Proteome profiling and vector yield optimization in a recombinant adeno‐associated virus‐producing yeast model

Aponte-Ubillus, Juan Jose; Barajas, Daniel; Sterling, Harry J.; Aghajanirefah, Ali; Bardliving, Cameron; Peltier, Joseph; Shamlou, P. Ayazi; Roy, Mimi; Gold, Daniel

doi:10.1002/mbo3.1136

Cited by 7 publications

(8 citation statements)

References 35 publications

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“…Given that viral proteins and viral nucleic acids are perceived as exogenous entities within the host cell milieu, their presence has the potential to trigger a series of host cell stress reactions, including but not limited to autophagy, endoplasmic reticulum (ER) stress, and programmed cell death (apoptosis). Our investigation unveiled an increased activation of these stress responses during phases associated with viral production 4,7,26,37–41 (Fig. 5-7).…”

Section: Discussionmentioning

confidence: 52%

Temporal Insights into Molecular and Cellular Responses during rAAV Production in HEK293T Cells

Patra,

Tan,

Kok

et al. 2023

Preprint

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The gene therapy field is actively pursuing cost-effective, large-scale production of recombinant adeno-associated virus (rAAV) vectors for therapeutic applications, which demand high dosages. Enhanced yield is essential but presents technical and cost challenges. Strategies like suspension cell culture, transfection optimization, and cultivation conditions have shown moderate success but fall short for large-scale applications. In this study, we explore the host cell proteome of HEK293T cell lines used for AAV2 production under different transfection conditions (standard, sub-optimal, optimal) using SWATH-MS. To understand molecular and cellular mechanisms, we created a tailored spectral library for HEK293T-AAV interactions. Our gene ontology and pathway analysis revealed significant protein expression variations, particularly in processes related to cellular homeostasis, metabolic regulation, vesicular transport, ribosomal biogenesis, and cellular proliferation under optimal transfection conditions. These conditions increased rAAV titre by 50% compared to standard protocols. Furthermore, we identified alterations in host cell proteins crucial for AAV mRNA stability and gene translation, particularly regarding AAV capsid transcripts in optimal transfection conditions. This study provides insights into cellular mechanisms during rAAV production in HEK293T cells and offers potential advancements in scalability and cost-efficiency for gene therapy vector production.SignificanceGenerating AAV is challenging and the amount produced is limited, despite the success of gene therapy. Thus, it is necessary to enhance the productivity of host cells through various engineering techniques. The replication and transduction of viruses cause numerous modifications to the host cell’s proteome. Viruses exploit the processes of the host cell, influencing the availability, post-translational adjustments, associations, or placement of its proteins during the replication/growth cycle. Comprehending the fluctuations in biological processes during AAV replication is essential to grasping how virus-host relations shape the result of infection and viral protein expression.

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Section: Discussionmentioning

confidence: 52%

Temporal Insights into Molecular and Cellular Responses during rAAV Production in HEK293T Cells

Patra,

Tan,

Kok

et al. 2023

Preprint

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“…On the more contemporary side, newer synthetic biology technologies have shown promise for upending current paradigms of AAV production. Yeast-based AAV production systems are under development, though these are still limited in their production capacity and may require extensive further engineering to achieve their full potential. − Synthetic biology has recently been used to create a self-attenuating helper adenovirus system as a novel approach for AAV production . To address challenges in AAV plasmid production, an in vitro technology utilizing enzymatic machinery has been developed for synthesis of large quantities of GMP-grade linear AAV DNA constructs called doggybone DNA (dbDNA). , These designs represent some examples of synthetic biology for AAV manufacturing and may help inspire and inform future research in the field.…”

Section: Introductionmentioning

confidence: 99%

“…Unfortunately, the AAVs made using these approaches were less infectious even than those produced via BV systems and the overall yield was around 1000-fold lower than standard mammalian cell methods. Aponte-Ubillus et al took partial steps toward ameliorating the low yield by leveraging liquid chromatography mass spectrometry to analyze proteomic differences between yeast producing AAVs and a nonproducing control . Bioinformatic analysis of the results identified several proteins which could each be overexpressed to enhance AAV yields.…”

Section: Introductionmentioning

confidence: 99%

“…Aponte-Ubillus et al took partial steps toward ameliorating the low yield by leveraging liquid chromatography mass spectrometry to analyze proteomic differences between yeast producing AAVs and a nonproducing control. 58 Bioinformatic analysis of the results identified several proteins which could each be overexpressed to enhance AAV yields. Though this overexpression strategy only enhanced AAV yield by up to 3-fold, Aponte-Ubillus suggested that more dramatic improvements might arise through the synergistic effects of overexpression of multiple carefully chosen proteins at once.…”

Section: Introductionmentioning

confidence: 99%

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Synthetic Biology Design as a Paradigm Shift toward Manufacturing Affordable Adeno-Associated Virus Gene Therapies

2023

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Gene therapy has demonstrated enormous potential for changing how we combat disease. By directly engineering the genetic composition of cells, it provides a broad range of options for improving human health. Adeno-associated viruses (AAVs) represent a leading gene therapy vector and are expected to address a wide range of conditions in the coming decade. Three AAV therapies have already been approved by the FDA to treat Leber’s congenital amaurosis, spinal muscular atrophy, and hemophilia B. Yet these therapies cost around $850,000, $2,100,000, and $3,500,000, respectively. Such prices limit the broad applicability of AAV gene therapy and make it inaccessible to most patients. Much of this problem arises from the high manufacturing costs of AAVs. At the same time, the field of synthetic biology has grown rapidly and has displayed a special aptitude for addressing biomanufacturing problems. Here, we discuss emerging efforts to apply synthetic biology design to decrease the price of AAV production, and we propose that such efforts could play a major role in making gene therapy much more widely accessible.

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“…Proteomic analysis was performed by Aponte-Ubillus and co-workers to study cellular rAAV production in yeast, and they reported differential expression of proteins involved in protein folding, cell metabolism, and cellular stress responses. They subsequently used their observations for targeted overexpression of various proteins, which resulted in increased vector yields [ 11 ]. However, the most widely used expression systems for rAAV are HEK293 and Sf9 cells [ 1 ].…”

Section: Introductionmentioning

confidence: 99%

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

Strasser

Boi

Guapo

et al. 2021

IJMS

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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.

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Proteome profiling and vector yield optimization in a recombinant adeno‐associated virus‐producing yeast model

Abstract: This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Cited by 7 publications

References 35 publications

Temporal Insights into Molecular and Cellular Responses during rAAV Production in HEK293T Cells

Temporal Insights into Molecular and Cellular Responses during rAAV Production in HEK293T Cells

Synthetic Biology Design as a Paradigm Shift toward Manufacturing Affordable Adeno-Associated Virus Gene Therapies

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

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