Main Methods of RCR Testing

What is RCR Testing?

RCR testing refers to a series of methods used during the preparation of CAR-T cell therapy products to detect the presence of Replication Competent Retrovirus (RCR) or Replication Competent Lentivirus (RCL). As these replication-competent viruses (RCV) can arise through revertant mutations of viral vectors and possess replication capabilities, they potentially pose a safety risk to patients. The purpose of RCR testing is to ensure the safety of CAR-T products and prevent RCV contamination.

Main Methods of RCR Testing

Indicator Cell Cultivation Method

This method involves co-cultivating the sample with indicator cells and observing for cytopathic effects (CPE) or signs of viral replication. This method can visually detect the presence of RCR but is time-consuming.

ELISA Method (p24 Protein Assay)

This method determines the presence of RCR contamination by detecting the presence of the viral core protein p24. The p24 protein is a core protein of retroviruses and lentiviruses, and its presence generally indicates the presence of virus particles.

PCR/qPCR Method

This method uses specific primers to amplify specific regions in the viral genome (such as the psi-gag or VSV-G genes) using Polymerase Chain Reaction (PCR) or quantitative PCR (qPCR) technology to detect the presence of RCR. Due to its short detection time, high sensitivity, and strong reproducibility, the qPCR method is widely used for the rapid release testing of CAR-T products.

Reverse Transcriptase Activity Assay (PERT)

This method determines the presence of RCR by detecting the activity of reverse transcriptase. Reverse transcriptase is an enzyme essential for the replication of retroviruses and lentiviruses, and its activity can serve as a marker for viral replication.

Among these methods, the qPCR method is preferred by many CAR-T application companies due to its efficiency and sensitivity. With the qPCR method, RCR testing results can be obtained in a relatively short time, ensuring the safety and quality of CAR-T products.

In general, RCR testing is an indispensable part of the production process for CAR-T cell therapy products, aiming to ensure that the product does not contain replication-competent viruses, thereby reducing the risk of secondary tumors and other potential safety issues for patients receiving the treatment.