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.
Optic Neuropathies