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Related Experiment Video

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

L Zehetner1, D Széliová2, B Kraus3

  • 1Department for Analytical Chemistry, University of Vienna, Vienna, 1090, Austria; Doctoral School of Chemistry, University of Vienna, Vienna, 1090, Austria.

Metabolic Engineering
|April 12, 2025
PubMed
Summary

Investigating HEK293 cell strains for Adeno-associated virus (AAV) production revealed that inhibiting hypoxia-inducible factor 1-alpha (HIF-1α) boosts capsid production but impairs genome packaging. This highlights a critical trade-off in optimizing viral vector yield.

Keywords:
AAVBioprocessGSMMHEK293HIF-1α

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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.