EP3952923A1 - Thérapies géniques pour troubles lysosomaux - Google Patents

Thérapies géniques pour troubles lysosomaux

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Publication number
EP3952923A1
EP3952923A1 EP20787456.1A EP20787456A EP3952923A1 EP 3952923 A1 EP3952923 A1 EP 3952923A1 EP 20787456 A EP20787456 A EP 20787456A EP 3952923 A1 EP3952923 A1 EP 3952923A1
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
raav
gene
protein
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP20787456.1A
Other languages
German (de)
English (en)
Other versions
EP3952923A4 (fr
Inventor
Asa Abeliovich
Laura Heckman
Herve Rhinn
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Prevail Therapeutics Inc
Original Assignee
Prevail Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Prevail Therapeutics Inc filed Critical Prevail Therapeutics Inc
Priority claimed from PCT/US2020/027658 external-priority patent/WO2020210615A1/fr
Publication of EP3952923A1 publication Critical patent/EP3952923A1/fr
Publication of EP3952923A4 publication Critical patent/EP3952923A4/fr
Pending legal-status Critical Current

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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
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    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
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    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01045—Glucosylceramidase (3.2.1.45), i.e. beta-glucocerebrosidase
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/20—Animals treated with compounds which are neither proteins nor nucleic acids
    • A—HUMAN NECESSITIES
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    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • A01K2267/0318—Animal model for neurodegenerative disease, e.g. non- Alzheimer's
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2750/14011—Parvoviridae
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    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • Gaucher disease is a rare inborn error of glycosphingolipid metabolism due to deficiency of lysosomal acid b-glucocerebrosidase (Gcase,“GBA”).
  • Gcase,“GBA” lysosomal acid b-glucocerebrosidase
  • Patients suffer from non- CNS symptoms and findings including hepatosplenomegly, bone marrow insufficiency leading to pancytopenia, lung disorders and fibrosis, and bone defects.
  • a significant number of patients suffer from neurological manifestations, including defective saccadic eye movements and gaze, seizures, cognitive deficits, developmental delay, and movement disorders including Parkinson’s disease.
  • the present disclosure relates, in part, to compositions and methods for treating certain central nervous system (CNS) diseases, for example neurodegenerative diseases (e.g., neurodegenerative diseases listed in Table 2), synucleinopathies (e.g., synucleinopathies listed in Table 3), tauopathies (tauopathies listed in Table 4), or lysosomal storage diseases (e.g., lysosomal storage diseases listed in Table 5).
  • CNS central nervous system
  • Gaucher disease patients who possess mutations in both chromosomal alleles of GBA1 gene
  • patients with mutations in only one allele of GBA1 are at highly increased risk of Parkinson’s disease (PD).
  • PD Parkinson’s disease
  • Gaucher disease patients have the most severe course, whereas patients with a single mild mutation in GBA1 typically have a more benign course.
  • Mutation carriers are also at high risk of other PD-related disorders, including Lewy Body Dementia, characterized by executive dysfunction, psychosis, and a PD- like movement disorder, and multi-system atrophy, with characteristic motor and cognitive impairments. No therapies exist that alter the inexorable course of these disorders.
  • Gcase e.g., the gene product of GBA1 gene
  • LIMP Lysosomal Membrane Protein 1
  • SCARB2 Lysosomal Membrane Protein 1
  • Gaucher disease e.g., neuronopathic Gaucher disease, such as Type 2 Gaucher disease or Type 3 Gaucher disease.
  • the disclosure is based, in part, on expression constructs (e.g., vectors) encoding one or more genes, for example Gcase, GBA2, prosaposin, progranulin, LIMP2, GALC, CTSB, SMPD1, GCH1, RAB7, VPS35, IL-34, TREM2, TMEM106B, or a combination of any of the foregoing (or portions thereof), associated with central nervous system (CNS) diseases, for example Gaucher disease, PD, etc.
  • CNS central nervous system
  • combinations of gene products described herein act together (e.g., synergistically) to reduce one or more signs and symptoms of a CNS disease when expressed in a subject.
  • the disclosure provides an isolated nucleic acid comprising an expression construct encoding a Gcase (e.g., the gene product of GBA1 gene).
  • the isolated nucleic acid comprises a Gcase-encoding sequence that has been codon optimized (e.g., codon optimized for expression in mammalian cells, for example human cells).
  • the nucleic acid sequence encoding the Gcase encodes a protein comprising an amino acid sequence as set forth in SEQ ID NO: 14 (e.g., as set forth in NCBI Reference Sequence NP_000148.2).
  • the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 15.
  • the expression construct comprises adeno-associated virus (AAV) inverted terminal repeats (ITRs), for example AAV ITRs flanking the nucleic acid sequence encoding the Gcase protein.
  • AAV adeno-associated virus
  • ITRs inverted terminal repeats
  • the disclosure provides an isolated nucleic acid comprising an expression construct encoding Prosaposin (e.g ., the gene product of PSAP gene).
  • the isolated nucleic acid comprises a prosaposin-encoding sequence that has been codon optimized (e.g., codon optimized for expression in mammalian cells, for example human cells).
  • the nucleic acid sequence encoding the prosaposin encodes a protein comprising an amino acid sequence as set forth in SEQ ID NO: 16 (e.g., as set forth in NCBI Reference Sequence NP_002769.1).
  • the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 17.
  • the expression construct comprises adeno-associated virus (AAV) inverted terminal repeats (ITRs), for example AAV ITRs flanking the nucleic acid sequence encoding the prosaposin protein.
  • AAV adeno-associated virus
  • ITRs inverted terminal repeats
  • the disclosure provides an isolated nucleic acid comprising an expression construct encoding LIMP2/SCARB2 (e.g., the gene product of SCARB2 gene).
  • the isolated nucleic acid comprises a SCARB2-encoding sequence that has been codon optimized (e.g., codon optimized for expression in mammalian cells, for example human cells).
  • the nucleic acid sequence encoding the LIMP2/SCARB2 encodes a protein comprising an amino acid sequence as set forth in SEQ ID NO: 18 (e.g., as set forth in NCBI Reference Sequence NP_005497.1).
  • the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 19.
  • the expression construct comprises adeno-associated virus (AAV) inverted terminal repeats (ITRs), for example AAV ITRs flanking the nucleic acid sequence encoding the SCARB2 protein.
  • AAV adeno-associated virus
  • ITRs inverted terminal repeats
  • the disclosure provides an isolated nucleic acid comprising an expression construct encoding GBA2 protein (e.g., the gene product of GBA2 gene).
  • the isolated nucleic acid comprises a GBA2-encoding sequence that has been codon optimized (e.g., codon optimized for expression in mammalian cells, for example human cells).
  • the nucleic acid sequence encoding the GBA2 encodes a protein comprising an amino acid sequence as set forth in SEQ ID NO: 30 (e.g., as set forth in NCBI Reference Sequence NP_065995.1).
  • the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 31.
  • the expression construct comprises adeno-associated vims (AAV) inverted terminal repeats (ITRs), for example AAV
  • the disclosure provides an isolated nucleic acid comprising an expression construct encoding GALC protein (e.g ., the gene product of GALC gene).
  • the isolated nucleic acid comprises a GALC-encoding sequence that has been codon optimized (e.g., codon optimized for expression in mammalian cells, for example human cells).
  • the nucleic acid sequence encoding the GALC encodes a protein comprising an amino acid sequence as set forth in SEQ ID NO: 33 (e.g., as set forth in NCBI Reference Sequence NP_000144.2).
  • the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 34.
  • the expression construct comprises adeno-associated vims (AAV) inverted terminal repeats (ITRs), for example AAV ITRs flanking the nucleic acid sequence encoding the GALC protein.
  • AAV adeno-associated vims
  • ITRs inverted terminal repeats
  • the disclosure provides an isolated nucleic acid comprising an expression construct encoding CTSB protein (e.g., the gene product of CTSB gene).
  • the isolated nucleic acid comprises a CTSB-encoding sequence that has been codon optimized (e.g., codon optimized for expression in mammalian cells, for example human cells).
  • the nucleic acid sequence encoding the CTSB encodes a protein comprising an amino acid sequence as set forth in SEQ ID NO: 35 (e.g., as set forth in NCBI Reference Sequence NP_001899.1).
  • the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 36.
  • the expression construct comprises adeno-associated vims (AAV) inverted terminal repeats (ITRs), for example AAV ITRs flanking the nucleic acid sequence encoding the CTSB protein.
  • AAV adeno-associated vims
  • ITRs inverted terminal repeats
  • the disclosure provides an isolated nucleic acid comprising an expression construct encoding SMPD1 protein (e.g., the gene product of SMPD1 gene).
  • the isolated nucleic acid comprises a SMPD1 -encoding sequence that has been codon optimized (e.g., codon optimized for expression in mammalian cells, for example human cells).
  • the nucleic acid sequence encoding the SMPD1 encodes a protein comprising an amino acid sequence as set forth in SEQ ID NO: 37 (e.g., as set forth in NCBI Reference Sequence NP_000534.3).
  • the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 38.
  • the expression construct comprises adeno-associated vims (AAV) inverted terminal repeats (ITRs), for example AAV ITRs flanking the nucleic acid sequence encoding the SMPD1 protein.
  • AAV adeno-associated vims
  • ITRs inverted terminal repeats
  • the disclosure provides an isolated nucleic acid comprising an expression construct encoding GCH1 protein (e.g., the gene product of GCH1 gene).
  • the isolated nucleic acid comprises a GCH1 -encoding sequence that has been codon optimized (e.g., codon optimized for expression in mammalian cells, for example human cells).
  • the nucleic acid sequence encoding the GCH1 encodes a protein comprising an amino acid sequence as set forth in SEQ ID NO: 45 (e.g., as set forth in NCBI Reference Sequence NP_000534.3).
  • the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 46.
  • the expression construct comprises adeno-associated vims (AAV) inverted terminal repeats (ITRs), for example AAV ITRs flanking the nucleic acid sequence encoding the GCH1 protein.
  • AAV adeno-associated vims
  • ITRs inverted terminal repeats
  • the disclosure provides an isolated nucleic acid comprising an expression construct encoding RAB7L protein (e.g., the gene product of RAB7L gene).
  • the isolated nucleic acid comprises a RAB7L-encoding sequence that has been codon optimized (e.g., codon optimized for expression in mammalian cells, for example human cells).
  • the nucleic acid sequence encoding the RAB7L encodes a protein comprising an amino acid sequence as set forth in SEQ ID NO: 47 (e.g., as set forth in NCBI Reference Sequence NP_003920.1).
  • the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 48.
  • the expression construct comprises adeno-associated vims (AAV) inverted terminal repeats (ITRs), for example AAV ITRs flanking the nucleic acid sequence encoding the RAB7L protein.
  • AAV adeno-associated vims
  • ITRs inverted terminal repeats
  • the disclosure provides an isolated nucleic acid comprising an expression construct encoding VPS35 protein (e.g., the gene product of VPS35 gene).
  • the isolated nucleic acid comprises a VPS35-encoding sequence that has been codon optimized (e.g., codon optimized for expression in mammalian cells, for example human cells).
  • the nucleic acid sequence encoding the VPS35 encodes a protein comprising an amino acid sequence as set forth in SEQ ID NO: 49 (e.g., as set forth in NCBI Reference Sequence NP_060676.2).
  • the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 50.
  • the expression construct comprises adeno-associated vims (AAV) inverted terminal repeats (ITRs), for example AAV ITRs flanking the nucleic acid sequence encoding the VPS35 protein.
  • AAV adeno-associated vims
  • ITRs inverted terminal repeats
  • the disclosure provides an isolated nucleic acid comprising an expression construct encoding IL-34 protein (e.g., the gene product of IL34 gene).
  • the isolated nucleic acid comprises a IL-34-encoding sequence that has been codon optimized (e.g., codon optimized for expression in mammalian cells, for example human cells).
  • the nucleic acid sequence encoding the IL-34 encodes a protein comprising an amino acid sequence as set forth in SEQ ID NO: 55 (e.g., as set forth in NCBI
  • the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 56.
  • the expression construct comprises adeno-associated virus (AAV) inverted terminal repeats (ITRs), for example AAV ITRs flanking the nucleic acid sequence encoding the IL-34 protein.
  • AAV adeno-associated virus
  • ITRs inverted terminal repeats
  • the disclosure provides an isolated nucleic acid comprising an expression construct encoding TREM2 protein (e.g., the gene product of TREM gene).
  • the isolated nucleic acid comprises a TREM2-encoding sequence that has been codon optimized (e.g., codon optimized for expression in mammalian cells, for example human cells).
  • the nucleic acid sequence encoding the TREM2 encodes a protein comprising an amino acid sequence as set forth in SEQ ID NO: 57 (e.g., as set forth in NCBI Reference Sequence NP_061838.1).
  • the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 58.
  • the expression construct comprises adeno-associated virus (AAV) inverted terminal repeats (ITRs), for example AAV ITRs flanking the nucleic acid sequence encoding the TREM2 protein.
  • AAV adeno-associated virus
  • ITRs inverted terminal repeats
  • the disclosure provides an isolated nucleic acid comprising an expression construct encoding TMEM106B protein (e.g., the gene product of TMEM106B gene).
  • the isolated nucleic acid comprises a TMEM106B -encoding sequence that has been codon optimized (e.g., codon optimized for expression in mammalian cells, for example human cells).
  • the nucleic acid sequence encoding the TMEM106B encodes a protein comprising an amino acid sequence as set forth in SEQ ID NO: 63 (e.g., as set forth in NCBI Reference Sequence NP_060844.2).
  • the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 64.
  • the expression construct comprises adeno-associated virus (AAV) inverted terminal repeats (ITRs), for example AAV ITRs flanking the nucleic acid sequence encoding the TMEM106B protein.
  • AAV adeno-associated virus
  • ITRs inverted terminal repeats
  • the disclosure provides an isolated nucleic acid comprising an expression construct encoding progranulin (e.g., the gene product of PGRN gene, also referred to as GRN gene).
  • the isolated nucleic acid comprises a prosaposin- encoding sequence that has been codon optimized (e.g., codon optimized for expression in mammalian cells, for example human cells).
  • the nucleic acid sequence encoding the progranulin (PRGN also referred to as GRN) encodes a protein comprising an amino acid sequence as set forth in SEQ ID NO: 67 (e.g., as set forth in NCBI Reference Sequence NP_002078.1).
  • the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 68.
  • the expression construct comprises adeno-associated virus (AAV) inverted terminal repeats (ITRs), for example AAV ITRs flanking the nucleic acid sequence encoding the prosaposin protein.
  • AAV inverted terminal repeats
  • aspects of the disclosure relate to isolated nucleic acids and expression constructs (e.g., rAAV vectors) encoding one or more inhibitory nucleic acids.
  • one or more inhibitory nucleic acids target a gene associated with certain central nervous system (CNS) diseases (e,g, SNCA, TMEM106B, RPS2 or MAPT).
  • CNS central nervous system
  • the inhibitory nucleic acids are expressed alone, or in combination with one or more gene products described herein (e.g., GBA1, PSAP, PRGN, etc.).
  • an isolated nucleic acid encodes 1) an inhibitory nucleic acid targeting SNCA, and 2) GBA1 protein.
  • an isolated nucleic acid encodes 1) an inhibitory nucleic acid targeting SNCA, and 2) PSAP protein.
  • an isolated nucleic acid encodes 1) an inhibitory nucleic acid targeting SNCA, and 2) PGRN protein (e.g., GRN protein).
  • an isolated nucleic acid encodes 1) an inhibitory nucleic acid targeting MAPT, and 2) GBA1 protein.
  • an isolated nucleic acid encodes 1) an inhibitory nucleic acid targeting MAPT, and 2) PSAP protein. In some embodiments, an isolated nucleic acid encodes 1) an inhibitory nucleic acid targeting MAPT, and 2) PGRN protein (e.g., GRN protein). In some embodiments, an isolated nucleic acid encodes 1) an inhibitory nucleic acid targeting TMEM106B, and 2) GBA1 protein. In some embodiments, an isolated nucleic acid encodes 1) an inhibitory nucleic acid targeting TMEM106B, and 2) PSAP protein. In some embodiments, an isolated nucleic acid encodes 1) an inhibitory nucleic acid targeting TMEM106B, and 2) PGRN protein (e.g., GRN protein).
  • an isolated nucleic acid encodes 1) an inhibitory nucleic acid targeting RPS25, and 2) GBA1 protein. In some embodiments, an isolated nucleic acid encodes 1) an inhibitory nucleic acid targeting RPS25, and 2) PSAP protein. In some embodiments, an isolated nucleic acid encodes 1) an inhibitory nucleic acid targeting RPS25, and 2) PGRN protein (e.g., GRN protein).
  • the disclosure provides an isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid that inhibits expression or activity of a- Syn flanked by AAV inverted terminal repeats (ITRs).
  • the inhibitory nucleic acid is complementary to at least six contiguous nucleotides of the sequence set forth in SEQ ID NO: 90.
  • the inhibitory nucleic acid is an inhibitory RNA comprising the nucleic acid sequence set forth in any one of SEQ ID NOs: 20-25.
  • the inhibitory nucleic acid comprises the sequence set forth in any one of SEQ ID NOs: 94-99.
  • the disclosure provides an isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid that inhibits expression or activity of
  • the inhibitory nucleic acid is complementary to at least six contiguous nucleotides of the sequence set forth in SEQ ID NO: 91.
  • the inhibitory nucleic acid is an inhibitory RNA comprising the nucleic acid sequence set forth in SEQ ID NO: 92 or 93.
  • the inhibitory nucleic acid comprises the sequence set forth in SEQ ID NO: 65 or 66.
  • the disclosure provides an isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid that inhibits expression or activity of MAPT flanked by AAV inverted terminal repeats (ITRs).
  • the inhibitory nucleic acid is complementary to at least six contiguous nucleotides of the sequence set forth in SEQ ID NO: 114.
  • the inhibitory nucleic acid is an inhibitory RNA comprising the nucleic acid sequence set forth in SEQ ID NO: 123, 124, 127, 128, 131, 132, 135 or 136).
  • the inhibitory nucleic acid comprises the sequence set forth in SEQ ID NO: 125, 126, 129, 130, 133, 134, 137 or 138.
  • the disclosure provides an isolated nucleic acid comprising an expression construct encoding a first gene product and a second gene product, wherein each gene product independently is selected from the gene products, or portions thereof, set forth in Table 1 or an inhibitory nucleic acid targeting a gene or gene product set forth in Table 1.
  • the first gene product is a protein
  • the second gene product is a protein.
  • the first gene product is an inhibitory nucleic acid and the second gene product is a protein.
  • the first gene product is an inhibitory nucleic acid and the second gene product is an inhibitory nucleic acid.
  • the first gene product is a Gcase protein, or a portion thereof.
  • the second gene product is an inhibitory nucleic acid that targets SNCA.
  • the interfering nucleic acid is a siRNA, shRNA, miRNA, or dsRNA, optionally wherein the interfering nucleic acid inhibits expression of a-Syn protein.
  • the isolated nucleic acid further comprises one or more promoters, optionally wherein each of the one or more promoters is independently a chicken-beta actin (CBA) promoter, a CAG promoter, a CD68 promoter, or a JeT promoter.
  • the isolated nucleic acid further comprising an internal ribosomal entry site (IRES), optionally wherein the IRES is located between the first gene product and the second gene product.
  • IRES internal ribosomal entry site
  • the isolated nucleic acid further comprising a self-cleaving peptide coding sequence, optionally wherein the self-cleaving peptide is T2A.
  • the expression construct comprises two adeno-associated virus (AAV) inverted terminal repeat
  • the disclosure provides an isolated nucleic acid comprising an expression construct encoding a first gene product and a second gene product, wherein each gene product independently is selected from the gene products, or portions thereof, set forth in Table 1 or an inhibitory nucleic acid targeting a gene or gene product set forth in Table 1.
  • a first gene product or a second gene product is a Gcase protein, or a portion thereof.
  • a first gene product is a Gcase protein and a second gene product is selected from GBA2, prosaposin, progranulin, LIMP2, GALC, CTSB, SMPD1, GCH1, RAB7, VPS35, IL-34, TREM2, and TMEM106B.
  • an expression construct encodes (e.g ., alone or in addition to another gene product) an interfering nucleic acid (e.g., shRNA, miRNA, dsRNA, etc.).
  • an interfering nucleic acid inhibits expression of a-Synuclein (a-Synuclein).
  • an expression construct encodes an inhibitory nucleic acid targeting SNCA, and encodes one or more gene product selected from GBA1, GBA2, PSAP, PRGN, LIMP2, GALC, CTSB, SMPD1, GCH1, RAB7, VPS35, IL-34, TREM2, and TMEM106B.
  • an interfering nucleic acid that targets a-Synuclein comprises a sequence set forth in any one of SEQ ID NOs: 20-25. In some embodiments, an interfering nucleic acid that targets a-Synuclein binds to (e.g., hybridizes with) a sequence set forth in any one of SEQ ID NO: 20-25.
  • an interfering nucleic acid inhibits expression of TMEM106B.
  • an expression construct encodes an inhibitory nucleic acid targeting TMEM106B, and encodes one or more gene product selected from GBA1, GBA2, PSAP,
  • an interfering nucleic acid that targets TMEM106B comprises a sequence set forth in SEQ ID NO: 65 or 66. In some embodiments, an interfering nucleic acid that targets TMEM106B binds to (e.g., hybridizes with) a sequence set forth in SEQ ID NO: 65 or 66.
  • an interfering nucleic acid inhibits expression of MAPT.
  • an expression construct encodes an inhibitory nucleic acid targeting MAPT, and encodes one or more gene product selected from GBA1, GBA2, PSAP, PRGN, LIMP2, GALC, CTSB, SMPD1, GCH1, RAB7, VPS35, IL-34, TREM2, and TMEM106B.
  • an interfering nucleic acid that targets MAPT comprises a sequence set forth in any one of SEQ ID NOs: 123-138. In some embodiments, an interfering nucleic acid that targets MAPT binds to (e.g., hybridizes with) a sequence set forth in any one of SEQ ID NO: 123-138. In some embodiments, an interfering nucleic acid inhibits expression of RPS25. In some embodiments, an expression construct encodes an inhibitory nucleic acid targeting RPS25, and encodes one or more gene product selected from GBA1, GBA2, PSAP, PRGN, LIMP2, GALC, CTSB, SMPD1, GCH1, RAB7, VPS35, IL-34, TREM2, and TMEM106B. In some
  • an interfering nucleic acid that targets RPS25 comprises a sequence set forth in any one of SEQ ID NOs: 115-122. In some embodiments, an interfering nucleic acid that targets RPS25 binds to ( e.g ., hybridizes with) a sequence set forth in any one of SEQ ID NO:
  • an expression construct further comprises one or more promoters.
  • a promoter is a chicken-beta actin (CBA) promoter, a CAG promoter, a CD68 promoter, or a JeT promoter.
  • a promoter is a RNA pol II promoter (e.g., or an RNA pol III promoter (e.g., U6, etc.).
  • an expression construct further comprises an internal ribosomal entry site (IRES).
  • IRES internal ribosomal entry site
  • an IRES is located between a first gene product and a second gene product.
  • an expression construct further comprises a self-cleaving peptide coding sequence.
  • a self-cleaving peptide is a T2A peptide.
  • an expression construct comprises two adeno-associated virus (AAV) inverted terminal repeat (ITR) sequences.
  • ITR sequences flank a first gene product and a second gene product (e.g., are arranged as follows from 5’-end to 3’- end: ITR-first gene product- second gene product-ITR).
  • one of the ITR sequences of an isolated nucleic acid lacks a functional terminal resolution site (trs).
  • one of the ITRs is a AITR.
  • the disclosure relates, in some aspects, to rAAV vectors comprising an ITR having a modified“D” region (e.g., a D sequence that is modified relative to wild-type AAV2 ITR, SEQ ID NO: 29).
  • the ITR having the modified D region is the 5’ ITR of the rAAV vector.
  • a modified“D” region comprises an“S” sequence, for example as set forth in SEQ ID NO: 26.
  • the ITR having the modified “D” region is the 3’ ITR of the rAAV vector.
  • a modified“D” region comprises a 3’ITR in which the“D” region is positioned at the 3’ end of the ITR (e.g., on the outside or terminal end of the ITR relative to the transgene insert of the vector).
  • a modified“D” region comprises a sequence as set forth in SEQ ID NO: 26 or 27.
  • an isolated nucleic acid (e.g., an rAAV vector) comprises a TRY region.
  • a TRY region comprises the sequence set forth in SEQ ID NO: 28.
  • an isolated nucleic acid described by the disclosure comprises or consists of, or encodes a peptide having, the sequence set forth in any one of SEQ ID NOs: 1- 149.
  • the disclosure provides a vector comprising an isolated nucleic acid as described by the disclosure.
  • a vector is a plasmid, or a viral vector.
  • a viral vector is a recombinant AAV (rAAV) vector or a Baculovims vector.
  • rAAV recombinant AAV
  • Baculovims vector recombinant AAV
  • an rAAV vector is single- stranded (e.g., single-stranded DNA).
  • the disclosure provides a host cell comprising an isolated nucleic acid as described by the disclosure or a vector as described by the disclosure.
  • the disclosure provides a recombinant adeno-associated virus (rAAV) comprising a capsid protein and an isolated nucleic acid or a vector as described by the disclosure.
  • rAAV recombinant adeno-associated virus
  • a capsid protein is capable of crossing the blood-brain barrier, for example an AAV9 capsid protein or an AAVrh.10 capsid protein.
  • an rAAV transduces neuronal cells and non-neuronal cells of the central nervous system (CNS).
  • the disclosure provides a method for treating a subject having or suspected of having or suspected of having a central nervous system (CNS) disease, the method comprising administering to the subject a composition (e.g., a composition comprising an isolated nucleic acid or a vector or a rAAV) as described by the disclosure.
  • a composition e.g., a composition comprising an isolated nucleic acid or a vector or a rAAV
  • the CNS disease is a neurodegenerative disease, such as a neurodegenerative disease listed in Table 2.
  • the CNS disease is a synucleinopathy, such as a synucleinopathy listed in Table 3.
  • the CNS disease is a tauopathy, such as a tauopathy listed in Table 4.
  • the CNS disease is a lysosomal storage disease, such as a lysosomal storage disease listed in Table 5.
  • the lysosomal storage disease is neuronopathic Gaucher disease, such as Type 2 Gaucher disease or Type 3 Gaucher disease.
  • the disclosure relates to methods of treating a disease selected from Parkinson’s Disease (e.g., Parkinson’s Disease with GBA1 mutation (PD-GBA), sporadic Parkinson’s Disease (sPD)), Gaucher Disease (e.g., neuronopathic Gaucher disease (nGD), Type I Gaucher Disease (T1GD), Type II Gaucher Disease (T2GD), and Type III Gaucher Disease (T3GD)), Dementia with Lewy Bodies (DLB), Amyotrophic lateral sclerosis (ALS), and Niemann-Pick Type C disease (NPC) by administering to a subject in need thereof an isolated nucleic acid (e.g., an rAAV vector or rAAV comprising an isolated nucleic acid) that encodes GBA1.
  • Parkinson’s Disease e.g., Parkinson’s Disease with GBA1 mutation (PD-GBA), sporadic Parkinson’s Disease (sPD)
  • Gaucher Disease e.g., neuronopathic Gaucher disease (nGD), Type I Gaucher Disease
  • the disclosure relates to methods of treating Frontotemporal Dementia (e.g., Frontotemporal Dementia with GRN mutation (FTD-GRN), Frontotemporal Dementia with MAPT mutation (FTD-tau), and Frontotemporal Dementia with C90RF72 mutation (FTD-C9orf72)), Parkinson’s Disease (PD), Alzheimer’s Disease (AD), Neuronal Ceroid Lipofuscinosis (NCL), Corticobasal Degeneration (CBD), Motor Neuron Disease (MND), or Gaucher Disease (GD) by administering to a subject in need thereof an isolated nucleic acid (e.g., an rAAV vector or rAAV comprising an isolated nucleic acid) that encodes PGRN (e.g. GRN).
  • FTD-GRN Frontotemporal Dementia with GRN mutation
  • FTD-tau Frontotemporal Dementia with MAPT mutation
  • FTD-C9orf72 Frontotemporal Dementia with
  • the disclosure relates to methods of treating Synucleinopathies (e.g., multiple system atrophy (MSA), Parkinson’s Disease (PD), Parkinson's disease with GBA1 mutation (PD-GBA), Dementia with Lewy Bodies (DLB), Dementia with Lewy Bodies with GBA1 mutation, and Lewy Body Disease) by administering to a subject in need thereof an isolated nucleic acid (e.g., an rAAV vector or rAAV comprising an isolated nucleic acid) that encodes GBA1 gene product, and an inhibitory nucleic acid targeting SNCA.
  • Synucleinopathies e.g., multiple system atrophy (MSA), Parkinson’s Disease (PD), Parkinson's disease with GBA1 mutation (PD-GBA), Dementia with Lewy Bodies (DLB), Dementia with Lewy Bodies with GBA1 mutation, and Lewy Body Disease
  • an isolated nucleic acid e.g., an rAAV vector or r
  • the disclosure relates to methods of treating a disease selected from Parkinson’s Disease (PD), Frontotemporal Dementia (e.g., Frontotemporal Dementia with GRN mutation (FTD-GRN)), Lysosomal Storage Diseases (LSDs), or Gaucher Disease (GD) by administering to a subject in need thereof an isolated nucleic acid (e.g., an rAAV vector or rAAV comprising an isolated nucleic acid) that encodes PSAP.
  • PD Parkinson’s Disease
  • Frontotemporal Dementia e.g., Frontotemporal Dementia with GRN mutation (FTD-GRN)
  • LSDs Lysosomal Storage Diseases
  • GD Gaucher Disease
  • the disclosure relates to methods of treating Alzheimer’s Disease (AD), Nasu-Hakola Disease (NHD) or Parkinson’s Disease (PD), by administering to a subject in need thereof an isolated nucleic acid (e.g., an rAAV vector or rAAV comprising an isolated nucleic acid) that encodes TREM2.
  • an isolated nucleic acid e.g., an rAAV vector or rAAV comprising an isolated nucleic acid
  • the disclosure relates to methods of treating Alzheimer’s disease (AD) or Frontotemporal Dementia (Frontotemporal Dementia with MAPT mutation (FTD-Tau), Progressive supranuclear palsy (PSP), neurodegenerative disease, Lewy Body Disease (LBD) or Parkinson’s Disease by administering to a subject in need thereof an isolated nucleic acid (e.g., an rAAV vector or rAAV comprising an isolated nucleic acid) that encodes inhibitory nucleic acids targeting MAPT.
  • an isolated nucleic acid e.g., an rAAV vector or rAAV comprising an isolated nucleic acid
  • the disclosure provides a method for treating a subject having or suspected of having Parkinson’s disease, the method comprising administering to the subject a composition (e.g., a composition comprising an isolated nucleic acid or a vector or a rAAV) as described by the disclosure.
  • a composition comprises a nucleic acid (e.g ., an rAAV genome, for example encapsidated by AAV capsid proteins) that encodes two or more gene products (e.g., CNS disease-associated gene products), for example 2, 3, 4, 5, or more gene products described in this application.
  • a composition comprises two or more (e.g., 2, 3, 4, 5, or more) different nucleic acids (e.g., two or more rAAV genomes, for example separately encapsidated by AAV capsid proteins), each encoding one or more different gene products.
  • two or more different compositions are administered to a subject, each composition comprising one or more nucleic acids encoding different gene products.
  • different gene products are operably linked to the same promoter type (e.g., the same promoter).
  • different gene products are operably linked to different promoters.
  • administration comprises direct injection to the CNS of a subject.
  • direct injection is intracerebral injection, intraparenchymal injection, intrathecal injection, intra-cistema manga injection, or any combination thereof.
  • direct injection to the CNS of a subject comprises convection enhanced delivery (CED).
  • CED convection enhanced delivery
  • administration comprises peripheral injection.
  • peripheral injection is intravenous injection.
  • the present disclosure provides a method for treating a subject having or suspected of having a central nervous system (CNS) disease, the method comprising
  • an isolated nucleic acid comprising: (i) an expression construct comprising a transgene encoding one or more gene products listed in Table 1 or an inhibitory nucleic acid targeting a gene or gene product set forth in Table 1; and (ii) two adeno-associated vims (AAV) inverted terminal repeats (ITRs) flanking the expression construct.
  • AAV adeno-associated vims
  • the present disclosure provides a method for treating a subject having or suspected of having a central nervous system (CNS) disease, the method comprising administering to the subject two or more types of isolated nucleic acids encoding different gene products, where each type of isolated nucleic acid comprises: (i) an expression construct comprising a transgene encoding one or more gene products listed in Table 1 or an inhibitory nucleic acid targeting a gene or gene product set forth in Table 1; and (ii) two adeno-associated virus (AAV) inverted terminal repeats (ITRs) flanking the expression construct.
  • AAV adeno-associated virus
  • the transgene encodes one or more proteins selected from: GBA1,
  • GBA2 PGRN (e.g., GRN), TREM2, PSAP, SCARB2, GALC, SMPD1, CTSB, RAB7L,
  • the transgene encoding one or more gene products comprises a codon-optimized protein coding sequence.
  • the transgene encodes one or more inhibitory nucleic acids targeting SNCA, MAPT, RPS25, and/or TMEM106B.
  • the AAV ITRs are AAV2 ITRs.
  • the isolated nucleic acid is packaged into a recombinant adeno- associated virus (rAAV).
  • rAAV adeno- associated virus
  • the rAAV comprises an AAV9 capsid protein.
  • the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the CNS disease is a neurodegenerative disease, synucleinopathy, tauopathy, and/or lysosomal storage disease (LSD). In some embodiments, the CNS disease is listed in Table 2, Table 3, Table 4, or Table 5.
  • the administration comprises direct injection to the CNS of the subject, optionally wherein the direct injection is intracerebral injection, intraparenchymal injection, intrathecal injection, intra-cistema magna injection or any combination thereof.
  • the intra-cisterna magna injection is suboccipital injection into the cistema magna.
  • the direct injection to the CNS of the subject comprises convection enhanced delivery (CED).
  • the administration comprises peripheral injection, optionally wherein the peripheral injection is intravenous injection.
  • the subject is administered about 1 x 10 10 vg to about 1 x 10 16 vg of the rAAV.
  • FIG. 1 is a schematic depicting one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof).
  • Gcase e.g., GBA1 or a portion thereof.
  • FIG. 2 is a schematic depicting one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof) and LIMP2 (SCARB2) or a portion thereof.
  • Gcase e.g., GBA1 or a portion thereof
  • LIMP2 LIMP2
  • FIG. 2 is a schematic depicting one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof) and LIMP2 (SCARB2) or a portion thereof.
  • the coding sequences of Gcase and LIMP2 are separated by an internal ribosomal entry site (IRES).
  • IRS internal ribosomal entry site
  • FIG. 3 is a schematic depicting one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof) and LIMP2 (SCARB2) or a portion thereof. Expression of the coding sequences of Gcase and LIMP2 are each driven by a separate promoter.
  • Gcase e.g., GBA1 or a portion thereof
  • LIMP2 SCARB2
  • FIG. 4 is a schematic depicting one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof), LIMP2 (SCARB2) or a portion thereof, and an interfering RNA for a-Syn.
  • FIG. 5 is a schematic depicting one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof), Prosaposin (e.g., PSAP or a portion thereof), and an interfering RNA for a-Syn.
  • FIG. 6 is a schematic depicting one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof) and Prosaposin (e.g., PSAP or a portion thereof).
  • Gcase e.g., GBA1 or a portion thereof
  • Prosaposin e.g., PSAP or a portion thereof
  • the coding sequences of Gcase and Prosaposin are separated by an internal ribosomal entry site (IRES).
  • IRS internal ribosomal entry site
  • FIG. 7 is a schematic depicting one embodiment of a vector comprising an expression construct encoding a Gcase (e.g., GBA1 or a portion thereof).
  • the vector comprises a CBA promoter element (CBA), consisting of four parts: the CMV enhancer
  • CMVe CBA promoter
  • CBAp CBA promoter
  • Exon 1 intron 1
  • int intron 1 to constitutively express the codon optimized coding sequence of human GBA1.
  • the 3’ region also contains a WPRE regulatory element followed by a bGH polyA tail.
  • Three transcriptional regulatory activation sites are included at the 5’ end of the promoter region: TATA, RBS, and YY1.
  • the flanking ITRs allow for the correct packaging of the intervening sequences. Two variants of the 5’ ITR sequence (inset box) were evaluated; these have several nucleotide differences within the 20-nucleotide “D” region of wild-type AAV2 ITR.
  • an rAAV vector contains the“D” domain nucleotide sequence shown on the top line.
  • a rAAV vector comprises a mutant“D” domain (e.g., an“S” domain, with the nucleotide changes shown on the bottom line).
  • FIG. 8 is a schematic depicting one embodiment of the vector described in FIG. 6
  • FIG. 9 shows representative data for delivery of an rAAV comprising a transgene encoding a Gcase (e.g., GBA1 or a portion thereof) in a CBE mouse model of Parkinson’s disease.
  • Daily IP delivery of PBS vehicle, 25 mg/kg CBE, 37.5 mg/kg CBE, or 50 mg/kg CBE (left to right) initiated at P8. Survival (top left) was checked two times a day and weight (top right) was checked daily. All groups started with n 8. Behavior was assessed by total distance traveled in Open Field (bottom left) at P23 and latency to fall on Rotarod (bottom middle) at P24. Levels of the GCase substrates were analyzed in the cortex of mice in the PBS and 25 mg/kg CBE treatment groups both with (Day 3) and without (Day 1) CBE withdrawal.
  • a Gcase e.g., GBA1 or a portion thereof
  • GluSph and GalSph levels (bottom right) are shown as pmol per mg wet weight of the tissue. Means are presented. Error bars are SEM. *p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.001, nominal p-values for treatment groups by linear regression.
  • FIG. 10 is a schematic depicting one embodiment of a study design for maximal rAAV dose in a CBE mouse model. Briefly, rAAV was delivered by ICV injection at P3, and daily CBE treatment was initiated at P8. Behavior was assessed in the Open Field and Rotarod assays at P24-25 and substrate levels were measured at P36 and P38.
  • FIG. 11 shows representative data for in-life assessment of maximal rAAV dose in a CBE mouse model.
  • mice were treated with either excipient or 8.8e9 vg rAAV-GBAl via ICV delivery.
  • Daily IP delivery of either PBS or 25 mg/kg CBE was initiated at P8.
  • half the mice were sacrificed one day after their last CBE dose at P36 (Day 1) while the remaining half went through 3 days of CBE withdrawal before sacrifice at P38 (Day3).
  • FIG. 12 shows representative data for biochemical assessment of maximal rAAV dose in a CBE mouse model.
  • FIG. 15 shows representative data for in-life assessment of rAAV dose ranging in a CBE mouse model.
  • Mice received excipient or one of three different doses of rAAV-GBAl by ICV delivery at P3: 3.2e9 vg, l.OelOvg, or 3.2el0 vg.
  • ICV delivery at P3: 3.2e9 vg, l.OelOvg, or 3.2el0 vg.
  • P8 daily IP treatment of 25 mg/kg CBE was initiated.
  • Mice that received excipient and CBE or excipient and PBS served as controls.
  • n 10 (5M/5F) per group. All mice were sacrificed one day after their final CBE dose (P38-P40). All treatment groups were weighed daily, and their weight was analyzed at P36. Motor performance was assessed by latency to fall on Rotarod at P24 and latency to traverse the Tapered Beam at P30.
  • FIG. 16 shows representative data for biochemical assessment of rAAV dose ranging in a CBE mouse model.
  • GCase activity is shown as ng of GCase per mg of total protein.
  • GluSph and GluCer levels are shown as pmol per mg wet weight of the tissue.
  • Biodistribution is shown as vector genomes per 1 pg of genomic DNA.
  • Vector genome presence was quantified by quantitative PCR using a vector reference standard curve; genomic DNA concentration was evaluated by A260 optical density measurement. Vector genome presence was also measured in the liver (E). Means are presented. Error bars are SEM.
  • FIG. 17 shows representative data for tapered beam analysis in maximal dose rAAV- GBAl in a genetic mouse model.
  • the total slips and active time are shown as total over 5 trials on different beams.
  • Speed and slips per speed are shown as the average over 5 trials on different beams. Means are presented. Error bars are SEM.
  • PGRN progranulin
  • GRN protein also referred to as GRN protein
  • the left panel shows a standard curve of progranulin (PGRN) ELISA assay.
  • the bottom panel shows a dose-response of PGRN expression measured by ELISA assay in cell lysates of HEK293T cells transduced with rAAV.
  • MOI multiplicity of infection (vector genomes per cell).
  • FIG. 19 shows representative data for in vitro expression of rAAV constructs encoding GBA1 in combination with Prosaposin ( PSAP ), SCARB2, and/or one or more inhibitory nucleic acids. Data indicate transfection of HEK293 cells with each construct resulted in overexpression of the transgenes of interest relative to mock transfected cells.
  • FIG. 20 is a schematic depicting an rAAV vectors comprising a“D” region located on the“outside” of the ITR (e.g., proximal to the terminus of the ITR relative to the transgene insert or expression construct) (top) and a wild-type rAAV vectors having ITRs on the“inside” of the vector (e.g., proximal to the transgene insert of the vector).
  • FIG. 21 a schematic depicting one embodiment of a vector comprising an expression construct encoding GBA2 or a portion thereof, and an interfering RNA for a-Syn.
  • FIG. 22 is a schematic depicting one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof) and Galactosylceramidase (e.g., GALC or a portion thereof). Expression of the coding sequences of Gcase and
  • Galactosylceramidase are separated by a T2A self-cleaving peptide sequence.
  • FIG. 23 is a schematic depicting one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof) and Galactosylceramidase (e.g., GALC or a portion thereof). Expression of the coding sequences of Gcase and
  • Galactosylceramidase are separated by a T2A self-cleaving peptide sequence.
  • FIG. 24 is a schematic depicting one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof), Cathepsin B (e.g., CTSB or a portion thereof), and an interfering RNA for a-Syn. Expression of the coding sequences of Gcase and Cathepsin B are separated by a T2A self-cleaving peptide sequence.
  • Gcase e.g., GBA1 or a portion thereof
  • Cathepsin B e.g., CTSB or a portion thereof
  • interfering RNA for a-Syn interfering RNA for a-Syn.
  • FIG. 25 is a schematic depicting one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof), Sphingomyelin phosphodiesterase 1 (e.g., SMPD1 a portion thereof, and an interfering RNA for a-Syn.
  • Gcase e.g., GBA1 or a portion thereof
  • Sphingomyelin phosphodiesterase 1 e.g., SMPD1 a portion thereof
  • interfering RNA for a-Syn interfering RNA for a-Syn.
  • FIG. 26 is a schematic depicting one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof) and Galactosylceramidase (e.g., GALC or a portion thereof). The coding sequences of Gcase and Galactosylceramidase are separated by an internal ribosomal entry site (IRES).
  • FIG. 27 is a schematic depicting one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof) and Cathepsin B (e.g., CTSB or a portion thereof). Expression of the coding sequences of Gcase and Cathepsin B are each driven by a separate promoter.
  • FIG. 28 is a schematic depicting one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof), GCH1 (e.g., GCH1 or a portion thereof), and an interfering RNA for a-Syn.
  • Gcase e.g., GBA1 or a portion thereof
  • GCH1 e.g., GCH1 or a portion thereof
  • interfering RNA for a-Syn e.g., T2A self-cleaving peptide sequence
  • FIG. 29 is a schematic depicting one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof), RAB7L1 (e.g., RAB7L1 or a portion thereof), and an interfering RNA for a-Syn .
  • Gcase e.g., GBA1 or a portion thereof
  • RAB7L1 e.g., RAB7L1 or a portion thereof
  • interfering RNA for a-Syn e.g., T2A self-cleaving peptide sequence.
  • FIG. 30 is a schematic depicting one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof), GCH1 (e.g., GCH1 or a portion thereof), and an interfering RNA for a-Syn.
  • Gcase e.g., GBA1 or a portion thereof
  • GCH1 e.g., GCH1 or a portion thereof
  • interfering RNA for a-Syn e.g., GCH1 or a portion thereof
  • Expression of the coding sequences of Gcase and GCH1 are an internal ribosomal entry site (IRES).
  • FIG. 31 is a schematic depicting one embodiment of a vector comprising an expression construct encoding VPS35 (e.g., VPS35 or a portion thereof) and interfering RNAs for a-Syn and TMEM106B.
  • VPS35 e.g., VPS35 or a portion thereof
  • TMEM106B interfering RNAs for a-Syn and TMEM106B.
  • FIG. 32 is a schematic depicting one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof), IL-34 (e.g., IL34 or a portion thereof), and an interfering RNA for a-Syn.
  • Gcase e.g., GBA1 or a portion thereof
  • IL-34 e.g., IL34 or a portion thereof
  • interfering RNA for a-Syn The coding sequences of Gcase and IL-34 are separated by T2A self-cleaving peptide sequence.
  • FIG. 33 is a schematic depicting one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof) and IL-34 (e.g., IL34 or a portion thereof).
  • Gcase e.g., GBA1 or a portion thereof
  • IL-34 e.g., IL34 or a portion thereof.
  • the coding sequences of Gcase and IL-34 are separated by an internal ribosomal entry site (IRES).
  • IRS internal ribosomal entry site
  • FIG. 34 is a schematic depicting one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof) and TREM2 (e.g., TREM2 or a portion thereof). Expression of the coding sequences of Gcase and TREM2 are each driven by a separate promoter.
  • Gcase e.g., GBA1 or a portion thereof
  • TREM2 e.g., TREM2 or a portion thereof
  • FIG. 35 is a schematic depicting one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof) and IL-34 (e.g., IL34 or a portion thereof). Expression of the coding sequences of Gcase and IL-34 are each driven by a separate promoter.
  • Gcase e.g., GBA1 or a portion thereof
  • IL-34 e.g., IL34 or a portion thereof
  • FIGs. 36A-36B show representative data for overexpression of TREM2 and GBA1 in HEK293 cells relative to control transduced cells, as measured by qPCR and ELISA.
  • FIG. 36A shows data for overexpression of TREM2.
  • FIG. 36B shows data for overexpression of GBA1 from the same construct.
  • FIG. 37 shows representative data indicating successful silencing of SNCA in vitro by GFP reporter assay (top) and a-Syn assay (bottom).
  • FIG. 38 shows representative data indicating successful silencing of TMEM106B in vitro by GFP reporter assay (top) and a-Syn assay (bottom).
  • FIG. 39 is a schematic depicting one embodiments of a vector comprising an expression construct encoding PGRN (also referred to as GRN).
  • PGRN also referred to as GRN
  • FIG. 40 shows data for transduction of HEK293 cells using rAAVs having ITRs with wild-type (circles) or alternative (e.g.,“outside”; squares) placement of the“D” sequence.
  • the rAAVs having ITRs placed on the“outside” were able to transduce cells as efficiently as rAAVs having wild-type ITRs.
  • FIG. 41 is a schematic depicting one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof).
  • Gcase e.g., GBA1 or a portion thereof.
  • FIG. 42 is a schematic depicting one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof).
  • Gcase e.g., GBA1 or a portion thereof.
  • FIG. 43 is a schematic depicting one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof) and an interfering RNA for a-Syn.
  • Gcase e.g., GBA1 or a portion thereof
  • FIG. 44 is a schematic depicting one embodiments of a vector comprising an expression construct encoding PGRN (also referred to as GRN).
  • PGRN also referred to as GRN
  • FIG. 45 is a schematic depicting one embodiments of a vector comprising an expression construct encoding PGRN (also referred to as GRN).
  • PGRN also referred to as GRN
  • FIG. 46 is a schematic depicting one embodiments of a vector comprising an expression construct encoding PGRN (also referred to as GRN) and an interfering RNA for microtubule- associated protein tau (MAPT).
  • PGRN also referred to as GRN
  • MTT microtubule-associated protein tau
  • FIG. 47 is a schematic depicting one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof) and an interfering RNA for a-Syn.
  • Gcase e.g., GBA1 or a portion thereof
  • interfering RNA for a-Syn e.g., interfering RNA for a-Syn.
  • FIG. 48 is a schematic depicting one embodiments of a vector comprising an expression construct encoding PSAP.
  • FIG. 49 is a schematic depicting one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof).
  • FIG. 50 is a schematic depicting one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof) and Galactosylceramidase (e.g., GALC or a portion thereof).
  • Gcase e.g., GBA1 or a portion thereof
  • Galactosylceramidase e.g., GALC or a portion thereof
  • FIG. 51 is a schematic depicting one embodiment of a plasmid comprising an rAAV vector that includes an expression construct encoding Gcase (e.g., GBA1 or a portion thereof), Prosaposin (e.g., PSAP or a portion thereof), and an interfering RNA for a-Syn.
  • Gcase e.g., GBA1 or a portion thereof
  • Prosaposin e.g., PSAP or a portion thereof
  • interfering RNA for a-Syn a-Syn.
  • FIG. 52 is a schematic depicting one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof) and an inhibitory RNA targeting SNCA.
  • Gcase e.g., GBA1 or a portion thereof
  • inhibitory RNA targeting SNCA an inhibitory RNA targeting SNCA.
  • FIG. 53 is a schematic depicting one embodiment of a plasmid comprising an rAAV vector that includes an expression construct encoding SNCA.
  • FIG. 54 is a schematic depicting one embodiment of a plasmid comprising an rAAV vector that includes an expression construct encoding an inhibitory RNA targeting SNCA.
  • the inhibitory RNA is positioned within an intron between the promoter sequence and the Gcase encoding sequence.
  • FIG. 55 is a schematic depicting one embodiment of a plasmid comprising an rAAV vector that includes an expression construct encoding progranulin (PGRN, also referred to as GRN) and an inhibitory RNA targeting SNCA.
  • PGRN progranulin
  • the inhibitory RNA is positioned within an intron between the promoter sequence and the Gcase encoding sequence.
  • FIG. 56 is a schematic depicting one embodiment of a plasmid comprising an rAAV vector that includes an expression construct encoding Gcase (GBA1) and an inhibitory RNA targeting SNCA.
  • the inhibitory RNA is positioned within an intron between the promoter sequence and the Gcase encoding sequence.
  • FIG. 57 is a schematic depicting one embodiment of a plasmid comprising an rAAV vector that includes an expression construct encoding Gcase (GBA1) and an inhibitory RNA targeting SNCA.
  • the inhibitory RNA is positioned within an intron between the promoter sequence and the Gcase encoding sequence.
  • FIG. 58 is a schematic depicting one embodiment of a plasmid comprising an rAAV vector that includes an expression construct encoding Gcase (GBA1) and an inhibitory RNA targeting SNCA.
  • The“D” sequence of the 3’ITR is positioned on the“outside” of the vector.
  • FIG. 59 is a schematic depicting one embodiment of a plasmid comprising an rAAV vector that includes an expression construct encoding Gcase (GBA1) and an inhibitory RNA targeting SNCA.
  • the inhibitory RNA is positioned within an intron between the promoter sequence and the Gcase encoding sequence.
  • FIG. 60 is a schematic depicting one embodiment of a plasmid comprising an rAAV vector that includes an expression construct encoding Gcase (GBA1) and an inhibitory RNA targeting SNCA.
  • the inhibitory RNA is positioned within an intron between the promoter sequence and the Gcase encoding sequence.
  • FIG. 61 is a schematic depicting one embodiment of a plasmid comprising an rAAV vector that includes an expression construct encoding Gcase (GBA1) and an inhibitory RNA targeting SNCA.
  • FIG. 62 is a schematic depicting one embodiment of a plasmid comprising an rAAV vector that includes an expression construct encoding Gcase (GBA1) and an inhibitory RNA targeting SNCA.
  • the inhibitory RNA is positioned within an intron between the promoter sequence and the Gcase encoding sequence.
  • FIG. 63 is a schematic depicting one embodiment of a plasmid comprising an rAAV vector that includes an expression construct encoding Gcase (GBA1) and an inhibitory RNA targeting SNCA.
  • the inhibitory RNA is positioned within an intron between the promoter sequence and the Gcase encoding sequence.
  • FIG. 64 is a schematic depicting one embodiment of a plasmid comprising an rAAV vector that includes an expression construct encoding Gcase (GBA1) and progranulin (PGRN, also referred to as GRN), and an inhibitory RNA targeting TMEM106B.
  • GAA1 expression construct encoding Gcase
  • PGRN progranulin
  • TMEM106B inhibitory RNA targeting TMEM106B.
  • the inhibitory RNA is positioned within an intron between the promoter sequence and the Gcase encoding sequence.
  • FIG. 65 is a schematic depicting one embodiment of a plasmid comprising an rAAV vector that includes an expression construct encoding an inhibitory RNA targeting RPS25.
  • FIG. 66 is a schematic depicting one embodiment of a plasmid comprising an rAAV vector that includes an expression construct encoding an inhibitory RNA targeting RPS25.
  • FIG. 67 is a schematic depicting one embodiment of a plasmid comprising an rAAV vector that includes an expression construct encoding an inhibitory RNA targeting MAPI.
  • FIG. 68 is a schematic depicting one embodiment of a plasmid comprising an rAAV vector that includes an expression construct encoding an inhibitory RNA targeting MAPT.
  • FIG. 69 is a schematic depicting one embodiment of a plasmid comprising an rAAV vector that includes an expression construct encoding progranulin (PGRN, also referred to as GRN) and an inhibitory RNA targeting MAPT.
  • the inhibitory RNA is positioned within an intron between the promoter sequence and the PGRN (also referred to as GRN) encoding sequence.
  • FIG. 70 is a schematic depicting one embodiment of a plasmid comprising an rAAV vector that includes an expression construct encoding an inhibitory RNA targeting MAPI.
  • FIG. 71 is a schematic depicting one embodiment of a plasmid comprising an rAAV vector that includes an expression construct encoding progranulin (PGRN, also referred to as GRN) and an inhibitory RNA targeting MAPT.
  • PGRN progranulin
  • the inhibitory RNA is positioned within an intron between the promoter sequence and the PGRN (also referred to as GRN) encoding sequence.
  • FIG. 72 is a schematic depicting one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof) and an inhibitory RNA targeting SNCA. Nucleic acid sequence of this vector is set forth in SEQ ID NO: 141.
  • FIG. 73 is a schematic depicting one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof) and an inhibitory RNA targeting SNCA. Nucleic acid sequence of this vector is set forth in SEQ ID NO: 143.
  • FIG. 74 is a schematic depicting one embodiment of a plasmid comprising an rAAV vector that includes an expression construct encoding Gcase (GBA1) and prosaposin (PSAP), and an inhibitory RNA targeting SNCA. Nucleic acid sequence of this vector is set forth in SEQ ID NO: 144.
  • FIG. 75A-75C are charts showing MAPT knockdown in SY5Y Cells by RNA interference.
  • FIG. 75A shows that immunofluorescent stationing of the AAV vectors using a probe directed to BGHpA.
  • FIG. 75B shows RT-PCR results of MAPT expression 3 and 7 days post transduction.
  • FIG. 75C shows the general information of the rAAV virus stocks used for transduction.
  • a gene product can be a protein, a fragment (e.g., portion) of a protein, an interfering nucleic acid that inhibits a CNS disease-associated gene, etc.
  • a gene product is a protein or a protein fragment encoded by a CNS disease-associated gene.
  • a gene product is an interfering nucleic acid (e.g., shRNA, siRNA, miRNA, amiRNA, etc.) that inhibits a CNS disease-associated gene.
  • a CNS disease-associated gene refers to a gene encoding a gene product that is genetically, biochemically or functionally associated with a CNS disease, such as PD.
  • a CNS disease such as PD.
  • individuals having mutations in the GBA1 gene which encodes the protein Gcase
  • synucleinopathies e.g., PD, etc.
  • SNCA which encodes a-Syn
  • an expression cassette described herein encodes a wild-type or non-mutant form of a CNS disease- associated gene, for example a PD-associated gene (or coding sequence thereof).
  • CNS diseases-associated genes e.g., PD-associated genes, AD-associated genes, FTD- associated genes, etc.
  • Table 1 Examples of CNS disease-associated genes and gene products
  • An isolated nucleic acid may be DNA or RNA.
  • the term“isolated” means artificially produced.
  • An“isolated nucleic acid”, as used herein, refers to nucleic acids (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; (iii) purified, as by cleavage and gel separation; or (iv) synthesized by, for example, chemical synthesis.
  • An isolated nucleic acid is one which is readily manipulable by recombinant DNA techniques well known in the art.
  • an isolated nucleic acids e.g ., rAAV vectors
  • an expression construct encoding one or more CNS disease-associated genes (e.g., PD-associated genes), for example a Gcase, a Prosaposin, a LIMP2/SCARB2, a GBA2, GALC protein, a CTSB protein, a SMPD1, a GCH1 protein, a RAB7L protein, a VPS35 protein, a IL- 34 protein, a TREM2 protein, or a TMEM106B protein.
  • CNS disease-associated genes e.g., PD-associated genes
  • the disclosure also provides, in some aspects, isolated nucleic acids (e.g., rAAV vectors) encoding one or more inhibitory nucleic acids that target one or more CNS disease-associated gene, for example SNCA, TMEM106B, RPS25, and MAPT.
  • isolated nucleic acid encoding the CNS disease-associated genes may further comprises coding sequences for inhibitory nucleic acids targeting one or more CNS disease-associated genes.
  • the CNS disease-associated genes and the inhibitory nucleic acids targeting CNS disease-associated genes are encoded on different nucleic acids.
  • the disclosure provides an isolated nucleic acid comprising an expression construct encoding Gcase (e.g., the gene product of GBA1 gene).
  • Gcase also referred to as b-glucocerebrosidase or GBA, refers to a lysosomal protein that cleaves the beta-glucosidic linkage of the chemical glucocerebroside, an intermediate in glycolipid metabolism.
  • Gcase is encoded by the GBA1 gene, located on chromosome 1.
  • GBA1 encodes a peptide that is represented by NCBI Reference Sequence NCBI Reference Sequence NP_000148.2 (SEQ ID NO: 14).
  • an isolated nucleic acid comprises a Gcase-encoding sequence that has been codon optimized (e.g., codon optimized for expression in mammalian cells, for example human cells), such as the sequence set forth in SEQ ID NO:
  • the disclosure provides an isolated nucleic acid comprising an expression construct encoding Prosaposin (e.g., the gene product of PSAP gene).
  • Prosaposin is a precursor glycoprotein for sphingolipid activator proteins (saposins) A, B, C, and D, which facilitate the catabolism of glycosphingolipids with short oligosaccharide groups.
  • the PSAP gene is located on chromosome 10.
  • PSAP encodes a peptide that is represented by NCBI Reference Sequence NP_002769.1 (e.g., SEQ ID NO: 16).
  • an isolated nucleic acid comprises a prosaposin-encoding sequence that has been codon optimized (e.g., codon optimized for expression in mammalian cells, for example human cells), such as the sequence set forth in SEQ ID NO: 17.
  • an isolated nucleic acid comprising an expression construct encoding LIMP2/SCARB2 (e.g., the gene product of SCARB2 gene).
  • SCARB2 refers to a membrane protein that regulates lysosomal and endosomal transport within a cell.
  • SCARB2 gene is located on chromosome 4.
  • the SCARB2 gene encodes a peptide that is represented by NCBI Reference Sequence NP_005497.1 (SEQ ID NO: 18).
  • an isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 19.
  • the isolated nucleic acid comprises a SC ARB 2-encoding sequence that has been codon optimized.
  • GBA2 protein refers to non-lysosomal glucosylceramidase.
  • GBA2 gene is located on chromosome 9.
  • the GBA2 gene encodes a peptide that is represented by NCBI Reference Sequence NP_065995.1 (SEQ ID NO: 30).
  • an isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 31.
  • the isolated nucleic acid comprises a GBA2-encoding sequence that has been codon optimized.
  • GALC protein refers to galactosylceramidase (or galactocerebrosidase), which is an enzyme that hydrolyzes galactose ester bonds of galactocerebroside, galactosylsphingosine, lactosylceramide, and
  • an isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 34. In some embodiments the isolated nucleic acid comprises a GALC-encoding sequence that has been codon optimized.
  • CTSB protein refers to cathepsin B, which is a lysosomal cysteine protease that plays an important role in intracellular proteolysis.
  • CTSB gene is located on chromosome 8.
  • the CTSB gene encodes a peptide that is represented by NCBI Reference Sequence NP_001899.1 (SEQ ID NO: 35).
  • an isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 36.
  • the isolated nucleic acid comprises a CTSB- encoding sequence that has been codon optimized.
  • SMPD1 protein refers to sphingomyelin phosphodiesterase 1, which is a hydrolase enzyme that is involved in sphingolipid metabolism.
  • SMPD1 gene is located on chromosome 11.
  • the SMPD1 gene encodes a peptide that is represented by NCBI Reference Sequence NP_000534.3 (SEQ ID NO: 37).
  • an isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 38.
  • the isolated nucleic acid comprises a SMPD1 -encoding sequence that has been codon optimized.
  • GCH1 protein refers to GTP cyclohydrolase I, which is a hydrolase enzyme that is part of the folate and biopterin biosynthesis pathways.
  • GCH1 gene is located on chromosome 14.
  • the GCH1 gene encodes a peptide that is represented by NCBI Reference Sequence NP_000152.1 (SEQ ID NO: 45).
  • an isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 46.
  • the isolated nucleic acid comprises a GCH1 -encoding sequence that has been codon optimized.
  • RAB7L protein refers to RAB7, member RAS oncogene family-like 1, which is a GTP binding protein.
  • RAB7L gene is located on chromosome 1.
  • the RAB7L gene encodes a peptide that is represented by NCBI Reference Sequence NP_003920.1 (SEQ ID NO: 47).
  • an isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 48.
  • the isolated nucleic acid comprises a RAB7L-encoding sequence that has been codon optimized.
  • VPS35 protein refers to vacuolar protein sorting- associated protein 35, which is part of a protein complex involved in retrograde transport of proteins from endosomes to the trans-Golgi network.
  • VPS35 gene is located on chromosome 16.
  • the VPS35 gene encodes a peptide that is represented by NCBI Reference Sequence NP_060676.2 (SEQ ID NO: 49).
  • an isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 50.
  • the isolated nucleic acid comprises a VPS 35 -encoding sequence that has been codon optimized.
  • an isolated nucleic acid comprising an expression construct encoding IL-34 protein (e.g., the gene product of IL34 gene).
  • IL-34 protein refers to interleukin 34, which is a cytokine that increases growth and survival of monocytes.
  • IL34 gene is located on chromosome 16.
  • the IL34 gene encodes a peptide that is represented by NCBI Reference Sequence NP_689669.2 (SEQ ID NO: 55).
  • an isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 56.
  • the isolated nucleic acid comprises a IL-34-encoding sequence that has been codon optimized.
  • TREM2 protein refers to triggering receptor expressed on myeloid cells 2, which is an immunoglobulin superfamily receptor found on myeloid cells. In humans, TREM2 gene is located on
  • the TREM2 gene encodes a peptide that is represented by NCBI Reference Sequence NP_061838.1 (SEQ ID NO: 57).
  • the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 58.
  • an isolated nucleic acid comprises a TREM2-encoding sequence that has been codon optimized.
  • aspects of the disclosure relate to an isolated nucleic acid comprising an expression construct encoding TMEM106B protein (e.g ., the gene product of TMEM106B gene).
  • TMEM106B protein refers to transmembrane protein 106B, which is a protein involved in dendrite morphogenesis and regulation of lysosomal trafficking.
  • TMEM106B gene is located on chromosome 7.
  • the TMEM106B gene encodes a peptide that is represented by NCBI Reference Sequence NP_060844.2 (SEQ ID NO: 63).
  • an isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 64.
  • the isolated nucleic acid comprises a TMEM106B -encoding sequence that has been codon optimized.
  • PGRN protein refers to progranulin, which is a protein involved in development, inflammation, cell proliferation and protein homeostasis.
  • PGRN also referred to as GRN
  • the GRN gene encodes a peptide that is represented by NCBI Reference Sequence NP_002078.1 (SEQ ID NO: 67).
  • an isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 68.
  • the isolated nucleic acid comprises a PGRN-encoding sequence (GRN- encoding sequence) that has been codon optimized.
  • the disclosure provides an isolated nucleic acid comprising an expression construct encoding a first gene product and a second gene product, wherein each gene product independently is selected from the gene products, or portions thereof, set forth in Table 1 or an inhibitory nucleic acid targeting a gene or gene product set forth in Table 1.
  • a gene product is encoded by a coding portion (e.g., a cDNA) of a naturally occurring gene.
  • a first gene product is a protein (or a fragment thereof) encoded by the GBA1 gene.
  • a gene product is a protein (or a fragment thereof) encoded by another gene listed in Table 1, for example the SCARB2/LIMP2 gene or the PSAP gene.
  • a first gene product e.g., Gcase
  • a second gene product e.g., LIMP2, etc.
  • a gene product is a fragment (e.g., portion) of a gene listed in Table 1.
  • a protein fragment may comprise about 50%, about 60%, about 70%, about 80% about 90% or about 99% of a protein encoded by the genes listed in Table 1.
  • a protein fragment comprises between 50% and 99.9% (e.g ., any value between 50% and 99.9%) of a protein encoded by a gene listed in Table 1.
  • a-Syn a-Synuclein
  • isolated nucleic acids described herein comprise an inhibitory nucleic acid that reduces or prevents expression of a-Syn protein.
  • the disclosure provides an isolated nucleic acid comprising an expression construct encoding one or more interfering nucleic acids (e.g., dsRNA, siRNA, miRNA, amiRNA, etc.) that target an microtubule-associated protein tau, MAPT (e.g., the gene product of MAPT gene), which is involved in Alzheimer’s disease and FTD-tau.
  • interfering nucleic acids e.g., dsRNA, siRNA, miRNA, amiRNA, etc.
  • MAPT microtubule-associated protein tau
  • an isolated nucleic acid as described herein may encode 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more inhibitory nucleic acids (e.g., dsRNA, siRNA, shRNA, miRNA, amiRNA, etc.). In some embodiments, an isolated nucleic acid encodes more than 10 inhibitory nucleic acids. In some embodiments, each of the one or more inhibitory nucleic acids targets a different gene or a portion of a gene (e.g., a first miRNA targets a first target sequence of a gene and a second miRNA targets a second target sequence of the gene that is different than the first target sequence). In some embodiments, each of the one or more inhibitory nucleic acids targets the same target sequence of the same gene (e.g., an isolated nucleic acid encodes multiple copies of the same miRNA).
  • inhibitory nucleic acids e.g., dsRNA, siRNA, shRNA, miRNA, amiRNA, etc.
  • an isolated nucleic acid encodes more than 10 inhibitory nucle
  • the disclosure provides relate to an isolated nucleic acid comprising an expression construct encoding one or more interfering nucleic acids (e.g., dsRNA, siRNA, miRNA, amiRNA, etc.) that target an a-Synuclein protein (e.g., the gene product of SNCA gene).
  • a-Synuclein protein refers to a protein found in brain tissue, which is plays a role in maintaining a supply of synaptic vesicles in presynaptic terminals by clustering synaptic vesicles and regulating the release of dopamine.
  • SNCA gene is located on chromosome 4.
  • the SNCA gene encodes a peptide that is represented by NCBI Reference Sequence NP_001139527.1.
  • a SNCA gene comprises the sequence set forth in SEQ ID NO: 90.
  • An inhibitory nucleic acid targeting SNCA may comprise a region of complementarity (e.g., a region of the inhibitory nucleic acid that hybridizes to the target gene, such as SNCA) that is between 6 and 50 nucleotides in length.
  • an inhibitory nucleic acid comprises a region of complementarity with SNCA that is between about 6 and 30, about 8 and 20, or about 10 and 19 nucleotides in length.
  • an inhibitory nucleic acid is complementary with at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous nucleotides of a SNCA sequence.
  • the disclosure provides an isolated nucleic acid comprising an expression construct encoding one or more interfering nucleic acids (e.g., dsRNA, siRNA, miRNA, amiRNA, etc.) that target an TMEM106B protein (e.g., the gene product of
  • TMEM106B gene refers to transmembrane protein 106B, which is a protein involved in dendrite morphogenesis and regulation of lysosomal trafficking. In humans, TMEM106B gene is located on chromosome 7. In some embodiments, the TMEM106B gene encodes a peptide that is represented by NCBI Reference Sequence NP_060844.2. In some embodiments, a TMEM106B gene comprises the sequence set forth in SEQ ID NO: 91.
  • An inhibitory nucleic acid targeting TMEM106B may comprise a region of
  • an inhibitory nucleic acid comprises a region of complementarity with TMEM106B that is between about 6 and 30, about 8 and 20, or about 10 and 19 nucleotides in length.
  • an inhibitory nucleic acid is complementary with at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous nucleotides of a TMEM106B sequence.
  • the disclosure provides an isolated nucleic acid comprising an expression construct encoding one or more interfering nucleic acids (e.g., dsRNA, siRNA, miRNA, amiRNA, etc.) that target an ribosomal protein s25 (RPS25) (e.g., the gene product of RPS25).
  • RPS25 protein refers to a ribosomal protein which is a subunit of the s40 ribosome, a protein complex involved in protein synthesis. In humans, RPS25 gene is located on
  • the RPS25 gene encodes a peptide that is represented by NCBI Reference Sequence NP_001019.1.
  • a RPS25 gene comprises the sequence set forth in SEQ ID NO: 113.
  • An inhibitory nucleic acid targeting RPS25 may comprise a region of complementarity (e.g., a region of the inhibitory nucleic acid that hybridizes to the target gene, such as RPS25) that is between 6 and 50 nucleotides in length.
  • an inhibitory nucleic acid comprises a region of complementarity with RPS25 that is between about 6 and 30, about 8 and 20, or about 10 and 19 nucleotides in length.
  • an inhibitory nucleic acid is complementary with at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous nucleotides of a RPS25 sequence.
  • the disclosure provides an isolated nucleic acid comprising an expression construct encoding one or more interfering nucleic acids (e.g., dsRNA, siRNA, miRNA, amiRNA, etc.) that target an microtubule-associated protein tau, MAPT (e.g., the gene product of MAPT gene).
  • MAPT protein refers to microtubule-associated protein tau, which is a protein involved in microtubule stabilization.
  • MAPT gene is located on chromosome 17.
  • the MAPT gene encodes a peptide that is represented by NCBI Reference Sequence NP_005901.2.
  • a MAPT gene comprises the sequence set forth in SEQ ID NO: 114.
  • An inhibitory nucleic acid targeting MAPT may comprise a region of complementarity (e.g., a region of the inhibitory nucleic acid that hybridizes to the target gene, such as MAPT) that is between 6 and 50 nucleotides in length.
  • an inhibitory nucleic acid comprises a region of complementarity with MAPT that is between about 6 and 30, about 8 and 20, or about 10 and 19 nucleotides in length.
  • an inhibitory nucleic acid is complementary with at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous nucleotides of a MAPT sequence.
  • aspects of the disclosure relate to isolated nucleic acids encoding one or more gene products (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more gene products).
  • the one or more gene products are two or more proteins.
  • the one or more gene products are two or more inhibitory nucleic acids.
  • the one or more gene products are one or more protein and one or more inhibitory nucleic acid.
  • the disclosure provides an isolated nucleic acid comprising an expression construct encoding a first gene product and a second gene product, wherein each gene product independently is selected from the gene products, or portions thereof, set forth in Table 1 or an inhibitory nucleic acid targeting a gene or gene product set forth in Table 1.
  • a sequence encoding an inhibitory nucleic acid may be placed in an untranslated region (e.g., intron, 5’UTR, 3’UTR, etc.) of the expression vector.
  • a gene product is encoded by a coding portion (e.g., a cDNA) of a naturally occurring gene.
  • a first gene product is a protein (or a fragment thereof) encoded by the GBA1 gene.
  • a gene product is an inhibitory nucleic acid that targets (e.g., hybridizes to, or comprises a region of complementarity with) a
  • PD-associated gene e.g., SNCA
  • Gcase a gene product
  • second gene product e.g., inhibitory RNA targeting
  • SNCA can generally be reversed (e.g., the inhibitory RNA is the first gene product and Gcase is the second gene product).
  • a gene product is a fragment (e.g., portion) of a gene listed in Table 1.
  • a protein fragment may comprise about 50%, about 60%, about 70%, about 80% about 90% or about 99% of a protein encoded by the genes listed in Table 1.
  • a protein fragment comprises between 50% and 99.9% (e.g., any value between 50% and 99.9%) of a protein encoded by a gene listed in Table 1.
  • a gene product hybridizes to portion of a target gene (e.g., is complementary to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more contiguous nucleotides of a target gene, for example S/VCA).
  • a target gene e.g., is complementary to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more contiguous nucleotides of a target gene, for example S/VCA.
  • an expression construct is monocistronic (e.g., the expression construct encodes a single fusion protein comprising a first gene product and a second gene product).
  • an expression construct is polycistronic (e.g., the expression construct encodes two distinct gene products, for example two different proteins or protein fragments).
  • a polycistronic expression vector may comprise a one or more (e.g., 1, 2, 3, 4, 5, or more) promoters.
  • Any suitable promoter can be used, for example, a constitutive promoter, an inducible promoter, an endogenous promoter, a tissue-specific promoter (e.g., a CNS- specific promoter), etc.
  • a promoter is a chicken beta-actin promoter (CBA promoter), a CAG promoter (for example as described by Alexopoulou et al. (2008) BMC Cell Biol. 9:2; doi: 10.1186/1471-2121-9-2), a CD68 promoter, or a JeT promoter (for example as described by Tornpc et al.
  • a promoter is operably-linked to a nucleic acid sequence encoding a first gene product, a second gene product, or a first gene product and a second gene product.
  • an expression cassette comprises one or more additional regulatory sequences, including but not limited to transcription factor binding sequences, intron splice sites, poly(A) addition sites, enhancer sequences, repressor binding sites, or any combination of the foregoing.
  • a nucleic acid sequence encoding a first gene product and a nucleic acid sequence encoding a second gene product are separated by a nucleic acid sequence encoding an internal ribosomal entry site (IRES).
  • IRES sites are described, for example, by Mokrejs et al. (2006) Nucleic Acids Res. 34(Database issue):D125-30.
  • a nucleic acid sequence encoding a first gene product and a nucleic acid sequence encoding a second gene product are separated by a nucleic acid sequence encoding a self cleaving peptide.
  • self-cleaving peptides include but are not limited to T2A, P2A, E2A, F2A, BmCPV 2A, and BmIFV 2A, and those described by Liu et al. (2017) Sci Rep. 7: 2193.
  • the self-cleaving peptide is a T2A peptide.
  • an inhibitory nucleic acid is positioned in an intron of an expression construct, for example in an intron upstream of the sequence encoding a first gene product.
  • An inhibitory nucleic acid can be a double stranded RNA (dsRNA), siRNA, micro
  • RNA RNA
  • amiRNA artificial miRNA
  • an inhibitory nucleic acid binds to (e.g ., hybridizes with) between about 6 and about 30 (e.g., any integer between 6 and 30, inclusive) contiguous nucleotides of a target RNA (e.g., mRNA).
  • the inhibitory nucleic acid molecule is an miRNA or an amiRNA, for example an miRNA that targets SNCA (the gene encoding a-Syn protein) or TMEM106B (e.g.. the gene encoding TMEM106B protein).
  • the miRNA does not comprise any mismatches with the region of SNCA mRNA to which it hybridizes (e.g., the miRNA is “perfected”).
  • the inhibitory nucleic acid is an shRNA (e.g., an shRNA targeting SNCA or TMEM106B).
  • an inhibitory nucleic acid is an artificial miRNA (amiRNA) that includes a miR-155 scaffold and a SNCA or TMEM106B targeting sequence.
  • an inhibitory nucleic acid is an artificial microRNA (amiRNA).
  • amiRNA artificial microRNA
  • a microRNA typically refers to a small, non-coding RNA found in plants and animals and functions in transcriptional and post-translational regulation of gene expression.
  • MiRNAs are transcribed by RNA polymerase to form a hairpin-loop structure referred to as a pri-miRNAs which are subsequently processed by enzymes (e.g., Drosha, Pasha, spliceosome, etc.) to for a pre-miRNA hairpin structure which is then processed by Dicer to form a miRNA/miRNA* duplex (where * indicates the passenger strand of the miRNA duplex), one strand of which is then incorporated into an RNA-induced silencing complex (RISC).
  • an inhibitory RNA as described herein is a miRNA targeting SNCA or TMEM106B.
  • an inhibitory nucleic acid targeting SNCA comprises a miRNA/miRNA* duplex.
  • the miRNA strand of a miRNA/miRNA* duplex comprises or consists of the sequence set forth in any one of SEQ ID NOs: 20-25.
  • the miRNA* strand of a miRNA/miRNA* duplex comprises or consists of the sequence set forth in any one of SEQ ID NOs: 20-25.
  • an inhibitory nucleic acid targeting TMEM106B comprises a miRNA/miRNA* duplex.
  • the miRNA strand of a miRNA/miRNA* duplex comprises or consists of the sequence set forth in SEQ ID NO: 92 or 93.
  • the miRNA* strand of a miRNA/miRNA* duplex comprises or consists of the sequence set forth in SEQ ID NOs: 92 or 93.
  • an artificial microRNA is derived by modifying native miRNA to replace natural targeting regions of pre-mRNA with a targeting region of interest.
  • a naturally occurring, expressed miRNA can be used as a scaffold or backbone (e.g., a pri-miRNA scaffold), with the stem sequence replaced by that of an miRNA targeting a gene of interest.
  • An artificial precursor microRNA pre-amiRNA is normally processed such that one single stable small RNA is preferentially generated.
  • scAAV vectors and scAAVs described herein comprise a nucleic acid encoding an amiRNA.
  • the pri- miRNA scaffold of the amiRNA is derived from a pri-miRNA selected from the group consisting of pri-MIR-21, pri-MIR-22, pri-MIR-26a, pri-MIR-30a, pri-MIR-33, pri-MIR-122, pri-MIR-375, pri-MIR-199, pri-MIR-99, pri-MIR-194, pri-MIR-155, and pri-MIR-451.
  • an amiRNA comprises a nucleic acid sequence targeting SNCA or TMEM106B and an eSIBR amiRNA scaffold, for example as described in Fowler et al. Nucleic Acids Res. 2016 Mar 18; 44(5): e48.
  • an amiRNA targeting SNCA comprises or consists of the sequence set forth in any one of SEQ ID NOs: 94-99.
  • an amiRNA targeting TMEM106B comprises or consists of the sequence set forth in SEQ ID NOs: 65-66.
  • an amiRNA targeting RPS25 comprises or consists of the sequence set forth in SEQ ID NOs: 115 to 122.
  • an amiRNA targeting MAPT comprises or consists of the sequence set forth in SEQ ID NOs: 123-138.
  • an isolated nucleic acid or vector (e.g., rAAV vector) described by the disclosure comprises or consists of a sequence set forth in any one of SEQ ID NOs: 1-13, 15, 17, 19-29, 31, 32, 34, 36, 38-44, 46, 48, 50-54, 56, 58-62, 64-66, and 68-145.
  • an isolated nucleic acid or vector (e.g., rAAV vector) described by the disclosure comprises or consists of a sequence that is complementary (e.g., the complement of) a sequence set forth in any one of SEQ ID NOs: 1-13, 15, 17, 19-29, 31, 32, 34, 36, 38-44, 46, 48, 50-54,
  • an isolated nucleic acid or vector (e.g., rAAV vector) described by the disclosure comprises or consists of a sequence that is a reverse complement of a sequence set forth in any one of SEQ ID NOs: 1-13, 15, 17, 19-29, 31, 32, 34, 36, 38-44, 46, 48, 50-54, 56, 58-62, 64-66, and 68-145.
  • an isolated nucleic acid or vector (e.g., rAAV vector) described by the disclosure comprises or consists of a portion of a sequence set forth in any one of SEQ ID NOs: 1-13, 15, 17, 19-29, 31, 32, 34, 36, 38-44, 46, 48, 50-54, 56, 58-62, 64-66, and 68-145.
  • a portion may comprise at least 25%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of a sequence set forth in any one of SEQ ID NOs: 1-13,
  • a nucleic acid sequence described by the disclosure is a nucleic acid sense strand (e.g., 5’ to 3’ strand), or in the context of a viral sequences a plus (+) strand.
  • a nucleic acid sequence described by the disclosure is a nucleic acid antisense strand (e.g., 3’ to 5’ strand), or in the context of viral sequences a minus (-) strand.
  • any one or more thymidine (T) nucleotides or uridine (U) nucleotides in a sequence provided herein may be replaced with any other nucleotide suitable for base pairing (e.g., via a Watson-Crick base pair) with an adenosine nucleotide.
  • T may be replaced with U
  • U may be replaced with T.
  • a vector can be a plasmid, cosmid, phagemid, bacterial artificial chromosome (BAC), or a viral vector (e.g., adenoviral vector, adeno-associated virus (AAV) vector, retroviral vector, baculoviral vector, etc.).
  • the vector is a plasmid (e.g., a plasmid comprising an isolated nucleic acid as described herein).
  • an rAAV vector is single- stranded (e.g., single- stranded DNA).
  • the vector is a recombinant AAV (rAAV) vector.
  • a vector is a Baculovirus vector (e.g., an Autographa californica nuclear polyhedrosis (AcNPV) vector).
  • an rAAV vector (e.g., rAAV genome) comprises a transgene (e.g., an expression construct comprising one or more of each of the following: promoter, intron, enhancer sequence, protein coding sequence, inhibitory RNA coding sequence, polyA tail sequence, etc.) flanked by two AAV inverted terminal repeat (ITR) sequences.
  • the transgene of an rAAV vector comprises an isolated nucleic acid as described by the disclosure.
  • each of the two ITR sequences of an rAAV vector is a full-length ITR (e.g., approximately 145 bp in length, and containing functional Rep binding site (RBS) and terminal resolution site (trs)).
  • one of the ITRs of an rAAV vector is truncated (e.g., shortened or not full-length).
  • a truncated ITR lacks a functional terminal resolution site (trs) and is used for production of self-complementary AAV vectors (scAAV vectors).
  • scAAV vectors self-complementary AAV vectors
  • a truncated ITR is a AITR, for example as described by McCarty et al. (2003) Gene Ther. 10(26):2112-8.
  • nucleic acids e.g., rAAV vectors
  • isolated nucleic acids comprising an ITR having one or more modifications (e.g., nucleic acid additions, deletions, substitutions, etc.) relative to a wild-type AAV ITR, for example relative to wild-type AAV2 ITR (e.g., SEQ ID NO: 1
  • wild-type AAV2 ITR The structure of wild-type AAV2 ITR is shown in FIG. 20. Generally, a wild-type
  • ITR comprises a 125 nucleotide region that self-anneals to form a palindromic double- stranded
  • T-shaped, hairpin structure consisting of two cross arms (formed by sequences referred to as
  • the“D” region of an ITR is positioned between the stem region formed by the A/A' sequences and the insert containing the transgene of the rAAV vector (e.g., positioned on the“inside” of the ITR relative to the terminus of the ITR or proximal to the transgene insert or expression construct of the rAAV vector).
  • a“D” region comprises the sequence set forth in SEQ ID NO: 27. The“D” region has been observed to play an important role in encapsidation of rAAV vectors by capsid proteins, for example as disclosed by Ling et al. (2015) J Mol Genet Med 9(3).
  • rAAV vectors comprising a“D” region located on the“outside” of the ITR are efficiently encapsidated by AAV capsid proteins than rAAV vectors having ITRs with unmodified (e.g., wild-type) ITRs
  • rAAV vectors having a modified“D” sequence e.g., a“D” sequence in the“outside” position
  • a modified“D” sequence comprises at least one nucleotide substitution relative to a wild-type“D” sequence (e.g., SEQ ID NO: 27).
  • a modified“D” sequence may have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 nucleotide substitutions relative to a wild-type“D” sequence (e.g., SEQ ID NO: 27).
  • a modified “D” sequence comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 nucleic acid
  • substitutions relative to a wild-type“D” sequence e.g., SEQ ID NO: 27.
  • a modified“D” sequence is between about 10% and about 99% (e.g., 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%) identical to a wild-type“D” sequence (e.g., SEQ ID NO: 27).
  • a modified “D” sequence comprises the sequence set forth in SEQ ID NO: 26, also referred to as an“S” sequence as described in Wang et al. (1995) J Mol Biol 250(5):573-80.
  • An isolated nucleic acid or rAAV vector as described by the disclosure may further comprise a“TRY” sequence, for example as set forth in SEQ ID NO: 28 or as described by Francois, et al. 2005.
  • a TRY sequence is positioned between an ITR (e.g. a 5’ ITR) and an expression construct (e.g. a transgene-encoding insert) of an isolated nucleic acid or rAAV vector.
  • aspects of the disclosure relate to constructs which are configured to express one or more transgenes in myeloid cells (e.g., CNS myeloid cells, such as microglia) of a subject.
  • myeloid cells e.g., CNS myeloid cells, such as microglia
  • a construct (e.g., gene expression vector) comprises a protein coding sequence that is operably linked to a myeloid cell-specific promoter.
  • myeloid cell-specific promoters include CD68 promoter, lysM promoter, csflr promoter, CD 11c promoter, c-fes promoter, and F4/80 promoter, for example as described in Lin et al. Adv Exp Med Biol. 2010;706:149-56.
  • a myeloid cell-specific promoter is a CD68 promoter or a F4/80 promoter.
  • the disclosure relates to Baculovims vectors comprising an isolated nucleic acid or rAAV vector as described by the disclosure.
  • the disclosure relates to Baculovims vectors comprising an isolated nucleic acid or rAAV vector as described by the disclosure.
  • Baculovims vector is an Autographa californica nuclear polyhedrosis (AcNPV) vector, for example as described by Urabe et al. (2002) Hum Gene Ther 13(16): 1935-43 and Smith et al. (2009) Mol Ther 17(11): 1888-1896.
  • AcNPV Autographa californica nuclear polyhedrosis
  • the disclosure provides a host cell comprising an isolated nucleic acid or vector as described herein.
  • a host cell can be a prokaryotic cell or a eukaryotic cell.
  • a host cell can be a mammalian cell, bacterial cell, yeast cell, insect cell, etc.
  • a host cell is a mammalian cell, for example a HEK293T cell.
  • a host cell is a bacterial cell, for example an E. coli cell. rAAVs
  • the disclosure relates to recombinant AAVs (rAAVs) comprising a transgene that encodes one or more isolated nucleic acids as described herein (e.g ., an rAAV vector encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more gene products described herein and/or inhibitory nucleic acids targeting gene products described herein).
  • rAAVs generally refers to viral particles comprising an rAAV vector encapsidated by one or more AAV capsid proteins.
  • An rAAV described by the disclosure may comprise a capsid protein having a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10, or a variant thereof.
  • a capsid protein is an AAV9 capsid protein or a variant thereof.
  • an AAV9 capsid protein variant comprises a mutation at one or more positions corresponding to T492, Y705, and Y731 of SEQ ID NO: 147 (e.g., corresponding to those positions of AAV6).
  • the one or more mutations are selected from T492V, Y705F, Y73 IF, or a combination thereof.
  • an AAV9 capsid protein variant comprises the amino acid sequence set forth in SEQ ID NO: 149.
  • an rAAV comprises a capsid protein from a non-human host, for example a rhesus AAV capsid protein such as AAVrh.10, AAVrh.39, etc.
  • a rhesus AAV capsid protein such as AAVrh.10, AAVrh.39, etc.
  • an rAAV described by the disclosure comprises a capsid protein that is a variant of a wild-type capsid protein, such as a capsid protein variant that includes at least 1, 2, 3, 4, 5,
  • an AAV capsid protein variant is an AAV1RX capsid protein, for example as described by Albright et al. Mol Ther. 2018 Feb 7;26(2):510-523.
  • a capsid protein is AAV1RX and comprises the amino acid sequence set forth in SEQ ID NO: 146 (or is encoded by the nucleic acid sequence set forth in SEQ ID NO: 145).
  • a capsid protein variant is an AAV TM6 capsid protein, for example as described by Rosario et al. Mol Ther Methods Clin Dev. 2016; 3: 16026.
  • an AAV6 capsid protein variant is AAV-TM6 capsid protein and comprises the amino acid sequence set forth in SEQ ID NO: 148.
  • rAAVs described by the disclosure readily spread through the CNS, particularly when introduced into the CSF space or directly into the brain parenchyma. Accordingly, in some embodiments, rAAVs described by the disclosure comprise a capsid protein that is capable of crossing the blood-brain barrier (BBB).
  • BBB blood-brain barrier
  • an rAAV comprises a capsid protein having an AAV9 or AAVrh.lO serotype. Production of rAAVs is described, for example, by Samulski et al. (1989) J Virol. 63(9):3822-8 and Wright (2009) Hum Gene Ther. 20(7): 698-706.
  • an rAAV comprises a capsid protein that specifically or preferentially targets myeloid cells, for example microglial cells.
  • an rAAV transduces microglial cells.
  • an rAAV as described by the disclosure e.g ., comprising a recombinant rAAV genome encapsidated by AAV capsid proteins to form an rAAV capsid particle
  • BEVS Baculovirus vector expression system
  • Production of rAAVs using BEVS are described, for example by Urabe et al. (2002) Hum Gene Ther 13(16): 1935-43, Smith et al. (2009) Mol Ther 17(11): 1888-1896, U.S. Patent No. 8,945,918, U.S. Patent No. 9,879,282, and International PCT Publication WO 2017/184879.
  • an rAAV can be produced using any suitable method (e.g., using recombinant rep and cap genes).
  • an rAAV as disclosed herein is produced in HEK293 (human embryonic kidney) cells.
  • the disclosure provides pharmaceutical compositions comprising an isolated nucleic acid or rAAV as described herein and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, e.g., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
  • the term“pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the invention within or to the patient such that it may perform its intended function. Additional ingredients that may be included in the pharmaceutical compositions used in the practice of the invention are known in the art and described, for example in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.
  • compositions e.g ., pharmaceutical compositions
  • enteral e.g., oral
  • parenteral intravenous
  • intramuscular intra-arterial
  • intramedullary intrathecal
  • subcutaneous intraventricular
  • transdermal transdermal
  • interdermal interdermal
  • rectal intravaginal
  • intraperitoneal topical
  • mucosal nasal, bucal, sublingual
  • the most appropriate route of administration will depend upon a variety of factors including the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), and/or the condition of the subject (e.g., whether the subject is able to tolerate oral administration).
  • the compound or pharmaceutical composition described herein is suitable for topical administration to the eye of a subject.
  • a composition comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) different rAAVs, each rAAV comprising an isolated nucleic acid that encodes a different gene product (e.g., a different protein or inhibitory nucleic acid).
  • the different rAAVs may comprise a capsid protein of the same serotype or different serotypes.
  • compositions for expression of one or more CNS disease-associated gene products in a subject to treat CNS -associated diseases relate to compositions for expression of one or more CNS disease-associated gene products in a subject to treat CNS -associated diseases.
  • the one or more CNS disease-associated gene products relate to compositions for expression of one or more CNS disease-associated gene products in a subject to treat CNS -associated diseases.
  • CNS disease-associated gene products may be encoded by one or more isolated nucleic acids or rAAV vectors.
  • a subject is administered a single vector (e.g., isolated nucleic acid, rAAV, etc.) encoding one or more (1, 2, 3, 4, 5, or more) gene products.
  • a subject is administered a plurality ( e.g ., 2, 3, 4, 5, or more) vectors (e.g., isolated nucleic acids, rAAVs, etc.), where each vector encodes a different CNS disease- associated gene product.
  • a CNS -associated disease may be a neurodegenerative disease, synucleinopathy, tauopathy, or a lysosomal storage disease. Examples of neurodegenerative diseases and their associated genes are listed in Table 2.
  • A“synucleinopathy” refers to a disease or disorder characterized by accumulation, overexpression or activity of alpha-Synuclein (the gene product of SNCA ) in a subject (e.g., relative to a healthy subject, for example a subject not having a synucleinopathy). Examples of synucleinopathies and their associated genes are listed in Table 3.
  • A“tauopathy” refers to a disease or disorder characterized by accumulation
  • tauopathies overexpression or activity of Tau protein in a subject (e.g., a healthy subject not having a tauopathy).
  • a subject e.g., a healthy subject not having a tauopathy.
  • tauopathies and their associated genes are listed in Table 4.
  • A“lysosomal storage disease” refers to a disease characterized by abnormal build-up of toxic cellular products in lysosomes of a subject. Examples of lysosomal storage diseases and their associated genes are listed in Table 5.
  • the disclosure relates to methods of treating a disease selected from Parkinson’s Disease (e.g., Parkinson’s Disease with GBA1 mutation (PD-GBA), sporadic Parkinson’s Disease (sPD)), Gaucher Disease (e.g., neuronopathic Gaucher disease (nGD), Type I Gaucher Disease (T1GD), Type II Gaucher Disease (T2GD), and Type III Gaucher Disease (T3GD)), Dementia with Lewy Bodies (DLB), Amyotrophic lateral sclerosis (ALS), and Niemann-Pick Type C disease (NPC) by administering to a subject in need thereof an isolated nucleic acid (e.g., an rAAV vector or rAAV comprising an isolated nucleic acid) that encodes GBA1.
  • Parkinson’s Disease e.g., Parkinson’s Disease with GBA1 mutation (PD-GBA), sporadic Parkinson’s Disease (sPD)
  • Gaucher Disease e.g., neuronopathic Gaucher disease (nGD), Type I Gaucher Disease
  • the disclosure relates to methods of treating Frontotemporal Dementia (e.g., Frontotemporal Dementia with GRN mutation (FTD-GRN), Frontotemporal Dementia with MAPT mutation (FTD-tau), and Frontotemporal Dementia with C90RF72 mutation (FTD-C9orf72)), Parkinson’s Disease (PD), Alzheimer’s Disease (AD), Neuronal Ceroid Lipofuscinosis (NCL), Corticobasal Degeneration (CBD), Motor Neuron Disease (MND), or Gaucher Disease (GD) by administering to a subject in need thereof an isolated nucleic acid (e.g., an rAAV vector or rAAV comprising an isolated nucleic acid) that encodes PGRN (also referred to as GRN).
  • FTD-GRN Frontotemporal Dementia with GRN mutation
  • FTD-tau Frontotemporal Dementia with MAPT mutation
  • FTD-C9orf72 Frontotemporal Dementi
  • the disclosure relates to methods of treating Synucleinopathies
  • an isolated nucleic acid e.g., an rAAV vector or rAAV comprising an isolated nucleic acid
  • an inhibitory nucleic acid targeting SNCA e.g., SNCA
  • the disclosure relates to methods of treating a disease selected from Parkinson’s Disease (PD), Frontotemporal Dementia (e.g., Frontotemporal Dementia with GRN mutation (FTD-GRN)), Lysosomal Storage Diseases (LSDs), or Gaucher Disease (GD) by administering to a subject in need thereof an isolated nucleic acid (e.g., an rAAV vector or rAAV comprising an isolated nucleic acid) that encodes PSAP.
  • PD Parkinson’s Disease
  • Frontotemporal Dementia e.g., Frontotemporal Dementia with GRN mutation (FTD-GRN)
  • LSDs Lysosomal Storage Diseases
  • GD Gaucher Disease
  • the disclosure relates to methods of treating Alzheimer’s Disease (AD), Nasu-Hakola Disease (NHD) Frontotemporal Dementia with MAPT mutation (FTD-Tau), or Parkinson’s Disease (PD), by administering to a subject in need thereof an isolated nucleic acid (e.g., an rAAV vector or rAAV comprising an isolated nucleic acid) that encodes TREM2.
  • AD Alzheimer’s Disease
  • NHS Nasu-Hakola Disease
  • FTD-Tau Frontotemporal Dementia with MAPT mutation
  • PD Parkinson’s Disease
  • the disclosure relates to methods of treating Alzheimer’s disease (AD) or Frontotemporal Dementia (Frontotemporal Dementia with MAPT mutation (FTD-Tau), a tauopathy, Progressive supranuclear palsy (PSP), neurodegenerative disease, Lewy Body Disease (LBD) or Parkinson’s Disease by administering to a subject in need thereof an isolated nucleic acid (e.g., an rAAV vector or rAAV comprising an isolated nucleic acid) that encodes inhibitory nucleic acids targeting MAPT.
  • an isolated nucleic acid e.g., an rAAV vector or rAAV comprising an isolated nucleic acid
  • “treat” or“treating” refers to (a) preventing or delaying onset of a CNS disease; (b) reducing severity of a CNS disease; (c) reducing or preventing development of symptoms characteristic of a CNS disease; (d) and/or preventing worsening of symptoms characteristic of a CNS disease.
  • Symptoms of CNS disease may include, for example, motor dysfunction (e.g., shaking, rigidity, slowness of movement, difficulty with walking, paralysis), cognitive dysfunction (e.g., dementia, depression, anxiety, psychosis), difficulty with memory, emotional and behavioral dysfunction.
  • compositions for expression of combinations of CNS diseases-associated genes e.g., PD-associated gene products
  • a subject that act together (e.g., synergistically) to treat the disease.
  • the disclosure provides a method for treating a subject having or suspected of having CNS-associated diseases (e.g., Parkinson’s disease, AD, FTD, etc.), the method comprising administering to the subject a composition (e.g., a composition comprising an isolated nucleic acid or a vector or a rAAV) as described by the disclosure.
  • a composition e.g., a composition comprising an isolated nucleic acid or a vector or a rAAV
  • a subject has one or more signs or symptoms, or has a genetic predisposition (e.g., a mutation in a gene listed in Table 1) to a neurodegenerative disease listed in Table 2.
  • a subject has one or more signs or symptoms, or has a genetic predisposition (e.g., a mutation in a gene listed in Table 1) to a synucleinopathy listed in Table 3.
  • a subject has one or more signs or symptoms, or has a genetic predisposition (e.g., a mutation in a gene listed in Table 1) to a tauopathy listed in Table 4.
  • a subject has one or more signs or symptoms, or has a genetic predisposition (e.g., a mutation in a gene listed in Table 1) to a tauopathy listed in Table 4.
  • a subject has one or more signs or symptoms, or has a genetic predisposition
  • predisposition e.g., a mutation in a gene listed in Table 1
  • lysosomal storage disease listed in Table 5.
  • the disclosure is based, in part, on compositions for expression of one or more CNS- disease associated gene products in a subject to treat Gaucher disease.
  • the Gaucher disease is a neuronopathic Gaucher disease, for example Type 2 Gaucher disease or Type 3 Gaucher disease.
  • a subject does not have PD or PD symptoms.
  • the disclosure provides a method for treating a subject having or suspected of having neuronopathic Gaucher disease, the method comprising administering to the subject a composition (e.g., a composition comprising an isolated nucleic acid or a vector or a rAAV) as described by the disclosure.
  • a composition e.g., a composition comprising an isolated nucleic acid or a vector or a rAAV
  • the subject does not have Alzheimer’s disease.
  • the disclosure provides a method for treating a subject having or suspected of having FTD, the method comprising administering to the subject a composition (e.g., a composition comprising an isolated nucleic acid or a vector or a rAAV) as described by the disclosure.
  • a composition e.g., a composition comprising an isolated nucleic acid or a vector or a rAAV
  • FTD fronto-temporal dementia
  • PGRN Progranulin
  • GRN Progranulin
  • the disclosure provides a method for delivering a transgene to microglial cells, the method comprising administering an rAAV as described herein to a subject.
  • a rAAV encoding a Gcase protein for treating Type 2 or Type 3 Gaucher disease or Parkinson’s disease with a GBA1 mutation is administered to a subject as a single dose, and the rAAV is not administered to the subject subsequently.
  • a rAAV encoding a Gcase protein is administered via a single suboccipital injection into the cistema magna.
  • the injection into the cisterna magna is performed under radiographic guidance.
  • a subject is typically a mammal, preferably a human.
  • a subject is between the ages of 1 month old and 10 years old (e.g., 1 month, 2 months, 3 months, 4, months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 3, years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or any age therebetween).
  • a subject is between 2 years old and 20 years old.
  • a subject is between 30 years old and 100 years old.
  • a subject is older than 55 years old.
  • a composition is administered directly to the CNS of the subject, for example by direct injection into the brain and/or spinal cord of the subject.
  • CNS-direct administration modalities include but are not limited to intracerebral injection, intraventricular injection, intracisternal injection, intraparenchymal injection, intrathecal injection, and any combination of the foregoing.
  • a composition is administered to a subject by intra-cistema magna (ICM) injection.
  • ICM intra-cistema magna
  • direct injection into the CNS of a subject results in transgene expression (e.g., expression of the first gene product, second gene product, and if applicable, third gene product) in the midbrain, striatum and/or cerebral cortex of the subject.
  • direct injection into the CNS results in transgene expression (e.g., expression of the first gene product, second gene product, and if applicable, third gene product) in the spinal cord and/or CSF of the subject.
  • direct injection to the CNS of a subject comprises convection enhanced delivery (CED).
  • CED convection enhanced delivery
  • Convection enhanced delivery is a therapeutic strategy that involves surgical exposure of the brain and placement of a small-diameter catheter directly into a target area of the brain, followed by infusion of a therapeutic agent (e.g., a composition or rAAV as described herein) directly to the brain of the subject.
  • a therapeutic agent e.g., a composition or rAAV as described herein
  • a composition is administered peripherally to a subject, for example by peripheral injection.
  • peripheral injection include subcutaneous injection, intravenous injection, intra-arterial injection, intraperitoneal injection, or any combination of the foregoing.
  • the peripheral injection is intra-arterial injection, for example injection into the carotid artery of a subject.
  • a composition e.g., a composition comprising an isolated nucleic acid or a vector or a rAAV as described by the disclosure is administered both peripherally and directly to the CNS of a subject.
  • a subject is administered a composition by intra-arterial injection (e.g., injection into the carotid artery) and by intraparenchymal injection (e.g., intraparenchymal injection by CED).
  • the direct injection to the CNS and the peripheral injection are simultaneous (e.g., happen at the same time).
  • the direct injection occurs prior (e.g., between 1 minute and 1 week, or more before) to the peripheral injection.
  • the direct injection occurs after (e.g., between 1 minute and 1 week, or more after) the peripheral injection.
  • a subject is administered an immunosuppressant prior to (e.g., between 1 month and 1 minute prior to) or at the same time as a composition as described herein.
  • the immunosuppressant is a corticosteroid (e.g., prednisone, budesonide, etc.), an mTOR inhibitor (e.g., sirolimus, everolimus, etc.), an antibody (e.g., adalimumab, etanercept, natalizumab, etc.), or methotrexate.
  • composition e.g., a composition comprising an isolated nucleic acid or a vector or a rAAV
  • amount of composition as described by the disclosure administered to a subject will vary depending on the administration method.
  • a rAAV as described herein is administered to a subject at a titer between about 10 9 Genome copies (GC)/kg and about 10 14 GC/kg (e.g., about 10 9 GC/kg, about 10 10 GC/kg, about 10 11 GC/kg, about 10 12 GC/kg, about 10 12 GC/kg, or about 10 14 GC/kg).
  • GC Genome copies
  • a subject is administered a high titer (e.g., >10 12 Genome Copies GC/kg of an rAAV) by injection to the CSF space, or by intraparenchymal injection.
  • a rAAV as described herein is administered to a subject at a dose ranging from about 1 x 10 10 vector genomes (vg) to about 1 x 10 17 vg by intravenous injection.
  • a rAAV as described herein is administered to a subject at a dose ranging from about 1 x 10 10 vg to about 1 x 10 16 vg by injection into the cisterna magna.
  • a composition e.g., a composition comprising an isolated nucleic acid or a vector or a rAAV
  • a composition can be administered to a subject once or multiple times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or more) times.
  • a composition is administered to a subject continuously (e.g., chronically), for example via an infusion pump.
  • Example 1 rAAV vectors AAV vectors are generated using cells, such as HEK293 cells for triple-plasmid transfection.
  • the ITR sequences flank an expression construct comprising a promoter/enhancer element for each transgene of interest, a 3’ polyA signal, and posttranslational signals such as the WPRE element.
  • Multiple gene products can be expressed simultaneously such as GBA1 and
  • LIMP2 and/or Prosaposin by fusion of the protein sequences; or using a 2A peptide linker, such as T2A or P2A, which leads 2 peptide fragments with added amino acids due to prevention of the creation of a peptide bond; or using an IRES element; or by expression with 2 separate expression cassettes.
  • a 2A peptide linker such as T2A or P2A, which leads 2 peptide fragments with added amino acids due to prevention of the creation of a peptide bond
  • IRES element or by expression with 2 separate expression cassettes.
  • shRNAs and other regulatory RNAs can potentially be included within these sequences. Examples of expression constructs described by the disclosure are shown in FIGs. 1-8, 21-35, 39 and 41-51, and in Table 6 below.
  • Example 2 Cell based assays of viral transduction into GBA-deficient cells
  • Cells deficient in GBA1 are obtained, for example as fibroblasts from GD patients, monocytes, or hES cells, or patient-derived induced pluripotent stem cells (iPSCs). These cells 5 accumulate substrates such as glucosylceramide and glucosylsphingosine (GlcCer and GlcSph).
  • Gcase inhibitors such as CBE
  • lysosomal defects are quantified in terms of accumulation of protein aggregates, such as of a-Synuclein with an antibody for this protein or phospho-aSyn,
  • Imaging for lysosomal abnormalities by ICC for protein markers such as LAMP1, LAMP2, LIMP1, LIMP2, or using dyes such as Lysotracker, or by uptake through the endocytic compartment of fluorescent dextran or other markers is also performed.
  • Imaging for autophagy marker accumulation due to defective fusion with the lysosome, such as for LC3, can also be performed.
  • Western blotting and/or ELISA is 15 used to quantify abnormal accumulation of these markers. Also, the accumulation of glycolipid substrates and products of GBA1 is measured using standard approaches.
  • Therapeutic endpoints e.g ., reduction of PD-associated pathology
  • Gcase can is also quantified using protein ELISA measures, or by standard Gcase 0 activity assays.
  • Example 3 In vivo assays using mutant mice
  • This example describes in vivo assays of AAV vectors using mutant mice.
  • In vivo studies of AAV vectors as above in mutant mice are performed using assays described, for example, by Liou et al. (2006) J. Biol. Chem. 281(7): 4242-4253, Sun el al. (2005) J. Lipid Res. 46:2102-2113, and Farfel-Becker et al. (2011) Dis. Model Mech. 4(6):746-752.
  • intrathecal or intraventricular delivery of vehicle control and AAV vectors are performed using concentrated AAV stocks, for example at an injection volume between 5-10 pL.
  • Intraparenchymal delivery by convection enhanced delivery is performed.
  • Treatment is initiated either before onset of symptoms, or subsequent to onset.
  • Endpoints measured are the accumulation of substrate in the CNS and CSF, accumulation of Gcase enzyme by ELISA and of enzyme activity, motor and cognitive endpoints, lysosomal dysfunction, and accumulation of a-Synuclein monomers, protofibrils or fibrils.
  • This example describes in vivo assays of AAV vectors using a chemically-induced mouse model of Gaucher disease (e.g., the CBE mouse model). In vivo studies of these AAV vectors are performed in a chemically-induced mouse model of Gaucher disease, for example as described by Vardi et al. (2016) J Pathol. 239(4):496-509.
  • a chemically-induced mouse model of Gaucher disease e.g., the CBE mouse model
  • Intrathecal or intraventricular delivery of vehicle control and AAV vectors are performed using concentrated AAV stocks, for example with injection volume between 5-10 pL.
  • Intraparenchymal delivery by convection enhanced delivery is performed.
  • Peripheral delivery is achieved by tail vein injection.
  • Treatment is initiated either before onset of symptoms, or subsequent to onset.
  • Endpoints measured are the accumulation of substrate in the CNS and CSF, accumulation of Gcase enzyme by ELISA and of enzyme activity, motor and cognitive endpoints, lysosomal dysfunction, and accumulation of a-Synuclein monomers, protofibrils or fibrils.
  • Example 5 Clinical trials in PD, LBD, Gaucher disease patients
  • patients having certain forms of Gaucher disease have an increased risk of developing Parkinson’s disease (PD) or Lewy body dementia (LBD).
  • GDI Gaucher disease
  • PD Parkinson’s disease
  • LBD Lewy body dementia
  • Example describes clinical trials to assess the safety and efficacy of rAAVs as described by the disclosure, in patients having Gaucher disease, PD and/or LBD.
  • Clinical trials of such vectors for treatment of Gaucher disease, PD and/or LBD are performed using a study design similar to that described in Grabowski et al. (1995) Ann. Intern. Med. 122(l):33-39.
  • patients having certain forms of Gaucher disease exhibit symptoms of peripheral neuropathy, for example as described in Biegstraaten et al. (2010) Brain 133(10):2909-2919.
  • This example describes in vivo assays of AAV vectors as described herein for treatment of peripheral neuropathy associated with Gaucher disease (e.g., Type 1 Gaucher disease).
  • Gaucher disease e.g., Type 1 Gaucher disease
  • Type 1 Gaucher disease patients identified as having signs or symptoms of peripheral neuropathy are administered a rAAV as described by the disclosure.
  • the peripheral neuropathic signs and symptoms of the subject are monitored, for example using methods described in Biegstraaten et al., after administration of the rAAV.
  • Levels of transduced gene products as described by the disclosure present in patients are assayed, for example by Western blot analysis, enzymatic functional assays, or imaging studies.
  • This example describes in vivo assays of rAAVs as described herein for treatment of CNS forms of Gaucher disease.
  • Gaucher disease patients identified as having a CNS form of Gaucher disease e.g., Type 2 or Type 3 Gaucher disease
  • a rAAV as described by the disclosure are administered a rAAV as described by the disclosure.
  • Levels of transduced gene products as described by the disclosure present in the CNS of patients e.g., in serum of the CNS of a patient, in cerebrospinal fluid (CSF) of a patient, or in CNS tissue of a patient
  • CSF cerebrospinal fluid
  • Example 8 Gene therapy of Parkinson’ s Disease in subjects having mutations in GBA1
  • This example describes administration of a recombinant adeno-associated virus (rAAV) encoding GBA1 to a subject having Parkinson’s disease characterized by a mutation in
  • the rAAV-GBAl vector insert contains the CBA promoter element (CBA), consisting of four parts: the CMV enhancer (CMVe), CBA promoter (CBAp), Exon 1, and intron (int) to constitutively express the codon optimized coding sequence (CDS) of human GBA1 (maroon).
  • CBA CBA promoter element
  • the 3’ region also contains a Woodchuck hepatitis virus Posttranscriptional Regulatory Element (WPRE) posttranscriptional regulatory element followed by a bovine Growth Hormone polyA signal (bGH polyA) tail.
  • WPRE Woodchuck hepatitis virus Posttranscriptional Regulatory Element
  • bGH polyA bovine Growth Hormone polyA
  • the rAAV-GBAl vector product contains the“D” domain nucleotide sequence shown in FIG. 7 (inset box, top sequence).
  • a variant vector harbors a mutant“D” domain (termed an“S” domain herein, with the nucleotide changes shown by shading), performed similarly in preclinical studies.
  • the backbone contains the gene to confer resistance to kanamycin as well as a stuffer sequence to prevent reverse packaging.
  • a schematic depicting a rAAV-GBAl vector is shown in FIG. 8. The rAAV-GBAl vector is packaged into an rAAV using AAV9 serotype capsid proteins.
  • rAAV-GBAl is administered to a subject as a single dose via a fluoroscopy guided sub- occipital injection into the cisterna magna (intracistemal magna; ICM).
  • ICM intracistemal magna
  • “D” domain within the 145 bp 5’ ITR is thought to be necessary for optimal viral vector production, but mutations within the“D” domain have also been reported to increase transgene expression in some cases.
  • a second vector form with a mutant D domain was also evaluated.
  • Both rAAV-GBAl and the variant express the same transgene. While both vectors produced virus that was efficacious in vivo as detailed below, rAAV-GBAl, which contains a wild-type“D” domain, was selected for further development.
  • mice were dosed with CBE, a specific inhibitor of GCase. Mice were given CBE by IP injection daily, starting at postnatal day 8 (P8). Three different CBE doses (25 mg/kg, 37.5 mg/kg, 50 mg/kg) and PBS were tested to establish a model that exhibits a behavioral phenotype (FIG. 9). Higher doses of CBE led to lethality in a dose-dependent manner. All mice treated with 50 mg/kg CBE died by P23, and 5 of the 8 mice treated with 37.5 mg/kg CBE died by P27. There was no lethality in mice treated with 25 mg/kg CBE. Whereas CBE-injected mice showed no general motor deficits in the open field assay (traveling the same distance and at the same velocity as mice given PBS), CBE- treated mice exhibited a motor coordination and balance deficit as measured by the rotarod assay.
  • mice surviving to the end of the study were sacrificed on the day after their last CBE dose (P27,“Day 1”) or after three days of CBE withdrawal (P29,“Day 3”) ⁇ Lipid analysis was performed on the cortex of mice given 25 mg/kg CBE to evaluate the accumulation of GCase substrates in both the Day 1 and Day 3 cohorts.
  • GluSph and GalSph levels were significantly accumulated in the CBE-treated mice compared to PBS-treated controls, consistent with GCase insufficiency.
  • rAAV-GBAl or excipient was delivered by intracerebroventricular (ICV) injection at postnatal day 3 (P3) followed by daily IP CBE or PBS treatment initiated at P8 (FIG. 10).
  • mice that received rAAV-GBAl performed statistically significantly better on the rotarod than those that received excipient (FIG. 11).
  • Mice in the variant treatment group did not differ from excipient treated mice in terms of other behavioral measures, such as the total distance traveled during testing (FIG. 11).
  • mice that received both CBE and rAAV-GBAl had GCase activity levels that were similar to the PBS-treated group, indicating that delivery of rAAV-GBAl is able to overcome the inhibition of GCase activity induced by CBE treatment.
  • Lipid analysis was performed on the motor cortex of the mice to examine levels of the substrates GluCer and GluSph. Both lipids accumulated in the brains of mice given CBE, and rAAV-GBAl treatment significantly reduced substrate accumulation.
  • Lipid levels were negatively correlated with both GCase activity and performance on the Rotarod across treatment groups.
  • FIG. 13 preliminary biodistribution was assessed by vector genome presence, as measured by qPCR (with >100 vector genomes per 1 pg genomic DNA defined as positive). Mice that received rAAV-GBAl, both with and without CBE, were positive for rAAV-GBAl vector genomes in the cortex, indicating that ICV delivery results in rAAV-GBAl delivery to the cortex. Additionally, vector genomes were detected in the liver, few in spleen, and none in the heart, kidney or gonads. For all measures, there was no statistically significant difference between the Day 1 and Day 3 groups.
  • mice were sacrificed for biochemical analysis (FIG. 16). GCase activity in the cortex was assessed in biological triplicates by a fluorometric assay.
  • CBE-treated mice showed reduced GCase activity whereas mice that received a high rAAV-GBAl dose showed a statistically significant increase in GCase activity compared to CBE treatment.
  • CBE-treated mice also had accumulation of GluCer and GluSph, both of which were rescued by administering a high dose of rAAV-GBAl.
  • mice In addition to the established chemical CBE model, rAAV-GBAlis also evaluated in the 4L/PS-NA genetic model, which is homozygous for the V394L GD mutation in Gbal and is also partially deficient in saposins, which affect GCase localization and activity. These mice exhibit motor strength, coordination, and balance deficits, as evidenced by their performance in the beam walk, rotarod, and wire hang assays. Typically the lifespan of these mice is less than 22 weeks. In an initial study, 3 pi of maximal titer virus was delivered by ICV at P23, with a final dose of 2.4el0 vg (6.0el0 vg/g brain). With 6 mice per group, the treatment groups were:
  • BD biodistribution
  • NS nonsignificant
  • T trend
  • S significant
  • N/A not applicable
  • + positive
  • - negative.
  • Example 9 In vitro analysis of r AAV vectors
  • FIG. 18 shows representative data for in vitro expression of rAAV constructs encoding progranulin (PGRN, also referred to as GRN) protein.
  • the left panel shows a standard curve of progranulin (PGRN) ELISA assay.
  • the bottom panel shows a dose-response of PGRN expression measured by ELISA assay in cell lysates of HEK293T cells transduced with rAAV.
  • MOI multiplicity of infection (vector genomes per cell).
  • FIG. 19 shows representative data indicating that transfection of HEK293 cells with each of the constructs resulted in overexpression of the corresponding gene product compared to mock transfected cells.
  • FIGs. 36A-36B show representative data indicating that transfection of HEK293 cells with each of the constructs resulted in overexpression of the corresponding gene product compared to mock transfected cells.
  • HEK293 Human embryonic kidney 293 cell line (HEK293) were used in this study (#85120602, Sigma- Aldrich). HEK293 cells were maintained in culture media (D-MEM [#11995065,
  • Thermo Fisher Scientific supplemented with 10% fetal bovine serum [FBS] [#10082147, Thermo Fisher Scientific]) containing 100 units/ml penicillin and 100 pg/ml streptomycin (#15140122, Thermo Fisher Scientific).
  • Plasmid transfection was performed using Fipofectamine 2000 transfection reagent (#11668019, Thermo Fisher Scientific) according to the manufacture’s instruction. Briefly, HEK293 cells (#12022001, Sigma- Aldrich) were plated at the density of 3xl0 5 cells/ml in culture media without antibiotics. On the following day, the plasmid and Fipofectamine 2000 reagent were combined in Opti-MEM solution (#31985062, Thermo Fisher Scientific). After 5 minutes, the mixtures were added into the HEK293 culture. After 72 hours, the cells were harvested for RNA or protein extraction, or subjected to the imaging analyses. For imaging analyses, the plates were pre-coated with 0.01% poly-F-Fysine solution (P8920, Sigma-Aldrich) before the plating of cells.
  • P8920 poly-F-Fysine solution
  • Relative gene expression levels were determined by quantitative real-time PCR (qRT- PCR) using Power SYBR Green Cells-to-CT Kit (#4402955, Thermo Fisher Scientific) according to the manufacturer’s instruction.
  • the candidate plasmids were transiently transfected into HEK293 cells plated on 48-well plates (7.5 xlO 4 cells/well) using Fipofectamine 2000 transfection reagent (0.5 pg plasmid and 1.5 pi reagent in 50 m ⁇ Opti-MEM solution). After 72 hours, RNA was extracted from the cells and used for reverse transcription to synthesize cDNA according to the manufacturer’s instruction.
  • 2 ⁇ 5 m ⁇ of cDNA products were amplified in duplicates using gene specific primer pairs (250 nM final concentration) with Power SYBR Green PCR Master Mix (#4367659, Thermo Fisher
  • the primer sequences for SNCA, TMEM106B, and GAPDH genes were: 5’- AAG AGG GTG TTC TCT ATG TAG GC -3’ (SEQ ID NO: 71), 5’- GCT CCT CCA ACA TTT GTC ACT T -3’ (SEQ ID NO: 72) for SNCA, 5’-ACA CAG TAC CTA CCG TTA TAG CA-3’ (SEQ ID NO: 73), 5’-TGT TGT CAC AGT AAC TTG CAT CA-3’ (SEQ ID NO: 74) for TMEM106B, and 5’- CTG GGC TAC ACT GAG CAC C -3’ (SEQ ID NO: 75), 5’- AAG TGG TCG TTG AGG GCA ATG -3’ (SEQ ID NO: 76) for GAPDH. Quantitative PCR was performed in a QuantStudio 3 Real-Time PCR system (Thermo Fisher Scientific). Expression levels were normalized by the housekeeping gene
  • EGFP reporter plasmids which contain 3’-UTR of human SNCA gene at downstream of EGFP coding region, were used for the validation of SNCA and TMEM106B knockdown plasmids.
  • EGFP reporter plasmids and candidate knockdown plasmids were simultaneously transfected into HEK293 cells plated on poly-L- Lysine coated 96-well plates (3.0 xlO 4 cells/well) using Lipofectamine 2000 transfection reagent (0.04 pg reporter plasmid, 0.06 pg knockdown plasmid and 0.3 m ⁇ reagent in 10 m ⁇ Opti-MEM solution).
  • the fluorescent intensities of EGFP signal were measured at excitation 488 nm/emission 512 nm using Varioskan LUX multimode reader (Thermo Fisher Scientific). Cells were fixed with 4% PFA at RT for 10 minutes, and incubated with D-PBS containing 40 pg/ml 7-aminoactinomycin D (7-AAD) for 30 min at RT. After washing with D-PBS, the fluorescent intensities of 7-AAD signal were measured at excitation 546 nm/emission 647 nm using Varioskan reader to quantify cell number. Normalized EGFP signal per 7-AAD signal levels were compared with the control knockdown samples.
  • oc-Synuclein reporter plasmids which contain 3’-UTR of human SNCA gene or TMEM106B gene downstream of SNCA coding region, were used for the validation of knockdown plasmids at the protein level.
  • Levels of oc-synuclein protein were determined by ELISA (#KHB0061, Thermo Fisher Scientific) using the lysates extracted from HEK293 cells.
  • the candidate plasmids were transiently transfected into HEK293 cells plated on 48-well plates (7.5 xlO 4 cells/well) using Lipofectamine 2000 transfection reagent (0.1 pg reporter plasmid, 0.15 pg knockdown plasmid and 0.75 pi reagent in 25 pi Opti-MEM solution). After 72 hours, cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (#89900, Thermo Fisher Scientific) supplemented with protease inhibitor cocktail (#P8340, Sigma- Aldrich), and sonicated for a few seconds.
  • RIPA radioimmunoprecipitation assay
  • FIG. 37 and Table 9 show representative data indicating successful silencing of SNCA in vitro by GFP reporter assay (top) and a-Syn assay (bottom).
  • FIG. 38 and Table 10 show representative data indicating successful silencing of TMEM106B in vitro by GFP reporter assay (top) and a-Syn assay (bottom).
  • ITR“D” sequence The effect of placement of ITR“D” sequence on cell transduction of rAAV vectors was investigated.
  • HEK293 cells were transduced with Gcase-encoding rAAVs having 1) wild-type ITRs (e.g.,“D” sequences proximal to the transgene insert and distal to the terminus of the ITR) or 2) ITRs with the“D” sequence located on the“outside” of the vector (e.g.,“D” sequence located proximal to the terminus of the ITR and distal to the transgene insert), as shown in FIG. 20.
  • Data indicate that rAAVs having the“D” sequence located in the“outside” position retain the ability to be packaged and transduce cells efficiently (FIG. 40).
  • Example 12 In vitro testing of Prog ranulin rAAVs
  • FIG. 39 is a schematic depicting one embodiments of a vector comprising an expression construct encoding PGRN (also referred to as GRN).
  • Progranulin is overexpressed in the CNS of rodents deficient in GRN, either heterozygous or homozygous for GRN deletion, by injection of an rAAV vector encoding PGRN (e.g., codon-optimized PGRN, also referred to as codon- optimized GRN), either by intrap arenchymal or intrathecal injection such as into the cistema magna.
  • PGRN also referred to as GRN
  • mice are injected at 2 months or 6 months of age, and aged to 6 months or 12 months and analyzed for one or more of the following: expression level of GRN at the RNA and protein levels, behavioral assays (e.g., improved movement), survival assays (e.g., improved survival), microglia and inflammatory markers, gliosis, neuronal loss, Lipofuscinosis, and/or Lysosomal marker accumulation rescue, such as LAMP1.
  • Assays on GRN-deficient mice are described, for example by Arrant et al. (2017) Brain 140: 1477-1465; Arrant et al. (2016) J. Neuroscience 38(9):2341-2358; and Amado et al. (2016) doi:https://doi.org/10.1101/30869; the entire contents of which are incorporated herein by reference.
  • Example 13 In vitro testing ofMAPT rAAVs
  • SY5Y cells were plated at 4xl0 4 cells per well in a 96-well plate. The following day, cells were transduced with two vims stocks (Intronic_eSIBR_MAPT_MiR615 conserveed vector) encoding inhibitory RNA targeting MAPT (J00130 produced in a mammalian cell-based system, and J00122 produced in a Baculovims-based system; shown in FIG. 75C) in triplicates at MOI of 2x 10 5 in media containing luM Hoechst. Excipient alone was used as negative control. The cells were harvested 72 hours later, and stained with a probe to detect AAV vectors expressing inhibitory RNA for MAPT. The probe targets BGHpA. FIG. 75A shows that both vims stocks successfully transduced SY5Y cells.
  • SY5Y cells were plated at 4xl0 4 cells per well in a 96-well plate. The following day, cells were transduced with two vims stocks ((Intronic_eSIBR_MAPT_MiR615 conserveed vector) encoding inhibitory RNA targeting MAPT (J00130 and J00122; shown in FIG. 75C) in triplicates at MOI 2xl0 6 in media containing luM Hoechst. Excipient alone was used as negative control. SY5Y cells were lysed for RNA extraction 72 hours or 7 days after
  • FIG.75B shows data for knockdown of MAPT expression by J00130 and J00122.
  • a reference to“A and/or B,” when used in conjunction with open-ended language such as“comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • “or” should be understood to have the same meaning as“and/or” as defined above.
  • “or” or“and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as“only one of’ or“exactly one of,” or, when used in the claims,“consisting of,” will refer to the inclusion of exactly one element of a number or list of elements.
  • the phrase“at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
  • This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase“at least one” refers, whether related or unrelated to those elements specifically identified.
  • “at least one of A and B” can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
  • an expression cassette encoding one or more gene products comprises or consists of (or encodes a peptide having) a sequence set forth in any one of SEQ ID NOs: 1-149.
  • a gene product is encoded by a portion (e.g., fragment) of any one of SEQ ID NOs: 1-149.

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  • Orthopedic Medicine & Surgery (AREA)
  • Cell Biology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne, selon certains aspects, des compositions et des méthodes pour le traitement de maladies du système nerveux central (SNC) par exemple la maladie de Parkinson (MP) et la maladie de Gaucher. Dans certains modes de réalisation, l'invention concerne des constructions d'expression comprenant un transgène codant pour un ou plusieurs produits géniques associés à une maladie du SNC et/ou un ou plusieurs acides nucléiques inhibiteurs ciblant un gène ou un produit génique associé à une maladie du SNC. Dans certains modes de réalisation, l'invention concerne des méthodes de traitement de maladies du SNC par administration de telles constructions d'expression à un sujet en ayant besoin.
EP20787456.1A 2019-04-10 2020-04-10 Thérapies géniques pour troubles lysosomaux Pending EP3952923A4 (fr)

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US201962831840P 2019-04-10 2019-04-10
US201962832223P 2019-04-10 2019-04-10
US201962831856P 2019-04-10 2019-04-10
US201962831846P 2019-04-10 2019-04-10
US201962934450P 2019-11-12 2019-11-12
US201962954089P 2019-12-27 2019-12-27
US202062960471P 2020-01-13 2020-01-13
US202062988665P 2020-03-12 2020-03-12
US202062990246P 2020-03-16 2020-03-16
PCT/US2020/027658 WO2020210615A1 (fr) 2019-04-10 2020-04-10 Thérapies géniques pour troubles lysosomaux

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JP (1) JP2022527015A (fr)
KR (1) KR20210150487A (fr)
CN (1) CN114025806A (fr)
IL (1) IL286582A (fr)
MX (1) MX2021012467A (fr)

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US10648001B2 (en) * 2012-07-11 2020-05-12 Sangamo Therapeutics, Inc. Method of treating mucopolysaccharidosis type I or II
SG11201509419QA (en) * 2013-05-15 2015-12-30 Univ Minnesota Adeno-associated virus mediated gene transfer to the central nervous system
US10967073B2 (en) * 2015-05-07 2021-04-06 The Mclean Hospital Corporation Glucocerebrosidase gene therapy for Parkinson's disease
TWI832036B (zh) * 2017-08-03 2024-02-11 美商航海家醫療公司 用於aav之遞送之組合物及方法

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KR20210150487A (ko) 2021-12-10
CN114025806A (zh) 2022-02-08
MX2021012467A (es) 2021-12-10
IL286582A (en) 2021-12-01
EP3952923A4 (fr) 2023-10-18

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