Gutless Adenovirus Mediated Targeted Therapeutics for Glioma

描述（申请人提供）：胶质母细胞瘤是成人中最具破坏性的脑肿瘤，没有一种传统的抗癌疗法成功地延长了患者的寿命。超过80%的胶质瘤原位表达大量突变的IL-13受体（IL13a2R），不同于正常细胞中表达的IL-13受体。一种由IL-13融合到突变假单胞菌外毒素（PE）的嵌合毒素已被证明可以杀死表达IL-13a2R的细胞。PE缺乏内源性蛋白结合域，使其能够与普遍表达的ct2-巨球蛋白受体结合，但保留其抑制蛋白质合成的能力，从而导致细胞死亡。因此，它与IL-13的融合将细胞毒性分子重新靶向表达il13a2r的胶质瘤细胞。IL-13的突变体，比如IL-13。与IL-13相比，E13K （mulL-13）与正常细胞的结合可以忽略不计，而与胶质瘤特异性IL13a2R的亲和力更高。然而，嵌合毒素的全身治疗需要高浓度，可能会引起不良副作用，而由于毒素的半衰期短，肿瘤内治疗需要反复给药。尽管存在这些局限性，但这种方法目前被认为是治疗这种毁灭性疾病最令人兴奋的新方法之一，并在各个临床中心进行的早期临床试验中提供了非常令人鼓舞的结果。我们假设，将表达与PE融合的嵌合蛋白mulL-13的高容量腺病毒载体（HC- Ad）注入人胶质瘤裸鼠瘤内，将导致嵌合毒素在瘤内的有效浓度，杀死人胶质瘤细胞，但保留正常脑组织。HC-Ad载体具有高克隆能力的优势，并且由于其抗原性极低，可以在体内诱导稳定的转基因表达，毒性低。为了提高我们方法的特异性，我们将引入突变IL-4的cDNA （mulL4）到载体中。mulL4作为拮抗剂，结合正常细胞中存在的IL13R/IL4R，但不与胶质瘤细胞中表达的IL13a2R相互作用，也不影响嵌合毒素在GBM细胞中的细胞毒性作用。为了进一步提高这种治疗的安全性，我们将调节TetON系统整合到载体中，允许严格调节治疗性转基因的表达，当需要添加或停用多西环素（DOX）时，通过“打开”和“关闭”它。我们之前的数据提供了强有力的证据，证明HC-Ad载体即使在人体存在系统性抗腺病毒免疫的情况下，也能长期调节嵌合毒素的表达。因此，我们假设提出的靶向胶质瘤基因治疗方法将在没有不良副作用的情况下获得高治疗效果。

DESCRIPTION (provided by applicant): Gliobastoma is the most devastating brain tumor in adults and none of the conventional anticancer therapies has been successful in significantly prolonging the lifespan of the patients. More than 80% of gliomas in situ express high amounts of a mutated IL-13 receptor (IL13a2R), different from the one expressed in normal cells. A chimeric toxin consisting of IL-13 fused to a mutated Pseudomonas exotoxin, (PE) has been shown to kill IL-13a2R expressing cells. PE lacks its endogenous protein binding domain, which allows it to bind to the ubiquitous expressed ct2-macroglobulin receptor, but retains its ability to inhibit protein synthesis, which leads to cell death. Thus, its fusion to IL-13 retargets the cytotoxic molecule to IL13a2R-expressing glioma cells. Mutants of IL-13, such as IL13.E13K (mulL-13), have shown negligible binding to normal cells and higher affinity to the glioma-specific IL13a2R when compared to IL-13. However, the systemic treatment with the chimeric toxin requires high concentrations that can induce adverse side effects while the intratumoral treatment requires repeated administration due to the short half life of the toxin. In spite of these limitations this approach is currently considered one of the most exciting novel treatments for this devastating disease, and has provided very encouraging results in early clinical trials performed at various clinical centers. We hypothesize that intratumoral administration of a high-capacity adenoviral vector (HC- Ad) expressing the chimeric protein mulL-13 fused to PE, to human glioma bearing nude mice, will lead to effective intratumoral concentration of the chimeric toxin, killing human glioma cells but preserving normal brain tissue. HC-Ad vectors have the advantage of high cloning capacity and, due to their minimal antigenicity, they elicit stable transgene expression in vivo with low toxicity. To increase the specificity of our approach, we will introduce into the vector the cDNA of mutated IL-4, (mulL4). mulL4 acts as an antagonist, binding to IL13R/IL4R present in normal cells, but it does not interact with IL13a2R expressed on glioma cells, nor does it affect the cytotoxic effects of the chimeric toxin in GBM cells. To further improve the safety of this treatment we incorporated the regulatory TetON system into the vector that allows tight regulation of expression of the therapeutic transgene, by switching it "on" and "off" as and when needed by the addition or withdrawal of doxycycline (DOX). Our previous data provide strong evidence that HC-Ad vectors provide long term, regulated expression of the chimeric toxin even in the presence of systemic anti-adenoviral immunity as present in humans. Thus, we hypothesize that the targeted glioma gene therapy approach proposed will elicit high therapeutic efficacy in the absence of adverse side effects.