The biggest need is for kidneys. According to The New York Times: “More than 800,000 Americans have kidney failure, and over 100,000 are on a waiting list for a transplant.” However, due to the shortage of available human donor organs, the annual count of kidney transplants conducted remains below 25,000. This scarcity leads to the unfortunate outcome of thousands of individuals on the waiting list with one estimate claiming that as many as 40% of listed patients die within 5 years while waiting for a kidney transplant.
Given the shortage of human organs for transplant, scientists have explored the idea of using organs from other mammals such as pigs that are more readily available. Transplants that occur between different species are referred to as xenotransplants. The choice of pigs over other mammalian alternatives such as nonhuman primates (NHPs) has to do with organ size (not too small, not too big, Figure 1), abundant supply (more readily available than from nonhuman primates), and more compatible physiology and immunology compared to distantly-related mammals.
The two main challenges to xenotransplantation with pig organs have been immune rejection and viral infection. Organ transplant between humans suffers from rejection because the donor organ is considered non-self by the recipient immune system. Pig organs have the added disadvantage of being even more likely to be recognized as foreign because of nonhuman antigens. In addition, because they are often maintained in unsanitary conditions, pigs contain numerous viruses which can be transmitted from donated organ to host especially when the recipient is immunosuppressed. The most insidious of these are porcine endogenous retroviruses (PERV) which are embedded in the genome of the pig, and can only be removed by genetic means.
There are three types of immune rejection of organs; two of which are mediated by the humoral immune system, i.e. antibodies, and the third by cell-mediated immunity, i.e. T cells. The first is hyperacute rejection which is "mediated by the xenoreactive natural antibodies (XNAs) from the recipient and occur within minutes or hours after the restoration of xenograft blood circulation. XNAs bind to the xenoantigens of the xenograft and activate the classical complement pathway in the recipient, resulting in interstitial hemorrhage, edema and thrombosis of the xenograft, and finally leading to inactivation and necrosis of the xenograft within a few minutes or hours.”
The second is acute vascular rejection which "usually occurs two to three days after xenotransplantation.... This rejection may be caused by the interaction between the xenograft and the recipient’s xenoantibodies, macrophages, or platelets. The combination of XNAs and xenograft endothelial causes the activation of xenograft endothelial cells and receptor of macrophage, which induces the expression of various specific proteins, including cytokines, endothelial adhesion molecules, and blood coagulation factors. These factors can cause inflammation, thrombosis, cellulose precipitation or diffuse blood clotting in the xenograft, which lead to xenograft loss or inactivation." Thus, one of the key characteristics of acute vascular rejection is inflammation.
Finally acute cellular rejection is the result of cell-mediated immunity by NK cells and T lymphocytes as opposed to the antibody-mediated immunity of the first two rejection responses: "After xenotransplantation, antigen presenting cells present xenoantigen epitopes to recipient CD4+ T Cells via xenogeneic MHC class Ⅱ molecules. The xenoantigen of the graft can also be presented to CD8+ T cells via the MHC class I molecules of the recipient. The proliferation of CD4+ T cells and CD8+ T cells induces the production of interleukin-2 (IL-2) and interferon-γ (IFN-γ), which induces a series of immune rejection reactions." The activated NK cells and CD8+ T cells can directly attack the transplanted pig organ.
Because hyperaccute rejection is the fastest and most deadly, a large body of research has been devoted to ameliorating this response. Importantly, scientists have identified galactose-α1,3-galactose, often abbreviated as α-Gal, as the culprit xenoantigen recognized by human antibodies as foreign. α-Gal is a disaccharide (two simple sugar molecules linked together by a glycosidic bond) carbohydrate found in the cells of many mammals, but is not produced by humans and other primates. It is synthesized by the enzyme α-1, 3-galactosyltransferase (GGTA1) and then incorporated into glycoproteins and glycolipids on the cell surface where it can be recognized by antibodies.
Fortuitously, it is possible to genetically engineer pigs in which the GGTA1 gene is deleted so that no α-Gal is produced. Organs from these genetically modified pigs termed GTKO (for GGTA1 knock out) survive for much longer times when transplanted into non-human primates (NHPs) mainly because the occurrence of hyperacute rejection was dramatically decreased or even completely eliminated. Further genetic engineering has resulted in pigs containing deletions or transgenes of additional genes involved in acute vascular rejection and acute cellular rejection.
Thanks to these genetic modifications, the use of pig organs for xenotransplantion has been gaining momentum although they have never been placed in a live human recipient yet. A new article in The New York Times described how two different groups have reported transplanted pig kidneys able to function in brain dead patients for a week or longer:
“Researchers at the University of Alabama at Birmingham published a peer-reviewed study showing that modified pig kidneys performed complex life-sustaining functions in a brain-dead patient for a full week.In an apparent response, surgeons at NYU Langone Health announced that a kidney from a genetically modified pig continued to function well after 32 days in a brain-dead patient maintained on a ventilator, the longest period for such an experiment.”
The use of brain-dead patients is less risky than using live patients while still being a rigorous test for the ability of the transplanted kidney to function in humans with a still active immune system. The brain-dead patient is placed on a ventilator which oxygenates the blood and helps keep the heart beating (the heart does not need nerve impulses from the brain to beat).
The kidney is a critical organ performing a wide range of essential functions from removing waste in the blood to be excreted as urine to maintaining fluid and electrolyte balance to blood pressure control. The research group from UAB assessed kidney function in terms of urine production and the level of creatine in the blood. Creatine is a waste product that results from the breakdown of creatine phosphate, an energy storage molecule found in muscle cells, and it can build up in the blood over time. The ability to secrete creatine into the urine from the blood is an indicator of the general filtration capacity of the kidney.
The results were encouraging (JAMA Surgery):
"[T]he xenografts made urine, producing more than 37 L in the first 24 hours. Urine concentrated over time, with concurrent decreases in urine volume to a median of 14.1 L (IQR, 13.8-20 L) on PODs 1 to 3 and a median of 5.1 L (IQR, 5-6 L) on PODs 4 to 7. Before xenotransplant, serum creatinine was 3.9 mg/dL after cessation of dialysis and bilateral native nephrectomy. After xenotransplant, serum creatinine decreased to 1.9 mg/dL within the first 24 hours, normalized to 1.1 mg/dL at 48 hours, remained within normal limits through study duration, and was 0.9 mg/dL on POD 7 at study completion."
Interestingly, the UAB transplanted pig kidney contained 10 gene modifications (10-GE), whereas the kidneys used at N.Y.U. Langone Health had only one genetic modification. The 10-GE kidney including the following genetic changes: "[T]argeted insertion of two human complement inhibitor genes (hDAF, hCD46), two human anticoagulant genes (hTBM, hEPCR), and two immunomodulatory genes (hCD47, hHO1), as well as deletion (knockout) of 3 pig carbohydrate antigens and the pig growth hormone receptor gene." Adding human complement inhibitor genes may help with the hyperacute rejection while the anticoagulant genes can reduce excessive coagulation from inflammation during acute vascular rejection. The CD47 gene plays a role in T cell maturation which can influence the cell-mediated rejection by perhaps making the pig cells appear more human to the recipient immune system.
The GGTA1 knockout (GTKO) described above was one of the three deletions of pig carbohydrate antigens, and was most likely the single genetic modification in the NYU pig kidney.
These exciting results come on the heels of the announcement in 2022 that a medical team at the University of Maryland conducted a heart transplant procedure using a genetically modified pig's heart on a 57-year-old individual suffering from heart failure. The patient survived for two months before passing away when the pig heart failed. A number of factors contributed to the deterioration of the pig heart including immune rejection and potentially viral infection in the heart, but the fact it lasted two months in a human host was an amazing accomplishment.
The steady progress made with xenotransplantation using genetically modified pig organs is impressive. An alternate approach is to grow human or humanized organs in pigs for transplantation. The procedure is to inject human stem cells into the early animal embryo to create a hybrid or chimeric embryo consisting of both human and animal cells (QH). The advantage is that immune rejection from nonhuman antigens would be minimized, and it may even be possible to use stem cells from the recipient to further increase compatibility.
Figure 1. A pig kidney being prepared for transplantation into a brain-dead human subject (Reuters).
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