NOVEL E1 CELL LINE FOR RCA-FREE ADENOVIRAL GENE THERAPY

用于无 RCA 腺病毒基因治疗的新型 E1 细胞系

• 批准号: 6211901
• 负责人: RICHARD W PELUSO
• 金额: $ 8.48万
• 依托单位: GENOVO, INC.
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-04 至 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6211901
• 关键词: Adenoviridae; biotechnology; cell line; complementary DNA; gene delivery system; gene therapy; interferon beta; introns; nucleic acid sequence; technology /technique development; transfection /expression vector; virus replication

DESCRIPTION: Local delivery of interferon-beta causes regression of experimental human prostate tumors. However, effectiveness is limited by rapid clearance of the protein. Genovo will use a recombinant adenoviral vector (rAdIFNbeta) to deliver the human beta-interferon gene locally to produce more substantial anti-tumor effects. Commercial production of rAd is limited by replication competent adenovirus (RCA) present in 40-60 percent of rAd lots. Although the degree of risk associated with RCA is unknown, regulators view it as a biological "contaminant" to be minimized/eliminated. Genovo will create a new helper cell line with a novel E1 complementing sequence, containing a heterologous intron plus multiple silent mutations in the ~300 nt region of potential sequence overlap with rAd vectors which will eliminate homologous recombination and therefore RCA. Feasibility will be demonstrated if the new E1 helper cell line allows production of > 103 rAdIFNbeta particles/cell rAd pfu in 10/10 small scale lots. The helper cells will be derived from a human diploid cell line with a known regulatory and safety profile. We propose to develop a manufacturing process for rAdIFNbeta that: 1) eliminates /substantially reduces RCA by minimizing homologous recombination between E1 helper cells and E1-deleted rAd vectors using a novel E1 sequence construct; 2) avoids re-engineering current rAd vectors: 3) utilizes an E1 helper cell line, recognized as safe by the FDA, which can be readily scaled to >10 L bioreactors. PROPOSED COMMERCIAL APPLICATION: Genovo is using adenoviral vectors expressing the beta-interferon gene for the treatment of localized malignant tumors of the brain, ovaries, and prostate, for which there are no curative remedies. Current production systems for gene therapy adenoviral vectors have batch rejection rates of 40-60% due to contamination by replication competent adenovirus (RCA). The proposed cell line will eliminate this contamination, thereby increasing production efficiency and significantly decreasing overall cost. This cell line will be used to produce adenoviral vectors to deliver INF-beta as an anti-tumor drug and thereby decrease the overall cost of these diseases to the national healthcare system.

描述：干扰素-β的局部递送导致 实验性人类前列腺肿瘤然而，有效性受到快速 清除蛋白质。Genovo将使用重组腺病毒载体 （rAdIFN β）在局部递送人β-干扰素基因， 显著的抗肿瘤作用。rAd的商业生产受到以下因素的限制： 有复制能力的腺病毒（RCA）存在于40- 60%的rAd批次中。 尽管与RCA相关的风险程度尚不清楚，但监管机构认为， 作为生物“污染物”被最小化/消除。 Genovo将创建一个新的辅助细胞系，具有新的E1互补序列， 含有一个异源内含子加上多个沉默突变的~300 nt区域， 与rAd载体的潜在序列重叠，这将消除同源 重组，因此RCA。可行性将得到证明，如果新E1 辅助细胞系允许产生> 103 rAdIFN β颗粒/细胞rAd pfu 在10/10的小规模批次中。辅助细胞将来自人类 具有已知的监管和安全性特征的二倍体细胞系。 我们建议开发rAdIFN β的生产工艺，该工艺： 1)通过最小化同源重组消除/显著降低RCA 使用新的E1序列在E1辅助细胞和E1缺失的rAd载体之间进行比较 建设; 2)避免重新设计当前的rAd载体： 3)利用E1辅助细胞系，被FDA认为是安全的， 易于放大至>10 L生物反应器。 拟定商业应用： Genovo正在使用表达β-干扰素基因的腺病毒载体进行治疗 脑、卵巢和前列腺的局部恶性肿瘤， 治疗药物 目前用于基因治疗的腺病毒载体的生产系统 由于可复制腺病毒的污染，批次拒收率为40- 60 （RCA）。 拟议的细胞系将消除这种污染，从而提高产量 效率和显著降低整体成本。 该细胞系将用于生产 腺病毒载体来递送作为抗肿瘤药物的INF-β，从而减少肿瘤细胞的增殖。 这些疾病对国家医疗保健系统的总成本。