State-of-the-art of the production of retroviral vectors

Abstract

Retroviral vectors are still the vectors that are used in the majority of gene therapy trials for treatment of acquired or inherited diseases. In this review, the present state-of-the-art of the production of retroviral vectors and the most important parameters, such as the choice of the producer cell line, stability issues, medium additives, serum, type of bioreactor, that influence production issues is presented and discussed in light of an optimal vector production. The available literature data clearly indicate that, on one hand, the choice of the producer cell line is of utmost importance for obtaining a high level producer cell line, and that, on the other hand, the optimization of the medium, e.g. the replacement of glucose by fructose, has a potential for improving vector production rates and titers. Finally, the use of high-density perfusion culture systems for adherent as well as for suspension cells presents the best choice for a production system, because high cell densities imply high reactor specific production rates, which must be associated with a rapid harvest of the produced vector, thus avoiding vector inactivation due to an extended residence time. The overall optimization of the cultivation and production parameters will have a significant impact on the use of retroviral vectors for gene therapy purposes.

Cell Culture Processes for the Production of Viral Vectors for Gene Therapy Purposes

“…There was a potential to double this volume of higher-titer supernatant by increasing the frequency of harvest. Virus titers eight times greater than T-flask cultures were observed using a TE Fly clone (12.8 · 10 6 IP ml À1 ); however, this result was inferior to titers obtained in fixed bed bioreactors (Merten 2004).…”

“…The performance of the CellCube is reported to be comparable with the Celligen packed bed reactor, with each systems producing an average of 2.1 · 10 7 and 0.55 · 10 7 IP ml À1 , respectively (Merten 2004). The yield from the CellCube was also reported to be comparable with the automated roller bottle system, RollerCell 40 (Wikstro¨m et al 2004).…”

“…To date, the majority of clinical trials have used vectors derived from murine leukemia virus (MLV) (Merten 2004), which belong to the oncovirus sub-family, although some believe that adenovirus is currently the most commonly used virus. As a result, the majority of studies on optimization of culture and process conditions have focused on retroviruses.…”

“…Thus, an increase in total viral yield can only be accomplished by an increase in unit number or longer culture periods. Preliminary data recently published by Merten (2004) have also shown the potential of the Cytopilot; the PG13 packaging cell line was cultured on 150 ml of Cytoline 1 for 336 h and produced a total vector harvest of 1.33 · 10 10 infectious particles. This was comparable with the standard CellCube process.…”

“…The typical characteristics of the main viral vectors are summarized in Table 1. Although vectors derived from adenovirus, adenoassociated virus and herpes virus are receiving more attention in the field of gene therapy (Vos 1995;Parasrampuria 1998;Benihoud et al 1999) recombinant retroviruses are currently used in the majority of clinical trials (Kang et al 2000;Wu and Ataai 2000;Wu et al 2002;Merten 2004). The main limitation of viral vectors is the relatively low harvest from non-optimized processes.…”