Abstract：
OBJECTIVE: Ex vivo lung perfusion has emerged as a platform for organ preservation, evaluation, and restoration. Gene delivery using a clinically relevant adeno-associated vector during ex vivo lung perfusion may be useful in optimizing donor allografts while the graft is maintained physiologically active. We evaluated the feasibility of adeno-associated vector-mediated gene delivery during ex vivo lung perfusion in a rat transplant model. Additionally, we assessed off-target effects and explored different routes of delivery.
METHODS: Rat heart-lung blocks were procured and underwent 1-hour ex vivo lung perfusion. Before ex vivo lung perfusion, 4e11 viral genome luciferase encoding adeno-associated vector 9 was administered via the left bronchus (Br group, n = 4), via the left pulmonary artery (PA group, n = 3), or directly into the circuit (Circuit group, n = 3). Donor lungs in the Control group (n = 3) underwent ex vivo lung perfusion without adeno-associated vector 9. Only the left lung was transplanted. Animals underwent bioluminescence imaging weekly before being killed at 2 weeks. Tissues were collected for luciferase activity measurement.
RESULTS: All recipients tolerated the transplant well. At 2 weeks post-transplant, luciferase activity in the transplanted lung was significantly higher among animals in the Br group compared with the other 3 groups (Br: 1.1 × 106 RLU/g, PA: 8.3 × 104 RLU/g, Circuit: 3.8 × 103 RLU/g, Control: 2.5 × 103 RLU/g, P = .0003). No off-target transgene expression was observed.
CONCLUSIONS: In this work, we demonstrate that a clinically relevant adeno-associated vector 9 vector mediates gene transduction during ex vivo lung perfusion in rat lung grafts when administered via the airway and potentially the pulmonary artery. Our preliminary results suggest a higher transduction efficiency when adeno-associated vector 9 was delivered via the airway, and delivery during ex vivo lung perfusion reduces off-target effects after graft implant.
METHODS: Rat heart-lung blocks were procured and underwent 1-hour ex vivo lung perfusion. Before ex vivo lung perfusion, 4e11 viral genome luciferase encoding adeno-associated vector 9 was administered via the left bronchus (Br group, n = 4), via the left pulmonary artery (PA group, n = 3), or directly into the circuit (Circuit group, n = 3). Donor lungs in the Control group (n = 3) underwent ex vivo lung perfusion without adeno-associated vector 9. Only the left lung was transplanted. Animals underwent bioluminescence imaging weekly before being killed at 2 weeks. Tissues were collected for luciferase activity measurement.
RESULTS: All recipients tolerated the transplant well. At 2 weeks post-transplant, luciferase activity in the transplanted lung was significantly higher among animals in the Br group compared with the other 3 groups (Br: 1.1 × 106 RLU/g, PA: 8.3 × 104 RLU/g, Circuit: 3.8 × 103 RLU/g, Control: 2.5 × 103 RLU/g, P = .0003). No off-target transgene expression was observed.
CONCLUSIONS: In this work, we demonstrate that a clinically relevant adeno-associated vector 9 vector mediates gene transduction during ex vivo lung perfusion in rat lung grafts when administered via the airway and potentially the pulmonary artery. Our preliminary results suggest a higher transduction efficiency when adeno-associated vector 9 was delivered via the airway, and delivery during ex vivo lung perfusion reduces off-target effects after graft implant.
摘要：
目的：离体肺灌注（EVLP）已成为器官保存的平台，评估，和恢复。在EVLP期间使用临床相关腺相关载体(AAV)的基因递送可用于优化供体同种异体移植物,同时移植物保持生理活性。在这里，我们评估了在大鼠移植模型中EVLP期间AAV介导的基因递送的可行性。此外,我们评估了脱靶效应,并探索了不同的给药途径.
方法：获得大鼠心肺阻滞并接受1小时EVLP。在EVLP之前，通过左支气管施用编码AAV9的4e11病毒基因组荧光素酶（luc）（Br组，n=4),通过左肺动脉（PA组，n=3),或直接进入电路(电路组，n=3)。对照组（n=3）的供体肺接受了无AAV9的EVLP。仅移植左肺。动物在两周处死之前每周进行生物发光成像。收集组织用于luc活性测量。
结果：所有受者对移植耐受良好。移植后2周,在Br组的动物中，移植肺中的luc活性显着升高，与其他三组相比（Br：1.1x106RLU/g，PA:8.3x104RLU/g,电路:3.8x103RLU/g,控制：2.5x103RLU/g，p=0.0003)。没有观察到脱靶转基因表达。
结论：在这项工作中，我们证明，当通过气道和潜在的PA给药时，临床相关的AAV9载体在大鼠肺移植物的EVLP过程中介导基因转导。我们的初步结果表明，当通过气道递送AAV9时，转导效率更高，而在EVLP期间递送可减少移植物植入后的脱靶效应。
方法：获得大鼠心肺阻滞并接受1小时EVLP。在EVLP之前，通过左支气管施用编码AAV9的4e11病毒基因组荧光素酶（luc）（Br组，n=4),通过左肺动脉（PA组，n=3),或直接进入电路(电路组，n=3)。对照组（n=3）的供体肺接受了无AAV9的EVLP。仅移植左肺。动物在两周处死之前每周进行生物发光成像。收集组织用于luc活性测量。
结果：所有受者对移植耐受良好。移植后2周,在Br组的动物中，移植肺中的luc活性显着升高，与其他三组相比（Br：1.1x106RLU/g，PA:8.3x104RLU/g,电路:3.8x103RLU/g,控制：2.5x103RLU/g，p=0.0003)。没有观察到脱靶转基因表达。
结论：在这项工作中，我们证明，当通过气道和潜在的PA给药时，临床相关的AAV9载体在大鼠肺移植物的EVLP过程中介导基因转导。我们的初步结果表明，当通过气道递送AAV9时，转导效率更高，而在EVLP期间递送可减少移植物植入后的脱靶效应。
