Dataset Information

Improvement of Precision in Recombinant Adeno-Associated Virus Infectious Titer Assay with Droplet Digital PCR as an Endpoint Measurement.

ABSTRACT: 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.

SUBMITTER: Duong T

PROVIDER: S-EPMC10457655 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature

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Improvement of Precision in Recombinant Adeno-Associated Virus Infectious Titer Assay with Droplet Digital PCR as an Endpoint Measurement.

Duong Tam T McAllister James J Eldahan Khalid K Wang Jennifer J Onishi Eric E Shen Kate K Schrock Robert R Gu Bingnan B Wang Peng P

Human gene therapy 20230704 15-16

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 ...[more]

PMID: 37276150

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Project description:Precise, reproducible characterization of AAV is critical for comparing preclinical results between laboratories and determining a safe and effective clinical dose for gene therapy applications. In this study, we systematically evaluated numerous parameters to produce a simple and robust ddPCR protocol for AAV characterization. The protocol uses a low ionic strength buffer containing Pluronic-F68 and polyadenylic acid to dilute the AAV into the ddPCR concentration range and a 10-minute thermal capsid lysis prior to assembling ddPCR reactions containing MspI. A critical finding is that the buffer composition affected the ITR concentration of AAV but not the ITR concentration of a double stranded plasmid, which has implications when using a theoretical, stoichiometric conversion factor to obtain the titer based on the ITR concentration. Using this protocol, a more comprehensive analysis of an AAV vector formulation was demonstrated with multiple ddPCR assays distributed throughout the AAV vector genome. These assays amplify the ITR, regulatory elements, and eGFP transgene to provide a more confident estimate of the vector genome concentration and a high-resolution characterization of the vector genome identity. Additionally, we compared two methods of genome integrity analysis for three control sample types at eight different concentrations for each sample. The genome integrity was independent of sample concentration and the expected values were obtained when integrity was determined based on the excess number of positive droplets relative to the number of double positive droplets expected by chance co-encapsulation of two DNA targets. The genome integrity was highly variable and produced unexpected values when the double positive droplet percentage was used to calculate the genome integrity. A protocol using a one-minute thermal capsid lysis prior to assembling ddPCR reactions lacking a restriction enzyme used the non-ITR assays in a duplex ddPCR milepost experiment to determine the genome integrity using linkage analysis.

Dataset Information

Improvement of Precision in Recombinant Adeno-Associated Virus Infectious Titer Assay with Droplet Digital PCR as an Endpoint Measurement.

Publications

Improvement of Precision in Recombinant Adeno-Associated Virus Infectious Titer Assay with Droplet Digital PCR as an Endpoint Measurement.

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