Utility of Commercially Available Cationic Lipid-based Delivery of CRISPR-Cas9 to the Retina in vivo

Abstract

Purpose : Given its accessibility and the number of inherited diseases caused by mutations in genes with very distinct spatial and stoichiometric expression, the eye is a particularly attractive organ for the clinical translation of Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated mutation correction in vivo. Cationic-lipid based delivery has been successfully used for CRISPR knockout in mouse inner ear (Zuris et al. Nat Biotech; 2015: 33: 73-80). Here, we investigate the efficiency and efficacy of a cationic lipid-based CRISPR-Cas9 delivery system to the retina in vivo.

Methods : A YFP mouse model was used given the ability to readily detect and monitor CRISPR activity in vivo through observed knockout of YFP fluorescence in the retina. CRISPR guide RNAs (gRNA) were designed against the YFP sequence and synthesized using T7 polymerase. gRNA against LacZ was used as a negative control. Cas9 recombinant protein was generated from His-tagged Cas9 synthesized in E. coli BL21 STAR (DE3)-competent cells and isolated through a His-Pur Ni-NTA resin. YFP gRNA and recombinant Cas9 was packaged in cationic lipids- Lipofectamine 2000, RNAiMAX, invivofectamine 2.0. Intravitreal injections were performed in anesthetised (60:5 mg/kg, ketamine:xylazine) adult Tg(Thy1-YFP) 16Jrs mice. Electroretinography and optical coherence tomography measurements were conducted in anesthetised mice 3 weeks post-injection. Eyes were dissected post-mortem and retinal whole mounts examined using fluorescent microscopy.

Results : Cas9 precipitation was observed with invivofectamine 2.0; however concentrated Cas9 (200mM) could be used with lipofectamine 2000 and RNAiMAX. Following in vitro validation, intravitreal delivery of lipofectamine 2000 and RNAiMAX packaged or nude Cas9 and sgRNA was undertaken for at least 5 mice per experimental arm. Overall poor knockout efficiencies were observed with in delivery conditions with ~32% YFP:DAPI ratios observed in all cases. Packaging using RNAiMAX was found to cause retino-uveitis

Conclusions : Our current studies suggest the use of commercial cationic lipid delivery systems do not result in marked retinal cell gene knockout out. In particular RNAiMAX is not recommended for intravitreal injections in this context. Ongoing work investigating alternative cationic lipid packages as well as different routes, such as subretinal delivery, should be explored.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.