Corneal allografts have an average survival time of12.4 years, with the major cause of failure being immunological rejection. Immune modulation of corneal allografts using gene therapy has been successfully demonstrated in animal models. Using a lentiviral vector, we aimed to compare two constitutively active promoters, controlling expression of a therapeutic transgene encoding interleukin-10 (IL-10).A549 cells and excised ovine corneas were transduced with a self-inactivating HIV-1 lentivirus expressing ovine IL-10under the control of either the simian virus 40 (SV40) or cytomegalovirus (CMV) promoter. After ten days in culture,IL-10 was measured at protein and mRNA levels by ELISA and qRT-PCR respectively. Each treatment had an n-value of 3. Further ovine corneas were treated with lentivirus at varying multiplicities of infection (MOI): 20, 40 and 100.IL-10 expression was measured over ten days at the protein level by ELISA. In cell culture, CMV induced stronger initial transgene expression, being significantly higher thanSV40 treatment at the mRNA level at day 2 (p= 0.01),with expression then declining until day 10 where SV40expression was significantly higher than CMV (p= 0.005).SV40 gave more consistent expression over the ten day culture at the mRNA level. These trends were reflected in the protein levels. In the cornea culture at the mRNA level, the SV40 promoter induced significantly higher transgene expression than the mock-treated corneas (p= 0.03), however CMV gave approximately 14-fold higher expression thanSV40. The CMV promoter showed higher, stronger expression of IL10 throughout the culture at the protein level:480× higher than untreated corneas and 70x higher thanSV40 induced expression at day 10. Across all time points and at all MOIs, the CMV promoter induced significantly higher protein expression than SV40. These results indicate CMV is the constitutive promoter of choice to drive transgene expression for gene therapy in ovine corneas.
|Number of pages||2|
|Journal||Journal of Gene Medicine|
|Publication status||Published - Sep 2009|
|Event||6th Australasian Gene Therapy Society Meeting - Sydney, Australia|
Duration: 29 Apr 2009 → …