Superinfection arising in stable lentiviral vector producer cell lines bearing Cocal-G envelope proteins

Conference Dates

June 17-22, 2018


Lentiviral vectors (LV) have been shown to successfully transfer therapeutic genes into dividing and non-dividing cells in laboratory and clinical environments for the benefits of cell and gene therapies. Current LV production chiefly relies on a transient transfection method, wherein HEK 293T cells are transfected with 3-4 plasmids. Such methods have shown batch to batch variability, and increased costs due to the requirements of considerable quantities of cGMP plasmids at clinical stages [1]. This can be circumvented using stable producer cell lines, such as the WinPac cell line, that stably harbor all constructs required for vector production and reliably output LV vectors over long periods of time [2]. However, the commonly pseudotyped LV envelope protein, VSV-G, has difficulty in long term expression and is inactivated by complement [3] and therefore alternatives must be sought. Such alternatives can be found in the Cocal-G envelope protein, which can be expressed long term, is resistant to complement, and bears similarity to VSV-G whereby both derive within the same vesiculovirus genus [4].

A stable LV producer using Cocal-G envelope in the WinPac cell line was produced. Results have illustrated that Cocal-G envelope protein expression leads to superinfection of the LV producing cell line, creating long term instability due to accumulation of the GFP transgene as determined by qPCR. Such superinfection can be prevented by the addition of the non-nucleoside reverse transcriptase inhibitor nevirapine to the cell culture media, leading to protection from superinfection in long term culture. The antiviral can subsequently be removed by buffer exchange in Vivaspin 4 ultrafiltration cassettes (100,000 MWCO), regenerating infectious titre of LV and suggests antiviral addition in upstream production does not negatively impact downstream purification. The cocal enveloped producer cell line was therefore robust enough to be scaled up for large scale LV harvesting as indicated from scaling to a Corning HYPERFlask system.

This work increases our understanding of how LV envelope design may impact superinfection and ultimately specific productivity once cell progress further down a development pathway. In identifying the importance of envelope choice and necessary precautions as a result, work can continue to improve stable LV producers, leading to scalable solutions to address demand for vectors in cell and gene therapies.

[1] Cornetta K, Reeves L, Cross S. Production of Retroviral Vectors for Clinical Use. Methods in Molecular Biology. 2008;:17-32.

[2] Sanber K, Knight S, Stephen S, Bailey R, Escors D, Minshull J et al. Construction of stable packaging cell lines for clinical lentiviral vector production. Scientific Reports. 2015;5(1).

[3] DePolo N, Reed J, Sheridan P, Townsend K, Sauter S, Jolly D et al. VSV-G Pseudotyped Lentiviral Vector Particles Produced in Human Cells Are Inactivated by Human Serum. Molecular Therapy. 2000;2(3):218-222.

[4] Humbert O, Gisch D, Wohlfahrt M, Adams A, Greenberg P, Schmitt T et al. Development of Third-generation Cocal Envelope Producer Cell Lines for Robust Lentiviral Gene Transfer into Hematopoietic Stem Cells and T-cells. Molecular Therapy. 2016;24(7):1237-1246.

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