Replication Competent Lentiviruses (RCL) qPCR test
Case study
Challenge
Chimeric antigen receptor T (CAR T) cell therapy requires a retrovirus or lentivirus to introduce the chimeric antigen receptor gene into activated T cells. A safety concern with using lentiviral vectors for this treatment is the potential for replication competent lentiviruses (RCL) to arise. Recent efforts in gene therapy have improved the lentiviral vector design to reduce the likelihood of generating RCLs during the manufacture of CAR T cells by using a split plasmid design, where the vector genome, envelope, and packaging components are on separate plasmids. In addition, these lentiviral vectors have been modified by removing genes encoding essential regulatory functions. The FDA has posed safety concerns with the possibility of RCLs being created and requires monitoring of both CAR T products as well as patients undergoing therapy. The RCL qPCR test will be used to detect the presence of RCL in DNA isolated from peripheral blood mononuclear cells, whole blood and formalin-fixed paraffin-embedded tissues from patients enrolled in relevant clinical trials.
Exposure to RCL is a theoretical safety concern for individuals receiving lentiviral gene therapy, as no positive RCL results from samples obtained from patients as part of monitoring have been reported. Detection of a vector- associated RCL is complicated due to the similarities between vector and native lentiviruses (i.e. HIV+ carrier). In addition, the FDA requirements include the development of a molecular assay with a sensitivity of 50 copies/ug of DNA.
ICON Specialty Laboratories partners with clients to design, verify and validate clinical trial assays (CTA) for patient selection, therapeutic efficacy, and monitoring. We are at the forefront of precision medicine diagnostic technology and our expertise ensures reliable and accurate results for pivotal clinical end points.
When you partner with ICON you have access to:
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Scientific expertise for planning and risk mitigation, starting in pre-clinical/phase 1
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Advanced technology platforms such as NGS, droplet digital PCR, and IHC to support emerging biomarkers
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Biomarker assay specialists to design a durable, fit-for-purpose test
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RCL, RCR & VCN safety testing via ddPCR and qPCR methods
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Platform partnerships with key IVD and application suppliers
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Novel approaches that leverage advanced genomic platforms such as targeted RNA sequencing, tumour mutation load, liquid biopsy, and tumour infiltrating lymphocytes
Solution
The RCL qPCR test targets the Vesicular Stomatitis Virus G gene (VSV-G), an envelope protein found on engineered lentiviral particles (Figure 1). The VSV-G is a universal gene used in lentiviral packaging vectors. This allows detection of all constructs utilising the lentiviral platform.
Outcome
The qualitative qPCR assay validated by ICON is able to detect the VSV-G in a variety of sample matrices, such as whole blood, PBMC (as per FDA guidelines) and FFPE, with high specificity and high sensitivity, meeting the FDA guidelines for RCL molecular testing. We are now offering an off the shelf (OTS) RCL qPCR assay to all the sponsors that are engineering lentiviral vectors for their cell and gene therapy.