Raav vectors for treatment of GM1 and GM2 gangliosidosis

Abstract

To provide a recombinant adeno-associated virus (rAAV), pharmaceutical compositions and methods for treating GM1 gangliosidosis.SOLUTION: Provided is a recombinant AAV (rAAV) comprising a capsid containing a nucleic acid having a specific sequence. In some embodiments, at least one capsid protein of the rAAV described herein is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, or AAVrh10 capsid protein. In some embodiments, at least one ITR of the rAAV described herein is selected from the group consisting of AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, or AAV6 ITR.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application is filed on January 1, 2018, the entire contents of each application being incorporated herein by reference. The application was filed on October 5th and is entitled "RAAV VECTORS FOR THE TREATMEN T OF GM1 AND GM2 GANGLIOSIDOSIS" Provisional Patent Application No. 62 / 741,848, filed March 6, 2019, entitled “RAA V VECTORS FOR THE TREATMENT OF GM1 AND G Specification No. 62 / 814,587 entitled "M2 GANGLIOSIDOSIS" , filed on March 8, 2019, entitled "RAAV VECTORS FOR THE TR "EATMENT OF GM1 AND GM2 GANGLIOSIDOSIS" 62 / 815,996, filed on March 29, 2019, AV VECTORS FOR THE TREATMENT OF GM1 AND Specifications No. 62 / 826,863 entitled "GM2 GANGLIOSIDOSIS" The document was filed on April 29, 2019, and is entitled "RAAV VECTORS FOR THE TREATMENT OF GM1 AND GM2 GANGLIOSIDOSIS” 62 / 840,359, entitled "Compound for a cellular endothelial cell," and "RAAV VECTORS FOR THE TREATMENT OF GM1 AND GM2 GANGLIOSIDOSIS” The benefit under 35 U.S.C. § 119(e) of the 58 specification as of the filing date of the application is Assert.

[0002] References to sequence listings submitted electronically via EFS-Web This application was filed electronically via EFS-Web and is incorporated by reference in its entirety. The sequence listing incorporated herein (Title: "Sequence Listing"; Size: 80,237 bytes; and and creation software: Patent-In 3.5).

[0003] Field In some aspects, the present disclosure provides recombinant adeno-associated antibodies useful for treating lysosomal storage disorders. In some embodiments, lysosomal viruses (rAAV), compositions, and kits are provided. Somatic storage disorders include GM1 gangliosidosis or GM2 gangliosidosis (e.g. For example, Tay-Sachs disease.

[0004] Federally sponsored research This invention was made in accordance with grant number HD060576 awarded by the National Institutes of Health. This invention was made with government support under the Federal Register. The United States Federal Government has certain rights in this invention. do. [Background technology]

[0005] GM1 gangliosidosis is caused by a mutation in the human gene GLB1. It is an autosomal recessive neuropathic lysosomal storage disorder caused by the human GLB1 gene. It is involved in the synthesis of numerous molecules in the central nervous system (e.g., GM1 ganglioside) and in peripheral tissues. Numerous molecules (e.g., oligosaccharides, glycoproteins, and glycosaminoglycans) are The enzyme β-D-galactosidase (βgal), which removes terminal galactose residues, was used to The lack of lysosomal β-gal activity is primarily due to the fact that lysosomal synthesis is at its maximal rate. In the central nervous system, accumulation of GM1 ganglioside and its asialo derivative, GA1, is promoted. GM1 gangliosidosis is generally fatal and there is no effective treatment. stomach.

[0006] Tay-Sachs disease (GM2 gangliosidosis) and Sandhoff disease are resulting in a deficiency of acetyl-D-hexosaminidase (Hex) enzyme activity, respectively. Autosomal recessive leukemia caused by mutations in the HEXA and HEXB genes Hex enzymes are composed of two protein subunits, HexA and HexB. Hex is a heterodimer containing the protein subunits β and β. Deficiency of Hex activity results in central nervous system This leads to progressive accumulation of GM2 gangliosides and subsequent neurodegeneration in Tay-Sachs disease is characterized by relentlessly progressive neurological deterioration and eventual death. Symptoms of Tay-Sachs disease include frequent seizures, difficulty swallowing, loss of motor control, and and frequent respiratory infections. Sandhoff disease also causes progressive weakening of the CNS. It is characterized by gait abnormalities, swallowing and speech difficulties, peripheral neuropathy, psychiatric symptoms, and finally Currently, there is no evidence that Tay-Sachs or Sandhoff disease However, there are no disease-modifying treatment options and only symptomatic treatments, including anticonvulsants and anticonvulsants, are available. Injuries are available. Summary of the Invention [Means for solving the problem]

[0007] Aspects of the present disclosure relate to recombinant AAV vectors for gene delivery. AV vectors may result in adverse events (e.g., cytotoxicity) in subjects. The present disclosure provides a method for the manipulation of regulatory elements to deliver high levels of gene expression that reduces adverse events. The expression level of the transgene is adjusted to provide a therapeutic amount of the transgene without induction. This is based in part on the recognition that it may be possible to modulate

[0008] In some aspects, the present disclosure provides a method for the detection of lysosomal storage disorder-associated tags via chimeric introns. to transgenes encoding proteins (e.g., HEXA, HEXB, GLB1, etc.) A recombinant AAV comprising a capsid containing a nucleic acid comprising a promoter operably linked to In some aspects, the present disclosure provides a method for the delivery of recombinant AAV (rAAV) to a mammalian animal via a chimeric intron. The capsid contains a nucleic acid comprising a promoter operably linked to a gene encoding the The promoter and the transgene are separated by a chimeric intron, and the nucleic acid is The transgene does not contain an enhancer element and encodes a lysosomal storage disorder-associated protein. Encoding proteins (e.g., HEXA and / or HEXB and / or GLB1) In some embodiments, the promoter is selected from the group consisting of chicken AAV and chicken hamster ovarian cancer. In some embodiments, the chimeric intro The introns are derived from chicken beta-actin and / or rabbit beta-globin introns. In some embodiments, the nucleic acid contains one or more untranslated sequences, e.g., The untranslated sequences derived from exon 1 and / or exon 2 of rabbit beta-globin In some embodiments, the chimeric intron further comprises two untranslated sequences (e.g., One untranslated sequence is located between the promoter and the chimeric intron, and a second untranslated sequence is located between the promoter and the chimeric intron. The region is located between the chimeric intron and the first codon of the transgene. More pinched.

[0009] In some embodiments, the βgal is human βgal. The rAAV described by In some embodiments, the transgene comprises a transgene encoding HEXA. In some embodiments, the transgene is represented by the sequence set forth in SEQ ID NO:20. - encoding hexosaminidase subunit beta (HexB). HEXB is represented by the sequence set forth in SEQ ID NO:21.

[0010] In some embodiments, at least one capsid of the rAAV described herein is Proteins include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, and AAV7. , AAV8, AAVrh8, AAV9, AAV10, or AAVrh10 capsid In some embodiments, at least one of the rAAVs described herein is a The four ITRs are AAV1 ITR, AAV2 ITR, AAV3 ITR, and AAV4 ITR. TR, AAV5 ITR, or AAV6 ITR. In some embodiments, at least one ITR is a full-length ITR. The AV contains two ITRs, in this case a hybrid promoter and a transgene The child is placed between two ITRs. In some embodiments, the rA AV includes the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, and AAV6. , AAV7, AAV8, AAVrh8, AAV9, AAV10, or AAVrh10 I have one of them.

[0011] In some embodiments, the disclosure provides vectors encoding components of the rAAV described herein. For example, in some embodiments, the present disclosure relates to nucleic acids having the sequences set forth in SEQ ID NOs: 1-6 or 16-19. In some embodiments, the present disclosure provides an isolated nucleic acid comprising a sequence selected from SEQ ID NO:2. An isolated nucleic acid containing a sequence encoding a peptide having an amino acid sequence designated 0 or 21. Present the acid.

[0012] In some embodiments, nucleic acids encoding components of an rAAV (e.g., SEQ ID NOs: 1-6) are or 16 to 19, or a part of a sequence derived from SEQ ID NO: 1 to 6 or 16 to 19 In some embodiments, the nucleic acid comprising a sequence selected from the group consisting of a nucleic acid ... The cell is a eukaryotic cell. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the host cell is a bacterial cell. It is a cell.

[0013] In some embodiments, the host cell is adapted to express AAV capsid proteins (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV17, AAV18, AAV19, AAV20, AAV21, AAV30, AAV40, AAV50, AAV51, AAV61, AAV72, AAV8, AAV9, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV17, AAV18, AAV19, AAV20, AAV30, AAV40, AAV51, AAV52, AAV63, AAV74, AAV8, AAV9, AAV15, AAV16, AV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8 A single fragment encoding the capsid protein of AAV10, AAV20, AAV30, AAV40, AAV50, AAV60, AAV70, AAV80, AAV90, AAV101, AAV102, AAV103, AAV104, AAV105, AAV106, AAV107, AAV108, AAV109, AAV2010, AAV20110, AAV20121, AAV20122, AAV20123, AAV20124, AAV20125, AAV20126, AAV20127, AAV20128, AAV20129, AAV302, AAV303, AAV304, AAV305, AAV306, AAV307, AAV308, AAV309, AAV409, AAV401 It further comprises an isolated nucleic acid.

[0014] In some aspects, the present disclosure provides a method for the production of a chimeric intron comprising: (i) isolating a beta-hexosaminyltransferase (TAT) from a beta-hexosaminyltransferase (TAT) via a chimeric intron; operably linked to a transgene encoding hexidase subunit alpha (HexA). a first rAAV comprising a capsid containing a first nucleic acid comprising a promoter linked thereto; ii) via a chimeric intron, the beta-hexosaminidase subunit beta (H A second promoter operably linked to a transgene encoding exB. and a second rAAV comprising a capsid containing the nucleic acid of In embodiments, the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier. In one embodiment, the first rAAV and the second rAAV are present in the composition in a 1:1 ratio.

[0015] In some aspects, the present disclosure provides a method for treating a lysosomal storage disorder, the method comprising: The recombinant AAV (rAAV) or pharmaceutical compositions described herein can be administered to lysosomal storage Disorders (e.g., Tay-Sachs disease, GM2 gangliosidosis, Sandhoff disease, type B chromosome Some embodiments of the present invention provide a method for treating a chronic inflammatory bowel disease, comprising administering the method to a subject suffering from chronic inflammatory bowel disease, such as Lukio's syndrome. In an embodiment, the lysosomal storage disorder is Tay-Sachs disease or Sandhoff disease. In some embodiments, the rAAV or pharmaceutical composition is administered to a subject by intracranial injection, intracerebral injection, or It is administered by injection into the CSF via the ventricular system, cisterna magna, or intrathecal cavity. In embodiments, the present disclosure provides a method for treating GM2 gangliosidosis, the method comprising: The rAAV or pharmaceutical composition described herein may be administered to a subject with GM2 gangliosidosis. A method is provided comprising administering to a subject comprising administering to said subject a therapeutically effective amount of said compound.

[0016] In some aspects, the present disclosure provides a method for storing recombinant AAV (rAAV) as described herein. or a composition comprising recombinant AAV (rAAV), as described herein. A kit is provided. In some embodiments, the kit includes a pharma- ceutically acceptable carrier. In some embodiments, the rAAV or a composition comprising the rAAV and a pharmaceutical In some embodiments, the container is a silicone container. It's Nji.

[0017] In some aspects, the present disclosure provides a transfected human GLB1 encoding vector set forth in SEQ ID NO:23. A recombinant AAV vector comprising a nucleic acid encoding a promoter operably linked to a transgene. In some embodiments, the present disclosure provides a vector for the human GLB1 as set forth in SEQ ID NO:23. An isolated nucleic acid encoding a promoter operably linked to a transgene encoding In some embodiments, the pharmaceutical composition comprises rAAV and a pharma- ceutical acceptable carrier. In some embodiments, the present disclosure provides a method for treating GM1 gangliosidosis. The method of claim 1, further comprising administering the rAAV or pharmaceutical composition to a subject having GM1 gangliosidosis. A method is presented comprising administering to an elephant. [Brief description of the drawings]

[0018] [Figure 1]Design of rAAV vector, CBA-mβgal-WPRE. Figure 1A shows a schematic diagram of rAAV vector, CBA-mβgal-WPRE. Two inverted terminal repeats (ITRs) from AAV2 flank the vector at each end. The CBA promoter is composed of a chimeric chicken beta-actin / rabbit beta globin intron (CBA), mouse lysosomal acid beta-galactosidase cDNA (mβgal), woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), and two tandem polyA signals from bovine growth hormone (BGH) and SV40, followed by a cytomegalovirus immediate early enhancer (CMV) fused to the chicken beta-actin promoter. This vector was then packaged into an AAVrh8 capsid. Figure 1B shows a recombinant AAV (rAAV) vector containing a transgene encoding hGLB1 under the control of the CBA promoter. [Diagram 2] Figure 2 shows that AAVrh8-mβgal injected intracranially into βgal- / - mice results in dose-dependent enzyme distribution. βgal expression in the brains of representative AAVrh8-injected animals and age-matched controls was analyzed by histochemical staining of 20 μm coronal brain sections with X-gal and counterstaining with Nuclear Fast Red at 2 weeks (4×1010vg) or 3 months (2.6×1010vg and 2.6×109vg) after injection. Figures 2A-2B show 4×1010vg; Figures 2E-2F show 2.6×1010vg; Figures 2I-2J show 2.6×109vg; Figures 2C-2D show naive βgal- / - mice; Figures 2G-2H show naive βgal+ / - mice. Images represent N≧3 mice per group. [Diagram 3]Figure 3: βgal- / - mice intracranially injected with AAV retain significant motor function on the rotarod. Animals were assessed for motor function in an accelerating rotarod test (4-40 rpm over 5 min). The best score from three trials was recorded. Using a nonparametric, unpaired, Student's T-test and Walsh correction, βgal- / - animals treated with a total dose of AAVrh8 vector (Fig. 3A) 4x1010vg; (Fig. 3B) 2.6x1010vg; and (Fig. 3C) 2.6x109 all retained significantly (P=0.006, 0.0009, 0.005, respectively) better motor function than naive βgal- / - controls at 6 months after injection. N=3-15 animals per group at each time point, N=6-10 animals per group at 6 months after treatment. [Figure 4] Figure 1 shows that βgal- / - mice intracranially injected with AAV achieve a significant lifespan extension. Kaplan-Meier survival curves for βgal- / - mice treated with intracranial AAVrh8. Using the log-rank (Mantel-Cox) test, mice treated with AAVrh8 vector at total doses of 4x1010vg, 2.6x1010vg, and 2.6x109vg all showed a significant (p=0.0004, p=0.002, p<0.0001, respectively) extension in lifespan compared to naive βgal- / - controls. Median survival was extended from naive βgal− / − controls (245.5 days, N=13) to the 4×1010vg cohort (293.5 days, N=12), 2.6×1010vg cohort (349.0 days, N=13), and 2.6×109vg cohort (389.0 days, N=13). [Diagram 5]Figure 5 shows that intracranial injection of AAV in βgal- / - mice results in aberrant filipin staining in the region of highest expression of the enzyme. Figure 5A shows a coronal section of mouse brain stained with Xgal for the presence of βgal enzyme and counterstained with Nuclear Fast Red 12 weeks after injection of a representative βgal- / - mouse (knockout+AAV) injected bilaterally into the thalamus with 1 μl of AAVrh8-CBA-mβgal-WPRE (total dose of 2.6×1010 vg). Figure 5B shows an untreated βgal- / - mouse (untreated knockout). Boxes indicate the location of images depicted in Figures 5C-5F. Flipin staining of adjacent brain sections in βgal- / - animals treated with 2.6×1010 vg of AAVrh8-CBA-mβgal-WPRE is shown in Figures 5C and 5E. Figures 5D and 5F show untreated βgal− / − mice (untreated knockout). Filipina images taken at 10x magnification. Images represent N≧3 mice per group. [Figure 6] 6A and 6B show persistent lysosomal accumulation in the spinal cord of long-lived βgal− / − mice intracranially injected with AAV. Spinal cord sections cut at 20 μm were stained with filipin for accumulation of GM1 ganglioside. All long-lived animals showed some amount of intraspinal clearance, but also contained areas where clearance did not occur. FIG. 6A and FIG. 6B show 621 days after injection of 4×1010 vg. FIG. 6C and FIG. 6D show 547 days after injection of 2.6×1010 vg. FIG. 6E and FIG. 6F show 495 days after injection of 2.6×109 vg. FIG. 6G and FIG. 6H show generalized accumulation approximately 250 days after injection in naive βgal− / −. Images represent N≧2 mice per group. [Figure 7]Figure 7 shows that intracranial injection of AAV in βgal- / - mice results in shape changes at the injection site in the thalamus. Intracranial injected βgal- / - mice were analyzed by H&E staining of 20 μm coronal brain sections at 2 weeks (4×1010vg) or 3 months (2.6×1010vg and 2.6×109vg) after injection. Shape changes in the injected area of ​​the thalamus: thick arrows indicate cellular infiltration into blood vessels and thin arrows point to inflammation. The left panel is taken at 10x and the right panel is a 40x picture (from the area on the left panel). Shape changes and neuronal loss appear to be attenuated even with the lowest injected dose. Figures 7A and 7B show 4×1010vg injections 2 weeks after treatment. Figures 7E and 7F show 2.6×1010vg injections 3 months after treatment. Figures 7I and 7J show injections of 2.6x109vg 3 months after treatment. Figures 7G and 7H show untreated naive βgal+ / - at the age of treated animals. Figures 7C and 7D show untreated naive βgal- / - at the age of treated animals. Figure 7K shows a cerebrum injected with AAVrh8-mβgal. Red arrows indicate the site of injection in βgal animals and the location of the photograph shown in this figure. Scale bar represents 100um. Images represent N>3 mice per group. [Figure 8]Figure 8 shows that intracranial injection of AAV in βgal- / - mice results in shape changes at the injection site in the deep cerebellar nuclei. Intracranial injected βgal- / - mice were analyzed by H&E staining of 20 μm coronal brain sections at 2 weeks (4×1010vg) or 3 months (2.6×1010vg and 2.6×109vg) after injection. Shape changes were observed within the injected area of ​​DCN. Thick arrows indicate cellular infiltration into blood vessels. Arrows point to infiltration suspected to be neurophagocytosis. Left panel is taken at 10x and right panel is 40x (from the area depicted on the left panel). Shape changes and neuronal loss appear to be attenuated even with the lowest injected dose. Figures 8A and 8B show 4×1010vg injections 2 weeks after treatment. Figures 8E and 8F show injection of 2.6x1010vg 3 months after treatment. Figures 8I and 8J show injection of 2.6x109vg 3 months after treatment. Figures 8G and 8H show untreated naive βgal+ / - at the age of treated animals. Figures 8C and 8D show injection into naive βgal- / - at the age of treated animals. Figure 8K shows DCN injected with AAVrh8-mβgal. Arrows indicate injection site in βgal animals and location of photographs shown in this figure. Scale bar represents 100um. Images represent N>3 mice per group. [Figure 9]9A-9I show that intracranial injection of AAV in βgal+ / − and βgal− / − mice results in aberrant filipin staining within the region of maximal expression of the enzyme. Sagittal sections of mouse brain stained with Xgal for the presence of βgal enzyme and counterstained with Nuclear Fast Red 10 weeks after injection of representative (FIG. 9A) βgal+ / − mice (chimera+AAV) and (FIG. 9B) βgal− / − mice (knockout+AAV) and (FIG. 9C) untreated βgal− / − mice (untreated knockout) injected with 1 μl of AAVrh8-CBA-mβgal-WPRE bilaterally into the thalamus at 1.7×1012 vg / μl, 2 ul into the ventricles, and 0.3 ul into the deep cerebellar nuclei. Boxes indicate the location of the images depicted in FIGS. 9D-9I. Filipin staining was positive within the region of maximal expression of the enzyme in both chimera+AAV-injected animals (Figure 9D, G, and J; βgal+ / -) and βgal- / - knockout+AAV-injected animals (Figure 9E, H, and K). Figure 9F, I, and L show that filipin content was unchanged in untreated βgal- / - mice (untreated knockout). Filipin staining on brain sections taken at 10x magnification. Images represent N>3 mice per group. [Figure 10]10A shows that vector design changes result in reduced βgal protein presence and / or βgal enzyme activity in βgal+ / − mice. FIG. 10A shows βgal enzyme activity within the injected structures of 2 mm×2 mm biopsy punches. 1 μl of AAVrh8 vector (total dose of 3.4×109 vg or 2.0×1010 vg for high dose CB6 alone) or mock-treated PBS was injected bilaterally into the thalamus as determined by 4-MU assay approximately 6 weeks after injection. Enzyme activity was normalized to protein concentration by Bradford reagent and reported as nanomoles per hour per mg protein. Error bars represent mean + SD, N=3 per group, * indicates significant difference of βgal+ / −+AAV (chimera + vector name) versus untreated βgal+ / − (untreated chimera) or significant difference indicated by connecting line. P values ​​calculated using an unpaired multiple T test (Holm-Sydak), where *=p<0.05, **=p<0.01, and ***=p<0.001. FIG. 10B shows the presence of endogenous βgal protein as determined by Western blot, which appears at 67 kd, whereas expression of the transgene from the AAV vector appears as a high-weight band. The loading control is actin, which appears at 42 kd. Western blots shown represent N=3 per trial. [Figure 11]Figure 1 shows the presence of vector genomes within the injected structure of a biopsy punch in βgal+ / − mice. Number of vector genomes per diploid genome within the injected structure of a 2 mm x 2 mm biopsy punch when 1 μl of AAVrh8 vector (3.4 x 109 vg or 2.0 x 1010 vg total dose in high dose CBA only) was injected into the thalamus bilaterally in βgal+ / − mice (chimera + vector name) as determined by qPCR for SV40 polyA on the transgene. Samples were taken 6 weeks after injection. Error bars represent mean + SD, N = 3 per group, * indicates significant differences using unpaired multiple T-test (Holm-Sydac) indicated by connecting lines, where * = p < 0.05, ** = p < 0.01, and *** = p < 0.001. [Figure 12] Figure 12A, 12D, 12G, 12J, 12M, and 12P show coronal sections of mouse brains stained with Xgal for the presence of βgal enzyme and counterstained with Nuclear Fast Red 6 weeks after injection of representative βgal+ / - (chimera+vector name) or uninjected βgal+ / - (untreated chimera) injected bilaterally into the thalamus with 1 μl of AAVrh8 vector (3.4×109 vg or 2.0×1010 vg total dose in high dose CBA only). 12B, 12E, 12H, 12K, 12N, and 12Q show filipin staining in parallel sections of the same animals in 12A, 12D, 12G, 12J, 12M, and 12P. 12C, 12F, 12I, 12L, 12O, and 12R show ToPro3 nuclear dye in parallel sections of the same animals in 12A, 12D, 12G, 12J, 12M, and 12P. Boxes represent approximate locations of filipin and ToPro3 staining on brain sections taken at 20x magnification. Scale bar=250 μm. Images represent N≧2 mice per group. [Figure 13]Figure 13: Clustering heatmaps and Venn diagrams for all differentially expressed genes in βgal+ / - mice confirm transgene expression-dependent variation. Clustering heatmaps and Venn diagrams for all differentially expressed genes were generated using data from 2mm x 2mm biopsy punches of thalamus injected with 1μl AAVrh8 vector (total dose of 3.4x109vg) or mock-treated with PBS in both sides of βgal+ / - mice (chimera+vector name) at approximately 6 weeks post-injection. Microarray results were determined by Affymetrix Mouse Gene 2.0 ST, N=3 per group, P<0.05, >1.8-fold change. [Figure 14] 1 shows that βgal enzyme activity in the CNS of treated βgal- / - mice results in variable enzyme distribution across structures. βgal enzyme activity in the cerebrum, cerebellum + brainstem or spinal cord of βgal- / - mice or untreated (untreated knockout) or untreated βgal+ / + mice (untreated wild type) injected with 1 μl of AAVrh8 vector (total dose of 3.4×109 vg or 2.0×1010 vg in high dose CBA only) into the thalamus and 0.3 ul into the DCN, as determined by 4-MU assay, approximately 6 weeks after injection. Enzyme activity is normalized to protein concentration by Bradford reagent and reported as nanomoles per hour per mg of protein. Values ​​represent mean + SD, N = 3 per group, * indicates significant difference between βgal- / - + AAVrh8-CB6 vector (total dose of 3.4 x 109 vg; knockout + low dose CB6) versus βgal- / - + AAVrh8-CB6 vector (total dose of 2.0 x 1010 vg; knockout + high dose CB6). P values ​​calculated using unpaired T-test (Holm-Sydac), *** = p < 0.001. [Figure 15]FIG. 15: Xgal staining for the presence of β-gal enzyme in β-gal− / − mice confirms dose-dependent spread of expression of the enzyme throughout the brain. Sagittal sections of mouse brain stained with Xgal for β-gal enzyme activity and counterstained with Nuclear Fast Red approximately 6 weeks after injection of representative β-gal− / − or untreated (untreated knockout; FIG. 15E) or untreated β-gal+ / − mice (untreated chimera; FIG. 15F) injected bilaterally into the thalamus with 1 μl of AAVrh8 vector (3.4×109 vg or 2.0×1010 vg total dose for high dose CBA only) (FIGS. 15A-15C) and 0.3 ul into the DCN. N=2-3 per group. Scale bar=10 mm. FIG. 15D shows knockout mice treated with empty vector. [Figure 16] Figure 1 shows filipin staining for GM1 ganglioside content in βgal- / - mouse brains after treatment with therapeutic AAVrh8. Sagittal sections of mouse brains stained with filipin for GM1 content or nuclear dye ToPro3 (untreated knockout; ToPro3; descending) 6 weeks after injection of representative βgal- / - or untreated (untreated knockout) mice injected with 1 μl of AAVrh8 vector (3.4×109 vg or 2.0×1010 vg total dose in high dose CBA only) into the thalamus bilaterally and 0.3 ul into the DCN. N=2-3 per group. Images taken at 5x, scale=100mm. [Figure 17]Figure 17 shows filipin staining for GM1 content in the spinal cord of βgal− / − mice after therapeutic treatment with AAVrh8 vector. Cervical (FIG. 17A, FIG. 17C, FIG. 17E, FIG. 17G, FIG. 17I, and FIG. 17K) and thoracic (FIG. 17B, FIG. 17D, FIG. 17F, FIG. 17H, FIG. 17J, and FIG. 17L) sections of spinal cord stained with filipin for GM1 content or nuclear dye ToPro3 (untreated knockout; ToPro3; descending) 6 weeks after representative βgal− / − or untreated (untreated knockout) injected with 1 μl of AAVrh8 vector (3.4×109 vg or 2.0×1010 vg total dose in high dose CBA only) into the thalamus bilaterally and 0.3 ul into the DCN. N=2-3 per group. Image taken at 5x magnification, scale = 100mm. [Figure 18] 1 shows that therapeutic treatment with AAVrh8 results in normalization of GM1 content in the CNS of βgal- / - mice when treated with a high dose of a low-expressing promoter. GM1 content quantified by LC-MS / MS in the cerebrum, cerebellum + brainstem or spinal cord of βgal- / -, untreated (untreated knockout), or untreated βgal+ / + (untreated wild type) mice 6 weeks after injection with 1 μl of AAVrh8 vector (3.4×109 vg or 2.0×1010 vg total dose for high dose CBA group only) injected into the thalamus bilaterally and 0.3 ul injected into the DCN. GM1 content expressed as ng of GM1 per μg of protein. Values ​​represent mean + SD, N>3 per group, * indicates significant difference of knockout+AAVrh8 versus knockout+PBS using multiple T-test (Holm-Sydac) or significant difference indicated by connecting lines, where *=p<0.05, **=p<0.01, and ***=p<0.001. [Figure 19] FIG. 1 depicts the structure of the AAVrh8 vector. [Figure 20]FIG. 1 shows the vector map for 2140.8 pAAV-cb-mβgal. [Figure 21] FIG. 1 shows the structure of a monocistronic AAV vector encoding the HexA alpha and beta subunits. [Figure 22] Neuropathology in monkey thalamus. In FIG. 22A, mononuclear cell infiltration around blood vessels (arrows) and necrotic areas (*) within the white matter are seen. FIG. 22B shows a section stained with Luxol Fast Blue, where the arrows indicate one large area of ​​necrosis, vascular proliferation, and white matter loss (note the pale color compared to the adjacent dark blue). FIG. 22C shows a necrotic area (arrow) within the thalamus with vacuolization. FIG. 22D is an example (40x) of cellular infiltration into blood vessels within the thalamus. Similar neuropathology was seen with the lowest dose (1 / 30) (FIG. 22E). FIG. 22F shows normal thalamus in PBS-injected animals (1x dose: FIG. 22A-22D; 1 / 30 dose: FIG. 22E; PBS injection: FIG. 22F). [Diagram 23] Figure 1 shows hexosaminidase activity in the CNS. Hex activity was measured in the thalamus and thoracic spinal cord of NHPs injected with different doses of 1:1 formulations of AAVrh8 vectors encoding cynomolgus Hex-alpha and cynomolgus Hex-beta. In the biochemical assay, two artificial substrates, namely MUG and MUGS, were used. The first substrate is cleaved by all Hex isoenzymes (HexA, HexB, HexS), whereas the latter is cleaved only by HexA and HexS isoenzymes. [Figure 24] (A) Pre-necropsy brain MRI revealing edema in the thalamus (arrows) in NHPs injected with a 30-fold dose of AAVrh8 vector formulations encoding cynomolgus Hex-alpha and cynomolgus Hex-beta at pre-necropsy. No changes were observed in NHPs injected with the transgene-empty AAVrh8 vector. [Diagram 25]Figure 25 shows protein expression in monkey thalamus. Hex-β staining (green) is shown proximal (Fig. 25A) and distal (Fig. 25B) to the injection site in the thalamus of a 1x dose animal. Hex-α staining (green) is shown proximal (Fig. 25C) and distal (Fig. 25D) to the injection site in the thalamus of a 1 / 30 dose animal. H&E staining is shown proximal (Fig. 25E) and distal (Fig. 25F) to the injection site in the thalamus of a 1 / 30 dose animal shown in Figs. 25C-25D. Figure 25G shows a magnified view of the boxed area in Fig. 25E. Arrows point to eosinophilic material within neurons. [Figure 26] Figure 26 presents a panel of new AAV vectors with a gradient of predicted HexA expression levels. Figure 26A shows the systematic removal of expression elements from the latest version (top) to its most basic form (bottom vector) with ITR-flanked cDNA with polyadenylation signal and no classical promoter elements, while Figure 26B shows ITR expression-based vectors that progressively incorporate more elements known to increase ITR-driven gene expression. Abbreviations: CMV Enh: cytomegalovirus immediate early enhancer; CB: chicken beta-actin promoter; HexA / B: for each vector design, a pair of vectors carrying cynomolgus HexA and cynomolgus HexB cDNAs is generated; pA: polyadenylation signal; ITR: AAV2 inverted terminal repeat; P1: promoter 1, which is believed to increase ITR-mediated gene expression approximately 10-fold; P2: promoter that is believed to increase ITR-mediated gene expression approximately 50-fold. [Figure 27] FIG. 1 shows the plasmid map for the pAAV-cb-ci-cmHexA-2158 vector. [Figure 28] FIG. 1 shows the plasmid map for the pAAV-P2Int-cmHexA_2194 (modified) vector. [Figure 29]Figure 29 shows the gradient of hexosaminidase activity in transiently transfected 293T cells. Hexosaminidase activity was measured in cell lysates 72 hours after transfection using artificial substrates 4MUG (Figure 29A) and 4MUGS (Figure 29B), which are cleaved by all beta-hexosaminidase isoforms (HexA, HexB, and HexS), or only isoforms containing the alpha-subunit (HexA, HexS), respectively. [Diagram 30] FIG. 1 shows the change in body weight over the one-month study period. Error bars represent one standard deviation. [Diagram 31] FIG. 31 shows that mouse brains expressing the highest levels of Cynomolgus Hex protein contain eosinophilic neurons. H&E dye indicates the presence of neurons containing eosinophil granules in the hippocampus and thalamus, which correlates with the expression level of Cynomolgus Hex (40x magnification). Figures for mice of group 1 (FIG. 31A, 31K), group 2 (FIG. 31B, 31L), group 3 (FIG. 31C, 31M), group 4 (FIG. 31D, 31N), group 5 (FIG. 31E, 31O), group 6 (FIG. 31F, 31P), group 7 (FIG. 31G, 31Q), group 8 (FIG. 31H, 31R), group 9 (FIG. 31I, 31S), and group 10 (FIG. 31J, 31T). [Diagram 32] Figure 32 shows that expression of cynomolgus monkey Hexα in the brain of athymic nude mice varies among AAV vectors. Hexα staining (green); nuclei counterstained with DAPI (blue) in the thalamus and hippocampus of mice from group 1 (Figure 32A, 32K), group 2 (Figure 32B, 32L), group 3 (Figure 32C, 32M), group 4 (Figure 32D, 32N), group 5 (Figure 32E, 32O), group 6 (Figure 32F, 32P), group 7 (Figure 32G, 32Q), group 8 (Figure 32H, 32R), group 9 (Figure 32I, 32S), and group 10 (Figure 32J, 32T). Arrows indicate enzyme-positive cells. [Diagram 33]Figure 33 shows that reduced expression of Cynomolgus Hex protein leads to reduced microglial activation. Iba-1 staining indicates reduced inflammation in groups with reduced expression of Cynomolgus Hex protein in the hippocampus and thalamus (20x). Figures for Group 1 (Figure 33A, 33K), Group 2 (Figure 33B, 33L), Group 3 (Figure 33C, 33M), Group 4 (Figure 33D, 33N), Group 5 (Figure 33E, 33O), Group 6 (Figure 33F, 33P), Group 7 (Figure 33G, 33Q), Group 8 (Figure 33H, 33R), Group 9 (Figure 33I, 33S), and Group 10 (Figure 33J, 33T). [Diagram 34] Figure 34 shows that the reduction of Cynomolgus Hex protein expression leads to the reduction of reactive astrogliosis. GFAP staining indicates the reduction of inflammation in the hippocampus and thalamus in the group with reduced Cynomolgus Hex protein expression (20x). Figures for Group 1 (Figure 34A, 34K), Group 2 (Figure 34B, 34L), Group 3 (Figure 34C, 34M), Group 4 (Figure 34D, 34N), Group 5 (Figure 34E, 34O), Group 6 (Figure 34F, 34P), Group 7 (Figure 34G, 34Q), Group 8 (Figure 34H, 34R), Group 9 (Figure 34I, 34S), and Group 10 (Figure 34J, 34T). [Diagram 35] FIG. 1 shows new AAV vectors that were selected for further testing in non-human primates. [Diagram 36] Figure 1 shows relative total Hex activity in athymic nude mice intracranially injected with AAV vectors encoding cynomolgus Hexα and cynomolgus Hexβ. Enzyme activity of HexB, HexA, and HexS in vitro (C1: olfactory bulb and first 3 mm coronal slice of cerebrum, C2: subsequent 2 mm coronal slice of cerebrum, C3: subsequent 3 mm coronal slice of cerebrum containing injection site, C4: subsequent 2 mm coronal slice of cerebrum, CRBL: cerebellum, BS: brainstem, SC: spinal cord) measured by MUG substrate and normalized to naive. [Figure 37]Figure 1 shows relative HexA and HexS activity in athymic nude mice intracranially injected with AAV vectors encoding cynomolgus Hexα and cynomolgus Hexβ. HexA and HexS enzyme activity in vitro (C1: olfactory bulb and first 3 mm coronal slice of cerebrum, C2: subsequent 2 mm coronal slice of cerebrum, C3: subsequent 3 mm coronal slice of cerebrum containing injection site, C4: subsequent 2 mm coronal slice of cerebrum, CRBL: cerebellum, BS: brainstem, SC: spinal cord) measured by MUGS substrate and normalized to naive. [Figure 38] MRI analysis of targeting and distribution after intraparenchymal injection. Figure 38A shows pre- and post-operative intracerebral MRI using different sequences. The AAVrh8 vector formulation contains 2 mM gadolinium to analyze the distribution of the injected solution using T1-weighted MRI; the volume of distribution of gadolinium (Gd-enhanced volume) in this animal was 1.67 mL. Figure 38B shows the volume of distribution (Vd) and mean + SD for each NHP; the total injected volume (Vi) in the thalamus was 0.3 mL. [Figure 39] Figure 1 shows intracerebral MRI of AAVrh8-injected NHPs over the course of the 90-day experiment. Arrows indicate areas of hyperintense signal within the thalamus in three NHPs. [Diagram 40] Figure 40 shows brain MRI hyperintensities detectable in the thalamus of some AAVrh8-injected NHPs 90 days after injection. One NHP in cohort 1 (Figure 40A) showed a large hyperintensity in the left thalamus at the 90 day imaging time point, which was undetectable at earlier imaging time points. Figure 40B shows an NHP in cohort 2 in which bilateral hyperintense signals were detected beginning at day 30 and remained unchanged through day 90. In Figure 40C, an NHP in cohort 3 showed a hyperintense signal in the left thalamus from days 30 to 90. [Diagram 41]Figure 1. Biopsy sampling of coronal brain sections. Higher than normal hexosaminidase was measured in the right (R) or left (L) thalamus, in dorsal (D) and ventral (V) samples. [Diagram 42] FIG. 1 shows neuropathological findings in the brains of Cohort 1 NHPs, including intraneuronal accumulation of eosinophil granules, neurodegeneration, and neuronal erosion. [Diagram 43] Figure 43 shows severe focal cavernostomy with perivascular cellular infiltration in the left thalamus of one monkey injected with AAVrh8 in cohort 1. Figure 43A was taken at 4x magnification; Figure 43B was taken at 10x magnification. [Diagram 44] Figure 44: Observations of neuropathology in monkeys from cohort 3. Neurodegeneration was rare in these monkeys (Figure 44A), although monkey 295709 showed evidence of perivascular lattice cells, likely related to the injection route (Figure 44B). [Diagram 45] FIG. 1 shows the assessment of intraneuronal accumulation of eosinophilic material in the right and left thalamus of monkeys in cohorts 1 and 3. [Figure 46] FIG. 1 shows the assessment of neurodegeneration and necrosis in the right and left thalamus of monkeys in cohorts 1 and 3. [Figure 47] FIG. 1 shows inflammation ratings in the right and left thalamus of monkeys in cohorts 1 and 3. [Figure 48]Figure 1 shows that intracranial injection of AAVrh8-cmHex vector results in increased Hex expression and reduced GM2 ganglioside content in the brain of Sandhoff mice. Six to eight week old Sandhoff mice received intracranial injections of PBS (n=1; purple bar; n=1), AAVrh8-CBA-cmHex-W (green bar (A), n=3, 4.68x109 vg), AAVrh8-CBA-cmHex (orange bar (B), n=3, 4.68x109 vg), or AAVrh8-CB-I-cmHex (blue bar (C), 4.68x109 vg: n=3; C (5x dose): 2.34x1010 vg: n=4) vectors. Brains were harvested 4 weeks after injection and divided into 2-3 mm coronal blocks as shown in the schematic above; vertical arrows indicate the injection sites. In Figures 48A-48C, total Hex enzyme activity (HexA, HexB, and HexS) was measured using an artificial substrate, MUG, and expressed as fold over wild-type levels (WT, black bars, n=1). Asterisks (*) represent animals with no detectable Hex activity. Figures 48D and 48F are bar graphs for the qualitative LC-MS / MS used to measure GM2 ganglioside content. [Figure 49-1] 49A shows a structural rendering of the promoters described by the present disclosure, and FIG 49B shows a structural rendering of the CB promoter, represented by SEQ ID NO:1. [Figure 49-2] 49A-49C show structural renderings of the promoters described in the present disclosure, and FIG 49B shows structural renderings of the CB(6)-I promoter, represented by SEQ ID NO:4. [Figure 49-3] 49A-49C show structural renderings for the promoters described in the present disclosure. [Figure 50] FIG. 1 shows a schematic representation of the dual promoter AAV9-Syn1-GFP-2×miRSO D1 / GFAP-2×miRSOD1-mCherry vector. [Figure 51]Figure 1 shows the transduction profile for the dual promoter AAV9-Syn1-GFP-2xmiRSOD1 / GFAP-2xmiRSOD1-mCherry after intrastriatal injection in adult SOD1G93A mice. The AAV9 vector contained GFP driven by the intraneuronal synapsin 1 promoter and mCherry driven by the astrocyte GFAP promoter. The shapes of GFP (green) and mCherry (red) expressing cells match those of neurons and astrocytes, respectively. [Figure 52] Figure 1 shows that human SOD1 mRNA levels are reduced in adult SOD1G93A mice after injection of the dual promoter AAV9-Syn1-GFP-2xmiRSO D1 / GFAP-2xmiRSOD1-mCherry vector. The artificial microRNA (miR) targets human SOD1. After intravenous injection of 1x1012 total vector genomes, we observed a maximum 25% reduction in human SOD1 mRNA expression in the spinal cord. After direct intrastriatal injection of 8x109 total vector genomes, we observed a maximum 55% reduction in human SOD1 mRNA expression in the striatum. [Diagram 53] Figure 53 shows that intracranial treatment with AAVrh8-mHEXA / B extends the life span of Sandhoff disease (SD) mice and results in the preservation of motor function. SD mice treated with 4.68x109vg of AAVrh8-mHexA / B vector formulations via TH / ICV or TH / DCN delivery were assessed for performance in (Figure 53A) the inversion screen test, (Figure 53B) the rotarod test, and (Figure 53C) the wire hanging test at 60, 120, and 180 days of age. FIG. 53D shows Kaplan-Meier survival plots for SD mice treated intracranially with 4.68×109 vg of AAVrh8-mHexA / B, showing that intracranial treatment in combination with bilateral intrathalamic (TH) injections, bilateral deep cerebellar nuclei (DCN) injections, or a single intracerebroventricular (ICV) injection results in comparable survival outcomes. [Figure 54]Figure 54 shows that intracranial AAVrh8-mHexA / B treatment in SD mice results in dose-dependent extension of life span and improves motor function. Figure 54A shows Kaplan-Meier survival plots for a dose-escalation experiment of AAVrh8-mHexA / B injected into TH / ICV intracranially in 1-month-old SD mice. SD mice intracranially treated with 4.68x109vg and 1.17x1010vg AAVrh8-mHexA / B vector formulations via TH / ICV delivery were assessed for performance in (Figure 54B) the inversion screen test, (Figure 54C) the rotarod test, and (Figure 54D) the wire hanging test at 60, 120, and 180 days of age. [Figure 55] Figure 55: Intracranial delivery of AAVrh8-mHexA / B has minimal effect on motor performance in normal mice. Heterozygous (HexB+ / -, HZ) mice treated with intracranial injection of 4.68x109vg of AAVrh8-mHexA / B via TH / ICV or TH / DCN delivery were assessed for performance at 60, 120, and 180 days of age in (Figure 55A) the reversal screen test, (Figure 55B) the rotarod test, and (Figure 55C) the hanging wire test. [Figure 56] Figure 56 shows that intracranial delivery of AAVrh8-mHexA / B treatment reduces GM2 ganglioside accumulation and results in widespread hexosaminidase expression in the central nervous system of SD mice. SD mice were treated with TH / ICV injection of 1.17x1010vg of AAVrh8-mHexA / B and neurochemistry was assessed at the same age as the humane endpoint of untreated SD mice. The cerebrum, cerebellum, and spinal cord were analyzed for (Figure 56A) GM2 ganglioside content, and (Figure 56B) hexosaminidase activity using two artificial substrates: MUG (cleaved by HexA, HexB, and HexS isozymes), and MUGS substrate (cleaved by HexA and HexB isozymes). [Figure 57]Figure 57 shows survival and biodistribution of AAV in SD cats after TH injection + ICV injection. Figure 57A shows that survival of untreated SD cats is 4.4 ± 0.6 months. Figure 57B shows that the onset of clinical disease is delayed and clinical signs are alleviated in SD cats after AAV treatment. Figure 57C shows a crude image of the brain showing the location of the section (left). Coronal sections of the right hemisphere of the brain and spinal cord in situ illustrating the location analyzed. Figure 57D shows naphthol staining for histochemistry showing the distribution of Hex in normal cats, SD cats, and representative cats treated with AAV. Figure 57E shows HexA enzyme activity in the brain and spinal cord of SD cats. [Figure 58] Figure 58 shows biomarkers for therapeutic efficacy of AAV-mediated gene therapy. Figure 58A shows 7T MRI of SD cat brain showing increased gray and white matter intensity due to increased demyelination in white matter and increased GM2 accumulation in gray matter. After AAV gene therapy TH / ICV delivery, there is complete normalization of gray and white matter intensity. Mild cortical atrophy is evident as the injection site in the thalamus is hyperintense. Figure 58B is MR spectroscopy of an untreated SD cat showing increased N-acetylhexosamine, a toxic metabolite previously reported in SD patients. After AAV gene therapy TH / ICV delivery, this is completely normalized. Figure 58C is an MR spectrum for a representative SD. Figure 58D is HexA activity in cerebrospinal fluid (CSF) in SD+AAV TH / ICV SD cats. Figures 58E and 58F are lactate dehydrogenase (Figure 58E) and aspartate aminotransferase (Figure 58F) levels in the CSF of normal (open circles), SD (closed circles), and SD+AAVTH / ICV cats (shaded circles). Figures 58G and 58H are serum antibody titers of SD+AAVTH / ICV cats 11-907 (Figure 58G) and 7-760 (Figure 58H). [Figure 59]Figure 59 shows the clinical therapeutic effect and biodistribution in SD+intracisternal (CM) AAV cats. Figure 59A is a Kaplan-Meier curve showing survival of 4.4±0.6 months in untreated SD cats. SD cats treated with AAV via the cisterna magna extended survival to 9.9±0.5 months. Figure 59B is a clinical assessment score showing the rapid decline in untreated SD cats. In SD+AAV cats, the decline was mitigated with a long plateau phenotype. The cat was euthanized before loss of ambulation due to visual and hearing loss as well as musculoskeletal abnormalities. The clinical assessment score was based on the following clinical signs: mild tremor, obvious tremor, hind limb weakness, drunken gait, ataxia, accidental falls, limited ambulation, front leg spasms, hind leg spasms, and inability to walk, with a normal score of 10 and 1 point subtracted for each symptom acquisition. Figure 59C shows a cat brain (left) with lines indicating the location of the brain section, right hemisphere with brain and spinal cord en bloc illustrating the location for biodistribution analysis. Figure 59D shows HexA activity in the brain and spinal cord of SD+AAV long-term cats (MUGS). The thick line at 1 indicates normal levels. Figures 59E and 59F show the expression of HexA in various brain regions as measured by qPCR. [Figure 60] Figure 1 shows accumulation clearance in SD+AAV cisterna magna (CM) cats. Representative periodic acid Schiff (PAS) staining for normal cats (left), SD cats (middle), and SD+AAV CM chronically treated cats. Normal cats show darker PAS staining in the white matter of the cerebrum and cerebellum than in the gray matter, and the reverse is true for the spinal cord. SD cats show darker PAS staining in the gray matter than in the white matter, with areas of loss of staining in the white matter (especially evident in the spinal cord). SD+AAV cats treated by injection via the cisterna magna showed increased accumulation and demyelination in the cerebral cortex, with normalization in the cerebellum, brainstem, and spinal cord. [Figure 61]Figure 1 shows normalization of tissue pathology after AAV gene therapy. SD cats exhibit degenerated neurons in the microgyrus, DCN, and brainstem. After 16 weeks of AAV gene therapy, SD cats had normalized morphology in all these regions, which was preserved in long-term AAV-treated animals, with the exception of Purkinje cell degeneration. Interestingly, within the dentate nucleus, a subset of neurons was found with grape-shaped eosinophilic inclusions (inset; scale bar: 5 μm). Scale bar: 10 μm. [Figure 62] Biomarkers of disease progression and improvement in SD+AAV CM cats. FIG. 62A is a 7T MRI of an SD cat showing cortical atrophy and hyperintense white matter and hypointense gray matter consistent with white matter demyelination and lipid accumulation in gray matter compared to normal cats. In SD+AAV CM cats, the intensity changes persisted, but cortical atrophy was ameliorated. MR spectroscopy of the thalamus of SD cats shows an increase in N-acetylhexosamine after 16 weeks of treatment, which is corrected by CM delivery of AAV. In one CM cat at the humane endpoint, NA Hex levels were further elevated. FIG. 62B shows that myoinositol (INS), a marker for gliosis, and glycerophosphocholine + phosphocholine (GPC+PCh), a marker for demyelination, were also increased in SD+AAV CM cats. FIG. 62C shows a representative MR spectrum. Figure 62D shows HexA in CSF in SD+AAV CM cats over time. Normal levels are indicated by dashed lines. Figures 62E-62H show aspartate aminotransferase (AST) activity, a marker of cytotoxicity, in CSF in normal cats (open circles), SD cats (filled circles), and SD+AAV CM cats (light filled circles). [Figure 63]63A shows that intracranial injection of AAVrh8-CB-CI-mHexA / B vector reduces the accumulation of GM2 ganglioside in the CNS while increasing hexosaminidase activity, improving survival of SD mice. SD mice received intracranial injection of AAVrh8-CH-CI-mHexA / B vector, or parent vector (4.68×109 vg), or PBS at a total dose of 4.68×1010 vg or 2.34×1010 vg. FIG. 63A shows the GM2 ganglioside content measured by LC-MS / MS 8 weeks after CNS injection. FIG. 63B shows the hexosaminidase activity in the cerebrum measured using MUG substrate 8 weeks after injection. The dashed line indicates the average activity of untreated wild-type animals. Results are shown as mean ± SD by Tukey's multiple comparison test, * (P ≤ 0.05), ** (P ≤ 0.01), *** (P ≤ 0.0001), n ​​= 4 for PBS, n = 4 for original CBA-mHexA / B, n = 6 for 2.34 x 1010 vg CB-CI-mHexA / B and 4.68 x 109 vg CB-CI-mHexA / B, nd (not detected). Figure 63C shows the survival and symptoms of SD mice (n = 8) injected with 2.34 x 1010 vg AAVrh8-CB-CI-mHexA / B (study endpoint 5 months). Median survival of untreated Sandhoff disease mice was 125.5 days (n = 6). [Figure 64] Figure 64 shows that increasing doses of AAVrh8-CB-CI-mHexA / B vector delivered to the CSF further reduces accumulation of GM2 ganglioside in the spinal cord and cerebellum. SD mice were injected with 1.17x1010vg of AAVrh8-CB-CI-mHexA / B in increasing CSF doses (1.17x1010vg, 2.34x1010vg, 5.85x1010vg, and 5.5x1011vg) into the thalamus. Four weeks after injection, GM2 ganglioside content was measured in the spinal cord (Figure 64A) and cerebellum (Figure 64B) by LC-MS / MS. Results are shown as mean ± SD, *(P≦0.05), **(P≦0.01), ***(P≦0.0001), n=3, by Dunnett's multiple comparison test. [Figure 65] 1 shows an example of the structure of an AAV-HexA vector genome and an AAV-HexB vector genome. [Figure 66] FIG. 1 shows the plasmid map of the pAAV-CB-ci-hHexA(v2) vector. [Figure 67] FIG. 1 shows the plasmid of pAAV-CB-ci-hHexB vector. [Figure 68] FIG. 1 shows HexA enzyme activity in serum and CSF before and after administration of rAAVrh8-HexA / B to a single subject with advanced Tay-Sachs disease. [Figure 69] FIG. 1 shows pre- and post-treatment MRI scans showing white matter within the frontal and parietal lobes compared to baseline MRI (approximately 2-year difference). [Figure 70] FIG. 1 shows the CD19 and CD20 counts of subjects after receiving a single dose of rituximab, and the IgG counts of subjects after IVIg infusion. [Figure 71] FIG. 1 shows a reduction of GM2 ganglioside in cerebrospinal fluid (CSF) by approximately 25% from baseline. [Figure 72] FIG. 1 shows Western blot staining for HEXA, HEXB, and TTR on day 0 and after 3 months of treatment. [Figure 73] FIG. 1 shows the treatment design of the clinical study. [Figure 74-1] FIG. 74A shows an example of catheter-based administration of AAVrh8 vectors at the cisterna magna and lumbar L2 level. [Figure 74-2] FIG. 74B shows an example of catheter-based administration of AAVrh8 vectors at the cisterna magna and lumbar L2 level. [Figure 74-3] FIG. 74C shows an example of catheter-based administration of AAVrh8 vectors at the cisterna magna and lumbar L2 level. [Figure 75-1]

[0013] Figure 75 shows one embodiment of a method for rAAV administration via a fluorescence microscope-guided intravascular microcatheter into the cisterna magna of a sheep. Figure 75A shows the fluorescence microscope-guided insertion of the intravascular microcatheter. Figure 75B shows the final placement of the microcatheter tip. [Figure 75-2] Figure 75C shows one embodiment of a method for rAAV administration via a fluorescent microscope-guided intravascular microcatheter into the cisterna magna of sheep. [Figure 75-3] FIG. 75D shows one embodiment of a method for rAAV administration via a fluorescence microscope-guided intravascular microcatheter into the cisterna magna of sheep. FIG. 75D shows GFP expression in spinal cord cross sections by immunohistochemistry. [Figure 75-4] FIG. 75D shows one embodiment of a method for rAAV administration via a fluorescence microscope-guided intravascular microcatheter into the cisterna magna of sheep. FIG. 75E shows GFP expression in brain sections by immunohistochemistry. [Figure 75-5] FIG. 75F shows one embodiment of a method for rAAV administration via a fluorescence microscope-guided intravascular microcatheter into the cisterna magna of sheep. FIG. 75F shows GFP expression in brain sections by immunohistochemistry. [Figure 75-6] FIG. 75G shows one embodiment of a method for rAAV administration via a fluorescence microscope-guided intravascular microcatheter into the cisterna magna of sheep. FIG. 75G shows GFP expression in brain sections by immunohistochemistry. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] In some cases, transgene delivery by current rAAV vectors requires the delivery of non-degradable substrates. accumulation of lysosomal proteins or dysregulation of the lysosomal compartment, which Responses that upregulate endothelial biogenesis, reduce substrates, or result in exocytosis Furthermore, physiological levels of transgene expression driven by rAAV vectors were observed. The introduction of therapeutic proteins that outperform the fold changes common in these disorders has also been shown to be It may also trigger deleterious protective cascades that may be associated with the response of unprotected proteins. .

[0020] Thus, in some aspects, the present disclosure provides methods for detecting lysosomal enzymes in tissues, such as CNS tissues. Compositions for expression of the gene (e.g., isolated nucleic acids, rAAV, rAAV vectors, etc.) are presented. In some aspects, the present disclosure provides a method for the transfection of GM1 gas using the rAAV described herein. Lysosomal storage disorders, such as glioside sphincterosis, Tay-Sachs disease, or Sandhoff disease The present disclosure relates to methods for treating therapeutically effective, but previously unused, The transgene was expressed at levels that did not cause vector-mediated genotoxicity associated with rAAV. Certain regulatory sequences and regulatory enzymes have been identified for use in rAAV to effect expression of genes. This is based, in part, on the discovery that components, such as promoter regions, can be manipulated.

[0021] Isolated Nucleic Acids In some aspects, the present disclosure provides a method for the preparation of a nucleic acid sequence comprising the steps of: The transgene contains a lysosomal storage disorder-associated protein ( For example, lysosomal storage proteins, such as HexA and / or HexB, Nucleic acids encoding the GLB1 and GLB2 are presented.

[0022] In some embodiments, the GLB1 transgene is the human GLB1 gene (GeneID The human GLB1 gene is located at NM_000404.4, NM_0010 79811.2, NM_001135602.2, or NM_001317040.1 The human GLB1 gene may comprise the nucleotide sequence of The human β-galactosidase protein is encoded by NP_000395.3. In NP_001073279.1, NP_001129074.1, or NP_00 In some embodiments, human GLB1 may comprise an amino acid sequence within 1303969.1. In some embodiments, the human HexA transactivator comprises a sequence as in SEQ ID NO: 23. The gene (GeneID:3073) is in NM_000520.5 or NM_001 In some embodiments, the HexA protein comprises a nucleotide sequence within 318825.1. The quality is based on NCBI reference sequence: NP_000511.2 (SEQ ID NO: 20) or NP_0 In some embodiments, the human He The xB transgene (GeneID:3074) is located in NM_000521.4 or NM_001292004.1. The xB protein is shown in NCBI Reference SEQ ID NO: NP_000512.1 (SEQ ID NO: 21) or the sequence set forth in NP_001278933.1.

[0023] The transgenes encoding proteins associated with lysosomal storage disorders were As used herein, an "operably linker" refers to a It refers to a promoter that is linked to a downstream transgene and drives its expression. In some embodiments, the promoter is a constitutive promoter, such as chicken beta-actin ( CBA) promoter, retroviral Rous sarcoma virus (RSV) LTR promoter activator (optionally with RSV enhancer), cytomegalovirus (CMV) promoter Motor (optionally with a CMV enhancer) [e.g., Boshart et al., Cell, 41:521-530 (1985)], SV40 promoter, dihydrofolate reductase β-actin promoter, phosphoglycerol kinase (PGK) promoter The promoters are the EF1α promoter [Invitrogen] and the EF1α promoter [Invitrogen]. In one embodiment, the promoter is an enhanced chicken β-actin promoter. In some embodiments, the promoter is a U6 promoter. The beta-actin promoter comprises the sequence set forth in SEQ ID NO:22.

[0024] In some embodiments, the promoter is an inducible promoter. - allows the regulation of gene expression and is regulated by exogenously supplied compounds, environmental factors such as temperature, or specific physiological states, such as the presence of an acute phase, a cell-specific differentiation state, or replication Inducible promoters and induction systems can be regulated by physiological conditions specific to the cell. including, without limitation, Invitrogen, Clontech, and Ariad. Many other systems have been described and are available in the art. The promoter can be easily selected by the person skilled in the art. Examples of inducible promoters include the zinc-inducible sheep metallothionein (MT) promoter, Dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, T 7 polymerase promoter system (WO98 / 10088); ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), tetracycline Inhibitory system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), tetra The cyclin inducible system (Gossen et al., Science, 268:1766-1769 (1995); see also Harvey et al. al., Curr. Opin. Chem. Biol., 2:512-518 (1998)), RU486-inducible system (Wang et al., Nat. Biotech., 15:239-243 (1997); and Wang et al., Gene Ther., 4:432-441 (1997)), and the rapamycin-inducible system (Magari et al., J. Clin. Invest., 1 00:2865-2872 (1997)). Still other types of inducible promoters that may be useful in this context include Promoters are regulated by specific physiological conditions, e.g., temperature, the presence of an acute phase, the differentiation state of a particular cell, or inducible promoters that are regulated by physiological conditions that are exclusive to replicating cells. be.

[0025] In another embodiment, the transgene (e.g., GLB1, HEXA, or HEXB) The native promoter will be used for the transgene. This may be preferred when expression of the gene is desired to mimic the native expression. The promoter of the present invention is capable of directing transgene expression in a temporal, developmental, or tissue-specific manner. can be used when a gene must be regulated differentially or in response to a specific transcriptional stimulus. In further embodiments, an enhancer element, a polyadenylation site, or a Koz Other native expression control elements, such as the ak consensus sequence, may also be used to mimic native expression. It can be used to

[0026] In some embodiments, the promoter drives expression of the transgene in neural tissue. In some embodiments, the present disclosure provides a tissue-specific The transgene includes a promoter and encodes a lysosomal storage disease protein. As used herein, a "tissue-specific promoter" refers to a promoter that is specific to a particular tissue. Preferentially regulate gene expression in a cell type relative to gene expression in other cell types (e.g. Cell type specific promoters refer to promoters that regulate, drive or upregulate the central nervous system. Central nervous system (CNS) cells, liver cells (e.g., hepatocytes), heart cells, kidney cells, eye cells, muscle The promoter may be specific for any cell type, such as the human synapsin 1 promoter ( The Syn1 promoter preferentially drives gene expression in neurons and is expressed by the GfaAB The C1D (also called GFAP) promoter directs expression preferentially in astrocytes. However, it is understood that some cell types may be resident within a particular type of tissue. For example, central nervous system tissues contain a wide variety of cells, including neuronal and non-neuronal cells (e.g., glial cells, astrocytes, etc.

[0027] In some embodiments, the isolated nucleic acid is a nucleic acid sequence that identifies a protein associated with a lysosomal storage disorder, e.g., The transgene comprises at least two transgenes encoding a chromosome 1 (e.g., HEXA and HEXB). The first transgene and the second transgene each include at least one promoter. In some embodiments, the first transgene can be operably linked to The second transgene encodes HEXB. and HEXB transgenes may be operably linked to the same promoter. , a first promoter, and a second promoter. The first promoter and / or the second promoter may be a constitutive (e.g., CBA) promoter. Alternatively, the first promoter and / or the second promoter The first promoter and / or the second promoter may be an inducible promoter. The promoter may be a tissue-specific promoter. The promoter is neuron specific and optionally includes the synapsin 1 promoter (S In some embodiments, the Syn1 promoter is represented by SEQ ID NO: 13. In some embodiments, the second promoter is specific for astrocytes. and optionally a GFAP promoter. The vector is represented by SEQ ID NO:14.

[0028] A further example of a tissue-specific promoter is the liver-specific thyroxine-binding globulin promoter. (TBG) promoter, insulin promoter, glucagon promoter, somatostatin tatin promoter, pancreatic polypeptide (PPY) promoter, synapsin 1 (Syn 1) Promoter, creatine kinase (MCK) promoter, mammalian desmin (D ES promoter, α-myosin heavy chain (a-MHC) promoter, or cardiac troponin (CTP) promoter. Other exemplary promoters include, but are not limited to, the cTnT promoter. The promoter may be, for example, the beta-actin promoter, among other promoters that will be apparent to those skilled in the art. , Hepatitis B virus core promoter (Sandig et al., Gene Ther., 3:1002-9 (1996)) alpha-fetoprotein (AFP) promoter (Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996)), the bone osteoclast promoter (Stein et al., Mol. Biology ol. Rep., 24:185-96 (1997)); bone sialoprotein promoter (Chen et al., J. Bo ne Miner. Res., 11:654-64 (1996)), CD2 promoter (Hansal et al., J. Immuno I., 161:1063-8 (1998)); immunoglobulin heavy chain promoter; T cell receptor α chain promoter , the neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)), Lament light chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88: 5611-5 (1991)), and the neuron-specific vgf gene promoter (Piccioli et al. , Neuron, 15:373-84 (1995)).

[0029] In some aspects, the present disclosure provides a method for the production of a nucleic acid sequence that is operably linked to a promoter via a chimeric intron. The ligated transgene (e.g., lysosomal storage protein, e.g., GLB1, H HexA, HexB, and / or HexA and HexB) In some embodiments, the chimeric intron is derived from the chicken beta-actin gene. Nucleic acid sequences that are non-coding, such as those derived from intron 1 of the chicken beta-actin gene. In some embodiments, the intron sequence of the chicken beta-actin gene is The intron sequence is about 50 to about 150 nucleotides in length (e.g., 50 to 150 nucleotides in length, including the end points). In some embodiments, the length of the chicken bait is in the range of 100-150 nucleotides. The intron sequence of the actin gene is about 100 to about 120 (for example, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, or 120) In some embodiments, the chimeric introns are one or more is placed between multiple non-translated sequences (e.g., a promoter sequence and a chimeric intron sequence) a first non-translated sequence, and / or a chimeric intron, and a transgene sequence, In some embodiments, one or more Each of the numerous untranslated sequences is identical to that found in the rabbit beta-globulin gene (e.g. For example, exon 1 and exon 2 of rabbit beta-globulin These are non-coding sequences derived from the ribosomal RNA (non-translated sequences which arise from the ribosomal RNA).

[0030] Recombinant AAV The isolated nucleic acid of the present disclosure can be a recombinant adeno-associated virus (rAAV). In one embodiment, the isolated nucleic acid described by the present disclosure is a first adeno-associated virus (AAV) reverse transcriptase. A region (e.g., the first region) containing an ITR or a variant thereof and a Transgenes associated with esosome storage disorders (e.g., GLB1, HEXA, and / or or HEXB), and a second region encoding the isolated nucleic acid (e.g., a recombinant AAV vector). The vector may be packaged into a capsid protein and administered to a subject. The transgene may also be inserted into and / or delivered to selected target cells. , e.g., protein and / or expression control, as described elsewhere in this disclosure. It may also include a region encoding sequences (eg, a polyA tail).

[0031] The present disclosure provides a vector that contains a single, cis-acting wild-type ITR. In some embodiments, the ITR is a 5' ITR. In some embodiments, the ITR is a 3' ITR. Generally, the ITR sequence is about 145 bp in length. Preferably, within the molecule, Substantially the entire sequence encoding the ITR(s) may be used, although references to these sequences may be made without limitation. However, some degree of minor modification is also tolerated. The ability to modify ITR sequences is within the skill of the art. (e.g., Sambrook et al., "Molecular Cloning. A Laboratory Manual ”, 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al. (See textbooks such as, for example, I., J. Virol., 70:520 532 (1996). For example, ITR is The vector ends where the TR is mutated, resulting in the formation of self-complementary AAV At its TR (terminal resolution) site, it inhibits replication in Another example of such a molecule that is utilized in the present disclosure is a trans A "cis-acting" plasmid containing the transgene of choice, in this case The gene sequence and associated regulatory elements are represented by the 5' AAV ITR sequences and the 3' and the apin-forming RNA sequence. The AAV ITR sequences are identified herein. It may be obtained from any known AAV, including mammalian AAV types. TR sequences are AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and AAV8. , AAVrh8, AAV9, AAV10, and / or AAVrh10 ITR sequences be.

[0032] The isolated nucleic acid and / or isolated rAAV of the present disclosure may be Modified and / or modified to enhance targeting of V to a target tissue (e.g., CNS). Non-limiting methods of modification and / or selection include modifying the AAV capsid serotype. (e.g., AAV8, AAV9), tissue-specific promoters (e.g., Syn1, GFA In some embodiments, the isolated nucleic acid and and rAAVs have been shown to have enhanced targeting to CNS tissues (e.g., AAV8, AAV9). In some embodiments, the isolated nucleic acids and rAAV capsid serotypes of the present disclosure include The AV includes a tissue-specific promoter (e.g., Syn1, GFAP). In certain embodiments, the isolated nucleic acids and rAAVs of the present disclosure are directed to CNS tissues and tissue-specific promoters. and AAV capsid serotypes that have enhanced targeting to the host.

[0033] In some aspects, the present disclosure provides isolated AAV. As used herein, the term "isolated" refers to an AAV that is artificially obtained or produced. The isolated AAV may be produced using recombinant methods. V is referred to as a "recombinant AAV." Recombinant AAV (rAAV) is preferably The V transgene is specifically delivered to one or more predetermined tissue(s). The AAV capsid has tissue-specific targeting ability, as shown in Fig. Therefore, targeting is an important factor in determining the targeting ability of a drug. In some embodiments, an rAAV can be selected that has a capsid appropriate for the tissue in which it is to be delivered. , rAAV, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV 7. AAV8, AAVrh8, AAV9, AAV10, or AAVrh10 capsid The term includes proteins having substantial homology thereto, including those of the In one embodiment, the rAAV comprises an AAVrh8 capsid protein.

[0034] In some embodiments, the rAAV of the present disclosure is a pseudotyped rAAV. The virus or viral vector is then combined with a foreign viral envelope protein. The result is a pseudotyped virus particle. Envelope proteins may alter host tropism or increase the stability of virus particles. In some embodiments, pseudotyped rAAVs can be used to increase / decrease the expression of two or more In one embodiment, the nucleic acid from one AAV is At least one other AAV nucleic acid encoding a capsid protein is also included in the other virus. In some embodiments, the pseudotyped rRNA encodes a rRNA protein and / or a viral genome. AAV consists of the inverted terminal repeats (ITRs) of one AAV serotype and the inverted terminal repeats (ITRs) of a different AAV serotype. For example, the term refers to an AAV that contains the capsid proteins of serotype Y together with the proteins of serotype Y. A pseudotyped AAV vector containing the serotype X ITRs encapsulated in the vector was designated AAVX / Y. (e.g., AAV2 / 1 is the AAV2 ITR and the AAV1 capsid. In some embodiments, the pseudotyped rAAV is derived from one AAV serotype. The tissue-specific targeting ability of the capsid protein was examined using a virus derived from another AAV serotype. This allows for the targeting of the transgene to the target tissue. It may be useful to enable targeted delivery of

[0035] The art provides methods for obtaining recombinant AAV with desired capsid proteins. are well known (e.g., the contents of which are incorporated herein by reference in their entirety). (See U.S. Patent Application Publication No. 2003 / 0138772 for examples of such methods.) Typically, The method includes: a nucleic acid sequence encoding an AAV capsid protein or a fragment thereof; Gene; a recombinant gene consisting of an AAV inverted terminal repeat (ITR) and a transgene. AAV vectors; and packaging of recombinant AAV vectors into AAV capsid proteins. Culturing the host cells containing sufficient helper functions to allow for cloning. Typically, the capsid protein is a structure encoded by the cap gene of AAV. In some embodiments, the AAV is a recombinant protein, all of which are alternative splice proteins. Three capsid proteins are transcribed from a single cap gene via isoforms It contains virion proteins 1 to 3 (designated VP1, VP2, and VP3), which are In an embodiment, the molecular weights of VP1, VP2, and VP3 are about 87 kDa and about 7 kDa, respectively. In some embodiments, the capsid protein is about 2 kDa, and about 62 kDa. The protein forms a spherical 60-mer protein shell around the viral genome. In this embodiment, the capsid protein protects the viral genome, delivers the genome, and In some embodiments, the capsid protein is a viral protein that interacts with the host cell. The genome is delivered to the host in a tissue-specific manner.

[0036] In some embodiments, the AAV capsid protein is AAV3, AAV4, AAV5, From AAV6, AAV8, AAVrh8, AAV9, AAV10, and AAVrh10 In some embodiments, the capsid protein is an AAV serotype selected from the group consisting of: The AAV capsid protein is a member of the AAVrh8 or AAVrh10 serotype capsid. In some embodiments, the AAV capsid protein is AAVrh8. The capsid protein of the serotype.

[0037] In some embodiments, the rAAV vector is prepared by injecting a ribozyme into an AAV capsid. To this end, components that are cultured within the host cell can be brought in trans into the host cell. Alternatively, any one or more of the required components (e.g., recombinant AA V vector, rep sequence, cap sequence, and / or helper function) are known to those skilled in the art. The method of the present invention is used to engineer the compound to contain one or more of the required components. Such a stable host cell may also be produced by a stable host cell that has been induced Most preferably, the desired component(s) are under the control of a specific promoter. However, the required component(s) may be under the control of a constitutive promoter. As used herein, suitable inducible and constitutive promoters may be Examples of such elements are provided in the discussion of regulatory elements suitable for use with transgenes. In yet another alternative, the selected stable host cell is a cell that expresses a constitutive promoter. The selected component(s) are under the control of one or more inducible promoters. and other selected component(s) under the control of the controller. 3 cells (containing E1 helper functions under the control of a constitutive promoter) However, the rep protein and / or the cap protein are under the control of an inducible promoter. Stable host cells containing the protein can be produced. Still other stable host cells can be produced by those skilled in the art. Cells may also be produced.

[0038] In some embodiments, the present disclosure provides a method for the preparation of a nucleic acid sequence comprising the steps of: 6. In embodiments, the present disclosure relates to a composition comprising the host cell described above. In an embodiment, the composition comprising the host cell further comprises a cryoprotectant.

[0039] Recombinant AAV vectors, rep sequences, ca sequences, and p sequences, and helper functions can be expressed using any suitable genetic element (vector). The selected genetic element can be delivered to the packaging host cell by the The delivery may be by any suitable method, including those described in any of the embodiments of the present disclosure. The methods used to construct the morphology are based on nucleic acid manipulation techniques. The methods are well known to those skilled in the art and include genetic engineering techniques, recombinant techniques, and the like. See, e.g., Sambrook et al, Molecular Biology, 1999, 144:1311-1321, 1999. lar Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, See, NY. Similarly, methods for producing rAAV virions are well known and suitable methods may be used. The choice of method is not a limitation of this disclosure. See, e.g., K. Fisher et al., J. Virol., 70:5 20-532 (1993) and U.S. Pat. No. 5,478,745.

[0040] In some embodiments, the recombinant AAV is produced using a triple transfection method (U.S. Patent No. 6, The present invention can be prepared using a method for producing a fluororesin (such as those described in detail in US Pat. No. 5,550,313). Typically, recombinant AAV is packaged into AAV particles in a host cell. AV vector (containing the transgene), AAV helper function vector, and accessor It is produced by transfecting an AAV helper function vector. The vector functions in trans for productive AAV replication and encapsidation. It encodes the "AAV helper function" sequences (i.e., rep and cap). , AAV helper function vectors produce detectable wild-type AAV virions (i.e., functional Without producing AAV virions (containing the rep and cap genes), Supporting the production of AAV vectors. Non-limiting examples of vectors suitable for use according to the present disclosure include: No. 6,001,650, the entire contents of which are incorporated herein by reference. The pHLP19 and the pHLP20 compounds described in U.S. Pat. No. 6,156,303 are also disclosed. The accessory function vector includes the pRep6cap6 vector, which is described in detail in the specification. These vectors are required to express non-AAV proteins that AAVs depend on for replication (i.e., "accessory functions"). Encoding nucleotide sequences for AV-derived viral and / or cellular functions Accessory functions are functions required for AAV replication, including, but not limited to: Activation of AAV gene transcription, stage-specific splicing of AAV mRNA, and AAV D Parts involved in NA replication, synthesis of cap expression product, and assembly of AAV capsids Virus-based accessory functions include adenovirus, herpes Viruses (other than herpes simplex type 1 virus) and vaccinia virus, among others. It may be derived from any of the luper viruses.

[0041] In some aspects, the disclosure provides a transfected host cell. The term "transfection" is used to refer to the uptake of foreign DNA by a cell, A cell is "transfected" when infectious DNA is introduced through the cell membrane. A number of transfection techniques are generally known in the art. aham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (198 6) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:1 See, e.g., 97. Such techniques include nucleotide integration vectors and other nucleic acid molecules. can be used to introduce one or more exogenous nucleic acids, such as a nucleic acid sequence, into a suitable host cell. .

[0042] "Host cell" means any cell capable of harboring or carrying a substance of interest. Host cells are often mammalian cells. Host cells are the host cells in which recombinant AAV is produced. Production and related AAV helper constructs, AAV minigene plasmids, and accessory functions The term can be used as a recipient for vectors or other introduced DNA. Thus, as used herein, "transfected" includes any progeny of the original cell that has been transfected. "Host cell" may refer to a cell that has been transfected with an exogenous DNA sequence. The descendants of a single parent cell may have natural mutations, accidental mutations, or deliberate mutations. Due to mutations, there may be differences in shape or genome or total DNA complement from the original parent, It is understood that they may not be exactly the same.

[0043] As used herein, the term "cell line" refers to a cell line that is capable of proliferation and differentiation in vitro. A cell lineage is a population of cells that are capable of sustained or extended division and differentiation. The term "clonal population" is often a clonal population that is a single clone. It is also known that upon accumulation or introduction, spontaneous or induced changes can occur in the karyotype. Therefore, cells derived from the cell lineages referred to are not necessarily related to the progenitor cells or cultured cells. and the cell lines referred to include such variants.

[0044] As used herein, the term "recombinant cell" refers to a cell that is capable of expressing biologically active polypeptides. A DNA segment that results in the transcription or production of a biologically active nucleic acid, such as RNA. etc. refers to a cell into which an exogenous DNA segment has been introduced.

[0045] As used herein, the term "vector" refers to a vector that is associated with appropriate control elements. and plasmids, phages, and transfectants that can replicate and transfer genetic sequences between cells. Any gene, including transposons, cosmids, chromosomes, artificial chromosomes, viruses, and virions Thus, the term includes cloning and expression vehicles, as well as In some embodiments, useful vectors include vectors that contain a nucleic acid segment to be transcribed. It is assumed that the target gene is a vector placed under the transcriptional control of a promoter. A "promoter" is a molecule that is recognized by the cell's synthetic machinery or by introduced synthetic machinery and that functions as a gene promoter. "Operably positioned" refers to a DNA sequence required to induce specific transcription of a gene. The phrases "under control" or "under transcriptional control" refer to a promoter that is capable of directing the transcription of an RNA polymerase It is in the correct location and orientation relative to the nucleic acids that control induction and gene expression. The term "expression vector or expression construct" refers to a portion of a nucleic acid coding sequence. It refers to any type of genetic construct that contains a nucleic acid that is transcribable in whole or in part. In some embodiments, expression can refer to, for example, the production of a biologically active polypeptide from a transcribed gene. tide products, or inhibitory RNA (e.g., shRNA, miRNA, miRNA inhibitors) The method includes transcription of a nucleic acid, which results in

[0046] The recombinant vector is packaged into a desired AAV capsid to produce the rAAV of the present disclosure. The foregoing methods for making the Other suitable methods will be apparent.

[0047] Recombinant AAV vectors The isolated nucleic acids of the disclosure can be recombinant AAV (rAAV) vectors. A "rAAV" vector typically contains, at a minimum, a transgene and This regulatory sequence, as well as the 5' and 3' AAV inverted terminal repeats (ITRs), constitute It is packaged into a capsid protein and delivered to the selected target cell. In some embodiments, the transgene is a recombinant AAV vector. Heterologous to the vector sequence, it can be used to produce a polypeptide, protein, or functional RNA molecule (e.g. Nucleic acid sequences encoding gene products of interest (e.g., miRNAs, miRNA inhibitors), or other gene products of interest. The nucleic acid encoding the sequence is a sequence that is capable of inducing a regulatory component to express the regulatory component in trans, intracellularly, in cells of a target tissue. Operatively linked in a manner that allows for the transcription, translation, and / or expression of the gene.

[0048] An embodiment of the present disclosure is that modifications of the regulatory sequences of rAAV are therapeutically effective, but not previously used. The transgene was expressed at levels that did not cause vector-mediated toxicity associated with rAAV. Thus, in some embodiments, the present disclosure relates to the discovery that a gene that is capable of expressing a nucleotide sequence similar to that of the nucleotide sequence of the present invention can be expressed by a nucleotide sequence similar to that of the nucleotide sequence of the present invention. In some embodiments, the present invention relates to recombinant AAV (rAAV) that includes modified gene regulatory elements. In one embodiment, the modified gene regulatory element is a hybrid promoter.

[0049] As used herein, the term "hybrid promoter" refers to a promoter that directs the transcription of an RNA transcript ( For example, driving transcription of a transcript (including a transcript encoded by a transgene) A regulatory construct capable of carrying out the above-mentioned functions, comprising two or more artificially arranged regulatory elements. Hybrid promoters typically refer to regulatory constructs that contain a minimal set of At least one element that is a promoter and one or more enhancer sequences or multiple transcriptional regulatory elements, intronic sequences, exon sequences, or UTs and at least one element having an R sequence. In embodiments that include exon, intron, or UTR sequences, such sequences The (s) may encode an upstream portion of an RNA transcript (e.g., as depicted in FIG. 1). On the other hand, regulatory elements that modulate (e.g., enhance) the transcription of a transcript also In some embodiments, the hybrid promoter comprises two or more The elements are derived from heterologous sources relative to each other. In some embodiments, hybrid Two or more elements of the promoter of the transgene are distinct from those of the transgene. In some embodiments, a hybrid promoter is a promoter derived from two or more species. The excess elements are from different loci. Two or more elements of the promoter are derived from the same locus, but In some embodiments, the hybridization promoter is located in a position not found in the locus. The promoter can incorporate promoter or enhancer elements from different sources. a first promoter fused to one or more nucleic acid sequences, In some embodiments, the hybrid promoter comprises the nucleic acid sequence of - the first sequence is derived from the actin promoter and the second sequence is derived from the CMV enhancer In some embodiments, the hybrid promoter comprises a chicken beta- The first sequence is derived from the actin promoter and the second sequence is derived from the chicken beta actin gene. and a second sequence derived from a chromosomal promoter. The chicken beta-actin promoter fused to the CMV enhancer sequence and a sequence derived from an intron of the chicken beta-actin gene. Includes:

[0050] In some embodiments, the rAAV comprises an enhancer element. The term "enhancer element" refers to an element that, when bound by an activator protein, or a nucleic acid sequence that activates or increases the transcription of multiple genes. Enhancer sequences are located upstream (i.e., 5') of the genes they regulate. Enhancer sequences can be located upstream (i.e., 3') of the ribosome or downstream (i.e., 3') of the ribosome. Tomegalovirus (CMV) enhancer sequence and simian vacuolating virus 40 (SV4 0) Enhancer sequence. In some embodiments, the rAAV comprises a CMV enhancer sequence. As used herein, the term "a portion thereof" includes any of the following: It is desirable to have a desired functional characteristic of the entire nucleotide or amino acid sequence from which it is derived. For example, the term "CMV enhancer" refers to a fragment of a nucleotide sequence or amino acid sequence that "Sequence or portion thereof" refers to a wild-type C sequence or portion thereof that is capable of increasing transcription of a transgene. Refers to a nucleotide sequence derived from the MV enhancer.

[0051] In some aspects, the rAAV comprises a post-transcriptional response element. The term "post-transcriptional response element" refers to a tertiary structure that, when transcribed, enhances the expression of a gene. An example of a post-transcriptional regulatory element is the woodchuck hepatitis virus mouse posttranscriptional regulatory element (WPRE), mouse RNA transport element (RTE), type 1 Constitutive transport element (CTE) of simian retrovirus 1 (SRV-1), Mason-Farber CTE from MPMV and human heat shock protein 7 0 (Hsp70 5'UTR). In one embodiment, the rAAV vector is a vector encoding the woodchuck hepatitis virus post-transcriptional regulatory element. Includes WPRE.

[0052] In some aspects, the present disclosure provides a rRNA containing a hybrid intron or a chimeric intron. As used herein, the term "chimeric intron" refers to an AAV vector. refers to an intron that has sequences derived from two or more different sources. In some embodiments, the chimeric intron is derived from a first source (e.g., an organism or species). and a splice donor site from a second source (e.g., an organism or species). In some embodiments, the chimeric polypeptide further comprises a nucleic acid encoding a price acceptor site. The transcription factor contains one or more transcriptional regulatory elements and / or enhancer sequences. In some embodiments, the chimeric intron is a hybrid promoter exon. In some embodiments, the present disclosure provides a method for the preparation of a transgene comprising the steps of: the transgene comprises a promoter operably linked to a lysosomal The present invention presents an rAAV that encodes a chromosome accumulation protein and further contains a chimeric intron.

[0053] In certain embodiments, the present disclosure relates to rAAV vectors comprising artificial transcriptional elements. In some embodiments, the term "artificial transcription element" as used herein is the transcription of DNA by RNA polymerase in a regulated manner to produce RNA transcripts. The transcriptionally active element of the present disclosure generally refers to a synthetic sequence that is less than 500 bp long. less than 200 bp, more preferably less than 100 bp, and most preferably In some embodiments, the artificial transcriptional element is a transcriptionally active element. It is generally known in the art that a transcriptionally active Elements such as promoters, enhancer sequences, TATA boxes, etc. are recognized. box, G / C box, CCAAT box, specificity protein 1 (Sp1) binding site , Inr region, CRE (cAMP regulatory element), and transcription activator factor 1 (ATF1) binding binding site, ATF1-CRE binding site, APBβ box, APBα box, CArG Box, CCAC Box, and U.S. Pat. No. 6,346,415. Combinations of the foregoing transcriptionally active elements are also contemplated. can be.

[0054] In some embodiments, the artificial transcription element comprises a promoter sequence. In some embodiments, the artificial transcription element comprises an enhancer sequence. In some embodiments, the artificial transcription element comprises an ATF1-CRE binding site. In some embodiments, the artificial transcription element comprises an SP1 binding site. In some embodiments, the artificial transcription element comprises a TATA box. In some embodiments, the artificial transcription element comprises an ATF1-CRE binding site, an SP1 binding site, In some embodiments, the artificial transcription element comprises a transcription factor receptor 1 (TCR1), a transcription factor receptor 2 (TCR2), a transcription factor receptor 3 (TCR3), a transcription factor receptor 4 (TCR4), a transcription factor receptor 5 (TCR5), a transcription factor receptor 6 (TCR6), a transcription factor receptor 7 (TCR7), a transcription factor receptor 8 (TCR8), a transcription factor receptor 9 (TCR9), a transcription factor receptor 10 (TCR10), a transcription factor receptor 1 , represented by SEQ ID NO:2.

[0055] The expression control sequence includes an appropriate transcription initiation sequence, a transcription termination sequence, a promoter sequence, and an endonucleaser sequence. Hanser sequences; splicing signals and polyadenylation (polyA) signals, etc. Efficient RNA processing signals; sequences that stabilize mRNA in the cytoplasm; translation efficiency sequences that enhance the rate of synthesis (i.e., Kozak consensus sequences); and, if desired, sequences that enhance secretion of the encoded product. In the field of gene expression, natural promoters, constitutive promoters, inducible promoters, and / or Many expression control sequences, including gene-specific or tissue-specific promoters, are known and may be used.

[0056] As used herein, nucleic acid sequences (e.g., coding sequences) and regulatory sequences refer to sequences which , covalently linking a nucleic acid sequence to place the expression or transcription of the nucleic acid sequence under the influence or control of a regulatory sequence. When nucleic acid sequences are linked in a functional manner, they are said to be "operably" linked. If it is desired that the protein be translated, the two DNA sequences are Induction of the promoter results in transcription of the coding sequence and two DNA The nature of the linkage between the sequences resulted in (1) the introduction of frameshift mutations. (2) interfere with the ability of the promoter region to direct the transcription of the coding sequence; or (3) ) without interfering with the ability of the corresponding RNA transcript to be translated into protein. These are said to be operably linked. Thus, the resulting transcript is a desired transcript. The promoter region directs this DNA so that it can be translated into a protein or polypeptide. A promoter region is operatively associated with a nucleic acid sequence if it is capable of effecting transcription of the sequence. Similarly, two or more coding regions may be linked to a common promoter. These transcripts from the mouse contain two or more transcripts translated in frame. An operably linked protein is one that is linked to a protein in a manner that results in the expression of the protein. In some embodiments, the operably linked coding sequences encode a fusion protein. In some embodiments, the operably linked coding sequence produces a functional RNA (e.g., For example, shRNA, miRNA, miRNA inhibitors.

[0057] For nucleic acids encoding proteins, polyadenylation sequences are generally included in the transgene. The rAA sequence is inserted after the 3′ AAV ITR sequence and before the 3′ AAV ITR sequence. The V construct also comprises a promoter / enhancer sequence located between the promoter / enhancer sequence and the transgene. The nucleic acid sequence may also contain an intron, which may be desirable to incorporate into the nucleic acid sequence. One possible intron sequence is the SV- 40 and is referred to as the SV-40 T intron sequence.

[0058] Another vector element that can be used is an internal ribosome entry site (IRES). IRES sequences allow more than one polypeptide to be produced from a single gene transcript. IRES sequences are used to create proteins that contain more than one polypeptide chain. These general vector elements, as well as other general Selection of suitable vector elements is routine and many such sequences are available. [See, e.g., Sambrook et al., and, e.g., 3.183.26 and 1 6. References cited on page 1716.27; and Ausubel et al., Current Pr [See, for example, "Anatomy of Proteins in Molecular Biology," John Wiley & Sons, New York, 1989]. In some embodiments, the foot and mouth disease virus 2A sequence is included within the polyprotein; It is a small peptide (approximately 18 amino acids) that has been shown to mediate polyprotein cleavage. (Ryan, MD et al., EMBO, 1994; 4: 928-933;Mattion, NM et al. al., J Virology, November 1996; p. 8124-8127;Furler, S et al., Gene Therapy, 20 01; 8: 864-873; and Halpin, C et al., The Plant Journal, 1999; 4: 453-459). The cleavage activity of the 2A sequence has previously been demonstrated in plasmids and gene therapy vectors (AAV and RNAi). This has been demonstrated in artificial systems, including human toroviruses (Ryan, MD et al., EMBO, 1994; 4: 928-933;Mattion, NM et al., J Virology, November 1996; p. 8124-8127;Furler , S et al., Gene Therapy, 2001; 8: 864-873; and Halpin, C et al., The Plant Jo urnal, 1999; 4: 453-459;de Felipe, P et al., Gene Therapy, 1999; 6: 198-208;de Felipe, P et al., Human Gene Therapy, 2000; 11: 1921-1931; and Klump, H et al ., Gene Therapy, 2001; 8: 811-817).

[0059] The precise nature of the regulatory sequences required for gene expression in host cells can vary depending on the species, tissue, or Although this can vary between cell types, generally, a TATA box, capping sequence, etc., are included as needed. These include the transcription sequence, CAAT sequence, and enhancer element, which are involved in the initiation of transcription and translation, respectively. In particular, such sequences include 5' non-transcribed and 5' non-translated sequences that contribute to the Such 5' nontranscribed regulatory sequences act as promoters for transcriptional control of operably linked genes. The regulatory sequence may also include a promoter region, including an endogenous sequence, if desired. The vectors of the present disclosure may also optionally include an enhancer sequence, or an upstream activator sequence. Alternatively, it may include a 5' leader sequence or a 5' signal sequence.

[0060] Examples of constitutive promoters include, but are not limited to, the retroviral Rous Sarcoma Virus (RSV). Respiratory syncytial virus (RSV) long terminal repeat promoter (optionally with the RSV enhancer), cytomegalovirus (RSV) Gallovirus (CMV) promoter (optionally with a CMV enhancer) [e.g. See, for example, Boshart et al., Cell, 41:521-530 (1985)], SV40 promoter -, dihydrofolate reductase promoter, β-actin promoter, phosphoglyceryl Promoter of PGK and promoter of EF1α [Invitro gen].

[0061] Inducible promoters allow for the regulation of gene expression and can be regulated by exogenously supplied compounds, temperature, etc. Environmental factors such as the degree of differentiation or specific physiological conditions, e.g., the presence of an acute phase, cell specific differentiation Inducible promoters can be regulated by conditions, or physiological conditions that are specific to the replicating cell. Transducers and inducible systems are available from, but not limited to, Invitrogen, Clontech, Many other systems have been described. and can be readily selected by one of skill in the art. An example of an inducible promoter regulated by zinc is the zinc-inducible sheep metallothionein (M T) promoter, dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV ) promoter, T7 polymerase promoter system (WO98 / 10088); Insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)) , tetracycline repression system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-555 1 (1992)), tetracycline-inducible system (Gossen et al., Science, 268:1766-1769 (1995) (see also Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1998)). , RU486 inducible system (Wang et al., Nat. Biotech., 15:239-243 (1997); and Wang et al. al., Gene Ther., 4:432-441 (1997)), and the rapamycin inducible system (Magari et al., J Clin. Invest., 100:2865-2872 (1997)). Further examples that may be useful in this context include Other types of inducible promoters are those that respond to specific physiological conditions, such as temperature, the presence of an acute phase, Induction, regulated by cell-specific differentiation states or physiological conditions restricted to replicating cells It is a conducive promoter.

[0062] The AAV sequence of the vector typically contains 5' and 3' flanking cis-acting inverted terminal repeat sequences. columns (e.g., BJ Carter, in “Handbook of Parvoviruses”, ed., P. Tijsser , CRC Press, pp. 155-168 (1990). The ITR sequence is approximately 145 bp long. Preferably, in the molecule, substantially the entire sequence encoding the ITR(s) is Although some minor modifications to these sequences are permitted, these I The ability to modify TR sequences is within the skill of the art (see, e.g., Sambrook et al. , “Molecular Cloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laborat ory, New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996). (See textbooks.) Examples of such molecules used in this disclosure are transgenes. In this case, the selected transgene sequence is a "cis-acting" plasmid that contains the The sequence and associated regulatory elements are the 5' AAV ITR sequence and the 3' AAV ITR sequence. The AAV ITR sequences are flanked by the mammalian AAV ITR sequences identified herein. It may be derived from any known AAV, including V types.

[0063] In some embodiments, the rAAV of the present disclosure is a pseudotyped rAAV. For example, serotype Y Pseudotyped AAV vectors containing the ITRs of serotype X encapsidated with proteins AAVX / Y would be used (e.g., AAV2 / 1 would be the ITR of AAV2). and an AAV1 capsid. In some embodiments, the pseudotyped rAAV comprises one AAV1 capsid. The tissue-specific targeting ability of the capsid protein derived from AV serotypes was examined by comparing it with that of other AA V serotype-derived viral DNA, thereby This may be useful to allow targeted delivery to tissues.

[0064] In addition to the major elements identified above for a recombinant AAV vector, The target also contains cells transfected with a plasmid vector produced according to the present disclosure. The transcription, translation and expression of the virus in a cell or in a cell infected with a virus produced according to the present disclosure. and / or a necessary, operably linked to the transgene in a manner allowing expression. Conventional control elements are also included. As used herein, "operably linked" sequences expresses a gene of interest and adjacent expression control sequences, and the gene of interest, in trans or The expression control sequences include both those that act to regulate expression at a distance.

[0065] Recombinant AAV vectors: transgene coding sequences The composition of the transgene sequence in the rAAV vector determines whether the resulting vector is For example, one type of transgene sequence, when expressed, In another example, the transgene comprises a reporter sequence that provides a detectable signal. The gene encodes a therapeutic protein or a functional RNA for therapeutic use. The transgene can be, for example, a somatic transgenic animal model carrying the transgene. for research purposes, e.g., to study the function of transgene products In another example, the transcription factor encodes a protein or functional RNA that is intended to be The Lance gene is intended to be used to create animal models for the disease. , encoding a protein or functional RNA. Those skilled in the art will recognize suitable transgene codes. The code sequence will be clear.

[0066] In some aspects, the disclosure relates to rAAV vectors useful for treating lysosomal storage disorders. Lysosomal storage disorders (also called lysosomal storage diseases) are disorders that occur when the lysosomal A group of inherited metabolic disorders resulting from a defect in lysosomal function. The disease is due to the dysfunction of a single protein (e.g., enzyme) involved in lysosomal metabolism. For example, Tay-Sachs disease is characterized by a defect in the hexosaminidase A (HEXA) It is caused by a genetic mutation in the gene that causes the HEXA enzyme to react with GM2 ganglioside. Other examples of lysosomal storage disorders include: Their associated protein is aspartylglucosaminuria (aspartylglucosaminuria). Batten disease (palmitoyl protein thioesterase), infantile Batten disease (palmitoyl protein thioesterase), late infantile Batten disease Tenn disease (tripeptidyl peptidase), Fabry disease (α-galactosidase), Fuco sidosis (α-fucosidase), galactosialidosis (protective protein / cathepsin A), Gaucher disease (β-glucosidase), galactosialidosis (protective protein / Cathepsin A), Globoid cell leukodystrophy (Galactosylceramidase), GM 1 Gangliosidosis (β-galactosidase), α-mannosidosis (α-manno sidase), metachromatic leukodystrophy (arylsulfatase A), mucopolysaccharidosis I (α- L-iduronidase), Mucopolysaccharidosis II (iduronate sulfatase), Mucopolysaccharidosis II IA (heparin sulfatase), mucopolysaccharidosis IIIB (α-N-acetylglucosaminidase) Mucopolysaccharidosis IIIC (acetyl-CoA alpha-glucosaminidase), sulphasidase), mucopolysaccharidosis IIID (N-acetylglucosamine-6-sulphate sulphasidase), Mucopolysaccharidosis IVA (N-acetylgalactosamine 6-sulfatase), Mucopolysaccharidosis IVA (N-acetylgalactosamine 6-sulfatase), Mucopolysaccharidosis IVB (β-galactosidase), Mucopolysaccharidosis IX (hyaluronidase), Mucopolysaccharidosis Saccharidosis VI (arylsulfatase B), mucopolysaccharidosis VII (β-glucuronidase), Mucolipidosis type I (α-neuraminidase), mucolipidosis type II (Glc N Ac-1-phosphotransferase, mucolipidosis type III (N-acetylglucosamine) Cosamine-1-phosphotransferase), Niemann-Pick disease (acid sphingomyelinase) enzyme), Pompe disease (α-glucosidase), Sandhoff disease (β-hexosaminidase A and B), Schindler's disease (α-N-acetylgalactosaminidase), Tay-Sachs disease (β-hexosaminidase A / B), and Wolman disease (acid lipase), However, the present invention is not limited to the above.

[0067] In some embodiments, the present disclosure provides a transcription factor encoding a lysosomal storage disease-associated protein. In some embodiments, the present invention provides an rAAV comprising a transgene associated with a lysosomal storage disease. The associated protein is selected from the group consisting of HEXA, HEXB, and GLB1. In one embodiment, the lysosomal storage disease-associated protein is HEXA and is represented by SEQ ID NO:2. In some embodiments, the lysosomal storage disease associated protein comprises a sequence set forth in is HEXB and comprises the sequence set forth in SEQ ID NO: 21. The sosomal storage disease-associated protein is GLB1, which has the sequence set forth in SEQ ID NO:23. include.

[0068] Also provided herein is the use of the rAAV described herein to deliver transgenes to Methods for treating lysosomal storage diseases by delivery to elephants are also contemplated. In some embodiments, the present disclosure provides a method for treating a lysosomal storage disease, comprising: In some embodiments, the method comprises administering rAAV to a subject. The AV comprises a hybrid promoter. In some embodiments, the rAAV comprises a chimeric In some embodiments, the rAAV comprises an artificial transcription element. In an embodiment, the artificial transcription element comprises an ATF1-CRE binding site, an SP1 binding site, In some embodiments, the promoter, the chimeric intro In some embodiments, the transcriptional element is operably linked to the transgene. In some embodiments, the transgene is a transgene associated with a lysosomal storage disease. In some embodiments, the transgene consists of HEXA, HEXB, and GLB1. In some embodiments, the lysosomal storage disease is selected from the group consisting of GM2 gangliosides. dosis (Sandhoff disease and Tay-Sachs disease), and rAAV is In some embodiments, the method further comprises administering to the patient a transgene encoding a lysosomal storage disease. The disease is GM1 gangliosidosis and the transgene encodes GLB1.

[0069] In some aspects, the disclosure provides a method for producing a first cell line operably linked to a first transgene. A cell type specific promoter and a second cell type specific promoter linked to a second transgene. The first promoter and the second promoter are expressed in the same cell. The present invention presents an rAAV that contains a nucleic acid-containing capsid that is not cell type specific. Without wishing to be bound by, in some embodiments, such rAAV vectors - treat diseases that affect multiple cell types within a given tissue (e.g., CNS tissue) It is useful to

[0070] Thus, in some aspects, the present disclosure provides rAAV vectors useful for treating CNS-related diseases. As used herein, "CNS-related disease" refers to a disease or disorder of the central nervous system. CNS-related disorders refer to conditions that affect the spinal cord (e.g., myelopathy), brain (e.g., encephalopathy), or can affect the tissues surrounding the brain and spinal cord. CNS-related disorders are genetically inherited. It may be a genetic disorder acquired through somatic mutation or acquired through a genetically inherited disorder. Related disorders include psychological conditions or disorders, such as attention deficit hyperactivity disorder, autism spectrum disorder, and the like. Spectral disorders, mood disorders, schizophrenia, depression, Rett syndrome, etc. The CNS-related disorder may be an autoimmune disorder. CNS-related disorders also include cancers of the CNS, e.g. It can also be cancer. CNS-related disorders that are cancers are caused by primary cancers of the CNS, e.g., astrocyte Cancers that have spread to CNS tissues, such as glioblastomas and glioblastomas, may also spread to the brain. Further non-limiting examples of CNS-related disorders include Huntington's disease, Parkinson's disease, and Parkinson's disease. Kinesiology, lysosomal storage disease, ischemia, neuropathic pain, amyotrophic lateral sclerosis ( ALS), multiple sclerosis (MS), Canavan disease (CD), frontotemporal lobar degeneration (FTLD) , spinocerebellar ataxia, spinal-bulbar muscular atrophy, dentatorubral-pallidoluysian atrophy, and Fritz-Klein syndrome. -Including Reich's ataxia.

[0071] In some embodiments, the rAAV vectors described by the present disclosure target CNS disease-related genes. In some embodiments, the transgene encodes a CNS disease-associated gene. The transgene may encode a protein or an interfering RNA. Examples include dsRNA, siRNA, shRNA, miRNA, and artificial miRNA (am Examples of CNS disease-related genes include, but are not limited to, Alzheimer's disease, -related disease, DRD2, GRIA1, GRIA2, GRIN1, SLC1A1, SY P, SYT1, CHRNA7, 3R Tau / 4rTUS, APP, BAX, BCL-2, G RIK1, GFAP, IL-1, AGER; Parkinson's disease-related, UCH-L1; SKP1, EGLN1, Nurr-1, BDNF, TrkB, gstm1, S106β; Related to Huntington's disease: Huntington's disease, IT15, PRNP, JPH3, TBP, ATXN1, ATXN2, ATXN3, atrophin1, FTL, TITF-1 , Xbp1s, CRAG; associated with Friedreich's ataxia, FXN; associated with Canavan disease Associated with ASPA; Associated with muscular dystrophy; Associated with DMD; Associated with spinal muscular atrophy , SMN1, UBE1, DYNC1H1; associated with amyotrophic lateral sclerosis (ALS), A LS2, ANG, ATXN2, C9orf72, DCTN1, FIG4, FUS, NEF H, OPTN, PFN1, PRPH, SETX, SIGMAR1, SMN1, SOD1, SPG11, TARDBP, UBQLN2, VAPB, VCP; alpha-mannosid cis-associated, MAN2B1, MAN2B2, MAN2C1; aspartyl glucoside Associated with urinary disorders, AGA; Associated with Batten disease, CLN1, CLN2, CLN3, C LN5, CLN6, MFSD8, CLN8, CTSD; associated with beta-mannosidosis MANBA; associated with cystinosis, CTNS; associated with Danon disease, LAM P2; associated with Fabry disease, GLA; associated with Farber disease, ASAH1; fucosy associated with galactosialidosis, FUCA1; associated with galactosialidosis, CTSA; Associated with Scheier's disease, GBA; Associated with Krabbe's disease, GALC; Metachromatic leukodystrophy associated with ARSA; mucopolysaccharidoses (e.g., Hurler syndrome, Hunter syndrome, A- Sanfilippo syndrome type D, Morquio syndrome, hyaluronidase deficiency, Maroteaux-Lami Sly syndrome, sialidosis, I-cell disease, mucolipidosis types I-IV, Multiple sulfatase deficiency, Niemann-Pick disease types A to C, Pompe disease, pyknodysostosis, Sandhoff disease, Schrinder's disease, Tay-Sachs disease, Wolman disease) and IDU A, IDS, SGSH, NAGLU, HGSNAT, GNS, GALNS, ARSB, G USB, HYAL1, SMPD1, NPC1, NPC2, GAA, NAGA, SLCA1 7A5, and LAL(LIPA).

[0072] Useful transgene products also include miRNAs. miRNAs and other small molecule inhibitors Interfering nucleic acids act by cleaving / degrading the target RNA transcript or by targeting the messenger RNA (mRNA miRNAs regulate gene expression through translational repression of transcription factors (e.g., transcription factors that regulate transcription of genes). miRNAs are expressed as final, non-translated RNA products19-25 of the target mRNA. They exert their activity through sequence-specific interactions with the 3' untranslated region (UTR) of These endogenously expressed miRNAs form hairpin-shaped precursors, which The miRNA is then processed into a miRNA duplex and then into a "mature" single-stranded miRNA. The mature miRNA is processed into the A molecule. Identifying a target site, e.g., within the 3'UTR region, of the target mRNA based on complementarity; In some embodiments, the inhibitory In some embodiments, the RNA is a miRNA. , human SOD1 (e.g., SOD1 G93A ) to target the inhibitory RNA. The transgene comprises SEQ ID NO:15.

[0073] Administration of recombinant AAV The rAAV can be delivered to a subject in a composition according to any suitable method known in the art. Preferably, the composition is suspended in a physiologically compatible carrier (i.e., The rAAVs can be used to infect a variety of subjects, including humans, mice, rats, cats, dogs, sheep, rabbits, and rabbits. Ma, cow, goat, pig, guinea pig, hamster, chicken, turkey, or non- The antibody may be administered to a host animal, such as a human primate (e.g., a macaque monkey). In this context, host animals do not include humans.

[0074] Delivery of the rAAV to a mammalian subject can be, for example, by intramuscular injection, Administration into the bloodstream of a subject may be by administration into the bloodstream. Administration into the bloodstream may be via a vein, artery, or any other route. In some embodiments, the rAAV can be injected into a blood vessel using techniques well known in the surgical arts. and prior to administration of rAAV virions, one skilled in the art would isolate the affected limb from the general circulation. is administered into the bloodstream via isolated limb perfusion, a method that essentially makes it possible. A variation of the isolated limb perfusion technique described in US Pat. No. 6,177,403 is also used. Administering the lysine to the vascular system of an isolated affected limb to transduce muscle cells or muscle tissue; Further, in certain cases, the use of It may also be desirable to deliver the on to the CNS of a subject. "CNS" refers to the brain of a vertebrate. The term refers to all cells and tissues of the nervous system, nervous system, and spinal cord. These include, but are not limited to, apoptotic cells, astrocytes, cerebrospinal fluid (CSF), interstitial spaces, bone, cartilage, etc. Recombinant AAV can be delivered to the brain using neurosurgical techniques known in the art, such as stereotaxic injection (e.g., Stein et al., J Virol 73:3424-3429, 1999;Davidson et al., PNAS 97:3428-3432, 20 00; Davidson et al., Nat. Genet. 3:219-223, 1993; and Alisky and Davidson, Hum . Gene Ther. 11:2315-2329, 2000) to insert needles, catheters, or associated devices, for example, in the ventricular region as well as in the striatum (e.g. Injections into the caudate nucleus or putamen of the cerebellum, spinal cord, and neuromuscular junction, or into the cerebellar lobules, It can be delivered directly to the NS or to the brain.

[0075] In some embodiments, the rAAV of the present disclosure is administered to the CNS via lumbar intrathecal injection (LIT). In some embodiments, the rAAV of the present disclosure is administered to the In some embodiments, the rAAV is administered directly to the thalamus via a 2-dose infusion. In some embodiments, the rAAV of the present disclosure is administered bilaterally in the thalamic injection. In some embodiments, the present invention is administered by injection directly into the deep cerebellar nuclei (DCN). The indicated rAAV was administered bilaterally by injection into both the thalamus and DCN. In some embodiments, the rAAV of the present disclosure is administered directly by intracerebroventricular (ICV) injection. In some embodiments, the rAAV of the present disclosure is injected bilaterally into the thalamus and In some embodiments, the rAAV of the present disclosure is administered to both the host and the ICV via injection. Administered directly into the DCN and ICV.

[0076] In some embodiments, the rAAV described in this disclosure is administered by intravenous injection. In some embodiments, the rAAV is administered by intracerebral injection. The AAV is administered by intrathecal injection. In some embodiments, the rAAV is administered by intracranial injection. In some embodiments, the rAAV is delivered by intracisternal injection. In some embodiments, the rAAV is delivered by intraventricular injection. The rAAV of the present disclosure is administered into the spinal canal via a fluoroscopically guided lumbar intrathecal catheter. In some embodiments, the rAAV of the present disclosure is directed to the cisterna magna and the medullary cavity (e.g., spinal canal). In some embodiments, the rAAV of the present disclosure is administered to the cisterna magna, the medullary cavity (e.g., spinal canal), and the thalamus (eg, by intrathalamic injection).

[0077] An embodiment of the present disclosure comprises at least one modified gene regulatory sequence or modified gene regulatory element. In some embodiments, the composition comprises a recombinant AAV that contains the Further comprising an acceptable carrier.

[0078] The compositions of the present disclosure may contain rAAV alone or one or more other viruses (e.g., rAAV in combination with a second rAAV carrying one or more different transgenes. In some embodiments, the compositions each comprise one or more different AAVs. Lance gene, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , or more, different rAAVs.

[0079] In some aspects, the disclosure provides a composition comprising an rAAV that includes a nucleic acid encoding a HexA gene. In some aspects, the present disclosure relates to compositions (e.g., pharmaceutical compositions) that incorporate the HexB gene. The present invention relates to a composition (e.g., a pharmaceutical composition) comprising an rAAV that contains a nucleic acid encoding the rAAV. Thus, the present disclosure provides a first rAAV comprising a nucleic acid encoding a HexA gene, and a second rAAV comprising a nucleic acid encoding a HexB gene. and a second rAAV comprising a nucleic acid encoding a gene. do.

[0080] Ratio of rAAV encoding HexA:rAAV encoding HexB in the pharmaceutical composition For example, the ratio may vary from about 1:10 to about 10:1. The ratio of rAAV encoding HexB to rAAV encoding HexB was 1:1. In an embodiment, the ratio of rAAV encoding HexA to rAAV encoding HexB is 1 In some embodiments, the rAAV encoding HexA:HexB is in a ratio of 2. In some embodiments, the ratio of the rAAV encoding HexA to the rAAV encoding HexA is 1:5. The ratio of rAAV: rAAV encoding HexB is 1:10. The ratio of rAAV encoding HexA to rAAV encoding HexB was 2:1. In some embodiments, the ratio of rAAV encoding HexA to rAAV encoding HexB is In some embodiments, the ratio of rAAV to HexA is 5:1. The ratio of rAAV encoding V:HexB is 10:1.

[0081] In some embodiments, an rAAV encoding HexB: an rAAV encoding HexA In some embodiments, the ratio of rAAV encoding HexB:HexB is 1:2. In some embodiments, the ratio of rAAV encoding HexA to HexB is 1:5. The ratio of rAAV encoding HexA to rAAV encoding HexA was 1:10. In this embodiment, the ratio of rAAV encoding HexB:rAAV encoding HexA is In some embodiments, the rAAV encoding HexB:HexA is in a ratio of 2:1. In some embodiments, the ratio of rAAV encoding HexB is 5:1. The ratio of rAAV encoding HexA to rAAV encoding HexA was 10:1.

[0082] An appropriate carrier can be readily selected by one of skill in the art with the application for which the rAAV is intended in mind. For example, one suitable carrier is saline, which may be formulated with a variety of buffers. Other exemplary carriers include sterile, saline solution (e.g., phosphate buffered saline). , lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin The carriers include, for example, peanut oil, sesame oil, and water. The choice of carrier is not limited by the present disclosure. .

[0083] Optionally, the compositions of the present disclosure may contain, in addition to the rAAV and carrier(s), a preservative or The formulation may contain other conventional pharmaceutical ingredients, such as chemical stabilizers. Suitable exemplary preservatives include: Chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, Contains parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0084] rAAV transfects cells of the desired tissue and is sufficient to deliver the desired therapeutic effect without causing unwanted adverse effects. The amount administered is sufficient to result in sufficient levels of gene transfer and expression. Pharmaceutically acceptable routes of administration include direct delivery to a selected organ (e.g., to the CNS). injection), oral administration routes, inhalation administration routes (including intranasal and intratracheal delivery), intraocular administration route, intravenous route, intramuscular route, subcutaneous route, intradermal route, intratumoral route, and other parenteral routes of administration. Can be combined.

[0085] The dose of rAAV virions required to achieve a particular "therapeutic effect", e.g. For example, the dose in genome copies per kilogram of body weight (GC / kg) is , the route of administration of the rAAV virion, the gene or RNA required to achieve a therapeutic effect. The expression level of A, the particular disease or disorder being treated, and the gene or RNA product This will vary based on a number of factors, including but not limited to the stability of the The vendor provides a dosage of rAAV virions for treating a patient with a particular disease or disorder. The range can be readily determined based on the factors mentioned above, as well as other factors well known in the art. This can be done.

[0086] An effective amount of rAAV is an amount sufficient to infect an animal and target the desired tissue. In some embodiments, an effective amount of rAAV is administered to a stable somatic transgenic animal. The effective amount will depend primarily on the species, age, weight, and health of the subject. The results are dependent on factors such as the gene expression level and the tissue targeted, and therefore may vary between animals and tissues. For example, an effective amount of rAAV is generally about 10 6 ~10 16 Genome copies (e.g. For example, 1×10, including the endpoints 6 ~1×10 16 ) about 1ml to about 100ml In some cases, the solution is about 10 11 ~10 12 Injection between rAAV genome copies In some embodiments, the dosage is about 10 11 ~10 13 rAAV genome copies In some embodiments, a dosage of about 10 11 ~10 14 rAAV genome coding In some embodiments, a dose of about 10 11 ~10 15 rAAV In some embodiments, a dose of about 10 12 ~10 14 rA A suitable dosage of AV genome copies is about 10 13 ~10 14 r The dosage of AAV genome copies is appropriate. In some embodiments, the dosage is 100 mg / kg body weight (kg). Approximately 1 x 10 per g 12 , about 1.1×10 12 , about 1.2×10 12 , about 1.3×1 0 12 , about 1.4×10 12 , about 1.5×10 12 , about 1.6×10 12 , about 1.7×1 0 12 , about 1.8×10 12 , about 1.9×10 12 , about 1×10 13 , about 1.1×10 1 3 , about 1.2×10 13 , about 1.3×10 13, about 1.4×10 13 , about 1.5×10 1 3 , about 1.6×10 13 , about 1.7×10 13 , about 1.8×10 13 , about 1.9×10 1 3 , or approximately 2.0 × 10 14 The dosage of vector genome (vg) copies is appropriate. In some embodiments, about 4 x 10 12 ~2×10 13 Dosage between rAAV genome copies In some embodiments, 4.68×10 7 The dosage is appropriate. In this embodiment, 4.68×10 8 The dosage of genome copies is appropriate. , 4.68×10 9 A dose of 1.17 genome copies is appropriate. ×10 10 A dose of 2.34×10 genome copies is appropriate. 1 0 A dose of 3.20×10 genome copies is appropriate. 11 genome A dose of about 4.2×10 copies is appropriate. 12 Genome copies The dosage is appropriate. In some embodiments, 1.2×10 13 The appropriate dose of genome copies In some embodiments, the 13 The dosage of genome copies is appropriate. In some embodiments, 1.5×10 13 The dosage of vector genome (vg) copies is appropriate. In some embodiments, about 1×10 14 The dosage of vector genome (vg) copies is appropriate. In some embodiments, about 1.5×10 per kg of 13 v A dosage of about 1×10 g per kg of brain weight is appropriate. 14 vg In certain embodiments, a dosage of 10 12 ~10 13 rAAV genome coding The peptide targets tissue associated with a lysosomal storage disease, e.g., brain tissue or CNS tissue. In certain embodiments, the 10 13 ~10 14 rAAV The genome copies are located in tissues associated with lysosomal storage diseases, e.g., brain tissue or CNS tissue. In some embodiments, the composition is administered intravenously to a subject. The dose of rAAV administered is approximately 10 per kilogram (kg) of body weight. 11 ~10 14 rAA In some embodiments, the vector is delivered intravenously. The amount is 1 x 10 per kg. 11 ~1×10 14 vg, 1 x 10 per kg 12 ~1× 10 14 vg, or 1 x 10 per kg 13 ~1×10 14 vg. In embodiments, the dose delivered intravenously is about 1.2×10 per kg. 13 ~1.8× ×10 13 In some embodiments, the intravenously delivered dose is between 100 mg / kg and 150 mg / kg. Approximately 1.5 x 10 13 In some embodiments, administration of the dose is by infusion into the cerebrospinal fluid ( In some embodiments, the total dose is delivered to the (i) cisterna magna and / or CSF. delivered to the CSF by injection via the intrathecal space (e.g., spinal canal); and / or (ii ) delivered to the thalamus by intrathalamic injection. In some embodiments, about 75% of the total dose is delivered to the thalamus. % is delivered to the cisterna magna. In some embodiments, about 25% of the total dose is delivered to the intrathecal cavity. In some embodiments, about 75% of the total dose to the CSF is delivered to the cisterna magna. In some embodiments, about 25% of the total dose into the CSF is delivered to the intrathecal cavity. In some cases, stable transgenic animals can be generated by multiple injections of rAAV. It is manufactured.

[0087] In some embodiments, the dose delivered to the thalamus is about 1×10 11 ~1×10 14 vg , 1×10 12 ~1×10 14 vg, or 1×10 12 ~5×10 13 vg. In some embodiments, the dose delivered to the thalamus is about 2.8×10 12 ~1.1×10 13 vg. In some embodiments, the dose delivered to the thalamus is about 7.2×10 12 ~1 .4×10 13 vg.

[0088] In some embodiments, the volume delivered to the thalamus is about 0.5 mL to about 1.5 mL, about 0 .75mL to about 1.25mL, or about 0.8mL to about 1.2mL.

[0089] In some embodiments, the dose delivered to the cisterna magna and / or the intrathecal space (e.g., spinal canal) is 1 x 10 12 ~1×10 15 vg, 1×10 13 ~1×10 15 vg, or 1×1 0 13 ~1×10 14 In some embodiments, the cisterna magna and / or the medullary cavity (e.g., For example, the dose delivered to the spinal canal is approximately 2.0 × 10 13 ~9.0×10 13 vg In some embodiments, the dose delivered to the intrathecal cavity (e.g., spinal canal) is about 2.0×10 13 ~8.13×10 13 vg.

[0090] In some embodiments, the volume delivered to the intrathecal cavity (e.g., spinal canal) is from about 1 mL to about 1 In some embodiments, the intrathecal cavity (e.g., the spinal canal) is about 0 mL, or about 2 mL to about 8 mL. ) the volume delivered is approximately 2mL, 3mL, 4mL, 5mL, 6mL, 7mL, or It is 8mL.

[0091] In some embodiments, the subject is administered the antibody or antibody-containing agonist, such as into the CSF, the intrathecal cavity, and / or the cisterna magna of the subject. Delivery of 1×10 is performed via an intravascular microcatheter. 14 A dose of vg is delivered via an intravascular microcatheter. The dose was 14 mL of CSF administered by passive flow through an intravascular microcatheter. Following removal from the subject, 9 mL of AAV was administered into the subject at approximately 1 mL / min, up to the subject's cisternal level. In some embodiments, delivery to a subject is by administration into the blood. Intralumbar injection (e.g., administration into the intrathecal space within the lumbar region) via an intraluminal microcatheter In some embodiments, delivery is by intralumbar injection into the L2 region of the subject. In some embodiments, a volume of 3 ml of the rAAV vector is administered to a subject.

[0092] In some embodiments, the present disclosure provides a transcriptome encoding a lysosomal storage disease protein. The rAAV composition comprising the gene for treating Tay-Sachs disease is administered to a subject in need thereof (e.g., a patient suffering from Tay-Sachs disease). or have or are suspected of having a lysosomal storage disorder, such as Sandhoff disease In some embodiments, the subject is, e.g., , MUG or MUGS, and the rA Prior to administration of a composition comprising AV or rAAV, normal β-heterozygote was detected in the CSF of the subject. β-hexosaminidase A activity of ≦1.0%, ≦0.5%, or ≦0.1% In some embodiments, the subject has infantile Tay-Sachs disease. In some embodiments, the subject is 5 to 36 months old. In some embodiments, the subject is between 12 and 36 months of age.

[0093] In some aspects, the present disclosure provides a method for administering an AAV to a subject. Recognition that a potential side effect is an immune response, including inflammation, in a subject to AAV. In some embodiments, the subject is administered one or more of the rA The subjects are immunosuppressed prior to administration of AV.

[0094] As used herein, "immunosuppressed" or "immunosuppression" refers to the suppression of immune function in a subject. It refers to the reduction in activation or efficacy of the immune response. Immunosuppression is achieved by using anti-inflammatory drugs such as rituximab, methylprednisolone, Lon, Prednisolone, Sirolimus, Immunoglobulin Injection, Prednisone, Solu-M edrol, lansoprazole, trimethoprim / sulfamethoxazole, methotrexate cert, and any combination thereof, Medications (e.g., multiple medications, such as two, three, four, five, or more medications) In some embodiments, the immunosuppressive regimen can be induced in a subject using Limus, prednisolone, lansoprazole, trimethoprim / sulfamethoxazole or any combination thereof.

[0095] In some embodiments, the methods described by the present disclosure include administering to a subject an rAAV (e.g., Prior to administration of the rAAV or pharmaceutical composition described by the disclosure, the subject Inducing immunosuppression (e.g., administering one or more immunosuppressive agents In some embodiments, the subject is administered rAAV for about 30 days to about 30 days after administration of the rAAV to the subject. Any time during the 30 days period up to and including about 0 days prior to (e.g., administration of rAAV) In some embodiments, the subject is immunosuppressed (e.g., immunosuppression is induced in the subject). In certain conditions, the subject is treated with immunosuppressive agents (e.g., rituximab, sirolimus, and / or prednisolone). Patients will be pretreated with 5-fluroquinone (DMSO) for at least 7 days.

[0096] In some embodiments, the methods described herein include the use of rAAV or rAA The present invention further includes co-administration or pre-administration of a drug to a subject receiving a pharmaceutical composition comprising V. In some embodiments, the drug is miglustat, Keppra, Prevacid, Clonazepam, zepam, and any combination thereof.

[0097] In some embodiments, the subject's immunosuppression is achieved upon administration of the rAAV or pharmaceutical composition, and In some embodiments, the subject is administered rAAV or a pharmaceutical After administration of the composition, the subject is immunosuppressed for a period ranging from one day to one year (e.g., one or more (The patient is given immunosuppressants.)

[0098] In some embodiments, the rAAV composition is particularly useful in the treatment of cancer, particularly at high rAAV concentrations (e.g., about 10 13 GC / ml or more) in the composition to reduce aggregation of AAV particles. Methods for reducing aggregation of rAAV are widely known in the art. These include, for example, the addition of surfactants, correction of pH, correction of salt concentration, etc. Wright FR, et al., Molecular Thera (see, e.g., J. Py (2005) 12, 171-178).

[0099] Formulation with pharma- ceutically acceptable excipient and carrier solutions may be used in accordance with certain of the methods described herein. The compositions described herein are suitable for use in a variety of treatment regimens, and are described in terms of administration and treatment regimens. The development of dimenes is well known to those skilled in the art.

[0100] The percentage of active ingredient(s) may, of course, vary and may be determined by weight or by percentage of the total formulation. Approximately 1 or 2% to approximately 70% or 80% or more of the weight or capacity Typically, these formulations contain at least about 0.5% to about 10% of the active ingredient, although it may be convenient to vary the volume. Of course, each therapeutically useful composition may contain 0.1% or more of the active compound. The amount of active compound is adjusted so that a suitable dosage will be obtained in any given unit dose of the compound. The soluble, soluble, and soluble forms of the pharmaceutical preparations can be prepared by those skilled in the art of preparing such pharmaceutical preparations. Ioavailability, in vivo half-life, route of administration, shelf life of the product, as well as other pharmacological properties Factors such as clinical and therapeutic considerations are anticipated, and thus various dosages and treatment regimens may be considered. As may be desired.

[0101] In certain circumstances, the rAAV-based therapeutic constructs can be used in combination with suitable In a properly formulated pharmaceutical composition, the drug may be delivered subcutaneously, intrapancreatically, intranasally, parenterally, intravenously ... Intravenous, intramuscular, intrathecal, or oral, intraperitoneal, or by inhalation In some embodiments, it may be desirable to use the method disclosed in U.S. Pat. Specification; Specification No. 5,641,515; and Specification No. 5,399,363 (each (each of which is specifically incorporated by reference in its entirety). Any of the administration modalities described herein may be used to deliver the rAAV. The preferred mode of administration is by injection into the portal vein.

[0102] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions, and sterile injectable solutions or or sterile powders for the extemporaneous preparation of sterile injectable dispersions. Dispersions in ethylene glycol and mixtures thereof, as well as in oils, have also been prepared. Under ordinary conditions of storage and use, these preparations will prevent the growth of microorganisms. In most cases, the formulation is sterile and provides easy injectability. The form must be stable under the conditions of manufacture and storage. It must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, polyethylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and / or The appropriate fluidity may be a solvent or dispersion medium, such as a lecithin, which may contain oil or vegetable oil. This can be maintained by the use of coatings such as acrylic or the like, and in the case of dispersions, the required Prevention of microbial action can be achieved by maintaining particle size and by using surfactants. Various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sol In many cases, isotonic agents, such as sugars or It would be preferable for the composition to contain sodium chloride. Agents that delay absorption, such as aluminum monostearate and gelatin. It can be brought about by use.

[0103] For administration of an injectable aqueous solution, for example, the solution should be suitably buffered, if necessary. The liquid diluent must first be made isotonic with sufficient saline or glucose. These particular aqueous solutions are suitable for, inter alia, intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous media that can be employed will be known to those skilled in the art. For example, a dose for single administration is dissolved in 1 ml of isotonic NaCl solution and 1000 ml It may be added to subcutaneous injection fluids or injected into designated injection sites. (For example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035- (See, e.g., 1038 and 1570-1580.) Some variation in dosage may be necessary depending on the condition of the host. In any event, the person responsible for administration will determine the appropriate dose for the individual host. This will be done.

[0104] Sterile injectable solutions may be prepared by mixing the required amount of active rAAV in a suitable solvent, as desired, with the appropriate amount of rAAV, as desired, and / or with the appropriate concentration, as described herein. It is prepared by incorporating the above-listed ingredients with various other ingredients, as listed in the specification, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating the various active ingredients in a sterilized form into a basic dispersion medium. and other required ingredients from the ingredients listed in In the case of a sterile powder for preparing a sterile injectable solution, the preferred preparation is The method comprises the steps of: mixing the active ingredient, plus any additional desired ingredients, in a powder form that has already been sterile filtered. from a solution, vacuum drying and freeze drying.

[0105] The rAAV compositions disclosed herein may also be formulated in a neutral or salt form. Pharmaceutically acceptable salts are the acid addition salts (formed with the free amino groups of the protein). For example, inorganic acids such as hydrochloric acid or phosphoric acid, or acetic acid, oxalic acid, tartaric acid, mandelic acid, This includes acid addition salts formed with organic acids such as phosphoric acid. The salts may be, for example, sodium, potassium, ammonium, calcium, or dihydroxyl. Inorganic bases such as iron, and isopropylamine, trimethylamine, histidine, procaine When formulated, the solution may also be derived from an organic base such as ethanol. The formulations will be administered in a therapeutically effective manner, such as injectable solutions, drug release capsules, and the like. It is easy to administer in a variety of dosage forms, including as a capsule.

[0106] As used herein, "carrier" refers to any solvent, dispersion medium, vehicle, coating, diluent, or mixture thereof. agents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, The art includes the following: The use of such media and agents is well known. Additionally, auxiliary substances may also be incorporated into the compositions. The term "pharmaceutical acceptable" refers to an allergen that is compatible with the body when administered to a host. The term refers to molecular entities and compositions that do not produce adverse gastrointestinal or similar reactions.

[0107] The compositions of the present invention can be delivered to suitable host cells using liposomes, nanocapsules, or other suitable delivery systems. Delivery vehicles such as microparticles, microspheres, lipid particles, vesicles, etc. may be used. In addition, transgenes delivered by rAAV vectors can be delivered in lipid particles, liposomes, or vesicles. The compound may be formulated for delivery encapsulated in a nanosphere, nanoparticle, or the like.

[0108] Such formulations include pharma- ceutically acceptable formulations of the nucleic acids or rAAV constructs disclosed herein. Those skilled in the art will be familiar with the formation and use of liposomes. It is generally known. Recently, liposomes with improved serum stability and circulating half-life have been developed. In addition, liposomes have been shown to be useful as potential drug carriers (U.S. Patent No. 5,741,516). Various methods have also been described using liposome- and liposome-like preparations (U.S. Pat. No. 5,333,351). Specification No. 5,567,434; Specification No. 5,552,157; ​​Specification No. 5,565,2 Specification No. 13; Specification No. 5,738,868; and Specification No. 5,795,587 book).

[0109] Liposomes are a powerful tool to transfect many cells that are normally resistant to transfection by other procedures. In addition, liposomes have been used successfully with a variety of cell types. There is no restriction on DNA length, which is typical of other delivery systems. Liposomes can deliver genes, drugs, radioactive therapeutic agents, Viruses, transcription factors, and allosteric effectors are expressed in a variety of cultured cell lines and animal models. In addition, the efficacy of liposome-mediated drug delivery has been improved. Several clinical trials investigating this have also been successfully completed.

[0110] Liposomes are multilamellar concentric bilayer vesicles (also called liposomes) dispersed in an aqueous medium. It is made of phospholipids that spontaneously form multilamellar vesicles (also called MLVs). MLVs generally have diameters between 25 nm and 4 μm. Sonication of MLVs reduces their diameter. Small unilamellar vesicles with a diameter in the range of 200-500 Å and containing an aqueous solution in the core This results in the formation of small ultraviolet (SUV).

[0111] Alternatively, nanocapsule formulations of rAAV can also be used. Nanocapsules are generally The polymer can be stably and reproducibly taken up into cells. To avoid side effects caused by the use of in vivo degradable polymers, Such ultrafine particles (size of about 0.1 μm) should be designed. Biodegradable polyalkylcyanoacrylate nanoparticles that meet the requirements are envisaged for use. will be done.

[0112] In addition to the delivery methods described above, the following techniques also can be used to deliver rAAV compositions to a host. In U.S. Patent No. 5,656,016, sonophoresis is envisioned as an alternative method to achieve this. The drug may be administered by a stimulant such as ultrasound to measure the rate of drug penetration into and through the circulatory system. It is contemplated that the present invention may be used and described as a device for enhancing the efficacy of the drug. Other alternative drug delivery methods include intraosseous injection (U.S. Patent No. 5,779,708), microtubule injection, and ophthalmic formulations (Bourlais et al., 199 8), transdermal matrices (U.S. Pat. Nos. 5,770,219 and 5,78 No. 3,208), and feedback-controlled delivery (U.S. Pat. No. 5,697,89 (No. 9).

[0113] In some embodiments, the disclosure provides a rAAV or pharmaceutical composition described herein. The present invention relates to the administration of one or more additional therapeutic agents to the subject to which the present invention has been administered. For example, an enzyme Administration of isolated wild-type protein to alleviate the decline in enzyme activity during replacement therapy (ERT) However, Gaucher disease, Hunter syndrome, Fabry disease, Pompe disease, Maroteaux-Lamy syndrome, To treat lysosomal storage disorders, including Morquio syndrome type A and LAL deficiency. It has been observed that the treatment is effective. Thus, in some embodiments, the subject is In addition to the rAAV or pharmaceutical composition described herein, one or more enzyme replacement therapies (E RT) is also administered. ERT is generally known and is described in, for example, Li (2018) Pediatr Ann. 4 7(5):e191-e197.

[0114] In some embodiments, the compositions described herein further comprise an isolated protein. In some embodiments, the composition comprises an isolated wild-type HexA protein. In some embodiments, the composition comprises an isolated wild-type HexB protein. The composition includes an isolated wild-type HexA protein and an isolated wild-type HexB protein. In some embodiments, the composition comprises an rAAV and an isolated wild-type HexA protein. In some embodiments, the composition comprises an rAAV and an isolated wild-type HexB protein. In one embodiment, the composition comprises an rAAV and an isolated wild-type HexA protein and an isolated wild-type HexA protein. Contains live HexB protein.

[0115] Kits and Related Compositions In some embodiments, the agents described herein are useful for therapeutic applications, diagnostic applications, or research. To facilitate their use in research applications, pharmaceutical or diagnostic kits or The present disclosure may be assembled into a research kit. The kit may include the components of the present disclosure and a method for use. and instructions for administering the compound to the subject. The present invention relates to a method for treating a patient with one or more of the agents described herein, the method comprising administering to the patient a therapeutically effective amount of the agent in accordance with the intended application of the agent. and instructions describing proper use. The drugs in the list are pharmaceutical formulations and dosages appropriate for the particular application and method of administration of the drug. Kits for research purposes may contain components in appropriate concentrations or amounts to perform a variety of experiments. may be contained in quantity.

[0116] In some embodiments, the present disclosure provides a kit for producing an rAAV, comprising: A container for storing an isolated nucleic acid having any one of the sequences 1 to 6 or 16 to 19. In some embodiments, the kit comprises instructions for making the rAAV. In some embodiments, the kit further comprises at least one and at least one container, wherein the recombinant AAV vector comprises a transgene. include.

[0117] In some embodiments, the disclosure includes a container for storing a recombinant AAV, as described above. In some embodiments, the kit includes a pharma- ceutically acceptable carrier. For example, the kit may include a container for storing the rAAV and a container for administering the rAAV to a subject. and a second container containing a buffer suitable for injection of the rAAV. In the embodiment, the container is a syringe.

[0118] The kits are designed to facilitate use of the methods described herein by researchers. Each of the components of the kit may be, where appropriate, liquid It may be provided in a soluble form (e.g., in a solution) or in a solid form (e.g., a dry powder). In certain cases, some of the compositions may be provided, e.g., in a kit. A suitable solvent or other type of vehicle (e.g., Can be configured or otherwise engineered by the addition of water or cell culture medium (e.g. As used herein, "instructions" refers to instructions and / or or promotional components of the present disclosure, typically including instructions for packaging the present disclosure. or associated with the packaging of the present disclosure. The instructions also include instructions for use in the kit and In any manner that clearly identifies the user with the relevant information, e.g. audiovisual (e.g., videotape, DVD, etc.), Internet, and / or web-based It may also include any verbal or electronic instructions provided, such as by communication of a The document is issued by a government agency that regulates the manufacture, use, or sale of drugs or biological products. Such instructions may also be in the form of instructions for manufacturing the product for administration to an animal. It may also reflect approval by a regulatory agency for the product, use, or sale.

[0119] The kit may include any one or more of the components described herein. The kit may be contained in a container or containers. By way of example, in one embodiment, the kit may include mixing one or more components of the sample and / or isolating and mixing the sample, The kit may include a container for storing the agents described herein and instructions for applying the agent to the subject. The drug may be in liquid, gel, or solid (powder) form. The drug may be prepared in a syringe, packaged, and shipped frozen. The second container may be a sterile prepared Alternatively, the kit may be premixed and includes syringes, vials, The kit may include, among other things, an active agent shipped in a bottle, tube, or other container. In the case of a kit for producing an animal model using specific somatic cells, a syringe, a topical Application devices, or IV needles, tubing, and bags, are used to administer drugs to animals. The present invention may include one or more or all of the components required to provide the

[0120] In some cases, the method comprises providing a cell with a proviral AAV genome at very low abundance. transfecting whole cell DNA isolated from a tissue that may be involved; Transcription of the AAV rep and cap genes in transfected cells Helper virus functions (e.g., adenovirus) may be used to induce and / or boost the expression of Optionally, the step of replenishing the R from the transfected cells. NA provides templates for RT-PCR amplification of cDNA and detection of novel AAVs The cells were then incubated with whole cell cultures isolated from tissues that may harbor the proviral AAV genome. When transfecting cells with AAV DNA, the cells are provided with factors that promote transcription of the AAV genes. For example, it is often desirable to supplement cells with adenovirus or herpes. The cells may be infected with a helper virus, such as a virus. The function is provided by the adenovirus. The adenovirus is a wild-type adenovirus. It can be of human or non-human origin, but is preferably can be from a non-human primate (NHP). Similarly, in the methods of the disclosure, Adenoviruses known to infect animals (e.g., chimpanzees, mice) can also be used. (See, e.g., U.S. Patent No. 6,083,716). In addition to the virus, recombinant viral or non-viral vectors carrying the necessary helper functions are available. Alternatively, vectors (e.g., plasmids, episomes, etc.) may be used. Such recombinant viruses are known and can be prepared in accordance with published techniques. US Patent No. 5,871,982 and US Patent No. 5,871,982, which describe lid Ad / AAV viruses, See U.S. Patent No. 6,251,677. Various adenovirus strains are available from Amer ican Type Cuture Collection, Manassas, Va. or available upon request from a variety of commercial sources and research laboratories. Additionally, the sequences of many such strains are available from sources such as PubMed. The sequences are available from a variety of databases, including GenBank and GenBank.

[0121] The cells can also express vectors that provide helper functions to the AAV (e.g., helper vectors). Vectors providing helper functions can also be transfected, e.g., E1a These include E1b, E2a, and E4ORF6. The sequences of the adenovirus genes that confer functionality are 40, etc., of the human serotypes identified herein that are known in the art. The adenovirus may be derived from any known adenovirus serotype, including any of the following: In some embodiments, the method includes inducing a cell to express AAV for replication, transcription of AAV genes, and / or or a vector expressing one or more genes required for AAV packaging. This involves a transfecting step.

[0122] Optionally, the novel isolated capsid gene can be recombinantly synthesized using methods well known in the art. Construct and package recombinant AAV vectors to express novel caspases encoded by genes These methods can be used to determine functional characteristics associated with novel single nucleotide polymorphisms. The abscised capsid gene is a reporter gene (e.g., B-galactosidase, GFP, Luciferase, etc.). Construct and package recombinant AAV (rAAV) vectors containing the ribosomal enzyme (RIA) The rAAV vector can then be delivered to an animal (e.g., a mouse). The tissue targeting properties of the newly isolated capsid gene have been demonstrated in various animal tissues (e.g. This can be determined by examining the expression of a reporter gene in the liver, heart, and kidneys. Other methods for characterizing novel isolated capsid genes are disclosed herein, Still other methods are known in the art.

[0123] The kits are available in blister pouches, shrink wrap pouches, vacuum sealing pouches, and seals. Ring thermoformed trays or accessories may be placed in a pouch, in one or more tubes, Similar pouches or containers packaged loosely in a container, box, or bag The kits may be in various forms, such as in a box or tray format. After the accessories are added, the kits are sterilized. This allows individual accessories to be placed in a container without being otherwise wrapped. The kits may be sterilized by radiation, heat, or other sterilization methods known in the art. The kits may also be sterilized using any suitable sterilization method, such as sterilization of the cellular component, ... or the like, depending on the specific application. and other components, such as containers, cell culture media, salts, buffers, reagents, syringes, needles, sterilization Gauze or other woven fabrics for applying or removing the agent, disposable gloves, and a hand towel for administration It may also include a support for the drug, as mentioned above.

[0124] The instructions included in the kit may include methods for detecting latent AAV in cells. In addition, the kits of the present disclosure may include instructions, negative and / or positive controls, containers, and diluents and buffers, sample preparation tubes, and a reference AAV sequence for sequence comparison. The table may include a printed or electronic table of:

[0125] Methods for Treating Lysosomal Storage Disorders - Patent application Aspects of the present disclosure provide methods for treating lysosomal storage disorders. Lysosome storage disorders (LSDs) are characterized by a loss of function of resident enzymes, leading to the accumulation of undegraded substrates in the lysosomes. Over time, this accumulation leads to dementia. This often results in somatic dysfunction and, as a result, cell death. LSD, which affects the central nervous system (CNS), leads to a cascade of events. The enzyme is delivered to target cells to achieve resolution of the disease by crossing the blood-brain barrier or One therapeutic delivery method involves the use of AAV-mediated targets. It was targeted gene delivery.

[0126] In some embodiments, the lysosomal storage disorders disclosed herein are invariably fatal. GM1 gangliosidosis is a chromosomal recessive disorder. β-galactosidase (OMIM number: 230500) encodes the enzyme β-galactosidase. It is caused by a mutation in the GLB1 gene. The amount of remaining β-gal activity and the disease Based on the incidence of disease, types I to III of GM1 gangliosidosis have been described, with type I being Type I is present in infancy, type II is present in late infancy or juvenility, and type III In some embodiments, the present disclosure relates to a method for treating GM1 syndrome in which the GM1 type is or suspected of having the same. Patients with type II GM1 who present in late infancy generally have onset of symptoms between 1 and 3 years of age. The average life expectancy is 5-10 years. These subjects meet developmental goals within the first year of life. However, they then begin to lose previously acquired abilities, such as speech and movement. Patients with juvenile onset have onset of symptoms between 3 and 10 years of age and similarly experience speech and motor difficulties. etc., indicates a decline in capabilities already achieved.

[0127] In some embodiments, the GLB1 is human GLB1 (GeneID:2720), NM_000404.3, NM_001079811.2, NM_001135602. 2, or the sequence set forth in NM_00137040.1. GLB1 is human GLB1, NP_000395.2, NP_001073279 .1, NP_00129074.1, or NP_001303969.1 In some embodiments, GLB1 comprises the amino acid sequence of mouse GLB1 (GeneID :12091), which includes the sequence set forth in NM_009752.2. In the present embodiment, the GLB1 is human GLB1 and has the amino acid sequence specified in NP_033882.1. In some embodiments, GLB1 comprises the sequence P10L, R59C, R59H, R1 21S, G123R, M132T, G134V, P136S, R148S, D151V, D151Y and / or reduced lysosomal β-gal activity. Any other mutations in GLB1 that are associated with the The present invention relates to a method for the preparation of a nucleic acid sequence for a nucleic acid fragment having a nucleotide sequence similar to that presented in Uniprot-KB, P16278, which is incorporated herein by reference. (including

[0128] In some aspects, the present disclosure provides methods for treating GM1 gangliosidosis. GM1 gangliosidosis is classified into type I GM1 gangliosidosis and type II GM1 GM1 gangliosidosis or type III GM1 gangliosidosis. Gangliosidoses are caused by a deficiency of β-galactosidase (β-gal) protein activity. Since GM1 gangliosidosis is caused by β-gal activity, a method for treating GM1 gangliosidosis is Non-limiting examples of methods for increasing β-gal activity include intracellular or intracellular A method for expressing wild-type GLB1 in an animal, a method for expressing a mutant GLB1 in a cell or a subject, Methods for reducing expression of the GLB1 gene in a cell or subject In some embodiments, the method includes providing a GLB1 protein (e.g., enzyme replacement therapy). In one embodiment, GM1 contains a transgene encoding GLB1 (e.g., wild-type GLB1). In some embodiments, the treatment may be by administering an isolated nucleic acid or rAAV containing the The Lance gene contains the wild-type GLB1 sequence, set forth in SEQ ID NO:23.

[0129] GLB1 activity in cells or in subjects with GM1 gangliosidosis is By carrying out the methods of the present disclosure, it can be increased. GLB1 activity in subjects with glial schizophrenia can be increased by 50% to 500% GLB1 activity in cells or in subjects with GM1 gangliosidosis is It can be increased by at least 50%. GLB1 activity in a subject can be increased by at least 500%. In subjects with GM1 gangliosidosis, GLB1 activity was 100% to 40%. 0% increase in cells or in subjects with GM1 gangliosidosis. In vivo, GLB1 activity can be increased by 200% to 500%. In subjects with gliosidosis, GLB1 activity was 50%, 60%, 70%, 80%, 90%, 100%, 120%, 140%, 160%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 32 0%, 90%, 100%, 125%, 150%, 175%, 200%, 225%, 250 %, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450 %, 475%, or 500%.

[0130] The method for treating GM1 gangliosidosis in a subject comprises administering to a subject a human GLB1 antibody. Administering an isolated nucleic acid, rAAV, or composition of the disclosure that contains a transgene that encodes The subject may be a human, a mouse, a rat, a pig, a dog, a cat, or a non-human. The administering step may be performed in a cell or in a subject, by administering to the subject an isolated antibody or antibody of the present disclosure. The term "administering" refers to contacting with a nucleic acid, rAAV, or composition. Non-limiting examples include intravenous injection, intraarterial injection, intracranial injection, intrathecal injection, intracerebral injection, infusion, or includes inhalation.

[0131] In some embodiments, the lysosomal storage disorder is a gene, HEXA, HEXB, and autosomal, caused by mutations in at least one of GM2A Tay-Sachs disease (also known as GM2 gangliosidosis), a recessive disorder Tay-Sachs disease (TSD) (OMIM number: 272800) is a rare disease that affects infants, Present in the first year of life or in adults, and during the second or third year of life, mental retardation, dementia, blindness, It is a universally fatal disorder, with symptoms including nausea, vomiting, and death. Hexosaminidase A (HexA), hexosaminidase B (HexB), and GM2 ganglioside Wild-type levels of all three GM2A ganglioside activators inhibited the degradation of GM2 gangliosides. A decrease in the activity of any of these three enzymes results in the It is associated with increased accumulation of GM2 ganglioside. Patients with TSD have a central nervous system disorder. For ganglion cells that have lipid deposits while leaving behind the characteristic "cherry red spots" The retina is characterized by a gray-white area around the fovea. does not exist.

[0132] In some embodiments, the lysosomal storage disorder of the present disclosure is a mutation in the HEXB gene. Sandhoff disease (GM2 ganglioside), an autosomal recessive disorder caused by Sandhoff disease (SD) (OMIM:268800 ) is a universally fatal disorder. Patients with SD typically present within the first 6 months of life with Debilitation develops followed by an exaggerated startle response, early blindness, progressive motor and intellectual decline, Doll-like facial features, cherry red spots, and macrocephaly develop. Death typically occurs by age 3 years. This occurs in.

[0133] In some embodiments, HexA is human HexA (GeneID:3073), Contains the sequence specified in NM_000520.5 or NM_001318825.1. In some embodiments, the human HexA is NP_000511.2 or NP_00130 In some embodiments, HexA comprises the amino acid sequence set forth in The gene was HexA (GeneID:15211) and was identified in NM_010421.5. In some embodiments, the mouse HexA comprises the sequence set forth in NP_034551.2. In some embodiments, the HexB comprises an amino acid sequence selected from the group consisting of human HexB (GeneI D:3074) and is listed in NM_000521.3 or NM_001292004.1. In some embodiments, human HexB comprises the sequence set forth below. or the amino acid sequence set forth in NP_001278933.1. In this case, HexB is mouse HexB (GeneID: 15212) and NM_010 In some embodiments, the mouse HexB comprises the sequence set forth in NP_0. In some embodiments, the GM2A comprises the amino acid sequence set forth in SEQ ID NO: 34552.1. GM2A (GeneID:2760), NM_000405.4 or NM_0 In some embodiments, the human G2MA comprises the sequence set forth in Amino acid sequence specified in NP_000396.2 or NP_001161079.1 In some embodiments, the GM2A is mouse GM2A (GeneID: 14667 ) and comprises the sequence set forth in NM_010299.3. The gene GM2A contains the amino acid sequence set forth in NP_034429.1.

[0134] In some embodiments, the present disclosure provides a method for the preparation of a human GLB1, human HexA, or human HexB. Isolated nucleic acids and recombinant AAVs containing the encoding transgenes are provided. In one embodiment, the present disclosure provides a gene encoding mouse GLB1, mouse HexA, or mouse HexB. In some embodiments, the present invention provides an isolated nucleic acid and a recombinant AAV comprising a transgene comprising: GLB1, HexA, and / or HexB may be used as a promoter, optionally a specific operably linked to a tissue-specific promoter that drives expression of the transgene in the tissue. In some embodiments, the specific tissue is within the CNS. The method comprises administering to a subject in need thereof an isolated nucleic acid or a recombinant AAV, as described above. Methods of administering the composition are provided. In some embodiments, the subject to whom the composition is administered is a patient with G including, but not limited to, M1 gangliosidosis, Tay-Sachs disease, or Sandhoff disease have or are suspected of having an unspecified lysosomal storage disorder.

[0135] In some embodiments, the subject has a nucleic acid encoding human HexA and / or human HexB. The subject is administered a rAAV or pharmaceutical composition of the present disclosure that contains a transgene, in which case the subject at least 10% of normal enzyme activity 3 months after administration of the rAAV or pharmaceutical composition. 0.5%, at least 1.0%, or at least 1.4% of β-heterozygote in CSF. In some embodiments, the enzyme activity is increased in the MU In some embodiments, the subject is The present disclosure includes a transgene encoding human HexA and / or human HexB. The rAAV or pharmaceutical composition is administered to the subject, whereby the β-hexosaminidase inhibitor is present in the CSF of the subject. After 3 months, the enzyme activity of α-D-glucose dehydrogenase A was measured relative to the baseline enzyme activity of the subjects before administration. In some embodiments, the expression level of the IL-1 gene is increased by at least 2-fold, or at least 3-fold, compared to the expression level of the IL-1 gene. In the present invention, the subject has Tay-Sachs disease (eg, infantile Tay-Sachs disease).

[0136] In some embodiments, GM2 ganglioside is a component of the rAAV or pharmaceutical composition of the disclosure. By approximately three months after administration, or at this time, baseline cerebrospinal fluid (CSF) levels in the subject At least 10%, at least 15%, at least 20%, at least 2 1%, at least 22%, at least 23%, at least 24%, or at least 25% % reduction.

[0137] Thus, in some embodiments, the present disclosure provides compounds useful in treating lysosomal storage disorders. In some embodiments, the isolated nucleic acid, rAAV, composition, and method are provided. Nucleic acids, rAAV, compositions, and methods for treating GM1 gangliosidosis In some embodiments, the isolated nucleic acid, rAAV, compositions, and methods. The AAVs, compositions, and methods are useful in the treatment of Tay-Sachs disease. In embodiments, the isolated nucleic acids, rAAV, compositions, and methods are used in the treatment of Sandhoff disease. It is useful.

[0138] In some aspects, the present disclosure provides methods for treating GM2 gangliosidosis. GM2 gangliosidosis may be Tay-Sachs disease or Sandhoff disease. GM2 gangliosidoses are characterized by deficiency of HEXA enzyme activity and / or HEXB enzyme activity. Since GM2 gangliosidosis is caused by a deficiency in GM2 activity, a method for treating GM2 gangliosidosis includes: This results in increased HEXA and / or HEXB activity. Limited examples include wild-type HEXA and / or wild-type HEXA in a cell or in a subject. Methods for expressing mutant HEXA and / or B in a cell or a subject The present invention relates to a method for reducing expression of the HEXB gene or for inhibiting the expression of a wild-type HEXB gene in a cell or a subject. Methods for producing wild-type HEXA and / or wild-type HEXB proteins (e.g., enzyme substitutions) In some embodiments, GM2 is fused to at least one HEXA or HE A transfectant encoding XB (e.g., wild-type HEXA and / or wild-type HEXB) Treatment can be by administering an isolated nucleic acid or rAAV containing the gene. In one embodiment, the transgene comprises the wild-type HEXA sequence set forth in SEQ ID NO:20. In some embodiments, the transgene comprises the wild-type HEXB sequence set forth in SEQ ID NO:21. Includes.

[0139] HEXA and / or in subjects with GM2 gangliosidosis Alternatively, the activity of HEXB can be increased by practicing the methods of the present disclosure. or HEXA and / or H in subjects with GM2 gangliosidosis The activity of EXB can be increased by 50% to 500%. The activity of HEXA and / or HEXB in a subject with idiopathies is at least Intracellular or in subjects with GM2 gangliosidosis, In the present study, the activity of HEXA and / or HEXB can be increased by at least 500%. Intracellular or in subjects with GM2 gangliosidosis, HEXA and / or the activity of HEXB can be increased by 100% to 400%. HEXA and / or HEXB activity in subjects with 2 gangliosidoses can be increased by 200% to 500%. Intracellular or GM2 gangliosidosis In subjects with HEXA and / or HEXB activity was 50%, 60%, 70%, or 0%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 225% , 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425% , 450%, 475%, or 500%.

[0140] The method for treating GM2 gangliosidosis in a subject comprises administering human HEXA and The isolated nucleic acid of the present disclosure, comprising a transgene encoding human HEXB, rAA The subject may be a human, a mouse, a rat, a pig, or the like. The animal may be a human, dog, cat, or non-human primate. contacting a subject with an isolated nucleic acid, rAAV, or composition of the present disclosure. Non-limiting examples of the administering step include intravenous injection, intraarterial injection, intracranial injection, intrathecal injection, and the like. This includes injection, intracerebral injection, infusion, or inhalation. EXAMPLES

[0141] [Example 1] material and method Vector design, construction, and virus generation An AAV vector (SEQ ID NO: 3) was constructed, which encodes the chicken beta-actin promoter. Following the cytomegalovirus (CMV) immediate-early enhancer fused to the motor, Chimeric chicken beta-actin / rabbit beta-globin intron (CBA), mouse Sosomal acid β-galactosidase cDNA (mβgal), woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and bovine growth hormone (BGH) and A promoter consisting of two tandem polyA signals derived from SV40 This vector carries an expression cassette driven by the AAV-CBA-mβgal AAV-CBA-mβgal E269Q-WPRE is a recombinant vector containing the following promoter: Immer: Forward 1: AAA CGT CTC ACT AGT CCG CGG A AT TC (SEQ ID NO: 7), reverse 1: AAA CGT CTC ACT GAG A AT TGA TCA AA (SEQ ID NO: 8), forward 2: AAA GGT CTC CGG CCG CTA GCG TCA G (SEQ ID NO: 9), reverse 2: AAA G GT CTC ATC AGT TCT ATA CTG GC (SEQ ID NO: 10) The resulting PCR product was amplified by PCR mutagenesis using SpeI and The cDNA was digested with the restriction enzymes NotI and NotI and cloned in place of the wild-type βgal cDNA. All other AAV vectors were derived from the AAV-CBA-mβgal-WPRE vector (SEQ ID NO: All AAVrh8 vector strains were generated by removing different elements from the AAVrh8 vector strain No. was prepared by standard methods.

[0142] Animal procedures The neomycin cassette in exon 6 of the β-galactosidase gene, GLB1 GM1-gangliosidosis mice, a knockout mouse model created by inserting (βgal - / - ) βgal - / - 、 βgal + / - , and βgal + / + Ma The mouse is a male βgal - / - Mice and female βgal + / - By crossing with mice, or gal + / - It is produced by mating a male and a female of the same species.

[0143] Intracranial injection βgal, 6-8 weeks old - / - Mouse or βgal + / - Mice were given 0.9% saline. Ketamine (125 mg / kg) and xylazine (12.5 mg / kg) in saline solution. The animals were anesthetized via intraperitoneal injection and placed in a rodent stereotaxic frame. The hair was shaved and the skin was wiped with povidone-iodine pads and 70% EtOH. The skull was exposed through a small longitudinal incision (<1 cm) along the nasal passage. The periosteum was then removed from the surgical field. Using a high-speed drill, the periosteum was removed from the surgical field at the appropriate stereotaxic coordinates. A small burr hole (<1 mm) was then made, and a 10 μl airtight syringe with a 33G needle attached was then inserted. An Ultramicro Pump is used to drive a 0. 1 μl of AAV vector or PBS was added to βgal at a rate of 2 μl / min. - / - Mouse Or βgal + / - On both sides of the mouse, the thalamus (stereotaxic coordinates: anterior-posterior coordinate: -2.0 m m; mediolateral coordinates: ±1.5 mm from the bregma; dorsoventral coordinates: -3.5 mm from the brain surface). , 0.3 or 1 μl, βgal - / - Mouse deep cerebellar nucleus (anteroposterior coordinate: -6.0 mm ; mediolateral coordinates: ±1.5 mm from the bregma; dorsoventral coordinates: -3.5 mm from the brain surface) Injection began 1 min after needle placement in the target structure and ended 2.5 min after injection. The scalp was closed with sterile wound clips (9 mm).

[0144] Behavioral assays The rotarod test consisted of a rotarod test in which the rpm was accelerated from 4 to 40 over a period of 5 min. The test was conducted on a walking device and the latency to fall was recorded. followed by one training trial in which the speed was accelerated from 2 to 20 rpm over a period of 1 min, with 15 Three trials were conducted, with a rest period of ~20 min between each. The latency to fall for each subject was recorded and the longest fall on the rotarod was recorded among the three trials. Time reported.

[0145] Tissue processing βgal + / - For biochemical studies in mice, brains were removed and cultured on a Brain Mat. Slice into 2 mm coronal blocks using rices and freeze instantly on dry ice. The block containing the thalamus was then examined for shape and location of the needle insertion point on the dorsal brain surface. Biopsy punches with a diameter of 2 mm were used to sample the thalamus and were then used for analysis. For histological studies, the brain and spinal cord were isolated from the tissue plugs, which were then placed in an appropriate buffer. They were excised, placed in Neg 50 refrigerant, and frozen in a dry ice / 2-methylbutane bath. βgal - / - For biochemical studies in mice, cerebrum, cerebellum + brainstem, and spinal cord were They were excised and flash frozen on dry ice.

[0146] Histological analysis Brain (sagittal and coronal) and spinal cord (transverse) sections of 20 μm were cut using cryoscopy. The tissue was excised using Tat and stored at -80°C.

[0147] As previously described, but with modifications, To determine the extent of the staining, the brain sections were stained with X-gal. Briefly, slides were stained with 0.5% Fix in 100 mM glutaraldehyde and store in ice-cold citrate phosphate buffer (CPB) (50 mM C 6H8O, 50 mM Na2HPO4, 10 mM NaCl, pH = 4.2) The cells were washed for 10 min and stained with X-gal staining solution [20 mM K4Fe(CN)6, 20 mM K3F e(CN), 2 mM MgCl, 0.01% C 24 H 39 NaO4, 0.02% (C2H4O)nC 14 H 22 O (IGEPAL CA-630, Sigma Aldr ich), 2 mg in 97% CPB, HCON(CH3)2 at pH=4.2 / ml 5-bromo-4-chloro-3-indolyl-β-D-galactosidase (Xg The slides were incubated overnight in CPB (100 mL). Then, rinse in water and counterstain with Vector Nuclear Fast Red. The tissue was then dehydrated in a 50% to 100% ethanol series, cleared in CitriSolv, and I mounted it with rmount.

[0148] Lysosomal accumulation was assessed as previously described, with modifications. To detect the phenotype, brain and spinal cord sections were stained with filipin. Fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) and Wash with 100 mL of glycine in water, incubate with 1.5% glycine in water, wash with PBS, and 1 μg / ml filipin, and 1 μg / ml ToPro3 iodide (Life Technologies chnologies, Grand Island, NY) for 1-2 minutes. Incubate, wash with PBS, and mount in fluorescent mounting medium, PermaFlu. Mounted by or.

[0149] To assess morphological changes within the tissue, brain sections were stained with Mayer's hematoxylin / eosin. Briefly, slides were dried at room temperature and stained with phosphate buffered saline (PBS). The cells were fixed in 4% paraformaldehyde in PBS, washed with water, and then analyzed by Mayer's hematoxylin. Incubate with phosphorus, wash with drained tap water, counterstain with eosin, and demineralized water. The tissue was then rinsed, dehydrated in a 50% to 100% ethanol series, cleared in CitriSolv, and stored in a 5% CO2-freezer. Mounted with ermount.

[0150] Whole-brain slice images were taken using a Nikon Super C equipped with a medical slide holder. The microscopy images were captured on a LightScan 5000 ED using white light. ica DFC425 C digital camera and ica DFC365 FX digital camera The images were captured on a Leica DM550 B Microscope equipped with a 100mW IR microscope. The fluorescein was imaged at 405 nm, and ToPro3 iodide was imaged at 636 nm. H&E was imaged using bright field.

[0151] All histological analyses were performed on N≧2-3 animals, with representative photographs shown in the figures. The study was conducted as an unblinded, qualitative analysis.

[0152] Enzyme assay and immunoblotting for βgal Biopsy punches were lysed in lysis buffer (0.2 M CHCOONa, 0.1 M NaCl, pH In step 4.3, homogenize in 0.1% Triton X-100 and add β-gal enzyme. Briefly, βgal substrate = 1 mM 4-methylumbelliferyl The reaction with 4-methyl-β-D-galactoside (4-MUG) was performed in a 96-well plate format. The amount of 4-methylumbelliferyl (4-MU) released was measured using BioT Excitation at 360 nm and The enzyme activity was measured against a standard curve by fluorescence detection via emission at 460 nm. The quality was normalized according to protein content as determined by Bradford reagent assay. and reported as nmoles substrate converted per mg protein per hour. For immunoblotting, biopsy punches from the injection sites were taken at 100 μg / mL for 1 min. Homogenize in T-PER buffer supplemented with protease inhibitor cocktail and store on ice. Incubate at 4°C for 10 min, then at 10,000×g for 5 min. The supernatant was collected and the protein concentration was determined using the Bradford Reagent Assay. The degree of adhesion was determined using the Mini-PROTEAN TGX precast gel. Total protein (20 μg) was separated by acrylamide gel electrophoresis. The blots were transferred to NitroPure nitrocellulose membranes. Block with Tris-buffered saline-Tween-20 (TBST) and then , α-rabbit GLB1 (β-galactosidase antibody) (1:250) and α-mouse β The mice were incubated with primary antibody against actin (1:1000). The signals were detected using 1:4000 anti-rabbit and anti-mouse secondary antibodies. Detection was performed using Pierce ECL Western Blotting Substrate. te and the blots were exposed to Amersham Hyperfilm ECL. did.

[0153] Genome Copy Using the Qiagen DNeasy Blood and Tissue kit, Genomic DNA derived from a biopsy punch of the injection site was isolated and analyzed using a Nanodrop spectrophotometer. The concentration was determined using the following primers specific for the BGH polyA in the vector genome: mArs and Taqman probes: (TaqMan Probe, 6FAM-AGC A TT TTT TTC ACT GCA TTC TAG TTG TGG TTT G Genomic DNA 100n was analyzed by qPCR using TC-TAMRA (SEQ ID NO: 11). The AAV vector genome copy number per μg of DNA was determined. Samples where the .gamma.-to-.gamma.+-100 vg were considered positive for vector genome.

[0154] Microarrays Total RNA was isolated from biopsy punches using Trizol and RNeasy Plus s Use the Agilent Bioanalyze Mini Kit for further purification The quality of the samples was analyzed using the Bioanalyzer RIN (RNA inte The grity number values ​​were 8.7-9.5, indicating high quality RNA. Sample preparation and microarray hybridization were performed by Affymetrix. The experiment was performed using Mouse Gene 2.0ST Arrays. Three samples were collected per group. Independent samples for each case were analyzed. The resulting data were processed and analyzed for P values ​​of <0.05. and 1.5-fold change over the PBS control were considered to be differentially expressed genes.

[0155] Quantification of GM1 ganglioside content The GM1 content in the CNS was determined by liquid chromatography / tandem mass spectrometry (LC-MS / The amount of the tissue was quantified by MS. Briefly, 25 μl of tissue (0.01-0.04 mg / μl) was used. l was homogenized in 0.1 M NaCl, 0.2 M CH3COONa at pH 4.3. The internal standard, 3 μg of d3-GM1, was added and extracted by the modified Folch extraction method. The sample was then passed through a C18 column, dried, and purified using runnier. The samples were reconstituted in a dilution buffer and then analyzed by LC-MS / MS using a Waters Quattro o Processed by Premier XE. Samples were eluted at a mass / charge ratio (m / z) of 290. The GM1 molecular species with different fatty acid compositions were separated and quantified. The purified G was determined by calculating the ratio of the sum of all molecular species to the standard curve. The results are plotted against the ratio of M1 / d3-GM1. Samples were analyzed using the Bradford Reagent Assay. Normalized for protein content in the initial lysate, determined by Reported as ng of GM1 per sample.

[0156] Exemplary Constructs A recombinant AAV vector encoding the human GLB1 gene was generated (rAAV9hG LB1 vector) (SEQ ID NO: 23). This is a replication-deficient adeno-associated viral gene transfer vector. The single-stranded DNA genome contains splice donor sites and splice access sites. Chicken β-actin with an intron from rabbit β-globin containing the adapter site It is driven by the cytomegalovirus immediate-early enhancer fused to the promoter The expression cassette is composed of human β-galactosidase (βgal) cDNA. The construct also consists of inverted terminal repeat sequences derived from AAV2, simian virus 40. The genome also contains a polyadenylation signal derived from human AAV. The virus is packaged into the AAV serotype 9 capsid (Gao 2004) (Figure 1B). The pharmaceutical composition is formulated in a buffered aqueous suspension for intravenous delivery.

[0157] C57BL / 6 mice were treated with PBS (control) or with The treated mice were injected with 2.25 × 10 1 1 Each cohort (n=5) was administered 100 mg of AAV9-hGLB1 at 30 days post-treatment. The mice were euthanized after 30 days and 30 months. β-4-methyl-2-pyridyl-2-pyridyl, a fluorogenic substrate that emits fluorescence when cleaved by β-methyl-2-pyridyl ..., isoform of β-galactosidase activity was measured from the samples using umbelliferone (4-MU). Increased virulence was observed in the AAV9-hGLB1-treated group compared to PBS-injected control mice. (data not shown).

[0158] [Example 2] AAV dose-dependent distribution of βgal in the brain The AAVrh8-CBA-mβgal-WPRE vector (Figures 1 and 20) was diluted with 4 × 1 0 10 vg, 2.6×10 10 vg, and 2.6 × 10 9 In total dose of vg, thalamus Bilateral injections into the cerebellar and deep cerebellar nuclei of the 6- to 8-week-old GM1 gangliosidosis mice S(βgal - / - Animals in the highest dose cohort were injected into the brain with intrathalamic and D Animals in the other two cohorts received bilateral injections of 1 μl into the CN, while animals in the other two cohorts received intrathalamic The subjects were administered 1 μl in the nascent ... The distribution pattern of β-gal activity was dose-dependent, with β-gal activity at the injection site at its highest intensity. The highest dose (4 × 10 10 vg) in the cerebral cortex (Fig. 2A) and the subcerebral cortex The medium dose, 2.6×, resulted in enzyme activity across most sections in the brain (FIG. 2B). 10 10 vg showed similar activity levels in the cerebrum (Fig. 2E), but not in the cerebellum (Fig. 2F). Although activity was slightly reduced, the enzyme still spread over the structure. The low dose was 2.6 × 10 9 The vg is widespread in both the cerebrum and cerebellum. 2I and 2J, respectively.

[0159] AAV-treated animals retain motor function in a dose-dependent manner The motor function of AAVrh8-treated mice was assessed over time using the rotarod test. The effect of AAVrh8 on βgal expression was evaluated (Figure 3). - / - All cohorts were included in the six treatment periods. At the month later, untreated βgal - / - The results were significantly better than those of the control group (high use Quantity: 4×10 10 vg;p=0.006, medium dose: 2.6×10 10 vg;p=0.00 09, and low dose: 2.6 × 10 9 vg; p=0.005). Six months after treatment , N=6-10 animals per group.

[0160] [Example 3] AAV treatment extends life span AAVrh8-treated βgal - / - Mice longevity is similar to that of naive βgal - / - Comparison with control The time to maturation was significantly prolonged by the addition of β-gal - / - The median survival for controls was , 245.5 days (N = 18), 4 × 10 10 For the vg cohort, 293. 5 days (N = 12, p = 0.0004), and 2.6 × 10 10 About the vg cohort was 349 days (N = 13, p = 0.002), and 2.6 × 10 9 Regarding the vg cohort For the control group, the mean mean was 389 days (N=12, p<0.0001).

[0161] Accumulation of GM1 ganglioside at the injection site and spinal cord in long-lived AAV-treated animals Persist Lysosomal storage was detected by filipin staining in the CNS of animals 3 months after injection. Histological analysis of the cytoplasmic mass revealed no significant difference in the presence of the enzyme compared with Xgal staining at the same time point. The results showed that the cerebrum and cerebellum responded to the stimulation with almost complete correction (Figure 2). In contrast, filipin-positive cells were found only at the injection site or along the injection route. (Figures 5C and 5E).

[0162] βgal injected with AAVrh8 - / - Presence of filipin-positive cells in the mouse thalamus The present study also showed that the Xg expression level was highest in the brain, which is also a semiquantitative indicator of high β-gal enzyme activity. This is surprising, since these are also brain regions that exhibited α-histological staining (Figure 5A, boxed). In the spinal cord of long-lived AAV-treated mice (495–612 days), lysosomal The effect on accumulation was that the remaining filipin-positive cells were distributed from the areas with very few remaining filipin-positive cells to the untreated areas. Place βgal - / - Variable, with no obvious changes observed in the area compared to the control. (Figures 6A, 6D, and 6F, and 6B, 6C, and 6E, respectively).

[0163] [Example 4] Neuropathology at the injection site After early loss or 3 months after injection, animals were cultured using hematoxylin / eosin (H&E The animals showed dose-related changes in shape at the injection site. Two weeks after injection, βgal - / - + AAV4 x 1 0 10 vg was associated with cellular infiltration into blood vessels and the appearance of inflammation (Figures 7A and 7B, respectively, indicated by thick arrows). There were numerous changes in the thalamus, including vascular cellular infiltration, and clear Large endothelial cells in the DCN with extensive neuronal phagocytosis (arrows in Fig. 8A and Fig. 8B, respectively) Three months after treatment, the amount of βgal - / - +AAV2×10 10 v g also showed thalamic changes with cellular infiltration of blood vessels and inflammation (Figure 7 E, 7F, thick and thin arrows). However, the dose was 2 × 10 10 vgβgal - / - + AAV caused only low-grade cell infiltration into blood vessels in the DCN (Fig. 8E, Taiya (The symbol indicates the lowest dose of the representative animal, 2 × 10 9 vg βga l - / - In +AAV, only very slight inflammation was observed in the thalamus (Fig. 7J, This effect was not seen in the DCN (Figures 8I and 8J). - / - Control (Figures 7C, 7D, and Figures 8C, 8D) or untreated βgal + / - Contrast (Fig. 7G, 7H, and Fig. 8G, 8H), no cellular infiltration into blood vessels or inflammation was observed. .

[0164] High levels of βgal induce unexpected responses in injected brain structures The paradoxical presence of filipin-positive cells at the injection site (Figures 5C and 5E) was consistent with the βgal - / - This may be the result of an unexpected response to AAV gene transfer in mice. To understand the phenomenon, normal, non-diseased βgal + / - Littermates were then injected with the AAVrh8 vector. was injected intracranially. - / - Similar to the results in mice (Figures 5C and 5E), Vrh8 injected βgal + / - In mice (Fig. 9D, 9G, and 9J), the βgal staining was There were also numerous filipin-positive cells within the brain regions with the highest intensity (FIGS. 9A, 9B). These results suggest that the presence of filipin-positive cells in the targeted brain structures is a key factor in the pathogenesis of AAV pathology. This indicates an unexpected adverse response to certain aspects of gene transfer.

[0165] [Example 5] Enzyme activity, protein levels, and AAVrh8 capsid response to filipin detection Validation of the AAVrh8 vector series to assess the contribution of To investigate the nature of unexpected responses at the injection site, A series of vectors were designed (Table 1). Further vectors were designed and are shown in Figure 19. This AAVrh8 series is hereafter referred to as "CBA-WPRE". The transgene was inserted into the vector AAVrh8-CBA-mβgal-WPRE. The essence of this vector is the sequential removal of elements that affect the expression level of In "CBA", the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) is removed. Vector 3, "CBA-EI-WPRE", has exactly the same bone marrow as Vector 1. βgal protein, but carrying the E269Q mutation within the putative active site This vector encodes for the .alpha., ... This study was designed to assess whether the effects of the β-amyloid protein on the expression of β-amyloid proteins were mediated by the β-amyloid protein. Vector 4, "CB6", contained the mβgal cDNA but not the mβgal cDNA in another vector. This vector did not carry the WPRE or chimeric intron present. "Low-dose CB6" (same dose as all other vectors), and "High-dose CB6" (2.0 × 10 10 Two doses of vector 5, the "empty transgene" or "T.Empty" contained all the components of Vector 1, but not the mβgal cDNA. Missing A.

[0166] The newly constructed AAVrh8 vector was administered at a high dose (2.0 × 10 10 Total dose of vg In contrast, 1 μl per site was also injected, with the exception of the CB6 vector, which was injected bilaterally. And normal βgal + / - Injected into the thalamus of mice (3.4 × 10 9 vg total dose Controls were injected with phosphate buffered saline (PBS). βgal + / - Mice, and naïve βgal + / - The enzyme activity in the thalamus was Sex and protein production were measured 6 weeks after injection of 4-methylumbelliferyl (4-MU ) as measured by biochemical assays and Western blot (Figure 10).

[0167] [Table 1]

[0168] The CBA-WPRE vector expresses naive βgal + / - Enzyme activity in the thalamus of mice (Figure 10A), and the corresponding increase in protein (Fig. 10B, lane 1). The CBA vector was + / - 224 times the level of βga This resulted in β1 activity, which was not observed in the CBA-WPRE vector. The activity of the protein product was significantly lower than that of the gal activity (Fig. 10A, p=0.001). This resulted in a correspondingly clear decrease (Figure 10B, lane 2). In the thalamus of vector-injected mice, βgal activity was significantly higher than in naive control mice. βgal activity was comparable to that of CBA-WPRE-injected cells (Fig. 10A). The protein was expressed at levels comparable to those in the thalamus (Fig. 10B, lane 1). Thus, the E269Q mutation impairs the enzyme activity but not the protein expression level. Injection of the CB6 vector at the same dose as the other vectors (low dose CB 6) is βgal + / - βgal activity was 54-fold higher than in the control (Fig. 4A). Accordingly, this resulted in the presence of lower amounts of protein than with other vectors. Injection of this vector at a high dose (high dose CB6) is βgal + / - This resulted in 420-fold increased β-gal activity compared to the levels induced by This was significantly higher than the βgal activity in the low-dose CB6 cohort (Figure 1). 10A, 10B, p=0.03). βgal activity levels in the high-dose CB6 cohort were significantly higher than in the control cohort. The βgal activity levels were comparable to those measured in the BA-WPRE cohort, and the protein levels were The results were comparable for both the control and control groups (Figures 10A and 10B, lane 1 compared with lane 5). The thalamus of animals injected with empty transgene vector (T.Empty) or PBS was compared with that of naive mice. buβgal + / - Changes in β-gal activity or protein levels compared to levels in The vector genome colocalization in the AAV-injected thalamus was not observed (Fig. 10A, 10B). The number of samples was 3.4 × 10 except for the T.Empty cohort. 9 was injected with a total dose of vg. , and were shown to be comparable within most cohorts (Figure 11). As expected, the 2.0 × 1 0 10 The high-dose CB6 cohort injected with CB6 showed a significant increase in vector genome copy number. The results showed that the α-amino acid residues in the α-amino acid residues were significantly increased (Figure 11).

[0169] Filipin detection response correlates with protein levels βgal injected with AAVrh8 + / - Mice, and control βgal + / - Mouse Brains were analyzed for β-gal enzyme activity by Xgal staining (Figures 12A, 12D, 12E). G, 12J, 12M, 12P, 12S), the presence or absence of filipin-positive cells in the thalamus The presence of 12B, 12E, 12H, 12K, 12N, 12Q, and 12T was analyzed. The thalamic regions with the greatest intensity of βgal staining (Figures 11A, 11D) were also located in the CBA-WP RE- and CBA-injected animals also contained filipin-positive cells, but the latter Within the CBA-EI-WPR cohort, they were clearly underrepresented (Figures 11B, 11E). The thalamus in the E cohort contained numerous filipin-positive cells but no active β-gal enzyme. No filipin staining was observed in the T-Empty control (Fig. 11K and 11J, respectively). This was also evident in the thalamus of the low-dose CB6 cohort (Fig. 11K, 11Q). observed in the CBA-WPRE and CBA-EI-WPRE cohorts The filipin staining method is also widely used. The pattern was also observed in the thalamus of the CB6 cohort, and was consistent with that observed in the CBA-WPRE cohort. The pattern is a mix of what is expected and what is observed within the low-dose CB6 cohort. These results suggest that the abnormal accumulation of filipin at the injection site was These results indicate that the expression level of the protein is related to its expression level and not to its enzyme activity.

[0170] Transcriptome changes in the injected thalamus were consistent with abnormalities detected by filipin. Correlates with the expected response Microarray analysis was performed in the thalamus to assess physiological changes induced by gene transfer. We further characterized the tissue response to CBA-mβgal-WPRE. cohort, low-dose CB6 cohort, T.Empty cohort, and PBS-injected cohort. The transcriptome changes (fold changes > 1.8-fold, p<0.05) are shown in the heat map (Figure 13A). The T.Empty and T.Empty samples clustered together with the PBS samples, and were distinct from the CBA-WPRE samples. The number of genes with >2-fold change in expression level was duplicated in the CBA-WPRE samples. The number of genes was significantly larger than in the CB6 and T.Empty samples, which had a small number of genes. (p<0.05) (Fig. 13B). Genes characteristic of activated microglia and reactive astrocytes (Table 2). None of the genes showed significant changes in the CB6 and T.Empty samples. Ta.

[0171] [Table 2]

[0172] [Example 6] Therapeutic effects of different AAVrh8 vectors in GM1 gangliosidosis mice The above, βgal + / - Studies conducted in mice have shown that The reduction in protein expression reduces pathological changes in transcription levels in the injected structures. We then determined whether changes in AAV vector design could affect the expression of GM1 ganglionic vesicles. Osidosis mice (βgal - / - ) led to differences in treatment outcomes in We investigated the role of βgal in 6- to 8-week-old mice. - / - The AAV vector was injected into the thalamus (hematology) of mice. Bilateral injections were given into the deep cerebellar nuclei (1 μl per side) and deep cerebellar nuclei (0.3 μl per side). Outcomes were measured approximately 6 weeks after the study. The study cohort consisted of 4.4 × 10 9 Total dose of vg CBA-WPRE, CB6 (low dose CB6), and empty transgenes administered at Injected with vector, βgal - / - In addition, CB6 was added at 2.6 × 1 0 10 vg total dose (high dose CB6). - / - Animals and naive untreated βgal + / +The animals were used as controls. Assessment of βgal activity by MU assay (Figure 14) demonstrated that CBA-WPRE inhibited βgal activity in the brain. , 45-fold higher than wild-type levels (FIG. 14), whereas low-dose CB6 was 9-fold higher (FIG. 1 4) showed that high dose CB6 increased βga by 30-fold (Figure 14). l - / - The T.Empty and untreated cohorts of mice were not associated with any of the analyzed CNs. There was no detectable βgal activity in the S region (Figure 14). The induction of CBA-WPRE was 47-fold higher than the wild-type level (Figure 14), whereas the induction of CBA-WPRE was 47-fold higher than the wild-type level (Figure 14). CB6 was 6-fold (Figure 14), and high dose CB6 was 27-fold (Figure 14). Unfortunately, in the spinal cord, CBA-WPRE was only 50% of wild-type levels (Fig. 14). The high dose CB6 was 30% (Figure 14), and the low dose CB6 had no detectable activity. In all CNS tissues analyzed, high dose CB6 reduced βgal activity and significantly higher than low-dose CB6 of the same vector injected at 100 mg / kg / day (p<0.001). It was big.

[0173] The distribution pattern of β-gal in the brain, as confirmed by the histological dye X-gal (Figure 15) was determined by the 4-Mu assay to determine the activity level in the cerebrum or cerebellum + brainstem. The CBA-WPRE vector increased the expression of dark blue staining in the thalamus and DCN. This resulted in widespread distribution of color and detectable enzyme activity throughout the brain. (Fig. 15A). In contrast, the low-dose CB6 cohort (Fig. 15B) showed increased expression in the thalamus and DCs. Intense staining was seen in the N, but no detectable levels were observed throughout the cerebrum, cerebellum, or brainstem. In high-dose CB6 animals, the brain distribution pattern of βgal was low. The distribution pattern in the brain appeared to be more widespread in CB6 animals (Figure 15C). As expected, the T.Empty cohort (Figure 15D) and naive βgal - / - No evidence of increased βgal activity was seen in the mouse cohort (FIG. 15E).

[0174] Histology for lysosomal accumulation in the brain and spinal cord using filipin staining The biological analysis (Figure 16) confirmed the βgal activity (Figure 14) and distribution pattern ( In the CBA-WPRE-injected animals, the injection site and route were not significantly different from those in the control animals. There was near complete clearance of the accumulation throughout the brain (Figure 16), but not in the ventral hippocampus. , and filipin-positive cells were seen throughout the thalamus ( Fig. 16 ; row 1, column 4). Filipin-positive cells were detected in the anterior cingulate cortex, striatum, and brainstem (Figure 16; row 2, columns 2, 5, and 7), thus highlighting the accumulation criterion in the low-dose CB6 cohort. The effect of the CB6-high dose on the CB6-induced inflammatory response was considered to be low (Figure 16; row 2). The efficiency of the CB6 vector was extremely high, and the resolution of lysosomal storage was observed throughout the brain. As in the previous study, filipin-positive cells were detected in the ventral hippocampus and dorsal optic nerve. In the CBA-WPRE, the degree was low (Figure 16; third row, first and fourth rows). The T.Empty cohort (Figure 16, row 4) showed that lysosomal storage, βgal - / - No changes were observed compared to the untreated control (Figure 16; row 5).

[0175] The spinal cords of AAV-treated animals were also assessed for accumulation content by filipin staining. The spinal cord in BA-WPRE animals showed almost no accumulation in the cervical region, but a large amount in the thoracic region. Only in the 17A-17B region, the reduction was slight (arrows in each figure). The spinal cord in the control group showed almost no discernible reduction in accumulation compared to untreated controls. The spinal cord in high-dose CB6 animals was affected in the cervical and thoracic regions (Figures 17C-17D). The accumulation of lysosomes in the nuclei was largely absent (Figures 17E-17F; arrows). The spinal cord of T.Empty animals shows lysosomal accumulation of untreated βgal - / - Compared to the control No significant changes were observed (Figures 17G to 17H; 17I to 17J, respectively).

[0176] GM1 ganglioside levels in the CNS were quantified by LC-MS / MS (Figure 18 ) in the CBA-WPRE and low-dose CB6 cohorts, cerebral (p = 0.05, respectively). 0.0009 and p=0.002) and cerebellum+brainstem (p<0.0001 and p=0.002, respectively). and p=0.0001) in untreated βgal - / - A significant reduction in GM1 expression was observed in the spinal cord in both cohorts compared to controls. No significant changes in glycerol content were observed in the high-dose CB6 cohort. Glycoside levels were significantly higher in all CNS regions examined: cerebrum, cerebellum + brainstem, and spinal cord. (Figure 18; p=0.64, p=0.06, and p=0. 79) As expected, in the T.Empty cohort, there was no evidence of any CNS involvement. The GM1 ganglioside content of the native βgal - / - Changes were observed compared to the control (Figure 18).

[0177] [Example 7] AAV-mediated transmission to the CNS in an animal model for GM2 gangliosidosis gene delivery Prior to conducting clinical trials in patients with Tay-Sachs disease, final studies were performed in non-human primates. This study examined the safety and efficacy of AAVrh8 vector formulations in the thalamus and unilateral brain. A single study with different endpoints to support the safety of the injection procedure consisting of bilateral injections into the ventricles was performed. 1 dose (based on previous studies in GM2 mice and GM2 cats; 3.2 × 10 12 v The study was designed to measure 100-fold increase in total vector dose (g).

[0178] First, NHPs (N=3) injected with the AAVrh8 formulation showed mid-point mean morbidity within 28 days of injection. The NHPs developed mild to severe neurological symptoms. Clinical evaluation revealed large, necrotic areas and lesions in the thalamus along the area believed to be the injection route. In contrast, the injection of saline revealed areas of myelin loss (Fig. 22A-22D). Injected NHPs showed no symptoms and no evidence of neuropathology at necropsy (>90 days post-injection). In addition to histopathological evaluation of the brain, hexosaminidase was not detected (Fig. 22F). The enzyme activity was measured to be 6-48 times higher than normal (Figure 24).

[0179] Two additional cohorts of NHPs (N=2 per cohort) were administered AAVrh8 The vector formulations were administered at a total dose of 1 / 10 and 1 / 30 of the original dose, respectively, at 2 × 1 0 11 vg and 1.1×10 11 vg. In 3 of 4 NHPs injected with AAVrh8 vector formulations expressing the mitochondrial subunit, Neurological symptoms developed in the 100 subjects, but their onset was progressively delayed with dose reduction. Despite the delayed onset of symptoms (or, in one NHP, the absence of obvious symptoms), Neuropathological evaluation of the brain revealed no significant differences in all four NHPs in the low-dose cohort. , revealing widespread areas of necrosis, nerve loss, and cellular infiltration into blood vessels (Figure 22 E) In these NHPs, brain hexosaminidase activity was also significantly increased by saline injections. The hexosaminidase activity in the animals with Hex expression was elevated compared to that in the animals with Hex expression (Figure 23). Increases were also found in the spinal cord, which were more pronounced in the 1x dose animals. (Figure 23, bottom row).

[0180] Control for potential toxicity associated with the AAVrh8 capsid and / or preparation method In another cohort of NHPs (N = 2), 3.2 × 10 12 vg, transgene When treated with empty AAVrh8 vector, all animals showed no improvement throughout the study (> 1 h postinjection). All animals in the study exhibited normal behavior. , summarized in Table 3.

[0181] Prior to autopsy, brain MRI was performed on some NHPs in the low-dose cohort. In the thalamus, bilateral signal changes were found, probably due to edema (above, Fig. 24), but no changes were observed in NHPs injected with the AAVrh8 vector containing an empty transgene. None was observed (lower row, FIG. 24).

[0182] In addition to neuronal loss in the thalamus along the injection route, there was also intracellular eosinophil granules in the vicinity. Large, dense neuronal fields were also observed (Fig. 25E, 25G), which are not consistent with other areas of the brain. Within the region, no significant changes were evident (Figure 25F). Hexosaminidase alpha subunit Alternatively, immunofluorescence staining with an antibody specific for the hexosaminidase beta subunit was performed. These granules are likely to contain these two proteins ( These cells, loaded with HexA subunit-positive granules, were isolated from the thalamus. 25B, 25D), which correlates with the findings in the H&E stained sections. These observations reveal intralysosomal accumulation of the protein in several tissues. Findings in a GUSB transgenic mouse model (Vogler et al., 2003) It brings to mind...

[0183] Two key observations emerged from this safety study in healthy young NHPs: The thalamic regions containing HexA-positive neurons were stained with no evidence of significant inflammatory infiltrates. Although the lesion appeared intact (Figures 25E and 25G), there was extensive nerve loss along the injection route. and inflammation (Figure 22), and secondly, at the highest dose, the transgene-empty AAV It has been observed that there is no evidence of toxicity associated with the rh8 vector. As a result, neurotoxicity in NHP brains after AAVrh8-HexA-mediated gene transfer Massive overexpression of HexA in AAVrh8-transduced thalamic neurons This is caused by a β-amyloid β-blocking factor (AMB), which, when exceeded by an unknown threshold, induces cell death and secondary neuritis. It is thought that this may result in an inflammatory response (such as cellular infiltration into blood vessels).

[0184] [Table 3]

[0185] [Example 8] The new AAV vector plasmid (Figure 26) was transiently transfected into 293T cells. We investigated the expression level of the hexosaminidase (Hex) enzyme during the cleavage process. Two examples are shown in FIGS.

[0186] The original AAV vector plasmid pair (pAAV-CBA-CI-W delta6ATG) was The promoterless AAV vector plus produced the highest level of Hex activity. pAAV-NoP, or AAV vector plasmid without a transgene (empty transgene) in Hex-active, naive, untransfected 293T cells In contrast, the other AAV vector plasmids did not result in a detectable increase in the expression of the IgG1 gene (Figure 29). The six test AAV vector pairs were transfected into nude mice. Two AAV vector controls (promoterless control, For in vivo studies, the combined A total of 15 vector strains were generated (Table 4).

[0187] AAV vectors were transfected into 10- to 12-week-old male athymic nude mice (Charles Ri ver Labs) into the thalamus and left lateral ventricle on both sides, 1.32 × 10 10 B The control group was injected with a total dose of promoterless AAV vector (vg). AAV vectors without transgenes (empty transgenes), mice injected with PBS (a 5'-diaminobenzyl 2-methyltransferase gene), and finally, mice injected with non- Injected mice (N=8 for all experimental and control groups) were included (Table 4). , biochemical analysis for Hex expression, and histological studies were performed 1 month after injection. All groups exhibited identical increases in mean body weight over the course of the experiment (Figure 30). The animals were euthanized due to significant weight loss and the development of neurological symptoms. Unlike the preliminary experiments with the lower dose, there was no evidence of significant behavioral changes during this period. The dose used in this study (1.32 × 10 10 vg) for all subjects For quality and control, one of the vector pairs was used to normalize the total vector dose. Determined by the lowest titer.

[0188] Hex activity was measured using the artificial substrates MUG (Figure 36) and MUGS (Figure 37). The injection sites were measured in four coronal cerebral blocks, the cerebellum, the brainstem, and the spinal cord. The Hex activity in the coronal cerebral blocks is shown in Table 4, and the findings are summarized. The original AAV vector pair (group 1) produced 400-1,700-fold higher Hex activity than normal. Similar to the results in cell culture, other AAV vector pairs were shown to be more efficient than the original AAV vector in the brain. The 1 / 3 (group 2), 20-30 (group 4), and 50-10 times smaller (group 5) of the turpentine preparation (group 1) were used. 0-fold lower (group 6) Hex activity, predicting the activity of AAV-mediated Hex expression in the brain. The other AAV vector pairs (groups 3 and 5) yielded a 1-2 log range of It did not result in Hex activity above normal levels present in the brain of athymic nude mice. The control groups (groups 7, 8, and 9) showed no significant changes in Hex activity.

[0189] Neuropathological examination of the brain revealed numerous eosinophilic granule-containing granules in Group 1 animals. This finding was consistent with the AAV vector used in group 1. The number of these abnormal neurons was identical to that observed in the ct-injected monkeys, but In mice, the mean sigma-positive rate was significantly lower than in monkeys. This observation was also observed in animals of group 2. However, the number of abnormal neurons was significantly smaller than that in group 1 (Fig. 31L). We also observed neurons in the hippocampus of group 2 animals (Fig. 31B). , but not in any other animal group. Immunofluorescence staining with antibodies was performed in groups 1 (Figures 31A and 31K), 2 (Figures 31B and 31L), and 4 ( 31D, 31N), and 6 (Fig. 31F, 31P) in the hippocampus and thalamus of animals In the control group, presumably human and cynomolgus monkeys, The antibody species used in this study detects the monkey enzyme but not the mouse protein. Due to specificity, no evidence of expression of the Hex-alpha subunit was seen.

[0190] Intrahippocampal and intracellular IL-1 expression in animals injected with the original AAV vector formulation (group 1) (Figures 33A, 33K) and in the thalamus, Iba-1 staining (microglial activation) was significantly higher than in the control (Figure 33I, 33J). We observed a dramatic increase in microglial activation compared to the control group (33S, 33T). This evidence was provided by localization of HexA-positive cells to sites detected by immunofluorescence staining. (Fig. 32A, 32K), no obvious changes in microglia were observed in other parts of the brain. In groups 2 and 3, the increase in Iba-1 staining in the hippocampus and thalamus was minimal (Fig. 33B-33C, 33L-33M) and were essentially indistinguishable from controls in groups 4-7. (Figures 33D-33G; 33N-33Q).

[0191] Brains were also examined for evidence of reactive astrogliosis using GFAP immunostaining. In the hippocampus, evidence of reactive astrogliosis was also observed in the original AA This was found only in animals injected with the V vector formulation (Group 1) (Figure 34A). All animals were indistinguishable from controls. In the thalamus, some aspartate Trogliosis was observed (Figs. 34K-34M), but was either weak or clustered. The levels were considered indistinguishable from the controls within 4 to 7 (Figures 34N to 34Q). No evidence of astrogliosis was seen in other brain regions within the group.

[0192] Two new AAV vectors (Figure 35), AAV-CB6-I-cmHex (sequence number promoter represented by sequence number 4), and AAVP2-I-cmHex (SEQ ID NO: 5, construct represented by SEQ ID NO:6) were added prior to the start of the experiment. All of the study criteria defined herein (absence of major behavioral changes, control group the same rotarod performance as the control group, whether the body weight remained constant between 0 and 30 days after injection, and The presence of neuropathology and the presence of thalamic and rostral non-injection blocks were These new AAV vector formulations (group 4 and 6) have demonstrated that increased Hex expression in the brain compared to controls is evidence of neuropathological changes. with little or no evidence of eosinophilic neurons, microgliosis, or absence of astrogliosis).

[0193] From the original AAV vector, the wpre element has been removed, but the expression element ( The new AAV-CBA-I (promoter and artificial intron) is still the same. The -cmHex vector (Figure 35) was also examined. This AAV vector (Group 2) , and approximately one-third of the Hex activity level compared to the original AAV vector. The results showed that the gliomas in the 14-HT10 mice were significantly decreased in microgliosis compared to the 14-HT10 mice. in the AAV vectors used in the study and in the long-term study in Sandhoff cats. Considering that the expression elements in the V vectors are the same, AAV vectors are It is also possible that the reduction in Hex expression levels mediates long-term expression in microglia. Given the weakened activation of the .ALPHA. gene, this new AAV vector is suitable for the experiments in Example 9. , dose (3×10 11 vg) and duration of exposure significantly affected the behavior of NHPs. There is little chance of this happening.

[0194] [Table 4]

[0195] [Example 9] In athymic nude mice, the expression of beta-hexosaminidase in the brain was evaluated by the original AA V vector formulation, and increased inflammatory responses (astrogliosis and microgliosis) Three AAV vector designs (Figures 19 and 35) were developed to reduce the activation of ribosomal proteins. The AAVrh8 vector was selected in the absence or presence of neutralizing antibodies against the AAVrh8 capsid. or into NHPs screened for very low titers (Table 5).

[0196] 3.2×10 11 The total dose of vg was administered bilaterally to the thalamus (50% dose; 2 × 150 μg / kg). The vector formulations were also injected into the left lateral ventricle (50% dose in 300 μl) and the left lateral ventricle (50% dose in 300 μl). We will determine the targeting accuracy and distribution in the brain by MRI immediately after injection. The endothelial cells also contained 2 mM gadolinium (Figure 38A). All NHPs tolerated the surgical procedure well. The mean volume of distribution of gadolinium in the thalamus (Vd) was 1.2 mg / kg / dL. The injection volume in the thalamus (Vi) was 0.3 ± 0.48 mL (Figure 38B). mL, this corresponds to a Vd / Vi ratio of 4.9.

[0197] The behavior of all six NHPs injected with AAVrh8 was consistent throughout the 90-day study. As planned, brain MRI was performed monthly (Figure 39). After the 30th day, Signal changes at the injection site were recorded in two monkeys from cohorts 2 and 3. However, no obvious changes were observed over time (Fig. 40B, 40C). In one monkey in study 1, brain MRI at 30 and 60 days after injection showed no significant On day 90, no changes were observed in the left thalamus, but on day 90, a large signal change was detected in the left thalamus. Despite this abnormal signal, the behavior of this monkey at 90 days was Behavior remained constant throughout the study.

[0198] Brains were cut into 4 mm coronal blocks and assayed for hexosaminidase (Hex) activity. The enzyme distribution maps were used to assess neuropathology. A biopsy punch (3 mm diameter) was used to sample the brain to create (Figure 41). In all cohorts, there was above-normal total Hex activity only within the thalamic punch. The genus HexA, HexB, and HexS were detected (Table 6). ex activity was highest in cohort 1 (up to 87-fold above normal) and in cohorts 2 and 3. The results are consistent with those in athymic nude mice. Total Hex activity in most other brain regions sampled was consistent with that in uninjected control monkeys. The total Hex activity was similar in

[0199] Neuropathological assessment of the brain revealed that monkeys in cohort 1 had intraneuronal eosinophilic material. In animals from cohort 1, moderate to severe symptoms were observed. In one monkey (ID: 295851) in cohort 1, neurodegeneration was observed in the left optic nerve. In the bed, severe focal spongiosis and perivascular cellular infiltration were observed (Figures 43A and 43B ) The lesion was located in the left thalamus of one monkey in cohort 1 90 days after injection. This corresponded to the abnormal MRI signal that was evident (Figure 40A).

[0200] In cohort 2, no accumulation of eosinophilic material was observed, and rare neurodegeneration and satellite lesions were observed. Focal inflammation in the white matter, accompanied by neurodegeneration, likely associated with injection site / tract trauma. was observed in one monkey (ID: 295847).

[0201] In cohort 3, no accumulation of intraneuronal eosinophilic material was observed, and rare neurodegenerative and satellite diseases were not identified. In one monkey (ID: 295709), cannulation and Possibly associated foci of perivascular lattice cells were seen (Figure 44B). In particular, intraneuronal accumulation of eosinophilic material (Figure 45), neurodegeneration and necrosis (Figure 46), and For the quantification of inflammation (Figure 47), the left thalamus and Serial sections, spaced 150 μm apart, were prepared throughout the right and left thalamus. cohort showed non-negligible neurodegeneration, necrosis, cavernous state, and corresponding inflammation. With the exception of one animal in the left injection site, formulations 1 and 3 were These findings were consistent with 90-day brain MRI and The best fit is shown in Figure 40A.

[0202] The new vector, AAVrh8-CBA-cmHex (Figure 19), and AAV rh8-CB-I-cmHex (Figure 19) inhibits Sandhoff disease ( A small pilot study was conducted to determine the effectiveness of reducing GM2 content in the CNS of SD mice. All previous studies in SD mice and SD cats have shown that this vector Since the original AAVrh8-CBA-cmHex-W Δ6ATG Vectors, The AAV vector was administered to both sides of 4-6 week old SD mice. 4.68 × 10 per mouse into the thalamus and left lateral ventricle 9 A total dose of vg was injected. Therapeutic efficacy in SD mice injected with the original AAVrh8-CBA-Hex-W vector In efficacy studies, this was the test dose. Total Hex activity in the thalamus of NHPs (cohort 2 in Table 6) was significantly higher than that of AAVrh8-CB Approximately 1 in 20 of the NHPs injected with A-cmHex (cohort 1 in Table 6) Therefore, a 5x higher dose (5x dose: 2.34 × 1010 vg), AAVrh8-CB- An additional cohort of SD mice injected with the I-cmHex vector was also enrolled. To measure the Hex activity as well as the GM2 ganglioside content, Mice were sacrificed and the brains were divided into four coronal blocks of approximately 2 mm each (Figure 48). Vrh8-CBA-cmHex-W Δ6ATG (green bar, Figure 48), and AAVrh8 -CBA-cmHex vector (orange bar, Figure 48) was injected into one of three SD mice. The AAVrh8-CB-I-cmHex vector had no detectable hexosaminidase activity. In mice injected with β-actin (blue bars in Figure 12), the 1-fold and 5-fold cohorts showed No Hex activity was detected in 3 of 3 and 2 of 4 animals, respectively, within the study. In a comparative study in athymic nude (nu / nu) mice, the SD mice of this example In some cases, the AAVrh8 vectors tested in this study showed 2-3 orders of magnitude higher than normal. , resulting in non-negligible Hex overexpression in a subset of SD mice. It is possible that the lack of Hex expression may limit enzyme distribution via a humoral response or an adaptive response. Cynomolgus Hexagonal Transfection Mediated by CYNOMOLGUS ANTIGEN, Resulting in Loss of Transduced Cells Immunoglobulins against the fa (A) subunit or the cynomolgus monkey Hex beta (B) subunit Although the number of mice per group was small, the Hex activity was high. There appears to be a direct correlation between the levels of GM2 ganglioside and the reduction in GM2 ganglioside. In block 3, AAVrh8-CBA-cmHex-W Δ6ATG Note vector In SD mice injected with α-glucose, a maximum reduction of 96% in GM2 ganglioside content was observed. For AAVrh8-CBA-cmHex, a 92% reduction was observed, and Injection of 1x and 5x doses of Vrh8-CB-I-cmHex vector, respectively. In mice treated with 100 mg / kg / day, a 42% and 85% reduction was observed.

[0203] [Table 5]

[0204] [Table 6]

[0205] [Example 10] Simultaneous expression of the transgene of interest in both neurons and astrocytes, respectively. For efficient dual expression, the human synapsin 1 (also called "Syn1") promoter was used. The present invention relates to a method for the preparation of a medaka-based ... We constructed an AAV vector carrying Syn1-GFP-2×miR. SOD1 / GFAP- 2×miR SOD1 The vector, called -mCherry, targets synapsins in neurons. GFP driven by the GFAP promoter in astrocytes. The two anti-SOD1miRNAs contain mCherry, which is transduced with human SOD1. The RNA contains the final codon and polyadenylation signal of each of GFP and mCherry. The vector uses the AAV9 capsid protein. In some embodiments, the vector, Syn1- GFP-2×miR SOD1 / GFAP-2×miR SOD1 -mCherry sequence Represented by the number 12.

[0206] The transduction profile of the dual promoter vector was investigated. n1-GFP-2×miR SOD1 / GFAP-2×miR SOD1 -mCherry , SOD1 G93A Intrastriatal injections were performed into adult mice and fluorescence microscopy was performed. The morphology of GFP (green) expressing cells and mCherry (red) expressing cells is different from that of the control cells. This indicates that the shape of the cells corresponds to that of neurons and astrocytes (Figure 51).

[0207] The expression of SOD1 mRNA was measured. Data were obtained using the dual promoter AAV9 -Syn1-GFP-2×miRSOD1 / GFAP-2×miRSOD1-mCher Following injection of the ry vector, adult SOD1 G93A Human SOD1 mRNA in mice It indicates that the NA level is reduced (Figure 52). 1×10 12 The whole vector genome of After intravenous injection, it reduced human SOD1 mRNA expression in the spinal cord by up to 25%. Observed. 8×10 9 After direct intrastriatal injection of the whole vector genome, A maximum 55% reduction in human SOD1 mRNA expression was observed.

[0208] The data show that this dual promoter construct is ubiquitous while reducing toxicity to non-CNS tissues. This is a larger, more extensive, and more efficient method of expressing C Resulting in transduction of specific cell types (e.g., neurons and astrocytes) within the NS It points to what will be brought about.

[0209] [Example 11] Combined intrathalamic (TH) and deep cerebellar nucleus (DCN) injections in both sides (TH / DCN), or bilateral intrathalamic injections combined with a single intracerebroventricular injection (TH / ICV). The hexosaminidase A subunit-encoding rAAVrh was synthesized through two methods: 8 and rAAVrh8 encoding the hexosaminidase B subunit (respectively, 1:1 formulations (rows 20 and 21) were intracranially injected into 4-week-old SD mice. Vrh8-treated SD mice (4.68 × 10 9 vg), and controls (untreated SD littermates and Behavioral performance of mice (and wild-type littermates) was assessed over time, starting at 60 days of age (1 month after injection). The effect of TH / ICV AAVrh on the survival of mice at 120 days of age (P≦0.05) was evaluated (FIG. 53). The inversion screen performance of SD mice treated with 9 was significantly better than that of untreated SD mice. TH / ICV AAVrh8-treated mice at 180 days of age, and All outcomes in mice treated with TH / DCN AAVrh8 were comparable to those of wild-type controls. The results were comparable (Figure 53A). The rotarod test also showed that AAVrh8 mice at 120 days of age Both cohorts of SD mice treated with α-aminobutyric acid performed better than untreated SD controls (P≦0.01). 01), and their performance was comparable to that of wild-type controls for at least 180 days of age. (Fig. 53B). In the wire hanging test, the mice showed significant improvement compared to untreated SD mice. Not measured (Figure 53C). TH / DCN or TH / ICV resulted in 4.68 x 10 9 SD mice treated with the vg dose survived for 424 and 423 days, respectively. The median value was 0.01 (Figure 53D). Thus, both intracranial delivery methods were Similar life expectancy extension compared with TH / DCN (P ≤ 0.001; TH / IC V, P ≤ 0.0001) and were not significantly different from each other. (P>0.05).

[0210] Whether increasing the dose of AAVrh8 further extends survival in SD mice A dose escalation study was conducted to determine the intracranial TH Two days before ICV injection, SD mice were infected with AAV8-TRG-mHexB (3 × 10 11 vg) was intravenously injected into SD mice. did not enhance survival of the treated SD mice (median survival for untreated SD mice was 127 days). whereas median survival for SD mice treated with AAV8-TBG-mHexB was 1.1. The mean mean was 128.5 days (Figures 53D and 54A). Systemic therapy-mediated tolerability increased median survival by 4.68 × 10 8 Skull in vg The combination therapy did not significantly enhance survival compared with intravenous delivery alone (median survival was 448.5 The median survival with TH / ICV alone was 423 days; Figures 53D and 5 4A). The same tolerized protocol (AAVrh8-mHexA / B vector) delivered intravenously. Combined with AAV8-TBG (mHexB) prior to TH / ICV delivery of the vector In addition, dose escalation of intracranially delivered AAV (4.68 × 10 7 vg, 4.68×10 8 vg , 4.68×10 9 vg, 1.17×10 10 vg) at the two highest doses This resulted in an extension of (4.68×10 9vg and 1.17×10 10 vg, P ≤ 0.0001; median survival of SD mice treated systemically alone was 128.5 days. Yes; 4.68×10 7 : 110.5 days, 4.68 x 10 8 vg: 135 days, 4.6 8×10 9 vg: 448.5 days, 1.17×10 10 vg: 591 days; Fig. 5 4A). Despite maximum survival of 666 days, the high dose (1.17 × 10 10 Dosage of vg ) Treated animals ultimately presented with hind limb weakness and ataxia at the time of euthanasia, with some animals Loss of ability to bear weight and / or stand upright when placed in a supine position.

[0211] Inverted screen performance (Figure 54B), rotarod performance (Figure 54C), or wire-hitch performance At 120 days of age, 4.68 x 10 9 vg or 1.17×10 10 vg (intracranial TH / ICV administration) dose-escalating combination therapy mice There was no significant difference in behavioral performance in the treated SD mice compared to untreated SD mice. AVrh8-treated SD mice retained their locomotor capacity and were significantly more resistant to TH / ICV treatment at 180 days of age. SD mice performed comparable to wild-type mice in the reversal screen at all doses. At 180 days of age, TH / ICV-treated SD at any dose was also significantly higher than control (Figure 54B). Mice also performed comparable to wild-type mice on the rotarod (Figure 54C). In the Yahang test, no significant improvement was measured compared to intracranially untreated SD mice (Figure 1). 54D), which was close to the median survival age of untreated SD mice (120 days). will further conduct studies at 180 days of age, when no untreated SD mice survive. I was able to do it.

[0212] The potential impact of intracranial injection of AAVrh8 vectors in the absence of underlying processes of neurodegeneration Age-matched heterozygous (HZ) littermates (HexB + / - ) mouse, 4.68 × 10 9 Intracranial injection of vg AAVrh8 vector formulations The mice were treated with AAVrh8 and non-treated with HZ immunization. Treatment controls (wild type and HZ) are evaluated over a time course in different behavioral tests. In AAVrh8-injected HZ mice, the behavioral changes were clearly observed compared to the controls (Figure 55). No major changes or effects on survival were observed. However, no differences between the cohorts were evident (Figure 55A). Performance was slightly lower in the rotarod test at 180 days of age than untreated HZ and wild-type controls. The results of the wire hang test were significantly worse at 60 and 120 days of age. Again, no significant difference was observed between the cohorts (Figure 55C). In this study, the performance of TH / DCN-injected HZ mice was significantly higher than that of untreated HZ animals (P ≤ 0.05) and non-treated HZ animals (P ≤ 0.05). At 180 days of age, TH / ICV injections were significantly worse than those in wild-type animals (P ≤ 0.01). No significant differences were observed in the performance of the animals compared to untreated HZ or wild-type animals.

[0213] The content of GM2 ganglioside and expression of hexosaminidase in the CNS were analyzed by TH / ICV AAVrh8-treated (1.17×1010vg) SD mice, untreated SD mice, and in untreated wild-type controls (Figure 56). G M2 ganglioside was not detected in the cerebrum of AAVrh8-treated animals, but was detected in the cerebellum (91.1 %), brain stem (99.6%), and spinal cord (99.8%) also showed abundance of GM2 ganglioside. A significant reduction in the expression of β-lactamase (β-lactamase) was observed in AAVrh8-treated SD mice (P≦0.0001, FIG. 56A). In the central nervous system, total hexosaminidase activity was restored. Enzyme activity levels were , compared to wild type and were comparable in the cerebellum, brainstem, and spinal cord. When subjected to standard endpoints equivalent to those of D mice, they were indistinguishable from wild-type controls ( data not shown).

[0214] [Example 12] All SD cat experiments were performed using vector constructs (AAVrh) used for human application. 8-CB-CI-hHexA / B), which is slightly different from AAVrh8-CBA-mHex A / B-WPRE was used to conduct treatment efficacy studies in patients with rapidly progressing disease and median survival of 4 These studies were performed in a naturally occurring Sprague-Dawley cat model, with the animals being aged 0.3±0.2 months. In this study, the hexosaminidase alpha subunit and the hexosaminidase alpha subunit of wild-type cats were 1 by two AAVrh8 vectors encoding the β and β subunits, respectively: One formulation was used.

[0215] In this example, long-term therapeutic efficacy data are reported. SD cats were treated with unilateral ICV injection (ICV ; n = 5) combined with bilateral intrathalamic injections resulted in 4.6 × 10 11 Total dose of vg (both The ratio of the vector to the gene was 1:1 (Table 7). The survival time was significantly increased (p<0.0001; Figure 57A), with the oldest cat living to 29.9 months of age. Untreated SD cats showed severe tremor with obvious generalized rest and intention tremors. suffered from brain disease which was accompanied by loss of locomotion and subsequent euthanasia at approximately 4 months of age. After AAV gene therapy, there was a marked improvement in neurological symptoms ( (FIG. 57B). Tremors were completely resolved in all treated cats, with the majority of cats showing no improvement over the course of the study. Throughout the study, SD cats retained their ability to walk, demonstrating a musculoskeletal disease that is not a typical neurological phenotype. All animals were euthanized due to chronic or gastrointestinal disease (Table 7).

[0216] [Table 7]

[0217] Both ICV and combined intrathalamic injections (Th+ICV) of AAV vectors , resulting in widespread hexosaminidase distribution throughout the cerebral cortex, cerebellum, and spinal cord. (Figs. 57B and 57C). The only area of ​​the cerebrum where the distribution is low is the temporal lobe. lobes (Figure 57D, lateral view of Figure 57D, and Figure 57E). HexA levels were , cerebellum, and spinal cord, were near or above normal, and In the AAV-treated cats, the amount of Hex was increased by 50 times compared to normal (Fig. 57E). No histopathological abnormalities were observed despite supraphysiological levels at the site. I couldn't.

[0218] Humane endpoint MRI (T2W) of SD cats showed brain-wide, cerebrospinal fluid, cerebrospinal fluid, and cerebrospinal fluid. Darkening of the white matter (accumulation of GM2) and lightening of the white matter (demyelination) are shown (Figure 58A). After V gene therapy, gray and white matter intensity throughout the brain, with the exception of the temporal lobe A normalization of the distribution of Hex was observed, which was thought to be due to a reduction in the distribution of Hex (Figure 58D). Despite normalization of cerebral and white matter intensity, AAV-treated SD cats showed no significant differences in lateral ventricles and Cortical atrophy persisted, as exemplified by increased CSF in the pericerebral sulci and surrounding cerebral sulci. Using MR spectroscopy, we identified a number of MR images in the thalamus of untreated SD cats, including those of mice and humans with SD. In the study, N-acetylhexosamine (NA-H), a previously reported toxic metabolite, was detected in After AAV gene therapy, a significant increase in NA-Hex was detected in all cats. In AAV-treated cats, the HexA gene was completely reduced to normal levels (Fig. 58B). The level of HexA is elevated at or above normal levels, and the elevated HexA level is associated with a long-term The effect persisted throughout the cytotoxic period (Fig. 58D). Transferase and lactate dehydrogenase were elevated in the CSF of SD cats, After AAV gene therapy, the levels of these enzymes returned to normal (Figures 58D and 58E Serum antibody titers against the capsid were present for 1-2 months after treatment, but did not increase over time. The humane endpoint was reached before the age of 1 year. The only cat to do so (11-907) had a maximum serum antibody titer of 1:6 approximately 2 months after treatment. The end point titer was only 1:16 (Figure 58G). A cat that lived to 6 months of age (7-760) had a maximum titer of 1:16, 3.6 months after surgery. Due to the limited number of vector strains, it was not possible to analyze all time points. However, after approximately 20 months of treatment, serum titers decreased to 1:1 (Figure 5 8H).

[0219] Despite CNS-directed delivery of AAV, SD+AAV TH / ICV cats did not express peripheral HexA enzyme activity in the organs was increased. The sciatic nerve showed a 203% increase over normal. whereas normal liver and muscle achieved an increase to 53 and 20% of normal. (Table 8).

[0220] [Table 8]

[0221] In this study, we used the chicken beta actin (CBA) promoter and the woodchuck transcription factor β-actin (TNF-α) promoter. Encoding feline HEXA or feline HEXB, flanked by a post-regulatory element (WPRE) AAVrh8 delivery of two monocistronic vectors was investigated. Intracisternal injection (6.4×10 11 vg, approximately 0.5 kg at the time of injection) and A subset of subjects (n=3) was euthanized 16 weeks after treatment and assessed for biodistribution. A second cohort (n=2) was followed to a humane endpoint. The patient suffered from severe cerebellar disease and progressively debilitating, pronounced, generalized, resting tremor and and intention tremors, loss of ambulation, and subsequent euthanasia at approximately 4.4 ± 0.6 months of age. (Figure 59A). AAV gene therapy via intracisternal injection (SD+AAV CM) Treated SD cats had a similar humane endpoint to untreated SD cats. At time point, 16 weeks after treatment, there were minor neurological signs, including staggered gait and mild hind limb weakness. The two cats followed to a humane endpoint had a mean score of 9.9. They survived to ±0.5 months of age and retained the ability to stand and walk, but were unable to detect visual or auditory stimuli. It was unresponsive to stimulation.

[0222] The biodistribution of Hex in SD+AAV CM cats was consistent throughout the spinal cord and cerebellum. The expression of cerebral stimuli was widespread in the brain and was reduced in the protocerebral regions (Figs. 59C, 59D, and 59E). SD cats treated short-term (data not shown) and SD cats treated long-term with SD+AAV CM In cats, hexosaminidase A (HexA) activity is positive in the cerebellum and spinal cord. reached or exceeded normal levels, but included the rostral thalamus, striatum, and frontal cortex. In the blocks with cerebral infarction (brain blocks D to A, respectively), the results were comparable to those of untreated cats. The biodistribution of the kinetics ... contained the lowest vg copy number (Figure 59F), but qPCR showed that the HeLa cells in the spinal cord and brain x expression differences.

[0223] Accumulation in SD cats was confirmed by periodic acid staining, which stains for glycolipids (e.g., GM2). The area is stained with periodic acid sulfate (PAS; gray scale). Areas with dark staining, e.g., PAS positive, are In SD cats, the PAS region in the gray matter shows increased accumulation of gangliosides. There was increased staining in the white matter and decreased staining in the gangliosides of the cell bodies. In SD+AAV CM cats (long-term), accumulation and clearing of sid The effect of Hex on the distribution in the spinal cord and cerebellum was inversely related to the biodistribution of Hex. In the cerebral cortex, no activity was observed. PAS-positive material was detected in the cerebral cortex and deep brain structures. Demyelination, as indicated by a decrease in PAS-positive material, also occurred in the cerebral cortex of treated cats. Within the white matter, it was clearly represented.

[0224] The cerebellum of SD cats showed widespread neurodegeneration, evident in H&E staining ( Figure 61). Purkinje cells, deep cerebellar neurons, and brainstem neurons are severely degenerated. The patient presented with cell loss and abundant, foamy, vacuolated, intracellular accumulations, indicative of Sixteen weeks after gene therapy, AAV-CM-treated SD cats showed increased expression of Purkinje cells, deep cerebellar The results showed normalization of the nuclei and morphology of brain stem cells (Figure 61). In treated animals, DCN and brainstem showed morphological normalization, whereas Purkinje cells showed no morphological normalization. Interestingly, chronic AAV-treated cats showed a prolonged degeneration and loss of the cerebellar fastigial nucleus. Within the dorsal medial region, grape-shaped eosinophilic inclusions were observed (inset) (Figure 61). These inclusions have previously been shown to be immunopositive for Hex after intraparenchymal injection of AAV. do.

[0225] Ultra-high field MRI (7 Tesla), T2 weighted MRI, reflects the distribution of Hex. Normalization of MRI architecture in AV-treated animals is shown (Figure 62A). D In cats, there was an increase in water in the white matter (demyelination) and an increase in lipids in the grey matter (accumulation of GM2). As a result, gray matter intensity and white matter intensity were inverted. 16 weeks after intracisternal injection, MR Partial normalization of I intensity was observed, and the corpus callosum (Figure 62A; black arrow) and corona radiata (Figure 62A; white arrow) ), and the white matter intensity of the cerebellum (Figure 62A; black arrow) was darkened. SD+AAV CM The humane endpoint in cats was pathological intensity correlation between gray and white matter in the cerebral cortex. Although the decompensation persisted, the atrophy of the cerebral cortex was alleviated, and the strength of the cerebellar white matter was partially restored (black Arrows). The thalamus (Fig. 62C), parietal cortex (Fig. 62D), and cerebellum (Fig. 62D MR spectroscopy of the toxic metabolite, N-acetylhexosamine (NA-H) x, Figure 62B). In SD+AAV CM cats after 16 weeks, NA- Hex is reduced in the thalamus (FIG. 62C) and cerebellum (FIG. 62E), but not in the humane endpoint. The parietal cortex of one SD+AAV CM long-term cat (Figure 62E) evaluated in vivo (Fig. 62D). Other metabolite changes were observed in the cerebellum after AAV treatment. of myelination (choline + phosphocholine) and metabolic activity (creatine + phosphocreatine This endorses a partial correction of the Cr+PCr (Figure 62E).

[0226] CSF levels of HexA were above normal in all SD+AAV cats (Figure 62 F), one cat (11-1042) was 5 months of age and had a humane endpoint of C HexA activity in the CSF was rapidly attenuated. Aspartate aminotransferase (AST; Figure 62G), and lactate dehydrogenase LDH (Figure 62H) was measured in one long-term SD+AAV CM cat (11-1042). In the cat (11-1148), the levels were reduced to near normal levels, whereas in the other cat (11-1148), the levels were moderately increased. I was forced to do it.

[0227] [Example 13] mouse A short-term, biochemical analysis of the effects on GM2 ganglioside content in the CNS. For the analysis, SD mice were transfected with mouse HexA / B (mHexA / B) subunits. The AAVrh8-CB-CI-mHexA / B vector formulation was administered in two doses, 4. 68×10 9 vg, and 2.34 × 10 10 vg, 4.68 × 10 9 Note in vg The original AAVrh8 vector injected (AAVrh8-CBA-mHexA / B-WPRE mut6ΔATG) were compared with AAVrh8-CB-CI-mHexA at any dose. In the brains of SD mice injected with the / B vector, the GM2 ganglioside content was significantly higher than that of the PB At the highest dose (2.34 × 10 10 vg) A 92% reduction in the number of AAVrh8-CB-CI-mHexA / B vectors was observed. The reduction was greater than that recorded in SD mice injected with the original AAVrh8 vector (97 %. The GM2 ganglioside content was comparable to that of PBS-injected SD mice. A significant reduction was observed in SD mice injected with the AAVrh8-CB-CI-mHexA / B vector. It was also observed in the cerebellum and brainstem of mice, but at a lower level than the original AAVrh8 vector. The SD injected with the AAVrh8-CB-CI-mHexA / B vector showed a reduction in The extent of GM2 reduction in the CNS of mice was dose-dependent (Figure 63A). Hexo in the brain of SD mice treated with rh8-CB-CI-mHexA / B vector The saminidase activity was dose-dependent and 7-43-fold lower than that of the original AAVrh8 vector. However, the levels were still 2 to 12 times higher than normal (Figure 63B). Survival (>1 month beyond the median survival of untreated SD mice) was used as the outcome measure. To investigate the therapeutic efficacy, another set of SD mice (n=8) was ×10 10 vg, and injected with AAVrh8-CB-CI-mHexA / B. Most of the SD mice (6 out of 8) survived to 5 months of age. However, no symptoms were observed after 5 months. Only two of the six animals were considered to be endothelial (Fig. 63C). Mice presented with varying degrees of hind limb dysfunction or weakness. The new AAVrh8 vector formulation reduces the original C-terminal content of GM2 ganglioside in the spinal cord. This can be explained by a more modest reduction compared to the BA vector (Figure 63A; % versus 35%).

[0228] To improve the effect on the accumulation of GM2 ganglioside in the spinal cord and cerebellum, The intrathalamic dose was kept constant (1.17 × 10 10 vg), through the lateral ventricle, The dose delivered to the SF was increased. SD mice were given increased CSF doses: 1.1 7×10 10 vg, 2.34×10 10 vg, 5.85×10 10 vg, 5.00×10 11 vg, AAVrh8-CB-CI-mHexA / B (per cohort (n=3) One month after injection, in the spinal cord (Fig. 64A) and in the cerebellum (Fig. 64B) The reduction in GM2 ganglioside content was dose-dependent, with the highest dose (5.2 × 10 1 1 vg) is 4.68 × 10 9 vg injected with the original AAVrh8 vector and showed similar effects.

[0229] [Example 14] Designing a compassionate use study for a single subject with Tay-Sachs disease rAAVrh8-HexA / B was developed based on the preclinical data presented above and the It was administered based on the lack of treatment available for patients with Sachs' disease.

[0230] Three weeks prior to treatment, subjects (approximately 30 months of age) with progressive infantile Tay-Sachs disease were Several clinical safety studies were conducted and immunosuppressive regimens were administered for approximately 7 days prior to treatment. The subjects were two mutant HexA alleles: 1) HexA: the most common hexA allele; A 4-base pair insertion (coding DNA: 1274-1278), which is a neugenase mutation; and 2) HexA: coding DNA: 82C→T (protein: Gln28). In addition, the subject may already have one or more of the following disease progressions: Age 8 months: Excessive startle reflex Age 12 months: Macrocephaly with abnormal myelination on MRI, biomembrane on MRS. Car abnormality Age 14 months: First afebrile seizure → Diagnosis Age 17 months: G-tube insertion Age 20 months: Emergency visit due to recurrent seizures The following symptoms were observed.

[0231] Subjects' seizures were treated with medication at baseline and with intrarectal midazolam as needed. The subjects were placed on a ketogenic diet, administered miglustat via a G-tube, and placed under moderate control. , Keppra, Prevacid, and Clonazepam.

[0232] A composition comprising a biological drug derived from rAAVrh8-HexA / B rAAV is administered to a subject at a rate dictated by the subject's CSF pressure. The target dose of the exA / B formulation was 1.0 × 10 14 Vector genome ( vg), or 1.0×10 14 It was vg.

[0233] The total dose administered to patients was 1040 ± 130 g in females aged 19 to 30 months. The total dose was calculated based on brain weight of 1000 mg / kg. Approximately 75% of the total dose was delivered to the cisterna magna and approximately 25% was delivered to the fluorescent It was administered into the spinal canal via a fluoroscopically guided lumbar intrathecal catheter (Figure 74A). 14 mL Following passive flow removal of 100 ml of CSF, 9 ml of AAVrh8 vector was infused into approximately 1 ml of the L / min at the cisternal level (Figure 74B). 3 ml of AAVrh8 vector was administered at L2 For subjects with advanced disease, co-delivery of rAAVr No intrathalamic injections of h8-HexA / B were administered to subjects.

[0234] The biological product used in the study was a separately produced AAVrh8-CB-c i-HEXA and AAVrh8-CB-ci-HEXB (rAAVrh8-HexA / B) were two non-replicating single-stranded adeno-associated virus vectors. The molecular characteristics are described in Tables 9 and 10. The AAVrh8 vector is a chicken beta- The gene encoding human HEXA and human HEXB is under the control of the cytoplasmic promoter. The AAV vector plasmids are depicted in Figures 65-67. The formulation was in Ca2+-free phosphate buffered saline.

[0235] rAAVrh8-HexA / B was generally well tolerated, with no clinically significant improvement after 3 months. There were no treatment-related, serious adverse events reported in the study update. No clinically relevant laboratory abnormalities were observed 3 months after treatment. No significant immune responses were observed. The clinical status of the subjects improved from baseline to 3 months. Neurological examination showed no signs of deterioration and the condition was stable. Magnetic resonance imaging (MRI) of the brain also showed that No evidence of progression from baseline when compared to pre-treatment brain MRI These data are shown in Figure 69.

[0236] Data collected at three months also showed that a single dose of rituximab significantly increased the CD1 9 and CD20 counts were completely suppressed (<1% of peripheral T cells), and IgG These data also indicated that IVIg infusions maintained the blood glucose levels within a safe range. The data is shown in FIG.

[0237] Subjects were randomly assigned to receive the AAVrh8 capsid and were randomly assigned to receive the AAVrh8 capsid after 3 months. After 3 months, no neutralizing antibodies against the transgene product were observed. In addition, after 3 months, T cell responses to the transgene product and cytotoxicity to the capsid were also observed. No adverse T cell responses were observed. regulatory Stimulating responses are important for future immune responses. This demonstrates the successful induction of long-term immune tolerance to the capsid, preventing viral response.

[0238] Very slight increases in the enzymes AST (less than 2x baseline) and ALT (extremely elevated levels) A slight increase (less than twice the upper limit of normal) was observed over the first 21 days, but spontaneous There was no evidence of systemic inflammation based on high-sensitivity C-reactive protein or total complement activity. Liver ultrasound and pediatric gastroenterology consultation also did not reveal any specific pathology.

[0239] β-Hexosaminidase A activity was determined using the 4MUGS assay. In our study, the CSF activity of β-hexosaminidase A in the subjects was 0.46% of normal. After 3 months, the β-hexosaminidase A activity in the subject's CSF was 1 / 2 the normal enzyme activity. There was a clear increase to 1.44%, representing a more than three-fold increase from baseline. Serum and CS levels before and up to 3 months after AAVrh8-HexA / B administration The enzymatic activity of HexA in F is shown in FIG.

[0240] As shown in Figure 71, GM2 ganglioside in cerebrospinal fluid (CSF) was This was due to a reduction of approximately 25% in the enzyme activity of β-hexosaminidase A in CSF. A three-fold increase from baseline in Tay-Sachs disease patients was accumulated, This indicates that the decrease in GM2 ganglioside, which is thought to be the cause of the progression of In addition, β-hexosaminidase A protein was detected by Western blot. Expression of α-amyloides was also increased from baseline to 3 months. These data are shown in Figure 72. vinegar.

[0241] [Table 9]

[0242] [Table 10]

[0243] [Example 15] A second subject (approximately 3 months of age) with progressive infantile Tay-Sachs disease was administered rAAVrh8- HexA / B were administered. Three weeks prior to treatment, subjects underwent several clinical safety tests. The subjects were administered an immunosuppressive regimen for approximately seven days prior to treatment. HexA alleles: 1) HexA: 1.75 kb deletion of exons 11-13 and 2) HexA protein: exon 10, Val381 * Truncation mutations Yes.

[0244] Prior to administration of rAAVrh8-HexA / B, subjects had normal laboratory values ​​and showed no signs of growth or maturity. The child was clinically well, with normal development and growth and no evidence of seizures or hyperactive startle responses. The controls had approximately 1% hexosaminidase activity compared to wild-type activity.

[0245] [Example 16] We designed a clinical study of rAAVrh8-HEXA / B. HEXA and IL-1 in Tay-Sachs disease (TSD) or Sandhoff disease (SD) Bilateral intrathalamic administration of AAVrh8 vectors encoding HEXB separately The study was designed as a two-stage, dose-escalation, and safety / efficacy study for intrathecal and intrathecal administration. The clinical studies are summarized below. GM2 Gangliosidosis Due to the rapidly progressive and fatal nature of, and the lack of approved therapies, research has No placebo or active control groups will be utilized. Treatment will be unblinded.

[0246] The subjects in this study were those under 18 months of age (premature infants (gestational age < 18 months)) at the time of gene transfer. 37 weeks) based on gene mutation analysis and onset of symptoms Infants of either sex with infantile-onset TSD or SD. The HEXA gene encodes the α subunit, or the H Subjects must have the respective mutation in the EXB gene. Patients were considered suitable surgical candidates based on their ability to sit for 5 seconds (according to the study neurosurgeon). (confirmed based on physical examination and MRI findings) and serum HexA activity <5% of normal and do not carry the G269S or W574C mutation. In addition, patients were also seronegative for AAVrh8 neutralizing antibodies based on the test assay threshold. Other inclusion / exclusion criteria may also be applied.

[0247] The study was conducted using an open-label, non-randomized, viral vector, rAAVrh8-H Single-dose administration of EXA / B via bilateral intrathalamic (BiTh) and intrathecal (IT) administration Subjects will be enrolled in the study in two stages (Figure 73) as follows: Phase 1: Four subjects were randomized to a sequential, dose-escalating randomized controlled trial to determine the optimal dose. Dose selection will be based on safety, biomarker, and further data. Phase 2: To determine safety / efficacy, up to Approximately 10 subjects are treated with the optimal dose identified in the first phase.

[0248] All subjects will be followed for up to four years to determine the ongoing safety and efficacy of treatment. It is planned to participate in a long-term follow-up (LTFU).

[0249] Study procedures were administered over the course of 2 days to minimize the risk of prolonged anesthesia. At Visit 1a / Day 0, subjects will receive rAAVrh8-HEXA / B, At Visit 1b / Day 1, subjects received bilateral intraparenchymal injections into the thalamus on each side of the brain; h8-HEXA / B. All injections were It is composed of a 1:1 mixture with rh8AAV-HEXB.

[0250] To prevent the risk of an inflammatory response, AAV-transduced cells were immunocompetent to the AAVrh8 capsid. To protect against immune responses, subjects were immunosuppressed prior to administration of AAVrh8-HexA / B treatment. The immunosuppressive regimen will be maintained as planned long-term. Beginning on day -16 of the study, begin the following: ·Body surface area 1m 2 375 mg of rituximab per dose 10mg / kg Solu-Medrol ·Body surface area 1m 2 1 mg of sirolimus per Prednisolone 2mg / kg per day Lansoprazole 1 to 1.5 mg / kg per day Trimethoprim, 10 mg / kg per dose, given three times weekly (Monday, Wednesday, Friday) / Sulfamethoxazole Day -7: Continuation of sirolimus, prednisolone, and lansoprazole Continuation of trimethoprim / sulfamethoxazole Rituximab infusion required to sustain CD20 counts above 5% Day -2: Continuation of sirolimus, prednisolone, and lansoprazole Continuation of trimethoprim / sulfamethoxazole Day 0 / BiTh injection day Continuation of sirolimus, prednisolone, and lansoprazole Continuation of trimethoprim / sulfamethoxazole Day 1: IT injection day Continuation of sirolimus, prednisolone, and lansoprazole Continuation of trimethoprim / sulfamethoxazole Days 2-7: Continuation of sirolimus, prednisolone, and lansoprazole Continuation of trimethoprim / sulfamethoxazole Intravenous immunoglobulin (IVIG) administration: Immunoglobulin levels are 700 ml / The dose should be below 700-1000 mg / dL. Adjust the volume to maintain the desired level after vector injection. Day 14: Continuation of sirolimus, prednisolone, and lansoprazole Continuation of trimethoprim / sulfamethoxazole Day 21: Continuation of sirolimus, prednisolone, and lansoprazole Continuation of trimethoprim / sulfamethoxazole Week 4 / Month 1: Continuation of sirolimus, prednisolone, and lansoprazole Continuation of trimethoprim / sulfamethoxazole Administration of IVIG Week 4 / Month 2: Continuation of sirolimus, prednisolone, and lansoprazole Continuation of trimethoprim / sulfamethoxazole Administration of IVIG Week 12 / Month 3: Continuation of sirolimus Continuation of trimethoprim / sulfamethoxazole Administration of IVIG 24th week / 6th month - Suspension of sirolimus under the guidance of the hospital's consulting pharmacist

[0251] Intrathalamic (ITh) administration For the first phase, the clinical dose and injection are proposed to be delivered to the thalamus (Th). For example, the input capacity is 2.8 x 10 as shown in Table 11. 12 ~1.1×10 13 vg / ml.

[0252] [Table 11]

[0253] The amount of the thalamus that is filled with the vector, or the volume of distribution (Vd), is the volume of the target brain region (the thalamus). The product is the product of how far the vector will diffuse from the point source of delivery. It can be calculated based on the injected volume (Vi) multiplied by a correction factor. a From injection studies (Yin, Richardson et al. 2010) (Yin et al, 2010), the ratio of Vd:Vi was assumed to be 3.0. The volume to the human thalamus is 1.08, as shown in Table 12. Vi in mL, and 4.35 ml 3 Vd (77% of the total thalamic volume per side) was calculated as I calculated it.

[0254] [Table 12]

[0255] A Th injection volume of 1.08 ml was offered for subjects 1 to 3 in phase 1 of the clinical trial. The doses of injection were at or above the doses given. Low, medium, and high doses of thalamus The proposed adjusted clinical dose for intravenous administration is 7.2 x 10 12 ~1.4×10 13 vg It is.

[0256] Intrathecal (IT) administration The proposed human dose for intrathecal administration is 2.03 x 10 13 ~8.13×1013 vg range, with a maximum injection volume of 8 mL (see table below) The latter value is based on an IT dose of 8.0 × 10 across all treated subjects. 13 vg and set It is proposed for use in clinical studies.

[0257] [Table 13]

[0258] Throughout the procedure, the subject is placed under general anesthesia with intubation. While performing the infusion at 0.1 μL / min, the infusion cannula on the right side, while preventing blockage and vector primed and attached to a delivery syringe and pump. A flexible catheter tube is placed stereotaxically into the target area in the right thalamus. The left hand is then secured to the scalp with anchoring ligatures and an adhesive dressing. Repeat the same steps for placement of the second catheter on the side. Continuous cephalad catheterization is performed throughout the procedure. Perform intracapsular pressure monitoring.

[0259] The infusion rate begins at 0.5 μL / min and is increased every 5 min (1.0 μL / min → 2. 0 μl / min → 5 μl / min). The study drug was delivered to the microinfusion pump, and the study drug was delivered to the HexA Each vector was injected via a syringe containing a 1:1 mixture of the HexB vector and the HexB vector. Inject into the catheter.

[0260] IT administration: IT drug infusions are administered at a rate dictated by CSF pressure. A total of 6.0 ml is administered through a high-neck catheter. 1.0 ml was delivered to the vertebral level / cisterna magna, and 2.0 ml was delivered to the thoracic / lumbar level. Under guidance, the Tuohy needle is inserted into the subarachnoid space. Insertion into the catheter is verified by spontaneous CSF flow. A total of 6-7 mL of CSF is drained by gravity. The tissue was then reintroduced into the flexible Excelsior ( SL-10 (registered trademark) Microcatheter (Stryker Neurova scular, Stryker) near or into the cisterna magna. A syringe containing a 1:1 mixture of HexA and HexB is attached to the catheter. Following administration of 6.0 ml at the high cervical / cisternal level, the microcatheter was inserted inferiorly. Switch to the thoracic-lumbar level and administer a second bolus of 2.0 ml. Injection is complete. Then, the microcatheter (dead volume: approximately 0.3 mL) was flushed with 0.5 mL of physiological saline. The cerebrospinal fluid is then removed. The dose was administered by two separate IT injections at the high cervical / cisternal and thoracic / lumbar levels. It can be given.

[0261] Primary endpoint(s): Phase 1: TEAEs (tr Incidence and severity of food emergent adverse events , severity, and relevance to treatment Phase 2: Surrogate biological markers: blood from baseline to visit 7 (month 3) Changes in HexA activity in serum / CSF Clinical function: At 9 / 12 months of visit, Bayley Scales of Infant and Toddler 5.0, without assistance, as assessed by Section 22 of the BSID-III. Proportion of subjects who achieve the ability to sit for seconds ·LTFU: Adverse events

[0262] Secondary endpoint(s): Phase 1: Changes in vital signs, including weight Changes in general and neurological examination values Complete blood count, comprehensive metabolic panel, and high-sensitivity C-reactive protein with automated white blood cell differential Changes in laboratory values, including quality of life CD20 count ECG, EEG -AAVrh8 capsid and HEXA and HEXB proteins The cellular responses and neutralizing antibody titers associated with HEXA and HEXB proteins were also Antibody levels against Phase 2: Biological markers for disease: CSF HexA activity levels from baseline to visit 9 (month 12) Changes Serum HexA at baseline to visits 4, 5, 6, 7, 8, and 9 Changes in activity levels From baseline (by LC-MS / MS) to visit 9 (month 12), C Changes in GM2 ganglioside levels in SF CS at baseline, visit 7 (month 3) and visit 9 (month 12) Lactate dehydrogenase (LDH) and aspartate aminotransferase in F Changes in AST levels ·Clinical functions: 1. Assessed on BSID-III item 22 at Visits 5, 7, and 8 , the proportion of subjects who achieve the ability to sit for 5 seconds without assistance 2. Baseline to Visits 5, 7, 8, and 9 of CHOP-INTEN D Total Score Change 3. At Visits 5, 7, 8, and 9, the CHOP INTEND motor function schedule was The following criteria were met: score of 40 or above, score of 50 or above, and score of 64 or above. Percentage of subjects achieving the score 4. BSID-III C at baseline through visits 5, 7, 8, and 9 Change in composite score 5. At Visits 7, 8, and 9, Hammersmith Infant Assessed by Neurological Examination-2 (HINE-2) , the proportion of subjects who achieved any new motor goal (the new motor goal was to sit unassisted) crawling on hands and knees, standing with assistance, walking with assistance, standing upright independently, and (defined as walking independently and 6. Using BSID-III (Gross Motor Subset #43) The proportion of subjects who take at least five steps independently while demonstrating the coordination and balance required

[0263] Exploratory endpoint(s) (Phase 2): Brainstem auditory evoked responses (BAER) and and changes in visual evoked responses (VER) MRI brain volumetric analysis of myelination from baseline to visits 7 and 9 indices, and diffusion tensor imaging (DTI) indices, and changes in intracerebral water MRS indices of change in metabolite accumulation from baseline to visits 7 and 9 Change in eye tracking assessment from baseline to visits 4, 5, 6, 7, 8, and 9 The following will be assessed from screening through to the end of treatment: Adverse events will be assessed and graded according to NCI CTCAE v5.0 Physical Examination Vital signs Standard laboratory tests: hematology, clinical chemistry, coagulation, and urinalysis ·AESI(Adverse Events of Special Interes) t): Surgery-related complications Device-related complications Acute hypersensitivity reactions Elevated liver function test values ​​(AST / ALT, GGT) Disturbances in hematological parameters, with special attention to platelet counts Late adverse events, including but not limited to autoimmune-like reactions and malignancies

[0264] [Example 17] Further details regarding the present disclosure can be found in US Pat. No. 6,399,433, the entire contents of which are incorporated herein by reference. The present application is based on U.S. Provisional Patent Application No. 62 / 840,359, filed April 29, 2019. This is set out in Appendix A of the specification.

[0265] We investigated a method for administering AAV into the cisterna magna via a microcatheter. AAV9-CB-GFP was injected into 1 × 10 cells via an intravascular microcatheter. 14 In vg, The microcatheter was injected into sheep (30 kg, n=3). The tip of the catheter was placed in the thoracic region of the sheep until it was finally placed (Figure 75B, right panel, arrow). The endoscopic examination was guided by fluorescence microscopy through the pulmonary artery and cervical regions (Fig. 75A). After injection of the contrast agent, Then, 15 ml of scAAV-CB-GFP was injected at 1 ml / min, and the distribution pattern near the cerebellum was observed. The sheep were then sacrificed and macroscopic images of the postmortem spinal cord were observed. Immunohistochemical staining showed that GFP was expressed in motor neurons, sensory neurons, and sarcolemma in the spinal cord (Fig. 75C). Ron (Fig. 75D), and Purkinje cells, cerebellar peduncle, deep cerebellar nuclei, brain stem, cerebrum, occipital cortex was expressed within the substantia nigra, parietal cortex, thalamus, hippocampus, temporal cortex, motor cortex, internal capsule, and frontal cortex. These results indicate that the AA Administration of V into the cisterna magna expresses the transgene of interest (e.g., GLB1, HEXA, HEXB It is noted that the present invention can be used to efficiently deliver

[0266] The present disclosure relates to the construction and arrangement of the components set forth in the description or illustrated in the drawings. The present disclosure is not limited in its application to the details of the arrangement. Also, the words and terms used herein may be changed without departing from the spirit or scope of the present invention. The present invention is intended to be illustrative and not limiting. "including", "comprising" " or "having," "containing," "involving," and the like. The use of these variations includes the following enumerated items and their equivalents, as well as further variations. This is intended to encompass all items.

[0267] Thus, in some aspects of at least one embodiment of the present disclosure, Having described the above, various changes, modifications, and improvements will readily occur to those skilled in the art. It is to be understood that such alterations, modifications, and improvements are part of this disclosure. It is contemplated and intended to be within the spirit and scope of the present disclosure. The description and drawings are for the purposes of example only.

Claims

1. Lysosomal storage selected from HEXA and HEXB via chimeric introns A promoter operably linked to a transgene encoding a disorder-associated protein. A capsid containing a nucleic acid comprising A recombinant AAV (rAAV) comprising:

2. The recombinant AAV of claim 1 , wherein the nucleic acid does not contain an enhancer element.

3. The promoter comprises a chicken beta-actin (CB) promoter.

3. The rAAV described in 1 or 2.

4. The chimeric intron comprises an intron sequence of chicken beta actin, and / or The nucleic acid sequence according to any one of claims 1 to 3, which comprises an intron sequence of rabbit beta globin. rAAV.

5. The chimeric intron is flanked by non-translated sequences, and optionally, the non-translated sequences are The rAA according to any one of claims 1 to 4, which is a non-translated sequence of gi beta-globin. V.

6. The untranslated sequence of rabbit beta-globin is derived from exon 1 or exon 2. The rAAV of claim 5, which is a non-translated sequence.

7. The transgene is located between the chimeric intron and a polyadenylation signal. and optionally, the transgene is arranged to include the untranslated sequence and a polyadenylation signal. The rAAV of any one of claims 1 to 6, wherein the rAAV is located between the

8. 8. Any of claims 1 to 7, wherein the HexA comprises the amino acid sequence set forth in SEQ ID NO:

20. The rAAV described in any one of claims.

9. 8. Any of claims 1 to 7, wherein the HexB comprises the amino acid sequence set forth in SEQ ID NO:

21. The rAAV described in any one of claims.

10. AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 , AAVrh8, AAV9, AAV10, and AAVrhlO of a serotype selected from 10. An rAAV according to any one of claims 1 to 9, comprising a capsid protein.

11. 10. The method according to claim 1, comprising the capsid protein of the AAVrh8 serotype. rAAV as described above.

12. It includes one or more ITRs, each ITR being an AAV1 ITR, an AAV2 ITR, From the AAV3 ITR, the AAV4 ITR, the AAV5 ITR, and the AAV6 ITR 12. The rAAV of any one of claims 1 to 11, selected from the group consisting of:

13. An isolated nucleic acid comprising a sequence defined by SEQ ID NO:1-6 or 16-19.

14. A sequence encoding a peptide having the sequence set forth in SEQ ID NO: 20 or 21. isolated nucleic acid.

15. A host cell comprising the isolated nucleic acid construct of claim 13 or 14.

16. The host cell of claim 15 , which is a eukaryotic cell.

17. 17. The host cell of claim 15 or 16, which is a mammalian cell.

18. The host cell of claim 15 which is a prokaryotic cell.

19. The host cell of claim 18 which is a bacterial cell.

20. 20. The method of claim 15, further comprising the step of: A host cell according to any one of claims 1 to 4.

21. The capsid protein is an AAVrh8 capsid protein or an AAVrh10 capsid protein. The host cell of claim 20, wherein the gene is a psyllid protein.

22. (i) via a chimeric intron, the beta-hexosaminidase subunit alpha (HexA) and a capsid containing a first nucleic acid, and optionally said HexA is as set forth in SEQ ID NO:

20. a first rAAV comprising the amino acid sequence shown; (ii) via a chimeric intron, the beta-hexosaminidase subunit beta (HexB) operably linked to a transgene encoding the promoter and a capsid containing a second nucleic acid, and optionally said HexB is as set forth in SEQ ID NO:

21. A second rAAV comprising the amino acid sequence shown. Including, Optionally, the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier.

23. The first rAAV and / or the second rAAV has an AAVrh8 serotype. The pharmaceutical composition of claim 22, comprising a capsid protein comprising

24. The first nucleic acid and / or the second nucleic acid flank the transgene.

3. The method of claim 2, further comprising:

24. The pharmaceutical composition according to claim 2 or 23.

25. The first nucleic acid and / or the second nucleic acid does not contain an enhancer element.

25. The pharmaceutical composition according to any one of claims 22 to 24.

26. The promoter of (i) and / or the promoter of (ii) is 26. The method according to any one of claims 22 to 25, comprising a cytoplasmic β-actin (CB) promoter. A pharmaceutical composition comprising:

27. The chimeric intron of (i) and / or the chimeric intron of (ii) is Intron sequence of chicken beta actin and / or intron sequence of rabbit beta globin 27. The pharmaceutical composition of any one of claims 22 to 26, comprising a ribonucleic acid sequence.

28. The chimeric intron (i) and / or the chimeric intron (ii) may be provided with a non- flanked by translated sequences, and optionally the untranslated sequences are the untranslated sequences of rabbit beta-globin.

28. The pharmaceutical composition of any one of claims 22 to 27, which is a sequence.

29. The untranslated sequence of rabbit beta-globin is derived from exon 1 or exon 2.

29. The pharmaceutical composition of claim 28, which is a non-translated sequence.

30. The transgene (i) and / or the transgene (ii) is A chimeric intron is placed between the chimeric intron and a polyadenylation signal, and optionally, the intron A gene encoding the polypeptide is located between the non-translated sequence and the polyadenylation signal.

30. The pharmaceutical composition according to any one of claims 22 to 29.

31. The first rAAV and the second rAAV are present in the composition in a 1:1 ratio. The pharmaceutical composition according to any one of claims 22 to 30.

32. 13. A method for treating a lysosomal storage disorder comprising administering to a subject a therapeutically effective amount of any one of claims 1 to 12. or a recombinant AAV (rAAV) according to any one of claims 22 to 31. The method comprises administering a pharmaceutical composition of the formula (I) to a subject having a lysosomal storage disorder.

33. 3. The method of claim 2, wherein the lysosomal storage disorder is Tay-Sachs disease or Sandhoff disease.

2. The method according to claim 2.

34. The rAAV or pharmaceutical composition is administered by intracranial injection, intracerebral injection, or intravenous injection into the ventricular system, cisterna magna, or The method of claim 32 or 33, wherein the compound is administered by injection into the CSF, preferably via the intrathecal cavity. Law.

35. A container for storing the recombinant AAV (rAAV) according to any one of claims 1 to 12. Kit including:

36. (i) via a chimeric intron, the beta-hexosaminidase subunit alpha (HexA) and a capsid containing a first nucleic acid, and optionally said HexA is as set forth in SEQ ID NO:

20. a first container containing a first rAAV comprising the indicated amino acid sequence; (ii) via a chimeric intron, the beta-hexosaminidase subunit beta (HexB) operably linked to a transgene encoding the promoter and a capsid containing a second nucleic acid, and optionally said HexB is as set forth in SEQ ID NO:

21. a second container containing a second rAAV comprising the indicated amino acid sequence; and optionally, (iii) a third container comprising a pharma- ceutically acceptable carrier; and Kit including:

37. 4. The method of claim 1 , wherein the first container, the second container, and / or the third container are syringes.

37. The kit according to claim 35 or 36.

38. 32 or 33, wherein the administration comprises injection into the CSF via the cisterna magna and / or the intrathecal cavity.

35. The method according to any one of claims 34 to 35.

39. 39. The method of claim 38, wherein the administration further comprises an intrathalamic injection.

40. The administration may be by injection into the CSF via the cisterna magna and intrathecal cavity, and by intrathalamic injection.

40. The method of any one of claims 32 to 34 or 38 to 39, wherein

41. The administration is into the intrathecal space via a fluoroscopically guided lumbar intrathecal catheter.

41. The method of any one of claims 32 to 34 or 38 to 40.

42. The total rAAV administered to the subject is about 1×10 １１ ~1×10 １５ Vector genome 42. The method according to claim 32, wherein the (vg) copies are between the Method of posting.

43. in the CSF of the subject prior to administration of the rAAV or the pharmaceutical composition. β-hexosaminidase A activity is less than 0.5% of normal β-hexosaminidase A activity.

43. The method of any one of claims 32 to 34 or 38 to 42, having A activity.

44. The subject has normal enzyme activity about 3 months after administration of the rAAV or pharmaceutical composition. at least 0.5%, at least 1.0%, or at least 1.4% of the original activity 3. The method according to claim 32, wherein the enzyme activity of β-hexosaminidase A in CSF is increased.

44. The method of any one of claims 4 or 38 to 43.

45. The subject has a pre-administration, pre-treatment, and post-treatment, At least a two-fold increase, or at least a three-fold increase, compared to the subject's baseline.

45. The method of any one of claims 32 to 34 or 38 to 44, comprising:

46. operably linked to a transgene encoding the human GLB1 sequence in SEQ ID NO:23 Capsid containing nucleic acid containing a promoter A recombinant AAV (rAAV) comprising:

47. operably linked to a transgene encoding the human GLB1 sequence in SEQ ID NO:23 The promoter 2. An isolated nucleic acid comprising:

48. The promoter comprises a chicken beta-actin (CB) promoter.

47. An rAAV as described in claim 46, or an isolated nucleic acid as described in claim 47.

49. AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 AAVrh8, AAV9, AAV10, and AAVrhl0.

49. The rAAV of claim 46 or 48, comprising a capsid protein of a serotype selected from the group consisting of serotypes.

50. 50. The rAAV of claim 49, comprising an AAV9 capsid protein.

51. One or more ITRs, each ITR being an AAV1 ITR, an AAV2 ITR, From the AAV3 ITR, the AAV4 ITR, the AAV5 ITR, and the AAV6 ITR 51. The rAAV of any one of claims 46 or 48 to 50, selected from the group consisting of 。

52. 52. The rAAV of claim 51, comprising one or more AAV2 ITRs.

53. 53. The rAAV of claim 46 or 48 to 52, and a pharma- ceutically acceptable carrier.

23. A pharmaceutical composition comprising:

54. A method for treating GM1 gangliosidosis comprising administering to a patient of claim 46 or 48 52, or the pharmaceutical composition of claim 53, A method comprising administering to a subject having GM1 gangliosidosis.

55. 50. The method of claim 49, wherein the administration is intravenous administration.

56. The dose is about 10 mg / kg of the subject's body weight. １１ ~10 １４ rAAV 56. The method of claim 54 or 55, wherein the vector genome (vg) copies.

57. A method for treating GM2 gangliosidosis comprising administering to a patient a compound according to any one of claims 1 to 12. or a pharmaceutical composition according to any one of claims 22 to 31, A method comprising administering to a subject having GM2 gangliosidosis.

58. GM2 ganglioside is about 3 months after administration of the rAAV or the pharmaceutical composition, in the cerebrospinal fluid (CSF) of the subject, from baseline by at least at least 15%, The method according to claims 32 to 34, 38 to 39, wherein the amount of the ion exchange reaction is reduced by at least 20% or at least 25%. 45 or 57. The method of claim 45 or 57.

59. The dose administered to the thalamus is about 1×10 １１ ~1×10 １５ vg. The method of any one of 2 to 34, 38 to 45, or 57 to 58.

60. The dose administered into the cisterna magna and / or the intrathecal cavity is about 1×10 １１ ~1×10 １５ vg 60. The method of claim 32, 34, 38, 45, or 57, wherein method.

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