row of Tables 1, 2 or 3 or a sgRNA as shown in a single row of Table 7). In further embodiments, the DNA binding domain recognizes a target site in an AGA-related gene where the AGA-related gene is a human gene.

[0018] The DNA-binding domains, artificial TFs and/or artificial nucleases may bind to and/or cleave the target gene within the coding region of the gene or in a non-coding sequence within or adjacent to the gene, such as, for example, a leader sequence, trailer sequence or intron, or within a non-transcribed region, either upstream or downstream of the coding region. In certain embodiments, the DNA- binding domains as described herein bind to sequence within an intron of the target gene. In other embodiments, the DNA-binding domains as described herein bind to sequence within an exon of the target gene {e.g., exon 2, 3 or 4 of PTGDS).

[0019] In yet another aspect, a polynucleotide encoding one or more of the

DNA binding proteins or fusion molecules (or components thereof) described herein is provided. In certain embodiments, the polynucleotide is carried on a viral {e.g., AAV or Ad) vector and/or a non-viral {e.g., plasmid or mRNA vector). Host cells comprising these polynucleotides {e.g., AAV vectors) and/or pharmaceutical compositions comprising the polynucleotides, proteins and/or host cells as described herein are also provided. Host cells include but are not limited to T-cell and stem cells such as skin stem cells.

[0020] In some embodiments, the polynucleotide encoding the DNA binding protein is an mRNA. In some aspects, the mRNA may be chemically modified {See e.g. Kormann et al, (2011) Nature Biotechnology 29(2): 154-157). In other aspects, the mRNA may comprise an ARCA cap (see U.S. Patents 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).

[0021] In yet another aspect, a gene delivery vector comprising any of the polynucleotides described herein is provided. In certain embodiments, the vector is an adenovirus vector (e.g., an Ad5/F35 vector), a lentiviral vector (LV) including integration competent or integration-defective lentiviral vectors, or an adenovirus associated viral vector (AAV). In certain embodiments, the AAV vector is an AAV6 or AAV9 vector. Thus, also provided herein are adenovirus (Ad) vectors, LV or adenovirus associate viral vectors (AAV) comprising a sequence encoding at least one nuclease (ZFN or TALEN) and/or a donor sequence for targeted integration into a target gene. In certain embodiments, the Ad vector is a chimeric Ad vector, for example an Ad5/F35 vector. In certain embodiments, the lentiviral vector is an integrase-defective lentiviral vector (IDLV) or an integration competent lentiviral vector. In certain embodiments, the vector is pseudo-typed with a VSV-G envelope, or with other envelopes.

[0022] Additionally, pharmaceutical compositions comprising the nucleic acids and/or proteins (e.g., ZFPs, Cas or TALEs or fusion proteins comprising the ZFPs, Cas or TALEs) are also provided. For example, certain compositions include a nucleic acid comprising a sequence that encodes one of the ZFPs, Cas or TALEs described herein operably linked to a regulatory sequence, combined with a pharmaceutically acceptable carrier or diluent, wherein the regulatory sequence allows for expression of the nucleic acid in a cell. Protein based compositions include one of more ZFPs. CRISPR/Cas or TALEs as disclosed herein and a pharmaceutically acceptable carrier or diluent.

[0023] In yet another aspect also provided is an isolated cell comprising any of the proteins, polynucleotides and/or compositions as described herein.

[0024] In another aspect, described herein is a method for cleaving one or more AGA-related genes in a cell, the method comprising: (a) introducing, into the cell, one or more polynucleotides encoding one or more artificial nucleases that bind to a target site in the one or more genes under conditions such that the nuclease(s) is(are) expressed and the one or more AGA-related genes are cleaved. In one embodiment, the described method for cleaving one or more AGA-related genes is used to cleave human AGA-related genes. [0025] In another embodiment, described herein is a method for modifying one or more AGA-related gene sequence(s) (e.g, PTGDS, PTGDR2 and/or AR) in the genome of a cell, the method comprising (a) providing a cell comprising one or more AGA-related sequences; and (b) expressing one or more artificial transcription factors and/or artificial nucleases as described herein in the cell such that the gene(s) is(are) modified. In certain embodiments, one, two, three or more genes are modified. In certain embodiments, the modification comprises modifying expression of the gene at the transcriptional level (e.g., activation or repression). In other embodiments, modification comprises cleaving or the gene(s) and alteration of the target gene sequence (e.g., insertions and/or deletions and/or correction of mutations). In certain embodiments, a pair of nucleases is used to achieve cleavage. Optionally, cleavage results in insertion of an exogenous sequence (transgene) into the cell. In other embodiments, non-homologous end joining results in insertions and/or deletions ("indels") in the target gene(s), for example within or between the target site(s) and/or cleavage site(s) of the nucleases. In certain embodiments, a deletion is made by cleaving the target gene(s) in at least two locations and deleting the sequences between the first and second cleavage sites. The size of the deletion in the gene sequence is determined by the distance between the first and second cleavage sites. Accordingly, deletions of any size, in any genomic region of interest, can be obtained. Deletions of 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 nucleotide pairs, or any integral value of nucleotide pairs within this range, can be obtained. In addition, deletions of a sequence of any integral value of nucleotide pairs greater than 1,000 nucleotide pairs can be obtained using the methods and compositions disclosed herein.

[0026] In other aspects, the invention comprises delivery of a donor nucleic acid to a target cell. The donor may be delivered prior to, after, or along with the nucleic acid encoding the nuclease(s). The donor nucleic acid may comprise an exogenous sequence (transgene) to be integrated into the genome of the cell, for example, a sequence encoding a regulator of hair growth into the genome. In some embodiments, the donor may comprise a full-length gene or fragment thereof flanked by regions of homology with the targeted cleavage site. In certain embodiments, each homology arm comprises 50-350 or more base pairs. In some embodiments, the donor lacks homologous regions and is integrated into a target locus through homology independent mechanism (i.e. HEJ). The donor may comprise any nucleic acid sequence, for example a nucleic acid that, when used as a substrate for homology-directed repair of the nuclease-induced double-strand break, leads to a donor-specified deletion to be generated at the endogenous chromosomal locus or, alternatively (or in addition to), novel allelic forms of (e.g., point mutations that ablate a transcription factor binding site) the endogenous locus to be created. In some aspects, the donor nucleic acid is an oligonucleotide wherein integration leads to a gene correction event, or a targeted deletion.

[0027] In another aspect, provided herein are genetically modified cells in which one, two, three or more AGA-related gene(s) is(are) modified using the compositions and methods described herein. The genetically modified cells as described herein include at least one (1, 2, 3 or more) AGA-related gene(s) which is (are) partially or completely inactivated, for example via an insertion and/or deletion mediated by a nuclease. In certain embodiments, the insertion comprises a non- coding sequence or non-functional RNA sequence that is less than 100 base pairs in length (or any number less than 100 base pairs), including insertions of less than 50 base pairs and insertions of less than 25 base pairs (e.g., insertions of less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 base pairs).

[0028] Genetically modified cells as described herein may be used for a variety of purposes, including, but not limited to, in vitro, ex vivo and in vivo purposes such as producing a protein (e.g., using a TF activator to express a targeted gene or via targeted nuclease-mediated integration of a transgene that is expressed in vitro); assaying the impact of repression and/or inactivation of a target gene in vitro or in vivo (e.g., using a TF repressor or engineered nuclease to repress and/or inactivate the target gene); ex vivo production of a protein; ex vivo (e.g., of skin cells, including skin stem cells) for treating and/or preventing androgenic alopecia; ex vivo administration (e.g., of skin cells, including skin stem cells) for augmenting hair loss therapies; in vivo for treating and/or preventing androgenic alopecia; or for providing a genetically modified cell (e.g., stem cell) as described herein to a subject in which expression of the target gene is modulated (e.g., activated, repressed and/or inactivated). The proteins expressed in the cells may be secreted from the cells or the cells may be lysed and the protein isolated. In some embodiments, the genetically modified cells as described herein are administered to a subject using any suitable means (e.g., intravenous, intraperitoneal, mucosal, etc.) and, following administration, the cells stimulate hair growth in a subject and/or augment other hair growth therapies in the subject. In addition, the genetically modified cells as described herein may be generated in vivo via administration by any suitable method including but not limited to injection, topical application, mucosal administration, etc. using of the

compositions described herein such that the genetically modified cells are produced in a subject. The ex vivo and in vivo methods may be used for protein production as well as for the treatment and/or prevention androgenic alopecia in the subject. In addition to use as hair growth modulators, the ex vivo and in vivo methods may also be used for protein production {e.g., of a transgene inserted into a target AGA-related gene as described herein).

[0029] In certain embodiments, the cells described herein comprise a modification {e.g., nucleotide deletion and/or insertion, including a point mutation or insertion of a transgene) to one or more AGA-related genes in which the modification is within or near nuclease(s) binding and/or cleavage site(s), including but not limited to, modifications to sequences within the target site and/or between two paired target sites; modifications within 1-300 (or any number of base pairs therebetween) base pairs upstream, downstream and/or including 1 or more base pairs of the site(s) of cleavage and/or binding site; modifications within 1-100 base pairs (or any number of base pairs therebetween) of including and/or on either side of the binding and/or cleavage site(s); modifications within 1 to 50 base pairs (or any number of base pairs therebetween) including and/or on either side of the binding and/or cleavage site(s); and/or modifications to one or more base pairs within the nuclease binding site and/or cleavage site.

[0030] The modified cells of the invention may be a skin cells, a

stem/progenitor cell {e.g., an induced pluripotent stem cell (iPSC), an embryonic stem cell {e.g., human ES), a mesenchymal stem cell (MSC), a skin stem cell (HSC), or a mesenchymal stem cell). The stem cells may be totipotent or pluripotent {e.g., partially differentiated such as an HSC that is a pluripotent myeloid or lymphoid stem cell or a mesenchymal stem cell that differentiates into an epithelial stem cell and/or bulge stem cell that give rise to hair cells). Any of the modified stem cells described herein (modified at an AGA-related target gene) may then be differentiated to generate a differentiated {in vivo or in vitro) cell descended from a stem cell as described herein. Any of the modified stem cells described herein may be comprise further modifications in other genes of interest). Modified cells as described herein may be modified in vivo or may be isolated and modified in vitro.

[0031] In certain embodiments, the cells described herein comprise a modification {e.g., nucleotide deletion and/or insertion, including a point mutation) to a gene involved in AGA in which the modification is within or near nuclease(s) binding and/or cleavage site(s), including but not limited to, modifications to sequences within the target site and/or between two paired target sites; modifications within 1-300 (or any number of base pairs therebetween) base pairs upstream, downstream and/or including 1 or more base pairs of the site(s) of cleavage and/or binding site; modifications within 1-100 base pairs (or any number of base pairs therebetween) of including and/or on either side of the binding and/or cleavage site(s); modifications within 1 to 50 base pairs (or any number of base pairs therebetween) including and/or on either side of the binding and/or cleavage site(s); and/or modifications to one or more base pairs within the nuclease binding site and/or cleavage site.

[0032] The modified cells of the invention may be a stem/progenitor cell {e.g., an induced pluripotent stem cell (iPSC), an embryonic stem cell {e.g., human ES), a mesenchymal stem cell (MSC), a hematopoietic stem cell (HSC), or a mesenchymal stem cell). The stem cells may be totipotent or pluripotent {e.g., partially

differentiated such as an HSC that is a pluripotent myeloid or lymphoid stem cell or a mesenchymal stem cell that differentiates into a epithelial stem cell and/or bulge stem cell that give rise to hair cells). Any of the modified stem cells described herein (modified at a locus related to AGA) may then be differentiated to generate a differentiated {in vivo or in vitro) cell descended from a stem cell as described herein. Any of the modified stem cells described herein may be comprise further

modifications in other genes of interest). Modified cells as described herein may be modified in vivo or may be isolated and modified in vitro.

[0033] In another aspect, the compositions (modified cells, polynucleotides and/or proteins used to modulate AGA-related genes) and methods described herein can be used, for example, in the production of a protein {e.g., by activating

endogenous gene expression or by inserting a transgene that is expressed into the target gene), for the provision of in vivo or in vitro model systems {e.g., animals or cells with genetically modified cells can be used for drug discovery) and/or for the treatment or prevention or amelioration of a disorder such as aberrant hair growth. The methods typically can comprise any modification (up-regulation, down- regulation, cleaving, etc.) an endogenous gene associated with hair growth in an isolated cell or in the skin of a subject using an engineered transcription factor and/or nuclease (e.g., ZFN or TALEN) or nuclease system such as CRISPR/Cas or

Cfpl/CRISPR with an engineered crRNA/tracr RNA, or using an engineered transcription factor (e.g. ZFN-TF, TALE-TF, Cfpl-TF or Cas9-TF) such that the gene is modulated (up-regulated, down-regulated, inactivated); and (b) introducing the cell into the subject or applying the polynucleotides and/or proteins used to modulate AGA-related genes, thereby treating or preventing the disorder (aberrant hair growth). In certain embodiments, modulation of the target gene(s) results in increased hair growth (as compared to subjects in which the target gene(s) is(are) not modulated). In some embodiments, modulation of the target gene(s) results in decreased hair loss (as compared to subjects in which the target gene(s) is(are) not modulated). In other embodiments, modulation of the target gene(s) results in decreased hair growth (as compared to subjects in which the target gene(s) is(are) not modulated), while is some embodiments, modulation of the target gene(s) results in increased hair loss (as compared to subjects in which the target gene(s) is(are) not modulated). The compositions may be a pharmaceutical composition, for example, a topical composition comprising an engineered transcription factor, nuclease and/or cell as described herein for application to the area of skin in which hair growth is to be altered (e.g., head, face, back, etc.).

[0034] In any of the compositions and methods described herein, the nuclease or transcription factor may comprise one or more zinc finger proteins (ZFP-TFs or ZFNs), one or more TAL-effector domain nucleases (TALE-TFs or TALENs), and/or one or more components of a TtAgo or CRISPR/Cas transcription factor or nuclease system (e.g. the Cas protein and/or the sgRNA). In addition, the compositions and methods described herein may be made or practiced in vivo or ex vivo, including, but not limited to, mammalian cells such as K562 cells, Hepal-6 cells, CD4+ T cells, CD8+ T cells, CD34+ hematopoietic stem cells (HPSCs), and in vivo (e.g., skin cells, bulge stem cells); yeast cells such as S. cerevisiae or S. pichia; insect cells such as SF- 9 cells, and plant cells derived from maize, wheat or canola.

[0035] In another aspect, the invention provides kits that are useful for altering hair growth comprising engineered transcription factors and/or nucleases (e.g., ZFP- TFs, TALE-TFs, ZFNs, TAL-effector domain nuclease fusion proteins, engineered homing endonucleases, Ttago, CRISPR/Cas transcription factor, sgRNA or nuclease systems). The kits typically include one or more nucleases that bind to a target site in a gene associate with hair growth.

[0036] The nuclease(s) and/or transcription factor(s) can be introduced as mRNA, in protein form and/or as a DNA sequence encoding the nuclease(s). In some embodiments, the nuclease(s) or transcription factor(s) are introduced into the skin as mRNAs, while in others, they are introduced as DNA. In some embodiments, delivery is accomplished via a microneedle, a microneedle array (Deng et al (2016) Sci Reports 6:21422), nanoparticle (for example only see McCaffrey et al (2016) J Contr Release 226: 238) or liposome (see Desmet et al (2016) Int J Pharm. 500(1- 2):268-74). In certain embodiments, isolated stem cells comprising the nuclease or transcription factor are introduced into the subject, while in others, the isolated stem cells are treated first ex vivo and then introduced into the subject (see Mistriotis and Andreadis (2013) Tiss Engineer 19(4):265). In preferred embodiments, the stem cells are introduced into the hair follicle (Sugiyama-Nakagiri et al (2006) Gene Therapy 13 :732-737).

[0037] Any of the proteins described herein may further comprise a functional domain, such as a transcriptional regulatory domain (activation domain, repression domain) or a nuclease domain (cleavage domain and/or a cleavage half-domain {e.g., a wild-type or engineered Fokl cleavage half-domain)). Thus, in any of the transcription factors and/or nucleases described herein, the nuclease domain may comprise a wild-type functional domain or an engineered functional domain {e.g., engineered Fokl cleavage half domains that form obligate heterodimers). See, e.g., U.S. Patent Publication No. 2008/0131962.

[0038] In another aspect, the disclosure provides a polynucleotide encoding any of the proteins described herein. Any of the polynucleotides described herein may also comprise sequences (donor or patch sequences) for targeted insertion into a safe harbor gene {e.g. CCR5 or AAVS1). In yet another aspect, a gene delivery vector comprising any of the polynucleotides described herein is provided. In certain embodiments, the vector is an adenoviral vector {e.g., an Ad5/F35 vector) or a lentiviral vector (LV) including integration competent or integration-defective lentiviral vectors or an adeno-associated vector (AAV). Thus, also provided herein are viral vectors comprising a sequence encoding a transcription factor and/or nuclease {e.g. ZFN or TALEN and/or a nuclease system (CRISPR/Cas or Ttago) and/or a donor sequence for targeted integration into a target gene. In some embodiments, the donor sequence and the sequences encoding the nuclease are on different vectors. In other embodiments, the nucleases are supplied as polypeptides. In preferred embodiments, the polynucleotides are mRNAs. In some aspects, the mRNA may be chemically modified {See e.g. Kormann et al, (2011) Nature

Biotechnology 29(2): 154-157). In other aspects, the mRNA may comprise an ARCA cap (see U.S. Patents 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 2012/0195936).

[0039] In yet another aspect, the disclosure provides an isolated cell comprising any of the proteins, polynucleotides and/or vectors described herein. In certain embodiments, the cell is a stem/progenitor cell, for example a hair stem cell (mesenchymal stem cell, bulge stem cells, etc.). In a still further aspect, the disclosure provides a cell or cell line which is descended from a cell or line comprising any of the proteins, polynucleotides and/or vectors described herein, namely a cell or cell line descended {e.g., in culture) from a cell in which a gene involved in hair growth has been modulated by a engineered transcription factor and/or engineered nuclease {e.g., in which a donor polynucleotide has been stably integrated into the genome of the cell). Thus, descendants of cells as described herein may not themselves comprise the proteins, polynucleotides and/or vectors described herein, but, in these cells, at least one gene involved in hair growth is modulated.

[0040] In another aspect, described herein are methods of modulating

(inactivating, down regulating or up regulating) a gene related to androgenic alopecia in a cell by introducing one or more proteins, polynucleotides and/or vectors into the cell as described herein. In any of the methods described herein the engineered transcription factor may up or down-regulate expression of one or more genes associated with hair growth and the nucleases may induce targeted mutagenesis, deletions of cellular DNA sequences, and/or facilitate targeted recombination at a predetermined chromosomal locus. Thus, in certain embodiments, the nucleases delete or insert one or more nucleotides of the target gene. In some embodiments the gene is inactivated by nuclease cleavage followed by non-homologous end joining. In other embodiments, a genomic sequence in the target gene is replaced, for example using a nuclease (or vector encoding said nuclease) as described herein and a "donor" sequence that is inserted into the gene following targeted cleavage with the nuclease. The donor sequence may be present in the nuclease vector, present in a separate vector (e.g., AAV, Ad or LV vector) or, alternatively, may be introduced into the cell using a different nucleic acid delivery mechanism.

[0041] Furthermore, any of the methods described herein can be practiced in vitro, in vivo and/or ex vivo. In certain embodiments, the methods are practiced ex vivo, for example to modify stem cells, to make them useful as therapeutics in an allogenic setting to treat a subject {e.g., a subject with AGA).

DETAILED DESCRIPTION

[0042] Disclosed herein are compositions and methods for preventing or treating androgenic alopecia, including pharmaceutical composition comprising one or more engineered transcription factors and/or nuclease that modulate expression of one or more genes involved in androgenic alopecia. Cells modified by these transcription factors and/or nucleases can be used as therapeutics, for example, transplants, to alter {e.g., restore) hair growth. Additionally, other genes of interest may be inserted into cells in which the gene(s) has been manipulated and/or other genes of interest may be knocked out.

General

[0043] Practice of the methods, as well as preparation and use of the compositions disclosed herein employ, unless otherwise indicated, conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, computational chemistry, cell culture, recombinant DNA and related fields as are within the skill of the art. These techniques are fully explained in the literature. See, for example, 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; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, "Chromatin" (P.M. Wassarman and A. P. Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, "Chromatin Protocols" (P.B. Becker, ed.) Humana Press, Totowa, 1999. Definitions

[0044] The terms "nucleic acid," "polynucleotide," and "oligonucleotide" are used interchangeably and refer to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double-stranded form. For the purposes of the present disclosure, these terms are not to be construed as limiting with respect to the length of a polymer. The terms can encompass known analogues of natural nucleotides, as well as nucleotides that are modified in the base, sugar and/or phosphate moieties (e.g. , phosphorothioate backbones). In general, an analogue of a particular nucleotide has the same base-pairing specificity; i.e. , an analogue of A will base-pair with T.

[0045] 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 analogues or modified derivatives of corresponding naturally-occurring amino acids.

[0046] "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. , contacts with phosphate residues in a DNA backbone), as long as the interaction as a whole is sequence-specific. Such interactions are generally characterized by a dissociation constant (Kd) of 10^"6 M^"1 or lower. "Affinity" refers to the strength of binding:

increased binding affinity being correlated with a lower Kd.

[0047] A "binding protein" is a protein that is able to bind non-covalently to another molecule. A binding protein can bind to, for example, a DNA molecule (a DNA- binding protein), an RNA molecule (an RNA-binding protein) and/or a protein molecule (a protein-binding protein). In the case of a protein-binding protein, it can bind to itself (to form homodimers, homotrimers, etc.) and/or it can bind to one or more molecules of a different protein or proteins. A binding protein can have more than one type of binding activity. For example, zinc finger proteins have DNA-binding, RNA-binding and protein- binding activity.

[0048] A "zinc finger DNA binding protein" (or binding domain) is a protein, or a domain within a 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 whose structure is stabilized through coordination of a zinc ion. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP. [0049] A "TALE DNA binding domain" or "TALE" is a polypeptide comprising one or more TALE repeat domains/units. The repeat domains are 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 with other TALE repeat sequences within a naturally occurring TALE protein. See, e.g., U.S. Patent No. 8,586,526, incorporated by reference herein in its entirety.

[0050] Zinc finger and TALE DNA-binding domains can be "engineered" to bind to a predetermined nucleotide sequence, for example via engineering (altering one or more amino acids) of the recognition helix region of a naturally occurring zinc finger protein or by engineering of the amino acids involved in DNA binding (the repeat variable diresidue or RVD region). Therefore, engineered zinc finger proteins or TALE proteins are proteins that are non-naturally occurring. Non-limiting

examples of methods for engineering zinc finger proteins and TALEs are design and selection. A designed protein is a protein not occurring in nature whose

design/composition results principally from rational criteria. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP or TALE designs and binding data. See, for example, U.S. Patent Nos. 8,586,526; 6, 140,081;

6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059; WO 98/53060;

WO 02/016536 and WO 03/016496.

[0051] A "selected" zinc finger protein or TALE is a protein not found in nature whose production results primarily from an empirical process such as phage display, interaction trap or hybrid selection. See e.g., U.S. 5,789,538; U.S. 5,925,523;

U.S. 6,007,988; U.S. 6,013,453; U.S. 6,200,759; 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.

[0052] "TtAgo" is a prokaryotic Argonaute protein thought to be involved in gene silencing. TtAgo is derived from the bacteria Thermus thermophilus. See, e.g. Swarts et al, ibid, G. Sheng et al, (2013) Proc. Natl. Acad. Sci. U.S.A. I l l, 652). A "TtAgo system" is all the components required including e.g. guide DNAs for

cleavage by a TtAgo enzyme.

[0053] "Recombination" refers to a process of exchange of genetic

information between two polynucleotides. For the purposes of this disclosure,

"homologous recombination (HR)" refers to the specialized form of such exchange that takes place, for example, during repair of double-strand breaks in cells via homology-directed repair mechanisms. This process requires nucleotide sequence homology, uses a "donor" molecule to template repair of a "target" molecule (i.e., the one that experienced the double-strand break), and is variously known as "non- crossover gene conversion" or "short tract gene conversion," because it leads to the transfer of genetic information from the donor to the target. Without wishing to be bound by any particular theory, such transfer can involve mismatch correction of heteroduplex DNA that forms between the broken target and the donor, and/or "synthesis-dependent strand annealing," in which the donor is used to resynthesize genetic information that will become part of the target, and/or related processes. Such specialized HR often results in an alteration of the sequence of the target molecule such that part or all of the sequence of the donor polynucleotide is incorporated into the target polynucleotide.

[0054] In the methods of the disclosure, one or more targeted nucleases as described herein create a double-stranded break in the target sequence (e.g., cellular chromatin) at a predetermined site, and a "donor" polynucleotide, having homology to the nucleotide sequence in the region of the break, can be introduced into the cell. The presence of the double-stranded break has been shown to facilitate integration of the donor sequence. The donor sequence may be physically integrated or, alternatively, the donor polynucleotide is used as a template for repair of the break via homologous recombination, resulting in the introduction of all or part of the nucleotide sequence as in the donor into the cellular chromatin. Thus, a first sequence in cellular chromatin can be altered and, in certain embodiments, can be converted into a sequence present in a donor polynucleotide. Thus, the use of the terms

"replace" or "replacement" can be understood to represent replacement of one nucleotide sequence by another, (i.e., replacement of a sequence in the informational sense), and does not necessarily require physical or chemical replacement of one polynucleotide by another.

[0055] In any of the methods described herein, additional pairs of zinc-finger proteins can be used for additional double-stranded cleavage of additional target sites within the cell.

[0056] In certain embodiments of methods for targeted recombination and/or replacement and/or alteration of a sequence in a region of interest in cellular chromatin, a chromosomal sequence is altered by homologous recombination with an exogenous "donor" nucleotide sequence. Such homologous recombination is stimulated by the presence of a double-stranded break in cellular chromatin, if sequences homologous to the region of the break are present.

[0057] In any of the methods described herein, the first nucleotide sequence (the "donor sequence") can contain sequences that are homologous, but not identical, to genomic sequences in the region of interest, thereby stimulating homologous recombination to insert a non-identical sequence in the region of interest. Thus, in certain embodiments, portions of the donor sequence that are homologous to sequences in the region of interest exhibit between about 80 to 99% (or any integer therebetween) sequence identity to the genomic sequence that is replaced. In other embodiments, the homology between the donor and genomic sequence is higher than 99%, for example if only 1 nucleotide differs as between donor and genomic sequences of over 100 contiguous base pairs. In certain cases, a non-homologous portion of the donor sequence can contain sequences not present in the region of interest, such that new sequences are introduced into the region of interest. In these instances, the non-homologous sequence is generally flanked by sequences of 50- 1,000 base pairs (or any integral value therebetween) or any number of base pairs greater than 1,000, that are homologous or identical to sequences 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 non-homologous recombination mechanisms.

[0058] Any of the methods described herein can be used for partial or complete inactivation of one or more target sequences in a cell by targeted integration of donor sequence that disrupts expression of the gene(s) of interest. Cell lines with partially or completely inactivated genes are also provided.

[0059] Furthermore, the methods of targeted integration as described herein can also be used to integrate one or more exogenous sequences. The exogenous nucleic acid sequence can comprise, for example, one or more genes or cDNA molecules, or any type of coding or noncoding sequence, as well as one or more control elements (e.g., promoters). In addition, the exogenous nucleic acid sequence may produce one or more RNA molecules (e.g., small hairpin RNAs (shRNAs), inhibitory RNAs (RNAis), microRNAs (miRNAs), etc.).

[0060] "Cleavage" refers to the breakage of the covalent backbone of a DNA molecule. Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-stranded cleavage and double-stranded cleavage are possible, and double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events. DNA cleavage can result in the production of either blunt ends or staggered ends. In certain embodiments, fusion polypeptides are used for targeted double-stranded DNA cleavage.

[0061] A "cleavage half-domain" is a polypeptide sequence which, in conjunction with a second polypeptide (either identical or different) forms a complex having cleavage activity (preferably double-strand cleavage activity). The terms "first and second cleavage half-domains;" "+ and - cleavage half-domains" and "right and left cleavage half-domains" are used interchangeably to refer to pairs of cleavage half- domains that dimerize.

[0062] An "engineered cleavage half-domain" is a cleavage half-domain that has been modified so as to form obligate heterodimers with another cleavage half- domain (e.g., another engineered cleavage half-domain). See, also, U.S. Patent Nos. 7,888,121; 7,914,796; 8,034,598; 8,623,618 and U.S. Patent Publication No.

2011/0201055, incorporated herein by reference in their entireties.

[0063] The term "sequence" refers to a nucleotide sequence of any length, which can be DNA or RNA; can be linear, circular or branched and can be either single-stranded or double stranded. The term "donor sequence" refers to a nucleotide sequence that is inserted into a genome. A donor sequence can be of any length, for example between 2 and 10,000 nucleotides in length (or any integer value

therebetween or thereabove), preferably between about 100 and 1,000 nucleotides in length (or any integer therebetween), more preferably between about 200 and 500 nucleotides in length.

[0064] "Chromatin" is the nucleoprotein structure comprising the cellular genome. Cellular chromatin comprises nucleic acid, primarily DNA, and protein, including histones and non-histone chromosomal proteins. The majority of eukaryotic cellular chromatin exists in the form of nucleosomes, wherein a 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 nucleosome cores. A molecule of histone HI is generally associated with the linker DNA. For the purposes of the present disclosure, the term "chromatin" is meant to encompass all types of cellular nucleoprotein, both prokaryotic and eukaryotic. Cellular chromatin includes both chromosomal and episomal chromatin.

[0065] A "chromosome," is a chromatin complex comprising all or a portion of the genome of a cell. The genome of a cell is often characterized by its karyotype, which is the collection of all the chromosomes that comprise the genome of the cell. The genome of a cell can comprise one or more chromosomes.

[0066] An "episome" is a replicating nucleic acid, 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.

[0067] 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 sufficient conditions for binding exist. For example, the sequence 5' GAATTC 3' is a target site for the Eco RI restriction endonuclease.

[0068] 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. "Normal presence in the cell" is determined with respect to the particular developmental stage and environmental conditions of the cell. Thus, for example, a molecule that is present only during embryonic development of muscle is an exogenous molecule with respect to an adult muscle cell. Similarly, a molecule induced by heat shock is an exogenous molecule with respect to a non-heat- shocked cell. An exogenous molecule can comprise, for example, a functioning version of a malfunctioning endogenous molecule or a malfunctioning version of a normally- functioning endogenous molecule.

[0069] An exogenous molecule can be, among other things, a small molecule, such as is generated by a combinatorial chemistry process, or a macromolecule such as a protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein,

polysaccharide, any modified derivative of the above molecules, or any complex comprising one or more of the above molecules. Nucleic acids include DNA and RNA, can be single- or double-stranded; can be linear, branched or circular; and can be of any length. Nucleic acids include those capable of forming duplexes, as well as triplex-forming nucleic acids. See, for example, U.S. Patent 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, acetylases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, gyrases and helicases.

[0070] An exogenous molecule can be the same type of molecule as an endogenous molecule, e.g., an exogenous protein or nucleic acid. For example, an exogenous nucleic acid can comprise an infecting viral genome, a plasmid or episome introduced into a cell, or a chromosome that is not normally present in the cell.

Methods for the introduction of 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. They may also include cargo delivery by mechanical forces resulting in cell squeezing in a microfluidic system. An exogenous molecule can also be the same type of molecule as an endogenous molecule but derived from a different species than the cell is derived from. For example, a human nucleic acid sequence may be introduced into a cell line originally derived from a mouse or hamster.

[0071] By contrast, an "endogenous" molecule is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions. For example, an endogenous nucleic acid can comprise a chromosome, the genome of a mitochondrion, chloroplast or other organelle, or a naturally- occurring episomal nucleic acid. Additional endogenous molecules can include proteins, for example, transcription factors and enzymes.

[0072] A "fusion" molecule is a molecule in which two or more subunit molecules are linked, preferably covalently. The subunit molecules can be the same chemical type of molecule, or can be different chemical types of molecules.

Examples of the first type of fusion molecule include, but are not limited to, fusion proteins (for example, a fusion between a ZFP or TALE DNA-binding domain and one or more activation domains) and fusion nucleic acids (for example, a nucleic acid encoding the fusion protein described supra). Examples of the second type of fusion molecule include, but are not limited to, a fusion between a triplex-forming nucleic acid and a polypeptide, and a fusion between a minor groove binder and a nucleic acid. The term also includes systems in which a polynucleotide component associates with a polypeptide component to form a functional molecule (e.g., a CRISPR/Cas system in which a single guide RNA associates with a functional domain to modulate gene expression).

[0073] Expression of a fusion protein in a cell can result from delivery of the fusion protein to the cell or by delivery of a polynucleotide encoding the fusion protein to a cell, wherein the polynucleotide is transcribed, and the transcript is translated, to generate the fusion protein. Trans-splicing, polypeptide cleavage and polypeptide ligation can also be involved in expression of a protein in a cell. Methods for polynucleotide and polypeptide delivery to cells are presented elsewhere in this disclosure.

[0074] A "gene," for the purposes of the present disclosure, includes a DNA region encoding a gene product (see infra), as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. Accordingly, a gene includes, but is 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, replication origins, matrix attachment sites and locus control regions.

[0075] A "safe harbor" locus is a locus within the genome wherein a gene may be inserted without any deleterious effects on the host cell. Most beneficial is a safe harbor locus in which expression of the inserted gene sequence is not perturbed by any read-through expression from neighboring genes. Non-limiting examples of safe harbor loci that are targeted by nuclease(s) include CCR5, CCR5, HPRT, AAVS1, Rosa and albumin. See, e.g., U.S. Patent Nos. 7,951,925; 8,771,985;

8, 110,379; 7,951,925; U.S. Publication Nos. 2010/0218264; 2011/0265198;

2013/0137104; 2013/0122591; 2013/0177983; 2013/0177960; 2015/0056705 and 2015/0159172.

[0076] "Gene expression" refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct

transcriptional product of a gene {e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of an mRNA. Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation. [0077] "Modulation" of gene expression refers to a change in the activity of a gene. Modulation of expression can 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 as described herein. Thus, gene inactivation may be partial or complete.

[0078] 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, in which it is desirable to bind an exogenous molecule. Binding can be for the purposes of targeted DNA cleavage and/or targeted recombination. A region of interest can be present in a chromosome, an episome, an organellar genome (e.g., mitochondrial, chloroplast), or an infecting viral genome, for example. A region of interest can be within the coding region of a gene, within transcribed non-coding regions such as, for example, leader sequences, trailer sequences or introns, or within non-transcribed regions, either upstream or downstream of the coding region. A region of interest can be as small as a single nucleotide pair or up to 2,000 nucleotide pairs in length, or any integral value of nucleotide pairs. In some embodiments, a region of interest can be up to 3000, 4000, 5000, 7000 or 10000 base pairs in length, or any integral value of nucleotide pairs.

[0079] "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).

[0080] The terms "operative linkage" and "operatively linked" (or "operably linked") are used interchangeably with reference to a juxtaposition of two or more components (such as sequence elements), in which the components are arranged such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components. By way of illustration, a transcriptional regulatory sequence, such as a promoter, is operatively 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. A

transcriptional regulatory sequence is generally operatively linked in cis with a coding sequence, but need not be directly adjacent to it. For example, an enhancer is a transcriptional regulatory sequence that is operatively linked to a coding sequence, even though they are not contiguous. [0081] With respect to fusion polypeptides, the term "operatively linked" can refer to the fact that each of the components performs the same function in linkage to the other component as it would if it were not so linked. For example, with respect to a fusion polypeptide in which a DNA-binding domain (e.g., ZFP, TALE) is fused to an activation domain, the DNA-binding domain and the activation domain are in operative linkage if, in the fusion polypeptide, the DNA-binding domain portion is able to bind its target site and/or its binding site, while the activation domain is able to up-regulate gene expression. When a fusion polypeptide in which a DNA-binding domain is fused to a cleavage domain, the DNA-binding domain and the cleavage domain are in operative linkage if, in the fusion polypeptide, the DNA-binding domain portion is able to bind its target site and/or its binding site, while the cleavage domain is able to cleave DNA in the vicinity of the target site. Similarly, with respect to a fusion polypeptide in which a DNA-binding domain is fused to an activation or repression domain, the DNA-binding domain and the activation or repression domain are in operative linkage if, in the fusion polypeptide, the DNA-binding domain portion is able to bind its target site and/or its binding site, while the activation domain is able to upregulate gene expression or the repression domain is able to downregulate gene expression.

[0082] A "functional fragment" of a protein, polypeptide or nucleic acid is a protein, polypeptide or nucleic acid whose sequence is not identical to the full-length protein, polypeptide or nucleic acid, yet retains the same function as the full-length protein, polypeptide or nucleic acid. A functional fragment can possess more, fewer, or the same number of residues as the corresponding native molecule, and/or can contain one or more amino acid or nucleotide substitutions. Methods for determining the function of a nucleic acid (e.g., coding function, ability to hybridize to another nucleic acid) are well-known in the art. Similarly, methods for determining protein function 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 assayed 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, for example, Fields et al. (\9 9) Nature 340:245-246; U.S. Patent No. 5,585,245 and PCT WO 98/44350. [0083] A "vector" is capable of transferring gene sequences to target cells.

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 which can transfer gene sequences to target cells. Thus, the term includes cloning, and expression vehicles, as well as integrating vectors.

[0084] A "reporter gene" or "reporter sequence" refers to any sequence that produces a protein product that is easily measured, preferably although not necessarily in a routine assay. Suitable reporter genes include, but are not limited to, sequences encoding proteins that mediate antibiotic resistance (e.g., ampicillin resistance, neomycin resistance, G418 resistance, puromycin resistance), sequences encoding colored or fluorescent or luminescent proteins (e.g., green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein, luciferase), and proteins which mediate enhanced cell growth and/or gene amplification (e.g., dihydrofolate reductase). Epitope tags include, for example, one or more copies of FLAG, His, myc, Tap, HA or any detectable amino acid sequence. "Expression tags" include sequences that encode reporters that may be operably linked to a desired gene sequence in order to monitor expression of the gene of interest.

[0085] The terms "treating" and "treatment" as used herein refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms and/or their underlying cause, and improvement or remediation of damage. Cancer and graft versus host disease are non-limiting examples of conditions that may be treated using the compositions and methods described herein. DNA-binding domains

[0086] Described herein are compositions comprising a DNA-binding domain that specifically binds to a target site in any gene associated with AGA. Any DNA- binding domain can be used in the compositions and methods disclosed herein, including but not limited to a zinc finger DNA-binding domain, a TALE DNA binding domain, the DNA-binding portion (sgRNA) of a CRISPR/Cas nuclease, or a DNA-binding domain from a meganuclease.

[0087] In certain embodiments, the DNA binding domain comprises a zinc finger protein. Preferably, the zinc finger protein is non-naturally occurring in that it is engineered to bind to a target site of choice. See, for example, Beerli et al. (2002) Nature Biotechnol. 20: 135-141; Pabo et a/. (2001) Ann. Rev. Biochem. 70:313-340; Isalan et a/. (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. Patent Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,030,215;

6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S. Patent Publication Nos. 2005/0064474; 2007/0218528; 2005/0267061, all incorporated herein by reference in their entireties. In certain embodiments, the DNA-binding domain comprises a zinc finger protein disclosed in U.S. Patent Publication No.

2012/0060230 (e.g., Table 1), incorporated by reference in its entirety herein.

[0088] An engineered zinc finger binding domain can have a novel binding specificity, compared to a naturally-occurring zinc finger protein. Engineering methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, using databases comprising triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence. See, for example, U.S. Patents 6,453,242 and 6,534,261, incorporated by reference herein in their entireties.

[0089] Exemplary selection methods, including phage display and two-hybrid systems, are disclosed in U.S. Patent Nos 5,789,538; 5,925,523; 6,007,988;

6,013,453; 6,410,248; 6, 140,466; 6,200,759; and 6,242,568; as well as

WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB 2,338,237. In addition, enhancement of binding specificity for zinc finger binding domains has been described, for example, in U.S. Patent No. 6,794, 136.

[0090] In addition, as disclosed in these and other references, zinc finger domains and/or multi-fingered zinc finger proteins may be linked together using any suitable linker sequences, including for example, linkers of 5 or more amino acids in length. See, also, U.S. Patent Nos. 6,479,626; 6,903,185; 7,153,949; and 7,972,854 and Application No. 15/380,784 for exemplary linker sequences. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein. In addition, enhancement of binding specificity for zinc finger binding domains has been described, for example, in U.S. Patent No. 6,794,136. [0091] Selection of target sites; ZFPs and methods for design and construction of fusion proteins (and polynucleotides encoding same) are known to those of skill in the art and described in detail in U.S. Patent Nos. 6, 140,081; 5,789,538; 6,453,242; 6,534,261; 5,925,523; 6,007,988; 6,013,453; 6,200,759; 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.

[0092] In addition, as disclosed in these and other references, zinc finger domains and/or multi-fingered zinc finger proteins may be linked together using any suitable linker sequences, including for example, linkers of 5 or more amino acids in length. See, also, U.S. Patent Nos. 6,479,626; 6,903,185; and 7, 153,949 for exemplary linker sequences 6 or more amino acids in length. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein.

[0093] In certain embodiments, the DNA binding domain is an engineered zinc finger protein that binds (in a sequence-specific manner) to a target site in an AR gene or hair growth regulatory gene and modulates expression of a hair growth gene. In some embodiments, the zinc finger protein binds to a target site in PTGDS or the GPR44 receptor (also known as PTGDR2, or DP2S).

[0094] Usually, the ZFPs include at least three fingers. Certain of the ZFPs include four, five or six fingers. The ZFPs that include three fingers typically recognize a target site that includes 9 or 10 nucleotides; ZFPs that include four fingers typically recognize a target site that includes 12 to 14 nucleotides; while ZFPs having six fingers can recognize target sites that include 18 to 21 nucleotides. The ZFPs can also be fusion proteins that include one or more regulatory domains, which domains can be transcriptional activation or repression domains.

[0095] In some embodiments, the DNA-binding domain may be derived from a nuclease. For example, the recognition sequences of homing endonucleases and meganucleases such as I-Scel, I-Ceul, PI-i¾pI, PI-Sce, I-SceIV, I-Csml, I-Panl, I- Seen, I-Ppol, I-SceIII, I-Crel, I-Tevl, I-TevII and I-JevIII are known. See also U.S. Patent No. 5,420,032; U.S. Patent No. 6,833,252; Belfort et a/. (1997) Nucleic Acids Res. 25:3379-3388; Dujon et a/. (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: 163-180; Argast et al. (1998) J. Mol. Biol. 280:345- 353 and the New England Biolabs catalogue. In addition, the DNA-binding specificity of homing endonucleases and meganucleases can be engineered to bind non-natural target sites. See, for example, Chevalier et al. (2002) Molec. Cell 10:895- 905; Epinat et a/. (2003) Nucleic Acids Res. 31:2952-2962; Ashworth et al. (2006) Nature 441:656-659; Paques et al. (2007) Current Gene Therapy 7:49-66; U.S. Patent Publication No. 2007/0117128.

[0096] In other embodiments, the DNA binding domain comprises an engineered domain from a TAL effector similar to those derived from the plant pathogens Xanthomonas (see Boch et al, (2009) Science 326: 1509-1512 and Moscou and Bogdanove, (2009) Science 326: 1501) and Ralstonia (see Heuer et al (2007) Applied and Environmental Microbiology 73(13): 4379-4384); U.S. Patent

Publication Nos. 2011/0301073 and 2011/0145940. The plant pathogenic bacteria of the genus Xanthomonas are known to cause many diseases in important crop plants. Pathogenicity of Xanthomonas depends on a conserved type III secretion (T3S) system which injects more than 25 different effector proteins into the plant cell.

Among these injected proteins are transcription activator-like effectors (TALE) which mimic plant transcriptional activators and manipulate the plant transcriptome (see Kay et al (2007) Science?) 18:648-651). These proteins contain a DNA binding domain and a transcriptional activation domain. One of the most well characterized TALEs is AvrBs3 from Xanthomonas campestgris pv. Vesicatoria (see Bonas et al (1989) Mol Gen Genet 218: 127-136 and WO2010079430). TALEs contain a centralized domain of tandem repeats, each repeat containing approximately 34 amino acids, which are key to the DNA binding specificity of these proteins. In addition, they contain a nuclear localization sequence and an acidic transcriptional activation domain (for a review see Schornack S, et al (2006) J Plant Physiol 163(3): 256-272). In addition, in the phytopathogenic bacteria Ralstonia solanacearum two genes, designated brgl 1 and hpxl7 have been found that are homologous to the AvrBs3 family of

Xanthomonas in the R. solanacearum biovar 1 strain GMI1000 and in the biovar 4 strain RSI 000 (See Heuer et al (2007) Appl and Envir Micro 73(13): 4379-4384). These genes are 98.9% identical in nucleotide sequence to each other but differ by a deletion of 1,575 bp in the repeat domain of hpxl7. However, both gene products have less than 40% sequence identity with AvrBs3 family proteins of Xanthomonas.

[0097] Specificity of these TAL effectors depends on the sequences found in the tandem repeats. The repeated sequence comprises approximately 102 base pairs and the repeats are typically 91-100% homologous with each other (Bonas et al, ibid). Polymorphism of the repeats is usually located at positions 12 and 13 and there appears to be a one-to-one correspondence between the identity of the hypervariable diresidues (the repeat variable diresidue or RVD region) at positions 12 and 13 with the identity of the contiguous nucleotides in the TAL-effector's target sequence (see Moscou and Bogdanove, (2009) Science 326: 1501 and Boch et al (2009) Science 326: 1509-1512). Experimentally, the natural code for DNA recognition of these TAL-effectors has been determined such that an HD sequence at positions 12 and 13 (Repeat Variable Diresidue or RVD) leads to a binding to cytosine (C), NG binds to T, NI to A, C, G or T, NN binds to A or G, and ING binds to T. These DNA binding repeats have been assembled into proteins with new combinations and numbers of repeats, to make artificial transcription factors that are able to interact with new sequences and activate the expression of a non-endogenous reporter gene in plant cells (Boch et al, ibid). Engineered TAL proteins have been linked to a Fokl cleavage half domain to yield a TAL effector domain nuclease fusion (TALEN), including TALENs with atypical RVDs. See, e.g., U.S. Patent No. 8,586,526.

[0098] In some embodiments, the TALEN comprises an endonuclease {e.g.,

Fokl) cleavage domain or cleavage half-domain. In other embodiments, the TALE- nuclease is a mega TAL. These mega TAL nucleases are fusion proteins comprising a TALE DNA binding domain and a meganuclease cleavage domain. The

meganuclease cleavage domain is active as a monomer and does not require dimerization for activity. (See Boissel et al., (2013) Nucl Acid Res: 1-13, doi:

10.1093/nar/gktl224).

[0099] In still further embodiments, the nuclease comprises a compact TALEN. These are single chain fusion proteins linking a TALE DNA binding domain to a Tevl nuclease domain. The fusion protein can act as either a nickase localized by the TALE region, or can create a double strand break, depending upon where the TALE DNA binding domain is located with respect to the Tevl nuclease domain (see Beurdeley et al (2013) Nat Comm: 1-8 DOI: 10.1038/ncomms2782). In addition, the nuclease domain may also exhibit DNA-binding functionality. Any TALENs may be used in combination with additional TALENs {e.g., one or more TALENs (c TALENs or Fokl-TALENs) with one or more mega- TALEs.

[0100] In addition, as disclosed in these and other references, zinc finger domains and/or multi-fingered zinc finger proteins or TALEs may be linked together using any suitable linker sequences, including for example, linkers of 5 or more amino acids in length. See, also, U.S. Patent Nos. 6,479,626; 6,903, 185; and

7, 153,949 for exemplary linker sequences 6 or more amino acids in length. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein. In addition, enhancement of binding specificity for zinc finger binding domains has been described, for example, in U.S. Patent No. 6,794, 136.

[0101] In certain embodiments, the DNA-binding domain is part of a

CRISPR/Cas nuclease system, including a single guide RNA (sgRNA) that binds to DNA. See, e.g., U.S. Patent No. 8,697,359 and U.S. Patent Publication Nos.

2015/0056705 and 2015/0159172. The CRISPR (clustered regularly interspaced short palindromic repeats) locus, which encodes RNA components of the system, and the cas (CRISPR-associated) locus, which encodes proteins (Jansen et al, 2002. Mol. Microbiol. 43 : 1565-1575; Makarova et al, 2002. Nucleic Acids Res. 30: 482-496; Makarova et al, 2006. Biol. Direct 1 : 7; Haft et al., 2005. PLoS Comput. Biol. 1 : e60) make up the gene sequences of the CRISPR/Cas nuclease system. CRISPR loci in microbial hosts contain a combination of CRISPR-associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR- mediated nucleic acid cleavage.

[0102] The Type II CRISPR is one of the most well characterized systems and carries out targeted DNA double-strand break in four sequential steps. First, two non- coding RNA, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the repeat regions of the pre-crRNA and mediates the processing of pre-crRNA into mature crRNAs containing individual spacer sequences. Third, the mature crRNA: tracrRNA complex directs functional domain {e.g., nuclease such as Cas) to the target DNA via Watson-Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA next to the protospacer adjacent motif (PAM), an additional requirement for target recognition. Finally, Cas9 mediates cleavage of target DNA to create a double-stranded break within the protospacer. Activity of the CRISPR/Cas system comprises of three steps: (i) insertion of alien DNA sequences into the CRISPR array to prevent future attacks, in a process called 'adaptation', (ii) expression of the relevant proteins, as well as expression and processing of the array, followed by (iii) RNA-mediated interference with the alien nucleic acid. Thus, in the bacterial cell, several of the so-called 'Cas' proteins are involved with the natural function of the CRISPR/Cas system and serve roles in functions such as insertion of the alien DNA etc.

[0103] In certain embodiments, Cas protein may be a "functional derivative" of a naturally occurring Cas protein. A "functional derivative" of a native sequence polypeptide is a compound having a qualitative biological property in common with a native sequence polypeptide. "Functional derivatives" include, but are not limited to, fragments of a native sequence and derivatives of a native sequence polypeptide and its fragments, provided that they have a biological activity in common with a corresponding native sequence polypeptide. A biological activity contemplated herein is the ability of the functional derivative to hydrolyze a DNA substrate into fragments. The term "derivative" encompasses both amino acid sequence variants of polypeptide, covalent modifications, and fusions thereof such as derivative Cas proteins. Suitable derivatives of a Cas polypeptide or a fragment thereof include but are not limited to mutants, fusions, covalent modifications of Cas protein or a fragment thereof. Cas protein, which includes Cas protein or a fragment thereof, as well as derivatives of Cas protein or a fragment thereof, may be obtainable from a cell or synthesized chemically or by a combination of these two procedures. The cell may be a cell that naturally produces Cas protein, or a cell that naturally produces Cas protein and is genetically engineered to produce the endogenous Cas protein at a higher expression level or to produce a Cas protein from an exogenously introduced nucleic acid, which nucleic acid encodes a Cas that is same or different from the endogenous Cas. In some case, the cell does not naturally produce Cas protein and is genetically engineered to produce a Cas protein. In some embodiments, the Cas protein is a small Cas9 ortholog for delivery via an AAV vector (Ran et al (2015) Nature 510, p. 186).

[0104] In some embodiments, the DNA binding domain is part of a TtAgo system (see Swarts et al, ibid; Sheng et al, ibid). In eukaryotes, gene silencing is mediated by the Argonaute (Ago) family of proteins. In this paradigm, Ago is bound to small (19-31 nt) RNAs. This protein-RNA silencing complex recognizes target RNAs via 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 likely function 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, ibid). Exemplary prokaryotic Ago proteins include those from Aquifex aeolicus,

Rhodobacter sphaeroides, and Thermus thermophilics.

[0105] One of the most well-characterized prokaryotic Ago protein is the one from T. thermophilus (TtAgo; Swarts et al. ibid). TtAgo associates with either 15 nt or 13-25 nt single-stranded DNA fragments with 5' phosphate groups. This "guide DNA" bound by TtAgo serves to direct the protein-DNA complex to bind a Watson- Crick complementary DNA sequence in a third-party molecule of DNA. Once the sequence information in these guide DNAs has allowed identification of the target DNA, the TtAgo-guide DNA complex cleaves the target DNA. Such a mechanism is also supported by the structure of the TtAgo-guide DNA complex while bound to its target DNA (G. Sheng et al, ibid). Ago from Rhodobacter sphaeroides (RsAgo) has similar properties (Olivnikov et al. ibid).

[0106] Exogenous guide DNAs of arbitrary DNA sequence can be loaded onto the TtAgo protein (Swarts et al. ibid.). Since the specificity of TtAgo cleavage is directed by the guide DNA, a TtAgo-DNA complex formed with an exogenous, investigator-specified guide DNA will therefore direct TtAgo target DNA cleavage to a complementary investigator-specified target DNA. In this way, one may create a targeted double-strand break in DNA. Use of the TtAgo-guide DNA system (or orthologous Ago-guide DNA systems from other organisms) allows for targeted cleavage of genomic DNA within cells. Such cleavage can be either single- or double- stranded. For cleavage of mammalian genomic DNA, it would be preferable to use of a version of TtAgo codon optimized for expression in mammalian cells. Further, it might be preferable to treat cells with a TtAgo-DNA complex formed in vitro where the TtAgo protein is fused to a cell-penetrating peptide. Further, it might be preferable to use a version of the TtAgo protein that has been altered via mutagenesis to have improved activity at 37°C. Ago-RNA-mediated DNA cleavage could be used to affect a panopoly of outcomes including gene knock-out, targeted gene addition, gene correction, targeted gene deletion using techniques standard in the art for exploitation of DNA breaks.

[0107] Thus, any DNA-binding domain can be used.

Fusion molecules

[0108] Fusion molecules comprising DNA-binding domains (e.g., ZFPs or

TALEs, CRISPR/Cas components such as single guide RNAs) as described herein and a heterologous regulatory (functional) domain (or functional fragment thereof) are also provided. Common domains include, e.g., transcription factor domains

(activators, repressors, co-activators, co-repressors), silencers, oncogenes (e.g., myc, jun, fos, myb, max, mad, rel, ets, bcl, myb, mos family members etc.); DNA repair enzymes and their associated factors and modifiers; DNA rearrangement enzymes and their associated factors and modifiers; chromatin associated proteins and their modifiers (e.g. kinases, acetylases and deacetylases); and DNA modifying enzymes (e.g., methyltransferases, topoisomerases, helicases, ligases, kinases, phosphatases, polymerases, endonucleases) and their associated factors and modifiers. U.S. Patent Publication Nos. 2005/0064474; 2006/0188987 and 2007/0218528 for details regarding fusions of DNA-binding domains and nuclease cleavage domains, incorporated by reference in their entireties herein.

[0109] Suitable domains for achieving activation include the HSV VP 16 activation domain (see, e.g., Hagmann et al, J. Virol. 71, 5952-5962 (1997)) nuclear hormone receptors (see, e.g., Torchia et al, Curr. Opin. Cell. Biol. 10:373-383

(1998)); the p65 subunit of nuclear factor kappa B (Bitko & Barik, J. Virol. 72:5610- 5618 (1998) and Doyle & Hunt, Neuroreport 8:2937-2942 (1997)); Liu et al, Cancer Gene Ther. 5:3-28 (1998)), or artificial chimeric functional domains such as VP64 (Beerli et al, (1998) Proc. Natl. Acad. Sci. USA 95: 14623-33), and degron (Molinari et al, (1999) EMBO J. 18, 6439-6447). Additional exemplary activation domains include, Oct 1, Oct-2A, Spl, AP-2, and CTF1 (Seipel et al, EMBO J. 11, 4961-4968 (1992) as well as p300, CBP, PCAF, SRC1 PvALF, AtHD2A and ERF -2. See, for example, Robyr et a/. (2000) Mol. Endocrinol. 14:329-347; Collingwood et al. (1999) J. Mol. Endocrinol. 23 :255-275; Leo et al. (2000) Gene 245: 1-11; Manteuffel- Cymborowska (1999) Acta Biochim. Pol. 46:77-89; McKenna et al. (1999) J. Steroid Biochem. Mol. Biol. 69:3-12; Malik et al. (2000) Trends Biochem. Sci. 25:277-283; and Lemon et al. (1999) Curr. Opin. Genet. Dev. 9:499-504. Additional exemplary activation domains include, but are not limited to, OsGAI, HALF-1, CI, API, ARF- 5,-6,-7, and -8, CPRFl, CPRF4, MYC-RP/GP, and TRAB l . See, for example, Ogawa et al. (2000) Gene 245:21-29; Okanami et al. (1996) Genes Cells 1 :87-99; Goff et al. (1991) Genes Dev. 5:298-309; Cho et al. (1999) Plant Mol. Biol. 40:419-429;

Ulmason et a/. (1999) Proc. Natl. Acad. Sci. USA 96:5844-5849; Sprenger-Haussels et al. (2000) Plant J. 22: 1-8; Gong et al. (1999) Plant Mol. Biol. 41 :33-44; and Hobo et al. (1999) Proc. Natl. Acad. Sci. USA 96: 15,348-15,353. [0110] It will be clear to those of skill in the art that, in the formation of a fusion protein (or a nucleic acid encoding same) between a DNA-binding domain and a functional domain, either an activation domain or a molecule that interacts with an activation domain is suitable as a functional domain. Essentially any molecule capable of recruiting an activating complex and/or activating activity (such as, for example, histone acetylation) to the target gene is useful as an activating domain of a fusion protein. Insulator domains, localization domains, and chromatin remodeling proteins such as ISWI-containing domains and/or methyl binding domain proteins suitable for use as functional domains in fusion molecules are described, for example, in U.S. Patent Publication Nos. 2002/0115215 and 2003/0082552 and in WO

02/44376.

[0111] Exemplary repression domains include, but are not limited to, KRAB

A/B, KOX, TGF-beta-inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3, members of the DNMT family (e.g., DNMT1, DNMT3A, DNMT3B), Rb, and MeCP2. See, for example, Bird et al. (1999) Cell 99:451-454; Tyler et al. (1999) Cell 99:443-446; Knoepfler et al. (1999) Cell 99:447-450; and Robertson et al. (2000) Nature Genet. 25:338-342. Additional exemplary repression domains include, but are not limited to, ROM2 and AtHD2A. See, for example, Chem et al. (1996) Plant Cell 8:305-321; and Wu et al. (2000) Plant J. 22: 19-27.

[0112] Fusion molecules are constructed by methods of cloning and biochemical conjugation that are well known to those of skill in the art. Fusion molecules comprise a DNA-binding domain and a functional domain {e.g., a transcriptional activation or repression domain). Fusion molecules also optionally comprise nuclear localization signals (such as, for example, that from the SV40 medium T-antigen) and epitope tags (such as, for example, FLAG and

hemagglutinin). Fusion proteins (and nucleic acids encoding them) are designed such that the translational reading frame is preserved among the components of the fusion.

[0113] Fusions between a polypeptide component of a functional domain (or a functional fragment thereof) on the one hand, and a non-protein DNA-binding domain (e.g., antibiotic, intercalator, minor groove binder, nucleic acid) on the other, are constructed by methods of biochemical conjugation known to those of skill in the art. See, for example, the Pierce Chemical Company (Rockford, IL) Catalogue. Methods and compositions for making fusions between a minor groove binder and a polypeptide have been described. Mapp et al. (2000) Proc. Natl. Acad. Sci. USA 97:3930-3935. Furthermore, single guide RNAs of the CRISPR/Cas system associate with functional domains to form active transcriptional regulators and nucleases.

[0114] In certain embodiments, the target site is present in an accessible region of cellular chromatin. Accessible regions can be determined as described, for example, in U.S. Patent Nos. 7,217,509 and 7,923,542. If the target site is not present in an accessible region of cellular chromatin, one or more accessible regions can be generated as described in U.S. Patent Nos. 7,785,792 and 8,071,370. In additional embodiments, the DNA-binding domain of a fusion molecule is capable of binding to cellular chromatin regardless of whether its target site is in an accessible region or not. For example, such DNA-binding domains are capable of binding to linker DNA and/or nucleosomal DNA. Examples of this type of "pioneer" DNA binding domain are found in certain steroid receptor and in hepatocyte nuclear factor 3 (HNF3) (Cordingley et al. (1987) Cell 48:261-270; Pina et al. (1990) Cell 60: '19-731; and Cirillo et a/. (1998) EMBO J. 17:244-254).

[0115] The fusion molecule may be formulated with a pharmaceutically acceptable carrier, as is known to those of skill in the art. See, for example,

Remington's Pharmaceutical Sciences, 17th ed., 1985; and U.S. Patent Nos. 6,453,242 and 6,534,261.

[0116] The functional component/domain of a fusion molecule can be selected from any of a variety of different components capable of influencing transcription of a gene once the fusion molecule binds to a target sequence via its DNA binding domain. Hence, the functional component can include, but is not limited to, various transcription factor domains, such as activators, repressors, co-activators, co- repressors, and silencers. In some embodiments, the functional domain enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. In some aspects, the functional domain comprises cytidine deaminase activity, and mediates the direct conversion of a cytidine to a uridine, thereby effecting a C to T (or G to A) substitution. The resulting 'base editors' convert cytidines within a window of approximately five nucleotides of the site of DNA binding, and can efficiently cause a variety of point mutations relevant to human disease (see Komor et al, (2016) Nature Apr 20. doi: 10.1038/naturel7946). [0117] Additional exemplary functional domains are disclosed, for example, in U.S. Patent Nos. 6,534,261 and 6,933,113.

[0118] Functional domains that are regulated by exogenous small molecules or ligands may also be selected. For example, RheoSwitch® technology may be employed wherein a functional domain only assumes its active conformation in the presence of the external RheoChem™ ligand (see for example U.S. Publication No. 2009/0136465). Thus, the ZFP may be operably linked to the regulatable functional domain wherein the resultant activity of the ZFP-TF is controlled by the external ligand. Additional regulation can be accomplished through the use of transcriptional switches (e.g. small RNA or other types of controllable molecular switches (Aschrafi et al (2016) J Psychiatry NeurosciA 1 (3): 150154)).

Nucleases

[0119] In certain embodiments, the fusion protein comprises a DNA-binding binding domain and cleavage (nuclease) domain. As such, gene modification can be achieved using a nuclease, for example an engineered nuclease. Engineered nuclease technology is based on the engineering of naturally occurring DNA-binding proteins.

For example, engineering of homing endonucleases with tailored DNA-binding specificities has been described. Chames et al. (2005) Nucleic Acids Res 33(20):el78; Arnould et al. (2006) J. Mol. Biol. 355:443-458. In addition, engineering of ZFPs has also been described. See, e.g., U.S. Patent Nos. 6,534,261; 6,607,882; 6,824,978;

6,979,539; 6,933,113; 7,163,824; and 7,013,219.

[0120] In addition, ZFPs and/or TALEs have been fused to nuclease domains to create ZFNs and TALENs - a functional entity that is able to recognize its intended nucleic acid target through its engineered (ZFP or TALE) DNA binding domain and cause the DNA to be cut near the DNA binding site via the nuclease activity. See, e.g., Kim et al. (1996) Proc Nat'lAcadSci USA 93(3): 1156-1160. More recently, such nucleases have been used for genome modification in a variety of organisms. See, for example, U.S. Patent Publication Nos. 2003/0232410; 2005/0208489;

2005/0026157; 2005/0064474; 2006/0188987; 2006/0063231; and International Publication WO 07/014275.

[0121] Thus, the methods and compositions described herein are broadly applicable and may involve any nuclease of interest. Non-limiting examples of nucleases include meganucleases, TALENs and zinc finger nucleases. The nuclease may comprise heterologous DNA-binding and cleavage domains (e.g., zinc finger nucleases; meganuclease DNA-binding domains with heterologous cleavage domains) or, alternatively, the DNA-binding domain of a naturally-occurring nuclease may be altered to bind to a selected target site (e.g., a meganuclease that has been engineered to bind to site different than the cognate binding site).

[0122] In any of the nucleases described herein, the nuclease can comprise an engineered TALE DNA-binding domain and a nuclease domain (e.g., endonuclease and/or meganuclease domain), also referred to as TALENs. Methods and

compositions for engineering these TALEN proteins for robust, site specific interaction with the target sequence of the user's choosing have been published (see U.S. Patent No. 8,586,526). In some embodiments, the TALEN comprises an endonuclease (e.g., Fokl) cleavage domain or cleavage half-domain. In other embodiments, the TALE-nuclease is a mega TAL. These mega TAL nucleases are fusion proteins comprising a TALE DNA binding domain and a meganuclease cleavage domain. The meganuclease cleavage domain is active as a monomer and does not require dimerization for activity. (See Boissel et al., (2013) Nucl Acid Res: 1-13, doi: 10.1093/nar/gktl224). In addition, the nuclease domain may also exhibit DNA-binding functionality.

[0123] In still further embodiments, the nuclease comprises a compact TALEN (cTALEN). These are single chain fusion proteins linking a TALE DNA binding domain to a Tevl nuclease domain. The fusion protein can act as either a nickase localized by the TALE region, or can create a double strand break, depending upon where the TALE DNA binding domain is located with respect to the Tevl nuclease domain (see Beurdeley et al (2013) Nat Comm: 1-8 DOI:

10.1038/ncomms2782). Any TALENs may be used in combination with additional TALENs (e.g., one or more TALENs (c TALENs or Fokl-TALENs) with one or more mega-TALs) or other DNA cleavage enzymes.

[0124] In certain embodiments, the nuclease comprises a meganuclease

(homing endonuclease) or a portion thereof that exhibits cleavage activity. Naturally- occurring meganucleases recognize 15-40 base-pair cleavage sites and are commonly grouped into four families: the LAGLIDADG family, the GIY-YIG family, the His- Cys box family and the UNH family. Exemplary homing endonucleases include I- Scel, I-Ceul, PI-PspI, ΡΙ-Sce, 1-SceIV, I-Csml, I-Panl, I-Scell, I-Ppol, 1-SceIII, I- Crel, I-TevI, I-TevII and I-TevIII. Their recognition sequences are known. See also U.S. Patent No. 5,420,032; U.S. Patent No. 6,833,252; Belfort et a/. (1997) Nucleic Acids Res. 25:3379-3388; Dujon et a/. (1989) Gene 82: 115-118; Perler et a/. (1994) Nucleic Acids Res. 22, 1125-1127; Jasin (1996) Trends Genet. 12:224-228; Gimble et al. (1996) 7. Mol. Biol. 263 : 163-180; Argast et a/. (1998) 7 Mol. Biol. 280:345- 353 and the New England Biolabs catalogue.

[0125] DNA-binding domains from naturally-occurring meganucleases, primarily from the LAGLIDADG family, have been used to promote site-specific genome modification in plants, yeast, Drosophila, mammalian cells and mice, but this approach has been limited to the modification of either homologous genes that conserve the meganuclease recognition sequence (Monet et al. (1999), Biochem. Biophysics. Res. Common. 255: 88-93) or to pre-engineered genomes into which a recognition sequence has been introduced (Route et al. (1994), Mol. Cell. Biol. 14: 8096-106; Chilton et al. (2003), Plant Physiology . 133 : 956-65; Puchta et al. (1996), Proc. Natl. Acad. Sci. USA 93 : 5055-60; Rong et al. (2002), Genes Dev. 16: 1568-81; Gouble et al. (2006), 7. Gene Med. 8(5):616-622). Accordingly, attempts have been made to engineer meganucleases to exhibit novel binding specificity at medically or biotechnologically relevant sites (Porteus et al. (2005), Nat. Biotechnol. 23 : 967-73; Sussman et al. (2004), 7. Mol. Biol. 342: 31-41; Epinat et al. (2003), Nucleic Acids Res. 31 : 2952-62; 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 :656-659;

Paques et al. (2007) Current Gene Therapy 7:49-66; U.S. Patent Publication Nos. 2007/0117128; 2006/0206949; 2006/0153826; 2006/0078552; and 2004/0002092). In addition, naturally-occurring or engineered DNA-binding domains from

meganucleases can be operably linked with a cleavage domain from a heterologous nuclease {e.g., Fokl) and/or cleavage domains from meganucleases can be operably linked with a heterologous DNA-binding domain {e.g., ZFP or TALE).

[0126] In other embodiments, the nuclease is a zinc finger nuclease (ZFN) or

TALE DNA binding domain-nuclease fusion (TALEN). ZFNs and TALENs comprise a DNA binding domain (zinc finger protein or TALE DNA binding domain) that has been engineered to bind to a target site in a gene of choice and cleavage domain or a cleavage half-domain {e.g., from a restriction and/or meganuclease as described herein).

[0127] As described in detail above, zinc finger binding domains and TALE

DNA binding domains can be engineered to bind to a sequence 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 a/. (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. An engineered zinc finger binding domain or TALE protein can have a novel binding specificity, compared to a naturally-occurring protein.

Engineering methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, using databases comprising triplet (or quadruplet) nucleotide sequences and individual zinc finger or TALE amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers or TALE repeat units which bind the particular triplet or quadruplet sequence. See, for example, U.S. Patents 6,453,242 and 6,534,261, incorporated by reference herein in their entireties.

[0128] Selection of target sites; and methods for design and construction of fusion proteins (and polynucleotides encoding same) are known to those of skill in the art and described in detail in U.S. Patent Nos. 7,888,121 and 8,409,861, incorporated by reference in their entireties herein.

[0129] In addition, as disclosed in these and other references, zinc finger domains, TALEs and/or multi-fingered zinc finger proteins may be linked together using any suitable linker sequences, including for example, linkers of 5 or more amino acids in length. See, e.g., U.S. Patent Nos. 6,479,626; 6,903, 185; and

7, 153,949 for exemplary linker sequences 6 or more amino acids in length. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein. See, also, U.S. Patent No. 8,772,453.

[0130] Thus, nucleases such as ZFNs, TALENs and/or meganucleases can comprise any DNA-binding domain and any nuclease (cleavage) domain (cleavage domain, cleavage half-domain). As noted above, the cleavage domain may be heterologous to the DNA-binding domain, for example a zinc finger or TAL-effector DNA-binding domain and a cleavage domain from a nuclease or a meganuclease DNA-binding domain and cleavage domain from a different nuclease. Heterologous cleavage domains can be obtained from any endonuclease or exonuclease. Exemplary endonucleases from which a cleavage domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, for example, 2002-2003 Catalogue, New England Biolabs, Beverly, MA; and Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes which cleave DNA are known (e.g., SI Nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn et al. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One or more of these enzymes (or functional fragments thereof) can be used as a source of cleavage domains and cleavage half-domains.

[0131] Similarly, a cleavage half-domain can be derived from any nuclease or portion thereof, as set forth above, that requires dimerization for cleavage activity. In general, two fusion proteins are required for cleavage if the fusion proteins comprise cleavage half-domains. Alternatively, a single protein comprising two cleavage half- domains can be used. The two cleavage half-domains can be derived from the same endonuclease (or functional fragments thereof), or each cleavage half-domain can be derived from a different endonuclease (or functional fragments thereof). In addition, the target sites for the two fusion proteins are preferably disposed, 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 to each other that allows the cleavage half-domains to form a functional cleavage domain, e.g., by dimerizing. Thus, in certain embodiments, the near edges of the target sites are separated by 5-8 nucleotides or by 15-18 nucleotides. However, any integral number of nucleotides or nucleotide pairs can intervene between two target sites {e.g., from 2 to 50 nucleotide pairs or more). In general, the site of cleavage lies between the target sites.

[0132] Restriction endonucleases (restriction enzymes) are present in many species and are capable of sequence-specific binding to DNA (at a recognition site), and cleaving DNA at or near the site of binding. Certain restriction enzymes {e.g., Type IIS) cleave DNA at sites removed from the recognition site and have separable binding and cleavage domains. For example, the Type IIS enzyme Fok I catalyzes double-stranded cleavage of DNA, at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other. See, for example, U.S. Patent Nos. 5,356,802; 5,436, 150 and 5,487,994; as well as Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; 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, fusion proteins comprise the 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. [0133] An exemplary Type IIS restriction enzyme, whose cleavage domain is separable from the binding domain, is Fok I. This particular enzyme is active as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10,570-10,575.

Accordingly, for the purposes of the present disclosure, the portion of the Fok I enzyme used in the disclosed fusion proteins is considered a cleavage half-domain. Thus, for targeted double-stranded cleavage and/or targeted replacement of cellular sequences using zinc finger-Fok I 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 containing a zinc finger binding domain and two Fok I cleavage half-domains can also be used. Parameters for targeted cleavage and targeted sequence alteration using zinc finger-Fok I fusions are provided elsewhere in this disclosure.

[0134] A cleavage domain or cleavage half-domain can be any portion of a protein that retains cleavage activity, or that retains the ability to multimerize (e.g., dimerize) to form a functional cleavage domain.

[0135] Exemplary Type IIS restriction enzymes are described in International

Publication WO 07/014275, incorporated herein in its entirety. Additional restriction enzymes also contain separable binding and cleavage domains, and these are contemplated by the present disclosure. See, for example, Roberts et al. (2003) Nucleic Acids Res. 31 :418-420.

[0136] In certain embodiments, the cleavage domain comprises one or more engineered cleavage half-domain (also referred to as dimerization domain mutants) that minimize or prevent homodimerization, as described, for example, in U.S. Patent Nos. 7,914,796; 8,034,598 and 8,623,618; and U.S. Patent Publication No.

2011/0201055, the disclosures of all of which are incorporated by reference in their entireties herein. Amino acid residues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 of Fok I are all targets for influencing dimerization of the Fok I cleavage half-domains.

[0137] Exemplary engineered cleavage half-domains of Fok I that form obligate heterodimers include a pair in which a first cleavage half-domain includes mutations at amino acid residues at positions 490 and 538 of Fok I and a second cleavage half-domain includes mutations at amino acid residues 486 and 499.

[0138] Thus, in one embodiment, a mutation at 490 replaces Glu (E) with Lys

(K); the mutation at 538 replaces Iso (I) with Lys (K); the mutation at 486 replaced Gin (Q) with Glu (E); and the mutation at position 499 replaces Iso (I) with Lys (K). Specifically, the engineered cleavage half-domains described herein were prepared by mutating positions 490 (E→K) and 538 (I→K) in one cleavage half-domain to produce an engineered cleavage half-domain designated "E490K:I538K" and by mutating positions 486 (Q→E) and 499 (I→L) in another cleavage half-domain to produce an engineered cleavage half-domain designated "Q486E:I499L". The engineered cleavage half-domains described herein are obligate heterodimer mutants in which aberrant cleavage is minimized or abolished. See, e.g., U.S. Patent

Publication No. 2008/0131962, the disclosure of which is incorporated by reference in its entirety for all purposes. In certain embodiments, the engineered cleavage half- domain comprises mutations at positions 486, 499 and 496 (numbered relative to wild-type Fokl), for instance mutations that replace the wild type Gin (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 position 496 with an Asp (D) or Glu (E) residue (also referred to as a "ELD" and "ELE" domains, respectively). In other embodiments, the engineered cleavage half-domain comprises mutations at positions 490, 538 and 537 (numbered relative to wild-type Fokl), for instance 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). In other

embodiments, the engineered cleavage half-domain comprises mutations at positions 490 and 537 (numbered relative to wild-type Fokl), for instance 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, e.g., U.S. Patent Nos. 7,914,796; 8,034,598 and 8,623,618, the disclosures of which are incorporated by reference in its entirety for all purposes. In other embodiments, the engineered cleavage half domain comprises the "Sharkey" and/or "Sharkey mutations" (see Guo et al, (2010) J. Mol. Biol. 400(1):96-107).

[0139] Alternatively, nucleases may be assembled in vivo at the nucleic acid target site using so-called "split-enzyme" technology (see e.g. U.S. Patent Publication No. 2009/0068164). Components of such split enzymes may be expressed either on separate expression constructs, or can be linked in one open reading frame where the individual components are separated, for example, by a self-cleaving 2A peptide or IRES sequence. Components may be individual zinc finger binding domains or domains of a meganuclease nucleic acid binding domain.

[0140] Nucleases (e.g., ZFNs and/or TALENs) can be screened for activity prior to use, for example in a yeast-based chromosomal system as described in as described in U.S. Patent No. 8,563,314.

[0141] In certain embodiments, the nuclease comprises a CRISPR/Cas system.

The CRISPR (clustered regularly interspaced short palindromic repeats) locus, which encodes RNA components of the system, and the cas (CRISPR-associated) locus, which encodes proteins (Jansen et al, 2002. Mol Microbiol. 43 : 1565-1575;

Makarova et al, 2002. Nucleic Acids Res. 30: 482-496; Makarova et al, 2006. Biol. Direct 1 : 7; Haft et al, 2005. PLoS Comput. Biol. 1 : e60) make up the gene sequences of the CRISPR/Cas nuclease system. CRISPR loci in microbial hosts contain a combination of CRISPR-associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage.

[0142] The Type II CRISPR is one of the most well characterized systems and carries out targeted DNA double-strand break in four sequential steps. First, two non- coding RNA, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the repeat regions of the pre-crRNA and mediates the processing of pre-crRNA into mature crRNAs containing individual spacer sequences. Third, the mature crRNA: tracrRNA complex directs Cas9 to the target DNA via Watson-Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA next to the protospacer adjacent motif (PAM), an additional requirement for target recognition. Finally, Cas9 mediates cleavage of target DNA to create a double-stranded break within the protospacer. Activity of the CRISPR/Cas system comprises of three steps: (i) insertion of alien DNA sequences into the CRISPR array to prevent future attacks, in a process called 'adaptation', (ii) expression of the relevant proteins, as well as expression and processing of the array, followed by (iii) RNA-mediated interference with the alien nucleic acid. Thus, in the bacterial cell, several of the so-called 'Cas' proteins are involved with the natural function of the CRISPR/Cas system and serve roles in functions such as insertion of the alien DNA etc. [0143] Guide RNAs are often produced in vitro using a promoter system such as the T7, T3 or SP6 phage promoters. Those methods require specific primers synthesized for each guide RNA, and are not suitable for large scale applications. Additionally, efficient transcription initiation starts preferably with "G" nucleotide, which poses a constraint on the first nucleotide of target sequence in the guide RNA if efficiency is a concern. Alternatively, and especially for in vivo use, guide RNAs are designed to be driven by the U3/U6 snoRNA promoters, especially in mammalian cells and in plants. Transcription must be done using the RNA polymerase III system. The RNA polymerase II system in vivo can add extraneous structural RNA features such as a 5' cap, potentially 5' or 3' untranslated sequences (UTRs), or poly A tracts, each of which could potentially interfere with the activity of the guide RNA in the CRISPR/Cas system. Additionally, RNAs made by the RNA polymerase III system are often rapidly exported from the nucleus into the cytoplasm for translation, and thus may be less available for use in gene editing with the Cas nuclease (see e.g.

Sander and Joung, (2014) Nat Biotechnol. 32(4): 347-355).

[0144] In some embodiments, the CRISPR-Cpfl system is used. The

CRISPR-Cpfl system, identified in Francisella spp, is a class 2 CRISPR-Cas system that mediates robust DNA interference in human cells. Although functionally conserved, Cpf 1 and Cas9 differ in many aspects including in their guide RNAs and substrate specificity (see Fagerlund et al, (2015) Genom Bio 16:251). A major difference between Cas9 and Cpfl proteins is that Cpfl does not utilize tracrRNA, and thus requires only a crRNA. The FnCpfl crRNAs are 42-44 nucleotides long (19- nucleotide repeat and 23-25-nucleotide spacer) and contain a single stem-loop, which tolerates sequence changes that retain secondary structure. In addition, the Cpfl crRNAs are significantly shorter than the ~100-nucleotide engineered sgRNAs required by Cas9, and the PAM requirements for FnCpfl are 5'-TTN-3' and 5'-CTA-3' on the displaced strand. Although both Cas9 and Cpfl make double strand breaks in the target DNA, Cas9 uses its RuvC- and HNH-like domains to make blunt-ended cuts within the seed sequence of the guide RNA, whereas Cpfl uses a RuvC-like domain to produce staggered cuts outside of the seed. Because Cpfl makes staggered cuts away from the critical seed region, NHEJ will not disrupt the target site, therefore ensuring that Cpfl can continue to cut the same site until the desired HDR

recombination event has taken place. Thus, in the methods and compositions described herein, it is understood that the term '"Cas" includes both Cas9 and Cfpl proteins. Thus, as used herein, a "CRISPR/Cas system" refers both CRISPR/Cas and/or CRISPR/Cfpl systems, including both nuclease and/or transcription factor systems.

[0145] In certain embodiments, Cas protein may be a "functional derivative" of a naturally occurring Cas protein. A "functional derivative" of a native sequence polypeptide is a compound having a qualitative biological property in common with a native sequence polypeptide. "Functional derivatives" include, but are not limited to, fragments of a native sequence and derivatives of a native sequence polypeptide and its fragments, provided that they have a biological activity in common with a corresponding native sequence polypeptide. A biological activity contemplated herein is the ability of the functional derivative to hydrolyze a DNA substrate into fragments. The term "derivative" encompasses both amino acid sequence variants of polypeptide, covalent modifications, and fusions thereof. Suitable derivatives of a Cas polypeptide or a fragment thereof include but are not limited to mutants, fusions, covalent modifications of Cas protein or a fragment thereof. Cas protein, which includes Cas protein or a fragment thereof, as well as derivatives of Cas protein or a fragment thereof, may be obtainable from a cell or synthesized chemically or by a combination of these two procedures. The cell may be a cell that naturally produces Cas protein, or a cell that naturally produces Cas protein and is genetically engineered to produce the endogenous Cas protein at a higher expression level or to produce a Cas protein from an exogenously introduced nucleic acid, which nucleic acid encodes a Cas that is same or different from the endogenous Cas. In some case, the cell does not naturally produce Cas protein and is genetically engineered to produce a Cas protein.

[0146] In some embodiments, the nuclease is a self-inactivating (see Epstein and Schaffer, (2016) ASGCT poster abstract 119).

[0147] The nuclease(s) may make one or more double-stranded and/or single- stranded cuts in the target site. In certain embodiments, the nuclease comprises a catalytically inactive cleavage domain (e.g., Fokl and/or Cas protein). See, e.g., U.S. Patent No. 9,200,266; 8,703,489 and Guillinger et al. (2014) Nature Biotech.

32(6):577-582. The catalytically inactive cleavage domain may, in combination with a catalytically active domain act as a nickase to make a single-stranded cut.

Therefore, two nickases can be used in combination to make a double-stranded cut in a specific region. Additional nickases are also known in the art, for example, McCaffery et al. (2016) Nucleic Acids Res. 44(2):el l . doi: 10.1093/nar/gkv878. Epub 2015 Oct 19.

Delivery

[0148] The proteins {e.g., nucleases and/or transcription factors),

polynucleotides and/or compositions comprising the proteins and/or polynucleotides described herein may be delivered to a target cell by any suitable means, including, for example, by administration of the protein and/or polynucleotide {e.g., mRNA) components.

[0149] Suitable cells include but are not limited to eukaryotic and prokaryotic cells and/or cell lines. Non-limiting examples of such cells or cell lines generated from such cells include T-cells, COS, CHO (e.g., CHO-S, CHO-Kl, CHO-DG44, CHO-DUXB 11, CHO-DUKX, CHOK1 SV), VERO, MDCK, WI38, V79, B14AF28- G3, BHK, HaK, NS0, SP2/0-Agl4, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), and perC6 cells as well as insect cells such as Spodoptera fugiperda (Sf), or fungal cells such as Saccharomyces, Pichia and Schizosaccharomyces. In certain embodiments, the cell line is a CHO-Kl, MDCK or HEK293 cell line. Suitable cells also include stem cells such as, by way of example, embryonic stem cells, induced pluripotent stem cells (iPS cells), hematopoietic stem cells, neuronal stem cells, mesenchymal stem cells and bulge stem cells.

[0150] Methods of delivering proteins comprising DNA-binding domains as described herein are described, for example, in U.S. Patent Nos. 6,453,242;

6,503,717; 6,534,261; 6,599,692; 6,607,882; 6,689,558; 6,824,978; 6,933, 113;

6,979,539; 7,013,219; and 7,163,824, the disclosures of all of which are incorporated by reference herein in their entireties.

[0151] DNA binding domains and fusion proteins comprising these DNA binding domains as described herein may also be delivered using vectors containing sequences encoding one or more of the DNA-binding protein(s). Additionally, additional nucleic acids {e.g., donors) also may be delivered via these vectors. Any vector systems may be used including, but not limited to, plasmid vectors, retroviral vectors, lentiviral vectors, adenovirus vectors, poxvirus vectors; herpesvirus vectors and adeno-associated virus vectors, etc. See, also, U.S. Patent Nos. 6,534,261;

6,607,882; 6,824,978; 6,933, 113; 6,979,539; 7,013,219; and 7, 163,824, incorporated by reference herein in their entireties. Furthermore, it will be apparent that any of these vectors may comprise one or more DNA-binding protein-encoding sequences and/or additional nucleic acids as appropriate. Thus, when one or more DNA-binding proteins as described herein are introduced into the cell, and additional DNAs as appropriate, they may be carried on the same vector or on different vectors. When multiple vectors are used, each vector may comprise a sequence encoding one or multiple DNA-binding proteins and additional nucleic acids as desired.

[0152] Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding engineered DNA-binding proteins in cells (e.g., mammalian cells) and target tissues and to co-introduce additional nucleotide sequences as desired. Such methods can also be used to administer nucleic acids (e.g., encoding DNA-binding proteins and/or donors) to cells in vitro. In certain embodiments, nucleic acids are administered for in vivo or ex vivo gene therapy uses. Non-viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel & Feigner, TIBTECH 11 :211-217 (1993); Mitani & Caskey, TIBTECH 11 : 162-166 (1993);

Dillon, TIBTECH 11 : 167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10): 1 149-1154 (1988); Vigne, Restorative Neurology and

Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiology and

Immunology Doerfler and Bohm (eds.) (1995); and Yu et al., Gene Therapy 1 : 13-26 (1994).

[0153] Methods of non-viral delivery of nucleic acids include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, mRNA, artificial virions, and agent-enhanced uptake of DNA. Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) can also be used for delivery of nucleic acids. In a preferred embodiment, one or more nucleic acids are delivered as mRNA. Also preferred is the use of capped mRNAs to increase translational efficiency and/or mRNA stability.

Especially preferred are ARCA (anti-reverse cap analog) caps or variants thereof. See U.S. Patent Nos. 7,074,596 and 8,153,773, incorporated by reference herein. [0154] Additional exemplary nucleic acid delivery systems include those provided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Maryland), BTX Molecular Delivery Systems (Holliston, MA) and Copernicus Therapeutics Inc, (see for example U.S. Patent No. 6,008,336). Lipofection is described in e.g., U.S. Patent Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™, Lipofectin™, and Lipofectamine™ RNAiMAX). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424, WO 91/16024. Delivery can be to cells (ex vivo administration) or target tissues (in vivo administration).

[0155] The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill 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. Patent 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).

[0156] Additional methods of delivery include the use of packaging the nucleic acids to be delivered into EnGeneIC delivery vehicles (EDVs). These EDVs are specifically delivered to target tissues using bispecific antibodies where one arm of the antibody has specificity for the target tissue and the other has specificity for the EDV. The antibody brings the EDVs to the target cell surface and then the EDV is brought into the cell by endocytosis. Once in the cell, the contents are released (see MacDiarmid et al (2009) Nature Biotechnology 27(7) p. 643).

[0157] The use of RNA or DNA viral based systems for the delivery of nucleic acids encoding engineered DNA-binding proteins, and/or donors (e.g. CARs or ACTRs) as desired takes advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro and the modified cells are administered to patients (ex vivo).

Conventional viral based systems for the delivery of nucleic acids include, but are not limited to, retroviral, lentivirus, adenoviral, adeno-associated, vaccinia and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.

[0158] The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system depends on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon 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., J. Virol. 66:2731-2739 (1992); Johann e/ a/., J. Virol. 66: 1635-1640 (1992); Sommerfelt et al, Virol. 176:58-59 (1990); Wilson et al, J. Virol. 63 :2374-2378 (1989); Miller et al, J. Virol. 65:2220- 2224 (1991); PCT/US94/05700).

[0159] In applications in which transient expression is preferred, adenoviral based systems can be used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and high levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus ("AAV") vectors are also used to transduce 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 procedures (see, e.g., West et al, Virology 160:38-47 (1987); U.S. Patent No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994);

Muzyczka, J. Clin. nvest. 94: 1351 (1994). Construction of recombinant AAV vectors are described in a number of publications, including U.S. Patent No.

5, 173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al, Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS USA 81 :6466- 6470 (1984); and Samulski et al, J. Virol. 63 :03822-3828 (1989). [0160] At least six viral vector approaches are currently available for gene transfer in clinical trials, which utilize approaches that involve complementation of defective vectors by genes inserted into helper cell lines to generate the transducing agent.

[0161] pLASN and MFG-S are examples of retroviral vectors that have been used in clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn et al., Nat. Med. 1 : 1017-102 (1995); Malech et a/., PNAS USA 94:22 12133-12138 (1997)). PA317/pLASN was the first therapeutic vector used in a gene therapy trial. (Blaese et al., Science 270:475-480 (1995)). Transduction efficiencies of 50% or greater have been observed for MFG-S packaged vectors. (Ellem et al., Immunol Immunother. 44(1): 10-20 (1997); Dranoff et al, Hum. Gene Ther. 1 : 111-2 (1997).

[0162] Recombinant adeno-associated virus vectors (rAAV) are a promising alternative gene delivery systems based on the defective and nonpathogenic parvovirus adeno-associated type 2 virus. All vectors are derived from a plasmid that retains only the AAV 145 bp inverted terminal repeats flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery due to integration into the genomes of the transduced cell are key features for this vector system. (Wagner et al., Lancet 351 :9117 1702-3 (1998), Kearns et al., Gene Ther. 9:748-55 (1996)). Other AAV serotypes, including AAV1, AAV3, AAV4, AAV5, AAV6, AAV8, AAV8.2, AAV9 and AAVrhlO and pseudotyped AAV such as AAV2/8, AAV2/5 and AAV2/6 can also be used in accordance with the present invention.

[0163] Replication-deficient recombinant adenoviral vectors (Ad) can be produced at high titer and readily infect a number of different cell types. Most adenovirus vectors are engineered such that a transgene replaces the Ad El a, Elb, and/or E3 genes; subsequently the replication defective vector is propagated in human 293 cells that supply deleted gene function in trans. Ad vectors can transduce multiple types of tissues in vivo, including nondividing, differentiated cells such as those found in liver, kidney and muscle. Conventional Ad vectors have a large carrying capacity. An example of the use of an Ad vector in a clinical trial involved polynucleotide therapy for antitumor immunization with intramuscular injection (Sterman et al., Hum. Gene Ther. 7: 1083-9 (1998)). Additional examples of the use of adenovirus vectors for gene transfer in clinical trials include Rosenecker et al., Infection 24: 1 5-10 (1996); Sterman et al., Hum. Gene Ther. 9:7 1083-1089 (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).

[0164] Packaging cells are used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by a producer cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host (if applicable), other viral sequences being replaced by an expression cassette encoding the protein to be expressed. The missing viral functions are supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess inverted terminal repeat (ITR) sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line is also infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV.

[0165] In many gene therapy applications, it is desirable that the gene therapy vector be delivered with a high degree of specificity to a particular tissue type.

Accordingly, a viral vector can be modified to have specificity for a given cell type by expressing a ligand as a fusion protein with a viral coat protein on the outer surface of the virus. The ligand is chosen 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 murine leukemia virus can be modified to express human heregulin fused to gp70, and the recombinant virus infects certain human breast cancer cells expressing human epidermal growth factor receptor. This principle can be extended to other virus-target cell pairs, in which the target cell expresses a receptor and the virus expresses a fusion protein comprising a ligand for the cell-surface receptor. For example, filamentous phage can be engineered to display antibody fragments (e.g., FAB or Fv) having specific binding affinity for virtually any chosen cellular receptor. Although the above description applies primarily to viral vectors, the same principles can be applied to nonviral vectors. Such vectors can be engineered to contain specific uptake sequences which favor uptake by specific target cells.

[0166] Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration {e.g., intravenous,

intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application, as described below. In certain embodiments, the proteins and/or polynucleotides described herein are formulated in a pharmaceutical composition for topical delivery to the skin. Any regime may be used for in vivo administration {e.g., topical), including but not limited to a one-time administration, daily, twice daily, every other day, weekly, etc.

[0167] Alternatively, vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient {e.g., lymphocytes, bone marrow aspirates, tissue biopsy, skin grafts) or universal donor hematopoietic stem cells, followed by re- implantation of the cells into a patient, usually after selection for cells which have incorporated the vector. Any regime can used for ex vivo administration {e.g., skin graft), for example a one-time graft or any multiple administration of such grafts.

[0168] Ex vivo cell transfection for diagnostics, research, transplant or for gene therapy {e.g., via re-infusion of the transfected cells into the host organism) is well known to those of skill in the art. In a preferred embodiment, cells are isolated from the subject organism, transfected with a DNA-binding proteins nucleic acid (gene or cDNA), and re-infused back into the subject organism {e.g., patient).

Various cell types suitable for ex vivo transfection are well known to those of skill in the art (see, e.g., Freshney et al, Culture of Animal Cells, A Manual of Basic Technique (3rd ed. 1994)) and the references cited therein for a discussion of how to isolate and culture cells from patients).

[0169] In one embodiment, stem cells are used in ex vivo procedures for cell transfection and gene therapy. The advantage to using stem cells is that they can be differentiated into other cell types in vitro, or can be introduced into a mammal (such as the donor of the cells) where they will engraft, for example in the bone marrow or in the skin. Methods for differentiating CD34+ cells in vitro into clinically important immune cell types using cytokines such a GM-CSF, IFN-γ and T F-a are known (see Inaba et al, J. Exp. Med. 176: 1693-1702 (1992)). [0170] Stem cells are isolated for transduction and differentiation using known methods. For example, stem cells are isolated from bone marrow cells by panning the bone marrow cells with antibodies which bind unwanted cells, such as CD4+ and CD8+ (T cells), CD45+ (panB cells), GR-1 (granulocytes), and lad (differentiated antigen presenting cells) (see Inaba et al., J. Exp. Med. 176: 1693-1702 (1992)).

[0171] Stem cells that have been modified may also be used in some embodiments. For example, skin stem cells that have been made resistant to apoptosis may be used as therapeutic compositions where the stem cells also contain modifications that induce resistance to apoptosis, for example, by knocking out BAX and/or BAK using BAX- or BAK-specific ZFNs (see, U.S. Patent No. 8,597,912) in the stem cells, or those that are disrupted in a caspase, again using caspase-6 specific ZFNs for example. Methods to isolate hair follicle mesenchymal stem cells are known in the art (see e.g. EP 1509597).

[0172] Vectors {e.g., retroviruses, adenoviruses, liposomes, etc.) containing therapeutic DNA-binding proteins (or nucleic acids encoding these proteins) can also be administered directly to an organism for transduction of cells in vivo.

Alternatively, naked DNA can be administered. Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells including, but not limited to, injection, infusion, topical application and electroporation. Heat may be used to increase delivery in conjunction with various administration methods. In preferred embodiments, topical administration directly to the site of treatment {e.g., scalp) is performed. Suitable methods of administering {e.g., by topical application) such nucleic acids, proteins and cells as described herein are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.

[0173] Methods for introduction of DNA into hematopoietic stem cells are disclosed, for example, in U.S. Patent No. 5,928,638. Vectors useful for introduction of transgenes into hematopoietic stem cells, e.g., CD34+ cells, include adenovirus Type 35.

[0174] Vectors suitable for introduction of transgenes into immune cells {e.g.,

T-cells) include non-integrating lentivirus vectors. See, for example, 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. 72:9873-9880; Follenzi et al. (2000) Nature

Genetics 25:21 '-222.

[0175] In some embodiments, the therapeutic DNA-binding proteins can be delivered as polypeptides. In some instances, the therapeutic DNA-binding proteins can be delivered as polypeptides complexed to anionic nucleic acids. In some aspects, the proteins with or without bound nucleic acids are delivered using cationic lipid transfection reagents (Zuris et al (2015) Nat Biotechnol 33 :73-80).

[0176] Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. In certain embodiments, pharmaceutically acceptable carriers for topical administration are used. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions available, as described below (see, e.g., Remington's Pharmaceutical Sciences, 17th ed., 1989).

[0177] As noted above, the disclosed methods and compositions can be used in any type of cell including, but not limited to, prokaryotic cells, fungal cells,

Archaeal cells, plant cells, insect cells, animal cells, vertebrate cells, mammalian cells and human cells, including T-cells and stem cells of any type. Suitable cell lines for protein expression are known to those of skill in the art and include, but are not limited to COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NS0, SP2/0-Agl4, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), perC6, insect cells such as Spodoptera fugiperda (Sf), and fungal cells such as Saccharomyces, Pichia and

Schizosaccharomyces. Progeny, variants and derivatives of these cell lines can also be used.

[0178] Colloidal nanostructured lipid carriers (NLCs) represent a relatively new type of colloidal drug delivery system that consists of solid lipid and liquid lipid, and offers the advantage of improved drug loading capacity and release properties compared with solid lipid nanoparticles. Currently, there is an increasing interest in follicular delivery of drugs using nanocolloidal lipid-based delivery systems for treatment of various disorders (acne, alopecia, and other sebaceous gland dysfunction) associated with the pilosebaceous structure. Follicular targeting of drugs offers the advantages of reducing the drug dose along with decreasing potential systemic toxicity associated with oral drug administration. For example, NLCs have been used to perform follicle targeted delivery of spironolactone in mice in a model of androgenic alopecia (Shamma and Aburhama (2014) Int. J Nanomed 9: 5449-5460). In addition, nucleic acids have been delivered by topical application and by intradermal injection resulted in genotypic and phenotypic correction of an albino mutation in mice (Alexeev et al (2000) Nat Biotechnol 19:43). Another method of follicle delivery is the use of microneedles for delivery into the follicle and also the use of nanoincapsulation of therapeutic compounds followed by the delivery of the nanoincapsulated compounds via microneedles (Gomaa et al (2014) Eur. J Biopharm 86(2): 145-155). Injector devices such as RCI-02 device (RepliCel™) for interdermal injections may also be used for delivery (see e.g. EP2623146). Yet another delivery modality involves the preparation of a fusion protein comprising the targeting nuclease and a collagen binding domain (CBD). The CBD is obtained from a collagenase (e.g. Clostridium histolyticum collagenase) and binds to type I collagen, thereby delivering the directly to the hair follicle. See U.S. Patent No. 8,450,273. Applications

[0179] The disclosed compositions and methods can be used for any application in which it is desired to modulate gene expression and/or functionality, including but not limited to, therapeutic and research applications in which gene modulation is desirable for the prevention or treatment of androgenic alopecia. For example, the disclosed compositions can be used in vivo and/or ex vivo (cell therapies) to disrupt or repress the expression of endogenous AR or signaling through it in cells modified for adoptive cell therapy thereby treating and/or preventing the alopecia.

[0180] Methods and compositions also include stem cell compositions wherein the gene within the stem cells has been modulated (modified) and the cells further comprise an additional transgene. These altered stem cells are introduced into the patient through methods known in the art (e.g., through introduction into the follicle) to allow the engraftment of the cells in the patient. The introduced cells may also have other alterations to help during subsequent therapy.

[0181] The methods and compositions of the invention are also useful for the design and implementation of in vitro and in vivo models, for example, animal models of androgenic alopecia and associated disorders, which allows for the study of these disorders. EXAMPLES

Example 1: Design of specific nucleases

[0182] AGA related transcription factors and nucleases are constructed to enable modulation of one or more of the following genes: androgen receptor (AR), the ectodysplasin A2 receptor (EDA2R), histone deactylases HDAC4 and/or histone deacetylase HDAC9, prostaglandin D2 synthase (PTGDS), GPR44 receptor

(PTGDR2, or DP2), the TWIST 1 and TWIST2 transcription factors, WntlOA, WNT3, ITPR2, TARDBP, SUCNR1, MBNL1, EBF1, AUTS2, IMP5, and SSETBP1. Further preferred genes include Hic5/ARA55, TGFpl and 2, DKK1, and SRD5A2.

[0100] In particular, ZFNs and ZFP-TFs were designed essentially as described in Urnov et al. (2005) Nature 435(7042):646-651, Lombardo et al (2007) Nat Biotechnol. Nov;25(l l): 1298-306, and U.S. Patent Publication Nos.

2008/0131962, 2015/016495, 2014/0120622, 2014/0301990 and U.S. Patent No. 8,956,828. The recognition helices for exemplary ZFN pairs as well as the target sequence are shown below in Tables 1, 2 and 3. Target sites are shown in the first column. Nucleotides in the target site that are targeted by the ZFP recognition helices are indicated in uppercase letters; non-targeted nucleotides indicated in lowercase. Linkers used to join the Fokl nuclease domain and the ZFP DNA binding domain are also shown in the first column (see U.S. Patent Publication No. 2015/0132269). For example, the amino acid sequence of the domain linker L0 is DNA binding domain- QLVKS-Fokl nuclease domain (SEQ ID NO: 1). Similarly, the amino acid sequences for the domain linker N7a is Fokl nuclease domain-SGTPHEVGVYTL-DNA binding domain (SEQ ID NO:2) and the sequence for N6a is SGAQGSTLDF (SEQ ID NO:3). ZFNs were made in either ZFP-Fok or Fok-ZFP orientation (U.S. Patent No. 7,972,854 and Application No. 15/380,784) and pairs with obligate heterodimers

(e.g., ELD and KKR) were used. See, e.g., U.S. Patent Nos. 7,914,796; 8,034,598 and 8,623,618.

Table 1: PTGDS Zinc-finger Designs

ctGCTCTTCG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID CATGcTGTGG NO : 15 ) NO : 16 ) NO: 17) NO : 18 ) NO : 19 ) NO : 20 ) Atgggttt

(SEQ ID

NO: 4)

N6a

63470 DRSDLSR RSHHLKA RSDNLSE ASKTRTN RSDNLAR QKVNLMS ccCAAGAGAC (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID CCAGgAGGAC NO : 21 ) NO: 22) NO: 23) NO: 24) NO: 25) NO : 26 ) Caaaccca

(SEQ ID

NO: 5)

N6a

63752* RSDVLSE SPSSRRT RSDHLSR RSDDLTR ERGTLAR QSGDLTR CtGCAGCCtG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID CGGGGgCTCC NO : 27 ) NO : 28 ) NO : 29 ) NO: 30) NO: 31) NO: 32) TGgacact

(SEQ ID

NO: 6)

N7a

63758 TSGHLSR QSGSLTR QSSDLSR HRSTRNR RSDHLSE RNDTRKT tcACGGGGGC (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID TGCTGTAGGT NO: 33) NO: 34) NO: 35) NO : 36 ) NO: 37) NO: 38) gtagtgtc

(SEQ ID

NO: 7)

N7a

63504 RSDHLTT DGYYLPT DRSALAR RSDVLSE SPSSRRT NA tcCTCCTGgG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID

TCTCTTGGga NO : 39 ) NO : 40 ) NO : 41 ) NO: 27) NO : 28 )

ttcccaca

(SEQ ID

NO: 8)

N7a

63508 RSDVLSE TSGHLSR RSDHLSQ DSSHRTR RSDVLSE NA ccCTGGGCTG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID

GGGTCTGtgg NO : 27 ) NO: 33) NO: 42) NO: 43) NO: 27)

gaatccca

(SEQ ID

NO: 9)

N7a

63750* RSDVLSE SPSSRRT RSDHLSR RSDDLTR ERGTLAR QSGDLTR CtGCAGCCtG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID CGGGGgCTCC NO : 27 ) NO : 28 ) NO : 29 ) NO: 30 NO: 31) NO: 32) TGgacact (SEQ ID

NO: 6)

N6a

63756 RSHSLLR RSDYLAK QSSDLSR HRSTRNR RSDHLSE RNDTRKT tcACGGGGGC (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID TGCTgTAGGT NO : 44 ) NO: 45) NO: 35) NO : 36 ) NO: 37) NO: 38) Gtagtgtc

(SEQ ID

NO: 7)

N6a

63466* QSGHLAR WRSSLMA RSDVLST CRRNLRN YKHVLSD TSGSLTR ctGCTCTTCG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID CATGcTGTGG NO : 15 ) NO : 16 ) NO: 17) NO : 18 ) NO : 19 ) NO : 20 ) Atgggttt

(SEQ ID

NO: 4)

N7a

63471* DRSDLSR RSDNLTR RSDNLSE ASKTRTN RSDNLAR QKVNLMS ccCAAGAGAC (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID CCAGGAGGAC NO : 21 ) NO : 46 ) NO: 23) NO: 24) NO: 25) NO : 26 ) caaaccca

(SEQ ID

NO: 5)

N7a

63718 RSDVLSE QKCCLRS ARSTRIT QSGSLTR RSDSLSV DRSALAR agATCATGGT (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID ACTGcAGCCT NO : 27 ) NO: 47) NO : 48 ) NO: 34) NO : 49 ) NO : 41 ) Gcgggggc

(SEQ ID

NO: 10)

N7a

63731* RSDHLSN DSRSRIN RPYTLRL QSGSLTR TSGHLSR QSGSLTR ctGTAGGTGT (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID AGTGTCCAGG NO: 50) NO: 51) NO: 52) NO: 34) NO: 33) NO: 34) agcccccg

(SEQ ID

NO: 11)

N7a

63467 TSGNLTR RSDALAR QSSDLSR LRHNLRA NRHDRAK NA ctTCGCATGC (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID

TGTGGATggg NO: 53) NO: 54) NO: 35) NO: 55) NO: 56)

tttggtcc

N6a

63472* DRSDLSR RSDNLTR RSDNLSE ASKTRTN RSDNLAR QKVNLMS ccCAAGAGAC (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID CCAGGAGGAC NO : 21 ) NO : 46 ) NO: 23) NO: 24) NO: 25) NO : 26 ) caaaccca

(SEQ ID

NO: 5)

N6a

63732* RSDHLSN DSRSRIN RPYTLRL QSGSLTR TSGHLSR QSGSLTR ctGTAGGTGT (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID AGTGTCCAGG NO: 50) NO: 51) NO: 52) NO: 34) NO: 33) NO: 34) agcccccg

(SEQ ID

NO: 11)

N7a

63720 TSGNLTR TLQNRMS QSGSLTR QSGDLTR DRSHLTR QSGDLTR ccGCAGGCtG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID CAGTAcCATG NO: 53) NO: 57) NO: 34) NO: 32) NO: 58) NO: 32) ATcttggt

(SEQ ID

NO: 12)

L0

63571 DRSNLSR VAEYRYK QSGNLAR AKWNLDA DRSDLSR RRTDLRR caGCGCCCCA (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID GGAAcTGCGA NO: 59) NO: 60) NO: 61) NO: 62) NO : 21 ) NO: 63) Ccagtgga

(SEQ ID

NO: 13)

L0

63575 QSSDLSR FRYYLKR ERGTLAR RSDNLTR DRSHLTR QSGHLSR ttGGAGGCGA (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID GGCCCGCGCT NO: 35) NO: 64) NO: 31) NO : 46 ) NO: 58) NO: 65) gtaccagc

(SEQ ID

NO: 14)

N6a

*ZFNs comprising 63465 or 63466; 63752 or 63750; 63471 or 63472; and 63731 or 63732 differ in linker used between ZFP and Fokl domain

Table 2: PTGDR Designs

ZFN Name Fl F2 F3 F4 F5 F6 target

sequence

linker

62601 QSSDLSR RSDNLTR RSDDLTR QSGSLTR TSGNLTR DRSALAR tgGTCGATGT (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID AGCGGAGGCT NO: 35) NO : 46 ) NO: 30) NO: 34) NO: 53) NO : 41 ) agagttgc

(SEQ ID

NO: 66)

N6a

62600 RSDVLSE KHSTRRV RSDHLST HSNTRKN RSDSLLR QSSDLTR ttGCTGTGGT (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID TTGGAAGCTG NO : 27 ) NO : 81 ) NO: 82) NO: 83) NO: 84) NO: 85) gaccatct

(SEQ ID

NO: 67)

L0

62884 QSGSLTR QSGSLTR MGHHLTQ QNATRTK QSGDLTR QRTHLKA ccAGAGCAGC (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID AAGTtGTAGT NO: 34) NO: 34) NO : 86 ) NO: 87) NO: 32) NO : 88 ) Agcacatg

(SEQ ID

NO: 68)

N7a

62883 QSGSLTR ERGTLAR RSDALTQ RPYTLRL DSSHRTR NA gcGGCTTGAT (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID

GGCCGCAtca NO: 34) NO: 31) NO : 89 ) NO: 52) NO: 43)

tgtgctac

(SEQ ID

NO: 69)

N7a

62720 RSDSLSV QSGHLSR QRNHRTT DRSNLTR QSANRTK QSGNLAR agGAAGAAGA (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID CCGAGGAATG NO : 49 ) NO: 65) NO: 90) NO: 91) NO: 92) NO: 61) tagcttgc

(SEQ ID

NO: 70)

N6a

62717 RSDNLSE ASKTRKN LQQNLSD TSGHLSR QSGNLAR QSGDLTR ttGCAGAAGG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID TAGTGCCCAG NO : 23 ) NO: 93) NO: 94) NO: 33) NO: 61) NO: 32) ctcccacg

(SEQ ID

NO: 71)

L0

62677* LRHHLTR QSGALAR QSGNLAR RSDNLSE SKQYLIK NA acTGCCAGGA (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID

AGTAGGTgaa NO: 95) NO: 96) NO: 61) NO: 23) NO: 97)

gaaaggca (SEQ ID

NO: 72)

N6a

62675 DRSHLSR DRSHLAR DRSHLTR QSGHLSR RSDNLST RSTHRTQ aaAGGCAGGG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID AGGCGGCGGC NO: 98) NO: 99) NO: 58) NO: 65) NO: 100) NO: 101) taacaagt

(SEQ ID

NO: 73)

LO

62676 LRHHLTR QSGALAR QSGNLAR RSDNLSE SKQYLIK NA acTGCCAGGA (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID

AGTAGGTgaa NO: 95) NO: 96) NO: 61) NO: 23) NO: 97)

gaaaggca

(SEQ ID

NO: 72)

N7a

62674 QSSDLSR RSDDLTR RSDDLTR RSDDLTR RSDHLSQ NSRNLRN aaGGCAGGGA (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID GGCGGCGGCT NO: 35) NO: 30) NO: 30) NO: 30) NO: 42) NO: 102) aacaagtc

(SEQ ID

NO: 74)

LO

62705 DRSNLSS RSHSLLR QSGALAR RKDQLVA RSDNLST RQWSLRI gcTTGCAGAA (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID GGTAGTGCCC NO: 103) NO: 44) NO: 96) NO: 104) NO: 100) NO: 105) agctccca

(SEQ ID

NO: 75)

N7a

62704 RSDDLSK DNSNRIK DRSHLTR RSDALAR QSGDLTR RRDWLPQ tcCTGGCAGT (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID GGGCCACTCG NO : 106 ) NO: 107) NO: 58) NO: 54) NO: 32) NO: 108) tgggagct

(SEQ ID

NO: 76)

N7a

62659 DRSNLSR LKQHLTR LRHHLTR QSYDRFQ QSSDLSR QWSTRKR gtGCAGCAcC (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID CAGGTGGTGA NO: 59) NO: 109) NO: 95) NO : 110 ) NO: 35) NO : 111 ) Ccactgtc

(SEQ ID

NO: 77) N6a

62657* QSGDLTR RRADLSR QSGDLTR DTGARLK QSSDLSR RKYYLAK ggTGGGCTGT (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID CGCAtGCGCC NO: 32) NO: 112) NO: 32) NO: 113) NO: 35) NO: 114) Agacagtg

(SEQ ID

NO: 78)

N7a

62656* QSGDLTR RRADLSR QSGDLTR DTGARLK QSSDLSR RKYYLAK ggTGGGCTGT (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID CGCAtGCGCC NO: 32) NO: 112) NO: 32) NO: 113) NO: 35) NO: 114) Agacagtg

(SEQ ID

NO: 78)

N6a

62714 QSSDLSR QSGSLTR QSSNLAR RSDNLTR DRSNLSR QSGNLAR aaGAAGACcG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID AGGAAtGTAG NO: 35) NO: 34) NO: 115) NO : 46 ) NO: 59) NO: 61) CTtgcaga

(SEQ ID

NO: 79)

N6a

62710 DSSNRHK DRSNLTR RSDHLSR QSSDLRR QSSHLTR RSDALAR tcGTGGGAGC (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID TGGGCACTAC NO : 116 ) NO: 91) NO : 29 ) NO: 117) NO : 118 ) NO: 54) cttctgca

(SEQ ID

NO: 80)

N7a

*ZFNs comprising 62677 or 62676 and 62657 or 62656 differ in lin cer used between ZFP and Fokl domain

Table 3: AR Designs

ZFN Name Fl F2 F3 F4 F5 F6 target

sequence

linker

61819 QNAHRKT LRHHLTR RSHSLLR HRKSLSR RSDHLSE RKDARIT ggATGGGGGC (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID CGTGGGTAGA NO: 135) NO: 95) NO: 44) NO: 136) NO: 37) NO: 137) cccttccc

(SEQ ID

NO: 119) N7a

61812 QSSDLSR RSDHLTQ TSGSLSR QSGDLTR TSGHLSR QSGHLSR atGGAGGTGC (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID AGTTAGGGCT NO: 35) NO: 138) NO: 139) NO: 32) NO: 33) NO: 65) gggaaggg

(SEQ ID

NO: 120)

N7a

61970 TSGSLSR QSGSLTR DRSHLTR DSSDRKK RSDHLSA QHGALQT ccATAAGGTC (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID CGGAGTAGTT NO : 139 ) NO: 34) NO: 58) NO: 140) NO: 141) NO: 142) ctccatcc

(SEQ ID

NO: 121)

N7a

61969 YPQVLES DRSNLTR RSDHLSQ QSANRTT RSDSLSV QNANRKT caAAAGTGAA (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID ATGGGACCTT NO : 143 ) NO: 91) NO: 42) NO: 144) NO : 49 ) NO: 145) ggatggag

(SEQ ID

NO: 122)

N7a

61828 QSGALAR RKYYLAK ERGTLAR RSDHLSR RSDVLST RYAYLTS ctTGGATGGG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID GGCCgTGGGT NO : 96 ) NO: 114) NO: 31) NO : 29 ) NO: 17) NO: 146) Agaccctt

(SEQ ID

NO: 123)

N6a

61820 RSDHLSR QSSDLRR RSDHLSE TSGSLTR QSGDLTR NKHHRNR gaGGTGCAGT (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID TAGGGCTGGG NO : 29 ) NO: 117) NO: 37) NO : 20 ) NO: 32) NO: 147) aagggtct

(SEQ ID

NO: 124)

N6a

61831 TSGHLSR RSDALAR ERGTLAR RSDHLSR RSDVLST RYAYLTS ctTGGATGGG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID GGCCGTGGGT NO: 33) NO: 54) NO: 31) NO : 29 ) NO: 17) NO: 146) agaccctt

(SEQ ID

NO: 123)

N6a

61824 QSGHLAR RKWTLQG RSDHLSE TSGSLTR QSGDLTR NKHHRNR gaGGTGCAGT (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID TAGGgCTGGG NO : 15 ) NO: 147) NO: 37) NO : 20 ) NO: 32) NO: 147) Aagggtct

(SEQ ID

NO: 124)

N6a

61984 LRHHLTR DRSTLRQ ERGTLAR QSSDLRR QSGDLTR YRWLRNN acCTTGCAGC (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID TGCCACAAGT NO: 95) NO: 148) NO: 31) NO: 117) NO: 32) NO: 149) gagagctc

(SEQ ID

NO: 125)

N7a

61983 RSDTLSA ANSTRTN DRSALAR QSSDLSR QSSDLSR RRDALLM ttCTGGCTGT (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID CACTACGgag NO: 150) NO: 151) NO : 41 ) NO: 35) NO: 35) NO: 152) ctctcact

(SEQ ID

NO: 126)

N7a

61835 RSHSLLR HRKSLSR RSDHLSE RKDARIT SLTYLPT DRSALAR agGTCTTGgA (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID TGGGGGCCGT NO : 44 ) NO: 136) NO: 37) NO: 137) NO: 153) NO : 41 ) Gggtagac

(SEQ ID

NO: 127)

N7a

61832 RSDNLSV RSAHLSR QSSDLSR RSDHLTQ TSGSLSR QSGDLTR gtGCAGTTAG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID GGCTGGGAAG NO: 154) NO: 155) NO: 35) NO: 138) NO: 139) NO: 32) ggtctacc

(SEQ ID

NO: 128)

N7a

61823 RSDNLST RSDHLSR QMHHLSA TSGHLSR RSDHLSR TSGNLTR tgGATGGGGG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID CCGTGGGTAG NO : 100 ) NO : 29 ) NO: 156) NO: 33) NO : 29 ) NO: 53) acccttcc

(SEQ ID

NO: 129)

N7a

61816 RSDVLSE RSAHLSR TSGSLSR QSGDLTR TSGHLSR QSGHLSR atGGAGGTGC (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID AGTTaGGGCT NO : 27 ) NO: 155) NO: 139) NO: 32) NO: 33) NO: 65) Gggaaggg (SEQ ID

NO: 120)

N7a

61817 DRSNLSR QKTSRDN RSHSLLR HRKSLSR RSDHLSE RKDARIT ggATGGGGGC (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID CGTGgGTAGA NO: 59) NO: 156) NO: 44) NO: 136) NO: 37) NO: 137) Cccttccc

(SEQ ID

NO: 119)

N7a

61810 DRSHLTR RSDYLAK RSDNLSE RSAALAR RSANLAR RSDALTQ ggATGGAGGT (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID GCAGtTAGGG NO: 58) NO: 45) NO: 23) NO: 157) NO: 158) NO : 89 ) Ctgggaag

(SEQ ID

NO: 130)

N7a

61867 LPQTLQR QNATRTK RSDHLSR TSGNLTR RSDSLLR DRSNRNQ acAACGTGGA (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID TGGGGCAGCT NO: 159) NO: 87) NO : 29 ) NO: 53) NO: 84) NO: 160) gagtcatc

(SEQ ID

NO: 131)

N7a

61866 RLDNRTA TSGNLTR QSNDLNS IRSTLRD HRSSLRR NA caGCTCCTCC (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID

AGATCAGgat NO: 161) NO: 53) NO: 162) NO: 163) NO: 164)

gactcagc

(SEQ ID

NO: 132)

N7a

61931 TSGNLTR RRDWRRD QSGHLAR QLTHLNS RSDNLSQ QRQHRKT agAGACAGAG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID AGGACGGGAT NO: 53) NO: 165) NO: 15) NO: 166) NO: 167) NO: 168) ctcaagtg

(SEQ ID

NO: 133)

N7a

61925 QSGHLAR RKWTLQG QSSDLSR QSGSLTR RSDNLSE KRCNLRC tgAAGCAGGT (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID AGCTCTGGGA NO: 15) NO: 147) NO: 35) NO: 34) NO: 23) NO: 169) cacttgag

(SEQ ID

NO: 134)

N7a [0183] In addition, TALE or single guide RNAs are designed to target sites comprising at least 12 nucleotides (e.g., 12, 13, 14, 15, 16, 17, 18 or more

nucleotides) of the target sequences shown herein. DNA-binding domains (ZFPs, TALEs or sgRNAs) are also designed to homologous target sequences (e.g., in different species such as human), for example DNA-binding domains that bind to target sequences exhibiting 80%, 90%, or even 95%-99% homology to the target sites shown above (e.g., homologous target sites in PTGDS, PTGDR, AR, etc.).

[0184] All ZFNs were tested and found to be active as shown below in Tables

4 to 6.

Table 4: PTGDS Nuclease Activity

62705_62704 11.90

62659_62657 11.48

62659_62656 11.46

62676_62675 11.20

62714_62710 10.77

Table 6: AR Nuclease Activity

[0185] Guide RNAs for the S. pyogenes CRISPR/Cas9 system are also constructed to target the gene (see U.S. Patent Publication No. 2015/0056705). All guide RNAs are tested in the CRISPR/Cas9 system and are found to be active in K562 cells and in human stem (skin stem) cells. Exemplary Guide RNAs for use with a S. pyogenes Cas 9 (spCas9) by expression using a U6 promoter system are shown below in Table 7.

Table 7: Exemplary Guide RNAs

Gf2449bl

AR- gCAGGATGTCTTTAAGGTCAG 172 CAGGATGTCTTTAAGGTCAGCGG 202

Gr2651cl

AR- gTAAGGCAGTGTCGGTGTCCA 173 TAAGGCAGTGTCGGTGTCCATGG 203

Gf2854dl

AR- gTCTCTGCTAGACGACAGCGC 174 TCTCTGCTAGACGACAGCGCAGG 204

Gf3011el

AR- gAGTTGTAGTAGTCGCGACTC 175 AGTTGTAGTAGTCGCGACTCTGG 205

Gr3195fl

AR- gCACCACACGGTCCATACAAC 176 CACCACACGGTCCATACAACTGG 206

Gr3450gl

AR- GTGCGGTGAAGTCGCTTTCC 177 GTGCGGTGAAGTCGCTTTCCTGG 207

Gr3594hl

AR- GGTGGAAAGTAATAGTCAAT 178 GGTGGAAAGTAATAGTCAATGGG 208

Grl00256il

AR- GCACTATTGATAAATTCCGA 179 GCACTATTGATAAATTCCGAAGG 209

Gfl43034jl

PTGDR2-targeted sgRNAs

PTGDR2- GAGTGGCTTCAGTGTGGCGT 180 GAGTGGCTTCAGTGTGGCGTTGG 210 Gr3260al

PTGDR2- GTGGTCGATGTAGCGGATGC 181 GTGGTCGATGTAGCGGATGCTGG 211 Gr3329bl

PTGDR2- gCATCCGCTACATCGACCACG 182 CATCCGCTACATCGACCACGCGG 212 Gf3349cl

PTGDR2- gCGCCAGGTGCAGCACCCAGG 183 CGCCAGGTGCAGCACCCAGGTGG 213 Gr3452dl

PTGDR2- gCCTGCTCAGCGCCATCAGCC 184 CCTGCTCAGCGCCATCAGCCTGG 214 Gf3628el

PTGDR2- gCGCGGCGCACAAAGTCTGCC 185 CGCGGCGCACAAAGTCTGCCTGG 215 Gf3706fl

PTGDR2- gACACCATCTCGCGGCTGGAC 186 ACACCATCTCGCGGCTGGACGGG 216 Gf3780gl

PTGDR2- gCACCTGCCGCGAGTTGCACG 187 CACCTGCCGCGAGTTGCACGTGG 217 Gr3842hl

PTGDR2- GATCATCGCCTCGAGCCACG 188 GATCATCGCCTCGAGCCACGCGG 218 Gf3928il

PTGDR2- gAGGCCGCTTCGTGCGCCTGG 189 AGGCCGCTTCGTGCGCCTGGTGG 219 Gf3991jl

PTGDS-targeted sgRNAs

PTGDS-

GGGCCTCCGGTGCTGCCTGC 190 GGGCCTCCGGTGCTGCCTGCAGG 220 Grll35al

PTGDS- gCGACCTGCAGGCAGCACCGG 191 CGACCTGCAGGCAGCACCGGAGG 221 Gfll48al

PTGDS- gCACCTTGTCCTGCTGGAAGT 192 CACCTTGTCCTGCTGGAAGTTGG 222 Grll73al

PTGDS-

GCCCAACTTCCAGCAGGACA 193 GCCCAACTTCCAGCAGGACAAGG 223 Gfll87al

PTGDS-

GTCGCTCGCCGCAGTTCCTG 194 GTCGCTCGCCGCAGTTCCTGGGG 224 Gf2495al

PTGDS-

GCCTCGCCTCCAACTCGAGC 195 GCCTCGCCTCCAACTCGAGCTGG 225 Gf2534al

PTGDS- gACAACGCCGCCTTCTTCTCC 196 ACAACGCCGCCTTCTTCTCCCGG 226 Gr2543al

PTGDS-

GGCCACCACAGACTTGCACA 197 GGCCACCACAGACTTGCACATGG 227 Gr2566al

PTGDS- gCCCTCGGCTCCTACAGCTAC 198 CCCTCGGCTCCTACAGCTACCGG 228 Gf2786al PTGDS- gCAGCGCGTACTGGTCGTAGT 199 CAGCGCGTACTGGTCGTAGTCGG 229

Gr3482al

[0186] TALENs and TALE-TFs are made to target the AGA-related gene(s) in any species (see U.S. Patent 8,586,526), including TALENs and TALE-TFs that bind to any of the target sites shown in Tables 1-3 or 7, and are tested in K562 cells and human stem cells (skin stem cells) and found to be active.

Example 2: Topical Administration

[0187] The proteins, polynucleotides and/or cells (e.g., skin cells or skin stem cells) as described in Example 1 are formulated for topical administration (including but not limited to LNP formulation) for injection and applied at varying dosage regimes (daily, every other day, weekly, etc.) to the areas of skin where hair regrowth is desired.

[0188] Hair regrowth is seen in subjects receiving proteins, polynucleotides and/or cells as described in Example 1. In addition, ex vivo therapy using cells also results in increased hair growth.

Example 3: Activity of AGA related ZFNs transcription factors and nucleases in vivo

[0189] The proteins, polynucleotides, LNP and/or cells of Example 2 are tested in a mouse model of AGA (Crabtree et al (2010) Endocrinology 151(5):2373- 2380). In brief, transgenic mice expressing human AR in the basal epidermis and the follicle outer root sheath display an androgen-dependent delay in hair regrowth. Hair is removed from the animal's back using hair clippers and wax strips. Compounds formulated as above are delivered via topical administration and applied. Hair begins to regrow after about 11 days, and by 15 days, there is a clear difference between control and treated mice.

[0190] All patents, patent applications and publications mentioned herein are hereby incorporated by reference in their entirety.

[0191] Although disclosure has been provided in some detail by way of illustration and example for the purposes of clarity of understanding, it will be apparent to those skilled in the art that various changes and modifications can be practiced without departing from the spirit or scope of the disclosure. Accordingly, the foregoing description and examples should not be construed as limiting.

Claims

What is claimed is: 1. A genetically modified cell in which expression of an AGA-related gene partially or completely inactivated, the AGA-related gene comprising an insertion and/or deletion.

2. The genetically modified cell of claim 1, wherein the AGA-related gene is prostaglandin D2 synthase (PTGDS), a GPR44 receptor (PTGDR) gene and/or an androgen receptor (AR) gene.

3. The genetically modified cell of claim 1 or claim 2, wherein insertion and/or deletion is within a target site as shown in any of Tables 1, 2, 3 or 7 or within 300 nucleotides of a target site as shown in any of Tables 1, 2, 3 or 7.

4. The genetically modified cell of any of claims 1 to 3, wherein the insertion and/or deletion is induced by a nuclease.

5. The genetically modified cell of claim 4, wherein the nuclease is a zinc finger protein.

6. A pharmaceutical composition comprising a genetically modified cell of any of claims 1 to 5.

7. A method of treating or preventing androgenic alopecia in a subject, the method comprising:

administering a nuclease that modifies an AGA-related gene to generate a genetically modified cell according to any of claims 1 to 4, wherein the cell is in the subject and androgenic alopecia is treated or prevented.

8. The method of claim 7, wherein the genetically modified cell is generated in vitro and administered to the subject.

9. The method of claim 8, wherein cell is administered via topical application or via injection into the area to be treated.

10. A composition for modifying one or more AGA-related genes in a cell of a mammalian subject, the composition comprising one or more nucleases, each nuclease comprising a DNA-binding domain that binds to the endogenous AGA- related gene and an endonuclease cleavage domain, wherein the nuclease cleaves the one or more endogenous AGA-related gene.

11. The composition of claim 10, wherein the modification is selected from the group consisting of an insertion, a deletion, a substitution and combinations thereof.

12. The composition of claim 10 or claim 11, wherein the one or more AGA- related genes are partially or completely inactivated.

13. A composition according to any of claims 10 to 12, characterized in that the compositions modifies an endogenous AGA-related gene in one or more cells of the subject.

14. The composition according to any of claims 10 to 13, wherein the subject is suffering from androgenic alopecia.

15. The composition of any of claims 10 to 14, wherein the AGA-related genes are PTGDS, PTGDR and/or AR.