Optimization of Lentivirus Production for Cancer Therapy

Vectors based on lentivirus backbones have revolutionized our ability to transfer genesinto many cell types. Lentiviral vectors integrate into the chromatin of target cells and do not transfer any viral genes causing vector replication. Both of these features arecommonly used in gene therapy and have been used clinically in individuals sufferingfrom cancer, infections and genetic diseases. It has been discovered that T-cells can be genetically modified to be used as effective weapons against cancer: therefore virus mustbe produced to deliver the gene of interest into the T-cells. In this project, lentiviralvectors have been produced to transfer the gene coding for a chimeric antigen receptor(CAR) which is directed to CD19 on B-cells. The vectors will, hence, be used to generateCD19 retargeted T-cells in purpose to kill CD19 cells such as B-cell lymphoma andleukemia. We have evaluated two production protocols to determine a feasible method toculture these vectors. We have also stimulate T-cells with two different antibodies (anti-CD3 and anti-CD28) and transduced T-cells. Our results demonstrate that theconcentration of virus was higher after prolonged incubation in 4˚C, which can not beexplained. The stimulation demonstrated that bound anti-CD3 was the best stimulator,and moreover the FACS-analysis showed that addition of anti-CD28 gave a highertransduction level. In conclusion, the viral vectors may be kept in 4˚C for two days beforeconcentrating the virus, and bound anti-CD3 is a better choice than soluble anti-CD3 forstimulation of T-cells