Cellular Transduction with Replication-Competent Retrovirus Vectors

多形性胶质母细胞瘤（GBM）是成人中最常见的原发性脑肿瘤， 预后仅12-15个月，尽管进行了积极的手术、放疗和化疗。缺乏 有效的治疗选择使这种疾病成为新策略如基因治疗的目标。然而，在这方面， 唯一的主要III期临床试验的基因治疗，涉及使用常规的复制缺陷型 逆转录病毒载体在GBM患者中，导致令人不安的低和治疗上的不充分转导 水平仅为0.02%。标准的复制缺陷型逆转录病毒载体无法实现 因此，体内肿瘤的有效转导是神经胶质瘤基因治疗的主要障碍。使用 用于基因转移的可复制载体将更有效，因为每个肿瘤细胞成功地 转导的细胞本身将成为一个病毒产生细胞，即使在最初的转导后， 局我们以前已经证明，直接瘤内注射小鼠白血病病毒， 基于MLV的可复制型逆转录病毒（RCR）载体制剂可以实现非常有效的逆转录病毒复制。 胶质瘤中的自杀基因转移，转导严格限制在活跃分裂的肿瘤细胞中 无明显扩散至瘤外部位的证据，并导致生存期显著延长 而没有可检测的全身副作用。在此，我们建议进一步改善 通过将同种异体反应性细胞毒性T淋巴细胞（alloCTL）工程化为RCR， 载体生产细胞，其然后可以充当能动的细胞递送平台，其可以渗透到细胞中。 肿瘤块，并促进复制载体的多灶性扩散。同种异体反应性CTL表现出 这为该目的提供了独特优势：异源CTL可以穿过组织，自身可以杀死肿瘤 并且可以产生诱导细胞凋亡或可以启动内源性免疫的细胞因子 反应在CMS神经胶质瘤中，免疫抑制环境位于免疫特权部位， 免疫系统对它们自身的破坏可以被规避足够长的时间， 作用，但alloCTL应迅速破坏泄漏到全身循环。我们将测试他们的 活性、生物分布、安全性和作为细胞递送载体的效用，以增强RCR载体的传播， 治疗效果，比较体内免疫缺陷和免疫活性胶质瘤模型。

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.