rAAV vectors for the treatment of GM1 gangliosidosis and GM2 gangliosidosis

Abstract

Aspects of the present disclosure relate to compositions and methods for treating lysosomal storage disorders, such as GM1 gangliosidosis, Tay-Sachs disease, and Sandhoff disease. In some embodiments, the compositions comprise a viral vector encoding beta-galactosidase. In some embodiments, the compositions comprise a viral vector encoding a beta-hexosaminidase subunit (e.g., HEXA, HEXB, or a combination thereof).

Description

Technical Field

[0001] Related Applications This application claims the benefit, under 35 U.S.C.§119(e), as of the filing date, of U.S. Provisional Patent Application No. 62 / 741,848, filed Oct. 5, 2018, entitled "RAAV VECTORS FOR THE TREATMENT OF GM1 AND GM2 GANGLIOSIDOSIS", U.S. Provisional Patent Application No. 62 / 814,587, filed Mar. 6, 2019, entitled "RAAV VECTORS FOR THE TREATMENT OF GM1 AND GM2 GANGLIOSIDOSIS", U.S. Provisional Patent Application No. 62 / 815,996, filed Mar. 8, 2019, entitled "RAAV VECTORS FOR THE TREATMENT OF GM1 AND GM2 GANGLIOSIDOSIS", U.S. Provisional Patent Application No. 62 / 826,863, filed Mar. 29, 2019, entitled "RAAV VECTORS FOR THE TREATMENT OF GM1 AND GM2 GANGLIOSIDOSIS", U.S. Provisional Patent Application No. 62 / 840,359, filed Apr. 29, 2019, entitled "RAAV VECTORS FOR THE TREATMENT OF GM1 AND GM2 GANGLIOSIDOSIS", and U.S. Provisional Patent Application No. 62 / 848,858, filed May 16, 2019, entitled "RAAV VECTORS FOR THE TREATMENT OF GM1 AND GM2 GANGLIOSIDOSIS", the entire contents of each of which are incorporated herein by reference.

[0002] Reference to a Sequence Listing Submitted Electronically via EFS-Web This application includes a Sequence Listing (name: "Sequence Listing"; size: 80,237 bytes; and creation software: Patent-In 3.5) that was submitted electronically via EFS-Web and is incorporated herein by reference in its entirety.

[0003] Field In some aspects, the present disclosure presents recombinant adeno-associated viruses (rAAVs), compositions, and kits useful for treating lysosomal storage disorders. In some embodiments, the lysosomal storage disorder is GM1 gangliosidosis or GM2 gangliosidosis (e.g., Tay-Sachs disease).

[0004] Research supported by the federal government This invention was made with government support under grant number: HD060576, awarded by the National Institutes of Health. The United States federal government has certain rights in this invention.

Background Art

[0005] GM1 gangliosidosis is an autosomal recessive neurodegenerative lysosomal storage disease caused by mutations in the human gene GLB1. The human GLB1 gene encodes β-D-galactosidase (βgal), an enzyme that removes terminal galactose residues from a number of molecules in the central nervous system (e.g., GM1 ganglioside) and a number of molecules in peripheral tissues (e.g., oligosaccharides, glycoproteins, and glycosaminoglycans). Deficiency of βgal activity in lysosomes results in the accumulation of GM1 ganglioside and its asialo derivative, GA1, mainly in the central nervous system where its synthesis rate is maximal. GM1 gangliosidosis is generally lethal and there is no effective treatment.

[0006] Tay-Sachs disease (GM2 gangliosidosis) and Sandhoff disease are autosomal recessive lysosomal storage disorders caused by mutations in the HEXA and HEXB genes, respectively, resulting in a deficiency of β-N-acetyl-D-hexosaminidase (Hex) enzyme activity. The Hex enzyme is a heterodimer containing the HexA protein subunit and the HexB protein subunit. Deficiency of Hex activity leads to progressive accumulation of GM2 ganglioside in the central nervous system and subsequent neurodegeneration. Tay-Sachs disease is characterized by relentless progression, neurological deterioration, and eventual death. The symptoms of Tay-Sachs disease include frequent seizures, difficulty swallowing, loss of motor control, and frequent respiratory infections. Sandhoff disease is also characterized by progressive deterioration of the CNS, leading to abnormal gait, difficulty swallowing and speaking, peripheral neuropathy, psychiatric symptoms, and eventual death. Currently, there are no disease-modifying treatment options for either Tay-Sachs disease or Sandhoff disease, and only symptomatic treatments, including anticonvulsants and antispasmodics, are available.

Summary of the Invention

Means for Solving the Problems

[0007] Aspects of the present disclosure relate to recombinant AAV vectors for gene delivery. Some current AAV vectors deliver high levels of gene expression that result in adverse events (e.g., cytotoxic effects) in a subject. The present disclosure is based in part on the recognition that the expression level of a transgene can be modulated such that manipulation of regulatory elements results in a therapeutic amount of the transgene without inducing adverse events.

[0008] In some embodiments, the present disclosure provides a recombinant adeno-associated virus (rAAV) comprising a capsid containing a nucleic acid comprising a promoter operably linked to a transgene encoding a lysosomal storage disorder-related protein (e.g., HEXA, HEXB, GLB1, etc.) via a chimeric intron. In some embodiments, the present disclosure provides a recombinant adeno-associated virus (rAAV) comprising a capsid containing a nucleic acid comprising a promoter operably linked to a transgene via a chimeric intron, wherein the promoter and the transgene are separated by the chimeric intron, the nucleic acid does not contain an enhancer element, and the transgene encodes a lysosomal storage disorder-related protein (e.g., HEXA and / or HEXB and / or GLB1). In some embodiments, the promoter comprises a chicken beta-actin (CB) promoter. In some embodiments, the chimeric intron comprises an intron of chicken beta-actin and / or an intron of rabbit beta-globin. In some embodiments, the nucleic acid further comprises one or more untranslated sequences, e.g., untranslated sequences derived from exon 1 and / or exon 2 of rabbit beta-globin. In some embodiments, the chimeric intron is flanked by two untranslated sequences (e.g., a first untranslated sequence is located between the promoter and the chimeric intron, and a second untranslated region is located between the chimeric intron and the first codon of the transgene).

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

[0010] In some embodiments, at least one capsid protein of the rAAV described herein is a capsid protein of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, or AAVrh10. 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. In some embodiments, at least one ITR is a full-length ITR. In some embodiments, rAAV contains two ITRs, in which case the hybrid promoter and the transgene are disposed between the two ITRs. In some embodiments, the rAAV described herein has one of the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, or AAVrh10.

[0011] In some embodiments, the present disclosure relates to a nucleic acid encoding a component of the rAAV described herein. For example, in some aspects, the present disclosure presents an isolated nucleic acid comprising a sequence selected from SEQ ID NOs: 1-6 or 16-19. In some aspects, the present disclosure presents an isolated nucleic acid comprising a sequence encoding a peptide having the amino acid sequence set forth in SEQ ID NO: 20 or 21.

[0012] In some embodiments, a nucleic acid encoding a component of rAAV (e.g., a nucleic acid comprising a sequence selected from SEQ ID NOs: 1-6 or 16-19, or a sequence derived from a portion of a sequence selected from SEQ ID NOs: 1-6 or 16-19) is contained within a host cell. In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the host cell is a bacterial cell.

[0013] In some embodiments, the host cell further comprises an isolated nucleic acid encoding an AAV capsid protein (e.g., the capsid protein of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, or AAVrh10).

[0014] In some aspects, the present disclosure presents a pharmaceutical composition comprising: (i) a first rAAV comprising a capsid containing a first nucleic acid containing a promoter operably linked to a transgene encoding beta-hexosaminidase subunit alpha (HexA) via a chimeric intron; and (ii) a second rAAV comprising a capsid containing a second nucleic acid containing a promoter operably linked to a transgene encoding beta-hexosaminidase subunit beta (HexB) via a chimeric intron. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the first rAAV and the second rAAV are present in the composition in a 1:1 ratio.

[0015] In some aspects, the present disclosure presents a method for treating a lysosomal storage disorder, the method comprising administering to a subject having a lysosomal storage disorder (e.g., Tay-Sachs disease, GM2 gangliosidosis, Sandhoff disease, Morquio syndrome type B, etc.) a recombinant AAV (rAAV) or a pharmaceutical composition as described herein. In some embodiments, the lysosomal storage disorder is Tay-Sachs disease or Sandhoff disease. In some embodiments, the rAAV or the pharmaceutical composition is administered to the subject by intracranial injection, intracerebral injection, or injection into the CSF via the ventricular system, cisterna magna, or spinal canal. In some embodiments, the present disclosure presents a method for treating GM2 gangliosidosis, the method comprising administering to a subject having GM2 gangliosidosis an rAAV or a pharmaceutical composition as described herein.

[0016] In some embodiments, the present disclosure provides a container for storing a recombinant adeno-associated virus (rAAV) as described herein; or a kit comprising a composition comprising a recombinant adeno-associated virus (rAAV) as described herein. In some embodiments, the kit further comprises a container for storing a pharmaceutically acceptable carrier. In some embodiments, the rAAV or the composition comprising rAAV and the pharmaceutically acceptable carrier are stored in the same container. In some embodiments, the container is a syringe.

[0017] In some embodiments, the present disclosure provides a recombinant AAV vector comprising a nucleic acid encoding a promoter operably linked to a transgene encoding human GLB1 as set forth in SEQ ID NO: 23. In some embodiments, the present disclosure provides an isolated nucleic acid encoding a promoter operably linked to a transgene encoding human GLB1 as set forth in SEQ ID NO: 23. 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 comprising administering rAAV or the pharmaceutical composition to a subject having GM1 gangliosidosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018]

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[0019] In some cases, delivery of a transgene by current rAAV vectors causes accumulation of non-degradable substrates or dysregulation of the lysosomal compartment, which leads to responses that upregulate lysosomal biogenesis, substrate reduction, or exocytosis. Additionally, introduction of therapeutic proteins above physiological levels via expression of a transgene driven by an rAAV vector can also induce harmful protective cascades that may be associated with responses to unfolded proteins, which are common in these disorders.

[0020] Accordingly, in some aspects, the present disclosure presents compositions (e.g., isolated nucleic acids, rAAV, rAAV vectors, etc.) for the expression of lysosomal enzymes in tissues such as CNS tissue. In some aspects, the present disclosure relates to methods for treating lysosomal storage disorders such as GM1 gangliosidosis, Tay-Sachs disease, or Sandhoff disease using the rAAV described herein. The present disclosure is based in part on the discovery that certain regulatory sequences and regulatory elements, such as promoter regions, can be engineered in rAAV to provide transgene expression at levels that are therapeutically effective but do not cause vector-mediated genetic toxicity associated with previously used rAAV.

[0021] Isolated nucleic acid In some aspects, the present disclosure presents nucleic acids comprising at least one transgene operably linked to a promoter, wherein the transgene encodes a protein associated with a lysosomal storage disorder (e.g., a lysosomal storage protein such as HexA and / or HexB, and GLB1).

[0022] In some embodiments, the GLB1 transgene is the human GLB1 gene (GeneID: 2720). The human GLB1 gene may include the nucleotide sequence of NM_000404.4, NM_001079811.2, NM_001135602.2, or NM_001317040.1. The human GLB1 gene encodes a β-galactosidase protein. The human β-galactosidase protein may include the amino acid sequence within NP_000395.3, within NP_001073279.1, within NP_001129074.1, or within NP_001303969.1. In some embodiments, human GLB1 includes the sequence as set forth in SEQ ID NO: 23. In some embodiments, the human HexA transgene (GeneID: 3073) includes the nucleotide sequence within NM_000520.5 or within NM_001318825.1. In some embodiments, the HexA protein is represented by the sequence set forth in NCBI reference sequence number: NP_000511.2 (SEQ ID NO: 20) or NP_001305754.1. In some embodiments, the human HexB transgene (GeneID: 3074) includes the nucleotide sequence within NM_000521.4 or within NM_001292004.1. In some embodiments, the HexB protein is represented by the sequence set forth in NCBI reference sequence number: NP_000512.1 (SEQ ID NO: 21) or NP_001278933.1.

[0023] A transgene encoding a protein associated with lysosomal storage disorder can be operably linked to a promoter. As used herein, an "operable linker" refers to a promoter that is linked to a downstream transgene and promotes its expression. In some embodiments, the promoter is a constitutive promoter, such as the chicken beta-actin (CBA) promoter, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, for example, Boshart et al., Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the beta-actin promoter, the phosphoglycerate kinase (PGK) promoter, and the EF1α promoter [Invitrogen]. In some embodiments, the promoter is the enhanced chicken beta-actin promoter. In some embodiments, the promoter is the U6 promoter. In some embodiments, the chicken beta-actin promoter comprises the sequence set forth in SEQ ID NO: 22.

[0024] In some embodiments, the promoter is an inducible promoter. Inducible promoters allow for the regulation of gene expression and can be 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 a physiological state limited to replicating cells. Inducible promoters and induction systems are commercially available from a variety of vendors, including, without limitation, Invitrogen, Clontech, and Ariad. Many other systems have also been described and can be readily selected by those of skill in the art. Examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO98 / 10088); the ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline repression system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), the tetracycline induction system (Gossen et al., Science, 268:1766-1769 (1995); see also, Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1998)), the RU486 induction system (Wang et al., Nat. Biotech., 15:239-243 (1997); and Wang et al., Gene Ther., 4:432-441 (1997)), and the rapamycin induction system (Magari et al., J. Clin. Invest., 100:2865-2872 (1997)). Still other types of inducible promoters that may be useful in this context are inducible promoters regulated by specific physiological states, such as temperature, the presence of an acute phase, a cell-specific differentiation state, or a physiological state limited to replicating cells.

[0025] In another embodiment, a native promoter will be used for the transgene (e.g., GLB1, HEXA, or HEXB). The native promoter may be preferred when it is desired for the expression of the transgene to mimic native expression. The native promoter can be used when the expression of the transgene must be regulated transiently, developmentally, or tissue-specifically, or in response to specific transcriptional stimuli. In further embodiments, other native expression control elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, can also be used to mimic native expression.

[0026] In some embodiments, the promoter drives the expression of the transgene within neural tissue. In some embodiments, the disclosure presents a nucleic acid comprising a tissue-specific promoter operably linked to a transgene, wherein the transgene encodes a lysosomal storage disease protein. As used herein, a "tissue-specific promoter" refers to a promoter that preferentially regulates (e.g., drives or upregulates) gene expression within a particular cell type as compared to gene expression within other cell types. A cell-type specific promoter can be specific to any cell type, such as central nervous system (CNS) cells, liver cells (e.g., hepatocytes), heart cells, kidney cells, eye cells, muscle cells, etc. For example, the human synapsin 1 promoter (Syn1 promoter) preferentially drives gene expression within neurons, and the GfaABC1D (also referred to as GFAP) promoter preferentially drives expression within astrocytes. However, it should be understood that some cell types may be resident within a particular type of tissue. For example, central nervous system tissue includes nerve cells and non-nerve cells (e.g., glial cells, astrocytes, etc.).

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

[0028] Further examples of tissue-specific promoters include, but are not limited to, the liver-specific thyroxine-binding globulin (TBG) promoter, the insulin promoter, the glucagon promoter, the somatostatin promoter, the pancreatic polypeptide (PPY) promoter, the synapsin 1 (Syn1) promoter, the creatine kinase (MCK) promoter, the mammalian desmin (DES) promoter, the α-myosin heavy chain (a-MHC) promoter, or the cardiac troponin T (cTnT) promoter. Other exemplary promoters include, among other promoters apparent to those skilled in the art, the beta-actin promoter, the hepatitis B virus core promoter (Sandig et al., Gene Ther., 3:1002-9 (1996)); the alpha-fetoprotein (AFP) promoter (Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996)), the bone osteoclast promoter (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)); the bone sialoprotein promoter (Chen et al., J. Bone Miner. Res., 11:654-64 (1996)), the CD2 promoter (Hansal et al., J. Immunol., 161:1063-8 (1998)); the immunoglobulin heavy chain promoter; the T cell receptor alpha chain promoter, neuronal promoters such as the neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)), the neurofilament 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 one aspect, the present disclosure relates to an isolated nucleic acid comprising a transgene (e.g., a lysosomal storage protein such as GLB1, HexA, HexB, and / or HexA and HexB) operably linked to a promoter via a chimeric intron. In some embodiments, the chimeric intron comprises a nucleic acid sequence derived from the chicken beta-actin gene, e.g., a non-coding intron sequence derived from intron 1 of the chicken beta-actin gene. In some embodiments, the intron sequence of the chicken beta-actin gene ranges in length from about 50 to about 150 nucleotides (e.g., any length between 50 and 150 nucleotides, including the endpoints). In some embodiments, the intron sequence of the chicken beta-actin gene ranges in length from 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) nucleotides. In some embodiments, the chimeric intron is adjacent to one or more untranslated sequences (e.g., an untranslated sequence located between the promoter sequence and the chimeric intron sequence, and / or an untranslated sequence located between the chimeric intron and the first codon of the transgene sequence). In some embodiments, each of the one or more untranslated sequences is a non-coding sequence derived from the rabbit beta-globulin gene (e.g., an untranslated sequence derived from exon 1, exon 2, etc. of rabbit beta-globulin).

[0030] Recombinant AAV The isolated nucleic acids of the present disclosure can be recombinant adeno-associated virus (rAAV). In some embodiments, the isolated nucleic acids described by the present disclosure include a first adeno-associated virus (AAV) inverted terminal repeat (ITR), or a region containing a variant thereof (e.g., a first region), and a second region encoding a transgene associated with lysosomal storage disorder (e.g., GLB1, HEXA, and / or HEXB). The isolated nucleic acid (e.g., recombinant AAV vector) may be packaged into a capsid protein and administered to a subject and / or delivered to a selected target cell. The transgene may also include, as described elsewhere in the present disclosure, a region encoding, for example, a protein and / or an expression control sequence (e.g., polyA tail).

[0031] The present disclosure presents a vector containing a single, cis - acting wild - type ITR. In some embodiments, the ITR is a 5′ - side ITR. In some embodiments, the ITR is a 3′ - side ITR. Generally, the ITR sequence is about 145 bp in length. Preferably, substantially the entire sequence encoding the ITR(s) is used within the molecule, although some minor modifications to these sequences are also tolerated. The ability to modify the ITR sequence is within the scope of the art (see, for example, textbooks such as Sambrook et al, “Molecular Cloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520 - 532 (1996)). For example, the ITR can be mutated at its TR (terminal resolution) site, which inhibits replication at the vector terminus, resulting in the formation of self - complementary AAV. Another example of such a molecule utilized in the present disclosure is a “cis - acting” plasmid containing a transgene, where the selected transgene sequence and associated regulatory elements are flanked by a 5′ - side AAV ITR sequence and a 3′ - side hairpin - forming RNA sequence. The AAV ITR sequence can be obtained from any known AAV, including the mammalian AAV types identified herein. In some embodiments, the ITR sequence is the ITR sequence of AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, and / or AAVrh10.

[0032] The isolated nucleic acids and / or isolated rAAV of the present disclosure can be modified and / or selected to enhance the targeting of the isolated nucleic acids and / or isolated rAAV to a target tissue (e.g., the CNS). Non-limiting methods of modification and / or selection include AAV capsid serotypes (e.g., AAV8, AAV9), tissue-specific promoters (e.g., Syn1, GFAP), and / or targeting peptides. In some embodiments, the isolated nucleic acids and rAAV of the present disclosure include AAV capsid serotypes with enhanced targeting to CNS tissue (e.g., AAV8, AAV9). In some embodiments, the isolated nucleic acids and rAAV of the present disclosure include a tissue-specific promoter (e.g., Syn1, GFAP). In some embodiments, the isolated nucleic acids and rAAV of the present disclosure include AAV capsid serotypes with enhanced targeting to CNS tissue and tissue-specific promoters.

[0033] In some aspects, the present disclosure presents an isolated AAV. With respect to AAV, as used herein, the term "isolated" refers to an AAV that is obtained or produced artificially. Isolated AAV can be produced using recombinant methods. As used herein, such AAV is referred to as "recombinant AAV" (rAAV). Recombinant AAV (rAAV) preferably has tissue-specific targeting ability such that the transgene of the rAAV is specifically delivered to one or more predetermined tissues (s). The AAV capsid is an important element in determining these tissue-specific targeting abilities. Thus, an rAAV having an appropriate capsid for the tissue to be targeted can be selected. In some embodiments, the rAAV includes the capsid protein of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, or AAVrh10, or a protein having substantial homology thereto. In some embodiments, the rAAV includes the AAVrh8 capsid protein.

[0034] In some embodiments, the rAAV of the present disclosure is a pseudotyped rAAV. Pseudotyping is the process of creating a virus or viral vector by combining it with a foreign viral envelope protein. The result is a pseudotyped virus particle. By this method, the foreign viral envelope protein can be used to alter host tropism or increase / decrease the stability of the virus particle. In some aspects, the pseudotyped rAAV contains nucleic acids derived from two or more different AAVs, in which case the nucleic acid derived from one AAV encodes a capsid protein and the nucleic acid of at least one other AAV encodes other viral proteins and / or a viral genome. In some embodiments, a pseudotyped rAAV refers to an AAV that contains the inverted terminal repeat sequence (ITR) of one AAV serotype and a capsid protein of a different AAV serotype. For example, a pseudotyped AAV vector containing the ITR of serotype X and capsid-encapsulated with the protein of serotype Y would be designated AAVX / Y (e.g., AAV2 / 1 has the ITR of AAV2 and the capsid of AAV1). In some embodiments, pseudotyped rAAV combines the tissue-specific targeting ability of a capsid protein derived from one AAV serotype with viral DNA derived from another AAV serotype, which may be useful to enable targeted delivery of a transgene to a target tissue.

[0035] In the art, methods for obtaining recombinant AAVs having a desired capsid protein are well known (see, e.g., US Patent Application Publication No. 2003 / 0138772, the contents of which are incorporated herein by reference in their entirety). Typically, the method comprises culturing a recombinant AAV vector composed of a nucleic acid sequence encoding an AAV capsid protein or a fragment thereof; a functional rep gene; AAV inverted terminal repeats (ITRs), and a transgene; and a host cell containing helper functions sufficient to enable packaging of the recombinant AAV vector into the AAV capsid protein. Typically, the capsid protein is a structural protein encoded by the cap gene of AAV. In some embodiments, AAV comprises virion proteins 1-3 (designated VP1, VP2, and VP3), all of which are three capsid proteins transcribed from a single cap gene via alternative splicing. In some embodiments, the molecular weights of VP1, VP2, and VP3 are approximately 87 kDa, approximately 72 kDa, and approximately 62 kDa, respectively. In some embodiments, upon translation, the capsid protein forms a spherical 60-mer protein shell around the viral genome. In some embodiments, the capsid protein protects the viral genome, delivers the genome, and / or interacts with the host cell. In some aspects, the capsid protein delivers the viral genome to the host in a tissue-specific manner.

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

[0037] In some embodiments, for packaging an rAAV vector within an AAV capsid, the components that are cultured within a host cell can be provided to the host cell in trans. Alternatively, one or more of the required components (e.g., a recombinant AAV vector, a rep sequence, a cap sequence, and / or a helper function) can also be provided by a stable host cell engineered to contain one or more of the required components using methods known to those of skill in the art. Such stable host cells would most appropriately contain the required component(s) under the control of an inducible promoter. However, the required component(s) may be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are presented in the discussion of regulatory elements suitable for use with a transgene herein. In yet another alternative, the selected stable host cell can contain a selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters. For example, stable host cells can be generated that are derived from 293 cells (containing an E1 helper function under the control of a constitutive promoter) but contain a rep protein and / or a cap protein under the control of an inducible promoter. Other stable host cells can also be generated by those of skill in the art.

[0038] In some embodiments, the disclosure relates to a host cell containing a nucleic acid comprising a coding sequence selected from the group consisting of SEQ ID NOs: 1-6 operably linked to a promoter. In some embodiments, the disclosure relates to a composition comprising the host cell described above. In some embodiments, the composition comprising the host cell further comprises a cryoprotectant.

[0039] The recombinant AAV vectors, rep sequences, cap sequences, and helper functions useful for generating the rAAV of the present disclosure can be delivered into packaging host cells using the elements (vectors) of any suitable gene. The selected gene elements can be delivered by any suitable method, including the methods described herein. The methods used to construct any embodiment of the present disclosure are known to those of ordinary skill in the art of nucleic acid manipulation and include genetic engineering methods, recombinant engineering methods, and synthetic methods. See, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Similarly, methods for generating rAAV virions are also well known, and the selection of a suitable method is not a limitation to the present disclosure. See, for example, K. Fisher et al, J. Virol., 70:520-532 (1993) and U.S. Patent No. 5,478,745.

[0040] In some embodiments, recombinant AAV can be produced using the triple transfection method (described in detail in U.S. Patent No. 6,001,650). Typically, recombinant AAV is produced by transfecting a host cell with a recombinant AAV vector (containing a transgene) that is packaged into AAV particles, an AAV helper function vector, and an accessory function vector. The AAV helper function vector encodes "AAV helper function" sequences (i.e., rep and cap) that function in trans for the replication and capsid encapsidation of productive AAV. Preferably, the AAV helper function vector supports the efficient production of AAV vectors without producing detectable wild-type AAV virions (i.e., AAV virions containing functional rep and cap genes). Non-limiting examples of vectors suitable for use according to the present disclosure include pHLP19, described in U.S. Patent No. 6,001,650, and the pRep6cap6 vector, described in U.S. Patent No. 6,156,303, each of which is incorporated herein by reference in its entirety. The accessory function vector encodes nucleotide sequences for viral and / or cellular functions that AAV depends on for replication (i.e., "accessory functions"). Accessory functions are functions required for the replication of AAV and include, without limitation, functions involved in the activation of AAV gene transcription, the stage-specific splicing of AAV mRNA, the replication of AAV DNA, the synthesis of cap expression products, and the assembly of the AAV capsid. Viral-based accessory functions can be derived from any of the known helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type 1), and vaccinia virus.

[0041] In some embodiments, the present disclosure presents transfected host cells. The term "transfection" is used to refer to the uptake of foreign DNA by a cell, and a cell is "transfected" when exogenous DNA has been introduced through the cell membrane. In the art generally, a number of transfection techniques are known. See, for example, Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous nucleic acids, such as nucleotide integration vectors, and other nucleic acid molecules, into a suitable host cell.

[0042] "Host cell" refers to any cell that is capable of harboring or possessing a substance of interest. Host cells are often mammalian cells. Host cells can be used as recipients of AAV helper constructs, AAV mini gene plasmids, accessory function vectors, or other introduced DNA related to the production of recombinant AAV. The term includes progeny cells of the original cell that have been transfected. Thus, "host cell" as used herein may refer to a cell that has been transfected with an exogenous DNA sequence. It is understood that progeny cells of a single parental cell may not necessarily be identical in form or genomic or total DNA complement to the original parent due to natural, accidental, or intentional mutations.

[0043] As used herein, the term "cell line" refers to a population of cells capable of continued or extended growth and division in vitro. A cell line is often a clonal population derived from a single progenitor cell. It is also known in the art that spontaneous or induced changes can occur in the karyotype upon accumulation or introduction of such clonal populations. Thus, cells derived from a cell line referred to may not be exactly identical to the progenitor or cultured cells, and the cell line referred to includes such variants.

[0044] As used herein, the term "recombinant cell" refers to a cell into which an exogenous DNA segment, such as a DNA segment, has been introduced, resulting in the transcription of a biologically active polypeptide or the production of a biologically active nucleic acid, such as RNA.

[0045] As used herein, the term "vector" includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., that is capable of replication when associated with appropriate regulatory elements and can introduce a gene sequence between cells. Thus, the term includes viral vectors in addition to cloning and expression media. In some embodiments, a useful vector is envisioned to be a vector in which the nucleic acid segment to be transcribed is placed under the transcriptional control of a promoter. A "promoter" refers to a DNA sequence that is recognized by the synthetic machinery of a cell or an introduced synthetic machinery and is required to induce specific transcription of a gene. The phrases "operatively disposed," "under control," or "under transcriptional control" mean that the promoter is in the correct position and orientation in relation to a nucleic acid that controls the induction of RNA polymerase and the expression of a gene. The term "expression vector or expression construct" means any type of genetic construct containing a nucleic acid in which some or all of the nucleic acid coding sequences are transcribable. In some embodiments, expression includes transcription of a nucleic acid that results in, for example, a biologically active polypeptide product or an inhibitory RNA (e.g., shRNA, miRNA, miRNA inhibitor) from a transcribed gene.

[0046] The foregoing methods for packaging a recombinant vector into a desired AAV capsid to generate the rAAV of the present disclosure are not intended to be limiting, and other suitable methods will be apparent to those skilled in the art.

[0047] Recombinant AAV vector The isolated nucleic acids of the present disclosure can be recombinant AAV (rAAV) vectors. The "recombinant AAV (rAAV) vector" of the present disclosure typically consists minimally of a transgene and its regulatory sequences, as well as AAV inverted terminal repeat sequences (ITRs) on the 5' and 3' sides. It is this recombinant AAV vector that is packaged into capsid proteins and delivered to selected target cells. In some embodiments, the transgene is heterologous to the vector sequence and is a nucleic acid sequence encoding a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor), or other gene product of interest. The nucleic acid encoding the sequence is operably linked to regulatory components in a manner that enables transcription, translation, and / or expression of the transgene within the cells of the target tissue.

[0048] Aspects of the present disclosure relate to the discovery that modification of the regulatory sequences of rAAV results in transgene expression at levels that are therapeutically effective but do not cause vector-mediated toxicity associated with conventionally used rAAV. Accordingly, in some embodiments, the present disclosure relates to recombinant AAV (rAAV) comprising a modified gene regulatory element. In some embodiments, the modified gene regulatory element is a hybrid promoter.

[0049] As used herein, the term "hybrid promoter" refers to a regulatory construct capable of driving the transcription of an RNA transcript (e.g., a transcript including a transcript encoded by a transgene), the regulatory construct being artificially arranged and including two or more regulatory elements. Typically, a hybrid promoter includes at least one element that is a minimal promoter, and at least one element having an enhancer sequence, or an intron sequence, exon sequence, or UTR sequence including one or more transcriptional regulatory elements. In embodiments where the hybrid promoter includes an exon sequence, intron sequence, or UTR sequence, such sequence(s) can encode the upstream portion of an RNA transcript (e.g., as depicted in FIG. 1), and also contains regulatory elements that modulate (e.g., enhance) the transcription of the transcript. In some embodiments, two or more elements of the hybrid promoter are from heterologous sources compared to each other. In some embodiments, two or more elements of the hybrid promoter are from heterologous sources compared to the transgene. In some embodiments, two or more elements of the hybrid promoter are from different loci. In some embodiments, two or more elements of the hybrid promoter are from the same locus but are arranged in a form not found at the locus. In some embodiments, the hybrid promoter includes a first nucleic acid sequence derived from one promoter that is fused to one or more nucleic acid sequences including promoter elements or enhancer elements from different sources. In some embodiments, the hybrid promoter includes a first sequence derived from the chicken beta-actin promoter and a second sequence by the CMV enhancer. In some embodiments, the hybrid promoter includes a first sequence derived from the chicken beta-actin promoter and a second sequence derived from the intron of the chicken beta-actin gene.In some embodiments, the hybrid promoter comprises a first sequence derived from the chicken beta-actin promoter fused to a CMV enhancer sequence and a sequence derived from the intron of the chicken beta-actin gene.

[0050] In some aspects, the rAAV comprises an enhancer element. As used herein, the term "enhancer element" refers to a nucleic acid sequence that, when bound by an activator protein, activates one or more genes or increases their transcription. Enhancer sequences can be upstream (i.e., 5' side) or downstream (i.e., 3' side) of the gene they regulate. Examples of enhancer sequences include the cytomegalovirus (CMV) enhancer sequence and the simian vacuolating virus 40 (SV40) enhancer sequence. In some embodiments, the rAAV comprises a CMV enhancer element or a portion thereof. As used herein, the term "portion thereof" refers to a fragment of a nucleotide sequence or amino acid sequence that retains the desired functional characteristics of the full nucleotide sequence or full amino acid sequence from which it is derived. For example, "CMV enhancer sequence or a portion thereof" refers to a nucleotide sequence derived from the wild-type CMV enhancer that is capable of increasing the transcription of a transgene.

[0051] In some aspects, the rAAV comprises a post-transcriptional response element. As used herein, the term "post-transcriptional response element" refers to a nucleic acid sequence that, when transcribed, adopts a tertiary structure that enhances gene expression. Examples of post-transcriptional regulatory elements include, but are not limited to, the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), the mouse RNA transport element (RTE), the constitutive transport element (CTE) of type 1 simian retrovirus (SRV-1), the CTE derived from Mason-Pfizer monkey virus (MPMV), and the 5' untranslated region of human heat shock protein 70 (Hsp70 5'UTR). In some embodiments, the rAAV vector comprises the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0052] In some embodiments, the present disclosure presents an rAAV vector comprising a hybrid intron or a chimeric intron. As used herein, the term "chimeric intron" refers to an intron having sequences derived from two or more different sources. In some embodiments, the chimeric intron comprises a nucleic acid encoding a splice donor site derived from a first source (e.g., an organism or species) and a splice acceptor site derived from a second source (e.g., an organism or species). In some embodiments, the chimeric intron comprises one or more transcriptional regulatory elements and / or enhancer sequences. In some embodiments, the chimeric intron is positioned between an exon of a hybrid promoter and an exon of a transgene. In some embodiments, the present disclosure presents an rAAV comprising a promoter operably linked to a transgene, wherein the transgene encodes a lysosomal storage protein and further comprises a chimeric intron.

[0053] In certain embodiments, the present disclosure relates to an rAAV vector comprising an artificial transcription element. In some embodiments, as used herein, the term "artificial transcription element" refers to a synthetic sequence that enables the transcription of DNA by RNA polymerase, which is controlled to produce an RNA transcript. The transcriptionally active elements of the present disclosure are generally less than 500 bp, preferably less than 200 bp, more preferably less than 100 bp, and most preferably less than 50 bp. In some embodiments, the artificial transcription element comprises two or more nucleic acid sequences derived from a transcriptionally active element. Transcriptionally active elements are generally recognized in the art and include, for example, promoters, enhancer sequences, TATA boxes, G / C boxes, CCAAT boxes, specificity protein 1 (Sp1) binding sites, Inr regions, CRE (cAMP regulatory element), transcription activator 1 (ATF1) binding sites, ATF1-CRE binding sites, APBβ boxes, APBα boxes, CArG boxes, CCAC boxes, and transcriptionally active elements disclosed by U.S. Patent No. 6,346,415. Combinations of the foregoing transcriptionally active elements are also envisioned.

[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 C box. 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, and a TATA box. In some embodiments, the artificial transcription element is represented by SEQ ID NO: 2.

[0055] Expression control sequences include appropriate transcription start sequences, transcription termination sequences, promoter sequences, and enhancer sequences; efficient RNA processing signals such as splicing signals and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, if desired, sequences that enhance the secretion of the encoded product. In the art, numerous expression control sequences, including natural promoters, constitutive promoters, inducible promoters, and / or tissue-specific promoters, are known and can be utilized.

[0056] As used herein, a nucleic acid sequence (e.g., a coding sequence) and a regulatory sequence are said to be "operably" linked when they are covalently linked such that the expression or transcription of the nucleic acid sequence is placed under the influence or control of the regulatory sequence. When it is desired that the nucleic acid sequence be translated into a functional protein, two DNA sequences are operably linked if induction of the promoter within the 5'-regulatory sequence results in transcription of the coding sequence and the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frameshift mutation, (2) interfere with the ability of the promoter region to direct transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region is operably linked to a nucleic acid sequence if the promoter region is capable of effecting transcription of this DNA sequence such that the resulting transcript can be translated into the desired protein or polypeptide. Similarly, two or more coding regions are operably linked if they are linked such that their transcription from a common promoter results in the expression of two or more proteins that are translated in-frame. In some embodiments, the operably linked coding sequences result in a fusion protein. In some embodiments, the operably linked coding sequences result in a functional RNA (e.g., shRNA, miRNA, miRNA inhibitor).

[0057] For nucleic acids encoding proteins, generally, a polyadenylation sequence is inserted after the transgene sequence and before the 3' AAV ITR sequence. rAAV constructs useful in the present disclosure may also contain an intron, which is desirably placed between the promoter / enhancer sequence and the transgene. One possible intron sequence is derived from SV-40 and is referred to as the SV-40 T intron sequence.

[0058] Another vector element that can be used is an internal ribosome entry site (IRES). IRES sequences are used to produce more than one polypeptide from a single gene transcript. IRES sequences will be used to produce proteins containing more than one polypeptide chain. The selection of these common vector elements, as well as other common vector elements, is routine and many such sequences are available. [See, for example, Sambrook et al, and the references cited therein, for example, on pages 3.183.26 and 16.1716.27; and also see Ausubel et al., Current 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; this is a small peptide (about 18 amino acids in length) that has been shown to mediate cleavage of the polyprotein (Ryan, M D et al., EMBO, 1994; 4: 928-933; Mattion, N M 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 Journal, 1999; 4: 453-459).The cleavage activity of the 2A sequence has previously been demonstrated in artificial systems, including plasmids and gene therapy vectors (AAV and retroviruses) (Ryan, M D et al., EMBO, 1994; 4: 928-933; Mattion, N M 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 Journal, 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 exact nature of the regulatory sequences required for gene expression in a host cell can vary between species, tissues, or cell types, but generally, as needed, will include 5' non-transcribed and 5' untranslated sequences involved in the initiation of transcription and translation, such as a TATA box, capping sequence, CAAT sequence, enhancer element, etc., respectively. In particular, such 5' non-transcribed regulatory sequences will include a promoter region that includes a promoter sequence for transcriptional control of an operably linked gene. The regulatory sequences may also optionally include enhancer sequences, or upstream activator sequences. The vectors of the present disclosure may optionally include a 5' leader sequence or a 5' signal sequence.

[0060] Examples of constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al., Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerate kinase (PGK) promoter, and the EF1α promoter [Invitrogen].

[0061] Inducible promoters enable the regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or specific physiological states, such as the presence of an acute phase, the differentiation state specific to a cell, or a physiological state limited to replicating cells. Inducible promoters and induction systems are commercially available from various vendors, including, without limitation, Invitrogen, Clontech, and Ariad. Many other systems have also been described and can be readily selected by those skilled in the art. Examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO98 / 10088); the ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline repression system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), the tetracycline induction system (Gossen et al., Science, 268:1766-1769 (1995); see also, Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1998)), the RU486 induction system (Wang et al., Nat. Biotech., 15:239-243 (1997); and Wang et al., Gene Ther., 4:432-441 (1997)), and the rapamycin induction system (Magari et al., J. Clin. Invest., 100:2865-2872 (1997)). Still other types of inducible promoters that may be useful in this context are inducible promoters regulated by specific physiological states, such as temperature, the presence of an acute phase, the differentiation state specific to a cell, or a physiological state limited to replicating cells.

[0062] The AAV sequences of the vector typically include cis - acting inverted terminal repeat sequences on the 5' and 3' sides (see, e.g., B. J. Carter, in “Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155 - 168 (1990)). The ITR sequences are approximately 145 bp in length. Preferably, substantially the entire sequences encoding the ITR(s) are used within the molecule, although some minor modifications to these sequences are also tolerated. The ability to modify these ITR sequences is within the scope of the art (see, e.g., textbooks such as Sambrook et al, “Molecular Cloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520 - 532 (1996)). An example of such a molecule utilized in the present disclosure is a “cis - acting” plasmid containing a transgene, in which case the selected transgene sequence and associated regulatory elements are flanked by a 5' AAV ITR sequence and a 3' AAV ITR sequence. The AAV ITR sequences can be obtained from any known AAV, including the mammalian AAV types identified herein.

[0063] In some embodiments, the rAAV of the present disclosure is a pseudotyped rAAV. For example, a pseudotyped AAV vector containing the ITR of serotype X and capsid - encapsulated with the protein of serotype Y would be designated AAVX / Y (e.g., AAV2 / 1 has the ITR of AAV2 and the capsid of AAV1). In some embodiments, pseudotyped rAAV combines the tissue - specific targeting ability of capsid proteins from one AAV serotype with viral DNA from another AAV serotype, and thus can be useful to enable targeted delivery of a transgene to a target tissue.

[0064] In addition to the major elements identified above for the recombinant AAV vector, the vector also includes the necessary conventional control elements operably linked to the transgene in a form that allows its transcription, translation, and / or expression within cells transfected with the plasmid vector produced by the present disclosure or within cells infected with the virus produced by the present disclosure. As used herein, an "operably linked" sequence includes both an expression control sequence adjacent to the gene of interest and an expression control sequence that acts to control the gene of interest, either in trans or at a distance.

[0065] Recombinant AAV vector: Transgene coding sequence The composition of the transgene sequence of the rAAV vector will depend on the use to which the resulting vector is put. For example, one type of transgene sequence includes a reporter sequence that, when expressed, provides a detectable signal. In another example, the transgene encodes a therapeutic protein or a therapeutic functional RNA. In another example, the transgene is intended for use for research purposes, such as creating a somatic transgenic animal model carrying the transgene and studying the function of the transgene product, for example, by encoding a protein or a functional RNA. In another example, the transgene is intended to be used to create an animal model for a disease, by encoding a protein or a functional RNA. Appropriate transgene coding sequences will be apparent to those skilled in the art.

[0066] In one aspect, the present disclosure relates to rAAV vectors useful for the treatment of lysosomal storage disorders. Lysosomal storage disorders (also referred to as lysosomal storage diseases) are a group of hereditary metabolic disorders resulting from lysosomal dysfunction. Generally, lysosomal storage diseases are characterized by a functional impairment of a single protein (e.g., an enzyme) involved in lysosomal metabolism. For example, Tay-Sachs disease is caused by a genetic mutation in the hexosaminidase A (HEXA) gene, resulting in a state where the HEXA enzyme cannot hydrolyze GM2 ganglioside.Other examples of lysosomal storage diseases, and their related proteins, include, but are not limited to, aspartylglucosaminuria (aspartylglucosaminidase), infantile Batten disease (palmitoylprotein thioesterase), late infantile Batten disease (tripeptidyl peptidase), Fabry disease (α-galactosidase), fucosidosis (α-fucosidase), galactosialidosis (protective protein / cathepsin A), Gaucher disease (β-glucosidase), galactosialidosis (protective protein / cathepsin A), globoid cell leukodystrophy (galactosylceramidase), GM1 gangliosidosis (β-galactosidase), α-mannosidosis (α-mannosidase), metachromatic leukodystrophy (arylsulfatase A), mucopolysaccharidosis I (α-L-iduronidase), mucopolysaccharidosis II (iduronate sulfatase), mucopolysaccharidosis IIIA (heparan sulfatase), mucopolysaccharidosis IIIB (α-N-acetylglucosaminidase), mucopolysaccharidosis IIIC (acetyl-CoA α-glucosaminide acetyltransferase), mucopolysaccharidosis IIID (N-acetylglucosamine-6-sulfate sulfatase), mucopolysaccharidosis IVA (N-acetylgalactosamine 6-sulfatase), mucopolysaccharidosis IVB (β-galactosidase), mucopolysaccharidosis IX (hyaluronidase), mucopolysaccharidosis VI (arylsulfatase B), mucopolysaccharidosis VII (β-glucuronidase), type I mucolipidosis (α-neuraminidase), type II mucolipidosis (GlcNAc-1-phosphotransferase), type III mucolipidosis (N-acetylglucosamine-1-phosphotransferase), Niemann-Pick disease (acid sphingomyelinase), Pompe disease (α-glucosidase), Sandhoff disease (β-hexosaminidase A and B), Schindler disease (α-N-acetylgalactosaminidase), Tay-Sachs disease (β-hexosaminidase A / B), and Wolman disease (acid lipase).

[0067] In some embodiments, the present disclosure presents an rAAV comprising a transgene encoding a lysosomal storage disease-related protein. In some embodiments, the lysosomal storage disease-related protein is selected from the group consisting of HEXA, HEXB, and GLB1. In some embodiments, the lysosomal storage disease-related protein is HEXA and comprises the sequence set forth in SEQ ID NO: 20. In some embodiments, the lysosomal storage disease-related protein is HEXB and comprises the sequence set forth in SEQ ID NO: 21. In some embodiments, the lysosomal storage disease-related protein is GLB1 and comprises the sequence set forth in SEQ ID NO: 23.

[0068] Also contemplated herein are methods for treating lysosomal storage diseases by delivering a transgene to a subject using the rAAV described herein. In some embodiments, the present disclosure relates to a method for treating a lysosomal storage disease, the method comprising administering rAAV to a subject. In some embodiments, the rAAV comprises a hybrid promoter. In some embodiments, the rAAV comprises a chimeric intron. In some embodiments, the rAAV comprises an artificial transcription element. In some embodiments, the artificial transcription element comprises an ATF1-CRE binding site, an SP1 binding site, and a TATA box. In some embodiments, the promoter, chimeric intron, or artificial transcription 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 is selected from the group consisting of HEXA, HEXB, and GLB1. In some embodiments, the lysosomal storage disease is GM2 gangliosidosis (Sandhoff disease and Tay-Sachs disease), and the rAAV comprises transgenes encoding HEXA and HEXB. In some embodiments, the lysosomal storage disease is GM1 gangliosidosis, and the transgene encodes GLB1.

[0069] In some embodiments, the disclosure provides an rAAV capsid containing a nucleic acid having a first promoter specific for a first cell type operably linked to a first transgene and a second promoter specific for a second cell type linked to a second transgene, wherein the first promoter and the second promoter are not specific for the same cell type. Without wishing to be bound by any particular theory, in some embodiments, such rAAV vectors are useful for treating diseases that affect multiple cell types within a given tissue (e.g., CNS tissue).

[0070] Accordingly, in some embodiments, the disclosure relates to rAAV vectors useful for treating CNS-related diseases. As used herein, "CNS-related disease" refers to a disease or condition of the central nervous system. CNS-related disorders can affect the spinal cord (e.g., myelopathy), the brain (e.g., encephalopathy), or the tissues surrounding the brain and spinal cord. CNS-related disorders can be genetic disorders, either inherited or acquired through somatic mutations. CNS-related disorders can be psychological states or psychological disorders, such as attention deficit hyperactivity disorder, autism spectrum disorder, mood disorders, schizophrenia, depression, Rett syndrome, etc. CNS-related disorders can be autoimmune disorders. CNS-related disorders can also be cancers of the CNS, such as brain cancer. CNS-related disorders that are cancers can be primary cancers of the CNS, such as astrocytomas, glioblastomas, etc., or cancers that have metastasized to CNS tissue, such as lung cancer that has metastasized to the brain. Further non-limiting examples of CNS-related disorders include Huntington's disease, Parkinson's disease, 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 Friedreich's ataxia.

[0071] In some embodiments, the rAAV vectors described by the present disclosure contain a transgene encoding a CNS disease-related gene. In some embodiments, the transgene encoding a CNS disease-related gene encodes a protein or interfering RNA. Examples of interfering RNAs include, but are not limited to, dsRNA, siRNA, shRNA, miRNA, and artificial miRNA (amiRNA). Examples of CNS disease-related genes include DRD2, GRIA1, GRIA2, GRIN1, SLC1A1, SYP, SYT1, CHRNA7, 3R tau / 4rTUS, APP, BAX, BCL-2, GRIK1, GFAP, IL-1, AGER, which are associated with Alzheimer's disease; UCH-L1, SKP1, EGLN1, Nurr-1, BDNF, TrkB, gstm1, S106β, which are associated with Parkinson's disease; huntingtin (Htt), IT15, PRNP, JPH3, TBP, ATXN1, ATXN2, ATXN3, atrophin 1, FTL, TITF-1, Xbp1s, CRAG, which are associated with Huntington's disease; FXN, which is associated with Friedreich's ataxia; ASPA, which is associated with Canavan disease; DMD, which is associated with muscular dystrophy; SMN1, UBE1, DYNC1H1, which are associated with spinal muscular atrophy; ALS2, ANG, ATXN2, C9orf72, DCTN1, FIG4, FUS, NEFH, OPTN, PFN1, PRPH, SETX, SIGMAR1, SMN1, SOD1, SPG11, TARDBP, UBQLN2, VAPB, VCP, which are associated with amyotrophic lateral sclerosis (ALS); MAN2B1, MAN2B2, MAN2C1, which are associated with alpha-mannosidosis; AGA, which is associated with aspartylglucosaminuria; CLN1, CLN2, CLN3, CLN5, CLN6, MFSD8, CLN8, CTSD, which are associated with Batten disease; MANBA, which is associated with beta-mannosidosis; CTNS, which is associated with cystinosis; LAMP2, which is associated with Danon disease; GLA, which is associated with Fabry disease; ASAH1, which is associated with Farber disease; FUCA1, which is associated with fucosidosis; CTSA, which is associated with galactosialidosis; GBA, which is associated with Gaucher disease; GALC, which is associated with Krabbe disease; ARSA, which is associated with metachromatic leukodystrophy;Associated with mucopolysaccharidoses (such as Hurler syndrome, Hunter syndrome, Sanfilippo syndromes A - D, Morquio syndrome, hyaluronidase deficiency, Maroteaux - Lamy syndrome, Sly syndrome, sialidosis, I - cell disease, mucolipidoses I - IV, multiple sulfatase deficiency, Niemann - Pick diseases A - C, Pompe disease, pycnodysostosis, Sandhoff disease, Schindler disease, Tay - Sachs disease, Wolman disease), including but not limited to IDUA, IDS, SGSH, NAGLU, HGSNAT, GNS, GALNS, ARSB, GUSB, HYAL1, SMPD1, NPC1, NPC2, GAA, NAGA, SLC17A5, and LAL (LIPA).;

[0072] Useful transgene products also include miRNAs. miRNAs and other small interfering nucleic acids regulate gene expression through cleavage / degradation of target RNA transcripts or translational repression of target messenger RNA (mRNA). miRNAs are naturally typically expressed as final, 19 - 25 non - translated RNA products. miRNAs exert their activity through sequence - specific interactions with the 3' - untranslated region (UTR) of the target mRNA. These endogenously expressed miRNAs form hairpin precursors, which are then processed into miRNA duplexes and further processed into'mature' single - stranded miRNA molecules. This mature miRNA induces miRISC, a multi - protein complex that identifies target sites within, for example, the 3'UTR region of the target mRNA, based on their complementarity to the mature miRNA. In some embodiments, the inhibitory RNA is an miRNA. In some embodiments, the rAAV described by the present disclosure contains a transgene encoding an inhibitory RNA that targets human SOD1 (e.g., SOD1 G93A ) and includes SEQ ID NO: 15.

[0073] Methods of administering recombinant AAV rAAV can be delivered to a subject by a composition according to any suitable method known in the art. Preferably, rAAV suspended in a physiologically compatible carrier (i.e., a composition) can be administered to a subject, i.e., a host animal such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or non-human primate (e.g., cynomolgus monkey). In some embodiments, the host animal does not include humans.

[0074] Delivery of rAAV to a mammalian subject can be, for example, by intramuscular injection or by administration to the bloodstream of the mammalian subject. Administration to the bloodstream can be by injection into a vein, artery, or any other blood vessel. In some embodiments, rAAV is administered to the bloodstream by isolated limb perfusion, a technique well known in the field of surgical techniques that essentially enables one of ordinary skill in the art to separate the affected limb from the systemic circulation prior to administration of the rAAV virions. Variations of the isolated limb perfusion method described in U.S. Patent No. 6,177,403 can also be utilized by one of ordinary skill in the art to administer virions to the vasculature of the isolated affected limb to potentially enhance transduction of muscle cells or muscle tissue. Additionally, in certain cases, it may be desirable to deliver virions to the CNS of the subject. "CNS" means all cells and tissues of the brain and spinal cord of a vertebrate. Thus, the term includes, but is not limited to, neurons, glial cells, astrocytes, cerebrospinal fluid (CSF), interstitial spaces, bone, cartilage, and the like. Recombinant AAV can be directly delivered to the CNS or brain, for example, to ventricular regions, the striatum (e.g., the caudate or putamen of the striatum), the spinal cord, and the neuromuscular junction, or cerebellar lobules, using neurosurgical methods known in the art such as stereotactic injection (see, e.g., Stein et al., J Virol 73:3424-3429, 1999; Davidson et al., PNAS 97:3428-3432, 2000; Davidson et al., Nat. Genet. 3:219-223, 1993; and Alisky and Davidson, Hum. Gene Ther. 11:2315-2329, 2000) with a syringe needle, catheter, or device attached thereto.

[0075] In some embodiments, the rAAV of the present disclosure is administered into the central nervous system (CNS) by lumbar intrathecal injection (LIT). In some embodiments, the rAAV of the present disclosure is directly administered into the thalamus by injection (e.g., intracerebral injection). In some embodiments, two intracerebral injections of rAAV are performed bilaterally. In some embodiments, the rAAV of the present disclosure is directly administered into the deep cerebellar nuclei (DCN) by injection. In some embodiments, the rAAV of the present disclosure is administered bilaterally by injection into both the thalamus and DCN. In some embodiments, the rAAV of the present disclosure is directly administered by intracerebroventricular (ICV) injection. In some embodiments, the rAAV of the present disclosure is administered bilaterally by injection into both the thalamus and ICV. In some embodiments, the rAAV of the present disclosure is directly administered by injection into both DCN and ICV.

[0076] In some embodiments, the rAAV described in the present disclosure is administered by intravenous injection. In some embodiments, rAAV is administered by intracerebral injection. In some embodiments, rAAV is administered by intrathecal injection. In some embodiments, rAAV is delivered by intracranial injection. In some embodiments, rAAV is delivered by cisternal injection. In some embodiments, rAAV is delivered by lateral ventriculostomy injection. In some embodiments, 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 administered into both the cistern and the spinal canal (e.g., the spinal canal). In some embodiments, the rAAV of the present disclosure is administered into the cistern, the spinal canal (e.g., the spinal canal), and the thalamus (e.g., by intracerebral injection).

[0077] Aspects of the present disclosure relate to a composition comprising a recombinant AAV comprising at least one modified gene regulatory sequence or modified gene regulatory element. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0078] The compositions of the present disclosure can include rAAV alone or rAAV in combination with one or more other viruses (e.g., a second rAAV having one or more different transgenes). In some embodiments, the composition includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different rAAVs, each having one or more different transgenes.

[0079] In some aspects, the present disclosure relates to a composition (e.g., a pharmaceutical composition) comprising an rAAV comprising a nucleic acid encoding the HexA gene. In some aspects, the present disclosure relates to a composition (e.g., a pharmaceutical composition) comprising an rAAV comprising a nucleic acid encoding the HexB gene. In some aspects, the present disclosure relates to a composition (e.g., a pharmaceutical composition) comprising a first rAAV comprising a nucleic acid encoding the HexA gene and a second rAAV comprising a nucleic acid encoding the HexB gene.

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

[0081] In some embodiments, the ratio of rAAV encoding HexB:rAAV encoding HexA is a ratio of 1:2. In some embodiments, the ratio of rAAV encoding HexB:rAAV encoding HexA is a ratio of 1:5. In some embodiments, the ratio of rAAV encoding HexB:rAAV encoding HexA is a ratio of 1:10. In some embodiments, the ratio of rAAV encoding HexB:rAAV encoding HexA is a ratio of 2:1. In some embodiments, the ratio of rAAV encoding HexB:rAAV encoding HexA is a ratio of 5:1. In some embodiments, the ratio of rAAV encoding HexB:rAAV encoding HexA is a ratio of 10:1.

[0082] A suitable carrier can be readily selected by one of ordinary skill in the art, bearing in mind the adaptation to which the rAAV is directed. For example, one suitable carrier includes a physiological saline solution (e.g., phosphate buffered saline) that can be formulated with various buffers. Other exemplary carriers include sterile, physiological saline solution, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, 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 can contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers, in addition to the rAAV and carrier(s). Suitable, exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0084] rAAV is administered in an amount sufficient to transfect cells of the desired tissue and provide sufficient levels of gene introduction and expression without undue adverse effects. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the selected organ (e.g., injection into the CNS), oral route of administration, inhalation route of administration (including intranasal delivery and intratracheal delivery), intraocular route of administration, intravenous route of administration, intramuscular route of administration, subcutaneous route of administration, intradermal route of administration, intratumoral route of administration, and other parenteral routes of administration. Routes of administration may be combined, if desired.

[0085] The dose of rAAV virions required to achieve a particular “therapeutic effect,” for example, with the number of genome copies per kilogram of body weight (GC / kg) as the dose unit, will vary based on several factors including, but not limited to, the route of administration of the rAAV virions, the level of gene or RNA expression required to achieve the therapeutic effect, the specific disease or disorder being treated, and the stability of the gene or RNA product. One of ordinary skill in the art can readily determine the dosage range of rAAV virions for treating a patient having a particular disease or disorder based on the aforementioned factors as well as other factors well known in the art.

[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 an amount sufficient to generate a stable somatic transgenic animal model. The effective amount can vary between animals and between tissues because it depends primarily on factors such as species, age, body weight, the health of the subject, and the tissue being targeted. For example, an effective amount of rAAV is generally about 10 6 ~10 16 genome copies (e.g., including endpoints, 1×10 6 ~1×10 16 ) in a solution in the range of about 1 ml to about 100 ml. In some cases, a dosage between about 10 11 ~10 12 rAAV genome copies is appropriate. In some embodiments, about 10 11 ~1013 The dosage between rAAV genome copies is appropriate. In some embodiments, about 10 11 ~10 14 The dosage between rAAV genome copies is appropriate. In some embodiments, about 10 11 ~10 15 The dosage between rAAV genome copies is appropriate. In some embodiments, about 10 12 ~10 14 The dosage of rAAV genome copies is appropriate. In some embodiments, about 10 13 ~10 14 The dosage of rAAV genome copies is appropriate. In some embodiments, per kilogram (kg) of body weight, about 1×10 12 , about 1.1×10 12 , about 1.2×10 12 , about 1.3×10 12 , about 1.4×10 12 , about 1.5×10 12 , about 1.6×10 12 , about 1.7×10 12 , about 1.8×10 12 , about 1.9×10 12 , about 1×10 13 , about 1.1×10 13 , about 1.2×10 13 , about 1.3×10 13 , about 1.4×10 13 , about 1.5×10 13 , about 1.6×10 13 , about 1.7×10 13 , about 1.8×10 13 , about 1.9×10 13 , or about 2.0×10 14 The dosage of vector genome (vg) copies is appropriate. In some embodiments, about 4×10 12 ~2×10 13 The dosage between rAAV genome copies is appropriate. In some embodiments, 4.68×10 7 The dosage is appropriate. In some embodiments, 4.68×10 8 The dosage of genome copies is appropriate. In some embodiments, 4.68×10 9The dosage of genomic copies is appropriate. In some embodiments, 1.17×10 10 The dosage of genomic copies is appropriate. In some embodiments, 2.34×10 10 The dosage of genomic copies is appropriate. In some embodiments, 3.20×10 11 The dosage of genomic copies is appropriate. In some embodiments, about 4.2×10 12 The dosage of genomic copies is appropriate. In some embodiments, 1.2×10 13 The dosage of genomic copies is appropriate. In some embodiments, 1.3×10 13 The dosage of genomic 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, by intravenous administration, about 1.5×10 per kg 13 The dosage of vg is appropriate. In some embodiments, about 1×10 per kg of brain weight 14 The dosage of vg is appropriate. In certain embodiments, 10 12 ~10 13 rAAV genomic copies are effective for targeting tissues associated with lysosomal storage diseases, such as brain tissue or CNS tissue. In certain embodiments, 10 13 ~10 14 rAAV genomic copies are effective for targeting tissues associated with lysosomal storage diseases, such as brain tissue or CNS tissue. In some embodiments, the dosage of rAAV administered intravenously to a subject is about 10 per kilogram (kg) of body weight 11 ~10 14 among rAAV vector genome (vg) copies. In some embodiments, the dosage delivered intravenously is 1×10 per kg 11 ~1×10 14 vg, 1×10 per kg 12 ~1×10 14 vg, or 1×10 per kg 13 ~1×10 14It is between vg. In some embodiments, the dose delivered intravenously is about 1.2×10 per kg 13 ~1.8××10 13 It is between vg. In some embodiments, the dose delivered intravenously is about 1.5×10 per kg 13 vg. In some embodiments, the administration of the dose includes delivery to cerebrospinal fluid (CSF). In some embodiments, the total dose is delivered to the CSF by (i) injection via the cisterna magna and / or the spinal canal (e.g., the vertebral canal) and / or (ii) by thalamic injection to the thalamus. In some embodiments, about 75% of the total dose is delivered to the cisterna magna. In some embodiments, about 25% of the total dose is delivered to the spinal canal. In some embodiments, about 75% of the total dose to the CSF is delivered to the cisterna magna. In some embodiments, about 25% of the total dose to the CSF is delivered to the spinal canal. Optionally, stable transgenic animals are produced by multiple administrations of rAAV.

[0087] In some embodiments, the dose delivered to the thalamus is about 1×10 11 ~1×10 14 vg, 1×10 12 ~1×10 14 vg, or 1×10 12 ~5×10 13 vg. In some embodiments, the dose delivered to the thalamus is about 2.8×10 12 ~1.1×10 13 vg. In some embodiments, the dose delivered to the thalamus is about 7.2×10 12 ~1.4×10 13 vg.

[0088] In some embodiments, the volume delivered to the thalamus is about 0.5 mL to about 1.5 mL, about 0.75 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 spinal canal (e.g., the vertebral canal) is 1×10 12 ~1×10 15vg, 1×10 13 ~1×10 15 vg, or 1×10 13 ~1×10 14 vg. In some embodiments, the dose delivered to the cistern and / or the spinal canal (e.g., the spinal canal) is about 2.0×10 13 ~9.0×10 13 vg. In some embodiments, the dose delivered to the spinal canal (e.g., the spinal canal) is about 2.0×10 13 ~8.13×10 13 vg.

[0090] In some embodiments, the volume delivered to the spinal canal (e.g., the spinal canal) is about 1 mL to about 10 mL, or about 2 mL to about 8 mL. In some embodiments, the volume delivered to the spinal canal (e.g., the spinal canal) is about 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, or 8 mL.

[0091] In some embodiments, delivery to the subject (e.g., to the subject's CSF, spinal canal, and / or cistern) is performed by an intravascular microcatheter. In some embodiments, a dose of 1×10 14 vg is delivered via an intravascular microcatheter. In some embodiments, the dose is delivered by inserting an intravascular microcatheter and removing 14 mL of CSF from the subject by passive flow, followed by administering 9 ml of AAV to the subject at a rate of about 1 mL / min to the cistern level of the subject. In some embodiments, delivery to the subject is performed by, for example, intralumbar injection (e.g., administration to the spinal canal within the lumbar region) via an intravascular microcatheter. In some embodiments, the delivery is an intralumbar injection to the L2 region of the subject. In some embodiments, a volume of 3 ml of rAAV vector is administered to the subject.

[0092] In some embodiments, the present disclosure relates to a method for administering an rAAV composition comprising a transgene encoding a lysosomal storage disease protein to a subject in need thereof (e.g., a subject having or suspected of having a lysosomal storage disorder such as Tay-Sachs disease or Sandhoff disease). In some embodiments, the subject has, as measured using an artificial substrate such as MUG or MUGS, ≦1.0%, ≦0.5%, or ≦0.1% of normal β-hexosaminidase A activity in the subject's CSF prior to administration of the rAAV or composition comprising rAAV disclosed herein. In some embodiments, the subject has infantile Tay-Sachs disease. In some embodiments, the subject is between 5 and 36 months old. In some embodiments, the subject is between 12 and 36 months old. In some embodiments, the subject is between 18 and 30 months old.

[0093] In some aspects, the present disclosure relates to the recognition that one potential side effect of administering AAV to a subject is an immune response, including inflammation, to AAV in the subject. In some embodiments, the subject is immunosuppressed prior to administration of one or more of the rAAVs described herein.

[0094] As used herein, "immunosuppressed" or "immunosuppression" refers to a decrease in the activation or efficacy of the immune response in a subject. Immunosuppression can be induced in a subject using one or more agents (e.g., multiple agents such as two, three, four, five, or more agents) including, but not limited to, rituximab, methylprednisolone, prednisone, sirolimus, immune globulin injection, prednisolone, Solu-Medrol, lansoprazole, trimethoprim / sulfamethoxazole, methotrexate, and any combination thereof. In some embodiments, the immunosuppression regimen includes administration of sirolimus, prednisone, lansoprazole, trimethoprim / sulfamethoxazole, or any combination thereof.

[0095] In some embodiments, the methods described by the present disclosure further include inducing immunosuppression (e.g., administering one or more immunosuppressive agents) in a subject prior to the subject being administered rAAV (e.g., rAAV or a pharmaceutical composition described by the present disclosure). In some embodiments, the subject is immunosuppressed (e.g., immunosuppression is induced in the subject) between about 30 days and about 0 days prior to administration of rAAV to the subject (e.g., at any point during the 30 days, including the endpoints, up to administration of rAAV). In some embodiments, the subject is pretreated with an immunosuppressive agent (e.g., rituximab, sirolimus, and / or prednisone) for at least 7 days.

[0096] In some embodiments, the methods described by the present disclosure further include co-administering or pre-administering an agent to a subject to whom the rAAV or pharmaceutical composition comprising rAAV of the present disclosure is administered. In some embodiments, the agent is selected from the group consisting of miglustat, Keppra, Prevacid, clonazepam, and any combination thereof.

[0097] In some embodiments, immunosuppression of the subject is maintained at the time of and / or after administration of the rAAV or pharmaceutical composition. In some embodiments, the subject is immunosuppressed (e.g., administered one or more immunosuppressive agents) for between 1 day and 1 year after administration of the rAAV or pharmaceutical composition.

[0098] In some embodiments, the rAAV composition is formulated to reduce aggregation of AAV particles in the composition, particularly when a high rAAV concentration (e.g., about 10 13 GC / ml or more) is present. Methods for reducing aggregation of rAAV are well known in the art and include, for example, addition of a surfactant, correction of pH, correction of salt concentration, etc. (see, e.g., Wright FR, et al., Molecular Therapy (2005) 12, 171-178, the content of which is incorporated herein by reference).

[0099] Formulations with pharmaceutically acceptable excipient solutions and carrier solutions are well known to those skilled in the art, similar to the development of dosing regimens and treatment regimens suitable for using the specific compositions described herein in various treatment regimens.

[0100] The percentage of the active ingredient(s) will, of course, vary and may conveniently be between about 1 or 2% to about 70% or 80% or more by weight or volume of the total weight or volume of the formulation, but typically these formulations may contain at least about 0.1% or more of the active compound. Of course, the amount of the active compound in each therapeutically useful composition can be prepared such that an appropriate dosage is obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, in vivo half-life, route of administration, shelf life of the product, as well as other pharmacological considerations are envisioned by those skilled in the art of preparing such pharmaceutical formulations, and in that way, various dosages and treatment regimens may be desired.

[0101] In certain circumstances, it may be desired to deliver an rAAV-based therapeutic construct in a pharmaceutically appropriate formulated composition disclosed herein by subcutaneous delivery, intra-pancreatic delivery, intranasal delivery, parenteral delivery, intravenous delivery, intramuscular delivery, intrathecal delivery, or oral delivery, intraperitoneal delivery, or by inhalation. In some embodiments, the dosing modalities described in U.S. Patent Nos. 5,543,158; 5,641,515; and 5,399,363 (each specifically incorporated herein by reference in its entirety) may be used to deliver rAAV. In some embodiments, the preferred mode of administration is by intraportal injection.

[0102] Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions in glycerol, liquid polyethylene glycols, and mixtures thereof, and oils may also be prepared. Under normal storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms. In many cases, the form is sterile and fluid to the extent that easy injectability exists. The form must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycols, etc.), suitable mixtures thereof, and / or vegetable oils. Appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, and in the case of dispersions, can be maintained by the maintenance of the required particle size and the use of surfactants. Prevention of microbial action can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of injectable compositions can be brought about by the use in the composition of agents that delay absorption, for example, aluminum monostearate and gelatin.

[0103] For administration of an aqueous injection solution, for example, the solution may be appropriately buffered if necessary, and the liquid diluent can first be made isotonic with sufficient saline or glucose. These particular aqueous solutions are suitable, inter alia, for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, the sterile aqueous media that can be utilized will be known to those skilled in the art. For example, the dose for a single administration may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion fluid and may be injected into the proposed injection site (see, for example, "Remington’s Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in the dosage will necessarily occur depending on the condition of the host. In any event, it is the responsibility of the administering person to determine the appropriate dosage for the individual host.

[0104] Sterile injectable solutions are prepared by incorporating the required amount of active rAAV, optionally together with various other ingredients enumerated herein, in a suitable solvent, and then subjecting the mixture to filtration sterilization. Generally, dispersions are prepared by incorporating the sterilized, various active ingredients into a sterile medium that contains a basic dispersion medium and the required other ingredients derived from the ingredients enumerated above. In the case of sterile powders for preparing sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, in which a powder containing the active ingredient and any additional desired ingredients is obtained from its already sterile-filtered solution.

[0105] The rAAV compositions disclosed herein can be formulated either in the neutral form or in salt form. Pharmaceutically acceptable salts are acid addition salts (formed with the free amino groups of the protein) and include, for example, acid addition salts formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed from free carboxyl groups can also be derived from, for example, inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc. When formulated, the solution will be administered in a therapeutically effective amount in a manner compatible with the dosage formulation. The formulations are readily administered in various dosage forms, such as injection solutions, drug release capsules, etc.

[0106] As used herein, "carrier" includes any solvent, dispersion medium, vehicle, coating, diluent, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffer solutions, carrier solutions, suspensions, colloids, etc., and all combinations thereof. The use of such media and agents for pharmaceutical active substances is well known in the art. Also, adjuvants to the active ingredient can be incorporated into the composition. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to a host.

[0107] Delivery media such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc. can be used to introduce the compositions herein into appropriate host cells. In particular, the transgene delivered by the rAAV vector can be encapsulated in lipid particles, liposomes, vesicles, nanospheres, or nanoparticles, etc. and formulated for delivery.

[0108] Such formulations may be preferred for the introduction of pharmaceutically acceptable formulations of nucleic acids or rAAV constructs as disclosed herein. The formation and use of liposomes are generally known to those skilled in the art. In recent years, liposomes with improved serum stability and circulating half-life have been developed (U.S. Patent No. 5,741,516). Additionally, various methods using liposomes and liposome-like preparations as potential drug carriers have also been described (U.S. Patent Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868; and 5,795,587).

[0109] Liposomes have been successfully used with a number of cell types that are normally resistant to transfection by other procedures. In addition, liposomes do not have the DNA length constraints typical of virus-based delivery systems. Liposomes have been effectively used to introduce genes, drugs, radiotherapeutic agents, viruses, transcription factors, and allosteric effectors into a variety of cultured cell lines and animals. Additionally, several clinical trials examining the effectiveness of liposome-mediated drug delivery have been successfully completed.

[0110] Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form vesicles with multi-lamellar concentric bilayers (also referred to as multilamellar vesicles (MLV)). MLV generally have a diameter of 25 nm to 4 μm. Sonication of MLV results in the formation of small unilamellar vesicles (SUV) with a diameter in the range of 200 - 500 Å and containing an aqueous solution within the core.

[0111] Alternatively, nano-capsule formulations of rAAV can also be used. Nano-capsules can generally take up substances in a stable and reproducible form. In order to avoid side effects caused by excessive input of polymers into cells, biodegradable polymers should be used in vivo to design such ultra-fine particles (about 0.1 μm in size). Biodegradable polyalkylcyanoacrylate nanoparticles that meet these requirements are envisioned for use.

[0112] In addition to the delivery methods described above, the following techniques are also envisioned as alternative methods for delivering rAAV compositions to a host. In U.S. Patent No. 5,656,016, sonophoresis (e.g., ultrasound) is used and described as a device for enhancing the penetration of drugs into the circulatory system and the rate and efficacy of drug penetration through the circulatory system. Other envisioned alternative drug delivery methods are intraosseous injection (U.S. Patent No. 5,779,708), microchip devices (U.S. Patent No. 5,797,898), ophthalmic formulations (Bourlais et al., 1998), transdermal matrices (U.S. Patent Nos. 5,770,219; and 5,783,208), and feedback-controlled delivery (U.S. Patent No. 5,697,899).

[0113] In some embodiments, the disclosure relates to the administration of one or more additional therapeutic agents to a subject administered an rAAV or pharmaceutical composition described herein. For example, the administration of wild-type isolated proteins that mitigate a decrease in enzyme activity in enzyme replacement therapy (ERT) has been observed to be effective in treating lysosomal storage disorders, including Gaucher disease, Hunter syndrome, Fabry disease, Pompe disease, Maroteaux-Lamy syndrome, Morquio syndrome type A, and LAL deficiency. Thus, in some embodiments, the subject is administered one or more enzyme replacement therapies (ERT) in addition to the rAAV or pharmaceutical composition described by the disclosure. ERT is generally known and is described, for example, by Li (2018) Pediatr Ann. 47(5):e191-e197.

[0114] In some embodiments, the compositions described herein may 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. In some embodiments, the composition comprises an isolated wild-type HexA protein and an isolated wild-type HexB protein. In some embodiments, the composition comprises rAAV and an isolated wild-type HexA protein. In some embodiments, the composition comprises rAAV and an isolated wild-type HexB protein. In some embodiments, the composition comprises rAAV, an isolated wild-type HexA protein, and an isolated wild-type HexB protein.

[0115] Kits and Related Compositions In some embodiments, the agents described herein can be assembled into a pharmaceutical kit, a diagnostic kit, or a research kit to facilitate their use in therapeutic applications, diagnostic applications, or research applications. The kit can include one or more containers that store the components of the present disclosure and instructions for use. Specifically, such a kit can include one or more of the agents described herein, along with instructions that describe the intended applications and proper use of these agents. In certain embodiments, the agents in the kit can be pharmaceutical formulations and dosages that are suitable for a particular application and method of administration of the agent. A kit for research purposes can contain the components in concentrations or amounts suitable for performing a variety of experiments.

[0116] In some embodiments, the present disclosure relates to a kit for producing rAAV, the kit comprising a container storing an isolated nucleic acid having any one of the sequences of SEQ ID NOs: 1-6 or 16-19. In some embodiments, the kit further comprises instructions for producing rAAV. In some embodiments, the kit further comprises at least one container storing a recombinant AAV vector, where the recombinant AAV vector comprises a transgene.

[0117] In some embodiments, the present disclosure relates to a kit comprising a container storing the recombinant AAV described above. In some embodiments, the kit further comprises a container storing a pharmaceutically acceptable carrier. For example, the kit can include one container storing rAAV and a second container storing a buffer suitable for injection of the rAAV into a subject. In some embodiments, the container is a syringe.

[0118] The kit may be designed to facilitate use of the methods described herein by a researcher and can take many forms. Each of the compositions of the kit may, where appropriate, be provided in liquid form (e.g., in solution) or in solid form (e.g., as a dry powder). In certain cases, some of the compositions may be configured or otherwise processable (e.g., into an active form) by the addition of a suitable solvent or other type of medium (e.g., water or cell culture medium), which may or may not be provided by the kit. As used herein, "instructions" define the components of the instructions and / or promotion, and typically accompany an instruction sheet for the packaging of the present disclosure or an instruction sheet associated with the packaging of the present disclosure. The instructions may also include any oral or electronic instructions provided in any manner such that the user clearly recognizes that the instructions are associated with the kit, such as by visual means (e.g., videotape, DVD, etc.), the Internet, and / or web-based communication. The instruction sheet may be in a form mandated by a government agency that regulates the manufacture, use, or sale of a pharmaceutical or biopharmaceutical, and such an instruction sheet may also reflect approval by the regulatory agency for manufacture, use, or sale for administration to animals.

[0119] The kit can contain any one or more of the components described herein within one or more containers. By way of example, in one embodiment, the kit can include instructions for mixing one or more components of the kit and / or isolating and mixing a sample for application to a subject. The kit can include a container for storing the agent described herein. The agent can be in the form of a liquid, gel, or solid (powder). The agent can be prepared sterile, packaged in a syringe, and shipped frozen. Alternatively, the agent can be stored in a vial or other container for storage. The second container can have another agent prepared sterile. Alternatively, the kit can include an active agent that is pre-mixed and shipped in a syringe, vial, tube, or other container. The kit can have one or more or all of the components required for administering the agent to an animal, such as a syringe, topical application device, or iv injection needle, tube, and bag, particularly in the case of a kit for creating an animal model with specific somatic cells.

[0120] In some cases, the method involves transfecting a cell with whole cell DNA isolated from a tissue that may possess the proviral AAV genome at very low abundance, and supplementing helper virus functions (e.g., adenovirus) to induce and / or boost the transcription of the AAV rep gene and the AAV cap gene within the transfected cell. In some cases, RNA derived from the transfected cell provides a template for RT-PCR amplification of cDNA and detection of novel AAVs. When transfecting a cell with whole cell DNA isolated from a tissue that may possess the proviral AAV genome, it is often desirable to supplement the cell with factors that promote transcription of the AAV genes. For example, the cell may be infected with a helper virus such as adenovirus or herpesvirus. In a specific embodiment, the helper function is provided by adenovirus. The adenovirus can be a wild-type adenovirus, which may be of human origin or non-human origin, but preferably can be of non-human primate (NHP) origin. Similarly, in the methods of the present disclosure, adenoviruses known to infect non-human animals (e.g., chimpanzees, mice) can also be utilized. (See, e.g., U.S. Patent No. 6,083,716). In addition to wild-type adenoviruses, recombinant viruses or non-viral vectors (e.g., plasmids, episomes, etc.) that possess the necessary helper functions can also be utilized. Such recombinant viruses are known in the art and can be prepared according to published techniques. See, e.g., U.S. Patent Nos. 5,871,982 and 6,251,677, which describe hybrid Ad / AAV viruses. Various adenovirus strains are available from the American Type Cuture Collection, Manassas, Va., or can be obtained upon request from various commercial sources and sources supplied by research institutions. Furthermore, the sequences of many such strains are available from various databases, including, for example, PubMed and GenBank.

[0121] The cells can also be transfected with a vector (e.g., a helper vector) that confers a helper function to AAV. The vector that confers a helper function can confer an adenovirus function, for example, including E1a, E1b, E2a, E4 ORF6. The sequences of the adenovirus genes that confer these functions can be from any known adenovirus serotype, such as serotypes 2, 3, 4, 7, 12, and 40, and further include any of the human serotypes identified herein that are known in the art. Thus, in some embodiments, the method involves transfecting the cells with a vector that expresses one or more genes necessary for AAV replication, AAV gene transcription, and / or AAV packaging.

[0122] Optionally, the novel isolated capsid gene can be used to construct and package a recombinant AAV vector using methods well known in the art and to determine the functional characteristics associated with the novel capsid protein encoded by the gene. For example, the novel isolated capsid gene can be used to construct and package a recombinant AAV (rAAV) vector that includes a reporter gene (e.g., B-galactosidase, GFP, luciferase, etc.). The rAAV vector is then delivered to an animal (e.g., a mouse), and the tissue targeting properties of the novel isolated capsid gene can be determined by examining the expression of the reporter gene in various tissues of the animal (e.g., heart, liver, kidney). Other methods for characterizing the novel isolated capsid gene are disclosed herein, and still other methods are well known in the art.

[0123] The kit can have various forms, such as a similar pouch or tray form, which is packaged without stuffing a blister pouch, a shrink wrap pouch, a vacuum sealing pouch, a sealing thermoformed tray, or accessories into the pouch, within one or more tubes, within a container, within a box, or within a bag. After the accessories are added, the kit is sterilized, which enables the individual accessories within the container to be placed without wrapping them in other forms. The kit can be sterilized using any suitable sterilization method, such as radiation sterilization, heat sterilization, or other sterilization methods known in the art. The kit can also include other components, such as containers, cell culture media, salts, buffers, reagents, syringes, needles, woven fabrics such as gauze for applying or removing sterilizing agents, disposable gloves, supports for drugs before administration, etc., depending on the specific application.

[0124] The instructions included in the kit can be accompanied by a method for detecting latent AAV within cells. In addition, the kits of the present disclosure can include instructions, negative and / or positive controls, containers, diluents and buffers for samples, sample preparation tubes, and printed or electronic tables of reference AAV sequences for sequence comparison.

[0125] Method for treating lysosomal storage disorders Aspects of the present disclosure provide a method for treating lysosomal storage disorders. Lysosomal storage disorders (LSDs) are a class of >50 disorders associated with the dysfunction of resident enzymes that result in the accumulation of non-degraded substrates within lysosomes. Over time, this accumulation leads to lysosomal dysfunction, which in turn often results in a cascade of events that lead to cell death. LSDs affecting the central nervous system (CNS) require therapies intended to cross or avoid the blood-brain barrier to deliver functional enzymes to target cells to achieve disease resolution. One therapeutic delivery method has been AAV-mediated target gene delivery.

[0126] In some embodiments, the lysosomal storage disorder of the present disclosure is GM1 gangliosidosis, an autosomal recessive disorder that is invariably lethal. GM1 gangliosidosis (OMIM number: 230500) is caused by mutations in the GLB1 gene, which encodes the enzyme β-galactosidase. Based on the residual amount of β-gal activity and the onset of the disease, types I-III of GM1 gangliosidosis have been described, with type I present in infancy, type II present in late infancy or childhood, and type III present in adults. In some embodiments, the present disclosure presents methods and compositions for administration to a subject having or suspected of having type II GM1. Patients with type II GM1 who develop in late infancy generally develop symptoms between 1 and 3 years of age and have a mean life expectancy of 5 to 10 years. These subjects meet developmental goals within the first year of life but then begin to lose already acquired abilities such as speech and movement. Patients with type II GM1 and juvenile onset develop symptoms between 3 and 10 years of age and similarly show a decline in already achieved abilities such as speech and movement.

[0127] In some embodiments, GLB1 is human GLB1 (GeneID: 2720) and includes the sequences set forth in NM_000404.3, NM_001079811.2, NM_001135602.2, or NM_00137040.1. In some embodiments, GLB1 is human GLB1 and includes the amino acid sequences set forth in NP_000395.2, NP_001073279.1, NP_00129074.1, or NP_001303969.1. In some embodiments, GLB1 is mouse GLB1 (GeneID: 12091) and includes the sequence set forth in NM_009752.2. In some embodiments, GLB1 is human GLB1 and includes the amino acid sequence set forth in NP_033882.1. In some embodiments, GLB1 includes mutations such as P10L, R59C, R59H, R121S, G123R, M132T, G134V, P136S, R148S, D151V, D151Y, and / or any other mutations in GLB1 associated with a decrease in β-gal activity within the lysosome (e.g., those presented in Uniprot-KB, P16278, the contents of which are incorporated herein in their entirety).

[0128] In one aspect, the present disclosure presents a method for treating GM1 gangliosidosis. GM1 gangliosidosis can be type I GM1 gangliosidosis, type II GM1 gangliosidosis, or type III GM1 gangliosidosis. Since GM1 gangliosidosis is caused by a deficiency in β-galactosidase (β-gal) protein activity, a method for treating GM1 gangliosidosis results in an increase in β-gal activity. Non-limiting examples of methods for increasing β-gal activity include methods of expressing wild-type GLB1 intracellularly or in a subject, methods of reducing the expression of a mutant GLB1 gene intracellularly or in a subject, or methods of providing a wild-type GLB1 protein intracellularly or in a subject (e.g., enzyme replacement therapy). In some embodiments, GM1 can be treated by administering an isolated nucleic acid or rAAV comprising a transgene encoding GLB1 (e.g., wild-type GLB1). In some embodiments, the transgene comprises the wild-type GLB1 sequence set forth in SEQ ID NO: 23.

[0129] Within cells, or in a subject having GM1 gangliosidosis, GLB1 activity can be increased by practicing the methods of the present disclosure. Within cells, or in a subject having GM1 gangliosidosis, GLB1 activity can be increased by 50% to 500%. Within cells, or in a subject having GM1 gangliosidosis, GLB1 activity can be increased by at least 50%. Within cells, or in a subject having GM1 gangliosidosis, GLB1 activity can be increased by at least 500%. Within cells, or in a subject having GM1 gangliosidosis, GLB1 activity can be increased by 100% to 400%. Within cells, or in a subject having GM1 gangliosidosis, GLB1 activity can be increased by 200% to 500%. Within cells, or in a subject having GM1 gangliosidosis, GLB1 activity can be increased by 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, or 500%.

[0130] A method for treating GM1 gangliosidosis in a subject can include administering an isolated nucleic acid, rAAV, or composition of the present disclosure that comprises a transgene encoding human GLB1. The subject can be a human, mouse, rat, pig, dog, cat, or non-human primate. The step of administering means contacting the isolated nucleic acid, rAAV, or composition of the present disclosure with the cells or in the subject. Non-limiting examples of the step of administering include intravenous injection, intra-arterial injection, intracranial injection, intrathecal injection, intracerebral injection, infusion, or inhalation.

[0131] In some embodiments, the lysosomal storage disorder is Tay-Sachs disease (also known as GM2 gangliosidosis), an autosomal recessive disorder caused by a mutation in at least one of the genes HEXA, HEXB, and GM2A. Tay-Sachs disease (TSD) (OMIM number: 272800) is a uniformly lethal disorder that presents in infants, children, or adults and develops symptoms such as mental retardation, dementia, blindness, and death by the second or third year of life. All three wild-type levels of the enzymes hexosaminidase A (HexA), hexosaminidase B (HexB), and GM2 ganglioside activator (GM2A) are required for the breakdown of GM2 ganglioside, and a decrease in the activity of any of these three enzymes is associated with an increase in the accumulation of GM2 ganglioside within lysosomes. Patients with TSD develop a gray-to-white area around the fovea centralis due to lipid-laden ganglion cells, leaving a "cherry red spot," which is characteristic of the disorder, in the center. There is no current treatment for patients with TSD.

[0132] In some embodiments, the lysosomal storage disorder of the present disclosure is Sandhoff disease (also known as GM2 gangliosidosis), an autosomal recessive disorder caused by a mutation within the HEXB gene. Sandhoff disease (SD) (OMIM: 268800) is a uniformly lethal disorder. Patients with SD typically develop weakness within the first six months of life, followed by an exaggerated startle response, early blindness, progressive motor and intellectual decline, a doll-like facial appearance, a cherry red spot, and macrocephaly. Death typically occurs by the age of three.

[0133] In some embodiments, HexA is human HexA (GeneID: 3073) and includes the sequences set forth in NM_000520.5 or NM_001318825.1. In some embodiments, human HexA includes the amino acid sequences set forth in NP_000511.2 or NP_001305754.1. In some embodiments, HexA is mouse HexA (GeneID: 15211) and includes the sequence set forth in NM_010421.5. In some embodiments, mouse HexA includes the amino acid sequence set forth in NP_034551.2. In some embodiments, HexB is human HexB (GeneID: 3074) and includes the sequences set forth in NM_000521.3 or NM_001292004.1. In some embodiments, human HexB includes the amino acid sequences set forth in NP_000512.1 or NP_001278933.1. In some embodiments, HexB is mouse HexB (GeneID: 15212) and includes the sequence set forth in NM_010422.2. In some embodiments, mouse HexB includes the amino acid sequence set forth in NP_034552.1. In some embodiments, GM2A is human GM2A (GeneID: 2760) and includes the sequences set forth in NM_000405.4 or NM_001167607.1. In some embodiments, human G2MA includes the amino acid sequences set forth in NP_000396.2 or NP_001161079.1. In some embodiments, GM2A is mouse GM2A (GeneID: 14667) and includes the sequence set forth in NM_010299.3. In some embodiments, mouse GM2A includes the amino acid sequence set forth in NP_034429.1.

[0134] In some embodiments, the present disclosure presents an isolated nucleic acid and a recombinant AAV comprising a transgene encoding human GLB1, human HexA, or human HexB. In some embodiments, the present disclosure presents an isolated nucleic acid and a recombinant AAV comprising a transgene encoding mouse GLB1, mouse HexA, or mouse HexB. In some embodiments, GLB1, HexA, and / or HexB are operably linked to a promoter, optionally a tissue-specific promoter that drives expression of the transgene within a specific tissue. In some embodiments, the specific tissue is within the CNS. In some embodiments, the present disclosure presents a method of administering to a subject in need thereof a composition comprising the isolated nucleic acid or recombinant AAV described above. In some embodiments, the subject to which the composition is administered has or is suspected of having a lysosomal storage disorder including, but not limited to, GM1 gangliosidosis, Tay-Sachs disease, or Sandhoff disease.

[0135] In some embodiments, the subject is administered the rAAV or pharmaceutical composition of the present disclosure comprising a transgene encoding human HexA and / or human HexB, wherein the subject has an increase in the enzyme activity of β-hexosaminidase A in the CSF that is at least 0.5%, at least 1.0%, or at least 1.4% of normal enzyme activity at 3 months after administration of the rAAV or pharmaceutical composition. In some embodiments, the enzyme activity is measured using an artificial substrate such as MUG or MUGS. In some embodiments, the subject is administered the rAAV or pharmaceutical composition of the present disclosure comprising a transgene encoding human HexA and / or human HexB, wherein the enzyme activity of β-hexosaminidase A in the CSF of the subject is increased by at least 2-fold, or at least 3-fold, compared to the baseline enzyme activity of the subject prior to administration at 3 months after administration. In some embodiments, the subject has Tay-Sachs disease (e.g., infantile Tay-Sachs disease).

[0136] In some embodiments, GM2 ganglioside is reduced by at least 10%, at least 15%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, or at least 25% from baseline in the cerebrospinal fluid (CSF) of a subject by about 3 months after or at the time of administration of the rAAV or pharmaceutical composition of the present disclosure.

[0137] Accordingly, in some embodiments, the present disclosure provides isolated nucleic acids, rAAVs, compositions, and methods useful in the treatment of lysosomal storage disorders. In some embodiments, the isolated nucleic acids, rAAVs, compositions, and methods are isolated nucleic acids, rAAVs, compositions, and methods for treating GM1 gangliosidosis. In some embodiments, the isolated nucleic acids, rAAVs, compositions, and methods are useful in the treatment of Tay-Sachs disease. In some embodiments, the isolated nucleic acids, rAAVs, compositions, and methods are useful in the treatment of Sandhoff disease.

[0138] In one aspect, the present disclosure presents a method for treating GM2 gangliosidosis. GM2 gangliosidosis can be Tay-Sachs disease or Sandhoff disease. Since GM2 gangliosidosis is caused by a deficiency in HEXA enzyme activity and / or a deficiency in HEXB enzyme activity, a method for treating GM2 gangliosidosis results in an increase in HEXA activity and / or HEXB activity. Non-limiting examples of methods for increasing activity include methods of expressing wild-type HEXA and / or wild-type HEXB intracellularly or in a subject, methods of reducing the expression of mutant HEXA and / or HEXB genes intracellularly or in a subject, or methods that result in wild-type HEXA and / or wild-type HEXB proteins intracellularly or in a subject (e.g., enzyme replacement therapy). In some embodiments, GM2 can be treated by administering an isolated nucleic acid or rAAV comprising a transgene encoding at least one HEXA or HEXB (e.g., wild-type HEXA and / or wild-type HEXB). In some embodiments, the transgene comprises the wild-type HEXA sequence as set forth in SEQ ID NO: 20. In some embodiments, the transgene comprises the wild-type HEXB sequence as set forth in SEQ ID NO: 21.

[0139] The activity of HEXA and / or HEXB within cells or in a subject having GM2 gangliosidosis can be increased by practicing the methods of the present disclosure. The activity of HEXA and / or HEXB within cells or in a subject having GM2 gangliosidosis can be increased by 50% to 500%. The activity of HEXA and / or HEXB within cells or in a subject having GM2 gangliosidosis can be increased by at least 50%. The activity of HEXA and / or HEXB within cells or in a subject having GM2 gangliosidosis can be increased by at least 500%. The activity of HEXA and / or HEXB within cells or in a subject having GM2 gangliosidosis can be increased by 100% to 400%. The activity of HEXA and / or HEXB within cells or in a subject having GM2 gangliosidosis can be increased by 200% to 500%. The activity of HEXA and / or HEXB within cells or in a subject having GM2 gangliosidosis can be increased by 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, or 500%.

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

Example

[0141] [Example 1] Materials and Methods Design, construction of vectors, and generation of viruses An AAV vector (SEQ ID NO: 3) was constructed, which contains an expression cassette driven by a promoter composed of a cytomegalovirus immediate early enhancer (CMV) followed by a chimeric chicken beta-actin / rabbit beta-globin intron (CBA) fused to a chicken beta-actin promoter, mouse lysosomal acid beta-galactosidase cDNA (mβgal), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and tandem, two polyA signals derived from bovine growth hormone (BGH) and SV40. This vector is referred to as AAV-CBA-mβgal-WPRE. AAV-CBA-mβgal E269Q-WPRE was generated by PCR mutagenesis using the following primers: Forward 1: AAA CGT CTC ACT AGT CCG CGG AAT TC (SEQ ID NO: 7), Reverse 1: AAA CGT CTC ACT GAG AAT 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 GGT CTC ATC AGT TCT ATA CTG GC (SEQ ID NO: 10). The resulting PCR product was digested with the restriction enzymes SpeI and NotI and cloned in place of the wild-type βgal cDNA. All other AAV vectors were generated by removal of different elements from the AAV-CBA-mβgal-WPRE vector (SEQ ID NO: 3). All AAVrh8 vector stocks were prepared by standard methods.

[0142] Animal procedures GM1-gangliosidosis mice (βgal), which are knockout forms created by insertion of a neomycin cassette within exon 6 of GLB1, a β-galactosidase gene - / - ) is. βgal - / - 、 βgal + / - , and βgal + / + mice are male βgal - / - mice and female βgal + / -By mating with mice or by mating male and female + / - + / - to produce.

[0143] Intracranial injection 6 - 8 - week - old - / - - / - mice or + / - + / - mice were anesthetized by intraperitoneal injection of ketamine (125 mg / kg) and xylazine (12.5 mg / kg) in 0.9% saline and attached to a stereotaxic frame for rodents. The body hair around the incision site was shaved, and the skin was wiped with povidone - iodine pads and 70% EtOH. The skull was exposed by a small longitudinal incision (<1 cm) along the midline. The periosteum was removed from the surgical area with a sterile cotton swab. At appropriate stereotaxic coordinates, a small burr hole (<1 mm) was made using a high - speed drill. An Ultramicro Pump was used to drive a 10 - μl air - tight glass syringe fitted with a 33G injection needle to inject 1 μl of AAV vector or PBS at a rate of 0.2 μl / min into - / - - / - mice or + / - + / - both sides of - / - - / - mice into the thalamus (stereotaxic coordinates: anteroposterior coordinate: - 2.0 mm; mediolateral coordinate: ±1.5 mm from bregma; dorsoventral coordinate: - 3.5 mm from the brain surface), injecting 0.3 or 1 μl, and into the deep cerebellar nuclei (anteroposterior coordinate: - 6.0 mm; mediolateral coordinate: ±1.5 mm from bregma; dorsoventral coordinate: - 3.5 mm from the brain surface) of - / -

[0144] Behavioral assay The rotor rod test was performed on a rotor rod apparatus that was accelerated from 4 to 40 rpm over 5 minutes, and the waiting time until falling was recorded. The test consisted of one training trial that was accelerated from 2 to 20 rpm over 1 minute at the start of the session, followed by three trials with breaks of 15 - 20 minutes each in between. The waiting time until falling for each mouse in the test session was recorded, and the longest time on the rotor rod among the three trials was reported.

[0145] Tissue processing βgal + / - For biochemical research in mice, the brain was removed, sliced into 2 mm coronal blocks using Brain Matrices, and instantaneously frozen on dry ice. Blocks containing the thalamus were identified by the shape and presence of the injection needle insertion points on the dorsal brain surface. A biopsy punch with a diameter of 2 mm was used to sample the thalamus, and the tissue plug was placed in an appropriate buffer for analysis. For histological research, the brain and spinal cord were removed, placed in Neg 50 refrigerant, and frozen in a dry ice / 2 - methylbutane bath. βgal - / - For biochemical research in mice, the cerebrum, cerebellum + brainstem, and spinal cord were removed and instantaneously frozen on dry ice.

[0146] Histological analysis Brain sections (sagittal and coronal sections) and spinal cord sections (transverse sections) of 20 μm were cut out with a cryostat and stored at - 80 °C.

[0147] As previously described, with modifications, brain sections were stained with X - gal to evaluate the distribution of βgal. Briefly, slides were fixed in 0.5% glutaraldehyde in PBS and washed three times in ice - cold citrate phosphate buffer (CPB) (50 mM C6H8O, 50 mM Na2HPO4, 10 mM NaCl, pH = 4.2), and then incubated in X - gal staining solution [20 mM K4Fe(CN)6, 20 mM K3Fe(CN)6, 2 mM MgCl2, 0.01% C 24 H 39NaO4, 0.02% (C2H4O)nC 14 H 22 O (IGEPAL CA - 630, Sigma Aldrich), in 97% CPB, HCON(CH3)2 at pH = 4.2, with 2 mg / ml 5 - bromo - 4 - chloro - 3 - indolyl - β - D - galactosidase (X - gal), incubated overnight at 37°C. The next day, the slides were rinsed in CPB, then in water, counterstained with Vector Nuclear Fast Red, dehydrated in a series of 50% - 100% ethanol, cleared with CitriSolv, and mounted with Permount.

[0148] As previously described, with modifications, to evaluate lysosomal accumulation, brain and spinal cord sections were stained with filipin. Briefly, the slides were fixed in 4% paraformaldehyde in phosphate - buffered saline (PBS), washed with PBS, incubated in water with 1.5% glycine, washed with PBS, incubated for 1 - 2 minutes with 100 μg / ml filipin and 1 μg / ml ToPro3 iodide (Life Technologies, Grand Island, NY), washed with PBS, and mounted with PermaFluor, a fluorescent mounting medium.

[0149] To evaluate morphological changes in the tissue, brain sections were stained with Mayer's hematoxylin / eosin. Briefly, the slides were air - dried at room temperature, fixed in 4% paraformaldehyde in phosphate - buffered saline (PBS), washed with water, incubated with Mayer's hematoxylin, washed with running tap water, counterstained with eosin, rinsed with deionized water, dehydrated in a series of 50% - 100% ethanol, cleared with CitriSolv, and mounted with Permount.

[0150] Whole brain slice images were captured on a Nikon Super CoolScan 5000 ED with a medical slide holder using white light. Microscopic images were captured on a Leica DM550 B Microscope equipped with a Leica DFC425 C digital camera and a DFC365 FX digital camera. Filipin was imaged at 405 nm and ToPro3 iodide was imaged at 636 nm. H&E was imaged using bright field.

[0151] All histological analyses were performed as non-blinded, qualitative analyses on N ≥ 2 - 3 animals, based on representative photographs shown in the figures.

[0152] Enzyme assay and immunoblotting for βgal The biopsy punch was homogenized in lysis buffer (0.2 M CH3COONa, 0.1 M NaCl, 0.1% Triton X-100 in pH 4.3) and assayed for β-gal enzyme activity. Briefly, the reaction with β-gal substrate = 1 mM 4-methylumbelliferyl-β-D-galactoside (4-MUG) was carried out in a 96-well plate format, and the amount of 4-methylumbelliferyl (4-MU) released was measured against a standard curve by fluorescence detection via excitation at 360 nm and emission at 460 nm using a BioTek Synergy HT plate reader. Enzyme activity was normalized against the protein content determined by Bradford reagent assay and reported as nanomoles (converted substrate) per hour per milligram of protein. For immunoblotting, the biopsy punch from the injection site was homogenized in T-PER buffer supplemented with Complete Mini protease inhibitor cocktail, incubated on ice for 10 minutes, and then centrifuged at 10,000×g for 5 minutes. The supernatant was collected and the protein concentration was determined using Bradford reagent assay. Total protein (20 μg) was separated by polyacrylamide gel electrophoresis using a Mini-PROTEAN TGX precast gel and transferred to a NitroPure nitrocellulose membrane. The blot was blocked with Tris-buffered saline-Tween-20 (TBST) with 5% non-fat dry milk and then incubated with primary antibodies against α-rabbit GLB1 (β-galactosidase antibody) (1:250) and α-mouse β-actin (1:1000). HRP-conjugated anti-rabbit secondary antibody and anti-mouse secondary antibody were used (1:4000), and signal detection was performed with Pierce ECL Western Blotting Substrate, and the blot was exposed to Amersham Hyperfilm ECL.

[0153] Genomic copy Genomic DNA was isolated from biopsy punches at the injection site using the Qiagen DNeasy Blood and Tissue kit, and the concentration was determined using a Nanodrop spectrophotometer. The copy number of AAV vector genomes in 100 ng of genomic DNA was determined by qPCR using the following primers and TaqMan probe specific for BGH polyA within the vector genome: (TaqMan Probe, 6FAM-AGC ATT TTT TTC ACT GCA TTC TAG TTG TGG TTT GTC-TAMRA: SEQ ID NO: 11). Samples with a genome copy number of ≥100 vg per μg of DNA were considered positive for the vector genome.

[0154] Microarray Total RNA was isolated from biopsy punches using Trizol, further purified using the RNeasy Plus Mini kit, and its quality was analyzed on an Agilent Bioanalyzer. The RIN (RNA integrity number) value by the Bioanalyzer was 8.7 - 9.5, indicating high-quality RNA. Sample preparation and microarray hybridization were performed using Affymetrix Mouse Gene 2.0ST Arrays. Three independent samples per group were analyzed. The resulting data were processed, and a P-value < 0.05 and a 1.5-fold change relative to the PBS control were considered differentially expressed genes.

[0155] Quantification of GM1 ganglioside content The GM1 content in the CNS was quantified by liquid chromatography / tandem mass spectrometry (LC-MS / MS). Briefly, 25 μl of tissue at 0.01 - 0.04 mg per μl 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 gangliosides were isolated by a modified Folch extraction method. The sample was then passed through a C18 column, dried, reconstituted in running buffer, and then processed by Waters Quattro Premier XE, an LC-MS / MS. The sample was separated at a mass / charge ratio (m / z) of 290, and each GM1 molecular species with different fatty acid compositions was quantified. The GM1 content was determined by calculating the ratio of the sum of all molecular species to d3-GM1 and plotted against the ratio of purified GM1 / d3-GM1 by a calibration curve. The sample was normalized for the protein content in the initial lysate determined by Bradford reagent assay and reported as ng of GM1 per μg of protein.

[0156] Exemplary constructs A recombinant AAV vector encoding the human GLB1 gene (rAAV9hGLB1 vector) (SEQ ID NO: 23) was generated. This is a replication-deficient adeno-associated virus gene delivery vector. The single-stranded DNA genome consists of an expression cassette driven by the cytomegalovirus immediate early enhancer, fused to the chicken β-actin promoter with an intron of rabbit β-globin containing splice donor and splice acceptor sites, flanked by the human β-galactosidase (βgal) cDNA, and composed of inverted terminal repeats derived from AAV2. The construct also contains a polyadenylation signal derived from simian virus 40. The genome is packaged within the capsid AAV serotype 9, which is of human origin (Gao 2004) (Figure 1B). This was formulated into a buffered aqueous suspension for intravenous delivery.

[0157] C57BL / 6 mice were injected with PBS (control) or AAV9-hGLB1 under the transcriptional control of the CAG promoter (Figure 1B). Treated mice were administered 2.25×10 11 vg of AAV9-hGLB1. Each cohort (n = 5) was euthanized 7 days, 30 days, and 30 months after treatment. Enzymatic activity of β-galactosidase was measured from samples using the fluorogenic substrate β-4-methylumbelliferone (4-MU), which fluoresces when cleaved by β-galactosidase. An increase in β-galactosidase activity was observed within the AAV9-hGLB1 treatment group compared to PBS-injected control mice (data not shown).

[0158] [Example 2] AAV dose-dependent distribution of βgal in the brain The AAVrh8-CBA-mβgal-WPRE vector (Figures 1 and 20) was injected into the brains of 6- to 8-week-old GM1 gangliosidosis mice (βgal 10 ) by bilateral injections into the thalamus and deep cerebellar nuclei at a total dose of 4×10 10 vg, 2.6×10 9 vg, and 2.6×10 - / - vg. Animals in the highest-dose cohort received bilateral injections of 1 μl each into the thalamus and DCN, while animals in the other two cohorts received 1 μl into the thalamus and 0.3 μl into the DCN. The distribution pattern of βgal in the brain 3 months after injection was considered dose-dependent, with the highest intensity of βgal activity at the injection site (Figure 2). The highest dose (4×10 10 vg) resulted in enzymatic activity across most cross-sections in the cerebrum (Figure 2A) and cerebellum (Figure 2B). The intermediate dose, 2.6×10 10 vg, showed similar activity levels in the cerebrum (Figure 2E), but in the cerebellum (Figure 2F), activity was slightly reduced but still appeared to result in spread of the enzyme across the structure. The low dose, 2.6×10 9 vg, reduced spread in both the cerebrum and cerebellum (Figures 2I and 2J, respectively).

[0159] AAV-treated animals retain motor function in a dose-dependent manner The motor function of AAVrh8-treated mice was evaluated over time using a rotarod test (Figure 3). AAVrh8-treated βgal - / - All cohorts of βgal had significantly better performance than untreated βgal - / - controls at 6 months after treatment (high dose: 4×10 10 vg; p = 0.006, medium dose: 2.6×10 10 vg; p = 0.0009, and low dose: 2.6×10 9 vg; p = 0.005). N = 6 - 10 animals per group at 6 months after treatment.

[0160] [Example 3] AAV treatment extends lifespan AAVrh8-treated βgal - / - The lifespan of mice was significantly extended compared to naive βgal - / - controls (Figure 4). The median survival for naive βgal - / - controls was 245.5 days (N = 18), and for the 4×10 10 vg cohort it was 293.5 days (N = 12, p = 0.0004), for the 2.6×10 10 vg cohort it was 349 days (N = 13, p = 0.002), and for the 2.6×10 9 vg cohort it was 389 days (N = 12, p < 0.0001).

[0161] Accumulation of GM1 ganglioside persists at the injection site and in the spinal cord of long-lived AAV-treated animals Histological analysis of lysosome accumulation by Filipin staining in the CNS of animals 3 months after injection revealed near-complete correction in the cerebrum and cerebellum corresponding to the presence of the enzyme seen in Xgal staining at the same time point (Figure 2). Surprisingly, Filipin-positive cells were found only at the injection site or along the injection path (Figures 5C, 5E).

[0162] βgal injected with AAVrh8 - / - The presence of Filipin-positive cells in the thalamus of the mouse was also, surprisingly, the brain region (Figure 5A, framed) that presented the maximum intensity of X-gal histochemical staining in the brain, which is also a semi-quantitative indicator of high βgal enzyme activity. In the spinal cord of long-lived AAV-treated mice (495 - 612 days), the effect on lysosomal accumulation was from regions where the remaining Filipin-positive cells were extremely few to non-treated βgal - / - It was variable in the range of regions where no obvious changes were seen compared to the control (Figure 6A, 6D, 6F, and 6B, 6C, 6E, respectively).

[0163] [Example 4] Neuropathology at the injection site After early loss or 3 months after injection, the animals were histologically evaluated with hematoxylin / eosin (H&E). These animals were found to have morphological changes at the injection site that correlated with the dose. At 2 weeks after injection, βgal - / - +AAV4×10 10 vg demonstrated extensive changes in the thalamus, such as cell infiltration into blood vessels and the appearance of inflammation (thick arrow and thin arrow in Figures 7A and 7B, respectively), as well as extensive changes in the DCN accompanied by cell infiltration into blood vessels and obvious neuronophagia (thick arrow in Figure 8A and arrow in Figure 8B, respectively). At 3 months after treatment, βgal - / - +AAV2×10 10 vg also showed alterations in the thalamus accompanied by cell infiltration into blood vessels and inflammation (thick arrow and thin arrow in Figures 7E and 7F, respectively). However, βgal at a dose of 2×10 10 vg - / - +AAV resulted in only low-grade cell infiltration into blood vessels in the DCN (thick arrow in Figure 8E). In a representative animal at the lowest dose at 3 months after injection, βgal 9 vg - / - +AAV showed only extremely mild inflammation in the thalamus (thin arrow in Figure 7J), and this effect was not seen in the DCN (Figures 8I and 8J). Non-treated βgal - / -Controls (Figs. 7C, 7D, and Figs. 8C, 8D), or non-treated βgal + / - In the controls (Figs. 7G, 7H, and Figs. 8G, 8H), neither cell infiltration into blood vessels nor inflammation was observed.

[0164] High levels of βgal induce an unexpected response within the injected brain structure The paradoxical presence of Filipin-positive cells at the injection site (Figs. 5C, 5E) may be the result of an unexpected response to AAV gene transfer in mice. To understand this phenomenon, normal, non-affected βgal - / - Littermates were intracranially injected with the AAVrh8 vector. βgal + / - Similar to the results in mice (Figs. 5C, 5E), a large number of Filipin-positive cells were also present within the brain regions (Figs. 9A, 9B) with the highest βgal staining intensity in AAVrh8-injected βgal - / - mice (Figs. 9D, 9G, 9J). This result indicates that the presence of Filipin-positive cells within the targeted brain structure is an unexpected adverse response to certain aspects of AAV gene transfer. + / - In [Example 5]

[0165] Validation of the AAVrh8 vector series to evaluate the contribution of enzyme activity, protein level, and the AAVrh8 capsid to the Filipin detection response ​To study the nature of unexpected responses at the injection site, a series of AAVrh8 vectors were designed (Table 1). Additional vectors designed are shown in Figure 19. This series of AAVrh8, henceforth referred to as "CBA-WPRE" in this section, is based on the original vector, AAVrh8-CBA-mβgal-WPRE, and involves the sequential removal of elements that affect transgene expression levels. In "CBA", which is vector 2, the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) was removed. "CBA-EI-WPRE", which is vector 3, has exactly the same backbone as vector 1 but encodes a βgal protein that carries an E269Q mutation within the putative active site. This vector was designed to evaluate whether the observed response was caused by enzyme activity or protein production. "CB6", which is vector 4, contained mβgal cDNA but did not carry the WPRE or chimeric intron present in the other vectors. This vector was examined at two doses, "low-dose CB6" (the same dose as all other vectors) and "high-dose CB6" (2.0×10 10 vg). "Empty transgene" or "T.Empty", which is vector 5, contained all the components of vector 1 but lacked the mβgal cDNA.

[0166] The newly constructed AAVrh8 vectors were injected bilaterally into the thalamus of normal βgal 10 mice (1 μl per site for a total dose of 2.0×10 + / - vg), except for the CB6 vector which was also injected at a high dose (1 μl per site for a total dose of 2.0×10 9 vg). Controls were βgal + / - mice injected with phosphate-buffered saline (PBS) and naive βgal + / - mice. Enzyme activity and protein production in the thalamus were measured 6 weeks after injection by 4-methylumbelliferyl (4-MU) biochemical assay and Western blot (Figure 10).

[0167]

Table 1

[0168] The CBA-WPRE vector had the highest enzyme activity, 686-fold that of the naive βgal + / - enzyme activity in the thalamus of mice (Figure 10A), and a corresponding increase in the protein (Figure 10B, lane 1). The CBA vector resulted in a βgal + / - activity that was 224-fold that of the βgal level, but this was significantly lower than the βgal activity obtained with the CBA-WPRE vector (Figure 10A, p = 0.001), resulting in a corresponding, obvious decrease in the protein product (Figure 10B, lane 2). In the thalamus of mice injected with the CBA-EI-WPRE vector, the βgal activity was equivalent to that in naive control mice (Figure 10A), but the protein was expressed at a level equivalent to that in the thalamus injected with CBA-WPRE (lane 3 compared to lane 1 in Figure 10B). Thus, the E269Q mutation impairs enzyme activity but does not affect the protein expression level. Injection of the CB6 vector at the same dose as the other vectors (low-dose CB6) resulted in a βgal + / - activity that was 54-fold that of the level in βgal (Figure 4A), and, correspondingly, a lower amount of protein present than the presence of the protein by the other vectors (Figure 10B, lane 4). Injection of this vector at a high dose (high-dose CB6) resulted in a βgal + / - activity that was 420-fold that of the level by βgal, but this was significantly higher than the βgal activity within the low-dose CB6 cohort (Figure 10A, 10B, p = 0.03). The βgal activity level within the high-dose CB6 cohort was considered equivalent to the βgal activity level measured within the CBA-WPRE cohort, and also equivalent at the protein level (lane 1 compared to lane 5 in Figure 10A, 10B). The thalamus of animals injected with the empty transgene vector (T.Empty) or PBS was naive βgal + / -showed no change in βgal activity or protein level compared to the levels in 9 (Figure 10A, 10B). Vector genome copy numbers in the thalamus injected with AAV were shown to be equivalent in most cohorts injected with a total dose of 3.4 × 10 10 vg, except within the T.Empty cohort (Figure 11). As predicted, the high-dose CB6 cohort injected with 2.0 × 10

[0169] The filipin detection response correlates with the protein level The brains of βgal + / - mice injected with AAVrh8, and control βgal + / - mice were analyzed for βgal enzyme activity by Xgal staining (Figure 12A, 12D, 12G, 12J, 12M, 12P, 12S), and for the presence or absence of filipin-positive cells in the thalamus (Figure 12B, 12E, 12H, 12K, 12N, 12Q, 12T). The thalamic regions with the highest intensity of βgal staining (Figure 11A, 11D) also contained filipin-positive cells in CBA-WPRE injected animals, and CBA injected animals, although clearly fewer in the latter cohort (Figure 11B, 11E). The thalamus within the CBA-EI-WPRE cohort had numerous filipin-positive cells but no active βgal enzyme (Figures 11K, 11J respectively). Similar filipin staining was also evident within the thalamus of the T-Empty cohort, and the low-dose CB6 cohort (Figures 11K, 11Q), but was thought to be small dots that were significantly different from the patterns observed within the CBA-WPRE cohort, and the CBA-EI-WPRE cohort. Filipin staining was also observed within the thalamus of the high-dose CB6 cohort, and the pattern was thought to be a mix of the pattern observed within the CBA-WPRE cohort and the pattern observed within the low-dose CB6 cohort (Figure 11H). These results indicate that abnormal filipin accumulation at the injection site is associated with the protein expression level and not with the enzyme activity.

[0170] Changes in the transcriptome within the injected thalamus correlate with the abnormal responses detected by the Philippines Microarray analysis was performed within the thalamus to further characterize the tissue response to the physiological changes induced by gene transfer. Total thalamic RNA was isolated from the CBA-mβgal-WPRE cohort, low-dose CB6 cohort, T.Empty cohort, and PBS injection cohort. Changes in the transcriptome (fold change > 1.8-fold, p < 0.05) for all analyzed samples are represented in a heatmap (Figure 13A). The low-dose CB6 samples and T.Empty samples clustered together with PBS and were different from the CBA-WPRE samples. The number of genes with a change in expression level > 2-fold was significantly larger in the CBA-WPRE samples (with a small number of overlapping genes) compared to the CB6 samples and T.Empty samples (p < 0.05) (Figure 13B). A number of genes upregulated in the CBA-WPRE samples are characteristic of activated microglia and reactive astrocytes (Table 2). None of these genes showed significant changes in the CB6 samples and T.Empty samples.

[0171]

Table 2

[0172] [Example 6] Therapeutic effects of different AAVrh8 vectors in GM1 gangliosidosis mice βgal as described above + / - From studies performed in mice, the correlation was determined that a reduction in protein expression from the transgene could reduce changes in the pathological transcription levels within the injected structure. Next, it was explored whether changes in the AAV vector design led to differences in the treatment outcome in GM1 gangliosidosis mice (βgal - / - ). βgal mice at 6 - 8 weeks of age - / -Mice were bilaterally injected with an AAV vector into the thalamus (1 μl per side) and the deep cerebellar nuclei (0.3 μl per side), and the outcome was measured approximately 6 weeks after the injection. The study cohort consisted of βgal mice injected with CBA-WPRE, CB6 (low-dose CB6), and an empty transgene vector, administered at a total dose of 4.4×10 9 vg. In addition, CB6 was injected at a total dose of 2.6×10 - / - vg (high-dose CB6). Naïve untreated βgal 10 animals and naïve untreated βgal - / - animals were used as controls. Assay of βgal activity by 4-MU assay for the CNS (Figure 14) showed that in the cerebrum, CBA-WPRE was 45-fold that of wild-type levels (Figure 14), while low-dose CB6 was 9-fold (Figure 14) and high-dose CB6 was 30-fold (Figure 14). As predicted, the T.Empty cohort and the untreated cohort of βgal + / + mice had no detectable βgal activity in any of the CNS regions analyzed (Figure 14). The trend was the same in the cerebellum + brainstem, where CBA-WPRE was 47-fold that of wild-type levels (Figure 14), low-dose CB6 was 6-fold (Figure 14), and high-dose CB6 was 27-fold (Figure 14). Interestingly, in the spinal cord, CBA-WPRE was only 50% of wild-type levels (Figure 14), high-dose CB6 was 30% (Figure 14), and low-dose CB6 had no detectable activity (Figure 14). In all CNS tissues analyzed, high-dose CB6 had significantly (p<0.001) greater βgal activity than low-dose CB6, which was the same vector injected at a low dose. - / -

[0173] ​The distribution pattern of βgal in the brain (Figure 15), verified by X-gal, a histological dye, correlated with the activity levels in the cerebral or cerebellar + brainstem determined by the 4-Mu assay (Figure 14). The CBA-WPRE vector resulted in intense blue staining in the thalamus and DCN, and a widespread distribution of detectable enzyme activity throughout the brain (Figure 15A). In contrast, in the low-dose CB6 cohort (Figure 15B), intense staining was seen in the thalamus and DCN, but the detectable levels were low throughout the cerebrum, cerebellum, or brainstem. In high-dose CB6 animals, the distribution pattern of βgal in the brain was thought to be more extensive than that in low-dose CB6 animals (Figure 15C). As expected, no evidence of increased βgal activity was seen in the T.Empty cohort (Figure 15D), and naive βgal - / - mouse cohort (Figure 15E).

[0174] Histological analysis of lysosomal accumulation in the brain and spinal cord using Filipin staining (Figure 16) correlated with the βgal activity (Figure 14) and distribution pattern (Figure 15) described above. In CBA-WPRE-injected animals, almost complete clearance of accumulation was seen throughout the brain except at the injection site and along the injection path (Figure 16), but Filipin-positive cells were seen within the ventral hippocampus and throughout the thalamus (Figure 16; first row, fourth column). Since Filipin-positive cells still existed in the anterior cingulate cortex, striatum, and brainstem (Figure 16; second row, second, fifth, and seventh columns respectively), the clearance of accumulation in the low-dose CB6 cohort was considered inefficient (Figure 16; second row). At high dose (high-dose CB6), the efficiency of the CB6 vector was extremely high, and the dissipation of lysosomal accumulation occurred throughout the brain (Figure 16; third row). As before, Filipin-positive cells were present in the ventral hippocampus and dorsal thalamus, but were less prominent in CBA-WPRE (Figure 16; third row, first and fourth columns). The T.Empty cohort (Figure 16, fourth row) showed no change compared to the untreated control (Figure 16; fifth row). - / -

[0175] ​ The spinal cords of AAV-treated animals were also assayed for storage content by Filipin staining. In CBA-WPRE animals, spinal cord accumulation in the cervical region was almost absent, while only in the thoracic region was the reduction slight (Figures 17A-17B; respective arrows). The spinal cords of low-dose CB6 animals showed almost no distinguishable reduction in accumulation compared to untreated controls (Figures 17C-17D). The spinal cords of high-dose CB6 animals were almost devoid of lysosomal accumulation in both the cervical and thoracic regions (Figures 17E-17F; arrows). As expected, the spinal cords of T.Empty animals showed no change in lysosomal accumulation compared to untreated βgal - / - controls (Figures 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, significant reductions in GM1 ganglioside content were seen in the cerebrum (p = 0.0009 and p = 0.002, respectively) and cerebellum + brainstem (p < 0.0001 and p = 0.0001, respectively) compared to untreated βgal - / - controls. No significant change in GM1 ganglioside content was seen in the spinal cord in any cohort. In the high-dose CB6 cohort, GM1 ganglioside levels were normalized in all explored CNS regions: cerebrum, cerebellum + brainstem, and spinal cord (Figure 18; p = 0.64, p = 0.06, and p = 0.79, respectively). As expected, in the T.Empty cohort, no change in GM1 ganglioside content compared to naive βgal - / - controls was seen in any region of the CNS (Figure 18).

[0177] [Example 7] AAV-Mediated Gene Delivery to the CNS in an Animal Model of GM2 Gangliosidosis In patients with Tay-Sachs disease, prior to conducting clinical trials, a final safety study was conducted in non-human primates. This study involved different evaluation items to support the safety of the injection procedure consisting of bilateral injections of the AAVrh8 vector formulation into the thalamus and one lateral ventricle, and was designed to be a single-dose study (based on prior studies in GM2 mice and GM2 cats; total vector dose of 3.2×10 12 vg).

[0178] First, the NHPs (N = 3) injected with the AAVrh8 formulation developed moderate to severe neurological symptoms within 28 days after injection. When the NHPs were euthanized, histological evaluation of the CNS revealed large necrotic and myelin loss areas in the thalamus along the area considered to be the injection path (Figures 22A - 22D). In contrast, the NHPs injected with saline showed no symptoms and no evidence of neuropathy was found even at autopsy (> 90 days after injection) (Figure 22F). In addition to the histopathological evaluation of the brain, hexosaminidase activity was measured to be 6 to 48 times the normal activity (Figure 24).

[0179] Two additional cohorts of NHPs (N = 2 per cohort) were administered the AAVrh8 vector formulation at 1 / 10 and 1 / 30 of the original total dose, 2×10 11 vg and 1.1×10 11Injected with vg. Neurological symptoms developed in 3 out of 4 NHPs injected with an AAVrh8 vector formulation expressing low-dose cynomolgus HexA subunit, but with decreasing dose, the onset was progressively delayed. Despite the delay in symptom onset (or the absence of obvious symptoms in 1 NHP), neuropathological evaluation of the brain revealed extensive areas of necrosis, neuronal loss, and cellular infiltration into blood vessels in all 4 NHPs within the low-dose cohort (Figure 22E). In these NHPs, hexosaminidase activity in the brain was also increased compared to that in animals injected with saline (Figure 23). The increase in Hex expression was also found in the spinal cord, which was more prominent in the 1× dose animals (Figure 23, bottom).

[0180] As a control for potential toxicity associated with the AAVrh8 capsid and / or preparation method, another cohort of NHPs (N = 2) was administered 3.2×10 12 vg of an AAVrh8 vector with an empty transgene, and all animals presented normal behavior throughout the study (>90 days post-injection). The kinematic observations for all animals in the study are summarized in Table 3.

[0181] Prior to necropsy, brain MRI was performed on some NHPs within the low-dose cohort, and bilateral signal changes, presumably due to edema, were found in the thalamus (top, Figure 24), but no changes were observed in NHPs injected with an AAVrh8 vector with an empty transgene (bottom, Figure 24).

[0182] In addition to nerve loss in the thalamus along the injection path, in the vicinity, a large neuronal field filled with intracellular eosinophilic granules was also observed (Figs. 25E, 25G), but this was not evident in other regions of the brain (Fig. 25F). Immunofluorescent staining with antibodies specific for the hexosaminidase alpha subunit or the hexosaminidase beta subunit revealed that these granules were likely to contain these two proteins (Figs. 25A, 25C). These cells filled with HexA subunit-positive granules were observed only within the thalamus (Figs. 25B, 25D), which correlates with the findings in H&E-stained sections. These observations recall the findings in the GUSB transgenic mouse model (Vogler et al., 2003) where lysosomal accumulation of proteins became apparent in some tissues.

[0183] From this safety study in normal young NHPs, two key observations were made: First, the thalamic region containing a large number of HexA-positive neurons presented an intact appearance without evidence of significant inflammatory infiltrates (Figs. 25E and 25G), but along the injection path, there was extensive nerve loss and inflammation (Fig. 22). Second, no evidence was observed regarding the toxicity associated with the AAVrh8 vector with an empty transgene at the highest dose. As a result, the neurotoxicity in the NHP brain after AAVrh8-HexA-mediated gene transfer was caused by the massive overexpression of HexA in the thalamic neurons transduced with AAVrh8, which, when exceeding an unknown threshold, was thought to induce cell death and lead to a secondary neuroinflammatory response (such as cell infiltration into blood vessels).

[0184] [Table 3]

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

[0186] The original AAV vector plasmid pair (pAAV-CBA-CI-W Δ6ATG) yielded the highest level of Hex activity, but the AAV vector plasmid without a promoter (pAAV-NoP), or the AAV vector plasmid without a transgene (empty transgene) did not result in a detectable increase in Hex activity over naive non-transfected 293T cells (Figure 29). Other AAV vector plasmid pairs yielded a gradient of Hex activity. Six test AAV vector pairs were selected for further testing in nude mice, along with two AAV vector controls (control without a promoter, and control without a transgene). A total of 15 vector stocks were generated for the in vivo test (Table 4).

[0187] AAV vectors were injected bilaterally into the thalamus and left ventricle of 10 - 12-week-old male athymic nude mice (Charles River Labs) at a total dose of 1.32×10 10 vector genomes (vg). Control groups included mice injected with an AAV vector preparation without a promoter (AAV-NoP), an AAV vector without a transgene (empty transgene), phosphate-buffered saline (PBS), and finally, non-injected mice (N = 8 for all experimental and control groups) (Table 4). Mice were sacrificed one month after injection for biochemical analysis of Hex expression and histological studies. All groups showed the same increase in average body weight over the course of the experiment (Figure 30), and unlike in preliminary experiments with high AAV vector doses that euthanized animals due to significant weight loss and the development of neurological symptoms, no evidence of major behavioral changes was seen during this period. The dose used in this experiment (1.32×10 10vg) was determined by the lowest titer of one of the vector pairs for all test substances and controls to normalize the total vector dose.

[0188] Hex activity was measured in four coronal brain blocks, cerebellum, brainstem, and spinal cord using the artificial substrates MUG (Figure 36) and MUGS (Figure 37). Hex activity within the coronal brain block containing the injection site is presented in Table 4 and summarized for the findings. The original AAV vector pair (Group 1) resulted in Hex activity 400 - 1,700 times normal. Similar to the results in cell culture, the other AAV vector pairs resulted in Hex activity in the brain that was one-third (Group 2), one-twentieth to one-thirtieth (Group 4), and one-fiftieth to one-hundredth (Group 6) of the original AAV vector preparation (Group 1), resulting in the expected 1 - 2 log range of AAV-mediated Hex expression activity in the brain. The other AAV vector pairs (Groups 3 and 5) did not result in Hex activity above normal levels present in the brains of athymic nude mice. The control groups (Groups 7, 8, and 9) did not show a significant change in Hex activity.

[0189] Neuropathological examination of the brain revealed numerous thalamic neurons containing eosinophilic granules in the animals of Group 1 (Figure 31K). This finding was identical to the observations in the monkeys injected with the AAV vector used in Group 1, although the number of these abnormal neurons was considered to be significantly smaller in mice than in monkeys. This observation was also made in the animals of Group 2, but the number of abnormal neurons was significantly smaller than in Group 1 (Figure 31L). The same neurons were also observed in the hippocampus of the animals in Group 2 (Figure 31B). Evidence of such neurons was not seen in any other animal group. Immunofluorescent staining with an antibody against the alpha-subunit of HexA revealed numerous enzyme-expressing cells in the hippocampus and thalamus of the animals in Groups 1 (Figure 31A, 31K), 2 (Figure 31B, 31L), 4 (Figure 31D, 31N), and 6 (Figure 31F, 31P). In the control group, evidence of the expression of Hex-alpha subunit was not seen, probably due to the species specificity of the antibody used in this study, which detects the enzyme in humans and cynomolgus monkeys but not the mouse protein.

[0190] In the animals injected with the original AAV vector preparation (Group 1) (Figure 33A, 33K), a dramatic increase in Iba-1 staining (activation of microglia) was observed in the hippocampus and thalamus compared to the control (Figure 33I, 33J, 33S, 33T). This evidence of microglial activation was localized to the sites where HexA-positive cells were detected by immunofluorescent staining (Figure 32A, 32K), and no obvious changes in microglia were seen elsewhere in the brain. In Groups 2 and 3, the increase in Iba-1 staining in the hippocampus and thalamus was minimal (Figure 33B - 33C, 33L - 33M) and essentially indistinguishable from the control within Groups 4 - 7 (Figure 33D - 33G; 33N - 33Q).

[0191] The brain was also analyzed for evidence of reactive astrogliosis using GFAP immunostaining (Figure 34). In the hippocampus, evidence of reactive astrogliosis was found only in animals injected with the original AAV vector preparation (Group 1) (Figure 34A). All other animals were indistinguishable from controls. In the thalamus, some astrogliosis was seen in Groups 1 - 3 (Figure 34K - 34M), but was considered to be at a weak level or indistinguishable from controls within Groups 4 - 7 (Figure 34N - 34Q). No evidence of astrogliosis was seen in any other brain regions within any of the groups.

[0192] Two new AAV vectors (Figure 35), AAV - CB6 - I - cmHex (promoter represented by SEQ ID NO: 4), and AAVP2 - I - cmHex (promoter represented by SEQ ID NO: 5, construct represented by SEQ ID NO: 6), met all of the test criteria herein defined prior to the start of the experiment (absence of major behavioral changes, rotarod ability equivalent to the control group, body weight remaining constant or increasing between 0 - 30 days post - injection, absence of neuropathology, and over - normal enzyme expression in the thalamic block and rostral non - injected block). These new AAV vector preparations (Groups 4 and 6) resulted in an increase in Hex expression in the brain with little or no evidence of neuropathological changes (absence of eosinophilic neurons, microgliosis, or astrogliosis).

[0193] From the original AAV vector, the wpre element has been removed, but the expression elements (promoter and artificial intron) remain the same. The new AAV-CBA-I-cmHex vector (Figure 35) was also examined. This AAV vector (Group 2) showed approximately one-third of the Hex activity level compared to the original AAV vector and a decrease in microgliosis compared to the original AAV vector. Considering that the expression elements are the same in the AAV vector used in the long-term experiment in Sandhoff mice and the AAV vector used in the long-term experiment in Sandhoff cats, the AAV vector is also considered to be able to mediate long-term expression. Based on the reduction in Hex expression level and the attenuation of microglial activation, this new AAV vector is less likely to have a significant impact on the behavior of NHP in terms of the dose (3×10 11 vg) and duration of the experiment in Example 9.

[0194] [Table 4]

[0195] [Example 9] In athymic nude mice, three AAV vector designs (Figures 19 and 35) were selected to increase the expression of beta-hexosaminidase in the brain compared to the original AAV vector formulation and to reduce the inflammatory response (astroglial activation and microglial activation). The AAVrh8 vector was injected into NHPs (Table 5) that were screened for the absence or very low titer of neutralizing antibodies against the AAVrh8 capsid.

[0196] 3.2×10 11The total dose of vg was injected bilaterally into the thalamus (50% of the dose; 2 × 150 μl) and the left ventricle (50% of the dose in 300 μl). The vector formulation also contained 2 mM gadolinium to determine targeting accuracy and distribution by in - brain MRI immediately after injection (Figure 38A). All NHPs tolerated the surgical procedure well and had no complications. The mean distribution volume (Vd) of gadolinium in the thalamus was 1.47 ± 0.48 mL (Figure 38B), and the injection volume (Vi) in the thalamus was 0.3 mL, which corresponded to a Vd / Vi ratio of 4.9.

[0197] The behavior of all 6 NHPs injected with AAVrh8 remained normal throughout the 90 - day study. Intracranial MRI was performed monthly as planned (Figure 39). After day 30, signal changes at the injection site were recorded in 2 monkeys from cohorts 2 and 3, but no obvious changes were seen over time (Figure 40B, 40C). In 1 monkey within cohort 1, no signal changes were seen on intracranial MRI at days 30 and 60 after injection, but a large signal change was detected in the left thalamus at day 90 (Figure 40A). Despite this abnormal signal at day 90, the behavior of this monkey remained unchanged throughout the study.

[0198] The brain was sectioned into 4 - mm coronal blocks and used to map hexosaminidase (Hex) activity and evaluate neuropathology. A biopsy punch (3 - mm diameter) was used to sample the brain to create maps of enzyme distribution (Figure 41). In all cohorts, elevated total Hex activity (HexA, HexB, and HexS) above normal was detected only within the thalamic punches (Table 6). The total Hex activity within the thalamic punches was highest in cohort 1 (up to 87 - fold above normal) and similar in cohorts 2 and 3 (up to 9 - fold above normal), which is consistent with studies in athymic nude mice. Total Hex activity in most other sampled brain regions was similar to that in non - injected control monkeys.

[0199] Neuropathological assessment of the brain revealed a large accumulation of eosinophilic substances in the nerves of the monkeys in cohort 1 (Figure 42, black arrows). Moderate to severe neurodegeneration was observed in the animals of cohort 1. In the left thalamus of one monkey (ID: 295851) in cohort 1, severe focal spongiosis and perivascular cell infiltration were observed (Figures 43A, 43B). This lesion corresponded to an abnormal MRI signal that became apparent in the left thalamus of one monkey in cohort 1 90 days after injection (Figure 40A).

[0200] In cohort 2, no accumulation of eosinophilic substances was observed, and rare neurodegeneration and satellite lesions were seen. Focal inflammation of white matter with neurodegeneration, which was highly likely to be associated with injection site / route trauma, was observed in one monkey (ID: 295847).

[0201] In cohort 3, no accumulation of eosinophilic substances in the nerves was observed, and rare neurodegeneration and satellite lesions were seen (Figure 44A). In one monkey (ID: 295709), a lesion of perivascular lattice cells, which was highly likely to be associated with cannula insertion, was seen (Figure 44B). For histological findings, namely, intraneural accumulation of eosinophilic substances (Figure 45), neurodegeneration and necrosis (Figure 46), and in addition, for quantification of inflammation (Figure 47), serial sections were prepared at 150-μm intervals throughout the left and right thalamus of the monkeys in cohorts 1 and 3. These findings indicate that formulations 1 and 3 had equivalent effects in the brain, with the exception of the left injection site in one animal in cohort 1 where non-negligible neurodegeneration, necrosis, spongiosis, and corresponding inflammation were seen. These findings are in excellent agreement with the in-brain MRI on day 90 (Figure 40A).

[0202] A small pilot study was conducted to determine the efficacy of two new vectors, AAVrh8-CBA-cmHex (Figure 19) and AAVrh8-CB-I-cmHex (Figure 19), in reducing GM2 content in the CNS of Sandhoff disease (SD) after intracranial delivery. In all prior studies in SD mice and cats, the original AAVrh8-CBA-cmHex-W Δ6ATG vector was used as an efficacy reference. The AAV vectors were injected bilaterally into the thalamus and the left ventricle of 4- to 6-week-old SD mice at a total dose of 4.68×10 9 vg per mouse. This was used as the test 1-fold dose in a therapeutic efficacy study in SD mice injected with the original AAVrh8-CBA-Hex-W vector. Since the total Hex activity in the thalamus of NHPs injected with AAVrh8-CB-I-cmHex (cohort 2 in Table 6) was about 1 / 20 of that in NHPs injected with AAVrh8-CBA-cmHex (cohort 1 in Table 6), an additional cohort of SD mice injected with a 5-fold higher dose (5-fold dose: 2.34×10 10 vg) of the AAVrh8-CB-I-cmHex vector was also included. In addition to Hex activity, to measure GM2 ganglioside content, mice were sacrificed 1 month after injection and the brain was divided into four coronal blocks approximately 2 mm thick (Figure 48). AAVrh8-CBA-cmHex-W Δ6ATGIn one out of three SD mice injected with the green bar (Figure 48) and the AAVrh8-CBA-cmHex vector (orange bar, Figure 48), hexosaminidase was undetectable. In mice injected with the AAVrh8-CB-I-cmHex vector (blue bar in Figure 12), Hex activity was not detected in 3 out of 3 and 2 out of 4 animals, respectively, within the 1-fold cohort and the 5-fold cohort. In a comparative study in athymic nude (nu / nu) mice, the test AAVrh8 vectors in the SD mice of this example resulted in a non-negligible Hex overexpression in some cases, which was 2 to 3 orders of magnitude above normal. This indicates the possibility that the lack of Hex expression in a subset of SD mice is related to an immune response against the cynomolgus monkey Hex alpha (A) subunit or the cynomolgus monkey Hex beta (B) subunit, which restricts enzyme distribution via a humoral response or results in the loss of transduced cells mediated by an acquired response. Although the number of mice per group is small, a direct correlation is considered to exist between the Hex activity level and the reduction of GM2 ganglioside. In Block 3, in SD mice injected with the AAVrh8-CBA-cmHex-W Δ6ATG vector, a maximum reduction of 96% in GM2 ganglioside content was observed, for AAVrh8-CBA-cmHex, a 92% reduction was observed, and for the 1-fold dose and 5-fold dose of the AAVrh8-CB-I-cmHex vector injected into mice, reductions of 42% and 85% were observed, respectively.

[0203] [Table 5]

[0204] [Table 6]

[0205] [Example 10] AAV vectors carrying the human synapsin 1 (also referred to as "Syn1") promoter and the human GfaABC1D (also referred to as "GFAP") promoter were generated for simultaneous dual expression of the transgene of interest in both neurons and astrocytes, respectively. Syn1-GFP-2xmiR SOD1 / GFAP-2×miR SOD1 The vector, designated Syn1-GFP-2×miR-mCherry, contains GFP driven by the synapsin 1 promoter in neurons and mCherry driven by the GFAP promoter in astrocytes; two anti-SOD1 miRNAs, encoding human SOD1, are placed between the final codon and polyadenylation signal of each of GFP and mCherry ( FIG. 50 ). The vector was packaged using AAV9 capsid protein ( FIG. 50 ). In some embodiments, the vector, Syn1-GFP-2×miR SOD1 / GFAP-2×miR SOD1 -mCherry is represented by SEQ ID NO: 12.

[0206] We investigated the transduction profile of the dual promoter vector, AAV9-Syn1-GFP-2×miR. SOD1 / GFAP-2×miR SOD1 -mCherry, SOD1 G93A Intrastriatal injections were performed into adult mice, and fluorescence microscopy was performed. The data indicate that the morphology of GFP (green) and mCherry (red) expressing cells corresponds to that of neurons and astrocytes, respectively (FIG. 51).

[0207] The expression of SOD1 mRNA was measured. Data show that after injection of the dual promoter AAV9-Syn1-GFP-2×miRSOD1 / GFAP-2×miRSOD1-mCherry vector, adult SOD1 G93A This indicates that human SOD1 mRNA levels are reduced in mice (Figure 52). 12After intravenous injection of the entire vector genome, a maximum 25% decrease in human SOD1 mRNA expression was observed in the spinal cord. 8×10 9 After direct intrastriatal injection of the entire vector genome, a maximum 55% decrease in human SOD1 mRNA expression was observed in the striatum.

[0208] Data indicate that this dual promoter construct results in transduction of a greater, broad range of specific cell types within the CNS (e.g., neurons and astrocytes) than current methods using ubiquitous promoters (e.g., CBA, U6), while reducing toxicity to non-CNS tissues.

[0209] [Example 11] A 1:1 formulation of rAAVrh8 encoding the hexosaminidase A subunit and rAAVrh8 encoding the hexosaminidase B subunit (SEQ ID NOs: 20 and 21, respectively) was intracranially injected into 4-week-old SD mice via two methods: a combination of bilateral intrathalamic (TH) injection and intradentate nucleus (DCN) injection (TH / DCN), or bilateral intrathalamic injection (TH / ICV) combined with a single intracerebroventricular injection. AAVrh8-treated SD mice (4.68×10 9vg), and the behavioral performance of the controls (non-treated SD littermates and wild-type littermates) was evaluated over time starting at 60 days of age (1 month after injection) (Figure 53). At 120 days of age (P≤0.05), the reversal screen performance of SD mice treated with TH / ICV AAVrh9 was significantly better than that of non-treated SD mice, and at 180 days of age, the performance of mice treated with TH / ICV AAVrh8 and mice treated with TH / DCN AAVrh8 was equivalent to that of wild-type controls (Figure 53A). In the rotarod test, the performance of any cohort of SD mice treated with AAVrh8 at 120 days of age was also better (P≤0.01) than that of non-treated SD controls, and their performance was equivalent to that of wild-type controls until at least 180 days of age (Figure 53B). In the wire hang test, no significant improvement was measured compared to non-treated SD mice (Figure 53C). SD mice treated with TH / DCN or TH / ICV at a dose of 4.68×10 9 vg had median survival times of 424 days and 423 days, respectively (Figure 53D). Thus, both intracranial delivery methods resulted in a similar extension of lifespan (P≤0.001 for TH / DCN and P≤0.0001 for TH / ICV) compared to non-treated SD mice and were not significantly different from each other (P>0.05).

[0210] To determine whether increasing the dose of AAVrh8 further extends survival in SD mice, a dose escalation study was performed. Two days before intracranial TH / ICV injection of AAVrh-mHexA / B, SD mice were injected intravenously with AAV8-TRG-mHexB (3×10 11 vg). Intravenous injection of AAV8-TBG-mHexB alone did not enhance the survival of SD mice (the median survival time for non-treated SD mice was 127 days, while the median survival time for SD mice treated with AAV8-TBG-mHexB was 128.5 days; Figures 53D and 54A). Tolerance mediated by systemic therapy combined with intracranial therapy increased the median survival time to 4.68×10 8Compared to intracranial delivery alone in vg, there was no significant enhancement (the median survival of the combination therapy was 448.5 days, and the median survival of TH / ICV alone was 423 days; Figures 53D and 54A). Intravenous delivery, combined with the same tolerance protocol (AAV8-TBG mHexB before TH / ICV delivery of the AAVrh8-mHexA / B vector), with a dose escalation of intracranial delivery of AAV (4.68×10 7 vg, 4.68×10 8 vg, 4.68×10 9 vg, 1.17×10 10 vg) resulted in an extension of survival at the two highest doses (4.68×10 9 vg and 1.17×10 10 vg, P≤0.0001; the median survival of SD mice treated only systemically was 128.5 days; 4.68×10 7 : 110.5 days, 4.68×10 8 vg: 135 days, 4.68×10 9 vg: 448.5 days, 1.17×10 10 vg: 591 days; Figure 54A). Despite a maximum survival of 666 days, animals treated with a high dose (the dose of 1.17×10 10 vg) ultimately presented with hindlimb weakness and ataxia at the time of euthanasia, and some animals lost weight and / or the ability to right themselves when laid on their backs.

[0211] The inverted screen performance (Figure 54B), rotarod performance (Figure 54C), or wire hang test (Figure 54D) measured at 120 days of age, 4.68×10 9 vg or 1.17×10 10In the dose-escalating combination therapy mice with vg (intracranial TH / ICV administration), no significant differences in behavioral performance were observed compared to untreated SD mice. However, AAVrh8-treated SD mice retained motor function, and at 180 days of age, TH / ICV-treated SD mice were equivalent to wild-type mice in the inverted screen performance at any dose (Figure 54B). At 180 days of age, TH / ICV-treated SD mice at any dose were also equivalent to wild-type mice in the rotarod performance (Figure 54C). In the wire hang test, no significant improvement was measured compared to intracranial untreated SD mice (Figure 54D), and the median survival age approximated that of untreated SD mice (120 days). However, the mice were able to be further tested at 180 days of age when untreated SD mice had not survived.

[0212] To evaluate the potential effects of intracranial injection of the AAVrh8 vector in the absence of underlying processes of neurodegeneration, age-matched, heterozygous (HZ) littermate (HexB + / - ) mice were intracranially injected with 4.68×10 9 vg of an AAVrh8 vector formulation via TH / ICV or TH / DCN. AAVrh8-treated HZ mice and untreated controls (wild-type and HZ) were evaluated over time in different behavioral tests (Figure 55). No obvious and large changes in behavior or any effect on survival were seen in AAVrh8-injected HZ mice compared to controls. In the inverted screen test, differences between cohorts were not obvious (Figure 55A). The performance of TH / DCN-injected HZ mice was slightly worse than that of untreated HZ and wild-type controls in the 180-day rotarod test (Figure 55B). In the wire hang performance, no significant differences between cohorts were seen at either 60 or 120 days of age (Figure 55C). However, at 180 days of age, the performance of TH / DCN-injected HZ mice was worse than that of untreated HZ animals (P≤0.05) and untreated wild-type animals (P≤0.01). At 180 days of age, no significant differences were seen in the performance of TH / ICV-injected animals compared to untreated HZ or wild-type animals.

[0213] The content of GM2 ganglioside and the expression of hexosaminidase in the CNS were measured in TH / ICV AAVrh8-treated (1.17×10¹⁰ vg) SD mice, untreated SD mice, and untreated wild-type controls (Figure 56). GM2 ganglioside measured by LC-MS / MS was not detected in the brains of AAVrh8-treated animals, and a significant reduction in GM2 ganglioside was also seen in the cerebellum (91.1%), brainstem (99.6%), and spinal cord (99.8%) (P≤0.0001, Figure 56A). In the central nervous system of AAVrh8-treated SD mice, the total hexosaminidase activity was restored. The enzyme activity levels were higher than those of wild-type in the cerebrum and equivalent in the cerebellum, brainstem, and spinal cord. When the animals were subjected to the same predetermined evaluation items as untreated SD mice, they were indistinguishable from wild-type controls (data not shown).

[0214] [Example 12] All SD cat experiments utilized AAVrh8-CBA-mHexA / B-WPRE, which is slightly different from the vector construct (AAVrh8-CB-CI-hHexA / B) used for human application. Efficacy studies were conducted in a naturally occurring SD cat model where the disease progresses rapidly and the mean survival is 4.3±0.2 months of age. In these studies, a 1:1 formulation with two AAVrh8 vectors that individually encode the hexosaminidase alpha subunit and the hexosaminidase beta subunit of wild-type cats was used.

[0215] In this example, long-term efficacy data are reported. SD cats were subjected to bilateral thalamic injections combined with unilateral ICV injection (ICV; n = 5) with 4.6×10 11Treated with the total dose of vg (ratio of both vectors 1:1; Table 7). Gene therapy significantly prolonged the survival of SD cats (p<0.0001; Figure 57A), and the oldest cat survived up to 29.9 months of age. Untreated SD cats suffered from severe cerebellar disease with obvious generalized static tremors and intentional tremors, which became a wasting disease associated with loss of walking and subsequent euthanasia at approximately 4 months of age. After AAV gene therapy, a marked improvement in neurological signs was seen (Figure 57B). In all treated cats, the tremors were completely improved, and most cats retained the ability to walk throughout the course of the study and were euthanized due to non-neurological skeletal or gastrointestinal diseases, which are not the normal neurological disease phenotype of SD cats (Table 7).

[0216]

Table 7

[0217] Intrathalamic injection combined with ICV injection (Th+ICV) of both AAV vectors resulted in a widespread hexosaminidase distribution throughout the cerebral cortex, cerebellum, and spinal cord (Figures 57B and 57C), and the only region of the brain with low distribution was the temporal lobe (Figures 57D, lateral view of Figure 57D, and Figure 57E). HexA levels were near or above normal throughout the brain, cerebellum, and spinal cord, and reached nearly 50 times normal at the intrathalamic injection site (Figure 57E). No histopathological abnormalities were observed in AAV-treated cats despite levels of Hex above physiological levels at the injection site.

[0218] In MRI (T2W) at the humane endpoint of SD cats, darkening of gray matter (accumulation of GM2) and brightening of white matter (demyelination) are shown throughout the brain (Figure 58A). After AAV gene therapy, normalization of gray matter intensity and white matter intensity was seen throughout the brain, with the exception of the temporal lobe, which was thought to be due to a reduction in the distribution of Hex (Figure 58D). Despite the normalization of gray matter intensity and white matter intensity, in AAV-treated SD cats, cortical atrophy was delayed, as exemplified by an increase in CSF in the lateral ventricles and in the cerebral sulci around the brain. When MR spectroscopy was used, a significant increase in N-acetylhexosamine (NA-Hex), a previously reported toxic metabolite, was detected in the thalamus of untreated SD cats, in mice and humans with SD. After AAV gene therapy, in all cats, NA-Hex was completely reduced to normal levels (Figure 58B). In AAV-treated cats, HexA levels increased to normal levels or above normal levels, and the increase in HexA was delayed throughout the life of the cats (Figure 58D). Aspartate aminotransferase and lactate dehydrogenase, markers of cytotoxicity, were elevated in the CSF of SD cats, but 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 to 2 months after treatment but decreased gradually over time (Figures 58G and 58H). The only cat (11-907) that reached the humane endpoint before 1 year of age showed a maximum serum antibody titer of 1:6 approximately 2 months after treatment, and the endpoint titer was only 1:16 (Figure 58G). In contrast, the cat (7-760) that survived until 26 months of age had a maximum titer of 1:16 3.6 months after surgery (Figure 58H). Due to the limited number of vector stocks, not all time points could be analyzed, but at approximately 20 months after treatment, the serum titer decreased to 1:1 (Figure 58H).

[0219] Despite the CNS-directed delivery of AAV, in SD + AAV TH / ICV cats, the HexA enzyme activity in peripheral organs was increased. The sciatic nerve achieved a 203% increase relative to normal, while normal liver and muscle had increases to 53 and 20% of normal, respectively (Table 8).

[0220]

Table 8

[0221] In this study, we investigated the AAVrh8 delivery of two single-cistronic vectors encoding feline HEXA or feline HEXB, flanked by the chicken beta-actin (CBA) promoter and the woodchuck post-transcriptional regulatory element (WPRE). Cats were treated by intracisternal injection (6.4 × 10 11 vg, approximately 0.5 kg at the time of injection), and a subset of animals (n = 3) was euthanized 16 weeks after treatment to evaluate biodistribution; a second cohort (n = 2) was followed to the humane endpoint. Untreated SD cats suffered from severe cerebellar disease and became progressively cachectic, with obvious, generalized, resting and intention tremors, loss of gait, and subsequent euthanasia at approximately 4.4 ± 0.6 months of age (Figure 59A). SD cats treated with AAV gene therapy (SD + AAV CM) via intracisternal injection had minor neurological signs, including ataxic gait and mild hindlimb weakness, at 16 weeks after treatment, a time point equivalent to the humane endpoint in untreated SD cats (Figure 59B). Two cats followed to the humane endpoint survived to 9.9 ± 0.5 months of age and retained the ability to stand and walk but were non-responsive to visual or auditory stimuli.

[0222] In the SD+AAV CM cats, the in vivo distribution of Hex was extensive throughout the spinal cord and cerebellum, but reduced within the telencephalic region (Figs. 59C, 59D, 59E). In short-term treated SD cats (data not shown) and long-term treated SD cats with SD+AAV CM, hexosaminidase A (HexA) activity reached or exceeded normal levels in the cerebellum and spinal cord, but was equivalent to that in untreated cats within the blocks containing the rostral thalamus, striatum, and frontal cortex (brain blocks D to A, respectively). The in vivo distribution of the vector also showed a similar trend, with brain block D (the most anterior block analyzed) containing the lowest vg copy number (Fig. 59F), but qPCR did not reflect the difference in Hex expression between the spinal cord and the brain.

[0223] Accumulation in SD cats was represented by periodic acid Schiff (PAS; grayscale) staining that stains for glycolipids (e.g., GM2). Regions with dark staining, e.g., PAS-positive regions, indicate increased ganglioside accumulation. In SD cats, increased PAS staining in the gray matter and reduced staining in the white matter were seen, which is consistent with ganglioside accumulation and demyelination in the cell bodies. In SD+AAV CM cats (long-term), the clearance of accumulation inversely reflected the in vivo distribution of Hex and was effective in the spinal cord and cerebellum, but not in the cerebral cortex. PAS-positive material remained in the cerebral cortex and deep brain structures. Demyelination, represented by a decrease in PAS-positive material, was also evident in the white matter of the cerebral cortex of treated cats.

[0224] In the cerebellum of SD cats, extensive neurodegeneration, evident in H&E staining, was seen (Figure 61). Purkinje cells, deep cerebellar neurons, and brainstem neurons showed cell loss, a sign of severe degeneration, and numerous, foamy, vacuolated, intracellular accumulations. Sixteen weeks after gene therapy, AAV-CM-treated SD cats showed normalization of the shape of Purkinje cells, deep cerebellar nuclei, and brainstem cells (Figure 61). In animals followed to the humane endpoint, normalization of shape was seen in the DCN and brainstem, but Purkinje cell degeneration and loss were delayed. Interestingly, long-term AAV-treated cats presented with grape-like eosinophilic inclusions within the dorsomedial part of the cerebellar fastigial nucleus (insert) (Figure 61). These inclusions have previously been shown to be immunopositive for Hex after parenchymal injection of AAV.

[0225] T2-weighted MRI, which is ultra-high-field MRI (7 Tesla), shows normalization of the MRI architecture in AAV-treated animals that reflects the distribution of Hex (Figure 62A). In untreated SD cats, inversion of gray matter intensity and white matter intensity is seen due to increased water (demyelination) in the white matter and increased lipids (GM2 accumulation) in the gray and white matter. Sixteen weeks after intracisternal injection, partial normalization of MRI intensity is seen, with darkening of the white matter intensity in the corpus callosum (Figure 62A; black arrow), corona radiata (Figure 62A; white arrow), and cerebellar white matter (Figure 62A; black arrow). At the humane endpoint of SD+AAV CM cats, the pathological intensity inversion of gray and white matter in the cerebral cortex was delayed, but cortical atrophy was alleviated and the intensity of the cerebellar white matter was partially restored (black arrow). MR spectroscopy of the thalamus (Figure 62C), parietal cortex (Figure 62D), and cerebellum (Figure 62D) in SD cats showed an increase in the toxic metabolite N-acetylhexosamine (NA-Hex, Figure 62B). In SD+AAV CM cats at 16 weeks, NA-Hex is reduced in the thalamus (Figure 62C) and cerebellum (Figure 62E), but continues to increase in the parietal cortex (Figure 62D) of one SD+AAV CM long-term cat (Figure 62E) evaluated at the humane endpoint. Changes in other metabolites underscore the partial correction of myelination (choline + phosphocholine) and metabolic activity (creatine + phosphocreatine, Cr+PCr) in the cerebellum after AAV treatment (Figure 62E).

[0226] CSF levels of HexA were elevated above normal in all SD+AAV cats (Figure 62F), and one cat (11-1042) underwent a rapid attenuation of HexA activity in the CSF from 5 months of age to the humane endpoint. The CSF markers of cytotoxicity, aspartate aminotransferase (AST; Figure 62G) and lactate dehydrogenase (LDH; Figure 62H), were reduced to near-normal levels in one long-term SD+AAV CM cat (11-1042), but moderately increased in another cat (11-1148).

[0227] [Example 13] Mouse For short-term, biochemical analysis of the effect on GM2 ganglioside content in the CNS, SD mice were injected with an AAVrh8-CB-CI-mHexA / B vector preparation encoding the mouse HexA / B (mHexA / B) subunits at two doses, 4.68×10 9 vg, and 2.34×10 10 vg, and compared with the original AAVrh8 vector (AAVrh8-CBA-mHexA / B-WPREmut6ΔATG) injected at 4.68×10 9 vg. In the brains of SD mice injected with either dose of the AAVrh8-CB-CI-mHexA / B vector, the GM2 ganglioside content was significantly lower compared to PBS-injected SD mice, and the 92% reduction observed for the highest dose (2.34×10 10 vg) of the AAVrh8-CB-CI-mHexA / B vector was equivalent to the reduction (97%) recorded in SD mice injected with the original AAVrh8 vector. A significant reduction in GM2 ganglioside content compared to PBS-injected SD mice was also observed in the cerebellum and brainstem of SD mice injected with the AAVrh8-CB-CI-mHexA / B vector, but it was a lower reduction than that of the original AAVrh8 vector. The degree of GM2 reduction in the CNS of SD mice injected with the AAVrh8-CB-CI-mHexA / B vector was dose-dependent (Figure 63A). The hexosaminidase activity in the brains of SD mice treated with the AAVrh8-CB-CI-mHexA / B vector was dose-dependent and was 1 / 7 to 1 / 43 of that of the original AAVrh8 vector, yet it was still 2 to 12 times normal (Figure 63B). To examine the therapeutic efficacy using survival up to 5 months of age (the median survival of untreated SD mice exceeds 1 month) as an outcome measure, another set of SD mice (n = 8) was injected with 2.34×10 10For vg, AAVrh8-CB-CI-mHexA / B was injected. Most (6 out of 8) of the AAV-treated SD mice survived until 5 months of age. However, only 2 out of 6 animals were considered asymptomatic after 5 months (Figure 63C). The remaining 4 mice presented with varying degrees of hindlimb dysfunction or debilitation. The hindlimb dysfunction can be explained by a more modest reduction in GM2 ganglioside content in the spinal cord by the new AAVrh8 vector formulation compared to the original CBA vector (Figure 63A; 35% compared to 70%).

[0228] To improve the effect on the accumulation of GM2 ganglioside in the spinal cord and cerebellum, while maintaining the dose in the thalamus at a constant (1.17×10 10 vg), the dose delivered to the CSF via the lateral ventricle was increased. For SD mice, the CSF dose was increased: 1.17×10 10 vg, 2.34×10 10 vg, 5.85×10 10 vg, 5.00×10 11 vg of AAVrh8-CB-CI-mHexA / B was injected (n = 3 per cohort). The reduction in GM2 ganglioside content in the spinal cord (Figure 64A) and cerebellum (Figure 64B) one month after injection was dose-dependent, and the highest dose (total dose of 5.2×10 11 vg) showed an effect similar to that of the original AAVrh8 vector injected with 4.68×10 9 vg.

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

[0230] Three weeks prior to treatment, several clinical safety tests were conducted on a subject (approximately 30 months old) with progressive infantile Tay-Sachs disease, and an immunosuppressive regimen was administered for approximately 7 days prior to treatment. The subject had two mutant HexA alleles: 1) HexA: a 4-base pair insertion (coding DNA: 1274 - 1278), the most common Ashkenazi mutation; and 2) HexA: coding DNA: 82C→T (protein: Gln28). In addition, the subject had already presented the following disease progression: 8 months old: Excessive startle reflex 12 months old: Macrocephaly with abnormal myelination on MRI, abnormal biomarkers on MRS 14 months old: First afebrile seizure → diagnosis 17 months old: Insertion of a G-tube 20 months old: Emergency hospital visit due to recurrence of seizures had presented.

[0231] The subject's seizures were kept under reasonable control with baseline medication and rectal midazolam as needed. Through the G-tube, the subject was made to ingest a ketogenic diet, miglustat, Keppra, Prevacid, and clonazepam.

[0232] A composition containing a biological pharmaceutical of rAAVrh8-HexA / B rAAV was injected into the subject at a rate indicated by the subject's CSF pressure. The target dose of the rAAVrh8-HexA / B preparation to be administered was 1.0×10 14 vector genomes (vg) per kg of brain weight, or 1.0×10 14 vg.

[0233] The total dose administered to the patient was calculated based on a brain weight of 1040 ± 130 g in female infants aged 19 - 30 months. Approximately 75% of the total dose was delivered to the cisterna magna, and approximately 25% was administered to the spinal canal via a fluoroscopically guided lumbar intrathecal catheter (Figure 74A). Following the removal of 14 mL of CSF by passive flow, 9 mL of the AAVrh8 vector was administered at a rate of approximately 1 mL / min to the cisterna magna level (Figure 74B). 3 mL of the AAVrh8 vector was injected at the L2 level (Figure 74C). Due to the progressive disease of the subject, the intracerebral injection of rAAVrh8-HexA / B, which was co-delivered, was not administered to the subject.

[0234] The biological pharmaceuticals used in the study were two non-replicating single-stranded adeno-associated virus vectors, individually produced, designated AAVrh8-CB-ci-HEXA and AAVrh8-CB-ci-HEXB (rAAVrh8-HexA / B). The molecular characteristics of the vectors are described in Tables 9 and 10. The AAVrh8 vectors encode human HEXA and human HEXB under the chicken beta-actin promoter. The AAV vector plasmids used are depicted in Figures 65 - 67. The vectors were formulated in phosphate-buffered saline without Mg2+ and Ca2+.

[0235] rAAVrh8-HexA / B was generally well tolerated and was not associated with any serious adverse events as reported in the updated clinical trial data at 3 months. No clinically relevant laboratory abnormalities were observed 3 months after treatment, and no significant immune response was observed after treatment. The clinical status of the subject remained stable from baseline to 3 months without worsening in neurological examinations. Furthermore, in magnetic resonance imaging (MRI) of the brain at 3 months after treatment, no evidence of worsening from baseline was revealed when compared to the pre-treatment MRI of the brain at baseline. These data are shown in Figure 69.

[0236] Data collection at 3 months also indicated that a single dose of rituximab completely suppressed the counts of target CD19 and CD20 (<1% of peripheral T cells), and IgG was maintained within a safe range by IVIg infusion. These data are shown in Figure 70.

[0237] Subjects showed neutralizing antibody levels near baseline against the AAVrh8 capsid at 3 months, and no neutralizing antibodies against the transgene product were seen at 3 months. In addition, no T cell responses against the transgene product or cytotoxic T cell responses against the capsid were seen at 3 months. T regulatory Stimulation of the response supports the success of inducing long-term immune tolerance to the capsid that prevents future immune responses.

[0238] Very slight increases in the enzyme AST (less than 2-fold above baseline) and ALT (less than 2-fold above the upper limit of normal) were seen over the first 21 days but resolved spontaneously. No signs of systemic inflammation based on high-sensitivity C-reactive protein or total complement activity were seen. Liver ultrasound and pediatric gastrointestinal consultations also showed no specific pathology.

[0239] β-Hexosaminidase A activity was determined using the 4MUGS assay. At baseline, β-hexosaminidase A activity in the subjects' CSF was 0.46% of normal. At 3 months, β-hexosaminidase A activity in the subjects' CSF increased significantly to 1.44% of normal enzyme activity, representing an increase of more than 3-fold from baseline. Enzyme activity of HexA in serum and CSF before and up to 3 months after administration of rAAVrh8-HexA / B is shown in Figure 68.

[0240] As shown in FIG. 71, GM2 ganglioside in cerebrospinal fluid (CSF) was reduced by approximately 25% from the baseline. This indicates that a three-fold increase in the enzyme activity of β-hexosaminidase A in CSF from the baseline is associated with a decrease in GM2 ganglioside, which is considered to accumulate in Tay-Sachs disease patients and cause disease progression. In addition, the expression of β-hexosaminidase A protein measured by Western blot also increased from the baseline to the third month. These data are shown in FIG. 72.

[0241] [Table 9]

[0242] [Table 10]

[0243] [Example 15] A second subject (about 3 months old) with progressive infantile Tay-Sachs disease was administered rAAVrh8-HexA / B. Three weeks before the treatment, several clinical safety tests were performed on the subject, and an immunosuppressive regimen was administered for about 7 days before the treatment. The subject had two mutant HexA alleles: 1) HexA: a 1.75 kb deletion spanning exons 11-13; and 2) HexA protein: Val381 * a cleavage mutation within exon 10.

[0244] Before the administration of rAAVrh8-HexA / B, the subject had normal test values, normal growth and development, no evidence of seizures or exaggerated startle responses, and was clinically well. The subject had approximately 1% hexosaminidase activity compared to wild-type activity.

[0245] [Example 16] A clinical study of rAAVrh8-HEXA / B was designed. The study was designed as a two-stage, dose-escalating, and safety / efficacy study of bilateral intrathalamic parenchymal and intrathecal administration of an AAVrh8 vector encoding HEXA and HEXB individually, in infantile-onset Tay-Sachs disease (TSD) or Sandhoff disease (SD). The clinical study is outlined as described below. Due to the rapidly progressive and lethal nature of GM2 gangliosidosis and the lack of an approved therapy, the study does not utilize a placebo control group or an active comparator group. The treatment is not blinded.

[0246] The subjects in this study will be infants, male and female, with infantile-onset TSD or SD, less than 18 months of age (corrected for premature infants (gestational age < 37 weeks)) at the time of gene transfer, based on genetic mutation analysis and manifestation of symptoms. TSD subjects and SD subjects must each have a mutation in the HEXA gene encoding the α subunit or the HEXB gene encoding the β subunit. Subjects must be able to sit unaided for 5 seconds, be considered suitable surgical candidates (confirmed by the study neurosurgeon based on physical examination and MRI findings), have serum HexA activity less than 5% of normal, and not carry the G269S or W574C mutations. Subjects must also be seronegative for AAVrh8 neutralizing antibodies based on the assay threshold. Other inclusion / exclusion criteria may also apply.

[0247] The study will be a single-dose, open-label, non-randomized administration of rAAVrh8-HEXA / B, a viral vector, by bilateral intrathalamic (BiTh) and intrathecal (IT) administration. Subjects will enroll in the study in two stages (Figure 73) as follows: Phase 1: For the purpose of determining the optimal dose, four subjects shall be treated sequentially and with escalating doses. The dose selection shall be determined from safety, biomarkers, and further data; Phase 2: For the purpose of determining safety / efficacy, up to about ten subjects shall be treated at the optimal dose identified in Phase 1.

[0248] All subjects shall participate in long-term follow-up (LTFU) which is planned to determine the ongoing safety and efficacy of the treatment over a maximum of four years.

[0249] The study treatment shall be administered over a two-day period as follows to minimize the risk of prolonged anesthesia: On Visit 1a / 0 day, the subject shall receive bilateral parenchymal injections of rAAVrh8-HEXA / B into the thalamus on each side of the brain; On Visit 1b / 1 day, the subject shall receive an IT injection of rAAVrh8-HEXA / B. All injections shall consist of a 1:1 mixture of rAAVrh8-HEXA and rh8AAV-HEXB.

[0250] To prevent the risk of an inflammatory response and protect AAV-transduced cells from the immune response to the AAVrh8 capsid, the subject shall be immunosuppressed prior to administration of AAVrh8-HexA / B treatment. The immunosuppression regimen shall be maintained as previously determined over the long term. Starting on Day -16 of the study, initiate the following: · Rituximab at 375 mg per square meter of body surface area 2 · Solu-Medrol at 10 mg / kg · Sirolimus at 1 mg per square meter of body surface area · Prednisolone at 2 mg / kg per day 2 · Lansoprazole at 1 - 1.5 mg / kg per day · Trimethoprim / sulfamethoxazole at 10 mg / kg per administration, administered three times per week (Monday, Wednesday, Friday) Day -7: · Continue administration of sirolimus, prednisolone, and lansoprazole · Continuation of trimethoprim / sulfamethoxazole · Infusion of rituximab required for continuation of CD20 count of 5% or more - Day 2: · Continuation of administration of sirolimus, prednisolone, and lansoprazole · Continuation of trimethoprim / sulfamethoxazole Day 0 / BiTh infusion day · Continuation of administration of sirolimus, prednisolone, and lansoprazole · Continuation of trimethoprim / sulfamethoxazole Day 1: IT infusion day · Continuation of administration of sirolimus, prednisolone, and lansoprazole · Continuation of trimethoprim / sulfamethoxazole Days 2 - 7: · Continuation of administration of sirolimus, prednisolone, and lansoprazole · Continuation of trimethoprim / sulfamethoxazole · Administration of intravenous immunoglobulin (IVIG): The immunoglobulin level must not fall below 700 ml / dL. The dose is adjusted to maintain a serum trough level of 700 - 1000 mg / dL. It is performed after vector infusion. Day 14: · Continuation of administration of sirolimus, prednisolone, and lansoprazole · Continuation of trimethoprim / sulfamethoxazole Day 21: · Continuation of administration of sirolimus, prednisolone, and lansoprazole · Continuation of trimethoprim / sulfamethoxazole Week 4 / Month 1: · Continuation of administration of sirolimus, prednisolone, and lansoprazole · Continuation of trimethoprim / sulfamethoxazole · Administration of IVIG Week 4 / Month 2: · Continuation of administration 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 Week 24 / Month 6 · Discontinuation of sirolimus under the guidance of the in - hospital consulting pharmacist

[0251] Intrathalamic (ITh) administration For the first stage, the clinical dose and infusion volume proposed to be delivered to the thalamus (Th) are, for example, as shown in Table 11, 2.8×10 12 ~1.1×10 13 vg / ml.

[0252]

Table 11

[0253] The amount of thalamus filled with the vector, or the distribution volume (Vd), can be calculated based on the infusion volume (Vi) multiplied by a correction factor for how far the vector diffuses from the point source of delivery to the target brain region (thalamus), which is the volume of the target brain region (thalamus). From other AAV IPa injection studies (Yin, Richardson et al. 2010) (Yin et al, 2010), the ratio of Vd:Vi was assumed to be 3.0. The volume to the human thalamus was calculated as 1.08 mL of Vi and 4.35 ml 3 of Vd (77% of the total thalamus volume per side).

[0254]

Table 12

[0255] The Th injection volume of 1.08 ml is equal to or greater than the injection volume proposed for Subjects 1 to 3 in the first stage of the clinical trial. The corrected clinical doses proposed for low, medium, and high dose intracerebral administrations are 7.2×10 12 ~1.4×10 13 vg.

[0256] Intrathecal (IT) administration The human doses proposed for intrathecal administration are doses in the range of 2.03×10 13 ~8.13×10 13 vg, with a maximum injection volume of 8 mL (see the table below). The latter value is proposed to be used in clinical studies where the IT dose is set at 8.0×10 13 vg across all treatment subjects.

[0257]

Table 13

[0258] Throughout the procedure, the subject is placed under general anesthesia with intubation. Then, while injecting at 0.1 μL / min to prevent occlusion of the cannula tip, the right injection cannula and the junction line primed with the vector and joined to the delivery syringe and pump are placed into the target area in the right thalamus by stereotactic fixation. The flexible catheter tube is fixed on the scalp with an anchoring ligature and an adhesive dressing. Then, the same steps are repeated for the placement of the second catheter on the left side. Continuous intracranial pressure monitoring is performed throughout the procedure.

[0259] The injection rate starts at 0.5 μL / min and is increased every 5 minutes (1.0 μl / min → 2.0 μl / min → 5 μl / min). The study drug is injected into each catheter via a microinjection pump and a syringe containing the study drug as a 1:1 mixture of HexA vector and HexB vector.

[0260] IT administration: IT drug injection is performed at a rate directed by the CSF pressure. A total of 6.0 ml is delivered to the high cervical level / cistern, and 2.0 ml is delivered to the thoracolumbar level. Under the guidance of a fluorescence microscope, a Tuohy subarachnoid puncture needle is inserted into the subarachnoid space. Insertion into the subarachnoid space is verified by spontaneous CSF flow. Approximately 6 - 7 mL of CSF is drained by gravity, and then, under direct guidance by a fluorescence microscope, a flexible Excelsior (registered trademark) SL-10 (registered trademark) microcatheter (Stryker Neurovascular, Stryker) is passed near or into the cistern. A syringe containing the study drug (a 1:1 mixture of HexA and HexB) is joined to the catheter, and following administration of 6.0 ml at the high cervical / cistern level, the microcatheter is replaced to the lower thoracolumbar level and a second bolus of 2.0 ml is administered. Once the injection is complete, the microcatheter (dead volume: approximately 0.3 mL) is flushed with 0.5 mL of saline and then removed. Based on the surgeon's discretion, which is based on spinal anatomy and other considerations, the dose can be administered by two separate IT injections at the high cervical / cistern and thoracolumbar levels.

[0261] Primary evaluation item(s) (multiple possible): Phase 1: Incidence, severity, seriousness, and relationship to treatment of TEAE (treatment emergent adverse event) graded by NCI CTCAE v5.0 Phase 2: · Surrogate biological marker: Change in HexA activity in serum / CSF from baseline to visit 7 (3 months) · Clinical function: Ratio of subjects achieving the ability to sit for 5 seconds without assistance, as evaluated by item 22 of the Bayley Scales of Infant and Toddler Development, Third Edition (BSID-III) at visit 9 / 12 months · LTFU: Adverse events

[0262] Secondary evaluation item(s) (multiple possible): Phase 1: · Changes in vital signs, including body weight · Changes in general examination values and neurological examination values · Changes in clinical examination values, including complete blood count, comprehensive metabolic panel, and high-sensitivity C-reactive protein, with automated white blood cell differential · CD20 count · ECG, EEG · Cellular responses and neutralizing antibody titers against AAVrh8 capsid and HEXA protein and HEXB protein, as well as antibody levels against HEXA protein and HEXB protein Phase 2: · Biological markers for the disease: · Changes in HexA activity levels in CSF from baseline to Visit 9 (12 months) · Changes in HexA activity levels in serum from baseline to Visits 4, 5, 6, 7, 8, and 9 · Changes in GM2 ganglioside levels in CSF from baseline (by LC-MS / MS) to Visit 9 (12 months) · Changes in levels of lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) in CSF from baseline to Visit 7 (3 months) and Visit 9 (12 months) · Clinical functions: 1. At Visits 5, 7, and 8, the proportion of subjects achieving the ability to sit for 5 seconds without assistance, as evaluated by Item 22 of BSID-III 2. Changes in CHOP-INTEND Total Score from baseline to Visits 5, 7, 8, and 9 3. At Visits 5, 7, 8, and 9, the proportion of subjects achieving scores of 40 or above, 50 or above, and 64 on the CHOP INTEND motor function scale 4. Changes in BSID-III Composite Score from baseline to Visits 5, 7, 8, and 9 5. At visits 7, 8, and 9, the proportion of subjects who achieved any new motor milestones, as evaluated by the Hammersmith Infant Neurological Examination-2 (HINE-2), where new motor milestones are defined as sitting without support, crawling on hands and knees, standing with support, walking with support, standing independently, and walking independently 6. The proportion of subjects who independently took at least 5 steps while demonstrating coordinated movement and balance sense using the BSID-III (Gross Motor Subset #43)

[0263] Exploratory assessment item(s) (multiple possible) (Phase 2): · Changes in brainstem auditory evoked response (BAER) and visual evoked response (VER) from baseline to visits 7, 8, and 9 · Brain volume index by MRI and diffusion tensor imaging (DTI) index for myelination, and changes in brain water from baseline to visits 7 and 9 · MRS index for changes in metabolite accumulation from baseline to visits 7 and 9 · Changes in visual target tracking assessment from baseline to visits 4, 5, 6, 7, 8, and 9 The following are evaluated from screening through the end of treatment: · Adverse events are assessed and graded according to NCI CTCAE v5.0 · Physical examination · Vital signs · Standard clinical tests: hematology, clinical chemistry, coagulation, and urine tests · AESI (Adverse Events of Special Interest): Complications related to surgery Complications related to devices Acute allergic reactions Elevated liver function test values (AST / ALT, GGT) Hematological parameter disorders that require particular attention to platelet count Late-onset adverse events including, but not limited to, autoimmune-like reactions and malignancies

[0264] [Example 17] For further details regarding the present disclosure, all of its content is presented in Appendix A of U.S. Patent Provisional Application No. 62 / 840,359, filed on April 29, 2019, which is incorporated herein by reference in its entirety.

[0265] The method of administering AAV into the cisterna magna via a microcatheter was investigated. scAAV9-CB-GFP was injected into sheep (30 kg, n = 3) at 1×10 14 vg via an intravascular microcatheter. The microcatheter was guided through the thoracic and cervical regions of the sheep (Figure 75A) by a fluorescence microscope until the tip of the microcatheter was finally placed (Figure 75B, right panel, arrow). After injection of the contrast agent, 15 ml of scAAV-CB-GFP was injected at 1 ml / min to image the distribution pattern near the cerebellum. The sheep was then sacrificed and macroscopic images of the postmortem spinal cord were observed (Figure 75C). Immunohistochemical staining showed that GFP was expressed in the motor neurons, sensory neurons (Figure 75D), and Purkinje cells, cerebellar peduncles, deep cerebellar nuclei, brainstem, cerebral, occipital cortex, parietal cortex, thalamus, hippocampus, temporal cortex, motor cortex, internal capsule, and prefrontal cortex (Figures 75E - 75G). These results indicate that administration of AAV into the cisterna magna via a microcatheter can be used to efficiently deliver the transgene of interest (e.g., GLB1, HEXA, HEXB, etc.).

[0266] The present disclosure is not limited to the application to the details of the construction and arrangement of the components explicitly described in this description or illustrated in the drawings. The present disclosure is also capable of other embodiments, implementations, or executions in various forms. Also, the phrases and terms used in this specification are for the purpose of description and should not be considered limiting. The use of "including", "comprising", "having", "containing", "accompanying", and variations thereof in this specification is intended to include the items listed hereinafter, and their equivalents, as well as further items.

[0267] In this way, some aspects of at least one embodiment of the present disclosure have been described, but it should be understood by those skilled in the art that various changes, modifications, and improvements can be easily conceived. Such changes, modifications, and improvements are intended to be part of the present disclosure and are intended to be within the spirit and scope of the present disclosure. Therefore, the foregoing description and drawings are for illustrative purposes only.

Claims

1. A recombinant AAV (rAAV) comprising a capsid containing a nucleic acid, wherein the nucleic acid comprises the sequence shown in SEQ ID NO: 18 or 19, said recombinant AAV.

2. The rAAV according to claim 1, wherein the rAAV comprises a capsid protein of a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, and AAVrh10.

3. The rAAV according to claim 1 or 2, wherein the rAAV comprises a capsid protein of the AAVrh8 serotype.

4. The rAAV according to any one of claims 1 to 3, wherein the rAAV comprises one or more ITRs, and each ITR is selected from the group consisting of AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, and AAV6 ITR.

5. The rAAV according to any one of claims 1 to 4, wherein the nucleic acid comprises the sequence shown in SEQ ID NO:

18.

6. The rAAV according to any one of claims 1 to 4, wherein the nucleic acid comprises the sequence shown in SEQ ID NO:

19.

7. (i) a first rAAV comprising a capsid containing a first nucleic acid comprising the sequence shown in SEQ ID NO: 18; and (ii) a second rAAV comprising a capsid containing a second nucleic acid comprising the sequence shown in SEQ ID NO: 19 A pharmaceutical composition comprising the same.

8. The pharmaceutical composition according to claim 7, further comprising a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 7 or 8, wherein the first rAAV and / or the second rAAV comprises a capsid protein of the AAVrh8 serotype.

10. The pharmaceutical composition according to any one of claims 7 to 9, wherein the first rAAV and the second rAAV are present in the composition in a ratio of 1:

1.

11. A pharmaceutical composition for treating lysosomal storage disorders, comprising the recombinant AAV (rAAV) according to any one of claims 1 to 6, or the pharmaceutical composition according to any one of claims 7 to 10, said pharmaceutical composition.

12. The pharmaceutical composition according to claim 11, wherein the lysosomal storage disorder is Tay-Sachs disease or Sandhoff disease.

13. The rAAV or pharmaceutical composition according to claim 11 or 12, which is administered by intracranial injection, intracerebral injection, or injection into the CSF via the ventricular system, cistern, or spinal canal.

14. A kit comprising a container containing a recombinant AAV (rAAV) according to any one of claims 1 to 6.

15. (i) A first container containing a first rAAV, comprising a capsid containing a first nucleic acid comprising the sequence shown in SEQ ID NO: 18; (ii) A second container containing a second rAAV, comprising a capsid containing a second nucleic acid comprising the sequence shown in SEQ ID NO: 19 A kit comprising.

16. The kit according to claim 15, wherein the first rAAV and / or the second rAAV comprises a capsid protein of the AAVrh8 serotype.

17. (iii) The kit according to claim 15 or 16, further comprising a third container containing a pharmaceutically acceptable carrier.

18. The kit according to any one of claims 14 to 16, wherein the first container and / or the second container is a syringe.

19. The kit according to any one of claims 14 to 17, wherein the first container, the second container and / or the third container is a syringe.

20. The pharmaceutical composition according to any one of claims 11 to 13, wherein the AAV is administered by injection into the CSF via the cistern and / or spinal canal.

21. The pharmaceutical composition according to claim 20, wherein the AAV is administered by intracothalamic injection.

22. The pharmaceutical composition according to any one of claims 11 to 13 or 20 or 21, wherein the AAV is administered by injection into the CSF via the cistern and spinal canal and intracothalamic injection.

23. The pharmaceutical composition according to any one of claims 11 to 13 or 20 to 22, wherein the AAV is administered via a fluoroscopy-guided lumbar intrathecal catheter.

24. All rAAV administered to the subject is between 1×10 １１ and 1×10 １５ vector genome (vg) copies, the pharmaceutical composition according to any one of claims 11 to 13 or 20 to 23.

25. The pharmaceutical composition according to any one of claims 11 to 13 or 20 to 24, wherein the subject has a β-hexosaminidase A activity of less than 0.5% of the normal β-hexosaminidase A activity in the CSF of the subject prior to administration of the rAAV or the pharmaceutical composition.

26. The pharmaceutical composition according to any one of claims 11 to 13 or 20 to 25, wherein the subject has an increase in the enzyme activity of β-hexosaminidase A in the CSF that is at least 0.5% of normal enzyme activity 3 months after administration of the rAAV or the pharmaceutical composition.

27. The pharmaceutical composition according to any one of claims 11 to 13 or 20 to 26, wherein the subject has an increase in the enzyme activity of β-hexosaminidase A in the CSF that is at least 2-fold compared to the baseline of the subject prior to administration.

28. A pharmaceutical composition for treating GM2 gangliosidosis, comprising the rAAV according to any one of claims 1 to 6, or the pharmaceutical composition according to any one of claims 7 to 10.

29. The pharmaceutical composition according to any one of claims 11 to 13, 20 to 27, or 28, wherein GM2 ganglioside is reduced by at least 15% from baseline in the cerebrospinal fluid (CSF) of the subject 3 months after administration of the rAAV or the pharmaceutical composition.

30. The dosage administered to the thalamus is between 1×10 １１ and 1×10 １５ vg, and the pharmaceutical composition according to any one of claims 11 to 13, 20 to 27, or 28 or 29.

31. The dosage administered to the large groove and / or the medullary cavity is between 1×10 １１ and 1×10 １５ vg, and the pharmaceutical composition according to any one of claims 11 to 13, 20 to 27, or 28 to 30.

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