Lentivirus-based positive/negative selection in minimally ablative transplants

描述(由申请人提供)：同种异体移植是治疗许多遗传疾病的唯一方法，受到供体缺乏、调节/免疫抑制的毒性以及移植物抗宿主病(GVHD)的限制。在自体造血干细胞(HSC)中的基因转移已经显示出希望，但由于HSC转导效率低下和免疫反应取消了基因表达，长期的纠正一直是困难的。因此，使用基因转移策略来改善同种异体移植的结果可能被证明是一个强有力的替代方案。我们的总体目标是利用一种新的“阳性/阴性”选择方法，为遗传性疾病的异基因移植开发更安全的方案。通过转导含有P140K-O6-甲基鸟嘌呤-甲基转移酶(MGMTP140K)、单纯疱疹病毒胸苷激酶(TKHSV)和绿色荧光蛋白(EGFP)的三顺反子慢病毒载体，供体HSC将具有竞争优势。这些载体的表达使干细胞水平的体内化学选择(正选择)能够对内源性MGMT(而不是MGMTP140K)的抑制剂苯基鸟嘌呤(BG)和氯乙基化试剂(如BCNU)产生抗性。用更昔洛韦(GCV)表达TKHSV能够耗尽(阴性选择)介导GVHD的供体T细胞，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体内选择同种异体供体细胞建立了概念验证，而不需要清髓或免疫抑制，以及不需要移植物消融的GCV介导的供体细胞耗竭。目前修订的R01应用的重点是将这种阳性/阴性选择方法应用于临床前同种异体小鼠模型和翻译大动物研究。要测试的假设是：1)用白花丹或BG/BCNU进行非消融性预适应，以及移植后BG/BCNU治疗，将使异基因HSC能够在体内植入和扩增，并抑制供体和宿主来源的同种反应性、未转导的细胞；2)应用GCV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，将首先在同基因小鼠模型(修订的AIM1A)和非人类灵长类动物的自体移植(修订的AIM3)中开发条件化方案。随着最近NHLBI试点项目(2011年1月)(加州大学戴维斯分校胎儿猴子基因转移中心)的授予，非人类灵长类动物的研究现在正在开始，这将在当前的目标中继续下去。为了加强这些研究的翻译效果，将测试EF？定向的MGMTP140K/TKHSV载体(并入第一阶段试验)(修订的AIMS 1B-C)。在异基因移植中应用基于MGMTP140K/TKHSV的选择所需的关键参数，然后将在MHC不匹配的小鼠模型中进行检查(AIM 2)。这种正/负选择方法的成功应用将对该领域产生变革，将在正常基因组环境中携带治疗性基因的同种异体HSC的优势结合起来，并使用基因转移方法来减少细胞消融和GVHD的风险。

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.