Implementation and Evaluation of Marker Rescue and qPCR Assays for Detection of Replication-Competent Retrovirus in Gene Therapy Products

Abstract

Quality control of retroviral gene therapy vectors requires reliable detection of replication-competent retrovirus (RCR) particles. Cell-based assays remain the standard for detecting RCR, while PCR-based assays serve as rapid, complementary methods. Despite their widespread use, few studies provide comprehensive protocols and performance data to guide assay setup. Here we provide a detailed description and performance assessment of a cell-based firefly luciferase marker rescue assay for RCR testing. Additionally, we describe the setup and assessment of a qPCR-based RCR assay targeting retroviral pol gene sequence. Different viral copy numbers of MuLV were used to determine the final sensitivity of the marker rescue assay with and without initial amplification. Different q-PCR strategies were investigated for RCR testing using different types of primers, including env and pol genes. The marker rescue assay detected 73.2 RCR genome particles without amplification, while the amplification phase enabled detection of functional RCR particles and increased sensitivity, with the amplification step increasing viral particles by up to 5.23x103 fold. The PCR-based RCR assay using retroviral pol gene could detect approximately 7.32x103 viral particles. This study offers a resource for establishing RCR testing procedures for clinical-grade retroviral vector products.