Vinay P. Rao

Proteomic identification of non-Gal antibody targets after pig-to-primate cardiac xenotransplantation.

Abstract:  Background:  Experience with non‐antigenic galactose α1,3 galactose (αGal) polymers and development of αGal deficient pigs has reduced or eliminated the significance of this antigen in xenograft rejection. Despite these advances, delayed xenograft rejection (DXR) continues to occur most likely due to antibody responses to non‐Gal endothelial cell (EC) antigens.

Increased immunosuppression, not anticoagulation, extends cardiac xenograft survival

Background. Cardiac xenograft function is lost due to delayed xenograft rejection (DXR) characterized by microvascular thrombosis and myocardial necrosis. The cause of DXR is unknown but may result from thrombosis induced by antibody-mediated activation of endothelial cells and/or by incompatibilities in thromboregulatory interactions. Methods. To examine these issues, a series (Groups 1–6) of previous transgenic CD46 pig-to-baboon heterotopic cardiac transplants were reanalyzed for baseline immunosuppressive levels, graft survival and infectious complications with and without systemic anticoagulation. Groups 1–4 received low dose tacrolimus and sirolimus maintenance therapy, with splenectomy, anti-CD20 and daily α-Gal polymer. Group 1 recipients received no anticoagulation. Groups 2–4 were anticoagulated with aspirin and Plavix, Lovenox, or Coumadin, respectively. Group 5 was treated with Lovenox and high dose tacrolimus and sirolimus maintenance therapy. Group 6 recipients received no postoperative anticoagulation but the same immunosuppression as group 5. Results. Median survival (15–22 days) within groups 1–4 was not significantly different. At rejection all tissues exhibited microvascular thrombosis, coagulative necrosis and similar levels of platelet and fibrin deposition. Groups 5 and 6 median survival (76 days) was significantly increased compared to groups 1–4. There was no significant difference in median survival between Lovenox treated recipients (68 days) and anticoagulant free recipients (96 days). Rejected tissues showed vascular antibody deposition, microvascular thrombosis, and myocyte necrosis. Conclusion. Significant prolongation in xenograft survival is achieved by improved immunosuppression. These results suggest that ongoing immune responses remain the major stimulus for DXR.

T‐cell responses during pig‐to‐primate xenotransplantation

Abstract: Xenotransplantation using porcine organs may resolve a chronic shortage of donor organs for clinical transplantation if significant immunological barriers can be overcome. To determine the potential role of T lymphocytes in Xenograft (Xg) rejection, we transplanted transgenic hCD46 porcine hearts heterotopically into baboon recipients. Methods: Recipients were treated to deplete anti‐Gal antibody with a non‐antigenic α‐Gal polyethylene glycol polymer (TPC) (n=2), TPC plus rituximab (anti‐CD20) (n=1) or were untreated (n=1). None of the recipients received T‐cell immunosuppression. Results: All Xgs failed within 7 days and showed evidence of a mixed humoral and cellular rejection process. Cellular infiltration consisting primarily of CD4+ T cells and few CD8+ T cells. Proliferation and cytotoxicity assays showed sensitization of CD4+ and CD8+ T cells that reacted with porcine IFN‐γ (pIFN‐γ)‐stimulated porcine aortic endothelial cells (PAEC). The CD4+ lymphocytes displayed greater cytotoxicity than CD8+ cells. An increased frequency of PAEC‐specific interleukin (IL) 2 and IFN‐γ‐secreting T cells was observed, suggesting a Th1 cytokine bias. An increase in the percentage of circulating CD4+CD28− cells was observed at the time of rejection and over 50% of the CD4+ cells recovered from residual pig tissue at necropsy lacked CD28 expression. Conclusions: These findings show that lymphocytes are efficiently stimulated by PAEC antigens and can mediate direct tissue destruction. These studies (1) provide an insight into the potential of cellular‐mediated cardiac Xg rejection, (2) show for the first time the induction of cytotoxic pig‐specific CD4+CD28− lymphocytes and (3) provide a rational basis for determining different modes of immunosuppression to treat Xg rejection.

Efficient and durable gene transfer to transplanted heart using adeno-associated virus 9 vector.

BACKGROUND In this investigation we studied the efficacy and durability of recombinant adeno-associated virus serotype 9 (rAAV9) vector-mediated gene transfer to the transplanted rat heart. METHODS A rAAV9-CMV-lacZ vector diluted in cold (4 degrees C) University of Wisconsin solution was used to perfuse the rat coronary vasculature for 20 minutes prior to syngeneic heterotopic transplantation. Perfusion experiments (six groups, n = 3/group) were performed without rAAV9 and at four separate doses ranging from 2 x 10(9) to 2 x 10(12) viral genomes/ml. The transplanted heart was recovered 10 days or 3 months after transplantation and expression of lacZ assessed by histology, enzyme-linked immunoassay and real-time reverse transcript-polymerase chain reaction (RT-PCR). In a final group (n = 3), rAAV9 was administered systemically to compare the cardiac transduction efficiency and viral distribution to other organs. RESULTS Transduction efficiency of perfused virus correlated with vector dose (p < 0.0001), with myocardial transduction ranging up to 71.74% at the highest dose. Cardiac expression of lacZ was equivalent at 10 days and 3 months. There was no evidence of viral gene transfer to other organs after heart transplantation. CONCLUSIONS Our findings demonstrate efficient and durable rAAV9-mediated gene transfer to the transplanted heart after ex vivo perfusion and suggest that AAV9 is a promising vector for cardiac gene therapy.

Recombinant adeno-associated virus vector for gene transfer to the transplanted rat heart

Efficient durable viral vector transduction of the transplanted heart remains elusive. This study assesses the potential of recombinant adeno‐associated virus (rAAV) mediated gene delivery to the transplanted rat heart. rAAV serotype 1, 2 and 5 vectors encoding the green fluorescent protein (GFP) gene (1 × 1011 viral particles/ml) were diluted in cold University of Wisconsin solution and circulated through the coronary vasculature of the donor organs for 30 min before syngeneic rat heterotopic heart transplantation was performed. Study 1: animals (n = 5 each serotype) were killed at 21 days post‐transplant to evaluate the efficiency of GFP transduction using RT‐PCR and expression by fluorescence microscopy. Study 2: using rAAV‐1, animals (n = 5 each group) were killed at 7, 21 and 84 days to evaluate the durability of GFP expression. The maximum cardiac GFP expression at 21 days was observed in rAAV‐1. GFP expression by rAAV‐1 was detectable at 7 days, improved at 21 days, and was still evident at 84 days. This study demonstrates cardiac rAAV gene transduction with a cold perfusion preservation system of the donor heart. These data show that AAV‐1 is superior to AAV‐2 and AAV‐5 for this purpose and that durable expression is achievable.

Sodium iodide symporter (hNIS) permits molecular imaging of gene transduction in cardiac transplantation.

Background. We evaluated the feasibility of noninvasive micro-single photon emission computed tomography (SPECT)/computed tomography (CT) imaging and quantification of cardiac gene expression after sodium iodide symporter (hNIS) gene transfer in cardiac transplantation. Methods. Donor rat hearts were perfused ex vivo with adenovirus expressing hNIS (Ad-hNIS), Ad-Null, or University of Wisconsin (UW) solution prior to heterotopic transplantation into syngeneic recipients. In the first group of recipients, imaging of the transplanted hearts with micro-SPECT/CT on day 5 was followed by immediate explant of the organs for ex vivo analyses. Radioactivity counts in the explanted hearts were obtained ex vivo and expressed as a percentage of the injected dose per gram of tissue (%ID/g). Intensities of the SPECT images of the transplanted hearts were quantified and converted to radioactive counts using a standard equation. The second group of recipients was imaged sequentially after injection of I123 on days 2 to 14 after transplantation. Results. Higher ex vivo radioiodine counts were noted in the hearts perfused with Ad-hNIS (1.04±0.2) compared to either the UW group (0.31±0.11, P<0.001) or the Ad-Null group (0.32±0.08, P<0.001). Image intensity in the Ad-NIS group (0.9±0.2) was also significantly higher than in the UW group (0.4±.03, P=0.003) or the Ad-Null group (0.5±0.1, P<0.05). Sequential imaging of Ad-NIS-perfused hearts between postoperative days 2 and 14 revealed peak image intensity at day 5. Overall, image intensities correlated with ex vivo counts of radioactivity (&rgr;=0.74, P<0.05). Conclusions. These data demonstrate that hNIS gene transfer permits sequential real-time detection and quantification of reporter gene expression in the transplanted heart with micro-SPECT/CT imaging.

Prolonged cardiac allograft survival using iodine 131 after human sodium iodide symporter gene transfer in a rat model.

BACKGROUND Radioiodine is efficiently concentrated by tissues expressing the human sodium iodide symporter (hNIS). OBJECTIVE To analyze the effects of iodine 131 on acute cardiac allograft rejection after ex vivo hNIS gene transfer in a rat model of cardiac allotransplantation. MATERIALS AND METHODS Hearts from Brown Norway rats were perfused ex vivo either with UW (University of Wisconsin) solution (n = 9) or UW solution containing 1 x 10(9) pfu/mL of adenovirus 5 plus NIS (Ad-NIS) (n = 18). Donor hearts were transplanted heterotopically into the abdomen of Lewis rats, and recipients were treated on postoperative day 3 with either 15,000 microCi of (131)I or saline solution. The hearts were explanted when no longer beating, and were evaluated histologically for evidence of rejection and other changes. RESULTS Grafts perfused with the Ad-NIS vector survived significantly longer in recipients injected with (131)I (mean [SD], 11.3 [1.9] days) compared with control animals not treated with (131)I (5.7 [0.65] days) (P < .001). Treatment with (131)I did not prolong graft survival in recipients of hearts that were not perfused with Ad-NIS (5.5 [1.0] vs 5.3 [0.8] days). In Ad-NIS (131)I-treated transplants, the level of myocardial damage on day 6 after surgery, when control hearts were rejected, was significantly lower (60.8 [28.0] vs 99.7 [0.8]; P < .05). CONCLUSION Our findings indicate that (131)I, after NIS gene transfer, can effectively prolong cardiac allograft survival. To our knowledge, this is the first report of the use of NIS-targeted (131)I therapy in cardiac transplantation. Further studies are required to determine the mechanism of this effect and its potential for clinical application.

(1) Gene‐knockout (GT‐KO) heterotopic cardiac xenotransplantation: are GT‐KO pigs essential for successful clinical xenotransplantation?

One of the fundamental advantages of xenotransplantation is that the donor organ, because it is derived from a pig, can be genetically modified to relieve the patient of as much of the immunosuppressive burden as possible. The first example of the utility of this approach was the development of human complement regulatory protein pigs which essentially eliminated hyperacute rejection (HAR) and extended cardiac xenograft survival from less than a few hours to 5–7 days. These donor organs eliminated the need for systemic complement inhibition and thereby significantly reduced the immunosuppressive burden of the recipient. With the advent of nuclear transfer technology, and the extension of that technique to agricultural species, it became possible to produce pigs with targeted mutations. This lead to the disruption of the porcine a-galactosyltransferase gene which catalyzes the terminal addition of UDP-galactose to produce the a-Gal carbohydrate. As optimal control of a-Gal antibodies using daily infusions of Gal-polymers had resulted in median cardiac xenograft survival beyond 3 months, the creation of pigs deficient in the a-Gal antigen was widely anticipated to eliminate the need for a-Gal polymers and perhaps extend graft survival by reducing the intensity of the overall immune response. Before the utility of gene-knockout (GT-KO) donors for clinical xenotransplantation can be determined, there are three major questions that should be addressed: First, will these animals be phenotypically normal and will they reproduce through natural breeding? Secondly, will using GT-KO pig hearts eliminate the need for infusion of a-Gal polymers and achieve similar survival times? Finally, what form of rejection will be observed using GT-KO organs and how will this new hurdle be overcome? The Mayo group, in collaboration with Nextran, developed GT-KO pigs from porcine fetal fibroblasts containing a neomycin insertion that disrupts exon 9 of the a-galactosyltransferase gene [1]. The initial heterozygous female animals were bred to a series of unrelated male pigs carrying an hDAF transgene and the offspring were backcrossed to produce homozygous GTKO and GT-KO; hDAF pigs. We presented the lineage of GT heterozygous by heterozygous and GT-KO homozygous boar by heterozygous sow matings. All of the GT-KO animals produced to date were healthy and showed no evidence of cataracts, a phenotype initially reported in GTKO mice. Production of the GT-KO genotype has been at or near the predicted Mendelian frequencies. We also reported on a series of eight GT-KO pig to baboon heterotopic heart transplants that used a maintenance immunosuppressive regimen that matched a previous study using GT+ CD46 transplants in which the recipients were successfully treated with daily infusion of a-Gal polymers to block anti-Gal function and induction. These GT-KO transplants were designed to test the efficacy of GT-KO donor hearts as a substitute for a-Gal polymers. The recipients were not pre-selected for low levels of preformed non-Gal antibody as this form of selection was not possible in previous transplants. Unexpectedly, one of these hearts underwent HAR. While immediate graft function after reperfusion was excellent, within 10 min the graft exhibited discoloration which progressed globally until graft failure 90 min after reperfusion. Flow cytometry using GT-KO porcine aortic endothelial cells confirmed the presence of preformed non-Gal IgM and IgG and the cytolytic nature of this preformed antibody in the recipient. Histological analysis of the graft showed typical HAR including widespread edema and hemorrhage with strong vascular deposition of IgM and C5b. The remaining transplants survived for a median of 27 days (range: 2–128 days) with five out of seven grafts exhibiting graft failure and delayed xenograft rejection. All grafts had vascular antibody deposition at the time of explant with variable levels of complement deposition. Survival of these GT-KO organs was comparable with the survival of GT+ CD46 transgenic hearts in recipients treated with an a-Gal polymer to control induction of anti-Gal antibody. We believe that the GT-KO donors may be clinically useful as they may eliminate the need for intravenous a-Gal polymers, however, the Ethical aspects of xenotransplantation

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