815. 293 FLEX a New Cell Line for Fast, Reproducible and Efficient Production ofRetroviral Gene Therapy Vectors
Authors: Ana S. Coroadinha, Paula M. Alves, Manuel J.T. Carrondo
Journal: Molecular Therapy (2006)

This work describes the establishment of a pioneering packaging cell line for the production of retroviral Gene Therapy Vectors. The development of this 293 FLEX packaging cell line was performed not only by generating a flexible exchange system for the transgene, but also, taking into account the optimal retroviral components stoichiometry, for the enhancement of the cell-specific productivity.Currently, a retroviral vector producer cell line must be established for the manufacturing of each therapeutic gene. This is performed by transfection of a packaging cell line with the gene of interest. However, the cell productivities are generally low and thus, in order to find a high titer retroviral vector producer clone, exhaustive and time consuming clone screening is necessary.In this work the establishment of a new generation retroviral packaging cell line is reported in which the transgene was expressed using Flp/FRT (Flipase/Flipase Recombination Targets) cassette exchange system, allowing easy exchange of the gene of interest but maintaining the production vector titers. The first step consisted in the tagging of 293 cells with a LacZ reporter gene, which allowed screening and choosing a high expressing chromosomal locus. This strategy, based on the introduction of the transgene in the first step, allowed to eliminate transgene limitations in the production of infectious particles and to maximize the infectious vector productivity by the levels of expression of Gag-Pol and envelope, which were sub-sequentially stably transfected. The ratios of the retroviral components had also significant impact on the proportion of defective particles produced, although no effect was observed on the vector half-life. Thus, a balanced stoichiometry of the viral components have not only shown to improve the cell-productivity but also to increase the quality of vector preparations, by reducing the amount of defective particles.Finally, the use of the Flp/FRT cassette exchange in the retroviral packaging cell line was validated by maintaining the vector titer production while replacing the LacZ by a GFP transgene.The retroviral packaging cell line herein developed is highly advantageous since, after its validation for clinical applications, any therapeutic gene can be used without altering the properties of either the cell line or the vector. Keeping the cell line properties unchanged allows the establishment of a single cost-effective large scale production process for several clinical applications. Keeping the vector properties constant will guarantee reproducibility in clinical protocols, as the vector titer and efficiency of transduction will remain unaltered.Coroadinha, AS; Schucht, R; Gama-Norton, L; Wirth, D; Hauser, H; Carrondo, MJT. The use of recombinase Cassette Exchange in retroviral Vector Producer Cell Lines: predictability and efficiency in transgene replacement. J.Biotechnol. (in press). This work describes the establishment of a pioneering packaging cell line for the production of retroviral Gene Therapy Vectors. The development of this 293 FLEX packaging cell line was performed not only by generating a flexible exchange system for the transgene, but also, taking into account the optimal retroviral components stoichiometry, for the enhancement of the cell-specific productivity. Currently, a retroviral vector producer cell line must be established for the manufacturing of each therapeutic gene. This is performed by transfection of a packaging cell line with the gene of interest. However, the cell productivities are generally low and thus, in order to find a high titer retroviral vector producer clone, exhaustive and time consuming clone screening is necessary. In this work the establishment of a new generation retroviral packaging cell line is reported in which the transgene was expressed using Flp/FRT (Flipase/Flipase Recombination Targets) cassette exchange system, allowing easy exchange of the gene of interest but maintaining the production vector titers. The first step consisted in the tagging of 293 cells with a LacZ reporter gene, which allowed screening and choosing a high expressing chromosomal locus. This strategy, based on the introduction of the transgene in the first step, allowed to eliminate transgene limitations in the production of infectious particles and to maximize the infectious vector productivity by the levels of expression of Gag-Pol and envelope, which were sub-sequentially stably transfected. The ratios of the retroviral components had also significant impact on the proportion of defective particles produced, although no effect was observed on the vector half-life. Thus, a balanced stoichiometry of the viral components have not only shown to improve the cell-productivity but also to increase the quality of vector preparations, by reducing the amount of defective particles. Finally, the use of the Flp/FRT cassette exchange in the retroviral packaging cell line was validated by maintaining the vector titer production while replacing the LacZ by a GFP transgene. The retroviral packaging cell line herein developed is highly advantageous since, after its validation for clinical applications, any therapeutic gene can be used without altering the properties of either the cell line or the vector. Keeping the cell line properties unchanged allows the establishment of a single cost-effective large scale production process for several clinical applications. Keeping the vector properties constant will guarantee reproducibility in clinical protocols, as the vector titer and efficiency of transduction will remain unaltered. Coroadinha, AS; Schucht, R; Gama-Norton, L; Wirth, D; Hauser, H; Carrondo, MJT. The use of recombinase Cassette Exchange in retroviral Vector Producer Cell Lines: predictability and efficiency in transgene replacement. J.Biotechnol. (in press).