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

治疗 I 型神经纤维瘤病 (NF1) 的基因疗法

基本信息

批准号：
10669801
负责人：
Madeleine Janette Sitton
金额：
$ 3.42万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-01 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10669801
关键词：
Address Affect Biological Cancer Biology Cell Line Cell Proliferation Cell model Cells Clinical Clustered Regularly Interspaced Short Palindromic Repeats Complementary DNA Cre-LoxP Development Diagnosis Disease Dose Doxycycline Exhibits Exons Fellowship Foundations GTPase-Activating Proteins Genes Genetic Genetic Engineering Genetic Transcription Guide RNA Histologic Knowledge Magnetic Resonance Imaging Malignant Neoplasms Measures Mitogen-Activated Protein Kinase Kinases Mitogen-Activated Protein Kinases Modeling Monitor Morbidity - disease rate Mus Mutate Mutation NF1 gene Neurofibromatosis 1 Neurofibrosarcoma Pathogenicity Pathway interactions Patients Persons Phase Phenotype Phosphorylation Physiological Plexiform Neurofibroma Positioning Attribute Postdoctoral Fellow Proteins RNA Splicing Ras Signaling Pathway Research Research Personnel Research Project Grants Schwann Cells Series Signal Transduction Symptoms Syndrome System Testing Therapeutic Time Training Tumor Cell Line Tumor Suppression Western Blotting Work autosomal dominant mutation cancer predisposition cell growth design driver mutation effective therapy experimental study functional restoration gene therapy homologous recombination improved in vitro Model kinase inhibitor mouse model mutation correction neurofibroma novel novel therapeutics patient population post-doctoral training postmitotic prevent reduce symptoms response restoration tumor uncontrolled cell growth

项目摘要

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.

项目摘要/摘要神经纤维瘤病I型（NF1）是由常染色体显性突变引起的癌症易感综合征在基因NF1中，全球2500人中有1人。 NF1编码神经纤维蛋白，GTPase激活负调节RAS信号通路的蛋白质，因此NF1的损失导致RAS过度激活导致不受控制的细胞生长和增殖。 NF1的特征是多种症状，但大多数关于丛状神经纤维瘤，影响30-50％的患者。此外，8-13％的患者还将发展恶性周围神经护套肿瘤（MPNST），这是发病率的主要原因 NF1患者，只有20-50％的患者诊断5年。而治疗策略靶向RAS信号传导可以通过缩小神经纤维瘤，基因来为NF1患者提供一些好处编辑是唯一可以通过纠正NF1中的突变来解决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。另外，实现临床改善所需的神经纤维蛋白恢复量尚不清楚。差距在阻碍NF1最佳治疗策略发展的知识中。 F99阶段的目的该奖学金是开发基于CRISPR的系统，以纠正约90％NF1的致病突变通过将4.5 kb的副翼序列插入突变的NF1基因中以恢复正确的患者人群 NF1序列。 HEK293T细胞中的实验已显示出成功的整合和转录在内源性外显子上脱离了巨型丝网。预计这会创建功能神经纤维蛋白，将RAS信号降低至基础水平，并降低小鼠中丛状神经纤维瘤的大小 NF1的模型。该研究金的K00阶段的目的是询问所需的神经纤维蛋白的量在两个不同的生物学环境中恢复功能：丛状神经纤维瘤和mpnsts。新颖的强力霉素 - 将生成诱导型细胞和鼠标模型，以检验以下假设。恢复肿瘤的抑制，但此数量会根据瘤。这项工作将直接解决对丛生神经纤维瘤新型治疗的需求（F99 阶段），并通过询问所需的神经纤维蛋白的量来填补关键的知识差距在丛状神经纤维瘤和MPNST（K00相）的背景下恢复功能。一起，这个知识不仅将为NF1及相关的新型治疗剂的开发提供基础癌症，但也是其他癌症前置综合症。拟议的培训计划还将提供基因工程和癌症生物学领域领导者的特殊培训，定位申请人成为这两个领域界面上成功的独立研究人员。