Ppackh1 lentivector packaging mix

DNA encoding the DCLK1 CAR was synthesized and subcloned into a third-generation lentiviral vector, Lenti CMV-MCS-EF1a-puro by Syno Biological (Beijing, China) [39 (link),42 (link)]. Ten million growth-arrested HEK293FT cells (ThermoFisher) were seeded into T75 flasks. Cells were cultured overnight and transfected using the CalPhos Transfection Kit (Takara, Mountain View, CA, USA) and pPACKH1 Lentivector Packaging Mix (System Biosciences, Palo Alto, CA, USA) containing 10 µg of the lentiviral vector. The media was changed the following day and 48 h later, media containing the lentivirus was collected and clarified by centrifugation at 2100× g for 30 min. The virus particles were collected by centrifugation at 112,000× g for 100 min, suspended in AIM V medium, aliquoted, and frozen at −80 °C. The titers of the virus preparations were determined by quantitative Reverse transcription polymerase chain reaction (RT-PCR)using the Lenti-X qRT-PCR kit (Takara) according to the manufacturer’s protocol and the 7900HT thermal cycler (ThermoFisher). The lentiviral titers were >1 × 10⁸ plaque forming units (pfu)/mL [39 (link),42 (link)].

A DNA encoding the BCMA CAR was synthesized and subcloned into a third-generation lentiviral vector, Lenti CMV-MCS-EF1a-puro by Syno Biological (Beijing, China). Three different humanized BCMA ScFv (BCMA-h1, h2 and h3) were generated as described in [22 (link)] and used for CAR lentivirus. Ten million growth-arrested HEK293FT cells (Thermo Fisher) were seeded into T75 flasks and cultured overnight, then transfected with the pPACKH1 Lentivector Packaging Mix (System Biosciences, Palo Alto, CA, USA) and 10 µg of the lentiviral vector using the CalPhos Transfection Kit (Takara, Mountain View, CA, USA). The next day the medium was replaced with fresh medium, and 48 h later the lentivirus-containing medium was collected. The medium was cleared of cell debris by centrifugation at 2100× g for 30 min. The virus particles were collected by centrifugation at 112,000× g for 100 min, suspended in AIM V medium, aliquoted and frozen at −80 °C. The titers of the virus preparations were determined by quantitative RT-PCR using the Lenti-X qRT-PCR kit (Takara) according to the manufacturer’s protocol and the 7900HT thermal cycler (Thermo Fisher). The lentiviral titers were >1 × 10⁸ pfu/mL.

Ten million growth-arrested HEK293FT cells were seeded into T75 flasks and cultured overnight, then transfected with the pPACKH1 Lentivector Packaging mix (System Biosciences, Palo Alto, CA, USA) and 10 μg of either the CD19-FLAG [16 (link)] or BCMA 4C8A [18 (link)] lentiviral vector using the CalPhos Transfection Kit (Takara, Mountain View, CA, USA). The next day the medium was replaced with fresh medium, and 48 h later the lentivirus-containing medium was collected. The medium was cleared of cell debris by centrifugation at 2100× g for 30 min. The virus particles were collected by centrifugation at 110,000× g for 100 min, suspended in AIM V medium (Thermo Fisher), aliquoted and frozen at −80 °C, as described [16 (link),37 (link),38 (link),39 (link)].