GALV-Based Retroviral Replicating Vectors for Glioma Gene Therapy

多形性胶质母细胞瘤(GBM)是成人最常见的原发脑肿瘤，预后很差。 非复制型逆转录病毒载体用于GBM基因治疗的临床试验表明，基因转移不足， 没有治疗上的好处。因此，我们开发了一种独特的逆转录病毒复制载体(RRV)策略，用于 更有效、更有肿瘤选择性地传递前药物激活剂(‘自杀’)基因，而不会扩散到正常组织。 在颅内胶质瘤模型中，这使得抗癌药物能够从系统的 使用无毒前药，实现长期存活，没有全身骨髓毒性。由先前版本支持 NINDS U01资助，表达酵母胞嘧啶脱氨酶自杀基因(RRV-CD，TocA)的优化RRV 511‘)被开发出来，第一个人类复发性高级别胶质瘤的I期试验已经显示出放射学 与同期的外部对照相比，完全应答和显著提高的存活率， 导致FDA被指定为“突破性治疗”，以及目前正在进行的关键的第三阶段试验。 理想情况下，这种方法可以通过使用RRV联合传递2个或更多自杀基因来增强， 类似于联合化疗，但在肿瘤内部产生，没有全身骨髓毒性。 由于病毒包装的容量限制了我们每个病毒只能有一个自杀基因，我们开发了第二种RRV递送 另一种自杀基因，单纯疱疹病毒胸苷激酶(TK)，可以激活抗疱疹前药，如 更昔洛韦通过磷酸化作用，作为超抗原发挥作用。同样，作为RRV，包裹着相同的信封 竞争结合相同的细胞表面受体，我们用长臂猿白血病病毒包被RRV-TK (Galv)包络，以避免与当前临床载体RRV-CD的此类干扰，并允许联合使用 这两种病毒都有。然而，由于Galv包被的RRV可能显示不想要的造血细胞转导， 在这里，我们建议进行研究，以验证这种新的RRV的安全性和有效性。因为Galv信封绑定了 细胞受体在人和大鼠(而不是小鼠)细胞上的保守性，用于评估颅内肿瘤模型 肿瘤的转导、生物分布和遗传毒性，我们建议建立(A)人脑胶质瘤异种移植瘤 在人源化造血的免疫缺陷小鼠中，以及(B)免疫活性大鼠中的同基因胶质瘤。 为了减轻潜在的遗传毒性，我们还建议评估一种通过掺入来限制RRV的新策略。 已知在造血细胞中高度丰富的microRNA miR-142-3p的靶序列。 在R21阶段，我们将首先检查新开发的Galv-2的复制效率和选择性。 体外脑胶质瘤与造血细胞的RRV包衣实验(Aim 1)，并对上述体内模型进行优化(Aim 2)。 在R33期，我们将使用这些模型来评估肿瘤的选择性和转导效率 GalV包被的RRV(Aim 1)，并评价其对自杀基因治疗和抗肿瘤激活的作用 豁免(目标2)。如果成功，我们将为Galv包衣的进一步临床翻译建立一个主要候选者 RRV-TK作为单独的代理和/或通过Create计划与RRV-CD结合。

Glioblastoma multiforme (GBM), the most common primary brain tumor in adults, carries a dismal prognosis. Clinical trials with non-replicating retroviral vectors for gene therapy of GBM showed inadequate gene transfer, without therapeutic benefit. Hence we developed a unique strategy with retroviral replicating vectors (RRV) for more efficient & tumor-selective delivery of prodrug activator (`suicide') genes, without spread to normal tissues. In intracranial glioma models, this enabled tumor-localized activation of an anti-cancer drug from a systemically administered non-toxic prodrug, achieving long-term survival without systemic myelotoxicity. Supported by prior NINDS U01 funding, an optimized RRV expressing the yeast cytosine deaminase suicide gene (RRV-CD, `Toca 511') was developed, and first-in-human Phase I trials for recurrent high-grade glioma have shown radiographic complete responses and significantly increased survival compared to contemporaneous external controls, leading to FDA `Breakthrough Therapy' designation, and a pivotal Phase III trial that is now underway. Ideally, this approach may be augmented by using RRV to deliver 2 or more suicide genes in combination, analogous to combination chemotherapy, but generated within the tumor itself, without systemic myelotoxicity. As viral packaging capacity limits us to a single suicide gene per virus, we developed a second RRV delivering another suicide gene, Herpes simplex thymidine kinase (TK), which activates anti-herpetic prodrugs such as ganciclovir by phosphorylation, and acts as a super-antigen. Also, as RRV encoated with the same envelope compete for binding to the same cell surface receptors, we encoated RRV-TK with Gibbon ape leukemia virus (GALV) envelope to avoid such interference with the current clinical vector RRV-CD, and enable combined use of both viruses. However, since GALV-encoated RRV may show unwanted transduction of hematopoietic cells, here we propose studies to validate the safety and efficacy of this new RRV. As the GALV envelope binds a cellular receptor conserved on human and rat (but not mouse) cells, for intracranial tumor models to evaluate tumor transduction, biodistribution, and genotoxicity, we propose to establish both (a) human glioma xenografts in immunodeficient mice with humanized hematopoiesis, and (b) syngeneic gliomas in immunocompetent rats. To mitigate potential genotoxicity, we also propose to evaluate a novel strategy to restrict RRV by incorporation of the target sequence for microRNA miR-142-3p, which is known to be highly abundant in hematopoietic cells. In the R21 Phase, we will first examine replicative efficiency and selectivity of the newly developed GALV- encoated RRV in glioma vs. hematopoietic cells in vitro (Aim 1), and optimize the above in vivo models (Aim 2). In the R33 Phase, we will employ these models to evaluate tumor selectivity and transduction efficiency of GALV-encoated RRV (Aim 1), and to evaluate its efficacy for suicide gene therapy and activation of anti-tumor immunity (Aim 2). If successful, we will establish a lead candidate for further clinical translation of GALV-encoated RRV-TK, as a single agent by itself and/or combined with RRV-CD, through the CREATE Program.