Deep Intronic NF1 Mutations and Possible Therapeutic Interventions

Abstract

A significant proportion of germline mutations causing neurofibromatosis type 1 affect the correct splicing of the NF1 gene. Splicing is a complex mechanism by which introns from a pre-mRNA are removed. This process is finely regulated at the cellular level, and it has been shown that it can be modulated by using antisense oligonucleotides (AONs). Multiple studies have demonstrated the power of AONs in modulating abnormal splicing caused by constitutive DNA mutations, first in cultured cells, later in preclinical animal models, and currently in several promising clinical trials. A subset of splicing mutations, known as deep intronic mutations, create or strengthen acceptor or donor splice sites that, using a wild-type counterpart sequence, lead to the inclusion of a cryptic exon in the mRNA that will eventually produce an aberrant protein. In NF1, deep intronic mutations account for approximately 2 % of all NF1 germline mutations identified to date. Here we review different examples in which phosphorodiamidate morpholino oligomers (PMOs, a class of AONs) have been used to efficiently correct the abnormal splicing produced by NF1 deep intronic mutations. Reestablishment of normal splicing after PMO treatment has been analyzed at the mRNA level in different patient-derived primary cell cultures. Further, indirect evidence of neurofibromin function restoration has been assessed by quantifying the levels of active Ras before and after PMO treatment. In vitro results collected so far have demonstrated the considerable potential of this type of antisense therapy for deep intronic NF1-causing mutations, although further experimental studies, particularly in preclinical animal models, will be required in order to safely translate these results into clinical trials and eventually to the clinic. The success of clinical trials using AON technology for other mis-splicing mutations causing Duchenne muscular dystrophy (DMD) is encouraging and delineates a possible path from bench to bedside for these types of mutations causing neurofibromatosis type 1.