基于水疱性口炎病毒糖蛋白和慢病毒 RNA 的快速准确定量有活力慢病毒载体的稳健方法

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/ L).

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.