NOVEL E1 CELL LINE FOR RCA-FREE ADENOVIRAL GENE THERAPY

描述：局部注射β-干扰素可导致 实验性的人类前列腺癌。然而，有效性受到快速反应的限制。 清除蛋白质。Genovo将使用重组腺病毒载体 (RAdIFNbeta)局部传递人β-干扰素基因以产生更多 具有显著的抗肿瘤作用。Rad的商业化生产受到以下限制 增殖型腺病毒(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 作为一种生物“污染物”，需要最大限度地减少/消除。 Genovo将创造一种具有新的E1互补序列的新辅助细胞系， 含有一个异源内含子和在~300nT区域的多个沉默突变 潜在序列与Rad载体重叠，将消除同源 重组，因此是RCA。如果新的E1 辅助细胞系允许生产&gt；103 rAdIFNbeta粒子/cell Rad PFU 在10/10的小规模批次中。辅助细胞将来自人类 具有已知调控和安全特征的二倍体细胞系。 我们建议开发一种用于rAdIFNbeta的制造工艺，该工艺： 1)通过最小化同源重组来消除/显著减少RCA 使用一种新的E1序列在E1辅助细胞和E1缺失的Rad载体之间 建设； 2)避免重新设计当前Rad向量： 3)使用被FDA认可为安全的E1辅助细胞系，该细胞系可以 很容易扩展到10个L生物反应器。 建议的商业应用： Genovo正在使用表达β-干扰素基因的腺病毒载体进行治疗 脑、卵巢和前列腺的局限性恶性肿瘤，目前尚无治疗方法 治愈性疗法。目前用于基因治疗腺病毒载体的生产系统有 40%-60%的批次排斥率由可复制性腺病毒污染引起 (RCA)。拟建的细胞系将消除这种污染，从而提高产量 提高效率，显著降低总体成本。该细胞系将用于生产 腺病毒载体携带干扰素-β作为抗肿瘤药物，从而降低 这些疾病给国家医疗系统带来的总成本。

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.