EXPERIMENTAL ISCHEMIA AND REPERFUSION INJURY

S. Hirayama¹, M. Sato¹, M. Liu¹, G. Zehong¹, S. Loisel-meyer², D. Wagnetz¹, J.C. Yeung¹, M. Cypel¹, J.A. Medin², S. Keshavjee^1
1Thoracic Surgery, University Health Network, University of Toronto, Toronto/ON/CANADA, ², Ontario Cancer Institute and the University of Toronto, Toronto/CANADA

Body: Introduction: Interleukin-10 (IL-10), a key immunoregulatory cytokine, might play a key role in achieving alloantigen-specific immunosuppression after lung transplantation. Pseudotyped recombinant lentiviral vectors (LV) manifest stable and persistent gene transduction in various tissues and organs without induction of host immune responses. The protective roles of consistent, low-level IL-10 generation in the lung allograft on the process of allograft airway obliteration have never been examined and could be ideal therapeutics of the lung graft by local allo-immune modulation. Herein, we introduce lentivirus as a gene delivery tool through the airway and demonstrate a protective role of IL-10 in an experimental airway graft transplantation model by repeated administration of the vectors to genetically modify local allo-immune responses. Methods: For transduction studies, C57BL6 mice received LV carrying the human IL-10 (huIL-10) cDNA by intra-tracheal delivery. ELISA assay was performed with lung tissue to examine the levels of huIL-10 up to 56 days post-transduction. For the single-dose administration study, recipient mice received LV-huIL-10 or LV-GFP by intra-tracheal delivery 7 days before transplantation. A mouse intra-pulmonary tracheal transplant model of obliterative bronchiolitis (BALBc→C57BL6) was then used to examine a protective role of IL-10 on the process of allograft airway obliteration. Recipient mice were allocated to three groups receiving LV-huIL-10, LV-GFP, or no treatment. Animals were sacrificed on post-operative days 28 and 42. For the repeated administration study, recipients in the treatment group received LV-huIL-10 for 3 consecutive days ending 7 days before transplantation and were compared to non-treated animals. Animals were sacrificed at post-operative day 42. In both settings, the left lung with tracheal allograft were divided into two parts; one for histological assessment and the other for determination of huIL-10 levels in the lung tissue by ELISA assay to confirm transduction. Results: LV-huIL-10 airway delivery resulted in no huIL-10 expression in the serum, but stably persistent expression of huIL-10 in the lung tissue (average 6.23 pg/mg protein at day 14, 7.58 at day 28, and 3.77 at day 56). In the single-dose administration study, histology at post-operative day 28 showed no significant difference in degree of luminal occulusion among the groups, but at post-operative day 42, LV-huIL-10 significantly reduced luminal occulusion compared to LV-GFP and no treatment (mean±SEM; 71.8±5.2 vs 86.4±2.3 and 90.7±2.1%, p<0.05, p<0.01 respectively). HuIL-10 expression in the lung tissue remained at a detectable level (average 4.9 pg/ mg protein) in the LV-huIL-10 treatment group. In the repeated administration study, huIL-10 significantly reduced luminal occulusion compared to no treatment (62.9±4.9 vs 91.6±5.3%, p=0.0159). HuIL-10 expression in the lung tissue also reached higher levels (average 11.8 pg/ mg protein) in the LV-huIL-10 treatment. Conclusion: We demonstrated long-term and stable gene expression of huIL-10 in mouse lungs using lentivirus-mediated gene transduction via airway administration; achieving a protective effect on allografted airways in the lung. Furthermore, unlike conventional viral vectors such as adenovirus, lentiviral vectors allow for repeated application to increase transgene expression levels. These properties will facilitate investigation to genetically modify and improve allografts in organ transplantation.

Disclosure: All authors have declared no conflicts of interest.