Intrathecal delivery of a bicistronic AAV9 vector expressing ß-hexosaminidase A corrects Sandhoff disease in a murine model: A dosage study.

The pathological accumulation of GM2 ganglioside associated with Tay-Sachs disease (TSD) and Sandhoff disease (SD) occurs in individuals who possess mutant forms of the heterodimer ß-hexosaminidase A (Hex A) because of mutation of the HEXA and HEXB genes, respectively. With a lack of approved therapies, patients experience rapid neurological decline resulting in early death. A novel bicistronic vector carrying both HEXA and HEXB previously demonstrated promising results in mouse models of SD following neonatal intravenous administration, including significant reduction in GM2 accumulation, increased levels of Hex A, and a 2-fold extension of survival. The aim of the present study was to identify an optimal dose of the bicistronic vector in 6-week-old SD mice by an intrathecal route of administration along with transient immunosuppression, to inform possible clinical translation. Three doses of the bicistronic vector were tested: 2.5e11, 1.25e11, and 0.625e11 vector genomes per mouse. The highest dose provided the greatest increase in biochemical and behavioral parameters, such that treated mice lived to a median age of 56 weeks (>3 times the lifespan of the SD controls). These results have direct implications in deciding a human equivalent dose for TSD/SD and have informed the approval of a clinical trial application (NCT04798235).

Efficacy of Adeno-Associated Virus Serotype 9-Mediated Gene Therapy for AB-Variant GM2 Gangliosidosis.

GM2 gangliosidoses are a group of neurodegenerative lysosomal storage disorders that are characterized by the accumulation of GM2 gangliosides (GM2), leading to rapid neurological decline and death. The hydrolysis of GM2 requires the specific synthesis, processing, and combination of products of three genes-HEXA, HEXB, and GM2A-within the cell's lysosomes. Mutations in these genes result in Tay-Sachs disease, Sandhoff disease, or AB-variant GM2 gangliosidosis (ABGM2), respectively. ABGM2, the rarest of the three types, is characterized by a mutation in the GM2A gene, which encodes the GM2 activator (GM2A) protein. Being a monogenic disease, gene therapy is a plausible and likely effective method of treatment for ABGM2. This study aimed at assessing the effects of administering a one-time intravenous treatment of single-stranded Adeno-associated virus serotype 9 (ssAAV9)-GM2A viral vector at a dose of 1 × 1014 vector genomes (vg) per kilogram per mouse in an ABGM2 mouse model (Gm2a-/-). ssAAV9-GM2A was administered at 1-day (neonatal) or 6-weeks of age (adult-stage). The results demonstrated that, in comparison to Gm2a-/- mice that received a vehicle injection, the treated mice had reduced GM2 accumulation within the central nervous system and had long-term persistence of vector genomes in the brain and liver. This proof-of-concept study is a step forward towards the development of a clinically therapeutic approach for the treatment of patients with ABGM2.

Efficacy of a Bicistronic Vector for Correction of Sandhoff Disease in a Mouse Model.

GM2 gangliosidoses are a family of severe neurodegenerative disorders resulting from a deficiency in the ß-hexosaminidase A enzyme. These disorders include Tay-Sachs disease and Sandhoff disease, caused by mutations in the HEXA gene and HEXB gene, respectively. The HEXA and HEXB genes are required to produce the α and ß subunits of the ß-hexosaminidase A enzyme, respectively. Using a Sandhoff disease mouse model, we tested for the first time the potential of a comparatively lower dose (2.04 × 1013 vg/kg) of systemically delivered single-stranded adeno-associated virus 9 expressing both human HEXB and human HEXA cDNA under the control of a single promoter with a P2A-linked bicistronic vector design to correct the neurological phenotype. A bicistronic design allows maximal overexpression and secretion of the Hex A enzyme. Neonatal mice were injected with either this ssAAV9-HexB-P2A-HexA vector or a vehicle solution via the superficial temporal vein. An increase in survival of 56% compared with vehicle-injected controls and biochemical analysis of the brain tissue and serum revealed an increase in enzyme activity and a decrease in brain GM2 ganglioside buildup. This is a proof-of-concept study showing the "correction efficacy" of a bicistronic AAV9 vector delivered intravenously for GM2 gangliosidoses. Further studies with higher doses are warranted.

Systemic Gene Transfer of a Hexosaminidase Variant Using an scAAV9.47 Vector Corrects GM2 Gangliosidosis in Sandhoff Mice.

GM2 gangliosidosis is a group of neurodegenerative diseases caused by ß-hexosaminidase A (HexA) enzyme deficiency. There is currently no cure. HexA is composed of two similar, nonidentical subunits, α and ß, which must interact with the GM2 activator protein (GM2AP), a substrate-specific cofactor, to hydrolyze GM2 ganglioside. Mutations in either subunit or the activator can result in the accumulation of GM2 ganglioside within neurons throughout the central nervous system. The resulting neuronal cell death induces the primary symptoms of the disease: motor impairment, seizures, and sensory impairments. This study assesses the long-term effects of gene transfer in a Sandhoff (ß-subunit knockout) mouse model. The study utilized a modified human ß-hexosaminidase α-subunit (µ-subunit) that contains critical sequences from the ß-subunit that enables formation of a stable homodimer (HexM) and interaction with GM2AP to hydrolyze GM2 ganglioside. We investigated a self-complementary adeno-associated viral (scAAV) vector expressing HexM, through intravenous injections of the neonatal mice. We monitored one cohort for 8 weeks and another cohort long-term for survival benefit, behavioral, biochemical, and molecular analyses. Untreated Sandhoff disease (SD) control mice reached a humane endpoint at approximately 15 weeks, whereas treated mice had a median survival age of 40 weeks, an approximate 2.5-fold survival advantage. On behavioral tests, the treated mice outperformed their knockout age-matched controls and perform similarly to the heterozygous controls. Through the enzymatic and GM2 ganglioside analyses, we observed a significant decrease in the GM2 ganglioside level, even though the enzyme levels were not significantly increased. Molecular analyses revealed a global distribution of the vector between brain and spinal cord regions. In conclusion, the neonatal delivery of a novel viral vector expressing the human HexM enzyme is effective in ameliorating the SD mouse phenotype for long-term. Our data could have implications not only for treatment of SD but also for Tay-Sachs disease (α-subunit deficiency) and similar brain disorders.