rAAV vectors for the 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 titled "RAAV VECTORS FOR THE TREATMEN T OF GM1 AND GM2 GANGLIOSIDOSIS,” U.S. 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 March 29, 2019, entitled "RA 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 titled "RAAV VECTORS FOR THE TREATMENT OF GM1 AND GM2 GANGLIOSIDOSIS” 62 / 840,359, filed May 16, 2019, entitled RAAV VECTORS FOR THE TREATMENT OF GM1 AND GM2 GANGLIOSIDOSIS" No. 62 / 848,8 58 as of the filing date, benefit under 35 U.S.C. § 119(e) Assert.

[0002] Reference to a sequence listing 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. Viruses (rAAV), compositions, and kits are provided. In some embodiments, lysosomal Some telomere storage disorders include GM1 gangliosidosis or GM2 gangliosidosis (e.g., For example, Tay-Sachs disease.

[0004] Federally sponsored research This invention was made in part through 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. is expressed in numerous molecules in the central nervous system (e.g., GM1 ganglioside) and in peripheral tissues. Numerous molecules (e.g., oligosaccharides, glycoproteins, and glycosaminoglycans) The enzyme β-D-galactosidase (βgal), which removes terminal galactose residues, was used to The lack of β-gal activity in lysosomes is primarily due to the loss of β-gal activity during the period when its synthesis rate is maximal. In the central nervous system, accumulation of GM1 ganglioside and its asialo derivative, GA1, GM1 gangliosidosis is generally fatal and there is no effective treatment. stomach.

[0006] Tay-Sachs disease (GM2 gangliosidosis) and Sandhoff disease are β-N- 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 Hex and Hex. Deficiency of Hex activity results in central nervous system This leads to progressive accumulation of GM2 gangliosides and subsequent neurodegeneration in the brain. 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 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 Currently, there are no known causes of either Tay-Sachs or Sandhoff disease. However, there are no disease-modifying treatment options, and only symptomatic treatments are available, including anticonvulsants and anticonvulsants. 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., cytotoxic effects) in subjects. This disclosure demonstrates that manipulation of regulatory elements can reduce adverse events and deliver high levels of gene expression. 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 ATP can modulate

[0008] In some aspects, the present disclosure provides a method for the treatment of lysosomal storage disorder-associated tumors 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 AAVs via chimeric introns. a capsid containing a nucleic acid comprising a promoter operably linked to a transgene gene; 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 a chicken promoter. In some embodiments, the chimeric intro The introns are located in the chicken beta-actin intron and / or the rabbit beta-globin intron. 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. region 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. -encodes 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 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 protein. The four ITRs are AAV1 ITR, AAV2 ITR, AAV3 ITR, and AAV4 ITR. 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 present disclosure provides vectors encoding the components of the rAAVs described herein. For example, in some embodiments, the present disclosure relates to nucleic acids set forth in SEQ ID NOS: 1-6 or 16-19. In some aspects, 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 the amino acid sequence specified in 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 used. 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: 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. It is a cell.

[0013] In some embodiments, the host cell contains an AAV capsid protein (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV17, AAV18, AAV19, AAV20, AAV21, AAV22, AAV23, AV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8 A single protein encoding the capsid protein of AAV10, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAVrh10 It further comprises an isolated nucleic acid.

[0014] In some aspects, the present disclosure provides a method for the production of beta-hexosaminidase-binding proteins (e.g., ribozymes ... operably linked to a transgene encoding hexidase subunit alpha (HexA). a first rAAV comprising a capsid containing a first nucleic acid comprising a promoter ligated 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 pharmaceutically acceptable carrier. In this case, 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 monocyte Some embodiments provide methods for administering the compound to a subject with a condition such as Lukio's syndrome. In embodiments, 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, comprising: The rAAV or pharmaceutical compositions described herein can be administered to patients with GM2 gangliosidosis. A method is presented comprising administering to a subject

[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 pharmaceutically acceptable carrier. In some embodiments, the rAAV or a composition comprising the rAAV and a pharmaceutical In some embodiments, the container is a silicone-based container. It's a ninja.

[0017] In some aspects, the present disclosure provides a transfectant encoding human GLB1 as 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 gene 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 pharmaceutically acceptable carrier. In some embodiments, the present disclosure provides a method for treating GM1 gangliosidosis. The method of claim 1, wherein the rAAV or pharmaceutical composition is administered to a subject having GM1 gangliosidosis. A method is presented that includes administering to an elephant. [Brief explanation of the drawings]

[0018] [Figure 1]Design of the rAAV vector, CBA-mβgal-WPRE. Figure 1A shows a schematic diagram of the rAAV vector, CBA-mβgal-WPRE. Two inverted terminal repeats (ITRs) derived from AAV2 flank the vector at each end. The CBA promoter consists of a chimeric chicken beta-actin / rabbit beta-globin intron (CBA) followed by a cytomegalovirus immediate-early enhancer (CMV) fused to the chicken beta-actin promoter, mouse lysosomal acid beta-galactosidase cDNA (mβgal), a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), and two tandem poly(A) signals derived from bovine growth hormone (BGH) and SV40. 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. [Figure 2] Figure 2 shows that intracranial injection of AAVrh8-mβgal 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 × 10 vg) or 3 months (2.6 × 10 vg and 2.6 × 10 vg) after injection. Figures 2A-2B show 4 × 10 vg; Figures 2E-2F show 2.6 × 10 vg; Figures 2I-2J show 2.6 × 10 vg; Figures 2C-2D show naive βgal- / - mice; and Figures 2G-2H show naive βgal+ / - mice. Images represent N≥3 mice per group. [Figure 3]Figure 3 shows that 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 (Figure 3A) 4 x 1010 vg; (Figure 3B) 2.6 x 1010 vg; and (Figure 3C) 2.6 x 109 retained significantly better motor function than naive βgal- / - controls at 6 months post-injection (P = 0.006, 0.0009, and 0.005, respectively). N = 3-15 animals per group at each time point, and N = 6-10 animals per group at 6 months post-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 increased from naive βgal controls (245.5 days, N=13) to the 4x1010vg cohort (293.5 days, N=12), 2.6x1010vg cohort (349.0 days, N=13), and 2.6x109vg cohort (389.0 days, N=13). [Figure 5]Figure 5A shows that intracranial injection of AAV in βgal- / - mice results in abnormal filipin staining within the region of highest enzyme expression. Figure 5A shows a coronal section of the 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) bilaterally injected into the thalamus with 1 μl of AAVrh8-CBA-mβgal-WPRE (total dose of 2.6 × 10 vg). Figure 5B shows an untreated βgal- / - mouse (untreated knockout). Boxes indicate the location of images depicted in Figures 5C-5F. Flipin staining in adjacent brain sections from a βgal- / - animal treated with 2.6 × 10 vg of AAVrh8-CBA-mβgal-WPRE is shown in Figures 5C and 5E. Figures 5D and 5F show untreated βgal mice (untreated knockout). Images of filipinas taken at 10x magnification. Images represent N≧3 mice per group. [Figure 6] These figures show that lysosomal accumulation persists 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 GM1 ganglioside accumulation. All long-lived animals showed some amount of intraspinal clearance, but also contained areas where clearance did not occur. Figures 6A and 6B show data 621 days after injection of 4×1010 vg. Figures 6C and 6D show data 547 days after injection of 2.6×1010 vg. Figures 6E and 6F show data 495 days after injection of 2.6×109 vg. Figures 6G and 6H show generalized accumulation approximately 250 days after injection in naive βgal- / - mice. Images represent N≧2 mice per group. [Figure 7]Figure 7 shows that intracranial injection of AAV in βgal- / - mice results in morphological changes at the injection site in the thalamus. Intracranially injected βgal- / - mice were analyzed by H&E staining of 20 μm coronal brain sections 2 weeks (4 × 10 vg) or 3 months (2.6 × 10 vg and 2.6 × 10 vg) after injection. Shape changes within the injected area of ​​the thalamus: thick arrows indicate cellular infiltration into blood vessels, and thin arrows indicate inflammation. The left panel is a 10x magnification, and the right panel is a 40x magnification (from the area above the left panel). Shape changes and neuronal loss appear to be attenuated even with the lowest injected dose. Figures 7A and 7B show the 4 × 10 vg injection after 2 weeks of treatment. Figures 7E and 7F show the 2.6 × 10 vg injection after 3 months of treatment. Figures 7I and 7J show injections of 2.6 x 109 vg 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 injection site in βgal animals and the location of the photographs shown in this figure. Scale bar represents 100 um. Images represent N > 3 mice per group. [Figure 8]Figure 8 shows that intracranial injection of AAV in βgal- / - mice results in morphological changes at the injection site within the deep cerebellar nuclei. Intracranial-injected βgal- / - mice were analyzed by H&E staining of 20 μm coronal brain sections 2 weeks (4 × 10 vg) or 3 months (2.6 × 10 vg and 2.6 × 10 vg) after injection. Morphological changes were observed within the injected area of ​​the DCN. The thick arrow indicates cellular infiltration into blood vessels. Arrows point to infiltration suspected to be phagocytic neurons. The left panel is a 10x magnification, and the right panel is a 40x magnification photograph (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 × 10 vg injections 2 weeks after treatment. Figures 8E and 8F show injections of 2.6 x 10 vg 3 months after treatment. Figures 8I and 8J show injections of 2.6 x 10 vg 3 months after treatment. Figures 8G and 8H show untreated naive βgal+ / - mice at the age of treated animals. Figures 8C and 8D show injections into naive βgal- / - mice at the age of treated animals. Figure 8K shows a DCN injected with AAVrh8-mβgal. Arrows indicate the injection site in βgal animals and the location of the photographs shown in this figure. Scale bar represents 100 um. Images represent N > 3 mice per group. [Figure 9]Figure 9 shows that intracranial injection of AAV in βgal+ / − and βgal− / − mice results in abnormal filipin staining within the region of maximal expression of the enzyme. Sagittal sections of mouse brains stained with Xgal for the presence of βgal enzyme and counterstained with Nuclear Fast Red 10 weeks after injection of representative (Figure 9A) βgal+ / − mice (chimera + AAV) and (Figure 9B) βgal− / − mice (knockout + AAV), and (Figure 9C) untreated βgal− / − mice (untreated knockout), were injected with 1 μl of AAVrh8-CBA-mβgal-WPRE bilaterally into the thalamus at 1.7 × 1012 vg / μl, 2 μl into the ventricles, and 0.3 μl into the deep cerebellar nuclei. Boxes indicate the locations of images depicted in Figures 9D-9I. Filipin staining was positive within the region of maximal enzyme expression in both chimera and AAV-injected animals (Figures 9D, 9G, and 9J; βgal+ / −) and βgal− / − knockout and AAV-injected animals (Figures 9E, 9H, and 9K). Figures 9F, 9I, and 9L 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]These figures show that changes in vector design result in the presence of β-gal protein and / or reduced β-gal enzyme activity in β-gal mice. Figure 10A shows β-gal enzyme activity within the injected structures of 2 mm × 2 mm biopsy punches. Approximately 6 weeks after injection, 1 μl of AAVrh8 vector (3.4 × 10 vg, or 2.0 × 10 vg total dose for high-dose CB6 alone) or mock-treated PBS was injected bilaterally into the thalamus, as determined by 4-MU assay. Enzyme activity was normalized to protein concentration by Bradford reagent and is reported as nanomoles per hour per mg of protein. Error bars represent mean + SD, N = 3 per group. * indicates significant differences between β-gal + / - AAV (chimera + vector name) and untreated β-gal + / - (untreated chimera), or as indicated by a connecting line. P values ​​calculated using an unpaired multiple t-test (Holm-Sydac), where *=p<0.05, **=p<0.01, and ***=p<0.001. Figure 10B shows the presence of endogenous β-gal protein, as determined by Western blot, which appears at 67 kd, while expression of the transgene from the AAV vector appears as a higher-mass band. The loading control is actin, which appears at 42 kd. Western blots shown represent N=3 per run. [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 alone) was injected bilaterally into the thalamus of 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 an unpaired multiple t-test (Holm-Sydac), indicated by a connecting line, 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 β-gal enzyme and counterstained with Nuclear Fast Red 6 weeks after injection of a representative β-gal (chimera + vector name) or uninjected β-gal (untreated chimera) bilaterally into the thalamus with 1 μl of AAVrh8 vector (3.4 × 10 vg, or a total dose of 2.0 × 10 vg in high-dose CBA alone). Figures 12B, 12E, 12H, 12K, 12N, and 12Q show filipin staining in parallel sections of the same animals in Figures 12A, 12D, 12G, 12J, 12M, and 12P. Figures 12C, 12F, 12I, 12L, 12O, and 12R show ToPro3 nuclear staining in parallel sections of the same animals in Figures 12A, 12D, 12G, 12J, 12M, and 12P. Boxes represent the 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 1 shows that clustering heat maps and Venn diagrams for all differentially expressed genes in βgal+ / − mice confirm transgene expression-dependent variation. Clustering heat maps and Venn diagrams for all differentially expressed genes were generated using data from 2mm x 2mm biopsy punches from the thalamus of βgal+ / − mice (chimera + vector name) injected with 1 μl of AAVrh8 vector (total dose of 3.4 x 109 vg) or mock-treated with PBS approximately 6 weeks after injection. Microarray results were determined using Affymetrix Mouse Gene 2.0 ST, N = 3 per group, P < 0.05, > 1.8-fold change. [Figure 14] Figure 1 shows that β-gal enzyme activity within the CNS of treated β-gal mice results in variable enzyme distribution across structures. β-gal enzyme activity in the cerebrum, cerebellum + brainstem, or spinal cord of β-gal mice, or untreated (untreated knockout) or untreated β-gal mice (untreated wild-type) injected bilaterally with 1 μl of AAVrh8 vector (3.4 × 10 vg, or 2.0 × 10 vg total dose in high-dose CBA alone) into the thalamus and 0.3 μl into the DCN, as determined by 4-MU assay approximately 6 weeks after injection. Enzyme activity was normalized to protein concentration by Bradford reagent and is reported as nanomoles per hour per mg of protein. Values ​​represent the mean + SD, N = 3 per group, * indicates a significant difference between βgal- / - + AAVrh8-CB6 vector (3.4 x 109 vg total dose; knockout + low-dose CB6) and βgal- / - + AAVrh8-CB6 vector (2.0 x 1010 vg total dose; knockout + high-dose CB6). P values ​​calculated using an unpaired t-test (Holm-Sydac), *** = p < 0.001. [Figure 15]Figure 15A-15C. Xgal staining for the presence of β-gal enzyme in β-gal mice confirms the dose-dependent spread of enzyme expression throughout the brain. Sagittal sections of mouse brains stained with Xgal for β-gal enzyme activity and counterstained with Nuclear Fast Red approximately 6 weeks after injection of representative β-gal mice, or untreated (untreated knockout; Figure 15E), or untreated β-gal mice (untreated chimera; Figure 15F) bilaterally injected with 1 μl of AAVrh8 vector (3.4 × 10 vg, or 2.0 × 10 vg total dose for high-dose CBA alone) into the thalamus and 0.3 μl into the DCN. N = 2-3 per group. Scale bar = 10 mm. Figure 15D shows a knockout mouse treated with empty vector. [Figure 16] Figure 1 shows filipin staining for GM1 ganglioside content in the brains of βgal- / - mice after treatment with therapeutic AAVrh8. Sagittal sections of representative βgal- / - or untreated (untreated knockout) mouse brains were stained with filipin for GM1 content or the nuclear dye ToPro3 (untreated knockout; ToPro3; descending row) 6 weeks after injection of 1 μl of AAVrh8 vector (3.4 × 109 vg, or a total dose of 2.0 × 1010 vg in high-dose CBA alone) into the thalamus bilaterally and 0.3 μl into the DCN. N = 2-3 per group. Images taken at 5x magnification, scale = 100 mm. [Figure 17]Figure 17 shows filipin staining for GM1 content in the spinal cord of βgal- / - mice after therapeutic treatment with AAVrh8 vector. Cervical (Figures 17A, 17C, 17E, 17G, 17I, and 17K) and thoracic (Figures 17B, 17D, 17F, 17H, 17J, and 17L) spinal cord sections were stained with filipin for GM1 content or the nuclear dye ToPro3 (untreated knockout; ToPro3; descending) 6 weeks after injection of representative βgal- / - or untreated (untreated knockout) mice, injected bilaterally with 1 μl of AAVrh8 vector (3.4 × 109 vg, or a total dose of 2.0 × 1010 vg for high-dose CBA alone) into the thalamus and 0.3 μl into the DCN. N = 2-3 per group. The image was taken at 5x magnification and the scale is 100mm. [Figure 18]

[0023] Figure 1 shows that therapeutic treatment with AAVrh8 in the CNS of βgal- / - mice results in normalization of GM1 content when treated with a high dose of a low-expressing promoter. GM1 content was 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 of 1 μl of AAVrh8 vector (3.4 × 109 vg, or a total dose of 2.0 × 1010 vg for the high-dose CBA group only) into the thalamus bilaterally and 0.3 μl into the DCN. GM1 content is 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 a multiple t-test (Holm-Sydac) or significant difference indicated by a connecting line, 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 a 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 the monkey thalamus. Figure 22A shows mononuclear cell infiltration around blood vessels (arrows) and areas of necrosis (*) within the white matter. Figure 22B shows a cross section stained with Luxol Fast Blue, where the arrow highlights one large area of ​​necrosis, vascular proliferation, and white matter loss (note the pale color compared to the adjacent dark blue). Figure 22C shows a necrotic area (arrow) within the thalamus with vacuolization. Figure 22D shows an example of cellular infiltration into blood vessels within the thalamus (40x magnification). Similar neuropathology was also seen with the lowest dose (1 / 30) (Figure 22E). Figure 22F shows a normal thalamus in a PBS-injected animal (1x dose: Figures 22A-22D; 1 / 30 dose: Figure 22E; PBS injection: Figure 22F). [Figure 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 a 1:1 formulation of AAVrh8 vectors encoding cynomolgus monkey Hex-alpha and cynomolgus monkey Hex-beta. Two artificial substrates, MUG and MUGS, were used in the biochemical assay. The first substrate is cleaved by all Hex isozymes (HexA, HexB, and HexS), whereas the latter is cleaved only by HexA and HexS isozymes. [Figure 24] (Figure 1) Pre-necropsy brain MRI revealing edema within the thalamus. Pre-necropsy thalamic edema (arrow) in an NHP injected with a 1 / 30th dose of a formulation of AAVrh8 vector encoding cynomolgus Hex-alpha and cynomolgus Hex-beta. No changes were observed in NHP injected with an empty transgene AAVrh8 vector. [Figure 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 1× 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 an enlarged view of the boxed area in Figure 25E. Arrows indicate 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), which consists of ITR-flanked cDNA with a 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 monkey HexA cDNA and cynomolgus monkey HexB cDNA is generated; pA: polyadenylation signal; ITR: AAV2 inverted terminal repeat; P1: promoter 1, which is thought to increase ITR-mediated gene expression approximately 10-fold; P2: promoter that is thought 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 the alpha-subunit-containing isoforms (HexA, HexS), respectively. [Figure 30] Figure 1 shows the change in body weight over the one-month study period. Error bars represent 1 standard deviation. [Figure 31] Figure 31 shows that mouse brains expressing the highest levels of cynomolgus monkey Hex protein contain eosinophilic neurons. H&E staining indicates the presence of neurons containing eosinophil granules in the hippocampus and thalamus, which correlates with the expression level of cynomolgus monkey Hex (40x magnification). Figures for mice from Group 1 (Figures 31A, 31K), Group 2 (Figures 31B, 31L), Group 3 (Figures 31C, 31M), Group 4 (Figures 31D, 31N), Group 5 (Figures 31E, 31O), Group 6 (Figures 31F, 31P), Group 7 (Figures 31G, 31Q), Group 8 (Figures 31H, 31R), Group 9 (Figures 31I, 31S), and Group 10 (Figures 31J, 31T). [Figure 32] Figure 32 shows that expression of cynomolgus monkey Hexα in the brains of athymic nude mice varies among AAV vectors. Hexα staining (green) in the thalamus and hippocampus of mice from Group 1 (Figures 32A, 32K), Group 2 (Figures 32B, 32L), Group 3 (Figures 32C, 32M), Group 4 (Figures 32D, 32N), Group 5 (Figures 32E, 32O), Group 6 (Figures 32F, 32P), Group 7 (Figures 32G, 32Q), Group 8 (Figures 32H, 32R), Group 9 (Figures 32I, 32S), and Group 10 (Figures 32J, 32T) is shown; nuclei are counterstained with DAPI (blue). Arrows indicate enzyme-positive cells. [Figure 33]Figure 33A-33K shows that reduced expression of cynomolgus monkey Hex protein results in reduced microglial activation. Iba-1 staining indicates reduced inflammation in the hippocampus and thalamus in groups with reduced expression of cynomolgus monkey Hex protein (20x magnification). Figures for Group 1 (Figures 33A-33K), Group 2 (Figures 33B-33L), Group 3 (Figures 33C-33M), Group 4 (Figures 33D-33N), Group 5 (Figures 33E-33O), Group 6 (Figures 33F-33P), Group 7 (Figures 33G-33Q), Group 8 (Figures 33H-33R), Group 9 (Figures 33I-33S), and Group 10 (Figures 33J-33T). [Figure 34] Figure 34 shows that reduced expression of cynomolgus monkey Hex protein results in reduced reactive astrogliosis. GFAP staining indicates reduced inflammation in the hippocampus and thalamus in groups with reduced expression of cynomolgus monkey Hex protein (20x magnification). Figures for Group 1 (Figures 34A, 34K), Group 2 (Figures 34B, 34L), Group 3 (Figures 34C, 34M), Group 4 (Figures 34D, 34N), Group 5 (Figures 34E, 34O), Group 6 (Figures 34F, 34P), Group 7 (Figures 34G, 34Q), Group 8 (Figures 34H, 34R), Group 9 (Figures 34I, 34S), and Group 10 (Figures 34J, 34T). [Figure 35] FIG. 1 shows new AAV vectors selected for further testing in non-human primates. [Figure 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 with MUG substrate and normalized to naive mice. [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 with MUGS substrate and normalized to naive mice. [Figure 38] Figure 38 shows 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 gadolinium distribution volume (Gd-enhanced volume) in this animal was 1.67 mL. Figure 38B shows the distribution volume (Vd) and mean + SD for each NHP; the total injected volume (Vi) within the thalamus was 0.3 mL. [Figure 39] 1 shows brain MRI of NHPs injected with AAVrh8 over the course of the 90-day experiment. Arrows indicate areas of hyperintense signal within the thalamus in three NHPs. [Figure 40] Figure 40 shows intracerebral 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 timepoint, which was undetectable at earlier imaging timepoints. 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. [Figure 41]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. [Figure 42] FIG. 1 shows neuropathological findings in the brains of Cohort 1 NHPs, including intraneuronal accumulation of eosinophil granules, neurodegeneration, and neuronal erosion. [Figure 43] Figure 43 shows severe focal spongiosis with perivascular cellular infiltration in the left thalamus of one monkey injected with AAVrh8 within cohort 1. Figure 43A was taken at 4x magnification; Figure 43B was taken at 10x magnification. [Figure 44] Figure 44 shows 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). [Figure 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 the 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 brains 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, 4.68 × 10 vg), AAVrh8-CBA-cmHex (orange bar (B), n=3, 4.68 × 10 vg), or AAVrh8-CB-I-cmHex (blue bar (C), 4.68 × 10 vg; C (5x dose): 2.34 × 10 vg; n=4) vectors. Brains were harvested 4 weeks after injection and sectioned 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 increase relative to wild-type levels (WT, black bars, n=1). An asterisk (*) represents an animal with no detectable Hex activity. Figures 48D and 48F are bar graphs of the qualitative LC-MS / MS used to measure GM2 ganglioside content. [Figure 49-1] 49A and 49B show structural renderings for the promoters described by the present disclosure. Figure 49A shows a structural rendering for the CB promoter, represented by SEQ ID NO:1. [Figure 49-2] Figure 49A shows a structural rendering of the promoters described by the present disclosure. Figure 49B shows a structural rendering of the CB(6)-I promoter, represented by SEQ ID NO:4. [Figure 49-3] Figure 49C shows a structural rendering for the promoters described by the present disclosure. Figure 49C shows a structural rendering for the P2-I promoter, represented by SEQ ID NO:5. [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 astrocytic GFAP promoter. The shapes of GFP (green)- and mCherry (red)-expressing cells correspond to those of neurons and astrocytes, respectively. [Figure 52] This figure shows that human SOD1 mRNA levels are reduced in adult SOD1G93A mice after injection of the dual-promoter AAV9-Syn1-GFP-2×miRSO D1 / GFAP-2×miRSOD1-mCherry vector. The artificial microRNA (miR) targets human SOD1. After intravenous injection of 1×10 12 total vector genomes, a maximum 25% reduction in human SOD1 mRNA expression in the spinal cord was observed. After direct intrastriatal injection of 8×10 9 total vector genomes, a maximum 55% reduction in human SOD1 mRNA expression in the striatum was observed. [Figure 53]

[0049] Figure 53 shows that intracranial treatment with AAVrh8-mHEXA / B extends the lifespan of Sandhoff disease (SD) mice, resulting in the preservation of motor function. SD mice treated with 4.68 x 10 vg of AAVrh8-mHexA / B vector formulations via TH / ICV or TH / DCN delivery were assessed for performance in the (Figure 53A) reversal screen test, (Figure 53B) rotarod test, and (Figure 53C) wire hang test at 60, 120, and 180 days of age. Figure 53D shows Kaplan-Meier survival plots for SD mice treated intracranially with 4.68 x 10 vg of AAVrh8-mHexA / B, demonstrating 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 54A shows that intracranial AAVrh8-mHexA / B treatment in SD mice results in a dose-dependent extension of lifespan and improves motor function. Figure 54A shows Kaplan-Meier survival plots for a dose-escalation experiment of AAVrh8-mHexA / B injected intracranially via TH / ICV into 1-month-old SD mice. SD mice intracranially treated with 4.68 x 10 vg and 1.17 x 10 vg AAVrh8-mHexA / B vector formulations via TH / ICV delivery were assessed for performance in the (Figure 54B) reversal screen test, (Figure 54C) rotarod test, and (Figure 54D) 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. The performance of heterozygous (HexB+ / −, HZ) mice treated with intracranial injection of 4.68×109 vg of AAVrh8-mHexA / B via TH / ICV or TH / DCN delivery was assessed at 60, 120, and 180 days of age in (Figure 55A) the reversal screen test, (Figure 55B) the rotarod test, and (Figure 55C) the wire-hanging 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 1.17 x 10 vg of AAVrh8-mHexA / B via TH / ICV injection, and neurochemistry was assessed at the same age as the humane endpoint for untreated SD mice. Using two artificial substrates: MUG (cleaved by HexA, HexB, and HexS isozymes) and MUGS substrate (cleaved by HexA and HexB isozymes), the cerebrum, cerebellum, and spinal cord were analyzed for (Figure 56A) GM2 ganglioside content and (Figure 56B) hexosaminidase activity. [Figure 57]Figures 57A and 57B show survival and biodistribution of AAV in SD cats after TH and ICV injections. Figure 57A shows that survival of untreated SD cats was 4.4±0.6 months. Figure 57B shows that the onset of clinical disease was delayed and clinical signs were alleviated in SD cats after AAV treatment. Figure 57C shows a crude image of the brain (left) indicating the location of the section. Figure 57C shows a coronal section of the right hemisphere of the brain and spinal cord in situ illustrating the location of the analysis. Figure 57D shows naphthol staining for histochemistry showing the distribution of Hex in normal cats, SD cats, and a representative SD cat treated with AAV. Figure 57E shows HexA enzyme activity in the brain and spinal cord of an SD cat. [Figure 58] Figure 58 shows biomarkers for the therapeutic efficacy of AAV-mediated gene therapy. Figure 58A shows 7T MRI of an SD cat brain, demonstrating increased gray and white matter intensity due to increased demyelination in the white matter and increased GM2 accumulation in the gray matter. Following TH / ICV delivery with AAV gene therapy, complete normalization of gray and white matter intensity is observed. Mild cortical atrophy is evident as the injection site in the thalamus is hyperintense. Figure 58B shows MR spectroscopy of an untreated SD cat, demonstrating increased N-acetylhexosamine, a toxic metabolite previously reported in SD patients. Following TH / ICV delivery with AAV gene therapy, this is completely normalized. Figure 58C shows an MR spectrum for a representative SD cat. Figure 58D shows HexA activity in cerebrospinal fluid (CSF) in an SD+AAV TH / ICV SD cat. Figures 58E and 58F show lactate dehydrogenase (Figure 58E) and aspartate aminotransferase (Figure 58F) levels in the CSF of normal (open circles), SD (filled circles), and SD+AAV TH / ICV cats (shaded circles). Figures 58G and 58H show serum antibody titers in SD+AAV TH / 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 a survival of 4.4 ± 0.6 months in untreated SD cats. SD cats treated with intracisternal AAV extended survival to 9.9 ± 0.5 months. Figure 59B is a clinical assessment score showing a rapid decline in untreated SD cats. In SD+AAV cats, decline was attenuated, with a prolonged plateau phenotype. Cats were euthanized due to visual and hearing loss as well as musculoskeletal abnormalities before loss of ambulation. The clinical assessment score was based on the following clinical signs: mild tremor, pronounced tremor, hindlimb weakness, drowsy gait, ataxia, accidental falls, limited ambulation, front leg spasms, hind leg spasms, and inability to walk. Normal score was 10, with 1 point subtracted for each symptom. Figure 59C shows a cat brain (left), with lines indicating the location of the brain cross-section, and the right hemisphere with the brain and spinal cord en bloc illustrating the location for biodistribution analysis. Figure 59D shows HexA activity in the brain and spinal cord of a SD+AAV long-term cat (MUGS). The thick line at 1 indicates normal levels. Figures 59E and 59F show HexA expression 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 a normal cat (left), an SD cat (middle), and a cat chronically treated with SD+AAV CM. Normal cats exhibited darker PAS staining in the white matter of the cerebrum and cerebellum than in the gray matter, and the reverse was true for the spinal cord. SD cats exhibited darker PAS staining in the gray matter than in the white matter, with areas of loss of staining within the white matter (particularly 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 histopathology after AAV gene therapy. SD cats exhibit degenerated neurons in the microgyrus, DCN, and brainstem. Sixteen weeks after AAV gene therapy, SD cats showed normalized morphology in all of these regions, and in long-term AAV-treated animals, morphology was preserved, with the exception of Purkinje cell degeneration. Interestingly, within the dentate nucleus, a subset of neurons was found to contain grape-shaped eosinophilic inclusions (inset; scale bar: 5 μm). Scale bar: 10 μm. [Figure 62] Figure 62A shows biomarkers of disease progression and improvement in SD+AAV CM cats. Figure 62A is a 7T MRI of an SD cat showing cerebral cortical atrophy and hyperintense white and hypointense gray matter, consistent with white matter demyelination and lipid accumulation within the gray matter, compared to a normal cat. Intensity changes persisted in SD+AAV CM cats, but cortical atrophy improved. MR spectroscopy of the thalamus of an SD cat 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. Figure 62B shows that myo-inositol (INS), a marker for gliosis, and glycerophosphocholine + phosphocholine (GPC + PCh), a marker for demyelination, also increased in SD+AAV CM cats. Figure 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 (lightly filled circles). [Figure 63]Figure 63 shows that intracranial injection of the AAVrh8-CB-CI-mHexA / B vector reduces GM2 ganglioside accumulation in the CNS while increasing hexosaminidase activity, thereby improving survival in SD mice. SD mice received intracranial injections of the AAVrh8-CH-CI-mHexA / B vector at a total dose of 4.68 x 10 vg or 2.34 x 10 vg, or the original vector (4.68 x 10 vg), or PBS. Figure 63A shows the GM2 ganglioside content measured by LC-MS / MS 8 weeks after CNS injection. Figure 63B shows the hexosaminidase activity in the cerebrum measured using the 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 2.34×1010 vg CB-CI-mHexA / B and 4.68×109 vg CB-CI-mHexA / B; nd (not detected). Figure 63C shows the survival and symptoms of SD mice (n=8) injected with 2.34×1010 vg AAVrh8-CB-CI-mHexA / B (study endpoint: 5 months). The median survival of untreated Sandhoff disease mice was 125.5 days (n=6). [Figure 64] Figure 64 shows that increasing the dose of AAVrh8-CB-CI-mHexA / B vector delivered to the CSF further reduces GM2 ganglioside accumulation in the spinal cord and cerebellum. SD mice were injected with 1.17×10 vg of AAVrh8-CB-CI-mHexA / B at increasing CSF doses (1.17×10 vg, 2.34×10 vg, 5.85×10 vg, and 5.5×10 vg) 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 with Dunnett's multiple comparison test; *(P≦0.05), **(P≦0.01), ***(P≦0.0001), n=3. [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 the pAAV-CB-ci-hHexB vector. [Figure 68] FIG. 1 shows the enzymatic activity of HexA 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 an AAVrh8 vector 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]Figures 75A and 75B show 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 fluorescence microscope-guided intravascular microcatheter into the cisterna magna of sheep. Figure 75D shows a macroscopic photograph of a post-mortem spinal cord. [Figure 75-3] Figure 75D shows one embodiment of a method for rAAV administration via a fluorescence microscope-guided intravascular microcatheter into the cisterna magna of sheep. Figure 75E shows GFP expression in spinal cord cross sections by immunohistochemistry. [Figure 75-4] Figure 75A shows one embodiment of a method for rAAV administration via a fluorescence microscope-guided intravascular microcatheter into the cisterna magna of sheep. Figure 75B shows GFP expression in brain cross sections by immunohistochemistry. [Figure 75-5] Figure 75F shows one embodiment of a method for rAAV administration via a fluorescence microscope-guided intravascular microcatheter into the cisterna magna of sheep. Figure 75F shows GFP expression in brain cross sections by immunohistochemistry. [Figure 75-6] Figure 75G shows one embodiment of a method for rAAV administration via a fluorescence microscope-guided intravascular microcatheter into the cisterna magna of sheep. Figure 75G shows GFP expression in brain cross sections by immunohistochemistry. DETAILED DESCRIPTION OF THE INVENTION

[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 substrate availability, or result in exocytosis Furthermore, physiological levels of transgene expression driven by rAAV vectors are also observed. The introduction of therapeutic proteins that exceed the target protein also addresses the fold defects common in these disorders. It can also trigger harmful 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.

[0023] Compositions for expression of genes (e.g., isolated nucleic acids, rAAV, rAAV vectors, etc.) are presented. In some aspects, the present disclosure provides methods for transfecting GM1 gas using the rAAVs described herein. Lysosomal storage disorders, such as gliosides, Tay-Sachs disease, or Sandhoff disease The present disclosure relates to methods for treating therapeutically effective, but conventionally used, The transgene was expressed at levels that did not cause vector-mediated genotoxicity associated with rAAV. Certain regulatory sequences and regulatory elements are used for use in rAAV to direct gene expression. This is based in part on the discovery that elements, such as promoter regions, can be manipulated.

[0021] Isolated nucleic acids In some aspects, the present disclosure provides a method for the production of a gene encoding at least one transcription factor operably linked to a promoter. 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 :2720). The human GLB1 gene is located at NM_000404.4 and NM_0010. 79811.2, NM_001135602.2, or NM_001317040.1 The human GLB1 gene may contain the nucleotide sequence 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. , comprising a sequence as in SEQ ID NO: 23. In some embodiments, the human HexA trans 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 number: 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. In some embodiments, He The xB protein is identified by NCBI reference sequence number: NP_000512.1 (SEQ ID NO: 21) or or the sequence specified in NP_001278933.1.

[0023] The transgenes encoding proteins associated with lysosomal storage disorders were As used herein, "operably linker" refers to a A promoter that is linked to and drives the expression of a downstream transgene. In some embodiments, the promoter may be a constitutive promoter, such as chicken beta-actin ( CBA promoter, retroviral Rous sarcoma virus (RSV) LTR promoter promoter (optionally with an 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 promoter and EF1α promoter [Invitrogen]. In some embodiments, 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 for 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, but not limited to, 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); also see 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, such as temperature, the presence of an acute phase, and the cell's specific differentiation state. or inducible promoters that are regulated by physiological conditions 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 natural expression. The promoter of the present invention may be used to ensure that transgene expression is temporally, developmentally, or tissue-specific. 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 natural expression control elements, such as the ak consensus sequence, may also be used to mimic natural 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 a promoter, and the transgene 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 within a cell type relative to gene expression within 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 a human synapsin 1 promoter ( The Syn1 promoter drives gene expression preferentially in neurons and is expressed by the GfaAB The C1D (also called GFAP) promoter directs expression preferentially within astrocytes. However, it is understood that some cell types may reside within a particular type of tissue. For example, central nervous system tissues contain both neuronal and non-neuronal cells (e.g., glial cells, astrocytes, etc.

[0027] In some embodiments, the isolated nucleic acid is a nucleic acid sequence encoding a protein associated with a lysosomal storage disorder, e.g., The transgene contains at least two transgenes encoding a nucleotide sequence (e.g., HEXA and HEXB). The first transgene and the second transgene each comprise at least one promoter. In some embodiments, the first transgene may 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 13. In some embodiments, the second promoter is astrocyte-specific. 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, somatos tatin promoter, pancreatic polypeptide (PPY) promoter, synapsin 1 (Syn 1) Promoter, creatine kinase (MCK) promoter, mammalian desmin (D ES) promoter, α-myosin heavy chain (α-MHC) promoter, or cardiac troponin Other exemplary promoters include, but are not limited to, the cTnT promoter. The promoter may be a 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)), bone osteoclast promoter (Stein et al., Mol. Bi 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 promoter, neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)), The lambda 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 operably linking a promoter to a gene encoding a target gene 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 contain non-codons, 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 included. The nucleotide sequence is about 50 to about 150 nucleotides in length (e.g., 50 to 150 nucleotides, including the endpoints). In some embodiments, the length of the nucleotide sequence is in the range of 100 to 150 nucleotides (any length between 100 to 150 nucleotides). The intron sequence of the actin gene is about 100 to about 120 (e.g., 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 nucleotides in length. is placed between multiple untranslated sequences (e.g., promoter sequences and chimeric intron sequences) a first non-translated sequence and / or a chimeric intron and a transgene sequence, In some embodiments, one or more Each of the several untranslated sequences is a sequence of a rabbit beta-globulin gene (e.g. For example, it is derived from exon 1 and exon 2 of rabbit beta-globulin. It is a non-coding sequence derived from a gene (a non-translated sequence that arises from a gene).

[0030] Recombinant AAV The isolated nucleic acid of the present disclosure can be a recombinant adeno-associated virus (rAAV). In some embodiments, the isolated nucleic acids described by the present disclosure are derived from a first adeno-associated virus (AAV) reverse transcriptase. a region (e.g., the first region) containing an inter-terminal repeat (ITR) or a variant thereof, and a Transgenes associated with esosome storage disorders (e.g., GLB1, HEXA, and / or and a second region encoding a recombinant AAV vector (e.g., HEXB). The vector may be packaged into a capsid protein and administered to a subject. The transgene may also be transduced 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 vectors containing a single, cis-acting wild-type ITR. In some embodiments, the ITR is the 5' ITR. In some embodiments, the ITR is the 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 the relative position of these sequences may be varied. The ability to modify ITR sequences is within the skill of the art. (See, e.g., Sambrook et al., "Molecular Cloning. A Laboratory Manual"). ”, 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et a (See textbooks such as 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 utilized in the present disclosure is a trans A "cis-acting" plasmid containing the transgene, in this case The gene sequence and associated regulatory elements are located between the 5' AAV ITR sequence and the 3' The AAV ITR sequences are flanked by the ribosomal RNA sequences and the ribosomal RNA sequences. It can 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 an isolated nucleic acid and / or an isolated rAA Modified and / or modified to enhance targeting of V to target tissues (e.g., CNS). Non-limiting methods of modification and / or selection include modifying or selecting AAV capsid serotypes. (e.g., AAV8, AAV9), tissue-specific promoters (e.g., Syn1, GFA In some embodiments, the isolated nucleic acids 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 rAAVs of the present disclosure comprise AAV capsid serotypes. The AV comprises a tissue-specific promoter (e.g., Syn1, GFAP). In some embodiments, the isolated nucleic acids and rAAVs of the present disclosure are directed to CNS tissues and tissue-specific promoters. and AAV capsid serotypes with enhanced targeting to HIV-1.

[0033] In some aspects, the present disclosure provides isolated AAVs. As used herein, the term "isolated" refers to an AAV that has been artificially obtained or created. The isolated AAV may be produced using recombinant methods. V is referred to as a "recombinant AAV." Recombinant AAV (rAAV) is preferably V transgene is specifically delivered to one or more predetermined tissue(s). AAV capsids have tissue-specific targeting capabilities, such as Therefore, targeting is an important factor in determining the targeting ability. 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 This includes proteins that are substantially homologous to the above-mentioned proteins. In one embodiment, the rAAV comprises an AAVrh8 capsid protein.

[0034] In some embodiments, the rAAV of the present disclosure is a pseudotyped rAAV. A virus or viral vector is created by combining it with a foreign viral envelope protein. The result is a pseudotyped virus particle. Envelope proteins may alter host tropism or increase the stability of viral particles. In some embodiments, pseudotyped rAAVs can be used to increase / decrease the number of nucleotides in a given gene. and nucleic acids from different AAVs, where the nucleic acids from one AAV are 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 viral 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, an AAV containing a capsid protein of serotype Y together with a protein of serotype Y. Pseudotyped AAV vectors containing the serotype X ITRs are 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 confirmed by using a virus derived from another AAV serotype. This allows the transgene to be targeted to the target tissue. These may be useful for enabling targeted delivery.

[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, which is incorporated herein by reference.) 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 host cells containing sufficient helper functions to allow 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 splicers. Three capsid proteins are transcribed from a single cap gene via isolating It contains virion proteins 1 to 3 (designated VP1, VP2, and VP3). In an embodiment, the molecular weights of VP1, VP2, and VP3 are approximately 87 kDa and 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 interacts with the viral 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: AAV capsid proteins are expressed in the capsids of AAVrh8 or AAVrh10 serotypes. 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 rAAV vector into an AAV capsid. Thus, components that are cultured within the host cell can be brought into the host cell in trans. Alternatively, any one or more of the required components (e.g., recombinant AA V vector, rep sequence, cap sequence, and / or helper functions) are known to those skilled in the art. engineered to contain one or more of the required components using the method of Such stable host cells may also be derived from the Most preferably, the desired component(s) are under the control of a promoter. However, the required component(s) may be under the control of a constitutive promoter. Suitable inducible and constitutive promoters are used herein. 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 contains 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) but the rep protein and / or cap protein under the control of an inducible promoter Still other stable host cells containing the protein can be generated by those skilled in the art. Cells may also be produced.

[0038] In some embodiments, the present disclosure provides a nucleic acid sequence comprising SEQ ID NO: 1 to 3 operably linked to a promoter. 6. A host cell containing a nucleic acid comprising a coding sequence selected from the group consisting of: In embodiments, the present disclosure relates to a composition comprising the host cells described above. In some embodiments, the composition comprising the host cells further comprises a cryoprotectant.

[0039] Recombinant AAV vectors, rep sequences, ca, and dsRNAs useful for generating rAAV of the present disclosure. p sequences, and helper functions can be used with any suitable genetic element (vector). The selected genetic element can be delivered to a packaging host cell by the The delivery may be by any suitable method, including those described in the literature. The methods used to construct the morphology are based on nucleic acid manipulation techniques. These methods are well known to those skilled in the art and include genetic engineering techniques, recombinant techniques, and the like. This includes engineering and synthetic methods. See, e.g., Sambrook et al., Molecules 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 to 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, recombinant AAV is produced using a triple transfection method (U.S. Patent No. 6, 001,650). Typically, recombinant AAV is packaged into AAV particles in host cells. AV vector (containing the transgene), AAV helper function vector, and accessor It is produced by transfecting an AAV helper function vector. The activator functions in trans for productive AAV replication and encapsidation. Preferably, the vector encodes "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 in accordance with the present disclosure include: No. 6,001,650, the entire contents of which are incorporated herein by reference. pHLP19, as described in the specification, and U.S. Pat. No. 6,156,303 The accessory function vectors include the pRep6cap6 vector, which is described in the specification. These vectors are non-AAV-dependent proteins that AAVs rely 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, AAV D Parts involved in NA replication, cap expression product synthesis, and AAV capsid assembly 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 present 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 transgenic DNA is introduced through the cell membrane. Many transfection techniques are generally known in the art. For example, Gr 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 cells in which recombinant AAV is produced. Manufactured and associated AAV helper constructs, AAV minigene plasmids, and accessory functions It 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 naturally occurring mutations, accidental mutations, or deliberate mutations. Because of the mutation, the original parent may not necessarily differ in shape or genome or total DNA complement. It is understood that they may not be exactly the same.

[0043] As used herein, the term "cell line" refers to a cell line capable of growing and culturing in vitro. A cell line is a population of cells that are capable of sustained or extended division and differentiation. The art further describes the structure of such clonal populations. It is also known that spontaneous or induced changes can occur within the karyotype upon accumulation or introduction. Therefore, cells derived from the cell line 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 cells" refers to cells that express biologically active polypeptides. a DNA segment, resulting in the transcription of, or production of, a biologically active nucleic acid, such as RNA 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 are capable of replicating and transferring 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 nucleic acid segments 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 "Operatively 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 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 kind of genetic construct that contains a nucleic acid that is transcribable in whole or in part. In some embodiments, expression may be, for example, the production of biologically active polypeptides from transcribed genes. Tide products, or inhibitory RNAs (e.g., shRNAs, miRNAs, miRNA inhibitors) This involves transcription of the nucleic acid, resulting 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 present disclosure can be recombinant AAV (rAAV) vectors. A "rAAV (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), constitutes It is packaged into capsid proteins and delivered to selected target cells. In some embodiments, the transgene is a recombinant AAV vector. Heterologous to the vector sequence and can be used to produce polypeptides, proteins, functional RNA molecules (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 used to transfect the regulatory components into the cells of the target tissue. They are operably linked in a manner that allows for the transcription, translation, and / or expression of the gene.

[0048] Aspects of the present disclosure demonstrate that modifications of regulatory sequences of rAAV are therapeutically effective but not previously used. levels of transgenes that do not cause vector-mediated toxicity associated with rAAV Thus, in some embodiments, the present disclosure relates to the discovery that Some embodiments relate to recombinant AAV (rAAV) containing 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 ( driving transcription of a gene (e.g., a transcript, including a transcript encoded by a transgene) a regulatory construct that can be used to manipulate two or more artificially positioned regulatory elements; Typically, hybrid promoters are regulatory constructs that contain minimal At least one element that is a promoter and one or more enhancer sequences or multiple transcriptional regulatory elements, including intronic sequences, exon sequences, or UTs and at least one element having an R sequence. In embodiments involving exon, intron, or UTR sequences, such sequences The (s) may encode an upstream portion of an RNA transcript (e.g., as depicted in Figure 1). On the other hand, regulatory elements that modulate (e.g., enhance) transcription of a transcript also exist. In some embodiments, a hybrid promoter contains 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 are distinct from the transgene. In some embodiments, hybrid promoters are derived from two or more species. The excess elements are from different loci. Two or more elements of a promoter are derived from the same locus, but In some embodiments, the hybridization promoter is located in a manner not found in the locus. The promoter may contain 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 chicken beta - 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 nucleotide ... - chicken beta-actin promoter fused to a 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 gene they regulate. Enhancer sequences can be located upstream or downstream (i.e., 3'). 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 the retain the desired functional characteristics of the entire nucleotide or amino acid sequence from which it is derived. For example, "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. It 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 (WHBV) gene. Mouse posttranscriptional regulatory element (WPRE), mouse RNA transport element (RTE), type 1 Constitutive transport element (CTE) of simian retrovirus (SRV-1), Mason-Fa CTE from MPMV, and human heat shock protein 7 0 (Hsp70 5'UTR) In this embodiment, the rAAV vector contains a woodchuck hepatitis virus post-transcriptional regulatory element. Includes (WPRE).

[0052] In some aspects, the present disclosure provides a method for detecting a gene that contains 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). a splice donor site from a second source (e.g., organism or species) In some embodiments, the chimeric nucleic acid 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 determining whether a transgene is a the transgene comprises a promoter operably linked to a transgene, The rAAV encodes a rAAV protein and further contains a chimeric intron.

[0053] In certain embodiments, the present disclosure relates to rAAV vectors comprising artificial transcription elements. In some embodiments, the term "artificial transcription element" as used herein is the transcription of DNA by RNA polymerase in a controlled 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), transcription activator factor 1 (ATF1) binding Binding site, ATF1-CRE binding site, APBβ box, APBα box, CArG Box, CCAC Box, and as disclosed in 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 transcriptional site, a transcriptional site, a TATA box, and a TATA box. , represented by SEQ ID NO:2.

[0055] The expression control sequences include appropriate transcription initiation sequences, transcription termination sequences, promoter sequences, and endogenous sequences. 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 engineering, there are naturally occurring promoters, constitutive promoters, inducible promoters, and / or Numerous expression control sequences, including promoters specific to the host or tissue, are known and may be used.

[0056] As used herein, nucleic acid sequences (e.g., coding sequences) and regulatory sequences refer to sequences that , covalently linked to place the expression or transcription of a nucleic acid sequence under the influence or control of a regulatory sequence. Nucleic acid sequences are said to be "operably" linked when they are effectively linked. If it is desired that the protein be translated, the two DNA sequences should be Induction of the promoter results in transcription of the coding sequence and the two DNA The nature of the linkage between the sequences resulted in (1) the introduction of frameshift mutations. (2) interfere with the promoter region's ability to direct transcription of the coding sequence; ) without interfering with the ability of the corresponding RNA transcript to be translated into protein. Thus, the resulting transcript is said to be operably linked to the desired target gene. The promoter region directs this DNA so that it can be translated into a protein or polypeptide. A promoter region is operably 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 together in a common promoter. These transcripts from the target gene are translated in frame into two or more transcripts. An operably linked protein is one that is operably linked when it is linked in a manner that results in 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 used in transgenesis. The rAA sequences useful in the present disclosure are inserted after the 3' AAV ITR sequences and before the 3' AAV ITR sequences. The V construct also contains a promoter / enhancer sequence located between the promoter / enhancer sequence and the transgene. The SV-1000 sequence may also contain an intron, which may be desirable. One possible intron sequence is the SV-1000 sequence. 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 containing 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 therein, e.g., 3.18, 3.26, and 1 6.1716.27; and Ausubel et al., Current Pr [See "Protocols 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. amino acid length) (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, 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 recombinant). has been documented in artificial systems, including the avian torovirus (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 may vary between cell types, it generally contains a TATA box, capping sequence, etc., as needed. These include the nucleotide sequence, CAAT sequence, and enhancer element, which are involved in the initiation of transcription and translation, respectively. In particular, the term "antisense oligonucleotide" includes 5' untranscribed and 5' untranslated sequences that contribute to the translation of the nucleic acid. Such 5' non-transcribed 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 contain an enhancer sequence, or an upstream activator sequence. Optionally, 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 promoter. 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, phosphoglycerin The promoter of PGK and the 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 It can be regulated by the physiological conditions present within the replicating cell. Transducers and inducible systems are available from manufacturers including, but not limited to, Invitrogen, Clontech, Many other systems are commercially available from various sources, including Sigma and Ariad. Exogenously supplied promoters are listed in the literature and can be readily selected by one skilled in the art. An example of an inducible promoter regulated by zinc is the zinc-inducible ovine 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 sequences of the vector typically contain 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 these sequences are used, some minor modifications to these sequences are also permitted. 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 textbook.) Examples of such molecules utilized in this disclosure are transgenes. In this case, the selected transgene sequence is a "cis-acting" plasmid containing 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 type V.

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

[0064] In addition to the key elements identified above for a recombinant AAV vector, the vector The target also includes cells transfected with a plasmid vector produced according to the present disclosure. The transcription, translation, and and / or a necessary, operably linked to the transgene in a manner allowing expression. Conventional regulatory elements are also included. As used herein, "operably linked" sequences expresses a gene of interest and its flanking expression control sequences, and the gene of interest, in trans or It includes both expression control sequences 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 The gene encodes a therapeutic protein or a functional therapeutic RNA. 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 transfected into a target cell. The transgene is intended to be used to create animal models for the disease. , a protein, or a functional RNA. Those skilled in the art will recognize suitable transgene codes. The code sequence will be clear.

[0066] In some aspects, the present disclosure provides rAAV vectors useful for treating lysosomal storage disorders. Lysosomal storage disorders (also called lysosomal storage diseases) are disorders in which 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 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 proteins are aspartylglucosaminuria (aspartylglucosaminuria) Batten disease (palmitoyl protein thioesterase), infantile Batten disease (palmitoyl protein thioesterase), late infantile Batten disease Marten disease (tripeptidyl peptidase), Fabry disease (α-galactosidase), fucoprotein 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), Mucopolysaccharidosis IIID (N-acetylglucosamine-6-sulfate sulfa Mucopolysaccharidosis IVA (N-acetylgalactosamine 6-sulfatase), ... Mucopolysaccharidosis IVB (β-galactosidase), Mucopolysaccharidosis IX (hyaluronidase), Mucopolysaccharide Mucopolysaccharidosis VI (arylsulfatase B), mucopolysaccharidosis VII (β-glucuronidase), Type I mucolipidosis (α-neuraminidase), type II mucolipidosis (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), Not limited to:

[0067] In some embodiments, the present disclosure provides a transcription factor encoding a lysosomal storage disease-associated protein. In some embodiments, rAAVs containing transgenes associated with lysosomal storage diseases are presented. 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 genomic storage disease-related protein is GLB1, which has the sequence set forth in SEQ ID NO: 23. include.

[0068] Also described 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. In some embodiments, the rAAV comprises a chimeric promoter. 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, chimeric intro In some embodiments, the transcriptional element, or artificial 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 a subject a transgene encoding HEXB. The disease is GM1 gangliosidosis and the transgene encodes GLB1.

[0069] In some aspects, the disclosure provides 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. We present a rAAV that contains nucleic acid-containing capsids that are not cell type specific. Without wishing to be bound by any of the foregoing, in some embodiments, such rAAV vectors - Treat diseases that affect multiple cell types within a given tissue (e.g., CNS tissue) It is useful for

[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 CNS-related disorders can affect the tissues surrounding the brain and spinal cord. It can be a genetic disorder acquired through somatic mutation or acquired through a genetic mutation. Related disorders include psychological conditions or disorders, such as attention deficit hyperactivity disorder, autism spectrum disorder, and These may include spectral disorders, mood disorders, schizophrenia, depression, Rett syndrome, etc. The CNS-related disorder can be an autoimmune disorder. CNS-related disorders also include cancers of the CNS, e.g., brain tumors. 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 metastasized to CNS tissues, such as glioblastomas and glioblastomas, may also be present. Further non-limiting examples of CNS-related disorders include Huntington's disease, Parkinson's disease, and rheumatoid arthritis. Kinesiology, lysosomal storage diseases, 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 Fritzl -Including Reich's ataxia.

[0071] In some embodiments, the rAAV vectors described by the present disclosure target CNS disease-associated 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, iRNAs. -related to disease, DRD2, GRIA1, GRIA2, GRIN1, SLC1A1, SY P, SYT1, CHRNA7, 3R Tau / 4rTUS, APP, BAX, BCL-2, G RIK1, GFAP, IL-1, AGER; associated with Parkinson's disease, UCH-L1, SKP1, EGLN1, Nurr-1, BDNF, TrkB, gstm1, S106β; Huntington's disease-associated Huntingtin (Htt), IT15, PRNP, JPH3, TBP, ATXN1, ATXN2, ATXN3, atrophin 1, 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 Associated with cis, MAN2B1, MAN2B2, MAN2C1; aspartyl cyclosami 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; GBA, associated with Sher disease; GALC, associated with Krabbe disease; metachromatic leukodystrophy associated with ARSA; mucopolysaccharidoses (e.g., Hurler syndrome, Hunter syndrome, A- Sanfilippo syndrome type D, Morquio syndrome, hyaluronidase deficiency, Maroteaux-Lami syndrome 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 other related diseases. 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 messenger RNA (mRNA miRNAs regulate gene expression through translational repression of genes. miRNAs are expressed as final, non-coding RNA products of target mRNAs. They exert their activity through sequence-specific interactions with the 3' untranslated region (UTR) of These endogenously expressed miRNAs form hairpin-shaped precursors, which is then processed into a miRNA duplex, which is then further processed into a "mature" single-stranded miRNA. This mature miRNA is then processed into a molecule called A. 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 ) and inhibitory RNA that targets The transgene comprises SEQ ID NO: 15.

[0073] Recombinant AAV administration method The rAAV can be delivered to a subject in a composition according to any suitable method known in the art. Preferably, the compound is suspended in a physiologically compatible carrier (i.e., composition). rAAV 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 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 by isolated limb perfusion, a method that essentially allows The isolated limb perfusion variation described in U.S. Pat. No. 6,177,403 also utilizes Administering the lysine 100 into the vasculature of an isolated affected limb to transduce muscle cells or muscle tissue; Further, in certain cases, 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 spinal cord and the nervous system. These include, but are not limited to, astrocytes, cerebrospinal fluid (CSF), interstitial spaces, bone, and cartilage. 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 the striatum (e.g., striatum injection into the caudate nucleus or putamen of the spinal cord, the neuromuscular junction, or the cerebellar lobule It can be delivered directly to the NS or 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 by injection (e.g., intrathalamic injection). In some embodiments, the rAAV is administered twice. In some embodiments, the rAAV of the present disclosure is administered bilaterally via intrathalamic 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 patient 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 into the cisterna magna, the intrathecal cavity (e.g., spinal canal), and the thalamus (e.g., by intrathalamic injection).

[0077] Embodiments of the present disclosure include at least one modified gene regulatory sequence or modified gene regulatory element. In some embodiments, the composition is a pharmaceutically acceptable carrier. Further included is 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 may each comprise one or more different AAVs. lance gene, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , or more, containing different rAAVs.

[0079] In some aspects, the present disclosure provides a composition comprising an rAAV comprising a nucleic acid encoding a HexA gene. In some aspects, the present disclosure relates to compositions (e.g., pharmaceutical compositions) containing the HexB gene. Some embodiments relate to compositions (e.g., pharmaceutical compositions) comprising an rAAV containing a nucleic acid encoding the rAAV. In this disclosure, a first rAAV comprising a nucleic acid encoding a HexA gene and a second rAAV comprising a nucleic acid encoding a HexB gene is described. and a second rAAV containing a nucleic acid encoding a gene. do.

[0080] Ratio of rAAV encoding HexA:rAAV encoding HexB in the pharmaceutical composition The ratio can vary, for example, from about 1:10 to about 10:1. The ratio of rAAV encoding HexB to rAAV encoding HexB was 1:1. In one 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 1:2. In some embodiments, the ratio of the rAAV encoding HexA to the rAAV encoding HexA is 1:5. The ratio of rAAV to rAAV encoding HexB is 1:10. In this case, the ratio of rAAV encoding HexA to rAAV encoding HexB was 2:1. In some embodiments, the ratio of a rAAV encoding HexA to a rAAV encoding HexB is In some embodiments, the ratio of rAAV to rAA encoding HexA is 5:1. The ratio of V:HexB-encoding rAAV is 10:1.

[0081] In some embodiments, an rAAV encoding HexB: an rAAV encoding HexA In some embodiments, the ratio of HexB-encoding rAAV:He 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 to 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 to 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, bearing in mind the application for which the rAAV is intended. For example, one suitable carrier is saline, which may be formulated with a variety of buffers. Other exemplary carriers include sterile saline (e.g., phosphate buffered saline). , lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin The carriers include 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 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 induce the desired cell death without 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 administration route, intramuscular administration route, subcutaneous administration route, intradermal administration route, intratumoral administration 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 several 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 used to generate stable somatic transgenic animals. The effective amount will depend primarily on the species, age, weight, and health of the subject. The results may vary between animals and tissues, depending on factors such as the type of antibody and the tissue being targeted. For example, an effective amount of rAAV is generally about 10 6 ~10 16 Genome copies (e.g. For example, 1 x 10, including the endpoints 6 ~1×10 16 ) containing about 1 ml to about 100 ml In some cases, the solution is in the range of about 10 11 ~10 12 Injection between rAAV genome copies In some embodiments, about 10 11 ~10 13 rAAV genome copies In some embodiments, a dose of about 10 11 ~10 14 rAAV genome code In some embodiments, a dose between about 10 11 ~10 15 rAAV In some embodiments, a dose of about 10 12 ~10 14 rA A suitable dosage is about 10 AV genome copies. 13 ~10 14 r A suitable dosage of AAV genome copies is given per kilogram (kJ) of body weight. 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 A dose of 100 genome copies is appropriate. , 4.68×10 9 A dose of 1.17 genome copies is appropriate. x10 10 A dose of 2.34 x 10 genome copies is appropriate. 1 0 A dose of 3.20 x 10 genome copies is appropriate. 11 genome A dose of about 4.2 x 10 copies is appropriate. 12 Genome copies In some embodiments, the dosage is 1.2 x 10 13 The dose of genome copies is appropriate. In some embodiments, 1.3 x 10 13 The dose 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 x 10 per kg by intravenous administration. 13 v In some embodiments, a dose of about 1 x 10 g / kg of brain weight is appropriate. 14 vg In certain embodiments, a dosage of 10 12 ~10 13 rAAV genome code The PI targets tissue associated with lysosomal storage disorders, e.g., brain tissue or CNS tissue. In certain embodiments, the 10 13 ~10 14 rAAV The genome copy is located in tissue associated with a lysosomal storage disease, e.g., brain tissue or CNS tissue. In some embodiments, the compound is administered intravenously to a subject and is effective in targeting the tissue. The dose of rAAV administered is approximately 10 mg / 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 Between vg. In embodiments, the intravenously delivered dose is about 1.2 x 10 per kg. 13 ~1.8× x10 13 In some embodiments, the intravenously delivered dose is between 100 mg / kg and 100 mg / kg. Approximately 1.5 x 10 13 In some embodiments, administration of the dose is via cerebrospinal fluid ( In some embodiments, the total dose is delivered to (i) the 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. In some embodiments, about 25% of the total dose is delivered to the cisterna magna. In some embodiments, about 75% of the total dose into the CSF is delivered into 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 are 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 between about 0.5 mL and about 1.5 mL, about 0 0.75 mL to about 1.25 mL, or about 0.8 mL to about 1.2 mL.

[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., The dose delivered to the spinal canal (e.g., spinal canal) is approximately 2.0 x 10 13 ~9.0×10 13 vg In some embodiments, the dose delivered to the intrathecal space (e.g., spinal canal) is about 2.0 x 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 volume of the intrathecal cavity (e.g., spinal canal) is about 0 mL, or about 2 mL to about 8 mL. ) the volume delivered to the It is 8mL.

[0091] In some embodiments, the subject (e.g., into the CSF, the intrathecal cavity, and / or the cisterna magna of the subject) Delivery of 1 x 10 is performed by an intravascular microcatheter. 14 In some embodiments, a dose of 100 mg 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 transcript encoding a lysosomal storage disease protein. The rAAV composition containing the gene is administered to a subject in need thereof (e.g., a patient with Tay-Sachs disease). or have or are suspected of having a lysosomal storage disorder such as Sandhoff disease In some embodiments, the subject is, for example, rA as disclosed herein, as measured using artificial substrates such as , MUG, or MUGS. Prior to administration of a composition comprising AV or rAAV, normal β-heterozygotes are 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 18 to 30 months of age.

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

[0094] As used herein, "immunosuppressed" or "immunosuppression" refers to the immunosuppression in a subject. It refers to the reduction in activation or efficacy of the immune response. Immunosuppression is mediated by rituximab, methylprednisolone, Lon, prednisolone, sirolimus, immunoglobulin injection, prednisone, Solu-M edrol, lansoprazole, trimethoprim / sulfamethoxazole, methotrexate one or more of, but not limited to, a sigma-based ... 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 involve administering to a subject an rAAV (e.g., In a subject, before being administered an rAAV or pharmaceutical composition described by the disclosure, Inducing immunosuppression (e.g., administering one or more immunosuppressive agents) In some embodiments, the subject is administered rAAV for about 30 days to about 40 days after administration of the rAAV to the subject. Any time during the 30 days prior to (e.g., up to and including) administration of rAAV, including the endpoint, for approximately 0 days. In some embodiments, the subject is immunosuppressed (e.g., immunosuppression is induced in the subject). In this condition, the subject is receiving immunosuppressive drugs (e.g., rituximab, sirolimus, and / or prednisolone). Patients are pretreated with fluconazole (prescription doxorubicin) for at least 7 days.

[0096] In some embodiments, the methods described by the present disclosure 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, or fluticasone. 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 agent. After administration of the composition, the patient 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 suitable for use in high rAAV concentrations (e.g., about 10 13 GC / ml or more) in the composition to reduce aggregation of AAV particles. There are many methods in the art for reducing rAAV aggregation. These include, for example, adding surfactants, correcting pH, correcting salt concentration, etc. Wright FR, et al., Molecular Thera py (2005) 12, 171-178).

[0099] Formulations with pharmaceutically acceptable excipient and carrier solutions are specific, as described herein. The compositions described herein are suitable for use in a variety of treatment regimens, including 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 calculated by weight or by percentage of the total formulation. Approximately 1 or 2% to approximately 70% or 80% or more of the weight or Typically, these formulations contain at least about 0.5g of ethanol, although it may be convenient to use a volume between 0.5g and 100g. Of course, each therapeutically useful composition may contain 0.1% or more of the active compound. The amount of active compound is determined so that a suitable dosage will be obtained in any given unit dose of the compound. Those skilled in the art of preparing such pharmaceutical formulations can prepare soluble, soluble, bioavailability, in vivo half-life, route of administration, product shelf life, as well as other pharmacological Factors such as clinical considerations are anticipated, and thus various dosages and treatment regimens may be used. This may be desirable.

[0101] Under certain circumstances, rAAV-based therapeutic constructs can be used in combination with suitable recombinant vectors, as disclosed herein. In appropriately formulated pharmaceutical compositions, the drug may be administered subcutaneously, intrapancreatically, intranasally, parenterally, intravenously ... Intravenous, intramuscular, intrathecal, or oral, intraperitoneal, or inhalation delivery In some embodiments, it may be desirable to use the method described in U.S. Pat. Nos. 5,641,515 and 5,399,363 (each (each of which is incorporated herein by reference in its entirety). Any of the administration modalities described herein may be used to deliver rAAV. The preferred administration route 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 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 easy to inject. 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), or the like. polyethylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and / or The appropriate fluidity may be, for example, a solvent or dispersion medium containing lecithin or vegetable oil. This can be maintained by the use of coatings such as acrylic and, in the case of dispersions, the required This 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, e.g., sugars or It would be preferable for the composition to contain sodium chloride. Agents that delay absorption include 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 a single administration may be dissolved in 1 ml of isotonic NaCl solution and 1000 ml of It may be added to subcutaneous infusion fluids or injected into designated infusion sites. (See, for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035- (See, for example, 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 active rAAV, as desired, as described herein. It is prepared by incorporating the above-listed ingredients with various other ingredients, followed by filter sterilization. Generally, dispersions are prepared by sterilizing various active ingredients in a basic dispersion medium containing the above-mentioned components. 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 involves adding a powder of the active ingredient plus any additional desired ingredients to a sterile filtered powder of the active ingredient. 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 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 dihydroxy Inorganic bases such as iron, and isopropylamine, trimethylamine, histidine, procaine Once formulated, the solution may be prepared in a manner compatible with the dosage formulation. The formulations may be administered in a therapeutically effective amount in a manner suitable for injection, drug release capsules, or the like. It is easy to administer in a variety of dosage forms, including acetaminophen.

[0106] As used herein, "carrier" refers to any solvent, dispersion medium, vehicle, coating, diluent, or antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, The art is well known for its use in pharmaceutical active substances. The use of such media and agents is well known. In addition, auxiliary substances may also be incorporated into the compositions. The phrase "pharmaceutically acceptable" means that the allergen can be effectively tolerated when administered to a host. "anti-inflammatory" 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 are delivered via lipid particles, liposomes, and vesicles. The compound may be formulated for delivery encapsulated in a nanosphere, nanoparticle, or the like.

[0108] Such formulations may be used to provide pharmaceutically acceptable carriers of the nucleic acids or rAAV constructs disclosed herein. Those skilled in the art will appreciate the ease with which liposomes can be formed and used. Recently, liposomes with improved serum stability and circulation half-life have been developed. Furthermore, liposomes as potential drug carriers have been reported (U.S. Patent No. 5,741,516). Various methods have also been described using liposome- and liposome-like preparations (U.S. Pat. No. 5,629,393). Specification No. 5,567,434; Specification No. 5,552,157; ​​Specification No. 5,565,2 13; 5,738,868; and 5,795,587 book).

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

[0110] Liposomes are multilamellar concentric bilayer vesicles (also known as 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 the diameter. Small unilamellar vesicles with a diameter in the range of 200-500 Å and containing aqueous solution in the core This results in the formation of SUVs.

[0111] Alternatively, nanocapsule formulations of rAAV can also be used. Nanocapsules generally contain The polymer can be stably and reproducibly taken up into the cells. To avoid side effects, in vivo degradable polymers are used. Such ultrafine particles (size of about 0.1 μm) should be designed. Biodegradable polyalkylcyanoacrylate nanoparticles that meet the requirements are envisioned 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 (e.g., ultrasound) to modulate the rate of drug penetration into and through the circulatory system. It is envisioned that the device may be used and described as a device for enhancing the efficacy of Other alternative drug delivery methods include intraosseous injection (U.S. Patent No. 5,779,708), microtubule injection, and ophthalmic formulations (Bourlais et al., 1999); 8), transdermal matrices (U.S. Pat. Nos. 5,770,219; and 5,78 3,208), and feedback-controlled delivery (U.S. Pat. No. 5,697,899 No. 9).

[0113] In some embodiments, the present disclosure provides a method for the production of an rAAV or pharmaceutical composition described herein. The administration of one or more additional therapeutic agents to the administered subject, such as enzymes, Administration of wild-type isolated proteins to alleviate the decline in enzyme activity during replacement therapy (ERT) However, Gaucher disease, Hunter syndrome, Fabry disease, Pompe disease, Maroteaux-Lamy syndrome, For treating lysosomal storage disorders, including Morquio syndrome type A and LAL deficiency Thus, in some embodiments, subjects may be administered a In addition to the rAAV or pharmaceutical composition described herein, one or more enzyme replacement therapies (E ERT is also administered. ERT is generally known and is described, for example, in 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 this embodiment, the composition comprises rAAV and isolated wild-type HexA protein and 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 components of the present disclosure may be assembled into a research kit. and instructions for using the kit. The present invention relates to the administration of one or more of the agents described herein to a subject in accordance with the intended application of these agents. 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 at concentrations or concentrations appropriate for performing a variety of experiments. may be contained in quantity.

[0116] In some embodiments, the present disclosure provides a kit for producing 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 producing rAAV. In some embodiments, the kit further comprises at least one and further comprising at least one container, wherein the recombinant AAV vector contains a transgene. include.

[0117] In some embodiments, the present disclosure includes a container for storing a recombinant AAV, as described above. In some embodiments, the kit includes a pharmaceutically acceptable carrier. For example, the kit may include a container for storing the rAAV and a container for delivering the rAAV to a subject. and a second container containing a buffer solution suitable for injection of the rAAV. In the example, 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 liquid, where appropriate. It may be provided in a liquid 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 in a kit, e.g. A suitable solvent or other type of vehicle (e.g., water or cell culture medium) or otherwise processable (e.g. As used herein, "instructions" means instructions and / or and instructions for packaging of the present disclosure. or instructions associated with the packaging of the present disclosure. In any way that clearly identifies the user with the relevant information, for example, audiovisually. (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 the manufacture of 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 contain any one or more of the components described herein. The kit may be contained in one or more containers. 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 patient. 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 solution. Alternatively, the kit may be premixed and includes syringes, vials, The kit may include 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 Applicator devices, or IV needles, tubing, and bags, are used to administer medications to animals. The device may have one, more, or all of the components required to provide the device.

[0120] In some cases, the method comprises providing a cell containing a proviral AAV genome at very low abundance. transfecting whole cell DNA isolated from a tissue that may be involved in the cell death; Transcription of the AAV rep gene and AAV cap gene in transfected cells helper virus functions (e.g., adenovirus) to induce and / or boost the Optionally, the R derived from the transfected cells is supplemented with the R NA provides a template for RT-PCR amplification of cDNA and detection of novel AAVs The cells were then transfected with whole cells isolated from tissues that may harbor the proviral AAV genome. When transfecting cells with AAV DNA, factors that promote transcription of AAV genes are added to the cells. For example, cells may be infected with adenovirus or herpes. In a specific embodiment, 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 derived from a non-human primate (NHP). Similarly, in the methods of the disclosure, Adenoviruses known to infect animals (e.g., chimpanzees, mice) are also available. (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 also available. Alternatively, vectors (e.g., plasmids, episomes, etc.) may be used. Such recombinant viruses are known and can be prepared according to published techniques. U.S. Patent No. 5,871,982 and U.S. 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. It is available from a variety of databases, including ed and GenBank.

[0121] The cells may also contain vectors that provide helper functions to the AAV (e.g., helper vectors). Vectors that provide helper functions can also be transfected, for example, E1a These genes can provide adenoviral functions, including E1b, E2a, and E4 ORF6. The sequences of the adenoviral genes that confer functionality are identified for serotypes 2, 3, 4, 7, 12, and 40, etc., and any 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 cells to induce AAV replication, AAV gene transcription, 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 engineered using methods well known in the art. A recombinant AAV vector is constructed and packaged to express the novel cloning gene. These methods can be used to determine functional characteristics associated with novel single-stranded proteins. The capsid gene is a reporter gene (e.g., B-galactosidase, GFP, Luciferase). Construct and package recombinant AAV (rAAV) vectors containing the rAAV gene (e.g., ribosomal enzymes). 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 can be placed in pouches, in one or more tubes, Loosely packaged pouches or similar containers in a container, box, or bag The kit may have various forms, such as a tray or a container. After the accessories are added, the kit is sterilized. This allows individual accessories to be placed in a container without other wrapping. The kits may be sterilized by radiation, heat, or other sterilization methods known in the art. The kit may also be sterilized using any suitable sterilization method, such as a syringe or syringe, depending on the specific application. and other components, such as containers, cell culture media, salts, buffers, reagents, syringes, needles, disinfectants, etc. Gauze or other woven fabrics for applying or removing the antimicrobial agent, disposable gloves, and a The aforementioned support for the drug may also be included.

[0124] 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 reference AAV sequences for sequence comparison This may include printed or electronic tables of:

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

[0126] In some embodiments, the lysosomal storage disorders of the present disclosure are invariably fatal. GM1 gangliosidosis is a chromosomal recessive disorder. seq (OMIM number: 230500) encodes the enzyme β-galactosidase It is caused by a mutation in the GLB1 gene. The amount of residual β-gal activity and the disease Based on the incidence of the disease, types I to III of GM1 gangliosidoses have been described, with type I being Type II is present in late infancy or juvenile period, and type III In some embodiments, the present disclosure provides a method for treating a patient with type II GM1 or or suspected of having the same. Patients with type II GM1 who present in late infancy typically develop symptoms between the ages of 1 and 3 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 the ages of 3 and 10 years, and similarly experience speech and motor deficits. etc., indicates a decline in abilities 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) and includes the sequence set forth in NM_009752.2. In this embodiment, 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, associated with mutations such as 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 detection of HIV-1-associated ... (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 It may be gangliosidosis or type III GM1 gangliosidosis. Gangliosidoses are caused by a deficiency of β-galactosidase (β-gal) protein activity. Because GM1 gangliosidosis is caused by β-gal activity, methods for treating GM1 gangliosidosis include Non-limiting examples of methods for increasing β-gal activity include intracellular or intracellular A method for expressing wild-type GLB1 in a mammal, 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 a subject, or for reducing expression of the wild-type GLB1 gene in a cell or a subject In some embodiments, methods for providing GLB1 protein (e.g., enzyme replacement therapy) are included. In the present invention, 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 practicing the methods of the present disclosure, the amount of GM1 gangliosides in cells or in GM1 ganglia can be increased. GLB1 activity in subjects with gliosis 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 GLB1 activity can be increased by 200% to 500% in cells or in GM1 cancer cells. In subjects with gliosidosis, GLB1 activity was 50%, 60%, 70%, 80%, and 90%. 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 includes administering human GLB1 to a subject. administering an isolated nucleic acid, rAAV, or composition of the disclosure containing a transgene that encodes The subject may be a human, mouse, rat, pig, dog, cat, or non-human. The administering step may be performed in a cell or in a subject, in which the isolated antibody of the present disclosure is administered. The term "administering" refers to contacting a subject with a nucleic acid, rAAV, or composition. Non-limiting examples include intravenous injection, intra-arterial 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, children, and young children. Present in the first year of life or in adults, and in the second or third year of life, mental retardation, dementia, blindness, It is a universally fatal disorder, with symptoms including glaucoma and death. Hexosaminidase A (HexA), hexosaminidase B (HexB), and GM2 gangliosides Wild-type levels of all three ganglioside activators (GM2A) inhibit the degradation of GM2 ganglioside. A decrease in the activity of any of these three enzymes results in the This is associated with increased accumulation of GM2 ganglioside. Patients with TSD have a central nervous system with for lipid-deposited ganglion cells, leaving behind the characteristic "cherry red" spots. , a gray-white area appears around the fovea of ​​the retina. 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 a mutation Sandhoff disease (SD) (OMIM:268800 ) is a universally fatal disorder. Patients with SD typically present with: Developing weakness is 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. It occurs immediately.

[0133] In some embodiments, the 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 SEQ ID NO: 5754.1. HexA (GeneID: 15211) and 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, the human HexB comprises the sequence shown. 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), and 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 GM2A gene contains the amino acid sequence set forth in NP_034429.1.

[0134] In some embodiments, the present disclosure provides a method for the production of human GLB1, human HexA, or human HexB. Isolated nucleic acids and recombinant AAVs containing the encoding transgenes are provided. In some embodiments, the present disclosure provides a gene encoding mouse GLB1, mouse HexA, or mouse HexB. In some embodiments, the present invention provides isolated nucleic acids and recombinant AAVs comprising a transgene. GLB1, HexA, and / or HexB may be used in conjunction with a promoter, optionally a specific gene 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. to a subject in need thereof, the isolated nucleic acid or recombinant AAV described above. Methods of administering the compositions are provided. In some embodiments, the subject receiving the composition has G including, but not limited to, M1 gangliosidosis, Tay-Sachs disease, or Sandhoff disease have or are suspected of having an undetermined lysosomal storage disorder.

[0135] In some embodiments, the subject has a gene encoding human HexA and / or human HexB. The subject is administered a rAAV or pharmaceutical composition of the present disclosure containing 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-hemoglobin in CSF. In some embodiments, the enzyme activity is increased MU In some embodiments, the subject is monitored using an artificial substrate such as G or MUGS. The present disclosure also includes a transgene encoding human HexA and / or human HexB. The subject is administered an rAAV or pharmaceutical composition, in which case the subject's CSF contains β-hexosaminidase inhibitors. The enzyme activity of α-D-aspartate was measured at 3 months after administration. ... In some embodiments, the expression level is increased by at least two-fold, or at least three-fold, compared to the expression level of the control. 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 levels of cerebrospinal fluid (CSF) in the subject From the line, 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 the treatment of lysosomal storage disorders. In some embodiments, isolated nucleic acids, rAAVs, compositions, and methods for use in the treatment of cancer 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 can be Tay-Sachs disease or Sandhoff disease GM2 gangliosidosis is characterized by a deficiency of HEXA enzyme activity and / or HEXB enzyme activity. Since GM2 gangliosidosis is caused by a deficiency in GM2 activity, the method for treating GM2 gangliosidosis is This results in increased HEXA and / or HEXB activity. A limiting example is the expression of wild-type HEXA and / or wild-type HEX in a cell or in a subject. A method for expressing mutant HEXA and / or B in a cell or a subject. The present invention relates to a method for reducing expression of a HEXB gene or for increasing 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 substitution) In some embodiments, GM2 is fused to at least one of 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 GM2 gangliosidosis in subjects with intracellular or 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%. In subjects with rhesus mastitis, the activity of HEXA and / or HEXB is at least Intracellular or in subjects with GM2 gangliosidosis, In this study, the activity of HEXA and / or HEXB was increased by at least 500%. HEXA and GM2 gangliosidosis in cells or in subjects with GM2 gangliosidosis / 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 The activity of HEXA and / or HEXB in subjects with 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 includes administering human HEXA and and / or a transgene encoding human HEXB. The subject may be a human, a mouse, a rat, a pig, or a mammal. 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 disclosure. Non-limiting examples of administering include intravenous injection, intraarterial injection, intracranial injection, intrathecal injection, and the like. injection, intracerebral injection, infusion, or inhalation. [Example]

[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 virus post-transcriptional regulatory element (WPRE), and bovine growth hormone (BGH) and A promoter consisting of two tandem poly(A) signals derived from SV40 This vector carries an expression cassette driven by the AAV-CBA-mβgal AAV-CBA-mβgal E269Q-WPRE is a vector containing the following promoter: Timer: 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 vector strain No. 3. 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 male is 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 by 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 vertebral column. The periosteum was then removed from the surgical area. Using a high-speed drill, the periosteum was removed from the surgical area at the appropriate stereotaxic coordinates. A small burr hole (<1 mm) was then made. A 10 μl airtight syringe with a 33G needle was inserted. An Ultramicro Pump is used to drive a 0. 1 μl of AAV vector or PBS was added to the βgal - / - 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) Infusion began 1 minute after needle placement in the target structure and was completed 2.5 minutes 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 rotation speed was accelerated from 4 to 40 rpm over a period of 5 minutes. The test was conducted on a rig and the latency to fall was recorded. followed by one training trial, accelerating from 2 to 20 rpm over 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 placed on a Brain Mat. Slice into 2 mm coronal blocks using rice cookers and store immediately on dry ice. The block containing the thalamus was then frozen. The shape and location of the needle insertion point on the dorsal brain surface were examined. A 2 mm diameter biopsy punch was used to sample the thalamus and was used for analysis. For histological studies, the brain and spinal cord were 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, the cerebrum, cerebellum + brainstem, and spinal cord They were excised and flash frozen on dry ice.

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

[0147] As previously described, but with modifications, to assess the distribution of βgal To determine the staining time, the brain sections were stained with X-gal. Briefly, the slides were stained with 0.5% X-gal in PBS. Fixation was performed in glutaraldehyde and ice-cold citrate phosphate buffer (CPB) (50 mM C 6H8O, 50 mM Na2HPO4, 10 mM NaCl, pH = 4.2) The cells were washed extensively and stained with X-gal staining solution [20 mM KFe(CN), 20 mM KF e(CN)6, 2 mM MgCl2, 0.01% C 24 H 39 NaO4, 0.02% (C2H4O)nC 14 H 22 O(IGEPAL CA-630, Sigma Aldrich ich), 2 mg in 97% CPB, HCON(CH3)2 at pH = 4.2 / ml 5-bromo-4-chloro-3-indoyl-β-D-galactosidase (Xg The slides were incubated overnight in CPB (at 37°C). Then rinse in water and counterstain with Vector Nuclear Fast Red. Then, the tissue was 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, but with modifications. To detect the phenotype, brain and spinal cord sections were stained with filipin. Fixation in 4% paraformaldehyde in phosphate-buffered saline (PBS) was performed. Washed with 100 ml of PBS, incubated with 1.5% glycine in water, washed with PBS, and 1 μg / ml filipin, and 1 μg / ml ToPro3 iodide (Life Te technologies, Grand Island, NY) for 1-2 minutes. Incubate, wash with PBS, and then mount in PermaFlu, a fluorescent mounting medium. Mounted by or.

[0149] To assess for shape changes within the tissue, brain sections were stained with Mayer's hematoxylin / eosin. Briefly, the 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-200 (MS). Incubate with phosphorus, wash with drained tap water, counterstain with eosin, and demineralized water. The tissue was then dehydrated in a 50% to 100% ethanol series, cleared in CitriSolv, and then stored at RT. Mounted it with ermount.

[0150] Whole-brain slice images were taken using a Nikon Super C with a medical slide holder. Microscopic images were captured using white light on a CoolScan 5000 ED. ica DFC425 C digital camera and ica DFC365 FX digital camera The images were captured on a Leica DM550 B Microscope equipped with a 1000-megapixel IR microscope. The iodide was imaged at 405 nm and the 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 7.0). 4.3) homogenized in 0.1% Triton X-100 and β-gal enzyme Briefly, βgal substrate = 1 mM 4-methylumbelliferol The reaction with 4-methyl-β-D-galactoside (4-MUG) was carried out 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 nanomoles (substrate converted) per hour per mg of protein. For immunoblotting, biopsy punches from the injection sites were taken using Complete Minute Homogenize in T-PER buffer supplemented with protease inhibitor cocktail and store on ice. Incubate at 4°C for 10 minutes, then at 10,000 x g for 5 minutes. The supernatant was collected and analyzed for protein concentration using the Bradford reagent assay. The degree of cleavage was determined using Mini-PROTEAN TGX precast gel. Total protein (20 μg) was separated by acrylamide gel electrophoresis. The blot was transferred to a NitroPure nitrocellulose membrane. Blocking was performed with Tris-buffered saline-Tween-20 (TBST), followed by , α-rabbit GLB1 (β-galactosidase antibody) (1:250) and α-mouse β The primary antibody against actin (1:1000) was incubated with HRP conjugate. The signal was detected using a 1:4000 dilution of anti-rabbit and anti-mouse secondary antibody. Detection was performed using Pierce ECL Western Blotting Substrate The blots were exposed to Amersham Hyperfilm ECL. did.

[0153] Genome copying Using the Qiagen DNeasy Blood and Tissue kit, Genomic DNA derived from injection site biopsy punches 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: Chromatin and TaqMan probe: (TaqMan Probe, 6FAM-AGC A TT TTT TTC ACT GCA TTC TAG TTG TGG TTT G 100n of genomic DNA was analyzed by qPCR using TC-TAMRA (SEQ ID NO: 11). The AAV vector genome copy number per μg of DNA was determined. Samples with a ≥ 100 vg were considered positive for vector genome.

[0154] Microarray Total RNA was isolated from biopsy punches using Trizol and RNeasy Plus s Use the Mini Kit for further purification and analysis using the Agilent Bioanalyze The quality of the samples was analyzed using the Bioanalyzer RIN (RNA intensity). The chromatin (gritity 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. 3 per group Independent samples for each case were analyzed. The resulting data were processed and a P value of <0.05 was used. and 1.5-fold change relative to the PBS control were considered to be differentially expressed genes.

[0155] Quantitation of GM1 ganglioside content The GM1 content in the CNS was determined by liquid chromatography / tandem mass spectrometry (LC-MS / The amount of tissue was quantified by MS. Briefly, 25 μL of tissue (0.01-0.04 mg / μL) was used. 1 was homogenized in 0.1 M NaCl, 0.2 M CH3COONa at pH 4.3. 3 μg of d3-GM1, an internal standard, was added and extracted by the modified Folch extraction method. The sample was then passed through a C18 column, dried, and runnylidene iodide was isolated. The samples were reconstituted in a quenching buffer and then analyzed by LC-MS / MS using a Waters Quattro The sample was processed by Premier XE. 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 were plotted against the ratio of M1 / d3-GM1. Normalized for protein content in the initial lysate, determined by Reported as ng of GM1 per 1000 μg.

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

[0157] C57BL / 6 mice were treated with PBS (control) or phenotype-specific ... The treated mice were injected with 2.25 × 10 AAV9-hGLB1 (Figure 1B). 1 1 Each cohort (n=5) received 30 mg of AAV9-hGLB1 at 7 days post-treatment. The mice were euthanized after 30 days and 30 months. β-4-methyl-2-pyridyl-2-methylpropanol, a fluorogenic substrate that emits fluorescence when cleaved by β-methyl-2-pyridyl-2-methylpropanol β-galactosidase activity was measured in the samples using umbelliferone (4-MU). Increased virility was observed within 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 (Fig. 1 and 20) was diluted 4 × 1 0 10 vg, 2.6 × 10 10 vg, and 2.6 × 10 9 Thalamus in total dose of vg Bilateral injections into the cerebellar and deep cerebellar nuclei produced GM1 gangliosidosis in 6- to 8-week-old mice. S (βgal - / - ) into the brain. Animals in the highest dose cohort received 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 injections of 1 μl each. 1 μl of the β-glucan in the brain and 0.3 μl of the β-glucan in the DCN were administered. 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 subcortex The medium dose, 2.6x, resulted in enzyme activity across most sections of the brain (Figure 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 throughout the structure. The low dose was 2.6 x 10 9 vg is widespread in both the cerebrum and cerebellum. The results showed that the α-glucan-containing α-glucan derivatives of α-glucan (Fig. 2I and 2J, respectively) reduced the risk of α-glucan-containing α-glucan (Fig. 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 results were evaluated using the AAVrh8-treated βgal - / - All cohorts were included in the 6 months of treatment. At the month later time point, 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 lifespan AAVrh8-treated βgal - / - Mice lifespan is significantly longer than that of naive βgal - / - Control and comparison The time to maturation was significantly prolonged (Fig. 4). - / - 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] GM1 ganglioside accumulation at the injection site and spinal cord of long-lived AAV-treated animals persist Lysosomal storage by filipin staining in the CNS of animals 3 months after injection Histological analysis of the amount of erythrocytes revealed no significant difference in the presence of the enzyme compared with Xgal staining at the same time point. In response to this, we found almost complete correction in the cerebrum and cerebellum (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 showed that the maximum intensity of Xg in the brain, which is also a semiquantitative indicator of high β-gal enzyme activity, was This is surprising, since this is also the brain region that exhibits the most histochemical staining (Figure 5A, boxed). In the spinal cord of long-lived AAV-treated mice (495-612 days), lysosomal The effect on accumulation was observed in areas with very few remaining filipin-positive cells, and in areas with no treatment. Place βgal - / - The range of areas where no obvious changes were observed compared to the control was variable. (Figures 6A, 6D, 6F, and 6B, 6C, 6E, respectively).

[0163] [Example 4] Neuropathology at the injection site After early loss or 3 months after injection, animals were analyzed by hematoxylin / eosin (H&E) These animals showed dose-related changes in shape at the injection site. Two weeks after injection, βgal - / - +AAV4×1 0 10 vg was associated with cell infiltration into blood vessels and the appearance of inflammation (Figures 7A and 7B, respectively, indicated by thick arrows). Massive changes in the thalamus, such as vascular cellular infiltration, and clear Large phagocytic lesions in the DCN with extensive neuronal phagocytosis (thick arrows in Figure 8A and Figure 8B, respectively). Three months after treatment, the changes in the amount of βgal - / - +AAV2×10 10 v g also showed thalamic changes accompanied by cellular infiltration of blood vessels and inflammation (Fig. 7 E, 7F, thick and thin arrows). However, a dose of 2 × 10 10 vg βgal - / - + AAV resulted in only low-grade cell infiltration into blood vessels in the DCN (Fig. 8E, Taiya (See the symbol). 3 months after injection, the lowest dose 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 (Fig. 8I, 8J). - / - Control (Figures 7C, 7D, and Figures 8C, 8D), or untreated βgal + / - Control (Fig. 7G, 7H, and Fig. 8G, 8H), neither cellular infiltration into blood vessels nor inflammation was observed. .

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

[0165] [Example 5] Enzyme activity, protein levels, and filipin-detection response of AAVrh8 capsid Validation of the AAVrh8 vector series to assess its contribution to To investigate the nature of unexpected responses at the injection site, we used the AAVrh8 vector. A series of vectors were designed (Table 1). Further vectors designed are shown in Figure 19. This AAVrh8 series will be referred to as "CBA-WPRE" from this section onwards. 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", contains exactly the same bone structure as Vector 1. βgal protein, but carrying the E269Q mutation within the putative active site This vector encodes the . This vector allows for the determination of whether the observed response is caused by enzymatic activity. This study was designed to assess whether the effects of the β-glucan on the oxidative stress response were caused by the production of a protein. Vector 4, "CB6," contained mβgal cDNA but not in other vectors. 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, an "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 injected into the cells at a high dose (2.0 × 10 10 vg total dose In contrast, 1 μl per site was also injected, except for the CB6 vector, which 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 naive 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 is a naive βgal + / - Enzyme activity in the thalamus of mice (Fig. 10A), the highest enzyme activity and corresponding protein increase (Fig. 10B, Lane 1). The CBA vector + / - 224 times the level of βga This resulted in β1 activity, which was not observed with the CBA-WPRE vector. gal activity (Fig. 10A, p=0.001), and the protein product This resulted in a correspondingly clear decrease (Fig. 10B, lane 2). In the thalamus of vector-injected mice, βgal activity was significantly higher than in naive control mice. βgal activity (Fig. 10A), but the protein was The protein was expressed at a level comparable to that in the thalamus (Fig. 10B, lane 1). Therefore, the E269Q mutation impairs the enzyme activity but not the protein expression level. injection of the CB6 vector (low dose CB6) at the same dose as other vectors. 6) is βgal + / - βgal activity was 54-fold higher than in the control group (Fig. 4A). Accordingly, this results in lower amounts of protein than with other vectors. (Fig. 10B, lane 4). Injection of this vector at a high dose (high dose CB6) is βgal + / - This resulted in 420-fold higher β-gal activity than the levels induced by This was significantly higher than the β-gal activity in the low-dose CB6 cohort (Figure 1). 10A, 10B, p=0.03). β-gal activity levels within the high-dose CB6 cohort were significantly higher than those in the C The β-gal activity levels were comparable to those measured within the BA-WPRE cohort, and the protein levels The results were comparable for both the bell and the bell (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 coding region in the AAV-injected thalamus showed no expression (Fig. 10A, 10B). The number of peaks was 3.4 × 10 except for the T.Empty cohort. 9 injected with a total dose of vg , were shown to be comparable within most cohorts (Figure 11). As expected, 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-glucan was 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 X-gal staining (Figs. 12A, 12D, 12E). G, 12J, 12M, 12P, 12S), the presence or absence of filipin-positive cells in the thalamus The presence of α-glucan was analyzed (Figures 12B, 12E, 12H, 12K, 12N, 12Q, and 12T). The thalamic regions with the greatest intensity of βgal staining (Figures 11A, 11D) were also CBA-WP RE- and CBA-injected animals also contained filipin-positive cells, but the latter Within the cohort, CBA-EI-WPR were clearly underrepresented (Fig. 11B, 11E). The thalamus in the E cohort contained numerous filipin-positive cells but no active β-gal enzyme. Similar filipin staining was not observed in the T-Empty control cells (Fig. 11K and 11J, respectively). This was also evident in the thalamus of the low-dose CB6 cohort (Fig. 11K, 11Q). observed within the CBA-WPRE and CBA-EI-WPRE cohorts The filipin staining was also highly used. The pattern was also observed within the thalamus of the CB6 cohort, and was consistent with that observed within the CBA-WPRE cohort. This is a mix of patterns expected and those observed within the low-dose CB6 cohort. These results suggest that abnormal filipin accumulation occurred at the injection site (Fig. 11H). This indicates that the expression level of the protein is correlated with its activity, but not with its enzyme activity.

[0170] Transcriptome changes in the injected thalamus were consistent with abnormalities detected by filipin. correlates with a positive 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 > 100%) for all analyzed samples were The difference between the low-dose CB6 sample and the control sample (1.8-fold, p<0.05) is shown in the heat map (Fig. 13A). The T.Empty and PBS samples clustered together, and were distinct from the CBA-WPRE samples. The number of genes with a >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 the pathological changes in transcription levels within the injected structures. We then determined the correlation between the ability of AAV vector design to target GM1 ganglionic acid and the ability to target GM1 ganglionic acid. Osidotic mice (βgal - / - ) led to differences in treatment outcomes We investigated the effects of βgal on the 6- to 8-week-old - / - AAV vectors were injected into mice into the thalamus (hematological Bilateral injections were given into the 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 vg total dose CBA-WPRE, CB6 (low dose CB6), and empty transgene injected with vector, βgal - / - In addition, CB6 was added to the mice at 2.6 × 1 0 10 The total dose of βgal was 100 mg / kg (high dose CB6). - / - Animals and naive untreated βgal + / +The animals were used as controls. Assessment of βgal activity by MU assay (Fig. 14) showed 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), high dose CB6 showed a 30-fold increase in βga (Figure 14). l - / - The T.Empty and untreated cohorts of mice were not affected by any of the analyzed CNs. Even within the S region, there was no detectable β-gal activity (Fig. 14). The CBA-WPRE expression levels were 47-fold higher than the wild-type level (Fig. 14), and the low dose 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 β-gal activity and significantly higher than the same vector, low-dose CB6 (p<0.001). It was big.

[0173] The distribution pattern of β-gal in the brain, as confirmed by the histological dye X-gal (Fig. 15) was determined by the 4-Mu assay, and activity levels in the cerebrum or cerebellum plus brainstem were The CBA-WPRE vector increased the dark blue staining in the thalamus and DCN (Fig. 14). 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 intrathalamic and DC Intense staining was observed within the N, but no detectable levels were observed throughout the cerebrum, cerebellum, or brainstem. In high-dose CB6 animals, the brain distribution pattern of β-gal was low. The distribution pattern in the brain appeared more widespread in CB6 animals (Fig. 15C). As expected, the T.Empty cohort (Figure 15D) and naive βgal - / - In the mouse cohort (FIG. 15E), there was no evidence of increased βgal activity.

[0174] Histology for lysosomal accumulation in the brain and spinal cord using filipin staining The biological analysis (Fig. 16) confirmed the βgal activity (Fig. 14) and distribution pattern ( In CBA-WPRE-injected animals, the injection site and route were not significantly different. Near-complete clearance of accumulation was observed throughout the brain (Fig. 16), but not within the ventral hippocampus. , and filipin-positive cells were observed throughout the thalamus (Figure 16; row 1, column 4). Filipin-positive cells were detected in the anterior cingulate cortex, striatum, and brainstem (Fig. 16; respectively). row 2, columns 2, 5, and 7), suggesting a clear accumulation in the low-dose CB6 cohort. The effect of CB6 on the steroid hormone was considered to be low (Figure 16; row 2). The efficiency of the CB6 vector is extremely high, and the dissipation of lysosomal accumulation is widespread throughout the brain. As before, filipin-positive cells were detected in the ventral hippocampus and dorsal visual cortex (Fig. 16; row 3). Although it was present in the bed, it was low in CBA-WPRE (Figure 16; 3rd row, 1st, 4th The T.Empty cohort (Figure 16, row 4) showed a significant increase in lysosomal accumulation of 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 the thoracic region Only the area where the CB6 activity was decreased was slightly reduced (Figures 17A-17B; arrows). 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 showed a significant increase in the cervical and thoracic regions (Figures 17C-17D). The accumulation of lysosomes within 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 (Figs. 17G-17H; 17I-17J, respectively).

[0176] GM1 ganglioside levels in the CNS were quantified by LC-MS / MS (Figure 18 ) in the CBA-WPRE cohort and the low-dose CB6 cohort, cerebral (p = 0.0009 and p=0.002) and cerebellum+brainstem (p<0.0001 and p<0.0002, respectively). and p=0.0001) of GM1 ganglioside content in untreated βgal - / - In the spinal cord, GM1 gas was significantly reduced in both cohorts compared to controls. No significant changes in guanosine glioside content were observed in the high-dose CB6 cohort. Glycoside levels were measured in all CNS regions investigated: cerebrum, cerebellum + brainstem, and spinal cord. (Figure 18; p=0.64, p=0.06, and p=0.001, respectively) 79). As expected, in the T.Empty cohort, no significant changes were observed in any region of the CNS. The GM1 ganglioside content of the native βgal - / - Changes compared to the control were observed (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 conducted in non-human primates. This study evaluated the safety 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 3.2 × 10 doses (based on previous studies in GM2 mice and GM2 cats) 12 v The study was designed to be a 200-day (1000-2000 mg / kg total vector dose) study.

[0178] First, NHPs (N=3) injected with the AAVrh8 formulation showed a median age of 14 days after injection. The NHPs developed mild to severe neurological symptoms. The NHPs were euthanized, and CNS histology was performed. Clinical evaluation revealed large, necrotic areas and lesions within the thalamus along the area of ​​the suspected 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 also detected. The enzyme activity was measured to be 6 to 48 times higher than normal activity (Figure 24).

[0179] Two additional cohorts of NHPs (N=2 per cohort) were administered AAVrh8 The vector formulations were administered at 1 / 10 and 1 / 30 of the original dose, respectively, for a total dose of 2 × 1 0 11 vg and 1.1 × 10 11 vg. In 3 of 4 NHPs injected with AAVrh8 vector formulations expressing the subunit, Neurological symptoms developed, but the onset was progressively delayed with decreasing dose. 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 necrosis, nerve loss, and cell infiltration into blood vessels (Figure 22 E) In these NHPs, brain hexosaminidase activity was also significantly increased by saline injection. Hexosaminidase activity was elevated compared to that in 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, descending 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 received empty AAVrh8 vector, none of the animals showed any signs of aging throughout the study (>10 days post-injection). 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, possibly due to edema (ascending, Fig. 24), but no changes were observed in NHPs injected with the AAVrh8 vector containing an empty transgene. None was observed (lower row, Figure 24).

[0182] In addition to neuronal loss within the thalamus along the injection route, intracellular eosinophil granules were also observed in the vicinity. Large, dense neuronal fields were also observed (Fig. 25E, 25G), which are similar to those in other parts of the brain. No significant changes were evident within the region (Fig. 25F). Hexosaminidase alpha subunit Alternatively, immunofluorescence staining with an antibody specific for the hexosaminidase beta subunit revealed These granules likely contain these two proteins ( These cells, loaded with HexA subunit-positive granules, were found in the thalamus. This 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 normal young NHPs: first, many The thalamic regions containing HexA-positive neurons were not associated with evidence of significant inflammatory infiltrates. Although the lesion appears intact (Figs. 25E and 25G), there is extensive nerve loss along the injection route. and inflammation (Figure 22), and secondly, the transgene-empty AAV at the highest dose. 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-amyloid complex, which, above an unknown threshold, induces cell death and secondary neuritis. It is thought that this may result in an inflammatory response (such as cell infiltration into blood vessels).

[0184] [Table 3]

[0185] [Example 8] The new AAV vector plasmid (Figure 26) was transiently transfected into 293T cells. The expression level of the hexosaminidase (Hex) enzyme was examined during the cleavage. Two examples are shown in Figures 27 and 28.

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

[0187] AAV vectors were transfected into 10-12 week old male athymic nude mice (Charles Ri ver Labs) into the thalamus and left lateral ventricle on both sides, 1.32 × 10 10 Be The control group was injected with a total dose of promoterless AAV vector (vg). vector formulation (AAV-NoP), AAV vector without a transgene (empty transgene), mice injected with phosphate-buffered saline (PBS), 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 the same increase 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 higher doses, there was no evidence of significant behavioral changes during this period. The dose used in this study (1.32 × 10 10 vg) indicates all test items For quality and control, to normalize the total vector dose, one of the vector pairs Determined by the lowest titer.

[0188] Hex activity was measured using the artificial substrates MUG (Figure 36) and MUGS (Figure 37). Measurements were taken in four coronal cerebral blocks, the cerebellum, the brainstem, and the spinal cord. The Hex activity in coronal cerebral blocks containing the serotonin A and serotonin B is shown in Table 4, and the findings are summarized. The original AAV vector pair (group 1) produced 400-1,700 times higher Hex activity than normal. Similar to the results in cell culture, other AAV vector pairs were shown to be more potent than the original AAV vector in the brain. The 1 / 3 (Group 2), 1 / 20-1 / 30 (Group 4), and 50-10 times smaller (Group 5) of the turpentine formulation (Group 1) were used. 0-fold lower Hex activity (group 6), demonstrating 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 responses. It did not result in Hex activity above the 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 tumors in Group 1 animals. This finding was consistent with the AAV vector used in Group 1. Although the number of these abnormal neurons was identical to that observed in the ct-injected monkeys, In mice, the spleen size was significantly smaller than in monkeys. This observation was also observed in Group 2 animals. Although the number of abnormal neurons was significantly smaller than that of group 1 (Fig. 31L). Neurons were also observed in the hippocampus of group 2 animals (Fig. 31B). , which was not observed in any other animal group. Immunofluorescence staining with antibodies showed that the IgG4-dependent markers were significantly different in groups 1 (Fig. 31A, 31K), 2 (Fig. 31B, 31L), and 4 (Fig. 31C). 31D, 31N), and 6 (Fig. 31F, 31P) animals in the hippocampus and thalamus. In the control group, a large number of enzyme-expressing cells were found. The antibody species used in this study detects the monkey enzyme but not the mouse protein. Due to specificity, no evidence of Hex-alpha subunit expression was seen.

[0190] Intrahippocampal and intracellular signals 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 (Fig. 33I, 33J). We observed a dramatic increase in microglial activation compared to the control group (33S, 33T). This evidence is based on the localization of HexA-positive cells to the site detected by immunofluorescence staining. (Fig. 32A, 32K), no obvious changes in microglia were observed elsewhere in the brain. In groups 2 and 3, the increase in Iba-1 staining in the hippocampus and thalamus was very weak (Fig. 33B-33C, 33L-33M) and were essentially indistinguishable from controls in groups 4-7. (Figures 33D-33G; 33N-33Q).

[0191] Using GFAP immunostaining, brains were also examined for evidence of reactive astrogliosis. 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 ascites were observed in groups 1-3. Trogliosis was observed (Figs. 34K-34M), but was either weak or clustered. The levels were considered indistinguishable from the control within 4 to 7 (Figs. 34N to 34Q). No other brain regions in the group showed evidence of astrogliosis.

[0192] Two new AAV vectors (Figure 35) were identified: AAV-CB6-I-cmHex (sequence number 1111111); 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 significant behavioral changes, control group and whether the body weight remained stable between 0 and 30 days after injection. The presence of neuropathology and the presence of thalamic and rostral non-injection blocks were associated with increased 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 promoter and artificial intron) are still the same in the new AAV-CBA-I The -cmHex vector (Figure 35) was also investigated. This AAV vector (Group 2) , approximately one-third of the Hex activity level compared to the original AAV vector, and A long-term study in Sandhoff mice showed a reduction in microgliosis compared to control mice. in the AAV vectors used in the study and in the long-term study in Sandhoff cats. Considering that the expression elements are the same in the AAV vector and the V vector, It is also possible that the reduction in Hex expression levels may mediate long-term expression. Given the attenuation of the activation of the α-amyloid β-amyloid complex, this new AAV vector is suitable for the experiments in Example 9. , dose (3×10 11 vg) and duration 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 Increased inflammatory response (astrogliosis and microgliosis) compared to V vector preparations Three AAV vector designs (Figures 19 and 35) were developed to mitigate the effects of HIV-1 and HIV-2. 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 μ The vector formulation was also injected into the left lateral ventricle (50% dose in 300 μl). Immediately after injection, targeting accuracy and distribution in the brain were determined by MRI. The endoscopic procedure also contained 2 mM gadolinium (Figure 38A). All NHPs tolerated the procedure well. The mean volume of distribution of gadolinium in the thalamus (Vd) was The injection volume in the thalamus (Vi) was 0.3 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). From day 30 onwards, Signal changes at the injection site were recorded in two monkeys from cohorts 2 and 3. However, no significant changes were observed over time (Figs. 40B and 40C). In one monkey in Study 1, brain MRI at 30 and 60 days after injection showed no significant changes in the signal intensity. On day 90, no significant signal changes were observed in the left thalamus. Despite this abnormal signal, the behavior of this monkey at 90 days was Movement 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 genotypes (HexA, HexB, and HexS) were detected (Table 6). ex activity was highest in cohort 1 (up to 87 times 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 comparable to that in uninjected control monkeys. The total Hex activity was similar to that of the control.

[0199] Neuropathological assessment of the brain revealed intraneuronal eosinophilic material in monkeys in cohort 1. In animals in cohort 1, moderate to severe symptoms were observed. In cohort 1, one monkey (ID: 295851) showed severe neurodegeneration in the left optic nerve. In the bed, severe focal spongiosis and perivascular cellular infiltration were observed (Figures 43A and 43B ) This lesion was observed 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 neurodegenerative 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 observed. In one monkey (ID: 295709), cannulation and Foci of perivascular lattice cells were seen, likely associated with the lesions (Fig. 44B). eosinophilic material accumulation in the nerve (Fig. 45), nerve degeneration and necrosis (Fig. 46), as well as For quantification of inflammation (Figure 47), the left thalamus and Serial sections were prepared at 150 μm intervals throughout the right and left thalamus. cohort showed significant neurodegeneration, necrosis, spongiosis, and corresponding inflammation. With the exception of one animal at the left injection site, formulations 1 and 3 significantly increased intracerebral These findings were consistent with the 90-day brain MRI and the The best fit was obtained (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. 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 4-6 week-old SD mice in both the thyroid and thyroid glands. 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 treated with 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 x 1010 vg), AAVrh8-CB- An additional cohort of SD mice injected with the I-cmHex vector was also enrolled. To measure Hex activity as well as GM2 ganglioside content, 1 month after injection, 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) in one of three SD mice injected In the AAVrh8-CB-I-cmHex vector, hexosaminidase was undetectable. In mice injected with acetaminophen (blue bars in Figure 12), the 1x and 5x cohorts showed significant differences. Hex activity was not 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 vector tested in this study exceeded normal levels by 2-3 orders of magnitude. This resulted in significant Hex overexpression in a subset of SD mice. The lack of Hex expression may limit enzyme distribution via a humoral response or adaptive response. Cynomolgus monkey Hexalin mediated by α-glucan, resulting in loss of transduced cells Immunization 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 There appears to be a direct correlation between the sex level and the reduction of GM2 ganglioside. Block 3 contains AAVrh8-CBA-cmHex-W Δ6ATG Note vector In SD mice injected with α-glucan, a maximum 96% reduction 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 IFN-γ, 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 referred to as "Syn1") promoter was used. and the human GfaABC1D (also called "GFAP") promoter. 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. Two anti-SOD1mi antibodies encoding human SOD1 and mCherry, which are transduced with 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 AAV9-Sy 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 shapes of GFP (green) and mCherry (red) expressing cells are different from each other. This indicates that the shape of the cells corresponds to that of neurons and astrocytes (Figure 51).

[0207] SOD1 mRNA expression was measured. Data were obtained using the dual promoter AAV9 -Syn1-GFP-2×miRSOD1 / GFAP-2×miRSOD1-mCher After injection of the ry vector, adult SOD1 G93A Human SOD1 mRNA in mice This indicates that the NA level is reduced (Figure 52). 12 of the whole vector genome 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 demonstrate that this dual promoter construct is ubiquitous while reducing toxicity to non-CNS tissues. This is larger and more widespread than current approaches that use heterologous promoters (e.g., CBA, U6). 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 (TH / DCN), or bilateral intrathalamic injections combined with a single intracerebroventricular injection (TH / ICV). rAAVrh encoding the hexosaminidase A subunit was synthesized through two methods. 8 and rAAVrh8 encoding the hexosaminidase B subunit (respectively, 1:1 formulations (columns 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 the 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 performance of SD mice treated with 9 on the inverted screen was significantly better than that of untreated SD mice. TH / ICV AAVrh8-treated mice at 180 days of age, and The performance of both TH / DCN AAVrh8-treated mice was comparable to that of wild-type controls. The results were comparable (Fig. 53A). The rotarod test also showed that AAVrh8 mice at 120 days of age Both cohorts of SD mice treated with α-glucan-1-phosphate dehydrogenase (α-glucan-1) performed better than untreated SD controls (P≦0.01). .01), and their performance was comparable to that of wild-type controls up to at least 180 days of age. In the wire hanging test, the treated SD mice showed significant improvement compared to the 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 1.07 (Figure 53D). Therefore, both intracranial delivery methods were Compared with TH / DCN, similar lifespan extension (P ≤ 0.001 for 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 efficacy of AAVrh-mHexA / B. SD mice were injected with AAV8-TRG-mHexB (3 × 10 11 vg) were intravenously injected. AAV8-TBG-mHexB systemic injection alone was administered to SD mice. did not enhance survival of the untreated SD mice (median survival was 127 days for untreated SD mice). whereas median survival for SD mice treated with AAV8-TBG-mHexB was The mean value was 128.5 days (Figs. 53D and 54A). Systemic therapy-mediated tolerability improved 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 years). The median survival with TH / ICV alone was 423 days; Figures 53D and 5 4A). The same tolerization protocol (AAVrh8-mHexA / B vector) delivered intravenously. AAV8-TBG (mHexB) in combination with HIV-1 / HIV-2 (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 x 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 vg dose ) Treated animals ultimately presented with hind limb weakness and ataxia at the time of euthanasia, with some animals Loss of weight bearing and / or ability to stand upright when placed supine.

[0211] Inverted screen performance (Figure 54B), rotarod performance (Figure 54C), or wire-hugging At 120 days of age, 4.68 x 10 9 vg or 1.17 x 10 10 vg (intracranial TH / ICV administration) dose-escalating combination therapy mice There was no significant difference in behavioral performance compared to untreated SD mice. AVrh8-treated SD mice retained their motor function and were significantly more active than TH / ICV-treated SD mice at 180 days of age. SD mice performed comparable to wild-type mice in reversal screen performance at all doses. At 180 days of age, TH / ICV-treated SD at any dose also showed a significant improvement (Fig. 54B). Mice also performed comparable to wild-type mice on the rotarod (Fig. 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). Further studies will be performed at 180 days of age, when no untreated SD mice survive. I was able to do it.

[0212] Potential effects of intracranial injection of AAVrh8 vectors in the absence of underlying neurodegenerative processes Age-matched heterozygous (HZ) littermates (HexB + / - ) mouse, 4.68 × 10 9 Intracranial injection of vg of AAVrh8 vector formulation The AAVrh8-treated HZ mice and non-treated HZ mice were administered TH / ICV or TH / DCN. Treated controls (wild type and HZ) are evaluated over a time course in different behavioral tests. In AAVrh8-injected HZ mice, there was a clear improvement in behavior compared to controls (Fig. 55). No major changes or effects on survival were observed. However, no differences between the cohorts were apparent (Fig. 55A). Performance was slightly superior to untreated HZ and wild-type controls in the rotarod test at 180 days of age. The results of wire hanging were significantly poorer at either 60 or 120 days of age (Fig. 55B). In this case, 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 injection was significantly worse than that in treated 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 × 1010 vg) SD mice, untreated SD mice, and untreated wild-type controls (Figure 56). G measured by LC-MS / MS M2 ganglioside was not detected in the cerebrum of AAVrh8-treated animals, but in the cerebellum (91.1 %, brainstem (99.6%), and spinal cord (99.8%) also showed the presence of GM2 ganglioside. A significant reduction in the expression of IFN-γ was observed in AAVrh8-treated SD mice (P≦0.0001, Figure 56A). In the central nervous system, total hexosaminidase activity was restored. , exceeded wild type and were comparable in the cerebellum, brainstem, and spinal cord. When subjected to routine 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 the 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 a median survival of 4 years. These studies were conducted in a naturally occurring Sprague-Dawley cat model, with the cats 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 zeta subunit and the zeta subunit separately: 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 vg total dose (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 tremors with pronounced 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, significant improvement in neurological symptoms was observed ( (Figure 57B). Tremors were completely resolved in all treated cats, with the majority remaining stable over the course of the study. Throughout the study, the ability to walk was preserved, and the musculoskeletal disease was not a typical neurological phenotype in SD cats. All animals were euthanized due to gastrointestinal or respiratory illness (Table 7).

[0216] [Table 7]

[0217] Both ICV injections and combined intrathalamic injections (Th+ICV) of AAV vectors , resulting in widespread hexosaminidase distribution throughout the cerebral cortex, cerebellum, and spinal cord. (Figures 57B and 57C). The only area of ​​the brain where it is less distributed is the temporal lobe. The HexA levels were significantly higher in the brain than in the control group (Figure 57D, lateral view of Figure 57D, and Figure 57E). , cerebellum, and spinal cord, were near or above normal, and In AAV-treated cats, the increase in Hex was nearly 50 times higher than normal (Fig. 57E). No histopathological abnormalities were observed despite supraphysiological levels at the site. I couldn't.

[0218] MRI (T2W) at the humane endpoint in SD cats showed whole-brain, 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 decrease in the distribution of Hex (Figure 58D). Despite normalization of cerebral and white matter intensity, AAV-treated SD cats showed significant increases in the lateral ventricles and Cortical atrophy persisted, as exemplified by increased CSF in the pericerebral sulci and pericerebral cortex. Using MR spectroscopy, we identified a number of thalamic sites in untreated SD cats, including those in mice and humans with SD. In this study, the previously reported toxic metabolite, N-acetylhexosamine (NA-H), After AAV gene therapy, a significant increase in NA-Hex was detected in all cats. In AAV-treated cats, the HexA level was completely reduced to normal levels (Fig. 58B). The level of HexA is elevated at or above normal levels, and elevated HexA is associated with a lifetime 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 decrease over time. The humane endpoint was reached before age 1 year. Only one cat (11-907) had a maximum serum antibody titer of 1:6 approximately 2 months after treatment. The endpoint titer was only 1:16 (Figure 58G). A cat (7-760) that lived to 6 months of age 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 showed peripheral HexA enzyme activity in the organs was increased. The sciatic nerve showed a 203% increase over normal. whereas normal liver and muscle showed increases to 53 and 20% of normal. (Table 8).

[0220] [Table 8]

[0221] In this study, we investigated the chicken beta-actin (CBA) promoter and the woodchuck transcription factor (WTF) 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. He suffered from severe cerebellar disease, manifested by a progressively debilitating, generalized resting tremor and The animals were diagnosed with tremors and intention tremors, loss of ambulation, and subsequent euthanasia at approximately 4.4 ± 0.6 months of age. 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. Two cats followed to a humane endpoint had a 9.9% mortality rate (Figure 59B). They survived to ±0.5 months of age and retained the ability to stand and walk, but were unable to respond to visual or auditory stimuli. It was unresponsive to stimulation.

[0222] The biodistribution of Hex in SD+AAV CM cats was observed throughout the spinal cord and cerebellum. The cerebral cortex was occluded and widespread, but was reduced within the protocerebral regions (Figs. 59C, 59D, 59E). Short-term treatment of SD cats (data not shown) and long-term treatment of SD cats with SD+AAV CM In cats, hexosaminidase A (HexA) activity is normal 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 steroid showed a similar trend, with brain block D (the most anterior analyzed block) showing a similar distribution. contained the lowest vg copy number (Fig. 59F), but qPCR showed that the He x expression differences.

[0223] Accumulation in SD cats was assessed using periodic acid staining, which stains for glycolipids (e.g., GM2). The area is shown by PAS (gray scale) staining. Dark staining areas, e.g., PAS positive, In SD cats, the PAS region in the gray matter shows increased accumulation of gangliosides. There was increased staining and decreased staining in the white matter, which may be due to gangliosides within the cell bodies. In SD+AAV CM cats (long-term), accumulation and clearance of steroids were observed. The effect of Hex on the body distribution was inversely reflected by its efficacy in the spinal cord and cerebellum, but It was not effective in the cerebral cortex. PAS-positive substances were 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 extensive 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 numerous foamy, vacuolated, intracellular deposits, which are signs of Sixteen weeks after gene therapy, AAV-CM-treated SD cats showed increased activity in Purkinje cells, deep cerebellum, and The nuclei and morphology of brain stem cells were normalized (Figure 61). In treated animals, normalization of the DCN and brainstem morphology was observed, but the Purkinje cells Interestingly, the degeneration and loss of the cerebellar fastigial nucleus was prolonged in long-term AAV-treated cats. Grape-shaped eosinophilic inclusions were observed within the dorsal medial side (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 gray matter (accumulation of GM2). As a result, a reversal of gray matter intensity and white matter intensity was observed. 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 cerebellar white matter (Figure 62A; black arrows) were darkened. At humane endpoint in cats, pathological intensity correlations between gray and white matter in the cerebral cortex were observed. Although the degeneration 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) of an SD cat. MR spectroscopy of ) has been shown to detect the toxic metabolite, N-acetylhexosamine (NA-He). 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 situ. (Fig. 62D). Other metabolite changes were observed in the cerebellum after AAV treatment. of myelination (choline + phosphocholine) and metabolic activity (creatine + phosphocreatine) This endorses the 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, but in the other cat (11-1148), the levels were moderately increased. I was forced to do it.

[0227] [Example 13] mouse Short-term biochemical analysis of the effects on GM2 ganglioside content in the CNS For the analysis, SD mice were coated 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 x 10 9 Note: vg The original AAVrh8 vector injected (AAVrh8-CBA-mHexA / B-WPRE mut6ΔATG). In the brains of SD mice injected with the / B vector, the GM2 ganglioside content was significantly higher than that of PB 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 comparable to that recorded in SD mice injected with the original AAVrh8 vector (97 % of the GM2 ganglioside content in the SD mice injected with PBS. 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 (Fig. 63B). Survival to 1 month (exceeding 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 treated with 2.34 x10 10 vg of AAVrh8-CB-CI-mHexA / B. Most of the SD mice (6 out of 8) survived to 5 months of age. Only two of the six animals were considered to be rhesus-like (Fig. 63C). Mice presented with varying degrees of hind limb dysfunction or weakness. The new AAVrh8 vector formulation reduces the GM2 ganglioside content in the spinal cord by 20%. This can be explained by a more modest reduction compared to the BA vector (Fig. 63A; 70 35% compared to %.

[0228] To improve the effect on the accumulation of GM2 ganglioside in the spinal cord and cerebellum, The intrathalamic dose was determined as a constant (1.17 × 10 10 vg), through the lateral ventricle, The dose delivered to the SF was increased. SD mice received increased CSF doses: 1.1 7×10 10 vg, 2.34 × 10 10 vg, 5.85×10 10 vg, 5.00 x 10 11 vg of AAVrh8-CB-CI-mHexA / B were injected (per cohort) (n=3) One month after injection, the intraspinal (Fig. 64A) and intracerebellar (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 a similar effect.

[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 due to the lack of treatment available for patients with Sachs' disease.

[0230] Three weeks before treatment, subjects (approximately 30 months of age) with advanced infantile Tay-Sachs disease were given several Several clinical safety studies have been conducted, and immunosuppressive regimens have been 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 the following disease progression: 8 months: Excessive startle reflex Age 12 months: Macrocephaly with abnormal myelination on MRI, biomechanical changes on MRS Car abnormality Age 14 months: First afebrile seizure → diagnosis 17 months old: 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 rectal midazolam as needed. The subjects were placed under moderate control and administered a ketogenic diet, miglustat, via a G-tube. , Keppra, Prevacid, and clonazepam.

[0232] A composition containing a biological drug based on rAAVrh8-HexA / B rAAV is administered to a subject. The administered rAAVrh8-H was infused into the spleen at a rate dictated by the subject's CSF pressure. The target dose of the exA / B formulation was 1.0 × 10 per kg of brain weight. 14 Vector genome ( vg), or 1.0 × 10 14 It was vg.

[0233] The total dose administered to patients was 1040±130 g in 19-30 month old girls. The total dose was calculated based on brain weight. Approximately 75% 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 (Fig. 74A). Following passive flow removal of 100 ml of CSF, 9 ml of AAVrh8 vector was infused into approximately 1 ml of the spleen. 3 ml of AAVrh8 vector was administered at the L2 For subjects with advanced disease, co-delivery of rAAVr No intrathalamic injections of h8-HexA / B were administered to subjects.

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

[0235] rAAVrh8-HexA / B was generally well tolerated, with clinical outcomes at 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 remained unchanged from baseline to 3 months. Neurological examination showed no signs of deterioration and the condition was stable. Brain magnetic resonance imaging (MRI) also showed no significant changes at baseline. No evidence of progression from baseline when compared with pre-treatment MRI of the brain These data are shown in Figure 69.

[0236] Data collected at 3 months also showed that a single dose of rituximab significantly reduced the CD1 9 and CD20 counts were completely suppressed (<1% of peripheral T cells), and IgG These data also indicated that IVIg infusion maintained the blood pressure within a safe range. The data is shown in Figure 70.

[0237] Subjects were randomly assigned to receive the AAVrh8 capsid at 3 months and had near-baseline neutralizing antibody levels against the AAVrh8 capsid. 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 a response is a key factor in future immune responses. This confirms the successful induction of long-term immune tolerance to the capsid, preventing response.

[0238] Very slight increases in the enzymes AST (less than 2x baseline) and ALT A slight increase (less than twice the upper limit of normal) was observed over the first 21 days, but spontaneous There was also evidence of systemic inflammation based on high-sensitivity C-reactive protein or total complement activity. Liver ultrasound and pediatric gastroenterology examinations also revealed no specific pathology.

[0239] β-Hexosaminidase A activity was determined using the 4MUGS assay. In this study, the CSF β-hexosaminidase A activity of the subject was 0.46% of normal. After 3 months, the β-hexosaminidase A activity in the subject's CSF was 1 / 2 of the normal enzyme activity. This significantly increased 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 observed, suggesting that the disease This indicates that a decrease in GM2 ganglioside, which is thought to be the cause of the progression of In addition, β-hexosaminidase A protein, as measured by Western blot, Expression of α-amyloid 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 received rAAVrh8- HexA / B was 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 in exons 11-13 and 2) HexA protein: Val381 in exon 10. * truncation mutations Has.

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

[0245] [Example 16] A clinical study of rAAVrh8-HEXA / B was designed. HEXA and erythropoietin in Tay-Sachs disease (TSD) or Sandhoff disease (SD) Bilateral intrathalamic administration of AAVrh8 vectors encoding HEXB individually Designed as a two-stage, dose-escalation, and safety / efficacy study for intrathecal and intrathecal administration. 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 not be blinded.

[0246] The subjects in this study were 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 symptom onset Infants of either sex with infantile-onset TSD or SD. The HEXA gene encodes the α subunit, or the H gene encodes the β subunit. Subjects must have the respective mutation in the EXB gene. The patient was considered a suitable surgical candidate (according to the study neurosurgeon) based on his ability to sit for 5 seconds. (confirmed based on physical examination and MRI findings), serum HexA activity less than 5% of normal and do not carry the G269S or W574C mutation. Based on the test assay threshold, patients were also seronegative for AAVrh8 neutralizing antibodies. Other inclusion / exclusion criteria may also be applied.

[0247] The study was an open-label, non-randomized, viral vector-based study using rAAVrh8-H A single dose of EXA / B administered bilaterally intrathalamic (BiTh) and intrathecally (IT) Subjects will be enrolled in the study in two stages (Figure 73) as follows: Phase 1: Four subjects were randomly assigned to a sequential, dose-escalating study 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 will be 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 of rAAVr All injections were performed with rAAVrh8-HEXA and rAAVrh8-HEXA / B. 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 against the AAVrh8 capsid. To protect against an immune response, subjects were immunosuppressed prior to administration of AAVrh8-HexA / B treatment. The immunosuppressive regimen is maintained long-term as planned. Beginning on day -16 of the study, begin the following: ·Body surface area 1m 2 375 mg of rituximab per 10 mg / kg Solu-Medrol ·Body surface area 1m 2 1 mg of sirolimus per 2 mg / kg of prednisolone per day 1 to 1.5 mg / kg of lansoprazole 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 a CD20 count 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 - Withdrawal 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 thalamus filled with vector, or the volume of distribution (Vd), is determined by the volume of the target brain region (thalamus). The product is the distance 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. aVd:Vi ratio from injection studies (Yin, Richardson et al. 2010) (Yin et al, 2010) was assumed to be 3.0. The volume to the human thalamus is 1.08, as shown in Table 12. mL Vi, and 4.35 ml 3 Calculated as Vd (77% of total thalamic volume per side) I calculated.

[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. Low-, medium-, and high-dose thalamus injections are at or exceed the doses infused. The proposed adjusted clinical dose for intravenous administration is 7.2 x 10 12 ~1.4×10 13 vg is.

[0256] Intrathecal (IT) administration The proposed human dose for intrathecal administration is 2.03 x 10 13 ~8.13×1013 vg dose range, with a maximum injection volume of 8 mL (see table below) The latter value represents 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, remove the right infusion cannula, preventing blockage. and vector primed and attached to a delivery syringe and pump. The catheter is placed stereotaxically into the target area in the right thalamus. The left arm is fixed on the scalp with anchoring ligatures and adhesive dressings. Repeat the same steps for placement of the second catheter on the side. Monitor the intralid pressure.

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

[0260] IT administration: IT drug infusion is 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 thoracolumbar level. Under this 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 delivered by gravity. The tissue was then extracted with a 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 into the inferior The patient is then transferred to the thoracic-lumbar level and a second bolus of 2.0 ml is administered. Then, the microcatheter (dead volume: approximately 0.3 mL) was flushed with 0.5 mL of physiological saline. The surgeon's discretion, based on spinal anatomy and other considerations, The dose was administered by two separate IT injections at the high cervical / cisternal and thoracolumbar levels. It can be given.

[0261] Primary endpoint(s): Phase 1: TEAEs (tr Incidence and severity of 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: Bayley Scales of Infant and Toddler at visits 9 / 12 months 5, without assistance, as assessed by Section 22 of the BSID-III. Percentage of subjects achieving 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 test values, including quality of life CD20 count ECG, EEG -AAVrh8 capsid and HEXA and HEXB proteins The cellular responses and neutralizing antibody titers, as well as HEXA and HEXB proteins Antibody levels against Phase 2: Biological markers for disease: CSF HexA activity levels from baseline to visit 9 (month 12) Changes in Serum HexA at baseline to visits 4, 5, 6, 7, 8, and 9 Changes in activity levels C from baseline (by LC-MS / MS) to visit 9 (month 12) Changes in GM2 ganglioside levels in SF CS from baseline to 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 by 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. CHOP-INTEN at baseline through visits 5, 7, 8, and 9 D Change in Total Score 3. At Visits 5, 7, 8, and 9, the CHOP INTEND motor function schedule was completed. In the rule, scores of 40 or above, scores of 50 or above, and scores of 64 Percentage of subjects achieving the score 4. BSID-III C at baseline through visits 5, 7, 8, and 9 Change in composite score 5. 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 upright with assistance, walking with assistance, standing upright independently, and and walking independently) 6. Use 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 schizophrenia at baseline through visits 7, 8, and 9 and changes in visual evoked responses (VER) MRI brain volumetric myelination from baseline to visits 7 and 9 index, and diffusion tensor imaging (DTI) index, and changes in intracerebral water MRS indices of changes 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 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) Disturbed hematological parameters, with particular attention to platelet counts Late adverse events, including but not limited to autoimmune-like reactions and malignancies

[0264] [Example 17] Further details regarding this disclosure can be found in the entire contents of which are incorporated herein by reference. U.S. Provisional Patent Application No. 62 / 840,359, filed April 29, 2019, discloses This is presented 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 x 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 examination was guided by fluorescence microscopy through the rectum and neck area (Fig. 75A). After injection of contrast agent 15 ml of scAAV-CB-GFP was injected at 1 ml / min, and the distribution pattern near the cerebellum was The sheep were then sacrificed and macroscopic images of the spinal cord were observed post-mortem. Immunohistochemical staining showed that GFP was expressed in motor neurons, sensory neurons, and neurites in the spinal cord. Ron (Fig. 75D), and Purkinje cells, cerebellar peduncle, deep cerebellar nuclei, brainstem, cerebrum, occipital scalp expressed within the substantia nigra, parietal cortex, thalamus, hippocampus, temporal cortex, motor cortex, internal capsule, and frontal cortex These results indicate that AA Administration of V into the cisterna magna expresses the transgene of interest (e.g., GLB1, HEXA, HEXB This indicates 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 device. Also, the phrases and terms used herein may be used interchangeably. The present invention is intended to be illustrative and not to be construed as limiting. , "including", "comprising ", or "having," "containing," "involving," and The use of these variations includes the subsequently listed items and their equivalents, as well as further It is intended to encompass items.

[0267] Thus, in some aspects of at least one embodiment of the present disclosure, Having described the invention, 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 intended that the foregoing statements be interpreted as including all such statements and should be construed as being within the spirit and scope of the present disclosure. The description and drawings are for purposes of example only.

Claims

1. An isolated nucleic acid comprising the sequence set forth in SEQ ID NO: 18 or 19.

2. A host cell comprising the isolated nucleic acid construct of claim 1.

3. The host cell of claim 2 , which is a eukaryotic cell.

4. The host cell of claim 2 or 3, which is a mammalian cell.

5. The host cell of claim 2 , which is a prokaryotic cell.

6. The host cell of claim 5 which is a bacterial cell.

7. 7. The host cell of claim 2, further comprising an isolated nucleic acid encoding an AAV capsid protein.

8. The host cell of claim 7, wherein the capsid protein is an AAVrh8 capsid protein or an AAVrhlO capsid protein.

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