Abstract:
Medical compositions and methods of treating or preventing neurodegeneration in a human suffering from or that is at risk of or susceptible to neurodegeneration or cellular dysfunction associated with expression or impaired cellular function of a neuronal protein encoded by one or more genes that code for alpha-synuclein (SNCA), Parkin RBR E3 ubiquitin protein ligase, (PARK2), Leucine-rich repeat kinase 2 (LRRK2), PTEN-induced putative kinase/(PINK1), Daisuke-Junko 1, (DJ-1) and ATPase type 13A2 (ATP13A2), are disclosed. Methods of treatment for these disorders is also provided, comprising administering a vector into a cell, wherein the vector facilitates expression of a molecular component that alters one of the aforementioned genes in the cell or expression of the gene in the cell, the gene being implicated in an etiology of the neurological deficit.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    The present application claims priority to U.S. Provisional Application Ser. No. 62/142,243, filed Apr. 2, 2015. The entire contents of U.S. Provisional Application Ser. No. 62/142,243 is incorporated herein in its entirety. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The present invention generally relates to the field of therapeutics and therapeutic methods using genetic manipulation, as therapeutics and therapeutic methods that provide for genetically modifying one or several gene sequences in a population of human cells to be used as a therapeutic and/or therapeutic method for the treatment of a neurological disorder (e.g., Parkinson&#39;s disease) are disclosed. The invention also relates to the field of protein expression modulation, as methods for providing altered protein expression through selected techniques and modifications at the DNA level are demonstrated that provide for the peunanent arrest of disease progression. 
         [0003]    The invention also generally relates to the compositions and methods of genetically modifying one or several gene sequences in human cells in vivo; either in coding sequences of a gene (creating random mutations at defined positions or insert exogenous genomic sequence to introduce defined changes in the gene), or alteration of regulatory elements that regulate gene expression for selected genes causative for or which increase the risk for neurodegeneration. The gene modification methods provided in the present invention may permanently alter or transiently affect the genomic sequence of a gene. Hence, a method to provide modified gene sequences and altered protein expression is provided, wherein progression of disease is permanently arrested. 
       BACKGROUND OF THE INVENTION 
       [0004]    Parkinson&#39;s disease (PD) and Alzheimer&#39;s disease (AD) are the two most common neurodegenerative diseases of aging. Approximately 1-2% of the population over 65 years of age are affected by PD, and it is estimated that the number of prevalent cases of this disease will double by the year 2030 (Dorsey et al., 2007). PD has also been observed in younger patients, possibly due to specific genetic susceptibility and/or exposure to environmental factors, drugs, or trauma. PD is a slowly progressive debilitating disease with no known current cure. The cause (or causes) of PD also remain largely unknown. While the classical clinical features of the disease include tremor, rigidity, bradykinesia, and postural instability (Gelb et al., 1999) it is now increasingly clear that the disease comprises a whole spectrum of clinical symptoms that go far beyond motor deficits, including reduction in sense of smell, sleep problems, autonomic dysfunction, psychiatric abnormalities, and cognitive decline, which are all part of the clinical syndrome (Langston, 2006). Furthermore, the disease exhibits a remarkable degree of clinical variability in terms of severity, age at onset, and gender difference, with a male/female ratio of approximately 2:1 (Van Den Eeden et al., 2003). 
         [0005]    Although PD represents mostly a sporadic disease, familial forms of parkinsonism and several causative genes have been described and investigated (reviewed in Klein and Schlossmacher, 2007; Klein and Westenberger, 2012; Puschmann, 2013 Table 1 and 2). Neuropathologically, the cardinal features of PD include loss of dopaminergic neurons in the substantia nigra of the brain, and the buildup of intracellular inclusions known as Lewy bodies, the neuropathological hallmark of PD. The presence of Lewy bodies is classically associated with dementia (DLB)(Poulopoulos et al., 2012; Schulz-Schaeffer, 2010). A major component of Lewy bodies is the protein, alpha-synuclein. Alpha-synuclein (approved gene symbol is SNCA) was the first gene in which a causative mutation, SNCA p. A53T, was discovered in 1997 (Polymeropoulos et al., 1997). With the advent of alpha-synuclein based immunohistochemistry, a staining method to detect this protein within a cell, it has been possible to better characterize not only Lewy bodies, but also the wide-spread alpha-synuclein positive Lewy neuritic pathology that exists in both the central and peripheral nervous system. These tools have provided an increasingly well-defined neuroanatomical basis for the many non-motor features of this disease (Forman et al., 2005; Langston, 2006; Litvan et al., 2007a; Litvan et al., 2007b). 
         [0006]    Other alpha-synucleinopathies have been described that present a spectrum of disorders having a range of clinical symptoms, such as Parkinson&#39;s disease with dementia (PDD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), atypical parkinsonism, and atypical Parkinson&#39;s disease (Table 2). 
         [0007]    The use of small interference RNAs (siRNA) in the brain has recently been shown to be effective against endogenous murine alpha-synuclein (Gorbatyuk et al., 2010; Khodr et al., 2014; Lewis et al., 2008) serotonin transporter (SERT) (Ferres-Coy et al., 2013), and mutant human Huntingtin (Chen et al., 2005; DiFiglia et al., 2007; Pfister et al., 2009). siRNA knockdown of alpha-synuclein has also been shown in an MPTP-exposed non-human primate (McCormack et al., 2010). Alpha-synuclein siRNA knockdown in animals has been reported to provide neuroprotection in non-human primates against MPTP. In this model, downregulation of alpha-synuclein mRNA and protein was observed to protect neurons from degeneration and cell death (Maraganore, 2011). 
         [0008]    The advent of alpha-synuclein based immunohistochemistry, a staining method to detect protein within a cell, resulted in the report that the protein, alpha-synuclein, is directly linked to the pathogenesis of certain forms of familial parkinsonism, as well as sporadic PD. 
         [0009]    The protein name is alpha-synuclein, whereas the corresponding approved gene name abbreviation is SNCA. Genetic changes of the SNCA gene, which are point mutations and large genomic multiplications, can cause familial PD (Chartier-Harlin et al., 2004; Fuchs et al., 2007a; Ibanez et al., 2004; Krüger et aL, 1998; Nishioka et al., 2006; Polymeropoulos et al., 1997; Singleton et al., 2003; Zarranz et al., 2004). Two point mutations have been described to be causative for PD, SNCA p.H50G, and SNCA, p.G51D. This raises the number of SNCA point mutations associated with PD to five (Appel-Cresswell et al., 2013; Kiely et al., 2013; Lesage et al., 2013; Proukakis et al., 2013). The dominant inheritance of SNCA mutations, or the mere overexpression of wild-type alpha-synuclein, suggest that parkinsonism is caused by a toxic gain-of-function mechanism. This means that in the disease, the gene/protein takes on a different function that is harmful to the cell. Also, the finding that both qualitative and quantitative alterations in the SNCA gene are associated with the development of a parkinsonian phenotype indicates that amino acid substitutions and overexpression of wild-type alpha-synuclein are capable of triggering a clinicopathological process that is very similar to typical PD (Deng and Yuan, 2014), including nigrostriatal cell death and intracellular protein aggregations, called Lewy bodies. 
         [0010]    One hypothesis regarding the cause of PD is that subtle to moderate overexpression of alpha-synuclein due to other genetic risk variants over many decades can either predispose or even cause the neurodegenerative changes that characterize PD similar to SNCA gene multiplications linked to PD. Several association studies investigated specific polymorphisms within the SNCA gene that might be expected to alter its expression, and have been reported to be associated with PD (Chiba-Falek et al., 2005; Chiba-Falek and Nussbaum, 2001; Chiba-Falek et al., 2003; Farrer et al., 2001; Holzmann et al., 2003; Maraganore et al., 2006; Mizuta et al., 2006; Pals et al., 2004; Wang et al., 2006). The NACP-Rep1 polymorphism of the SNCA promoter, a mixed dinucleotide repeat, was studied in multiple study populations, and reports an association of the NACP-Rep1 allele with PD (Farrer et al., 2001; Hadjigeorgiou et al., 2006; Krüger et al., 1999; Mellick et al., 2005; Pals et al., 2004; Tan et al., 2003). It was also reported that the NACP-Rep1 alleles differed in frequency for cases and controls (P&lt;0.001) and the long allele, 263 bp, was associated with PD (odds ratio, 1.43) (Maraganore et al. et al., 2006). 
         [0011]    The DNA binding protein and transcriptional regulator, PARP-1, showed specific binding to SNCA-Rep1. The PARPs catalyze the transfer of ADP-ribose to various nuclear proteins, and are involved in several cellular processes (e.g., DNA repair, regulation of chromatin structure, transcriptional regulation, trafficking, cell death activation (Beneke and Burkle, 2007)). These data were confirmed by a transgenic mouse model and demonstrated regulatory translational activity (Cronin et al., 2009). Functionally, SNCA expression levels in postmortem brains suggest that the Rep-1 allele and SNPs in the 3′ region of the SNCA gene have a significant effect on SNCA mRNA levels in the substantia nigra and the temporal cortex. 
         [0012]    The promoter region of the SNCA gene has been examined in cancer cell lines and in rat cortical neurons. Regulatory regions in intron 1 and the 5′ region of exon 1 have been shown to exhibit transcriptional activation (Clough et al., 2011; Clough et al., 2009; Clough and Stefanis, 2007), as well as the NACP-Rep-1 region upstream of the SNCA gene (Chiba-Falek et al., 2005; Chiba-Falek and Nussbaum, 2001; Chiba-Falek et al., 2003; Cronin et al., 2009; Touchman et al., 2001). Several transcription factors have been identified, such as PARP-1 (Chiba-Falek et al., 2005), GATA (Scherzer et al., 2008), ZIPRO1, and ZNF219 (Clough et al., 2009) to have an effect on regulating the SNCA promoter region. 
         [0013]    Gene isoforms of alpha-synuclein: Alpha-synuclein has three smaller gene isoforms, which have been described as a result of alternative mRNA splicing. Alpha-synuclein 140 is the main complete protein, alpha-synuclein 112 and alpha-synuclein 126 are shorter proteins lacking exon 5 (C-terminus) and exon 3 (N-terminus), respectively (Campion et al., 1995; Ueda et al., 1993). A third isoform is lacking both exon 3 and exon 5, resulting in a protein product of 96 amino acids (Beyer et al., 2008). It has been shown that each of the three alternative isoforms aggregates significantly less than the canonical isoform SNCA140 (Bungeroth, M., Appenzeller, S., Regulin, A., Volker, W., Lorenzen, I., Grotzinger, J., Pendziwiat, M., and Kuhlenbaumer, G. (2014). Differential aggregation properties of alpha-synuclein isoforms. Neurobiol Aging 35, 1913-1919.)). 
         [0014]    Beyer and colleagues examined expression levels of the alpha-synuclein isoforms 112, 126, and 140 in patients with Diffuse Lewy body disease (DLB), Alzheimer disease (AD), and controls by quantitative RT-PCR. Overall, total alpha-synuclein was upregulated in patients with DLB compared to controls. The relative expression of alpha-synuclein 112 in patients with DLB showed a significant increase compared to controls, whereas isoform 126 had surprisingly lower levels in synucleinopathies and AD compared to controls (Beyer, 2006; Beyer et al., 2006; Beyer et al., 2004). The shift expression ratios for the alpha-synuclein isoforms could be crucial for the disease process and neurodegeneration as shown for the tau gene (Hutton et al., 1998). The importance of isoform ratios shows the misregulation of the 4R to 3R isoforms of tau in familial fronto-temporal dementia with parkinsonism (FTDP-17). A balance between these isoforms seems to be highly important for neuronal function. Mutations of exon 10 of the tau gene either include or exclude exon 10 and alter the 3R14R ratio leading to the disease phenotype (Hong et al., 1998; Hutton et al., 1998). Specht and collaborators evaluated alpha-synuclein—eGFP fusion proteins in vitro to identify functional protein motifs. Cytoplasmic distribution was observed in two N-terminal constructs, which showed some similarity to the alpha-synuclein isoform 112. Constructs of the C-terminal domain of alpha-synuclein caused almost exclusively nuclear localization (Specht et al., 2005). 
         [0015]    Kontopoulos and coworkers report that a vector construct expressing alpha-synuclein tagged with a nuclear localization signal may mediate neurotoxicity, whereas its cytoplasmic localization might be protective in an in vitro system and transgenic  Drosophila  (Kontopoulos et al., 2006). 
         [0016]    Specific subcellular distribution of alpha-synuclein can exhibit differences in toxicity. Predominately cytoplasmic distribution is neuroprotective whereas nuclear distribution is neurotoxic. Shifting the distribution of alpha-synuclein towards the cytoplasm by creating a specific alpha-synuclein isoform with 112 amino acids is a neuroprotective or neuroregenerative approach. 
         [0017]    Gene editing technologies: Recent developments of technologies to permanently alter the human genome and to introduce site-specific genome modifications in disease relevant genes lay the foundation for therapeutic applications in CNS disorders such as Parkinson&#39;s disease (PD) or Alzheimer disease (AD). These technologies are now commonly known as “genome editing.” Current gene editing technologies comprise zinc-finger nucleases (ZFN), TAL effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system or a combination of nucleases (e.g. mutated Cas9 with Fok1) (Tsai, S. Q., Wyvekens, N., Khayter, C., Foden, J. A., Thapar, V., Reyon, D., Goodwin, M. J., Aryee, M. J., and Joung, J. K. (2014). Dimeric CRISPR RNA-guided Fold nucleases for highly specific genome editing. Nature biotechnology 32, 569-576.)) All three technologies create site-specific double-strand breaks. The imprecise repair of a double strand break by non-homologous end joining (NHEJ) has been used to attempt targeted gene alteration (nucleotide insertion, nucleotide deletion, and/or nucleotide substitution mutation). A double-strand break increases the frequency of homologous recombination (HR) at the targeted locus by 1,000 fold, an event that introduces homologous sequence at a target site, such as from a donor DNA fragment. Another approach to minimize off-target effects is to only introduce single strand breaks or nicks using Cas9 nickase (Chen et al., 2014; Fauser et al., 2014; Rong et al., 2014; Shen et al., 2014). 
         [0018]    The CRISPR/Cas9 nuclease system can be targeted to specific genomic sites by complexing with a synthetic guide RNA (sgRNA) that hybridizes a 20-nucleotide DNA sequence (protospacer) immediately preceding an NGG motif (PAM, or protospacer-adjacent motif) recognized by Cas9. CRISPR-Cas9 nuclease generates double-strand breaks at defined genomic locations that are usually repaired by non-homologous end-joining (NHEJ). This process is error-prone and results in frameshift mutation that leads to knock-out alleles of genes and dysfunctional proteins (Gilbert et al., 2013; Heintze et al., 2013; Jinek et al., 2012). Studies on off-target effects of CRISPR show high specificity of editing by next-generation sequencing approaches (Smith et al., 2014; Veres et al., 2014) ( FIG. 1 , panel  1 ). 
         [0019]    Other applications for heart disease, HIV, and Rett syndrome have been described. (Ding et al., 2014; Swiech et al., 2014; Tebas et al., 2014). For heart disease, permanent alteration of a gene called PCSK9 using CRIPR technology reduces blood cholesterol levels in mice (Ding et al., 2014). This approach was based on the observation that individuals with naturally occurring loss-of-function PCSK9 mutations experience reduced blood low-density lipoprotein cholesterol (LDL-C) levels and protection against cardiovascular disease (Ding et al., 2014). A second example for the feasibility of this approach is HIV. Individuals carrying the inherited Delta 32 mutation in the C-C chemokine receptor type 5, also known as CCR5 or CD195 are resistant to HIV-1 infection. Gene modification in CD4 T cells were tested in a safety trial of 12 patients and has shown a significant downregulation of CCR5 in human (Tebas et al., 2014). Another recent study showed the successful use of CRISPR/Cas9 technology in CNS in a mouse model for the editing of the methyl-binding protein 2 (MecP2) gene. Mutation in this gene causes Rett syndrome, a condition in young children—mostly girls—with mental retardation and failure to thrive. In this approach an adeno-associated virus (AAV) was used as the delivery vehicle for the Cas9 enzyme in vivo. Overall, 75% transfection efficiency was described with a high targeting efficiency that almost completely abolished the expression of MecP2 protein and functionally altered that arborization of the neurons similar to what has been described for Rett syndrome (Swiech et al., 2014). This shows the proof of concept that gene editing using CRISPR/Cas9 technology is achievable in the adult brain in vivo. 
         [0020]    Despite reports in the literature describing the use of genetic editing techniques, none have been described or suggested for genes associated with neurodegenerative disorders. A strong need continues to exist in the medical arts for a method for treating and/or inhibiting diseases associated with neurodegenerative disorders, such as materials and techniques useful for the treatment of Parkinson&#39;s Disease. 
       SUMMARY OF THE INVENTION 
       [0021]    In a general and overall sense, the present invention provides for the arrest and/or prevention of neurodegeneration associated with neurodegernative disease in vivo. In some embodiments, arrest and/or prevention of neurodegeneration is accomplished using gene editing methodologies and molecular tools to manipulate specific gene(s) and/or gene regulatory elements, to provide a modification of the gene and/or genomic regions associated with neurodegeneration and neurodegenerative disease, such as Parkinson&#39;s Disease. 
         [0022]    In some aspects, the present invention provides a method of treating a neurological deficit associated with neuropathological disease comprising administering a genetically engineered vector comprising a gene for a nuclease and a promoter for the nuclease, as well as an appropriate molecular “guide” into a cell. Following the administration, the vector facilitates an expression of a molecular component that alters a gene in the cell or expression of a targeted gene associated with the neuropathology in the cell. The affected gene would be implicated in an etiology of the neurological deficit. 
         [0023]    In other embodiments, a medical composition for treating a neurological deficit in a patient is provided. The medical composition includes a nuclease that introduces double strand break in a gene implicated a neurological deficit, a guide RNA that targets a gene implicated in neurological disease, and a delivery system that delivers the nuclease and guide RNA to a cell. 
         [0024]    For purposes of the description of the present invention, the term “modification of gene and/or genomic region” may be interpreted to include one or more of the following events ( FIG. 1 ): 
         [0025]    a) Targeted introduction of a double-strand break by a composition disclosed, resulting in targeted alterations (random mutations e.g. insertions, deletions and/or substitution mutations) in one or more exons of one or more genes. This modification in some embodiments provides a permanent mutation in a cell or population of cells having the modified gene. 
         [0026]    b) Targeted binding of non-functional mutant Cas9 to non-coding regions (e.g. promoters, evolutionary conserved functional regions, enhancer or repressor elements). Binding is induced by compositions disclosed. Sterical hindrance of binding of other proteins (e.g. transcription factors, polymerases or other proteins involved in transcription) may also result as a consequence of binding. 
         [0027]    1. CRISPR sgRNA introduces small insertions or deletions through non-homologous end joining (NHEJ), in general several nucleotides, rarely larger fragments (Swiech et al., 2014). 
         [0028]    2. Homology-directed repair (HDR) to correct point mutations by introducing a non-natural, but partially homologous template. 
         [0029]    3. Double Genome editing of splice-sites or splicing related non-coding elements to eliminate certain gene regions, e.g. exon 5 of SNCA gene. 
         [0030]    4. Double Genome editing of non-coding or intronic gene regions to eliminate regulatory elements that increase or decrease gene expression, e.g. D6 or I12 regulatory region in SNCA gene. 
         [0031]    5. sgRNA guides mutant Cas9 to physically inhibit binding of transcription factors in promoter region, 
         [0032]    6. sgRNA guides mutant Cas9 to physically inhibit binding of transcription factors in regulatory regions or intronically. 
         [0033]    Gene editing or modification can be achieved by use of any variety of techniques, including zinc-finger nuclease (ZFN) or TAL effector nuclease (TALEN) technologies or by use of clustered, regularly interspaced, short palindromic repeat (CRIPSR)/Cas9 technologies or through the use of a catalytically inactive programmable RNA-dependent DNA binding protein (dCas9) fused to VP16 tetramer activation domain, or a Krueppel-associated box (KRAB) repressor domain, or any variety of related nucleases employed for gene editing. These can be seen as existing tools to sever the genomic region in question. 
         [0034]    The tools mentioned above, are general in their application. Aspects of the present methods and compositions provide the design of custom CRISPR single-guide RNA (sgRNA) sequences specific for coding gene regions and regulatory sequences in genes implicated in neurodegeneration. In this manner, an exact genomic location for precise gene alteration in humans may be accomplished, with a resulting improvement and/or elimination of a neurodegenerative disorder pathology or symptom. 
         [0035]    Other aspects of the invention provide for the use and accomplishment of the following molecular events, with the expected phenotypic results for neurodegenerative disease treatment: 
         [0036]    1. CRISPR sgRNA leading to non-homologous end joining (NHEJ) introducing insertion or deletions (e.g. small nucleotide insertions or deletions resulting in frameshifts and non-functional protein) ( FIG. 1 , panel A). 
         [0037]    2. CRISPR sgRNA with donor to achieve homology directed repair (HDR) to introduce a specific mutation or sequence change ( FIG. 1 , panel B). 
         [0038]    3. Mutant Cas9/CRISPR sgRNA to target regulatory elements activating or repressing SNCA and/or MAPT gene expression, including promoter, enhancer elements, silencer elements or other like regulatory regions in cis or trans, or introns ( FIG. 1 , panel C). 
         [0039]    4. CRISPR sgRNA to target splice sites to induce nonsense mediated decay or alteration of isoforms ( FIG. 1 , panel D). 
         [0040]    5. sgRNA guides mutant Cas9 to physically inhibit binding of transcription factors in promoter region ( FIG. 1 , panel E). 
         [0041]    6. sgRNA guides mutant Cas9 to physically inhibit binding of transcription factors in regulatory regions or intronically ( FIG. 1 , panel F). 
         [0042]    Specific compositions of nucleotide sequences that can serve or be transcribed in vivo or in vitro into sgRNAs and can introduce changes. 
         [0043]    In another aspect, specifically designed sgRNAs are provided that enhance promoter activity or other regulatory elements within a gene to enhance or repress expression of SNCA and/or MAPT or other genetic factors related to neurodegenerative disorders in vivo. 
         [0044]    The sgRNA constructs may be applied alone or in combination to multiple specific sequences. 
         [0045]    In some embodiments, specifically designed sgRNA sequences are provided that target or disrupt specific splice sites to preferentially create specific protein forms (isoforms) which prevent the promotion of protein aggregation, such as specifically targeted to the SNCA gene and/or MAPT gene. 
         [0046]    In other embodiments, specifically designed donor constructs with specific, defined mutations to introduce frameshift mutations of genes implicated in neurodegenerative disease, leading to loss-of-function introduced by homology-directed repair (HDR), as provided. 
         [0047]    The invention also provides specific promoter-driven expression of gene editing or modification constructs in defined cell populations, e.g. tyrosine hydroxylase promoter to deliver construct in defined cell populations, for selective delivery in targeted cell types that exhibit disease pathology. 
         [0048]    In yet other embodiments, specific inducible/silencing constructs to turn on/off the expression of a gene modification construct are provided. 
         [0049]    The invention includes various delivery systems for a vector, such as an adeno-associated virus (AAV) delivery system for in vivo/in human expression of gene editing modification machinery. 
         [0050]    Editing of SNCA and tau patient-derived stem cells or human embryonic stem cells followed by transplantation of disease gene modified differentiated neurons (progenitors) from stem cells into human/patient is included in the present invention as part of a treatment method. 
         [0051]    In some embodiments, gene editing or modification constructs described will be cloned in a single vector or multiple vectors. 
         [0052]    Techniques and materials for gene editing or modification constructs delivered separately based on size of the constructs are also provided. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0053]      FIG. 1 . Scheme of CRISPR Single-guide RNA design against gene coding and regulatory regions.  1 A. CRISPR. sgRNA introduces small insertions or deletions through non-homologous end joining (NHEJ), in general several nucleotides, rarely larger fragments (Swiech et al., 2014),  1 B. Homology-directed repair (HDR) to correct point mutations by introducing a non-natural, but partially homologous template (green),  1 C. Double Genome editing of splice-sites or splicing related non-coding elements to eliminate certain gene regions, e.g. exon 5 of SNCA gene.  1 D. Double Genome editing of non-coding or intronic gene regions to eliminate regulatory elements that increase or decrease gene expression, e.g. D6 or 112 regulatory region in SNCA gene.  1 E. sgRNA guides mutant Cas9 to physically inhibit binding of transcription factors in promoter region,  1 F. sgRNA guides mutant Cas9 to physically inhibit binding of transcription factors in regulatory regions or intronically. 
           [0054]      FIG. 2 . Overview and workflow of CRISPR/Cas9 construct validation.  2 A. Single guide RNAs are designed in silico;  2 B. sgRNA oligonucleotides are cloned into the pGS-U6-gRNA vector which contains a human U6 promoter;  2 C. double transfection of both vectors into HEK293 cells;  2 D. results of gene modification are shown in gel electrophoresis,  FIGS. 3 and 4 . 
           [0055]      FIG. 3 . SNCA CRISPR/Cas9 constructs for exon 2. Columns: SNCA 1, SNCA 2, SNCA 3 tested in HEK293 cells. Arrows in lane 2, 4, 6, and 8 point to bands that resulted from cleavage by Cel-1 which allows to detect mismatch changes between DNA strands indicating a double strand break induced insertion/deletion in a pool of cells. 2% AgaroseEX on E-gel iBase Power System, 11 min electrophoresis time. DNA was extracted by GeneArt QE DNA kit. 
           [0056]      FIG. 4 . MAPT CRISPR/Cas9 constructs for exon 2. Columns: MAPT  1 , MAPT  2 , MAPT  3  tested in HEK293 cells. Arrows in lane 2, 4, 6, and 8 point to bands that resulted from cleavage by Cel-1 which allows to detect mismatch changes between DNA strands indicating a double strand break induced insertion/deletion in a pool of cells. 2% AgaroseEX on E-gel iBase Power System, 11 min electrophoresis time. DNA was extracted by GeneArt QE DNA kit. 
           [0057]      FIG. 5 . Schematic overview of testing non-coding regulation regions via luciferase assay.  5 A. In silico comparison of evolutionary conserved regions;  5 B. Cloning of identified ncECR elements into pGL3 luc+ vector;  5 C. transfection into HEK293 or neuroblastoma cells;  5 D. Luciferase assay shows regulation of luciferin gene. 
           [0058]      FIG. 6 . Overview of all ncECR regions in the SNCA locus. Arrows indicate the ncECRs on the genomic locus 4q21. Exons are indicated abbreviated as Ex, D, In, U distinguish regions downstream (D), intronic (In), and upstream (U) of the SNCA gene (UCSC Genome Browser on Human Mar. 2006 Assembly, NCBI36/HG18 (Sterling et al., 2014)). 
           [0059]      FIG. 7 . Functionally confirmed non-coding regulatory elements in the a-synuclein gene (Sterling et al., 2014). Panels A-C show results of ncECRs upstream ( 7 A), intragenic ( 7 B), and downstream ( 7 C) of the SNCA gene. The X-axis shows the ncECRs, the Y-axis shows the ratio of luciferase and  renilla  expression as percentage. Bas=pGL3 basic, Con=pGL3 control, prom=pGL3 promoter construct. All red or green box plot elements represent ncECRs that modulate expression significantly. The box plots show the median (horizontal line within box), the 25 and 75% tiles (horizontal borders of box), and the whiskers show the minimal and maximal values.  7 C: Luciferase assay results of D6 element cloned into the pGL3 control vector construct. The X-axis shows the D6 and pGL3 control in green and red, respectively, the Y-axis shows the ratio of luciferase and  renilla  expression as percentage. 
           [0060]      FIG. 8 . Schematic overview of testing Cas9 inhibition of non-coding regulatory regions via luciferase assay and SNCA expression in vitro.  8 A. Single guide RNAs are designed in silico, which is further discussed at Example 2 herein, examples of sgRNA for selected ncECRs D6 and I12 and listed in Table 6; 8B. sgRNA oligonucleotides are cloned into pdCas9/hU6 vector which contains a human U6 promoter and the mutant dead Cas9 protein, second vector is the pGL3 promoter with cloned ncECR shown in  FIG. 6 ;  8 C. double transfection of both vectors into HEK293 or neuroblastoma; and  8 D. Luciferase assay shows regulation of luciferin gene and expression analysis of SNCA gene. 
           [0061]      FIG. 9 . Predicted intracellular distribution of alpha-synuclein isoforms.  9 A. Alpha-synuclein gene isoform 112, lacking exon 5, has a similar structure as compared to the two published constructs 1-102aa-eGFP (Specht et al., 2005) and 140aa-NES (Kontopoulos et al., 2006) which promote cytoplasmic distribution. Cytoplasmic distribution of alpha-synuclein has shown to be neuroprotective. The Alpha-synuclein gene isoform 112 can be created by double CRISPR editing thus releasing exon 5.  9 B. Alpha-synuclein gene isoform 126, lacking exon 3, has some aspects of structure of two published constructs 103-140aa-eGFP (Specht et al., 2005) And 140aa-NES (Kontopoulos et al., 2006) which promote nuclear distribution. Nuclear distribution of alpha-synuclein has been shown to be cytotoxic. aa: amino acid, NES: nuclear export sequence, NLS: nuclear localization signal. 
           [0062]      FIG. 10 . Options in the treatment of neurological deficits. Different combined delivery and transplantation therapeutic approaches based on disease stage, genetics (e.g. GBA mutations or SNCA risk factors), and additional symptoms (e.g. cognitive decline). In early stages of disease, only viral gene delivery could be sufficient to stop disease progression. In later stages of the disease, gene edited or modified HLA-typed iPS cells, human embryonic stem cells, or patient-derived iPS cells gene modified for SNCA and/or MAPT are transplanted as pre-differentiated cells. Abbreviations: PD=Parkinson&#39;s disease, H&amp;Y=Hoehn and Yahr P D disease stage. 
           [0063]      FIG. 11 . Treatment Options of Ex vivo gene editing and In vivo gene editing. Illustration of ex vivo gene editing in combination with cell transplantation by gene disruption, gene correction, gene modification, or gene repression/activation. Alternativley, direct in vivo gene editing via viral vectors, e.g. AAV, may be used. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Definitions 
       [0064]    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 tern&#39;s 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”. 
         [0065]    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. 
         [0066]    “Binding” refers to a sequence-specific, non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), as long as the interaction as a whole is sequence-specific. Such interactions are generally characterized by a dissociation constant (Kd) of 10-6 M-1 or lower. 
         [0067]    The following definitions and abbreviations are used in the description of the present invention. 
         [0068]    A “binding protein” relates to 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, nucleases have DNA-binding, RNA-binding and protein-binding activity. 
         [0069]    The term “sequence” relates 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. 
         [0070]    The term “donor sequence” relates 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 “homologous, non-identical sequence” relates to a first sequence which shares a degree of sequence identity with a second sequence, but whose sequence is not identical to that of the second sequence. For example, a polynucleotide comprising the wild-type sequence of a mutant gene is homologous and non-identical to the sequence of the mutant gene. In certain embodiments, the degree of homology between the two sequences is sufficient to allow homologous recombination therebetween, utilizing normal cellular mechanisms. Two homologous non-identical sequences can be any length and their degree of non-homology can be as small as a single nucleotide (e.g., for correction of a genomic point mutation by targeted homologous recombination) or as large as 10 or more kilobases (e.g., for insertion of a gene at a predetermined ectopic site in a chromosome). Two polynucleotides comprising the homologous non-identical sequences need not be the same length. For example, an exogenous polynucleotide (i.e., donor polynucleotide) of between 20 and 10,000 nucleotides or nucleotide pairs can be used. 
         [0072]    The term “identity” relates to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their percent identity. The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100. 
         [0073]    “Sequence similarity” between polynucleotides can be determined by hybridization of polynucleotides under conditions that allow formation of stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments. 
         [0074]    Two nucleic acids, or two polypeptide sequences are substantially “homologous” to each other when the sequences exhibit at least about 70%-75%, preferably 80%-82%, more preferably 85%-90%, even more preferably 92%, still more preferably 95%, and most preferably 98% sequence identity over a defined length of the molecules, as determined using the methods above. 
         [0075]    “Recombination” relates to a process of exchange of genetic information between two polynucleotides. For the purposes of this disclosure, “homologous recombination (HR)” relates 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. 
         [0076]    In the methods of the disclosure, one or more targeted “nucleases”, e.g. CRIPSR/Cas9, TALEN or ZFN, 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. 
         [0077]    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. 
         [0078]    In any of the methods described herein, the first nucleotide sequence (the “donor sequence”) can contain sequences that are homologous, but not identical, to genomic sequences in the region of interest, thereby stimulating homologous recombination to insert a non-identical sequence in the region of interest. Thus, in certain embodiments, portions of the donor sequence that are homologous to sequences in the region of interest exhibit between about 80 to 99% (or any integer therebetween) sequence identity to the genomic sequence that is replaced. In 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), 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. 
         [0079]    “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. 
         [0080]    “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 H1 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. 
         [0081]    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. 
         [0082]    A “target site” or “target sequence” is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind, provided sufficient conditions for binding exist. For example, the sequence 5′-GAATTC-3′ is a target site for the Eco RI restriction endonuclease. 
         [0083]    An “exogenous” molecule relates to a molecule that is not noanally 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 also be a molecule normally found in another species, for example, a human sequence introduced into an animal&#39;s genome. 
         [0084]    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 acidsProteins 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. 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. 
         [0085]    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. 
         [0086]    A “fusion” molecule relates 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 and fusion nucleic. Examples of the second type of fusion molecule include, but are not limited to, a fusion between a triplex-foiming nucleic acid and a polypeptide, and a fusion between a minor groove binder and a nucleic acid. 
         [0087]    A “gene,” for the purposes of the present disclosure, relates to 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, 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. 
         [0088]    A “gene regulatory sequence” is a part of a gene that is non-coding for a protein product and can recruit DNA binding protein that can modulate gene expression, either up or downregulation. It can be located upstream or downstream of a gene as well as intragenic. 
         [0089]    “Gene expression” or “expression” relates 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 a 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. 
         [0090]    “Modulation” of gene expression relates to a change in the activity of a gene. Modulation of expression can include, but is not limited to, gene activation and gene repression. Genome editing (e.g., cleavage, alteration, inactivation, random mutation) can be used to modulate expression. Gene inactivation refers to any reduction in gene expression as compared to a cell that does not include a ZFP as described herein. Thus, gene inactivation may be partial or complete. 
         [0091]    A “region of interest” relates 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. 
         [0092]    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. 
         [0093]    The CRISPR/Cas9 constructs described herein may be “delivered” or “introduced” into a target cell by any suitable means, including, for example, by injection of mRNA or accordingly nucleic acid, for example, a CDNA, CRNA, or IRNA. See, Hamrnerschmidt et al. (1999) Methods Cell Biol. 59:87-115. Methods of delivering proteins comprising zinc-fingers are described, for example, in U.S. Pat. Nos. 6,453,242; 6,503,717; 6,534,261; 6,599,692; 6,607,882; 6,689,558; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, the disclosures of all of which are incorporated by reference herein in their entireties. 
         [0094]    CRISPR/Cas9 as described herein may also be delivered using “vectors” containing sequences encoding one or more of the CRISPR/Cas9s variations. 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. Pat. 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 CRISPR/Cas9 encoding sequences. Thus, when one or more pairs of CRISPR/Cas9 are introduced into the cell, the CRISPR/Cas9 may be carried on the same vector or on different vectors. 
         [0095]    When multiple vectors are used, each vector may comprise a sequence encoding one or multiple CRISPR/Cas9. Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding engineered CRISPR/Cas9 in cells. Such methods can also be used to administer nucleic acids encoding CRISPR/Cas9 to cells in vitro. In certain embodiments, nucleic acids encoding CRISPR/Cas9 are administered for in vivo or ex vivo uses. Non-viral vector delivery systems 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 can also be used for delivery of nucleic acids. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. Additional exemplary nucleic acid delivery systems include those provided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.) and Copernicus Therapeutics Inc, (see for example U.S. Pat. No. 6,008,336). Lipofection is described in e.g., U.S. Pat. No. 5,049,386, U.S. Pat. No. 4,946,787; and U.S. Pat. No. 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 Felgner, WO 91/17424, WO 91/16024. Delivery can be to cells (ex vivo administration) or target tissues (in vivo administration). 
         [0096]    The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. 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 vol 27(7) p. 643). 
         [0097]    As noted above, the disclosed methods and compositions can be used in any type of cell, progeny, variants and derivatives of animal cells can also be used. 
         [0098]    By “Integration” relates to both physical insertion (e.g., into the genome of a host cell) and, in addition, integration by copying of the donor sequence into the host cell genome via the nucleic acid replication processes. Donor sequences can also comprise nucleic acids such as CDNA, CRNA, IRNA, shRNAs, miRNAs, etc. Additional donor sequences of interest may be human genes which encode proteins relevant to disease models. 
         [0099]    “Genomic editing” (e.g., inactivation, integration and/or targeted or random mutation) of an animal gene can be achieved, for example, by a single cleavage event, by cleavage followed by non-homologous end joining, by cleavage followed by homology-directed repair mechanisms, by cleavage followed by physical integration of a donor sequence, by cleavage at two sites followed by joining so as to delete the sequence between the two cleavage sites, by targeted recombination of a missense or nonsense codon into the coding region, by targeted recombination of an irrelevant sequence (i.e., a “stuffer” sequence) into the gene or its regulatory region, so as to disrupt the gene or regulatory region, or by targeting recombination of a splice acceptor sequence into an intron to cause mis-splicing of the transcript. See, U.S. Patent Publication Nos. 20030232410; 20050208489; 20050026157; 20050064474; 20060188987; 20060063231; and International Publication WO 07/014,275, the disclosures of which are incorporated by reference in their entireties for all purposes. 
         [0100]    “Stem cell” as used herein, refers to an undifferentiated cell that is capable of self-renewal and differentiation into any tissue type of an organism (e.g. neuron, neuroprogenitor cell). 
         [0101]    “Brain-related cell” as used herein, refers to a cell of the central nervous system and can comprise of a neural stem cell, neuroprogenitor cell, neuron, glia cell, astrocyte, oligodendrocyte and their progeny. 
       Example 1 
     Neurodegenerative Targets Disease and Associated Gene 
       [0102]    The compositions and methods disclosed have been described with respect to at least one gene, such as SNCA or alpha-synuclein gene (SNCA: synuclein, alpha (non A4 component of amyloid precursor) chromosomal location 4q21.3-q22), and at least one disorder, Parkinson&#39;s disease (Online Mendelian Inheritance in Man, OMIM #607060). However, others skilled in the art will appreciate that the methods of gene editing and modification and delivery of the constructs described herein are generally applicable to those neurodegenerative diseases in which over expression or decreased expression of the noimal wildtype sequence or specific genetic variants causes neurodegenerative disease or syndromes The neurodegenerative diseases and genes are listed in Tables 1 and 2. 
         [0103]    An overview of the different targeting strategies is visualized in  FIG. 1 . Gene editing or genome editing with engineered nucleases relies on the introduction of double-strand breaks in the DNA by specific enzymes, called nucleases or ‘molecular scissors’. Currently, there are four known such nucleases; zinc-finger nuclease (ZFN), TAL effector nuclease (TALEN), clustered, regularly interspaced, short palindromic repeat (CRIPSR/Cas9) or meganuclease. These nucleases are use alone or in combination, e.g. Cas9 and Fok1. These double strand breaks are not random events, but can be placed very specifically in the genome with guided sequences complementary to the target sequence of the genome. 
         [0104]    To further increase the efficiency for the system by multiplexing, additional guide RNAs are designed against the specific SNCA exons 3, 4, or 5, which are listed in Table 4. Multiplexing relates to combining multiple constructs in one transfection by delivering several guide RNAs simultaneously. 
       Example 2 
     CRISPR sgRNA in Silico Design 
       [0105]    Several publicly available bioinformatics tools are used to design oligonucleotides for small guide RNAs. 
         [0106]    The in silico design workflow requires input the target sequence (e.g. SNCA coding region, Human Genome Build hg19, length &lt;250 bp) into the CRISPR Design Tool with the following settings: 1) “other region (25-500 nt)” for sequence type and 2) “human (hg19)” for target genome. The CRISPR Design Tool output list of sgRNA designs is ranked according to their “quality scores”. Selection criteria are: 1) high quality guides or/and 2) a variety of sgRNA designs that are far apart from one another and on both strands (+/−) for non-coding ECRs. The candidate designs have to be subsequently assessed for off-target analysis. 
         [0107]    To identify the sgRNA sequence for the SNCA gene, the consensus mRNA was used for the SNCA gene, transcript variant 1 (longest isoform, containing all 6 exons) NM_000345.3 (Genome Reference Assembly hg19/GRCh37) and individually searched exons for sgRNA target sequences. The designs with the highest predictive score and least mis-matches using Cas-OFFinder and GGGenome are selected for further validation. Targeting exonic (coding) sequence will introduce random mutations that will lead to novel allelic forms of the gene. These mutations (insertions, deletions) can alter the reading frame of the transcribed RNA and lead to a non-functional protein. 
         [0108]    To identify the sgRNA sequence for the MAPT gene, the consensus mRNA was used for the MAPT gene, transcript variant 1 (longest isoform, containing all exons) NM_016835.4 (Genome Reference Assembly hg19/GRCh37) and individually searched exons for sgRNA target sequences. These mutations (insertions, deletions) can alter the reading frame of the transcribed RNA and lead to a non-functional protein. The three designs with the highest predictive score and least mis-matches using Cas-OFFinder and GGGenome are selected for further validation. Targeting exonic (coding) sequence will introduce random mutations that will lead to novel allelic forms of the gene. 
         [0109]    These sgRNA sequences will be experimentally proven and verified. A schematic overview illustrates the workflow ( FIG. 2 ). Table 3 shows SNCA CRISPR guide RNA design in exon 2 for three target guide sequences.  FIG. 3  shows the experimental verification of the SNCA CRISPR/Cas9 constructs, U6 guide RNA vector and CMV humanized Cas9 vector, in human HEK293 cells. The HEK293 cells were plated the day before transfection. On the day of transfection the cells were 80-90% confluent and then transfected with 500 ng each of DNA construct with Lipofectamine (Life Technologies). 48 hours later the HEK293 cells were harvested using GeneArt QE DNA kit. DNA was amplified with custom-designed primers to amplify exon 2. The PCR product was subjected to an enzyme Cel-1 that cleaves the DNA where there are heterozygous mutations present. The main PCR amplification band was about 500 bp. The arrows in lane 2, 4, 6, and 8 point to bands that resulted from cleavage by Cel-1 which allows us to detect mismatch changes between DNA strands indicating a double strand break induced insertion/deletion in a pool of cells. Three microliter of PCR reaction was run on 2% AgaroseEX on E-gel iBase Power System. 
         [0110]    The sgRNA sequences for the MAPT gene will be to experimentally proven and verified in parallel with the SNCA studies. Table 5 shows MAPT CRISPR guide RNA design in exon 2 for three target guide sequences.  FIG. 4  shows the experimental verification of the MAPT CRISPR/Cas9 constructs, U6 guide RNA vector and CMV humanized Cas9 vector, in human HEK293 cells. The HEK293 cells were plated the day before transfection. On the day of transfection the cells were 80-90% confluent and then transfected with 500 ng each of DNA constructs with Lipofectamine (Life Technologies). 48 hours later the HEK293 cells were harvested using by GeneArt QE DNA kit. DNA was amplified with custom-designed primers for amplifying exon 2 and PCR product was subjected to an enzyme Cel-1 that cleaves the DNA when there are heterozygous mutations present. The main PCR amplification band was about 500 bp. The arrows in lane 2, 4, 6, and 8 point to bands that resulted from cleavage by Cel-1 which allows to detect mismatch changes between DNA strands indicating a double strand break induced insertion/deletion in a pool of cells. Three microliter of PCR reaction was run on 2% AgaroseEX on E-gel iBase Power System. 
       Example 3 
     Gene Regulation to Downregulate Alpha-Synuclein 
       [0111]    Gene regulation can be achieved by a mutant dCas9 construct, a catalytically inactive programmable RNA-dependent DNA binding protein, fused to VP16 tetramer activation domain or a Krueppel-associated box (KRAB) repressor domain. Twelve regulatory domains were identified within the SNCA gene (Sterling et al., 2014) ( FIGS. 5-7 ) that can be targeted similar to the guide RNAs within the exons of the SNCA and MAPT gene. By mutant CRISPR/dCas9 binding to specific regulatory sites fused to a repressor domain, alpha-synuclein expression can be down regulated. 
         [0112]    The catalytically dead Cas-9 (dCas9) lacking endonuclease from type II CRISPR system can control gene expression when co-expressed with sgRNA. The dCas9 generates a DNA recognition complex that does not cleave the DNA, but that can specifically interfere with transcriptional elongation, RNA polymerase binding, and/or transcription factor binding (Jinek et al., 2012; Qi et al., 2013). This system does not alter the genome but modifies gene expression by steric hindrance and is reversible (Kearns et al., 2014; Larson et al., 2013; Qi et al., 2013; Sampson and Weiss, 2014; Xu et al., 2014). The system also does not need the host machinery to function, whereas with genome editing, the cell needs proper function of certain protein and pathway functions e.g. NHEJ or HDR (Xu et al., 2014)  FIG. 1 , panel C). 
         [0113]    Besides the inhibition of gene expression, Cas9 can also be combined with a transactivator and and sgRNAs to induce specific expression of endogenous target genes, which could be a versatile approach for RNA-guided gene activation (Farzadfard et al., 2013). 
         [0114]    The dCas9 can be fused to either a CP16 tetramer activation domain (VP64) or a Krueppel-associated box (KRAB) repressor domain. The CRISPR/dCas9 effector system can either be used to modulate or block transcription (CRISPR interference) (Bikard et al., 2013; Kearns et al., 2014; Qi et al., 2013) or activate gene expression using and incorporated effector domain (CRISPR activation) targeting e.g. the promoter region of a gene (Gilbert et al., 2013; Kearns et al., 2014; Konermann et al., 2013; Mali et al., 2013) ( FIG. 1 , panel C). 
         [0115]    Gene Regulation Through Evolutionary Conserved Genomics Regions. 
         [0116]    Two main mechanisms are involved in the transcriptional regulation of genes. The first mechanism is gene accessibility, which is regulated through chromatin structure, nucleosome positioning, histone modifications, and DNA methylation. The second factor involves the initiation of transcription through distinct transcription factors such as activators or repressors, and other modulating factors. Transcription factors bind to genomic regions with specific recognition sites. 
         [0117]    Identification of 32 Evolutionary Conserved Non-Coding Genomic Regions (ncECRs) within the SNCA Gene 
         [0118]    To identify evolutionary conserved non-coding regions, two complementary genome browsers were used (Vista browser (http://pipeline.lb1.gov/cgi-bin/gateway2) and ECR browser (http://ecrbrowser.dcode.org/)) to establish a genetic conservation profile of the SNCA gene by aligning the human SNCA gene with mouse in a pair-wise fashion. Established selection parameters were &gt;100 bp in length and &gt;75% identity (Dubchak et al., 2000; Loots et aL, 2000). In addition to 111.4 kb SNCA genomic region, a 44.5 kb upstream and a 50 kb downstream intergenic region to capture surrounding promoter and regulatory elements (Sterling et al., 2014) ( FIG. 6 ). 
         [0119]    32 non-coding evolutionary conversed regions (ncECRs) in the SNCA gene region of 206 kb on chromosome 4q21 (Chr.4: 90, 961056-91, 167082) were identified by pair-wise comparison between human and mouse ( FIG. 6 ). In comparison to similar screens, where conserved regions range from 8-45 elements (Grice et al., 2005; Liu and Francke, 2006; Sabherwal et al., 2007), a similar number of elements was found that show a high evolutionary conservation. (Sterling et al., 2014). 
         [0120]    The Promega pGL3 luciferase reporter vectors and the neuroblastoma cell line SK-N-SH were used as a tool to determine the SNCA regulatory elements. NcECRs identified through the comparative analysis were cloned upstream of a SV-40 promoter in the pGL3 promoter construct, transfected in neuroblastoma cells and assayed in a dual luciferase assay by measuring firefly luciferase and  renilla  luciferase as an internal control to normalize the firefly signal ( FIG. 5 ) (Sterling et al., 2014). 
         [0121]    Cells were transfected at 90-95% confluency. All transfections were performed in quadruplicates and each experiment was repeated three times. For the luciferase assay, the Dual-Luciferase® Reporter (DLR™) Assay System (Promega) was used, in which activities of firefly and  Renilla  luciferases can be measured sequentially in one sample (Sterling et al., 2014). 
         [0122]    Overall, 12 of 32 constructs exhibited either an enhancement or reduction of the expression of the reporter gene ( FIG. 2 ). Three elements upstream of the SNCA gene displayed an approx. 1.5 fold (p&lt;0.009) increase in expression (U3, U4-1, U4-3). Of the intronic regions, three showed a 1.5 fold increase (12, 16, 18) and two others caused a 2 and 2.5 fold increase (I5, I12) in expression (p&lt;0.002). Two elements downstream of the SNCA gene showed 1.5 fold (D1, D2) and 2.5 fold increase (D3; p&lt;0.0009). One element downstream of SNCA had a reduced expression of the reporter gene of 0.35 fold (D6; p&lt;0.0009) of normal activity that was also continued in a pGL3 control vector ( FIG. 2C , insert). The D6 element reduced the expression of the pGL3 control construct by ˜50%, confirming that this element represents a strong repressor. These data provide experimental evidence that a significant proportion of the ncECRs show a regulatory function in the luciferase reporter assay ( FIG. 7 ) (Sterling et al., 2014). 
         [0123]    The NACP-Rep1 polymorphism of the SNCA promoter, a mixed dinucleotide repeat, that increases the expression of alpha-synuclein when the long allele is present. A mutant Cas9 repressor/CRISPR guide RNA against this region will facilitate downregulation of gene expression of alpha-synuclein ( FIG. 1C ). 
         [0124]    With the identification of regulatory elements in the SNCA gene, CRISPR guide RNAs will be designed against these regulatory regions to modestly downregulate the expression of alpha-synuclein. Gene downregulation will be achieved by a mutant Cas9 construct that can be fused to a Krueppel-associated box (KRAB) repressor domain. The advantage of the system is that the regulation is modest versus a complete knockdown and at the same time abolishing its normal function ( FIG. 8 ). 
         [0125]    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, gene or chromatin structure analysis, computational biology, cell culture, recombinant DNA methods and related methods or techniques within the skill of the art. These techniques and principles are fully explained in the literature. 
         [0126]    Although the 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. 
       Example 4 
     Predicted Intracellular Distribution of Alpha-Synuclein Isoforms 
       [0127]    The full length alpha-synuclein has 140 amino acids. However, there are several smaller alpha synuclein proteins that lack certain exon(s) and therefore are shorter in nature, isoform 112, isoform 126, and isoform 96. SNCA gene isoform 112, lacking exon 5, has a similar structure as compared to two published constructs 1-102aa-eGFP (Specht et al., 2005) and 140aa-NES (Kontopoulos et al., 2006), which promote cytoplasmic distribution. Cytoplasmic distribution of alpha-synuclein has shown to be neuroprotective. The Alpha-synuclein gene isoform 112 can be created by double CRISPR editing thus releasing exon 5. Using a double gene editing approach as discussed in  FIG. 1C or 1D  will result in specific splice forms of the alpha-synuclein protein and potentially be neuroprotective. 
       Prophetic Example 5 
     Virus Delivery System for In Vivolin Human Gene Editing for Neurodegeneration Disease Progression 
       [0128]    The delivery vehicle that will be utilized for Cas9/CRISPR delivery will be an adeno-associated virus (AAV) (Bartus et al., 2013; Bartus et al., 2011b; Marks et al., 2010) or lentiviral (Palfi et al., 2014) vector that have been previously used for gene delivery in human brain for PD. Both viruses have been used in clinical trials in human for brain delivery and have been proven in phase I/II clinical trials to be safe and well tolerated (Bartus et al., 2014; Kaufmann et al., 2013). Other viral delivery options for the gene editing technology proposed in this application will be considered when available and if deemed to be more advantageous over the aforementioned approaches. 
         [0129]    Over the last several years, a total of 9 gene therapy clinical trials have been performed for the treatment of PD. Even though the clinical benefits were small or clinical endpoints were not reached, these clinical trials were necessary and important to demonstrate that gene therapy in the central nervous system is not producing an untoward risk or harm after administration of different viral vector delivery constructs. The gene therapy trials have also paved the road for new clinical trial designs, patient selection, and outcome measures based on treatment specific differences with gene therapy in the CNS (Bartus et al., 2014). 
         [0130]    Adeno-associated viral vectors are excellent vehicles to transfer genes into the nervous system due to their property to transduce also post-mitotic cells, their ability to be grown to very high titers (up to 10 13  virion particles per ml), and their relatively large insert capacity (insert capacity of ˜7.5 kb). Adenoviral vectors can express the transgene(s) for a long time in the CNS in vivo and in cell culture such as neurons and glia (Southgate et al., 2008). 
         [0131]    Lentiviral vectors have shown to have a low immunogenicity, can transduce neurons, and can carry large inserts which allows for the introduction of multiple sgRNAs against several locations of one gene or multiple genes concurrently (Cockrell and Kafri, 2007). 
         [0132]    The CRISPR gene editing or gene regulation approach will be delivered in the brain via stereotactic surgery into the substantia nigra pars compacta (Kells et al., 2012; Salegio et al., 2012). Depending on the state of the art at the time of entering into clinical trials, stereotactic administration into the substantia nigra or other basal ganglia will be determined. 
         [0133]    Gene therapy delivery systems discussed for in vivo gene editing application in human. These do not form part of the instant application but are provided for information only. 
         [0134]    Gene editing in patient-derived stem cells or human embryonic stem cells followed by neuronal transplantation as a therapeutic approach to prevent neurodegeneration and disease progression. 
         [0135]    For more advanced stages of neurodegeneration in PD or related neurodegenerative disease different approaches as proposed ( FIG. 10 ). Besides the direct treatment with AAV CRISPR constructs including sgRNA targeting SNCA or MAPT, human stem cells from different sources, either human embryonic stem cells (hESC) or HLA-typed induced pluripotent stem cells (iPSC) or individual patient-derived pluripotent stem cell clones are being gene modified with CRISPR/sgRNA SNCA to specifically lower the expression of alpha-synuclein in a controlled fashion. Correctly targeted stem cell clones are then differentiated into neuronal cultures (neuroprogenitor cells) and subsequently transplanted into the basal ganglia in the CNS. 
         [0136]    Fetal Transplant Trials in Parkinson&#39;s Disease. 
         [0137]    Estimates from pre-clinical models suggest that one dopaminergic neurons in the substantia nigra in the brainstem can innervate up to 75,000 neurons in the neostriatum, on the other hand one striatal neuron can be under the influence of 100-200 dopaminergic neurons (Matsuda et al., 2009). This suggests that here must be an enormous energy requirement for dopaminergic neurons, but there is also major redundancy of the system which could explain why motor symptoms of PD only present when dopaminergic demise reaches a critical threshold of loss of 70-80% of striatal nerve terminals and 50-60% of the nuclei in the SNc (Bernheimer et al., 1973; Riederer and Wuketich, 1976). 
         [0138]    If dopaminergic neurons could be replenished and transplanted neuronal cells would survive, re-innervate the lesions in the midbrain and improve symptoms, the disease could potentially be cured. This has been the working hypothesis for cellular replacement in PD for the last 35 years and is a major hope for patients to overcome the disease (Bjorklund et al., 2003; Freed et al., 2011). 
         [0139]    The first report on cell replacement in a parkinsonian pre-clinical model was published in 1979, when rats were transplanted with fetal stem cells which survived, had axonal outgrowth and motor abnormalities were reduced (Perlow et al., 1979). The first fetal transplant surgery in human was performed in 1987 and since then an estimated 300 PD patients have received implantation of human fetal tissue from 6-9 week old aborted fetuses (Clarkson, 2001). Initial open-label studies have been promising and provided proof of principle that grafted DA neurons can improve motor symptoms of PD, however, the interpretation of the functional improvements seen in these open-label studies has been questioned because placebo effects and/or natural history of some mild forms of PD (Freed et al., 1993; Freed et al., 1992a; Freed et al., 1990a; Freed et al., 1990b; Freed et al., 1992b; Lindvall et al., 1990; Lindvall et al., 1989). 
         [0140]    Long-term outcome of fetal transplants were first reported in 2008 with several publications reporting alpha-synuclein positive Lewy body pathology in grafts of autopsies from brain donors, showing that the disease process continues and the healthy graft also degenerates over time (Kordower et al., 2008; Li et al., 2008; Mendez et al., 2008). 
         [0141]    The gene editing approach of alpha-synuclein modulation and repression will ‘shield’ or protect the dopaminergic neurons in the brain as well as the transplanted neurons described above. Similarly this approach can be utilized for protein aggregation of tau or other proteins aggregating in the cell. 
       Example 6 
     Virus Delivery System 
       [0142]    Viral vectors: The delivery vehicle that will be utilized for Cas9/CRISPR delivery will be an adeno-associated virus (AAV) (Bartus et al., 2013; Bartus et al., 2011b; Marks et al., 2010) or lentiviral (Palfi et al., 2014) vector that have been previously used for gene delivery in human brain for PD. Both viruses have been used in clinical trials in human for brain delivery and have been proven in phase I/II clinical trials to be safe and well tolerated (Bartus et al., 2014; Kaufmann et al., 2013). Other viral delivery options for the gene editing technology proposed in this application will be considered when available and if deemed to be more advantageous over the aforementioned approaches. 
         [0143]    AAV vectors: Adeno-associated viral vectors are excellent vehicles to transfer genes into the nervous system due to their property to transduce also post-mitotic cells, their ability to be grown to very high titers (up to 10 13  virion particles per ml), and their relatively large insert capacity (insert capacity of ˜7.5 kb). Adenoviral vectors can express the transgene(s) for a long time in the CNS in vivo and in cell culture such as neurons and glia (Southgate et al., 2008). 
         [0144]    Lentiviral vectors: Lentiviral vectors have shown to have a low immunogenicity, can transduce neurons, and can carry large inserts which allows for the introduction of multiple sgRNAs against several locations of one gene or multiple genes concurrently (Cockrell and Kafri, 2007). 
         [0145]    Gene editing design: The CRISPR/Cas9 gene will be cloned into the delivery vector such as AAV or lentivirus together with the designed sgRNA against selected regions of the SNCA or MAPT gene. The sgRNA can comprise of any one or combination of the sgRNA designs in this application, either exonic, intronic, promoter region, or regulatory regions. 
         [0146]    Stereotactic surgery: The CRISPR gene editing or gene regulation approach will be delivered in the brain via stereotactic surgery into the substantia nigra pars compacta (Kells et al., 2012; Salegio et al., 2012). Depending on the state of the art at the time of entering into clinical trials, stereotactic administration into the substantia nigra or other basal ganglia will be determined. The clinical trial design will be based on prior designs of neuromodulating therapies (Bartus et al., 2011a; Marks et al., 2010; Marks et al., 2008). 
         [0147]    Patient population: The patient populations envisioned for this treatment of gene editing of the SNCA genes are patients with Parkinson&#39;s disease in early disease stages, such as Hoehn and Yahr 1 and 2, when the symptoms are unilateral or bilateral, but no gait instability or balance problems are present. Patients with early signs of cognitive decline or mild cognitive impairment can be candidates for the MAPT in vivo gene editing. 
       Example 7 
     Gene Editing in Patient-Derived Stem Cells or Human Embryonic Stem Cells Followed by Neuronal Transplantation 
       [0148]    Replenishment of dopaminergic neurons and the survival of transplanted neuronal cells, it is expected that re-innervation of the lesions in the midbrain and improvement of symptoms, would be treated. This has been the working hypothesis for cellular replacement in PD. (Bjorklund et al., 2003; Freed et al., 2011). 
         [0149]    Stem cell derivation and characterization: Different stem cell lines can be used for this approach: (1) Well-characterized human embryonic stem cell (ESC) lines; (2) Human derived induced pluripotent stem cells (iPSCs) from the patient; or (3) Matched HLA-type iPSCs. Human ESCs or matched HLA-type iPSCs can be obtained from a cell bank whereas patient&#39;s own iPSCs can be grown and reprogrammed in animal free conditions and under good manufacturing practices (GMP). Stem cells are derived and they passed quality control of gene expression for pluripotency markers, markers for all three germ layers, and potential to differentiate in target tissue of interest (e.g. neurons). Cells are vigorously tested for genetic changes during in vitro culture. 
         [0150]    CRISPR/Cas9 cloning: After tissue derivation, the CRISPR/Cas9 gene will be cloned into the delivery vector separate or together with the designed sgRNA against selected regions of the SNCA or MAPT gene. The sgRNA can comprise of any one or combination of the sgRNA designs in this application, either exonic, intronic, promoter region, or regulatory regions. Designs for direct viral delivery or ex vivo editing use the same sgRNA sequences. The vector design can vary. 
         [0151]    Ex vivo editing: Human stem cells from different sources (hESCs, iPSCs) are being gene modified with CRISPR/sgRNA SNCA to specifically lower the expression of alpha-synuclein in a controlled fashion. Gene editing vectors are delivered via electroporation or nucleofection. Stem cells recover from nucelofection of one passage before they are subcloned and single clones are tested for gene modification by various genetic amplification methods (T7 endonuclease treatment, gene sequencing or droplet PCR). Correctly targeted stem cell clones are then differentiated into neuronal cultures (neuroprogenitor cells) either as embryoid body/neural rosette cultures (Ebert et al., 2013; Shelley et al., 2014; Swistowski et al., 2009), small molecule-induced direct differentiation (Ganat et al., 2012; Kriks et al., 2011) (Cooper et al., 2010; Hargus et al., 2010) and subsequently transplanted into the basal ganglia in the CNS (Clarkson, 2001; Freeman et al., 1995; Kordower et al., 1998; Olanow et al., 2001; Olanow et al., 2003). 
         [0152]    Patient population: Ex vivo gene modification with subsequence transplantation is proposed from move advance stages of Parkinson&#39;s disease, disease stage Hoehn and Yahr 2 and higher. 
       Example 8 
     Viral Gene Delivery and Gene Edited Cell Transplantation 
       [0153]    A third alternative approach is the combination of viral gene delivery and transplantation of gene edited cells to preserve the existing neurons and replenish with the new neurons that are less likely develop disease due to its gene modification which promotes neuroprotection. 
       Tables 
       [0154]      
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Neurodegenerative diseases 
               
               
                 A non-exhaustive list of neurodegenerative diseases that can be 
               
               
                 targeted in the brain with the proposed invention is presented in 
               
               
                 Table 1. The examples of diseases listed can be targeted with 
               
               
                 this method when a specific gene is known to cause the disease. 
               
             
          
           
               
                   
                 Phenotype 
               
               
                 Disease; Disease Symbol 
                 MIM Number 
               
               
                   
               
               
                 Dementia with Lewy bodies; DLB 
                 #127750 
               
               
                 Parkinson&#39;s disease 
                 #168600 
               
               
                 Parkinson&#39;s disease 1, autosomal dominant; PARK1 
                 #168601 
               
               
                 Parkinson&#39;s disease, juvenile, type 2; PARK2 
                 #600116 
               
               
                 Parkinson&#39;s disease, juvenile, type 6; PARK6 
                 #605909 
               
               
                 Parkinson disease 7, autosomal recessive early-onset; 
                 #606324 
               
               
                 PARK7 
               
               
                 Parkinson&#39;s disease 8, autosomal dominant; PARK8 
                 #607060 
               
               
                 Frontotemporal dementia, Pick complex included; FTD 
                 #600274 
               
               
                 Kufor-Rakeb Syndrome; KRS 
                 #606693 
               
               
                 Parkinson disease 14, autosomal recessive; PARK14 
                 #612953 
               
               
                 Heterogeneous phenotype: dystonia-parkinsonism, 
                 #256600 
               
               
                 dementia, frontotemporal atrophy, w/wo brain 
               
               
                 iron accumulation 
               
               
                 Neurodegeneration with brain iron accumulation 2A; 
                 #256600 
               
               
                 NBIA2A 
               
               
                 Parkinsonian-Pyramidal Syndrome; PARK15 
                 #260300 
               
               
                 Parkinson&#39;s disease 17; PARK17 
                 #614203 
               
               
                 Parkinson&#39;s disease 18; PARK18 
                 #614251 
               
               
                 Parkinson disease 19, Juvenile-onset; PARK19 
                 #615528 
               
               
                 Parkinson&#39;s disease 20; PARK20 
                 #615530 
               
               
                 Dystonia-Parkinsonism, X-Linked; DYT3 
                 #314250 
               
               
                 Dopamine transporter deficiency syndrome; DTDS 
                 #613135 
               
               
                 Gaucher disease; GD1 
                 #230800 
               
               
                 Tay-Sachs disease; TSD 
                 #272800 
               
               
                 Niemann-Pick disease, type A 
                 #257200 
               
               
                 Niemann-Pick disease, type B 
                 #607616 
               
               
                 Alzheimer disease; AD 
                 #104300 
               
               
                 Alzheimer disease, type 3; AD3 
                 #607822 
               
               
                 Pick disease 
                 #172700 
               
               
                 Multiple system atrophy; MSA1 
                 #146500 
               
               
                 Dystonia, dopa-responsive; DRD, DYT5 
                 #128230 
               
               
                 Perry syndrome, parkinsonism with alveolar 
                 #168605 
               
               
                 hypoventilation and mental depression 
               
               
                 Dystonia-Parkinsonism, X-linked, Lubag disease, 
                 #314250 
               
               
                   
               
             
          
         
       
     
         [0155]    The database Online Mendelian Inheritance of Man (MIM) Ahttp://www.ncbi.nlm.nih.gov/omim)&gt;, the contents of which are incorporated herein by reference, describes genetic causation and association factors for a range of disease states and conditions, indexing the genes according to a “Gene/Locus MIM Number” and the disease states and conditions according to a “Phenotype MIM Number”. Table 1 below provides non-limiting examples of genetically linked adult-onset neurodegeneration diseases listed in the Online MIM. For further details of each condition, including the chromosomal locations and names of the genes, as well as literature references and discussion of the underlying research, please refer to Table 2 below and the OMIM website. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 A non-exhaustive list of examples of genes 
               
               
                 A non-exhaustive list of examples of genes that can be targeted for Parkinsonian 
               
               
                 disorders, memory function and related disorders recessive disorder is provided below. 
               
             
          
           
               
                 Symbol 
                   
                   
                   
                 Inheritance 
                   
               
               
                 for the 
                   
                   
                   
                 (AD = autosomal 
                 Gene/Locus 
               
               
                 Disease, 
                 HUGO gene name, 
                   
                   
                 dominant; 
                 Number or 
               
               
                 HUGO 
                 symbol, locus, reference 
                 Gene 
                   
                 AR = autosomal 
                 other MIM 
               
               
                 Gene 
                 sequence 
                 locus 
                 Disorder 
                 recessive) 
                 Reference 
               
               
                   
               
               
                 PARK1, 
                 Synuclein, alpha (non A4 
                 4q21-22 
                 Parkinson&#39;s disease, 
                 AD 
                 #168601 
               
               
                 SNCA 
                 component of amyloid 
                   
                 Dementia with Lewy 
               
               
                   
                 precursor), SNC4, chr4q21-22, 
                   
                 bodies 
               
               
                   
                 NM_000345.3 
               
               
                 MAPT 
                 microtubule-associated 
                   
                 Frontotemporal 
                 AD 
                 #157140 
               
               
                   
                 protein tau, 17q21.31 
                   
                 dementia, with or 
               
               
                   
                   
                   
                 without parkinsonism, 
               
               
                   
                   
                   
                 Alzheimer disease 
               
               
                 PSEN1 
                 Presinilin 1, 14q24.2 
                 14q24.2 
                 Frontotemporal 
                 AD 
                 #104311 
               
               
                   
                   
                   
                 dementia, Alzheimer 
               
               
                   
                   
                   
                 disease 
               
               
                 PARK 2, 
                 Parkin RBR E3 ubiquitin 
                 6q25.2-q27 
                 Parkinson&#39;s disease, 
                 AR 
                 #602544 
               
               
                 PARKIN 
                 protein ligase, PARK2, 
                   
                 juvenile, type 2 
               
               
                   
                 chr6q25.2-p27 
               
               
                   
                 NM_004562.2 
               
               
                 PARK4, 
                 Synuclein, alpha (non A4 
                 4q21-22 
                 Parkinson&#39;s disease, 
                 AD 
                 #605543 
               
               
                 SNCA 
                 component of amyloid 
                   
                 Dementia with Lewy 
               
               
                   
                 precursor) 
                   
                 bodies 
               
               
                 PARK6, 
                 PTEN induced putative 
                 1p35-p36 
                 Parkinson&#39;s disease, 
                 AR 
                 #608309 
               
               
                 PINK1 
                 kinase 1, PINK1, chr1p35-36, 
                   
                 juvenile, type 6 
               
               
                   
                 NM_032409.2 
               
               
                 PARK7, 
                 Daisuke-Junko 1, DJ-1 
                 1p36 
                 Parkinson&#39;s disease 7, 
                 AR 
                 #602533 
               
               
                 DJ-1 
                 chr1p36 
                   
                 autosomal recessive 
               
               
                   
                 NM_001123377.1 
                   
                 early-onset 
               
               
                 PARK8, 
                 Leucine-rich repeat kinase 
                 12q12 
                 Parkinson&#39;s disease, 
                 AD 
                 #609007 
               
               
                 LRRK2 
                 2, LRRK2, chr12q12, 
                   
                 Parkinson&#39;s disease 
               
               
                   
                 NM_198578.3 
                   
                 dementia, Dementia 
               
               
                   
                   
                   
                 with Parkinson&#39;s 
               
               
                   
                   
                   
                 disease 
               
               
                 PARK9, 
                 ATPase type 13A2, 
                 1p36 
                 Kufor-Rakeb 
                 AR 
                 #610513 
               
               
                 ATP13A2 
                 ATP13A2, 
                   
                 syndrome 
               
               
                   
                 chr1p36, NM_022089.3 
               
               
                 PARK14, 
                 Phospholipase A2, group VI 
                 22q13.1 
                 Dystonia- 
                 AR 
                 *603604 
               
               
                 PLA2G6 
                 (cytosolic, calcium- 
                   
                 parkinsonism, 
               
               
                   
                 independent), PLA2G6, 
                   
                 dementia, 
               
               
                   
                 chr22q13.1, NM_003560.2 
                   
                 frontotemporal 
               
               
                   
                   
                   
                 atrophy, w/wo brain 
               
               
                   
                   
                   
                 iron accumulation, 
               
               
                   
                   
                   
                 pallido-pyramidal 
               
               
                   
                   
                   
                 syndrome 
               
               
                 PARK15, 
                 F-box protein 7, FBXO7, 
                 22q12-q13 
                 Pallido-pyramidal 
                 AR 
                 #605648 
               
               
                 FBX07 
                 chr22q12-q13, 
                   
                 syndrome 
               
               
                   
                 NM_012179.3 
               
               
                 PARK17, 
                 Vacuolar protein sorting 35 
                 16q11.2 
                 Parkinson&#39;s disease 17 
                 AD 
                 *601501 
               
               
                 VPS35 
                 homolog ( S. cerevisiae ), 
               
               
                   
                 VPS35, chr16q11.2, 
               
               
                   
                 NM_018206.4 
               
               
                 PARK18, 
                 Eukaryotic translation 
                 3q27.1 
                 Parkinson&#39;s disease 18 
                 AD 
                 *600495 
               
               
                 EIF4G1 
                 initiation factor 4 gamma, 1, 
               
               
                   
                 EIF4G1, chr3q27.1, 
               
               
                   
                 NM_001291157.1 
               
               
                 PARK19, 
                 DnaJ (Hsp40) homolog, 
                 1p31.3 
                 Neurodegenerative 
                 AR 
                 #608375 
               
               
                 DNAJC6 
                 subfamily C, member 6, 
                   
                 disorder including 
               
               
                   
                 DNAJC6 
                   
                 parkinsonism and 
               
               
                   
                 chr1p31.3, 
                   
                 additional features 
               
               
                   
                 NM_001256864.1 
               
               
                 PARK20, 
                 Synaptojanin 1, SYNJ1 
                 21q22.11 
                 Parkinson disease 20, 
                 AR 
                 #604297 
               
               
                 SYNJ-1 
                 chr21q22.11, NM_203446.2 
                   
                 early-onset 
               
               
                 DNAJC13 
                 DnaJ (Hsp40) homolog, 
                 3q22.1 
                 Parkinson&#39;s disease, 
                 AD 
                 *614334 
               
               
                   
                 subfamily C, member 13, 
                   
                 parkinsonism, 
               
               
                   
                 DNAJC13, chr3q22.1, 
                   
                 DNAJ/HSP40 
               
               
                   
                 NM_015268.3 
                   
                 HOMOLOG, 
               
               
                   
                   
                   
                 SUBFAMILY C, 
               
               
                   
                   
                   
                 MEMBER 13 
               
               
                 GCH-1 
                 GTP cyclohydrolase 1, 
                 14q22.1- 
                 GTP 
                 AD 
                 *600225 
               
               
                   
                 GCH1, 
                 14q22.2 
                 CYCLOHYDROLASE 
               
               
                   
                 Chr14q22.1-22.2, 
                   
                 I, Dopa-responsive 
               
               
                   
                 NM_000161.2 
                   
                 dystonia, parkinsonism 
               
               
                 DCTN1 
                 Dynactin 1, DCTN1, 
                 2p13 
                 Dynactin 1, Perry 
                 AD 
                 *601143 
               
               
                   
                 chr2p13, NM_004082.4 
                   
                 syndrome, 
               
               
                   
                   
                   
                 parkinsonism 
               
               
                 DYT3, 
                 TAF1 RNA polymerase II, 
                 Xq13.1 
                 Severe progressive 
                 X-linked 
                 #313650 
               
               
                 TAF1 
                 TATA box binding protein 
                   
                 torsion dystonia 
               
               
                   
                 (TBP)-associated factor, 
                   
                 followed by 
               
               
                   
                 250 kDa, TAF1, chrXq13.1, 
                   
                 parkinsonism 
               
               
                   
                 NM_004606.4 
               
               
                 SLC6A3 
                 Solute carrier family 6 
                 5p15.33 
                 Dopamine transporter 
                 AR 
                 *126455 
               
               
                   
                 (neurotransmitter 
                   
                 deficiency syndrome 
               
               
                   
                 transporter), member 3, 
                   
                 (DTDS) 
               
               
                   
                 SLC6A3chr5p15.33, 
               
               
                   
                 NM_001044.4 
               
               
                 GBA 
                 Glucosidase, beta, acid, 
                 1q22 
                 Parkinson&#39;s disease 
                 AR 
                 #606463 
               
               
                   
                 GBA, chr1q22, 
               
               
                   
                 NM_000157.3 
               
               
                 HEXA 
                 Beta-hexosaminidase A, 
                 15q23 
                 Tay-Sachs disease 
                 AR 
                 *606869 
               
               
                   
                 HEXA, 15q23, NM_000520 
               
               
                 SMPD1 
                 Sphingomyelin 
                 11p15.4 
                 Niemann Pick Type 
                 AR 
                 #607608 
               
               
                   
                 phosphodiesterase 1, acid 
                   
                 A/B 
               
               
                   
                 lysosomal, SMPD1, 
               
               
                   
                 11p15.4, NM_000543 
               
               
                 POLG 
                 POLYMERASE, DNA, 
                 15q26.1 
                 Complex neurological 
                 AR 
                 *174763 
               
               
                   
                 GAMMA, POLG, 15q26.1, 
                   
                 phenotype including 
               
               
                   
                 NM_001126131 
                   
                 Dopa-responsive 
               
               
                   
                   
                   
                 Parkinsonism 
               
               
                 SNCB 
                 Synuclein, beta, SNCB, 
                 5q35.2 
                 Alzheimer disease 
                 AD 
                 #602569 
               
               
                   
                 5q35.2, NM_001001502 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 SNCA CRISPR guide RNA design in exon 2 
               
               
                 SNCA CRISPR Design 
               
               
                 SpCas9 from  Streptococcus pyogenes : 5′-NGG-3′ 
               
               
                 Genome Reference Assembly:  Homo sapiens  (hg19/GRCh37), February 2009 
               
               
                 SNCA Gene Reference Sequence:  Homo sapiens  synuclein (SNCA), transcript variant 1, mRNA 
               
               
                 (NM_000345.3) 
               
             
          
           
               
                   
                   
                   
                   
                   
                 GC 
               
               
                 SEQ ID 
                 Name 
                 Target Sequence (5′→3′) 
                 Strand 
                 Genomic Location 
                 Content 
               
               
                   
               
             
          
           
               
                 SEQ ID NO: 1 
                 SNCA_E2_1 
                 GTAAAGGAATTCATTAGCCA TGG   
                 − 
                 chr4: 90756815- 
                 39% 
               
               
                   
                   
                   
                   
                 90756837 
                   
               
               
                   
               
               
                 SEQ ID NO: 2 
                 SNCA_E2_2 
                 GCCATGGATGTATTCATGAA AGG   
                 − 
                 chr4: 90756799- 
                 43% 
               
               
                   
                   
                   
                   
                 90756821 
                   
               
               
                   
               
               
                 SEQ ID NO: 3 
                 SNCA_E2_3 
                 TCCTTTCATGAATACATCCA TGG   
                 + 
                 chr4: 90756798- 
                 39% 
               
               
                   
                   
                   
                   
                 90756820 
                   
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 SNCA CRISPR guide RNA design in exon 3, 4, 5 
               
               
                 SNCA CRISPR Design 
               
               
                 SpCas9 from  Streptococcus pyogenes : 5′-NGG-3′ 
               
               
                 Genome Reference Assembly:  Homo sapiens  (hg19/GRCh37), February 2009 
               
               
                 SNCA Gene Reference Sequence:  Homo sapiens  synuclein (SNCA), transcript variant 1, mRNA 
               
               
                 (NM_000345.3) 
               
             
          
           
               
                   
                   
                   
                   
                   
                 GC 
               
               
                 SEQ ID 
                 Name 
                 Target Sequence (5′→3′) 
                 Strand 
                 Genomic Location 
                 Content 
               
               
                   
               
             
          
           
               
                 SEQ ID NO: 4 
                 SNCA_E3_1 
                 ACCAAGGAGGGAGTGGTGCATGG 
                 − 
                 chr4: 90749305-90749327 
                 60% 
               
               
                   
               
               
                 SEQ ID NO: 5 
                 SNCA_E3_3 
                 ACCATGCACCACTCCCTCCTTGG 
                 + 
                 chr4: 90749304-90749326 
                 60% 
               
               
                   
               
               
                 SEQ ID NO: 6 
                 SNCA_E4_1 
                 CTTTGTCAAAAAGGACCAGTTGG 
                 − 
                 chr4: 90743402-90743424 
                 40% 
               
               
                   
               
               
                 SEQ ID NO: 7 
                 SNCA_E4_2 
                 GAGCAAGTGACAAATGTTGGAGG 
                 − 
                 chr4: 90743500-90743522 
                 45% 
               
               
                   
               
               
                 SEQ ID NO: 8 
                 SNCA_E5_1 
                 GCCTCATTGTCAGGATCCACAGG 
                 + 
                 chr4: 90650364-90650386 
                 55% 
               
               
                   
               
               
                 SEQ ID NO: 9 
                 SNCA_E5_3 
                 GCCTGTGGATCCTGACAATGAGG 
                 − 
                 chr4: 90650365-90650387 
                 55% 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 MAPT CRISPR guide RNA design in exon 2 
               
               
                 MAPT CRISPR Design 
               
               
                 SpCas9 from  Streptococcus pyogenes : 5′-NGG-3′ 
               
               
                 Genome Reference Assembly:  Homo sapiens  (hg19/GRCh37), February 2009 
               
               
                 MAPT Gene: microtubule-associated protein tau (MAPT), transcript variant 1, mRNA 
               
               
                 (NM_016835.4) 
               
             
          
           
               
                   
                   
                   
                   
                   
                   
                 GC 
               
               
                 SEQ ID 
                 Identifier 
                 Target Sequence (5′→3′) 
                 Strand 
                 Genomic Location 
                 Exon 
                 Content 
               
               
                   
               
             
          
           
               
                 SEQ ID NO: 10 
                 MAPT_E2_1 
                 CACGCTGGGACGTACGGGTTGGG 
                 + 
                 chr17: 44039743- 
                 2 
                 70% 
               
               
                   
                   
                   
                   
                 44039765 
                   
                   
               
               
                   
               
               
                 SEQ ID NO: 11 
                 MAPT_E2_2 
                 AAGATCACGCTGGGACGTACGGG 
                 + 
                 chr17: 44039738- 
                 2 
                 61% 
               
               
                   
                   
                   
                   
                 44039760 
                   
                   
               
               
                   
               
               
                 SEQ ID NO: 12 
                 MAPT_E2_5 
                 ATCACTTCGAACTCCTGGCGGGG 
                 − 
                 chr17: 44039713- 
                 2 
                 61% 
               
               
                   
                   
                   
                   
                 44039735 
                   
                   
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
               
                   
               
               
                 sgRNA designs for non-coding evolutionary conserved regions in SNCA locus 
               
               
                   
               
             
             
               
                 6A. NcECR SNCA D6 sgRNA designs 
               
               
                 Chr4: 90636868 + 90637447 
               
               
                 Genome Reference Assembly:  Homo sapiens  (hg19/GRCh37), February 2009 
               
               
                 Genomic region covers 582 base pairs 
               
             
          
           
               
                   
                   
                   
                   
                   
                 GC 
               
               
                 SEQ ID 
                 Identifier 
                 Target Sequence (5′→3′) 
                 Genomic Location 
                 Strand 
                 Content 
               
               
                   
               
               
                 SEQ ID NO: 13 
                 SNCA_D6_1 
                 TGTGAAAGACCGTAAGTTGCTGG 
                 chr4: 90636884-90636906 
                 + 
                 45% 
               
               
                   
               
               
                 SEQ ID NO: 14 
                 SNCA_D6_2 
                 GGATCAAGACAACTTGGCCATGG 
                 chr4: 90637178-90637200 
                 + 
                 50% 
               
               
                   
               
               
                 SEQ ID NO: 15 
                 SNCA_D6_3 
                 AAGAGGTGAGTTCTAATCATTGG 
                 chr4: 90637114-90637136 
                 − 
                 35% 
               
               
                   
               
               
                 SEQ ID NO: 16 
                 SNCA_D6_4 
                 CTTGATGGCAGTCACTTGAAAGG 
                 chr4: 90637246-90637268 
                 + 
                 45% 
               
               
                   
               
               
                 SEQ ID NO: 17 
                 SNCA_D6_5 
                 ATGGTGTCCTGAGATGACTGAGG 
                 chr4: 90637056-90637078 
                 + 
                 50% 
               
               
                   
               
             
          
           
               
                 6B. NcECR SNCA I12 sgRNA designs 
               
               
                 Chr4: 90721247 + 90721915 
               
               
                 Genome Reference Assembly:  Homo sapiens  (hg19/GRCh37), February 2009 
               
               
                 Genomic region covers 669 base pairs 
               
             
          
           
               
                   
                   
                   
                   
                   
                 GC 
               
               
                 SEQ ID 
                 Identifier 
                 Target Sequence (5′→3′) 
                 Genomic Location 
                 Strand 
                 Content 
               
               
                   
               
               
                 SEQ ID NO: 18 
                 SNCA_I12_1 
                 GTCAAGTTTACTATCTTACGTGG 
                 chr4: 90721433-90721455 
                 + 
                 35% 
               
               
                   
               
               
                 SEQ ID NO: 19 
                 SNCA_I12_2 
                 TTTCACTTACGTACTATGAAAGG 
                 chr4: 90721479-90721501 
                 − 
                 30% 
               
               
                   
               
               
                 SEQ ID NO: 20 
                 SNCA_I12_3 
                 GAATTGTACTGACTACACCACGG 
                 chr4: 90721881-90721903 
                 + 
                 40% 
               
               
                   
               
               
                 SEQ ID NO: 21 
                 SNCA_I12_4 
                 TACTCATATTACAGTCAGTCTGG 
                 chr4: 90721588-90721610 
                 − 
                 35% 
               
               
                   
               
               
                 SEQ ID NO: 22 
                 SNCA_I12_5 
                 ATCCTTGTATAAACCCCACAAGG 
                 chr4: 90721634-90721656 
                 + 
                 40% 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 SNCA promoter region sgRNA designs 
               
               
                 Genome Reference Assembly:  Homo sapiens  (hg19/GRCh37), February 2009; 
               
               
                 Genomic region comprises of 996 base pairs on Chr.4: 90756741-90757736 
               
             
          
           
               
                 SEQ ID 
                 Identifier 
                 sgRNA Designs 
                 Genomic Location 
                 Strand 
               
               
                   
               
               
                 SEQ ID NO: 23 
                 SNCA_Promoter_1 
                 TAGCCACCTAACCACGGATTAGG 
                 chr4: 90757040-90757062 
                 + 
               
               
                   
               
               
                 SEQ ID NO: 24 
                 SNCA_Promoter_2 
                 GGTTAACAAGTGCTGGCGCGGGG 
                 chr4: 90757456-90757478 
                 − 
               
               
                   
               
               
                 SEQ ID NO: 25 
                 SNCA_Promoter_3 
                 GGGTTAACAAGTGCTGGCGCGGG 
                 chr4: 90757457-90757479 
                 − 
               
               
                   
               
               
                 SEQ ID NO: 26 
                 SNCA_Promoter_4 
                 CATCCTAATCCGTGGTTAGGTGG 
                 chr4: 90757043-90757065 
                 − 
               
               
                   
               
               
                 SEQ ID NO: 27 
                 SNCA_Promoter_5 
                 CGGGTTAACAAGTGCTGGCGCGG 
                 chr4: 90757458-90757480 
                 − 
               
               
                   
               
               
                 SEQ ID NO: 28 
                 SNCA_Promoter_6 
                 AGTTGGATGCTCACGCTCCATGG 
                 chr4: 90757598-90757620 
                 + 
               
               
                   
               
               
                 SEQ ID NO: 29 
                 SNCA_Promoter_7 
                 TGGAGCATCCTCGCGTTTCCCGG 
                 chr4: 90757618-90757640 
                 + 
               
               
                   
               
               
                 SEQ ID NO: 30 
                 SNCA_Promoter_8 
                 GAGCATCCTCGCGTTTCCCGGGG 
                 chr4: 90757620-90757642 
                 + 
               
               
                   
               
               
                 SEQ ID NO: 31 
                 SNCA_Promoter_9 
                 GCTTTTCCCCGGGAAACGCGAGG 
                 chr4: 90757626-90757648 
                 − 
               
               
                   
               
               
                 SEQ ID NO: 32 
                 SNCA_Promoter_10 
                 ATTCATCCTAATCCGTGGTTAGG 
                 chr4: 90757046-9075706 
                 − 
               
               
                   
               
               
                 SEQ ID NO: 33 
                 SNCA_Promoter_11 
                 AGAACGCCCCCTCGGGTGGCTGG 
                 chr4: 90757414-90757436 
                 − 
               
               
                   
               
               
                 SEQ ID NO: 34 
                 SNCA_Promoter_13 
                 TGCTGGCGCGGGGTCCGCTAGGG 
                 chr4: 90757446-90757468 
                 − 
               
               
                   
               
               
                 SEQ ID NO: 35 
                 SNCA_Promoter_14 
                 TGGCGCGGGGTCCGCTAGGGTGG 
                 chr4: 90757443-90757465 
                 − 
               
               
                   
               
               
                 SEQ ID NO: 36 
                 SNCA_Promoter_15 
                 AGAGGATTCATCCTAATCCGTGG 
                 chr4: 90757051-90757073 
                 − 
               
               
                   
               
               
                 SEQ ID NO: 37 
                 SNCA_Promoter_16 
                 GGTAACGGGTTAACAAGTGCTGG 
                 chr4: 90757463-90757485 
                 − 
               
               
                   
               
               
                 SEQ ID NO: 38 
                 SNCA_Promoter_17 
                 GCAGGCGCCGCTCACACTCGCGG 
                 chr4: 90757373-9075739 
                 + 
               
               
                   
               
               
                 SEQ ID NO: 39 
                 SNCA_Promoter_18 
                 CAGGCGCCGCTCACACTCGCGGG 
                 chr4: 90757374-90757396 
                 + 
               
               
                   
               
               
                 SEQ ID NO: 40 
                 SNCA_Promoter_19 
                 TCCCGGGGAAAAGCGGATCCCGG 
                 chr4: 90757635-90757657 
                 + 
               
               
                   
               
               
                 SEQ ID NO: 41 
                 SNCA_Promoter_20 
                 GGAGCATCCTCGCGTTTCCCGGG 
                 chr4: 90757619-90757641 
                 + 
               
               
                   
               
               
                 SEQ ID NO: 42 
                 SNCA_Promoter_21 
                 ACTGTTACCCTTTAGACCCCGGG 
                 chr4: 90757137-90757159 
                 − 
               
               
                   
               
               
                 SEQ ID NO: 43 
                 SNCA_Promoter_22 
                 TTTAAATTCCCGGGGTCTAAAGG 
                 chr4: 90757129-90757151 
                 + 
               
               
                   
               
               
                 SEQ ID NO: 44 
                 SNCA_Promoter_23 
                 GCTGAGAACGCCCCCTCGGGTGG 
                 chr4: 90757418-90757440 
                 − 
               
               
                   
               
               
                 SEQ ID NO: 45 
                 SNCA_Promoter_24 
                 CTAACTGCTCACTCGGGGTGTGG 
                 chr4: 90757266-90757288 
                 + 
               
               
                   
               
               
                 SEQ ID NO: 46 
                 SNCA_Promoter_25 
                 AGACGGCCCGCGAGTGTGAGCGG 
                 chr4: 90757380-90757402 
                 − 
               
               
                   
               
               
                 SEQ ID NO: 47 
                 SNCA_Promoter_26 
                 TTAAATTCCCGGGGTCTAAAGGG 
                 chr4: 90757130-90757152 
                 + 
               
               
                   
               
               
                 SEQ ID NO: 48 
                 SNCA_Promoter_27 
                 CAGAACTAACTGCTCACTCGGGG 
                 chr4: 90757261-90757283 
                 + 
               
               
                   
               
               
                 SEQ ID NO: 49 
                 SNCA_Promoter_28 
                 TCGGGGTGTGGTTCAAACTCAGG 
                 chr4: 90757278-90757300 
                 + 
               
               
                   
               
               
                 SEQ ID NO: 50 
                 SNCA_Promoter_29 
                 TAAAGTAAGCAAGCTGCGTTTGG 
                 chr4: 90757528-90757550 
                 − 
               
               
                   
               
               
                 SEQ ID NO: 51 
                 SNCA_Promoter_30 
                 GGAAACGCGAGGATGCTCCATGG 
                 chr4: 90757615-90757637 
                 − 
               
               
                   
               
               
                 SEQ ID NO: 52 
                 SNCA_Promoter_31 
                 TCGCGTTTCCCGGGGAAAAGCGG 
                 chr4: 90757628-90757650 
                 + 
               
               
                   
               
               
                 SEQ ID NO: 53 
                 SNCA_Promoter_32 
                 TACTGTTACCCTTTAGACCCCGG 
                 chr4: 90757138-90757160 
                 − 
               
               
                   
               
               
                 SEQ ID NO: 54 
                 SNCA_Promoter_33 
                 ACCCCGCGCCAGCCACCCGAGGG 
                 chr4: 90757406-90757428 
                 + 
               
               
                   
               
               
                 SEQ ID NO: 55 
                 SNCA_Promoter_34 
                 AAAGTCTAGCCACCTAACCACGG 
                 chr4: 90757034-90757056 
                 + 
               
               
                   
               
               
                 SEQ ID NO: 56 
                 SNCA_Promoter_35 
                 GTGTACACTCATTTAACCATTGG 
                 chr4: 90756947-90756969 
                 + 
               
               
                   
               
               
                 SEQ ID NO: 57 
                 SNCA_Promoter_36 
                 TGAATTCCTTTACACCACACTGG 
                 chr4: 90756825-90756847 
                 + 
               
               
                   
               
               
                 SEQ ID NO: 58 
                 SNCA_Promoter_37 
                 GGAGGCTGAGAACGCCCCCTCGG 
                 chr4: 90757422-90757444 
                 − 
               
               
                   
               
               
                 SEQ ID NO: 59 
                 SNCA_Promoter_38 
                 GAGGCTGAGAACGCCCCCTCGGG 
                 chr4: 90757421-90757443 
                 − 
               
               
                   
               
               
                 SEQ ID NO: 60 
                 SNCA_Promoter_39 
                 CCCCTCGGGTGGCTGGCGCGGGG 
                 chr4: 90757407-90757429 
                 − 
               
               
                   
               
               
                 SEQ ID NO: 61 
                 SNCA_Promoter_40 
                 GGATTAGAACCATCACACTTGGG 
                 chr4: 90757312-90757334 
                 − 
               
               
                   
               
               
                 SEQ ID NO: 62 
                 SNCA_Promoter_41 
                 AATCTGTCTGCCCGCTCTCTTGG 
                 chr4: 90757561-90757583 
                 − 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
             
               
               
               
               
               
             
           
               
                 TABLE 8 
               
               
                   
               
               
                 SNCA intron 4 and 5 region sgRNA designs 
               
               
                   
               
             
             
               
                 SNCA intron 4 guide RNAs 
               
               
                 Genome Reference Assembly:  Homo sapiens  (hg19/GRCh37), February 2009. 
               
             
          
           
               
                 SEQ ID 
                 Identifier 
                 sgRNA Designs 
                 Genomic Location 
                 Strand 
               
               
                   
               
               
                 SEQ ID NO: 63 
                 SNCA_Intron 4_1 
                 TTGCCGCAATGTTTCCCCGGAGG 
                 Chr4: 90650595-90650617 
                 − 
               
               
                   
               
               
                 SEQ ID NO: 64 
                 SNCA_Intron 4_2 
                 GCACACCTTAATATTACTACTGG 
                 Chr4: 90650553-90650575 
                 + 
               
               
                   
               
               
                 SEQ ID NO: 65 
                 SNCA_Intron 4_3 
                 GGGAAACATTGCGGCAACATGGG 
                 Chr4: 90650601-90650623 
                 + 
               
               
                   
               
               
                 SEQ ID NO: 66 
                 SNCA_Intron 4_4 
                 GGGGAAACATTGCGGCAACATGG 
                 Chr4: 90650600-90650622 
                 + 
               
               
                   
               
               
                 SEQ ID NO: 67 
                 SNCA_Intron 4_5 
                 TGTGCCATTTTCAAGATCCGTGG 
                 Chr4: 90650535-90650557 
                 − 
               
               
                   
               
               
                 SEQ ID NO: 68 
                 SNCA_Intron 4_6 
                 TAAACTCCACAATGCCTCCGGGG 
                 Chr4: 90650581-90650603 
                 + 
               
               
                   
               
               
                 SEQ ID NO: 69 
                 SNCA_Intron 4_7 
                 ATGTTGCCGCAATGTTTCCCCGG 
                 Chr4: 90650598-90650620 
                 − 
               
               
                   
               
               
                 SEQ ID NO: 70 
                 SNCA_Intron 4_8 
                 ATGCCTCCGGGGAAACATTGCGG 
                 Chr4: 90650592-90650614 
                 + 
               
               
                   
               
               
                 SEQ ID NO: 71 
                 SNCA_Intron 4_1 
                 TTGGCCACGGATCTTGAAAATGG 
                 Chr4: 90650531-90650553 
                 + 
               
               
                   
               
               
                 SEQ ID NO: 72 
                 SNCA_Intron 4_10 
                 AATCTTACATATAGGGATGTTGG 
                 Chr4: 90650512-90650534 
                 + 
               
               
                   
               
               
                 SEQ ID NO: 73 
                 SNCA_Intron 4_11 
                 TCTAAACTCCACAATGCCTCCGG 
                 Chr4: 90650579-90650601 
                 + 
               
               
                   
               
               
                 SEQ ID NO: 74 
                 SNCA_Intron 4_12 
                 ATGTTTCCCCGGAGGCATTGTGG 
                 Chr4: 90650587-90650609 
                 − 
               
               
                   
               
               
                 SEQ ID NO: 75 
                 SNCA_Intron 4_13 
                 CTAAACTCCACAATGCCTCCGGG 
                 Chr4: 90650580-90650602 
                 + 
               
               
                   
               
               
                 SEQ ID NO: 76 
                 SNCA_Intron 4_14 
                 GAATGCCAGTAGTAATATTAAGG 
                 Chr4: 90650558-90650580 
                 − 
               
               
                   
               
               
                 SEQ ID NO: 77 
                 SNCA_Intron 4_15 
                 ACATATAGGGATGTTGGCCACGG 
                 Chr4: 90650518-90650540 
                 + 
               
               
                 1. 
                 2. 
                 3. 
                 4. 
                  5. 
               
               
                   
               
               
                 6. 
                 7. 
                 8. 
                 9. 
                 10. 
               
             
          
           
               
                 SNCA intron 5 guide RNAs 
               
               
                 Genome Reference Assembly:  Homo sapiens  (hg19/GRCh37), February 2009 
               
             
          
           
               
                 SEQ ID 
                 Identifier 
                 sgRNA Designs 
                 Genomic Location 
                 Strand 
               
               
                   
               
               
                 SEQ ID NO: 78 
                 SNCA_Intron 5_1 
                 CTCTCTACATGCTCATTACGTGG 
                 Chr4: 90650228-90650250 
                 − 
               
               
                   
               
               
                 SEQ ID NO: 79 
                 SNCA_Intron 5_2 
                 CTTACTACTTGACCCTTTACAGG 
                 Chr4: 90650077-90650099 
                 − 
               
               
                   
               
               
                 SEQ ID NO: 80 
                 SNCA_Intron 5_3 
                 AGTAAGTATATTAAGCTATGTGG 
                 Chr4: 90650094-90650116 
                 + 
               
               
                   
               
               
                 SEQ ID NO: 81 
                 SNCA_Intron 5_4 
                 GTCATCCCATTGGACATGTATGG 
                 Chr4: 90649933-90649955 
                 + 
               
               
                   
               
               
                 SEQ ID NO: 82 
                 SNCA_Intron 5_5 
                 TTTACTATGCGTCATCCCATTGG 
                 Chr4: 90649923-90649945 
                 + 
               
               
                   
               
               
                 SEQ ID NO: 83 
                 SNCA_Intron 5_6 
                 TTTTGCCATACATGTCCAATGGG 
                 Chr4: 90649938-90649960 
                 − 
               
               
                   
               
               
                 SEQ ID NO: 84 
                 SNCA_Intron 5_7 
                 TTTTTGCCATACATGTCCAATGG 
                 Chr4: 90649939-90649961 
                 − 
               
               
                   
               
               
                 SEQ ID NO: 85 
                 SNCA_Intron 5_8 
                 GGAAATCAATGGAATGCCAAAGG 
                 Chr4: 90649973-90649995 
                 − 
               
               
                   
               
               
                 SEQ ID NO: 86 
                 SNCA_Intron 5_9 
                 TGGAAAGTTAGGGAAATCAATGG 
                 Chr4: 90649984-90650006 
                 − 
               
               
                   
               
               
                 SEQ ID NO: 87 
                 SNCA_Intron 5_10 
                 ATCAATGGAATGCCAAAGGAAGG 
                 Chr4: 90649969-90649991 
                 − 
               
               
                   
               
             
          
         
       
     
       SEQUENCE LISTING 
       [0156]    The sequences referred to in the above description by Identification Numbers are as follows: 
         [0000]    
       
         
               
               
               
             
           
               
                   
                   
               
               
                   
                 SEQ ID 
                 Target Sequence (5′→3′) 
               
               
                   
                   
               
             
             
               
                   
                 SEQ ID NO: 1 
                 GTAAAGGAATTCATTAGCCATGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 2 
                 GCCATGGATGTATTCATGAAAGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 3 
                 TCCTTTCATGAATACATCCATGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 4 
                 ACCAAGGAGGGAGTGGTGCATGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 5 
                 ACCATGCACCACTCCCTCCTTGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 6 
                 CTTTGTCAAAAAGGACCAGTTGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 7 
                 GAGCAAGTGACAAATGTTGGAGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 8 
                 GCCTCATTGTCAGGATCCACAGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 9 
                 GCCTGTGGATCCTGACAATGAGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 10 
                 CACGCTGGGACGTACGGGTTGGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 11 
                 AAGATCACGCTGGGACGTACGGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 12 
                 ATCACTTCGAACTCCTGGCGGGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 13 
                 TGTGAAAGACCGTAAGTTGCTGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 14 
                 GGATCAAGACAACTTGGCCATGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 15 
                 AAGAGGTGAGTTCTAATCATTGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 16 
                 CTTGATGGCAGTCACTTGAAAGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 17 
                 ATGGTGTCCTGAGATGACTGAGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 18 
                 GTCAAGTTTACTATCTTACGTGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 19 
                 TTTCACTTACGTACTATGAAAGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 20 
                 GAATTGTACTGACTACACCACGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 21 
                 TACTCATATTACAGTCAGTCTGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 22 
                 ATCCTTGTATAAACCCCACAAGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 23 
                 TAGCCACCTAACCACGGATTAGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 24 
                 GGTTAACAAGTGCTGGCGCGGGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 25 
                 GGGTTAACAAGTGCTGGCGCGGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 26 
                 CATCCTAATCCGTGGTTAGGTGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 27 
                 CGGGTTAACAAGTGCTGGCGCGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 28 
                 AGTTGGATGCTCACGCTCCATGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 29 
                 TGGAGCATCCTCGCGTTTCCCGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 30 
                 GAGCATCCTCGCGTTTCCCGGGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 31 
                 GCTTTTCCCCGGGAAACGCGAGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 32 
                 ATTCATCCTAATCCGTGGTTAGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 33 
                 AGAACGCCCCCTCGGGTGGCTGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 34 
                 TGCTGGCGCGGGGTCCGCTAGGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 35 
                 TGGCGCGGGGTCCGCTAGGGTGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 36 
                 AGAGGATTCATCCTAATCCGTGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 37 
                 GGTAACGGGTTAACAAGTGCTGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 38 
                 GCAGGCGCCGCTCACACTCGCGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 39 
                 CAGGCGCCGCTCACACTCGCGGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 40 
                 TCCCGGGGAAAAGCGGATCCCGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 41 
                 GGAGCATCCTCGCGTTTCCCGGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 42 
                 ACTGTTACCCTTTAGACCCCGGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 43 
                 TTTAAATTCCCGGGGTCTAAAGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 44 
                 GCTGAGAACGCCCCCTCGGGTGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 45 
                 CTAACTGCTCACTCGGGGTGTGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 46 
                 AGACGGCCCGCGAGTGTGAGCGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 47 
                 TTAAATTCCCGGGGTCTAAAGGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 48 
                 CAGAACTAACTGCTCACTCGGGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 49 
                 TCGGGGTGTGGTTCAAACTCAGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 50 
                 TAAAGTAAGCAAGCTGCGTTTGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 51 
                 GGAAACGCGAGGATGCTCCATGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 52 
                 TCGCGTTTCCCGGGGAAAAGCGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 53 
                 TACTGTTACCCTTTAGACCCCGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 54 
                 ACCCCGCGCCAGCCACCCGAGGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 55 
                 AAAGTCTAGCCACCTAACCACGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 56 
                 GTGTACACTCATTTAACCATTGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 57 
                 TGAATTCCTTTACACCACACTGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 58 
                 GGAGGCTGAGAACGCCCCCTCGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 59 
                 GAGGCTGAGAACGCCCCCTCGGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 60 
                 CCCCTCGGGTGGCTGGCGCGGGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 61 
                 GGATTAGAACCATCACACTTGGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 62 
                 AATCTGTCTGCCCGCTCTCTTGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 63 
                 TTGCCGCAATGTTTCCCCGGAGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 64 
                 GCACACCTTAATATTACTACTGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 65 
                 GGGAAACATTGCGGCAACATGGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 66 
                 GGGGAAACATTGCGGCAACATGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 67 
                 TGTGCCATTTTCAAGATCCGTGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 68 
                 TAAACTCCACAATGCCTCCGGGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 69 
                 ATGTTGCCGCAATGTTTCCCCGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 70 
                 ATGCCTCCGGGGAAACATTGCGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 71 
                 TTGGCCACGGATCTTGAAAATGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 72 
                 AATCTTACATATAGGGATGTTGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 73 
                 TCTAAACTCCACAATGCCTCCGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 74 
                 ATGTTTCCCCGGAGGCATTGTGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 75 
                 CTAAACTCCACAATGCCTCCGGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 76 
                 GAATGCCAGTAGTAATATTAAGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 77 
                 ACATATAGGGATGTTGGCCACGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 78 
                 CTCTCTACATGCTCATTACGTGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 79 
                 CTTACTACTTGACCCTTTACAGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 80 
                 AGTAAGTATATTAAGCTATGTGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 81 
                 GTCATCCCATTGGACATGTATGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 82 
                 TTTACTATGCGTCATCCCATTGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 83 
                 TTTTGCCATACATGTCCAATGGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 84 
                 TTTTTGCCATACATGTCCAATGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 85 
                 GGAAATCAATGGAATGCCAAAGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 86 
                 TGGAAAGTTAGGGAAATCAATGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 87 
                 ATCAATGGAATGCCAAAGGAAGG 
               
               
                   
                   
               
             
          
         
       
     
       BIBLIOGRAPHY 
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