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疾病的唯一治疗方法，受到缺乏捐助者的限制，调理/免疫抑制的毒性以及移植物与宿主疾病（GVHD）。自体造血干细胞（HSC）中的基因转移已显示出希望，但是由于无效的HSC转导和免疫反应废除了基因表达，因此很难实现长期校正。因此，采用基因转移策略改善同种异体移植结果可能被证明是一种有力的选择。我们的总体目标是使用一种新型的“正/负”选择方法为遗传疾病的同种异体移植制定更安全的方案。供体HSC将通过与含有P140K-O6-甲基鸟嘌呤 - 甲基转移酶（MGMTP140K），HSV-硫酰胺激酶（TKHSV）（TKHSV）和EGFP的Tricistronic Lentiviral载体转导的竞争优势。这些矢量的表达通过赋予内源性MGMT的抑制剂而非MGMTP140K的抑制剂和氯乙基化剂（如BCNU），可以在干细胞水平上进行体内化学选择（阳性选择）。用ganciclovir（GCV）给药的TKHSV表达介导GVHD的供体T细胞的耗竭（负选择），并消除插入突变引起的潜在恶性克隆。我们的初步研究为新生儿中同种异体供体细胞的体内选择，没有骨髓化或免疫抑制，以及GCV介导的供体细胞的耗竭而没有移植消融。当前修订的R01应用的重点是在临床前的同种异体鼠模型和翻译大型动物研究中应用这种正/阴性选择方法。要测试的假设是：1）使用Busulfan或BG/BCNU的非富开动性调节，以及移植后BG/BCNU治疗，将使同种异体HSC的植入和体内扩展，并抑制同种反应性的，无反应性的供体供体和宿主和宿主的细胞； 2）GCV给药会耗尽转导的异常反应供体T细胞，从而导致GVHD并减轻移植后免疫抑制的需求。为了消除同种免疫响应的混杂变量，最初将在同步鼠模型（修订后的AIM1A）和非人类灵长类动物的自体移植物（修订AIM 3）中开发调节方案。随着NHLBI试点项目的最新奖项（2011年1月）（胎儿猴子基因转移中心，加州大学戴维斯分校），非人类灵长类动物研究正在开始，这将继续以目前的目的。为了增强这些研究的翻译影响，将测试EF？指导的MGMTP140K/TKHSV矢量（在第1阶段试验中纳入）（修订后的AIMS 1B-C）。然后将在Allo-Transplant中应用MGMTP140K/TKHSV选择所需的关键参数，然后在MHC不匹配的鼠模型中检查（AIM 2）。成功应用这种正/阴性选择方法将使该领域具有变化，将同种异体HSC携带治疗基因的优势结合在其正常基因组环境中，并使用基因转移方法来减轻细胞控制和GVHD的风险。