Using chemical transfection, iBET scientists have developed a scalable and constitutive lentiviral production method to meet the clinically expanding demand for lentiviral vectors in gene therapy applications.
Persistent gene expression in both diving and non-dividing cells, larger packaging capacity and lower immunogenicity have all made lentiviral (LV) vector a favourable choice for use in gene therapy. Tisagenlecleucel (marketed as Kymriah™), the first FDA approved gene therapy treatment uses LV vectors to modify T cells for treating acute lymphoblastic leukaemia. Growing interest in the use of these vectors for gene therapy has led to an increase in the demand for their scalable production. However, scaling up LV vector production still faces several challenges, hindering its application clinically.
Transient co-transfection of HEK293T cells using four plasmids – Gag-Pro-Pol, Rev, Envelope and vector genome carrying the gene of interest- was the traditional method used for LV production. However, the approach was costly, highly variable between batches and difficult to scale-up, thus promoting the generation of stable LV cell lines constitutively producing high titer LVs. To date, three stable cell lines have been reported constitutively expressing all the LV components. But these stable cell lines also present several challenges including, the need to do several transductions/transfections and selection steps and the difficulty in finding the best LV producer clones.
In the present study published in Scientific Reports, Dr Ana Sofia Coroadinha and team at Portugal’s Instituto de Biologia Experimental e Tecnológica (iBET) and Universidade Nova de Lisboa developed an alternative methodology to accelerate the generation of LV producer cell lines. The approach used polyethylenimine to trasfect HEK293T cells with 3rd generation viral plasmids-gag-pro-pol/rev split cassette approach and maintaining the self-inactivating design of the vector genome. To generate more robust cell lines and minimize potential cytotoxicity problems associated to the viral protease activity, a Gag-Pro-Pol construct harbouring the less active T26S mutated protease was used. The non-toxic MLV amphotropic envelope was used instead of the traditional VSV-G. Antibiotic selection steps was used during the entire cell line development process and the resulting LentiPro26-derived LV producer stable cell line resulted in an yield of 106 TU.mL−1.day−1.
LentiPro26-A59 cell line showed a stable LV production for nearly one month in the absence of antibiotics and for at least two months in the presence of antibiotics. These results show the reproducibility and scalability of the LentiPro26-A59 cell line, evidencing its potential to be adapted to continuous large-scale production systems.
Through further optimization to increase productivity and safety, the team is hopeful that LentiPro26 could be a valuable addition to the LV vector toolkit to supply its demand in gene therapy clinical applications.
Source: Tomas HA et al., LentiPro26: novel stable cell lines for constitutive lentiviral vector production. Scientific Reports March 2018. DOI