Restoring Vision with High-Fidelity Nonsense Codon Correction

PROJECT SUMMARY/ABSTRACT Nonsense mutations cause approximately 15% of genetically inherited retinopathies and inherited human diseases in general, accounting for 2.5 to 3 million patients in the U.S. For certain specific genes, nonsense mutation incidences can be as high as 40%. Because nonsense mutations cause premature termination (PTC)

of protein translation, the disease phenotype is often severe. Currently, there are only a limited number of therapies for nonsense mutations being tested in human clinical trials, including gene therapy, small molecule read-through drugs, or genome editing. Associated challenges equal the promises of each of these therapeutic

options. Looking forward, newer technologies may address these hurdles and provide more safe and efficacious treatments for patients. During protein translation, tRNA functions at the ribosomal site to incorporate a specific amino acid into the polypeptide sequence. We aim to develop the next generation of nucleic acid therapy based

on anticodon encoding transfer RNA (ace-tRNA) that incorporates the correct wild type amino acid at the site of a disease-causing nonsense mutation. Because of the many anatomical advantages afforded by the eye, we seek to test the broad applicability of ace-tRNA therapeutics for nonsense mutations that cause retinopathies

and related blindness due to defects in a variety of genes, including those encoding ion channel proteins. Specifically we will focus on nonsense mutation in ion channels expressed in photoreceptors (PR) which convert retinal light inputs and retinal pigment epithelium (RPE), which provide support for PR. These two cell types are

primarily the site of blindness pathogenesis. In this project, we will: 1) Develop ace-tRNA therapeutics that target specific nonsense mutations across several PR and RPE ion channels. 2) Engineer both viral and non-viral ace-tRNA delivery systems for long-term editing. Using these we will determine the functional outcome of ace-tRNA treatment using cultured cells and human iPSC-derived

RPE and iPSC-PR retinal organoids. 3) Test both our viral and non-viral ace-tRNA in vivo using mice harboring genetic defects that cause blindness in humans; and 4) Assess the safety and bioavailability of ace-tRNA therapeutics in our preclinical NHP model systems. There are no FDA-approved therapeutic drugs that target channelopathies because of the complexities

associated with precise post-translational modifications, carefully regulated expression, and assembly. Our team’s combined expertise in ace-tRNA development, nanomaterial synthesis, human pluripotent stem cell biology, ion-channel physiology, and pathophysiological model systems is unique and ideally suited to advance

ace-tRNA technology toward clinical trials for a wide range of genetic diseases that cause blindness.