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

项目概要/摘要 I型神经纤维瘤病（NF 1）是一种由常染色体显性突变引起的癌症易感综合征 在基因中，NF 1，影响全世界2,500人中的1人。NF 1编码神经纤维蛋白，一种GTP酶激活蛋白 一种负调节Ras信号通路的蛋白质，例如NF 1的缺失导致Ras过度活化 导致不受控制的细胞生长和增殖。虽然NF 1的特征是广泛的症状， 最令人关注的是丛状神经纤维瘤，其影响30-50%的患者。此外，8-13%的患者 也会发展成恶性外周神经鞘瘤（MPNSTs），这是发病的主要原因， NF 1患者，只有20-50%的患者在诊断后存活5年。虽然治疗策略 靶向Ras信号可以通过缩小丛状神经纤维瘤，基因， 编辑是唯一可以通过纠正NF 1中的突变来解决NF 1的根本原因的策略。然而，在这方面， 许多现有的基因编辑和基因治疗策略不能适用于NF 1的治疗。 此外，实现临床改善所需的神经纤维蛋白恢复量尚不清楚; 这阻碍了NF 1最佳治疗策略的发展。F99阶段的目标 该奖学金的目的是开发一种基于CRISPR的系统，以纠正约90%的NF 1中的致病性突变。 通过将4.5kb的superexon序列插入突变的NF 1基因， NF 1序列。在HEK 293 T细胞中的实验已经显示了成功的整合和转录， superexon以及正确剪接到内源性外显子。据预测， 神经纤维蛋白，将Ras信号传导降低至基础水平，并减小小鼠中丛状神经纤维瘤的大小。 NF 1的型号。该研究金的K 00阶段的目的是询问所需的神经纤维蛋白的量 在两种不同的生物学背景下恢复功能：丛状神经纤维瘤和MPNST。新型强力霉素- 将产生诱导型细胞和小鼠模型，以检验以下假设： 恢复肿瘤抑制，但该量将根据肿瘤的遗传和生物学背景而变化。 肿瘤这项工作将直接解决对丛状神经纤维瘤（F99）新疗法的需求 阶段），并将通过询问所需的神经纤维蛋白的量来填补关键的知识空白， 在丛状神经纤维瘤和MPNST（K 00期）的背景下恢复功能。在一起，这 这些知识不仅为开发NF 1和相关疾病的新疗法提供了基础， 癌症，以及其他癌症易感综合征。培训计划还将提供 在基因工程和癌症生物学领域的领导者的特殊培训，定位 申请人成为一个成功的独立研究人员在这两个领域的接口。

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.