Christopher Burlak

• PhD
• Professor at University of Miami

Objective To evaluate the clinically relevant anti‐CD40 antibody iscalimab for baseline immunosuppression in a preclinical pig‐to‐rhesus renal xenograft model. Summary Background Data CD40/CD40L co‐stimulation blockade‐based immunosuppression has been more successful than calcineurin‐based protocols in prolonging xenograft survival in preclinical...

Attack of donor tissues by pre‐formed anti‐pig antibodies is well known to cause graft failure in xenotransplantation. Genetic engineering of porcine donors to eliminate targets of these pre‐formed antibodies coupled with advances in immunosuppressive medicines have now made it possible to achieve extended survival in the pre‐clinical pig‐to‐non‐hu...

Pig liver xenotransplantation is limited by a thrombocytopenic coagulopathy that occurs immediately following graft reperfusion. In vitro and ex vivo studies from our lab suggested that the thrombocytopenia may be the result of a species incompatibility in platelet glycosylation. Realization that platelet α‐granules contain antibodies caused us to...

Organ supply remains inadequate to meet the needs of many patients who could benefit from allotransplantation. Xenotransplantation, the use of animals as organ donors, provides an opportunity to alleviate this challenge. Pigs are widely accepted as the ideal organ donor, but humans and nonhuman primates have strong humoral immune responses to porci...

Genetically engineered pigs with multiple gene deletions and insertions are predicted to extend porcine to human xenograft survival. Several genes have been successfully knocked out and inserted, yet more have failed to produce viable animals for unexplained reasons. The effects of gene editing on cellular homeostasis may be the cause of reduced em...

A safe, efficacious, and clinically applicable immunosuppressive regimen is necessary for islet xenotransplantation to become a viable treatment option for diabetes. We performed intraportal transplants of wildtype adult porcine islets in 25 streptozotocin‐diabetic cynomolgus monkeys. Islet engraftment was good in 21, partial in 3 and poor in 1 rec...

The human leukocyte antigen G1 (HLA-G1), a non-classical class I major histocompatibility complex (MHC-I) protein, is a potent immunomodulatory molecule at the maternal/fetal interface and other environments to regulate the cellular immune response. We created GGTA1⁻/HLAG1⁺ pigs to explore their use as organ and cell donors that may extend xenograf...

Objective: Pig-to-primate renal xenotransplantation is plagued by early antibody mediated graft loss which precludes clinical application of renal xenotransplantation. We evaluated whether temporary complement inhibition with anti-C5 antibody Tesidolumab could minimize the impact of early antibody mediated rejection in rhesus monkeys receiving pig...

Understanding the anti-carbohydrate antibody response toward epitopes expressed on porcine cells, tissues, and organs is critical to advancing xenotransplantation toward clinical application. In this study, we determined IgM and IgG antibody specificities and relative concentrations in five cynomolgus monkeys at baseline and at intervals following...

Objectives Islet transplantation is an emerging treatment option for type 1 diabetes but its application is limited by the shortage of human pancreas donors. Characterization of the N- and O-glycan surface antigens that vary between human and genetically engineered porcine islet donors could shed light on targets of antibody mediated rejection. Me...

Background Genetically engineered porcine donors are a potential solution for the shortage of human organs for transplantation. Incompatibilities between humans and porcine donors are largely due to carbohydrate xenoantigens on the surface of porcine cells, provoking an immune response which leads to xenograft rejection. Materials and Methods Mult...

Introduction: Macrophages contribute to xenograft rejection by direct cytotoxicity and by amplifying T cell-mediated immune responses. It has been shown that transgenic expression of hCD47 protects porcine cells from human macrophages by restoring the CD47-SIRPα self-recognition signal. It has also been reported that the long 3' untranslated regio...

Progress has been made in overcoming antibody‐mediated rejection of porcine xenografts by deleting pig genes that produce unique carbohydrate epitopes. Pigs deficient in galactose α‐1,3 galactose (gene modified: GGTA1) and neu5Gc (gene modified: CMAH) have reduced levels of human antibody binding. Previously we identified α‐fucose as a glycan that...

Pigs are especially useful large animal models, however, limited availability of commercially available antibodies for immunoblotting presents a significant obstacle facing preclinical xenotransplantation research. Major histocompatibility complex class I (MHC‐I) molecule expression enhancement by nucleotide‐binding oligomerization domain (NOD)‐lik...

Background: Xenotransplantation of porcine islets has emerged in recent decades as a potential treatment for type 1 diabetes (T1D). Current methods of detection, indicative of successful engraftment, occur downstream of actual islet death. Epigenetic biomarkers can be detected in circulating cell-free DNA (cfDNA) to provide an earlier indication o...

The ever‐increasing disparity between the lack of organ donors and patients on the transplant waiting list is increasing worldwide. For the past several decades xenotransplantation has led the way to correct this deficit and remains clearly the only feasible option to provide a means to meet the demand for patients in need of an organ transplant. X...

The CRISPR/Cas9 gene editing system has enhanced the development of genetically engineered animals for use in xenotransplantation. Potential limitations to the CRISPR/Cas9 system impacting the development of genetically engineered cells and animals include the creation of off-target mutations. We sought to develop a method to reduce the likelihood...

Background: Engineering of α-Galactosyltransferase gene-knockout pigs circumvented hyperacute rejection of pig organs after xenotransplantation in non-human primates. Overcoming this hurdle revealed the importance of non-α-Gal carbohydrate antigens in the immunobiology of acute humoral xenograft rejection. Methods: This study analyzed serum from...

Handmade cloning is a zona-free nuclear transfer approach and an economical, efficient, and simple micromanipulation-free alternative to dolly based traditional cloning (TC). In this study, based on handmade cloning with minor modifications, an optimized bi-oocyte fusion (BOF) cloning method was established to produce GGTA1 KO porcine embryos using...

Immune tolerance to allografts has been pursued for decades as an important goal in transplantation. Administration of apoptotic donor splenocytes effectively induces antigen-specific tolerance to allografts in murine studies. Here we show that two peritransplant infusions of apoptotic donor leukocytes under short-term immunotherapy with antagonist...

Tick-borne flaviviruses (TBFVs), including Powassan virus and tick-borne encephalitis virus cause encephalitis or hemorrhagic fevers in humans with case-fatality rates ranging from 1–30%. Despite severe disease in humans, TBFV infection of natural rodent hosts has little noticeable effect. Currently, the basis for resistance to disease is not known...

Recent advances in xenotransplantation explore immunologic protection of islets that may deliver the first pig tissue to the clinic. The following summarizes the advancements that may help to enhance long term survival of xenografts with special reference to pig islets as a therapy for type 1 and type 2 diabetic patients

Significance The aggregation of α-synuclein (aSyn) is a pathological hallmark of Parkinson’s disease. Here we show that the enzymatic component of the innate inflammation system, known as caspase-1, hydrolyzes aSyn, rendering it aggregation-prone.

Ib;heart xenotransplantation

CD46;porcine endogenous retroviruses;xenotransplantation

Gal;islets;non-human primate;pig; SLA ;xenotransplantation

CRISPR;cytomegalovirus;islet;lung;xenotransplantation

Asialoglycoprotein receptor-1 (ASGR1) mediates capture and phagocytosis of platelets in pig-to-primate liver xenotransplantation. However, thrombocytopenia is also observed in xenotransplantation or xenoperfusion of other porcine organs than liver. We therefore assessed ASGR1 expression as well as ASGR1-mediated xenogeneic platelet phagocytosis in...

Background The lethal thrombocytopenia that accompanies liver xenotransplantation is a barrier to clinical application. Human platelets are bound by the asialoglycoprotein receptor (ASGR) on pig sinusoidal endothelial cells and phagocytosed. Inactivation of the ASGR1 gene in donor pigs may prevent xenotransplantation-induced thrombocytopenia.Method...

Background The origins of neointimal smooth muscle cells that arise following vascular injury remains controversial. Studies have suggested that these cells may arise from previously differentiated medial vascular smooth muscle cells, resident stem cells or blood born progenitors. In the current study we examined the contribution of the previously...

Background Manipulating the pig genome to increase compatibility with human biology may facilitate the clinical application of xenotransplantation. Genetic modifications to pig cells have been made by sequential recombination in fetal fibroblasts and liver‐derived cells followed by cross‐breeding or somatic cell nuclear transfer. The generation of...

Kupffer cells (KC) play a critical role in both liver physiology and the pathogenesis of various liver diseases. Isolated primary KC have a limited lifespan in culture, and due to the relatively low number obtained, limit their study in vitro. Here, a cytokine-producing immortalized KC (ImKC) line was established from transgenic mice that express t...

Significance Many complex diseases are caused by aberrant protein–protein interactions or protein misfolding, which present immense challenges for drug discovery. Here, we present the design, synthesis, and usage of a diverse library of potential pharmacological chaperones that could interfere with undesirable protein–protein interactions and preve...

Background: Pig erythrocytes are potentially useful to solve the worldwide shortage of human blood for transfusion. Domestic pig erythrocytes, however, express antigens that are bound by human preformed antibodies. Advances in genetic engineering have made it possible to rapidly knock out the genes of multiple xenoantigens, namely galactose α1,3 g...

Xenotransplantation using genetically modified pig organs could solve the donor organ shortage problem. Two inactivated genes that make humans unique from pigs are GGTA1 and CMAH, the products of which produce the carbohydrate epitopes, aGal and Neu5Gc that attract preformed human antibody. When the GGTA1 and CMAH genes were deleted in pigs, human...

Significance Mutations in the gene encoding for leucine-rich-repeat kinase 2 (LRRK2) are a common cause of Parkinson disease (PD). To understand how LRRK2 causes PD, we need to understand its normal functions and how they are altered by disease-causing mutations. This effort has been hampered by the lack of appropriate samples, which led to some co...

The temporary or long-term xenotransplantation of pig organs into people would save thousands of lives each year if not for the robust human antibody response to pig carbohydrates. Genetically engineered pigs deficient in galactose α1,3 galactose (gene modified: GGTA1) and N-glycolylneuraminic acid (gene modified: CMAH) have significantly improved...

Patients in fulminant hepatic failure currently do not have a temporary means of support while awaiting liver transplantation. A potential therapeutic approach for such patients is the use of extracorporeal perfusion with porcine livers as a form of "liver dialysis". During a 72-h extracorporeal perfusion of porcine livers with human blood, porcine...

Background: Antibody-mediated rejection continues to be an obstacle for xenotransplantation despite development of α1,3-galactosyltransferase knockout (GTKO) pigs. Fibronectin (Fn) from GTKO pigs was identified as a xenoantigen in baboons. N-glycolylneuraminic acid (Neu5Gc), similar to galactose α1,3-galactose, is an antigenic carbohydrate found i...

Clinical xenotransplantation is not possible because humans possess antibodies that recognize antigens on the surface of pig cells. Galα-1,3-Gal (Gal) and N-glycolylneuraminic acid (Neu5Gc) are two known xenoantigens. We report the homozygous disruption of the α1, 3-galactosyltransferase (GGTA1) and the cytidine monophosphate-N-acetylneuraminic aci...

Pigs are currently the preferred species for future organ xenotransplantation. With advances in the development of genetically modified pigs, clinical xenotransplantation is becoming closer to reality. In preclinical studies (pig-to-nonhuman primate), the xenotransplantation of livers from pigs transgenic for human CD55 or from α1,3-galactosyltrans...

Xenotransplantation has the potential to solve the critical shortage of human organs available for allotransplantation. The major barrier to porcine liver xenotransplantation is sequestration of human platelets causing thrombocytopenia. Porcine liver sinusoidal endothelial cells (LSEC) bind and phagocytose human platelets at least in part through b...

Cooper DKC, Ekser B, Burlak C, Ezzelarab M, Hara H, Paris L, Tector AJ, Phelps C, Azimzadeh AM, Ayares D, Robson SC, Pierson RN III. Clinical lung xenotransplantation – what donor genetic modifications may be necessary? Xenotransplantation 2012; 19: 144–158. © 2012 John Wiley & Sons A/S. Abstract: Barriers to successful lung xenotransplantation app...

Methods and techniques used to detect apoptosis have benefited from advances in technologies such as flow cytometry. With a large arsenal of lasers, fluorescent labels, and readily accessible biological targets, it is possible to detect multiple targets with unique combinations of fluorescent spectral signatures from a single sample. Traditional fl...

Human preformed antibodies continue to recognize porcine xenografts, despite the advent of α-galactosyltransferase knockout (GTKO) pigs. This study examined the potential reactivity of human preformed IgG and IgM antibodies toward antigens in the GTKO pig liver. Human serum was analyzed for the concentration of IgG, IgM, anti-αgal antibody, anti-no...

Acute thrombocytopenia was revealed as a limiting factor to porcine liver xenotransplantation from in vitro and in vivo studies using porcine liver in human and baboon transplant models. The asialoglycoprotein receptor 1 (ASGR1) on liver sinusoidal endothelial cells (LSEC) and macrophage antigen complex-1 (Mac-1) on Kupffer cells (KC) mediate plate...

Hepatic failure has been treated successfully with clinical extracorporeal perfusions of porcine livers. However, dog-to-pig and pig-to-baboon liver xenotransplant models have resulted in severe bleeding secondary to liver xenograft-induced thrombocytopenia. Kupffer cells (KC) are abundant phagocytic cells in the liver. KC express the CD11b/CD18 re...

Porcine liver xenografts represent a potential solution to the organ shortage, but thrombocytopenia occurs within minutes to hours after xenotransplantation, preventing clinical application. Recently, it was discovered that porcine liver sinusoidal endothelial cells (LSEC) bind and phagocytose human platelets. We examined the role of ASGR1 in bindi...