Efficiency of Cytokine Gene Transfer in Corneal Endothelial Cells and Organ-Cultured Corneas Mediated by Liposomal Vehicles and Recombinant Adenovirus

Abstract

Purpose: Gene transfer of immunoregulatory cytokines could contribute to reduce rejection of corneal grafts. The aim of our study was to examine the gene transfer efficiency of liposomal vehicles compared to adenoviral vectors for transferring the Epstein-Barr-virus-derived interleukin 10 homologue (viral IL-10, vIL-10) into corneal endothelial cells and organ-cultured human corneas (HC) in vitro. Method: To test liposomal efficiency, 2 lipid formulations (SP-Chol/DOPE 20/80 and DDAB/DOPE 30/70 in various concentrations) were complexed with a plasmid containing the vIL-10 cDNA in an eukaryotic expression vector (pcDSRα-BCRF-I). The complexes were transferred to (1) subconfluent bovine corneal endothelial cells (BCEC) after 1 passage and to (2) HC stored in organ culture. In addition, BCEC and HC were transduced with the recombinant adenoviral vector encoding for vIL-10 (AdvIL-10). Secretion of vIL-10 in the supernatants from both transfected BCEC and HC was measured by specific ELISA. Results: For gene transfer in BCEC, both transfection methods (liposomes and adenovirus) led to high secretion of vIL-10 [>2 ng/ml (liposomes) and <150 ng/ml (adenovirus) per 5,000 initially planted BCEC]. Expression levels in BCEC were dependent on the concentration of applied liposomes. For gene transfer in HC, only the adenoviral transduction technique achieved a high production of vIL-10, whereas liposomal transfection led only to low vIL-10 secretion (4.8 µg/ml vs. 95 pg/ml per quarter of cornea). Conclusion: For transfection of corneal endothelial cells in culture, liposomes can be considered as a safe and useful alternative method of gene transfer avoiding side-effects of viral vectors. However, for transfection of organ-cultured HC, adenoviral vectors are superior to liposomal vehicles.

Effects of local and systemic viral interleukin-10 gene transfer on corneal allograft survival

“…To investigate this we used an adenoviral gene transfer system for the ex vivo transduction of corneas which leads to high levels of therapeutic gene expression. 19 However, similar to the results obtained after liposomal gene transfer local IL-10 gene transfer mediated by AdvIL-10 did not lead to prolonged allograft survival (Figure 2a). Recently, it has been described that Ad-mediated gene transfer of ovine IL-10 prolongs graft survival in a sheep transplant model.…”

“…20 However, the efficiency of gene transferparticularly in solid tissues -seems to be low compared with viral vectors. 19 Nevertheless, we have obtained significant amounts of vIL-10 in the supernatants of cultured corneas after liposomal gene transfer (Figure 1a) beginning from day 4 until the end of the observation period. However, transplantation of corneas, which have been transfected with vIL-10 did not lead to prolonged survival (Figure 1b).…”

“…19 Donor corneas were transduced either with the AdvIL-10 (1 Â 10 8 PFU (plaque forming units)) or Adb-Gal (1.0 Â 10 8 PFU) construct as vector control and transplanted in high-responder MHC class I/II disparate recipients. The survival data are shown in Figure 2a.…”

“…19,32,33 Ex vivo transduction of corneal transplants with AdvIL-10 and local and systemic administration of AdvIL-10 into recipients…”

Control of TNF-Induced Dendritic Cell Maturation by Hybrid-Type N-Glycans

“…BC015265) was cloned into pAd/CMV/V5-DEST adenoviral vector using Gateway cloning (Invitrogen). Amplification and purification of adenoviral constructs were described previously (14). DCs were transduced at a multiplicity of infection of 100 in 0.5 ml/well RPMI 1640 (with 8 mg/ml polybrene) by 1 h centrifugation (2000 3 g) and additional incubation for 2 h at 37˚C.…”