Lentivector Producer Cell Lines with Stably Expressed Vesiculovirus Envelopes

Area of Science:

• * Virology
• * Molecular Biology
• * Gene Therapy

Background:

• * Lentiviral vectors (LVs) commonly utilize the vesicular stomatitis virus Indiana strain G protein (VSVind.G) for entry.
• * Cytotoxicity of VSVind.G has hindered the development of stable LV producer cell lines and scalable production.

Purpose of the Study:

• * To investigate alternative vesiculovirus G proteins for stable lentivector production.
• * To overcome limitations associated with VSVind.G in LV manufacturing.
• * To enhance the robustness and stability of lentiviral particles.

Main Methods:

• * Constitutive expression of VSVind.G, VSV New Jersey strain G protein (VSVnj), Cocal virus G protein (COCV.G), and Piry virus G protein (PIRYV.G) in producer cells.
• * Assessment of lentivector production, particle stability, and resistance to complement inactivation.
• * Generation of chimeric G proteins to map complement inactivation determinants.
• * Evaluation of strategies to prevent vector superinfection and reconstitution methods.

Main Results:

• * Constitutive expression of VSVind.G, VSVnj.G, COCV.G, and PIRYV.G supported LV production for up to 10 weeks.
• * All G-enveloped lentiviral particles demonstrated robustness, allowing concentration and freeze-thawing.
• * COCV.G and PIRYV.G exhibited resistance to complement inactivation, with a mapped determinant in VSVind.G.
• * Vector superinfection was observed but preventable with nevirapine; reconstitution by mixing supernatants was successful.

Conclusions:

• * Alternative G proteins offer a viable strategy for stable lentivector producer cell lines.
• * Robust and complement-resistant lentiviral particles can be generated, improving manufacturing and therapeutic potential.
• * Nevirapine treatment or supernatant mixing are effective countermeasures for LV production challenges.