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Background: Transgenic expression of human complement regulatory proteins reduces the frequency of hyperacute rejection (HAR) in Gal-positive cardiac xenotransplantation. In this study, we examined the impact of human CD55 (hCD55) expression on a Gal knockout (GTKO) background using pig-to-primate heterotopic cardiac xenotransplantation., Methods: Cardiac xenotransplantation was performed with GTKO (group 1; n=6) and GTKO.hCD55 (group 2; n=5) donor pigs using similar immunosuppression. Cardiac biopsies were obtained 30 min after organ reperfusion. Rejection was characterized by histology and immunohistology. Intragraft gene expression, serum non-Gal antibody, and antibody recovered from rejected hearts were analyzed., Results: HAR of a GTKO heart was observed. Remaining grafts developed delayed xenograft rejection. Median survival was 21 and 28 days for groups 1 and 2, respectively. Vascular antibody deposition was uniformly detected 30 min after organ reperfusion and at explant. A higher frequency of vascular C5b deposition was seen in GTKO organs at explant. Serum non-Gal antibody, antibody recovered from the graft, and intragraft gene expression were similar between the groups., Conclusion: HAR of GTKO hearts without hCD55 may occur. Expression of hCD55 seemed to restrict local complement activation but did not improve graft survival. Chronic vascular antibody deposition with evidence of protracted endothelial cell activation was seen. These observations suggest that non-Gal antibody-induced chronic endothelial cell activation coupled to possible hemostatic incompatibilities may be the primary stimulus for delayed xenograft rejection of GTKO hearts. To avoid possible HAR, future clinical studies should use donors expressing human complement regulatory proteins in the GTKO background.

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.

Objective: We studied the concordance of transgene expression in the transplanted heart using bicistronic adenoviral vector coding for a transgene of interest (human carcinoembryonic antigen: hCEA - beta human chorionic gonadotropin: betahCG) and for a marker imaging transgene (human sodium iodide symporter: hNIS)., Methods: Inbred Lewis rats were used for syngeneic heterotopic cardiac transplantation. Donor rat hearts were perfused ex vivo for 30 min prior to transplantation with University of Wisconsin (UW) solution (n=3), with 10(9) pfu/ml of adenovirus expressing hNIS (Ad-NIS; n=6), hNIS-hCEA (Ad-NIS-CEA; n=6) and hNIS-betahCG (Ad-NIS-CG; n=6). On postoperative day (POD) 5, 10, 15 all animals underwent micro-single photon emission computed tomography/computed tomography (SPECT/CT) imaging of the donor hearts after tail vein injection of 1000 microCi (123)I and blood sample collection for hCEA and betahCG quantification., Results: Significantly higher image intensity was noted in the hearts perfused with Ad-NIS (1.1+/-0.2; 0.9+/-0.07), Ad-NIS-CEA (1.2+/-0.3; 0.9+/-0.1) and Ad-NIS-CG (1.1+/-0.1; 0.9+/-0.1) compared to UW group (0.44+/-0.03; 0.47+/-0.06) on POD 5 and 10 (p<0.05). Serum levels of hCEA and betahCG increased in animals showing high cardiac (123)I uptake, but not in those with lower uptake. Above this threshold, image intensities correlated well with serum levels of hCEA and betahCG (R(2)=0.99 and R(2)=0.96, respectively)., Conclusions: These data demonstrate that hNIS is an excellent reporter gene for the transplanted heart. The expression level of hNIS can be accurately and non-invasively monitored by serial radioisotopic SPECT imaging. High concordance has been demonstrated between imaging and soluble marker peptides at the maximum transgene expression on POD 5.

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 I 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 (rho=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.

Background: Adenovirus serotype 5 has remained the pre-eminent vector in pre-clinical gene therapy applications in cardiac transplantation. Concerns over the potential effects of adenoviral vectors on the later development of cardiac allograft vasculopathy (CAV) are addressed in this study., Methods: Hearts (n = 22) harvested from Brown Norway rats were perfused ex vivo with either University of Wisconsin (UW) solution with no virus, Ad-CMV-LacZ or Ad-CMV-Null. Donor hearts were transplanted heterotopically into the abdomen of Lewis rats. All recipients received cyclosporine for the duration of the experiment. Transplanted hearts were recovered for analysis at 120 days. Sections of the heart were stained with elastic-van Gieson stain for morphometric analysis of the vessels to ascertain the degree of vascular luminal occlusion. Hematoxylin-eosin staining facilitated diagnosis of chronic rejection., Results: Seventy-seven percent of transplanted hearts showed signs of chronic rejection with no difference in the proportion of animals between groups (p = 0.797). No difference was noted in the degree of vascular luminal occlusion between the Ad-Null (0.57 +/- 0.22), Ad-LacZ (0.62 +/- 0.19) and UW (0.47 +/- 0.29) groups (p = 0.653)., Conclusions: Vascularized cardiac allografts transplanted from Brown Norway to Lewis rats demonstrated cardiac allograft vasculopathy CAV at 120 days. Adenoviral perfusion of the donor heart ex vivo did not affect the development of CAV.

Introduction: Endomyocardial biopsy is the standard means of establishing cardiac allograft rejection diagnosis. The efficacy of this procedure in xenotransplantation has not been determined. In this study we compare the histology of right ventricular endomyocardial biopsy specimens with the corresponding full cross sections of explanted right ventricle (RV). We also compare RV with the related left ventricle (LV) cross sections., Methods: Heterotopic CD46 pig-to-baboon cardiac xenotransplants (n = 64) were studied. RV endomyocardial biopsy specimens were taken at cardiac explant by using a standard bioptome (n = 24) or by sharp dissection (n = 40). Hematoxylin and eosin stained sections of RV and LV cross-section and RV endomyocardial biopsy specimens were compared in a blinded fashion. Characteristics of delayed xenograft rejection and a global assessment of ischemia were scored from 0 to 4 according to the percentage of myocardium involved (0, 0%; 1, 1%-25%; 2, 26%-50%; 3, 51%-75%; and 4, 76%-100%)., Results: Median graft survival was 30 days (range, 3-137 days). Linear regression analysis of histology scores demonstrated that specimens from both bioptome and sharp dissection equally represented the histology of the RV cross section. Global ischemic injury was strongly correlated between RV and RV endomyocardial biopsy (R(2) = 0.84) and between RV and LV cross sections (R(2) = 0.84). Individual characteristics of delayed xenograft rejection showed no significant variation between RV and RV endomyocardial biopsy or between RV and LV (p < 0.05)., Conclusions: These results indicate that delayed xenograft rejection is a widespread process involving both right and left ventricles similarly. This study shows that histologic assessment of RV endomyocardial biopsy specimens is an effective method for the monitoring of delayed xenograft rejection after cardiac xenotransplantation.

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 x 10(11) 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.