In Vivo Stem Cell Selection in Neonatal Allo-Transplants

描述（由申请人提供）：我们在本申请中的总体目标是开发一种非清髓性同种异体移植方法，该方法需要最低限度的或不需要移植后免疫抑制。这种方法独特地结合了新生儿期的造血干细胞（HSC）移植，用于体内扩增基因工程耐药供体HSC的阳性选择策略，以及在发生不良事件时消除这些细胞的阴性选择策略。选择新生儿模型用于这种方法，因为T细胞个体发育在这个阶段是不完整的，并且免疫耐受可能比在成人中移植更容易实现。我们的阳性选择策略涉及用P140 K-MGMT（甲基鸟嘌呤-甲基转移酶）慢病毒介导的HSC转导，P140 K-MGMT是一种变体DMA烷基转移酶，其赋予对内源性MGMT抑制剂如苄基鸟嘌呤（BG）和氯乙基化剂如BCNU的抗性，允许体内阳性选择和干细胞水平的供体细胞富集。新生儿移植后富集P140 K-MGMT转导的供体HSC和诱导对转基因编码的新抗原的免疫耐受的关键参数将首先在同基因移植模型中建立（Aim 1）。然后将这种新生儿移植/体内阳性选择策略应用于一种新的半同种异体非清髓性模型，该模型模拟HLA-半相合供体移植（Aim 2），使我们能够研究供体嵌合体水平的提高，以及在宿主免疫系统发育不成熟的阶段进行移植，是否会诱导对移植物同种异体抗原的耐受性。我们还将测试胎肝来源的HSC作为脐带血的替代物，以确定供体细胞个体发育的阶段是否影响涉及移植物抗宿主病（GVHD）的细胞免疫反应，并检查在体内选择过程中使用连续周期的BCNU是否可以提供足以限制甚至消除GVHD的免疫抑制。最后，阳性/阴性选择载体，其中P140 K-MGMT连接到单纯疱疹病毒胸苷激酶（HSV TK）自杀基因，将用于评估更昔洛韦治疗是否可以限制GVHD通过选择性消除增殖供体细胞。这种阴性选择策略也可能在体内强制富集和扩增后消除转导供体细胞的自主克隆增殖，这是一个重要的潜在安全性问题。

DESCRIPTION (provided by applicant): Our overall objectives in this application are to develop a non-myeloablative allogeneic transplantation approach that requires minimal or no post-transplant immunosuppression. This approach uniquely combines hematopoietic stem cell (HSC) transplantation during the neonatal period, a positive selection strategy for in vivo amplification of genetically engineered drug-resistant donor HSC, and a negative selection strategy to eliminate these cells should an adverse event occur. The neonatal model was chosen for this approach because T cell ontogeny is incomplete at this stage and immune tolerance may be more readily achieved than with transplantation in adults. Our positive selection strategy involves lentivirus-mediated transduction of HSCs with P140K-MGMT (methylguanine-methyltransferase), a variant DMA alkyltransferase that confers resistance to endogenous MGMT inhibitors such as benzylguanine (BG) and to chloroethylating agents such as BCNU, allowing positive selection in vivo and donor cell enrichment at the stem cell level. Critical parameters for enrichment of P140K-MGMT-transduced donor HSCs after neonatal transplantation, and for induction of immunotolerance to transgene-encoded neoantigens, will first be established in a syngeneic transplant model (Aim 1). This neonatal transplantation/ in vivo positive selection strategy will then be applied to a novel semi-allogeneic non-myeloablative model that mimics HLA-haploidentical donor transplantation (Aim 2), allowing us to investigate whether enhanced levels of donor chimerism, coupled with transplantation at a stage when the host immune system is developmentally immature, will induce tolerance to graft allo-antigens. We will also test fetal liver-derived HSCs as surrogates for cord blood to determine whether the stage of donor cell ontogeny affects cellular immune responses involved in graft vs. host disease (GVHD), and examine whether the use of successive cycles of BCNU during the in vivo selection process might provide immunosuppression sufficient to limit or even abrogate GVHD. Finally, positive/negative selection vectors, in which P140K-MGMT is linked to the Herpes simplex virus thymidine kinase (HSV TK) suicide gene, will be used to assess whether ganciclovir treatment can limit GVHD by selective elimination of proliferating donor cells. This negative selection strategy may also abrogate autonomous clonal proliferation of transduced donor cells after forced enrichment and expansion in vivo, an important potential safety issue.