Genetically engineered pig heart transplantation in non-human primates

Abstract

Background: Improvement in gene modifications of donor pigs has led to the prevention of early cardiac xenograft rejection and significantly prolonged cardiac xenograft survival in both heterotopic and orthotopic preclinical non-human primate (NHP) models. This progress formed the basis for FDA approval for compassionate use transplants in two patients.

Methods: Based on our earlier report of 9-month survival of seven gene-edited (7-GE) hearts transplanted (life-supporting orthotopic) in baboons, we transplanted 10 gene-edited pig hearts into baboons (n = 4) using non-ischemic continuous perfusion preservation (NICP) and immunosuppression regimen based on co-stimulation blockade by anti-CD40 monoclonal antibody. This pivotal study expands on the 7-GE backbone, with 3 additional gene edits, using 10-GE pigs as donors to baboon recipients.

Results: 10 GE cardiac xenografts provide life-supporting function up to 225 days (mean 128 ± 36 days) in a non-human primate model. Undetectable or latent porcine cytomegalovirus (PCMV) does not influence cardiac xenograft survival in this study but still needs more exploration with a larger cohort. Xenograft histology demonstrates adipose (Fat) deposition (n = 1), chronic vasculopathy (n = 1), micro and macro thrombosis, and acute cellular rejection (n = 1).

Conclusions: These data demonstrate that 10 GE cardiac xenografts have variable cardiac xenograft survival in NHP due to perhaps presence of 4th antigen and require further study. However, these 10GE organs may be suitable for clinical cardiac xenotransplantation and have already been utilized in two human cases.

Fig. 1. Cardiac xenograft survival.

Kaplan–Meir survival curve of cardiac xenograft recipients receiving 7, 9, and 10 GE edit donor pigs.

Fig. 2. Hematological parameter.

Hematology (A) White blood counts (WBC), hematocrit (HCT), platelets, and coagulation parameter of cardiac xenograft recipient receiving from 10GE donor pig.

Fig. 3. Immunological monitoring.

A Immunophenotyping of T and B cells. B Non‐Gal IgG and IgM levels; anti‐pig non‐Gal IgG and IgM antibodies levels in mean fluorescence intensity (MFI) were measured in cardiac xenograft grafts baboon recipient’s serum by flow cytometry using 10GE porcine endothelial cells.

Fig. 4. Serum cytokines levels.

IL-6, TNF-α, IFN-γ, IL-12p40, IL-17, IL-23, IL-1β, IFN-β, IP-10 (CXCL10) levels in the serum of cardiac xenotransplant recipients.

Fig. 5. 10GE cardiac xenograft measurements post-transplantation according to transthoracic echocardiogram (TTE).

A LV ejection fraction was measured longitudinally after transplantation. B Septal thickness, left ventricular (LV) dimension in end-diastole, relative wall thickness (rWT), posterior wall thickness, LV mass. LV mass percent change was calculated according to the average LV mass in the 30 days after transplantation and 30 days before euthanasia.

Fig. 6. Histological and Immunohistochemistry evaluation of the 10 GE cardiac xenografts.

A Hematoxylin and Eosin (H&E) staining of a section from the explanted heart from the necropsy demonstrating mononuclear cell infiltrate (i), arterial intimal thickening (ii), chronic vasculopathy (iii), and microvascular thrombosis (iv). B Immunohistochemistry of right ventricle (RV) section demonstrating complement (C4d & C3) and immunoglobulin (IgG&IgM) staining.

Fig. 7. Non-gal anti-pig antibodies in cardiac xenotransplantation recipients.

A Cumulative mean non-gal anti-pig antibodies (IgG and IgM antibodies) after cardiac xenotransplantation in cardiac xenograft recipients receiving 7, 9, and 10GE donor pig xenograft. B Pre-existing donor-specific anti-pig IgG and IgM antibodies in the baboon recipient before transplantation and cardiac xenograft outcomes.