Gene Therapy for the Treatment of Neurofibromatosis Type I (NF1)

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PROJECT SUMMARY/ABSTRACT Neurofibromatosis Type I (NF1) is a cancer predisposition syndrome caused by an autosomal dominant mutation in the gene, NF1, that effects 1 in 2,500 people worldwide. NF1 encodes for neurofibromin, a GTPase-activating protein that negatively regulates the Ras signaling pathway, such that loss of NF1 results in Ras hyperactivation leading to uncontrolled cell growth and proliferation. While NF1 is characterized by a wide host of symptoms, the most concerning are plexiform neurofibromas, which affects 30-50% of patients. Furthermore, 8-13% of patients will also develop malignant peripheral nerve sheath tumors (MPNSTs), which is the main cause of morbidity in NF1 patients, with only 20-50% of patients surviving 5 years post-diagnosis. While therapeutic strategies targeting Ras signaling can provide some benefit for NF1 patients by shrinking plexiform neurofibromas, gene editing is the only strategy that can address the root cause of NF1 by correcting mutations in NF1. However, many of the existing gene editing and gene therapy strategies cannot be adapted to the treatment of NF1. Additionally, the amount of neurofibromin restoration required to achieve clinical improvement is unknown; a gap in knowledge that hinders the development of optimal therapeutic strategies for NF1. The aim of the F99 phase of this fellowship is to develop a CRISPR-based system to correct pathogenic mutations in ~90% of the NF1 patient population by inserting a 4.5 kb superexon sequence into the mutated NF1 gene to restore the correct NF1 sequence. Experiments in HEK293T cells have shown successful integration and transcription of the superexon as well as correct splicing to the endogenous exon. This is predicted to create functional neurofibromin, reduce Ras signaling to basal levels, and reduce the size of plexiform neurofibromas in a mouse model of NF1. The aim of the K00 phase of this fellowship is to interrogate the amount of neurofibromin required to restore function in two different biological contexts: plexiform neurofibromas and MPNSTs. Novel doxycycline- inducible cell and mouse models will be generated to test the hypothesis that there is a threshold required to restore tumor suppression, but that this amount will vary depending on the genetic and biological context of the tumor. This work will directly address the need for novel therapeutics for plexiform neurofibromas (F99 phase) and will fill a critical knowledge gap by interrogating the amount of neurofibromin needed to restore function in the context of plexiform neurofibromas and MPNSTs (K00 phase). Together, this knowledge will not only provide a foundation for the development of novel therapeutics for NF1 and related cancers, but also other cancer pre-disposition syndromes. The proposed training plan will also provide exceptional training by leaders in the genetic engineering and cancer biology fields, positioning the applicant to become a successful independent researcher at the interface of these two fields.