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 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.

Cardiac xenotransplantation: recent preclinical progress with 3-month median survival.

OBJECTIVES: Transplantation is limited by a lack of human organ donors. Organs derived from animals, most likely the pig, represent a potential solution to this problem. For the heart, 90-day median graft survival of life-supporting pig hearts transplanted to nonhuman primates has been considered a reasonable standard for entry into the clinical arena. Overcoming the immune barrier to successful cardiac xenotransplantation is most appropriately first explored with the non-life-supporting heterotopic model. METHODS: We performed a series of 7 heterotopic heart transplantations from CD46 transgenic pigs to baboons using a combination of therapeutic agents largely targeted at controlling the synthesis of anti-pig antibodies. Rituximab (anti-CD20) and Thymoglobulin (rabbit antithymocyte globulin [ATG]; SangStat Medical Corp, Fremont, Calif) were used as induction therapy. Baseline immunosuppression consisted of splenectomy, tacrolimus, sirolimus, steroids, and TPC (an anti-Gal antibody therapeutic). Rejection events were not treated. RESULTS: By using Kaplan-Meier analysis, median graft survival was 96 days (range, 15-137 days; 95% confidence interval, 38-99 days). Only 2 grafts were lost as a result of rejection, as defined by cessation of graft palpation. There was no evidence of a consumptive coagulopathy, infectious complications were treatable, and no posttransplantation lymphoproliferative disorders occurred. No cellular infiltration was observed. CONCLUSIONS: This study reports the longest median survival to date (96 days) of pig hearts transplanted heterotopically into baboons. Duplication of these results in the orthotopic life-supporting position could bring cardiac xenotransplantation to the threshold of clinical application.

Warfarin or low-molecular-weight heparin therapy does not prolong pig-to-primate cardiac xenograft function.

Microvascular thrombosis is a prominent feature in cardiac delayed xenograft rejection (DXR). We investigated the impact of warfarin or low-molecular-weight heparin (LMWH) anti-coagulation on xenograft function using a heterotopic pig-to-primate model. Donor hearts were from CD46 transgenic pigs and baboon immunosuppression included tacrolimus, sirolimus, anti-CD20 and TPC, an alpha-galactosyl-polyethylene glycol conjugate. Three groups of animals were studied. Group 1 (n = 9) was treated with warfarin, Group 2 (n = 13) with LMWH and Group 3, received no anti-coagulant drugs. The median duration of xenograft function was 20 days (range 3-62 days), 18 days (range 5-109 days) and 15 days (range 4-53 days) in Groups 1 to 3 respectively. Anti-coagulation achieved the targeted international normalized prothrombin ratio (INR) and anti-factor Xa levels consistent with effective in vivo therapy yet, no significant impact on median xenograft function was observed. At rejection, a similar histology of thrombosis and ischemia was apparent in each group and the levels of fibrin deposition and platelet thrombi in rejected tissue was the same. Anti-coagulation with warfarin or LMWH did not have a significant impact on the onset of DXR and microvascular thrombosis. However, a role for specific anti-coagulant strategies to achieve long-term xenograft function cannot be excluded.

Prevention, detection, and management of early bacterial and fungal infections in a preclinical cardiac xenotransplantation model that achieves prolonged survival.

BACKGROUND: We analyzed bacterial and fungal infectious complications in a cohort of 16 consecutive experiments with the longest surviving cardiac xenografts to date. METHODS: Transgenic, porcine-to-baboon, heterotopic (abdomen) cardiac xenotransplantation was performed in 16 consecutive experiments, using rapamycin, tacrolimus, corticosteroids, anti-CD20 monoclonal antibody, and an alpha-Gal-PEG polymer, as immunosuppression. Prophylactic anti-microbials included i.v. trimethoprim/sulfamethoxazole, oral ganciclovir/valganciclovir, and oral itraconazole. An episode of bacterial infection was defined as a positive blood and/or wound culture with: leukocytosis, fever >101.5 degrees F, and/or clinical deterioration. RESULTS: Mean graft survival was 71 +/- 29 days; the longest was 113 days. There were 23 episodes of bacterial infection; 14 resolved with treatment. The mean time to the first episode of infection was 44 +/- 21 days (n=12). Eight of 16 deaths were due to infection: two bacterial-only, two cytomegalovirus (CMV) only, four both bacterial and CMV, and none fungal. The frequency of infection was 1, 2.8, and 1.8 episodes/100 survival days, respectively, for animals whose grafts survived for 30 to 59, 60 to 89, and >90 days. CMV infection (reviewed in detail in a separate communications) was due to baboon CMV, and was associated with low serum levels of ganciclovir. CONCLUSION: In a cardiac xenograft model that achieved prolonged (>3 months) survival, bacteremia was common, but usually reversible, and fungal infection was prevented with prophylaxis. The level of immunosuppression required to achieve clinically meaningful xenograft survival is associated with a level of bacterial and fungal infectious complications that is manageable and similar to the early clinical experiences in human transplantation. Further research will determine if the viral infectious complications observed in these experiments can be reduced by optimizing blood levels of anti-viral prophylaxis and monitoring viral polymerase chain reaction levels.

Cardiac xenotransplantation: progress toward the clinic.

BACKGROUND: Animal organs could satisfy the demand for solid organ transplants, which currently exceeds the limited human donor supply. Hyperacute rejection, the initial immune barrier to successful xenotransplantation, has been overcome with pig donors transgenic for human complement regulatory proteins. Delayed xenograft rejection, thought to be mediated by anti-pig antibodies predominantly to Gal antigens, is currently regarded as the major barrier to successful xenotransplantation. A median graft survival of 90 days in the life-supporting position is considered a reasonable initial standard for consideration of entry to the clinic. METHODS: A series of 10 heterotopic heart transplants from CD46 transgenic pigs to baboons was completed. Immunosuppression consisted of splenectomy, Rituximab (Anti-CD20), tacrolimus, sirolimus, corticosteroids, and TPC. Thymoglobulin (Rabbit Anti-Thymocyte Globulin) was used to treat putative rejection episodes. RESULTS: Median graft survival was 76 days (range 56-113 days, n = 9). Only three grafts were lost to rejection. The remaining grafts lost were due to recipient mortality with baboon cytomegalovirus (BCMV) being the major cause (n = 4). No cellular infiltrates were present as a manifestation of rejection. Three hearts showed chronic graft vasculopathy. CONCLUSIONS: The median survival of 76 days in this group of heterotopic porcine-to-baboon cardiac xenografts represents a major advance over the median 27-day survival reported in the literature. Cellular rejection may not constitute a direct major barrier to xenotransplantation. A median survival of 90 days may be achievable with better control of BCMV infection. If further studies in the orthotopic position replicate these outcomes, criteria considered appropriate for clinical application of cardiac xenotransplantation would be approached.

Effective antiplatelet therapy does not prolong transgenic pig to baboon cardiac xenograft survival.

BACKGROUND: Microvascular thrombosis is a prominent characteristic of delayed xenograft rejection, therefore the effects of antiplatelet therapy with aspirin and clopidogrel on long-term cardiac xenograft function was investigated in a heterotopic pig-to-baboon cardiac transplant model. METHODS: Donor hearts from human CD46 transgenic pigs were transplanted heterotopically to baboons. The recipients received immunosuppression that included tacrolimus, sirolimus, corticosteroids, anti-CD20 monoclonal antibody and TPC, an alpha-galactosyl-polyethylene glycol conjugate. In group 1 (n = 9) in addition to immunosuppression, the recipients received combination therapy consisting of aspirin (80 mg/day) and clopidogrel (75 mg/day) beginning 2 days after transplant and continuing until cessation of graft function. Antiaggregatory efficacy was evaluated by platelet aggregation assay. In group 2 (n = 9) antiplatelet drugs were not given. RESULTS: Functional assays confirmed inhibition of platelet aggregation in group 1 suggesting sufficient systemic effects of the treatment. However, anticoagulant therapy did not result in significant prolongation of xenograft function (group 1: median survival 22 days, range 15 to 30 days; group 2: median survival 15 days, range 4 to 53 days). Histologic analysis at rejection revealed no difference in the level of platelet containing thrombi between the groups. CONCLUSIONS: Inhibition of platelet aggregation by a combination of aspirin and clopidogrel did not have a significant impact on the length of xenograft survival or on the development of microvascular thrombosis in this pig-to-primate model.