A cell line for high-yield production of E1-deleted adenovirus vectors without the emergence of replication-competent virus.

Production of E1-deleted adenovirus vectors for gene therapy has been plagued by the emergence of replication-competent adenovirus. A number of investigators have minimized homologous sequences between the vector and transfected E1 DNA in an attempt to avoid replication-competent adenovirus. We describe a HeLa-based cell line called GH329 that stably expresses the E1 locus from a promoter derived from the phosphoglycerate kinase gene. Overlap sequences with a standard E1-deleted vector that retains a full pIX transcriptional unit have been eliminated at the 5' end and minimized at the 3' end. The GH329 cell line plaques and produces E1-deleted adenovirus as well as 293 cells. Replication-competent virus has emerged after 5 passages of vector on 293 cells but was not detected after 20 passages on GH329 cells.

High-titer adeno-associated viral vectors from a Rep/Cap cell line and hybrid shuttle virus.

Adeno-associated virus (AAV) is a potential vector for in vivo gene therapy. A critical analysis of its utility has been hampered by methods of production that are inefficient, difficult to scale up, and that often generate substantial quantities of replication-competent AAV. We describe a novel method for producing AAV that addresses these problems. A cell line, called B50, was created by stably transfecting into HeLa cells a rep/cap-containing plasmid utilizing endogenous AAV promoters. Production of AAV occurs in a two-step process. B50 is infected with an adenovirus defective in E2b, to induce Rep and Cap expression and provide helper functions, followed by a hybrid virus in which the AAV vector is cloned in the E1 region of a replication-defective adenovirus. This results in a 100-fold amplification and rescue of the AAV genome, leading to a high yield of recombinant AAV that is free of replication-competent AAV. Intramuscular injection of vector encoding erythropoietin into skeletal muscle of mice resulted in supraphysiologic levels of hormone in serum that was sustained and caused polycythemia. This method of AAV production should be useful in scaling up for studies in large animals, including humans.