NOVEL E1 CELL LINE FOR RCA-FREE ADENOVIRAL GENE THERAPY

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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.