Proteomic Landscape of Adeno-Associated Virus (AAV)-Producing HEK293 Cells.

Lisa Strasser,Jonathan Bones,Felipe Guapo,Alana Rainbow-Fletcher,Niall Barron,Nicholas Donohue,Stefano Boi

doi:10.3390/ijms222111499

Abstract

Adeno-associated viral (AAV) vectors are widely used for gene therapy, providing treatment for diseases caused by absent or defective genes. Despite the success of gene therapy, AAV manufacturing is still challenging, with production yields being limited. With increased patient demand, improvements in host cell productivity through various engineering strategies will be necessary. Here, we study the host cell proteome of AAV5-producing HEK293 cells using reversed phase nano-liquid chromatography and tandem mass spectrometry (RPLC-MS/MS). Relative label-free quantitation (LFQ) was performed, allowing a comparison of transfected vs. untransfected cells. Gene ontology enrichment and pathway analysis revealed differential expression of proteins involved in fundamental cellular processes such as metabolism, proliferation, and cell death. Furthermore, changes in expression of proteins involved in endocytosis and lysosomal degradation were observed. Our data provides highly valuable insights into cellular mechanisms involved during recombinant AAV production by HEK293 cells, thus potentially enabling further improvements of gene therapy product manufacturing.

Highlights

Gene therapy using adeno-associated viral (AAV) vector delivery is a rapidly evolving field of the biotherapeutics industry
HEK293 cells maintained in suspension were either transfected for AAV5 expression via triple transfection or mock transfected as a negative control
Cells were harvested to determine the AAV viral genome titer using qPCR, and samples were processed for proteomic analysis

Summary

Introduction

Gene therapy using adeno-associated viral (AAV) vector delivery is a rapidly evolving field of the biotherapeutics industry. While the first recombinant AAV product, Glybera, was approved only in 2012, there are over 150 clinical trials involving AAV registered on ClinicalTrials.gov [1]. Despite this clinical activity, production of AAV-based therapeutics faces several significant challenges. Viral particle yield is low, resulting in high manufacturing costs. Zolgensma, which is used for treating spinal muscular atrophy, is still the most expensive biotherapeutic product on the market, with a cost close to USD.

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Results

Conclusion