CRISPR-Cas9 RNP complex, delivered via lipid nanoparticle, is effective in disruption of RHO gene in vitro

Abstract

Purpose : Retinitis pigmentosa (RP) is a hereditary retinal disease that presents with nyctalopia and visual field defect and leads to central vision loss in late stages. Currently, the only approved treatment for RP is the use of retinal prosthesis, which is only suitable for patients with advanced disease and profound vision loss. Gene therapy is a promising approach that could potentially modify the course of the disease. However, gene therapy for autosomal dominant RP (ADRP) is challenging as it requires elimination of toxic products produced by dominant negative effects of the mutated allele. RHO is the most common gene involved in ADRP. Our approach uses CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated system9 (Cas9)) gene editing technology to silence the human RHO gene. We used lipofectamine CRISPRMAX lipid nanoparticle to directly deliver CRISPR-Cas9 Ribonucleoprotein (RNP) complex into cells. When optimized, this approach may enable intravitreal delivery of CRISPR-Cas9 for gene editing in ADRP without the need for viral vectors or electroporation. Following disruption of the mutated gene, a normal copy of the gene can be inserted via viral vector delivery.

Methods : 24 hours after seeding, Human Embryonic Kidney 293 (HEK293) cells were transfected with CRISPR-Cas9 RNP complexed with Lipofectamine CRISPRMAX. Guide RNA was designed to target a region of exon 1, encoding for amino acids 98 through 104, with high on-target and off-target scores of 73 and 74 respectively. Genomic Cleavage Detection Kit was used to verify the gene editing efficacy 48 hours after transfection.

Results : Using PCR, with the amplicon length of 421 bp, two bands with 307 bp and 114 bp length were detected on the agarose gel. The presence of the 2 expected bands confirmed that the RHO gene was successfully disrupted at the desired region.

Conclusions : CRISPR-Cas9 RNP complex, delivered via lipofectamine CRISPRMAX lipid nanoparticle can successfully disrupt the RHO gene in vitro in HEK293 Cells. This mutation independent gene editing may allow silencing of both normal and mutated alleles of the RHO gene, paving the way for a successful treatment of RP patients with mutations in RHO gene.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.