Patent Publication Number: US-2012030778-A1

Title: Genomic editing of genes involved with parkinsons disease

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation-in-part of U.S. patent application Ser. No. 12/842,217, filed Jul. 23, 2010, which claims the benefit of U.S. provisional application No. 61/343,287, filed Apr. 26, 2010, U.S. provisional application No. 61/323,702, filed Apr. 13, 2010, U.S. provisional application No. 61/323,719, filed Apr. 13, 2010, U.S. provisional application No. 61/323,698, filed Apr. 13, 2010, U.S. provisional application No. 61/309,729, filed Mar. 2, 2010, U.S. provisional application No. 61/308,089, filed Feb. 25, 2010, U.S. provisional application No. 61/336,000, filed Jan. 14, 2010, U.S. provisional application No. 61/263,904, filed Nov. 24, 2009, U.S. provisional application No. 61/263,696, filed Nov. 23, 2009, U.S. provisional application No. 61/245,877, filed Sep. 25, 2009, U.S. provisional application No. 61/232,620, filed Aug. 10, 2009, U.S. provisional application No. 61/228,419, filed Jul. 24, 2009, and is a continuation in part of U.S. non-provisional application Ser. No. 12/592,852, filed Dec. 3, 2009, which claims priority to U.S. provisional application No. 61/200,985, filed Dec. 4, 2008 and U.S. provisional application No. 61/205,970, filed Jan. 26, 2009, all of which are hereby incorporated by reference in their entirety. 
    
    
     FIELD OF THE INVENTION 
     The invention generally relates to genetically modified animals or cells comprising at least one edited chromosomal sequence encoding proteins associated with Parkinson&#39;s disease. In particular, the invention relates to the use of a zinc finger nuclease-mediated process to edit chromosomal sequences encoding proteins associated with Parkinson&#39;s disease. 
     BACKGROUND OF THE INVENTION 
     Parkinson&#39;s disease (PD) is a degenerative disease caused by the death of neurons that produce dopamine, a neurotransmitter essential for proper muscle coordination, movement, and balance. PD symptoms vary from person to person, but the most evident symptoms include resting tremors, slow movement, instability, stiffness, problems walking, and reduced facial expression. Other symptoms include mild to severe cognitive dysfunction and mood disorders such as depression and apathy, difficulty sleeping, loss of the sense of smell, constipation, difficulty speaking and swallowing, low blood pressure, and drooling. At least one million Americans and six million people worldwide are believed to have PD. 
     Several proteins have been associated with the development of PD in humans. What are needed are animal models with these proteins genetically modified to provide research tools that allow the elucidation of mechanisms underlying development and progression of PD. 
     SUMMARY OF THE INVENTION 
     One aspect of the present disclosure encompasses a genetically modified animal comprising an edited chromosomal sequence encoding Leucine-rich repeat kinase 1 (LRRK1) protein, wherein the animal is other than a mouse. 
     A further aspect encompasses a genetically modified cell comprising an edited chromosomal sequence encoding LRRK1 protein, wherein the cell is derived from an animal other than a mouse. 
     A further aspect encompasses a method for assessing the role of LRRK1 protein on the development of Parkinson&#39;s disease in an animal. The method comprises comparing a wild type animal to a genetically modified animal comprising an edited chromosomal sequence encoding LRRK1 protein, and measuring a selected parameter. The selected parameters are chosen from (a) amyloidogenesis; (b) protein aggregation; (c) response to dopamine; (d) neurodegeneration; (e) mitochondrial dysfunction; (f) coordination; (g) balance; (h) gait; (i) motor impairment; (j) tremors and twitches; (k) rigidity; (l) hypokinesia; and (m) cognitive impairment. 
     An additional aspect encompasses a method for assessing the effect of an agent on progression or symptoms of Parkinson&#39;s disease. The method comprises contacting a first genetically modified animal comprising an edited chromosomal sequence encoding LRRK1 protein with the agent, and comparing the results of a selected parameter to results obtained from a second genetically modified animal comprising an edited chromosomal sequence encoding LRRK1 protein that is not contacted with the agent, wherein the edited chromosomal sequence of the first and second genetically modified animals are identical. The selected parameters are chosen from: (a) amyloidogenesis; (b) protein aggregation; (c) response to dopamine; (d) neurodegeneration; (e) mitochondrial dysfunction; (f) coordination; (g) balance; (h) gait; (i) motor impairment; (j) tremors and twitches; (k) rigidity; (l) hypokinesia; and (m) cognitive impairment. 
     Other aspects and features of the disclosure are described more thoroughly below. 
     REFERENCE TO COLOR FIGURES 
     The application file contains at least one figure executed in color. Copies of this patent application publication with color figures will be provided by the Office upon request and payment of the necessary fee. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1  presents the DNA sequences of two edited LRRK2 loci. A. presents a LRRK2 sequence (SEQ ID NO:1) that has a 10 bp deletion in the target sequence of exon 30. B. Present a sequence (SEQ ID NO:2) having a 8 bp deletion in the target sequence of exon 30. The exon is highlighted in green; the target site is highlighted in yellow, and the deletions are highlighted in dark blue. 
         FIG. 2  illustrates Cel-1 nuclease analysis of ZFN mediated cleavage of LRRK1 locus in rat cells. Cells were exposed to DNA or mRNA encoding the targeted ZDN pair. Control cells were exposed to a nucleic acid encoding GFP. Cel-1 cleavage bands (of 193 bp and 134 bp) are visible in the lanes that were exposed to the ZFNs. 
         FIG. 3  presents the wild type sequence (SEQ ID NO:24) of LRRK1 near the target site. The target site is highlighted in yellow, and exon 4 is shown in bold. 
         FIG. 4  illustrates the edited LRRK1 locus in animal #28. A. Presents the DNA sequence (SEQ ID NO:24) in which 19 bp has been deleted (highlighted in blue) in the target sequence of exon 4. B. Presents translation of the sequence having the 19 bp deletion (SEQ ID NO:25). The target site is highlighted in yellow, and exon 4 is shown in bold. 
         FIG. 5  presents the edited LRRK1 locus in animal #33. There is a 610 bp deletion (highlighted in blue), along with 7 bp insertion (GTTACTT) that includes portions of intron 3-4 and exon 4 (SEQ ID NO:26). Since the mutation in animal #33 extends into an intron/exon boundary, possible alternative splicing or exon skipping may occur, and thus no translation is presented. The target site is highlighted in yellow, and exon 4 is shown in bold. 
         FIG. 6  presents the edited LRRK1 locus in animal #42 that has a 1000 bp deletion (highlighted in blue) including portions of intron 3-4, all of exon 4, and portions of intron 4-5 (SEQ ID NO:27). No translation is shown as the mutation extends into intron/exon boundaries, and thus possible alternative splicing or exon skipping may occur. Exon 4 is shown in bold. 
         FIG. 7  illustrates the edited LRRK1 locus in animal #46. A. Presents the DNA sequence (SEQ ID NO:24) in which 7 bp has been deleted (highlighted in blue) within exon 4. B. Presents translation of the sequence having the 7 bp deletion (SEQ ID NO:28). The target site is highlighted in yellow, and exon 4 is shown in bold. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present disclosure provides a genetically modified animal or animal cell comprising at least one edited chromosomal sequence encoding a protein associated with PD. In particular, the genetically modified animal or cell comprises an edited chromosomal sequence encoding Leucine-rich repeat kinase 1 (LRRK1) protein. The edited chromosomal sequence may comprise a deletion of at least one nucleotide, an insertion of at least one nucleotide, a substitution of at least one nucleotide, or combinations thereof. Thus, the edited chromosomal sequence may be (1) inactivated, (2) modified, or (3) comprise an integrated sequence. An inactivated chromosomal sequence is altered such that a functional protein is not made. Thus, a genetically modified animal comprising an inactivated chromosomal sequence may be termed a “knock out” or a “conditional knock out.” Similarly, a genetically modified animal comprising an integrated sequence may be termed a “knock in” or a “conditional knock in.” As detailed below, a knock in animal may be a humanized animal. Furthermore, a genetically modified animal comprising a modified chromosomal sequence may comprise a targeted point mutation(s) or other modification such that an altered protein product is produced. The chromosomal sequence encoding the protein associated with PD generally is edited using a zinc finger nuclease-mediated process. Briefly, the process comprises introducing into an embryo or cell at least one nucleic acid encoding a targeted zinc finger nuclease and, optionally, at least one accessory polynucleotide. The method further comprises incubating the embryo or cell to allow expression of the zinc finger nuclease, wherein a double-stranded break introduced into the targeted chromosomal sequence by the zinc finger nuclease is repaired by an error-prone non-homologous end-joining DNA repair process or a homology-directed DNA repair process. The method of editing chromosomal sequences encoding a protein associated with PD using targeted zinc finger nuclease technology is rapid, precise, and highly efficient. 
     (I) Genetically Modified Animals 
     One aspect of the present disclosure provides a genetically modified animal in which at least one chromosomal sequence encoding a protein associated with PD has been edited. For example, the edited chromosomal sequence may be inactivated such that the sequence is not transcribed and/or a functional protein associated with PD is not produced. Alternatively, the chromosomal sequence may be edited such that the sequence is over-expressed and a functional protein associated with PD is over-produced. The edited chromosomal sequence may also be modified such that it codes for an altered protein associated with PD. For example, the chromosomal sequence may be modified such that at least one nucleotide is changed and the expressed protein associated with PD comprises at least one changed amino acid residue (missense mutation). The chromosomal sequence may be modified to comprise more than one missense mutation such that more than one amino acid is changed. Additionally, the chromosomal sequence may be modified to have a three nucleotide deletion or insertion such that the expressed PD-related protein comprises a single amino acid deletion or insertion, provided such a protein is functional. The modified protein associated with PD may have altered substrate specificity, altered enzyme activity, altered kinetic rates, and so forth. Furthermore, the edited chromosomal sequence encoding a protein associated with PD may comprise a sequence encoding a protein associated with PD integrated into the genome of the animal. The chromosomally integrated sequence may encode an endogenous protein associated with PD normally found in the animal, or the integrated sequence may encode an orthologous protein associated with PD, or combinations of both. The genetically modified animal disclosed herein may be heterozygous for the edited chromosomal sequence encoding a protein associated with PD. Alternatively, the genetically modified animal may be homozygous for the edited chromosomal sequence encoding a protein associated with PD. 
     In one embodiment, the genetically modified animal may comprise at least one inactivated chromosomal sequence encoding a protein associated with PD. The inactivated chromosomal sequence may include a deletion mutation (i.e., deletion of one or more nucleotides), an insertion mutation (i.e., insertion of one or more nucleotides), or a nonsense mutation (i.e., substitution of a single nucleotide for another nucleotide such that a stop codon is introduced). As a consequence of the mutation, the targeted chromosomal sequence is inactivated and a functional protein associated with PD is not produced. The inactivated chromosomal sequence comprises no exogenously introduced sequence. Such an animal may be termed a “knock-out.” Also included herein are genetically modified animals in which two, three, or more chromosomal sequences encoding proteins associated with PD are inactivated. 
     In another embodiment, the genetically modified animal may comprise at least one edited chromosomal sequence encoding a protein associated with PD such that the sequence is over-expressed and a functional protein associated with PD is over-produced. For example, the regulatory regions controlling the expression of the protein associated with PD may be altered such that the protein associated with PD is over-expressed. 
     In yet another embodiment, the genetically modified animal may comprise at least one chromosomally integrated sequence encoding a protein associated with PD. For example, an exogenous sequence encoding an orthologous or an endogenous protein associated with PD may be integrated into a chromosomal sequence encoding a protein associated with PD such that the chromosomal sequence is inactivated, but wherein the exogenous sequence encoding the orthologous or endogenous protein associated with PD may be expressed or over-expressed. In such a case, the sequence encoding the orthologous or endogenous protein associated with PD may be operably linked to a promoter control sequence. Alternatively, an exogenous sequence encoding an orthologous or endogenous protein associated with PD may be integrated into a chromosomal sequence without affecting expression of a chromosomal sequence. For example, an exogenous sequence encoding a protein associated with PD may be integrated into a “safe harbor” locus, such as the Rosa26 locus, HPRT locus, or AAV locus, wherein the exogenous sequence encoding the orthologous or endogenous protein associated with PD may be expressed or over-expressed. An animal comprising a chromosomally integrated sequence encoding a protein associated with PD may be called a “knock-in,” and it should be understood that in such an iteration of the animal no selectable marker is present. The present disclosure also encompasses genetically modified animals in which 2, 3, 4, 5, 6, 7, 8, 9, 10 or more sequences encoding proteins associated with PD are integrated into the genome. 
     The chromosomally integrated sequence encoding a protein associated with PD may encode the wild type form of the protein associated with PD. Alternatively, the chromosomally integrated sequence encoding a protein associated with PD may comprise at least one modification such that an altered version of the protein associated with PD is produced. In some embodiments, the chromosomally integrated sequence encoding a protein associated with PD comprises at least one modification such that the altered version of the protein causes PD. In other embodiments, the chromosomally integrated sequence encoding a protein associated with PD comprises at least one modification such that the altered version of the protein associated with PD protects against PD. 
     In an additional embodiment, the genetically modified animal may be a “humanized” animal comprising at least one chromosomally integrated sequence encoding a functional human protein associated with PD. The functional human protein associated with PD may have no corresponding ortholog in the genetically modified animal. Alternatively, the wild-type animal from which the genetically modified animal is derived may comprise an ortholog corresponding to the functional human protein associated with PD. In this case, the orthologous sequence in the “humanized” animal is inactivated such that no functional protein is made and the “humanized” animal comprises at least one chromosomally integrated sequence encoding the human protein associated with PD. For example, a humanized animal may comprise an inactivated LRRK2 sequence and a chromosomally integrated human LRRK2 sequence. Those of skill in the art appreciate that “humanized” animals may be generated by crossing a knock out animal with a knock in animal comprising the chromosomally integrated sequence. 
     In yet another embodiment, the genetically modified animal may comprise at least one edited chromosomal sequence encoding a protein associated with PD such that the expression pattern of the protein is altered. For example, regulatory regions controlling the expression of the protein, such as a promoter or transcription factor binding site, may be altered such that the protein associated with PD is over-produced, or the tissue-specific or temporal expression of the protein is altered, or a combination thereof. Alternatively, the expression pattern of the protein associated with PD may be altered using a conditional knockout system. A non-limiting example of a conditional knockout system includes a Cre-lox recombination system. A Cre-lox recombination system comprises a Cre recombinase enzyme, a site-specific DNA recombinase that can catalyze the recombination of a nucleic acid sequence between specific sites (lox sites) in a nucleic acid molecule. Methods of using this system to produce temporal and tissue specific expression are known in the art. In general, a genetically modified animal is generated with lox sites flanking a chromosomal sequence, such as a chromosomal sequence encoding a protein associated with PD. The genetically modified animal comprising the lox-flanked chromosomal sequence encoding a protein associated with PD may then be crossed with another genetically modified animal expressing Cre recombinase. Progeny animals comprising the lox-flanked chromosomal sequence and the Cre recombinase are then produced, and the lox-flanked chromosomal sequence encoding a protein associated with PD is recombined, leading to deletion or inversion of the chromosomal sequence encoding the protein. Expression of Cre recombinase may be temporally and conditionally regulated to effect temporally and conditionally regulated recombination of the chromosomal sequence encoding a protein associated with PD. 
     (a) Proteins Associated with the Development of Parkinson&#39;s Disease 
     The present disclosure comprises editing of any chromosomal sequences that encode proteins associated with Parkinson&#39;s disease. The PD-related proteins are typically selected based on an experimental association of the PD-related protein to PD. For example, the production rate or circulating concentration of a PD-related protein may be elevated or depressed in a population having a cognitive disorder relative to a population lacking the cognitive disorder. Differences in protein levels may be assessed using proteomic techniques including but not limited to Western blot, immunohistochemical staining, enzyme linked immunosorbent assay (ELISA), and mass spectrometry. Alternatively, the PD-related proteins may be identified by obtaining gene expression profiles of the genes encoding the proteins using genomic techniques including but not limited to DNA microarray analysis, serial analysis of gene expression (SAGE), and quantitative real-time polymerase chain reaction (Q-PCR). By way of non-limiting example, proteins associated with Parkinson&#39;s disease include but are not limited to α-synuclein, DJ-1, LRRK1, LRRK2, PINK1, Parkin, UCHL1, Synphilin-1, and NURR1. 
     The normal cellular functions of α-synuclein protein, encoded by the α-synuclein gene, have not been determined. Several mutations in α-synuclein have been associated with early-onset familial PD and the mutant protein aggregates abnormally in Parkinson&#39;s disease, Alzheimer&#39;s disease, Lewy body disease, and other neurodegenerative diseases. Non-limiting examples of mutations in α-synuclein that may cause PD include A30P (i.e. alanine at position 30 is changed to proline), A30T (i.e. alanine at position 30 is changed to threonine), and E46K (i.e. glutamate at position 46 is changed to lysine). 
     The DJ-1 protein encoded by the PARK7 gene (Parkinson disease (autosomal recessive, early onset) 7) belongs to the peptidase C56 family of proteins. It acts as a positive regulator of androgen receptor-dependent transcription. It may also function as a redox-sensitive chaperone, as a sensor for oxidative stress, and it apparently protects neurons against oxidative stress and cell death. A variety of defects in this gene cause autosomal recessive early-onset Parkinson&#39;s disease. Non-limiting examples of mutations in DJ-1 that may cause PD include L166P (i.e. leucine at position 166 is changed to proline), M26I (i.e. methionine at position 26 is changed to isoleucine), E64D (i.e. glutamate at position 64 is changed to aspartate), A104T (i.e. alanine at position 104 is changed to threonine), and D149A (i.e. aspartate at position 149 is changed to alanine). 
     Leucine-rich repeat kinase 1 also known as LRRK1 is a protein member of the leucine-rich repeat kinase family which in humans is encoded by the LRRK1 gene. A high degree of sequence identity exists between LRRK1 and LRRK2. LRRK1 has a similar domain structure as LRRK2. LRRK1 is a GTP-regulated protein kinase harboring both the kinase effector domain and the GTP-binding regulatory domain. Binding of GTP to LRRK1 is specific, requires the GTPase-like Roc domain, and leads to a stimulation of LRRK1 kinase activity. LRRK1 also regulates EGFR (epidermal growth factor receptor) transport from early to late endosomes and regulates the motility of EGF-containing early endosomes in a manner dependent on its kinase activity. LRRK1 serves as a scaffold facilitating the interaction of EGFR with the endosomal sorting complex, thus enabling efficient sorting of EGFR to the inner vesicles of multivesicular bodies. In rat, LRRK1 is present in adult brain, adrenal gland, liver, lung, spleen and kidney and also in embryonic brain, with declining gene activity after birth. A variety of single amino acid changes in LRRK1 that may be associated with PD have been characterized. Non-limiting mutations in LRRK1 include M1030V (i.e., methionine at position 1030 is changed to valine); V1074D (i.e., valine at position 1074 is changed to aspartate); T1178M (i.e., threonine at position 1178 is changed to methionine); G1232D (i.e., glycine at position 1232 is changed to aspartate); and E1297K (glutamate at position 1297 is changed to lysine). 
     Leucine-rich repeat kinase 2, also known as LRRK2 or as dardarin, is a protein member of the leucine-rich repeat kinase family which in humans is encoded by the LRRK2 gene. The LRRK2 protein comprises an ankyrin repeat region, a leucine-rich repeat (LRR) domain, a kinase domain, a DFG-like motif, a RAS domain, a GTPase domain, an MLK-like domain, and a WD40 domain. The protein is present largely in the cytoplasm but also associates with the mitochondrial outer membrane. Non-limiting examples of mutations in LRRK2 that may cause PD include G2019S (i.e. glycine at position 2019 is changed to serine), I2020T (i.e. isoleucine at position 2020 is changed to threonine), I1371V (i.e. isoleucine at position 1371 is changed to valine), R1441H (i.e. arginine at position 144 is changed to histidine), I2012T (i.e. isoleucine at position 2012 is changed to threonine). 
     The PINK1protein (mitochondrial serine/threonine-protein kinase), is an enzyme that in humans is encoded by the PINK1 gene. This gene encodes a serine/threonine protein kinase that localizes to mitochondria. It is thought to protect cells from stress-induced mitochondrial dysfunction. Mutations in this gene cause one form of autosomal recessive early-onset Parkinson disease. Non-limiting examples of mutations in PINK1 that may cause PD include C92F (i.e. cysteine at position 92 is changed to phenyl alanine), A168P (i.e. alanine at position 168 is changed to proline), Q239X (i.e. glutamine at position 239 is changed to another amino acid), R246X (i.e. arginine at position 246 is changed to another amino acid), H271Q (i.e. histidine at position 271 is changed to glutamine), G309D (i.e. glycine at position 309 is changed to aspartate), L347P (i.e. leucine at position 347 is changed to proline), E417G (i.e. glutamate at position 417 is changed to glycine), W437X (i.e. tryptophan at position 437 is changed to another amino acid), R464H (i.e. arginine at position 464 is changed to histidine), R492X (i.e. arginine at position 492 is changed to another amino acid). 
     Parkin is a protein which in humans is encoded by the PARK2 gene. The precise function of this protein is unknown; however, the protein is a component of a multiprotein E3 ubiquitin ligase complex which in turn is part of the ubiquitin-proteasome system that mediates the targeting of substrate proteins for proteasomal degradation. Mutations in this gene are known to cause a familial form of Parkinson&#39;s disease known as autosomal recessive juvenile Parkinson&#39;s disease. This form of genetic mutation may be one of the most common known genetic causes of early-onset Parkinson&#39;s disease. Non-limiting examples of mutations in Parkin that may cause PD include V15M (i.e. valine at position 15 is changed to methionine), P37L (i.e. proline at position 37 is changed to leucine), R42P (i.e. arginine at position 42 is changed to proline), A46P (i.e. alanine at position 46 is changed to proline), A82E (i.e. alanine at position 82 is changed to glutamate), K161N (i.e. lysine at position 161 is changed to asparagine), M192V (i.e. methionine at position 192 is changed to valine), K211R (i.e. lysine at position 211 is changed to arginine), K211N (i.e. lysine at position 211 is changed to asparagine), C212Y (i.e. cysteine at position 212 is changed to tyrosine), T240R (i.e. threonine at position 240 is changed to arginine), T240M (i.e. threonine at position 240 is changed to methionine), C253W (i.e. cysteine at position 253 is changed to tryptophan), R256C (i.e. arginine at position 256 is changed to cysteine), R275W (i.e. arginine at position 275 is changed to tryptophan), D280N (i.e. aspartate at position 280 is changed to asparagine), G284R (i.e. glycine at position 248 is changed to arginine), C289G (i.e. cysteine at position 289 is changed to glycine), G328E (i.e. glycine at position 328 is changed to glutamate), R334C (i.e. arginine at position 334 is changed to cysteine), T351P (i.e. threonine at position 351 is changed to proline), A398T (i.e. alanine at position 398 is changed to threonine), T415N (i.e. threonine at position 415 is changed to asparagine), G430D (i.e. glycine at position 430 is changed to aspartate), C431F (i.e. cysteine at position 431 is changed to phenylalanine), P437L (i.e. proline at position 437 is changed to leucine), and C441R (i.e. cysteine at position 441 is changed to arginine). 
     Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) encoded by the UCHL1 gene is a deubiquitinating enzyme. UCHL1 is a member of a protein family whose products hydrolyze small C-terminal adducts of ubiquitin to generate the ubiquitin monomer. Expression of UCHL1 is highly specific to neurons and to cells of the diffuse neuroendocrine system and their tumors. It is present in all neurons. Mutations in the gene encoding this protein are implicated as the cause of Parkinson&#39;s disease. Furthermore, a polymorphism in this gene has been found to be associated with a reduced risk for PD. A non-limiting example of a mutation in UCHL1 that may cause PD includes 193M (i.e. isoleucine at position 93 is changed to methionine). A non-limiting example of a mutation in UCHL1 that may reduce the risk for PD includes S18Y (i.e. serine at position 18 is changed to tyrosine). 
     Synphilin-1 is a protein that in humans is encoded by the SNCAIP gene. This gene encodes a protein containing several protein-protein interaction domains, including ankyrin-like repeats, a coiled-coil domain, and an ATP/GTP-binding motif. The encoded protein interacts with α-synuclein in neuronal tissue and may play a role in the formation of cytoplasmic inclusions and neurodegeneration. At least one mutation in this gene has been associated with Parkinson&#39;s disease. A non-limiting example of a mutation in Synphilin-1 that may cause PD includes R621C (i.e. arginine at position 621 is changed to cysteine). 
     The Nuclear receptor related 1 (nuclear receptor subfamily 4, group A, member 2, or NURR1) protein is a member of the nuclear receptor family of intracellular transcription factors and is encoded by the NR4A2 gene. NURR1 plays a key role in the maintenance of the dopaminergic system of the brain. Mutations in this gene have been associated with disorders related to dopaminergic dysfunction, including Parkinson&#39;s disease, schizophrenia, and manic depression. A non-limiting example of a mutation in NURR1 that may cause PD includes S125C (i.e. serine at position 125 is changed to cysteine). 
     The identity of the proteins associated with PD whose chromosomal sequence is edited can and will vary. For example, the edited chromosomal sequence may encode any of the foregoing proteins detailed herein that are associated with Parkinson&#39;s disease or any combination of the proteins. In this regard, the genetically modified animal or cell may comprise one, two, three, four, five, six, seven, eight, nine, or ten or more edited chromosomal sequences encoding a protein associated with PD, and zero, one, two, three, four, five, six, seven, eight, nine or more chromosomally integrated sequences encoding proteins associated with PD. 
     Table A details preferred combinations of inactivated chromosomal sequences and integrated sequences. For example, those rows having no entry in the “Integrated Sequence” column indicate a genetically modified animal in which the sequence specified in that row under “Edited Chromosomal Sequence” is inactivated (i.e., a knock-out). Subsequent rows indicate single or multiple knock-outs with knock-ins of one or more integrated orthologous sequences, as indicated in the “Integrated Sequence” column 
     
       
         
           
               
               
             
               
                 TABLE A 
               
               
                   
               
               
                 Edited Chromosomal Sequence 
                 Integrated Sequence 
               
               
                   
               
             
            
               
                 α-synuclein 
                 none 
               
               
                 PARK7 
                 none 
               
               
                 LRRK1 
                 none 
               
               
                 LRRK2 
                 none 
               
               
                 PINK1 
                 none 
               
               
                 PARK2 
                 none 
               
               
                 α-synuclein 
                 α-synuclein 
               
               
                 PARK7 
                 PARK7 
               
               
                 LRRK1 
                 LRRK1 
               
               
                 LRRK2 
                 LRRK2 
               
               
                 PINK1 
                 PINK1 
               
               
                 PARK2 
                 PARK2 
               
               
                 α-synuclein, PARK7 
                 α-synuclein, PARK7 
               
               
                 α-synuclein, LRRK1 
                 α-synuclein, LRRK1 
               
               
                 α-synuclein, LRRK2 
                 α-synuclein, LRRK2 
               
               
                 α-synuclein, PINK1 
                 α-synuclein, PINK1 
               
               
                 α-synuclein, PARK2 
                 α-synuclein, PARK2 
               
               
                 PARK7, LRRK1 
                 PARK7, LRRK1 
               
               
                 PARK7, LRRK2 
                 PARK7, LRRK2 
               
               
                 PARK7, PINK1 
                 PARK7, PINK1 
               
               
                 PARK7, PARK2 
                 PARK7, PARK2 
               
               
                 LRRK1, LRRK2 
                 LRRK1, LRRK2 
               
               
                 LRRK1, PINK1 
                 LRRK1, PINK1 
               
               
                 LRRK1, PARK2 
                 LRRK1, PARK2 
               
               
                 LRRK2, PARK2 
                 LRRK2, PARK2 
               
               
                 PINK1, PARK2 
                 PINK1, PARK2 
               
               
                 α-synuclein, PARK7, LRRK1 
                 α-synuclein, PARK7, LRRK1 
               
               
                 α-synuclein, PARK7, LRRK2 
                 α-synuclein, PARK7, LRRK2 
               
               
                 α-synuclein, PARK7, PINK1 
                 α-synuclein, PARK7, PINK1 
               
               
                 α-synuclein, PARK7, PARK2 
                 α-synuclein, PARK7, PARK2 
               
               
                 α-synuclein, LRRK1, LRRK2 
                 α-synuclein, LRRK1, LRRK2 
               
               
                 α-synuclein, LRRK1, PINK1 
                 α-synuclein, LRRK1, PINK1 
               
               
                 α-synuclein, LRRK1, PARK2 
                 α-synuclein, LRRK1, PARK2 
               
               
                 α-synuclein, LRRK2, PINK1 
                 α-synuclein, LRRK2, PINK1 
               
               
                 α-synuclein, LRRK2, PARK2 
                 α-synuclein, LRRK2, PARK2 
               
               
                 α-synuclein, PINK1, PARK2 
                 α-synuclein, PINK1, PARK2 
               
               
                 PARK7, LRRK1, LRRK2 
                 PARK7, LRRK1, LRRK2 
               
               
                 PARK7, LRRK1, PINK1 
                 PARK7, LRRK1, PINK1 
               
               
                 PARK7, LRRK1, PARK2 
                 PARK7, LRRK1, PARK2 
               
               
                 PARK7, LRRK2, PINK1 
                 PARK7, LRRK2, PINK1 
               
               
                 PARK7, LRRK1, PARK2 
                 PARK7, LRRK1, PARK2 
               
               
                 LRRK1, LRRK2, PINK1 
                 LRRK1, LRRK2, PINK1 
               
               
                 LRRK1, LRRK2, PARK2 
                 LRRK1, LRRK2, PARK2 
               
               
                 PARK7, PINK1, PARK2 
                 PARK7, PINK1, PARK2 
               
               
                 LRRK2, PINK1, PARK2 
                 LRRK2, PINK1, PARK2 
               
               
                 α-synuclein, LRRK1, PINK1, 
                 α-synuclein, LRRK1, PINK1, PARK2 
               
               
                 PARK2 
               
               
                 α-synuclein, LRRK2, PINK1, 
                 α-synuclein, LRRK2, PINK1, PARK2 
               
               
                 PARK2 
               
               
                 α-synuclein, PARK7, PINK1, 
                 α-synuclein, PARK7, PINK1, PARK2 
               
               
                 PARK2 
               
               
                 α-synuclein, LRRK1, PINK1, 
                 α-synuclein, LRRK1, PINK1, PARK2 
               
               
                 PARK2 
               
               
                 α-synuclein, PARK7, LRRK2, 
                 α-synuclein, PARK7, LRRK2, 
               
               
                 PARK2 
                 PARK2 
               
               
                 α-synuclein, PARK7, LRRK2, 
                 α-synuclein, PARK7, LRRK2, PINK1 
               
               
                 PINK1 
               
               
                 PARK7, LRRK2, PINK1, 
                 PARK7, LRRK2, PINK1, PARK2 
               
               
                 PARK2 
               
               
                 α-synuclein, PARK7, LRRK2, 
                 α-synuclein, PARK7, LRRK2, 
               
               
                 PINK1, PARK2 
                 PINK1, PARK2 
               
               
                   
               
            
           
         
       
     
     (b) Animals 
     The term “animal,” as used herein, refers to a non-human animal. The animal may be an embryo, a juvenile, or an adult. Suitable animals include vertebrates such as mammals, birds, reptiles, amphibians, and fish. Examples of suitable mammals include without limit rodents, companion animals, livestock, and primates. Non-limiting examples of rodents include mice, rats, hamsters, gerbils, and guinea pigs. Suitable companion animals include but are not limited to cats, dogs, rabbits, hedgehogs, and ferrets. Non-limiting examples of livestock include horses, goats, sheep, swine, cattle, llamas, and alpacas. Suitable primates include but are not limited to capuchin monkeys, chimpanzees, lemurs, macaques, marmosets, tamarins, spider monkeys, squirrel monkeys, and vervet monkeys. Non-limiting examples of birds include chickens, turkeys, ducks, and geese. Alternatively, the animal may be an invertebrate such as an insect, a nematode, and the like. Non-limiting examples of insects include  Drosophila  and mosquitoes. An exemplary animal is a rat. Non-limiting examples of suitable rat strains include Dahl Salt-Sensitive, Fischer 344, Lewis, Long Evans Hooded, Sprague-Dawley, and Wistar. In another iteration of the invention, the animal does not comprise a genetically modified mouse. In each of the foregoing iterations of suitable animals for the invention, the animal does not include exogenously introduced, randomly integrated transposon sequences. 
     (c) Proteins Associated with PD 
     The protein associated with PD may be from any of the animals listed above. Furthermore, the protein associated with PD may be a human protein associated with PD. The type of animal and the source of the protein can and will vary. The protein may be endogenous or exogenous (such as an orthologous protein) to the animal. As an example, the genetically modified animal may be a rat, cat, dog, or pig, and the orthologous protein associated with PD may be human. One of skill in the art will readily appreciate that numerous combinations are possible. 
     Additionally, the protein associated with PD may be modified to include a tag or reporter gene, which are well-known. Reporter genes include those encoding selectable markers such as chloramphenicol acetyltransferase (CAT) and neomycin phosphotransferase (neo), and those encoding a fluorescent protein such as green fluorescent protein (GFP), red fluorescent protein, or any genetically engineered variant thereof that improves the reporter performance. Non-limiting examples of known such FP variants include EGFP, blue fluorescent protein (EBFP, EBFP2, Azurite, mKalama1), cyan fluorescent protein (ECFP, Cerulean, CyPet) and yellow fluorescent protein derivatives (YFP, Citrine, Venus, YPet). For example, in a genetic construct containing a reporter gene, the reporter gene sequence can be fused directly to the targeted gene to create a gene fusion. A reporter sequence can be integrated in a targeted manner in the targeted gene, for example the reporter sequences may be integrated specifically at the 5′ or 3′ end of the targeted gene. The two genes are thus under the control of the same promoter elements and are transcribed into a single messenger RNA molecule. Alternatively, the reporter gene may be used to monitor the activity of a promoter in a genetic construct, for example by placing the reporter sequence downstream of the target promoter such that expression of the reporter gene is under the control of the target promoter, and activity of the reporter gene can be directly and quantitatively measured, typically in comparison to activity observed under a strong consensus promoter. It will be understood that doing so may or may not lead to destruction of the targeted gene. 
     (d) Exemplary Embodiments 
     In preferred embodiments, the genetically modified animal comprises an edited chromosomal sequence encoding Leucine-rich repeat kinase 1 (LRRK1) protein. In an exemplary embodiment, the chromosomal sequence encoding LRRK1 is inactivated such that no functional LRRK1 protein is produced. In another exemplary embodiment, the chromosomal sequence encoding LRRK1 is modified such that a modified version of LRRK1 is produced. The modified LRRK1 protein may have at least one amino acid change, a short deletion, or a short insertion such that the structure and/or activity of the protein is modified. The modified LRRK1 may have altered (i.e., decreased, increased) enzyme activity, altered substrate specificity, altered kinetic properties, altered binding activity, altered stability, and the like. The animal may be heterozygous or homozygous for the edited chromosomal sequence. Homozygous animals may be created by cross-breeding animals heterozygous for the edited chromosomal sequence. 
     In another preferred embodiment, the genetically modified animal comprising the edited chromosomal sequence encoding LRRK1 may further comprise at least one additional edited chromosomal sequence encoding a protein associated with Parkinson&#39;s disease chosen from α-synuclein, DJ-1, LRRK2, PINK1, Parkin, UCHL1, Synphilin-1, and NURR1. Non-limiting examples include the following: 1) an animal comprising an inactivated chromosomal sequence encoding LRRK1 and an inactivated chromosomal sequence encoding α-synuclein, DJ-1, LRRK2, PINK1, Parkin, UCHL1, Synphilin-1, or NURR1; and 2) an animal comprising an inactivated chromosomal sequence encoding LRRK1 and a modified chromosomal sequence encoding α-synuclein, DJ-1, LRRK2, PINK1, Parkin, UCHL1, Synphilin-1, or NURR1. Animals comprising edited chromosomal sequences encoding LRRK1 and another protein associated with PD may be created by cross-breeding the appropriate animals. 
     In an exemplary embodiment, the animal comprising the edited chromosomal sequence encoding LRRK1 may be a rat. Preferably, the chromosomal sequence encoding LRRK1 is inactivated such that no functional LRRK1 is produced in the rat. 
     (II) Genetically Modified Cells 
     A further aspect of the present disclosure provides genetically modified cells or cell lines comprising at least one edited chromosomal sequence encoding a protein associated with PD. The genetically modified cell or cell line may be derived from any of the genetically modified animals disclosed herein. Alternatively, the chromosomal sequence coding a protein associated with PD may be edited in a cell as detailed below. The disclosure also encompasses a lysate of said cells or cell lines. 
     In general, the cells will be eukaryotic cells. Suitable host cells include fungi or yeast, such as  Pichia, Saccharomyces , or  Schizosaccharomyces ; insect cells, such as SF9 cells from  Spodoptera frugiperda  or S2 cells from  Drosophila melanogaster ; and animal cells, such as mouse, rat, hamster, non-human primate, or human cells. Exemplary cells are mammalian. The mammalian cells may be primary cells. In general, any primary cell that is sensitive to double strand breaks may be used. The cells may be of a variety of cell types, e.g., fibroblast, myoblast, T or B cell, macrophage, epithelial cell, and so forth. 
     When mammalian cell lines are used, the cell line may be any established cell line or a primary cell line that is not yet described. The cell line may be adherent or non-adherent, or the cell line may be grown under conditions that encourage adherent, non-adherent or organotypic growth using standard techniques known to individuals skilled in the art. Non-limiting examples of suitable mammalian cell lines include Chinese hamster ovary (CHO) cells, monkey kidney CVI line transformed by SV40 (COS7), human embryonic kidney line 293, baby hamster kidney cells (BHK), mouse sertoli cells (TM4), monkey kidney cells (CVI-76), African green monkey kidney cells (VERO), human cervical carcinoma cells (HeLa), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT), rat hepatoma cells (HTC), HIH/3T3 cells, the human U2-OS osteosarcoma cell line, the human A549 cell line, the human K562 cell line, the human HEK293 cell lines, the human HEK293T cell line, and TRI cells. For an extensive list of mammalian cell lines, those of ordinary skill in the art may refer to the American Type Culture Collection catalog (ATCC®, Mamassas, Va.). 
     In still other embodiments, the cell may be a stem cell. Suitable stem cells include without limit embryonic stem cells, ES-like stem cells, fetal stem cells, adult stem cells, pluripotent stem cells, induced pluripotent stem cells, multipotent stem cells, oligopotent stem cells, and unipotent stem cells. 
     In a preferred embodiment, the genetically modified cell comprises an edited chromosomal sequence encoding LRRK1 protein. In an exemplary embodiment, the edited chromosomal sequence encoding LRRK1 is inactivated such that no functional protein is produced. In another exemplary embodiment, the genetically modified cell is a rat cell. 
     (III) Zinc Finger-Mediated Genomic Editing 
     In general, the genetically modified animal or cell detailed above in sections (I) and (II), respectively, is generated using a zinc finger nuclease-mediated genome editing process. The process for editing a chromosomal sequence comprises: (a) introducing into an embryo or cell at least one nucleic acid encoding a zinc finger nuclease that recognizes a target sequence in the chromosomal sequence and is able to cleave a site in the chromosomal sequence, and, optionally, (i) at least one donor polynucleotide comprising a sequence for integration that is flanked by an upstream sequence and a downstream sequence that share substantial sequence identity with either side of the cleavage site, or (ii) at least one exchange polynucleotide comprising a sequence that is substantially identical to a portion of the chromosomal sequence at the cleavage site and which further comprises at least one nucleotide change; and (b) culturing the embryo or cell to allow expression of the zinc finger nuclease such that the zinc finger nuclease introduces a double-stranded break into the chromosomal sequence, and wherein the double-stranded break is repaired by (i) a non-homologous end-joining repair process such that an inactivating mutation is introduced into the chromosomal sequence, or (ii) a homology-directed repair process such that the sequence in the donor polynucleotide is integrated into the chromosomal sequence or the sequence in the exchange polynucleotide is exchanged with the portion of the chromosomal sequence. 
     Components of the zinc finger nuclease-mediated method are described in more detail below. 
     (a) Zinc Finger Nuclease 
     The method comprises, in part, introducing into an embryo or cell at least one nucleic acid encoding a zinc finger nuclease. Typically, a zinc finger nuclease comprises a DNA binding domain (i.e., zinc finger) and a cleavage domain (i.e., nuclease). The DNA binding and cleavage domains are described below. The nucleic acid encoding a zinc finger nuclease may comprise DNA or RNA. For example, the nucleic acid encoding a zinc finger nuclease may comprise mRNA. When the nucleic acid encoding a zinc finger nuclease comprises mRNA, the mRNA molecule may be 5′ capped. Similarly, when the nucleic acid encoding a zinc finger nuclease comprises mRNA, the mRNA molecule may be polyadenylated. An exemplary nucleic acid according to the method is a capped and polyadenylated mRNA molecule encoding a zinc finger nuclease. Methods for capping and polyadenylating mRNA are known in the art. 
     (i) Zinc Finger Binding Domain 
     Zinc finger binding domains may be engineered to recognize and bind to any nucleic acid sequence of choice. See, for example, Beerli et al. (2002) Nat. Biotechnol. 20:135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) Nat. Biotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416; Zhang et al. (2000) J. Biol. Chem. 275(43):33850-33860; Doyon et al. (2008) Nat. Biotechnol. 26:702-708; and Santiago et al. (2008) Proc. Natl. Acad. Sci. USA 105:5809-5814. An engineered zinc finger binding domain may have a novel binding specificity compared to a naturally-occurring zinc finger protein. Engineering methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, using databases comprising doublet, triplet, and/or quadruplet nucleotide sequences and individual zinc finger amino acid sequences, in which each doublet, triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence. See, for example, U.S. Pat. Nos. 6,453,242 and 6,534,261, the disclosures of which are incorporated by reference herein in their entireties. As an example, the algorithm of described in U.S. Pat. No. 6,453,242 may be used to design a zinc finger binding domain to target a preselected sequence. Alternative methods, such as rational design using a nondegenerate recognition code table may also be used to design a zinc finger binding domain to target a specific sequence (Sera et al. (2002) Biochemistry 41:7074-7081). Publically available web-based tools for identifying potential target sites in DNA sequences and designing zinc finger binding domains may be found at http://www.zincfingertools.org and http://bindr.gdcb.iastate.edu/ZiFiT/, respectively (Mandell et al. (2006) Nuc. Acid Res. 34:W516-W523; Sander et al. (2007) Nuc. Acid Res. 35:W599-W605). 
     A zinc finger binding domain may be designed to recognize a DNA sequence ranging from about 3 nucleotides to about 21 nucleotides in length, or from about 9 to about 18 nucleotides in length. In general, the zinc finger binding domains of the zinc finger nucleases disclosed herein comprise at least three zinc finger recognition regions (i.e., zinc fingers). In one embodiment, the zinc finger binding domain may comprise four zinc finger recognition regions. In another embodiment, the zinc finger binding domain may comprise five zinc finger recognition regions. In still another embodiment, the zinc finger binding domain may comprise six zinc finger recognition regions. A zinc finger binding domain may be designed to bind to any suitable target DNA sequence. See for example, U.S. Pat. Nos. 6,607,882; 6,534,261 and 6,453,242, the disclosures of which are incorporated by reference herein in their entireties. 
     Exemplary methods of selecting a zinc finger recognition region may include phage display and two-hybrid systems, and are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; as well as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB 2,338,237, each of which is incorporated by reference herein in its entirety. In addition, enhancement of binding specificity for zinc finger binding domains has been described, for example, in WO 02/077227. 
     Zinc finger binding domains and methods for design and construction of fusion proteins (and polynucleotides encoding same) are known to those of skill in the art and are described in detail in U.S. Patent Application Publication Nos. 20050064474 and 20060188987, each incorporated by reference herein in its entirety. Zinc finger recognition regions and/or multi-fingered zinc finger proteins may be linked together using suitable linker sequences, including for example, linkers of five or more amino acids in length. See, U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949, the disclosures of which are incorporated by reference herein in their entireties, for non-limiting examples of linker sequences of six or more amino acids in length. The zinc finger binding domain described herein may include a combination of suitable linkers between the individual zinc fingers of the protein. 
     In some embodiments, the zinc finger nuclease may further comprise a nuclear localization signal or sequence (NLS). A NLS is an amino acid sequence which facilitates targeting the zinc finger nuclease protein into the nucleus to introduce a double stranded break at the target sequence in the chromosome. Nuclear localization signals are known in the art. See, for example, Makkerh et al. (1996) Current Biology 6:1025-1027. 
     (ii) Cleavage Domain 
     A zinc finger nuclease also includes a cleavage domain. The cleavage domain portion of the zinc finger nucleases disclosed herein may be obtained from any endonuclease or exonuclease. Non-limiting examples of endonucleases from which a cleavage domain may be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, for example, 2002-2003 Catalog, New England Biolabs, Beverly, Mass.; and Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388 or www.neb.com. Additional enzymes that cleave DNA are known (e.g., S1 Nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease). See also Linn et al. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993. One or more of these enzymes (or functional fragments thereof) may be used as a source of cleavage domains. 
     A cleavage domain also may be derived from an enzyme or portion thereof, as described above, that requires dimerization for cleavage activity. Two zinc finger nucleases may be required for cleavage, as each nuclease comprises a monomer of the active enzyme dimer. Alternatively, a single zinc finger nuclease may comprise both monomers to create an active enzyme dimer. As used herein, an “active enzyme dimer” is an enzyme dimer capable of cleaving a nucleic acid molecule. The two cleavage monomers may be derived from the same endonuclease (or functional fragments thereof), or each monomer may be derived from a different endonuclease (or functional fragments thereof). 
     When two cleavage monomers are used to form an active enzyme dimer, the recognition sites for the two zinc finger nucleases are preferably disposed such that binding of the two zinc finger nucleases to their respective recognition sites places the cleavage monomers in a spatial orientation to each other that allows the cleavage monomers to form an active enzyme dimer, e.g., by dimerizing. As a result, the near edges of the recognition sites may be separated by about 5 to about 18 nucleotides. For instance, the near edges may be separated by about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotides. It will however be understood that any integral number of nucleotides or nucleotide pairs may intervene between two recognition sites (e.g., from about 2 to about 50 nucleotide pairs or more). The near edges of the recognition sites of the zinc finger nucleases, such as for example those described in detail herein, may be separated by 6 nucleotides. In general, the site of cleavage lies between the recognition sites. 
     Restriction endonucleases (restriction enzymes) are present in many species and are capable of sequence-specific binding to DNA (at a recognition site), and cleaving DNA at or near the site of binding. Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removed from the recognition site and have separable binding and cleavage domains. For example, the Type IIS enzyme Fok I catalyzes double-stranded cleavage of DNA, at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other. See, for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; as well as Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem. 269:31, 978-31, 982. Thus, a zinc finger nuclease may comprise the cleavage domain from at least one Type IIS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered. Exemplary Type IIS restriction enzymes are described for example in International Publication WO 07/014,275, the disclosure of which is incorporated by reference herein in its entirety. Additional restriction enzymes also contain separable binding and cleavage domains, and these also are contemplated by the present disclosure. See, for example, Roberts et al. (2003) Nucleic Acids Res. 31:418-420. 
     An exemplary Type IIS restriction enzyme, whose cleavage domain is separable from the binding domain, is Fok I. This particular enzyme is active as a dimmer (Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10, 570-10, 575). Accordingly, for the purposes of the present disclosure, the portion of the Fok I enzyme used in a zinc finger nuclease is considered a cleavage monomer. Thus, for targeted double-stranded cleavage using a Fok I cleavage domain, two zinc finger nucleases, each comprising a FokI cleavage monomer, may be used to reconstitute an active enzyme dimer. Alternatively, a single polypeptide molecule containing a zinc finger binding domain and two Fok I cleavage monomers may also be used. 
     In certain embodiments, the cleavage domain may comprise one or more engineered cleavage monomers that minimize or prevent homodimerization, as described, for example, in U.S. Patent Publication Nos. 20050064474, 20060188987, and 20080131962, each of which is incorporated by reference herein in its entirety. By way of non-limiting example, amino acid residues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 of Fok I are all targets for influencing dimerization of the Fok I cleavage half-domains. Exemplary engineered cleavage monomers of Fok I that form obligate heterodimers include a pair in which a first cleavage monomer includes mutations at amino acid residue positions 490 and 538 of Fok I and a second cleavage monomer that includes mutations at amino-acid residue positions 486 and 499. 
     Thus, in one embodiment, a mutation at amino acid position 490 replaces Glu (E) with Lys (K); a mutation at amino acid residue 538 replaces Iso (I) with Lys (K); a mutation at amino acid residue 486 replaces Gln (Q) with Glu (E); and a mutation at position 499 replaces Iso (I) with Lys (K). Specifically, the engineered cleavage monomers may be prepared by mutating positions 490 from E to K and 538 from I to K in one cleavage monomer to produce an engineered cleavage monomer designated “E490K:I538K” and by mutating positions 486 from Q to E and 499 from I to L in another cleavage monomer to produce an engineered cleavage monomer designated “Q486E:I499L.” The above described engineered cleavage monomers are obligate heterodimer mutants in which aberrant cleavage is minimized or abolished. Engineered cleavage monomers may be prepared using a suitable method, for example, by site-directed mutagenesis of wild-type cleavage monomers (Fok I) as described in U.S. Patent Publication No. 20050064474 (see Example 5). 
     The zinc finger nuclease described above may be engineered to introduce a double stranded break at the targeted site of integration. The double stranded break may be at the targeted site of integration, or it may be up to 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, or 1000 nucleotides away from the site of integration. In some embodiments, the double stranded break may be up to 1, 2, 3, 4, 5, 10, 15, or 20 nucleotides away from the site of integration. In other embodiments, the double stranded break may be up to 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides away from the site of integration. In yet other embodiments, the double stranded break may be up to 50, 100, or 1000 nucleotides away from the site of integration. 
     (b) Optional Donor Polynucleotide 
     The method for editing a chromosomal sequence encoding a protein associated with PD may further comprise introducing at least one donor polynucleotide comprising a sequence encoding a protein associated with PD into the embryo or cell. A donor polynucleotide comprises at least three components: the sequence coding the protein associated with PD, an upstream sequence, and a downstream sequence. The sequence coding the protein associated with PD may be altered such that it codes for a modified version of the protein. The sequence encoding the protein is flanked by the upstream and downstream sequence, wherein the upstream and downstream sequences share sequence similarity with either side of the site of integration in the chromosome. 
     Typically, the donor polynucleotide will be DNA. The donor polynucleotide may be a DNA plasmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a viral vector, a linear piece of DNA, a PCR fragment, a naked nucleic acid, or a nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer. An exemplary donor polynucleotide comprising the sequence encoding the protein associated with PD may be a BAC. 
     The sequence of the donor polynucleotide that encodes the protein associated with PD may include coding (i.e., exon) sequence, as well as intron sequences and upstream regulatory sequences (such as, e.g., a promoter). Depending upon the identity and the source of the sequence encoding the protein associated with PD, the size of the sequence encoding the protein associated with PD will vary. For example, the sequence encoding the protein associated with PD may range in size from about 1 kb to about 5,000 kb. 
     The donor polynucleotide also comprises upstream and downstream sequence flanking the sequence encoding the protein associated with PD. The upstream and downstream sequences in the donor polynucleotide are selected to promote recombination between the chromosomal sequence of interest and the donor polynucleotide. The upstream sequence, as used herein, refers to a nucleic acid sequence that shares sequence similarity with the chromosomal sequence upstream of the targeted site of integration. Similarly, the downstream sequence refers to a nucleic acid sequence that shares sequence similarity with the chromosomal sequence downstream of the targeted site of integration. The upstream and downstream sequences in the donor polynucleotide may share about 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with the targeted chromosomal sequence. In other embodiments, the upstream and downstream sequences in the donor polynucleotide may share about 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the targeted chromosomal sequence. In an exemplary embodiment, the upstream and downstream sequences in the donor polynucleotide may share about 99% or 100% sequence identity with the targeted chromosomal sequence. 
     An upstream or downstream sequence may comprise from about 50 bp to about 2500 bp. In one embodiment, an upstream or downstream sequence may comprise about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, or 2500 bp. An exemplary upstream or downstream sequence may comprise about 200 bp to about 2000 bp, about 600 bp to about 1000 bp, or more particularly about 700 bp to about 1000 bp. 
     In some embodiments, the donor polynucleotide may further comprise a marker. Such a marker may make it easy to screen for targeted integrations. Non-limiting examples of suitable markers include restriction sites, fluorescent proteins, or selectable markers. 
     One of skill in the art would be able to construct a donor polynucleotide as described herein using well-known standard recombinant techniques (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996). 
     In the method detailed above for integrating a sequence encoding the protein associated with PD, a double stranded break introduced into the chromosomal sequence by the zinc finger nuclease is repaired, via homologous recombination with the donor polynucleotide, such that the sequence encoding the protein associated with PD is integrated into the chromosome. The presence of a double-stranded break facilitates integration of the sequence encoding the protein associated with PD. A donor polynucleotide may be physically integrated or, alternatively, the donor polynucleotide may be used as a template for repair of the break, resulting in the introduction of the sequence encoding the protein associated with PD as well as all or part of the upstream and downstream sequences of the donor polynucleotide into the chromosome. Thus, endogenous chromosomal sequence may be converted to the sequence of the donor polynucleotide. 
     (c) Optional Exchange Polynucleotide 
     The method for editing chromosomal sequences encoding a protein associated with PD may further comprise introducing into the embryo or cell at least one exchange polynucleotide comprising a sequence that is substantially identical to the chromosomal sequence at the site of cleavage and which further comprises at least one specific nucleotide change. 
     Typically, the exchange polynucleotide will be DNA. The exchange polynucleotide may be a DNA plasmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a viral vector, a linear piece of DNA, a PCR fragment, a naked nucleic acid, or a nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer. An exemplary exchange polynucleotide may be a DNA plasmid. 
     The sequence in the exchange polynucleotide is substantially identical to a portion of the chromosomal sequence at the site of cleavage. In general, the sequence of the exchange polynucleotide will share enough sequence identity with the chromosomal sequence such that the two sequences may be exchanged by homologous recombination. For example, the sequence in the exchange polynucleotide may have at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with a portion of the chromosomal sequence. 
     Importantly, the sequence in the exchange polynucleotide comprises at least one specific nucleotide change with respect to the sequence of the corresponding chromosomal sequence. For example, one nucleotide in a specific codon may be changed to another nucleotide such that the codon codes for a different amino acid. In one embodiment, the sequence in the exchange polynucleotide may comprise one specific nucleotide change such that the encoded protein comprises one amino acid change. In other embodiments, the sequence in the exchange polynucleotide may comprise two, three, four, or more specific nucleotide changes such that the encoded protein comprises one, two, three, four, or more amino acid changes. In still other embodiments, the sequence in the exchange polynucleotide may comprise a three nucleotide deletion or insertion such that the reading frame of the coding reading is not altered (and a functional protein is produced). The expressed protein, however, would comprise a single amino acid deletion or insertion. 
     The length of the sequence in the exchange polynucleotide that is substantially identical to a portion of the chromosomal sequence at the site of cleavage can and will vary. In general, the sequence in the exchange polynucleotide may range from about 50 bp to about 10,000 bp in length. In various embodiments, the sequence in the exchange polynucleotide may be about 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, or 5000 bp in length. In other embodiments, the sequence in the exchange polynucleotide may be about 5500, 6000, 6500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, or 10,000 bp in length. 
     One of skill in the art would be able to construct an exchange polynucleotide as described herein using well-known standard recombinant techniques (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996). 
     In the method detailed above for modifying a chromosomal sequence, a double stranded break introduced into the chromosomal sequence by the zinc finger nuclease is repaired, via homologous recombination with the exchange polynucleotide, such that the sequence in the exchange polynucleotide may be exchanged with a portion of the chromosomal sequence. The presence of the double stranded break facilitates homologous recombination and repair of the break. The exchange polynucleotide may be physically integrated or, alternatively, the exchange polynucleotide may be used as a template for repair of the break, resulting in the exchange of the sequence information in the exchange polynucleotide with the sequence information in that portion of the chromosomal sequence. Thus, a portion of the endogenous chromosomal sequence may be converted to the sequence of the exchange polynucleotide. The changed nucleotide(s) may be at or near the site of cleavage. Alternatively, the changed nucleotide(s) may be anywhere in the exchanged sequences. As a consequence of the exchange, however, the chromosomal sequence is modified. 
     (d) Delivery of Nucleic Acids 
     To mediate zinc finger nuclease genomic editing, at least one nucleic acid molecule encoding a zinc finger nuclease and, optionally, at least one exchange polynucleotide or at least one donor polynucleotide are delivered to the embryo or the cell of interest. Typically, the embryo is a fertilized one-cell stage embryo of the species of interest. 
     Suitable methods of introducing the nucleic acids to the embryo or cell include microinjection, electroporation, sonoporation, biolistics, calcium phosphate-mediated transfection, cationic transfection, liposome transfection, dendrimer transfection, heat shock transfection, nucleofection transfection, magnetofection, lipofection, impalefection, optical transfection, proprietary agent-enhanced uptake of nucleic acids, and delivery via liposomes, immunoliposomes, virosomes, or artificial virions. In one embodiment, the nucleic acids may be introduced into an embryo by microinjection. The nucleic acids may be microinjected into the nucleus or the cytoplasm of the embryo. In another embodiment, the nucleic acids may be introduced into a cell by nucleofection. 
     In embodiments in which both a nucleic acid encoding a zinc finger nuclease and a donor (or exchange) polynucleotide are introduced into an embryo or cell, the ratio of donor (or exchange) polynucleotide to nucleic acid encoding a zinc finger nuclease may range from about 1:10 to about 10:1. In various embodiments, the ratio of donor (or exchange) polynucleotide to nucleic acid encoding a zinc finger nuclease may be about 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In one embodiment, the ratio may be about 1:1. 
     In embodiments in which more than one nucleic acid encoding a zinc finger nuclease and, optionally, more than one donor (or exchange) polynucleotide are introduced into an embryo or cell, the nucleic acids may be introduced simultaneously or sequentially. For example, nucleic acids encoding the zinc finger nucleases, each specific for a distinct recognition sequence, as well as the optional donor (or exchange) polynucleotides, may be introduced at the same time. Alternatively, each nucleic acid encoding a zinc finger nuclease, as well as the optional donor (or exchange) polynucleotides, may be introduced sequentially. 
     (e) Culturing the Embryo or Cell 
     The method of inducing genomic editing with a zinc finger nuclease further comprises culturing the embryo or cell comprising the introduced nucleic acid(s) to allow expression of the zinc finger nuclease. An embryo may be cultured in vitro (e.g., in cell culture). Typically, the embryo is cultured at an appropriate temperature and in appropriate media with the necessary O 2 /CO 2  ratio to allow the expression of the zinc finger nuclease. Suitable non-limiting examples of media include M2, M16, KSOM, BMOC, and HTF media. A skilled artisan will appreciate that culture conditions can and will vary depending on the species of embryo. Routine optimization may be used, in all cases, to determine the best culture conditions for a particular species of embryo. In some cases, a cell line may be derived from an in vitro-cultured embryo (e.g., an embryonic stem cell line). 
     Alternatively, an embryo may be cultured in vivo by transferring the embryo into the uterus of a female host. Generally speaking the female host is from the same or similar species as the embryo. Preferably, the female host is pseudo-pregnant. Methods of preparing pseudo-pregnant female hosts are known in the art. Additionally, methods of transferring an embryo into a female host are known. Culturing an embryo in vivo permits the embryo to develop and may result in a live birth of an animal derived from the embryo. Such an animal would comprise the edited chromosomal sequence encoding the protein associated with PD in every cell of the body. 
     Similarly, cells comprising the introduced nucleic acids may be cultured using standard procedures to allow expression of the zinc finger nuclease. Standard cell culture techniques are described, for example, in Santiago et al. (2008) PNAS 105:5809-5814; Moehle et al. (2007) PNAS 104:3055-3060; Urnov et al. (2005) Nature 435:646-651; and Lombardo et al (2007) Nat. Biotechnology 25:1298-1306. Those of skill in the art appreciate that methods for culturing cells are known in the art and can and will vary depending on the cell type. Routine optimization may be used, in all cases, to determine the best techniques for a particular cell type. 
     Upon expression of the zinc finger nuclease, the chromosomal sequence may be edited. In cases in which the embryo or cell comprises an expressed zinc finger nuclease but no donor (or exchange) polynucleotide, the zinc finger nuclease recognizes, binds, and cleaves the target sequence in the chromosomal sequence of interest. The double-stranded break introduced by the zinc finger nuclease is repaired by an error-prone non-homologous end-joining DNA repair process. Consequently, a deletion, insertion or nonsense mutation may be introduced in the chromosomal sequence such that the sequence is inactivated. 
     In cases in which the embryo or cell comprises an expressed zinc finger nuclease as well as a donor (or exchange) polynucleotide, the zinc finger nuclease recognizes, binds, and cleaves the target sequence in the chromosome. The double-stranded break introduced by the zinc finger nuclease is repaired, via homologous recombination with the donor (or exchange) polynucleotide, such that the sequence in the donor polynucleotide is integrated into the chromosomal sequence (or a portion of the chromosomal sequence is converted to the sequence in the exchange polynucleotide). As a consequence, a sequence may be integrated into the chromosomal sequence (or a portion of the chromosomal sequence may be modified). 
     The genetically modified animals disclosed herein may be crossbred to create animals comprising more than one edited chromosomal sequence or to create animals that are homozygous for one or more edited chromosomal sequences. For example, two animals comprising the same edited chromosomal sequence may be crossbred to create an animal homozygous for the edited chromosomal sequence. Alternatively, animals with different edited chromosomal sequences may be crossbred to create an animal comprising both edited chromosomal sequences. 
     For example, animal A comprising an inactivated PARK7 chromosomal sequence may be crossed with animal B comprising a chromosomally integrated sequence encoding a human DJ-1 protein to give rise to a “humanized” PARK7 offspring comprising both the inactivated PARK7 chromosomal sequence and the chromosomally integrated human PARK7 gene. Similarly, an animal comprising an inactivated α-synuclein chromosomal sequence may be crossed with an animal comprising chromosomally integrated sequence encoding the human α-synuclein protein to generate “humanized” α-synuclein offspring. Moreover, a humanized PARK7 animal may be crossed with a humanized α-synuclein animal to create a humanized PARK7/α-synuclein animal. Those of skill in the art will appreciate that many combinations are possible. Exemplary combinations are presented above in Table A. 
     In other embodiments, an animal comprising an edited chromosomal sequence disclosed herein may be crossbred to combine the edited chromosomal sequence with other genetic backgrounds. By way of non-limiting example, other genetic backgrounds may include wild type genetic backgrounds, genetic backgrounds with deletion mutations, genetic backgrounds with another targeted integration, and genetic backgrounds with non-targeted integrations. 
     (IV) Applications 
     A further aspect of the present disclosure encompasses a method for using the genetically modified animals. In one embodiment, the animals may be used to study the effects of mutations on the animal and development and/or progression of the disease using measures commonly used in the study of PD. Methods for measuring and studying progression of PD in animals are known in the art. Commonly used measures in the study of PD include without limit, amyloidogenesis or protein aggregation, dopamine response, neurodegeneration, development of mitochondrial related dysfunction phenotypes, as well as functional, pathological or biochemical assays. Other relevant indicators regarding development or progression of PD include coordination, balance, gait, motor impairment, tremors and twitches, rigidity, hypokinesia, and cognitive impairments. Such assays may be made in comparison to wild type littermates. 
     In another embodiment, the genetically modified animals may be used for assessing the effect(s) of a therapeutic agent in the development or progression of PD. For example, the effect(s) of a PD therapeutic agent may be measured in a “humanized” genetically modified rat, such that the information gained therefrom may be used to predict the effect of the agent in a human. In general, the method comprises contacting a genetically modified animal comprising at least one edited chromosomal sequence encoding a protein associated with PD, and comparing results of a selected parameter to results obtained from contacting a control genetically modified animal with the same agent. Non-limiting examples of parameters used to assess the effect of an agent on PD may include response to dopamine. 
     Also provided are methods to assess the effect(s) of an agent in an isolated cell comprising at least one edited chromosomal sequence encoding a protein associated with PD, as well as methods of using lysates of such cells (or cells derived from a genetically modified animal disclosed herein) to assess the effect(s) of an agent. For example, the role of a particular protein associated with PD in the metabolism of a particular agent may be determined using such methods. Similarly, substrate specificity and pharmacokinetic parameter may be readily determined using such methods. Those of skill in the art are familiar with suitable tests and/or procedures. 
     Yet another aspect encompasses a method for assessing the efficacy of a potential gene therapy strategy. That is, a chromosomal sequence encoding a protein associated with PD may be modified such that PD development and/or progression is inhibited or reduced. In particular, the method comprises editing a chromosomal sequence encoding a protein associated with PD such that an altered protein product is produced and the animal has an altered response. Accordingly, the genetically modified animal may be compared with an animal predisposed to development of PD such that the effect of the gene therapy event may be assessed. 
     Still yet another aspect encompasses a method of generating a cell line or cell lysate using a genetically modified animal comprising an edited chromosomal sequence encoding protein associated with PD. An additional other aspect encompasses a method of producing purified biological components using a genetically modified cell or animal comprising an edited chromosomal sequence encoding an protein associated with PD. Non-limiting examples of biological components include antibodies, cytokines, signal proteins, enzymes, receptor agonists and receptor antagonists. 
     DEFINITIONS 
     Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale &amp; Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise. 
     The term “chromosomal sequence involved in PD” refers to a chromosomal sequence which has been identified to be a cause or factor in the development of PD and related complications. Exemplary chromosomal sequences involved in PD are identified in Section (I)(a) herein. Any chromosomal sequence known to be involved in PD is included within the scope of the present invention. 
     The term “a protein encoded by a chromosomal sequence involved in PD” or “a protein involved in PD” refers to a protein that has been encoded by a chromosomal sequence identified to be a cause or factor in the development of PD and related complications. Exemplary proteins involved in PD are identified in Section (I)(a) herein. Any type of protein involved in PD is included in the scope of the present invention including, but not limited to, structural proteins, enzyme and catalytic proteins, transport proteins, hormonal proteins, contractile proteins, storage proteins, genetic proteins, defense proteins, and receptor proteins. 
     A “gene,” as used herein, refers to a DNA region (including exons and introns) encoding a gene product, as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, and locus control regions. 
     The terms “nucleic acid” and “polynucleotide” refer to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double-stranded form. For the purposes of the present disclosure, these terms are not to be construed as limiting with respect to the length of a polymer. The terms can encompass known analogs 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 analog of a particular nucleotide has the same base-pairing specificity; i.e., an analog of A will base-pair with T. 
     The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues. 
     The term “recombination” refers to a process of exchange of genetic information between two polynucleotides. For the purposes of this disclosure, “homologous recombination” refers to the specialized form of such exchange that takes place, for example, during repair of double-strand breaks in cells. This process requires sequence similarity between the two polynucleotides, uses a “donor” or exchange 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 being 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 homologous recombination 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. 
     As used herein, the terms “target site” or “target sequence” refer to a nucleic acid sequence that defines a portion of a chromosomal sequence to be edited and to which a zinc finger nuclease is engineered to recognize and bind, provided sufficient conditions for binding exist. 
     Techniques for determining nucleic acid and amino acid sequence identity are known in the art. Typically, such techniques include determining the nucleotide sequence of the mRNA for a gene and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Genomic sequences can also be determined and compared in this fashion. In general, identity refers 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. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986). An exemplary implementation of this algorithm to determine percent identity of a sequence is provided by the Genetics Computer Group (Madison, Wis.) in the “BestFit” utility application. Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR. Details of these programs can be found on the GenBank website. With respect to sequences described herein, the range of desired degrees of sequence identity is approximately 80% to 100% and any integer value there between. Typically the percent identities between sequences are at least 70-75%, preferably 80-82%, more preferably 85-90%, even more preferably 92%, still more preferably 95%, and most preferably 98% sequence identity. 
     Alternatively, the degree of sequence similarity between polynucleotides can be determined by hybridization of polynucleotides under conditions that allow formation of stable duplexes between regions that share a degree of sequence identity, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments. Two nucleic acid, or two polypeptide sequences are substantially similar 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. As used herein, substantially similar also refers to sequences showing complete identity to a specified DNA or polypeptide sequence. DNA sequences that are substantially similar can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; Nucleic Acid Hybridization: A Practical Approach, editors B. D. Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL Press). 
     Selective hybridization of two nucleic acid fragments can be determined as follows. The degree of sequence identity between two nucleic acid molecules affects the efficiency and strength of hybridization events between such molecules. A partially identical nucleic acid sequence will at least partially inhibit the hybridization of a completely identical sequence to a target molecule. Inhibition of hybridization of the completely identical sequence can be assessed using hybridization assays that are well known in the art (e.g., Southern (DNA) blot, Northern (RNA) blot, solution hybridization, or the like, see Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.). Such assays can be conducted using varying degrees of selectivity, for example, using conditions varying from low to high stringency. If conditions of low stringency are employed, the absence of non-specific binding can be assessed using a secondary probe that lacks even a partial degree of sequence identity (for example, a probe having less than about 30% sequence identity with the target molecule), such that, in the absence of non-specific binding events, the secondary probe will not hybridize to the target. 
     When utilizing a hybridization-based detection system, a nucleic acid probe is chosen that is complementary to a reference nucleic acid sequence, and then by selection of appropriate conditions the probe and the reference sequence selectively hybridize, or bind, to each other to form a duplex molecule. A nucleic acid molecule that is capable of hybridizing selectively to a reference sequence under moderately stringent hybridization conditions typically hybridizes under conditions that allow detection of a target nucleic acid sequence of at least about 10-14 nucleotides in length having at least approximately 70% sequence identity with the sequence of the selected nucleic acid probe. Stringent hybridization conditions typically allow detection of target nucleic acid sequences of at least about 10-14 nucleotides in length having a sequence identity of greater than about 90-95% with the sequence of the selected nucleic acid probe. Hybridization conditions useful for probe/reference sequence hybridization, where the probe and reference sequence have a specific degree of sequence identity, can be determined as is known in the art (see, for example, Nucleic Acid Hybridization: A Practical Approach, editors B. D. Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL Press). Conditions for hybridization are well-known to those of skill in the art. 
     Hybridization stringency refers to the degree to which hybridization conditions disfavor the formation of hybrids containing mismatched nucleotides, with higher stringency correlated with a lower tolerance for mismatched hybrids. Factors that affect the stringency of hybridization are well-known to those of skill in the art and include, but are not limited to, temperature, pH, ionic strength, and concentration of organic solvents such as, for example, formamide and dimethylsulfoxide. As is known to those of skill in the art, hybridization stringency is increased by higher temperatures, lower ionic strength and lower solvent concentrations. With respect to stringency conditions for hybridization, it is well known in the art that numerous equivalent conditions can be employed to establish a particular stringency by varying, for example, the following factors: the length and nature of the sequences, base composition of the various sequences, concentrations of salts and other hybridization solution components, the presence or absence of blocking agents in the hybridization solutions (e.g., dextran sulfate, and polyethylene glycol), hybridization reaction temperature and time parameters, as well as, varying wash conditions. A particular set of hybridization conditions may be selected following standard methods in the art (see, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.). 
     EXAMPLES 
     The following examples are included to illustrate the invention. 
     Example 1 
     Identification of ZFNs that Edit the LRRK2 Locus 
     The LRRK2 gene in rat was chosen for zinc finger nuclease (ZFN) mediated genome editing. ZFNs were designed, assembled, and validated using strategies and procedures previously described (see Geurts et al. Science (2009) 325:433). ZFN design made use of an archive of pre-validated 1-finger and 2-finger modules. The LRRK2 gene region (XM — 235581) was scanned for putative zinc finger binding sites to which existing modules could be fused to generate a pair of 4-, 5-, or 6-finger proteins that would bind a 12-18 bp sequence on one strand and a 12-18 bp sequence on the other strand, with about 5-6 bp between the two binding sites. 
     Capped, polyadenylated mRNA encoding each pair of ZFNs was produced using known molecular biology techniques. The mRNA was transfected into rat cells. Control cells were injected with mRNA encoding GFP. Active ZFN pairs were identified by detecting ZFN-induced double strand chromosomal breaks using the Cel-1 nuclease assay. This assay detects alleles of the target locus that deviate from wild type as a result of non-homologous end joining (NHEJ)-mediated imperfect repair of ZFN-induced DNA double strand breaks. PCR amplification of the targeted region from a pool of ZFN-treated cells generates a mixture of WT and mutant amplicons. Melting and reannealing of this mixture results in mismatches forming between heteroduplexes of the WT and mutant alleles. A DNA “bubble” formed at the site of mismatch is cleaved by the surveyor nuclease Cel-1, and the cleavage products can be resolved by gel electrophoresis. This assay revealed that the ZFN pair targeted to bind 5′-tgGGTCATGAAGTGGGGGTGagtgctgt-3′ (SEQ ID NO:3; contact sites in uppercase) and 5′-gaGCCCTGTACCTGGCTGTCtacgacct&#39;3′ (SEQ ID NO:4) cleaved within the LRRK2 locus. 
     Example 2 
     Editing the LRRK2 Locus in Rat Embryos 
     Capped, polyadenylated mRNA encoding the active pair of ZFNs was microinjected into fertilized rat embryos using standard procedures (e.g., see Geurts et al. (2009) supra). The injected embryos were either incubated in vitro, or transferred to pseudopregnant female rats to be carried to parturition. The resulting embryos/fetus, or the toe/tail clip of live born animals were harvested for DNA extraction and analysis. DNA was isolated using standard procedures. The targeted region of the LRRK2 locus was PCR amplified using appropriate primers. The amplified DNA was subcloned into a suitable vector and sequenced using standard methods.  FIG. 1  illustrates edited LRRK2 loci in two founder animals. One animal had a 10 bp deletion in the target sequence of exon 30, and the second animal had an 8 bp deletion in the target sequence of exon 30. These deletions disrupt the reading frame of the LRRK2 coding region. 
     Example 3 
     Identification of ZFNs that Edit the SNCA Locus 
     ZFNs that may edit the SNCA (α-synuclein) locus were designed by scanning the rat SNCA locus (NM — 019169) for putative zinc finger binding sites. The ZFNs were assembled and tested essentially as described in Example 1. This analysis revealed that the ZFN pair targeted to bind 5′-agTCAGCACAGGCATGTccatgttgagt-3′ (SEQ ID NO:5) and 5′-ccTCTGGGGTAGTGAACAGGtctcccac-3′ (SEQ ID NO:6) cleaved within SNCA gene. 
     Example 4 
     Identification of ZFNs that Edit the DJ-1 Locus 
     ZFNs with activity at the DJ-1 locus were identified as described above. That is, the rat DJ-1 gene (NM — 019169) was scanned for putative zinc finger binding sites, and ZFNs were assembled and tested essentially as described in Example 1. It was found that the ZFN pair targeted to bind 5′-aaGCCGACTAGAGAGAGaacccaaacgc-3′ (SEQ ID NO:7) and 5′-gtGAAGGAGATcCTCAAGgagcaggaga-3′ (SEQ ID NO:8) edited the DJ-1 locus. 
     Example 5 
     Identification of ZFNs that Edit the Parkin Locus 
     To identify ZFNs that target and cleave the Parkin locus, the rat Parkin gene (NM — 020093) was scanned for putative zinc finger binding sites. The ZFNs pairs were assembled and tested essentially as described in Example 1. This analysis revealed that the ZFN pair targeted to bind 5′-gaACTCGGaGTTTCCCAGgctggacctt-3′ (SEQ ID NO:9) and 5′-gtGCGGCACCTGCAGACaagcaaccctc-3′ (SEQ ID NO:10) cleaved within the Parkin gene. 
     Example 6 
     Identification of ZFNs that Edit the PINK1 Locus 
     ZFNs with activity at the PINK1 locus were identified essentially as described above. The rat PINK1 gene (NM — 020093) was scanned for putative zinc finger binding sites. The ZFNs were assembled and tested essentially as described in Example 1. This analysis revealed that the ZFN pair targeted to bind 5′-ggGTAGTAGTGTGGGGGtagcatgtcag-3′ (SEQ ID NO:11) and 5′-aaGGCCTGgGCCACGGCCGCAcactctt-3′ (SEQ ID NO:12) edited the PINK1 gene. 
     The table below presents the amino acid sequences of helices of the active ZFNs. 
     
       
         
           
               
               
               
             
               
                   
               
               
                   
                   
                 SEQ 
               
               
                   
                   
                 ID 
               
               
                 Name 
                 Sequence of Zinc Finger Helices 
                 NO: 
               
               
                   
               
             
            
               
                 SNCA 
                 WRSCRSA QSGSLTR RSDNLRE QSGSLTR QSADRTK 
                 13 
               
               
                   
               
               
                 SNCA 
                 RSDHLSA DRSNRKT RSAALSR QSGSLTR RSDHLSE 
                 14 
               
               
                   
                 RKHDRTK 
                   
               
               
                   
               
               
                 DJ-1 
                 RSDALSV QSQHRTT RSDNLSV DRSNLTR DRSDLSR 
                 15 
               
               
                   
               
               
                 DJ-1 
                 RSDNLST DNSSRIT TSSNLSR QSGHLQR QSGNLAR 
                 16 
               
               
                   
               
               
                 LRRK2 
                 ASTGLIR RSDHLSR RSDALSR QSGNLAR NNTQLIE 
                 17 
               
               
                   
                 TSSILSR 
                   
               
               
                   
               
               
                 LRRK2 
                 DRSALSR QSSDLRR RSDVLSA DRSNRIK RSDSLSA 
                 18 
               
               
                   
                 DRSSRTK 
                   
               
               
                   
               
               
                 Parkin 
                 RSDNLSQ ASNDRKK HRSSLRR RSDHLSE ARSTRTN 
                 19 
               
               
                   
               
               
                 Parkin 
                 DRSNLSR QSGDLTR HKTSLKD QSGDLTR RSDDLTR 
                 20 
               
               
                   
               
               
                 PINK1 
                 RSSHLSR RSDHLST ASSARKT QSGALAR QSGSLTR 
                 21 
               
               
                   
               
               
                 PINK1 
                 QSGDLTR DRSDLSR RSDTLSV DNSTRIK RSDALSV 
                 22 
               
               
                   
                 DSSHRTR 
               
               
                   
               
            
           
         
       
     
     Example 7 
     Identification of ZFNs that Edit the LRRK1 Locus in Rat 
     The LRRK1 gene in rat was chosen for zinc finger nuclease (ZFN) mediated genome editing. ZFNs were designed, assembled, and validated using strategies and procedures previously described (see Geurts et al. Science (2009) 325(5939):433). ZFN design made use of an archive of pre-validated 2-finger modules. The LRRK1 gene ZFN target site genomic location is 64285-64464. 
     Capped, polyadenylated mRNA encoding the ZFNs was produced using known molecular biology techniques. The mRNA was transfected into rat cells (Long Evans Hooded, LEH). Plasmid DNA encoding the ZFNs was also transfected into rat cells. Control cells were injected with mRNA encoding GFP. Active ZFNs were identified by detecting ZFN-induced double strand chromosomal breaks using the Cel-1 nuclease assay. This assay detects alleles of the target locus that deviate from wild type as a result of non-homologous end joining (NHEJ)-mediated imperfect repair of ZFN-induced DNA double strand breaks. PCR amplification of the targeted region from a pool of ZFN-treated cells generates a mixture of WT and mutant amplicons. Melting and reannealing of this mixture results in mismatches forming between heteroduplexes of the WT and mutant alleles. A DNA “bubble” formed at the site of mismatch is cleaved by the surveyor nuclease Cel-1, and the cleavage products can be resolved by gel electrophoresis. This assay revealed that the ZFN pair targeted to bind 5′-ACCCCCTCCAGGTCCCTGtgcctcGCAGCGGCTGCTGAACTG-3′ (SEQ ID NO:23; contact sites in uppercase) within the LRRK1 locus was effective at cleaving and editing the site ( FIG. 2 ). ZFNs introduced as either DNA or mRNA were equally effective at mediating genomic editing, although DNA had a slightly higher efficiency. 
     Example 8 
     Editing the LRRK1 Locus in Rat 
     Capped, polyadenylated mRNA encoding the active ZFN pair was introduced into fertilized one-cell rat embryos via pronuclear microinjection using standard procedures (e.g., see Geurts et al. (2009) supra). The injected embryos were either incubated in vitro and/or transferred to pseudopregnant female rats to be carried to parturition. Toe/tail clips of resultant live born animals were harvested for DNA extraction and analysis. DNA was isolated using standard procedures. The targeted region of the LRRK1 locus was PCR amplified using appropriate primers. The amplified DNA was subcloned into a suitable vector and sequenced using standard methods.  FIG. 3  to  FIG. 7  illustrate edited LRRK1 loci in four founder animals. One animal (#28) had a 19 bp deletion in the target sequence of exon 4 ( FIG. 4 ), which results in the introduction of premature stop codons. A second animal (#33) had a 610 bp deletion, along with 7 bp insertion (GTTACTT), including portions of intron 3-4 and exon 4 ( FIG. 5 ). Since the mutation in animal #33 extends into the intron/exon boundary, possible alternative splicing or exon skipping may occur, and thus no translation is shown. In founder animal #42, there was a 1000 bp deletion including portions of intron 3-4, all of exon 4, and portions of intron 4-5 ( FIG. 6 ). No translation was shown as the mutation extends into intron/exon boundary, and thus possible alternative splicing or exon skipping may occur. In founder animal #46, there was a 7 bp deletion within exon 4 ( FIG. 7 ), which results in the introduction of premature stop codons. 
     Example 9 
     Genotyping LRRK1 Locus in Rat after ZFN-Mediated Genome Editing 
     The DNA of rats comprising edited LRRK1 sequences may be extracted and genotyped through PCR and gel analysis to determine the allele composition at the mutant locus. For example, wild-type, heterozygote and homozygote animals may be identified using Lrrk1 Cel-1 F (5′-ATAATATGGAGCCGTGCTGG-3′; SEQ ID NO:29) and Lrrk1 Cel-1 R (5′-TGACCGGGAACTCATTCTTC-3′; SEQ ID NO:30) primers. The PCR conditions for genotyping may be 95° C. for 5 minutes, then 35 cycles of 95° C. for 30 seconds, 60° C. for 30 seconds, and 68° C. for 40 seconds, and lastly 68° C. for 5 minutes. PCR products may be resolved on 10% TBE polyacrylamide gels. Homozygous deletion animals are expected to have PCR fragments of one size which are derived from the two deletion alleles in the DNA. Heterozygous deletion animals are expected to have PCR fragments of two sizes, one of which is derived from the deletion allele, and the other is derived from the wild type non-deletion allele in the animal. The wild-type PCR amplification product is expected to be 327 bp in size. 
     PCR products may also be treated with Exo-SAP and sent directly to sequencing. SAP will dephosphorylate any residual primers and dNTPs (from the PCR) and ExoI will degrade the single stranded molecules so that the DNA can be used directly for sequencing with the specific sequencing primer. 
     For animals with larger deletions, primers Lrrk1 Lg D Fwd (5′-CTCCAGCCCTTTACCTTCAA-3′; SEQ ID NO:31) and Lrrk1 Lg D Rev (5′-TGGCAACTTCCAACTGTCTG-3′; SEQ ID NO:32) may be used for determining wild-type, heterozygote and homozygote animals. The PCR condition for genotyping may be 95° C. for 5 minutes, then 35 cycles of 95° C. for 30 seconds, 60° C. for 30 seconds, and 68° C. for 2 minutes, and lastly 68° C. for 5 minutes. PCR products may be resolved on 1% agarose gels. The wild-type PCR amplification product is expected to be 1506 bp in size. 
     Example 10 
     Confirmation that Founder Rats with Edited LRRK1 Loci are Knockout Animals 
     Each of the founder rats detailed above has a deletion in or near exon 4 of the LRRK1 coding sequence, suggesting that chromosomal sequence encoding LRRK1 is inactivated or knocked out. To verify that the chromosomal sequence is inactivated such that transcription of the LRRK1 gene is disrupted and no LRRK1 protein is made, the levels of LRRK1 mRNA and LRRK1 protein may be determined. For this, mRNA or protein may be isolated using standard procedures. Levels of LRRK1 mRNA may be analyzed using quantitative RT-PCR. Levels of LRRK1 protein may be analyzed using a Western blot assay or an ELISA-based assay.