EA046777B1 - GENE THERAPY FOR LYSOSOMAL DISORDERS - Google Patents

Description

Cross-reference to related applications

This application claims priority to U.S. Provisional Patent Application No. 62/988,665, filed March 12, 2020, U.S. Provisional Patent Application No. 62/960,471, filed January 13, 2020, U.S. Provisional Patent Application No. 62/954,089, filed December 27, 2019, U.S. Provisional Patent Application No. 62/934,450, filed November 12, 2019, and U.S. Provisional Patent Application No. 62/831,846, filed April 10, 2019. The disclosures of each of these applications are incorporated herein by reference in their entirety.

Description of a text file presented in electronic form

The contents of the text file provided in electronic form are incorporated herein by reference in their entirety: copy of the sequence listing in machine-readable format (file name: PRVL_010_05WO_SeqList.txt, Date of entry: April 10, 2020; file size: ~612902 bytes).

Field of technology

The present invention relates to the field of gene therapy and methods of using it.

State of the art

Gaucher disease is a rare inborn error of glycosphingolipid metabolism due to deficiency of lysosomal acid β-glucocerebrosidase (G-case, GBA). Patients suffer from non-CNS manifestations 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 options available to treat the peripheral disease and underlying clinical manifestations of the bone marrow and visceral hematopoietic dysfunction, including enzyme replacement therapy as described below, chaperone-like small molecule drugs that bind to the defective G-case and increase stability, and substrate-reducing therapy that blocks the production of the substrate that accumulates in Gaucher disease, leading to symptoms and pathology. However, other aspects of Gaucher disease (especially those associated with skeletal and brain involvement) appear to be refractory to treatment.

Progranulin (PGRN) is an accessory protein associated with lysosomal function. PGRN is encoded by the GRN gene. Haploinsufficiency of GRN in humans results in an approximately 90% risk of developing FTD-GRN (GRN-mutated frontotemporal dementia), a neurodegenerative disease characterized by executive function impairment, behavioral changes, and language difficulties, accompanied by atrophy of the frontal and temporal lobes. There is no disease-modifying treatment for patients with FTD.

The essence of the invention

The present invention provides a method of treating a subject having or suspected of having frontotemporal dementia with a GRN mutation, said method comprising administering to the subject a recombinant adeno-associated virus (rAAV) comprising (i) an rAAV vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a transgene insert encoding a PGRN protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 68; and (ii) an AAV9 capsid protein. In some embodiments, the rAAV is administered to the subject at a dose of about 1 x 10 ¹³ vector genomes (vg) to about 7 x 10 ¹⁴ vg. In some embodiments, the rAAV is administered by injection into the cerebellomedullary cistern.

In some embodiments, the promoter operably linked to the transgene insert encoding the PGRN protein is the 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 with or suspected of having frontotemporal dementia with a GRN mutation, said method comprising administering to the subject an rAAV comprising: (i) an rAAV vector comprising a nucleic acid comprising, in order from 5' to 3': (a) an AAV2 ITR; (b) a CMV enhancer; (c) a CBA promoter; (d) a transgene insert encoding a PGRN protein, said transgene insert comprising the nucleotide sequence

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SEQ ID NO: 68; (e) WPRE; (f) bovine growth hormone polyA signal tail; and (g) AAV2 ITR; and (ii) AAV9 capsid protein. In some embodiments, rAAV is administered to the subject at a dose of about 1x10 ¹³ vg to about 7x10 ¹⁴ vg. In some embodiments, rAAV is administered by injection into the cisterna cerebellomedullaris.

In some embodiments, the rAAV is administered in a formulation comprising about 20 mM Tris, pH 8.0, about 1 mM _MgCl2 , about 200 mM NaCl, and about 0.001% poloxamer 188 (w/v). The present invention provides a pharmaceutical composition comprising (i) a rAAV comprising: (a) an rAAV vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a transgene insert encoding a PGRN protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 68; and (b) an 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 comprising: (a) an rAAV vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a transgene insert encoding a PGRN protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 68; and (b) an AAV9 capsid protein for use in a method of treating frontotemporal dementia with a GRN mutation in a subject.

This document provides a method for quantifying the level of PGRN protein in a cerebrospinal fluid (CSF) sample, comprising: (1) diluting the CSF sample in a master mixture containing dithiothreitol (DTT) and sample buffer; (2) loading the diluted CSF sample, anti-progranulin antibody, a secondary antibody that detects anti-progranulin antibody, luminol, and peroxide into the wells of a capillary cartridge; (3) loading the capillary cartridge into an automated Western blot immunoassay instrument; (4) using the automated Western blot immunoassay instrument to calculate signal intensity, peak area, and signal-to-noise ratio; and (5) quantifying the level of progranulin protein in the CSF sample as the peak area of immunoreactivity to the anti-progranulin antibody.

Brief description of graphic materials

Fig. 1 is a schematic representation of one embodiment of a vector containing an expression construct encoding a G-case (e.g., GBA1 or a portion thereof).

Fig. 2 is a schematic representation of one embodiment of a vector comprising an expression construct encoding a G-case (e.g., GBA1 or a portion thereof) and LIMP2 (SCARB2) or a portion thereof. The coding sequences of G-case and LIMP2 are separated by an internal ribosome entry site (IRES).

Fig. 3 is a schematic representation of one embodiment of a vector comprising an expression construct encoding a G-case (e.g., 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 embodiment of a vector comprising an expression construct encoding a G-case (e.g., 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 embodiment of a vector comprising an expression construct encoding a G-case (e.g., GBA1 or a portion thereof), a prosaposin (e.g., PSAP or a portion thereof), and an interfering RNA for α-Syn.

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

Fig. 7 is a schematic representation of one embodiment of a vector comprising an expression construct encoding a G-case (e.g., GBA1 or a portion thereof). In this embodiment, the vector comprises a CBA promoter element (CBA) consisting of four parts: the CMV enhancer (CMVe), the CBA promoter (CBAp), exon 1, and an intron (int) for constitutive expression of a codon-optimized human GBA1 coding sequence. The 3' region also comprises a WPRE regulatory element followed by a bGH polyA tail. Three transcriptional activation sites are included at the 5' end of the promoter region: TATA, RBS, and YY1. Flanking ITRs allow for proper packaging of insert sequences. Two variants of the 5' ITR sequence (insert) were evaluated; they have several nucleotide differences in the 20-nucleotide region of the D ITR of wild-type AAV2. In some embodiments, the rAAV vector comprises the nucleotide sequence of the D domain shown in the top row. In some embodiments, the rAAV vector comprises a mutant D domain (e.g., S domain, nucleotide changes are shown in the bottom row).

Fig. 8 is a schematic representation of one embodiment of the vector

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Fig. 9 shows representative data for delivery of rAAV containing a transgene encoding a G-case (e.g., GBA1 or a portion thereof) in the VES mouse model of Parkinson's disease. Daily IP delivery of PBS vehicle, 25 mg/kg VES, 37.5 mg/kg VES, or 50 mg/kg VES (left to right) began at P8. Survival (upper left) was monitored twice daily and weight (upper right) was monitored daily. All groups started with n=8. Behavior was assessed by total distance walked in an open field (lower left) at P23 and ability to remain on a rotating drum (lower center) at P24. Levels of G-case substrates were analyzed in the cortex of mice in the PBS and 25 mg/kg VES treatment groups with and without VES withdrawal (Day 3) and without VES withdrawal (Day 1). Cumulative GluSph and GalSph levels (bottom right) are given in pmol per mg fresh tissue weight. Values are mean. Error bars represent SEM. *p<0.05; **p<0.01; ***p<0.001, nominal p-values for treatment groups by linear regression.

Fig. 10 is a schematic representation of one design for a maximum rAAV dose study in a CBE mouse model. Briefly, rAAV was administered by ICV injection at P3, and daily CBE treatment was initiated at P8. Behavior was assessed in open field and drum retention assays at P24–25, and substrate levels were measured at P36 and P38.

Fig. 11 shows representative data for the assessment of the maximum lifetime rAAV dose in the VES mouse model. At P3, mice were administered either excipient or 8,8e9 vg rAAV-GBA1 via ICV delivery. Daily IP delivery of PBS or 25 mg/kg VES was initiated at P8. At the end of the study, half of the mice were sacrificed one day after receiving the last dose of VES at P36 (day 1), while the remaining half were off VES for 3 days before being sacrificed at P38 (day 3). All treatment groups (excipient+PBS n=8, rAAV-GBA1+PBS n=7, excipient+VES n=8, and variant+VES n=9) were weighed daily (top left) and weight analyzed at P36 (top right). Behavior was assessed by total distance traveled in the open field at P23 (bottom left) and time to fall from the rotating drum at P24 (bottom right), estimated for each animal as the median value over 3 trials. Considering lethality, n=7 for the adjuvant+SBE group for behavioral analyses and n=8 for all other groups. Mean values are presented for animals. Error bars represent SEM. *p<0.05; ***p<0.001, nominal p-values for treatment groups from linear regression in SBE-treated animals.

Fig. 12 shows representative data for the biochemical assessment of the maximum lifetime rAAV dose in the CBE mouse model. Cortices from all treatment groups (excipient+PBSn=8, variant+PBSn=7, excipient+CBE n=7, and variant+CBE n=9) were used to measure G-case activity (upper left), GluSph levels (upper right), GluCer levels (lower left), and vector genomes (lower right) in the groups before (Day 1) or after (Day 3) CBE withdrawal. Biodistribution is presented as vector genomes per 1 μg genomic DNA. Mean values are shown. Error bars represent SEM. (*) p<0.1; **p<0.01; ***p<0.001, nominal p-values for treatment groups by linear regression in SVE-treated animals, adjusted for collection days and sex as covariates.

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

Fig. 14 shows representative biodistribution data of the variant in the CBE mouse model. The presence of vector genomes was assessed in the liver, spleen, kidney, and gonads for all treatment groups (excipient+PBS n=8, variant+PBS n=7, excipient+CBE n=7, and variant+CBE n=9). Biodistribution is presented as vector genomes per 1 μg genomic DNA. The presence of vector genome was quantified by qPCR using a vector reference standard curve; genomic DNA concentration was estimated by measuring the A260 optical density. Values are shown as mean. Error bars represent SEM. (*)p<0.05; **p<0.01; ***p<0.001, nominal p-values for treatment groups from linear regression in CBE-treated animals adjusted for collection days and sex as covariates.

Fig. 15 shows representative data for the maximum lifetime rAAV dose assessment in the CBE mouse model. Mice received excipient or one of three different doses of rAAVGBA1 by ICV delivery at P3: 3.2e9 vg, 1.0e10 vg, or 3.2e10 vg. Daily IP dosing at 25 mg/kg CBE was started at P8. Mice treated with excipient and CBE or excipient and PBS served as controls. All treatment groups were started with n=10 (5M/5F) per group. All mice were sacrificed one day after the last dose of CBE (P38–P40). All treatment groups were weighed daily and their weights were analyzed at P36. Locomotor activity was assessed by the time to fall from a rotating drum at P24 and by the time to turning on a tapered beam at

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P30. Due to early lethality, the numbers of mice used in behavioral assays were: excipient+PBS n=10, excipient+CBEn n=9, and 3.2e9 vg rAAV-GBA1+CBEn n=6, 1.0e10 vg rAAV-GBA1+CBEn n=10, 3.2e10 vg rAAV-GBA1+CBEn=7. Methods are shown. Error bars represent SEM. *p<0.05; **p<0.01 for nominal p-values for CBEn treatment groups by linear regression adjusting for sex as a covariate.

Fig. 16 shows representative data for the biochemical assessment of the maximum lifetime dose of rAAV in the CBE mouse model. G-case activity, GluSph levels, GluCer levels, and vector genomes were measured in the cerebral cortex of animals from all treatment groups (excipient+PBS n=10, excipient+CBE n=9, and 3.2e9 vg rAAV-GBA1+CBE n=6, 1.0e10 vg rAAV-GBA1+CBE n=10, 3.2e10 vg rAAV-GBA1+CBE n=7). G-case activity is given as ng G-case/mg total protein. GluSph and GluCer levels are given as pmol/mg fresh tissue weight. Biodistribution is given as vector genomes/μg genomic DNA. The presence of the vector genome was quantified by qPCR using a vector reference standard curve; genomic DNA concentration was estimated by measuring the optical density A260. The presence of the vector genome was also measured in the liver (E). Values are mean. Error bars represent SEM. **p<0.01; ***p<0.001 for nominal p-values for the SVE treatment groups by linear regression, adjusting for sex as a covariate.

Fig. 17 shows representative data for the tapered beam behavior assay at the highest rAAV-GBA1 dose in a genetic mouse model. The locomotor activity of the treatment groups (WT+excipient, n=5), 4L/PS-NA+excipient (n=6), and 4L/PSNA+rAAV-GBA1 (n=5)) was assessed using the beam walking test 4 weeks after rAAV-GBA1 administration. Total slip counts and active time are presented as the total of 5 trials on different beams. Speed and slip-to-speed are presented as the mean of 5 trials on different beams. Mean values are shown. Error bars represent SEM.

Fig. 18 shows representative data on the in vitro expression of rAAV constructs encoding the progranulin (PGRN) protein. The left panel shows a standard curve of the progranulin (PGRN) ELISA. The bottom panel shows the dose-response of PGRN expression measured by ELISA in cell lysates of rAAV-transduced HEK293T cells. MOI=multiplicity of infection (vector genomes per cell).

Fig. 19 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. The data indicate that transfection of HEK293 cells with each construct resulted in overexpression of the transgenes of interest compared to mock-transfected cells.

Fig. 20 is a schematic depicting rAAV vectors having a 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 an ITR on the inside of the vector (e.g., proximal to the transgene insertion into the vector).

Fig. 21 is a schematic representation of one embodiment of a vector comprising an expression construct encoding GBA2 or a portion thereof and interfering RNA for α-Syn.

Fig. 22 is a schematic representation of one embodiment of a vector comprising an expression construct encoding a G-case (e.g., GBA1 or a portion thereof) and a galactosylceramidase (e.g., GALC or a portion thereof). Expression of the G-case and galactosylceramidase coding sequences is separated by a T2A self-cleaving peptide sequence.

Fig. 23 is a schematic representation of one embodiment of a vector comprising an expression construct encoding a G-case (e.g., GBA1 or a portion thereof) and a galactosylceramidase (e.g., GALC or a portion thereof). Expression of the G-case and galactosylceramidase coding sequences is separated by a T2A self-cleaving peptide sequence.

Fig. 24 is a schematic representation of one embodiment of a vector comprising an expression construct encoding a G-case (e.g., GBA1 or a portion thereof), a cathepsin B (e.g., CTSB or a portion thereof), and an interfering RNA for α-Syn. The expression of the G-case and cathepsin B coding sequences is separated by a T2A self-cleaving peptide sequence.

Fig. 25 is a schematic representation of one embodiment of a vector comprising an expression construct encoding a G-case (e.g., GBA1 or a portion thereof), sphingomyelin phosphodiesterase 1 (e.g., SMPD1 or a portion thereof), and interfering RNA for αSyn.

Fig. 26 is a schematic representation of one embodiment of a vector comprising an expression construct encoding a G-case (e.g., GBA1 or a portion thereof) and a galactosidase

- 4 046777 tosylceramidase (e.g., GALC or part thereof). The coding sequences of G-case and galactosylceramidase are separated by an internal ribosome entry site (IRES).

Fig. 27 is a schematic representation of one embodiment of a vector comprising an expression construct encoding a G-case (e.g., GBA1 or a portion thereof) and a cathepsin B (e.g., CTSB or a portion thereof). Expression of the G-case and cathepsin B coding sequences is each driven by a separate promoter.

Fig. 28 is a schematic representation of one embodiment of a vector comprising an expression construct encoding a G-case (e.g., GBA1 or a portion thereof), a GCH1 (e.g., GCH1 or a portion thereof), and an interfering RNA for α-Syn. The coding sequences for G-case and GCH1 are separated by a T2A self-cleaving peptide sequence.

Fig. 29 is a schematic representation of one embodiment of a vector comprising an expression construct encoding a G-case (e.g., GBA1 or a portion thereof), a RAB7L1 (e.g., a RAB7L1 or a portion thereof), and an interfering RNA for α-Syn. The coding sequences for the G-case and RAB7L1 are separated by a T2A self-cleaving peptide sequence.

Fig. 30 is a schematic representation of one embodiment of a vector comprising an expression construct encoding a G-case (e.g., GBA1 or a portion thereof), a GCH1 (e.g., GCH1 or a portion thereof), and an interfering RNA for α-Syn. Expression of the coding sequences for G-case and GCH1 is an internal ribosome entry site (IRES).

Fig. 31 is a schematic representation of one embodiment of a vector comprising an expression construct encoding VPS35 (e.g., VPS35 or a portion thereof) and interfering RNA for α-Syn and TMEM106B.

Fig. 32 is a schematic representation of one embodiment of a vector comprising an expression construct encoding a G-case (e.g., GBA1 or a portion thereof), IL-34 (e.g., IL34 or a portion thereof), and interfering RNA for α-Syn. The coding sequences for G-case and IL-34 are separated by a T2A self-cleaving peptide sequence.

Fig. 33 is a schematic representation of one embodiment of a vector comprising an expression construct encoding a G-case (e.g., GBA1 or a portion thereof) and IL-34 (e.g., IL34 or a portion thereof). The coding sequences of G-case and IL-34 are separated by an internal ribosome entry site (IRES).

Fig. 34 is a schematic representation of one embodiment of a vector comprising an expression construct encoding a G-case (e.g., GBA1 or a portion thereof) and a TREM2 (e.g., TREM2 or a portion thereof). Expression of the G-case and TREM2 coding sequences is each driven by a separate promoter.

Fig. 35 is a schematic representation of one embodiment of a vector comprising an expression construct encoding a G-case (e.g., GBA1 or a portion thereof) and IL-34 (e.g., IL34 or a portion thereof). Expression of the G-case and IL-34 coding sequences is each driven by a separate promoter.

Fig. 36A-Fig. 36B show representative data for TREM2 and GBA1 overexpression in HEK293 cells compared to control transduced cells as measured by qPCR and ELISA.

Fig. 36A shows data regarding TREM2 overexpression.

Fig. 36B shows data on overexpression of GBA1 from the same construct.

Fig. 37 shows representative data indicating successful silencing of SNCA in vitro using a GFP reporter assay (top) and an α-Syn assay (bottom).

Fig. 38 shows representative data indicating successful silencing of TMEM106B in vitro by GFP reporter assay (top) and α-Syn assay (bottom).

Fig. 39 is a schematic representation of one embodiment of a vector containing an expression construct encoding PGRN.

Fig. 40 shows data on transduction of HEK293 cells using rAAVs having ITRs with wild-type (circles) or alternative (e.g., on the outside; squares) placement of the D sequence. rAAVs having ITRs on the outside were able to transduce cells as efficiently as rAAVs having wild-type ITRs.

Fig. 41 is a schematic representation of one embodiment of a vector containing an expression construct encoding a G-case (e.g., GBA1 or a portion thereof).

Fig. 42 is a schematic representation of one embodiment of a vector containing an expression construct encoding a G-case (e.g., GBA1 or a portion thereof).

Fig. 43 is a schematic representation of one embodiment of a vector comprising an expression construct encoding a G-case (e.g., GBA1 or a portion thereof) and interfering RNA for α-Syn.

Fig. 44 is a schematic representation of one embodiment of a vector,

- 5 046777 containing an expression construct encoding PGRN.

Fig. 45 is a schematic representation of one embodiment of a vector containing an expression construct encoding PGRN.

Fig. 46 is a schematic representation of one embodiment of a vector comprising an expression construct encoding PGRN and interfering RNA for microtubule-associated protein tau (MAPT).

Fig. 47 is a schematic representation of one embodiment of a vector comprising an expression construct encoding a G-case (e.g., GBA1 or a portion thereof) and interfering RNA for α-Syn.

Fig. 48 is a schematic representation of one embodiment of a vector containing an expression construct encoding a PSAP.

Fig. 49 is a schematic representation of one embodiment of a vector comprising an expression construct encoding a G-case (e.g., GBA1 or a portion thereof). Fig. 50 is a schematic representation of one embodiment of a vector comprising an expression construct encoding a G-case (e.g., GBA1 or a portion thereof) and a galactosylceramidase (e.g., GALC or a portion thereof).

Fig. 51 is a schematic representation of one embodiment of a plasmid containing an rAAV vector that includes an expression construct encoding a G-case (e.g., GBA1 or a portion thereof), a prosaposin (e.g., PSAP or a portion thereof), and an interfering RNA for α-Syn.

Fig. 52A shows that iPSC-derived neural stem cell (NSC) lines from patients with FTD-GRN mutations secreted less progranulin than NSC lines derived from healthy controls. Statistics were determined by unpaired t-test; *=p<0.05, **=p<0.01, ***=p<0.001. Data are presented as mean ± SEM.

Fig. 52B shows the results of PR006A transduction over a range of doses in FTD-GRN mutation carrier neuronal cultures. NSCs were seeded at equal densities and differentiated into neurons. On day 7, neurons were transduced with excipient or the indicated amounts of PR006A for 72 h. Secreted progranulin expression was measured in the cell medium by ELISA and normalized by volume (n=3-4; mean ± SEM). The black dotted line represents endogenous levels of progranulin secreted by control (excipient-treated) neurons. Secreted progranulin was undetectable in excipient-treated FTDGRN neurons. Statistics were determined using ANOVA followed by Tukey's HSD test, and statistical comparisons of each condition with vehicle-treated control neurons are shown in the graph, *=p<0.05, ***=p<0.001. LLOQ=lower limit of quantification; MOI=multiplicity of infection.

Fig. 52C shows that PR006 treatment of neuronal cultures rescued the defective maturation of a key lysosomal protease, cathepsin D, in FTD-GRN neuronal cultures. NSCs were seeded at equal concentrations and differentiated into neurons. On day 7, neurons were transduced with auxiliary or PR006A at an MOI of 5.3x105 for 72 h. Neurons were lysed and lysates were analyzed on the Protein Simple Western Jess system with an anti-cathepsin D (CTSD) primary antibody. Bands corresponding to both mature cathepsin D (matCTSD) and pro-cathepsin D (proCTSD) were detected and the area under the curve was quantified for each band and normalized to the internal normalization signal of total protein. The matCTSD/proCTSD ratio in auxiliary- or PR006A-treated FTD-GRN neurons was determined; The ordinate axis shows the matCTSD/proCTSD ratio as a percentage of the ratio of control neurons treated with the excipient (n=3; mean ± SEM). Statistics were determined using a paired t-test; *=p<0.05.

Fig. 52D and Fig. 52F show that PR006A reduces TDP-43 pathology in FTD-GRN neuronal cultures. NSCs were seeded at equal concentrations and differentiated into neurons. On day 7, neurons were transduced with auxiliary or PR006A at an MOI of 5.3x105 and harvested 21 days post-transduction. Fig. 52D: Neurons were lysed and the Triton-X insoluble protein fraction was isolated and analyzed on the Protein Simple Western Jess system with anti-TDP-43 antibody (#12892-AP-1). A band corresponding to TDP-43 was detected and the area under the curve was quantified for each band and normalized to the total protein concentration in the insoluble fraction. The y-axis shows the amount of insoluble TDP-43 as a percentage of excipient-treated levels, normalized separately for each FTD-GRN cell line (n=3; mean ± SEM). Fig. 52D shows that PR006 treatment reduces insoluble TDP-43, a hallmark of FTD-GRN pathology, in FTD-GRN neuron cultures. Fig. 52F: Quantification of nuclear TDP-43 signal from immunofluorescence images of PR006A-treated iPSC-derived neurons. TDP-43 signal intensity per nucleus in excipient- or PR006A-treated FTD-GRN neurons was determined; the y-axis shows TDP-43 signal intensity per nucleus as

- 6,046,777 percent of TDP-43 signal intensity per nucleus in vehicle-treated control neurons (n=145-306 cells; mean ± SEM). TDP-43 was measured using anti-TDP-43 antibody (#12892-AP-1) and nuclear area was determined by DAPI staining. Fig. 52F shows that PR006 treatment increased TDP-43 nuclear expression levels in FTD-GRN neuron cultures to levels close to wild-type control levels. Statistics were determined by unpaired t-test; **=p<0.01, ***=p<0.001.

Fig. 52E shows that iPSC-derived NSC lines from patients with FTDGRN mutations expressed less progranulin than NSC lines derived from healthy controls. Statistics were determined by unpaired t-test; *=p<0.05, **=p<0.01, ***=p<0.001. Data are presented as mean ± SEM.

Fig. 52G is a series of images showing that neural stem cell (NSC) lines from human FTD-GRN lines and human control cell lines were successfully differentiated into neuronal cultures. Control and FTD-GRN NSC lines (FTD-GRN#1 and FTD-GRN#2) differentiated into neurons after 7 days, as indicated by cell morphology and immunofluorescence staining for neuronal markers (NeuN [red]; MAP2 or Tau, as noted on the left [green]). DAPI (blue) was used to stain the nucleus.

Figs. 53A-53C are a series of histograms depicting the results of experiments analyzing the biodistribution and expression of progranulin in the CNS in a dose-finding study in the adult PR006A FTD-GRN mouse model with a dose range. Four-month-old Grn KO mice were administered PR006A or excipient by ICV administration. Mice were sacrificed 3 months after treatment with excipient (red) or PR006A at a dose of 1.1x109 vg (2.7x109 vg/g brain), 1.1x10 ¹⁰ vg (2.7x10 ¹⁰ vg/g brain), or 1.1x1011 vg (blue) for biochemical endpoints in the CNS. Fig. 53A: The presence of vector genomes was assessed in the cerebral cortex and spinal cord, and the biodistribution is shown as vector genomes per μg gDNA on a log scale (n=8-10/group; mean ± SEM). The presence of vector genomes was quantified by qPCR using a vector reference standard curve. The dotted line (50 vector genomes/μg g/THK) represents the threshold for positive vector presence. Fig. 53B: The expression of PR006A-encoded GRN RNA was assessed by quantitative RT-PCR (qRT-PCR) in the cerebral cortex (n=8-10/group; mean ± SEM). The number of GRN copies (specific for our codon-optimized PR006A sequence) was normalized to 1 μg total RNA and shown on a log scale. Fig. 53C: Progranulin protein levels were measured using a human-specific progranulin ELISA in brain and spinal cord (n=8-10/group; mean ± SEM). Tissue progranulin levels were normalized to total protein concentration. The lower limit of quantification (LLOQ) is indicated by the dotted gray line. For tissue ELISA assays, LLOQ values (ng/mg) were determined by dividing the assay LLOQ value (ng/mL) by the mean total protein concentration for all samples. A simple line matching the color of the experimental group legend on the x-axis without error bars indicates that all animals in that group were 0. Statistical analysis was performed by ANOVA followed by Dunnett's test for comparisons with the excipient-treated Grn KO mice group; *= p < 0.05, **= p < 0.01, ***= p < 0.001. vg=vector genomes; LLOQ=lower limit of quantification; SC=spinal cord.

Fig. 53D-53E are a series of histograms depicting the results of progranulin biodistribution and expression analysis in peripheral tissues in a dose-finding study in the adult PR006A FTD-GRN mouse model. Four-month-old Grn KO mice were treated with PR006A or excipient by ICV. Mice were sacrificed 3 months after treatment with excipient (red) or PR006A at a dose of 1.1x109 vg (2.7x109 vg/g brain), 1.1x10 ¹⁰ vg (2.7x10 ¹⁰ vg/g brain), or 1.1x1011 vg (blue) for biochemical endpoints in the liver, heart, lung, kidney, spleen, and gonads. 53D: The presence of vector genomes was assessed and biodistribution is shown as vector genomes per µg gDNA on a log scale (n=8-10/group; mean ± SEM). The presence of vector genomes was quantified by qPCR using a vector reference standard curve. The dotted line (50 vector genomes/µg gDNA) represents the threshold for positive vector presence. Fig. 53E: Progranulin protein levels were measured by ELISA (n=810/group; mean ± SEM). Tissue progranulin levels were normalized to total protein concentration. A simple line matching the color of the experimental group legend on the x-axis without error bars indicates that all animals in that group were 0. Statistical analysis was performed by ANOVA followed by Dunnett's test for comparison with the excipient-treated Grn KO mice group; *=p<0.05, *** =p<0.001. vg= vector genomes.

Fig. 53F is a histogram depicting the results of experiments analyzing plasma progranulin levels in a dose-finding study in the PR006A FTD-GRN adult mouse model. Four-month-old Grn KO mice were treated with PR006A or excipient

- 7 046 777 by ICV administration. Mice were sacrificed 3 months after treatment with excipient (red) or PR006A at a dose of 1.1x109 vg (2.7x109 vg/g brain), 1.1x101° vg (2.7x10 ¹⁰ vg/g brain), or 1.1x1011 vg (2.7x1011 vg/g brain) (blue) for plasma biochemical end points. Progranulin protein levels were measured using a human-specific progranulin ELISA in plasma (n=8-10/group; mean ± SEM). Plasma levels are shown on a log scale. The lower limit of quantification (LLOQ) is indicated by the dotted gray line. Statistical analysis was performed using ANOVA followed by Dunnett's test for comparison with the adjuvant-treated Gm KO mice group; *=p < 0.05, **=p < 0.01, ***=p < 0.001. LLOQ=lower limit of quantification, vg=vector genomes.

Fig. 53G-53H are a series of histograms depicting the results of experiments demonstrating the amelioration of lysosomal and neuropathological defects in a dose-finding study in the PR006A FTD-GRN adult mouse model. Four-month-old Gm KO mice were treated with PR006A or excipient by ICV. Mice were sacrificed 3 months after treatment with excipient (red) or PR006A at a dose of 1.1x109 vg (2.7x109 vg/g brain), 1.1x10 ¹⁰ vg ( ^2.7x1011 vg/g brain), or 1.1x1011 vg (blue). Lipofuscinosis was analyzed by two independent methods: (1) evaluation of hematoxylin and eosin-stained brain sections by a pathologist and (2) quantitative evaluation of lipofuscin autofluorescence from IHC sections. Fig. 53G: Lipofuscin accumulation (autofluorescent lipofuscin granules) was scored semiquantitatively in H&E-stained sections from different brain regions by a blinded board-certified pathologist according to the following scoring scheme: 0=no lipofuscin detected; 1=very small lipofuscin granules (<2 μm) scattered throughout the region; 2=increased density of small granule clusters and/or formation of larger granules (>2-3 μm); 3=multifocal areas of high lipofuscin granule density visible at low power objective; 4=extensive lipofuscin accumulation. Lipofuscin accumulation scores in the cerebral cortex, hippocampus, and thalamus/hypothalamus brain regions are shown (n=8-10/group). Fig. 53H: IHC analysis for ubiquitin and quantification of ubiquitin presence in the cerebral cortex, hippocampus, and thalamus. The size of immunoreactive objects above the threshold (immunoreactive object size [ ^μm2 ] is shown for ubiquitin (n=8-10/group; mean ± SEM). Statistical analysis was performed by ANOVA followed by Dunnett's test for comparison with vehicle-treated Gm KO mice; *=p<0.05, **=p<0.01, ***=p<0.001. vg=vector genomes; WT=wild type.

Fig. 53I-53K are a series of histograms depicting the results of experiments demonstrating the reduction of neuroinflammatory markers in a dose-finding study in the PR006A FTD-GRN adult mouse model. Four-month-old Grn KO mice were treated with PR006A or excipient by ICV. Mice were sacrificed 3 months after treatment with excipient (red) or PR006A at a dose of ^1.1x109vg ( ^2.7x109 vg/g brain), 1.1x1010 vg (2.7x1011 vg/g brain), or 1.1x1011 vg (blue). Fig. 53I: Gene expression (mRNA levels) of Tnf Cd68 was measured by qRT-PCR in the somatosensory cortex (mean ± SEM; n = 8-10/group). Gene expression was normalized to the housekeeping gene Ppib. Figs. 53J-53K: Iba1 (Fig. 53J) and GFAP (Fig. 53K) were analyzed by IHC and quantified in fixed brain sections in the cortex, hippocampus, and thalamus. The percentage of the region of interest covered by objects above the threshold (immunoreactive area [%]) is shown (mean ± SEM; n = 8-10/group). Statistical analysis was performed by ANOVA with Dunnett's correction comparing each group with the vehicle-treated Grn KO mice group; *=p <0.05, ***=p <0.001. vg=vector genomes; WT=wild type.

Fig. 53L-53N are a series of histograms depicting the results of experiments demonstrating the downregulation of lysosomal and immune pathway genes in a dose-finding study in the PR006A FTD-GRN adult mouse model. Four-month-old Grn KO mice were treated with PR006A or excipient by ICV. Mice were sacrificed 3 months after treatment with excipient (red) or PR006A at a dose of 1.1x109 vg (2.7x109 vg/g brain), 1.1x10 ¹⁰ vg ( ^2.7x1011 vg/g brain), or 1.1x1011 vg (blue). RNA-seq was performed on cortex samples from ICV-treated Grn KO mice and age-matched WT C57BL/6J mice (gray). Gene set variation analysis (GSVA) methodology was used to compare mRNA expression levels of previously published gene signatures that are dysregulated in excipient-treated Grn KO mice compared to WT mice. Data shown represent GSVA activity scores for curated gene sets from two published studies and one HALLMARK pathway. Fig. 53L: Cellular component: vacuole (GO: 0005773), Fig. 53M: lysosome, and Fig. 53N: complement system (HALLMARK pathway) (median ± range; n=8-10/group). Statistical analysis was performed using ANOVA followed by Dunnett's test for comparisons with the excipient-treated Grn KO group while controlling for the type I error rate adjusted for

- 8 046777 effect of multiple comparisons, ***=p<0.001. GSVA=gene set variation analysis; vg=vector genomes; WT=wild type.

Fig. 54A is a series of histograms depicting the results of experiments analyzing the biodistribution of the PR006A transgene quantified by qPCR. Transgene levels were analyzed by qPCR techniques in NHPs 182 days after ICM injection with either adjuvant, low-dose PR006A (6.5x109 vg/g brain), or high-dose PR006A (6.5x10 ¹⁰ vg/g brain). Each bar represents the mean ± SEM of 3 animals per group; the yellow line indicates the lower limit of quantification at 50 μg/μg DNA.

Fig. 54B is a series of histograms depicting the results of experiments analyzing levels of anti-drug antibodies to human progranulin. Antibodies to progranulin in serum and CSF samples from NHPs on Day 29 and Day 182 after treatment with either excipient, low dose PR006A ( ^6.5x109 vg/g brain), or high dose PR006A (6.5x1010 vg/g brain). Data are presented as mean ± SEM.

Fig. 54C is a series of histograms depicting the results of experiments analyzing the expression of the PR006A (GRN) transgene. GRN expression levels were determined in the cerebral cortex, hippocampus, and ventral midbrain of NHPs collected at Day 183 by RT-qPCR. Data are presented as mean ± SEM.

Fig. 54D is a histogram depicting the results of experiments analyzing CSF progranulin levels quantified using the Simple Western™ platform (Jess). Progranulin levels were determined in CSF samples from NHPs that were collected on Day 183 using the Simple Western™ assay (Jess). CSF samples were from NHPs that received adjuvant, low dose PR006A ( ^6.5x109 vg/g brain), or high dose PR006A (6.5x1010 vg/g brain). Data shown are mean ± SEM; P-value: *p < 0.05, by one-way dose-response analysis using William's test for trend.

Fig. 55 is a graph showing the selectivity and specificity results for the automated Western Jess assay. Progranulin protein levels in CSF samples from FTD patients were determined to be 58 kDa by Jess. Group (A): heterozygous FTD patients and groups (B) and (C): familial non-carriers or normal individuals. Data are presented as mean ± SEM. SEM values are shown as vertical error bars.

Fig. 56 is a graph showing progranulin levels in CSF samples from a patient with FTD detected by ELISA. Group (A): heterozygous FTD patients and groups (B) and (C): familial non-carriers or normal individuals. Data are presented as mean ± standard error of the mean (SEM). SEM values are shown as vertical error bars.

Fig. 57 is a gel image of each CSF sample run in duplicate on the Jess Western automated platform. Samples were analyzed at 4-fold dilution using the Adipogen PG-359-7 primary antibody. The first lane is the molecular weight standards, and on the right is the band identification used to calculate the immunoreactivity presented in Example 14.

Figs. 58A-58B are a series of graphs showing the measurement of human PGRN expression levels. Human PGRN expression levels were determined in non-human primate (NHP) CSF samples collected on day 180 using the Simple Western™ assay (Jess). CSF samples from NHPs treated with excipient (excipient), low dose PR006A ( ^6.5x109 vg/g brain, low dose), or high dose PR006 (6.5x1010 vg/g brain, high dose). Data are expressed as the mean peak area of immunoreactivity (Fig. 58A) or fold change for excipient-treated animals (Fig. 58B). Each point represents a CSF sample from one NHP (mean of technical duplicates), and the cell represents the mean +/- standard error from three individual NHPs.

Fig. 59A-59C are a series of histograms depicting the results of experiments analyzing the biodistribution and expression of progranulin in the CNS in the mature FTD-GRN mouse model following PR006A administration. Tissue samples were collected from 18-month-old Grn KO mice 2 months after ICV administration of excipient (red) or ^9.7x1010 vg (2.4x1011 vg/g brain) PR006A (blue). Fig. 59A: The presence of vector genomes was assessed in the cerebral cortex and spinal cord (mean ± SEM; n = 4/group). Biodistribution is presented as vector genomes per 1 μg gDNA on a log scale. The presence of vector genomes was quantified by qPCR using a vector reference standard curve. The dotted line (50 vector genomes/µg gDNA) represents the threshold for positive vector presence. Fig. 59B-59C: Progranulin protein levels were measured with

- 9 046 777 by ELISA in CNS tissues (brain and spinal cord (Fig. 59B)) and in CSF (Fig. 59C) (mean ± SEM; n = 4/group). Tissue progranulin levels were normalized to total protein concentration, and CSF progranulin levels were normalized to fluid volume. The lower limit of quantification (LLOQ) is indicated by the dotted gray line. For tissue ELISA assays, LLOQ values (ng/mL) were determined by dividing the assay LLOQ value (ng/mL) by the mean total protein concentration for all samples. A plain red line on the x-axis without error bars indicates that all animals in that group were 0. Statistical analysis was performed using the Kruskal-Wallis method; * = p < 0.05, ** = p < 0.01, *** = p < 0.001. vg=vector genomes; LLOQ=lower limit of quantification; SC=spinal cord.

Fig. 59D-59E are a series of histograms and images showing experimental results demonstrating amelioration of lysosomal and neuropathological defects in the mature FTD-GRN mouse model following PR006A administration. Tissue samples were collected from 18-month-old Grn KO mice 2 months after ICV administration of excipient (red) or ^9.7x1010 vg (2.4x1011 vg/g brain) PR006A (blue). Lipofuscinosis was analyzed by a pathologist by scoring hematoxylin and eosin-stained brain sections. Fig. 59D: Representative images of lipofuscin from the thalamic/hypothalamic region of brain sections. White arrows point to examples of lipofuscin accumulation. Summary data on the degree of lipofuscin accumulation in the cerebral cortex, hippocampus, and thalamus/hypothalamus are shown in H&E-stained microscopic preparations from brain sections that were assessed for the presence of autofluorescent lipofuscin granules. Lipofuscin accumulation was scored semiquantitatively by a blinded board-certified pathologist according to the following scoring scheme: 0 = no lipofuscin detected; 1 = very small lipofuscin granules (<2 μm) scattered throughout the area; 2 = increased density of small granule clusters and/or formation of larger granules (>2-3 μm); 3 = multifocal areas of high lipofuscin granule density visible at low power; 4 = extensive lipofuscin accumulation. Fig. 59E: IHC analysis for ubiquitin (n=4/group) and quantification of ubiquitin presence in the cerebral cortex, hippocampus and thalamus. Positive cell density (cells/ ^mm2 ) for each region is demonstrated (mean ± SEM). Statistics were determined by t-test; *=p<0.05, **=p<0.01. vg=vector genomes.

Fig. 59F-59I are a series of histograms depicting the results of experiments demonstrating the reduction of neuroinflammatory markers in the mature FTD-GRN mouse model following PR006A administration. Tissue samples were collected from 18-month-old Grn KO mice 2 months after ICV administration of excipient (red) or ^9.7x1010 vg (2.4x1011 vg/g brain) PR006A (blue). Fig. 59F: Tnf and Cd68 gene expression was measured by qRT-PCR in the somatosensory cortex (mean ± SEM; n= 4/group). Gene expression was normalized to the housekeeping gene Ppib. (Fig. 59G) Protein expression of the proinflammatory cytokine TNFa was measured in the cerebral cortex using the Mesoscale Discovery Mouse Proinflammatory Cytokine Assay (mean ± SEM; n = 4/group). The cerebral cortex was homogenized, and protein expression levels were normalized to total protein concentration in tissue lysates. Figs. 59H–59I: IHC analysis of Iba1 (Fig. 59H) and GFAP (Fig. 59I) were performed and their levels were quantified in fixed brain sections. Compilation of positive cell density (cells/mm ² ) from the three brain regions analyzed (cerebral cortex, hippocampus, and thalamus) is shown (mean ± SEM; n = 3-4/group). Statistics were determined using t-test; * = p < 0.05. vg = vector genomes.

Fig. 60 is a graph depicting the dose-response curve for HEK293T cells transduced with PR006A (n=2; mean ± SEM). Equal numbers of cells were transduced with varying amounts of PR006A. After 72 hours, progranulin protein levels in the cell medium were measured by ELISA.

Fig. 61 is a schematic of the design of the maximum dose study of PR006A in the mature FTD-GRN mouse model. 10 μL of excipient (control) or PR006A at a dose of 9.7x10 ¹⁰ vg (2.4x1011 vg/g brain) were delivered via ICV injection to two groups of Grn KO mice: (1) 16 months of age at the time of injection (n=4-5/group; PRV-2018-027) and (2) 14 months of age at the time of injection (n=1/excipient-treated group; n=3/PR006A-treated group; PRV-2019-002). Animals were sacrificed two months after injection. CNS and peripheral tissues were collected for PR006A biodistribution analysis (qPCR), progranulin protein expression (ELISA), and histopathological characterization (H&E). Expression of proinflammatory markers, lipofuscin accumulation, and ubiquitin accumulation were assessed in the brain.

Fig. 62A-62B are histograms depicting the results of peripheral tissue biodistribution and progranulin expression in the mature FTD-GRN mouse model following PR006A administration. Tissue samples were collected from 18-month-old Grn KO mice 2 months after ICV administration of excipient (red) or 9.7x10 ¹⁰ vg (2.4x1011 vg/g brain) PR006A (blue). Fig. 62A: The presence of vector genomes was assessed in the liver, heart, lung, kidney, spleen, and gonads (mean ± SEM; n = 4/group). Biodistribution is presented as vector genomes

- 10,046,777 per µg dDNA on a log scale. The presence of vector genomes was quantified by qPCR using a vector reference standard. Fig. 62B: Progranulin protein levels were measured by ELISA (mean ± SEM; n = 4/group). Tissue progranulin levels were normalized to total protein concentration. The plain red line on the x-axis without error bars indicates that all animals in that group were 0. Statistical analysis was performed using the Kruskal-Wallis method; * = p < 0.05, ** = p < 0.01, *** = p < 0.001. vg = vector genomes.

Fig. 63 is a schematic of the design of a dose ranging study for PR006A in the adult FTD-GRN mouse model. 10 μL of excipient (control) or PR006A at a dose of 1.1 x 109 vg (2.7 x 109 vg/g brain), 1.1 x ^{10 10} vg (2.7 x 10 ¹⁰ vg/g brain), or 1.1 x 1011 vg (2.7 x 1011 vg/g brain) of PR006A were administered by ICV injection to 4-month-old Gm KO mice (n = 10/group). Animals were sacrificed three months after injection, when the mice were 7 months old. CNS and peripheral tissues were collected for PR006A biodistribution analysis (qPCR), progranulin protein expression (ELISA), and histopathological characterization (H&E). Expression of proinflammatory markers, lipofuscin accumulation, ubiquitin accumulation, and large-scale changes in gene expression were assessed in the brain.

Fig. 64 is a schematic representation of one embodiment of a recombinant adeno-associated virus (PR006A) vector comprising an expression construct encoding human progranulin, bp refers to base pairs, kan refers to a gene that confers resistance to kanamycin. GRN refers to progranulin. ITR refers to the inverted terminal repeat sequence of adeno-associated virus. TRY refers to a sequence containing three transcriptional regulatory sites: TATA, RBS and YY1. CBP refers to the chicken β-actin promoter. CMVe refers to the cytomegalovirus enhancer. WPRE refers to the woodchuck hepatitis virus post-transcriptional regulatory element. bGH refers to the bovine growth hormone polyA signal tail, int refers to an intron. The nucleotide sequences of the two chains of PR006A are shown in SEQ ID NOs: 90 and 91.

Detailed description of the invention

The present invention is based in part on compositions and methods for expressing combinations of certain gene products (e.g., gene products associated with a CNS disease) in a subject. The gene product can be a protein, a fragment (e.g., a portion) of a protein, an interfering nucleic acid that inhibits a gene associated with a CNS disease, 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 (e.g., 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 FTD (frontotemporal dementia) or PD (Parkinson's disease). For example, individuals carrying a pathogenic mutation in the GRN gene (which encodes the PGRN protein) have an increased risk of developing FTD compared to individuals who do not have a mutation in GRN. Similarly, individuals carrying 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 PD-associated gene. In some embodiments, the expression cassette described herein encodes a wild-type or non-mutant form of a gene associated with a CNS disease (or its coding sequence). Examples of genes associated with a CNS disease are listed in Table 1.

- 11 046777

Table 1

Examples of genes associated with CNS disease

Name Gene Function Accession number NCBI Lysosomal membrane protein 2 SCARB2ILIMP2 lysosomal glucosylceramidase receptor (targeting GBA) NP_005497.1 (Isoform 1), NP 001191184.1 (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 cleaves the beta- glucosidic bond of glucocerebroside ΝΡ_001005742.1 (Isoform 1), ΝΡ_001165282.1 (Isoform 2), ΝΡ_001165283.1 (Isoform 3) Non-lysosomal glucosylceramidase GBA2 catalyzes the conversion of glucosylceramide into free glucose and ceramide NP 065995.1 (Isoform 1), NP_001317589.1 (Isoform 2) Galactosylceramidase GALC removes galactose from ceramide derivatives EAW81359.1 (CRAa isoform), EAW81360.1 (CRAb isoform), EAW81362.1 (CRAc isoform) Sphingomyelin phosphodiesterase 1 SMPD1 converts sphingomyelin into ceramide EAW68726.1 (Isoform CRAa), EAW68727.1 (Isoform CRAb), EAW68728.1 (Isoform CRAc), EAW68729.1 (Isoform CRAd)

- 12 046777

Cathepsin B CTSB thiol protease believed to be involved in intracellular protein degradation and turnover; also involved in tumor invasion and metastasis AAC37547.1, AAN95408.1, AAN10240.1 RAB7, RAS oncogene family member-like protein 1 RAB7L1 regulates vesicular transport AAN02585.1 Vacuolar sorting protein family-associated protein 35 VPS35 component of the retromer cargo-selective complex NP 060676.2 GTP cyclohydrolase 1 GCH1 responsible for the hydrolysis of guanosine triphosphate to form 7,8-dihydronopterin triphosphate AAN25415.1 Interleukin 34 IL34 stimulates growth or increases survival of monocytes; induces activity by binding to colony-stimulating factor 1 receptor AAN29804.1 Myeloid cell-expressed trigger receptor 2 TREM2 forms a receptor signaling complex with protein tyrosine kinase binding protein TYRO; participates in the immune response and may be involved in the pathogenesis of chronic inflammation AAF69824.1 Progranulin PGRN plays a role in development, inflammation, cell proliferation and protein homeostasis NP_002087.1

In addition to patients with Gaucher disease (who have mutations in both chromosomal alleles of the GBA1 gene), patients with mutations in only one GBA1 allele are at increased risk of Parkinson's disease (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 decline. 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 of other PD-related disorders, including dementia with Lewy bodies, characterized by executive dysfunction, psychosis, and PD-like movement disorder, and multiple system atrophy, with characteristic motor and cognitive impairment. There are no treatments that would alter the

- 13 046777 the inevitably progressive course of these disorders does not exist.

Deficiencies of enzymes such as G-case (eg, the gene product of the GBA1 gene), as well as common variants in many genes involved in lysosome function or the transport of macromolecules into lysosomes (eg, lysosomal membrane protein 1 (LIMP), also called SCARB2), have been associated with an increased risk of PD and/or the risk of Gaucher disease (eg, neuronopathic Gaucher disease such as Gaucher disease type 2 or Gaucher disease type 3). The present invention relies in part on expression constructs (e.g., vectors) encoding one or more genes, e.g., G-case, GBA2, prosaposin, progranulin (PGRN), LIMP2, GALC, CTSB, SMPD1, GCH1, RAB7, VPS35, IL-34, TREM2, TMEM106B, or a combination of any of the foregoing (or portions thereof), associated with central nervous system (CNS) diseases, e.g., Gaucher disease, PD, etc. In some embodiments, the gene product combinations described herein act together (e.g., synergistically) to reduce one or more signs and symptoms of a CNS disease when expressed in a subject.

Accordingly, in some aspects of the present invention, there is provided an isolated nucleic acid comprising an expression construct encoding a G-case (e.g., a gene product of the GBA1 gene). In some embodiments, the isolated nucleic acid comprises a sequence encoding a G-case that has been codon-optimized (e.g., codon-optimized for expression in mammalian cells, such as 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 (e.g., as set forth in NCBI reference sequence NP_000148.2). 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), such as AAV ITRs, flanking a nucleic acid sequence encoding a G-case protein.

In some aspects, the invention provides an isolated nucleic acid comprising an expression construct encoding prosaposin (e.g., a gene product of the PSAP gene). In some embodiments, the isolated nucleic acid comprises a prosaposin encoding sequence that has been codon-optimized (e.g., 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 (e.g., 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), such as AAV ITRs, flanking a nucleic acid sequence encoding the prosaposin protein.

In some aspects, the invention provides an isolated nucleic acid comprising an expression construct encoding LIMP2/SCARB2 (e.g., a gene product of the SCARB2 gene). In some embodiments, the isolated nucleic acid comprises a SCARB2 encoding sequence that has been codon-optimized (e.g., 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 (e.g., 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), such as AAV ITRs, flanking a nucleic acid sequence encoding the SCARB2 protein.

In some aspects, the invention provides an isolated nucleic acid comprising an expression construct encoding a GBA2 protein (e.g., a gene product of a GBA2 gene). In some embodiments, the isolated nucleic acid comprises a GBA2 encoding sequence that has been codon-optimized (e.g., codon-optimized for expression in mammalian cells, such as human cells). In some embodiments, the nucleic acid sequence encoding GBA2 encodes a protein comprising an amino acid sequence set forth in SEQ ID NO: 30 (e.g., as set forth in NCBI reference sequence NP065995.1). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 31. In some embodiments, the expression construct comprises adeno-associated virus (AAV) inverted terminal repeats (ITRs), such as AAV ITRs, flanking the nucleic acid sequence encoding the GBA2 protein.

In some aspects of the present invention, an isolated nucleic acid is provided comprising

- 14 046 777 containing an expression construct encoding a GALC protein (e.g., a gene product of a GALC gene). In some embodiments, the isolated nucleic acid comprises a GALC encoding sequence that has been codon optimized (e.g., codon optimized for expression in mammalian cells, such as human cells). In some embodiments, the GALC encoding nucleic acid sequence encodes a protein comprising an amino acid sequence set forth in SEQ ID NO: 33 (e.g., as set forth in NCBI reference sequence NP000144.2). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 34. In some embodiments, the expression construct comprises adeno-associated virus (AAV) inverted terminal repeats (ITRs), such as AAV ITRs, flanking the GALC encoding nucleic acid sequence.

In some aspects, the invention provides an isolated nucleic acid comprising an expression construct encoding a CTSB protein (e.g., a gene product of a CTSB gene). In some embodiments, the isolated nucleic acid comprises a CTSB encoding sequence that has been codon-optimized (e.g., codon-optimized for expression in mammalian cells, such as human cells). In some embodiments, the CTSB encoding nucleic acid sequence encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 35 (e.g., as set forth in NCBI reference sequence NP001899.1). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 36. In some embodiments, the expression construct comprises adeno-associated virus (AAV) inverted terminal repeats (ITRs), such as AAV ITRs, flanking the CTSB protein encoding nucleic acid sequence.

In some aspects, the invention provides an isolated nucleic acid comprising an expression construct encoding an SMPD1 protein (e.g., a gene product of the SMPD1 gene). In some embodiments, the isolated nucleic acid comprises a sequence encoding SMPD1 that has been codon-optimized (e.g., codon-optimized for expression in mammalian cells, such as human cells). In some embodiments, the nucleic acid sequence encoding SMPD1 encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 37 (e.g., as set forth in NCBI reference sequence NP_000534.3). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 38. In some embodiments, the expression construct comprises adeno-associated virus (AAV) inverted terminal repeats (ITRs), such as AAV ITRs, flanking a nucleic acid sequence encoding the SMPD1 protein.

In some aspects, the invention provides an isolated nucleic acid comprising an expression construct encoding a GCH1 protein (e.g., a gene product of a GCH1 gene). In some embodiments, the isolated nucleic acid comprises a GCH1 encoding sequence that has been codon-optimized (e.g., codon-optimized for expression in mammalian cells, such as human cells). In some embodiments, the nucleic acid sequence encoding GCH1 encodes a protein comprising an amino acid sequence set forth in SEQ ID NO: 45 (e.g., as set forth in NCBI reference sequence NP000534.3). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 46. In some embodiments, the expression construct comprises adeno-associated virus (AAV) inverted terminal repeats (ITRs), such as AAV ITRs, flanking the nucleic acid sequence encoding the GCH1 protein.

In some aspects, the invention provides an isolated nucleic acid comprising an expression construct encoding a RAB7L protein (e.g., a gene product of the RAB7L gene). In some embodiments, the isolated nucleic acid comprises a RAB7L encoding sequence that has been codon-optimized (e.g., codon-optimized for expression in mammalian cells, such as human cells). In some embodiments, the nucleic acid sequence encoding RAB7L encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 47 (e.g., as set forth in NCBI reference sequence NP003920.1). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 48. In some embodiments, the expression construct comprises adeno-associated virus (AAV) inverted terminal repeats (ITRs), such as AAV ITRs, flanking a nucleic acid sequence encoding the RAB7L protein.

In some aspects, the present invention provides an isolated nucleic acid comprising an expression construct encoding a VPS35 protein (e.g., a gene product of the VPS35 gene). In some embodiments, the isolated nucleic acid comprises the sequence

- 15 046 777 a VPS35 encoding nucleic acid that has been codon optimized (e.g., codon optimized for expression in mammalian cells, such as human cells). In some embodiments, the VPS35 encoding nucleic acid sequence encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 49 (e.g., as set forth in NCBI reference sequence NP_060676.2). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 50. In some embodiments, the expression construct comprises adeno-associated virus (AAV) inverted terminal repeats (ITRs), such as AAV ITRs, flanking the VPS35 encoding nucleic acid sequence.

In some aspects, the invention provides an isolated nucleic acid comprising an expression construct encoding an IL-34 protein (e.g., a gene product of the IL34 gene). In some embodiments, the isolated nucleic acid comprises an IL-34 encoding sequence that has been codon-optimized (e.g., codon-optimized for expression in mammalian cells, such as human cells). In some embodiments, the IL-34 encoding nucleic acid sequence encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 55 (e.g., as set forth in NCBI reference sequence NP_689669.2). In some embodiments, the isolated nucleic acid comprises a sequence set forth in SEQ ID NO: 56. 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 an IL-34 protein. In some aspects, the invention provides an isolated nucleic acid comprising an expression construct encoding a TREM2 protein (e.g., a gene product of the TKEM gene). In some embodiments, the isolated nucleic acid comprises a TREM2 encoding sequence that has been codon-optimized (e.g., codon-optimized for expression in mammalian cells, e.g., human cells). In some embodiments, the nucleic acid sequence encoding TREM2 encodes a protein comprising an amino acid sequence set forth in SEQ ID NO: 57 (e.g., as set forth in NCBI reference sequence NP_061838.1). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 58. In some embodiments, the expression construct comprises adeno-associated virus (AAV) inverted terminal repeats (ITRs), such as AAV ITRs, flanking a nucleic acid sequence encoding the TREM2 protein.

In some aspects, the invention provides an isolated nucleic acid comprising an expression construct encoding a TMEM106B protein (e.g., a gene product of the TMEM106B gene). In some embodiments, the isolated nucleic acid comprises a TMEM106B encoding sequence that has been codon-optimized (e.g., codon-optimized for expression in mammalian cells, such as human cells). In some embodiments, the TMEM106B encoding nucleic acid sequence encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 63 (e.g., as set forth in NCBI reference sequence NP060844.2). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 64. In some embodiments, the expression construct comprises adeno-associated virus (AAV) inverted terminal repeats (ITRs), such as AAV ITRs, flanking a nucleic acid sequence encoding the TMEM106B protein.

In some aspects, the invention provides an isolated nucleic acid comprising an expression construct encoding progranulin (e.g., a gene product of the PGRN gene). In some embodiments, the isolated nucleic acid comprises a prosaposin encoding sequence that has been codon-optimized (e.g., codon-optimized for expression in mammalian cells, such as human cells). In some embodiments, the nucleic acid sequence encoding progranulin (PGRN) encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 67 (e.g., as set forth in NCBI reference sequence NP_002078.1). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 68. In some embodiments, the expression construct comprises adeno-associated virus (AAV) inverted terminal repeats (ITRs), such as AAV ITRs, flanking a nucleic acid sequence encoding the prosaposin protein.

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, wherein each gene product is independently selected from the gene products or portions thereof shown 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

- 16 046 777 duct is a G-case protein, and the second gene product is selected from GBA2, prosaposin, progranulin, LIMP2, GALC, CTSB, SMPD1, GCH1, RAB7, VPS35, IL-34, TREM2, and TMEM106B. In some embodiments, the expression construct encodes (e.g., alone or in addition to another gene product) an interfering nucleic acid (e.g., shRNA, miRNA, dsRNA, etc.). In some embodiments, the interfering nucleic acid inhibits the expression of α-synuclein (α-synuclein). In some embodiments, the interfering nucleic acid targeting α-synuclein comprises a sequence set forth in any of SEQ ID NOs: 20-25. In some embodiments, an interfering nucleic acid targeting α-synuclein binds to (e.g., hybridizes to) a sequence set forth in any of SEQ ID NOs: 20-25.

In some embodiments, the interfering nucleic acid inhibits the expression of TMEM106B. In some embodiments, the interfering nucleic acid targeting TMEM106B comprises a sequence set forth in SEQ ID NO: 64 or 65. In some embodiments, the interfering nucleic acid targeting TMEM106B binds to (e.g., hybridizes to) a sequence set forth in SEQ ID NO: 64 or 65. In some embodiments, the expression construct further comprises one or more promoters. In some embodiments, the promoter is a chicken beta-actin (CBA) promoter, a CAG promoter, a CD68 promoter, or a JeT promoter. In some embodiments, the promoter is an RNA pol II promoter (or an RNA pol III (e.g., U6, etc.) promoter).

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, the ITR sequences flank a first gene product and a second gene product (e.g., arranged from 5' to 3' as follows: ITR-first gene product-second gene product-ITR). In some embodiments, one of the ITR sequences of the isolated nucleic acid does not have a functional terminal resolution site (trs). For example, in some embodiments, one of the ITRs is AITR.

In some aspects, the present invention provides rAAV vectors comprising an ITR having a modified D region (e.g., a D sequence that is modified relative to the wild-type AAV2 ITR, SEQ ID NO:29). In some embodiments, the ITR having a modified D region is the 5' ITR of the rAAV vector. In some embodiments, the modified D region comprises an S sequence, e.g., as set forth in SEQ ID NO:26. In some embodiments, the ITR having a modified D region is the 3' ITR of the rAAV vector. In some embodiments, the modified D region comprises a 3' ITR, wherein the D region is located at the 3' end of the ITR (e.g., at the outer or terminal end of the ITR relative to the transgene insert of the vector). 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 (e.g., rAAV vector) comprises a TRY region. In some embodiments, the TRY region comprises the sequence set forth in SEQ ID NO: 28.

In some embodiments, an isolated nucleic acid described herein comprises, consists of, or encodes a peptide having a sequence set forth in any of SEQ ID NOs: 1-91.

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

In some embodiments, the present invention provides a host cell comprising an isolated nucleic acid as described herein or a vector as described herein.

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

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In some embodiments, said capsid protein is capable of crossing the blood-brain barrier, such as the AAV9 capsid protein or the 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, said method comprising administering to the subject (e.g., a composition comprising an isolated nucleic acid or vector, or an rAAV) as described herein. In some embodiments, the CNS disease is a neurodegenerative disease, such as a neurodegenerative disease listed in Table 12. In some embodiments, the CNS disease is a synucleinopathy, such as a synucleinopathy listed in Table 13. In some embodiments, the CNS disease is a tautopathy, such as a tautopathy listed in Table 14. In some embodiments, the CNS disease is a lysosomal storage disease, such as a lysosomal storage disease listed in Table 15. In some embodiments, the lysosomal storage disease is a neuronopathic Gaucher disease, such as Gaucher disease type 2 or Gaucher disease type 3.

In some aspects, the present invention provides a method of treating a subject with an existing or suspected central nervous system (CNS) disease, said method comprising administering to the subject (e.g., a composition comprising an isolated nucleic acid or vector, or rAAV) as described herein.

In some embodiments, the present invention provides a method of treating a subject with existing or suspected frontotemporal dementia (FTD), FTD with a GRN mutation, FTD with a tau mutation, FTD with a C9orf72 mutation, ceroid lipofuscinosis, Parkinson's disease, Alzheimer's disease, corticobasal degeneration, motor neuron disease, or Gaucher's disease, said method comprising administering to the subject an rAAV encoding progranulin (PGRN), wherein PGRN is encoded by the nucleic acid sequence of SEQ ID NO: 68; and wherein the rAAV comprises a capsid protein having the AAV9 serotype.

In some embodiments, the present invention provides a method of treating a subject with existing or suspected FTD with a GRN mutation, said method comprising administering to the subject an rAAV encoding progranulin (PGRN), wherein PGRN is encoded by the nucleic acid sequence of SEQ ID NO: 68; and wherein the rAAV comprises a capsid protein having the AAV9 serotype. In some embodiments, the rAAV is administered to the subject at a dose of about 3.5 x 10 ¹³ vector genomes (vg), about 7.0 x ^{10 13} vg, or about 1.4 x 10 ¹⁴ vg. In some embodiments, the rAAV is administered by injection into the cerebellomedullary cistern.

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., gene products associated with a CNS disease), such as 2, 3, 4, 5, or more of the gene products described herein. In some embodiments, the composition comprises 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 encoding one or more different gene products. In some embodiments, two or more different compositions are administered to the subject, each comprising one or more nucleic acids encoding different gene products. In some embodiments, the different gene products are operably linked to the same type of promoter (e.g., 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, the present invention provides isolated nucleic acids (e.g., rAAV vectors) comprising an expression construct and encoding one or more PD-associated genes, such as a G-case (e.g., the gene product of the GBA1 gene) or a portion thereof. G-case, also referred to as β-glucocerebrosidase or GBA, refers to a lysosomal protein that cleaves the beta-glucosidic bond of the chemical glucocerebroside, an intermediate in glycolipid metabolism. 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 NCBI reference sequence NP_000148.2 (SEQ ID NO: 14). In some embodiments, the isolated nucleic acid comprises a sequence encoding a G-case that has been codon optimized (e.g., codon optimized for expression in mammalian cells, such as 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

- 18 046777 containing an expression construct encoding prosaposin (e.g., 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 short oligosaccharide glycosphingolipids. In humans, the PSAP gene is located on chromosome 10. In some embodiments, PSAP encodes a peptide that is represented by NCBI reference sequence NP_002769.1 (e.g., SEQ ID NO: 16). In some embodiments, the isolated nucleic acid comprises a prosaposin encoding sequence that has been codon-optimized (e.g., codon-optimized for expression in mammalian cells, such as human cells), such as the sequence set forth in SEQ ID NO: 17.

Aspects of the present invention relate to an isolated nucleic acid comprising an expression construct encoding LIMP2/SCARB2 (e.g., a gene product of the SCARB2 gene). SCARB2 refers to a membrane protein that regulates lysosomal and endosomal transport within a cell. 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 NP_005497.1 (SEQ ID NO: 18). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 19. In some embodiments, the isolated nucleic acid comprises a sequence encoding SCARB2 that has been codon optimized.

Aspects of the present invention relate to an isolated nucleic acid comprising an expression construct encoding a GBA2 protein (e.g., a gene product of the GBA2 gene). The GBA2 protein is a non-lysosomal glucosylceramidase. In humans, the GBA2 gene is located on chromosome 9. In some embodiments, the GBA2 gene encodes a peptide that is represented by the NCBI reference sequence NP_065995.1 (SEQ ID NO: 30). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 31. In some embodiments, the isolated nucleic acid comprises a sequence encoding GBA2 that has been codon optimized.

Aspects of the present invention relate to an isolated nucleic acid comprising an expression construct encoding a GALC protein (e.g., a gene product of the GALC gene). The GALC protein refers to galactosylceramidase (or galactocerebrosidase), which is an enzyme that hydrolyzes the galactose ester bonds of galactocerebroside, galactosylsphingosine, lactosylceramide, and monogalactosyl diglyceride. In humans, the GALC gene is located on chromosome 14. In some embodiments, the GALC gene encodes a peptide that is represented by NCBI reference sequence NP_000144.2 (e.g., SEQ ID NO: 33). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 34. In some embodiments, the isolated nucleic acid comprises a sequence encoding GALC that has been codon optimized.

Aspects of the present invention relate to an isolated nucleic acid comprising an expression construct encoding a CTSB protein (e.g., a gene product of the CTSB gene). The CTSB protein refers to cathepsin B, a lysosomal cysteine protease that plays an important role in intracellular proteolysis. In humans, the CTSB gene is located on chromosome 8. In some embodiments, the CTSB gene encodes a peptide that is represented by the NCBI reference sequence NP_001899.1 (SEQ ID NO: 35). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 36. In some embodiments, the isolated nucleic acid comprises a sequence encoding CTSB that has been codon optimized.

Aspects of the present invention relate to an isolated nucleic acid comprising an expression construct encoding an SMPD1 protein (e.g., a gene product of the SMPD1 gene). The SMPD1 protein is a sphingomyelin phosphodiesterase 1, which is a hydrolase enzyme involved in sphingolipid metabolism. In humans, the SMPD1 gene is located on chromosome 11. In some embodiments, the SMPD1 gene encodes a peptide that is represented by the NCBI reference sequence NP_000534.3 (e.g., SEQ ID NO: 37). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 38. In some embodiments, the isolated nucleic acid comprises a sequence encoding SMPD1 that has been codon optimized.

Aspects of the present invention relate to an isolated nucleic acid comprising an expression construct encoding a CCH1 protein (e.g., a gene product of the GCH1 gene). The GCH1 protein is a GTP cyclohydrolase I, which is a hydrolase enzyme involved in the folate and biopterin biosynthetic pathways. In humans, the GCH1 gene is located on chromosome 14. In some embodiments, the GCH1 gene encodes a peptide that is represented by the NCBI reference sequence NP_000152.1 (e.g., SEQ ID NO: 45). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 46. In some embodiments, the isolated nucleic acid comprises a sequence encoding

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GCH1, which has been codon optimized.

Aspects of the present invention relate to an isolated nucleic acid comprising an expression construct encoding a RAB7L protein (e.g., a gene product of the RAB7L gene). The RAB7L protein refers to RAB7, a member of the RAS 1 oncogene family, which is a GTP-binding protein. In humans, the RAB7L gene is located on chromosome 1. In some embodiments, the RAB7L gene encodes a peptide that is represented by the NCBI reference sequence NP_003920.1 (SEQ ID NO: 47). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 48. In some embodiments, the isolated nucleic acid comprises a sequence encoding RAB7L that has been codon optimized.

Aspects of the present invention relate to an isolated nucleic acid comprising an expression construct encoding a VPS35 protein (e.g., a VPS35 gene product). The VPS35 protein refers to vacuolar protein sorting associated protein 35, which is part of a protein complex involved in the retrograde transport of proteins from endosomes to the transGolgi network. In humans, the VPS35 gene is located on chromosome 16. In some embodiments, the VPS35 gene encodes a peptide that is represented by the NCBI reference sequence NP_060676.2 (e.g., SEQ ID NO: 49). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 50. In some embodiments, the isolated nucleic acid comprises a VPS35 encoding sequence that has been codon optimized. Aspects of the present invention relate to an isolated nucleic acid comprising an expression construct encoding an IL-34 protein (e.g., an IL34 gene product). The IL-34 protein refers to interleukin 34, which is a cytokine that increases the growth and survival of monocytes. In humans, the IL34 gene is located on chromosome 16. In some embodiments, the IL34 gene encodes a peptide that is represented by the NCBI reference sequence NP_689669.2 (e.g., SEQ ID NO: 55). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 56. In some embodiments, the isolated nucleic acid comprises a sequence encoding IL-34 that has been codon optimized.

Aspects of the present invention relate to an isolated nucleic acid comprising an expression construct encoding a TREM2 protein (e.g., a gene product of the TREM2 gene). The TREM2 protein refers to the trigger receptor expressed in myeloid cells 2, which is an immunoglobulin superfamily receptor found on myeloid cells. In humans, the TREM2 gene is located on chromosome 6. In some embodiments, the TREM2 gene encodes a peptide that is represented by the NCBI reference sequence NP061838.1 (SEQ ID NO: 57). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 58. In some embodiments, the isolated nucleic acid comprises a sequence encoding TREM2 that has been codon optimized.

Aspects of the present invention relate to an isolated nucleic acid comprising an expression construct encoding a TMEM106B protein (e.g., a gene product of the TMEM106B gene). The TMEM106B protein refers to the 106B transmembrane protein, which is a protein involved in dendritic morphogenesis and the regulation of lysosomal transport. In humans, the TMEM106B gene is located on chromosome 7. In some embodiments, the TMEM106B gene encodes a peptide that is represented by the NCBI reference sequence NP_060844.2 (SEQ ID NO: 62). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 63. In some embodiments, the isolated nucleic acid comprises a sequence encoding TMEM106B that has been codon optimized.

Aspects of the present invention relate to an isolated nucleic acid comprising an expression construct encoding a progranulin protein (e.g., a gene product of the PGRN gene). The PGRN protein refers to progranulin, which is a protein involved in development, inflammation, cell proliferation, and protein homeostasis. In humans, the PGRN gene is located on chromosome 17. In some embodiments, the PGRN gene encodes a peptide that is represented by the NCBI reference sequence NP_002078.1 (e.g., SEQ ID NO: 67). In some embodiments, the isolated nucleic acid comprises the sequence set forth in SEQ ID NO: 68. In some embodiments, the isolated nucleic acid comprises a sequence encoding PGRN that has been codon optimized. In some embodiments, the nucleic acid further comprises a chicken β-actin (CBA) promoter and a cytomegalovirus enhancer (CMVe).

In some aspects of the present invention, an automated Western blot immunoassay is provided for quantifying the level of PGRN protein in a cerebrospinal fluid (CSF) sample. In some embodiments, the immunoassay is a capillary-based automated Western blot immunoassay platform in which all steps such as protein separation, immunoprobing, washing, and chemiluminescence detection are performed in a capillary cartridge. In some embodiments, the CSF sample is taken

- 20 046 777 from a human or non-human primate. In some aspects, the immunoassay detects differences in PGRN protein levels in the presence of circulating antibodies. In some aspects, the present invention provides a method of quantifying the level of progranulin protein in a CSF sample, the method comprising: (1) diluting the CSF sample (e.g., a 4-fold dilution); (2) loading the CSF sample, an anti-progranulin antibody, a secondary antibody that detects the anti-progranulin antibody, luminol, and peroxide into the wells of a capillary cartridge; (3) loading the capillary cartridge into an automated Western blot immunoassay instrument; (4) using the automated Western blot immunoassay instrument to calculate one or more of the following parameters: signal intensity, peak area, signal-to-noise ratio, and total protein normalization parameters; and (5) quantifying the level of progranulin protein in the CSF sample as the peak area of immunoreactivity to the anti-progranulin antibody. In some embodiments, the CSF sample is diluted in a master mix comprising dithiothreitol (DTT) and sample buffer. The master mix may further comprise other specific components. In some aspects, the anti-progranulin antibody detects human progranulin. In some embodiments, the level of progranulin protein is quantified based on calculated parameters using software that controls the automated Western blot immunoassay. In some embodiments, the software is Compass software for Simple Western™ (ProteinSimple, San Jose, Calif.).

In some embodiments, the present invention provides a method for quantifying the level of progranulin protein in a cerebrospinal fluid (CSF) sample, comprising: (1) diluting the CSF sample (e.g., a 4-fold dilution) in a master mix comprising dithiothreitol (DTT) and sample buffer; (2) loading the diluted CSF sample, an anti-progranulin antibody, a secondary antibody that detects the anti-progranulin antibody, luminol, and peroxide into the wells of a capillary cartridge; (3) loading the capillary cartridge into an automated Western blot immunoassay instrument; (4) using the automated Western blot immunoassay instrument to calculate signal intensity, peak area, and signal-to-noise ratio; and (5) quantifying the level of progranulin protein in the CSF sample as the peak area of immunoreactivity to the anti-progranulin antibody.

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, wherein each gene product is independently selected from the gene products or portions thereof shown in Table 1.

In some embodiments, an isolated nucleic acid or vector (e.g., an rAAV vector) described herein comprises or consists of a sequence set forth in any of SEQ ID NOs: 1-91. In some embodiments, an isolated nucleic acid or vector (e.g., an rAAV vector) described herein comprises or consists of a sequence that is complementary to (e.g., complementary to) a sequence set forth in any of SEQ ID NOs: 1-91. In some embodiments, an isolated nucleic acid or vector (e.g., an rAAV vector) described herein comprises or consists of a sequence that is the reverse complement of a sequence set forth in any of SEQ ID NOs: 1-91. In some embodiments, an isolated nucleic acid or vector (e.g., an rAAV vector) described herein comprises or consists of a portion of a sequence set forth in any of SEQ ID NOs: 1-91. The portion may comprise at least 25%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the sequence set forth in any of SEQ ID NOs: 1-91. In some embodiments, the nucleic acid sequence described herein is a sense strand of a nucleic acid (e.g., 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 strand of a nucleic acid (e.g., the 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 (e.g., cDNA) of a naturally occurring gene. In some embodiments, the first gene product is a protein (or fragment thereof) encoded by the GBA1 gene. In some embodiments, the gene product is a protein (or fragment thereof) encoded by another gene listed in Table 1, such as the SCARB2/LIMP2 gene or the PSAP gene. However, one of skill in the art will appreciate that the order of expression of the first gene product (e.g., G-case) and the second gene product (e.g., LIMP2, etc.) may typically be reversed (e.g., LIMP2 is the first gene product and G-case is the second gene product). In some embodiments, the gene product is a fragment (e.g., part) of a gene listed in Table 1. The protein fragment may comprise 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

- 21 046 777 comprises from 50% to 99.9% (e.g., any value from 50% to 99.9%) of the protein encoded by the gene indicated in Table 1. In some embodiments, the expression construct is monocistronic (e.g., the expression construct encodes a single fusion protein comprising a first gene product and a second gene product). In some embodiments, the expression construct is polycistronic (e.g., the expression construct encodes two different gene products, such as two different proteins or protein fragments).

The polycistronic expression vector may comprise one or more (e.g., 1, 2, 3, 4, 5, or more) promoters. Any suitable promoter may be used, such as a constitutive promoter, an inducible promoter, an endogenous promoter, a tissue-specific promoter (e.g., a CNS-specific promoter), etc. In some embodiments, the promoter is the chicken beta-actin promoter (CBA promoter), the 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 (e.g., as described in Tornoe et al. (2002) Gene 297(1-2):21-32). In some embodiments, the promoter is operably linked to a nucleic acid sequence encoding a first gene product, a second gene product, or a first gene product and a second gene product. In some embodiments, the expression cassette comprises one or more additional regulatory sequences, including, but not limited to, transcription factor binding sequences, intron 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 IRESs are described, for example, in Mokrejs et al. (2006) Nucleic Acids Res. 34(Database issue):D125-30. In some embodiments, the nucleic acid sequence encoding the first gene product and the nucleic acid sequence encoding the second gene product are separated by a nucleic acid sequence encoding a self-cleaving peptide. Examples of self-cleaving peptides include, but are not limited to, T2A, P2A, E2A, F2A, BmCPV 2A, and BmIFV 2A, as well as the peptides 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's disease are associated with the accumulation of protein aggregates consisting primarily of the α-synuclein (α-Syn) protein. Accordingly, in some embodiments, the isolated nucleic acids described herein comprise an inhibitory nucleic acid that reduces or prevents expression of the α-Syn protein. The sequence encoding the inhibitory nucleic acid can be placed in the untranslated region (e.g., intron, 5'UTR, 3'UTR, etc.) of the expression vector.

In some embodiments, the inhibitory nucleic acid is located in an intron of the expression construct, such as in an intron upstream of the sequence encoding the first gene product. The inhibitory nucleic acid can be a double-stranded RNA (dsRNA), siRNA, shRNA, microRNA (miRNA), artificial miRNA (amiRNA), or an RNA aptamer. Typically, the inhibitory nucleic acid binds to (e.g., hybridizes to) about 6 to about 30 (e.g., any integer from 6 to 30, inclusive) contiguous nucleotides of the target RNA (e.g., mRNA). In some embodiments, the inhibitory nucleic acid molecule is a miRNA or amiRNA, such as a miRNA targeting SNCA (the gene encoding the α-Syn protein) or TMEM106B (e.g., the gene encoding the TMEM106B protein). In some embodiments, the miRNA does not contain any mismatches to the SNCA region of the mRNA to which it hybridizes (e.g., the miRNA is improved). In some embodiments, the inhibitory nucleic acid is an shRNA (e.g., an shRNA targeting SNCA or TMEM106B). In some embodiments, the inhibitory nucleic acid is an artificial miRNA (amiRNA) that includes a miR-155 backbone and a SNCA or TMEM106B target sequence. It will be understood by the skilled person that, with respect to nucleic acid sequences comprising or encoding inhibitory nucleic acids (e.g., dsRNA, siRNA, miRNA, amiRNA, etc.), any one or more thymidine (T) nucleotides or uridine (U) nucleotides in the sequence provided herein may be replaced by any other nucleotide suitable for base pairing (e.g., via Watson-Crick base pairing) with an adenosine nucleotide. For example, T may be replaced by U, and U may 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 (e.g., adenovirus vector, vector

- 22 046 777 adeno-associated virus (AAV), retroviral vector, baculovirus vector, etc.). In some embodiments, the vector is a plasmid (e.g., a plasmid comprising an isolated nucleic acid as described herein). In some embodiments, the rAAV vector is single-stranded (e.g., single-stranded DNA). In some embodiments, the vector is a recombinant AAV vector (rAAV). In some embodiments, the vector is a baculovirus vector (e.g., an Autographa californica nuclear polyhedrosis virus (AcNPV) vector).

Typically, an rAAV vector (e.g., an rAAV genome) comprises a transgene (e.g., an expression construct comprising one or more of the following: a promoter, an intron, an enhancer sequence, a protein encoding sequence, an inhibitory RNA encoding sequence, a polyA tail sequence, etc.) flanked by two AAV inverted terminal repeat (ITR) sequences. In some embodiments, the rAAV vector transgene comprises an isolated nucleic acid as described herein. In some embodiments, each of the two ITR sequences of the rAAV vector is a full-length ITR (e.g., approximately 145 bp in length and comprises a functional Rep binding site (RBS) and a terminal resolution site (trs)). In some embodiments, one of the ITRs of the rAAV vector is truncated (e.g., shortened or of limited length). In some embodiments, the truncated ITR lacks a functional terminal resolution site (trs) and is used to produce self-complementary AAV vectors (scAAV vectors). In some embodiments, the truncated ITR is an AITR, such 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) comprising an ITR having one or more modifications (e.g., additions, deletions, nucleic acid substitutions, etc.) relative to a wild-type AAV ITR, such as a wild-type AAV2 ITR (e.g., SEQ ID NO: 29). The structure of a wild-type AAV2 ITR is shown in Fig. 20. Typically, a wild-type ITR comprises a 125 nucleotide region that self-anneals to form a palindromic, double-stranded T-shaped hairpin structure consisting of two transverse arms (formed by sequences designated B/B' and C/C, respectively), a longer stem region (formed by sequences A/A'), and a single-stranded terminal region referred to as the D region (Fig. 20). 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 (e.g., 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, the D region comprises the sequence set forth in SEQ ID NO: 27. The D region has been found to play an important role in the encapsidation of rAAV vectors by capsid proteins, e.g., 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 (e.g., proximal to the end of the ITR relative to the transgene insertion or expression construct) efficiently encapsidate AAV capsid proteins, compared to rAAV vectors having ITRs with unmodified ITRs (e.g., wild-type) ITRs. In some embodiments, rAAV vectors having a modified D sequence (e.g., 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 comprises at least one nucleotide substitution relative to the wild-type D sequence (e.g., SEQ ID NO:27). The modified D sequence can have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 nucleotide substitutions relative to the wild-type D sequence (e.g., SEQ ID NO:27). In some embodiments, the modified D sequence comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 nucleic acid substitutions relative to the wild-type D sequence (e.g., SEQ ID NO:27). In some embodiments, the modified D sequence is 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 (e.g., SEQ ID NO: 27). In some embodiments, the modified D sequence comprises the sequence set forth in SEQ ID NO: 26, also referred to as the S sequence, as described in Wang et al. (1995) J Mol Biol 250(5):573-80.

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

- 23 046777

In some aspects, the present invention provides baculovirus vectors comprising an isolated nucleic acid or rAAV vector as described herein. In some embodiments, the baculovirus vector is an Autographa californica nuclear polyhedrosis vector (AcNPV), such 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 comprising 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 HEK293T cell. In some embodiments, the host cell is a bacterial cell, such as an E. coli cell.

rAAV.

In some aspects, the present invention provides recombinant AAVs (rAAVs) comprising a transgene that encodes a nucleic acid as described herein (e.g., an rAAV vector as described herein). The term rAAV generally refers to viral particles comprising an rAAV vector encapsidated with one or more AAV capsid proteins. A RAAV as described herein may comprise a capsid protein having a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10. In some embodiments, the rAAV comprises a capsid protein from a non-human host, such as a rhesus macaque AAV capsid protein, such as AAVrh.10, AAVrh.39, etc. In some embodiments, the rAAV described herein comprises a capsid protein that is a variant of a wild-type capsid protein, such as a variant capsid protein that comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 (e.g., 15, 20, 25, 50, 100, etc.) amino acid substitutions (e.g., mutations) relative to the wild-type AAV capsid protein from which it is derived. In some embodiments, the variant AAV capsid protein is the AAV1RX capsid protein, such as described in Albright et al. Mol Ther. 2018 Feb 7; 26(2):510-523. In some embodiments, the variant capsid protein is the AAV TM6 capsid protein, such as described in Rosario et al. Mol Ther Methods Clin Dev. 2016; 3: 16026. In some embodiments, the rAAVs described herein are readily distributed into the CNS, particularly when administered into the CSF space or directly into the brain parenchyma. Accordingly, in some embodiments, the rAAVs described herein comprise a capsid protein that is capable of crossing the blood-brain barrier (BBB). For example, in some embodiments, the rAAV comprises a capsid protein having the AAV9 or AAVrh serotype. 10. The production of rAAV is described, for example, in Samulski et al. (1989) J Virol. 63(9):3822-8 and Wright (2009) Hum Gene Ther. 20(7):698-706. In some embodiments, the rAAV comprises 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 PR006A. PR006A is an rAAV that delivers a functional human GRN gene resulting in increased expression of a functional human PGRN gene. The PR006A vector insert comprises a chicken β-actin (CBA) promoter element consisting of 4 parts: a cytomegalovirus (CMV) enhancer, a CBA promoter, exon 1, and an intron (int) for constitutive expression of a codon-optimized human GRN coding sequence (SEQ ID NO:68). The 3' region also comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), followed by a bovine growth hormone polyadenylation signal tail. Three well-characterized transcriptional regulatory activation sites are included at the 5' end of the promoter region: TATA, RBS, and YY1 (see, e.g., Francois et al., (2005) J. Virol. 79(17): 11082-11094). Flanking inverted terminal repeats (ITRs) allow correct packaging of the inserted sequences. The framework contains a gene conferring kanamycin resistance as well as a spacer sequence preventing refolding. A schematic of the rAAV vector is shown in Fig. 64. SEQ ID NO 90 is the nucleotide sequence of the first strand (in 5' to 3' order) of the PR006A vector shown in Fig. 64. SEQ ID NO 91 is the nucleotide sequence of the second strand (in 5' to 3' order) of the PR006A vector shown in Fig. 64. PR006A contains AAV9 capsid proteins.

In some embodiments, rAAV as described herein (e.g., comprising a recombinant rAAV genome encapsidated with AAV capsid proteins to form an rAAV capsid particle) is produced in a baculovirus vector expression system (BEVS). Production of rAAV using a BEVS is described, for example, in Urabe et al. (2002) Hum Gene Ther 13(16):1935-43, Smith et al. (2009) Mol Ther 17(11):1888-1896, U.S. Patent No. 8,945,918, U.S. Patent No. 9,879,282, and PCT International Publication No. WO 2017/184879. However, rAAV can be produced using any suitable method (e.g., using recombinant rep and

- 24 046777 cap). 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 abolish the biological activity or properties of the compound and is relatively non-toxic, e.g., the material can be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent, or encapsulating material, that is involved in carrying or transporting a compound useful in the context of the present invention into the body or to a patient so that it can perform its intended function. Additional ingredients that can 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 Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, Pa.), which is incorporated herein by reference.

The compositions (e.g., pharmaceutical compositions) of the present invention 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 (e.g., as powders, ointments, creams, and/or drops), mucosal, nasal, buccal, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol. Particularly contemplated routes are oral administration, intravenous administration (e.g., systemic intravenous injection), regional administration via the blood and/or lymph, and/or direct administration to the affected area. Typically, the most appropriate route of administration will depend on a variety of factors, including the nature of the agent (e.g., its stability in the gastrointestinal environment) and/or the condition of the subject (e.g., whether the subject can orally take the drug). In certain embodiments, the 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 PR006A finished drug product comprising the PR006A rAAV described above, provided in the form of an aqueous solution. In some embodiments, the final formulation buffer comprises about 20 mM Tris, pH 8.0, about 1 mM _MgCl2 , about 200 mM NaCl, and about 0.001% poloxamer 188 (w/v). In some embodiments, the finished drug product and the final formulation buffer are suitable for injection into the cerebellomedullary cistern (ICM).

Methods.

Aspects of the present invention relate to compositions for expressing one or more gene products associated with a CNS disease in a subject being treated for diseases involving the CNS. The one or more gene products associated with a CNS disease may be encoded by one or more isolated nucleic acids or rAAV vectors. In some embodiments, the subject is administered a single vector (e.g., an isolated nucleic acid, rAAV, etc.) encoding one or more (1, 2, 3, 4, 5, or more) gene products. In some embodiments, the subject is administered a plurality (e.g., 2, 3, 4, 5, or more) of vectors (e.g., isolated nucleic acids, rAAV, etc.), wherein each vector encodes a different gene product associated with a CNS disease.

A disease affecting the central nervous system may be a neurodegenerative disorder, a synucleinopathy, a tauopathy, or a lysosomal storage disorder. Examples of neurodegenerative disorders and their associated genes are listed in Table 12.

The term synucleinopathy refers to a disease or disorder characterized by accumulation of alpha-synuclein (the gene product of SNCA) in a subject (e.g., compared to a healthy subject, e.g., a subject without synucleinopathy). Examples of synucleinopathies and their associated genes are listed in Table 13.

The term tauopathy refers to a disease or disorder characterized by abnormal accumulation of tau protein in a subject (e.g., compared to a healthy subject who does not have a tauopathy). Examples of tauopathies and their associated genes are listed in Table 14.

A lysosomal storage disease refers to a disease characterized by the abnormal accumulation of toxic cellular products in the subject's lysosomes. Examples of lysosomal diseases

- 25,046,777 accumulations and associated genes are listed in Table 15.

As used in this document, the terms treat or affect refer to (a) preventing or delaying the onset of a CNS disease; (b) reducing the severity of a CNS disease; (c) reducing or preventing the development of symptoms characteristic of a CNS disease; (d) and/or preventing the worsening of symptoms characteristic of a CNS disease. Symptoms of a CNS disease may include, for example, motor dysfunction (e.g., tremors, rigidity, slowness of movement, difficulty walking, paralysis), cognitive dysfunction (e.g., dementia, depression, anxiety, psychosis), memory problems, emotional and behavioral dysfunction.

The present invention is based in part on compositions for expressing combinations of PD-associated gene products in a subject that act together (e.g., synergistically) to treat Parkinson's disease.

Accordingly, in some aspects of the present invention, there is provided a method of treating a subject with existing or suspected Parkinson's disease, said method comprising administering to the subject a composition (e.g., a composition comprising 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.

In some embodiments, the Gaucher disease is a neuronopathic Gaucher disease, such as Gaucher disease type 2 or Gaucher disease type 3. In some embodiments, the subject suffering from Gaucher disease does not have PD or does not have symptoms of PD.

Accordingly, in some aspects of the present invention, there is provided a method of treating a subject with existing or suspected neuronopathic Gaucher disease, said method comprising administering to the subject a composition (e.g., a composition comprising 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 treating Alzheimer's disease or frontotemporal dementia (FTD). In some embodiments, the subject has Alzheimer's disease. In some embodiments, the subject has FTD and does not have Alzheimer's disease. In some embodiments, the subject has FTD with a GRN (progranulin) mutation. In some embodiments, the subject has FTD with a GRN mutation, and the subject is heterozygous for a GRN mutation (e.g., a pathogenic GRN mutation). In some embodiments, the GRN mutation is a null mutation (e.g., nonsense mutations, frameshift mutations, or splice site mutations, or a complete or partial (exonic) deletion of the gene). In some embodiments, the GRN mutation is a pathogenic mutation with a proven functional deleterious effect. In some embodiments, the GRN mutation is a pathogenic missense mutation. In some embodiments, the GRN mutation is listed in the Molgen FTD database (molgen.ua.ac.be). In some embodiments, the GRN mutation results in low plasma PGRN levels (<70 ng/mL) in the subject.

In some embodiments, the subject has FTD, FTD with a GRN mutation, FTD with a may mutation, FTD with a C9orf72 mutation, neuronal ceroid lipofuscinosis, Parkinson's disease, Alzheimer's disease, corticobasal degeneration, motor neuron disease, or Gaucher disease. In some embodiments, the subject has symptomatic FTD (e.g., behavioral variant FTD (bvFTD), FTD with primary progressive aphasia (PPA) and corticobasal syndrome, or a variant with a combination of syndromes).

Accordingly, in some aspects, the present invention provides a method of treating a subject with existing or suspected FTD with a GRN mutation, said method comprising administering to the subject a composition (e.g., a composition comprising an isolated nucleic acid or vector, or rAAV) as described herein.

In some embodiments, a subject having Alzheimer's disease or FTD (e.g., FTD with a GRN mutation) is administered an rAAV encoding progranulin (PGRN), or a portion thereof. In some embodiments, a subject having Alzheimer's disease or FTD (e.g., FTD with a GRN mutation) is administered an rAAV encoding PGRN, or a portion thereof, wherein the PGRN protein is encoded by a codon-optimized nucleic acid sequence or the nucleic acid sequence of SEQ ID NO: 68. In some embodiments, the PGRN protein comprises the amino acid sequence of SEQ ID NO: 67, or a portion thereof. In some embodiments, the rAAV encoding PGRN comprises a capsid protein having the AAV9 serotype.

In some embodiments, a composition comprising a rAAV encoding PGRN for treating FTD (e.g., FTD with a GRN mutation) is administered to a subject at a dose of from about 1 x ^{10 12} vector genomes (vg) to about 1 x 10 ¹⁵ vg, or from about 1 x ^{10 13} vg to about 7 x 10 ¹⁴ vg, or from about 1 x ¹⁰ 13 vg to about 5 x 10 ¹⁴ vg, or from about 2 x 10 ¹³ vg to about 2 x 10 ¹⁴ vg, or from about 3 x 10 ¹³ vg to about 2 x 10 ¹⁴ vg, or from

- 26 046 777 about 3.5x1013 vg to about 1.4x10 ¹⁴ vg. In some embodiments, a composition comprising a rAAV encoding PGRN for treating FTD (e.g., FTD with a GRN mutation) is administered to a subject at a dose of about 2x10 ¹³ vg, about 3x10 ¹³ vg, about 4x10 ¹³ vg, about 5x10 ¹³ vg, about 6x10 ¹³ vg, about 7x1013 vg, about 8x10 ¹³ vg, about 9x10 ¹³ vg, about 1x10 ¹⁴ vg, or about 2x10 ¹⁴ vg.

In some aspects, the present invention provides a method of treating a subject with existing or suspected FTD (e.g., FTD with a GRN mutation), said method comprising administering to the subject a composition comprising rAAV encoding PGRN, said composition being administered at a dose of about 3.5x1013 vector genomes (vg), about 7.0x10 ¹³ vg, or about 1.4x10 ¹⁴ vg.

In some aspects, the present invention provides a method of treating a subject with existing or suspected FTD (e.g., FTD with a GRN mutation), said method comprising administering to the subject a composition comprising rAAV encoding PGRN, said composition being administered at a dose of about 1x10 ¹⁴ vector genomes (vg), about 2.0x10 ¹⁴ vg, or about 4.0x10 ¹⁴ vg.

In some embodiments, a composition comprising a rAAV encoding PGRN for treating FTD (e.g., FTD with a GRN mutation) is administered to a subject as a single dose, and the composition is not subsequently administered to the subject.

In some embodiments, the composition comprising rAAV is delivered by a single suboccipital injection into the cisterna magna. In some embodiments, the injection into the cisterna magna is performed under radiographic guidance. In some embodiments, the present invention provides a method of treating a symptom in a subject with known or suspected FTD with a GRN mutation, said method comprising administering to the subject a composition comprising rAAV, a coding sequence for a functional progranulin protein (PGRN), and wherein the PGRN protein is encoded by a codon-optimized nucleic acid sequence or the nucleic acid sequence of SEQ ID NO: 68. In some embodiments, the symptom of FTD with a GRN mutation may be personality change, executive dysfunction, disinhibition, apathy, slow speech production, poor grammar, multimodal agnosia, semantic aphasia, or impaired word comprehension. In some embodiments, the rAAV encoding PGRN comprises a capsid protein having the AAV9 serotype.

In some embodiments, the present invention provides a method of reducing lipofuscin accumulation in the brain of a subject with known or suspected FTD with a GRN mutation, said method comprising administering to the subject a composition comprising rAAV encoding progranulin (PGRN), and wherein the PGRN protein is encoded by a codon-optimized nucleic acid sequence or the nucleic acid sequence of SEQ ID NO: 68. In some aspects, the present invention provides a method of reducing ubiquitin accumulation in the brain of a subject with known or suspected FTD with a GRN mutation, said method comprising administering to the subject a composition comprising rAAV encoding progranulin (PGRN), and wherein the PGRN protein is encoded by a codon-optimized nucleic acid sequence or the nucleic acid sequence of SEQ ID NO: 68. In some aspects, the present invention provides a method of reducing gene expression and/or protein expression of TNFa and/or CD68 in the brain of a subject with existing or suspected FTD with a GRN mutation, wherein said method comprises administering to the subject a composition comprising rAAV encoding progranulin (PGRN), and wherein the PGRN protein is encoded by a codon-optimized nucleic acid sequence or the nucleic acid sequence of SEQ ID NO: 68. In some aspects, the present invention provides a method of increasing cathepsin D maturation in the brain of a subject with existing or suspected FTD with a GRN mutation, wherein said method comprises administering to the subject a composition comprising rAAV encoding progranulin (PGRN), and wherein the PGRN protein is encoded by a codon-optimized nucleic acid sequence or the nucleic acid sequence of SEQ ID NO: 68. In some aspects, the present invention provides a method of increasing the level of TDP-43 (43 kDa trans-action response DNA binding protein) nuclear protein in the brain of a subject with existing or suspected FTD with a GRN mutation, wherein wherein said method comprises administering to the subject a composition comprising a rAAV encoding progranulin (PGRN), and wherein the PGRN protein is encoded by a codon-optimized nucleic acid sequence or the nucleic acid sequence of SEQ ID NO: 68. In some embodiments, the present invention provides a method of reducing the level of neurofilament light chain (NFL) in the blood or CSF of a subject with existing or suspected FTD with a GRN mutation, wherein said method comprises administering to the subject a composition comprising a rAAV encoding progranulin (PGRN), and wherein the PGRN protein is encoded by a codon-optimized nucleic acid sequence or the nucleic acid sequence of SEQ ID NO: 68. In some embodiments, the rAAV encoding PGRN comprises a capsid protein having the AAV9 serotype.

The subject is typically a mammal, preferably a human. In some embodiments,

- 27 046 777 implementation, the subject is between 1 month and 10 years old (e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or any age in between). In some embodiments, the subject is between 2 years and 20 years old. In some embodiments, the subject is between 30 years and 100 years old. In some embodiments, the subject is over 55 years old.

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

In some embodiments, direct injection into the CNS of a subject involves convective enhanced delivery (CED). Convective enhanced delivery is a therapeutic strategy that involves surgically intervening in the brain and placing a small-bore catheter directly into the target area of the brain, followed by infusion of a therapeutic agent (e.g., a composition or rAAV as described herein) directly into the brain of the subject. 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 the subject peripherally, such as by peripheral injection. Examples of peripheral injection include subcutaneous injection, intravenous injection, intraarterial injection, intraperitoneal injection, or any combination of the above. In some embodiments, the peripheral injection is an intraarterial injection, such as an injection into the carotid artery of the subject.

In some embodiments, a composition (e.g., a composition comprising an isolated nucleic acid or vector or rAAV) as described herein is administered both peripherally and directly into the CNS of a subject. For example, in some embodiments, the composition is administered to the subject by intra-arterial injection (e.g., carotid injection) and by intraparenchymal injection (e.g., intraparenchymal injection via a CED). In some embodiments, the direct injection into the CNS and the peripheral injection are performed simultaneously (e.g., at the same time). In some embodiments, the direct injection is performed prior to the peripheral injection (e.g., within a period of time from 1 minute to 1 week or more prior to the peripheral injection). In some embodiments, the direct injection is performed after the peripheral injection (e.g., within a period of time from 1 minute to 1 week or more following the peripheral injection).

In some embodiments, the subject is administered an immunosuppressant prior to administration of the composition (e.g., within a period of 1 month to 1 minute prior to 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 (e.g., a composition comprising 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 at a titer of about 109 genome copies (GC/kg) to about 10 ¹⁴ GC/kg (e.g., about 109 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, a high titer (e.g., >10 ¹² GC genome copies/kg rAAV) is administered to the subject by injection into the CSF space ^or by intraparenchymal injection. In some embodiments, rAAV as described herein is administered to the 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 1x10 ¹⁰ vg to about 1x10 ¹⁶ vg by injection into the cisterna medullaris.

A composition (e.g., a composition comprising an isolated nucleic acid or vector,

- 28 046777 or rAAV) as described herein can be administered to a subject one or more times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 or more) times. In some embodiments, the composition is administered to the subject continuously (e.g., over an extended period), such as via 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 the 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 fusing protein sequences; or by using a 2A peptide linker such as T2A or P2A, resulting in 2 peptide fragments with added amino acids due to the prevention of peptide bond formation; or by 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 to be expressed can enhance expression levels. shRNA and other regulatory RNAs can potentially be included in these sequences. Examples of expression constructs described herein are shown in Figs. 1-8, 21-35, 39, 41-51, and 64 and in Table 2 below.

Table 2

Name About shPH CD By A Bicis About CD By Al MOT TO SI 1 three NI MOT S2 LI IN op 1 th or 2 A2 a eleme me NT railway U IT R CMVe_CBAp_GBAl_WPRE_bGH CB GB WPRE 37 A Al -bGH 41 LT 1 s_JetLong_mRNAiaSYn_SCARB2, JetL aSyn SC bGH T2A GB 42 T2A, GBAl bGH ong AR Al 15 B2 LIl_JetLong_SCARB2-IRES-GBAl_bGH JetL SC bGH IRES GB 43 ong AR Al 99 B2 FPl_JetLong_GBAl_bGH_JetLong_SCA JetL GB bGH JetL SC SV 44 RB2SV40L ong Al ong AR 40 64 B2 L PrevailVector_LT2s_JetLong_mRNAiaSY JetL aSyn PS bGH T2A - GB - 43 n PSAP, T2A, GBAl_bGH_4353nt ong AP Al 53

- 29 046777

PrevailVector_LI2_JetLong_PSAP_IRES_ GB Al_SymtheticpolyA_43 37nt JetLong PS AP Synthetic IRES GB Al 43 37 PrevailVector_10s_JetLong_mRNAiaSy_ GBA2_WPRE_bGH_4308nt JetLong aSyn GB A2 WPRE bGH - - - - 43 08 PrevailVector_FT4_JetLong_GBAl_T2A GALCSy ntheticpoly A_43 73nt JetLong GB Al Synthetic T2A GA LC 43 73 PrevailVector_LT4_JetLong_GALC_T2A _GBAl_SyntheticpolyA_4373nt JetLong GA LC Synthetic T2A GB Al 43 73 PrevailVector_LT5s_JetLong_mRNAiaSy nCTSB, T2A, GB Al_WPRE_bGH_43 92nt JetLong aSyn CT SB WPRE bGH T2A GB Al 43 92 PrevailVectorFTl ItJetLongmRNAiaS yn_GB A1T2SSMPD l_SyntheticpolyA_ 4477nt JetLong aSyn GB Al Synthetic T2A SM PD 1 44 77 PrevailVector_LI4_JetLong_GALC_IRES _GBAl_SymtheticpolyA_4820nt JetLong GA LC Synthetic rA IRES GB Al 48 20 PrevailVector_FP5_JetLong_GBAl_bGH _JetLong_CTSB_SV401_4108nt JetLong GB Al bGH JetLong CT SB SV 40 L 41 08 PrevailVector_FT6s_JetLong_mRNAiaSy n GBAl, T2A, GCH l_WPRE_bGH_4125nt JetLong aSyn GB Al WPRE bGH T2A GC Hl 41 25 PrevailVector_LT7 sJetLongmRNAiaSy n_RAB7Ll, T2A, GB Al_WPRE_bGH_3 984nt JetLong aSyn RA B7 LI WPRE bGH T2A GB Al 39 84 PrevailVector_FI6s_JetLong_mRNAiaSY n_GB Al -IRES-GCH l_bGH_3 978nt JetLong aSyn GB Al bGH IRES - GC Hl - 39 78 PrevailVector_9st_JetLong_mRNAiaSyn_ mRNAiTMEM106B_VPS35_WPRE_bG H_4182nt JetLong aSyn & TME MIO 6B VP S35 WPRE bGH 41 82 PrevailVector_FT12s_JetLong_mRNAiaS yn GBAl, T2A, IL3 4_WPRE_bGH_4104nt JetLong aSyn GB Al WPRE bGH T2A IL3 4 41 04 PrevailVector_FI12s_JetLong_mRNAiaS Yn_GBAl-IRES-IL34_bGH_3957nt JetLong aSyn GB Al bGH IRES - IL3 4 - 39 57 PrevailVector_FP8_JetLong_GBAl_bGH _CD68_TREM2_SV401_4253nt JetLong GB Al bGH CD6 8 TR EM 2 SV 40 L 42 53 PrevailVector_FP12_CMVe_CBA_GBAl _bGH_JetLong_IL34_SV401_4503nt CB A GB Al bGH JetLong IL3 4 SV 40 L 45 03

Example 2. Cellular assays of viral transduction in GBA-deficient cells.

GBA1-deficient cells have been 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 (GlcCer and GlcSph). Treatment of cultured wild-type or mutant cell lines with G-case inhibitors such as CBE can also be used to obtain GBA-deficient cells.

Using such cellular models, lysosomal defects are quantified in terms of accumulation of protein aggregates such as α-synuclein with antibody to this protein or phospho

- 30 046777 aSyn, followed by visualization by fluorescence microscopy. Visualization of lysosomal abnormalities is also performed by ICC for protein markers such as LAMP1, LAMP2, LIMP1, LIMP2, or by using dyes such as Lysotracker, or by uptake through the endocytic compartment of fluorescent dextran or other markers. Visualization of accumulation of autophagy markers due to defective fusion with lysosomes, such as LC3, can also be performed. Western blotting and/or ELISA are used to quantify abnormal accumulation of these markers. In addition, accumulation of glycolipid substrates and GBA1 products is measured by standard assays. Therapeutic criteria (e.g., reduction in PD-associated pathology) are assessed in the context of expression of transduced AAV vectors to confirm and quantify activity and function. G-case can also be quantified by protein ELISA or by standard G-case activity assays.

Example 3. In vivo assays using mutant mice.

This example describes in vivo assays of AAV vectors using mutant mice. In vivo assays of AAV vectors as described above in mutant mice are performed using assays described, for example, in 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 (e.g., at a dose of 2x1011 vg/mouse) is performed using concentrated AAV stocks, e.g., in an injection volume of 5-10 μl. Intraparenchymal convection enhanced delivery is performed.

Treatment is initiated either before or after the onset of symptoms. Measurable endpoints include substrate accumulation in the CNS and CSF, assessment of G-case enzyme accumulation by ELISA and enzyme activity, motor and cognitive function, 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 (e.g., the EDS mouse model). In vivo assays of these AAV vectors are performed in a chemically induced mouse model of Gaucher disease, e.g., as described in Vardi et al. (2016) J Pathol. 239(4):496–509.

Intrathecal or intraventricular delivery of control vehicles and AAV vectors (e.g., 2x1011 vg/mouse) is performed using concentrated AAV stocks, e.g., 5-10 μL injection volume. Intraparenchymal convection enhanced delivery is performed. Peripheral delivery is achieved by tail vein injection. Treatment is initiated either before or after symptom onset. Endpoints measured include CNS and CSF substrate accumulation, assessment of G-case enzyme accumulation by ELISA and enzyme activity, motor and cognitive function, 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 (e.g., 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 performed using a study design similar to that described in Grabowski et al. (1995) Ann. Intern. Med. 122(1):33-39.

Example 6. Treatment of peripheral disease.

In some embodiments, patients suffering from certain forms of Gaucher disease exhibit symptoms of peripheral neuropathy, such 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 (e.g., type 1 Gaucher disease). 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, such as by the methods described in Biegstraaten et al., following administration of rAAV.

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

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

This example shows in vivo rAAV assays as described herein for treatment

- 31 046 777 forms of Gaucher disease with CNS involvement. Briefly, patients with Gaucher disease identified as having a form of Gaucher disease with CNS involvement (e.g., type 2 or 3 Gaucher disease) are administered rAAV as described herein. The levels of transduced gene products, as described herein, present in the CNS of the 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 Western blotting, enzymatic function assays, or imaging studies.

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

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

The rAAV-GBA1 vector insert contains a CBA promoter element (CBA) consisting of four parts: the CMV enhancer (CMVe), the CBA promoter (CBAp), exon 1, and an intron (int) for constitutive expression of the codon-optimized coding sequence (CDS) of human GBA1 (maroon). 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 proper packaging of the insert sequences. Two 5' ITR sequence variants were evaluated (Fig. 7, inset, lower 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-GBA1 vector product contains the D domain nucleotide sequence shown in Fig. 7 (insert, upper sequence) The vector variant contains a mutant D domain (referred to herein as the S domain, nucleotide changes are shown as hatched), similar to that studied in preclinical studies. The scaffold contains a gene conferring resistance to kanamycin, as well as a spacer sequence that prevents refolding. A schematic of the rAAV-GBA1 vector is shown in Fig. 8. The rAAV-GBA1 vector is packaged into rAAV using capsid proteins of the AAV9 serotype. rAAV-GBA1 is administered to a subject as a single dose by suboccipital injection under fluoroscopic guidance into the cisterna magna (ICM injection). One embodiment of the rAAV-GBA1 administration regimen studied is as follows:

A single dose of rAAV-GBA1 is administered to patients (n=12) at one of two dose levels (Ze13 vg (low dose); 1e14 vg (high dose), etc.), which are determined based on the results of preclinical pharmacology and toxicology studies.

Initial studies were conducted in a chemical mouse model involving daily delivery of conduritol-b-epoxide (CBE), a GCase inhibitor, to evaluate the efficacy and safety of the rAAV-GBA1 vector and the rAAV-GBA1 S-variant construct (as described further below). Additionally, initial studies were performed in a mouse genetic model carrying a homozygous GBA1 mutation and partially deficient in saposins (4L/PS-NA). Additional dose-ranging studies are underway in mice and non-human primates (NHPs) to further evaluate the safety and efficacy of the vector.

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). The 20 bp D domain of the 145 bp 5′ ITR is thought to be necessary for optimal viral vector production, but mutations in the D domain have also been reported to increase transgene expression in some cases. Thus, in addition to the rAAV-GBA1 viral vector, which contains the intact D domain, a second form of the vector with a mutated D domain (referred to herein as the S domain) was also evaluated. Both rAAV-GBA1 and the variant express the same transgene. Although both vectors produced virus that was effective in vivo, as detailed below, rAAV-GBA1, which contains the wild-type D domain, was selected for further development. To establish a CBE model of GCase deficiency, young mice were treated with CBE, a specific GCase inhibitor. Mice were treated with CBE by intraperitoneal injection daily starting at postnatal day 8 (P8). Three different doses of CBE (25 mg/kg, 37.5 mg/kg, 50 mg/kg) and PBS were tested to establish a model that exhibits a behavioral phenotype (Fig. 9). Higher doses of CBE were lethal in a dose-dependent manner. All mice treated with 50 mg/kg CBE died before P23, and 5 of 8 mice treated with 37.5 mg/kg CBE died before P27. No lethality was observed in mice treated with 25 mg/kg CBE. While mice injected with CBE did not exhibit global motor impairment in the open field assay (moving the same distance and at the same speed as mice given PBS), mice given CBE exhibited deficits in motor coordination and balance as measured by the rotating drum test.

Mice that survived to the end of the study were sacrificed the day after they received their last dose of VES (P27, Day 1) or three days after VES withdrawal (P29, Day 3). Lipid analysis was performed on the cortex of mice treated with 25 mg/kg VES to assess the accumulation of GC-case substrates in both the Day 1 and Day 3 cohorts. GluSph and GalSph levels (measured together in this example) accumulated significantly in VES-treated mice compared to the con

- 32 046777 control animals treated with PBS, consistent with GCase deficiency.

Based on the study described above, a dose of 25 mg/kg CBE was chosen because it induced behavioral impairments without affecting survival. To achieve broad distribution of GBA1 in the brain and transgene expression during CBE administration, rAAV-GBA1 or adjuvant was administered via intracerebroventricular (ICV) injection on postnatal day 3 (P3) followed by daily intraperitoneal administration of CBE or PBS administration starting at P8 (Fig. 10). CBE-treated mice injected with rAAV-GBA1 performed statistically significantly better on the rotating drum than adjuvant-treated mice (Fig. 11). Mice in the treatment group did not differ from adjuvant-treated mice in other behavioral measures, such as total distance traveled during testing (Fig. 11).

At the end of the study, half of the surviving mice were sacrificed the day after the last dose of VES (P36, Day 1) or three days after VES withdrawal (P38, Day 3) for biochemical analysis (Fig. 12). Using a fluorometric enzyme assay performed in triplicate, GCase activity was assessed in the cortex. GCase activity was increased in rAAV-GBA1-treated mice, whereas VES administration decreased GCase activity. Furthermore, mice that received both VES and rAAV-GBA1 had GCase activity levels that were similar to the PBS-treated group, indicating that rAAV-GBA1 delivery is able to overcome the inhibition of GCase activity induced by VES administration. Lipid analysis was performed on the motor cortex of mice to examine the levels of GluCer and GluSph substrates. Both lipids accumulated in the brains of mice treated with VES, and rAAV-GBA1 administration significantly reduced substrate accumulation.

Lipid levels were negatively correlated with both GCase activity and successful performance on the rotating drum test in all treatment groups. Increased GCase activity after rAAV-GBA1 administration was associated with decreased substrate and increased 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 rAAV-GBA1, both with and without VES, were positive for rAAV-GBA1 vector genomes in the cortex, indicating that ICV delivery results in delivery of rAAV-GBA1 to the cortex. Additionally, vector genomes were detected in the liver, in some cases in the spleen, and were not detected in the heart, kidney, or gonads. There was no statistically significant difference between the Day 1 and Day 3 groups for all endpoints. A larger study in the CBE model further investigated the effective doses of rAAV-GBA1 in the CBE model. Using a 25 mg/kg CBE dose model, adjuvant or rAAV-GBA1 was delivered via ICV at P3, and daily IP administration of PBS or CBE was initiated at P8. Given the similarities between the CBE-withdrawn and non-CBE-withdrawn groups observed in previous studies, all mice were sacrificed the day after the last CBE dose (P38-40). The effect of three different doses of rAAV-GBA1 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 3.2e9 vg (2.13e10 vg/g brain) rAAV-GBA1 ICV+25 mg/kg CBE IP 1.0e10 vg (6.67e10 vg/g brain) rAAV-GBA1 ICV+25 mg/kg CBE IP 3.2e10 vg (2.13el 1 vg/g brain) rAAV-GBA1 ICV+25 mg/kg CBE IP.

The highest dose of rAAV-GBA1 rescued treatment-related failure to gain weight at P37. Additionally, this dose resulted in statistically significant increases in the rotating drum test and tapered beam test performance scores compared to the adjuvant+GBE group (Fig. 15). Mortality was observed in multiple groups, including those receiving both adjuvant and rAAV-GBA1 (adjuvant+PBS: 0; adjuvant+25 mg/kg GBE: 1; 3.2e9 vg rAAV-GBA1+ 25 mg/kg GBE: 4; 1.0e10 vg rAAV-GBA1+ 25 mg/kg GBE: 0; 3.2e10 vg rAAV-GBA1+ 25 mg/kg GBE: 3).

At the end of the study, all surviving mice were sacrificed for biochemical analysis (Fig. 16). GCase activity in the cerebral cortex was assessed in triplicate using a fluorometric assay. Mice treated with VES showed decreased GCase activity, whereas mice treated with high dose rAAV-GBA1 showed a statistically significant increase in GCase activity compared to VES. Mice treated with VES also showed accumulation of GluCer and GluSph, which was abrogated by high dose rAAV-GBA1.

In addition to the established chemical model of CBE, rAAV-GBA1 was also evaluated in the genetic model 4L/PS-NA, which is homozygous for the V394L GD mutation in Gba1 and is also partially deficient in saposins that affect GCase localization and activity. These mice exhibit deficits in motor strength, coordination, and balance, as evidenced by their performance in the beam walking test, rotating drum retention test, and cord suspension test. The lifespan of these mice is typically less than 22 weeks. In the initial study, 3 μL

- 33,046,777 peak titer viruses were delivered via ICV at P23, with a final dose of 2.4-10 μg (6.0-10 μg/g brain). Treatment groups were as follows (with 6 mice per group):

WT + excipient ICV

4L/PS-NA + excipient ICV

4L/PS-NA + 2.4e10 vg (6.0e10 vg/r brain) rAAV-GBAl ICV

Motor performance was assessed using the beam walking test 4 weeks after rAAV-GBA1 delivery. The group of rAAV-GBA1-treated mutant mice showed a trend toward fewer total slips and fewer slips per speed compared to the excipient-treated mutant mice, restoring motor function to near WT levels (Fig. 17). Because the motor phenotype impairments become more severe as these mice age, their performance in this and other behavioral tests is being assessed at later time points. Lipid levels, GCase activity, and biodistribution are assessed in these mice at the conclusion of the study.

Additional lower doses of rAAV-GBA1 are currently being tested using the CBE model, corresponding to 0.03x, 0.1x and 1x the proposed high clinical Phase 1 dose. Each group includes 10 mice (5M/5F) per group:

Excipient ICV

Excipient ICV + 25 mg/kg SVE IP

3.2e8 vg (2.13e9 vg/r brain) rAAV-GBAl ICV + 25 mg/kg SVE IP 1.0e9 vg (6.67e9 vg/r brain) rAAV-GBAl ICV + 25 mg/kg SVE IP 1.0е10 vg (6.67е10 vg/r brain) rAAV-GBAl ICV + 25 mg/kg SBE IP

In addition to motor phenotypes, lipid levels and GCase activity were assessed in the cerebral cortex. Treatments and analyses were also performed.

To evaluate efficacy and safety data, a larger dose-ranging study was initiated. 10 4L/PS-NA mice (5M/5F per group) were injected with 10 µL rAAV-GBA1. Using allometric calculation of brain weight, the doses correlated with 0.15x, 1.5x, 4.4x, and 14.5x of the proposed high clinical phase 1 dose. Injection groups consisted of:

WT + excipient ICV

4L/PS-NA + excipient ICV

4L/PS-NA + 4.3е9 vg (1.1е10 vg/r brain) rAAV-GBAl ICV

4L/PS-NA + 4.3el0 vg (1, lei 1 vg/r brain) rAAV-GBAl ICV

4L/PS-NA + l.3el 1 vg (3.2ell vg/r brain) rAAV-GBAl ICV

4L/PS-NA + 4.3el 1 vg (1, lel2 vg/r brain) rAAV-GBAl ICV

Example 9. In vitro analysis of rAAV vectors.

The rAAV constructs were tested in vitro and in vivo. Figure 18 shows representative in vitro expression data for rAAV constructs encoding the progranulin (PGRN) protein. The left panel shows a standard curve of the progranulin (PGRN) ELISA. The bottom panel shows the dose response of PGRN expression measured by ELISA in cell lysates of rAAV-transduced HEK293T cells. MOI = multiplicity of infection (vector genomes per cell). A pilot study was performed to evaluate the in vitro activity of rAAV vectors encoding prosaposin (PSAP) and SCARB2, alone or in combination with GBA1 and/or one or more inhibitory RNAs. One construct encoding PSAP and progranulin (PGRN) was also tested. The vectors tested included those listed in Table 1. 3. Opt refers to a nucleic acid sequence that is codon optimized for expression in mammalian cells (e.g., human cells). Fig. 19 shows representative data indicating that transfection of HEK293 cells with each of the constructs resulted in overexpression of the respective gene product compared to mock-transfected cells. A pilot study was performed to evaluate the activity of rAAV vectors encoding TREM2 alone or in combination with one or more inhibitory RNAs. The vectors tested include those listed in Table 3. Opt refers to a nucleic acid sequence that is codon optimized for expression in mammalian cells (e.g., human cells). Figs. 36A-36B show representative data indicating that transfection of HEK293 cells with each of the constructs resulted in overexpression of the respective gene product compared to mock-transfected cells.

- 34 046777

Table 3

ID Promoter Inhibitory RNA Promoter Transgene 100015 ZVintronny SNCA letLong Opt- PSAPGBAl 100039 - - letLong Opt-PSAP-GRN 100046 - - SVA Opt-PSAP 100014 letLong SNCA letLong Opt- SCARB2_GBA1 100040 IL, CD68 opt-GBAl, TREM2

Example 10. Testing of SNCA and TMEM106B shPHK designs.

HEK293 cells.

The human embryonic kidney 293 (HEK293) cell line (#85120602, Sigma-Aldrich) was used in this study. HEK293 cells were maintained in culture medium (D-MEM [#1995065, Thermo Fisher Scientific] supplemented with 10% fetal bovine serum [FBS] [#10082147, Thermo Fisher Scientific]) containing 100 units/mL penicillin and 100 μg/mL streptomycin (#15140122, Thermo Fisher Scientific).

Plasmid transfection.

Plasmid transfection was performed using Lipofectamine 2000 transfection reagent (#11668019, Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, HEK293 cells (#12022001, Sigma-Aldrich) were seeded in antibiotic-free culture medium at a density of 3x105 cells/mL. The next day, plasmid and Lipofectamine 2000 reagent were combined in Opti-MEM solution (#31985062, Thermo Fisher Scientific). After 5 min, the mixtures were added to the HEK293 culture. After 72 h, cells were harvested for RNA or protein extraction or visualization analysis. For visualization analysis, plates were precoated with 0.01% poly-L-lysine (P8920, Sigma-Aldrich) before cell seeding.

Gene expression analysis was performed using quantitative real-time PCR (qRTPCR).

Relative gene expression levels were determined by quantitative real-time PCR (qRT-PCR) using the Power SYBR Green Cells-to-CT kit (#4402955, Thermo Fisher Scientific) according to the manufacturer's instructions. Candidate plasmids were transiently transfected into HEK293 cells seeded in 48-well plates (7.5 ^x104 cells/well) using Lipofectamine 2000 transfection reagent (0.5 μg plasmid and 1.5 μl reagent in 50 μl Opti-MEM solution). After 72 h, RNA was extracted from the cells and used for reverse transcription to synthesize cDNA according to the manufacturer's instructions. For quantitative PCR analysis, 2~5 µl of cDNA products were amplified in duplicate using gene-specific primer pairs (final concentration 250 nM) with Power SYBR Green PCR Master Mix (#4367659, Thermo Fisher Scientific). The primer sequences for the SNCA, TMEM106B, and GAPDH genes were as follows: 5'AAG AGG GTG TTC TCT ATG TAG GC -3' (SEQ ID NO: 71), 5'- GCT CCT CCA ACA TTT GTC ACT T -3' (SEQ ID NO: 72) for SNCA, 5'-ACA CAG TAC CTA CCG TTA TAG CA-3' (SEQ ID NO: 73), 5'-TGT TGT CAC AGT AAC TTG CAT CA-3' (SEQ ID NO: 74) for TMEM106B, and 5'- CTG GGC TAC ACT GAG CAC C -3' (SEQ ID NO: 75), 5'- AAG TGG TCG TTG AGG GCA ATG -3' (SEQ ID NO: 76) for GAPDH. Quantitative PCR was performed in a QuantStudio 3 real-time PCR system (Thermo Fisher Scientific). Expression levels were normalized using the housekeeping gene GAPDH and calculated using the comparative CT method.

Fluorescence imaging analysis.

EGFP reporter plasmids, which contain the 3'-UTR of the human SNCA gene located downstream of the EGFP coding region, were used to validate the SNCA and TMEM106B knockdown plasmids. EGFP reporter plasmids and candidate knockdown plasmids were simultaneously transfected into HEK293 cells plated on poly-L-lysine-coated 96-well plates (3.0 x 104 cells/well) using Lipofectamine 2000 transfection reagent (0.04 μg reporter plasmid, 0.06 μg knockdown plasmid, and 0.3 μl reagent in 10 μl Opti-MEM solution). After 72 h, the EGFP signal fluorescence intensity was measured at excitation 488 nm/emission 512 nm using a Varioskan LUX multi-mode plate reader (Thermo Fisher Scientific). Cells were fixed with 4% PFA at room temperature for 10 min and incubated with D-PBS containing 40 μg/mL 7-aminoactinomycin D (7-AAD) for 30 min at room temperature. After washing with D-PBS, the fluorescence intensity of the 7-AAD signal was measured at excitation 546 nm/emission 647 nm using a Varioskan LUX plate reader.

- 35,046,777 kan for cell number determination. Normalized EGFP signal to 7-AAD signal levels were compared to knockdown control samples.

Enzyme-linked immunosorbent assay (ELISA).

α-Synuclein reporter plasmids containing the 3'-UTR of the human SNCA gene or the TMEM106B gene downstream of the SNCA coding region were used to validate the knockdown plasmids at the protein level. α-Synuclein protein levels were determined by ELISA (#KHB0061, Thermo Fisher Scientific) using lysates extracted from HEK293 cells. Candidate plasmids were transiently transfected into HEK293 cells seeded in 48-well plates (7.5 x ¹⁰⁴ cells/well) using Lipofectamine 2000 transfection reagent (0.1 μg reporter plasmid, 0.15 μg knockdown plasmid, and 0.75 μl reagent in 25 μl Opti-MEM solution). After 72 h, cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (#89900, Thermo Fisher Scientific) supplemented with protease inhibitor cocktail (#P8340, Sigma-Aldrich) and sonicated for a few seconds. After incubation on ice for 30 min, lysates were centrifuged at 20,000 x g at 4°C for 15 min and the supernatant was collected. Protein levels were quantified. Plates were read on a Varioskan plate reader at 450 nm and concentrations were calculated using SoftMax Pro 5 software. Measured protein concentrations were normalized to the total protein concentration determined by the bicinchoninic acid assay (#23225, Thermo Fisher Scientific).

Fig. 37 and Table 4 show representative data indicating successful silencing of SNCA in vitro by the GFP reporter assay (top) and the α-Syn assay (bottom). Fig. 38 and Table 5 show representative data indicating successful silencing of TMEM106B in vitro by the GFP reporter assay (top) and the α-Syn assay (bottom).

_____________________________Table 4______________________________

ID Promoter Knockdown Promoter Overexpression 100007 SMUIntronic SNCA_mi CMV opt-GBAl 100008 N1 SNCAsh CMV opt-GBAl 100009 N1 SNCA_Pubsh4 CMV opt-GBAl 100014 Shintronny SNCA_mi JetLong opt- SCARB2GBA 100015 Uintronic SNCA_mi JetLong opt-PSAP_GBA 100016 •Tintronic SNCA_mi JetLong opt-CTSB_GBA 100019 •Tintronic SNCATMEMmi JetLong opt-VPS35 100023 •Tintronic SNCAmi JetLong opt-GBAl_IL34 100024 •Tintronic SNCAmi JetLong opt-GBA2 100028 intronic SNCABroadsh CMV opt-GBAl 100029 intronic SNCA_Pubsh4 CMV opt-GBAl

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Table 5

ID Promoter Novdown Promoter Overexpression 100010 N1 TMEMPubsh CMV opt-GRN 100011 Gintronny TMEMmi JetLong opt-GBAl_GRN 100012 N1 TMEMsh CMV opt-GRN 100019 Gintronny SNCATMEMmi JetLong opt-VPS35

Example 11. ITR D sequence placement and cellular transduction.

The effect of ITR D sequence placement on cellular transduction of rAAV vectors was investigated. HEK293 cells were transduced with rAAV viruses encoding G-case and having either 1) wild-type ITRs (e.g., D sequences proximal to the transgene insertion and distal to the ITR terminus) or 2).

ITR with the D sequence located outside the vector (e.g., the D sequence located proximal to the end of the ITR and distal to the transgene insertion), as shown in Fig. 20. Surprisingly, it was found that rAAVs having the D sequence located in the external position retain the ability to efficiently package and transduce cells (Fig. 40).

Example 12. In vitro testing of rAAV progranulin.

Fig. 39 is a schematic representation of one embodiment of a vector comprising an expression construct encoding PGRN. Progranulin was overexpressed in the CNS of rodents deficient in GRN, heterozygous, or homozygous for a GRN deletion by administration of an rAAV vector encoding PGRN (e.g., a codon-optimized PGRN) via either intraparenchymal or intrathecal injection, e.g., into the cisterna magna. Mice were injected at 2 or 6 months of age and at up to 6 or 12 months of age and analyzed for one or more of the following: GRN expression levels at the RNA and protein levels, behavioral test results (e.g., improved movement), survival assays (e.g., increased survival), microglia and inflammatory markers, gliosis, neuronal loss, lipofuscinosis, and/or restoration of lysosomal marker accumulation such as LAMP1. Assays in PGRN-deficient mice are described, for example, in Arrant et al. (2017) Brain 140: 1477–1465; Arrant et al. (2018) J. Neuroscience 38(9):2341–2358; and Amado et al. (2018) doi:https://doi.org/10.1101/30869; the entire contents of which are incorporated herein by reference.

Example 13. In vitro and in vivo testing of rAAV progranulin.

In vitro and in vivo assays were performed to evaluate the effects of an rAAV construct (PR006 (also referred to as PR006A); see Fig. 64) encoding the progranulin (PGRN) protein. PR006 contains a capsid of the AAV9 serotype.

Preclinical in vitro studies.

Expression of PR006A-derived progranulin in HEK293T cells.

The ability of PR006A to induce progranulin protein production in a cellular context was examined. HEK293T cells were transduced with PR006A at a range of multiplicities of infection (MOI) from 2.1 x 10 ⁵ to 3.3 x 10 ⁶ vector genomes (vg)/cell. Transduction with PR006A resulted in a robust, dose-dependent increase in progranulin protein expression and secretion into the cellular environment (Fig. 6O). Significantly lower levels of progranulin protein, reflecting endogenous human GRN gene-derived expression, were detected in the negative control group receiving adjuvant alone (putative clinical vehicle).

Efficacy in FTD-GRN iPSC-derived neurons.

We performed an in vitro analysis of the rAAV construct efficacy in human FTDGRN (frontotemporal dementia with a GRN mutation) neuronal cultures. Cell lines were obtained from the National Institute of Neurological Disorders and Stroke (NINDS) Human Cell and Data Repository (NHCDR): ND50015 (FTD-GRN, M1L), ND50060 (FTD-GRN, R493X), and ND38555 (control, wild type) (see Table 6).

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Table 6

General characteristics of iPSC cell line

Cell line NINDS Cell line ID# Clinical diagnosis of FTD? GRN mutation Age Floor Original cell / reprogramming method FTD-GRN #1 ND50015 Yes MIL 54 F Fibroblasts / episomal plasmids FTD-GRN #2 ND50060 At risk (brother or sister affected at age 62) R493X 60 M Fibroblasts / episomal plasmids Control ND38555 No n.d. 48 F Fibroblasts / retroviral plasmids

To define a cell model that would be pathologically relevant to FTD-GRN, iPSCs from each line were differentiated into neuronal cells using a two-step protocol. In the first step, iPSCs were differentiated into proliferating neural stem cell (NSC) lines that lacked expression of pluripotency markers (e.g., Oct4 and SSEA1) and overexpressed neuronal stem cell markers (e.g., SOX2, Nestin, SOX1, and PAX6) as determined by immunofluorescence labeling.

Control and FTD-GRN NSC lines were seeded at the same density and after 48 hours, progranulin expression was measured by enzyme-linked immunosorbent assay (ELISA) in cell lysates (intracellular progranulin) (Fig. 52E) and cell medium (secreted progranulin) (Fig. 52A). Progranulin expression was normalized to total protein concentration to account for differences in cell numbers (n=3; mean ± SEM). NSC lines with heterozygous GRN mutations had significantly lower levels of intracellular and secreted progranulin compared to control NSCs, with FTD-GRN NSCs expressing ~25-50% of endogenous progranulin levels. This suggests that this FTD-GRN cell model recapitulates the clinical progranulin deficiency observed in FTD-GRN patients, who express one-third to one-half of the normal plasma progranulin levels (Finch et al., Brain 132, 583-591 (2009); Ghidoni et al., Neurology 71, 1235-1239, (2008); Sleegers et al., Ann Neurol 65, 603-609 (2009)).

NSCs from all cell lines were differentiated into neuronal cultures. After establishing that iPSC-derived NSCs exhibit reduced progranulin expression, the lines were differentiated into neurons to provide a clinically representative cell type for preclinical studies of PR006A efficacy. NSCs were seeded in neuronal differentiation medium, terminally differentiated into postmitotic neurons for 7 days, and then assessed for neuronal marker expression (i.e., MAP2, NeuN, Tau, Tuj1, NF-H) by immunofluorescence (Fig. 52G). Both control and FTD-GRN iPSC-derived NSC lines efficiently differentiated into neurons according to this protocol.

FTD-GRN iPSC-derived neuronal cultures were used to evaluate the efficacy of PR006A in vitro. FTD-GRN neurons were treated with vehicle or PR006A at an MOI of 2.7x105, 5.3x105, or 1.1x106 vg/cell. PR006 transduction resulted in robust, dose-dependent expression of secreted progranulin, as measured by ELISA, in all cell lines (Fig. 52B). Vehicle-treated control and FTD-GRN neurons were assessed for endogenous progranulin levels. Control neurons expressed endogenously secreted progranulin, whereas secreted progranulin was undetectable in FTD-GRN neurons (Fig. 52B). Linear regression analysis confirmed a significant correlation between PR006A dose and progranulin levels in both FTD-GRN cell lines (p= ^3.5x10-13 ). These results demonstrate that PR006A administration results in increased progranulin secretion in the FTD-GRN neuronal model.

Progranulin is known to stimulate the maturation of the lysosomal protease cathepsin D (CTSD), the loss of function of which is also associated with lysosomal storage disorders and neurodegeneration. CTSD is expressed as an inactive full-length protein (proCTSD), which undergoes proteolytic processing to an enzymatically active mature protease (matCTSD). Progranulin has been reported to act as a molecular chaperone that binds to proCTSD to enhance its maturation to matCTSD protease. In FTD-GRN neuronal cultures, transduction of PR006 with

- 38 046777 retained defective cathepsin D maturation (Fig. 52C). Control, FTD-GRN#1 and FTD-GRN#2 neurons were transduced with PR006A or auxiliary. An MOI of 5.3x105 PR006A was used for efficacy experiments, as it restored progranulin levels to at least 2-fold that of control cells (Fig. 52B). To assess efficacy, proCTSD and matCTSD expression levels were measured in cell lysates using the Simple Western™ automated platform (Jess) (Fig. 52C). Assistant-treated FTDGRN neurons had a lower matCTSD to proCTSD ratio compared to assistant-treated control neurons; PR006A treatment significantly increased the ratio in both FTD-GRN neuronal lines (Fig. 52C). In control neurons, the matCTSD to proCTSD ratio was not significantly altered by PR006A treatment. These data demonstrate that PR006A restores the lysosomal function-related phenotype in FTDGRN neurons.

In normal neurons, TDP-43 (trans-active response protein 43 kDa DNA binding protein) is localized to the nucleus. In postmortem brains of FTD-GRN patients, TDP-43 aggregation is observed in the cytoplasm of neurons, and nuclear accumulation of TDP-43 is reduced. FTD neurons have reduced nuclear TDP-43, leading to aggregation and toxicity in neurons. Because Grn KO mice do not fully recapitulate this TDP-43 pathology, induced pluripotent stem cell (iPSC)-derived neurons represent a valuable FTD-GRN model for studying TDP-43 biology. Decreased nuclear accumulation of TDP-43 and increased accumulation of insoluble TDP-43 were reported in neurons derived from iPSCs of FTD-GRN patients compared to control neurons that do not carry the GRN mutation, as described by Valdez et al. (Human Molecular Genetics 26, 4861–4872 (2017)). PR006A transduction of neuronal cultures from both FTD-GRN mutation carrier lines reversed the TDP-43 abnormalities, resulting in decreased insoluble TDP-43 (measured by the Simple Western™ (Jess) platform (Fig. 52D)) and increased nuclear localization of TDP-43 (measured by immunofluorescence (Fig. 52F)). Thus, PR006 transduction restored defective maturation of the lysosomal enzyme, cathepsin D, and reduced the manifestations of abnormal TDP-43 pathology in FTD-GRN neurons.

Preclinical in vivo studies.

Efficacy and biodistribution in mature Grn knockout mice.

The in vivo efficacy of PR006A and the maximum dose of PR006A were evaluated in a Grn knockout (KO) mouse model. In the Grn KO mouse model used in these studies (B6(Cg)-Gm ^tmUAldl /J (Jackson Laboratory, Bar Harbor, ME), exons 1-4 are deleted from the target gene progranulin (Grn) (Yin et al., J Exp Med 207, 117-128 (2010)). These animals have complete loss of progranulin and exhibit age-dependent phenotypes including lysosomal changes, neuronal lipofuscin accumulation, ubiquitin accumulation, microgliosis, and neuroinflammation, and are therefore widely used to model FTD-GRN. Every attempt was made to eliminate bias; mice were assigned to treatment groups that were balanced for sex and body weight, and blinded assessment of experimental efficacy endpoints was performed by trained personnel.

In initial studies, PR006A was administered to mature Grn KO mice at a dose of ^9.7x1010 vg (2.4x1011 vg/g brain), which was the highest achievable dose at the time of the study due to limitations in injection volume and the physical titer of the virus lot used for the study. Mature mice were used because many of the phenotypes associated with FTD-GRN, including CNS inflammation and microgliosis, develop in an age-dependent manner, with the most pronounced phenotypes occurring between 12 and 24 months of age.

In studies in mature Grn KO mice, PR006A was administered via a single intracerebroventricular (ICV) injection. 10 μl of excipient (putative clinical vehicle; 20 mM Tris pH 8.0, 200 mM NaCl, and 1 mM _MgCl2 +0.001% Pluronic F68) or ^9.7x1010 vg PR006A (2.4x1011 vg/g brain [based on 400 mg mature mouse brain]) were delivered by ICV injection into two groups of mature Grn KO mice: (1) 16 months of age at injection (n=4/group; PRV-2018-027; Fig. 6 1) and (2) 14 months of age at injection (planned n=3/group; PRV-2019-002; Fig. 6 1). Animals were sacrificed two months after injection.

In study PRV-2018-027, a single dose of PR006A was administered to 16-month-old mice in the following treatment groups:

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Model ICV ICV dose N Grn KO Excipient N.d. 4(2M/2F) Grn KO PR006A 9.7 x 10 ¹⁰ vg (2.4 x 10 ¹¹ vg/r brain) 5(3M/2F)

Due to unexpected study biases (genotyping errors and premature loss of animals), only 1 mouse in the adjuvant-treated group was included in study PRV-2019-002 (14-month cohort) instead of the planned n=3. The small sample size made statistical analysis impossible and therefore this study is excluded from further discussion. However, the results of this study were comparable to those of study PRV-2018-027.

Biodistribution and progranulin expression: Biodistribution was determined by measuring the presence of vector genome using a qPCR assay that meets current U.S. Food and Drug Administration Center for Biologics Evaluation and Research (CBER)/Office of Tissue and Advanced Therapies (OTAT) standards for PCR sensitivity (with >50 vector genomes per 1 μg genomic DNA defined as positive). All PR006A-treated mice were positive for vector genomes in the cerebral cortex and spinal cord, indicating that ICV administration successfully resulted in PR006A transduction in the brain and CNS (Fig. 59A). ICV administration of PR006A promoted the production of significant levels of human progranulin protein in the CNS (brain, spinal cord) of Grn KO mice, whereas, as expected, human progranulin was undetectable in mice receiving the excipient (Fig. 59B). Because progranulin is primarily a secreted protein, expression in the CSF can be considered a surrogate for protein production in the brain, representing a potential translational endpoint for FTDGRN patients who have reduced CSF progranulin levels. We were able to detect human progranulin in the CSF of PR006A-treated mice, but due to small sample volumes and technical limitations in obtaining sufficient CSF volume from mice, CSF progranulin measurements were below the lower limit of quantification (LLOQ) of the assay (Fig. 59C).

ICV administration also resulted in widespread dissemination of the vector genome and progranulin protein levels in peripheral tissues including liver, heart, lung, kidney, spleen, and gonads (Figs. 62A-62B). In addition, significant levels of human progranulin were detected in the plasma of PR006A-treated Grn KO mice. As expected, human progranulin was undetectable in adjuvant-treated Grn KO mice.

Lipofuscin accumulation: Accumulation of neuronal lipofuscin, an electron-dense, autofluorescent material that progressively accumulates in the lysosomes of postmitotic cells and is an indicator of lysosomal dysfunction, is a hallmark of the age-dependent phenotype of Grn KO mice. Lipofuscin accumulation was assessed by two independent methods in adjacent brain sections: (1) in a more clinical approach, lipofuscin accumulation in the brain was scored by a blinded pathologist on a scale of 0 (no lipofuscin observed) to 4 (widespread lipofuscin accumulation) and (2) in a more quantitative approach, lipofuscin autofluorescence was detected by immunohistochemistry (IHC) and automatically quantified. Grn KO mice showed significant lipofuscinosis throughout the brain, and ICV administration of PR006A reduced the lipofuscin accumulation score in the cerebral cortex, hippocampus, and thalamus (Fig. 59D). Quantification of lipofuscin accumulation by IHC images also revealed a reduction in lipofuscinosis with PR006A administration in all three brain regions. Since ubiquitin-positive inclusions are a defining pathological feature in FTD-GRN patients, which also accumulate in the Grn KO mouse model in an age-dependent manner; IHC was performed and quantified in brain regions of interest (cerebral cortex, hippocampus, thalamus) to assess ubiquitin accumulation. PR006A administration significantly reduced ubiquitin accumulation in Grn KO mice (Fig. 59E). These data suggest that PR006A attenuates lysosomal dysfunction in the Grn KO mouse model of FTD-GRN. Neuroinflammation: Chronic CNS inflammation is a pathological characteristic of the brains of patients with FTD-GRN, which is recapitulated in Grn KO mice in an age-dependent manner. Progranulin has anti-inflammatory effects in mouse models of FTDGRN, and loss of progranulin results in upregulation of proinflammatory cytokines, including TNFa. In this study, PR006A administration suppressed the levels of inflammatory markers in mature Grn KO mice. ICV administration of PR006A reduced the expression of the proinflammatory cytokine Tnf(TNFa) gene and Cd68 (CD68), a microglia marker, in the cerebral cortex (Fig. 59F). TNFa protein levels were also reduced in cortex samples from PROO6A-treated Grn KO mice, as assessed by

- 40 046777 using the Mesoscale Discovery mouse proinflammatory cytokine assay (Fig. 59G). To further assess neuroinflammation, immunohistochemistry (IHC) for Iba1, a marker of microgliosis, and GFAP, a marker of astrocytosis, were performed and quantified in brain regions of interest (cerebral cortex, hippocampus, thalamus). PR006A treatment resulted in a trend toward decreased microgliosis (Iba1) but had no effect on astrocytosis (GFAP) in Grn KO mice (Fig. 59H; Fig. 59I). Taken together, these results demonstrate that PR006A treatment reduces neuroinflammation in the FTD-GRN model of mature Grn KO mice. Histopathology: Detailed histopathological analysis of hematoxylin and eosin (H&E) staining of brain, thoracic spinal cord, liver, heart, spleen, lung, and kidney from all mice by a blinded board-certified pathologist revealed no adverse effects related to PR006A treatment. Administration of PR006A to Grn KO mice resulted in a reduction in the incidence and/or severity scores of events characteristic of the model, including a reduction in the incidence and/or severity scores of neuronal necrosis in the medulla and pons. In addition, both the incidence and severity of axonal degeneration in the thoracic spinal cord were reduced with PR006A treatment. These results are discussed in detail in the Toxicology section below.

Conclusion: ICV administration of PR006A at a dose of 9.7x10 ¹⁰ vg (2.4x1011 vg/g brain) resulted in widespread distribution of the vector genome throughout the brain and peripheral tissues in mature Grn KO mice. PR006A administration increased global progranulin expression. Furthermore, PR006A reduced lipofuscin and ubiquitin accumulation in the brain, pathological conditions known to occur in both the Grn KO mouse model and FTD-GRN patients. PR006A also reduced proinflammatory cytokine expression and immune cell activation in the cerebral cortex, which are phenotypes indicative of chronic CNS inflammation.

Dose-varying efficacy in adult Grn knockout mice.

To further evaluate the effective doses of PR006A, a larger dose-ranging study was conducted in adult Grn KO mice. In PRV-2019-004, 10 μL of the excipient (putative clinical vehicle; 20 mM Tris pH 8.0, 200 mM NaCl, and 1 mM _MgCl2 +0.001% Pluronic F68) or PR006A were administered to 4-month-old animals via ICV. These adult mice were used instead of mature Grn KO mice, as the latter were not available in sufficient numbers to conduct the dose-ranging study. Although adult Grn KO mice have a milder phenotype than mature mice, they still exhibit lysosomal defects and neuroinflammatory changes and are therefore suitable for evaluating the effective dose range of PR006A. To evaluate the efficacy of PR006A over a wide range of viral doses, PR006A was administered at a dose of 1.1 x 1011 vg (2.7 x 1011 vg/g brain), the highest achievable dose at the time of the study due to injection volume limitations and the physical titer of the virus lot used for the study, at a mid-dose of 1.1 x ¹⁰¹⁰ vg (2.7 x ¹⁰¹⁰ vg/g brain), or at a low dose of 1.1 x ¹⁰⁹ vg (2.7 x 109 vg/g brain), with the full difference on a logarithmic scale covering each dose. Details of the experimental design are shown in Fig. 63.

Three doses of PR006A were evaluated with 10 mice (4M/6F) per group:____________________

Model ICV ICV dose N Grn KO Excipient N.d. 10(4M/6F) Grn KO PR006A 1.1 x 10 ⁹ vg (2.7 x 10 ⁹ vg/g brain) 10(4M/6F) Grn KO PR006A 1.1 x 10 ¹⁰ vg (2.7 x 10 ¹⁰ vg/g brain) 10(4M/6F) Grn KO PR006A 1.1 x 10 ¹¹ vg (2.7 x 10 ¹¹ vg/g brain) 10(4M/6F)

Age-matched mice of the same parental strain as the Grn KO mice with wild-type (WT) Grn alleles (7-month-old C57BL/6J mice) served as controls for selected performance criteria in this study. _____________________________________________

Model ICV ICV dose N WT (C57BL/6J) n.d. N.D. 10(5M/5F)

Progranulin Biodistribution and Expression: Biodistribution was determined by measuring the presence of vector genome using a qPCR assay that meets current US Food and Drug Administration CBER/OTAT standards for PCR sensitivity (with >50 vector genomes per 1 μg genomic DNA defined as positive). Mice receiving PR006A were positive for vector genomes in the cerebral cortex and spinal cord.

- 41 046777 human brain in a dose-dependent manner, indicating that ICV administration results in successful transduction of PR006A in the CNS (Fig. 53A). qRT-PCR analysis of PR006A-encoded GRN demonstrated that ICV administration of PR006A doses resulted in dose-dependent induction of human GRN mRNA expression in the cerebral cortex (Fig. 53B). PR006A administration increased human progranulin protein levels in the brain and spinal cord (Fig. 53C). In brain tissue, human progranulin levels were detected and quantified at the highest dose of PR006A; at lower doses, progranulin levels were below the limit of detection due to high background in the brain. However, based on the logarithmic difference between doses, the proportional estimate of expected progranulin levels at lower doses would be well below the lower limit of quantification (LLOQ) of the assay in brain tissue. Endogenous mouse progranulin levels were measured in age- and strain-matched mice with wild-type (WT) Grn alleles; in both cortex and spinal cord, human progranulin levels in PR006A-treated Grn KO mice did not exceed endogenous progranulin levels in WT mice at any dose. Because different detection methods were used to measure human and mouse progranulin, using non-species-cross-reactive anti-progranulin antibodies, absolute numbers could not be compared with appropriate precision.

PR006A administration also resulted in widespread distribution of the vector genome and progranulin protein levels in peripheral tissues including liver, heart, lung, kidney, spleen, and gonads (Fig. 53D; Fig. 53E).

Significant levels of human progranulin were detected in plasma of PR006A-treated Grn KO mice at all dose levels (Fig. 53F). As expected, human progranulin was undetectable in excipient-treated Grn KO mice. Human progranulin levels in animals receiving the middling dose of PR006A were in the same range as mouse progranulin levels measured in mice with WT Grn alleles. Because different detection methods were used to measure human and mouse progranulin, which utilized non-species cross-reactive anti-progranulin antibodies, absolute numbers could not be compared with adequate precision. Lipofuscin accumulation: Lipofuscin accumulation was assessed by two independent methods in adjacent brain sections: (1) in a more clinical approach, lipofuscin accumulation in the brain was scored by a blinded pathologist on a scale of 0 (no lipofuscin observed) to 4 (widespread lipofuscin accumulation) and (2) in a more quantitative approach, lipofuscin autofluorescence was detected by IHC and automatically quantified. Grn KO mice had lipofuscinosis throughout the brain, whereas WT mice had no detectable lipofuscin in the brain (Fig. 53G). ICV administration of PR006A resulted in a dose-dependent reduction in the intracellular lipofuscin accumulation score in the brains of Grn KO mice (Fig. 53G). The efficacy of PR006A in reducing lipofuscinosis can be most readily quantified in brain regions that exhibit the most robust lipofuscinosis phenotype in the Grn KO mouse model of FTD-GRN, including the hippocampus and thalamus. In addition to pathologist assessment of lipofuscin, IHC performed in the brain regions of interest (e.g., cortex, hippocampus, thalamus) to quantify lipofuscinosis revealed a dose-dependent reduction in the amount of lipofuscin accumulation in the cortex and thalamic brain regions, with significant reductions occurring at medium and high doses of PR006A. IHC testing was also performed to assess brain ubiquitin accumulation, an additional pathology associated with FTD-GRN that occurs in Grn KO mice. Compared with WT mice, Grn KO mice exhibited increased ubiquitin accumulation in all brain regions (Fig. 53H). PR006A significantly reduced the size of ubiquitin immunoreactive objects to levels close to WT at all three doses (Fig. 53H).

Neuroinflammation: PR006A administration suppressed levels of inflammatory markers in the brain of adult Grn KO mice. ICV administration of PR006A reduced gene expression of the proinflammatory cytokine Tnf (TNFa) and Cd68 (CD68), a microglia marker, in the cerebral cortex over a dose range from 2.7x109 vg/g brain to 2.7x1011 vg/g brain (Fig. 53I). Consistent with published data, we observed increased gene expression of these neuroinflammatory markers in adjuvant-treated Grn KO mice compared with age-matched mice with wild-type Grn alleles (Fig. 53I). In contrast to the observations in 18-month-old Grn KO mice from PRV-2018-027 and reports of TNFa abnormalities in the literature, there was no significant increase in TNFa protein levels in the cortex of 7-month-old Grn KO mice receiving excipient; furthermore, no significant changes were observed with PR006A in Grn KO mice. These data are consistent with previously published data that persistent neuroinflammatory phenotypes do not occur in the Grn KO mouse model until 12–24 months of age. To further assess neuronal inflammation, immunohistochemistry (IHC) was performed by staining for Iba1, a marker of microgliosis, and GFAP, a marker of astrocytosis, and quantified in brain regions of interest (cerebral cortex, hippocampus, and thalamus). A significant increase in micro

- 42,046,777 gliosis (Iba1) and astrocytosis (GFAP) in all brain regions in Gm KO mice compared with WT mice (Figs. 53J-53K). PR006A administration significantly reduced microgliosis (Iba1) at all three doses (Fig. 53J). A trend toward decreased astrocytosis (GFAP) was observed at the middle dose of PR006A, and a significant decrease in astrocytosis (GFAP) was observed at the high dose of PR006A in the thalamic region of the brain (Fig. 53K).

Although many of the phenotypes of the Gm KO mouse model emerge late in life, previous studies have demonstrated that Grn KO mice exhibit widespread gene expression changes as early as 4 months of age, including alterations in lysosomal and immune pathways. Therefore, in addition to the targeted qRT-PCR analysis described above, we used a transcriptomics approach to assess changes in mRNA levels that can be assessed globally using sensitive, high-throughput technologies (RNA sequencing) and require minimal sample material. We performed RNA sequencing on the cortex and used Gene Set Variation Analysis (GSVA) (Hanzelmann et al., BMC Bioinformatics 14, 7 (2013)) to determine which gene expression pathways are altered in 7-month-old excipient-treated Gm KO mice compared to age-matched WT mice of the same strain. We confirmed deficiencies in lysosomal and immune pathways in Gm-null mice as reported in previously published studies. Significant changes were reported in the Vacuole GO TERM (GO:0005773) gene subset (containing 4 genes reported to be dysregulated in Gm KO mice as described by Lui et al (Cell 165, 921–935 (2016)) , the Lysosomal gene set (a subset of 25 lysosome-related genes shown to be dysregulated in Grn KO mice as described by Evers et al (Cell Reports 20, 2565–2574 (2017) ), and the Complement gene set from the HALLMARK gene set enrichment analysis database (containing genes encoding components of the complement system, parts of the innate immune system). We then measured and compared the activity levels of these gene sets upon administration of PR006A (Figs. 53L–53N). Administration of PR006A dose-dependently rescued the gene set deficits observed in Gm KO mice.

Histopathology: Detailed histopathology analysis by a blinded board-certified pathologist using hematoxylin and eosin (H&E) staining of the brain, thoracic spinal cord, liver, heart, spleen, lungs, kidneys, and gonads of all mice revealed no evidence of PR006A-related toxicity. Details of the toxicity analysis are provided in the section below.

Conclusion: ICV administration of PR006A at doses ranging from 2.7x109 vg/g brain to 2.7x1011 vg/g brain resulted in widespread distribution of the vector genome throughout the brain and peripheral tissues in a dose-dependent manner. PROO6A administration also resulted in production of progranulin mRNA and protein in the CNS. A clear dose-response relationship was observed between PR006A and reduction in lipofuscinosis, an indicator of lysosomal dysfunction, in many brain regions. A consistent and statistically significant reduction in lipofuscinosis was observed at the intermediate and highest doses of PR006A. All doses of PR006A reduced ubiquitin accumulation in the brain. Starting from the lowest dose of 2.7x109 vg/g brain, PR006A reduced the expression of proinflammatory markers in the brain at the RNA and protein levels.

Abstract: Preclinical in vivo studies.

PR006A efficiently transduced Grn KO mice, resulting in robust, dose-dependent transgene biodistribution and progranulin mRNA and protein production in the CNS. PR006A dose-dependently reversed gene expression abnormalities in lysosomal and neuroinflammatory pathways. PR006A ameliorated multiple phenotypes that occur in the brain of this FTD-GRN mouse model, including lipofuscinosis, ubiquitin accumulation, and microgliosis. In a dose-ranging study, the lowest dose of PR006A at 2.7x109 vg/g brain significantly suppressed cortical inflammatory marker expression. The intermediate dose of PR006A at 2.7x10 ¹⁰ vg/g brain attenuated both lysosomal defects (e.g., lipofuscinosis) and neuroinflammation in a robust and statistically significant manner. A high dose of PR006A, 2.7x1011 vg/g brain, further increased progranulin expression without any signs of toxicity.

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Table 7

Summary of biodistribution data

Study Dose Cerebral cortex Spinal cord Pen Spleen Heart Kidney It's easy Gon adas PRV-2018-027 9,7xl0 ¹⁰ vg PR006A + + + + + + + + PRV-2019-004 1.1 x 10 ⁹ vg PR006A + + + + + + + + 1.1 x 10 ¹⁰ vg PR006A + + + + + + + + 1.1 x 10 ¹¹ vg PR006A + + + + + + + +

Positive biodistribution is defined as > 50 vg/µg genomic DNA.

Safety pharmacology.

No adverse events attributable to the study drug were identified during these studies. Safety findings from in vivo and histopathological analyses of animals in PRV-2018-027, PRV-2019-002, and PRV-2019-004 are discussed in the section below.

Single dose toxicity.

A series of preclinical studies of PR006A were conducted to investigate safety criteria in mice and monkeys. Three studies were performed in the Grn KO mouse model, with safety criteria including neuropathological assessments and analyzing both protective activity and potential toxicity resulting from the administration of PR006A by intracerebroventricular (ICV) injection; ICM administration in mice is technically more difficult. These mouse models are representative of FTD-GRN, in which patients have a mutation in the GRN gene resulting in decreased progranulin levels. In cynomolgus monkeys, a pilot study also included neuropathological assessments, with PR006A administered into the cisterna magna (ICM). A GLP study was conducted in cynomolgus monkeys injected with PR006A into the ICM and sacrificed on Day 7, Day 30, or Day 183. The GLP study included a comprehensive list of clinical criteria in addition to an assessment of anatomical pathology in a comprehensive list of tissues. To support single-dose administration in the clinic, the following single-dose studies were conducted.

Maximum dose of PR006A in the mature FTD-GRN mouse model (PRV-2018-027 and PRV-2019-002).

As part of these efficacy studies in Grn KO mice, neuropathological assessments were performed in mice treated with either excipient or PR006A via ICV. Grn KO mice exhibit complete loss of progranulin and are widely used as models of FTD-GRN due to their age-dependent phenotypes, which include lysosomal alterations, neuronal lipofuscin accumulation, microgliosis, and neuroinflammation. Aspects of the pharmacological portion of the study are provided in the sections above, while the toxicological criteria assessed in this study are summarized below. Two studies of PR006A were conducted in the mature Grn KO mouse model. In the first study (PRV-2018-027), 9 Grn KO mice of mixed sexes, 16 months of age, were treated via ICV with either PR006A or excipient. Animals were sacrificed 9 weeks post-treatment. This study included a single-dose PR006A group: 10 µL of undiluted virus for a total dose of 9.7x10 ¹⁰ vg (2.4x1011 vg/g brain), and a control group received 10 µL of adjuvant.

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Table 8

Study design PRV-2018-027

Model Treatment RoA (Dose Volume) Dose PR006A (vg/r BRAIN) Total PR006A Dose (vg) Number of mice Necropsy after treatment Grn KO Excipient ICV(IOmkt) 0 0 4(2M/2F) 9 weeks Grn KO PR006A ICV (10 µl) 2.4 x 10 ¹¹ 9.7 x 10 ¹⁰ 5(3M/2F) 9 weeks

RoA: route of administration.

This study protocol assessed the achievement of various postmortem endpoints such as biodistribution, lysosomal changes, and inflammatory markers (see section above). Animals were also monitored twice daily for survival and body weight was measured once daily. Following euthanasia 2 months post-treatment, target tissues were collected, drop-fixed in chilled 4% paraformaldehyde, and stored at 4°C. Tissues from the 8 animals that completed the study were excised, processed, and embedded in paraffin blocks. They were then sectioned at ~5 μm, stained with hematoxylin and eosin (H&E), and slides were sent to a board-certified veterinary pathologist.

During this study, 1 mouse in the experimental group died prematurely; no abnormalities were noted in the deceased animal at necropsy, so the cause of death is unknown. No other deaths or abnormalities were observed. All treatment groups were monitored equally for body weight, with no significant differences observed.

Histopathological examination revealed no adverse findings related to PR006A. Lipofuscin accumulation in the brain was consistent with the expected findings in Grn KO mice. Animals treated with PR006A showed a reduction in the degree of lipofuscin accumulation in all brain regions. Morphological changes also showed a slight reduction in the incidence and/or severity score, particularly for neuronal necrosis in the medulla and pons, with PR006A administration. However, these trends in morphological changes were not as consistent as those in the lipofuscin scoring.

In the thoracic spinal cord, axonal degeneration and, very rarely (1 of 4 animals in each group), minimal neuronal necrosis were observed. There was also a slight reduction in both the incidence and severity of axonal degeneration in animals treated with PR006A.

The following findings, which were thought to be associated with homozygous Grn knockout mice, demonstrated a reduction in the incidence and/or severity of the following events in animals treated with PR006A: dilated tubules in the renal medulla, glomerulopathy in the kidneys, and foreign material in the lungs (characterized by linear, acellular, dusky pink structures typically found in the airways and often associated with foreign body giant cells and/or macrophages). A larger cohort of animals will be required to draw more definitive conclusions. All other observed histopathological findings were considered to be incidental and/or of similar incidence and severity in excipient- and test substance-treated animals and, therefore, were considered unrelated to PR006A administration.

In the second study (PRV-2019-002), 5 male and female Grn KO mice aged 14 months were treated with either PR006A or adjuvant via ICV. Animals were sacrificed 8 weeks post-treatment. This study included a single-dose PR006A group: 10 μL of undiluted virus for a total dose of 9.7x10 ¹⁰ vg (2.4x10 ¹¹ vg/g brain), and a control group received 10 μL of adjuvant.

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Table 9

Study design PRV-2019-002

Model Treatment RoA (Dose Volume) PR006A dose (brain vg/r) Total Dose PR006A (vg) Number of mice Necropsy after treatment Grn KO Excipient ICV (10 µl) 0 0 2 (0M/2F)* 8 weeks Grn KO PR006A ICV (10 µl) 2.4 x 10 ¹¹ 9.7 x 10 ¹⁰ 3(1M/2F) 8 weeks

* Genotyping results at the end of the study confirmed that n=1 animal in the adjuvant-treated group was Grn heterozygous KO instead of the expected Grn homozygous KO.

Animals were analyzed similarly to the PRV-2018-027 study. Animals were checked twice daily for survival and body weight was measured once daily. Following euthanasia 2 months post-treatment, target tissues were collected, droplet-fixed in chilled 4% paraformaldehyde, and stored at 4°C until evaluation.

In the CNS, data consistent with those previously observed in Grn KO mice were observed in the brain (Yin et al., J Exp Med 207(l):l 17-128 (2010)). In particular, a widespread increase in lipofuscin accumulation was observed throughout the brain. Rarely, minimal neuronal necrosis was also observed (in one untreated animal with early death and in one excipient-treated animal).

Due to the small sample size, it was not possible to demonstrate a consistent trend in drug-related outcomes. There was no consistent difference in response between the study drug (PR006A) and the excipient.

In non-CNS tissues, findings thought to be consistent with the Grn KO mouse phenotype were observed in the kidney (tubular dilation and mononuclear inflammatory cell infiltrates) and liver (Kupffer cell/sinusoidal lining cell vacuolization and Kupffer cell microgranulomas) (Yin et al., J Exp Med 207(1): 117–128 (2010)).

All animals that underwent surgery and were included in the study developed glomerulopathy. While there have been no published reports of this finding as a change associated with standard, untreated Grn knockout mice, one study demonstrated that progranulin-deficient mice fed a diet that induces hyperhomocysteinemia developed thickening of the glomerular basement membrane and thinning of the podocyte processes (Fu et al., Hypertension 69(2):259–266 (2017)).

All other results were consistent with those typically observed in laboratory mice. Due to the small sample size, it was not possible to demonstrate convincing differences related to the drug's effects.

PR006A dose range in the adult FTD-GRN mouse model (PRV-2019-004).

To further evaluate the safety of PR006A, a larger dose-ranging study was conducted in adult Grn KO mice. A total of 40 mice of either sex were divided into 4 groups and administered either excipient or one of three doses of PR006A by a single unilateral ICV injection into the left hemisphere; all animals, regardless of treatment group, received a total dose volume of 10 μL. Mice were administered either excipient or study drug at 4 months of age and were sacrificed 3 months after administration. An additional control group of wild-type (WT) mice, which included untreated C57BL/6J mice (same parental strain) at approximately 7 months of age, were also sacrificed and subjected to similar necropsy. The study was conducted according to the study design described below:

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Table 10

Study design PRV-2019-004

Group Model Treatment RoA (Dose Volume) Dose PR006A (brain Vg/r) Total PR006 A Dose (vg) Number of mice Necropsy after treatment 1 Grn KO Excipient ICV(10 µl) 0 0 10(4M/ 6F) Week 13 2 Grn KO PR006A ICV (10 µl) 2.7 x 10 ¹¹ 1D ^x U ¹¹ 10(4M/ 6F) Week 13 3 Grn KO PR006A ICV (10 µl) 2.7 x 10 ¹⁰ 1.1 x ¹⁰ 10(4M/ 6F) Week 13 4 Grn KO PR006A ICV (10 µl) 2.7 x 10 ⁹ 1.1 x y ⁹ 10 (4M/6F) Week 13 n.d. WT (C57BL/6J) None N.d. 0 0 10 (5M/5F) n.d.

During the study, animals were checked for survival twice daily and weighed once weekly. Mice were sacrificed 3 months after exposure, and various postmortem examinations were performed to evaluate the efficacy of PR006A (see section above). In addition, H&E-stained sections of brain, thoracic spinal cord, liver, heart, spleen, lung, kidney, and gonads were evaluated by a board-certified pathologist.

Histopathological examination revealed no adverse effects related to PR006A in any of the mice, regardless of treatment group.

Phenotypes consistent with the Grn KO mouse model were observed, such as intracellular lipofuscin accumulation in various brain regions: cerebral cortex, cerebral nuclei, hippocampus, thalamus/hypothalamus, cerebellum and brainstem (especially in the pons and medullary substance). No clear evidence of morphological changes on H&E stained sections (neuronal vacuolization and gliosis) was observed. Lipofuscin pigment accumulation may precede easily detectable morphological changes and therefore serves as an adequate biomarker of efficacy. While all Grn homozygous KO groups demonstrated lipofuscin accumulation, differences in the severity of this phenomenon were observed between treatment groups. The frequency of higher lipofuscin accumulation scores was highest for the group of animals receiving the excipient (Group 1). Of the PR006A-treated animals, the highest frequency of scores was observed in Group 4 (low dose PR006A; ^2.7x109 vg/g brain) followed by Group 3 (medium dose PR006A; ^2.7x1010 vg/g brain). The lowest lipofuscin accumulation scores were observed in Group 2 (high dose PR006A; ^2.7x1011 vg/g brain). These data demonstrate a dose-dependent reduction in the extent of intracellular lipofuscin accumulation in the brain of Grn homozygous knockout mice. All other histopathological findings observed were considered to be coincidental and/or of similar frequency and severity between excipient- and test substance-treated animals and, therefore, were considered unrelated to PR006A administration.

Single-dose GLP study in monkeys (PRV-2018-028).

Study design.

The objective of this GLP study was to evaluate the toxicity and biodistribution of study drug PR006A following a single administration via ICM injection to cynomolgus monkeys with 6-day, 29-day, or 182-day post-dose observation periods; animals were sacrificed on Day 7, Day 30, or Day 183 of the study. The study was designed to evaluate 2 dose levels: the highest dose was the maximum achievable dose achieved with a volume of 1.2 mL (the highest volume administered) of undiluted PR006A, and a lower dose that was equivalent to one log unit lower than the high dose. The doses corresponded to a low dose of 4.8x10 ¹¹ vg and a high dose of 4.8x10 ¹² vg; with an estimated brain weight of 74 g in the cynomolgus macaque, the NHP species used in this study, this corresponds to approximately 6.5x10 ⁹ vg/g brain and 6.5x10 ¹⁰ vg/c brain. The study also included a control group in which animals received only 1.2 ml of the excipient (20 mM Tris pH 8.0, 200 mM NaCl, and 1 mM MgCl ₂ +0.001% [w/v] Pluronic F68). Both male and female animals were used in this study.

- 47,046,777 cynomolgus macaques. The Day 7 group included 1 female treated with the highest dose and was designed as a sentinel for the early toxicity study; and the remaining two 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 study design is presented in Table 11.

Table 11

Review of the GLP study PRV-2018-028 in NHP

Target Assessment of tolerability and biodistribution of PR006A in NHPs Regulatory Compliance GLP Study drug PR006A Total number of animals 19 Javanese macaque monkeys Weight (age) 2-5 kg (25-50 months) Research design Assignments to groups: Group Dose (vg/r brain) Number of animals Necropsy I (Day 7) Necropsy I (Day 30) Necropsy I (Day 183) 1 0 0 2M/1F 2M/1F 2 6.5 x 10 ⁹ 0 2M/1F 2M/1F 3 6.5 x 1010 1F 2M/1F 2M/1F Method and frequency of administration Into the large cistern using a 1-3 cm polypropylene syringe and a lumbar puncture needle (Pencan 25 G x 2.5 cm BBraun); a single slow bolus is administered at a maximum rate of 0.5 cc/min. Compositions The administered solution is provided at a concentration of 4.01 x 10 ¹² vg/ml.

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Clinical signs Daily (including food intake); Close observations weekly Body weight Weekly Neurological, ophthalmological, ECG examination Once before the dose and during Weeks 2 and 26. Clinical pathology All groups of hematology, clinical chemistry, coagulation parameters Hematology Red blood cell count average corpuscle, volume Hemoglobin platelet count Hematocrit white blood cell count Average corpuscular abs. neutrophil count Hemoglobin abs. lymphocyte count Average corpuscular abs. monocyte count Hemoglobin concentration abs. reticulocyte count Abs. eosinophil count white blood cell count Abs. basophil count blood smear Clinical biochemistry glucose alanine aminotransferase urea nitrogen alkaline phosphatase creatinine gamma-glutamyl transferase 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 formalin (unless otherwise specified) or recorded as missing, if applicable.

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Tissue Preservation, continued Adrenal gland ³ Injection site Rectum Aorta (overlying skin) Salivary gland Bone, femur Jejunum Sciatic nerve bone with Kidney ³ Seminal vesicle ³ bone marrow Lesions Skin/subcutaneous layer Bone, sternum Liver ³ Spinal cord with bone Lung with large (cervical, thoracic, brain, bronchi Brain ³ Lymph node lumbar) Cecum (mandibular) Spleena Cervix Lymph node Stomach Colon (mesenteric) Testiclea Duodenum Mammary gland Thymus Epididymis ³ Muscle, bicipital Thyroid gland Esophagus thigh muscle ^Eye Optic nerve Parathyroid ³ Gallbladder Ovary ³ Tongue GALT (Peyer's patch) Fallopian tube Trachea Pancreas Urinary bladder Heart ³ Pituitary gland ³ Uterus ³ Ileum Prostate gland ³ Vagina ³ Organs (if present) will be weighed or marked as missing; ^ь Collected in modified Davidson's fixative and stored in 10% neutral buffered formalin. Histopathology All groups - all fabrics Biodistribution The following tissues/biofluids will be analyzed for biodistribution using qPCR: Frontal Cortex Liver Hippocamp DRG (cervical) Ventral Midbrain DRG (thoracic) Periventricular Gray DRG (lumbar) matter Spinal Cord (thoracic) Putamen Spinal Cord (lumbar) Testicle Spinal Cord (cervical) Ovary Spleen Kidney Heart (apex) Stomach (pyloric) CSF Blood Lung Transgene expression All qPCR positive samples will be assessed for progranulin expression.

Abbreviations: F - female; ICM - intra cisterna magna; M - male; MgCl2 - magnesium chloride; NaCl sodium chloride; vg - vector genome(s); DRG - dorsal root ganglia; GALT - intestinal lymphoid tissue.

NHP cynomolgus macaques were assessed through several life-course 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 at Weeks 2 and 26), indirect ophthalmoscopy (baseline and at Weeks 2 and 26), and electrocardiography (ECG) (baseline and at Weeks 2 and 26). AAV9 capsid neutralizing antibody (nAb) analysis was performed at baseline and at sacrifice on Days 7, 30, or 183. Clinical evaluations including hematology, coagulation, clinical chemistry, and urinalysis were performed twice at baseline (blood tests; once urine tests) and once at Weeks 1 and 13 of the dosing phase.

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Animals were sacrificed and tissues were collected on Days 7, 30, or 183. Tissues listed in Table 11, if present, were collected from all animals, weighed (if applicable), and divided into replicates. One replicate was preserved in 10% neutral-buffered formalin (except when special fixatives were required for optimal fixation) for histopathological evaluation (all animals). Additional replicates were collected for qPCR and transgene expression analysis.

Safety and toxicology.

There were no unplanned deaths and all animals survived to scheduled necropsy. There were no PR006A-related adverse clinical events, changes in body weight, ophthalmologic findings, or physical or neurologic examination results; gross examination at necropsy revealed no drug-related abnormalities in either group. In addition, there were no PR006A-related changes in PR interval, QRS duration, QT interval, corrected QT interval (QTc), or heart rate observed in male or female mice dosed with 6.5x109 or 6.5x101° vg/g brain. No abnormal ECG waveforms or arrhythmias were observed during qualitative ECG evaluation.

Biodistribution.

Analysis of the biodistribution of the PR006A transgene was performed using a qPCR-based assay. On Day 183, the high dose group (6.5 x 10 ¹⁰ vg/g brain) showed widespread transduction throughout the CNS and periphery, with all tissues tested positive for the presence of vector at a cutoff of 50 vg/μg DNA, the lower limit of quantification for the qPCR assay. Data from selected representative regions from Day 183 are shown in Fig. 54A; Day 30 data not shown. On Day 30, in the high dose group (6.5 x 1010 vg/g brain), all CNS tissues tested were positive for transduction except the putamen. Tissues from animals receiving the low dose (6.5 x 109 vg/g brain) were positive in the CNS at Day 183, but of the peripheral tissues, only the spleen and liver were positive. Additionally, one female NHP receiving the high dose of PR006A was positive for transduction in the ovaries at Day 7, and males receiving the high dose were positive for transduction in the testes at Day 30 to Day 183. PR006A transduction was most robust in the liver and nervous system tissues and consistently lower in the other peripheral organs examined. In the brain, vector transduction stabilized at Day 183 compared to Day 30, demonstrating robust and persistent transduction of the transgene.

NHPs treated with ICM-treated PR006A mount a significant allogeneic immune response to the transgene product, progranulin, with anti-progranulin antibodies detected in serum and CSF samples collected at Day 30 and Day 183 post-treatment; this immune response indicates that human progranulin protein is expressed in NHPs. Anti-drug antibody (ADA) levels were determined using commonly used immunoassay technologies. The data are shown in Fig. 54B.

PR006A (GRN) expression was measured at the mRNA level using an RT-qPCR-based assay and at the protein level using the Simple Western™ assay (Jess). Concurrent with PR006A transduction levels, transgene expression was monitored by measuring mRNA by RT-qPCR in selected regions of brain (Fig. 54C), liver, gonads, spinal cord, and DRG collected at Day 183.

Transgene expression was measurable in brain and liver at both doses of PR006A, and expression levels were dose-dependent and robust. In the gonads, expression was measurable in males only at the high dose; at both doses, expression was measurable in females on Day 7 and Day 30, but not on Day 183.

To confirm that human progranulin was produced in treated NHPs, protein levels in CSF were assessed using the Simple Western™ platform (Jess). Details of the method are provided in Example 14. The method was validated by measuring progranulin levels in CSF samples from FTD-GRN patients and finding that they were approximately half the levels measured in CSF samples from healthy controls and from FTD patients without the GRN mutation. The results in CSF samples demonstrate that progranulin levels are increased in a dose-dependent manner in animals treated with both low and high doses of PR006A (Fig. 54D). These results indicate that efficient and broad transduction of PR006A in NHPs following ICM administration results in elevated progranulin levels.

Progranulin protein measurements were focused on CSF samples because the Simple Western™ assay (Jess) is not suitable for measuring progranulin levels in brain tissue due to high levels of non-specific background bands. Currently available assays do not reliably measure transgenic human progranulin levels in NHP tissues due to high levels of non-specific background. CSF levels are thought to reflect corresponding concentrations in the brain and are of particular value as translational biomarkers for clinical research.

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Resume.

There were no adverse safety findings or toxicity concerns that would preclude initiation of a clinical trial in any of the preclinical studies, including the small non-GLP pilot study in NHP and the GLP study in NHP, through Day 183. Pathology findings in the GLP study were consistently minimal in severity, with low lesion cell counts in both dose groups. There were no other adverse antemortem or postmortem findings related to PR006A.

Phase 1/2 study in human subjects with FTD-GRN.

Human subjects (n=15) will be enrolled in an open-label study of recombinant AAV PR006. Subject inclusion criteria are: 30-80 years of age (inclusive), presence of a pathogenic GRN mutation, symptomatic disease stage, and stable background therapy prior to study drug administration. Each subject will receive study drug via a single ICM (intra-cisterna magna) injection. 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: progranulin, NfL (neurofilament light chain), and volumetric MRI; and (3) performance: CDR plus NACC FTLD (Clinical Dementia Rating Scale plus National Alzheimer's Disease Coordinating Center Frontotemporal Dementia Rating Scale); measures of behavior, cognition, language, function, and quality of life.

Table 12

Examples of neurodegenerative 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, prionopathy with variable protease sensitivity) PRNP

Table 13

Examples of synucleinopathies

Disease Associated genes Parkinson's disease LRRK2, PARK7, PINK1, PRKN, SNCA, GBA, UCHL1, ATP13A2, VPS35 Dementia with Lewy bodies APOE, GBA, SNCA, SNCB Multiple system atrophy COQ2, SNCA

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Table 14

Examples of tauopathies

Disease Associated genes Alzheimer's disease APP, PSEN1, PSEN2, APOE Primary age-related tauopathy MARCH Progressive supranuclear palsy MARCH Corticobasal degeneration MARCH, GRN, C9orf72, VCP, CHMP2B, TARDBP, FUS Frontotemporal dementia in parkinsonism-17 MARCH Subacute sclerosing panencephalitis SCN1A Lytico-Bodig disease Ganglioglioma, gangliocytoma Meningioangiomatosis Postencephalitic Parkinsonism Chronic traumatic encephalopathy

Table 15

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 Metachromacic leukodystrophy ARSA, PSAP Farber's disease ASAHI Galactosialidosis CTSA Schindler's disease NAGA GM1 gangliosidosis GLB1 GM2 gangliosidosis GM2A Sandhoff's 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 GUSB 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 AGA Fucosidosis FUG Al

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Example 14. Automated Western analysis for the detection of progranulin in cerebrospinal fluid.

The objective of this experiment was to quantify progranulin (PGRN) protein levels in cerebrospinal fluid (CSF) using the Jess ProteinSimple automated Western platform (San Jose, CA). This assay can be used to analyze CSF samples from non-human primates (NHPs). To determine the expression levels of human progranulin protein, a PR006A transgene product, CSF samples from non-human primate subjects were analyzed on the Simple Western™ platform (Jess) using an antibody that specifically detects human progranulin protein. The Simple Western™ platform is a capillary-based automated Western blot immunoassay platform in which all steps, including protein separation, immunoprobe, wash, and chemiluminescence detection, are performed in a capillary cartridge. Samples (at 4-fold dilution) and primary antibody to human progranulin (Adipogen PG-359-7, at 10-fold dilution), in addition to secondary antibodies and all buffers provided by ProteinSimple, were loaded into a custom cartridge and run on the Jess platform. Semi-quantitative data analysis was performed automatically after each run, with parameters such as signal intensity, peak area, and signal-to-noise ratio calculated by the Jess instrument. For each individual sample, progranulin levels were measured as the peak area of immunoreactivity to the antibody. All analyses were performed using blinded samples.

The assay described herein was performed on CSF samples from a non-human primate study. CSF samples were tested for the presence and levels of progranulin protein to examine the efficacy of gene therapy using an rAAV construct (PR006; see Fig. 64) encoding the progranulin protein (PGRN). In this study, either adjuvant or PR006 was administered at a low dose of PR006 ( ^1.8x1010 vg/g brain weight) or at a high dose of PR006 (1.8x1011 vg/g brain weight) by injection into the cerebellomedullary cistern (ICM) of NHP animals. Each group consisted of 3 animals. Nine NHP animals were sacrificed at Day 180 post-infection (Table 16) and CSF samples were analyzed using the Jess platform-based assay.

Table 16

NHP Animal Data Summary by Group and Dose

Group PR006 Dose (vg/r brain weight) Number of animals Necropsy (Day 180) 1 0 2M/1F 2 1.8 x 10 ¹⁰ 2M/1F 3 1.8 x 10 ¹¹ 2M/1F

Table 17

Materials for automated Western analysis

Description of the material Manufacturer Product number Jess Separation Module 12-230 kDa, 25 Capillary Cartridges ProteinSimple SM-W004 EZ Standard Pack 1, 12-230 kDa ProteinSimple PS-ST01EZ-8 Anti-Mouse Antibody Detection Module for Jess ProteinSimple DM-002 Monoclonal antibody to progranulin (human), clone PG359-7 (primary antibody) Adipogen AG-20 A-0052-C100

Note: Allow reagents to warm to room temperature before opening vials.

The following procedures were followed in performing this method: Preparation of stock solutions.

1. Prepare a 400 mM DTT solution by adding 40 µL of water to a clear tube in the EZ Standard Pack Separation Module. Mix gently.

2. To prepare the master mix, add 20 µl 10x sample buffer and 20 µl 400 mM DTT to a pink EZ master mix tube. Mix gently.

3. To prepare the biotinylated ladder, pipette 20 µl of water into the clear biotinylated EZ ladder tube containing the pink precipitate. Mix gently.

4. Prepare a mixture of luminol and peroxide by adding them in equal amounts. For one cycle, add 200 µl of luminol to 200 µl of peroxide.

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5. Prepare a dilution of primary antibodies (10-fold dilution) by mixing 25 µl of primary antibodies and 225 µl of Antibody Diluent 2.

Preparation of samples.

1. Samples are diluted with 0.1x sample buffer. Prepare 0.1x sample buffer by adding 10 µl of 10x sample buffer to 990 µl of water.

2. Dilute samples if necessary. For example, NHP CSF samples are diluted 4-fold before adding the master mix. Add 5 µl NHP CSF to 15 µl 0.1X sample buffer.

3. Prepare samples by adding 1X master mix to 4X sample. To run technical duplicates, prepare a total of 15 µL of sample plus master mix per sample. For example, add 3 µL of master mix to 12 µL of diluted sample. Mix gently.

4. Boil samples at 95°C for 5 minutes.

5. Briefly spin the samples using a benchtop mini centrifuge. Vortex the sample before loading.

Load reagents and samples into the cartridge.

1. Pipette all samples according to the cartridge card.

a. Pipette 15 µl of the luminol+peroxide mixture into each well of lane E.

b. Pipette 10 µl of streptavidin into the first well of lane D.

c. Pipette 10 µl of secondary antibody into the remaining 24 wells of lane D.

d. Pipette 10 µl of the antibody dilution into the first well of lane C.

e. Pipette 10 µl of the primary antibody dilution into the remaining 24 wells of lane C.

f. Pipette 10 µl of the antibody dilution into all wells of lane B.

g. Pipette 10 µl of the prepared EZ ladder into the first well of lane A.

h. Pipette 5 µl of sample and master mix solution to duplicate lanes in lane A.

2. Rotate the cartridge at room temperature at 2500 rpm for 5 minutes.

Load the capillaries and cartridge into the device.

1. Insert the capillaries into the slot. Make sure the light turns blue.

2. Load the cartridge into the tool after rotating.

3. Press the start button after the blue indicator on the device stops flashing.

The performance of the assay system was considered acceptable if the percentage CV (coefficient of variation) for duplicates was < 30%.

Before using the assay to detect progranulin in NHP CSF samples, the assay was validated as follows. Qualification of the Jess assays included evaluation of dilution linearity, selectivity, and specificity. Normal CSF samples from BioIVT were used to determine the dilution linearity of the Jess assay. CSF samples from patients with frontotemporal dementia (FTD) with a PGRN mutation (obtained from the National Centralized Repository for Alzheimer's Disease and Related Dementias (NCRAD; Indianapolis, IN)) were used to determine the selectivity and specificity of the Jess assay.

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Table 18 Summary of results

Elements Compliance criteria Results Dilution linearity - Analysis of endogenous PGRN levels in unprocessed CSF samples (BioIVT). - Perform blank analysis in matrix. - Minimum required dilution (MRD) is determined by diluting blank matrix in 2-fold serial dilutions. - If endogenous PGRN levels in matrix are too low, dilutions will be performed using spiked matrix. - The MRD is defined as the lowest dilution required when a linear raw signal or concentration is observed. Within the linear range, corrected observed concentrations should be ±30% of the MRD. Compliant - All tested matrices passed the tests successfully with a linear dilution range of ±30% of the MRD (see Results and Discussion, Dilution Linearity). Selectivity and Specificity - Analysis of PGRN levels in CSF samples from patients with FTD. - MRD is determined using a dilution linearity test. Compliant - All tested matrices passed the tests successfully with a % CV of the technical replicate of 20% (see Results and Discussion, Selectivity and Specificity).

Results and discussion.

Linearity of dilution.

The dilution linearity of PGRN protein detected by the Jess platform was tested in commercially available (BioIVT) CSF samples from healthy individuals. To determine dilution linearity, endogenous PGRN levels in CSF samples were measured. Samples from two individuals were tested in 2-fold serial dilutions that ranged from 2- to 64-fold dilution. Table 19 shows the peak area of PGRN protein at 58 kDa obtained by the Jess platform and the % difference of each dilution compared to 16-fold dilution. Results within the linearity range are highlighted in bold (difference within 100 ± 30%). Dilution linearity was found to be within the 4- to 16-fold dilution range.

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Table 19

Linearity of dilution in CSF samples

SMZh#1 SMZh#2 Dilution factor Peak area 58 kDa (adjusted for dilution) % difference Peak area 58 kDa (adjusted for dilution) % difference 1:2 3915099 -41.2 6392991 -38.8 1:4 6040885 -9.2 8020821 -23.2 1:8 5773987 -13.3 12615004 20.8 1:16 6656474 0,0 10446186 0,0 1:32 8911479 33.9 11782404 12.8 1:64 12056943 81.1 6795118 -35.0

In summary, all matrices tested had an acceptable linear range that met the acceptance criteria of a % difference of 0±-30%, although the size of the range and the amount of dilution varied between matrices. The MRD of sample linearity was found to be 4-fold dilution. Dilution linearity was found to be within the range of 4-16-fold dilution. A summary of the MRD and the linear dilution range that met the acceptance criteria for CSF is presented in Table 20.

Table 20

MRD and CSF linear dilution range

Textile Linearity MRD Linear dilution range CMS 1:4 1:4 -1:16

Selectivity and specificity.

The selectivity and specificity of PGRN protein detected by the Jess assay were tested in CSF samples from PR006 FTD patients from NCRAD. Three groups (group A, B, and C) of CSF samples were collected from heterozygous FTD patients (group A), familial noncarriers (group B or C), and healthy individuals (group B or C). Six samples were analyzed in each group. The sample groups are listed in Table 16.

Information on CSF samples from patients with FTD.

CSF samples were diluted 4-fold with 0.1x sample buffer provided by ProteinSimple and tested in technical duplicates. Duplicate samples with % CV greater than 20% were re-assayed. Results with % CV less than 20% are presented in Table 22. Table 22 lists the PGRN protein peak area at 58 kDa obtained by the Jess platform and the % CV between duplicates. The results demonstrated that PGRN levels were approximately two-fold higher in groups B and C compared to group A, indicating the selectivity and specificity of the Jess assay in detecting PGRN levels in CSF samples (Fig. 55).

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Table 21

Information on CSF samples from patients with FTD

Barcode MRN variant Visit Set number Sample type Box name Regulations Group 000335559 8 ST- 20000108 Cycle 2 - CSF 25728 2 CMS 27488 CSF 1 WITH 000477733 8 ST- 20000118 Cycle 2 - CSF 26763 3 CMS 27488 CSF 2 WITH 000477732 9 ST- 20000306 Cycle 1 - CSF 26055 1 CMS 27488 CSF 3 A 000477732 6 ST- 20000328 Cycle 2 CSF 26054 4 CMS 27488 CSF 4 WITH 000477733 5 ST- 20000386 Cycle 1 - CSF 26711 0 CMS 27488 CSF 5 A 000477734 5 ST- 20000590 Cycle 2 - CSF 26785 9 CMS 27488 CSF 6 IN 000477733 2 ST- 20000621 Cycle 1 - CSF 26641 3 CMS 27488 CSF 7 IN 000462892 3 ST- 20000757 Cycle 1 - CSF 26981 7 CMS 27488 CSF 8 IN 000469510 3 ST- 20001142 Cycle 1 - CSF 30814 9 CMS 27488 CSF 9 A 000407462 9 ST- 20000107 Cycle 2 - CSF 25821 2 CMS 27488 CSF 10 WITH 000335847 5 ST- 20000110 Cycle 2 - CSF 25821 0 CMS 27488 CSF 11 With 000335846 3 ST- 20000274 Cycle 2 CSF 25729 2 CMS 27488 CSF 12 A 000478882 8 ST- 20000309 Cycle 2 - CSF 30309 3 CMS 27488 CSF 13 WITH 000335878 1 ST- 20000615 Cycle 1 - CSF 25727 8 CMS 27488 CSF 14 V 000335879 3 ST- 20000616 Cycle 1 - CSF 25730 5 CMS 27488 CSF 15 A 000477732 1 ST- 20000637 Cycle 1 - CSF 25730 7 CMS 27488 CSF 16 IN 000477734 1 ST- 20000768 Cycle 1 - CSF 26785 7 CMS 27488 CSF 17 V 000469510 6 ST- 20001165 Cycle 1 - CSF 31739 6 CMS 27488 CSF 18 A

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Table 22

Selectivity and specificity results

Groups Sample barcode Peak area 58 kDa (adjusted for dilution) %CV Group(A) Heterozygous patients with FTD 0004777329 2838645 5.08 0004777335 4293344 1,20 0004695103 6738165 1.08 0003358463 3594249 11,10 0003358793 5992434 2.49 0004695106 2472462 10.40 Group (B) Healthy or familial non-carriers 0004777345 3836185 11,18 0004777332 6006224 3.05 0004628923 3758940 1.44 0003358781 7860294 17.08 0004777321 7187172 0.69 0004777341 8450410 0,50 Group (C) Healthy or familial non-carriers 0003355598 2981005 1.70 0004777338 6803428 0.18 0004777326 5030695 3.56 0004074629 5448863 3.47 0003358475 7892529 1D7 0004788828 6944800 1.85

CSF samples from the FTD patient study (Table 21) were also analyzed with the human PGRN ELISA kit (Adipogen, AG-45A-0018YEK-KI01). The ELISA results (Fig. 56) revealed similar trends in PGRN levels between groups as the Jess assay and demonstrated that the Jess assay is suitable for use in assessing PGRN levels in CSF samples.

In conclusion, the ProteinSimple automated Western assay Jess was found to be suitable for use in assessing PGRN levels in CSF samples from NHPs.

Jess assay data for CSF samples from NHPs are shown in Table 23. Each sample is the average of two technical replicates. The peak area for the 58 kDa band in the sample lane is shown. Data are presented as the average peak area of the technical replicate and corrected dilution factors.

Table 23

Jess assay data for CSF samples from NHP

Sample ID Dose group Peak area (58 kDa) PRV-028 dl80 CSF 101 Low dose 4944754 PRV-028 dl80 CSF 102 Control 4449066 PRV-028 dl80 CSF 103 Low dose 6222881 PRV-028 dl80 CSF 104 High dose 5499901 PRV-028 dl80 CSF 105 Low dose 4293853 PRV-028 dl80 CSF 106 High dose 10149400 PRV-028 dl80 CSF 107 Control 1360173 PRV-028 dl80 CSF 108 Control 5742081 PRV-028 dl80 CSF 109 High dose 9658597

The aim of this assay was to confirm the expression level of progranulin protein (PGRN) following PR006 transduction in tissue regions of interest in NHPs. This was accomplished using an automated Western platform in which progranulin protein was detected using monoclonal antibodies. Progranulin expression could be measured in the CSF of both control and PR006-treated NHPs; however, the assay does not differentiate between endogenous progranulin protein and PR006A-specific progranulin protein.

This application incorporates by reference the contents of the following documents in their entireties: PCT International Application Publication No. WO 2019/070893; PCT International Application Publication No. WO 2019/070891; U.S. Provisional Patent Applications: Application Serial No. 62/567,296, filed October 3, 2017, and entitled GENE THERAPY FOR LYSOSOMAL DISORDERS; Application Serial No. 62/567,311, filed October 3, 2017, and entitled GENE

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GENE THERAPY FOR LYSOSOMAL DISORDERS; Application Serial No. 62/567,319, filed October 3, 2017, and entitled GENE THERAPY FOR LYSOSOMAL DISORDERS; Application Serial No. 62/567,301, filed October 3, 2018, and entitled GENE THERAPY FOR LYSOSOMAL DISORDERS; Application Serial No. 62/567,310, filed October 3, 2017, and entitled GENE THERAPY FOR LYSOSOMAL DISORDERS; Application Serial No. 62/567,303, filed October 3, 2017, and entitled GENE THERAPY FOR LYSOSOMAL DISORDERS; and Application Serial No. 62/567,305, 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 easily made by those skilled in the art. It is believed that such changes, modifications and optimizations are part of this description and are within the spirit and scope of the invention. Accordingly, the above description and drawings are provided by way of example only.

Although several embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily conceive of many other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications will be deemed 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 by way of example and that the 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 ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is therefore 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 otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and/or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, and/or methods, if such features, systems, articles, materials, and/or methods are not mutually incompatible, is within the scope of the present invention.

As used herein in the specification and claims, unless expressly stated otherwise, singular terms 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 either or both of co-occurring elements, that is, elements that are present together in some instances and disjointly in others. Other elements other than the elements specifically identified by the phrase and/or may optionally be present, whether related or unrelated to those elements specifically identified, unless expressly stated otherwise. Thus, as a non-limiting example, reference to A and/or B, when used in combination with an open-ended language term such as comprising, 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); and so on.

As used herein in the description and claims, the term or shall be understood to have the same meaning as and/or, as defined above. For example, when separating elements in a list, the terms or or and/or shall be interpreted as inclusive, i.e., including at least one, but also more than one, of the number or list of elements, and optionally additional elements not listed. Only terms that clearly indicate the contrary, such as only one of or exactly one of or, when used in a claim, consisting of, shall refer to the inclusion of exactly one element of the number or list of elements. In general, the term or, as used herein, shall be interpreted only as indicating exclusive alternatives (i.e., one or the other, but not both) when preceded by exclusivity terms, such as any, one of, only one of, or exactly one of. As used in the description and claims herein, the phrase "at least one" with respect to a list of one or more elements shall 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 not excluding any combination of elements in the list of elements. This definition also allows for elements to be optionally present in addition to the elements specifically identified in the list of elements to which the phrase "at least one" refers.

- 60 046 777 at least one, whether or not related to these specifically identified elements. Thus, as a non-limiting example, at least one of A and B (or, equivalently, at least one of A or B or, equivalently, at least one of A and/or B) may refer, in one embodiment, to at least one, optionally including more than one, of A, without B (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, of B, without A (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, of A, and at least one, optionally including more than one, of B (and optionally including other elements); and so on.

The use of ordinal terms such as first, second, third, etc. in a claim to modify an element of a claim does not in itself imply any priority, preference or dominant order of one claim element over another or the temporal order in which the actions of a method are performed, and these terms are used merely as labels to distinguish one claim element having a certain name from another element having the same name (but for the use of an ordinal term) to differentiate the elements of the claim.

It should also be understood that, unless expressly stated otherwise, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are stated. Each of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referenced in this application 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) comprises or consists of (or encodes a peptide having) a sequence set forth in any of SEQ ID NOs: 1-91. In some embodiments, the gene product is encoded by a portion (e.g., a fragment) of any of SEQ ID NOs: 1-91.

Numbered embodiments.

Notwithstanding the appended claims, the following numbered embodiments are disclosed herein.

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

2. The isolated nucleic acid of embodiment 1, wherein the Gcase protein comprises the amino acid sequence indicated 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 indicated in SEQ ID NO: 15.

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

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

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

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

8. The isolated nucleic acid of embodiment 7, wherein the prosaposin protein comprises the amino acid sequence indicated 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, not

- 61 046777 necessarily with the nucleic acid sequence indicated in SEQ ID NO: 17.

10. The isolated nucleic acid of any one of embodiments 7-9, wherein the modified region D 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 comprising the modified D sequence 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 comprising an expression construct encoding a SCARB2 protein flanked by two inverted terminal repeats (ITRs) of an adeno-associated virus (AAV), wherein (i) at least one of the ITRs comprises a modified D region relative to the wild-type AAV2 ITR (SEQ ID NO: 29); and/or (ii) the SCARB2 protein is encoded by a codon-optimized nucleic acid sequence.

14. The isolated nucleic acid of embodiment 13, wherein the SCARB2 protein comprises the amino acid sequence set forth in SEQ ID NO: 18, or a portion thereof. 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 of any of embodiments 13-15, wherein the modified D region is a D sequence located outside the ITR with respect to the expression construct.

17. The isolated nucleic acid of any one of embodiments 13-16, wherein the ITR comprising the modified D sequence 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 comprising an expression construct encoding a GBA2 protein flanked by two inverted terminal repeats (ITRs) of an adeno-associated virus (AAV), wherein (i) at least one of the ITRs comprises a modified D region relative to the wild-type AAV2 ITR (SEQ ID NO: 29); and/or (ii) the GBA2 protein is encoded by a codon-optimized nucleic acid sequence.

20. The isolated nucleic acid of embodiment 19, wherein the GBA2 protein comprises the amino acid sequence set forth in SEQ ID NO: 30, or a portion thereof.

21. The isolated nucleic acid of embodiment 19 or 20, wherein the GBA2 protein is encoded by a codon-optimized nucleic acid sequence, optionally the nucleic acid sequence set forth in SEQ ID NO: 31.

22. The isolated nucleic acid of any one of embodiments 19-21, wherein the modified region D is a D sequence located outside the ITR relative to the expression construct.

23. The isolated nucleic acid of any one of embodiments 19-22, wherein the ITR comprising the modified D sequence is a 3'ITR.

24. The isolated nucleic acid of any one of embodiments 19-23, further comprising a TRY sequence, optionally wherein the TRY sequence is set forth in SEQ ID NO: 28.

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

26. The isolated nucleic acid of embodiment 25, wherein the GALC protein comprises the amino acid sequence set forth in SEQ ID NO: 33, or a portion thereof.

27. The isolated nucleic acid of embodiment 25 or 26, wherein the GALC protein is encoded by a codon-optimized nucleic acid sequence, optionally the nucleic acid sequence set forth in SEQ ID NO: 34.

28. The isolated nucleic acid of any one of embodiments 25-27, wherein the modified region D is a D sequence located outside the ITR relative to the expression construct.

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29. The isolated nucleic acid of any one of embodiments 25-28, wherein the ITR comprising the modified D sequence is a 3'ITR.

30. The isolated nucleic acid of any one of embodiments 25-29, further comprising a TRY sequence, optionally wherein the TRY sequence is set forth in SEQ ID NO: 28.

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

32. The isolated nucleic acid of embodiment 31, wherein the CTSB protein comprises the amino acid sequence set forth in SEQ ID NO: 30, or a portion thereof.

33. The isolated nucleic acid of embodiment 31 or 32, wherein the CTSB protein is encoded by a codon-optimized nucleic acid sequence, optionally the nucleic acid sequence set forth in SEQ ID NO: 36.

34. The isolated nucleic acid of any one of embodiments 31-33, wherein the modified region D is a D sequence located outside the ITR relative to the expression construct.

35. The isolated nucleic acid of any one of embodiments 31-34, wherein the ITR comprising the modified D sequence is a 3'ITR.

36. The isolated nucleic acid of any one of embodiments 31-35, further comprising a TRY sequence, optionally wherein the TRY sequence is set forth in SEQ ID NO: 28.

37. An isolated nucleic acid comprising an expression construct encoding an SMPD1 protein flanked by two inverted terminal repeats (ITRs) of an adeno-associated virus (AAV), wherein (i) at least one of the ITRs comprises a modified D region relative to the wild-type AAV2 ITR (SEQ ID NO: 29); and/or (ii) the SMPD1 protein is encoded by a codon-optimized nucleic acid sequence.

38. The isolated nucleic acid of embodiment 37, wherein the SMPD1 protein comprises the amino acid sequence set forth in SEQ ID NO: 37, or a portion thereof.

39. The isolated nucleic acid of embodiment 37 or 38, wherein the SMPD1 protein is encoded by a codon-optimized nucleic acid sequence, optionally the nucleic acid sequence set forth in SEQ ID NO: 38.

40. The isolated nucleic acid of any one of embodiments 37-39, wherein the modified region D is a D sequence located outside the ITR relative to the expression construct.

41. The isolated nucleic acid of any one of embodiments 37-40, wherein the ITR comprising the modified D sequence is a 3'ITR.

42. The isolated nucleic acid of any one of embodiments 37-41, further comprising a TRY sequence, optionally wherein the TRY sequence is set forth in SEQ ID NO: 28.

43. An isolated nucleic acid comprising an expression construct encoding a GCH1 protein flanked by two inverted terminal repeats (ITRs) of an adeno-associated virus (AAV), wherein (i) at least one of the ITRs comprises a modified D region relative to the wild-type AAV2 ITR (SEQ ID NO: 29); and/or (ii) the GCH1 protein is encoded by a codon-optimized nucleic acid sequence.

44. The isolated nucleic acid of embodiment 43, wherein the GCH1 protein comprises the amino acid sequence set forth in SEQ ID NO: 45, or a portion thereof.

45. The isolated nucleic acid of embodiment 43 or 44, wherein the GCH1 protein is encoded by a codon-optimized nucleic acid sequence, optionally the nucleic acid sequence set forth in SEQ ID NO: 46.

46. The isolated nucleic acid of any one of embodiments 43-45, wherein the modified region D is a D sequence located outside the ITR relative to the expression construct.

47. The isolated nucleic acid of any one of embodiments 43-46, wherein the ITR comprising the modified D sequence is a 3'ITR.

48. The isolated nucleic acid of any one of embodiments 43-47, further comprising a TRY sequence, optionally wherein the TRY sequence is set forth in SEQ

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ID NO: 28.

49. An isolated nucleic acid comprising an expression construct encoding a RAB7L protein flanked by two inverted terminal repeats (ITRs) of an adeno-associated virus (AAV), wherein (i) at least one of the ITRs comprises a modified D region relative to the wild-type AAV2 ITR (SEQ ID NO: 29); and/or (ii) the RAB7L protein is encoded by a codon-optimized nucleic acid sequence.

50. The isolated nucleic acid of embodiment 49, wherein the RAB7L protein comprises the amino acid sequence set forth in SEQ ID NO: 47, or a portion thereof.

51. The isolated nucleic acid of embodiment 49 or 50, wherein the RAB7L protein is encoded by a codon-optimized nucleic acid sequence, optionally the nucleic acid sequence set forth in SEQ ID NO: 48.

52. The isolated nucleic acid of any one of embodiments 49-51, wherein the modified region D is a D sequence located outside the ITR relative to the expression construct.

53. The isolated nucleic acid of any one of embodiments 49-52, wherein the ITR comprising the modified D sequence is a 3'ITR.

54. The isolated nucleic acid of any one of embodiments 49-53, further comprising a TRY sequence, optionally wherein the TRY sequence is set forth in SEQ ID NO: 28.

55. An isolated nucleic acid comprising an expression construct encoding a VPS35 protein flanked by two inverted terminal repeats (ITRs) of an adeno-associated virus (AAV), wherein (i) at least one of the ITRs comprises a modified D region relative to the wild-type AAV2 ITR (SEQ ID NO: 29); and/or (ii) the VPS35 protein is encoded by a codon-optimized nucleic acid sequence.

56. The isolated nucleic acid of embodiment 55, wherein the VPS35 protein comprises the amino acid sequence set forth in SEQ ID NO: 49, or a portion thereof.

57. The isolated nucleic acid of embodiment 55 or 56, wherein the VPS35 protein is encoded by a codon-optimized nucleic acid sequence, optionally the nucleic acid sequence set forth in SEQ ID NO: 50.

58. The isolated nucleic acid of any one of embodiments 55-57, wherein the modified region D is a D sequence located outside the ITR relative to the expression construct.

59. The isolated nucleic acid of any one of embodiments 55-58, wherein the ITR comprising the modified D sequence is a 3'ITR.

60. The isolated nucleic acid of any one of embodiments 55-59, further comprising a TRY sequence, optionally wherein the TRY sequence is set forth in SEQ ID NO: 28.

61. An isolated nucleic acid comprising an expression construct encoding an IL-34 protein flanked by two inverted terminal repeats (ITRs) of an adeno-associated virus (AAV), wherein (i) at least one of the ITRs comprises a modified D region relative to the wild-type AAV2 ITR (SEQ ID NO: 29); and/or (ii) the IL-34 protein is encoded by a codon-optimized nucleic acid sequence.

62. The isolated nucleic acid of embodiment 61, wherein the IL-34 protein comprises the amino acid sequence set forth in SEQ ID NO: 55, or a portion thereof.

63. The isolated nucleic acid of embodiment 61 or 62, wherein the IL-34 protein is encoded by a codon-optimized nucleic acid sequence, optionally the nucleic acid sequence set forth in SEQ ID NO: 56.

64. The isolated nucleic acid of any one of embodiments 61-63, wherein the modified region D is a D sequence located outside the ITR relative to the expression construct.

65. The isolated nucleic acid of any one of embodiments 61-64, wherein the ITR comprising the modified D sequence is a 3'ITR.

66. The isolated nucleic acid of any one of embodiments 61-65, further comprising a TRY sequence, optionally wherein the TRY sequence is set forth in SEQ ID NO: 28.

67. An isolated nucleic acid comprising an expression construct encoding a TREM2 protein flanked by two inverted terminal repeats (ITRs) of an adeno-associated virus (AAV), wherein (i) at least one of the ITRs comprises a modified D region relative to the ITR

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wild-type AAV2 (SEQ ID NO: 29); and/or (ii) the TREM2 protein is encoded by a codon-optimized nucleic acid sequence.

68. The isolated nucleic acid of embodiment 67, wherein the TREM2 protein comprises the amino acid sequence set forth in SEQ ID NO: 57, or a portion thereof.

69. The isolated nucleic acid of embodiment 67 or 68, wherein the TREM2 protein is encoded by a codon-optimized nucleic acid sequence, optionally the nucleic acid sequence set forth in SEQ ID NO: 58.

70. The isolated nucleic acid of any one of embodiments 67-69, wherein the modified region D is a D sequence located outside the ITR relative to the expression construct.

71. The isolated nucleic acid of any one of embodiments 67-70, wherein the ITR comprising the modified D sequence is a 3'ITR.

72. The isolated nucleic acid of any one of embodiments 67-71, further comprising a TRY sequence, optionally wherein the TRY sequence is set forth in SEQ ID NO: 28.

73. An isolated nucleic acid comprising an expression construct encoding a TMEM106B protein flanked by two inverted terminal repeats (ITRs) of an adeno-associated virus (AAV), wherein (i) at least one of the ITRs comprises a modified D region relative to the ITR of wild-type AAV2 (SEQ ID NO: 29); and/or (ii) the TMEM106B protein is encoded by a codon-optimized nucleic acid sequence.

74. The isolated nucleic acid of embodiment 73, wherein the TMEM106B protein comprises the amino acid sequence set forth in SEQ ID NO: 63, or a portion thereof.

75. The isolated nucleic acid of embodiment 73 or 74, wherein the TMEM106B protein is encoded by a codon-optimized nucleic acid sequence, optionally the nucleic acid sequence set forth in SEQ ID NO: 64.

76. The isolated nucleic acid of any one of embodiments 73-75, wherein the modified region D is a D sequence located outside the ITR relative to the expression construct.

77. The isolated nucleic acid of any one of embodiments 73-76, wherein the ITR comprising the modified D sequence is a 3'ITR.

78. The isolated nucleic acid of any one of embodiments 73-77, further comprising a TRY sequence, optionally wherein the TRY sequence is set forth in SEQ ID NO: 28.

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

80. The isolated nucleic acid of embodiment 79, wherein the PGRN protein comprises the amino acid sequence set forth in SEQ ID NO: 67, or a portion thereof.

81. The isolated nucleic acid of embodiment 79 or 80, wherein the PGRN protein is encoded by a codon-optimized nucleic acid sequence, optionally the nucleic acid sequence set forth in SEQ ID NO: 68.

82. The isolated nucleic acid of any one of embodiments 79-81, wherein the modified region D is a D sequence located outside the ITR relative to the expression construct.

83. The isolated nucleic acid of any one of embodiments 79-82, wherein the ITR comprising the modified D sequence is a 3'ITR.

84. The isolated nucleic acid of any one of embodiments 79-83, further comprising a TRY sequence, optionally wherein the TRY sequence is set forth in SEQ ID NO: 28.

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

86. The isolated nucleic acid of embodiment 85, wherein the first gene product is a G-case protein or a portion thereof.

87. The isolated nucleic acid of embodiment 85 or 86, wherein the second gene product is LIMP2 or a portion thereof, or prosaposin or a portion thereof.

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88. The isolated nucleic acid of any one of embodiments 85-87, further encoding an interfering nucleic acid (e.g., shRNA, miRNA, dsRNA, etc.), optionally wherein the interfering nucleic acid inhibits the expression of α-Syn or TMEM106B.

89. The isolated nucleic acid of any one of embodiments 85-88, further comprising one or more promoters, optionally wherein each of the one or more promoters is independently a chicken beta-actin (CBA) promoter, a CAG promoter, a CD68 promoter, or a JeT promoter.

90. The isolated nucleic acid of any one of embodiments 85-89, further comprising an internal ribosome entry site (IRES), optionally wherein said IRES is located between the first gene product and the second gene product.

91. The isolated nucleic acid of any one of embodiments 85-90, further comprising a sequence encoding a self-cleaving peptide, optionally wherein said self-cleaving peptide is T2A.

92. The isolated nucleic acid of any one of embodiments 85-91, 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 wherein one of the ITR sequences lacks a functional terminal resolution site.

93. The isolated nucleic acid of embodiment 92, wherein at least one of the ITRs comprises a modified D region relative to the wild-type AAV2 ITR (SEQ ID NO: 29).

94. The isolated nucleic acid of embodiment 93, wherein the modified region D is a D sequence located outside the ITR relative to the expression construct.

95. The isolated nucleic acid of embodiment 93 or 94, wherein the ITR comprising the modified D sequence is a 3' ITR.

96. The isolated nucleic acid of any one of embodiments 85-95, further comprising a TRY sequence, optionally wherein the TRY sequence is set forth in SEQ ID NO: 28.

97. An isolated nucleic acid having a sequence as set forth in any one of SEQ ID NOs: 1-91.

98. A vector comprising the isolated nucleic acid of any one of embodiments 1-97.

99. The vector according to embodiment 98, characterized in that said vector is a plasmid.

100. The vector of embodiment 98, wherein said vector is a viral vector, optionally wherein the viral vector is a recombinant AAV vector (rAAV) or a baculovirus vector.

101. A composition comprising an isolated nucleic acid of any one of embodiments 1-97 or a vector of any one of embodiments 98-100.

102. A host cell comprising the isolated nucleic acid of any one of embodiments 1-97 or the vector of any one of embodiments 98-100.

103. Recombinant adeno-associated virus (rAAV) containing:

(i) a capsid protein; and (ii) an isolated nucleic acid according to any one of embodiments 1-97 or a vector according to any one of embodiments 98-100.

104. The rAAV of embodiment 103, wherein the capsid protein is capable of crossing the blood-brain barrier, wherein said capsid protein is optionally an AAV9 capsid protein or an AAVrh.10 capsid protein.

105. The rAAV of embodiment 103 or 104, wherein said rAAV transduces neuronal cells and non-neuronal cells of the central nervous system (CNS).

106. A method of treating a subject with existing or suspected Parkinson's disease, or, wherein said method comprises administering to the subject an isolated nucleic acid of any one of embodiments 1-97, a vector of any one of embodiments 98-100, a composition of embodiment 101, or an rAAV of any one of embodiments 103-105.

107. The method of embodiment 106, wherein the administration comprises a direct injection into the CNS of the subject, optionally wherein the direct injection is an intracerebral injection, an intraparenchymal injection, an intrathecal injection, an intracisternal injection, or any combination thereof.

108. The method of embodiment 107, wherein said direct injection into the CNS of the subject comprises convective enhanced delivery (CED).

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109. The method according to any one of embodiments 106-108, characterized in that the administration comprises a peripheral injection, wherein the peripheral injection is not necessarily an intravenous injection.

110. A method of treating a subject having or suspected of having frontotemporal dementia with a GRN mutation, comprising administering to said subject a recombinant adeno-associated virus (rAAV) comprising:

(i) an rAAV vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a transgene insert encoding a PGRN protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 68; and (ii) an AAV9 capsid protein.

111. The method of embodiment 110, wherein the rAAV is administered to the subject at a dose of from about 1x10 ¹³ vector genomes (vg) to about 7x10 ¹⁴ vg.

112. The method according to embodiment 110 or 111, characterized in that the rAAV is administered by injection into the cerebellomedullary cistern.

113. The method according to any one of embodiments 110-112, characterized in that the promoter is the chicken beta-actin (CBA) promoter.

114. The method according to any one of embodiments 110-113, wherein the rAAV vector further comprises a cytomegalovirus (CMV) enhancer.

115. The method according to any one of embodiments 110-114, wherein the rAAV vector further comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

116. The method according to any one of embodiments 110-115, characterized in that the rAAV vector further comprises a bovine growth hormone polyA signal tail.

117. The method according to any one of embodiments 110-116, wherein the nucleic acid comprises two adeno-associated virus inverted terminal repeat (ITR) sequences flanking the expression construct.

118. The method of embodiment 117, wherein each ITR sequence is a wild-type AAV2 ITR sequence.

119. The method according to any one of embodiments 110-118, 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.

120. A method of treating a subject having or suspected of having frontotemporal dementia with a GRN mutation, comprising administering to said subject an rAAV comprising:

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

(a) AAV2 ITR;

(b) CMV enhancer;

(c) SVA promoter;

(d) a transgene insert encoding a PGRN protein, wherein said transgene insert comprises the nucleotide sequence of SEQ ID NO: 68;

(e) WPRE;

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

121. The method of embodiment 120, wherein the rAAV is administered to the subject at a dose of from about 1x10 ¹³ vg to about 7x10 ¹⁴ vg.

122. The method according to embodiment 120 or 121, characterized in that the rAAV is administered by injection into the cerebellomedullary cistern.

123. The method of any one of embodiments 110-122, wherein the rAAV is administered in a formulation comprising about 20 mM Tris, pH 8.0, about 1 mM _MgCl2 , about 200 mM NaCl, and about 0.001% w/v poloxamer 188.

124. A pharmaceutical composition comprising (i) an rAAV comprising:

(a) an rAAV vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a transgene insert encoding a PGRN protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 68; and (b) an 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.

125. rAAV containing:

(a) an rAAV vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a transgene insert encoding a PGRN protein, wherein

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Claims (14)

wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 68; and (b) an AAV9 capsid protein, for use in a method of treating frontotemporal dementia with a GRN mutation in a subject. 126. A method for quantitatively determining the level of PGRN protein in a cerebrospinal fluid (CSF) sample, comprising: 1. A method of treating a subject with or suspected of having frontotemporal dementia with a GRN mutation, comprising administering to said subject a recombinant adeno-associated virus (rAAV) comprising: (i) an rAAV vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a transgene insert encoding a progranulin protein (PGRN), wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 68; and (ii) an AAV9 capsid protein. (1) dilution of the CSF sample in a master mix containing dithiothreitol (DTT) and sample buffer; 2. The method of claim 1, wherein the rAAV is administered to the subject at a dose of from 1x10 ¹³ vector genomes (vg) to about 7x10 ¹⁴ vg. (2) loading the diluted CSF sample, anti-progranulin antibody, a secondary antibody that detects anti-progranulin antibody, luminol, and peroxide into the wells of a capillary cartridge; 3. The method according to claim 1 or 2, wherein the rAAV is administered by injection into the cerebellomedullary cistern. (3) loading the capillary cartridge into an automated Western blot immunoassay instrument; 4. The method according to any one of claims 1 to 3, wherein the promoter is the chicken beta-actin (CBA) promoter. (4) use of an automated Western blot immunoassay instrument to calculate signal intensity, peak area, and signal-to-noise ratio; and (5) quantification of the progranulin protein level in the CSF sample as the peak area of immunoreactivity to anti-progranulin antibody. CLAUSE OF INVENTION 5. The method according to any one of claims 1 to 4, wherein the rAAV vector further comprises a cytomegalovirus (CMV) enhancer. 6. The method according to any one of claims 1 to 5, wherein the rAAV vector further comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). 7. The method according to any one of claims 1 to 6, wherein the rAAV vector further comprises a bovine growth hormone polyA signal tail. 8. The method according to any one of claims 1 to 7, wherein the nucleic acid comprises two adeno-associated virus inverted terminal repeat (ITR) sequences flanking the expression construct, wherein the first ITR sequence is a 5' ITR and the second ITR sequence is a 3' ITR. 9. The method of claim 8, wherein each of said two ITR sequences is a wild-type AAV2 ITR sequence. 10. The method according to any one of claims 1 to 9, wherein the rAAV vector further comprises a TRY region between the 5' ITR and the expression construct, wherein the TRY region comprises SEQ ID NO: 28. 11. A method of treating a subject with or suspected of having frontotemporal dementia with a GRN mutation, comprising administering to the subject an rAAV comprising: (i) an rAAV vector containing a nucleic acid comprising, in order from 5' to 3': (a) AAV2 ITR; (b) CMV enhancer; (c) SVA promoter; (d) a transgene insert encoding a PGRN protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 68; (e) WPRE; (f) the bovine growth hormone polyA signal tail; and (g) the AAV2 ITR; and (ii) the AAV9 capsid protein. 12. The method of claim 11, wherein the rAAV is administered to the subject at a dose of 1x10 ¹³ to 7x10 ¹⁴ vg. 13. The method according to claim 11 or 12, wherein the rAAV is administered by injection into the cerebellomedullary cistern. 14. The method according to any one of claims 1 to 13, wherein the rAAV is administered in a formulation containing 20 mM Tris, pH -