Gene Therapy for Lysosomal Disorders

Abstract

The present disclosure relates to compositions and methods for treating diseases associated with abnormal lysosomal function, such as Parkinson's disease or Gaucher disease. The present disclosure provides expression constructs comprising a transgene encoding beta-glucocerebrosidase, an inhibitory RNA targeting alpha-synuclein, or a combination of the foregoing. The present disclosure further provides methods for treating Gaucher disease, Parkinson's disease, or other synucleinopathies by administering such expression constructs to a subject in need thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 990,246, filed March 16, 2020, and U.S. Provisional Patent Application No. 62 / 831,840, filed April 10, 2019, the disclosures of each of which are incorporated herein by reference in their entireties.

[0002] Description of electronically submitted text files The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: Computer-readable format copy of the Sequence Listing (Filename: PRVL_009_02WO_SeqList.txt, Recorded on: April 10, 2020, File size approximately 361,154 bytes). [Background technology]

[0003] Abnormal expression of proteins such as lysosomal acid β-glucocerebrosidase (Gcase) and α-synuclein is involved in many central nervous system disorders. Gaucher disease is a rare congenital abnormality of glycosphingolipid metabolism due to deficiency of Gcase. Patients suffer from non-CNS symptoms and findings including hepatosplenomegaly, bone marrow failure leading to pancytopenia, pulmonary damage and fibrosis, and bone defects. In addition, a significant number of patients suffer from neurological symptoms including saccadic eye movement and gaze defects, seizures, cognitive impairment, developmental delay, and movement disorders including Parkinson's disease.

[0004] Several therapies exist to address peripheral disease and major clinical symptoms in hematopoietic bone marrow and visceral organs, including enzyme replacement therapy, chaperone-like small molecule drugs that bind to defective Gcase and improve its stability, and substrate reduction therapy that blocks 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.

[0005] In addition to Gaucher disease patients (who have mutations in both chromosomal alleles of the GBA1 gene), patients with mutations in only one allele of GBA1 are at greatly increased risk for Parkinson's disease (PD). Elevated levels of α-synuclein also underpin synucleinopathies such as PD. The severity of PD symptoms, including gait difficulties, resting tremors, rigidity, and frequent depression, sleep disorders, and cognitive decline, correlates with the degree of decrease in enzyme activity. Thus, Gaucher disease patients have the most severe course, while patients with a single mild mutation in GBA1 typically have a milder course. Mutation carriers are also at increased risk for other PD-related disorders, including Lewy body dementia, characterized by executive dysfunction, psychosis, and PD-like movement disorders, and multiple system atrophy, which has characteristic movement disorders and cognitive impairment. No therapy exists to alter the inevitable course of these disorders and other synucleinopathies.

[0006] FIELD OF THE DISCLOSURE This disclosure relates to the field of gene therapy and methods of use thereof. Summary of the Invention

[0007] Provided herein are methods for treating a subject having type 2 Gaucher disease or type 3 Gaucher disease, the methods comprising administering to the subject a recombinant adeno-associated virus (rAAV) comprising: (i) an rAAV vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a transgene insert encoding a glucocerebrosidase (Gcase) protein, the transgene insert comprising the nucleotide sequence of SEQ ID NO: 15, and (ii) an AAV9 capsid protein. In some embodiments, the rAAV is administered in an amount of about 5×10 10 vg / g brain ~ approx. 5 × 10 11 In some embodiments, the rAAV is administered to a subject at a dose in the range of about 1.3×10 11 The subject is administered a dose of 1000 mg / g brain.

[0008] Provided herein are methods for treating a subject with Parkinson's disease having a glucocerebrosidase 1 (GBA1) mutation, the methods comprising administering to the subject a recombinant adeno-associated virus (rAAV) comprising: (i) an rAAV vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a transgene insert encoding a Gcase protein, the transgene insert comprising the nucleotide sequence of SEQ ID NO: 15, and (ii) an AAV9 capsid protein. In some embodiments, the rAAV is administered in an amount of about 5×10 13 vg~approx. 5×10 14 In some embodiments, the rAAV is administered to a subject at a dose ranging from about 1×10 14 vg or approximately 2×10 14 vg dose is administered to the subject.

[0009] In some embodiments, rAAV is administered via suboccipital injection into the cisterna magna.

[0010] Provided herein are methods for treating a subject with type 1 Gaucher disease, the methods comprising administering to the subject a recombinant adeno-associated virus (rAAV) comprising: (i) a rAAV vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a transgene insert encoding a Gcase protein, the transgene insert comprising the nucleotide sequence of SEQ ID NO: 15, and (ii) an AAV9 capsid protein. In some embodiments, the rAAV is administered at a concentration of about 5×10 13 vg~approx. 5×10 14 In some embodiments, the rAAV is administered to the subject at a dose in the range of 0.1 to 1.0 mg / kg.

[0011] Provided herein are methods for treating a subject having a synucleinopathy or parkinsonism, the methods comprising administering to the subject a recombinant adeno-associated virus (rAAV) comprising: (i) a rAAV vector comprising a nucleic acid comprising an expression construct comprising a transgene comprising (a) a Gcase protein coding sequence comprising the nucleotide sequence of SEQ ID NO: 15, and (b) an inhibitory nucleic acid coding sequence comprising the nucleotide sequence of SEQ ID NO: 20, and (ii) an AAV9 capsid protein.

[0012] Provided herein are methods for treating a subject having a synucleinopathy or parkinsonism, the methods comprising administering to the subject a recombinant adeno-associated virus (rAAV) vector comprising: (i) a nucleic acid comprising an expression construct comprising a transgene comprising an inhibitory nucleic acid coding sequence comprising the nucleotide sequence of SEQ ID NO:20; and (ii) an AAV9 capsid protein.

[0013] In some embodiments, the synucleinopathy or parkinsonism is multiple system atrophy, Parkinson's disease, Parkinson's disease with GBA1 mutations, Lewy body disease, dementia with Lewy bodies, dementia with Lewy bodies with GBA1 mutations, progressive supranuclear palsy, or corticobasal syndrome.

[0014] In some embodiments, the promoter is a chicken beta actin (CBA) promoter. In some embodiments, the rAAV vector further comprises a cytomegalovirus (CMV) enhancer. In some embodiments, the rAAV vector further comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In some embodiments, the rAAV vector further comprises a bovine growth hormone polyA signal tail. In some embodiments, the nucleic acid comprises two adeno-associated virus inverted terminal repeat (ITR) sequences flanking the expression construct. In some embodiments, each ITR sequence is a wild-type AAV2 ITR sequence. In some embodiments, the rAAV vector further comprises a TRY region between the 5'ITR and the expression construct, the TRY region comprising SEQ ID NO:28.

[0015] Provided herein is a method for treating a subject having type 2 Gaucher disease or type 3 Gaucher disease, the method comprising administering to the subject an rAAV comprising: (i) in the order of 5' to 3', (a) AAV2 ITR, (b) CMV enhancer, (c) CBA promoter, (d) a transgene insert encoding a Gcase protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 15; (e)WPRE, (f) bovine growth hormone polyA signal tail, and (g) an rAAV vector comprising a nucleic acid comprising an AAV2 ITR; and (ii) AAV9 capsid protein. rAAV is approximately 5 × 10 10 vg / g brain ~ approx. 5 × 10 11 The subject is administered a dose in the range of vg / g brain.

[0016] Provided herein is a method for treating a subject having Parkinson's disease with a GBA1 mutation, the method comprising administering to the subject an rAAV comprising: (i) in the order of 5' to 3', (a) AAV2 ITR, (b) CMV enhancer, (c) CBA promoter, (d) a transgene insert encoding a Gcase protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 15; (e)WPRE, (f) bovine growth hormone polyA signal tail, and (g) an rAAV vector comprising a nucleic acid comprising an AAV2 ITR; and (ii) AAV9 capsid protein. rAAV is approximately 5 × 10 13 vg~approx. 5×10 14 The subject is administered a dose in the range of vg.

[0017] In some embodiments, the rAAV is administered via suboccipital injection into the cisterna magna. In some embodiments, the rAAV is administered in a formulation comprising about 20 mM Tris (pH 8.0), about 1 mM MgCl2, about 200 mM NaCl, and about 0.001% w / v poloxamer 188.

[0018] Provided herein is a pharmaceutical composition comprising: (i) (a) a rAAV vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a transgene insert Gcase protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 15; and (b) rAAV containing AAV9 capsid protein; (ii) about 20 mM Tris (pH 8.0); (iii) about 1 mM MgCl2, (iv) about 200 mM NaCl, and (v) about 0.001% w / v of poloxamer 188.

[0019] In this specification, (a) an rAAV vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a transgene insert encoding a Gcase protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 15; (b) AAV9 capsid protein, Provided is an rAAV for use in a method of treating Gaucher disease type 1, Gaucher disease type 2, Gaucher disease type 3, or GBA1-mutated Parkinson's disease in a subject.

[0020] In this specification, (i) (a) a Gcase protein coding sequence comprising the nucleotide sequence of SEQ ID NO: 15, and (b) an rAAV vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a transgene insert comprising an inhibitory nucleic acid coding sequence comprising the nucleotide sequence of SEQ ID NO: 20; (ii) AAV9 capsid protein, Provided is an rAAV for use in a method of treating a synucleinopathy or parkinsonism in a subject.

[0021] In this specification, (i) a rAAV vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a transgene insert comprising an inhibitory nucleic acid coding sequence comprising the nucleotide sequence of SEQ ID NO: 20; (ii) AAV9 capsid protein, Provided is an rAAV for use in a method of treating a synucleinopathy or parkinsonism in a subject. [Brief description of the drawings]

[0022] [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]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 LIMP2 (SCARB2) or a portion thereof. The coding sequences for Gcase and LIMP2 are separated by an internal ribosome entry site (IRES). [Diagram 3] Schematic diagram showing an embodiment of a plasmid comprising an rAAV vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof) and LIMP2 (SCARB2) or a portion thereof. Expression of the coding sequences for Gcase and LIMP2 are each driven by separate promoters. [Figure 4] FIG. 1 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. 1 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), prosaposin (e.g., PSAP or a portion thereof), and α-Syn. [Figure 6] 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). [Figure 7]Schematic diagram showing one embodiment of a plasmid containing a rAAV vector containing an expression construct encoding Gcase (e.g., GBA1 or a portion thereof). In this embodiment, the vector contains a four-part CBA promoter element (CBA): a CMV enhancer (CMVe), a CBA promoter (CBAp), exon 1, and an intron (int) for constitutive expression of a codon-optimized coding sequence of human GBA1. The 3' region also contains a WPRE regulatory element followed by a bGH polyA tail. Three transcriptional regulatory activation sites are included at the 5' end of the promoter region: TATA, RBS, and YY1 containing sequences. The flanking ITR allows for correct packaging of the intervening sequence. Two variants (insertion boxes) of the 5'ITR sequence were evaluated, which 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 in the bottom row). [Figure 8] FIG. 8 is a schematic diagram showing one embodiment of a plasmid encoding the rAAV vector described in FIG. [Figure 9-1]Representative data for CBE mouse model validation are shown. Viability (A) was checked twice daily, body weight (B) was recorded daily and analyzed at P27 (C). All groups were started with n=8. Behavior was assessed by latency to fall on the rotarod at P24 (D) and total distance traveled in the open field (F). Due to early lethality, the number of animals for each group was different: n=8 for PBS and 25 mg / kg CBE, and n=4 for 37.5 mg / kg CBE. Due to complete lethality by P24, the 50 mg / kg CBE treatment group was not evaluated on the rotarod. Statistical results are shown for comparisons to the PBS group using ANOVA followed by Tukey HSD test. Levels of GCase substrate were analyzed in the cerebral cortex of mice in the PBS and 25 mg / kg CBE treatment groups. Aggregated GluSph and GalSph levels (E) are shown as pmol per mg wet weight of tissue. Statistical results are presented using Student's t-test. Means are shown. Error bars are standard error of the mean (SEM). *P<0.05, **P<0.01, ***P<0.001. [Figure 9-2]Representative data for CBE mouse model validation are shown. Viability (A) was checked twice daily, body weight (B) was recorded daily and analyzed at P27 (C). All groups were started with n=8. Behavior was assessed by latency to fall on the rotarod at P24 (D) and total distance traveled in the open field (F). Due to early lethality, the number of animals for each group was different: n=8 for PBS and 25 mg / kg CBE, and n=4 for 37.5 mg / kg CBE. Due to complete lethality by P24, the 50 mg / kg CBE treatment group was not evaluated on the rotarod. Statistical results are shown for comparisons to the PBS group using ANOVA followed by Tukey HSD test. Levels of GCase substrate were analyzed in the cerebral cortex of mice in the PBS and 25 mg / kg CBE treatment groups. Aggregated GluSph and GalSph levels (E) are shown as pmol per mg wet weight of tissue. Statistical results are presented using Student's t-test. Means are shown. Error bars are standard error of the mean (SEM). *P<0.05, **P<0.01, ***P<0.001. [Figure 10] FIG. 1 is a schematic showing one embodiment of a study design for the maximum dose of rAAV encoding GCase in the CBE mouse model. 4 μL of PR001B or dPBS was delivered by ICV injection at P3, and daily 25 mg / kg CBE treatment was initiated at P8. Behavior was assessed in a rotarod assay at P24. Half of the animals were sacrificed at P36, 1 day after the final CBE dose at P35, and the other half were sacrificed at P38, 3 days after the final CBE dose at P35. "vg" refers to the vector genome. [Figure 11]Representative data are shown for in-life evaluation of maximum PR001B 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 either PBS or 25 mg / kg CBE was initiated at P8. At the end of the study, half of the mice were sacrificed 1 day after the final CBE dose on P36 (day 1), and the other half were sacrificed on P38 (day 3) after a 3-day CBE withdrawal. All treatment groups (vehicle+PBS (n=8), rAAV+PBS (n=7), vehicle+CBE (n=8), and rAAV+CBE (n=9)) were weighed daily (A) and weights were analyzed at P33 (B). Behavior was assessed by total distance traveled in the open field at P23 (D) and latency to fall on the rotarod at P24 (C) and was assessed for each animal as the median across three trials. Due to lethality, n=7 for the vehicle+CBE group for behavioral assays and n=8 for all other groups. Means 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 12-1]A-B show representative data for biochemical evaluation of maximum PR001B rAAV dose in the CBE mouse model. Cerebral cortex from all treatment groups was used to measure vector genomes (A) and GCase activity (B). Biodistribution is shown as vector genomes per μg of genomic DNA (gDNA). The dashed line (at 100 vector genomes / μg gDNA) represents the detection threshold for positive vector presence. Enzyme activity was assessed by measuring the rate of glucose production by GCase using Amplex Red (Invitrogen; #A22189) and then converting to effective GCase activity levels using a recombinant GCase reference standard curve. One unit was defined as 1 ng / mL activity of recombinant purified GCase normalized to mg of total protein. n=6-9 / group. Means are shown. Error bars are SEM. *P<0.05, nominal p-value for treatment groups in CBE-treated animals corrected for day of collection and sex as covariates. [Figure 12-2] C-D show representative data for glycolipid analysis of the maximum PR001B rAAV dose in the CBE mouse model. Cerebral cortex from all treatment groups (PBS+dPBS [left bar in each graph] (n=4), CBE+dPBS [middle bar in each graph] (n=6), and CBE+PR001B [right bar in each graph] (n=9)) was used to measure GluSph (C) and GluCer (D) levels in groups before (day 1) or after (day 3) CBE withdrawal. GluSph and GluCer levels are shown as pmol per nmol of phosphate. Means are shown. Error bars are SEM. *p<0.1, **p<0.01, ***p<0.001, nominal p-values ​​of treatment groups in CBE-treated animals from multiple linear regression, corrected for collection day and sex as covariates. [Figure 13]Representative data for behavioral and biochemical correlates in the CBE mouse model following administration of vehicle+PBS, vehicle+CBE, and PR001B rAAV+CBE treatment groups are shown. Across treatment groups, performance on the rotarod 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). [Figure 14] Representative data for biodistribution of PR001B rAAV in the CBE mouse model are shown. Presence of vector genomes was quantified by quantitative PCR using a vector reference standard curve, and genomic DNA concentration was assessed by A260 optical density measurement. The dashed line (at 100 vector genomes / μg gDNA) represents the detection threshold for positive vector presence. Means are shown. Error bars are SEM. n=7-9 per group. [Figure 15]A is a schematic diagram showing one embodiment of a study design for a dose range of rAAV encoding GCase in the CBE mouse model. PR001A was delivered by ICV injection at P3 and daily 25 mg / kg CBE treatment was initiated at P8. Behavior was assessed in open field and rotarod assays at P21-P22 and by tapered beam at P28. Animals were sacrificed at P38-P40, 1 day after the final CBE dose. Cortex was analyzed for GluSph and GluCer substrate levels and GCase activity. There were 10 mice in each treatment group (5 males, 5 females). B-E show representative data for in-life evaluation of PR001 rAAV dose range in the CBE mouse model. Mice received vehicle or one of three different doses of PR001A, low (middle bar), medium (second bar from the right), or high (far right bar), by ICV delivery of 4 μL at P3. Daily IP treatments of 25 mg / kg CBE were initiated at P8. Mice receiving vehicle and CBE (second bar from the left) or vehicle and PBS (far left bar) served as controls. All treatment groups were initiated with n=10 (5 males / 5 females) per group. All mice were sacrificed 1 day after the final CBE dose (P38-P40). All treatment groups were weighed daily (B) and their body weights were analyzed at P37 (C). Motor performance was assessed by latency to fall on the rotarod at P24 (D) and latency to traverse the tapered beam at P30 (E). Due to early lethality, the number of mice participating in the behavioral assays was n=10 for vehicle+PBS (leftmost bar), n=9 for vehicle+CBE (second bar from the left), n=6 for low-dose PR001A+CBE (middle bar), n=10 for medium-dose PR001A+CBE (second bar from the right), and n=7 for high-dose PR001A+CBE (rightmost bar). Means are shown. Error bars are SEM. *p<0.05, **p<0.01 for nominal p-values ​​in the CBE-treated group, corrected for sex as a covariate. [Figure 16A]Representative data for biodistribution in a dose-ranging CBE model study of PR001A are shown. Mice received one of three different doses of PR001A by ICV delivery at P3: low (middle bar), medium (second bar from the right), or high (far right bar). Daily IP treatments of 25 mg / kg CBE were initiated at P8. Mice receiving vehicle and CBE (second bar from the left) or vehicle and PBS (far left bar) served as controls. All mice were sacrificed at P38-P40, 1 day after the final CBE dose. The presence of vector genomes was assessed in each tissue and in all treatment groups and expressed as the number of vector copies per μg of genomic DNA. The presence of vector genomes was quantified by qPCR using a vector reference standard curve. N=10, 9, 6, 10, 7 per group, respectively. Dashed lines represent the detection threshold for positive vector presence. Means are shown. Error bars are SEM. [Figure 16B] Representative data for GCase enzyme activity in a dose-ranging CBE model study of PR001A are shown. Effective enzyme GCase activity is shown for each tissue and all treatment groups. Activity is shown as units per mg of total protein with 1 unit defined as the activity of 1 ng / mL of recombinant purified GCase. Means are shown. Error bars are SEM. Statistical results are shown for comparison to the vehicle+CBE group (second bar from the left). N=10, 9, 6, 10, 7 per group, respectively. *P<0.05, **P<0.01, ***P<0.001 by ANOVA followed by Tukey HSD multiple testing correction. [Figure 16C] Representative data for glycolipid analysis in a dose-ranging CBE model study of PR001A are shown. GluSph levels are shown as pmol per nmol of phosphate. Means are shown. Error bars are SEM. **P<0.01, ***P<0.001 by ANOVA followed by Tukey HSD multiple testing correction. [Figure 16D]Representative data for glycolipid analysis in a dose-ranging CBE model study of PR001A are shown. GluCer levels are shown as pmol per nmol of phosphate. Means are shown. Error bars are SEM. **P<0.01, ***P<0.001 by ANOVA followed by Tukey HSD multiple testing correction. [Figure 16E] Representative data for hematoxylin and eosin staining analysis in a dose-ranging CBE model study of PR001A are shown. Brain tissue was processed for hematoxylin and eosin (H&E) staining, and tissue sections were evaluated for pathological changes. The percentage of animals positive for cortical glial scarring, a sign of neuroinflammation, is shown. CBE treatment resulted in a significant increase in glial scarring compared to vehicle-treated controls. PR001A significantly reduced CBE-induced glial scarring in a dose-dependent manner. Statistical results are shown for comparison against the CBE+vehicle group (left bar). N=10, 9, 6, 10, 7 per group, respectively. *: P<0.05, **: P<0.01, ***: P<0.001 for Fisher's exact test. [Figure 16F] Representative data for cerebral cortex immunohistochemistry analysis in a dose-ranging CBE model study of PR001A are shown. Graph shows the mean immunoreactive area measured within the cerebral cortex (n=5-10 per group). Iba1 (ionized calcium-binding adaptor molecule 1) immunoreactive area was significantly higher in CBE+vehicle treated animals (second bar from the left) than in all other groups of mice studied. Mean values ​​are shown and error bars are SEM. Data were analyzed by one-way ANOVA and Sidak's post-hoc test for multiple comparisons. **: P<0.01, ***: P<0.001. [Figure 17-1]Representative data for tapered beam analysis in maximum dose GBA1 rAAV in genetic mouse model are shown. Locomotor performance of treatment groups (WT+vehicle (n=5), 4L / PS-NA+vehicle (n=6), and 4L / PS-NA+rAAV (n=5)) was assayed by beam walk 4 weeks after rAAV administration. Total slips and activity time are shown as the sum of five trials on different beams. Velocity and slips per speed are shown as the average of five trials on different beams. Means are shown. Error bars are SEM. [Figure 17-2] Representative data for tapered beam analysis in maximum dose GBA1 rAAV in genetic mouse model are shown. Locomotor performance of treatment groups (WT+vehicle (n=5), 4L / PS-NA+vehicle (n=6), and 4L / PS-NA+rAAV (n=5)) was assayed by beam walk 4 weeks after rAAV administration. Total slips and activity time are shown as the sum of five trials on different beams. Velocity and slips per speed are shown as the average of five trials on different beams. Means are shown. Error bars are SEM. [Figure 18-1] Representative data are shown 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 relative to GFP-transfected cells. [Figure 18-2] Representative data are shown 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 relative to GFP-transfected cells. [Figure 19]FIG. 1 is a schematic diagram showing an rAAV vector (top) containing a "D" region located "outside" the ITRs (e.g., proximal to the end of the ITR relative to a transgene insert or expression construct), and a wild-type rAAV vector having an ITR on the "inside" of the vector (e.g., proximal to the transgene insert of the vector). [Figure 20] Data are shown for transduction of HEK293 cells using rAAV with ITRs with wild-type (circles) or alternative (e.g., "outside", square) configurations of the "D" sequence. rAAV with ITRs positioned on the "outside" were able to transduce cells as efficiently as rAAV with wild-type ITRs. [Figure 21] 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). [Figure 22] 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 23] FIG. 1 is a schematic diagram showing one embodiment of a plasmid comprising an rAAV vector comprising an expression construct encoding an interfering RNA for Gcase (e.g., GBA1 or a portion thereof) or a portion thereof, and α-Syn. [Figure 24] FIG. 1 is a schematic diagram showing one embodiment of a plasmid comprising an rAAV vector comprising an expression construct encoding an interfering RNA for Gcase (e.g., GBA1 or a portion thereof) or a portion thereof, and α-Syn. [Diagram 25] FIG. 1 is a schematic diagram showing one embodiment of a plasmid comprising an rAAV vector that includes an expression construct encoding prosaposin (e.g., PSAP or a portion thereof). [Figure 26] 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). [Figure 27]FIG. 1 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), prosaposin (e.g., PSAP or a portion thereof), and α-Syn. [Figure 28] Representative data are shown showing that administration of rAAV vectors encoding Gcase reduces glial scarring in vivo. Tissues were processed for staining with hematoxylin and eosin (H&E) and slides were evaluated for pathological changes. The percentage of animals positive for glial scarring reflecting astrogliosis in each group is shown in light shading, while animals negative for glial scarring are shown in black. CBE treatment resulted in a significant increase in glial scarring compared to vehicle-treated controls. rAAV-GBA1 significantly reduced CBE-induced glial scarring in a dose-dependent manner. Statistics are shown for comparison against vehicle+CBE group (red). N=10, 9, 6, 10, 7 per group, respectively. *: p<0.05, **: p<0.01, ***: p<0.001 for Fisher's exact test. [Figure 29] Representative data are shown for the mean values ​​(n = 6–10 per group) of immunofluorescence signals measured within the cortex of mice administered rAAV-GBA1 or vehicle. Quantification of GCase (A) immunolabeling revealed the strongest immunofluorescence labeling in high-dose rAAV-GBA1-treated animals, followed by medium- and low-dose rAAV-GBA1-treated animals. Iba1 (B) immunoreactive area was significantly higher in CBE / vehicle-treated animals than in all other groups of mice investigated. Data were analyzed with one-way ANOVA and Sidak's post-hoc test for multiple comparisons. Bar graphs represent group means + SEM. [Diagram 30]1 is a bar graph showing representative data for biodistribution of the PR001A transgene in study PRV-2018-016 at day 183. This study is described in Example 12. Transgene levels were analyzed using qPCR in NHPs (non-human primates) 183 days after intracisternomagnathus (ICM) injection of either vehicle, low dose PR001A (6.2×1010 vg / g brain), or high dose PR001A (2.3×1011 vg / g brain). Each bar represents the mean ± SEM of three animals per group, with values ​​below the limit of quantification plotted as zero. The qPCR values ​​for vehicle-treated animals were zero for the region, so the vehicle bar is not shown on this scale of the graph. [Diagram 31] Graph showing representative data for human GCase expression on day 183 in study PRV-2018-016. This study is described in Example 12. GCase expression levels were determined in NHP (non-human primate) cortex, hippocampus, and midbrain samples collected on day 183 by Simple Western™ (Jess) analysis. GCase expression levels from NHPs treated with vehicle (left in each panel), low dose PR001A (6.2×1010vg / g brain weight, center in each panel), or high dose PR001A (2.3×1011vg / g brain weight, right in each panel) are shown. A shows data from individual cortex, hippocampus, and midbrain regions. B shows percent change from vehicle-treated (left), low dose (center), and high dose (right) groups. Data for this plot was mean normalized within tissues and combined for cortex, hippocampus, and midbrain. Each bar represents the percent of the vehicle group median for each dose of the mean normalized data. To calculate significance, a one-way ANOVA was performed to examine the significance of the combined treatment group, which included both low- and high-dose treated animals in the vehicle group. P value = 0.014 (*<0.05). [Diagram 32]1 is a set of plots showing representative data of PR001A transgene biodistribution quantified by qPCR in study PRV-2019-005. This study is described in Example 12. Transgene levels were analyzed using qPCR in NHPs (non-human primates) 30 and 90 days after intracisternal (ICM) injection of either vehicle or PR001A (7.0×1011 vg / g brain). Each plot represents each individual animal (n=3 / group) with the mean±SEM. [Diagram 33] Figure 1 is a line graph showing representative data for GCase activity following in vitro transduction of HEK293T cells with PR001A. HEK293T cells transduced with PR001A at different multiplicities of infection (MOI) were assayed for GCase activity. Activity was measured by hydrolysis of the profluorescent substrate resorufin β-D-glucopyranoside. Fluorescence of the cleaved substrate was determined using a plate reader with excitation at 573 nm and emission at 610 nm. Values ​​are mean ± SEM, n=2, and units correspond to an activity of 1 ng / mL of recombinant purified GCase. [Diagram 34] Bar graphs showing representative data for GCase activity (A) and α-synuclein levels (B) after in vitro transduction of HeLa cells with PR001A. HeLa cells treated with vehicle (left bar) or transduced with 2×105 vg / cell of PR001A (middle bar) or 2×106 vg / cell of PR001A (right bar) were harvested 72 hours after treatment and analyzed for GCase activity levels (A) by fluorometric enzymatic assay or for α-synuclein levels (B) by ELISA. Effective enzymatic GCase activity is shown as units per mg of total protein with 1 unit defined as the activity of 1 ng / mL of recombinant purified GCase. α-synuclein concentrations are shown as ng / mL per mg of total protein. Tests were performed in biological triplicates. Means are shown. Error bars are SEM. One-way ANOVA followed by Dunnett's multiple comparison test was used. [Diagram 35]Bar graphs showing representative data for GCase activity (A) and α-synuclein levels (B) after in vitro transduction of mouse hippocampal neurons with PR001A. Primary cultures of mouse hippocampal neurons were treated with vehicle (left bar) or transduced with 1.3×105 vg / cell of PR001A (middle bar) or 1.3×106 vg / cell of PR001A (right bar) on day in vitro (DIV) 2. At DIV9, cells were harvested and analyzed for GCase activity levels (A) by fluorometric enzyme assay or for α-synuclein levels (B) by ELISA. GCase activity is shown as relative fluorescent units (RFU) per time per mg of total protein. α-synuclein concentrations are shown as ng / mL per mg of total protein. Studies were performed in biological duplicates. Means are shown. Error bars are SEM. One-way ANOVA followed by Dunnett's multiple comparison test was used. [Diagram 36] FIG. 1 is a schematic diagram showing one embodiment of a study design for chronic treatment with rAAV encoding GCase in the CBE mouse model. PR001A was delivered by ICV injection at P3, and daily CBE treatments were initiated at P8. Behavior was assessed in rotarod assays at weeks 3, 6, and 15, and tapered beam assays at weeks 4, 7, and 13. Animals were sacrificed around week 26, 1 day after the final CBE dose. Cerebral cortex was analyzed for GluSph and GluCer substrate levels and GCase activity. There were 10-11 animals per treatment group, each of which included male and female mice. [Figure 37]Representative data for the evaluation of chronic PR001A treatment in the CBE model are shown. Cortex from all treatment groups (PBS+vehicle: left bar, CBE+vehicle: middle bar, CBE+2.0x1010vg PR001A: right bar) was used to measure vector genome (A), GCase activity (B), GluSph levels (C), and GluCer levels (D). The presence of vector genome was assessed in each tissue and in all treatment groups and presented as the number of vector copies per μg of genomic DNA. The presence of vector genome was quantified by qPCR using a vector reference standard curve. Effective enzyme GCase activity is presented as units per mg of total protein with 1 unit defined as the activity of 1 ng / mL of recombinant purified GCase. GluSph and GluCer levels are presented as pmol per nmol of phosphate. Means are shown. Error bars are SEM. N=10, 11, 10 per group, respectively. (*)P<0.1, *P<0.05, **P<0.01, ***P<0.001 by ANOVA followed by Tukey HSD multiple testing correction. [Figure 38] Representative data are shown for the in-life evaluation of additional dose range PR001A in the CBE model. All treatment groups were weighed daily (A) and their body weights were analyzed at P45 (B). Motor performance was assessed by latency to fall on the rotarod at weeks 3 (C) and 5 (D) and latency to traverse a tapered beam at week 4 (E). n=8-11 per group. Means are shown. Error bars are SEM. Statistical results are shown for comparison against the CBE+vehicle group (second bar from the left). ***P<0.001 by ANOVA followed by Tukey HSD test. [Figure 39]Representative data for biodistribution in an additional dose-ranging CBE model study are shown. The presence of vector genomes was assessed in each tissue and in all treatment groups and is expressed as the number of vector copies per μg of gDNA. The presence of vector genomes was quantified by qPCR using a vector reference standard curve. n=9-11 per group. The dashed black line (at 100 vector genomes / μg gDNA) represents the detection threshold for positive vector presence. Means are shown. Error bars are SEM. [Diagram 40] Representative data for GCase enzyme activity in an additional dose range CBE model study are shown. Effective enzymatic GCase activity was measured and shown for cerebral cortex of all treatment groups. Activity is shown as units per mg of total protein with 1 unit defined as the activity of 1 ng / mL of recombinant purified GCase. Means are shown. Error bars are SEM. Statistical results are shown for comparison against the CBE+vehicle group (second bar from the left). n=9-11 per group. (*)P<0.1 by ANOVA followed by Tukey HSD test. [Diagram 41] Representative data for glycolipid analysis in an additional dose-ranging CBE model study are shown. GluSph (A) and GluCer (B) levels are shown as pmol per nmol of phosphate. Means are shown. Error bars are SEM. Statistical results are shown for comparison to the CBE+vehicle group (second bar from the left). n=9-11 per group. **P<0.01, ***P<0.001 by ANOVA followed by Tukey HSD test. [Diagram 42]Representative data are shown for the in-life evaluation of further dose range PR001A in the CBE model study. All treatment groups were weighed daily (A) and their body weights were analyzed at P37 (B). Motor performance was assessed by latency to fall on the rotarod at week 3 (C) and latency to traverse the tapered beam at week 4 (D). n=9-10 per group. Means are shown. Error bars are SEM. Statistical results are shown for comparison against the CBE+vehicle group (second bar from the left). ***P<0.001 by ANOVA followed by Tukey HSD test. [Diagram 43] Representative data for biodistribution and GCase enzymatic activity in the additional dose range PR001A in the CBE model study are shown. Vector genomes were measured in the cerebral cortex of all treatment groups (A) and are shown as vector copy number per μg of genomic DNA (gDNA). Presence of vector genomes was quantified by qPCR using a vector reference standard curve. The black dashed line represents the detection threshold for positive vector presence (at 100 vector genomes / μg gDNA). Effective enzymatic GCase activity was measured in the cerebral cortex (B) and is shown as units per mg of total protein with 1 unit defined as the activity of 1 ng / mL of recombinant purified GCase. Means are shown. Error bars are SEM. N=9-10 per group. ***P<0.001 by ANOVA followed by Tukey HSD test. [Diagram 44] Representative data for glycolipid analysis in the additional dose range PR001A in the CBE model study are shown. GluSph (A) and GluCer (B) levels are shown as pmol per nmol of phosphate. Means are shown. Error bars are SEM. Statistical results are shown for comparison to the CBE+vehicle group (second bar from the left). n=9-10 per group. ***P<0.001 by ANOVA followed by Tukey HSD test. [Diagram 45]FIG. 1 is a schematic diagram showing one embodiment of the study design for treatment with rAAV encoding GCase in the 4L / PS-NA genetic mouse model. PR001A was delivered by ICV injection to 3-4 week old 4L / PS-NA mice. Beamwalk was tested at 8, 12 and 18 weeks of age (5, 9 and 15 weeks after ICV treatment) and rotarod was tested at 12 and 18 weeks of age (9 and 15 weeks after ICV treatment). Mice were sacrificed at 18 weeks. Cerebral cortex was analyzed for GCase enzyme activity and cerebellum for GluSph and GluCer substrate levels. There were 3 male and 3 female mice in each treatment group. [Figure 46] Representative data for biodistribution at the highest dose PR001A in the 4L / PS-NA genetic mouse model are shown. The presence of vector genomes was assessed in each tissue and in all treatment groups and expressed as the number of vector copies per μg of genomic DNA (gDNA). The presence of vector genomes was quantified by qPCR using a vector reference standard curve. Means are shown. Error bars are SEM. n=4-5 per group. The dashed line represents the detection threshold for positive vector presence (at 100 vector genomes / μg gDNA). [Figure 47] Representative data for GCase enzyme activity at maximum dose PR001A in the 4L / PS-NA genetic mouse model are shown. Effective enzymatic GCase activity was measured and shown for each tissue and all treatment groups. Activity is shown as units per mg of total protein with 1 unit defined as the activity of 1 ng / mL of recombinant purified GCase. Means are shown. Error bars are SEM. N=4-5 per group. *: P<0.05, **: P<0.01, ***: P<0.001 by ANOVA followed by Tukey HSD multiple testing correction. [Figure 48]Representative data for glycolipid analysis of PR001A in the 4L / PS-NA genetic mouse model are shown. 4L / PS-NA mice received vehicle (middle bar) or 1.5×1010vg PR001A (right bar), and control mice received vehicle (left bar) by ICV delivery at postnatal day P23. Cerebellum was used to measure GluSph (A) and GluCer (B) levels. Levels are shown as pmol per nmol of phosphate. Means are shown. Error bars are SEM. n=4, 5, 5 per group, respectively. *: P<0.05, **: P<0.01, ***: P<0.001 by ANOVA followed by Tukey HSD multiple testing correction. [Figure 49] Representative data are shown for biochemical assessment of cerebral cortex α-synuclein accumulation in the 4L / PS-NA genetic mouse model. 4L / PS-NA mice received ICV vehicle (middle bar) or 1.5×1010vg PR001A (right bar), and control mice received ICV vehicle (left bar) on postnatal day 23. Triton X-soluble and Triton X-insoluble fractions of brain lysates from cerebral cortex were analyzed for α-synuclein protein levels using a customized immunosorbent assay. Insoluble α-synuclein (A) and the ratio of insoluble to soluble α-synuclein (B) are shown. Means are shown. Error bars are SEM. N=3–5 per group. (*): P<0.20, ANOVA followed by Tukey HSD multiple testing correction. [Figure 50] FIG. 1 is a schematic showing one embodiment of the study design for dose-ranging PR001A rAAV in the 4L / PS-NA genetic mouse model. PR001A was delivered by ICV injection to 4L / PS-NA mice for 3-4 weeks. Beamwalk was assessed at 8, 12, and 18 weeks of age (5, 9, and 15 weeks after ICV treatment) and rotarod was assessed at 12 and 18 weeks of age (9 and 15 weeks after ICV treatment). Mice were sacrificed at 18 weeks. Cerebral cortex was analyzed for GCase enzyme activity and cerebellum for GluSph and GluCer substrate levels. There were 10-11 mice per treatment group. [Figure 51] Representative data for behavioral analysis at 18 weeks in dose serum PR001A in the 4L / PS-NA genetic mouse model. Motor performance was assessed in the beam walk and the average total slips per speed were shown across two trials on different beams. N=10, 6, 5, 7, 4, 8 per group, respectively. Means are shown. Error bars are SEM, *: P<0.05, ***: P<0.001, by ANOVA followed by Tukey HSD multiple testing correction. [Figure 52] Representative data for biodistribution and GCase enzymatic activity in the dose range PR001A in the 4L / PS-NA genetic mouse model are shown. Vector genomes were measured in the cerebral cortex of all treatment groups (A) and are shown as vector copy number per μg of genomic DNA (gDNA). The presence of vector genomes was quantified by qPCR using a vector reference standard curve. The dashed line represents the detection threshold for the presence of positive vector genomes (at 100 vector genomes / μg gDNA). Effective enzymatic GCase activity was measured in the cerebral cortex (B) and is shown as units per mg of total protein with 1 unit defined as the activity of 1 ng / mL of recombinant purified GCase. Means are shown. Error bars are SEM. n=10, 10, 10, 10, 7, 8 per group, respectively. ***P<0.001 by ANOVA followed by Tukey HSD multiple testing correction. [Diagram 53] Representative data for glycolipid analysis in the dose range PR001A in the 4L / PS-NA genetic mouse model are shown. Cerebellum was used to measure GluSph (A) and GluCer (B) levels. Levels are shown as pmol per nmol of phosphate. Means are shown. Error bars are SEM. n=10, 10, 10, 10, 7, 8 per group, respectively. ***: P<0.001 by ANOVA followed by Tukey HSD multiple test correction. (#): P<0.1, #: P<0.05 by multiple linear regression for genotype and dose across all animals. [Figure 54]Representative data for biochemical assessment of α-synuclein protein levels in CBE-treated α-synuclein transgenic mice are shown. Hippocampal brain lysates were analyzed for α-synuclein concentration using the Simple Western™ (Jess) automated capillary Western blot system and MJFR-14-6-4-2 α-synuclein antibody. Multiple bands were observed at 48 kDa to 230 kDa and were grouped as "high molecular weight" (HMW). A single band was present at 18 kDa, consistent with the predicted molecular weight of α-synuclein monomer. The mean fold change relative to the normalized mean of the A53T + vehicle group is shown. Error bars are SEM. N=3-5 per group. *: P<0.05 by ANOVA followed by Tukey HSD multiple testing correction. [Figure 55] Schematic diagram of one embodiment of a plasmid encoding a recombinant adeno-associated virus vector (PR001A) containing an expression construct encoding human Gcase. "bp" refers to "base pairs." "kan" refers to a gene that confers resistance to kanamycin. "ORF1" refers to the open reading frame for Gcase. "ITR" refers to the adeno-associated virus inverted terminal repeat. "TRY" refers to a sequence that contains three transcriptional control activation sites, namely TATA, RBS, and YY1. "CBAp" refers to chicken β-actin promoter. "CMVe" refers to the cytomegalovirus enhancer. "WPRE" refers to woodchuck hepatitis virus posttranscriptional regulatory element. "bGH" refers to bovine growth hormone polyA signal tail. "int" refers to intron. The double-stranded nucleotide sequence of PR001A is provided in SEQ ID NOs: 39 and 40. [Figure 56]Schematic diagram showing one embodiment of a plasmid encoding a recombinant adeno-associated virus vector (PR004X) containing an expression construct encoding an shRNA targeting human Gcase and α-synuclein. "bp" refers to "base pair." "kan" refers to a gene that confers resistance to kanamycin. "aSyn_MshRNA" refers to a region encoding an shRNA that inhibits α-synuclein. "GBA_CDSopt" refers to the open reading frame for Gcase. "ITR" refers to the adeno-associated virus inverted terminal repeat. "TRY" refers to a sequence that contains three transcriptional control activation sites, namely TATA, RBS, and YY1. "CBAp" refers to chicken β-actin promoter. "CMVe" refers to the cytomegalovirus enhancer. "WPRE" refers to woodchuck hepatitis virus posttranscriptional regulatory element. "bGH" refers to the bovine growth hormone polyA signal tail. "int" refers to an intron. The double stranded nucleotide sequence of PR004X (sequence verified) is provided in SEQ ID NOs:41 and 42. [Figure 57] Schematic diagram showing one embodiment of a plasmid encoding a recombinant adeno-associated virus vector (PR004Y) containing an expression construct encoding an shRNA targeting human Gcase and α-synuclein. "bp" refers to "base pair." "kan" refers to a gene that confers resistance to kanamycin. "shSNCA" refers to a region encoding an shRNA that inhibits α-synuclein. "GBA_CDSopt" refers to the open reading frame for Gcase. "ITR" refers to the adeno-associated virus inverted terminal repeat. "TRY" refers to a sequence that contains three transcriptional control activation sites, namely TATA, RBS, and YY1. "CBAp" refers to chicken β-actin promoter. "CMVe" refers to the cytomegalovirus enhancer. "WPRE" refers to woodchuck hepatitis virus posttranscriptional regulatory element. "bGH" refers to the bovine growth hormone polyA signal tail. "int" refers to an intron. The theoretical nucleotide sequence of the two strands of PR004Y is provided in SEQ ID NOs:43 and 44. [Figure 58]Schematic diagram of one embodiment of a plasmid encoding a recombinant adeno-associated virus vector (PR014X) containing an expression construct encoding an shRNA targeting alpha-synuclein. "bp" refers to "base pair." "kan" refers to a gene that confers resistance to kanamycin. "aSyn_MshRNA" refers to a region encoding an shRNA that inhibits alpha-synuclein. "ITR" refers to adeno-associated virus inverted terminal repeat. "TRY" refers to a sequence that contains three transcriptional control activation sites, namely TATA, RBS, and YY1. "CBAp" refers to chicken β-actin promoter. "CMVe" refers to cytomegalovirus enhancer. "WPRE" refers to woodchuck hepatitis virus posttranscriptional regulatory element. "bGH" refers to bovine growth hormone polyA signal tail. "int" refers to intron. The nucleotide sequence (theoretical) of the double strand of PR014X is provided in SEQ ID NOs: 45 and 46. The theoretical nucleotide sequence of the double stranded shRNA coding region is provided in SEQ ID NOs:47 and 48. [Figure 59] FIG. 1 is a schematic diagram showing one embodiment of a study design for dose-ranging PR001 rAAV in the D409V Hom genetic mouse model. PR001 was delivered to D409V Hom mice by intravenous (IV) injection. The parameters listed in the figure were assessed after 5 weeks. [Figure 60] Representative data for liver biochemistry of PR001 intravenous (IV) administration in the D409V Hom genetic mouse model are shown. Five weeks after IV injection, mice were sacrificed. Cytokine levels (A) and glycolipid levels (B, C) were quantified. Statistics were determined using D409V ANOVA followed by Dunnett's test compared to Hom+vehicle group. Means are shown as ± SEM (n=8-10 / group). ****: p<0.0001, ***: p<0.001, **: p<0.01, *: p<0.05, (*): p=0.10. GluCer=glucosylceramide. GluSph=glucosylsphingosine. WT=wild type. [Figure 61]Representative data for brain biochemistry of PR001 intravenous (IV) administration in the D409V Hom genetic mouse model are shown. Five weeks after IV injection, mice were sacrificed. Glycolipid levels were quantified. Statistics were determined using ANOVA followed by Dunnett's test compared to the D409V Hom+vehicle group. Means are shown as ± SEM (n=8-10 / group). ****: p<0.0001, *: p<0.05. GluCer=glucosylceramide. GluSph=glucosylsphingosine. WT=wild type. [Figure 62] Representative data for lung biochemistry of PR001 intravenous (IV) administration in the D409V Hom genetic mouse model are shown. Five weeks after IV injection, mice were sacrificed. Cytokine levels were quantified. Statistics were determined using ANOVA followed by Dunnett's test compared to the D409V Hom+vehicle group. Means are shown as ±SEM (n=8-10 / group). *: P<0.05. WT=wild type. [Figure 63] FIG. 1 is a schematic diagram showing one embodiment of the study design for dose-range PR001 rAAV in the 4L / PS-NA genetic mouse model. PR001 was delivered to 4L / PS-NA mice by intravenous (IV) injection. Parameters described in the diagram were evaluated at the indicated time points. [Figure 64] Representative data for liver biochemistry of PR001 intravenous (IV) administration in the 4L / PS-NA genetic mouse model are shown. Mice were sacrificed 15 weeks after IV injection. Glycolipid levels were quantified. Statistics were determined using ANOVA followed by Dunnett's test compared to the 4L / PS-NA+vehicle group. Means are shown as ±SEM (n=10 / group). ****: p<0.0001, ***: p<0.001, *: p<0.05. GluCer=glucosylceramide. GluSph=glucosylsphingosine. [Figure 65]Representative data for brain biochemistry of PR001 intravenous (IV) administration in the 4L / PS-NA genetic mouse model are shown. Mice were sacrificed 15 weeks after IV injection. Glycolipid levels were quantified. Statistics were determined using ANOVA followed by Dunnett's test compared to the 4L / PS-NA+vehicle group. Means are shown as ±SEM (n=10 / group). ****: p<0.0001, **: p<0.01, *: p<0.05. GluCer=glucosylceramide. GluSph=glucosylsphingosine. [Figure 66] Representative data for α-synuclein protein levels and Gcase activity in HeLa cells after transduction with PR004 or PR014 are shown. HeLa cells were treated with PR004, PR014 or vehicle, and α-synuclein levels (A) and GCase activity (B) in cell lysates were measured 72 hours later. Data are shown as mean ± SEM (n=3 / condition). [Figure 67] Representative data are shown for PR004 efficacy in neuronal cultures derived from induced pluripotent stem cells (iPSCs) from Parkinson's disease patients. Induced pluripotent stem cells derived from Parkinson's disease patients with SNCA triple were differentiated into neurons (A). iPSC-derived neurons were treated with PR004 or vehicle, and GCase activity (B) and α-synuclein levels (C) were measured in cell lysates after 2 weeks. Statistics were determined using unpaired t-test, *=p<0.05, **=p<0.01. Data are presented as mean ± SEM (n=2-3 / group). [Figure 68A] Representative data is shown for a study evaluating shRNA targeting SNCA from the PR004 vector in HEK293 cells by qRT-PCR. HEK293 cells were transfected with PR004 or control, and RNA was extracted 72 hours later. qRT-PCR was performed for various genes and normalized to GAPDH expression. Data are normalized to the control condition and shown as mean ± SEM (n=3 / group). [Figure 68B]Representative data is shown for a study evaluating shRNA targeting SNCA from the PR004 vector in HEK293 cells by qRT-PCR. HEK293 cells were transfected with PR004 or control, and RNA was extracted 72 hours later. qRT-PCR was performed for various genes and normalized to GAPDH expression. Data are normalized to the control condition and shown as mean ± SEM (n=3 / group). [Figure 69] FIG. 1 is a schematic diagram showing one embodiment of a study design investigating gastrointestinal, motor behavior, and biochemical endpoints in the SNCA-A53T PAC mouse model following administration of PR004. ICV = intracerebroventricular. [Figure 70] FIG. 1 is a schematic diagram showing one embodiment of a study design investigating locomotor behavior and biochemical analysis in an AAV2-SNCA-A53T IPa-injected mouse model following administration of PR004. IPa=intraparenchymal. SN=substantia nigra. ICV=intracerebroventricular. [Figure 71] Representative data are shown for a study evaluating the motor phenotype following PR004 administration in the AAV2-SNCA-A53T mouse model. 10-week-old mice were dosed with (1) AAV-null or AAV-SNCA-A53T via IPa injection into the SN, and (2) vehicle or PR004 via ICV injection. Microkinetic gait analysis (MotoRater) was performed 4 weeks (A) and 9 weeks (B) after treatment. Statistics were determined by ANOVA followed by Dunnett's multiple test correction, *=p<0.05, **=p<0.01. Data are presented as mean ± SEM (n=10 / group). IPa=intraparenchymal. SN=substantia nigra. ICV=intracerebroventricular. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The present disclosure is based, in part, on compositions and methods for the expression of certain combinations of gene products (e.g., gene products associated with CNS disease) in a subject. The gene products can be proteins, protein fragments (e.g., portions), interfering nucleic acids that inhibit CNS disease-associated genes, and the like. In some embodiments, the gene products are proteins or protein fragments encoded by CNS disease-associated genes. In some embodiments, the gene products are interfering nucleic acids (e.g., shRNA, siRNA, miRNA, amiRNA, and the like) that inhibit CNS disease-associated genes.

[0024] CNS disease-associated genes refer to genes that encode gene products that are genetically, biochemically, or functionally related to central nervous system (CNS) diseases, such as Parkinson's disease (PD), Gaucher's disease (GD), or synucleinopathies. For example, it has been observed that individuals with mutations in the GBA1 gene (encoding the protein Gcase) are at increased risk of developing PD compared to individuals without mutations in GBA1. In another example, PD is associated with the accumulation of protein aggregates that include alpha-synuclein (alpha-Syn) protein, and thus SNCA (encoding alpha-Syn) is a CNS disease-associated gene. In some embodiments, the expression cassettes described herein encode wild-type or non-mutant forms of CNS disease-associated genes (or their coding sequences). Examples of CNS disease-associated genes are listed in Table 1. [Table 1]

[0025] Deficiencies in enzymes such as lysosomal acid β-glucocerebrosidase (e.g., the gene product of the GBA1 gene, also referred to as GCase), as well as common variants in many genes involved in lysosomal function or transport of macromolecules to lysosomes (e.g., lysosomal membrane protein 1 (LIMP), also referred to as SCARB2), are associated with increased risk of PD and / or increased risk of Gaucher disease (e.g., neuropathic Gaucher disease, such as Gaucher disease type 2 or Gaucher disease type 3). The present disclosure is based, in part, on expression constructs (e.g., vectors) encoding a combination of Gcase (or a portion thereof), prosaposin (or a portion thereof), LIMP2 (or a portion thereof), or Gcase (or a portion thereof) with one or more additional gene products from genes associated with central nervous system (CNS) diseases, such as PD, Gaucher disease, etc. (e.g., LIMP2, prosaposin, and / or α-synuclein (α-Syn)). In some embodiments, a combination of gene products described herein, when expressed in a subject, act together (e.g., synergistically) to reduce one or more signs and symptoms of a CNS disorder.

[0026] Thus, in some aspects, the disclosure provides an isolated nucleic acid comprising 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, e.g., a human cell) Gcase coding sequence. In some embodiments, the nucleic acid sequence encoding Gcase encodes a protein comprising an amino acid sequence set forth in SEQ ID NO: 14 (e.g., set forth in NCBI reference sequence NP_000148.2). 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.

[0027] In some aspects, the disclosure provides an isolated nucleic acid comprising 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, e.g., a human cell) prosaposin coding sequence. In some embodiments, the nucleic acid sequence encoding prosaposin encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 16 (e.g., set forth in NCBI reference sequence NP_002769.1). In some embodiments, the isolated nucleic acid comprises the 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.

[0028] In some aspects, the disclosure provides an isolated nucleic acid comprising 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, e.g., a human cell) SCARB2 coding sequence. In some embodiments, the nucleic acid sequence encoding LIMP2 / SCARB2 encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 18 (e.g., set forth in NCBI reference sequence NP_005497.1). In some embodiments, the isolated nucleic acid comprises the 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.

[0029] In some embodiments, the disclosure provides an isolated nucleic acid comprising an expression construct encoding a first gene product and a second gene product, each gene product independently selected from the gene products set forth in Table 1, or a portion thereof.

[0030] 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.

[0031] In some embodiments, the expression construct encodes (e.g., alone or in addition to another gene product) 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 comprises a sequence set forth in SEQ ID NO: 20. In some embodiments, the interfering nucleic acid targeting alpha-synuclein binds (e.g., hybridizes) to a sequence set forth in any one of SEQ ID NOs: 20-25. In some embodiments, the interfering nucleic acid targeting alpha-synuclein binds (e.g., hybridizes) to a sequence set forth in SEQ ID NO: 20.

[0032] 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. -- or an RNA pol III promoter (eg, U6, etc.).

[0033] 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.

[0034] 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.

[0035] In some embodiments, the expression construct comprises two adeno-associated virus (AAV) inverted terminal repeat (ITR) sequences. In some embodiments, the ITR sequences flank a first gene product and a second gene product (e.g., arranged from the 5' end to the 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 release site (trs). For example, in some embodiments, one of the ITRs is a ΔITR.

[0036] The present disclosure relates in some embodiments to rAAV vectors comprising an ITR with a modified "D" region (e.g., a D sequence modified relative 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, e.g., 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 in which the "D" region is located at the 3' end of the ITR (e.g., outside or terminal to the ITR relative to the transgene insert of the vector). In some embodiments, the modified "D" region comprises a sequence set forth in SEQ ID NO: 26 or 27.

[0037] 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.

[0038] 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, 19, and 32-48. 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.

[0039] In some aspects, the present disclosure provides a vector comprising the isolated nucleic acid described herein.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).

[0040] In some aspects, the present disclosure provides a host cell comprising an isolated nucleic acid according to the present disclosure or a vector according to the present disclosure.

[0041] 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 herein.

[0042] In some embodiments, the capsid protein, e.g., AAV9 capsid protein or AAVrh.10 capsid protein, can cross the blood-brain barrier. In some embodiments, the rAAV transduces neuronal and non-neuronal cells of the central nervous system (CNS).

[0043] In some embodiments, the disclosure provides a method for treating a subject having or suspected of having a central nervous system (CNS) disease, the method comprising administering to the subject a composition according to the disclosure (e.g., a composition comprising an isolated nucleic acid or vector or rAAV). In some embodiments, the CNS disease is a neurodegenerative disease, such as a neurodegenerative disease listed in Table 4. In some embodiments, the CNS disease is a synucleinopathy, such as a synucleinopathy listed in Table 5. In some embodiments, the CNS disease is a tauopathy, such as a tauopathy listed in Table 6. In some embodiments, the CNS disease is a lysosomal storage disease, such as a lysosomal storage disease listed in Table 7. In some embodiments, the lysosomal storage disease is a neuropathic Gaucher disease, such as Gaucher disease type 1, Gaucher disease type 2, or Gaucher disease type 3.

[0044] In some embodiments, the present disclosure provides a method for treating a subject having or suspected of having Parkinson's disease, the method comprising administering to the subject a composition described herein (e.g., a composition comprising an isolated nucleic acid or vector or rAAV).

[0045] In some embodiments, the disclosure provides a method for treating a subject with type 2 Gaucher disease or type 3 Gaucher disease, the method comprising administering to the subject an rAAV comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a sequence encoding a Gcase protein, the sequence encoding the Gcase protein comprising SEQ ID NO: 15, and the rAAV comprising a capsid protein having an AAV9 serotype. In some embodiments, the rAAV is administered to a subject with type 2 Gaucher disease or type 3 Gaucher disease at a dose of about 1.3×10 11 The dose is given at vector genome (vg) / g brain.

[0046] In some embodiments, the disclosure provides a method for treating a subject with Parkinson's disease having a glucocerebrosidase-1 (GBA1) mutation, the method comprising administering to the subject an rAAV comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a sequence encoding a Gcase protein, the sequence encoding the Gcase protein comprising SEQ ID NO: 15, and the rAAV comprising a capsid protein having an AAV9 serotype. In some embodiments, the rAAV is administered to a subject at a concentration of about 1×10 14 Vector genome (vg) or approximately 2 × 10 14 vg of the compound is administered to a subject with Parkinson's disease.

[0047] In some embodiments, rAAV is administered via suboccipital injection into the cisterna magna.

[0048] In some embodiments, the composition comprises a nucleic acid (e.g., an rAAV genome, packaged by an AAV capsid protein) encoding two or more gene products (e.g., CNS disease-related gene products), e.g., two, three, four, five, or more gene products described in this application. In some embodiments, the composition comprises two or more (e.g., two, three, four, five, or more) different nucleic acids (e.g., two or more rAAV genomes packaged separately by AAV capsid proteins), each encoding one or more different gene products. In some embodiments, two or more different compositions are administered to the subject, each composition comprising one or more nucleic acids encoding different gene products. In some embodiments, the different gene products are operably linked to the same promoter type (e.g., the same promoter). In some embodiments, the different gene products are operably linked to different promoters.

[0049] Isolated Nucleic Acids and Vectors The isolated nucleic acid can be DNA or RNA. In some aspects, the disclosure provides an isolated nucleic acid comprising an expression construct encoding Gcase (e.g., the gene product of the GBA1 gene) or a portion thereof. Gcase, also referred to as beta-glucocerebrosidase or GBA, refers to a lysosomal protein that cleaves the β-glucosidic bond of the chemical glucocerebroside, an intermediate in glycolipid metabolism. Deficiency of Gcase, the main lysosomal enzyme required for normal metabolism of glycolipids, results in the accumulation of the Gcase glycolipid substrates glucosylceramide (GluCer) and glucosylsphingosine (GluSph). In humans, Gcase is encoded by the GBA1 gene located on chromosome 1. In some embodiments, GBA1 is 、N It encodes a peptide represented by the CBI 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 a mammalian cell, e.g., a human cell) Gcase coding sequence, such as the sequence set forth in SEQ ID NO: 15.

[0050] In some aspects, the disclosure provides an isolated nucleic acid comprising 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 sequence encoding a codon-optimized prosaposin (e.g., codon-optimized for expression in mammalian cells, e.g., human cells), such as the sequence set forth in SEQ ID NO: 17.

[0051] Aspects of the present disclosure relate to an isolated nucleic acid comprising an expression construct encoding LIMP2 / SCARB2 (e.g., a gene product of the SCARB2 gene). SCARB2 refers to a membrane protein that regulates lysosomal and endosomal trafficking within 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 set forth in SEQ ID NO: 19. In some embodiments, the isolated nucleic acid comprises a codon-optimized SCARB2 coding sequence.

[0052] In some embodiments, the disclosure provides an isolated nucleic acid comprising an expression construct encoding a first gene product and a second gene product, each gene product independently selected from the gene products set forth in Table 1, or a portion thereof.

[0053] In some embodiments, the isolated nucleic acid or vector described according to the present disclosure (e.g., rAAV vector) comprises or consists of a sequence set forth in any one of SEQ ID NOs: 1-48. In some embodiments, the isolated nucleic acid or vector described according to the present disclosure (e.g., rAAV vector) comprises or consists of a sequence that is complementary to (e.g., is the complement of) a sequence set forth in any one of SEQ ID NOs: 1-48. In some embodiments, the isolated nucleic acid or vector described according to the present disclosure (e.g., rAAV vector) comprises or consists of a sequence that is the reverse complement of a sequence set forth in any one of SEQ ID NOs: 1-48. In some embodiments, the isolated nucleic acid or vector described according to the present disclosure (e.g., rAAV vector) comprises or consists of a portion of a sequence set forth in any one of SEQ ID NOs: 1-48. The portion may comprise at least 25%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of a sequence set forth in any one of SEQ ID NOs: 1-48. In some embodiments, the nucleic acid sequences described herein are nucleic acid sense strands (e.g., 5' to 3' strands) or, in the context of a viral sequence, are plus (+) strands. In some embodiments, the nucleic acid sequences described herein are nucleic acid antisense strands (e.g., 3' to 5' strands) or, in the context of a viral sequence, are minus (-) strands.

[0054] 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 comprise 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% (eg, any value between 50% and 99.9%) of a protein encoded by a gene listed in Table 1.

[0055] In some embodiments, the expression construct is monocistronic (e.g., the expression construct encodes a single fusion protein that includes 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).

[0056] A polycistronic expression vector may include one or more (e.g., one, two, three, four, five, or more) promoters. Any suitable promoter may be used, such as a constitutive promoter, an inducible promoter, an endogenous promoter, a tissue-specific promoter (e.g., a CNS-specific promoter), etc. In some embodiments, the promoter is a chicken beta actin promoter (CBA promoter), a CAG promoter (e.g., as described by Alexopoulou et al. (2008) BMC Cell Biol. 9:2; doi:10.1186 / 1471-2121-9-2), a CD68 promoter, or a JeT promoter (e.g., as described by 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 gene product 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 of the foregoing.

[0057] 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, by 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, which are described in Liu et al. (2017) Sci Rep. 7: 2193. In some embodiments, the self-cleaving peptide is a T2A peptide.

[0058] Pathologically, disorders such as PD and Gaucher's disease are associated with the accumulation of protein aggregates that primarily contain alpha-synuclein (alpha-Syn) protein. Thus, in some embodiments, the isolated nucleic acids described herein comprise inhibitory nucleic acids that reduce or prevent expression of alpha-Syn protein. The sequence encoding the inhibitory nucleic acid can be placed in a non-translated region (e.g., an intron, 5'UTR, 3'UTR, etc.) of the expression vector.

[0059] In some embodiments, the inhibitory nucleic acid is placed in an intron of the expression construct, for example, in an intron upstream of a sequence encoding a first gene product. The inhibitory nucleic acid can be a double-stranded RNA (dsRNA), siRNA, shRNA, microRNA (miRNA), artificial miRNA (amiRNA), or RNA aptamer. Generally, the inhibitory nucleic acid binds to (e.g., hybridizes with) about 6 to about 30 (e.g., any integer between 6 and 30, inclusive) consecutive nucleotides of a target RNA (e.g., mRNA). In some embodiments, the inhibitory nucleic acid molecule is an miRNA or amiRNA, for example, 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 SNCA mRNA 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). In some embodiments, the shRNA that targets SNCA is encoded by SEQ ID NO:47. In some embodiments, the shRNA targeting SNCA is encoded by a sequence comprising SEQ ID NO:20.

[0060] Those skilled in the art will recognize that when referring to a nucleic acid sequence that contains or encodes an inhibitory nucleic acid (e.g., dsRNA, siRNA, shRNA, miRNA, amiRNA, etc.), any one or more thymidine (T) or uridine (U) nucleotides in the sequences provided herein may be replaced with any other nucleotide suitable for base pairing with an adenosine nucleotide (e.g., via Watson-Crick base pairing). For example, T may be replaced with U, and U may be replaced with T.

[0061] The isolated nucleic acid described herein may exist by itself or as part of a vector. In general, the vector may be 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 the 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).

[0062] Typically, an rAAV vector (e.g., an rAAV genome) comprises a transgene (e.g., an expression construct comprising one or more of each of the following) flanked by two AAV inverted terminal repeat (ITR) sequences. In some embodiments, the transgene of the rAAV vector comprises an isolated nucleic acid as described herein. 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 including a functional Rep binding site (RBS) and terminal release 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 release site (trs) and is used for the production of a self-complementary AAV vector (scAAV vector). In some embodiments, the truncated ITR is, for example, a ΔITR as described by McCarty et al. (2003) Gene Ther. 10(26):2112-8.

[0063] 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., nucleic acid additions, deletions, substitutions, etc.) relative to a wild-type AAV ITR, e.g., relative 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, the wild-type ITR includes 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 designated 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 insert containing the transgene of the rAAV vector (e.g., located "inside" the ITR relative to the end of the ITR or located 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, as disclosed, for example, by Ling et al. (2015) J Mol Genet Med 9(3).

[0064] This disclosure is based, in part, on the surprising discovery that rAAV vectors containing a "D" region located "outside" the ITRs (e.g., proximal 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 having ITRs with unmodified (e.g., wild-type) ITRs. In some embodiments, rAAV vectors having modified "D" sequences (e.g., "D" sequences at "outside" positions) have reduced toxicity relative to rAAV vectors having wild-type ITR sequences.

[0065] In some embodiments, the modified "D" sequence contains at least one nucleotide substitution relative 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 than 10 nucleotide substitutions relative 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 nucleic acid substitutions relative 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 set forth in Wang et al. (1995) J Mol Biol 250(5):573-80.

[0066] The isolated nucleic acid or rAAV vector described herein may further comprise a "TRY" sequence, e.g., as set forth in SEQ ID NO: 28, or as described in Francois et al., (2005) J. Virol. 79(17):11082-11094. In some embodiments, the TRY sequence is located between the ITR (e.g., the 5'ITR) of the isolated nucleic acid or rAAV vector and the expression construct (e.g., the transgene coding insert).

[0067] In some aspects, the present disclosure relates to a baculovirus vector comprising an isolated nucleic acid or rAAV vector according to the present disclosure. In some embodiments, the baculovirus vector is an Autographa californica nuclear polyhedrosis (AcNPV) vector, e.g., as described by Urabe et al. (2002) Hum Gene Ther 13(16):1935-43 and Smith et al. (2009) Mol Ther 17(11):1888-1896.

[0068] In some aspects, the present disclosure provides a host cell comprising an 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, e.g., a HEK293T cell. In some embodiments, the host cell is a bacterial cell, e.g., an E. coli cell.

[0069] rAAV In some aspects, the present disclosure relates to a recombinant AAV (rAAV) comprising a transgene encoding a nucleic acid described herein (e.g., a rAAV vector described herein). The term "rAAV" generally refers to a viral particle comprising a rAAV vector enclosed by one or more AAV capsid proteins. The rAAV described according to the present 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, etc. In some embodiments, the rAAV described according to the present disclosure comprises a capsid protein that is a variant of a wild-type capsid protein, such as a capsid protein variant that comprises 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. In some embodiments, the AAV capsid protein variant is, for example, the AAV1RX capsid protein described by Albright et al. Mol Ther. 2018 Feb 7; 26(2): 510-523. In some embodiments, the capsid protein variant is, for example, the AAV TM6 capsid protein described by Rosario et al. Mol Ther Methods Clin Dev. 2016; 3: 16026.

[0070] In some embodiments, the rAAV described herein spreads easily through the CNS, especially when introduced directly into the CSF space or into the brain parenchyma. Thus, in some embodiments, the rAAV described herein comprises a capsid protein that can cross the blood-brain barrier (BBB). For example, in some embodiments, the rAAV comprises a capsid protein having an AAV9 or AAVrh.10 serotype. The production of rAAV is described, for example, by Samulski et al. (1989) J Virol. 63(9):3822-8 and Wright (2009) Hum Gene Ther. 20(7):698-706. In some embodiments, the rAAV comprises a capsid protein that specifically or preferentially targets myeloid cells, such as microglia.

[0071] In some embodiments, the disclosure provides an rAAV designated "PR001." This rAAV expresses the codon-optimized coding sequence of human GBA1 (SEQ ID NO: 15). In some embodiments, the disclosure provides an rAAV designated "PR001A." PR001A (AAV9.CBA.GBA1.A) is an rAAV that delivers a functional human GBA1 gene and results in increased expression of functional human Gcase. The PR001A vector insert contains a chicken beta-actin (CBA) promoter element, including four parts, a cytomegalovirus (CMV) enhancer, a CBA promoter, exon 1, and an intron (int), to constitutively express the codon-optimized coding sequence of human GBA1 (SEQ ID NO: 15). The 3' region also contains a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), followed by a bovine growth hormone polyadenylation signal tail. Three well-described transcriptional regulatory activation sites are contained at the 5' end of the promoter region: TATA, RBS, and YY1 (see, e.g., Francois et al., (2005) J. Virol. 79(17):11082-11094). Adjacent inverted terminal repeats (ITRs) allow for correct packaging of intervening sequences. Two variants of the 5'ITR sequence (Figure 7, inset box, lower sequence) are provided that have several nucleotide differences within the 20 nucleotide "D" region of the ITR that are believed to affect the efficiency of packaging and expression. PR001A contains the "D" domain nucleotide sequence shown in Figure 7 (inset box, upper sequence, SEQ ID NO:30). In some embodiments, the disclosure provides a variant vector designated "PR001B" that carries a mutant "D" domain (referred to herein as the "S" domain, with the nucleotide changes indicated by shading in SEQ ID NO:31 of Figure 7). Except for the different 5'ITR sequences, PR001B is identical to PR001A. The backbone contains a gene that confers resistance to kanamycin and a stuffer sequence to prevent reverse packaging. A schematic showing the plasmid encoding the rAAV vector is shown in Figure 55.SEQ ID NO:39 provides the nucleotide sequence (in 5' to 3' order) of the first strand of a plasmid encoding the PR001A vector shown in Figure 55. SEQ ID NO:40 provides the nucleotide sequence (in 5' to 3' order) of the second strand of a plasmid encoding the PR001A vector shown in Figure 55. PR001A contains the AAV9 capsid protein.

[0072] In some embodiments, the disclosure provides an rAAV designated "PR004", which expresses a codon-optimized coding sequence of human GBA1 (SEQ ID NO: 15) and an inhibitory nucleic acid coding sequence that targets and reduces alpha-synuclein and comprises the nucleotide sequence of SEQ ID NO: 20. In some embodiments, the disclosure provides an rAAV designated "PR004X". In some embodiments, the disclosure provides an rAAV designated "PR004Y". Each of PR004X and PR004Y is an rAAV that (i) delivers a functional human GBA1 gene, resulting in increased expression of functional human Gcase, and (ii) encodes an shRNA that reduces alpha-synuclein levels via RNA interference. The PR004 vector insert contains a chicken β-actin (CBA) promoter element, including four parts, a cytomegalovirus (CMV) enhancer, a CBA promoter, exon 1, and an intron (int), for constitutive expression of an inhibitory nucleic acid coding sequence, including the codon-optimized coding sequence of human GBA1 (SEQ ID NO: 15) and the nucleotide sequence of SEQ ID NO: 20. The 3' region also contains a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), followed by a bovine growth hormone polyadenylation signal tail. Three well-described transcriptional regulatory activation sites, TATA, RBS, and YY1, are included at the 5' end of the promoter region (see, e.g., Francois et al., (2005) J. Virol. 79(17):11082-11094). Flanking inverted terminal repeats (ITRs) allow for correct packaging of the intervening sequence. The backbone contains a gene conferring resistance to kanamycin, and a stuffer sequence to prevent reverse packaging. A schematic diagram showing the rAAV PR004X vector is shown in Figure 56. SEQ ID NO:41 provides the nucleotide sequence (5' to 3' order) of the first strand of a plasmid encoding the PR004X vector shown in Figure 56. SEQ ID NO:42 provides the nucleotide sequence (5' to 3' order) of the second strand of a plasmid encoding the PR004X vector shown in Figure 56. A schematic diagram showing the rAAV PR004Y vector is shown in Figure 57.SEQ ID NO:43 provides the nucleotide sequence (5' to 3' order) of the first strand of a plasmid encoding the PR004Y vector shown in Figure 57. SEQ ID NO:44 provides the nucleotide sequence (5' to 3' order) of the second strand of a plasmid encoding the PR004Y vector shown in Figure 57. PR004X and PR004Y each contain the AAV9 capsid protein. The PR004X and PR004Y vectors are designed to reduce accumulation of all forms of alpha-synuclein, including aggregated and extracellular forms.

[0073] In some embodiments, the disclosure provides an rAAV designated "PR014", which targets and reduces alpha-synuclein and expresses an inhibitory nucleic acid coding sequence comprising the nucleotide sequence of SEQ ID NO:20. In some embodiments, the disclosure provides an rAAV designated "PR014X". PR014X is an rAAV encoding an shRNA that reduces alpha-synuclein levels via RNA interference. The PR014X vector insert contains a chicken beta-actin (CBA) promoter element, including four parts, a cytomegalovirus (CMV) enhancer, a CBA promoter, exon 1, and an intron (int), to constitutively express an inhibitory nucleic acid coding sequence comprising the nucleotide sequence of SEQ ID NO:20. The 3' region also contains a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), followed by a bovine growth hormone polyadenylation signal tail. Three well-described transcriptional regulatory activation sites are contained at the 5' end of the promoter region: TATA, RBS, and YY1 (see, e.g., Francois et al., (2005) J. Virol. 79(17):11082-11094). Flanking inverted terminal repeats (ITRs) allow for correct packaging of the intervening sequence. The backbone contains a gene that confers resistance to kanamycin, and a stuffer sequence to prevent reverse packaging. A schematic diagram showing the rAAV vector is shown in FIG. 58. SEQ ID NO: 45 provides the nucleotide sequence (in 5' to 3' order) of the first strand of the plasmid encoding the PR014X vector shown in FIG. 60. SEQ ID NO: 46 provides the nucleotide sequence (in 5' to 3' order) of the second strand of the plasmid encoding the PR014X vector shown in FIG. 60. SEQ ID NO: 47 provides the nucleotide sequence (in 5' to 3' order) of the first strand of the shRNA in the plasmid encoding the PR014X vector shown in FIG. 58. SEQ ID NO: 48 provides the nucleotide sequence (5' to 3' order) of the second strand of the shRNA in a plasmid encoding the PR014X vector shown in Figure 58. PR014X contains the AAV9 capsid protein.The PR014X vector is designed to reduce the accumulation of all forms of α-synuclein, including aggregated and extracellular forms.

[0074] In some embodiments, the rAAV described herein (e.g., comprising a recombinant rAAV genome encapsidated by AAV capsid proteins to form rAAV capsid particles) is produced in a baculovirus vector expression system (BEVS). Production of rAAV using BEVS is described, for example, by Urabe et al. (2002) Hum Gene Ther 13(16):1935-43, Smith et al. (2009) Mol Ther 17(11):1888-1896, U.S. Patent No. 8,945,918, U.S. Patent No. 9,879,282, and International PCT Publication No. WO2017 / 184879. However, rAAV can be produced using any suitable method (e.g., using recombinant rep and cap genes). In some embodiments, the rAAV disclosed herein is produced in HEK293 (human embryonic kidney) cells.

[0075] Pharmaceutical Compositions In some embodiments, the present disclosure provides a pharmaceutical composition comprising an 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 deleterious manner with any of the components of the composition in which it is contained.

[0076] As used herein, the term "pharmaceutical acceptable carrier" refers to a pharma- ceutically acceptable material, composition, or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent, or encapsulating material, that is involved in carrying or transporting a compound useful in the present invention in or to a patient so that it can perform its intended function. Additional components that may be included in pharmaceutical compositions 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.

[0077] The compositions (e.g., pharmaceutical compositions) provided herein may be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, intradermal, rectal, intravaginal, intraperitoneal, topical (by powder, ointment, cream, and / or drop), mucosal, nasal, buccal, sublingual; by 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), topical administration via the blood and / or lymphatic supply, and / or direct administration to the affected area. In general, the most appropriate route of administration will depend on a variety of factors, including the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract) and / or the condition of the subject (e.g., whether the subject can tolerate oral administration). In certain embodiments, the compounds or pharmaceutical compositions described herein are suitable for topical administration to the eye of a subject.

[0078] In some embodiments, the disclosure provides a PR001 (e.g., PR001A) finished drug product comprising the above-described PR001 rAAV present in an aqueous solution. In some embodiments, the final formulation buffer comprises about 20 mM Tris [pH 8.0], about 1 mM MgCl2, about 200 mM NaCl, and about 0.001% [w / v] poloxamer 188. In some embodiments, the finished drug product and the final formulation buffer are suitable for intracisternal (ICM) injection or intravenous administration.

[0079] method Aspects of the present disclosure relate to the delivery of compositions (e.g., isolated nucleic acids, rAAVs, etc.) engineered to express a CNS disease-associated gene product to a cell (e.g., a cell(s) of a subject).

[0080] As further described in the Examples section, aspects of the disclosure relate to compositions that express gene products that inhibit or prevent glial scar formation (e.g., gliosis).Thus, in some aspects, the disclosure provides a method for inhibiting glial scar formation in a subject, the method comprising administering to the subject a composition (e.g., an isolated nucleic acid or rAAV) described herein.

[0081] In some embodiments, the subject has or is suspected of having a central nervous system (CNS) disease. In some embodiments, the subject has Gaucher disease (GD). In some embodiments, the subject has neuropathic GD (nGD) (e.g., GD type 2 or GD type 3). In some embodiments, the subject has GD type 1. In some embodiments, the subject with GD does not have PD or PD symptoms. In some embodiments, the subject has parkinsonism. In some embodiments, the subject has Parkinson's disease (PD). In some embodiments, the subject has atypical Parkinson's disease. In some embodiments, the atypical Parkinson's disease is dementia with Lewy bodies, progressive supranuclear palsy, multiple system atrophy, or corticobasal syndrome.

[0082] The present disclosure is based, in part, on a composition for the expression of one or more CNS disease-related gene products in a subject to treat a CNS-related disease. The one or more CNS disease-related gene products may be encoded by one or more isolated nucleic acids or rAAV vectors. In some embodiments, a subject is administered a single vector (e.g., isolated nucleic acid, rAAV, etc.) encoding one or more (one, two, three, four, five, or more) gene products. In some embodiments, a subject is administered multiple (e.g., two, three, four, five, or more) vectors (e.g., isolated nucleic acid, rAAV, etc.), each vector encoding a different CNS disease-related gene product. In some embodiments, the composition expresses GBA or a portion thereof. In some embodiments, the composition expresses an interfering RNA that targets alpha-synuclein. In some embodiments, the composition expresses GBA or a portion thereof and an interfering RNA that targets alpha-synuclein.

[0083] The CNS-related disease may be a neurodegenerative disease, a synucleinopathy, a tauopathy, or a lysosomal storage disease. Examples of neurodegenerative diseases and their associated genes are listed in Table 4.

[0084] "Synucleinopathy" refers to a disease or disorder characterized by the accumulation of alpha-synuclein (the gene product of SNCA) in a subject (e.g., relative to a healthy subject, e.g., a subject without a synucleinopathy). Examples of synucleinopathies and their associated genes are listed in Table 5.

[0085] "Tauopathy" refers to a disease or disorder characterized by the accumulation of abnormal Tau protein in a subject (e.g., relative to a healthy subject without a tauopathy). Examples of tauopathies and their associated genes are listed in Table 6.

[0086] "Lysosomal storage disease" refers to a disease characterized by the abnormal accumulation of toxic cellular products in the lysosomes of a subject. Examples of lysosomal storage diseases and their associated genes are listed in Table 7.

[0087] As used herein, "treat" or "treating" refers to (a) preventing or delaying the onset of a CNS disease, (b) reducing the severity of a CNS disease, (c) reducing or preventing the onset of symptoms characteristic of a CNS disease, and (d) and / or preventing the worsening of symptoms characteristic of a CNS disease. Symptoms of a CNS disease can include, for example, motor dysfunction (e.g., tremors, stiffness, slowness of movement, difficulty walking, paralysis), cognitive dysfunction (e.g., dementia, depression, anxiety, psychosis), impaired memory, and emotional and behavioral dysfunction.

[0088] The present disclosure is based, in part, on compositions for the expression of one or more PD-related gene products in a subject that act together (eg, synergistically) to treat Parkinson's disease.

[0089] Thus, in some embodiments, the present disclosure provides a method for treating a subject having or suspected of having Parkinson's disease, the method comprising administering to the subject a composition described herein (e.g., a composition comprising an isolated nucleic acid or vector or rAAV).

[0090] The present disclosure is based, in part, on compositions for the expression of one or more CNS disease-related gene products in a subject to treat Gaucher disease (GD). Diagnosis of GD is established by the presence of biallelic pathogenic mutations in GBA1 or by the finding of less than 15% of normal GCase activity in peripheral blood leukocytes. GBA1 mutations causing more severe enzyme deficiencies are associated with earlier onset of disease, faster progression of symptoms, and a higher likelihood of developing neurological symptoms (Svennerholm et al., Clin Genet. 1986; 30(2): 131-5; Cox, Biologics. 2010; 4: 299-313). GD has traditionally been subdivided into three broader phenotypes (neuropathic [GD type 2 and GD type 3, nGD] or non-neuropathic [GD type 1]) differentiated by the presence of neurological symptoms.

[0091] Within nGD, the distinction between type 2 and type 3 GD may represent a phenotypic continuum from acute to chronic presentation of CNS and visceral symptoms. Infants with type 2 GD, known as the acute neuropathic form, classically present with early bulbar signs (such as strabismus and / or dysphagia), opisthotonosis or spasticity, supranuclear gaze palsy, and failure to achieve motor, behavioral, and cognitive milestones. Most children die by age 2 years. (Goker-Alpan et al., J Pediatr. 2003;143(2):273-6; Roshan and Sidransky, Diseases. 2017;5(1):pii:E10). In type 3 GD, the characteristic clinical sign is a slow horizontal supranuclear gaze palsy, accompanied by other neurological symptoms ranging from cognitive impairment to ataxia, seizures, and death in childhood or early adolescence (Goker-Alpan et al., J Pediatr. 2003;143(2):273-6; Tylki-Szymanska et al., J Inherit Metab Dis. 2010;33(4):339-46).

[0092] Thus, in some embodiments, the present disclosure provides a method for treating a subject having or suspected of having neuropathic Gaucher disease, the method comprising administering to the subject a composition described herein (e.g., a composition comprising an isolated nucleic acid or vector or rAAV).

[0093] In some embodiments, the disclosure provides a method for treating a subject with type 2 Gaucher disease or type 3 Gaucher disease, the method comprising administering to the subject an rAAV comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a sequence encoding a Gcase protein, the sequence encoding the Gcase protein comprising SEQ ID NO: 15, and the rAAV comprising a capsid protein having an AAV9 serotype. In some embodiments, the disclosure provides a method for treating a neurological symptom of a subject with type 2 Gaucher disease or type 3 Gaucher disease, the method comprising administering to the subject an rAAV comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a sequence encoding a Gcase protein, the sequence encoding the Gcase protein comprising SEQ ID NO: 15, and the rAAV comprising a capsid protein having an AAV9 serotype. In some embodiments, the neurological symptom of type 2 Gaucher disease or type 3 Gaucher disease is supranuclear gaze palsy, hypotension, seizures, spasticity, motor hypofunction, motor or behavioral developmental delay or impairment, cognitive delay or impairment, ataxia, intention tremor, or rigidity.

[0094] In some embodiments, patients with certain forms of Gaucher disease exhibit symptoms of peripheral neuropathy, for example, as described in Biegstraaten et al. (2010) Brain 133(10):2909-2919. In some embodiments, the disclosure provides a method for treating peripheral neuropathy in a subject with Gaucher disease (e.g., type 1 Gaucher disease), the method comprising administering to the subject an rAAV comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a sequence encoding a Gcase protein, the sequence encoding the Gcase protein comprising SEQ ID NO: 15, and the rAAV comprising a capsid protein having an AAV9 serotype. In some embodiments, the disclosure provides a method for treating type 1 Gaucher disease in a subject, the method comprising administering to the subject an rAAV comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a sequence encoding a Gcase protein, the sequence encoding the Gcase protein comprising SEQ ID NO: 15, and the rAAV comprising a capsid protein having an AAV9 serotype. In some embodiments, the rAAV is administered intravenously to a subject to treat type 1 Gaucher disease.

[0095] In some embodiments, the disclosure provides a method for treating a subject with Parkinson's disease (PD) having a glucocerebrosidase-1 (GBA1) mutation (e.g., a pathogenic GBA1 mutation), the method comprising administering to the subject an rAAV comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a sequence encoding a Gcase protein, the sequence encoding the Gcase protein comprising SEQ ID NO: 15, and the rAAV comprising a capsid protein having an AAV9 serotype. In some embodiments, the disclosure provides a method for treating a symptom of a subject with PD having a GBA1 mutation, the method comprising administering to the subject an rAAV comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a sequence encoding a Gcase protein, the sequence encoding the Gcase protein comprising SEQ ID NO: 15, and the rAAV comprising a capsid protein having an AAV9 serotype. In some embodiments, the motor symptom of PD is resting tremor, bradykinesia, rigidity, or difficulty walking. In some embodiments, the non-motor symptoms of PD are cognitive impairment / dementia, depression, delusion / hallucination, psychosis, sleep disorder, constipation, urinary symptoms, pain, olfactory disorder, dysphagia, or hypotension.In some embodiments, the subject with PD has one GBA1 mutation.In some embodiments, the subject with PD has two GBA1 mutations.

[0096] In some embodiments, an rAAV encoding a Gcase protein for treating Gaucher disease type 1, Gaucher disease type 2, Gaucher disease type 3, or Parkinson's disease with a GBA1 mutation is administered to a subject at a dose of about 1×10 12 Vector genome (vg) ~ approx. 1 × 10 15 vg, or approximately 1 × 10 13 vg~approx. 5×10 14 vg, or approximately 5 × 10 13 vg~approx. 5×10 14 vg, or approximately 3.4 × 10 13 vg~approx. 1×10 14 vg, or approximately 1 × 10 14 vg~approx. 5×10 14 vg, or approximately 1 × 1014 vg ~ approx. 3×10 14 vg, or approximately 1 × 10 14 ~about 2×10 14 vg range. The total dose assumes an adult brain mass of 1.3 kg (Hakim and Mathieson, Neurology, 1979; 29(9 Pt 1): 1209-14). For pediatric subjects, the dose may be adjusted accordingly. In some embodiments, the dose for pediatric subjects may be adjusted using age-dependent brain weight estimates, for example, based on a combined dataset including brain weights from 21 anatomy and neuroimaging publications (Vannucci and Vannucci, Am J Phys Anthropol. 2019; 168(2): 247-61).

[0097] In some embodiments, the rAAV encoding the Gcase protein for treating Parkinson's disease with a GBA1 mutation is about 1×10 14 vg, approx. 2×10 14 vg, approx. 3×10 14 vg, approx. 4×10 14 vg, or approximately 5 × 10 14 In some embodiments, the rAAV for treating Parkinson's disease with a GBA1 mutation is administered to a subject (e.g., a human adult subject) at a dose of about 1×10 vg. 14 vg(approx. 7.7×10 10 vg / g brain), approximately 2 × 10 14 vg(approx. 1.5×10 11 vg / g brain), or approximately 3 × 10 14 vg(approx. 1.9×10 11 vg / g brain) to a subject (e.g., an adult human subject).

[0098] In some embodiments, an rAAV encoding a Gcase protein for treating type 2 or type 3 Gaucher disease is administered in an amount of about 5×10 10 vg / g brain ~ approx. 5 × 10 11In some embodiments, the rAAV for treating Gaucher type 2 or Gaucher type 3 is administered to a subject (e.g., a human pediatric subject) at a dose in the range of about 1.3×10 11 vg / g brain (approximately 5.9 × 10 13 vg~approx. 1.7×10 14 In some embodiments, the rAAV for treating type 2 Gaucher disease or type 3 Gaucher disease is administered to a subject (e.g., a human pediatric subject) at a dose of about 1.9×10 11 vg / g brain (approximately 8.6 × 10 13 vg~approx. 2.5×10 14 In some embodiments, the rAAV for treating type 2 Gaucher disease or type 3 Gaucher disease is administered to a subject (e.g., a human pediatric subject) at a dose of about 7.7×10 10 vg / g brain (approximately 3.4 × 10 13 vg~approx. 1×10 14 vg) or approximately 2.3 × 10 11 vg / g brain (approximately 1 × 10 14 vg ~ approx. 3×10 14 vg) to a subject (e.g., a human pediatric subject).

[0099] In some embodiments, an rAAV encoding a Gcase protein for treating type 1, type 2 or type 3 Gaucher disease or Parkinson's disease with a GBA1 mutation is administered to a subject as a single dose and no rAAV is subsequently administered to the subject.

[0100] In some embodiments, the rAAV encoding the Gcase protein is delivered via a single suboccipital injection into the cisterna magna. In some embodiments, the injection into the cisterna magna is performed under radiation guidance.

[0101] In some embodiments, the disclosure provides a method for treating a subject having a synucleinopathy or parkinsonism, the method comprising administering to the subject an rAAV comprising a nucleic acid comprising an expression construct comprising a transgene comprising (a) a Gcase protein coding sequence comprising the nucleotide sequence of SEQ ID NO: 15, and (b) an inhibitory nucleic acid coding sequence comprising the nucleotide sequence of SEQ ID NO: 20 or SEQ ID NO: 47, wherein the rAAV comprises a capsid protein having an AAV9 serotype.

[0102] In some embodiments, the disclosure provides a method for treating a subject having multiple system atrophy, Parkinson's disease, Parkinson's disease with GBA1 mutations, Lewy body disease, dementia with Lewy bodies, dementia with Lewy bodies with GBA1 mutations, progressive supranuclear palsy, or corticobasal syndrome, the method comprising administering to the subject an rAAV comprising a nucleic acid comprising an expression construct comprising a transgene comprising (a) a Gcase protein coding sequence comprising a nucleotide sequence of SEQ ID NO: 15, and (b) an inhibitory nucleic acid coding sequence comprising a nucleotide sequence of SEQ ID NO: 20 or SEQ ID NO: 47, wherein the rAAV comprises a capsid protein having an AAV9 serotype.

[0103] In some embodiments, the disclosure provides a method for treating a subject having a synucleinopathy or parkinsonism, the method comprising administering to the subject an rAAV comprising a nucleic acid comprising an expression construct comprising a transgene comprising an inhibitory nucleic acid coding sequence comprising a nucleotide sequence of SEQ ID NO:20 or SEQ ID NO:47, wherein the rAAV comprises a capsid protein having an AAV9 serotype.

[0104] In some embodiments, the disclosure provides a method for treating a subject having multiple system atrophy, Parkinson's disease, Parkinson's disease with GBA1 mutations, Lewy body disease, dementia with Lewy bodies, dementia with Lewy bodies with GBA1 mutations, progressive supranuclear palsy, or corticobasal syndrome, the method comprising administering to the subject a rAAV comprising a nucleic acid comprising an expression construct comprising a transgene comprising an inhibitory nucleic acid coding sequence comprising a nucleotide sequence of SEQ ID NO:20 or SEQ ID NO:47, wherein the rAAV comprises a capsid protein having an AAV9 serotype.

[0105] The subject is typically a mammal, preferably a human. In some embodiments, the subject is 1 month to 10 years old (e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or any age in between). In some embodiments, the subject is 2 years to 20 years old. In some embodiments, the subject is 30 years to 100 years old. In some embodiments, the subject is older than 55 years old.

[0106] In some embodiments, the composition is administered directly to the CNS of the subject, for example, by direct injection into the brain and / or spinal cord of the subject. Examples of CNS direct administration modes 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, the composition is administered to the subject by intracisternal (ICM) injection. In some embodiments, direct injection into the CNS of the 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 the subject.

[0107] In some embodiments, direct injection into the CNS of a subject comprises convection-enhanced delivery (CED). Convection-enhanced delivery is a therapeutic strategy that involves surgical exposure of the brain and direct placement of a small-diameter catheter into a target region of the brain, followed by infusion of a therapeutic agent (e.g., a composition described herein or an rAAV) directly into the subject's brain. CED is described, for example, by Debinski et al. (2009) Expert Rev Neurother. 9(10):1519-27.

[0108] In some embodiments, the composition is administered peripherally to the subject, for example, by peripheral injection. Examples of peripheral injection include subcutaneous injection, intravenous injection, intraarterial injection, intraperitoneal injection, or any combination of the above. In some embodiments, the peripheral injection is intraarterial injection, for example injection into the carotid artery of the subject.

[0109] In some embodiments, a composition described herein (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, a 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 performed simultaneously (e.g., simultaneously). In some embodiments, the direct injection is performed prior to (e.g., one minute to one week or more before) the peripheral injection. In some embodiments, the direct injection is performed after (e.g., one minute to one week or more after) the peripheral injection.

[0110] In some embodiments, the subject is administered an immunosuppressant prior to (e.g., one month to one minute prior to) or concomitantly with the compositions described herein. In some embodiments, the immunosuppressant is a corticosteroid (e.g., prednisone, budesonide, etc.), an mTOR inhibitor (e.g., sirolimus, everolimus, etc.), an antibody (e.g., adalimumab, etanercept, natalizumab, etc.), or methotrexate.

[0111] 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 in an amount of about 10 9 Genome copies (GC) / kg ~ approx. 10 14 GC / kg (e.g., about 10 9 GC / kg, approx. 10 10 GC / kg, approx. 10 11 GC / kg, approx. 10 12 GC / kg, approx. 10 12 GC / kg, or approximately 10 14 In some embodiments, subjects are administered a high titer (e.g., 10 GC / kg) by injection into the CSF space or by intraparenchymal injection. 12In some embodiments, the rAAV described herein is administered at a dose of about 1×10 10 Vector genome (vg) ~ approx. 1 × 10 17 In some embodiments, the rAAV described herein is administered to a subject at a dose ranging from about 1×10 to about 1×10 vg by injection into the cisterna magna. 10 vg~approx. 1×10 16 The subject is administered a dose in the range of vg.

[0112] A composition according to the present disclosure (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 times). In some embodiments, the composition is administered to a subject continuously (e.g., chronically), e.g., via an infusion pump. EXAMPLES

[0113] Example 1: rAAV Vectors Cells such as HEK293 cells are used to generate AAV vectors for triple plasmid transfection. The ITR sequences flank the expression construct, which contains a promoter / enhancer element for each transgene of interest, a 3' polyA signal, and a post-translational signal such as a WPRE element. Multiple gene products such as GBA1 and LIMP2, and / or prosaposin, can be expressed simultaneously by fusion of protein sequences, or by using a 2A peptide linker such as T2A or P2A leading to two peptide fragments with added amino acids by preventing the formation of a peptide bond, or by using an IRES element, or by expression in two separate expression cassettes. The presence of a short intron sequence that is efficiently spliced ​​upstream of the expressed gene can improve expression levels. shRNAs and other regulatory RNAs can potentially be included within these sequences. Examples of plasmids containing the described rAAV vectors according to the present disclosure are shown in Figures 1-6, 21-27, and 55-58, as well as in Table 2 below. [Table 2]

[0114] Example 2: Cell-based assay of viral transduction into GBA-deficient cells GBA1-deficient cells are obtained, for example, from GD patients, monocytes or hES cell-derived fibroblasts, or patient-derived induced pluripotent stem cells (iPSCs). These cells accumulate substrates such as glucosylceramide and glucosylsphingosine (GluCer and GluSph). Treatment with Gcase inhibitors such as CBE in wild-type or mutant cell lines can also be used to obtain GBA-deficient cells.

[0115] Using such cell models, lysosomal defects are quantified in terms of accumulation of protein aggregates such as α-synuclein with antibodies for this protein or phospho-αSyn followed by imaging with fluorescence microscopy. Lysosomal abnormalities are also imaged by ICC for 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 for autophagy marker accumulation due to defective fusion with lysosomes, such as for 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 techniques.

[0116] Therapeutic endpoints (e.g., reduction in PD-related pathology) are measured in terms of expression of AAV vector transduction, activity and function are confirmed and quantified. Gcase can also be quantified using protein ELISA measurement or by standard Gcase activity assay.

[0117] Example 2.1: In vitro pharmacology studies with rAAV encoding Gcase Transduction and potency In vitro studies to evaluate the ability of PR001A (AAV9.CBA.GBA1.A) (schematic representation of the plasmid encoding vector provided in Figure 55), which contains the codon-optimized coding sequence of human GBA1 (sequence number 15), to express the GBA1 transgene in HEK293T cells showed a dose-dependent increase in GCase activity following transduction of PR001A in HEK293T cells (Figure 33).

[0118] Efficacy measurement (α-synuclein) In vitro studies were also performed in the human cell line HeLa cells and primary mouse hippocampal neurons. 6 A roughly two-fold increase in GCase activity levels and a decrease in total α-synuclein levels were observed in HeLa cells treated with PR001A at vg / cell compared to vehicle-treated control cells (FIGS. 34A and 34B). Similar effects were not observed with lower doses of PR001A.

[0119] 1.3×10 5 vg / cell or 1.3 × 10 6 Mouse hippocampal neurons transduced with PR001A at vg / cell showed increased levels of GCase activity and a trend towards decreased levels of total α-synuclein (FIGS. 35A and 35B).

[0120] In summary, transduction of PR001A in cell lines and primary neuronal cultures resulted in increased GCase activity. In HeLa cells and mouse hippocampal neurons, PR001A transduction also resulted in decreased α-synuclein levels, supporting a link between GCase activity and α-synuclein levels (Mazzulli et al., Cell. 2011;146(1):37-52).

[0121] Example 3: In vivo assays using mutant mice This example describes the in vivo assay of AAV vectors using mutant mice. The in vivo testing of the above-mentioned AAV vectors in mutant mice is carried out using the assays described, for example, by 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.

[0122] Vehicle control and intrathecal or intracerebroventricular delivery of AAV vectors (e.g., 2×10 11 The dose of 1000 mg / mouse is performed in an injection volume of, for example, 5-10 µL using concentrated AAV stocks. Perform intraparenchymal delivery by convection-enhanced delivery.

[0123] Treatment is initiated either prior to or after the onset of symptoms. Endpoints measured are accumulation of substrate in the CNS and CSF, accumulation of Gcase enzyme and enzyme activity by ELISA, motor and cognitive endpoints, lysosomal dysfunction, and accumulation of α-synuclein monomers, protofibrils, or fibrils.

[0124] Example 4: Chemical models of disease This example describes an in vivo assay of AAV vectors using a chemically induced mouse model of Gaucher disease (e.g., CBE mouse model). In vivo testing of these AAV vectors is performed in a chemically induced mouse model of Gaucher disease, e.g., as described by Vardi et al. (2016) J Pathol. 239(4):496-509.

[0125] Vehicle control and intrathecal or intracerebroventricular delivery of AAV vectors (e.g., 2×10 11Intraparenchymal delivery is performed using a concentrated AAV stock, e.g., in an injection volume of 5-10 µL. Intraparenchymal delivery is performed by convection-enhanced delivery. Peripheral delivery is achieved by tail vein injection.

[0126] Treatment is initiated either prior to or after the onset of symptoms. Endpoints measured are accumulation of substrate in the CNS and CSF, accumulation of Gcase enzyme and enzyme activity by ELISA, motor and cognitive endpoints, lysosomal dysfunction, and accumulation of α-synuclein monomers, protofibrils, or fibrils.

[0127] Example 5: Clinical trials in 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). This example describes a clinical trial to evaluate the safety and efficacy of rAAV as described herein in patients with Gaucher disease, PD and / or LBD.

[0128] Clinical trials of such vectors for the treatment of Gaucher disease, PD and / or LBD will be conducted using a trial design similar to that described in Grabowski et al. (1995) Ann. Intern. Med. 122(1):33-39.

[0129] Example 6: Treatment of peripheral diseases In some embodiments, patients with certain forms of Gaucher disease exhibit symptoms of peripheral neuropathy, for example, as described in Biegstraaten et al. (2010) Brain 133(10):2909-2919.

[0130] This example describes an in vivo assay of an 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 an rAAV described in accordance with the present disclosure. In some embodiments, the subject's signs and symptoms of peripheral neuropathy are monitored following administration of the rAAV, e.g., using the methods described in Biegstraaten et al.

[0131] The levels of the transduced gene products described herein present in the patient (e.g., in the patient's serum, in peripheral tissues (e.g., liver tissue, spleen tissue, etc.)) are assayed, for example, by Western blot analysis, enzyme function assays, or imaging studies.

[0132] Example 7: Treatment of CNS Morphology This example describes an in vivo assay of the rAAV described herein for the treatment of CNS forms of Gaucher disease. Briefly, a Gaucher disease patient identified as having a CNS form of Gaucher disease (e.g., Gaucher disease type 2 or type 3) is administered a rAAV described in accordance with the present disclosure. The level of the transduced gene product described in accordance with the present 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 study.

[0133] 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 a subject with Parkinson's disease, which is characterized by mutations in the GBA1 gene.

[0134] The rAAV vector insert contains a CBA promoter element (CBA) consisting of four parts, the CMV enhancer (CMVe), the CBA promoter (CBAp), exon 1, and an intron (int) to constitutively express the codon-optimized coding sequence (CDS) of human GBA1 (maroon). The 3' region also contains the Woodchuck Hepatitis Virus posttranscriptional regulatory element (WPRE )、 It contains a bovine growth hormone polyA signal (bGH polyA) tail followed by the 5'ITR. The flanking ITRs allow for correct packaging of the intervening sequence. Two variants of the 5'ITR sequence (Figure 7, insert box, lower sequence) were evaluated, and these variants have several nucleotide differences within the 20 nucleotide "D" region of the ITR that are likely to affect the efficiency of packaging and expression. The rAAV vector product contains the "D" domain nucleotide sequence shown in Figure 7 (insert box, upper sequence). strange having a variant "D" domain (referred to herein as an "S" domain, with nucleotide changes indicated by shading) The mutant vector was also studied in preclinical studies. The backbone contains a gene that confers resistance to kanamycin, and a stuffer sequence to prevent reverse packaging. A schematic showing the rAAV vector is shown in Figure 8. The rAAV vector is packaged into rAAV using the AAV9 serotype capsid protein.

[0135] GBA1-rAAV is administered to subjects as a single dose via fluoroscopically guided suboccipital injection into the cisterna magna (intracisternae, ICM). One embodiment of a dosing regimen study is as follows:

[0136] Example 8.1: In vivo pharmacology studies with rAAV encoding Gcase Initial studies were conducted in a chemical mouse model with daily delivery of conduritol-β-epoxide (CBE), an inhibitor of GCase, to evaluate the efficacy and safety of the PR001A rAAV vector (AAV9.CBA.GBA1.A) (schematic representation of the plasmid encoding the vector provided in FIG. 55) containing the codon-optimized coding sequence of human GBA1 (SEQ ID NO: 15) and the PR001B rAAV S-variant construct (described further below). In addition, initial studies were 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 vector.

[0137] These mouse models exhibit characteristic phenotypes of nGD (neuropathic Gaucher disease) and PD-GBA (Parkinson's disease characterized by mutations in the GBA1 gene), including reduced GCase activity, accumulation of glycolipid substrates for GCase, deficits in motor behavior, and neuropathological changes including astrogliosis and microgliosis that reflect inflammation. Intracerebroventricular injection of PR001A suppressed all of these disease-associated phenotypes. Furthermore, the 4L / PS-NA model showed accumulation of α-synuclein, and ICV administration of PR001A in the 4L / PS-NA model reduced α-synuclein accumulation.

[0138] Two slightly different versions of the 5' inverted terminal repeat (ITR) in 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 production, however, mutations within the "D" domain have also been reported to increase transgene expression in some cases. Thus, in addition to the PR001A viral vector that retains an intact "D" domain, a second vector form (PR001B) with a mutant D domain (referred to herein as the "S" domain) was also evaluated. Both PR001A rAAV and variant PR001B rAAV express the same transgene. Although both vectors produced effective virus in vivo as detailed below, the PR001A rAAV containing the wild-type "D" domain was selected for further development.

[0139] Nonclinical in vivo pharmacology (efficacy) studies are summarized in Table 14. A total of 10 studies were completed. The four pivotal studies are discussed in detail in the subsequent sections.

[0140] Example 8.1.1: CBE Mouse Model Study Overview of the CBE model Pharmacological inhibition of GCase activity has been achieved using selective and irreversible covalent competitive inhibitors of GCase in the CBE chemical mouse model, resulting in accumulation of glycolipids (GluCer and GluSph), neuropathological changes including astrogliosis and microgliosis, and motor behavior deficits (Manning-Bog et al., Neurotoxicology. 2009;30(6):1127-32; Farfel-Becker et al., Dis Model Mech. 2011;4(6):746-52; Rocha et al., Antioxid Redox Signal. 2015;23(6):550-64).

[0141] CBE is a pharmacological inhibitor of GCase, and mice treated with CBE exhibit a phenotype consistent with loss of function of GCase. By varying the dosage of CBE, and thus the degree of GCase inhibition in vivo, it is possible to recapitulate the various degrees of enzyme deficiency seen in different GBA1-related disorders, thereby modulating the severity of the resulting phenotype. This makes the CBE mouse model an attractive model for PD-GBA, with significant technical advantages over genetic models of GCase deficiency. The systemic reduction of GCase activity in the CBE model recapitulates the human disease, as in patients with PD-GBA, which is present with reduced GCase activity throughout the CNS and peripheral organs. Because CBE inhibits both endogenous GCase activity and exogenous GCase activity resulting from PR001A treatment, this model is expected to underestimate the effects of PR001A.

[0142] Study PRV-2017-001: CBE dose ranging study To establish a CBE model of GCase deficiency, young mice were dosed with CBE, a specific inhibitor of GCase. Starting at postnatal day 8 (P8), mice were given CBE by daily IP injection. Three different CBE doses (25 mg / kg, 37.5 mg / kg, 50 mg / kg) or daily intraperitoneal (IP) vehicle (PBS) were tested to establish a model exhibiting a behavioral phenotype (Figure 9A-F). High doses of CBE caused lethality in a dose-dependent manner. All mice treated with 50 mg / kg CBE died by P23, and five of eight mice treated with 37.5 mg / kg CBE died by P27 (Figure 9A). There was no lethality in mice treated with 25 mg / kg CBE. Mice treated with CBE showed an inability to gain weight that correlated with CBE dose. At P27, the end of the in-life portion of the study, the weight differences were statistically significant between control animals and those treated with either 25 or 37.5 mg / kg CBE, and none of the mice treated with 50 mg / kg CBE survived to P27 (Figure 9B,C). Whereas CBE-injected mice did not exhibit generalized motor deficits in the open field assay (moving the same distance and at the same speed as PBS-fed mice, Figure 9F), CBE-treated mice exhibited deficits in motor coordination and balance as measured by rotarod assay (Figure 9D).

[0143] Mice that survived to the end of the study were sacrificed the day after the last CBE administration (P27, "day 1") or 3 days after CBE withdrawal (P29, "day 3"). Lipid analysis was performed on the cortex of mice given 25 mg / kg CBE to assess the accumulation of GCase substrates in both day 1 and day 3 cohorts. GluSph and GalSph levels (measured in aggregates in this example) were significantly accumulated in CBE-treated mice compared to PBS-treated controls, consistent with GCase insufficiency (Figure 9E).

[0144] In summary, a daily dose of 25 mg / kg CBE injected IP resulted in deficits in motor behavior and accumulation of GCase substrates (aggregates of GluSph and GalSph levels) consistent with inhibition of GCase activity. Thus, the 25 mg / kg dose was selected for subsequent studies as it recapitulated the core features of the human disease while allowing for longer studies to assess vector persistence.

[0145] Study PRV-2018-002: Efficacy of PR001B in CBE Models Based on the studies mentioned above, a CBE dose of 25 mg / kg was selected because it produced behavioral deficits without affecting survival. In all non-clinical mouse studies, intracerebroventricular (ICV) injection was selected as the route of administration (ROA). Because intracisternal (ICM) injection (the intended clinical ROA) is technically challenging in mice, ICV injection was deemed the most appropriate alternative approach to recapitulate ICM delivery of therapeutics to the cerebrospinal fluid (CSF). To achieve broad GBA1 distribution and transgene expression throughout the brain during CBE treatment, 4 μL of vehicle (dPBS + 0.001% Pluronic F68, "dPBS") or 8.8 × 10 9 vg (based on a brain weight of 150 mg, 5.9 × 10 10 PR001B (100 mg / g brain) was delivered by ICV injection on P3 and daily IP injections of PBS or 25 mg / kg CBE treatment were initiated on P8 (Figure 10). To determine whether CBE treatment completely masked the effects of PR001B, half of the animals were sacrificed on P36, 1 day after the final CBE injection, and the other half underwent CBE withdrawal and were sacrificed on P38, 3 days after the final CBE injection. All measurements were analyzed by combining groups and adjusting for collection day as a covariate.

[0146] CBE-treated mice showed a reduction in weight gain that was attenuated by PR001B treatment (FIG. 11A, FIG. 11B). CBE-treated mice receiving rAAV performed statistically significantly better on the rotarod than mice receiving vehicle (FIG. 11C). Mice in the variant vector-treated group did not differ from vehicle-treated mice in terms of total distance traveled during the test (FIG. 11D).

[0147] Upon completion of the in-life study, half of the mice were sacrificed the day after the last CBE administration (P36, "day 1") or 3 days after CBE withdrawal (P38, "day 3") for biochemical analysis (Figures 12B-D). GCase activity in the cortex was assessed using a fluorometric enzyme assay performed in biological triplicates. GCase activity was increased in mice treated with PR001B rAAV, whereas CBE treatment reduced GCase activity (Figure 12B). In addition, mice receiving both CBE and PR001B rAAV had similar GCase activity levels to the PBS-treated group, indicating that delivery of rAAV can overcome the inhibition of GCase activity induced by CBE treatment. Lipid analysis was performed on the motor cortex of the mice to investigate the levels of the substrates GluCer and GluSph. Both lipids accumulated in the brains of mice given CBE and rAAV treatment, which significantly reduced GluCer accumulation and tended to reduce GluSph accumulation (Figures 12C, 12D).

[0148] 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 substrates and enhanced motor function (Figure 13).

[0149] Preliminary biodistribution was assessed by the presence of vector genomes (>100 vector genomes per μg of genomic DNA was defined as positive) as measured by qPCR, as shown in Figure 14. Mice receiving PR001B rAAV were positive for rAAV vector genomes in the cortex, both with and without CBE (Figure 12A), indicating that ICV delivery results in rAAV delivery to the cortex. Furthermore, vector genomes were detected in the liver, spleen, heart, and lungs, with low levels in the kidneys, and not detected in the gonads (Figure 14). There were no statistically significant differences between the day 1 and day 3 groups for all measurements (data not shown).

[0150] In summary, 8.8 x 10 9 vg(5.9×10 10 PR001B, injected ICV at a dose of 100 mg / g brain (vg / g brain), distributed to brain and peripheral tissues and expressed enzymatically active GCase in the brain. PR001B improved biochemical (i.e., glycolipid levels) deficits and performance on the rotarod. CBE withdrawal was not required to see the effect of PR001B, so in all subsequent studies, mice were sacrificed 1 day after the final CBE dose.

[0151] Study PRV-2018-005: Dose ranging PR001A in the CBE model A schematic showing the design of an exemplary dose ranging study is shown in FIG. 15A.

[0152] A larger study in the CBE model further explored the effective dose of PR001 rAAV in the CBE model. Using a 25 mg / kg CBE dose model, vehicle or PR001 rAAV was delivered via ICV on P3, with daily IP PBS or CBE treatments initiated on P8. Given the similarity between groups with and without CBE withdrawal observed in previous studies, all mice were sacrificed 1 day after the final CBE dose (P38-40). The effect of three different rAAV doses was evaluated, resulting in the following results for five groups with 10 mice per group (5M / 5F): Vehicle ICV+PBS IP Vehicle ICV + 25mg / kg CBE IP 2.0e9vg (1.3e10vg / g brain) of rAAV ICV + 25mg / kg of CBE IP. 6.2e10vg (4.2e10vg / g brain) of rAAV ICV + 25mg / kg of CBE IP. 2.0e10vg (1.3e11vg / g brain) of rAAV ICV + 25mg / kg of CBE IP.

[0153] CBE-treated animals gained weight at a lower rate than control animals, a typical observation in this animal model. At the highest dose, PR001A corrected the failure to gain weight associated with CBE treatment. Furthermore, this dose produced statistically significant improvements in the rotarod and tapered beam tasks compared to the CBE-treated group that did not receive PR001A (Figures 15B-E). Brain GCase activity was positively correlated with performance on the rotarod. Lethality was observed in several groups, including both vehicle- and rAAV-treated groups.

[0154] Upon completion of the in-life study, mice were sacrificed for biodistribution and biochemical analysis (Figure 16A-D). Of the tissues examined, brain, spinal cord, liver, spleen, heart, kidney, and lung were positive for vector genome at the mid and highest doses. Brain, spinal cord, lung, and heart were also positive at the lower dose (Figure 16A). Gonads were not positive at any dose. Effective GCase activity, assessed by measuring enzyme activity in all tissues using a fluorometric assay, was reduced by up to 60% after treatment with CBE (Figure 16B). At the highest dose of PR001A, GCase activity was significantly increased in brain, spinal cord, and heart. It should be noted that the CBE model study likely underestimates the efficacy of PR001A as measured by GCase activity by about 2-fold, since CBE treatment inhibits the activity of PR001A-encoded GCase by about 50% to the same extent as endogenous GCase. CBE-treated mice showed accumulation of GluCer and GluSph in the brain cortex, and high doses of PR001A reduced their accumulation (Figures 16C and 16D).

[0155] Reactive astrogliosis and microglial activation are prominent inflammatory aspects of CNS pathology described in neuropathic GD and PD-GBA patients (Wong et al., 2004; Ginns et al., 2014). In this study, CBE-treated mice showed glial scar formation, a symptom of reactive astrogliosis, in the cerebral cortex, consistent with previous studies showing CNS activation in the context of CBE (Sun et al., 2011). PR001A treatment resulted in a statistically significant dose-dependent reduction in the glial scar phenotype (Figure 16E). Thus, PR001 treatment suppresses neuropathology associated with GCase deficiency in the CNS. A full description of the histopathological findings from this study is discussed in the toxicity section.

[0156] Immunohistochemistry was performed in the cortex for GCase and ionized calcium-binding adaptor molecule 1 (Iba1, a marker of microgliosis) expression (Figure 16F) (Wong et al., 2004; Vitner et al., 2016). GCase expression was significantly increased in all mice treated with PR001A compared to CBE+vehicle-treated animals and correlated with the dose delivered. Iba1 staining was significantly decreased in a dose-dependent manner in mice treated with PR001A compared to mice receiving CBE+vehicle, which had significantly increased Iba1 staining compared to mice receiving PBS.

[0157] In summary, results from the PRV-2018-005 study show that ICV administration of PR001A at three dose levels led to widespread vector genome biodistribution, increased GCase activity, improved motor behavior endpoints, and reduced glycolipid accumulation. Two distinct measures of neuroinflammation (microgliosis and astrogliosis) showed dose-dependent, statistically significant reductions in mice treated with PR001A. The CBE model inherently underestimates the efficacy of PR001A because CBE also inhibits enzyme activity upon PR001A treatment. Collectively, these results demonstrate that in the CBE mouse model, a 2.0 × 10 10 vg(1.3×10 11 Results show that ICV administration of PR001A at 100 mg / g brain (p < 0.05) was effective. A trend toward efficacy was observed with the lower dose of PR001A in a subset of endpoints.

[0158] Study PRV-2018-007: Long-term effects of PR001A in a CBE model This study evaluated the persistence of PR001A vector copy number biodistribution and durability of PR001A-mediated GCase expression in a CBE mouse model. A single dose of vehicle or PR001A was delivered via ICV on P3, and daily IP PBS or CBE treatments were initiated on P8 and continued from P183 to P185 (Figure 36). All mice were sacrificed 1 day after the final CBE dose. No lethality was observed in any group.

[0159] A single ICV dose of PR001A in CBE-treated mice resulted in the presence of vector genome copies 6 months after dosing at levels comparable to those seen approximately 1 month after ICV dosing (see study PRV-2018-005) (Figure 37A). Chronic CBE treatment resulted in a reduction in GCase activity levels, and GCase activity was nearly normalized in mice receiving PR001A, indicating that a single dose of PR001A results in durable expression of GCase (Figure 37B). Mice treated with 6 months CBE showed significant accumulation of glycolipid substrates GluCer and GluSph in the cerebral cortex compared to mice receiving 1 month CBE treatment. A single dose of PR001A at P3 resulted in a significant reduction in GluCer and GluSph levels to near wild-type levels (Figure 37C, Figure 37D).

[0160] Study PRV-2018-008: Additional dose range PR001A in the CBE model This study was intended to evaluate additional doses of PR001A to determine the minimal effective dose and to explore the tolerability of higher doses. However, there was an unexpected dosing deviation, so this study replicated the doses from PRV-2018-005. Following a similar design to PRV-2018-005 (Figure 15A), 4 µL of vehicle or PR001A was delivered via ICV on P3, followed by daily IP PBS or CBE treatments, starting on P8 and continuing until P51–P53.

[0161] Unlike previous studies, CBE treatment did not result in significant changes in body weight. CBE treatment resulted in significantly decreased performance on the rotarod and tapered beam, whereas treatment with PR001A did not significantly alter this performance (Figures 38A-E).

[0162] Of the tissues examined, brain, spinal cord, liver, heart, and lungs were positive for PR001A at all dose levels. Kidneys were also positive at the mid and highest doses, while spleen was positive only at the highest dose. Gonads were also examined but were not positive at any dose level (Figure 39). GCase activity was assessed only in selected organs. In the cerebral cortex, low and mid doses of PR001A restored GCase activity levels to above PBS+vehicle levels, although this did not reach statistical significance. High doses of PR001A tended to significantly increase GCase activity compared to levels in CBE+vehicle treated animals (Figure 40).

[0163] Consistent with other studies in this model, CBE-treated mice showed accumulation of GluSph and GluCer in the brain, which was reduced by administering PR001A (Figure 41, Figure 41B). In a dose-dependent manner, all doses of PR001A significantly reduced GluSph levels, while the medium and high doses significantly reduced GluCer levels.

[0164] This study confirmed findings from PRV-2018-005 and showed that PR001A treatment resulted in widespread biodistribution and a robust increase in GCase activity that markedly reduced the accumulation of glycolipid substrates caused by CBE treatment. This study did not reproduce the behavioral phenotypes observed with PRV-2018-005, however these phenotypes are known to be variable and unreliable in mice.

[0165] PRV-2018-025: Further dose ranging in the CBE model PR001A Given the study deviations in PRV-2018-008, an additional study was conducted in the CBE model to expand on the previous dose-ranging study. ICV administration of PR001A and IP injection of PBS or CBE followed the same protocol as PRV-2018-005. However, this study included a lower PR001A dose to explore the minimal effective dose, and a higher dose to explore tolerability.

[0166] In this study, CBE treatment did not result in an inability to gain weight over time, but a statistically significant decrease in motor performance was observed in CBE + vehicle animals on both the rotarod and tapered beam. 10 Treatment with 1.7×10 vg of PR001A significantly improved motor performance to nearly the same level as PBS+vehicle animals. 10 Improvement was also observed in animals treated with vg PR001A, although this did not reach significance (Figures 42A-D).

[0167] The cerebral cortex of animals treated with PR001A was positive for vector genomes at all doses, with 5.2 × 10 10 Treatment of vg with PR001A resulted in a significant increase in GCase activity. 10 Treatment of vg with PR001A restored activity to near wild-type levels (Figures 43A-B), although this did not reach statistical significance.

[0168] Consistent with other studies in this model, CBE-treated mice showed accumulation of GluSph and GluCer in the brain, which increased by 1.7 × 10 10 vg or 5.2×10 10 vg, the increase in the IL-1 expression level was significantly reduced by administration of PR001A (Figures 44A to 44B).

[0169] This study confirmed and extended findings from previous studies in the CBE model. Although this study did not fully recapitulate the behavioral phenotype observed in PRV-2018-005, it did show a 1.7 × 10 10 A non-significant improvement of 5.2 × 10 was observed in both the rotarod and tapered beam with the PR001A vg. 10 Treatment with vg PR001A significantly improved performance in both tasks. Moreover, both doses of treatment reduced the accumulation of glycolipid substrates, confirming results from other CBE studies.

[0170] CBE Model Test Summary Results from the CBE model study indicate that PR001A can be effectively delivered to the CNS and also to peripheral tissues by ICV injection. Within the CNS, ICV delivery of PR001A resulted in a consistent increase in GCase activity, a decrease in the glycolipid substrates GluCer and GluSph, a decrease in glial scar formation, and an improvement in several motor deficits. These effects were assessed and persisted up to 6 months after treatment.

[0171] Example 8.1.2: 4L / PS-NA Genetic Mouse Model Study Overview of the 4L / PS-NA model 4L / PS-NA mice are an established genetic model of GD and PD-GBA (Sun et al., J Lipid Res. 2005; 46(10):2102-13; Mazzulli et al., Cell. 2011; 146(1):37-52; Xu et al., Mol Genet Metab. 2011; 102(4):436-47). These mice are homozygous for the V394L mutation in GBA1 and further harbor a mutation in PSAP, which encodes saposin C, an activator of GCase; the presence of mutant GCase enzyme and low levels of the GCase activator saposin C together result in severely reduced GCase activity, accumulation of glycolipid substrates, and motor behavior deficits. These mice exhibit deficits in motor strength, coordination, and balance, as evidenced by their performance in beam-walk, rotarod, and wire-hanging assays. Typically, the life span of these mice is less than 22 weeks. The "control" mice in this study are homozygous for the V394L mutation in Gba1, but wild-type for the endogenous prosaposin gene, and therefore have a more moderate reduction in GCase activity. It should be noted that treatment with PR001A does not affect saposin C, so the results obtained in 4L / PS-NA mice likely underestimate the predicted effect in humans. Two studies were performed with PR001A in these mice.

[0172] Study PRV-2018-006:PR001A in the 4L / PS-NA genetic model In study PRV-2018-006, PR001A or vehicle was delivered ICV to 3-4 week-old 4L / PS-NA mice, and animals were sacrificed 15 weeks after administration of PR001A. A dose of 3 µL of undiluted vector (total of 1.5 × 10 10 vg, 3.7×10 10 vg / g brain) were administered (Figure 45).

[0173] Progressive motor impairment was observed in 4L / PS-NA mice, and treatment with PR001A resulted in non-significant improvements in beamwalk after 5 and 9 weeks of treatment. After 15 weeks of treatment, there were no statistically significant differences between groups. Biodistribution of PR001A vector genome in 4L / PS-NA mice was quantified approximately 15 weeks after dosing. All tissues examined were positive for vector genome, including cerebral cortex, spinal cord, liver, kidney, heart, lung, spleen, and gonads. (Figure 46). Analysis of GCase activity in tissue lysates, assessed using a fluorometric assay, revealed a significant increase in effective GCase activity in the cortex and liver (Figure 47).

[0174] There was a statistically significant accumulation of GluSph and GluCer in brain lysates from 4L / PS-NA mice compared to lysates from control animals. Treatment with PR001A resulted in a statistically significant decrease in the accumulation of GluSph and a trend toward a decrease in GluCer (P=0.16) in 4L / PS-NA mice (Figure 48A,B).

[0175] Previous studies have demonstrated increased accumulation of α-synuclein protein in the cortex of the 4L / PS-NA mouse model, consistent with the proposed role of GCase in α-synuclein pathology (Sun et al., J Lipid Res. 2005;46(10):2102-13; Mazzulli et al., Cell. 2011;146(1):37-52; Xu et al., Mol Genet Metab. 2011;102(4):436-47). Soluble and insoluble α-synuclein cerebral cortical levels were biochemically investigated. In vehicle-treated 4L / PS-NA mice, there was a non-significant increase in insoluble α-synuclein and the ratio of insoluble to soluble α-synuclein in the cerebral cortex, and treatment with ICV PR001A reversed these effects (P=0.19, P=0.87, respectively) (Figure 49A, Figure 49B). These data are consistent with the in vitro studies described in Example 2.1 demonstrating a decrease in α-synuclein accumulation.

[0176] Locomotor performance using the beam-walk test was assessed 4 weeks after rAAV delivery. The group of mutant mice that received PR001A rAAV showed less total slips and a trend towards less slips per speed compared to vehicle-treated mutant mice, restoring motor function to near wild-type levels (Figure 17).

[0177] Study PRV-2018-011: Dose-ranging PR001A in the 4L / PS-NA genetic model A second study in 4L / PS-NA mice explored a range of PR001A doses using a similar design to that used in study PRV-2018-006 (Figure 50).

[0178] In the beam-walk test, 4L / PS-NA mice performed significantly worse than control mice. 11 vg, 9.3×10 10 vg, or 2.9 × 10 104L / PS-NA mice treated with vg PR001A showed significant improvement compared to vehicle-treated 4L / PS-NA mice at week 18 (Figure 51). There was no effect of PR001A at the previous time point. There was no difference in rotarod test results between 4L / PS-NA and control mice, and PR001A treatment did not appear to affect this outcome.

[0179] All PR001A-treated groups were positive for vector genomes in the cortex. Effective GCase activity, assessed using a fluorometric assay, was measured in the cortex and was 2.9 × 10 11 vg was found to be significantly increased in PR001A-treated mice (Fig. 52A,B).

[0180] Soluble and insoluble α-synuclein levels in the cerebral cortex and hippocampus were investigated biochemically, and there was no difference in these levels between 4L / PS-NA mice and control animals, with published reports indicating a variable α-synuclein phenotype.

[0181] There was a statistically significant accumulation of GluSph and GluCer in the cerebellum of vehicle-treated 4L / PS-NA mice. Treatment with PR001A led to a trend toward a dose-dependent decrease in the levels of GluSph and a statistically significant dose-dependent decrease in GluCer (Figures 53A, B).

[0182] Summary of the 4L / PS-NA genetic mouse model Although 4L / PS-NA mice showed variability in the phenotypes measured across the two studies, the overall data was consistent with the findings of the CBE model and published data: GCase deficiency was associated with increased levels of glycolipid substrates and motor behavior deficits. Treatment with ICV PR001A strongly attenuated these phenotypes. In study PRV-2018-006, insoluble α-synuclein levels in the cerebral cortex were non-significantly increased in 4L / PS-NA compared to control mice, as reported in published studies (Sun et al., J Lipid Res. 2005; 46(10): 2102-13; Mazzulli et al., Cell. 2011; 146(1): 37-52; Xu et al., Mol Genet Metab. 2011; 102(4): 436-47). Treatment with ICV PR001A reversed such accumulation, consistent with the in vitro analyses disclosed herein. Taken together, these studies support the clinical development of PR001A.

[0183] Example 8.1.3: In vivo α-synuclein model testing Studies PRV-2018-019 and PRV-2019-001:PR001A in α-synuclein transgenic mice treated with CBE To further explore the effect of PR001A on α-synuclein pathology, two studies were conducted: dbl-PAC-Tg (SNCAA53T), which is homozygous for the human PD-associated α-synuclein A53T mutation transgene in a Snca knockout background; - / -This study was performed in mice (Snca encodes the mouse α-synuclein protein). These mice have been reported to exhibit a gastrointestinal phenotype and subtle motor abnormalities between 6 and 12 months of age, but do not exhibit widespread α-synuclein pathology in the brain (Kuo et al., Hum Mol Genet. 2010; 19(9): 1633-50). Previous studies in a human α-synuclein A53T transgenic mouse model have reported that treatment of such mice with CBE results in elevated α-synuclein levels (Rockenstein et al., Hum Mol Genet. 2016; 25(13): 2645-60, Papadopoulos et al., Hum Mol Genet. 2018; 27(10): 1696-1710). Because of these published findings, and to verify the effect of GCase deficiency in this model, these mice were treated with CBE. At 9–10 weeks of age, mice were inoculated with 10 µL of vehicle or 2.9 × 10 11 vg(7.4×10 11 After 2 weeks of ICV treatment, mice were given IP PBS or 100 mg / kg CBE daily for 1 week.

[0184] The presence of vector genomes and GCase activity in the cerebral cortex were evaluated. To confirm the increase in cortical glycolipid substrates with CBE treatment for PRV-2018-019, α-synuclein levels from hippocampal lysates were evaluated using an automated capillary Simple Western™ immunoblot system on a Jess instrument. Multiple α-synuclein immunoreactive bands were observed, consistent with the presence of monomeric and high molecular weight (HMW) species. A statistically significant decrease in the ratio of HMW α-synuclein species to monomeric α-synuclein levels was observed with PR001A treatment of α-synuclein transgenic mice administered CBE (Figure 54A, Figure 54B).

[0185] Summary of non-clinical efficacy studies The above studies show that a single ICV injection of PR001A effectively delivers GBA1 to the CNS and peripheral tissues of mice. In two animal models of PD-GBA and nGD, PR001A increased GCase activity in the CNS. Increased GCase activity reduced accumulation of glycolipid substrates in the brain. These glycolipid substrates are proposed as biomarker outcome measures for intended clinical trials. Importantly, these benefits persist for at least 6 months after a single treatment with PR001A. The CBE model presents reactive astrogliosis and microgliosis, typical histopathological findings in patients with PD-GBA, nGD, and in animal models of these disorders (Hamby and Sofroniew, Neurotherapeutics. 2010;7(4):494-506; Farfel-Becker et al., Dis. Model Mech. 2011;4(6):746-752; Farfel-Becker et al., Hum Mol Genet. 2011;20(7):1375-86; Booth et al., Trends Neurosci. 2017;40(6):358-70; McMahon et al., Mol Genet Metab. 2018;123(2):S93). PR001A can prevent or reverse CBE-induced reactive gliosis and microgliosis. Both models display motor deficits, and treatment with PR001A improves some of these deficits in both models. Along with these two models, additional mouse models were used to investigate α-synuclein pathology. Although α-synuclein phenotypes are variable in mouse models, PR001A can suppress or reverse phenotypes when they are observed, and additional in vitro studies support the efficacy of PR001A in reducing α-synuclein levels. Taken together, these studies support the efficacy of PR001A in models of PD-GBA and nGD.

[0186] Example 8.1.4: Toxicity Single-dose mouse study Safety and toxicity studies performed with PR001A in mouse models are summarized in Table 15. Two of the mouse model efficacy studies (PRV-2018-005 and PRV-2018-006) also included selected safety endpoints such as histopathology to evaluate the safety of PR001A in the disease models.

[0187] Study PRV-2018-005: Dose ranging PR001A in the CBE model Histopathological analysis was performed by hematoxylin and eosin (H&E) staining of the brain, thoracic spinal cord, heart, liver, spleen, lungs, and kidneys, and the results were evaluated by a board-certified veterinary pathologist. In mice treated with CBE, findings in the CNS included glial scarring and neuronal necrosis in the cerebral cortex, brainstem, and thoracic spinal cord. 11 Intracerebroventricular PR001A at a dose of 10 ...

[0188] Example 9: In vitro analysis of rAAV vectors Pilot studies were conducted 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 3. "Opt" refers to a nucleic acid sequence that is codon-optimized for expression in mammalian cells (e.g., human cells). Figure 18 shows representative data showing that transfection of HEK293 cells with each of the constructs resulted in overexpression of the corresponding gene product compared to mock-transfected cells. [Table 3]

[0189] Example 10: Placement of ITR "D" sequences and cell transduction The effect of the positioning of the ITR "D" sequence on the cell transduction of rAAV vectors was investigated. As shown in FIG. 19, HEK293 cells were transduced with Gcase-encoding rAAVs with 1) wild-type ITRs (e.g., the "D" sequence proximal to the transgene insert and distal to the end of the ITR) or 2) ITRs with the "D" sequence located "outside" the vector (e.g., the "D" sequence proximal to the end of the ITR and distal to the transgene insert). Surprisingly, the data show that rAAVs with the "D" sequence located in the "outside" position retain the ability to be packaged and efficiently transduce cells (FIG. 20).

[0190] Example 11: In vivo 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 the treatment group from postnatal day 8 until the end of the study. Animals were euthanized 24 hours after the last IP administration. After euthanasia, target tissues were harvested, drop-fixed in chilled 4% paraformaldehyde, stored at 4°C, and then sent for histopathological processing and evaluation.

[0191] Tissues from 42 animals euthanized on days 38–40 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.

[0192] There were no histopathological findings or evidence of toxicity due to treatment with rAAV. In mice treated with conduritol B-epoxide (CBE), there were findings in the central nervous system (CNS) including glial scarring and neuronal necrosis in the cerebral cortex, as well as neuronal necrosis in the brainstem and thoracic spinal cord. High-dose rAAV treatment resulted in a marked reduction in the expression of these CNS findings, while low- and medium-dose virus had a dose-dependent reduction in the expression of glial scarring in the cerebral cortex and equivocal effects on other CNS findings (Figure 28).

[0193] Immunohistochemistry was performed to assess GCase and Iba1 expression in the cortex (Figures 29A-29B). GCase expression was significantly increased in all animals treated with rAAV-GBA1 compared to CBE / vehicle treated animals. The increase in GCase expression correlated with the dose delivered, with the highest GCase expression observed in high dose treated animals, followed by medium and low dose treated animals. Iba1, a marker of microgliosis, was significantly increased in animals treated with CBE / vehicle. All doses of rAAV-GBA1 reduced Iba1 staining and ameliorated microgliosis in the CBE model. Microgliosis is a well-described endpoint in neuropathic GD and models of this disorder. [Table 4] [Table 5] [Table 6] [Table 7]

[0194] Example 12: Non-human primate testing with rAAV encoding Gcase The safety of PR001A (AAV9.CBA.GBA1.A), which contains the codon-optimized coding sequence of human GBA1 (SEQ ID NO: 15), was evaluated in vivo in non-human primates (NHPs). Additional details of the PR001A components are provided above. The NHP brain is most similar to that of humans, and the anatomical characteristics of the NHP spinal cord and CSF volume and flow rate allow for ICM (intracisternal) injections. Due to the anatomical similarities with humans, NHP studies were expected to provide reliable biodistribution data to support clinical dosing of PR001A.

[0195] The safety and biodistribution of PR001A was evaluated in three toxicity studies in cynomolgus macaques (Table 8), two non-GLP studies (PRV-2018-015 and PRV-2019-005) and a larger 21 CFR58 GLP-compliant study (PRV-2018-016). [Table 8]

[0196] A pilot non-GLP study (PRV-2018-015) was conducted in NHPs to confirm that the PR001A final product was delivered to the NHP brain following ICM administration. A GLP toxicity and biodistribution study in NHPs (PRV-2018-016) evaluated the safety and biodistribution of PR001A.

[0197] Doses tested in NHPs include the maximum feasible dose as determined by the volume administered and test article potency. In addition, lower doses were also evaluated in GLP studies. Time points in GLP studies were post-treatment but selected prior to peak onset (day 7), onset of peak onset (day 30), and prolonged post-peak onset (day 183) to assess safety.

[0198] Study PRV-2018-015: A Non-GLP NHP Study of PR001A A non-GLP pilot tolerability and biodistribution study of PR001A was conducted in male cynomolgus monkeys. The objective of this study was to examine the biodistribution of PR001A to various brain regions and major peripheral organs following ICM delivery. The time points for sacrifice were selected to inform planned GLP NHP toxicity studies, with early in-life observations measured, most notably by a functional observation battery (FOB), as they were expected to allow meaningful measurements of potential early toxicity. Studies of intrathecal AAV delivery have demonstrated that transgene expression peaks 2-3 weeks after injection (Hinderer et al., Mol Ther. 2014;22(12):2018-27; Hinderer et al., Mol Ther Methods Clin Dev. 2014;1:14051; Hinderer et al., Mol Ther. 2015;23(8)1298-307; Hinderer et al., Mol Genet Metab. 2016;119(1-2):124-30). Thus, evaluation on day 18 should provide information on transgene biodistribution and expression at a time point corresponding to the earlier peak expression, as well as detect any immediate toxicity due to the injection procedure or innate inflammatory response to the test article. The study design included a group of rapamycin treatment (0.3 mg / kg orally, days -3 to 18) in combination with PR001A to determine if immunosuppression would be beneficial in mitigating potential toxicity. To increase transgene expression in the brain, one group in the study included intraparenchymal (IPa) administration of PR001A directly to the midbrain targeting the substantia nigra pars compacta bilaterally in combination with ICM delivery. The ICM administration volume was 0.5 mL, the largest volume experienced in administration, and the IPa dose was 10 μL bilaterally, a dose of 1.47 × 10 for ICM alone. 13 vg, 1.53 × 10 for both ICM and IPa treatments 13 With an estimated brain weight of 74 g, this would be a dose of 2.0 × 10 11 vg / g brain and 2.1 × 10 11vg / g brain. A tabular summary of the study design is shown in Table 9. [Table 9] Abbreviations: FOB, functional observational battery; GLP, good laboratory practice; H&E, hematoxylin and eosin; ICM, intracisternae; Immunosupp, immunosuppression; IPa, intraparenchymal; MgCl2, magnesium chloride; NaCl, sodium chloride; NHP, non-human primate; qPCR, quantitative polymerase chain reaction; ROA, route of administration; vg, vector genome(s).

[0199] H&E analysis was performed by two independent board-certified veterinary pathologists, who both concluded that there were no PR001A-related toxicity findings. The spinal cord changes observed were likely the result of trauma at the time of ICM injection and were not considered PR001A-related. All histopathological findings in non-nervous system tissues were considered spontaneous or incidental changes commonly seen in control monkeys. Overall, there were no definitive adverse PR001A effects in the brain or spinal cord.

[0200] The reviewing pathologist noted that nonspecific changes (variable infiltration of mainly mononuclear cells) in the meninges, brain or spinal cord parenchyma, and / or injection sites (in these tissues) were likely related to the test article, but the pathologist did not consider these changes to be adverse. Similar infiltration of the severity described could reasonably be expected to be observed in any monkey having an experimental procedure that disrupts the meninges and / or blood-brain barrier. Furthermore, some infiltration (particularly within the choroid plexus and occasionally in the parenchyma) is typically observed in control monkeys (Butt et al., Toxicol Pathol. 2015;43:513-8). All other histopathological findings observed were deemed to be incidental and / or of similar incidence and severity in vehicle and PR001A-treated animals and therefore unrelated to administration of PR001A. A second independent board-certified veterinary pathologist who reviewed the same tissue samples noted that all findings were non-specific across all groups, including the control group that received vehicle only, and therefore could not be distinguished from incidental findings or trauma occurring during the injection procedure. Additionally, another board-certified veterinary pathologist reviewed the non-GLP tissues and concluded that there were no PR001A-related effects.

[0201] Overall, there were no changes in FOB scores, weight gain, or food consumption over the course of the study, regardless of group and across time points. Microglial morphology in the midbrain did not appear to differ between treatment groups (as determined by Iba1 staining). Midbrain tyrosine hydroxylase-positive neuron expression and morphology did not differ across treatment groups. By day 18, AAV9-nAb titers increased in all PR001A-treated animals, while vehicle-treated control animals showed only minor changes compared to baseline. One of the monkeys in the group receiving oral rapamycin had a lower AAV9 nAb titer on day 18 (1:64) compared to the other PR001A-treated animals (>1:256), and the difference in titers does not appear to have affected biodistribution, although the sample size is too small to be conclusive.

[0202] Biodistribution was assessed in all test samples collected using quantitative polymerase chain reaction (qPCR). Tissues were considered positive with at least 100 vg / μg of DNA (these criteria were also used to evaluate positive tissues in the mouse efficacy study). All tissues tested were positive in all groups treated with PR001A, indicating widespread distribution throughout the CNS and periphery. Additionally, animals receiving ICM administration of PR001A in combination with bilateral IPa administration to the midbrain had increased regional expression. Treatment with rapamycin did not appear to have any effect on safety or biodistribution (selected representative regions shown in Figure 30). Of note, several tissues from control animals (pons, spinal cord, paraventricular, dorsal root ganglion, and lung) were also positive as determined by qPCR. Several issues were noted with the necropsy procedure indicating increased risk of cross-contamination across animals in different treatment groups and between different organs within each animal. Changes have been implemented to the necropsy procedure to minimize contamination for future studies. However, there were no adverse toxicity findings in any of the animals that were qPCR positive. Analysis of transgene expression (GCase activity) showed no significant increase in GCase activity in PR001A-treated animals compared to controls. GCase activity was explored in more detail in the GLP NHP toxicity study PRV-2018-016.

[0203] Taken together, the results of the non-GLP NHP clinical trial PRV-2018-015 demonstrated no safety or toxicity concerns in either in-life or post-mortem evaluations. All animals survived to the scheduled necropsy date, and post-mortem pathological analysis showed no adverse toxicity concerns. The study also demonstrated homogenous biodistribution of PR001A in the brain.

[0204] Study PRV-2018-016: A GLP NHP Study of PR001A Test Design The objective of this GLP study was to evaluate the toxicity and biodistribution of PR001A administered once via ICM injection to cynomolgus monkeys with observation periods of 7, 30, or 183 days after dosing. The study was designed to evaluate two dose levels, the highest dose being the maximum feasible dose achievable in a 1.2 mL volume of undiluted test article (the highest volume administered), and the lower dose being 1 / 2 log unit lower than the higher dose. The doses were 4.6 x 10 12 vg, and 1.7 × 10 13 vg, the estimated brain weight in cynomolgus monkeys was 74 g, which is approximately 6.2 × 10 10 vg / g brain and 2.3 × 10 11 The results are converted to vg / g brain. The study also included a control group in which animals received 1.2 mL of vehicle only (20 mM Tris (pH 8.0), 200 mM NaCl, 1 mM MgCl2, and 0.001% [w / v] poloxamer 188). The study utilized both male and female cynomolgus macaques. The day 7 group contained one male at the highest dose and was designed as a sentinel for early toxicity, while the remaining two time points (days 30 and 183) contained two males and one female at each dose. In addition to samples from multiple brain regions, peripheral tissue samples were collected for qPCR analysis. All samples that were positive by qPCR were analyzed for transgene expression. A tabular summary of the study design is shown in Table 10. [Table 10-1] [Table 10-2] [Table 10-3] Abbreviations: CSF, cerebrospinal fluid; DRG, dorsal root ganglion; F, female; GALT, gut-associated lymphoid tissue; GLP, good laboratory practice; ICM, intracisternae; M, male; MgCl2, magnesium chloride; NaCl, sodium chloride; NHP, non-human primate; qPCR, quantitative polymerase chain reaction; vg, vector genome(s). a20 mM Tris (pH 8.0), 200 mM NaCl, 1 mM MgCl2, and 0.001% (w / v) poloxamer 188.

[0205] Cynomolgus NHPs were assessed by multiple in-life observations and measurements including mortality / morbidity (daily), clinical observations (daily), body weight (baseline and weekly thereafter), visual inspection of food consumption (daily), neurological observations (baseline and between weeks 2 and 26), indirect ophthalmoscopy (baseline and between weeks 2 and 26), and electrocardiogram (ECG) measurements (baseline and between weeks 2 and 26).

[0206] Analysis of nAbs to the AAV9 capsid was performed at baseline and at sacrifice on days 7, 30, or 183. Clinical pathology studies consisting of hematology, coagulation, clinical chemistry, and urinalysis were performed twice at baseline (once for blood tests, once for urinalysis) and once during weeks 1 and 13 of the dosing phase.

[0207] Animals were euthanized and tissues were collected on days 7, 30, or 183. Tissues were collected from all animals, weighed (if applicable), and divided into replicates. One replicate was stored (all animals) in 10% neutral buffered formalin (unless special fixatives were required for optimal fixation) for histopathological evaluation. Additional replicates were collected for qPCR and transgene expression analysis.

[0208] Safety and Toxicity All animals survived to the scheduled necropsy date with no unexpected deaths. There were no concerns or problems in in-life assessments for either group, and gross examination at necropsy revealed no PR001A-related abnormalities in either cohort.

[0209] No PR001A-related organ weight differences or gross or microscopic findings were present in any of the groups at interim sacrifice on days 7 or 30, or at terminal sacrifice on day 183. Hemorrhage, characterized by focal areas of perivascular hemorrhage primarily in the region of the brain stem, was present across all groups, including controls, and was therefore considered to be procedure-related (CSF collection prior to necropsy) and not PR001A-related. All other microscopic findings, including minimal mononuclear infiltrates in the brain or spinal cord, were considered to be spontaneous and / or incidental as they occurred at a low incidence, were randomly distributed among groups (including concurrent controls), and / or their severity was as expected for monkeys of this age, and therefore they were considered not PR001A-related.

[0210] No PR001A-related findings were observed in clinical pathology test results, and increased fibrinogen was seen in animals showing the highest anti-AAV9 titers consistent with an immune response to the vector. Positive titers for anti-AAV9 antibodies were observed by day 7 in all animals receiving PR001A. There were no PR001A-related clinical observations, weight changes, ophthalmologic observations, or physical or neurologic examination findings. No PR001A-related differences in mean PR interval, QRS duration, QT interval, corrected QT (QTc) interval, or heart rate were observed in males alone or in male-female combinations receiving any dose of PR001A. No PR001A-related arrhythmias or abnormal waveforms were observed.

[0211] 0, 6.2×10 10 , or 2.3 × 10 11 PR001A dose levels of vg / g brain were well tolerated when administered via a single injection in the cisterna magna to male and female monkeys. No in-life, clinical pathologic, or anatomo-pathologic observations that could be related to the gene product were observed with PR001A.

[0212] Biodistribution and immune response Biodistribution analysis of vector genome copies was performed using a qPCR-based assay (vector present) and expression of the transgene (GBA1) was measured in samples that were positive for vector genome presence. At days 30 and 183, all tissues examined (including CNS and periphery) were positive for the high dose (2.3 × 10 11 vg / g brain) by qPCR analysis (representative areas were selected from day 183 shown in Figure 30). At day 30, tissue samples taken from testes and ovaries showed positive results after treatment with high dose PR001A (2.3 x 10 11 vg / g brain), all NHPs treated with PR001A at a low dose (6.2 × 10 10 One male NHP treated with 100 mg / g brain (vg / g brain) was positive in testes on day 30. At day 183, one male and one female were positive for PR001A transduction in the gonads after treatment with the high dose, and two males treated with the low dose were positive in testes.

[0213] To confirm that human GCase was produced in treated NHPs, protein levels were assessed with a Simple Western™ immunoblot system on a Jess instrument. Results from cortex, hippocampus, and midbrain samples obtained from PR001A-dosed NHPs showed elevated levels of GCase expression when analyzed in aggregates compared to samples from normal NHPs that received vehicle only, combining both low- and high-dose groups for statistical comparison with the control group (Figures 31A and 31B). These results indicate that effective and widespread transduction of PR001A in NHPs following ICM administration leads to increased GCase levels.

[0214] In conclusion, the biodistribution findings indicate that ICM administration of PR001A in NHPs results in robust and widespread transduction of the human GBA1 transgene in the brain and peripheral organs. In summary of the NHP biodistribution data, ICM administration of PR001A results in widespread biodistribution throughout the brain comparable to levels shown to be effective in mouse models, and this transduction leads to elevated GCase protein levels in the brain.

[0215] Study PRV-2019-005: A Non-GLP NHP Study of PR001A Test Design A non-GLP study was conducted in 12 male cynomolgus macaques to evaluate the toxicity and biodistribution of PR001A administered once via ICM injection with observation periods of 30 and 90 days post-dose. The study was designed to evaluate a single dose level: 5.2 × 10, assuming an average brain weight of 74 g for cynomolgus macaques. 13 vg, or 7.0×10 11 vg / g brain. The dose administered is the maximum feasible dose achievable with undiluted PR001A product in a 1.2 mL volume (the highest volume experienced in administration). The study also included a control group in which animals received 1.2 mL of vehicle only (20 mM Tris (pH 8.0), 200 mM NaCl, and 1 mM MgCl2 + 0.001% [w / v] Pluronic F68). Samples from multiple brain regions and peripheral organs were collected for qPCR analysis to measure biodistribution, and clinical pathology measurements and histopathology were performed to assess safety. A tabular summary of the study design is shown in Table 11. [Table 11-1] [Table 11-2] Abbreviations: CSF, cerebrospinal fluid; FOB, functional observational battery; GLP, good laboratory practice; H&E, hematoxylin and eosin; NHP, non-human primate; qPCR, quantitative polymerase chain reaction; vg, vector genome(s).

[0216] As part of this study, tissues were fixed in 10% formalin, embedded in paraffin, and processed to generate H&E stained slides. Digital slides were prepared and examined by an independent board-certified veterinary pathologist. At both 30 and 90 days after treatment, none of the findings in the PR001A-treated animals were attributable to treatment with PR001A, as they were either consistent with findings commonly observed in cynomolgus macaques (Chamanza et al., Toxicol Pathol. 2010;38(4):642-57) and / or observed in both vehicle control and PR001A-treated animals and were therefore considered incidental.

[0217] PR001A administered into the cisterna magna did not affect weight gain or food consumption, as there were no statistical differences between treatment and control groups during the course of the study. Furthermore, there was no change in FOB scores regardless of group and across time points, indicating that there were no issues or concerns during the in-life phase of the study. Plasma levels of nAbs against AAV9 were measured using an in vitro assay. Samples were prepared from animals in the study at baseline (before ICM administration) and at sacrifice (either day 30 or day 90). Treatment with PR001A resulted in an increase in AAV9 nAb titers at both days 30 and 90 between baseline and the time of necropsy, while titers in vehicle-treated animals remained stable or declined overall.

[0218] Biodistribution and expression of PR001A Biodistribution of the PR001A transgene was assessed using qPCR in all test samples collected, with tissues considered positive with at least 50 vg / μg DNA, the lower limit of quantification for the assay. All tissues tested were positive in all groups treated with PR001A, indicating widespread distribution throughout the CNS and periphery. Data from selected representative regions from both the 30-day and 90-day cohorts are shown in Figure 32.

[0219] Taken together, the results of the non-GLP NHP clinical trial PRV-2019-005 demonstrated no safety or toxicity concerns in either in-life or post-mortem evaluations. All animals survived to the scheduled necropsy date, and post-mortem pathological analysis revealed no adverse toxicity concerns.

[0220] Safety and toxicity studies conducted with PR001A in NHPs are summarized in Table 16.

[0221] Example 13: Phase 1 / 2 study in human subjects Parkinson's disease with GBA1 mutations Human subjects will be enrolled in a randomized, double-blind, sham-controlled trial of PR001A rAAV. Subject inclusion criteria include mono- or biallelic GBA1 mutations, moderate to severe Parkinson's disease, and stable use of background Parkinson's disease medications prior to dosing with the study drug. Subjects will be divided into two groups: (1) PR001 low dose (1 × 10 14 vg) vs placebo (N=8, 6:2), and (2) high dose PR001 (2 × 10 14 vg) vs placebo (N=8, 6:2). Each subject will receive the investigational drug as a single ICM (intracisternal) injection. The study will include a 3-month biomarker readout, a 12-month clinical readout, and a 5-year safety and clinical follow-up. The study will analyze: (1) safety and tolerability, (2) primary biomarkers including: Gcase, GluCer, and GluSph (CSF and blood), (3) additional biomarkers including: α-synuclein, NfL (neurofilament light chain), DAT (dopamine transporter), SPECT (single-photon emission computed tomography), and MRI (magnetic resonance imaging), and (4) efficacy: MDS-UPDRS (Movement Disorders Society Unified Parkinson's disease Rating Scale), cognition, and ADL (activities of daily living).

[0222] Type 2 Gaucher disease Human subjects (n=15) will be enrolled in an open-label study of PR001A rAAV. Subject inclusion criteria include infants aged 0-24 months, biallelic GBA1 mutations, neurological signs and symptoms consistent with type 2 Gaucher disease, and stable standard of care background medication. Each subject will receive the investigational drug as a single ICM (intracisternal) injection. The study will include a 3-month biomarker readout, a 12-month clinical readout, and 5-year safety and clinical follow-up. The study will analyze: (1) safety and tolerability, (2) key biomarkers including: Gcase, GluCer, and GluSph (CSF and blood), (3) time to clinical events (e.g., tracheostomy, PEG (percutaneous endoscopic gastrostomy) placement, death), and (4) efficacy: behavior, cognition, total motor, function, QoL (quality of life).

[0223] Example 14: Testing intravenous administration of rAAV encoding Gcase A PR001 intravenous dose-ranging study was conducted in the D409V Hom mouse model. D409V / D409V (D409V Hom) mice (The Jackson Laboratory, Bar Harbor, ME) display a Gaucher disease-associated phenotype, including reduced GCase activity (see, e.g., Sardi et al., Proc Natl Acad Sci US A. 2011;108(29):12101-6). The study design is provided in Figure 59. Groups and doses are provided in Table 12. [Table 12] † Wild-type animals, not littermates, were purchased from The Jackson Laboratory (Bar Harbor, ME).

[0224] Intravenous administration of PR001 reduced inflammation in the liver (Figure 60A). D409V Hom mice showed glycolipid accumulation in the liver, which was dose-dependently suppressed by PR001 treatment (Figure 60B, Figure 60C). D409V Hom mice showed accumulation of GluSph in the brain, which was reduced by PR001 treatment (Figure 61B). Intravenous administration of PR001 reduced inflammation in the lung (Figure 62).

[0225] A PR001 intravenous dose-ranging study was also conducted in the 4L / PS-NA mouse model. The study design is provided in Figure 63. Groups and doses are provided in Table 13. [Table 13]

[0226] 4L / PS-NA mice showed accumulation of glycolipids in the liver, which was reduced by PR001 treatment (Fig. 64A, Fig. 64B). 4L / PS-NA mice showed accumulation of glycolipids in the brain, which was reduced by PR001 treatment (Fig. 65A, Fig. 65B).

[0227] Example 15: Testing of rAAV encoding inhibitory RNA targeting alpha-synuclein HeLa cells were transduced with PR004 or PR014 at several multiplicities of infection (MOI). Both PR004 and PR014 dose-dependently reduced α-synuclein protein levels (Figure 66A). PR004 dose-dependently increased GCase activity (Figure 66B).

[0228] The efficacy of PR004 was evaluated in neuronal cultures derived from Parkinson's disease patient-derived induced pluripotent stem cells (iPSCs). Parkinson's disease patient-derived induced pluripotent stem cells with SNCA triple were differentiated into neurons (Figure 67A). Neurons transduced with PR004 had increased GCase activity (Figure 67B) and decreased α-synuclein protein levels (Figure 67C).

[0229] No off-target effects of PR004 rAAV vector were observed. Off-target effects of shRNA targeting SNCA from PR004 vector were evaluated in HEK293 cells by qRT-PCR. Expression of 15 genes with the most sequence similarity to the target region of SNCA was evaluated (Figure 68A). Expression of SNCA family members β and γ synuclein (SNCB and SNCG, respectively) was also evaluated (Figure 68B). The mRNA levels of these genes were not affected by PR004.

[0230] PR004 efficacy was evaluated in the AAV2-SNCA-A53T AAV mouse model of Parkinson's disease (Figure 69, Figure 70). AAV2 encoding human SNCA with the A53T mutation is directly injected into the substantia nigra of adult wild-type mice. Starting 4 weeks after injection, animals show gait abnormalities, altered dopamine metabolism, loss of dopaminergic neurons, neuroinflammation, and phosphorylated α-synuclein expression. Automated locomotor gait analysis (MotoRater) was performed 4 weeks (Figure 71A) and 9 weeks (Figure 71B) after PR004 intracerebroventricular injection. A trend of PR004 treatment effect was observed at both time points.

[0231] This application incorporates by reference the entire contents of the following documents: International PCT Publication No. WO2019 / 070894, International PCT Publication No. WO2019 / 070891, U.S. Provisional Patent Application No. 62 / 567,311, filed October 3, 2017, and entitled "GENE THERAPIES FOR LYSOSOMAL DISORDERS", U.S. Provisional Patent Application No. 62 / 567,319, filed October 3, 2017, and entitled "GENE THERAPIES FOR LYSOSOMAL DISORDERS", U.S. Provisional Patent Application No. 62 / 567,301, filed October 3, 2018, and entitled "GENE THERAPIES FOR LYSOSOMAL DISORDERS", U.S. Provisional Patent Application No. 62 / 567,301, filed October 3, 2017, and entitled "GENE THERAPIES FOR LYSOSOMAL DISORDERS", No. 62 / 567,310, filed on October 3, 2017 and entitled "GENE THERAPIES FOR LYSOSOMAL DISORDERS," No. 62 / 567,303, filed on October 3, 2017 and entitled "GENE THERAPIES FOR LYSOSOMAL DISORDERS," and No. 62 / 567,305, filed on October 3, 2017 and entitled "GENE THERAPIES FOR LYSOSOMAL DISORDERS."

[0232] Having thus described several aspects of at least one embodiment of this invention, it will be appreciated 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.

[0233] 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 one or more of the results and / or 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 intended to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application or applications in 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 equivalents thereof, 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.

[0234] As used herein in the specification and claims, the indefinite articles "a" and "an" should be understood to mean "at least one," unless clearly indicated to the contrary.

[0235] As used herein in the specification and claims, the term "and / or" should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are present in conjunction in some cases and disjunction in other cases. Other elements, other than the elements specifically identified by the "and / or" clause, may optionally be present, whether associated with the elements specifically identified or not, unless expressly indicated to the contrary. Thus, as a non-limiting example, when used in conjunction with open-ended language such as "comprising," a reference to "A and / or B" may refer in one embodiment to A without B (optionally including elements other than B), in another embodiment to B without A (optionally including elements other than A), in yet another embodiment to both A and B (optionally including other elements), etc.

[0236] As used herein in the specification and 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 being inclusive, i.e., including at least one of the elements of the number or list, but more than one of them, and optionally including additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of" or "exactly one of," or "consisting of," when used in the claims, refer to including exactly one element of the elements of the number or list. In general, when used herein, the term "or" should be interpreted as indicating exclusive alternatives (i.e., "one or the other, but not both") only when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of."

[0237] As used herein in the specification and claims, the phrase "at least one" in connection with 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 of the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows for 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, may optionally be present. 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") can refer in one embodiment to at least one, optionally including one or more A, and no B (and optionally including elements other than B), in another embodiment to at least one, optionally including more than one B, and no A (and optionally including elements other than A), and in yet another embodiment to at least one, optionally including more than one A, and at least one, optionally including one or more B (and optionally including other elements).

[0238] The use of ordinal terms such as "first," "second," "third," etc. in the claims to modify claim elements does not, in itself, imply any importance, priority, or order of a claim element relative to other elements, or the temporal order in which acts of a method are performed, but is used solely as a label to distinguish one claim element having a particular name from another element having the same name (other than with respect to the use of ordinal terms) to distinguish the claim elements.

[0239] It should also be understood that, unless expressly stated to the contrary, in any method claimed herein that includes more than one step or act, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are recited.

[0240] Each of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referenced in this application are hereby incorporated by reference in their entirety.

[0241] array In some embodiments, an expression cassette encoding one or more gene products (e.g., the 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, 25, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48. 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, 25, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48.

[0242] Numbered embodiments Without limiting the scope of the appended claims, the present disclosure describes the following numbered embodiments of the present disclosure.

[0243] 1. An isolated nucleic acid comprising an expression construct encoding a Gcase protein flanked by two adeno-associated virus (AAV) inverted terminal repeats (ITRs), (i) at least one of the ITRs comprises a modified "D" region relative to the wild-type AAV2 ITR (SEQ ID NO:29); and / or (ii) The isolated nucleic acid, wherein the Gcase is encoded by a codon-optimized nucleic acid sequence.

[0244] 2. The isolated nucleic acid of embodiment 1, wherein the Gcase protein comprises the amino acid sequence set forth in SEQ ID NO: 14 or a portion thereof.

[0245] 3. The isolated nucleic acid of embodiment 1 or 2, wherein the Gcase protein is encoded by a codon-optimized nucleic acid sequence, optionally the nucleic acid sequence set forth in SEQ ID NO: 15.

[0246] 4. The isolated nucleic acid of any one of embodiments 1-3, wherein the modified "D" region is a "D" sequence located outside the ITRs relative to the expression construct.

[0247] 5. The isolated nucleic acid of any one of embodiments 1-4, wherein the ITR comprising the modified "D" sequence is a 3'ITR.

[0248] 6. The isolated nucleic acid according to any one of embodiments 1 to 5, further comprising a TRY sequence, optionally wherein said TRY sequence is set forth in SEQ ID NO: 28.

[0249] 7. An isolated nucleic acid comprising an expression construct encoding a prosaposin protein flanked by two adeno-associated virus (AAV) inverted terminal repeats (ITRs), (i) at least one of the ITRs comprises a modified "D" region relative to the wild-type AAV2 ITR (SEQ ID NO:29); and / or (ii) the isolated nucleic acid, wherein the prosaposin is encoded by a codon-optimized nucleic acid sequence.

[0250] 8. The isolated nucleic acid of embodiment 7, wherein the prosaposin protein comprises the amino acid sequence set forth in SEQ ID NO: 16, or a portion thereof.

[0251] 9. The isolated nucleic acid of embodiment 7 or 8, wherein the prosaposin protein is encoded by a codon-optimized nucleic acid sequence, optionally as set forth in SEQ ID NO: 17.

[0252] 10. The isolated nucleic acid of any one of embodiments 7-9, wherein the modified "D" region is a "D" sequence located outside the ITRs relative to the expression construct.

[0253] 11. The isolated nucleic acid of any one of embodiments 7 to 10, wherein the ITR comprising the modified "D" sequence is a 3'ITR.

[0254] 12. The isolated nucleic acid according to any one of embodiments 7 to 11, further comprising a TRY sequence, optionally wherein said TRY sequence is set forth in SEQ ID NO: 28.

[0255] 13. An isolated nucleic acid comprising an expression construct encoding a SCARB2 protein flanked by two adeno-associated virus (AAV) inverted terminal repeats (ITRs), (i) at least one of the ITRs comprises a modified "D" region relative to the wild-type AAV2 ITR (SEQ ID NO:29); and / or (ii) the isolated nucleic acid, wherein the SCARB2 is encoded by a codon-optimized nucleic acid sequence.

[0256] 14. The isolated nucleic acid of embodiment 13, wherein the SCARB2 protein comprises the amino acid sequence set forth in SEQ ID NO: 18, or a portion thereof.

[0257] 15. The isolated nucleic acid of embodiment 13 or 14, wherein said SCARB2 protein is encoded by a codon-optimized nucleic acid sequence, or the nucleic acid sequence set forth in SEQ ID NO: 19.

[0258] 16. The isolated nucleic acid of any one of embodiments 13-15, wherein the modified "D" region is a "D" sequence located outside the ITRs relative to the expression construct.

[0259] 17. The isolated nucleic acid of any one of embodiments 13-16, wherein the ITR comprising the modified "D" sequence is a 3'ITR.

[0260] 18. The isolated nucleic acid according to any one of embodiments 13 to 17, further comprising a TRY sequence, optionally wherein said TRY sequence is set forth in SEQ ID NO: 28.

[0261] 19. 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.

[0262] 20. The isolated nucleic acid of embodiment 19, wherein the first gene product is a Gcase protein or a portion thereof.

[0263] 21. The isolated nucleic acid of embodiment 19 or embodiment 20, wherein the second gene product is LIMP2 or a portion thereof, or prosaposin or a portion thereof.

[0264] 22. The isolated nucleic acid of any one of embodiments 19 to 21, further encoding an interfering nucleic acid (e.g., shRNA, miRNA, dsRNA, etc.), optionally wherein said interfering nucleic acid inhibits expression of α-Syn.

[0265] 23. The isolated nucleic acid of any one of embodiments 19-22, further comprising one or more promoters, optionally each of said one or more promoters independently being a chicken beta actin (CBA) promoter, a CAG promoter, a CD68 promoter, or a JeT promoter.

[0266] 24. The isolated nucleic acid of any one of embodiments 19 to 23, further comprising an internal ribosome entry site (IRES), optionally wherein said IRES is located between said first gene product and said second gene product.

[0267] 25. The isolated nucleic acid of any one of embodiments 19 to 23, further comprising a self-cleaving peptide coding sequence, optionally wherein said self-cleaving peptide is T2A.

[0268] 26. The isolated nucleic acid of any one of embodiments 19 to 24, wherein the expression construct comprises two adeno-associated virus (AAV) inverted terminal repeat (ITR) sequences flanking the first gene product and the second gene product, and optionally, one of the ITR sequences lacks a functional terminal release site.

[0269] 27. The isolated nucleic acid of embodiment 26, wherein at least one of the ITRs comprises a modified "D" region relative to the wild-type AAV2 ITR (sequence number 29).

[0270] 28. The isolated nucleic acid of embodiment 27, wherein the modified "D" region is a "D" sequence located outside the ITRs relative to the expression construct.

[0271] 29. The isolated nucleic acid of embodiment 27 or 28, wherein the ITR comprising the modified "D" sequence is a 3'ITR.

[0272] 30. The isolated nucleic acid according to any one of embodiments 27 to 29, further comprising a TRY sequence, optionally wherein said TRY sequence is set forth in SEQ ID NO: 28.

[0273] 31. An isolated nucleic acid according to any one of embodiments 1 to 30, having a sequence according to any one of SEQ ID NOs: 1 to 12, 14, 16, and 18.

[0274] 32. A vector comprising the isolated nucleic acid according to any one of embodiments 1 to 31.

[0275] 33. The vector of embodiment 32, wherein the vector is a plasmid.

[0276] 34. The vector of embodiment 32, wherein the vector is a viral vector, optionally wherein the viral vector is a recombinant AAV (rAAV) vector or a baculovirus vector.

[0277] 35. A composition comprising an isolated nucleic acid according to any one of embodiments 1 to 31, or a vector according to any one of embodiments 32 to 34.

[0278] 36. A host cell comprising an isolated nucleic acid according to any one of embodiments 1 to 31 or a vector according to any one of embodiments 32 to 34.

[0279] 37. (i) a capsid protein, and (ii) A recombinant adeno-associated virus (rAAV) comprising an isolated nucleic acid according to any one of embodiments 1 to 31, or a vector according to any one of embodiments 32 to 34.

[0280] 38. The rAAV of embodiment 37, wherein the capsid protein is capable of crossing the blood-brain barrier, and optionally, the capsid protein is an AAV9 capsid protein or an AAVrh.10 capsid protein.

[0281] 39. The rAAV of embodiment 37 or embodiment 38, wherein the rAAV transduces neuronal and non-neuronal cells of the central nervous system (CNS).

[0282] 40. A method for treating a subject having or suspected of having Parkinson's disease, comprising administering to the subject an isolated nucleic acid described in any one of embodiments 1 to 31, a vector described in any one of embodiments 32 to 34, a composition described in embodiment 35, or an rAAV described in any one of embodiments 37 to 39.

[0283] 41. The method of embodiment 40, wherein the administration comprises direct injection into the CNS of the subject, and optionally, the direct injection is intracerebral injection, intraparenchymal injection, intrathecal injection, intracisternal injection, or any combination thereof.

[0284] 42. The method of embodiment 41, wherein the direct injection into the CNS of the subject comprises convection-enhanced delivery (CED).

[0285] 43. The method of any one of embodiments 40-42, wherein the administration comprises a peripheral injection, and optionally, the peripheral injection is an intravenous injection.

[0286] 44. A method for treating a subject having type 2 Gaucher disease or type 3 Gaucher disease, comprising: (i) a recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a transgene insert encoding a glucocerebrosidase (Gcase) protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 15; and (ii) administering to the subject an rAAV comprising an AAV9 capsid protein.

[0287] 45. The rAAV is about 5 × 10 10 vg / g brain ~ approx. 5 × 10 11 The method of embodiment 44, wherein the subject is administered a dose in the range of vg / g brain.

[0288] 46. ​​The rAAV is about 1.3 × 10 11 The method of embodiment 44, wherein the subject is administered a dose of 100 mg / g brain.

[0289] 47. A method for treating a subject with Parkinson's disease having a glucocerebrosidase 1 (GBA1) mutation, comprising: (i) a recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a transgene insert encoding a Gcase protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 15; and (ii) administering to the subject an rAAV comprising an AAV9 capsid protein.

[0290] 47. The rAAV is about 5 × 10 13 vg~approx. 5×1014 The method of embodiment 46, wherein the subject is administered a dose in the range of vg.

[0291] 48. The rAAV is about 1 × 10 14 vg or approximately 2×10 14 The method of embodiment 46, wherein the subject is administered a dose of 0.5 mg / kg.

[0292] 49. The method of any one of embodiments 44 to 48, wherein the rAAV is administered via suboccipital injection into the cisterna magna.

[0293] 1. A method for treating a subject having Gaucher disease type 50.1, comprising: (i) a recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a transgene insert encoding a Gcase protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 15; and (ii) administering to the subject an rAAV comprising an AAV9 capsid protein.

[0294] 51. The rAAV is about 5 × 10 13 vg~approx. 5×10 14 The method of embodiment 50, wherein the subject is administered a dose in the range of vg.

[0295] 52. The method of embodiment 50 or 51, wherein the rAAV is administered intravenously.

[0296] 53. A method for treating a subject having a synucleinopathy or parkinsonism, comprising: (i) (a) a Gcase protein coding sequence comprising the nucleotide sequence of SEQ ID NO: 15, and (b) a recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid comprising an expression construct comprising a transgene comprising an inhibitory nucleic acid coding sequence comprising the nucleotide sequence of SEQ ID NO: 20; (ii) administering to the subject an rAAV comprising an AAV9 capsid protein.

[0297] 54. A method for treating a subject having a synucleinopathy or parkinsonism, comprising: (i) a recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid comprising an expression construct comprising a transgene comprising an inhibitory nucleic acid coding sequence comprising the nucleotide sequence of SEQ ID NO: 20; (ii) administering to the subject an rAAV comprising an AAV9 capsid protein.

[0298] 55. The method of embodiment 53 or 54, wherein the synucleinopathy or parkinsonism is multiple system atrophy, Parkinson's disease, Parkinson's disease with GBA1 mutations, Lewy body disease, dementia with Lewy bodies, dementia with Lewy bodies with GBA1 mutations, progressive supranuclear palsy, or corticobasal syndrome.

[0299] 56. The method of any one of embodiments 44 to 55, wherein the promoter is a chicken beta actin (CBA) promoter.

[0300] 57. The method of any one of embodiments 44 to 56, wherein the rAAV vector further comprises a cytomegalovirus (CMV) enhancer.

[0301] 58. The method of any one of embodiments 44 to 57, wherein the rAAV vector further comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0302] 59. The method of any one of embodiments 44 to 58, wherein the rAAV vector further comprises a bovine growth hormone polyA signal tail.

[0303] 60. The method of any one of embodiments 44 to 59, wherein the nucleic acid comprises two adeno-associated virus inverted terminal repeat (ITR) sequences flanking the expression construct.

[0304] 61. The method of embodiment 60, wherein each ITR sequence is a wild-type AAV2 ITR sequence.

[0305] 62. The method of any one of embodiments 44 to 61, wherein the rAAV vector further comprises a TRY region between the 5'ITR and the expression construct, and the TRY region comprises SEQ ID NO: 28.

[0306] 63. A method for treating a subject having type 2 Gaucher disease or type 3 Gaucher disease, said method comprising: (i) in the order of 5' to 3', (a) AAV2 ITR, (b) CMV enhancer, (c) CBA promoter, (d) a transgene insert encoding a Gcase protein, said transgene insert comprising the nucleotide sequence of SEQ ID NO: 15; (e)WPRE, (f) bovine growth hormone polyA signal tail, and (g) an rAAV vector comprising a nucleic acid comprising an AAV2 ITR; (ii) administering to the subject an rAAV comprising an AAV9 capsid protein; The rAAV is about 5×10 10 vg / g brain ~ approx. 5 × 10 11 The method comprises administering to the subject a dose in the range of 100 to 200 mg / g brain.

[0307] 64. A method for treating a subject with Parkinson's disease having a GBA1 mutation, the method comprising: (i) in the order of 5' to 3', (a) AAV2 ITR, (b) CMV enhancer, (c) CBA promoter, (d) a transgene insert encoding a Gcase protein, said transgene insert comprising the nucleotide sequence of SEQ ID NO: 15; (e)WPRE, (f) bovine growth hormone polyA signal tail, and (g) an rAAV vector comprising a nucleic acid comprising an AAV2 ITR; (ii) administering to the subject an rAAV comprising an AAV9 capsid protein; The rAAV is about 5×10 13 vg~approx. 5×10 14 The method comprises administering to the subject a dose in the range of 0.1 mg / kg.

[0308] 65. The method of embodiment 63 or 64, wherein the rAAV is administered via suboccipital injection into the cisterna magna.

[0309] 66. The method of any one of embodiments 44-52 and 63-65, wherein the rAAV is administered in a formulation comprising about 20 mM Tris (pH 8.0), about 1 mM MgCl2, about 200 mM NaCl, and about 0.001% w / v poloxamer 188.

[0310] 67. (i) (a) a rAAV vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a transgene insert Gcase protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 15; and (b) a rAAV comprising an AAV9 capsid protein; (ii) about 20 mM Tris (pH 8.0); (iii) about 1 mM MgCl2; (iv) about 200 mM NaCl; (v) about 0.001% w / v of poloxamer 188.

[0311] 68. (i) a recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a transgene insert encoding a Gcase protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 15; and (ii) AAV9 capsid protein, 1. An rAAV for use in a method for treating Gaucher disease type 1, Gaucher disease type 2, Gaucher disease type 3 or GBA1 mutated Parkinson's disease in a subject.

[0312] 69. (i) (a) a Gcase protein coding sequence comprising the nucleotide sequence of SEQ ID NO: 15, and (b) an rAAV vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a transgene insert comprising an inhibitory nucleic acid coding sequence comprising the nucleotide sequence of SEQ ID NO: 20; (ii) AAV9 capsid protein, rAAV for use in a method for treating a synucleinopathy or parkinsonism in a subject.

[0313] 70. (i) a rAAV vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a transgene insert comprising an inhibitory nucleic acid coding sequence comprising the nucleotide sequence of SEQ ID NO: 20; (ii) AAV9 capsid protein, rAAV for use in a method for treating a synucleinopathy or parkinsonism in a subject. [Table 14-1] [Table 14-2] [Table 15] [Table 16]

Claims

1. 1. A pharmaceutical composition for treating a subject with Gaucher disease type 2 or Gaucher disease type 3, comprising: (i) a recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a transgene insert encoding a glucocerebrosidase (Gcase) protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 15; and (ii) AAV9 capsid protein; and Including; (a) Approximately 5 × 10 １０ vg / g brain ~ approx. 5 x 10 １１ vg / g brain range, or (b) Approximately 1.3×10 １１ vg / g brain The pharmaceutical composition comprising a dose of the rAAV.

2. 1. A pharmaceutical composition for treating a subject with Parkinson's disease having a glucocerebrosidase 1 (GBA1) mutation, comprising: (i) a rAAV vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a transgene insert encoding a Gcase protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 15; (ii) AAV9 capsid protein; and Including; (a) Approximately 1 × 10 １３ vg ~ approx. 5×10 １４ Take the range of vg, (b) Approximately 5 x 10 １３ vg ~ approx. 5×10 １４ vg range, or (c) Approximately 1 x 10 １４ vg or about 2 x 10 １４ vg The pharmaceutical composition comprising a dose of the rAAV.

3. 1. A pharmaceutical composition for treating a subject with type 1 Gaucher disease, comprising: (i) a rAAV vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a transgene insert encoding a Gcase protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 15; (ii) AAV9 capsid protein; and Including; (a) Approximately 5 × 10 １３ vg ~ approx. 5×10 １４ vg range, or (b) Approximately 1×10 １３ vg ~ approx. 5×10 １４ Take the range of vg The pharmaceutical composition comprising a dose of the rAAV.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the promoter is a chicken beta actin (CBA) promoter.

5. The pharmaceutical composition of any one of claims 1 to 4, wherein the rAAV vector further comprises a cytomegalovirus (CMV) enhancer.

6. The pharmaceutical composition of any one of claims 1 to 5, wherein the rAAV vector further comprises a Woodchuck Hepatitis Virus post-transcriptional regulatory element (WPRE).

7. The pharmaceutical composition of any one of claims 1 to 6, wherein the rAAV vector further comprises a bovine growth hormone polyA signal tail.

8. 8. The pharmaceutical composition of any one of claims 1 to 7, wherein the nucleic acid comprises two adeno-associated virus inverted terminal repeat (ITR) sequences flanking the expression construct, wherein a first ITR sequence is the 5'ITR and a second ITR sequence is the 3'ITR.

9. 9. The pharmaceutical composition of claim 8, wherein each of the two ITR sequences is a wild-type AAV2 ITR sequence.

10. The pharmaceutical composition of claim 8 or 9, wherein the rAAV vector further comprises a TRY region between the 5' ITR and the expression construct, wherein the TRY region comprises SEQ ID NO:

28.

11. 1. A pharmaceutical composition for treating a subject with Gaucher disease type 2 or Gaucher disease type 3, comprising: (i) in order from 5' to 3', (a) AAV2 ITR, (b) CMV enhancer, (c) the CBA promoter; (d) a transgene insert encoding a Gcase protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 15; (e) WPRE; (f) a bovine growth hormone polyA signal tail, and (g) AAV2 ITR and an rAAV vector comprising a nucleic acid comprising: (ii) AAV9 capsid protein; and Including, The rAAV is about 5×10 １０ vg / g brain ~ approx. 5 x 10 １１ The pharmaceutical composition is administered in a dose range of 100 mg / g brain.

12. A pharmaceutical composition for treating a subject having Parkinson's disease with a glucocerebrosidase 1 (GBA1) mutation, comprising: (i) in order from 5' to 3', (a) AAV2 ITR, (b) CMV enhancer, (c) the CBA promoter; (d) a transgene insert encoding a Gcase protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 15; (e) WPRE; (f) a bovine growth hormone polyA signal tail, and (g) AAV2 ITR and an rAAV vector comprising a nucleic acid comprising: (ii) AAV9 capsid protein; and Including, The rAAV is about 1×10 １３ vg ~ approx. 5×10 １４ vg range or about 5×10 １３ vg ~ approx. 5×10 １４ The pharmaceutical composition is administered in a dose range of 0.5 mg / kg.

13. 1. A pharmaceutical composition for treating a subject with type 1 Gaucher disease, comprising: (i) in order from 5' to 3', (a) AAV2 ITR, (b) CMV enhancer, (c) the CBA promoter; (d) a transgene insert encoding a Gcase protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 15; (e) WPRE; (f) a bovine growth hormone polyA signal tail, and (g) AAV2 ITR and an rAAV vector comprising a nucleic acid comprising: (ii) AAV9 capsid protein; and Including, The rAAV is about 1×10 １３ vg ~ approx. 5×10 １４ vg range or about 5×10 １３ vg ~ approx. 5×10 １４ The pharmaceutical composition is administered in a dose range of 0.5 mg / kg.

14. The rAAV is dissolved in about 20 mM Tris (pH 8.0), about 1 mM MgCl ２ 14. The pharmaceutical composition of claim 1, comprising about 200 mM NaCl, and about 0.001% w / v poloxamer 188.

15. (i) (a) a rAAV vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a transgene insert Gcase protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 15; and (b) a rAAV comprising an AAV9 capsid protein; and (ii) about 20 mM Tris (pH 8.0); (iii) about 1 mM MgCl2; and (iv) about 200 mM NaCl; (v) about 0.001% w / v of poloxamer 188; and 1. A pharmaceutical composition for treating a subject with type 2 Gaucher disease, type 3 Gaucher disease, Parkinson's disease with a glucocerebrosidase 1 (GBA1) mutation, and / or type 1 Gaucher disease, comprising:

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