Optimized large-scale production of high titer lentivirus vector pseudotypes
Introduction
Lentivirus vectors have become an extremely valuable tool for in vivo gene delivery because they can stably transduce dividing and non-dividing cells by integrating into the host genome. In addition, these vectors are non-toxic and minimally immunogenic since no viral genes are encoded in the vector genome. As a result, it has been possible to obtain long-term gene expression in many different tissues and organs. Another important characteristic of lentiviruses is that they form a number of different pseudotypes by incorporating into the vector particles envelopes derived from other viruses. Recently, it has become apparent that certain envelopes, when compared to VSV-G, can dramatically enhance their ability to transduce certain organs (Duisit et al., 2002, Kang et al., 2002, Kobinger et al., 2001, MacKenzie et al., 2002), subsets of cells in a tissue (Desmaris et al., 2001, Duisit et al., 2002, Kang et al., 2002, Watson et al., 2002, Wong et al., 2004), or impart the vector with neural retrograde transport properties (Mazarakis et al., 2001). Lentivirus vectors can also be selectively targeted to certain cells or organs by using chimeric envelopes that display peptides recognized by cell or tissue-specific surface receptors (Peng et al., 2001, Verhoeyen et al., 2003). Testing this ever-increasing variety of natural and chimeric envelopes in vivo requires high titer lentivirus stocks. This can only be accomplished in a relatively short time for such a large variety of envelopes by transient transfection of 293T cells, which is the fastest and most efficient option for this purpose. Although several stable lentivirus packaging cell lines have been developed over the past few years, most have been designed to generate VSV-G pseudotypes and the efficiency of vector production, defined as the number of transducing units (tu)/mL of unconcentrated supernatant, is still lower than that obtained by transient transfection. Thus, most experiments with lentivirus vectors have used transient transfection of 293T cells to produce lentivirus vector stocks.
The goal of the present study was to optimize the transient transfection of 293T cells and concentration procedure to develop an efficient method for large-scale production of high titer vector stocks of lentivirus vectors pseudotyped with the VSV-G, Mokola, Rabies, MLV-Ampho, MLV-10A1, LCMV-WE, or LCMV-Arm53b envelopes.
Section snippets
Materials and methods
293T cells generated by Dr. Michele P. Calos (Department of Genetics, Stanford University School of Medicine, Stanford, CA) were obtained from the American Type Culture Collection (Rockville, MD). The cells were grown in Dulbecco's Modified Eagle Medium (DMEM) (Life Technologies/Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 100 units/mL penicillin/100 μg/mL streptomycin (Life Technologies/Invitrogen) at 37 °C in a 5% CO2 humidified atmosphere.
Effect of different envelope expression vectors on titers
The ability to produce different pseudotypes was tested initially by transfection of 293T cells plated in 60 mm dishes. The envelope genes were expressed from the original expression vectors in which they where obtained (VSV-G, Mokola, Rabies, MLV-Ampho, MLV-10A1) or newly cloned into (LCMV-WE and LCMV-Am53b) (Fig. 1). The titers obtained for the VSV-G (1.3 × 108 ± 0.6 × 108 tu/mL), Mokola (3.0 × 106 ± 0.8 × 106 tu/mL), and Rabies (1.3 × 106 ± 0.7 × 106 tu/mL) pseudotypes were comparable to previously published
Discussion
The optimized transfection and production parameters established in this study consistently yielded concentrated lentivirus vector stocks with titers above 1 × 109 tu/mL for most pseudotypes, and higher than 1 × 1010 tu/mL for VSV-G. Also, the unconcentrated VSV-G pseudotyped lentivirus vector titers were 10–100-fold higher than previously reported (Karolewski et al., 2003, Reiser, 2000, Scherr et al., 2002, Zufferey and Trono, 2000). Thus, the large-scale method described here is well suited for
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