Lentivirus-based positive/negative selection in minimally ablative transplants

微量切除移植中基于慢病毒的阳性/阴性选择

• 批准号: 8390413
• 负责人: KARIN L GAENSLER
• 金额: $ 57.7万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-12-01 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8390413
• 关键词: Ablation; Achievement; Address; Adult; Alkylating Agents; Allogenic; Animal Model; Animals; Autologous; Autologous Transplantation; Award; Basic Science; Bone Marrow Purging; Busulfan; CD34 gene; Carmustine; Cells; Childhood; Chimerism; Clinical Data; Clinical Trials; Clonality; Confounding Factors (Epidemiology); Disease; Donor Selection; Dose; Engraftment; Ganciclovir; Gene Expression; Gene Silencing; Gene Transfer; Genes; Genetic Engineering; Genomics; Goals; Guanine; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Hereditary Disease; Homologous Transplantation; Immune response; Immunosuppression; In Vitro; Knowledge; Length; Lentivirus Vector; MGMT gene; Macaca mulatta; Malignant - descriptor; Mediating; Methods; Methyltransferase; Modeling; Monkeys; Morbidity - disease rate; Mus; Mutation; National Heart, Lung, and Blood Institute; Outcome; Phase I Clinical Trials; Pilot Projects; Population; Proliferating; Protocols documentation; Regimen; Resistance; Risk; SIV; Safety; Simplexvirus; Stem cell transplant; Stem cells; Subfamily lentivirinae; T-Lymphocyte; Testing; Thalassemia intermedia; Thymidine Kinase; Toxic effect; Toxicology; Translations; Transplantation; base; cellular transduction; clinical application; clinically relevant; conditioning; expression cloning; expression vector; fetal; gene replacement; gene therapy; genotoxicity; graft vs host disease; head-to-head comparison; improved; in vivo; inhibitor/antagonist; mortality; neonate; nonhuman primate; novel; pre-clinical; preconditioning; promoter; senescence; suicide gene; telomere; temozolomide; therapeutic gene; transduction efficiency; translational study; validation studies; vector; viral gene delivery

DESCRIPTION (provided by applicant): Allogeneic transplantation, the only treatment for many genetic disorders, is limited by the lack of donors, toxicity of conditioning/immunosuppression, and graft vs. host disease (GVHD). Gene transfer in autologous hematopoietic stem cells (HSC) has shown promise, but long-term correction has been difficult to achieve due to inefficient HSC transduction and immune responses abrogating gene expression. Thus, employing gene transfer strategies to improve allogeneic transplant outcomes may prove to be a powerful alternative. Our overall goals are to develop safer protocols for allogeneic transplantation for genetic disorders using a novel "positive/negative" selection approach. Donor HSC will be endowed with a competitive advantage by transduction with tricistronic lentiviral vectors containing P140K-O6-methylguanine-methyltransferase (MGMTP140K), HSV-Thymidine kinase (TKHSV) and eGFP. Expression of these vectors enables in vivo chemo selection (positive selection) at the stem cell level by conferring resistance to benzyl guanine (BG), an inhibitor of endogenous MGMT but not MGMTP140K, and to chloroethylating agents such as BCNU. Expression of TKHSV with Ganciclovir (GCV) administration enables depletion (negative selection) of donor T cells mediating GVHD, and elimination of potential malignant clones arising from insertional mutations. Our preliminary studies establish proof-of-concept for in vivo selection of allogeneic donor cells in neonates, without myeloablation or immunosuppression, and for GCV mediated donor cell depletion without graft ablation. The focus of the current, revised R01 application is to apply this positive/negative selection approach in pre-clinical allogeneic murine models and in translational large animal studies. The hypotheses to be tested are: 1) non-ablative conditioning with busulfan or BG/BCNU, and post-transplant BG/BCNU treatment, will enable engraftment and in vivo expansion of allogeneic HSC, and suppress allo-reactive, untransduced cells of donor and host origin; 2) GCV administration will deplete transduced allo-reactive donor T cells causing GVHD and mitigate the need for post-transplant immunosuppression. To eliminate confounding variables of allo-immune responses, conditioning protocols will initially be developed in syngeneic murine models (revised AIM1A) and in autologous transplants in non-human primates (revised AIM 3). With the recent award of an NHLBI pilot project (January, 2011) (Center for Fetal Monkey Gene Transfer, UC Davis), non-human primate studies are now beginning, that will continue in the current AIMS. To enhance the translational impact of these studies, an EF?-directed-MGMTP140K/TKHSV vector (incorporated in a Phase 1 trial) will be tested (revised AIMs 1B-C). Critical parameters, required to apply MGMTP140K/TKHSV-based selection in allo-transplants, will then be examined in an MHC-mismatched murine model (AIM 2). Successful application of this positive/negative selection approach would be transformative for the field, combining advantages of allogeneic HSC carrying therapeutic genes in their normal genomic context, with the use of gene transfer methods to mitigate risks of cytoablation and GVHD.

描述（由申请人提供）：同种异体移植是许多遗传性疾病的唯一治疗方法，但由于缺乏供体、调理/免疫抑制的毒性以及移植物抗宿主病（GVHD）而受到限制。自体造血干细胞 (HSC) 中的基因转移已显示出希望，但由于 HSC 转导效率低下和免疫反应消除基因表达，因此很难实现长期纠正。因此，采用基因转移策略来改善同种异体移植结果可能被证明是一种强有力的替代方案。我们的总体目标是使用新颖的“阳性/阴性”选择方法开发更安全的遗传性疾病同种异体移植方案。通过转导含有 P140K-O6-甲基鸟嘌呤甲基转移酶 (MGMTP140K)、HSV-胸苷激酶 (TKHSV) 和 eGFP 的三顺反子慢病毒载体，供体 HSC 将获得竞争优势。这些载体的表达通过赋予对苄基鸟嘌呤 (BG)（内源性 MGMT 的抑制剂但不是 MGMTP140K 的抑制剂）和氯乙基化剂（如 BCNU）的抗性，在干细胞水平上实现体内化学选择（正选择）。更昔洛韦 (GCV) 给药后 TKHSV 的表达能够消除（阴性选择）介导 GVHD 的供体 T 细胞，并消除因插入突变而产生的潜在恶性克隆。我们的初步研究为新生儿体内同种异体供体细胞的选择（无需清髓或免疫抑制）以及无需移植物消融的 GCV 介导的供体细胞耗竭建立了概念验证。当前修订后的 R01 应用的重点是将这种正/负选择方法应用于临床前同种异体小鼠模型和转化大型动物研究。要测试的假设是：1）用白消安或 BG/BCNU 进行非消融调节，以及移植后 BG/BCNU 治疗，将能够实现同种异体 HSC 的植入和体内扩增，并抑制供体和宿主来源的同种异体反应性、未转导的细胞； 2) GCV 给药将耗尽转导的同种异体反应性供体 T 细胞，导致 GVHD，并减轻移植后免疫抑制的需要。为了消除同种免疫反应的混杂变量，首先将在同基因小鼠模型（修订版 AIM1A）和非人类灵长类动物自体移植（修订版 AIM 3）中开发调节方案。随着最近获得 NHLBI 试点项目（2011 年 1 月）（加州大学戴维斯分校胎猴基因转移中心），非人类灵长类动物研究现已开始，并将在当前的 AIMS 中继续进行。为了增强这些研究的转化影响，将测试 EF? 导向的 MGMTP140K/TKHSV 载体（纳入一期试验）（修订后的 AIM 1B-C）。然后，将在 MHC 不匹配的小鼠模型 (AIM 2) 中检查在同种异体移植中应用基于 MGMTP140K/TKHSV 的选择所需的关键参数。这种正/负选择方法的成功应用将对该领域产生变革，结合了在正常基因组环境中携带治疗基因的同种异体 HSC 的优势，以及使用基因转移方法来降低细胞消融和 GVHD 的风险。