Cellular Transduction with Replication-Competent Retrovirus Vectors

描述（由申请人提供）：多形性胶质母细胞瘤（GBM）是成人中最常见的原发性脑肿瘤，尽管进行了积极的手术、放疗和化疗，但预后仅为12-15个月。由于缺乏有效的治疗方案，这种疾病成为基因治疗等新策略的目标。然而，基因治疗的唯一主要III期临床试验，涉及在GBM患者中使用常规的复制缺陷型逆转录病毒载体，导致仅0.02%的数量级的令人不安的低和治疗上不充分的转导水平。因此，标准的复制缺陷型逆转录病毒载体不能在体内实现肿瘤的有效转导是神经胶质瘤基因治疗的主要障碍。使用有复制能力的载体进行基因转移会更有效，因为每个成功转导的肿瘤细胞本身都会成为病毒产生细胞，即使在初次给药后也能维持进一步的转导事件。我们先前已经证明，直接瘤内注射基于鼠白血病病毒（MLV）的可复制逆转录病毒（RCR）载体制剂可以在神经胶质瘤中实现非常有效的自杀基因转移，转导严格限于活跃分裂的肿瘤细胞，而没有证据表明显著扩散到瘤外部位，并且导致前药给药后显著延长的生存期，没有可检测到的全身副作用。在这里，我们建议进一步提高这种方法的效率，通过工程同种异体反应性细胞毒性T淋巴细胞（allocCTL）成为RCR载体生产细胞，然后可以作为能动的细胞传递平台，可以渗透到肿瘤块，促进多灶性扩散的复制载体。同种异体反应性CTL表现出为此目的提供独特优势的特征：同种异体CTL可以穿过组织，在接触时可以自身杀死肿瘤，并且可以产生诱导细胞凋亡或可以启动内源性免疫应答的细胞因子。在CMS神经胶质瘤内，免疫特权部位内的免疫抑制环境，它们自身被免疫系统破坏的时间可能足够长，以使它们具有有益的效果，但是异源CTL在泄漏到全身循环中时应被迅速破坏。我们将测试它们的生存能力，生物分布，安全性和实用性作为细胞运载工具，以提高RCR载体的传播和治疗效果，比较免疫缺陷和免疫活性胶质瘤模型在体内。

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.