Video analysis of ex vivo beating hearts during preservation on the TransMedics® organ care system.

Background: Reliable biomarkers for assessing the viability of the donor hearts undergoing ex vivo perfusion remain elusive. A unique feature of normothermic ex vivo perfusion on the TransMedics® Organ Care System (OCS™) is that the donor heart is maintained in a beating state throughout the preservation period. We applied a video algorithm for an in vivo assessment of cardiac kinematics, video kinematic evaluation (Vi.Ki.E.), to the donor hearts undergoing ex vivo perfusion on the OCS™ to assess the feasibility of applying this algorithm in this setting. Methods: Healthy donor porcine hearts (n = 6) were procured from Yucatan pigs and underwent 2 h of normothermic ex vivo perfusion on the OCS™ device. During the preservation period, serial high-resolution videos were captured at 30 frames per second. Using Vi.Ki.E., we assessed the force, energy, contractility, and trajectory parameters of each heart. Results: There were no significant changes in any of the measured parameters of the heart on the OCS™ device over time as judged by linear regression analysis. Importantly, there were no significant changes in contractility during the duration of the preservation period (time 0-30 min, 918 ± 430 px/s; time 31-60 min, 1,386 ± 603 px/s; time 61-90 min, 1,299 ± 617 px/s; time 91-120 min, 1,535 ± 728 px/s). Similarly, there were no significant changes in the force, energy, or trajectory parameters. Post-transplantation echocardiograms demonstrated robust contractility of each allograft. Conclusion: Vi.Ki.E. assessment of the donor hearts undergoing ex vivo perfusion is feasible on the TransMedics OCS™, and we observed that the donor hearts maintain steady kinematic measurements throughout the duration.

Ex Vivo Gene Delivery to Porcine Cardiac Allografts Using a Myocardial-Enhanced Adeno-Associated Viral Vector.

Transplantation, the gold standard intervention for organ failure, is a clinical field that is ripe for applications of gene therapy. One of the major challenges in applying gene therapy to this field is the need for a method that achieves consistent and robust gene delivery to allografts. Normothermic ex vivo perfusion is a growing organ preservation method and a device for cardiac preservation was recently approved by the Food and Drug Administration (FDA) (Organ Care System, OCS™; TransMedics, Inc., Andover, MA); this device maintains donor hearts in a near physiologic state while they are transported from the donor to the recipient. This study describes the administration of recombinant adeno-associated viral vectors (rAAVs) during ex vivo normothermic perfusion for the delivery of transgenes to porcine cardiac allografts. We utilized a myocardial-enhanced AAV3b variant, SASTG, assessing its transduction efficiency in the OCS perfusate relative to other AAV serotypes. We describe the use of normothermic ex vivo perfusion to deliver SASTG carrying the Firefly Luciferase transgene to porcine donor hearts in four heterotopic transplant procedures. Durable and dose-dependent transgene expression was achieved in the allografts in 30 days, with no evidence of off-target transgene expression. This study demonstrates the feasibility and efficiency of delivering genes to a large animal allograft utilizing AAV vectors during ex vivo perfusion. These findings support the idea of gene therapy interventions to enhance transplantation outcomes.

Ex Vivo Delivery of Viral Vectors by Organ Perfusion for Cardiac Transplantation Gene Therapy.

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]).

A Porcine Heterotopic Heart Transplantation Protocol for Delivery of Therapeutics to a Cardiac Allograft.

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts and 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 intraabdominal position serves as an excellent model for assessing the effects of novel interventions and 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.

A normothermic ex vivo organ perfusion delivery method for cardiac transplantation gene therapy.

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.

Ex vivo heart perfusion after cardiocirculatory death; a porcine model.

BACKGROUND: 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). METHODS: 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 pressure <50 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. RESULTS: 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. CONCLUSIONS: 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.