Product Enhanced Reverse Transcriptase for assessing replication competent virus in vectors retroviral vectors pseudotyped with GALV and VSV-G envelopes.

We evaluated the use of the Product Enhanced Reverse Transcriptase (PERT) assay as a means of detecting virus in retroviral vectors products pseudotyped with Gibbon Ape Leukemia Virus (GALV) and Vesicular Stomatitis Virus G (VSVG) envelopes. PERT provides greater standardization than the S+/L- assay which has been used extensively in virus detection. A challenge is that PERT will also detect residual retroviral vectors as vector particles contain reverse transcriptase. Vector products were cultured for 3 weeks on HEK293 cells to amplify any potential virus. In addition, vector supernatant and end-of-production cells were spiked with GALV to evaluate for inhibition by the test article. Results of PERT and the S+/L- assay were compared. PERT and S+/L- assays were both effective in detecting virus. Vector supernatants were negative at the end of 3 weeks of culture by PERT for both GAVL and VSVG pseudotyped vector. In contrast, end-of-production cells were positive by PERT due to persistent vector producing cells. A one-week culture of cell-free media obtained at the 3 weeks timepoint allowed distinction of virus-free test articles from those with virus. The PERT assay is suitable for detecting replication competent retrovirus in vector products pseudotyped with GALV and VSVG envelopes.

Replication-Competent Lentivirus Analysis of Vector-Transduced T Cell Products Used in Cancer Immunotherapy Clinical Trials.

Lentiviral vectors are being used in a growing number of clinical applications, including T cell immunotherapy for cancer. As this new technology moves forward, a safety concern is the inadvertent recombination and subsequent development of a replication-competent lentivirus (RCL) during the manufacture of the vector material. To assess this risk, regulators have required screening of T cell products infused into patients for RCL. Since vector particles have many of the proteins and nucleotide sequences found in RCL, a biologic assay has proven the most sensitive method for RCL detection. As regulators have required screening of up to 108 cells per T cell product, this method described a procedure for assessing RCL contamination of large-volume T cell products.