Oligonucleotide Repair of a Retinal Degenerarion Model

Emory University, Atlanta GA

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Abstract

DESCRIPTION (provided by applicant): Mutations in the gene encoding the beta subunit of rod photoreceptor cGMP phosphodiesterase (beta-PDE) are associated with retinitis pigmentosa (RP), a disease family that leads to blindness in humans. The mouse rd1 (Pde6b rd1) and rd10 (Pde6b rd10) retinal degeneration phenotypes are models for the study of human RP because these phenotypes result from mutations in the gene encoding beta-PDE. Oligonucleotide-directed gene correction is a strategy in which a mismatch between a therapeutic oligonucleotide and its genomic target sequence is recognized by endogenous DNA repair mechanisms, inducing them to convert the mutation to wild type. As opposed to other gene therapies, this strategy is intended to permanently repair the defect in the chromosomal gene. The broad objective of this proposed research is to determine whether oligonucleotide-directed gene correction can be used to repair the rd1 or rd10 mutations, prevent photoreceptor degeneration, and preserve vision. In Specific Aim 1, the existence of oligonucleotide-inducible DNA repair proteins will be determined by immunohistology and immunoblotting. A cell-free assay will be used to determine whether oligonucleotides induce repair of test DNA targets in the presence of retina and photoreceptor proteins. The same protocol will be coupled with inhibitors of individual repair proteins to test for their importance in the repair reaction. In Specific Aim 2, oligonucleotides will be injected and iontophoresed into eyes of neonatal mice. Photoreceptor rescue will be assessed by immunoblotting and immunohistochemical examination of retinas for rhodopsin and beta-PDE. Since rhodopsin is the most abundantly expressed photoreceptor marker, monitoring its levels should provide an exquisitely sensitive and robust assessment of photoreceptor rescue. DNA repair will be assessed by immunohistochemical examination of retinas for (-PDE and by assaying retinal extracts for PDE6 enzymatic activity. Noninvasive measures of visual function (e.g., ERG) will be taken also. Preventing vision loss in these animal models should direct our efforts for gene therapy of inherited retinal diseases such as autosomal recessive RP in humans.