This project will develop a gene therapy strategy to treat hearing loss associated with Usher Syndrome type 3A (USH3A) in human. This will involve optimizing viral vectors in mouse models of USH3A, testing the best vectors for efficacy and cochlear toxicity in non-human primates and in human hair cells, and testing sequence variants in the human USH3A gene (CLRN1) for pathogenicity. At the completion of the project, we will be able to request a pre-IND meeting with the Food and Drug Administration to determine specific requirements for preclinical testing.
Aim 1 is to test different AAV vectors for gene delivery to cochlear hair cells in mouse. Three new AAV- related vectors have shown promise in transducing cochlear hair cells, and we will test them all to identify the best. We will then test AAV-mediated restoration of hair cell function in the Clrn1-/- and N48K mouse models. Finally, we will test toxicity to hearing of AAV-mediated Clrn1 expression.
Aim 2 is to test the efficacy of novel AAV vectors in human hair cells in vitro, and to test, in non-human primates, the efficiency, toxicity and immunogenicity of these vectors administered by round window membrane injection. We will test the two best vectors from Aim 1, expressing GFP to assess the degree of viral transduction of hair cells. We will also describe toxicity and systemic distribution of GFP- and CLRN1- encoding AAV vectors. An immune response might interfere with subsequent AAV injections or could lead to toxicity in the inner ear, so we will determine whether AAV injection into the ear induces neutralizing antibody or T-cell response.
In Aim 3 we will characterize human pre-existing immunity against applicable serotypes in perilymph, using perilymph collected during surgery for other auditory disorders. We will also determine whether there is a correlation between antibody titers in perilymph and serum. Many variants have been identified in the CLRN1 gene but not all are known to be pathogenic. To determine which variants in CLRN1 in USH3A patients are pathogenic, we will set up a complementation assay using the Clrn1-/- mouse, use AAV to express Clrn1 with specific mutations equivalent to human variants of unknown significance, and assess degree of functional rescue with electrophysiology and electron microscopy.
Gene therapy offers the promise of treatment for a variety of inherited disorders, but has not been used successfully for human hereditary deafness. This project will target an inherited deafness/blindness, Usher syndrome type 3a, developing new gene therapy vectors to treat the deafness. The project will provide data necessary for discussions with the FDA to design preclinical testing.