Effect of Temperature, Medium Composition, and Cell Passage on Production of Herpes-Based Viral Vectors James B. Wechuck, 1 Ali Ozuer, 1 William F. Goins, 2 Darren Wolfe, 2 Thomas Oligino, 2 Joseph C. Glorioso, 2 Mohammad M. Ataai 1 1 Department of Chemical Engineering & Center for Biotechnology and Bioengineering, University of Pittsburgh, 300 Technology Drive, Pittsburgh, Pennsylvania 15219; fax: 412-383-9710; e-mail: Ataai@engrng.pitt.edu 2 Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pensylvania 15261 Received 31 August 2001; accepted 21 February 2002 DOI: 10.1002/bit.10310 Abstract: Our work uses replication-defective genomic herpes simplex virus type-1 (HSV-1)-based vectors to transfer therapeutic genes into cells of the central ner- vous system and other tissues. Obtaining highly puri®ed high-titer vector stocks is one of the major obstacles remaining in the use of these vectors in gene therapy applications. We have examined the effects of tempera- ture and media conditions on the half-life of HSV-1 vectors. The results reveal that HSV stability is 2.5-fold greater at 33°C than at 37°C and is further stabilized at 4°C. Additionally, a signi®cantly higher half-life was measured for the vector in infection culture conditioned serum medium compared to fresh medium with or without serum. Synchronous infections incubated at 33°C produced 2-fold higher amounts of vector than in- fected cells incubated at 37°C, but with a lag of 16±24 h. Vector production yielded 3-fold higher titers and re- mained stable at peak levels for a longer period of time in cultures incubated at 33°C than 37°C. A pronounced negative effect of increased cell passage number on vector yield was observed. Vector production at 33°C yielded similar levels regardless of passage number but was reduced at 37°C as passage number increased. To- gether, these results contribute to improved methods for high-titer HSV vector production. ã 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 79: 112±119, 2002. Keywords: HSV-1; gene therapy; viral vectors; half-life; passage effect INTRODUCTION Delivery of genes to various tissues for the prevention and treatment of a wide range of genetic and acquired diseases is emerging as an important route to thera- peutics (Anderson, 2000; Kay et al., 2001; Somia and Verma, 2000). Viral vectors, which have been used ex- tensively for gene delivery, include retroviral (Cavaz- zana-Calvo et al., 2000; Park et al., 2000), adenoviral (Balague et al., 2000; Rosengart et al., 1999), and adeno- associated virus vectors (Kay et al., 2000; Wagner et al., 1999). More recently, herpes simplex virus-based vectors (Akkarajuetal.,1999;Gossetal.,2001;Ozueretal.,2002; Wolfe et al., 2001) have emerged for several gene therapy applications. Herpes simplex virus type 1 (HSV-1) is a double-stranded linear DNA virus of 152 Kb and its virion diameter is 150±200 nm. HSV-1 structure is comprised of a DNA core surrounded by a capsid and a tegument of viral proteins that connect the capsid to an envelope that possesses at least 10 dierent virus- encoded glycoproteins. Some relevant characteristics of HSV-1 that suggest it will prove useful as a gene delivery vehicle include its large genome, which can accommo- date large (Akkaraju, et al., 1999) or multiple transgenes (Krisky et al., 1998a), ease of gene manipulation (Krisky, et al., 1998a, 1998b), a wide host cell range, short reproductive cycle, rapid spread in culture, a natural tropism for neurons, and the ability to establish a latent or quiescent state in these cells for the life of the host. Replication-defective HSV-based viral vectors deleted for one or several essential immediate-early (IE) regu- latory gene products can be propagated in vitro in complementing cell lines engineered to express the missing essential functions resulting in virus replication (DeLuca et al., 1985) and ultimately leading to lysis of the host cell. Vector-associated cytotoxicity in non- complementing cells is minimized through deactivating the cytotoxic IE genes in the HSV-1 vector (Johnson et al., 1994; Krisky, et al., 1998b; Marconi et al., 1996; Samaniego et al., 1995, 1997, 1998; Wu et al., 1996). The progressive deactivation of the HSV-1 regulatory genes not only progressively reduces the cytotoxicity of the virus, but it also reduces vector yield. For example, the yield achieved with replication-defective vectors deleted for multiple IE genes is only about 0.1% of that obtained Correspondence to: M. M. Ataai Contract grant sponsor: National Science Foundation Contract grant number: DTE9554576 ã 2002 Wiley Periodicals, Inc.