HIGH-TITER ADENOASSOCIATED VIRAL VECTORS FROM A REP CAP CELL-LINE ANDHYBRID SHUTTLE VIRUS/

Adeno-associated virus (AAV) is a potential vector for in vivo gene th erapy. A critical analysis of its utility has been hampered by methods of production that are inefficient, difficult to scale up, and that o ften generate substantial quantities of replication-competent AAV, We describe a novel method for producing AAV that addresses these problem s, A cell line, called B50, was created by stably transfecting into He La cells a rep/cap-containing plasmid utilizing endogenous AAV promote rs, Production of AAV occurs in a two-step process. B50 is infected wi th an adenovirus defective in E2b, to induce Rep and Cap expression an d provide helper functions, followed by a hybrid virus in which the AA V vector is cloned in the El region of a replication-defective adenovi rus. This results in a 100-fold amplification and rescue of the AAV ge nome, leading to a high yield of recombinant AAV that is free of repli cation-competent AAV, Intramuscular injection of vector encoding eryth ropoietin into skeletal muscle of mice resulted in supraphysiologic le vels of hormone in serum that was sustained and caused polycythemia, T his method of AAV production should be useful in scaling up for studie s in large animals, including humans.