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

项目摘要/摘要 I型神经纤维瘤病(NF1)是一种由常染色体显性突变引起的癌症易感综合征 在NF1基因中，全球每2500人中就有一人受到影响。NF1编码神经纤维蛋白，一种GTP酶激活蛋白 负性调节RAS信号通路的蛋白质，使NF1的缺失导致RAS的过度激活 导致细胞生长和增殖失控。虽然NF1的特点是有一系列症状，但 最令人担忧的是丛状神经纤维瘤，影响30%-50%的患者。此外，8%-13%的患者 还会发展成恶性周围神经鞘瘤(MPNSTs)，这是在 NF1患者，只有20%-50%的患者在诊断后5年内存活。虽然治疗策略 靶向RAS信号可通过收缩丛状神经纤维瘤基因为NF1患者提供一些好处 编辑是唯一可以通过纠正NF1突变来解决NF1根本原因的策略。然而， 许多现有的基因编辑和基因治疗策略不能适应NF1的治疗。 此外，达到临床改善所需的神经纤维蛋白修复的量是未知的；缺口 这些知识阻碍了NF1最佳治疗策略的发展。F99阶段的目标是 该奖学金的目的是开发一种基于CRISPR的系统来纠正~90%的NF1的致病突变 通过在突变的NF1基因中插入4.5kb的超级外显子序列来恢复正确的 NF1序列。在HEK293T细胞中的实验表明成功地整合和转录了 超外显子以及与内源外显子的正确剪接。预计这将创建功能 神经纤维素，将RAS信号降低到基础水平，并缩小小鼠丛状神经纤维瘤的大小 NF1的模型。这项研究的K00阶段的目的是询问所需的神经纤维素量。 在两种不同的生物学环境下恢复功能：丛状神经纤维瘤和MPNSTs。新型强力霉素-- 将生成可诱导的细胞和小鼠模型，以测试需要阈值的假设 恢复肿瘤抑制，但这一数量将根据基因和生物学背景的不同而不同 肿瘤。这项工作将直接解决对丛状神经纤维瘤的新疗法的需求(F99 阶段)，并将通过询问所需的神经纤维素量来填补关键的知识空白 在丛状神经纤维瘤和MPNSTs的情况下恢复功能(K00期)。总而言之，这 知识不仅将为NF1和相关疾病的新疗法的开发提供基础 癌症，但也有其他癌症易感综合征。拟议的培训计划还将提供 由基因工程和癌症生物学领域的领导者提供的非凡培训，将 申请者将成为这两个领域的一名成功的独立研究员。

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.