Conclusion: MAESTRO software was very efficient in detecting single point mutations that increase or reduce fold-stability. Thermal stability correlated well with the speed of proteolytic degradation and presentation of peptides on the surface of dendritic cells in vitro. This change in processing kinetics significantly influenced the polarization of T cell responses in vivo. Modulating the fold-stability of proteins thus has the potential to optimize and polarize immune responses, which opens the door to more efficient design of molecular vaccines.
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.
Adeno-associated
virus (AAV)-based gene therapy is a rapidly developing
field, requiring analytical methods for detailed product characterization.
One important quality attribute of AAV products that requires monitoring
is the amount of residual empty capsids following downstream processing.
Traditionally, empty and full particles are quantified via analytical
ultracentrifugation as well as anion exchange chromatography using
ultraviolet or fluorescence detection. Here, we present a native mass
spectrometry-based approach to assess the ratio of empty to full AAV-capsids
without the need for excessive sample preparation. We report the rapid
determination of the relative amount of empty capsids in AAV5 and
AAV8 samples. The results correlate well with more conventional analysis
strategies, demonstrating the potential of native mass spectrometry
for the characterization of viral particles.
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