LentiPro26: novel stable cell lines for constitutive lentiviral vector production

H A Tomás,A S Coroadinha,A F Rodrigues,M J T Carrondo

doi:10.1038/s41598-018-23593-y

Abstract

Lentiviral vectors (LVs) are excellent tools to promote gene transfer and stable gene expression. Their potential has been already demonstrated in gene therapy clinical trials for the treatment of diverse disorders. For large scale LV production, a stable producer system is desirable since it allows scalable and cost-effective viral productions, with increased reproducibility and safety. However, the development of stable systems has been challenging and time-consuming, being the selection of cells presenting high expression levels of Gag-Pro-Pol polyprotein and the cytotoxicity associated with some viral components, the main limitations. Hereby is described the establishment of a new LV producer cell line using a mutated less active viral protease to overcome potential cytotoxic limitations. The stable transfection of bicistronic expression cassettes with re-initiation of the translation mechanism enabled the generation of LentiPro26 packaging populations supporting high titers. Additionally, by skipping intermediate clone screening steps and performing only one final clone screening, it was possible to save time and generate LentiPro26-A59 cell line, that constitutively produces titers above 106 TU.mL−1.day−1, in less than six months. This work constitutes a step forward towards the development of improved LV producer cell lines, aiming to efficiently supply the clinical expanding gene therapy applications.

Highlights

Lentiviral vectors (LVs) are remarkable tools for gene transfer, both in vivo and in vitro, due to their ability to permanently integrate into the cell genome of dividing and non-dividing cells, sustaining long-term stable expression
The production of LVs has relied on transient co-transfection of HEK293T cells with four expression cassettes: (i) Gag-Pro-Pol, coding for the viral structural proteins and enzymes; (ii) Rev, encoding Rev accessory protein which plays an important role on viral genome nuclear exportation; (iii) Envelope, coding for the glycoproteins that interact with target cell receptors to mediate virus cell entry; and (iv) Vector genome, carrying the gene of interest to be packaged into produced viral particles
The Long Terminal Repeats (LTRs) and packaging (Ψ) sequences of the γ-retroviral vectors (γ-RVs) used in STAR cell line development are present in the genome of the LV producer cells, which could promote the generation of replication competent lentivirus (RCL), raising safety concerns[12]

Summary

Introduction

Lentiviral vectors (LVs) are remarkable tools for gene transfer, both in vivo and in vitro, due to their ability to permanently integrate into the cell genome of dividing and non-dividing cells, sustaining long-term stable expression. The selection of a clone with stable and high expression of Gag-Pro-Pol seems to be the major challenge, being difficult to develop a stable high titer LV producer cell line exclusively by traditional plasmid cell transfection. The RD2-MolPack-Chim[3] LV producer cell line was developed using a recombinant hybrid baculo-AAV vector to successfully integrate the gag-pro-pol and rev genes into the cell genome, avoiding the usage of γ-RVs13. In WinPac derived cell lines development, a different approach used γ-RVs to integrate a reporter expression cassette into the cells genome to identify a clone supporting high reporter expression levels[16] This reporter expression cassette was replaced by a new one containing a codon-optimized LV gag-pro-pol sequence, by means of Cre recombinase mediated cassette exchange (RMCE). Is described an alternative methodology to accelerate the establishment of LV producer cell lines presenting high titers, exclusively by using chemical transfections followed antibiotic selection steps during the entire cell line development process

Methods

Results

Conclusion