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 的局部递送会导致 实验性人类前列腺肿瘤。然而，其有效性受到快速的限制 蛋白质的清除。 Genovo 将使用重组腺病毒载体 (rAdIFNbeta) 局部递送人类 β-干扰素基因以产生更多 显着的抗肿瘤作用。 rAd 的商业生产受到以下因素的限制 40-60% 的 rAd 批次中存在具有复制能力的腺病毒 (RCA)。 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”予以最小化/消除。 Genovo 将创建一种具有新颖 E1 互补序列的新辅助细胞系， 含有一个异源内含子以及在约 300 nt 区域的多个沉默突变 与 rAd 载体潜在的序列重叠，这将消除同源 重组，因此 RCA。如果新的E1 辅助细胞系可产生 > 103 个 rAdIFNbeta 颗粒/细胞 rAd pfu 10/10 小规模批次。辅助细胞将来自人类 具有已知监管和安全特征的二倍体细胞系。 我们建议开发 rAdIFNbeta 的制造工艺： 1) 通过最小化同源重组消除/显着减少 RCA 使用新的 E1 序列在 E1 辅助细胞和 E1 删除的 rAd 载体之间进行分析 构造; 2) 避免重新设计当前的rAd向量： 3) 使用被 FDA 认定为安全的 E1 辅助细胞系，可 易于扩展到 >10 L 生物反应器。 拟议的商业应用： Genovo 正在使用表达 β-干扰素基因的腺病毒载体进行治疗 脑部、卵巢和前列腺的局部恶性肿瘤，目前尚无治疗方法 治疗方法。 目前用于基因治疗腺病毒载体的生产系统 由于具有复制能力的腺病毒污染，批次废品率为 40-60% （美国无线电公司）。 拟议的细胞系将消除这种污染，从而提高产量 效率并显着降低总体成本。 该细胞系将用于生产 腺病毒载体可传递 INF-β 作为抗肿瘤药物，从而降低 这些疾病给国家医疗保健系统带来的总体成本。