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

Shruti V Patil; Balasankara Reddy Kaipa; Sujata Ranshing; Yogapriya Sundaresan; J Cameron Millar; Bhavani Nagarajan; Charles Kiehlbauch; Qihong Zhang; Ankur Jain; Charles C Searby; Todd E Scheetz; Abbot F Clark; Val C Sheffield; Gulab S Zode

doi:10.21203/rs.3.rs-3740880/v1

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

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

Shruti V Patil, Balasankara Reddy Kaipa, Sujata Ranshing, Yogapriya Sundaresan, J Cameron Millar, Bhavani Nagarajan, Charles Kiehlbauch, Qihong Zhang, Ankur Jain, Charles C Searby, …

Research square

12/19/2023

DOI: 10.21203/rs.3.rs-3740880/v1

PMCID: PMC10775399

PMID: 38196579

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https://doi.org/10.21203/rs.3.rs-3740880/v1View

Preprint (Author's original)This preprint has not been evaluated by subject experts through peer review. Preprints may undergo extensive changes and/or become peer-reviewed journal articles. Open Access

Abstract

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.

Details

Title: Subtitle: Lentiviral mediated delivery of CRISPR/Cas9 reduces intraocular pressure in a mouse model of myocilin glaucoma
Creators: Shruti V Patil - University of North Texas Health Science Center
Balasankara Reddy Kaipa - University of California System
Sujata Ranshing - University of North Texas
Yogapriya Sundaresan - University of California System
J Cameron Millar
Bhavani Nagarajan - University of North Texas Health Science Center
Charles Kiehlbauch - University of North Texas
Qihong Zhang - University of Iowa
Ankur Jain - University of Iowa
Charles C Searby - University of Iowa
Todd E Scheetz - University of Iowa
Abbot F Clark - University of North Texas Health Science Center
Val C Sheffield - University of Iowa
Gulab S Zode - University of California System
Resource Type: Preprint
Publication Details: Research square
DOI: 10.21203/rs.3.rs-3740880/v1
PMID: 38196579
PMCID: PMC10775399
Language: English
Date posted: 12/19/2023
Academic Unit: Roy J. Carver Department of Biomedical Engineering; Stead Family Department of Pediatrics; Iowa Neuroscience Institute; Medical Genetics and Genomics; Ophthalmology and Visual Sciences
Record Identifier: 9984544959402771

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