WO2015153760A2 - Methods and compositions for prevention or treatment of a nervous system disorder - Google Patents

Methods and compositions for prevention or treatment of a nervous system disorder Download PDF

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WO2015153760A2
WO2015153760A2 PCT/US2015/023882 US2015023882W WO2015153760A2 WO 2015153760 A2 WO2015153760 A2 WO 2015153760A2 US 2015023882 W US2015023882 W US 2015023882W WO 2015153760 A2 WO2015153760 A2 WO 2015153760A2
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gene
cell
dna
protein
sequence
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WO2015153760A3 (en
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Fyodor Urnov
H. Steve Zhang
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Sangamo Biosciences, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Definitions

  • the present disclosure is in the field of genome engineering of cells, especially for the treatment of a disorder of the nervous system.
  • 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:
  • Another area of gene therapy that is especially promising is the ability to genetically engineer a cell to cause that cell to express a product not previously being produced in that cell.
  • uses of this technology include the insertion of a gene encoding a novel therapeutic protein, insertion of a coding sequence encoding a protein that is lacking in the cell or in the individual, insertion of a wild type gene in a cell containing a mutated gene sequence, and insertion of a sequence that encodes a structural nucleic acid such as a microRNA or siRNA.
  • Transgenes can be delivered to a cell by a variety of ways such that the transgene becomes integrated into the cell's own genome and is maintained there.
  • a strategy for transgene integration has been developed that uses cleavage with site-specific nucleases (e.g., zinc finger nucleases (ZFNs), TALE nucleases (TALENs), Ttago nuclease systems and CRISPR/Cas nuclease systems) for targeted insertion into a chosen genomic locus.
  • site-specific nucleases e.g., zinc finger nucleases (ZFNs), TALE nucleases (TALENs), Ttago nuclease systems and CRISPR/Cas nuclease systems
  • Nucleases specific for targeted genes can be utilized such that the transgene construct is inserted by either homology directed repair (HDR) or by end capture during non-homologous end joining (NHEJ) driven processes. See, for example, 8,623,618; 8,034,598; 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;
  • HDR homology directed repair
  • NHEJ non-homologous end joining
  • Nuclease-mediated integration offers the prospect of improved transgene expression, increased safety and expressional durability, as compared to classic integration approaches that rely on random integration of the transgene, since it allows exact transgene positioning for a minimal risk of gene silencing or activation of nearby oncogenes.
  • nucleases can be used to direct gene correction or gene modification, where segments of an endogenous gene are replaced with sequences supplied in concert with the specific nuclease(s).
  • This technology can be used to correct sequences known to be associated with disease (e.g. sickle cell anemia), or to alter gene sequences to confer new characteristics.
  • nucleases can include zinc finger nucleases (ZFN), transcription activator like effector nucleases (TALENs), mega or homing
  • nuclease systems such as CRISPR/Cas that use a guide RNA to determine specificity
  • fusions between nucleases such as mega-TALs.
  • Engineered nucleases can also be used to silence or knock out a gene through targeted double strand cleavage at a desired locus.
  • the cell will rely on error- prone non-homologous end joining (NHEJ) to heal the break.
  • NHEJ often results in the insertion or deletions of nucleotides at or near the break ("indels"), causing missense mutations within the coding sequence.
  • Targeted cleavage can also result in the knockout of splicing sequences such that resulting transcripts are improperly spliced. Both of these approaches can result in the silencing of the targeted gene following cleavage by the nuclease.
  • NS diseases and disorders represent the largest and fastest growing area of unmet medical need: 1.5 billion people worldwide, including over 100 million people in the U.S., suffer from NS diseases or disorders.
  • CNS central nervous system
  • PNS peripheral nervous system
  • the pathogenesis of NS disease is often the most difficult to understand, although for more and more of these disorders, genetic clues are being discovered.
  • 1 in 8 people over the age of 65 has Alzheimer's disease, and 45% of people over the age of 85 are afflicted. In fact, it is currently the sixth leading cause of death.
  • Alzheimer's disease By the year 2050, there may be 13.2 million people in the United States alone will have Alzheimer's disease. See, e.g., Alzheimer's and Dementia (2012) 8(2). Statistics and predictions for other types of NS diseases demonstrate that these diseases are also very pervasive in the population. For most NS disorders, treatments are inadequate, and often physicians can only attempt to alleviate symptoms rather than achieve functional cures.
  • NS nervous system
  • RNAs a disease or disorder of the central nervous system (CNS) and/or peripheral nervous system (PNS).
  • CNS central nervous system
  • PNS peripheral nervous system
  • Alteration of genomic sequence or expression levels of such genes can result in the treatment of these nervous system disorders.
  • genome editing is used to correct an aberrant gene, insert a wild type gene, or change the expression of an endogenous gene.
  • a mutated gene encoding a protein involved in a NS disorder may be corrected in a cell to produce a wild type protein to prevent and/or treat the NS disorder.
  • One approach further involves the use of gene modification in a stem cell, which stem cell can then be used to engraft into a patient, for treatment of a nervous system disease.
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • CMT Charcot-Marie-Tooth disease
  • DMD Duchenne's muscular dystrophy
  • ALS Friedrich's Ataxia
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • CMT Charcot-Marie-Tooth disease
  • DMD Duchenne's muscular dystrophy
  • ALS Friedrich's Ataxia
  • familial familial
  • frontotemporal dementia Fragile X disease, myotonic dystrophy, Freidreich's ataxia, multiple sclerosis, Huntington's disease (HD), Tourette's syndrome, Rett syndrome, Canavan syndrome, Crigler Najjir syndrome, certain lysosomal storage diseases, stroke, migraine, concussion, major depressive disorder, epilepsy, bipolar disorder, borderline personality disorder, opiate addiction, and schizophrenia.
  • genes that may be modified include microtubule associated protein tau (MAPT, encoding Tau proteins) and/or
  • apolipoprotein E (APOE) alleles (e.g., apoE2, apoE3 or apoE4, for stroke, concussion and/or AD, and epilepsy); LRRK2 and alpha-synuclein (for PD); amyloid precursor protein (APP), presenilin 1 and/or presenilin 2 ⁇ e.g., for AD); SLC6A4, HTR2A, alpha- 1 subunit of a voltage-dependent calcium channel (CACNAIC) and/or calcium channel, voltage-dependent, beta 2 subunit (CACNB2) ⁇ e.g., for depression and/or migraines); dystrophia myotonica-protein kinase (DMPK for myotonic dystrophy); CACNA1 A, ATP1 A2, SCN1A ⁇ e.g., for migraine); FXN (for Friedrich's Ataxia); HTT genes ⁇ e.g., HD); PMP22 (CMT); dystrophin and/or
  • TGFBR2, PHACTR1, FHM5, c7orfl0, MMP16, ASTN2, TSPAN2, GFRA2 and LRP1 (Migraine); HLA-DRB1, IL7Ra, IL2Ra, CYP27Ba, TYK2 (multiple sclerosis); NRXN1, A AD AC, CTNNA3, FSCB, KCHE1, KCHE2, RCAN1 (Tourette syndrome); CAMSAP1LK1, NMDA receptor subunit 1, GAMA-A receptor subunit alpha- 1, GAD65, adenosine kinase, GCNF, BDNF, IGF, neuropeptide Y, galanin (epilepsy); . See, also, Cross-Disorder Group of the Psychiatric Genomics
  • a fusion protein comprising a DNA- binding domain ⁇ e.g., a ZFP, TALE, or single guide RNA) that binds to a target site in a selected gene and a functional domain.
  • the functional domain comprises a transcriptional regulatory domain, for example an activation domain or a repression domain.
  • the resulting artificial transcriptional factor ⁇ e.g., ZFP-TF, TALE-TF, CRISPR-TF
  • the functional domain comprises a nuclease (cleavage) domain that cleaves DNA at or near the target site(s), for example, within 1-300 (or any value therebetween) base pairs upstream or downstream of the site(s) of cleavage, more preferably within 1-100 base pairs (or any value therebetween) of either side of the binding and/or cleavage site(s), even more preferably within 1 to 50 base pairs (or any value therebetween) on either side of the binding and/or cleavage site(s).
  • a nuclease (cleavage) domain that cleaves DNA at or near the target site(s), for example, within 1-300 (or any value therebetween) base pairs upstream or downstream of the site(s) of cleavage, more preferably within 1-100 base pairs (or any value therebetween) of either side of the binding and/or cleavage site(s), even more preferably within 1 to 50 base pairs (or any value therebetween) on either side of the binding and/or cleavage site(s).
  • fusion proteins can be used to cleave DNA at or near the target site.
  • the fusion protein used will depend on the gene targeted. For example, some genes involved in NS disorders can be activated to treat and/or prevent the NS disorder ⁇ e.g., C9orf72 in frontotemporal dementia) while some genes can be repressed and/or inactivated (via cleavage) to treat and/or prevent the NS disorder ⁇ e.g., MAPT encoding Tau, ApoE4, PMP22, C90RF72, FMR1, DMPK, FXN, presenilin, CACNAIC, CACNB2, etc.)
  • nuclease ⁇ e.g., ZFN, TALEN, mega or homing endonuclease, mega-TAL or a CRISPR/Cas system
  • the nuclease comprises one or more engineered domains.
  • the nuclease is a zinc-finger nuclease (ZFN) that cleaves a target genomic region of interest, wherein the ZFN comprises one or more engineered zinc-finger binding domains and a nuclease cleavage domain or cleavage half-domain.
  • the nuclease is a TALE nuclease (TALEN) that cleaves a target genomic region of interest, wherein the TALEN comprises one or more engineered TALE DNA binding domains and a nuclease cleavage domain or cleavage half-domain.
  • the nuclease is a CRISPR/Cas system wherein the specificity of the CRISPR/Cas is determined by an engineered single guide mRNA.
  • the nuclease is a Ttago nuclease system. Cleavage domains and cleavage half domains can be obtained, for example, from various restriction endonucleases and/or homing endonucleases.
  • the cleavage half-domains are derived from a Type IIS restriction endonuclease (e.g., Fok l).
  • a CRISPR/Cas system that binds to target site in a region of interest ⁇ e.g., a highly expressed gene, a disease associated gene or a safe harbor gene) in a genome, wherein the CRISPR/Cas system comprises a CRIPSR/Cas nuclease and an engineered crRNA/tracrRNA (or single guide RNA).
  • the CRISPR/Cas system recognizes a target site in a highly expressed, disease associated, or safe harbor gene.
  • the fusion proteins and systems may bind to and/or cleave the region of interest in a coding or non-coding region 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.
  • the fusion protein and/or system binds to (and/or activates, represses or cleaves) a safe-harbor gene, for example a CCR5 gene, a CXCR4 gene, an HPRT gene, a PPP1R12C (also known as AAVS1) gene, an albumin gene or a Rosa gene.
  • a safe-harbor gene for example a CCR5 gene, a CXCR4 gene, an HPRT gene, a PPP1R12C (also known as AAVS1) gene, an albumin gene or a Rosa gene. See, e.g., U.S. Patent Nos. 7,888,121; 7,972,854;
  • compositions comprising one or more of the fusion proteins or one or more components of a CRISPR/Cas system as described herein.
  • fusion proteins and/or system bind to (and/or cleave, activate or repress) a gene encoding a gene product involved in a NS disorder.
  • compositions comprising one or more of the zinc-finger, TALE, single guide RNAs, and/or Cas nucleases as described herein.
  • polynucleotide encoding one or more fusion proteins (activators, repressors and/or nucleases) and/or systems (or components thereof) as described herein.
  • the polynucleotide may be, for example, mRNA.
  • the mRNA may be chemically modified (See e.g. Kormann et al, (2011) Nature Biotechnology 29(2):154-157).
  • Any of the polynucleotides described herein may include a promoter, for example where the fusion protein is operably linked to a promoter.
  • the expression vector is a viral vector.
  • the invention described herein comprises one or more NS gene-modulating transcription factors, such as a NS gene -modulating transcription factors comprising one or more of a zinc finger protein (ZFP TFs), a TALEs (TALE-TF), and a CRISPR/Cas-TFs for example, ZFP-TFs, TALE-TFs or CRISPR/Cas-TFs.
  • the NS gene -modulating transcription factor can repress expression of a NS gene in one or more cells of a subject. The repression can be about 85% or greater, about 90% or greater, about 92% or greater, or about 95% or greater repression of NS gene in the one or more cells of the subject.
  • the NS gene-modulating transcription factor can be used to achieve one or more of the methods described herein.
  • a ZFN, TALEN and/or CRISPR/Cas system that is used to cleave a target DNA.
  • a method for inserting a sequence into an endogenous gene in which the endogenous gene encodes a gene product that is involved in a NS disorder in a cell (e.g. stem cell), the method comprising cleaving the endogenous gene using one or more nucleases and inserting a sequence into the cleavage site.
  • the insertion results in a replacement of a genomic sequence in any target gene, for example using a ZFN or TALEN pair, or a CRIPSR/Cas system (or vector or polynucleotide encoding said ZFN, TALEN and/or CRIPSR/Cas system) as described herein using a "donor" sequence (also known as a "transgene”) that is inserted into (and replaces at least part of) the gene following targeted cleavage with the ZFN, TALEN and/or a CRIPSR/Cas system.
  • a "donor" sequence also known as a "transgene”
  • the donor sequence may be present in the ZFN or TALEN vector, present in a separate vector (e.g., Ad, AAV or LV vector) or, alternatively, may be introduced into the cell using a different nucleic acid delivery mechanism.
  • a donor nucleotide sequence into the target locus (e.g., a safe -harbor gene, etc.) results in the expression of the transgene under control of the target locus 's genetic control elements.
  • the donor nucleotide sequence includes one or more control elements that drive expression of one or more transgenes.
  • the transgene encodes a non-coding RNA (e.g., an shRNA).
  • a non-coding RNA e.g., an shRNA
  • the cells and/or cells lines are used for treatment, prevention and/or amelioration of a nervous system disorder by administration to a subject with a nervous system disorder.
  • the transgenic cell and/or animal includes a transgene that encodes a human gene.
  • the cells comprises a mutation (e.g., "indel" which comprises one or more insertions and/or deletions), for example an indel that results in production of a product involved in a NS disorder.
  • the modification corrects a mutant allele such that the wild-type gene product is produced.
  • the transgenic animal comprises a knock-out at the endogenous locus corresponding to exogenous transgene (e.g., the mouse gene involved in the NS disorder is knocked out and the human gene involved in the NS disorder is inserted into a mouse), thereby allowing the development of an in vivo system where the human gene may be studied in isolation.
  • exogenous transgene e.g., the mouse gene involved in the NS disorder is knocked out and the human gene involved in the NS disorder is inserted into a mouse
  • Such transgenic models may be used for screening purposes to identify small molecules or large biomolecules or other entities which may interact with or modify the human protein or gene product of interest.
  • the transgene is integrated into the selected locus (e.g., NS gene, safe-harbor) into a stem cell (e.g., an embryonic stem cell, an induced pluripotent stem cell, a hematopoietic stem cell, etc.) or animal embryo obtained by any of the methods described herein, and then the embryo is implanted such that a live animal is born. The animal is then raised to sexual maturity and allowed to produce offspring wherein at least some of the offspring comprise edited endogenous gene sequence or the integrated transgene.
  • a stem cell e.g., an embryonic stem cell, an induced pluripotent stem cell, a hematopoietic stem cell, etc.
  • animal embryo obtained by any of the methods described herein
  • a method for site specific integration of a nucleic acid sequence into an endogenous locus of a chromosome for example into the chromosome of a non-human embryo, which locus encodes a gene involved in a NS disorder.
  • the method comprises: (a) injecting a non-human embryo with (i) at least one DNA vector, wherein the DNA vector comprises an upstream sequence and a downstream sequence flanking the nucleic acid sequence to be integrated, and (ii) at least one RNA molecule encoding a zinc finger (e.g., ZFN), TALE (e.g., TALEN) or Cas9 protein (e.g., nuclease component of a CRISPR/Cas system)).
  • a zinc finger e.g., ZFN
  • TALE e.g., TALEN
  • Cas9 protein e.g., nuclease component of a CRISPR/Cas system
  • an engineered single guide (sg)RNA is also introduced.
  • the nuclease or nuclease system recognizes the target site in the target locus (e.g., NS gene or safe harbor locus), and then (b) the non-human embryo is cultured to allow expression of the zinc finger nuclease, TALE nuclease and/or CRISPR/Cas system, wherein a double stranded break is introduced into the target by the zinc finger nuclease, TALEN or
  • CRISPR/Cas system is then repaired, via homologous recombination with the DNA vector, so as to integrate the nucleic acid sequence into the chromosome.
  • a method of treating, preventing and/or ameliorating a nervous system disorder in a subject comprising genetically modifying a cell of the subject, such that the cell treats, prevents and/or ameliorates the nervous system disorder.
  • the modification comprises inserting an exogenous sequence that into a specified genomic locus of a cell of the subject.
  • the exogenous sequence can comprise, for example, one or more transgenes that express proteins involved in the nervous system disorder, one or more RNAs and combinations thereof.
  • the modification comprises alteration of an endogenous sequence such that a beneficial gene product is produced.
  • the cell may be modified (exogenous sequence integration) in the subject (in vivo); may be isolated from the subject or from another source (e.g., cell line), modified to include the exogenous sequence and re-introduced into the subject (ex vivo); and combinations thereof.
  • the genetically modified cells as described herein may be delivered by any suitable means to a subject in need thereof.
  • the cells are delivered directly to the nervous system, for example by administration directly into the CNS or PNS (e.g., grafting).
  • the polynucleotide encoding the fusion protein and/or system (or component thereof) can comprise DNA, R A or combinations thereof.
  • the polynucleotide comprises a plasmid.
  • the polynucleotide encoding the nuclease comprises mRNA.
  • kits comprising the fusion proteins and/or systems of the invention.
  • the kit may comprise nucleic acids encoding the compositions described herein, (e.g. RNA molecules or ZFP-TF,TAL-TF, ZFNJALEN or Cas9 encoding genes contained in a suitable expression vector) and engineered sg RNA if needed , or aliquots of the nuclease proteins, transcription factors, donor molecules, suitable host cell lines, instructions for performing the methods of the invention, and the like.
  • the invention describes genomic editing of a target cell such that there is a favorable change in the expression of one or more genes, which in turn results in treatment and/or prevention of a disorder of the nervous system in a subject in need thereof.
  • Favorable changes in the expression of a gene includes, but is not limited, correction of an aberrant endogenous gene sequence (i.e., an endogenous gene with one or more mutations).
  • delivery of altered stem cells can be similarly beneficial in treating a nervous system disorder.
  • the methods and compositions of the invention can be used to alter the expression of one or more genes in a cell wherein those genes are related to a nervous system disease.
  • the cells may be modified in vivo or ex vivo (i.e., isolated cells, including patient derived cells, patient derived induced pluripotent stem cells or isolated stem cells) can be modified and re-introduced into a subject with the nervous system disease.
  • alteration of the expression of the Tau gene and/or an allele of ApoE may be beneficial.
  • Alzheimer's Disease pathogenesis is thought to be triggered by accumulation of the amyloid beta peptide due to over production of this protein and failure of its natural clearance mechanisms.
  • Amyloid beta self-aggregates and accumulates in plaques thought to be synaptotoxins. The plaques also may interfere with phosphorylation of tau, leading to its hyperphosphorylation and loss of normal function.
  • Tau is a microtubule associated protein involved in axonal transport, and an alteration of its normal function leads to accumulation of neurofibrillary tangles (Medeiros et al, (2011) CNS Neurosci Ther 17(5): p. 514).
  • Another potentially important target for Alzheimer's disease is the APOE4 allele of apolipoprotein E (apoE).
  • the APOE4 allele is the greatest genetic risk factor for AD and a person with two APOE4 alleles has 15 times the risk of developing AD than a person with APOE3 alleles.
  • ApoE is thought to bind amyloid beta and the soluble forms of this complex may modulate levels of neurotoxic amyloid beta. Clearance of soluble amyloid beta appears to be slower in the presence of the APOE4 encoded apoE, and apoE may also serve a role in the aggregation and deposition of amyloid beta (Tai et al (2014) Molecular Neurodegeneration 9(2)).
  • fusion proteins of the invention comprising transcription factor regulatory domains may be used to down regulate expression of either or both Tau and APOE4. Fusion proteins of the invention may also be used to increase the expression beneficial apoE alleles ⁇ e.g. APOE2) in a heterozygous subject.
  • CMT Charcot-Marie-Tooth Disease
  • CMT is the most common inherited neuro muscular disorder, with a prevalence of approximately 17-40 per 100,000 in the population.
  • the disease is characterized clinically by wasting and weakness in the distal limb muscles, skeletal deformities and a decrease or absence of deep tendon reflexes.
  • the disease can be linked to mutations in a number of different genes, but the various mutations all lead to axonal degeneration where the longer axon fibers are affected first and more severely resulting in the observed impairment of the feet and lower legs (Pareyson et al, (2006) Neuromolecular Medicine 8:3).
  • CMT1A The most frequent subtype of CMT, CMT1A is usually caused by a 1.5 Mb duplication of chromosome 17pl 1.2 which leads to an increase in gene dosage or overexpression of the PMP22 gene.
  • PMP22 is an integral membrane protein that is an important component of compact peripheral nervous system myelin sheath. A more severe form of CMT is seen in patients with missense mutations in one copy of PMP22, indicating a toxic gain of function mutation.
  • DMD Duchenne Muscular Dystrophy
  • DMD Prevalence of DMD is approximately 1 in 3500 male births and follows an X-linked recessive inheritance pattern. It arises out of mutations in the dystrophin gene, which is the largest gene in the human genome.
  • Dystrophin is a 427 kDa cytoskeletal protein that is required for muscle fiber stability, and loss of the protein results in necrosis and diminished regenerative capacity of muscle, ultimately leading to fibrosis of the muscle tissue.
  • a similar protein, utrophin is also a cytoskeletal protein that may be able to compensate for defective dystrophin levels.
  • a zinc finger-based transcription factor designed to upregulate utrophin was disclosed in Onori et al (2013, BMC Molecular Biology 14(3)) and U.S. Patent 8,304,235.
  • utrophin In adult muscle, utrophin localizes to the neuromuscular and myotendinous junctions while dystrophin localizes to the entire sarcolemma. In developing muscle however, utrophin can be found along the sacrolemma. In some DMD patients, utropin levels are upregulated, and there appears to be a positive correlation between the level of utrophin expression and disease progression (Kleopa et al (2006) Hum Mol Genet 15(10):1623). Current therapies for patients afflicted with DMD have increased life expectancy by decreasing respiratory complications.
  • the methods and compositions of the invention can be used to treat, delay and/or prevent the onset of symptoms associated with DMD.
  • Engineered DNA binding proteins e.g. ZFP, TALEs, CRIPSR/Cas systems
  • ZFP ZFP
  • TALEs TALEs
  • ALS Amyotrophic Lateral Sclerosis
  • the C9orf72 mutations are typically hexanucleotide expansions of GGGGCC in the first intron of the C9orf72 gene.
  • the pathology associated with this expansion appears to be related to the formation of unusual structures in the DNA and to some type of RNA- mediated toxicity (Taylor (2014) Nature 507: 175).
  • Incomplete RNA transcripts of the expanded GGGGCC form nuclear foci in fALS patient cells and also the RNAs can also undergo repeat-associate non-ATP -dependent translation, resulting in the production of three proteins that are prone to aggregation (Gendron et al (2013) Acta Neuropathol 126:829).
  • C9orf72 mutations are also the most common genetic cause of fronototemporal dementia (FTD), a common form of early-onset dementia.
  • FTD fronototemporal dementia
  • Other genetic causes of FTD include mutations in the progranulin gene (GRN) and in the gene encoding Tau (Mahoney et al (2012) Brain 135:736).
  • GNN progranulin gene
  • Tau Tau
  • Zinc finger proteins linked to repression domains have been successfully used to preferentially repress the expression of expanded Htt alleles in cells derived from Huntington patients by binding to expanded tracts of CAG. See, also, U.S. Patent Publication Nos.
  • the methods and compositions of the invention can be used to treat, delay or prevent ALS and FTD.
  • engineered DNA binding proteins e.g. ZFPs, TALEs, Cas
  • a wild type version of C9orf72 lacking the abnormally expanded GGGGCC tract, may be inserted into the genome to allow for the normal expression of the gene product.
  • the fusion proteins of the invention can be used to modulate the expression of other genes (e.g. GRN, Tau) or disrupt them via use of the engineered nucleases of the invention to prevent or treat FTD.
  • Parkinson's disease is a neurodegenerative disease that afflicts approximately 4-6 million people worldwide. In the United States, approximately one to two hundred people per 100,000 have PD. It appears that many factors can play a role in disease onset and/or progression of PD. For example, genetic mutations in the leucine rich repeat kinase 2 gene (LRRK2, also known as PAR 8) and alpha- synuclein have been identified to be involved in both familial and sporatic forms of PD and have been targeted by nucleases. See, also, U.S. Patent Publication Nos. 20120192301 and 20120214241.
  • LRRK2 leucine rich repeat kinase 2 gene
  • Fragile X syndrome is the leading monogenic cause of intellectual disability and autism.
  • the FMR1 gene encoding the fragile X mental retardation protein, FMRP
  • FMRP fragile X mental retardation protein
  • Subjects with 45-54 repeats are considered to be at risk for Fragile X syndrome, and people with 55-200 repeats are considered to have a pre-mutation for the syndrome.
  • Patients afflicted with Fragile X have between 200 and 1000 CGG repeats.
  • these repeats are also capable of forming the unusual structures in the DNA (G-quadruplexes, see Kettani et al (1995) J Mol Biol 254(4):638)
  • the Fragile X-associated FMR1 gene is methylated and is silenced or improperly expressed. Without sufficient FMRP, mental retardation results.
  • the methods and compositions of the invention can be used to insert a wild type copy of the gene in a safe harbor location to supply the subject with FMRP lacking due to the repeat expansion mutation.
  • Myotonic dystrophy is another muscle wasting disease associated with neuropathy that affects approximately 1 in 8000 people worldwide. Patients often have prolonged muscle contractions and may not be able to relax specific muscles after use and there may be cardiac conduction defects leading to abnormalities in the electric signals that control heartbeat.
  • DM1 (type 1) and DM2 (type 2) are inherited autosomal dominant diseases caused by unstable expanded sequences (CTG and CCTG, respectively) in the non-coding regions of DMPK and ZNF9 (also known as CNBP), respectively.
  • Engineered transcription factors can be used to down regulate expression of a diseased allele associated with either DM1 or DM2, or the diseased allele can be knocked out via cleavage by an engineered nuclease.
  • wild type versions of these genes can be inserted into an endogenous location in the genome to allow for expression of the normal gene product.
  • Rett syndrome is a neurodevelopmental disorder that affects 1 in 10,000 live female births. The symptoms of RTT appear after an early period (approximately 6- 18 months of life) of apparently normal development in an infant. Initially there is a slowing down or stagnation of learning skills followed by a loss of communication skills and purposeful use of the hands. RTT can present with a wide range of disability ranging from mild to severe and causes problems in brain function that are associated with cognitive, sensory, emotional, motor and autonomic functions leading to effects on speech, sensation, mood, movement, breathing, cardiac function, and digestion. Life expectancy is thought to be 40 to 50 years, with patients requiring intensive care for daily life. RTT is usually caused by a mutation in the Methyl CpG binding protein 2 (MECP2) where 35% of RTT cases are cause by nonsense mutations in MECP2 (see Pitcher et al, (2015) Hum Mol Genet 1-11, doi:
  • MECP2 Methyl CpG binding protein 2
  • RTT 6.1093/hmg/ddv030.
  • 60% of RTT cases are caused by 8 different mutations in the MECP2 protein as follows: R106W; R133C; T158M; R168X; R255X; R270X; R294X and R306C.
  • Engineered transcription factors can be designed to shut off the expression of mutant MECP2 genes and/or to upregulate a wild type MECP2 gene in a heterozygous individual.
  • Engineered nucleases can be used to introduce a double strand break in the 5 ' end of the mutant MECP2 gene followed by targeted integration of a cDNA comprising a sequence encoding the MECP2 gene and a poly A signaling sequence.
  • the MECP2 promoter will thus drive normal expression of the inserted transgene while silencing the mutant copy.
  • Viral vectors comprising either the engineered transcription factor or engineered nuclease can be introduced into the brain to edit the cells therein. Similarly, these viral vectors can be introduced into other target tissue such as the heart and/or lung tissue to cause the expression of the wild type protein in these tissues. Additionally, altered regulation (e.g. up regulation of gene expression) of other factors which interact with MECP2 are also
  • BDNF brain derived neutrotrophic factor
  • IGF insulin like growth factor
  • Canavan's disease is a hereditary leukodystrophy caused by mutations in the aspartoacylase gene (ASPA) that lead to a loss of enzyme activity and an increase in concentrations of the enzyme's substrate N-acetylaspartate (NAA) in the brain.
  • ASPA aspartoacylase gene
  • NAA N-acetylaspartate
  • an engineered transcription factor can be used to increase the expression of a wild type ASPA allele in situ in the brain.
  • engineered nucleases can be used to knock out expression of an aberrant ASPA allele, or can be used to introduce a short oligonucleotide to correct a mutated gene and/or to introduce a wild type cDNA.
  • wild type ASPA protein may be made hepatically (e.g. from the albumin locus) where the protein comprises a peptide allowing it to cross the blood brain barrier.
  • the invention contemplates the correction of an aberrant ASPA gene in an iPSC derived from a Canavan patient that can be forced along the pathway to becoming a neuronal stem cell. These altered neuronal stem cells are then reintroduced into the brain of the patient and correct and/or treat the disease.
  • Dravet syndrome also known as Severe Myoclonic Epilepsy of
  • SCN1A mutations are found in 79% of diagnosed DS patients).
  • the alpha subunits form the transmembrane pore in the channel.
  • the SCN1A mutations cluster in the C-terminus of the protein and cause a loss of function, resulting in decreased activity in GABAergic inhibitory neurons (see Rossi (2014) Epil Curr 14(4): 189-190).
  • the activity of the Navl .6 sodium channels is reduced in DS.
  • DS is refractory to most current anti-epileptic medications. Symptoms appear in the first year of life as prolonged seizure events. At two years of age, patients begin suffering from a variety of other seizure types and developmental milestones begin to plateau and then regress during this phase.
  • the symptoms include motor and balance issues, delayed language and speech, growth and nutrition issues, sleeping difficulties, and chronic infections.
  • the SCN8A gene may also be involved in DS, as it has been found that increased seizure resistance is found in mice with mutations in the mouse Scn8A gene.
  • SCN8A may be a genetic modifier of DS (see Martin et al (2007) Hum Mol Gene 16(23):2892-2899).
  • the methods and compositions of the invention can be used to prevent or treat DS.
  • Engineered transcription factors can be used to down regulate expression of SCN8A in the brain to decrease seizure frequency.
  • Engineered nucleases could be designed to cleave the mutant SCN1 A alleles to knock out expression.
  • a wild type cDNA encoding the SCN1A gene could be introduced into the brain using various methods known in the art ⁇ e.g. through a viral vector such as AAV2) in addition to mutant SNC1A specific nucleases to cause expression of the wild type gene in place of the mutant.
  • Stem cells corrected by the methods of the invention can also be introduced into specific seizure centers to treat or prevent Canavan disease.
  • Another genetic disease of the nervous system is Spinal Muscular
  • SMA Atrophy
  • SMA survival motor neuron 1
  • SMA is associated with a homozygous loss of the SMNl gene
  • some researchers have tried introducing the SMNl gene via an AAV9 viral vector in animal models of SMA (see Bevan et al (2011) Mol Ther 19(11): 1971-1980). This early work showed that the gene could be delivered either through IV administration or through direct injection into the cerebral spinal fluid. However, penetration of the virus and complications relating to the crossing of the blood brain barrier still exist.
  • the methods and compositions of the invention can be used to prevent or treat SMA.
  • Engineered transcription factors specific for SNM2 may be designed to increase the expression of this gene.
  • Engineered nucleases can also be used to cleave and correct the SMN2 mutation and cause stable expression by essentially turning it into the SMNl gene.
  • a wild type SMNl cDNA may be inserted into the genome by targeted insertion using an engineered nuclease.
  • the wild type SMNl gene may be inserted into the endogenous SMNl gene and thus be expressed under the regulation of the SMNl promoter, or it may be inserted into a safe harbor gene ⁇ e.g. AAVSl).
  • the gene may also be inserted via nuclease directed targeted integration into neuronal stem cells, where the engineered stem cells are then re-introduced into the patient such that the neurons that are derived from these stem cells function normally.
  • Crigler Najjir syndrome is a disease related to hyperbilirubinemia caused by an excess of unconjugated bilirubin in the blood which leads to severe neurological damage.
  • the disease is tied to deficiency in the uridine-diphosphate (UDP) glucuronosyltransferase 1 Al enzyme encoded by the UGT1 Al gene.
  • UDP uridine-diphosphate
  • UGT1 Al Mutations in the UGT1 Al gene that result in no expression cause the disease, and current treatment is limited to phototherapy treatment for 10-12 hours per day.
  • the lack of UDP1A1 leads to high concentrations of unconjugated bilirubin in the blood. Normally the enzyme acts on bilirubin to make it more water soluble and thus allows the molecule to be more easily eliminated from the body.
  • Mutations in the promoter region of UGT1A1 may also be tied to a closely related, but milder disease known as Gilbert syndrome. UGT1A1 is most highly expressed in the liver.
  • adenoviral or lentiviral vectors have been able to deliver the UCT1A1 gene to the liver and normalize plasma bilirubin levels (see van der Wegen (2005) Mol Ther 13(2) p. 374). Additional studies using delivery by AAV have also shown promise (see Bortolussi et al (2014) Hum Gen Ther 25:844-855).
  • the methods and compositions of the invention provide a treatment for both Crigler Najjir and Gilbert syndromes.
  • Engineered nucleases can be used to insert the UGT1A1 gene into the albumin promoter in the liver and cause expression of the enzyme such that serum bilirubin levels are dropped.
  • the gene can be inserted into other safe harbor loci in the liver ⁇ e.g. AAVS1).
  • Opiate addiction is undergoing a resurgence and has emerged as an epidemic, especially in parts of the United States. It is estimated that between 26 and 36 million people worldwide participate in opiate abuse, with an estimated 2.1 million people in the United States suffering from substance use disorders related to prescription opioid pain relievers in 2012, and an estimated 467,000 people in the US are addicted to heroin (see United Nations Office on Drugs and Crime, World Report 2012 and Substance Abuse and Mental Health Services Administration, NSDUG Series H-46, HHS Publication No (SMA) 13-4795). In the brain, the primary site of action of nearly all analgesic and addictive opiates is the mu opioid receptor (MOP-r, encoded by the OPRM1 gene).
  • MOP-r mu opioid receptor
  • the endogenous ligands for this receptor are the peptide products of the precursor proteins proopiomelanocortin (POMC) and proenkephalin (PENK).
  • POMC proopiomelanocortin
  • PENK proenkephalin
  • the endogenous opioid system also includes two other receptors, delta (DOP-r, encoded by OPRD1) and kappa (KOP-r, encoded by
  • the MOP-r that is produced also has an increased binding affinity for its natural ligand.
  • the Al 18G SNP results in an altered response in the stress and hypothalamic-pituitary-adrenal axis in humans. This alone is thought to be a major factor in the vulnerability to specific addictions. In fact, the Al 18G SNP is associated with a substantial increase in the vulnerability to heroin addiction (Reed, ibid). Polymorphisms in other genes associated with the endogenous opiate system such as OPRK1 and OPRDlmay also prove to be factors leading to susceptibility to opiate addiction. Additionally, transcription factors that are elevated in response to stimulation of MOP-r such as
  • ELKl (tied to many cellular processes such as cell division, differentiation, migration and apoptosis) are elevated to a greater extent in people with the Al 18G SNP than in controls (Sillivan et al (2013) Biol Pyschiatry 74(7):5110519).
  • MOP-r The mu opiate receptor
  • BPD borderline personality disorder
  • MOP-r is implicated in emotional and stress response regulation (see Prossin et al (2010) Am J Psychiatry 167:925-933).
  • BPD patients have a marked increase in morbidity and mortality that includes risk for suicide and over the long term, they have severe and persistent impairment in social functioning. BPD patients also display an increased susceptibility to opiate addiction as nearly 40% have a co-diagnosis of drug use disorder (see Panagopoulos et al (2013) Drug Alcohol Depend 128(3)). Both opiate addiction and BPD represent chronic states that have enormous impact on patients over a lifetime.
  • the methods and compositions of the invention can be used to prevent or treat dysfunction in the endogenous opiate system that are associated with opiate addiction and/or BPD.
  • transcription factors comprising DNA binding domains specific for OPRM1 may be used to repress the expression of dysfunctional receptors in opiate addicts and BPD patients.
  • engineered nucleases may be used to knock out or correct specific genes encoding mutant receptors such as the Al 18G OPRM1 variant.
  • Nucleic acids encoding these engineered proteins may be delivered to the brain via viral delivery systems.
  • wild type genes encoding MOP-r may be delivered to specific areas of the brain known to be highly active in the pathology of addiction and/or BPD and inserted into the genome via nuclease targeted integration to treat or prevent these disorders.
  • stem cells corrected by the methods of the invention can be introduced into the brain that are associated with addiction and/or BPD to treat or prevent these conditions.
  • MDD Major depressive disorder
  • SLC6A4 serotonin transporter
  • HTR2A serotonin receptor 2A
  • BDNF brain derived neurotrophic factor
  • TPH2 tryptophan hydroxylase
  • the methods and compositions of the invention may be used to prevent or treat MDD in a patient.
  • Mutant alleles in SLC6A4, HTR2A and/or TPH2 may be targeted by the engineered transcription factors or nucleases of the invention to repress or knock out expression. Wild type genes for these loci may also be introduced via nuclease-dependent targeted integration.
  • stem cells corrected by the methods of the invention can be introduced into the brain. Corrective measures (i.e. engineered transcription factors, nucleases, donor DNAs, stem cells) can be targeted in areas of the brain that are associated with MDD.
  • Schizophrenia does not appear to be linked to a single gene, yet while it occurs in 1% of the general population, a family history of psychosis increases the risk to about 10% of people with a parent or sibling with SZ. Twin studies have also shown that an identical twin has about a 50% chance of having SZ if their twin has it. Overall, 80% of SZ patients appear to have a genetic component to their disease. Early studies showed evidence of SNPs in the DTNBP1 (dystrobrevin- binding protein 1 or dysbindin) were strongly associated with SZ (Straub et al (2002) Am J Hum Genet 71 :337-348).
  • neutrotransmission and synaptic plasticity e.g. GRM3, GRIN2A, SRR, GRIAl. Associations were also seen with voltage gated calcium channel subunits (e.g.
  • SZ also may be associated with epigenetic regulation.
  • GADl glutamic acid decarboxylase67
  • RELN reelin
  • DNMT1 and TETl encode enzymes that methylate and hydroxymethylate cytosines near and within cytosine phosphodiester guanine (CpG) islands of many gene promoters.
  • CpG cytosine phosphodiester guanine
  • nucleases of the invention may be designed to target disease associated SNPs such as in DTNBP1 to knock out disease associated alleles. Further, specific transcription factors and/or nucleases can be engineered to modulate specific alleles of DRD2, GRM3, GRIN2A, SRR, GRIAl, CACNA1C, CACNB2, CACN1 II, GADl, RELN, BDNF, TET1, and DTNBP1. Nucleic acids encoding these modulators can be injected into the brain in the regions of the brain known to be most affected by SZ, or they can be delivered via viral vectors. Similarly, stem cells corrected by the methods of the invention can be introduced into the brain and used to treat or prevent SZ.
  • Bipolar disorder is another extremely debilitating psychiatric disorder affecting about 1% of the population worldwide, and is associated with increased morbidity and mortality that appears to have a strong genetic component.
  • BD is associated with the highest risk of suicide and is characterized by cyclothymic and irritable temperaments, leading to a rate of suicide that is up to 20 times that of the average population (see Dwivedi and Zhang (2015) Front Neurosci 8(457)).
  • Genome wide association studies have been performed and have identified a number of SNPs in specific chromosomal regions in the genome.
  • BDNF expression also seems to play a role in BD.
  • BD patients often respond favorably to therapeutic lithium treatment, and it appears that one of the mechanisms of lithium is to cause hypomethylation of the BDNF exon IV promoter resulting in increased BDNF expression.
  • compositions of the invention can be used to prevent or treat BD.
  • Specific nucleases of the invention may be designed to target disease associated SNPs such as in ANK3, ODZ4, TRANK1 , ADCY2, and CACNA1 C to knock out disease associated alleles. Further, specific transcription factors can be engineered to modulate and increase the expression of BDNF. Nucleic acids encoding these modulators or stem cells corrected by the methods of the invention can be introduced into the brain in the regions of the brain known to be most affected by BD, or they can be delivered via viral vectors.
  • Migraine is the most common disorder in the NS, affecting
  • the methods and compositions of the invention may be used to prevent or treat chronic migraine in patients.
  • Engineered transcription factors and nucleases may be designed to repress or knockout mutant genes known to induced recurrent migraine.
  • wild type genes may be inserted in to safe harbor loci of cells in the brain via nuclease dependent targeted integration to cause expression of wild type proteins.
  • stem cells corrected by the methods of the invention can be introduced into the brain in the areas know to be associated with migraine to treat or prevent the disease.
  • MS multiple sclerosis
  • the methods and compositions of the invention may be used to prevent or treat MS.
  • Engineered transcription factors and/or nucleases can be used to repress or knockout mutant alleles associated with MS such as HLA-DRB1 * 1501.
  • wild type genes can be inserted into the genome via nuclease assisted targeted integration in safe harbor genes where the genes and nucleases are delivered to the brain in the regions most associated with MS phenotype.
  • Stem cells corrected by the methods of the invention can also be used. Delivery can be via direct introduction of cells, or via viral vectors.
  • Tourette syndrome is a neuropsychiatric disorder characterized by repetitive, involuntary movements and vocalizations called tics and is often accompanied by obsessive-compulsive disorder and/or attention-deficit/hyperactivity disorder.
  • tics repetitive, involuntary movements and vocalizations
  • tics is often accompanied by obsessive-compulsive disorder and/or attention-deficit/hyperactivity disorder.
  • TS Tourette syndrome
  • NRXN 1 The five loci were in the NRXN 1 , AADAC, CTNNA3 , FSCB and KCHE 1 , KCHE2, RCAN 1 genes.
  • a deletion of the 5' exon of the neurexin 1 gene (NRXN1) is associated with TS (also with autism, schizophrenia) as is deletion of the 5' region of microsomal arylacetamide deacetylase (AADAC). Deletion of 450 Kb of the FSCB gene
  • TS encoding fibrous sheath CABYR-binding protein
  • KCNE 1 voltage-gated potassium channels
  • RCANl regulator of calcineutin 1
  • Some of these genes encode cell adhesion molecules (e.g. NRXN1) which may play a role in linking synaptic cell adhesion to cognition (Sundaram et al (2010) Neurology 74(20):1583).
  • NRXN1 cell adhesion molecules
  • the methods and compositions of the invention can be used to prevent or treat TS.
  • Engineered transcription factors and/or nucleases can be used to repress or knockout mutant alleles associated with TS such as those in NRXN1, AADAC, CTNNA3, FSCB and KCHEl, KCHE2, RCANl .
  • wild type genes can be inserted into the genome via nuclease assisted targeted integration in safe harbor genes or corrected stem cell, where the genes, nucleases and/or stem cells are delivered to the brain in the regions most associated with TS phenotype. Delivery can be via direct introduction or via viral vectors.
  • Epilepsy affects about three percent of the population and comprises a wide constellation of primary and syndromic neurological disorders.
  • the epileptic encephalopathies (EE) are the most severe of the epilepsies, distinguished by multiple refractory seizures, cognitive deficit, and poor developmental outcome. De novo mutations in several genes are known causes of EE, but the genetic etiology of the vast majority of these encephalopathies is unknown. People with epilepsy tend to suffer from recurrent seizures due to excessive neuronal discharge. It appears that genetic factors play a role in 40% of epilepsies, however it has been difficult to identify the specific genes involved.
  • CAMSAP1L1 encodes a cytoskeletal protein (Tan et al, 2014, ibid).
  • Some approaches to epilepsy embrace the idea of using gene therapy to treat the symptoms of the disease such as anti-seizure approaches ⁇ e.g. to decrease the effect of excitatory neurotransmitters such as antisense approaches against NMDA receptor subunit 1 , or increase the inhibition of neurotransmission such as GAMA-A receptor subunit alpha- 1, GAD65 or adenosine kinase) or disease modifying approaches.
  • NPY neuropeptides and as galanin and neuropeptide Y (NPY) could be promising candidates for such a purpose ⁇ see Noe et al, 2012. Noebels et al, editors. Jaspar's Basic Mechanisms of the Epilepsies. 4 th Edition. Bethesda (MD): National Center for Biotechnology
  • the methods and compositions of the invention can be used to prevent or treat epilepsy.
  • Approaches that are helpful to decrease excitatory neurotransmitters can be carried out where the engineered transcription factors and/or nucleases of the invention are used to repress or knock out NMDA receptor subunit 1 , GAMA-A receptor subunit alpha- 1, GAD65 or adenosine kinase.
  • Other uses of these methods and compositions can be to repress or knock out specific mutant alleles associated with SNPs in the CAMSAP1LK1 gene.
  • genes encoding transcription factors to increase the expression of GCNF, BDNF and IGF may be administered in a localized fashion at the seizure centers within the brain.
  • cDNAs encoding anti-convulsant peptides such as NPY and galanin can be inserted via nuclease driven targeted integration in to seizure centers to inhibit the frequency and magnitude of seizures.
  • alterations to genes encoding proteins involved in NS disorders using the methods and compositions as described herein can be used to correct an aberrant endogenous gene or insert a wild type gene at a desired location in the genome of a cell ⁇ e.g., into a "safe harbor" gene of an HSC).
  • Precursor cells can be derived from subjects in need, modified ex vivo, and then given back to the subject either in a bone marrow graft.
  • polynucleotides encoding the fusion proteins of the invention can be delivered to the neural tissue for correction, modification of expression, gene silencing and/or targeted insertion of a gene for the treatment or prevention of a nervous system disorder.
  • 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,
  • nucleic acid refers to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double-stranded form.
  • polynucleotide refers to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double-stranded form.
  • 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).
  • an analogue of a particular nucleotide has the same base-pairing specificity; i.e., an analogue of A will base-pair with T.
  • polypeptide peptide
  • protein protein
  • amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of corresponding naturally-occurring amino acids.
  • 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 (Kj) of 10 ⁇ 6 M “1 or lower.
  • Kj dissociation constant
  • Affinity refers to the strength of binding:
  • 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).
  • 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.
  • 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.
  • 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.
  • Zinc finger and TALE 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 or TALE protein. Therefore, engineered DNA binding proteins (zinc fingers or
  • TALEs are proteins that are non-naturally occurring.
  • methods for engineering DNA-binding proteins are design and selection.
  • a designed DNA binding protein is a protein not occurring in nature whose design/composition results principally from rational criteria. Rational criteria for design include
  • 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. Patent Nos. 8,586,526; 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,200,759; 6,242,568; 6,733,970;7,297,491;
  • 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, for example, guide DNAs for cleavage by a TtAgo enzyme.
  • Recombination refers to a process of exchange of genetic information between two polynucleotides, including but not limited to, donor capture by non-homologous end joining (NHEJ) and homologous recombination.
  • NHEJ non-homologous end joining
  • 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.
  • 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.
  • homologous recombination 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.
  • 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 re-synthesize 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.
  • 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.
  • a "donor" polynucleotide having homology to the nucleotide sequence in the region of the break
  • 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.
  • a first sequence in cellular chromatin can be altered and, in certain embodiments, can be converted into a sequence present in a donor polynucleotide.
  • 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.
  • additional pairs of zinc-finger or TALEN proteins can be used for additional double-stranded cleavage of additional target sites within the cell.
  • a CRISPR/Cas system may be similarly employed to induce additional double strand breaks.
  • a chromosomal sequence is altered by homologous recombination with an exogenous "donor" nucleotide sequence.
  • 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.
  • the exogenous nucleotide 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the donor sequence is non-homologous to the first sequence, and is inserted into the genome by non-homologous recombination mechanisms.
  • exogenous nucleic acid sequence can comprise, for example, one or more genes or cDNA
  • the exogenous nucleic acid sequence may produce one or more R A molecules (e.g., small hairpin R As (shR As), inhibitory RNAs (RNAis), microRNAs (miRNAs), etc.).
  • R A molecules e.g., small hairpin R As (shR As), inhibitory RNAs (RNAis), microRNAs (miRNAs), etc.
  • 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.
  • a "cleavage half-domain” is a polypeptide sequence which, in
  • 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.
  • 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 and 8,823,618, incorporated herein by reference in their entireties.
  • 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.
  • donor sequence refers to a nucleotide sequence
  • 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.
  • a "NS-disease associated gene” is one that is defective in some
  • 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.
  • chromatin is meant to encompass all types of cellular nucleoprotein, both prokaryotic and eukaryotic. Cellular chromatin includes both chromosomal and episomal chromatin.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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,
  • 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.
  • an exogenous molecule can be the same type of molecule as an endogenous molecule, e.g., an exogenous protein or nucleic acid.
  • 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.
  • 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.
  • 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.
  • a human nucleic acid sequence may be introduced into a cell line originally derived from a mouse or hamster.
  • an "endogenous" molecule is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions.
  • 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.
  • 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 DNA-binding domain (e.g., ZFP, TALE and/or meganuclease DNA-binding domains) and a functional domain (e.g., endonuclease, meganuclease, ZFP-transcription factor, (ZFP-TF), TALE- transcription factor (TALE-TF), CRIPSR/Cas transcription factor (CRISPR/Cas-TF) etc.) and fusion nucleic acids (for example, a nucleic acid encoding a fusion protein described supra).
  • a DNA-binding domain e.g., ZFP, TALE and/or meganuclease DNA-binding domains
  • a functional domain e.g., endonuclease, meganuclease, ZFP-transcription factor, (ZFP-TF), TALE- transcription factor (TALE-TF
  • 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.
  • 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.
  • Gene expression refers to the conversion of the information, contained in a gene, into a gene product.
  • a gene product can be the direct
  • 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.
  • 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 or TALE. . Thus, gene inactivation may be partial or complete.
  • 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.
  • 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).
  • 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.
  • a transcriptional regulatory sequence such as a promoter
  • a transcriptional regulatory sequence 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.
  • transcriptional regulatory sequence is generally operatively linked in cis with a coding sequence, but need not be directly adjacent to it.
  • an enhancer is a transcriptional regulatory sequence that is operatively linked to a coding sequence, even though they are not contiguous.
  • 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.
  • a fusion polypeptide in which a DNA-binding domain (ZFP, TALE) is fused to a cleavage domain e.g., endonuclease domain such as Fokl, meganuclease domain, etc.
  • 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 (nuclease) domain is able to cleave DNA in the vicinity of the target site.
  • the nuclease domain may also exhibit DNA-binding capability (e.g., a nuclease fused to a ZFP or TALE domain that also can bind to DNA).
  • DNA-binding capability e.g., a nuclease fused to a ZFP or TALE domain that also can bind to DNA.
  • 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 up regulate gene expression or the repression domain is able to down regulate gene expression.
  • a fusion polypeptide in which a Cas DNA-binding domain is fused to an activation domain the Cas DNA-binding domain and the activation domain are in operative linkage if, in the fusion polypeptide, the Cas 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.
  • the Cas DNA-binding domain and the cleavage domain are in operative linkage if, in the fusion polypeptide, the Cas 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.
  • 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.
  • 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. (1989) Nature 340:245-246; U.S. Patent No. 5,585,245 and PCT WO 98/44350.
  • a "vector" is capable of transferring gene sequences to target cells.
  • vector construct means any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells.
  • vector construct means any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells.
  • the term includes cloning, and expression vehicles, as well as integrating vectors.
  • subject and patient are used interchangeably and refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, dogs, cats, rats, mice, rabbits and other animals. Accordingly, the term “subject” or “patient” as used herein means any patient or subject (e.g., mammalian) having a NS disorder.
  • compositions for example transcriptional regulators and/or nucleases, which are useful targeting a gene that encodes a protein involved in a NS disorder, for example nucleases that facilitate targeted correction of a mutant gene, targeted inactivation of a gene and/or targeted integration (e.g., of a gene encoding a protein that is aberrantly expressed in the subject with the NS disorder).
  • the compositions can comprise fusion proteins, for example DNA-binding domains fused to functional domains (e.g., transcriptional activation domains, transcriptional repression domains and/or nucleases) or nuclease or transcription factor systems such as the CRISPR/Cas system.
  • compositions comprising cells that are genetically modified using the proteins and systems described herein and methods of using these cell compositions to treat and/or prevent a nervous system disorder.
  • Any DNA-binding domain can be used in the nucleases used in the compositions and methods disclosed herein, including but not limited to a zinc finger DNA-binding domain, a TALE DNA binding domain, or a DNA-binding domain from a meganuclease, or a CRIPSR/Cas DNA binding complex.
  • the composition comprises a DNA-binding domain and/or nuclease (cleavage) domain from a meganuclease (homing
  • 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-Cyst box family and the HNH family.
  • Exemplary homing endonucleases include l-Scel, l-Ceul, Fl-Pspl, Fl-Sce, 1-SceTV, I- Csml, l-Panl, l-Scell, l-Ppol, l-Scelll, l-Crel, l-Tevl, l-Tevll and I-73 ⁇ 4vIII.
  • 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 i?t3 ⁇ 4.25:3379-3388; Dujon et a/.
  • the homing endonuclease (meganuclease) is engineered (non-naturally occurring).
  • the recognition sequences of homing endonucleases and meganucleases such as l-Scel, l-Ceul, Fl-Pspl, Fl-Sce, l-SceTV, I- Csml, l-Panl, l-Scell, l-Ppol, 1-SceIII, l-Crel, l-Tevl, l-Tevll and I- ZevIII are known. See also U.S. Patent No. 5,420,032; U.S. Patent No.
  • DNA- binding specificity of homing endonucleases and meganucleases can be engineered to bind non-natural target sites. See, for example, U.S. Patent No. 8,021,867; Chevalier et al. (2002) Molec. Cell 10:895-905; Epinat et a/. (2003) Nucleic Acids Rey.31:2952- 2962; Ashworth et al. (2006) Nature 441:656-659; and Paques et al. (2007) Current Gene Therapy! :49-66.
  • DNA-binding domains of the homing endonucleases and meganucleases may be altered in the context of the nuclease as a whole (i.e., such that the nuclease includes the cognate cleavage domain) or may be fused to a heterologous DNA-binding domain (e.g., zinc finger protein or TALE) or to a heterologous cleavage domain.
  • DNA-binding domains derived from meganucleases may also exhibit DNA-binding activity.
  • the DNA-binding domain comprises a naturally occurring or engineered (non-naturally occurring) TAL effector DNA binding domain.
  • TAL effector DNA binding domain comprises a naturally occurring or engineered (non-naturally occurring) TAL effector DNA binding domain.
  • T3S conserved type III secretion
  • TALE transcription activator-like effectors
  • TALEs contain a DNA binding domain and a transcriptional activation domain.
  • 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).
  • 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) Appland 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 .
  • the DNA binding domain that binds to a target site in a target locus is 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
  • the DNA binding domain comprises a zinc finger protein ⁇ e.g., a zinc finger protein that binds to a target site in a gene involved in a NS disorder or safe-harbor gene).
  • the zinc finger protein is non- naturally occurring in that it is engineered to bind to a target site of choice. See, for example, See, for example, Beerli et al. (2002) Nature Biotechnol. 20: 135-141; Pabo et al. (2001) Ann. Rev. Biochem 0:3 l3-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr. Opin.
  • An engineered zinc finger binding or TALE domain can have a novel binding specificity, compared to a naturally-occurring zinc finger protein.
  • 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, co-owned U.S. Patents 8,586,526; 6,453,242 and 6,534,261 , incorporated by reference herein in their entireties.
  • Exemplary selection methods including phage display and two-hybrid systems, are disclosed in U.S. Patents 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;
  • telomere sequences 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 DNA binding proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein.
  • enhancement of binding specificity for zinc finger binding domains has been described, for example, in co-owned WO 02/077227.
  • WO 98/5431 1 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.
  • DNA-binding domains 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.
  • the DNA binding domain comprises a single-guide R A in combination with a CRISPR/Cas nuclease system or a
  • the DNA-binding domains may be operably linked to any functional domain.
  • the functional domain comprises a
  • 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.,
  • 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 al. (2000) Mol. Endocrinol. 14:329-347; Collingwood et al. (1999) J. Mol.
  • Additional exemplary activation domains include, but are not limited to, OsGAI, HALF-1, CI, API, ARF-5,-6,-7, and -8,
  • a fusion protein (or a nucleic acid encoding same) between a DNA-binding domain as described herein and a functional domain
  • an activation domain or a molecule that interacts with an activation domain is suitable as a functional domain.
  • 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 co-owned U.S. Patent Nos. 6,919,204 and 7,053,264.
  • Exemplary repression domains include, but are not limited to, KRAB
  • 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.
  • the target site bound by the DNA-binding domain is present in an accessible region of cellular chromatin. Accessible regions can be determined as described, for example, in U.S. Patent No. 6,511 ,808. 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 co-owned WO 01/83793.
  • 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.
  • the functional (regulatory) domain comprises a nuclease (cleavage) domain.
  • Any suitable cleavage domain can be operatively linked to any DNA-binding domain to form a nuclease.
  • ZFP DNA-binding domains have been fused to nuclease domains to create ZFNs - a functional entity that is able to recognize its intended nucleic acid target through its engineered (ZFP) DNA binding domain and cause the DNA to be cut near the ZFP binding site via the nuclease activity. See, e.g., Kim et al. (1996) Proc N at ⁇ Acad Sci USA 93(3):1156- 1160. See, for example, U.S. Patent Nos. 7,888,121 ; 7,972,854; 7,914,796;
  • TALE DNA-binding domains have been fused to nuclease domains to create TALENs. See, e.g., U.S. Patent No. 8,586,526.
  • the cleavage domain may be heterologous to the
  • DNA-binding domain for example a zinc finger DNA-binding domain and a cleavage domain from a nuclease or a TALEN DNA-binding domain and a cleavage domain, or 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.
  • the nuclease is naturally occurring.
  • the nuclease is non-naturally occurring, i.e., engineered in the DNA- binding domain and/or cleavage domain.
  • 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).
  • the nuclease comprises heterologous DNA- binding and cleavage domains (e.g., zinc finger nucleases; TAL-effector nucleases; meganuclease DNA-binding domains with heterologous cleavage domains), or a generic nuclease guided by a specific guide RNA (e.g. a CRPISR/Cas).
  • heterologous DNA- binding and cleavage domains e.g., zinc finger nucleases; TAL-effector nucleases; meganuclease DNA-binding domains with heterologous cleavage domains
  • a generic nuclease guided by a specific guide RNA e.g. a CRPISR/Cas
  • a cleavage half-domain can be derived from any nuclease or portion thereof, as set forth above, that requires dimerization for cleavage activity.
  • two fusion proteins are required for cleavage if the fusion proteins comprise cleavage half-domains.
  • 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).
  • 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.
  • the near edges of the target sites are separated by 5-8 nucleotides or by 15-18 nucleotides.
  • any integral number of nucleotides or nucleotide pairs can intervene between two target sites (e.g., from 2 to 50 nucleotide pairs or more).
  • the site of cleavage lies between the target sites.
  • a Cas protein may be linked to a heterologous nuclease domain.
  • the Cas protein is a Cas9 protein devoid of nuclease activity linked to a Fokl nuclease domain such that double strand cleavage is dependent on dimerization of the Fokl nuclease domains.
  • Restriction endonucleases 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
  • 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.
  • 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.
  • 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. USA95: 10,570-10,575.
  • the portion of the Fok I enzyme used in the disclosed fusion proteins is considered a cleavage half-domain.
  • two fusion proteins, each comprising a Fokl cleavage half-domain can be used to reconstitute a catalytically active cleavage domain.
  • a single polypeptide molecule containing a DNA binding domain and two Fok I cleavage half-domains can also be used.
  • 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.
  • 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. 7,888,121; 8,409,861; 7,914,796; and 8,034,598, 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.
  • 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.
  • a mutation at 490 replaces Glu (E) with Lys
  • 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 Nos. 7,888,121; 8,409,861; 7,914,796; and 8,034,598 and U.S. Patent Publication No. 20120040398, the disclosures of which are incorporated by reference in their entireties for all purposes. .
  • 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).
  • 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 "KK " and "KKR” domains, respectively).
  • 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).
  • E wild type Glu
  • H His
  • R Arg
  • Engineered cleavage half-domains described herein can be prepared using any suitable method, for example, by site-directed mutagenesis of wild-type cleavage half-domains (Fok l) as described in U.S. Patent Nos. 7,888,121; 7,914,796 and 8,034,598.
  • nucleases may be assembled in vivo at the nucleic acid target site using so-called “split-enzyme” technology (see, e.g., U.S. Patent
  • 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.
  • the nuclease domain may also be derived from a homing endonuclease
  • exemplary homing endonucleases include l-Scel, l-Ceul, Fl-Pspl, ⁇ -Sce, I-SceW, l-Csml, l-Panl, l-Scell, l-Ppol, l-Scelll, l-Crel, I-Tevl, l-Tevll and I- Tevlll.
  • nuclease as described herein can comprise any DNA-binding domain and any nuclease.
  • the nuclease comprises a zinc finger DNA- binding domain and a restriction endonuclease nuclease domain, also referred to as a zinc finger nuclease (ZFN).
  • ZFN zinc finger nuclease
  • the nuclease comprises an engineered TALE
  • DNA-binding domain and a nuclease domain ⁇ e.g., endonuclease and/or
  • the TALEN comprises an endonuclease ⁇ e.g., Fokl) cleavage domain or cleavage half-domain.
  • 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.
  • the nuclease comprises a compact
  • TALEN cTALEN
  • TALENs may be used in combination with additional TALENs ⁇ e.g., one or more TALENs (cTALENs or Fokl-TALENs) with one or more mega-TALs).
  • Nucleases can be screened for activity prior to use, for example in a yeast-based chromosomal system as described in U.S. Patent No. 8,563,314. Nuclease expression constructs can be readily designed using methods known in the art. See, e.g., United States Patent Nos. 7,888,121 and 8,409,861; 20030232410;
  • Expression of the nuclease may be under the control of a constitutive promoter or an inducible promoter, for example the galactokinase promoter which is activated (de-repressed) in the presence of raffinose and/or galactose and repressed in presence of glucose.
  • a constitutive promoter or an inducible promoter for example the galactokinase promoter which is activated (de-repressed) in the presence of raffinose and/or galactose and repressed in presence of glucose.
  • the nuclease or transcription factor comprises a CRISPR/Cas system. See, e.g., U.S. Patent No. 8,697,359.
  • the CRISPR (clustered regularly interspaced short palindromic repeats) locus which encodes RNA components of the system
  • 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.
  • 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.
  • the Type II CRISPR is one of the most well characterized systems and carries out targeted DNA double-strand break in four sequential steps.
  • 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.
  • the tracrRNA must also be present as it base pairs with the crRNA at its 3' end, and this association triggers Cas9 activity.
  • 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.
  • '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.
  • Type II CRISPR systems have been found in many different bacteria.
  • Cas9 refers to an RNA guided DNA nuclease comprising a DNA binding domain and two nuclease domains, where the gene encoding the Cas9 may be derived from any suitable bacteria.
  • the Cas9 protein has at least two nuclease domains: one nuclease domain is similar to a HNH endonuclease, while the other resembles a Ruv endonuclease domain.
  • the HNH-type domain appears to be responsible for cleaving the DNA strand that is complementary to the crRNA while the Ruv domain cleaves the non-complementary strand.
  • the Cas 9 nuclease can be engineered such that only one of the nuclease domains is functional, creating a Cas nickase (see Jinek et al, ibid).
  • Nickases can be generated by specific mutation of amino acids in the catalytic domain of the enzyme, or by truncation of part or all of the domain such that it is no longer functional. Since Cas 9 comprises two nuclease domains, this approach may be taken on either domain. A double strand break can be achieved in the target DNA by the use of two such Cas 9 nickases. The nickases will each cleave one strand of the DNA and the use of two will create a double strand break.
  • sgRNA single-guide RNA
  • tracrRNA: crRNA fusion guides the functional domain (e.g., Cas9 or other functional domain) to modify the target DNA, for example when a double strand RNA:DNA heterodimer forms between the Cas associated RNAs and the target DNA in the case of a nuclease.
  • This system comprising the Cas9 protein and an engineered sgRNA containing a PAM sequence has been used for RNA guided genome editing (see Ramalingam ibid ) and has been useful for zebrafish embryo genomic editing in vivo (see Hwang et al (2013) Nature Biotechnology 31 (3):227) with editing efficiencies similar to ZFNs and TALENs.
  • Chimeric or sgRNAs can be engineered to comprise a sequence complementary to any desired target.
  • a guide sequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length.
  • a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length.
  • the RNAs comprise 22 bases of complementarity to a target and of the form G[nl9], followed by a protospacer-adjacent motif (PAM) of the form NGG.
  • PAM protospacer-adjacent motif
  • sgRNAs can be designed by utilization of a known ZFN target in a gene of interest by (i) aligning the recognition sequence of the ZFN heterodimer with the reference sequence of the relevant genome (human, mouse, or of a particular plant species); (ii) identifying the spacer region between the ZFN half-sites; (iii) identifying the location of the motif G[N20]GG that is closest to the spacer region (when more than one such motif overlaps the spacer, the motif that is centered relative to the spacer is chosen); (iv) using that motif as the core of the sgRNA.
  • This method advantageously relies on proven nuclease targets.
  • sgRNAs can be designed to target any region of interest simply by identifying a suitable target sequence the conforms to the G[n20]GG formula.
  • an sgRNA may comprise additional nucleotides to extend to tail region of the tracrRNA portion of the sgRNA (see Hsu et al (2013) Nature Biotech doi: 10.1038/nbt.2647). Tails may be of +67 to +85 nucleotides, or any number therebetween with a preferred length of +85 nucleotides.
  • Truncated sgRNAs may also be used, "tru-gRNAs" (see Fu et al, (2014) Nature Biotech 32(3): 279). In tru-gRNAs, the complementarity region is diminished to 17 or 18 nucleotides in length.
  • PAM sequence can be NAG as an alternative to NGG (Hsu 2014, ibid) using a S. pyogenes Cas9. Additional PAM sequences may also include those lacking the initial G (Sander and Joung (2014) Nature Biotech 32(4):347). In addition to the S.
  • PAM sequences can be used that are specific for Cas9 proteins from other bacterial sources.
  • PAM sequences shown below are specific for these Cas9 proteins:
  • CRISPR/Cas system can be chosen according to the following guideline: [nl7, nl8, nl9, or n20](G/A)G.
  • the PAM sequence can follow the guideline G[nl7, nl8, nl9, n20](G/A)G.
  • G[nl7, nl8, nl9, n20](G/A)G For Cas9 proteins derived from non-S. pyogenes bacteria, the same guidelines may be used where the alternate PAMs are substituted in for the S. pyogenes PAM sequences.
  • a target sequence with the highest likelihood of specificity that avoids potential off target sequences can be identified by considering the following attributes: i) similarity in the target sequence that is followed by a PAM sequence known to function with the Cas9 protein being utilized; ii) a similar target sequence with fewer than three mismatches from the desired target sequence; iii) a similar target sequence as in ii), where the mismatches are all located in the PAM distal region rather than the PAM proximal region (there is some evidence that nucleotides 1-5 immediately adjacent or proximal to the PAM, sometimes referred to as the 'seed' region (Wu et al (2014) Nature Biotech doi: 10.1038/nbt2889) are the most critical for recognition, so putative off target sites with mismatches located in the seed region may be the least likely be recognized by the sg RNA); and iv) a similar target sequence where the mismatch
  • 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.
  • a functional derivative may comprise a single biological property of a naturally occurring Cas protein.
  • a function derivative may comprise a subset of biological properties of a naturally occurring Cas protein. Suitable derivatives of a Cas polypeptide or a fragment thereof include but are not limited to mutants, fusions, covalent
  • 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.
  • the cell does not naturally produce Cas protein and is genetically engineered to produce a Cas protein.
  • the nuclease comprises a DNA-binding domain in that specifically binds to a target site in any gene into which it is desired to insert a donor (transgene) in combination with a nuclease domain that cleaves DNA.
  • DNA-binding domains e.g., ZFPs
  • TALEs single-guide RNAs
  • TALEs single-guide RNAs
  • the DNA-binding domains bind to a sequence in a gene encoding a protein that is involved in a NS disorder.
  • genes involved in NS disorders include microtubule associated protein tau (MAPT, encoding Tau proteins), apolipoprotein E (APOE) alleles(e.g., apoE2, apoE3 or apoE4), amyloid precursor protein (APP), presenilin 1, presenilin 2, SLC6A4, HTR2A, CACNAIC, CACNB2, dystrophia myotonica-protein kinase (DMPK), CACNA1A, ATP1A2, SCN1A; FXN; HTT, PMP22, dystrophin, utrophin; C9orf72, SODl, TARDBP, FUS, ANG, ALS
  • FHM5 FHM5, MMP16, ASTN2, of WT transgene, TSPAN2, GFRA2, LRP1 corrected stem cell
  • AMSAP1K1 NMDA Transcriptional receptor 1
  • GAMA-A modulation KO of
  • GAD1 GAD1, RELN, BDNF, stem cell
  • the DNA-binding domain binds to a sequence in a safe -harbor gene.
  • safe harbor genes include, for example, a CCR5 gene, a CXCR4 gene, an HPRT gene, a PPP1R12C (also known as AAVSl) gene, an albumin gene or a Rosa gene. See, e.g., U.S. Patent Nos. 7,951,925 and 8,110,379; U.S. Publication Nos. 20080159996; 201000218264; 20100291048; 20120017290; 20110265198; 20130137104; 20130122591;
  • donor sequence or “donor” or “transgene”
  • donor sequence for example for correction of a mutant gene or for increased expression of a wild-type gene. It will be readily apparent that the donor sequence need not be identical to the genomic sequence where it is placed.
  • a donor sequence can contain a non-homologous sequence flanked by two regions of homology to allow for efficient HDR at the location of interest.
  • donor sequences can comprise a vector molecule containing sequences that are not homologous to the region of interest in cellular chromatin.
  • a donor molecule can contain several, discontinuous regions of homology to cellular chromatin.
  • sequences not normally present in a region of interest can be present in a donor nucleic acid molecule and flanked by regions of homology to sequence in the region of interest.
  • a donor molecule may be integrated into a cleaved target locus via non-homologous end joining (NHEJ) mechanisms.
  • NHEJ non-homologous end joining
  • polynucleotides for insertion into a chosen location Polynucleotides for insertion can also be referred to as "exogenous" polynucleotides, "donor” polynucleotides or molecules or “transgenes.”
  • the donor polynucleotide can be DNA or RNA, single- stranded and/or double-stranded and can be introduced into a cell in linear or circular form. See, e.g., U.S. Patent Publication Nos. 20100047805; 20110281361; and 20110207221.
  • the donor sequence(s) can be contained within a DNA MC, which may be introduced into the cell in circular or linear form. If introduced in linear form, the ends of the donor sequence can be protected ⁇ e.g., from exonucleo lytic
  • one or more dideoxynucleotide residues are added to the 3' terminus of a linear molecule and/or self-complementary oligonucleotides are ligated to one or both ends. See, for example, Chang et al. (1987) Proc. Natl. Acad. Sci. t/&484:4959-4963; Nehls et al. (1996) Science 272:886-889.
  • Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified internucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues.
  • a polynucleotide can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance.
  • donor polynucleotides can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by viruses ⁇ e.g. , adenovirus, AAV, herpesvirus, retrovirus, lentivirus and integrase defective lentivirus (IDLV)).
  • the double-stranded donor includes sequences ⁇ e.g., coding sequences, also referred to as transgenes) greater than 1 kb in length, for example between 2 and 200 kb, between 2 and 10 kb (or any value therebetween).
  • the double-stranded donor also includes at least one nuclease target site, for example.
  • the donor includes at least 1 target site, for example, for use with a CRISPR/Cas, or 2 target sites, for example for a pair of ZFNs and/or TALENs.
  • the nuclease target sites are outside the transgene sequences, for example, 5' and/or 3' to the transgene sequences, for cleavage of the transgene.
  • the nuclease cleavage site(s) may be for any nuclease(s).
  • the nuclease target site(s) contained in the double-stranded donor are for the same nuclease(s) used to cleave the endogenous target into which the cleaved donor is integrated via homo logy-independent methods.
  • the donor is generally inserted so that its expression is driven by the endogenous promoter at the integration site, namely the promoter that drives expression of the endogenous gene into which the donor is inserted.
  • the donor may comprise a promoter and/or enhancer, for example a constitutive promoter or an inducible or tissue specific promoter.
  • the donor molecule may be inserted into an endogenous gene such that all, some or none of the endogenous gene is expressed.
  • a transgene as described herein may be inserted into a selected locus such that some or none of the endogenous sequences are expressed, for example as a fusion with the transgene.
  • the transgene is integrated into any endogenous locus, for example a safe-harbor locus.
  • exogenous sequences may also include transcriptional or translational regulatory sequences, for example, promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides and/or polyadenylation signals.
  • the transgenes carried on the donor sequences described herein may be isolated from plasmids, cells or other sources using standard techniques known in the art such as PCR.
  • Donors for use can include varying types of topology, including circular supercoiled, circular relaxed, linear and the like. Alternatively, they may be chemically synthesized using standard oligonucleotide synthesis techniques. In addition, donors may be methylated or lack methylation.
  • Donors may be in the form of bacterial or yeast artificial chromosomes (BACs or YACs).
  • the double-stranded donor polynucleotides described herein may include one or more non-natural bases and/or backbones.
  • insertion of a donor molecule with methylated cytosines may be carried out using the methods described herein to achieve a state of transcriptional quiescence in a region of interest.
  • exogenous (donor) polynucleotide may comprise any sequence of interest (exogenous sequence).
  • exogenous sequences include, but are not limited to any polypeptide coding sequence (e.g., cDNAs), promoter sequences, enhancer sequences, epitope tags, marker genes, cleavage enzyme recognition sites and various types of expression constructs.
  • Marker 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.
  • the exogenous sequence comprises a polynucleotide encoding any polypeptide of which expression in the cell is desired, including, but not limited to any polypeptide involved in a NS disorder, antibodies, antigens, enzymes, receptors (cell surface or nuclear), hormones, lymphokines, cytokines, reporter polypeptides, growth factors, and functional fragments of any of the above.
  • the coding sequences may be, for example, cDNAs.
  • Non-limiting examples of polypeptides that may be encoded by the exogenous (donor) sequences include growth factors (e.g., growth hormone, insulin-like growth factor- 1, platelet-derived growth factor, epidermal growth factor, acidic and basic fibroblast growth factors, transforming growth factor-(3, etc.), to treat growth disorders or wasting syndromes; and antibodies (e.g., human or humanized), to provide passive immunization or protection of a subject against foreign antigens or pathogens (e.g., H. Pylori), or to provide treatment of cancer, arthritis or
  • cytokines interferons (e.g., interferon (INF), INF-a2b and 2a, INF-aNl, INF-(31b, INF-gamma), interleukins (e.g., IL-1 to IL 10), tumor necrosis factor (TNF-a TNF-R), chemokines, granulocyte macrophage colony stimulating factor (GM-CSF), polypeptide hormones, antimicrobial polypeptides (e.g., antibacterial, antifungal, antiviral, and/or antiparasitic polypeptides), enzymes (e.g., adenosine deaminase), gonadotrophins, chemotactins, lipid-binding proteins, filgastim (Neupogen), hemoglobin, erythropoietin, insulinotropin, imiglucerase, sarbramostim, tissue plasminogen activator (WA), urokinase, streptokin
  • PTH protein-like, hormone, soluble CD4, and antibodies and/or antigen-binding fragments (e.g, FAbs) thereof (e.g., orthoclone OKT-3 (anti-CD3), GPllb/lla monoclonal antibody).
  • FAbs antigen-binding fragments
  • antigen-binding fragments e.g., orthoclone OKT-3 (anti-CD3), GPllb/lla monoclonal antibody.
  • the exogenous sequences can comprise a marker gene (described above), allowing selection of cells that have undergone targeted integration, and a linked sequence encoding an additional functionality.
  • marker genes include GFP, drug selection marker(s) and the like.
  • Additional gene sequences that can be inserted may include, for example, wild-type genes to replace mutated sequences.
  • a wild-type gene sequence may be inserted into the genome of a stem cell in which the endogenous copy of the gene is mutated. The wild-type copy may be inserted at the endogenous locus, or may alternatively be targeted to a safe harbor locus.
  • Construction of such expression cassettes following the teachings of the present specification, utilizes methodologies well known in the art of molecular biology (see, for example, Ausubel or Maniatis). Before use of the expression cassette to generate a transgenic animal, the responsiveness of the expression cassette to the stress-inducer associated with selected control elements can be tested by introducing the expression cassette into a suitable cell line (e.g., primary cells, transformed cells, or immortalized cell lines).
  • a suitable cell line e.g., primary cells, transformed cells, or immortalized cell lines.
  • exogenous sequences may also transcriptional or translational regulatory sequences, for example, promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides and/or polyadenylation signals.
  • control elements of the genes of interest can be operably linked to reporter genes to create chimeric genes (e.g., reporter expression cassettes).
  • Targeted insertion of non-coding nucleic acid sequence may also be achieved. Sequences encoding antisense R As, RNAi, shRNAs and micro RNAs (miRNAs) may also be used for targeted insertions.
  • the donor nucleic acid may comprise non- coding sequences that are specific target sites for additional nuclease designs.
  • nucleases may be expressed in cells such that the original donor molecule is cleaved and modified by insertion of another donor molecule of interest.
  • additional nucleases may be expressed in cells such that the original donor molecule is cleaved and modified by insertion of another donor molecule of interest.
  • reiterative integrations of donor molecules may be generated allowing for trait stacking at a particular locus of interest or at a safe harbor locus.
  • nucleases, polynucleotides encoding these nucleases, donor polynucleotides and compositions comprising the proteins and/or polynucleotides described herein may be delivered in vivo or ex vivo by any suitable means.
  • Nucleases and/or donor constructs as described herein may also be delivered using vectors containing sequences encoding one or more of compositions described herein.
  • 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.
  • any of these vectors may comprise one or more of the sequences needed for treatment.
  • the nucleases and/or donor polynucleotide may be carried on the same vector or on different vectors.
  • each vector may comprise a sequence encoding one or multiple nucleases and/or donor constructs.
  • 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.
  • Methods of non- viral delivery of nucleic acids include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, 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.
  • Additional exemplary nucleic acid delivery systems include those provided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc.
  • 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., TransfectamTM and LipofectinTM).
  • Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424, WO 91/16024.
  • lipid:nucleic acid complexes including targeted liposomes such as immuno lipid complexes
  • Boese et al Cancer Gene Ther. 2:291-297 (1995); Behr et al, Bioconjugate Chem. 5:382-389 (1994); Remy et al, Bioconjugate Chem. 5:647-654 (1994); Gao et al, Gene Therapy 2:710-722 (1995); Ahmad et al, Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).
  • 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):643).
  • EDVs EnGeneIC delivery vehicles
  • RNA or DNA viral based systems for the delivery of nucleic acids encoding engineered ZFPs take 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 subjects (in vivo) or they can be used to treat cells in vitro and the modified cells are administered to subjects (ex vivo).
  • Conventional viral based systems for the delivery of ZFPs 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.
  • 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 cz ' s-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum czs-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);
  • 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);
  • 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.
  • 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 al, PNAS 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).
  • Vectors suitable for introduction of polynucleotides described herein also include non-integrating lentivirus vectors (IDLV). See, for example, Ory et al. (1996) Proc. Natl. Acad. Sci. USA93: l 1382-11388; Dull et al. (1998) J. Virol.
  • IDLV non-integrating lentivirus vectors
  • Recombinant adeno-associated virus vectors may also be used to deliver the compositions described herein. All vectors are derived from a plasmid that retains only the AAV inverted terminal repeats flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery 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)).
  • AAV serotypes including AAV1, AAV3, AAV4, AAV5, AAV6,AAV8, AAV9 and AAVrhlO, pseudotyped AAV such as AAV2/8, AAV2/5 and AAV2/6 and all variants thereof, can also be used in accordance with the present invention.
  • Ad 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 Ela, 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 non-dividing, differentiated cells such as those found in liver, kidney and muscle. Conventional Ad vectors have a large carrying capacity.
  • Ad vector An example of the use of an Ad vector in a clinical trial involved polynucleotide therapy for anti-tumor 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).
  • 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 ⁇ 2 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.
  • 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.
  • ITR inverted terminal repeat
  • 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.
  • the gene therapy vector be delivered with a high degree of specificity to a particular tissue type.
  • 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.
  • 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.
  • filamentous phage can be engineered to display antibody fragments ⁇ e.g., FAB or Fv) having specific binding affinity for virtually any chosen cellular receptor.
  • Gene therapy vectors can be delivered in vivo by administration to an individual subject, typically by systemic administration ⁇ e.g., intravenous,
  • vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient ⁇ e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector.
  • cells ex vivo such as cells explanted from an individual patient ⁇ e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector.
  • Vectors ⁇ e.g., retroviruses, adenoviruses, liposomes, etc.
  • nucleases and/or donor constructs can also be administered directly to an organism for transduction of cells in vivo.
  • 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. Suitable methods of administering such nucleic acids 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.
  • compositions including fusion proteins,
  • compositions as described herein ⁇ e.g., polynucleotides and/or proteins are delivered directly in vivo.
  • compositions are delivered directly in vivo.
  • polynucleotides and/or proteins may be administered directly into the CNS, including but not limited to direct injection (including grafting of cells) into the brain or spinal cord. See, e.g., U.S. Patent No. 5,529,774 regarding in vivo administration of polynucleotide vectors to the CNS and U.S. Patent No. 5,082,670 and 6,451,306 regarding cell grafting.
  • One or more areas of the brain may be targeted, including but not limited to, the hippocampus, the substantia nigra, the nucleus basalis of Meynert (NBM), the striatum and/or the cortex.
  • compositions may be administered systemically ⁇ e.g., intravenous, intraperitoneal, intracardial, intramuscular, intrathecal, subdermal, and/or intracranial infusion).
  • Cell- containing compositions may be administered into the nervous system directly, for example by grafting.
  • Methods and compositions for delivery of compositions as described herein directly to a subject include but are not limited to direct injection (e.g., stereotactic injection) via needle assemblies. Such methods are described, for example, in U.S. Patent Nos. 7,837,668; 8,092,429, relating to a needle assembly for delivery of compositions to the brain and U.S. Patent Publication No.
  • compositions are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. 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).
  • nuclease-encoding sequences and donor constructs can be delivered using the same or different systems.
  • a donor polynucleotide can be carried by a plasmid
  • the one or more nucleases can be carried by a AAV vector.
  • the different vectors can be administered by the same or different routes (intramuscular injection, tail vein injection, other intravenous injection, intraperitoneal administration and/or intramuscular injection.
  • the vectors can be delivered simultaneously or in any sequential order.
  • the instant disclosure includes in vivo or ex vivo treatment of diseases and conditions that are amenable to insertion of a transgenes encoding a therapeutic protein, for example treatment of NS disorders via nuclease-mediated integration of a gene encoding a protein aberrantly expressed in a subject with the NS disorder.
  • cells are isolated from the subject organism, transfected with a ZFP nucleic acid (gene or cDNA), and re -infused back into the subject organism (e.g., patient).
  • Methods of cell therapy to the NS are known.
  • 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).
  • Suitable cells include but 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 COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44, CHO- DUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NSO, SP2/0-Agl4, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), and perC6 cells, any plant cell (differentiated or undifferentiated) as well as insect cells such as Spodopterafugiperda (Sf), or fungal cells such as
  • COS COS
  • CHO e.g., CHO-S, CHO-K1, CHO-DG44, CHO- DUXB11, CHO-DU
  • the cell line is a CHO-K1, MDCK or HEK293 cell line.
  • primary cells may be isolated and used ex vivo for reintroduction into the subject to be treated following treatment with the nucleases (e.g. ZFNs or TALENs) or nuclease systems (e.g.
  • Suitable primary cells include neuronal cells, peripheral blood mononuclear cells (PBMC), and other blood cell subsets such as, but not limited to, CD4+ T cells or CD8+ T cells.
  • PBMC peripheral blood mononuclear cells
  • Suitable cells also include stem cells such as, by way of example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells (CD34+), neuronal stem cells and mesenchymal stem cells.
  • 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 in the bone marrow.
  • Stem cells that have been modified may also be used in some embodiments.
  • stem cells that have been made resistant to apoptosis may be used as therapeutic compositions where the stem cells also contain the ZFPs, TALEs, ZFNs, TALENs, CRISPR/Cas systems and/or donors of the invention.
  • Resistance to apoptosis may come about, for example, by knocking out BAX and/or BAK using BAX- or BAK-specific nucleases (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.
  • resistance to apoptosis can also be achieved by the use of caspase inhibitors like Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-[0- methylj-fluoromethylketone).
  • Vectors e.g., retroviruses, adenoviruses, liposomes, etc.
  • therapeutic ZFPs, TALEs, ZFNs, TALENs, CRISPR/Cas system and/or donor nucleic acids can also be administered directly to an organism for transduction of cells in vivo.
  • naked DNA or mR A 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. Suitable methods of administering such nucleic acids 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.
  • nuclease(s) and donor to be administered will vary from patient to patient and according to the therapeutic polypeptide of interest. Accordingly, effective amounts are best determined by the physician administering the compositions and appropriate dosages can be determined readily by one of ordinary skill in the art. After allowing sufficient time for integration and expression (typically 4-15 days, for example), analysis of the serum or other tissue levels of the therapeutic polypeptide and comparison to the initial level prior to administration will determine whether the amount being administered is too low, within the right range or too high. Suitable regimes for initial and subsequent administrations are also variable, but are typified by an initial administration followed by subsequent administrations if necessary. Subsequent administrations may be administered at variable intervals, ranging from daily to annually to every several years.
  • Formulations for both ex vivo and in vivo administrations include suspensions in liquid or emulsified liquids.
  • the active ingredients often are mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient.
  • Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof.
  • the composition may contain minor amounts of auxiliary substances, such as, wetting or emulsifying agents, pH buffering agents, stabilizing agents or other reagents that enhance the effectiveness of the pharmaceutical composition.
  • the methods and compositions disclosed herein are for modifying expression of protein, correcting an aberrant gene sequence that encodes a gene product expressed in a NS disorder or insertion of a transgene whose gene product is known to be helpful in treatment or prevention of a NS disorder.
  • the methods and compositions provide for the treatment and/or prevention of such disorders.
  • Genome editing for example of stem cells, is used to correct an aberrant gene, insert a wild type gene, or change the expression of an endogenous gene.
  • a wild type gene may be inserted into a cell to provide the proteins deficient and/or lacking in the subject and thereby treat a NS disorder caused by faulty gene product expression.
  • genomic editing with or without administration of the appropriate donor can correct the faulty endogenous gene, e.g., correcting the point mutation in gene encoding a gene product involved in a NS disorder, to restore expression of the gene and/or treat a the disorder.
  • nuclease comprises a zinc finger nuclease (ZFN) or TALEN.
  • ZFN zinc finger nuclease
  • TALEN zinc finger nuclease
  • additional TALENs ⁇ e.g., Mega-TALs and/or compact TALENs
  • homing endonucleases meganucleases
  • meganucleases homing endonucleases
  • engineered transcription factor e.g. ZFP-TF, TALE-TF, CRISPR/Cas- TF
  • Example 1 Design, Construction and general characterization of compositions that alter NS-related genes
  • Zinc finger proteins and TALEs that bind to NS-related genes ⁇ e.g.,
  • Table 1 operably linked to transcriptional regulatory or nuclease domains are designed and incorporated into plasmids, AAV or adenoviral vectors essentially as described in Urnov et al. (2005) Nature 435(7042):646-651, Perez et al (2008) Nature Biotechnology 26(7):808-816, and as described in U.S. Patent Nos. 8,586,526 and 6,534,261.
  • sgRNAs for use in the CRISPR/Cas system are made synthetically by methods known in the art (see Hsu et al, ibid or Sternberg et al, (2014) Nature 507: 62)).
  • the sgRNAs are engineered as described above and are designed to target a sequence in an NS-related gene.
  • Table II below shows the genomic coordinates of the NS genes of interest. These coordinates are derived from the UCSC Genome
  • sgRNAs, ZFPs or TALEs are designed to target regions within the bounds of the NS genes.
  • ZFNs and TALENs targeting the selected locus are made as described above.
  • the Cel-I assay (SurveyorTM, Transgenomics) as described in Perez et al. (2008) Nat. Biotechnol. 26: 808-816 and Guschin et al. (2010) Methods Mol Biol. 649:247-56), is used to detect nuclease-induced modifications of the target gene in K562 cells or HSCs.
  • PCR-amplification of the target site is followed by quantification of insertions and deletions (indels) using the mismatch detecting enzyme Cel-I (Yang et al. (2000) Biochemistry 39: 3533-3541) which provided a lower- limit estimate of DSB frequency.
  • genomic DNA is isolated cells using the DNeasy® kit (Qiagen) and show the nucleases cleave their target genes.
  • iPSC/ESCs are passaged with accutase and cultured on matrigel coated plates in E8 media (Life Technologies). Neural stem cells are derived using StemPro Neural Induction Medium (Life Technologies). Briefly, iPSC/ESCs are seeded into geltrex coated 6 well dish with 200,000 cells/well and when 10-20% confluent the medium is changed to StemPro Neural Induction Medium. Medium is changed every 2 days and NSC are harvested and expanded on day 7. StemPro NSC SFM medium (Life Technologies) is used to culture NSCs. Human NSCs are transfected with 1.5 or 0.5 ⁇ g ZFP mRNA using nucleofection.
  • Mouse models of a NS disease preferably carrying a human transgene with the mutant gene receive stereotactic, bilateral striatal injections of 3el0 vector genomes of recombinant AAV2/6 encoding the ZFP-TF of the invention driven by a CMV promoter. Mice are injected at 5 weeks of age and sacrificed for molecular analysis at 8 weeks of age. Left and right striata are dissected from each hemisphere and snap frozen. To assess repression of the mutant transgene, total RNA is extracted from each striatum with TRIzol Plus (Life Technologies) followed by cDNA synthesis using High Capacity RT (Life Technologies).
  • mutant transgene expression is measured by qPCR and normalized to the geometric mean of three reference genes (Atp5b, Eif4a2, UbC) as previously described by Benn et al. ((2008) Molecular Neurodegeneration: 3, 17). Repression of the mutant transgene is observed.
  • K562 cells are obtained from the American Type Culture Collection and grown as recommended in F-12 medium (Invitrogen) supplemented with 10% qualified fetal calf serum (FCS, Cyclone). Cells are disassociated from plastic ware using TrypLE SelectTM protease (Invitrogen). For transfection, one million K562 cells are mixed with 2 ⁇ g of the zinc-finger nuclease plasmid and ⁇ Amaxa Solution T. Cells are transfected in an Amaxa
  • Genomic DNA is harvested and a portion of the mutant locus encompassing the intended cleavage site is PCR amplified.
  • PCR using the Accuprime HiFi polymerase from InVitrogen is performed as follows: after an initial 3 minute denaturation at 94°C, 30 cycles of PCR are performed with a 30 second denaturation step at 94°C followed by a 30 second annealing step at 58°C followed by a 30 second extension step at 68°C. After the completion of 30 cycles, the reaction was incubated at 68°C for 7 minutes, then at 10°C indefinitely.
  • the ZFNs are demonstrated to be capable of targeting the mutant genes with a gene modification efficiency of between 8-40%>, assayed as described previously by the amount of indels observed.
  • ZFN modification of a NS mutant allele is performed in patient- derived iPSCs.
  • DNA sequences e.g., plasmids
  • mutant allele-specific ZFNs e.g., the green fluorescent protein (GFP)
  • GFP green fluorescent protein
  • the donor construct contains 1 kb of wild type (wt) gene sequence (500 bps in each direction of the mutation).
  • wt wild type gene sequence
  • the donor is used as a template to repair the DSB as well as the mutation; the majority of the cells will use NHEJ to resolve the DSB.
  • Transfected (GFP-positive) cells are enriched through fluorescence-based cell sorting and replated on MEF feeder layers for single-cell cloning.
  • Genomic DNA is isolated from clones, the regions of interest are amplified by PCR. Clones are subject to sequencing analysis to confirm the presence of 2 wild type alleles for corrected cells, and to identify those having mutations on the mutant allele and unmodified wild type allele.
  • cytogenetic analysis is performed to confirm normal karyotype, and their pluripotent state is verified by expression of the pluripotency markers (OCT4, NANOG and SOX2), as well as ability to form all three developmental germ layers in teratoma- formation assays.
  • the teratoma-formation assay is performed as follows. Cell culture and transfection: The iPSC were grown in mTeSR®l medium (STEM CELLTM
  • a Cell assay was performed using primers adjacent to the area of correction.
  • the Cel I assay is performed using 1-3 ⁇ of a 1/1000 dilution of the PCR described above.
  • the donor is designed to correct the mutation in the mutated allele and is transfected along with the nucleases as described above.
  • To analyze the insertion of the donor individual clones treated with the nuclease pair and donor are examined for the presence of the donor sequences and to confirm gene correction at the mutation site.
  • Genomic DNA is extracted (using Qiagen QIAamp DNA micro kit) and analyzed for ZFN activity as follows. Briefly, the region comprising the cleavage site is amplified by PCR by standard methods, and following amplification, the PCR product is sequenced via MiSeq high throughput sequencing analysis according to manufacturer's instructions (Ilumina). To quantitate the percent of edited alleles, the genomic region of interest is PCR amplified using primers which add the standard Illumina sequencing adapter sequences.
  • a second group of 13 rounds of PCR is performed to add barcode and bridge adapter sequences to both ends. Sequencing is performed on an Illumina MiSeq according to manufacturer's protocols for amplicon sequencing. The MiSeq generates paired-end reads, which are merged and adapter- trimmed using a standard alignment software. Reads are then demultiplexed by sample via barcode sequence pairs using custom scripts. Amplicon sequences are then globally aligned to a reference sequence via an implementation of the Needleman- Wunsch algorithm (Needleman, Saul B.; and Wunsch, Christian D. (1970). Jour Mol Bio 48 (3): 443-53). Gaps or insertions in the alignment were counted as % NHEJ events, and compared to an untreated control sample sequence to determine sequence- specific background rates .
  • Amplicon sequences are globally aligned to a reference sequence via a biopython implementation of the Needleman-Wunsch algorithm (Needleman, Saul B.; and Wunsch, Christian D.
  • Sequence changes generated via experimental treatments were searched for, counted, and compared to counts in control samples.
  • Known single feature polymorphisms SFPs
  • SFPs single feature polymorphisms
  • the percentage of NHEJ is calculated by determining the percentage of sequences that contain insertions or deletions. Samples treated only with GFP vector are used to assess the PCR and sequencing error based background frequency of insertions and deletions. Background frequencies of less than 1% are observed.
  • compositions are administered to a subject with a NS disorder, for example directly to the NS, essentially as described in U.S. Patent Nos. 7,837,668; 8,092,429; U.S. Patent Publication No. 20060239966; U.S. Patent Nos. 6,180,613; 6,503,888 and /or U.S. Patent Nos. 6,998, 118 and 7,101 ,540 to provide therapy for a subject in need thereof.

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Abstract

Methods and compositions for nervous system (NS) disorders are provided.

Description

METHODS AND COMPOSITIONS FOR PREVENTION OR TREATMENT OF A NERVOUS SYSTEM DISORDER
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of U.S. Provisional
Application No. 61/973,471, filed April 1 , 2014 and U.S. Provisional Application No. 61/992,656, filed May 13, 2014, the disclosures of which are hereby incorporated by reference in their entireties.
TECHNICAL FIELD
[0002] The present disclosure is in the field of genome engineering of cells, especially for the treatment of a disorder of the nervous system. BACKGROUND
[0003] Modulation of gene expression holds enormous potential for a new era in human medicine. These methodologies will allow treatment for conditions that heretofore have not been addressable by standard medical practice.
[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] Another area of gene therapy that is especially promising is the ability to genetically engineer a cell to cause that cell to express a product not previously being produced in that cell. Examples of uses of this technology include the insertion of a gene encoding a novel therapeutic protein, insertion of a coding sequence encoding a protein that is lacking in the cell or in the individual, insertion of a wild type gene in a cell containing a mutated gene sequence, and insertion of a sequence that encodes a structural nucleic acid such as a microRNA or siRNA.
[0006] Transgenes can be delivered to a cell by a variety of ways such that the transgene becomes integrated into the cell's own genome and is maintained there. In recent years, a strategy for transgene integration has been developed that uses cleavage with site-specific nucleases (e.g., zinc finger nucleases (ZFNs), TALE nucleases (TALENs), Ttago nuclease systems and CRISPR/Cas nuclease systems) for targeted insertion into a chosen genomic locus. Nucleases specific for targeted genes (including "safe harbor" loci such as CCR5, CXCR4, AAVSl, albumin or Rosa) can be utilized such that the transgene construct is inserted by either homology directed repair (HDR) or by end capture during non-homologous end joining (NHEJ) driven processes. See, for example, 8,623,618; 8,034,598; 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 20030232410;
20050208489; 20050026157; 20060063231; 20080159996; 201000218264;
20120017290; 20110265198; 20130137104; 20130122591; 20130177983;
20130177960 and 20150056705, the disclosures of which are incorporated by reference in their entireties for all purposes. Nuclease-mediated integration offers the prospect of improved transgene expression, increased safety and expressional durability, as compared to classic integration approaches that rely on random integration of the transgene, since it allows exact transgene positioning for a minimal risk of gene silencing or activation of nearby oncogenes.
[0007] In addition, specific nucleases can be used to direct gene correction or gene modification, where segments of an endogenous gene are replaced with sequences supplied in concert with the specific nuclease(s). This technology can be used to correct sequences known to be associated with disease (e.g. sickle cell anemia), or to alter gene sequences to confer new characteristics. In both transgene addition, and gene correction, nucleases can include zinc finger nucleases (ZFN), transcription activator like effector nucleases (TALENs), mega or homing
endonucleases, nuclease systems such as CRISPR/Cas that use a guide RNA to determine specificity, and fusions between nucleases such as mega-TALs.
[0008] Engineered nucleases can also be used to silence or knock out a gene through targeted double strand cleavage at a desired locus. In the absence of a donor, the cell will rely on error- prone non-homologous end joining (NHEJ) to heal the break. NHEJ often results in the insertion or deletions of nucleotides at or near the break ("indels"), causing missense mutations within the coding sequence. Targeted cleavage can also result in the knockout of splicing sequences such that resulting transcripts are improperly spliced. Both of these approaches can result in the silencing of the targeted gene following cleavage by the nuclease. Diseases of the nervous system (NS), including central nervous system (CNS) and peripheral nervous system (PNS) disorders, are threatening to come to the forefront of causes of mortality in humans, especially as our population ages. It is said that NS diseases and disorders represent the largest and fastest growing area of unmet medical need: 1.5 billion people worldwide, including over 100 million people in the U.S., suffer from NS diseases or disorders. Unfortunately, the pathogenesis of NS disease is often the most difficult to understand, although for more and more of these disorders, genetic clues are being discovered. As an example, in the United States, 1 in 8 people over the age of 65 has Alzheimer's disease, and 45% of people over the age of 85 are afflicted. In fact, it is currently the sixth leading cause of death. By the year 2050, there may be 13.2 million people in the United States alone will have Alzheimer's disease. See, e.g., Alzheimer's and Dementia (2012) 8(2). Statistics and predictions for other types of NS diseases demonstrate that these diseases are also very pervasive in the population. For most NS disorders, treatments are inadequate, and often physicians can only attempt to alleviate symptoms rather than achieve functional cures.
[0009] Thus, there remains a significant need for additional methods and compositions that can be used for genome editing, to correct an aberrant gene or alter the expression of one or more genes to treat and/or prevent a disease of the nervous system.
SUMMARY
[0010] Disclosed herein are methods and compositions for altering the expression of, or for correcting, one or more genes whose gene products are involved in a nervous system (NS) disorder {e.g., producing proteins lacking, deficient or aberrant in the nervous system disorder and/or proteins that regulate these proteins or gene products that are toxic RNAs) such as a disease or disorder of the central nervous system (CNS) and/or peripheral nervous system (PNS). Alteration of genomic sequence or expression levels of such genes can result in the treatment of these nervous system disorders. In particular, genome editing is used to correct an aberrant gene, insert a wild type gene, or change the expression of an endogenous gene. By way of non-limiting example, a mutated gene encoding a protein involved in a NS disorder may be corrected in a cell to produce a wild type protein to prevent and/or treat the NS disorder. One approach further involves the use of gene modification in a stem cell, which stem cell can then be used to engraft into a patient, for treatment of a nervous system disease.
[0011] Examples of NS disorders that may be treated by genetic modifications
(including by in vivo genetic modifications and by administration of genetically modified cells) include, but are not limited to, Alzheimer's disease (AD), Parkinson's disease (PD), Charcot-Marie-Tooth disease (CMT), Duchenne's muscular dystrophy (DMD), Friedrich's Ataxia; amyotrophic lateral sclerosis (ALS), familial
frontotemporal dementia, Fragile X disease, myotonic dystrophy, Freidreich's ataxia, multiple sclerosis, Huntington's disease (HD), Tourette's syndrome, Rett syndrome, Canavan syndrome, Crigler Najjir syndrome, certain lysosomal storage diseases, stroke, migraine, concussion, major depressive disorder, epilepsy, bipolar disorder, borderline personality disorder, opiate addiction, and schizophrenia. Non-limiting examples of particular genes that may be modified (including for correction of mutant genes and/or for targeted inactivation or integration into such genes) include microtubule associated protein tau (MAPT, encoding Tau proteins) and/or
apolipoprotein E (APOE) alleles(e.g., apoE2, apoE3 or apoE4, for stroke, concussion and/or AD, and epilepsy); LRRK2 and alpha-synuclein (for PD); amyloid precursor protein (APP), presenilin 1 and/or presenilin 2 {e.g., for AD); SLC6A4, HTR2A, alpha- 1 subunit of a voltage-dependent calcium channel (CACNAIC) and/or calcium channel, voltage-dependent, beta 2 subunit (CACNB2) {e.g., for depression and/or migraines); dystrophia myotonica-protein kinase (DMPK for myotonic dystrophy); CACNA1 A, ATP1 A2, SCN1A {e.g., for migraine); FXN (for Friedrich's Ataxia); HTT genes {e.g., HD); PMP22 (CMT); dystrophin and/or utrophin(e.g., for DMD); C9orf72, SOD1, TARDBP, FUS, ANG, ALS2, SETX, progranulin gene (GRN) and/or VAPB {e.g., for ALS and/or dementia); FMR1 {e.g., for Fragile X) and/or HPRT {e.g., for Lesch-Nyhan Disease); MECP2 (Rett syndrome); ASPA (Canavan Disease); SCN1A, SCN8A (Dravet syndrome); SMN1, SMN1 (SMA); UGT1A1 (Crigler Najjir); OPRM1, OPRK1, OPRD1 (opiate addiction); OPRM1 (Borderline personality disorder); SLC6A4, HTR2a, TPH2 (Major depressive disorder); DRD2, GRM3, GRIN2A, SRR, GRIA1, CACNAIC, CACNB2, CACN11I, GAD1, RELN, BDNF, TET1, and DTNBP1 (Schizophrenia); ANK3, ODZ4, TRANK1, ADCY2, CACNA1C, BDNF (Bipolar disorder); PRDM16, AJAP1, MEF2D, TRPM8,
TGFBR2, PHACTR1, FHM5, c7orfl0, MMP16, ASTN2, TSPAN2, GFRA2 and LRP1 (Migraine); HLA-DRB1, IL7Ra, IL2Ra, CYP27Ba, TYK2 (multiple sclerosis); NRXN1, A AD AC, CTNNA3, FSCB, KCHE1, KCHE2, RCAN1 (Tourette syndrome); CAMSAP1LK1, NMDA receptor subunit 1, GAMA-A receptor subunit alpha- 1, GAD65, adenosine kinase, GCNF, BDNF, IGF, neuropeptide Y, galanin (epilepsy); . See, also, Cross-Disorder Group of the Psychiatric Genomics
Consortium (2013)7¾e Lancet - 381( 9875): 1371-1379.
[0012] In one aspect, described herein is a fusion protein comprising a DNA- binding domain {e.g., a ZFP, TALE, or single guide RNA) that binds to a target site in a selected gene and a functional domain. In certain embodiments, the functional domain comprises a transcriptional regulatory domain, for example an activation domain or a repression domain. When linked to a specific DNA-binding domain, the resulting artificial transcriptional factor {e.g., ZFP-TF, TALE-TF, CRISPR-TF) alters gene expression of the targeted gene. In other embodiments, the functional domain comprises a nuclease (cleavage) domain that cleaves DNA at or near the target site(s), for example, within 1-300 (or any value therebetween) base pairs upstream or downstream of the site(s) of cleavage, more preferably within 1-100 base pairs (or any value therebetween) of either side of the binding and/or cleavage site(s), even more preferably within 1 to 50 base pairs (or any value therebetween) on either side of the binding and/or cleavage site(s). By selecting either an activation domain or repression domain for fusion with a DNA-binding domain, such fusion proteins can be used either to activate or to repress gene expression. Similarly, by selecting a nuclease domain for fusion with a DNA-binding domain, such fusion proteins can be used to cleave DNA at or near the target site. It will be apparent that the fusion protein used will depend on the gene targeted. For example, some genes involved in NS disorders can be activated to treat and/or prevent the NS disorder {e.g., C9orf72 in frontotemporal dementia) while some genes can be repressed and/or inactivated (via cleavage) to treat and/or prevent the NS disorder {e.g., MAPT encoding Tau, ApoE4, PMP22, C90RF72, FMR1, DMPK, FXN, presenilin, CACNAIC, CACNB2, etc.)
[0013] In another aspect, described herein is nuclease {e.g., ZFN, TALEN, mega or homing endonuclease, mega-TAL or a CRISPR/Cas system) that binds to target site in a region of interest of a gene encoding a protein involved in a NS disorder in a genome, wherein the nuclease comprises one or more engineered domains. In one embodiment, the nuclease is a zinc-finger nuclease (ZFN) that cleaves a target genomic region of interest, wherein the ZFN comprises one or more engineered zinc-finger binding domains and a nuclease cleavage domain or cleavage half-domain. In another embodiment, the nuclease is a TALE nuclease (TALEN) that cleaves a target genomic region of interest, wherein the TALEN comprises one or more engineered TALE DNA binding domains and a nuclease cleavage domain or cleavage half-domain. In another embodiment, the nuclease is a CRISPR/Cas system wherein the specificity of the CRISPR/Cas is determined by an engineered single guide mRNA. In yet another embodiment, the nuclease is a Ttago nuclease system. 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 a Type IIS restriction endonuclease (e.g., Fok l).
[0014] In another aspect, described herein is a CRISPR/Cas system that binds to target site in a region of interest {e.g., a highly expressed gene, a disease associated gene or a safe harbor gene) in a genome, wherein the CRISPR/Cas system comprises a CRIPSR/Cas nuclease and an engineered crRNA/tracrRNA (or single guide RNA). In certain embodiments, the CRISPR/Cas system recognizes a target site in a highly expressed, disease associated, or safe harbor gene.
[0015] The fusion proteins and systems {e.g., transcriptional activators, transcriptional repressors, ZFNs, TALENs and/or CRISPR/Cas system) as described herein may bind to and/or cleave the region of interest in a coding or non-coding region 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 other embodiments, the fusion protein and/or system binds to (and/or activates, represses or cleaves) a safe-harbor gene, for example a CCR5 gene, a CXCR4 gene, an HPRT gene, a PPP1R12C (also known as AAVS1) gene, an albumin gene or a Rosa gene. See, e.g., U.S. Patent Nos. 7,888,121; 7,972,854;
7,914,796; 7,951,925; 8,110,379; 8,409,861; 8,586,526; U.S. Patent Publications 20030232410; 20050208489; 20050026157; 20060063231; 20080159996;
201000218264; 20120017290; 20110265198; 20130137104; 20130122591;
20130177983; 20130177960 and 20150056705. In addition, to aid in selection, the HPRT locus may be used (see U.S. Patent Publication No. 20130122591). [0016] In another aspect, described herein are compositions comprising one or more of the fusion proteins or one or more components of a CRISPR/Cas system as described herein. In some embodiments, fusion proteins and/or system bind to (and/or cleave, activate or repress) a gene encoding a gene product involved in a NS disorder. In another aspect, described herein are compositions comprising one or more of the zinc-finger, TALE, single guide RNAs, and/or Cas nucleases as described herein.
[0017] In another aspect, described herein is a polynucleotide encoding one or more fusion proteins (activators, repressors and/or nucleases) and/or systems (or components thereof) as described herein. The polynucleotide may be, for example, mRNA. In some aspects, the mRNA may be chemically modified (See e.g. Kormann et al, (2011) Nature Biotechnology 29(2):154-157). Any of the polynucleotides described herein may include a promoter, for example where the fusion protein is operably linked to a promoter. In one embodiment, the expression vector is a viral vector.
[0018] In further aspects, the invention described herein comprises one or more NS gene-modulating transcription factors, such as a NS gene -modulating transcription factors comprising one or more of a zinc finger protein (ZFP TFs), a TALEs (TALE-TF), and a CRISPR/Cas-TFs for example, ZFP-TFs, TALE-TFs or CRISPR/Cas-TFs. In certain embodiments, the NS gene -modulating transcription factor can repress expression of a NS gene in one or more cells of a subject. The repression can be about 85% or greater, about 90% or greater, about 92% or greater, or about 95% or greater repression of NS gene in the one or more cells of the subject. In certain embodiments, the NS gene-modulating transcription factor can be used to achieve one or more of the methods described herein.
[0019] In one aspect, described herein is a ZFN, TALEN and/or CRISPR/Cas system that is used to cleave a target DNA.
[0020] In another aspect, described herein is a method for inserting a sequence into an endogenous gene, in which the endogenous gene encodes a gene product that is involved in a NS disorder in a cell (e.g. stem cell), the method comprising cleaving the endogenous gene using one or more nucleases and inserting a sequence into the cleavage site. In certain embodiments, the insertion results in a replacement of a genomic sequence in any target gene, for example using a ZFN or TALEN pair, or a CRIPSR/Cas system (or vector or polynucleotide encoding said ZFN, TALEN and/or CRIPSR/Cas system) as described herein using a "donor" sequence (also known as a "transgene") that is inserted into (and replaces at least part of) the gene following targeted cleavage with the ZFN, TALEN and/or a CRIPSR/Cas system. The donor sequence may be present in the ZFN or TALEN vector, present in a separate vector (e.g., Ad, AAV or LV vector) or, alternatively, may be introduced into the cell using a different nucleic acid delivery mechanism. In certain embodiments, such insertion of a donor nucleotide sequence into the target locus (e.g., a safe -harbor gene, etc.) results in the expression of the transgene under control of the target locus 's genetic control elements. In other embodiments, the donor nucleotide sequence includes one or more control elements that drive expression of one or more transgenes. In some
embodiments, the transgene encodes a non-coding RNA (e.g., an shRNA).
[0021] In yet another aspect, provided herein are cells, cell lines and/or transgenic animal models (systems). In certain embodiments, the cells and/or cells lines are used for treatment, prevention and/or amelioration of a nervous system disorder by administration to a subject with a nervous system disorder. In some embodiments, the transgenic cell and/or animal includes a transgene that encodes a human gene. In other embodiments, the cells comprises a mutation (e.g., "indel" which comprises one or more insertions and/or deletions), for example an indel that results in production of a product involved in a NS disorder. In some instances, the modification corrects a mutant allele such that the wild-type gene product is produced. In some instances, the transgenic animal comprises a knock-out at the endogenous locus corresponding to exogenous transgene (e.g., the mouse gene involved in the NS disorder is knocked out and the human gene involved in the NS disorder is inserted into a mouse), thereby allowing the development of an in vivo system where the human gene may be studied in isolation. Such transgenic models may be used for screening purposes to identify small molecules or large biomolecules or other entities which may interact with or modify the human protein or gene product of interest. In some aspects, the transgene is integrated into the selected locus (e.g., NS gene, safe-harbor) into a stem cell (e.g., an embryonic stem cell, an induced pluripotent stem cell, a hematopoietic stem cell, etc.) or animal embryo obtained by any of the methods described herein, and then the embryo is implanted such that a live animal is born. The animal is then raised to sexual maturity and allowed to produce offspring wherein at least some of the offspring comprise edited endogenous gene sequence or the integrated transgene. [0022] In a still further aspect, provided herein is a method for site specific integration of a nucleic acid sequence into an endogenous locus of a chromosome, for example into the chromosome of a non-human embryo, which locus encodes a gene involved in a NS disorder. In certain embodiments, the method comprises: (a) injecting a non-human embryo with (i) at least one DNA vector, wherein the DNA vector comprises an upstream sequence and a downstream sequence flanking the nucleic acid sequence to be integrated, and (ii) at least one RNA molecule encoding a zinc finger (e.g., ZFN), TALE (e.g., TALEN) or Cas9 protein (e.g., nuclease component of a CRISPR/Cas system)). In the case of using a Cas9 protein, an engineered single guide (sg)RNA is also introduced. The nuclease or nuclease system recognizes the target site in the target locus (e.g., NS gene or safe harbor locus), and then (b) the non-human embryo is cultured to allow expression of the zinc finger nuclease, TALE nuclease and/or CRISPR/Cas system, wherein a double stranded break is introduced into the target by the zinc finger nuclease, TALEN or
CRISPR/Cas system is then repaired, via homologous recombination with the DNA vector, so as to integrate the nucleic acid sequence into the chromosome.
[0023] In another aspect, described herein is a method of treating, preventing and/or ameliorating a nervous system disorder in a subject, the method comprising genetically modifying a cell of the subject, such that the cell treats, prevents and/or ameliorates the nervous system disorder. In certain embodiments, the modification comprises inserting an exogenous sequence that into a specified genomic locus of a cell of the subject. The exogenous sequence can comprise, for example, one or more transgenes that express proteins involved in the nervous system disorder, one or more RNAs and combinations thereof. In other embodiments, the modification comprises alteration of an endogenous sequence such that a beneficial gene product is produced. In any of these methods, the cell may be modified (exogenous sequence integration) in the subject (in vivo); may be isolated from the subject or from another source (e.g., cell line), modified to include the exogenous sequence and re-introduced into the subject (ex vivo); and combinations thereof. For ex vivo methods, the genetically modified cells as described herein may be delivered by any suitable means to a subject in need thereof. In certain embodiments, the cells are delivered directly to the nervous system, for example by administration directly into the CNS or PNS (e.g., grafting). [0024] In any of the methods described herein, the polynucleotide encoding the fusion protein and/or system (or component thereof) can comprise DNA, R A or combinations thereof. In certain embodiments, the polynucleotide comprises a plasmid. In other embodiments, the polynucleotide encoding the nuclease comprises mRNA.
[0025] A kit, comprising the fusion proteins and/or systems of the invention, is also provided. The kit may comprise nucleic acids encoding the compositions described herein, (e.g. RNA molecules or ZFP-TF,TAL-TF, ZFNJALEN or Cas9 encoding genes contained in a suitable expression vector) and engineered sg RNA if needed , or aliquots of the nuclease proteins, transcription factors, donor molecules, suitable host cell lines, instructions for performing the methods of the invention, and the like.
[0026] These and other aspects will be readily apparent to the skilled artisan in light of disclosure as a whole.
DETAILED DESCRIPTION
[0027] Disclosed herein are methods and compositions for studying and/or treating a disease or disorder of the nervous system. The invention describes genomic editing of a target cell such that there is a favorable change in the expression of one or more genes, which in turn results in treatment and/or prevention of a disorder of the nervous system in a subject in need thereof. Favorable changes in the expression of a gene includes, but is not limited, correction of an aberrant endogenous gene sequence (i.e., an endogenous gene with one or more mutations). Additionally, delivery of altered stem cells (altered to express a desired protein product and/or correct an aberrant endogenous gene sequence) can be similarly beneficial in treating a nervous system disorder.
[0028] Thus, the methods and compositions of the invention can be used to alter the expression of one or more genes in a cell wherein those genes are related to a nervous system disease. The cells may be modified in vivo or ex vivo (i.e., isolated cells, including patient derived cells, patient derived induced pluripotent stem cells or isolated stem cells) can be modified and re-introduced into a subject with the nervous system disease. For example, for the treatment or prevention of Alzheimer's disease, alteration of the expression of the Tau gene and/or an allele of ApoE (e.g., ApoE4) may be beneficial. Alzheimer's Disease (AD) pathogenesis is thought to be triggered by accumulation of the amyloid beta peptide due to over production of this protein and failure of its natural clearance mechanisms. Amyloid beta self-aggregates and accumulates in plaques thought to be synaptotoxins. The plaques also may interfere with phosphorylation of tau, leading to its hyperphosphorylation and loss of normal function. Tau is a microtubule associated protein involved in axonal transport, and an alteration of its normal function leads to accumulation of neurofibrillary tangles (Medeiros et al, (2011) CNS Neurosci Ther 17(5): p. 514). Another potentially important target for Alzheimer's disease is the APOE4 allele of apolipoprotein E (apoE). The APOE4 allele is the greatest genetic risk factor for AD and a person with two APOE4 alleles has 15 times the risk of developing AD than a person with APOE3 alleles. ApoE is thought to bind amyloid beta and the soluble forms of this complex may modulate levels of neurotoxic amyloid beta. Clearance of soluble amyloid beta appears to be slower in the presence of the APOE4 encoded apoE, and apoE may also serve a role in the aggregation and deposition of amyloid beta (Tai et al (2014) Molecular Neurodegeneration 9(2)). The interaction sites on the apoE and amyloid beta proteins have been identified studies have shown that blockage of that interaction by use of a peptide Αβ12-28Ρ reduced the behavioral and biochemical hallmarks of AD in a mouse model (Liu et al (2014) Neurochemistry 128:577). Thus, the methods and compositions of the invention can be used to treat or prevent AD. Engineered nucleases against Tau or APOE4 can cause a knock out of the endogenous genes and prevent accumulation of the toxic amyloid beta aggregates. In addition, fusion proteins of the invention comprising transcription factor regulatory domains may be used to down regulate expression of either or both Tau and APOE4. Fusion proteins of the invention may also be used to increase the expression beneficial apoE alleles {e.g. APOE2) in a heterozygous subject.
[0029] The methods and compositions described herein can also be used to treat and/or prevent Charcot-Marie-Tooth Disease (CMT). CMT is the most common inherited neuro muscular disorder, with a prevalence of approximately 17-40 per 100,000 in the population. The disease is characterized clinically by wasting and weakness in the distal limb muscles, skeletal deformities and a decrease or absence of deep tendon reflexes. The disease can be linked to mutations in a number of different genes, but the various mutations all lead to axonal degeneration where the longer axon fibers are affected first and more severely resulting in the observed impairment of the feet and lower legs (Pareyson et al, (2006) Neuromolecular Medicine 8:3). The most frequent subtype of CMT, CMT1A is usually caused by a 1.5 Mb duplication of chromosome 17pl 1.2 which leads to an increase in gene dosage or overexpression of the PMP22 gene. PMP22 is an integral membrane protein that is an important component of compact peripheral nervous system myelin sheath. A more severe form of CMT is seen in patients with missense mutations in one copy of PMP22, indicating a toxic gain of function mutation. In mouse models where PMP22 is overexpressed via a regulatable tetracycline operator and causes dysmyelination, reduction of gene expression lead to reversal of the CMT phenotype and myelination of previously unmyelinated nerve fibers (Perea et al (2001) Human Mol Gen 10(10): 1007) . Thus, the methods and compositions of the invention can be used to treat CMT. Specific nucleases of the invention can be designed to knock out or correct specific PMP22 missense mutations, or specific transcription factors of the invention may be designed to reduce expression of PMP22.
[0030] Duchenne Muscular Dystrophy (DMD), a disease with nervous system implications, and the most common severe form of muscular dystrophy can also be treated and/or prevented using the methods and compositions described herein.
Prevalence of DMD is approximately 1 in 3500 male births and follows an X-linked recessive inheritance pattern. It arises out of mutations in the dystrophin gene, which is the largest gene in the human genome. Dystrophin is a 427 kDa cytoskeletal protein that is required for muscle fiber stability, and loss of the protein results in necrosis and diminished regenerative capacity of muscle, ultimately leading to fibrosis of the muscle tissue. A similar protein, utrophin, is also a cytoskeletal protein that may be able to compensate for defective dystrophin levels. A zinc finger-based transcription factor designed to upregulate utrophin was disclosed in Onori et al (2013, BMC Molecular Biology 14(3)) and U.S. Patent 8,304,235. In adult muscle, utrophin localizes to the neuromuscular and myotendinous junctions while dystrophin localizes to the entire sarcolemma. In developing muscle however, utrophin can be found along the sacrolemma. In some DMD patients, utropin levels are upregulated, and there appears to be a positive correlation between the level of utrophin expression and disease progression (Kleopa et al (2006) Hum Mol Genet 15(10):1623). Current therapies for patients afflicted with DMD have increased life expectancy by decreasing respiratory complications. However, as patients live longer than in the past, cardiac function complications are becoming more prevalent as a result of cardiomyopathies that occur later in the disease progression (Malik et al (2012) Expert Opin Emerg Drugs 17(2):261). Thus, the methods and compositions of the invention can be used to treat, delay and/or prevent the onset of symptoms associated with DMD. Engineered DNA binding proteins (e.g. ZFP, TALEs, CRIPSR/Cas systems) are fused to transcriptional regulator domains to cause an increase in expression of the utrophin and/or dystrophin protein. See, also, U.S. Publication No. 20140140969.
[0031] Amyotrophic Lateral Sclerosis (ALS) is the most common adult-onset motor neuron disorder and is fatal for most patients less than three years from when the first symptoms appear. Generally, it appears that the development of ALS in approximately 90-95% of patients is completely random (sporadic ALS, sALS), with only 5-10% of patients displaying any kind of identified genetic risk (familial ALS, fALS). Mutations in several genes, including the C9orf72, SOD1, TARDBP, FUS, ANG, ALS2, SETX, and VAPB genes, cause familial ALS and contribute to the development of sporadic ALS. Mutations in the C9orf72gene are responsible for 30 to 40 percent of familial ALS in the United States and Europe. The C9orf72 mutations are typically hexanucleotide expansions of GGGGCC in the first intron of the C9orf72 gene. The pathology associated with this expansion (from approximately 30 copies in the wild type human genome to hundreds in fALS patients) appears to be related to the formation of unusual structures in the DNA and to some type of RNA- mediated toxicity (Taylor (2014) Nature 507: 175). Incomplete RNA transcripts of the expanded GGGGCC form nuclear foci in fALS patient cells and also the RNAs can also undergo repeat-associate non-ATP -dependent translation, resulting in the production of three proteins that are prone to aggregation (Gendron et al (2013) Acta Neuropathol 126:829). C9orf72 mutations are also the most common genetic cause of fronototemporal dementia (FTD), a common form of early-onset dementia. Other genetic causes of FTD include mutations in the progranulin gene (GRN) and in the gene encoding Tau (Mahoney et al (2012) Brain 135:736). Zinc finger proteins linked to repression domains have been successfully used to preferentially repress the expression of expanded Htt alleles in cells derived from Huntington patients by binding to expanded tracts of CAG. See, also, U.S. Patent Publication Nos.
20110082093 and 20130253040. Thus, the methods and compositions of the invention can be used to treat, delay or prevent ALS and FTD. For example, engineered DNA binding proteins (e.g. ZFPs, TALEs, Cas) can be constructed to bind to the expansion tract of the C9orf72 disease associated allele and repress its expression. Alternatively or in addition, a wild type version of C9orf72, lacking the abnormally expanded GGGGCC tract, may be inserted into the genome to allow for the normal expression of the gene product. Additionally, the fusion proteins of the invention can be used to modulate the expression of other genes (e.g. GRN, Tau) or disrupt them via use of the engineered nucleases of the invention to prevent or treat FTD.
[0032] Parkinson's disease (PD) is a neurodegenerative disease that afflicts approximately 4-6 million people worldwide. In the United States, approximately one to two hundred people per 100,000 have PD. It appears that many factors can play a role in disease onset and/or progression of PD. For example, genetic mutations in the leucine rich repeat kinase 2 gene (LRRK2, also known as PAR 8) and alpha- synuclein have been identified to be involved in both familial and sporatic forms of PD and have been targeted by nucleases. See, also, U.S. Patent Publication Nos. 20120192301 and 20120214241.
[0033] Fragile X syndrome is the leading monogenic cause of intellectual disability and autism. The FMR1 gene (encoding the fragile X mental retardation protein, FMRP) is found on the X chromosome and comprises a CGG trinucleotide repeat track, which in normal FMR1 genes, contains about 5-44 CGG repeats.
Subjects with 45-54 repeats are considered to be at risk for Fragile X syndrome, and people with 55-200 repeats are considered to have a pre-mutation for the syndrome. Patients afflicted with Fragile X have between 200 and 1000 CGG repeats. As discussed above, these repeats are also capable of forming the unusual structures in the DNA (G-quadruplexes, see Kettani et al (1995) J Mol Biol 254(4):638) As a result, the Fragile X-associated FMR1 gene is methylated and is silenced or improperly expressed. Without sufficient FMRP, mental retardation results. Thus the methods and compositions of the invention can be used to insert a wild type copy of the gene in a safe harbor location to supply the subject with FMRP lacking due to the repeat expansion mutation.
[0034] Myotonic dystrophy is another muscle wasting disease associated with neuropathy that affects approximately 1 in 8000 people worldwide. Patients often have prolonged muscle contractions and may not be able to relax specific muscles after use and there may be cardiac conduction defects leading to abnormalities in the electric signals that control heartbeat. There are two types: type 1 and type 2. Type one is apparent at birth (congenital) with muscle impairment generally in the lower legs, hands, neck and face while type 2 has muscle impairment in the neck, shoulders, elbows and hips. DM1 (type 1) and DM2 (type 2) are inherited autosomal dominant diseases caused by unstable expanded sequences (CTG and CCTG, respectively) in the non-coding regions of DMPK and ZNF9 (also known as CNBP), respectively. Radvanszky et al. (2013) Neuromuscul Disord. 23(7):591-8. The mutations in both genes result in the intranuclear accumulation of mutant transcripts and misplaced transcripts, resulting in R A-mediated toxicity. In addition, the presence of neurotangles in the brain of DM patients suggests that DM is also a degenerative brain disease belonging to the class of tauopathies (Calliet-Boudin et al (2013) Front Mol Neurosci 6(57)). Thus the methods and compositions of the invention can be used to treat or prevent myotonic dystrophy. Engineered transcription factors can be used to down regulate expression of a diseased allele associated with either DM1 or DM2, or the diseased allele can be knocked out via cleavage by an engineered nuclease. In addition, wild type versions of these genes can be inserted into an endogenous location in the genome to allow for expression of the normal gene product.
[0035] Rett syndrome (RTT) is a neurodevelopmental disorder that affects 1 in 10,000 live female births. The symptoms of RTT appear after an early period (approximately 6- 18 months of life) of apparently normal development in an infant. Initially there is a slowing down or stagnation of learning skills followed by a loss of communication skills and purposeful use of the hands. RTT can present with a wide range of disability ranging from mild to severe and causes problems in brain function that are associated with cognitive, sensory, emotional, motor and autonomic functions leading to effects on speech, sensation, mood, movement, breathing, cardiac function, and digestion. Life expectancy is thought to be 40 to 50 years, with patients requiring intensive care for daily life. RTT is usually caused by a mutation in the Methyl CpG binding protein 2 (MECP2) where 35% of RTT cases are cause by nonsense mutations in MECP2 (see Pitcher et al, (2015) Hum Mol Genet 1-11, doi:
10.1093/hmg/ddv030). In fact, 60% of RTT cases are caused by 8 different mutations in the MECP2 protein as follows: R106W; R133C; T158M; R168X; R255X; R270X; R294X and R306C. Thus, the methods and compositions of the invention can be used to treat RTT. Engineered transcription factors can be designed to shut off the expression of mutant MECP2 genes and/or to upregulate a wild type MECP2 gene in a heterozygous individual. Engineered nucleases can be used to introduce a double strand break in the 5 ' end of the mutant MECP2 gene followed by targeted integration of a cDNA comprising a sequence encoding the MECP2 gene and a poly A signaling sequence. The MECP2 promoter will thus drive normal expression of the inserted transgene while silencing the mutant copy. Viral vectors comprising either the engineered transcription factor or engineered nuclease can be introduced into the brain to edit the cells therein. Similarly, these viral vectors can be introduced into other target tissue such as the heart and/or lung tissue to cause the expression of the wild type protein in these tissues. Additionally, altered regulation (e.g. up regulation of gene expression) of other factors which interact with MECP2 are also
contemplated, including brain derived neutrotrophic factor (BDNF) and insulin like growth factor (IGF), both of which have been found to partially reverse some of the phenotypes associated with RTT in rodent models (Pitcher ibid).
[0036] Canavan's disease (CD) is a hereditary leukodystrophy caused by mutations in the aspartoacylase gene (ASPA) that lead to a loss of enzyme activity and an increase in concentrations of the enzyme's substrate N-acetylaspartate (NAA) in the brain. There are over 54 characterized loss of function mutations in ASPA that lead to CD, and the disease is characterized by dysmyelination, intramyelinic edema, and extensive vacuolization of the CNS white matter (see Ahmed and Gao (2013) Mol Ther 21(3): 505-506). Clinically, CD leads to macrocephaly, severe cognitive and motor delays, epilepsy and death, typically after about thirty years of life. Symptoms appear in the first 3 to 6 months of life and progress rapidly. Gene therapy has been attempted for this disease were AAV comprising the ASPA gene was introduced into the brain with some success (see Leone et al (2012) Sci TranslMed 4(165): 165ral63. Doi: 10.1126/scitranslmed.3003454). Additionally, stem cell therapy where corrected neuronal stem cells derived from patent iPSC has also been proposed (see Goldman et al, (2008) Hum Mol Genet 17(1) R76-R83). Thus, the methods and compositions of the invention can be used to prevent or treat Canavan's disease. Since the
accumulation of the ASPA precursor NAA is tied to many of the symptoms of the disease, an engineered transcription factor can be used to increase the expression of a wild type ASPA allele in situ in the brain. Similarly, engineered nucleases can be used to knock out expression of an aberrant ASPA allele, or can be used to introduce a short oligonucleotide to correct a mutated gene and/or to introduce a wild type cDNA. Additionally, wild type ASPA protein may be made hepatically (e.g. from the albumin locus) where the protein comprises a peptide allowing it to cross the blood brain barrier. Thus the methods and compositions of the invention can be utilized to cause a decrease in the accumulation of NAA and thus decrease the symptoms of the disease. In addition, the invention contemplates the correction of an aberrant ASPA gene in an iPSC derived from a Canavan patient that can be forced along the pathway to becoming a neuronal stem cell. These altered neuronal stem cells are then reintroduced into the brain of the patient and correct and/or treat the disease.
[0037] Dravet syndrome (DS, also known as Severe Myoclonic Epilepsy of
Infancy) is a genetic epilepsy syndrome associated with loss of function mutations in SCN1A, the gene that encodes the alpha 1 subunit of the voltage dependent sodium channels (SCN1A mutations are found in 79% of diagnosed DS patients). The alpha subunits form the transmembrane pore in the channel. Typically, the SCN1A mutations cluster in the C-terminus of the protein and cause a loss of function, resulting in decreased activity in GABAergic inhibitory neurons (see Rossi (2014) Epil Curr 14(4): 189-190). Additionally the activity of the Navl .6 sodium channels is reduced in DS. Unfortunately, DS is refractory to most current anti-epileptic medications. Symptoms appear in the first year of life as prolonged seizure events. At two years of age, patients begin suffering from a variety of other seizure types and developmental milestones begin to plateau and then regress during this phase.
Eventually the symptoms include motor and balance issues, delayed language and speech, growth and nutrition issues, sleeping difficulties, and chronic infections. In addition to SCN1 A, the SCN8A gene may also be involved in DS, as it has been found that increased seizure resistance is found in mice with mutations in the mouse Scn8A gene. Thus, SCN8A may be a genetic modifier of DS (see Martin et al (2007) Hum Mol Gene 16(23):2892-2899). Hence, the methods and compositions of the invention can be used to prevent or treat DS. Engineered transcription factors can be used to down regulate expression of SCN8A in the brain to decrease seizure frequency. Engineered nucleases could be designed to cleave the mutant SCN1 A alleles to knock out expression. Alternatively or in addition, a wild type cDNA encoding the SCN1A gene could be introduced into the brain using various methods known in the art {e.g. through a viral vector such as AAV2) in addition to mutant SNC1A specific nucleases to cause expression of the wild type gene in place of the mutant. Stem cells corrected by the methods of the invention can also be introduced into specific seizure centers to treat or prevent Canavan disease. [0038] Another genetic disease of the nervous system is Spinal Muscular
Atrophy (SMA). SMA is the most frequent genetic cause of death in infants and toddlers (approximately 1 in 6-10,000 births) and involves progressive and symmetric muscle weakness involving the upper arm and leg muscles as well as the muscles of the head and trunk and intercostal muscles. Additionally there is degeneration of the motor neurons in the spinal cord. SMA onset has been divided into three categories as follows: Type I, the most common, has an onset at about 6 months of age and results in death by about 2 years; Type II has an onset between 6 and 18 months where the patient can have the ability to sit up, but not walk; and type III, which has an onset after 18 months, where the patients have some ability to walk for some amount of time. 95% of all types of SMA are tied to a homozygous loss of the survival motor neuron 1 (SMNl) protein. The severity of SMA can be offset by the expression of the SMN2 protein, which is nearly identical to SMNl except for a single mutation that plays a role in the splicing of the RNA message. SMN2 is truncated however and rapidly degraded so while high expression of SMN2 may partially alleviate the loss of SMNl, it is not fully able to compensate (see lascone et al (2015) F1000 Pri Rep 7:04). In fact, there appears to be an inverse correlation with the amount of SMN2 mRNA and the severity of the SMA disease. Since SMA is associated with a homozygous loss of the SMNl gene, some researchers have tried introducing the SMNl gene via an AAV9 viral vector in animal models of SMA (see Bevan et al (2011) Mol Ther 19(11): 1971-1980). This early work showed that the gene could be delivered either through IV administration or through direct injection into the cerebral spinal fluid. However, penetration of the virus and complications relating to the crossing of the blood brain barrier still exist. The methods and compositions of the invention can be used to prevent or treat SMA. Engineered transcription factors specific for SNM2 may be designed to increase the expression of this gene. Engineered nucleases can also be used to cleave and correct the SMN2 mutation and cause stable expression by essentially turning it into the SMNl gene. Furthermore, a wild type SMNl cDNA may be inserted into the genome by targeted insertion using an engineered nuclease. The wild type SMNl gene may be inserted into the endogenous SMNl gene and thus be expressed under the regulation of the SMNl promoter, or it may be inserted into a safe harbor gene {e.g. AAVSl). The gene may also be inserted via nuclease directed targeted integration into neuronal stem cells, where the engineered stem cells are then re-introduced into the patient such that the neurons that are derived from these stem cells function normally.
[0039] Crigler Najjir syndrome is a disease related to hyperbilirubinemia caused by an excess of unconjugated bilirubin in the blood which leads to severe neurological damage. The disease is tied to deficiency in the uridine-diphosphate (UDP) glucuronosyltransferase 1 Al enzyme encoded by the UGT1 Al gene.
Mutations in the UGT1 Al gene that result in no expression cause the disease, and current treatment is limited to phototherapy treatment for 10-12 hours per day. The lack of UDP1A1 leads to high concentrations of unconjugated bilirubin in the blood. Normally the enzyme acts on bilirubin to make it more water soluble and thus allows the molecule to be more easily eliminated from the body. Mutations in the promoter region of UGT1A1 may also be tied to a closely related, but milder disease known as Gilbert syndrome. UGT1A1 is most highly expressed in the liver. In rat models of Crigler Najjir disease (the so-called Gunn rat) adenoviral or lentiviral vectors have been able to deliver the UCT1A1 gene to the liver and normalize plasma bilirubin levels (see van der Wegen (2005) Mol Ther 13(2) p. 374). Additional studies using delivery by AAV have also shown promise (see Bortolussi et al (2014) Hum Gen Ther 25:844-855). The methods and compositions of the invention provide a treatment for both Crigler Najjir and Gilbert syndromes. Engineered nucleases can be used to insert the UGT1A1 gene into the albumin promoter in the liver and cause expression of the enzyme such that serum bilirubin levels are dropped. Alternatively, the gene can be inserted into other safe harbor loci in the liver {e.g. AAVS1).
[0040] Opiate addiction is undergoing a resurgence and has emerged as an epidemic, especially in parts of the United States. It is estimated that between 26 and 36 million people worldwide participate in opiate abuse, with an estimated 2.1 million people in the United States suffering from substance use disorders related to prescription opioid pain relievers in 2012, and an estimated 467,000 people in the US are addicted to heroin (see United Nations Office on Drugs and Crime, World Report 2012 and Substance Abuse and Mental Health Services Administration, NSDUG Series H-46, HHS Publication No (SMA) 13-4795). In the brain, the primary site of action of nearly all analgesic and addictive opiates is the mu opioid receptor (MOP-r, encoded by the OPRM1 gene). The endogenous ligands for this receptor are the peptide products of the precursor proteins proopiomelanocortin (POMC) and proenkephalin (PENK). The endogenous opioid system also includes two other receptors, delta (DOP-r, encoded by OPRD1) and kappa (KOP-r, encoded by
OPR 1). It has been reported that mutations in the endogenous opiate system may play a role in the susceptibility of an individual to opiate addiction, and the success of treatment from addiction (see Reed et al (2014) Curr Psychiatry Rep 16:504). For example, the Al 18G SNP in OPRMl results in the Asn40Asp amino acid substitution in MOP-r and is associated with a substantial reduction of OPRMl mRNA
expression, and thus reduced levels of MOP-r. The MOP-r that is produced also has an increased binding affinity for its natural ligand. Importantly, the Al 18G SNP results in an altered response in the stress and hypothalamic-pituitary-adrenal axis in humans. This alone is thought to be a major factor in the vulnerability to specific addictions. In fact, the Al 18G SNP is associated with a substantial increase in the vulnerability to heroin addiction (Reed, ibid). Polymorphisms in other genes associated with the endogenous opiate system such as OPRK1 and OPRDlmay also prove to be factors leading to susceptibility to opiate addiction. Additionally, transcription factors that are elevated in response to stimulation of MOP-r such as
ELKl (tied to many cellular processes such as cell division, differentiation, migration and apoptosis) are elevated to a greater extent in people with the Al 18G SNP than in controls (Sillivan et al (2013) Biol Pyschiatry 74(7):5110519).
[0041] The mu opiate receptor (MOP-r) has also been tied to other disorders of the brain. For example, borderline personality disorder (BPD, shown to be present by epidemiological data in 1-5% of the population) may be tied to a dysregulation of the endogenous opiate system. Patients with BPD suffer from a dysregulation of emotional processing and MOP-r is implicated in emotional and stress response regulation (see Prossin et al (2010) Am J Psychiatry 167:925-933). Studies have demonstrated that BPD patients at baseline have an increase in MOP-r availability then controls, although the OPRMl genotype was not investigated. This increased MOP-r availability is thought to cause a greater activation of stress and emotional responses leading to the dysregulation observed in BPD patients. BPD patients have a marked increase in morbidity and mortality that includes risk for suicide and over the long term, they have severe and persistent impairment in social functioning. BPD patients also display an increased susceptibility to opiate addiction as nearly 40% have a co-diagnosis of drug use disorder (see Panagopoulos et al (2013) Drug Alcohol Depend 128(3)). Both opiate addiction and BPD represent chronic states that have enormous impact on patients over a lifetime. Thus the methods and compositions of the invention can be used to prevent or treat dysfunction in the endogenous opiate system that are associated with opiate addiction and/or BPD. Engineered
transcription factors comprising DNA binding domains specific for OPRM1 may be used to repress the expression of dysfunctional receptors in opiate addicts and BPD patients. Similarly, engineered nucleases may be used to knock out or correct specific genes encoding mutant receptors such as the Al 18G OPRM1 variant. Nucleic acids encoding these engineered proteins may be delivered to the brain via viral delivery systems. In addition, wild type genes encoding MOP-r may be delivered to specific areas of the brain known to be highly active in the pathology of addiction and/or BPD and inserted into the genome via nuclease targeted integration to treat or prevent these disorders. Similarly, stem cells corrected by the methods of the invention can be introduced into the brain that are associated with addiction and/or BPD to treat or prevent these conditions.
[0042] Major depressive disorder (MDD) is another common psychiatric illness with high levels of morbidity and mortality. Approximately 10 to 15 percent of the population will experience clinical depression during their lifetime, and twin studies suggest that MDD has a heritability of 40 to 50%. Due to the response of MDD patients to medications related to serotonin, several investigators have examined the serotonin system in MDD. Some studies have implicated variants in the serotonin transporter (5HTT/SLC6A4) and serotonin receptor 2A (HTR2A) as potential targets for the treatment of MDD. For example, a 44 bp repeat
polymorphism in the promoter region of SLC6A4 may be implicated in MDD (Lohoff (2010) Curr Psychiatry Rep 12(6):539-546). BDNF (brain derived neurotrophic factor) is another gene that has been suggested to have a tie to MDD. For example, the Val66Met mutation in the BDNF protein may be implicated in MDD. Other genes, associated with the synthesis of serotonin in the brain such as tryptophan hydroxylase (encoded by TPH2) may be targeted for the treatment of MDD. The Arg441His variant in TPH2 results in an 80% reduction of serotonin production in vitro, and so may play a similar role in MDD in vivo (Lohoff, ibid). Thus, the methods and compositions of the invention may be used to prevent or treat MDD in a patient. Mutant alleles in SLC6A4, HTR2A and/or TPH2 may be targeted by the engineered transcription factors or nucleases of the invention to repress or knock out expression. Wild type genes for these loci may also be introduced via nuclease- dependent targeted integration. Similarly, stem cells corrected by the methods of the invention can be introduced into the brain. Corrective measures (i.e. engineered transcription factors, nucleases, donor DNAs, stem cells) can be targeted in areas of the brain that are associated with MDD.
[0043] Schizophrenia (SZ) does not appear to be linked to a single gene, yet while it occurs in 1% of the general population, a family history of psychosis increases the risk to about 10% of people with a parent or sibling with SZ. Twin studies have also shown that an identical twin has about a 50% chance of having SZ if their twin has it. Overall, 80% of SZ patients appear to have a genetic component to their disease. Early studies showed evidence of SNPs in the DTNBP1 (dystrobrevin- binding protein 1 or dysbindin) were strongly associated with SZ (Straub et al (2002) Am J Hum Genet 71 :337-348). More recently, genome-wise association studies have found 108 independent associated loci that are associated with SZ, and of these 108, 75%) were located in protein coding genes (see Ripke et al (2014) Nature 511(7510): 421-427). Notably, associations were found with the DRD2 gene (currently the target of all effective anti-psychotic drugs), and genes involved in glutamatergic
neutrotransmission and synaptic plasticity (e.g. GRM3, GRIN2A, SRR, GRIAl). Associations were also seen with voltage gated calcium channel subunits (e.g.
CACNA1C, CACNB2, CACN1 II). SZ also may be associated with epigenetic regulation. An observed down regulation of glutamic acid decarboxylase67 (GADl), reelin (RELN) and BDNF expression in the brain of patients suffering from SZ is associated with overexpression of DNA methyltransferase 1 (DNMT1) and ten-eleven translocase methylcytosine dioxygenase 1 (TET1). DNMT1 and TETl encode enzymes that methylate and hydroxymethylate cytosines near and within cytosine phosphodiester guanine (CpG) islands of many gene promoters. Other evidence suggests that expression of GADl is epigenetically regulated in specific regions of the brain by epigenetic mechanisms (see Mitchell (2014) Schizophr Res
doi: 10.1016/j.schres 2014). Thus, the methods and compositions of the invention can be used to prevent or treat SZ. Specific nucleases of the invention may be designed to target disease associated SNPs such as in DTNBP1 to knock out disease associated alleles. Further, specific transcription factors and/or nucleases can be engineered to modulate specific alleles of DRD2, GRM3, GRIN2A, SRR, GRIAl, CACNA1C, CACNB2, CACN1 II, GADl, RELN, BDNF, TET1, and DTNBP1. Nucleic acids encoding these modulators can be injected into the brain in the regions of the brain known to be most affected by SZ, or they can be delivered via viral vectors. Similarly, stem cells corrected by the methods of the invention can be introduced into the brain and used to treat or prevent SZ.
[0044] Bipolar disorder (BD) is another extremely debilitating psychiatric disorder affecting about 1% of the population worldwide, and is associated with increased morbidity and mortality that appears to have a strong genetic component. Among mental disorders, BD is associated with the highest risk of suicide and is characterized by cyclothymic and irritable temperaments, leading to a rate of suicide that is up to 20 times that of the average population (see Dwivedi and Zhang (2015) Front Neurosci 8(457)). Genome wide association studies have been performed and have identified a number of SNPs in specific chromosomal regions in the genome. These include ANK3, ODZ4, TRANK1, ADCY2, CACNA1C and a region of the genome between MIR2113 and POU3F2 (see Muhleisen et al (2014) Nat Com 5, article number 3339; and Ferreira et al (2008) Nat Genet 40(9): 1056-1058).
Epigenetic regulation of BDNF expression also seems to play a role in BD. BD patients often respond favorably to therapeutic lithium treatment, and it appears that one of the mechanisms of lithium is to cause hypomethylation of the BDNF exon IV promoter resulting in increased BDNF expression. Thus, the methods and
compositions of the invention can be used to prevent or treat BD. Specific nucleases of the invention may be designed to target disease associated SNPs such as in ANK3, ODZ4, TRANK1 , ADCY2, and CACNA1 C to knock out disease associated alleles. Further, specific transcription factors can be engineered to modulate and increase the expression of BDNF. Nucleic acids encoding these modulators or stem cells corrected by the methods of the invention can be introduced into the brain in the regions of the brain known to be most affected by BD, or they can be delivered via viral vectors.
[0045] Migraine is the most common disorder in the NS, affecting
approximately 14% of the adult population. Genome wide association studies pooling data from several studies (considering over 23,000 individual migraine suffers) have identified 12 loci that have an association with migraine. These include SNP rs2651899 in PRDM16, rsl0915437 near AJAPl, rsl2134493 in MEF2D, rs7577262 in TRPM8, rs6790925 near TGFBR2, rs9349379 in PHACTR1, rsl3208321 in FHM5, rs4379368 in c7orfl0, rsl0504861 near MMP16, rs64782541 in ASTN2, rsl2134493 near TSPAN2, rs701567 near GFRA2 and rsl 1172113 in LRP1 (See Anittila et al (2013) Nat Genet 45(8):912-917). Interestingly, eight of the 12 identified loci are located in or immediately outside genes with known function in synaptic or neuronal regulation and several exert regulatory control on one another, suggesting a common circuitry. Thus, the methods and compositions of the invention may be used to prevent or treat chronic migraine in patients. Engineered transcription factors and nucleases may be designed to repress or knockout mutant genes known to induced recurrent migraine. In addition or alternatively, wild type genes may be inserted in to safe harbor loci of cells in the brain via nuclease dependent targeted integration to cause expression of wild type proteins. Similarly, stem cells corrected by the methods of the invention can be introduced into the brain in the areas know to be associated with migraine to treat or prevent the disease.
[0046] Genetic factors contributing to multiple sclerosis (MS) are not well characterized although there is strong evidence that a genetic link exists. MS is a chronic inflammatory autoimmune disease believed to arise from a complex interaction of both environmental and genetic factors and manifests in different forms and severity. The genes encoding the major histocompatibility complex (MHC) are associated with susceptibility to MS, in particular the HLA-DRB1 allele HLA- DRB1 * 1501 (SNP rs3135388, Lincoln et al (2005) Nat Genet 37(10): 1108-12) as are the genes IL7Ra and IL2Ra (Tan et al (2014) Ann Trans I Med 2(12): 124). Other genome wide association studies have found two mutant alleles in two additional genes that may be candidates for the methods and compositions of the invention. In the gene CYP27Ba, which encodes the vitamin-D activating 1 -alpha hydroxylase enzyme, a loss of function variant has been identified (R389H) which seems to be associated with susceptibility to MS. Another SMP (rs55762744) found in TYK2, also may be associated with a high frequency of developing MS. TYK2 encodes a tyrosine kinase that modulates the function of multiple immune related genes, and mutations in this gene may lead to MS (see Jiang et al (2014) Ann Trans I Med 2(12): 125). Thus, the methods and compositions of the invention may be used to prevent or treat MS. Engineered transcription factors and/or nucleases can be used to repress or knockout mutant alleles associated with MS such as HLA-DRB1 * 1501. Additionally, wild type genes can be inserted into the genome via nuclease assisted targeted integration in safe harbor genes where the genes and nucleases are delivered to the brain in the regions most associated with MS phenotype. Stem cells corrected by the methods of the invention can also be used. Delivery can be via direct introduction of cells, or via viral vectors. [0047] Tourette syndrome (TS) is a neuropsychiatric disorder characterized by repetitive, involuntary movements and vocalizations called tics and is often accompanied by obsessive-compulsive disorder and/or attention-deficit/hyperactivity disorder. Worldwide prevalence of TS is estimated to be between 0.3 and 1% of the population and the heritability behind TS appears to be complex. Several researchers have identified multiple rare copy number variants (CNVs) including genomic deletions and duplications that may play a role in TS. Several exon-affecting rare CNVs were found in one study and three had previously been implicated in studies related to schizophrenia, autism and attention-deficit hyperactivity disorder. The five loci were in the NRXN 1 , AADAC, CTNNA3 , FSCB and KCHE 1 , KCHE2, RCAN 1 genes. A deletion of the 5' exon of the neurexin 1 gene (NRXN1) is associated with TS (also with autism, schizophrenia) as is deletion of the 5' region of microsomal arylacetamide deacetylase (AADAC). Deletion of 450 Kb of the FSCB gene
(encoding fibrous sheath CABYR-binding protein) was also associated with TS as was a duplication of genes involved with voltage-gated potassium channels (KCNE 1 , KCNE2) and a regulator of calcineutin 1 (RCANl). Some of these genes encode cell adhesion molecules (e.g. NRXN1) which may play a role in linking synaptic cell adhesion to cognition (Sundaram et al (2010) Neurology 74(20):1583). Thus, the methods and compositions of the invention can be used to prevent or treat TS.
Engineered transcription factors and/or nucleases can be used to repress or knockout mutant alleles associated with TS such as those in NRXN1, AADAC, CTNNA3, FSCB and KCHEl, KCHE2, RCANl . Additionally, wild type genes can be inserted into the genome via nuclease assisted targeted integration in safe harbor genes or corrected stem cell, where the genes, nucleases and/or stem cells are delivered to the brain in the regions most associated with TS phenotype. Delivery can be via direct introduction or via viral vectors.
[0048] Epilepsy affects about three percent of the population and comprises a wide constellation of primary and syndromic neurological disorders. The epileptic encephalopathies (EE) are the most severe of the epilepsies, distinguished by multiple refractory seizures, cognitive deficit, and poor developmental outcome. De novo mutations in several genes are known causes of EE, but the genetic etiology of the vast majority of these encephalopathies is unknown. People with epilepsy tend to suffer from recurrent seizures due to excessive neuronal discharge. It appears that genetic factors play a role in 40% of epilepsies, however it has been difficult to identify the specific genes involved. In a study of Han Chinese subjects, two highly correlated SNPs, rs2292096 and rs6660197 located in the CAMSAP1LK1 gene showed a relationship with epilepsy. CAMSAP1L1 encodes a cytoskeletal protein (Tan et al, 2014, ibid). Some approaches to epilepsy embrace the idea of using gene therapy to treat the symptoms of the disease such as anti-seizure approaches {e.g. to decrease the effect of excitatory neurotransmitters such as antisense approaches against NMDA receptor subunit 1 , or increase the inhibition of neurotransmission such as GAMA-A receptor subunit alpha- 1, GAD65 or adenosine kinase) or disease modifying approaches. For example, overexpression of an endogenous molecule with anticonvulsant properties at the area in the brain associated with seizure onset could modify the frequency and intensity of seizures. Enhanced release of neurotrophic factors such as GDNF, BDNF, and FGF also could be helpful. Neuropeptides and as galanin and neuropeptide Y (NPY) could be promising candidates for such a purpose {see Noe et al, 2012. Noebels et al, editors. Jaspar's Basic Mechanisms of the Epilepsies. 4th Edition. Bethesda (MD): National Center for Biotechnology
Information). Thus the methods and compositions of the invention can be used to prevent or treat epilepsy. Approaches that are helpful to decrease excitatory neurotransmitters can be carried out where the engineered transcription factors and/or nucleases of the invention are used to repress or knock out NMDA receptor subunit 1 , GAMA-A receptor subunit alpha- 1, GAD65 or adenosine kinase. Other uses of these methods and compositions can be to repress or knock out specific mutant alleles associated with SNPs in the CAMSAP1LK1 gene. In addition, genes encoding transcription factors to increase the expression of GCNF, BDNF and IGF may be administered in a localized fashion at the seizure centers within the brain. Similarly, cDNAs encoding anti-convulsant peptides such as NPY and galanin can be inserted via nuclease driven targeted integration in to seizure centers to inhibit the frequency and magnitude of seizures.
[0049] Thus, alterations to genes encoding proteins involved in NS disorders using the methods and compositions as described herein can be used to correct an aberrant endogenous gene or insert a wild type gene at a desired location in the genome of a cell {e.g., into a "safe harbor" gene of an HSC). Precursor cells can be derived from subjects in need, modified ex vivo, and then given back to the subject either in a bone marrow graft. Alternatively, polynucleotides encoding the fusion proteins of the invention can be delivered to the neural tissue for correction, modification of expression, gene silencing and/or targeted insertion of a gene for the treatment or prevention of a nervous system disorder.
General
[0050] 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
[0051] 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.
[0052] 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.
[0053] "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 (Kj) of 10~6 M"1 or lower. "Affinity" refers to the strength of binding:
increased binding affinity being correlated with a lower IQ.
[0054] 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.
[0055] 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.
[0056] 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.
[0057] Zinc finger and TALE 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 or TALE protein. Therefore, engineered DNA binding proteins (zinc fingers or
TALEs) are proteins that are non-naturally occurring. Non-limiting examples of methods for engineering DNA-binding proteins are design and selection. A designed DNA binding 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 and/or TALE designs and binding data. See, for example, U.S. Patents 8,586,526; 6,140,081; 6,453,242; 6,746,838; 7,241,573; 6,866,997; 7,241,574 and 6,534,261; see also and
WO 03/016496.
[0058] 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. Patent Nos. 8,586,526; 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,200,759; 6,242,568; 6,733,970;7,297,491;
WO 98/53057; WO 02/099084.
[0059] "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, for example, guide DNAs for cleavage by a TtAgo enzyme. "Recombination" refers to a process of exchange of genetic information between two polynucleotides, including but not limited to, donor capture by non-homologous end joining (NHEJ) and homologous recombination. For the purposes of this disclosure, "homologous recombination (HR)" refers to 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.
[0060] "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 re-synthesize 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.
[0061] 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.
[0062] In any of the methods described herein, additional pairs of zinc-finger or TALEN proteins can be used for additional double-stranded cleavage of additional target sites within the cell. In addition, a CRISPR/Cas system may be similarly employed to induce additional double strand breaks.
[0063] 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.
[0064] In any of the methods described herein, the exogenous nucleotide sequence (the "donor sequence" or "transgene") 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.
[0065] 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. Cells and cell lines with partially or completely inactivated genes are also provided.
[0066] 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 non-coding sequence, as well as one or more control elements (e.g., promoters). In addition, the exogenous nucleic acid sequence may produce one or more R A molecules (e.g., small hairpin R As (shR As), inhibitory RNAs (RNAis), microRNAs (miRNAs), etc.).
[0067] "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.
[0068] 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.
[0069] 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 and 8,823,618, incorporated herein by reference in their entireties.
[0070] 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.
[0071] A "NS-disease associated gene" is one that is defective in some
manner in subject with a NS disorder.
[0072] "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.
[0073] 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.
[0074] 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.
[0075] 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.
[0076] 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.
[0077] 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.
[0078] 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. 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.
[0079] 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.
[0080] 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 DNA-binding domain (e.g., ZFP, TALE and/or meganuclease DNA-binding domains) and a functional domain (e.g., endonuclease, meganuclease, ZFP-transcription factor, (ZFP-TF), TALE- transcription factor (TALE-TF), CRIPSR/Cas transcription factor (CRISPR/Cas-TF) etc.) and fusion nucleic acids (for example, a nucleic acid encoding a 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. [0081] 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.
[0082] 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.
[0083] "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.
[0084] "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 or TALE. . Thus, gene inactivation may be partial or complete.
[0085] 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.
[0086] "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).
[0087] 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.
[0088] 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 (ZFP, TALE) is fused to a cleavage domain (e.g., endonuclease domain such as Fokl, meganuclease domain, etc.), 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 (nuclease) domain is able to cleave DNA in the vicinity of the target site. The nuclease domain may also exhibit DNA-binding capability (e.g., a nuclease fused to a ZFP or TALE domain that also can bind to DNA). 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 up regulate gene expression or the repression domain is able to down regulate gene expression. In addition, a fusion polypeptide in which a Cas DNA-binding domain is fused to an activation domain, the Cas DNA-binding domain and the activation domain are in operative linkage if, in the fusion polypeptide, the Cas 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 Cas DNA-binding domain is fused to a cleavage domain, the Cas DNA-binding domain and the cleavage domain are in operative linkage if, in the fusion polypeptide, the Cas 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.
[0089] 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. (1989) Nature 340:245-246; U.S. Patent No. 5,585,245 and PCT WO 98/44350.
[0090] 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.
[0091] The terms "subject" and "patient" are used interchangeably and refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, dogs, cats, rats, mice, rabbits and other animals. Accordingly, the term "subject" or "patient" as used herein means any patient or subject (e.g., mammalian) having a NS disorder. Compositions for Altering Gene expression
[0092] Described herein are compositions, for example transcriptional regulators and/or nucleases, which are useful targeting a gene that encodes a protein involved in a NS disorder, for example nucleases that facilitate targeted correction of a mutant gene, targeted inactivation of a gene and/or targeted integration (e.g., of a gene encoding a protein that is aberrantly expressed in the subject with the NS disorder). The compositions can comprise fusion proteins, for example DNA-binding domains fused to functional domains (e.g., transcriptional activation domains, transcriptional repression domains and/or nucleases) or nuclease or transcription factor systems such as the CRISPR/Cas system. Also described are compositions comprising cells that are genetically modified using the proteins and systems described herein and methods of using these cell compositions to treat and/or prevent a nervous system disorder.
A. DNA-binding domains
[0093] Any DNA-binding domain can be used in the nucleases used in the compositions and methods disclosed herein, including but not limited to a zinc finger DNA-binding domain, a TALE DNA binding domain, or a DNA-binding domain from a meganuclease, or a CRIPSR/Cas DNA binding complex.
[0094] In certain embodiments, the composition comprises a DNA-binding domain and/or nuclease (cleavage) domain from a meganuclease (homing
endonuclease). 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-Cyst box family and the HNH family.
Exemplary homing endonucleases include l-Scel, l-Ceul, Fl-Pspl, Fl-Sce, 1-SceTV, I- Csml, l-Panl, l-Scell, l-Ppol, l-Scelll, l-Crel, l-Tevl, l-Tevll and I-7¾vIII. 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 i?t¾.25:3379-3388; Dujon et a/. (1989) GeneSl: 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. 5/o/.280:345-353 and the New England Biolabs catalogue.
[0095] In certain embodiments, the homing endonuclease (meganuclease) is engineered (non-naturally occurring). The recognition sequences of homing endonucleases and meganucleases such as l-Scel, l-Ceul, Fl-Pspl, Fl-Sce, l-SceTV, I- Csml, l-Panl, l-Scell, l-Ppol, 1-SceIII, l-Crel, l-Tevl, l-Tevll and I- ZevIII 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.25:3319-33%%; Dujon et a/. (1989) GeneSl: 115-118; Perler et a/.(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.
5z'o/.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, U.S. Patent No. 8,021,867; Chevalier et al. (2002) Molec. Cell 10:895-905; Epinat et a/. (2003) Nucleic Acids Rey.31:2952- 2962; Ashworth et al. (2006) Nature 441:656-659; and Paques et al. (2007) Current Gene Therapy! :49-66. The DNA-binding domains of the homing endonucleases and meganucleases may be altered in the context of the nuclease as a whole (i.e., such that the nuclease includes the cognate cleavage domain) or may be fused to a heterologous DNA-binding domain (e.g., zinc finger protein or TALE) or to a heterologous cleavage domain. DNA-binding domains derived from meganucleases may also exhibit DNA-binding activity.
[0096] In other embodiments, the DNA-binding domain comprises a naturally occurring or engineered (non-naturally occurring) TAL effector DNA binding domain. See, e.g., U.S. Patent No. 8,586,526, incorporated by reference in its entirety herein. 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 318:648-651).
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) Appland 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] Thus, in some embodiments, the DNA binding domain that binds to a target site in a target locus is 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 Nos.8,586,526; 8,420,782 and 8,440,431.
[0098] In certain embodiments, the DNA binding domain comprises a zinc finger protein {e.g., a zinc finger protein that binds to a target site in a gene involved in a NS disorder or safe-harbor gene). 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, See, for example, Beerli et al. (2002) Nature Biotechnol. 20: 135-141; Pabo et al. (2001) Ann. Rev. Biochem 0:3 l3-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416; U.S. Patent Nos. 7,888,121; 7,972,854; 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, all incorporated herein by reference in their entireties.
[0099] An engineered zinc finger binding or TALE 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, co-owned U.S. Patents 8,586,526; 6,453,242 and 6,534,261 , incorporated by reference herein in their entireties.
[0100] Exemplary selection methods, including phage display and two-hybrid systems, are disclosed in U.S. Patents 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 co-owned WO 02/077227.
[0101] In addition, as disclosed in these and other references, DNA domains
(e.g., multi-fingered zinc finger proteins or TALE domains) 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 DNA binding 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 co-owned WO 02/077227.
[0102] Selection of target sites; DNA-binding domains 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. 8,586,526; 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 98/5431 1 ; 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.
[0103] In addition, as disclosed in these and other references, DNA-binding domains (e.g., 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. [0104] In still further embodiments, the DNA binding domain comprises a single-guide R A in combination with a CRISPR/Cas nuclease system or a
CRISPR/Cas transcription factor.
B. Functional Domains
[0105] The DNA-binding domains may be operably linked to any functional domain.
[0106] In certain embodiments, the functional domain comprises a
transcriptional regulatory domain, including an activation domain or a repression domain. 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 al. (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,
CPRF1, CPRF4, MYC-RP/GP, and TRAB1. See, for example, Ogawa et al. (2000) Gene 245:21-29; Okanami et a/.(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 al. (1999) Proc. Natl. Acad. Sci. USA 96:5844-5849; Sprenger-Haussels et al. (2000) Plant J. 22: 1-8; Gong et a/.(1999) Plant Mol. Biol. 41 :33-44; and Hobo et al. (1999) Proc. Natl. Acad. Sci. USA 96: 15,348-15,353.
[0107] 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 as described herein 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 co-owned U.S. Patent Nos. 6,919,204 and 7,053,264.
[0108] 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.
[0109] In certain embodiments, the target site bound by the DNA-binding domain is present in an accessible region of cellular chromatin. Accessible regions can be determined as described, for example, in U.S. Patent No. 6,511 ,808. 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 co-owned WO 01/83793. 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 60:719- 731; and Cirillo et al. (1998) EMBO J. 17:244-254.
[0110] In other embodiments, the functional (regulatory) domain comprises a nuclease (cleavage) domain. Any suitable cleavage domain can be operatively linked to any DNA-binding domain to form a nuclease. For example, ZFP DNA-binding domains have been fused to nuclease domains to create ZFNs - a functional entity that is able to recognize its intended nucleic acid target through its engineered (ZFP) DNA binding domain and cause the DNA to be cut near the ZFP binding site via the nuclease activity. See, e.g., Kim et al. (1996) Proc N at Ί Acad Sci USA 93(3):1156- 1160. See, for example, U.S. Patent Nos. 7,888,121 ; 7,972,854; 7,914,796;
7,951,925; 8,110,379; 8,409,861; 8,586,526; U.S. Patent Publications 20030232410; 20050208489; 20050026157; 20060063231; 20100218264; 20120017290;
20110265198; 20130137104; 20130122591; 20130177983 and 20130177960.
Likewise, TALE DNA-binding domains have been fused to nuclease domains to create TALENs. See, e.g., U.S. Patent No. 8,586,526.
[0111] As noted above, the cleavage domain may be heterologous to the
DNA-binding domain, for example a zinc finger DNA-binding domain and a cleavage domain from a nuclease or a TALEN DNA-binding domain and a cleavage domain, or 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 i?t¾.25:3379-3388. 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.
[0112] In certain embodiments, the nuclease is naturally occurring. In other embodiments, the nuclease is non-naturally occurring, i.e., engineered in the DNA- binding domain and/or cleavage domain. For example, 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). In other embodiments, the nuclease comprises heterologous DNA- binding and cleavage domains (e.g., zinc finger nucleases; TAL-effector nucleases; meganuclease DNA-binding domains with heterologous cleavage domains), or a generic nuclease guided by a specific guide RNA (e.g. a CRPISR/Cas).
[0113] 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.
[0114] In some embodiments, a Cas protein may be linked to a heterologous nuclease domain. In some aspects, the Cas protein is a Cas9 protein devoid of nuclease activity linked to a Fokl nuclease domain such that double strand cleavage is dependent on dimerization of the Fokl nuclease domains.
[0115] 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, US Patents 5,356,802; 5,436,150 and 5,487,994; as well as Li et a/.(1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. t/&490:2764- 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.
[0116] 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. USA95: 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 fmger- o 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 DNA binding domain and two Fok I cleavage half-domains can also be used.
[0117] 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.
[0118] Exemplary Type IIS restriction enzymes are described in U.S.
Publication No. 20070134796, 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 R&y.31:418-420.
[0119] 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. 7,888,121; 8,409,861; 7,914,796; and 8,034,598, 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.
[0120] 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.
[0121] 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 Nos. 7,888,121; 8,409,861; 7,914,796; and 8,034,598 and U.S. Patent Publication No. 20120040398, the disclosures of which are incorporated by reference in their entireties for all purposes. .
[0122] 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 "KK " 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 U.S. Patent No. 8,623,618, incorporated by reference herein).
[0123] Engineered cleavage half-domains described herein can be prepared using any suitable method, for example, by site-directed mutagenesis of wild-type cleavage half-domains (Fok l) as described in U.S. Patent Nos. 7,888,121; 7,914,796 and 8,034,598.
[0124] 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. 20090068164). 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.
[0125] The nuclease domain may also be derived from a homing endonuclease
(meganuclease). Exemplary homing endonucleases include l-Scel, l-Ceul, Fl-Pspl, ΡΙ-Sce, I-SceW, l-Csml, l-Panl, l-Scell, l-Ppol, l-Scelll, l-Crel, I-Tevl, l-Tevll and I- Tevlll.
[0126] Thus, the nuclease as described herein can comprise any DNA-binding domain and any nuclease.
[0127] In certain embodiments, the nuclease comprises a zinc finger DNA- binding domain and a restriction endonuclease nuclease domain, also referred to as a zinc finger nuclease (ZFN).
[0128] In other embodiments, the nuclease comprises 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.
[0129] 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
meganuclease {e.g., 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 (cTALENs or Fokl-TALENs) with one or more mega-TALs).
[0130] Nucleases can be screened for activity prior to use, for example in a yeast-based chromosomal system as described in U.S. Patent No. 8,563,314. Nuclease expression constructs can be readily designed using methods known in the art. See, e.g., United States Patent Nos. 7,888,121 and 8,409,861; 20030232410;
20050208489; 20050026157; 20060063231; and 20070134796. Expression of the nuclease may be under the control of a constitutive promoter or an inducible promoter, for example the galactokinase promoter which is activated (de-repressed) in the presence of raffinose and/or galactose and repressed in presence of glucose.
[0131] In certain embodiments, the nuclease or transcription factor comprises a CRISPR/Cas system. See, e.g., U.S. Patent No. 8,697,359. 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.
[0132] The Type II CRISPR, initially described in S. pyogenes, 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 where processing occurs by a double strand-specific RNase III in the presence of the Cas9 protein. 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. In addition, the tracrRNA must also be present as it base pairs with the crRNA at its 3' end, and this association triggers Cas9 activity. 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.
[0133] Type II CRISPR systems have been found in many different bacteria.
BLAST searches on publically available genomes by Fonfara et al ((2013) Nuc Acid Res 42(4):2377-2590) found Cas9 orthologs in 347 species of bacteria. Additionally, this group demonstrated in vitro CRISPR/Cas cleavage of a DNA target using Cas9 orthologs from S. pyogenes, S. mutans, S. therophilus, C. jejuni, N. meningitides, P. multocida and F. novicida. Thus, the term "Cas9" refers to an RNA guided DNA nuclease comprising a DNA binding domain and two nuclease domains, where the gene encoding the Cas9 may be derived from any suitable bacteria.
[0134] The Cas9 protein has at least two nuclease domains: one nuclease domain is similar to a HNH endonuclease, while the other resembles a Ruv endonuclease domain. The HNH-type domain appears to be responsible for cleaving the DNA strand that is complementary to the crRNA while the Ruv domain cleaves the non-complementary strand. The Cas 9 nuclease can be engineered such that only one of the nuclease domains is functional, creating a Cas nickase (see Jinek et al, ibid). Nickases can be generated by specific mutation of amino acids in the catalytic domain of the enzyme, or by truncation of part or all of the domain such that it is no longer functional. Since Cas 9 comprises two nuclease domains, this approach may be taken on either domain. A double strand break can be achieved in the target DNA by the use of two such Cas 9 nickases. The nickases will each cleave one strand of the DNA and the use of two will create a double strand break.
[0135] The requirement of the crRNA-tracrRNA complex can be avoided by use of an engineered "single-guide RNA" (sgRNA) that comprises the hairpin normally formed by the annealing of the crRNA and the tracrRNA (see Jinek et al (2012) Science 337:816 and Cong et al (2013)
Sciencexpress/10.1126/science.1231143). In S. pyrogenes, the engineered
tracrRNA: crRNA fusion, or the sgRNA, guides the functional domain (e.g., Cas9 or other functional domain) to modify the target DNA, for example when a double strand RNA:DNA heterodimer forms between the Cas associated RNAs and the target DNA in the case of a nuclease. This system comprising the Cas9 protein and an engineered sgRNA containing a PAM sequence has been used for RNA guided genome editing (see Ramalingam ibid ) and has been useful for zebrafish embryo genomic editing in vivo (see Hwang et al (2013) Nature Biotechnology 31 (3):227) with editing efficiencies similar to ZFNs and TALENs.
[0136] Chimeric or sgRNAs can be engineered to comprise a sequence complementary to any desired target. In some embodiments, a guide sequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. In certain embodiments, the RNAs comprise 22 bases of complementarity to a target and of the form G[nl9], followed by a protospacer-adjacent motif (PAM) of the form NGG. Thus, in one method, sgRNAs can be designed by utilization of a known ZFN target in a gene of interest by (i) aligning the recognition sequence of the ZFN heterodimer with the reference sequence of the relevant genome (human, mouse, or of a particular plant species); (ii) identifying the spacer region between the ZFN half-sites; (iii) identifying the location of the motif G[N20]GG that is closest to the spacer region (when more than one such motif overlaps the spacer, the motif that is centered relative to the spacer is chosen); (iv) using that motif as the core of the sgRNA. This method advantageously relies on proven nuclease targets. Alternatively, sgRNAs can be designed to target any region of interest simply by identifying a suitable target sequence the conforms to the G[n20]GG formula. Along with the complementarity region, an sgRNA may comprise additional nucleotides to extend to tail region of the tracrRNA portion of the sgRNA (see Hsu et al (2013) Nature Biotech doi: 10.1038/nbt.2647). Tails may be of +67 to +85 nucleotides, or any number therebetween with a preferred length of +85 nucleotides. Truncated sgRNAs may also be used, "tru-gRNAs" (see Fu et al, (2014) Nature Biotech 32(3): 279). In tru-gRNAs, the complementarity region is diminished to 17 or 18 nucleotides in length.
[0137] Further, alternative PAM sequences may also be utilized, where a
PAM sequence can be NAG as an alternative to NGG (Hsu 2014, ibid) using a S. pyogenes Cas9. Additional PAM sequences may also include those lacking the initial G (Sander and Joung (2014) Nature Biotech 32(4):347). In addition to the S.
pyogenes encoded Cas9 PAM sequences, other PAM sequences can be used that are specific for Cas9 proteins from other bacterial sources. For example, the PAM sequences shown below (adapted from Sander and Joung, ibid, and Esvelt et al, (2013) Nat Meth 10(11): 1116) are specific for these Cas9 proteins:
Species PAM
S. pyogenes NGG
S. pyogenes NAG
S. mutans NGG S. thermophilius NGGNG
S.thermophilius NAAAW
S. thermophilius NAGAA
S. thermophilius NNGATT
C. jejuni NNNACA
N. meningitides NNNGATT
P. multocida G NNC NA
F. novicida NG
[0138] Thus, a suitable target sequence for use with a S. pyogenes
CRISPR/Cas system can be chosen according to the following guideline: [nl7, nl8, nl9, or n20](G/A)G. Alternatively the PAM sequence can follow the guideline G[nl7, nl8, nl9, n20](G/A)G. For Cas9 proteins derived from non-S. pyogenes bacteria, the same guidelines may be used where the alternate PAMs are substituted in for the S. pyogenes PAM sequences.
[0139] Most preferred is to choose a target sequence with the highest likelihood of specificity that avoids potential off target sequences. These undesired off target sequences can be identified by considering the following attributes: i) similarity in the target sequence that is followed by a PAM sequence known to function with the Cas9 protein being utilized; ii) a similar target sequence with fewer than three mismatches from the desired target sequence; iii) a similar target sequence as in ii), where the mismatches are all located in the PAM distal region rather than the PAM proximal region (there is some evidence that nucleotides 1-5 immediately adjacent or proximal to the PAM, sometimes referred to as the 'seed' region (Wu et al (2014) Nature Biotech doi: 10.1038/nbt2889) are the most critical for recognition, so putative off target sites with mismatches located in the seed region may be the least likely be recognized by the sg RNA); and iv) a similar target sequence where the mismatches are not consecutively spaced or are spaced greater than four nucleotides apart (Hsu 2014, ibid). Thus, by performing an analysis of the number of potential off target sites in a genome for whichever CRIPSR/Cas system is being employed, using these criteria above, a suitable target sequence for the sgRNA may be identified.
[0140] 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. In some aspects, a functional derivative may comprise a single biological property of a naturally occurring Cas protein. In other aspects, a function derivative may comprise a subset of biological properties of a naturally occurring Cas protein. 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.
[0141] Exemplary CRISPR/Cas nuclease systems targeted to specific genes are disclosed for example, in U.S. Publication No. 20150056705.
[0142] Thus, the nuclease comprises a DNA-binding domain in that specifically binds to a target site in any gene into which it is desired to insert a donor (transgene) in combination with a nuclease domain that cleaves DNA.
Target Sites
[0143] As described in detail above, DNA-binding domains (e.g., ZFPs,
TALEs, single-guide RNAs) can be engineered to bind to any sequence of choice in a locus, for example a gene encoding a protein that is involved in a NS disorder or a safe harbor gene.
[0144] In certain embodiments, the DNA-binding domains bind to a sequence in a gene encoding a protein that is involved in a NS disorder. Non-limiting examples of particular genes involved in NS disorders that may be targeted (including for alteration of gene expression via transcriptional regulation, correction of mutant genes and/or for targeted inactivation or integration into such genes) include microtubule associated protein tau (MAPT, encoding Tau proteins), apolipoprotein E (APOE) alleles(e.g., apoE2, apoE3 or apoE4), amyloid precursor protein (APP), presenilin 1, presenilin 2, SLC6A4, HTR2A, CACNAIC, CACNB2, dystrophia myotonica-protein kinase (DMPK), CACNA1A, ATP1A2, SCN1A; FXN; HTT, PMP22, dystrophin, utrophin; C9orf72, SODl, TARDBP, FUS, ANG, ALS2, SETX, progranulin gene (GRN), VAPB; FMR1, LRRK2, alpha-synuclein and/or HPRT. See, also, Table 1 below, where "KO" refers to knock-out (inactivation) of the gene using one or more
Table 1: Exemplary NS targets
Figure imgf000055_0001
Transcriptional
modulation or KO of
Dravet Syndrome SCN1A, SCN8A
mutant alleles, insertion of WT transgene
Transcriptional modulation or KO of
Spinal Muscular Atrophy
SMN1, SMN2 mutant alleles, insertion (SMA)
of WT transgene, corrected stem cell
Insertion of a WT
Crigler Najjar Syndrome UGT1A1
transgene
Transcriptional
Borderline Personality modulation or KO of
OPRM1
Disorder mutant alleles, corrected stem cell
ANK3, ODZ4, TRANK1, Transcriptional
Bipolar Disorder ADCY2, CACNA1C, modulation or KO of
BDNF mutant alleles
Transcriptional modulation or KO of
CYP27B1, TYK2, HLA-
Multiple Sclerosis mutant alleles, insertion
DRB1, IL7Ra, IL2Ra
of WT transgene, corrected stem cell
Transcriptional
NRXN1, AADAC, modulation or KO of
Tourette's Syndrome CTNNA3, FSCB, KCHE1, mutant alleles, insertion
KCHE2, RCAN1 of WT transgene,
corrected stem cell
ORDM16, AJAP1, Transcriptional
MEF2D, TRPM8, modulation or KO of
Migraine TGFBR2, PHACTR1, mutant alleles, insertion
FHM5, MMP16, ASTN2, of WT transgene, TSPAN2, GFRA2, LRP1 corrected stem cell
Transcriptional modulation or KO of
Major Depressive Disorder SLC6A4, HTR2a, TPH2 mutant alleles, insertion of WT transgene, corrected stem cell
AMSAP1K1, NMDA Transcriptional receptor 1, GAMA-A modulation, KO of
Epilepsy
receptor, GAD65, mutant alleles adenosine kinase
Transcriptional
Epilepsy GDNF, BDNF, FGF, NPY modulation, insertion of transgene
DRD2, GRM3, GRIN2A, Transcriptional
Schizophrenia
SRR, GRIA1, CACNA1C, modulation, KO of CACNB2, CACN11I, mutant alleles, corrected
GAD1, RELN, BDNF, stem cell
TET1, and DTNBP1
[0145] In other embodiments, the DNA-binding domain binds to a sequence in a safe -harbor gene. Non-limiting examples of safe harbor genes (including for targeted of exogenous molecules such as sequences encoding therapeutic NS proteins) include, for example, a CCR5 gene, a CXCR4 gene, an HPRT gene, a PPP1R12C (also known as AAVSl) gene, an albumin gene or a Rosa gene. See, e.g., U.S. Patent Nos. 7,951,925 and 8,110,379; U.S. Publication Nos. 20080159996; 201000218264; 20100291048; 20120017290; 20110265198; 20130137104; 20130122591;
20130177983 and 20130177960 and U.S. Provisional Application No. 61/823,689.
Donors
[0146] As noted above, insertion of an exogenous sequence (also called a
"donor sequence" or "donor" or "transgene"), for example for correction of a mutant gene or for increased expression of a wild-type gene. It will be readily apparent that the donor sequence need not be identical to the genomic sequence where it is placed. A donor sequence can contain a non-homologous sequence flanked by two regions of homology to allow for efficient HDR at the location of interest. Additionally, donor sequences can comprise a vector molecule containing sequences that are not homologous to the region of interest in cellular chromatin. A donor molecule can contain several, discontinuous regions of homology to cellular chromatin. For example, for targeted insertion of sequences not normally present in a region of interest, said sequences can be present in a donor nucleic acid molecule and flanked by regions of homology to sequence in the region of interest. Alternatively, a donor molecule may be integrated into a cleaved target locus via non-homologous end joining (NHEJ) mechanisms. See, e.g., U.S. Patent Publication Nos. 20110207221 and 20130326645.
[0147] Described herein are methods of targeted insertion of any
polynucleotides for insertion into a chosen location. Polynucleotides for insertion can also be referred to as "exogenous" polynucleotides, "donor" polynucleotides or molecules or "transgenes." The donor polynucleotide can be DNA or RNA, single- stranded and/or double-stranded and can be introduced into a cell in linear or circular form. See, e.g., U.S. Patent Publication Nos. 20100047805; 20110281361; and 20110207221. The donor sequence(s) can be contained within a DNA MC, which may be introduced into the cell in circular or linear form. If introduced in linear form, the ends of the donor sequence can be protected {e.g., from exonucleo lytic
degradation) by methods known to those of skill in the art. For example, one or more dideoxynucleotide residues are added to the 3' terminus of a linear molecule and/or self-complementary oligonucleotides are ligated to one or both ends. See, for example, Chang et al. (1987) Proc. Natl. Acad. Sci. t/&484:4959-4963; Nehls et al. (1996) Science 272:886-889. Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified internucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues.
[0148] A polynucleotide can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance. Moreover, donor polynucleotides can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by viruses {e.g. , adenovirus, AAV, herpesvirus, retrovirus, lentivirus and integrase defective lentivirus (IDLV)).
[0149] In certain embodiments, the double-stranded donor includes sequences {e.g., coding sequences, also referred to as transgenes) greater than 1 kb in length, for example between 2 and 200 kb, between 2 and 10 kb (or any value therebetween). The double-stranded donor also includes at least one nuclease target site, for example. In certain embodiments, the donor includes at least 1 target site, for example, for use with a CRISPR/Cas, or 2 target sites, for example for a pair of ZFNs and/or TALENs. Typically, the nuclease target sites are outside the transgene sequences, for example, 5' and/or 3' to the transgene sequences, for cleavage of the transgene. The nuclease cleavage site(s) may be for any nuclease(s). In certain embodiments, the nuclease target site(s) contained in the double-stranded donor are for the same nuclease(s) used to cleave the endogenous target into which the cleaved donor is integrated via homo logy-independent methods.
[0150] The donor is generally inserted so that its expression is driven by the endogenous promoter at the integration site, namely the promoter that drives expression of the endogenous gene into which the donor is inserted. However, it will be apparent that the donor may comprise a promoter and/or enhancer, for example a constitutive promoter or an inducible or tissue specific promoter.
[0151] The donor molecule may be inserted into an endogenous gene such that all, some or none of the endogenous gene is expressed. For example, a transgene as described herein may be inserted into a selected locus such that some or none of the endogenous sequences are expressed, for example as a fusion with the transgene. In other embodiments, the transgene is integrated into any endogenous locus, for example a safe-harbor locus. Furthermore, although not required for expression, exogenous sequences may also include transcriptional or translational regulatory sequences, for example, promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides and/or polyadenylation signals.
[0152] The transgenes carried on the donor sequences described herein may be isolated from plasmids, cells or other sources using standard techniques known in the art such as PCR. Donors for use can include varying types of topology, including circular supercoiled, circular relaxed, linear and the like. Alternatively, they may be chemically synthesized using standard oligonucleotide synthesis techniques. In addition, donors may be methylated or lack methylation. Donors may be in the form of bacterial or yeast artificial chromosomes (BACs or YACs).
[0153] The double-stranded donor polynucleotides described herein may include one or more non-natural bases and/or backbones. In particular, insertion of a donor molecule with methylated cytosines may be carried out using the methods described herein to achieve a state of transcriptional quiescence in a region of interest.
[0154] The exogenous (donor) polynucleotide may comprise any sequence of interest (exogenous sequence). Exemplary exogenous sequences include, but are not limited to any polypeptide coding sequence (e.g., cDNAs), promoter sequences, enhancer sequences, epitope tags, marker genes, cleavage enzyme recognition sites and various types of expression constructs. Marker 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. [0155] In a preferred embodiment, the exogenous sequence (transgene) comprises a polynucleotide encoding any polypeptide of which expression in the cell is desired, including, but not limited to any polypeptide involved in a NS disorder, antibodies, antigens, enzymes, receptors (cell surface or nuclear), hormones, lymphokines, cytokines, reporter polypeptides, growth factors, and functional fragments of any of the above. The coding sequences may be, for example, cDNAs. Non-limiting examples of polypeptides that may be encoded by the exogenous (donor) sequences include growth factors (e.g., growth hormone, insulin-like growth factor- 1, platelet-derived growth factor, epidermal growth factor, acidic and basic fibroblast growth factors, transforming growth factor-(3, etc.), to treat growth disorders or wasting syndromes; and antibodies (e.g., human or humanized), to provide passive immunization or protection of a subject against foreign antigens or pathogens (e.g., H. Pylori), or to provide treatment of cancer, arthritis or
cardiovascular disease; cytokines, interferons (e.g., interferon (INF), INF-a2b and 2a, INF-aNl, INF-(31b, INF-gamma), interleukins (e.g., IL-1 to IL 10), tumor necrosis factor (TNF-a TNF-R), chemokines, granulocyte macrophage colony stimulating factor (GM-CSF), polypeptide hormones, antimicrobial polypeptides (e.g., antibacterial, antifungal, antiviral, and/or antiparasitic polypeptides), enzymes (e.g., adenosine deaminase), gonadotrophins, chemotactins, lipid-binding proteins, filgastim (Neupogen), hemoglobin, erythropoietin, insulinotropin, imiglucerase, sarbramostim, tissue plasminogen activator (WA), urokinase, streptokinase, phenylalanine ammonia lyase, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), thrombopoietin (TPO), superoxide dismutase (SOD), adenosine deamidase, catalase calcitonin, endothelian, L-asparaginase pepsin, uricase trypsin, chymotrypsin elastase, carboxypeptidase lactase, sucrase intrinsic factor, calcitonin parathyroid hormone
(PTH)-like, hormone, soluble CD4, and antibodies and/or antigen-binding fragments (e.g, FAbs) thereof (e.g., orthoclone OKT-3 (anti-CD3), GPllb/lla monoclonal antibody).
[0156] In certain embodiments, the exogenous sequences can comprise a marker gene (described above), allowing selection of cells that have undergone targeted integration, and a linked sequence encoding an additional functionality. Non-limiting examples of marker genes include GFP, drug selection marker(s) and the like. [0157] Additional gene sequences that can be inserted may include, for example, wild-type genes to replace mutated sequences. For example, a wild-type gene sequence may be inserted into the genome of a stem cell in which the endogenous copy of the gene is mutated. The wild-type copy may be inserted at the endogenous locus, or may alternatively be targeted to a safe harbor locus.
[0158] Construction of such expression cassettes, following the teachings of the present specification, utilizes methodologies well known in the art of molecular biology (see, for example, Ausubel or Maniatis). Before use of the expression cassette to generate a transgenic animal, the responsiveness of the expression cassette to the stress-inducer associated with selected control elements can be tested by introducing the expression cassette into a suitable cell line (e.g., primary cells, transformed cells, or immortalized cell lines).
[0159] Furthermore, although not required for expression, exogenous sequences may also transcriptional or translational regulatory sequences, for example, promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides and/or polyadenylation signals. Further, the control elements of the genes of interest can be operably linked to reporter genes to create chimeric genes (e.g., reporter expression cassettes).
[0160] Targeted insertion of non-coding nucleic acid sequence may also be achieved. Sequences encoding antisense R As, RNAi, shRNAs and micro RNAs (miRNAs) may also be used for targeted insertions.
[0161] In additional embodiments, the donor nucleic acid may comprise non- coding sequences that are specific target sites for additional nuclease designs.
Subsequently, additional nucleases may be expressed in cells such that the original donor molecule is cleaved and modified by insertion of another donor molecule of interest. In this way, reiterative integrations of donor molecules may be generated allowing for trait stacking at a particular locus of interest or at a safe harbor locus.
Delivery
[0162] The nucleases, polynucleotides encoding these nucleases, donor polynucleotides and compositions comprising the proteins and/or polynucleotides described herein may be delivered in vivo or ex vivo by any suitable means.
[0163] Methods of delivering nucleases as described herein are described, for example, in U.S. Patent Nos. 8,586,526; 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.
[0164] Nucleases and/or donor constructs as described herein may also be delivered using vectors containing sequences encoding one or more of compositions described herein. 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 of the sequences needed for treatment. Thus, when one or more nucleases and a donor construct are introduced into the cell, the nucleases and/or donor polynucleotide 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 nucleases and/or donor constructs.
[0165] Conventional viral and non- viral based gene transfer methods can be used to introduce nucleic acids encoding nucleases and donor constructs in cells {e.g., mammalian cells) and target tissues. 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): 1149-1154 (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).
[0166] Methods of non- viral delivery of nucleic acids include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, 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. [0167] 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 US6008336). 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™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424, WO 91/16024.
[0168] The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immuno lipid 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. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).
[0169] 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):643).
[0170] The use of RNA or DNA viral based systems for the delivery of nucleic acids encoding engineered ZFPs take 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 subjects (in vivo) or they can be used to treat cells in vitro and the modified cells are administered to subjects (ex vivo). Conventional viral based systems for the delivery of ZFPs 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.
[0171] 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 cz's-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum czs-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 et al, J. Virol. 66: 1635-1640 (1992); Sommerfelt et al, Virol. 176:58-59 (1990); Wilson et al, J. iro/.63:2374-2378 (1989); Miller et al, J. Virol. 65:2220- 2224 (1991); PCT/US94/05700).
[0172] 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. Invest. 94: 1351 (1994). Construction of recombinant AAV vectors is described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81 :6466-6470 (1984); and Samulski et al. , J. Virol. 63:03822-3828 (1989).
[0173] 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.
[0174] 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 al, PNAS 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).
[0175] Vectors suitable for introduction of polynucleotides described herein also include non-integrating lentivirus vectors (IDLV). See, for example, Ory et al. (1996) Proc. Natl. Acad. Sci. USA93: l 1382-11388; Dull et al. (1998) J. Virol.
72:8463-8471; Zuffery et a/. (1998) J. Virol. 72:9873-9880; Follenzi et al. (2000) Nature Genetics 25:217-222; U.S. Patent Publication No 20090117617.
[0176] Recombinant adeno-associated virus vectors (rAAV) may also be used to deliver the compositions described herein. All vectors are derived from a plasmid that retains only the AAV inverted terminal repeats flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery 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, AAV9 and AAVrhlO, pseudotyped AAV such as AAV2/8, AAV2/5 and AAV2/6 and all variants thereof, can also be used in accordance with the present invention.
[0177] 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 Ela, 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 non-dividing, 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 anti-tumor 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).
[0178] 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.
[0179] 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.
[0180] Gene therapy vectors can be delivered in vivo by administration to an individual subject, typically by systemic administration {e.g., intravenous,
intraperitoneal, intramuscular, intrathecal, subdermal, or intracranial infusion) or topical application, as described below. 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) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector.
[0181] Vectors {e.g., retroviruses, adenoviruses, liposomes, etc.) containing nucleases and/or donor constructs 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. Suitable methods of administering such nucleic acids 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.
[0182] In certain embodiments, the compositions (including fusion proteins,
CRISPR/Cas systems and/or modified cells) as described herein {e.g., polynucleotides and/or proteins) are delivered directly in vivo. The compositions (cells,
polynucleotides and/or proteins) may be administered directly into the CNS, including but not limited to direct injection (including grafting of cells) into the brain or spinal cord. See, e.g., U.S. Patent No. 5,529,774 regarding in vivo administration of polynucleotide vectors to the CNS and U.S. Patent No. 5,082,670 and 6,451,306 regarding cell grafting. One or more areas of the brain may be targeted, including but not limited to, the hippocampus, the substantia nigra, the nucleus basalis of Meynert (NBM), the striatum and/or the cortex. Alternatively or in addition to CNS delivery, the compositions may be administered systemically {e.g., intravenous, intraperitoneal, intracardial, intramuscular, intrathecal, subdermal, and/or intracranial infusion). Cell- containing compositions may be administered into the nervous system directly, for example by grafting. Methods and compositions for delivery of compositions as described herein directly to a subject (including directly into the CNS) include but are not limited to direct injection (e.g., stereotactic injection) via needle assemblies. Such methods are described, for example, in U.S. Patent Nos. 7,837,668; 8,092,429, relating to a needle assembly for delivery of compositions to the brain and U.S. Patent Publication No. 20060239966 as well as U.S. Patent Nos. 6,180,613 and 6,503,888 (AAV-mediated delivery of DNA to cells of the nervous system) and U.S. Patent Nos. 6,998,118 and 7,101,540 (gene delivery to neuronal cells), incorporated herein by reference in their entireties.
[0183] 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. 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).
[0184] It will be apparent that the nuclease-encoding sequences and donor constructs can be delivered using the same or different systems. For example, a donor polynucleotide can be carried by a plasmid, while the one or more nucleases can be carried by a AAV vector. Furthermore, the different vectors can be administered by the same or different routes (intramuscular injection, tail vein injection, other intravenous injection, intraperitoneal administration and/or intramuscular injection. The vectors can be delivered simultaneously or in any sequential order.
[0185] Thus, the instant disclosure includes in vivo or ex vivo treatment of diseases and conditions that are amenable to insertion of a transgenes encoding a therapeutic protein, for example treatment of NS disorders via nuclease-mediated integration of a gene encoding a protein aberrantly expressed in a subject with the NS disorder.
[0186] Ex vivo cell transfection for diagnostics, research, 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 ZFP nucleic acid (gene or cDNA), and re -infused back into the subject organism (e.g., patient). Methods of cell therapy to the NS are known. 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).
[0187] Suitable cells include but 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 COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44, CHO- DUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NSO, SP2/0-Agl4, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), and perC6 cells, any plant cell (differentiated or undifferentiated) as well as insect cells such as Spodopterafugiperda (Sf), or fungal cells such as
Saccharomyces, Pichia and Schizosaccharomyces . In certain embodiments, the cell line is a CHO-K1, MDCK or HEK293 cell line. Additionally, primary cells may be isolated and used ex vivo for reintroduction into the subject to be treated following treatment with the nucleases (e.g. ZFNs or TALENs) or nuclease systems (e.g.
CRISPR/Cas). Suitable primary cells include neuronal cells, peripheral blood mononuclear cells (PBMC), and other blood cell subsets such as, but not limited to, CD4+ T cells or CD8+ T cells. Suitable cells also include stem cells such as, by way of example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells (CD34+), neuronal stem cells and mesenchymal stem cells.
[0188] 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 in the bone marrow.
[0189] Stem cells that have been modified may also be used in some embodiments. For example, stem cells that have been made resistant to apoptosis may be used as therapeutic compositions where the stem cells also contain the ZFPs, TALEs, ZFNs, TALENs, CRISPR/Cas systems and/or donors of the invention.
Resistance to apoptosis may come about, for example, by knocking out BAX and/or BAK using BAX- or BAK-specific nucleases (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. Alternatively, resistance to apoptosis can also be achieved by the use of caspase inhibitors like Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-[0- methylj-fluoromethylketone).
[0190] Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containing therapeutic ZFPs, TALEs, ZFNs, TALENs, CRISPR/Cas system and/or donor nucleic acids can also be administered directly to an organism for transduction of cells in vivo. Alternatively, naked DNA or mR A 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. Suitable methods of administering such nucleic acids 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.
[0191] The effective amount of nuclease(s) and donor to be administered will vary from patient to patient and according to the therapeutic polypeptide of interest. Accordingly, effective amounts are best determined by the physician administering the compositions and appropriate dosages can be determined readily by one of ordinary skill in the art. After allowing sufficient time for integration and expression (typically 4-15 days, for example), analysis of the serum or other tissue levels of the therapeutic polypeptide and comparison to the initial level prior to administration will determine whether the amount being administered is too low, within the right range or too high. Suitable regimes for initial and subsequent administrations are also variable, but are typified by an initial administration followed by subsequent administrations if necessary. Subsequent administrations may be administered at variable intervals, ranging from daily to annually to every several years. One of skill in the art will appreciate that appropriate immunosuppressive techniques may be recommended to avoid inhibition or blockage of transduction by immunosuppression of the delivery vectors, see e.g., Vilquin et al., (l995)Human Gene Ther. 6: 1391-1401.
[0192] Formulations for both ex vivo and in vivo administrations include suspensions in liquid or emulsified liquids. The active ingredients often are mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof. In addition, the composition may contain minor amounts of auxiliary substances, such as, wetting or emulsifying agents, pH buffering agents, stabilizing agents or other reagents that enhance the effectiveness of the pharmaceutical composition. Applications
[0193] The methods and compositions disclosed herein are for modifying expression of protein, correcting an aberrant gene sequence that encodes a gene product expressed in a NS disorder or insertion of a transgene whose gene product is known to be helpful in treatment or prevention of a NS disorder. Thus, the methods and compositions provide for the treatment and/or prevention of such disorders.
Genome editing, for example of stem cells, is used to correct an aberrant gene, insert a wild type gene, or change the expression of an endogenous gene. By way of non- limiting example, a wild type gene may be inserted into a cell to provide the proteins deficient and/or lacking in the subject and thereby treat a NS disorder caused by faulty gene product expression. Alternatively or in addition, genomic editing with or without administration of the appropriate donor, can correct the faulty endogenous gene, e.g., correcting the point mutation in gene encoding a gene product involved in a NS disorder, to restore expression of the gene and/or treat a the disorder.
[0194] The following Examples relate to exemplary embodiments of the present disclosure in which the nuclease comprises a zinc finger nuclease (ZFN) or TALEN. It will be appreciated that this is for purposes of exemplification only and that other nucleases can be used, for instance additional TALENs {e.g., Mega-TALs and/or compact TALENs), homing endonucleases (meganucleases) with engineered DNA-binding domains and/or fusions of naturally occurring of engineered homing endonucleases (meganucleases) and DNA-binding domains and heterologous cleavage domains and/or a CRISPR/Cas system comprising an engineered single guide RNA. It will also be appreciated that these examples serve as exemplification for use of an engineered transcription factor (e.g. ZFP-TF, TALE-TF, CRISPR/Cas- TF) as well.
EXAMPLES
Example 1: Design, Construction and general characterization of compositions that alter NS-related genes
[0195] Zinc finger proteins and TALEs that bind to NS-related genes {e.g.,
Table 1) operably linked to transcriptional regulatory or nuclease domains are designed and incorporated into plasmids, AAV or adenoviral vectors essentially as described in Urnov et al. (2005) Nature 435(7042):646-651, Perez et al (2008) Nature Biotechnology 26(7):808-816, and as described in U.S. Patent Nos. 8,586,526 and 6,534,261.
[0196] sgRNAs for use in the CRISPR/Cas system are made synthetically by methods known in the art (see Hsu et al, ibid or Sternberg et al, (2014) Nature 507: 62)). The sgRNAs are engineered as described above and are designed to target a sequence in an NS-related gene. Table II below shows the genomic coordinates of the NS genes of interest. These coordinates are derived from the UCSC Genome
Browser, hgl9 assembly. Thus, sgRNAs, ZFPs or TALEs are designed to target regions within the bounds of the NS genes.
Table II- Genomic Coordinates of exemplary NS genes
Figure imgf000072_0001
Example 2: Nuclease Activity
[0197] ZFNs and TALENs targeting the selected locus are made as described above. The Cel-I assay (Surveyor™, Transgenomics) as described in Perez et al. (2008) Nat. Biotechnol. 26: 808-816 and Guschin et al. (2010) Methods Mol Biol. 649:247-56), is used to detect nuclease-induced modifications of the target gene in K562 cells or HSCs. In this assay, PCR-amplification of the target site is followed by quantification of insertions and deletions (indels) using the mismatch detecting enzyme Cel-I (Yang et al. (2000) Biochemistry 39: 3533-3541) which provided a lower- limit estimate of DSB frequency. Three days following transfection of the nuclease expression vector at either standard conditions (37°C) or using a
hypothermic shock (30°C, U.S. Patent Publication No. 20110041195), genomic DNA is isolated cells using the DNeasy® kit (Qiagen) and show the nucleases cleave their target genes.
Example 3: Allele specific repression in human neural stem cells (NSCs)
[0198] Human iPSC/ESCs are passaged with accutase and cultured on matrigel coated plates in E8 media (Life Technologies). Neural stem cells are derived using StemPro Neural Induction Medium (Life Technologies). Briefly, iPSC/ESCs are seeded into geltrex coated 6 well dish with 200,000 cells/well and when 10-20% confluent the medium is changed to StemPro Neural Induction Medium. Medium is changed every 2 days and NSC are harvested and expanded on day 7. StemPro NSC SFM medium (Life Technologies) is used to culture NSCs. Human NSCs are transfected with 1.5 or 0.5 μg ZFP mRNA using nucleofection. Forty-eight hours post transfection cells are harvested and expression quantified by RT-PCR. Allele- specific detection of expression is performed using a SNP based genotyping assay. At the ZFP doses that are tested, allele-specific repression of mutant target genes is observed.
Example 4: Allele repression in mice
[0199] Mouse models of a NS disease, preferably carrying a human transgene with the mutant gene receive stereotactic, bilateral striatal injections of 3el0 vector genomes of recombinant AAV2/6 encoding the ZFP-TF of the invention driven by a CMV promoter. Mice are injected at 5 weeks of age and sacrificed for molecular analysis at 8 weeks of age. Left and right striata are dissected from each hemisphere and snap frozen. To assess repression of the mutant transgene, total RNA is extracted from each striatum with TRIzol Plus (Life Technologies) followed by cDNA synthesis using High Capacity RT (Life Technologies). Subsequently, mutant transgene expression is measured by qPCR and normalized to the geometric mean of three reference genes (Atp5b, Eif4a2, UbC) as previously described by Benn et al. ((2008) Molecular Neurodegeneration: 3, 17). Repression of the mutant transgene is observed.
Example 5: Cleavage activity of mutant allele-specific ZFNs
[0200] To test cleavage activity, plasmids encoding the pairs of human mutant allele-specific ZFNs are transfected into K562 cells. K562 cells are obtained from the American Type Culture Collection and grown as recommended in F-12 medium (Invitrogen) supplemented with 10% qualified fetal calf serum (FCS, Cyclone). Cells are disassociated from plastic ware using TrypLE Select™ protease (Invitrogen). For transfection, one million K562 cells are mixed with 2μg of the zinc-finger nuclease plasmid and ΙΟΟμί Amaxa Solution T. Cells are transfected in an Amaxa
Nucleofector II™ using program U-23 and recovered into 1.4mL warm F-12 medium + 10% FCS.
[0201] Genomic DNA is harvested and a portion of the mutant locus encompassing the intended cleavage site is PCR amplified. PCR using the Accuprime HiFi polymerase from InVitrogen is performed as follows: after an initial 3 minute denaturation at 94°C, 30 cycles of PCR are performed with a 30 second denaturation step at 94°C followed by a 30 second annealing step at 58°C followed by a 30 second extension step at 68°C. After the completion of 30 cycles, the reaction was incubated at 68°C for 7 minutes, then at 10°C indefinitely.
[0202] The genomic DNA from the mutant allele-specific ZFN treated cells is examined by the Surveyor™ nuclease (Transgenomic) as described, for example, in U.S. Patent No. 7,951 ,925 and U.S. Patent Publication Nos. 20080015164 and 20080131962.
[0203] The ZFNs are demonstrated to be capable of targeting the mutant genes with a gene modification efficiency of between 8-40%>, assayed as described previously by the amount of indels observed.
Example 6: Targeted Modification in patient derived iPSC
[0204] ZFN modification of a NS mutant allele is performed in patient- derived iPSCs. DNA sequences (e.g., plasmids) encoding mutant allele-specific ZFNs, the green fluorescent protein (GFP) and a donor DNA construct are co- delivered by nucleofection to patient-derived iPSCs that are heterozygous for the mutation. The donor construct contains 1 kb of wild type (wt) gene sequence (500 bps in each direction of the mutation). In a subpopulation of cells that contain ZFN- mediated DSB, the donor is used as a template to repair the DSB as well as the mutation; the majority of the cells will use NHEJ to resolve the DSB. Transfected (GFP-positive) cells are enriched through fluorescence-based cell sorting and replated on MEF feeder layers for single-cell cloning. [0205] Genomic DNA is isolated from clones, the regions of interest are amplified by PCR. Clones are subject to sequencing analysis to confirm the presence of 2 wild type alleles for corrected cells, and to identify those having mutations on the mutant allele and unmodified wild type allele. Before further functional
characterization of gene-corrected or mutant iPSC clones, cytogenetic analysis is performed to confirm normal karyotype, and their pluripotent state is verified by expression of the pluripotency markers (OCT4, NANOG and SOX2), as well as ability to form all three developmental germ layers in teratoma- formation assays.
[0206] The teratoma-formation assay is performed as follows. Cell culture and transfection: The iPSC were grown in mTeSR®l medium (STEM CELL™
Technologies) on Matrigel coated dishes (BD BioCoat™, BD Biosciences) and the media changed daily. One day before transfection, lOuM of Rho-associated protein kinase (ROCK) Inhibitor (Calbiochem Y-27632) is added to the culture. Transfection is carried out with the Amaxa (Lonza) 96-well shuttle using P4 Primary Cell 96-well Nucleofector Kit following manufacturer's instructions: 2e5 cells are transfected with ZFN-encoding plasmid DNA or mRNA in the presence of donor DNA to trigger homologous recombination and gene correction.
[0207] To determine the level of gene modification, a Cell assay was performed using primers adjacent to the area of correction. The Cel I assay is performed using 1-3 μΐ of a 1/1000 dilution of the PCR described above. The donor is designed to correct the mutation in the mutated allele and is transfected along with the nucleases as described above. To analyze the insertion of the donor, individual clones treated with the nuclease pair and donor are examined for the presence of the donor sequences and to confirm gene correction at the mutation site.
[0208] In addition, the clones are examined to determine if donor integration occurs through NHEJ or HR. Sequence analysis is performed to verify the percent of alleles comprising a targeted integration and/or indels introduced following nuclease cleavage. Genomic DNA is extracted (using Qiagen QIAamp DNA micro kit) and analyzed for ZFN activity as follows. Briefly, the region comprising the cleavage site is amplified by PCR by standard methods, and following amplification, the PCR product is sequenced via MiSeq high throughput sequencing analysis according to manufacturer's instructions (Ilumina). To quantitate the percent of edited alleles, the genomic region of interest is PCR amplified using primers which add the standard Illumina sequencing adapter sequences. A second group of 13 rounds of PCR is performed to add barcode and bridge adapter sequences to both ends. Sequencing is performed on an Illumina MiSeq according to manufacturer's protocols for amplicon sequencing. The MiSeq generates paired-end reads, which are merged and adapter- trimmed using a standard alignment software. Reads are then demultiplexed by sample via barcode sequence pairs using custom scripts. Amplicon sequences are then globally aligned to a reference sequence via an implementation of the Needleman- Wunsch algorithm (Needleman, Saul B.; and Wunsch, Christian D. (1970). Jour Mol Bio 48 (3): 443-53). Gaps or insertions in the alignment were counted as % NHEJ events, and compared to an untreated control sample sequence to determine sequence- specific background rates .
[0209] For calculation of targeted integration, Amplicon sequences are globally aligned to a reference sequence via a biopython implementation of the Needleman-Wunsch algorithm (Needleman, Saul B.; and Wunsch, Christian D.
ibid). Sequence changes generated via experimental treatments were searched for, counted, and compared to counts in control samples. Known single feature polymorphisms (SFPs) may be masked out during this process and excluded from further counts {e.g., 1-bp deletion SFPs close to the ZFN target site). The percentage of NHEJ (also referred to as indels) is calculated by determining the percentage of sequences that contain insertions or deletions. Samples treated only with GFP vector are used to assess the PCR and sequencing error based background frequency of insertions and deletions. Background frequencies of less than 1% are observed.
[0210] These results demonstrate that the ZFNs are able to cause specific gene correction in iPSCs derived from patient cells. Example 7: In vivo Administration
[0211] Compositions (cells, proteins and or polynucleotides) as described herein are administered to a subject with a NS disorder, for example directly to the NS, essentially as described in U.S. Patent Nos. 7,837,668; 8,092,429; U.S. Patent Publication No. 20060239966; U.S. Patent Nos. 6,180,613; 6,503,888 and /or U.S. Patent Nos. 6,998, 118 and 7,101 ,540 to provide therapy for a subject in need thereof.
[0212] All patents, patent applications and publications mentioned herein are hereby incorporated by reference in their entirety. [0213] 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 descriptions and examples should not be construed as limiting.

Claims

What is claimed is: 1. A method of modifying an endogenous presenilin, alpha- 1 subunit of a voltage-dependent calcium channel (CACNAIC), voltage-dependent, beta 2 subunit (CACNB2), TET1, ODZ4, TRANK1, PRDM16, PHACTR1, FHM5, c7orfl0, AADAC, FSCB, KCHEl, KCHE2, CAMSAPILKI, GAMA-A receptor subunit alpha- 1 or C9orf72 gene in a cell, the method comprising altering expression of the endogenous gene by administering a transcription factor or nuclease that targets the endogenous gene, wherein the transcription factor or nuclease is selected from the group consisting of a protein comprising a zinc finger protein, a protein comprising a TAL-effector protein and a CRISPR/Cas system.
2. The method of claim 1, wherein expression of the endogenous gene is up-regulated.
3. The method of claim 1, wherein expression of the endogenous gene is down-regulated.
4. The method of claim 3, wherein expression of the endogenous gene is inactivated.
5. The method of claim 1 or claim 3, wherein the modification comprises integration of an exogenous sequence into the endogenous gene.
6. The method of claim 5, wherein the exogenous sequence corrects a mutation in the endogenous gene.
7. The method of claim 6, wherein the exogenous sequence comprises a sequence of the endogenous gene.
8. The method of any of claims 1 to 7, wherein the cell is a stem cell.
9. The method of claim 8, wherein the stem cell is a neuronal stem cell, an induced pluripotent stem cell or a mesenchymal stem cell.
10. An isolated modified cell made by the method of any of claims 1 to 9.
11. A method of treating or preventing a nervous system disorder in a subject in need thereof, the method comprising administering a cell according to claim 10 to the subject such that the cell treats or prevents the nervous system disorder.
12. The method of claim 11 , wherein the nervous system disorder is a central nervous system (CNS) disorder and the cell is administered to the CNS.
13. The method of claim 11, wherein the nervous system disorder is a peripheral nervous system (PNS) disorder and the cell is administered to the PNS.
14. A method of treating or preventing a nervous system disorder in a subject in need thereof, the method comprising modifying a cell in the subject according to the method of any of claims 1 to 9 such that the nervous system disorder is treated or prevented.
15. The method of claim 14, wherein the nervous system disorder is a central nervous system (CNS) disorder and the cell in the subject is in the CNS.
16. The method of claim 14, wherein the nervous system disorder is a peripheral nervous system (PNS) disorder and the cell in the subject is in the PNS.
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