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Inducible HEK293 AAV packaging cell lines expressing Rep proteins.

Lovro Jalšić1,2, Viktoria Lytvyn2, Seyyed Mehdy Elahi2

  • 1Département de Génie Chimique, Université Laval, Québec, QC G1V0A6, Canada.

Molecular Therapy. Methods & Clinical Development
|August 10, 2023
PubMed
Summary

Developing stable producer cell lines for recombinant adeno-associated virus (rAAV) production is challenging. This study successfully created inducible REP-expressing HEK293SF packaging cells, achieving scalable rAAV titers comparable to standard methods.

Keywords:
AAVHEK293Repgene therapyinducible expressionmanufacturingpackaging cellserum-freesuspensiontransfection

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Cell Line Engineering

Background:

  • Scalable production of recombinant adeno-associated virus (rAAV) is crucial for gene therapy applications.
  • Creating stable packaging cell lines for rAAV production is difficult due to the cytostatic Rep proteins.
  • HEK293 cells are a preferred mammalian platform for rAAV production due to constitutive E1A expression.

Purpose of the Study:

  • To develop stable, inducible packaging cell lines for scalable rAAV production.
  • To overcome challenges associated with Rep protein expression in producer cell lines.
  • To establish a robust system for large-scale rAAV manufacturing.

Main Methods:

  • Engineered HEK293SF cells with a cumate/coumermycin-inducible gene expression system.
  • Utilized lentiviral vector transduction to integrate Rep 40 and Rep 68 genes.
  • Employed inducible promoters with varying expression levels for Rep protein control.
  • Cultured cells in suspension, serum-free media for scalability.

Main Results:

  • Generated stable HEK293SF clones expressing inducible Rep proteins.
  • Achieved rAAV titers comparable to standard triple-plasmid transfection methods.
  • Demonstrated clone stability for up to 7 weeks in continuous culture.
  • Validated scalable rAAV production (1L wave bioreactor) using serum-free suspension culture.

Conclusions:

  • Successfully created inducible AAV packaging cell lines using HEK293SF cells.
  • The developed cell lines enable scalable and stable rAAV production.
  • This represents a significant advancement for efficient rAAV manufacturing in gene therapy.