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Brian Rossmiller, Takeshi Iwata; Optimized Homology Directed Repair for Treatment of Inherited Retinal Diseases Using the CRISPR/Cas9 System. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4469.
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© ARVO (1962-2015); The Authors (2016-present)
Nearly 1.5 million people, worldwide, are affected by hereditary vision impairment each year. For many of these, there is little to no effective treatment. Advancements in CRISPR/Cas mediated site specific gene editing provide a new and powerful tool for disease treatment but relies on inefficient homology directed recombination (HDR). The purpose of this project is to treat three models of inherited retinal degeneration using CRISPR/Cas9 mediated HDR. Prior to treatment, we will assess optimal conditions for use of the CRISPR/Cas9 system in the retina. These include age of injection, delivery vehicle (nanoparticle or adeno-associated virus (AAV)), and method of increasing homologous integration of gene replacement (ligase IV inhibitors and neurotrophic factors).
LPD lipid ratios were tested through transfection of 2.5 ug of plasmid containing control cytomegalovirus promoter driven firefly luciferase DNA into HEK293 cells. Luciferase activity was measured 48 hours post-transfection. Assessment of sgRNA cleavage efficiency was performed using a novel dual luciferase approach with a molar ratio of 1:4 target to H1-sgRNA expression vector. Luciferase activity was measured 48 hours post-transfection. Each vector is assessed in three animal models of inherited retinal degeneration. First, a mouse model of retinitis pigmentosa, Rho (I307N), kindly provided by Dr. Nishina of Jackson laboratory. The second model is of normal tension glaucoma, OTPN (E50K) and produced in the Iwata lab. Finally, we created a third model featuring a premature truncation of KCNJ13 (W53X) resulting in Leber’s congenital amaurosis.
Here, we have confirmed transfection, of CMV-GFP plasmid, by LPDs, into HEK293 cells by GFP expression. These results confirm that a ratio of 1:2:3:15, DNA, nuclear localization signal and trans-acting transcription peptide, protamine sulfate, and lipids, respectively, provided the best DNA transfection rates in tissue culture and will be used moving forward in mouse models. Cleavage of KCNJ13, by CRISPR/Cas9, resulted in a significant reduction in luciferase activity (70%, p < 0.05).
Our current LPD mixture and sgRNAs targeting KCNJ13 have shown to provide significant transfection and targeted knock-down in vitro.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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