Optimizing lentivirus production for CAR T cells development
Optimizing lentivirus production for CAR T cells development
Zahra Sharifzadeh,1,*Shafieeh Mansoori,2Soheila Ajdari,3Mohammadali Shokrgozar,4Shahriyar Abdoli,5Mohsen Basiri,6
1. Immunology Department, Pasteur Institute of Iran, Tehran, Iran 2. Immunology Department, Pasteur Institute of Iran, Tehran, Iran 3. Immunology Department, Pasteur Institute of Iran, Tehran, Iran 4. National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran 5. School of Advanced Medical Technologies, Golestan University of Medical Sciences, Gorgan, Iran 6. Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
Introduction: Immunotherapy with chimeric antigen receptor T (CAR T) cells is one of the most successful approaches of cancer therapy. Recently, five CAR T cell drugs are approved for relapsed or refractory hematological malignancy and multiple myeloma treatment. To produce CAR T cells, T lymphocytes are genetically modified ex vivo to express a CAR receptor, conferring them the ability to identify and destroy cancerous cells. Although there are some techniqies for gene engineering of T cells, viral vectors are the most efficient one. All marketed CAR T cell therapies rely on viral vectors (lenti or retro virus) to transduce the therapeutic chimeric antigen receptor (CAR) into T lymphocytes. However, high titer virus production for transduction to Jurkat or primary T cells is a challenge. To achieve this goal Primarily, transfection rate must be high. In addition, gene editing with viral vectors is more expensive than the other methods because it requires a large amount of an efficient transfection reagent which typically are expensive. Therefore, the choice of the desired transfection reagent and optimizing transfection parameters can tackle the challenges. Here, we have used polyethylenimine (PEI) 25000 as a cheap transfection reagent and considered some parameters such as pre-coating plate, DNA: PEI ration, and DNA/PEI complex volume for optimization. A TGFb CAR T cell, which was already developed in our lab, used for the experiments.
Methods: The TGFb CAR contains a C-myc tag in the outer part of the CAR, which is used to assess the surface expression of the TGFb CAR, and a mcherry marker on the cytosolic part. The TGFb CAR was replaced at GFP site of the pLOX plasmid. pLOX is a viral vector plasmid containing GFP marker that was used as a control. The second generation viral vector including the TGFb CAR/or pLOX, as a transfer vector, pSPAX2 and pMD2G plasmids were used for virus production. The total DNA of 4.5 μg was considered as a constant parameter. The used PEI reagent volumes were 4.5 μl and 9 μl, and DNA/PEI complex volume was set on 100 μl and 200 μl. To pre-coating, the 6-well plates were coated with FBS 10% one day before transfection. On the transfection day, 1.2X106 LentiX 293T cells counted and co-transfected with all three plasmids according to the designed test. 48 h after, CAR expression were assessed using fluorescent microscope and flowcytometery. The virus titration was determined using the control cell and the following formula:
Results: Hek293T cells were co-transfected with viral plasmids and PEI 25000 according to the corresponding test set. No significant toxicity was observed when PEI 25000 with 1:1 and 1:2 DNA:PEI ratio was used. TGFb CAR was successfully expressed in Hek293 T cells well after transduction. The best transfection rate for pLOX and TGFb CAR were included 1 DNA: 2 Reagent ratio, with the pre-coating plate, and 200 μl DNA/PEI complex. These conditions exhibited high titer virus production compared to other ones as well.
Conclusion: In this study, the optimized conditions were determined for co-transfection of TGFb CAR using PEI 25000 reagent to reach high titer virus production and reduce the costs of CAR T cells development using viral-based gene editing systems. Several transfection reagents such as Lipofectamin, Screenfect A and Turbofect have been used that have shown high transfection efficiency and less toxicity. However, these reagents were expensive and it is a limiting factor for gene delivery via viral vectors. PEI25000 can be recruited as a cheap reagent with no toxicity under the optimized conditions.