PO.ET04.01 · 实验与分子治疗
克服半复制型逆转录病毒载体中的重组：一种用于胶质母细胞瘤的新型双自杀基因疗法
Overcoming recombination in semi-replicating retroviral vectors: A novel double suicide gene therapy for glioblastoma
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摘要 Abstract
中文摘要
半复制型逆转录病毒载体（sRRV）系统开发中长期存在的挑战之一，是在分裂的肿瘤细胞中逆转录期间由于非同源重组导致治疗基因丢失。为解决这一问题，我们开发了一种经遗传稳定化的sRRV平台，能够进行高效的组合基因治疗，在仅在肿瘤内复制和扩散的同时保持多个治疗基因的完整性。我们的系统由两个反式互补、复制缺陷的逆转录病毒载体组成：一个编码MuLV-Gag-Pol和胞嘧啶脱氨酶（CD），另一个编码GaLV-Env和HSV1-胸苷激酶（TK）。这些载体共感染肿瘤细胞，并在肿瘤微环境内选择性增殖。通过消除载体之间的同源序列，我们实现了抗重组设计，确保在连续复制过程中长期遗传稳定性。该平台的抗肿瘤疗效在两种胶质母细胞瘤模型中得到验证。首先，在同基因大鼠原位胶质瘤模型中，将C6胶质瘤细胞颅内植入Wistar大鼠。将治疗性载体直接注射至肿瘤部位，随后全身给予前体药物5-氟胞嘧啶（5-FC）和更昔洛韦（GCV）。第98天的组织学分析显示剂量依赖性肿瘤消退，高剂量双前药队列中肿瘤完全根除。生存分析证实治疗组生存期显著延长。其次，在使用无胸腺裸鼠的颅内异种移植模型中，将人胶质瘤细胞植入脑实质，随后立体定向递送sRRV载体并全身给予前药治疗。值得注意的是，尽管缺乏适应性免疫，联合治疗组再次观察到肿瘤完全缓解，凸显了该平台的内在疗效。对从肿瘤组织回收的载体基因组分析显示无重组或基因丢失的证据，证实了系统稳健的遗传稳定性。载体维持了两个治疗基因的持续共表达，这对于协同前药激活和肿瘤细胞杀伤至关重要。总之，本研究提出了一种新一代sRRV平台，解决了一个重大技术障碍——重组介导的基因丢失——同时实现了多个治疗基因的稳定且肿瘤选择性递送。它在免疫功能正常和免疫缺陷脑肿瘤模型（包括颅内异种移植）中的强效疗效，使其成为胶质母细胞瘤及其他难治性实体瘤临床转化的有前景候选者。
查看英文原文 English abstract
One of the longstanding challenges in developing semi-replicating retroviral vector (sRRV) systems has been the loss of therapeutic genes due to non-homologous recombination during reverse transcription in dividing tumor cells. To address this, we developed a genetically stabilized sRRV platform capable of efficient combinatorial gene therapy, preserving the integrity of multiple therapeutic genes while replicating and spreading exclusively within tumors. Our system consists of two trans-complementing, replication-defective retroviral vectors: one encoding MuLV-Gag-Pol and cytosine deaminase (CD), and the other encoding GaLV-Env and HSV1-thymidine kinase (TK). These vectors co-infect tumor cells and propagate selectively within the tumor microenvironment. By eliminating homologous sequences between vectors, we achieved a recombination-resistant design, ensuring long-term genetic stability during serial replication. The antitumor efficacy of this platform was validated in two glioblastoma models. First, in a syngeneic rat orthotopic glioma model, C6 glioma cells were implanted intracranially into Wistar rats.The therapeutic vectors were directly injected into the tumor site, followed by systemic administration of prodrugs 5-fluorocytosine (5-FC) and ganciclovir (GCV). Histological analysis on day 98 revealed dose-dependent tumor regression, with complete tumor eradication in the high-dose, dual-prodrug cohort. Survival analysis confirmed significantly extended survival in treated groups. Second, in an intracranial xenograft model using athymic nude mice, human glioma cells were implanted in the brain parenchyma, followed by stereotactic delivery of the sRRV vectors and systemic prodrug treatment. Remarkably, complete tumor remission was again observed in the combination treatment group, despite the absence of adaptive immunity, highlighting the platform's intrinsic efficacy. Analysis of vector genomes recovered from tumor tissues revealed no evidence of recombination or gene loss, confirming the system's robust genetic stability. The vectors maintained persistent coexpression of both therapeutic genes, which are essential for synergistic prodrug activation and tumor cell killing. In conclusion, this study presents a next-generation sRRV platform that resolves a major technical barrier-recombination-mediated gene loss-while enabling stable and tumor-selective delivery of multiple therapeutic genes. Its potent efficacy in both immunocompetent and immunodeficient brain tumor models, including intracranial xenografts, positions it as a promising candidate for clinical translation in glioblastoma and other refractory solid tumors.
利益披露 Disclosure
S. Kim,
Articure Employment, Soojin Kim is an employee of Articure.
M. Kang,
Articure Employment, Moonkyung Kang is an employee of Articure.
Y. Kim,
Articure g., Board of Directors, non-salaried role), Stock, As a professor, I founded artiCure, a university holding company.