EA046909B1 - GENE THERAPY FOR LYSOSOMAL DISORDERS - Google Patents

Abstract

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

Description

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 description of each of these applications is incorporated herein by reference in its entirety.

Description of the text file presented in electronic form

The contents of the text file, provided electronically, are incorporated herein in their entirety by reference: Copy of the Sequence Listing in machine-readable format (file name: PRVL_009_02WO_SeqList.txt, Date of entry: April 10, 2020; file size: ~361154 bytes).

State of the art

Aberrant expression of proteins such as lysosomal acidic β-glucocerebrosidase (G-case) and α-synuclein is characteristic of the pathogenesis of many diseases of the central nervous system. Gaucher disease is a rare inborn error of glycosphingolipid metabolism due to G-case deficiency. Patients suffer from non-CNS symptoms including hepatosplenomegaly, bone marrow failure leading to pancytopenia, pulmonary disease and fibrosis, and bone defects. In addition, a significant number of patients suffer from neurological manifestations, including abnormal saccadic eye and gaze movements, seizures, cognitive impairment, developmental delay and movement disorders, including Parkinson's disease.

There are several therapeutic agents available to treat peripheral diseases and major clinical manifestations of bone marrow and visceral hematopoietic dysfunction, including enzyme replacement therapy, chaperone-like small molecule drugs that bind to the defective G-case and increase stability, and substrate-reducing therapy. , which blocks the production of substrates that accumulate in Gaucher disease, which leads to the appearance of symptoms and pathologies. However, other aspects of Gaucher disease cannot be treated.

In addition to patients with Gaucher disease (who have mutations in both chromosomal alleles of the GBA1 gene), patients with mutations in only one allele of GBA1 have an increased risk of Parkinson's disease (PD). Elevated levels of α-synuclein also underlie synucleinopathies such as PD. The severity of PD symptoms, which include gait disturbance, resting tremor, rigidity and often depression, sleep disturbances and cognitive decline, correlates with the degree of enzyme activity reduction. Thus, patients with Gaucher disease have the most severe course, whereas patients with a single minor mutation in GBA1 usually have a more benign course. Mutation carriers are also at high risk for other disorders associated with PD, including dementia with Lewy bodies, characterized by executive dysfunction, psychosis and movement disorders similar to PD, and multiple system atrophy with characteristic motor and cognitive impairments. There are no treatments that alter the inexorably progressive course of these disorders and other synucleinopathies.

Field of technology

The present invention relates to the field of gene therapy and methods of its use.

Summary

The present invention provides a method of treating a subject with Gaucher disease type 2 or Gaucher disease type 3, the method comprising administering to the subject a recombinant adeno-associated virus (rAAV) containing: (i) an rAAV vector containing a nucleic acid containing an expression construct containing a promoter , operably linked to a transgene insert encoding a glucocerebrosidase (G-case) protein, wherein the transgene insert contains the nucleotide sequence SEQ ID NO: 15; and (ii) AAV9 capsid protein. In some embodiments, rAAV is administered to a subject at a dose of from about 5x10 ¹⁰ vg/g brain to about 5x1ο ¹¹ vg/g brain. In some embodiments, rAAV is administered to a subject at a dose of about 1.3 x 1011 vg/g brain.

The present invention provides a method of treating a subject suffering from Parkinson's disease with a glucocerebrosidase-1 (GBA1) mutation, comprising administering to the subject a recombinant adeno-associated virus (rAAV) containing: (i) an rAAV vector containing a nucleic acid containing an expression construct containing a promoter, operably linked to a transgene insert encoding a G-case protein, wherein the transgene insert contains the nucleotide sequence SEQ ID NO: 15; and (ii) AAV9 capsid protein. In some embodiments, rAAV is administered to a subject at a dose of about 5x10 ¹³ vg to about 5x10 ¹⁴ vg. In some embodiments, rAAV is administered to a subject at a dose of about 1x 1014 vg or about 2x 10 ¹⁴ vg.

In some embodiments, the rAAV is administered by suboccipital injection into the cisterna magna.

The present invention provides a method of treating a subject suffering from type 1 Gaucher disease, comprising administering to said subject a recombinant adeno-associated virus

- 1 046909 (rAAV), containing: (i) an rAAV vector containing a nucleic acid containing an expression construct containing a promoter operably linked to a transgene insert encoding a G-case protein, wherein the transgene insert contains the nucleotide sequence SEQ ID NO: 15 ; and (ii) AAV9 capsid protein. In some embodiments, rAAV is administered to a subject at a dose of about 5x10 ¹³ vg to about 5x10 ¹⁴ vg. In some embodiments, rAAV is administered intravenously. The present invention provides a method of treating a subject suffering from synucleinopathy or parkinsonism, the method comprising administering to the subject a recombinant adeno-associated virus (rAAV) containing: (i) an rAAV vector containing a nucleic acid containing an expression construct containing a transgene containing (a) a G-case protein coding sequence containing the nucleotide sequence SEQ ID NO: 15; and (b) a sequence encoding an inhibitory nucleic acid comprising the nucleotide sequence of SEQ ID NO: 20; and (ii) AAV9 capsid protein.

The present invention provides a method of treating a subject suffering from synucleinopathy or parkinsonism, comprising administering to said subject a recombinant adeno-associated virus (rAAV) comprising: (i) an rAAV vector containing a nucleic acid containing an expression construct containing a transgene containing a sequence encoding an inhibitory nucleic acid containing the nucleotide sequence SEQ ID NO: 20; and AAV9 capsid protein.

In some embodiments, the synucleinopathy or parkinsonism is multiple system atrophy, Parkinson's disease, GBA1 mutated Parkinson's disease, Lewy body disease, Lewy body dementia, GBA1 mutated Lewy body dementia, progressive supranuclear palsy, or corticobasal syndrome.

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, wherein the TRY region comprises SEQ ID NO: 28.

The present invention provides a method of treating a subject suffering from type 2 Gaucher disease or type 3 Gaucher disease, comprising administering to the subject an rAAV comprising:

(i) an rAAV vector containing a nucleic acid containing, in order 5' to 3':

(a) AAV2 ITR;

(b) CMV enhancer;

(c) CBA promoter;

(d) a transgene insert encoding a G-case protein, wherein the transgene insert contains the nucleotide sequence of SEQ ID NO: 15;

(e) WPRE;

(f) bovine growth hormone polyA signal tail; and (g) AAV2 ITR; and (ii) AAV9 capsid protein;

wherein the rAAV is administered to the subject at a dose of about ^5x10 vg/g brain to about 5x10 ^vg /g brain.

The present invention provides a method of treating a subject suffering from Parkinson's disease with a GBA1 mutation, comprising administering to the subject an rAAV comprising:

(i) an rAAV vector containing a nucleic acid containing, in order 5' to 3':

(a) AAV2 ITR;

(b) CMV enhancer;

(c) CBA promoter;

(d) a transgene insert encoding a G-case protein, wherein the transgene insert contains the nucleotide sequence of SEQ ID NO: 15;

(e) WPRE;

(f) bovine growth hormone polyA signal tail; and (g) AAV2 ITR; and (ii) AAV9 capsid protein;

wherein the rAAV is administered to the subject at a dose of about 5x 10 ¹³ vg to about 5x 10 ¹⁴ vg.

In some embodiments, the rAAV is administered by suboccipital injection into the large

- 2 046909 tank. In some embodiments, rAAV is administered in a formulation containing about 20 mM Tris, pH 8.0, about 1 mM MgCl ₂ , about 200 mM NaCl, and about 0.001% poloxamer 188.

The present invention provides a pharmaceutical composition containing:

(i) rAAV containing:

(a) an rAAV vector containing a nucleic acid containing an expression construct containing a promoter operably linked to a transgene insert encoding a G-case protein, wherein the transgene insert contains the nucleotide sequence of SEQ ID NO: 15; and (b) AAV9 capsid protein; and (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. poloxamer 188. The present invention provides an rAAV containing:

(a) an rAAV vector containing a nucleic acid containing an expression construct containing a promoter operably linked to a transgene insert encoding a G-case protein, wherein the transgene insert contains the nucleotide sequence of SEQ ID NO: 15; and (b) an AAV9 capsid protein, for use in a method of treating Gaucher disease type 1, Gaucher disease type 2, Gaucher disease type 3 or Parkinson's disease with a GBA1 mutation in a subject.

The present invention provides an rAAV comprising:

(i) an rAAV vector containing a nucleic acid containing an expression construct containing a promoter operably linked to the transgene insert, containing:

(a) a G-case protein coding sequence comprising the nucleotide sequence of SEQ ID NO: 15; and (b) a sequence encoding an inhibitory nucleic acid comprising the nucleotide sequence of SEQ ID NO: 20; and (ii) an AAV9 capsid protein, for use in a method of treating synucleinopathy or parkinsonism in a subject. The present invention provides an rAAV comprising:

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

Brief description of graphic materials

Fig. 1 is a schematic representation of one embodiment of a plasmid containing an rAAV vector that includes an expression construct encoding a G-case (eg, GBA1 or a portion thereof).

Fig. 2 is a schematic representation of one embodiment of a plasmid containing an rAAV vector that includes an expression construct encoding a G case (eg, GBA1 or a portion thereof) and LIMP2 (SCARB2) or a portion thereof. The coding sequences of G-case and LIMP2 are separated by the internal ribosome entry site (IRES).

Fig. 3 is a schematic representation of one embodiment of a plasmid containing an rAAV vector that includes an expression construct encoding G-case (eg, GBA1 or a portion thereof) and LIMP2 (SCARB2) or a portion thereof. Expression of the G-case and LIMP2 coding sequences is each driven by a separate promoter.

Fig. 4 is a schematic representation of one variant of a plasmid containing an rAAV vector that includes an expression construct encoding G-case (eg, GBA1 or a portion thereof), LIMP2 (SCARB2) or a portion thereof, and interfering RNA for α-Syn.

Fig. 5 is a schematic representation of one variant of a plasmid containing an rAAV vector that includes an expression construct encoding G-case (eg, GBA1 or a portion thereof), prosaposin (eg, PSAP or a EHB portion), and interfering RNA for α-Syn.

Fig. 6 is a schematic representation of one embodiment of a plasmid containing an rAAV vector that includes an expression construct encoding G-case (eg, GBA1 or a portion thereof) and prosaposin (eg, PSAP or a portion thereof). The coding sequences of G-case and prosaposin are separated by the internal ribosome entry site (IRES).

Fig. 7 is a schematic representation of one embodiment of a rAAV vector that includes an expression construct encoding G-case (eg, GBA1 or a portion thereof).

In this embodiment, the vector contains a CBA promoter element (CBA), consisting of

- 3 046909 four parts: CMV enhancer (CMVe), CBA promoter (CBAp), exon 1 and intron (int) for continuous expression of the codon-optimized coding sequence of human GBA1. The 3' region also contains a WPRE regulatory element followed by a bGH polyA tail. The 5' end of the promoter region includes three transcriptional activation sites: TATA, RBS, and YY1. Flanking ITRs allow insertion sequences to be packaged correctly. Two variants of the 5' ITR sequence (insert) were evaluated; they have several nucleotide differences in the 20-nt 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 contains a mutant D domain (eg, S domain, nucleotide changes are shown in the bottom row).

Fig. 8 is a schematic representation of one embodiment of the plasmid encoding the rAAV vector shown in FIG. 7.

In fig. 9A-Fig. 9F shows representative data regarding the validation of the SVE mouse model. Survival (Fig. 9A) was checked twice daily, and weight (Fig. 9B) was recorded daily and analyzed at P27 (Fig. 9C). All groups started with n=8. Behavior was assessed by retention time on the rotating drum (Fig. 9D) at P24 and by total distance traveled in the open field (Fig. 9F). Due to early mortality, the number of animals in each group is different: n=8 for PBS and 25 mg/kg SBE, n=4 for 37.5 mg/kg SBE. The 50 mg/kg SBE group was not evaluated on the rotating drum due to absolute mortality at P24. Statistical results are presented for comparison with the PBS group using ANOVA followed by Tukey's HSD test. Levels of G-case substrates were analyzed in the cerebral cortex of mice in the PBS and 25 mg/kg SBE treatment groups. Cumulative levels of GluSph and GalSph (Fig. 9E) are reported in pmol per mg tissue weight. Statistical results are presented using Student's t test. Average values are shown. Error bars represent standard error of the mean (SEM). * P<0.05; ** P <0.01; *** P <0.001.

Fig. 10 is a schematic representation of one embodiment of a maximum dose study design for rAAV encoding G-case in a mouse model of SVE. 4 μl of PR001B or dPBS was administered by ICV injection at P3, and daily administration of 25 mg/kg SBE was started at P8. Behavior was assessed in the rotating drum test at P24. Half of the animals were sacrificed at P36, 1 day after their last dose of SBE at P35, while the remaining half were sacrificed at P38, 3 days after their last dose of SBE at P35. vg stands for vector genomes.

Fig. 11A-Fig. 11D shows representative data regarding the estimated lifetime maximum dose of PR001B rAAV in a murine model of SVE. At P3, mice were treated with either vehicle or 8.8e9 vg rAAV via ICV administration. Daily IP delivery of PBS or 25 mg/kg SBE was started at P8. At the end of the study, half of the mice were sacrificed the day after they received their last dose of SBE on P36 (Day 1), while the remaining half were withdrawn from SBE for 3 days before sacrifice at P38 (Day 3). All treatment groups (excipient+PBS n=8, rAAV+PBS n=7, excipient+SBE n=8, and rAAV+SBE n=9) were weighed daily (Fig. 11 A) and weights were analyzed at P33 (Fig. 11B). Behavior was assessed by the total distance traveled in the open field at P23 (Fig. 11D) and by the time period before falling from the rotating drum at P24 (Fig. 11C), which was estimated for each animal as the median value over the 3 trials. Considering mortality, n=7 for the adjuvant+SVE group for behavioral analyses, and n=8 for all other groups. Average values for animals are presented. Error bars represent SEM. *p<0.05; ***p<0.001, nominal p-values for treatment groups by linear regression in SVE-treated animals.

Fig. 12A, FIG. 12B shows representative data regarding the biochemical assessment of the maximum dose of PR001B rAAV in a mouse model of SVE. The cerebral cortex of mice from all treatment groups was used to measure vector genomes (Fig. 12A) and G-case activity (Fig. 12B). Biodistribution is presented as vector genomes per 1 μg of genomic DNA (gDNA). The dotted line (100 vector genomes/μg gDNA) represents the threshold for determining positive vector presence. Enzyme activity was assessed by measuring the Gcase rate of glucose production using Amplex Red (Invitrogen; #A22189) and then converted to an effective level of Gcase activity using a recombinant Gcase reference standard curve. One unit was defined as the activity of 1 ng/ml of recombinant purified G-case normalized per mg of total protein, n=6-9 per group. Average values are shown. Error bars represent SEM. *P < 0.05, nominal P values for treatment groups in SVE-treated animals, adjusted for days of collection and sex as a covariate.

Fig. 12C, fig. 12D shows representative data from a maximum dose glycolipid analysis of PR001B rAAV in a mouse model of SVE. Cortices from all treatment groups (PBS+dPBS [left bars on each graph] n=4, CBE+dPBS [centre bars on each graph] n=6, and CBE+PROOIB [right bars on each graph] n=9) were used to measure GluSph levels (Fig. 12C) and GluCer levels (Fig. 12D) in groups before (day 1) or after (day 3) SVE withdrawal.

- 4 046909

GluSph and GluCer levels are given in pmol per nmol of phosphate. Average values are shown. Error bars represent SEM. *P<0.1; **P<0.01; ***p < 0.001, nominal P -values for treatment groups in SVE-treated animals, adjusted for collection days and sex as covariates.

Fig. 13 shows representative data regarding behavioral and biochemical correlations in the BES mouse model following administration for the following groups: excipient+PBS, excipient+BES, and PR001B rAAV+BES. In all treatment groups, ability to remain on a rotating drum was negatively correlated with GluCer accumulation (A, p=0.0012 by linear regression), and GluSph accumulation was negatively correlated with increased G-case activity (B, p=0.0086 by linear regression). regression).

Fig. 14 shows representative data regarding the biodistribution of PR001B rAAV in a mouse model of SVE. The presence of the vector genome was quantified by qPCR using a vector reference standard curve; Genomic DNA concentration was assessed by measuring the absorbance of A260. The dotted line (100 vector genomes/μg g DNA) represents the threshold for determining positive vector presence. Average values are shown. Error bars represent SEM. n=7-9 per group.

Fig. 15A is a schematic representation of one study design to determine the dose range of rAAV encoding G-case in a murine model of VES. PROOIA was administered by ICV injection at P3, and daily administration of 25 mg/kg SBE began at P8. Behavior was assessed in the open field and rotating drum tests at P21-P22, and in the tapering beam test at P28. Animals were sacrificed at P38-P40, 1 day after they received their last dose of SVE. The cortex was analyzed for levels of the substrates GluSph and GluCer and G-case activity. There were 10 mice (5 males, 5 females) in each treatment group.

Fig. 15B-fig. 15E shows representative data regarding the estimated lifetime dose range of PR001 rAAV in a murine model of SVE. Mice received excipient or 1 of 3 different doses of PR001A via 4 μl ICV at P3: low dose (middle column), medium dose (second from right column), or high dose (far right column). At P8, daily IP administration was started at a dose of 25 mg/kg SBE. Mice that received excipient and SBE (second column from left) or excipient and PBS (far left column) served as controls. All treatment groups started with n=10 (5M/5F) per group. All mice were sacrificed 1 day after the last dose of SBE (P38-P40). All treatment groups were weighed daily (Fig. 15B) and their weights were analyzed at P37 (Fig. 15C). Locomotor activity was assessed by the time period before falling from the rotating drum at P24 (Fig. 15D) and by the time period before turning around on the tapering beam at P30 (Fig. 15E). Due to early lethality, the number of mice participating in behavioral assays was: excipient+PBS (far left column) n=10; excipient+SBE (second column from left) n=9; low dose PROOIA+CBE (middle column) n=6; mean dose PROOIA+CBE (second column from right) n=10; high dose PROOIA+CBE (far right column) n=7. Average values are presented. Error bars represent SEM. **p<0.05; ***p<0.01 for nominal p-values for SVE treatment groups, adjusted for sex as a covariate.

Fig. 16A shows representative biodistribution data from the PR001A study using the dose range SBE model. Mice received the excipient or 1 of 3 different doses of PR001A by ICV administration at P3: low dose (middle column), medium dose (second from right column), or high dose (far right column). At P8, daily IP administration was started at a dose of 25 mg/kg SBE. Mice that received excipient and SBE (second column from left) or excipient and PBS (far left column) served as controls. All mice were sacrificed at P38–P40, 1 day after the last dose of SBE. The presence of vector genomes was assessed in each tissue and across all treatment groups, as demonstrated by the number of vector copies per 1 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. The dotted lines represent the detection threshold for positive vector presence. Average values are shown. Error bars represent SEM.

Fig. 16B shows representative data regarding Gcase enzymatic activity in the PR001A study using a dose range SBE model. Efficient G-case enzymatic activity was demonstrated for each tissue and all treatment groups. Activity is given in units per mg of total protein, with one unit defined as the activity of 1 ng/ml of recombinant purified G-case. Average values are shown. Error bars represent SEM. Statistical results are presented for comparison with the excipient+SBE groups (second column from left). n=10, 9.6,10.7 per group, respectively. * P<0.05; ** P <0.01; ***P<0.001 using ANOVA followed by Tukey's HSD multiple comparison test.

Fig. 16C-fig. 16D shows representative data for glycolipid analysis in the PR001A study using the dose-ranging CBE model. Levels of GluSph (Fig. 16C) and GluCer

- 5 046909 (Fig. 16D) are given in pmol per nmol of phosphate. Average values are shown. Error bars represent SEM. ** P <0.01; ***P<0.001 using ANOVA followed by Tukey's HSD multiple comparison test.

Fig. 16E shows representative data from the hematoxylin and eosin staining analysis of the PR001A study using a dose-ranging SBE model. Brain tissue was processed for hematoxylin and eosin (H&E) staining, and tissue sections were evaluated for pathological changes. The percentage of animals that tested positive for the presence of cerebrocortical glial scars, a sign of neuroinflammation, is shown. Administration of SBE resulted in a significant increase in glial scars compared with the control group receiving the excipient. PR001A significantly reduced SES-induced glial scarring in a dose-dependent manner. Statistical results are presented for comparison with the SBE + excipient groups (left column). n=10, 9.6, 10.7 per group, respectively. * P<0.05; ** P <0.01; *** P <0.001 for Fisher's exact test.

Fig. 16F shows representative data from cerebrocortical immunohistochemical analysis in the PR001A study using a dose-ranging SBE model. The graph shows the average values of the immunoreactive area measured in the cerebral cortex (n=5-10 per group). The immunoreactive area of Ibal (ionizing potassium-binding adapter molecule 1) was significantly larger in animals treated with CBE + excipient (second column from left) than in mice of all other groups studied. Means are shown and error bars represent SEM. Data were analyzed using one-way ANOVA and Sidak's post hoc test for multiple comparisons. **:.P<0.01;***:.P<0.001.

Fig. 17 shows representative data from a tapering bar behavior assay at the highest dose of GBA1 rAAV in a genetic mouse model. The motor activity of the treatment groups (WT+adjuvant, n=5), 4L/PS-NA+adjuvant (n=6) and 4L/PS-NA+rAAV (n=5)) was assessed using a beam walk test across 4 weeks after rAAV administration.

The total numbers of slips and active times are given as the total number of 5 tests on different beams. Velocity and velocity slip are given as the average of 5 tests on different beams. Average values are shown. Error bars represent SEM.

Fig. 18 shows representative in vitro expression data of rAAV constructs encoding GBA1 in combination with prosaposin (PSAP), SCARB2, and/or one or more inhibitory nucleic acids. Data indicate that transfection of HEK293 cells with each construct resulted in overexpression of the transgenes of interest compared to GFP-transfected cells.

Fig. 19 is a diagram depicting rAAV vectors containing the D region located on the outside of the ITR (e.g., proximal to the end of the ITR relative to the transgene insertion or expression construct) (top) and wild-type rAAV vectors having the ITR on the inside of the vector (e.g., proximal to the transgene insertion into the vector).

Fig. 20 shows data regarding transduction of HEK293 cells using rAAVs having ITRs with wild-type (circles) or alternative (eg, outer side; squares) placement of the D sequence. rAAVs having ITRs located outer side were able to transduce cells as efficiently , like rAAVs that have a wild-type ITR.

Fig. 21 is a schematic representation of one embodiment of a plasmid containing an rAAV vector that includes an expression construct encoding a G case (eg, GBA1 or a portion thereof).

Fig. 22 is a schematic representation of one embodiment of a plasmid containing an rAAV vector that includes an expression construct encoding a G-case (eg, GBA1 or a portion thereof).

Fig. 23 is a schematic representation of one variant of a plasmid containing an rAAV vector that includes an expression construct encoding a G-case (eg, GBA1 or a portion thereof) as well as interfering RNA for α-Syn.

Fig. 24 is a schematic representation of one variant of a plasmid containing an rAAV vector that includes an expression construct encoding a G-case (eg, GBA1 or a portion thereof) as well as interfering RNA for α-Syn.

Fig. 25 is a schematic representation of one embodiment of a plasmid containing an rAAV vector that includes an expression construct encoding prosaposin (eg, PSAP or a portion thereof).

Fig. 26 is a schematic representation of one embodiment of a plasmid containing an rAAV vector that includes an expression construct encoding a G-case (eg, GBA1 or a portion thereof).

Fig. 27 is a schematic representation of one embodiment of a plasmid containing

- 6 046909 rAAV vector, which includes an expression construct encoding G-case (eg, GBA1 or part thereof), prosaposin (eg, PSAP or part thereof), and interfering RNA for α-Syn.

In fig. 28 provides representative data demonstrating that administration of the rAAV vector encoding Ga3 reduces glial scarring in vivo. Tissues were processed for hematoxylin and eosin (H&E) staining, and slides were assessed for pathological changes. The percentage of animals positive for the presence of glial scars, which confirms reactive astrogliosis, in each group is shown in light shading, and the percentage of animals negative for the presence of glial scars is shown in black. The use of SBE resulted in a significant increase in glial scars compared with the control group receiving the excipient. rAAVGBAl significantly reduced VES-induced glial scarring in a dose-dependent manner. Statistical results are presented for comparison with the excipient+SBE groups (red). n=10, 9, 6, 10, 7 per group, respectively. *: p<0.05; **: p<0.01; ***: p<0.001 for Fisher's exact test.

Fig. 29A, fig. 29B shows representative data regarding the average immunofluorescence signal measured in the cerebral cortex (n=6-10 per group) of mice administered rAAV-GBAl or excipient. Quantification of Gcase immunolabeling (Fig. 19A) revealed the strongest immunofluorescent labeling in high-dose rAAV-GBAl-treated animals, followed by medium- and low-dose rAAVGBAl-treated animals. The immunoreactive area of Iba1 (Fig. 29B) was significantly larger in animals treated with SBE/excipient than in mice of all other groups tested. Data were analyzed using one-way ANOVA and Sidak's multiple comparison post hoc test. G histograms represent group means+SEM.

Fig. 30 is a histogram showing representative data regarding the biodistribution of the PR001A transgene in study PRV-2018-016 on day 183. This study is described in Example 12 Transgene levels were analyzed using qPCR techniques in NHP (non-human primate) organisms after 183 days after injection into the cisterna magna (ICM) of either the excipient, either a low dose of PR001A ( ^6.2x1010 vg/g brain) or a high dose of PROOIA (2.3x1011 vg/g brain). Each bar represents the mean ± SEM of 3 animals per group; values that were below the limit of quantitation were reported as zero. Because qPCR values for excipient-treated animals were zero for each region, bars for excipient are not shown in the graph with this scale.

Fig. 31A, fig. 31B are graphs showing representative data regarding human G-case expression on Day 183 in study PRV-2018-016. This study is described in Example 12. G-case expression levels were determined using the Simple Western™ assay (Jess) in NHP (non-human primate) cortical, hippocampal and midbrain samples that were collected on Day 183. Shown are G-case expression levels from NHPs treated with adjuvant (left in each panel), low dose PR001A (6.2 x ¹⁰ vg/g brain; center in each panel), or high dose PR001A (2.3 x 10 vg/g brain; on the right side of each panel). Fig. 31A presents data for selected regions of the cortex, hippocampus, and midbrain. Fig. 31B represents the percentage change for the excipient group (left), low dose group (center), and high dose group (right). Data for this plot were normalized to the mean within tissue and pooled for the cerebral cortex, hippocampus, and midbrain. Each column represents the percentage of the excipient group median for each dose of the mean normalized data. To calculate significance, a one-way ANOVA was performed to assess significance for the combination treatment group, which included both low- and high-dose animals, compared with the excipient group. P value=0.014 (*<0.05).

Fig. 32 is a series of graphs showing representative data regarding the biodistribution of the PR001A transgene quantified by qPCR in the PRV-2019-005 study. This study is described in Example 12. Transgene levels were analyzed using qPCR techniques in NHP organisms 30 and 90 days after injection into the cisterna magna (ICM) of either excipient or PR001A (7.0 x 1011 vg/g brain). Each graph represents each individual animal (n=3/group) with mean ± SEM.

Fig. 33 is a line graph showing representative data regarding G-case activity following in vitro transduction of HEK293T cells with PR001A. HEK293T cells transduced with PR001A at different multiplicities of infection (MOI) were analyzed for G-case activity. Activity was measured by hydrolysis of the profluorescent substrate resorufin P-D-glucopyranoside. The fluorescence of the digested substrate was determined using a plate reader at excitation 573 nm and emission 610 nm. Values are means ± SEM, n=2; unit is equivalent to the activity of 1 ng/ml recombinant

- 7 046909 purified G-case.

Fig. 34A, fig. 34B are histograms showing representative data regarding G-case activity (FIG. 34A) and α-synuclein levels (FIG. 34B) following in vitro transduction of HeLa cells using PR001A. HeLa cells treated with excipient (left column) or transduced with 2x10 ⁵ vg/cell PR001A (center column) or ^2x10 vg/cell PR001A (right column) were harvested 72 hours after treatment and analyzed for levels of G-case activity (Fig. . 34A) using a fluorometric enzyme assay or α-synuclein levels (Fig. 34B) using ELISA. Effective G-case enzymatic activity is given in units per mg of total protein, with one unit defined as the activity of 1 ng/ml of recombinant purified G-case. The concentration of α-synuclein is presented in ng/ml per mg of total protein. The studies were carried out in three biological replicates. Average values are shown. Error bars represent SEM. One-way ANOVA followed by Dunnett's multiple comparison test.

Fig. 35A, fig. 35B are histograms showing representative data regarding G-case activity (FIG. 35A) and α-synuclein levels (FIG. 35B) following in vitro transduction of mouse hippocampal neurons using PR001A. Primary cultures of mouse hippocampal neurons were treated with vehicle (left column) or transduced with 1.3 x 10 ⁵ vg/cell PR001A (central column) or 1.3 x 10 ⁶ vg/cell PR001A (right column) on Day 2 in vitro (DIV). At DIV9, cells were collected and analyzed for levels of G-case activity (Fig. 35A) using a fluorometric enzyme assay or for levels of α-synuclein (Fig. 35B) using ELISA. G-case activity is reported as relative fluorescent units (RFU) per hour per mg of total protein. The concentration of α-synuclein is presented in ng/ml per mg of total protein. The studies were carried out in two biological replicates. Average values are shown. Error bars represent SEM. One-way ANOVA followed by Dunnett's multiple comparison test.

Fig. 36 is a schematic representation of one long-term study design of rAAV encoding G-case in a murine model of SVE. PR001A was administered by ICV injection at P3, and daily VES administration began at P8. Behavior was assessed in the rotating drum test at Weeks 3, 6 and 15, and in the tapering beam test at Weeks 4, 7, 13. Animals were sacrificed at approximately Week 26, 1 day after the last dose of VES. The brain cortex was analyzed for levels of the substrates GluSph and GluCer and G-case activity. Each experimental group consisted of 10-11 animals, each of which included male and female mice.

Fig. 37A-Fig. 37D shows representative data regarding the evaluation of long-term use of PR001A in the SVE model. Cerebral cortex from mice of all treatment groups (PBS+excipient: left column, SBE+excipient: central column, SBE+2.0 x 10 ¹⁰ vg PR001A: right column) was used to measure vector genomes (Fig. 37A), activity G-case (Fig. 37B), GluSph levels (Fig. 37C) and GluCer levels (Fig. 37D). The presence of vector genomes was assessed in each tissue and across all treatment groups, as demonstrated by the number of vector copies per 1 μg of genomic DNA. The presence of vector genomes was quantified by qPCR using a vector reference standard curve. Effective Gcase enzymatic activity is given in units per mg of total protein, with one unit defined as the activity of 1 ng/ml recombinant purified Gcase. GluSph and GluCer levels are given as pmol per nmol of phosphate. Average values are shown. Error bars represent SEM. n=10, 11, 10 per group, respectively. (*) P<0.1, * P<0.05; ** P <0.01;***P<0.001 using ANOVA followed by Tukey's HSD multiple comparison test.

Fig. 38A-Fig. 38E shows representative data regarding the lifetime assessment of an additional dose range of PR001A in the SBE model. Mice from all treatment groups were weighed daily (FIG. 38A) and their weights were analyzed at P45 (FIG. 38B). Motor performance was assessed by the period of time remaining on the rotating drum in Week 3 (Fig. 38C) and in Week 5 (Fig. 38D), as well as by the period of time before turning on the tapering beam (Fig. 38E) in Week 4. n=8 -11 per group. Average values are shown. Error bars represent SEM. Statistical results are presented for comparison with the SBE + excipient group (second column from left). *** P < 0.001 by ANOVA followed by Tukey's HSD test.

Fig. 39 shows representative biodistribution data from a complementary study using a dose-ranging SBE model. The presence of vector genomes was assessed in each tissue and across all treatment groups, as demonstrated by the number of vector copies per 1 μg of gDNA. The presence of vector genomes was quantified by qPCR using a vector reference standard curve; n=9-11 per group. The black dotted line (100 vector genomes/μg gDNA) represents the threshold for determining positive vector presence. Average values are shown. Error bars represent SEM.

- 8 046909

Fig. 40 shows representative data regarding Gcase enzymatic activity in a complementary study using a dose range SBE model. Efficient G-case enzymatic activity was measured and demonstrated for the cerebral cortex of all treatment groups. Activity is given in units per mg of total protein, with one unit defined as the activity of 1 ng/ml of recombinant purified G-case. Average values are shown. Error bars represent SEM. Statistical results are presented for comparison with the SBE+adjuvant group (second column from left), n=9-11 per group. (*)P<0.1 by ANOVA followed by Tukey's HSD test.

Fig. 41A, fig. 41B shows representative data from a glycolipid assay in a complementary study using a dose range SBE model. The levels of GluSph (Fig. 41A) and GluCer (Fig. 41B) are given in pmol per nmol of phosphate. Average values are shown. Error bars represent SEM. Statistical results are presented for comparison with the SBE+adjuvant group (second column from left), n=9-11 per group. **P<0.01; ***p < 0.001, by ANOVA followed by Tukey's HSD test.

Fig. 42A - fig. 42D shows representative data regarding the lifetime assessment of an additional dose range of PR001A in the SBE model. Mice from all treatment groups were weighed daily (FIG. 42A) and their weights were analyzed at P37 (FIG. 42B). Motor performance was assessed by the period of time remaining on the rotating drum in Week 3 (FIG. 42C), as well as the period of time before turning on the tapering beam (FIG. 42D) in Week 4. n=9-10 per group. Average values are shown. Error bars represent SEM. Statistical results are presented for comparison with the SBE + excipient group (second column from left). *** P < 0.001 by ANOVA followed by Tukey's HSD test.

Fig. 43A, fig. 43B shows representative data regarding the biodistribution and enzymatic activity of G-case in a complementary dose-ranging study of PROOIA in the SBE model. Vector genomes were measured in the cerebral cortex (Fig. 43 A) in all treatment groups and shown as the number of vector copies per 1 μg of genomic DNA (gDNA). The presence of vector genomes was quantified by qPCR using a vector reference standard curve. The black dotted line represents the detection threshold for positive vector presence (at 100 vector genomes/μg gDNA). Efficient G-case enzymatic activity was measured in the cerebral cortex (FIG. 43B) and shown in units per mg of total protein, with one unit defined as the activity of 1 ng/ml recombinant purified G-case. Average values are shown. Error bars represent SEM. n=9-10 per group. *** P <0.001; using ANOVA analysis followed by correction for Tukey's HSD multiple comparison test.

Fig. 44A, fig. 44B shows representative data for glycolipid analysis of an additional dose range of PR001A in the SBE model. The levels of GluSph (Fig. 44A) and GluCer (Fig. 44B) are given in pmol per nmol of phosphate. Average values are shown. Error bars represent SEM. Statistical results are presented for comparison with the SBE + excipient group (second column from left), n=9-10 per group. ***P < 0.001 by ANOVA followed by Tukey's HSD test.

Fig. 45 is a schematic representation of one study design using rAAV encoding G-case in the 4L/PS-NA genetic mouse model. PR001A was administered via ICV injection to 4L/PS-NA mice at 3–4 weeks of age. Behavior on the tapered beam was analyzed at Weeks 8, 12 and 18 of life (5, 9 and 15 weeks after ICV administration), and behavior on the rotating drum was analyzed at Weeks 12 and 18 of life (9 and 15 weeks after ICV administration). Mice were sacrificed at Week 18. The cerebral cortex was analyzed for Gcase enzymatic activity, and the cerebellum was analyzed for levels of the substrates GluSph and GluCer. There were 3 males and 3 females in each experimental group.

Fig. 46 shows representative data regarding the biodistribution of the highest dose of PR001A in the 4L/PS-NA genetic mouse model. The presence of vector genomes was assessed in each tissue and across all treatment groups, as demonstrated by the number of vector copies per 1 μg of genomic DNA (gDNA). The presence of vector genomes was quantified by qPCR using a vector reference standard curve. Average values are shown. Error bars represent SEM. n=4-5 per group. The dotted lines represent the detection threshold for positive vector presence (at 100 vector genomes/μg g DNA).

Fig. 47 shows representative data regarding Gcase enzymatic activity at the highest dose of PR001A in the 4L/PS-NA mouse genetic model. Effective G-case enzymatic activity was measured and demonstrated for each tissue and all treatment groups. Activity is given in units per mg of total protein, with one unit defined as the activity of 1 ng/ml of recombinant purified G-case. Average values are shown. Error bars represent SEM. n=4-5 per group. *: P<0.05; ** P <0.01; ***P<0.001 using ANOVA followed by Tukey's HSD multiple comparison test.

Fig. 48A, fig. 48B shows representative glycolipid assay data for PR001A in

- 9 046909 genetic mouse model 4L/PS-NA. 4L/PS-NA mice received excipient (center column) or 1.5 ± 10 ¹⁰ vg PR001A (right column), and control mice received excipient (right column) by ICV administration on postnatal Day P23. In the cerebellum, levels of GluSph (FIG. 48A) and GluCer (FIG. 48B) were measured. Levels are given in pmol per nmol of phosphate. Average values are shown. Error bars represent SEM. n=4, 5, 5 per group, respectively. * P<0.05; ** P <0.01;***P<0.001 using ANOVA followed by Tukey's HSD multiple comparison test.

Fig. 49A, fig. 49B shows representative data regarding the biochemical assessment of α-synuclein accumulation in the cerebral cortex in the 4L/PS-NA genetic mouse model. 4L/PSNA mice received excipient by ICV (center column) or 1.5 x ^{10 10} vg PR001A (right column), and control mice received excipient (right column) by ICV on postnatal Day 23. Brain lysate fractions from the cerebral cortex, Triton X soluble and Triton X insoluble were analyzed for α-synuclein levels using a specific immunosorbent assay. Insoluble alpha-synuclein (FIG. 49A) and the ratio of insoluble to soluble alpha-synuclein (FIG. 49B) are shown. Average values are shown. Error bars represent SEM. n=3-5 per group. (*): P < 0.20, using ANOVA followed by Tukey's HSD multiple comparison test.

Fig. 50 is a schematic representation of one dose-ranging study design of PR001A rAAV in the 4L/PS-NA genetic mouse model. PR001A was administered via ICV injection to 4L/PS-NA mice at 3–4 weeks of age. Behavior on the tapered beam was analyzed at Weeks 8, 12 and 18 of life (5, 9 and 15 weeks after ICV administration), and behavior on the rotating drum was analyzed at Weeks 12 and 18 of life (9 and 15 weeks after ICV administration). Mice were sacrificed at Week 18. The cerebral cortex was analyzed for G-case enzymatic activity, and the cerebellum was analyzed for levels of the substrates GluSph and GluCer. There were 10-11 mice in each treatment group.

Fig. 51 shows representative data from behavioral assays at Week 18 for the PR001A dose range in the 4L/PS-NA genetic mouse model. Motor performance on a tapered beam was assessed, with the average of the total number of slips relative to speed demonstrated across two trials on different beams. n=10, 6, 5, 7, 4, 8 for the group, respectively. Average values are shown. Error bars represent SEM. *: P<0.05; ***: P < 0.001 using ANOVA followed by Tukey's HSD multiple comparison test.

Fig. 52A, fig. 52B shows representative data regarding the biodistribution and enzymatic activity of G-case in a dose-ranging study of PR001A in the 4L/PS-NA genetic mouse model. Vector genomes were measured in the cerebral cortex (Fig. 52A) in all treatment groups and shown as the number of vector copies per 1 μg of genomic DNA (gDNA). The presence of vector genomes was quantified by qPCR using a vector reference standard curve. The dotted line represents the detection threshold for positive vector genome presence (at 100 vector genomes/μg gDNA). Efficient G-case enzymatic activity was measured in the cerebral cortex (FIG. 52B) and shown in units per mg of total protein, with one unit defined as the activity of 1 ng/ml recombinant purified G-case. Average values are shown. Error bars represent SEM. n=10, 10, 10, 10, 7, 8 per group, respectively.*** P < 0.001 using ANOVA followed by Tukey's HSD multiple comparison test.

Fig. 53 A, fig. 53B shows representative data for glycolipid analysis in a dose-ranging study of PR001A in the 4L/PS-NA genetic mouse model. In the cerebellum, levels of GluSph (FIG. 53A) and GluCer (FIG. 53B) were measured. Levels are given in pmol per nmol of phosphate. Average values are shown. Error bars represent SEM. n=10, 10, 10, 10, 7, 8 per group, respectively.*** P < 0.001 using ANOVA followed by Tukey's HSD multiple comparison test. (#): P<0.1; #: P < 0.05 using multiple linear regression for genotype and dose for all animals.

Fig. 54A, fig. 54B shows representative data regarding the biochemical assessment of α-synuclein protein levels in transgenic mice treated with SBE. Hippocampal brain lysates were analyzed for α-synuclein concentration using the Simple Western™ automated capillary Western blotting system (Jess) and using the α-synuclein antibody MJFR-146-4-2. Multiple bands between 48 kDa and 230 kDa were observed and were grouped as high molecular weight (HMW). One band was present at 18 kDa, which corresponded to the predicted molecular mass of the α-synuclein monomer. The mean fold change compared to the normalized mean for the A53T+excipient group is presented. Error bars represent SEM. n=3-5 per group. *: P < 0.05, using ANOVA followed by Tukey's HSD multiple comparison test.

Fig. 55 is a schematic representation of one embodiment of a plasmid encoding a recombinant adeno-associated virus vector (PR001A) containing

- 10 046909 expressive construct encoding human G-case, p.o. means base pairs, kan means the gene that confers resistance to kanamycin. ORF1 stands for open reading frame for G-case. ITR refers to the inverted terminal repeat sequence of an adeno-associated virus. TRY refers to a sequence containing three transcriptional regulatory sites: TATA, RBS, and YY1. SVAr refers to the chicken β-actin promoter. CMVe refers to the cytomegalovirus enhancer. WPRE refers to the post-transcriptional regulatory element of woodchuck hepatitis virus. bGH refers to the bovine growth hormone polyA signal tail, int refers to the intron. The nucleotide sequences of the two chains of PR001A are shown in SEQ ID NO: 39 and 40.

Fig. 56 is a schematic representation of one embodiment of a plasmid encoding a recombinant adeno-associated virus vector (PR004X) containing an expression construct encoding human G-case and shRNA targeting α-synuclein. By. means base pairs, kan means the gene that confers resistance to kanamycin. aSynMshRNA refers to the region encoding shRNA that inhibits α-synuclein. GBACDSopt stands for G-case open reading frame. ITR refers to the inverted terminal repeat sequence of an adeno-associated virus. TRY refers to a sequence containing three transcriptional regulatory sites: TATA, RBS, and YY1. SVAr refers to the chicken β-actin promoter. CMVe refers to the cytomegalovirus enhancer. WPRE refers to the post-transcriptional regulatory element of woodchuck hepatitis virus. bGH refers to the bovine growth hormone polyA signal tail, int refers to the intron. The nucleotide sequences (sequence verified) of the two chains of PR004X are provided in SEQ ID NOs: 41 and 42.

Fig. 57 is a schematic representation of one embodiment of a plasmid encoding a recombinant adeno-associated virus vector (PR004Y) containing an expression construct encoding human G-case and shRNA targeting α-synuclein. By. means base pairs, kan means the gene that confers resistance to kanamycin. shSNCA refers to the region encoding shRNA that inhibits α-synuclein. GBACDSopt stands for G-case open reading frame. ITR refers to the inverted terminal repeat sequence of an adeno-associated virus. TRY refers to a sequence containing three transcriptional regulatory sites: TATA, RBS, and YY1. SVAr refers to the chicken β-actin promoter. CMVe refers to the cytomegalovirus enhancer. WPRE refers to the post-transcriptional regulatory element of woodchuck hepatitis virus. bGH refers to the bovine growth hormone polyA signal tail, int refers to the intron. The nucleotide sequences (theoretical) of the two chains of PR004Y are provided in SEQ ID NO: 43 and 44.

Fig. 58 is a schematic representation of one embodiment of a plasmid encoding a recombinant adeno-associated virus vector (PR014X) containing an expression construct encoding shRNA targeting α-synuclein. By. means base pairs, kan means the gene that confers resistance to kanamycin. aSyn_MshRNA refers to the shRNA coding region that inhibits α-synuclein. ITR refers to the inverted terminal repeat sequence of an adeno-associated virus. TRY refers to a sequence containing three transcriptional regulatory sites: TATA, RBS, and YY1. SVAr refers to the chicken β-actin promoter. CMVe refers to the cytomegalovirus enhancer. WPRE refers to the post-transcriptional regulatory element of woodchuck hepatitis virus. bGH refers to the bovine growth hormone polyA signal tail, int refers to the intron. The nucleotide sequences (theoretical) of the two strands of PR014X are presented in SEQ ID NOs: 45 and 46. The nucleotide sequences (theoretical) of the two strands of the shRNA coding region are presented in SEQ ID NOs: 47 and 48.

Fig. 59 is a schematic representation of one dose-ranging study design of PR001 rAAV in the D409V Not genetic mouse model. PR001 was administered via intravenous (IV) injection to D409V Not mice. The parameters shown in the figure were assessed after 5 weeks.

Fig. 60A-Fig. 60C demonstrate representative data on liver biochemistry following intravenous (IV) administration of PR001 in the D409V Not genetic mouse model. Mice were sacrificed 5 weeks after i.v. injection. Cytokine levels (FIG. 60A) and glycolipid levels (FIG. 60B; FIG. 60C) were quantified. Statistics were determined by ANOVA followed by Dunnett's test for comparison with the D409V Hom+excipient group. Mean values are presented +/- SEM (n=8-10/group). ****: p 0.0001; ***: p<0.001; **: p<0.01; *: p<0.05; ⁽ *) p=0.10. GluCer=glucosylceramide. GluSph=glucosylsphingosine. \UT=wild type.

Fig. 61A, fig. 61B shows representative data regarding brain biochemistry upon intravenous (IV) administration of PR001 in the D409V Hom genetic mouse model. Mice were sacrificed 5 weeks after i.v. injection. Glycolipid levels were quantified. Statistics were determined using ANOVA followed by Dunnett's test for comparison with

- 11 046909 group D409V Hom+excipient. Mean values are presented +/- SEM (n=8-10/group). ****: p<0.0001; *: p<0.05. GluCer=glucosylceramide. GluSph=glucosylsphingosine. WT^^Uk™ type.

Fig. 62 shows representative data on lung biochemistry following intravenous (IV) administration of PR001 in the D409V Hom genetic mouse model. Mice were sacrificed 5 weeks after i.v. injection. Cytokine levels were quantified. Statistics were determined by ANOVA followed by Dunnett's test for comparison with the D409V Hom+excipient group. Mean values are presented +/- SEM (n=8-10/group). *: p<0.05. WT=wild type.

Fig. 63 is a schematic representation of one dose range study design of PR001 rAAV in the 4L/PS-NA genetic mouse model. PR001 was administered via intravenous (IV) injection into 4L/PS-NA mice. The parameters shown in the figure were evaluated at the indicated time points.

Fig. 64A, fig. 64B shows representative data regarding liver biochemistry upon intravenous (IV) administration of PR001 in the 4L/PS-NA genetic mouse model. Mice were sacrificed 15 weeks after i.v. injection. Glycolipid levels were quantified. Statistics were determined by ANOVA followed by Dunnett's test for comparison with the 4L/PSNA+excipient group. Mean values are presented +/- SEM (n=10/group). ****: p<0.0001; ***: p<0.001; *: p<0.05. GluCer= glucosylceramide. GluSph=glucosylsphingosine.

Fig. 65A, fig. 65B shows representative data regarding brain biochemistry upon intravenous (IV) administration of PR001 in the 4L/PS-NA genetic mouse model. Mice were sacrificed 15 weeks after i.v. injection. Glycolipid levels were quantified. Statistics were determined by ANOVA followed by Dunnett's test for comparison with the 4L/PS-NA+adjuvant group. Mean values are presented +/- SEM (n=10/group). ****: p<0.0001; **: p<0.01; *: p<0.05. GluCer= glucosylceramide. GluSph=glucosylsphingosine.

Fig. 66A, fig. 66B shows representative data regarding α-synuclein protein levels and G-case activity in HeLa cells after transduction with PR004 or PR014. HeLa cells were treated with PR004, PR014, or excipient, and levels of α-synuclein (FIG. 66A) and G-case activity (FIG. 66B) in cell lysates were measured after 72 hours. Data are presented as mean ± SEM (n=3/arb).

Fig. 67A-Fig. 67C demonstrates representative data regarding the efficacy of PR004 in neuronal cultures from induced pluripotent stem cells (iPSCs) from a patient with Parkinson's disease (iPSC). Induced pluripotent stem cells obtained from a Parkinson's disease patient with SNCA triplication were differentiated into neurons (Fig. 67A). iPSC-derived neurons were treated with PR004 or excipient, and G-case activity (Figure 67B) and α-synuclein levels (Figure 67C) in cell lysates were measured two weeks later. Statistics were determined using an unpaired t test; *=p<0.05, **=p<0.01. Data are presented as mean ± SEM (n = 2-3/group).

Fig. 68A, fig. 68B shows representative data from studies evaluating shRNA targeting of SNCA from the PR004 vector in HEK293 cells by qRT-PCR. HEK293 cells were transfected with PR004 or control, and RNA was extracted after 72 h. qRT-PCR for various genes was performed and normalized to GAPDH expression. Data were normalized to control conditions and presented as mean ± SEM (u=3/group).

Fig. 69 is a schematic representation of one study design examining gastrointestinal, motor, and biochemical endpoints in the SNCA-A53T mouse model of ASD following administration of PR004. ICV=intracerebroventricular.

Fig. 70 is a schematic representation of one study design examining motor and biochemical endpoints in the AAV2-SNCA-A53T mouse model following IP administration of PR004. IPa = intraparenchymal. Sn=substantia nigra. ICV=inracerebroventricular.

Fig. 71A, fig. 71B shows representative data regarding the assessment of motor phenotypes following PR004 administration in the AAV2-SVG4-A53T mouse model. Mice at 10 weeks of age were administered (1) AAV-Null or AAV-SNCA-A53T via IPa injection into the SN and (2) excipient or PR004 via ICV injection. Kinematic analysis of fine motor skills (MotoRater) was performed 4 weeks (Fig. 71A) and 9 weeks (Fig. 71B) after treatment. Statistics were determined by ANOVA followed by Dunnett's multiple comparison test; *=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 Invention

The present invention is based in part on compositions and methods for expressing combinations of certain gene products (eg, gene products associated with a CNS disease) in a subject. The gene product may be a protein, a fragment (e.g., part) of a protein, an interfacial

- 12 046909 rying nucleic acid, which inhibits a gene associated with a disease of the central nervous system, etc. In some embodiments, the gene product is a protein or protein fragment encoded by a gene associated with a CNS disease. In some embodiments, the gene product is an interfering nucleic acid (eg, shRNA, siRNA, miRNA, amiRNA, etc.) that inhibits a gene associated with a CNS disease.

A CNS disease-associated gene refers to a gene encoding a gene product that is genetically, biochemically, or functionally associated with a CNS disease, such as Parkinson's disease (PD), Gaucher disease (GD), or synucleinopathy. For example, individuals who have mutations in the GBA1 gene (which encodes the Gcase protein) have an increased risk of developing PD compared to individuals who do not have a mutation in GBA1. In another example, PD is associated with the accumulation of protein aggregates containing the protein α-synuclein (α-Syn); Accordingly, SNCA (which encodes α-Syn) is a gene associated with CNS disease. In some embodiments, an expression cassette described herein encodes a wild-type or non-mutant form of a gene associated with a CNS disease (or a coding sequence thereof). Examples of genes associated with CNS disease are listed in Table. 1.

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Table 1

Examples of genes associated with CNS disease

Name Gene Function NCBI Accession Number Lysosomal protein membranes 2 SCARB2ILIMP2 lysosomal receptor glucosylceramidase (targeting GBA) NP 005497.1 (Isoform 1), NP001191184.1 (Isoform 2) Prosaposin PSAP precursor of saposins A, B, C and D, which are localized in the lysosomal compartment and promote the catabolism of glycosphingolipids with short oligosaccharide groups AAN01503.1, AAN07612.1, AAN04275.1, AAA60303.1 beta-glucocerebrosidase GBA1 breaks down beta glucoside linkage of glucocerebroside ΝΡ 001005742.1 (Isoform 1), ΝΡ 001165282.1 (Isoform 2), ΝΡ 001165283.1 (Isoform 3) alpha-synuclein SNCA plays a role in maintaining supplies synaptic vesicles in presynaptic endings due to clustering synaptic vesicles and may contribute regulation of dopamine release NP001139527.1

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

- 14 046909

Accordingly, some aspects of the present invention provide an isolated nucleic acid comprising an expression construct encoding G-case (eg, the gene product of the GBA1 gene). In some embodiments, the isolated nucleic acid comprises a G-case coding sequence that has been codon-optimized (eg, codon-optimized for expression in mammalian cells, eg, human cells). In some embodiments, the nucleic acid sequence encoding a G-case encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 14 (eg, as set forth in NCBI reference sequence NP000148.2). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 15. In some embodiments, the expression construct comprises adeno-associated virus (AAV) inverted terminal repeats (ITRs), e.g., AAV ITRs flanking a nucleic acid sequence encoding G- kazoo.

In some aspects of the present invention, an isolated nucleic acid is provided comprising an expression construct encoding prosaposin (eg, the gene product of the PSAP gene). In some embodiments, the isolated nucleic acid comprises a prosaposin coding sequence that has been codon optimized (eg, codon optimized for expression in mammalian cells, such as human cells). In some embodiments, the nucleic acid sequence encoding prosaposin encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 16 (eg, as set forth in NCBI reference sequence NP002769.1). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 17. In some embodiments, the expression construct comprises adeno-associated virus (AAV) inverted terminal repeats (ITRs), e.g., AAV ITRs flanking a nucleic acid sequence encoding prosaposin.

In some aspects of the present invention, an isolated nucleic acid is provided comprising an expression construct encoding LIMP2/SCARB2 (eg, the gene product of the SCARB2 gene). In some embodiments, the isolated nucleic acid comprises a SCARB2 coding sequence that has been codon optimized (eg, codon optimized for expression in mammalian cells, such as human cells). 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 (eg, as set forth in NCBI reference sequence NP005497.1). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 29. In some embodiments, the expression construct comprises adeno-associated virus (AAV) inverted terminal repeats (ITRs), e.g., AAV ITRs, flanking a nucleic acid sequence encoding SCARB2.

In some aspects of the present invention, there is provided an isolated nucleic acid comprising an expression construct encoding a first gene product and a second gene product, each gene product being independently selected from the gene products or portions thereof set forth in Table. 1.

In some embodiments, the first gene product or the second gene product is a G-case protein or a portion thereof. In some embodiments, the first gene product or the second gene product is a LIMP2 protein 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 a LIMP2 protein or a portion thereof, or prosaposin or a portion thereof. In some embodiments, the expression construct encodes (eg, alone or in addition to another gene product) an interfering nucleic acid (eg, shRNA, miRNA, dsRNA, etc.). In some embodiments, the interfering nucleic acid inhibits the expression of α-synuclein (α-synuclein). In some embodiments, the α-synuclein-targeting interfering nucleic acid comprises the sequence set forth in any of SEQ ID NOs: 20-25. In some embodiments, the α-synuclein-targeting interfering nucleic acid comprises the sequence set forth in SEQ ID NO: 20. In some embodiments, the α-synuclein-targeting interfering nucleic acid binds to (e.g., hybridizes to) a sequence set forth in any from SEQ ID NO: 20-25. In some embodiments, the interfering nucleic acid targeting α-synuclein binds to (e.g., hybridizes to) the sequence set forth in SEQ ID NO: 20.

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 II promoter

- 15 046909 pol III (for example, U6, etc.).

In some embodiments, the expression construct further comprises an internal ribosome entry site (IRES). In some embodiments, the IRES is located between the first gene product and the second gene product.

In some embodiments, the expression construct further comprises a coding sequence for a self-cleaving peptide. In some embodiments, the self-cleaving peptide is a T2A peptide.

In some embodiments, the expression construct comprises two adeno-associated virus (AAV) inverted terminal repeat (ITR) sequences. In some embodiments, ITR sequences flank the first gene product and the second gene product (e.g., arranged as follows from 5' end to 3' end: ITR-first gene product-second gene product-ITR) In some embodiments, one of the sequences The ITR of the isolated nucleic acid does not have a functional terminal resolution site (trs). For example, in some embodiments, one of the ITRs is an AITR.

In some aspects, the present invention provides rAAV vectors containing an ITR having a modified D region (eg, a D sequence that is modified relative to the wild-type AAV2 ITR, SEQ ID NO: 29). In some embodiments, the ITR having the modified D region is the 5' ITR of the rAAV vector. In some embodiments, the modified D region comprises an S sequence, for example, as set forth in SEQ ID NO: 26. In some embodiments, the ITR having the modified D region is the 3' ITR of the rAAV vector. In some embodiments, the modified D region includes a 3'ITR, in which the D region is located at the 3' end of the ITR (eg, at the outer or terminal end of the ITR relative to the transgene vector insert). In some embodiments, the modified D region comprises the sequence set forth in SEQ ID NO: 26 or 27. In some embodiments, the isolated nucleic acid (eg, rAAV vector) contains a TRY region. In some embodiments, the TRY region comprises the sequence set forth in SEQ ID NO: 28.

In some embodiments, the isolated nucleic acid described herein comprises the sequence set forth in any of SEQ ID NOs: 1 to 13, 15, 17, 19, and 32-48. In some embodiments, the isolated nucleic acid described herein encodes a peptide containing or consisting of the sequence identified in any of SEQ ID NOs: 14, 16, and 18.

In some aspects, the present invention provides a vector containing isolated nucleic acid, as described herein. In some embodiments, the vector is a plasmid or viral vector. In some embodiments, the viral vector is a recombinant AAV vector (rAAV). In some embodiments, the rAAV vector is single-stranded (eg, single-stranded DNA).

In some aspects, the present invention provides a host cell containing an isolated nucleic acid, as described herein, or a vector, as described herein. In some aspects, the present invention provides a recombinant adeno-associated virus (rAAV) comprising a capsid protein and an isolated nucleic acid or vector as described herein.

In some embodiments, said capsid protein is capable of crossing the blood-brain barrier, for example, AAV9 capsid protein or AAVrh.10 capsid protein. In some embodiments, rAAV transduces neuronal cells and non-neuronal cells of the central nervous system (CNS).

In some aspects, the present invention provides a method of treating a subject with an existing or suspected central nervous system (CNS) disease, the method comprising administering to the subject (eg, a composition containing an isolated nucleic acid or vector, or rAAV) as described herein. 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 the synucleinopathy listed in table. 5. In some embodiments, the CNS disease is a tauopathy, such as the tauopathy listed in table. 6. In some embodiments, the CNS disease is a lysosomal storage disease, such as the lysosomal storage disease specified in table. 7. In some embodiments, the lysosomal storage disease is a neuronopathic Gaucher disease, such as Gaucher disease type 1, Gaucher disease type 2, or Gaucher disease type 3. In some aspects, the present invention provides a method of treating a subject with known or suspected Parkinson's disease, the method comprising administering to the subject a composition (eg, a composition containing an isolated nucleic acid or vector, or rAAV) as described herein.

- 16 046909

In some embodiments, a method is provided for treating a subject suffering from 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 G-case protein coding sequence , wherein the sequence encoding the G-case protein contains SEQ ID NO: 15; and wherein rAAV contains a capsid protein having the serotype AAV9. In some embodiments, rAAV is administered to a subject suffering from type 2 Gaucher disease or type 3 Gaucher disease at a dose of about 1.3 x 1011 vector genomes (vg)/p brain.

In some embodiments, a method is provided for treating a subject suffering from Parkinson's disease with 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 G protein coding sequence -kazoo, wherein the sequence encoding the G-kazoo protein contains SEQ ID NO: 15; and wherein rAAV contains a capsid protein having the serotype AAV9. In some embodiments, rAAV is administered to a subject suffering from Parkinson's disease at a dose of about 1x10 ¹⁴ vector genomes (vg) or about 2x10 ¹⁴ vg. In some embodiments, the rAAV is administered by suboccipital injection into the cisterna magna.

In some embodiments, the composition comprises a nucleic acid (e.g., an rAAV genome, e.g., encapsidated by AAV capsid proteins) that encodes two or more gene products (e.g., CNS disease-associated gene products), e.g., 2, 3, 4, 5 or more gene products described in this document. In some embodiments, the composition contains two or more (e.g., 2, 3, 4, 5 or more) different nucleic acids (e.g., two or more rAAV genomes, e.g., separately encapsidated by AAV capsid proteins), each of which encodes one or more different gene products. In some embodiments, a subject is administered two or more different compositions, each containing one or more nucleic acids encoding different gene products. In some embodiments, different gene products are operably linked to the same type of promoter (eg, the same promoter). In some embodiments, different gene products are operably linked to different promoters.

Isolated nucleic acids and vectors

The isolated nucleic acid may be DNA or RNA. In some aspects of the present invention, an isolated nucleic acid is provided that contains an expression construct encoding a G-case (eg, the gene product of the GBA1 gene), or a portion thereof. G-case, also called β-glucocerebrosidase or GBA, refers to a lysosomal protein that cleaves the beta-glucosidic bond of the chemical glucocerebroside, an intermediate in glycolipid metabolism. Deficiency of G-case, a key lysosomal enzyme required for normal glycolipid metabolism, results in the accumulation of the glycolipid substrates of G-case: glucosylceramide (GluCer) and glucosylsphingosine (GluSph). In humans, G-case is encoded by the GBA1 gene located on chromosome 1. In some embodiments, GBA1 encodes a peptide that is represented by the NCBI reference sequence NP_000148.2 (SEQ ID NO: 14). In some embodiments, the isolated nucleic acid comprises a G-case coding sequence that has been codon optimized (e.g., codon optimized for expression in mammalian cells, e.g., human cells), such as the sequence set forth in SEQ ID NO: 15 .

In some aspects of the present invention, an isolated nucleic acid is provided comprising an expression construct encoding prosaposin (eg, the gene product of the PSAP gene). Prosaposin is a precursor glycoprotein for sphingolipid activator proteins (saposins) A, B, C and D, which promote the catabolism of glycosphingolipids with short oligosaccharide groups. In humans, the PSAP gene is located on chromosome 10. In some embodiments, PSAP encodes a peptide that is represented by the NCBI reference sequence NP002769.1 (eg, SEQ ID NO: 16). In some embodiments, the isolated nucleic acid comprises a prosaposin coding sequence that has been codon optimized (e.g., codon optimized for expression in mammalian cells, e.g., human cells), such as the sequence set forth in SEQ ID NO: 17.

Aspects of the present invention relate to an isolated nucleic acid containing an expression construct encoding LIMP2/SCARB2 (eg, the gene product of the SCARB2 gene). SCARB2 is a membrane protein that regulates lysosomal and endosomal transport within cells. In humans, the SCARB2 gene is located on chromosome 4. In some embodiments, the SCARB2 gene encodes a peptide that is represented by the NCBI reference sequence NP005497.1 (SEQ ID NO: 18). In some embodiments, the isolated nucleic acid contains the sequence set forth in SEQ ID NO: 19. In some embodiments, you

- 17 046909 the divided nucleic acid contains a SCARB2 coding sequence that has been codon optimized.

In some aspects of the present invention, there is provided an isolated nucleic acid comprising an expression construct encoding a first gene product and a second gene product, each gene product being independently selected from the gene products or portions thereof set forth in Table. 1.

In some embodiments, an isolated nucleic acid or vector (eg, an rAAV vector) described herein contains or consists of a sequence set forth in any of SEQ ID NOs: 1-48. In some embodiments, the isolated nucleic acid or vector (eg, rAAV vector) described herein contains or consists of a sequence that is complementary to (eg, complementary to) the sequence set forth in any of SEQ ID NO: 1-48. In some embodiments, the isolated nucleic acid or vector (eg, rAAV vector) described herein contains or consists of a sequence that is the reverse complement of the sequence specified in any of SEQ ID NO: 1-48. In some embodiments, the isolated nucleic acid or vector (eg, rAAV vector) described herein contains or consists of a portion of the sequence identified in any of SEQ ID NO: 1-48. The portion may contain at least 25%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the sequence specified in any of SEQ ID NO: 1-48. In some embodiments, the nucleic acid sequence described herein is a sense nucleic acid strand (eg, the 5' to 3' strand) or, in the context of viral sequences, a plus (+) strand. In some embodiments, the nucleic acid sequence described herein is an antisense nucleic acid strand (eg, a 3' to 5' strand) or, in the context of viral sequences, a minus (-) strand.

In some embodiments, the gene product is encoded by a coding portion (eg, cDNA) of a naturally occurring gene. In some embodiments, the first gene product is a protein (or fragment thereof) encoded by the GBAIm gene. In some embodiments, the gene product is a protein (or fragment thereof) encoded by the SCARB2/LIMP2 gene or the PSAP gene. However, one skilled in the art will appreciate that the order of expression of the first gene product (eg, G-case) and the second gene product (eg, LIMP2) may typically be reversed (eg, LIMP2 is the first gene product and G-case is the second gene product). In some embodiments, the gene product is a fragment (eg, part) of a gene listed in Table. 1. The protein fragment may contain about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the protein encoded by the genes listed in table. 1. In some embodiments, the protein fragment contains from 50% to 99.9% (eg, any value from 50% to 99.9%) of the protein encoded by the gene specified in table. 1.

In some embodiments, the expression construct is monocistronic (eg, the expression construct encodes a single fusion protein comprising a first gene product and a second gene product). In some embodiments, the expression construct is polycistronic (eg, the expression construct encodes two different gene products, such as two different proteins or protein fragments).

A polycistronic expression vector may contain one or more (eg, 1, 2, 3, 4, 5, or more) promoters. Any suitable promoter may be used, for example, a constitutive promoter, an inducible promoter, an endogenous promoter, a tissue-specific promoter (eg, 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 in Alexopoulou et al. (2008) BMC Cell Biol. 9:2; doi: 10.1186/1471-2121-9 -2), the CD68 promoter or the JeT promoter (eg, as described in Tornoe et al. (2002) Gene 297(1-2):21-32). In some embodiments, the promoter is operably linked to a nucleic acid sequence encoding a first gene product, a second gene product, or a first 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, intronic splice sites, poly(A) attachment sites, enhancer sequences, repressor binding sites, or any combination of the foregoing.

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 regions are described, for example, in Mokrejs et al. (2006) Nucleic Acids Res. 34(Database issue):D125-30. In some embodiments, the nucleic acid sequence encoding the first gene product and the nucleic acid sequence encoding the second gene product are separated by the nucleic acid sequence encoding

- 18 046909 cabbage soup self-cleaving peptide. Examples of self-cleaving peptides include, but are not limited to, T2A, P2A, E2A, F2A, BmCPV 2A and BmIFV 2A, as well as those described in Liu et al. (2017) Sci Rep. 7: 2193. In some embodiments, the self-cleaving peptide is a T2A peptide.

Pathogenetically, disorders such as Parkinson's disease and Gaucher disease are associated with the accumulation of protein aggregates consisting mainly of the protein α-synuclein (α-Syn). Accordingly, in some embodiments, the isolated nucleic acids described herein contain an inhibitory nucleic acid that reduces or prevents the expression of a-Syn protein. The sequence encoding the inhibitory nucleic acid may be placed in the untranslated region (eg, intron, 5UTR, 3'UTR, etc.) of the expression vector. In some embodiments, the inhibitory nucleic acid is located in an intron of the expression construct, for example, in an intron upstream of the sequence encoding the first gene product. The inhibitory nucleic acid may be double-stranded RNA (dsRNA), siRNA, shRNA, microRNA (miRNA), artificial miRNA (amiRNA), or an RNA aptamer. Typically, the inhibitory nucleic acid binds to (eg, hybridizes to) about 6 to about 30 (eg, any integer from 6 to 30, inclusive) contiguous nucleotides of the target RNA (eg, mRNA). In some embodiments, the inhibitory nucleic acid molecule is a miRNA or amiRNA, for example, a miRNA targeting SNCA (the gene encoding the α-Syn protein). In some embodiments, the miRNA does not contain any mismatches with the SNCA region of the mRNA to which it hybridizes (eg, the miRNA is an improved miRNA). In some embodiments, the inhibitory nucleic acid is a shRNA (eg, an SNCA-targeting shRNA). In some embodiments, the shRNA that targets SNCA is encoded by SEQ ID NO: 47. In some embodiments, the shRNA that targets SNCA is encoded by the sequence comprising SEQ ID NO: 20.

One skilled in the art will appreciate that when it comes to nucleic acid sequences containing or encoding inhibitory nucleic acids (e.g., dsRNA, siRNA, shRNA, miRNA, amiRNA, etc.), any one or more thymidine (T) nucleotides or The uridine (U) nucleotides in the sequence presented herein can be replaced by any other nucleotide suitable for base pairing (eg, by Watson-Crick base pairing) with the adenosine nucleotide. For example, T can be replaced by U, and U can be replaced by T.

The isolated nucleic acid described herein may exist on its own or as part of a vector. Typically, the vector may be a plasmid, cosmid, phagemid, bacterial artificial chromosome (BAC), or viral vector (eg, adenoviral vector, adeno-associated virus (AAV) vector, retroviral vector, baculovirus vector, etc.). In some embodiments, the vector is a plasmid (eg, a plasmid containing an isolated nucleic acid, as described herein). In some embodiments, said vector is a recombinant AAV vector (rAAV). In some embodiments, the rAAV vector is single-stranded (eg, single-stranded DNA). In some embodiments, the vector is a baculovirus vector (eg, Autographa californica nuclear polyhedrosis virus (AcNPV) vector).

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

Aspects of the present invention relate to isolated nucleic acids (e.g., rAAV vectors) containing an ITR having one or more modifications (e.g., additions, deletions, nucleic acid substitutions, etc.) relative to the wild-type AAV ITR, e.g., relative to the ITR Wild-type AAV2 (eg, SEQ ID NO: 29). The structure of the wild-type AAV2 ITR is shown in Fig. 19. Typically, the wild-type ITR contains a 125 nucleotide region that self-anneals to form a palindromic double-stranded T-hairpin structure, consisting

- 19 046909 cabbage soup from two transverse arms (formed by the sequences designated as B/B' and C/C, respectively), a longer stem region (formed by the sequences A/A') and a single-stranded terminal region called the D region (Fig. 19 ). Typically, the D region of the ITR is located between the stem region formed by the A/A' sequences and the insert containing the rAAV vector transgene (eg, located within the ITR relative to the end of the ITR or proximal to the transgene insert or rAAV vector expression construct). In some embodiments, region D comprises the sequence set forth in SEQ ID NO: 27. Region D has been found to play an important role in the encapsidation of rAAV vectors by capsid proteins, for example, as described in Ling et al. (2015) J Mol Genet Med 9(3).

The present invention is based in part on the unexpected discovery that rAAV vectors containing a D region located outside the ITR (eg, proximal to the end of the ITR relative to the transgene insertion or expression construct) are effectively encapsidated AAV capsid proteins, compared to rAAV vectors having an ITR with unmodified ITR (eg wild type). In some embodiments, rAAV vectors having a modified D sequence (eg, a D sequence in an external position) have reduced toxicity compared to rAAV vectors having wild-type ITR sequences.

In some embodiments, the modified D sequence contains at least one nucleotide substitution relative to the wild type D sequence (eg, SEQ ID NO: 27). The modified D sequence may have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 nucleotide substitutions relative to the wild type D sequence (eg, 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 (eg, SEQ ID NO: 27). In some embodiments, the modified sequence D is from 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 (eg, SEQ ID NO: 27). In some embodiments, the modified sequence D comprises the sequence set forth in SEQ ID NO: 26, also referred to as sequence S, as described in Wang et al. (1995) J Mol Biol 250(5):573–80.

The isolated rAAV nucleic acid or vector as described herein may further contain a TRY sequence, for example, as described 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 an ITR (eg, a 5' ITR) and an expression construct (eg, an insert encoding a transgene) of the isolated rAAV nucleic acid or vector.

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

In some aspects, the present invention provides a host cell containing an isolated nucleic acid or vector, as described herein. The host cell may be a prokaryotic cell or a eukaryotic cell. For example, the host cell may be a mammalian cell, a bacterial cell, a yeast cell, an insect cell, etc. In some embodiments, the host cell is a mammalian cell, such as a cell

NEK293T. In some embodiments, the host cell is a bacterial cell, such as an E. coli cell.

rAAV

In some aspects, the present invention relates to recombinant AAV (rAAV) containing a transgene that encodes a nucleic acid as described herein (eg, an rAAV vector as described herein). The term rAAV generally refers to viral particles containing the rAAV vector encapsidated by one or more AAV capsid proteins. RAAV described herein may contain 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, for example, a rhesus monkey AAV capsid protein such as AAVrh.10, AAVrh.39, etc. In some embodiments, the rAAV described herein comprises a capsid protein that is a wild-type variant capsid protein, such as a variant capsid protein that includes at least 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, or more than 10 (e.g., 15, 20, 25, 50, 100, etc.) amino acid substitutions (e.g., mutations) relative to the wild-type AAV capsid protein from which it is derived. In some embodiments, the variant AAV capsid protein is the AAV1RX capsid protein, for example, as described in Albright et al. Mol Ther. Feb 2018

- 20 046909

7;26(2):510-523. In some embodiments, the variant capsid protein is an AAV TM6 capsid protein, for example, as described in Rosario et al. Mol Ther Methods Clin Dev. 2016; 3: 16026. In some embodiments, the rAAVs described herein spread readily into the CNS, especially when introduced into spaces where CSF circulates or directly into the brain parenchyma. Accordingly, in some embodiments, the rAAVs described herein contain a capsid protein that is capable of crossing the blood-brain barrier (BBB). For example, in some embodiments, rAAV comprises a capsid protein having a serotype of AAV9 or AAVrh. 10. The production of rAAV is described, for example, in the publications of Samulski et al. (1989) J Virol. 63(9):3822-8 and Wright (2009) Hum Gene Ther. 20(7): 698-706. In some embodiments, rAAV contains a capsid protein that specifically or preferentially targets myeloid cells, such as microglial cells.

In some embodiments, the present invention provides an rAAV referred to as PR001. This rAAV expresses the codon-optimized coding sequence of human GBA1 (SEQ ID NO:15). In some embodiments, the present invention provides an rAAV referred to as PR001A. PR001A (AAV9.CBA.GBA1.A) is an rAAV that delivers a functional human GBA1 gene, resulting in increased expression of a functional human G-case gene. The PR001A vector insert contains a chicken β-actin (CBA) promoter element consisting of 4 parts: cytomegalovirus (CMV) enhancer, CBA promoter, exon 1 and intron (int) for continuous expression of the codon-optimized human GBA1 coding sequence (SEQ ID NO: 15). The 3' region also contains the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) followed by the bovine growth hormone polyadenylation signal tail. Three well-characterized regulatory transcriptional activation sites are included at the 5' end of the promoter region: TATA, RBS and YY1 (see, for example, Francois et al., (2005) J. Virol. 79(17): 11082-11094).

Flanking inverted terminal repeats (ITRs) allow insertion sequences to be packaged correctly. Two variants of the 5' ITR sequence are shown (Fig. 7, insert, bottom sequence); these variants have several nucleotide differences within the 20 nucleotide D region of the ITR, which are thought to affect packaging and expression efficiency. PR001A contains the D domain nucleotide sequence shown in FIG. 7 (insert, top sequence; SEQ ID NO:30). In some embodiments, the present invention provides a variant vector called PROOIB that carries a mutant D domain (referred to herein as the S domain, with nucleotide changes shown by shading in SEQ ID NO: 31 in FIG. 7). Except for a different 5'ITR sequence, PR001B is identical to PROOIA. The framework contains a gene that confers resistance to kanamycin, as well as a spacer sequence that prevents refolding. A schematic representation of the plasmid encoding the rAAV vector is shown in FIG. 55. SEQ ID NO: 39 is the first strand nucleotide sequence (in order 5' to 3') of the plasmid encoding the PR001A vector shown in FIG. 55. SEQ ID NO: 40 is the second strand nucleotide sequence (in order 5' to 3') of the plasmid encoding the PR001A vector shown in FIG. 55. PR001A contains AAV9 capsid proteins.

In some embodiments, the present invention provides an rAAV referred to as PR004. This rAAV expresses a codon-optimized coding sequence of human GBA1 (SEQ ID NO: 15) and an inhibitory nucleic acid coding sequence that targets the reduction of α-synuclein levels and includes the nucleotide sequence of SEQ ID NO: 20. In some embodiments, the present invention provides rAAV, called PR004X. In some embodiments, the present invention provides an rAAV referred to as PR004Y. PR004X and PR004Y are each an rAAV that (i) delivers a functional human GBA1 gene, resulting in increased expression of functional human G-case, and (ii) encodes a shRNA that reduces α-synuclein levels via RNA interference. The PR004 vector insert contains a chicken β-actin (CBA) promoter element consisting of 4 parts: cytomegalovirus (CMV) enhancer, CBA promoter, exon 1 and intron (int) for continuous expression of the codon-optimized human GBA1 coding sequence (SEQ ID NO: 15), as well as a sequence encoding an inhibitory nucleic acid containing the nucleotide sequence of SEQ ID NO: 20. The 3' region also contains a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) followed by a bovine growth hormone polyadenylation signal tail. The 5' end of the promoter region includes three well-characterized transcriptional activation regulatory sites: TATA, RBS and YY1 (see, for example, Francois et al., (2005) J. Virol. 79(17): 11082-11094). Flanking inverted terminal repeats (ITRs) allow insertion sequences to be packaged correctly. The framework contains a gene that confers resistance to kanamycin, as well as a spacer sequence that prevents refolding. Schematic representation of the plasmid encoding the rAAV PR004X vector,

- 21 046909 denominated in Fig. 56. SEQ ID NO: 41 is the first strand nucleotide sequence (in order 5' to 3') of the plasmid encoding the PR004X vector shown in FIG. 56. SEQ ID NO: 42 is the second strand nucleotide sequence (in order 5' to 3') of the plasmid encoding the PR004X vector shown in FIG. 56. A schematic representation of the plasmid encoding the rAAV PR004Y vector is shown in FIG. 57. SEQ ID NO: 43 is the first strand nucleotide sequence (in order 5' to 3') of the plasmid encoding the PR004Y vector shown in FIG. 57. SEQ ID NO: 44 is the second strand nucleotide sequence (in order 5' to 3') of the plasmid encoding the PR004Y vector shown in FIG. 57. PR004X and PR004Y each contain AAV9 capsid proteins. The PR004X and PR004Y vectors are designed to reduce the accumulation of all forms of α-synuclein, including aggregated and extracellular forms.

In some embodiments, the present invention provides an rAAV referred to as PR014. This rAAV expresses a sequence encoding an inhibitory nucleic acid that is targeted to reduce the level of α-synuclein and contains the nucleotide sequence SEQ ID NO: 20. In some embodiments, the present invention provides an rAAV referred to as PR014X. PR014X is an rAAV that encodes a shRNA that reduces α-synuclein levels through RNA interference. The PR014X vector insert contains a chicken β-actin (CBA) promoter element consisting of 4 parts: cytomegalovirus (CMV) enhancer, CBA promoter, exon 1 and intron (int) for constant expression of an inhibitory nucleic acid coding sequence that contains the nucleotide sequence SEQ ID NO: 20. The 3' region also contains a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) followed by a GH polyadenylation signal tail. The 5' end of the promoter region includes three well-characterized regulatory transcriptional activation sites: TATA, RBS and YY1 (see, for example, Francois et al., (2005) J. Virol. 79(17): 11082-11094). Flanking inverted terminal repeats (ITRs) allow insertion sequences to be packaged correctly. The framework contains a gene that confers resistance to kanamycin, as well as a spacer sequence that prevents refolding. A schematic representation of the plasmid encoding the rAAV vector is shown in FIG. 58. SEQ ID NO: 45 is the first strand nucleotide sequence (in order 5' to 3') of the plasmid encoding the PR014X vector shown in FIG. 60. SEQ ID NO: 46 is the second strand nucleotide sequence (in order 5' to 3') of the plasmid encoding the PR014X vector shown in FIG. 60. SEQ ID NO: 47 is the nucleotide sequence of the first strand (in order 5' to 3') of shRNA in the plasmid encoding the PR014X vector shown in FIG. 58. SEQ ID NO: 48 is the nucleotide sequence of the second strand (in order 5' to 3') of shRNA in the plasmid encoding the PR014X vector shown in FIG. 58. PR014X contains AAV9 capsid proteins. The PR014X vector is designed to reduce the accumulation of all forms of α-synuclein, including aggregated and extracellular forms. In some embodiments, rAAV as described herein (eg, comprising a recombinant rAAV genome encapsidated by AAV capsid proteins to form an rAAV capsid particle) is produced in a baculovirus vector expression system (BEVS). The production of rAAV using BEVS is described, for example, in the publications of Urabe et al. (2002) Hum Gene Ther 13(16):1935–43, Smith et al. (2009) Mo/ Ther 17(11):1888-1896, US Patent No. 8945918, US Patent 9879282 and PCT International Publication WO 2017/184879. However, rAAV can be produced using any suitable method (eg using recombinant rep and cap genes). In some embodiments, the rAAV described herein is produced in HEK293 (human embryonic kidney) cells.

Pharmaceutical compositions

In some aspects, the present invention provides pharmaceutical compositions comprising an isolated nucleic acid or rAAV as described herein and a pharmaceutically acceptable carrier. As used herein, the term pharmaceutically acceptable refers to a material, such as a carrier or diluent, that does not interfere with the biological activity or properties of the compound and is relatively non-toxic, e.g., the material can be administered to a subject without causing undesirable biological effects or interacting harmfully. manner with any of the components of the composition in which it is contained.

As used herein, the term pharmaceutically acceptable carrier means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid excipient, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickener, solvent or encapsulating material, involved in carrying or transporting a compound , suitable in the context of the present invention, into the body or to a patient, so that it can perform its intended function. Additional ingredients that may be included in the pharmaceutical compositions used in the practice of the present invention are known in the art and are described, for example, in Remington's Pharma.

- 22 046909 ceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

The compositions (e.g., pharmaceutical compositions) provided herein can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (for example, in the form of powders, ointments, creams and/or drops), mucosal, nasal, buccal, sublingual route; by intratracheal instillation, bronchial installation and/or inhalation; and/or in the form of an oral spray, nasal spray and/or aerosol. Specifically contemplated routes are oral administration, intravenous administration (eg, systemic intravenous injection), regional administration through the blood and/or lymph, 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 (eg, its stability in the gastrointestinal tract environment) and/or the condition of the subject (eg, whether the subject can take the drug orally). In certain embodiments, a compound or pharmaceutical composition described herein is suitable for topical administration to the eye of a subject.

In some embodiments, the present invention provides a PR001 finished drug product (eg, PR001A) containing the PR001 rAAV described above, presented in the form of an aqueous solution. In some embodiments, the final formulation buffer contains about 20 mM Tris, pH 8.0, about 1 mM MgCl ₂ , about 200 mM NaCl, and about 0.001% poloxamer 188 (w/v). In some embodiments, the finished drug product and the final formulation buffer are suitable for intravenous injection (ICM) or intravenous administration.

Methods

Aspects of the present invention relate to the delivery of compositions (eg, isolated nucleic acids, rAAV, etc.) designed to express CNS disease-associated gene products into a cell or cells (eg, a cell or cells of a subject).

As described below in the Examples section, aspects of the present invention relate to compositions expressing gene products that inhibit or prevent glial scarring (eg, gliosis). Accordingly, some aspects of the present invention provide a method of inhibiting glial scarring in a subject, the method comprising administering to the subject a composition (eg, an isolated nucleic acid or rAAV) as described herein.

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 neuronopathic GD (nGD) (eg, GD type 2 or GD type 3). In some embodiments, the subject has GD type 1. In some embodiments, the subject suffering from GD disease does not have PD or does not have symptoms of PD. In some embodiments, the subject has parkinsonism. In some embodiments, the subject has Parkinson's disease (PD). In some embodiments, the subject has an atypical parkinsonian disorder. In some embodiments, the atypical parkinsonian disorder is dementia with Lewy bodies, progressive supranuclear palsy, multiple system atrophy, or corticobasal syndrome.

The present invention is based in part on compositions for expressing one or more CNS disease-associated gene products in a subject for the treatment of CNS-associated diseases. One or more gene products associated with CNS disease may be encoded by one or more isolated rAAV nucleic acids or vectors. In some embodiments, a single vector (eg, isolated nucleic acid, rAAV, etc.) encoding one or more (1.2, 3,4.5, or more) gene products is administered to the subject. In some embodiments, a plurality (eg, 2,3,4,5 or more) of vectors (eg, isolated nucleic acids, rAAV, etc.) are administered to a subject, each vector encoding a different gene product associated with a CNS disease. In some embodiments, the composition expresses GBA or a portion thereof. In some embodiments, the composition expresses interfering RNA that targets alpha-synuclein. In some embodiments, the composition expresses GBA or a portion thereof and interfering RNA targeting alpha-synuclein.

The disease involves the central nervous system and may be a neurodegenerative disease, synucleinopathy, tauopathy, or lysosomal storage disease. Examples of neurodegenerative diseases and their associated genes are listed in Table. 4.

The term synucleinopathy refers to a disease or disorder characterized by the accumulation of alpha-synuclein (SNCA gene product) in a subject (eg, compared to a healthy subject, eg, a subject not suffering from synucleinopathy). Examples of synucleinopathies and their associated genes are listed in Table. 5.

- 23 046909

The term tauopathy refers to a disease or disorder characterized by the accumulation of abnormal tau protein in a subject (eg, compared to a healthy subject not suffering from tauopathy). Examples of tauopathies and genes associated with them are given in table. 6. Lysosomal storage disease refers to a disease characterized by the abnormal accumulation of toxic cellular products in a subject's lysosomes. Examples of lysosomal storage diseases and their associated genes are listed in Table. 7.

As used herein, the terms treat or influence refer to (a) preventing or delaying the onset of a CNS disease; (b) reducing the severity of the CNS disease; (c) reducing or preventing the development of symptoms characteristic of a central nervous system disease; (d) and/or preventing worsening of symptoms characteristic of a CNS disease. Symptoms of CNS disease may include, for example, motor dysfunction (eg, trembling, rigidity, slowness of movement, difficulty walking, paralysis), cognitive dysfunction (eg, dementia, depression, anxiety, psychosis), memory problems, emotional and behavioral dysfunction. The present invention is based in part on compositions for expressing one or more PD-associated gene products in a subject that act together (eg, synergistically) to treat Parkinson's disease.

Accordingly, some aspects of the present invention provide a method of treating a subject with known or suspected Parkinson's disease, the method comprising administering to the subject a composition (eg, a composition containing an isolated nucleic acid or vector, or rAAV) as described herein.

The present invention is based in part on compositions for expressing one or more gene products associated with a CNS disease in a subject for the treatment of Gaucher disease (GD). The diagnosis of GD is made by the presence of biallelic pathogenic mutations in GBA1 or by the detection of less than 15% of normal G-case activity in peripheral blood leukocytes. GBA1 mutations, which cause more severe enzyme deficiencies, are associated with earlier onset of disease, more rapid progression of symptoms, and a higher likelihood of developing neurological symptoms (Svennerholm et al., Clin Genet. 1986;30(2):131-5; Cox, Biologies . 2010;4:299313). GD has traditionally been classified into three broader phenotypes distinguished by the presence of neurological manifestations (neuronopathic [GD type 2 and GD type 3; nGD] or non-neuronopathic [GD type 1]). Among nGDs, differences between GD type 2 and type 3 may represent a phenotypic continuum from acute to chronic manifestations of CNS and visceral symptoms. In infants, GD type 2, known as the acute neuronopathic form, typically presents with early bulbar signs (such as strabismus and/or difficulty swallowing), opisthotonus or spasticity, supranuclear gaze palsy, and inability to achieve motor, behavioral, and cognitive activities. Most children die by age 2 (Goker-Alpan et al., J Pediatr. 2003;143(2):273-6; Roshan and Sidransky, Diseases. 2017;5(1):pii:E10). The characteristic clinical feature of GD type 3 is slow horizontal supranuclear gaze palsy with other neurological manifestations ranging from cognitive impairment to ataxia and seizures to death in childhood or early adolescence (GokerAlpan et al., J Pediatr. 2003;143(2):273- 6; Tylki-Szymanska et al., J Inherit Metab Dis. 2010;33(4):339-46).

Accordingly, some aspects of the present invention provide a method of treating a subject with known or suspected neuronopathic Gaucher disease, the method comprising administering to the subject a composition (eg, a composition comprising an isolated nucleic acid or vector, or rAAV). as described in this document.

In some aspects, the present invention provides a method of treating a subject suffering from Gaucher disease type 2 or Gaucher disease type 3, the method comprising administering to the subject an rAAV containing a nucleic acid containing an expression construct containing a promoter operably linked to a G-protein coding sequence kazoo, wherein the G-kazoo protein coding sequence contains SEQ ID NO: 15; and wherein rAAV contains a capsid protein having the serotype AAV9. In some embodiments, the present invention provides a method of treating a neurological symptom in a subject suffering from Gaucher disease type 2 or Gaucher disease type 3, 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 the G-case protein, wherein the sequence encoding the G-case protein contains SEQ ID NO: 15; and wherein rAAV contains a capsid protein having the serotype AAV9. In some embodiments, the neurological symptom of Gaucher disease type 2 or type 3 is supranuclear gaze palsy, hypotonia, seizures, spasticity, hypokinesia, delayed or impaired motor or behavioral development, delayed or impaired cognitive function, ataxia, intention tremor, or rigidity.

In some embodiments, patients suffering from 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 present invention provides a method of treating peripheral neuropathy in a subject suffering from Ho's disease.

- 24 046909 more (for example, Gaucher disease type 1), wherein the method includes administering to the subject an rAAV containing a nucleic acid containing an expression construct containing a promoter operably linked to a G-case protein coding sequence, wherein the protein coding sequence G-Kazoo, contains SEQ ID NO: 15; and wherein rAAV contains a capsid protein having the serotype AAV9. In some embodiments, the present invention provides a method of treating Gaucher disease type 1 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 G-case protein coding sequence, wherein the sequence , encoding the G-case protein, contains SEQ ID NO: 15; and wherein rAAV contains a capsid protein having the serotype AAV9. In some embodiments, rAAV is administered intravenously to a subject to treat Gaucher disease type 1.

In some embodiments, a method is provided for treating a subject suffering from Parkinson's disease (PD) with a glucocerebrosidase-1 (GBA1) mutation (e.g., a pathogenic GBA1 mutation), the method comprising administering to the subject an rAAV containing a nucleic acid comprising an expression construct, containing a promoter operably linked to a sequence encoding a G-case protein, wherein the sequence encoding a G-case protein comprises SEQ ID NO: 15; and wherein rAAV contains a capsid protein having the serotype AAV9. In some embodiments, a method is provided for treating a symptom in a subject having PD with 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 G-case protein coding sequence, wherein the sequence , encoding the G-case protein, contains SEQ ID NO: 15; and wherein rAAV contains a capsid protein having the serotype AAV9. In some embodiments, the motor symptoms of PD are resting tremor, bradykinesia, rigidity, or difficulty gait. In some embodiments, non-motor symptoms of PD are cognitive impairment/dementia, depression, delusions/hallucinations, psychosis, sleep disturbances, constipation, urinary symptoms, pain, anosmia, difficulty swallowing, or hypotension. In some embodiments, the subject has PD with a single GBA1 mutation. In some embodiments, the subject has PD with two GBA1 mutations.

In some embodiments, an rAAV encoding the G-casoo protein for the treatment of 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 1x 10 ¹² vector genomes (vg) to about 1x 10 ¹⁵ vg, or from about 1x 10 ¹³ vg to about 5x10 ¹⁴ vg, or from about 5x10 ¹³ vg to about 5x10 ¹⁴ vg, or from about 3.4x10 ¹³ vg to about 1x 10 ¹⁴ vg, or from about 1x 10 ¹⁴ vg to about 5x 10 ¹⁴ vg, or from about 1x 10 ¹⁴ vg to about 3x 10 ¹⁴ vg, or from about 1x 10 ¹⁴ vg to about 2x 10 ¹⁴ vg. 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 increased accordingly. In some embodiments, the dose for pediatric subjects may be adjusted based on an age-specific estimate of brain weight, for example, based on a composite data set that includes derived brain weight data from 21 autopsy and neuroimaging publications (Vannucci and Vannucci, Am JPhys Anthropol 2019;168(2):247-61).

In some embodiments, rAAV encoding the G-case protein is administered to a subject for the treatment of Parkinson's disease with a GBA1 mutation (eg, an adult human subject) at a dose of about 1 x 10 ¹⁴ vg, about 2 x 10 ¹⁴ vg, about 3 x 10 ¹⁴ vg, about 4 x 10 ¹⁴ vg, or about 5x10 ¹⁴ vg. In some embodiments, rAAV encoding the G-case protein is administered to a subject for the treatment of Parkinson's disease with a GBA1 mutation (eg, an adult human subject) at a dose of about 1x 10 ¹⁴ vg (about 7.7 x ^{10 10} vg/g brain), about 2x10 ¹⁴ vg (about 1.5x1011 vg/g brain), or about 3x10 ¹⁴ vg (about 1.9x 10 ¹¹ vg/g brain).

In some embodiments, a rAAV encoding the G-kazoo protein is administered to a subject (e.g., a child subject) to treat Gaucher disease type 2 or type 3, at a dose of about 5 x 10 ¹⁰ vg/g brain to about 5 x 10 ¹¹ vg/g brain. In some embodiments, rAAV is administered to a subject (eg, a child subject) for the treatment of Gaucher disease type 2 or Gaucher disease type 3, at a dose of about 1.3 x 10 ¹¹ vg/g brain (from about 5.9 x ^{10 13} vg to about 1.7x10 ¹⁴ vg). In some embodiments, rAAV is administered to a subject (e.g., a child subject) for the treatment of Gaucher disease type 2 or Gaucher disease type 3, at a dose of about 1.9 x 1011 vg/g brain (from about 8.6 x ^{10 13} vg to about 2.5 x 10 ¹⁴ vg). In some embodiments, rAAV is administered to a subject (eg, a child subject) for the treatment of Gaucher disease type 2 or Gaucher disease type 3, at a dose of about 7.7 x ^{10 10} vg/g brain (from about 3.4 x 10 ¹³ vg to about 1 x 10 ¹⁴ vg) or at a dose of about 2.3x1011 vg/g brain (from about 1x 10 ¹⁴ vg to about 3K10 ¹⁴ Vg).

In some embodiments, rAAV encoding the G-case protein for the treatment of Gaucher disease type 1, type 2, or type 3 or Parkinson's disease with a GBA1 mutation is administered to the subject as a single dose, and the subject is subsequently not administered rAAV.

In some embodiments, the rAAV encoding the G-case protein is administered by suboccipito

- 25 046909 tal injection into the cistern magna. In some embodiments, the injection into the cisterna magna is performed under x-ray guidance.

In some embodiments, the present invention provides a method of treating a subject suffering from synucleinopathy or parkinsonism, the method comprising administering to the subject an rAAV containing a nucleic acid containing an expression construct containing a transgene containing (a) a G-case protein coding sequence containing a nucleotide sequence SEQ ID NO: 15; and (b) a sequence encoding an inhibitory nucleic acid comprising the nucleotide sequence of SEQ ID NO: 20; or SEQ ID NO: 47; rAAV contains a capsid protein of the AAV9 serotype.

In some embodiments, the present invention provides a method of treating a subject having multiple system atrophy, Parkinson's disease, Parkinson's disease with a GBA1 mutation, Lewy body disease, dementia with Lewy bodies, dementia with Lewy bodies with a GBA1 mutation, progressive supranuclear palsy, or corticobasal syndrome, wherein said method involves administering to the subject an rAAV containing a nucleic acid containing an expression construct containing a transgene containing (a) a sequence encoding a Gcase protein containing the nucleotide sequence of SEQ ID NO: 15; and (b) a sequence encoding an inhibitory nucleic acid comprising the nucleotide sequence of SEQ ID NO: 20; or SEQ ID NO: 47; rAAV contains a capsid protein of the AAV9 serotype.

In some embodiments, the present invention provides a method of treating a subject suffering from 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 sequence encoding an inhibitory nucleic acid comprising the nucleotide sequence of SEQ ID NO : 20 or SEQ ID NO: 47; rAAV contains a capsid protein of the AAV9 serotype.

In some embodiments, the present invention provides a method of treating a subject having multiple system atrophy, Parkinson's disease, Parkinson's disease with a GBA1 mutation, Lewy body disease, dementia with Lewy bodies, dementia with Lewy bodies with a GBA1 mutation, progressive supranuclear palsy, or corticobasal syndrome, wherein the method comprises administering to the subject an rAAV containing a nucleic acid containing an expression construct containing a transgene containing a sequence encoding an inhibitory nucleic acid containing the nucleotide sequence of SEQ ID NO: 20 or SEQ ID NO: 47; rAAV contains a capsid protein of the AAV9 serotype.

The subject is usually a mammal, preferably a human. In some embodiments, the age of the subject is between 1 month and 10 years (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's age is between 2 years and 20 years. In some embodiments, the subject's age is between 30 years and 100 years. In some embodiments, the subject's age is greater than 55 years.

In some embodiments, the composition is administered directly to the subject's CNS, for example, by direct injection into the subject's brain and/or spinal cord. Examples of methods of direct administration to the CNS 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 a subject by intracranial injection (ICM). In some embodiments, direct injection into the CNS of a subject results in expression of the transgene (e.g., expression of a first gene product, a second gene product, and, if applicable, a third gene product) in the midbrain, striatum, and/or cortex of the subject. In some embodiments, direct injection into the CNS results in expression of the transgene (eg, expression of a first gene product, a second gene product, and, if applicable, a third gene product) in the spinal cord and/or CSF of the subject.

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

In some embodiments, the composition is administered to a subject peripherally, for example, by peripheral injection. Examples of peripheral injection include subcutaneous injection, intravenous injection, intra-arterial injection, intraperitoneal injection, or any

- 26 046909 combination of the above injections. In some embodiments, the peripheral injection is an intra-arterial injection, such as an injection into the carotid artery of a subject.

In some embodiments, a composition (eg, a composition comprising an isolated nucleic acid or vector or rAAV) as described herein is administered either peripherally or directly into the CNS of a subject. For example, in some embodiments, the subject is administered the composition by intra-arterial injection (eg, carotid injection) and by intraparenchymal injection (eg, intraparenchymal injection by CED). In some embodiments, the direct CNS injection and the peripheral injection are performed simultaneously (eg, at the same time). In some embodiments, the direct injection is performed before the peripheral injection (eg, between 1 minute and 1 week or more before the peripheral injection). In some embodiments, the direct injection is performed after the peripheral injection (eg, between 1 minute and 1 week or more after the peripheral injection).

In some embodiments, the subject is administered an immunosuppressant prior to administration of the composition (eg, between 1 month and 1 minute before administration of the composition) or concurrently with the composition, as 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.

The amount of a composition (eg, a composition containing an isolated nucleic acid or vector or rAAV) as described herein administered to a subject will vary depending on the route of administration. For example, in some embodiments, rAAV, as described herein, is administered to a subject with a titer of from about 10 ⁹ genome copies (GC)/kt to about 10 ¹⁴ GC/kg (e.g., about 10 ⁹ GC/kg, about 10 ¹⁰ GC /kg, about 10 ¹¹ GC/kg, about 10 ¹² GC/kg, about 10 ¹² GC/kg, or about 10 ¹⁴ GC/kg). In some embodiments, the subject is administered a high titer (eg, >10 ¹² GC genome copies/kg rAAV) by injection into the space where CSF circulates or by intraparenchymal injection. In some embodiments, rAAV, as described herein, is administered to a subject at a dose of about 1 x ^{10 10} vector genomes (vg) to about 1 x 10 ¹⁷ vg by intravenous injection. In some embodiments, rAAV, as described herein, is administered to a subject at a dose of about 1x 10 ¹⁰ vg to about 1x 10 ¹⁶ vg by injection into the cerebellomedullary cistern.

A composition (e.g., a composition containing an isolated nucleic acid or vector or rAAV) as described herein may 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 (eg, over an extended period), for example, using an infusion pump.

Examples

Example 1: rAAV vectors.

AAV vectors are generated using cells such as HEK293 cells for triple plasmid transfection. ITR sequences flank an expression construct containing a promoter/enhancer element for each transgene of interest, a 3' polyA signal, and post-translational signals such as a WPRE element. Multiple gene products can be expressed simultaneously, such as GBA1 and LIMP2 and/or prosaposin, by fusion of protein sequences; or using a 2A peptide linker such as T2A or P2A, which results in the formation of 2 peptide fragments with added amino acids due to the prevention of peptide bond formation; or using an IRES element; or by expression with 2 separate expression cassettes. The presence of a short intronic sequence that is efficiently spliced upstream of the gene being expressed can increase expression levels. shRNA and other regulatory RNAs could potentially be included in these sequences. Examples of plasmids containing the rAAV vectors described herein are shown in FIG. 1-6, Fig. 21-27 and Fig. 55-58, as well as in table. 2 below.

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table 2

Name Promoto pi shPH TO CDS1 PolyA 1 Bicistric element Prom otor 2 CDS2 By whether A2 Length on me WAITING FOR ITR CMVe_CBAp_GBAl_ WPREbGH C.B.A. GBA1 WPRE- bGH 374 1 LT 1 sJetLongmPHKia SYn_SCARB2-T2AGBAlbGH JetLong aSyn SCARB2 bGH T2A GBA1 421 5 LIl_JetLong_SCARB2- IRES-GBALbGH JetLong SCARB2 bGH IRES GBA1 439 9 FP IJetLongGB AlbG H_JetLong_SCARB2_S V40L JetLong GBA1 bGH JetLo ng SCAR B2 SV 40 L 446 4 PrevailBeKTop_LT2s_Jet LongmPHKiaSY n_PS A P-T2AGBAl_bGH_4353nt JetLong aSyn PSAP bGH T2A GBA1 435 3 PrevailVector_LI2_JetLo ng_PSAP_IRES_GBAl_ Sy mtheticpoly A_43 37nt JetLong PSAP Synthetic pA IRES GBA1 433 7

Example 2: Cell-based assays of viral transduction in GBA-deficient cells.

GBA1-deficient cells were obtained, for example, as fibroblasts from GD patients, monocytes or hES cells, or patient-derived induced pluripotent stem cells (iPSCs). These cells accumulate substrates such as glucosylceramide and glucosylsphingosine (GluCer and GluSph). Treatment of cultured wild-type or mutant cell lines with G-case inhibitors such as CBE can also be used to generate GBA-deficient cells.

Using such cellular models, lysosomal defects are quantified in terms of the accumulation of protein aggregates, such as α-synuclein with an antibody to this protein or phosphoaSyn, followed by visualization using fluorescence microscopy. Imaging of lysosomal abnormalities is also performed using ICC for protein markers such as LAMP1, LAMP2, LIMP1, LIMP2, or using dyes such as Lysotracker, or by uptake through the endocytic compartment of fluorescent dextran or other markers. It is also possible to visualize the accumulation of autophagy markers due to defective fusion with lysosomes, for example for LC3. Western blotting and/or ELISA are used to quantify the abnormal accumulation of these markers. In addition, the accumulation of glycolipid substrates and GBA1 products is measured using standard approaches. Therapeutic endpoints (eg, reduction of PD-associated disease process) are assessed in the context of expression of AAV transduction vectors to confirm and quantify activity and function. G-case can also be quantified by protein ELISA or standard G-case activity assays.

Example 2.1. In vitro pharmacological studies with rAAV encoding G-case.

Transduction and activity.

In vitro study to evaluate the ability of PR001A (AAV9.CBA.GBA1.A) (a schematic representation of the vector encoding plasmid is shown in FIG. 55) containing the codon-optimized human GBA1 coding sequence (SEQ ID NO: 15) for expression of the GBA1 transgene in HEK293T cells, showed a dose-dependent increase in G-case activity after PR001A transduction in HEK293T cells (Fig. 33).

Efficiency indicators (a-synuclein).

In vitro studies were also performed in HeLa cells, a human cell line, and in primary mouse hippocampal neurons. In HeLa cells treated with PR001A at 2x10 ⁶ vg/cell, there was an approximately 2-fold increase in G-case activity levels and a decrease in total α-synuclein levels compared to vehicle-treated control cells (FIG. 34A and FIG. 34B). No similar effect was observed at the lower dose of PR001A.

Mouse hippocampal neurons transduced with PROOIA in an amount of 1.3x10 ⁵ vg/cell or

- 28 046909

1.3x 10 ⁶ vg/cell, showed increased levels of G-case activity and a trend toward decreased overall α-synuclein levels (FIG. 35A and FIG. 35B).

Thus, transduction of PROOIA in cell lines and cultures of primary neurons leads to increased G-case activity. In HeLa cells and mouse hippocampal neurons, PR001A transduction also resulted in a decrease in α-synuclein levels, supporting a link between G-case activity and α-synuclein levels. (Mazzulli et al., Cell. 2011;146(l):37-52 ).

Example 3: In vivo assays using mutant mice.

This example describes in vivo assays of AAV vectors using mutant mice. In vivo studies of AAV vectors, as described above, in mutant mice are carried out using assays described, for example, in the publications of 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.

Intrathecal or intraventricular delivery of control vehicles and AAV vectors (eg, at a dose of 2x1ο ¹¹ vg/mouse) is performed using concentrated stocks of AAV, for example, an injection volume of 5-10 μl. Intraparenchymal convection improved delivery is carried out.

Treatment begins either before symptoms appear or after they appear. The criteria measured are substrate accumulation in the CNS and CSF, assessment of G-case enzyme accumulation by ELISA and enzyme activity, motor and cognitive functions, lysosomal dysfunction, and accumulation of α-synuclein monomers, protofibrils, or fibrils.

Example 4: Chemical models of disease.

This example describes in vivo assays of AAV vectors using a chemically induced mouse model of Gaucher disease (eg, the SVE mouse model). In vivo studies of these AAV vectors are performed in a chemically induced Gaucher disease mouse model, for example as described in Vardi et al. (2016) J Pathol. 239(4):496-509.

Intrathecal or intraventricular delivery of control vehicles and AAV vectors (eg, at a dose of 2x1011 vg/mouse) is performed using concentrated AAV stocks, for example, in an injection volume of 5-10 μl. Intraparenchymal convection improved delivery is carried out. Peripheral delivery is achieved through tail vein injection. Treatment begins either before symptoms appear or after they appear. The criteria measured are substrate accumulation in the CNS and CSF, assessment of G-case enzyme accumulation by ELISA and enzyme activity, motor and cognitive functions, lysosomal dysfunction, and accumulation of α-synuclein monomers, protofibrils, or fibrils.

Example 5: Clinical studies involving patients with PD, LBD and Gaucher disease.

In some embodiments, patients suffering from certain forms of Gaucher disease (eg, GD1) have an increased risk of developing Parkinson's disease (PD) or Lewy body dementia (LBD). This example describes clinical studies to evaluate the safety and efficacy of rAAV, as described herein, in patients with Gaucher disease, PD and/or LBD. Clinical studies of such vectors for the treatment of Gaucher disease, PD and/or LBD are being performed using a study design similar to that described in Grabowski et al. (1995) Ann. Intern. Med. 122(l):33-39.

Example 6 Treatment of a peripheral disease.

In some embodiments, patients suffering from certain forms of Gaucher disease exhibit symptoms of peripheral neuropathy, for example, as described in Biegstraaten et al. (2010) Brain 133(10):2909–2919.

This example describes in vivo assays of AAV vectors, as described herein, for the treatment of peripheral neuropathy associated with Gaucher disease (eg, Gaucher disease type 1). Briefly, patients with type 1 Gaucher disease who exhibit signs or symptoms of peripheral neuropathy are administered rAAV as described herein. In some embodiments, peripheral neuropathic signs and symptoms in a subject are monitored, for example, through the methods described in Biegstraaten et al., following rAAV administration.

The levels of transduced gene products, as described herein, present in patients (eg, in the patient's serum, in peripheral tissue (eg, liver tissue, spleen tissue, etc.)) of the patient are analyzed, for example, using Western blotting, enzymatic functional assays or imaging studies.

Example 7. Treatment of forms of diseases with damage to the central nervous system.

This example provides in vivo rAAV assays as described herein for the treatment of CNS forms of Gaucher disease. Briefly, patients with Gaucher disease identified as having the CNS form of Gaucher disease (eg, Gaucher disease type 2 or 3) are administered rAAV as described herein. Levels of transduced gene products, as described herein, present in the CNS of patients (e.g., in the patient's CNS serum, in the patient's cerebrospinal fluid (CSF), or in the patient's CNS tissue) are analyzed, for example, by

- 29 046909

Western blotting, enzymatic functional assays or imaging studies.

Example 8 Gene therapy for Parkinson's disease in subjects having mutations in GBA1.

This example describes the administration of a recombinant adeno-associated virus (rAAV) encoding GBA1 to a subject suffering from Parkinson's disease characterized by a mutation in the GBA1 gene.

The rAAV vector insert contains a CBA promoter element (CBA), consisting of four parts: CMV enhancer (CMVe), CBA promoter (CBAp), exon 1, and intron (int) for continuous expression of the codon-optimized coding sequence (CDS) of human GBA1 (dark -burgundy). The 3' region also contains the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) followed by the bovine growth hormone polyA signal tail (bGH polyA). Flanking ITRs allow insertion sequences to be packaged correctly. Two variants of the 5' ITR sequence were evaluated (Fig. 7, inset, bottom sequence); these variants have several nucleotide differences within the 20-nucleotide D region of the ITR, which are thought to affect packaging and expression efficiency. The rAAV vector product contains the D domain nucleotide sequence shown in FIG. 7 (insert, top sequence) The vector variant contains a mutant D domain (referred to herein as the S domain, nucleotide changes shown in shaded) similarly studied in preclinical studies. The framework contains a gene that confers resistance to kanamycin, as well as a spacer sequence that prevents refolding. A diagram of the rAAV vector is shown in Fig. 8. The rAAV vector is packaged into rAAV using serotype AAV9 capsid proteins.

GBA1-rAAV is administered to a subject as a single dose via fluoroscopically guided suboccipital injection into the cisterna magna (injection into the cisterna magna; ICM). One of the options for studying the administration regimen is the following:

Example 8.1. In vivo pharmacological studies with rAAV encoding G-case.

Initial studies were conducted in a chemical mouse model involving daily administration of conduritol-β-epoxide (CBE), a G-case inhibitor, to evaluate the efficacy and safety of the rAAV vector PR001A (AAV9.CBA.GBA1.A) (schematic illustration of the plasmid encoding the vector , shown in FIG. 55) containing the codon optimized human GBA1 coding sequence (SEQ ID NO: 15) and the rAAV PR001B S variant construct (as described below). In addition, initial studies were performed in a genetic mouse model carrying a homozygous GBA1 mutation and partially deficient in saposins (4L/PS-NA). To further evaluate the safety and efficacy of the vector, additional dose-ranging studies are being conducted in mice and non-human primates (NHPs).

These mouse models exhibit phenotypes characteristic of nGD (neuronopathic Gaucher disease) and PD-GBA (Parkinson's disease characterized by a mutation in the GBA1 gene), including reduced G-case activity, accumulation of G-case glycolipid substrates, motor deficits, and neuropathological changes. including astrogliosis and microgliosis, reflecting inflammation. Intracerebroventricular injection of PR001A suppressed all of these disease-associated phenotypes. Additionally, the 4L/PS-NA mouse model demonstrated α-synuclein accumulation, and ICV administration of PR001A in the 4L/PS-NA model reduced α-synuclein accumulation.

Two slightly different versions of the 5' inverted terminal repeat (ITR) in the AAV backbone were tested to evaluate the feasibility of transgene production and expression (Fig. 7). Domain D is believed to be 20 bp long. in 5' ITR of 145 bp. is necessary for optimal viral vector production, but mutations in domain D have also been reported to increase transgene expression in some cases. Thus, in addition to the viral vector PR001A, which contains an intact D domain, a second form of the vector (PR001B) with a mutant D domain (referred to herein as the S domain) was also evaluated. Both the PR001A rAAV and the PR001B rAAV variant express the same transgene. Although both vectors produced virus that was effective in vivo, as detailed below, rAAV PR001A, which contains the wild-type D domain, was selected for further development.

Preclinical in vivo pharmacology (efficacy) studies are presented in Table. 14. A total of 10 studies were performed; The 4 main studies are discussed in detail in the following sections.

Example 8.1.1. Studies on a mouse model of SVE.

Overview of the SBE model.

In a chemical mouse model of WES, pharmacological inhibition of G-case activity is achieved using a selective and irreversible covalent competitive G-case inhibitor, resulting in accumulation of glycolipids (GluCer and GluSph), neuropathological changes including astrogliosis and microgliosis, and motor deficits (Manning- Bog et al., Neurotoxicology. 2009;30(6): 1127-32; Farfel-Becker et al., Dis Model Mech. 2011;4(6):746-52; ;23(6):550-64).

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SBE is a pharmacological inhibitor of G-case, and mice treated with SBE exhibit phenotypes consistent with loss of G-case function. By varying the dose of SBE and thus the degree of G-case inhibition in vivo, it is possible to determine the different degrees of enzyme deficiency observed in different GBA1-associated disorders, thereby modulating the severity of the resulting phenotype. For this reason, the mouse model of VES has significant technical advantages over genetic models of G-case deficiency, making it an attractive model for PD-GBA. The systemic decrease in G-case activity in the SBE model recapitulates the human disease, as patients with PD-GBA exhibit decreased G-case activity throughout the central nervous system and peripheral organs. This model is expected to underestimate the effects of PR001A because VES will inhibit both endogenous G-case activity and exogenous G-case activity resulting from PR001A administration.

Study PRV-2017-001: SVE dose range study.

To create a SBE model of GCase deficiency, young mice were administered SBE, a specific GCase inhibitor. Mice were administered VES by intraperitoneal injection daily beginning on Postnatal Day 8 (P8). Three different doses of SBE (25 mg/kg, 37.5 mg/kg, 50 mg/kg) or daily intraperitoneal (IP) administration of vehicle (PBS) were tested to create a model that demonstrates the behavioral phenotype (Figure 9A-Figure 9F ). Higher doses of VES were fatal in a dose-dependent manner. All mice treated with 50 mg/kg SBE died before P23, and 5 of 8 mice treated with 37.5 mg/kg SBE died before P27 (Fig. 9A). No lethality was observed in mice treated with SBE at a dose of 25 mg/kg. Mice treated with SVE did not gain weight, which correlated with the dose of SVE. At P27, at the end of the mice's lifetime portion of the study, the difference in weight was statistically significant between control animals and animals receiving 25 or 37.5 mg/kg SBE; no mice treated with 50 mg/kg SBE survived to P27 (Fig. 9B, Fig. 9C). While SBE-treated mice did not show overall motor deficits in the open field assay (moving the same distance and speed as PBS-treated mice; Fig. 9F), SBE-treated mice showed deficits motor coordination and balance, as measured by the rotating drum test (Fig. 9D). Mice surviving to the end of the study were sacrificed the day after they received their last dose of SBE (P27, Day 1) or three days after discontinuation of SBE (P29, Day 3). Lipid analysis was performed on the cerebral cortex of mice fed 25 mg/kg SBE to assess the accumulation of GCase substrates in both Day 1 and Day 3 cohorts. GluSph and GalSph levels (measured collectively in this example) were significantly degrees accumulated in SBE-treated mice compared with PBS-treated controls, consistent with G-case deficiency (Fig. 9E).

Thus, a daily intraperitoneal dose of 25 mg/kg SBE resulted in deficits in motor behavior and accumulation of G-case substrates (the sum of GluSph and GalSph levels), consistent with inhibition of G-case activity. Therefore, the 25 mg/kg dose was chosen for subsequent studies because it reproduces the main features of the human disease, allowing longer-term studies to evaluate vector stability. Study PRV2018-002: Efficacy of PR001B in the SBE model

Based on the study described above, a 25 mg/kg SBE dose was chosen because it induced behavioral impairment without affecting survival. For all preclinical studies in mice, intracerebroventricular (ICV) injection was selected as the route of administration (ROA). Because intracisterna magna (ICM) injection (the proposed clinical route of administration) is technically difficult in mice, ICV injection was considered the most suitable alternative approach to replicate ICM delivery of a therapeutic agent into the cerebrospinal fluid (CSF). To achieve wide distribution of GBA1 in the brain and transgene expression during VES administration, it is necessary to use 4 μl of vehicle (dPBS+0.001% Pluronic F68, dPBS), or 8.8x109 vg ( ^5.9x10101 vg/g brain, based on brain weight brain 150 mg) PR001B was delivered by ICV injection at P3, and daily PBS IP or SBE 25 mg/kg was started at P8 (Fig. 10). To determine whether SBE treatment would completely mask the effect of PR001B, half of the animals were sacrificed at P36, 1 day after their last SBE injection, while the other half were subjected to SBE withdrawal and sacrificed at P38, 3 days after their last SBE injection. For all measures, groups were pooled for analysis, adjusting for day of collection as a covariate.

Mice treated with SBE showed a reduction in body weight gain, which was attenuated by PR001B (Fig. 11A; Fig. 11B). SBE-treated mice administered rAAV performed statistically significantly better on the rotating drum than vehicle-treated mice (Figure 11C). Mice in the variant vector group did not differ from vehicle-treated mice in terms of metrics such as total distance walked during testing (Figure 11D).

At the end of the study, half of the surviving mice were sacrificed the day after the last dose of SBE (P36, Day 1) or three days after withdrawal of SBE (P38, Day 3) to

- 31 046909 biochemical analysis (Fig. 12B-Fig. 12D). Using fluorometric enzyme assays performed in three biological replicates, G-case activity was assessed in the cerebral cortex. G-case activity was increased in mice treated with PR001B rAAV, whereas SBE administration decreased G-case activity (Fig. 12B). In addition, mice that received both SBE and PR001B rAAV had levels of G-case activity that were similar to the PBS-treated group, indicating that rAAV delivery was able to overcome the inhibition of G-case activity caused by SBE administration. Lipid analysis was performed on the mouse motor cortex to examine the levels of the substrates GluCer and GluSph. Both lipids accumulated in the brains of SBE-treated mice and rAAV administration significantly reduced GluCer accumulation and tended to reduce GluSph accumulation (Fig. 12C; Fig. 12D).

Lipid levels were negatively correlated with both G-case activity and success in the rotating drum test in all treatment groups. Increased G-case activity after rAAV administration was associated with a decrease in substrate and an increase in motor function (Fig. 13). As shown in FIG. 14, preliminary biodistribution was assessed by the presence of vector genome measured by qPCR (with >100 vector genomes per 1 μg of genomic DNA defined as positive). Mice that received PR001B rAAV, both with and without SVE, were positive for rAAV vector genomes in the cerebral cortex (Fig. 12A), indicating that ICV delivery results in delivery of rAAV to the cortex. In addition, vector genomes were detected in the liver, spleen, heart and lungs, with lower levels in the kidneys and absent in the gonads (Fig. 14). For all measures, there was no statistically significant difference between the Day 1 and Day 3 groups (data not shown).

Thus, at a dose of 8.8 x ^{10 9} vg (5.9 x 10 ¹⁰ vg/g brain) administered by ICV, PR001B was distributed in the brain and peripheral tissues, and enzymatically active G-case was expressed in the brain. PR001B reduced biochemical deficits (i.e., glycolipid levels) and improved performance in the rotating drum test. Because withdrawal of SVE was not necessary to see the effects of PR001B, mice were sacrificed 1 day after the last dose of SVE in all future studies.

Study PRV-2018-005: PR001A dose range in the SBE model.

A diagram showing an illustrative dose range study design is shown in FIG. 15 A.

A larger study of the WES model further examined the effective doses of PR001 rAAV in the WES model. Using a 25 mg/kg SBE dose model, excipient or PR001 rAAV was delivered via ICV at P3, and daily IP administration of PBS or SBE was initiated at P8. Given the similarity between the SBE withdrawal and non-SBE withdrawal groups observed in previous studies, all mice were sacrificed one day after the last dose of SBE (P38–40). The effect of three different doses of rAAV was assessed, resulting in the following five groups of 10 mice (5M/5F) each: Excipient ICV + PBS IP Excipient ICV + 25 mg/kg CBE IP 2.0e9 vg (1.3e10 vg/g brain) rAAV ICV+25 mg/kg SVE IP 6.2e10 vg (4.2e10 vg/g brain) rAAV ICV+25 mg/kg SVE IP 2.0e10 vg (1.3e11 vg/g brain brain) rAAV ICV+25 mg/kg SVE IP.

Animals receiving SBE gained weight at a slower rate than control animals, which is a typical characteristic for this animal model. At the highest dose, PR001A corrected the failure to gain weight associated with VES administration. In addition, this dose resulted in a statistically significant improvement in the results of the rotating drum and tapered beam test compared to the SBE group that did not receive PR001A (Fig. 15B to Fig. 15E). Brain G-case activity was positively correlated with performance on the rotating drum test. Mortality was observed in several groups, including both the excipient group and the rAAV group.

At the conclusion of the study, all surviving mice were sacrificed for biodistribution characterization and biochemical analysis (FIG. 16A-FIG. 16D). Of the tissues examined, brain, spinal cord, liver, spleen, heart, kidney and lung were positive for vector genomes at medium and maximum doses. The brain, spinal cord, lungs and heart were also positive at the low dose (Fig. 16A). Gonads were not positive at any dose. Effective G-case activity, assessed by measuring enzymatic activity in all tissues using a fluorometric assay, was reduced by up to 60% after administration of SBE (Fig. 16B). At the highest dose of PR001A, G-case activity was significantly increased in the brain, spinal cord and heart. It should be noted that because administration inhibits PR001A-encoded G-case activity to the same extent as endogenous G-case activity by approximately 50%, studies in the SBE model likely underestimate the potency of PR001A as measured by G-case activity by approximately 2 times. Mice treated with SBE showed accumulation of GluCer and GluSph in the cerebral cortex. A high dose of PR001A reduced their accumulation (Fig. 16C; Fig. 16D).

Reactive astrogliosis and microglial activation are prominent inflammatory aspects of CNS pathology described in patients with neuronopathic GD and PD-GBA (Wong et al 2004; Ginns

- 32 046909 et al 2014). In this study, mice treated with SES exhibited glial scars, a manifestation of reactive astrogliosis in the cerebral cortex, consistent with previous studies demonstrating CNS activation in the context of SES (Sun et al 2011). Administration of PR001A resulted in a statistically significant dose-dependent reduction in the glial scarring phenotype (Fig. 16E). Thus, the use of PR001 suppresses the neuropathology associated with G-case deficiency in the central nervous system. A full description of the histopathological findings of this study is discussed in the toxicology section. Immunohistochemical analysis was performed to characterize the expression of G-case and ionizing calcium-binding adapter molecule 1 (Ibal; a marker of microgliosis) in the cortex (Fig. 16F) (Wong et al 2004; Vitner et al 2016). G-ase expression was significantly increased in all PR001A-treated mice compared with CBE+excipient-treated animals and correlated with the dose administered. The intensity of Ibal staining was significantly reduced in a dose-dependent manner in PR001A-treated mice compared with CBE+excipient-treated mice, in which the intensity of Ibal staining was significantly increased compared with PBS-treated mice. Thus, the results of the PRV-2018-005 study demonstrate that ICV administration of PR001A in three doses led to a wide biodistribution of the vector genome, an increase in G-case activity, improved motor functions and a decrease in glycolipid accumulation. Two different measures of neuroinflammation (microgliosis and astrogliosis) showed dose-dependent, statistically significant reductions in PR001A-treated mice. The SBE model inherently underestimates the efficacy of PR001A because SBE also inhibits enzyme activity due to the administration of PR001A. Taken together, these results demonstrate that ICV administration of PR001A at a dose of 2.Ox1O ¹⁰ vg (1.3x1011 vg/g brain) was effective in a mouse model of SVE. A trend toward efficacy was observed at lower doses of PR001A in a subset of endpoints.

Study PRV-2018-007: Long-term effects of PR001A in the SVE model.

This study assessed the persistence of PR001A vector copy number biodistribution and the persistence of PROOlA-mediated G-case expression in a mouse model of VES. A single dose of excipient or PR001A was delivered via ICV at P3, and daily IP administration of PBS or SBE began at P8 and continued until P183-P185 (FIG. 36). All mice were sacrificed 1 day after the last dose of SBE. Mortality was not observed in any group.

A single ICV dose of PR001A in SBE-treated mice resulted in the presence of vector genome copies 6 months post-dose (Figure 37 A) at levels comparable to those observed approximately 1 month post-ICV dose (see study PRV-2018-005). Long-term administration of VES led to a decrease in the level of G-case activity; G-case activity was nearly normalized in PR001A-treated mice, indicating that a single dose of PR001A resulted in sustained G-case expression (Figure 37B). Six-month-old mice treated with SBE showed significant accumulation of the glycolipid substrates GluCer and GluSph in the cerebral cortex compared to mice treated with SBE for 1 month. A single injection of PR001A into P3 resulted in a significant reduction in GluCer and GluSph levels to levels similar to those of the wild type (Fig. 37C; Fig. 37D).

Study PRV-2018-008: Additional dose range of PR001A in the SBE model.

This study was designed to evaluate additional doses of PR001A to determine the minimum effective dose and examine higher doses for tolerability. However, due to an unexpected dose deviation, doses from PRV-2018-005 were replicated in this study. In a design similar to PRV-2018-005 (Figure 15A), 4 μl of excipient or PROOIA was delivered via ICV at P3, and daily IP administration of PBS or SBE began at P8 and continued until P51-P53. In contrast to previous studies, VES administration did not result in significant changes in body weight. Although the use of CBE resulted in significantly worse rotating drum and tapered beam test results, the addition of PR001A did not significantly change these performances (FIG. 38A-FIG. 38E).

Of the tissues examined, brain, spinal cord, liver, heart, and lung were positive for PR001A at all dose levels. The kidneys were also positive at the intermediate and maximum doses, while the spleen was positive only at the maximum dose. Gonads were also tested but did not yield positive results at any dose level (Fig. 39). Gcase activity was assessed only in individual organs. In the cerebral cortex, low and medium doses of PR001A restored G-case activity levels to levels equivalent to or higher than PBS+excipient levels, although this effect did not reach statistical significance. The high dose of PR001A tended to significantly increase G-case activity compared to levels in CBE+excipient-treated animals (FIG. 40).

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

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This study confirmed the results of PRV-2018-005, demonstrating that administration of PR001A resulted in broad biodistribution and a significant increase in G-case activity, which significantly reduced the accumulation of glycolipid substrate caused by VES administration. This study did not replicate the behavioral phenotypes observed with PRV-2018-005; however, these phenotypes are known to be variable and less stable in mice.

PRV-2018-025: Additional PR001A dose range in the CBE model.

Given the study deviation in PRV-2018-008, an additional study was conducted in the SBE model to expand on the previous dose range studies. ICV administration of PR001A and intraperitoneal administration of PBS or SVE were performed using the same protocol as PRV-2018-005. However, this study included a lower dose of PR001A to determine the minimum effective dose and a higher dose to determine tolerability.

In this study, VES administration did not result in weight loss over time; however, a statistically significant decrease in locomotor activity was observed in animals receiving SBE + excipient, both on the rotating drum and on the tapering beam. Application of PROOIA at a dose of 5.2 x ^{10 10} vg significantly improved motor functions to almost the same level as in animals receiving PBS + excipient. Improvement was also observed in animals treated with 1.7 x ^{10 10} vg PR001A, although this effect did not reach statistical significance (Fig. 42A-Fig. 42D). The cerebral cortex of animals treated with PR001A was positive for vector genomes at all doses, and application of 5.2x10 ¹⁰ vg PR001A led to a significant increase in G-case activity. Application of 1.7 x ^{10 10} vg PR001A restored activity to levels similar to those of the wild type (Fig. 43A-Fig. 43B), although this effect did not reach statistical significance. Consistent with other studies in this model, SBE-treated mice exhibited accumulation of GluSph and GluCer in the brain, which was significantly reduced by administration of PR001A at doses of 1.7 x ^{10 10} vg or 5.2 x ^{10 10} vg (Fig. 44A-Fig. 44B ).

This study confirmed and extended the results of previous studies of the SBE model. Although this study did not fully replicate the behavioral phenotypes observed in PRV-2018005, minor improvements were seen in both the rotating drum and tapered beam tests with a dose of 1.7 x ^{10 10} vg PR001A, and administration of 5.2 x ^{10 10} vg PR001A significantly improved results of both tests. In addition, treatment at any dose reduced the accumulation of glycolipid substrate, which confirms the results of other studies of SBE.

Brief description of research on the SVE model.

Results from the VES model demonstrate that PR001A can be effectively delivered to the CNS as well as peripheral tissues by ICV injection. Within the CNS, delivery of PR001A via ICV resulted in a consistent increase in G-case activity, a decrease in the glycolipid substrates GluCer and GluSph, a reduction in glial scars, and an improvement in several motor characteristics. These effects, when assessed, were maintained 6 months after treatment.

Example 8.1.2. Studies using genetic models of 4L/PS-NA mice.

Review of the 4L/PS-NA model.

4L/PS-NA mice are a well-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(l ):37-52; Xu et al., Mol Genet Metab 2011;102(4):436-47). These mice are homozygous for the V394L mutation in GBA1 and additionally carry mutations in PSAP, which encodes saposin C, a G-case activator; the presence of a mutant G-case enzyme and low levels of the G-case activator saposin C together lead to severely reduced G-case activity, accumulation of glycolipid substrates, and motor deficits. These mice exhibit deficits in motor strength, coordination, and balance, as evidenced by their performance on the beam walk test, rotating drum retention test, and cord suspension test. These mice typically have a lifespan of less than 22 weeks. Control mice in this study are homozygous for the V394L mutation in Gbal but are wild-type for the endogenous prosaposin gene, and thus have a more modest reduction in G-case activity. Note that since PR001A does not affect saposin C, the results obtained in 4L/PS-NA mice likely underestimate the predicted effect in humans. Two PR001 A studies were conducted in these mice.

Study PRV-2018-006: PR001A in the 4L/PS-NA genetic model.

In study PRV-2018-006, PR001A or excipient was administered via ICV to 34-week-old 4L/PS-NA mice, and the animals were sacrificed 15 weeks after PR001A administration. A dose of 3 μl of undiluted vector was administered (total 1.5x 10 ¹⁰ vg; 3.7x 10 ¹⁰ vg/g brain) (Fig. 45).

Progressive motor deficits were observed in 4L/PS-NA mice, and treatment with PR001A resulted in modest improvements in beam walking at 5 and 9 weeks post-treatment. At 15 weeks after treatment, there was no statistically significant difference between the groups. The biodistribution of PR001A vector genomes in 4L/PS-NA mice was quantified approximately 15 weeks after administration.

- 34 046909 dose information. All tissues examined, including cerebral cortex, spinal cord, liver, kidney, heart, lung, spleen, and gonads, were positive for vector genomes. (Fig. 46). Analysis of G-case activity in tissue lysates, assessed by fluorometric assay, revealed a significant increase in effective G-case activity in the cortex and liver (Fig. 47).

There was a statistically significant accumulation of GluSph and GluCer in brain lysates of 4L/PS-NA mice compared to lysates of control animals. In 4L/PS-NA mice, PR001A treatment resulted in a statistically significant decrease in GluSph accumulation and a trend (P=0.16) for a decrease in GluCer (FIG. 48A; FIG. 48B).

Previous studies have demonstrated increased accumulation of α-synuclein protein in the cerebral cortex in the 4L/PS-NA mouse model, consistent with the proposed role of G-case in α-synuclein pathology (Sun et al., J Lipid Res. 2005;46(10 ):2102-13; Mazzulli et al., Cell. 2011;146(l):37-52; Xu et al., Mol Genet Metab. 2011;102(4):436-47). The levels of soluble and insoluble α-synuclein in the cerebral cortex were studied biochemically. Excipient-treated 4L/PS-NA mice exhibited a slight increase in insoluble α-synuclein levels and the ratio of insoluble to soluble α-synuclein in the cerebral cortex; ICV administration of PR001A reversed these effects (P=0.19, P=0.87, respectively) (FIG. 49A; FIG. 49B). These data are consistent with the in vitro studies described in Example 2.1, which demonstrate reduced α-synuclein accumulation.

Motor performance was assessed using a beam walk test 4 weeks after rAAV administration. A group of mutant mice administered PR001A rAAV showed a trend towards fewer total slips and fewer slips per velocity compared to the adjuvant-treated mutant mice, restoring motor function to near wild-type levels (Fig. 17). Study PRV-2018-011: PROOIA dose range in the 4L/PS-NA genetic model.

A second study in 4L/PS-NA mice examined a dose range of PR001A with a design similar to that used in the PRV-2018-006 study (Figure 50).

In the tapered beam test, 4L/PS-NA mice performed significantly worse than control mice. 4L/PS-NA mice treated with 2.9 x 1ο ¹¹ vg, 9.3 x ^{10 10} vg, or 2.9 x ^{10 10} vg PR001A showed significant improvement compared to 4L/PS-NA mice treated with adjuvant at Week 18 (Figure 51 ). No effect of PR001A was observed at earlier time points. There was no difference in performance on the rotating drum test between 4L/PSNA mice and control mice, and administration of PR001A did not appear to influence this result. All PR001A-treated groups were positive for vector genomes in the cerebral cortex. Efficient G-case activity, assessed by fluorometric assay, was measured in the cerebral cortex and was found to be significantly increased in mice treated with 2.9x1011 vg PR001A (Fig. 52A; Fig. 52B).

Hippocampal levels of soluble and insoluble α-synuclein in the cerebral cortex were studied biochemically. There were no differences in these levels between 4L/PS-NA mice and controls; Data published in the literature have demonstrated variable α-synuclein phenotypes. A statistically significant accumulation of GluSph and GluCer was observed in the cerebellum of 4L/PS-NA mice treated with the excipient. Administration of PR001A resulted in a dose-dependent trend toward decreased GluSph levels and a statistically significant dose-dependent decrease in GluCer (FIG. 53A; FIG. 53B).

Brief characteristics of the 4L/PS-NA mouse genetic model

Although 4L/PS-NA mice showed variability in the measured phenotypes across 2 studies, the overall findings were consistent with the SBE model and published data: G-case deficiency was associated with increased levels of glycolipid substrates and motor behavioral deficits. ICV administration of PR001A strongly attenuated these phenotypes. In the PRV-2018-006 study, cortical insoluble α-synuclein levels were not significantly increased in 4L/S-NA mice 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). ICV administration of PR001A reversed this accumulation according to the in vitro assays described herein. Taken together, these studies provide the rationale for clinical development of PR001A.

Example 8.1.3. In vivo α-synuclein model studies.

Study of PRV-2018-019 and PRV-2019-001: PR001A in α-synuclein transgenic mice treated with SVE.

To further investigate the effect of PR001A on α-synuclein pathology, 2 studies were conducted in db1-PAC-Tg(SNCAA53T);Snca ^-/- mice, which are homozygous for the human PD-associated mutant α-synuclein transgene A53T in Snca knockout mice (Snca encodes the mouse α-synuclein protein). These mice are reported to exhibit gastrointestinal

- 35 046909 intestinal phenotypes and subtle motor abnormalities between 6 and 12 months of age, but no widespread α-synuclein pathology in the brain (Kuo et al., Hum Mol Genet. 2010;19(9):1633-50 ). Previous studies in human α-synuclein A53T transgenic mouse models have demonstrated that administration of SBE to such mice results in increased 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). Based on these published results and to confirm the effects of G-case deficiency in this model, we administered SBE to these mice. At 9 to 10 weeks of age, mice were administered 10 μl of excipient or 2.9 x 1ο ¹¹ vg (7.4 x 1011 vg/g brain, based on 400 mg brain weight) PR001A via ICV injection. Two weeks after ICV administration, PBS or 100 mg/kg SBE was administered IP daily for 1 week.

The presence of vector genomes and G-case activity were assessed in the cerebral cortex. For PRV-2018019, an increase in cortical glycolipid substrates was confirmed with VES, and α-synuclein levels in hippocampal lysates were assessed using the Simple Western™ automated capillary immunoblotting system on a Jess instrument. Multiple α-synuclein immunoreactive bands were observed, consistent with the presence of monomers and high molecular weight (HMW) species. A statistically significant reduction in the ratio of HMW α-synuclein species to monomeric α-synuclein levels was observed when PROOIA was administered to SBE-treated α-synuclein transgenic mice (Figure 54A; Figure 54B).

Brief description of preclinical efficacy studies.

The above studies demonstrate 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 G-case activity in the CNS. Increased G-case activity reduces the accumulation of glycolipid substrates in the brain; these glycolipid substrates are proposed as biomarkers of outcome in a prospective clinical trial. Importantly, these benefits persist for at least 6 months after a single dose of PR001A. The SBE model represents reactive astrogliosis as well as microgliosis, which are common histopathological findings in patients with PD-GBA, nGD, as well as in animal models of these diseases (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; Trends Neurosci 2017;40(6):358-70; McMahon et al. Mol Genet Metab 2018;123(2):S93). PR001A is able to prevent or reverse SVE-induced reactive gliosis and microgliosis. Both models demonstrate motor deficits, and PR001A improves some of these manifestations in both models. Along with these two models, an additional mouse model was used to study α-synuclein pathology. While α-synuclein phenotypes are variable in mouse models, PR001A was able to suppress or reverse the phenotypes when observed; additional in vitro studies support the effectiveness of PR001A in reducing alpha-synuclein levels. Together, these studies support the efficacy of PR001A in PD-GBA and nGD models.

Example 8.1.4. Toxicology.

Single dose mouse studies.

Safety and toxicology studies conducted with PR001A in mouse models are summarized in Table 15. Two mouse model efficacy studies (PRV-2018-005 and PRV-2018-006) also included evaluations of individual safety endpoints, such as histopathology, to assess safety PR001A in a disease model.

Study PRV-2018-005: PR001A dose range in the SBE model.

Histopathological analysis was performed by hematoxylin and eosin (H&E) staining of the brain, thoracic spinal cord, heart, liver, spleen, lungs and kidneys; results were assessed by a board-certified veterinary pathologist. In mice treated with VES, changes in the central nervous system included glial scars and neuronal necrosis in the cerebral cortex, brainstem, and thoracic spinal cord. Intracerebroventricular PR001A at doses up to 1.3x1011 vg/g was well tolerated in these mice, and this highest dose resulted in a marked reduction in the incidence of these CNS changes; Low and moderate doses of PR001A caused a dose-dependent reduction in the number of animals with glial scars in the cerebral cortex, with ambiguous effects on other CNS parameters such as neuronal necrosis. No adverse effects of VES or PR001A were observed in peripheral tissues. In summary, no adverse histopathological findings or signs of toxicity were observed in association with PR001A in studies in the murine model of SVE.

Example 9: In vitro analysis of rAAV vectors.

A pilot study was performed to evaluate the in vitro activity of rAAV vectors encoding prosaposin (PSAP) and SCARB2, alone or in combination with GBA1 and/or one or more inhibitory RNAs. One construct encoding PSAP and progranulin was also tested.

- 36 046909 (PGRN). Vectors tested include the vectors shown in Table 1. 3. Opt refers to a nucleic acid sequence that is codon optimized for expression in mammalian cells (eg, human cells). Fig. 18 shows representative data indicating that transfection of HEK293 cells with each construct resulted in overexpression of the corresponding gene product compared to mock-transfected cells.

Table 3

ID Promoter Inhibitory RNA Promoter Transgene 100015 Shintronny SNCA JetLong Opt-PSAP_GBA1 100039 - - JetLong Opt-PSAP-GRN 100046 - - Opt-PSAP 100014 JetLong SNCA JetLong Opt- SCARB2GBA1

Example 10 ITR D sequence placement and cellular transduction.

The effect of ITR D sequence placement on cellular transduction of rAAV vectors was examined. HEK 293 cells were transduced with rAAV viruses encoding G-case and having 1) a wild-type ITR (e.g., D sequences proximal to the transgene insertion and distal to the end of the ITR) or 2) an ITR with a D sequence located outside the vector (e.g., D sequence located proximal end of the ITR and distal to the transgene insertion), as demonstrated in FIG. 19. Surprisingly, it was found that rAAVs having the D sequence located in the outer position retain the ability to efficiently package and transduce cells (Fig. 20).

Example 11: In vivo toxicity studies.

Fifty (50) mice received rAAV encoding GBA1 via intracerebroventricular (ICV) injection of 4 μl on postpartum day 3. All mice received daily intraperitoneal (IP) injections of conduritol B-epoxide (CBE) or PBS, depending on treatment group, from postnatal day 8 until the end of the study. Animals were sacrificed 24 hours after the last IP dose. After euthanasia, target tissues were collected, the drop was fixed in refrigerated 4% paraformaldehyde and stored at 4°C, and then sent for histopathological processing and evaluation.

Tissue from forty-two (42) animals sacrificed after 38 to 40 days was trimmed, processed, and embedded in paraffin blocks. ~5 μm sections were then cut, stained with hematoxylin and eosin (H&E), and mounted on glass slides for further evaluation.

There were no histopathological changes or signs of toxicity associated with rAAV use.

In mice treated with conduritol B-epoxide (CBE), the central nervous system (PNS) showed glial scars and neuronal necrosis in the cerebral cortex, as well as neuronal necrosis in the brainstem and thoracic spinal cord. High doses of rAAV resulted in a marked reduction in the incidence of these changes in the CNS, while low and medium doses of the virus caused a dose-dependent reduction in the incidence of glial scar formation in the cerebral cortex with ambiguous effects on other changes in the CNS (Fig. 28).

Immunohistochemical analysis was performed to evaluate the expression of G-case and Ibal in the cerebral cortex (FIGS. 29A-29B). G-case expression was significantly increased in all rAAV-GBA1-treated animals compared with SBE/excipient-treated animals. The increase in G-case expression correlated with the administered dose, with the highest G-case expression observed in high-dose animals, followed by medium- and low-dose animals. The level of Iba1, a marker of microgliosis, was significantly increased in SBE/excipient-treated animals. All doses of rAAV-GBA1 decreased the intensity of Ibal staining, thereby reducing microgliosis in the SVE model. Microgliosis is a well-characterized endpoint in neuronopathic GD and in models of this disease.

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Examples of neurodegenes Table 4 of therapeutic diseases Disease Associated genes Alzheimer's disease APP, PSEN1, PSEN2, APOE Parkinson's disease LRRK2, PARK7, PINK1, PRKN, SNCA, GBA, UCHL1, ATP13A2, VPS35 Huntington's disease NTT Amyotrophic lateral sclerosis ALS2, ANG, ATXN2, C9orf72, CHCHD10, CHMP2B, DCTN1, ERBB4, FIG4, FUS, HNRNPA1, MATR3, NEFH, OPTN, PFN1, PRPH, SETX, SIGMARI, SMN1, SOD1, SPG11, SQSTM1, TARDBP, ТВК1, TRPM7, TUBA4A, UBQLN2, VAPB, VCP Batten disease (neuronal ceroid lipofuscinosis) PPT1, TPP1, CLN3, CLN5, CLN6, MFSD8, CLN8, CTSD, DNAJC5, CTSF, ATP13A2, GRN, KCTD7 Friedreich's ataxia FXN Lewy body disease APOE, GBA, SNCA, SNCB Spinal muscular atrophy SMN1, SMN2 Multiple sclerosis CYP27B1, HLA-DRB1, IL2RA, IL7R, TNFRSF1A Prion disease (Creutzfeldt-Jakob disease, fatal familial insomnia, Hertzmann-Straussler-Scheinker syndrome, variable protease sensitivity prionopathy) PRNP Examples of syn Disease Parkinson's disease Dementia with Lewy bodies Multiple system atrophy Examples Disease Alzheimer's disease Primary age-related tauopathy Progressive supranuclear palsy Corticobasal degeneration Frontotemporal dementia with parkinsonism-17 Subacute sclerosing panencephalitis Lytico-Bodig disease Table 5 cleinopathies Associated genes LRRK2, PARK7, PINK1, PRKN, SNCA, GBA, UCHL1, ATP13A2, VPS35 APOE, GBA, SNCA, SNCB COQ2, SNCA Table 6 tauopathies Associated genes APP, PSEN1, PSEN2, APOE MARCH MARCH MARCH, GRN, C9orf72, VCP, CHMP2V, TARDBP, FUS MARCH SCN1A Ganglioglioma, gangliocytoma Meningioangiomatosis Postencephalitic parkinsonism Chronic traumatic encephalopathy

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Table 7

Examples of lysosomal storage diseases

Disease Associated genes Niemann-Pick disease NPC1, NPC2, SMPD1 Fabry disease GLA Krabbe disease GALC Gaucher disease GBA Tay-Sachs disease NEHA Metachromatic leukodystrophy ARSA, PSAP Farber's disease ASAHI Galactosialidosis CTSA Schindler's disease NAGA Gangliosidosis GM1 GLB1 Gangliosidosis GM2 GM2A Sandhoff disease HEXB Lysosomal acid lipase deficiency LIPA Multiple sulfatase deficiency SUMF1 Mucopolysaccharidosis type I IDEA Mucopolysaccharidosis type II IDS Mucopolysaccharidosis type III GNS, HGSNAT, NAGLU, SGSH Mucopolysaccharidosis type IV GALNS, GLB1 Mucopolysaccharidosis type VI ARSB Mucopolysaccharidosis type VII G.U.S.B. Mucopolysaccharidosis type IX HYAL1 Mucolipidosis type II GNPTAB Mucolipidosis type III alpha/beta GNPTAB Mucolipidosis type III gamma GNPTG Mucolipidosis type IV MCOLN1 Neuronal ceroid lipofuscinosis PPT1, TPP1, CLN3, CLN5, CLN6, MFSD8, CLN8, CTSD, DNAJC5, CTSF, ATP13A2, GRN, KCTD7 Alpha-mannosidosis MAN2B1 Beta-mannosidosis MANBA Aspartylglucosaminuria A.G.A. Fucosidosis FUG Al

Example 12 Non-human primate studies with rAAV encoding G-kasoo.

The safety of PR001A (AAV9.CBA.GBA1.A), containing the codon-optimized coding sequence of human GBA1 (SEQ ID NO: 15), was assessed in vivo in non-human primates (NHP). Additional information about PR001A components is provided above. The NHP brain is most similar to the human brain, and the anatomical features of the NHP spinal cord and CSF volume and flow allow for ICM injections (intra cisterna magna injections). Due to the anatomical similarity to humans, studies in NHP were expected to provide reliable biodistribution data that would be the basis for clinical dose determination of PROOIA.

The safety and biodistribution of PR001A were assessed in three toxicology studies in cynomolgus monkeys (Table 8): two non-GLP (Good Laboratory Practice) studies (PRV-2018-015 and PRV-2019-005), and a larger study 21CFR58. compliant with GLP requirements. (PRV-2018-016).

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Table 8

Review of Preclinical Safety Studies in NHP Using PR001A

Study number Regulatory supervision Species (age ) R.O. A Dose Group (vg/r head About the brain) Total PR001 A Dose (vg) Necropsy I Time moments Criteria for evaluation PRV- 2018-015 He-GLP Cynomolgus macaque (2-3 years) ICM ICM + 1Ra 0 2.0 x 10 ¹¹ 2.1 x 10 ¹¹ 0 1.47 x 10 ¹³ 1.53 x 10 ¹³ D18 Safety during life; Biodistribution; G istopathology PRV- 2018-016 GLP Cynomolgus macaque (2-4 years) ICM 0 6.2 x Yu ¹⁰ 2.3 x Yu ¹¹ 0 4.6 x 10 ¹² 1.7 x 10 ¹³ D7, D30, D183 Safety during life; Biodistribution; G istopathology; SBC; Vector shedding PRV- 2019-005 He-GLP Javanese macaques (2-3 ICM 0 7.0 x 10 ¹¹ 0 5.2 x 10 ¹³ D30, D90 Safety during life; of the year) Biodistribution e; G istopathology

Abbreviations: CBC - complete blood count;

D - day; GLP - Good Laboratory Practice;

ICM - inside a large tank;

IPa - intraparenchymal;

NHP - non-human primate;

ROA - method of administration; vg - vector genome

A non-GLP pilot study (PRV-2018-015) was conducted in NHPs to confirm that the final product PR001A is delivered to the NHP brain after ICM administration. The GLP toxicology and biodistribution study on NHP (PRV-2018-016) assessed the safety and biodistribution of PR001A.

Doses tested in NHPs include the maximum possible dose determined by the volume administered and the titer of the test product. In addition, the GLP study also evaluated a lower dose. GLP study time points were selected to assess safety after treatment but before peak expression (Day 7), onset of peak expression (Day 30), and after peak long-term expression (Day 183).

Study PRV-2018-015: He-GLP NHP study PR001A.

A pilot non-GLP study of tolerability and biodistribution of PR001A was conducted in male cynomolgus macaques. The purpose of this study was to verify the biodistribution of PR001A in various brain regions and major peripheral organs after ICM administration. The timing of killing was chosen based on prediction and in a manner that would allow for meaningful measurements of potential early toxicity to inform the planned GLP NHP toxicology study, particularly with early lifetime observations measured through the functional test battery (FOB). Studies of intrathecal delivery of AAV 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;l:14051; Hinderer et al., Mol Ther. 2015;23(8 )1298-307; Hinderer et al., Mol Genet Metab. 2016;119(1-2): 124-30). Thus, Day 18 assessments should reveal immediate toxicity caused by the injection procedure or the innate inflammatory response to the study drug, as well as provide information on the biodistribution and expression of the transgene at a time point corresponding to the early peak of expression. The study design included a rapamycin treatment arm (0.3 mg/kg PO, Day -3 to Day

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18) in combination with PR001A to determine whether immunosuppression would be beneficial in reducing potential toxicity. To increase transgene expression in the brain, one arm of the study involved intraparenchymal (IPa) administration of PR001A directly into the midbrain, targeting the bilateral pars compacta of the substantia nigra, in combination with ICM delivery. The ICM dose volume was 0.5 mL, the largest volume ever previously administered, and the IPa dose was 10 μL bilaterally, corresponding to doses of 1.47 x 1013 vg when administered as ICM alone and 1.53 x 10 vg when administered as ICM. so is IPa. With an estimated brain weight of 74 g, this corresponds to an ICM dose of 2.0 x 1011 vg/g brain and a dose of 2.1 x 10 vg/g brain for the ICM plus IPa group. A summary of the design of this study is presented in Table. 9.

Table 9

Review of the non-GLP study PRV-2018-015 in NHPs

Biodistribution and Safety Study Following Administration of PR001A to NHPs Target Evaluation of tolerability and biodistribution of PR001A in NHPs Regulatory Compliance requirements He-GLP Study drug PR001A Total number of animals 8 male cynomolgus macaques Weight (age) 3-4 kg (2-3 years) Number of animals/group 2/group Study design Group assignments: Group Dose (vg/r brain) ROA Immunosupp. Number of animals 1 0 ICM No 2 2 2.0 x 10 ¹¹ ICM No 2 3 2.0 x 10 ¹¹ ICM Dd 2 4 2.1 x 10 ¹¹ ICM+ IPa No 2 Method and frequency of administration A syringe was used for ICM administration; a single injection of 0.5 ml IPa was performed using a Hamilton syringe; bilateral injection of 10 μl into each hemisphere Compositions The solution for administration is provided in a concentration of 2.9 x 10 ¹³ vg/ml; the excipient used in the control group is a formulation similar to that intended for clinical use (20 mM Tris, pH 8.0.200 MMNaCl, 1 mM MgCE and 0.001% [w/v] poloxamer 188) FOB Weekly Body weight Weekly Necropsy Day 18 H&E and qPCR The following tissues were examined in all animals in all groups: liver frontal cortex paraventricular lung parietal cortex nucleus kidney occipital cortex pons gonads insular cortex entorhinal cortex heart cingulate gyrus medullary substance

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spleen hippocampus cerebellum ganglia dorsal roots putamen midbrain cervical region spinal cord For H&E staining of the midbrain, at least 12 sections are provided, which includes 6 sections around the infusion site in the IPa group; groups with only 1 cm injection (1-3) include the same anatomical levels

Abbreviations: FOB - functional test set;

GLP - Good Laboratory Practice;

H&E - hematoxylin and eosin;

ICM - inside a large tank;

Immunosuppression. - immunosuppressive;

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)

The H&E analysis was performed by two independent board-certified veterinary pathologists and both concluded that no toxicity associated with PR001A was detected. The observed spinal cord changes were likely the result of injury during ICM injection and were not considered related to PR001A. All histopathological changes in non-nervous system tissues were considered to be spontaneous or incidental changes commonly observed in control monkeys. Overall, there were no apparent adverse effects of PR001A in the brain or spinal cord. The reviewing pathologist noted that nonspecific changes (primarily variable mononuclear cell infiltrates) in the meninges, brain or spinal cord parenchyma, and/or injection site (in these tissues) were likely related to the study drug and did not evaluate these changes as unfavorable. Given the severity noted, similar infiltrates would reasonably be expected in any monkey undergoing an experimental procedure that disrupts the meninges and/or the blood-brain barrier. In addition, some infiltrates (especially within the choroid plexus and occasionally in the parenchyma) are commonly observed in control monkeys (Butt et al., Toxicol Pathol. 2015;43:513-8). All other observed histopathological changes were considered random and/or of similar frequency and severity in animals treated with excipient and PR001A, and were therefore considered not to be related to PR001A administration. A second independent board-certified veterinary pathologist who examined the same tissue samples noted that all changes were indistinguishable from incidental changes or damage incurred during the injection procedure, as these changes were nonspecific and across all groups, including the control group receiving only the excipient. Additionally, another board-certified veterinary pathologist examined non-GLP tissue and concluded that there were no effects associated with PR001A.

Overall, there were no changes in FOB scores, weight gain, or food intake over the course of the study, regardless of group or time point. Microglial morphology in the midbrain did not appear to differ between treatment groups (as determined by Ibal staining).

The expression and morphology of tyrosine hydroxylase-positive midbrain neurons did not appear to differ between treatment groups. By Day 18, AAV9-nAb titers were increased in all PR001A-treated animals, while vehicle-treated control animals showed only modest changes from baseline. One of the monkeys in the oral rapamycin group had a lower AAV9 nAb titer (1:64) on Day 18 compared to the other PR001A-treated animals (>1:256); the difference in titers did not affect biodistribution, but the sample size was too small to draw a definitive conclusion. Biodistribution was assessed in all test samples collected by quantitative polymerase chain reaction (qPCR); tissues were considered positive with at least 100 vg/μg DNA (these criteria were also used to evaluate positive tissues in mouse efficacy studies). All tissues tested were positive in all PR001A-treated groups, indicating widespread distribution of the drug in the CNS and periphery. In addition, animals administered PR001A via ICM in combination with bilateral midbrain IPa injection had increased localized expression. Administration of rapamycin did not appear to have any effect on safety or biodistribution (selected representative

- 42 046909 areas are shown in Fig. thirty). It should be noted that some tissues from control animals (pons, spinal cord, paraventricular ganglia, dorsal root ganglia and lung) were also positive as determined by qPCR. Several concerns were noted with necropsy procedures that indicated an increased risk of cross-contamination between animals within different treatment groups and between different organs within each animal. Modifications were made to the necropsy procedure to minimize contamination for future studies. However, no adverse toxic effects were detected in any of the qPCR-positive animals. Analysis of transgene expression (G-case activity) demonstrated no significant increase in G-case activity in PR001A-treated animals compared to controls; G-case activity was studied in more detail in the GLP NHP toxicity study PRV-2018-016.

Taken together, the results of the non-GLP NHP trial PRV-2018-015 demonstrated no safety or toxicity concerns in any in-life or post-mortem assessments. All animals survived to the scheduled date of necropsy, and postmortem analysis revealed no concerns for adverse toxicity. The study also demonstrated the uniform biodistribution of PR001A in the brain.

Study PRV-2018-016: GLP NHP study PR001A.

Study design.

The purpose of this GLP study was to evaluate the toxicity and biodistribution of PR001A when administered single dose via ICM injection to cynomolgus monkeys with a 7-30 day or 183 day post-administration observation period. The study was designed to evaluate 2 dose levels: the highest dose being the highest possible dose achievable with a volume of 1.2 mL (the highest volume that occurred during administration) of undiluted test product, and a lower dose of ^1/2 log unit lower than high dose. The doses corresponded to a low dose of 4.6x 10 ¹² vg and a high dose of 1.7x 10 ¹³ vg; with an estimated brain weight of 74 g in the cynomolgus monkey, this corresponds to approximately 6.2 x ^{10 10} vg /g brain and 2.3 x 10 11 vg /g brain. The study also included a control group in which animals received only 1.2 ml of excipient (20 mM Tris pH 8.0, 200 mM NaCl and 1 mM MgCl2 and 0.001% [w/v] Poloxamer 188). Both male and female cynomolgus macaques were used in this study. The Day 7 group included 1 male receiving the highest dose and was designed as a sentinel group for early toxicity studies; and the remaining 2 time points (Day 30 and Day 183) included 2 males and 1 female at each dose. In addition to samples from several brain regions, peripheral tissue samples were collected for qPCR analysis. All qPCR-positive samples were analyzed for transgene expression. A summary of the design of this study is presented in Table. 10.

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Table 10

Review of GLP study PRV-2018-016 in NHP________________

Toxicity and biodistribution study of a single dose of intracisternal administration in cynomolgus monkeys with a 7-day, 30-day or 183-day observation period Target Evaluation of tolerability and biodistribution of PR001A in NHPs Regulatory Compliance requirements GLP Study drug PR001A Total number of animals 19 cynomolgus monkeys Weight (age) 2-5 kg (25-50 months) Study design Group assignments: Group Dose (vg/r BRAIN) Number of animals Necropsy (Day 7) Necropsy (Day 30) Necropsy (Day 183) 1 0 0 2M/1F 2M/1F 2 6.2 x Yu ¹⁰ 0 2M/1F 2M/1F 3 2.3 x 10 ¹¹ i/m 2M/1F 2M/1F Method and frequency of administration ICM administration using a syringe; 1-3 cu. see and needles for spinal puncture (Pencan 25 G x 2.5 cm BBraun); a single slow bolus is administered at a maximum speed of 0.5 cc. cm/min. Compositions The injected solution is provided at a concentration of 1.42 x 10 ¹³ vg/ml. Clinical signs Daily (including food consumption); careful observations weekly Body weight Weekly Neurological, ophthalmic examination and electrocardiogram Once before dosing and during Weeks 2 and 26. Clinical pathology All groups of hematology, clinical chemistry, coagulation parameters Hematology number of red blood cells average corpuscle, volume

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hemoglobin platelet count hematocrit number of leukocytes average corpuscular hemoglobin blood smear hemoglobulin abs. number of lymphocytes average corpuscle. number of leukocytes hemoglobin concentration differential number of cells blood Clinical biochemistry glucose alanine aminotransferase Urea nitrogen alkaline phosphatase creatinine gamma-glutamyltransferase total protein aspartate aminotransferase albumin calcium globulin inorganic phosphorus albumin/globulin ratio sodium cholesterol potassium total bilirubin chloride creatine kinase triglycerides Coagulation prothrombin time fibrinogen activated partial thromboplastin time Vector shedding (urine/feces) When killed Necropsy Day 7, Day 30, Day 183 Preservation of tissues for histopathological analysis The following tissues from each animal will be collected in 10% neutral buffered formaldehyde (unless otherwise stated) or recorded as missing if applicable: G istopathology All groups - all fabrics Biodistribution The following tissues will be analyzed for biodistribution using qPCR: Frontal cortex Liver Hippocampus DRG (cervical) Ventral midbrain DRG (thoracic region) Periventricular gray matter DRG (lumbar region) Putamen Spinal cord (thoracic region) Testicles Spinal cord (lumbar department) Ovary Spinal cord (cervical) Kidney Spleen Stomach (pyloric region) Heart (apex) Blood CSF

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Expression Skazy All samples positive by qPCR will be assessed for progranulin expression. Tissue preservation Adrenal gland ³ Injection site Rectum Aorta (covering skin) Salivary glands Bone, femur with Jejunum Sciatic nerve bone marrow Kidney ³ Seminal vesicles ³ with bone marrow Liver ³ Spinal cord Brain ³ Lung with large (cervical, thoracic, cecum bronchi lumbar) Cervix Lymph node Spleen ³ Large intestine (mandibular) Stomach Twelve. intestine Lymph node Testicles ³ Epididymis ³ (mesenteric) Thymus ³ Esophagus Mammary gland Thyroid gland with Eye ^Muscle , biceps parathyroid glands ³ Gallbladder of the thigh Tongue GALT (Peyer's optic nerve Tracheal plaque) Ovary ³ Bladder Heart ³ Fallopian tube Uterus ³ Ileum pancreas gland Vagina Pituitary gland Prostate gland ^{3 3} Organs (if present) will be weighed or marked as missing; ^b Collected in modified Davidson's fixative and stored in 10% neutral buffered formalin.

Abbreviations: CSF - cerebrospinal fluid;

DRG - dorsal root ganglia;

F - female;

GALT - gut-associated lymphoid tissue;

GLP - Good Laboratory Practice;

ICM - inside a large tank;

M - male;

MgCl2 - magnesium chloride;

NaCl - sodium chloride;

NHP - non-human primate;

qPCR - quantitative polymerase chain reaction;

vg - vector genome(s) mM Tris pH 8.0, 200 mM NaCl, 1 mM MgCl ₂ and 0.001% (w/v) poloxamer 188

NHP cynomolgus monkeys were assessed through several lifetime observations and measurements, including mortality/morbidity (daily), clinical observations (daily), body weight (baseline and weekly thereafter), visual inspection of food intake (daily), neurological observations ( baseline and Weeks 2 and 26), indirect ophthalmoscopy (baseline and Weeks 2 and 26), and electrocardiography (baseline and Weeks 2 and 26).

AAV9 capsid nAb assays were performed at baseline and at Days 7, 30, or 183 sacrifice.

Clinical tests, including hematology, coagulation, clinical chemistry, and urinalysis, were performed twice at baseline (blood tests; once urinalysis) and once at Weeks 1 and 13 of the dosing phase.

Animals were sacrificed 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 preserved in 10% formalin with neutral buffer (except in cases where special fixation is required for optimal fixation).

- 46 046909 nal fixatives) for histopathological evaluation (all animals). Additional replicates were collected for qPCR and transgene expression analysis.

Safety and toxicology.

All animals survived to the scheduled necropsy date without unexpected death. There were no concerns or problems with lifetime assessments in either group; Gross examination at necropsy revealed no abnormalities associated with PR001A in either group.

No differences in PR001A-related organ weights or macroscopic or microscopic changes were detected in either group at intermediate sacrifices on Day 7 or 30 or at terminal sacrifice at Day 183. Hemorrhage, characterized by foci of perivascular hemorrhage, mainly in the area brainstem was present in all groups, including controls, and was therefore considered procedure related (CSF collection before necropsy) and not related to PR001A. All other microscopic changes, including minimal mononuclear cell infiltrates in the brain or spinal cord, were considered spontaneous and/or incidental because they occurred at low frequency, were randomly assigned to groups (including parallel controls), and/or their severity was as follows: expected for monkeys of this age; therefore, they were considered unrelated to PR001A.

No changes associated with PR001 A were observed in clinical laboratory diagnostic test results; elevated fibrinogen levels were observed in the animal exhibiting the highest anti-AAV9 titer consistent with an immune response against the vector. Positive anti-AAV9 antibody titers were observed by Day 7 in all PR001A-treated animals. There were no clinical changes associated with PR001A, changes in body weight, ophthalmic abnormalities, or abnormal physical or neurological examination findings. No PR001A-related differences in mean PR interval, QRS duration, QT interval, corrected QT interval (QTc), or heart rate were observed in males or mixed-sex individuals administered any dose of PR001A. There were also no arrhythmias or abnormal waveforms associated with PR001A.

Dose levels of 0, 6.2x1010, or 2.3x1011 vg/g brain PR001A were well tolerated when administered by a single injection into the cisterna magna in male and female monkeys. No lifetime changes, abnormal clinical laboratory test results, or pathologic findings were observed that were considered related to the gene product upon administration of PR001A.

Biodistribution and immune response.

Analysis of the biodistribution of vector genome copies was performed using a qPCR-based assay (vector presence); transgene expression (GBA1) was measured in samples that were positive for the presence of the vector genome. On Days 30 and 183, all tissues examined (including CNS and peripheral) were qPCR positive after high dose (2.3 x 1011 vg/g brain) (selected representative areas from Day 183 are shown in FIG. 30). At Day 30, tissues collected from testes and ovaries were positive for transduction in all NHPs receiving the high dose of PR001A (2.3x1011 vg/g brain). Additionally, 1 male NHP treated with a low dose of PR001A (6.2x1010 vg/g brain) was positive for testicular transduction on Day 30. On Day 183, 1 male and 1 female were positive for PR001A transduction in the gonads after administration high dose and 2 males receiving low dose were testicular positive.

To confirm that human G-case was produced in treated NHPs, protein levels were assessed using the Simple Western™ immunoblotting system on a Jess instrument. Results from analysis of cortical, hippocampal and midbrain samples obtained from NHPs treated with PR001 A demonstrated increased levels of G-case expression when analyzed together compared to samples from normal NHPs who received excipient alone; Both the low and high dose groups were combined for statistical comparison with the control group (Fig. 31A and Fig. 31B). These results indicate that efficient and widespread transduction of PR001A in NHPs following ICM administration results in increased levels of G-case expression. In conclusion, biodistribution results demonstrate that administration of PR001A to NHPs via ICM results in robust and widespread transduction of the human GBA1 transgene in the brain and peripheral organs. Summarizing the biodistribution data in NHPs, administration of PR001A via ICM resulted in a broad biodistribution throughout the brain comparable to levels demonstrated to be effective in mouse models; this transduction results in increased levels of G-case protein in the brain. Study PRV-2019-005: He-GLP NHP study PR001A

Study design.

To evaluate the toxicity and biodistribution of PR001A following a single dose of ICM injection, a non-GLP study was conducted in 12 male cynomolgus monkeys with 30- and 90-day follow-up periods after administration. The study was designed to estimate a single dose level of 5.2 x 101 ³ vg or 7.0 x 1011 vg/g brain, assuming that the average brain weight in

- 47 046909 cynomolgus monkey is 74 g. The administered dose represents the maximum possible dose achievable with a volume of 1.2 ml (the highest volume that occurred during administration) of undiluted product PR001A. The study also included a control group in which animals received only 1.2 ml of excipient (20 mM Tris pH 8.0, 200 mM NaCl and 1 mM MgCl ₂ + and 0.001% [w/v] Pluronic F68). Samples from multiple brain regions and peripheral organs were collected for qPCR analysis to measure biodistribution, and clinical laboratory tests and histopathological examination were performed to assess safety. A summary of the design of this study is presented in Table. eleven.

Table 11

Review of the non-GLP study PRV-2019-005 in NHPs

He-GLP toxicology and biodistribution study following administration of PR001A to the cisterna magna in non-human primates Target Evaluation of tolerability and biodistribution of PR001A in NHPs Regulatory Compliance requirements He-GLP Study drug PR001A Total number of animals 12 cynomolgus monkeys Weight (age) 2-4 kg (2-3 years) Study design Group assignments: Group Dose (vg/r Brain) Number of animals Day 30 Day 90 1 0 3 3 2 7.0 x 10 ¹¹ 3 3 Method and frequency of administration inside a large tank; one slow bolus administered over 3 minutes Compositions The injected solution is provided at a concentration of 4.3 x 10 ¹³ vg/ml. Clinical signs Daily (including food consumption) Body weight Daily FOB Day -14, 7, 30, 60, 90 Clinical laboratory tests (chemistry and hematology) Day -14, 7, 30, 60, 90 Hematology number of red blood cells average corpuscle, volume hemoglobin platelet count hematocrit number of leukocytes average corpuscular hemoglobin blood smear number of reticulocytes medium body differential number of cells blood hemoglobin concentration Clinical biochemistry glucose alkaline alanine aminotransferase urea nitrogen phosphatase creatinine gamma-glutamyltransferase total protein aspartate aminotransferase albumin calcium globulin inorganic phosphorus albumin/globulin sodium ratio cholesterol potassium total bilirubin chloride

- 48 046909

triglycerides Necropsy Day 30 and Day 90 Tissue preservation The following tissues were examined in all animals in all groups: frontal cortex liver hippocampus kidney ventral midbrain heart (apex) periventricular nucleus spleen putamen stomach (pyloric section) dorsal root ganglia (cervical spine) testicles dorsal root ganglia (thoracic region) spinal cord (cervical region) dorsal root ganglia (lumbar region) spinal cord (thoracic region) CSF spinal cord (lumbar) G istopathology Specimens from the above tissue list will be preserved and embedded in paraffin wax for histological examination by H&E. Biodistribution Fabrics from the list above will be collected and stored. Samples from all animals will be analyzed for biodistribution using quantitative PCR (qPCR).

Abbreviations: CSF - cerebrospinal fluid;

FOB - set of functional tests;

GLP - Good Laboratory Practice;

H&E - hematoxylin and eosin;

NHP - non-human primate;

qPCR - quantitative polymerase chain reaction;

vg - vector genome(s)

For this study, tissues were fixed in 10% formaldehyde, embedded in paraffin, and processed to produce hematoxylin and eosin-stained slides. Digital slides were prepared and reviewed by an independent board-certified veterinary pathologist. At both 30 and 90 days post-administration, no changes associated with PR001A were observed, as findings in PR001A-treated animals were either consistent with those typically observed in cynomolgus monkeys (Chamanza et al., Toxicol Pathol. 2010 ;38(4):642-57), and/or were observed in both control animals receiving the excipient and animals receiving PR001 A, and were therefore considered to be due to chance.

There was no effect of PR001A administered into the cisterna magna on weight gain or food intake, as there was no statistical difference between the experimental and control groups during the study. Additionally, there were no changes in FOB scores across groups or time points, indicating no problems or concerns during the lifetime phase of the study. Anti-AAV9 nAb levels in plasma were measured using an in vitro assay. Samples were obtained from study animals at baseline (before ICM administration) and at the time of sacrifice (Day 30 or Day 90). Treatment with PR001A resulted in increased AAV9 nAb titers between baseline and necropsy time at both Day 30 and Day 90, whereas titers in vehicle-treated animals generally remained stable or decreased.

Biodistribution and expression of PR001A.

The biodistribution of the PR001A transgene was assessed in all test samples collected by qPCR; tissues were considered positive when DNA content was at least 50 μg/μg, which is the lower limit of quantitation for the assay. All tissues tested were positive in all PR001A-treated groups, indicating widespread distribution of the drug in the CNS and periphery. Data from selected representative areas for both Day 30 and Day 90 cohorts are shown in FIG. 32.

Taken together, the results of the non-GLP NHP trial PRV-2019-005 demonstrated no safety or toxicity concerns in any in-life or post-mortem assessments. All animals survived to the scheduled date of necropsy, and the postmortem pathologist

- 49 046909 mic analysis revealed no concerns about adverse toxicity.

Safety and toxicology studies conducted with PR001A in NHPs are summarized in Table. 16.

Example 13: Phase 1/2 Human Studies.

Parkinson's disease with GBA1 mutation.

Human subjects will be enrolled in the randomized, double-blind, sham-controlled PR001A rAAV trial. Subject inclusion criteria include single or biallelic GBA1 mutations, moderate to severe Parkinson's disease, and stable use of background Parkinson's disease medications prior to study drug administration. Subjects will be divided into two groups: (1) PR001 low dose (1 x 10 ¹⁴ vg) versus placebo (n=8, 6:2); and (2) PR001 high dose (2x10 ¹⁴ vg) versus Placebo (n=8, 6:2). Each subject will receive study drug via a single ICM injection (intra cisterna magna). The study will include 3 months of biomarker monitoring, 12 months of clinical monitoring, and 5 years of safety and clinical follow-up. The study will analyze: (1) safety and tolerability; (2) key biomarkers including: G-kasoo, GluCer and GluSph (CSF and blood); (3) additional biomarkers including: αsynuclein, NfL (neurofilament light proteins), DAT (dopamine transporter), SPECT (single photon emission computed tomography); and MRI (magnetic resonance imaging); and (4) effectiveness: MDS-UPDRS (International Movement Disorder Society Unified Parkinson's Disease Rating Scale); cognition; as well as ADLs (activities of daily living).

Gaucher disease type 2.

Human subjects (n=15) will be enrolled in the open-label PR001A rAAV study. Subject inclusion criteria include: infants 0-24 months; biallelic GBA1 mutations; neurological signs and symptoms consistent with Gaucher disease type 2; and ongoing standard drug treatment. Each subject will receive study drug via a single ICM injection (intra cisterna magna). The study will include 3 months of biomarker monitoring, 12 months of clinical monitoring, and 5 years of safety and clinical follow-up. The study will analyze: (1) safety and tolerability; (2) key biomarkers including: G-kasoo, GluCer and GluSph (CSF and blood); (3) time to clinical event (eg, tracheostomy, PEG (percutaneous endoscopic gastrostomy) placement, death); and (4) performance: behavior, cognition, gross motor, function, quality of life (QoL).

Example 14 Intravenous administration studies of rAAV encoding G-case.

An intravenous dose range study of PR001 was performed in the D409V Hom mouse model. Homozygous Gbal ^D409V/D409Y (D409V Hom) mice (The Jackson Laboratory, Bar Harbor, Maine) exhibit phenotypes associated with Gaucher disease, including decreased G-case activity (see, e.g., Sardie et al., Proc NatlAcad Sci USA. 2011 ;108(29):12101-6). The study design is shown in Fig. 59. Groups and doses are given in table. 12.

Table 12

Groups and doses for the intravenous study

^Wild-type animals purchased from The Jackson Laboratory (Bar Harbor, Maine), not from the same litter

Intravenous administration of PR001 reduced inflammation in the liver (Fig. 60A). D409V Hom mice exhibited glycolipid accumulation in the liver, which was suppressed in a dose-dependent manner by PR001 administration (Figure 60B; Figure 60C). D409V Hom mice exhibited GluSph accumulation in the brain, which was reduced by PR001 administration (Figure 61B). Intravenous administration of PR001 reduced

- 50 046909 inflammation in the lung (Fig. 62).

An intravenous dose range study of PR001 was also performed in the 4L/PS-NA mouse model. The study design is shown in FIG. 63. Groups and doses are given in table. 13.

Table 13

Groups and doses for the intravenous study

PR001 to 4L/PS-NA mice

Group Vector genomes/kg Control + Auxiliary, substance N.d. 4L/PS-NA + Auxiliary substance N.d. 4L/PS-NA + PR001 Dose 1 12 9.5 x 10 4L/PS-NA + PR001 Dose 2 3.0 x Yu ¹³ 4L/PS-NA + PR001 Dose 3 13 9.5 x 10 4L/PS-NA + PR001 Dose 4 14 3.0 X 10

4L/PS-NA mice showed glycolipid accumulation in the liver, which was reduced by PR001 administration (FIG. 64A; FIG. 64B). 4L/PS-NA mice showed glycolipid accumulation in the brain, which was reduced by PR001 administration (FIG. 65A; FIG. 65B).

Example 15: Studies of rAAVs encoding inhibitory RNA targeting α-synuclein.

HeLa cells were transduced with PR004 or PR014 at several multiplicities of infection (MOI). Both PR004 and PR014 reduced α-synuclein protein levels in a dose-dependent manner (Fig. 66A). PR004 increased G-case activity in a dose-dependent manner (Fig. 66B).

The efficacy of PR004 was assessed in neuronal cultures from induced pluripotent stem cells from a patient with Parkinson's disease (iPSC). Induced pluripotent stem cells obtained from a patient with Parkinson's disease with SNCA triplication differentiated into neurons (Fig. 67A). Neurons transduced with PR004 had increased G-case activity (Fig. 67B) and decreased levels of α-synuclein protein (Fig. 67C).

No off-target effects of the PR004 rAAV vector were observed. The off-target effects of shRNA targeting SNCA from the PR004 vector were assessed in HEK293 cells by qRT-PCR. The expression of 15 genes most similar in sequence to the SNCA target region was assessed (Fig. 68A). The expression of beta and gamma synucleins of members of the SNCA family (SNCB and SNCG, respectively) was also assessed (Fig. 68B). The mRNA levels of these genes were independent of PR004.

The efficacy of PR004 was assessed in the AAV2-55VG4-A53T AAV mouse model of Parkinson's disease (FIG. 69; FIG. 70). AAV2, encoding human SNCA with the A53T mutation, was injected directly into the substantia nigra of adult wild-type mice. Beginning at 4 weeks post-injection, animals exhibited gait abnormalities, changes in dopamine metabolism, loss of dopaminergic neurons, neuroinflammation, and phosphorylated α-synuclein expression. Automated kinematic gait analysis (MotoRater) was performed 4 weeks (FIG. 71A) and 9 weeks (FIG. 71B) after intracerebroventricular injection of PR004. A trend in the treatment effect of PR004 was observed at both time points.

The present invention incorporates by reference the contents of the following documents in their entirety: PCT International Application Publication No. WO 2019/070894; Publication of international application PCT No. WO 2019/070891; US Provisional Patent Applications: Serial No. 62/567311, filed October 3, 2017, entitled Gene Therapy for Lysosomal Disorders; application serial number 62/567319, filed October 3, 2017, entitled Gene Therapy for Lysosomal Disorders; application serial number 62/567301, filed October 3, 2018, entitled Gene Therapy for Lysosomal Disorders; application serial number 62/567310, filed October 3, 2017, entitled Gene Therapy for Lysosomal Disorders; application serial number 62/567303, filed October 3, 2017, entitled Gene Therapy for Lysosomal Disorders; and application serial number 62/567305, filed October 3, 2017, entitled Gene Therapy for Lysosomal Disorders. Having thus described several aspects of at least one embodiment of this invention, it should be appreciated that various changes, modifications and optimizations will be readily accomplished by those skilled in the art. It is intended that such changes, modifications and optimizations form part of this specification and are within the spirit and scope of the invention. Accordingly, the above description and graphics are provided for illustrative purposes only.

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Although several embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily envision many other means and/or structures for performing the functions and/or producing the results and/or one or more of those described herein. advantages, and each of such variations and/or modifications will be considered to be within the scope of the present invention. In a more general context, those skilled in the art will readily understand that all parameters, dimensions, materials and configurations described herein are intended to be exemplary, and that actual parameters, dimensions, materials and/or configurations will depend on the particular application or applications to which the teachings of the present invention are applied. Those skilled in the art will understand or be able to determine, using no more than routine experimentation to this effect, many equivalents to the specific embodiments of the invention described herein. Accordingly, it is to be understood that the above embodiments are presented by way of example only and that within the scope of the appended claims and their equivalents, the present invention may be practiced other than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material and/or method described herein. Moreover, any combination of two or more such features, systems, products, materials and/or methods, unless such features, systems, products, materials and/or methods are mutually incompatible, is within the scope of the present invention.

The singular terms used herein in the description and claims, unless expressly stated otherwise, are to be understood to mean at least one. The phrase and/or, as used herein in the specification and claims, is to be understood to mean one or both of combining elements, that is, elements that are present together in some cases and separate in others. Optionally, other elements other than those specifically identified by the phrase and/or may be present, whether or not related to those elements specifically identified, unless explicitly stated otherwise. Thus, as a non-limiting example, a reference to A and/or B, when used in conjunction with a non-limited language term such as containing, 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.

As used herein in the specification and claims, the term or is to be understood to have the same meaning as and/or as defined above. For example, when separating items in a list, the terms either or and/or should be interpreted as inclusive, that is, including at least one, but also more than one, of a number or list of items, and optionally additional items not listed. Only terms clearly indicating the contrary, such as only one of or exactly one of or, when used in a claim, consisting of will refer to the inclusion of exactly one element from a number or list of elements. In general, the term or, as used herein, should be interpreted only as indicating exclusive alternatives (i.e., one or the other, but not both) when preceded by exclusivity terms such as either, one of, only one of, or exactly one of. As used herein in the specification and claims, the phrase at least one, with respect to a list of one or more elements, is to be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of each element specifically listed in the list of elements, and without excluding any combinations of elements in the list of elements. This definition also allows that elements may not necessarily be present beyond those specifically identified in the list of elements to which at least one phrase refers, whether or not they are related to those specifically identified elements. Thus, by way of 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) may be, in one in an embodiment, to at least one, optionally including more than one, A, without B (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, without A (and optionally including elements other than A); in another embodiment, at least one, optionally including more than one A, and at least one, optionally including more than one B (and optionally including other elements); etc.

The use of ordinal terms such as first, second, third, etc., in a claim to modify a claim element does not of itself imply any priority, preference or dominant order of one claim element over another or a temporal order in which the actions of the method are performed, and these terms are used simply

- 52 046909 as marks to distinguish one element of the claims having a certain name from another element having the same name (but to use an ordinal term) to differentiate the elements of the claims.

It should also be understood that, unless expressly stated otherwise, in any methods claimed herein that include more than one step or action, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are stated.

Each of the US patents, US patent application publications, US patent applications, foreign patents, foreign patent applications and non-patent publications mentioned in this invention is incorporated herein by reference in its entirety.

Sequences.

In some embodiments, an expression cassette encoding one or more gene products (e.g., a first, second, and/or third gene product) contains or consists of (or encodes a peptide having) the sequence set forth in any of SEQ ID NO: 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., fragment) of any of SEQ ID NO: 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.

Numbered Embodiments

Notwithstanding the appended claims, the following numbered embodiments are set forth herein:

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

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

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

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

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

6. The isolated nucleic acid of any one of embodiments 1-5, further comprising a TRY sequence, optionally the TRY sequence being set forth in SEQ ID NO: 28.

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

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

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

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

11. The isolated nucleic acid of any one of embodiments 7-10, wherein the ITR containing the modified sequence D is a 3' ITR.

12. The isolated nucleic acid of any one of embodiments 7-11, further comprising a TRY sequence, optionally wherein the TRY sequence is set forth in SEQ ID NO: 28.

13. An isolated nucleic acid containing an expression construct encoding a SCARB2 protein flanked by two adeno-associated virus (AAV) inverted terminal repeats (ITRs), wherein (i) at least one of the ITRs contains a modified D region relative to the wild-type AAV2 ITR (SEQ ID NO: 29); and/or

- 53 046909 (ii) the SCARB2 protein is encoded by a codon-optimized nucleic acid sequence.

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

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

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

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

18. The isolated nucleic acid of any one of embodiments 13-17, further comprising a TRY sequence, optionally wherein the TRY sequence is set forth in SEQ ID NO: 28.

19. An isolated nucleic acid containing an expression construct encoding a first gene product and a second gene product, each gene product being independently selected from the gene products or portions thereof presented in Table. 1.

20. The isolated nucleic acid of embodiment 19, wherein the first gene product is a G-case protein or a portion thereof.

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.

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

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

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

25. The isolated nucleic acid of any one of embodiments 19-23, further comprising a sequence encoding a self-cleaving peptide, optionally said self-cleaving peptide being T2A.

26. The isolated nucleic acid of any one of embodiments 19-24, 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, optionally one of the sequences ITR does not have a functional terminal resolution site.

27. The isolated nucleic acids of embodiment 26, wherein at least one of the ITRs contains a modified D region relative to the wild-type AAV2 ITR (SEQ ID NO: 29).

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

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

30. The isolated nucleic acid of any one of embodiments 27-29, further comprising a TRY sequence, optionally wherein the TRY sequence is set forth in SEQ ID NO: 28.

31. The isolated nucleic acid of any one of embodiments 1-30, having the sequence specified in any of SEQ ID NOs: 1-12, 14, 16 and 18.

32. A vector containing the isolated nucleic acid of any one of embodiments 1-31.

33. The vector of embodiment 32, wherein said vector is a plasmid.

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

35. A composition comprising the isolated nucleic acid of any one of embodiments 1-31 or the vector of any of embodiments 32-34.

- 54 046909

36. A host cell comprising the isolated nucleic acid of any one of embodiments 1-31 or the vector of any of embodiments 32-34.

37. Recombinant adeno-associated virus (rAAV), containing:

(i) capsid protein; and (ii) the isolated nucleic acid of any one of embodiments 1-31 or the vector of any of embodiments 32-34.

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

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

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

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

42. The method of embodiment 41, wherein said direct injection into the subject's CNS comprises convection enhanced delivery (CED).

43. The method of any one of embodiments 40-42, wherein the administration involves a peripheral injection, wherein the peripheral injection is not necessarily an intravenous injection.

44. A method of treating a subject suffering from Gaucher disease type 2 or Gaucher disease type 3, comprising administering to the subject a recombinant adeno-associated virus (rAAV) containing:

(i) an rAAV vector containing a nucleic acid containing an expression construct containing a promoter operably linked to a transgene insert encoding a glucocerebrosidase (G-case) protein, wherein the transgene insert contains the nucleotide sequence of SEQ ID NO: 15; and (ii) AAV9 capsid protein.

45. The method of embodiment 44, wherein the rAAV is administered to the subject at a dose of about 5x10 ¹⁰ vg/g brain to about 5x1ο ¹¹ vg/g brain.

46. The method of embodiment 44, wherein the rAAV is administered to the subject at a dose of about 1.3 x 1011 vg/g brain.

47. A method of treating a subject suffering from Parkinson's disease with a glucocerebrosidase 1 (GBA1) mutation, comprising administering to said subject a recombinant adeno-associated virus (rAAV) containing:

(i) an rAAV vector containing a nucleic acid containing an expression construct containing a promoter operably linked to a transgene insert encoding a G-case protein, wherein the transgene insert contains the nucleotide sequence of SEQ ID NO: 15; and (ii) AAV9 capsid protein.

47. The method of embodiment 46, wherein the rAAV is administered to the subject at a dose of about 5x10 ¹³ vg to about 5x10 ¹⁴ vg.

48. The method of embodiment 46, wherein the rAAV is administered to the subject at a dose of about 1x10 ¹⁴ vg or about 2x10 ¹⁴ vg.

49. The method of any one of embodiments 44-48, wherein the rAAV is administered by suboccipital injection into the cisterna magna.

50. A method of treating a subject suffering from type 1 Gaucher disease, comprising administering to said subject a recombinant adeno-associated virus (rAAV) containing:

(i) an rAAV vector containing a nucleic acid containing an expression construct containing a promoter operably linked to a transgene insert encoding a G-case protein, wherein the transgene insert contains the nucleotide sequence of SEQ ID NO: 15; and (ii) AAV9 capsid protein.

51. The method of embodiment 50, wherein the rAAV is administered to the subject at a dose of about 5x10 ¹³ vg to about 5x10 ¹⁴ vg.

52. The method of embodiment 50 or 51, wherein the rAAV is administered intravenously. 53. A method of treating a subject suffering from synucleinopathy or parkinsonism, comprising administering to said subject a recombinant adeno-associated virus (rAAV) containing:

(i) an rAAV vector containing a nucleic acid containing an expression construct,

- 55 046909 containing a transgene containing (a) a sequence encoding a G-case protein containing the nucleotide sequence SEQ ID NO: 15; and (b) a sequence encoding an inhibitory nucleic acid comprising the nucleotide sequence of SEQ ID NO: 20; and (ii) AAV9 capsid protein.

54. A method of treating a subject suffering from synucleinopathy or parkinsonism, comprising administering to said subject a recombinant adeno-associated virus (rAAV) containing:

(i) an rAAV vector containing a nucleic acid containing an expression construct containing a transgene containing a sequence encoding an inhibitory nucleic acid containing the nucleotide sequence of SEQ ID NO: 20; and (ii) AAV9 capsid protein.

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 mutation, Lewy body disease, dementia with Lewy bodies, dementia with Lewy bodies with GBA1 mutation, progressive supranuclear palsy or corticobasal syndrome.

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

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

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

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

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

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

62. The method of any one of embodiments 44-61, 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.

63. A method of treating a subject suffering from type 2 Gaucher disease or type 3 Gaucher disease, comprising administering to the subject an rAAV containing:

(i) an rAAV vector containing a nucleic acid containing, in order 5' to 3':

(a) AAV2 ITR;

(b) CMV enhancer;

(c) CBA promoter;

(d) a transgene insert encoding a G-case protein, wherein the transgene insert contains the nucleotide sequence of SEQ ID NO: 15;

(f) WPRE;

(f) bovine growth hormone polyA signal tail; and (g) AAV2 ITR; and (ii) AAV9 capsid protein;

wherein rAAV is administered to a subject at a dose of from about 5x10 ¹⁰ vg/g brain to about 5x1ο ¹¹ vg/g brain.

64. A method of treating a subject suffering from Parkinson's disease with a GBA1 mutation, comprising administering to the subject an rAAV containing: (i) an rAAV vector containing a nucleic acid containing, in the order 5' to 3':

(a) AAV2 ITR;

(b) CMV enhancer;

(c) CBA promoter;

(d) a transgene insert encoding a G-case protein, wherein the transgene insert contains the nucleotide sequence of SEQ ID NO: 15;

(e) WPRE;

(f) bovine growth hormone polyA signal tail; and (g) AAV2 ITR; and (ii) AAV9 capsid protein;

wherein the rAAV is administered to the subject at a dose of about 5x 101 ³ vg to about 5x 101 ⁴ vg.

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

- 56 046909

66. The method of any one of embodiments 44-52 and 63-65, wherein rAAV is administered in a formulation containing about 20 mM Tris, pH 8.0, about 1 mM MgCl ₂ , about 200 mM NaCl, and about 0.001% w/v poloxamer 188.

67. Pharmaceutical composition containing (i) rAAV containing:

(a) an rAAV vector containing a nucleic acid containing an expression construct containing a promoter operably linked to a transgene insert encoding a G-case protein, wherein the transgene insert contains the nucleotide sequence of SEQ ID NO: 15; and (b) AAV9 capsid protein; and (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. poloxamer 188.

68. rAAV containing:

(i) an rAAV vector containing a nucleic acid containing an expression construct containing a promoter operably linked to a transgene insert encoding a G-case protein, wherein the transgene insert contains the nucleotide sequence of SEQ ID NO: 15; and (ii) an AAV9 capsid protein, for use in a method of treating Gaucher disease type 1, Gaucher disease type 2, Gaucher disease type 3 or Parkinson's disease with a GBA1 mutation in a subject.

69. rAAV containing:

(i) an rAAV vector containing a nucleic acid containing an expression construct containing a promoter operably linked to the transgene insert, containing:

(a) a G-case protein coding sequence comprising the nucleotide sequence of SEQ ID NO: 15; and (b) a sequence encoding an inhibitory nucleic acid comprising the nucleotide sequence of SEQ ID NO: 20; and (ii) an AAV9 capsid protein, for use in a method of treating synucleinopathy or parkinsonism in a subject.

70. rAAV containing:

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

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Summary of preclinical in vivo pharmacology (efficacy) studies

Table 14

Study number Target Status results PRV-2017-001 Validation of the SVE mouse model for studying Scaza deficiency Completed 25 mg/kg SBE reproduces the main characteristics of Skaza deficiency Accumulation of glycolipids (the combination of GluSph and galactosylsphingosine [GalSph]) relative to the control Motor activity deficit in the rotating drum test PRV-2018-002 Demonstrate the effectiveness of the highest possible dose of PR001B ³ in SBE mice Completed Wide biodistribution of the PR001B vector genome Increasing the activity of Skaza correlated with a decrease in glycolipid substrate accumulation. Improved behavioral performance when tested on a rotating drum. No adverse effects associated with PR001B were observed. PRV-2018-005 Determine effective doses of ICV PR001A in a mouse model of SES. Completed Wide biodistribution of the PR001A vector genome Increased activity of Skaza and decreased accumulation of pathological glycolipid substrate Reduction of astrogliosis and microgliosis Elimination of motor behavior deficits No adverse effects associated with PR001A were observed.

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PRV-2018-007 Long term (6 months) persistence of ICV PR001A in a mouse model of SVE Completed Preservation of the vector genome, persistent increase in Scaza activity and decrease in glycolipid levels 6 months after treatment No adverse effects associated with PR001A were observed. PRV-2018-008 Additional ICV Dose Range PR001A in a mouse model of FES Completed Wide biodistribution of the PR001A vector genome Increased cortical activity and decreased storage abnormal glycolipid substrate No adverse effects associated with PR001A were observed. PRV-2018-025 Additional ICV Dose Range PR001A in a mouse model of FES Ongoing Biodistribution of the PR001A vector genome in the cortex Increased cortical activity and decreased storage pathological glycolipid substrate Elimination of motor behavior deficits PRV-2018-006 Demonstrate the effectiveness of ICV PR001A in mouse models 4L/PS-NA Completed Wide biodistribution of the PR001A vector genome Increased activity of Skase in the central nervous system and periphery, associated with decreased accumulation of glycolipid substrate Trend towards improvement of motor functions Reduced accumulation of insoluble ossynuclein No adverse effects associated with PR001A were observed. PRV-2018-011 Dose range of ICV PR001A in a genetic mouse model 4L/PS-NA Completed Wide biodistribution of the PR001A vector genome Increased activity of Skaza and decreased accumulation of pathological glycolipid substrate

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Addressing motor behavior deficits No adverse effects associated with PR001A were observed. PRV-2018-019 Effect of ICV PR001A on α-synuclein in SBE-treated transgenic mice. Ongoing Biodistribution of the genome of the PR001A vector in the cortex and a decrease in the level of glycolipids. A decrease in the accumulation of insoluble α-synuclein and the ratio insoluble and soluble accumulation of insoluble α-synuclein No adverse effects associated with PR001A were observed. PRV-2019-001 Effect of ICV PR001A on α-synuclein in SBE-treated transgenic mice. Ongoing Biodistribution of the PR001A vector genome in the cortex and increased Skaze activity in the cortex No adverse effects associated with PR001A were observed.

Abbreviations: CBE - conduritol-v-epoxide;

CNS - central nervous system;

G-case - glucocerebrosidase;

GluSph - glucosylsphingosine;

ICV - intracerebroventricular;

vg - vector genome

PR001B is a version of PR001A with an altered D domain;

PR001A and PR001B are otherwise identical

Table 15

Summary of Safety Assessments in PR001A Efficacy Studies in Mice

Purpose of the study Study number Route of administration View Dose (vg/r brain) General dose PR001 A(vg) Injection volume (µl) Moment necropsy time A Efficiency with selected safety assessment criteria PRV-2018- 005 ICV C57BL/6J mice treated with eSBE 1.3 x ¹⁰ 4.2 x ¹⁰ 1.3 x 10 ¹¹ 2.0 x 10 ⁹ 6.2 x 10 ⁹ 2.0 x ¹⁰ 4 Days 35-37 Efficiency with selected criteria PRV-2018- 006 ICV Mice 4L/PS-NA 3.7 x Yu ¹⁰ 1.5 x Yu ¹⁰ 3 Week 15 assessments security

Abbreviations: CBE - conduritol-v-epoxide;

ICV - intracerebroventricular;

vg - genome of vector ^a After application of PR001A

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Claims (23)

Table 16 Summary of safety assessments in PR001A safety studies in NHPs Target Number Path View Dose General Volume Moment of research research introduction (vg/r dose injections time I I I cephalopod PR001 and (µl) brain necropsy) A(vg) A Pilot Non-GLP Toxicology Study PRV-2018-015 ICM ICM + IPa (Midbrain) Cynomolgus 2.0 x 10 ¹¹ 2.1 x 10 ¹¹ 1.47 x 10 ¹³ 1.53 x 10 ¹³ 500 520 Day 18 GLP toxicology study PRV-2018-016 ICM Cynomolgus macaque 6.2 x ¹⁰ 2.3 x 10 ¹¹ 4.6 x 10 ¹² 1.7 x 10 ¹³ 1200 Days 7, 30, 183 He-GLP toxicology study PRV-2019-005 ICM Cynomolgus macaque 7.0 x 10 ¹¹ 5.2 x 10 ¹³ 1200 Day 30.90 Abbreviations: GLP - Good Laboratory Practice; ICM - inside a large tank; IPa - intraparenchymal; NHP - non-human primate; vg - vector genome CLAIM 1. A method of treating a subject suffering from Gaucher disease type 2 or Gaucher disease type 3, comprising administering to the subject a recombinant adeno-associated virus (rAAV) containing: (i) an rAAV vector containing a nucleic acid containing an expression construct containing a promoter operably linked to a transgene insert encoding a glucocerebrosidase (G-case) protein, wherein the transgene insert contains the nucleotide sequence of SEQ ID NO: 15; and (ii) AAV9 capsid protein. 2. The method according to claim 1, characterized in that rAAV is administered to the subject at a dose of 5x10 ¹⁰ vg/g brain to 5x 10 vg/g brain. 3. The method according to claim 1, characterized in that rAAV is administered to the subject at a dose of 1.3x1011 vg/g brain. 4. A method of treating a subject suffering from Parkinson's disease with a glucocerebrosidase-1 (GBA1) mutation, comprising administering to said subject a recombinant adeno-associated virus (rAAV) containing: (i) an rAAV vector containing a nucleic acid containing an expression construct containing a promoter operably linked to a transgene insert encoding a G-case protein, wherein the transgene insert contains the nucleotide sequence of SEQ ID NO: 15; and (ii) AAV9 capsid protein. 5. The method according to claim 4, characterized in that rAAV is administered to the subject at a dose of 5x10 ¹³ vg to 5x10 ¹⁴ vg. 6. The method according to claim 4, characterized in that the rAAV is administered to the subject at a dose of 1x10 vg or 2x10 vg. 7. Method according to any one of claims 1 to 6, characterized in that the rAAV is administered by suboccipital injection into the cisterna magna. 8. A method of treating a subject suffering from type 1 Gaucher disease, comprising administering to said subject a recombinant adeno-associated virus (rAAV) containing: (i) an rAAV vector containing a nucleic acid containing an expression construct containing a promoter operably linked to a transgene insert encoding a G-case protein, wherein the transgene insert contains the nucleotide sequence of SEQ ID NO: 15; and (ii) AAV9 capsid protein. 9. The method according to claim 8, characterized in that rAAV is administered to the subject at a dose of 5x10 ¹³ vg to 5x10 ¹⁴ vg. 10. Method according to claim 8 or 9, characterized in that rAAV is administered intravenously. 11. The method according to any one of claims 1 to 10, wherein the promoter is a chicken beta actin (CBA) promoter. - 61 046909 12. The method according to any one of claims 1 to 11, wherein the rAAV vector further comprises a cytomegalovirus (CMV) enhancer. 13. The method according to any one of claims 1 to 12, wherein the rAAV vector further comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). 14. The method according to any one of claims 1 to 13, wherein the rAAV vector further comprises a bovine growth hormone polyA signal tail. 15. The method according to any one of claims 1 to 14, wherein the nucleic acid contains two adeno-associated virus inverted terminal repeat (ITR) sequences flanking the expression construct, wherein the first ITR sequence is a 5' ITR and the second ITR sequence is a 3' ITR. 16. The method of claim 15, wherein each of the two ITR sequences is a wild-type AAV2 ITR sequence. 17. The method of claim 15 or 16, 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. 18. A method of treating a subject suffering from type 2 Gaucher disease or type 3 Gaucher disease, comprising administering to the subject an rAAV containing: (i) an rAAV vector containing a nucleic acid containing, in order 5' to 3': (a) AAV2 ITR; (b) CMV enhancer; (c) CBA promoter; (d) a transgene insert encoding a G-case protein, wherein the transgene insert contains the nucleotide sequence of SEQ ID NO: 15; (e) WPRE; (f) bovine growth hormone polyA signal tail; and (g) AAV2 ITR; and (ii) AAV9 capsid protein; wherein rAAV is administered to the subject at a dose of 5x 10 ¹⁰ vg/g brain to 5x 10 ¹¹ vg/g brain. 19. A method of treating a subject suffering from Parkinson's disease with a GBA1 mutation, comprising administering to the subject an rAAV containing: (i) an rAAV vector containing a nucleic acid containing, in order 5' to 3': (a) AAV2 ITR; (b) CMV enhancer; (c) CBA promoter; (d) a transgene insert encoding a G-case protein, wherein the transgene insert contains the nucleotide sequence of SEQ ID NO: 15; (e) WPRE; (f) bovine growth hormone polyA signal tail; and (g) AAV2 ITR; and (ii) AAV9 capsid protein; wherein rAAV is administered to the subject at a dose of 5x 10 ¹³ vg to 5x 10 ¹⁴ vg. 20. The method according to claim 18 or 19, characterized in that the rAAV is administered by suboccipital injection into the cisterna magna. 21. Method according to any one of claims 1-10 and 18-20, characterized in that rAAV is administered in a composition containing 20 mM Tris, pH 8.0, 1 mM MgCl ₂ , 200 mM NaCl and 0.001% w/v . poloxamer 188. 22. Pharmaceutical composition containing: (i) rAAV containing: (a) an rAAV vector containing a nucleic acid containing an expression construct containing a promoter operably linked to a transgene insert encoding a G-case protein, wherein the transgene insert contains the nucleotide sequence of SEQ ID NO: 15; and (b) AAV9 capsid protein; and (ii) 20 mM Tris, pH 8.0, (iii) about 1 mM MgCl ₂ , (iv) 200 mM NaCl, (v) 0.001% w/v. poloxamer 188. 23. Use of rAAV containing: (a) an rAAV vector containing a nucleic acid containing an expression construct containing a promoter operably linked to a transgene insert encoding a G-case protein, wherein the transgene insert contains the nucleotide sequence of SEQ ID NO: 15; and (b) AAV9 capsid protein, for the treatment of Gaucher disease type 1, Gaucher disease type 2, Gaucher disease type 3 or Parkinson's disease with a GBA1 mutation in a subject. -