Production of adeno-associated virus vectors for in vitro and in vivo applications

Kimura, Tamizo; Ferrán, Beatriz; Tsukahara, Yusuke; Shang, Qifan; Desai, Suveer; Fedoce, Alessandra; Pimentel, David R.; Luptak, Ivan; Adachi, Takeshi; Ido, Yasuo; Matsui, Reiko; Bachschmid, Markus

doi:10.1038/s41598-019-49624-w

Cited by 113 publications

(110 citation statements)

References 65 publications

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“…After the study of additional serotypes, it was reported that the transport of AAV into the media can be highly dependent on serotype, with a majority of AAV expressed in the media for all serotypes except AAV2 and AAV5 (Vandenberghe et al, 2010). Additionally the transport of AAV into media is dependent on expression conditions, such as a mildly alkaline pH or low serum levels (Atkinson et al, 2003;Kimura et al, 2019;Vandenberghe et al, 2010), as well as time post infection/transfection (Piras et al, 2016).…”

Section: Harvest and Clarification Of Aav Cell Culturementioning

confidence: 99%

“…Use of detergents is concentration and time dependent, with reported concentrations of Triton X-100 between 0.1% (2 hr and 37°C; Nass et al, 2018) it poses a potential concern for the downstream impact on chromatographic unit operations exists. The use of acidic buffers for cell lysis, such as citrate at pH 4.2, has also been explored (Kimura et al, 2019;Shuhei Sakamoto et al, 2018) and could prove an alternative to detergents that could mitigate the impact on downstream unit operations as well as the need to show clearance of the additive. Citrate complexes bivalent ions and the low pH may trigger a protease activity of viral capsid proteins that aides with the release of AAV vectors.…”

Section: Harvest and Clarification Of Aav Cell Culturementioning

confidence: 99%

“…Citrate complexes bivalent ions and the low pH may trigger a protease activity of viral capsid proteins that aides with the release of AAV vectors. Additionally, lysis of cells using an acidic buffer such as citrate could promote AAV release with less contamination (Kimura et al, 2019).…”

Section: Harvest and Clarification Of Aav Cell Culturementioning

confidence: 99%

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Moving from the bench towards a large scale, industrial platform process for adeno‐associated viral vector purification

Adams

Bak

Tustian

2020

Biotech & Bioengineering

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In recent years, there has been a strong interest in the development and production of gene therapy products, especially those utilizing adeno‐associated virus (AAV) particles. This is evident with the growing number of clinical successes and agency approvals for AAV therapeutics. Due to this increased investment in this technology, a need exists for scalable commercial production methods to ensure adequate product supply as research in AAV shifts from bench‐scale development to clinical production. The purpose of this review is to summarize current scalable purification techniques that can be employed during the commercial manufacturing of AAV as well as highlight certain development considerations, such as adventitious agent removal and process development using the principals of quality by design.

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Section: Harvest and Clarification Of Aav Cell Culturementioning

confidence: 99%

Section: Harvest and Clarification Of Aav Cell Culturementioning

confidence: 99%

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Moving from the bench towards a large scale, industrial platform process for adeno‐associated viral vector purification

Adams

Bak

Tustian

2020

Biotech & Bioengineering

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“…This strategy does have advantages over gradient-based purification of intracellular rAAV but still requires large volumes of media and ultracentrifuges that may be cost-prohibitive to smaller laboratories. Another method purifies rAAV from the media via polyethylene glycol precipitation with subsequent aqueous twophase partitioning [48]. This method can be completed without an ultracentrifuge or iodixanol gradient but still requires several additional time-consuming steps such as media changes and incubations with chemical reagents.…”

Section: Discussionmentioning

confidence: 99%

Utilizing minimally purified secreted rAAV for rapid and cost-effective manipulation of gene expression in the CNS

Goodwin

Croft

Futch

et al. 2020

Mol Neurodegeneration

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Background: Recombinant adeno-associated virus (rAAV) is widely used in the neuroscience field to manipulate gene expression in the nervous system. However, a limitation to the use of rAAV vectors is the time and expense needed to produce them. To overcome this limitation, we evaluated whether unpurified rAAV vectors secreted into the media following scalable PEI transfection of HEK293T cells can be used in lieu of purified rAAV. Methods:We packaged rAAV2-EGFP vectors in 30 different wild-type and mutant capsids and subsequently collected the media containing secreted rAAV. Genomic titers of each rAAV vector were assessed and the ability of each unpurified virus to transduce primary mixed neuroglial cultures (PNGCs), organotypic brain slice cultures (BSCs) and the mouse brain was evaluated.Results: There was~40-fold wide variance in the average genomic titers of the rAAV2-EGFP vector packaged in the 30 different capsids, ranging from a low~4.7 × 10 10 vector genomes (vg)/mL for rAAV2/5-EGFP to a high of2 .0 × 10 12 vg/mL for a capsid mutant of rAAV2/8-EGFP. In PNGC studies, we observed a wide range of transduction efficiency among the 30 capsids evaluated, with the rAAV2/6-EGFP vector demonstrating the highest overall transduction efficiency. In BSC studies, we observed robust transduction by wild-type capsid vectors rAAV2/6, 2/8 and 2/9, and by capsid mutants of rAAV2/1, 2/6, and 2/8. In the in vivo somatic brain transgenesis (SBT) studies, we found that intra-cerebroventricular injection of media containing unpurified rAAV2-EGFP vectors packaged with select mutant capsids resulted in abundant EGFP positive neurons and astrocytes in the hippocampus and forebrain of non-transgenic mice. We demonstrate that unpurified rAAV can express transgenes at equivalent levels to lysate-purified rAAV both in vitro and in vivo. We also show that unpurified rAAV is sufficient to drive tau pathology in BSC and neuroinflammation in vivo, recapitulating previous studies using purified rAAV. Conclusions: Unpurified rAAV vectors secreted into the media can efficiently transduce brain cells in vitro and in vivo, providing a cost-effective way to manipulate gene expression. The use of unpurified virus will greatly reduce costs of exploratory studies and further increase the utility of rAAV vectors for standard laboratory use.

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“…In these applications, the GOI replaces the natural AAV genome for delivery to tissues or cells to treat a monogenic disease. In addition, rAAVs are widely used research tools for transgene expression in tissue culture and preclinical animal models ( 18 ).…”

Section: Introductionmentioning

confidence: 99%

Adeno-associated virus capsid assembly is divergent and stochastic

Wörner

Bennett²,

Habka

et al. 2020

Preprint

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Adeno-associated viruses (AAVs) are small, non-enveloped, and have a T=1 icosahedral capsid. They belong to the Parvoviridae, genus Dependoparvovirus. Interest in AAVs has grown over recent years as they have emerged as promising gene therapy vectors. The AAV capsid, encapsulating the transgene, consists, in total, of 60 subunits made up from three distinct viral proteins (VPs) originating from the same cap gene (VP1, VP2, and VP3), which vary only in their N-terminus. While all three VPs play a crucial and specific role in cell-entry and transduction, their exact stoichiometry and organization in AAV capsids has, despite the availability of several high-resolution structures remained elusive. Here we obtained a set of native mass spectra of intact AAV capsids (Mw ≈ 3.8 MDa) that display both highly resolved regions and regions wherein interferences occur. Through spectrum simulation we resolved and elucidated this spectral complexity, allowing us to directly assess the VP stoichiometries in a panel of serotypes from different production platforms. The data reveals an extremely heterogeneous population of capsids of variable composition. The relative abundance for each of the hundreds of co-occurring capsid compositions is accurately described by a model based upon stochastic assembly from a mixed pool of expressed VP1, VP2, and VP3. We show that even the single-most abundant VP stoichiometry represents only a few percent of the total AAV population. We estimate that virtually every AAV capsid in a particular preparation has a unique composition and arrangement, i.e. no particle is identical. The systematic scoring of the stochastic assembly model against experimental high-resolution native MS data offers a sensitive and accurate new method to characterize these exceptionally heterogeneous gene-delivery vectors.

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Production of adeno-associated virus vectors for in vitro and in vivo applications

Cited by 113 publications

References 65 publications

Moving from the bench towards a large scale, industrial platform process for adeno‐associated viral vector purification

Moving from the bench towards a large scale, industrial platform process for adeno‐associated viral vector purification

Utilizing minimally purified secreted rAAV for rapid and cost-effective manipulation of gene expression in the CNS

Adeno-associated virus capsid assembly is divergent and stochastic

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