Engineering Stable Lentiviral Vector Producer Cells Improves LVV Production

Area of Science:

• Biotechnology
• Molecular Biology
• Gene Therapy Manufacturing

Background:

• Stable lentiviral vector (LVV) producer cell lines are crucial for scalable and cost-effective manufacturing.
• However, intrinsic host-cell limitations often restrict LVV productivity.

Purpose of the Study:

• To identify cellular factors constraining LVV production in stable producer cell lines.
• To assess if targeted host-cell engineering can enhance LVV yield.

Main Methods:

• Comparative transcriptomic analysis of high- and low-producing clones.
• CRISPR/Cas9-mediated gene knockout and overexpression screening in a low-producing clone.
• Functional analysis of candidate genes impacting LVV productivity.

Main Results:

• Transcriptomic analysis revealed differences in pathways like transcription, translation, metabolism, and stress response.
• Single-gene knockouts of H1-2, ADAMTS1, INSIG1, GADD45B, and HSPA1B increased cell-specific LVV productivity up to 2.6-fold.
• Combinatorial gene disruption further boosted productivity, while overexpression did not enhance yield.

Conclusions:

• Transcriptomics-guided host-cell engineering is effective for identifying and alleviating bottlenecks in LVV production.
• Targeted gene knockout strategies can significantly improve vector yield from stable producer cell lines.
• This approach provides a pathway for optimizing scalable lentiviral vector manufacturing.