CRISPR-based Genome Editing for treatment of CEP290-IVS26-associated Leber Congenital Amaurosis (LCA)

Abstract

Purpose : Completely blind patients with CEP290-associated LCA often have a small zone of seemingly viable cone photoreceptor cells that are visible with optical coherence tomography (OCT). Some LCA patients with similar OCT anatomy have very useful central vision raising the possibility that just a small amount of improved CEP290 function could have a large therapeutic benefit in blind CEP290 patients. Unfortunately, overexpression of CEP290 using a conventional gene augmentation approach is toxic to photoreceptors. The advent of CRISPR-based genome editing raises the possibility of correcting the most common human mutation in CEP290 (an IVS26 cryptic splice site) while leaving the gene under the control of its natural promoter.

Methods : CRISPR guides specific for the IVS26 mutation were cloned into an AAV transgene cassette containing a U6 driven small guide and tracrRNA sequence upstream of a CBA driven human codon-optimized SA-Cas9. These constructs were validated in HEK cells. Targeting efficiency was evaluated via a T7E1 assay and quantified using TA-cloning and Sanger sequencing. Patient-specific iPSCs and photoreceptor precursor cells were generated using clinical grade differentiation protocols.

Results : CEP290-specific CRISPR constructs were found to efficiently delete the IVS26 mutation in human cells. These constructs were successfully packaged into AAV5 under cGMP conditions. Primary dermal fibroblasts were isolated from ten LCA patients, each of whom harbored at least one IVS26 allele. Human 3D eye cups were successfully generated from a subset of these patient-derived cell lines. In contrast to photoreceptor precursors derived from normal individuals, cells generated from IVS26 CEP290 patients were found to rapidly degenerate as they progressed from the early retinal progenitor cell stage to the more mature photoreceptor precursor cell state.

Conclusions : AAV-mediated CRISPR-based genome editing is capable of destroying the cryptic splice site in CEP290 IVS26 in vitro, suggesting that this may be a clinically meaningful way to restore CEP290 function without the risk of overexpression toxicity. The phenotype of CEP290-patient-derived retinal precursor cells recapitulates the human disease, and as such, these cells will be ideal for the preclinical testing of CRISPR-based therapeutics.

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