Development of sd-rxRNAs as Therapy for Retinoblastoma and Other Malignancies

描述（由申请人提供）：对视网膜母细胞瘤（一种儿童肿瘤）的研究为癌症生物学提供了许多见解。也许最重要的是，这项工作揭示了癌症可以对肿瘤抑制基因的突变做出反应。肿瘤抑制基因突变占所有癌症突变的约80%，但与癌基因中的激活突变不同，不能被抑制突变基因产物的药物靶向。Cobrinik实验室最近表明，视网膜母细胞瘤的生长不仅由视网膜母细胞瘤蛋白（RB）突变驱动，而且还由视网膜锥前体的“正常”信号通路驱动。该正常回路包括极高表达的鼠双微小癌基因（MDM 2）、V-myc骨髓细胞瘤病病毒相关癌基因（MYCN）、甲状腺激素受体2（THB 2）和视黄酸受体γ（RXRG）核受体。这些基因的表达对视网膜母细胞瘤细胞的生长和存活至关重要，这表明它们可能是有效的治疗靶点，尽管它们在视网膜母细胞瘤中没有遗传改变。MYCN是一个特别有吸引力的发展目标，因为它驱动各种癌症的肿瘤发生，包括神经母细胞瘤，髓母细胞瘤和其他CNS恶性肿瘤。此外，瞬时MYC下调似乎在生物体水平上耐受良好，同时显著抑制肿瘤发生。然而，MYCN蛋白目前被认为是“不可用的”。基于siRNA的药物代表了用于治疗多种疾病（包括癌症和视网膜病症）的新的且潜在重要的治疗范例。由于缺乏有效和无毒的体内递送系统，将这种技术应用于人类疾病的能力受到阻碍。我们已经开发了一类新的疏水修饰的RNAi化合物，“自递送rxRNA”或sd-rxRNATM，其不需要递送载体进入细胞，并且与传统siRNA相比具有改进的药理学。在啮齿动物眼睛中玻璃体内施用sd-rxRNA导致完全视网膜穿透和有效的肿瘤摄取。此外，有效的持久沉默（高达21 单次注射后30天）。该提案的目的是开发用于治疗视网膜母细胞瘤和其他癌症的基于sd-rxRNA的新疗法。我们认为，基于sd-rxRNA的视网膜母细胞瘤治疗是可行的，并且将具有最小的全身毒性，这在儿科患者的情况下特别重要。作为该提案的一部分，将在体内评价通过不同给药途径（玻璃体内和动脉内注射）的sd-rxRNA分布以及接种到眼内的视网膜母细胞瘤细胞肿瘤中的靶mRNA沉默。同时，将鉴定针对MYCN和其他视网膜母细胞瘤靶点（作为备份）的sd-rxRNA化合物，并证实其在体外有效。第二阶段的应用将集中在视网膜母细胞瘤模型中MYCN和这些其他靶点的沉默。将评价沉默对小鼠眼中人肿瘤异种移植物进展的影响。最有效的化合物将进入临床前开发阶段，重点是开发足以支持提交IND的数据包。 视网膜母细胞瘤是眼睛的癌症，通常始于视网膜，主要影响幼儿。目前的治疗方法包括化疗、放疗、激光治疗和可能的手术。我们最近开发了一类新的疏水修饰的RNAi化合物，并在啮齿动物眼中证明了有效和持久的沉默。这项拨款申请的重点是评估d-rxRNA在体内视网膜母细胞瘤模型中沉默基因的适用性。

DESCRIPTION (provided by applicant): The study of retinoblastoma, a childhood tumor, has provided numerous insights into cancer biology. Perhaps most significantly, this work has revealed that cancers can develop in response to mutations in tumor suppressor genes. Tumor suppressor mutations account for ~80% of all cancer mutations, but unlike activating mutations in oncogenes, cannot be targeted by drugs that inhibit a mutant gene product. The Cobrinik lab has recently shown that retinoblastoma growth is driven not only by retinoblastoma protein (RB) mutations, but also by "normal" signaling circuitry of retinal cone precursors. This normal circuitry includes extremely high expression of murine double minute oncogene (MDM2), V-myc myelocytomatosis viral related oncogene (MYCN), thyroid hormone receptor ¿2 (THRB2) and retinoic acid receptor gamma (RXRG) nuclear receptor. The expression of these genes is critical to retinoblastoma cell growth and survival, suggesting that they may be effective therapeutic targets despite the fact that they are not genetically altered in retinoblastoma. MYCN is an especially attractive target for development because it drives tumorigenesis in diverse cancers, including neuroblastoma, medulloblastoma, and other CNS malignancies. Moreover, transient MYC down-regulation appears to be well tolerated at the organismal level, while dramatically suppressing tumorigenesis. However, MYCN proteins are considered to be 'undruggable' at the present time. siRNA-based drugs represent a new and potentially significant therapeutic paradigm for the treatment of multiple diseases including cancer and retinal disorders. The ability to apply this technology to human disease has been impeded by the absence of efficient and non-toxic in vivo delivery systems. We have developed a novel class of hydrophobically modified RNAi compounds, "self-delivering rxRNA" or sd-rxRNATM, that do not require a delivery vehicle to enter cells and have improved pharmacology compared to traditional siRNAs. Intravitreal administration of sd-rxRNA in a rodent eye results in complete retina penetration and efficient tumor uptake. In addition, potent long lasting silencing (up to 21 days post single injection) was demonstrated. The objective of this proposal is to develop novel sd-rxRNA based therapeutics for treatment of retinoblastoma and other cancers. We believe that sd-rxRNA based treatment of retinoblastoma is feasible and will have minimal systemic toxicity which is specifically important in the context of pediatric patients. As part of this proposal, sd-rxRNA distribution via different routes of administration (intravitreal and intra-arterial injection) and target mRNA silencing in retinoblastoma cell tumors seeded into the eye will be evaluated in vivo. In parallel, sd-rxRNA compounds against MYCN and other retinoblastoma targets (as back up) will be identified and confirmed to be efficacious in vitro. A phase II application will focus on silencing of MYCN and these other targets in a retinoblastoma model. The impact of silencing on progression of a human tumor xenograft in mouse eye will be evaluated. The most potent compounds will be moved into preclinical development with a focus on developing a data package sufficient to support filing an IND. PUBLIC HEALTH RELEVANCE: Retinoblastoma is cancer of the eye that usually begins in the retina and primarily affects young children. Current treatments include chemotherapy, radiation therapy, laser therapy and possible surgery. We have recently developed a novel class of hydrophobically modified RNAi compounds and have demonstrated potent and long lasting silencing in a rodent eye. The focus of this grant application is to evaluate the applicability of d-rxRNA to silence genes in a retinoblastoma model in vivo.