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Abstract #117442 Published in IGR 24-4
Lentiviral mediated delivery of CRISPR/Cas9 reduces intraocular pressure in a mouse model of myocilin glaucoma
Patil SV; Kaipa BR; Kaipa BR; Ranshing S; Ranshing S; Sundaresan Y; Millar JC; Nagarajan B; Kiehlbauch C; Zhang Q; Jain A; Searby CC; Scheetz TE; Clark AF; Sheffield VC; Zode GSResearch square 2023; 0:
Mutations in myocilin () are the leading known genetic cause of primary open-angle glaucoma, responsible for about 4% of all cases. Mutations in cause a gain-of-function phenotype in which mutant myocilin accumulates in the endoplasmic reticulum (ER) leading to ER stress and trabecular meshwork (TM) cell death. Therefore, knocking out myocilin at the genome level is an ideal strategy to permanently cure the disease. We have previously utilized CRISPR/Cas9 genome editing successfully to target using adenovirus 5 (Ad5). However, Ad5 is not a suitable vector for clinical use. Here, we sought to determine the efficacy of adeno-associated viruses (AAVs) and lentiviruses (LVs) to target the TM. First, we examined the TM tropism of single-stranded (ss) and self-complimentary (sc) AAV serotypes as well as LV expressing GFP via intravitreal (IVT) and intracameral (IC) injections. We observed that LV_ expression was more specific to the TM injected via the IVT route. IC injections of Trp-mutant scAAV2 showed a prominent expression of GFP in the TM. However, robust GFP expression was also observed in the ciliary body and retina. We next constructed lentiviral particles expressing Cas9 and guide RNA (gRNA) targeting () and transduction of TM cells stably expressing mutant myocilin with LV_cr significantly reduced myocilin accumulation and its associated chronic ER stress. A single IVT injection of LV_cr in - mice decreased myocilin accumulation in TM and reduced elevated IOP significantly. Together, our data indicates, LV_cr targets gene editing in TM and rescues a mouse model of myocilin-associated glaucoma.
University of North Texas Health Science Center at Fort Worth.
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