Gene therapies for lysosomal disorders

Abstract

To provide, in some aspects, compositions and methods for treatment of diseases associated with the aberrant lysosomal function, such as Parkinson disease and Gaucher disease.SOLUTION: In some embodiments, the disclosure provides expression constructs comprising a transgene encoding beta-glucocerebrosidase (GBA) or a portion thereof, Lysosome membrane protein 2 (LIMP2), prosaposin, or any combination of them. In some embodiments, the disclosure provides methods for Parkinson disease by administering such an expression construct to a subject in need thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. 119(e) of the filing date of U.S. Provisional Application No. 62 / 567,296, entitled "Gene Therapy for Lysosomal Storage Disorders," filed on October 3, 2017; the entire contents of which are incorporated herein by reference. [Background technology]

[0002] background Gaucher disease is a rare congenital abnormality of glycosphingolipid metabolism due to deficiency of lysosomal acid beta-glucocerebrosidase (Gcase, "GBA"). Patients suffer from non-CNS manifestations and findings including hepatosplenomegaly and bone marrow failure leading to pancytopenia, pulmonary damage / fibrosis, and bone defects. In addition, a significant number of patients suffer from neurological symptoms, including saccadic eye movement and gaze abnormalities, seizures, cognitive impairment, developmental delay, and movement disorders including Parkinson's disease.

[0003] Several therapies exist to address the major clinical manifestations of peripheral disease and hematopoietic bone marrow and visceral organs, including enzyme replacement therapy, chaperone-like small molecule drugs that bind to and improve the stability of the defective Gcase, and substrate inhibition therapy that blocks the production of the substrate that accumulates in Gaucher disease and leads to symptoms and pathology. However, other aspects of Gaucher disease appear to be refractory to treatment. Summary of the Invention

[0004] overview In addition to Gaucher disease patients (those with mutations in both chromosomal alleles of the GBA1 gene), patients with mutations in only one allele of GBA1 are at a significantly increased risk of Parkinson's disease (PD). The severity of PD symptoms - difficulty walking, resting tremor, rigidity, and often depression, sleep difficulties, and cognitive decline - correlates with the degree of reduction in enzyme activity. Thus, Gaucher disease patients have the most severe course, while patients with a single mild mutation in GBA1 typically have a more benign course. Mutation carriers are also at increased risk of other PD-related diseases, including dementia with Lewy bodies, characterized by executive dysfunction, psychosis, and PD-like movement disorders, and multiple system atrophy with characteristic motor and cognitive impairments. No treatment exists to alter the relentless course of these diseases.

[0005] Deficiencies in enzymes such as Gcase (e.g., the gene product of the GBA1 gene) and common variants in many genes associated with lysosomal function or transport of macromolecules to lysosomes (e.g., lysosomal membrane protein 1 (LIMP), also known as SCARB2) have been associated with increased PD risk. The present disclosure is based in part on expression constructs (e.g., vectors) encoding Gcase (or a portion thereof), prosaposin (or a portion thereof), LIMP2 (or a portion thereof), or a combination of Gcase (or a portion thereof) and one or more additional gene products from PD-associated genes (e.g., LIMP2, prosaposin, and / or alpha-synuclein (alpha-Syn)). In some embodiments, the combination of gene products described herein, when expressed in a subject, act together (e.g., synergistically) to alleviate one or more signs and symptoms of PD.

[0006] Thus, in some aspects, the disclosure provides an isolated nucleic acid, including an expression construct, encoding Gcase (e.g., a gene product of the GBA1 gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized (e.g., codon-optimized for expression in a mammalian cell, such as a human cell) Gcase coding sequence. In some embodiments, the nucleic acid sequence encoding 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_0014148.2). In some embodiments, the isolated nucleic acid comprises a sequence set forth in SEQ ID NO: 15. In some embodiments, the expression construct comprises an adeno-associated virus (AAV) inverted terminal repeat (ITR), e.g., an AAV ITR adjacent to the nucleic acid sequence encoding Gcase.

[0007] In some aspects, the disclosure provides an isolated nucleic acid, including an expression construct, encoding prosaposin (e.g., a gene product of a PSAP gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized (e.g., codon-optimized for expression in a mammalian cell, such as a human cell) prosaposin coding sequence. In some embodiments, the nucleic acid sequence encoding 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). In some embodiments, the isolated nucleic acid comprises a sequence set forth in SEQ ID NO: 17. In some embodiments, the expression construct comprises an adeno-associated virus (AAV) inverted terminal repeat (ITR), e.g., an AAV ITR adjacent to the nucleic acid sequence encoding prosaposin.

[0008] In some aspects, the disclosure provides an isolated nucleic acid, including an expression construct, encoding LIMP2 / SCARB2 (e.g., a gene product of the SCARB2 gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized (e.g., codon-optimized for expression in a mammalian cell, such as a human cell) SCARB2 coding sequence. In some embodiments, the nucleic acid sequence encoding 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). In some embodiments, the isolated nucleic acid comprises a sequence set forth in SEQ ID NO: 29. In some embodiments, the expression construct comprises an adeno-associated virus (AAV) inverted terminal repeat (ITR), e.g., an AAV ITR adjacent to the nucleic acid sequence encoding SCARB2.

[0009] In some aspects, 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 is independently selected from the gene products set forth in Table 1, or a portion thereof. In some embodiments, the first gene product or the second gene product is a Gcase protein or a portion thereof. In some embodiments, the first gene product or the second gene product is LIMP2 or a portion thereof, or prosaposin or a portion thereof. In some embodiments, the first gene product is a Gcase protein and the second gene product is LIMP2 or a portion thereof, or prosaposin or a portion thereof.

[0010] In some embodiments, the expression construct further encodes an interfering nucleic acid (e.g., shRNA, miRNA, dsRNA, etc.). In some embodiments, the interfering nucleic acid inhibits expression of alpha-synuclein (α-synuclein). In some embodiments, the interfering nucleic acid targeting alpha-synuclein comprises a sequence set forth in any one of SEQ ID NOs: 20-25. In some embodiments, the interfering nucleic acid targeting alpha-synuclein binds to (e.g., hybridizes to) a sequence set forth in any one of SEQ ID NOs: 20-25. In some embodiments, the expression construct further comprises one or more promoters. In some embodiments, the promoter is a chicken beta actin (CBA) promoter, a CAG promoter, a CD68 promoter, or a JeT promoter. In some embodiments, the promoter is an RNA pol II promoter (e.g., or an RNA pol III promoter (e.g., U6, etc.)).

[0011] In some embodiments, the expression construct further comprises an internal ribosome entry site (IRES). In some embodiments, the IRES is located between the first gene product and the second gene product. In some embodiments, the expression construct further comprises a self-cleaving peptide coding sequence. In some embodiments, the self-cleaving peptide is a T2A peptide. In some embodiments, the expression construct comprises two adeno-associated virus (AAV) inverted terminal repeat (ITR) sequences. In some embodiments, the ITR sequences flank the first gene product and the second gene product (e.g., arranged from 5' to 3' end as follows: ITR-first gene product-second gene product-ITR). In some embodiments, one of the ITR sequences of the isolated nucleic acid lacks a functional terminal resolution site (trs). For example, in some embodiments, one of the ITRs is a ΔITR.

[0012] The present disclosure relates in some aspects to an rAAV vector comprising an ITR with a modified "D" region (e.g., a modified D sequence compared to the wild-type AAV2 ITR, SEQ ID NO: 29). In some embodiments, the ITR with the modified D region is the 5'ITR of the rAAV vector. In some embodiments, the modified "D" region comprises an "S" sequence, for example, as set forth in SEQ ID NO: 26. In some embodiments, the ITR with the modified "D" region is the 3'ITR of the rAAV vector. In some embodiments, the modified "D" region comprises a 3'ITR, where the "D" region is located at the 3' end of the ITR (e.g., outside or at the end of the ITR relative to the transgene insert of the vector). In some embodiments, the modified "D" region comprises a sequence as set forth in SEQ ID NO: 26 or 27. In some embodiments, the isolated nucleic acid (e.g., a rAAV vector) comprises a TRY region. In some embodiments, the TRY region comprises the sequence set forth in SEQ ID NO:28.

[0013] In some embodiments, the isolated nucleic acid described herein comprises or consists of a sequence set forth in any one of SEQ ID NOs: 1-13, 15, 17, and 19. In some embodiments, the isolated nucleic acid described herein encodes a peptide comprising or consisting of a sequence set forth in any one of SEQ ID NOs: 14, 16, and 18. In some aspects, the present disclosure provides a vector comprising the isolated nucleic acid described in the present disclosure.In some embodiments, the vector is a plasmid or a viral vector.In some embodiments, the viral vector is a recombinant AAV (rAAV) vector.In some embodiments, the rAAV vector is single-stranded (e.g., single-stranded DNA).

[0014] In some aspects, the present disclosure provides a host cell comprising an isolated nucleic acid described in this disclosure or a vector described in this disclosure. In some aspects, the present disclosure provides a recombinant adeno-associated virus (rAAV) comprising a capsid protein and an isolated nucleic acid or vector described in this disclosure. In some embodiments, the capsid protein is capable of crossing the blood-brain barrier, e.g., AAV9 capsid protein or AAVrh.10 capsid protein. In some embodiments, the rAAV transduces neural and non-neuronal cells of the central nervous system (CNS). In some aspects, the disclosure provides a method of treating a subject having or suspected of having Parkinson's disease, the method comprising administering to the subject a composition described in this disclosure (e.g., a composition comprising an isolated nucleic acid or vector or rAAV).

[0015] In some embodiments, administering comprises direct injection into the CNS of the subject.In some embodiments, direct injection is intracerebral injection, intraparenchymal injection, intrathecal injection, intracisternal injection, or any combination thereof.In some embodiments, direct injection into the CNS of the subject comprises convection-enhanced delivery (CED). In some embodiments, the administration comprises a peripheral injection. In some embodiments, the peripheral injection is an intravenous injection. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 is a schematic diagram showing one embodiment of a plasmid comprising an rAAV vector containing an expression construct encoding Gcase (e.g., GBA1 or a portion thereof). [Diagram 2] 2 is a schematic diagram showing one embodiment of a plasmid comprising an rAAV vector containing an expression construct encoding Gcase (e.g., GBA1 or a portion thereof) and LIMP2 (SCARB2) or a portion thereof. The coding sequences for Gcase and LIMP2 are separated by an internal ribosome entry site (IRES). [Diagram 3]3 is a schematic diagram showing one embodiment of a plasmid comprising an rAAV vector containing an expression construct encoding Gcase (e.g., GBA1 or a portion thereof) and LIMP2 (SCARB2) or a portion thereof. Expression of the coding sequences for Gcase and LIMP2 is driven by separate promoters.

[0017] [Figure 4] FIG. 4 is a schematic diagram showing one embodiment of a plasmid comprising a rAAV vector containing expression constructs encoding interfering RNA for Gcase (e.g., GBA1 or a portion thereof), LIMP2 (SCARB2) or a portion thereof, and α-Syn. [Diagram 5] FIG. 5 is a schematic diagram showing one embodiment of a plasmid comprising an rAAV vector containing expression constructs encoding interfering RNA for Gcase (e.g., GBA1 or a portion thereof), prosaposin (e.g., PSAP or a portion thereof), and α-Syn. [Figure 6] 6 is a schematic diagram showing one embodiment of a plasmid comprising an rAAV vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof) and prosaposin (e.g., PSAP or a portion thereof). The coding sequences for Gcase and prosaposin are separated by an internal ribosome entry site (IRES).

[0018] [Figure 7]FIG. 7 is a schematic diagram showing one embodiment of a rAAV vector containing an expression construct encoding Gcase (e.g., GBA1 or a portion thereof). In this embodiment, the vector contains a CBA promoter element (CBA) consisting of four parts: CMV enhancer (CMVe), CBA promoter (CBAp), exon 1, and intron (int), and constitutively expresses the codon-optimized coding sequence of human GBA1. The 3' region also contains a WPRE regulatory element followed by a bGH polyA tail. The 5' end of the promoter region contains three transcriptional regulatory activation sites: TATA, RBS, and YY1. The adjacent ITRs allow for correct packaging of the intervening sequence. Two variants of the 5'ITR sequence (insertion box) were evaluated; these have several nucleotide differences within the 20-nucleotide "D" region of the wild-type AAV2 ITR. In some embodiments, the rAAV vector contains the "D" domain nucleotide sequence shown in the top row. In some embodiments, the rAAV vector comprises a mutant "D" domain (e.g., an "S" domain, with the nucleotide changes shown at the bottom line).

[0019] [Figure 8] FIG. 8 is a schematic diagram showing one embodiment of a plasmid encoding the rAAV vector described in FIG. [Figure 9-1]FIG. 9 shows representative data for delivery of rAAV containing a transgene encoding Gcase (e.g., GBA1 or a portion thereof) in the 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) was initiated at P8. Viability (top left) was checked twice daily and body weight (top right) was checked daily. All groups were initiated at n=8. Behavior was assessed at P23 for total distance traveled in the open field (bottom left) and at P24 for latency to fall on the rotarod (bottom center). Levels of GCase substrate were analyzed in the cortex of mice in PBS and 25 mg / kg CBE treatment groups, both with (day 3) and without (day 1) CBE withdrawal. Total GluSph&GalSph levels (bottom right) are expressed as pmol / mg wet weight of tissue. Mean values ​​are presented. Error bars are SEM. *p<0.05; **p<0.01; ***p<0.001, nominal p-values ​​for treatment groups from linear regression. [Figure 9-2] FIG. 9 shows representative data for delivery of rAAV containing a transgene encoding Gcase (e.g., GBA1 or a portion thereof) in the 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) was initiated at P8. Viability (top left) was checked twice daily and body weight (top right) was checked daily. All groups were initiated at n=8. Behavior was assessed at P23 for total distance traveled in the open field (bottom left) and at P24 for latency to fall on the rotarod (bottom center). Levels of GCase substrate were analyzed in the cortex of mice in PBS and 25 mg / kg CBE treatment groups, both with (day 3) and without (day 1) CBE withdrawal. Total GluSph&GalSph levels (bottom right) are expressed as pmol / mg wet weight of tissue. Mean values ​​are presented. Error bars are SEM. *p<0.05; **p<0.01; ***p<0.001, nominal p-values ​​for treatment groups from linear regression.

[0020] [Figure 10]Figure 10 is a schematic diagram showing one embodiment of the study design for maximum rAAV dose in the CBE mouse model. Briefly, rAAV was delivered by ICV injection at P3 and daily CBE treatments began at P8. Behavior was assessed by open field and rotarod assays at P24-25 and substrate levels were measured at P36 and P38. [Figure 11-1] FIG. 11 shows representative data for survival assessment of maximum rAAV dose in the CBE mouse model. At P3, mice were treated with either vehicle or 8.8e9vg of rAAV via ICV delivery. Daily IP delivery of PBS or 25 mg / kg CBE was initiated at P8. At the end of the study, half of the mice were sacrificed at P36 (day 1), one day after the last CBE administration, while the remaining half underwent a 3-day withdrawal of CBE before being sacrificed at P38 (day 3). All treatment groups (vehicle+PBS: n=8, rAAV+PBS: n=7, vehicle+CBE: n=8, and rAAV+CBE: n=9) were weighed daily (top left) and body weights at P36 were analyzed (top right). Behavior was assessed as total distance traveled in the open field at P23 (bottom left) and latency to fall on the rotarod at P24 (bottom right) and was assessed as the median of three trials for each animal. n=7 in vehicle+CBE group for behavioral assays due to lethality, n=8 in all other groups. Mean values ​​across animals are shown. Error bars are SEM. *p<0.05; ***p<0.001, nominal p-values ​​for treatment groups by linear regression in CBE-treated animals. [Figure 11-2]FIG. 11 shows representative data for survival assessment of maximum rAAV dose in the CBE mouse model. At P3, mice were treated with either vehicle or 8.8e9vg of rAAV via ICV delivery. Daily IP delivery of PBS or 25 mg / kg CBE was initiated at P8. At the end of the study, half of the mice were sacrificed at P36 (day 1), one day after the last CBE administration, while the remaining half underwent a 3-day withdrawal of CBE before being sacrificed at P38 (day 3). All treatment groups (vehicle+PBS: n=8, rAAV+PBS: n=7, vehicle+CBE: n=8, and rAAV+CBE: n=9) were weighed daily (top left) and body weights at P36 were analyzed (top right). Behavior was assessed as total distance traveled in the open field at P23 (bottom left) and latency to fall on the rotarod at P24 (bottom right) and was assessed as the median of three trials for each animal. n=7 in vehicle+CBE group for behavioral assays due to lethality, n=8 in all other groups. Mean values ​​across animals are shown. Error bars are SEM. *p<0.05; ***p<0.001, nominal p-values ​​for treatment groups by linear regression in CBE-treated animals. [Figure 11-3]FIG. 11 shows representative data for survival assessment of maximum rAAV dose in the CBE mouse model. At P3, mice were treated with either vehicle or 8.8e9vg of rAAV via ICV delivery. Daily IP delivery of PBS or 25 mg / kg CBE was initiated at P8. At the end of the study, half of the mice were sacrificed at P36 (day 1), one day after the last CBE administration, while the remaining half underwent a 3-day withdrawal of CBE before being sacrificed at P38 (day 3). All treatment groups (vehicle+PBS: n=8, rAAV+PBS: n=7, vehicle+CBE: n=8, and rAAV+CBE: n=9) were weighed daily (top left) and body weights at P36 were analyzed (top right). Behavior was assessed as total distance traveled in the open field at P23 (bottom left) and latency to fall on the rotarod at P24 (bottom right) and was assessed as the median of three trials for each animal. n=7 in vehicle+CBE group for behavioral assays due to lethality, n=8 in all other groups. Mean values ​​across animals are shown. Error bars are SEM. *p<0.05; ***p<0.001, nominal p-values ​​for treatment groups by linear regression in CBE-treated animals.

[0021] [Figure 12-1] Figure 12 shows representative data for biochemical evaluation of maximum rAAV dose in the CBE mouse model. Cortex from all treatment groups (vehicle+PBS: n=8, rAAV+PBS: n=7, vehicle+CBE: n=7, rAAV+CBE: n=9) was used to measure GCase activity (top left), GluSph levels (top right), GluCer levels (bottom left), and vector genomes (bottom right) in groups before (day 1) or after (day 3) cessation of CBE. Biodistribution is displayed as vector genomes per μg of genomic DNA. Mean values ​​are shown. Error bars are SEM. (*)p<0.1; **p<0.01; ***p<0.001, nominal p-values ​​for treatment groups by linear regression in CBE-treated animals, corrected for day of collection and sex as covariates. [Figure 12-2]Figure 12 shows representative data for biochemical evaluation of maximum rAAV dose in the CBE mouse model. Cortex from all treatment groups (vehicle+PBS: n=8, rAAV+PBS: n=7, vehicle+CBE: n=7, rAAV+CBE: n=9) was used to measure GCase activity (top left), GluSph levels (top right), GluCer levels (bottom left), and vector genomes (bottom right) in groups before (day 1) or after (day 3) cessation of CBE. Biodistribution is displayed as vector genomes per μg of genomic DNA. Mean values ​​are shown. Error bars are SEM. (*)p<0.1; **p<0.01; ***p<0.001, nominal p-values ​​for treatment groups by linear regression in CBE-treated animals, corrected for day of collection and sex as covariates. [Figure 13] Figure 13 shows representative data of behavioral and biochemical correlates in the CBE mouse model following administration of vehicle+PBS, vehicle+CBE, and rAAV+CBE treatment groups. Across treatment groups, rotarod performance was negatively correlated with GluCer accumulation (A, p=0.0012 by linear regression), and GluSph accumulation was negatively correlated with increased GCase activity (B, p=0.0086 by linear regression).

[0022] [Figure 14-1] Figure 14 shows representative data of GBA1rAAV biodistribution in the CBE mouse model. The presence of vector genomes was assessed in liver, spleen, kidney, and gonads of all treatment groups (vehicle+PBS: n=8, rAAV+PBS: n=7, vehicle+CBE: n=7, and rAAV+CBE: n=9). Biodistribution is expressed as vector genomes per μg of genomic DNA. The presence of vector genomes was quantified by quantitative PCR using a vector reference standard curve; genomic DNA concentrations were assessed by A260 optical density measurements. Mean values ​​are shown. Error bars are SEM. *p<0.05; **p<0.01; ***p<0.001, nominal p-values ​​for treatment groups by linear regression in CBE-treated animals, corrected for harvest day and sex as covariates. [Figure 14-2]Figure 14 shows representative data of GBA1rAAV biodistribution in the CBE mouse model. The presence of vector genomes was assessed in liver, spleen, kidney, and gonads of all treatment groups (vehicle+PBS: n=8, rAAV+PBS: n=7, vehicle+CBE: n=7, and rAAV+CBE: n=9). Biodistribution is expressed as vector genomes per μg of genomic DNA. The presence of vector genomes was quantified by quantitative PCR using a vector reference standard curve; genomic DNA concentrations were assessed by A260 optical density measurements. Mean values ​​are shown. Error bars are SEM. *p<0.05; **p<0.01; ***p<0.001, nominal p-values ​​for treatment groups by linear regression in CBE-treated animals, corrected for harvest day and sex as covariates.

[0023] [Figure 15-1] Figure 15 shows representative data for survival assessment of rAAV dose ranges in the CBE mouse model. Mice were administered one of three different doses of vehicle or GBA1 rAAV: 3.2e9vg, 1.0e10vg, or 3.2e10vg by ICV delivery at P3. Daily IP treatments of 25mg / kg CBE were initiated at P8. Mice administered vehicle plus CBE or vehicle plus PBS served as controls. All treatment groups were initiated with n=10 (5M / 5F) per group. All mice were sacrificed 1 day after the last dose of CBE (P38-P40). Body weights of all treatment groups were measured daily and their weights were analyzed at P36. Motor performance was assessed by latency to fall on the rotarod at P24 and latency to traverse a tapered beam at P30. Due to early lethality, the following numbers of mice participated in behavioral assays: vehicle+PBS: n=10, vehicle+CBE: n=9, and rAAV+CBE at 3.2e9vg: n=6, rAAV+CBE at 1.0e10vg: n=10, and rAAV+CBE at 3.2e10vg: n=7. Mean values ​​are shown. Error bars are SEM; *p<0.05; **p<0.01, nominal p-values ​​from linear regression in CBE-treated animals, corrected for sex as a covariate. [Figure 15-2]Figure 15 shows representative data for survival assessment of rAAV dose ranges in the CBE mouse model. Mice were administered one of three different doses of vehicle or GBA1 rAAV: 3.2e9vg, 1.0e10vg, or 3.2e10vg by ICV delivery at P3. Daily IP treatments of 25mg / kg CBE were initiated at P8. Mice administered vehicle plus CBE or vehicle plus PBS served as controls. All treatment groups were initiated with n=10 (5M / 5F) per group. All mice were sacrificed 1 day after the last dose of CBE (P38-P40). Body weights of all treatment groups were measured daily and their weights were analyzed at P36. Motor performance was assessed by latency to fall on the rotarod at P24 and latency to traverse a tapered beam at P30. Due to early lethality, the following numbers of mice participated in behavioral assays: vehicle+PBS: n=10, vehicle+CBE: n=9, and rAAV+CBE at 3.2e9vg: n=6, rAAV+CBE at 1.0e10vg: n=10, and rAAV+CBE at 3.2e10vg: n=7. Mean values ​​are shown. Error bars are SEM; *p<0.05; **p<0.01, nominal p-values ​​from linear regression in CBE-treated animals, corrected for sex as a covariate.

[0024] [Figure 16-1]Figure 16 shows representative data for biochemical evaluation of a range of rAAV doses in the CBE mouse model. Cortex from all treatment groups (vehicle+PBS: n=10, vehicle+CBE: n=9, and rAAV+CBE at 3.2e9vg: n=6, rAAV+CBE at 1.0e10vg: n=10, rAAV+CBE at 3.2e10vg: n=7) was used to measure GCase activity, GluSph levels, GluCer levels, and vector genomes. GCase activity is shown as ng of GCase per mg of total protein. GluSph and GluCer levels are shown as pmol per mg of wet weight of tissue. Biodistribution is shown as vector genomes per μg of genomic DNA. Vector genome presence was quantified using a vector reference standard curve by quantitative PCR; genomic DNA concentration was assessed by A260 optical density measurements. Vector genome presence was also measured in the liver (E). Mean values ​​are shown. Error bars are SEM. **p<0.01; ***p<0.001, nominal p-values ​​from linear regression in CBE-treated animals, corrected for sex as a covariate. [Figure 16-2] Figure 16 shows representative data for biochemical evaluation of a range of rAAV doses in the CBE mouse model. Cortex from all treatment groups (vehicle+PBS: n=10, vehicle+CBE: n=9, and rAAV+CBE at 3.2e9vg: n=6, rAAV+CBE at 1.0e10vg: n=10, rAAV+CBE at 3.2e10vg: n=7) was used to measure GCase activity, GluSph levels, GluCer levels, and vector genomes. GCase activity is shown as ng of GCase per mg of total protein. GluSph and GluCer levels are shown as pmol per mg of wet weight of tissue. Biodistribution is shown as vector genomes per μg of genomic DNA. Vector genome presence was quantified using a vector reference standard curve by quantitative PCR; genomic DNA concentration was assessed by A260 optical density measurements. Vector genome presence was also measured in the liver (E). Mean values ​​are shown. Error bars are SEM. **p<0.01; ***p<0.001, nominal p-values ​​from linear regression in CBE-treated animals, corrected for sex as a covariate.

[0025] [Figure 17-1] Figure 17 shows representative data of tapered beam analysis at maximum dose GBA1rAAV in genetic mouse model. The motor performance of treatment groups (WT+vehicle, (n=5), 4L / PS-NA+vehicle (n=6), and 4L / PS-NA+rAAV (n=5)) was analyzed by beam walking 4 weeks after rAAV administration. Total slips and active time are shown as the average of 5 trials on different beams. Velocity and slips per velocity are shown as the average of 5 trials on different beams. Mean values ​​are displayed. Error bars are SEM. [Figure 17-2] Figure 17 shows representative data of tapered beam analysis at maximum dose GBA1rAAV in genetic mouse model. The motor performance of treatment groups (WT+vehicle, (n=5), 4L / PS-NA+vehicle (n=6), and 4L / PS-NA+rAAV (n=5)) was analyzed by beam walking 4 weeks after rAAV administration. Total slips and active time are shown as the average of 5 trials on different beams. Velocity and slips per velocity are shown as the average of 5 trials on different beams. Mean values ​​are displayed. Error bars are SEM. [Figure 18-1] 18 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. The data show that transfection of HEK293 cells with each construct resulted in overexpression of the transgene of interest compared to cells transfected with GFP. [Figure 18-2] 18 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. The data show that transfection of HEK293 cells with each construct resulted in overexpression of the transgene of interest compared to cells transfected with GFP.

[0026] [Figure 19] FIG. 19 is a schematic diagram showing an rAAV vector (top) that includes a "D" region located "outside" the ITRs (e.g., proximal to the end of the ITR compared to the transgene insert or expression construct), and a wild-type rAAV vector that has the ITRs "inside" the vector (e.g., proximal to the transgene insert of the vector). [Figure 20] Figure 20 shows data for transduction of HEK293 cells using rAAV with wild-type (circles) ITRs or ITRs in alternative (e.g., "outside"; square) configurations of the "D" sequence. rAAV with ITRs in the "outside" configuration were able to transduce cells as efficiently as rAAV with wild-type ITRs.

[0027] Detailed Description The present disclosure is based in part on compositions and methods for expressing combinations of PD-related gene products in subjects.Gene products can be proteins, protein fragments (e.g., parts), interference nucleic acids that inhibit PD-related genes, etc. In some embodiments, gene products are proteins or protein fragments that are encoded by PD-related genes.In some embodiments, gene products are interference nucleic acids (e.g., shRNA, siRNA, miRNA, amiRNA, etc.) that inhibit PD-related genes.

[0028] PD-associated gene refers to a gene that encodes a gene product that is genetically, biochemically or functionally related to PD. For example, it has been observed that individuals with mutations in the GBA1 gene (which encodes the protein Gcase) are at higher risk of developing PD than individuals without mutations in GBA1. In another example, PD is associated with the accumulation of protein aggregates that contain alpha-synuclein (alpha-Syn) protein; thus, SCNA (which encodes alpha-Syn) is a PD-associated gene. In some embodiments, the expression cassette described herein encodes a wild-type or non-mutated form of a PD-associated gene (or its coding sequence). Examples of PD-associated genes are listed in Table 1.

[0029] [Table 1]

[0030] Isolated Nucleic Acids and Vectors The isolated nucleic acid may be DNA or RNA. In some aspects, the present disclosure provides an isolated nucleic acid, including an expression construct encoding Gcase (e.g., the gene product of the GBA1 gene) or a portion thereof. Gcase, also called β-glucocerebrosidase or GBA, refers to a lysosomal protein that cleaves the β-glucosidic bond of the chemical glucocerebroside, an intermediate in glycolipid metabolism. In humans, Gcase is encoded by the GBA1 gene located on chromosome 1. In some embodiments, GBA1 encodes a peptide represented by NCBI reference sequence NP_000148.2 (SEQ ID NO: 14). In some embodiments, the isolated nucleic acid comprises a codon-optimized (e.g., codon-optimized for expression in mammalian cells, such as human cells) Gcase coding sequence, such as the sequence shown in SEQ ID NO: 15.

[0031] In some aspects, the disclosure provides an isolated nucleic acid, including an expression construct, encoding prosaposin (e.g., a gene product of the PSAP gene). Prosaposin is a precursor glycoprotein of sphingolipid activator proteins (saposins) A, B, C, and D, which promotes the catabolism of sphingoglycolipids with short oligosaccharide groups. In humans, the PSAP gene is located on chromosome 10. In some embodiments, PSAP encodes a peptide represented by NCBI reference sequence NP_002769.1 (e.g., SEQ ID NO: 16). In some embodiments, the isolated nucleic acid comprises a codon-optimized (e.g., codon-optimized for expression in mammalian cells, such as human cells) prosaposin coding sequence, such as the sequence set forth in SEQ ID NO: 17.

[0032] Aspects of the present disclosure relate to isolated nucleic acids, including expression constructs encoding LIMP2 / SCARB2 (e.g., gene products of the SCARB2 gene). SCARB2 refers to a membrane protein that regulates lysosomal and endosomal trafficking in cells. In humans, the SCARB2 gene is located on chromosome 4. In some embodiments, the SCARB2 gene encodes a peptide represented by NCBI reference sequence NP_005497.1 (SEQ ID NO: 18). In some embodiments, the isolated nucleic acid comprises a sequence shown in SEQ ID NO: 19. In some embodiments, the isolated nucleic acid comprises a codon-optimized SCARB2 coding sequence.

[0033] In some aspects, 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 is independently selected from the gene products set forth in Table 1, or a portion thereof.

[0034] In some embodiments, the gene product is encoded by a coding portion (e.g., cDNA) of a naturally occurring gene. In some embodiments, the first gene product is a protein (or a fragment thereof) encoded by the GBA1 gene. In some embodiments, the gene product is a protein (or a fragment thereof) encoded by the SCARB2 / LIMP2 gene and / or the PSAP gene. However, those skilled in the art will recognize that the order of expression of the first gene product (e.g., Gcase) and the second gene product (e.g., LIMP2) can generally be reversed (e.g., LIMP2 is the first gene product and Gcase is the second gene product). In some embodiments, the gene product is a fragment (e.g., a portion) of a gene listed in Table 1. The protein fragment can include about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the protein encoded by the gene listed in Table 1. In some embodiments, the protein fragment comprises between 50% and 99.9% (e.g., any value between 50% and 99.9%) of the protein encoded by a gene listed in Table 1.

[0035] In some embodiments, the expression construct is monocistronic (e.g., the expression construct encodes a single fusion protein comprising a first gene product and a second gene product). In some embodiments, the expression construct is polycistronic (e.g., the expression construct encodes two different gene products, e.g., two different proteins or protein fragments).

[0036] A polycistronic expression vector can include one or more (e.g., 1, 2, 3, 4, 5, or more) promoters. Any suitable promoter can be used, such as a constitutive promoter, an inducible promoter, an endogenous promoter, a tissue-specific promoter (e.g., a CNS-specific promoter), and the like. In some embodiments, the promoter is a chicken beta-actin promoter (CBA promoter), a CAG promoter (e.g., as described in Alexopoulou et al. (2008) BMC Cell Biol. 9:2; doi: 10.1186 / 1471-2121-9-2), a CD68 promoter, or a JeT promoter (e.g., as described in Tornoe et al. (2002) Gene 297(1-2):21-32). In some embodiments, the promoter is operably linked to a nucleic acid sequence encoding a first gene product, a second gene product, or a first and a second gene product. In some embodiments, the expression cassette contains 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 thereof.

[0037] In some embodiments, the nucleic acid sequence encoding the first gene product and the nucleic acid sequence encoding the second gene product are separated by a nucleic acid sequence encoding an internal ribosome entry site (IRES). Examples of IRES sites are described, for example, in Mokrejs et al. (2006) Nucleic Acids Res. 34(Database issue):D125-30. In some embodiments, the nucleic acid sequence encoding the first gene product and the nucleic acid sequence encoding the second gene product are separated by a nucleic acid sequence encoding a self-cleaving peptide. Examples of self-cleaving peptides include, but are not limited to, T2A, P2A, E2A, F2A, BmCPV 2A, and BmIFV 2A, and those described in Liu et al. (2017) Sci Rep. 7: 2193. In some embodiments, the self-cleaving peptide is a T2A peptide.

[0038] Pathologically, diseases such as PD and Gaucher's disease are associated with the accumulation of protein aggregates that are mainly composed of alpha-synuclein (alpha-Syn) protein. Thus, in some embodiments, the isolated nucleic acid described herein comprises an inhibitory nucleic acid that reduces or prevents the expression of alpha-Syn protein. The sequence encoding the inhibitory nucleic acid can be placed in the untranslated region (e.g., intron, 5'UTR, 3'UTR, etc.) of the expression vector.

[0039] In some embodiments, the inhibitory nucleic acid is placed in an intron of the expression construct, e.g., an intron upstream of a sequence encoding a first gene product. The inhibitory nucleic acid can be a double-stranded RNA (dsRNA), siRNA, microRNA (miRNA), artificial miRNA (amiRNA), or RNA aptamer. In general, the inhibitory nucleic acid binds to (e.g., hybridizes with) about 6 to about 30 (e.g., any integer between 6 and 30, inclusive) nucleotides adjacent to a target RNA (e.g., mRNA). In some embodiments, the inhibitory nucleic acid molecule is an miRNA or amiRNA, e.g., an miRNA that targets SNCA (the gene encoding the α-Syn protein). In some embodiments, the miRNA does not contain any mismatches with the region of the mRNA of SNCA to which it hybridizes (e.g., the miRNA is "perfect"). In some embodiments, the inhibitory nucleic acid is an shRNA (e.g., an shRNA that targets SNCA).

[0040] The isolated nucleic acid described herein can be present by itself or as part of a vector. Generally, the vector is a plasmid, a cosmid, a phagemid, a bacterial artificial chromosome (BAC), or a viral vector (e.g., an adenovirus vector, an adeno-associated virus (AAV) vector, a retrovirus vector, a baculovirus vector, etc.). In some embodiments, the vector is a plasmid (e.g., a plasmid comprising an isolated nucleic acid described herein). In some embodiments, the vector is a recombinant AAV (rAAV) vector. In some embodiments, the rAAV vector is single-stranded (e.g., single-stranded DNA). In some embodiments, the vector is a baculovirus vector (e.g., an Autographa californica nuclear polyhedrosis (AcNPV) vector).

[0041] Typically, a rAAV vector (e.g., a rAAV genome) comprises a transgene (e.g., an expression construct comprising one or more of each of the following: a promoter, an intron, an enhancer sequence, a protein coding sequence, an inhibitory RNA coding sequence, a polyA tail sequence, etc.) flanked by two AAV inverted terminal repeat (ITR) sequences. In some embodiments, the transgene of the rAAV vector comprises an isolated nucleic acid described in this disclosure. In some embodiments, each of the two ITR sequences of the rAAV vector is a full-length ITR (e.g., about 145 bp in length and comprising a functional Rep binding site (RBS) and a terminal resolution site (trs)). In some embodiments, one of the ITRs of the rAAV vector is truncated (e.g., shortened or not full-length). In some embodiments, the truncated ITR lacks a functional terminal resolution site (trs) and is used for the generation of self-complementary AAV vectors (scAAV vectors). In some embodiments, the truncated ITR is a ΔITR, e.g., as described in McCarty et al. (2003) Gene Ther. 10(26):2112-8.

[0042] Aspects of the present disclosure relate to isolated nucleic acids (e.g., rAAV vectors) that include an ITR that has one or more modifications (e.g., additions, deletions, substitutions, etc. of nucleic acids) compared to a wild-type AAV ITR, e.g., compared to a wild-type AAV2 ITR (e.g., SEQ ID NO: 29). The structure of the wild-type AAV2 ITR is shown in FIG. 19. Generally, wild-type ITRs include 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 designated B / B' and C / C', respectively), a longer stem region (formed by sequences A / A'), and a single-stranded terminal region designated the "D" region (FIG. 19). Generally, the "D" region of the ITR is located between the stem region formed by the A / A' sequences and the transgene-containing insert of the rAAV vector (e.g., located "inside" the ITR relative to the end of the ITR or proximal to the transgene insert or expression construct of the rAAV vector). In some embodiments, the "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, e.g., as disclosed in Ling et al. (2015) J Mol Genet Med 9(3).

[0043] This disclosure is based, in part, on the surprising discovery that rAAV vectors containing a "D" region located "outside" the ITRs (e.g., closer to the end of the ITR relative to a transgene insert or expression construct) are more efficiently encapsidated by AAV capsid proteins than rAAV vectors with unmodified (e.g., wild-type) ITRs. In some embodiments, rAAV vectors with modified "D" regions have reduced toxicity compared to rAAV vectors with wild-type ITR sequences.

[0044] In some embodiments, the modified "D" sequence contains at least one nucleotide substitution compared to the wild-type "D" sequence (e.g., SEQ ID NO:27). The modified "D" sequence can have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotide substitutions compared to the wild-type "D" sequence (e.g., SEQ ID NO:27). In some embodiments, the modified "D" sequence contains at least 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 nucleotide substitutions compared to the wild-type "D" sequence (e.g., SEQ ID NO:27). In some embodiments, the modified "D" sequence is about 10% to 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 the wild-type "D" sequence (e.g., SEQ ID NO: 27). In some embodiments, the modified "D" sequence comprises the sequence set forth in SEQ ID NO: 26, also referred to as the "S" sequence, as described in Wang et al. (1995) J Mol Biol 250(5):573-80.

[0045] An isolated nucleic acid or rAAV vector described in this disclosure can further comprise a "TRY" sequence, e.g., as set forth in SEQ ID NO:28, or as described in Francois, et al. The Cellular TATA Binding Protein Is Required for Rep-Dependent Replication of a Minimal Adeno-Associated Virus Type 2 p5 Element. J Virol. 2005. In some embodiments, the TRY sequence is located between the ITR (e.g., 5'ITR) of the isolated nucleic acid or rAAV vector and the expression construct (e.g., an insert encoding a transgene).

[0046] In some aspects, the present disclosure relates to a baculovirus vector, comprising the isolated nucleic acid or rAAV vector described in the present disclosure.In some embodiments, the baculovirus vector is an Autographa californica nuclear polyhedrosis (AcNPV) vector, for example, as described in Urabe et al. (2002) Hum Gene Ther 13(16):1935-43 and Smith et al. (2009) Mol Ther 17(11):1888-1896.

[0047] In some aspects, the present disclosure provides a host cell comprising the isolated nucleic acid or vector described herein.The host cell can be a prokaryotic cell or a eukaryotic cell.For example, the host cell can be a mammalian cell, a bacterial cell, a yeast cell, an insect cell, etc.In some embodiments, the host cell is a mammalian cell, for example, a HEK293T cell.In some embodiments, the host cell is a bacterial cell, for example, an E. coli cell.

[0048] rAAV In some aspects, the present disclosure relates to recombinant AAV (rAAV) comprising a transgene encoding a nucleic acid described herein (e.g., an rAAV vector described herein). The term "rAAV" generally refers to a viral particle comprising a rAAV vector encapsidated by one or more AAV capsid proteins. The rAAV described in this disclosure may comprise a capsid protein having a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 and AAV10. In some embodiments, the rAAV comprises a capsid protein from a non-human host, e.g., a rhesus AAV capsid protein such as AAVrh.10, AAVrh.39. In some embodiments, the rAAV described herein comprises a capsid protein that is a variant of a wild-type capsid protein, e.g., a capsid protein variant that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 (e.g., 15, 20, 25, 50, 100, etc.) amino acid substitutions (e.g., mutations) relative to the wild-type AAV capsid protein from which it is derived.

[0049] In some embodiments, the rAAV described in the present disclosure spreads easily through the CNS, especially when introduced into the CSF space or directly into the brain parenchyma.Thus, in some embodiments, the rAAV described in the present disclosure comprises a capsid protein that can cross the blood-brain barrier (BBB).For example, in some embodiments, the rAAV comprises a capsid protein having AAV9 or AAVrh.10 serotype.The generation of rAAV is described, for example, in Samulski et al. (1989) J Virol. 63(9):3822-8 and Wright (2009) Hum Gene Ther. 20(7): 698-706.

[0050] In some embodiments, the rAAV described in this disclosure (e.g., comprising a recombinant rAAV genome that is encapsidated by AAV capsid proteins to form rAAV capsid particles) is produced in a baculovirus vector expression system (BEVS). The production of rAAV using BEVS is described, for example, in 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. However, rAAV can be produced using any suitable method (e.g., using recombinant rep and cap genes).

[0051] Pharmaceutical Compositions In some aspects, the present disclosure provides a pharmaceutical composition comprising the isolated nucleic acid or rAAV described herein and a pharma- ceutically acceptable carrier.As used herein, the term "pharma-ceutically acceptable" refers to a material, such as a carrier or diluent, that does not abolish the biological activity or properties of a 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 harmful manner with any of the components of the composition in which it is contained.

[0052] As used herein, the term "pharmaceutical acceptable carrier" refers to a pharmaceutical acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent or encapsulating material, which is associated with carrying or transporting a compound useful within the present invention into or to a patient so that it can perform its intended function. Additional components that can be included in the pharmaceutical composition used in the practice of the present 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.

[0053] The compositions (e.g., pharmaceutical compositions) provided herein can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intracerebroventricular, transdermal, intradermal, rectal, intravaginal, intraperitoneal, topical (by powder, ointment, cream, and / or drops), mucosal, nasal, buccal, sublingual; intratracheal instillation, bronchial instillation, and / or inhalation; and / or as oral spray, nasal spray, and / or aerosol. Specifically contemplated routes are oral administration, intravenous administration (e.g., systemic intravenous injection), local administration via blood and / or lymphatic supply, and / or direct administration to the affected site. In general, the most appropriate route of administration will depend on various factors, such as 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 can tolerate oral administration). In certain embodiments, the compounds or pharmaceutical compositions described herein are suitable for topical administration to the subject's eye.

[0054] method The present disclosure is based in part on the composition for the expression of a combination of PD-related gene products in a subject, which act together (e.g., synergistically) to treat Parkinson's disease.As used herein, "treat" or "treating" refers to (a) preventing or delaying the onset of Parkinson's disease; (b) reducing the severity of Parkinson's disease; (c) reducing or preventing the onset of symptoms characteristic of Parkinson's disease; (d) preventing the worsening of symptoms characteristic of Parkinson's disease.Symptoms of Parkinson's disease include, for example, motor dysfunction (e.g., tremors, stiffness, slowness, difficulty walking), cognitive dysfunction (e.g., dementia, depression, anxiety), emotional and behavioral dysfunction.

[0055] Thus, in some aspects, the disclosure provides a method of treating a subject having or suspected of having Parkinson's disease, the method comprising administering to the subject a composition described in the disclosure (e.g., a composition comprising an isolated nucleic acid or vector or rAAV).

[0056] In some embodiments, the composition is administered directly to the CNS of a subject, for example, by direct injection into the brain and / or spinal cord of a subject. Examples of CNS direct administration methods include, but are not limited to, intracerebral injection, intraventricular injection, intracisternal injection, intraparenchymal injection, intrathecal injection, and any combination of the above. In some embodiments, direct injection into the CNS of a subject results in transgene expression (e.g., expression of the first gene product, the second gene product, and, if applicable, the third gene product) in the midbrain, striatum, and / or cerebral cortex of the subject. In some embodiments, direct injection into the CNS results in transgene expression (e.g., expression of the first gene product, the second gene product, and, if applicable, the third gene product) in the spinal cord and / or CSF of a subject.

[0057] In some embodiments, direct injection into the CNS of the subject comprises convection-enhanced delivery (CED).Convection-enhanced delivery is a therapeutic strategy that comprises surgical exposure of the brain and placing a small-diameter catheter directly into the target area of ​​the brain, followed by injecting a therapeutic agent (e.g., the composition described herein or rAAV) directly into the brain of the subject.CED is described, for example, in Debinski et al. (2009) Expert Rev Neurother. 9(10):1519-27.

[0058] In some embodiments, the composition is administered to the subject's periphery, for example by peripheral injection.Examples of peripheral injection include subcutaneous injection, intravenous injection, intraarterial injection, intraperitoneal injection, or any combination thereof.In some embodiments, the peripheral injection is intraarterial injection, for example injection into the carotid artery of the subject.

[0059] In some embodiments, the composition described in this disclosure (e.g., a composition comprising an isolated nucleic acid or vector or rAAV) is administered both peripherally and directly to the CNS of a subject. For example, in some embodiments, the subject is administered the composition by intra-arterial injection (e.g., injection into the carotid artery) and intraparenchymal injection (e.g., intraparenchymal injection by CED). In some embodiments, the direct injection into the CNS and the peripheral injection are simultaneous (e.g., occur simultaneously). In some embodiments, the direct injection is performed before the peripheral injection (e.g., between 1 minute and 1 week, or more). In some embodiments, the direct injection is performed after the peripheral injection (e.g., between 1 minute and 1 week, or more).

[0060] The amount of a composition described herein (e.g., a composition comprising an isolated nucleic acid or vector or rAAV) administered to a subject will vary depending on the method of administration. For example, in some embodiments, the rAAV described herein is administered to a subject at a dose of about 10 9 genome copies (GC) / kg and approximately 10 14 Between GC / kg (e.g., about 10 9 GC / kg, approx. 10 10 GC / kg, approx. 10 11GC / kg, approx. 10 12 GC / kg, approx. 10 12 GC / kg, or approximately 10 14 In some embodiments, the subject is administered a high titer (e.g., >10 12 The rAAV (genome copies GC / kg) is administered by injection into the CSF space or by intraparenchymal injection.

[0061] A composition described herein (e.g., a composition comprising an isolated nucleic acid or vector or rAAV) can be administered to a subject one or more times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or more). In some embodiments, the composition is administered to a subject continuously (e.g., chronically), e.g., via an infusion pump. EXAMPLES

[0062] Example 1: rAAV vector AAV vectors are generated using cells such as HEK293 cells for triple plasmid transfection. ITR sequences flank the expression construct, which contains the promoter / enhancer elements, 3' polyA signal, and post-translational signals such as WPRE elements for each transgene of interest. Multiple gene products such as GBA1, LIMP2, and prosaposin can be expressed simultaneously by: fusion of protein sequences; or by use of 2A peptide linkers such as T2A and P2A (which prevent the creation of a peptide bond, resulting in two peptide fragments with additional amino acids); or by use of IRES elements; or by expression in two separate expression cassettes. The presence of short intron sequences that are efficiently spliced ​​upstream of the expressed gene can improve expression levels. shRNAs and other regulatory RNAs may be included within these sequences. Examples of plasmids containing the rAAV vectors described in this disclosure are shown in Figures 1-6 and Table 2 below.

[0063] [Table 2]

[0064] Example 2: Cell-based assay of viral transduction into GBA-deficient cells Cells deficient in GBA1 are obtained, for example, as fibroblasts, monocytes, or hES cells from GD patients, or patient-derived induced pluripotent stem cells (iPSCs). These cells accumulate substrates such as glucosylceramide and glucosylsphingosine (GluCer and GluSph). Treatment of wild-type or mutant cultured cell lines with Gcase inhibitors such as CBE is also used to obtain GBA-deficient cells.

[0065] Using such cell models, lysosomal defects are quantified in terms of accumulation of protein aggregates such as α-synuclein using antibodies against this protein or phospho-αSyn, followed by imaging using a fluorescent microscope. Lysosomal abnormalities are also imaged by ICC of protein markers such as LAMP1, LAMP2, LIMP1, LIMP2, or by using dyes such as Lysotracker, or by uptake of fluorescent dextran or other markers via endocytic compartments. Imaging of accumulation of autophagy markers due to defects in fusion with lysosomes such as LC3 can also be performed. Western blotting and / or ELISA are used to quantify abnormal accumulation of these markers. Accumulation of glycolipid substrates and products of GBA1 is also measured using standard approaches. Therapeutic endpoints (such as reduction in PD-related pathology) are measured in the context of expression of the AAV vector transduction to confirm and quantify activity and function. Gcase can also be quantified using protein ELISA measurements or by standard Gcase activity assays.

[0066] Example 3: In vivo assay using mutant mice In this example, the in vivo assay of AAV vector using mutant mice is described.The in vivo study of AAV vector as described above in mutant mice is carried out using the assay described in, for example, Liou et al. (2006) J.Biol.Chem.281(7): 4242-4253, Sun et al. (2005) J.Lipid Res.46:2102-2113, and Farfel-Becker et al. (2011) Dis.Model Mech.4(6):746-752. Vehicle control and intrathecal or intracerebroventricular delivery of AAV vectors (e.g., 2 × 10 11 vg / mouse) using concentrated AAV stocks with an injection volume of, for example, 5-10 µL. Perform intraparenchymal delivery via convection-enhanced delivery. Treatment is initiated before or after the onset of symptoms. Endpoints measured include accumulation of substrate in the CNS and CSF, accumulation of Gcase enzyme by ELISA, and enzyme activity, motor and cognitive endpoints, lysosomal dysfunction, and accumulation of α-synuclein monomers, protofibrils, or fibrils.

[0067] Example 4: Chemical models of disease In this example, we describe in vivo assays of AAV vectors using a chemically induced mouse model of Gaucher disease (such as the CBE mouse model). These in vivo studies of AAV vectors are performed in a chemically induced mouse model of Gaucher disease, for example, as described in Vardi et al. (2016) J Pathol. 239(4):496-509. Vehicle control and intrathecal or intracerebroventricular delivery of AAV vectors (e.g., 2 × 10 11 Dose of 100 mg / vg / mouse) is performed using concentrated AAV stocks, e.g., with an injection volume of 5-10 µL. Intraparenchymal delivery is performed by convection-enhanced delivery. Peripheral delivery is achieved by tail vein injection. Treatment is initiated before or after the onset of symptoms. Endpoints measured include accumulation of substrate in the CNS and CSF, accumulation of Gcase enzyme by ELISA, and enzyme activity, motor and cognitive endpoints, lysosomal dysfunction, and accumulation of α-synuclein monomers, protofibrils, or fibrils.

[0068] Example 5: Clinical trials for patients with PD, LBD, and Gaucher disease In some embodiments, patients with certain forms of Gaucher disease (e.g., GD1) are at increased risk of developing Parkinson's disease (PD) or Lewy body dementia (LBD). In this example, a clinical trial is described to evaluate the safety and efficacy of the rAAV described in this disclosure in patients with Gaucher disease, PD and / or LBD. Clinical trials of such vectors for the treatment of Gaucher disease, PD and / or LBD will be conducted using a study design similar to that described in Grabowski et al. (1995) Ann. Intern. Med. 122(1):33-39.

[0069] Example 6: Treatment of peripheral diseases In some embodiments, patients with certain forms of Gaucher disease exhibit symptoms of peripheral neuropathy, e.g., as described in Biegstraaten et al. (2010) Brain 133(10):2909-2919. This example describes the in vivo assay of the AAV vector described herein for the treatment of peripheral neuropathy associated with Gaucher disease (e.g., type 1 Gaucher disease). Briefly, a type 1 Gaucher disease patient identified as having signs or symptoms of peripheral neuropathy is administered rAAV as described in this disclosure. In some embodiments, the subject's signs and symptoms of peripheral neuropathy are monitored after administration of rAAV, for example, using the method described in Biegstraaten et al. The levels of the transduced gene products described in this disclosure present in the patient (e.g., in the patient's serum, peripheral tissues (e.g., liver tissue, spleen tissue, etc.)) are assayed, for example, by Western blot analysis, enzyme function assays, or imaging studies.

[0070] Example 7: Treatment of CNS type In this example, the in vivo assay of rAAV described herein for the treatment of CNS-type Gaucher disease is described. Briefly, a Gaucher disease patient identified as having a CNS-type Gaucher disease (e.g., Gaucher disease type 2 or 3) is administered rAAV as described in this disclosure. The level of the transduced gene product described in this disclosure present in the patient's CNS (e.g., in the serum of the patient's CNS, in the patient's cerebrospinal fluid (CSF), or in the patient's CNS tissue) is assayed, for example, by Western blot analysis, enzyme function assay, or imaging.

[0071] Example 8: Gene therapy for Parkinson's disease in subjects with mutations in GBA1 This example describes the administration of a recombinant adeno-associated virus (rAAV) encoding GBA1 to patients with Parkinson's disease, which is characterized by mutations in the GBA1 gene. The rAAV vector insert is composed of four parts: the CMV enhancer (CMVe), the CBA promoter (CBAp), exon 1, and an intron (int), which contains the CBA promoter element (CBA) that constitutively expresses the codon-optimized coding sequence (CDS) of human GBA1 (maroon). The 3' region also contains the posttranscriptional regulatory element of the woodchuck hepatitis virus (WPRE) followed by the bovine growth hormone polyA signal (bGH polyA) tail. The flanking ITRs allow for correct packaging of the intervening sequences. Two variants of the 5'ITR sequence (Figure 7, insert box, lower sequence) were evaluated; these variants have several nucleotide differences within the 20 nucleotide "D" region of the ITR, which are believed to affect the efficiency of packaging and expression. The rAAV product contains the "D" domain nucleotide sequence shown in Figure 7 (insert box, upper sequence). The variant vectors feature a mutated "D" domain (herein referred to as the "S" domain, with nucleotide changes shown shaded) that behaves similarly in preclinical trials. The backbone includes a gene that confers kanamycin resistance and a stuffer sequence to prevent reverse packaging. A schematic showing rAAV vectors is shown in Figure 8. rAAV vectors are packaged into rAAV using AAV9 serotype capsid proteins.

[0072] GBA1-rAAV is administered to subjects as a single dose via fluoroscopically guided suboccipital injection into the cisterna magna (intracisternal cytometer; ICM). One aspect of the dosing regimen study is as follows: Administer a single dose of rAAV to patients (N=12) at one of two dose levels (3e13vg (low dose); 1e14vg (high dose), etc.) that will be determined based on the results of nonclinical pharmacology and toxicology studies. Initial studies were conducted in a chemical mouse model involving daily delivery of conduritol-b-epoxide (CBE), an inhibitor of GCase, to evaluate the efficacy and safety of rAAV vectors and variant rAAV S-variant constructs (as described further below). Initial studies were also conducted in a genetic mouse model carrying a homozygous GBA1 mutation and partially deficient in saposin (4L / PS-NA). Additional dose-ranging studies in mice and non-human primates (NHPs) will be conducted to further evaluate the safety and efficacy of the vectors.

[0073] Two slightly different versions of the 5' inverted terminal repeat (ITR) of the AAV backbone were tested to evaluate manufacturability and transgene expression (Figure 7). The 20 bp "D" domain within the 145 bp 5' ITR is believed to be necessary for optimal viral vector generation, however mutations within the "D" domain have also been reported to increase transgene expression in some cases. Thus, in addition to viral vectors possessing an intact "D" domain, a second vector form with a mutated D domain (referred to herein as the "S" domain) was also evaluated. Both rAAV and variant rAAV express the same transgene. Although both vectors generated effective virus in vivo as detailed below, the rAAV containing the wild-type "D" domain was selected for further development.

[0074] To establish a CBE model of GCase deficiency, young mice were administered CBE, a specific inhibitor of GCase. Mice were given CBE by daily IP injection 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 exhibiting a behavioral phenotype (Figure 9). High doses of CBE caused lethality in a dose-dependent manner. All mice treated with 50 mg / kg CBE died by P23, and 5 of 8 mice treated with 37.5 mg / kg CBE died by P27. There was no lethality in mice treated with 25 mg / kg CBE. Although mice injected with CBE did not show general motor impairments in the open field assay (moving the same distances and at the same speed as mice administered PBS), mice treated with CBE showed impaired motor coordination and balance as measured by the rotarod assay.

[0075] Mice that survived to the end of the study were sacrificed the day after the last CBE dose (P27, "day 1") or 3 days after CBE cessation (P29, "day 3"). Lipid analysis was performed in the cortex of mice treated with 25 mg / kg CBE to assess accumulation of GCase substrates in both day 1 and day 3 cohorts. GluSph and GalSph levels (measured in total in this example) were significantly accumulated in CBE-treated mice compared to PBS-treated controls, consistent with GCase insufficiency. Based on the above studies, a CBE dose of 25 mg / kg was selected due to its ability to induce behavioral deficits without affecting survival. To achieve brain-wide GBA1 distribution and transgene expression during CBE treatment, rAAV or vehicle was delivered by intracerebroventricular (ICV) injection on postnatal day 3 (P3), followed by daily IP CBE or PBS treatments starting at P8 (Figure 10).

[0076] CBE-treated mice administered rAAV performed statistically significantly better on the rotarod than vehicle-treated mice (Figure 11). Mice in the variant vector-treated group did not differ from vehicle-treated mice in other behavioral measures, such as total distance traveled during the test (Figure 11). Upon completion of the survival study, half of the mice were sacrificed for biochemical analysis, either the day after the last dose of CBE (P36, "day 1") or 3 days after CBE cessation (P38, "day 3") (Figure 12). GCase activity was assessed in the cortex using a fluorescent enzyme assay performed in biological triplicates. GCase activity was increased in mice treated with GBA1-rAAV, whereas CBE treatment reduced GCase activity. Furthermore, mice administered both CBE and GBA1-rAAV showed similar GCase activity levels to the PBS-treated group, indicating that rAAV delivery can overcome the inhibition of GCase activity induced by CBE treatment. Lipid analysis was performed in the motor cortex of mice to examine the levels of the substrates GluCer and GluSph. Both lipids accumulated in the brains of mice administered CBE, and rAAV treatment significantly reduced the accumulation of the substrates.

[0077] Lipid levels were negatively correlated with both GCase activity and performance on the rotarod across treatment groups. Increased GCase activity after rAAV administration was associated with reduced substrate and enhanced motor function (Figure 13). Preliminary biodistribution was assessed by the presence of vector genomes using qPCR measurements (>100 vector genomes per μg of genomic DNA defined as positive), as shown in Figure 14. Mice administered GBA1-rAAV both with and without CBE were positive for rAAV vector genomes in the cortex, indicating that ICV delivery results in rAAV delivery to the cortex. Furthermore, vector genomes were detected in the liver, barely detectable in the spleen, and not detected at all in the heart, kidney, or gonads. There were no statistically significant differences between the day 1 and day 3 groups for all measurements.

[0078] A larger study in the CBE model further explored the effective dose of GBA1-rAAV in the CBE model. Using a 25 mg / kg CBE dose model, vehicle or GBA1-rAAV was delivered via ICV on P3, and daily IP PBS or CBE treatments were initiated on P8. Given the similarity between groups with and without CBE cessation observed in previous studies, all mice were sacrificed 1 day after the final CBE administration (P38-40). The effects of three different rAAV doses were evaluated, resulting in the following five groups, with 10 mice per group (5M / 5F): Excipient ICV+PBS IP Vehicle ICV + 25mg / kg CBE IP 3.2e9vg (2.13e10vg / g brain) of rAAV ICV + 25mg / kg of CBE IP 1.0e10vg (6.67e10vg / g brain) of rAAV ICV + 25mg / kg of CBE IP 3.2e10vg (2.13e11vg / g brain) of rAAV ICV + 25mg / kg of CBE IP.

[0079] The highest dose of rAAV rescued the failure to gain weight at P37 associated with CBE treatment. Furthermore, this dose resulted in a statistically significant increase in performance on the rotarod and tapered beam compared to the vehicle+CBE treated group (Figure 15). Lethality was observed in several groups, including both vehicle and rAAV treated groups (vehicle+PBS: 0; vehicle+25mg / kg CBE: 1; 3.2e9vg rAAV+25mg / kg CBE: 4; 1.0e10vg rAAV+25mg / kg CBE: 0; 3.2e10vg rAAV+25mg / kg CBE: 3).

[0080] Upon completion of the survival study, mice were sacrificed for biochemical analysis (Figure 16). Cortical GCase activity was assessed by fluorescent assay in biological triplicates. CBE-treated mice showed a decrease in GCase activity, whereas mice administered high-dose rAAV 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 administration of high-dose rAAV.

[0081] In addition to the established chemical CBE model, GBA1-rAAV will also be evaluated in the 4L / PS-NA genetic model; which is homozygous for the V394L GD mutation in Gba1 and partially deficient in saposins affecting GCase localization and activity. These mice exhibit impaired motor strength, coordination, and balance as evidenced by performance in beam walking, rotarod, and wire-hanging assays. Typically, these mice have a life span of less than 22 weeks. In initial studies, 3 μl of maximum titer virus was delivered by ICV at P23 with a final dose of 2.4e10vg (6.0e10vg / g brain). Treatment groups with 6 mice per group are as follows: WT + vehicle ICV 4L / PS-NA+Excipient ICV 4L / PS-NA+2.4e10vg (6.0e10vg / g brain) rAAV ICV

[0082] Motor performance with the beam walking test was assessed 4 weeks after rAAV delivery. The group of mutant mice administered GBA1-rAAV showed a trend towards fewer total slips and fewer slips per speed compared to vehicle-treated mutant mice, restoring motor function to near WT levels (Figure 17). As the motor phenotype becomes more severe as these mice age, their performance in this and other behavioral tests will be assessed at later time points. Upon completion of survival studies, lipid levels, GCase activity, and biodistribution will be assessed in these mice.

[0083] Additional lower doses of rAAV are currently being tested using the CBE model, corresponding to 0.03x, 0.1x, and 1x the proposed Phase 1 high clinical dose. Each group includes 10 mice per group (5M / 5F): Excipient ICV Vehicle ICV + 25mg / kg CBE IP 3.2e8vg (2.13e9vg / g brain) of rAAV ICV + 25mg / kg of CBE IP 1.0e9vg (6.67e9vg / g brain) of rAAV ICV + 25mg / kg of CBE IP 1.0e10vg (6.67e10vg / g brain) of rAAV ICV + 25mg / kg of CBE IP.

[0084] In addition to the motor phenotype, lipid levels and GCase activity will be assessed in the cortex. A time course of treatment and analysis will also be performed. A larger dose range study was initiated to evaluate efficacy and safety data. Ten 4L / PS-NA mice (5M / 5F per group) were injected with 10 μl of rAAV. Using allometric brain weight calculations, doses correlate to 0.15x, 1.5x, 4.4x, and 14.5x the proposed Phase 1 high clinical dose. Injection groups consisted of: WT + vehicle ICV 4L / PS-NA+Excipient ICV 4L / PS-NA+4.3e9vg (1.1e10vg / g brain) rAAV ICV 4L / PS-NA+4.3e10vg (1.1e11vg / g / brain) rAAV ICV 4L / PS-NA+1.3e11vg (3.2e11vg / g brain) rAAV ICV rAAV ICV in 4L / PS-NA+4.3e11vg (1.1e12vg / g brain).

[0085] A summary of the non-clinical studies of the CBE model is shown in Table 3 below. [Table 3] Note that positive biodistribution is defined as greater than 100 vg / 1 μg genomic DNA. Abbreviations: BD = biodistribution; NS = not significant; T = trend; S = significant; N / A = not applicable; + = positive; - = negative.

[0086] Example 9: In vitro analysis of rAAV vectors A pilot study was performed to evaluate the in vitro activity of rAAV vectors encoding prosaposin (PSAP) and SCARB2, alone or in combination with GBA1 and / or one or more inhibitory RNAs. One construct encoding PSAP and progranulin (PGRN) was also tested. The vectors tested include those shown in Table 4. "Opt" refers to the nucleic acid sequence codon optimized for expression in mammalian cells (e.g., human cells). Figure 18 shows representative data showing that transfection of HEK293 cells with each construct resulted in overexpression of the corresponding gene product compared to mock-transfected cells. [Table 4]

[0087] Example 10: Placement of ITR "D" sequences and transduction of cells The effect of ITR "D" sequence placement on rAAV vector transduction of cells was examined. HEK293 cells were transduced with rAAV encoding Gcase with either 1) a wild type ITR (e.g., the "D" sequence proximal to the transgene insert and distal to the end of the ITR) or 2) an ITR with the "D" sequence located "outside" the vector (e.g., the "D" sequence located proximal to the end of the ITR and distal to the transgene insert) as shown in FIG. 19. Surprisingly, the data show that rAAV with the "D" sequence in the "outside" position retains the ability to be packaged and efficiently transduce cells (FIG. 20).

[0088] Example 11: In vitro toxicity testing Fifty mice were administered rAAV encoding GBA1 via 4 μl intracerebroventricular (ICV) injection on postnatal day 3. All mice received daily intraperitoneal (IP) injections of conduritol B epoxide (CBE) or PBS depending on treatment group from postnatal day 8 until the end of the study. Animals were euthanized 24 h after the last IP administration. Target tissues were harvested after euthanasia, drop-fixed in chilled 4% paraformaldehyde, stored at 4°C, and then sent for histopathological processing and evaluation. There were eight early deaths in the course of the study that were not sent or analyzed.

[0089] Tissues from 42 animals euthanized at 38–40 days were trimmed, processed, and embedded in paraffin blocks, which were then sectioned at approximately 5 μm, stained with hematoxylin and eosin (H&E), and mounted on slides for evaluation. There were no histopathological findings or evidence of toxicity from treatment with rAAV. Mice treated with conduritol B epoxide (CBE) had central nervous system (CNS) findings including glial scarring and neuronal necrosis in the cerebral cortex, and neuronal necrosis in the brainstem and thoracic spinal cord. High-dose rAAV treatment significantly reduced the incidence of these CNS findings, whereas low and intermediate doses of virus produced a dose-dependent reduction in the incidence of glial scarring in the cerebral cortex and had variable effects on other CNS findings.

[0090] Equivalent This application incorporates by reference the entire contents of the following documents: International PCT application referenced by Attorney Docket No. P1094.70003WO00, filed October 3, 2018; International PCT application referenced by Attorney Docket No. P1094.70004WO00, filed October 3, 2018; U.S. Provisional Application No. 62 / 567,311, filed October 3, 2017, entitled "Gene Therapy for Lysosomal Storage Disorders"; U.S. Provisional Application No. 62 / 567, No. 319, entitled "Gene Therapy for Lysosomal Storage Disorders"; No. 62 / 567,301, filed on October 3, 2018, entitled "Gene Therapy for Lysosomal Storage Disorders"; No. 62 / 567,310, filed on October 3, 2017, entitled "Gene Therapy for Lysosomal Storage Disorders"; No. 62 / 567,303, filed on October 3, 2017, entitled "Gene Therapy for Lysosomal Storage Disorders"; and No. 62 / 567,305, filed on October 3, 2017, entitled "Gene Therapy for Lysosomal Storage Disorders".

[0091] Having thus described several aspects of at least one embodiment of this invention, it should be understood that various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawings are by way of example only.

[0092] Although several embodiments of the invention have been described and illustrated herein, those skilled in the art will readily envision various other means and / or structures for performing the functions and / or obtaining the results and / or one or more advantages described herein, and each such variation and / or modification is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application(s) for which the teachings of the present invention are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Thus, the foregoing embodiments are presented by way of example only, and it will be understood that within the scope of the appended claims and their equivalents, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, and / or methods is included within the scope of the present invention, if such features, systems, articles, materials, and / or methods are not mutually inconsistent.

[0093] The indefinite articles "a" and "an," as used in the specification and claims, unless clearly indicated to the contrary, should be understood to mean "at least one." The term "and / or" as used in the specification and claims should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Unless expressly indicated to the contrary, optionally other elements may also be present, whether related to the specifically identified elements or not, other than the elements specifically identified in the "and / or" clause. Thus, as a non-limiting example, a reference to "A and / or B" when used in conjunction with open-ended language such as "comprising" can, in one embodiment, refer to A without B (optionally including elements other than B); in another embodiment, refer to B without A (optionally including elements other than A); in yet another embodiment, refer to both A and B (optionally including other elements); and so forth.

[0094] As used herein and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as inclusive, i.e., including at least one of, and also including a plurality of, a number or list of elements, and optionally including additional items not listed. When terms to the contrary are clearly indicated, such as "only one" or "exactly one," or when used in the claims, "consisting of" refers to including exactly one of, a number or list of elements. In general, the term "or" as used herein, when preceded by terms of exclusivity, such as "either," "one," or "only one," or "exactly one," should be interpreted as referring to exclusive alternatives (i.e., one or the other, but not both). When used in the claims, "consisting essentially of" shall have its ordinary meaning as used in the field of patent law.

[0095] As used herein and in the claims, 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 every respective element specifically listed in the list of elements, nor excluding any combination of elements in the list of elements. This definition allows for the optional presence of elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether or not related to the specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") means, in one embodiment, at least one, optionally more than one, A, and no B (and optionally including elements other than B); in another embodiment, at least one, optionally more than one, B, and no A (optionally including elements other than A); in yet another embodiment, at least one, optionally more than one, A, and at least one, optionally more than one, B (and optionally including other elements); and so forth.

[0096] In the claims and the above specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," and the like, are to be understood as open-ended, i.e., to mean inclusive without limitation. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as set forth in Section 2111.03 of the United States Patent Office Manual of Patent Examining Procedure. The use of ordinal terms such as "first," "second," "third," etc. in the claims to modify claim elements does not, in itself, indicate a priority, precedence, or order of one claim element over another, or the temporal order in which acts of a method are performed, but is used solely as a label to distinguish a given claim element having a particular name from another element of the same name (but differing only in ordinal terms) to distinguish between the claim elements. It should also be understood that, unless expressly stated to the contrary, in any method claimed herein that includes one or more steps or acts, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are presented.

[0097] array In some embodiments, an expression cassette encoding one or more gene products (e.g., a first, second and / or third gene product) comprises or consists of (or encodes a peptide having) a sequence set forth in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, the gene product is encoded by a portion (e.g., a fragment) of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25.

Claims

1. An isolated nucleic acid comprising an expression construct encoding a Gcase protein flanked by two adeno-associated virus (AAV) inverted terminal repeats (ITRs), wherein: (i) at least one ITR comprises a modified "D" region relative to the wild-type AAV2 ITR (SEQ ID NO:29); and / or (ii) Gcase is encoded by a codon-optimized nucleic acid sequence; The isolated nucleic acid.

2. The isolated nucleic acid of claim 1 , wherein the Gcase protein comprises the amino acid sequence set forth in SEQ ID NO: 14 or a portion thereof.

3. 3. The isolated nucleic acid of claim 1 or 2, wherein the Gcase protein is encoded by a codon-optimized nucleic acid sequence, optionally by the nucleic acid sequence shown in SEQ ID NO:

15.

4. 4. The isolated nucleic acid of any one of claims 1 to 3, wherein the modified "D" region is a "D" sequence located outside the ITRs relative to the expression construct.

5. The isolated nucleic acid of any one of claims 1 to 4, wherein the ITR containing a modified "D" sequence is a 3'ITR.

6. 6. The isolated nucleic acid of any one of claims 1 to 5, further comprising a TRY sequence, optionally wherein the TRY sequence is set forth in SEQ ID NO:

28.

7. 1. An isolated nucleic acid comprising an expression construct encoding a prosaposin protein flanked by two adeno-associated virus (AAV) inverted terminal repeats (ITRs), wherein: (i) at least one ITR comprises a modified "D" region relative to the wild-type AAV2 ITR (SEQ ID NO:29); and / or (ii) prosaposin is encoded by a codon-optimized nucleic acid sequence; The isolated nucleic acid.

8. The isolated nucleic acid of claim 7, wherein the prosaposin protein comprises the amino acid sequence set forth in SEQ ID NO: 16 or a portion thereof.

9. 9. The isolated nucleic acid of claim 7 or 8, wherein the prosaposin protein is encoded by a codon-optimized nucleic acid sequence, optionally by the nucleic acid sequence shown in SEQ ID NO:

17.

10. 10. The isolated nucleic acid of any one of claims 7 to 9, wherein the modified "D" region is a "D" sequence located outside the ITR relative to the expression construct.

11. The isolated nucleic acid of any one of claims 7 to 10, wherein the ITR comprising a modified "D" sequence is a 3'ITR.

12. 12. The isolated nucleic acid of any one of claims 7 to 11, further comprising a TRY sequence, optionally wherein the TRY sequence is set forth in SEQ ID NO:

28.

13. 1. An isolated nucleic acid comprising an expression construct encoding a SCARB2 protein flanked by two adeno-associated virus (AAV) inverted terminal repeats (ITRs), wherein: (i) at least one ITR comprises a modified "D" region relative to the wild-type AAV2 ITR (SEQ ID NO:29); and / or (ii) SCARB2 is encoded by a codon-optimized nucleic acid sequence; The isolated nucleic acid.

14. 14. The isolated nucleic acid of claim 13, wherein the SCARB2 protein comprises the amino acid sequence set forth in SEQ ID NO: 18 or a portion thereof.

15. 15. The isolated nucleic acid of claim 13 or 14, wherein the SCARB2 protein is encoded by a codon-optimized nucleic acid sequence or the nucleic acid sequence shown in SEQ ID NO:

19.

16. 16. The isolated nucleic acid of any one of claims 13 to 15, wherein the modified "D" region is a "D" sequence located outside the ITRs relative to the expression construct.

17. The isolated nucleic acid of any one of claims 13 to 16, wherein the ITR comprising a modified "D" sequence is a 3'ITR.

18. 18. The isolated nucleic acid of any one of claims 13 to 17, further comprising a TRY sequence, optionally wherein the TRY sequence is set forth in SEQ ID NO:

28.

19. 1. An isolated nucleic acid comprising an expression construct encoding a first gene product and a second gene product, wherein each gene product is independently selected from the gene products set forth in Table 1, or a portion thereof.

20. 20. The isolated nucleic acid of claim 19, wherein the first gene product is a Gcase protein or a portion thereof.

21. 21. The isolated nucleic acid of claim 19 or 20, wherein the second gene product is LIMP2 or a portion thereof, or prosaposin or a portion thereof.

22. 22. The isolated nucleic acid of any one of claims 19 to 21, further encoding an interfering nucleic acid (e.g., shRNA, miRNA, dsRNA, etc.), optionally wherein the interfering nucleic acid inhibits expression of α-Syn.

23. 23. The isolated nucleic acid of any one of claims 19 to 22, further comprising 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.

24. 24. The isolated nucleic acid of any one of claims 19 to 23, further comprising an internal ribosome entry site (IRES), optionally wherein the IRES is located between the first gene product and the second gene product.

25. 24. The isolated nucleic acid of any one of claims 19 to 23, further comprising a self-cleaving peptide coding sequence, optionally wherein the self-cleaving peptide is T2A.

26. 25. The isolated nucleic acid of any one of claims 19 to 24, wherein the expression construct comprises two adeno-associated virus (AAV) inverted terminal repeat (ITR) sequences flanking a first gene product and a second gene product, optionally wherein one of the ITR sequences lacks a functional terminal resolution site.

27. 27. The isolated nucleic acid of claim 26, wherein at least one ITR comprises a modified "D" region compared to a wild-type AAV2 ITR (SEQ ID NO:29).

28. 28. The isolated nucleic acid of claim 27, wherein the modified "D" region is a "D" sequence located outside the ITR relative to the expression construct.

29. 29. The isolated nucleic acid of claim 27 or 28, wherein the ITR containing a modified "D" sequence is a 3' ITR.

30. 30. The isolated nucleic acid of any one of claims 27 to 29, further comprising a TRY sequence, optionally wherein the TRY sequence is set forth in SEQ ID NO:

28.

31. 31. The isolated nucleic acid of any one of claims 1 to 30, having a sequence as set forth in any one of SEQ ID NOs: 1 to 12, 14, 16, and 18.

32. A vector comprising the isolated nucleic acid of any one of claims 1 to 31.

33. The vector of claim 32, wherein the vector is a plasmid.

34. 33. The vector of claim 32, wherein the vector is a viral vector, optionally wherein the viral vector is a recombinant AAV (rAAV) vector or a baculovirus vector.

35. A composition comprising an isolated nucleic acid according to any one of claims 1 to 31 or a vector according to any one of claims 32 to 34.

36. A host cell comprising an isolated nucleic acid according to any one of claims 1 to 31 or a vector according to any one of claims 32 to 34.

37. Recombinant adeno-associated virus (rAAV), including: (i) a capsid protein; and (ii) an isolated nucleic acid according to any one of claims 1 to 31, or a vector according to any one of claims 32 to 34.

38. 38. The rAAV of claim 37, wherein the capsid protein is capable of crossing the blood-brain barrier, optionally wherein the capsid protein is an AAV9 capsid protein or an AAVrh.10 capsid protein.

39. The rAAV of claim 37 or claim 38, wherein the rAAV transduces neuronal and non-neuronal cells of the central nervous system (CNS).

40. 40. A method of treating a subject having or suspected of having Parkinson's disease, comprising administering to the subject an isolated nucleic acid of any one of claims 1-31, a vector of any one of claims 32-34, a composition of claim 35, or an rAAV of any one of claims 37-39.

41. 41. The method of claim 40, wherein the administration comprises direct injection into the CNS of the subject, optionally wherein the direct injection is intracerebral injection, intraparenchymal injection, intrathecal injection, intracisternal injection, or any combination thereof.

42. 42. The method of claim 41, wherein direct injection into the CNS of the subject comprises convection-enhanced delivery (CED).

43. 43. The method of any one of claims 40 to 42, wherein the administration comprises a peripheral injection, optionally wherein the peripheral injection is an intravenous injection.

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