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

项目摘要 使用自体CD 34+细胞的基因治疗是治疗原发性免疫缺陷的有希望的治疗方法， 特别是对于没有最佳同种异体供体的个体。SCID-X1由IL 2 RG突变引起， 编码多种细胞因子受体的共同γ链（γc）。患有SCID-X1的男孩缺乏T和NK 细胞，它们的B细胞分别由于缺乏IL-7、IL-15和IL-21功能而不能产生抗体。 该项目旨在测试一种新的自失活慢病毒（LV）载体治疗SCID的有效性和安全性。 X1。我们假设，这项试验将通过引入低剂量的 白消安调节（目标1），并通过改变γ逆转录病毒（γRV）载体来提高安全性 将先前试验中使用的左心室向量用于本试验（目标2）。 先前的SCID-X1基因治疗试验在没有化疗条件的情况下注入细胞， 导致T细胞恢复和基因标记稳健，但B细胞中的基因标记可忽略不计， 体液免疫重建NK细胞的初始发育和标记没有持续。在目标1中， 检查低剂量白消安调节对1）细胞类型特异性植入和基因标记的影响， 2)通过深度测序的体内T细胞重建、T细胞表型和TRB库，3）体内体液免疫， 免疫重建，B细胞数量，表型，IL-21依赖性功能和IGH库 测序; 4）NK细胞数量、表型和功能。 先前的SCID-X1基因治疗试验使用了具有完整病毒启动子/增强子的γRV载体， 这导致5/20的患者由于插入性肿瘤发生而发展为T细胞白血病。基因治疗使用 删除了病毒增强子的自失活γRV载体显示了令人鼓舞的证据， 减少了淋巴癌基因附近的插入位点，但初始插入位点模式仍有风险。的 在本申请中提出的试验将通过使用自失活LV载体进一步提高安全性。在目标2中， 将研究患者CD 34+转导细胞中的初始插入位点模式，并比较样本 从拟定试验到使用γRV的历史试验，分析基因治疗后外周血中的插入位点谱 进行谱系追踪，并将聚类与来自先前试验的样品进行比较。

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.