Abstract
Purpose :
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 two 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).
Methods :
An array of nanoparticles were screened first utilizing transfection of 2.5 mg of plasmid containing control cytomegalovirus promoter driven GFP in 661W cells and secondly in B6 mice. Mice were injected with the same plasmid at either postnatal day 5 or 15. 2 weeks post injection, retinas were disassociated and measured via FACS. 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 will be assessed in two 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.
Results :
Here, we have confirmed optimal transfection, of CMV-GFP plasmid, by two commercially available nanoparticles, into 661W cells, by GFP expression. These results were confirmed in B6 mice and currently show p5 injections to be optimal for DNA delivery. The sgRNA cleavage showed H1 driven sgRNA to result in the highest knockdown, 77% reduction in OPTN. Whereas RNA mediated knockdown resulted in a 50% and 63% knockdown of OPTN and RHO respectively. These results will be confirmed in vivo.
Conclusions :
Our current nanoparticles have shown significant transfection of 661W cells. This is coupled with a significant reduction in target gene expression by both DNA and RNA derived sgRNA in both RHO and OPTN models in vitro.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.