Joshua L. Chan

Selection of Patients for Initial Clinical Trials of Solid Organ Xenotransplantation

Several groups have reported extended survival of genetically engineered pig organs in nonhuman primates, varying from almost 10 months for life-supporting kidney grafts and more than 2 years for non-life-supporting heart grafts to less than 1 month for life-supporting liver and lung grafts. We have attempted to define groups of patients who may not have an option to wait for an allograft. These include kidney, heart, and lung candidates who are highly-allosensitized. In addition, some kidney candidates (who have previously lost at least 2 allografts from rapid recurrence of native kidney disease) have a high risk of further recurrence and will not be offered a repeat allotransplant. Patients with complex congenital heart disease, who may have undergone previous palliative surgical procedures, may be unsuitable for ventricular assist device implantation. Patients dying of fulminant hepatic failure, for whom no alternative therapy is available, may be candidates for a pig liver, even if only as a bridge until an allograft becomes available. When the results of pig organ xenotransplantation in nonhuman primates suggest a realistic potential for success of a pilot clinical trial, highly selected patients should be offered participation.

Robotic-assisted real-time MRI-guided TAVR: from system deployment to in vivo experiment in swine model

PurposeReal-time magnetic resonance imaging (rtMRI) guidance provides significant advantages during transcatheter aortic valve replacement (TAVR) as it provides superior real-time visualization and accurate device delivery tracking. However, performing a TAVR within an MRI scanner remains difficult due to a constrained procedural environment. To address these concerns, a magnetic resonance (MR)-compatible robotic system to assist in TAVR deployments was developed. This study evaluates the technical design and interface considerations of an MR-compatible robotic-assisted TAVR system with the purpose of demonstrating that such a system can be developed and executed safely and precisely in a preclinical model.MethodsAn MR-compatible robotic surgical assistant system was built for TAVR deployment. This system integrates a 5-degrees of freedom (DoF) robotic arm with a 3-DoF robotic valve delivery module. A user interface system was designed for procedural planning and real-time intraoperative manipulation of the robot. The robotic device was constructed of plastic materials, pneumatic actuators, and fiber-optical encoders.ResultsThe mechanical profile and MR compatibility of the robotic system were evaluated. The system-level error based on a phantom model was 1.14 ± 0.33 mm. A self-expanding prosthesis was successfully deployed in eight Yorkshire swine under rtMRI guidance. Post-deployment imaging and necropsy confirmed placement of the stent within 3 mm of the aortic valve annulus.ConclusionsThese phantom and in vivo studies demonstrate the feasibility and advantages of robotic-assisted TAVR under rtMRI guidance. This robotic system increases the precision of valve deployments, diminishes environmental constraints, and improves the overall success of TAVR.

Circulating cell-free DNA as a biomarker of tissue injury: Assessment in a cardiac xenotransplantation model

BACKGROUND Observational studies suggest that cell-free DNA (cfDNA) is a biomarker of tissue injury in a range of conditions including organ transplantation. However, the lack of model systems to study cfDNA and its relevance to tissue injury has limited the advancements in this field. We hypothesized that the predictable course of acute humoral xenograft rejection (AHXR) in organ transplants from genetically engineered donors provides an ideal system for assessing circulating cfDNA as a marker of tissue injury. METHODS Genetically modified pig donor hearts were heterotopically transplanted into baboons (n = 7). Cell-free DNA was extracted from pre-transplant and post-transplant baboon plasma samples for shotgun sequencing. After alignment of sequence reads to pig and baboon reference sequences, we computed the percentage of xenograft-derived cfDNA (xdcfDNA) relative to recipient by counting uniquely aligned pig and baboon sequence reads. RESULTS The xdcfDNA percentage was high early post-transplantation and decayed exponentially to low stable levels (baseline); the decay half-life was 3.0 days. Post-transplantation baseline xdcfDNA levels were higher for transplant recipients that subsequently developed graft loss than in the 1 animal that did not reject the graft (3.2% vs 0.5%). Elevations in xdcfDNA percentage coincided with increased troponin and clinical evidence of rejection. Importantly, elevations in xdcfDNA percentage preceded clinical signs of rejection or increases in troponin levels. CONCLUSION Cross-species xdcfDNA kinetics in relation to acute rejection are similar to the patterns in human allografts. These observations in a xenotransplantation model support the body of evidence suggesting that circulating cfDNA is a marker of tissue injury.

Encouraging experience using multi-transgenic xenografts in a pig-to-baboon cardiac xenotransplantation model

Innovations in transgenic technology have facilitated improved xenograft survival. Additional gene expression appears to be necessary to overcome the remaining immune and biologic incompatibilities. We report for the first time the novel use of six‐gene modifications within a pig‐to‐baboon cardiac xenotransplantation model.

CD4+CD25HiFoxP3+ regulatory T cells in long-term cardiac xenotransplantation

CD4+CD25HiFoxP3+ T (Treg) cells are a small subset of CD4+ T cells that have been shown to exhibit immunoregulatory function. Although the absolute number of Treg cells in peripheral blood lymphocytes (PBL) is very small, they play an important role in suppressing immune reactivity. Several studies have demonstrated that the number of Treg cells, rather than their intrinsic suppressive capacity, may contribute to determining the long‐term fate of transplanted grafts. In this study, we analyzed Treg cells in PBL of long‐term baboon recipients who have received genetically modified cardiac xenografts from pig donors.

Surgical management of adult endocardial fibroelastosis

From the Cardiothoracic Surgery Research Program, and Cardiovascular and Pulmonary Branch, National Heart, Lung, and Blood Institute, and Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md. Disclosures: This study was funded by the Division of Intramural Research of the National Institutes of Health; National Heart, Lung, and Blood Institute; and National Institute of Allergy and Infectious Diseases. The subject was evaluated on a clinical research protocol to evaluate the natural history of eosinophilic disorders (NCT no. 00001406). Authors have nothing to disclose with regard to commercial support. Received for publication Jan 30, 2017; revisions received April 28, 2017; accepted for publication May 12, 2017; available ahead of print June 28, 2017. Address for reprints: Keith A. Horvath, MD, Building 10-CRC, Room B2-3701, 10 Center Dr, Bethesda, MD 20892 (E-mail: khorvath@aamc.org). J Thorac Cardiovasc Surg 2017;154:e81-4 0022-5223/

Cardiac xenografts show reduced survival in the absence of transgenic human thrombomodulin expression in donor pigs: XXXX

36.00 Copyright 2017 Published by Elsevier Inc. on behalf of The American Association for Thoracic Surgery http://dx.doi.org/10.1016/j.jtcvs.2017.05.050 Histologic demonstration of extensive endocardial fibroelastosis with Russell–Movat staining.

Expression of Human Thrombomodulin on GTKO. CD46 Donor Pigs and Costimulation Blockade by Anti CD40 Antibody is Critical for Extending Cardiac Xenograft Survival in Non-human Primates

A combination of genetic manipulations of donor organs and target‐specific immunosuppression is instrumental in achieving long‐term cardiac xenograft survival. Recently, results from our preclinical pig‐to‐baboon heterotopic cardiac xenotransplantation model suggest that a three‐pronged approach is successful in extending xenograft survival: (a) α‐1,3‐galactosyl transferase (Gal) gene knockout in donor pigs (GTKO) to prevent Gal‐specific antibody‐mediated rejection; (b) transgenic expression of human complement regulatory proteins (hCRP; hCD46) and human thromboregulatory protein thrombomodulin (hTBM) to avoid complement activation and coagulation dysregulation; and (c) effective induction and maintenance of immunomodulation, particularly through co‐stimulation blockade of CD40‐CD40L pathways with anti‐CD40 (2C10R4) monoclonal antibody (mAb). Using this combination of manipulations, we reported significant improvement in cardiac xenograft survival. In this study, we are reporting the survival of cardiac xenotransplantation recipients (n = 3) receiving xenografts from pigs without the expression of hTBM (GTKO.CD46). We observed that all grafts underwent rejection at an early time point (median 70 days) despite utilization of our previously reported successful immunosuppression regimen and effective control of non‐Gal antibody response. These results support our hypothesis that transgenic expression of human thrombomodulin in donor pigs confers an independent protective effect for xenograft survival in the setting of a co‐stimulation blockade‐based immunomodulatory regimen.

Consideration of appropriate clinical applications for cardiac xenotransplantation

Introduction Heterotopic cardiac transplant model is being used in xenotransplantation to investigate xenograft survival of multi-genie modified donor pig organs and optimizing immunosuppression (IS) regimen in non-human primate model. We have shown earlier that significant graft survival can be achieved in alpha 1,3 alpha[MM1] Galactosidase transferase enzyme knockout pigs (GTKO) also expressing human complement regulatory transgene CD46 (CD46Tg) and thrombomodulin (hTBM). In this study, we evaluated the necessity of hTBM expression in donors hearts for the long-term graft survival using our costimulation blockade-based IS regimen. Materials and Methods Heterotopic cardiac xenotransplantation were performed in specific pathogen-free baboons from genetically engineered (GE) two gene pig donors which do not express hTBM (i.e.GTKO.CD46 only) (n=3) and three gene pigs (GTKO.CD46.hTBM; (n=5). Recipient baboons were treated with a short course of anti CD20 antibody, cobra venom factor, anti-thymocyte globulin, and were maintained on anti CD40 antibody (clone 2C10R4), mycophenolate mofetil and tapering doses of steroids. All baboons received continuous intravenous heparin. The dose of heparin was adjusted based on ACT levels designed to maintain the ACT at twice the baseline level. Graft survival was monitored with continuous telemetry, periodic echocardiography, and manual palpation. Blood work (CBC, chemistry, troponin, and ACT) was performed at 1-2 week intervals. Results Cardiac xenograft survival in recipient baboon receiving xenograft from GE donor pig without hTBM expression was significantly less (Median 70 days) as compared to 3 genes expressing pig (Median 298 days). All the three xenografts from non-hTBM donor pigs failed on 9, 98 and 70 days respectively. Increased troponin release and poor contractility of the transplanted heart were noted at the time of rejection. Non-Gal IgG and IgM antibody levels in recipient baboons from non-TBM GE donor pigs were at the baseline level at the time of rejection. The animals receiving the grafts expressing TBM also maintained their coagulation profile (PTT, PT and Fibrinogen levels) better and had fewer episodes of bleeding. Also, there was considerably less inflammation observed in 3 gene grafts. Conclusion Cardiac xenograft rejection is a complex phenomenon and a combination of manipulations are required to overcome this process. The study described above further supports this notion that hTBM gene expression on donors hearts is required along with the anti-CD40 mAb-based IS regimen and this regimen alone is insufficient to achieve long-term graft survival. This study further reveals that each mechanism of xenograft rejection is needed to be addressed separately. These very critical findings will further help us advance our journey towards clinical xenotransplantation.

Routine Use of Topical Bacitracin to Prevent Sternal Wound Infections After Cardiac Surgery

The field of cardiac xenotransplantation has entered an exciting era due to recent advances in the field. Although several hurdles remain, the use of rapidly evolving transgenic technology has the potential to address current allogeneic donor pool constraints and mechanical circulatory system device limitations. The success of xenotransplantation will undoubtedly be dependent on specific patient selection criteria. Defining these particular indications for xenotransplantation is important as we approach the possibility of clinical applications.