Highly Sensitive ddPCR Method For Detection Of Replication-Competent Lentiviral Vectors In Gene And Cell Therapies

By Rebecca Van Orden, M.S.F.S.; Danielle Walton, B.S.; Rebecca Noll, Ph.D.; Klaudia Shick, M.Sc. Ph.D.

The growing use of lentiviral vectors in cell and gene therapies for treating various diseases, including cancer and chronic illnesses, necessitates rigorous safety testing. Regulatory agencies recommend monitoring for Replication-Competent Lentiviral Vectors (RCL) during manufacturing and post-infusion to mitigate human health risks.

To meet this critical need, a droplet digital PCR (ddPCR) assay has been designed and validated for the detection of RCL. This method specifically targets the VSV-g sequence, which is the gene encoding the envelope protein, to indicate the presence of a vector with replication capabilities. Utilizing ddPCR technology offers significant advantages over real-time PCR, providing absolute quantitation without a standard curve and superior reproducibility at low target concentrations. The assay is a duplex ddPCR that also uses IPO8 as a reference gene.

The developed method offers a highly sensitive and specific solution for RCL testing in gene and cell therapies without requiring product-specific qualification (PSQ). With a Quantitation Limit (QL) of 2 copies/µL and a Detection Limit (DL) of 0.25 copies/µL, the assay provides a robust, GMP-compliant way to ensure product safety.

Discover the full validation details and performance summary of this advanced ddPCR assay.