qPCR and qRT-PCR analysis: Regulatory points to consider when conducting biodistribution and vector shedding studies

Abstract

Gene and cell therapy fields have experienced remarkable growth during the past decade. Demands for preclinical and clinical safety assessments of these cell and gene therapy test articles (TAs) have effectively increased the necessity for regulated biodistribution, vector shedding, gene expression, and/or pharmacokinetics bioanalysis studies. Guidance documents issued from numerous international regulatory authorities recommend the use of quantitative polymerase chain reaction (qPCR) and/or quantitative reverse transcriptase PCR (qRT-PCR) assays due to their highly sensitive and robust target-specific detection. However, only preclinical biodistribution assay sensitivity is specified in these documents. Criteria such as accuracy, precision, and repeatability are not yet defined. This guidance void has resulted in several conflicting institutional interpretations of essential parameters necessary for the development and validation of robust assays to support safety assessments of gene and cell therapy TAs. There is an urgent need for an ongoing discussion among bioanalytical scientists in this field to generate a “best practice” consensus around preclinical and clinical qPCR/qRT-PCR assay design. With regard to this need, we offer critical points to consider when developing, validating, running sample analysis, and reporting qPCR/qRT-PCR assays. Gene and cell therapy fields have experienced remarkable growth during the past decade. Demands for preclinical and clinical safety assessments of these cell and gene therapy test articles (TAs) have effectively increased the necessity for regulated biodistribution, vector shedding, gene expression, and/or pharmacokinetics bioanalysis studies. Guidance documents issued from numerous international regulatory authorities recommend the use of quantitative polymerase chain reaction (qPCR) and/or quantitative reverse transcriptase PCR (qRT-PCR) assays due to their highly sensitive and robust target-specific detection. However, only preclinical biodistribution assay sensitivity is specified in these documents. Criteria such as accuracy, precision, and repeatability are not yet defined. This guidance void has resulted in several conflicting institutional interpretations of essential parameters necessary for the development and validation of robust assays to support safety assessments of gene and cell therapy TAs. There is an urgent need for an ongoing discussion among bioanalytical scientists in this field to generate a “best practice” consensus around preclinical and clinical qPCR/qRT-PCR assay design. With regard to this need, we offer critical points to consider when developing, validating, running sample analysis, and reporting qPCR/qRT-PCR assays. Although the first clinical trial of gene therapy only started in 1990 for treatment of adenosine deaminase severe combined immunodeficiency disease,1Wirth T. Parker N. Ylä-Herttuala S. History of gene therapy.Gene. 2013; 525: 162-169Crossref PubMed Scopus (262) Google Scholar 30 cell and gene therapy products had received market authorization worldwide by the end of 20182Shukla V. Seoane-Vazquez E. Fawaz S. Brown L. Rodriguez-Monguio R. The landscape of cellular and gene therapy products: authorization, discontinuations, and cost.Hum. Gene Ther. Clin. Dev. 2019; 30: 102-113Crossref PubMed Scopus (24) Google Scholar with numerous others currently in the development pipeline, ranging from the initial stages of research and discovery to phase III human clinical trials. Gene therapy test articles typically consist of a vector formulation containing a genetically engineered construct that is introduced to the host primarily through injection. These constructs have been designed to affect host cells in highly specific ways, and of the of gene are the several of vector have been and and the of gene 2019; PubMed Scopus Google Scholar to gene cell therapy typically of cellular the This genetically cells from products from the past regulatory have been and by the and and the for of and clinical as as of of gene and cell therapy V. Seoane-Vazquez E. Fawaz S. Brown L. Rodriguez-Monguio R. The landscape of cellular and gene therapy products: authorization, discontinuations, and cost.Hum. Gene Ther. Clin. 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