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

A firefly luciferase marker rescue assay for replication-competent MuLV detected 73.2 RCR genome particles without amplification, and amplification increased sensitivity up to 5.23x103-fold.

• Why it matters: Cell-based RCR testing is standard, but PCR methods are faster and complementary; however, detailed protocols and performance benchmarks are limited for setting up reliable assays.
• What they did: The study optimized a marker rescue workflow using defined MuLV viral copy numbers, with and without an initial amplification phase, and evaluated qPCR RCR assays targeting the retroviral pol gene using different primer strategies.
• The result: The marker rescue assay detected functional RCR after amplification, while the pol-targeted qPCR assay detected approximately 7.32x103 viral particles, supporting clinical-grade RCR testing procedure development.

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