Restoration of Normal NF1 Function with Antisense Morpholino Treatment of Recurrent Pathogenic Patient-Specific Variant c.1466A>G; p.Y489C.

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disorder with almost 3000 different disease-causing variants within the NF1 gene identified. Up to 44% of these variants cause splicing errors to occur within pre-mRNA. A recurrent variant in exon 13, c.1466A>G; p.Y489C (Y489C) results in the creation of an intragenic cryptic splice site, aberrant splicing, a 62 base pair deletion from the mRNA, and subsequent frameshift. We investigated the ability of phosphorodiamidate morpholino oligomers (PMOs) to mask this variant on the RNA level, thus restoring normal splicing. To model this variant, we have developed a human iPS cell line homozygous for the variant using CRISPR/Cas9. PMOs were designed to be 25 base pairs long, and to cover the mutation site so it could not be read by splicing machinery. Results from our in vitro testing showed restoration of normal splicing in the RNA and restoration of full length neurofibromin protein. In addition, we observe the restoration of neurofibromin functionality through GTP-Ras and pERK/ERK testing. The results from this study demonstrate the ability of a PMO to correct splicing errors in NF1 variants at the RNA level, which could open the door for splicing corrections for other variants in this and a variety of diseases.