Patent Application: US-201615089174-A

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 , parkin rbr e3 ubiquitin protein ligase , , leucine - rich repeat kinase 2 , pten - induced putative kinase /, daisuke - junko 1 , and atpase type 13a2 , 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:
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 ”. 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 . “ 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 . the following definitions and abbreviations are used in the description of the present invention . 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 . 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 . 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 . 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 . 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 . “ 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 . 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 . “ 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 . 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 . 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 . 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 . “ 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 . “ 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . “ 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 . “ 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 . 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 . 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 . 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 . 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 . 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 ). 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 ). 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 . 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 . “ 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 . “ 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 ). “ 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 . 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 . an overview of the different targeting strategies is visualized in fig1 . 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 . 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 . several publicly available bioinformatics tools are used to design oligonucleotides for small guide rnas . 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 . 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 . 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 . these sgrna sequences will be experimentally proven and verified . a schematic overview illustrates the workflow ( fig2 ). table 3 shows snca crispr guide rna design in exon 2 for three target guide sequences . fig3 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 . 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 . fig4 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 . 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 ) ( fig5 - 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 . 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 ) fig1 , panel c ). 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 ). 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 ) ( fig1 , panel c ). 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 . identification of 32 evolutionary conserved non - coding genomic regions ( ncecrs ) within the snca gene 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 ) ( fig6 ). 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 ( fig6 ). 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 ). 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 ( fig5 ) ( sterling et al ., 2014 ). 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 ). overall , 12 of 32 constructs exhibited either an enhancement or reduction of the expression of the reporter gene ( fig2 ). 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 ( fig2 c , 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 ( fig7 ) ( sterling et al ., 2014 ). 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 ( fig1 c ). 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 ( fig8 ). 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 . 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 . 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 fig1 c or 1d will result in specific splice forms of the alpha - synuclein protein and potentially be neuroprotective . virus delivery system for in vivolin human gene editing for neurodegeneration disease progression 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 . 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 ). 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 ). 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 ). 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 . 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 . 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 . for more advanced stages of neurodegeneration in pd or related neurodegenerative disease different approaches as proposed ( fig1 ). 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 . 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 ). 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 ). 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 ). 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 ). 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 . 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 . 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 ). 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 ). 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 . 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 ). 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 . gene editing in patient - derived stem cells or human embryonic stem cells followed by neuronal transplantation 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 ). 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 . 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 . 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 ). 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 . 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 . 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 . 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