Browsing by Subject "gene therapy"
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Item GENE DELIVERY OF GRP78/BIP PROMOTES RETINAL GANGLION CELL SURVIVAL FOLLOWING OPTIC NERVE CRUSH(2014-03) Liu, Yang; Sharma, Tasneem; Wordinger, Robert J.; Gorbatyuk, Marina S.; Clark, Abbot F.We delivered GRP78/BiP gene into the mouse eye and promoted retinal ganglion cell survival after optic nerve injury. This study suggests a potential therapeutic target for central nervous system neurodegenerative diseases such as glaucoma and optic nerve trauma. Purpose (a): Optic nerve injury triggers endoplasmic reticulum (ER) stress and activates the unfolded protein response (UPR), leading to retinal ganglion cell (RGC) degeneration. Glucose-regulated protein (GRP78/BiP) is a sensor of ER homeostasis and plays a role in ER stress alleviation. In this study, we evaluated the involvement of GRP78/BiP in RGC degeneration induced by optic nerve crush (ONC) and the neuroprotective effects of gene delivery of GRP78/BiP. Methods (b): ONC was performed unilaterally in adult BALB/cJ mice. The expression of GRP78/BiP was evaluated by real time PCR and fluorescent in situ hybridization (FISH). To evaluate the potential neuroprotective effect of BiP , an AAV2 vector harboring the human BiP gene (AAV2-hBiP) or green fluorescent protein (AAV2-GFP) (2 x 109 P) was intravitreally injected 4 weeks prior to the ONC. Seven and fourteen days after the ONC, RGC survival was determined by RBPMS immunofluorescence staining of retinal flat mounts. Retinal function was assessed using full field flash ERG. Expression of UPR related proteins was evaluated by western blotting. Results (c): Three days after ONC, GRP78/BiP expression was significantly up-regulated in RGCs (p<0.01). Intravitreal administration of AAV2-hBiP significantly reduced RGC loss at 7 and 14 days post-ONC compared to AAV2-GFP injected group (n=5, p<0.01). ERG analysis showed partial protection of pSTR amplitudes in AAV2-hBiP injected eyes (n=5, p<0.05). Retina levels of cleaved ATF6 in AAV2-hBiP injected eyes were much lower than those of AAV2-GFP injected eyes. Conclusions (d): Gene delivery of GRP78/BiP promotes RGC survival and preserves RGC function following optic nerve injury. This study suggests a potential therapeutic target for central nervous system neurodegenerative diseases.Item Identifying Potential Biomarkers and Clinical Trial Outcomes for Future Clinical Trials for SLC13A5 Deficiency(2022-12) Cooper, Sydney; Simecka, Jerry W.; Cihlar, Jennifer C.Item Lentiviral mediated delivery of CRISPR/Cas9 reduces intraocular pressure in a mouse model of myocilin glaucoma(Springer Nature Limited, 2024-03-24) Patil, Shruti V.; Kaipa, Balasankara R.; Ranshing, Sujata; Sundaresan, Yogapriya; Millar, J. Cameron; Nagarajan, Bhavani; Kiehlbauch, Charles; Zhang, Qihong; Jain, Ankur; Searby, Charles C.; Scheetz, Todd E.; Clark, Abbot F.; Sheffield, Val C.; Zode, Gulab S.Mutations 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.