(64b) Cloning Feline β-Hexosaminidase for Gene Therapy in a Feline Model of Neurologic Disease

The GM2 Gangliosidoses are a class of lysosomal storage diseases caused by a deficiency of β-hexosaminidase. This enzyme is made up of two subunits, the α subunit and the β subunit. The gene HEXA codes for the α subunit, while HEXB codes for the β subunit. A human example of GM2 Gangliosidosis is Tay-Sachs disease, caused by mutations in the HEXA gene. This disease currently has no cure, and is always fatal by about 4 to 5 years of age. Other GM2 Gangliosidoses have effectively the same symptoms and outcomes. The principal investigator has treated cats with the human genes that code for the subunits of this enzyme. The treatment did reduce storage material, but the cats showed a significant immune response. The scope of the project for the research assistant was to isolate the two feline HEXA and HEXB genes in plasmids and verify enzyme expression in mammalian cells. The two genes both had unverified expressive sequences, but homology between species provided confidence that the suspected sequences were correct. The HEXA gene was isolated from RNA. The HEXB gene was isolated by primer overlap extension of a shorter sequence that was previously thought to be expressive to obtain the longer expressive gene. Both genes were loaded into plasmids so that the sequence could be verified by sequencing and so that gene expression could be verified in mammalian cells. A collaborator loaded the genes into adeno-associated viral vectors that will be used for treatment.