Proteome profiling and vector yield optimization in a recombinant adeno‐associated virus‐producing yeast model. Issue 12 (9th November 2020)

• Record Type: Journal Article
• Authors: Aponte‐Ubillus, Juan Jose; Barajas, Daniel; Sterling, Harry; Aghajanirefah, Ali; Bardliving, Cameron; Peltier, Joseph; Shamlou, Parviz; Roy, Mimi; Gold, Daniel
• Is Part Of: MicrobiologyOpen. Volume 9:Issue 12(2020)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2020-11-09
• Subjects: adeno‐associated virus -- gene therapy -- mass spectrometry -- Saccharomyces; Microbiology -- Periodicals; 579
• DOI: 10.1002/mbo3.1136
• Languages: English
• ISSNs: 2045-8827

Abstract

ABSTRACT: Recent studies on recombinant adeno‐associated viral (rAAV) vector production demonstrated the generation of infectious viral particles in Saccharomyces cerevisiae . Proof‐of‐concept results showed low vector yields that correlated with low AAV DNA encapsidation rates. In an attempt to understand the host cell response to rAAV production, we profiled proteomic changes throughout the fermentation process by mass spectrometry. By comparing an rAAV‐producing yeast strain with a respective non‐producer control, we identified a subset of yeast host proteins with significantly different expression patterns during the rAAV induction period. Gene ontology enrichment and network interaction analyses identified changes in expression patterns associated mainly with protein folding, as well as amino acid metabolism, gluconeogenesis, and stress response. Specific fold change patterns of heat shock proteins and other stress protein markers suggested the occurrence of a cytosolic unfolded protein response during rAAV protein expression. Also, a correlative increase in proteins involved in response to oxidative stress suggested cellular activities to ameliorate the effects of reactive oxygen species or other oxidants. We tested the functional relevance of the identified host proteins by overexpressing selected protein leads using low‐ and high‐copy number plasmids. Increased vector yields up to threefold were observed in clones where proteins SSA1, SSE1, SSE2, CCP1, GTT1, and RVB2 were overexpressed. Recombinant expression of SSA1 and YDJ insect homologues (HSP40 and HSC70, respectively) in Sf9 cells led to a volumetric vector yield increase of 50% relative to control, which validated the importance of chaperone proteins in rAAV‐producing systems. Overall, these results highlight the utility of proteomic‐based tools for the understanding and optimization of rAAV‐producing recombinant strains. Abstract : Proteomic changes in yeast recombinant and control strains were profiled to identify cellular events associated with adeno‐associated viral (AAV) vector production. Bioinformatics data analysis suggested cytoplasmic unfolded protein response was primarily taking place in the AAV‐producing yeast strain. Additional changes related to response to stress and amino acid and glucose metabolism were identified. Host protein overexpression was hypothesized as a means for molecular optimization. Overexpression of SSA1, YDJ, SSE1, and other selected proteins in AAV‐producing yeast strains and insect cells resulted in increased vector yield.