Production of rAAV by plasmid transfection induces antiviral and inflammatory responses in suspension HEK293 cells

Chung, Cheng-Han; Murphy, Christopher M.; Wingate, Vincent P. M.; Pavlicek, Jeffrey W.; Nakashima, Reiko; Wei, Wei; McCarty, Douglas M.; Rabinowitz, Joseph E.; Barton, Erik S.

doi:10.1016/j.omtm.2023.01.002

Cited by 18 publications

(24 citation statements)

References 58 publications

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“…A transcriptomic analysis of triple transfection also identified genes involved in defense and immune responses, such as C3, FOS, IL‐17, that were differentially expressed. The expression of type I interferon and multiple interferon‐stimulated genes, including IFIT2 and OASL, also increased during rAAV production 23 …”

Section: Resultsmentioning

confidence: 99%

“…The expression of type I interferon and multiple interferon-stimulated genes, including IFIT2 and OASL, also increased during rAAV production. 23…”

Section: Cellular Responses-integrated Viewmentioning

confidence: 99%

“…22 RNAseq studies reported transcriptional upregulation of inflammatory and antiviral response during rAAV production by plasmid transfection, again on host cell genes. 23,24 This work examined the kinetics of rAAV production in plasmidtransfected HEK293 cells by transcriptomic analysis and quantitative proteomics to enable a thorough profiling of viral gene expression and global functional alterations at the host cell level. The absolute quantification (AQUA) through targeted quantitative proteomics allowed for a first quantitative look at the kinetics of all major viral components, while tandem mass tags (TMTs) based proteomic lent us corroboration of host functional response at the protein level (Figure 1).…”

Section: Introductionmentioning

confidence: 99%

“…To that end, a proteomic study using label‐free quantification on host cell proteins showed altered proliferation and metabolism upon rAAV production using triple plasmid transfection 22 . RNAseq studies reported transcriptional upregulation of inflammatory and antiviral response during rAAV production by plasmid transfection, again on host cell genes 23,24 …”

Section: Introductionmentioning

confidence: 99%

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Multi‐omics kinetic analysis of recombinant adeno‐associated virus production by plasmid transfection of HEK293 cells

Lu,

Lee,

2024

Biotechnology Progress

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Recombinant adeno‐associated virus (rAAV) is among the most commonly used vectors for gene therapy. It is commonly produced by transfection of HEK293 cells with three plasmids each containing the vector genome including gene of interest (GOI), helper functions, and rep and cap genes for genome replication and capsid formation. To meet the potential clinical needs, the productivity of the production system needs to be enhanced. A better process characterization of the production system will further advance our insights into ways to enhance productivity. Here, we employed transcriptomic analysis to quantify the dynamics of different isoforms of viral transcripts and to assess the shift of cellular physiology, and deployed targeted proteomic analysis for absolute quantification of viral proteins and tandem mass tags (TMTs) for assessing cellular responses at the protein level. Functional analysis at transcriptome and proteome levels identified defense and immune response, unfolded protein response, p53 signaling as enriched. The small molecule additive intervention study based on functional analysis showed the potential of such omics‐guided productivity enhancement. Together, multi‐omics analysis advanced understanding of rAAV production and provided insight into enhancing rAAV production by plasmid transfection.

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Section: Resultsmentioning

confidence: 99%

“…The expression of type I interferon and multiple interferon-stimulated genes, including IFIT2 and OASL, also increased during rAAV production. 23…”

Section: Cellular Responses-integrated Viewmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Multi‐omics kinetic analysis of recombinant adeno‐associated virus production by plasmid transfection of HEK293 cells

Lu,

Lee,

2024

Biotechnology Progress

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“…Similarly, to address the rAAV productivity limitation, a transcriptomics study can be conducted. Recently, one RNA-seq study revealed that a robust antiviral and inflammatory response was triggered by transient transfection mediated rAAV production (Chung et al, 2023). In addition to comparing producer/non-producer states, different cell lines, clones from the same cell line but with varying productivities can be further investigated using transcriptomics to understand the cellular features for high productivity.…”

Section: Multi-omics As a Tool To Address Productivity And Bioprocess...mentioning

confidence: 99%

Critical challenges and advances in recombinant adeno‐associated virus (rAAV) biomanufacturing

Polanco

Lee

et al. 2023

Biotech & Bioengineering

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Gene therapy is a promising therapeutic approach for genetic and acquired diseases nowadays. Among DNA delivery vectors, recombinant adeno‐associated virus (rAAV) is one of the most effective and safest vectors used in commercial drugs and clinical trials. However, the current yield of rAAV biomanufacturing lags behind the necessary dosages for clinical and commercial use, which embodies a concentrated reflection of low productivity of rAAV from host cells, difficult scalability of the rAAV‐producing bioprocess, and high levels of impurities materialized during production. Those issues directly impact the price of gene therapy medicine in the market, limiting most patients’ access to gene therapy. In this context, the current practices and several critical challenges associated with rAAV gene therapy bioprocesses are reviewed, followed by a discussion of recent advances in rAAV‐mediated gene therapy and other therapeutic biological fields that could improve biomanufacturing if these advances are integrated effectively into the current systems. This review aims to provide the current state‐of‐the‐art technology and perspectives to enhance the productivity of rAAV while reducing impurities during production of rAAV.

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Tuning capsid formation dynamics in recombinant adeno‐associated virus producing synthetic cell lines to enhance full particle productivity

Lu,

Lin,

Kuo

et al. 2024

Biotechnology Journal

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Recombinant adeno‐associated virus (rAAV) is widely used as an in vivo delivery vector for gene therapy. It is used in a very large dose, and the large quantities required for broad applications present manufacturing challenges. We have developed a synthetic biology platform of constructing cell lines integrated with essential viral genes which can be induced to produce rAAV without plasmid transfection or virus transduction. Through iterative design‐construct‐characterization cycles, we have showcased the potential of this synthetic cell production system. Systems characterization of the dynamics of viral transcripts and proteins as well as virus assembly and packaging revealed that the expression level and balance of viral genome and capsid protein are keys to not only the productivity but also the full particle content, an important product quality attribute. Boosting cap gene expression by sequential transfection and integration of multiple copies of the cap gene elevated the rAAV titer to levels on a par with traditional plasmid transfection and virus infection. However, overexpression of the cap gene shifted the balance and kinetics of the genome and capsid. We independently tuned the dynamics of genome amplification and capsid protein synthesis by modulating the induction concentration as well as the time profile, and significantly enhanced full particle content while maintaining a high productivity. This strategy of constructing an inducible stable producer cell line is readily adaptable to rAAV vectors of different serotypes and payloads. It can greatly facilitate scalable production of gene therapy vectors.

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Production of rAAV by plasmid transfection induces antiviral and inflammatory responses in suspension HEK293 cells

Cited by 18 publications

References 58 publications

Multi‐omics kinetic analysis of recombinant adeno‐associated virus production by plasmid transfection of HEK293 cells

Multi‐omics kinetic analysis of recombinant adeno‐associated virus production by plasmid transfection of HEK293 cells

Critical challenges and advances in recombinant adeno‐associated virus (rAAV) biomanufacturing

Tuning capsid formation dynamics in recombinant adeno‐associated virus producing synthetic cell lines to enhance full particle productivity

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