Dataset Information

Generation of infectious recombinant Adeno-associated virus in Saccharomyces cerevisiae.

ABSTRACT: The yeast Saccharomyces cerevisiae has been successfully employed to establish model systems for a number of viruses. Such model systems are powerful tools to study the virus biology and in particular for the identification and characterization of host factors playing a role in the viral infection cycle. Adeno-associated viruses (AAV) are heavily studied due to their use as gene delivery vectors. AAV relies on other helper viruses for successful replication and on host factors for several aspects of the viral life cycle. However the role of host and helper viral factors is only partially known. Production of recombinant AAV (rAAV) vectors for gene delivery applications depends on knowledge of AAV biology and the limited understanding of host and helper viral factors may be precluding efficient production, particularly in heterologous systems. Model systems in simpler eukaryotes like the yeast S. cerevisiae would be useful tools to identify and study the role of host factors in AAV biology. Here we show that expression of AAV2 viral proteins VP1, VP2, VP3, AAP, Rep78, Rep52 and an ITR-flanked DNA in yeast leads to capsid formation, DNA replication and encapsidation, resulting in formation of infectious particles. Many of the AAV characteristics observed in yeast resemble those in other systems, making it a suitable model system. Future findings in the yeast system could be translatable to other AAV host systems and aid in more efficient production of rAAV vectors.

SUBMITTER: Barajas D

PROVIDER: S-EPMC5371294 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Generation of infectious recombinant Adeno-associated virus in Saccharomyces cerevisiae.

Barajas Daniel D Aponte-Ubillus Juan Jose JJ Akeefe Hassibullah H Cinek Tomas T Peltier Joseph J Gold Daniel D

PloS one 20170329 3

The yeast Saccharomyces cerevisiae has been successfully employed to establish model systems for a number of viruses. Such model systems are powerful tools to study the virus biology and in particular for the identification and characterization of host factors playing a role in the viral infection cycle. Adeno-associated viruses (AAV) are heavily studied due to their use as gene delivery vectors. AAV relies on other helper viruses for successful replication and on host factors for several aspect ...[more]

PMID: 28355224

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Project description:Recombinant adeno-associated virus (rAAV) has been utilized successfully for in vivo gene delivery for treatment of a variety of human diseases. To sustain the growth of recombinant AAV gene therapy products, there is a critical need for the development of accurate and robust analytical methods. Fifty percent tissue culture infectious dose (TCID50) assay is an in vitro cell-based method widely used to determine AAV infectivity, and this assay is historically viewed as a challenge due to its high variability. Currently, quantitative PCR (qPCR) serves as the endpoint method to detect the amount of replicated viral genome after infection. In this study, we optimize the TCID50 assay by adapting endpoint detection with droplet digital PCR (ddPCR). We performed TCID50 assays using ATCC AAV-2 reference standard stock material across 18 independent runs. The cell lysate from TCID50 assay was then analyzed using both qPCR and ddPCR endpoint to allow for direct comparison between the two methods. The long-term 1-year side-by-side comparison between qPCR and ddPCR as endpoint measurement demonstrated improved interassay precision when the ddPCR method was utilized. In particular, after the addition of a novel secondary set threshold for infectivity scoring of individual wells, the average infectious titer of 18 runs is 6.45E+08 with % coefficient of variation (CV) of 42.5 and 5.63E+08 with % CV of 34.9 by qPCR and ddPCR, respectively. In this study, we offer improvements of infectious titer assay with (1) higher interassay precision by adapting ddPCR as an endpoint method without the need of standard curve preparation; (2) identification of a second "set threshold" value in infectivity scoring that improves assay precision; and (3) application of statistical analysis to identify the acceptance range of infectious titer values. Taken together, we provide an optimized TCID50 method with improved interassay precision that is important for rAAV infectious titer testing during process development and manufacturing.

Dataset Information

Generation of infectious recombinant Adeno-associated virus in Saccharomyces cerevisiae.

Publications

Generation of infectious recombinant Adeno-associated virus in Saccharomyces cerevisiae.

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