Identification of a Human Clinical Candidate ASO Selectively Targeting the P23H Variant of Rhodopsin for the Treatment of P23H Rhodopsin-mediated ADRP

Abstract

Purpose: To identify an allele-specific antisense oligonucleotide (ASO) targeting the P23H variant of human rhodopsin mRNA (hRHO), the most common cause of ADRP in American patients. P23H RHO ASO will be selective for the P23H allele, sparing normal (WT) mRNA expression, resulting in a reduction of the mutant protein and allowing WT protein expression and processing, and maintenance of rod survival and function.

Methods: Cell lines expressing human WT and P23H RHO sequences were used to identify potent and selective P23H RHO ASOs. Leads were evaluated in vivo using transgenic (Tg) mice expressing one allele of the P23H hRHO gene or Tg mice expressing one allele of the human WT RHO gene after administration of either a human P23H specific ASO or a control non-rhodopsin ASO by intravitreal injection (IVT). To assess therapeutic potential of the ASO, the lead P23H RHO ASO was evaluated for activity and selectivity in cynomolgus monkeys.

Results: ISIS 664844 had an in vitro IC₅₀ of 2uM in P23H hRHO expressing cells and >40uM in cells expressing WT hRHO. The ASO targeting the human P23H RHO sequence achieved a 42±5% reduction in mutant RHO mRNA expression 7d following a 50 µg IVT injection in the P23H RHO Tg mice. There was no reduction in the control non-rhodopsin ASO treated eyes (0±3%). ISIS 664844, did not significantly reduce human WT RHO expression. Evaluation of 664844 in monkeys demonstrated no significant reduction in monkey WT RHO expression at 150 µg (3 ± 3%; p>0.05) 10 wk after a single IVT injection as compared to PBS-injected eyes. As a positive control, a monkey active ASO targeted against the WT RHO sequence achieved a 60±7% reduction after a single IVT injection at 400 µg. No structural or functional changes were observed in the eyes treated with 664844, as determined by ophthalmological exam, histological exam or ERG analysis.

Conclusions: We have identified and characterized a human RHO P23H allele-specific ASO, ISIS 664844, which targets the P23H RHO allele without significantly affecting the expression of the WT RHO allele. The P23H mutation causes a nonfunctional protein with a toxic gain of function and a dominant negative effect on normal protein. Therefore, reduction of the mutant protein, while sparing the RHO WT protein, should diminish the rate of degeneration associated with adRP and preserve vision for a longer period of time.