Gene therapy for gangliosidosis type 2 (GM2)

Abstract

Provided herein is a recombinant adeno-associated virus (rAAV) comprising an AAV capsid and a multicistronic expression cassette comprising an engineered nucleic acid sequence encoding an β-N-acetylhexosaminidase alpha subunit (HexA), and β-N-acetylhexosaminidase beta subunit (HexB), and optionally ganglioside GM2 activator (GM2A). Also provided are a pharmaceutical composition comprising a rAAV, as described herein, in a formulation buffer, nucleic acid molecule, packaging host cells, rAAV production system, and a method of treating one or more of GM2 gangliosidosis disease, including GM2 activator deficiency, Tay-Sachs disease and / or Sandhoff disease.

Description

[0001] GENE THERAPY FOR GANGLIOSIDOSIS TYPE 2 (GM2)

[0002] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The electronic sequence listing filed herewith named "UPN-23- 10176PCT” with size of 210,554 bytes, created on date of April 2, 2025, and the contents of the electronic sequence listing (e.g., the sequences and text therein) are incorporated herein by reference in entirety.

[0004] BACKGROUND OF THE INVENTION

[0005] GM2 gangliosidoses are a group of rare recessively inherited disorders, which are classified as lysosomal storage disorders (LSD), and which result in progressive destroying of the nerve cells in brain and spinal cord (childrenshospital.org / conditions / gm2-gangliosidosis). GM2 gangliosidosis is characterized by accumulation of the GM2 ganglioside in the lysosome due to a deficiency in the P-hexosaminidase activity resulting in a central nervous system disfunction, including neurodevelopment alterations, neuroinflammation, neuronal apoptosis, motor deficits, progressive weakness and hypotonia, decreased responsiveness, vision deterioration, and seizures (Leal, A.F.. et al.. Int J Mol Sci. 2020 Sep; 21(17): 6213; Lawson C.A., and Martin D.R., The Application of Clinical Genetics 2016:9 111-120).

[0006] GM2 gangliosidosis is often caused by mutations in genes encoding two major isozymes of P-hexosaminidases: P-hexosaminidase S composed of two alpha subunits (HexA or HEXA), P-hexosaminidase A which is composed of alpha subunit (HexA or HEXA) and beta subunit (HexB or HEXB), P-hexosaminidase B which is composed of two beta (HEXB) subunits, or tire GM2 activator protein (GM2A) (Lawson, C.A., and Martin, D.R., The Application of Clinical Genetics, 111-120). There are three major forms of GM2 gangliosidosis including Tay-Sachs disease and its variants caused by HexA deficiency (also called B type). Sandhoff disease (also called tire 0 type) caused by HexB deficiency, and the AB variant caused by an abnormality of the GM2-activator protein (Singer, H.S., et al., 15 - Inherited Metabolic Disorders Associated with Extrapyramidal Symptoms, Movement Disorders in Childhood, W.B. Saunders, 2010, Pages 164-204). Clinical phenotypical presentation of GM2 gangliosidoses ranges from onset as acuteinfantile, sub-acute juvenile, and late-onset adult GM2 gangliosidoses (Toro, C., et al., Neurosci Lett. 2021 Nov 1; 764: 136195). i The current standard of care for Tay-Sachs and Sandhoff disease relieves symptoms, but does not alter the progression of disease. Investigational therapies for GM2 gangliosidosis include hematopoietic stem cell transplantation, enzyme replacement therapy, substrate reduction therapy, pharmacological chaperones, and gene therapy, although the blood brain barrier (BBB) represents a challenge for therapies and therapeutic development.

[0007] While current therapies have led to some control of the disease via management of tire specific symptoms, treatment of gangliosidosis type 2 is a therapeutic challenge and requires improvement as there are no currently approved therapies to reverse the effects of GM2 gangliosidosis.

[0008] SUMMARY OF THE INVENTION

[0009] In one aspect, provided herein is a recombinant adeno-associated virus (rAAV) useful for preventing accumulation of GM2 ganglioside, the rAAV comprising an adeno-associated virus (AAV) capsid and a vector genome in the AAV capsid, wherein the vector genome is a nucleic acid molecule which comprises an expression cassette comprising a nucleic acid sequence encoding 0-N-acetylhexosaminidase alpha subunit (HexA), a nucleic acid sequence encoding 0- N-acetylhexosaminidase beta subunit (HexB), wherein the I lex A subunit and HexB subunit coding sequences are each operably linked to regulatory sequences which permit expression of the HexA subunit and the HexB subunit proteins in a target cell, wherein the expression cassette is a multicistronic expression cassette and comprises: (a) the HexA coding sequence, a 2A linker, and the HexB coding sequence; or (b) the HexA coding sequence, a bi-directional promoter, and the HexB coding sequence. In certain embodiments, tire multicistronic expression cassette is bi- cistronic or tri-cistronic, wherein the tri-cistronic cassette optionally further comprises a nucleic acid sequence encoding ganglioside GM2 activator (GM2A). In certain embodiments, the nucleic acid sequence encoding for HexA comprises nucleic acid sequence of SEQ ID NO: 15, or a sequence at least 95% identical to SEQ ID NO: 15 encoding an amino acid sequence of SEQ ID NO: 16. In certain embodiments, the nucleic acid sequence encoding HexB comprises nucleic acid sequence of SEQ ID NO: 17, or a sequence at least 95% identical to SEQ ID NO: 17 encoding an amino acid sequence of SEQ ID NO: 18. In certain embodiments, the nucleic acid sequence encoding GM2A comprises nucleic acid sequence of SEQ ID NO: 19, or a sequence at least 95% identical to SEQ ID NO: 19 encoding an amino acid sequence of SEQ ID NO: 20. In certain embodiments, the AAV capsid is a clade F AAV capsid, optionally the AAV capsid is an AAVhu68 capsid, AAV9 capsid or a mutant AAV9 capsid.

[0010] In certain embodiment, the multicistronic expression cassette of (a) comprises the 2A linker, wherein the 2A linker is Thosea asigna virus GSG linker (GT2A), Porcine teschovirus- 1 GSG linker (GP2A), or Foot-and-mouth disease virus GSG linker (GF2A). In certain embodiments, the rAAV comprises tire multicistronic expression cassette of (a) comprising: (i) a CAG promoter comprising cytomegalovirus immediate early enhancer, a chicken beta actin promoter, and a chicken beta actin intron, (ii) the HexA coding sequence, (iii) a GT2A linker or GP2A linker, (iv) the HexB coding sequence, and (v) a rabbit beta globin (rBG) polyA signal sequence. In certain embodiments, the expression cassette comprises CAG promoter comprising nucleic acid sequence of SEQ ID NO: 21, a HexA coding sequence of SEQ ID NO: 15, a GP2A linker comprising a nucleic acid sequence of SEQ ID NO: 22, a HexB coding sequence comprising a nucleic acid sequence of SEQ ID NO: 17, and a rBG polyA comprising a nucleic acid sequence of SEQ ID NO: 24, optionally tire expression cassette comprises nucleic acid sequence of SEQ ID NO: 1. In certain embodiment, the expression cassette comprises CAG promoter comprising nucleic acid sequence of SEQ ID NO: 21, a HexA coding sequence of SEQ ID NO: 15, a GT2A linker comprising a nucleic acid sequence of SEQ ID NO: 23, a HexB coding sequence comprising a nucleic acid sequence of SEQ ID NO: 17, and a rBG polyA comprising a nucleic acid sequence of SEQ ID NO: 24, optionally the expression cassette comprises the nucleic acid sequence of SEQ ID NO: 3.

[0011] In certain embodiments. rAAV comprises tire multicistronic expression cassette of (b) comprising: (i) a rabbit beta globin (rBG) polyA signal sequence, (ii) the HexB coding sequence, (iii) a bi-directional promoter, (iv) tire HexA coding sequence, (v) a bovine growth hormone (bGH) polyA signal sequence. In certain embodiments, the bi-directional promoted is A1RC- GPAT bi-directional promoter, POFUT1-PLAGL2 bi-directional promoter, CB-CMV-CB bidirectional promoter, or RH0N1-CMV-F0XM1 bi-directional promoter.

[0012] In certain embodiments, tire expression cassette comprises a rabbit beta globin (rBG) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 24, a HexB coding sequence comprising nucleic acid sequence of SEQ ID NO: 17, a AIRC-GPAT bi-directional promoter comprising nucleic acid sequence of SEQ ID NO: 37, a HexA coding sequence comprising nucleic acid sequence of SEQ ID NO: 15, a bovine growth hormone (bGH) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 25, optionally the expression cassette comprises nucleic acid sequence of SEQ ID NO: 5. In certain embodiments, the expression cassette comprises a rabbit beta globin (rBG) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 24, a HexB coding sequence comprising nucleic acid sequence of SEQ ID NO: 17, a POFUT1-PLAGL2 bi-directional promoter comprising nucleic acid sequence of SEQ ID NO: 31, a HexA coding sequence comprising nucleic acid sequence of SEQ ID NO: 15, a bovine growth hormone (bGH) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 25, optionally the expression cassette comprises nucleic acid sequence of SEQ ID NO: 9. In certain embodiments, the expression cassette comprises a rabbit beta globin (rBG) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 24, a HexB coding sequence comprising nucleic acid sequence of SEQ ID NO: 17, a CB-CMV-CB bidirectional promoter comprising nucleic acid sequence of SEQ ID NO: 28, a HexA coding sequence comprising nucleic acid sequence of SEQ ID NO: 15, a bovine growth hormone (bGH) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 25, optionally the expression cassette comprises nucleic acid sequence of SEQ ID NO: 7. In certain embodiments, the expression cassette comprises a rabbit beta globin (rBG) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 24. a HexB coding sequence comprising nucleic acid sequence of SEQ ID NO: 17, a RHONl-CMV-FOXMlbi-directional promoter comprising nucleic acid sequence of SEQ ID NO: 34, a HexA coding sequence comprising nucleic acid sequence of SEQ ID NO: 15, a bovine growth hormone (bGH) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 25, optionally the expression cassette comprises nucleic acid sequence of SEQ ID NO: 11.

[0013] In certain embodiments, the rAAV comprises the tri-cistronic expression cassette comprising: (i) a bovine growth hormone (bGH) polyA signal sequence, (ii) the GM2A coding sequence, (iii) the bi-directional promoter, (iv) the HexA coding sequence, (v) the GT2A linker, (vi) the HexB coding sequence, and (vii) a rabbit beta globin (rBG) polyA signal sequence, optionally the bi-directional promoter is POFUT1-PLAGL2 bi-directional promoter. In certain embodiments, the expression cassette comprises a bovine growth hormone (bGH) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 25, a GM2A coding sequence comprising nucleic acid sequence of SEQ ID NO: 19, a POFUT1-PLAGL2 bi-directional promoter comprising nucleic acid sequence of SEQ ID NO: 31, a HexA coding sequence comprising nucleic acid sequence of SEQ ID NO: 15, a GT2A linker comprising nucleic acid sequence of SEQ ID NO: 23, a HexB coding sequence comprising nucleic acid sequence of SEQ ID NO: 17, and a rabbit beta globin (rBG) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 24, optionally the expression cassette comprises nucleic acid sequence of SEQ ID NO: 13.

[0014] In a further aspect, provided herein is a composition and pharmaceutical composition comprising a rAAV or a vector as described herein and an aqueous suspension media. In certain embodiments, the rAAV or the composition thereof is for use in tire treatment GM2 gangliosidosis disease, GM2 activator deficiency, Tay-Sachs disease and / or Sandhoff disease. In certain embodiments, the suspension is formulated for intravenous or intrathecal delivery, optionally wherein the intrathecal delivery is an intracerebroventricular (1CV) injection or an intracistemal magna (ICM) injection. In certain embodiments, the rAAV, the composition, and the pharmaceutical composition is for use in preparing a medicament for treatment one or more of GM2 gangliosidosis disease, GM2 activator deficiency, Tay-Sachs disease and / or Sandhoff disease.

[0015] In another aspect, provided herein is a recombinant nucleic acid molecule comprising a vector genome comprising an adeno-associated virus (AAV) 5' inverted terminal repeat (ITR). and expression cassette, and an AAV 3' ITR, wherein the expression cassette comprises nucleic acid sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, or 13. In certain embodiments, tire vector genome comprises nucleic acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, or 14. In certain embodiments, the recombinant nucleic acid molecule is a plasmid.

[0016] In another aspect, provided herein is a packaging host cell comprising a recombinant nucleic acid molecule as described herein. In certain embodiments, the packaging host cell further comprises AAV rep coding sequences operably linked to sequences which express rep protein in the packaging host cell, an AAV capsid coding sequences operably linked to sequences which express AAV capsid proteins in the packaging host cell, and helper virus functions necessary to permit packaging of the expression cassette and AAV ITRs into the AAV capsid. In certain embodiments, a packaging cell is provided which comprises the expression cassette, vector genome or plasmid.

[0017] In another aspect provided herein is a production system useful for producing rAAV as described herein, wherein the production system comprises a cell culture comprising: (a) a nucleic acid sequence encoding a AAV capsid protein; (b) the vector genome; and (c) sufficient AAV rep functions and helper functions to permit packaging of the vector genome into the AAV capsid. In another aspect provided herein is a method for treating GM2 gangliosidosis disease, GM2 activator deficiency, Tay-Sachs disease, and / or Sandhoff disease, said method comprising administrating to the subject a suspension of a rAAV as described herein.

[0018] These and other aspects and embodiments of the invention are apparent from the following detailed description.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1 shows a schematic overview of tire designed rAAV vectors comprising multicistronic expression cassettes.

[0021] FIG. 2A shows a schematic rAAV vector genome (comprising CB-CMV-CB bidirectional promoters). FIG. 2B shows representative microscopy image (mCherry) of liver tissue samples which confirm expression kinetics of rAAV vectors containing CB-CMV-CB bidirectional promoters in adult mouse tissue. FIG. 2C shows representative microscopy image (GFP) of liver tissue samples which confirm expression kinetics of rAAV vectors containing CB- CMV-CB bi-directional promoters in adult mouse tissue. FIG. 2D shows representative microscopy image (in Cherry / GFP overlay) of liver tissue samples which confirm expression kinetics of rAAV vectors containing CB-CMV-CB bi-directional promoters in adult mouse tissue.

[0022] FIG. 3A shows a schematic rAAV vector genome (comprising AI-GP bi-directional promoters). FIG. 3B shows representative microscopy image (mCherry) of liver tissue samples which confirm expression kinetics of rAAV vectors containing AI-GP bi-directional promoters in adult mouse tissue. FIG. 3C shows representative microscopy image (GFP) of liver tissue samples which confirm expression kinetics of rAAV vectors containing AI-GP bi-directional promoters in adult mouse tissue. FIG. 3D shows representative microscopy image (mCherry / GFP overlay) of liver tissue samples which confirm expression kinetics of rAAV vectors containing AI-GP bi-directional promoters in adult mouse tissue.

[0023] FIG. 4A shows a schematic rAAV vector genome (comprising PO-PL bi-directional promoters). FIG. 4B shows representative microscopy image (mCherry) of liver tissue samples which confirm expression kinetics of rAAV vectors containing PO-PL bi-directional promoters in adult mouse tissue. FIG. 4C shows representative microscopy image (GFP) of liver tissue samples which confirm expression kinetics of rAAV vectors containing PO-PL bi-directional promoters in adult mouse tissue. FIG. 4D shows representative microscopy image (mCherry / GFP overlay) of liver tissue samples which confirm expression kinetics of rAAV vectors containing PO-PL bi-directional promoters in adult mouse tissue.

[0024] FIG. 5A shows a schematic rAAV vector genome (comprising RH-CMVe-FO bidirectional promoters). FIG. 5B shows s representative microscopy image (mChcrry) of liver tissue samples which confirm expression kinetics of AAV vectors containing RH-CMVe-FO bidirectional promoters in adult mouse tissue. FIG. 5C shows s representative microscopy image (GFP) of liver tissue samples which confirm expression kinetics of rAAV vectors containing RH- CMVe-FO bi-directional promoters in adult mouse tissue. FIG. 5D shows s representative microscopy image (mCherry / GFP overlay) of liver tissue samples which confirm expression kinetics of rAAV vectors containing RH-CMVe-FO bi-directional promoters in adult mouse tissue.

[0025] FIG. 6A shows a schematic rAAV vector genome (comprising AI-GP bi-directional promoters). FIG. 6B shows representative microscopy image (m Cherry) of quadricep muscle tissue samples which confirm expression kinetics of rAAV vectors containing AI-GP bidirectional promoters in adult mouse tissue. FIG. 6C shows representative microscopy image (GFP) of quadricep muscle tissue samples which confirm expression kinetics of rAAV vectors containing AI-GP bi-directional promoters in adult mouse tissue. FIG. 6D shows representative microscopy image (mCherry / GFP overlay) of quadricep muscle tissue samples which confirm expression kinetics of rAAV vectors containing AI-GP bi-directional promoters in adult mouse tissue.

[0026] FIG. 7A shows a schematic rAAV vector genome (comprising AI-GP bi-directional promoters). FIG. 7B shows representative microscopy image (mCherry) of brain tissue samples which confirm expression kinetics of rAAV vectors containing AI-GP bi-directional promoters in adult mouse tissue. FIG. 7C shows representative microscopy image (GFP) of brain tissue samples which confirm expression kinetics of rAAV vectors containing AI-GP bi-directional promoters in adult mouse tissue. FIG. 7D shows representative microscopy image (mCherry / GFP overlay) of brain tissue samples which confirm expression kinetics of rAAV vectors containing AI-GP bi-directional promoters in adult mouse tissue.

[0027] FIG. 8A shows a schematic representation of evaluated rAAV vectors.

[0028] FIG. 8B shows in vitro hexosaminidase activity analysis from rAAV vectors containing various bi-directional promoters. FIG. 8C shows in vitro hexosaminidase expression (HEXA / HEXB) analysis (western blot) from rAAV vectors containing various bi-directional promoters.

[0029] FIG. 8D shows in vitro hexosaminidase expression (HEXA / HEXB) analysis (quantified from western blot) from rAAV vectors containing various bi-directional promoters.

[0030] FIG. 8E shows hexosaminidase A activity following in vitro transfection of HEXA / B-KO 293T cells with rAAV comprising various BD promoters.

[0031] FIG. 9A shows schematic representation of evaluated rAAV vectors.

[0032] FIG. 9B shows in vitro hexosaminidase activity analysis from rAAV vectors containing various 2A cleavage peptides.

[0033] FIG. 9C shows in vitro hexosaminidase expression analysis (western blot) from rAAV vectors containing various 2A cleavage peptides.

[0034] FIG. 9D shows in vitro hexosaminidase expression analysis (quantified from western blot) from rAAV vectors containing various 2A cleavage peptides.

[0035] FIG. 9E shows hexosaminidase A activity of following in vitro transfection of HEXA / BKO cells with rAAV comprising various 2A peptides.

[0036] FIG. 10A shows results of the behavior study as measured latency to fall (rotarod averages) of WT and HexB homozygous (HOM) mice.

[0037] FIG. 10B shows results of the survival study plotted as percent probability of survival of WT and HexB HOM mice.

[0038] FIG. 11A shows analysis of hexosaminidase activity in serum.

[0039] FIG. 1 IB shows analysis of hexosaminidase activity in brain and liver.

[0040] FIG. 12A shows hexosaminidase A enzyme activity following administration of bicistronic rAAV vectors in mice.

[0041] FIG. 12B shows Kaplan-Meier survival analysis of probability of survival in mice following administration of bicistronic rAAV vectors.

[0042] FIG. 13 shows quantified histological analysis of SD mice brains stained with an anti- GM2 antibody.

[0043] FIG. 14A is a representative image of histological analysis of SD mice brain stained with an anti-GM2 antibody in WT mice. FIG. 14B is a representative image of histological analysis of SD mice brain (midbrain) stained with an anti-GM2 antibody in WT mice. FIG. 14C is a representative image of histological analysis of SD mice brain (cerebellum) stained with an anti- GM2 antibody in WT mice. FIG. 14D is a representative image of histological analysis of SD mice brain (cortex) stained with an anti-GM2 antibody in WT mice. FIG. 14E is a representative image of histological analysis of SD mice brain (hippocampus) stained with an anti-GM2 antibody in WT mice. FIG. 14F is a representative image of histological analysis of SD mice brain (brain stem) stained with an anti-GM2 antibody in WT mice. FIG. 14G is a representative image of histological analysis of SD mice brain (thalamus) stained with an anti-GM2 antibody in WT mice.

[0044] FIG. 15A shows a representative image of histological analysis of SD mice brain stained with an anti-GM2 antibody in KO mice treated with PBS. FIG. 15B is a representative image of histological analysis of SD mice brain (midbrain) stained with an anti-GM2 antibody in KO mice treated with PBS. FIG. 15C is a representative image of histological analysis of SD mice brain (cerebellum) stained with an anti-GM2 antibody in KO mice treated with PBS. FIG. 15D is a representative image of histological analysis of SD mice brain (cortex) stained with an anti-GM2 antibody in KO mice treated with PBS. FIG. 15E is a representative image of histological analysis of SD mice brain (hippocampus) stained with an anti-GM2 antibody in KO mice treated with PBS. FIG. 15F is a representative image of histological analysis of SD mice brain (brain stem) stained with an anti-GM2 antibody in KO mice treated with PBS. FIG. 15G is a representative image of histological analysis of SD mice brain (thalamus) stained with an anti- GM2 antibody in KO mice treated with PBS.

[0045] FIG. 16A shows a representative image of histological analysis of SD mice brain stained with an anti-GM2 antibody in Hexb knock out mice treated with AAVhu68-HEXA-[2Al]-HEXB (GP2A peptide). FIG. 16B is a representative image of histological analysis of SD mice brain (midbrain) stained with an anti-GM2 antibody in Hexb knock out mice treated with AAVhu68- HEXA-[2A1]-HEXB (GP2A peptide). FIG. 16C is a representative image of histological analysis of SD mice brain (cerebellum) stained with an anti-GM2 antibody in Hexb knock out mice treated with AAVhu68-HEXA-[2Al]-HEXB (GP2A peptide). FIG. 16D is a representative image of histological analysis of SD mice brain (cortex) stained with an anti-GM2 antibody in Hexb knock out mice treated with AAVhu68-HEXA-[2Al]-HEXB (GP2A peptide). FIG. 16E is a representative image of histological analysis of SD mice brain (hippocampus) stained with an anti-GM2 antibody in Hexb knock out mice treated with AAVhu68-HEXA-[2Al]-HEXB (GP2A peptide). FIG. 16F is a representative image of histological analysis of SD mice brain (brain stem) stained with an anti-GM2 antibody in Hexb knock out mice treated with AAVhu68-HEXA- [2A1]-HEXB (GP2A peptide). FIG. 16G is a representative image of histological analysis of SD mice brain (thalamus) stained with an anti-GM2 antibody in Hexb knock out mice treated with AAVhu68-HEXA-[2Al]-HEXB (GP2A peptide).

[0046] FIG. 17A shows a representative image of histological analysis of SD mice brain stained with an anti-GM2 antibody in Hexb knock out mice treated with AAVhu68-HEXB-[BD-3]- HEXA (RH-CMVe-FO). FIG. 17B shows a representative image of histological analysis of SD mice brain (midbrain) stained with an anti-GM2 antibody in Hexb knock out mice treated with AAVhu68-HEXB-[BD-3]-HEXA (RH-CMVe-FO). FIG. 17C shows representative image of histological analysis of SD mice brain (cerebellum) stained with an anti-GM2 antibody in Hexb knock out mice treated with AAVhu68-HEXB-(BD-3J-HEXA (RH-CMVe-FO). FIG. 17D is a representative image of histological analysis of SD mice brain (cortex) stained with an anti-GM2 antibody in Hexb knock out mice treated with AAVhu68-HEXB-[BD-3]-HEXA (RH-CMVe- FO). FIG. 17E is a representative image of histological analysis of SD mice brain (hippocampus) stained with an anti-GM2 antibody in Hexb knock out mice treated with AAVhu68-HEXB-[BD- 3]-HEXA (RH-CMVe-FO). FIG. 17F is a representative image of histological analysis of SD mice brain (brain stem) stained with an anti-GM2 antibody in Hexb knock out mice treated with AAVhu68-HEXB-[BD-3]-HEXA (RH-CMVe-FO). FIG. 17G shows representative image of histological analysis of SD mice brain (thalamus) stained with an anti-GM2 antibody in Hexb knock out mice treated with AAVhu68-HEXB-[BD-3]-HEXA (RH-CMVe-FO).

[0047] FIG. 18A shows a representative image of histological analysis of SD mice brain stained with an anti-GM2 antibody in Hexb knock out mice treated with AAV-HexB-CB-CMVe-CB- HexA. FIG. 18B is a representative image of histological analysis of SD mice brain (midbrain) stained with an anti-GM2 antibody in Hexb knock out mice treated with AAV-HexB-CB-CMVe- CB-HexA. FIG. 18C is a representative image of histological analysis of SD mice brain (cerebellum) stained with an anti-GM2 antibody in Hexb knock out mice treated with AAV- HexB-CB-CMVe-CB-HexA. FIG. 18D is a representative image of histological analysis of SD mice brain (cortex) stained with an anti-GM2 antibody in Hexb knock out mice treated with AAV-HexB-CB-CMVe-CB-HexA. FIG. 18E is a representative image of histological analysis of SD mice brain (hippocampus) stained with an anti-GM2 antibody in Hcxb knock out mice treated with AAV-HexB-CB-CMVe-CB-HexA. FIG. 18F is a representative image of histological analysis of SD mice brain (brain stem) stained with an anti-GM2 antibody in Hexb knock out mice treated with AAV-HexB-CB-CMVe-CB-HexA. FIG. 18G is a representative image of histological analysis of SD mice brain (thalamus) stained with an anti-GM2 antibody in Hexb knock out mice treated with AAV-HexB-CB-CMVe-CB-HexA.

[0048] DETAILED DESCRIPTION OF THE INVENTION

[0049] Provided herein are recombinant, replication-defective adeno-associated virus (rAAV) vectors having an AAV capsid and a vector genome comprising an expression cassette which is a multicistronic (also referred to as multi-ci stronic) expression cassette comprising a nucleic acid sequence encoding P-N-acetylhexosaminidase subunit alpha (HexA) and -N- acctylhcxosaminidasc subunit beta (HcxB), and compositions containing the same which arc useful in preventing accumulation of GM2 ganglioside, and / or are suitable for treatment of GM2 gangliosidosis disease, Tay-Sachs disease and / or Sandhoff disease. Additionally, provided herein are recombinant, replication-defective adeno-associated vims (rAAV) vectors having an AAV capsid and a vector genome comprising an expression cassette comprising a nucleic acid sequence encoding P-N-acetylhexosaminidase subunit alpha (HexA), P-N-acetylhexosaminidase submit beta (HexB), and ganglioside GM2 activator (GM2A), and compositions containing the same which are useful in preventing accumulation of GM2 ganglioside, and / or are suitable for treatment of GM2 gangliosidosis disease, and / or GM2 activator deficiency. Various methods of administration are provided, including systemic administration (e.g.. intravenous), and administration to the central nervous system (e.g.. intrathecal administration) for treatment of GM2 gangliosidosis disease including GM2 activator deficiency, Tay-Sachs disease and / or Sandhoff disease. Also provided are pharmaceutical compositions, formulations containing same, and in particularly, an aqueous liquid suspension. Uses of these compositions are also provided. Also provided are method of compositions useful for the treatment and / or prevention of GM2 gangliosidosis disease including GM2 activator deficiency, Tay-Sachs disease and / or Sandhoff disease, and / or alleviating symptoms thereof.

[0050] In certain embodiments, the compositions and regimens described herein are useful for delivery of HexA and HexB to the central nervous system (CNS). In certain embodiments, the compositions and regimens described herein are useful for delivery of HexA, HexB and GM2A to the central nervous system (CNS). In certain embodiments, the nucleic acid sequences provided herein are useful for packaging HexA, HexB, GM2A coding sequence into suitable vector (e.g., rAAV) or a genetic element useful for manufacture (e.g., plasmid). In certain embodiments, an rAAV as provided herein contains a vector genome with a multicistronic expression cassette which has a self-cleaving peptide linker (e.g., a 2A peptide linker). In certain embodiments, an rAAV as provided herein contains a vector genome with a multicistronic expression cassette which has a bi-directional promotor, optionally with a linker. The multi-cistronic expression cassette may be a bi-cistronic (also, referred to as bicistronic) expression cassette. In certain embodiments, the multi-cistronic expression cassette may be a tri- cistronic (also, referred to as tricistronic) expression cassette, optionally with a linker between two subunit coding sequences, to enable expression of two protein subunits in the same reading frame from the bi-directional (also referred to as bidirectional) promoter. In certain embodiments, an rAAV as provided herein contains a vector genome with a multicistronic expression cassette comprising a HexB coding sequence, a bi-directional promoter, a HexA coding sequence. In certain embodiments, an rAAV as provided herein contains a vector genome with a multicistronic expression cassette comprising a HexA coding sequence, a bi-directional promoter, a HexB coding sequence. In certain, embodiments, an rAAV as provided herein contains a vector genome with a multicistronic expression cassette comprising a GM2A coding sequence, a bi-directional promoter, a HexA coding sequence, a 2A peptide linker, a HexB coding sequence. In certain, embodiments, an rAAV as provided herein contains a vector genome with a multicistronic expression cassette comprising a GM2A coding sequence, a bi-directional promoter, a HexB coding sequence, a 2A peptide linker, a HexA coding sequence. In certain, embodiments, an rAAV as provided herein contains a vector genome with a multicistronic expression cassette comprising a polyA signal sequence, a I IcxA coding sequence, a bidirectional promoter, a HexB coding sequence, a 2A peptide linker, a GM2A coding sequence. In certain, embodiments, an rAAV as provided herein contains a vector genome with a multicistronic expression cassette comprising a HexA coding sequence, a bi-directional promoter, a GM2A coding sequence, a 2A peptide linker, a HexB coding sequence. In certain, embodiments, an rAAV as provided herein contains a vector genome with a multicistronic expression cassette comprising a HexB coding sequence, a bi-directional promoter, a HexA coding sequence, a 2A peptide linker, a GM2A coding sequence. In certain, embodiments, an rAAV as provided herein contains a vector genome with a multicistronic expression cassette comprising a HexB coding sequence, a bi-directional promoter, a GM2A coding sequence, a 2A peptide linker, a HexA coding sequence. As used herein, the term “bi-directional promoter”, or “bidirectional promoter” refers to a “promoter” capable of directing transcription in both the forward and reverse orientations. “Bidirectional promoters” can direct the transcription of two transcripts placed in either orientation (i.e., downstream or upstream) of the promoter simultaneously (e.g., the “sense” and “antisense” strands of a gene). In other words, a “bi-directional promoter” directs transcription from either strand of the “promoter” region. Examples of bi-directional promoters are provided herein. In certain embodiments, the bi-directional promoter may further comprise an enhancer.

[0051] In certain embodiments, the bi-directional promoter is AIRC-GPAT (also referred to as AI-GP. or GP-A1. or Al / GP-BDP). A1RC and GPAT encode enzymes in the pathway for de novo purine nucleotide synthesis in vertebrates. The human GPAT and AIRC genes are divergently transcribed from a 778 bp intergenic promoter region. In certain embodiments, the bi-directional promoter is RHN01-F0XM1 (also referred to as RH-FO, or FO-RH, or FO / RH-BDP). F0XM1 (an oncoprotein) and RHN01 (a gene involved in the ATR-Chkl signaling pathway that functions in the DNA replication stress response) are head-to-head oncogenes (i.e., bidirectional) regulated by a bidirectional promoter (named F / R-BDP). In certain embodiments, the bidirectional promoter is RHN01-F0XM1 which further comprises an enhancer. In certain embodiments, the bi-directional promoter is RHN01-F0XM1 which further comprises an enhancer which is a cytomegalovirus (CMV) enhancer (RH-CMVe-FO). In certain embodiments, the bi-directional promoter is POFUT1-PLAGL2 (also referred to as PO-PL, or PL-PO, or PL / PO-BDP). PLAGL2 and POFUT1 are regulated by an evolutionarily conserved bidirectional promoter and are collaboratively involved in colorectal cancer.

[0052] In certain embodiments, a multicistronic nucleic acid include, e.g., at least one of a HexA, HexB or GM2 subunit protein coding sequence (in any order) separated by a bidirectional promoter and / or an intervening 2A peptide coding sequence.

[0053] Multicistronic expression constructs simultaneously express two or more separate proteins from the same mRNA (i.e., a transcript produced from the same promoter). For example, such nucleic acids can comprise coding sequences for two or more reporter proteins separated by an intervening internal ribosome entry site (IRES) or an intervening 2A peptide coding sequence. As one example, such multicistronic vectors can use a bidirectional promoter to allow for initiation of translation in each direction (5‘ and 3’) to the promoter. Additionally or alternatively, such multicistronic vectors can use one or more 2A peptides. These peptides are small “self-cleaving” peptides, generally having a length of 18-22 amino acids and produce approximately equimolar levels of multiple genes from the same mRNA. The "‘cleavage’" occurs between the glycine and proline residues found on the C -terminus, meaning the upstream cistron will have a few additional residues added to the end, while the downstream cistron will start with the proline. As a result, the “cleaved-off ’ downstream peptide may have proline at its N-terminus. 2A-mediated cleavage is a universal phenomenon in all eukaryotic cells. Examples of suitable 2A peptides are provided herein. In certain embodiments, tire 2A peptides derived from foot-and- mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A). See also, Jake Chng, et al. (2015) Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells, mAbs, 7:2, 403-412. which is incorporated herein by reference in its entirety. In certain embodiments, the 2A peptide linker further comprises GSG linker at the N-terminus of 2A peptide. In certain embodiments, the 2A peptide linker further comprises a furin recognition site. A furin recognition site that consists of arginine-lysine-arginine-arginine (RKRR) amino acid sequence may be used. Due to tire mechanism of furin-mediated cleavage, vector-expressed protein may contain an additional arginine (R) residue added to the last position of the amino acid sequence. In certain embodiments, tire 2A peptide linker is a 2A peptide from foot-and-mouth disease vims further comprising a furin recognition site with the GSG linker (GF2A). In certain embodiments, the 2A peptide linker is a 2A peptide from porcine teschovirus- 1 further comprising a furin recognition site with the GSG linker (GP2A). In certain embodiments, the 2A peptide linker is a 2A peptide from Thosea asigna vims further comprising furin recognition site with the GSG linker (GT2A). Other furin cleavage sites can be used (arginine-X-X-arginine. or arginine-X-lysine or argininearginine), which can also generate C-tenninal heterogeneity. In certain embodiments, the vector expressed HexA and HexB may be a heterogeneous population of the proteins in which the HexA and HexB subunits have 0, 1. 2. 3, or 4 amino acids at its C-terminus as a result of the linker processing. In certain embodiments, the vector expressed HexA and HexB may be a heterogeneous population of the proteins in which the HexA and HexB subunits may have independently have 0, 1, and / or 2 amino acids truncation from the self-cleaving 2A peptide, and still forms a functional hexosaminidase A enzyme.

[0054] In certain embodiments, provided herein are coding sequence(s), expression cassette(s), and a vector genome(s) comprising a nucleic acid sequence which encodes HexA amino acid sequence of SEQ ID NO: 16 or an amino acid sequence at least 95% identical thereto. In certain embodiments, the expression cassette comprises a nucleic acid sequence which encodes HexB amino acid sequence of SEQ ID NO: 18 or an amino acid sequence at least identical thereto. In certain embodiments, the expression cassette comprises a nucleic acid sequence which encodes GM2A amino acid sequence of SEQ ID NO: 20 or an amino acid sequence at least identical thereto. In certain embodiments, provided herein are coding sequence(s), expression cassette(s), and a vector genome(s) comprising a nucleic acid sequence which encodes HexA amino acid sequence of SEQ ID NO: 16 or an amino acid sequence at least 95% identical thereto and HexB amino acid sequence of SEQ ID NO: 18 or an amino acid sequence at least identical thereto. In certain embodiments, provided herein are coding sequence(s). expression cassette(s), and a vector genome(s) comprising a nucleic acid sequence which encodes HexA amino acid sequence of SEQ ID NO: 16 or an amino acid sequence identical thereto, HexB amino acid sequence of SEQ ID NO: 18 or an amino acid sequence at least identical thereto, and GM2A amino acid sequence of SEQ ID NO: 20 or an amino acid sequence at least) identical thereto. Provided herein are also coding sequence(s), expression cassette(s), and a vector genome(s) encoding the HexA, wherein the sequence(s), expression cassette(s), or a vector genome(s) comprises engineered nucleic acid sequence comprising SEQ ID NO: 15, or a sequence at least 95% identical to SEQ ID NO: 15 encoding an amino acid sequence of SEQ ID NO: 16. Provided herein are also coding sequence(s), expression cassette(s), and a vector genome(s) encoding the HexB, wherein the sequence(s), expression cassette(s), or a vector genome(s) comprises engineered nucleic acid sequence comprising SEQ ID NO: 17, or a sequence at least 95% identical to SEQ ID NO: 17 encoding an amino acid sequence of SEQ ID NO: 18. Provided herein are also coding sequence(s), expression cassette(s), and a vector genome(s) encoding the GM2A wherein the sequence(s), expression cassette(s), or a vector genome(s) comprises engineered nucleic acid sequence comprising SEQ ID NO: 19, or a sequence at least 95% identical to SEQ ID NO: 19 encoding an amino acid sequence of SEQ ID NO: 20.

[0055] Provided herein are also coding sequence(s), expression cassette(s), and a vector genome(s) encoding the HexA and HexB, wherein the sequence(s), expression cassette(s), or a vector genome(s) comprises nucleic acid sequence comprising SEQ ID NO: 15, or a sequence at least 95% identical to SEQ ID NO: 15 encoding an amino acid sequence of SEQ ID NO: 16, and SEQ ID NO: 17, or a sequence at least 95% identical to SEQ ID NO: 17 encoding an amino acid sequence of SEQ ID NO: 18. Provided herein are also coding sequence(s). expression cassette(s), and a vector genome(s) encoding the HexA. HexB, and GM2A wherein the sequence(s). expression cassette(s), or a vector genome(s) comprises nucleic acid sequence comprising SEQ ID NO: 15, or a sequence at least 95% identical to SEQ ID NO: 15 encoding an amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 17, or a sequence at least 95% identical to SEQ ID NO: 17 encoding an amino acid sequence of SEQ ID NO: 18, and SEQ ID NO: 19, or a sequence at least 95% identical to SEQ ID NO: 19 encoding an amino acid sequence of SEQ ID NO: 20.

[0056] In certain embodiments, provided herein is an rAAV comprising an adeno-associated virus (AAV) capsid and a vector genome in the AAV capsid, wherein the vector genome is a nucleic acid molecule which comprises an expression cassette comprising a nucleic acid sequence encoding 0-N-acetylhexosaminidase alpha subunit (HexA), a nucleic acid sequence encoding 0- N -acetylhexosaminidase beta subunit (HexB). wherein the HexA subunit and HexB subunit coding sequences are each operably linked to regulatory sequences which permit expression of the HexA subunit and the HexB subunit proteins in a target cell, w herein the expression cassette is a multicistronic (bi-cistronic) expression cassette and comprises tire HexA coding sequence, a 2A linker, and the HexB coding sequence.

[0057] In certain embodiments, provided herein is an rAAV comprising an adeno-associated virus (AAV) capsid and a vector genome in the AAV capsid, wherein the vector genome is a nucleic acid molecule w hich comprises an expression cassette comprising a nucleic acid sequence encoding 0-N-acetylhexosaminidase alpha subunit (HexA), a nucleic acid sequence encoding 0- N-acetylhexosaminidase beta subunit (HexB), wherein the HexA subunit and HexB subunit coding sequences are each operably linked to regulatory sequences which permit expression of the HexA subunit and tire HexB subunit proteins in a target cell, wherein the expression cassette is a multicistronic (bi-cistronic) expression cassette and comprises tire HexA coding sequence, a bi-directional promoter, and the HexB coding sequence.

[0058] In certain embodiments, provided herein is an rAAV comprising an adeno-associated virus (AAV) capsid and a vector genome in the AAV capsid, wherein the vector genome is a nucleic acid molecule w hich comprises an expression cassette comprising a nucleic acid sequence encoding 0-N-acetylhexosaminidase alpha subunit (HexA), a nucleic acid sequence encoding 0- N-acetylhexosaminidase beta subunit (HexB), and a nucleic acid sequence encoding ganglioside GM2 activator (GM2A), wherein the HexA subunit, HexB subunit and GM2A coding sequences are each operably linked to regulatory sequences which permit expression of the HexA subunit, the HexB subunit and GM2A proteins in a target cell, wherein the expression cassette is a multicistronic expression (i.e., tri-cistronic) cassette and comprises the GM2A coding sequence, a bidirectional promoter, the HexA coding sequence, a 2A linker, and the HexB coding sequence. In certain embodiments, the 2A linker comprises a furin recognition site and Thosea asigna virus peptide connected via a GSG linker (GT2A), a furin recognition site and Porcine teschovirus- 1 peptide connected via a GSG linker (GP2A), or a furin recognition site and Foot- and-mouth disease virus peptide connected GSG linker (GF2A). In certain embodiments, the GT2A linker comprises nucleic acid sequence of SEQ ID NO: 23, or a sequence at least 95% identical to SEQ ID NO: 23 encoding an amino acid sequence of SEQ ID NO: 39. In certain embodiments, tire GP2A linker comprises nucleic acid sequence of SEQ ID NO: 22, or a sequence at least 95% identical to SEQ ID NO: 22 encoding an amino acid sequence of SEQ ID NO: 38.

[0059] With regard to the description of these various embodiments, it is intended that each of the compositions herein described, is useful, in another embodiment, in the methods of the invention. In addition, it is also intended that each of the compositions herein described as useful in the methods, is, in another embodiment, itself an embodiment of the invention.

[0060] Unless defined otherwise in this specification, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and by reference to published texts, which provide one skilled in the art with a general guide to many of the terms used in the present application.

[0061] Nucleic Acid Molecules

[0062] In one aspect, a recombinant nucleic acid molecule comprising an engineered nucleic acid sequence encoding P-N-acetylhexosaminidase subunit alpha (HexA), and P-N- acetylhexosaminidase subunit beta (HexB) is provided. In another aspect, a recombinant nucleic acid molecule comprising an engineered nucleic acid sequence encoding P-N- acetylhexosaminidase subunit alpha (HexA), P-N-acetylhexosaminidase subunit beta (HexB), and ganglioside GM2 activator (GM2A) is provided. In one embodiment, the engineered sequence is useful to improve production, transcription, expression or safety in a subject. In another embodiment, the engineered sequence is useful to increase efficacy of the resulting therapeutic compositions or treatment. In a further embodiment, the engineered sequence is useful to increase the efficacy of tire HexA and HexB proteins being expressed, but may also pennit a lower dose of a therapeutic reagent that delivers the proteins to increase safety. In a further embodiment, the engineered sequence is useful to increase the efficacy of the HexA, HexB. and GM2A proteins being expressed, but may also permit a lower dose of a therapeutic reagent that delivers the proteins to increase safety.

[0063] In one aspect, provided herein is a recombinant nucleic acid molecule comprising an engineered nucleic acid sequence encoding HexA. In certain embodiments, the recombinant nucleic acid molecule comprises nucleic acid sequence of SEQ ID NO: 15, or a sequence at least 95%identical to SEQ ID NO: 15. In certain embodiments, tire recombinant nucleic acid molecule comprises nucleic acid sequence of SEQ ID NO: 15, or a sequence at least 95% identical to SEQ ID NO: 15 encoding an amino acid sequence of SEQ ID NO: 16. In certain embodiments, the recombinant nucleic acid molecule comprises nucleic acid sequence of SEQ ID NO: 15.

[0064] In one aspect, provided herein is a recombinant nucleic acid molecule comprising an engineered nucleic acid sequence encoding HexB. In certain embodiments, the recombinant nucleic acid molecule comprises nucleic acid sequence of SEQ ID NO: 17, or a sequence at least 95%identical to SEQ ID NO: 17. In certain embodiments, tire recombinant nucleic acid molecule comprises nucleic acid sequence of SEQ ID NO: 17, or a sequence at least 95% identical to SEQ ID NO: 17 encoding an amino acid sequence of SEQ ID NO: 18. In certain embodiments, the recombinant nucleic acid molecule comprises nucleic acid sequence of SEQ ID NO: 17.

[0065] In one aspect, provided herein is a recombinant nucleic acid molecule comprising an engineered nucleic acid sequence encoding GM2A. In certain embodiments, the recombinant nucleic acid molecule comprises nucleic acid sequence of SEQ ID NO: 19, or a sequence at least 95%identical to SEQ ID NO: 19. In certain embodiments, the recombinant nucleic acid molecule comprises nucleic acid sequence of SEQ ID NO: 19, or a sequence at least 95% identical to SEQ ID NO: 19 encoding an amino acid sequence of SEQ ID NO: 20. In certain embodiments, the recombinant nucleic acid molecule comprises nucleic acid sequence of SEQ ID NO: 19.

[0066] In one aspect, provided herein is a recombinant nucleic acid molecule comprising an engineered nucleic acid sequence encoding HexA and HexB, wherein the HexA coding sequence comprises nucleic acid sequence of SEQ ID NO: 15, or a sequence at least 95% identical to SEQ ID NO: 15 encoding an amino acid sequence of SEQ ID NO: 16, and HexB coding sequence comprises nucleic acid sequence of SEQ ID NO: 17, or a sequence at least 95% identical to SEQ ID NO: 17 encoding an amino acid sequence of SEQ ID NO: 18.

[0067] In one aspect, provided herein is a recombinant nucleic acid molecule comprising an engineered nucleic acid sequence encoding HexA, HexB and GM2A, wherein the HexA coding sequence comprises nucleic acid sequence of SEQ ID NO: 15, or a sequence at least 95% identical to SEQ ID NO: 15 encoding an amino acid sequence of SEQ ID NO: 16, HexB coding sequence comprises nucleic acid sequence of SEQ ID NO: 17, or a sequence at least 95% identical to SEQ ID NO: 17 encoding an amino acid sequence of SEQ ID NO: 18, and GM2A coding sequence comprises nucleic acid sequence of SEQ ID NO: 19, or a sequence at least 95% identical to SEQ ID NO: 19 encoding an amino acid sequence of SEQ ID NO: 20.

[0068] In certain embodiments, tire recombinant nucleic acid molecules encoding a HexA, and HexB, or HexA, HexB and GM2A, and oilier constructs encompassed by the present invention and useful in generating expression cassettes and vector genomes may be engineered for expression in yeast cells, insect cells or mammalian cells, such as human cells. Methods are known and have been described previously (e.g., WO 96 / 09378). In certain embodiments, the nucleic acid sequences encoding a HexA, HexB, and / or GM2A described herein are engineered (i.e., assembled and placed) into any suitable genetic element, e.g., naked DNA, phage, transposon, cosmid, episome, etc., which transfers the HexA, HexB, and / or GM2A carried thereon to a host cell, e.g., for generating non-viral delivery systems (e.g., RNA-based systems, naked DNA, or the like), or for generating viral vectors in a packaging host cell, and / or for deli ven’ to a host cells in a subject. In one embodiment, the genetic element is a vector. In one embodiment, the genetic element is a plasmid. The methods used to make such engineered constructs are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0069] In certain embodiments, the recombinant nucleic acid molecule comprises a vector genome comprising an adeno-associated virus (AAV) 5' inverted terminal repeat (ITR), and expression cassette, and an AAV 3' ITR, wherein the expression cassette is a multicistronic expression cassette.

[0070] In certain embodiments, provided herein is a recombinant nucleic acid molecule comprising multicistronic expression cassette comprising the HexA coding sequence, a 2A linker, and HexB coding sequence. In certain embodiments, provided herein is a recombinant nucleic acid molecule comprising a multicistronic expression cassette comprising the HexB coding sequence, a bi-directional promoter, and tire HexA coding sequence. In certain embodiments, provided herein is a recombinant nucleic acid molecule comprising the GM2A coding sequence, a bi-directional promoter, the HexA coding sequence, a 2A linker, and the HexB coding sequence. In certain embodiments, the 2A linker is Thosea asigna virus GSG linker with a furin recognition site (GT2A), Porcine teschovirus- 1 GSG linker with a furin recognition site (GP2A), or Foot-and-mouth disease virus GSG linker with a furin recognition site (GF2A). In certain embodiments, the multicistronic expression cassette comprises a CAG promoter. In certain embodiments, the multicistronic expression cassette comprises a bidirectional promoter, wherein the bidirectional promoter is: (i) an AIRC-GPAT bi-directional promoter, (ii) a POFUT1- PLAGL2 bidirectional promoter comprising a protein O-fucosyltransferase 1 (POFUT1) promoter and pleomorphic adenoma gene-like 2 (PLAGL2) promoter, (iii) a CB-CMV-CB bidirectional promoter comprising a first copy of a chicken beta actin (CB) promoter, a cytomegalovirus (CMV) enhancer, and a second copy of a chicken beta actin (CB) promoter, or (iv) RH0N1-CMV-F0XM1 bidirectional promoter comprising RAD9-HUS1-RAD1 Interacting Nuclear Orphan 1 (RHN01) promoter, a cytomegalovirus (CMV) enhancer, and forkhead box protein Ml (F0XM1) promoter. In certain embodiments, the multicistronic expression cassette comprises a polyadenylation (poly A) signal sequence which is a rabbit beta globin polyA and / or bovine growth hormone (bGH) polyA.

[0071] In certain embodiments, the nucleic acid molecule comprises multicistronic expression cassette comprising a CAG promoter comprising nucleic acid sequence of SEQ ID NO: 21, a HexA coding sequence of SEQ ID NO: 15, a GP2A linker comprising a nucleic acid sequence of SEQ ID NO: 22, a HexB coding sequence comprising a nucleic acid sequence of SEQ ID NO: 17, and a rBG polyA comprising a nucleic acid sequence of SEQ ID NO: 24.

[0072] In certain embodiments, tire nucleic acid molecule comprises multicistronic expression cassette comprising a CAG promoter comprising nucleic acid sequence of SEQ ID NO: 21, a HexA coding sequence of SEQ ID NO: 15, a GT2A linker comprising a nucleic acid sequence of SEQ ID NO: 23, a HexB coding sequence comprising a nucleic acid sequence of SEQ ID NO: 17. and a rBG polyA comprising a nucleic acid sequence of SEQ ID NO: 24.

[0073] In certain embodiments, tire nucleic acid molecule comprises multicistronic expression cassette comprising a rabbit beta globin (rBG) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 24, a HexB coding sequence comprising nucleic acid sequence of SEQ ID NO: 17, a AIRC-GPAT bi-directional promoter comprising nucleic acid sequence of SEQ ID NO: 37, a HexA coding sequence comprising nucleic acid sequence of SEQ ID NO: 15, a bovine growth hormone (bGH) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 25. In certain embodiments, the nucleic acid molecule comprises multicistronic expression cassette comprising a rabbit beta globin (rBG) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 24, a HexB coding sequence comprising nucleic acid sequence of SEQ ID NO: 17, a POFUT1-PLAGL2 bi-directional promoter comprising nucleic acid sequence of SEQ ID NO: 31, a HexA coding sequence comprising nucleic acid sequence of SEQ ID NO: 15, a bovine growth hormone (bGH) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 25.

[0074] In certain embodiments, the nucleic acid molecule comprises multicistronic expression cassette comprising a rabbit beta globin (rBG) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 24, a HexB coding sequence comprising nucleic acid sequence of SEQ ID NO: 17, a CB-CMV-CB bi-directional promoter comprising nucleic acid sequence of SEQ ID NO: 28, a HexA coding sequence comprising nucleic acid sequence of SEQ ID NO: 15, a bovine growth hormone (bGH) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 25.

[0075] In certain embodiments, the nucleic acid molecule comprises multicistronic expression cassette comprising a rabbit beta globin (rBG) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 24, a HexB coding sequence comprising nucleic acid sequence of SEQ ID NO: 17, a RHONl-CMV-FOXMlbi-directional promoter comprising nucleic acid sequence of SEQ ID NO: 34, a HexA coding sequence comprising nucleic acid sequence of SEQ ID NO: 15, a bovine growth hormone (bGH) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 25.

[0076] In certain embodiments, the nucleic acid molecule comprises multicistronic expression cassette comprising a bovine growth hormone (bGH) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 25. a GM2A coding sequence comprising nucleic acid sequence of SEQ ID NO: 19, a POFUT1-PLAGL2 bi-directional promoter comprising nucleic acid sequence of SEQ ID NO: 31, a HexA coding sequence comprising nucleic acid sequence of SEQ ID NO: 15, a GT2A linker comprising nucleic acid sequence of SEQ ID NO: 23, a HexB coding sequence comprising nucleic acid sequence of SEQ ID NO: 17, and a rabbit beta globin (rBG) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 24.

[0077] In certain embodiments, the recombinant nucleic acid molecule comprises an expression cassette comprising nucleic acid sequence of SEQ ID NOs: 1, 3. 5, 7, 9, 11, or 13. In certain embodiments, the recombinant nucleic acid molecule comprises a vector genome comprising nucleic acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, or 14.

[0078] In certain embodiments, the recombinant nucleic acid molecule optionally further comprises miRNA. In certain embodiments, the miRNA is a dorsal root ganglion (drg)-specific miRNA target sequence. In certain embodiments, the nucleic acid sequence further comprises at least one, at least two, at least three or preferably at least four tandem repeats of dorsal root ganglion (drg)-specific miRNA target sequences. In certain embodiments, the nucleic acid sequence further comprises at least one. at least two, at least three, at least four, at least five, at least six, at least seven or preferably at least eight tandem repeats of dorsal root ganglion (drg)- specific miRNA target sequences. See, e.g., PCT / US 19 / 67872, filed December 20, 2019, and now published as WO 2020 / 132455, which are incorporated herein by reference. See, also, International Patent Application No. PCT / US21 / 32003, filed May 12, 2021, and now published WO2021 / 231579A1, which are incorporated herein by reference. See also, US Provisional Patent Application No. 63 / 279,561, filed November 15, 2021, which is incorporated herein by reference in its entirety.

[0079] A ‘"nucleic acid” or “nucleotide sequence”, as described herein, are nucleotides which are linked to each other repeatedly (e.g., in strands by formation of bonds, such as phosphodiester bonds), and which can be RNA, DNA, or a modification thereof, and can be single or double stranded, and can be selected, for example, from a group including nucleic acid encoding a protein of interest, oligonucleotides, nucleic acid analogues, for example peptide -nucleic acid (PNA), pseudocomplementary PNA (pc-PNA), locked nucleic acid (LNA) etc. A “nucleotide” refers to a ribonucleotide, deoxy nucleotide or a modified form of either type of nucleotide (e.g., a peptide nucleic acid oligomer). The skilled man will appreciate that functional variants of these nucleic acid molecules are also intended to be a part of the present invention. Functional variants are nucleic acid sequences that can be directly translated, using the standard genetic code, to provide an amino acid sequence identical to that translated from the parental nucleic acid molecules. Such nucleic acid sequences include, for example, but are not limited to, nucleic acid sequence encoding proteins, for example that act as transcriptional repressors, antisense molecules, ribozymes, small inhibitory nucleic acid sequences, for example but are not limited to RNAi, shRNAi, siRNA, micro RNAi (mRNAi). antisense oligonucleotides etc.

[0080] It will be understood to one of skill in the art that when the nucleotide sequences are provided such as DNA (i.e., sense DNA), it also includes and encompasses sequences of complimentary DNA (i.e., anti-sense) thereof, complimentary RNA thereof, including mRNA. tRNA, miRNA. A term '‘complementary” refers to the ability of nucleotide sequence to hybridize and form base pairs (e.g., DNA duplex) in the Watson-Crick manner (e.g., adenine (A) to thymine (T), A to uracil (U) (i.e., in RNA), cytosine (C) to guanine (G), or in any other manner that allows for the formation of duplexes). Base pairs are typically formed by hydrogen bonds between nucleotide units forming antiparallel nucleotide strands. For example, in addition to providing the coding sequence of SEQ ID NO: 15, 17 or 19 complimentary DNA sequences thereof and complementary RNA sequences therefor are included and incorporated herein.

[0081] A sequence is considered engineered if the design of at least one codon is intentionally changed as compared to a native or reference (e.g., wild type (WT)) sequence is replaced by a codon that is more preferred for use in the expression cassette, e.g., for expression from the vector genome. Unless otherwise specified, a “nucleic acid sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleic acid sequences can be cloned using routine molecular biology techniques, or generated de novo by DNA synthesis, which can be performed using routine procedures by service companies having business in the field of DNA synthesis and / or molecular cloning (e.g., GeneArt, GenScript, Life Technologies, Eurofins).

[0082] It should be understood that the HexA, HexB, and / or GM2A coding sequences described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.

[0083] Expression Cassette and Vector Genome

[0084] A gene therapy vector is provided herein which comprises an expression cassette which is a multicistronic expression comprising an engineered nucleic acid sequence for P-N - acetylhexosaminidase alpha subunit (HexA), and a nucleic acid sequence encoding p-N- acetylhexosaminidase beta subunit (HexB), wherein the HexA and HexB coding sequences are each operably linked to regulatory sequences which permit expression of HexA subunit and HexB subunit proteins in a target cell. Also provided herein is a gene therapy vector which comprises an expression cassette which is a multicistronic comprising P-N-acetylhexosaminidase alpha subunit (HexA), a nucleic acid sequence encoding P-N-acetylhexosaminidase beta subunit (HexB), and ganglioside GM2 activator (GM2A). wherein the HexA, HexB and GM2A coding sequences are each operably linked to regulatory sequences which permit expression of HexA subunit, HexB subunit, and GM2A proteins in a target cell. Provided herein also is a recombinant nucleic acid molecule comprising expression cassette as described herein. Provided herein also is a recombinant nucleic acid molecule comprising vector genome as described herein.

[0085] In certain embodiments, tire expression cassette is a multicistronic expression cassette comprising nucleic acid sequence comprising the HexA coding sequence, a 2A linker, and tire HexB coding sequence. In certain embodiments, the expression cassette is a multicistronic expression cassette comprising nucleic acid sequence comprising the HexA coding sequence, a bi-directional promoter, and the HexB coding sequence. In certain embodiments, the expression cassette is a multicistronic expression cassette comprising nucleic acid sequence comprising the GM2A coding sequence, a bi-directional promoter, the HexA coding sequence, a 2A linker, and the HexB coding sequence.

[0086] In certain embodiments, tire regulatory sequences comprise a promoter. In certain embodiments the selected promoter is a constitutive promoter. In certain embodiments, the promoter is a ubiquitous promoter. For example such promoters may include chicken beta-actin (CB) promoter, human cytomegalovirus (CMV) promoter, ubiquitin C promoter (UbC), the early and late promoters of simian virus 40 (SV40). U6 promoter, metallothionein promoters, EFla promoter, ubiquitin promoter, hypoxanthine phosphoribosyl transferase (HPRT) promoter, dihydrofolate reductase (DHFR) promoter (Scharfmann et al., Proc. Natl. Acad. Sci. USA 88:4626-4630 (1991), adenosine deaminase promoter, phosphoglycerol kinase (PGK) promoter, pyruvate kinase promoter phosphoglycerol mutase promoter, the P-actin promoter (Lai et al., Proc. Natl. Acad. Sci. USA 86: 10006-10010 (1989)), tire long terminal repeats (LTR) of Moloney Leukemia Virus and other retroviruses, tire thymidine kinase promoter of Herpes Simplex Virus and other constitutive promoters known to those of skill in the art. In certain embodiment, the promoter is a chicken P-actin (also referred to as chicken beta-actin, CB or CBA) promoter. A variety of chicken beta-actin promoters have been described alone, or in combination with various enhancer elements (e.g., CB7, also referred to as hybrid CB7) promoter comprising a cytomegalovirus immediate-early (CMV IE) enhancer (CMVe) and the chicken P- actin promoter, optionally with spacer sequence, optionally with a chimeric intron comprising chicken beta actin intron and further comprising a chicken beta-actin splicing donor (including the exon sequence, chicken beta actin intron) and rabbit beta-globin splicing acceptor), or a CBh promoter |SJ Gray et al, Hu Gene Ther, 201 1 Sep; 22(9): 143-1 153], In certain embodiments, a chicken beta actin promoter is a CAG promoter comprising cytomegalovirus (CMV) enhancer, a chicken beta actin promoter, and a hybrid intron comprising a chicken beta actin intron. In certain embodiments, the promoter is a tissue specific promoter. In certain embodiments, the promoter is a Synapsin 1 promoter (see, e.g., Kugler S et al, Human synapsin 1 gene promoter confers highly neuron-specific long-term transgene expression from an adenoviral vector in the adult rat brain depending on tire transduced area. Gene Ther. 2003 Feb;10(4):337-47), a neuron- specific enolase (NSE) promoter (see, e.g., Kim J et al, Involvement of cholesterol-rich lipid rafts in interleukin-6- induced neuroendocrine differentiation of LNCaP prostate cancer cells. Endocrinology . 2004 Feb;145(2):613-9. Epub 2003 Oct 16), or a CB6 promoter (see, e.g.. Large-Scale Production of Adeno-Associated Viral Vector Serotype-9 Carrying the Human Survival Motor Neuron Gene, Mol Biotechnol. 2016 Jan;58(l):30-6. doi: 10. 1007 / s 12033-015-9899-5). In still other embodiments, multiple enhancers and / or promoters may be included.

[0087] In certain embodiments, the regulatory sequences comprise one or more of a promoter, an enhancer, an intron, a transcription factor, a transcription terminator, an efficient RNA processing signals such as splicing and polyadenylation site signals (poly A), a sequences that stabilize cytoplasmic mRNA, for example Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE). and sequences that enhance translation efficiency (i.e., Kozak consensus sequence). In certain embodiments, the polyA is a synthetic polyA or from bovine growth hormone (bGH), human growth hormone (hGH), SV40, rabbit 0-globin (RBG or rBG), or modified RBG (mRBG). Optionally, one or more sequences may be selected to stabilize mRNA. An example of such a sequence is a modified WPRE sequence, which may be engineered upstream of the polyA sequence and downstream of tire coding sequence [see, e.g., MA Zanta- Boussif, et al. Gene Therapy (2009) 16: 605-619],

[0088] In certain embodiments, the expression cassette comprises one or more expression enhancers. In one embodiment, the expression cassette contains two or more expression enhancers. These enhancers may be the same or may differ from one another. In a further embodiment, the enhancer(s) is selected from one or more of an APB enhancer, an ABPS enhancer, an alpha mic / bik enhancer, a TTR enhancer, an en34 enhancer, an ApoE enhancer, a CMV IE enhancer, or an RSV enhancer. In yet another embodiment, the regulatory’ elements comprise an intron. In a further embodiment, the intron is selected from chicken beta actin intron (CBA), human beta globin, IVS2, SV40 (Promega), chimeric intron available from Promega®, bGH. alpha-globulin, beta-globulin, collagen, ovalbumin, or p53. See. e.g., WO 2011 / 126808. In certain embodiments, the expression cassettes may include one or more expression enhancers such as post-transcriptional regulatory element from hepatitis viruses of woodchuck (WPRE), human (HPRE), ground squirrel (GPRE) or arctic ground squirrel (AGSPRE), or a synthetic post-transcriptional regulatory element. These expression-enhancing elements are particularly advantageous when placed in a 3' UTR and can significantly increase mRNA stability and / or protein yield. In certain embodiments, the expressions cassettes provided include a regulator sequence that is a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) or a variant thereof. Suitable WPRE sequences are provided in the vector genomes described herein and are known in the art (e.g., such as those are described in US Patent Nos. 6,136,597, 6,287,814, and 7,419,829, which are incorporated by reference). In certain embodiments, the WPRE is a variant that has been mutated to eliminate expression of the woodchuck hepatitis B virus X (WHX) protein, including, for example, mutations in tire start codon of the WHX gene. See also, Kingsman S.M., Mitrophanous K., & Olsen J.C. (2005), “Potential Oncogene Activity of the Woodchuck Hepatitis Post-Transcriptional Regulatory Element (Wpre)” Gene Ther. 12(l):3-4: and Zanta-Boussif M.A., Charrier S., Brice-Ouzet A., Martin S., Opolon P.. Thrasher A.J., Hope T.J., & Galy A. (2009). Validation of a Mutated PreSequence Allowing High and Sustained Transgene Expression While Abrogating Whv-X Protein Synthesis: Application to the Gene Therapy of Was, Gene Ther. 16(5): 605- 19, both of which are incorporated herein by reference in its entirety. In other embodiments, enhancers are selected from a non-viral source. In certain embodiments, no WPRE sequence is present.

[0089] In certain embodiments, tire regulatory sequence further comprises an intron. In a further embodiment, the intron is a chicken beta-actin intron (GenBank # X00182. 1). In certain embodiments, the intron is a hybrid intron consisting of a human beta-globin splice donor and immunoglobulin G (IgG) splice acceptor elements. In certain embodiments, the intron is a hybrid intron comprising a chicken beta actin intron, and rabbit betal globin acceptor and rabbit beta globin exon sequence. In another embodiment, a chimeric intron available from Promega® is used. Other suitable introns include those known in the art may by a human P-globulin intron, and / or a commercially available intron, and those described in WO 2011 / 126808.

[0090] In certain embodiments, the promoter is a CAG promoter comprising a Cytomegalovirus (CMV) enhancer (CMVe), a chicken beta actin (CB) promoter, and a hybrid intron comprising a chicken beta actin intron, and optionally comprising linking sequences. In certain embodiments, the CMV enhancer comprises nucleic acid sequence of SEQ ID NO: 27. In certain embodiments, the CB promoter comprises nucleic acid sequence of SEQ ID NO: 26. In certain embodiments, the chicken beta actin intron comprises nucleic acid sequence of SEQ ID NO: 35. In certain embodiments, the hybrid intron comprises nucleic acid sequence of SEQ ID NO: 36. In certain embodiments, the CAG promoter comprises nucleic acid sequence of SEQ ID NO: 21.

[0091] In certain embodiments, tire promoter is a bidirectional promoter. In certain embodiments, the bidirectional promoter AIRC-GPAT bidirectional promoter. In certain embodiments, tire AIRC-GPAT (AI-GP, or GP-AI) bidirectional promoter comprises nucleic acid sequence of SEQ ID NO: 37. In certain embodiments, bi-directional promoter is a POFUT1-PLAGL2 (PO-PL or PL-PO) bidirectional promoter comprising a protein O-fucosyltransferase 1 (POFUT 1) promoter and pleomorphic adenoma gene-like 2 (PLAGL2) promoter. In certain embodiments, the POFUT1-PLAGL2 bidirectional promoter comprises nucleic acid sequence of SEQ ID NO: 31.

[0092] In certain embodiments, the bidirectional promoter is a hybrid bidirectional promoter further comprising an enhancer. In certain embodiments, the bidirectional promoter is a CB- CMV-CB bidirectional promoter comprising a first copy of a chicken beta actin (CB) promoter, a cytomegalovirus (CMV) enhancer, and a second copy of a chicken beta actin (CB) promoter. In certain embodiments, the CB-CMV-CB bidirectional promoter comprises nucleic acid sequence of SEQ ID NO: 28. In certain embodiments, the bidirectional promoter is a RH0N1-CMV- F0XM1 (RH-CMVe-FO) bidirectional promoter comprising RAD9-HUS1-RAD1 Interacting Nuclear Orphan 1 (RHN01) promoter, a cytomegalovirus (CMV) enhancer, and forkhead box protein Ml (F0XM1) promoter. In certain embodiments, the RH0N1-CMV-F0XM1 bidirectional promoter comprises nucleic acid sequence of SEQ ID NO: 34.

[0093] In certain embodiments, the polyA is a rabbit beta globin polyA. In certain embodiments, the rabbit beta globin polyA comprises nucleic acid sequence of SEQ ID NO: 24. In certain embodiments, tire polyA is a bovine growth hormone polyA. In certain embodiments, the bovine growth hormone polyA comprises nucleic acid sequence of SEQ ID NO: 25.

[0094] In certain embodiments, tire expression cassette comprises a HexA and HexB coding sequences and may include other regulatory sequences therefor. The regulator) sequences necessary arc operably linked to the HexA and HexB coding sequences in a manner which permits its transcription, translation and / or expression in target cell. In certain embodiments, the expression cassette comprises a HexA, HexB and GM2A coding sequences and may include other regulatory sequences therefor. The regulatory sequences necessary are operably linked to the HexA, HexB and GM2A coding sequences in a manner which permits its transcription, translation and / or expression in target cell.

[0095] In certain embodiment, the target cell may be a central nervous system cell. In certain embodiments, the target cell is one or more of an excitatory neuron, an inhibitory neuron, a glial cell, a cortex cell, a frontal cortex cell, a cerebral cortex cell, a spinal cord cell. In certain embodiments, the target cell is in leptomeninges (LM) of the CNS. In certain embodiments, the target cell is in parenchyma of CNS.

[0096] In certain embodiments, the expression cassette is a multicistronic expression cassette (i.e. , bi-cistronic) comprising a promoter, optionally an enhancer, optionally an intron, a hexA coding sequence, a 2A linker, a HexB coding sequence, and a polyA signal sequence. In certain embodiments, the expression cassette comprises CAG promoter, a Kozak sequence, a HexA coding sequence, a 2A linker, a HexB coding sequence, and a rBG polyA. In certain embodiments, the expression cassette comprises CAG promoter, a Kozak sequence, a HexA coding sequence, a GT2A linker, a HexB coding sequence, and a rBG polyA. In certain embodiments, tire expression cassette comprises CAG promoter, a Kozak sequence, a HexA coding sequence, a GP2A linker, a HexB coding sequence, and a rBG polyA. In certain embodiments, the expression cassette comprises CAG promoter comprising nucleic acid sequence of SEQ ID NO: 21, a Kozak sequence, a HexA coding sequence of SEQ ID NO: 15, a GP2A linker comprising a nucleic acid sequence of SEQ ID NO: 22, a HexB coding sequence comprising a nucleic acid sequence of SEQ ID NO: 17, and a rBG polyA comprising a nucleic acid sequence of SEQ ID NO: 24. In certain embodiments, the expression cassette comprises CAG promoter comprising nucleic acid sequence of SEQ ID NO: 21, a HexA coding sequence of SEQ ID NO: 15, a GP2A linker comprising a nucleic acid sequence of SEQ ID NO: 22, a HexB coding sequence comprising a nucleic acid sequence of SEQ ID NO: 17, and a rBG polyA comprising a nucleic acid sequence of SEQ ID NO: 24. In certain embodiments, the expression cassette comprises CAG promoter comprising nucleic acid sequence of SEQ ID NO: 21, a HexA coding sequence of SEQ ID NO: 15, a GT2A linker comprising a nucleic acid sequence of SEQ ID NO: 23, a HexB coding sequence comprising a nucleic acid sequence of SEQ ID NO: 17, and a rBG polyA comprising a nucleic acid sequence of SEQ ID NO: 24. In certain embodiments, the expression cassette comprises CAG promoter comprising nucleic acid sequence of SEQ ID NO: 21, a Kozak sequence, a HexA coding sequence of SEQ ID NO: 15. a GT2A linker comprising a nucleic acid sequence of SEQ ID NO: 23, a HexB coding sequence comprising a nucleic acid sequence of SEQ ID NO: 17, and a rBG polyA comprising a nucleic acid sequence of SEQ ID NO: 24.

[0097] In certain embodiments, the expression cassette is a multicistronic (i.e., bi-cistronic) expression cassette comprising a polyA signal sequence, a HexB coding sequence, a bidirectional promoter, a HexA coding sequence, and a polyA signal sequence. In certain embodiments, the expression cassette comprises a rBG polyA signal sequence, a HexB coding sequence, a bidirectional promoter, a HexA coding sequence, and a bGH polyA signal sequence.

[0098] In certain embodiments, the expression cassette is a multicistronic expression (i.e.. bi- cistronic) cassette comprising a rBG polyA signal sequence, a HexB coding sequence, an A1RC- GPAT bidirectional promoter, a HexA coding sequence, and a bGH polyA signal sequence. In certain embodiments, the expression cassette comprises a rBG polyA signal sequence, a HexB coding sequence, a Kozak sequence, an AIRC-GPAT bidirectional promoter, a Kozak sequence, a HexA coding sequence, and a bGH polyA signal sequence. In certain embodiments, tire expression cassette comprises a rabbit beta globin (rBG) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 24, a HexB coding sequence comprising nucleic acid sequence of SEQ ID NO: 17, an AIRC-GPAT bi-directional promoter comprising nucleic acid sequence of SEQ ID NO: 37, a HexA coding sequence comprising nucleic acid sequence of SEQ ID NO: 15, a bovine growth hormone (bGH) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 25. In certain embodiments, the expression cassette comprises a rabbit beta globin (rBG) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 24, a HexB coding sequence comprising nucleic acid sequence of SEQ ID NO: 17, a Kozak sequence, an AIRC-GPAT bi-directional promoter comprising nucleic acid sequence of SEQ ID NO: 37, a Kozak sequence, a HexA coding sequence comprising nucleic acid sequence of SEQ ID NO: 15, a bovine growth hormone (bGH) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 25.

[0099] In certain embodiments, tire expression cassette is a multicistronic (i.e., bi-cistronic) expression cassette comprising a rBG polyA signal sequence, a HexB coding sequence, a POFUT1-PLAGL2 bidirectional promoter, a HexA coding sequence, and a bGH polyA signal sequence. In certain embodiments, tire expression cassette comprises a rBG polyA signal sequence, a HexB coding sequence, a Kozak sequence, a POFUT 1-PLAGL2 bidirectional promoter, a Kozak sequence, a HexA coding sequence, and a bGH polyA signal sequence. In certain embodiments, the expression cassette comprises a rabbit beta globin (rBG) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 24, a HexB coding sequence comprising nucleic acid sequence of SEQ ID NO: 17, a POFUT1-PLAGL2 bi-directional promoter comprising nucleic acid sequence of SEQ ID NO: 31, a HexA coding sequence comprising nucleic acid sequence of SEQ ID NO: 15, a bovine growth hormone (bGH) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 25. In certain embodiments, the expression cassette comprises a rabbit beta globin (rBG) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 24, a HexB coding sequence comprising nucleic acid sequence of SEQ ID NO: 17, a Kozak sequence, a POFUT1-PLAGL2 bi-directional promoter comprising nucleic acid sequence of SEQ ID NO: 31. a Kozak sequence, a HexA coding sequence comprising nucleic acid sequence of SEQ ID NO: 15, a bovine growth hormone (bGH) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 25.

[0100] In certain embodiments, tire expression cassette is a multicistronic (i.e., bi-cistronic) expression cassette comprising a rBG polyA signal sequence, a HexB coding sequence, a bidirectional promoter comprising an enhancer, a HexA coding sequence, and a bGH polyA signal sequence. In certain embodiments, the expression cassette comprises a rBG polyA signal sequence, a HexB coding sequence, a Kozak sequence, a bidirectional promoter comprising an enhancer, a Kozak sequence, a HexA coding sequence, and a bGH polyA signal sequence.

[0101] In certain embodiments, the expression cassette is a multicistronic (i.e., bi-cistronic) expression cassette comprising a rBG polyA signal sequence, a HexB coding sequence, a RHON 1-CMV-F0XM1 bidirectional promoter, a HexA coding sequence, and a bGH polyA signal sequence. In certain embodiments, the expression cassette comprises a rBG polyA signal sequence, a HexB coding sequence, a Kozak sequence, a RHON 1-CMV-F0XM1 bidirectional promoter, a Kozak sequence, a HexA coding sequence, and a bGH polyA signal sequence. In certain embodiments, the expression cassette comprises a rabbit beta globin (rBG) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 24, a HexB coding sequence comprising nucleic acid sequence of SEQ ID NO: 17, a RHONl-CMV-FOXMlbidirectional promoter comprising nucleic acid sequence of SEQ ID NO: 34, a HexA coding sequence comprising nucleic acid sequence of SEQ ID NO: 15, a bovine growth hormone (bGH) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 25. In certain embodiments, the expression cassette comprises a rabbit beta globin (rBG) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 24. a HexB coding sequence comprising nucleic acid sequence of SEQ ID NO: 17, a Kozak sequence, a RH0N1-CMV-F0XM1 bidirectional promoter comprising nucleic acid sequence of SEQ ID NO: 34, a Kozak sequence, a HexA coding sequence comprising nucleic acid sequence of SEQ ID NO: 15, a bovine growth hormone (bGH) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 25.

[0102] In certain embodiments, tire expression cassette is a multicistronic (i.e., bi-cistronic) expression cassette comprising a rBG polyA signal sequence, a HexB coding sequence, a CB- CMV-CB bidirectional promoter, a HexA coding sequence, and a bGH polyA signal sequence. In certain embodiments, the expression cassette comprises a rBG polyA signal sequence, a HexB coding sequence, a Kozak sequence, a CB-CMV-CB bidirectional promoter, a Kozak sequence, a HexA coding sequence, and a bGH polyA signal sequence. In certain embodiments, the expression cassette comprises a rabbit beta globin (rBG) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 24, a HexB coding sequence comprising nucleic acid sequence of SEQ ID NO: 17, a CB-CMV-CB bi-directional promoter comprising nucleic acid sequence of SEQ ID NO: 28, a HexA coding sequence comprising nucleic acid sequence of SEQ ID NO: 15, a bovine growth hormone (bGH) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 25. In certain embodiments, the expression cassette comprises a rabbit beta globin (rBG) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 24, a HexB coding sequence comprising nucleic acid sequence of SEQ ID NO: 17, a Kozak sequence, a CB-CMV-CB bi-directional promoter comprising nucleic acid sequence of SEQ ID NO: 28, a Kozak sequence, a HexA coding sequence comprising nucleic acid sequence of SEQ ID NO: 15, a bovine growth hormone (bGH) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 25.

[0103] In certain embodiments, the expression cassette is a multicistronic (i.e., tri-cistronic) expression cassette comprising a polyA signal sequence, a GM2A coding sequence, a bidirectional promoter, a HexA coding sequence, a 2A linker, a HexB coding sequence, and a polyA signal sequence. In certain embodiments, the expression cassette comprises a bovine growth hormone (bGH) polyA signal sequence, a GM2A coding sequence, a bi-directional promoter, a HexA coding sequence, a GT2A linker, a HexB coding sequence, and a rabbit beta globin (rBG) polyA signal sequence. In certain embodiments, the expression cassette comprises a bovine growth hormone (bGH) polyA signal sequence, a GM2A coding sequence, a POFUT1- PLAGL2 bidirectional promoter, a HexA coding sequence, a GT2A linker, a HexB coding sequence, and a rabbit beta globin (rBG) polyA signal sequence. In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 1 or a sequence at least 95%identical thereto. In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 1 or a sequence at least 99% identical thereto. In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 1.

[0104] In certain embodiments, tire expression cassette comprises nucleic acid sequence of SEQ ID NO: 3 or a sequence at least 95%identical thereto. In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 3 or a sequence at least 99% identical thereto. In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 3.

[0105] In certain embodiments, tire expression cassette comprises nucleic acid sequence of SEQ ID NO: 5 or a sequence at least 95%identical thereto. In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 5 or a sequence at least 99% identical thereto. In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 5.

[0106] In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 7 or a sequence at least 95%identical thereto. In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 7 or a sequence at least 99% identical thereto. In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 7.

[0107] In certain embodiments, tire expression cassette comprises nucleic acid sequence of SEQ ID NO: 9 or a sequence at least 95%identical thereto. In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 9 or a sequence at least 99% identical thereto. In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 9.

[0108] In certain embodiments, tire expression cassette comprises nucleic acid sequence of SEQ ID NO: 11 or a sequence at least 95%identical thereto. In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 11 or a sequence at least 99% identical thereto. In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 11.

[0109] In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 13 or a sequence at least 95%identical thereto. In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 13 or a sequence at least 99% identical thereto. In certain embodiments, the expression cassette comprises nucleic acid sequence of SEQ ID NO: 13.

[0110] In a further aspect, provided herein is a vector genome comprising an AAV 5' inverted terminal repeat (ITR), an expression cassette as described herein, and an AAV3' ITR.

[0111] As used herein, a ‘‘vector genome’’ refers to the nucleic acid sequence packaged inside a parvovirus (e.g., rAAV) capsid which forms a viral particle. Such a nucleic acid sequence contains AAV inverted terminal repeat sequences (ITRs). In the examples herein, a vector genome contains, at a minimum, from 5' to 3'. an AAV 5’ ITR (also referred to as "AAV 5’ ITR”. ■‘5’ ITR”, “AAV 5' ITR”, or “5' ITR”), coding sequence(s) (i.e., transgene(s)), and an AAV 3’ ITR (also referred to as “AAV 3’ ITR”, “3’ ITR”, “AAV 3' ITR”, or “3' ITR”). ITRs from AAV2, a different source AAV than the capsid, or other than full-length ITRs may be selected. In certain embodiments, tire ITRs are from tire same AAV source as the AAV which provides the rep function during production or a transcomplementing AAV. Further, other ITRs, e.g., self- complementary (scAAV) ITRs, may be used. Both single -stranded AAV and self-complementary (sc) AAV are encompassed with the rAAV. The transgene is a nucleic acid coding sequence, heterologous to the vector sequences, which encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor) or other gene product, of interest. The nucleic acid coding sequence is operatively linked to regulatory components in a maimer which permits transgene transcription, translation, and / or expression in a cell of a target tissue. Suitable components of a vector genome are discussed in more detail herein. In one example, a “vector genome” contains, at a minimum, from 5’ to 3’, a vector-specific sequence, a nucleic acid sequence encoding HexA, HexB, GM2A operably linked to regulatory control sequences (which direct their expression in a target cell), where the vector-specific sequence may be a terminal repeat sequence which specifically packages the vector genome into a viral vector capsid or envelope protein. For example, AAV inverted terminal repeats are utilized for packaging into AAV and certain other parvovirus capsids. In certain embodiments, the vector genome is an expression cassette having inverted terminal repeat (ITR) sequences necessary for packaging the vector genome into the AAV capsid at the extreme 5’ and 3‘ end and containing therebetween a HexA, HexB. GM2A genes as described herein operably linked to sequences which direct expression thereof. In certain embodiments, a vector genome may comprise at a minimum from 5’ to 3’, an AAV 5’ ITR, coding sequence(s), and an AAV 3’ ITR. In certain embodiments, the ITRs are from AAV2, a different source AAV than the capsid, or other than full-length ITRs may be selected. In certain embodiments, the ITRs are from the same AAV source as the AAV which provides the rep function during production or a transcomplementing AAV. Further, other ITRs may be used.

[0112] The AAV sequences of the vector typically comprise the cis-acting 5' and 3' inverted terminal repeat sequences (See, e.g., B. J. Carter, in “Handbook of Parvoviruses'’, ed., P. Tijsser, CRC Press, pp. 155 168 (1990)). The ITR sequences are about 145 bp in length. Preferably, substantially the entire sequences encoding the ITRs are used in the molecule, although some degree of minor modification of these sequences is permissible. The ability to modify these ITR sequences is within the skill of the art. (See, e.g., texts 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 employed is a “cis- acting” plasmid containing the transgene, in which the selected transgene sequence and associated regulatory elements are flanked by the 5' and 3' AAV ITR sequences. In one embodiment, the ITRs are from an AAV different than that supplying a capsid. In one embodiment, the ITR sequences from AAV2. However. ITRs from other AAV sources may be selected. A shortened version of the 5 ’ ITR, termed AITR, has been described in which the D- sequence and terminal resolution site (trs) are deleted. In certain embodiments, the vector genome includes a shortened AAV2 ITR of 130 base pairs, wherein the external A elements is deleted. Without wishing to be bound by theory, it is believed that the shortened ITR reverts back to the wild-type length of 145 base pairs during vector DNA amplification using the internal (A’) element as a template. In other embodiments, full-length AAV 5’ and 3‘ ITRs are used. Where the source of the ITRs is from AAV2 and the AAV capsid is from another AAV source, the resulting vector may be termed pseudotyped. However, other configurations of these elements may be suitable.

[0113] It should be understood that the compositions in the expression cassette and vector genome described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.

[0114] Recombinant Adeno-associated Virus (rAAV)

[0115] Provided herein is a recombinant adeno-associated virus (rAAV) comprising an engineered nucleic acid sequence encoding P-N-acetylhexosaminidase alpha subunit (HexA), and an engineered nucleic acid sequence encoding P-N-acetylhexosaminidase beta subunit. Provided herein also is a recombinant adeno-associated virus (rAAV) comprising an engineered nucleic acid sequence encoding P-N-acetylhexosaminidase alpha subunit (HexA), an engineered nucleic acid sequence encoding P-N-acetylhexosaminidase beta subunit (HexB), and engineered nucleic acid sequence encoding ganglioside GM2 activator (GM2A). Provided herein also is a recombinant adeno-associated virus (rAAV) useful for preventing accumulation of GM2 ganglioside Provided herein also is a recombinant adeno-associated virus (rAAV) useful for treating GM2 gangliosidosis disease including GM2 activator deficiency, Tay-Sachs disease and / or Sandhoff disease. The rAAV comprises (a) an AAV capsid; and (b) a vector genome packaged in the AAV capsid of (a). Suitably, the AAV capsid selected targets the cells to be treated. In certain embodiments, the capsid is from Clade F. However, in certain embodiments, another AAV capsid source may be selected, i.e., Clade A. In certain embodiments, the AAV capsid is an AAVhu68 capsid. In certain embodiments, the AAV capsid is an AAVrh91 capsid. In certain embodiments, the AAV capsid is an AAVhu95 capsid. In certain embodiments, tire AAV capsid is an AAV9 capsid. In certain embodiments, the AAV capsid is a mutant AAV9 capsid. In certain embodiments, the AAV capsid is AAVhu96 capsid.

[0116] In certain embodiments, the provided herein is rAAV comprising a nucleic acid molecule comprising a vector genome comprising at least one AAV ITR at the extreme 5' and / or extreme 3' end of the nucleic acid molecule which is the vector genome and an expression cassette. In certain embodiments, the vector genome is a nucleic acid molecule which comprises a 5' - AAV ITR (AAV 5' ITR), tire expression cassette and a 3'- AAV ITR (AAV 3' ITR). In certain embodiments, the orientation of the ITRs may change from the orientation presented in tire vector genome of the nucleic acid used in production (e.g., a plasmid). Thus, in certain embodiments, the rAAV may comprise a vector genome flanked by 3' and 5' AAV ITRs. respectively. In certain embodiments, the rAAV may comprise a vector genome flanked by two 5' AAV ITRs. In certain embodiments, the rAAV may comprise a vector genome flanked by two 3' AAV ITRs. In other embodiments, an rAAV as provided herein may be partially truncated such that the 5' AAV ITR and / or the 3' AAV ITR is not detectable in the vector genome packaged in a final rAAV product.

[0117] The vector genome comprises an AAV 5’ inverted terminal repeat (ITR), an expression cassette comprising engineered coding sequences comprising sequences encoding HexA subunit and HexB subunit proteins and nucleic acid sequences operably linked thereto which regulate expression of the HexA and HexB subunit proteins, and an AAV 3’ ITR. The vector genome comprises an AAV 5’ inverted terminal repeat (ITR), an expression cassette comprising engineered coding sequences comprising sequences encoding HexA subunit, HexB subunit and GM2A proteins and nucleic acid sequences operably linked thereto which regulate expression of the HexA subunit, HexB subunit, and GM2A proteins, and an AAV 3’ ITR.

[0118] In one embodiment, rAAV comprises an AAV capsid and a vector genome packaged therein which comprises at least one element heterologous to AAV capsid. In one embodiment, the vector genome comprises, from 5' to 3': an AAV ITR, an expression cassette as describes herein, and an AAV ITR.

[0119] In certain embodiments, the rAAV comprises vector genome comprising a nucleic acid molecule comprising, 5' to 3', an AAV ITR, a promoter, a HexA coding sequence, a 2A linker, a HexB coding sequence, a polyA signal sequence, and an AAV ITR. In certain embodiments, tire rAAV comprises vector genome comprising a nucleic acid molecule comprising an AAV ITR, a CAG promoter, a HexA coding sequence, a 2A linker, a HexB coding sequence, a polyA signal sequence, and an AAV ITR. In certain embodiments, tire rAAV comprises vector genome comprising a nucleic acid molecule comprising an AAV ITR, a CAG promoter, a Kozak sequence, a HexA coding sequence, a 2A linker, a HexB coding sequence, a polyA signal sequence, and an AAV ITR. In certain embodiments, the rAAV comprises vector genome comprising a nucleic acid molecule comprising an AAV ITR, a CAG promoter, a Kozak sequence, a HexA coding sequence, a GT2A linker, a HexB coding sequence, a polyA signal sequence, and an AAV ITR. In certain embodiments, the rAAV comprises vector genome comprising a nucleic acid molecule comprising an AAV ITR, a CAG promoter, a Kozak sequence, a HexA coding sequence, a GP2A linker, a HexB coding sequence, a polyA signal sequence, and an AAV ITR.

[0120] In certain embodiments, the rAAV comprises vector genome comprising a nucleic acid molecule comprising an AAV ITR, a polyA, a HexB coding sequence, a bidirectional promoter, a HexB coding sequence, a polyA signal sequence, and an AAV ITR. In certain embodiments, the rAAV comprises vector genome comprising a nucleic acid molecule comprising an AAV ITR, an rBG polyA, a HexB coding sequence, a bidirectional promoter, a HexB coding sequence, a bGH polyA signal sequence, and an AAV ITR. In certain embodiments, the rAAV comprises vector genome comprising a nucleic acid molecule comprising an AAV ITR, an rBG polyA, a HexB coding sequence, a Kozak sequence, a bidirectional promoter, a Kozak sequence, a HexB coding sequence, a bGH polyA signal sequence, and an AAV ITR. In certain embodiments, the rAAV comprises vector genome comprising a nucleic acid molecule comprising an AAV ITR, an rBG polyA, a HexB coding sequence, a Kozak sequence, an AIRC-GPAT bidirectional promoter, a Kozak sequence, a HexB coding sequence, a bGH polyA signal sequence, and an AAV ITR. In certain embodiments, the rAAV comprises vector genome comprising a nucleic acid molecule comprising an AAV ITR, an rBG polyA, a HexB coding sequence, a Kozak sequence, a POFUT1-PLAGL2 bidirectional promoter, a Kozak sequence, a HexB coding sequence, a bGH polyA signal sequence, and an AAV ITR. In certain embodiments, the rAAV comprises vector genome comprising a nucleic acid molecule comprising an AAV ITR. an rBG polyA, a HexB coding sequence, a Kozak sequence, a CB-CMV-CB bidirectional promoter, a Kozak sequence, a HexB coding sequence, a bGH polyA signal sequence, and an AAV ITR. In certain embodiments, the rAAV comprises vector genome comprising a nucleic acid molecule comprising an AAV ITR, an rBG polyA, a HexB coding sequence, a Kozak sequence, a RH0N1- CMV-F0XM1 bidirectional promoter, a Kozak sequence, a HexB coding sequence, a bGH polyA signal sequence, and an AAV ITR. In certain embodiments, the rAAV comprises vector genome comprising a nucleic acid molecule comprising an AAV ITR, an bGH polyA, a GM2A coding sequence, a bidirectional promoter, a HexA coding sequence, a GT2A linker, a HexB coding sequence, a rBG polyA signal sequence, and an AAV ITR.

[0121] In certain embodiments, the rAAV comprises a vector genome comprising nucleic acid molecule comprising nucleic acid sequence of SEQ ID NO: 2 or a sequence of at least 95%identical to SEQ ID NO: 2. In certain embodiments, the rAAV comprises a vector genome comprising nucleic acid molecule comprising nucleic acid sequence of SEQ ID NO: 2 or a sequence of at least 99% identical to SEQ ID NO: 2. In certain embodiments, the rAAV comprises a vector genome comprising nucleic acid molecule comprising nucleic acid sequence of SEQ ID NO: 2.

[0122] In certain embodiments, the rAAV comprises a vector genome comprising nucleic acid molecule comprising nucleic acid sequence of SEQ ID NO: 4 or a sequence of at least 95%, identical to SEQ ID NO: 4. In certain embodiments, the rAAV comprises a vector genome comprising nucleic acid molecule comprising nucleic acid sequence of SEQ ID NO: 4 or a sequence of at least 99% identical to SEQ ID NO: 4. In certain embodiments, the rAAV comprises a vector genome comprising nucleic acid molecule comprising nucleic acid sequence of SEQ ID NO: 4. In certain embodiments, the rAAV comprises a vector genome comprising nucleic acid molecule comprising nucleic acid sequence of SEQ ID NO: 6 or a sequence of at least 95%identical to SEQ ID NO: 6. In certain embodiments, the rAAV comprises a vector genome comprising nucleic acid molecule comprising nucleic acid sequence of SEQ ID NO: 6 or a sequence of at least 99% identical to SEQ ID NO: 6. In certain embodiments, the rAAV comprises a vector genome comprising nucleic acid molecule comprising nucleic acid sequence of SEQ ID NO: 6.

[0123] In certain embodiments, the rAAV comprises a vector genome comprising nucleic acid molecule comprising nucleic acid sequence of SEQ ID NO: 8 or a sequence of at least 95%identical to SEQ ID NO: 8. In certain embodiments, the rAAV comprises a vector genome comprising nucleic acid molecule comprising nucleic acid sequence of SEQ ID NO: 8 or a sequence of at least 99% identical to SEQ ID NO: 8. In certain embodiments, the rAAV comprises a vector genome comprising nucleic acid molecule comprising nucleic acid sequence of SEQ ID NO: 8.

[0124] In certain embodiments, the rAAV comprises a vector genome comprising nucleic acid molecule comprising nucleic acid sequence of SEQ ID NO: 10 or a sequence of at least 95%identical to SEQ ID NO: 10. In certain embodiments, the rAAV comprises a vector genome comprising nucleic acid molecule comprising nucleic acid sequence of SEQ ID NO: 10 or a sequence of at least 99% identical to SEQ ID NO: 10. In certain embodiments, tire rAAV comprises a vector genome comprising nucleic acid molecule comprising nucleic acid sequence of SEQ ID NO: 10.

[0125] In certain embodiments, the rAAV comprises a vector genome comprising nucleic acid molecule comprising nucleic acid sequence of SEQ ID NO: 12 or a sequence of at least 95%identical to SEQ ID NO: 12. In certain embodiments, the rAAV comprises a vector genome comprising nucleic acid molecule comprising nucleic acid sequence of SEQ ID NO: 12 or a sequence of at least 99% identical to SEQ ID NO: 12. In certain embodiments, tire rAAV comprises a vector genome comprising nucleic acid molecule comprising nucleic acid sequence of SEQ ID NO: 12.

[0126] In certain embodiments, the rAAV comprises a vector genome comprising nucleic acid molecule comprising nucleic acid sequence of SEQ ID NO: 14 or a sequence of at least 95%identical to SEQ ID NO: 14. In certain embodiments, the rAAV comprises a vector genome comprising nucleic acid molecule comprising nucleic acid sequence of SEQ ID NO: 14 or a sequence of at least 99% identical to SEQ ID NO: 14. In certain embodiments, the rAAV comprises a vector genome comprising nucleic acid molecule comprising nucleic acid sequence of SEQ ID NO: 14.

[0127] In certain embodiments, tire rAAV comprises AAV capsid which is a clad F AAV capsid.

[0128] As used herein, the term “clade” as it relates to groups of AAV refers to a group of AAV which are phylogenetically related to one another as determined using a Neighbor-Joining algorithm by a bootstrap value of at least 75% (of at least 1000 replicates) and a Poisson correction distance measurement of no more than 0.05, based on alignment of the AAV vpl amino acid sequence. The Neighbor- Joining algorithm has been described in tire literature. See. e.g., M. Nei and S. Kumar, Molecular Evolution and Phylogenetics (Oxford University Press, New York (2000). Computer programs are available that can be used to implement this algorithm. For example, the MEGA v2.1 program implements the modified Nei-Gojobori method. Using these techniques and computer programs, and the sequence of an AAV vpl capsid protein, one of skill in tire art can readily determine whether a selected AAV is contained in one of the clades identified herein, in another clade, or is outside these clades. See, e.g., G Gao, et al, J Virol, 2004 Jun; 78(10): 6381-6388, which identifies Clades A. B, C, D, E and F, and provides nucleic acid sequences of novel AAV, GenBank Accession Numbers AY530553 to AY530629. See, also, WO 2005 / 033321 .

[0129] In certain embodiments, the Clade F AAV capsid is an AAVhu68 capsid [See, e.g., US2020 / 0056159; PCT / US21 / 55436; SEQ ID NO: 40 and 41 for nucleic acid sequence; SEQ ID NO: 42 for amino acid sequence], an AAVhu95 capsid [See, e.g., US Provisional Application No. 63 / 251,599, filed October 2, 2201, International Patent Application No. PCT / US2022 / 077315, filed September 30. 2022: SEQ ID NOs: 43 and 44 (hu95 nucleic acid sequence)] and SEQ ID NO: 45 (hu95 amino acid sequence), or an AAVhu96 capsid [See. e.g., US Provisional Application No. 63 / 251,599, filed October 2, 2201, and International Patent Application No. PCT / US2022 / 077315, filed September 30, 2022; SEQ ID NOs: 46 and 47 (hu96 nucleic acid sequence) and SEQ ID NO: 48 (hu96 amino acid sequence)], AAV9 capsid (SEQ ID NO: 49 for nucleic acid sequence; SEQ ID NO: 50 for amino acid sequence) [Sec, e.g., US 7,906,111] or engineered mutants and variants thereof [see, e.g., W02020 / 200499; W02003 / 054197], See also, International Patent Application No. PCT / US2022 / 077315, filed September 30, 2022, which is incorporated herein by reference in its entirety. See also, US Provisional Patent Application No. 63 / 387,941, filed December 17, 2022, and US Provisional Patent Application No. 63 / 514,404 filed July 19, 2023, now WO 2024 / 130067, and US Provisional Application No. 63 / 598.718, now PCT / US24 / 55910, WO 2022159892, WO_2021092300, W0_2022020616, WO_2023196967, WO 2024173802, which are incorporated herein by reference in its entirety.

[0130] In certain embodiment, the AAV capsid is an AAVhu68 capsid. In certain embodiments the AAV capsid is an AAV9 capsid. In certain embodiments the AAV capsid is an AAVhu95 capsid. In certain embodiments, tire AAV capsid is an AAVhu96 capsid. In certain embodiments the AAV capsid is a mutant AAV9 capsid. See also. International Patent Application No. PCT7US2021 / 061312, filed December 1, 2021 (now published as WO 2022 / 119871 A2), and International Patent Application No. PCT / US2022 / 025879, filed April 22. 2022 (now published as WO 2022 / 226263 Al) which are all incorporated herein by reference in their entireties.

[0131] In certain embodiments, the AAV capsid for tire compositions and methods described herein is chosen based on the target cell. In certain embodiment, the AAV capsid transduces a CNS cell and / or a PNS cell. In certain embodiments, other AAV capsid may be chosen, the AAV capsid is selected from a cy02 capsid, a rh43 capsid, an AAV8 capsid, a rhOl capsid, an AAV9 capsid, a rh8 capsid, a rhlO capsid, a bbOl capsid, a hu37 capsid, a rh02 capsid, a rh20 capsid, a rh39 capsid, a rh64 capsid, an AAV6 capsid, an AAV 1 capsid, a hu44 capsid, a hu48 capsid, a cy05 capsid a hul 1 capsid, a hu32 capsid, a pi2 capsid, or a variation thereof. In certain embodiments, the AAV capsid is a Clade F capsid, such as AAV9 capsid, AAVhu68 capsid, hu31 capsid, hu32 capsid, or a variation thereof. See, e.g., WO 2005 / 033321 published April 14, 2015, WO 2018 / 160582, and US 2015 / 0079038, each of which is incorporated herein by reference in its entirety. In certain embodiments, the AAV capsid is a non-clade F capsid, for example a Clade A, B, C, D, or E capsid. In certain embodiment, the non-Clade F capsid is an AAV 1 or a variation thereof. In certain embodiment, the AAV capsid transduces a target cell other than tire nervous system cells. In certain embodiments, the AAV capsid is a Clade A capsid (e.g.. AAV1, AAV6, AAVrh91), a Clade B capsid (e.g., AAV 2), a Clade C capsid (e.g., hu53), a Clade D capsid (e.g., AAV7), or a Clade E capsid (e.g., rhlO). In certain embodiments, the AAV capsid is an AAVrh32 / 33 capsid. See also, US 10,947,561 B2, which is incorporated herein by reference in its entirety.

[0132] A rAAV is composed of an AAV capsid and a vector genome. An AAV capsid is an assembly of a heterogeneous population of vpl, a heterogeneous population of vp2, and a heterogeneous population of vp3 proteins. As used herein when used to refer to vp capsid proteins, the term '‘heterogeneous’’ or any grammatical variation thereof, refers to a population consisting of elements that are not the same, for example, having vpl, vp2 or vp3 (also referenced as VP1, VP2, VP3, or Vpl, Vp2, Vp3) monomers (proteins) with different modified amino acid sequences. The term “heterogeneous population” as used in connection with vpl, vp2 and vp3 proteins (alternatively termed isoforms), refers to differences in the amino acid sequence of the vpl, vp2 and vp3 proteins within a capsid. The AAV capsid contains subpopulations within the vpl proteins, within the vp2 proteins and within tire vp3 proteins which have modifications from the predicted amino acid residues. These subpopulations include, at a minimum, certain deamidated asparagine (N or Asn) residues. For example, certain subpopulations comprise at least one. two, three or four highly deamidated asparagines (N) positions in asparagine - glycine pairs and optionally further comprising other deamidated amino acids, wherein the deamidation results in an amino acid change and other optional modifications.

[0133] In certain embodiments, AAV capsids are provided which have a heterogeneous population of AAV capsid isofonns (i.e., VP1, VP2, VP3) which contain multiple highly deamidated “NG” positions. In certain embodiments, the highly deamidated positions are in the locations identified below, with reference to the predicted full-length VP1 amino acid sequence. In other embodiments, the capsid gene is modified such that the referenced “NG” is ablated and a mutant “NG” is engineered into another position.

[0134] In certain embodiments, the mutant capsids described herein are characterized by having a deamidation pattern similar to their parental AAV, e.g., such as described in US 2020 / 0056159, published Feb 20, 2020 (AAVhu68; highly deamidated in N57, N329, N452 and N512), with minor optional amounts of deamidation); US 2020 / 0407750, published Dec 31, 2020 (AAV9, highly deamidated in N57, N329, N452 andN512), each of which is incorporated herein by reference.

[0135] In certain embodiments, the mutant AAV capsid comprises an exogenous targeting peptide which is immediately preceded by flanking amino acids which are mutated, as compared to parental AAV capsid (e.g., AAV9 capsid). In certain embodiments, the mutated flanking amino acids, together with 1, 2, 3, 4, 5, or 6 inserted amino acids comprise the exogenous targeting peptide. In certain embodiments, the entirety of the exogenous targeting peptide is inserted into the parental AAV capsid. In still other embodiments, the sequence inserted into a capsid may comprise all or a fragment of the exogenous targeting peptide at the carboxy (COO-) or amino terminus (N-) (i.e., via insertion of the 5' or 3' coding sequences therefor) and further comprises 0-3 flanking amino acid residues as provided in the above formulae. In certain embodiments, engineered rAAV capsids comprising the targeting peptides, as provided herein, demonstrate reduced transduction (i.e., de-targeted / ing) to liver as compared to its parental capsid (e.g., AAV9 or another clade F capsid (e.g., AAVhu68)).

[0136] As used herein, the terms “target cell” and “target tissue” can refer to any cell or tissue which is intended to be transduced by the subject AAV vector. The term may refer to any one or more of muscle, liver, lung, airway epithelium, central nervous system, neurons, eye (ocular cells), or heart. In certain embodiments, the target cells is a central nervous system (CNS) cell.

[0137] Additionally, provided herein, is an rAAV production system useful for producing a rAAV as described herein. The production system comprises a cell culture comprising (a) a nucleic acid sequence encoding an AAV capsid protein; (b) the vector genome; and (c) sufficient AAV rep functions and helper functions to permit packaging of the vector genome into the AAV capsid. In certain embodiments, tire vector genome is SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14. In certain embodiments, the cell culture is bacterial cell culture. In certain embodiments, the cell culture is mammalian cell culture. In certain embodiments, tire cell culture is a human embry onic kidney 293 (HEK293) cell culture. In certain embodiments, the cell culture is a suspension cell culture. In certain embodiments, the AAV rep is from a different AAV. In certain embodiments, wherein the AAV rep is from AAV2. In certain embodiments, the AAV rep coding sequence and cap genes are on the same nucleic acid molecule, wherein there is optionally a spacer between the rep sequence and cap gene.

[0138] For use in producing an AAV viral vector (e.g., a recombinant (r) AAV), the vector genomes can be carried on any suitable vector, e.g., a plasmid, which is delivered to a packaging host cell. The plasmids useful in this invention may be engineered such that they are suitable for replication and packaging in vitro in prokaryotic cells, insect cells, mammalian cells, among others. Suitable transfection techniques and packaging host cells are known and / or can be readily designed by one of skill in the art.

[0139] In certain embodiments, a recombinant nucleic acid molecule is a plasmid. In certain embodiments, a recombinant nucleic acid molecule (e.g., plasmid) is useful in rAAV production. In certain embodiments, a recombinant nucleic acid molecule (e.g., a plasmid) useful in rAAV production comprises a vector genome comprising expression cassette comprising nucleic acid sequence of SEQ ID NO: 1. 3, 5, 7, 9. 11, or 13. In certain embodiments, a recombinant nucleic acid molecule (e.g.. a plasmid) useful in rAAV production comprises a vector genome comprising nucleic acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14. Methods for generating and isolating AAVs suitable for use as vectors are known in the art. See generally, e.g., Grieger & Samulski, 2005, Adeno-associated virus as a gene therapy vector: Vector development, production and clinical applications, Adv. Biochem. Engin / Biotechnol. 99: 119-145; Buning et al., 2008, Recent developments in adeno-associated virus vector technology, J. Gene Med. 10:717-733; and the references cited below, each of which is incorporated herein by reference in its entirety. As used herein, a gene therapy vector refers to a rAAV as described herein, which is suitable for use in treating a patient. For packaging a gene into virions, the ITRs are the only AAV components required in cis in the same construct as the nucleic acid molecule containing the gene. The cap and rep genes can be supplied in trans.

[0140] In one embodiment, the expression cassettes described herein are engineered into a genetic element (e.g., a shuttle plasmid) which transfers the immunoglobulin construct sequences carried thereon into a packaging host cell for production of a viral vector. In one embodiment, the selected genetic element may be delivered to an AAV packaging cell by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity- DNA-coated pellets, viral infection and protoplast fusion. Stable AAV packaging cells can also be made. The methods used to make such constructs are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Molecular Cloning: A Laboratory' Manual, ed. Green and Sambrook, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0141] The term “AAV intermediate” or “AAV vector intermediate” refers to an assembled rAAV capsid which lacks tire desired genomic sequences packaged therein. These may also be termed an “empty” capsid. Such a capsid may contain no detectable genomic sequences of an expression cassette, or only partially packaged genomic sequences which are insufficient to achieve expression of the gene product. These empty capsids are non-functional to transfer the gene of interest to a host cell.

[0142] The recombinant adeno-associated virus (AAV) described herein may be generated using techniques which are known. See, e.g., WO 2003 / 042397; WO 2005 / 033321, WO 2006 / 110689; US 7588772 B2. Such a method involves culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid protein; a functional rep gene; an expression cassette composed of, at a minimum, AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to permit packaging of the expression cassette into the AAV capsid protein. Methods of generating the capsid, coding sequences therefor, and methods for production of rAAV viral vectors have been described. See, e.g., Gao, et al. Proc. Natl. Acad. Sci. U.S.A. 100 (10), 6081-6086 (2003) and US 2013 / 0045186A1. In certain embodiment, the rAAV are generated (manufactured) using triple transfection techniques. In certain embodiments the rAAV are generated using a stable mammalian cell line. In certain embodiments, the stable cell line comprises one or more of: (a) a first plurality of polynucleotide molecules which comprise a coding sequence for at least one adeno-associated virus (AAV) replicase (Rep) protein necessary for production of a replication-defective rAAV vector (Rep52 and Rep78), wherein said rep proteins coding sequences are operably linked to a doxycycline-inducible promoter which directs expression of the rep proteins in the cell line; (b) at least a second plurality of polynucleotide molecules each encoding adenovirus (Ad) helper proteins necessary for production of a replication-defective rAAV vector comprising at least an Ad E2A DNA Binding Protein (DBP) coding sequence, and Ad E4ORF6 coding sequence, wherein the Ad E2A DBP coding sequences and tire Ad E4ORF6 coding sequences are operably linked to a doxycycline- inducible promoter which direct expression of the Ad helper proteins in tire cell line; (c) a nucleic acid molecule comprising an Ad E 1 coding sequence operably linked to a constitutive promoter which directs expression of the Ad El in the cell line; (d) at least a third plurality of nucleic acid molecules each of which comprises an AAV VP 1 coding sequence which encodes AAV VP 1 proteins, AAV VP2 proteins and AAV VP3 proteins which self-assemble to form an AAV capsid following expression in the cell, said AAV VP 1 coding sequence being operably linked to a promoter which directs expression of the VP1 coding sequences in the cell line.

[0143] In one embodiment, a production cell culture useful for producing a recombinant AAV having a capsid selected from an AAVhu68, an AAV9, a mutant AAV9. an AAVhu95 or an AAVhu96 is provided. Such a cell culture contains a nucleic acid which expresses tire AAVhu68 capsid protein (or alternatively AAV9 capsid, mutant AAV9 capsid. AAVhu95 capsid, or AAVhu96 capsid) in the host cell (e.g., SEQ ID NO: 40 or SEQ ID NO: 41 for AAVhu68; SEQ ID NO: 43 or SEQ ID NO: 44 for AAVhu95; SEQ ID NO: 46 or SEQ ID NO: 47 for AAVhu96); a nucleic acid molecule suitable for packaging into the AAVhu68 capsid, e.g., a vector genome which contains AAV ITRs and a non-AAV nucleic acid sequence encoding a gene operably linked to regulatory sequences which direct expression of the gene in a host cell: and sufficient AAV rep functions and adenovirus helper functions to pennit packaging of the vector genome into tire recombinant AAVhu68 (or AAV9, mutant AAV9, AAVhu95. AAVhu96 capsid. In one embodiment, the cell culture is composed of mammalian cells (e.g., human embryonic kidney 293 cells, among others) or insect cells (e.g., Spodoptera frugiperda (Sf9) cells). In certain embodiments, baculovirus provides the helper functions necessary for packaging the vector genome into the recombinant AAVhu68 capsid, AAV9 capsid, mutant AAV9 capsid, AAVhu95 capsid or AAVhu96 capsid. Optionally the rep functions are provided by an AAV other than AAV2, selected to complement the source of the ITRs.

[0144] In one embodiment, cells are manufactured in a suitable cell culture (e g., HEK 293 or Sf9) or suspension. Methods for manufacturing the gene therapy vectors described herein include methods well known in the art such as generation of plasmid DNA used for production of the gene therapy vectors, generation of the vectors, and purification of tire vectors. In some embodiments, the gene therapy vector is an AAV vector and tire plasmids generated are an AAV cis-plasmid encoding the AAV vector genome and the gene of interest, an AAV trans-plasmid containing AAV rep and cap genes, and an adenovirus helper plasmid. The vector generation process can include method steps such as initiation of cell culture, passage of cells, seeding of cells, transfection of cells with the plasmid DNA, post-transfection medium exchange to serum free medium, and the harvest of vector-containing cells and culture media. The harvested vectorcontaining cells and culture media are referred to herein as crude cell harvest. In yet another system, the gene therapy vectors are introduced into insect cells by infection with baculovirus- based vectors. For reviews on these production systems, see generally, e g., Zhang et al., 2009, Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production, Human Gene Therapy 20:922-929, the contents of each of which is incorporated herein by reference in its entirety. Methods of making and using these and other AAV production systems are also described in the following US patents, the contents of each of which is incorporated herein by reference in its entirety: US Patent Nos. 5, 139,941 ; 5,741,683; 6,057.152; 6.204,059; 6.268.213; 6,491,907; 6.660,514; 6,951.753: 7,094,604; 7.172.893; 7,201,898; 7,229,823; and 7,439,065.

[0145] The crude cell harvest may thereafter be subject method steps such as concentration of the vector harvest, diafiltration of the vector harvest, microfluidization of the vector harvest, nuclease digestion of the vector harvest, filtration of microfluidizcd intermediate, crude purification by chromatography, crude purification by ultracentrifugation, buffer exchange by tangential flow filtration, and / or formulation and filtration to prepare bulk vector. An affinity chromatography purification followed anion exchange resin chromatography are used to purify the vector drug product and to remove empty capsids. These methods are described in more detail in International Patent Application No. PCT / US2016 / 065970, filed December 9. 2016, and US 11,098,286 B2, entitled “Scalable Purification Method for AAV9”, which are incorporated by reference. Purification methods for AAV8, International Patent Application No.

[0146] PCT / US2016 / 065976, filed December 9, 2016, and US 11,015,174 B2, entitled “Scalable Purification Method for AAV8”, which are incorporated herein by reference. Purification methods for rhlO, International Patent Application No. PCT / US 16 / 066013, filed December 9, 2016, and US 11,028,372 B2, entitled “Scalable Purification Method for AAVrhlO’', which are incorporated herein by reference. Purification methods for AAV1. International Patent Application No. PCT / US2016 / 065974, filed December 9, 2016. and US 11,015.173 B2. entitled “Scalable Purification Method for AAV 1”, which are incorporated herein by reference. Other suitable methods may be selected.

[0147] To calculate empty and full particle content, VP3 band volumes for a selected sample (e.g., in examples herein an iodixanol gradient-purified preparation where # of genome copies (GC) = # of particles) are plotted against GO particles loaded. The resulting linear equation (y = mx+c) is used to calculate tire number of particles in tire band volumes of the test article peaks. The number of particles (pt) per 20 uL loaded is then multiplied by 50 to give particles (pt) / mL. Pt / mL divided by GC / mL gives the ratio of particles to genome copies (pt / GC). Pt / mL-GC / mL gives empty pt / mL. Empty pt / mL divided by pt / mL and x 100 gives the percentage of empty particles.

[0148] Generally, methods for assaying for empty capsids and AAV vector particles with packaged genomes have been known in tire art. See, e g., Grimm et al., Gene Therapy (1999) 6: 1322-1330; Sommer et al., Molec. Ther. (2003) 7:122-128. To test for denatured capsid, the methods include subjecting the treated AAV stock to SDS-poly acrylamide gel electrophoresis, consisting of any gel capable of separating the three capsid proteins, for example, a gradient gel containing 3-8% Tris-acetate in the buffer, then running the gel until sample material is separated, and blotting the gel onto nylon or nitrocellulose membranes, preferably nylon. Anti-AAV capsid antibodies are then used as the primary antibodies that bind to denatured capsid proteins, preferably an anti-AAV capsid monoclonal antibody, most preferably the Bl anti-AAV-2 monoclonal antibody (Wobus et al., J. Virol. (2000) 74:9281-9293). A secondary antibody is then used, one that binds to tire primary antibody and contains a means for detecting binding with the primary antibody, more preferably an anti-IgG antibody containing a detection molecule covalently bound to it, most preferably a sheep anti-mouse IgG antibody covalently linked to horseradish peroxidase. A method for detecting binding is used to semi-quantitatively determine binding between the primary and secondary antibodies, preferably a detection method capable of detecting radioactive isotope emissions, electromagnetic radiation, or colorimetric changes, most preferably a chemiluminescence detection kit. For example, for SDS-PAGE, samples from column fractions can be taken and heated in SDS-PAGE loading buffer containing reducing agent (e.g., DTT), and capsid proteins were resolved on pre-cast gradient polyacrylamide gels (e.g., Novex). Silver staining may be performed using SilverXpress (Invitrogen, CA) according to the manufacturer's instructions or other suitable staining method, i.e., SYPRO ruby or coomassie stains. In one embodiment, the concentration of AAV vector genomes (vg) in column fractions can be measured by quantitative real time PCR (Q-PCR). Samples are diluted and digested with DNase I (or another suitable nuclease) to remove exogenous DNA. After inactivation of the nuclease, the samples are further diluted and amplified using primers and a TaqMan™ Anorogenic probe specific for tire DNA sequence betw een tire primers. The number of cycles required to reach a defined level of Auorescence (threshold cycle, Ct) is measured for each sample on an Applied Biosystems Prism 7700 Sequence Detection System. Plasmid DNA containing identical sequences to that contained in the AAV vector is employed to generate a standard curve in the Q-PCR reaction. The cycle threshold (Ct) values obtained from the samples are used to determine vector genome titer by normalizing it to the Ct value of the plasmid standard curve. End-point assays based on the digital PCR can also be used.

[0149] In one aspect, an optimized q-PCR method is used which utilizes a broad spectrum serine protease, e.g., proteinase K (such as is commercially available from Qiagen). More particularly, the optimized qPCR genome titer assay is similar to a standard assay, except that after the DNase I digestion, samples are diluted with proteinase K buffer and treated with proteinase K followed by heat inactivation. Suitably samples are diluted with proteinase K buffer in an amount equal to the sample size. The proteinase K buffer may be concentrated to 2 -old or higher. Typically, proteinase K treatment is about 0.2 mg / mL, but may be varied from 0. 1 mg / mL to about 1 mg / mL. The treatment step is generally conducted at about 55 °C for about 15 minutes, but may be performed at a lower temperature (e.g., about 37 °C to about 50 °C) over a longer time period (e.g., about 20 minutes to about 30 minutes), or a higher temperature (e.g.. up to about 60 °C) for a shorter time period (e.g., about 5 to 10 minutes). Similarly, heat inactivation is generally at about 95 °C for about 15 minutes, but the temperature may be lowered (e.g.. about 70 to about 90 °C) and the time extended (e.g.. about 20 minutes to about 30 minutes). Samples are then diluted (e.g., 1000-fold) and subjected to TaqMan analysis as described in the standard assay.

[0150] Additionally, or alternatively, droplet digital PCR (ddPCR) may be used. For example, methods for determining single-stranded and self-complementary AAV vector genome titers by ddPCR have been described. See, e.g., M. Lock et al, Hu Gene Therapy Methods, Hum Gene Ther Methods. 2014 Apr;25(2): 115-25. doi: 10. 1089 / hgtb.2013.131. Epub 2014 Feb 14.

[0151] In brief, the method for separating the rAAV capsid (clade F, mutant Clade F, AAVhu68, AAV9, mutant AAV9, AAVhu95 or AAVhu96) particles having packaged genomic sequences from genome-deficient AAV intermediates involves subjecting a suspension comprising recombinant AAV viral particles and AAV capsid intermediates to fast performance liquid chromatography, wherein the AAV viral particles and AAV intermediates are bound to a strong anion exchange resin equilibrated (e.g., at a high pH of about 10.2), and subjected to a salt gradient while monitoring eluate for ultraviolet absorbance at about 260 nanometers (nm) and about 280 nm. In other embodiments, isocratic methods may be used. Although less optimal for the mutant Clade F rAAV, or for hu68 or AAV9, the pH may be in tire range of about 10 to 10.4. In this method, the AAV full capsids are collected from a fraction which is eluted when the ratio of A260 / A280 reaches an inflection point. In one example, for the Affinity Chromatography step, the diafiltered product may be applied to an affinity resin (Life Technologies) that efficiently captures the AAV serotype. Under these ionic conditions, a significant percentage of residual cellular DNA and proteins flow through the column, while AAV particles are efficiently captured.

[0152] The produced rAAV is suspended in a suitable physiologically compatible composition (e.g., a buffered saline). This composition may be frozen for storage, later thawed and optionally diluted with a suitable diluent. Alternatively, the vector may be prepared as a composition which is suitable for delivery to a patient without proceeding through the freezing and thawing steps.

[0153] Unless defined otherwise in this specification, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art and by reference to published texts, which provide one skilled in the art with a general guide to many of the terms used in the present application.

[0154] Pharmaceutical Composition In one aspect, provided herein is a pharmaceutical composition comprising a rAAV as described herein in a formulation buffer.

[0155] In one embodiment, the rAAV is formulated at about 1 x 109genome copies (GC) / mL to about 1 x 1014GC / mL. In a further embodiment, the rAAV is formulated at about 3 x 109GC / mL to about 3 x 10BGC / mL. In yet a further embodiment, the rAAV is formulated at about 1 x 109GC / mL to about 1 x 1013GC / mL. In one embodiment, the rAAV is formulated at least 1 x 1011GC / mL.

[0156] Provided herein also is a composition comprising an rAAV as described herein and an aqueous suspension media. In certain embodiments, the suspension is formulated for intravenous delivery, intrathecal administration, or intracerebroventricular administration. In one aspect, the compositions contain at least one rAAV stock and an optional carrier, excipient and / or preservative.

[0157] As used herein, “carrier'’ includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase “pharmaceutically-acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host. Delivery vehicles such as liposomes, nanocapsules, nanoparticles, lipid nanoparticle (LNP), microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present invention into suitable host cells. In particular, the rAAV vector delivered vector genomes may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.

[0158] In one embodiment, a composition includes a final formulation suitable for delivery to a subject, e.g., is an aqueous liquid suspension buffered to a physiologically compatible pH and salt concentration. Optionally, one or more surfactants are present in the formulation. In another embodiment, the composition may be transported as a concentrate which is diluted for administration to a subject. In other embodiments, tire composition may be lyophilized and reconstituted at the time of administration.

[0159] A suitable surfactant, or combination of surfactants, may be selected from among nonionic surfactants that are nontoxic. In one embodiment, the composition includes a carrier, diluent, excipient and / or adjuvant. Suitable carriers may be readily selected by one of skill in the art in view of the indication for which the transfer virus is directed. For example, one suitable carrier includes saline, which may be formulated with a variety' of buffering solutions (e.g., phosphate buffered saline). Other exemplary' carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The buffer / carrier should include a component that prevents the rAAV, from sticking to tire infusion tubing but does not interfere with the rAAV binding activity in vivo. A suitable surfactant, or combination of surfactants, may be selected from among non-ionic surfactants that are nontoxic. In one embodiment, a difunctional block copolymer surfactant terminating in primary' hydroxyl groups is selected, e.g., such as Poloxamer 188 (also known under the commercial names Pluronic® F68 [BASF], Lutrol® F68, Synperonic® F68, Kolliphor® P188) which has a neutral pH, has an average molecular weight of 8400. Other surfactants and other Poloxamers may be selected, i.e., nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (polypropylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (polyethylene oxide)), SOLUTOL HS 15 (Macrogol-15 Hydroxystearate), LABRASOL (Polyoxy capryllic glyceride), poly oxy -oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are commonly named w ith the letter "P" (for poloxamer) followed by three digits: the first two digits x 100 give the approximate molecular mass of the polyoxypropylene core, and the last digit x 10 gives the percentage polyoxyethylene content. In one embodiment Poloxamer 188 is selected. The surfactant may be present in an amount up to about 0.0005 % to about 0.001% of the suspension.

[0160] In certain embodiments, the composition containing the rAAV is delivered at a pH in tire range of 6 to 8. or 7.2 to 7.8, or 7.5 to 8. For intrathecal delivery, a pH above 7.5 may be desired, e.g., 7.5 to 8, or 7.8. For intravenous delivery, a pH of about 6.8 to about 7.2 may be desired.

[0161] In certain embodiments, tire formulation may contain a buffered saline aqueous solution not comprising sodium bicarbonate. Such a formulation may contain a buffered saline aqueous solution comprising one or more of sodium phosphate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride and mixtures thereof, in water, such as a Harvard’s buffer. In one embodiment, the buffer is PBS. In another embodiment, the buffer is an artificial cerebrospinal fluid (aCSF), e.g., Eliott’s formulation buffer: or Harvard apparatus perfusion fluid (an artificial CSF with final Ion Concentrations (in mM): Na 150; K 3.0; Ca 1.4; Mg 0.8; P 1.0; Cl 155). The aqueous solution may further contain Kolliphor® P188, a poloxamer which is commercially available from BASF which was formerly sold under the trade name Lutrol® F68. The aqueous solution may have a pH of 7.2.

[0162] In another embodiment, the formulation may contain a buffered saline aqueous solution comprising 1 mM Sodium Phosphate (NasPCh), 150 mM sodium chloride (NaCl), 3mM potassium chloride (KC1), 1.4 mM calcium chloride (CaC12), 0.8 mM magnesium chloride (MgCh), and 0.001% poloxamer (e.g., Kolliphor®) 188, pH 7.2. See, e.g., harvardapparatus.com / harvard-apparatus-perfusion-fluid.html. In certain embodiments, Harvard’s buffer is preferred due to better pH stability observed with Harvard’s buffer.

[0163] In certain embodiments, the formulation buffer is artificial CSF with Pluronic F68. In other embodiments, the formulation may contain one or more permeation enhancers. Examples of suitable permeation enhancers may include, e.g., mannitol, sodium glycocholate, sodium taurocholate, sodium deoxycholate, sodium salicylate, sodium caprylate, sodium caprate, sodium lauryl sulfate, polyoxyethylene-9-laurel ether, or EDTA.

[0164] Optionally, the compositions of the invention may contain, in addition to tire rAAV and carrier(s). other conventional pharmaceutical ingredients, such as preservatives, or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0165] The compositions according to the present invention may comprise a pharmaceutically acceptable carrier, such as defined above. Suitably, tire compositions described herein comprise an effective amount of one or more AAV suspended in a pharmaceutically suitable carrier and / or admixed with suitable excipients designed for delivery to the subject via injection, osmotic pump, intrathecal catheter, or for delivery by another device or route. In certain embodiments, an ommaya reservoir is used for delivery. In one example, the composition is formulated for intrathecal delivery. In one example, the composition is formulated for intravenous (iv) delivery .

[0166] In one embodiment, a therapeutically effective amount of said vector is included in the pharmaceutical composition. The selection of tire carrier is not a limitation of the present invention. Other conventional pharmaceutically acceptable carrier, such as preservatives, or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin. As used herein, the term 'dosage or "amount can refer to the total dosage or amount delivered to the subject in the course of treatment, or the dosage or amount delivered in a single unit (or multiple unit or split dosage) administration.

[0167] In certain embodiments, tire compositions can be formulated in dosage units to contain an amount of rAAV that is in the range of about 1 x 109GC per gram of brain mass to about 1 x 1013genome copies (GC) per gram (g) of brain mass, including all integers or fractional amounts within the range and tire endpoints. In another embodiment, the dosage is 1 x IO10GC per gram of brain mass to about 1 x 1013GC per gram of brain mass. In specific embodiments, the dose of the vector administered to a patient is at least about 1.0 x 109GC / g, about 1.5 x 109GC / g, about 2.0 x 109GC / g, about 2.5 x 109GC / g, about 3.0 x 109GC / g, about 3.5 x 109GC / g, about 4.0 x 109GC / g, about 4.5 x 109GC / g, about 5.0 x 109GC / g, about 5.5 x 109GC / g, about 6.0 x 109GC / g, about 6.5 x 109GC / g, about 7.0 x 109GC / g, about 7.5 x 109GC / g, about 8.0 x 109GC / g, about 8.5 x 109GC / g, about 9.0 x 109GC / g, about 9.5 x 109GC / g, about 1.0 x IO10GC / g, about

[0168] 1.5 x 1010GC / g, about 2.0 x 1010GC / g, about 2.5 x 1010GC / g, about 3.0 x 1010GC / g, about 3.5 x 1010GC / g, about 4.0 x 1010GC / g. about 4.5 x 1010GC / g, about 5.0 x 1010GC / g. about 5.5 x 1010GC / g, about 6.0 x IO10GC / g, about 6.5 x 1010GC / g, about 7.0 x IO10GC / g, about 7.5 x 1010GC / g, about 8.0 x IO10GC / g, about 8.5 x 1010GC / g, about 9.0 x IO10GC / g, about 9.5 x 1010GC / g, about 1.0 x 1011GC / g, about 1.5 x 1011GC / g, about 2.0 x 1011GC / g, about 2.5 x 1011GC / g, about 3.0 x 1011GC / g, about 3.5 x 1011GC / g, about 4.0 x 1011GC / g, about 4.5 x 1011GC / g, about 5.0 x 1011GC / g, about 5.5 x 1011GC / g, about 6.0 x 1011GC / g, about 6.5 x 1011GC / g, about 7.0 x 1011GC / g, about 7.5 x 1011GC / g, about 8.0 x 1011GC / g, about 8.5 x 1011GC / g, about 9.0 x 10” GC / g, about 9.5 x 10” GC / g, about 1.0 x 1012GC / g, about 1.5 x 1012GC / g, about 2.0 x 1012GC / g, about 2.5 x 1012GC / g, about 3.0 x 1012GC / g, about 3.5 x 1012GC / g, about 4.0 x 1012GC / g, about 4.5 x 1012GC / g, about 5.0 x 1012GC / g, about 5.5 x 1012GC / g, about 6.0 x 1012GC / g, about 6.5 x 1012GC / g, about 7.0 x 1012GC / g, about 7.5 x 1012GC / g, about 8.0 x 1012GC / g, about 8.5 x 1012GC / g, about 9.0 x 1012GC / g, about 9.5 x 1012GC / g, about 1.0 x 1013GC / g, about 1.5 x 1013GC / g, about 2.0 x 1013GC / g, about 2.5 x 1013GC / g, about 3.0 x 1013GC / g, about 3.5 x 1013GC / g, about 4.0 x 1013GC / g, about 4.5 x 1013GC / g, about 5.0 x IO1GC / g, about 5.5 x 1013GC / g, about 6.0 x 101GC / g, about 6.5 x 1013GC / g, about 7.0 x 1013GC / g, about 7.5 x 1013GC / g, about 8.0 x 1013GC / g, about 8.5 x 1013GC / g, about 9.0 x 1013GC / g, about 9.5 x 1013GC / g, or about 1.0 x 1014GC / g brain mass. Also, the replication-defective virus compositions can be formulated in dosage units to contain an amount of replication-defective virus that is in the range of about 1.0 x 109GC to about 1 .0 x 1016GC (to treat an average subject of 70 kg in body weight) including all integers or fractional amounts within the range, and preferably 1.0 x 1012GC to 1.0 x 1014GC for a human patient. In one embodiment, the compositions are formulated to contain at least 1x109, 2x109, 3xl09, 4xl09, 5xl09, 6xl09, 7xl09, 8xl09, or 9xl09GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO10, 2xlO10, 3xl010, 4xlO10, 5xl010, 6xlO10. 7xlO10. 8xl010, or 9xlO10GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO11, 2xlOn, 3x10", 4x10", 5xl0n, 6xlOn, 7x10", 8x10", or 9x 10 GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO12, 2xl012, 3xl012, 4xl012, 5xl012, 6xl012, 7xl012, 8xl012, or 9xl012GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO13, 2xl013, 3xl013, 4xl013, 5xl013, 6xl013. 7xl013, 8xl013, or 9x10’3GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO14, 2xl014, 3xl014, 4x1014, 5xl014, 6xl014, 7xl 014, 8xl 014, or 9xl014GC per dose including all integers or fractional amounts within the range. In another embodiment, tire compositions are formulated to contain at least IxlO15, 2xl015, 3xl015, 4xl015, 5xl015, 6xl015, 7xl015, 8xl015, or 9xl015GC per dose including all integers or fractional amounts within tire range. In one embodiment, for human application the dose can range from IxlO10to about IxlO12GC per dose including all integers or fractional amounts within the range.

[0169] In certain embodiments, the composition is delivered intrathecally, optionally via intra- cistema magna (ICM) injection. In certain embodiments, the composition is delivered via intraparenchymal administration. In certain embodiments, the composition is delivered via Ommaya Reservoir delivery system.

[0170] In one embodiment, the pharmaceutical composition comprising a rAAV as described herein is administrable at a dose of about 1 x 109GC per gram of brain mass to about 1 x 1013GC per gram of brain mass.

[0171] The rAAV, preferably suspended in a physiologically compatible carrier, may be administered to a human or non-human mammalian patient. It should be understood that tire compositions in tire pharmaceutical composition described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.

[0172] Uses and Regimens

[0173] In one aspect, provided herein is a method for treating a human subject diagnosed with GM2 gangliosidosis disease. In one aspect, provided herein is a method for treating a human subject diagnosed GM2 activator deficiency, Tay-Sachs disease and / or Sandhoff disease. In another aspect, provided herein is a method for preventing accumulation of GM2 ganglioside. In certain embodiments, the method of treating GM2 gangliosidosis disease comprises ameliorating one or more symptoms of GM2 gangliosidosis disease in a subject.

[0174] A method provided herein comprises administering to a subject a suspension of an rAAV vector as described herein. In one embodiment, tire method comprises administering to a subject having a GM2 gangliosidosis disease, a suspension of a rAAV as described herein in a formulation buffer at a dose of about 1 x 109GC per gram of brain mass to about 1 x 1014GC per gram of brain mass. In another embodiment, the method comprises administering to a subject a suspension of a rAAV as described herein in a formulation buffer at a dose of about I x 109GC per kg of body weight to about 1 x 1014GC per kg of body weight. Suitable doses for systemic administration can be determined by one of skill in the art.

[0175] In one embodiment, the subject is delivered a therapeutically effective amount of the rAAV described herein. As used herein, a “therapeutically effective amount' ’ refers to the amount of the composition comprising the nucleic acid sequence encoding HexA and HexB, or HexA, HexB and GM2A which delivers and expresses in the target cells an amount of protein sufficient to achieve efficacy. The dosage is adjusted to balance the therapeutic benefit against any side effects and such dosages may vary depending upon the therapeutic application for which the recombinant vector is employed. The levels of expression of the transgene product can be monitored to determine the frequency of dosage resulting in viral vectors, preferably AAV vectors containing the transgcnc. Optionally, dosage regimens similar to those described for therapeutic purposes may be utilized for immunization using the compositions described herein.

[0176] Suitable, conventional, and pharmacally acceptable routes of administration include, but are not limited to. direct delivery to a desired organ (e.g., brain, CSF, intranasal, intrathecal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, intraparenchymal, intracerebroventricular, intrathecal. ICM. lumbar puncture and other parenteral routes of administration. Routes of administration may be combined, if desired.

[0177] Suitable volumes for delivery of these doses and concentrations may be determined by one of skill in tire art. For example, volumes of about 1 pL to 150 mL may be selected for CNS del i \ ery . with tire higher volumes being selected for adults. Typically, for newborn infants a suitable volume is about 0.5 mL to about 10 mL, for older infants, about 0.5 mL to about 15 mL may be selected. For toddlers, a volume of about 0.5 mL to about 20 mL may be selected. For children, volumes of up to about 30 mL may be selected. For pre-teens and teens, volumes up to about 50 mL may be selected. In still other embodiments, a patient may receive an intrathecal administration in a volume of about 5 mL to about 15 mL are selected, or about 7.5 mL to about 10 mL. Other suitable volumes and dosages may be determined for CNS delivery, intratumoral delivery, and / or for systemic delivery (e.g., IV). The dosage will be adjusted to balance the therapeutic benefit against any side effects and such dosages may vary depending upon the therapeutic application for which the recombinant vector is employed.

[0178] In one embodiment, the method comprises administering rAAV as described herein is to the subject in need. The above-described recombinant vectors may be administered or delivered to host or target cells according to published methods. The rAAV, preferably suspended in a physiologically compatible carrier, may be administered to a subject, human or non-human mammalian patient. In certain embodiments, for administration to a human patient, the rAAV is suitably suspended in an aqueous solution containing saline, a surfactant, and a physiologically compatible salt or mixture of salts. Suitably, the formulation is adjusted to a physiologically acceptable pH, e.g., in tire range of pH 6 to 9, or pH 6.5 to 7.5, pH 7.0 to 7.7, or pH 7.2 to 7.8. As the pH of the cerebrospinal fluid is about 7.28 to about 7.32, for intrathecal delivery, a pH within this range may be desired; whereas for intravenous delivery, a pH of about 6.8 to about 7.2 may be desired. For intravenous delivery', a pH of about 6.8 to about 7.2 may be desired. However, other pHs within the broadest ranges and these subranges may be selected for other route of delivery'.

[0179] Suitably, the pharmaceutical compositions, as described herein, and the uses thereof comprise delivering to the subject via injection, osmotic pump, intrathecal catheter, or for delivery by another device or route. In one example, the composition is formulated for intrathecal delivery. As used herein, the terms "‘intrathecal delivery” or “intrathecal administration” refer to a route of administration for drugs via an injection into the spinal canal, more specifically into the subarachnoid space so that it reaches the cerebrospinal fluid (CSF). Intrathecal delivery may include lumbar puncture, intraventricular (including intracerebroventricular (ICV)), suboccipital / intracistemal, and / or C 1-2 puncture. For example, material may be introduced for diffusion throughout the subarachnoid space by means of lumbar puncture. In another example, injection may be into the cistema magna. In certain embodiment, a rAAV, or composition as described herein is administrated to a subject in need via tire intrathecal administration. In certain embodiments, tire intrathecal administration is performed as described in US Patent Publication No. 2018 / 0339065 Al, published November 29, 2019, which is incorporated herein by reference in its entirety. In certain embodiments, the CNS administration is performed using Ommaya Reservoir (also referred to as Ommaya device or Ommaya system).

[0180] As used herein, the terms “intracistemal delivery” or “intracistemal administration” refer to a route of administration for drugs directly into the cerebrospinal fluid of the cistema magna cerebellomedularis, more specifically via a suboccipital puncture or by direct injection into tire cistema magna or via permanently positioned tube.

[0181] As used herein, the term “intraparenchymal”, “dentate nucleus” or IDN refers to a route of administration of a composition directly into dentate nuclei. IDN allows for targeting of dentate nuclei and / or cerebellum. In certain embodiments, the IDN administration is performed using ClearPoint® Neuro Navigation System (MRI Interventions, Inc., Memphis, TN) and ventricular cannula, which allows for MRI-guided visualization and administration. Alternatively, other devices and methods may be selected.

[0182] In still other embodiments, the rAAV compositions provided herein may be used in a regimen which further comprises co-administration with an immunosuppressive regimen.

[0183] In one embodiment, the method further comprises the subject receives an immunosuppressive co-therapy. Immunosuppressants for such co-therapy include, but are not limited to, a glucocorticoid, corticosteroids, antimetabolites, T-cell inhibitors, a macrolide (e.g., a rapamycin or rapalog), and cytostatic agents including an alkylating agent, an anti-metabolite, a cytotoxic antibiotic, an antibody, or an agent active on immunophilin. The immune suppressant may include a nitrogen mustard, nitrosourea, platinum compound, methotrexate, azathioprine, mercaptopurine, fluorouracil, dactinomycin, an anthracycline. mitomycin C. bleomycin, mithramycin, IL-2 receptor- (CD25-) or CD3-directed antibodies, anti-IL-2 antibodies, ciclosporin. tacrolimus, sirolimus. IFN-|3, IFN-y, an opioid, or TNF-a (tumor necrosis factoralpha) binding agent.

[0184] In certain embodiments, the immunosuppressive therapy may be started 0, 1 , 2, 7, or more days prior to the gene therapy administration. Such therapy may involve co-administration of two or more drugs, the (e.g., prednelisone, micophenolate mofetil (MMF) and / or sirolimus (i.e., rapamycin)) on the same day. One or more of these drugs may be continued after gene therapy administration, at the same dose or an adjusted dose. Such therapy may be for about 1 week (7 days), about 60 days, or longer, as needed. In certain embodiments, a tacrolimus-free regimen is selected.

[0185] In one embodiment, the method further comprises administering to a subject anti-AAV neutralizing antibodies (NAb) to reduce peripheral transduction, and mitigate the potential risk of HexA, HexB, Gm2A-induced toxicity. In certain embodiments, the method further comprises detect the presence of systemic AAV NAb prior to treating with anti-AAV NAb, wherein patients with levels of anti-AAV NAb in excess of a predetermined level against the rAAV capsid (or a sero-crossreactive capsid) do not require pretreatment. Such levels may be, e.g., in excess of about 1: 10, about 1:20, about 1:50, about 1: 100. about 1:250, or higher or lower levels. In certain embodiments, the method further comprises intravenously administering human anti-AAV polyclonal antibodies (e.g., plasma-derived, pooled human immunoglobulin (IVIG)), an anti- AAV monoclonal antibody, or a cocktail of anti-AAV antibodies, to a patient about 1 day to about 2 hours before treatment with a rAAV as described herein.

[0186] In certain embodiments, a combination regimen is provided for preventing off-target delivery rAAV, the regimen comprising (a) pretreating the patient by systemically administering a composition comprising anti-AAV capsid neutralizing antibodies directed against an AAV capsid in a recombinant AAV vector, and (b) administering to the central nervous system (CN S) rAAV as described herein (e.g., rAAV). See also, US Provisional Patent Application No. 63 / 328,227, filed April 6, 2022, and International Patent Application No. PCT / US2023 / 065422, filed April 6, 2023, now Publication No. WO2023 / 196892A1 which are incorporated herein by reference in their entirety.

[0187] Still other co-therapeutics may include, e.g., anti-IgG enzymes, which have been described as being useful for depleting anti-AAV antibodies (and thus may permit administration to patients testing above a threshold level of antibody for the selected AAV capsid), and / or delivery of anti-FcRN antibodies and / or one or more of a) a steroid or combination of steroids and / or (b) an IgG-cleaving enzyme, (c) an inhibitor of Fc-IgE binding; (d) an inhibitor of Fc-IgM binding; (e) an inhibitor of Fc-IgA binding; and / or (f) gamma interferon. anti-FcRN antibodies include, e.g., rozanolixizumab (UCB7665) (UCB SA); IMVT-1401, RVT-1401 (HL161), HBM9161 (all form HanAll BioPhrma Co. Ltd), Nipocalimab (M281) (Momenta Pharmaceuticals Inc), ARGX-113 (efgartigimod) (Argenx S.E.), orilanolimab (ALXN 1830, SYNT001, Alexion Pharmaceuticals Inc), SYNT002, ABY-039 (Affibody AB), or DX-2507 (Takeda Pharmaceutical Co. Ltd). In certain embodiments, a combinations of anti-FcRN antibodies is administered. In certain embodiments, an anti-FcRN antibody is administered in combination with a suitable anti- FcRn ligand (i.e., a peptide or protein construct binding human FcRn so as to inhibit IgG binding).

[0188] The methods and compositions described herein may be used for treatment of any of the stages of GM2 gangliosidosis disease. In certain embodiments, the patient is an infant, a toddler, or the patient is from 3 years to 6 years of age, from 3 years to 12 years of age, from 3 years to 18 years of age, from 3 years to 20 years of age. In certain embodiments, patients are older than 18 years of age.

[0189] In certain embodiments, the methods, rAAV and compositions may be used for treatment of GM2 gangliosidosis disease. In certain embodiments, the methods, rAAV and compositions may be used for treatment of GM2 activator deficiency. In certain embodiments, the methods, rAAV and compositions may be used for treatment of Tay-Sachs disease. In certain embodiments, the methods, rAAV and compositions may be used for treatment of Sandhoff disease. In certain embodiments, the methods, rAAV and compositions may be used for treatment of disease associated with mutation in genes encoding 0-N-acetylhexosaminidase alpha subunit, 0-N-acetylhexosaminidase beta subunit, and / or ganglioside GM2 activator (see also. (OMIM #272800. OMIM #268800, OMIM #272750). In certain embodiments, the methods, rAAV and compositions may be used for treatment of GM2 gangliosidosis disease at various onset of the disease, including infantile acute onset (i.e., at <1 year of age), juvenile subacute onset (i.e., with onset in early childhood), and adult chronic onset (i.e., with initial symptoms in early- to midtccns). See also, Lawson, C.A. and Martin, D.R., “Animal models of GM2 gangliosidosis: utility and limitations”, The Application of Clinical Genetics 2016:9 111-120, which is incorporated herein by reference in its entirety.

[0190] Symptoms of GM2 gangliosidosis include progressive neurological impairment, motor deficits, progressive weakness, hypotonia, decreased responsiveness, vision deterioration, and seizures, developmental arrest, neuronal death, regression in developmental milestones, motor regression, psychotic episodes, intellectual disability, dysphagia, cerebellar ataxia, muscle weakness, and manic depression. See also. Leal, A.F., et al., “GM2 Gangliosidoses: Clinical Features, Pathophysiological Aspects, and Current Therapies”, Int. J. Mol. Sci. 2020, 21, 6213, which is incorporated herein by reference in its entirety. Symptoms of Tay-Sachs disease (i.e., infantile acute onset) include seizures, axial hypotonia, cherry -red spot, regression in developmental milestones, exaggerated startle response. Symptoms of Sandhoff Disease (i.e., juvenile subacute onset) include ataxia, myoclonus, motor regression, psychotic episodes, intellectual disability, progressive clumsiness, loss of vision due to optic atrophy and retinitis pigmentosa, additionally wherein patients may also present systemic manifestations as organomegalies, such as cardiomegaly, hepatosplenomegaly, macroglossia, and skeletal abnormalities (i.e., as a consequence of the impaired HexB activity, which is also involved in the degradation of glycosaminoglycans such as keratan sulfate and chondroitin sulfate. Symptoms of GM2 activator deficiency (i.e., adult onset) include dysphagia, muscle atrophy, cerebellar ataxia, dysarthric speech, manic depression, muscle weakness, psychotic episodes.

[0191] In certain embodiments, the methods and compositions described herein are used to ameliorate or improve one or more symptoms of GM2 gangliosidosis disease. In certain embodiments, the methods and compositions described herein are used to ameliorate or improve one or more symptoms of GM2 activator deficiency. In certain embodiments, the methods and compositions described herein are used to ameliorate or improve one or more symptoms of Tay- Sachs disease. In certain embodiments, the methods and compositions described herein are used to ameliorate or improve one or more symptoms of Sandhoff disease.

[0192] The compositions described herein may be used in a regimen involving co-administration of other active agents. Any suitable method or route can be used to administer such other agents. Routes of administration include, for example, systemic, oral, intravenous, intraperitoneal, subcutaneous, or intramuscular administration. Optionally, the AAV compositions described herein may also be administered by one of these routes.

[0193] In certain embodiments, co-thcrapics or co-trcatmcnts may be utilized, which comprise co-administration with another active agent. In certain embodiments, the co-therapy may further comprise physical therapy.

[0194] In one embodiment, the rAAV as described herein is administrated once to the subject in need. In another embodiment, the rAAV is administrated more than once to the subject in need. It should be understood that the compositions in the method described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.

[0195] Kit

[0196] In certain embodiments, a kit is provided which includes a concentrated vector suspended in a formulation (optionally frozen), optional dilution buffer, and devices and components required for intrathecal, intracerebroventricular or intracistemal administration. In another embodiment, the kit may additional or alternatively include components for intravenous delivery. In one embodiment, the kit provides sufficient buffer to allow for injection. Such buffer may allow for about a 1: 1 to a 1:5 dilution of the concentrated vector, or more. In other embodiments, higher or lower amounts of buffer or sterile water are included to allow for dose titration and other adjustments by the treating clinician. In still other embodiments, one or more components of the device are included in the kit. Suitable dilution buffer is available, such as, a saline, a phosphate buffered saline (PBS) or a glycerol / PBS.

[0197] It should be understood that the compositions in kit described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.

[0198] Apparatus and Method for Delivery of a Pharmaceutical Composition

[0199] In one aspect, tire vectors, rAAV or compositions thereof provided herein may be administered intrathecally via the method and / or tire device provided in this section and described in WO 2017 / 136500 and WO 2018 / 160582. which are incorporated by reference herein. Alternatively, other devices and methods may be selected. In certain embodiments, the method comprises the steps of CT-guided sub-occipital injection via spinal needle into the cistema magna of a patient. As used herein, the term Computed Tomography (CT) refers to radiography in which a three-dimensional image of a body structure is constructed by computer from a series of plane cross-sectional images made along an axis. In certain embodiments, the apparatus is described in US Patent Publication No. 2018-0339065 Al, published November 29, 2019, which is incorporated herein by reference in its entirety. In certain embodiments, the vectors, rAAV or compositions thereof provided herein may be administered using Ommaya Reservoir. It should be understood that the compositions in the device described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.

[0200] As used herein, the term “biological sample” refers to any cell, biological fluid or tissue. Suitable samples for use in this invention may include, without limitation, whole blood, leukocytes, fibroblasts, serum, urine, plasma, saliva, bone marrow, cerebrospinal fluid, amniotic fluid, and skin cells. Such samples may further be diluted with saline, buffer or a physiologically acceptable diluent. Alternatively, such samples are concentrated by conventional means.

[0201] “Patient” or “subject” as used herein interchangeably, means a mammalian animal, including a human, a veterinary or farm animal, a domestic animal or pet, and animals normally used for clinical research. In one embodiment, the subject of these methods and compositions is a human patient. In one embodiment, the subject of these methods and compositions is a male or female human patient. In one embodiment, the subject of these methods and compositions is a human diagnosed with GM2 gangliosidosis disease including GM2 activator deficiency, Tay- Sachs disease and / or Sandhoff disease. In certain embodiments, the human subject of these methods and compositions is a prenatal, a newborn, an infant, a toddler, a preschool, a gradeschooler, a teen, a young adult or an adult.

[0202] As used herein, the phrases “ameliorate a symptom”, “improve a symptom” or any grammatical variants thereof, refer to reducing or reversal of symptoms associated with GM2 gangliosidosis disease including GM2 activator deficiency, Tay-Sachs disease and / or Sandhoff disease. In one embodiment, the amelioration or improvement refers to the total number of symptoms in a patient after administration of the described composition(s) or use of the described method, which is reduced by about 5%. about 10%. about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% compared to that before the administration or use. In another embodiment, tire amelioration or improvement refers to tire severity or progression of a symptom after administration of the described composition(s) or use of the described method, which is reduced by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% compared to that before the administration or use.

[0203] As used herein, a “neutralizing antibody” or “NAb” binds specifically to a viral capsid or envelope and interferes with the infectivity of the virus or a recombinant viral vector having the viral capsid or envelope, thus preventing the recombinant viral vector from delivering effective amounts of a gene product encoded by an expression cassette in its vector genome. Various methods for assessing neutralizing antibodies in a patient’s sera may be utilized. The term method and assay may be used interchangeably. As used herein, the term '‘neutralization assay” and “serum virus neutralization assay” refers to a serological test to detect the presence of systemic antibodies that may prevent infectivity of a virus. Such assays may also qualitatively or quantitatively discern the binding capacity (e.g.. magnitude) or efficiency of the antibodies to neutralize a target. Immunological assays may include enzyme immunoassay (EIA), radioimmunoassay (RIA), which uses radioactive isotopes, fluoroimmunoassay (FIA) which uses fluorescent materials, chemiluminescent immunoassay (CLIA) which uses chemiluminescent materials and counting immunoassay (CIA) which employs particle-counting techniques, other modified assays such as western blot, immunohistochemistry (IHC) and agglutination. One of the most common enzyme immunoassays is enzyme-linked immunosorbent assay (ELISA).

[0204] “Neutralizing antibody titer” (NAb titer) a measurement of how much neutralizing antibody (e.g., anti-AAV NAb) is produced which neutralizes the physiologic effect of its targeted epitope (e.g.. an AAV). Anti-AAV NAb titers may be measured as described in, e.g., Calcedo, R., et al., Worldwide Epidemiology of Neutralizing Antibodies to Adeno-Associated Viruses. Journal of Infectious Diseases, 2009, 199 (3): p. 381-390, which is incorporated by reference herein.

[0205] As used herein, a “subpopulation” of vp proteins refers to a group of vp proteins which has at least one defined characteristic in common and which consists of at least one group member to less than all members of the reference group, unless otherwise specified. For example, a “subpopulation” of vpl proteins is at least one (1) vpl protein and less than all vpl proteins in an assembled AAV capsid, unless otherwise specified. A “subpopulation” of vp3 proteins may be one (1) vp3 protein to less than all vp3 proteins in an assembled AAV capsid, unless otherwise specified. For example, vpl proteins may be a subpopulation of vp proteins; vp2 proteins may be a separate subpopulation of vp proteins, and vp3 are yet a further subpopulation of vp proteins in an assembled AAV capsid. In another example, vpl, vp2 and vp3 proteins may contain subpopulations having different modifications, e g., at least one, two, three or four highly deamidated asparagines, e.g., at asparagine - glycine pairs. Unless otherwise specified, highly deamidated refers to at least 45% deamidated, at least 50% deamidated, at least 60% deamidated, at least 65% deamidated, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 97%, 99%. up to about 100% deamidated, 50% to 100% deamidated, 70% to 100% deamidated, 75% to 100% deamidated, or 70% to 90% deamidated at a referenced amino acid position, as compared to the predicted amino acid sequence at the reference amino acid position. Such percentages may be determined using 2D-gel, mass spectrometry techniques, or other suitable techniques.

[0206] As used herein, a ‘‘stock” of rAAV refers to a population of rAAV. Despite heterogeneity in their capsid proteins due to deamidation, rAAV in a stock are expected to share an identical vector genome. A stock can include rAAV having capsids with, for example, heterogeneous deamidation patterns characteristic of the selected AAV capsid proteins and a selected production system. The stock may be produced from a single production system or pooled from multiple runs of the production system. A variety of production systems, including but not limited to those described herein, may be selected. See, e.g., WO 2019 / 168961, published September 6, 2019, including Table G providing the deamidation pattern for AAV9 and WO 2020 / 160582, fded September 7, 2018. See, also, e.g., WO 2020 / 223231, published November 5, 2020 (rh91, including table with deamidation pattern). US Provisional Patent Application No. 63 / 065,616, filed August 14, 2020, and US Provisional Patent Application No. 63 / 109.734, filed November 4, 2020, and International Patent Application No. PCT / US21 / 45945, filed August 13, 2021, which are all incorporated herein by reference in its entirety.

[0207] The abbreviation “sc” refers to self-complementary. “Self-complementary AAV” refers a construct in which a coding region carried by a recombinant AAV nucleic acid sequence has been designed to form an intra-molecular double-stranded DNA template. Upon infection, rather than waiting for cell mediated synthesis of the second strand, the two complementary halves of scAAV will associate to fonn one double stranded DNA (dsDNA) unit that is ready for immediate replication and transcription. See, e.g.. D M McCarty et al. “Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis”, Gene Therapy, (August 2001), Vol 8, Number 16, Pages 1248- 1254. Self-complementary AAVs are described in, e.g., U.S. Patent Nos. 6,596,535; 7,125,717; and 7,456,683, each of which is incorporated herein by reference in its entirety’.

[0208] The term “heterologous” when used with reference to a protein or a nucleic acid indicates that the protein or the nucleic acid comprises two or more sequences or subsequences which are not found in tire same relationship to each other in nature. For instance, the nucleic acid is ty pically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid. For example, in one embodiment, tire nucleic acid has a promoter from one gene arranged to direct the expression of a coding sequence from a different gene. Thus, with reference to the coding sequence, the promoter is heterologous.

[0209] As used herein, an “expression cassette” refers to a nucleic acid molecule which comprises a biologically useful nucleic acid sequence (e.g., a gene cDNA encoding a protein, enzy me or other useful gene product, mRNA, etc.) and regulatory sequences operably linked thereto which direct or modulate transcription, translation, and / or expression of the nucleic acid sequence and its gene product.

[0210] “Operable linkage” or being “operably linked” includes juxtaposition of two or more components (e.g., a promoter and another sequence element) such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of tire other components. For example, a promoter can be operably linked to a coding sequence if tire promoter controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. Operable linkage can include such sequences being contiguous with each other or acting in trans (e.g., a regulatory sequence can act at a distance to control transcription of the coding sequence).

[0211] In other embodiments, the term “transgene” refers to one or more DNA sequences from an exogenous source which are inserted into a target cell. Typically, an expression cassette contains the coding sequence for the gene product described herein flanked by packaging signals of the viral genome and other expression control sequences such as those described herein. In certain embodiments, a vector genome may contain two or more expression cassettes.

[0212] The term “exogenous” as used to describe a nucleic acid sequence or protein means that the nucleic acid or protein does not naturally occur in the position in which it exists in a chromosome, or host cell. An exogenous nucleic acid sequence also refers to a sequence derived from and inserted into the same host cell or subject, but which is present in a non-natural state, e.g., a different copy number, or under the control of different regulatory' elements.

[0213] A “replication-defective virus” or “viral vector” refers to a synthetic or artificial viral particle in which an expression cassette containing a gene of interest is packaged in a viral capsid or envelope, where any viral genomic sequences also packaged within the viral capsid or envelope are replication-deficient; i.e., they cannot generate progeny virions but retain the ability to infect target cells. In one embodiment, the genome of the viral vector does not include genes encoding the enzy mes required to replicate (the genome can be engineered to be “gutless” - containing only the transgene of interest flanked by the signals required for amplification and packaging of the artificial genome), but these genes may be supplied during production. Therefore, it is deemed safe for use in gene therapy since replication and infection by progeny virions cannot occur except in the presence of the viral enzyme required for replication.

[0214] As used herein, the terms “rAAV” and “artificial AAV” used interchangeably, mean, without limitation, a AAV comprising a capsid protein and a vector genome packaged therein, wherein tire vector genome comprising a nucleic acid heterologous to the AAV. In one embodiment, the capsid protein is a non-naturally occurring capsid. Such an artificial capsid may be generated by any suitable technique, using a selected AAV sequence (e.g., a fragment of a vpl capsid protein) in combination with heterologous sequences which may be obtained from a different selected AAV, non-contiguous portions of the same AAV, from a non-AAV viral source, or from a non-viral source. An artificial AAV may be, without limitation, a pseudotyped AAV, a chimeric AAV capsid, a recombinant AAV capsid, or a “humanized” AAV capsid. Pseudotyped vectors, wherein the capsid of one AAV is replaced with a heterologous capsid protein, are useful in the invention. In one embodiment. AAV2 / 5 and AAV2 / 8 are exemplary pseudotyped vectors. The selected genetic element may be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion. The methods used to make such constructs are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0215] The term “nuclease-resistant” indicates that the AAV capsid has assembled around the expression cassette which is designed to deliver a transgene to a host cell and protects these packaged genomic sequences from degradation (digestion) during nuclease incubation steps designed to remove contaminating nucleic acids which may be present from the production process.

[0216] In many instances, rAAV particles arc referred to as DNase resistant. However, in addition to this endonuclease (DNase), other endo- and exo- nucleases may also be used in the purification steps described herein, to remove contaminating nucleic acids. Such nucleases may be selected to degrade single stranded DNA and / or double- stranded DNA. and RNA. Such steps may contain a single nuclease, or mixtures of nucleases directed to different targets, and may be endonucleases or exonucleases.

[0217] As used herein, the term “host cell” may refer to the packaging cell line in which the rAAV is produced from tire plasmid. In the alternative, the term “host cell” may refer to the target cell in which expression of the transgene is desired.

[0218] As used herein, a “variant capsid” or a “variant AAV” or “variant AAV capsid” refers to a modified capsid, engineered capsid or a mutated capsid, wherein the capsid protein comprises an insertion of a tissue-specific targeting peptide, wherein modified insert is not a naturally occurring mutant.

[0219] The term “expression” is used herein in its broadest meaning and comprises the production of RNA or of RNA and protein. Expression may be transient or may be stable.

[0220] The term “substantial homology" or “substantial similarity,” when referring to a nucleic acid, or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95 to 99% of the aligned sequences. Preferably, the homology is over full-length sequence, or an open reading frame thereof, or another suitable fragment which is at least 15 nucleotides in length. Examples of suitable fragments are described herein.

[0221] The term “heterologous” as used to describe a nucleic acid sequence or protein means that the nucleic acid or protein was derived from a different organism or a different species of the same organism than tire host cell or subject in which it is expressed. The term "heterologous" when used with reference to a protein or a nucleic acid in a plasmid, expression cassette, or vector, indicates that the protein or the nucleic acid is present with another sequence or subsequence which with which the protein or nucleic acid in question is not found in the same relationship to each other in nature.

[0222] As described above, the terms “increase” “decrease” “reduce” “ameliorate” “improve” “delay” or any grammatical variation thereof, or any similar terms indication a change, means a variation of about 5 fold, about 2 fold, about 1 fold, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5 % compared to the corresponding reference (e.g., untreated control or a subject in normal condition without GM2 gangliosidosis disease, rmless otherwise specified.

[0223] As used herein, the term “administration” or any grammatical variations thereof refers to delivery' of composition described herein to a subject. The term "‘percent (%) identity”, “sequence identity”, “percent sequence identity”, or “percent identical” in the context of nucleic acid sequences and / or amino acid sequences refer to the residues in the two sequences which are the same when aligned for correspondence, often with corrections for missing or additional bases or amino acids as compared to a reference sequence. With respect to nucleic acids, the length of sequence identity may be specified to be over the full-length of the genome, the full-length of a gene coding sequence. In certain embodiments, a fragment of at least about 500 to 5000 nucleotides, or smaller fragments, e.g., of at least about nine nucleotides, usually at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides, may also be selected. Similarly, for amino acids, the identity may be over the full-length of a protein, or a specified peptide, polypeptide or region. A suitable amino acid fragment may be at least about 7 amino acids in length, and may be up to about 700 amino acids.

[0224] As used herein, the phrase “at least X% identity” includes the value of X and greater values. For example, at least 95% identity includes 95% or greater, at least 96%, at least 97%, at least 98%, at least 99%. or at least 99.9%. up to 100% or 95% to 100%, and values therebetween. In this example, 95% may also include decimals rounded to the nearest value of 95% in conformance with principles of direct rounding to an integer, including but not limited to round- toward-zero, round-away from zero, round to the nearest integer, round up, round down. E.g., if the decimal point of the integer starts with 5, 6, 7, 8, 9, the integer is rounded up to next full integer (i.e., 95.7% to 96%), and if the decimal point of the integer starts with 0, 1, 2, 3, 4, the integer is rounded down to next full integer (i.e., 95.3% to 95%).

[0225] Identity may be detennined by preparing an alignment of the sequences and through the use of a variety of algorithms and / or computer programs known in the art or commercially available (e.g., BLAST. ExPASy: Clustal Omega: FASTA; using, e.g., Needleman- Wunsch algorithm, Smith-Waterman algorithm). Alignments are performed using any of a variety of publicly or commercially available Multiple Sequence Alignment Programs. Multiple sequence alignment programs are available for nucleic acid sequences. Examples of such programs include, “Clustal Omega”, “Clustal W”, “MUSCLE”, “CAP Sequence Assembly”, “BLAST”, “MAP”, and “MEME”, which are accessible through Web Servers on the internet. Other sources for such programs are known to those of skill in the art. Alternatively, Vector NTI utilities are also used. There are also a number of algorithms known in the art that can be used to measure nucleotide sequence identity, including those contained in the programs described above. As another example, polynucleotide sequences can be compared using Fasta™, a program in GCG Version 10. 1. Fasta™ provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. For instance, percent sequence identity between nucleic acid sequences can be determined using Fasta™ with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) as provided in GCG Version 10. 1, herein incorporated by reference. Sequence alignment programs are also available for amino acid sequences, e.g., the “Clustal Omega”, “Clustal X”, “MUSCLE”, “MAP”, “PIMA”, “MSA”, “BLOCKMAKER”, “MEME”, and “Match-Box” programs. Generally, any of these programs are used at default settings, although one of skill in the art can alter these settings as needed. Alternatively, one of skill in the art can utilize another algorithm or computer program which provides at least the level of identity or alignment as that provided by the referenced algorithms and programs. See, e.g., J. D. Thomson et al, Nucl. Acids. Res., “A comprehensive comparison of multiple sequence alignments”, 27(13):2682-2690 (1999).

[0226] As used herein, the term “administration” or any grammatical variations thereof refers to delivery’ of composition described herein to a subject.

[0227] As used herein, an effective amount may be determined based on an animal model, rather than a human patient.

[0228] Designation of a range of values includes all integers within or defining the range, and all subranges defined by integers within the range.

[0229] As described above, tire tenn “about” when used to modify a numerical value means a variation of ±10%, (±10%, e.g., ±1, ±2, ±3, ±4, ±5, ±6, ±7, ±8, ±9, ±10, or values therebetween) from the reference given, unless otherwise specified.

[0230] In certain instances, the term “E±#” or the term “e±#” is used to reference an exponent. For example, “5E10” or “5el0” is 5 x 10lu. These terms may be used interchangeably.

[0231] As used throughout this specification and the claims, the terms “comprise” and “contain” and its variants including, “comprises”, “comprising”, “contains” and “containing”, among other variants, is inclusive of other components, elements, integers, steps and the like. The term “consists of’ or “consisting of’ arc exclusive of other components, elements, integers, steps and the like.

[0232] It is to be noted that the term “a” or “an”, refers to one or more, for example, “an enhancer”, is understood to represent one or more enhancer(s). As such, the terms “a” (or “an”), “one or more,” and “at least one” is used interchangeably herein. Unless defined otherw ise in this specification, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and by reference to published texts, which provide one skilled in the art with a general guide to many of the terms used in the present application.

[0233] EXAMPLES

[0234] The following examples are provided to illustrate certain aspects of the claimed invention. The invention is not limited to these examples.

[0235] EXAMPLE 1 : Production of rAAV comprising engineered HEXA, HEXB, and GM2A.

[0236] In the studies herein, various engineered sequences encoding P-N-acetylhexosaminidase alpha subunit (HexA), P-N-acetylhexosaminidase beta subunit (HexB), and GM2 activator (GM2A). and various rAAV comprising HexA and HexB coding sequences, or rAAV comprising HexA, HexB, and GM2A coding sequences were generated, and comparative studies were performed. rAAV are generated using triple transfection techniques, utilizing (1) a cis plasmid encoding AAV2 rep proteins and the AAVhu68 VP1 cap gene (alternatively AAVrh91, AAVhu95, AAV9, or a mutant AAV 9), (2) a cis plasmid comprising adenovirus helper genes not provided by the packaging cell line which expresses adenovirus El a, and (3) a trans plasmid containing the vector genome for packaging in the AAV capsid. See, e.g.. US 2020 / 0056159. The trans plasmid is designed to contain either tire vector genome comprising engineered HexA and HexB, or vector genome comprising engineered HexA, HexB and GM2A. The vector genomes include (among others) SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14.

[0237] EXAMPLE 2: Evaluation of rAAV encoding HexA, HexB and GM2A.

[0238] In this study, we evaluated novel compact multicistronic (bi- and tri-cistronic) expression cassettes applicable for the design of gene therapy drugs when several therapeutic proteins need to be expressed from a single AAV vector (including but not limited to GM2 gangliosidosis, AAV expressing monoclonal antibodies, AAV expressing a therapeutic protein along with a helper protein etc.). For gangliodisosis type 2 (GM2, also known as Tay-Sachs disease, Sandhoff disease, and GM2 activator deficiency), the disease is caused by mutation of either HEXA, HEXB, or GM2A genes encoding for 3 different proteins all responsible for a 0-N- acetylhexosaminidase A (hexosaminidase A) enzyme deficiency. Hexosaminidase A deficiency results in gangliosides lysosomal storage affecting the central nervous system. It manifests as a severe infantile neurodegenerative disease without any approved disease-modifying treatment currently available. Hexosaminidase A enzyme is a heterodimer composed of an alpha sub-unit (HexA or HEX-alpha encoded by the HEXA gene), a beta sub-unit (HexB or HEX-beta encoded by HEXB gene), with GM2A activator protein acting as a necessary co-factor. In order to provide disease prevention or correction using gene therapy, the therapeutic vector needs to co-express at minimal HEX-alpha and HEX-beta in similar proportions in same cell to generate a functional heterodimer enzyme. We also hypothesize that co-expressing the cofactor GM2A protein along with HEX-alpha and HEX-beta would provide a significant benefit. In this study, we describe several short bidirectional promoters alone or combined with 2A self-cleaving peptides used to produce multicistronic (bi-cistronic or tri-cistronic) expression cassette for use in AAV gene therapy. The bi-cistronic constructs encode HEX-alpha and HEX-beta from bi-directional promoters or from CAG promoter with self-cleaving peptides separating the two reading frames. The tri-cistronic construct encodes HEX-alpha, HEX-beta, and the GM2A protein. The ITR to 1TR sizes are comprised between 4.3 to 4.97 kb, which is amenable to AAV encapsidation with acceptable manufacturability.

[0239] Briefly, generating a functional hexosaminidase A heterodimers requires co-expression of the a- and 0-subunits within tire same cell; given the limited transgene capacity of AAV vectors, previous work has explored the co-delivery of two separate monocistronic AAV vectors, albeit with limited success. Our objective was to develop bicistronic AAV vectors that co-express both subunits of hexosaminidase A and identify the optimal design.

[0240] We developed novel multicistronic (i.e., bi-cistronic) AAV-hu68 vectors co-expressing the HEXA and HEXB complementary' DNA using a 2A peptide or bidirectional (BD) promoter strategy'. FIG. 1 show s a schematic overview of the designed AAV vectors comprising multicistronic expression cassettes. We evaluated several different viral 2A peptides (2A-1 to 2A- 5, including Foot-and-mouth disease virus peptide with furin recognition site connected via tire GSG linker (GF2A), Porcine teschovirus- 1 (P2A) peptide with furin recognition site connected via the GSG linker (GP2A). and Thosea asigna virus peptide with furin recognition site connected via the GSG linker (GT2A)), as well as novel and naturally occurring short bi-directional (bidirectional or BD) promoters with or without the cytomegalovirus enhancer, ranging in size from 131 bp to 869 bp (BD-1 to BD-6). BD promoters include: CB-CMV-CB bidirectional promoter (869 bp: wherein CB-CMV-CB comprises a first copy of a chicken beta actin (CB) promoter, a cytomegalovirus (CMV) enhancer, and a second copy of a chicken beta actin (CB) promoter); AIRC-GPAT bidirectional promoter (AI / GP-BDP; also GP-AI; 778 bp; AIRC and GPAT which encode enzy mes in the pathway for de novo purine nucleotide synthesis in vertebrates, wherein tire GPAT and AIRC genes are divergently transcribed from a 778 bp intergenic promoter region); RHN01-F0XM1 bidirectional promoter (FO / RH-BDP; also RH- FO; 131 bp; wherein F0XM1 (an oncoprotein) and RHN01 (a gene involved in the ATR-Chkl signaling pathway that functions in the DNA replication stress response) are head-to-head oncogenes (i.e., bidirectional) regulated by a bidirectional promoter (named F / R-BDP), optionally with CMV enhancer (RH-CMVe-FO); POFUT1-PLAGL2 bidirectional promoter (PL / PO-BDP; also PO-PL185 bp; wherein PLAGL2 and POFUT1 are regulated by an evolutionarily conserved bidirectional promoter and are collaboratively involved in colorectal cancer).

[0241] We identified vectors with tire most efficient 2A peptides following in vitro transfection and Western blot analysis of HEXA-HEXB double knockout (AB-KO) human embryonic kidney (HEK293) cells.

[0242] Pilot Study to Determine the Expression Kinetics of AAV Vectors Containing Bi-Directional Promoters in Adult Mouse Tissue

[0243] To test the BD promoters, we constructed AAV vectors with enhanced green fluorescent protein and mCherry fluorescent biomarkers flanking the promoters and tested these vectors in vitro in 293T cells and in vivo in C 7B16 mice.

[0244] Briefly, wild-type (WT) mice were administered intravenously (IV) with 4 x 1012GC (n=4 per group, 1-5 month mice), and mice were monitored for 4 weeks, following which necropsy was performed, and tissues were collected for histopathology (i.e., readout of GFP / mCherry signal in DRG, Brain, spinal cord, heart, liver, quad muscle tissue).

[0245] FIG. 2A shows a schematic rAAV vector genome (comprising CB-CMV-CB bidirectional promoters). FIG. 2B shows a representative microscopy image (mCherry) of liver tissue samples which confinn expression kinetics of rAAV vectors containing CB-CMV-CB bidirectional promoters in adult mouse tissue. FIG. 2C shows a representative microscopy image (GFP) of liver tissue samples which confirm expression kinetics of rAAV vectors containing CB- CMV-CB bi-directional promoters in adult mouse tissue. FIG. 2D shows a representative microscopy image (mCherry / GFP overlay) of liver tissue samples which confirm expression kinetics of rAAV vectors containing CB-CMV-CB bi-directional promoters in adult mouse tissue.

[0246] FIG. 3A shows a schematic rAAV vector genome (comprising AI-GP bi-directional promoters). FIG. 3B shows a representative microscopy image (mCherry) of liver tissue samples which confirm expression kinetics of rAAV vectors containing AI-GP bi-directional promoters in adult mouse tissue. FIG. 3C shows a representative microscopy image (GFP) of liver tissue samples which confirm expression kinetics of rAAV vectors containing AI-GP bi-directional promoters in adult mouse tissue. FIG. 3D shows a representative microscopy image (mCherry / GFP overlay) of liver tissue samples which confirm expression kinetics of rAAV vectors containing AI-GP bi-directional promoters in adult mouse tissue.

[0247] FIG. 4A shows a schematic rAAV vector genome (comprising PO-PL bi-directional promoters). FIG. 4B shows a representative microscopy image (mCherry) of liver tissue samples which confirm expression kinetics of rAAV vectors containing PO-PL bi-directional promoters in adult mouse tissue. FIG. 4C shows a representative microscopy image (GFP) of liver tissue samples which confirm expression kinetics of rAAV vectors containing PO-PL bi-directional promoters in adult mouse tissue. FIG. 4D shows a representative microscopy image (mCherry / GFP overlay) of liver tissue samples which confirm expression kinetics of rAAV vectors containing PO-PL bi-directional promoters in adult mouse tissue.

[0248] FIG. 5A shows a schematic rAAV vector genome (comprising RH-CMVe-FO bidirectional promoters). FIG. 5B shows a representative microscopy image (mCherry) of liver tissue samples which confinn expression kinetics of AAV vectors containing RH-CMVe-FO bidirectional promoters in adult mouse tissue. FIG. 5C shows a representative microscopy image (GFP) of liver tissue samples which confirm expression kinetics of rAAV vectors containing RH- CMVe-FO bi-directional promoters in adult mouse tissue. FIG. 5D shows a representative microscopy image (mCherry / GFP overlay) of liver tissue samples which confirm expression kinetics of rAAV vectors containing RH-CMVe-FO bi-directional promoters in adult mouse tissue.

[0249] FIG. 6A shows a schematic rAAV vector genome (comprising AI-GP bi-directional promoters). FIG. 6B shows a representative microscopy image (mCherry) of quadricep muscle tissue samples which confirm expression kinetics of rAAV vectors containing AI-GP bi- directional promoters in adult mouse tissue. FIG. 6C shows a representative microscopy image (GFP) of quadricep muscle tissue samples which confirm expression kinetics of rAAV vectors containing AI-GP bi-directional promoters in adult mouse tissue. FIG. 6D shows a representative microscopy image (mCherry / GFP overlay) of quadricep muscle tissue samples which confirm expression kinetics of rAAV vectors containing AI-GP bi-directional promoters in adult mouse tissue.

[0250] FIG. 7A shows a schematic rAAV vector genome (comprising AI-GP bi-directional promoters). FIG. 7B shows a representative microscopy image (mCherry) of brain tissue samples which confirm expression kinetics of rAAV vectors containing AI-GP bi-directional promoters in adult mouse tissue. FIG. 7C shows a representative microscopy image (GFP) of brain tissue samples which confirm expression kinetics of rAAV vectors containing AI-GP bi-directional promoters in adult mouse tissue. FIG. 7D shows a representative microscopy image (mCherry / GFP overlay) of brain tissue samples which confirm expression kinetics of rAAV vectors containing AI-GP bi-directional promoters in adult mouse tissue.

[0251] These results show that both AI-GP and RH-CMVE-FO promoters outperformed tire CBP-CMVE-CBP in liver. Additionally, these result show that AI-GP was the only one of the promoters that also expressed in quadricep muscle and brain.

[0252] In Vitro Hex Expression and Activity Analysis from AAV Vectors Containing Various 2A Peptides and Various Bi-Directional (BD) Promoters

[0253] Next, we evaluated the therapeutic efficacy of selected AAV-hu68 vectors with the various self-cleaving (e g., 2A) peptides or various bidirectional (bi-directional, BD) promoters in 4- to 6-week-old SD mice (Hexb ' ) following intracerebroventricular (ICV) administration of 1E11 (1 x 1011) genome copies (GC) / mouse.

[0254] We performed Western blot analysis of cell lysates from HEXA / BKO 293T cells transfected in vitro with the bicistronic AAV vectors engineered with different 2A peptides. Additionally, we analyzed HexA activity following in vitro transfection of HEXA / BKO cells. Hex activity’ was measured using two artificial substrates: MUG to determine total Hex activity’ (HexA, HexB, and HexS), and MUGS to determine HexA activity. For AAV vectors with novel bidirectional (BD) promoters. BD promoters from the human genome were used. For comparison, an AAV vector containing a BD promoter with a chicken beta-actin promoter (CBP) and CMVE was developed and tested in parallel. AAV vectors with enhanced green fluorescent protein (eGFP) and mCherry fluorescent transgenes flanking the promoters were tested in vitro and in vivo (see, FIGs. 2-7). The best performing BD promoters were used to generate bicistronic AAV vectors expressing HexA and HexB cDNAs and tested via in vitro transfected HEXA / B-KO 293T cells.

[0255] AAV vectors with 2A-1 (GP2A peptide) and 2A-2 (GT2A peptide) exhibited the highest expression, near-complete peptide cleavage, and highest enzyme activity in transfected AB-KO HEK293 cells. Transient expression in transfected 293T cells detected hexosaminidase expression and activity from all the constructs; strongest for CBP-CMVE-CBP promoter.

[0256] Among the BD promoters, AAV vectors with BD- 1 (GP-AL bidirectional promoter), BD- 2 (PO-PL bidirectional promoter), and BD-3 (RH-CMVe_FO) conferred the highest transgene expression and enzyme activity in transfected AB-KO HEK293 cells. Following ICV dosing in adult SD mice, we detected HexA enzyme activity' in the blood of all dosed animals on day 7 at levels that reached or exceeded wild-type levels. Moreover, all AAV vectors significantly improved survival compared to untreated SD mice. The median survival increased post dosing from 10.3 weeks for untreated SD mice to 15.7 weeks for AAVhu68-HEXB-[BD-3]-HEXA vector-dosed SD mice (p<0.01), 17.6 weeks for AAVhu68-HEXA-[2A2]-HEXB vector-dosed mice (p<0.005), and an undefined survival for AAVhu68-HEXA-[2Al]-HEXB vector-dosed mice, as 50% of the treated SD mice in this group survived past 22 weeks post-dosing (the experimental endpoint). Histological analysis of SD mice brains stained with an anti-GM2 antibody demonstrated that all vector-treated SD mice exhibited decreased GM2 buildup compared with untreated SD mice; we observed tire greatest reductions (85% and 67%) in GM2 accumulation in tire brain in SD mice treated with AAVhu68-HEXA-[2A2]-HEXB and AAVhu68-HEXB-[BD-3]-HEXA vector, respectively.

[0257] FIG. 8A shows a schematic representation of evaluated rAAV vectors. FIG. 8B shows in vitro hexosaminidase activity analysis from AAV vectors containing various bi-directional promoters. FIG. 8C shows in vitro hexosaminidase expression (HEXA / HEXB) analysis (western blot) from AAV vectors containing various bi-directional promoters. FIG. 8D shows in vitro hexosaminidase expression (HEXA / HEXB) analysis (quantified from western blot) from AAV vectors containing various bi-directional promoters. FIG. 8E shows hexosaminidase A activity following in vitro transfection of HEXA / B-KO 293T cells with AAV comprising various BD promoters. These results show that transient expression in transfected 293 T cells detected Hex expression and activity from all the constructs, with strongest for CBP-CMVE-CBP promoter. FIG. 9A is a schematic representation of evaluated rAAV vectors. FIG. 9B shows in vitro hexosaminidase activity analysis from AAV vectors containing various 2A cleavage peptides. FIG. 9C shows in vitro hexosaminidase expression analysis (western blot) from AAV vectors containing various 2A cleavage peptides. FIG. 9D shows in vitro hexosaminidase expression analysis (quantified from western blot) from AAV vectors containing various 2A cleavage peptides. FIG. 9E shows hexosaminidase A activity of following in vitro transfection of HEXA / BKO cells with AAV comprising various 2A peptides. These results show that vectors with GP2A and GT2A peptides performed the best in vitro.

[0258] These results show that bicistronic AAV vectors with the 2A-1 (GP2A) and 2A-2 (GT2A) peptides achieved the most efficient peptide cleavage and exhibited the highest hexosaminidase A activity following in vitro transfection of HEXA / B-KO 293T cells. Further, these results show7that bicistronic AAV vectors with BD-3B (RH-CMVe-FO) bidirectional promoter exhibited the highest and most proportional transgene expression in vitro.

[0259] In conclusion, upon ICV administration in SD mice, our novel bicistronic AAV vectors achieved high HexA expression and hexosaminidase A activity levels, ameliorated brain GM2 accumulation, and conferred significant survival benefits. These data highlight the potential efficacy of bicistronic AAV vectors for treating GM2 gangliosidoses such as TSD and SD.

[0260] In Vitro Hexosaminidase Activity Analysis of the Tricistronic AAV Vector

[0261] Additionally, we evaluated a tri-cistronic rAAV comprising HexA, HexB, and GM2A coding sequences for HexA, HexB, GM2A expression, hexosaminidase A activity levels, and effect on brain GM2 accumulation.

[0262] Mouse Models of GM2 Gangliosidosis

[0263] A moouse model for Tay-Sachs is commercially available from Jackson Laboratory (Jax stock # 002367; Hexa ' B6 mice; 129S-HexatmlRlp / J). Mice are viable and fertile, and present neuropathology7like human disease, however demonostrate a mild phenotype. Homozygous Hexa mice elicit partial catabolism of accumulated GM2 via GA2 through actions of sialidasc and - hexosaminidase B (Phaneuf et al.. 1996).

[0264] Mouse models for Sandhoff Disease are commercially available from Jackson Laboratory (Hexb Jax stock #002914; B6 mice; 129S4-HexbtmRlp / J), which mice present with severe phenoty pe closer to human disease, and that show motor defects by 3 months of age which progressively worsens, with abnormal gangliosidosis observed as well (GM2 / GA2 ganglioside accumulation), and with observed death by 4.5 months of age. See also, Phaneuf et al., 1996. Dramatically different phenotypes in mouse models of human Tay-Sachs and Sandhoff disease; and Seyrantepe et al., 2018. Murine sialidase Neu3 facilitates GM2 degradation and bypass in a mouse model of Tay-Sachs disease.

[0265] In this study, we evaluated a natural history of Sandhoff Disease for HexB mice with a goal to evaluate potential biomarkers of efficacy in Hexb mice. Treatment Groups included 2 groups: WT (8M / 8F) & Hexb HOM mice (10M / 10F), with age of enrollment at 1-1.5 months. The duration of the study is 150 days. The readouts included: behavior (open field, rotarod (BL, D30, D60, D90, D120)), bleeds (BL, D30, D60, D90, D120, D150); hexosaminidase activity (serum, liver, brain); histology: PAS, GM2 staining, CD68.

[0266] FIG. 10A shows results of the behavior study as measured latency to fall (rotarod averages) of WT and HexB HOM mice. FIG. 10B shows results of the survival study plotted as percent probability of survival of WT and HexB HOM mice. FIG. 11A shows analysis of hexosaminidase activity in serum. FIG. 1 IB shows analysis of hexosaminidase activity in brain and liver. These results show that all HexB HOM mice reached humane end point / FD between 4- 4.5 month of age.

[0267] In Vivo Efficacy of Bicistronic AAV Vector in Mice

[0268] In this study, the therapeutic efficacy of selected AAV-hu68 vectors with the bestperforming 2A peptides or BD promoters were evaluated in 4- to 6-week-old Sandhoff disease (SD) mice (Hexb / _) following intracerebroventricular (ICV) administration of IE 11 (1 xlO11) genome copies / mouse. FIG. 12A shows hexosaminidase A enzyme activity following administration of bicistronic AAV vectors in mice. Hexosaminidase A enzyme activity was detected in the blood of all dosed animals on day 7 at levels that reached (BD-3; RH-CMVe-FO) or exceeded (2A-1; GP2A) wild type levels. FIG. 12B shows Kaplan-Meier surv ival analysis of probability of survival in mice following administration of bicistronic AAV vectors. All AAV vectors significantly improved survival compared to untreated SD mice. The best survival was obtained for SD mice that received AAVhu68-HEXA-[2Al]-HEXB vector (GP2A peptide), with 50% surviving beyond 22 weeks post-dosing (the experimental endpoint). These results show that bicistronic AAV vectors with 2A-1 (GP2A) peptide and BD-3 (RH CMVe FO) promoter had the highest hexosaminidase A enzyme activity and conferred the longest survival to ICV- administered SD mice.

[0269] Additionally, we performed histological analysis of brains following bicistronic AAV vector administration. FIG. 13 shows quantified histological analysis of SD mice brains stained with an anti-GM2 antibody. These results demonstrated that all vector-treated SD mice exhibited decreased GM2 buildup compared with untreated SD mice.

[0270] FIG. 14A shows a representative image of histological analysis of SD mice brain stained with an anti-GM2 antibody in wild-type (WT) mice. FIG. 14B shows a representative image of histological analysis of SD mice brain (midbrain) stained with an anti-GM2 antibody in WT mice. FIG. 14C shows representative image of histological analysis of SD mice brain (cerebellum) stained with an anti-GM2 antibody in WT mice. FIG. 14D shows a representative image of histological analysis of SD mice brain (cortex) stained with an anti-GM2 antibody in WT mice. FIG. 14E shows a representative image of histological analysis of SD mice brain (hippocampus) stained with an anti-GM2 antibody in WT mice. FIG. 14F shows a representative image of histological analysis of SD mice brain (brain stem) stained with an anti-GM2 antibody in WT mice. FIG. 14G shows a representative image of histological analysis of SD mice brain (thalamus) stained with an anti-GM2 antibody in WT mice.

[0271] FIG. 15A shows a representative image of histological analysis of SD mice brain stained with an anti-GM2 antibody in KO mice treated with PBS. FIG. 15B shows a representative image of histological analysis of SD mice brain (midbrain) stained with an anti-GM2 antibody in KO mice treated with PBS. FIG. 15C shows a representative image of histological analysis of SD mice brain (cerebellum) stained with an anti-GM2 antibody in KO mice treated with PBS. FIG. 15D shows a representative image of histological analysis of SD mice brain (cortex) stained with an anti-GM2 antibody in KO mice treated with PBS. FIG. 15E shows representative image of histological analysis of SD mice brain (hippocampus) stained with an anti-GM2 antibody in KO mice treated with PBS. FIG. 15F shows a representative image of histological analysis of SD mice brain (brain stem) stained with an anti-GM2 antibody in KO mice treated with PBS. FIG. 15G shows a representative image of histological analysis of SD mice brain (thalamus) stained with an anti-GM2 antibody in KO mice treated with PBS.

[0272] FIG. 16A shows a representative image of histological analysis of SD mice brain stained with an anti-GM2 antibody in Hexb knock out mice treated with AAVhu68-HEXA-[2Al]-HEXB (GP2A peptide). FIG. 16B shows a representative image of histological analysis of SD mice brain (midbrain) stained with an anti-GM2 antibody in Hexb knock out mice treated with AAVhu68- HEXA-[2A1]-HEXB (GP2A peptide). FIG. 16C shows a representative image of histological analysis of SD mice brain (cerebellum) stained with an anti-GM2 antibody in Hexb knock out mice treated with AAVhu68-HEXA-[2Al]-HEXB (GP2A peptide). FIG. 16D shows a representative image of histological analysis of SD mice brain (cortex) stained with an anti-GM2 antibody in Hexb knock out mice treated with AAVhu68-HEXA-[2Al]-HEXB (GP2A peptide). FIG. 16E shows a representative image of histological analysis of SD mice brain (hippocampus) stained with an anti-GM2 antibody in Hexb knock out mice treated with AAVhu68-HEXA- [2A1J-HEXB (GP2A peptide). FIG. 16F shows a representative image of histological analysis of SD mice brain (brain stem) stained with an anti-GM2 antibody in Hexb knock out mice treated with AAVhu68-HEXA-[2Al]-HEXB (GP2A peptide). FIG. 16G shows a representative image of histological analysis of SD mice brain (thalamus) stained with an anti-GM2 antibody in Hexb knock out mice treated with AAVhu68-HEXA-[2Al]-HEXB (GP2A peptide).

[0273] FIG. 17A shows a representative image of histological analysis of SD mice brain stained with an anti-GM2 antibody in Hexb knock out mice treated with AAVhu68-HEXB-[BD-3]- HEXA (RH-CMVe-FO). FIG. 17B shows a representative image of histological analysis of SD mice brain (midbrain) stained with an anti-GM2 antibody in Hexb knock out mice treated with AAVhu68-HEXB-[BD-3]-HEXA (RH-CMVe-FO). FIG. 17C shows a representative image of histological analysis of SD mice brain (cerebellum) stained with an anti-GM2 antibody in Hexb knock out mice treated with AAVhu68-HEXB-[BD-3]-HEXA (RH-CMVe-FO). FIG. 17D shows a representative image of histological analysis of SD mice brain (cortex) stained with an anti- GM2 antibody in Hexb knock out mice treated with AAVhu68-HEXB-[BD-3]-HEXA (RH- CMVe-FO). FIG. 17E shows a representative image of histological analysis of SD mice brain (hipppocampus) stained with an anti-GM2 antibody in Hexb knock out mice treated with AAVhu68-HEXB-[BD-3]-HEXA (RH-CMVe-FO). FIG. 17F shows a representative image of histological analysis of SD mice brain (brain stem) stained with an anti-GM2 antibody in Hexb knock out mice treated with AAVhu68-HEXB-[BD-3]-HEXA (RH-CMVe-FO). FIG. 17G shows a representative image of histological analysis of SD mice brain (thalamus) stained with an anti- GM2 antibody in Hexb knock out mice treated with AAVhu68-HEXB-[BD-3]-HEXA (RH- CMVe-FO).

[0274] FIG. 18A shows a representative image of histological analysis of SD mice brain stained with an anti-GM2 antibody in Hexb knock out mice treated with AAV-HexB-CB-CMVe-CB- HexA. FIG. 18B shows a representative image of histological analysis of SD mice brain (midbrain) stained with an anti-GM2 antibody in Hexb knock out mice treated with AAV-HexB- CB-CMVe-CB-HexA. FIG. 18C shows a representative image of histological analysis of SD mice brain (cerebellum) stained with an anti-GM2 antibody in Hexb knock out mice treated with AAV-HexB-CB-CMVe-CB-HexA. FIG. 18D show s a representative image of histological analysis of SD mice brain (cortex) stained w ith an anti-GM2 antibody in Hexb knock out mice treated with AAV-HexB-CB-CMVe-CB-HexA. FIG. 18E shows a representative image of histological analysis of SD mice brain (hippocampus) stained with an anti-GM2 antibody in Hexb knock out mice treated with AAV-HexB-CB-CMVe-CB-HexA. FIG. 18F shows a representative image of histological analysis of SD mice brain (brain stem) stained with an anti-GM2 antibody in Hexb knock out mice treated with AAV-HexB-CB-CMVe-CB-HexA. FIG. 18G show s a representative image of histological analysis of SD mice brain (thalamus) stained with an anti- GM2 antibody in Hexb knock out mice treated with AA V-HexB-CB-CMVe-CB-HexA.

[0275] These results show that the greatest reductions (75% and 59%) in GM2 accumulation were observed in the brains of SD mice treated with AAVhu68-HEXA-[2A-l]-HEXB and AAVhu68-HEXB-|BD-3 l-HEXA vector, respectively. Additionally, these results show that bicistronic AAV vectors with 2A- 1 peptide and BD-3 promoter conferred the highest therapeutic efficacies, as reflected by the reduction of GM2 in brain of ICV-administered SD mice.

[0276] These studies show that upon ICV administration in SD mice, our novel bicistronic AAV vectors achieved high hexosaminidase A expression and activity levels, ameliorated brain GM2 accumulation, and conferred significant survival benefits. These data highlight the potential efficacy of bicistronic AAV vectors for treating GM2 gangliosidoses such as TSD and SD.

[0277] All documents cited in this specification are incorporated herein by reference. US Provisional Application No. 63 / 631,191, filed April 8, 2024, and US Provisional Patent Application No. 63 / 643,731, filed May 7, 2024 are incorporated herein by reference. While the invention has been described with reference to particular embodiments, it will be appreciated that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.

Claims

CLAIMS:

1. A recombinant adeno-associated virus (rAAV) useful for preventing accumulation of GM2 ganglioside, the rAAV comprising an adeno-associated virus (AAV) capsid and a vector genome in the AAV capsid. wherein the vector genome is a nucleic acid molecule which comprises an expression cassette comprising a nucleic acid sequence encoding P-N-acetylhexosaminidase alpha subunit (HexA), a nucleic acid sequence encoding P-N-acetylhcxosaminidase beta subunit (HcxB), wherein the HexA subunit and HcxB subunit coding sequences arc each operably linked to regulatory sequences which pennit expression of the HexA subunit and the HexB subunit proteins in a target cell, wherein the expression cassette is a multicistronic expression cassette and comprises:(a) the HexA coding sequence, a bi-directional promoter and the HexB coding sequence t; or(b)he HexA coding sequence, a 2A linker, and tire HexB coding sequence .

2. The rAAV according to claim 1. wherein tire multicistronic expression cassette is bi-cistronic or tri-cistronic, wherein the tri-cistronic cassette optionally further comprises a nucleic acid sequence encoding ganglioside GM2 activator (GM2A).

3. The rAAV according to claim 1 or claim 2, wherein the nucleic acid sequence encoding for HexA comprises nucleic acid sequence of SEQ ID NO: 15, or a sequence at least 95% identical to SEQ ID NO: 15 encoding an amino acid sequence of SEQ ID NO: 16.

4. The rAAV according to any one of claims 1 to 3, wherein the nucleic acid sequence encoding HexB comprises nucleic acid sequence of SEQ ID NO:

17. or a sequence at least 95% identical to SEQ ID NO: 17 encoding an amino acid sequence of SEQ ID NO: 18.

5. The rAAV according to any one of claims 1 to 4, wherein the expression cassette of (a) comprises the 2 A linker, wherein the 2A linker comprises:(A) a furin recognition site, GSG linker, and Porcine teschovirus- 1 peptide(GP2A),(B) a furin recognition site, GSG linker, and Thosea asigna virus peptide (GT2A) , or(C) a furin recognition site, GSG linker, and Foot-and-mouth disease virus peptide (GF2A).

6. The rAAV according to any one of claims 1 to 5, wherein the rAAV comprises the multicstronic expression cassette of (a) comprising:(i) a CAG promoter comprising cytomegalovirus enhancer, a chicken beta actin promoter, and a chicken beta actin intron,(ii) the HexA coding sequence,(iii) GP2A linker or a GT2A linker,(iv) the HexB coding sequence, and(v) a rabbit beta globin (rBG) polyA signal sequence.

7. The rAAV according to any one of claims 1 to 6, wherein the expression cassette comprises CAG promoter comprising nucleic acid sequence of SEQ ID NO: 21 , a HexA coding sequence of SEQ ID NO: 15, a GP2A linker comprising a nucleic acid sequence of SEQ ID NO:22, a HexB coding sequence comprising a nucleic acid sequence of SEQ ID NO: 17, and a rBG polyA comprising a nucleic acid sequence of SEQ ID NO: 24.

8. The rAAV according to claim 8. wherein tire expression cassette comprises nucleic acid sequence of SEQ ID NO: 1.

9. The rAAV according to any one of claims 1 to 6, wherein the expression cassette comprises CAG promoter comprising nucleic acid sequence of SEQ ID NO: 21, a HexA coding sequence of SEQ ID NO: 15, a GT2A linker comprising a nucleic acid sequence of SEQ ID NO:23, a HexB coding sequence comprising a nucleic acid sequence of SEQ ID NO:

17. and a rBG polyA comprising a nucleic acid sequence of SEQ ID NO: 24.

10. The rAAV according to claim 9. wherein the expression cassette comprises tire nucleic acid sequence of SEQ ID NO: 3.

11. The rAAV according to any one of claims 1 to 4, wherein the rAAV comprises the multicistronic expression cassette of (b) comprising:(i) a rabbit beta globin (rBG) polyA signal sequence.(ii) the HexB coding sequence,(iii) a bi-directional promoter,(iv) the HexA coding sequence,(v) a bovine growth hormone (bGH) polyA signal sequence.

12. The rAAV according to any one of claims 1 to 4 and 11, wherein the bidirectional promoter is AIRC-GPAT bidirectional promoter.

13. The rAAV according to any one of claims 1 to 4 and 11, wherein tire bidirectional promoter is a POFUT 1-PLAGL2 bidirectional promoter comprising a protein 0- fucosyltransferase 1 (P0FUT1) promoter and pleomorphic adenoma gene-like 2 (PLAGL2) promoter.

14. The rAAV according to any one of claims 1 to 4 and 11, wherein the bidirectional promoter is a CB-CMV-CB bidirectional promoter comprising a first copy of a chicken beta actin (CB) promoter, a cytomegalovirus (CMV) enhancer, and a second copy of a chicken beta actin (CB) promoter.

15. The rAAV according to any one of claims 1 to 4 and 11, wherein the bidirectional promoter is an RHON 1-CMV-F0XM1 bidirectional promoter comprising RAD9- HUS1-RAD1 Interacting Nuclear Orphan 1 (RHN01) promoter, a cytomegalovirus (CMV) enhancer, and forkhcad box protein Ml (F0XM1) promoter.

16. The rAAV according to any one of claims 1 to 4, 11 and 12, wherein the expression cassette of (b) comprises a rabbit beta globin (rBG) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 24, a HexB coding sequence comprising nucleic acidsequence of SEQ ID NO: 17, a AIRC-GPAT bi-directional promoter comprising nucleic acid sequence of SEQ ID NO: 37, a HexA coding sequence comprising nucleic acid sequence of SEQ ID NO: 15, a bovine growth hormone (bGH) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 25.

17. The rAAV according to claim 16, wherein the expression cassette of (b) comprises nucleic acid sequence of SEQ ID NO: 5.

18. The rAAV according to any one of claims I to 4, 11 and 13. wherein the expression cassette of (b) comprises a rabbit beta globin (rBG) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 24, a HexB coding sequence comprising nucleic acid sequence of SEQ ID NO: 17, a POFUT1-PLAGL2 bi-directional promoter comprising nucleic acid sequence of SEQ ID NO: 31, a HexA coding sequence comprising nucleic acid sequence of SEQ ID NO: 15, a bovine growth hormone (bGH) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 25.

19. The rAAV according to claim 18, wherein the expression cassette of (b) comprises nucleic acid sequence of SEQ ID NO: 9.

20. The rAAV according to any one of claims 1 to 4, 11 and 14, wherein the expression cassette of (b) comprises a rabbit beta globin (rBG) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 24, a HexB coding sequence comprising nucleic acid sequence of SEQ ID NO: 17, a CB-CMV-CB bi-directional promoter comprising nucleic acid sequence of SEQ ID NO: 28, a HexA coding sequence comprising nucleic acid sequence of SEQ ID NO: 15, a bovine growth hormone (bGH) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 25.

21. The rAAV according to claim 20, wherein the expression cassette of (b) comprises nucleic acid sequence of SEQ ID NO: 7.

22. The rAAV according to any one of claims 1 to 4, 11 and 15, wherein the expression cassette of (b) comprises a rabbit beta globin (rBG) polyA signal sequence comprisingnucleic acid sequence of SEQ ID NO:

24. a HexB coding sequence comprising nucleic acid sequence of SEQ ID NO: 17, a RHONl-CMV-FOXMlbi-directional promoter comprising nucleic acid sequence of SEQ ID NO: 34, a HexA coding sequence comprising nucleic acid sequence of SEQ ID NO: 15, a bovine growth hormone (bGH) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 25.

23. The rAAV according to claim 22, wherein the expression cassette of (b) comprises nucleic acid sequence of SEQ ID NO: 11.

24. The rAAV according to any one of claims 2 to 4, wherein the nucleic acid sequence encoding GM2A comprises nucleic acid sequence of SEQ ID NO: 19, or a sequence at least 95% identical to SEQ ID NO: 19 encoding an amino acid sequence of SEQ ID NO: 20.

25. The rAAV according to any one of claims 2 to 4, and 24, wherein tire multicistronic expression cassette is tri-cistronic expression cassette and comprises a linker, wherein tire linker comprises:(A) a furin recognition site, GSG linker, and Porcine teschovirus- 1 peptide (GP2A),(B) a furin recognition site, GSG linker, and Thosea asigna virus peptide (GT2A), or(C) a furin recognition site, GSG linker, and Foot-and-mouth disease virus peptide (GF2A).

26. The rAAV according to any one of claims 2 to 4, 24 and 25. wherein the rAAV comprises the tri-cistronic expression cassette comprising:(i) a bovine growth hormone (bGH) polyA signal sequence,(ii) the GM2A coding sequence,(iii) the bi-dircctional promoter,(iv) the HexA coding sequence,(v) the GT2A linker,(vi) the HexB coding sequence, and(vii) a rabbit beta globin (rBG) polyA signal sequence.

27. The rAAV according to any one of claims 2 to 4, and 24 to 26, wherein the bidirectional promoter is a POFUT1-PLAGL2 bi-directional promoter comprising a protein 0- fucosyltransferase 1 (P0FUT1) promoter and a pleomorphic adenoma gene-like 2 (PLAGL2) promoter.

28. The rAAV according to any one of claims 2 to 4, and 24 to 27. wherein the expression cassette of (b) comprises a bovine growth hormone (bGH) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO:

25. a GM2A coding sequence comprising nucleic acid sequence of SEQ ID NO: 19, a POFUT 1-PLAGL2 bi-directional promoter comprising nucleic acid sequence of SEQ ID NO: 31, a HexA coding sequence comprising nucleic acid sequence of SEQ ID NO: 15, a GT2A linker comprising nucleic acid sequence of SEQ ID NO: 23, a HexB coding sequence comprising nucleic acid sequence of SEQ ID NO: 17, and a rabbit beta globin (rBG) polyA signal sequence comprising nucleic acid sequence of SEQ ID NO: 24.

29. The rAAV according to any one of claims 2 to 4, and 24 to 28, w herein the expression cassette of (b) comprises nucleic acid sequence of SEQ ID NO: 1330. The rAAV according to any one of claims 1 to 29, wherein the AAV capsid is a clade F AAV capsid.

31. The rAAV according to any one of claims 1 to 30, wherein the AAV capsid is an AAVhu68 capsid, AAV9 capsid or a mutant AAV9 capsid.

32. The rAAV according to any one of claims 1 to 31, w hich is for use in treatment of GM2 gangliosidosis disease.

33. The rAAV according to any one of claims 2 to 4, and 24 to 32, w hich is for use in treatment of GM2 activator deficiency.

34. The rAAV according to any one of claims 1 to 32. which is for use in treatment of Tay-Sachs disease.

35. The rAAV according to any one of claims 1 to 32, which is for use in treatment of Sandhoff disease.

36. A composition comprising a stock of rAAV according to any of claims 1 to 35 and an aqueous suspension media.

37. The composition according to claim 36, wherein the suspension is formulated for systemic and / or central nervous system (CNS) injection.

38. The composition according to claim 36 or claim 37, wherein the suspension is formulated for an intrathecal administration, optionally wherein the intrathecal administration is an intracerebroventricular (ICV) injection, or an intracistemal magna (ICM) injection.

39. A pharmaceutical composition comprising a rAAV according to any of claims 1 to 35 in a formulation buffer.

40. The pharmaceutical composition according to claim 39, which is formulated for administration via intravenous (IV) injection.

41. The pharmaceutical composition according to claim 39, which is formulated for intrathecal administration.

42. The pharmaceutical composition according to claim 39 or claim 41, which is formulated for intrathecal administration via an intracerebroventricular (ICV) injection.

43. The pharmaceutical composition according to claim 39 or claim 41, which is formulated for intrathecal administration via an intracistemal magna (ICM) injection.

44. A recombinant nucleic acid molecule comprising a vector genome comprising an adeno-associated virus (AAV) 5' inverted terminal repeat (ITR), and expression cassette, and an AAV 3' ITR, wherein the expression cassette comprises nucleic acid sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, or 13.

45. The recombinant nucleic acid molecule according to claim 44, wherein tire vector genome comprises nucleic acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, or 14.

46. A packaging host cell in culture comprising a recombinant nucleic acid molecule according to claim 44 or claim 45.

47. The packaging host cell in culture according to claim 46, which further comprises AAV rep coding sequences operably linked to sequences which express rep protein in the packaging host cell, an AAV capsid coding sequences operably linked to sequences which express AAV capsid proteins in the packaging host cell, and helper virus functions necessary to permit packaging of tire expression cassette and AAV ITRs into the AAV capsid.

48. The packaging host cell in culture according to claim 46 or claim 47, wherein the AAV capsid is clad F AAV capsid.

49. The packaging host cell in culture according to any one of claims 46 to 48, wherein the AAV capsid is an AAVhu68 capsid.

50. An rAAV production system useful for producing the rAAV according to any of claims 1 to 35, wherein the rAAV production system comprises a cell culture comprising the packaging host cell of any of claims 46 to 48.

51. The rAAV production system according to claim 50, wherein the AAV capsid is AAVhu68.

52. A method for treating GM2 gangliosidosis disease, said method comprising administrating to the subject a suspension of a rAAV according to any of claims 1 to 32 in a formulation buffer.

53. A method for treating GM2 activator deficiency, said method comprising administrating to the subject a suspension of a rAAV according to any of claims 2 to 4, and 24 to 31 and 33 in a formulation buffer.

54. A method for treating Tay-Sachs disease, said method comprising administrating to the subject a suspension of a rAAV according to any of claims 1 to 31 and 34 in a formulation buffer.

55. A method for treating Sandhoff disease, said method comprising administrating to the subject a suspension of a rAAV according to any of claims 1 to 31 and 35 in a formulation buffer.

56. The method according to any one of claims 52 to 55, wherein the suspension is administered intrathecally.

57. The method according to any one of claims 52 to 56, wherein the suspension is administered via an Ommaya device.

58. A recombinant adeno-associated virus (rAAV) according to any one of claims 1 to 35 or a composition according to any one of claims 36 to 38 for use in preparing a medicament for treatment one or more of GM2 gangliosidosis disease including GM2 activator deficiency, Tay-Sachs disease and / or Sandhoff disease.

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