Multi-omics driven genome-scale metabolic modeling improves viral vector yield in HEK293.

Area of Science:

• Biotechnology
• Molecular Biology
• Cell Biology

Background:

• HEK293 cells are crucial for producing recombinant proteins and viral vectors like Adeno-associated virus (AAV).
• Optimizing AAV production yields remains a challenge despite high transfection efficiency of HEK293 cells.
• Understanding cellular and metabolic responses during AAV production is key to improving yields.

Purpose of the Study:

• To conduct a multi-omics analysis of high-producing (HP) and low-producing (LP) HEK293 strains during AAV production.
• To identify metabolic bottlenecks and cellular responses affecting AAV yield.
• To investigate the role of pseudohypoxia and hypoxia-inducible factor 1-alpha (HIF-1α) in AAV productivity.

Main Methods:

• Multi-omics profiling including lipidomics, exometabolomics, and transcriptomics.
• Reconstruction of genome-scale metabolic models (GSMMs) for HEK293 strains.
• Pharmacological inhibition of HIF-1α to assess its impact on AAV production.

Main Results:

• Significant metabolic and cellular differences were observed between HP and LP HEK293 strains, with LP strains exhibiting pseudohypoxia.
• Hypoxia-inducible factor 1-alpha (HIF-1α) was identified as a critical regulator in LP strains, correlating with reduced AAV productivity.
• HIF-1α inhibition increased viral capsid production by 2.5-fold but decreased viral genome numbers, negatively impacting the full-to-empty particle ratio.
• HIF-1α inhibition was linked to impaired nucleotide synthesis via the pentose phosphate pathway (PPP).

Conclusions:

• Pseudohypoxia, regulated by HIF-1α, is a significant factor limiting AAV production in certain HEK293 strains.
• Inhibiting HIF-1α presents a trade-off: enhanced capsid assembly versus compromised genome packaging due to reduced nucleotide synthesis.
• Targeting HIF-1α offers a potential strategy for AAV production enhancement, but requires careful balancing of capsid formation and genome replication for optimal functional vector yield.