Search

Recent advances in ex vivo perfusion have enabled an extended preservation time for solid organs prior to transplantation allowing for possible resuscitation of the donor organ during the preservation period. Opportunities to provide viral vector-mediated gene therapy to the entire cardiac graft during this extended preservation period may lead to improvements in cardiac transplantation outcomes. Here we describe how to achieve successful gene delivery using viral vectors to an entire cardiac graft by normothermic, ex vivo perfusion. This protocol has been confirmed with the most highly utilized viral vector types in gene therapy clinical studies (adenoviral [Ad] and adeno-associated viral vector [AAV]).

Cardiac transplantation remains the gold standard therapy for end stage heart failure. However, it remains limited by the number of available donor hearts and by complications such as primary graft dysfunction and graft rejection. The recent clinical use of an ex vivo perfusion device in cardiac transplantation introduces a unique opportunity for treating cardiac allografts with therapeutic interventions to improve function and avoid deleterious recipient responses. Establishing a translational, large-animal model for therapeutic delivery to the entire allograft is essential for testing novel therapeutic approaches in cardiac transplantation. The porcine, heterotopic heart transplantation model in the intra-abdominal position serves as an excellent model for assessing the effects of novel interventions as well as the immunopathology of graft rejection. This model additionally offers long-term survival for the pig given that the graft is not required to maintain the recipient’s circulation. The aim of this protocol is to provide a reproducible and robust approach for achieving ex vivo delivery of a therapeutic to the entire cardiac allograft prior to transplantation and provide technical details to perform a survival heterotopic transplant of the ex vivo perfused heart.

Clinically, both percutaneous and surgical approaches to deliver viral vectors to the heart either have resulted in therapeutically inadequate levels of transgene expression or have raised safety concerns associated with extra-cardiac delivery. Recent developments in the field of normothermic ex vivo cardiac perfusion storage have now created opportunities to overcome these limitations and safety concerns of cardiac gene therapy. This study examined the feasibility of ex vivo perfusion as an approach to deliver a viral vector to a donor heart during storage and the resulting bio distribution and expression levels of the transgene in the recipient post-transplant. The influence of components (proprietary solution, donor blood, and ex vivo circuitry tubing and oxygenators) of the Organ Care System (OC) (TransMedics, Inc., Andover MA) on viral vector transduction was examined using a cell-based luciferase assay. Our ex vivo perfusion strategy, optimized for efficient Adenoviral vector transduction, was utilized to deliver 5 × 1013 total viral particles of an Adenoviral firefly luciferase vector with a cytomegalovirus (CMV) promotor to porcine donor hearts prior to heterotopic implantation. We have evaluated the overall levels of expression, protein activity, as well as the bio distribution of the firefly luciferase protein in a series of three heart transplants at a five-day post-transplant endpoint. The perfusion solution and the ex vivo circuitry did not influence viral vector transduction, but the serum or plasma fractions of the donor blood significantly inhibited viral vector transduction. Thus, subsequent gene delivery experiments to the explanted porcine heart utilized an autologous blood recovery approach to remove undesired plasma or serum components of the donor blood prior to its placement into the circuit. Enzymatic assessment of luciferase activity in tissues (native heart, allograft, liver etc.) obtained post-transplant day five revealed wide-spread and robust luciferase activity in all regions of the allograft (right and left atria, right and left ventricles, coronary arteries) compared to the native recipient heart. Importantly, luciferase activity in recipient heart, liver, lung, spleen, or psoas muscle was within background levels. Similar to luciferase activity, the luciferase protein expression in the allograft appeared uniform and robust across all areas of the myocardium as well as in the coronary arteries. Importantly, despite high copy number of vector genomic DNA in transplanted heart tissue, there was no evidence of vector DNA in either the recipient’s native heart or liver. Overall we demonstrate a simple protocol to achieve substantial, global gene delivery and expression isolated to the cardiac allograft. This introduces a novel method of viral vector delivery that opens the opportunity for biological modification of the allograft prior to implantation that may improve post-transplant outcomes.

Donation after cardiocirculatory death (DCD) has lead to an increase in organ availability. However, because of medical, logistic, and ethical issues, the use of hearts from DCD donors for transplantation is not generally considered to be feasible. In this study, we investigated the feasibility of ex vivo resuscitation and assessment of the porcine heart after circulatory death using the organ care system (OCS).Cardiocirculatory death was induced in five pigs by cessation of mechanical ventilation. No heparin was administered. The agonal time (AT) was calculated as the time between a reduction of blood pressure50 mm Hg or a fall in saturation beneath 70% and the cessation of electrical activity. After a further 15 min of warm ischemia, hearts were procured and implanted into the OCS, mimicking the actual clinical scenario for other organs. Thus, procured grafts were assessed ex vivo over a period of 4 h.Four hearts were successfully resuscitated on the system (AT 8, 15, 20, and 34 min) Three grafts had excellent visual contractility and lactate trends and were considered to be transplantable. One graft (AT 34 min) had an increased lactate and abnormal contractility being unsuitable for transplantation. One heart with 48-min AT could not be resuscitated.Our data show that hearts from nonheparinized DCD porcine donors can be successfully resuscitated using the OCS in a scenario, which closely simulates clinical conditions.

s S119 Organ Care System (OCS) device for 8 hrs, which allowed ex vivo beating heart assessment in working mode in addition to the standard resting mode. Functional & metabolic parameters were evaluated. The hearts were maintained in resting and working modes for 4 hours each. Results: During resting perfusion, heart 1 had favourable lactate profiles (lactate extraction) suggestive of viable myocardium, however remained > 5mmol/l. Heart 2 had an adverse lactate profile suggestive of ischaemic myocardium (Figure 1). In a working state, a left atrial pressure challenge was conducted. Heart 1 functioned better than Heart 2 (figure 1), with superior cardiac output and generated pressure. The inferiority of Heart 2 was likely the result of dual BD + DCD insult, decreased post-conditioning activation, and an undiagnosed patent foramen ovale. Conclusion: We report the first 2 DCD human hearts recovered on the OCS. Although these were not the ideal donors for DCD heart donation (donor 1 age> 50, WIT > 30 mins; Donor 2 BD + DCD, PFO), both demonstrated viability and the potential for DCD cardiac allografts. Further studies of ideal criteria DCD donors are underway.