Gene therapy for neurodegenerative disorders

Abstract

The present disclosure relates to compositions and methods for the treatment of neurodegenerative disorders such as frontotemporal dementia (FTD). The present disclosure provides a method of treating FTD by administering to a subject in need thereof an expression construct comprising a transgene encoding progranulin or a portion thereof.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 063,852, filed August 10, 2020, the disclosures of which said Provisional Application are incorporated herein by reference in their entirety. Description of the electronically submitted text file

[0002] The contents of the following text file, submitted electronically together here, are incorporated herein by reference in their entirety: a computer-readable copy of the sequence listing (filename: PRVL_016_01WO_SeqList.txt, date recorded: August 10, 2021, file size approximately 612,834 bytes).

[0003] 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 acid 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 therapeutic agents exist to address peripheral diseases and major clinical manifestations in the hematopoietic bone marrow and viscera, including enzyme replacement therapy, chaperone-like small molecule drugs that bind to deficient Gcase and improve its stability, and substrate suppression therapies that block the production of substrates that accumulate and lead to symptoms and findings in Gaucher disease. However, other forms of Gaucher disease (particularly those affecting the skeleton and brain) are considered refractory to treatment.

[0005] Progranulin (PGRN) is an additional protein linked to lysosomal function. PGRN is encoded by the GRN gene. Haploinsufficiency of GRN in humans carries approximately a 90% risk of developing FTD-GRN (frontotemporal dementia with GRN mutations), a neurodegenerative disease characterized by executive function impairment, behavioral changes, and language difficulties, accompanied by atrophy of the frontal and temporal lobes. Disease-modifying therapies are not available for patients with FTD. [Overview of the project]

[0006] This specification provides a method for treating subjects who have or are suspected of having frontotemporal dementia with a GRN mutation, and the method is provided to the subjects, (i) an rAAV vector comprising nucleic acids comprising an expression construct comprising a promoter operably linked to an transgene insertion encoding a progranulin (PGRN) protein, wherein the transgene insertion comprises an rAAV vector comprising the nucleotide sequence of SEQ ID NO: 68, (ii) Recombinant adeno-associated virus (rAAV) containing adeno-associated virus (AAV) 9 capsid protein, and one or more of the following: (A) Sirolimus, (B) Methylprednisolone, (C) Rituximab, and (D) This includes administering prednisone and

[0007] This specification further provides a method for suppressing the immune response in subjects who have or are suspected of having frontotemporal dementia with a GRN mutation, the method being applied to the subjects, (i) an rAAV vector comprising nucleic acids comprising an expression construct comprising a promoter operably linked to an transgene insertion encoding a progranulin (PGRN) protein, wherein the transgene insertion comprises an rAAV vector comprising the nucleotide sequence of SEQ ID NO: 68, (ii) Recombinant adeno-associated virus (rAAV) containing adeno-associated virus (AAV) 9 capsid protein, and one or more of the following: (A) Sirolimus, (B) Methylprednisolone, (C) Rituximab, and (D) This includes administering prednisone and

[0008] In some embodiments of the methods provided herein, the promoter is a chicken beta-actin (CBA) promoter. In some embodiments of the methods provided herein, the rAAV vector further comprises a cytomegalovirus (CMV) enhancer. In some embodiments of the methods provided herein, the rAAV vector further comprises a woodchuck hepatitis virus post-transcriptional regulator (WPRE). In some embodiments of the methods provided herein, the rAAV vector further comprises a bovine growth hormone polyA signaling tail.

[0009] In some embodiments of the methods provided herein, the nucleic acid comprises two adeno-associated virus inverted terminal repeat (ITR) sequences adjacent to the expression construct. In some embodiments of the methods provided herein, each ITR sequence is an AAV2 ITR sequence.

[0010] In some embodiments of the methods provided herein, the rAAV vector further includes a TRY region between the 5'ITR and the expression construct, the TRY region including SEQ ID NO: 28.

[0011] This specification provides a method for treating subjects who have or are suspected of having frontotemporal dementia with a GRN mutation, and the method is provided to the subjects, (i) In the order from 5' to 3', (a) Adeno-associated virus (AAV) 2ITR and (b) Cytomegalovirus (CMV) enhancer, (c) Chicken beta-actin (CBA) promoter and (d) A transgene insertion encoding the progranulin (PGRN) protein, comprising the nucleotide sequence of SEQ ID NO: 68, (e) Woodchuck hepatitis virus post-transcriptional regulatory elements (WPREs), (f) Bovine growth hormone polyA signaling tail, (g) A nucleic acid containing AAV2 inverted terminal repeats (ITRs) and an rAAV vector, (ii) A recombinant adeno-associated virus (rAAV) containing the AAV9 capsid protein, and one or more of the following: (A) Sirolimus, (B) Methylprednisolone, (C) Rituximab, and (D) This includes administering prednisone and

[0012] This specification further provides a method for suppressing the immune response in subjects who have or are suspected of having frontotemporal dementia with a GRN mutation, the method being applied to the subjects, (i) In the order from 5' to 3', (a) Adeno-associated virus (AAV) 2ITR and (b) Cytomegalovirus (CMV) enhancer, (c) Chicken beta-actin (CBA) promoter and (d) A transgene insertion encoding the progranulin (PGRN) protein, comprising the nucleotide sequence of SEQ ID NO: 68, (e) Woodchuck hepatitis virus post-transcriptional regulatory elements (WPREs), (f) Bovine growth hormone polyA signaling tail, (g) A nucleic acid containing AAV2 inverted terminal repeats (ITRs) and an rAAV vector, (ii) A recombinant adeno-associated virus (rAAV) containing the AAV9 capsid protein, and one or more of the following: (A) Sirolimus, (B) Methylprednisolone, (C) Rituximab, and (D) This includes administering prednisone and

[0013] In some embodiments of the methods provided herein, rAAV is administered by injection into the cisterna magna.

[0014] In some embodiments of the methods provided herein, rAAV is about 1 × 10 13 Vector genome (vg) ~ approximately 7 × 10⁻⁶ 14 The subject is administered a dose in the range of vg. In some embodiments of the method provided herein, rAAV is administered at approximately 3.5 × 10 13 vg, approx. 7.0×10 13 vg, or approximately 1.4 × 10 14 It is administered to the target patient at a dose of VG.

[0015] In some embodiments of the methods provided herein, rAAV is administered in a formulation containing about 20 mM Tris, pH 8.0, about 1 mM MgCl2, about 200 mM NaCl, and about 0.001% w / v poloxamer 188.

[0016] In some embodiments of the methods provided herein, methylprednisolone is administered intravenously at a dose of approximately 1000 mg either the day before or on the same day as the administration of rAAV.

[0017] In some embodiments of the methods provided herein, prednisone is administered orally at a dose of approximately 30 mg / day for 14 days, starting the day following the administration of approximately 1000 mg of methylprednisolone (A), and then tapered over 7 days following the end of the 14-day period of (A).

[0018] In some embodiments of the methods provided herein, rituximab is administered intravenously at a dose of approximately 1000 mg on any one day between 14 days and 1 day prior to administration of rAAV.

[0019] In some embodiments of the methods provided herein, methylprednisolone is administered before rituximab is administered. In some embodiments of the methods provided herein, methylprednisolone is administered at least about 30 minutes before rituximab is administered. In some embodiments of the methods provided herein, both methylprednisolone and rituximab are administered the day before rAAV administration, with methylprednisolone administered at least about 30 minutes before rituximab is administered. In some embodiments of the methods provided herein, rituximab is administered on any day between 14 and 2 days before rAAV administration, and methylprednisolone is administered intravenously at a dose of about 100 mg at least 30 minutes before rituximab is administered on the same day as rituximab is administered.

[0020] In some embodiments of the methods provided herein, sirolimus is administered orally as a single dose of about 6 mg three, two, or one day before administration of rAAV, and (B) at a dose of about 2 mg / day to maintain a serum trough level of about 4 ng / ml to about 9 ng / mL for about 90 days after administration of rAAV, with the first dose of about 2 mg / day of sirolimus administered the day after the single dose of about 6 mg of sirolimus. In some embodiments of the methods provided herein, the administration of sirolimus is tapered off between 15 and 30 days after the end of the 90-day period after administration of rAAV.

[0021] In some embodiments of the methods provided herein, the method is (i) Administer methylprednisolone intravenously at a dose of approximately 1000 mg, (ii) Approximately 30 minutes after the administration of methylprednisolone in step (i), rituximab should be administered intravenously at a dose of approximately 1000 mg. (iii) The day after the administration of methylprednisolone in step (i), rAAV is administered into the cisterna magna by injection, (iv) Prednisone should be administered orally at a dose of approximately 30 mg / day for 14 days starting the day following the administration of methylprednisolone in step (i), (v) For 7 days after the completion of the 14-day period of step (iv), prednisone should be gradually tapered off. (vi) Three days, two days, or one day before the administration of rAAV in step (iii), sirolimus should be administered orally as a single dose of approximately 6 mg. (vii) Orally administer sirolimus at a dose of approximately 2 mg / day for approximately 90 days after rAAV administration in step (iii), maintaining a serum trough level of approximately 4 ng / ml to approximately 9 ng / mL, with the first dose of approximately 2 mg / day of sirolimus being administered the day after a single dose of approximately 6 mg of sirolimus. (viii) The administration of sirolimus is gradually reduced between 15 and 30 days after the end of the 90-day period of step (vii).

[0022] In some embodiments of the methods provided herein, the method is (i) Administer methylprednisolone intravenously at a dose of approximately 100 mg on any day between 14 and 2 days before the administration of rAAV in step (iv), (ii) Approximately 30 minutes after the administration of methylprednisolone in step (i), rituximab should be administered intravenously at a dose of approximately 1000 mg. (iii) Administer methylprednisolone intravenously at a dose of approximately 1000 mg either one day before or on the same day as the rAAV administration in step (iv), (iv) Administering rAAV into the cisterna magna via injection, (v) Prednisone shall be orally administered at a dose of approximately 30 mg / day for 14 days starting the day following the administration of methylprednisolone in step (iii), (vi) For 7 days following the completion of the 14-day period of step (v), the prednisone shall be administered in a gradually tapered manner. (vii) Three days, two days, or one day before the administration of rAAV in step (iv), sirolimus should be administered orally as a single dose of approximately 6 mg. (viii) Orally administer sirolimus at a dose of approximately 2 mg / day for approximately 90 days after rAAV administration in step (iv), to maintain a serum trough level of approximately 4 ng / ml to approximately 9 ng / mL, with the first dose of approximately 2 mg / day of sirolimus being administered the day after a single dose of approximately 6 mg of sirolimus. (ix) The administration of sirolimus is gradually reduced between 15 and 30 days after the end of the 90-day period of step (viii).

[0023] In some embodiments of the methods provided herein, the immune response is an immune response to rAAV. In some embodiments of the methods provided herein, the immune response is a T-cell response. In some embodiments of the methods provided herein, the immune response is a B-cell response. In some embodiments of the methods provided herein, the immune response is an antibody response. In some embodiments of the methods provided herein, the immune response is pleocytosis. In some embodiments of the methods provided herein, the pleocytosis is cerebrospinal fluid (CSF) pleocytosis. In some embodiments of the methods provided herein, the immune response is abnormal levels of CSF protein.

[0024] In some embodiments of the methods provided herein, an additional immunosuppressant other than sirolimus, methylprednisolone, rituximab, or prednisone is further administered to the subject.

[0025] This specification describes a method for treating frontotemporal dementia in which a subject has a GRN mutation, (i) an rAAV vector comprising nucleic acids comprising an expression construct comprising a promoter operably linked to an transgene insertion encoding a progranulin (PGRN) protein, wherein the transgene insertion comprises an rAAV vector comprising the nucleotide sequence of SEQ ID NO: 68, (ii) Recombinant adeno-associated virus (rAAV) containing adeno-associated virus (AAV) 9 capsid protein, and one or more of the following: (A) Sirolimus, (B) methylprednisolone, (C) rituximab, and (D) prednisone, and a combination therapy is provided.

[0026] Further provided herein, for use in a method of suppressing an immune response in a subject having or suspected of having frontotemporal dementia with a GRN mutation, (i) an rAAV vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a transgene insert encoding progranulin (PGRN) protein, wherein the transgene insert is an rAAV vector comprising the nucleotide sequence of SEQ ID NO: 68, and (ii) a recombinant adeno-associated virus (rAAV) comprising an adeno-associated virus (AAV)9 capsid protein, and one or more of the following: (A) sirolimus, (B) methylprednisolone, (C) rituximab, and (D) prednisone, and a combination therapy is provided.

[0027] In some embodiments, the combination therapy provided herein comprises about 1 × 10 13 vg to about 7 × 10 14 vg of said rAAV. In some embodiments, the combination therapy provided herein comprises about 3.5 × 10 13 vg, about 7.0 × 10 13 vg, or about 1.4 × 10 14 vg of said rAAV. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] [Figure 1] FIG. 1 is a schematic diagram depicting one embodiment of a vector comprising an expression construct encoding Gcase (e.g., GBA1 or a portion thereof).

[0029] [Figure 2]Figure 2 is a schematic diagram illustrating 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 for Gcase and LIMP2 are separated by an internal ribosome entry site (IRES).

[0030] [Figure 3] Figure 3 is a schematic diagram illustrating one embodiment of a vector containing expression constructs 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.

[0031] [Figure 4] Figure 4 is a schematic diagram illustrating 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.

[0032] [Figure 5] Figure 5 is a schematic diagram illustrating one embodiment of a vector comprising an expression construct encoding an interfering RNA for Gcase (e.g., GBA1 or a part thereof), a prosaposin (e.g., PSAP or a part thereof), and α-Syn.

[0033] [Figure 6] Figure 6 is a schematic diagram illustrating one embodiment of a vector containing expression constructs encoding Gcase (e.g., GBA1 or a portion thereof) and prosaposin (e.g., PSAP or a portion thereof). The coding sequences for Gcase and prosaposin are separated by an internal ribosome entry site (IRES).

[0034] [Figure 7]Figure 7 is a schematic diagram depicting one embodiment of a vector containing an expression construct encoding Gcase (e.g., GBA1 or a portion thereof). In this embodiment, the vector includes a CBA promoter element (CBA) consisting of 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 includes a WPRE regulator followed by a bGH poly-A tail. Three transcriptional regulatory activation sites are contained in TATA, RBS, and YY1, which are at the 5' end of the promoter region. Adjacent ITRs allow for the correct packaging of intervening sequences. Two variants of the 5' ITR sequence (insertion box) were evaluated. These have several nucleotide differences within the 20-nucleotide "D" region of the wild-type AAV2 ITR. In some embodiments, the rAAV vector includes the "D" domain nucleotide sequence shown in the upper row. In some embodiments, the rAAV vector includes a mutant "D" domain (for example, an "S" domain with nucleotide changes shown in the lower panel).

[0035] [Figure 8] Figure 8 is a schematic diagram illustrating one embodiment of the vector shown in Figure 6.

[0036] [Figure 9-1]Figure 9 shows representative data for rAAV delivery containing a transgene encoding Gcase (e.g., GBA1 or a portion thereof) in a mouse model of CBE for Parkinson's disease. Daily IP delivery of PBS vehicle, 25 mg / kg CBE, 37.5 mg / kg CBE, or 50 mg / kg CBE (left to right) started at P8. Survival (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 an open field at P23 (bottom left) and fall latency on a rotator 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) and (day 1). Aggregated GluSph and GalSph levels (bottom right) are shown as pmol per mg of wet tissue weight. Mean values ​​are presented. Error bars are SEM. Nominal p-values ​​for the treatment group based on linear regression: *p<0.05; **p<0.01; ***p<0.001. [Figure 9-2] Same as above.

[0037] [Figure 10] Figure 10 is a schematic diagram depicting one embodiment of a study design for the maximum dose of rAAV in a CBE mouse model. Briefly, rAAV was delivered by ICV injection at P3, and daily CBE treatment was initiated at P8. Behavior was assessed by open-field and rotorod assays at P24–25, and substrate levels were measured at P36 and P38.

[0038] [Figure 11-1]Figure 11 shows representative data for survival assessment of maximum rAAV dose in a CBE mouse model. In P3, mice were treated with either an excipient or 8.8 e9 vg 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 last CBE dose in P36 (day 1), and the other half underwent a 3-day CBE withdrawal before sacrificing in P38 (day 3). All treatment groups (excipient + PBS n=8, rAAV-GBA1 + PBS n=7, excipient + CBE n=8, and valiant + CBE n=9) were weighed daily (top left) and their weights were analyzed in P36 (top right). Behavior was evaluated by the total distance traveled in the open field at P23 (bottom left) and the fall latency on the rotorod at P24 (bottom right), and was evaluated as the median across the three trials for each animal. Due to lethality, the behavioral assays consisted of n=7 for the excipient + CBE group and n=8 for the other groups. Means across animals are presented. Error bars are SEM. Nominal p-values ​​for linear regression between treatment groups in CBE-treated animals: *p<0.05; ***p<0.001. [Figure 11-2] Same as above. [Figure 11-3] Same as above.

[0039] [Figure 12-1]Figure 12 shows representative data for the biochemical evaluation of the maximum rAAV dose in the CBE mouse model. GCase activity (top left), GluSph levels (top right), GluCer levels (bottom left), and vector genome (bottom right) were measured within the group before (day 1) or after (day 3) CBE withdrawal, using the cortex of 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. Mean values ​​are presented. Error bars are SEM. Nominal P-values ​​for treatment groups by linear regression in CBE-treated animals are shown, (*)P<0.1;**P<0.01;***P<0.001, adjusted for collection date and sex as covariates. [Figure 12-2] Same as above.

[0040] [Figure 13] Figure 13 shows representative data of behavioral and biochemical correlations in CBE mouse models after administration of excipient + PBS, excipient + CBE, and variant + CBE treatment groups. Across all treatment groups, rotarod performance was negatively correlated with GluCer accumulation (A, p=0.0012 by linear regression), and GluSph accumulation was negatively correlated with increased GCase activity (B, p=0.0086 by linear regression).

[0041] [Figure 14-1]Figure 14 shows representative data of the in vivo distribution of variants in the CBE mouse model. The presence of vector genomes was assessed in the liver, spleen, kidney, and gonads for all treatment groups (excipient + PBS n=8, variant + PBS n=7, excipient + CBE n=7, and variant + CBE n=9). 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 assessed by A260 optical density measurement. Mean values ​​are presented. Error bars are SEM. Nominal p-values ​​for treatment groups by linear regression in CBE-treated animals, corrected for sample date and sex as covariates: *p<0.05; **p<0.01; ***p<0.001. [Figure 14-2] Same as above.

[0042] [Figure 15-1]Figure 15 shows representative data for the survival assessment of rAAV dose ranges in a CBE mouse model. Mice received either an excipient or one of three different doses of rAAV-GBA1 (3.2 e9vg, 1.0 e10vg, or 3.2 e10vg) via ICV delivery at P3. Daily IP treatment with 25 mg / kg of CBE was initiated at P8. Mice administered with either an excipient and CBE or an excipient and PBS served as controls. All treatment groups were started with n=10 (5M / 5F) per group. All mice were sacrificed one day after the final dose of CBE (P38-P40). All treatment groups were weighed daily, and their weights were analyzed at P36. Motor capacity was assessed by fall latency on a rotor rod at P24 and transverse latency on a tapered beam at P30. Due to early lethality, the number of mice participating in the behavioral assay was as follows: 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. Mean values ​​are presented. Error bars are SEM, and nominal p-values ​​by linear regression in the CBE-treated groups, adjusted for sex as a covariate, were *p<0.05; **p<0.01. [Figure 15-2] Same as above.

[0043] [Figure 16-1]Figure 16 shows representative data for the biochemical evaluation of rAAV dose ranges in the CBE mouse model. 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 total protein mg. GluSph and GalSph 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 standard curve, and genomic DNA concentration was assessed by A260 optical density measurement. The presence of the vector genome was also measured in the liver (E). Mean values ​​are shown. Error bars are SEM. The nominal p-values ​​by linear regression in the CBE-treated group, after adjusting for sex as a covariate, were **p<0.01; ***p<0.001. [Figure 16-2] Same as above.

[0044] [Figure 17-1] Figure 17 shows representative data for tapered beam analysis at maximum dose rAAV-GBA1 in a genetic mouse model. Motor capacity in the treatment groups (WT + excipients, n=5), 4L / PS-NA + excipients (n=6), and 4L / PS-NA + rAAV-GBA1 (n=5)) was assessed by beam walk 4 weeks after rAAV-GBA1 administration. Total slip and effective 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. Mean values ​​are presented. Error bars are SEM. [Figure 17-2] Same as above.

[0045] [Figure 18]Figure 18 shows representative data for 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).

[0046] [Figure 19-1] Figure 19 shows representative in vitro expression data for rAAV constructs encoding GBA1 in combination with prosaposin (PSAP), SCARB2, and / or one or more inhibitory nucleic acids. The data show that transfection of HEK293 cells with each construct resulted in overexpression of the target transgene compared to mock-transfected cells. [Figure 19-2] Same as above. [Figure 19-3] Same as above.

[0047] [Figure 20] Figure 20 is a schematic diagram depicting an rAAV vector that includes a "D" region located "outside" the ITR (e.g., proximal to the terminal of the ITR compared to the transgene insertion or expression construct) (upper side) and a wild-type rAAV vector with ITRs "inside" the vector (e.g., proximal to the transgene insertion of the vector).

[0048] [Figure 21] Figure 21 is a schematic diagram illustrating one embodiment of a vector comprising GBA2 or a portion thereof, and an expression construct encoding an interfering RNA for α-Syn.

[0049] [Figure 22]Figure 22 is a schematic diagram illustrating one embodiment of a vector containing expression constructs encoding Gcase (GBA1 or a portion thereof) and galactosylceramidase (e.g., GALC or a portion thereof). Expression of the Gcase and galactosylceramidase encoding sequences is separated by a T2A self-cleaving peptide sequence.

[0050] [Figure 23] Figure 23 is a schematic diagram illustrating one embodiment of a vector containing expression constructs encoding Gcase (GBA1 or a portion thereof) and galactosylceramidase (e.g., GALC or a portion thereof). Expression of the Gcase and galactosylceramidase encoding sequences is separated by a T2A self-cleaving peptide sequence.

[0051] [Figure 24] Figure 24 is a schematic diagram depicting one embodiment of a vector comprising expression constructs encoding interfering RNA for Gcase (e.g., GBA1 or a portion thereof), cathepsin B (e.g., CTSB or a portion thereof), and α-Syn. Expression of the Gcase and cathepsin B coding sequences is separated by a T2A self-cleaved peptide sequence.

[0052] [Figure 25] Figure 25 is a schematic diagram illustrating one embodiment of a vector comprising an expression construct encoding an interfering RNA for Gcase (e.g., GBA1 or a part thereof), sphingomyelin phosphodiesterase 1 (e.g., SMPD1 or a part thereof), and α-Syn.

[0053] [Figure 26] Figure 26 is a schematic diagram illustrating one embodiment of a vector containing expression constructs encoding Gcase (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).

[0054] [Figure 27] Figure 27 is a schematic diagram illustrating one embodiment of a vector containing 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.

[0055] [Figure 28] Figure 28 is a schematic diagram depicting 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.

[0056] [Figure 29] Figure 29 is a schematic diagram illustrating one embodiment of a vector comprising an expression construct encoding interference 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.

[0057] [Figure 30] Figure 30 is a schematic diagram depicting 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).

[0058] [Figure 31] Figure 31 is a schematic diagram illustrating one embodiment of a vector comprising VPS35 (e.g., VPS35 or a portion thereof) and expression constructs encoding interfering RNAs for α-Syn and TMEM106B.

[0059] [Figure 32] Figure 32 is a schematic diagram depicting 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.

[0060] [Figure 33] Figure 33 is a schematic diagram illustrating 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 for Gcase and IL-34 are separated by an internal ribosome entry site (IRES).

[0061] [Figure 34] Figure 34 is a schematic diagram illustrating 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.

[0062] [Figure 35] Figure 35 is a schematic diagram illustrating 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.

[0063] [Figure 36] Figures 36A and 36B show representative data on the overexpression of TREM2 and GBA1 in HEK293 cells compared to control transduced cells, as measured by qPCR and ELISA. Figure 36A shows the data for TREM2 overexpression. Figure 36B shows the data for GBA1 overexpression from the same construct.

[0064] [Figure 37] Figure 37 shows representative data demonstrating the success of in vitro silencing of SNCAs using the GFP reporter assay (top) and the α-Syn assay (bottom).

[0065] [Figure 38] Figure 38 shows representative data demonstrating the success of in vitro silencing of TMEM106B using the GFP reporter assay (top) and the α-Syn assay (bottom).

[0066] [Figure 39] Figure 39 is a schematic diagram illustrating one embodiment of a vector containing an expression construct encoding a PGRN.

[0067] [Figure 40] Figure 40 shows the transduction data 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.

[0068] [Figure 41] Figure 41 is a schematic diagram illustrating one embodiment of a vector containing an expression construct encoding Gcase (e.g., GBA1 or a portion thereof).

[0069] [Figure 42] Figure 42 is a schematic diagram illustrating one embodiment of a vector containing an expression construct encoding Gcase (e.g., GBA1 or a portion thereof).

[0070] [Figure 43]Figure 43 is a schematic diagram illustrating one embodiment of a vector containing expression constructs encoding interfering RNAs for Gcase (e.g., GBA1 or a portion thereof) and α-Syn.

[0071] [Figure 44] Figure 44 is a schematic diagram illustrating one embodiment of a vector containing an expression construct encoding a PGRN.

[0072] [Figure 45] Figure 45 is a schematic diagram illustrating one embodiment of a vector containing an expression construct encoding a PGRN.

[0073] [Figure 46] Figure 46 is a schematic diagram illustrating one embodiment of a vector containing an expression construct encoding PGRN and interfering RNA of microtubule-associated protein tau (MAPT).

[0074] [Figure 47] Figure 47 is a schematic diagram illustrating one embodiment of a vector containing expression constructs encoding interfering RNAs for Gcase (e.g., GBA1 or a portion thereof) and α-Syn.

[0075] [Figure 48] Figure 48 is a schematic diagram illustrating one embodiment of a vector containing an expression construct encoding PSAP.

[0076] [Figure 49] Figure 49 is a schematic diagram illustrating one embodiment of a vector containing an expression construct encoding Gcase (e.g., GBA1 or a portion thereof).

[0077] [Figure 50] Figure 50 is a schematic diagram illustrating one embodiment of a vector comprising an expression construct encoding Gcase (GBA1 or a portion thereof) and galactosylceramidase (e.g., GALC or a portion thereof).

[0078] [Figure 51] Figure 51 is a schematic diagram illustrating one embodiment of a plasmid comprising an rAAV vector containing an expression construct encoding an interfering RNA for Gcase (e.g., GBA1 or a part thereof), a prosaposin (e.g., PSAP or a part thereof), and α-Syn.

[0079] [Figure 52A-D] Figure 52A shows that iPSC-derived neural stem cell (NSC) lines from patients with FTD-GRN mutations secrete less progranulin than NSC lines from healthy controls. Statistical analysis was performed using unpaired t-tests: *=p<0.05, **=p<0.01, ***=p<0.001. Data are expressed as mean ± SEM.

[0080] Figure 52B shows the results of dose-range PR006A gene transduction in FTD-GRN mutant carrier neuronal cultures. NSCs were seeded at equal density and differentiated into neurons. On day 7, neurons were transduced for 72 hours with either an excipient or an indicated dose of PR006A. Secretory 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 secretory progranulin from control neurons (excipient-treated). Secretory progranulin was not detected in excipient-treated FTD-GRN neurons. Statistics were determined using ANOVA followed by Tukey's test, and the statistical comparison of each condition with excipient-treated control neurons is shown in the graph. * =p<0.05, *** =p<0.001. LLOQ = lower limit of quantification, MOI = multiplicity of infection.

[0081] Figure 52C shows that treatment of nerve cultures with PR006 rescued poor maturation of cathepsin D, a key lysosomal protease, in FTD-GRN nerve cultures. NSCs were seeded at equal concentrations and differentiated into neurons. On day 7, 5.3 × 10⁶ neurons were obtained. 5Neurons were transduced for 72 hours using an excipient or PR006A at the MOI. 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 normalized signal. The matCTSD / proCTSD ratio in excipient or PR006A-treated FTD-GRN neurons was determined. The y-axis shows the matCTSD / proCTSD ratio as a percentage of the proportion (n=3; mean ± SEM) of excipient-treated control neurons. Statistics were determined using paired t-tests. * = p < 0.05.

[0082] Figures 52D and 52F show that PR006A reduces TDP-43 pathology in FTD-GRN neuronal cultures. NSCs were seeded at equal concentrations and differentiated into neurons. On day 7, 5.3 × 10⁶ 5Neurons were transduced using excipients or PR006A at MOI and collected 21 days after transduction. Figure 52D: Neurons were lysed, and the Triton-X insoluble protein fraction was isolated and analyzed using a simple Western Jess system with anti-TDP-43 antibody (#12892-AP-1). 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 cultures. Figure 52F: Quantification of nuclear TDP-43 signaling from immunofluorescence images of iPSC-derived neurons treated with PR006A. The TDP-43 signal intensity per nucleus was determined in FTD-GRN neurons treated with an excipient or PR006A. 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 the nuclear TDP-43 expression level in FTD-GRN neuronal cultures to nearly the wild-type control level. Statistics were determined by an unpaired t-test. * =p<0.01, ** = p < 0.001.

[0083] [Figure 52E-F] Figure 52E shows that NSC strains derived from iPSCs of patients with FTD-GRN mutations secrete less progranulin than NSC strains derived from healthy controls. Statistical analysis was performed using unpaired t-tests: *=p<0.05, **=p<0.01, ***=p<0.001. Data are expressed as mean ± SEM.

[0084] [Figure 52G]Figure 52G is a series of images showing that neural stem cell (NSC) lines from human FTD-GRN and human control cell lines successfully differentiated into neuronal cultures. NSC lines from control and FTD-GRN (FTD-GRN #1 and FTD-GRN #2) differentiated into neurons after 7 days, as indicated by immunofluorescence staining of cell morphology and neuronal markers (NeuN [red]; labeled MAP2 or Tau [green] on the left). Nuclei were stained using DAPI (blue).

[0085] [Figure 53A-C]Figures 53A–53C are a series of bar graphs showing the results of experiments analyzing the in vivo distribution and progranulin expression in the CNS in an adult dose-range PR006A FTD-GRN mouse model study. Four-month-old Grn KO mice were administered PR006A or excipients via ICV. They were sacrificed three months after treatment with excipients (red) or PR006A 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) (blue) for biochemical endpoints in the CNS. Figure 53A: The presence of vector genomes was evaluated in the cerebral cortex and spinal cord, and the in vivo distribution is shown as vector genomes per 1 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 the presence of a positive vector. Figure 53B: GRN RNA expression encoded by PR006A was evaluated by quantitative RT-PCR (qRT-PCR) in the cerebral cortex (n=8-10 / group, mean ± SEM). GRN copy number (specific to our codon-optimized PR006A sequence) is normalized to 1 μg of total RNA and shown on a logarithmic scale. Figure 53C: Progranulin protein levels were measured in the brain and spinal cord using human-specific progranulin ELISA (n=8-10 / group, mean ± SEM). Tissue progranulin levels were normalized to total protein concentration. The limit of quantification (LLOQ) is indicated by a gray dashed line. 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 simple line corresponding to the color of the treatment group legend on the x-axis without error bars indicates that all animals in that group had a value of 0. Statistical analysis was performed using ANOVA followed by Dunnett's test, and then compared with the excipient-treated Grn KO mouse group, with *=p<0.05, **=p<0.01, and ***=p<0.001. vg=vector genome; LLOQ=limit of quantification; SC=spinal cord.

[0086] [Figure 53D-E] Figures 53D–53E are a series of bar graphs showing the results of experiments analyzing peripheral tissue biodistribution and progranulin expression in an adult dose-range PR006A FTD-GRN mouse model study. 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 the liver, heart, lungs, kidneys, spleen, and gonads. Figure 53D: The presence of vector genomes was evaluated, and their in vivo distribution is shown on a logarithmic scale as vector genomes per 1 μg of gDNA (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 the presence of a positive vector. Figure 53E: Progranulin protein levels were measured using ELISA (n=8-10 / group, mean ± SEM). Tissue progranulin levels were normalized to total protein concentration. Simple lines corresponding to the legend colors of the treatment groups 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, compared with the excipient-treated Grn KO mouse group, *=p<0.05, ***=p<0.001. vg = vector genome.

[0087] [Figure 53F]Figure 53F is a bar graph showing the results of an experiment analyzing progranulin levels in plasma in an adult dose-range PR006A FTD-GRN mouse model study. 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 plasma biochemical endpoints. 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 limit of quantification (LLOQ) is indicated by the gray dashed line. Statistical analysis was performed using ANOVA followed by Dunnett's test, and compared with the excipient-treated Grn KO mouse group. *=p<0.05, **=p<0.01, ***=p<0.001. LLOQ = limit of quantification. vg = vector genome.

[0088] [Figure 53G-H]Figures 53G–53H are a series of bar graphs showing experimental results demonstrating reduced lysosomal and neuropathological deficiencies in adult-dose range PR006A FTD-GRN adult mouse model studies. Four-month-old Grn KO mice were administered PR006A or excipients via ICV. They were sacrificed for analysis three months after treatment with excipients (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). Lipofuscinosis was analyzed by two independent methods: (1) scoring of H&E-stained brain sections by pathologists, and (2) quantification of lipofuscin autofluorescence from IHC sections. Figure 53G: Lipofuscin accumulation (autofluorescent lipofuscin granules) was semi-quantitatively scored in H&E stained sections of different brain regions by blinded, committee-certified pathologists according to the following grading system: 0 = No lipofuscin observed; 1 = Very small granules of lipofuscin (<2 μm) scattered throughout the region; 2 = Increased density of small granule accumulation and / or development of larger granules (>2-3 μm); 3 = Multifocal region containing high density of lipofuscin granules visible from a low-magnification objective lens; 4 = Extensive accumulation of lipofuscin. Shows lipofuscin severity scores for the cerebral cortex, hippocampus, and thalamus / hypothalamic brain regions (n=8-10 / group). Figure 53H: IHC analysis of ubiquitin was performed and quantified in the cerebral cortex, hippocampus, and thalamus. The size of immune-responsive objects exceeding the threshold (immune-responsive object size [μm2]) is shown for ubiquitin (n=8-10 / group, mean ± SEM). Statistics were determined by ANOVA and then compared with the excipient-treated Grn KO mouse group by Dunnett's test, *=p<0.05, **=p<0.01, ***=p<0.001. vg=vector genome; WT=wild type.

[0089] [Figure 53I-K]Figures 53I–53K are a series of bar graphs showing experimental results demonstrating a reduction in neuroinflammatory markers in adult dose-range PR006A FTD-GRN mouse model studies. Four-month-old Grn KO mice were administered PR006A or excipients via ICV. They were sacrificed for analysis three months after treatment with excipients (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). Figure 53I: 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 to the housekeeping gene Ppib. Figures 53J-53K: IHC analysis of Iba1 (Figure 53J) and GFAP (Figure 53K) was performed and quantified in fixed brain sections of the cerebral cortex, hippocampus, and thalamus. The percentage of regions of interest covered by objects exceeding the threshold (immunely responsive areas [%]) is shown (mean ± SEM, n=8-10 / group). Statistics were determined by ANOVA with Dunnett adjustment, comparing each group to the excipient-treated Grn KO mouse group, *=p<0.05, ***=p<0.001. vg=vector genome; WT=wild type.

[0090] [Figure 53L-N]Figures 53L–53N are a series of bar graphs showing experimental results demonstrating decreased gene expression in lysosomes and immune pathways in an adult dose-range PR006A FTD-GRN mouse model study. Four-month-old Grn KO mice were administered PR006A or excipients via ICV. They were sacrificed for analysis three months after treatment with excipients (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). RNA sequencing was performed on cerebral cortical samples from ICV-treated Grn KO mice and WT C57BL / 6J mice of the same age (gray). Using gene set variation analysis (GSVA), mRNA expression levels of dysregulated, previously published gene signatures in excipient-treated Grn KO mice were compared to those in WT mice. The data shown are GSVA activity scores for curated gene sets from two published studies and one Hallmark pathway. Figure 53L: Cellular component: vacuole (GO:0005773), Figure 53M: Lysosome, and Figure 53N: Complement system (Hallmark pathway) (median ± range, n=8-10 / group). Statistical analysis was performed using ANOVA followed by Dunnett's test, comparing excipient-treated Grn KO mouse group while controlling for family-wise type I error rate, ***=p<0.001. GSVA = gene set variation analysis, vg = vector genome, WT = wild type.

[0091] [Figure 54A] Figure 54A is a series of bar graphs showing the results of an experiment analyzing the in vivo distribution of the PR006A transgene quantified by qPCR. Transgene levels were analyzed using the qPCR method in NHPs 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 limit of quantification at 50 vg / μg of DNA.

[0092] [Figure 54B] Figure 54B 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 in NHP serum and CSF samples at 29 and 182 days after administration using either an excipient, low-dose PR006A (6.5 x 10⁹ vg / g brain), or high-dose PR006A (6.5 x 10¹⁰ vg / g brain). Data represent mean ± SEM.

[0093] [Figure 54C] Figure 54C shows a series of bar graphs illustrating 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 expressed as mean ± SEM.

[0094] [Figure 54D] Figure 54D is a bar graph showing 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, determined by Simple Western® (Jess) analysis. CSF samples were from NHPs 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 p-values ​​from one-way dose-dependent response analysis using mean ± SEM, William's trend test: *p<0.05.

[0095] [Figure 55-56]Figure 55 is a graph showing the selectivity and specificity results of the automated Western Jess assay. Progranulin protein levels in CSF samples from FTD patients were detected by Jess at 58 kDa. Group (A): heterozygous FTD patients, and Groups (B) and (C): familial non-carriers or healthy controls. Data are expressed as mean ± mean standard error (SEM). SEM values ​​are shown as vertical error bars.

[0096] Figure 56 is a graph showing 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 healthy controls. Data are expressed as mean ± mean standard error (SEM). SEM values ​​are displayed as vertical error bars.

[0097] [Figure 57] Figure 57 shows gel images of each CSF sample performed in duplex on a Jess automated Western blotting platform. Samples were analyzed at a 4-fold dilution using the primary antibody AdipogenPG-359-7. The first lane is the molecular weight standard, and to the right is the band identification used to calculate the immunoresponsiveness reported in Example 14.

[0098] [Figure 58] Figures 58A and 58B are a series of plots showing the measurement of human PGRN expression levels. Human PGRN expression levels were determined in non-human primate CSF samples, which were collected at day 180 using Simple Western (Jess) analysis. CSF from NHPs treated with excipients ("Excipients"), low-dose PR006A (6.5 x 10⁹ vg / g brain weight, "Low"), or high-dose PR006 (6.5 x 10¹⁰ vg / g brain weight, "High") were analyzed. Data are presented as mean immune response peak area (Figure 58A) or magnification change on excipient-treated animals (Figure 58B). Each point represents a single CSF sample from one NHP (mean of technical overlap), and the boxes represent the mean ± standard error of the three individual NHPs.

[0099] [Figure 59A-C] Figures 59A–59C 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 administration of ICV excipient (red) or 9.7 × 10¹⁰ vg (2.4 × 10¹¹ vg / g brain) PR006A (blue). Figure 59A: The presence of vector genomes was assessed in the cerebral cortex and spinal cord (mean ± SEM, n=4 / group). In vivo distribution is shown on a logarithmic scale as vector genomes per 1 μg gDNA. 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 the presence of a positive vector. Figures 59B-59C: Progranulin protein levels were measured using ELISA in CNS tissues (brain and spinal cord (Figure 59B)) and CSF (Figure 59C) (mean ± SEM, n=4 / group). Tissue progranulin levels were normalized to total protein concentration, and progranulin CSF levels were normalized to fluid volume. The limit of quantification (LLOQ) is shown by the gray dashed line. 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 the Kruskal-Wallis test; *=p<0.05, **=p<0.01, ***=p<0.001. vg=vector genome; LLOQ=limit of quantification; SC=spinal cord.

[0100] [Figure 59D]Figures 59D–59E are a series of bar graphs and images showing experimental results demonstrating a reduction in lysosomal and neuropathological defects in an aged FTD-GRN mouse model after PR006A treatment. Tissue samples were taken from 18-month-old Grn KO mice two months after administration of ICV excipient (red) or 9.7 × 10¹⁰ vg (2.4 × 10¹¹ vg / g brain) PR006A (blue). Lipofuscinosis was analyzed by scoring of H&E-stained brain sections by a pathologist. Figure 59D: Representative lipofuscin image from the thalamic / hypothalamic region of a brain section. White arrows indicate examples of lipofuscin accumulation. A summary of lipofuscin severity scores for the cerebral cortex, hippocampus, and thalamus / thalamus of H&E-stained slides from brain sections evaluated for autofluorescent lipofuscin granules is provided. Lipofuscin accumulation was semi-quantitatively scored by blinded committee-certified pathologists according to the following grading system: 0 = No lipofuscin observed; 1 = Very small granules of lipofuscin (<2 μm) scattered throughout the region; 2 = Increased density of small granule accumulation and / or development of larger granules (>2-3 μm); 3 = Multifocal region containing high density of lipofuscin granules visible from a low-magnification objective lens; 4 = Extensive accumulation of lipofuscin. Figure 59E: 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.

[0101] [Figure 59E-G]Figures 59F–59I are a series of bar graphs showing experimental results illustrating 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 administration of ICV excipient (red) or 9.7 × 10¹⁰ vg (2.4 × 10¹¹ vg / g brain) PR006A (blue). Figure 59F: 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. (Figure 59G) Protein expression of the pro-inflammatory cytokine TNF, TNFα, was measured in the cerebral cortex using a 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. Figures 59H-59I: IHC analysis was performed on Iba1 (Figure 59H) and GFAP (Figure 59I), and quantified on fixed brain sections. The edited positive cell density (cells / mm2) from the three brain regions analyzed (cerebral cortex, hippocampus, and thalamus) is shown (mean ± SEM, n=3-4 / group). Statistical analysis was performed using t-tests, *=p<0.05. vg = vector genome. [Figure 59H-I] Same as above.

[0102] [Figure 60] Figure 60 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.

[0103] [Figure 61]Figure 61 shows the design of the study for the maximum dose of PR006A in an aged FTD-GRN mouse model. PR006A was delivered to two cohorts of Grn KO mice by ICV injection at a dose of 10 μl of excipient (control) or 9.7 × 10¹⁰ vg (2.4 × 10¹¹ vg / g brain): (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 two 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.

[0104] [Figure 62] Figures 62A and 62B are bar graphs showing the in vivo distribution and progranulin expression results in peripheral tissues of an aged FTD-GRN mouse model after PR006A treatment. Tissue samples were collected from 18-month-old Grn KO mice two months after administration of ICV excipient (red) or 9.7 × 10¹⁰ vg (2.4 × 10¹¹ vg / g brain) PR006A (blue). Figure 62A: The presence of the vector genome was assessed in the liver, heart, lung, kidney, spleen, and gonads (mean ± SEM, n=4 / group). In vivo distribution is shown as vector genome per 1 μg gDNA. The presence of the vector genome was quantified by qPCR using a vector reference standard. Figure 62B: Progranulin protein levels were measured using ELISA (mean ± SEM, n=4 / group). Tissue progranulin levels were normalized to total protein concentration. 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 the Kruskal-Wallis test; *=p<0.05, **=p<0.01, ***=p<0.001. vg = vector genome.

[0105] [Figure 63]Figure 63 shows the design of the study for the dose range of PR006A in an adult FTD-GRN mouse model. 10 μl of excipient (control) or 1.1 x 10⁹ vg (2.7 x 10⁹ vg / g brain), 1.1 x 10¹⁰ vg (2.7 x 10¹⁰ vg / g brain) of PR006A, or 1.1 x 10¹¹ vg (2.7 x 10¹¹ vg / g brain) of PR006A was delivered by ICV injection to 4-month-old Grn KO mice (n=10 / group). Animals were sacrificed three months after injection, when the mice were 7 months old. CNS and peripheral tissues were collected and analyzed for PR006A in vivo distribution (qPCR), progranulin protein expression (ELISA), and histopathology (H&E). We evaluated the expression of pro-inflammatory markers, lipofuscin accumulation, ubiquitin accumulation, and broad-spectrum gene expression changes in the brain.

[0106] [Figure 64] Figure 64 is a schematic diagram depicting 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" means "progranulin". "ITR" refers to the adeno-associated virus inverted terminal repeat sequence. "TRY" refers to the sequence containing three transcriptional regulatory activation sites: 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 signal tail. "int" refers to an intron. The double-stranded nucleotide sequences of PR006A are provided in SEQ ID NOs. 90 and 91. [Modes for carrying out the invention]

[0107] This disclosure relates to gene therapy for frontotemporal dementia (FTD). In particular, this disclosure relates to an immunosuppressive regimen administered in combination with recombinant adeno-associated virus (rAAV) that delivers a functional copy of the GRN gene encoding progranulin. Immunosuppressive regimens are necessary in patients receiving gene therapy to reduce the risk of immune-related adverse events.

[0108] This disclosure is, in part, based on compositions and methods for the expression of specific gene product combinations (e.g., gene products associated with CNS diseases) in a subject. The gene product may be a protein, a protein fragment (e.g., a moiety), or an interfering nucleic acid that inhibits a CNS disease-related gene. 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 that inhibits a CNS disease-related gene (e.g., shRNA, siRNA, miRNA, amiRNA, etc.).

[0109] CNS disease-related genes refer to genes that encode gene products genetically, biochemically, or functionally associated with CNS diseases such as feline neuropathy (FTD) and Parkinson's disease (PD). For example, individuals with a pathogenic mutation in the GRN gene (encoding the protein PGRN (progranulin)) have been observed to have an increased risk of developing FTD compared to individuals without the GRN mutation. 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 the GBA1 mutation. 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 wild-type or non-mutant versions of CNS disease-related genes (or their coding sequences). Examples of CNS disease-related genes are listed in Table 1. [Table 1-1] [Table 1-2] [Table 1-3]

[0110] In addition to patients with Gaucher disease (who have mutations in both chromosomal alleles of the GBA1 gene), patients with mutations in only one allele of GBA1 are at high risk of Parkinson's disease (PD). The severity of PD symptoms, including difficulty walking, resting tremor, rigidity, and often depression, dyssomnia, and cognitive decline, correlates with the degree of enzyme activity reduction. Therefore, patients with Gaucher disease have the most severe course, while patients with a single, mild mutation in GBA1 usually have a more benign course. Mutation carriers are at high 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, characterized by distinctive movement and cognitive impairments. There are no treatments that can alter the relentless course of these disorders.

[0111] Deficiencies in enzymes such as Gcase (e.g., the gene product of the GBA1 gene), and common variants in many genes involved in lysosome function or the transport of macromolecules to lysosomes (e.g., lysosomal membrane protein 1 (LIMP), also known as SCARB2), are 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, SMPD, GCH1, RAB7, VPS35, IL-34, TREM2, TMEM106B, or any combination (or part thereof) of the aforementioned, which are associated with the central nervous system (CNS), such as Gaucher disease and PD. In some embodiments, the combinations of gene products described herein act together (e.g., synergistically) to reduce one or more signs and symptoms of CNS diseases when expressed in a subject.

[0112] Accordingly, in some embodiments, the disclosure provides an isolated nucleic acid 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 Gcase coding sequence (e.g., codons optimized for expression in mammalian cells, e.g., human cells). 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 adeno-associated virus (AAV) inverted terminal repeats (ITRs), e.g., AAV ITRs adjacent to the nucleic acid sequence encoding the Gcase protein.

[0113] In some embodiments, the disclosure provides an isolated nucleic acid 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 prosaposin coding sequence (e.g., codons optimized for expression in mammalian cells, e.g., human cells). 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 adeno-associated virus (AAV) inverted terminal repeats (ITRs), e.g., AAV ITRs adjacent to the nucleic acid sequence encoding the prosaposin protein.

[0114] In some embodiments, the disclosure provides an isolated nucleic acid comprising an expression construct encoding LIMP2 / SCARB2 (e.g., the gene product of the SCRAB2 gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized SCRAB2 coding sequence (e.g., codons optimized for expression in mammalian cells, e.g., human cells). 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 adeno-associated virus (AAV) inverted terminal repeats (ITRs), e.g., AAV ITRs adjacent to the nucleic acid sequence encoding the SCRAB2 protein.

[0115] In some embodiments, the disclosure provides an isolated nucleic acid comprising an expression construct encoding the GBA2 protein (e.g., the gene product of the GBA2 gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized GBA2 coding sequence (e.g., codons optimized for expression in mammalian cells, e.g., human cells). 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 adeno-associated virus (AAV) inverted terminal repeats (ITRs), e.g., AAV ITRs adjacent to the nucleic acid sequence encoding the GBA2 protein.

[0116] In some embodiments, the disclosure provides an isolated nucleic acid comprising an expression construct encoding a GALC protein (e.g., the gene product of the GALC gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized GALC coding sequence (e.g., codons optimized for expression in mammalian cells, e.g., human cells). 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 adeno-associated virus (AAV) inverted terminal repeats (ITRs), e.g., AAV ITRs adjacent to the nucleic acid sequence encoding the GALC protein.

[0117] In some embodiments, the disclosure provides an isolated nucleic acid 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 CTSB coding sequence (e.g., codons optimized for expression in mammalian cells, e.g., human cells). 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 adeno-associated virus (AAV) inverted terminal repeats (ITRs), e.g., AAV ITRs adjacent to the nucleic acid sequence encoding the CTSB protein.

[0118] In some embodiments, the disclosure provides an isolated nucleic acid 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 SMPD1 coding sequence (e.g., codons optimized for expression in mammalian cells, e.g., human cells). 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 adeno-associated virus (AAV) inverted terminal repeats (ITRs), e.g., AAV ITRs adjacent to the nucleic acid sequence encoding the SMPD1 protein.

[0119] In some embodiments, the disclosure provides an isolated nucleic acid comprising an expression construct encoding the GCH1 protein (e.g., the gene product of the GCH1 gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized GCH1 coding sequence (e.g., codons optimized for expression in mammalian cells, e.g., human cells). 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 adeno-associated virus (AAV) inverted terminal repeats (ITRs), e.g., AAV ITRs adjacent to the nucleic acid sequence encoding the GCH1 protein.

[0120] In some embodiments, the disclosure provides an isolated nucleic acid 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 RAB7L coding sequence (e.g., codons optimized for expression in mammalian cells, e.g., human cells). 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 adeno-associated virus (AAV) inverted terminal repeats (ITRs), e.g., AAV ITRs adjacent to the nucleic acid sequence encoding the RAB7L protein.

[0121] In some embodiments, the disclosure provides an isolated nucleic acid comprising an expression construct encoding the VPS35 protein (e.g., the gene product of the VVPS35 gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized VPS35 coding sequence (e.g., codons optimized for expression in mammalian cells, e.g., human cells). 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 adeno-associated virus (AAV) inverted terminal repeats (ITRs), e.g., AAV ITRs adjacent to the nucleic acid sequence encoding the VPS35 protein.

[0122] In some embodiments, the disclosure provides an isolated nucleic acid comprising an expression construct encoding the IL-34 protein (e.g., the gene product of the IL34 gene). In some embodiments, the isolated nucleic acid comprises a codon-optimized IL-34 coding sequence (e.g., codons optimized for expression in mammalian cells, e.g., human cells). 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 adeno-associated virus (AAV) inverted terminal repeats (ITRs), e.g., AAV ITRs adjacent to the nucleic acid sequence encoding the IL-34 protein.

[0123] In some embodiments, the disclosure provides an isolated nucleic acid 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 TREM2 coding sequence (e.g., codons optimized for expression in mammalian cells, e.g., human cells). 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 adeno-associated virus (AAV) inverted terminal repeats (ITRs), e.g., AAV ITRs adjacent to the nucleic acid sequence encoding the TREM2 protein.

[0124] In some embodiments, the disclosure provides an isolated nucleic acid 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 TMEM106B coding sequence (e.g., codons optimized for expression in mammalian cells, e.g., human cells). 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 adeno-associated virus (AAV) inverted terminal repeats (ITRs), e.g., AAV ITRs adjacent to the nucleic acid sequence encoding the TMEM106B protein.

[0125] In some embodiments, the disclosure provides an isolated nucleic acid 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 prosaposin coding sequence (e.g., codons optimized for expression in mammalian cells, e.g., human cells). 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 adeno-associated virus (AAV) inverted terminal repeats (ITRs), e.g., AAV ITRs adjacent to the nucleic acid sequence encoding the prosaposin protein.

[0126] 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 independently selected from the gene products listed in Table 1, or a portion thereof.

[0127] 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.

[0128] 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 contains a sequence described in any one of SEQ ID NOs. 20-25. In some embodiments, the interfering nucleic acid targeting α-synuclein binds to (hybridizes with) a sequence described in any one of SEQ ID NOs. 20-25.

[0129] 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 with) the sequence described in SEQ ID NO: 64 or 65.

[0130] 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. - Alternatively, it could be an RNA pol III promoter (e.g., U6).

[0131] 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.

[0132] 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.

[0133] In some embodiments, the expression construct contains 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., from the 5' end to the 3' end: in the arrangement ITR-first gene product-second gene product-ITR). In some embodiments, one of the ITR sequences in the isolated nucleic acid lacks a functional end separation site (trs). For example, in some embodiments, one of the ITRs is a ΔITR.

[0134] 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 the wild-type AAV2 ITR, SEQ ID NO: 29). In some embodiments, the ITR having the modified D region is the 5' ITR of the rAAV vector. In some embodiments, the modified "D" region includes, 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 the 3' ITR, and 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 includes the sequence described in SEQ ID NO: 26 or 27.

[0135] 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.

[0136] In some embodiments, the isolated nucleic acids described herein encode a peptide comprising, consisting of, or having one of the sequences described in SEQ ID NOs: 1 to 91.

[0137] In some embodiments, the Disclosure provides a vector comprising an 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).

[0138] In some embodiments, the Disclosure provides a host cell containing an isolated nucleic acid described herein or a vector described herein.

[0139] In some embodiments, the Disclosure provides recombinant adeno-associated virus (rAAV) or vectors described herein, comprising a capsid protein and isolated nucleic acid.

[0140] In some embodiments, the capsid protein can cross the blood-brain barrier, for example, the AAV9 capsid protein or the AAVrh.10 capsid protein. In some embodiments, rAAV transducers neurons and non-neuronal cells of the central nervous system (CNS).

[0141] In some embodiments, the Disclosure provides a method for treating a subject having or suspected of having a central nervous system (CNS) disease, the method comprising administering to the subject a composition described in the Disclosure (e.g., a composition comprising isolated nucleic acid or vector or rAAV). 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.

[0142] 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 in the Disclosure (e.g., a composition comprising isolated nucleic acid or vector or rAAV) to the subject.

[0143] In some embodiments, the Disclosure provides a method for treating subjects who have or are suspected of having frontotemporal dementia (FTD), FTD with a GRN mutation, FTD with a tau mutation, FTD with a CC9orf72 mutation, ceroid lipofuscinosis, Parkinson's disease, Alzheimer's disease, corticobasal degeneration, motor neuron disease, or Gaucher disease, the method comprising administering to the subject an rAAV encoding progranulin (PGRN), PGRN being encoded by the nucleic acid sequence in SEQ ID NO: 68, and the rAAV comprising a capsid protein having the AAV9 serotype.

[0144] In some embodiments, the Disclosure provides a method for treating subjects having or suspected to have FTD with a GRN mutation, the method comprising administering to the subject an rAAV encoding progranulin (PGRN), PGRN being encoded by the nucleic acid sequence in 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 14 It is administered to the subject at a dose of vg. In some embodiments, rAAV is administered by injection into the cisterna magna.

[0145] In some embodiments, the composition comprises nucleic acids encoding two or more gene products (e.g., CNS disease-related gene products), e.g., 2, 3, 4, 5, or more gene products described in this application (e.g., an AAV genome capsidized by an AAV capsid protein). In some embodiments, the composition comprises two or more (e.g., 2, 3, 4, 5 or more) different nucleic acids (e.g., two or more rAAV genomes separately capsidized by an AAV capsid protein) 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, the different gene products are operably linked to the same promoter type (e.g., the same promoter). In some embodiments, the different gene products are operably linked to different promoters.

[0146] Isolated nucleic acids and vectors The isolated nucleic acid may be DNA or RNA. In some embodiments, this disclosure provides an isolated nucleic acid (e.g., an rAAV vector) comprising an expression construct encoding one or more PD-related genes, such as Gcase (e.g., the gene product of the GBA1 gene) or a portion thereof. Gcase, also called β-glucocerebrosidase or GBA, refers to a lysosomal protein that cleaves the beta-glucosidic bond of chemical glucocerebrosides, which are intermediates in glycolipid metabolism. In humans, Gcase is encoded by the GBA1 gene located on chromosome 1. In some embodiments, GBA1 is 、N It encodes a peptide represented by the CBI reference sequence NP_000148.2 (SEQ ID NO: 14). In some embodiments, the isolated nucleic acid includes a codon-optimized Gcase coding sequence (e.g., a codon optimized for expression in mammalian cells, e.g., human cells), such as the sequence described in SEQ ID NO: 15.

[0147] 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). 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 (SEQ ID NO: 16). In some embodiments, the isolated nucleic acid comprises a codon-optimized prosaposin-coding sequence (e.g., a codon optimized for expression in mammalian cells, e.g., human cells), such as the sequence described in SEQ ID NO: 17.

[0148] 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 endosome 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.

[0149] Aspects of this disclosure relate to isolated nucleic acids comprising expression constructs 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.

[0150] Aspects of this disclosure relate to isolated nucleic acids comprising expression constructs encoding GLAC proteins (e.g., gene products of GLAC genes). GALC proteins refer to galactosylceramidases (or galactocerebrosidases), which are enzymes that hydrolyze galactose ester bonds in 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.

[0151] Aspects of this disclosure relate to isolated nucleic acids comprising expression constructs encoding the 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.

[0152] Aspects of this disclosure relate to isolated nucleic acids comprising expression constructs encoding the SMPD1 protein (e.g., the gene product of the SMPD1 gene). The SMPD1 protein refers to sphingomyelin phosphodiesterase 1, a hydrolase enzyme involved in sphingolipid metabolism. In humans, the SMPD1 gene is located on chromosome 11. In some embodiments, the SMPD1 gene encodes a peptide 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.

[0153] Aspects of this disclosure relate to isolated nucleic acids comprising expression constructs encoding the GCH1 protein (e.g., the gene product of the GCH1 gene). The GCH1 protein refers to GTP cyclohydrolase I, a hydrolase 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.

[0154] Aspects of this disclosure relate to isolated nucleic acids comprising expression constructs encoding the RAB7L protein (e.g., the gene product of the RAB7L gene). The RAB7L protein refers to RAB7, a member of the RAS oncogene family-like 1, which is a GTP-binding protein. In humans, the RAB7L gene is located on chromosome 1. In some embodiments, the RAB7L gene encodes a peptide 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.

[0155] 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.

[0156] 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 growth 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.

[0157] 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 a trigger receptor, an immunoglobulin superfamily receptor present on bone marrow cells, expressed on bone marrow 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.

[0158] 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 lysosome 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.

[0159] Aspects of this disclosure relate to isolated nucleic acids comprising expression constructs encoding progranulin proteins (e.g., the gene product of the PGRN gene). PGRN proteins refer 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).

[0160] In some embodiments, the 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, where all steps, including protein separation, immunoprobing, washing, and chemiluminescent detection, occur within a capillary cartridge. In some embodiments, the CSF sample is derived from human or non-human primates. In some embodiments, the immunoassay enables the detection of differences in PGRN protein levels in the presence of circulating antibodies. In some embodiments, the Disclosure provides a method for quantifying progranulin protein levels in a CSF sample, the method comprising: (1) diluting the CSF sample (e.g., 4-fold dilution); (2) loading the CSF sample, an anti-progranulin antibody, a secondary antibody to detect the anti-progranulin antibody, luminol, and a peroxide into the wells of a capillary cartridge; (3) loading the capillary cartridge into an automated Western blot immunoassay instrument; (4) calculating one or more of the signal intensity, peak area, signal-to-noise ratio, and total protein normalization parameters using the automated Western blot immunoassay instrument; and (5) quantifying the progranulin protein levels in the CSF sample as the peak area of ​​the immune response to the anti-progranulin antibody. In some embodiments, the CSF sample is diluted with a master mix containing dithiothreitol (DTT) and sample buffer. The master mix may further contain other proprietary components. In some embodiments, the anti-progranulin antibody detects human progranulin. In some embodiments, progranulin protein levels are quantified from calculated parameters using software that controls an automated Western blot immunoassay instrument. In some embodiments, the software is Compass software for Simple Western® (ProteinSimple, San Jose, California).

[0161] 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 mix comprising dithiothreitol (DTT) and sample buffer (e.g., 4-fold dilution); (2) loading the diluted CSF sample, anti-progranulin antibody, secondary antibody to detect the anti-progranulin antibody, luminol, and peroxide into the wells of a capillary cartridge; (3) loading the capillary cartridge into an automated Western blot immunoassay instrument; (4) calculating 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 peak area of ​​the immune response to the anti-progranulin antibody.

[0162] 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 independently selected from the gene products listed in Table 1, or a portion thereof.

[0163] In some embodiments, the isolated nucleic acids or vectors described in this disclosure (e.g., rAAV vectors) include or consist of sequences described in any one of SEQ ID NOs: 1 to 91. In some embodiments, the isolated nucleic acids or vectors described in this disclosure (e.g., rAAV vectors) include or consist of sequences that are complementary (e.g., complementary) to the sequences described in any one of SEQ ID NOs: 1 to 91. In some embodiments, the isolated nucleic acids or vectors described in this disclosure (e.g., rAAV vectors) include or consist of sequences that are inversely complementary to the sequences described in any one of SEQ ID NOs: 1 to 91. In some embodiments, the isolated nucleic acids or vectors described in this disclosure (e.g., rAAV vectors) include or consist of a portion of the sequences described in any one of SEQ ID NOs: 1 to 91. The portion may include at least 25%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the sequences described in any one of SEQ ID NOs: 1 to 91. In some embodiments, the nucleic acid sequences described herein are the nucleic acid sense strand (e.g., 5' to 3' strand), or the positive (+) strand in the context of a viral sequence. In some embodiments, the nucleic acid sequences described herein are the nucleic acid antisense strand (e.g., 3' to 5' strand), or the negative (-) strand in the context of a viral sequence.

[0164] In some embodiments, the gene product is encoded by the coding portion of a native gene (e.g., cDNA). In some embodiments, the first gene product is a protein (or a fragment thereof) encoded by the GBA1 gene. In some embodiments, the gene product is a protein (or a fragment thereof) encoded by another gene listed in Table 1, such as the SCARB2 / LIMP2 gene or the PSAP gene. However, those skilled in the art will recognize that the order of expression of the first gene product (e.g., Gcase) and the second gene product (e.g., LIMP2) can generally be reversed (e.g., LIMP2 is the first gene product and Gcase is the second gene product). In some embodiments, the gene product is a fragment (e.g., a portion) of a gene listed in Table 1. A protein fragment may comprise about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the protein encoded by a gene listed in Table 1. In some embodiments, the protein fragment comprises 50% to 99.9% (e.g., any value between 50% and 99.9%) of the protein encoded by a gene listed in Table 1.

[0165] In some embodiments, the expression construct is monocistronic (e.g., the expression construct encodes a single fusion protein comprising the first gene product and the second gene product). In some embodiments, the expression construct is polycistronic (e.g., the expression construct encodes two different gene products, for example, two different proteins or protein fragments).

[0166] A polycistronic expression vector may comprise one or more (e.g., 1, 2, 3, 4, 5 or more) promoters. Any suitable promoter may be used, for example, a constitutive promoter, an inducible promoter, an endogenous promoter, a tissue-specific promoter (e.g., a CNS-specific promoter), and the like. In some embodiments, the promoter is a chicken beta-actin promoter (CBA promoter), a CAG promoter (described, for example, in Alexopoulou et al. (2008) BMC Cell Biol. 9:2; doi:10.1186 / 1471-2121-9-2), a CD68 promoter, or a JeT promoter (described, for example, in Tornoe et al. (2002) Gene 297(1-2):21-32). In some embodiments, the promoter is operably linked to a nucleic acid sequence encoding a first gene product, a second gene product, or both the first gene product and the second gene product. In some embodiments, the expression cassette comprises one or more additional regulatory sequences, including but not limited to a transcription factor binding sequence, an intron splice site, a poly(A) addition site, an enhancer sequence, a repressor binding site, or any combination of the foregoing.

[0167] In some embodiments, the nucleic acid sequence encoding the first gene product and the nucleic acid sequence encoding the second gene product are separated by a nucleic acid sequence encoding an internal ribosome entry site (IRES). Examples of IRES sites are described, for example, by Mokrejs et al. (2006) Nucleic Acids Res. 34(Database Issue):D125-30. In some embodiments, the nucleic acid sequence encoding the first gene product and the nucleic acid sequence encoding the second gene product are separated by a nucleic acid sequence encoding a self-cleaving peptide. Examples of self-cleaving peptides include, but are not limited to, T2A, P2A, E2A, F2A, BmCPV 2A, and BmIFV 2A, as well as those described in Liu et al. (2017) Sci Rep. 7:2193. In some embodiments, the self-cleaving peptide is a T2A peptide.

[0168] Pathologically, disorders such as PD and Gaucher disease are primarily associated with the accumulation of protein aggregates 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. The sequence encoding the inhibitory nucleic acid may be located within the untranslated region of the expression vector (e.g., introns, 5'UTR, 3'UTR, etc.).

[0169] In some embodiments, the inhibitory nucleic acid is located upstream of an intron in the expression construct, e.g., intron of the sequence encoding the first gene product. The inhibitory nucleic acid can be double-stranded RNA (dsRNA), siRNA, shRNA, microRNA (miRNA), artificial miRNA (miRNA), or RNA aptamer. Generally, the inhibitory nucleic acid binds to (e.g., hybridizes) about 6 to about 30 (e.g., any integer between 6 and 30) consecutive nucleotides of the target RNA (e.g., mRNA). In some embodiments, the inhibitory nucleic acid molecule is a miRNA or amiRNA, e.g., 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 targeting sequence.

[0170] 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 substituted with any other nucleotide suitable for base pairing with adenosine nucleotides (e.g., via Watson-Crick base pairing). For example, T may be substituted with U, and U may be substituted with T.

[0171] The isolated nucleic acids described herein may exist as themselves or as part of a vector. Generally, vectors may be plasmids, cosmids, phagemids, bacterial artificial chromosomes (BACs), or viral vectors (e.g., adenovirus vectors, adeno-associated virus (AAV) vectors, retroviral vectors, baculovirus vectors, 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., an Autographa californica nuclear polyhedron disease (AcNPV) vector).

[0172] Typically, an rAAV vector (e.g., an rAAV genome) contains a transgene (e.g., an expression construct comprising one or more promoters, such as introns, enhancer sequences, protein-coding sequences, inhibitory RNA-coding sequences, and poly-A tail sequences) adjacent to two AAV inverted terminal repeat (ITR) sequences. In some embodiments, the transgene of an rAAV vector contains an 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., about 145 bp in length, including a functional rep-binding site (RBS) and terminal separation 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 shortened ITR lacks a functional terminal separation site (trs) and is used to construct a self-complementary AAV vector (scAAV vector). In some embodiments, the shortened ITR is a ΔITR, as described, for example, in McCarty et al. (2003) Gene Ther. 10(26):2112-8. In some embodiments, each of the two ITR sequences is an AAV2 ITR sequence.

[0173] 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.) compared to wild-type AAVITR, for example, compared to wild-type AAV2 ITR (SEQ ID NO: 29). The structure of wild-type AAV2 ITR is shown in Figure 20. Generally, wild-type ITRs contain a 125-nucleotide region, which 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 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, either relative to the end of the ITR or 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 capsid formation of the rAAV vector by the capsid protein, as disclosed, for example, by Ling et al. (2015) J Mol Genet Med 9(3).

[0174] This disclosure is partly based on the surprising finding that rAAV vectors containing a “D” region located “outside” the ITR (e.g., proximal to the terminal of the ITR compared to an transgene insertion or expression construct) are more efficiently capsidated by the AAV capsid protein than rAAV vectors containing ITSs with unmodified (e.g., wild-type) ITRs. In some embodiments, rAAV vectors with a modified “D” sequence (e.g., a “D” sequence at an “outside” position) exhibit reduced toxicity compared to rAAV vectors with a wild-type ITR sequence.

[0175] In some embodiments, the modified “D” sequence includes 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 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 nucleotide substitutions relative to the wild-type “D” sequence (e.g., SEQ ID NO: 27). In some embodiments, the modified “D” sequence includes at least 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 nucleic acid substitutions relative to the wild-type “D” sequence (e.g., SEQ ID NO: 27). In some embodiments, the modified “D” sequence is identical to the wild-type “D” sequence (e.g., SEQ ID NO: 27) by approximately 10% to 99% (e.g., 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%). In some embodiments, the modified “D” sequence includes the sequence described in SEQ ID NO: 26, also known as the “S” sequence described in Wang et al. (1995) J Mol Biol 250(5):573-80.

[0176] The isolated nucleic acids or rAAV vectors described herein may further include a “TRY” sequence, for example, as described in SEQ ID NO: 28 or as described 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., transgene coding insertion) of the isolated nucleic acid or rAAV vector.

[0177] In some embodiments, this 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 in Urabe et al. (2002) Hum Gene Ther 13(16):1935-43 and Smith et al. (2009) Mol Ther 17(11):1888-1896.

[0178] In some embodiments, this disclosure provides host cells comprising isolated nucleic acids or vectors 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., Escherichia coli cells. rAAVs

[0179] In some embodiments, this disclosure relates to recombinant AAVs (rAAVs) comprising transgenes encoding nucleic acids described herein (e.g., rAAV vectors described herein). The term “rAAVs” generally refers to viral particles comprising an rAAV vector capsid-formed by one or more AAV capsid proteins. The rAAVs described herein may comprise capsid proteins having serotypes selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10. In some embodiments, the rAAVs comprise capsid proteins derived from non-human hosts, such as rhesus monkey AAV capsid proteins such as AAVrh.10 and AAVrh.39. In some embodiments, the rAAV described herein includes a capsid protein that is a variant of the wild-type capsid protein, such as a capsid protein variant that contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 (e.g., 15, 20, 25, 50, 100, etc.) amino acid substitutions (e.g., mutations) relative to the wild-type AAV capsid protein from which it is induced. In some embodiments, the AAV capsid protein variant is, for example, the AAV1RX capsid protein described in 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 in Rosario et al. Mol Ther Methods Clin Dev. 2016;3:16026.

[0180] In some embodiments, the rAAVs described herein spread readily through the CNS, particularly when introduced directly into the CSF lumen or brain parenchyma. Therefore, in some embodiments, the rAAVs described herein include a capsid protein capable of crossing the blood-brain barrier (BBB). For example, in some embodiments, the rAAV includes a capsid protein having the serotype AAV9 or AAVrh.10. 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 includes a capsid protein that specifically or preferentially targets bone marrow cells, such as microglia.

[0181] In some embodiments, this disclosure provides an rAAV called "PR006A". PR006A is an rAAV that delivers a functional human GRN gene, resulting in increased expression of a functional human PGRN. The PR006A vector insert contains a chicken β-actin (CBA) promoter element comprising four segments: a cytomegalovirus (CMV) enhancer, a CBA promoter, exon 1, and an intron (int), constitutively expressing 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 signal tail. Three well-described transcriptional regulatory activations

[0182] The sites are located 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 of the first strand (5'-3' order) of the PR006A vector shown in Figure 64. Sequence ID 91 provides the nucleotide sequence of the second strand (5'-3' order) of the PR006A vector shown in Figure 64. PR006A contains the AAV9 capsid protein.

[0183] In some embodiments, the rAAVs described herein (including, for example, recombinant rAAV genomes capsid-formed by AAV capsid proteins to form rAAV capsid particles) are produced in a baculovirus vector expression system (BEVS). Production of rAAVs 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 WO2017 / 184879. However, rAAVs can be produced using any suitable method (e.g., using recombinant rep and cap genes). In some embodiments, the rAAVs disclosed herein are produced in HEK293 (human embryonic kidney) cells. Pharmaceutical composition

[0184] In certain embodiments, the 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 impair the biological activity or properties of a compound and is relatively non-toxic, for example, allowing the material to be administered to an individual without causing undesirable biological effects or interacting in a harmful manner with any of the components of the composition it contains.

[0185] 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, which is involved in transporting or delivering a useful compound within or to a patient in the present invention so as to be able to 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.

[0186] The compositions (e.g., pharmaceutical compositions) described herein can be administered by any route including enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, intradermal, rectal, intravaginal, intraperitoneal, topical (powders, ointments, creams, and / or drops), mucosal, nasal, buccal, sublingual; intratracheal instillation, bronchial instillation, and / or inhalation; and / or as an oral spray, nasal spray, and / or aerosol. Specifically contemplated routes are oral administration, intravenous administration (e.g., systemic intravenous injection), local administration via blood and / or lymph supply, and / or direct administration to the affected site. In general, the most appropriate route of administration depends on a variety of factors including the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), and / or the condition of the subject (e.g., whether the subject is able to tolerate oral administration). In certain embodiments, the compound or pharmaceutical composition described herein is suitable for topical administration to the eye of a subject.

[0187] In some embodiments, the present disclosure provides a PR006A final drug product comprising the aforementioned PR006A rAAV presented in an aqueous solution. In some embodiments, the final formulation buffer comprises about 20 mM Tris [pH 8.0], about 1 mM MgCl2, about 200 mM NaCl, and about 0.001% [w / v] poloxamer 188. In some embodiments, the final drug product and the final formulation buffer are suitable for intracisternal magna (ICM) injection.

[0188] Provided herein is a combination therapeutic agent comprising: (A) an rAAV comprising (a) an rAAV vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a transgene insert encoding a PGRN protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 68, and (b) an AAV9 capsid protein; and (B) sirolimus for use in a method of treating frontotemporal dementia with a GRN mutation in a subject.

[0189] This specification provides a combination therapy comprising (i) an rAAV vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a transgene insert encoding a progranulin (PGRN) protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 68, and (ii) a recombinant adeno-associated virus (rAAV) comprising an adeno-associated virus (AAV) 9 capsid protein, and one or more immunosuppressants used in a method for treating frontotemporal dementia with a GRN mutation in a subject. This specification provides a combination therapy comprising (i) an rAAV vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a transgene insert encoding a progranulin (PGRN) protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 68, and (ii) a recombinant adeno-associated virus (rAAV) comprising an adeno-associated virus (AAV) 9 capsid protein, and one or more of the following: (A) Sirolimus, (B) Methylprednisolone, (C) Rituximab, and (D) Prednisone for use in the treatment of frontotemporal dementia with GRN mutations in the subject.

[0190] This specification provides a combination therapy agent comprising (i) a nucleic acid comprising an rAAV vector comprising an expression construct comprising a promoter operably linked to a transgene insertion encoding a progranulin (PGRN) protein, wherein the transgene insertion comprises an rAAV vector comprising the nucleotide sequence of SEQ ID NO: 68, and (ii) a recombinant adeno-associated virus (rAAV) comprising an adeno-associated virus (AAV) 9 capsid protein, and one or more of the following: (A) sirolimus, (B) methylprednisolone, (C) rituximab, and (D) prednisone, for use in a method of suppressing the immune response in subjects having or suspected of having frontotemporal dementia with a GRN mutation.

[0191] In some embodiments, the combination therapy agent is approximately 1 × 10 13 vg~approx. 7×10 14Includes vg rAAV. In some embodiments, the combination therapy agent is approximately 3.5 × 10 13 vg, approx. 7.0×10 13 vg, or approximately 1.4 × 10 14 Includes vg's rAAV.

[0192] In some embodiments, the combination therapy includes an additional immunosuppressant other than sirolimus, methylprednisolone, rituximab, or prednisone. method

[0193] Aspects of this disclosure are, in part, based on 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 (1, 2, 3, 4, 5, or more) gene products. In some embodiments, subjects are administered multiple (e.g., 2, 3, 4, 5, or more) vectors (e.g., isolated nucleic acids, rAAVs, etc.), each vector encoding a different CNS disease-related gene product.

[0194] 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.

[0195] "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, compared to a healthy subject, e.g., a subject without synuclein disease). Examples of synuclein diseases and their associated genes are listed in Table 13.

[0196] "Tauopathy" refers to a disease or disorder characterized by an abnormal accumulation of tau protein in a subject (for example, compared to a healthy subject without tauopathy). Examples of tauopathy and their associated genes are listed in Table 14.

[0197] "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 their associated genes are listed in Table 15.

[0198] As used herein, “to treat” or “to treat” means (a) to prevent or delay the onset of a CNS disorder, (b) to reduce the severity of a CNS disorder, (c) to reduce or prevent the onset of symptoms characteristic of a CNS disorder, and (d) and / or to prevent the exacerbation of symptoms characteristic of a CNS disorder. Symptoms of a CNS disorder may include, for example, motor impairment (e.g., shaking, rigidity, sluggishness, difficulty walking, paralysis), cognitive impairment (e.g., dementia, depression, anxiety, psychosis), memory problems, and emotional and behavioral impairments.

[0199] This disclosure is, in part, based on compositions for the expression of combinations of PD-related gene products in a subject that act together (e.g., synergistically) to treat Parkinson's disease.

[0200] Accordingly, in certain embodiments, the Disclosure provides a method for treating a subject who has or is suspected of having Parkinson's disease, the method comprising administering a composition described in the Disclosure (e.g., a composition comprising isolated nucleic acid or vector or rAAV) to the subject.

[0201] 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 a neuropathic Gaucher disease, such as type 2 Gaucher disease or type 3 Gaucher disease. In some embodiments, subjects having Gaucher disease do not have PD or symptoms of PD.

[0202] Accordingly, in certain embodiments, the Disclosure provides a method for treating a subject having or suspected to have neuropathic Gaucher disease, the method comprising administering a composition described in the Disclosure (e.g., a composition comprising isolated nucleic acid or vector or rAAV) to the subject.

[0203] 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 with a GRN (progranulin) mutation. In some embodiments, the subjects have FTD with 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 (exon) gene deletion). In some embodiments, the GRN mutation is a pathogenic mutation with demonstrated functional adverse 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 generates low plasma PGRN levels (<70 ng / mL) in the subject.

[0204] In some embodiments, the subjects include FTD, FTD with a GRN mutation, FTD with a tau mutation, FTD with a C9orf72 mutation, neuronal ceroid lipofuscinosis, Parkinson's disease, Alzheimer's disease, corticobasal degeneration, motor neuron disease, or Gaucher disease.

[0205] In some embodiments, the subjects have symptomatic FTD (e.g., behavioral variant FTD (bvFTD), primary progressive aphasia (PPA)-FTD, FTD with corticobasal syndrome, or a combination of syndromes).

[0206] Accordingly, in certain embodiments, the Disclosure provides a method for treating a subject having or suspected of having FTD with a GRN mutation, the method comprising administering a composition described in the Disclosure (e.g., a composition comprising isolated nucleic acid or vector or rAAV) to the subject.

[0207] In some embodiments, subjects with Alzheimer's disease or FTD (e.g., FTD with a GRN mutation) are administered an rAAV encoding progranulin (PGRN) or a portion thereof. In some embodiments, subjects with Alzheimer's disease or FTD (e.g., FTD with a GRN mutation) are administered an 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 encodes a PGRN comprising a capsid protein having the AAV9 serotype.

[0208] In some embodiments, a composition comprising rAAV encoding a PGRN for treating FTD (e.g., FTD with a GRN mutation) is approximately 1 × 10 12 Vector genome (vg) ~ approximately 1 × 10⁻⁶ 15 vg, or approximately 1 × 10⁻⁶ 13 vg~approx. 7×10 14 vg, or approximately 1 × 10⁻⁶ 13 vg~approx. 5×10 14 vg, or approximately 2 × 10 13 vg ~ approx. 2×10 14 , or approximately 3 x 10 13 ~Approx. 2×10 14 vg, or approximately 3.5 × 10 13 vg~approx. 1.4×10 14 It is administered to the subject in doses in the range of vg. In some embodiments, a composition containing rAAV encoding a PGRN to treat FTD (e.g., FTD with a GRN mutation) is about 2 × 10 13 vg, approx. 3×10 13 vg, approx. 4×1013 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 to the target patient at a dose of VG.

[0209] 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 a composition comprising an rAAV encoding a PGRN to the subject, the composition comprising approximately 3.5 × 10 13 Vector genome (vg), approximately 7.0 × 10⁻⁶ 13 vg, or approximately 1.4 × 10 14 It is administered at a dose of VG.

[0210] 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 a composition comprising an rAAV encoding a PGRN to the subject, 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.

[0211] In some embodiments, a composition containing an rAAV encoding a PGRN that treats FTD (e.g., FTD with a GRN mutation) is administered to a subject as a single dose, and the composition is not administered to the subject thereafter.

[0212] In some embodiments, the composition containing rAAV is delivered into the cisterna magna via a single suboccipital injection. In some embodiments, the injection into the cisterna magna is performed under radiographic guidance.

[0213] In some embodiments, the disclosure provides a method for treating symptoms of a subject having or suspected having FTD with a GRN mutation, the method comprising administering to the subject a composition comprising an rAAV encoding a functional progranulin (PGRN) protein sequence, 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, delayed speech, grammatical errors, multimode agnosia, semantic aphasia, or impaired speech comprehension. In some embodiments, the rAAV encodes a PGRN comprising a capsid protein having the AAV9 serotype.

[0214] In some embodiments, the disclosure provides a method for reducing lipofuscin accumulation in the brain of a subject having or suspected of having GRN mutation-containing 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 disclosure provides a method for reducing ubiquitin accumulation in the brain of a subject having or suspected of having GRN mutation-containing 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 disclosure provides a method for reducing gene expression and / or protein expression of TNFα and / or CD68 in the brain of a subject having or suspected of having GRN mutation-containing 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 Disclosure provides a method for increasing the maturation of cathepsin D in the brain of a subject having or suspected having GRN mutations in 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 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 or suspected having GRN mutations in 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 Sequence ID No. 68.In some embodiments, the disclosure provides a method for reducing the level of neurofilamentous light chains (NfLs) in the blood or CSF of a subject having or suspected having a GRN mutation in 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 encodes a PGRN comprising a capsid protein having the AAV9 serotype.

[0215] The subjects are typically mammals, preferably humans. In some embodiments, the subjects are between 1 month and 10 years of age (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 of age. In some embodiments, the subjects are between 30 and 100 years of age. In some embodiments, the subjects are 55 years of age or older.

[0216] 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 intracisricular cisterna magna (ICM) injection. In some embodiments, direct injection into the subject's CNS results in transgene expression (e.g., expression of the first gene product, the second gene product, and, if applicable, the third gene product) in the subject's midbrain, striatum, and / or cerebral cortex. In some embodiments, direct injection into the CNS results in transgene expression (e.g., expression of the first gene product, the second gene product, and, if applicable, the third gene product) in the subject's spinal cord and / or CSF.

[0217] In some embodiments, direct injection into the target CNS includes convective-enhanced delivery (CED). Convective-enhanced delivery is a therapeutic strategy involving 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 the 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.

[0218] 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, such as injection into the carotid artery of the subject.

[0219] In some embodiments, the compositions described herein (e.g., compositions comprising isolated nucleic acids or vectors or rAAV) are administered to the target CNS both peripherally and directly. For example, in some embodiments, the target 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 simultaneous (e.g., occur at the same time). In some embodiments, the direct injection occurs before the peripheral injection (e.g., 1 minute and 1 week or earlier). In some embodiments, the direct injection occurs after the peripheral injection (e.g., 1 minute and 1 week or later).

[0220] In some embodiments, the subject is administered an immunosuppressant before (e.g., one month to one minute before) or concurrently with the composition 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.

[0221] In some embodiments, subjects are administered an oral loading dose of approximately 6 mg of sirolimus on day -1 (window day -3 to day -1) (day 0 is administration of rAAV). For example, the sirolimus dose may be administered on day -3, day -2, or day -1. In some embodiments, a subsequent sirolimus maintenance dose of 2 mg is administered and adjusted as needed up to month 3 to maintain a serum trough level of approximately 4 ng / mL (ranging from approximately 2 ng / mL to approximately 8 ng / mL). In some embodiments, a subsequent sirolimus maintenance dose of 2 mg is administered and adjusted as needed up to month 3 to maintain a serum trough level of approximately 4 ng / mL to approximately 9 ng / mL. In some embodiments, sirolimus is then substantially tapered over the following 15 to 30 days (after the end of month 3). In some embodiments, trough levels are collected before administration of the sirolimus dose.

[0222] In some embodiments, subjects are administered an intravenous loading dose of approximately 1 g of methylprednisolone on day 0 (window - day 1 to day 0), followed by oral administration of approximately 30 mg of prednisone for 14 days starting the day after rAAV administration. In some embodiments, prednisone is tapered over the following 7 days. In some embodiments, prednisone is administered orally as a concomitant drug at a dose of 0.5 mg / kg daily from day 1 to day 14, and then 0.25 mg / kg daily for 4 days, followed by slow tapering over 4 days from 0.1 mg / kg to 0 mg / kg daily. In some embodiments, the administration of methylprednisolone and prednisone is combined with the administration of sirolimus as described above. In some embodiments, higher doses or longer tapering of prednisone may be used (e.g., in the case of elevated alanine aminotransferase (ALT) / aspartate aminotransferase (AST)).

[0223] In some embodiments, this specification provides a method for treating a subject having or suspected of having frontotemporal dementia with a GRN mutation, the method comprising: (i) in the order of 5' to 3', (a) AAV2 ITR, (b) CMV enhancer, (c) CBA promoter, (d) transgene insertion encoding the PGRN protein, comprising the nucleotide sequence of SEQ ID NO: 68, (e) WPRE, (f) bovine growth hormone poly(A) signaling tail, and (g) AAV2 (ii) an rAAV vector comprising an ITR and a nucleic acid, (ii) an rAAV comprising an AAV9 capsid protein, and (b) sirolimus, which is administered orally in a dose of approximately 6 mg within a range of 1 to 3 days prior to the administration of the rAAV, and in doses of approximately 2 mg to maintain a serum trough level of approximately 2 ng / mL to approximately 8 ng / mL for approximately 3 months after the administration of the rAAV, and the administration is gradually reduced between 15 and 30 days after the end of the 3-month period after the administration of the rAAV.

[0224] This disclosure provides a method for treating subjects having or suspected of having FTD-GRN, combining (1) administration of rAAV delivering a functional copy of the GRN gene encoding wild-type PGRN, and (2) administration of an immunosuppressive regimen. In some embodiments, the immunosuppressive regimen includes administration of one or more of sirolimus, methylprednisolone, anti-CD20 antibody, and prednisone. In some embodiments, the immunosuppressive regimen includes administration of all of sirolimus, methylprednisolone, anti-CD20 antibody, and prednisone. In some embodiments, the immunosuppressive regimen consists of administration of all of sirolimus, methylprednisolone, anti-CD20 antibody, and prednisone. In some embodiments, the anti-CD20 antibody is rituximab.

[0225] In some embodiments, the immunosuppressive regimen suppresses the AAV-related and / or transgene protein expression-related immune response in the subject. In some embodiments, the immunosuppressive regimen reduces the AAV9 capsid immune response in the subject. In some embodiments, the immunosuppressive regimen reduces the CSF inflammatory response in the subject.

[0226] This specification provides a method for treating subjects having or suspected to have frontotemporal dementia with a GRN mutation, the method comprising administering to a subject a recombinant adeno-associated virus (rAAV) comprising (i) an rAAV vector comprising an expression construct comprising a promoter operably linked to a transgene insert encoding a progranulin (PGRN) protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 68, and (ii) an adeno-associated virus (AAV) 9 capsid protein, and one or more of the following: (A) sirolimus, (B) methylprednisolone, (C) rituximab, and (D) prednisone.

[0227] Furthermore, this specification provides a method for treating subjects who have or are suspected of having frontotemporal dementia with a GRN mutation, the method comprising administering to the subject one or more of the following to: (i) a transgene insertion encoding a progranulin (AAV) protein in the order of 5' to 3', wherein the transgene insertion comprises a nucleic acid including (a) an adeno-associated virus (AAV) 2 ITR, (b) a cytomegalovirus (CMV) enhancer, (c) a chicken beta-actin (CBA) promoter, (d) a progranulin (PGRN) protein, the transgene insertion comprising the nucleic acid sequence of Sequence ID No. 68, (e) a woodchuck hepatitis virus post-transcriptional regulator (WPRE), (f) a bovine growth hormone polyA signaling tail, and (g) an AAV2 inverted terminal repeat (ITR), and (ii) a recombinant adeno-associated virus (rAAV) comprising an AAV9 capsid protein. (A) Sirolimus, (B) Methylprednisolone, (C) Rituximab, and (D) Prednisone.

[0228] This specification further provides a method for suppressing the immune response of subjects having or suspected to have frontotemporal dementia with a GRN mutation, the method comprising administering to a subject a recombinant adeno-associated virus (rAAV) comprising (i) an rAAV vector comprising an expression construct comprising a promoter operably linked to a transgene insert encoding a progranulin (PGRN) protein, wherein the transgene insert comprises a nucleotide sequence of SEQ ID NO: 68, and (ii) an adeno-associated virus (AAV) 9 capsid protein, and one or more of the following: (A) sirolimus, (B) methylprednisolone, (C) rituximab, and (D) prednisone.

[0229] Furthermore, this specification provides a method for suppressing the immune response of subjects having or suspected to have frontotemporal dementia with a GRN mutation, the method comprising administering to a subject one or more of the following: (i) a transgene insertion encoding a progranulin (AAV) protein in the order of 5' to 3', wherein the transgene insertion comprises a nucleic acid including (a) an adeno-associated virus (AAV) 2 ITR, (b) a cytomegalovirus (CMV) enhancer, (c) a chicken beta-actin (CBA) promoter, (d) a progranulin (PGRN) protein, the transgene insertion comprising the nucleic acid sequence of Sequence ID No. 68, (e) a woodchuck hepatitis virus post-transcriptional regulator (WPRE), (f) a bovine growth hormone polyA signal tail, and (g) an AAV2 inverted terminal repeat (ITR), and (ii) a recombinant adeno-associated virus (rAAV) comprising an AAV9 capsid protein. (A) Sirolimus, (B) Methylprednisolone, (C) Rituximab, and (D) Prednisone.

[0230] In the methods disclosed herein for suppressing the immune response in a subject, the immunosuppressant is produced by an immunosuppressant (e.g., sirolimus, methylprednisolone, anti-CD20 antibody, and prednisone) rather than by gene therapy (e.g., rAAV).

[0231] In some embodiments, methylprednisolone is administered intravenously at a dose of approximately 1000 mg the day before administration of rAAV. In some embodiments, methylprednisolone is administered intravenously at a dose of approximately 1000 mg on the same day as administration of rAAV.

[0232] In some embodiments, prednisone is administered orally at a dose of approximately 30 mg / day for 14 days, starting the day after administration of approximately 1000 mg of methylprednisolone (A), and then tapered over 7 days following the completion of the 14-day period in (A). In some embodiments, tapering of prednisone for a longer period is continued for an additional 4 weeks in subjects exhibiting ALT and / or AST > 3 × upper limit of normal (ULN) at the end of the initial 14-day tapering period.

[0233] In some embodiments, an anti-CD20 antibody (e.g., rituximab) is administered intravenously at a dose of approximately 1000 mg on any one day between 14 days and 1 day prior to the administration of rAAV.

[0234] In some embodiments, methylprednisolone is administered before the administration of an anti-CD20 antibody (e.g., rituximab). In some embodiments, methylprednisolone is administered at least about 30 minutes before the administration of an anti-CD20 antibody (e.g., rituximab). In some embodiments, both methylprednisolone and the anti-CD20 antibody (e.g., rituximab) are administered on the day before the administration of rAAV, with methylprednisolone administered at least about 30 minutes before the administration of the anti-CD20 antibody (e.g., rituximab). In some embodiments, an anti-CD20 antibody (e.g., rituximab) is administered on any day between 14 and 2 days prior to the administration of rAAV, and methylprednisolone is administered intravenously at a dose of approximately 100 mg at least 30 minutes before the administration of the anti-CD20 antibody (e.g., rituximab) on the same day as the administration of the anti-CD20 antibody (e.g., rituximab).

[0235] In some embodiments, sirolimus is administered orally as a single dose of approximately 6 mg three, two, or one day before administration of rAAV, and (B) at a dose of approximately 2 mg / day to maintain a serum trough level of approximately 4 ng / ml to approximately 9 ng / mL for approximately 90 days after administration of rAAV, with the first dose of approximately 2 mg / day of sirolimus administered the day after the single dose of approximately 6 mg of sirolimus. In some embodiments, the administration of sirolimus is gradually reduced between 15 and 30 days after the end of the 90-day period following administration of rAAV.

[0236] This specification provides a method for treating subjects who have or are suspected of having frontotemporal dementia with a GRN mutation, and the method is provided to the subjects, (i) Administer methylprednisolone intravenously at a dose of approximately 1000 mg, (ii) Approximately 30 minutes after the administration of methylprednisolone in step (i), rituximab should be administered intravenously at a dose of approximately 1000 mg. (iii) On the day following the administration of methylprednisolone in step (i), the rAAV disclosed herein is administered by injection into the cisterna magna, (iv) Prednisone should be administered orally at a dose of approximately 30 mg / day for 14 days starting the day following the administration of methylprednisolone in step (i), (v) For 7 days after the completion of the 14-day period of step (iv), prednisone should be gradually tapered off. (vi) Three days, two days, or one day before the administration of rAAV in step (iii), sirolimus should be administered orally as a single dose of approximately 6 mg. (vii) Orally administer sirolimus at a dose of approximately 2 mg / day for approximately 90 days after rAAV administration in step (iii), maintaining a serum trough level of approximately 4 ng / ml to approximately 9 ng / mL, with the first dose of approximately 2 mg / day of sirolimus being administered the day after a single dose of approximately 6 mg of sirolimus. (viii) The administration of sirolimus is gradually reduced between 15 and 30 days after the end of the 90-day period of step (vii).

[0237] This specification provides a method for suppressing the immune response in subjects who have or are suspected of having frontotemporal dementia with a GRN mutation, and the method applies to the subjects, (i) Administer methylprednisolone intravenously at a dose of approximately 1000 mg, (ii) Approximately 30 minutes after the administration of methylprednisolone in step (i), rituximab should be administered intravenously at a dose of approximately 1000 mg. (iii) The day following the administration of methylprednisolone in step (i), the rAAV is administered into the cisterna magna by injection, (iv) Prednisone should be administered orally at a dose of approximately 30 mg / day for 14 days starting the day following the administration of methylprednisolone in step (i), (v) For 7 days after the completion of the 14-day period of step (iv), prednisone should be gradually tapered off. (vi) Three days, two days, or one day before the administration of rAAV in step (iii), sirolimus should be administered orally as a single dose of approximately 6 mg. (vii) Orally administer sirolimus at a dose of approximately 2 mg / day for approximately 90 days after rAAV administration in step (iii), maintaining a serum trough level of approximately 4 ng / ml to approximately 9 ng / mL, with the first dose of approximately 2 mg / day of sirolimus being administered the day after a single dose of approximately 6 mg of sirolimus. (viii) The administration of sirolimus is gradually reduced between 15 and 30 days after the end of the 90-day period of step (vii).

[0238] This specification provides a method for treating subjects who have or are suspected of having frontotemporal dementia with a GRN mutation, and the method is provided to the subjects, (i) Administer methylprednisolone intravenously at a dose of approximately 100 mg on any day between 14 and 2 days before the administration of rAAV in step (iv), (ii) Approximately 30 minutes after the administration of methylprednisolone in step (i), rituximab should be administered intravenously at a dose of approximately 1000 mg. (iii) Administer methylprednisolone intravenously at a dose of approximately 1000 mg either one day before or on the same day as the rAAV administration in step (iv), (iv) Administering the rAAV described herein into the cisterna magna by injection, (v) Prednisone shall be orally administered at a dose of approximately 30 mg / day for 14 days starting the day following the administration of methylprednisolone in step (iii), (vi) For 7 days following the completion of the 14-day period of step (v), the prednisone shall be administered in a gradually tapered manner. (vii) Three days, two days, or one day before the administration of rAAV in step (iv), sirolimus should be administered orally as a single dose of approximately 6 mg. (viii) Orally administer sirolimus at a dose of approximately 2 mg / day for approximately 90 days after rAAV administration in step (iv), to maintain a serum trough level of approximately 4 ng / ml to approximately 9 ng / mL, with the first dose of approximately 2 mg / day of sirolimus being administered the day after a single dose of approximately 6 mg of sirolimus. (ix) The administration of sirolimus is gradually reduced between 15 and 30 days after the end of the 90-day period of step (viii).

[0239] This specification provides a method for suppressing the immune response in subjects who have or are suspected of having frontotemporal dementia with a GRN mutation, and the method applies to the subjects, (i) Administer methylprednisolone intravenously at a dose of approximately 100 mg on any day between 14 and 2 days before the administration of rAAV in step (iv), (ii) Approximately 30 minutes after the administration of methylprednisolone in step (i), rituximab should be administered intravenously at a dose of approximately 1000 mg. (iii) Administer methylprednisolone intravenously at a dose of approximately 1000 mg either one day before or on the same day as the rAAV administration in step (iv), (iv) Administering rAAV into the cisterna magna via injection, (v) Prednisone shall be orally administered at a dose of approximately 30 mg / day for 14 days starting the day following the administration of methylprednisolone in step (iii), (vi) For 7 days following the completion of the 14-day period of step (v), the prednisone shall be administered in a gradually tapered manner. (vii) Three days, two days, or one day before the administration of rAAV in step (iv), sirolimus should be administered orally as a single dose of approximately 6 mg. (viii) Orally administer sirolimus at a dose of approximately 2 mg / day for approximately 90 days after rAAV administration in step (iv), to maintain a serum trough level of approximately 4 ng / ml to approximately 9 ng / mL, with the first dose of approximately 2 mg / day of sirolimus being administered the day after a single dose of approximately 6 mg of sirolimus. (ix) The administration of sirolimus is gradually reduced between 15 and 30 days after the end of the 90-day period of step (viii).

[0240] In some embodiments, the immune response is an immune response to rAAV. In some embodiments, the immune response is a T-cell response. In some embodiments, the immune response is a B-cell response. In some embodiments, the immune response is an antibody response. In some embodiments, the immune response is pleocytosis. In some embodiments, the pleocytosis is cerebrospinal fluid (CSF) pleocytosis. In some embodiments, the immune response is an abnormal level of CSF protein. In some embodiments, the abnormal level of CSF protein is greater than 70 mg / dL.

[0241] In some embodiments, prophylactic IV corticosteroid treatment (targeting both T and B cells) is initiated the day before treatment with rAAV, with oral treatment continuing for 14 days, followed by tapering over 7 days. Sirolimus treatment, primarily targeting T cells, is initiated the day before treatment with rAAV, continues for 90 days, and then tapered. Rituximab, primarily targeting B cells, is administered once, preferably the day before treatment with rAAV, and its activity is expected to last for 6 months.

[0242] In some embodiments, subjects receive an immunosuppressive regimen consisting of a corticosteroid, rituximab, and sirolimus. Subjects receive a loading dose of 1000 mg of methylprednisolone via IV pulse on day -1 (acceptable on day -1 or day 0). Prednisone is administered orally at a dose of 30 mg / day as a concomitant medication for 14 days starting the day after the 1000 mg methylprednisolone IV pulse (day 0 or day 1), tapering over the following 7 days. Subjects receive a single 1000 mg rituximab IV dose on any day between day -14 and day -1. To reduce the risk and severity of rituximab-related infusion-related reactions (IRRs), subjects receive an IV dose of methylprednisolone before receiving the rituximab IV infusion. For rituximab administration on day -1, subjects receive rituximab infusion at least 30 minutes after the 1000 mg methylprednisolone IV pulse mentioned above. For rituximab administration between day -14 and day -2, subjects receive 100 mg methylprednisolone IV infusion approximately 30 minutes before rituximab IV administration. Subjects receive a 6 mg sirolimus oral loading dose on day -1 (window between day -3 and day -1). A subsequent 2 mg / day sirolimus oral maintenance dose is initiated as a concomitant medication on day 0 (or the day after the sirolimus loading dose, if the sirolimus loading dose is administered on day -3 or day -2) and adjusted as needed for 90 days to maintain a serum trough level of 6 ng / mL (range 4-9 ng / mL) for 90 days. Sirolimus is tapered over the following 15-30 days. High doses or longer tapering periods of corticosteroids and sirolimus may be used.

[0243] In some embodiments, a longer tapering period or restart of immunosuppressive therapy may be used (e.g., in cases of elevated AST or ALT, inflammatory changes in CSF, or other suspected immune system reactions).

[0244] In some embodiments, additional immunosuppressants other than sirolimus, methylprednisolone, rituximab, or prednisone are further administered to the target.

[0245] In some embodiments, the methods disclosed herein may include increasing the dose of an immunosuppressant, a long-term tapering regimen, the use of additional drugs, or initiating treatment based on signs or symptoms consistent with an immune response, such as: Asymptomatic pleocytosis with a white blood cell count (WBC) >30 mm³ and / or elevated cerebrospinal fluid (CSF) protein (>70 mg / dL). • CSF pleocytosis and / or protein elevation accompanied by clinical symptoms (including undercompensation of underlying FTD symptoms) • Appearance of sensory symptoms based on neurological examination and / or the Treatment-Induced Neuropathy Assessment Scale (TNAS) • Symptoms of hepatitis (e.g., macular degeneration, fatigue) are accompanied by elevated alanine aminotransferase (ALT) and / or aspartate aminotransferase (AST) levels > 5 × upper limit of normal (ULN). • Regardless of the presence or absence of clinical symptoms, ALT and / or AST elevation >10 × ULN.

[0246] The amount of the composition described herein (e.g., a composition comprising isolated nucleic acid or vector or rAAV) administered to a subject varies depending on the method of administration. For example, in some embodiments, the amount of rAAV described herein is about 10 9 10 genome copies (GC) / kg and approximately 10 14 GC / kg (for example, about 10 9 GC / kg, approximately 10 10 GC / kg, approximately 10 11 GC / kg, approximately 10 12 GC / kg, approximately 10 12 GC / kg, or about 10 14 The subject is administered a titer between 10 GC / kg. In some embodiments, the subject is administered a high titer (e.g., >10) by injection into the CSF lumen or intraparenchymal injection. 12 The rAAV (rAAV) is administered at a genome copy rate of GC / kg. In some embodiments, the rAAV described herein is approximately 1 × 10⁶ 10 Approximately 1 × 10⁻¹⁶ from the vector genome (vg)17 It is administered to the subject by intravenous injection in doses in the range of vg. In some embodiments, the rAAV described herein is approximately 1 × 10⁻⁶ 10 Approximately 1 × 10⁻¹⁶ from the vector genome (vg) 16 The dose is administered to the subject by intracerebral injection within the VG range.

[0247] The compositions described herein (e.g., compositions comprising isolated nucleic acids or vectors or rAAV) can be administered to a subject once or multiple times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or more). In some embodiments, the composition is administered to the subject continuously (e.g., chronically), for example, via an infusion pump. [Examples]

[0248] Example 1: rAAV vector AAV vectors are produced using cells such as HEK293 cells for triple plasmid transfection. The ITR sequence is adjacent to an expression construct containing post-translational signals such as promoter / enhancer elements, a 3' polyA signal, and a WPRE element for each transgene of interest. Multiple gene products, such as GBA1 and LIMP2 and / or prosaposins, can be expressed simultaneously by protein sequence fusion, by using a 2A peptide linker such as T2A or P2A leading to two peptide fragments with added amino acids to prevent peptide bond formation, by using an IRES element, or by expression of two separate expression cassettes. The presence of short intron sequences that are efficiently spliced ​​upstream of the expressed genes can enhance expression levels. shRNAs and other regulatory RNAs may potentially be contained within these sequences. Examples of expression constructs described herein are shown in Figures 1–8, 21–35, 39, 41–51 and Figure 64, and in Table 2 below. [Table 2-1] [Table 2-2]

[0249] Example 2: Cell-based assay for viral transduction into GBA-deficient cells GBA1-deficient cells are obtained, for example, as fibroblasts from GD patients, monocytes, or hES cells, or from 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.

[0250] Using such cell models, lysosome deficiencies are quantified in terms of the accumulation of protein aggregates, such as α-synuclein with antibodies against this protein or phospho-α-Syn, and subsequently visualized using fluorescence microscopy. Visualization of lysosome abnormalities is performed by ICC for protein markers such as LAMP1, LAMP2, LIMP1, and LIMP2, or by using dyes such as Lysotracker, or by uptake of fluorescent dextran or other markers through the endocytosis compartment. Visualization of autophagy marker accumulation due to poor fusion with lysosomes, such as LC3, can also be performed. Abnormal accumulation of these markers is quantified using Western blotting and / or ELISA. In addition, the accumulation of glycolipid substrates and GBA1 products is measured using standard approaches.

[0251] Therapeutic endpoints (e.g., reduction of PD-related pathology) are measured in relation to the expression of AAV vector transduction, and their activity and function are confirmed and quantified. Gcase can also be quantified using protein ELISA or standard Gcase activity assays.

[0252] Example 3: In vivo assay using mutant mice This example describes an in vivo assay of an AAV vector using mutant mice. In vivo testing of the above AAV vector 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.

[0253] Vehicle control and AAV vector (e.g., 2 × 10⁻¹⁰) 11 Subarachnoid or intraventricular delivery (at a dose equivalent to vg / mouse) is performed using concentrated AAV stock, for example, in an injection volume of 5-10 μL. Intraparent delivery is performed via convection-enhanced delivery.

[0254] Treatment is initiated before or after the onset of symptoms. Measured endpoints include substrate accumulation in the CNS and CSF, accumulation of Gcase enzyme and enzyme activity by ELISA, motor and cognitive endpoints, lysosomal dysfunction, and accumulation of α-synuclein monomers, prefibrillaries, or fibrillaries.

[0255] 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.

[0256] Vehicle control and AAV vector (e.g., 2 × 10⁻¹⁰) 11 Subarachnoid or intraventricular delivery (at the dose per mouse) is performed using concentrated AAV stock, for example, in an injection volume of 5-10 μL. Intraparenchymal delivery is performed by convective-enhanced delivery. Peripheral delivery is achieved by tail vein injection.

[0257] Treatment is initiated before or after the onset of symptoms. Measured endpoints include substrate accumulation in the CNS and CSF, accumulation of Gcase enzyme and enzyme activity by ELISA, motor and cognitive endpoints, lysosomal dysfunction, and accumulation of α-synuclein monomers, prefibrillaries, or fibrillaries.

[0258] 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 rAAVs described herein in patients with Gaucher disease, PD, and / or LBD.

[0259] Clinical trials of such vectors for the treatment of Gaucher disease, PD, and / or LBD were conducted using a study design similar to that described in Grabowski et al. (1995) Ann. Intern. Med. 122(1):33-39.

[0260] Example 6: Treatment of peripheral diseases In some embodiments, patients with a specific form of Gaucher disease exhibit symptoms of peripheral neuropathy, as described, for example, by Biegstraaten et al. (2010) Brain 133(10):2909-2919.

[0261] 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 rAAV as described herein. In some embodiments, the peripheral neuropathy signs and symptoms in question are observed after administration of rAAV using, for example, a method described by Biegstraaten et al.

[0262] The levels of the transdextrin products described herein present in the patient's (e.g., in the patient's serum, peripheral tissues (e.g., liver tissue, spleen tissue, etc.)) are assayed, for example, by Western blotting, enzyme function assays, or imaging studies.

[0263] Example 7: Treatment of CNS type This embodiment describes an in vivo assay of the rAAVs described herein for the treatment of CNS morphs of Gaucher disease. Briefly, a Gaucher disease patient identified as having a CNS morph of Gaucher disease (e.g., type 2 or type 3 Gaucher disease) is administered rAAVs as described herein. The levels of the transdextrin products described herein present in the patient's CNS (e.g., in the patient's CNS serum, in the patient's cerebrospinal fluid (CSF), in the patient's CNS tissue) are assayed, for example, by Western blotting, enzyme function assay, or imaging studies.

[0264] Example 8: Gene therapy for Parkinson's disease in subjects with GBA1 mutations This embodiment describes the administration of a recombinant adeno-associated virus (rAAV) encoding GBA1 to subjects with Parkinson's disease characterized by mutations in the GBA1 gene.

[0265] This 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 constitutive expression of the codon-optimized coding sequence (CDS) of human GBA1 (maroon). The 3' region also contains a woodchuck hepatitis virus posttranscriptional regulator (WPRE). )、Next, it contains a bovine growth hormone polyA signaling (bGH polyA) tail. Adjacent ITRs enable correct packaging of the intervening sequence. Two variants of the 5' ITR sequence (Figure 7, inset box, bottom sequence) were evaluated. These variants have several nucleotide differences within the 20-nucleotide "D" region of the ITR, which are thought to affect packaging and expression efficiency. The rAAV-GBA1 vector product contains the "D" domain nucleotide sequence shown in Figure 7 (inset box, upper sequence). The variant vectors were similarly tested in preclinical trials. , It carries a mutant "D" domain (referred to as the "S" region herein, with nucleotide changes indicated by shading). 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.

[0266] rAAV-GBA1 is administered as a single dose to the subject via fluoroscopy-guided suboccipital injection into the cisterna magna (intracommunicable cisterna magna, ICM). One embodiment of the rAAV-GBA1 administration regimen study is as follows:

[0267] A single dose of rAAV-GBA1 is administered to patients (N=12) at one of two dose levels (e.g., 3e13vg (low dose) or 1e14vg (high dose)) determined based on the results of nonclinical pharmacological and toxicity studies.

[0268] Initial studies were conducted in a chemical mouse model involving daily delivery of the GCase inhibitor conzlitol-β-epoxide (CBE) to evaluate the efficacy and safety of the rAAV-GBA1 vector and rAAV-GBA1 S-variant construct (detailed below). Further initial studies were performed in a genetic mouse model carrying a homozygous GBA1 mutation and partially lacking saposin (4L / PS-NA). Additional dose-range studies in mice and non-human primates (NHPs) will be conducted to further evaluate the safety and efficacy of the vectors.

[0269] Two slightly different versions of the 5' inverted terminal repeat (ITR) in the AAV backbone were tested to evaluate manufacturability and transgene expression (Figure 7). The 20 bp "D" domain within the 145 bp 5' ITR is considered necessary for optimal viral vector production, although mutations within the "D" domain have been reported to increase transgene expression in some cases. Therefore, in addition to the viral vector rAAV-GBA1 carrying the complete "D" domain, a second vector form with a variant "D" domain (referred to herein as the "S" domain) was also evaluated. Both rAAV-GBA1 and the variant express the same transgene. Both vectors produced a virus that was effective in vivo, as detailed below, but rAAV-GBA1 containing the wild-type "D" domain was selected for further development.

[0270] To establish a CBE model for GCase deficiency, young mice were administered CBE, a specific inhibitor of GCase. Mice were given CBE daily via IP injection starting at postnatal day 8 (P8). Three different CBE doses (25 mg / kg, 37.5 mg / kg, and 50 mg / kg) and PBS were tested to establish models exhibiting behavioral phenotypes (Figure 9). High doses of CBE resulted in dose-dependent lethality. 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. On the other hand, CBE-injected mice did not show general motor impairments in the open-field assay (moving the same distance at the same speed as mice given PBS), while CBE-treated mice exhibited motor coordination and balance defects as measured by the rotarod assay.

[0271] Mice that survived until the end of the study were sacrificed either 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 administered 25 mg / kg of CBE to assess GCase substrate accumulation 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.

[0272] Based on the aforementioned studies, a CBE dose of 25 mg / kg was selected because it induced behavioral disorders without affecting survival. To achieve broad GBA1 distribution throughout the brain and across transgene expression during CBE treatment, rAAV-GBA1 or excipients were delivered by intraventricular (ICV) injection on postnatal day 3 (P3), followed by daily IP CBE or PBS treatment at P8 (Figure 10).

[0273] CBE-treated mice administered with rAAV-GBA1 showed statistically significantly better performance in rotarod compared to mice administered with excipients (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 11D).

[0274] 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). GCase activity in the cortex was assessed using a bio-triple-duplication fluorescence enzyme assay. GCase activity increased in mice treated with rAAV-GBA1, while CBE treatment decreased it. Furthermore, 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 examine the levels of the substrates GluCer and GluSph. Both CBE-treated mice and rAAV-GBA1 treatment significantly reduced substrate accumulation in the brain.

[0275] Lipid levels were negatively correlated with both GCase activity and rotorod performance across all treatment groups. Increased GCase activity after rAAV-GBA1 administration was associated with reduced substrate and enhanced motor function (Figure 13). Preliminary in vivo distribution was assessed by the presence of vector genomes, as measured by qPCR (defined as having >100 vector genomes per 1 μg genomic DNA) as shown in Figure 14. Mice administered with rAAV-GBA1 were positive for rAAV-GBA1 vector genomes in the cortex, 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.

[0276] In larger studies using the CBE model, the effective dose of rAAV-GBA1 in the CBE model was further explored. Using a 25 mg / kg CBE dose model, excipients or rAAV-GBA1 were delivered via ICV at P3, and IP PBS or CBE treatment was performed daily, initiated at P8. Considering the similarities observed in previous studies between groups with and without CBE withdrawal, 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 groups of 10 mice per group (5M / 5F). Excipient ICV + PBS IP Excipient ICV + 25 mg / kg CBE IP 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

[0277] The highest dose of rAAV-GBA1 rescued the failure of weight gain at P37 associated with CBE treatment. Furthermore, this dose resulted in statistically significant improvements 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 treatment group and the rAAV-GBA1 treatment group (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).

[0278] At the completion of the survival test, mice were sacrificed for biochemical analysis (Figure 16). Cortical GCase activity was assessed by fluorescence analysis performed in biological triple duplication. CBE-treated mice showed decreased GCase activity, while mice administered a high dose of rAAV-GBA1 showed a statistically significant increase in GCase activity compared to CBE treatment. CBE-treated mice also had accumulations of GluCer and GluSph, both of which were relieved by administration of high doses of rAAV-GBA1.

[0279] In addition to the established chemical CBE model, rAAV-GBA1 is also evaluated in a 4L / PS-NA gene model that is homozygous for the V394L GD mutation in Gba1 and also partially lacks a saposin that affects GCase localization and activity. These mice exhibit deficits in motor intensity, coordination, and balance, as indicated 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 by ICV at P23 with a final dose of 2.4 e10 vg (6.0 e10 vg / g brain). Six mice were treated per group, as follows: WT + excipient ICV 4L / PS-NA + Excipient ICV 4L / PS-NA+2.4e10vg(6.0e10vg / g brain)rAAV-GBA1 ICV

[0280] Motor ability was assessed using a beam walking test four weeks after rAAV-GBA1 delivery. The group of mutant mice administered rAAV-GBA1 tended to have fewer slips and less total slip per velocity compared to mutant mice treated with excipients, and recovered motor function close to WT levels (Figure 17). Since the motor phenotype becomes more severe as these mice age, their performance in this and other behavioral tests will be assessed at later time points. Lipid levels, GCase activity, and in vivo distribution will be assessed in these mice at the completion of the survival test.

[0281] Currently, we are additionally testing lower doses of rAAV-GBA1 using CBE models corresponding to the proposed Phase 1 high clinical doses of 0.03x, 0.1x, and 1x. Each group contains 10 mice (5M / 5F). Excipient + CEB Excipient ICV + 25 mg / kg CBE IP 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

[0282] In addition to motor phenotype, lipid levels and GCase activity are evaluated in the cortex. The time course of treatment and analysis is also performed.

[0283] To evaluate efficacy and safety data, a larger dose-range study was initiated. Ten 4L / PS-NA mice (5M / 5F per group) were injected with 10 μl of rAAV-GBA1. Using allometric brain weight calculations, the dose correlated to 0.15x, 1.5x, 4.4x, and 14.5x of the proposed Phase 1 high clinical dose. The injection groups consisted of: WT + excipient ICV 4L / PS-NA + Excipient ICV 4L / PS-NA+4.3e9vg(1.1e10vg / g brain)rAAV-GBA1 ICV 4L / PS-NA+4.3e10vg(1.1e11vg / g / brain)rAAV-GBA1 ICV 4L / PS-NA+1.3e11vg(3.2e11vg / g brain)rAAV-GBA1 ICV 4L / PS-NA+4.3e11vg(1.1e12vg / g brain)rAAV-GBA1 ICV

[0284] Example 9: In vitro analysis of rAAV vectors The rAAV construct was tested in vitro and in vivo. Figure 18 shows representative data for 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).

[0285] A pilot study was 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 include those shown in Table 3. "Opt" refers to the 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.

[0286] A pilot study was 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 nucleic acid sequence codons optimized for expression in mammalian cells (e.g., human cells). Figures 36A–36B show representative data demonstrating that transfection of HEK293 cells with each construct resulted in overexpression of the corresponding gene product compared to mock-transfected cells. [Table 3]

[0287] Example 10: Testing of SNCA and TMEM106B shRNA constructs HEK293 cells The human embryonic kidney 293 cell line (HEK293) was used in this study (#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 penicillin and 100 μg / ml streptomycin (#15140122, Thermo Fisher Scientific).

[0288] 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 culture 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 combined in Opti-MEM solution (#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 image analysis. For image analysis, plates were pre-coated with 0.01% poly-L-lysine solution (P8920, Sigma-Aldrich) before seeding the cells.

[0289] Gene expression analysis using quantitative real-time PCR (qRT-PCR) Following the manufacturer's instructions, 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). Candidate plasmids were transfected using lipofectamine 2000 transfection reagent (0.5 μg plasmid and 1.5 μl reagent in 50 μl Opti-MEM solution) and plated in a 48-well plate (7.5 × 10⁶). 4 HEK293 cells seeded on cells / well were transiently transfected. After 72 hours, RNA was extracted from the cells and used for reverse transcription to synthesize cDNA according to the manufacturer's instructions. For quantitative PCR analysis, 2–5 μl of cDNA product was double-amplified using gene-specific primer pairs (250 nM final concentration) with Power SYBR Green PCR Master Mix (#4367659, Thermo Fisher Scientific). The primer sequences for the SNCA, TMEM106B, and GAPDH genes are as follows: Quantitative PCR was performed on the QuantStudio 3 real-time PCR system (Thermo Fisher Scientific) for 5'-AAG AGG GTG TTC TCT ATG TAG GC -3' (SEQ ID NO: 71), 5'-GCT CCT CCA ACA TTT GTC ACT T-3' (SEQ ID NO: 72) for SNCA, 5'-ACA CAG TAC CTA CCG TTA TAG CA-3' (SEQ ID NO: 73), 5'-TGT TGT CAC AGT AAC TTG CAT CA-3' (SEQ ID NO: 74) for TMEM106B, and 5'-CTG GGC TAC ACT GAG CAC C -3' (SEQ ID NO: 75), and 5'-AAG TGG TCG TTG AGG GCA ATG -3' (SEQ ID NO: 76) for GAPDH. Expression levels were normalized by the housekeeping gene GAPDH and calculated using the comparative CT method.

[0290] 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 reporter plasmid, 0.06 μg knockdown plasmid, and 0.3 μl reagent in 10 μl Opti-MEM solution) on a poly-L-lysine coated 96-well plate (3.0 x 10⁶). 4 HEK293 cells seeded on a cell / well were simultaneously transfected. 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 in 4% PFA at RT for 10 minutes and incubated in D-PBS containing 40 μg / ml 7-aminoactinomycin D (7-AAD) at RT 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.

[0291] Enzyme-linked immunosorbent assay (ELISA) An α-synuclein reporter plasmid containing the TMEM106B gene, located downstream of the 3'-UTR or SNCA coding region of the human SNCA gene, was used to validate 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 reporter plasmid, 0.15 μg knockdown plasmid, and 0.75 μl reagent in 25 μl Opti-MEM solution) into a 48-well plate (7.5 × 10⁶). 4HEK293 cells seeded on a cell / well were transiently transfected. After 72 hours, cells were lysed in radioactive immunoprecipitation (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 at 450 nm using a Varioskan plate reader, and concentrations were calculated using SoftMax Pro 5 software. The measured protein concentrations were normalized to the total protein concentration determined using a bicinchoninate assay (#23225, Thermo Fisher Scientific).

[0292] Figure 37 and Table 4 show representative data demonstrating the success of in vitro silencing of SNCA using the GFP reporter assay (top) and α-Syn assay (bottom). Figure 38 and Table 5 show representative data demonstrating the success of in vitro silencing of TMEM106B using the GFP reporter assay (top) and α-Syn assay (bottom). [Table 4] [Table 5]

[0293] Example 11: ITR "D" sequence placement and cell transduction The effect of ITR "D" sequence placement on rAAV vector cell transduction was investigated. HEK293 cells were transduced with Gcase-coded rAAVs having either 1) wild-type ITRs (e.g., "D" sequence located proximal to the transgene insertion and distal to the ITR terminal) or 2) ITRs with "D" sequences located "outside" the vector (e.g., "D" sequence located proximal to the ITR terminal and distal to the transgene insertion), as shown in Figure 20. Surprisingly, the data showed that rAAVs with "D" sequences located "outside" the vector were efficiently packaged and retained their ability to transduce cells (Figure 40).

[0294] Example 12: In vitro study of progranulin rAAVs Figure 39 is a schematic diagram illustrating one embodiment of a vector containing an expression construct encoding a PGRN. Progranulin is overexpressed in the CNS of GRN-deficient rodents that are either heterozygous or homozygous for GRN deletion by injection of an rAAV vector encoding a PGRN (e.g., a codon-optimized PGRN), for example, by intraparenchymal or subarachnoid injection into the cisterna magna.

[0295] Mice are injected at 2 or 6 months of age and analyzed at 6 or 12 months of age 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), microglia and inflammatory markers, gliosis, neuronal deficiency, lipofuscinosis, and / or accumulation of sososomal markers such as LAMP1. Assays on PGRN-deficient mice are described, for example, in Arrant et al. (2017) Brain 140:1477-1465; Arrant et al. (2018) J. Neuroscience 38(9):2341-2358; and Amado et al. (2018) doi:https: / / doi.org / 10.1101 / 30869; which are incorporated herein by reference.

[0296] Example 13: In vitro and in vivo studies of progranulin rAAV In vitro and in vivo assays were performed to analyze the effects of the rAAV construct encoding the progranulin (PGRN) protein (PR006 (also known as PR006A), see Figure 64). PR006 contains a capsid with the AAV9 serotype. In vitro nonclinical trials Progranulin expression derived from PR006A in HEK293T cells

[0297] The ability of PRPR006A to induce progranulin protein production in cellular conditions was investigated. HEK293T cells were subjected to 2.1 × 10⁶ 5 ~3.3×10 6 Transduction with PR006A was performed across a range of infection multiplicity (MOI) from vector genome (vg) / cell range. PR006A transduction resulted in a robust dose-dependent increase in progranulin protein expression and secretion into cell culture medium (Figure 60). Substantially low progranulin protein levels were detected in the negative control group treated with excipients (intended clinical vehicle) alone, reflecting expression derived from the endogenous human GRN gene.

[0298] Effectiveness of FTD-GRN in iPSC-derived neurons Assays were performed to analyze the efficacy of rAAV constructs in vitro in human FTD-GRN (frontotemporal dementia with GRN mutations) neuronal cultures. Cell lines were obtained from the National Institute of Neurological Disorders and the Stroke (NINDS) Human Cell and Data Repository (NHCDR). Materials used were ND50015 (FTD-GRN, M1L), ND50060 (FTD-GRN, R493X), and ND38555 (control, wild-type) (see Table 6). [Table 6]

[0299] To establish a cell model pathologically related to FTD-GRN, iPSCs from each strain were differentiated into neurons using a two-step protocol. In the first step, iPSCs were differentiated into proliferative neural stem cell (NSC) lines, which lacked the expression of pluripotency markers (i.e., Oct4 and SSEA1) and gained the expression of neural stem cell markers (i.e., SOX2, nestin, SOX1, and PAX6), as detected by immunofluorescence labeling.

[0300] Control and FTD-GRN NSC strains were seeded at equal densities, and after 48 hours, progranulin expression was measured by enzyme-linked immunosorbent assay (ELISA) in cell lysates (intracellular progranulin) (Figure 52E) and cell culture medium (secreted progranulin) (Figure 52A). 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, with FTD-GRN NSCs expressing approximately 25–50% of endogenous progranulin levels. This suggests that this FTD-GRN cell model replicates the clinical progranulin deficiency observed in FTD-GRN patients, who express one-third to one-half of normal progranulin levels in their 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)).

[0301] NSCs from all cell lines were differentiated into neuronal cultures. After establishing that iPSC-derived NSCs exhibit reduced progranulin expression, the lines were differentiated into neurons to generate clinically representative cell types for the preclinical efficacy study of PR006A. NSCs were seeded in a neuronal differentiation medium and ultimately differentiated into postmittal neurons over a 7-day period, and then evaluated for the expression of neuronal markers (i.e., MAP2, NeuN, Tau, Tuj1, NF-H) by immunofluorescence (Figure 52G). This protocol was used to efficiently differentiate both control and FTD-GRN iPSC-derived NSC lines into neurons.

[0302] The efficacy of PR006A in vitro was evaluated using neuronal cultures derived from FTD-GRN iPSCs. FTD-GRN neurons were cultured in a 2.7 x 10⁶ manner. 5 , 5.3 x 10 5 , or 1.1 x 10 6 Cells were treated with excipients or PR006A at the MOI of vg / cells. PR006 transduction resulted in robust, dose-dependent expression of secreted progranulin in all cell lines, as measured by ELISA (Figure 52B). Excipient-treated controls and FTD-GRN neurons were evaluated for endogenous progranulin levels. Control neurons expressed endogenous secreted progranulin, while secreted progranulin was not detected in FTD-GRN neurons (Figure 52B). Linear regression analysis revealed a significant correlation between PR006A dose and progranulin levels across both FTD-GRN cell lines (p = 3.5 × 10⁻⁶). -13 These results indicate that treatment with PR006A leads to increased progranulin secretion in the FTD-GRN neuron model.

[0303] Progranulin is known to stimulate the maturation of the lysosomal protease cathepsin D (CTSD), but its loss of function is also involved in lysosomal storage disorders and neurodegeneration. CTSD is expressed as an inactive full-length proprotein (proCTSD) that undergoes proteolytic processing 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 neuronal cultures, transduction with PR006 rescued cathepsin D maturation defects (Figure 52C). Control, FTD-GRN #1, and FTD-GRN #2 neurons were transduced with PR006A or excipients. 5.3 x 10 5 PR006A was used in efficacy experiments because it restored progranulin levels to at least twice the level of control cells (Figure 52B). To evaluate efficacy, proCTSD and matCTSD expression levels were assessed in cell lysates using an automated Simple Western (Jess) platform (Figure 52C). Excipient-treated FTD-GRN neurons had a lower matCTSD to proCTSD ratio compared to excipient-treated control neurons. PR006A treatment significantly increased the ratio in both FTD-GRN neuron lines (Figure 52C). In control neurons, PR006A treatment did not significantly alter the matCTSD to proCTSD ratio. These findings indicate that PR006A restores lysosomal function-related phenotypes in FTD-GRN neurons.

[0304] In normal neurons, the TDP-43 (transactive response DNA-binding protein 43 kDa) protein is localized in the nucleus. In 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 neuronal aggregation and downstream toxicity. 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 the biology of TDP-43. Compared to control neurons without the GRN mutation, 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, as described by Valdez et al. (Human Molecular Genetics 26, 4861-4872 (2017)). Transduction of neuronal cultures from both FTD-GRN mutant carrier strains into 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)).

[0305] In summary, PR006 transduction restored defective maturation in the lysosomal enzyme cathepsin D and improved abnormal TDP-43 pathology in FTD-GRN neurons.

[0306] In vivo nonclinical trials Efficacy and in vivo distribution in aged Grn knockout mice The efficacy of PR006A in vivo and at maximum dose PR006A was evaluated in a Grn knockout (KO) mouse model. These studies (B6(Cg)-Grn tm1.1AidiIn the Grn KO mouse model used in / J (Jackson Laboratory, Bar Harbor, ME), exons 1–4 are removed from the target progranulin (Grn) gene (Yin et al., J Exp Med 207, 117–128 (2010)). These animals have a complete loss of progranulin and exhibit 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 studies. Mice were assigned to treatment groups balanced by sex and weight, and blinded evaluation of experimental endpoints was performed by qualified personnel.

[0307] In the initial tests, PR006A measured 9.7 × 10 10 vg(2.4×10 11 The drug was delivered to aged Grn KO mice at a dose of vg / g brain. This was the highest dose achievable at the time of the study due to limitations in injection volume and physical titer of the virus lot used in the study. Aged mice were used because many FTD-GRN-related phenotypes, including CNS inflammation and microgliosis, occur in an age-dependent manner, with the most pronounced phenotypic expression occurring between 12 and 24 months of age.

[0308] In studies using aged Grn KO mice, PR006A was administered by a single intraventricular (ICV) injection. 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 x 10⁻¹⁰ 10 vg PR006A(2.4x10 11 vg / g brain (based on adult mouse brain weight of 400 mg) was delivered to aged Grn KO mice in two cohorts by ICV injection: (1) 16 months old at injection (n=4 / group, PRV-2018-027; Figure 61), and (2) 14 months old at injection (planned n=3 / group, PRV-2019-002, Figure 61). Animals were sacrificed two months after injection.

[0309] In study PRV-2018-027, a single dose of PR006A was delivered to 16-month-old mice in the following treatment groups. [Table 7]

[0310] Due to unexpected trial deviations (genotyping errors and early animal loss), the PRV-2019-002 trial (14-month-old cohort) enrolled only one animal in the excipient treatment group instead of the planned n=3. Statistical analysis is impossible due to the small sample size, and this trial is excluded from further consideration. However, the findings from this trial were comparable to those of the PRV-2018-027 trial.

[0311] In vivo distribution and progranulin expression In vivo distribution was determined by measuring the presence of vector genomes using a qPCR assay that meets current U.S. Food and Drug Administration Center for Biologics Evaluation and Research (CBER) / Tissue and Advanced Therapy (OTAT) standards for PCR sensitivity (defined as positive if >50 vector genomes per 1 μg genomic DNA). All mice administered with PR006A were positive for vector genomes in the cerebral cortex and spinal cord, demonstrating that ICV administration successfully resulted in PR006A transduction of 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 administered with excipients (Figure 59B). Since progranulin is primarily a secreted protein, its expression in CSF is considered a substitute for protein production in the brain and represents a potential translational endpoint in FTD-GRN patients with reduced CSF progranulin levels. We were able to detect human progranulin in the CSF of PR006A-treated mice; however, due to the small sample size and technical limitations in obtaining sufficient amounts of CSF in mice, the measurement of CSF progranulin levels was below the lower limit of quantification (LLOQ) of the assay (Figure 59C).

[0312] 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.

[0313] Lipofuscin accumulation: The accumulation of neuronal lipofuscin, an electron-dense autofluorescent substance that progressively accumulates over time in postmittal cell lysosomes, is an indicator of lysosomal dysfunction and a characteristic age-dependent phenotype in Grn KO mice. Lipofuscin accumulation was assessed using two independent methods in adjacent brain sections: (1) a more clinical approach, where lipofuscin accumulation in the brain was scored by blinded pathologists on a scale from 0 (no lipofuscin observed) to 4 (widespread lipofuscin accumulation); and (2) a more quantitative approach, where lipofuscin autofluorescence was detected by immunohistochemistry (IHC) and automatically quantified. Grn KO mice exhibited substantial lipofuscinopathy 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 in all three brain regions with PR006A treatment. Since ubiquitin-positive content is a definitive pathological feature of FTD-GRN patients that accumulates in an age-dependent manner in Grn KO mouse models, IHC was performed and quantified in the brain regions of interest (cerebral cortex, hippocampus, and thalamus) to assess ubiquitin accumulation. PR006A treatment significantly reduced ubiquitin accumulation in Grn KO mice (Figure 59E). These findings suggest that PR006A improves lysosomal dysfunction in Grn KO mouse models of FTD-GRN.

[0314] Neuroinflammation:Chronic CNS inflammation is a pathological feature in the brains of FTD-GRN patients and 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 the pro-inflammatory cytokine Tnf (TNFα) and the microglial marker Cd68 (CD68) in the cerebral cortex (Figure 59F). In addition, TNFα protein levels were reduced in cerebral cortical samples from PR006A-treated Grn KO mice using a mesoscale discovery mouse pro-inflammatory cytokine assay (Figure 59G). To further evaluate neuroinflammation, immunohistochemistry (IHC) was performed for Iba1, a marker of microgliosis, and GFAP, a marker of astrocytosis, and quantified in the brain regions of interest (cerebral cortex, hippocampus, and thalamus). PR006A treatment resulted in a trend toward reduced microgliosis (Iba1) in Grn KO mice, but did not affect astrocytosis (GFAP) (Figure 59H, Figure 59I). In summary, these results indicate that PR006A treatment reduces neuroinflammation in an aged Grn KO mouse model of FTD-GRN.

[0315] Histopathology: Thorough histopathological analysis by blinded, committee-certified pathologists of hematoxylin and eosin (H&E) stained brains, thoracic spinal cords, livers, hearts, spleens, lungs, and kidneys of all mice in these studies showed 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 findings characteristic of the model, including a reduction in the frequency and / or severity scores of nerve necrosis in the medulla and pons. Furthermore, PR006A treatment resulted in a reduction in both the incidence and severity of axonal degeneration in the thoracic spinal cord. These findings are described in detail in the toxicity section below.

[0316] conclusion : Administration of 9.7×10 10 vg (2.4×10 11 vg / g brain) via ICV resulted in extensive vector genome presence throughout the brain and peripheral tissues of aged Grn KO mice. PR006A treatment increased widespread progranulin expression. Furthermore, PR006A reduced the accumulation of lipofuscin and ubiquitin in the brain, a pathology known to occur in both the Grn KO mouse model and patients with FTD-GRN. PR006A also reduced the expression of pro-inflammatory cytokines and immune cell activation in the cerebral cortex, a phenotype indicative of chronic CNS inflammation.

[0317] 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 vehicle (intended clinical vehicle; 20 mM Tris pH 8.0, 200 mM NaCl, and 1 mM MgCl2 + 0.001% Pluronic F68) or PR006A was delivered via ICV to 4-month-old animals. These adult mice were used in place of aged Grn KO mice because a sufficient number of the latter were not available to conduct the dose-range study. Although adult Grn KO mice have a milder phenotype than aged mice, they still exhibit lysosomal defects and neuroinflammatory changes, and are therefore suitable for evaluating the effective dose range of PR006A. To evaluate the efficacy of PR006A across a wide range of viral doses, PR006A was administered at 1.1×10 11 vg (2.7×10 11 vg / g brain), the highest dose achievable given the injection volume constraints at the time of the study and the physical titer of the virus lot used in the study, the intermediate dose of 1.1×10 10 vg (2.7×10 10 vg / g brain), or the low dose of 1.1×10 9 vg (2.7×10 9 vg / brain). Details of the experimental design are shown in Figure 63.

[0318] Three doses of PR006A were evaluated in groups of 10 mice (4M / 6F). [Table 8]

[0319] In this study, age-matched mice from the same background strain as Grn KO mice (7-month-old C57BL / 6J) possessing the wild-type (WT) Grn allele were used as controls for the selective efficacy endpoint. [Table 9]

[0320] In vivo distribution and progranulin expression:The 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 CBER / OTAT criteria for PCR sensitivity (defined as positive if >50 vector genomes per 1 μg genomic DNA). Mice administered with PR006A showed dose-dependent positivity of vector genomes in the cerebral cortex and spinal cord, indicating that ICV administration successfully induces PR006A transduction in the CNS (Figure 53A). qRT-PCR analysis of GRN encoded by 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). In brain tissue, human progranulin levels were detected and quantified at the highest PR006A doses. At lower doses, progranulin levels were below the assay detection limit due to the high brain background. However, based on the logarithmic difference between doses, proportional estimates of expected progranulin levels at lower doses would fall well below the lower limit of quantification (LLOQ) of the assay in brain tissue. Endogenous mouse progranulin levels were measured in age- and strain-matched mice with wild-type (WT) Grn alleles. In both 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 given dose. Since human and mouse progranulins were measured using different detection assays with non-species cross-reactive anti-progranulin antibodies, absolute numbers cannot be compared to precision.

[0321] 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 (Figure 53D; Figure 53E).

[0322] In plasma, significant levels of human progranulin were detected at all dose levels in PRPR006A-treated Grn KO mice (Figure 53F). As expected, human progranulin was not detected in Grn KO mice treated with excipients. Levels of human progranulin in animals treated with intermediate doses of PRPR006A were within the same range as 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-species cross-reactive anti-progranulin antibodies, absolute numbers cannot be compared to precision.

[0323] Lipofuscin accumulation:Lipofuscin accumulation was assessed in adjacent brain sections using two independent methods. (1) In a more clinical approach, lipofuscin accumulation in the brain was scored by a blinded pathologist on a scale from 0 (no lipofuscin observed) to 4 (widespread lipofuscin accumulation), and (2) in a more quantitative approach, lipofuscin autofluorescence was detected by IHC and automatically quantified. Grn KO mice exhibited lipofuscinosis throughout the brain, while WT mice had no 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 lipofuscin scoring by pathologists, intravascular coagulation (IHC) performed to quantitatively assess lipofuscinosis in target brain regions (i.e., cerebral cortex, hippocampus, and thalamus) detected dose-dependent decreases in lipofuscin accumulation in cortical and thalamic brain regions, with significant decreases occurring at intermediate and high PR006A doses. IHC was also performed to assess ubiquitin accumulation in the brain, an additional 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 ubiquitin immune-responsive object size to near WT levels in all three doses (Figure 53H).

[0324] Neuroinflammation: Treatment with PR006A suppressed inflammatory marker levels in the brains of adult Grn KO mice. ICV PR006A was 2.7 x 10⁻¹⁰ 9 VG / G brain ~2.7x10 11Across doses ranging from vg / g brain, gene expression of the pro-inflammatory cytokines Tnf (TNFα) and Cd68 (CD68), markers of microglia in the cortex, was reduced (Figure 53I). In line with published data, we observed increased gene expression of these neuroinflammatory markers 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, no robust increase in cerebral cortical TNFα protein levels was observed in 7-month-old adult excipient-treated Grn KO mice, and furthermore, 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 develop in the Grn KO mouse model until 12–24 months of age. Immunohistochemistry (IHC) was performed and quantified in target brain regions (cerebral cortex, hippocampus, and thalamus) to further evaluate neuronal inflammation by staining for Iba1, a marker of microgliosis, and GFAP, a marker of astrocytosis. In Grn KO mice, there was a significant increase in microgliosis (Iba1) and astrocytosis (GFAP) throughout the brain compared to WT mice (Figures 53J-53K). PR006A treatment significantly reduced microgliosis (Iba1) in all three doses (Figure 53J). A trend toward reduction in astrocytosis (GFAP) was observed at intermediate PR006A doses, and a significant reduction in astrocytosis (GFAP) was observed at high PR006A doses in the thalamic brain region (Figure 53K).

[0325] While many Grn KO mouse model phenotypes are expressed later in life, studies have reported that Grn KO mice exhibit extensive gene expression changes as early as 4 months of age, including alterations in lysosomal and immune-related pathways. Therefore, in addition to the targeted qRT-PCR analysis described above, a transcriptomics approach was adopted to evaluate mRNA-level changes, which allows for comprehensive assessment using highly sensitive, high-throughput RNA sequencing and requires minimal sample material. RNA sequencing of the cerebral cortex was performed, and gene set variation analysis (GSVA) ​​(Hanzelmann et al., BMC Bioinformatics 14,7 (2013)) was used 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. As reported in previously published studies, we confirmed the deficiencies in lysosomal and immune-related pathways in Grn-deficient mice. 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 "complementary" gene set by gene set enrichment analysis using the Hallmark database (including genes encoding components of the complementary system, which is part of the innate immune system). Next, the activity levels of these gene sets were measured and compared with PR006A treatment (Figures 53L to 53N). Treatment with PR006A reversed the gene set deficiencies observed in Grn KO mice in a dose-dependent manner.

[0326] Histopathology:A thorough histopathological analysis conducted by a blinded board-certified pathologist, examining hematoxylin and eosin (H&E) staining of the brain, thoracic spinal cord, liver, heart, spleen, lung, kidney, and gonads from all mice in these studies, found no evidence of toxicity associated with PR006A treatment. Details of the toxicity analysis are provided in the following section.

[0327] conclusion :ICV PR006A, 2.7x10 9 vg / g brain to 2.7x10 11 vg / g brain, doses ranging from that resulted in dose-dependent widespread vector genome presence throughout the brain and peripheral tissues. PR006A treatment also led to the production of progranulin mRNA and protein in the CNS. A clear dose-response relationship between PR006A and reduced lipofuscinosis, a readout of lysosomal dysfunction, was observed across multiple brain regions. Robust and statistically significant reduction in lipofuscinosis was observed at the intermediate and highest dose levels of PR006A. All PR006A doses reduced ubiquitin accumulation in the brain. Starting from the lowest dose of 2.7x10 9 vg / g brain, PR006A reduced the expression of pro-inflammatory markers in the brain at the RNA and protein levels.

[0328] Summary of the invention: In vivo non-clinical trials PR006A effectively transduced Grn KO mice, resulting in robust, dose-dependent biodistribution of the transgene and production of progranulin mRNA and protein in the CNS. PR006A dose-dependently reversed abnormal gene expression in lysosomal and neuroinflammatory pathways. PR006A reduced many of the phenotypes that occur in the brain of this FTD-GRN mouse model, including lipofuscinosis, ubiquitin accumulation, and microgliosis. In the dose-ranging study, 2.7x10 9 vg / g brain, the lowest dose of PR006A significantly suppressed the expression of inflammatory markers in the cerebral cortex. 2.7×10 10Intermediate doses of vg / g brain PR006A robustly and statistically significantly improved both lysosomal deficiency (e.g., lipofuscinosis) and neuroinflammation. 2.7 × 10⁻⁶ 11 High doses of vg / g brain PR006A further increased progranulin expression without evidence of toxicity. [Table 10]

[0329] Safety pharmacology Throughout these tests, no adverse events attributable to the test substance were observed. Safety findings from animal survival and histopathological analysis in PRV-2018-027, PRV-2019-002, and PRV-2019-004 This will be explained in the following section.

[0330] Single-dose toxicity A series of non-clinical studies using PR006A were conducted, with safety endpoints investigated in mice and monkeys. Three studies were performed in a Grn KO mouse model, with endpoints including neuropathological endpoints, evaluating both protective activity and potential toxicity resulting from intraventricular (ICV) injection of PR006A. ICM administration is more technically challenging in mice. These mouse models are representative of FTD-GRN, where patients have mutations in the GRN gene, resulting in reduced progranulin levels. In cynomolgus monkeys, neuropathology was also performed as part of a pilot study in which PR006A was injected into the cisterna magna (ICM). GLP studies were conducted in cynomolgus monkeys to which PR006A was delivered via ICM, with monkeys sacrificed on days 7, 30, or 183. The GLP studies incorporated a comprehensive list of clinical endpoints in addition to anatomical and pathological endpoints on a complete list of tissues. The following single-dose studies were conducted to support single-dose administration in clinics.

[0331] Maximum dose PR006A (PRV-2018-027 and PRV-2019-002) in an aged FTD-GRN mouse model. As part of these efficacy studies in Grn KO mice, neuropathological evaluations were performed in mice treated with ICV with either the excipient or PR006A. Grn KO mice have complete progranulin loss and are widely used as a model for FTD-GRN due to their age-dependent phenotype, including lysosomal alterations, neuronal lipofuscin accumulation, microgliosis, and neuroinflammation. The aspects of the pharmacological portion of the study are summarized in the section above, while 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 were ICV-administered with either PR006A or the excipient. The animals were sacrificed 9 weeks after administration. A single PR006A dose group was included in this study: 10 μl of undiluted virus, total dose 9.7 × 10⁶ 10 vg(2.4×10 11 (vg / g brain), the control group was treated with 10 μl of excipient. [Table 11]

[0332] Various postmortem evaluation parameters, including in vivo distribution, lysosomal changes, and inflammatory markers, were assessed as part of the study protocol (see section above). Animals were checked twice daily for survival, and body weight was measured once daily. After euthanasia two months post-treatment, target tissue was collected, fixed dropwise in cooled 4% paraformaldehyde, and stored at 4°C. Tissue from eight animals that completed the study was trimmed, processed, and embedded in paraffin blocks. It was then sectioned into approximately 5 μm sections, stained with hematoxylin and eosin (H&E), and examined by a committee-certified veterinary pathologist.

[0333] During this study, one mouse in the treatment group died prematurely, but no abnormalities were recorded in the animal that died during necropsy, so there was no known cause of death. No other deaths or abnormalities were observed. All treatment groups were followed similarly in terms of body weight, and no significant differences were observed.

[0334] Histopathological examination revealed no adverse findings related to PR006A. Extensive lipofuscin accumulation was present in the brain, consistent with findings expected in Grn KO mice. PR006A-treated animals showed a reduction in the severity score of lipofuscin accumulation in all regions of the brain. Morphological changes also appeared to show a slight reduction in frequency and / or severity scores with respect to nerve necrosis, particularly in the medulla and pons, with PR006A treatment. However, these trends in morphological changes were not as consistent with lipofuscin scores.

[0335] In the thoracic spinal cord, axonal degeneration was observed, 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.

[0336] The following findings, likely associated with Grn homozygous knockout mice, appeared to be reduced in frequency and / or severity in animals treated with PR006A: dilated tubules in the renal medulla, glomerulosis in the kidneys, and foreign bodies in the lungs (characterized as linear, acellular, dark pink structures, usually within the airways, frequently associated with invasive giant cells and / or macrophages). A larger animal cohort is needed to draw more definitive conclusions.

[0337] All other histopathological findings observed were considered incidental and / or coincidentally similar, and the severity in excipient and test substance-treated animals was therefore considered unrelated to the administration of PR006A.

[0338] In the second trial (PRV-2019-002), five 14-month-old mixed-sex Grn KO mice were administered either PR006A or an excipient via ICV. The animals were sacrificed 8 weeks after administration. A single PR006A dose group was included in this study: 10 μl of undiluted virus, total dose 9.7 × 10⁶.10 vg(2.4×10 11 (vg / g brain), the control group was treated with 10 μl of excipient. [Table 12]

[0339] The animals were analyzed using the same method as in the PRV-2018-027 study. Animals were checked for viability twice daily, and body weight was measured once daily. After euthanasia two months post-treatment, target tissue was collected, fixed dropwise in cooled 4% paraformaldehyde, and stored at 4°C until evaluation.

[0340] 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, widespread increases in lipofuscin accumulation were observed throughout the brain. Rarely, minimal neuronal necrosis was also observed (in one untreated animal that died prematurely and in one animal treated with excipients).

[0341] Due to the small sample size, it was not possible to demonstrate consistent trends in treatment-related findings. There were no consistent differences in response between the test substance (PR006A) and the excipients.

[0342] 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 (vacuolation of Kupffer cells / sinoid lining cells and Kupffer cell microgranulomas) (Yin et al., J Exp Med 207(1):117-128(2010)).

[0343] All animals that underwent surgery and were enrolled in the study showed signs of "glomerulopathy." No published reports were found regarding this finding as a change associated with standard, unchallenged Grn knockout mice; however, one study demonstrated progranulin-deficient mice treated with a diet that induced hyperhomocysteinemia, glomerular basement membrane thickening, and podocyte foot atrophy (Fu et al., Hypertulation 69(2):259-266(2017)).

[0344] All other findings were consistent with those commonly observed in experimental mice. Due to the small sample size, no definitive differences related to treatment were observed.

[0345] 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 via a single unidirectional ICV injection into the left hemisphere of the brain. Regardless of the treatment group, all animals received a total dose volume of 10 μl. Mice were treated at 4 months of age and euthanized 3 months after treatment. An additional wild-type (WT) control group, including untreated C57BL / 6J mice (same background strain) that had grown to approximately 7 months of age, was also euthanized and subjected to similar necropsy.

[0346] The test was conducted according to the following test design. [Table 13]

[0347] During the study, animals were checked for survival twice daily and weighed once a week. Three months after treatment, mice were euthanized, and various postmortem evaluations were performed to assess the efficacy of PR006A (see section above). In addition, H&E-stained sections from the brain, thoracic spinal cord, liver, heart, spleen, lungs, kidneys, and gonads were evaluated by a committee-certified pathologist.

[0348] Histopathological examination revealed no harmful PR006A-related findings in any of the mice, regardless of the treatment group.

[0349] Findings consistent with the Grn KO mouse model phenotype were observed in various brain regions: intracellular lipofuscin accumulation in the cerebral cortex, cerebral nuclei, hippocampus, thalamus / hypothalamus, cerebellum, and brainstem (particularly the pons and medulla). No clear evidence of morphological changes was observed in H&E stained sections (neuronal and gliosis vacuolation). Lipofuscin dye accumulation can precede easily detectable morphological changes and therefore serves as a suitable biomarker of efficacy. All Grn homozygous KO groups showed lipofuscin accumulation, but the severity of this finding differed among treatment groups. The frequency of high lipofuscin accumulation scores was highest in the group of animals treated with excipients (Group 1). Among animals treated with PR006A, the frequency of higher scores was highest in Group 4 (low-dose PR006A; 2.7 × 10⁶). 9 Observed in vg / g brain, followed by the third group (medium dose PR006A; 2.7 × 10⁻¹⁰). 10 The lowest severity score was observed in group 2 (high-dose PR006A; 2.7 × 10⁻⁶). 11 These findings were observed in vg / g brains. 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 incidentally similar, and the severity in excipient and test substance-treated animals was therefore considered unrelated to the administration of PR006A.

[0350] 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 substance PR006A after a single dose via ICM injection in cynomolgus monkeys, with post-administration observation periods of 6, 29, or 182 days. Animals were sacrificed on days 7, 30, and 183. This study was designed to evaluate two dose levels. The maximum dose was the achievable maximum viable dose, with a volume of 1.2 mL of undiluted PR006A (the largest volume experienced in administration), while the lower dose was the equivalent of a dose one log unit lower than the maximum dose. The dose was 4.8 × 10⁶ in cynomolgus monkeys, a non-human primate used in this study with an estimated brain weight of 74 g. 11 Low dose of vg and 4.8 × 10 12 This was equivalent to a high dose of vg. This is approximately 6.5 × 10 9 vg / g brain and 6.5 × 10 10 This corresponds to vg / g brain. The 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 table summary of this study design is shown in Table 11. [Table 14-1] [Table 14-2] [Table 14-3]

[0351] Cynomolgus monkeys with NHP were assessed by multiple survival observations and measurements. These included death / illness (daily), clinical observation (daily), body weight (baseline and then weekly), visual inspection of food consumption (daily), neurological observation (baseline and weeks 2 and 26), indirect fundus examination (baseline and weeks 2 and 26), and electrocardiogram (ECG) measurements (baseline and weeks 2 and 26).

[0352] Analysis of neutralizing antibodies (nAbs) against the AAV9 capsid was performed at baseline and at sacrifice on days 7, 30, or 183. Clinicopathology, consisting of hematology, coagulation, clinical chemistry, and urinalysis, was performed twice at baseline (once with blood and urine tests) and once at weeks 1 and 13 of the administration period.

[0353] The animals were euthanized, and tissue was collected on days 7, 30, or 183. Where present, the tissues outlined in Table 11 were collected from all animals, weighed (where applicable), and divided into multiple copies. One copy was preserved in 10% neutral buffered formalin (unless a special fixative was required for optimal fixation) for histopathological evaluation (all animals). Additional copies were collected for qPCR and transgene expression analysis. Safety and Toxicology There were no unplanned deaths, and all animals survived until scheduled autopsies. There were no adverse PR006A-related clinical findings, weight changes, ocular observations, or physical or neurological findings, and general macroscopic examination at autopsy showed no drug-related abnormalities in any cohort. Furthermore, 6.5 × 10 9 or 6.5 × 10 10 In males or combined sexes administered vg / g brain, no PRPR006A-related changes in PR interval, QRS duration, QT interval, corrected QT (QTc) interval, or changes in heart rate were observed. No abnormal ECG waveforms or arrhythmias were observed during qualitative ECG evaluation.

[0354] In vivo distribution The in vivo distribution of the PR006A transgene was analyzed using a qPCR-based assay. High-dose group (6.5 × 10⁶) 10 On day 183, extensive transduction was observed throughout the CNS and surrounding areas in the vg / g brain, and vector presence was positive in all tissues at the 50 vg / μg DNA cutoff, which is the lower limit of quantification in the qPCR assay. Data from selected representative regions from day 183 are shown in Figure 54A, but data from day 30 are not shown. High-dose group (6.5 × 10⁻⁶) 10 On day 30 of the vg / g brain trial, all CNS tissues examined were transduction-positive, except for the putamen. 9 Tissues from animals treated with vg / g brain were positive in the CNS at day 183, but only the spleen and liver were positive from peripheral tissues. Furthermore, one female NHP treated with high-dose PR006A was positive in the ovaries at day 7, and a male treated with high doses was positive in the testes at days 30 and 183. PR006A gene transfer was most robust in liver and nervous system tissues and consistently low in other peripheral organs examined. In the brain, vector gene transfer was stable at day 183 compared to day 30, demonstrating robust and sustained gene transfer of the transgene.

[0355] NHP patients receiving PR006A ICM showed a significant alloimmune response to the transgene product progranulin, along with anti-progranulin antibodies detected in serum and CSF samples collected at 30 and 183 days post-treatment. This immune response indicates that the human progranulin protein was expressed in NHP patients. Anti-drug antibody (ADA) levels were determined using established immunoassay techniques. The data are shown in Figure 54B.

[0356] 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 the level of PR006A transduction, transgene expression was observed by mRNA measurement using RT-qPCR in selected brain regions (Figure 54C), liver, gonads, spinal cord, and DRG collected at day 183.

[0357] Transgene expression was measurable in the brain and liver at both doses of PR006A, and expression levels were both dose-dependent and persistent. In the gonads, expression was measurable only in high-dose males. In females at both doses, expression was measurable at days 7 and 30, but not at day 183.

[0358] To confirm that human progranulin was produced in treated NHP, protein levels in CSF were assessed using the J Simple Western® Jess platform. Detailed methodology is provided in Example 14. This method was qualified by measuring progranulin levels in CSF samples from FTD-GRN patients and establishing that they were approximately half the levels in CSF samples from healthy human controls and 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 indicate that the effective and widespread transduction of PR006A in NHP after ICM administration leads to increased progranulin levels.

[0359] 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 do not reliably measure the level of transgene-derived human progranulin 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 trials.

[0360] Summary of the Invention In any non-clinical studies, including small non-GLP trials in NHP and GLP trials in NHP up to day 183, no adverse safety findings or toxicity concerns precluding the initiation of clinical trials. Pathological findings in the GLP trials were consistently minimal in severity, with low numbers of affected cells across both dose groups. No other in-vivo or post-mortem PR006A-related adverse findings were reported.

[0361] Phase 1 / 2 trial in human subjects with FTD-GRN Human subjects (n=15) will be enrolled in an open-label trial of the PR006 recombinant AAV. Subject inclusion criteria include being 30–80 years old (inclusive), having a pathogenic GRN mutation, being in a symptomatic disease stage, and having stable use of background medication prior to administration of the investigational drug. Each subject will receive the investigational drug as a single ICM (intracisor macromolar) 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 (neuronal filament light chain), and volumetric MRI (magnetic resonance imaging); and (3) efficacy: CDR+NACC FTLD (Clinical Dementia Assessment + National Alzheimer's Disease Coordinating Center for Frontotemporal Dementia), behavioral scales, cognitive scales, language scales, functional scales, and QoL (quality of life). [Table 15] [Table 16] [Table 17] [Table 18]

[0362] 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 ProteinSimple's (San Jose, California) automated Western blot platform, Jess. This assay method can be used to analyze non-human primate (NHP) CSF samples. To determine the expression level of human progranulin protein, the PR006A transgene product, a CSF sample from a non-human primate subject, was analyzed on the Simple Western® (Jess) platform using an antibody that specifically detects human progranulin protein. The Simple Western® platform is a capillary-based automated Western blot immunoassay platform in which all steps, including protein separation, immunoprobing, washing, and chemiluminescent detection, occur within a capillary cartridge. In addition to secondary antibodies and all buffers manufactured by ProteinSimple, a sample (4-fold dilution) and primary antibody against human progranulin (Adipogen PG-359-7, 10-fold dilution) were loaded into a customized cartridge run on the Jess platform. Semi-quantitative data analysis was performed automatically after each run was completed, where parameters such as signal intensity, peak area, and signal-to-noise ratio were calculated using a Jess instrument. For each individual sample, the level of progranulin was measured as the peak area of ​​the immune response to the antibody. All analyses were performed using blinded samples.

[0363] The assays described herein were performed on CSF samples from non-human primate animal studies. CSF samples were tested for the presence and levels of progranulin protein to assess the efficacy of gene therapy using an rAAV construct encoding progranulin (PGRN) protein (PR006, see Figure 64). In this study, either an excipient or PR006 was added to a low dose of PR006 (1.8 × 10⁶). 10 (vg / g brain weight) or high dose PR006 (1.8 × 10⁻¹⁰) 11 The drug was delivered to NHP animals by intracisional cisterna magna (ICM) injection at a dose of 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 19] [Table 20]

[0364] In implementing this method, the following steps were followed. Preparation of stock solution 1. To clear the tubes in the EZ Standard Pack separation module, add 40 μL of water to prepare a 400 mM DTT solution. Mix gently. 2. To prepare the master mix, add 20 microliters of 10X sample buffer and 20 microliters of 400 mM DTT to the EZ pink master mix tube. Mix gently. 3. To prepare the biotinylated ladder, pipette 20 μL of water into an EZ Clear biotinylated ladder tube as a pink pellet. Mix gently. 4. Prepare a mixture of luminol and peroxide by adding equal amounts of each. For each run, add 200 μL of peroxide to 200 μL of luminol. 5. Prepare a 10-fold dilution of the primary antibody by mixing 25 μL of the primary antibody with 225 μL of antibody diluent 2. Sample preparation : 1. The sample is diluted in 0.1x sample buffer. Prepare the 0.1x sample buffer by adding 10 μL of 10x 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 mix. 5 μL of NHP CSF was added to 15 μL of 0.1x sample buffer. Prepare the sample by adding the 3.1X master mix to the 4X sample. To perform technical duplication, prepare a total of 15 μL of sample and master mix per sample. For example, add 3 μL of master mix to 12 μL of diluted sample. Mix gently. 4. Boil the sample at 95°C for 5 minutes. 5. Use a desktop mini centrifuge to quickly spin down the sample. Vortex before loading the sample. Load the reagents and samples into the cartridge. : 1. Dispense all samples using a pipette according to the cartridge map. a. Dispense 15 μL of the luminol + peroxide mixture into each well of lane E using a pipette. b. Dispense 10 μL of streptavidin into the first well of lane D. c. Pipette 10 μL of secondary antibody into the remaining 24 wells of lane D. d. Dispense 10 μL of antibody dilution into the first well in lane C. e. Pipette 10 μL of the secondary antibody dilution into the remaining 24 wells of lane C. Dispense 10 μL of antibody dilution into all wells of lane B. g. Pipette 10 μL of the prepared EZ ladder into the first well of lane A. Dispense 5 μL of sample and master mix solution into the replication lane for lane A. 2. Spin the cartridge at room temperature at 2500 RPM for 5 minutes. Load the capillary and cartridge into the device. : 1. Load the capillary into the slot. Confirm that the light turns blue. 2. Insert the spun cartridge into the device. 3. After the blue light on the device stops flashing, press the start button.

[0365] The suitability of the assay system was considered acceptable if the coefficient of variance (CV) ratio for overlap was 30% or less.

[0366] Before using the assay to detect progranulin in NHP CSF samples, the assay was tested as follows: Eligibility for the Jess assay included evaluation of dilution linearity, selectivity, and specificity. Dilution linearity of the Jess assay was determined using normal CSF samples from BioIVT. Selectivity and specificity of the Jess assay were determined using CSF samples from patients with frontotemporal dementia (FTD) who have PGRN mutations (obtained from the National Centralized Repository for Alzheimer's Disease and Related Dementias (NCRAD; Indianapolis, Indiana)). [Table 21]

[0367] Results and Discussion Dilution linearity The dilution linearity of PGRN proteins detected by Jess was tested using CSF samples from commercially available (BioIVT) general organisms. The endogenous level of PGRN in the CSF samples was measured to determine the dilution linearity. Two organisms were tested at 2x serial dilutions ranging from 2 to 64x.

[0368] Table 19 reports the peak area of ​​58 kDa PGRN proteins 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 a difference of 100 ± 30%). Dilution linearity was established to be within 4 to 16-fold dilutions. [Table 22]

[0369] In summary, all matrices tested had an acceptable linear range that passed the 0 ± 30% percent difference tolerance, although the size of this range and the dilution amount varied among matrices. Sample linearity (MRD) was established for a 4-fold dilution. Dilution linearity was established for dilutions between 4 and 16 times. Table 20 summarizes the MRDs and linear dilution ranges that passed the CSF tolerance criteria. [Table 23]

[0370] Selectivity and specificity The selectivity and specificity of PGRN proteins detected by Jess were tested in CSF samples 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 for each group. The sample groups are listed in Table 16 FTD patient CSF sample information.

[0371] CSF samples were diluted fourfold in 0.1X sample buffer provided by ProteinSimple and tested with technical duplication. Duplications of samples with results greater than 20% %CV were reanalyzed. Results with less than 20% %CV are reported in Table 22. Table 22 reports the peak area of ​​the 58kDa PGRN protein detected by %CV between Jess and duplication. The results showed approximately twice as high PGRN levels in groups B and C compared to group A, demonstrating the selectivity and specificity of the Jess assay in determining PGRN levels in CSF samples (Figure 55). [Table 24-1] [Table 24-2] [Table 25]

[0372] CSF samples from the FTD patient study (Table 21) were also analyzed using the human PGRN ELISA kit (Adipogen, AG-45A-0018YEK-KI01). The ELISA results (Figure 56) showed a similar trend in PGRN levels across the groups, indicating that the Jess assay is appropriate for evaluating PGRN levels in CSF samples.

[0373] In conclusion, the ProteinSimple automated Western Jess assay was determined to be suitable for evaluating PGRN levels in NHP CSF samples.

[0374] Table 23 shows the Jess data for NHP CSF samples. Each sample represents the average across two technical replicas. The peak area of ​​the 58kD band within the sample lane is reported. Data are presented as the average peak area for technical replicas and adjusted dilution ratios. [Table 26]

[0375] The objective of this assay was to confirm the progranulin (PGRN) protein expression level after transduction with PR006 in the target tissue region of the NHP study. This was performed using an automated Western blotting platform in which progranulin protein was detected using a monoclonal antibody. Progranulin expression was measurable in CSF in both control and PR006-treated NHP. The assay does not distinguish between endogenous progranulin protein and PR006A-induced progranulin protein.

[0376] Example 15: A Phase 1 / 2 study evaluating the safety and efficacy of PR006A and an immunosuppressive protocol with respect to progranulin levels in patients with FTD-GRN. PR006A is an investigational gene therapy that uses an AAV9 viral vector to deliver the DNA encoding wild-type GRN, i.e., the gene encoding PGRN, to the patient's cells (see Figure 64). Fifteen patients will receive a single dose of PR006A by suboccipital injection into the cisterna magna by a physician. Three dose-escalation cohorts are planned (3.5 × 10⁻⁶). 13 vg, approx. 7.0×10 13 VG or approximately 1.4 × 10 14 (vg PR006A). A single dose of rAAV(PR006A) is administered to the patient on day 0 of each regimen.

[0377] Each enrolled patient must have symptomatic FTD according to a healthcare professional's assessment (bvFTD, PPA-FTD, FTD with corticobasal syndrome, or a combination of syndromes are eligible for enrollment). Each enrolled patient must score ≥1 and ≤15 on the CDR+NACC FTLD SB (Clinical Dementia Assessment Staging Instrument + National Alzheimer's Coordinating Center Frontotemporal Degeneration Domain). Each enrolled patient must be a carrier of a pathogenic GRN mutation. Pathogenic mutations include all null mutations, including nonsense, frameshift, splice site mutations, and complete or partial (exon) gene deletions. • All previously published pathogenic mutations that have been proven to have adverse functional effects (selected missense mutations may be included if they are known to be pathogenic). • All pathogenic mutations listed in the Molgen FTD database • All new mutations with low plasma PGRN levels (<70 ng / mL) based on measurements at a central laboratory.

[0378] Administration of immunosuppressants

[0379] Administration of corticosteroids: Patients receive a loading dose of methylprednisolone (MPS) 1000 mg IV pulse on day -1 (acceptable on day -1 or day 0, depending on the laboratory setup). See the following section for the possibility of administering methylprednisolone 100 mg IV between day -14 and day -2 prior to rituximab (RTX) administration. Prednisone at a dose of 30 mg / day is administered orally as a concomitant medication for 14 days starting the day after the 1000 mg methylprednisolone IV pulse (day 0 or day 1), and then tapered over the following 7 days. Higher doses or longer tapering periods of corticosteroids may be used at the discretion of the healthcare professional.

[0380] Rituximab administration:Patients receive a single 1000 mg IV rituximab dose on any day between day -14 and day -1. To reduce the risk and severity of rituximab-related infusion-associated reactions (IRRs), patients receive an IV methylprednisolone dose before receiving the IV rituximab infusion. For the rituximab dose on day -1, patients receive the rituximab infusion at least 30 minutes after the 1000 mg IV methylprednisolone pulse mentioned above. For the rituximab dose on days -14 to -2, patients receive a 100 mg IV methylprednisolone infusion approximately 30 minutes before the IV rituximab infusion.

[0381] Depending on local practice and / or the discretion of the healthcare provider, acetaminophen and / or diphenhydramine may be provided in addition to prevent IRR.

[0382] Administration of sirolimus: The patient receives an oral loading dose of 6 mg of sirolimus on day -1 (within a window of day -3 to day -1). A subsequent oral maintenance dose of 2 mg / day of sirolimus is initiated on day 0 (or the day after the loading dose, if the loading dose is administered on day -3 or day -2) and is provided as a concomitant medication, adjusted as needed to maintain a serum trough level of 6 ng / mL (range 4–9 ng / mL) for 90 days. Sirolimus is then tapered over the following 15–30 days. Sirolimus trough levels are collected before administration of sirolimus for each visit. Higher doses or longer tapering periods of sirolimus may be used at the discretion of the healthcare provider.

[0383] Immunosuppression monitoring criteria: In addition to monitoring sirolimus struff levels, each patient's clinical status, laboratory findings, and potential adverse events are evaluated.

[0384] Furthermore, the need to increase the dose of immunosuppressants, extend tapering regimens, add additional medications, or restart treatment based on clinical signs or symptoms consistent with an immune response, including the following, should be considered: Asymptomatic pleocytosis with a white blood cell count (WBC) >30 mm³ and / or elevated cerebrospinal fluid (CSF) protein (>70 mg / dL). • CSF pleocytosis and / or protein elevation accompanied by clinical symptoms (including undercompensation of underlying FTD symptoms) • Appearance of sensory symptoms based on neurological examination and / or the Treatment-Induced Neuropathy Assessment Scale (TNAS) • Symptoms of hepatitis (e.g., macular degeneration, fatigue) are accompanied by elevated alanine aminotransferase (ALT) and / or aspartate aminotransferase (AST) levels > 5 × upper limit of normal (ULN). • Regardless of the presence or absence of clinical symptoms, elevated ALT and / or AST > 10 × ULN

[0385] Healthcare professionals should consider administering a longer, additional 4-week prednisone tapering in patients presenting with ALT and / or AST > 3 × ULN at the end of the initial 14-day tapering period. In cases of refractory AST / ALT elevation to prednisone treatment, healthcare professionals should seek expert advice from a hepatologist. In cases of CSF inflammatory changes requiring rescue immunosuppression, an unscheduled lumbar puncture should be performed 1–2 months after restarting / increasing the dose of immunosuppressants / introducing additional immunosuppressants.

[0386] Procedures before intracisional puncture

[0387] The patient undergoes a standard therapeutic medical evaluation for cisternotomy, including a consultation with an anesthesiologist. The physician and anesthesiologist review screening clinical laboratory analysis (including recorded negative pregnancy tests), MRI and MRA of the brain and cervical spine (if requested by the physician), and results of a local ECG. Medical history, currently prescribed medications, and over-the-counter medications are reviewed for any recent changes. Additional clinical evaluations may be performed at the anesthesiologist's discretion (specific to any associated medical conditions).

[0388] Intracisternal injection

[0389] On day 0, PR006A is administered as a single dose by a physician via suboccipital injection into the cisterna magna. Prior to the injection, a volume of fluid equivalent to the volume of PR006A administered is removed from the cisterna magna. The procedure is performed under general anesthesia or deep sedation, using imaging guidance. The patient remains under observation for 24 hours (overnight hospitalization) after PR006A administration.

[0390] Test design

[0391] This is a five-year clinical trial. In the first year, patients will be evaluated for the effects of PR006A on safety, tolerability, immunogenicity, biomarkers, and efficacy. Patients will be followed for another four years to continue monitoring safety, selected biomarkers, and efficacy parameters.

[0392] Effectiveness evaluation

[0393] The primary objective is to evaluate the safety, tolerability, and immunogenicity of three dose levels of PR006A administered via occipital injection into the cisterna magna, and to quantify PGRN levels in blood and CSF.

[0394] A secondary objective is to evaluate the effects of PR006A on the following: CDR+NACC FTLD and NfL (neuronal filament light chain) levels, blood and CSF levels.

[0395] The exploratory objective is to evaluate the effects of PR006A on: cognitive, behavioral, language, and daily living measures; viral shedding; imaging patterns based on quantification of vMRI and white matter lesions; and selected biomarkers of neuroinflammation, astrocyte lesions, and lysosomal function (e.g., glial cell fibrous acidic protein (GFAP), YKL-40, bis(monoacylglycero)phosphate (BMP)) in CSF, blood, and urine.

[0396] This application incorporates, by reference, the entire contents of the following documents. U.S. Patent Application Publication No. 2020 / 0332265, International PCT Application Publication No. WO2019 / 070893, International PCT Application Publication No. WO2019 / 070891, U.S. Provisional Application Serial Number 62 / 567,296, filed October 3, 2017, title "GENE THERAPIES FOR LYSOSOMAL DISORDERS", U.S. Provisional Application Serial Number 62 / 567,311, filed October 3, 2017, title "GENE THERAPIES FOR LYSOSOMAL DISORDERS", 62 / 567,319, filed October 3, 2017, title "GENE THERAPIES FOR LYSOSOMAL DISORDERS", 62 / 567,301, filed October 3, 2018, title "GENE THERAPIES FOR LYSOSOMAL "GENE THERAPIES FOR LYSOSOMAL DISORDERS", 62 / 567,310, filed October 3, 2017, title "GENE THERAPIES FOR LYSOSOMAL DISORDERS", 62 / 567,303, filed October 3, 2017, title "GENE THERAPIES FOR LYSOSOMAL DISORDERS", and 62 / 567,305, filed October 3, 2017, title "GENE THERAPIES FOR LYSOSOMAL DISORDERS".

[0397] Having described several aspects of at least one embodiment of the present invention, it should be understood that various changes, modifications, and improvements will readily arise for those skilled in the art. Such changes, modifications, and improvements are intended to be part of this disclosure and to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawings are for illustrative purposes only.

[0398] 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 advantages, and each such variation and / or modification will be considered within the scope of the embodiments 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 specific application in which the teachings of the present invention are used. Those skilled in the art will be able to recognize or confirm numerous equivalents to the specific embodiments of the present invention described herein by means of routine experimentation alone. Therefore, it will be understood that the embodiments described herein are presented only as examples, and within the scope of the appended claims and their equivalents, the present invention may be carried out in other ways other than those specifically described and described herein. The present invention covers each individual feature, system, article, material and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and / or methods is included within the scope of the present invention, provided that they are not contradictory to one another.

[0399] In this specification, the indefinite article “one (a and an)” in the specification and claims should be understood to mean “at least one” unless explicitly indicated otherwise.

[0400] In this specification and in the claims, the words “and / or” should be understood to mean “either or both” of the connected elements (i.e., the elements are sometimes connected and sometimes disjunctive). Other elements other than those specifically identified by the “and / or” clause may be present, whether related to those specifically identified elements or not, unless explicitly indicated otherwise. Thus, as a non-restrictive example, a reference to “A and / or B” when used with an unrestrictive word such as “comprising” could refer to A without B in one embodiment (optionally including elements other than B), to B without A in another embodiment (optionally including elements other than A), or to both A and B in another embodiment (optionally including other elements).

[0401] As used herein in the specification and claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, a separate item “or” or “and / or” in a list should be interpreted as inclusive, that is, including at least one of several elements or lists of elements, but also two or more, and any further items not listed at will. Only terms that explicitly indicate the opposite, such as “only one of” or “exactly one of” or, as used in the claims, “consisting of,” refer to including just one element of several elements or lists of elements. In general, as used herein, the term “or” should be interpreted only as indicating exclusive substitutes (i.e., “one or the other, but not both”) when preceded by exclusive terms such as “either,” “one of,” “only one of,” or “exactly one of.”

[0402] As used herein in the specification and claims, the expression “at least one” with respect to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of every element specifically listed in the list of elements, nor excluding any combination of elements in the list of elements. Furthermore, this definition allows for the presence of other elements in the list of elements referred to by the expression “at least one,” which may be of arbitrary choice, whether or not they are related to the specifically identified elements. Therefore, as a non-restrictive 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") could mean, in one embodiment, at least one A (optionally including two or more) where B is absent (optionally including elements other than B); in another embodiment, at least one B (optionally including two or more) where A is absent (optionally including elements other than A); and in yet another embodiment, at least one A (optionally including two or more) and at least one B (optionally including two or more) (optionally including other elements).

[0403] The use of sequential terms such as first, second, third, etc., in a claim for modifying elements of a claim does not in itself imply any priority, order, or temporal order in which any action of a particular claim element is performed relative to other claims, but is used solely as a mark to distinguish one claim element having a particular name from another element having the same name (except for the use of sequential terms).

[0404] Unless otherwise explicitly stated, it should be understood that in any method claimed herein, comprising two or more steps or actions, 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.

[0405] All U.S. patents, published U.S. patent applications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referenced herein are incorporated herein by reference in their entirety. array

[0406] In some embodiments, an expression cassette encoding one or more gene products (e.g., a first, second, and / or third gene product) includes or comprises a sequence described in any one of SEQ ID NOs: 1 to 91. In some embodiments, an expression cassette encoding one or more gene products includes or comprises a sequence complementary to the sequence described in any one of SEQ ID NOs: 1 to 91. In some embodiments, an expression cassette encoding one or more gene products includes or comprises a sequence that is inversely complementary to the sequence described in any one of SEQ ID NOs: 1 to 91. In some embodiments, a gene product is encoded by a part (e.g., a fragment) of any one of SEQ ID NOs: 1 to 91. In some embodiments, the nucleic acid sequence 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 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.

[0407] Numbered Embodiments Notwithstanding the attached claims, this disclosure provides for the following numbered embodiments:

[0408] 1. A method for treating a subject who has or is suspected of having frontotemporal dementia with a GRN mutation, wherein the subject (i) an rAAV vector comprising nucleic acids comprising an expression construct comprising a promoter operably linked to an transgene insertion encoding a progranulin (PGRN) protein, wherein the transgene insertion comprises an rAAV vector comprising the nucleotide sequence of SEQ ID NO: 68, (ii) Recombinant adeno-associated virus (rAAV) containing adeno-associated virus (AAV) 9 capsid protein, and one or more of the following: (A) Sirolimus, (B) Methylprednisolone, (C) Rituximab, and (D) A method comprising administering prednisone and

[0409] 2. A method for suppressing the immune response in subjects who have or are suspected of having frontotemporal dementia with a GRN mutation, wherein the subject (i) an rAAV vector comprising nucleic acids comprising an expression construct comprising a promoter operably linked to an transgene insertion encoding a progranulin (PGRN) protein, wherein the transgene insertion comprises an rAAV vector comprising the nucleotide sequence of SEQ ID NO: 68, (ii) Recombinant adeno-associated virus (rAAV) containing adeno-associated virus (AAV) 9 capsid protein, and one or more of the following: (A) Sirolimus, (B) Methylprednisolone, (C) Rituximab, and (D) A method comprising administering prednisone and

[0410] 3. rAAV is approximately 1 × 10 13 Vector genome (vg) ~ approximately 7 × 10⁻⁶ 14 The method according to Embodiment 1 or 2, administered to a subject in a dose within the range of vg.

[0411] 4. rAAV is approximately 3.5 × 10 13 vg, approx. 7.0×10 13 VG or approximately 1.4 × 10 14The method according to Embodiment 1 or 2, administered to the subject at a dose of VG.

[0412] 5. The method according to any one of Embodiments 1 to 4, wherein rAAV is administered by injection into the cisterna magna.

[0413] 6. The method according to any one of Embodiments 1 to 5, wherein the promoter is a chicken beta-actin (CBA) promoter.

[0414] 7. The method according to any one of Embodiments 1 to 6, wherein the rAAV vector further comprises a cytomegalovirus (CMV) enhancer.

[0415] 8. The method according to any one of Embodiments 1 to 7, wherein the rAAV vector further comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0416] 9. The method according to any one of Embodiments 1 to 8, wherein the rAAV vector further comprises a bovine growth hormone polyA signaling tail.

[0417] 10. The method according to any one of Embodiments 1 to 9, wherein the nucleic acid comprises two adeno-associated virus inverted terminal repeat (ITR) sequences adjacent to the expression construct.

[0418] 11. The method according to Embodiment 10, wherein each ITR sequence is an AAV2 ITR sequence.

[0419] 12. The method according to Embodiment 10 or 11, 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.

[0420] 13. A method for treating a subject who has or is suspected of having frontotemporal dementia with a GRN mutation, wherein the subject (i) In the order from 5' to 3', (a) Adeno-associated virus (AAV) 2ITR and (b) Cytomegalovirus (CMV) enhancer, (c) Chicken beta-actin (CBA) promoter and (d) A transgene insertion encoding the progranulin (PGRN) protein, comprising the nucleotide sequence of SEQ ID NO: 68, (e) Woodchuck hepatitis virus post-transcriptional regulatory elements (WPREs), (f) Bovine growth hormone polyA signaling tail, (g) A nucleic acid containing AAV2 inverted terminal repeats (ITRs) and an rAAV vector, (ii) A recombinant adeno-associated virus (rAAV) containing the AAV9 capsid protein, and one or more of the following: (A) Sirolimus, (B) Methylprednisolone, (C) Rituximab, and (D) A method comprising administering prednisone and

[0421] 14. A method for suppressing the immune response in subjects who have or are suspected of having frontotemporal dementia with a GRN mutation, wherein the subject (i) In the order from 5' to 3', (a) Adeno-associated virus (AAV) 2ITR and (b) Cytomegalovirus (CMV) enhancer, (c) Chicken beta-actin (CBA) promoter and (d) A transgene insertion encoding the progranulin (PGRN) protein, comprising the nucleotide sequence of SEQ ID NO: 68, (e) Woodchuck hepatitis virus post-transcriptional regulatory elements (WPREs), (f) Bovine growth hormone polyA signaling tail, (g) A nucleic acid containing AAV2 inverted terminal repeats (ITRs) and an rAAV vector, (ii) A recombinant adeno-associated virus (rAAV) containing the AAV9 capsid protein, and one or more of the following: (A) Sirolimus, (B) Methylprednisolone, (C) Rituximab, and (D) A method comprising administering prednisone and

[0422] 15. rAAV is approximately 1 × 10 13 vg~approx. 7×10 14 The method according to Embodiment 13 or 14, wherein the subject is administered a dose in the range of vg.

[0423] 16. rAAV is approximately 3.5 × 10 13 vg, approx. 7.0×10 13 VG or approximately 1.4 × 10 14 The method according to embodiment 13 or 14, administered to the subject at a dose of VG.

[0424] 17. The method according to any one of embodiments 13 to 16, wherein rAAV is administered by injection into the cisterna magna.

[0425] 18. rAAV is present in approximately 20 mM Tris, pH 8.0, and approximately 1 mM MgCl. 2、 The method according to any one of Embodiments 1 to 17, administered with a formulation containing approximately 200 mM NaCl and approximately 0.001% w / v poloxamer 188.

[0426] 19. The method according to any one of Embodiments 1 to 18, wherein in some embodiments, methylprednisolone is administered intravenously at a dose of about 1000 mg on either the day before or on the same day as the administration of rAAV.

[0427] 20. Prednisone, (A) For 14 days starting the day after administration of approximately 1000 mg of the methylprednisolone, at a dose of approximately 30 mg / day, and The method according to any one of Embodiments 1 to 19, wherein the dose is gradually reduced and administered orally over a 7-day period following the end of the 14-day period of (A).

[0428] 21. The method according to any one of Embodiments 1 to 20, wherein rituximab is administered intravenously at a dose of approximately 1000 mg on any one day between 14 days and 1 day prior to the administration of rAAV.

[0429] 22. The method according to Embodiment 21, wherein methylprednisolone is administered before rituximab is administered.

[0430] 23. The method according to Embodiment 22, wherein methylprednisolone is administered at least about 30 minutes before rituximab is administered.

[0431] 24. The method according to Embodiment 21, wherein both methylprednisolone and rituximab are administered the day before administration of rAAV, and methylprednisolone is administered at least about 30 minutes before administration of rituximab.

[0432] 25. The method according to Embodiment 21, wherein rituximab is administered on any day between 14 days and 2 days before administration of rAAV, and methylprednisolone is administered intravenously at a dose of approximately 100 mg at least 30 minutes before the administration of rituximab on the same day as the administration of rituximab.

[0433] 26. Sirolimus, (A) Three days, two days, or one day before administration of rAAV, as a single dose of approximately 6 mg, and (B) For approximately 90 days after rAAV administration, the drug is orally administered at a dose of approximately 2 mg / day to maintain a serum trough level of approximately 4 ng / mL to approximately 9 ng / mL. The method according to any one of Embodiments 1 to 25, wherein the first dose of sirolimus, approximately 2 mg / day, is administered the day following the single dose of approximately 6 mg of sirolimus.

[0434] 27. The method according to Embodiment 26, wherein the administration of sirolimus is gradually reduced between 15 and 30 days after the end of the 90-day period following the administration of rAAV. 28. (i) Administer methylprednisolone intravenously at a dose of approximately 1000 mg, (ii) Approximately 30 minutes after the administration of methylprednisolone in step (i), rituximab should be administered intravenously at a dose of approximately 1000 mg. (iii) The day after the administration of methylprednisolone in step (i), rAAV is administered into the cisterna magna by injection, (iv) Prednisone should be administered orally at a dose of approximately 30 mg / day for 14 days starting the day following the administration of methylprednisolone in step (i), (v) For 7 days after the completion of the 14-day period of step (iv), prednisone should be gradually tapered off. (vi) Three days, two days, or one day before the administration of rAAV in step (iii), sirolimus should be administered orally as a single dose of approximately 6 mg. (vii) Orally administer sirolimus at a dose of approximately 2 mg / day for approximately 90 days after rAAV administration in step (iii), maintaining a serum trough level of approximately 4 ng / ml to approximately 9 ng / mL, with the first dose of approximately 2 mg / day of sirolimus being administered the day after a single dose of approximately 6 mg of sirolimus. (viii) The method according to any one of Embodiments 1 to 27, comprising gradually reducing the dose of sirolimus between 15 and 30 days after the end of the 90-day period of step (vii).

[0435] 29. (i) Administer methylprednisolone intravenously at a dose of approximately 100 mg on any day between 14 and 2 days before the administration of rAAV in step (iv), (ii) Approximately 30 minutes after the administration of methylprednisolone in step (i), rituximab should be administered intravenously at a dose of approximately 1000 mg. (iii) Administer methylprednisolone intravenously at a dose of approximately 1000 mg either one day before or on the same day as the rAAV administration in step (iv), (iv) Administering rAAV into the cisterna magna via injection, (v) Prednisone shall be orally administered at a dose of approximately 30 mg / day for 14 days starting the day following the administration of methylprednisolone in step (iii), (vi) For 7 days following the completion of the 14-day period of step (v), the prednisone shall be administered in a gradually tapered manner. (vii) Three days, two days, or one day before the administration of rAAV in step (iv), sirolimus should be administered orally as a single dose of approximately 6 mg. (viii) Orally administer sirolimus at a dose of approximately 2 mg / day for approximately 90 days after rAAV administration in step (iv), to maintain a serum trough level of approximately 4 ng / ml to approximately 9 ng / mL, with the first dose of approximately 2 mg / day of sirolimus being administered the day after a single dose of approximately 6 mg of sirolimus. (ix) The method according to any one of Embodiments 1 to 27, comprising gradually reducing the dose of sirolimus between 15 and 30 days after the end of the 90-day period of step (viii).

[0436] 30. The method according to Embodiment 2 or 14, wherein the immune response is an immune response to rAAV.

[0437] 31. The method according to any one of Embodiments 2, 14, and 30, wherein the immune response is a T-cell reaction.

[0438] 32. The method according to any one of Embodiments 2, 14, and 30, wherein the immune response is a B-cell reaction.

[0439] 33. The method according to any one of Embodiments 2, 14, and 30, wherein the immune response is an antibody reaction.

[0440] 34. The method according to any one of Embodiments 2, 14, and 30, wherein the immune response is pleocytosis.

[0441] 35. The method according to Embodiment 34, wherein the pleocytosis is cerebrospinal fluid (CSF) pleocytosis.

[0442] 36. The method according to any one of Embodiments 2, 14, and 30, wherein the immune response is an abnormal level of CSF protein.

[0443] 37. The method according to any one of Embodiments 1 to 36, wherein an additional immunosuppressant other than sirolimus, methylprednisolone, rituximab, or prednisone is further administered to the target.

[0444] 38. A method for treating frontotemporal dementia with GRN mutations in the subject, (i) an rAAV vector comprising nucleic acids comprising an expression construct comprising a promoter operably linked to an transgene insertion encoding a progranulin (PGRN) protein, wherein the transgene insertion comprises an rAAV vector comprising the nucleotide sequence of SEQ ID NO: 68, (ii) Recombinant adeno-associated virus (rAAV) containing adeno-associated virus (AAV) 9 capsid protein, and one or more of the following: (A) Sirolimus, (B) Methylprednisolone, (C) Rituximab, and (D) Prednisone, It is a combination therapy agent.

[0445] 39. For use in suppressing the immune response in subjects with or suspected of having frontotemporal dementia with GRN mutations. (i) an rAAV vector comprising nucleic acids comprising an expression construct comprising a promoter operably linked to an transgene insertion encoding a progranulin (PGRN) protein, wherein the transgene insertion comprises an rAAV vector comprising the nucleotide sequence of SEQ ID NO: 68, (ii) Recombinant adeno-associated virus (rAAV) containing adeno-associated virus (AAV) 9 capsid protein, and one or more of the following: (A) Sirolimus, (B) Methylprednisolone, (C) Rituximab, and (D) Prednisone, A combination therapy agent.

[0446] 40. The number of combinations is approximately 1 x 10 13 vg~approx. 7×10 14 A combination therapy agent for use according to Embodiment 39, comprising vg rAAV.

[0447] 41. The combination is approximately 3.5 × 10 13 vg, approx. 7.0×10 13 VG or approximately 1.4 × 10 14 A combination therapy agent for use according to Embodiment 39, comprising vg rAAV.

Claims

1. For use in treating subjects with or suspected of having frontotemporal dementia with GRN mutations, (i) In the order of 5' to 3', (a) Adeno-associated virus (AAV) 2ITR and (b) Cytomegalovirus (CMV) enhancer, (c) Chicken beta-actin (CBA) promoter, (d) A transgene insertion encoding the progranulin (PGRN) protein, comprising the nucleotide sequence of Sequence ID No. 68, (e) Woodchuck hepatitis virus post-transcriptional regulatory elements (WPRE), (f) Bovine growth hormone polyA signal tail, (g) AAV2 inverted terminal repeat (ITR) and rAAV vectors containing nucleic acids, and (ii) AAV9 capsid protein including, A pharmaceutical composition containing recombinant adeno-associated virus (rAAV), Here, the rAAV is used in combination with methylprednisolone. The methylprednisolone is the only immunosuppressant administered to the subject together with the rAAV. The methylprednisolone is administered intravenously at a dose of 1000 mg either the day before or on the same day as the administration of the rAAV, and the rAAV is 1 × 10 １３ vg ~ 7 x 10 １４ A pharmaceutical composition characterized by being used to administer to the subject via injection into the cisterna magna in doses within the range of vg.

2. For use in suppressing the immune response in subjects with or suspected of having frontotemporal dementia with GRN mutations, (i) In the order of 5' to 3', (a) Adeno-associated virus (AAV) 2ITR and (b) Cytomegalovirus (CMV) enhancer, (c) Chicken beta-actin (CBA) promoter, (d) A transgene insertion encoding the progranulin (PGRN) protein, comprising the nucleotide sequence of Sequence ID No. 68, (e) Woodchuck hepatitis virus post-transcriptional regulatory elements (WPRE), (f) Bovine growth hormone polyA signal tail, (g) AAV2 inverted terminal repeat (ITR) and rAAV vectors containing nucleic acids, and (ii) AAV9 capsid protein including, A pharmaceutical composition containing recombinant adeno-associated virus (rAAV), Here, the rAAV is used in combination with methylprednisolone. The methylprednisolone is the only immunosuppressant administered to the subject together with the rAAV. The methylprednisolone is administered intravenously at a dose of 1000 mg on either the day before or the day of administration of the rAAV, and The aforementioned rAAV is 1 × 10 １３ vg ~ 7 x 10 １４ A pharmaceutical composition characterized by being used to administer to the subject via injection into the cisterna magna in doses within the range of vg.

3. The aforementioned rAAV is 3.5 × 10 １３ vg, 7.0 x 10 １３ vg or 1.4 × 10 １４ At a dose of VG, A pharmaceutical composition according to claim 1 or 2, characterized in that it is used to be administered to a target.

4. (a) The pharmaceutical composition further contains 20 mM Tris, pH 8.0, and 1 mM MgCl ２ , comprising 200 mM NaCl and 0.001% w / v poloxamer 188; and / or (b) The methylprednisolone is used in such a manner that it is administered intravenously at a dose of 1000 mg either one day before or on the same day as the administration of the rAAV. A pharmaceutical composition according to any one of claims 1 to 3.

5. The pharmaceutical composition according to claim 2, wherein the immune response is an immune response to the rAAV.

6. The pharmaceutical composition according to claim 2 or 5, wherein the immune response is a T-cell reaction, a B-cell reaction, an antibody reaction, cerebrospinal fluid (CSF) pleocytosis, or an abnormal level of cerebrospinal fluid (CSF) protein.

7. below: (a) White blood cell count (WBC) > 30 / mm ３ and / or asymptomatic pre-ococytosis with high cerebrospinal fluid (CSF) protein (> 70 mg / dL); (b) CSF pleocytosis and / or protein increase accompanied by clinical symptoms; (c) Appearance of sensory symptoms based on neurological examination and / or treatment-induced neurological impairment assessment scale (TNAS); (d) Symptoms of hepatitis accompanied by elevated alanine aminotransferase (ALT) and / or aspartate aminotransferase (AST) > 5 × upper limit of normal (ULN); and (e) ALT and / or AST elevation > 10 × ULN If one or more of the following conditions are present in the patient, the treatment may include increasing the dose of immunosuppressants, a long-term tapering regimen, the use of additional medications, or resuming treatment. A pharmaceutical composition according to any one of claims 1 to 6.

8. For use in methods for treating frontotemporal dementia with GRN mutations in the subject, (i) an rAAV vector comprising a nucleic acid comprising an expression construct comprising a chicken beta-actin (CBA) promoter operably linked to a transgene insert encoding a progranulin (PGRN) protein, wherein the transgene insert comprises the nucleotide sequence of Sequence ID No. 68; a cytomegalovirus (CMV) enhancer; a woodchuck hepatitis virus post-transcriptional regulator (WPRE); and a bovine growth hormone poly-A signal tail, and the nucleic acid comprises two adeno-associated virus 2-inverted terminal repeat (ITR) sequences adjacent to the expression construct, the first ITR sequence being a 5'ITR and the second ITR sequence being a 3'ITR, and the rAAV vector, and (ii) Adeno-associated virus (AAV) 9 capsid protein Recombinant adeno-associated virus (rAAV), including, With methylprednisolone, It is a combination therapy agent, The aforementioned rAAV is 1 × 10 １３ vg ~ 7 x 10 １４ The dose is administered to the subject via intracision into the cisterna magna at a dose in the range of vg. The methylprednisolone is the only immunosuppressant administered to the subject together with the rAAV, and The methylprednisolone is characterized in that it is administered intravenously at a dose of 1000 mg on either the day before or the day of administration of the rAAV. The aforementioned combination therapy agent.

9. For use in methods for suppressing the immune response in subjects with or suspected of having frontotemporal dementia with GRN mutations, (i) an rAAV vector comprising a nucleic acid comprising an expression construct comprising a chicken beta-actin (CBA) promoter operably linked to a transgene insert encoding a progranulin (PGRN) protein, wherein the transgene insert comprises the nucleotide sequence of Sequence ID No. 68; a cytomegalovirus (CMV) enhancer; a woodchuck hepatitis virus post-transcriptional regulator (WPRE); and a bovine growth hormone poly-A signal tail, and the nucleic acid comprises two adeno-associated virus 2-inverted terminal repeat (ITR) sequences adjacent to the expression construct, the first ITR sequence being a 5'ITR and the second ITR sequence being a 3'ITR, and the rAAV vector, and (ii) Adeno-associated virus (AAV) 9 capsid protein Recombinant adeno-associated virus (rAAV), including, With methylprednisolone, It is a combination therapy agent, The aforementioned rAAV is 1 × 10 １３ vg ~ 7 x 10 １４ The dose is administered to the subject via intracision into the cisterna magna at a dose in the range of vg. The methylprednisolone is the only immunosuppressant administered to the subject together with the rAAV, and The methylprednisolone is characterized in that it is administered intravenously at a dose of 1000 mg on either the day before or the day of administration of the rAAV. The aforementioned combination therapy agent.

10. 3.5 x 10 １３ vg, 7.0 x 10 １３ vg or 1.4 × 10 １４ vg including the rAAV, The combination therapy agent according to claim 8 or 9.

11. The aforementioned rAAV is 20 mM Tris, pH 8.0, 1 mM MgCl ２ The combination therapy agent according to any one of claims 8 to 10, characterized in that it is used to be administered in a formulation containing 200 mM NaCl and 0.001% w / v poloxamer 188.

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