Chimeric lentiviral vectors for gene therapy- Characterization of stable producer cells

Lentiviral vectors are overtaking gene therapy clinical trials and market, mainly due to their valuable characteristics, distinguishing them from other viral vectors. Bioprocesses relying on stable constitutive production systems are the best option to cope with increased gene therapy market demands. LentiPro26-4070A-mCPGFP, a stable constitutive lentiviral producer cell line, was recently developed using random viral cassette integration. However, alike other stable productions, still presents lower viral yields than transient systems. This work focused on bringing new insights on how lentiviral vector cassettes expression levels shape stable vector production yields and stability. To that end, gene copy number and expression levels of the four vector components were studied using LentiPro26 clones. Furthermore, top producer clones (#54 and #59) viral production and components expression stability over time without antibiotic selective pressure was evaluated. Clones rev and gag/pro/pol cassette expression levels did not restrict physical particles production, since all were able to sustain titers of 109 P.P./mL.day. Incongruent viral genome and functional titers suggested the envelope cassette expression as the main responsible factor of different viral particles functionalization (105-106 T.U./mL.day). Moreover, production stability behaviour was clone dependent. Specifically, clone #54 without antibiotics selective pressure, only decreased envelope expression levels below biological variability, associated to reduced functional titers (2 fold). Clone #59, without antibiotic pressure, decreased all viral components expression, surpassing intrinsic biological variability levels. Although physical particles titers were maintained, similar decline in viral genome (10 fold) and functional titers (8 fold) pointed to transgene cassette as the limiting component hampering viral genome incorporation into physical particles. This work identified the envelope and transgene viral cassettes as preeminent components shaping viral yields. Improved cassette expression strategies are worth to be pursue in search for high quality viral preparations. Overall, the knowledge generated contributes to advance development strategies for constitutive lentiviral vector systems