Vesicular Stomatitis Virus Glycoprotein- and Lentiviral RNA-Based Robust Approach for Rapid and Accurate Quantification of Viable Lentiviral Vectors

Chimeric antigen receptor (CAR)-T cell (CAR-T) therapy is often used for treating malignant tumors, including leukemia and lymphoma. Lentiviral vectors (LVs) and their dosages are the key determinants of efficient and stable CAR-T cell preparation, cell quality, and treatment costs. However, the rapid quantification of viable LVs remains challenging because of difficulties in determining LV’s viability. Herein, we developed a dual-label flow cytometry (FCM) method to rapidly and accurately quantify viable LVs. Notably, the viral genome was combined with the infectious vesicular stomatitis virus glycoprotein (VSV-G), rather than the structural protein P24. The developed RNA–VSV-G–FCM approach accurately reflected LV’s viability and exhibited considerable linearity with the traditional culture-dependent method (R 2 > 0.99) within only 1 h. Additionally, the developed method robustly quantified viable LVs in different viability statuses (detection limit: 1 viral particle/μL). Finally, using the developed RNA–VSV-G–FCM method, the recombinant CAR-Jurkat cells were stably prepared with a high positive rate (75%) and negligible interference due to the LV viability status. In conclusion, the developed RNA–VSV-G–FCM method enables the rapid and accurate quantification of viable LVs, offering a novel and robust approach for ensuring LV quality and guiding dosage adjustments in gene and cellular therapies.