Exon Skipping as a Therapeutic for Neurofibromatosis Type I

Abstract We investigated the feasibility of utilizing an exon skipping approach as a genotype-dependent therapeutic for neurofibromatosis type 1 (NF1) by determining which NF1 exons might be skipped while maintaining neurofibromin function. Human neurofibromin is well-known as a GTPase activating protein (GAP), but outside of its GAP-related domain (GRD), it is unclear how critical other regions are for function. Initial in silico analysis predicted exons that can be skipped with minimal loss of neurofibromin function. Utilizing a novel Nf1 cDNA system, we performed a functional screen to determine the effects of exon skipping on in vitro neurofibromin expression and GRD function. Loss of single exons 12, 17, 25, 41, 47, or 52 maintained significant GRD function in at least two Ras activity assays. Exons 18/19, 20 and 28 are critical for GRD function; deletion of exons 20, 41, or 47 led to significantly lower levels of neurofibromin. As suggested by in silico analysis, skipping of exons 17 or 52 resulted in both the highest neurofibromin levels and the greatest suppression of Ras activity. Assessment of NF1 patient databases indicates that pathogenic variants resulting in deletion or skipping of exons 17, 25, and 52 have not been reported; and truncating pathogenic variants in each exon account for ~0.91, 0.94, and 0.25% of unrelated NF1 cases, respectively. Hence, we designed antisense phosphodiamitate morpholino oligos (PMOs) to skip exon 17 and evaluated them in human cell lines that we generated via CRISPR/Cas9 with a patient-specific truncating pathogenic variant, c.1885G>A. We down-selected oligos that efficiently caused skipping of exon 17 and restored NF1 expression and function. Further, homozygous deletion of exon 17 in a novel mouse model is compatible with viable and grossly healthy animals with normal lifespan and no tumor development, providing proof-of-concept that exon 17 is not essential for murine neurofibromin function. Mild phenotypes observed include abnormal nesting behavior and lymphoid hyperplasia with increased numbers of both B- and T-cells. Hence, exon skipping should be further investigated as a therapeutic approach for NF1 patients with treatment of individuals with pathogenic variants in exon 17.