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Principal Investigator: JOHN M. SULLIVAN
Organization: STATE UNIVERSITY OF NEW YORK AT BUFFALO
Fiscal Year: 2024
Award: $540,069
Funding agency: National Eye Institute
Many mutations in the human rod opsin (hRHO) and peripherin genes cause autosomal dominant retinitis
pigmentosa (adRP) and macular degenerations. The folded mRNAs are targets for mutation-independent
hammerhead ribozyme (hhRz) gene therapy. Our long-range goal is to translate effective hhRz therapeutics
for hRHO adRP and other genes into human clinical trials. We have discovered a potent form of hhRz, which
we call Enhanced-hhRz (EhhRz) therapeutics. All or most known mutations in a given dominant disease gene
could be treated with a mutation-independent EhhRz strategy that pairs knockdown (KD) of mutant (and WT)
mRNA with reconstitution (RECON) of WT protein with expression of a cleavage-resistant mRNA. The current
stage of development of EhhRzs (against hRHO) lead to kinetic turnover rates greater than 2-log orders
improved over historical minimal hhRzs (mhhRz); one mhhRz partially rescued mutant RHO retinal
degeneration in rodent models. EhhRzs function under substrate (target) excess conditions and physiological
levels of cofactor Mg2+, which are optimum for intracellular KD therapeutics. If this exciting kinetic potential can
be harnessed for the photoreceptor, retinal, or ocular cells then there is potential for nucleic acid drugs
(injectable EhhRzs) as therapeutics without vector-based gene therapy, for diverse ocular diseases and
inherited retinal degenerations. Recent clinical success with intravitreal injection of antisense agents
incentivizes this development. The objective is to optimize EhhRzs for intracellular performance in cultured
human cells and mouse photoreceptor cells that are “humanized” for RHO mRNAs (same targets as in clinical
trials). The central hypothesis is that optimization of EhhRz kinetic performance and delivery will maximize
target mRNA/protein KD; optimization requires quantitation of biophysical variables that underlie target: EhhRz
interaction, rational and/or evolutionary engineering of RNA structure-related function, and identification of the
optimum delivery materials and approach. Our rationale is that EhhRzs can be optimized through synthetic
chemistry and/or natural modifications, while retaining or enhancing kinetic function, to support robust
intracellular KD. Treatment occurs through vector-mediated delivery (AAV) of expression constructs, or direct
delivery of synthetic EhhRz therapeutics (injectables). To test the central hypothesis the Specific Aims are:
Aim 1. Optimize current EhhRzs at lead hRHO target sites (266 CUC, 725 GUC, 1362 GUC) for rate
enhancement, cellular stability, target-colocalization, and knockdown performance. Aim 2. Optimize
EhhRz Knockdown strategy for: (i) rescue (efficacy) of retinal degeneration and (ii) lack of toxicity with
AAV-EhhRz gene therapy and injectable synthetic EhhRzs in transgenic mouse models humanized for
RHO. Innovation occurs through optimization of EhhRz design, and optimization of strategies for therapeutic
delivery in a humanized adRP model. Significance- The study optimizes EhhRz function and delivery as
an initial step toward a KD/RECON rescue strategy for dominant photoreceptor degenerations.
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