American Journal of Transplantation 2014; 14: 2275–2287 Wiley Periodicals Inc.
C
Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons doi: 10.1111/ajt.12868
Pig-to-Monkey Islet Xenotransplantation Using Multi-Transgenic Pigs R. Bottino1,*,z M. Wijkstrom2, D. J. van der Windt2, H. Hara2, M. Ezzelarab2, N. Murase2, S. Bertera1, J. He1, C. Phelps3, D. Ayares3, D. K. C. Cooper2, and M. Trucco1,
blood mononuclear cells; RIA, radioimmunoassay; Tx, transplantation; xenoTx, xenotransplantation Received 19 December 2013, revised 28 May 2014 and accepted for publication 07 June 2014
1
Division of Immunogenetics, Children’s Hospital of Pittsburgh, University of Pittsburgh Medical Center, Pittsburgh, PA 2 Thomas E. Starzl Transplantation Institute, University of Pittsburgh Medical Center, Pittsburgh, PA 3 Revivicor, Inc., Blacksburg, VA Corresponding author: Rita Bottino,
[email protected] y Joint senior authors. z Present address: Rita Bottino, Institute of Cellular Therapeutics, Allegheny Health Network, Pittsburgh, PA. The generation of pigs with genetic modifications has significantly advanced the field of xenotransplantation. New genetically engineered pigs were produced on an a1,3-galactosyltransferase gene-knockout background with ubiquitous expression of human CD46, with islet beta cell-specific expression of human tissue factor pathway inhibitor and/or human CD39 and/or porcine CTLA4-lg. Isolated islets from pigs with 3, 4 or 5 genetic modifications were transplanted intraportally into streptozotocin-diabetic, immunosuppressed cynomolgus monkeys (n ¼ 5). Immunosuppression was based on anti-CD154 mAb costimulation blockade. Monitoring included features of early islet destruction, glycemia, exogenous insulin requirement and histopathology of the islets at necropsy. Using these modified pig islets, there was evidence of reduced islet destruction in the first hours after transplantation, compared with two series of historical controls that received identical therapy but were transplanted with islets from pigs with either no or only one genetic modification. Despite encouraging effects on early islet loss, these multi-transgenic islet grafts did not demonstrate consistency in regard to long-term success, with only two of five demonstrating function beyond 5 months. Abbreviations: ACT, activated clotting time; alloTx, allotransplantation; AST, arginine stimulation test; GE, genetically engineered; GTKO, a1,3-galactosyltransferase gene-knockout; hTFPI, human tissue factor pathway inhibitor; IBMIR, instant blood-mediated inflammatory reaction; IEQ, islet equivalents; IVGTT, intravenous glucose tolerance test; MFI, mean fluorescence intensity; MMF, mycophenolate mofetil; PAEC, primary aortic endothelial cells; PBMC, peripheral
Introduction Xenotransplantation (xenoTx) of porcine islets is poised to become a therapeutic alternative to pancreas and islet allotransplantation (alloTx) for patients with Type 1 diabetes (1–5). A significant advance has been the ability to produce pigs with specific genetic modifications (6–13), which may prove useful in overcoming the metabolic and immunological barriers between species, and ultimately contribute to reduce the islet mass as well as the intensity of immunosuppressive therapy necessary to sustain islet graft survival. As also seen in the human islet alloTx setting, intraportal infusion of pig islets in monkeys results in an immediate loss of islets, along with the release of insulin and C-peptide, making it more difficult to achieve and maintain normoglycemia (14). Some of the mechanisms involved in early islet loss have been characterized as the instant bloodmediated inflammatory reaction (IBMIR) (15–17). Previously, our group achieved insulin-independence in diabetic, immunosuppressed cynomolgus monkeys following the transplantation (Tx) of islets from pigs expressing a single human complement-regulatory protein (hCD46) (18). However, while hCD46 was associated with successful engraftment when compared with WT pig islets, a reduction of early islet loss was not observed, suggesting that other modulatory transgenes would be beneficial to islet survival. Several studies have indicated that tissue factor, complement and coagulation activation, antibody binding and inflammation contribute to primary nonfunction (15,19–21). To reduce such effects, new genetically engineered (GE) pigs have been generated on a background of a1,3galactosyltransferase gene-knockout and ubiquitous expression of hCD46 (GTKO/hCD46 pigs). Specific transgenes were selected to target relevant mechanisms. Human tissue factor pathway inhibitor (hTFPI) was aimed at inhibition of coagulation and inflammation associated with IBMIR (22,23). Human CD39, through its ATPase 2275
Bottino et al
activity, has been shown to decrease platelet activation and prevent clotting in transgenic mouse models (24,25). In addition, the porcine CTLA4-Ig transgene was incorporated with the goal of inhibiting the cellular immune response (26,27). Upon reaching sufficient adult size and age, the pigs’ pancreases were harvested for isolation of the islets, which were infused into diabetic, immunosuppressed cynomolgus monkeys. We report the effects of these novel GE pig islets on early islet loss and Tx outcome in the first five experiments. Despite significant limitations, this study provides insights into the use of GE pigs in islet xenoTx.
Materials and Methods Sources of animals Six female pigs aged 16–36 months, weighing 350–400 lbs (159–181 kg) (Revivicor, Blacksburg, VA) were sources of islets (Table 1). The production of these GE pigs and their glucose metabolism are detailed in Wijkstrom et al (28) (Supplementary Methods). Five male cynomolgus monkeys (Macaca fascicularis; Three Springs Scientific, Perkasie, PA, and Alpha Genesis, Yemassee, SC) aged 2.5– 4.0 years, weighing 2.5–4.1 kg, were islet recipients (Table 2). One monkey received islets from two GTKO/CD46/hCD39 (3-GE) cloned pigs (P462-04 and P474-07). All procedures that impacted the care of animals were in compliance with guidelines in the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86-23, revised 2011), and approved by the University of Pittsburgh Institutional Animal Care and Use Committee.
islet equivalents (IEQ) (29). CIzymeTM Collagenase MA and BP Protease were used (VitaCyte, Indianapolis, IN) following the manufacturer’s guidelines. Viability was determined by double fluorescent calcein-AM/propidium iodide staining, a method validated for human islets (31) (Supplementary Methods). Islet preparations were stained with dithizone and the percent of dithizonepositive aggregates (at least 50) over whole tissue was used to express purity (32). For qualitative analysis, islets were subjected to dynamic secretagogue challenges (21) (Supplementary Methods). Table 1 summarizes islet graft characteristics.
Induction of diabetes and baseline metabolic studies in recipient monkeys To facilitate blood withdrawal and intravenous (i.v.) drug administration, catheters were inserted into the carotid artery and jugular vein and connected through a tether and jacket system to the exterior of the animal cage (33). The vascular lines were removed 5–6 weeks later (approximately 2 weeks after islet Tx) to minimize the risk of infection. Additional blood draws were obtained from the femoral vein, after ketamine sedation (Ketaset, Fort Dodge, IA; 10 mg/kg). Two to three weeks before islet Tx, diabetes was induced by i.v. streptozotocin (Zanosar, Sicor Pharmaceuticals, Irvine, CA; 125 mg/kg, never exceeding 1500 mg/m2 to avoid nephrotoxicity). Blood glucose levels were measured using Precision Xtra (Becton Dickinson, Franklin Lakes, NJ). Monkeys were considered diabetic if the following conditions were met: (i) hyperglycemia (>350 mg/dL) on at least two occasions, (ii) baseline primate C-peptide reduced by >75% (34), (iii) no increase in C-peptide after intravenous glucose tolerance test and arginine stimulation test (35) and (iv) need for exogenous insulin to prevent ketoacidosis. All monkeys met these criteria. Monkey C-peptide was measured by radioimmunoassay (RIA; Millipore, Billerica, MA) using anti-human antibodies, and concentrations were confirmed with Immulite System (UPMC Presbyterian Hospital Laboratory). Serum porcine C-peptide was measured by radioimmunoassay (RIA, Millipore) using antibodies that do not cross-react with monkey Cpeptide. The successful induction of diabetes was confirmed by histological immunostaining of pancreatic tissue at necropsy.
Pig islet isolation and islet quality assurance Pig pancreases were excised as nonsurvival procedures, as described (29,30). Pancreas was transported within 60 min to the laboratory for immediate islet isolation, purification, and culture. Islets were counted as
Islet transplantation and peri-transplant prophylaxis All islet preparations were cultured overnight prior to Tx, with the exception of islets from pig P462-04 that were cultured for 1 week and mixed with a
Table 1: Characteristics of islet-source pigs
Pig no.
Genetic manipulation1
Pig weight (kg)
Islet yield (IEQ)
Islet yield per gram pancreas (IEQ/g)
Viability (%)
Purity (%)
Stimulation index high/low g-high þ theoph/low g
P388-03 P384-02 P388-2 P462-04 P474-07 P388-01
4-GE2 4-GE2 5-GE2 3-GE1 3-GE1 5-GE2
160 160 160 160 160 180
338 083 399 083 331 846 325 420 300 750 149 606
1356 2237 1869 1574 1012 1202
91 94 94 953 953 90
85 75 75 803 803 75
2.0–8.1 2.3–6.3 3.1–5.3 2.6–6.93 2.6–6.93 2.2–10.8
g, glucose; IEQ, islet equivalents; theoph, theophylline. 3-GE ¼ GTKO/CD46/CD39; 4-GE ¼ GTKO/CD46/TFPI/CTL4-Ig; 5-GE ¼ GTKO/CD46/TFPI/CTL4-Ig/CD39. 2 All four of the 4-GE and 5-GE pigs are clones of each other with respect to the 4-GE background. The 4-GE cell line 548A.3 was used to clone P384-02 and P388-03. This same 4-GE cell line was subsequently transfected with the ins-CD39 vector, to generate the two 5-GE cloned animals, P388-01 and P388-2. While P388-03 was part of the cell pool transfected with ins-CD39, genotype analysis showed that this animal was a ‘‘no-take’’ with respect to integration of the CD39 transgene, and thus only 4-GE. 3 Islets from P462-04 and P474-07 were pooled and the combined prep was tested. 1
2276
American Journal of Transplantation 2014; 14: 2275–2287
Islet Xenotransplantation Table 2: Islet mass transplanted (IEQ/kg), number of recipient CD3þ T cells on day of transplantation, mean mycophenolate mofetil (MMF) trough levels after transplantation and graft survival Recipient monkey no. M2-11 M3-11 M1-11 M14-12 M12-12
Monkey weight at time of Tx (kg)
Pig islet donor no.
Islet mass transplanted (IEQ/kg)
Number of CD3þ T cells on day of Tx (cells/mL)
Mean ( SEM) MMF trough levels (mg/mL)
Graft survival (days)
3.3 2.8 4.1 2.5 2.8
P388-03 P384-02 P388-2 P462-04; P474-07 P388-01
100 000 100 000 75 000 100 000 50 000
491 340 441 183 91
0.59 0.15 1.25 0.17 2.78 0.53 1.63 0.40 1.86 0.47
0 365 160 5 3
IEQ, islet equivalents; Tx, transplantation.
second islet batch (P474-07) cultured overnight (Table 2). Culture was in CMRL-1066 (Life Technologies, Carlsbad, CA) supplemented with 10% heat-inactivated porcine serum, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 2 mmol/L L-glutamine (all from Life Technologies) at 248C in 5% CO2. Prior to Tx, islets were resuspended in fresh 20 mL CMRL-1066 medium with the addition of low molecular weight dextran sulfate (4.5 mg/kg of recipient; Sigma–Aldrich, St. Louis, MO) (36). The insulin content of the transplant medium was negligible (190 s (37). Postoperative treatment consisted of prophylactic cefazolin (10 mg/kg intramuscularly [i.m.] 2 daily) and buprenorphine (0.03 mg/kg i.m. 2 daily) for 3 days. Monkeys were allowed to eat on the evening of surgery.
Serum porcine C-peptide was measured 1, 2 and 24 h post-Tx, and expressed as ng/mL per 10 000 IEQ/kg to take into account differences in islet mass infused. Blood glucose was measured at least every 2 h during the first day and every 4 h on day 1. During the first 24 h post-Tx, the blood glucose target was 100–150 mg/dL. If the blood glucose rose >150 mg/dL, insulin was administered. If it fell
