Cellular pathways of recombinant adeno-associated virus production for gene therapy

Sha, Sha; Maloney, Andrew J.; Katsikis, Georgios; Nguyen, Tam; Neufeld, Caleb; Wolfrum, Jacqueline; Barone, Paul W.; Springs, Stacy L.; Manalis, Scott R.; Sinskey, Anthony J.; Braatz, Richard D.

doi:10.1016/j.biotechadv.2021.107764

Cited by 39 publications

(56 citation statements)

References 148 publications

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“…Adeno-associated viruses (AAVs) are being predominantly investigated and used as gene-delivery vectors because of their low immunogenic response, lack of pathogenesis, and broad tropism. 1 , 2 , 3 The AAV therapeutic potential can be illustrated by more than 200 clinical trials and 4 EMA (European Medicines Agency) and/or Food and Drug Administration-approved gene therapies. 4 …”

Section: Introductionmentioning

confidence: 99%

Assessing production variability in empty and filled adeno-associated viruses by single molecule mass analyses

Ebberink¹,

Ruisinger²,

Nuebel³

et al. 2022

Molecular Therapy - Methods & Clinical Development

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Section: Introductionmentioning

confidence: 99%

Assessing production variability in empty and filled adeno-associated viruses by single molecule mass analyses

Ebberink¹,

Ruisinger²,

Nuebel³

et al. 2022

Molecular Therapy - Methods & Clinical Development

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“…Suspension-based cell culture represents a more scalable alternative 18 since volume dimensions increase much more rapidly than surface area. Yet it is difficult to develop cell lines that efficaciously synthesize AAVs in suspension, per 19 (B) Some of the difficulties in upstream processing for AAV production at the clinical scale. Adherent cells are not scalable due to their growth on surfaces.…”

Section: ■ Introductionmentioning

confidence: 99%

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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“…rAAV is a complex macromolecular product requiring a diverse network of cellular processes and molecular interactions to enable coordinated cellular synthesis -host-cell proteins, transiently expressed viral helper, capsid and replicase genes as well as the single stranded therapeutic viral DNA payload itself [5]- [7] . Small molecule enhancers of recombinant protein production have demonstrated efficacy in a wide variety of mammalian cell lines [3,10,11] and the addition of chemicals as diverse as sodium chloride, sodium butyrate, and soy peptones have been shown in previous studies to improve rAAV production yields [11]- [13] .…”

Section: Introductionmentioning

confidence: 99%

Increased recombinant AAV production by HEK293 cells using small molecule chemical additives

Scarrott

Johari

Pohle

et al. 2022

Preprint

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Recombinant adeno-associated virus (rAAV) has established itself as a highly efficacious gene delivery vector with a well characterised safety profile allowing broad clinical application. Recent successes in rAAV-mediated gene therapy clinical trials will continue to drive demand for improved rAAV production processes to reduce costs. Here we demonstrate that small molecule bioactive chemical additives can significantly increase recombinant AAV vector production by HEK cells up to 3-fold. Nocodazole (an anti-mitotic agent) and M344 (a selective histone deacetylase inhibitor) were identified as positive regulators of rAAV8 genome titre in a microplate screening assay. Addition of nocodazole to triple-transfected HEK293 suspension cells producing rAAV arrested cells in G2/M phase, increased average cell volume, and reduced viable cell density relative to untreated rAAV producing cells at harvest.. Final crude genome vector titre from nocodazole treated cultures was >2-fold higher compared to non-treated cultures.. Further investigation showed nocodazole addition to cultures to be time critical.Genome titre improvement was found to be scalable and serotype independent across two distinct rAAV serotypes, rAAV8 and rAAV9. Furthermore, a combination of M344 and nocodazole produced a positive additive effect on rAAV8 genome titre, resulting in a 3-fold increase in genome titre compared to untreated cells.

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Cellular pathways of recombinant adeno-associated virus production for gene therapy

Cited by 39 publications

References 148 publications

Assessing production variability in empty and filled adeno-associated viruses by single molecule mass analyses

Assessing production variability in empty and filled adeno-associated viruses by single molecule mass analyses

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

Increased recombinant AAV production by HEK293 cells using small molecule chemical additives

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