Lentivirus-based positive/negative selection in minimally ablative transplants

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，但受到供体缺乏、预处理/免疫抑制毒性和移植物抗宿主病（GVHD）的限制。自体造血干细胞（HSC）中的基因转移已显示出前景，但由于HSC转导效率低下和免疫应答废除基因表达，长期校正一直难以实现。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一种新的“阳性/阴性”选择方法，为遗传性疾病的同种异体移植制定更安全的方案。通过用含有P140 K-O 6-甲基鸟嘌呤-甲基转移酶（MGMTP 140 K）、HSV-胸苷激酶（TKHSV）和eGFP的三顺反子慢病毒载体转导供体HSC将被赋予竞争优势。这些载体的表达通过赋予对苄基鸟嘌呤（BG）（内源性MGMT而非MGMTP 140 K的抑制剂）和氯乙基化剂（如BCNU）的抗性，使得能够在干细胞水平进行体内化学选择（阳性选择）。用更昔洛韦（GCV）施用表达TKHSV能够耗尽（阴性选择）介导GVHD的供体T细胞，并消除由插入突变产生的潜在恶性克隆。我们的初步研究建立了在新生儿体内选择同种异体供体细胞的概念验证，而无需骨髓消融或免疫抑制，以及GCV介导的供体细胞耗竭而无需移植消融。当前修订的R 01申请的重点是在临床前同种异体鼠模型和转化大动物研究中应用这种阳性/阴性选择方法。待检验的假设是：1）白消安或BG/BCNU的非消融性预处理以及移植后BG/BCNU治疗将使得同种异体HSC能够植入和体内扩增，并抑制供体和宿主来源的同种异体反应性未转导细胞; 2）GCV施用将耗尽引起GVHD的转导的同种异体反应性供体T细胞，并减轻对移植后免疫抑制的需要。为了消除同种免疫应答的混杂变量，将首先在同基因小鼠模型（修订的AIM 1A）和非人灵长类动物的自体移植（修订的AIM 3）中开发预处理方案。随着最近NHLBI试点项目（2011年1月）的授予（胎猴基因转移中心，加州大学戴维斯分校），非人灵长类动物研究现在开始，这将继续在当前的AIMS。为了增强这些研究的翻译影响，将检测EF 1 α导向的MGMTP 140 K/TKHSV载体（纳入I期试验）（修订的AIM 1B-C）。然后将在MHC错配小鼠模型（AIM 2）中检查在同种异体移植中应用基于MGMTP 140 K/TKHSV的选择所需的关键参数。这种阳性/阴性选择方法的成功应用将是该领域的变革，结合了在其正常基因组背景下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减轻细胞消融和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 EF1α 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.