Therapeutic potential of Cpf1-based gene editing for myeloproliferative neoplasms

基于 Cpf1 的基因编辑对骨髓增殖性肿瘤的治疗潜力

• 批准号: 9806809
• 负责人: WEN-SHU WU
• 金额: $ 17.39万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9806809
• 关键词: Adenovirus Vector; Adenoviruses; Allogenic; American; Autologous; Behavioral Research; Blood Cells; CD34 gene; Cause of Death; Cell Line; Cells; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; Development; Disease; Engraftment; Exons; Family; Future; Gene Delivery; Genes; Goals; Guide RNA; Health; Hematological Disease; Hematopoietic Neoplasms; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; High Dose Chemotherapy; Human; Immunosuppressive Agents; Introns; JAK2 gene; Joints; Lentivirus Vector; Mediating; Mission; Mus; Mutation; Myeloproliferative disease; NR5A2 gene; Names; Neoplasm Transplantation; Outcome; Patients; Personal Satisfaction; Pharmaceutical Preparations; Phenylalanine; Point Mutation; Positioning Attribute; Public Health; Publications; Research; Risk; Testing; Therapeutic; Transplantation; United States National Institutes of Health; Valine; Xenograft Model; base; curative treatments; endonuclease; experience; gain of function; gene transplantation for gene therapy; graft vs host disease; improved; in vivo; inhibitor/antagonist; mouse model; mutant; nuclease; public health relevance; social; stem cell therapy; therapeutic evaluation; therapeutic gene; therapeutic target

Project Summary Myeloproliferative neoplasms (MPNs) are groups of hematological diseases overproducing blood cells. A single gain-of-function point mutation in JAK2 gene (1849GèT in exon 14), named JAK2V617F, has been identified in the majority of MPN patients. MPNs are transplantable with hematopoietic stem cells (HSCs) carrying JAK2V617F gene. While JAK2 inhibitors are promising drugs for inhibiting MPN-induced complications, they cannot cure MPNs. Until now, allogeneic HSC transplantation (allo-HSCT) has been the only curative treatment for MPNs. However, a majority of patients suffer from a shortage of HLA-matched donors and some allo-HSCT recipients develop graft-versus-host disease (GvHD) that is a prominent cause of death and has important implications on immunosuppressive medications. Hence, it would be of immense clinical benefit to develop safe gene editing strategies to inactivate JAK2V617F mutant HSCs from MPN patients containing both healthy and mutant HSCs. If these strategies are successful, it will be possible to curatively treat MPN patients using autologous HSC transplantation (auto-HSCT), without a need to find a matching donor in the patient's family and to avoid the risk of GvHD. In this proposal, we hypothesize that JAK2V617F-specific guide RNA (gRNA) will direct Cpf1 nuclease-mediated DNA cleavage of JAK2V617F gene, leading to the inactivation of MPN-initiating HSCs. To test this hypothesis, we will pursue two specific aims: i) Inactivation of MPN cells by removing JAK2V617F exon 14 using CRISPR/Cpf1 gene editing, and ii) Assess the therapeutic potential of CRISPR/Cpf1-based auto-HSCT in a xenograft model of MPN. In aim 1, we will assess the effects of JAK2V617F-specific CRISPR/Cpf1 gene editing on the colony-forming capacity of MPN cell lines and on ex vivo inactivation of HSCs-harboring JAK2V617F in MPN CD34+ HSC pool. In aim 2, we will first generate xenograft models of MPN by engrafting MPN patient-derived CD34+ cells. Next, we will test the therapeutic potential of high-dose chemotherapy followed by auto-HSCT with MPN HSCs edited by JAK2V617F-CRISPR/Cpf1 gene editing. These studies will demonstrate the feasibility of CRISPR/Cpf1 gene editing-based auto-HSCT joint with high-dose chemotherapy for the treatment of patients with MPNs. Regarding expected outcomes, we anticipate that we will demonstrate effective gene editing strategies for inactivating MPN-initiating HSCs. Such results will have an important positive impact because these approaches are translatable for developing gene editing- based auto-HSCT therapeutic strategy for MPN patients and possible for other hematopoietic malignancies.

项目摘要 骨髓增生性肿瘤（MPN）是血液学疾病过量产生血细胞的组。一个 JAK2基因（外显子14中的1849gèt）中的单个功能点突变，名为JAK2V617F 在大多数MPN患者中发现。 MPN是用造血干细胞（HSC）移植的 携带JAK2V617F基因。尽管JAK2抑制剂是抑制MPN诱导并发症的有前途的药物，但 他们无法治愈MPN。到目前为止，同种异体HSC移植（Allo-HSCT）一直是唯一的治愈性 MPN的治疗。但是，大多数患者患有HLA匹配的供体短缺，有些患者 Allo-HSCT接受者患上移植物抗宿主病（GVHD），这是一个显着的死亡原因，已有 对免疫抑制药物的重要意义。因此，这将是巨大的临床益处 开发安全的基因编辑策略，以使来自包含两者 健康和突变的HSC。如果这些策略成功，可以适度治疗MPN患者 使用自体HSC移植（AUTO-HSCT），无需在患者的患者中找到匹配的供体 家庭并避免GVHD的风险。在此提案中，我们假设JAK2V617F特定指南RNA （GRNA）将指导CPF1核酸酶介导的JAK2V617F基因的DNA裂解，导致失活 MPN启动HSC。为了检验这一假设，我们将追求两个具体的目的：i）通过 使用CRISPR/CPF1基因编辑去除JAK2V617F外显子14，ii）评估治疗潜力 MPN的Xenograpony模型中的基于CRISPR/CPF1的自动-HSCT。在AIM 1中，我们将评估 JAK2V617F特异性CRISPR/CPF1基因编辑MPN细胞系和离体的菌落形成能力 MPN CD34+ HSC池中HSCS-HARBORING JAK2V617F的灭活。在AIM 2中，我们将首先生成异种移植 MPN模型通过雕刻MPN患者衍生的CD34+细胞。接下来，我们将测试 高剂量化学疗法，然后是由JAK2V617F-CRISPR/CPF1基因编辑的MPN HSC的自动HSCT 编辑。这些研究将证明CRISPR/CPF1基因编辑基于自动-HSCT关节的可行性 高剂量化疗用于治疗MPN患者。关于预期的结果，我们期望 我们将展示有效的基因编辑策略，以使MPN发射HSC灭活。这样的结果将会 具有重要的积极影响，因为这些方法是可以翻译用于开发基因编辑的 - 基于MPN患者的自动HSCT治疗策略，并可能用于其他造血恶性肿瘤。