GM2 Gangliosidosis Therapy Using Neurotropic Enzyme

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DESCRIPTION (provided by applicant): Neuronal storage disorders result from genetic deficiency in lysosomal enzymes, or accessory proteins, leading to substrate accumulation throughout the central nervous system (CNS), and remain incurable. Enzyme replacement (ERT), hematopoietic stem cell replacement (HSCRT) and gene replacement therapies variably rely on delivery of exogenous or secreted enzyme to lysosomes of host deficient neurons through endocytic uptake. Effectiveness of CNS therapy has been limited by the blood brain barrier, the need for global CNS delivery, and low neuronal endocytic rates. The atoxic C-fragment (Hc) of tetanus toxin is a potential solution to all these problems. Hc binds universally to neuronal membrane, is efficient endocytosed, can undergo axonal retrograde transport permitting CNS entry via peripheral terminals of motoneurons, and is retrogradely transsynaptically transferred to higher order neurons. We previously demonstrated that efficient neuronal uptake and depletion of lysosomal storage could be attained by chemically coupling beta- hexosaminidase (Hex), the deficient enzyme in GM2 gangliosidosis, to Hc. In recent studies on a 34 residue peptide (Hc1282) derived from Hc, we have found Hc1282 binds to neurons, enhances macromolecular uptake, can further enhance neuronal uptake when in multivalent form, and displays more efficient lysosomal delivery than Hc, We hypothesize that a fusion gene of the Hex beta-subunit (hexb) coding region and Hc or an Hc subsequence can yield a functional chimeric protein that will lead to an efficacious treatment for GM2 gangliosidosis. Hexb fusion genes will be constructed with Hc, the Hc C-terminal half, the Hc1282 peptide, and a multicopy version of Hc1282. Recombinant proteins will be screened for enzyme activity and enhanced lysosomal delivery using neuronal cultures from a mouse model of GM2 gangliosidosis, and for retrograde transport from the periphery using intramuscular injections. The best candidate construct(s) will then be tested on disease mice using: ERT initiated at birth; HSCRT, at 5 weeks of age, with ex vivo gene replacement where stem cell-derived microglia will secrete the high-uptake recombinant enzyme within the CNS; and potentially in a combined ERT/HSCRT regimen. The Hc-based strategy is applicable to numerous neuronal storage diseases that make up a significant portion of pediatric pathology and could be translated to human therapy. PUBLIC HEALTH RELEVANCE: This proposal seeks to develop a therapeutic strategy relevant to lysosomal storage diseases that affect 1 in 8000 children. About half of these disorders, like Tay Sachs disease, also affect the brain, frequently result in early death and remain without a cure. This investigation will explore a gene modification strategy of the enzyme deficient in Tay Sachs disease in the context of enzyme replacement therapy and in hematopoietic stem cell replacement therapy which are currently used in human patients. If successful this modification will allow entry of enzyme into brain from the blood, and promote both widespread and efficient delivery of active enzyme to lysosomes of neurons. In this manner it may make these therapeutic approaches highly effective for treating diseased brain in Tay Sachs and related diseases, and could be relatively quickly translated to human clinical trials.