Gene Editing and Hereditary Optic Neuropathies: CRISPR/Cas-Based Rescue of Missplicing Induced by OPA1 Mutation

Abstract

Hereditary optic neuropathies are characterized by progressive and bilateral loss of vision due to the degeneration of retinal ganglion cells whose axons form the optic nerve. With a prevalence of 1:10000 to 1:40000, dominant optic atrophy (DOA) is one of the most common inherited optic neuropathies. The majority of DOA patients experience isolated DOA restricted to the eye, but 20% of patients suffer from syndromic forms mainly including neurological dysfunctions. These conditions are also known as DOA plus or Behr syndrome. More than 60% of DOA cases are caused by mutations in the OPA1 gene, which encodes a GTPase crucial for mitochondrial function. A severe form of Behr syndrome has been observed in patients carrying an OPA1 deep intronic mutation (DIM) in trans with a missense variant that acts as an intralocus modifier. The DIM creates a cryptic acceptor splice site producing aberrant OPA1 transcripts, which are degraded by a cellular control mechanism, resulting in decreased expression of the OPA1 protein. The aim of my PhD project was to rescue the DIM-induced missplicing in patient-derived induced pluripotent stem cells (iPSCs) using a CRISPR/Cas-based approach. Genome editing using the endonuclease Cas12a, also known as Cpf1 reached splicing correction up to 80%. Interestingly, splice correction occurred despite retention of the DIM and the cryptic acceptor splice site assuming that the splice correction is associated with elimination or even introduction of a splicing regulatory element caused by the Cpf1-editings. Further characterization of Cpf1-edited iPSC clones also revealed a statistically significant increase of OPA1 protein expression compared to non-edited patient cells. The results of my PhD project demonstrate successful CRISPR/Cpf1-based rescue of missplicing caused by an OPA1 DIM in patient-derived iPSCs.