Optimization of viral protein ratios for production of rAAV serotype 5 in the baculovirus system

Bosma, Bas; Plessis, Francois Du; Ehlert, Erich; Nijmeijer, Bart; Haan, Martin de; Petry, Harald; Lubelski, Jacek

doi:10.1038/s41434-018-0034-7

Cited by 37 publications

(43 citation statements)

References 22 publications

(47 reference statements)

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“…Similar to the original Three-Bac, the Two-Bac [48] and the One-Bac systems, harboring the weak VP1 translational initiation codon, also showed suboptimal expression levels of VP1 proteins in certain serotypes other than AAV2. [49] Bosma et al [48] reported that the selection of a modified optimal VP1 translation initiation codon and the associated downstream nucleotide sequence resulted in optimal stoichiometric expression of all three VP proteins generating an AAV5 vector with improved functionality in the Two-Bac system. In One-Bac2.0, the second-generation One-Bac, the original strong VP1 codon, and a synthetic intron were introduced to restore the expression level of AAV5 VP1.…”

Section: Restoration Of Vp1 Expression Proportion For Improved Aav Fumentioning

confidence: 83%

“…Following this rationale, a modified version of the original Two-Bac, Duo-Bac was reported, which consists of Bac-Rep and Bac-CapX-GOI (gene of interest) baculoviruses. [48,87] Similarly, a modified version of One-Bac consisting of only the rep-expressing Sf9 cell line and Bac-CapX-GOI for AAVX (X = serotype) production has also been recently reported. [88] A study by Joshi et al [43] reported AAV5 production in high cell density fed-batch cultures using One-Bac3.0, with a final volumetric yield exceeding 2 × 10 14 VG per L of cell culture.…”

Section: Recent Advancements In the Aav Production Processmentioning

confidence: 99%

“…Similar to the original Three‐Bac, the Two‐Bac [ 48 ] and the One‐Bac systems, harboring the weak VP1 translational initiation codon, also showed suboptimal expression levels of VP1 proteins in certain serotypes other than AAV2. [ 49 ] Bosma et al.…”

Section: Molecular Design Of Baculovirus Expression Vectors and Cell mentioning

confidence: 99%

“…[ 49 ] Bosma et al. [ 48 ] reported that the selection of a modified optimal VP1 translation initiation codon and the associated downstream nucleotide sequence resulted in optimal stoichiometric expression of all three VP proteins generating an AAV5 vector with improved functionality in the Two‐Bac system. In One‐Bac2.0, the second‐generation One‐Bac, the original strong VP1 codon, and a synthetic intron were introduced to restore the expression level of AAV5 VP1.…”

Section: Molecular Design Of Baculovirus Expression Vectors and Cell mentioning

confidence: 99%

“…Following this rationale, a modified version of the original Two‐Bac, Duo‐Bac was reported, which consists of Bac‐Rep and Bac‐CapX‐GOI (gene of interest) baculoviruses. [ 48,87 ] Similarly, a modified version of One‐Bac consisting of only the rep‐ expressing Sf9 cell line and Bac‐CapX‐GOI for AAVX (X = serotype) production has also been recently reported. [ 88 ]…”

Section: Recent Advancements In the Aav Production Processmentioning

confidence: 99%

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Advancements in molecular design and bioprocessing of recombinant adeno‐associated virus gene delivery vectors using the insect‐cell baculovirus expression platform

Joshi

Venereo-Sánchez

Chahal

et al. 2021

Biotechnology Journal

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Despite rapid progress in the field, scalable high-yield production of adeno-associated virus (AAV) is still one of the critical bottlenecks the manufacturing sector is facing. The insect cell-baculovirus expression vector system (IC-BEVS) has emerged as a mainstream platform for the scalable production of recombinant proteins with clinically approved products for human use. In this review, we provide a detailed overview of the advancements in IC-BEVS for rAAV production. Since the first report of baculovirus-induced production of rAAV vector in insect cells in 2002, this platform has undergone significant improvements, including enhanced stability of Bac-vector expression and a reduced number of baculovirus-coinfections. The latter streamlining strategy led to the eventual development of the Two-Bac, One-Bac, and Mono-Bac systems. The one baculovirus system consisting of an inducible packaging insect cell line was further improved to enhance the AAV vector quality and potency. In parallel, the implementation of advanced manufacturing approaches and control of critical processing parameters have demonstrated promising results with process validation in large-scale bioreactor runs. Moreover, optimization of the molecular design of vectors to enable higher cell-specific yields of functional AAV particles combined with bioprocess intensification strategies may also contribute to addressing current and future manufacturing challenges.

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Section: Restoration Of Vp1 Expression Proportion For Improved Aav Fumentioning

confidence: 83%

Section: Recent Advancements In the Aav Production Processmentioning

confidence: 99%

Section: Molecular Design Of Baculovirus Expression Vectors and Cell mentioning

confidence: 99%

Section: Molecular Design Of Baculovirus Expression Vectors and Cell mentioning

confidence: 99%

Section: Recent Advancements In the Aav Production Processmentioning

confidence: 99%

See 3 more Smart Citations

Advancements in molecular design and bioprocessing of recombinant adeno‐associated virus gene delivery vectors using the insect‐cell baculovirus expression platform

Joshi

Venereo-Sánchez

Chahal

et al. 2021

Biotechnology Journal

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Production of adeno‐associated viral vector serotype 6 by triple transfection of suspension HEK293 cells at higher cell densities

Moço

Silva

et al. 2023

Biotechnology Journal

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In recent years, the use of adeno‐associated viruses (AAVs) as vectors for gene and cell therapy has increased, leading to a rise in the amount of AAV vectors required during pre‐clinical and clinical trials. AAV serotype 6 (AAV6) has been found to be efficient in transducing different cell types and has been successfully used in gene and cell therapy protocols. However, the number of vectors required to effectively deliver the transgene to one single cell has been estimated at 106 viral genomes (VG), making large‐scale production of AAV6 necessary. Suspension cell‐based platforms are currently limited to low cell density productions due to the widely reported cell density effect (CDE), which results in diminished production at high cell densities and decreased cell‐specific productivity. This limitation hinders the potential of the suspension cell‐based production process to increase yields. In this study, we investigated the improvement of the production of AAV6 at higher cell densities by transiently transfecting HEK293SF cells. The results showed that when the plasmid DNA was provided on a cell basis, the production could be carried out at medium cell density (MCD, 4 × 106 cells mL−1) resulting in titers above 1010 VG mL−1. No detrimental effects on cell‐specific virus yield or cell‐specific functional titer were observed at MCD production. Furthermore, while medium supplementation alleviated the CDE in terms of VG/cell at high cell density (HCD, 10 × 106 cells mL−1) productions, the cell‐specific functional titer was not maintained, and further studies are necessary to understand the observed limitations for AAV production in HCD processes. The MCD production method reported here lays the foundation for large‐scale process operations, potentially solving the current vector shortage in AAV manufacturing.

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Assessment of quality attributes for adeno‐associated viral vectors

Tustian

Bak

2021

Biotech & Bioengineering

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There is a strong and growing interest in the development and production of gene therapy products, including those utilizing adeno-associated virus (AAV) particles. This is evident with the increase in the number of clinical trials and agency approvals for AAV therapeutics. As bioproduction of AAV viral vectors matures, a quality by design (QbD) approach to process development can aid in process robustness and product quality. Furthermore, it may become a regulatory expectation. The first step in any QbD approach is to determine what physical, chemical, biological, or microbiological property or characteristic product attributes should be controlled within an appropriate limit, range, or distribution to ensure the desired product quality.Then predefined goals are set to allow proactive process development to design in quality. This review lists typical quality attributes used for release testing of AAV viral vectors and discusses these and selected attributes important to extended characterization studies in terms of safety, efficacy, and impact upon the patient immune response. K E Y W O R D S adeno-associated virus, analytics, critical quality attribute, gene therapy, quality by design 1 | INTRODUCTION Adeno-associated virus (AAV)-based viral vectors have shown enormous potential in recent years for use in gene therapy as a therapeutic modality to treat a wide range of genetic diseases, with more than 150 clinical trials specific to AAV (Penaud-Budloo et al., 2018).AAV treatments first gained regulatory approval in the European Union, with alipogene tiparvovec's (Glybera, uniQure) approval in 2012 for lipoprotein lipase deficiency. The first US regulatory ap-

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Optimization of viral protein ratios for production of rAAV serotype 5 in the baculovirus system

Cited by 37 publications

References 22 publications

Advancements in molecular design and bioprocessing of recombinant adeno‐associated virus gene delivery vectors using the insect‐cell baculovirus expression platform

Advancements in molecular design and bioprocessing of recombinant adeno‐associated virus gene delivery vectors using the insect‐cell baculovirus expression platform

Production of adeno‐associated viral vector serotype 6 by triple transfection of suspension HEK293 cells at higher cell densities

Assessment of quality attributes for adeno‐associated viral vectors

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