Two mammalian gene products, PC2 and PC3, have been proposed as candidate neuroendocrineprecursor processing enzymes based on the structural similarity of their catalytic domains to that of the yeast precursorprocessing endoprotease Kex2. In this report we demonstrate that these two proteases can cleave proopiomelanocortin (POMC) in the secretory pathway of mammalian cells. Similarly to pituitary corticotrophs, PC3 expressed in processingdeficient BSC-40 cells cleaved native mouse POMC at the -Lys-Arg-sites flanking corticotropin. The -Lys-Arg-within 13-lipotropin was less efficiently cleaved to release f-endorphin.Expression of PC2 together with PC3 resulted in efficient conversion of i-lipotropin, as occurs in pituitary melanotrophs. Furthermore, coexpression of PC2 together with mouse POMC in bovine adrenomedullary chromaffin cells resulted in conversion of j3-lipotropin to y-lipotropin and 8-endorphin in the regulated secretory pathway. Finally, the processing selectivities ofPC3 and PC2 expressed together in BSC-40 cells were determined by using a series of mutant mouse POMCs containing all possible pairs of basic residues at certain sites. The observed pattern of cleavage site selectivities mimicked that of the endogenous endoproteases of the insulinoma and bovine adrenomedullary chromaffmn cells, suggesting that PC2 and PC3 may represent important core endoproteases in the catalysis of prohormone processing in many neuroendocrine cell types.

Mammalian cell lines (BSC-40, NG108-15, and GH4C1) that cannot process the murine neuroendocrine peptide precursor prepro-opiomelanocortin (mPOMC) when its synthesis is directed by a vaccinia virus vector were coinfected with a second recombinant vaccinia virus carrying the yeast KEX2 gene, which encodes an endopeptidase that cleaves at pairs of basic amino acid residues. mPOMC was cleaved intracellularly to a set of product peptides normally found in vivo, including mature gamma-lipotropin and beta-endorphin1-31. In GH4C1 cells (a rat pituitary line), product peptides were incorporated into stored secretory granules. These results suggest that the inability of any particular cell line to process a prohormone precursor is due to the absence of a suitable endogenous processing enzyme.

A commercial rAAV manufacturing process needs to provide a safe product at high yield, be easily scalable, regulatory-compliant, and have reasonable cost of goods. Considerations for process development include not only product quantity and quality, but also ease of obtaining equipment, performing validation, and demonstrating control. In these regards, it is usually efficient to make use of proven technologies for more established areas of manufacturing, such as cell culture and purification methods used by the recombinant protein/monoclonal antibody industry. We have focused on stable mammalian producer cell lines with adenovirus type 5 helper virus as a means of achieving these goals. This review describes our current approach for generating producer cell clones and designing a scalable, regulatory-compliant vector production and purification process that addresses any product safety concerns relating to helper virus. To date, a producer cell line-based manufacturing process has been implemented at the 250-liter production scale, with no foreseeable impediments to scaling up to commercial vector manufacturing in 2000-liter bioreactors or larger.

No abstract