Anticodon Engineered tRNA for the Treatment of Hereditary Eye Disease

Summary Nonsense mutations are point mutations that convert an amino acid encoding codon into a premature termination codon (PTC). The consequences of PTCs are nonsense-mediated mRNA decay, and for transcripts that persist, premature translation termination. PTCs are a major cause of human disease, accounting for >10% of all described disease-causing genetic alterations, including nearly all forms of hereditary eye disease (retinal disease, corneal disease, glaucoma, congenital cataract, retinoblastoma, syndromes impacting vision, and others). There has been a longstanding appreciation that therapies promoting readthrough of PTCs might be broadly useful for treating many different diseases which are all fundamentally initiated by PTCs. In recent years there has been a surge in PTC therapy research using modified suppressor tRNA molecules. In this approach, tRNA with altered anticodons recognizing stop codons are utilized to base- pair to the PTC and allow translation. Remarkably, suppressor tRNAs appear to recognize PTCs and natural termination codons distinctly. Recently, we have conducted screens for anticodon engineered tRNA (ACE- tRNA) with a high suppression activity. Here, we utilize a newly generated strain of transgenic mice that ubiquitously express one of these, Arg-TGA ACE-tRNA, to assess its in vivo efficacy, safety, and activity in multiple ocular cell types. To achieve this, Specific Aim 1 will assess the ability of transgenic encoded ACE- tRNA-ArgUGA to rescue the pigment dispersing iris disease caused by the GpnmbR150X PTC mutation and the retinal degeneration caused by the Rd3R107X PTC mutation. Control cohorts will also be characterized with a variety of assays to test whether there is phenotypic evidence of damage induced in ocular tissues by presence of the ACE-tRNA-ArgUGA. To complement these experiments, Specific Aim 2 uses a reporter strain of mice to characterize whether there are any ocular cell types with unique features changing their sensitivity to ACE-tRNA-ArgUGA. Broadly, the results from SA1 and SA2 will form a rigorous initial test of in vivo efficacy, safety, and cellular activity of ACE-tRNA in the eye. The combined outcomes allow many opportunities to observe the degree to which this ACE-tRNA-ArgUGA can provided sustained correction of PTC mutations in the eye, whether it induces any additional detectable abnormalities, and whether there are any yet unknown biological processes regulating protein translation in some cell-types that impact how ACE-tRNA function.