Characterization of Viral Genome Encapsidated in Adeno-associated Recombinant Vectors Produced in Yeast Saccharomyces cerevisiae

Galli, Alvaro; Iaia, Ilenia; Milella, Maria Serena; Cipriani, Filippo; Latta, Veronica Della; Giacca, Mauro; Zentilin, Lorena; Cervelli, Tiziana

doi:10.1007/s12033-020-00294-4

Cited by 3 publications

(3 citation statements)

References 51 publications

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“…A newer platform utilizing baculovirus infection of Spodoptera frugiperda (Sf9) insect cells has recently gained popularity. Manufacturing approaches that use yeast cells to assemble rAAV also seem promising [25]. Currently, no platform appears to be preferred to any other.…”

Section: Aav Productionmentioning

confidence: 99%

PCR-Based Analytical Methods for Quantification and Quality Control of Recombinant Adeno-Associated Viral Vector Preparations

Shmidt

Егорова

2021

Pharmaceuticals

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Recombinant adeno-associated viral vectors (rAAV) represent a gene therapy tool of ever-increasing importance. Their utilization as a delivery vehicle for gene replacement, silencing and editing, among other purposes, demonstrate considerable versatility. Emerging vector utilization in various experimental, preclinical and clinical applications establishes the necessity of producing and characterizing a wide variety of rAAV preparations. Critically important characteristics concerning quality control are rAAV titer quantification and the detection of impurities. Differences in rAAV constructs necessitate the development of highly standardized quantification assays to make direct comparisons of different preparations in terms of assembly or purification efficiency, as well as experimental or therapeutic dosages. The development of universal methods for impurities quantification is rather complicated, since variable production platforms are utilized for rAAV assembly. However, general agreements also should be achieved to address this issue. The majority of methods for rAAV quantification and quality control are based on PCR techniques. Despite the progress made, increasing evidence concerning high variability in titration assays indicates poor standardization of the methods undertaken to date. This review summarizes successes in the field of rAAV quality control and emphasizes ongoing challenges in PCR applications for rAAV characterization. General considerations regarding possible solutions are also provided.

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Section: Aav Productionmentioning

confidence: 99%

PCR-Based Analytical Methods for Quantification and Quality Control of Recombinant Adeno-Associated Viral Vector Preparations

Shmidt

Егорова

2021

Pharmaceuticals

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“…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. 57 − 59 Synthetic biology has recently been used to create a self-attenuating helper adenovirus system as a novel approach for AAV production. 60 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).…”

Section: Introductionmentioning

confidence: 99%

“…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%

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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Commentary: Multiplex dPCR and SV-AUC are Promising Assays to Robustly Monitor the Critical Quality Attribute of AAV Drug Product Integrity

Hayes¹,

Dobnik

2022

Journal of Pharmaceutical Sciences

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Characterization of Viral Genome Encapsidated in Adeno-associated Recombinant Vectors Produced in Yeast Saccharomyces cerevisiae

Cited by 3 publications

References 51 publications

PCR-Based Analytical Methods for Quantification and Quality Control of Recombinant Adeno-Associated Viral Vector Preparations

PCR-Based Analytical Methods for Quantification and Quality Control of Recombinant Adeno-Associated Viral Vector Preparations

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

Commentary: Multiplex dPCR and SV-AUC are Promising Assays to Robustly Monitor the Critical Quality Attribute of AAV Drug Product Integrity

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