Ex vivo adenoviral vector gene delivery results in decreased vector-associated inflammation pre- and post-lung transplantation in the pig.

Acellular normothermic ex vivo lung perfusion (EVLP) is a novel method of donor lung preservation for transplantation. As cellular metabolism is preserved during perfusion, it represents a potential platform for effective gene transduction in donor lungs. We hypothesized that vector-associated inflammation would be reduced during ex vivo delivery due to isolation from the host immune system response. We compared ex vivo with in vivo intratracheal delivery of an E1-, E3-deleted adenoviral vector encoding either green fluorescent protein (GFP) or interleukin-10 (IL-10) to porcine lungs. Twelve hours after delivery, the lung was transplanted and the post-transplant function assessed. We identified significant transgene expression by 12 hours in both in vivo and ex vivo delivered groups. Lung function remained excellent in all ex vivo groups after viral vector delivery; however, as expected, lung function decreased in the in vivo delivered adenovirus vector encoding GFP (AdGFP) group with corresponding increases in IL-1ß levels. Transplanted lung function was excellent in the ex vivo transduced lungs and inferior lung function was seen in the in vivo group after transplantation. In summary, ex vivo delivery of adenoviral gene therapy to the donor lung is superior to in vivo delivery in that it leads to less vector-associated inflammation and provides superior post-transplant lung function.

Safety and Efficacy of Ex Vivo Donor Lung Adenoviral IL-10 Gene Therapy in a Large Animal Lung Transplant Survival Model.

Ex vivo normothermic lung perfusion (EVLP) is a novel platform and method developed to facilitate functional assessment and implementation of advanced therapies for donor lungs prior to transplantation. This study aimed to determine the safety and immunological and functional benefits of ex vivo adenoviral human interleukin-10 (AdhIL-10) gene delivery to prevent the development of primary graft dysfunction in a large animal survival model. Pig donor lungs were retrieved, preserved for 6 h at 4°C, and then randomly assigned to four groups: (1) AdhIL-10 gene therapy: 12 h EVLP + AdhIL-10 intra-bronchial delivery; (2) EVLP-control: 12 h EVLP; (3) Vector-control: 12 h EVLP + adenoviral vector intra-bronchial delivery; and (4) prolonged hypothermic preservation: additional 12 h of cold ischemia. The left lung was then transplanted and evaluated. The recipients were recovered and kept alive until day 7 post-transplant under standard triple immunosuppression. Plasma levels of hIL-10 were detected in the treatment group throughout the 7 days. Analysis of peripheral blood obtained after transplant showed no signs of hematological, renal, or hepatic toxicity in the AdhIL-10 group. The immediate post-transplant lung function was significantly better in the EVLP-control and AdhIL-10 groups. Gas exchange at day 7 was superior in allografts from the AdhIL-10 group, and the histologic inflammation score was significantly lower. Lymphocytes from AdhIL-10 group harvested from mediastinal lymph nodes at day 7 post-transplantation and co-cultured with donor lymphocytes showed significantly less interferon gamma production in an Enzyme-Linked ImmunoSpot assay when compared with non-treatment groups. It has been demonstrated in this preclinical large animal survival study that ex vivo treatment with AdhIL-10 is safe and improves post-transplant lung function over EVLP alone. Improved function and an immunological advantage in both the innate and adaptive immune responses have been demonstrated.

Continuous Normothermic Ex Vivo Kidney Perfusion Improves Graft Function in Donation After Circulatory Death Pig Kidney Transplantation.

BACKGROUND: Donation after circulatory death (DCD) is current clinical practice to increase the donor pool. Deleterious effects on renal graft function are described for hypothermic preservation. Therefore, current research focuses on investigating alternative preservation techniques, such as normothermic perfusion. METHODS: We compared continuous pressure-controlled normothermic ex vivo kidney perfusion (NEVKP) with static cold storage (SCS) in a porcine model of DCD autotransplantation. After 30 minutes of warm ischemia, right kidneys were removed from 30-kg Yorkshire pigs and preserved with 8-hour NEVKP or in 4°C histidine-tryptophan-ketoglutarate solution (SCS), followed by kidney autotransplantation. RESULTS: Throughout NEVKP, electrolytes and pH values were maintained. Intrarenal resistance decreased over the course of perfusion (0 hour, 1.6 ± 0.51 mm per minute vs 7 hours, 0.34 ± 0.05 mm Hg/mL per minute, P = 0.005). Perfusate lactate concentration also decreased (0 hour, 10.5 ± 0.8 vs 7 hours, 1.4 ± 0.3 mmol/L, P < 0.001). Cellular injury markers lactate dehydrogenase and aspartate aminotransferase were persistently low (lactate dehydrogenase < 100 U/L, below analyzer range; aspartate aminotransferase 0 hour, 15.6 ± 9.3 U/L vs 7 hours, 24.8 ± 14.6 U/L, P = 0.298). After autotransplantation, renal grafts preserved with NEVKP demonstrated lower serum creatinine on days 1 to 7 (P < 0.05) and lower peak values (NEVKP, 5.5 ± 1.7 mg/dL vs SCS, 11.1 ± 2.1 mg/dL, P = 0.002). The creatinine clearance on day 4 was increased in NEVKP-preserved kidneys (NEVKP, 39 ± 6.4 vs SCS, 18 ± 10.6 mL/min; P = 0.012). Serum neutrophil gelatinase-associated lipocalin at day 3 was lower in the NEVKP group (1267 ± 372 vs 2697 ± 1145 ng/mL, P = 0.029). CONCLUSIONS: Continuous pressure-controlled NEVKP improves renal function in DCD kidney transplantation. Normothermic ex vivo kidney perfusion might help to decrease posttransplant delayed graft function rates and to increase the donor pool.

Eight-Hour Continuous Normothermic Ex Vivo Kidney Perfusion Is a Safe Preservation Technique for Kidney Transplantation: A New Opportunity for the Storage, Assessment, and Repair of Kidney Grafts.

BACKGROUND: Hypothermic kidney storage causes preservation injury and is poorly tolerated by renal grafts. We investigated whether static cold storage (SCS) can be safely replaced with a novel technique of pressure-controlled normothermic ex vivo kidney perfusion (NEVKP) in heart-beating donor kidney transplantation. METHODS: Right kidneys were removed from 30 kg Yorkshire pigs in a model of heart-beating donation and either preserved in cold histidine-tryptophan-ketoglutarate solution for 8 hours (n = 5), or subjected to 8 hours of pressure-controlled NEVKP (n = 5) followed by renal heterotopic autotransplantation. RESULTS: During NEVKP, physiologic perfusion conditions were maintained with low intrarenal resistance and normal electrolyte and pH parameters. Aspartate aminotransferase and lactate dehydrogenase as injury markers were below the detectable analyzer range (<4 and <100 U/L, respectively). Perfusate lactate concentration decreased from baseline until the end of perfusion (10.38 ± 0.76 mmol/L vs 1.22 ± 0.26 mmol/L; P < 0.001). Posttransplantation, animals transplanted with NEVKP versus SCS grafts demonstrated similar serum creatinine peak levels (NEVKP, 2.0 ± 0.5 vs SCS 2.7 ± 0.7 mg/dL; P = 0.11) and creatinine clearance on day 10 (NEVKP, 65.9 ± 18.8 mL/min vs SCS 61.2 ± 15.6 mL/min; P = 0.74). After 10 days of follow-up, animals transplanted with NEVKP grafts had serum creatinine and blood urea nitrogen values comparable to their basal levels (P = 0.49 and P = 0.59), whereas animals transplanted with SCS grafts had persistently elevated serum creatinine and blood urea nitrogen when compared with basal levels (P = 0.01 and P = 0.03). CONCLUSIONS: Continuous pressure-controlled NEVKP is feasible and safe in good quality heart-beating donor kidney grafts. It maintains a physiologic environment and excellent graft function ex vivo during preservation without causing graft injury.

The role of the endothelin-1 pathway as a biomarker for donor lung assessment in clinical ex vivo lung perfusion.

BACKGROUND: Normothermic ex vivo lung perfusion (EVLP) is a preservation technique that allows reassessment of donor lungs before transplantation. We hypothesized that the endothelin-1 (ET-1) axis would be associated with donor lung performance during EVLP and recipient outcomes after transplantation. METHODS: ET-1, Big ET-1, endothelin-converting enzyme (ECE), and nitric oxide (NO) metabolites were quantified in the perfusates of donor lungs enrolled in a clinical EVLP trial. Lungs were divided into 3 groups: (I) Control: bilateral transplantation with good early outcomes defined as absence of primary graft dysfunction (PGD) Grade 3 (PGD3) ; (II) PGD3: bilateral lung transplantation with PGD3 any time within 72 hours; and (III) Declined: lungs rejected after EVLP. RESULTS: There were 25 lungs in Group I, 7 in Group II, and 16 in Group III. At 1 and 4 hours of EVLP, the perfusates of Declined lungs had significantly higher levels of ET-1 (3.1 ± 2.1 vs. 1.8±2.3 pg/ml, p = 0.01; 2.7 ± 2.2 vs. 1.3 ± 1.1 pg/ml, p = 0.007) and Big ET-1 (15.8 ± 14.2 vs. 7.0 ± 6.5 pg/ml, p = 0.001; 31.7 ± 17.4 vs. 19.4 ± 9.5 pg/ml, p = 0.007) compared with Controls. Nitric oxide metabolite concentrations were significantly higher in Declined and PGD3 lungs than in Controls. For cases of donation after cardiac death, PGD3 and Declined lungs had higher ET-1 and Big ET-1 levels at 4 hours of perfusion compared with Controls. At this time point, Big ET-1 had excellent accuracy to distinguish PGD3 (96%) and Declined (92%) from Control lungs. CONCLUSIONS: In donation after cardiac death lungs, perfusate ET-1 and Big ET-1 are potential predictors of lung function during EVLP and after lung transplantation. They were also associated with non-use of lungs after EVLP and thus could represent useful biomarkers to improve the accuracy of donor lungs selection.