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疾病的唯一治疗方法，由于缺乏供体、调节/免疫抑制的毒性和移植物抗宿主病（GVHD）而受到限制。自体造血干细胞（HSC）的基因转移已显示出希望，但由于低效率的HSC转导和废除基因表达的免疫反应，长期纠正一直难以实现。因此，采用基因转移策略来改善同种异体移植的结果可能被证明是一个强有力的选择。我们的总体目标是利用一种新的“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的三顺子慢病毒载体转导而具有竞争优势。这些载体的表达通过赋予对苄鸟嘌呤（一种内源性MGMT抑制剂，但不包括MGMTP140K）和氯乙基化剂（如BCNU）的抗性，可以在干细胞水平上进行体内化学选择（阳性选择）。用更昔洛韦（GCV）表达TKHSV可以耗尽（阴性选择）介导GVHD的供体T细胞，并消除由插入突变引起的潜在恶性克隆。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证明，不需要骨髓消融或免疫抑制，以及GCV介导的供体细胞消耗，不需要移植物消融。目前，修订后的R01申请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转化性大型动物研究。需要验证的假设是：1)用布苏凡或BG/BCNU进行非消融调节，以及移植后BG/BCNU治疗，将使同种异体HSC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给药会消耗引起GVHD的同种异体反应供体T细胞，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，条件调节方案将首先在同基因小鼠模型（修订的AIM1A）和非人灵长类动物的自体移植（修订的AIM 3）中开发。随着最近NHLBI试点项目（2011年1月）的奖励（加州大学戴维斯分校胎儿猴基因转移中心），非人类灵长类动物研究现在开始，这将在当前的AIMS中继续进行。为了增强这些研究的转化影响，EF？将测试靶向mgmtp140k /TKHSV载体（纳入1期试验）（修订AIMs 1B-C）。在同种异体移植中应用基于MGMTP140K/ tkhv的选择所需的关键参数，将在mhc错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革命性的影响，将同种异体HSC在其正常基因组背景下携带治疗基因的优势与使用基因转移方法来降低细胞消融和GVHD的风险结合起来。

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. PUBLIC HEALTH RELEVANCE: This proposal addresses central issues limiting application of allogeneic transplantation, the only curative therapy for many common genetic diseases of childhood, by using a novel viral gene delivery vector to genetically engineer donor stem cells. Expression of the two functional genes carried by this vector enables amplification of donor stem cells (positive selection) and depletion of derivative cells causing toxic effects (negative selection) by administration of different pharmacological agents. Positive/negative chemoselection represents a unique strategy that addresses current challenges for the safe application of gene therapy and stem cell transplantation. Achievement of the aims of this proposal will advance basic science knowledge and has the potential to expand the clinical application of curative transplantation for debilitating genetic diseases in childhood.