Gene therapy for SCID-X1 with low dose busulfan and a SIN-lentiviral vector

项目摘要 使用自体CD34细胞进行基因治疗是治疗原发免疫缺陷的一种很有前途的方法， 尤其是对于没有最佳同种异体捐献者的个人。SCID-X1是由IL2RG突变引起的 编码多种细胞因子受体的共同伽马链(γc)。患有SCID-X1的男孩缺乏T和NK 细胞及其B细胞由于缺乏IL-7、IL-15和IL-21功能而不能产生抗体。 本项目旨在测试一种新的自灭活慢病毒(LV)载体治疗SCID的有效性和安全性。 X1.我们推测，这项试验将通过引入低剂量的 白花丹调理(目标1)和通过改变伽玛逆转录病毒(γRV)载体来提高安全性 在之前的试验中使用的LV向量在本试验中(目标2)。 以前的SCID-X1基因治疗试验都是在没有化疗条件的情况下注入细胞，这 导致了强劲的T细胞恢复和基因标记，但B细胞的基因标记可以忽略不计， 体液免疫重建。NK细胞的初始发育和标记不能持续。在目标1中，我们将 检测低剂量白花丹处理对1)细胞类型特异性植入和基因标记的影响， 2)体内T细胞重建、T细胞表型和TRB基因序列的深度测序；3)体内体液 免疫重建、B细胞数量、表型、IL-21依赖功能和免疫球蛋白谱 测序，4)NK细胞数量、表型和功能。 以前针对SCID-X1的基因治疗试验使用了带有完整病毒启动子/增强子的γRV载体， 这导致5/20的患者由于插入癌而发展为T细胞白血病。使用基因疗法 一种自我失活的γRV载体，其中病毒增强剂已被删除，显示出令人鼓舞的证据 淋巴癌基因附近的插入部位减少，但初始插入部位模式仍有风险。这个 本申请中提议的试验将通过使用自失活的LV载体来进一步提高安全性。在《目标2》中，我们 将调查患者CD34转导细胞中的初始插入位点模式并比较样本 从建议的试验到使用γRV的历史试验，分析基因后外周血中的插入位点分布 进行血统追踪并与先前试验的样本进行比较的治疗。

Project Summary Gene therapy using autologous CD34+ cells is a promising treatment for primary immunodeficiency, particularly for individuals without optimal allogeneic donors. SCID-X1 is caused by mutations in IL2RG, which encodes the common gamma chain (γc) of multiple cytokine receptors. Boys with SCID-X1 lack T and NK cells, and their B cells fail to produce antibodies due to the lack of IL-7, IL-15 and IL-21 function respectively. This project seeks to test the efficacy and safety of a new self-inactivating lentiviral (LV) vector to treat SCID- X1. We hypothesize that this trial will improve immune reconstitution through the introduction of low dose busulfan conditioning (Aim 1) and improve safety through the change from a gammaretroviral (γRV) vector used in previous trials to the LV vector in this trial (Aim 2). Previous trials of gene therapy for SCID-X1 have infused cells without chemotherapy conditioning, which resulted in robust T cell recovery and gene marking, but negligible gene marking in B cells and failure of humoral immune reconstitution. Initial development and marking in NK cells was not sustained. In Aim 1 we will examine the impact of low dose busulfan conditioning on 1) cell type specific engraftment and gene marking, 2) in vivo T cell reconstitution, T cell phenotype and TRB repertoire by deep sequencing, 3) in vivo humoral immune reconstitution, B cell number, phenotype, IL-21 dependent function and IGH repertoire by deep sequencing, 4) NK cell number, phenotype and function. Previous trials of gene therapy for SCID-X1 have used a γRV vector with intact viral promoters/enhancers, which resulted in 5/20 patients developing T cell leukemia due to insertional oncogenesis. Gene therapy using a self-inactivating γRV vector in which viral enhancers have been deleted shows encouraging evidence of reduced insertion sites near lymphoid oncogenes, but an initial insertion site pattern that is still risky. The proposed trial in this application will further improve safety by using a self-inactivating LV vector. In Aim 2 we will investigate the initial insertion site pattern in the patients’ CD34+ transduced cells and compare samples from the proposed trial to historical trials using γRV, analyze insertion site profile in peripheral blood after gene therapy to perform lineage tracing and compare clustering with samples from previous trials.