Gene therapy for lysosomal disorders

Abstract

To provide compositions and methods for treatment of diseases associated with aberrant lysosomal functions, such as fronto-temporal dementia (FTD).SOLUTION: Provided are expression constructs comprising a transgene encoding progranulin or a portion thereof, and methods of treating FTD by administering such expression constructs to a subject in need thereof.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 988,665 filed on 12 March 2020, U.S. Provisional Patent Application No. 62 / 960,471 filed on 13 January 2020, U.S. Provisional Patent Application No. 62 / 954,089 filed on 27 December 2019, U.S. Provisional Patent Application No. 62 / 934,450 filed on 12 November 2019, and U.S. Provisional Patent Application No. 62 / 831,846 filed on 10 April 2019. The disclosures of each of these applications are incorporated herein by reference in their entirety.

[0002] Description of the electronically submitted text file The contents of the text files submitted electronically with this specification are incorporated herein by reference in their entirety: a computer-readable copy of the sequence listing (filename: PRVL_010_05WO_SeqList.txt, date recorded: April 10, 2020, file size approximately 612,902 bytes).

[0003] field This disclosure relates to the field of gene therapy and methods of using it. [Background technology]

[0004] Gaucher disease is a rare congenital anomaly of sphingoglycolipid metabolism due to a deficiency of lysosomal acidic β-glucocerebrosidase (Gcase, "GBA"). Patients suffer from non-CNS symptoms and findings, including hepatosplenomegaly, bone marrow failure leading to pancytopenia, pulmonary impairment and fibrosis, and bone defects. In addition, a significant number of patients suffer from neurological symptoms, including saccadic eye movement and gaze defects, seizures, cognitive impairment, developmental delay, and motor disorders, including Parkinson's disease. Several therapies exist to address peripheral diseases in the hematopoietic bone marrow and viscera, as well as the main clinical manifestations. These include enzyme replacement therapy, chaperone-like small molecule drugs that bind to the deficient Gcase to improve its stability, and substrate reduction therapy that blocks the production of substrates that accumulate in Gaucher disease and lead to symptoms and findings. However, other aspects of Gaucher disease (particularly those affecting the skeleton and brain) appear to be refractory to treatment.

[0005] Progranulin (PGRN) is an additional protein associated with lysosomal function. PGRN is encoded by the GRN gene. In humans, GRN haploy failure increases the risk of developing FTD-GRN (frontotemporal dementia with GRN mutations), a neurodegenerative disease characterized by executive function impairment, behavioral changes, and language disorders, accompanied by atrophy of the frontal and temporal lobes, to approximately 90%. There are no disease-modifying therapies for FTD patients. [Overview of the Initiative]

[0006] This specification provides a method for treating subjects having or suspected of having frontotemporal dementia with a GRN mutation, the 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 approximately 1 × 10⁻⁶ 13 Vector genome (vg) ~ approximately 7 × 10⁻⁶ 14It is administered to the subject in doses within the range of vg. In some embodiments, rAAV is administered by injection into the cisterna magna.

[0007] In some embodiments, the promoter operably linked to the transgene insert encoding the PGRN protein is a chicken beta-actin (CBA) promoter. In some embodiments, the rAAV vector further comprises a cytomegalovirus (CMV) enhancer. In some embodiments, the rAAV vector further comprises a woodchuck hepatitis virus post-transcriptional regulator (WPRE). In some embodiments, the rAAV vector further comprises a bovine growth hormone polyA signaling tail. In some embodiments, the nucleic acid comprises two adeno-associated virus inverted terminal repeat (ITR) sequences adjacent to the expression construct. In some embodiments, each ITR sequence is a wild-type AAV2 ITR sequence. In some embodiments, the rAAV vector further comprises a TRY region between the 5' ITR and the expression construct, the TRY region comprising SEQ ID NO: 28.

[0008] This specification provides a method for treating subjects having or suspected of having frontotemporal dementia with a GRN mutation, the method comprising administering to the subject an rAAV comprising: (i) a nucleic acid-containing rAAV vector 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, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 68, (e) a WPRE, (f) a bovine growth hormone poly(A) signaling tail, and (g) an AAV2 ITR; and (ii) AAV9 capsid protein. In some embodiments, rAAV is approximately 1 × 10⁻⁶ 13 vg~approx. 7×10 14 It is administered to the subject in doses within the range of vg. In some embodiments, rAAV is administered by injection into the cisterna magna.

[0009] In some embodiments, rAAV is administered in a formulation containing approximately 20 mM Tris (pH 8.0), approximately 1 mM MgCl2, approximately 200 mM NaCl, and approximately 0.001% w / v poloxamer 188.

[0010] Provided herein are pharmaceutical compositions comprising: (i) an rAAV vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to an transgene insert encoding a PGRN protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 68; and (b) an rAAV comprising an AAV9 capsid protein; (ii) about 20 mM Tris (pH 8.0); (iii) about 1 mM MgCl2; (iv) about 200 mM NaCl; and (v) about 0.001% w / v poloxamer 188.

[0011] This specification provides for rAAVs including: (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 for treating frontotemporal dementia having a GRN mutation in a subject.

[0012] This specification provides a method for quantifying PGRN protein levels in cerebrospinal fluid (CSF) samples, the method comprising: (1) diluting the CSF sample in a master mixture containing dithiothreitol (DTT) and sample buffer; (2) filling the wells of a capillary cartridge with the diluted CSF sample, anti-progranulin antibody, secondary antibody for detecting the anti-progranulin antibody, luminol, and peroxide; (3) loading the capillary cartridge into an automated Western blot immunoassay instrument; (4) calculating the signal intensity, peak area, and signal-to-noise ratio using the automated Western blot immunoassay instrument; and (5) quantifying the progranulin protein level in the CSF sample as the peak area of ​​immunoreactivity to the anti-progranulin antibody. [Brief explanation of the drawing]

[0013] [Figure 1] This is a schematic diagram showing one embodiment of a vector containing an expression construct that encodes Gcase (e.g., GBA1 or a portion thereof). [Figure 2] This is a schematic diagram showing one embodiment of a vector containing an expression construct encoding Gcase (e.g., GBA1 or a portion thereof) and LIMP2 (SCARB2) or a portion thereof. The coding sequences of Gcase and LIMP2 are separated by an internal ribosome entry site (IRES). [Figure 3] This is a schematic diagram showing one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof) and LIMP2 (SCARB2) or a portion thereof. The expression of the Gcase and LIMP2 coding sequences is driven by separate promoters, respectively. [Figure 4] This is a schematic diagram showing one embodiment of a vector comprising an expression construct encoding an interfering RNA for Gcase (e.g., GBA1 or a portion thereof), LIMP2 (SCARB2) or a portion thereof, and α-Syn. [Figure 5]This is a schematic diagram showing one embodiment of a vector comprising an expression construct encoding an interfering RNA for Gcase (e.g., GBA1 or a portion thereof), a prosaposin (e.g., PSAP or a portion thereof), and α-Syn. [Figure 6] This is a schematic diagram showing one embodiment of a vector containing an expression construct encoding Gcase (e.g., GBA1 or a portion thereof) and a prosaposin (e.g., PSAP or a portion thereof). The coding sequences for Gcase and the prosaposin are separated by an internal ribosome entry site (IRES). [Figure 7] This is a schematic diagram showing one embodiment of a vector containing an expression construct encoding Gcase (e.g., GBA1 or a portion thereof). In this embodiment, the vector comprises a CBA promoter element (CBA) consisting of the following four parts: a CMV enhancer (CMVe), a CBA promoter (CBAp), exon 1, and an intron (int) for constitutive expression of the codon-optimized coding sequence of human GBA1. The 3' region also contains a WPRE regulatory element, followed by a bGH poly-A tail. The following three transcriptional regulatory activation sites are included at the 5' end of the promoter region: TATA, RBS, and YY1. The adjacent ITR allows for the correct packaging of the intervening sequence. Two variants (insertion boxes) of the 5' ITR sequence were evaluated, and these have several nucleotide differences within the 20-nucleotide "D" region of the wild-type AAV2 ITR. In some embodiments, the rAAV vector contains the "D" domain nucleotide sequence shown in the upper row. In some embodiments, the rAAV vector includes a variant "D" domain (for example, an "S" domain having the nucleotide changes shown in the lower row). [Figure 8] Figure 6 is a schematic diagram showing one embodiment of the vector described. [Figure 9-1]Representative data on rAAV delivery containing transgenes encoding Gcase (e.g., GBA1 or a portion thereof) are shown in a mouse model of Parkinson's disease (CBE). IP delivery per day of PBS vehicle, 25 mg / kg CBE, 37.5 mg / kg CBE, or 50 mg / kg CBE (left to right) was initiated at P8. Survival rate (top left) was checked twice daily, and body weight (top right) was checked daily. All groups started with n=8. Behavior was assessed by total distance traveled in the open field at P23 (bottom left) and latency to fall on the rotorod at P24 (bottom center). GCase substrate levels were analyzed in the cortex of mice treated with PBS and 25 mg / kg CBE, both with and without CBE withdrawal (day 3). Aggregate GluSph and GalSph levels (bottom right) are shown as pmol per mg of wet tissue weight. Means are shown. Error bars represent SEM. *p<0.05, **p<0.01, ***p<0.001: Nominal p-values ​​for the treatment group based on linear regression. [Figure 9-2] Representative data on rAAV delivery containing transgenes encoding Gcase (e.g., GBA1 or a portion thereof) are shown in a mouse model of Parkinson's disease (CBE). IP delivery per day of PBS vehicle, 25 mg / kg CBE, 37.5 mg / kg CBE, or 50 mg / kg CBE (left to right) was initiated at P8. Survival rate (top left) was checked twice daily, and body weight (top right) was checked daily. All groups started with n=8. Behavior was assessed by total distance traveled in the open field at P23 (bottom left) and latency to fall on the rotorod at P24 (bottom center). GCase substrate levels were analyzed in the cortex of mice treated with PBS and 25 mg / kg CBE, both with and without CBE withdrawal (day 3). Aggregate GluSph and GalSph levels (bottom right) are shown as pmol per mg of wet tissue weight. Means are shown. Error bars represent SEM. *p<0.05, **p<0.01, ***p<0.001: Nominal p-values ​​for the treatment group based on linear regression. [Figure 10] This is a schematic diagram illustrating one embodiment of a study design for the maximum rAAV dose in a CBE mouse model. Briefly, rAAV was delivered by ICV injection at P3, and daily CBE treatment was initiated at P8. Behavior was evaluated in open-field and rotorod assays at P24–25, and substrate levels were measured at P36 and P38. [Figure 11-1] Representative data on the survival assessment of the maximum rAAV dose in a CBE mouse model are presented. In P3, mice were treated with either an excipient or 8.8e9vg of rAAV-GBA1 via ICV delivery. Daily IP delivery of either PBS or 25 mg / kg of CBE was initiated in P8. At the end of the study, half of the mice were sacrificed one day after the final CBE dose in P36 (day 1), and the other half were sacrificed in P38 (day 3) after a 3-day CBE withdrawal. All treatment groups (excipient + PBS, n=8, rAAV-GBA1 + PBS (n=7), excipient + CBE (n=8), and variant + CBE (n=9)) were weighed daily (top left) and weights were analyzed in P36 (top right). Behavior was evaluated by total distance traveled in the open field at P23 (bottom left) and latency to fall on the rotorod at p24 (bottom right), and was assessed for each animal as the median across the three trials. Due to lethality, there were n=7 for the excipient + CBE group for the behavioral assay and n=8 for all other groups. The mean for all animals is shown. Error bars are SEM. *p<0.05, ***p<0.001, nominal p-values ​​for treatment groups by linear regression in CBE-treated animals. [Figure 11-2]Representative data on the survival assessment of the maximum rAAV dose in a CBE mouse model are presented. In P3, mice were treated with either an excipient or 8.8e9vg of rAAV-GBA1 via ICV delivery. Daily IP delivery of either PBS or 25 mg / kg of CBE was initiated in P8. At the end of the study, half of the mice were sacrificed one day after the final CBE dose in P36 (day 1), and the other half were sacrificed in P38 (day 3) after a 3-day CBE withdrawal. All treatment groups (excipient + PBS, n=8, rAAV-GBA1 + PBS (n=7), excipient + CBE (n=8), and variant + CBE (n=9)) were weighed daily (top left) and weights were analyzed in P36 (top right). Behavior was evaluated by total distance traveled in the open field at P23 (bottom left) and latency to fall on the rotorod at p24 (bottom right), and was assessed for each animal as the median across the three trials. Due to lethality, there were n=7 for the excipient + CBE group for the behavioral assay and n=8 for all other groups. The mean for all animals is shown. Error bars are SEM. *p<0.05, ***p<0.001, nominal p-values ​​for treatment groups by linear regression in CBE-treated animals. [Figure 11-3]Representative data on the survival assessment of the maximum rAAV dose in a CBE mouse model are presented. In P3, mice were treated with either an excipient or 8.8e9vg of rAAV-GBA1 via ICV delivery. Daily IP delivery of either PBS or 25 mg / kg of CBE was initiated in P8. At the end of the study, half of the mice were sacrificed one day after the final CBE dose in P36 (day 1), and the other half were sacrificed in P38 (day 3) after a 3-day CBE withdrawal. All treatment groups (excipient + PBS, n=8, rAAV-GBA1 + PBS (n=7), excipient + CBE (n=8), and variant + CBE (n=9)) were weighed daily (top left) and weights were analyzed in P36 (top right). Behavior was evaluated by total distance traveled in the open field at P23 (bottom left) and latency to fall on the rotorod at p24 (bottom right), and was assessed for each animal as the median across the three trials. Due to lethality, there were n=7 for the excipient + CBE group for the behavioral assay and n=8 for all other groups. The mean for all animals is shown. Error bars are SEM. *p<0.05, ***p<0.001, nominal p-values ​​for treatment groups by linear regression in CBE-treated animals. [Figure 12-1] Representative data for the biochemical evaluation of the maximum rAAV dose in the CBE mouse model are shown. GCase activity (top left), GluSph levels (top right), GluCer levels (bottom left), and vector genome (bottom right) were measured in the cortex of all treatment groups (excipient + PBS (n=8), variant + PBS (n=7), excipient + CBE (n=7), and variant + CBE (n=9)) before CBE withdrawal (day 1) or after withdrawal (day 3). In vivo distribution is shown as vector genome per 1 μg of genomic DNA. Means are shown. Error bars are SEM. (*) p<0.1, ** p<0.01, *** p<0.001, nominal p-values ​​of treatment groups by linear regression in CBE-treated animals, collection days and sex were corrected as covariates. [Figure 12-2]Representative data for the biochemical evaluation of the maximum rAAV dose in the CBE mouse model are shown. GCase activity (top left), GluSph levels (top right), GluCer levels (bottom left), and vector genome (bottom right) were measured in the cortex of all treatment groups (excipient + PBS (n=8), variant + PBS (n=7), excipient + CBE (n=7), and variant + CBE (n=9)) before CBE withdrawal (day 1) or after withdrawal (day 3). In vivo distribution is shown as vector genome per 1 μg of genomic DNA. Means are shown. Error bars are SEM. (*) p<0.1, ** p<0.01, *** p<0.001, nominal p-values ​​of treatment groups by linear regression in CBE-treated animals, collection days and sex were corrected as covariates. [Figure 13] Representative data on behavioral and biochemical correlations in CBE mouse models after administration of excipient + PBS, excipient + CBE, and variant + CBE treatment groups are presented. Across the treatment groups, rotorod performance was negatively correlated with GluCer accumulation (A, p=0.0012 by linear regression), and GluSph accumulation was negatively correlated with increased GCase activity (B, p=0.0086 by linear regression). [Figure 14-1] This report presents representative data on the in vivo distribution of variants in CBE mouse models. The presence of vector genomes was evaluated 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)). In vivo distribution is shown as vector genome per 1 μg of genomic DNA. The presence of vector genomes was quantified by quantitative PCR using a vector reference standard curve, and genomic DNA concentration was evaluated by A260 optical density measurement. Means are shown. Error bars represent SEM. *p<0.05, **p<0.01, ***p<0.001: Nominal p-values ​​of treatment groups by linear regression in CBE-treated animals, collection days, and sex were corrected as covariates. [Figure 14-2]This report presents representative data on the in vivo distribution of variants in CBE mouse models. The presence of vector genomes was evaluated 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)). In vivo distribution is shown as vector genome per 1 μg of genomic DNA. The presence of vector genomes was quantified by quantitative PCR using a vector reference standard curve, and genomic DNA concentration was evaluated by A260 optical density measurement. Means are shown. Error bars represent SEM. *p<0.05, **p<0.01, ***p<0.001: Nominal p-values ​​of treatment groups by linear regression in CBE-treated animals, collection days, and sex were corrected as covariates. [Figure 15-1] Representative data on the survival assessment of rAAV dose ranges in a CBE mouse model are presented. Mice received either an excipient or one of three different doses of rAAV-GBA1 via ICV delivery at P3: 3.2e9vg, 1.0e10vg, or 3.2e10vg. Daily IP treatment with 25 mg / kg of CBE was initiated at P8. Mice receiving either the excipient and CBE or the excipient and PBS were used as controls. All treatment groups were started with n=10 (5 males / 5 females) per group. All mice were sacrificed one day after the final CBE dose (P38-P40). All treatment groups were weighed daily, and their body weight was analyzed at P36. Mobility was assessed by latency to fall on the rotor rod at P24 and latency to cross the tapered beam at P30. Due to early lethality, the number of mice participating in the behavioral assay was as follows: n=10 for excipient + PBS, n=9 for excipient + CBE, n=6 for 3.2e9vg rAAV-GBA1 + CBE, n=10 for 1.0e10vg rAAV-GBA1 + CBE, and n=7 for 3.2e10vg rAAV-GBA1 + CBE. Means are shown. Error bars are SEM, and *p<0.05 and **p<0.01 are nominal p-values ​​from linear regression in the CBE treatment groups, with sex adjusted as a covariate. [Figure 15-2]Representative data on the survival assessment of rAAV dose ranges in a CBE mouse model are presented. Mice received either an excipient or one of three different doses of rAAV-GBA1 via ICV delivery at P3: 3.2e9vg, 1.0e10vg, or 3.2e10vg. Daily IP treatment with 25 mg / kg of CBE was initiated at P8. Mice receiving either the excipient and CBE or the excipient and PBS were used as controls. All treatment groups were started with n=10 (5 males / 5 females) per group. All mice were sacrificed one day after the final CBE dose (P38-P40). All treatment groups were weighed daily, and their body weight was analyzed at P36. Mobility was assessed by latency to fall on the rotor rod at P24 and latency to cross the tapered beam at P30. Due to early lethality, the number of mice participating in the behavioral assay was as follows: n=10 for excipient + PBS, n=9 for excipient + CBE, n=6 for 3.2e9vg rAAV-GBA1 + CBE, n=10 for 1.0e10vg rAAV-GBA1 + CBE, and n=7 for 3.2e10vg rAAV-GBA1 + CBE. Means are shown. Error bars are SEM, and *p<0.05 and **p<0.01 are nominal p-values ​​from linear regression in the CBE treatment groups, with sex adjusted as a covariate. [Figure 16-1]Representative data for the biochemical evaluation of rAAV dose ranges in CBE mouse models are presented. GCase activity, GluSph levels, GluCer levels, and vector genome were measured using the cortex of all treatment groups (excipient + PBS (n=10), excipient + CBE (n=9), and 3.2e9vg rAAV-GBA1 + CBE (n=6), 1.0e10vg rAAV-GBA1 + CBE (n=10), and 3.2e10vg rAAV-GBA1 + CBE (n=7)). GCase activity is expressed as ng of GCase per mg of total protein. GluSph and GluCer levels are expressed as pmol per mg of wet tissue weight. In vivo distribution is expressed as vector genome per 1 μg of genomic DNA. The presence of vector genome was quantified by quantitative PCR using a vector reference curve, and genomic DNA concentration was evaluated by A260 optical density measurement. The presence of vector genomes was also measured in the liver (E). Means are shown. Error bars are SEM. **p<0.01, ***p<0.001 are nominal p-values ​​from linear regression in the CBE-treated group, with sex corrected as a covariate. [Figure 16-2]Representative data for the biochemical evaluation of rAAV dose ranges in CBE mouse models are presented. GCase activity, GluSph levels, GluCer levels, and vector genome were measured using the cortex of all treatment groups (excipient + PBS (n=10), excipient + CBE (n=9), and 3.2e9vg rAAV-GBA1 + CBE (n=6), 1.0e10vg rAAV-GBA1 + CBE (n=10), and 3.2e10vg rAAV-GBA1 + CBE (n=7)). GCase activity is expressed as ng of GCase per mg of total protein. GluSph and GluCer levels are expressed as pmol per mg of wet tissue weight. In vivo distribution is expressed as vector genome per 1 μg of genomic DNA. The presence of vector genome was quantified by quantitative PCR using a vector reference curve, and genomic DNA concentration was evaluated by A260 optical density measurement. The presence of vector genomes was also measured in the liver (E). Means are shown. Error bars are SEM. **p<0.01, ***p<0.001 are nominal p-values ​​from linear regression in the CBE-treated group, with sex corrected as a covariate. [Figure 17-1] Representative data for tapered beam analysis at maximum dose rAAV-GBA1 in a genetic mouse model are presented. Motor performance of treatment groups (WT + excipients (n=5), 4L / PS-NA + excipients (n=6), and 4L / PS-NA + rAAV-GBA1 (n=5)) was assayed by beam walk 4 weeks after rAAV-GBA1 administration. Total slip and activity time are shown as the sum of 5 trials with different beams. Velocity and slip per velocity are shown as the average of 5 trials with different beams. Averages are shown. Error bars represent SEM. [Figure 17-2]Representative data for tapered beam analysis at maximum dose rAAV-GBA1 in a genetic mouse model are presented. Motor performance of treatment groups (WT + excipients (n=5), 4L / PS-NA + excipients (n=6), and 4L / PS-NA + rAAV-GBA1 (n=5)) was assayed by beam walk 4 weeks after rAAV-GBA1 administration. Total slip and activity time are shown as the sum of 5 trials with different beams. Velocity and slip per velocity are shown as the average of 5 trials with different beams. Averages are shown. Error bars represent SEM. [Figure 18] Representative data on the in vitro expression of rAAV constructs encoding the progranulin (PGRN) protein are shown. The left panel shows the standard curve for the progranulin (PGRN) ELISA assay. The bottom panel shows the dose-response of PGRN expression measured by ELISA assay in cell lysates of HEK293T cells transduced with rAAV. MOI = Multiplicity of Infection (vector genome per cell). [Figure 19-1] Representative data on the in vitro expression of rAAV constructs encoding GBA1 in combination with prosaposin (PSAP), SCARB2, and / or one or more inhibitory nucleic acids are shown. The data indicate that transfection of HEK293 cells with each construct resulted in overexpression of the target transgene in mock transfected cells. [Figure 19-2] Representative data on the in vitro expression of rAAV constructs encoding GBA1 in combination with prosaposin (PSAP), SCARB2, and / or one or more inhibitory nucleic acids are shown. The data indicate that transfection of HEK293 cells with each construct resulted in overexpression of the target transgene in mock transfected cells. [Figure 19-3]Representative data on the in vitro expression of rAAV constructs encoding GBA1 in combination with prosaposin (PSAP), SCARB2, and / or one or more inhibitory nucleic acids are shown. The data indicate that transfection of HEK293 cells with each construct resulted in overexpression of the target transgene in mock transfected cells. [Figure 20] This schematic diagram shows an rAAV vector (top panel) containing a "D" region located "outside" the ITR (for example, proximal to the end of the ITR relative to the transgene insertion or expression construct), and a wild-type rAAV vector having the ITR "inside" the vector (for example, proximal to the transgene insertion of the vector). [Figure 21] A schematic diagram showing one embodiment of a vector comprising an expression construct encoding interference RNA for GBA2 or a portion thereof, and α-Syn. [Figure 22] This is a schematic diagram showing one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof) and galactosylceramidase (e.g., GALC or a portion thereof). The expression of the Gcase and galactosylceramidase encoding sequences is separated by a T2A self-cleaving peptide sequence. [Figure 23] This is a schematic diagram showing one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof) and galactosylceramidase (e.g., GALC or a portion thereof). The expression of the Gcase and galactosylceramidase encoding sequences is separated by a T2A self-cleaving peptide sequence. [Figure 24] This is a schematic diagram showing one embodiment of a vector comprising an expression construct encoding interfering RNA for Gcase (e.g., GBA1 or a portion thereof), cathepsin B (e.g., CTSB or a portion thereof), and α-Syn. The expression of the Gcase and cathepsin B coding sequences is separated by a T2A self-cleaved peptide sequence. [Figure 25]This is a schematic diagram showing one embodiment of a vector comprising an expression construct encoding an interfering RNA for Gcase (e.g., GBA1 or a portion thereof), sphingomyelin phosphodiesterase 1 (e.g., SMPD1 or a portion thereof), and α-Syn. [Figure 26] This is a schematic diagram showing one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof) and galactosylceramidase (e.g., GALC or a portion thereof). The coding sequences for Gcase and galactosylceramidase are separated by an internal ribosome entry site (IRES). [Figure 27] This is a schematic diagram showing one embodiment of a vector comprising expression constructs encoding Gcase (e.g., GBA1 or a portion thereof) and cathepsin B (e.g., CTSB or a portion thereof). The expression of the Gcase and cathepsin B coding sequences is driven by separate promoters, respectively. [Figure 28] This is a schematic diagram showing one embodiment of a vector comprising an expression construct encoding interfering RNA for Gcase (e.g., GBA1 or a portion thereof), GCH1 (e.g., GCH1 or a portion thereof), and α-Syn. The coding sequences for Gcase and GCH1 are separated by a T2A self-cleaving peptide sequence. [Figure 29] This is a schematic diagram showing one embodiment of a vector comprising an expression construct encoding interfering RNA for Gcase (e.g., GBA1 or a portion thereof), RAB7L1 (e.g., RAB7L1 or a portion thereof), and α-Syn. The coding sequences for Gcase and RAB7L1 are separated by a T2A self-cleaved peptide sequence. [Figure 30] This is a schematic diagram showing one embodiment of a vector comprising expression constructs encoding interference RNA for Gcase (e.g., GBA1 or a portion thereof), GCH1 (e.g., GCH1 or a portion thereof), and α-Syn. Expression of the Gcase and GCH1 coding sequences is performed at the internal ribosome entry site (IRES). [Figure 31]This is a schematic diagram showing one embodiment of a vector comprising expression constructs encoding interfering RNAs for VPS35 (e.g., VPS35 or a portion thereof) and α-Syn and TMEM106B. [Figure 32] This is a schematic diagram showing one embodiment of a vector comprising an expression construct encoding interfering RNA for Gcase (e.g., GBA1 or a portion thereof), IL-34 (e.g., IL34 or a portion thereof), and α-Syn. The coding sequences for Gcase and IL-34 are separated by a T2A self-cleaving peptide sequence. [Figure 33] This is a schematic diagram showing one embodiment of a vector containing expression constructs encoding Gcase (e.g., GBA1 or a portion thereof) and IL-34 (e.g., IL34 or a portion thereof). The coding sequences of Gcase and IL-34 are separated by an internal ribosome entry site (IRES). [Figure 34] This is a schematic diagram showing one embodiment of a vector containing expression constructs encoding Gcase (e.g., GBA1 or a portion thereof) and TREM2 (e.g., TREM2 or a portion thereof). The expression of the Gcase and TREM2 coding sequences is driven by separate promoters, respectively. [Figure 35] This is a schematic diagram showing one embodiment of a vector containing expression constructs encoding Gcase (e.g., GBA1 or a portion thereof) and IL-34 (e.g., IL34 or a portion thereof). The expression of the Gcase and IL-34 coding sequences is driven by separate promoters, respectively. [Figure 36] Figures A and B show representative data for the overexpression of TREM2 and GBA1 in HEK293 cells compared to control transduced cells, as measured by qPCR and ELISA. Figure A shows data for TREM2 overexpression. Figure B shows data for GBA1 overexpression from the same construct. [Figure 37] Representative data demonstrating successful in vitro silencing of SNCAs using the GFP reporter assay (top panel) and the α-Syn assay (bottom panel) are shown. [Figure 38] Representative data demonstrating successful in vitro silencing of TMEM106B using the GFP reporter assay (top panel) and the α-Syn assay (bottom panel) are shown. [Figure 39] This is a schematic diagram showing one embodiment of a vector containing an expression construct encoding a PGRN. [Figure 40] This report presents data on the transduction of HEK293 cells using rAAVs with ITRs having wild-type (circular) or alternative (e.g., "outer," square) arrangements of the "D" sequence. rAAVs with "outer" arranged ITRs were able to transduce cells as efficiently as rAAVs with wild-type ITRs. [Figure 41] This is a schematic diagram showing one embodiment of a vector containing an expression construct that encodes Gcase (e.g., GBA1 or a portion thereof). [Figure 42] This is a schematic diagram showing one embodiment of a vector containing an expression construct that encodes Gcase (e.g., GBA1 or a portion thereof). [Figure 43] This is a schematic diagram showing one embodiment of a vector comprising an expression construct encoding interfering RNA for Gcase (e.g., GBA1 or a portion thereof) and α-Syn. [Figure 44] This is a schematic diagram showing one embodiment of a vector containing an expression construct encoding a PGRN. [Figure 45] This is a schematic diagram showing one embodiment of a vector containing an expression construct encoding a PGRN. [Figure 46] This is a schematic diagram showing one embodiment of a vector containing expression constructs encoding interfering RNAs for PGRN and the microtubule-associated protein tau (MAPT). [Figure 47] This is a schematic diagram showing one embodiment of a vector comprising an expression construct encoding interfering RNA for Gcase (e.g., GBA1 or a portion thereof) and α-Syn. [Figure 48] This is a schematic diagram showing one embodiment of a vector containing an expression construct that encodes PSAP. [Figure 49]This is a schematic diagram showing one embodiment of a vector containing an expression construct that encodes Gcase (e.g., GBA1 or a portion thereof). [Figure 50] This is a schematic diagram showing one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof) and galactosylceramidase (e.g., GALC or a portion thereof). [Figure 51] This is a schematic diagram showing one embodiment of a plasmid comprising an rAAV vector containing an expression construct encoding interfering RNA for Gcase (e.g., GBA1 or a portion thereof), prosaposin (e.g., PSAP or a portion thereof), and α-Syn. [Figure 52A] This study shows that neural stem cell (NSC) lines derived from iPSCs of patients with FTD-GRN mutations secreted less progranulin than NSC lines derived from healthy controls. Statistical analysis was performed using unpaired t-tests, with *=p<0.05, **=p<0.01, and ***=p<0.001. Data are presented as mean ± SEM. [Figure 52B] This report presents the results of dose-range PR006A transduction in FTD-GRN mutant neuronal cultures. NSCs were seeded at equal density and differentiated into neurons. On day 7, neurons were transduced for 72 hours with either the excipient or the indicated amount of PR006A. Secreted progranulin expression was measured from cell culture by ELISA and normalized to volume (n=3-4, mean ± SEM). The black dashed line represents the endogenous level of progranulin secreted from control neurons (excipient-treated). Secreted progranulin was undetectable in excipient-treated FTD-GRN neurons. Statistics were determined using ANOVA followed by Tukey HSD, and statistical comparisons of each state against excipient-treated control neurons are shown in graphs, *=p<0.05, ***=p<0.001. LLOQ = lower limit of quantification, MOI = multiplicity of infection. [Figure 52C]In FTD-GRN neuron cultures, PR006 treatment of neuron cultures rescued defective maturation of the major lysosomal protease, cathepsin D. NSCs were seeded at equal concentrations and differentiated into neurons. On day 7, neurons were transduced with an excipient or PR006A at an MOI of 5.3 × 10⁵ for 72 hours. Neurons were lysed, and the lysates were analyzed using a Protein Simple Western Jess system with an anti-cathepsin D (CTSD) primary antibody. Bands corresponding to both mature cathepsin D (matCTSD) and procathepsin D (proCTSD) were detected, and the area under the curve was quantified for each band and normalized to the internal total protein normalization signal. The matCTSD / proCTSD ratio was determined in FTD-GRN neurons treated with excipients or PR006A, and the y-axis shows the matCTSD / proCTSD ratio as a percentage of the ratio of control neurons (n=3, mean ± SEM) treated with excipients. Statistics were determined using unpaired t-tests, *=p<0.05. [Figure 52D] This study demonstrates that PR006A reduces TDP-43 pathology in FTD-GRN neuron cultures. NSCs were seeded at equal concentrations and differentiated into neurons. On day 7, neurons were transduced with excipients or PR006A at a MOI of 5.3 × 10⁵ and collected 21 days after transduction. Neurons were lysed, and the Triton-X insoluble protein fraction was isolated and analyzed using an anti-TDP-43 antibody (#12892-AP-1) in a Protein Simple Western Jess system. Bands corresponding to TDP-43 were detected, and the area under the curve was quantified for each band and normalized to the total protein concentration of the insoluble fraction. The Y-axis shows the amount of insoluble TDP-43 as a percentage of the excipient treatment level, separately normalized for each FTD-GRN cell line (n=3, mean ± SEM). Figure 52D shows that PR006 treatment reduced insoluble TDP-43, a characteristic of FTD-GRN pathology, in FTD-GRN neuronal cell cultures. Statistical analysis was performed using unpaired t-tests, with **=p<0.01 and ***=p<0.001. [Figure 52E]This study shows that NSC strains derived from iPSCs of patients with FTD-GRN mutations expressed less progranulin than NSC strains derived from healthy controls. Statistical analysis was performed using unpaired t-tests, with *=p<0.05, **=p<0.01, and ***=p<0.001. Data are presented as mean ± SEM. [Figure 52F] This study demonstrates that PR006A reduces TDP-43 pathology in FTD-GRN neuron cultures. NSCs were seeded at equal concentrations and differentiated into neurons. On day 7, neurons were transduced with an excipient or PR006A at a MOI of 5.3 × 10⁵ and collected 21 days after transduction. This is a quantification of nuclear TDP-43 signaling from immunofluorescence images of iPSC-derived neurons treated with PR006A. The TDP-43 signal intensity per nucleus in FTD-GRN neurons treated with an excipient or PR006A was determined, and the y-axis shows the TDP-43 signal intensity per nucleus as a percentage of the TDP-43 signal intensity per nucleus in control neurons treated with the excipient (n=145–306 cells, mean ± SEM). TDP-43 was measured using an anti-TDP-43 antibody (#12892-AP-1), and the nuclear region was determined by DAPI staining. Figure 52F shows that PR006 treatment increased nuclear TDP-43 expression levels in FTD-GRN neuron cultures to levels close to wild-type control levels. Statistical analysis was performed using unpaired t-tests, with **=p<0.01 and ***=p<0.001. [Figure 52G] These images show a series of images demonstrating the successful differentiation of human FTD-GRN-derived neural stem cell (NSC) lines and human control cell lines into neuronal cell cultures. Control and FTD-GRN NSC lines (FTD-GRN#1 and FTD-GRN#2) were differentiated into neurons after 7 days, as indicated by immunofluorescence staining for cell morphology and neuronal markers (NeuN [red], MAP2 or Tau [green] labeled on the left). The nuclei were stained using DAPI (blue). [Figure 53-1]A-C are a series of bar graphs showing the results of experiments analyzing the in vivo distribution and progranulin expression in the CNS in a PR006A FTD-GRN mouse model study at adult dose ranges. Four-month-old Grn KO mice were administered PR006A or an excipient via ICV. They were sacrificed three months after treatment with either the excipient (red) or PR006A (blue) at doses of 1.1 × 10⁹ vg (2.7 × 10⁹ vg / g brain), 1.1 × 10¹⁰ vg (2.7 × 10¹⁰ vg / g brain), or 1.1 × 10¹¹ vg (2.7 × 10¹¹ vg / g brain) for biochemical endpoints in the CNS. A: The presence of the vector genome was evaluated in the cerebral cortex and spinal cord, and the in vivo distribution is shown as vector genome per μg of gDNA on a logarithmic scale (n=8-10 / group, mean ± SEM). The presence of vector genomes was quantified by qPCR using a vector reference standard curve. The dashed line (50 vector genomes / μg gDNA) represents the threshold for positive vector presence. B: GRN RNA expression encoding PR006A was evaluated by quantitative RT-PCR (qRT-PCR) in the cerebral cortex (n=8~10 / group, mean ± SEM). The number of GRN copies (specific to the inventors' codon-optimized PR006A sequence) is normalized to 1 μg of total RNA and shown on a logarithmic scale. C: Progranulin protein levels were measured using human-specific progranulin ELISA in the 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 shown in dashed gray. For tissue ELISA assays, the LLOQ (ng / mg) value is determined by dividing the assay LLOQ (ng / mL) by the mean total protein concentration from all samples. A single line corresponding to the color of the legend for the treatment group on the x-axis without error bars indicates that all animals in that group were 0. Statistical analysis was performed using ANOVA, followed by Dunnett's test, *=p<0.05, **=p<0.01, ***=p<0.001, to compare with the excipient-treated Grn KO mouse group. vg=vector genome, LLOQ=lower limit of quantification, SC=spinal cord. [Figure 53-2]D-E are a series of bar graphs showing the results of experiments analyzing the in vivo distribution of peripheral tissues and progranulin expression in a PR006A FTD-GRN mouse model study at adult dose ranges. Four-month-old Grn KO mice were administered PR006A or excipients via ICV. They were sacrificed three months after treatment with either the excipient (red) or PR006A (blue) at doses of 1.1 × 10⁹ vg (2.7 × 10⁹ vg / g brain), 1.1 × 10¹⁰ vg (2.7 × 10¹⁰ vg / g brain), or 1.1 × 10¹¹ vg (2.7 × 10¹¹ vg / g brain) for biochemical endpoints in the liver, heart, lungs, kidneys, spleen, and gonads. D: The presence of the vector genome was assessed, and the in vivo distribution is shown as vector genome per μg of gDNA on a logarithmic scale (n=8-10 / group, mean ± SEM). The presence of vector genomes was quantified by qPCR using a vector reference standard curve. The dashed line (50 vector genomes / μg gDNA) represents the threshold for positive vector presence. E: Progranulin protein levels were measured using ELISA (n=8-10 / group, mean ± SEM). Tissue progranulin levels were normalized against total protein concentration. Simple lines corresponding to the legend color of the treatment group on the x-axis without error bars indicate that all animals in that group had a value of 0. Statistical analysis was performed using ANOVA, followed by Dunnett's test, *=p<0.05, ***=p<0.001, to compare with the excipient-treated Grn KO mouse group. vg = vector genome. [Figure 53-3]F is a bar graph showing the results of an experiment analyzing progranulin levels in plasma in a PR006A FTD-GRN mouse model study within the adult dose range. Four-month-old Grn KO mice were administered PR006A or an excipient via ICV. They were sacrificed three months after treatment with either an excipient (red) or PR006A (blue) at doses of 1.1 × 10⁹ vg (2.7 × 10⁹ vg / g brain), 1.1 × 10¹⁰ vg (2.7 × 10¹⁰ vg / g brain), or 1.1 × 10¹¹ vg (2.7 × 10¹¹ vg / g brain) for biochemical endpoints in plasma. Progranulin protein levels were measured using human-specific progranulin ELISA in plasma (n=8-10 / group, mean ± SEM). Plasma levels are shown on a logarithmic scale. The lower limit of quantification (LLOQ) is indicated by a dashed gray line. To compare with the excipient-treated Grn KO mouse group, statistical analysis was performed using ANOVA followed by Dunnett's test, with *=p<0.05, **=p<0.01, and ***=p<0.001. LLOQ = lower limit of quantification. vg = vector genome. [Figure 53-4]G-H are a series of bar graphs showing experimental results demonstrating a reduction in lysosomal neuropathological deficiencies in a PR006A FTD-GRN adult mouse model study within the adult dose range. Four-month-old Grn KO mice were administered PR006A or an excipient via ICV. They were sacrificed three months after treatment with either the excipient (red) or PR006A (blue) at doses of 1.1 × 10⁹ vg (2.7 × 10⁹ vg / g brain), 1.1 × 10¹⁰ vg (2.7 × 10¹⁰ vg / g brain), or 1.1 × 10¹¹ vg (2.7 × 10¹¹ vg / g brain) for analysis. Lipofuscinosis was analyzed by two independent methods: (1) scoring of H&E-stained brain sections by a pathologist, and (2) quantification of lipofuscin autofluorescence from IHC sections. G: Lipofuscin accumulation (autofluorescent lipofuscin granules) was semi-quantitatively scored in H&E-stained sections in different brain regions by blinded, qualified pathologists according to the following grading scheme: 0 = No lipofuscin observed. 1 = Very small lipofuscin granules scattered throughout the region (<2 μm). 2 = Increased density of small granule accumulation and / or development of larger granules (>2-3 μm). 3 = Multifocal region with high density of lipofuscin granules visible from low objective lens power. 4 = Extensive lipofuscin accumulation. Lipofuscin severity scores are shown for the cerebral cortex, hippocampus, and thalamic / hypothalamic brain regions (n=8-10 / group). H: IHC analysis of ubiquitin was performed and quantified in the cerebral cortex, hippocampus, and thalamus. Size of immunoreactive objects exceeding the threshold (size of immunoreactive objects [μm2] is shown for ubiquitin (n=8-10 / group, mean ± SEM). Statistics were determined by ANOVA followed by Dunnett's test to compare with excipient-treated Grn KO mouse groups, *=p<0.05, **=p<0.01, ***=p<0.001. vg=vector genome, WT=wild type). [Figure 53-5]I-K are a series of bar graphs showing experimental results demonstrating the reduction of neuroinflammatory markers in a PR006A FTD-GRN mouse model study within the adult dose range. Four-month-old Grn KO mice were administered PR006A or an excipient via ICV. They were sacrificed three months after treatment with either the excipient (red) or PR006A (blue) at doses of 1.1 × 10⁹ vg (2.7 × 10⁹ vg / g brain), 1.1 × 10¹⁰ vg (2.7 × 10¹⁰ vg / g brain), or 1.1 × 10¹¹ vg (2.7 × 10¹¹ vg / g brain) for analysis. I: Gene expression (mRNA level) of Tnf and Cd68 was measured by qRT-PCR in the somatosensory cortex (mean ± SEM, n=8-10 / group). Gene expression was normalized relative to the housekeeping gene Ppib. J-K: IHC analysis of Iba1(J) and GFAP(K) was performed and quantified in fixed brain sections of the cerebral cortex, hippocampus, and thalamus. The percentage of the region of interest covered by substances exceeding the threshold (immune reaction area [%]) is shown (mean ± SEM, n=8-10 / group). Statistics were determined using Dunnett's adjusted ANOVA, *=p<0.05, ***=p<0.001, compared to the excipient-treated Grn KO mouse group. vg=vector genome, WT=wild-type. [Figure 53-6]L~N is a series of bar graphs showing experimental results illustrating the reduction in lysosomal and immune pathway gene expression in a PR006A FTD-GRN mouse model study within the adult dose range. Four-month-old Grn KO mice were administered PR006A or an excipient via ICV. They were sacrificed three months after treatment with either an excipient (red) or PR006A (blue) at doses of 1.1 × 10⁹ vg (2.7 × 10⁹ vg / g brain), 1.1 × 10¹⁰ vg (2.7 × 10¹⁰ vg / g brain), or 1.1 × 10¹¹ vg (2.7 × 10¹¹ vg / g brain) for analysis. RNA sequencing was performed on cerebral cortical samples from ICV-treated Grn KO mice and age-matched WT C57BL / 6J mice (gray). We compared mRNA expression levels of previously published, dysregulated gene features in excipient-treated Grn KO mice with those of wild-type mice using gene set variation analysis (GSVA). The data shown are GSVA activity scores for curated gene sets from two published studies and one Hallmark pathway. L: cellular component: vacuole (GO:0005773), M: lysosome, and N: complement system (Hallmark pathway) (median ± range, n=8-10 / group). Statistical analysis was performed using ANOVA followed by Dunnett's test, ***=p<0.001, to compare with the excipient-treated Grn KO mouse group while controlling for family-wise type I error rates. GSVA = gene set variation analysis, vg = vector genome, WT = wild-type. [Figure 54A] This is a series of bar graphs showing the results of experiments analyzing the in vivo distribution of the PR006A transgene quantified by qPCR. Transgene levels were analyzed using qPCR in NHP 182 days after ICM injection of either an excipient, a low dose of PR006A (6.5 × 10⁹ vg / g brain), or a high dose of PR006A (6.5 × 10¹⁰ vg / g brain). Each bar represents the mean ± SEM of 3 animals per group, and the yellow line indicates the lower limit of quantification at 50 vg / μg of DNA. [Figure 54B]This is a series of bar graphs showing the results of an experiment analyzing the levels of anti-drug antibodies against human progranulin. Antibodies against progranulin are shown in NHP serum and CSF samples at 29 and 182 days after treatment with either an excipient, low-dose PR006A (6.5 × 10⁹ vg / g brain), or high-dose PR006A (6.5 × 10¹⁰ vg / g brain). Data represent mean ± SEM levels. [Figure 54C] This is a series of bar graphs showing the results of an experiment analyzing the expression of the PR006A transgene (GRN). GRN expression levels were determined using RT-qPCR in the NHP cortex, hippocampus, and ventral midbrain collected on day 183. Data are shown as mean ± SEM. [Figure 54D] This bar graph shows the results of an experiment analyzing progranulin levels in CSF quantified by the Simple Western (JESS) platform. Progranulin levels were determined in NHP CSF samples collected on day 183 and analyzed using Simple Western (JESS) analysis. NHP-derived CSF samples were treated with excipients, low-dose PR006A (6.5 × 10⁹ vg / g brain weight) or high-dose PR006A (6.5 × 10¹⁰ vg / g brain weight). The data presented are mean ± SEM, p-value: *p<0.05, and based on one-way dose-dependent response analysis using the William trend test. [Figure 55] This graph shows the selectivity and specificity results of the automated Western JESS assay. Progranulin protein levels in CSF samples from FTD patients were detected at 58 kDa by Jess. Group (A): Heterozygous FTD patients, and Groups (B) and (C): Familial non-carriers or normal individuals. Data are expressed as mean ± mean standard error (SEM). SEM values ​​are displayed as vertical error bars. [Figure 56]This graph shows progranulin levels in CSF samples from FTD patients detected by ELISA. Group (A): Heterozygous FTD patients, and Groups (B) and (C): Familial non-carriers or normal individuals. Data are expressed as mean ± mean standard error (SEM). SEM values ​​are displayed as vertical error bars. [Figure 57] These are gel images of each CSF sample, performed in duplex mode on the Jess automated Western platform. Samples were analyzed at a 4-fold dilution using the primary antibody Adipogen PG-359-7. The first lane is the molecular weight criterion, and the right side is the identification of the bands used to calculate the immunoreactivity reported in Example 14. [Figure 58] A–B are a series of plots showing the measurement of human PGRN expression levels. Human PGRN expression levels were determined in non-human primate (NHP) CSF samples collected at day 180 using Simple Western® (Jess) analysis. CSF from NHPs treated with excipients ("Excipients"), low-dose PR006A (6.5 × 10⁹ vg / g brain weight, "Low"), or high-dose PR006 (6.5 × 10¹⁰ vg / g brain weight, "High") were analyzed. Data are presented as mean immunoreactivity peak area (A), or magnification change relative to animals treated with excipients (B). Each dot represents a single CSF sample from one NHP (mean of technical replication), and the boxes represent the mean + / - standard error of three individual NHPs. [Figure 59-1]A-C are a series of bar graphs showing the results of experiments analyzing the in vivo distribution and progranulin expression in the CNS of an aged FTD-GRN mouse model after PR006A treatment. Tissue samples were collected from 18-month-old Grn KO mice two months after receiving ICV with either the excipient (red) or 9.7 × 10¹⁰ vg (2.4 × 10¹¹ vg / g brain) of PR006A (blue). A: The presence of vector genomes in the cerebral cortex and spinal cord was evaluated (mean ± SEM, n=4 / group). In vivo distribution is shown as vector genomes per 1 μg gDNA on a logarithmic scale. The presence of vector genomes was quantified by qPCR using a vector reference standard curve. The dashed line (50 vector genomes / μg gDNA) represents the threshold for positive vector presence. B-C: Progranulin protein levels were measured using ELISA in CNS tissues (brain and spinal cord (B)) and CSF (C) (mean ± SEM, n=4 / group). Tissue progranulin levels were normalized to total protein concentration, and progranulin CSF levels were normalized to fluid volume. The lower limit of quantification (LLOQ) is shown in dashed gray. For tissue ELISA assays, the LLOQ (ng / mg) value was determined by dividing the assay LLOQ (ng / mL) by the mean total protein concentration from all samples. A simple red line on the x-axis without error bars indicates that all animals in that group had a value of 0. Statistical analysis was performed using Kruskal-Wallis, *=p<0.05, **=p<0.01, ***=p<0.001. vg=vector genome, LLOQ=lower limit of quantification, SC=spinal cord. [Figure 59-2]D–E are a series of bar graphs and images showing experimental results demonstrating a reduction in lysosomal neuropathological defects in an aged FTD-GRN mouse model after PR006A treatment. Tissue samples were collected from 18-month-old Grn KO mice two months after ICV treatment with either the excipient (red) or PR006A (blue) at 9.7 × 10¹⁰ vg (2.4 × 10¹¹ vg / g brain). Lipofuscinosis was analyzed by pathologist scoring of H&E-stained brain sections. D: Representative lipofuscin image from the thalamic / hypothalamic region of brain sections. White arrows indicate examples of lipofuscin accumulation. Provides an overview of lipofuscin severity scores in the cerebral cortex, hippocampus, and thalamus / hypothalamus of H&E-stained slides derived from brain sections, evaluated for autofluorescent lipofuscin granules. Lipofuscin accumulation was semi-quantitatively scored by blinded, qualified pathologists according to the following grading scheme: 0 = No lipofuscin observed. 1 = Very small granules of lipofuscin scattered throughout the region (<2 μm). 2 = Increased density of accumulation of small granules and / or development of larger granules (>2-3 μm). 3 = Multifocal region with high density of lipofuscin granules visible from low objective lens power. 4 = Extensive lipofuscin accumulation. E: IHC analysis of ubiquitin (n=4 / group) was performed and quantified in the cerebral cortex, hippocampus, and thalamus. Positive cell density (cells / mm2) for each region is shown (mean ± SEM). Statistics were determined using t-tests, *=p<0.05, **=p<0.01. vg = Vector genome. [Figure 59-3]D–E are a series of bar graphs and images showing experimental results demonstrating a reduction in lysosomal neuropathological defects in an aged FTD-GRN mouse model after PR006A treatment. Tissue samples were collected from 18-month-old Grn KO mice two months after ICV treatment with either the excipient (red) or PR006A (blue) at 9.7 × 10¹⁰ vg (2.4 × 10¹¹ vg / g brain). Lipofuscinosis was analyzed by pathologist scoring of H&E-stained brain sections. D: Representative lipofuscin image from the thalamic / hypothalamic region of brain sections. White arrows indicate examples of lipofuscin accumulation. Provides an overview of lipofuscin severity scores in the cerebral cortex, hippocampus, and thalamus / hypothalamus of H&E-stained slides derived from brain sections, evaluated for autofluorescent lipofuscin granules. Lipofuscin accumulation was semi-quantitatively scored by blinded, qualified pathologists according to the following grading scheme: 0 = No lipofuscin observed. 1 = Very small granules of lipofuscin scattered throughout the region (<2 μm). 2 = Increased density of accumulation of small granules and / or development of larger granules (>2-3 μm). 3 = Multifocal region with high density of lipofuscin granules visible from low objective lens power. 4 = Extensive lipofuscin accumulation. E: IHC analysis of ubiquitin (n=4 / group) was performed and quantified in the cerebral cortex, hippocampus, and thalamus. Positive cell density (cells / mm2) for each region is shown (mean ± SEM). Statistics were determined using t-tests, *=p<0.05, **=p<0.01. vg = Vector genome. F-I are a series of bar graphs showing experimental results demonstrating the reduction of neuroinflammatory markers in an aged FTD-GRN mouse model after PR006A treatment. Tissue samples were collected from 18-month-old Grn KO mice two months after ICV treatment with either the excipient (red) or 9.7 × 10¹⁰ vg (2.4 × 10¹¹ vg / g brain) of PR006A (blue). F: Gene expression of Tnf and Cd68 was measured by qRT-PCR in the somatosensory cortex (mean ± SEM, n=4 / group). Gene expression was normalized relative to the housekeeping gene Ppib.(G) Protein expression of the pro-inflammatory cytokine TNFα was measured in the cerebral cortex using the Mesoscale Discovery mouse pro-inflammatory cytokine assay (mean ± SEM, n=4 / group). The cerebral cortex was homogenized, and protein expression levels were normalized to the total protein concentration of tissue lysates. H~I: IHC analysis of Iba1(H) and GFAP(I) was performed and quantified in fixed brain sections. Edits of positive cell density (cells / mm2) from the three brain regions analyzed (cerebral cortex, hippocampus, and thalamus) are shown (mean ± SEM, n=3~4 / group). Statistical analysis was performed using the t-test, *=p<0.05. vg = vector genome. [Figure 59-4] F-I are a series of bar graphs showing experimental results demonstrating the reduction of neuroinflammatory markers in an aged FTD-GRN mouse model after PR006A treatment. Tissue samples were collected from 18-month-old Grn KO mice two months after ICV treatment with either the excipient (red) or 9.7 × 10¹⁰ vg (2.4 × 10¹¹ vg / g brain) of PR006A (blue). F: Gene expression of Tnf and Cd68 was measured by qRT-PCR in the somatosensory cortex (mean ± SEM, n=4 / group). Gene expression was normalized to the housekeeping gene Ppib. (G) Protein expression of the pro-inflammatory cytokine TNFα was measured in the cerebral cortex using the Mesoscale Discovery mouse pro-inflammatory cytokine assay (mean ± SEM, n=4 / group). The cerebral cortex was homogenized and protein expression levels were normalized to the total protein concentration of tissue lysates. H~I: IHC analysis of Iba1(H) and GFAP(I) was performed and quantified in fixed brain sections. Edits of positive cell density (cells / mm2) from the three analyzed brain regions (cerebral cortex, hippocampus, and thalamus) are shown (mean ± SEM, n=3~4 / group). Statistical analysis was performed using the t-test, *=p<0.05. vg = vector genome. [Figure 60]This graph shows the dose-response curve of 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 using an ELISA assay. [Figure 61] This is a diagram of the design for the maximum dose of PR006A in an aged FTD-GRN mouse model. 10 μL of excipient (control) or a dose of PR006A at 9.7 × 10¹⁰ vg (2.4 × 10¹¹ vg / g brain) was delivered by ICV injection to Grn KO mice in the following two cohorts: (1) 16 months of age at injection (n=4-5 / group, PRV-2018-027) and (2) 14 months of age at injection (n=1 / excipient-treated group, n=3 / PR006A-treated group, PRV-2019-002). Animals were sacrificed 2 months after injection. CNS and peripheral tissues were collected and analyzed for PR006A in vivo distribution (qPCR), progranulin protein expression (ELISA), and histopathology (H&E). The expression of pro-inflammatory markers, lipofuscin accumulation, and ubiquitin accumulation were evaluated in the brain. [Figure 62]Bar graphs A and B show the results of peripheral tissue biodistribution and progranulin expression in an aged FTD-GRN mouse model after PR006A treatment. Tissue samples were collected from 18-month-old Grn KO mice two months after ICV treatment with either excipient (red) or 9.7 × 10¹⁰ vg (2.4 × 10¹¹ vg / g brain) of PR006A (blue). A: The presence of vector genomes was evaluated in the liver, heart, lungs, kidneys, spleen, and gonads (mean ± SEM, n=4 / group). Biodistribution is shown as vector genomes per μg of gDNA on a logarithmic scale. The presence of vector genomes was quantified by qPCR using a vector reference standard. B: Progranulin protein levels were measured using ELISA (mean ± SEM, n=4 / group). Tissue progranulin levels were normalized relative to total protein concentration. A simple red line on the x-axis without error bars indicates that all animals in that group had a level of 0. Statistical analysis was performed using the Kruskal-Wallis protocol, with *=p<0.05, **=p<0.01, and ***=p<0.001. vg = vector genome. [Figure 63] This is a diagram of the design for a study on the dose range of PR006A in an adult FTD-GRN mouse model, in which PR006A was delivered by ICV injection to 4-month-old Grn KO mice (n=10 / group) at doses of 10 μl excipient (control) or 1.1 × 10⁹ vg (2.7 × 10⁹ vg / g brain), 1.1 × 10¹⁰ vg (2.7 × 10¹⁰ vg / g brain), or 1.1 × 10¹¹ vg (2.7 × 10¹¹ vg / g brain) of PR006A. The animals were sacrificed 3 months after injection when the mice were 7 months old. CNS and peripheral tissues were collected and analyzed for in vivo distribution of PR006A (qPCR), progranulin protein expression (ELISA), and histopathology (H&E). We evaluated the expression of pro-inflammatory markers, lipofuscin accumulation, ubiquitin accumulation, and overall gene expression changes in the brain. [Figure 64]This is a schematic diagram showing one embodiment of a recombinant adeno-associated virus vector (PR006A) containing an expression construct encoding human progranulin. "bp" refers to a "base pair". "kan" refers to the gene that confers resistance to kanamycin. "GRN" refers to "progranulin". "ITR" refers to the adeno-associated virus inverted terminal repeat sequence. "TRY" refers to the sequence containing three transcriptional regulatory activation sites, namely TATA, RBS, and YY1. "CBAp" refers to the chicken β-actin promoter. "CMVe" refers to the cytomegalovirus enhancer. "WPRE" refers to the woodchuck hepatitis virus post-transcriptional regulator. "bGH" refers to the bovine growth hormone polyA signaling tail. "int" refers to an intron. The double-stranded nucleotide sequence of PR006A is provided in SEQ ID NOs. 90 and 91. [Modes for carrying out the invention]

[0014] This disclosure is in part based on compositions and methods for the expression of a combination of specific gene products (e.g., gene products associated with CNS diseases) in a subject. The gene products may be proteins, protein fragments (e.g., parts), interfering nucleic acids that inhibit CNS disease-related genes, etc. In some embodiments, the gene product is a protein or protein fragment encoded by a CNS disease-related gene. In some embodiments, the gene product is an interfering nucleic acid (e.g., shRNA, siRNA, miRNA, amiRNA, etc.) that inhibits CNS disease-related genes.

[0015] CNS disease-related genes refer to genes that encode gene products genetically, biochemically, or functionally associated with CNS diseases such as FTD (frontotemporal dementia) or PD (Parkinson's disease). For example, individuals with a pathogenic mutation in the GRN gene (encoding the protein PGRN) have an increased risk of developing FTD compared to individuals without a mutation in GRN. Similarly, individuals with a mutation in the GBA1 gene (encoding the protein Gcase) have been observed to have an increased risk of developing PD compared to individuals without a mutation in GBA1. In another example, PD is associated with the accumulation of protein aggregates containing the α-synuclein (α-Syn) protein, and therefore SNCA (encoding α-Syn) is a PD-related gene. In some embodiments, the expression cassettes described herein encode CNS disease-related genes (or their coding sequences) in wild-type or non-mutant forms. Examples of CNS disease-related genes are listed in Table 1. Table 1: Examples of CNS disease-related genes [Table 1-1] [Table 1-2] [Table 1-3]

[0016] In addition to patients with Gaucher disease (mutations in both chromosomal alleles of the GBA1 gene), patients with mutations in only one allele of GBA1 have a significantly increased risk of Parkinson's disease (PD). The severity of PD symptoms, including difficulty walking, resting tremor, rigidity, and frequent depression, sleep disturbances, and cognitive decline, correlates with the degree of enzyme activity reduction. Therefore, Gaucher disease patients have the most severe course, while patients with a single mild mutation in GBA1 typically have a more benign course. Mutation carriers are also at higher risk of other PD-related disorders, including Lewy body dementia, characterized by executive dysfunction, psychosis, and PD-like movement disorders, as well as multiple system atrophy, which has characteristic movement and cognitive impairments. There are no therapies to alter the inevitable course of these disorders.

[0017] Defects in enzymes such as Gcase (e.g., the gene product of the GBA1 gene), as well as common variants in many genes involved in lysosomal function or the transport of macromolecules to lysosomes (e.g., lysosomal membrane protein 1 (LIMP), also known as SCARB2), have been associated with an increased risk of PD and / or Gaucher disease (e.g., neuropathic Gaucher diseases such as Gaucher disease type 2 or Gaucher disease type 3). This disclosure is based in part on expression constructs (e.g., vectors) encoding one or more genes, e.g., Gcase, GBA2, prosaposin, progranulin (PGRN), LIMP2, GALC, CTSB, SMPD1, GCH1, RAB7, VPS35, IL-34, TREM2, TMEM106B, or any combination of the foregoing (or parts thereof) associated with central nervous system (CNS) diseases, e.g., Gaucher disease, PD, etc. In some embodiments, the combinations of gene products described herein, when expressed in a subject, act together (e.g., synergistically) to reduce one or more signs and symptoms of CNS disease.

[0018] Accordingly, in some embodiments, the disclosure provides isolated nucleic acids comprising an expression construct encoding Gcase (e.g., the gene product of the GBA1 gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized (e.g., codon-optimized for expression in mammalian cells, e.g., human cells) Gcase coding sequence. In some embodiments, the nucleic acid sequence encoding Gcase encodes a protein comprising the amino acid sequence described in SEQ ID NO: 14 (e.g., described in NCBI reference sequence NP_000148.2). In some embodiments, the isolated nucleic acid comprises the sequence described in SEQ ID NO: 15. In some embodiments, the expression construct comprises an adeno-associated virus (AAV) inverted terminal repeat (ITR), e.g., an AAV ITR adjacent to the nucleic acid sequence encoding the Gcase protein.

[0019] In some embodiments, the disclosure provides isolated nucleic acids comprising an expression construct encoding a prosaposin (e.g., the gene product of the PSAP gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized (e.g., codon-optimized for expression in mammalian cells, e.g., human cells) prosaposin-coding sequence. In some embodiments, the nucleic acid sequence encoding the prosaposin encodes a protein comprising the amino acid sequence described in SEQ ID NO: 16 (e.g., described in NCBI reference sequence NP_002769.1). In some embodiments, the isolated nucleic acid comprises the sequence described in SEQ ID NO: 17. In some embodiments, the expression construct comprises an adeno-associated virus (AAV) inverted terminal repeat (ITR), e.g., an AAV ITR adjacent to the nucleic acid sequence encoding the prosaposin protein.

[0020] In some embodiments, the disclosure provides isolated nucleic acids comprising an expression construct encoding LIMP2 / SCARB2 (e.g., the gene product of the SCARB2 gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized (e.g., codon-optimized for expression in mammalian cells, e.g., human cells) SCARB2 coding sequence. In some embodiments, the nucleic acid sequence encoding LIMP2 / SCARB2 encodes a protein comprising the amino acid sequence described in SEQ ID NO: 18 (e.g., described in NCBI reference sequence NP_005497.1). In some embodiments, the isolated nucleic acid comprises the sequence described in SEQ ID NO: 29. In some embodiments, the expression construct comprises an adeno-associated virus (AAV) inverted terminal repeat (ITR), e.g., an AAV ITR adjacent to the nucleic acid sequence encoding the SCARB2 protein.

[0021] In some embodiments, the disclosure provides isolated nucleic acids comprising an expression construct encoding a GBA2 protein (e.g., the gene product of the GBA2 gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized (e.g., codon-optimized for expression in mammalian cells, e.g., human cells) GBA2 coding sequence. In some embodiments, the nucleic acid sequence encoding GBA2 encodes a protein comprising the amino acid sequence described in SEQ ID NO: 30 (e.g., described in NCBI reference sequence NP_065995.1). In some embodiments, the isolated nucleic acid comprises the sequence described in SEQ ID NO: 31. In some embodiments, the expression construct comprises an adeno-associated virus (AAV) inverted terminal repeat (ITR), e.g., an AAV ITR adjacent to the nucleic acid sequence encoding the GBA2 protein.

[0022] In some embodiments, the disclosure provides isolated nucleic acids comprising an expression construct encoding a GALC protein (e.g., the gene product of a GALC gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized (e.g., codon-optimized for expression in mammalian cells, e.g., human cells) GALC coding sequence. In some embodiments, the nucleic acid sequence encoding GALC encodes a protein comprising the amino acid sequence described in SEQ ID NO: 33 (e.g., described in NCBI reference sequence NP_000144.2). In some embodiments, the isolated nucleic acid comprises the sequence described in SEQ ID NO: 34. In some embodiments, the expression construct comprises an adeno-associated virus (AAV) inverted terminal repeat (ITR), e.g., an AAV ITR adjacent to the nucleic acid sequence encoding the GALC protein.

[0023] In some embodiments, the disclosure provides isolated nucleic acids comprising an expression construct encoding a CTSB protein (e.g., the gene product of the CTSB gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized (e.g., codon-optimized for expression in mammalian cells, e.g., human cells) CTSB coding sequence. In some embodiments, the nucleic acid sequence encoding CTSB encodes a protein comprising the amino acid sequence described in SEQ ID NO: 35 (e.g., described in NCBI reference sequence NP_001899.1). In some embodiments, the isolated nucleic acid comprises the sequence described in SEQ ID NO: 36. In some embodiments, the expression construct comprises an adeno-associated virus (AAV) inverted terminal repeat (ITR), e.g., an AAV ITR adjacent to the nucleic acid sequence encoding the CTSB protein.

[0024] In some embodiments, the disclosure provides isolated nucleic acids comprising an expression construct encoding the SMPD1 protein (e.g., the gene product of the SMPD1 gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized (e.g., codon-optimized for expression in mammalian cells, e.g., human cells) SMPD1 coding sequence. In some embodiments, the nucleic acid sequence encoding SMPD1 encodes a protein comprising the amino acid sequence described in SEQ ID NO: 37 (e.g., described in NCBI reference sequence NP_000534.3). In some embodiments, the isolated nucleic acid comprises the sequence described in SEQ ID NO: 38. In some embodiments, the expression construct comprises an adeno-associated virus (AAV) inverted terminal repeat (ITR), e.g., an AAV ITR adjacent to the nucleic acid sequence encoding the SMPD1 protein.

[0025] In some embodiments, the disclosure provides isolated nucleic acids comprising an expression construct encoding a GCH1 protein (e.g., the gene product of the GCH1 gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized (e.g., codon-optimized for expression in mammalian cells, e.g., human cells) GCH1 coding sequence. In some embodiments, the nucleic acid sequence encoding GCH1 encodes a protein comprising the amino acid sequence described in SEQ ID NO: 45 (e.g., described in NCBI reference sequence NP_000534.3). In some embodiments, the isolated nucleic acid comprises the sequence described in SEQ ID NO: 46. In some embodiments, the expression construct comprises an adeno-associated virus (AAV) inverted terminal repeat (ITR), e.g., an AAV ITR adjacent to the nucleic acid sequence encoding the GCH1 protein.

[0026] In some embodiments, the disclosure provides isolated nucleic acids comprising an expression construct encoding the RAB7L protein (e.g., the gene product of the RAB7L gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized (e.g., codon-optimized for expression in mammalian cells, e.g., human cells) RAB7L coding sequence. In some embodiments, the nucleic acid sequence encoding RAB7L encodes a protein comprising the amino acid sequence described in SEQ ID NO: 47 (e.g., described in NCBI reference sequence NP_003920.1). In some embodiments, the isolated nucleic acid comprises the sequence described in SEQ ID NO: 48. In some embodiments, the expression construct comprises an adeno-associated virus (AAV) inverted terminal repeat (ITR), e.g., an AAV ITR adjacent to the nucleic acid sequence encoding the RAB7L protein.

[0027] In some embodiments, the disclosure provides isolated nucleic acids comprising an expression construct encoding a VPS35 protein (e.g., the gene product of the VPS35 gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized (e.g., codon-optimized for expression in mammalian cells, e.g., human cells) VPS35 coding sequence. In some embodiments, the nucleic acid sequence encoding VPS35 encodes a protein comprising the amino acid sequence described in SEQ ID NO: 49 (e.g., described in NCBI reference sequence NP_060676.2). In some embodiments, the isolated nucleic acid comprises the sequence described in SEQ ID NO: 50. In some embodiments, the expression construct comprises an adeno-associated virus (AAV) inverted terminal repeat (ITR), e.g., an AAV ITR adjacent to the nucleic acid sequence encoding the VPS35 protein.

[0028] In some embodiments, the disclosure provides isolated nucleic acids comprising an expression construct encoding an IL-34 protein (e.g., the gene product of the IL34 gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized (e.g., codon-optimized for expression in mammalian cells, e.g., human cells) IL-34 coding sequence. In some embodiments, the nucleic acid sequence encoding IL-34 encodes a protein comprising the amino acid sequence described in SEQ ID NO: 55 (e.g., described in NCBI reference sequence NP_689669.2). In some embodiments, the isolated nucleic acid comprises the sequence described in SEQ ID NO: 56. In some embodiments, the expression construct comprises an adeno-associated virus (AAV) inverted terminal repeat (ITR), e.g., an AAV ITR adjacent to the nucleic acid sequence encoding the IL-34 protein.

[0029] In some embodiments, the disclosure provides isolated nucleic acids comprising an expression construct encoding the TREM2 protein (e.g., the gene product of the TREM gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized (e.g., codon-optimized for expression in mammalian cells, e.g., human cells) TREM2 coding sequence. In some embodiments, the nucleic acid sequence encoding TREM2 encodes a protein comprising the amino acid sequence described in SEQ ID NO: 57 (e.g., described in NCBI reference sequence NP_061838.1). In some embodiments, the isolated nucleic acid comprises the sequence described in SEQ ID NO: 58. In some embodiments, the expression construct comprises an adeno-associated virus (AAV) inverted terminal repeat (ITR), e.g., an AAV ITR adjacent to the nucleic acid sequence encoding the TREM2 protein.

[0030] In some embodiments, the disclosure provides isolated nucleic acids comprising an expression construct encoding the TMEM106B protein (e.g., the gene product of the TMEM106B gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized (e.g., codon-optimized for expression in mammalian cells, e.g., human cells) TMEM106B coding sequence. In some embodiments, the nucleic acid sequence encoding TMEM106B encodes a protein comprising the amino acid sequence described in SEQ ID NO: 63 (e.g., described in NCBI reference sequence NP_060844.2). In some embodiments, the isolated nucleic acid comprises the sequence described in SEQ ID NO: 64. In some embodiments, the expression construct comprises an adeno-associated virus (AAV) inverted terminal repeat (ITR), e.g., an AAV ITR adjacent to the nucleic acid sequence encoding the TMEM106B protein.

[0031] In some embodiments, the disclosure provides isolated nucleic acids comprising an expression construct encoding progranulin (e.g., the gene product of a PGRN gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized (e.g., codon-optimized for expression in mammalian cells, e.g., human cells) prosaposin-coding sequence. In some embodiments, the nucleic acid sequence encoding progranulin (PGRN) encodes a protein comprising the amino acid sequence described in SEQ ID NO: 67 (e.g., described in NCBI reference sequence NP_002078.1). In some embodiments, the isolated nucleic acid comprises the sequence described in SEQ ID NO: 68. In some embodiments, the expression construct comprises an adeno-associated virus (AAV) inverted terminal repeat (ITR), e.g., an AAV ITR adjacent to the nucleic acid sequence encoding the prosaposin protein.

[0032] In some embodiments, the Disclosure provides isolated nucleic acids comprising expression constructs encoding a first gene product and a second gene product, each gene product being independently selected from the gene products or a portion thereof listed in Table 1.

[0033] In some embodiments, the first or second gene product is a Gcase protein or a portion thereof. In some embodiments, the first gene product is a Gcase protein, and the second gene product is selected from GBA2, prosaposin, progranulin, LIMP2, GALC, CTSB, SMPD1, GCH1, RAB7, VPS35, IL-34, TREM2, and TMEM106B.

[0034] In some embodiments, the expression construct encodes an interfering nucleic acid (e.g., shRNA, miRNA, dsRNA, etc.) (e.g., alone or in addition to another gene product). In some embodiments, the interfering nucleic acid inhibits the expression of α-synuclein. In some embodiments, the interfering nucleic acid targeting α-synuclein comprises the sequence described in any one of SEQ ID NOs. 20-25. In some embodiments, the interfering nucleic acid targeting α-synuclein binds to (e.g., hybridizes) the sequence described in any one of SEQ ID NOs. 20-25.

[0035] In some embodiments, the interfering nucleic acid inhibits the expression of TMEM106B. In some embodiments, the interfering nucleic acid targeting TMEM106B includes the sequence described in SEQ ID NO: 64 or 65. In some embodiments, the interfering nucleic acid targeting TMEM106B binds to (e.g., hybridizes) the sequence described in SEQ ID NO: 64 or 65.

[0036] In some embodiments, the expression construct further comprises one or more promoters. In some embodiments, the promoter is a chicken beta-actin (CBA) promoter, a CAG promoter, a CD68 promoter, or a JeT promoter. In some embodiments, the promoter is an RNA pol II promoter (e.g., U6) or an RNA pol III promoter (e.g., U6).

[0037] In some embodiments, the expression construct further includes an internal ribosome entry site (IRES). In some embodiments, the IRES is located between the first and second gene products.

[0038] In some embodiments, the expression construct further comprises a self-cleaving peptide coding sequence. In some embodiments, the self-cleaving peptide is a T2A peptide.

[0039] In some embodiments, the expression construct includes two adeno-associated virus (AAV) inverted terminal repeat (ITR) sequences. In some embodiments, the ITR sequences are adjacent to the first and second gene products (e.g., arranged from the 5' end to the 3' end as follows: ITR-first gene product-second gene product-ITR). In some embodiments, one of the ITR sequences of the isolated nucleic acid lacks a functional terminal dissociation site (trs). For example, in some embodiments, one of the ITRs is a ΔITR.

[0040] This disclosure relates, in some embodiments, to an rAAV vector comprising an ITR having a modified "D" region (e.g., a D sequence modified relative to a wild-type AAV2 ITR, SEQ ID NO: 29). In some embodiments, the ITR having the modified "D" region is the 5' ITR of the rAAV vector. In some embodiments, the modified "D" region comprises, for example, the "S" sequence described in SEQ ID NO: 26. In some embodiments, the ITR having the modified "D" region is the 3' ITR of the rAAV vector. In some embodiments, the modified "D" region comprises a 3' ITR in which the "D" region is located at the 3' end of the ITR (e.g., outside or at the end of the ITR relative to the vector's transgene insertion). In some embodiments, the modified "D" region comprises the sequence described in SEQ ID NO: 26 or 27.

[0041] In some embodiments, the isolated nucleic acid (e.g., rAAV vector) includes a TRY region. In some embodiments, the TRY region includes the sequence described in SEQ ID NO: 28.

[0042] In some embodiments, the isolated nucleic acids described herein encode a peptide comprising, consisting of, or having the sequence described in any one of SEQ ID NOs: 1 to 91.

[0043] In some embodiments, the Disclosure provides a vector comprising the isolated nucleic acid described herein. In some embodiments, the vector is a plasmid or a viral vector. In some embodiments, the viral vector is a recombinant AAV (rAAV) vector or a baculovirus vector. In some embodiments, the rAAV vector is single-stranded (e.g., single-stranded DNA).

[0044] In some embodiments, the Disclosure provides a host cell comprising an isolated nucleic acid or a vector as described herein.

[0045] In some embodiments, the Disclosure provides recombinant adeno-associated virus (rAAV) comprising a capsid protein and an isolated nucleic acid or vector described herein.

[0046] In some embodiments, capsid proteins, such as AAV9 capsid protein or AAVrh.10 capsid protein, can cross the blood-brain barrier. In some embodiments, rAAV transducers neurons and non-neuronal cells of the central nervous system (CNS).

[0047] In some embodiments, the Disclosure provides methods for treating subjects having or suspected of having a central nervous system (CNS) disease, the methods comprising administering a composition described by the Disclosure (e.g., a composition comprising isolated nucleic acids or vectors or rAAV) to a subject. In some embodiments, the CNS disease is a neurodegenerative disease, such as the neurodegenerative diseases listed in Table 12. In some embodiments, the CNS disease is a synuclein disease, such as the synuclein disease listed in Table 13. In some embodiments, the CNS disease is a tauopathy, such as the tauopathy listed in Table 14. In some embodiments, the CNS disease is a lysosomal storage disorder, such as the lysosomal storage disorder listed in Table 15. In some embodiments, the lysosomal storage disorder is a neuropathic Gaucher disease, such as Gaucher disease type 2 or Gaucher disease type 3.

[0048] In some embodiments, the Disclosure provides a method for treating a subject having or suspected of having Parkinson's disease, the method comprising administering a composition described by the Disclosure (e.g., a composition comprising isolated nucleic acid or vector or rAAV) to the subject.

[0049] In some embodiments, the Disclosure provides methods for treating subjects having or suspected of having 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 disease, the methods comprising administering to the subject an rAAV encoding progranulin (PGRN), PGRN being encoded by the nucleic acid sequence of Sequence ID No. 68, and rAAV comprising a capsid protein having the AAV9 serotype.

[0050] In some embodiments, the Disclosure provides a method for treating subjects having or suspected of having FTD, having a GRN mutation, the method comprising administering to the subject an rAAV encoding progranulin (PGRN), PGRN being encoded by the nucleic acid sequence of SEQ ID NO: 68, and the rAAV comprising a capsid protein having the AAV9 serotype. In some embodiments, the rAAV is approximately 3.5 × 10⁻¹⁶ 13 Vector genome (vg), approximately 7.0 × 10⁻⁶ 13 vg, or approximately 1.4 × 10⁻⁶ 14 It is administered to the subject at a dose of vg. In some embodiments, rAAV is administered by injection into the cisterna magna.

[0051] In some embodiments, the composition comprises two or more gene products (e.g., CNS disease-related gene products), for example, nucleic acids encoding two, three, four, five, or more gene products described in this application (e.g., rAAV genomes encapsulated by AAV capsid proteins). In some embodiments, the composition comprises two or more (e.g., two, three, four, five, or more) different nucleic acids (e.g., two or more rAAV genomes separately encapsulated by AAV capsid proteins), each encoding one or more different gene products. In some embodiments, two or more different compositions are administered to a subject, each composition comprising one or more nucleic acids encoding different gene products. In some embodiments, different gene products are operably linked to the same promoter type (e.g., the same promoter). In some embodiments, different gene products are operably linked to different promoters.

[0052] Isolated nucleic acids and vectors The isolated nucleic acid may be DNA or RNA. In some embodiments, the disclosure provides an isolated nucleic acid (e.g., an rAAV vector) comprising an expression construct encoding one or more PD-related genes, e.g., Gcase (e.g., the gene product of the GBA1 gene) or a portion thereof. Gcase, also referred to as beta-glucocerebrosidase or GBA, refers to a lysosomal protein that cleaves the β-glucosidic bond of an intermediate in glycolipid metabolism. In humans, Gcase is encoded by the GBA1 gene located on chromosome 1. In some embodiments, GBA1 encodes a peptide represented by the NCBI reference sequence NP_000148.2 (SEQ ID NO: 14). In some embodiments, the isolated nucleic acid comprises a codon-optimized (e.g., codon-optimized for expression in mammalian cells, e.g., human cells) Gcase coding sequence, such as the sequence described in SEQ ID NO: 15.

[0053] In some embodiments, the disclosure provides isolated nucleic acids comprising expression constructs encoding prosaposins (e.g., the gene product of the PSAP gene). Prosaposins are precursor glycoproteins of sphingolipid-activating proteins (saposins) A, B, C, and D, which promote the catabolism of sphingoglycolipids having short oligosaccharide groups. In humans, the PSAP gene is located on chromosome 10. In some embodiments, PSAP encodes a peptide represented by the NCBI reference sequence NP_002769.1 (e.g., SEQ ID NO: 16). In some embodiments, the isolated nucleic acid comprises a sequence encoding a codon-optimized prosaposin (e.g., codon-optimized for expression in mammalian cells, e.g., human cells), such as the sequence described in SEQ ID NO: 17.

[0054] Aspects of this disclosure relate to isolated nucleic acids comprising an expression construct encoding LIMP2 / SCARB2 (e.g., the gene product of the SCARB2 gene). SCARB2 refers to a membrane protein that regulates intracellular lysosome and endosomal transport. In humans, the SCARB2 gene is located on chromosome 4. In some embodiments, the SCARB2 gene encodes a peptide represented by the NCBI reference sequence NP_005497.1 (SEQ ID NO: 18). In some embodiments, the isolated nucleic acid comprises the sequence described in SEQ ID NO: 19. In some embodiments, the isolated nucleic acid comprises a codon-optimized SCARB2 coding sequence.

[0055] Aspects of this disclosure relate to isolated nucleic acids comprising an expression construct encoding the GBA2 protein (e.g., the gene product of the GBA2 gene). The GBA2 protein refers to a non-lysosomal glucosylceramidase. In humans, the GBA2 gene is located on chromosome 9. In some embodiments, the GBA2 gene encodes a peptide represented by the NCBI reference sequence NP_065995.1 (SEQ ID NO: 30). In some embodiments, the isolated nucleic acid comprises the sequence described in SEQ ID NO: 31. In some embodiments, the isolated nucleic acid comprises a codon-optimized GBA2 coding sequence.

[0056] Aspects of this disclosure relate to isolated nucleic acids comprising expression constructs encoding a GALC protein (e.g., the gene product of a GALC gene). The GALC protein refers to galactosylceramidase (or galactocerebrosidase), an enzyme that hydrolyzes the galactose ester bonds of galactocerebrosides, galactosylsphingosine, lactosylceramides, and monogalactosyldiglycerides. In humans, the GALC gene is located on chromosome 14. In some embodiments, the GALC gene encodes a peptide represented by the NCBI reference sequence NP_000144.2 (SEQ ID NO: 33). In some embodiments, the isolated nucleic acid comprises the sequence described in SEQ ID NO: 34. In some embodiments, the isolated nucleic acid comprises a codon-optimized GALC coding sequence.

[0057] Aspects of this disclosure relate to isolated nucleic acids comprising an expression construct encoding a CTSB protein (e.g., the gene product of the CTSB gene). The CTSB protein refers to cathepsin B, a lysosomal cysteine ​​protease that plays a vital role in intracellular proteolysis. In humans, the CTSB gene is located on chromosome 8. In some embodiments, the CTSB gene encodes a peptide represented by the NCBI reference sequence NP_001899.1 (SEQ ID NO: 35). In some embodiments, the isolated nucleic acid comprises the sequence described in SEQ ID NO: 36. In some embodiments, the isolated nucleic acid comprises a codon-optimized CTSB coding sequence.

[0058] Aspects of this disclosure relate to isolated nucleic acids comprising an expression construct encoding the SMPD1 protein (e.g., the gene product of the SMPD1 gene). The SMPD1 protein refers to sphingomyelin phosphodiesterase 1, a hydrolytic enzyme involved in sphingolipid metabolism. In humans, the SMPD1 gene is located on chromosome 11. In some embodiments, the SMPD1 gene encodes a peptide represented by the NCBI reference sequence NP_000534.3 (SEQ ID NO: 37). In some embodiments, the isolated nucleic acid comprises the sequence described in SEQ ID NO: 38. In some embodiments, the isolated nucleic acid comprises a codon-optimized SMPD1 coding sequence.

[0059] Aspects of this disclosure relate to isolated nucleic acids comprising an expression construct encoding the GCH1 protein (e.g., the gene product of the GCH1 gene). The GCH1 protein refers to GTP cyclohydrolase I, a hydrolytic enzyme that is part of the folate and biopterin biosynthesis pathway. In humans, the GCH1 gene is located on chromosome 14. In some embodiments, the GCH1 gene encodes a peptide represented by the NCBI reference sequence NP_000152.1 (SEQ ID NO: 45). In some embodiments, the isolated nucleic acid comprises the sequence described in SEQ ID NO: 46. In some embodiments, the isolated nucleic acid comprises a codon-optimized GCH1 coding sequence.

[0060] Aspects of this disclosure relate to isolated nucleic acids comprising an expression construct encoding the RAB7L protein (e.g., the gene product of the RAB7L gene). The RAB7L protein refers to RAB7, a GTP-binding protein, member of the RAS oncogene family. In humans, the RAB7L gene is located on chromosome 1. In some embodiments, the RAB7L gene encodes a peptide represented by the NCBI reference sequence NP_003920.1 (SEQ ID NO: 47). In some embodiments, the isolated nucleic acid comprises the sequence described in SEQ ID NO: 48. In some embodiments, the isolated nucleic acid comprises a codon-optimized RAB7L coding sequence.

[0061] Aspects of this disclosure relate to isolated nucleic acids comprising an expression construct encoding the VPS35 protein (e.g., the gene product of the VPS35 gene). 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 trans-Golgi network. In humans, the VPS35 gene is located on chromosome 16. In some embodiments, the VPS35 gene encodes a peptide represented by the NCBI reference sequence NP_060676.2 (SEQ ID NO: 49). In some embodiments, the isolated nucleic acid comprises the sequence described in SEQ ID NO: 50. In some embodiments, the isolated nucleic acid comprises a codon-optimized VPS35 coding sequence.

[0062] Aspects of this disclosure relate to isolated nucleic acids comprising an expression construct encoding the IL-34 protein (e.g., the gene product of the IL34 gene). The IL-34 protein refers to interleukin 34, a cytokine that increases monocyte proliferation and survival. In humans, the IL34 gene is located on chromosome 16. In some embodiments, the IL34 gene encodes a peptide represented by the NCBI reference sequence NP_689669.2 (SEQ ID NO: 55). In some embodiments, the isolated nucleic acid comprises the sequence described in SEQ ID NO: 56. In some embodiments, the isolated nucleic acid comprises a codon-optimized IL-34 coding sequence.

[0063] Aspects of this disclosure relate to isolated nucleic acids comprising an expression construct encoding the TREM2 protein (e.g., the gene product of the TREM2 gene). The TREM2 protein refers to myeloid cell expression trigger receptor 2, an immunoglobulin superfamily receptor found in myeloid cells. In humans, the TREM2 gene is located on chromosome 6. In some embodiments, the TREM2 gene encodes a peptide represented by the NCBI reference sequence NP_061838.1 (SEQ ID NO: 57). In some embodiments, the isolated nucleic acid comprises the sequence described in SEQ ID NO: 58. In some embodiments, the isolated nucleic acid comprises a codon-optimized TREM2 coding sequence.

[0064] Aspects of this disclosure relate to isolated nucleic acids comprising expression constructs encoding the TMEM106B protein (e.g., the gene product of the TMEM106B gene). The TMEM106B protein refers to the transmembrane protein 106B, which is involved in the regulation of dendritic morphogenesis and lysosomal transport. In humans, the TMEM106B gene is located on chromosome 7. In some embodiments, the TMEM106B gene encodes a peptide represented by the NCBI reference sequence NP_060844.2 (SEQ ID NO: 62). In some embodiments, the isolated nucleic acid comprises the sequence described in SEQ ID NO: 63. In some embodiments, the isolated nucleic acid comprises a codon-optimized TMEM106B coding sequence.

[0065] Aspects of this disclosure relate to isolated nucleic acids comprising expression constructs encoding a progranulin protein (e.g., the gene product of the PGRN gene). The PGRN protein refers to progranulin, 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 represented by the NCBI reference sequence NP_002078.1 (SEQ ID NO: 67). In some embodiments, the isolated nucleic acid comprises the sequence described in SEQ ID NO: 68. In some embodiments, the isolated nucleic acid comprises a codon-optimized PGRN coding sequence. In some embodiments, the nucleic acid further comprises a chicken β-actin (CBA) promoter and a cytomegalovirus enhancer (CMVe).

[0066] In some embodiments, this disclosure provides an automated Western blot immunoassay for quantifying PGRN protein levels in cerebrospinal fluid (CSF) samples. In some embodiments, the immunoassay is a capillary-based automated Western blot immunoassay platform in which all steps, including protein separation, immunoprobing, washing, and chemiluminescent detection, are performed in a capillary cartridge. In some embodiments, the CSF sample is derived from human or non-human primates. In some embodiments, the immunoassay allows for the detection of differences in PGRN protein levels in the presence of circulating antibodies. In some embodiments, this disclosure provides a method for quantifying progranulin protein levels in CSF samples, the method comprising: (1) Diluting the CSF sample (e.g., 4-fold dilution), (2) Filling the wells of a capillary cartridge with the CSF sample, anti-progranulin antibody, secondary antibody to detect the anti-progranulin antibody, luminol, and peroxide, (3) Loading the capillary cartridge into an automated Western blot immunoassay instrument, (4) Calculating one or more of the following using the automated Western blot immunoassay instrument: signal intensity, peak area, signal-to-noise ratio, and total protein normalization parameter, and (5) Quantifying the progranulin protein level 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 mixture containing dithiothreitol (DTT) and sample buffer. The master mixture may further contain other proprietary components. In some embodiments, the anti-progranulin antibody detects human progranulin. In some embodiments, the progranulin protein level is quantified from the calculated parameters using software that controls the automated Western blot immunoassay instrument. In some embodiments, this software is Compas software for Simple Western® (ProteinSimple, San Jose, CA).

[0067] In some embodiments, the Disclosure provides a method for quantifying progranulin protein levels in cerebrospinal fluid (CSF) samples, the method comprising: (1) diluting the CSF sample in a master mixture containing dithiothreitol (DTT) and sample buffer (e.g., 4-fold dilution); (2) filling the wells of a capillary cartridge with the diluted CSF sample, an anti-progranulin antibody, a secondary antibody to detect the anti-progranulin antibody, luminol, and peroxide; (3) loading the capillary cartridge into an automated Western blot immunoassay instrument; (4) calculating the signal intensity, peak area, and signal-to-noise ratio using the automated Western blot immunoassay instrument; and (5) quantifying the progranulin protein levels in the CSF sample as the peak area of ​​immunoreactivity to the anti-progranulin antibody.

[0068] In some embodiments, the Disclosure provides isolated nucleic acids comprising expression constructs encoding a first gene product and a second gene product, each gene product being independently selected from the gene products or a portion thereof listed in Table 1.

[0069] In some embodiments, the isolated nucleic acid or vector described in this disclosure (e.g., an rAAV vector) comprises or consists of the sequence described in any one of SEQ ID NOs: 1 to 91. In some embodiments, the isolated nucleic acid or vector described in this disclosure (e.g., an rAAV vector) comprises or consists of a sequence that is complementary to (e.g., its complementary strand to) the sequence described in any one of SEQ ID NOs: 1 to 91. In some embodiments, the isolated nucleic acid or vector described in this disclosure (e.g., an rAAV vector) comprises or consists of a sequence that is the reverse complementary strand to the sequence described in any one of SEQ ID NOs: 1 to 91. In some embodiments, the isolated nucleic acid or vector described in this disclosure (e.g., an rAAV vector) comprises or consists of a portion of the sequence described in any one of SEQ ID NOs: 1 to 91. The portion may comprise at least 25%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the sequence described in any one of SEQ ID NOs: 1 to 91. In some embodiments, the nucleic acid sequence described herein is either the nucleic acid sense strand (e.g., the 5' to 3' strand) or, in the context of a viral sequence, the plus (+) strand. In some embodiments, the nucleic acid sequence described herein is either the nucleic acid antisense strand (e.g., the 3' to 5' strand) or, in the context of a viral sequence, the minus (-) strand.

[0070] In some embodiments, the gene product is encoded by a naturally occurring gene coding portion (e.g., cDNA). 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, e.g., the SCARB2 / LIMP2 gene or the PSAP gene. However, those skilled in the art will recognize that the expression order of the first gene product (e.g., Gcase) and the second gene product (e.g., LIMP2) can generally be reversed (e.g., LIMP2 is the first gene product and Gcase is the second gene product). In some embodiments, the gene product is a fragment (e.g., a portion) of a gene listed in Table 1. The protein fragment may contain about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the protein encoded by the gene listed in Table 1. In some embodiments, the protein fragment contains 50% to 99.9% (e.g., any value between 50% and 99.9%) of the protein encoded by the genes listed in Table 1.

[0071] In some embodiments, the expression construct is monocistronic (for example, the expression construct encodes a single fusion protein containing a first gene product and a second gene product). In some embodiments, the expression construct is polycistronic (for example, the expression construct encodes two different gene products, e.g., two different proteins or protein fragments).

[0072] A polycistron expression vector may contain one or more (e.g., one, two, three, four, five, or more) promoters. Any suitable promoter may be used, such as a constitutive promoter, an inducible promoter, an endogenous promoter, or a tissue-specific promoter (e.g., a CNS-specific promoter). In some embodiments, the promoter is a chicken beta-actin promoter (CBA promoter), a CAG promoter (e.g., described by Alexopoulou et al. (2008) BMC Cell Biol. 9:2;doi:10.1186 / 1471-2121-9-2), a CD68 promoter, or a JeT promoter (e.g., described by Tornoe et al. (2002) Gene 297(1-2):21-32). In some embodiments, the promoter is operably ligated to a nucleic acid sequence encoding a first gene product, a second gene product, or both the first and second gene products. In some embodiments, the expression cassette includes one or more additional regulatory sequences, including but not limited to transcription factor binding sequences, intron splice sites, poly(A) addition sites, enhancer sequences, repressor binding sites, or any combination thereof.

[0073] In some embodiments, the nucleic acid sequence encoding the first gene product and the nucleic acid sequence encoding the second gene product are separated by a nucleic acid sequence encoding an internal ribosome entry site (IRES). Examples of IRES sites are described, for example, by Mokrejs et al. (2006) Nucleic Acids Res. 34 (Database issue): D125-30. In some embodiments, the nucleic acid sequence encoding the first gene product and the nucleic acid sequence encoding the second gene product are separated by a nucleic acid sequence encoding a self-cleaving peptide. Examples of self-cleaving peptides, but not limited to, include T2A, P2A, E2A, F2A, BmCPV 2A, and BmIFV 2A, which are described by Liu et al. (2017) Sci Rep. 7: 2193. In some embodiments, the self-cleaving peptide is the T2A peptide.

[0074] Pathologically, disorders such as PD and Gaucher disease are associated with the accumulation of protein aggregates primarily composed of α-synuclein (α-Syn) protein. Therefore, in some embodiments, the isolated nucleic acids described herein include inhibitory nucleic acids that reduce or prevent the expression of α-Syn protein. Sequences encoding inhibitory nucleic acids may be placed in the untranslated region of the expression vector (e.g., introns, 5'UTR, 3'UTR, etc.).

[0075] In some embodiments, the inhibitory nucleic acid is positioned in an intron of the expression construct, for example, in an intron upstream of the sequence encoding the first gene product. The inhibitory nucleic acid may be double-stranded RNA (dsRNA), siRNA, shRNA, microRNA (miRNA), artificial miRNA (amiRNA), or RNA aptamer. Generally, the inhibitory nucleic acid binds to (e.g., hybridizes) about 6 to 30 consecutive nucleotides (e.g., any integer between 6 and 30, including 6 and 30) of the target RNA (e.g., mRNA). In some embodiments, the inhibitory nucleic acid molecule is a miRNA or amiRNA, for example, a miRNA targeting SNCA (a gene encoding the α-Syn protein) or TMEM106B (e.g., a gene encoding the TMEM106B protein). In some embodiments, the miRNA contains no mismatch whatsoever with the region of the SNCA mRNA it hybridizes to (e.g., the miRNA is "complete"). In some embodiments, the inhibitory nucleic acid is shRNA (e.g., shRNA targeting SNCA or TMEM106B). In some embodiments, the inhibitory nucleic acid is an artificial miRNA (amiRNA) containing a miR-155 scaffold and an SNCA or TMEM106B target sequence.

[0076] Those skilled in the art will recognize that, when referring to nucleic acid sequences containing 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 sequences provided herein may be replaced with any other nucleotides suitable for base pairing with adenosine nucleotides (e.g., via Watson-Crick base pairing). For example, T may be replaced with U, and U may be replaced with T.

[0077] The isolated nucleic acids described herein may exist as themselves or as part of a vector. Generally, the vector may be a plasmid, cosmid, phagemid, bacterial artificial chromosome (BAC), or viral vector (e.g., adenovirus vector, adeno-associated virus (AAV) vector, retrovirus vector, baculovirus vector, etc.). In some embodiments, the vector is a plasmid (e.g., a plasmid containing the isolated nucleic acids 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 (rAAV) vector. In some embodiments, the vector is a baculovirus vector (e.g., Autographa californica nuclear polyhedron disease (AcNPV) vector).

[0078] Typically, an rAAV vector (e.g., an rAAV genome) contains a transgene (e.g., an expression construct comprising one or more of the following: promoter, intron, enhancer sequence, protein-coding sequence, inhibitory RNA-coding sequence, polyA tail sequence, etc.) adjacent to two AAV inverted terminal repeat (ITR) sequences. In some embodiments, the transgene of an rAAV vector comprises the isolated nucleic acid described herein. In some embodiments, each of the two ITR sequences in an rAAV vector is a full-length ITR (e.g., approximately 145 bp in length and including a functional Rep-binding site (RBS) and terminal dissociation sites (trs)). In some embodiments, one of the ITRs in an rAAV vector is cleaved (e.g., shortened or not full length). In some embodiments, the cleaved ITR lacks a functional terminal dissociation site (trs) and is used for the production of a self-complementary AAV vector (scAAV vector). In some embodiments, the cleaved ITR is a ΔITR as described, for example, by McCarty et al. (2003) Gene Ther. 10(26):2112-8.

[0079] Aspects of this disclosure relate to isolated nucleic acids (e.g., rAAV vectors) containing ITRs having one or more modifications (e.g., nucleotide addition, deletion, substitution, etc.) to wild-type AAV ITRs, for example, to wild-type AAV2 ITRs (e.g., SEQ ID NO: 29). The structure of a wild-type AAV2 ITR is shown in Figure 20. Generally, a wild-type ITR contains a 125-nucleotide region that self-anneals to form a palindromic double-stranded T-shaped hairpin structure consisting of two cross arms (formed by sequences referred to as B / B' and C / C', respectively), a longer stem region (formed by a sequence referred to as sequence A / A'), and a single-stranded terminal region referred to as the "D" region (Figure 20). Generally, the "D" region of the ITR is located between the stem region formed by the A / A' sequence and the insert containing the rAAV vector's transgene (e.g., located "inside" the ITR relative to the ends of the ITR, or located proximal to the rAAV vector's transgene insert or expression construct). In some embodiments, the "D" region includes the sequence described in Sequence ID No. 27. The "D" region has been observed to play a crucial role in the capsidation of rAAV vectors by capsid proteins, as disclosed, for example, by Ling et al. (2015) J Mol Genet Med 9(3).

[0080] This disclosure is partly based on the surprising 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) are more efficiently capsidated by the AAV capsid protein than rAAV vectors having an unmodified (e.g., wild-type) ITR. In some embodiments, rAAV vectors having a modified "D" sequence (e.g., a "D" sequence at an "outside" position) exhibit reduced toxicity compared to rAAV vectors having a wild-type ITR sequence.

[0081] In some embodiments, the modified "D" sequence contains at least one nucleotide substitution relative to the wild-type "D" sequence (e.g., SEQ ID NO: 27). The modified "D" sequence may have at least one, two, three, four, five, six, seven, eight, nine, ten, or more than ten nucleotide substitutions relative to the wild-type "D" sequence (e.g., SEQ ID NO: 27). In some embodiments, the modified "D" sequence contains at least ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or nineteen nucleic acid substitutions relative to the wild-type "D" sequence (e.g., SEQ ID NO: 27). In some embodiments, the modified "D" sequence is approximately 10% to approximately 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 includes the sequence described in SEQ ID NO: 26, also referred to as the "S" sequence described in Wang et al. (1995) J Mol Biol 250(5):573-80.

[0082] The isolated nucleic acids or rAAV vectors described in this disclosure may further include, for example, the “TRY” sequence described in SEQ ID NO: 28 or by Francois et al., (2005) J. Virol. 79(17):11082-11094. In some embodiments, the TRY sequence is positioned between the ITR (e.g., 5'ITR) and the expression construct (e.g., the transgene coding insertion) of the isolated nucleic acid or rAAV vector.

[0083] In some embodiments, the present disclosure relates to baculovirus vectors, including isolated nucleic acids or rAAV vectors described herein. In some embodiments, the baculovirus vector is, for example, the Autographa californica nuclear polyhedron disease (AcNPV) vector described by Urabe et al. (2002) Hum Gene Ther 13(16):1935-43 and Smith et al. (2009) Mol Ther 17(11):1888-1896.

[0084] In some embodiments, this disclosure provides host cells comprising isolated nucleic acids or vectors as described herein. The host cells may be prokaryotic or eukaryotic cells. For example, the host cells may be mammalian cells, bacterial cells, yeast cells, insect cells, etc. In some embodiments, the host cells are mammalian cells, e.g., HEK293T cells. In some embodiments, the host cells are bacterial cells, e.g., E. coli cells.

[0085] rAAV In some embodiments, this disclosure relates to recombinant AAV (rAAV) comprising a transgene encoding a nucleic acid described herein (e.g., an rAAV vector described herein). The term “rAAV” generally refers to a viral particle comprising an rAAV vector encapsulated by one or more AAV capsid proteins. The rAAV 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 derived from a non-human host, e.g., rhesus monkey AAV capsid proteins such as AAVrh.10 and AAVrh.39. In some embodiments, the rAAV described by this disclosure includes a capsid protein that is a variant of the wild-type capsid protein, such as a capsid protein variant containing at least one, two, three, four, five, six, seven, eight, nine, ten, or more than ten (e.g., 15, 20, 25, 50, 100, etc.) amino acid substitutions (e.g., mutations) relative to the wild-type AAV capsid protein from which it is derived. In some embodiments, the AAV capsid protein variant is, for example, the AAV1RX capsid protein described by Albright et al. Mol Ther. 2018 Feb 7;26(2):510-523. In some embodiments, the capsid protein variant is, for example, the AAV TM6 capsid protein described by Rosario et al. Mol Ther Methods Clin Dev. 2016;3:16026.

[0086] In some embodiments, the rAAVs described herein readily diffuse through the CNS, particularly into the CSF space, or when introduced directly into the brain parenchyma. Therefore, in some embodiments, the rAAVs described herein comprise a capsid protein capable of crossing the blood-brain barrier (BBB). For example, in some embodiments, the rAAV comprises a capsid protein having the AAV9 or AAVrh.10 serotype. The production of rAAVs is described, for example, by Samulski et al. (1989) J Virol. 63(9):3822-8 and Wright (2009) Hum Gene Ther. 20(7):698-706. In some embodiments, the rAAV comprises a capsid protein that specifically or preferentially targets myeloid cells, such as microglia.

[0087] In some embodiments, this disclosure provides an rAAV referred to as “PR006A,” which is an rAAV that delivers a functional human GRN gene and results in increased expression of functional human PGRN. The PR006A vector insert contains a chicken β-actin (CBA) promoter element comprising four parts: a cytomegalovirus (CMV) enhancer, a CBA promoter, exon 1, and an intron, for constitutive expression of the codon-optimized coding sequence of human GRN (SEQ ID NO: 68). The 3' region also contains a woodchuck hepatitis virus post-transcriptional regulator (WPRE), followed by a bovine growth hormone polyadenylation signaling tail. Three well-described 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). Adjacent inverted terminal repeats (ITRs) enable correct packaging of intervening sequences. The backbone contains genes that confer resistance to kanamycin and stuffer sequences to prevent reverse packaging. A schematic diagram of the RAAV vector is shown in Figure 64. Sequence ID 90 provides the nucleotide sequence (5' to 3' order) of the first strand of the PR006A vector shown in Figure 64. Sequence ID 91 provides the nucleotide sequence (5' to 3' order) of the second strand of the PR006A vector shown in Figure 64. PR006A contains the AAV9 capsid protein.

[0088] In some embodiments, the rAAV described herein (including, for example, a recombinant rAAV genome capsidized by an AAV capsid protein to form rAAV capsid particles) is produced in a baculovirus vector expression system (BEVS). Production of rAAV using BEVS is described, for example, by Urabe et al. (2002) Hum Gene Ther 13(16):1935-43, Smith et al. (2009) Mol Ther 17(11):1888-1896, U.S. Patent No. 8,945,918, U.S. Patent No. 9,879,282, and International PCT Publication No. WO2017 / 184879. However, rAAV can be produced using any suitable method (for example, using recombinant rep and cap genes). In some embodiments, the rAAV disclosed herein is produced in HEK293 (human embryonic kidney) cells.

[0089] Pharmaceutical composition In some embodiments, this disclosure provides pharmaceutical compositions comprising isolated nucleic acids or rAAVs described herein and pharmaceutically acceptable carriers. As used herein, the term “pharmaceutically acceptable” means a material such as a carrier or diluent that does not negate the biological activity or properties of a compound and is relatively non-toxic, for example, the material can be administered to an individual without causing an undesirable biological effect or without interacting in a harmful manner with any of the components of the composition in which it is contained.

[0090] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent, or encapsulating material, that is involved in transporting or delivering a useful compound within the present invention to or within a patient so that it can perform its intended function. Additional components that may be included in the pharmaceutical compositions used in the practice of the present invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which are incorporated herein by reference.

[0091] The compositions provided herein (e.g., pharmaceutical compositions) may be administered by any route including enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, subarachnoid, subcutaneous, intraventricular, percutaneous, intercutaneous, rectal, vaginal, intraperitoneal, topical (by powder, ointment, cream, and / or droplet), mucous membrane, nasal cavity, oral cavity, and sublingual; by intratracheal infusion, bronchial infusion, and / or inhalation; and / or as oral spray, nasal spray, and / or aerosol. Specifically intended routes include oral administration, intravenous administration (e.g., systemic intravenous injection), topical administration via blood and / or lymphatic supply, and / or direct administration to the affected area. Generally, the most appropriate route of administration depends on various factors, including the properties of the drug (e.g., its stability in the gastrointestinal environment) and / or the condition of the subject (e.g., whether the subject can tolerate oral administration). In certain embodiments, the compounds or pharmaceutical compositions described herein are suitable for topical administration to the eye of the subject.

[0092] In some embodiments, the disclosure provides a finished PR006A drug comprising the aforementioned PR006A rAAV present in aqueous solution. In some embodiments, the final formulation buffer contains about 20 mM Tris [pH 8.0], about 1 mM MgCl2, about 200 mM NaCl, and about 0.001% [w / v] poloxamer 188. In some embodiments, the finished drug product and the final formulation buffer are suitable for intracisor chamber (ICM) injection.

[0093] method Aspects of this disclosure relate to compositions for the expression of one or more CNS disease-related gene products in subjects for the treatment of CNS-related diseases. One or more CNS disease-related gene products may be encoded by one or more isolated nucleic acids or rAAV vectors. In some embodiments, subjects are administered a single vector (e.g., isolated nucleic acid, rAAV, etc.) encoding one or more (one, two, three, four, five, or more) gene products. In some embodiments, subjects are administered multiple (e.g., two, three, four, five, or more) vectors (e.g., isolated nucleic acids, rAAV, etc.), each vector encoding a different CNS disease-related gene product.

[0094] CNS-related disorders may include neurodegenerative diseases, synuclein diseases, tauopathies, or lysosomal storage disorders. Examples of neurodegenerative diseases and their associated genes are listed in Table 12.

[0095] "Synuclein disease" refers to a disease or disorder characterized by the accumulation of alpha-synuclein (the gene product of SNCA) in a subject (for example, in a healthy subject, e.g., a subject without synuclein disease). Examples of synuclein disease and its related genes are listed in Table 13.

[0096] "Tauopathy" refers to a disease or disorder characterized by the accumulation of abnormal Tau protein in a subject (for example, in a healthy subject without tauopathy). Examples of tauopathy and its associated genes are listed in Table 14.

[0097] "Lysosomal storage disorders" refer to diseases characterized by the abnormal accumulation of toxic cell products in the affected lysosomes. Examples of lysosomal storage disorders and related genes are listed in Table 15.

[0098] As used herein, “to treat” or “to treat” means (a) preventing or delaying the onset of a CNS disorder, (b) reducing the severity of a CNS disorder, (c) reducing or preventing the onset of characteristic symptoms of a CNS disorder, and (d) and / or preventing the exacerbation of characteristic symptoms of a CNS disorder. Symptoms of a CNS disorder may include, for example, motor impairment (e.g., tremors, rigidity, slowness of movement, difficulty walking, paralysis), cognitive impairment (e.g., dementia, depression, anxiety, psychosis), memory impairment, and emotional and behavioral impairments.

[0099] This disclosure is based in part on compositions for the expression of combinations of PD-related gene products in subjects that act together (e.g., synergistically) to treat Parkinson's disease.

[0100] Accordingly, in some embodiments, the Disclosure provides a method for treating a subject having or suspected to have Parkinson's disease, the method comprising administering a composition described by the Disclosure (e.g., a composition comprising isolated nucleic acid or vector or rAAV) to the subject.

[0101] This disclosure is in part based on compositions for the expression of one or more CNS disease-related gene products in subjects for the treatment of Gaucher disease. In some embodiments, Gaucher disease is neuropathic Gaucher disease, e.g., type 2 Gaucher disease or type 3 Gaucher disease. In some embodiments, subjects having Gaucher disease do not have PD or PD symptoms.

[0102] Accordingly, in some embodiments, the Disclosure provides a method for treating a subject having or suspected of having neuropathic Gaucher disease, the method comprising administering a composition described by the Disclosure (e.g., a composition comprising isolated nucleic acid or vector or rAAV) to the subject.

[0103] This disclosure is in part based on compositions for the expression of one or more CNS disease-related gene products in subjects for the treatment of Alzheimer's disease or frontotemporal dementia (FTD). In some embodiments, the subjects do not have Alzheimer's disease. In some embodiments, the subjects have FTD and do not have Alzheimer's disease. In some embodiments, the subjects have FTD and have a GRN (progranulin) mutation. In some embodiments, the subjects have FTD and have a GRN mutation, and the subjects are heterozygous for the GRN mutation (e.g., a pathogenic GRN mutation). In some embodiments, the GRN mutation is a null mutation (e.g., a nonsense, frameshift, or splice site mutation, or a complete or partial (exonal) gene deletion). In some embodiments, the GRN mutation is a pathogenic mutation with demonstrated functionally harmful effects. In some embodiments, the GRN mutation is a missense pathogenic mutation. In some embodiments, the GRN mutation is listed in the Molgen FTD database (molgen.ua.ac.be). In some embodiments, the GRN mutation produces low plasma PGRN levels (less than 70 ng / mL) in the subject.

[0104] In some embodiments, the subjects have FTD, FTD with GRN mutation, FTD with tau mutation, FTD with C9Orf72 mutation, neuronal ceroid lipofuscinosis, Parkinson's disease, Alzheimer's disease, corticobasal degeneration, motor neuron disease, or Gaucher disease.

[0105] In some embodiments, the subject has symptomatic FTD (e.g., behavioral variant FTD (bvFTD), primary progressive aphasia (PPA)-FTD, FTD with corticobasal syndrome, or a combination of syndromes).

[0106] Thus, in some aspects, the present disclosure provides a method for treating a subject having or suspected of having FTD with a GRN mutation, the method comprising administering to the subject a composition as described by the present disclosure (e.g., a composition comprising an isolated nucleic acid or a vector or rAAV).

[0107] In some embodiments, a subject having Alzheimer's disease or FTD (e.g., FTD with a GRN mutation) is administered 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 rAAV encoding PGRN or a portion thereof, and 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.

[0108] In some embodiments, a composition comprising rAAV encoding PGRN for treating FTD (e.g., FTD with a GRN mutation) administers to the subject from about 1×10 12 vector genomes (vg) to about 1×10 15 vg, or from about 1×10 13 vg to about 7×10 14 vg, or from about 1×10 13 vg to about 5×10 14 vg, or from about 2×10 13 vg to about 2×10 14 vg, or from about 3×10 13 vg to about 2×10 14 vg, or from about 3.5×10 13 vg to about 1.4×1014 It is administered in doses in the range of vg. In some embodiments, a composition comprising rAAV encoding PGRN for treating FTD (e.g., FTD with GRN mutations) is administered to a target of about 2 × 10 13 vg, approx. 3×10 13 vg, approx. 4×10 13 vg, approx. 5×10 13 vg, approx. 6×10 13 vg, approx. 7×10 13 vg, approx. 8×10 13 vg, approx. 9×10 13 vg, approx. 1×10 14 vg, or approximately 2 × 10 14 It is administered at a dose of VG.

[0109] In some embodiments, the Disclosure provides a method for treating a subject having or suspected of having FTD (e.g., FTD with a GRN mutation), the method comprising administering to the subject a composition comprising an rAAV encoding a PGRN, the composition comprising approximately 3.5 × 10 13 Vector genome (vg), approximately 7.0 × 10⁻⁶ 13 vg, or approximately 1.4 × 10⁻⁶ 14 It is administered at a dose of VG.

[0110] In some embodiments, the Disclosure provides a method for treating a subject having or suspected of having FTD (e.g., FTD with a GRN mutation), the method comprising administering to the subject a composition comprising an rAAV encoding a PGRN, the composition comprising about 1 × 10 14 Vector genome (vg), approximately 2.0 × 10⁻⁶ 14 vg, or approximately 4.0 × 10 14 It is administered at a dose of VG.

[0111] In some embodiments, a composition comprising a single dose of an rAAV encoding a PGRN for treating a subject with FTD (e.g., FTD with a GRN mutation), wherein the composition is not subsequently administered to the subject.

[0112] In some embodiments, the composition containing rAAV is delivered via a single suboccipital injection into the cisterna magna. In some embodiments, the injection into the cisterna magna is performed under radiation guidance.

[0113] In some embodiments, the Disclosure provides methods for treating symptoms of subjects having or suspected of having FTD with a GRN mutation, the methods comprising administering to a subject a composition comprising an rAAV encoding a sequence for a functional progranulin (PGRN) protein, the PGRN protein being encoded by a codon-optimized nucleic acid sequence or the nucleic acid sequence of SEQ ID NO: 68. In some embodiments, symptoms of FTD with a GRN mutation may be personality changes, executive function impairment, disinhibition, apathy, stuttering, grammatical errors, various agnosia, semantic aphasia, or impaired word comprehension. In some embodiments, the rAAV encoding PGRN comprises a capsid protein having the AAV9 serotype.

[0114] In some embodiments, the Disclosure provides a method for reducing lipofuscin accumulation in the brain of subjects having a GRN mutation and having or suspected of having FTD, the method comprising administering to the subject a composition comprising rAAV encoding progranulin (PGRN), the PGRN protein being encoded by a codon-optimized nucleic acid sequence or the nucleic acid sequence of Sequence ID No. 68. In some embodiments, the Disclosure provides a method for reducing ubiquitin accumulation in the brain of subjects having a GRN mutation and having or suspected of having FTD, the method comprising administering to the subject a composition comprising rAAV encoding progranulin (PGRN), the PGRN protein being encoded by a codon-optimized nucleic acid sequence or the nucleic acid sequence of Sequence ID No. 68. In some embodiments, the Disclosure provides a method for reducing the gene and / or protein expression of TNFα and / or CD68 in the brain of a subject having a GRN mutation and having or suspected of having FTD, the method comprising administering to the subject a composition comprising rAAV encoding progranulin (PGRN), the PGRN protein being encoded by a codon-optimized nucleic acid sequence or the nucleic acid sequence of Sequence ID No. 68. In some embodiments, the Disclosure provides a method for increasing the maturation of cathepsin D in the brain of a subject having a GRN mutation and having or suspected of having FTD, the method comprising administering to the subject a composition comprising rAAV encoding progranulin (PGRN), the PGRN protein being encoded by a codon-optimized nucleic acid sequence or the nucleic acid sequence of Sequence ID No. 68. In some embodiments, the Disclosure provides a method for increasing the level of nuclear TDP-43 (transactive response DNA-binding protein 43kDa) protein in the brain of a subject having a GRN mutation and having or suspected of having FTD, the method comprising administering to the subject a composition comprising rAAV encoding progranulin (PGRN), the PGRN protein being encoded by a codon-optimized nucleic acid sequence or the nucleic acid sequence of Sequence ID No. 68.In some embodiments, the Disclosure provides a method for reducing levels of nerve filament light chains (NFLs) in the blood or CSF of a subject having a GRN mutation and having or suspected having FTD, the method comprising administering to the subject a composition comprising an rAAV encoding progranulin (PGRN), the PGRN protein being 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.

[0115] The subjects are typically mammals, preferably humans. In some embodiments, the subjects are 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 subjects are between 2 and 20 years old. In some embodiments, the subjects are between 30 and 100 years old. In some embodiments, the subjects are older than 55 years old.

[0116] In some embodiments, the composition is administered directly to the subject's CNS, for example, by direct injection into the subject's brain and / or spinal cord. Examples of direct CNS administration methods include, but are not limited to, intracerebral injection, intraventricular injection, intracisional injection, intraparenchymal injection, intraarachnoid injection, and any combination thereof. In some embodiments, the composition is administered to the subject by intracisional (ICM) injection. In some embodiments, direct injection into the subject's CNS results in transgene expression (e.g., expression of a first gene product, a second gene product, and, where applicable, a third gene product) in the subject's midbrain, striatum, and / or cerebral cortex. In some embodiments, direct injection into the CNS results in transgene expression (e.g., expression of a first gene product, a second gene product, and, where applicable, a third gene product) in the subject's spinal cord and / or CSF.

[0117] In some embodiments, direct injection into the target CNS includes convection-enhanced delivery (CED). Convection-enhanced delivery is a therapeutic strategy that includes surgical exposure of the brain and direct placement of a small-diameter catheter into a target area of ​​the brain, followed by direct injection of a therapeutic agent (e.g., the compositions described herein or rAAV) into the target brain. CED is described, for example, by Debinski et al. (2009) Expert Rev Neurother. 9(10):1519-27.

[0118] In some embodiments, the composition is administered peripherally to the subject, for example, by peripheral injection. Examples of peripheral injections include subcutaneous injection, intravenous injection, intra-arterial injection, intraperitoneal injection, or any combination thereof. In some embodiments, the peripheral injection is an intra-arterial injection, for example, into the carotid artery of the subject.

[0119] In some embodiments, the compositions described herein (e.g., compositions comprising isolated nucleic acids or vectors or rAAV) are administered to the CNS of a subject both peripherally and directly. For example, in some embodiments, the subject is administered the composition by intra-arterial injection (e.g., injection into the carotid artery) and intraparenchymal injection (e.g., intraparenchymal injection via CED). In some embodiments, the direct injection and peripheral injection into the CNS are performed simultaneously (e.g., simultaneously). In some embodiments, the direct injection is performed before the peripheral injection (e.g., 1 minute to 1 week or more before). In some embodiments, the direct injection is performed after the peripheral injection (e.g., 1 minute to 1 week or more after).

[0120] In some embodiments, subjects are administered an immunosuppressant before (e.g., 1 month to 1 minute before) or simultaneously with the compositions described herein. In some embodiments, the immunosuppressant is a corticosteroid (e.g., prednisone, budesonide, etc.), an mTOR inhibitor (e.g., sirolimus, everolimus, etc.), an antibody (e.g., adalimumab, etanercept, natalizumab, etc.), or methotrexate.

[0121] The composition described herein (e.g., isolated nucleic acid or vector) administered to the subject The amount of the composition containing rAAV (or rAAV) will vary depending on the method of administration. For example, in some embodiments, the amount of rAAV described herein is about 10 9 Genome copies (GC) / kg ~ approximately 10 14 GC / kg (for example, about 10 9 GC / kg, approx. 10 10 GC / kg, approx. 10 11 GC / kg, approx. 10 12 GC / kg, approx. 10 12 GC / kg, or approximately 10 14 The subject is administered a high potency (e.g., 10 GC / kg) by injection into the CSF space or by intraparenchymal injection. In some embodiments, the subject is administered a high potency (e.g., 10 GC / kg) by injection into the CSF space or by intraparenchymal injection. 12The rAAV (rAAV) with a genome copy GC / kg greater than 1 × 10¹⁶ is administered. In some embodiments, the rAAV described herein is administered by intravenous injection at a rate of approximately 1 × 10¹⁶ 10 Vector genome (vg) ~ approximately 1 × 10⁻⁶ 17 It is administered to the subject in doses in the range of vg. In some embodiments, the rAAV described herein is administered by injection into the cisterna magna, approximately 1 × 10⁻¹⁶ 10 vg~approx. 1×10 16 It is administered to the target population in doses within the VG range.

[0122] The compositions described herein (e.g., compositions comprising isolated nucleic acids or vectors or rAAV) may be administered to a subject once or multiple times (e.g., two, three, four, five, six, seven, eight, nine, ten, twenty, or more times). In some embodiments, the composition may be administered to the subject continuously (e.g., chronically), for example, via an infusion pump. [Examples]

[0123] Example 1: rAAV vector Using cells such as HEK293 cells, an AAV vector for triple plasmid transfection is generated. The ITR sequence is adjacent to an expression construct containing a promoter / enhancer element, a 3' polyA signal, and post-translational signals such as a WPRE element for each target transgene. Multiple gene products, such as GBA1 and LIMP2 and / or prosaposins, can be expressed simultaneously by protein sequence fusion, or by using a 2A peptide linker such as T2A or P2A that leads to two peptide fragments with added amino acids by preventing the formation of peptide bonds, or by using an IRES element, or by expression in two separate expression cassettes. The presence of short intron sequences that are efficiently spliced ​​upstream of the expressed genes may improve expression levels. shRNAs and other regulatory RNAs may potentially be contained within these sequences. Examples of expression constructs described in this disclosure are shown in Figures 1–8, 21–35, 39, 41–51, and 64, and in Table 2 below. [Table 2-1] [Table 2-2] [Table 2-3]

[0124] Example 2: Cell-based assay for viral transduction into GB GBA1-deficient cells can be obtained, for example, as fibroblasts derived from GD patients, monocytes, or hES cells, or as patient-derived induced pluripotent stem cells (iPSCs). These cells accumulate substrates such as glucosylceramide and glucosylsphingosine (GlcCer and GlcSph). Treatment of wild-type or mutant cultured cell lines with Gcase inhibitors such as CBE is also used to obtain GBA-deficient cells.

[0125] Using such cell models, lysosome deficiencies are quantified in terms of the accumulation of protein aggregates, such as α-synuclein, by imaging using antibodies against this protein or phospho-α-Syn, followed by fluorescence microscopy. Imaging of lysosomal abnormalities is also performed by ICC for protein markers such as LAMP1, LAMP2, LIMP1, and LIMP2, or by dyes such as Lysotracker, or by endocytosis compartment uptake of fluorescent dextran or other markers. Imaging of autophagy marker accumulation due to defective fusion with lysosomes, such as LC3, may also be performed. Abnormal accumulation of these markers is quantified using Western blotting and / or ELISA. In addition, the accumulation of glycolipid substrates and products of GBA1 is measured using standard methods.

[0126] Therapeutic endpoints (e.g., reduction of PD-related pathology) are measured in terms of the expression of AAV vector transduction, and their activity and function are confirmed and quantified. Gcase can also be quantified using protein ELISA or by standard Gcase activity assays.

[0127] Example 3: In vivo assay using mutant mice This example describes an in vivo assay of an AAV vector using mutant mice. The in vivo assay of the AAV vector described above in mutant mice is performed using assays described, for example, by Liou et al. (2006) J. Biol. Chem. 281(7):4242-4253, Sun et al. (2005) J. Lipid Res. 46:2102-2113, and Farfel-Becker et al. (2011) Dis. Model Mech. 4(6):746-752.

[0128] Subarachnoid or intraventricular delivery of vehicle control and AAV vector (e.g., 2 × 10⁻¹⁰ 11 The dose (vg / mouse dose) is performed using concentrated AAV stock, for example, in an injection volume of 5-10 μL. Substance delivery is performed via convection-enhanced delivery.

[0129] Treatment is initiated either before or after the onset of symptoms. Measured endpoints include substrate accumulation in the CNS and CSF, Gcase enzyme accumulation and enzyme activity accumulation by ELISA, motor and cognitive endpoints, lysosomal dysfunction, and accumulation of α-synuclein monomer, protofibril, or fibril.

[0130] Example 4: Chemical model of disease This example describes an in vivo assay of AAV vectors using a chemically induced mouse model of Gaucher disease (e.g., the CBE mouse model). These in vivo assays of AAV vectors are performed in a chemically induced mouse model of Gaucher disease, as described, for example, by Vardi et al. (2016) J Pathol. 239(4):496-509.

[0131] Subarachnoid or intraventricular delivery of vehicle control and AAV vector (e.g., 2 × 10⁻¹⁰ 11 The dose (vg / mouse dose) is performed using concentrated AAV stock, for example, in an injection volume of 5-10 μL. Intracellular delivery is performed via convection-enhanced delivery. Peripheral delivery is achieved by tail vein injection.

[0132] Treatment is initiated either before or after the onset of symptoms. Measured endpoints include substrate accumulation in the CNS and CSF, Gcase enzyme accumulation and enzyme activity accumulation by ELISA, motor and cognitive endpoints, lysosomal dysfunction, and accumulation of α-synuclein monomer, protofibril, or fibril.

[0133] Example 5: Clinical trial in patients with PD, LBD, and Gaucher disease In some embodiments, patients with certain forms of Gaucher disease (e.g., GD1) have an increased risk of developing Parkinson's disease (PD) or Lewy body dementia (LBD). This embodiment describes a clinical trial to evaluate the safety and efficacy of the rAAV described herein in patients with Gaucher disease, PD, and / or LBD.

[0134] Clinical trials of such vectors for the treatment of Gaucher disease, PD, and / or LBD will be conducted using a trial design similar to that described in Grabowski et al. (1995) Ann. Intern. Med. 122(1):33-39.

[0135] Example 6: Treatment of peripheral diseases In some embodiments, patients with a particular form of Gaucher disease present with symptoms of peripheral neuropathy, as described, for example, in Biegstraaten et al. (2010) Brain 133(10):2909-2919.

[0136] This embodiment describes an in vivo assay of the AAV vector described herein for the treatment of peripheral neuropathy associated with Gaucher disease (e.g., type 1 Gaucher disease). Briefly, a patient with type 1 Gaucher disease identified as having signs or symptoms of peripheral neuropathy is administered the rAAV described herein. In some embodiments, the signs and symptoms of peripheral neuropathy in question are monitored after administration of the rAAV using, for example, the method described by Biegstraaten et al.

[0137] The levels of the transdextrinsic products described herein, present in the patient (e.g., in the patient's serum, in peripheral tissues (e.g., liver tissue, spleen tissue, etc.)), are assayed, for example, by Western blot analysis, enzyme function assay, or imaging studies.

[0138] Example 7: Treatment of CNS morphology This embodiment describes an in vivo assay of the rAAV described herein for the treatment of the CNS morphology of Gaucher disease. Briefly, a Gaucher disease patient identified as having the CNS morphology of Gaucher disease (e.g., type 2 or type 3 Gaucher disease) is administered the rAAV described herein. The level of the transdextrin product described herein present in the patient's CNS (e.g., in the patient's CNS serum, in the patient's cerebrospinal fluid (CSF), or in the patient's CNS tissue) is assayed, for example, by Western blotting, enzyme function assay, or imaging study.

[0139] Example 8: Gene therapy for Parkinson's disease in subjects with mutations in GBA1 This embodiment describes the administration of recombinant adeno-associated virus (rAAV) encoding GBA1 to subjects with Parkinson's disease characterized by mutations in the GBA1 gene.

[0140] The rAAV-GBA1 vector insert contains a CBA promoter element (CBA) consisting of four parts: a CMV enhancer (CMVe), a CBA promoter (CBAp), exon 1, and an intron (int), for the constitutive expression of the codon-optimized coding sequence (CDS) of human GBA1 (chestnut). The 3' region also contains a woodchuck hepatitis virus posttranscriptional regulator (WPRE), followed by a bovine growth hormone polyA signaling (bGH polyA) tail. The adjacent ITR allows for correct packaging of the intervening sequence. Two variants of the 5' ITR sequence (Figure 7, insertion box, lower sequence) were evaluated, and these variants have several nucleotide differences within the 20-nucleotide "D" region of the ITR, which is thought to affect packaging and expression efficiency. The rAAV-GBA1 vector product contains the "D" domain nucleotide sequence shown in Figure 7 (insertion box, upper sequence). The mutant vector has a variant "D" domain (referred to herein as the "S" domain, where nucleotide changes are indicated by shading) similar to those performed in preclinical studies. The backbone contains a gene that confers resistance to kanamycin and a stuffer sequence to prevent reverse packaging. A schematic diagram of the rAAV-GBA1 vector is shown in Figure 8. The rAAV-GBA1 vector is packaged to rAAV using the AAV9 serotype capsid protein.

[0141] rAAV-GBA1 is administered to subjects as a single dose via fluoroscopy-guided suboccipital injection into the cisterna magna (intracerciome cisterna magna, ICM). One embodiment of an rAAV-GBA1 administration regimen study is as follows:

[0142] A single dose of rAAV-GBA1 administered to patients (N=12) at one of two dose levels (e.g., 3e13vg (low dose), 1e14vg (high dose)) determined based on the results of nonclinical pharmacological and toxicological studies.

[0143] Initial studies were conducted in a chemical mouse model with daily delivery of conzlitol-β-epoxide (CBE) and a GCase inhibitor to evaluate the efficacy and safety of the rAAV-GBA1 vector and the rAAV-GBA1 S-mutant construct (described further below). Furthermore, initial studies were performed in a (4L / PS-NA) gene mouse model carrying a homozygous GBA1 mutation and partially deficient in saposin. Additional dose-range studies in mice and non-human primates (NHPs) will be conducted to further evaluate the safety and efficacy of the vectors.

[0144] Two slightly different versions of the 5' inverted terminal repeat (ITR) in the AAV skeleton were tested to evaluate manufacturability and transgene expression (Figure 7). The 20 bp "D" domain within the 145 bp 5' ITR is considered necessary for optimal viral vector production, but mutations within the "D" domain have also been reported to increase transgene expression in some cases. Therefore, in addition to the viral vector rAAV-GBA1 possessing an intact "D" domain, a second vector form with a mutant D domain (referred to herein as the "S" domain) was also evaluated. Both rAAV-GBA1 and the mutant express the same transgene. Both vectors produced effective viruses in vivo, as detailed below, but rAAV-GBA1 containing the wild-type "D" domain was selected for further development.

[0145] To establish a GCase-deficient CBE model, young mice were administered CBE and a GCase-specific inhibitor. CBE was administered to the mice daily via IP injection, starting at postnatal day 8 (P8). Three different CBE doses (25 mg / kg, 37.5 mg / kg, 50 mg / kg) and PBS were tested to establish a model exhibiting a behavioral phenotype (Figure 9). Higher doses of CBE were dose-dependently lethal. All mice treated with 50 mg / kg of CBE died by P23, and 5 out of 8 mice treated with 37.5 mg / kg of CBE died by P27. Mice treated with 25 mg / kg of CBE were not lethal. While CBE-injected mice did not exhibit overall motor impairments in the open-field assay (moving the same distance and at the same speed as PBS-treated mice), CBE-treated mice showed impairments in motor coordination and balance, as measured by the rotorod assay.

[0146] Mice that survived until the end of the study were sacrificed the day after the last CBE administration (P27, "Day 1") or three days after CBE withdrawal (P29, "Day 3"). Lipid analysis was performed on the cortex of mice given 25 mg / kg of CBE to assess the accumulation of GCase substrates in both the Day 1 and Day 3 cohorts. GluSph and GalSph levels (measured in aggregates in this example) were significantly accumulated in CBE-treated mice compared to PBS-treated controls, consistent with GCase deficiency.

[0147] Based on the aforementioned studies, a CBE dose of 25 mg / kg was selected because it induced behavioral deficits without affecting survival. To achieve broad GBA1 distribution and transgene expression throughout the brain during CBE treatment, rAAV-GBA1 or excipients were delivered by intraventricular (ICV) injection on postnatal day 3 (P3), followed by the initiation of daily IP CBE or PBS treatment on P8 (Figure 10).

[0148] CBE-treated mice that received rAAV-GBA1 behaved statistically significantly better on the rotorod than mice treated with the excipient (Figure 11). The variant-treated mice did not differ from the excipient-treated mice in other behavioral measures, such as total distance traveled during the test (Figure 11).

[0149] At the completion of the survival study, half of the mice were sacrificed for biochemical analysis either the day after the last CBE administration (P36, "Day 1") or three days after CBE withdrawal (P38, "Day 3") (Figure 12). Cortical GCase activity was assessed using a bio-triple fluorescence enzyme assay. GCase activity was increased in mice treated with rAAV-GBA1, while CBE treatment reduced GCase activity. In addition, mice treated with both CBE and rAAV-GBA1 had similar GCase activity levels to the PBS-treated group, indicating that rAAV-GBA1 delivery can overcome the inhibition of GCase activity induced by CBE treatment. Lipid analysis was performed on the motor cortex of the mice to investigate the levels of the substrates GluCer and GluSph. Both lipids accumulated in the brains of mice treated with CBE and rAAV-GBA1 showed a significant reduction in substrate accumulation.

[0150] Lipid levels were negatively correlated with both GCase activity and performance on rotorods across treatment groups. Increased GCase activity after rAAV-GBA1 administration was associated with reduced substrate and enhanced motor function (Figure 13). As shown in Figure 14, preliminary in vivo distribution was measured by qPCR and assessed by the presence of vector genomes (positive was defined as over 100 vector genomes per 1 μg of genomic DNA). Mice treated with rAAV-GBA1 were positive for rAAV-GBA1 vector genomes in the cortex, both with and without CBE, indicating that ICV delivery results in rAAV-GBA1 delivery to the cortex. Furthermore, vector genomes were detected in the liver, barely detected in the spleen, and not detected in the heart, kidney, or gonads. There were no statistically significant differences between the day 1 and day 3 groups for all measurements.

[0151] Larger trials in the CBE model further explored the effective dose of rAAV-GBA1 in the CBE model. Using a 25 mg / kg CBE dose model, excipients or rAAV-GBA1 were delivered via ICV at P3, and daily IP PBS or CBE treatment was initiated at P8. Considering the intergroup similarity in CBE withdrawal observed in previous studies, all mice were sacrificed one day after the final CBE dose (P38-40). The effects of three different rAAV-GBA1 doses were evaluated, resulting in the following five group results with 10 mice per group (5M / 5F). Excipient ICV + PBS IP CBE IP with excipient ICV + 25 mg / kg 3.2e9vg (2.13e10vg / g brain) rAAV-GBA1 ICV + 25mg / kg CBE IP 1.0e10vg (6.67e10vg / g brain) rAAV-GBA1 ICV + 25mg / kg CBE IP 3.2e10vg (2.13e11vg / g brain) rAAV-GBA1 ICV + 25mg / kg CBE IP.

[0152] The highest dose of rAAV-GBA1 rescued CBE-associated weight gain failure at P37. Furthermore, this dose resulted in a statistically significant increase in rotorod and tapered beam performance compared to the excipient + CBE treatment group (Figure 15). Lethality was observed in several groups, including both the excipient and rAAV-GBA1 treatment groups (excipient + PBS: 0, excipient + 25 mg / kg CBE: 1, 3.2 e9 vg rAAV-GBA1 + 25 mg / kg CBE: 4, 1.0 e10 vg rAAV-GBA1 + 25 mg / kg CBE: 0, 3.2 e10 vg rAAV-GBA1 + 25 mg / kg CBE: 3).

[0153] At the completion of the survival study, the mice were sacrificed for biochemical analysis (Figure 16). Cortical GCase activity was evaluated biologically in triplicate by fluorescence assay. CBE-treated mice showed decreased GCase activity, while mice receiving high doses of rAAV-GBA1 showed a statistically significant increase in GCase activity compared to CBE-treated mice. CBE-treated mice also had accumulations of GluCer and GluSph, both of which were rescued by administration of high doses of rAAV-GBA1.

[0154] In addition to the established chemical CBE model, rAAV-GBA1 was also evaluated in a 4L / PS-NA genetic model that is homozygous for the V394L GD mutation in Gba1 and also partially lacks saposins that affect GCase localization and activity. These mice exhibit deficits in motor intensity, coordination, and balance, as demonstrated by their performance in beamwalk, rotarod, and wire-hang assays. Typically, these mice have a lifespan of less than 22 weeks. In the initial study, 3 μl of maximal titer virus was delivered via ICV at P23 with a final dose of 2.4 e10 vg (6.0 e10 vg / g brain). Six mice were used per group, and the treatment groups were as follows: WT + excipient ICV 4L / PS-NA + Excipient ICV rAAV-GBA1 ICV with 4L / PS-NA + 2.4e10vg (6.0e10vg / g brain)

[0155] Motor performance was evaluated using a beamwalk test four weeks after delivery of rAAV-GBA1. The mutant mice treated with rAAV-GBA1 tended to have less total slip and less slip per velocity compared to mutant mice treated with excipients, and their motor function recovered to near WT levels (Figure 17). Since the motor phenotype becomes more severe with age in these mice, their performance in this behavioral test and other behavioral tests will be evaluated at a later time. Lipid levels, GCase activity, and in vivo distribution were evaluated in these mice at the completion of the survival study.

[0156] Additional low doses of rAAV-GBA1 are currently being tested using the CBE model, corresponding to 0.03, 0.1, and 1 times the proposed Phase 1 high clinical doses. Each group contains 10 mice (5M / 5F). Excipient ICV CBE IP with excipient ICV + 25 mg / kg 3.2e8vg (2.13e9vg / g brain) rAAV-GBA1 ICV + 25mg / kg CBE IP 1.0e9vg (6.67e9vg / g brain) rAAV-GBA1 ICV + 25mg / kg CBE IP 1.0e10vg (6.67e10vg / g brain) rAAV-GBA1 ICV + 25mg / kg CBE IP.

[0157] In addition to motor phenotype, cortical lipid levels and GCase activity will be evaluated. The time course of treatment and analysis will also be recorded.

[0158] To evaluate efficacy and safety data, trials with a larger dose range were initiated. Ten 4L / PS-NA mice (5 males / 5 females per group) were injected with 10 μl of rAAV-GBA1. Using allometric brain weight calculations, the doses correlated with 0.15, 1.5, 4.4, and 14.5 times the proposed Phase 1 high clinical dose. The injection groups consisted of: WT + excipient ICV 4L / PS-NA + Excipient ICV rAAV-GBA1 ICV with 4L / PS-NA + 4.3e9vg (1.1e10vg / g brain) rAAV-GBA1ICV with 4L / PS-NA + 4.3e10vg (1.1e11vg / g brain) rAAV-GBA1 ICV of 4L / PS-NA+1.3e11vg (3.2e11vg / g brain) rAAV-GBA1 ICV with 4L / PS-NA + 4.3e11vg (1.1e12vg / g brain).

[0159] Example 9: In vitro analysis of rAAV vectors The rAAV construct was tested in vitro and in vivo. Figure 18 shows representative data for the in vitro expression of the rAAV construct encoding the progranulin (PGRN) protein. The left panel shows the standard curve for the progranulin (PGRN) ELISA assay. The bottom panel shows the dose-response of PGRN expression as measured by ELISA assay in cell lysates of HEK293T cells transduced with rAAV. MOI = Multiplicity of Infection (vector genome per cell).

[0160] Pilot studies were conducted to evaluate the in vitro activity of rAAV vectors encoding prosaposin (PSAP) and SCARB2, either 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 shown in Table 3. "Opt" refers to a nucleic acid sequence codon-optimized for expression in mammalian cells (e.g., human cells). Figure 19 shows representative data demonstrating that transfection of HEK293 cells with each construct resulted in overexpression of the corresponding gene product compared to mock-transfected cells.

[0161] Pilot studies were conducted to evaluate the in vitro activity of rAAV vectors encoding TREM2, either alone or in combination with one or more inhibitory RNAs. The vectors tested included those shown in Table 3. "Opt" refers to the nucleic acid sequence codon-optimized for expression in mammalian cells (e.g., human cells). Figures 36A–36B show representative data demonstrating that transfection of HEK293 cells in each construct resulted in overexpression of the corresponding gene product compared to mock-transfected cells. Table 3 [Table 3]

[0162] Example 10: Testing of SNCA and TMEM106B shRNA constructs HEK293 cells In this study, the human embryonic kidney 293 cell line (HEK293) was used (#85120602, Sigma-Aldrich). HEK293 cells were maintained in D-MEM [#11995065, Thermo Fisher Scientific] supplemented with 10% fetal bovine serum [FBS] [#10082147, Thermo Fisher Scientific], containing 100 units / mL of penicillin and 100 μg / mL of streptomycin (#15140122, Thermo Fisher Scientific).

[0163] 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 transfected in antibiotic-free medium at a rate of 3 × 10⁶ 5 Cells were seeded at a density of cells / ml. The following day, the plasmid and Lipofectamine 2000 reagent were mixed in Opti-MEM solution (No. 31985062, Thermo Fisher Scientific). After 5 minutes, the mixture was added to the HEK293 culture. After 72 hours, cells were harvested for RNA or protein extraction or subjected to imaging analysis. For imaging analysis, plates were pre-coated with 0.01% poly-L-lysine solution (P8920, Sigma-Aldrich) before cell seeding.

[0164] Gene expression analysis using quantitative real-time PCR (qRT-PCR) 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 transfected using Lipofectamine 2000 transfection reagent (0.5 μg plasmid and 1.5 μl reagent in 50 μl Opti-MEM solution) on HEK293 cells (7.5 × 10⁶) seeded on a 48-well plate. 4 Cells (per well) were transiently transfected. Following the manufacturer's instructions, RNA was extracted from the cells after 72 hours and used for reverse transcription to synthesize cDNA. For quantitative PCR analysis, 2–5 μL of cDNA product was double-amplified using gene-specific primer pairs (final concentration of 250 nM) in Power SYBR Green PCR Master Mix (#4367659, Thermo Fisher Scientific). The primer sequences for the SNCA, TMEM106B, and GAPDH genes are as follows: For SNCA, the gene names are 5'-AAG AGG GTG TTC TCT ATG TAG GC-3' (SEQ ID NO: 71) and 5'-GCT CCT CCA ACA TTT GTC ACT T-3' (SEQ ID NO: 72). For TMEM106B, the gene names are 5'-ACA CAG TAC CTA CCG TTA TAG CA-3' (SEQ ID NO: 73) and 5'-TGT TGT CAC AGT AAC TTG CAT CA-3' (SEQ ID NO: 74). For GAPDH, the gene names are 5'-CTG GGC TAC ACT GAG CAC C-3' (SEQ ID NO: 75) and 5'-AAG TGG TCG TTG AGG GCA ATG-3' (SEQ ID NO: 76). Quantitative PCR was performed using a QuantStudio 3 Real-Time PCR system (Thermo Fisher Scientific). Expression levels were normalized by the housekeeping gene GAPDH and calculated using the comparative CT method.

[0165] Fluorescence imaging analysis An EGFP reporter plasmid containing the 3'UTR of the human SNCA gene downstream of the EGFP coding region was used to validate SNCA and TMEM106B knockdown plasmids. The EGFP reporter plasmid and candidate knockdown plasmids were transfected using Lipofectamine 2000 transfection reagent (0.04 μg of reporter plasmid, 0.06 μg of knockdown plasmid, and 0.3 μl of reagent in 10 μl of Opti-MEM solution) on HEK293 cells (3.0 x 10⁶) seeded on poly-L-lysine coated 96-well plates. 4 Cells were simultaneously transfected (in wells). After 72 hours, the fluorescence intensity of the EGFP signal was measured using a Varioskan LUX multimode reader (Thermo Fisher Scientific) at excitation 488 nm / emission 512 nm. Cells were fixed with 4% PFA at room temperature for 10 minutes and incubated in D-PBS containing 40 μg / mL of 7-aminoactinomycin D (7-AAD) at room temperature for 30 minutes. After washing with D-PBS, the fluorescence intensity of the 7-AAD signal was measured using a Varioskan reader at excitation 546 nm / emission 647 nm, and the cell number was quantified. The normalized EGFP signal per 7-AAD signal level was compared to the control knockdown sample.

[0166] Enzyme-linked immunosorbent assay (ELISA) An α-synuclein reporter plasmid containing the 3'-UTR of either the human SNCA gene or the TMEM106B gene downstream of the SNCA coding region was used for validation of the knockdown plasmid 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 transfected using Lipofectamine 2000 transfection reagent (0.1 μg of reporter plasmid, 0.15 μg of knockdown plasmid, and 0.75 μl of reagent in 25 μl of Opti-MEM solution) on HEK293 cells (7.5 × 10⁶).4 Cells were transiently transfected (per well). After 72 hours, cells were lysed in radioimmunoprecipitation (RIPA) buffer (#89900, Thermo Fisher Scientific) supplemented with a protease inhibitor cocktail (#P8340, Sigma-Aldrich) and sonicated for several seconds. After incubation on ice for 30 minutes, the lysates were centrifuged at 20,000 × g at 4°C for 15 minutes, and the supernatant was collected. Protein levels were quantified. Plates were read with a 450 nm Varioskan plate reader, and concentrations were calculated using SoftMax Pro 5 software. The measured protein concentrations were normalized to the total protein concentration determined by bicinchoninate assay (#23225, Thermo Fisher Scientific).

[0167] Figure 37 and Table 4 show representative data demonstrating successful in vitro silencing of SNCA using the GFP reporter assay (top panel) and the α-Syn assay (bottom panel). Figure 38 and Table 5 show representative data demonstrating successful in vitro silencing of TMEM106B using the GFP reporter assay (top panel) and the α-Syn assay (bottom panel). Table 4 [Table 4] Table 5 [Table 5]

[0168] Example 11: Arrangement of ITR "D" sequence and cell transduction The effect of ITR "D" sequence placement on rAAV vector cell transduction was investigated. As shown in Figure 20, HEK293 cells were transduced with Gcase-encoded rAAVs having either 1) a wild-type ITR (e.g., a "D" sequence located proximal to the transgene insertion and distal to the end of the ITR) or 2) an ITR with a "D" sequence located "outside" the vector (e.g., a "D" sequence located proximal to the end of the ITR and distal to the transgene insertion). Surprisingly, the data showed that rAAVs with a "D" sequence located "outside" the vector retain their ability to be packaged and efficiently transduce cells (Figure 40).

[0169] Example 12: In vitro study of progranulin rAAV Figure 39 is a schematic diagram showing one embodiment of a vector containing an expression construct encoding PGRN. Progranulin is overexpressed in the CNS of rodents lacking GRN, either heterozygous or homozygous for GRN deletion, by injection of an rAAV vector encoding PGRN (e.g., codon-optimized PGRN) either intraparenchymal or intrathecal injection into the cisterna magna.

[0170] Mice are injected at 2 months or 6 months of age and aged to 6 months or 12 months of age, and analyzed for one or more of the following: GRN expression levels at the RNA and protein levels, behavioral assays (e.g., improved movement), survival assays (e.g., improved survival), microglial and inflammatory markers, gliosis, neuronal loss, lipofuscinosis, and / or lysosomal marker accumulation rescue such as LAMP1. Assays on PGRN-deficient mice are described, for example, by 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 full contents of which are incorporated herein by reference.

[0171] Example 13: In Vitro and In Vivo Tests of Progranulin rAAV To analyze the effect of an rAAV construct (PR006, also referred to as PR006A, see Figure 64) encoding progranulin (PGRN) protein, in vitro assays and in vivo assays were performed. PR006 contains a capsid with AAV9 serotype.

[0172] In Vitro Non-Clinical Tests Progranulin Expression Derived from PR006A in HEK293T Cells The ability to induce progranulin protein production of PR006A in a cellular environment was investigated. HEK293T cells were transduced with PR006A over a range of multiplicity of infection (MOI) of vector genomes (vg) / cell from 2.1×10 5 ~3.3×10 6 PR006A transduction resulted in a robust and dose-dependent increase in progranulin protein expression and secretion into the cell culture medium (Figure 60). Substantially low progranulin protein levels reflecting the expression from the endogenous human GRN gene were detected in the negative control group treated with only the excipient (intended clinical vehicle).

[0173] Effectiveness in FTD-GRN iPSC-Derived Neurons Assays were performed to analyze the in vitro effectiveness of the rAAV construct in human FTD-GRN (frontotemporal dementia with GRN mutation) neuron cultures. The cell lines were obtained from the National Institute of Neurological Disorders and Stroke (NINDS) Human Cell and Data Repository (NHCDR). Materials ND50015 (FTD-GRN, M1L), ND50060 (FTD-GRN, R493X) and ND38555 (control, wild type) (see Table 6). Table 6: Summary of Characteristics of iPSC Cell Lines

Table 6

[0174] To establish a cell model pathologically related to FTD-GRN, iPSCs derived from each strain were differentiated into neurons using a two-step protocol. In the first step, the iPSCs were differentiated into proliferating neuronal stem cell (NSC) lines that lacked the expression of pluripotency markers (i.e., OCT4 and SSEA1) and acquired the expression of neural stem cell markers (i.e., SOX2, Nestin, SOX1, and PAX6), as detected by immunofluorescence labeling.

[0175] Control and FTD-GRN NSC strains were seeded at equal densities, and after 48 hours, progranulin expression in cell lysates (intracellular progranulin) (Figure 52E) and cell culture medium (secreted progranulin) (Figure 52A) was measured by enzyme-linked immunosorbent assay (ELISA). Progranulin expression was normalized to total protein concentration to account for differences in cell number (n=3, mean ± SEM). NSC strains with heterozygous GRN mutations had significantly lower intracellular and secreted progranulin levels compared to control NSCs, and FTD-GRN NSCs expressed approximately 25-50% of the endogenous progranulin level. This suggests that this FTD-GRN cell model replicates the clinical progranulin deficiency observed in FTD-GRN patients expressing one-third to one-half of normal progranulin levels in plasma (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)).

[0176] NSCs derived from all cell lines were differentiated into neuronal cell cultures. After confirming that iPSC-derived NSCs exhibited reduced progranulin expression, the lines were differentiated into neurons to generate clinically representative cell types for the preclinical efficacy study of PR006A. The NSCs were seeded in neuronal differentiation medium and finally differentiated into postmittal neurons for 7 days, after which the expression of neuronal markers (i.e., MAP2, NeuN, Tau, Tuj1, NF-H) was evaluated by immunofluorescence (Figure 52G). Using this protocol, both control and FTD-GRN iPSC-derived NSC lines differentiated efficiently into neurons.

[0177] The efficacy of PR006A in vitro was evaluated using FTD-GRN iPSC-derived neuronal cultures. FTD-GRN neurons were cultured in a 2.7 × 10⁶ size. 5 , 5.3×10 5 , or 1.1 × 10 6 Cells were treated with excipients or PR006A at the MOI of vg / cell. PR006 transduction resulted in robust, dose-dependent expression of secreted progranulin in all cell lines, as measured by ELISA (Figure 52B). Endogenous progranulin levels were evaluated in excipient-treated control and FTD-GRN neurons. Control neurons expressed endogenous secreted progranulin, while secreted progranulin was not detected in FTD-GRN neurons (Figure 52B). Linear regression analysis confirmed a significant correlation between PR006A dose and progranulin levels across both FTD-GRN cell lines (p = 3.5 × 10⁻⁶). -13 These results demonstrate that treatment with PR006A leads to increased progranulin secretion in the FTD-GRN neuron model.

[0178] Progranulin is known to stimulate the maturation of the lysosomal protease cathepsin D (CTSD), and loss of its function is thought to be involved in lysosomal storage disorders and neurodegeneration. CTSD is expressed as an inactive full-length proprotein (proCTSD) that undergoes proteolytic treatment to the enzymatically active mature protease (matCTSD). Progranulin has been reported to act as a molecular chaperone that binds to proCTSD and enhances its maturation to the matCTSD protease. In FTD-GRN neuron cultures, PR006 transduction rescued defective cathepsin D maturation (Figure 52C). Control, FTD-GRN#1, and FTD-GRN#2 neurons were transduced with PR006A or excipients. Progranulin levels were restored to at least twice that of the control cells, resulting in a 5.3 × 10⁶ 5 PR006A with a MOI of [specified value] was used in efficacy experiments (Figure 52B). To evaluate efficacy, proCTSD and matCTSD expression levels were measured in cell lysates using an automated Simple Western® (Jess) platform (Figure 52C). Excipient-treated FTD-GRN neurons had a lower ratio of matCTSD to proCTSD compared to excipient-treated control neurons, and PR006A treatment significantly increased the ratio in both FTD-GRN neuron lines (Figure 52C). In control neurons, the ratio of matCTSD to proCTSD did not significantly change with PR006A treatment. These findings demonstrate that PR006A restores lysosomal function-related phenotypes in FTD-GRN neurons.

[0179] In normal neurons, the TDP-43 (transactive response DNA-binding protein 43kDa) protein is localized in the nucleus. In the postmortem brains of FTD-GRN patients, aggregation of TDP-43 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 downstream toxicity in neurons. Since Grn KO mice do not fully reproduce this TDP-43 pathology, induced pluripotent stem cell (iPSC)-derived neurons are a valuable FTD-GRN model for studying TDP-43 biology. As described by Valdez et al. (Human Molecular Genetics 26, 4861-4872 (2017)), reduced nuclear accumulation of TDP-43 and increased accumulation of insoluble TDP-43 have been reported in iPSC-derived neurons from patients with FTD-GRN compared to control neurons without the GRN mutation. Transduction of neuronal cultures from both FTD-GRN mutant strains with PR006A reversed TDP-43 abnormalities, resulting in a decrease in insoluble TDP-43 (measured using the Simple Western® (Jess) platform (Figure 52D)) and an increase in the nuclear localization of TDP-43 (measured using immunofluorescence (Figure 52F)).

[0180] In summary, transduction of PR006 restored defective maturation in the lysosomal enzyme cathepsin D and improved abnormal TDP-43 pathology in FTD-GRN neurons.

[0181] In vivo nonclinical trials Efficacy and in vivo distribution in aged Grn knockout mice The in vivo efficacy and maximum dose of PR006A were evaluated in a Grn knockout (KO) mouse model. The Grn KO mouse model used in these studies was B6(Cg)-Grrn tm1.1AidiIn the / J (Jackson Laboratory, Bar Harbor, ME), exons 1-4 were deleted from the target progranulin (Grn) gene (Yin et al., J Exp Med 207, 117-128 (2010)). These animals completely lost progranulin and exhibited age-dependent phenotypes including lysosomal alterations, neuronal lipofuscin accumulation, ubiquitin accumulation, microgliosis, and neuroinflammation, and are therefore widely used to model FTD-GRN. All attempts were made to eliminate bias from the study. Mice were assigned to treatment groups balanced in terms of sex and weight, and experimental endpoints were blinded and evaluated by qualified personnel.

[0182] In the initial tests, PR006A was measured at 9.7 × 10⁻⁶ 10 vg(2.4×10 11 The drug was delivered to aged GRN KO mice at a dose of vg / g brain, which was the highest dose achievable at the time of the study due to limitations on the injection volume and the physical titer of the viral lot used in the study. Aged mice were used because many FTD-GRN-related phenotypes, including CNS inflammation and microgliosis, develop in an age-dependent manner, and the most pronounced symptoms of the phenotype occur between 12 and 24 months of age.

[0183] In studies using aged Grn KO mice, PR006A was administered by a single intraventricular (ICV) injection. The dose was either 10 μl of excipients (intended clinical vehicle, 20 mM Tris (pH 8.0), 200 mM NaCl, and 1 mM MgCl2 + 0.001% Pluronic F68) or 9.7 × 10⁻¹⁴ 10 vg PR006A (2.4 x 10 11 vg / g brain (based on the brain weight of 400 mg of adult mouse brain) was delivered by ICV injection to the following two cohorts of aged Grn KO mice: (1) 16 months of age at injection (n=4 / group, PRV-2018-027, Figure 61) and (2) 14 months of age at injection (n=3 / group, PRV-2019-002, Figure 61). The animals were sacrificed 2 months after injection.

[0184] In the PRV-2018-027 trial, a single dose of PR006A was delivered to 16-month-old mice in the following treatment groups. [Table 7]

[0185] Due to unexpected trial deviations (errors in genotyping and premature loss of animals), the PRV-2019-002 trial (14-month cohort) enrolled only one mouse in the excipient treatment group instead of the planned n=3. Because statistical analysis is impossible due to the small sample size, this trial is excluded from further discussion. However, the findings from this trial were similar to those from trial PRV-2018-027.

[0186] In vivo distribution and progranulin expression: In vivo distribution was determined by measuring the presence of vector genomes using a qPCR assay that meets the current US Food and Drug Administration Center for Biologics Evaluation and Research (CBER) / Office of Tissues and Advanced Therapies (OTAT) criteria for PCR sensitivity (more than 50 vector genomes per 1 μg of genomic DNA were defined as positive). All mice treated with PR006A were positive for vector genomes in the cerebral cortex and spinal cord, indicating that ICV administration successfully results in PR006A transduction in the brain and CNS (Figure 59A). ICV PR006A resulted in significant levels of human progranulin protein in the CNS (brain, spinal cord) of Grn KO mice, but, as expected, human progranulin was undetectable in mice treated with excipients (Figure 59B). Since progranulin is primarily a secreted protein, its expression in CSF can be considered a surrogate for protein production in the brain, representing a potential translational endpoint in FTD-GRN patients with reduced CSF progranulin levels. While human progranulin could be detected in the CSF of PR006A-treated mice, the measurement of CSF progranulin levels fell below the lower limit of quantification (LLOQ) of the assay due to the small sample volume and technical limitations in obtaining sufficient amounts of CSF in mice (Figure 59C).

[0187] ICV administration also resulted in the presence of widespread vector genomes and progranulin protein levels in peripheral tissues, including the liver, heart, lungs, kidneys, spleen, and gonads (Figures 62A-62B). Furthermore, significant levels of human progranulin were detectable in the plasma of PR006A-treated Grn KO mice. As expected, human progranulin was not detected in excipient-treated Grn KO mice.

[0188] Lipofuscin accumulation: The accumulation of neuronal lipofuscin, an autofluorescent material with high electron density that gradually accumulates over time in the lysosomes of post-mitotic cells and is an indicator of lysosomal dysfunction, is a characteristic age-dependent phenotype of Grn KO mice. The accumulation of lipofuscin was evaluated in adjacent brain sections using the following two independent methods. (1) In a more clinical approach, the accumulation of lipofuscin in the brain was scored by a blinded pathologist on a scale of 0 (lipofuscin not observed) to 4 (extensive lipofuscin accumulation), and (2) in a more quantitative approach, the autofluorescence of lipofuscin was detected by immunohistochemistry (IHC) and automatically quantified. Grn KO mice exhibited substantial lipofuscinosis throughout the brain, and ICV PR006A treatment reduced the severity of lipofuscin scores in the cerebral cortex, hippocampus, and thalamus (Figure 59D). Quantification of lipofuscin accumulation from IHC images also detected a reduction in lipofuscinosis with PR006A treatment in all three brain regions. Since ubiquitin-positive inclusions are a definitive pathological feature of FTD-GRN patients that also accumulate in the Grn KO mouse model in an age-dependent manner, IHC was performed and quantified in the target brain regions (cerebral cortex, hippocampus, thalamus) to evaluate the accumulation of ubiquitin. PR006A treatment significantly reduced the accumulation of ubiquitin in Grn KO mice (Figure 59E). These findings suggest that PR006A improves lysosomal dysfunction in the Grn KO mouse model of FTD-GRN.

[0189] Neuroinflammation: Chronic CNS inflammation is a pathological feature in the brains of patients with FTD-GRN, which is reproduced in an age-dependent manner in Grn KO mice. Progranulin has anti-inflammatory effects in a mouse model of FTD-GRN, and progranulin loss leads to upregulation of pro-inflammatory cytokines, including TNFα. In this study, treatment with PR006A suppressed inflammatory marker levels in aged Grn KO mice. ICV PR006A reduced the gene expression of pro-inflammatory cytokines Tnf (TNFα) and Cd68 (CD68), microglial markers, in the cerebral cortex (Figure 59F). Furthermore, TNFα protein levels were also reduced in cerebral cortical samples from Grn KO mice treated with PR006A, using the Mesoscale Discovery mouse pro-inflammatory cytokine assay (Figure 59G). To further evaluate neuroinflammation, immunohistochemical (IHC) analysis was performed for Iba1, microgliosis markers, and GFAP and astrocytosis markers, and quantified in the target brain regions (cerebral cortex, hippocampus, and thalamus). PR006A treatment resulted in a tendency towards reduced microgliosis (Iba1) in Grn KO mice, but did not affect astrocytosis (GFAP) (Figure 59H, Figure 59I). Taken together, these results indicate that PR006A treatment reduces neuroinflammation in an aged Grn KO mouse model of FTD-GRN.

[0190] Histopathology: Thorough histopathological analysis of hematoxylin and eosin (H&E) stained brains, thoracic spinal cords, livers, hearts, spleens, lungs, and kidneys of all mice obtained from these studies, performed by blinded, committee-certified pathologists, revealed no adverse events associated with PR006A treatment. Administration of PR006A to Grn KO mice resulted in a reduction in the incidence and / or severity of model-specific findings, including a decrease in the frequency and / or severity scores of neuronal necrosis in the medulla and pons. Furthermore, PR006A treatment reduced both the incidence and severity of axonal degeneration in the thoracic spinal cord. These findings are discussed in detail in the following toxicology section.

[0191] Conclusion: 9.7×10 10 vg(2.4×10 11 ICV PR006A at a dose of vg / g brain resulted in the presence of a widespread vector genome throughout the brain and peripheral tissues in aged Grn KO mice. PR006A treatment increased overall progranulin expression. Furthermore, PR006A reduced the accumulation of lipofuscin and ubiquitin in the brain, a condition known to occur in both Grn KO mouse models and patients with FTD-GRN. PR006A also reduced the expression of pro-inflammatory cytokines and immune cell activation in the cerebral cortex, a phenotype exhibiting chronic CNS inflammation.

[0192] Efficacy of dose range in adult Grn knockout mice To further evaluate the effective dose of PR006A, a larger dose-range study was conducted in adult Grn KO mice. In PRV-2019-004, 10 μl of excipients (intended clinical vehicle, 20 mM Tris (pH 8.0), 200 mM NaCl, and 1 mM MgCl2 + 0.001% Pluronic F68) or PR006A was delivered to 4-month-old animals via ICV. These adult mice were used instead of aged Grn KO mice because the latter were not available in sufficient numbers to conduct the dose-range study. Adult Grn KO mice have a milder phenotype than aged mice, but they still exhibit lysosomal deficiency and neuroinflammatory changes, and are therefore suitable for evaluating the effective dose range of PR006A. To evaluate the efficacy of PR006A across a wide range of viral doses, PR006A was administered at 1.1 × 10⁶, the highest dose achievable at the time of the study, due to the injection volume constraints and physical potency of the viral lot used in the study. 11 vg(2.7×10 11 (vg / g brain), 1.1 × 10 10 vg(2.7×10 10 Moderate dose (vg / g brain), or 1.1 × 10 9 vg(2.7×10 9Low doses (vg / g brain) were administered in a complete logarithmic difference across each dose. Details of the experimental design are shown in Figure 63.

[0193] The following three doses of PR006A were evaluated in groups of 10 mice (4 males / 6 females). [Table 8]

[0194] In this study, Grn knockout mice (7-month-old C57BL / 6J) carrying the wild-type (WT) Grn allele, and age-matched mice of the same background strain, served as controls for the selected efficacy endpoints. [Table 9]

[0195] In vivo distribution and progranulin expression: In vivo distribution was determined by measuring the presence of the vector genome using a qPCR assay that meets the current US Food and Drug Administration (CBER / OTAT) criteria for PCR sensitivity (a positive result was defined as having more than 50 vector genomes per μg of genomic DNA). Mice treated with PR006A were dose-dependently positive for the vector genome in the cerebral cortex and spinal cord, indicating that ICV administration successfully induces PR006A transduction in the CNS (Figure 53A). qRT-PCR analysis of the GRN encoding PR006A revealed that ICV administration of PR006A resulted in dose-dependent induction of human GRN mRNA expression in the cerebral cortex (Figure 53B). PR006A treatment increased levels of human progranulin protein in the brain and spinal cord (Figure 53C). Human progranulin levels were detected and quantified in brain tissue at the highest dose of PR006A. At low doses, progranulin levels fell below the detection limit of the assay 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 fall far 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, and in both the cerebral 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. Since human and mouse progranulins were measured using different detection assays employing non-interspecies cross-reactive anti-progranulin antibodies, an accurate comparison of absolute numbers is not possible.

[0196] PR006A administration also resulted in the presence of widespread vector genomes and progranulin protein levels in peripheral tissues, including the liver, heart, lungs, kidneys, spleen, and gonads (Figures 53D, 53E).

[0197] In plasma, significant levels of human progranulin were detected at all dose levels in Grn KO mice treated with PR006A (Figure 53F). As expected, human progranulin was not detected in Grn KO mice treated with excipients. The levels of human progranulin in animals treated with moderate doses of PR006A were within the same range as the levels of mouse progranulin measured in mice with the WT Grn allele. Since human and mouse progranulin were measured using different detection assays employing non-interspecies cross-reactive anti-progranulin antibodies, an accurate comparison of absolute numbers is not possible.

[0198] Lipofuscin accumulation: Lipofuscin accumulation was evaluated in adjacent brain sections using two independent methods: (1) a more clinical approach, where lipofuscin accumulation in the brain was scored by a blinded pathologist on a scale of 0 (no lipofuscin observed) to 4 (extensive lipofuscin accumulation); and (2) a more quantitative approach, where lipofuscin autofluorescence was detected by IHC and automatically quantified. Grn KO mice exhibited lipofuscinosis throughout the brain, while WT mice did not have detectable lipofuscin in the brain (Figure 53G). ICV administration of PR006A resulted in a dose-dependent reduction in the severity score of intracellular lipofuscin accumulation in the brain of Grn KO mice (Figure 53G). The efficacy of PR006A in reducing lipofuscinosis can be most readily quantified in brain regions exhibiting the most robust lipofuscinosis phenotype in the Grn KO mouse model of FTD-GRN, including the hippocampus and thalamus. In addition to pathologist-mediated lipofuscin scoring, intravascular coagulation (IHC) performed in target brain regions (i.e., cerebral cortex, hippocampus, and thalamus) to quantitatively assess lipofuscinosis detected dose-dependent decreases in lipofuscin accumulation in the cerebral cortex and thalamic brain regions, with significant decreases observed at moderate and high doses of PR006A. IHC was also performed to assess ubiquitin accumulation in the brain, another FTD-GRN-related pathology occurring in Grn KO mice. Compared to WT mice, Grn KO mice showed increased ubiquitin throughout the brain (Figure 53H). PR006A significantly reduced the size of ubiquitin immunoreactive objects to near WT levels in all three doses (Figure 53H).

[0199] Neuroinflammation: Treatment with PR006A suppressed inflammatory marker levels in the brains of adult Grn KO mice. ICV PR006A suppressed 2.7 × 10⁻⁶ levels. 9 vg / g brain ~2.7 × 10 11Across the vg / g brain dose range, gene expression of the microglial markers, the pro-inflammatory cytokines Tnf (TNFα) and Cd68 (CD68), in the cerebral cortex was reduced (Figure 53I). Consistent with published data, increased gene expression of these neuroinflammatory markers was observed in excipient-treated Grn KO mice compared to age-matched mice with wild-type Grn alleles (Figure 53I). In contrast to observations in 18-month-old Grn KO mice from PRV-2018-027 and reports of TNFα abnormalities in the literature, there was no robust increase in cerebral cortical TNFα protein levels in 7-month-old adult excipient-treated Grn KO mice, and in addition, no significant changes in PR006A were observed in Grn KO mice. These findings are consistent with previously published findings that a robust neuroinflammatory phenotype does not occur in the Grn KO mouse model until 12–24 months of age. Immunohistochemical testing (IHC) was performed to further evaluate neuronal inflammation by quantifying in target brain regions (cerebral cortex, hippocampus, and thalamus) and staining for Iba1, a marker of microgliosis, and GFAP, a marker of astrocytosis. Compared to WT mice, Grn KO mice showed a significant increase in microgliosis (Iba1) and astrocytosis (GFAP) throughout the brain (Figures 53J-53K). PR006A treatment significantly reduced microgliosis (Iba1) in all three doses (Figure 53J). Moderate doses of PR006A showed a tendency to reduce astrocytosis (GFAP), and high doses of PR006A showed a significant reduction in astrocytosis (GFAP) in the thalamic brain region (Figure 53K).

[0200] While many phenotypes in Grn KO models appear later in life, studies have reported that Grn KO mice exhibit a wide range of gene expression changes, including alterations in lysosomal and immune-related pathways, as early as 4 months of age. Therefore, in addition to the targeted qRT-PCR analysis described above, we employed a transcriptomics approach to assess changes at the mRNA level. This can be comprehensively evaluated using a highly sensitive, high-throughput technique (RNA sequencing) and minimizes the required sample material. We performed RNA sequencing on the cerebral cortex and used gene set mutation analysis (GSVA) ​​(Hanzelmann et al., BMC Bioinformatics 14,7 (2013)) to determine which gene expression pathways were altered in 7-month-old excipient-treated Grn KO mice compared to age-matched WT mice of the same strain. We confirmed the deficiencies in lysosomal and immune-related pathways in Grn-deficient mice, as reported in previously published studies. Significant changes were reported in the GO TERM (GO:0005773) "vacuole" gene subset (including four genes reported to be dysregulated in Grn KO mice as described by Lui et al (Cell 165, 921-935 (2016))), the "lysosomal gene" set (a subset of 25 lysosomal-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 Gene Set Enrichment Analysis HALLMARK database (including genes encoding components of the complement system, which is part of the innate immune system). The activity levels of these gene sets were then measured and compared with PR006A treatment (Figures 53L-53N). Treatment with PR006A dose-dependently reversed the gene set deficiencies observed in Grn KO mice.

[0201] Histopathology: Thorough histopathological analysis of the brain, thoracic spinal cord, liver, heart, spleen, lungs, kidneys, and gonads of all mice obtained from these studies was performed by a blinded, committee-certified pathologist for hematoxylin and eosin (H&E) staining, and no evidence of toxicity associated with the PR006A treatment was found. Details of the toxicity analysis are described in the following section.

[0202] Conclusion: 2.7×10 9 vg / g brain ~2.7 × 10 11 ICV PR006A at doses in the vg / g brain range resulted in dose-dependent, widespread vector genome presence throughout the brain and peripheral tissues. PR006A treatment also resulted in progranulin mRNA and protein production in the CNS. A clear dose-response relationship between PR006A and reduced readout of lipofuscinosis and lysosomal dysfunction was observed across multiple brain regions. Robust and statistically significant reductions in lipofuscinosis were observed at moderate and maximum dose levels of PR006A. All doses of PR006A reduced ubiquitin accumulation in the brain. 2.7 × 10⁻⁶ 9 Starting with the lowest dose of vg / g brain, PR006A reduced the expression of pro-inflammatory markers in the brain at the RNA and protein levels.

[0203] Summary: In vivo nonclinical trials PR006A effectively transduced Grn KO mice, resulting in robust, dose-dependent in vivo distribution of the transgene and production of progranulin mRNA and protein in the CNS. PR006A dose-dependently reversed gene expression abnormalities in lysosomal and neuroinflammatory pathways. PR006A reduced many phenotypes occurring in the brain of this FTD-GRN mouse model, including lipofuscinosis, ubiquitin accumulation, and microgliosis. In dose-range studies, 2.7 × 10⁻⁶ 9 The lowest dose of vg / g brain PR006A significantly suppressed the expression of inflammatory markers in the cerebral cortex. The moderate dose of 2.7 × 10⁶ doses significantly suppressed the expression of inflammatory markers. 10PR006A in vg / g brain improved both lysosomal deficiency (e.g., lipofuscinosis) and neuroinflammation in a robust and statistically significant manner. High doses of 2.7 × 10⁻⁶ 11 PR006A in vg / g brains further increased progranulin expression without evidence of toxicity. Table 7: Summary of biodistribution [Table 10] The in vivo distribution of positive results is defined as genomic DNA exceeding 50 vg / μg.

[0204] Safety pharmacology Throughout these trials, no adverse events attributable to the test product were observed. Safety findings from in-animal and histopathological analyses in PRV-2018-027, PRV-2019-002, and PRV-2019-004 are discussed in the following sections.

[0205] Single-dose toxicity A series of non-clinical studies with PR006A were conducted to investigate safety endpoints in mice and monkeys. Three studies were conducted in a Grn KO mouse model, and endpoints included neuropathological evaluations and assessed both the protective activity and potential toxicity resulting from PR006A administration via intraventricular (ICV) injection. ICM administration is more technically challenging in mice. These mouse models are representative of FTD-GRN, in patients with mutations in the GRN gene that result in reduced progranulin levels. Neuropathological examinations were also performed as part of a pilot study in which PR006A was injected into the cisterna magna (ICM) in cynomolgus monkeys. GLP studies were conducted in cynomolgus monkeys to which PR006A was delivered via the ICM, and the monkeys were sacrificed on day 7, day 30, or day 183. GLP studies incorporated a comprehensive list of clinical endpoints in addition to anatomical and pathological evaluations of a complete list of tissues. The following single-dose studies were conducted to support single-dose administration in clinics.

[0206] Maximum dose of PR006A (PRV-2018-027 and PRV-2019-002) in aged FTD-GRN mouse models. As part of these efficacy studies in Grn KO mice, neuropathological evaluations were performed in mice treated with ICV with either an excipient or PR006A. Grn KO mice are widely used as a model for FTD-GRN due to their complete loss of progranulin and age-dependent phenotypes, including lysosomal alterations, neuronal lipofuscin accumulation, microgliosis, and neuroinflammation. The pharmacological aspects of the study are summarized in the section above, and the toxicology-related endpoints evaluated in this study are summarized below. Two studies of PR006A were conducted in an aged Grn KO mouse model. In the first study (PRV-2018-027), nine 16-month-old mixed-sex Grn KO mice received ICV with either PR006A or an excipient. Animals were sacrificed 9 weeks after administration. The following single PR006A dose group was included in this study: Total dose 9.7 × 10⁶ 10 vg(2.4×10 11 10 μl of undiluted virus (vg / g brain) was administered. The control group was treated with 10 μl of excipient. Table 8: Test design for PRV-2018-027 [Table 11] ROA: Route of administration

[0207] Various postmortem endpoints, including in vivo distribution, lysosomal changes, and inflammatory markers, were evaluated as part of this study protocol (see section above). Animal viability was checked twice daily, and body weight was measured once daily. After euthanasia two months after treatment, target tissue was collected, fixed by dropping into cooled 4% paraformaldehyde, and stored at 4°C. Unnecessary tissue portions from eight animals that completed the study were trimmed, processed, and embedded in paraffin blocks. These were then sectioned to approximately 5 μm, stained with hematoxylin and eosin (H&E), and examined by a board-accredited veterinary pathologist.

[0208] During this study, one mouse died early in the treatment group. No abnormalities were recorded in the deceased animal during necropsy, and therefore the cause of death is unknown. No other deaths or abnormalities were observed. All treatment groups were followed similarly in terms of body weight, and no significant differences were found.

[0209] Histopathological examination revealed no adverse findings related to PR006A. Extensive lipofuscin accumulation in the brain was consistent with expected findings in Grn KO mice. In animals treated with PR006A, there was a decrease in the severity score for lipofuscin accumulation in all brain regions. Morphological changes also appeared to show a slight decrease in frequency and / or severity scores, particularly with respect to neuronal necrosis in the medulla and pons, in PR006A treatment. However, these trends in morphological changes were not as consistent as the trends in lipofuscin scores.

[0210] Axonal degeneration was observed in the thoracic spinal cord, and very rarely (1 in 4 animals in each group) minimal neuronal necrosis was seen. In animals treated with PR006A, there was a slight reduction in both the incidence and severity of axonal degeneration.

[0211] The following findings, likely associated with Grn homozygous knockout mice, appeared to be reduced in incidence and / or severity in animals treated with PR006A: dilated tubules in the renal medulla, glomerulosis in the kidneys, and foreign bodies in the lungs (typically in the airways, characterized by linear, acellular, dark pink structures frequently associated with foreign giant cells and / or macrophages). A larger cohort of animals would be needed to draw more definitive conclusions.

[0212] All other histopathological findings observed were considered incidental and / or of similar incidence and severity in animals treated with the excipients and test samples, and were therefore considered unrelated to the administration of PR006A.

[0213] In the second study (PRV-2019-002), five 14-month-old mixed-sex Grn KO mice received ICV administration of either PR006A or an excipient. The animals were sacrificed 8 weeks after administration. A single PR006A dose group was included in this study: total dose 9.7 × 10⁶ 10 vg(2.4×10 11 For the VG / G brain, 10 μl of undiluted virus was used, and the control group was treated with 10 μl of excipient. Table 9: Test design for PRV-2019-002 [Table 12] * The genotyping results at the end of the study confirmed that the n=1 animal from the excipient group was Grn heterozygous KO instead of the expected Grn homozygous KO.

[0214] The animals were analyzed using the same method as in the PRV-2018-027 study. Animal survival was checked twice daily, and body weight was measured once daily. After euthanasia two months after treatment, target tissue was collected, fixed by dropping into cooled 4% paraformaldehyde, and stored at 4°C until evaluation.

[0215] In the central nervous system (CNS), findings consistent with those previously observed in Grn KO mice were observed in the brain (Yin et al., J Exp Med 207(1):117-128(2010)). Specifically, there was widespread increased accumulation of lipofuscin throughout the brain. Rarely, minimal neuronal necrosis was also observed (in one untreated animal that died prematurely and in one animal treated with excipients).

[0216] Due to the small sample size, it was not possible to show a consistent trend in findings related to the treatment. There was no consistent difference in response between the test sample (PR006A) and the excipients.

[0217] Regarding non-CNS tissues, findings consistent with the phenotype of Grn KO mice were observed in the kidney (tubular dilation and infiltration of mononuclear inflammatory cells) and liver (Kupffer cells / sinusoidal parallel cells, and vacuolation of Kupffer cell granulomas) (Yin et al., J Exp Med 207(1):117-128(2010)).

[0218] In all animals that underwent surgery and were enrolled in the study, findings of "glomerulopathy" were observed. No published reports were found of this finding as a change associated with standard, unloaded Grn knockout mice, however, one study demonstrated progranulin-deficient mice treated with a diet that induced hyperhomocysteinemia and developed thickening of the glomerular basement membrane and loss of podocyte foot processes (Fu et al., Hypertension 69(2):259-266(2017)).

[0219] All other findings were consistent with those commonly observed in laboratory mice. Due to the small sample size, no definitive differences related to the treatment were observed.

[0220] Dose range PR006A (PRV-2019-004) in adult FTD-GRN mouse model To further evaluate the safety of PR006A, a larger dose-range study was conducted in adult Grn KO mice. A total of 40 mixed-sex mice were divided into four groups and administered either the excipient or one of three doses of PR006A by a single unilateral ICV injection into the left hemisphere. All animals received a total dose volume of 10 μl, regardless of the treatment group. Mice were treated at 4 months of age and euthanized 3 months post-treatment. An additional wild-type (WT) control group, including untreated C57BL / 6J mice (same background strain) aged up to approximately 7 months, was also euthanized and subjected to similar necropsy.

[0221] The test was conducted according to the following test design: Table 10: Test design for PRV-2019-004 [Table 13]

[0222] During the experiment, the animals were checked for survival twice a day and weighed once a week. Three months after treatment, the mice were euthanized and various postmortem evaluations were performed to assess the efficacy of PR006A (see above section). Additionally, sections stained with H&E from the brain, spinal cord, liver, heart, spleen, lungs, kidneys, and gonads were evaluated by a board-certified pathologist.

[0223] In the histopathological examination, no harmful PR006A-related findings were observed in any of the mice, regardless of the treatment group.

[0224] Findings consistent with the phenotype of the Grn KO mouse model were present, such as intracellular lipofuscin accumulation in various regions of the brain, including the cerebral cortex, basal ganglia, hippocampus, thalamus / hypothalamus, cerebellum, and brainstem (especially the pons and medulla). No clear evidence of morphological changes (neuronal vacuolization and gliosis) was observed in the H&E-stained sections. Lipofuscin pigment accumulation can be detected before easily detectable morphological changes and thus functions as an appropriate biomarker for efficacy. All Grn homozygous KO groups showed lipofuscin accumulation, but there were differences in the severity of this finding across the treatment groups. The highest frequency of higher scores for lipofuscin accumulation was greatest in the group of animals treated with vehicle (group 1). Among the animals treated with PR006A, a higher frequency of higher scores was observed in group 4 (low-dose PR006A, 2.7×10 9 vg / g brain), followed by group 3 (moderate-dose PR006A, 2.7×10 10 vg / g brain). The lowest severity score was observed in group 2 (high-dose PR006A, 2.7×10 11 vg / g brain). These findings indicate a dose-dependent decrease in the severity score of intracellular lipofuscin accumulation in the brains of Grn homozygous knockout mice. All other histopathological findings were considered incidental and / or of similar incidence and severity in animals treated with vehicle and test article and were thus considered unrelated to the administration of PR006A.

[0225] GLP single-dose study in monkeys (PRV-2018-028) Test design The objective of this GLP study was to evaluate the toxicity and biodistribution of the test product, PR006A, after a single dose via ICM injection to cynomolgus monkeys at observation periods of 6, 29, or 182 days after administration. Animals were sacrificed on days 7, 30, or 183 of the study. This study was designed to evaluate two dose levels: the highest dose was the maximum viable dose achievable with 1.2 mL volume of undiluted PR006A (the highest volume previously administered), and the lower dose was equivalent to a dose one log unit lower than the higher dose. The dose was 4.8 × 10⁻⁶. 11 Low dose of vg, and 4.8 × 10 12 This is equivalent to a high dose of vg, and the estimated brain weight of the cynomolgus monkey (NHP species) used in this study is 74g, which is approximately 6.5 × 10⁻⁶. 9 vg / g brain and 6.5 × 10 10 The brain was vg / g. This study also included a control group in which animals received only 1.2 mL of excipients (20 mM Tris (pH 8.0), 200 mM NaCl, and 1 mM MgCl2 + 0.001% [w / v] Pluronic F68). This study utilized both male and female cynomolgus macaques. The day 7 group included one female at the highest dose and was designed as a sentinel for early toxicity, while the remaining two time points (days 30 and 183) included two males and one female at each dose. In addition to samples from multiple brain regions, peripheral tissue samples were collected for qPCR analysis. All samples that were positive by qPCR were analyzed for transgene expression. A tabular summary of the study design is shown in Table 11. Table 11: Summary of GLP NHP Test PRV-2018-028 [Table 14-1] [Table 14-2] [Table 14-3] Abbreviations: F, female; ICM, cisterna magna; M, male; MgCl2, magnesium chloride; NaCl, sodium chloride; vg, vector genome(s); DRG, dorsal root ganglion; GALT, intestinal lymphoid tissue.

[0226] Cynomolgus monkeys with NHP were evaluated through multiple survival observations and measurements, including mortality / morbidity (daily), clinical observation (daily), body weight (baseline and weekly thereafter), visual inspection of food consumption (daily), neurological observation (baseline and between weeks 2 and 26), indirect fundus examination (baseline and between weeks 2 and 26), and electrocardiogram (ECG) measurements (baseline and between weeks 2 and 26).

[0227] Neutralizing antibodies (nAb) against the AAV9 capsid were analyzed at baseline and at euthanasia on days 7, 30, or 183. Clinical pathology tests, consisting of hematological, coagulation, clinical chemistry, and urinalysis, were performed twice at baseline (once for blood and once for urine) and once between weeks 1 and 13 of the administration phase.

[0228] The animals were euthanized, and tissues were collected on day 7, day 30, or day 183. The tissues outlined in Table 11 were collected from all animals, where present, weighed (where applicable), and divided into replicas. For histopathological evaluation, one replica was stored in 10% neutral buffered formalin (unless a special fixative was required for optimal fixation) (all animals). Further replicas were collected for qPCR and transgene expression analysis.

[0229] Safety and Toxicology There were no unplanned deaths, and all animals survived until scheduled autopsies. There were no adverse PR006A-related clinical observations, weight changes, ophthalmic observations, or physical or neurological findings. No drug-related abnormalities were observed in any cohort on macroscopic examination at autopsy. Furthermore, 6.5 × 10 9 or 6.5 × 10 10There were no PR006A-related changes in the PR interval, QRS duration, QT interval, corrected QT (QTc) interval, or heart rate observed in male or male / female combinations administered vg / g brain. No abnormal ECG waveforms or arrhythmias were observed during qualitative evaluation of the ECG.

[0230] Biodistribution Biodistribution analysis of the PR006A transgene was performed using a qPCR-based assay. On day 183 in the high-dose group (6.5×10 10 vg / g brain), there was widespread transduction across the CNS and periphery, and all tissues were positive for the presence of the vector at the cutoff of 50 vg / μg of DNA, which is the lower limit of quantification of the qPCR assay. Data from selected representative regions from day 183 are shown in Figure 54A. Data from day 30 are not shown. On day 30 in the high-dose group (6.5×10 10 vg / g brain), all CNS tissues examined were positive for transduction except the putamen. Tissues from animals treated with low dose (6.5×10 9 vg / g brain) were positive in the CNS on day 183, but only the spleen and liver were positive from peripheral tissues. Additionally, one female NHP treated with high-dose PR006A was positive in the ovaries on day 7, and males treated with high dose were positive in the testes on days 30 and 183. PR006A transduction was most robust in the liver and nervous system tissues and consistently low in other peripheral organs examined. In the brain, vector transduction was stable on day 183 compared to day 30, demonstrating robust and persistent transduction of the transgene.

[0231] In NHPs receiving ICM administration of PR006A, there was a significant humoral immune response against the transgene product, progranulin, and anti-progranulin antibodies were detected in serum and CSF samples collected on days 30 and 183 after treatment, indicating that the human progranulin protein was expressed in NHPs. Anti-drug antibody (ADA) levels were determined using established immunoassay techniques. Data are shown in Figure 54B.

[0232] PR006A(GRN) expression was measured at the mRNA level using an RT-qPCR-based assay and at the protein level using Simple Western® (Jess) analysis. Simultaneously with PR006A transduction levels, transgene expression was observed in selected brain regions (Figure 54C), liver, gonads, spinal cord, and DRG collected at day 183 by mRNA measurement using RT-qPCR.

[0233] Transgene expression was measurable in the brain and liver at both doses of PR006A, and the expression levels were both dose-dependent and sustained. In the gonads, expression was measurable in males only at high doses, and in females at both doses, expression was measurable at days 7 and 30, but not at day 183.

[0234] To confirm the production of human progranulin in treated NHP, protein levels in CSF were assessed using the Simple Western® (Jess) platform. Detailed methodology is provided in Example 14. The method was limited to measuring progranulin levels in CSF samples from FTD-GRN patients and establishing that these levels were approximately half of those measured in CSF samples from healthy human controls and from FTD patients without GRN mutations. Results from CSF showed a dose-dependent increase in progranulin levels in animals treated with both low and high doses of PR006A (Figure 54D). These results suggest that effective and widespread transduction by PR006A in NHP after ICM administration leads to increased progranulin levels.

[0235] Because the Simple Western (Jess) assay is unsuitable for measuring progranulin levels in brain tissue due to high levels of nonspecific background banding, progranulin protein measurement has focused on CSF. Currently available assays are not reliable for measuring transgene-derived human progranulin levels in NHP tissue due to high levels of nonspecific background. CSF levels are generally considered to reflect relevant brain concentrations, and they are particularly valuable as translational biomarkers for clinical research.

[0236] summary In any of the non-clinical studies, including a small pilot non-GLP trial in NHP and a GLP trial in NHP up to day 183, no adverse safety findings or toxicity concerns were found that would preclude the initiation of clinical trials. Pathological findings in the GLP trial showed a low number of affected cells and consistently minimal severity across both dose groups. No other in-vivo or post-mortem PR006A-related adverse findings were reported.

[0237] Phase 1 / 2 trial in human subjects with FTD-GRN Human subjects (n=15) will be enrolled in an open-label trial of recombinant PR006 AAV. Inclusion criteria include: age 30–80 years (including upper and lower limits), presence of a pathogenic GRN mutation, symptomatic disease, and stable use of background medication prior to administration of the investigational drug. Each subject will receive the investigational drug as a single intracisional cell (ICM) injection. The trial will include a 3-month biomarker readout, a 12-month clinical readout, and a 5-year safety and clinical follow-up. The trial will analyze: (1) safety and tolerability; (2) key biomarkers including progranulin, NfL (neurofilament light chain), and volumetric MRI (magnetic resonance imaging); and (3) efficacy: CDR plus NACC FTLD (Clinical Dementia Rating plus National Alzheimer's Coordinating Center Frontal Temporal Lobar Dementia), behavioral, cognitive, language, functional, and QoL (quality of life) measures. Table 12: Examples of neurodegenerative diseases [Table 15] Table 13: Examples of synuclein disease [Table 16] Table 14: Examples of tauopathies [Table 17] Table 15: Examples of lysosomal storage disorders [Table 18]

[0238] Example 14: Automated Western Assay for Detection of Progranulin in Cerebrospinal Fluid The objective of this experiment was to quantify progranulin (PGRN) protein levels in cerebrospinal fluid (CSF) using the ProteinSimple (San Jose, CA) Automated Western Platform Jess. This assay method can be used to analyze non-human primate (NHP) CSF samples. To determine the expression level of the human progranulin protein, PR006A transgene product, CSF samples from non-human primate subjects were analyzed on the Simple Western (Jess) platform using an antibody that specifically detects human progranulin protein. The Simple Western (Jess) platform is a capillary-based automated Western blot immunoassay platform in which all steps, including protein separation, immunoprobing, washing, and chemiluminescent detection, are performed in a capillary cartridge. Samples (4-fold dilution), primary antibody against human progranulin (Adipogen PG-359-7, 10-fold dilution), secondary antibody, and all buffers prepared by ProteinSimple were loaded into customized cartridges and run on the Jess platform. Semi-quantitative data analysis was performed automatically after each run, and parameters such as signal intensity, peak area, and signal-to-noise ratio were calculated using the Jess instrument. For each individual sample, the level of progranulin was measured as the peak area of ​​immunoreactivity against the antibody. All analyses were performed using blinded samples.

[0239] The assays described herein were performed on CSF samples derived from non-human primate research. CSF samples were tested for the presence and levels of progranulin protein to evaluate the efficacy of gene therapy using the rAAV construct encoding progranulin (PGRN) protein (PR006; see Figure 64). In this study, either the excipient or PR006 was injected into NHP animals via intracisional cell mass injection (ICM) at a low dose of PR006 (1.8 × 10⁶). 10 (vg / g brain weight) or high dose of PR006 (1.8 × 10) 11The samples were delivered in vg / g brain weight. Each group consisted of three animals. Nine NHP animals were sacrificed 180 days post-infection (Table 16), and CSF samples were analyzed using a Jess-based assay. Table 16: Summary of NHP animals showing grouping and medication. [Table 19] Table 17: Materials for Automated Western Assay [Table 20] Note: All reagents must be warmed to room temperature before opening the vials.

[0240] When implementing this method, the following steps were followed: Preparation of stock solution: 1. Add 40 μL of water to the clear tube in the EZ Standard Pack separation module to prepare a 400 mM DTT solution. Mix gently. 2. To prepare the master mixture, add 20 μL of 10× sample buffer and 20 μL of 400 mM DTT to the EZ Pink Master Mixture tube. Mix gently. 3. To prepare the biotinylated ladder, pipette 20 μL of water into an EZ Clear biotinylated ladder tube containing a pink pellet. Mix gently. 4. Add equal amounts of each to prepare a mixture of luminol and peroxide. In one run, add 200 μL of luminol to 200 μL of hydrogen peroxide. A primary antibody diluent (10-fold dilution) is prepared by mixing 5.25 μL of primary antibody with 225 μL of antibody diluent 2. Sample preparation: 1. Dilute the sample in 0.1× sample buffer. Prepare 0.1× sample buffer by adding 10 μL of 10× sample buffer to 990 μL of water. 2. Dilute the sample as needed. For example, the NHP CSF sample was diluted fourfold before adding the master mixture. Add 5 μL of NHP CSF to 15 μL of 0.1X sample buffer. Prepare the sample by adding a 3.1X master mixture to a 4X sample. To perform technical replication, prepare a total of 15 μL of sample and a master mixture for each sample. For example, add 3 μL of master mixture to 12 μL of diluted sample. Mix gently. 4. Boil the sample at 95°C for 5 minutes. 5. Centrifuge the sample briefly using a benchtop mini centrifuge. Vortex before loading the sample. Fill the cartridge with reagents and samples: 1. Pipette all samples according to the cartridge map. a. Pipette 15 μL of luminol + peroxide mixture into each well in lane E. b. Pipette 10 μL of streptavidin into the first well in lane D. c. Pipette 10 μL of secondary antibody into the remaining 24 wells in lane D. d. Pipette 10 μL of antibody diluent into the first well of lane C. e. Pipette 10 μL of primary antibody diluent into the remaining 24 wells in lane C. f. Pipette 10 μL of antibody diluent into all wells in lane B. g. Pipette 10 μL of the prepared EZ Ladder into the first well in lane A. h. Pipette 5 μL of the sample and master mixture solution into the replication lane in lane A. 2. Rotate the cartridge at 2500 RPM for 5 minutes at room temperature. Load the capillary and cartridge into the instrument: 1. Load the capillary into the slot. Make sure the light is blue. 2. Load the spin cartridge into the device. 3. After the blue light on the device stops flashing, press the start button.

[0241] The assay system was considered acceptable if the coefficient of variation (CV) percentage for the replicates was 30% or less.

[0242] Before using the assay to detect progranulin in NHP CSF samples, the assay was tested as follows: Qualification testing of the Jess assay included evaluation of dilution linearity, selectivity, and specificity. Dilution linearity of the Jess assay was determined using typical CSF samples from BioIVT. Selectivity and specificity of the Jess assay were determined using CSF samples from frontotemporal dementia (FTD) patients with PGRN mutations (obtained from the National Centralized Repository for Alzheimer's Disease and Related Dementias (NCRAD, Indianapolis, Indiana)). Table 18: Summary of Results [Table 21]

[0243] Results and Discussion Dilution linearity The dilution linearity of PGRN proteins detected by Jess was tested in commercially available (BioIVT) CSF samples from normal individuals. The endogenous level of PGRN in the CSF samples was measured to determine the dilution linearity. Two individuals were tested at 2x serial dilutions ranging from 2x to 64x.

[0244] Table 19 reports the peak area of ​​PGRN proteins at 58 kDa detected by Jess, and the percentage difference for each dilution from a 16-fold dilution. Results within the linear range are shown in bold (within 100 ± 30% difference). Dilution linearity was established to be within 4 to 16 folds. [Table 22]

[0245] In summary, all tested matrices exhibited acceptable linear ranges, passing the tolerance criteria of 0 ± 30% difference, although range sizes and dilutions differed between matrices. Sample linearity (MRD) was established at a 4-fold dilution. Dilution linearity was established within the dilution range of 4 to 16 times. Table 20 summarizes the MRDs and linear dilution ranges that pass the tolerance criteria for CSF. Table 20: MRD and linear dilution range of CSF [Table 23]

[0246] Selectivity and specificity The selectivity and specificity of PGRN proteins detected by Jess were tested in CSF samples derived from PR006 FTD patient samples from NCRAD. Three groups of CSF samples (groups A, B, and C) were collected from heterozygous FTD patients (group A), familial non-carriers (group B or C), and normal individuals (group B or C). Six samples were analyzed from each group. The sample groups are listed in Table 16 under FTD patient CSF sample information.

[0247] CSF samples were diluted fourfold in 0.1X sample buffer provided by ProteinSimple and tested in technical replicas. Replicas of samples with a resulting %CV greater than 20% were reanalyzed. Results with a %CV less than 20% are reported in Table 22. Table 22 reports the peak area of ​​PGRN protein at 58 kDa detected by Jess, and the %CV between replicas. The results show that PGRN levels in groups B and C were approximately twice as high as in group A, demonstrating the selectivity and specificity of the Jess assay in determining PGRN levels for CSF samples (Figure 55). Table 21: CSF sample information from FTD patients [Table 24-1] [Table 24-2] Table 22: Selectivity and specificity results [Table 25]

[0248] CSF samples from FTD patients (Table 21) were also analyzed using a human PGRN ELISA kit (Adipogen, AG-45A-0018YEK-KI01). The results from the ELISA (Figure 56) showed a similar trend in PGRN levels between groups as in the Jess assay, demonstrating that the Jess assay is optimal for evaluating PGRN levels in CSF samples.

[0249] In conclusion, the ProteinSimple Automated Western Jess assay was determined to be optimal for evaluating PGRN levels in NHP CSF samples.

[0250] Table 23 shows the Jess data for NHP CSF samples. Each sample represents the average across two technical replicas. The peak area for the 58kD band in the sample lane is reported. The data is presented as the average peak area of ​​the technical replicas and the adjusted dilution factor. Table 23: Jess data for NHP CSF samples [Table 26]

[0251] The objective of this assay was to confirm the level of progranulin (PGRN) protein expression in a target tissue region for NHP testing after transduction with PR006. This was performed using an automated Western blotting platform that detects progranulin protein using a monoclonal antibody. Progranulin expression is measurable in CSF in both control and PR006-treated NHP, and this assay does not distinguish between endogenous progranulin protein and PR006A-induced progranulin protein.

[0252] This application incorporates, by reference, the entire contents of the following documents: International PCT Application Publication WO2019 / 070893, International PCT Application Publication WO2019 / 070891, U.S. Provisional Patent Application No. 62 / 567,296 filed on October 3, 2017, titled "GENE THERAPIES FOR LYSOSOMAL DISORDERS", U.S. Provisional Patent Application No. 62 / 567,311 filed on October 3, 2017, titled "GENE THERAPIES FOR LYSOSOMAL DISORDERS", U.S. Provisional Patent Application No. 62 / 567,319 filed on October 3, 2017, titled "GENE THERAPIES FOR LYSOSOMAL DISORDERS", and U.S. Provisional Patent Application No. 62 / 567,319 filed on October 3, 2018, titled "GENE THERAPIES FOR LYSOSOMAL Filings No. 62 / 567,301, titled "DISORDERS", No. 62 / 567,310, filed on October 3, 2017, titled "GENE THERAPIES FOR LYSOSOMAL DISORDERS", No. 62 / 567,303, filed on October 3, 2017, titled "GENE THERAPIES FOR LYSOSOMAL DISORDERS", and No. 62 / 567,305, filed on October 3, 2017, titled "GENE THERAPIES FOR LYSOSOMAL DISORDERS".

[0253] Having thus described several aspects of at least one embodiment of the present invention, it will be understood that those skilled in the art will readily conceive of various changes, modifications, and improvements. Such changes, modifications, and improvements are intended to be part of this disclosure and within the spirit and scope of the invention. Accordingly, the foregoing description and drawings are merely examples.

[0254] While several embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily conceive of various other means and / or structures to perform the functions described herein and / or to obtain one or more of the results and / or benefits, and each of such variations and / or modifications will be considered within the scope of the present invention. More generally, those skilled in the art will readily understand that all parameters, dimensions, materials and configurations described herein are intended to be illustrative, and that actual parameters, dimensions, materials and / or configurations will depend on the particular use or combination of uses in which the teachings of the present invention are used. Those skilled in the art will be able to recognize or confirm many equivalents of the particular embodiments of the present invention described herein by means of ordinary experimentation alone. Therefore, it should be understood that the embodiments described herein are presented merely as examples, and within the scope of the appended claims and their equivalents, the present invention may be carried out in ways other than those specifically described and claimed. The present invention covers each individual feature, system, article, material and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, and / or methods is included within the scope of the present invention if such features, systems, articles, materials, and / or methods are not mutually inconsistent.

[0255] As used in this specification and in the claims, the indefinite articles "a" and "an" should be understood to mean "at least one" unless explicitly stated otherwise.

[0256] As used herein in this specification and in the claims, the expression “and / or” should be understood to mean “either or both” of the elements thus combined, that is, elements that exist together in some cases and separately in others. Other elements other than those specifically identified by the “and / or” clause may exist, whether related to the specifically identified elements or not, unless otherwise explicitly indicated. Thus, as a non-restrictive example, when used in combination with unrestrictive language such as “including,” a reference to “A and / or B” may, in one embodiment, mean A without B (optionally including elements other than B), in another embodiment, mean B without A (optionally including elements other than A), in yet another embodiment, mean both A and B (optionally including other elements), and so on.

[0257] Where used herein and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” should be interpreted as inclusive, that is, including a number of items or at least one of the elements of the list, but more than one, and optionally, additional unlisted items. Only terms clearly indicated in the opposite way, such as “one of” or “exactly one of,” or “consisting of” where used in the claims, refer to including a number of items or exactly one of the elements of the list. In general, where used herein, the term “or” should be interpreted as indicating exclusive substitution (i.e., “one or the other, but not both”) only when preceded by terms of exclusivity such as “either,” “one of,” “one of,” or “exactly one of.”

[0258] As used herein in this specification and in the claims, the expression “at least one” relating to a list of one or more elements means at least one element selected from any one or more elements of the list of elements, but not necessarily including at least one of each and all elements specifically described in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows for the existence of elements other than those specifically identified in the list of elements to which the expression “at least one” refers, whether or not they relate to the specifically identified elements. Therefore, 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 mean, in one embodiment, that there is at least one, optionally one or more A, and no B (and optionally an element other than B); in another embodiment, that there is at least one, optionally more than one B, and no A (and optionally an element other than A); and in yet another embodiment, that there is at least one, optionally more than one A, and at least one, optionally one or more B (and optionally an other element).

[0259] The use of sequential terms such as “first,” “second,” and “third” in the claims to modify elements of the claims does not in itself imply importance, priority, or order of one element of a claim relative to other elements, or the temporal order in which the actions of the method are performed, but is used solely as a label to distinguish elements of the claims, to differentiate one element of a claim having a particular name from another element having the same name (apart from the use of sequential terms).

[0260] Conversely, unless explicitly stated otherwise, in any method claimed herein that includes more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are enumerated.

[0261] Each of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referenced in this application are incorporated herein by reference in their entirety.

[0262] array In some embodiments, an expression cassette encoding one or more gene products (e.g., a first, second, and / or third gene product) contains or consists of (or encodes a peptide having) the sequence described in any one of SEQ ID NOs: 1 to 91. In some embodiments, the gene product is encoded by any one portion (e.g., a fragment) of SEQ ID NOs: 1 to 91.

[0263] Numbered Embodiments Notwithstanding the attached claims, this disclosure describes the following numbered embodiments of the disclosure:

[0264] 1. An isolated nucleic acid comprising an expression construct encoding a Gcase protein adjacent to two adeno-associated virus (AAV) inverted terminal repeats (ITRs), (i) At least one of the ITRs includes a modified "D" region relative to the wild-type AAV2 ITR (SEQ ID NO: 29), and / or (ii) The isolated nucleic acid, wherein the Gcase protein is encoded by a codon-optimized nucleic acid sequence.

[0265] 2. The isolated nucleic acid according to Embodiment 1, wherein the Gcase protein comprises the amino acid sequence or a portion thereof described in SEQ ID NO: 14.

[0266] 3. The isolated nucleic acid according to Embodiment 1 or 2, wherein the Gcase protein is encoded by a codon-optimized nucleic acid sequence, optionally the nucleic acid sequence described in Sequence ID No. 15.

[0267] 4. The isolated nucleic acid according to any one of Embodiments 1 to 3, wherein the modified "D" region is a "D" sequence located outside the ITR with respect to the expression construct.

[0268] 5. The isolated nucleic acid according to any one of Embodiments 1 to 4, wherein the ITR containing the modified "D" sequence is a 3'ITR.

[0269] 6. An isolated nucleic acid according to any one of Embodiments 1 to 5, further comprising a TRY sequence, wherein the TRY sequence is optionally described in Sequence ID No. 28.

[0270] 7.An isolated nucleic acid comprising an expression construct encoding a prosaposin protein adjacent to two adeno-associated virus (AAV) inverted terminal repeats (ITRs), (i) At least one of the ITRs includes a modified "D" region relative to the wild-type AAV2 ITR (SEQ ID NO: 29), and / or (ii) The isolated nucleic acid, wherein the prosaposin protein is encoded by a codon-optimized nucleic acid sequence.

[0271] 8. The isolated nucleic acid according to Embodiment 7, wherein the prosaposin protein comprises the amino acid sequence or a portion thereof described in SEQ ID NO: 16.

[0272] 9. The isolated nucleic acid according to Embodiment 7 or 8, wherein the prosaposin protein is encoded by a codon-optimized nucleic acid sequence, optionally the nucleic acid sequence described in Sequence ID No. 17.

[0273] 10. The isolated nucleic acid according to any one of embodiments 7 to 9, wherein the modified "D" region is a "D" sequence located outside the ITR with respect to the expression construct.

[0274] 11. The isolated nucleic acid according to any one of embodiments 7 to 10, wherein the ITR containing the modified "D" sequence is a 3'ITR.

[0275] 12. An isolated nucleic acid according to any one of Embodiments 7 to 11, further comprising a TRY sequence, wherein the TRY sequence is optionally described in Sequence ID No. 28.

[0276] 13. An isolated nucleic acid comprising an expression construct encoding a SCARB2 protein adjacent to two adeno-associated virus (AAV) inverted terminal repeats (ITRs), (i) At least one of the ITRs includes a modified "D" region relative to the wild-type AAV2 ITR (SEQ ID NO: 29), and / or (ii) The isolated nucleic acid, wherein the SCARB2 protein is encoded by a codon-optimized nucleic acid sequence.

[0277] 14. The isolated nucleic acid according to Embodiment 13, wherein the SCARB2 protein comprises the amino acid sequence or a portion thereof described in SEQ ID NO: 18.

[0278] 15. The isolated nucleic acid according to Embodiment 13 or 14, wherein the SCARB2 protein is encoded by a codon-optimized nucleic acid sequence or the nucleic acid sequence described in Sequence ID No. 19.

[0279] 16. The isolated nucleic acid according to any one of embodiments 13 to 15, wherein the modified "D" region is a "D" sequence located outside the ITR with respect to the expression construct.

[0280] 17. The isolated nucleic acid according to any one of embodiments 13 to 16, wherein the ITR containing the modified "D" sequence is a 3'ITR.

[0281] 18. An isolated nucleic acid according to any one of embodiments 13 to 17, further comprising a TRY sequence, wherein the TRY sequence is optionally described in Sequence ID No. 28.

[0282] 19. An isolated nucleic acid comprising an expression construct encoding a GBA2 protein adjacent to two adeno-associated virus (AAV) inverted terminal repeats (ITRs), (i) At least one of the ITRs includes a modified "D" region relative to the wild-type AAV2 ITR (SEQ ID NO: 29), and / or (ii) The isolated nucleic acid, wherein the GBA2 protein is encoded by a codon-optimized nucleic acid sequence.

[0283] 20. The isolated nucleic acid according to Embodiment 19, wherein the GBA2 protein comprises the amino acid sequence or a portion thereof described in SEQ ID NO: 30.

[0284] 21. The isolated nucleic acid according to Embodiment 19 or 20, wherein the GBA2 protein is encoded by a codon-optimized nucleic acid sequence or the nucleic acid sequence described in Sequence ID No. 31.

[0285] 22. The isolated nucleic acid according to any one of embodiments 19 to 21, wherein the modified "D" region is a "D" sequence located outside the ITR with respect to the expression construct.

[0286] 23. The isolated nucleic acid according to any one of embodiments 19 to 22, wherein the ITR containing the modified "D" sequence is a 3'ITR.

[0287] 24. An isolated nucleic acid according to any one of embodiments 19 to 23, further comprising a TRY sequence, wherein the TRY sequence is optionally described in Sequence ID No. 28.

[0288] 25. An isolated nucleic acid comprising an expression construct encoding a GALC protein adjacent to two adeno-associated virus (AAV) inverted terminal repeats (ITRs), (i) At least one of the ITRs includes a modified "D" region relative to the wild-type AAV2 ITR (SEQ ID NO: 29), and / or (ii) The isolated nucleic acid, wherein the GALC protein is encoded by a codon-optimized nucleic acid sequence.

[0289] 26. The isolated nucleic acid according to Embodiment 25, wherein the GALC protein comprises the amino acid sequence or a portion thereof described in SEQ ID NO: 33.

[0290] 27. The isolated nucleic acid according to Embodiment 25 or 26, wherein the GALC protein is encoded by a codon-optimized nucleic acid sequence or the nucleic acid sequence described in Sequence ID No. 34.

[0291] 28. The isolated nucleic acid according to any one of embodiments 25 to 27, wherein the modified "D" region is a "D" sequence located outside the ITR with respect to the expression construct.

[0292] 29. The isolated nucleic acid according to any one of embodiments 25 to 28, wherein the ITR containing the modified "D" sequence is a 3'ITR.

[0293] 30. An isolated nucleic acid according to any one of embodiments 25 to 29, further comprising a TRY sequence, wherein the TRY sequence is optionally described in Sequence ID No. 28.

[0294] 31. An isolated nucleic acid comprising an expression construct encoding a CTSB protein adjacent to two adeno-associated virus (AAV) inverted terminal repeats (ITRs), (i) At least one of the ITRs includes a modified "D" region relative to the wild-type AAV2 ITR (SEQ ID NO: 29), and / or (ii) The isolated nucleic acid, wherein the CTSB protein is encoded by a codon-optimized nucleic acid sequence.

[0295] 32. The isolated nucleic acid according to Embodiment 31, wherein the CTSB protein comprises the amino acid sequence or a portion thereof described in SEQ ID NO: 30.

[0296] 33. The isolated nucleic acid according to Embodiment 31 or 32, wherein the CTSB protein is encoded by a codon-optimized nucleic acid sequence or the nucleic acid sequence described in Sequence ID No. 36.

[0297] 34. The isolated nucleic acid according to any one of embodiments 31 to 33, wherein the modified "D" region is a "D" sequence located outside the ITR with respect to the expression construct.

[0298] 35. The isolated nucleic acid according to any one of embodiments 31 to 34, wherein the ITR containing the modified "D" sequence is a 3'ITR.

[0299] 36. An isolated nucleic acid according to any one of embodiments 31 to 35, further comprising a TRY sequence, wherein the TRY sequence is optionally described in Sequence ID No. 28.

[0300] 37. An isolated nucleic acid comprising an expression construct encoding the SMPD1 protein adjacent to two adeno-associated virus (AAV) inverted terminal repeats (ITRs), (i) At least one of the ITRs includes a modified "D" region relative to the wild-type AAV2 ITR (SEQ ID NO: 29), and / or (ii) The isolated nucleic acid, wherein the SMPD1 protein is encoded by a codon-optimized nucleic acid sequence.

[0301] 38. The isolated nucleic acid according to Embodiment 37, wherein the SMPD1 protein comprises the amino acid sequence or a portion thereof described in SEQ ID NO: 37.

[0302] 39. The isolated nucleic acid according to Embodiment 37 or 38, wherein the SMPD1 protein is encoded by a codon-optimized nucleic acid sequence or the nucleic acid sequence described in Sequence ID No. 38.

[0303] 40. The isolated nucleic acid according to any one of embodiments 37 to 39, wherein the modified "D" region is a "D" sequence located outside the ITR with respect to the expression construct.

[0304] 41. The isolated nucleic acid according to any one of embodiments 37 to 40, wherein the ITR containing the modified "D" sequence is a 3'ITR.

[0305] 42. An isolated nucleic acid according to any one of embodiments 37 to 41, further comprising a TRY sequence, wherein the TRY sequence is optionally described in Sequence ID No. 28.

[0306] 43.An isolated nucleic acid comprising an expression construct encoding a GCH1 protein adjacent to two adeno-associated virus (AAV) inverted terminal repeats (ITRs), (i) At least one of the ITRs includes a modified "D" region relative to the wild-type AAV2 ITR (SEQ ID NO: 29), and / or (ii) The isolated nucleic acid, wherein the GCH1 protein is encoded by a codon-optimized nucleic acid sequence.

[0307] 44. The isolated nucleic acid according to Embodiment 43, wherein the GCH1 protein comprises the amino acid sequence or a portion thereof described in SEQ ID NO: 45.

[0308] 45. The isolated nucleic acid according to Embodiment 43 or 44, wherein the GCH1 protein is encoded by a codon-optimized nucleic acid sequence or the nucleic acid sequence described in Sequence ID No. 46.

[0309] 46. ​​The isolated nucleic acid according to any one of embodiments 43 to 45, wherein the modified "D" region is a "D" sequence located outside the ITR with respect to the expression construct.

[0310] 47. The isolated nucleic acid according to any one of embodiments 43 to 46, wherein the ITR containing the modified "D" sequence is a 3'ITR.

[0311] 48. An isolated nucleic acid according to any one of embodiments 43 to 47, further comprising a TRY sequence, wherein the TRY sequence is optionally described in Sequence ID No. 28.

[0312] 49.An isolated nucleic acid comprising an expression construct encoding the RAB7L protein adjacent to two adeno-associated virus (AAV) inverted terminal repeats (ITRs), (i) At least one of the ITRs includes a modified "D" region relative to the wild-type AAV2 ITR (SEQ ID NO: 29), and / or (ii) The isolated nucleic acid, wherein the RAB7L protein is encoded by a codon-optimized nucleic acid sequence.

[0313] 50. The isolated nucleic acid according to Embodiment 49, wherein the RAB7L protein comprises the amino acid sequence or a portion thereof described in SEQ ID NO: 47.

[0314] 51. The isolated nucleic acid according to Embodiment 49 or 50, wherein the RAB7L protein is encoded by a codon-optimized nucleic acid sequence or the nucleic acid sequence described in Sequence ID No. 48.

[0315] 52. The isolated nucleic acid according to any one of embodiments 49 to 51, wherein the modified "D" region is a "D" sequence located outside the ITR with respect to the expression construct.

[0316] 53. The isolated nucleic acid according to any one of embodiments 49 to 52, wherein the ITR containing the modified "D" sequence is a 3'ITR.

[0317] 54. An isolated nucleic acid according to any one of embodiments 49 to 53, further comprising a TRY sequence, wherein the TRY sequence is optionally described in Sequence ID No. 28.

[0318] 55. An isolated nucleic acid comprising an expression construct encoding the VPS35 protein adjacent to two adeno-associated virus (AAV) inverted terminal repeats (ITRs), (i) At least one of the ITRs includes a modified "D" region relative to the wild-type AAV2 ITR (SEQ ID NO: 29), and / or (ii) The isolated nucleic acid, wherein the VPS35 protein is encoded by a codon-optimized nucleic acid sequence.

[0319] 56. The isolated nucleic acid according to Embodiment 55, wherein the VPS35 protein comprises the amino acid sequence or a portion thereof described in Sequence ID No. 49.

[0320] 57. The isolated nucleic acid according to Embodiment 55 or 56, wherein the VPS35 protein is encoded by a codon-optimized nucleic acid sequence or the nucleic acid sequence described in Sequence ID No. 50.

[0321] 58. The isolated nucleic acid according to any one of embodiments 55 to 57, wherein the modified "D" region is a "D" sequence located outside the ITR with respect to the expression construct.

[0322] 59. The isolated nucleic acid according to any one of embodiments 55 to 58, wherein the ITR containing the modified "D" sequence is a 3'ITR.

[0323] 60. An isolated nucleic acid according to any one of embodiments 55 to 59, further comprising a TRY sequence, wherein the TRY sequence is optionally described in SEQ ID NO: 28.

[0324] 61. An isolated nucleic acid comprising an expression construct encoding an IL-34 protein adjacent to two adeno-associated virus (AAV) inverted terminal repeats (ITRs), (i) At least one of the ITRs includes a modified "D" region relative to the wild-type AAV2 ITR (SEQ ID NO: 29), and / or (ii) The isolated nucleic acid, wherein the IL-34 protein is encoded by a codon-optimized nucleic acid sequence.

[0325] 62. The isolated nucleic acid according to Embodiment 61, wherein the IL-34 protein comprises the amino acid sequence or a portion thereof described in SEQ ID NO: 55.

[0326] 63. The isolated nucleic acid according to Embodiment 61 or 62, wherein the IL-34 protein is encoded by a codon-optimized nucleic acid sequence or the nucleic acid sequence described in Sequence ID No. 56.

[0327] 64. The isolated nucleic acid according to any one of embodiments 61 to 63, wherein the modified "D" region is a "D" sequence located outside the ITR with respect to the expression construct.

[0328] 65. The isolated nucleic acid according to any one of embodiments 61 to 64, wherein the ITR containing the modified "D" sequence is a 3'ITR.

[0329] 66. An isolated nucleic acid according to any one of embodiments 61 to 65, further comprising a TRY sequence, wherein the TRY sequence is optionally described in SEQ ID NO: 28.

[0330] 67.An isolated nucleic acid comprising an expression construct encoding a TREM2 protein adjacent to two adeno-associated virus (AAV) inverted terminal repeats (ITRs), (i) At least one of the ITRs includes a modified "D" region relative to the wild-type AAV2 ITR (SEQ ID NO: 29), and / or (ii) The isolated nucleic acid, wherein the TREM2 protein is encoded by a codon-optimized nucleic acid sequence.

[0331] 68. The isolated nucleic acid according to Embodiment 67, wherein the TREM2 protein comprises the amino acid sequence or a portion thereof described in SEQ ID NO: 57.

[0332] 69. The isolated nucleic acid according to Embodiment 67 or 68, wherein the TREM2 protein is encoded by a codon-optimized nucleic acid sequence or the nucleic acid sequence described in Sequence ID No. 58.

[0333] 70. The isolated nucleic acid according to any one of embodiments 67 to 69, wherein the modified "D" region is a "D" sequence located outside the ITR with respect to the expression construct.

[0334] 71. The isolated nucleic acid according to any one of embodiments 67 to 70, wherein the ITR containing the modified "D" sequence is a 3'ITR.

[0335] 72. An isolated nucleic acid according to any one of embodiments 67 to 71, further comprising a TRY sequence, wherein the TRY sequence is optionally described in SEQ ID NO: 28.

[0336] 73. An isolated nucleic acid comprising an expression construct encoding the TMEM106B protein adjacent to two adeno-associated virus (AAV) inverted terminal repeats (ITRs), (i) At least one of the ITRs includes a modified "D" region relative to the wild-type AAV2 ITR (SEQ ID NO: 29), and / or (ii) The isolated nucleic acid, wherein the TMEM106B protein is encoded by a codon-optimized nucleic acid sequence.

[0337] 74. The isolated nucleic acid according to Embodiment 73, wherein the TMEM106B protein comprises the amino acid sequence or a portion thereof described in SEQ ID NO: 63.

[0338] 75. The isolated nucleic acid according to Embodiment 73 or 74, wherein the TMEM106B protein is encoded by a codon-optimized nucleic acid sequence or the nucleic acid sequence described in Sequence ID No. 64.

[0339] 76. The isolated nucleic acid according to any one of embodiments 73 to 75, wherein the modified "D" region is a "D" sequence located outside the ITR with respect to the expression construct.

[0340] 77. The isolated nucleic acid according to any one of embodiments 73 to 76, wherein the ITR containing the modified "D" sequence is a 3'ITR.

[0341] 78. An isolated nucleic acid according to any one of embodiments 73 to 77, further comprising a TRY sequence, wherein the TRY sequence is optionally described in SEQ ID NO: 28.

[0342] 79.An isolated nucleic acid comprising an expression construct encoding a progranulin (PGRN) protein adjacent to two adeno-associated virus (AAV) inverted terminal repeats (ITRs), (i) At least one of the ITRs includes a modified "D" region relative to the wild-type AAV2 ITR (SEQ ID NO: 29), and / or (ii) The isolated nucleic acid, wherein the PGRN protein is encoded by a codon-optimized nucleic acid sequence.

[0343] 80. The isolated nucleic acid according to Embodiment 79, wherein the PGRN protein comprises the amino acid sequence or a portion thereof described in SEQ ID NO: 67.

[0344] 81. The isolated nucleic acid according to Embodiment 79 or 80, wherein the PGRN protein is encoded by a codon-optimized nucleic acid sequence or the nucleic acid sequence described in Sequence ID No. 68.

[0345] 82. The isolated nucleic acid according to any one of embodiments 79 to 81, wherein the modified "D" region is a "D" sequence located outside the ITR with respect to the expression construct.

[0346] 83. The isolated nucleic acid according to any one of embodiments 79 to 82, wherein the ITR containing the modified "D" sequence is a 3'ITR.

[0347] 84. An isolated nucleic acid according to any one of embodiments 79 to 83, further comprising a TRY sequence, wherein the TRY sequence is optionally described in Sequence ID No. 28.

[0348] 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 a portion thereof listed in Table 1.

[0349] 86. The isolated nucleic acid according to Embodiment 85, wherein the first gene product is a Gcase protein or a portion thereof.

[0350] 87. The isolated nucleic acid according to Embodiment 85 or 86, wherein the second gene product is LIMP2 or a portion thereof, or a prosaposin or a portion thereof.

[0351] 88. An isolated nucleic acid according to any one of embodiments 85 to 87, further encoding an interfering nucleic acid (e.g., shRNA, miRNA, dsRNA, etc.), wherein the interfering nucleic acid optionally inhibits the expression of α-Syn or TMEM106B.

[0352] 89. The isolated nucleic acid according to any one of embodiments 85 to 88, further comprising one or more promoters, each of which optionally is independently a chicken beta-actin (CBA) promoter, a CAG promoter, a CD68 promoter, or a JeT promoter.

[0353] 90. An isolated nucleic acid according to any one of embodiments 85 to 89, further comprising an internal ribosome entry site (IRES), wherein the IRES is optionally located between a first gene product and a second gene product.

[0354] 91. An isolated nucleic acid according to any one of embodiments 85 to 90, further comprising a self-cleaving peptide coding sequence, wherein the self-cleaving peptide is optionally T2A.

[0355] 92. The isolated nucleic acid according to any one of embodiments 85 to 91, wherein the expression construct comprises two adeno-associated virus (AAV) inverted terminal repeat (ITR) sequences adjacent to the first gene product and the second gene product, and optionally one of the ITR sequences lacks a functional terminal dissociation site.

[0356] 93. The isolated nucleic acid according to Embodiment 92, wherein at least one of the ITRs includes a modified "D" region relative to the wild-type AAV2 ITR (SEQ ID NO: 29).

[0357] 94. The isolated nucleic acid according to Embodiment 93, wherein the modified "D" region is a "D" sequence located outside the ITR with respect to the expression construct.

[0358] 95. The isolated nucleic acid according to Embodiment 93 or 94, wherein the ITR containing the modified "D" sequence is a 3'ITR.

[0359] 96. An isolated nucleic acid according to any one of embodiments 85 to 95, further comprising a TRY sequence, wherein the TRY sequence is optionally described in Sequence ID No. 28.

[0360] 97. An isolated nucleic acid having the sequence described in any one of sequence numbers 1 to 91.

[0361] 98. A vector comprising an isolated nucleic acid as described in any one of Embodiments 1 to 97.

[0362] 99. The vector according to Embodiment 98, wherein the vector is a plasmid.

[0363] 100. The vector according to Embodiment 98, wherein the vector is a viral vector, and optionally the viral vector is a recombinant AAV (rAAV) vector or a baculovirus vector.

[0364] 101. A composition comprising an isolated nucleic acid according to any one of Embodiments 1 to 97, or a vector according to any one of Embodiments 98 to 100.

[0365] 102. A host cell comprising an isolated nucleic acid according to any one of Embodiments 1 to 97 or a vector according to any one of Embodiments 98 to 100.

[0366] 103. (i) Capsid protein, and (ii) comprising an isolated nucleic acid according to any one of Embodiments 1 to 97, or a vector according to any one of Embodiments 98 to 100, Recombinant adeno-associated virus (rAAV).

[0367] 104. The rAAV according to Embodiment 103, wherein the capsid protein is capable of crossing the blood-brain barrier, and optionally the capsid protein is AAV9 capsid protein or AAVrh.10 capsid protein.

[0368] 105. The rAAV according to Embodiment 103 or 104, wherein the rAAV transduces neurons and non-neuronal cells of the central nervous system (CNS).

[0369] 106. A method for treating a subject having or suspected to have Parkinson's disease, comprising administering to the subject an isolated nucleic acid according to any one of Embodiments 1 to 97, a vector according to any one of Embodiments 98 to 100, a composition according to Embodiment 101, or rAAV according to any one of Embodiments 103 to 105.

[0370] 107. The method according to Embodiment 106, wherein the administration comprises a direct injection into the CNS of the subject, and optionally the direct injection is an intracerebral injection, an intraparenchymal injection, an intraarachnoid injection, an intracisional injection, or any combination thereof.

[0371] 108. The method according to Embodiment 107, wherein the direct injection to the target CNS includes convection-enhanced delivery (CED).

[0372] 109. The method according to any one of embodiments 106 to 108, wherein the administration comprises a peripheral injection, and optionally the peripheral injection is an intravenous injection.

[0373] 110. A method for treating subjects who have or are suspected of having frontotemporal dementia with a GRN mutation, (i) A recombinant adeno-associated virus (rAAV) vector comprising nucleic acid comprising an expression construct comprising a promoter operably linked to an transgene insertion encoding a PGRN protein, wherein the transgene insertion comprises the nucleotide sequence of SEQ ID NO: 68, (ii) AAV9 capsid protein and, The method comprising administering rAAV containing to the subject.

[0374] 111. The rAAV is approximately 1 × 10 13 Vector genome (vg) ~ approximately 7 × 10⁻⁶ 14 The method according to Embodiment 110, administered to the subject in doses within the range of vg.

[0375] 112. The method according to embodiment 110 or 111, wherein the rAAV is administered by injection into the cisterna magna.

[0376] 113. The method according to any one of embodiments 110 to 112, wherein the promoter is a chicken beta-actin (CBA) promoter.

[0377] 114. The method according to any one of Embodiments 110 to 113, wherein the rAAV vector further comprises a cytomegalovirus (CMV) enhancer.

[0378] 115. The method according to any one of Embodiments 110 to 114, wherein the rAAV vector further comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0379] 116. The method according to any one of embodiments 110 to 115, wherein the rAAV vector further comprises a bovine growth hormone polyA signaling tail.

[0380] 117. The method according to any one of Embodiments 110 to 116, wherein the nucleic acid comprises two adeno-associated virus inverted terminal repeat (ITR) sequences adjacent to the expression construct.

[0381] 118. The method according to Embodiment 117, wherein each ITR sequence is a wild-type AAV2 ITR sequence.

[0382] 119. The method according to any one of Embodiments 110 to 118, wherein the rAAV vector further includes a TRY region between the 5'ITR and the expression construct, and the TRY region includes SEQ ID NO: 28.

[0383] 120. A method for treating subjects who have or are suspected of having frontotemporal dementia with a GRN mutation, (i) In order from 5' to 3', (a) AAV2 ITR, (b) CMV enhancer, (c) CBA Promoter, (d) A transgene insertion encoding a PGRN protein, wherein the transgene insertion contains the nucleotide sequence of SEQ ID NO: 68, (e)WPRE, (f) Bovine growth hormone polyA signaling tail, and (g) A nucleic acid containing AAV2 ITR is included in the rAAV vector, (ii) AAV9 capsid protein and, The method comprising administering rAAV containing to the subject.

[0384] 121. The rAAV is approximately 1 × 10 13 vg~approx. 7×10 14 The method according to Embodiment 120, administered to the subject in doses within the range of vg.

[0385] 122. The method according to embodiment 120 or 121, wherein the rAAV is administered by injection into the cisterna magna.

[0386] 123. The method according to any one of Embodiments 110 to 122, wherein the rAAV is administered in a formulation containing approximately 20 mM Tris (pH 8.0), approximately 1 mM MgCl2, approximately 200 mM NaCl, and approximately 0.001% w / v poloxamer 188.

[0387] 124. (i) (a) an rAAV vector comprising nucleic acid comprising an expression construct comprising a promoter operably linked to an transgene insert encoding a PGRN protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 68, and (b) Contains AAV9 capsid protein rAAV and, (ii) Tris at approximately 20 mM (pH 8.0), (iii) Approximately 1 mM MgCl2, (iv) Approximately 200 mM NaCl and (v) A pharmaceutical composition comprising approximately 0.001% w / v poloxamer 188.

[0388] 125. (a) an rAAV vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to an transgene insertion encoding a PGRN protein, wherein the transgene insertion comprises the nucleotide sequence of SEQ ID NO: 68, (b) AAV9 capsid protein, including rAAV for use in a method of treating frontotemporal dementia with GRN mutations in the target population.

[0389] 126. A method for quantifying PGRN protein levels in cerebrospinal fluid (CSF) samples, (1) Diluting the CSF sample in a master mixture containing dithiothreitol (DTT) and sample buffer, (2) Fill the wells of the capillary cartridge with the diluted CSF sample, the anti-progranulin antibody, the secondary antibody for detecting the anti-progranulin antibody, luminol, and peroxide. (3) Loading the capillary cartridge into an automated Western blot immunoassay instrument, (4) Using the automated Western blot immunoassay instrument, calculate the signal intensity, peak area, and signal-to-noise ratio, (5) The method comprising quantifying the progranulin protein level in the CSF sample as the peak area for the immunoreactive anti-progranulin antibody.

Claims

1. A method for quantifying progranulin (PGRN) protein levels in cerebrospinal fluid (CSF) samples, (a) Diluting the CSF sample in a master mixture containing dithiothreitol (DTT) and sample buffer, (b) Filling the wells of the capillary cartridge with the diluted CSF sample, anti-progranulin antibody, secondary antibody for detecting the anti-progranulin antibody, luminol, and peroxide, (c) Loading the capillary cartridge into an automated Western blot immunoassay instrument, (d) Using the automated Western blot immunoassay instrument, calculate the signal intensity, peak area, and signal-to-noise ratio, (e) The progranulin protein level in the CSF sample is quantified as the peak area for the immunoreactive anti-progranulin antibody, The aforementioned CSF sample (i) In order from 5' to 3', (a) Adeno-associated virus 2 (AAV2) ITR, (b) Cytomegalovirus (CMV) enhancer, (c) Chicken beta-actin (CBA) promoter, (d) A transgene insertion encoding the progranulin (PGRN) protein, wherein the transgene insertion contains the nucleotide sequence of Sequence ID No. 68, (e) Woodchuck hepatitis virus post-transcriptional regulatory elements (WPRE), (f) Bovine growth hormone polyA signaling tail, and (g) AAV2 ITR A recombinant adeno-associated virus (rAAV) vector comprising nucleic acids containing an expression construct, (ii) Adeno-associated virus (AAV) capsid protein, The method is based on subjects who have been previously administered recombinant adeno-associated virus (rAAV) containing the above.

2. The method according to claim 1, wherein the CSF sample is of human origin.

3. The method according to claim 1, wherein the CSF sample is from a subject having or suspected of having frontotemporal dementia (FTD).

4. The method according to claim 1, wherein the FTD is an FTD having a GRN mutation.

5. The method according to claim 1, wherein the CSF sample is derived from a primate other than a human.

6. The method according to claim 1, wherein the AAV capsid protein is the AAV9 capsid protein.

7. The method according to any one of claims 1 to 6, wherein the dilution step is a four-fold dilution.

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