Retroviral replicating vector for prodrug activator gene therapy

描述（由申请人提供）：许多实体瘤的基因治疗策略涉及使用基于复制缺陷的Moloney小鼠白血病（MLV）的逆转录病毒载体，但由于缺乏足够的肿瘤转导，其疗效受到限制。利用具有复制能力的逆转录病毒载体进行基因转移会更有效，因为每个转导的肿瘤细胞都会产生更多能够引发进一步感染事件的载体。我们设计了一种新的逆转录病毒复制载体（RRV），能够在体内高效地将基因传递到实体瘤中。我们已经证明，由于正常细胞的先天免疫的限制，载体传播被限制在肿瘤本身，以及病毒固有的无法感染有丝分裂前的正常细胞。我们在多中心I期试验中招募了17名复发性高级别胶质瘤（HGG）患者（www.clinicaltrials.gov:NCT01156584, NCT01470794）。这些研究中的RRV， Toca 511 (vocimagene amiretrorepvec，或本提案中的RRV-CD)，表达优化的酵母胞嘧啶脱氨酶（CD），将前药5-氟胞嘧啶（5- fc）转化为经典化疗药物5-氟尿嘧啶（5- FU）。5-FU常用于联合化疗，但直接抑制细胞增殖的药物会干扰载体的扩散。我们建议测试不同前药激活剂RRV的组合在不影响载体传播的情况下抑制肿瘤。这是可行的，我们发现，RRV感染的肿瘤细胞可以在体内再感染。我们现在构建了表达优化的单纯疱疹胸苷激酶（HSV-TK）前药激活因子基因（本提案中的RRV- tk）的RRV。我们建议用新开发的RRV-TK载体在体外和体内胶质瘤模型中进行验证研究。我们将评估联合RRV- cd和RRV- tk的双重RRV基因治疗的潜力。我们将在体外和体内测试RRV-TK的转导效率、转基因表达水平、复制稳定性和细胞毒性，以及2)在体外和体内胶质瘤模型中确定RRV-CD和RRV-TK联合治疗的最佳参数。来自这些研究的信息将用于制定未来的临床试验方案，并且与联合化疗一样，提供了潜在的协同治疗益处，并且比单独使用任何载体的单药治疗更有效地根除肿瘤。此外，由于受感染的肿瘤细胞自己产生化疗药物，避免了全身化疗的不良反应。

DESCRIPTION (provided by applicant): Many strategies for gene therapy of solid tumors involve the use of replication-defective Moloney murine leukemia (MLV)-based retroviral vectors, but efficacy has been limited due to lack of adequate tumor transduction. Gene transfer using replication-competent retroviral vectors would be more efficient, as each transduced tumor cell produces more vectors capable of initiating further infection events. We have devised novel retroviral replicating vectors (RRV) capable of highly efficient gene delivery to solid tumor in vivo. We have demonstrated that vector spread is restricted to the tumor itself due to restriction by innate immunity in normal cells, and the intrinsic inability of the virus to infect ost-mitotic normal cells. We have enrolled 17 subjects with recurrent high grade glioma (HGG) in multi-center Phase I trials (www.clinicaltrials.gov:NCT01156584, NCT01470794). The RRV in these studies, Toca 511 (vocimagene amiretrorepvec, or RRV-CD in this proposal), expresses an optimized yeast cytosine deaminase (CD) which converts the prodrug 5-fluorocytosine (5-FC) into the classic chemotherapy drug 5-fluorouracil (5- FU). 5-FU is often used in chemotherapy combinations, but agents that directly inhibit cell proliferation interfere with vecto spread. We propose to test combinations of different prodrug activator RRV to inhibit tumor without affecting vector spread. This is made feasible by our discovery that RRV infected tumor cells can be reinfected in vivo. We have now constructed RRV expressing an optimized Herpes simplex thymidine kinase (HSV-TK) prodrug activator gene (RRV-TK in this proposal). We propose to perform validation studies with the newly developed RRV-TK vector both in vitro and in glioma models in vivo. We will evaluate the potential for dual RRV gene therapy combining RRV-CD and RRV-TK. We will 1) test the transduction efficiency, transgene expression level, replicative stability, and cytotoxic potency of RRV-TK, in vitro and in vivo, and 2) determine the optimal parameters for combination therapy, using both RRV-CD and RRV-TK, in vitro and in glioma models in vivo. The information from these studies will be used to develop future clinical trial protocols and, like combination chemotherapy, offers the potential for synergistic therapeutic benefit and more efficient tumor eradication than single-agent therapy with either vector alone. Furthermore, as the infected tumor cells themselves produce their own chemotherapeutic drugs, the adverse effects of systemic chemotherapy are avoided. PUBLIC HEALTH RELEVANCE: Many researchers have used 'crippled' viruses (termed 'vectors') that can infect and modify tumor cells as cancer therapies. However such therapies have been shown to have only limited beneficial effects, because too many tumor cells never get infected. We have devised a new approach, using replicating viruses to achieve highly efficient gene transfer to tumor cells in a highly selective manner (i.e., the virus is selective fr cancer cells and will not infect normal cells). The infected tumor cell becomes a virus-producing cell, sustaining further infection. Our first vector is being tested in a multi-center clinical tril in patients with brain tumor. This proposal seeks to develop a second version of this type of vector to be used in combination with the first. These two gene therapy vectors cause the cancer cells to produce their own chemotherapy drugs and the second vector potentially enables non-invasive imaging of vector infection of brain tumors. If successful in these preclincial studies, this approach can be translated into the clinic and has the potential to reduce suffering and death in brain cancer patients. It may also be applicable to brain metastases caused by other malignancies.