Crispri targeting alpha-synuclein

Abstract

A composition and a method of treatment for treatment of diseases related to overexpression of SNCA gene in a subject such as Parkinson's disease comprising one or more clustered regularly interspaced short palindromic repeats associated protein (Cas9) or a variant thereof and one or more guide ribonucleic acids (RNAs). The present invention also provides a nanoparticle encapsulating any embodiment of the composition of the present invention.

Description

[0001] CRISPRI TARGETING ALPHA-SYNUCLEIN

[0002] FIELD OF THE INVENTION

[0003] The present invention is related to compositions and methods for inhibiting the expression of alpha-synuclein (SNCA) gene of a subject.

[0004] BACKGROUND OF THE INVENTION

[0005] Parkinson’s disease (PD) is a chronic degenerative disorder of the central nervous system that afflicts approximately 1-2% of the population over 65 years of age. The disease is progressive and affects both motor as well as non-motor functions. “The motor symptoms of the disease result from the nerve cell death in the substantia nigra, a midbrain region that supplies dopamine to the basal ganglia. The cause of this cell death is poorly understood but involves the aggregation of the protein alpha-synuclein into Lewy bodies within the neurons.” One possible treatment for PD and other diseases related to overexpression of the alpha synuclein (SNCA) gene may be to inhibit the expression of SNCA gene in subjects afflicted with PD using gene editing technology such as CRISPR. Therefore, there is a need for effective treatment for PD based on inhibition of SNCA gene expression using effective CRISPR Cas9 technology.

[0006] SUMMARY OF THE INVENTION

[0007] The present invention provides a composition comprising one or more clustered regularly interspaced short palindromic repeats associated protein (Cas9) or a variant thereof and one or more guide ribonucleic acids (RNAs) wherein each of the one or more guide RNAs is complexed with each of the one or more Cas9 protein or a variant thereof; wherein the nucleotide sequence of the one or more guide RNAs comprises at least 10, 12, 14, 16, 18 or 20 contiguous nucleotides that are complementary to a target polynucleotide sequence; wherein the target polynucleotide sequence comprises at least a part of synuclein alpha (SNCA) gene; and wherein the nucleotide sequence of each of the one or more guide RNAs is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical to SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 or SEQ ID NO. 10.

[0008] The present invention further provides a pharmaceutical composition comprising a nanoparticle encapsulating any embodiment of the guide RNA of the present invention and mRNA encoding any embodiment of the Cas9 protein or a variant thereof of the present invention.

[0009] The present invention also provides method of treatment for disorders related to increased expression or overexpression of the SNCA gene in a subject comprising the step of administration of any embodiment of the composition of the present invention to the subject.

[0010] BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 illustrates targeting sites and associated PAM sequence of three guide RNAs of the present invention.

[0012] Figure 2 illustrates in vitro suppression of alpha-synuclein protein expression in SH- SY5Y cells using Cas9 variant and guide RNAs of the present invention. As illustrated, dCas9 with gRNA G115 provides 50% reduction of a-synuclein in SH-SY5Y cells compared to other gRNA.

[0013] Figure 3 illustrates (A) flow cytometry and (B) western blot of in vitro suppresses of alpha-synuclein protein expression in SH-SY5Y cells using lipid nanoparticles encapsulating dCas9-KRAB with gRNA G115 guide RNAs of the present invention.

[0014] DETAILED DESCRIPTION OF THE INVENTION

[0015] As used in this specification and in claims which follow, the singular forms “a”, “an” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “an ingredient” includes mixtures of ingredients, reference to “an active pharmaceutical agent” includes more than one active pharmaceutical agent, and the like.

[0016] As used herein, the term “about” as a modifier to a quantity is intended to mean + or - 20%, + or - 15%, + or - 10% or + or - 5% inclusive of the quantity being modified.

[0017] As used herein, the term “subject,” “individual” or “patient” is used interchangeably herein, which refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets.

[0018] As used herein, the term “effective amount” or “a therapeutically effective amount” of a drug or pharmacologically active agent comprises administering an amount necessary to achieve a desired result. The exact amount required will vary from subject to subject, depending on the species, age, general condition of the subject, the severity of the disease, the particular anticancer agent, its mode of administration, the desired outcome, and the like. In certain embodiments of the present invention, a “therapeutically effective amount” of a compound or pharmaceutical composition is that amount effective for inhibiting progression or reversing of any disease disclosed herein in a subject or a biological sample (e.g., in cells). In certain embodiments, disease progression is inhibited by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99%. In certain embodiments, the compound inhibits disease progression by at least about 25%, at least about 50%, at least about 75%, or at least about 90%. In certain embodiments of the present invention, a “therapeutically effective amount” refers to an amount of a compound or composition sufficient to inhibit disease progression. In certain embodiments, the disease is reversed by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99% or any numbers and number ranges falling within these values.

[0019] As used herein, the term “nucleic acid”, “polynucleotide” or “oligonucleotide” refers to a DNA molecule, an RNA molecule, or analogs thereof. As used herein, the terms “nucleic acid”, “polynucleotide” and “oligonucleotide” include, but are not limited to DNA molecules such as cDNA, genomic DNA or synthetic DNA and RNA molecules such as a guide RNA, messenger RNA or synthetic RNA. Moreover, as used herein, the terms “nucleic acid” and “polynucleotide” include single-stranded and double-stranded forms.

[0020] As used herein, the term “target polynucleotide” or “target” refers to a polynucleotide comprising a nucleic acid sequence or the complement thereof to be bound to, nicked, or cleaved using a CRISPR system. A target polynucleotide may be single- stranded or doublestranded, and, in certain embodiments, is double-stranded DNA. In certain embodiments, the target polynucleotide is single-stranded RNA.

[0021] The term “modification” in the context of an oligonucleotide or polynucleotide includes but is not limited to (a) end modifications, e.g., 5’ end modifications or 3’ end modifications, (b) nucleobase (or “base”) modifications, including replacement or removal of bases, (c) sugar modifications, including modifications at the 2’, 3’, and / or 4’ positions, and (d) backbone modifications, including modification or replacement of the phosphodiester linkages. The term “modified nucleotide” generally refers to a nucleotide having a modification to the chemical structure of one or more of the base, the sugar, and the phosphodiester linkage or backbone portions, including nucleotide phosphates.

[0022] As used herein, the term “guide RNA” generally refers to an RNA molecule (or a group of RNA molecules collectively) that can bind to a Cas protein and aid in targeting the Cas protein to a specific location within a target polynucleotide (e.g., a DNA). A guide RNA can comprise a crRNA segment and a tracrRNA segment. As used herein, the term “crRNA” or “crRNA segment” refers to an RNA molecule or portion thereof that includes a polynucleotide-targeting guide sequence, a stem sequence, and, optionally, a 5 ’-overhang sequence. As used herein, the term “tracrRNA” or “tracrRNA segment” refers to an RNA molecule or portion thereof that includes a protein-binding segment (e.g., the protein-binding segment is capable of interacting with a CRISPR-associated protein, such as a Cas9). The term “guide RNA” encompasses a single guide RNA (sgRNA), where the crRNA segment and the tracrRNA segment are located in the same RNA molecule. The term “guide RNA” also encompasses, collectively, a group of two or more RNA molecules, where the crRNA segment and the tracrRNA segment are located in separate RNA molecules. The crRNA and tracrRNA can be expressed separately or engineered into an artificial fusion single guide RNA (sgRNA) via a synthetic stem loop to mimic the natural crRNA / tracrRNA duplex.

[0023] The term “scaffold” refers to the portions of guide RNA molecules comprising sequences which are substantially identical or are highly conserved across natural biological species. Scaffolds include the tracrRNA segment and the portion of the crRNA segment other than the polynucleotide-targeting guide sequence at or near the 5’ end of the crRNA segment, excluding any unnatural portions comprising sequences not conserved in native crRNAs and tracrRNAs.

[0024] A synthetic guide RNA that has “gRNA functionality” is one that has one or more of the functions of naturally occurring guide RNA, such as associating with a Cas protein, or a function performed by the guide RNA in association with a Cas protein. In certain embodiments, the functionality includes binding to a target polynucleotide. In certain embodiments, the functionality includes targeting a Cas protein or a gRNA:Cas protein complex to a target polynucleotide. In certain embodiments, the functionality includes nicking a target polynucleotide. In certain embodiments, the functionality includes cleaving a target polynucleotide. In certain embodiments, the functionality includes associating with or binding to a Cas protein. In certain embodiments, the functionality comprises any other known function of a guide RNA in a CRISPR-Cas system with a Cas protein, including an artificial CRISPR-Cas system with an engineered Cas protein. In certain embodiments, the functionality comprises any function of natural guide RNA. The synthetic guide RNA may have gRNA functionality to a greater or lesser extent than a naturally occurring guide RNA. In certain embodiments, a synthetic guide RNA may have greater functionality as to one property and lesser functionality as to another property in comparison to a similar naturally occurring guide RNA.

[0025] The term “CRISPR-associated protein” or “Cas protein” refers to a wild type Cas protein, a fragment thereof, or a mutant or variant thereof. The term “Cas mutant” or “Cas variant” refers to a protein or polypeptide derivative of a wild type Cas protein, e.g., a protein having one or more point mutations, insertions, deletions, truncations, a fusion protein, or a combination thereof. In certain embodiments, the “Cas mutant” or “Cas variant” substantially retains the nuclease activity of the Cas protein. In certain embodiments, the “Cas mutant” or “Cas variant” is mutated such that one or both nuclease domains are inactive. In certain embodiments, the “Cas mutant” or “Cas variant” has nuclease activity. In certain embodiments, the “Cas mutant” or “Cas variant” lacks some or all of the nuclease activity of its wild-type counterpart.

[0026] The term “nuclease domain” of a Cas protein refers to the polypeptide sequence or domain within the protein which possesses the catalytic activity for DNA cleavage. A nuclease domain can be contained in a single polypeptide chain, or cleavage activity can result from the association of two (or more) polypeptides. A single nuclease domain may consist of more than one isolated stretch of amino acids within a given polypeptide. Examples of these domains include RuvC-like motifs (amino acids 7-22, 759-766 and 982- 989 in SEQ ID NO: 1) and HNH motif (aa 837-863). Sec Gasiunas et al. (2012) Proc. Natl. Acad. Sci. USA, 109:39, E2579-E2586 and WO2013176772.

[0027] The term “hybridization” or “hybridizing” refers to a process where completely or partially complementary polynucleotide strands come together under suitable hybridization conditions to form a double- stranded structure or region in which the two constituent strands are joined by hydrogen bonds. As used herein, the term “partial hybridization” includes where the double-stranded structure or region contains one or more bulges or mismatches. Although hydrogen bonds typically form between adenine and thymine or adenine and uracil (A and T or A and U) or cytosine and guanine (C and G), other noncanonical base pairs may form (See e.g., Adams et al., “The Biochemistry of the Nucleic Acids,” 11th ed., 1992). It is contemplated that modified nucleotides may form hydrogen bonds that allow or promote hybridization.

[0028] The term “cleavage” or “cleaving” refers to breaking of the covalent phosphodiester linkage in the ribosylphosphodiester backbone of a polynucleotide. The terms “cleavage” or “cleaving” encompass both single- stranded breaks and double- stranded breaks. Double- stranded cleavage can occur as a result of two distinct single- stranded cleavage events. Cleavage can result in the production of either blunt ends or staggered ends.

[0029] The present invention provides a SNCA targeting composition comprising a Cas9 protein or a variant thereof and one or more guide RNAs wherein the nucleotide sequence of the guide RNA is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the nucleotide sequence of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, or SEQ ID NO. 10. In an embodiment, the one or more guide RNAs comprises a sequence or a 1, 2, 3, 4, or 5 nucleotide 5' truncation fragment thereof of the nucleotide sequences of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, or SEQ ID NO. 10.

[0030] SEQ ID NO. 1: ATTCATGAAAGGACTTTCAA

[0031] SEQ ID NO. 2: AAAGGCCAAGGAGGGAGTTG

[0032] SEQ ID NO. 3: ATGGATGTATTCATGAA

[0033] SEQ ID NO. 5: AGCAGCCACAACTCCCTCCTTGG

[0034] SEQ ID NO. 6: GCTGCTGAGAAAACCAAACAGGG

[0035] SEQ ID NO. 7: TGCTGCTGAGAAAACCAAACAGG SEQ ID NO. 8: GAAAGGACTTTCAAAGGCCAAGG SEQ ID NO. 9: AGGACTTTCAAAGGCCAAGGAGG SEQ ID NO. 10: GGACTTTCAAAGGCCAAGGAGGG

[0036] In an embodiment, each of the one or more guide RNAs of the present invention comprises a CRISPR RNA (crRNA) and a trans-encoded small RNA (tracrRNA), and wherein the crRNA comprises a nucleic acid sequence complementary to a target polynucleotide sequence. In an embodiment, the crRNA and tracrRNA can be expressed separately or engineered into an artificial fusion single guide RNA (sgRNA) via a synthetic stem loop to mimic the natural crRNA / tracrRNA duplex. In an embodiment, each of the one or more guide RNAs of the present invention comprises a single guide RNA comprising a nucleotide sequence that is complementary to a target polynucleotide sequence. In an embodiment, the one or more guide RNAs of the present invention comprises a nucleotide sequence comprising at least 10, 12, 14, 16, 18, 20 contiguous nucleotides that are complementary to the target polynucleotide sequence.

[0037] In an embodiment, the target polynucleotide sequence comprises at least a part of alpha- sy nuclein (SNCA) gene. In an embodiment, the at least a part of SNCA gene comprises at least a part of a sense strand, at least a part of an anti-sense strand, or a combination thereof of the SNCA gene. In an embodiment, the at least a part of SNCA gene comprises at least a part of a 5' UTR, at least a part of a 3' UTR, at least a part of an exon, at least a part of an intron, or a combination thereof of the SNCA gene. In an embodiment, the at least a part of SNCA gene comprises at least a part of an exon of the SNCA gene. In an embodiment, the at least a part of SNCA gene comprises at least a part of exon 2 of the SNCA gene. In an embodiment, the nucleotide sequence of the exon 2 of the SNCA gene is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 4.

[0038] SEQ ID NO. 4:

[0039] GTGTATTTTATGTTTTCCAGTGTGGTGTAAAGGAATTCATTAGCCATGGAT GTATTCATGAAAGGACTTTCAAAGGCCAAGGAGGGAGTTGTGGCTGCTGC TGAGAAAACCAAACAGGGTGTGGCAGAAGCAGCAGGAAAGACAAAAGA GGGTGTTCTCTATGTAGGTAGGTAAACCCCAAATGTCAGTTTGGTGCTTGT TCATGAG

[0040] In an embodiment, the SNCA gene comprises a mammalian SNCA gene. In an embodiment, the SNCA gene comprises a human SNCA gene, a monkey SNCA gene, a mouse SNCA gene, or a rat SNCA gene. In an embodiment, the guide RNA sequence can be a sense or anti-sense sequence. The guide RNA sequence generally includes a proto-spacer adjacent motif (PAM). The sequence of the PAM can vary depending upon the specificity requirements of the CRISPR endonuclease used. In the CRISPR-Cas system derived from S. pyogenes, the target DNA typically immediately precedes a 5’-NGG proto-spacer adjacent motif (PAM). Thus, for the S. pyogenes Cas9, the PAM sequence can be AGG, TGG, CGG or GGG. Other Cas9 orthologs may have different PAM specificities. For example, Cas9 from S. thermophilus requires 5’-NNAGAA for CRISPR 1 and 5’-NGGNG for CRISPR3 and Neiseria menigiditis requires 5’-NNNNGATT. The specific sequence of the guide RNA may vary, but, regardless of the sequence, useful guide RNA sequences will be those that minimize off-target effects while achieving high efficiency and complete ablation of the SNCA gene.

[0041] In an embodiment, the Cas9 protein or a variant thereof of the present invention is in a form comprising a polynucleotide, a polypeptide, or a recombinant protein. In an embodiment, the Cas9 or a variant thereof of the present invention comprises Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1 Cas9 (StlCas9), Steptococcus cants Cas9(ScCas9), or variant thereof. In an embodiment, the Cas9 protein or a variant thereof of the present invention comprises a wildtype Cas9, a high fidelity Cas9 variant, a nuclease inactivated Cas9 variant or a Cas9 nickase variant. In an embodiment, the Cas9 protein or a variant thereof of the present invention comprises a SpCas9 comprising one or more amino acid mutations at DIO, R691, D839, H840, N863. In an embodiment, the Cas9 protein or a variant thereof of the present invention comprises a SpCas9 comprising one or more amino acid mutations at D10A, R691 A, D839A, H840A, N863A, or a combination thereof. In an embodiment, the Cas9 protein or a variant thereof of the present invention comprises a sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% identical to the amino acid sequence of SEQ ID NO 11. In another embodiment, the Cas9 protein or a variant thereof of the present invention comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% similar to the amino acid sequence of SEQ ID NO 11.

[0042] The amino acid sequence of dCas9 of the present invention used in the Examples below is SEQ ID NO. 11 : DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETA EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERH PIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDL NPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGE KKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADL FLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKY KEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTF DNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW MTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVY NELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRN FMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVK VMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDK NRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIK RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKV REINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM PQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVV AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFE LENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

[0043] In an embodiment, the Cas9 protein or a variant thereof of the present invention further comprises a protospacer adjacent motif (PAM) specificity for a target polynucleotide sequence comprising 5’-NGG-3’, 5’-NAG-3’, 5’-NGA-3’, 5’-NAA-3’, 5'-NNAGGA-3', or 5’- NNACCA-3’ wherein N is selected from A, T, C, or G.

[0044] In an embodiment, the composition of the present invention is capable of creating a single strand break of the target polynucleotide sequence. In an embodiment, the composition of the present invention is capable of creating a double strand break of the target polynucleotide sequence. In an embodiment, the double strand break comprises a blunt end double strand break, or a sticky end double strand break. In an embodiment, the composition of the present invention does not alter the nucleotide sequence of its target polynucleotide. In an embodiment, the composition of the present invention does not alter the nucleotide sequence of its target polynucleotide while suppressing the expression of the target polynucleotide sequence. In an embodiment, the composition of the present invention is capable of creating a mutation in the target polynucleotide sequence comprising deletions on the target polynucleotide. In an embodiment, the composition of the present invention is capable of creating a mutation in the target polynucleotide sequence comprising insertions in its target polynucleotide. In an embodiment, the composition of the present invention suppresses the expression of the target polynucleotide sequence in a eukaryotic cell. In an embodiment, the composition of the present invention suppresses the expression of the target polynucleotide sequence in a subject such as a eukaryotic cell.

[0045] In an embodiment, the composition comprising a Cas9 protein or a variant thereof and one or more guide RNAs of the present invention further comprises a transcriptional regulator. In an embodiment, the Cas9 protein or a variant thereof is fused to the transcriptional regulator. In an embodiment, the fusion protein comprising the Cas9 protein or a variant thereof and the transcriptional regulator is in a form comprising a polynucleotide encoding the amino acid sequences of said fusion protein. In an embodiment, the fusion protein further comprises a linker that joins the Cas9 protein or a variant thereof and the transcriptional regulator. In an embodiment, the linker comprises an organic molecule, polymer, or chemical moiety. In an embodiment, the linker comprises a nucleic acid, a polynucleotide, a peptide, a protein, or an aptamer. In an embodiment, the transcriptional regulator comprises a transcriptional repressor. In an embodiment, the transcriptional regulator comprises a Kruppel associated box (KRAB) domain. In an embodiment, the transcriptional regulator enhances the transcriptional repression of the Cas9 protein or a variant thereof on the expression of its target polynucleotide sequence in a cell such as a eukaryotic cell.

[0046] The present invention also provides a composition comprising a nanoparticle encapsulating the mRNA (NP-mRNA) wherein the mRNA encodes any embodiment of the Cas9 protein or a variant thereof and / or one or more guide RNA of the present invention. In an embodiment, the nanoparticle comprises chemical nanoparticles such as but not limited to lipid nanoparticle, polymer nanoparticle, lipid-polymer hybrid nanoparticle or biological nanoparticles such as but not limited to liposome, exosome, virus, virus-like particle. In an embodiment, the biological nanoparticle comprises adeno-associated virus (AAV9) used to encapsulate Cas9 protein or a variant thereof and one or more guide RNA of the present invention. In yet another embodiment, the AAV9 encapsulating Cas9 protein or a variant thereof and one or more guide RNA of the present invention is administered via systemic injection, intracerebral injection, etc.

[0047] In an embodiment, the nanoparticle of the NP-mRNA of the present invention further comprises one or more conjugates for targeted delivery to a targeted site in a subject. In an embodiment, the target site for the targeted delivery comprises organs with cells that express the target polynucleotide. In an embodiment, the target site for the targeted delivery comprises cells that express the target polynucleotide. In an embodiment, the subject is diagnosed with a disease associated with increased SNCA gene expression comprising Parkinson’s disease (PD), PD dementia, dementia with Lewy bodies (DLB), or multiple system atrophy (MSA) including parkinsonian variant (MSA-P) and cerebellar variant (MSA- C). In an embodiment, the target site comprises a brain. In an embodiment, the target site comprises cells such as neuroblastoma-derived dopaminergic neuronal cells. In an embodiment, the nanoparticle further comprises a conjugate that facilitates the nanoparticle in crossing the blood brain barrier. In an embodiment, the nanoparticle comprises a conjugate comprising levodopa or a derivative of the levodopa that facilitates the nanoparticle in crossing the blood brain barrier.

[0048] In an embodiment, the composition comprising mRNA encoding the Cas9 protein or a variant thereof of the invention and one or more guide RNAs are encapsulated inside the nanoparticle. In an embodiment, more than about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% of the mRNA encoding Cas9 protein or a variant thereof and one or more guide RNAs are released from the nanoparticle at the target site. In an embodiment, more than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% of the mRNA encoding Cas9 protein or a variant thereof and the one or more guide RNAs are released from the nanoparticle within the cells that express the target polynucleotide. In an embodiment, less than about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% of the mRNA encoding the Cas9 protein or a variant thereof and one or more guide RNAs are released from the nanoparticle before reaching the target site.

[0049] The present invention further provides a method of treatment of any disease related to or caused by increased expression or overexpression of the SNCA gene including but not limited to Parkinson's disease (PD), PD dementia, dementia with Lewy bodies (DLB), or multiple system atrophy (MSA), including the parkinsonian variant (MSA-P) and cerebellar variant (MSA-C). In an embodiment, the method of treatment comprises the step of administration of any embodiment of the composition of the present invention to a subject. In an embodiment, the method of treatment comprises the step of administration of any embodiment of the NP-mRNA composition of the present invention to a subject. The present invention further provides a method of inhibiting expression of the SNCA gene comprising the step of administration of any embodiment of the SNCA targeting composition of the present invention to a subject. The present invention further provides a method of inhibiting expression of the SNCA gene comprising the step of administration of any embodiment of the NP-mRNA composition of the present invention to a subject. The present invention further provides a method of inhibiting expression of the SNCA gene comprising the step of administration of induced pluripotent stem cell (iPSCs) to a subject wherein the iPSCs are treated with any embodiment of the SNCA targeting composition of the present invention or any embodiment of the NP-mRNA composition of the present invention to a subject.

[0050] In an embodiment, the administration of any embodiment of the NP-mRNA of the present invention to a subject results in the expression of the subject’s targeted cell of an embodiment of the Cas9 protein or a variant thereof of the present invention. In an embodiment, the administration of any embodiment of the NP-mRNA of the present invention further results in the complex of the guide RNA of the present invention with the expressed Cas9 protein or a variant thereof of the present invention within the subject’s targeted cell. In an embodiment, more than about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the mRNA encoding Cas9 protein or a variant thereof are expressed in the subject’s targeted cell. In an embodiment, more than about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the expressed Cas9 protein or a variant thereof of the present invention complexes with the guide RNA of the present invention within a subject’s cell.

[0051] In an embodiment, the step of administration may comprise parenteral administration. For parenteral administration, any embodiment of the composition of the present invention may be administered by intravenous, subcutaneous, intramuscular, intraperitoneal, or intradermal injection, alone or in compositions further comprising pharmaceutically accepted carriers. For administration by injection, it is preferred to use any embodiment of the NP- mRNA of the present invention in a solution in a sterile aqueous vehicle which may also contain other solutes such as buffers or preservatives as well as sufficient quantities of pharmaceutically acceptable salts or of glucose to make the solution isotonic.

[0052] Pharmaceutical compositions suitable for an injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include but are not limited to physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany. N.J.) or phosphate buffered saline (PBS). In all cases, the composition should be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0053] In an embodiment, the method of treatment of the present invention results in contact between at least a part of a genome of a subject with the Cas9 protein or a variant thereof of the present invention complexed with the one or more guide RNAs of the present invention. In an embodiment, the guiding of the Cas9 protein or a variant thereof complexed with the one or more guide RNAs to the target site on the genome of a subject comprises the interaction between the one or more guide RNAs and the target polynucleotides. In an embodiment, the interaction between the one or more guide RNAs and their target polynucleotides comprises at least 10, 12, 14, 16, 18, 20 contiguous base pairing. In an embodiment, the interaction between the one or more guide RNAs complexed with the Cas9 protein or a variant thereof of the present invention with the target polynucleotide results in inhibition of expression of the gene comprising the target polynucleotide.

[0054] In an embodiment, the target polynucleotide sequence comprises at least a part of SNCA gene. In an embodiment, the at least a part of SNCA gene comprises at least a part of a sense strand, at least a part of an anti-sense strand, or a combination thereof of the SNCA gene. In an embodiment, the at least a part of SNCA gene comprises at least a part of a 5' UTR, at least a part of a 3' UTR, at least a part of an exon, at least a part of an intron, or a combination thereof of the SNCA gene. In an embodiment, the at least a part of SNCA gene comprises at least a part of an exon of the SNCA gene. In an embodiment, the at least a part of SNCA gene comprises at least a part of exon 2 of the SNCA gene. In an embodiment, the SNCA gene comprises a mammalian SNCA gene. In an embodiment, the SNCA gene comprises a human SNCA gene, a monkey SNCA gene, a mouse SNCA gene, or a rat SNCA gene.

[0055] In an embodiment, the guiding of the Cas9 protein or a variant thereof and the one or more guide RNAs to the target site on the genome of a subject further comprises the recognition of PAM sequence on the genome of a subject by the Cas9 protein or a variant thereof of the present invention. In an embodiment, the PAM sequence on the genome of a subject comprises a nucleotide sequence comprising 5'-NGG-3', 5'-NAG-3', 5'-NGA-3', 5'- NAA-3', 5'-NNAGGA-3', or 5'-NNACCA-3' wherein N is selected from A, T, C, or G.

[0056] In an embodiment, the method of treatment of the present invention results in the modification on the target site on the at least a part of the genome of a subject by the Cas9 protein or a variant thereof comprising creating a single strand break, a double strand break, or a combination thereof of the target polynucleotide sequence by the Cas9 protein or a variant thereof. In an embodiment, the double strand break comprises a blunt end double strand break, a sticky end double strand break, or a combination thereof. In an embodiment, the Cas9 protein or a variant thereof does not alter the nucleotide sequence of its target polynucleotide. In an embodiment, the Cas9 protein or a variant thereof creates a mutation comprising deletions, insertions, or a combination thereof on its target polynucleotide.

[0057] In an embodiment, the modification on the target site on the at least a part of the genome of a subject by the Cas9 protein or a variant thereof comprises modification of the nucleotide sequence of at least a part of the SNCA gene on the genome of a subject, modification of the expression level of the SNCA gene on the genome of a subject, or a combination thereof. In an embodiment, the at least a part of the SNCA gene comprises at least a part of a sense strand, at least a part of an anti-sense strand, or a combination thereof of the SNCA gene. In an embodiment, the at least a part of SNCA gene comprises at least a part of a 5' UTR, at least a part of a 3' UTR, at least a part of an exon, at least a part of an intron, or a combination thereof of the SNCA gene. In an embodiment, the at least a part of SNCA gene comprises at least a part of an exon of the SNCA gene. In an embodiment, the at least a part of SNCA gene comprises at least a part of exon 2 of the SNCA gene. In an embodiment, the SNCA gene comprises a mammalian SNCA gene. In an embodiment, the SNCA gene comprises a human SNCA gene, a monkey SNCA gene, a mouse SNCA gene, or a rat SNCA gene. In an embodiment, the modification of SNCA gene expression level of a subject comprises suppressing the expression of a SNCA mRNA isoform, a SNCA protein isoform, a non-coding fragment of the SNCA gene, or a combination thereof.

[0058] In an embodiment, the modification on the target site on the at least a part of the genome of a subject by the Cas9 protein or a variant thereof of the present invention further comprises a transcriptional regulation using a transcriptional regulator. In an embodiment, the transcriptional regulator comprises a transcriptional repressor. In an embodiment, the transcriptional regulator comprises a Kriippel associated box (KRAB) domain. In an embodiment, the transcriptional regulator enhances the transcriptional repression of the Cas9 protein or a variant thereof on the expression of its target polynucleotide sequence in a subject such as a eukaryotic cell.

[0059] In an embodiment, the method of treatment of the present invention to inhibit the expression of SNCA gene in a subject comprises administering to a subject in need any composition of the present invention. In an embodiment, the method of treatment of the present invention results in about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% inhibition of the expression of the SNCA gene in the subject. In an embodiment, the subject is diagnosed with a disease associated with increased SNCA gene expression comprising Parkinson's disease (PD), PD dementia, dementia with Lewy bodies (DLB), or multiple system atrophy (MSA) including the parkinsonian variant (MSA-P) and cerebellar variant (MSA-C). In an embodiment, the administration of the various compositions of the present invention to a cell of a subject to inhibits the expression of SNCA gene in the subject is for treating a subject diagnosed with a disease associated with increased SNCA gene expression comprising Parkinson’s disease (PD), PD dementia, dementia with Lewy bodies (DLB), or multiple system atrophy (MSA) including the parkinsonian variant (MSA-P) and cerebellar variant (MSA-C). In an embodiment, the expression of SNCA gene comprises expression of a SNCA mRNA isoform, expression of a SNCA protein isoform, expression of a non-coding fragment of the SNCA gene, or a combination thereof.

[0060] It is to be understood that both the foregoing general description and detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. In general, the terms used in the disclosure should not be construed to limit the technology to the specific embodiments disclosed in the specification, unless the above detailed description explicitly defines such terms. Accordingly, the actual scope of the technology encompasses the disclosed embodiments and all equivalent ways of practicing or implementing the technology.

[0061] EXAMPLES

[0062] Example 1 Preparation of gRNA

[0063] Guide RNA (gRNA) of the present invention were designed using a website (crisprscan.org) to target exon 2 of the SNCA gene, and an expression vector for the single-guide (sg) RNA was constructed with the following oligos: gRNA 2: 5 ' - CACCGGCAGGTGCTGTCCCGGCTGG-3 " (forward) ' , 5 ' - AAACCCAGCCGGGACAGCACCTGCC-3 " (reverse). For the cloning of sgRNA, 13 pL of forward and reverse gRNA oligos at an initial concentration of 100 pM were mixed. The gRNA oligos were annealed at 60°C for 1 min, followed by cooling to room temperature to make a double- stranded insertion. Single guide RNA (sgRNA) was cloned into CRISPRi SpCas9 (DIOA)-NGG plasmid (a gift from Academia Sinica). The restriction digestion reaction contained 1 pg plasmid, 1 pL BbsI (NEB, cat.# R0539S, Ipswich, MA, USA), 5 pL lOx reaction buffer (NEB, cat.# B7203S, Ipswich, MA, USA) and 43 pL ddH2O and was incubated at 37 °C for 2-3 h. Fifty microliters of vector was added to 150 pL ddH2O and 200 pL phenol (Sigma, cat.# P1037, St. Louis, MO, USA), followed by centrifugation at 15,000 rpm for 3 min at room temperature to remove the restriction enzymes. Isopropanol precipitation was then performed, where 200 pL vector, 20 pL 3 M sodium acetate, and 200 pL isopropanol were mixed followed by centrifugation at 15,000 rpm for 5 min at 4°C. Vectors were cleaned using 70% ethanol and subsequently dissolved in 12.5 pL ddlUC). The dephosphorylation reaction of the vector consisted of 1.5 pL lOx Antarctic phosphatase reaction buffer (NEB, cat.# B0289S, Ipswich, MA, USA), 1 pL Antarctic phosphatase (NEB, cat.# M0289S, Ipswich, MA, USA) and 12.5 pL vectors and was incubated at 37°C for 1 h followed by 70°C for 10 min for heat inactivation. Ligation was performed using 1 pL lOx reaction buffer (NEB, cat.# B0202S, Ipswich, MA, USA), 1 pL lOx PEG, 0.5 pL T4 DNA ligase (NEB, cat.# M0202S, Ipswich, MA, USA), 1.5 pL vector, 1 pL ddfUO and 5 pL insert oligos, which was incubated at 16°C for 1 h. Transformation of competent E. coli cells (strain JM109) with the ligation reaction was performed by the conventional calcium method. Three or more clones were picked for sequencing. The sequencing primer used was the human U6 promoter forward primer: 5 ' -GACTATCATATGCTTACCGT-3 ' . A QIAGEN Maxprep kit (REF. 12362, Hilden, Germany) was used to isolate plasmids from 200 mL of bacteria following the manufacturer’s instructions.

[0064] Example 2 In vitro Reduction of a-Synuclein Expression using CRISPRi

[0065] Culture 7xl05SH-SY65 cells in each well of a 6-well plate. The 6 wells comprise two control wells, two vector only wells and two puromycin wells. In each type of well, add 7|ig of either HiFi-Cas-9NGG or dCas9-NGG using 100 pL Neon™ Tip. Subsequently 1 .5pg of puromycin per mL is applied for 2 days followed by 0.75pg of puromycin per mL for maintaining the dCas9 groups.

[0066] Results are shown in FIG. 2. As illustrated in FIG. 2, both the G1602 and vector groups resulted in about 80% reduction a-synuclein in SH-SY5Y cells compared to the NT group while G15014 group did not differ from the NT group. However, the G115 group provided about 50% reduction a-synuclein in SH-SY5Y cells compared to the NT group.

[0067] Example 3 Preparation of LNP encapsulating mRNA Coding Cas9 and gRNA

[0068] LNP encapsulating mRNA coding the dCas9-KRAB protein (4980 nt) and G115 gRNA (107 nt) (LNP-mRNA) are prepared by first preparing the dCas9-KRAB mRNA and the guide RNA. The organic phase for preparing the LNP-mRNA of the present invention comprises ionizable lipid dissolved in 95% EtOH. The aqueous phase for preparing the LNP- mRNA of the present invention comprises the mRNA of the present invention in NaCl. The organic and aqueous phases are then injected into a microfluidic device. Dialysis is then performed on the resulting solution to form and isolate the LNP-mRNA of the present invention. The resulting LNP-mRNA characteristics such as particle size, PDI and charged are measured by a particle analyzer, which are shown below:

[0069] Table 1 nanoparticle characteristics

[0070] Example 4 In vitro Reduction of a-Synuclein Expression using LNP-mRNA Encapsulating CRISPRi spCas9 (DIOA)-NGG Plasmid with G115 gRNA.

[0071] G115 SH-SY5Y cells were treated with LNP encapsulated with mRNA of CRISPRi spCas9 (DIOA)-NGG with G115 gRNA of Example 3. SH-SY5Y cells were grown to confluence in tissue culture flasks in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 4500 mg / L D-glucose, 10 mg / mL penicillin / streptomycin and 10% iron- fortified new-born calf serum (Cosmic Calf Serum, Fisher, UK). Medium was replaced with fresh medium every 2 d. The cells were expanded twice followed by seeding in 24- well plates at a density of 80,000 cells per well. 24 h post seeding, the media was replaced with fresh media and incubated for an additional 24 h. Then the next day, SH-SY5Y cells were treated with the indicated concentrations of LNP containing dCas9-KRAB mRNA and G115 sgRNA for 24 hours, followed by flow cytometry analysis. For expression levels of a-synuclein, wild type SH-SY5Y cells were treated with the indicated concentrations of LNP containing dCas9- KRAB mRNA and G115 sgRNA for 24 hours. The cells were harvested and subjected to western blotting analysis.

[0072] Flow cytometry of FIG. 3A and Western Blot of FIG. 3B confirm that LNP-mRNA reduced amount of cellular a-synuclein.

[0073] It can be appreciated by those skilled in the art that changes could be made to the examples described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular examples disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the appended claims.

Claims

What is claimed is:

1. An alpha synuclein (SNCA) targeting composition comprising one or more clustered regularly interspaced short palindromic repeats associated protein 9 (Cas9) or a variant thereof and one or more guide ribonucleic acids (RNAs) wherein each of the one or more guide RNAs is complexed with each of the one or more Cas9 protein or a variant thereof; wherein the nucleotide sequence of the one or more guide RNAs comprises at least10, 12, 14, 16, 18 or 20 contiguous nucleotides that are complementary to a target polynucleotide sequence; wherein the target polynucleotide sequence comprises at least a part of SNCA gene; and wherein the nucleotide sequence of each of the one or more guide RNAs is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical to SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO.7, SEQ ID NO. 8, SEQ ID NO. 9 or SEQ ID NO. 10.

2. The composition of claim 1 , wherein the nucleotide sequence of each of the one or more guide RNAs is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical to SEQ ID NO. 1.

3. The composition of claim 1 , wherein the nucleotide sequence of each of the one or more guide RNAs is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical to SEQ ID NO. 2.

4. The composition of claim 1 , wherein the nucleotide sequence of each of the one or more guide RNAs is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical to SEQ ID NO. 3.

5. The composition of claim 1 , wherein the at least a part of SNCA gene comprises at least a part of a sense strand, at least a part of an anti-sense strand, or a combination thereof of the SNCA gene.

6. The composition of claim 1 , wherein the one or more Cas9 or a variant thereof comprises Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1 Cas9 (StlCas9), Streptococcus cants Cas9(ScCas9), or variants thereof.

7. The composition of claim 1 , wherein the one or more Cas9 protein or a variant thereof comprises a wildtype Cas9, a high fidelity Cas9 variant, a nuclease inactivated Cas9 variant or a Cas9 nickase variant.

8. The composition of claim 1 , wherein the one or more Cas9 protein or a variant thereof comprises a SpCas9 comprising one or more amino acid mutations at DIO, R691, D839, H840, N863.

9. The composition of claim 8, wherein the SpCas9 comprises mutations D10A, R691A, D839A, H840A, N863A, or a combination thereof.

10. The composition of claim 1, wherein the target polynucleotide sequence is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% identical to the nucleotide sequence of SEQ ID NO. 4.

11. The composition of claim 1 , wherein the Cas9 protein or a variant thereof comprises a protospacer adjacent motif (PAM) specificity for target polynucleotide sequence comprising 5'-NGG-3’, 5’-NAG-3', 5'-NGA-3', 5'-NAA-3', 5'-NNAGGA-3', or 5'- NNACCA-3’ wherein N is selected from A, T, C, or G.

12. The composition of claim 1, wherein the one or more Cas9 protein or a variant thereof is capable of creating a single strand break, a double strand break, or a combination in the target polynucleotide sequence.

13. The composition of claim 12, wherein the double strand break comprises a blunt end double strand break, a sticky end double strand break, or a combination thereof.

14. The composition of claim 1, wherein the one or more Cas9 protein or a variant thereof does not alter the nucleotide sequence of the target polynucleotide.

15. The composition of claim 1, wherein the one or more Cas9 protein or a variant thereof is capable of deleting one or more nucleotides from the target polynucleotide or inserting one or more nucleotides into the target polynucleotide.

16. The composition of claim 1, wherein the one or more Cas9 protein or a variant thereof is capable of suppressing expression of the target polynucleotide sequence in a subject.

17. The composition of claim 1 further comprises a transcriptional regulator, wherein the one of the one or more Cas9 protein or a variant thereof is fused to the transcriptional regulator.

18. The composition of claim 17, further comprising a linker that joins the one of the one or more Cas9 protein or a variant thereof and the transcriptional regulator.

19. The composition of claim 18, wherein the linker comprises an organic molecule, a polymer, or a chemical moiety.

20. The composition of claim 18, wherein the linker comprises a nucleic acid, a polynucleotide, a peptide, a protein, or an aptamer.

21. The composition of claim 17, wherein the transcriptional regulator comprises a transcriptional repressor.

22. The composition of claim 17, wherein the transcriptional regulator comprises a Kriippel associated box (KRAB) domain.

23. The composition of claim 17, wherein the transcriptional regulator enhances the transcriptional repression of the one or more Cas9 protein or a variant thereof on the expression of its target polynucleotide sequence in a cell such as a eukaryotic cell.

24. A method of treatment for disorders related to increased expression or overexpression of the SNCA gene in a subject comprising the step of administration of the composition of claim 1 to the subject.

25. The method of claim 24, wherein the disorders comprise Parkinson's disease (PD), PD dementia, dementia with Lewy bodies (DLB), multiple system atrophy (MSA), multiple system atrophy parkinsonian variant (MSA-P) or multiple system atrophy cerebellar variant (MSA-C).

26. The method of claim 24, wherein the administration step comprises parenteral administration comprising intravenous, subcutaneous, intramuscular, intraperitoneal, or intradermal injection, alone or in combination with pharmaceutically accepted carriers.

27. The method of claim 24, wherein the method inhibits the expression of the SNCA gene.

28. The method of claim 24, wherein the one or more Cas9 protein or a variant thereof comprises a wildtype Cas9, a high fidelity Cas9 variant, a nuclease inactivated Cas9 variant or a Cas9 nickase variant.

29. The method of claim 24, wherein the one or more Cas9 protein or a variant thereof comprises SpCas9 comprising one or more amino acid mutations at DIO, R691, D839, H840, N863.

30. The method of claim 24, wherein the SpCas9 comprises mutations D10A, R691A, D839A, H840A, N863A, or a combination thereof.

31. The method of claim 24, wherein the amino acid sequence of the one or more Cas9 protein or a variant thereof is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% identical or similar to the amino acid sequence of SEQ ID NO.i l.

32. The method of claim 24, wherein the one or more Cas9 protein or a variant thereof has a protospacer adjacent motif (PAM) specificity for a target polynucleotidesequence comprising 5’-NGG-3', 5’-NAG-3’, 5’-NGA-3’, 5’-NAA-3’, 5'-NNAGGA-3', or 5'-NNACCA-3' wherein N is selected from A, T, C, or G.

33. The method of claim 24, wherein the one or more Cas9 protein or a variant thereof creates a single strand break, a double strand break, or a combination thereof of the target polynucleotide sequence.

34. The method of claim 33, wherein the double strand break comprises a blunt end double strand break, a sticky end double strand break, or a combination thereof.

35. The method of claim 24, wherein the one or more Cas9 protein or a variant thereof does not alter the nucleotide sequence of its target polynucleotide.

36. The method of claim 24, wherein the one or more Cas9 protein or a variant thereof is capable of deleting one or more nucleotides from the target polynucleotide or inserting one or more nucleotides into the target polynucleotide.

37. The method of claim 24, wherein the target polynucleotide sequence comprises at least a part of the SNCA gene.

38. The method of claim 37, wherein the at least a part of the SNCA gene comprises at least a part of a sense strand, at least a part of an anti-sense strand, or a combination thereof of the SNCA gene.

39. The method of claim 37, wherein the at least a part of the SNCA gene comprises at least a part of a 5' UTR, at least a part of a 3' UTR, at least a part of an exon, at least a part of an intron, or a combination thereof of the SNCA gene.

40. The method of claim 37, wherein the at least a part of SNCA gene comprises at least a part of an exon of the SNCA gene.

41. The method of claim 37, wherein the at least a part of SNCA gene comprises at least a part of exon 2 of the SNCA gene.

42. The method of claim 37, wherein the target polynucleotide sequence is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% identical to the nucleotide sequence of SEQ ID NO. 4.

43. A pharmaceutical composition comprising a nanoparticle encapsulating the one or more guide RNA of claim 1 and mRNA encoding the Cas9 protein or a variant thereof of claim 1.

44. The pharmaceutical composition of claim 43, wherein the amino acid sequence of the Cas9 protein or a variant thereof is about 80%, 90% or 100% identical or similar to SEQ ID NO. 11.

45. The pharmaceutical composition of claim 43, wherein the nanoparticle comprises lipid nanoparticle, polymer nanoparticle, lipid-polymer hybrid nanoparticle, liposome, exosome, virus or virus-like particle.

46. The pharmaceutical composition of claim 43, wherein the nanoparticle further comprises a conjugate that targets cells that expresses the SNCA gene.

47. The pharmaceutical composition of claim 43, wherein the nanoparticle further comprises a conjugate that targets brain cells.

48. The pharmaceutical composition of claim 43, wherein the nanoparticle further comprises a conjugate capable of facilitating the nanoparticle in crossing the blood brain barrier.

49. The pharmaceutical composition of claim 48, wherein the conjugate comprises levodopa or a derivative thereof.

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