Lentiviral mediated delivery of CRISPR/Cas9 reduces intraocular pressure in a mouse model of myocilin glaucoma

dc.creatorPatil, Shruti V.
dc.creatorKaipa, Balasankara R.
dc.creatorRanshing, Sujata
dc.creatorSundaresan, Yogapriya
dc.creatorMillar, J. Cameron
dc.creatorNagarajan, Bhavani
dc.creatorKiehlbauch, Charles
dc.creatorZhang, Qihong
dc.creatorJain, Ankur
dc.creatorSearby, Charles C.
dc.creatorScheetz, Todd E.
dc.creatorClark, Abbot F.
dc.creatorSheffield, Val C.
dc.creatorZode, Gulab S.
dc.creator.orcid0000-0003-3594-6560 (Clark, Abbot F.)
dc.description.abstractMutations in myocilin (MYOC) are the leading known genetic cause of primary open-angle glaucoma, responsible for about 4% of all cases. Mutations in MYOC 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 MYOC 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_GFP 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 MYOC (crMYOC) and transduction of TM cells stably expressing mutant myocilin with LV_crMYOC significantly reduced myocilin accumulation and its associated chronic ER stress. A single IVT injection of LV_crMYOC in Tg-MYOC(Y437H) mice decreased myocilin accumulation in TM and reduced elevated IOP significantly. Together, our data indicates, LV_crMYOC targets MYOC gene editing in TM and rescues a mouse model of myocilin-associated glaucoma.
dc.description.sponsorshipThese studies were supported by the National Institutes of Health (EY026177 and EY030366) and facilitated by an NIH/NEI Center Support Grant to the University of Iowa (P30 EY025580). The authors acknowledge support from NIH grant P30EY034070 and from an unrestricted grant from Research to Prevent Blindness to the Gavin Herbert Eye Institute at the University of California, Irvine.
dc.identifier.citationPatil, S. V., Kaipa, B. R., Ranshing, S., Sundaresan, Y., Millar, J. C., Nagarajan, B., Kiehlbauch, C., Zhang, Q., Jain, A., Searby, C. C., Scheetz, T. E., Clark, A. F., Sheffield, V. C., & Zode, G. S. (2024). Lentiviral mediated delivery of CRISPR/Cas9 reduces intraocular pressure in a mouse model of myocilin glaucoma. Scientific reports, 14(1), 6958. https://doi.org/10.1038/s41598-024-57286-6
dc.publisherSpringer Nature Limited
dc.rights.holder© The Author(s) 2024
dc.rights.licenseAttribution 4.0 International
dc.sourceScientific Reports
dc.subjectER stress
dc.subjectgene therapy
dc.subjectgenome editing for glaucoma
dc.subjectintraocular pressure
dc.subjectlentiviral particles
dc.subjectmyocilin-associated glaucoma
dc.subjecttrabecular meshwork
dc.subjectviral vectors
dc.subject.meshGlaucoma, Open-Angle / genetics
dc.subject.meshGlaucoma, Open-Angle / therapy
dc.subject.meshGlaucoma, Open-Angle / metabolism
dc.subject.meshGlaucoma / metabolism
dc.subject.meshCytoskeletal Proteins
dc.titleLentiviral mediated delivery of CRISPR/Cas9 reduces intraocular pressure in a mouse model of myocilin glaucoma


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