Lentiviral vectors are generated by transfection of vectors with long terminal repeats along with a transgene in a vector construct called as transfer vector. The additional genes desired for the package like Gag/Pol/Pro and encapsulation gene, Env are included in another vector construct. In the next generation vectors, all the genes of the package are encoded by a separate vector that is transfected along with transfer vector and encapsulation vector. Further advanced construct was generated with the Rev gene encoded in a separate plasmid which is associated with the biosafety phenomenon.
To eliminate the impurities for efficient transduction of target cells, Lentiviral stocks are concentrated and their titer is increased. Many strategies were discovered for attaining high lentiviral titer with low infective capacity of the virus. As the quiescent T cells are difficult to be transduced, primary T cells of humans are problematic to be transduced by lenti virus. The integration of the gene into the genome by reverse transcription will not occur until TCR or proliferative cytokines activate T cells. In this study, the researchers were focused to establish superior transduction capacity along with high titre lenti virus in primary human T cells. Transduction efficiency increases when the infectivity of the viruses as well as the lentiviral titre are improved.
HEK293 T cells were grown on Dulbecco’s medium (modified Eagle’s medium) supplemented with high glucose and 10 percent of foetal calf serum. Human Jurkat cells derived from T lymphocytes were grown in RPMI 1640 medium added with 5percent FCS before transduction. Peripheral blood lymphocytes (PBLs) are isolated from human peripheral blood mononuclear cells by centrifugal elutriation. Negative bead isolation method is used for isolating CD4+CD45RA+ cells from PBLs. These cells were cultured in X-VIVO 15 supplemented with human serum, FCS and IL-2 and IL-7.
The vector MA1 was used for building a new pCCL empty plasmid vector with the transgenes called truncated form of nerve growth factor receptor (NGFR) and another gene transcribed with the help of human phosphoglycerate kinase promoter. One day before transfection, 293 T cells were grown in poly L-lysine coated flasks. The solutions were sterilized using filters.
Lenti virus production and concentration process
The transfer vector DNA of lenti virus, psPAX2 package and pMD2, and G envelope plasmid DNA were mixed at the ratio of 4:3:1 respectively. The third generation lentivirus vectors were constructed using package plasmid, transfer plasmid, pRSV-Rev plasmid and Env plasmid combined at the ratio of 4:2:1:1 respectively. The DNA is precipitated using water and calcium chloride. The precipitate was mixed with HEPES-buffered saline drop wise, vortexed slightly and incubated for half an hour at room temperature. The cells were periodically washed with PBS and then added with fresh medium. After 16 hrs of post transfection, RPMI medium replacement was made, incubated and then viral supernatant was collected. This collection was done for second time after 24hours. Lentiviral concentration was performed by ultracentrifugation using Sorval Discovery 100 SE centrifuge with AH-629 rotor.
Lentivirus was mixed with CD45RA+ lymphocytes grown on X-VIVO medium. Lentivirus purification was performed using Mustang Q Acrodisc and Optiprep purification method. The viral titres were determined after transducing 105 Jurkat cells in 100 microliters of RPMI 1640 medium.
The statistical analysis was performed using Graph pad prism 6 method. This study used one-way variance analysis followed by Bonferroni correction and Student’s t test to calculate the dfferences between the groups.
Results of the study
The lentiviral titres generated using lipofectamine mediated transfection in the absence of sodium butyrate were higher than those generated by calcium phosphate based procedure. Sodium butyrate supplementation for the transfection could increase the titres to the maximum at its concentration of 1mM. It is also determined from the study that second generation lentivirus vectors are found to be giving fifty fold higher viral yield than the third generation vectors. High viral yield with high titres is necessary for an efficient transduction into primary T cells. Third generation vector system was not found to have useful effect in efficiently transducing the T cells. As the viral yield was significantly higher due to second generation system, this system was used for preparing all further viral vectors.
There was 10 times reduction in viral yield by concentrating the lentivirus vectors indicating that the infectivity of the viruses was lost during ultracentrifugation. Transducing units with concentrated lentivirus used in transducing primary CD45RA+ T cells could show significantly higher expression of NGFR. The virus centrifuged at 20,000g showed higher viral titre and higher viral recovery indicating the increased transduction in CD45RA+ T cells. The infectivity and stability of virions was maintained at reduced centrifugation speeds. The viral recovery and titres were reduced using Acrodisc and Optiprep concentration techniques and these viral titres were correlated with diminished transduction of CD45RA+ T cells.
The transduction efficiency was enhanced by 1.94 times when the primary cells are activated with anti CD3/CD28 activation beads. Another feature that increased the transduction was by using human serum as supplement than in the cultured cells with added FCS.
High transduction efficiency
Optimization of culture conditions and preparing the "standard protocol" integrated with all the tested features in the study, lentiviral vector transduction was done using 3 viral preparations. The transduction efficiency was evaluated as higher with the measured NGFR expression of 79.81+/-14.68 percent. Therefore, this study identifies the importance of optimized production of lentivirus vectors and transduction methods.
Adam P Cribbs, Alan Kennedy, Bernard Gregory, Fionula M Brennan. Simplified production and concentration of lentiviral vectors to achieve high transduction in primary human T cells. BMC Biotechnology 2013,13:98.
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