Systemic Gene Transfer of a Hexosaminidase Variant Using an scAAV9.47 Vector Corrects G_M2Gangliosidosis in Sandhoff Mice

Abstract

GM2 gangliosidosis is a group of neurodegenerative diseases caused by β-hexosaminidase A (HexA) enzyme deficiency. There is currently no cure. HexA is composed of two similar, nonidentical subunits, α and β, 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 (β-subunit knockout) mouse model. The study utilized a modified human β-hexosaminidase α-subunit (μ-subunit) that contains critical sequences from the β-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.

Novel Vector Design and Hexosaminidase Variant Enabling Self-Complementary Adeno-Associated Virus for the Treatment of Tay-Sachs Disease

“…This paper provides information on the development of the reagents, along with evidence supporting the hypothesis that this has a likelihood of providing a therapeutic benefit. A companion article by Osmon and colleagues, 52 in this issue of Human Gene Therapy (see p. 497), describes the application of this approach and vector design with a mouse model of SD, which does display a severe disease phenotype, and is more appropriate for evaluating treatment efficacy.…”

GM2 Gangliosidoses: Clinical Features, Pathophysiological Aspects, and Current Therapies

“…Several papers have described a variety of studies of gene therapy applied to the brain of small and larger animal models of GM2 gangliosidoses, mostly using AAV vectors [ 4 ]. Early gene therapy studies for GM2 gangliosidoses were based on the intracranial co-administration of AAV1 or AAVrh8 vectors carrying the α and β subunits on SD mice [ 153 , 154 ] or cats [ 82 , 155 , 156 , 157 ]. These results showed a wide distribution of the enzyme through the brain, a significant increase in the life spam, the reduction of GM2 ganglioside levels, and an improvement on motor functions [ 82 , 155 , 156 , 157 , 158 , 159 ].…”

“…Moreover, treated SD cats showed an increase on the myelin-enriched cerebrosides and a decrease in the microglial activation, suggesting an attenuation of the neuroinflammation [ 79 , 160 ], which is an important physiopathology feature observed in GM2 patients [ 73 , 80 , 84 ]. As an alternative to the multi-vector injections, a self-complementary AAV9.47 vector was evaluated, which encoded for a hybrid µ subunit (HexM) that combine the α-subunit active site, the stable β-subunit interface, and unique areas of each subunit necessary for the interaction with the GM2AP [ 40 , 154 ]. The results showed that HexM was able reduce the GM2 ganglioside storage in thalamus, hypothalamus, and hippocampus; and led to an improvement on animal behavior [ 154 ].…”

“…As an alternative to the multi-vector injections, a self-complementary AAV9.47 vector was evaluated, which encoded for a hybrid µ subunit (HexM) that combine the α-subunit active site, the stable β-subunit interface, and unique areas of each subunit necessary for the interaction with the GM2AP [ 40 , 154 ]. The results showed that HexM was able reduce the GM2 ganglioside storage in thalamus, hypothalamus, and hippocampus; and led to an improvement on animal behavior [ 154 ].…”

Extracellular uridine diphosphate-mediated microglial inflammation in a mouse model of Sandhoff disease

“…In SD patients, an excessive accumulation of undegraded substrates, including GM2 ganglioside, is observed, particularly within lysosomes in the neuronal cells, due to the deficiencies of HexA and HexB, which leads to neurological symptoms in the central nervous system (CNS), such as mental retardation, spasms and quadriplegia. SD model mice (SD mice), established by means of Hex β-subunit gene disruption, exhibit the accumulation of GM2 gangliosides throughout the CNS and the abnormalities in motor functions, which are quite similar to those observed in SD patients Several therapeutic approaches for SD have been investigated for decades, including substrate reduction therapy [4][5][6] , bone marrow transplantation 7,8 , stem cell therapy 9,10 , enzyme replacement therapy [10][11][12][13][14] , gene therapy [15][16][17] and chaperon therapy 18 , where the aim is to reduce the accumulated substrates. However, the disease remains incurable.…”