AAV Gene Therapy for Sialidosis: from Mice to IND

Project summary Siladosis is a rare, lysosomal storage disorder that causes fatal neurological disease that has no effective therapy. It is caused by loss of function mutations in the NEU1 gene. The type 1 form affects young adults with progressive vision loss and myoclonic seizures that progresses to blindness and uncontrollable movements of

arms and legs requiring physical restraint. It is fatal in middle age. The Type 2 form affects children with developmental delay, seizures, blindness, hepatosplenomegaly, and bone deformities that progresses a semi- vegetative state and death in childhood. We have developed and tested three adeno associated virus (AAV)

constructs: AAV9 encoding human NEU1 (AAV9-Neu1), AAV9 encoding human NEU1 with an IUDA signal peptide(AAV_NEU1_IUDAsp) and an AAV9 with a bidirectional promoter encoding both NEU1 and its protective protein cathepsin A (CTSA; AAV9- CTSA-bici-NEU1). These vectors were tested in wild type and sialidosis

(Neu1 -/-) mice. All three vectors appeared equivalent until 10 months when one wild type and two sialidosis mice in the AAV9-Neu1 treatment group developed seizures. Histology of the AAV9-Neu1 cohort revealed a diffuse cytoplasmic subcellular localization as compared to from the AAV9-NEU1-bici-CTSA cohort where there

was a vacuolated appearance consistent with lysosomal localization. For these reasons we selected the AAV9- NEU1-bici-CTSA for clinical translation. Our team consists of Dr. Gray-Edwards (PI) is an expert in large animal AAV gene therapy and has taken several AAV gene therapies to the clinic through testing in large animal models

of human genetic diseases. Dr. Allison Keeler (co-I) who is an expert in the immune response to AAV in humans and is extending this expertise to better understand the immune response in AAV treated sialidosis sheep. Dr. Tifft (collaborator) is the director of the orphan disease center at the NIH and is conducting a natural history study

in sialidosis patients. While this human natural history study is out of scope for this proposal Dr. Tifft will help determine if changes in sheep and biomarkers evaluated correlate with human data. The experimental design of the proposal is as follows: Aim 1: Dose escalation, efficacy and safety in sialidosis mice. Neu1-/- and wild type mice will be used to

determine the minimum effective dose and maximum tolerated dose of AAV9-NEU1-bici-CTSA for the FDA. Aim 2: AAV testing in a sheep model of sialidosis. We have developed a novel sheep model of sialidosis using CRISPR Cas9 that recapitulates the human phenotype. We will use this model to refine our treatment

strategy, delivery route, immune responses and predict efficacy for future clinical trials. Aim 3: IND enabling safety studies in wild type mice and sheep. The FDA requires the toxicology studies using two species, including a non-rodent species. We will perform toxicology studies in sheep and rats to

establish safety before clinical trials.