Cellular Transduction with Replication-Competent Retrovirus Vectors

描述(申请人提供)：多形性胶质母细胞瘤(GBM)是成人最常见的原发脑肿瘤，尽管进行了积极的手术、放疗和化疗，但预后仅为12-15个月。缺乏有效的治疗选择使这种疾病成为基因治疗等新策略的目标。然而，基因治疗的唯一重大第三阶段临床试验，涉及在GBM患者中使用传统的复制缺陷逆转录病毒载体，导致令人失望的低转导水平和治疗不足，仅为0.02%。因此，标准复制缺陷逆转录病毒载体无法在体内实现对肿瘤的有效转导是胶质瘤基因治疗的主要障碍。使用具有复制能力的载体进行基因转移将更加有效，因为每个成功转导的肿瘤细胞本身都会成为产生病毒的细胞，即使在最初给药后也能维持进一步的转导事件。我们先前已经证明，直接瘤内注射基于小鼠白血病病毒(MLV)的复制能力强的逆转录病毒(RCR)载体制剂可以在胶质瘤中实现非常有效的自杀基因转移，转导严格限制在活跃分裂的肿瘤细胞而没有明显扩散到肿瘤外的证据，并导致前药给药显著延长生存期，没有检测到全身副作用。在这里，我们建议进一步提高这一方法的效率，通过工程同种异体反应性细胞毒性T淋巴细胞(AllCTL)成为RCR载体产生细胞，然后作为可穿透到肿瘤块的移动细胞输送平台，促进复制载体的多焦点扩散。同种异体反应性CTL的特性为这一目的提供了独特的优势：同种异体CTL可以在组织中移动，接触后能自身杀死肿瘤，并能产生诱导细胞凋亡的细胞因子或启动内源性免疫反应。在CMS胶质瘤中，免疫优势部位内的免疫抑制环境可能会在足够长的时间内避开免疫系统对它们的破坏，使它们产生有益的效果，但同种CTL一旦泄漏到一般循环中，就应该迅速被破坏。我们将测试它们作为细胞载体的活性、生物分布、安全性和实用性，以增强RCR载体的传播和治疗效果，比较体内免疫缺陷和免疫活性胶质瘤模型。

DESCRIPTION (provided by applicant): Glioblastoma multiforme (GBM), the most common primary brain tumor in adults, is associated with a dismal prognosis of only 12-15 months despite aggressive surgery, radiation, and chemotherapy. The lack of effective treatment options has made this disease a target for new strategies such as gene therapy. However, the only major Phase III clinical trial of gene therapy, involving the use of conventional replication-defective retrovirus vectors in GBM patients, resulted in disappointingly low and therapeutically inadequate transduction levels on the order of only 0.02%. The inability of standard replication-defective retroviral vectors to achieve effective transduction of tumors in vivo is therefore a major obstacle to gene therapy for gliomas. The use of replication-competent vectors for gene transfer would be more efficient, as each tumor cell that is successfully transduced would itself become a virus-producing cell, sustaining further transduction events even after initial administration. We have previously demonstrated that direct intratumoral injection of murine leukemia virus (MLV)-based replication-competent retrovirus (RCR) vector preparations can achieve tremendously efficient suicide gene transfer in gliomas, with transduction stringently restricted to the actively dividing tumor cells without evidence of significant spread to extratumoral sites, and resulting in significantly prolonged survival upon prodrug administration, without detectable systemic side effects. Here we propose to further improve the efficiency of this approach by engineering alloreactive cytotoxic T lymphocytes (alloCTLs) to become RCR vector producer cells, which can then serve as motile cellular delivery platforms that can penetrate into the tumor mass and facilitate multifocal spread of the replicating vectors. Alloreactive CTL exhibit characteristics that provide unique advantages for this purpose: alloCTLs can move through tissue, can themselves kill tumor upon contact, and can produce cytokines that induce apoptosis or can initiate an endogenous immune response. Within CMS gliomas, an immunosuppressive environment within an immunologically privileged site, their own destruction by the immune system may be circumvented long enough for them to have a beneficial effect, but alloCTLs should be rapidly destroyed upon leakage into the general circulation. We will test their viability, biodistribution, safety, and utility as cellular delivery vehicles to enhance RCR vector spread and therapeutic efficacy, comparing immunodeficient and immunocompetent glioma models in vivo.