Diabetes Volume 66, April 2017 987

Apolline Salama,1,2,3,4 Mathilde Mosser,1 Xavier Lévêque,1 Andrea Perota,5 Jean-Paul Judor,3,4 Corentin Danna,1 Sylvie Pogu,1 Anne Mouré,1 Dominique Jégou,1 Nicolas Gaide,6 Jérôme Abadie,6 Olivier Gauthier,7 Jean-Paul Concordet,8,9,10 Stéphanie Le Bas-Bernardet,3,4 David Riochet,3,4,11 Ludmilla Le Berre,3,4 Jérémy Hervouet,3,4 David Minault,3,4 Pierre Weiss,12,13 Jérôme Guicheux,12,13 Sophie Brouard,3,4,14 Steffi Bosch,1 Irina Lagutina,5 Roberto Duchi,5 Giovanna Lazzari,5,15 Emanuele Cozzi,16,17 Gilles Blancho,3,4,14 Sophie Conchon,3,4 Cesare Galli,5,15,18 Jean-Paul Soulillou,19 and Jean-Marie Bach1

Neu5Gc and a1-3 GAL Xenoantigen Knockout Does Not Affect Glycemia Homeostasis and Insulin Secretion in Pigs

Diabetes 2017;66:987–993 | DOI: 10.2337/db16-1060 MUOOYADTRANSPLANTATION AND IMMUNOLOGY

Xenocell therapy from neonate or adult pig pancreatic from control wild-type pigs. In contrast to wild-type islets, islets is one of the most promising alternatives to DKO islets did not induce anti-Neu5Gc antibody when allograft in type 1 diabetes for addressing organ short- grafted in cytidine monophosphate-N-acetylneuraminic age. In humans, however, natural and elicited anti- acid hydroxylase KO mice and exhibited in vitro normal bodies specific for pig xenoantigens, a-(1,3)-galactose insulin secretion stimulated by glucose and theophylline. (GAL) and N-glycolylneuraminic acid (Neu5Gc), are likely Adult DKO pancreata showed no histological abnormali- to significantly contribute to xenoislet rejection. We ties, and immunostaining of insulin and glucagon was obtained double-knockout (DKO) pigs lacking GAL and similar to that from wild-type pancreata. Blood glucose, Neu5Gc. Because Neu5Gc2/2 mice exhibit glycemic dys- insulin, C-peptide, the insulin-to-glucagon ratio, and HOMA- regulations and pancreatic b-cell dysfunctions, we eval- insulin resistance in fasted adult DKO pigs and blood uated islet function and glucose metabolism regulation in glucose and C-peptide changes after intravenous glu- DKO pigs. Isolation of islets from neonate piglets yielded cose or insulin administration were similar to wild-type identical islet equivalent quantities to quantities obtained pigs. This first evaluation of glucose homeostasis in

1IECM, Immuno-endocrinology, EA4644 Oniris, University of Nantes, USC1383 15Avantea Foundation, Cremona, Italy INRA, Oniris, Nantes, France 16Transplantation Immunology Unit, Department of Transfusion Medicine, Univer- 2Société d’Accélération du Transfert de Technologies Ouest Valorisation, Rennes, sity of Padua–Ospedale Giustinianeo, Padua, Italy France 17CORIT (Consortium for Research in Organ Transplantation), Padua, Italy 3INSERM CRTI UMR 1064, University of Nantes, Nantes, France 18Department of Veterinary Medical Sciences, University of Bologna, Ozzano 4Institute of Transplantation, Urology and Nephrology (ITUN), Nantes University Emilia, Italy Hospital–CHU de Nantes, Nantes, France 19University of Nantes, Nantes, France 5 Avantea Laboratory of Reproductive Technologies, Cremona, Italy Corresponding authors: Jean-Marie Bach, [email protected], 6 Animal cancers as Models for Research in comparative Oncology (AMaROC), Oniris, Jean-Paul Soulillou, [email protected], and Cesare Galli, [email protected]. Nantes, France Received 30 August 2016 and accepted 8 January 2017. 7Department of Experimental Surgery, Center for Research and Preclinical Investi- gation, Oniris, Nantes, France This article contains Supplementary Data online at http://diabetes 8Muséum National d’Histoire Naturelle, Paris, France .diabetesjournals.org/lookup/suppl/doi:10.2337/db16-1060/-/DC1. 9CNRS UMR 7196, Paris, France A.S., M.M., X.L., and A.P. contributed equally to this study. C.G., J.-P.S., and 10INSERM U1154, Paris, France J.-M.B. contributed equally to this study. 11Department of Pediatrics, Nantes University Hospital–CHU de Nantes, Nantes, France © 2017 by the American Diabetes Association. Readers may use this article as 12INSERM UMRS 791, Laboratoire d’ingénierie Ostéo-Articulaire et Dentaire long as the work is properly cited, the use is educational and not for profit, and the (LIOAD), Nantes, France; University of Nantes, UFR Odontologie, Nantes, France work is not altered. More information is available at http://www.diabetesjournals 13Nantes University Hospital–CHU de Nantes, PHU4 OTONN, Nantes, France .org/content/license. 14Centre d’investigation clinique (CIC) Biotherapy, Nantes University Hospital–CHU de Nantes, Nantes, France 988 Knockout Pig Islets for T1D Therapy Diabetes Volume 66, April 2017

DKO pigs for two major xenoantigens paves the way to 8–12 weeks old. Experiments were approved by the Ethics their use in (pre)clinical studies. Committees and performed in accordance with relevant Italian (DGL116/92) and French regulations (2001-464 and 2013-118, Approval 01074.01/02). All efforts were Pancreatic islet allotransplantation is a realistic alternative or made to minimize animal suffering and to restrict the complement to insulin therapy in type 1 diabetes (T1D) to number of experimental animals. prevent serious long-term complications but is limited by the Insulin and Glucagon Immunostaining and Content in lack of pancreas. Pig pancreas remains a promising comple- Pancreas mentary islet source. Reproducible evidence exists on the Formalin-fixed paraffin-embedded pancreas cross-sections long-term therapeutic benefit of islet xenotransplantation in were incubated overnight with rabbit anti-insulin (C27C9; pig to nonhuman primate preclinical models, using encapsu- Cell Signaling Technology, Beverly, MA) and mouse anti- lation for islet immunoprotection (1,2) or using immunosup- glucagon (G2654; Sigma-Aldrich, St. Louis, MO) IgGs and pressant treatments (3–6). Methods for pig islet purification then 1 h with secondary Alexa-Fluor 488 donkey anti-rabbit have significantly improved, in particular for neonatal pig IgG and Alexa-Fluor 555 donkey anti-mouse IgG antibodies islet-like cell clusters (NPCCs), which are easy to isolate and (Thermo Fisher Scientific, Rockford, IL). Pancreatic insulin functional in vivo in the long-term (4,6,7). However, strong and glucagon contents were evaluated by ELISA (Mercodia, humoral response to pig antigens, especially against the Uppsala, Sweden) after acid-ethanol extraction of three a-(1,3)-galactose (GAL) sugar, was detected in nonhuman 1cm3 frozen blocks per pancreas (head, body, and tail) primates after grafting of neonate (2) and adult (1) pig islets, according to the Animal Models of Diabetic Complications even encapsulated in alginate hydrogel, and may even affect Consortium (http://www.diacomp.org/shared/document.aspx? encapsulated islet survival (1). Humans have lost expression id=73&docType=Protocol - Ed. Leiter) protocol. of a-1,3 galactosyl transferase (GGTA1) enzyme generating the GAL epitope (8) and produce natural and elicited anti- Metabolic Investigations in Adult Pigs bodies to GAL (9). Among non-GAL, the N-glycolylneuraminic Glycemia (Stat-Strip-Glucometer; Nova Biomedical, Waltham, acid (Neu5Gc) sugar is also a major pig xenoantigen (10). MA), serum or plasma insulin, glucagon, and C-peptide Humans have also lost the ability to synthesize Neu5Gc (ELISA; Mercodia) were assessed in fasted pigs and after an from the N-acetylneuraminic form, after mutation of cytidine intravenous (I.V.) glucose tolerance test (IVGTT) and insulin monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) tolerance test (ITT). ELISA samples were run in duplicates (11). Pig islets express Neu5Gc, which reacts with human nat- with variation coefficient ,10%. HOMA of insulin resistance ural antibodies (12,13), and Neu5Gc knockout (KO) mice reject (HOMA-IR) was (glucose [mmol/L]*insulin [mIU/L])/22.5). pancreatic islets from wild-type (WT) counterparts (14). InIVGTT,theglucosedisappearanceratewas(Ln[Glucose Using transcription activator-like effector nucleases T5min] – Ln [Glucose T30min])*100/25 min. Acute insulin (TALEN), we obtained a double-knockout (DKO) pig line response to glucose (AIRglu) was the mean of serum insulin 2 2 for the CMAH and GGTA1 enzymes. However, CMAH / levels 3, 5, 10, and 15 min after glucose injection, minus the mice exhibited higher fasting blood glucose and insulin than prechallenge (t=0) level. Areas under the curve (AUC) were WTs, associated with mild glucose intolerance and significant calculated above t = 0 values using the trapezoidal rule. b-cell dysfunctions, including higher insulin secretion by iso- In the ITT, KITT measurement of glucose disappearance t t lated islets in response to glucose (15). As a prerequisite for was 0.693*100/ 1/2, where 1/2 is the time to serum glu- xenocell therapy for T1D and to study the metabolic effects cose level half the level at 2 min. of CMAH KO in a species more closely related to humans Isolated Neonatal Pig Islets than mice, we analyzed glucose homeostasis in our DKO Isolation from pancreas, culture, and capacity of NPCCs to adult pigs and insulin secretion of isolated DKO NPCCs. secrete insulin in response to acute glucose, with or without theophylline (potentiating insulin secretion), were performed RESEARCH DESIGN AND METHODS as described previously by Korbutt et al. (7) (Supplementary Animals and Ethical Statements Data). The insulin stimulatory index was the quantity of in- DKO pigs were obtained by targeting the swine CMAH sulin (ELISA) in the supernatant of NPCCs cultured with stim- 2 2 6 gene using TALEN on the GGTA1 / background (obtained ulus(20mmol/Lglucose 10 mmol/L theophylline), divided fromDr.D.H.Sachs,MassachusettsGeneralHospital,Boston, by the insulin level in the basal medium (RPMI, 2.8 mmol/L MA) and by somatic cell nuclear transfer (SCNT) (Supple- glucose, 2 mmol/L L-glutamine, 0.5% BSA). In the graft exper- m mentary Data). WT pigs were Landrace 3 Large-White iments, NPCCs were encapsulated into 200 L 2% silanized- (EARL Pont Romain, Surzur, and ANSES, Ploufragan, France). hydroxypropyl methylcellulose (Si-HPMC) hydrogel (16) and fl Adult and neonate pigs were males, respectively, 5–6months injected subcutaneously in 2% iso urane anesthetized mice. old weighing 37.90 6 3.47 kg and 3–10 days old weighing Statistical Analysis 2 2 1–3kg.CMAH / (B6.129 3 1-Cmahtm1Avrk/J; The Jack- Data are expressed as mean 6 SEM. P , 0.05 was con- son Laboratory, Bar Harbor, ME) and control mice (C57BL/6; sidered statistically different (Mann- Whitney test; Prism, Janvier Laboratories, Le Genest-Saint-Isle, France) were GraphPad Software, Inc., La Jolla, CA). diabetes.diabetesjournals.org Salama and Associates 989

RESULTS site (Supplementary Fig. 2). Eight living piglets from two 2/2 DKO Pig Clone Generation farrowings were tested to confirm CMAH genotype 2 2 GAL / pig primary fibroblasts were cotransfected with (four piglets originated from clone D1, two from A6, and plasmids encoding a TALEN pair targeting the HindIII site two from E8). We confirmed the absence of Neu5Gc (Fig. A B fi located in pig CMAH exon 4 fragment (Supplementary Fig. 1 ) and CMAH mRNA (Fig. 1 )inpig broblasts. 1), and the targeting donor DNA construct containing a Necropsy and Histological Characterization of Adult 1.65-kb fragment of the CMAH exon 4, and a phosphoglyc- DKO Pigs erate kinase promoter-PuroR-pA expression cassette in the DKO and WT adult pigs showed no obvious developmen- HindIII site in the CMAH exon 4 fragment. PCR genotyping tal abnormalities at necropsy (Supplementary Data). The of the 27 puromycin-resistant fibroblast clones identified DKO pancreas showed no evident macroscopic abnormal- 3 clones subsequently used for SCNT (A6, D1, E8) (Supple- ities. Pancreatic islets from four DKO and four WT pigs un- mentary Fig. 1). Sequence analysis showed deletions rang- derwent histological analyses (Fig. 2A). DKO islets seemed ing from 7 to 40 nucleotides around the targeted HindIII slightly larger and numerous than in WT pigs but without

Figure 1—DKO pig CMAH phenotype. A: After 2 weeks’ culture in DMEM medium with human serum to wash off exogenous Neu5Gc, fibroblasts from three pig clones (347, 349, and 361), human 293T cells, and fibroblasts from CMAH+/+ pigs were stained with anti-Neu5Gc- specific antibody (Poly21469) and analyzed by flow cytometry (LSR-II; BD Biosciences, Pont de Claix, France) and FlowJo software (Tree Star, Ashland, OR). Black and gray histograms are related to isotype control and Neu5Gc-specific antibodies, respectively. B: Pig fibroblast total RNA from the various clones selected was subjected to reverse transcription with Moloney murine leukemia virus reverse transcriptase (Thermo Fisher), and CMAH transcripts were then analyzed by quantitative PCR using Power SYBR Green Master Mix (Thermo Fisher) and normalized to expression detected in CMAH WT pig fibroblasts (Supplementary Data). 990 Knockout Pig Islets for T1D Therapy Diabetes Volume 66, April 2017

Figure 2—Analysis of adult DKO pig pancreas. A: Histological analyses of formalin-fixed paraffin-embedded pancreatic tissue 4-mm cross- sections stained with hematoxylin-eosin-saffron from one WT pig (right) and one DKO pig (left). B: Immunostaining of insulin (green, Alexa- Fluor 488) and glucagon (red, Alexa-Fluor 555) on 2.5-mm pancreatic tissue sections from one WT pig (right) and one DKO pig (left). Images (Zen 1.0 software; Carl Zeiss MicroImaging, Inc., Jena, Germany) are representative of the pancreas of 4 DKO and 4 WT pigs, and microscopic scales are indicated on the pictures. C–E: For islet number and size, at least 100 islets were investigated per animal (4 DKO and 4 WT pigs). For each animal, 5.28 mm2 ofthehead,thebody,andthetailofthe pancreas tissue were observed. C: Mean islet size (3103 mm2)inDKOandWT pigs. D: Percentage of islet area per pancreas area. E: Number of islets per mm2 of pancreatic area. F–H: For insulin and glucagon immunostaining analyses, at least 70 islets of the head, the body, and the tail of the pancreas tissue were analyzed per animal (4 DKO and 4 WT pigs). F: Mean insulin-positive area per islet (3103 mm2). G: Mean glucagon-positive area per islet (3103 mm2). H: Percentage of glucagon area per insulin area. Quantity of insulin (ng) (I) and glucagon (ng) (J) per protein quantity (mg) by Bradford protein assay (Fisher Scientific) of the pancreas tissue (4 DKO and 4 WT pigs). C–H: Image analyses were investigated using the ImageJ software (https://imagej.nih.gov/ij/index.html). All values are expressed as mean 6 SEM. Differences between DKOs and WTs are not significant by Mann-Whitney test.

asignificant difference (P = 0.1143) (Fig. 2C–E). Mean islet control pigs (4.73 6 0.03, n = 2) (Supplementary Table 1). volume density (Supplementary Data) did not differ (3.16 6 Fasting serum insulin was similar (1.74 6 0.24 and 1.73 6 0.13%forDKOpancreasand2.696 0.12% for WT pancreas, 0.21 pmol/L, respectively) (Supplementary Table 1). The P = 0.1143). Immunodetected (Fig. 2B)insulinandglucagon serum insulin-to-glucagon molar ratio (0.53 6 0.17 for areas on pancreas sections and percentage of glucagon DKOs vs. 0.55 6 0.24 for WTs) (Supplementary Table 1) area/insulin area exhibited no significant differences between and HOMA-IRs (2.04 6 0.33 vs. 2.52 6 0.32) after an DKO and WT pigs (P =0.4857[Fig.2F], P =0.8286[Fig.2G], overnight fast appeared almost identical in pig groups. and P =0.8286[Fig.2H], respectively). Insulin (Fig. 2I)and In IVGTT, blood glucose curves were very similar between glucagon (Fig. 2J) pancreatic contents did not differ between DKO and WT pigs. Blood glucose peaked 3 min after glucose pig groups (P = 0.8286). administration and then dropped to reach the fasting value Glycemic Homeostasis and Pancreatic Endocrine after 60–90 min (Fig. 3A). The rate of glucose disappearance Functions in Adult DKO Pigs did not differ (2.42 6 0.63 mmol/L/min for DKOs and Fasting blood glucose was slightly lower in adult DKO pigs 2.47 6 0.78 for WTs) (Supplementary Table 1). AUCglu (3.77 6 0.17 mmol/L, n = 4) than in age-matched WT were also almost identical (421.8 6 46.58 mmol*120 min/L diabetes.diabetesjournals.org Salama and Associates 991

Figure 3—Metabolic tests in adult DKO pigs. For IVGTT, adult DKO (n = 4, DKO 1 to DKO 4) and WT control (n = 2, WT 1 and WT 2) pigs fasted overnight received 0.5 g/kg I.V. glucose infusion (during 2 min). Blood glucose (mmol/L) (A), serum insulin concentration (pmol/L) (B), C-peptide plasma level (pmol/L) (C), and serum glucagon concentration (pmol/L) (D) were evaluated before glucose administration (t =0) and 1, 3, 5, 10, 15, 30, 60, 90, and 120 min after the end of the glucose infusion. For ITT, adult DKO (n = 3) and WT control (n = 2) pigs fasted overnight received 0.1 IU/kg I.V. Actrapid insulin (Novo Nordisk A/S, Bagsværd, Denmark). Blood glucose (mmol/L) (E) and C-peptide (pmol/L) (F) concentrations were evaluated, before (t = 0) and 2, 4, 6, 8, 10, 12, and 14 min after insulin administration. A–F: Results from individual animal are represented. and 435.5 6 19 for DKOs and WTs, respectively). Serum a chaotic waning of C-peptide after insulin injection insulin increased 8–17-fold in the first minutes after glucose (Fig. 3F). B injection (Fig. 3 ). C-peptide also showed rapid elevation (Fig. Pancreatic Islets From Neonate DKO Pigs 3C). The DKO pig number 3 (DKO3) had a delayed insulin The number of NPCC islet equivalent quantities (IEQs) (15 min after glucose administration) and C-peptide (10 min) obtained after isolation was comparable in DKOs (16.44 6 peak. Consistently, DKO3 presented a moderately elevated 1IEQs/g,n = 5) versus WTs (14.637 6 3.234, P = 0.5921, AUC associated with a slightly lower AIR and AUC glu glu ins n = 10). Viability of DKO NPCCs was not affected com- than WT and other DKO pigs, but not objectified by a differ- pared with WTs (data not shown). NPCCs exhibited a nor- ent AUCC-pep (Supplementary Table 1). Glucagon suppression was more rapid and intense in WTs and DKO3 than in the mal insulin secretion after glucose/theophylline stimulation A other DKOs (Fig. 3D). (Fig. 4 ). The average insulin secretion induced by glucose n During ITT, blood glucose in DKO pigs dropped rapidly alone appeared higher for DKO ( = 5) NPCCs than WT n fi P A and was similar to controls (Fig. 3E). Glucose disappear- ( = 6), without reaching signi cance ( = 0.1255) (Fig. 4 ). ance did not differ between pig groups (KITT, 11.36 6 As expected, theophylline increased insulin secretion by 1.50% for DKOs vs. 10.73 6 0.82 for WTs) (Supplemen- WT (P = 0.026) (Fig. 4A)andDKOislets(however,notsignifi- tary Table 1). C-peptide curves were also similar between cantly, probably because of the positive effect of glucose pigs, except for the DKO pig number 2 (DKO2) exhibiting alone on insulin secretion by DKO islets). The proportion 992 Knockout Pig Islets for T1D Therapy Diabetes Volume 66, April 2017

was even more rapid than for WT islets (34.73 6 5.58% at 24 h vs. 36.69 6 10.25 at 4–5days). Finally, we checked induction of anti-Neu5Gc antibodies 2 2 in CMAH / mice after a subcutaneous graft of pig NPCCs encapsulated in Si-HPMC to prolong their survival (unpub- 2 2 lished data). We confirmed that WT and GAL / NPCCs 2 2 induced anti-Neu5Gc antibody response in CMAH / mice unlike DKO NPCCs (Fig. 4B).

DISCUSSION Anti-GAL antibodies have been considered for years as the first obstacle to successful xenotransplantation. Natural anti-Neu5Gc antibodies are likely to be important non- GAL antibodies because they are present in most normal human sera (12,17) and are elicited by challenges with xenogenic tissues (18). Preexisting antibodies present in 2 2 human serum bind to GAL / pig islets (19). Moreover, WT syngeneic pancreatic islets are rapidly rejected by 2 2 2 2 CMAH / mice and not by GAL / mice (14). The methodology we described here to disable CMAH 2 2 gene expression in pig GAL / fibroblasts, using TALEN and SCNT, produces viable DKO pigs efficiently and rapidly (within 8 months from TALEN design). Others have also generated GAL and Neu5Gc DKO pigs using zinc finger nucleases (20), TALEN (21), or CRISPR-Cas9 (22). Only single GAL KO pigs have been investigated previously for glucose homeostasis and isolated islet functions (23). 2 2 Unlike the CMAH / mouse (15), fasting blood glu- cose and insulin and glucagon secretions appeared normal in adult DKO pigs. Furthermore, DKO pigs seemed to Figure 4—Pancreatic islets from neonate DKO pigs. A: Extracellular insulin was quantified by ELISA in the supernatant of 50 IEQ exhibit neither glucose intolerance nor insulin resistance NPCCs on day 1 after isolation, after 120 min culture in basal medium as indicated by changes in blood glucose levels after (2.8 mmol/L glucose), and after glucose (20 mmol/L) 6 theophylline IVGTT and ITT, respectively (confirmed by HOMA-IR). fi (20 mmol/L) stimulation. The NPCCs from ve DKO and six WT pigs Although the number of adult control pigs tested in our were investigated in three replicates for each islet preparation in three independent experiments. The results are given as mean insulin stimu- study was limited, the results with control WT pigs were latory index (S.I.) 6 SEM for the DKO and WT groups. *P = 0.026; **P = similar to those with DKOs, and values remained within 0.0043. Other differences between the groups were not significant the physiological range for fasting and IVGTT in pigs (24). (Mann-Whitney test). B:FivehundredIEQSi-HPMC–encapsulated NPCCs of DKO, GAL KO, and WT control neonate pigs were grafted After glucose administration, one DKO pig had delayed subcutaneously in WT or CMAH2/2 mice. The serum of grafted mice insulin secretion without this clearly affecting glycemia reg- was tested 15 to 20 days later for the presence of anti-Neu5Gc IgG. The ulation, probably as glucagon rapidly decreased in this pig. +/+ mouse serum was incubated with Neu5Gc thymocytes from C57BL/6 DKO pigs also displayed preserved islet architecture mouse and phycoerythrin-antibody anti-mouse IgG (BioLegend, San Diego, CA) as per Tahara et al. (14). Analyses were performed by Aria (islet number, area, volume density, and insulin and flow cytometry (BD Biosciences). Data were analyzed by FlowJo glucagon immunostaining). Further studies with a high- software. The black histograms represent phycoerythrin-isotype con- energy diet would be helpful to investigate the effect of trol staining and the gray histograms phycoerythrin-antibody anti- the CMAH KO on b-cell and islet compensation in re- mouse IgG (detecting IgG specific for Neu5Gc). Control pig islets in CMAH2/2 mouse (B1), GAL KO pig islets in CMAH2/2 mouse (B2), sponse to severe obesity-induced insulin resistance (15). control pig islets in WT mouse (B3), and DKO pig islets in CMAH2/2 Furthermore, isolation of NPCCs yielded an IEQs-to-g mouse (B4). One representative experiment of two independent ex- pancreas ratio similar to that resulting in control pigs. DKO periments is shown, for a total of two to three in each group. NPCCs were functional ex vivo and exhibited insulin secretion after stimulation with glucose and theophylline. Pig islet Neu5Gc and sialic antigens clearly contribute to pig islet of cellular insulin secreted in response to glucose plus the- antigenicity (12,13). Also, our results clearly confirmed that 2 2 ophylline stimulation by DKO islets increased between 24 h anti-Neu5GcantibodiesareinducedinCMAH / mice by and 4–5days’ in vitro culture (36.64 6 5.35% at 24 h vs. WT encapsulated NPCCs, probably mimicking what is 71.00 6 9.76% at 4–5days,P = 0.032) (Supplementary expected in the human context. CMAH editing in our pigs Table 2), suggesting the beginning of NPCC maturation prevented this humoral-specific response, consistent with diabetes.diabetesjournals.org Salama and Associates 993 earlier observations of reduced binding of human antibodies 6. Thompson P, Cardona K, Russell M, et al. CD40-specific costimulation to peripheral blood mononuclear cells from GGTA1/CMAH blockade enhances neonatal porcine islet survival in nonhuman primates. Am J KO pigs (25). CMAH editing will most likely be necessary Transplant 2011;11:947–957 when hydrogel-encapsulated pig islets are used for T1D xen- 7. Korbutt GS, Elliott JF, Ao Z, Smith DK, Warnock GL, Rajotte RV. Large scale isolation, growth, and function of porcine neonatal islet cells. J Clin Invest 1996; otherapy because encapsulation in hydrogel does not fully 97:2119–2129 prevent antisugar humoral responses (1,2). 8. Cooper DK, Koren E, Oriol R. Oligosaccharides and discordant xeno- In conclusion, our study opens the way for the use of transplantation. Immunol Rev 1994;141:31–58 DKO pig pancreatic islets in (pre)clinical studies on T1D 9. Galili U, Mandrell RE, Hamadeh RM, Shohet SB, Griffiss JM. Interaction cell therapy. between human natural anti-alpha-galactosyl immunoglobulin G and bacteria of the human flora. Infect Immun 1988;56:1730–1737 10. Salama A, Evanno G, Harb J, Soulillou JP. Potential deleterious role of Acknowledgments. The authors are very grateful to David H. Sachs anti-Neu5Gc antibodies in xenotransplantation. Xenotransplantation 2015;22: (Massachusetts General Hospital, Boston, MA) for providing the GAL KO pig. The 85–94 authors thank Odile Duvaux (Xenothera, Nantes, France) for the scientificdiscussions. 11. Irie A, Suzuki A. CMP-N-acetylneuraminic acid hydroxylase is exclusively Funding. This work was supported by Pays de la Loire Region (France) inactive in humans. Biochem Biophys Res Commun 1998;248:330–333 (Xenothera academic program and to A.S.), the Société d’Accélération du Trans- 12. Komoda H, Miyagawa S, Kubo T, et al. A study of the xenoantigenicity of fert de Technologies Ouest Valorisation (to A.S.), the European Center for Trans- adult pig islets cells. Xenotransplantation 2004;11:237–246 plantation and Immunotherapy Sciences (ECTIS IHU, Nantes, France), the National 13. Omori T, Nishida T, Komoda H, et al. A study of the xenoantigenicity of Research Agency “Investment Into The Future” programs (ANR-10-IBHU-005, neonatal porcine islet-like cell clusters (NPCC) and the efficiency of adenovirus- to X.L., and ANR-II-INSB-0014), the European Commission’s Xenome Sixth mediated DAF (CD55) expression. Xenotransplantation 2006;13:455–464 Framework Programme (LSHB-CT-2006-037377), and the European Seventh 14. Tahara H, Ide K, Basnet NB, et al. Immunological property of antibodies Framework Programme “Translink” research program (grant agreement 603049 against N-glycolylneuraminic acid epitopes in cytidine monophospho-N- and to L.L.B.). acetylneuraminic acid hydroxylase-deficient mice. J Immunol 2010;184:3269–3275 Duality of Interest. A.S. is currently an employee of the start-up 15. Kavaler S, Morinaga H, Jih A, et al. Pancreatic beta-cell failure in obese Xenothera. J.-P.S. and J.-M.B. are cofounders of the start-up Xenothera. No mice with human-like CMP-Neu5Ac hydroxylase deficiency. FASEB J 2011;25: other potential conflicts of interest relevant to this article were reported. 1887–1893 Author Contributions. A.S., M.M., X.L., A.P., J-.P.J., C.D., S.P., A.M., 16. Bourges X, Weiss P, Daculsi G, Legeay G. Synthesis and general properties D.J., N.G., J.A., O.G., S.L.B.-B., L.L.B., J.H., D.M., S.Bo., I.L., R.D., G.L., S.C., and of silated-hydroxypropyl methylcellulose in prospect of biomedical use. Adv J.-M.B. performed most of the experiments. A.S., M.M., X.L., C.D., S.P., A.M., Colloid Interface Sci 2002;99:215–228 D.J., S.Bo., S.C., C.G., J.-P.S., and J.-M.B. analyzed the data. A.S., M.M., X.L., 17. Padler-Karavani V, Yu H, Cao H, et al. Diversity in specificity, abundance, A.P., A.M., D.R., S.Br., E.C., S.C., C.G., J.-P.S., and J.-M.B. wrote or reviewed the and composition of anti-Neu5Gc antibodies in normal humans: potential impli- paper. A.P., J.-P.C., S.C., and C.G. generated the DKO pigs. D.R., S.Br., G.B., J.-P.S., cations for disease. Glycobiology 2008;18:818–830 and J.-M.B. helped acquire study funding. P.W. and J.G. provided Si-HPMC and 18. Scobie L, Padler-Karavani V, Le Bas-Bernardet S, et al. Long-term IgG helped with islet encapsulation design. C.G., J.-P.S., and J.-M.B. devised the response to porcine Neu5Gc antigens without transmission of PERV in burn pa- scientific strategy and experimental design. J.-M.B. is the guarantor of this work tients treated with porcine skin xenografts. J Immunol 2013;191:2907–2915 and, as such, had full access to all the data in the study and takes responsibility 19. van der Windt DJ, Marigliano M, He J, et al. Early islet damage after direct for the integrity of the data and the accuracy of the data analysis. exposure of pig islets to blood: has humoral immunity been underestimated? Cell Transplant 2012;21:1791–1802 References 20. Lutz AJ, Li P, Estrada JL, et al. Double knockout pigs deficient in 1. Dufrane D, Goebbels RM, Gianello P. Alginate macroencapsulation of N-glycolylneuraminic acid and galactose a-1,3-galactose reduce the humoral pig islets allows correction of streptozotocin-induced diabetes in primates barrier to xenotransplantation. Xenotransplantation 2013;20:27–35 up to 6 months without immunosuppression. Transplantation 2010;90: 21. Miyagawa S, Matsunari H, Watanabe M, et al. Generation of a1,3-galactosyl- 1054–1062 transferase and cytidine monophospho-N-acetylneuraminic acid hydroxylase gene 2. Kobayashi T, Harb G, Rajotte RV, et al. Immune mechanisms associated double-knockout pigs. J Reprod Dev 2015;61:449–457 with the rejection of encapsulated neonatal porcine islet xenografts. Xeno- 22. Li P, Estrada JL, Burlak C, et al. Efficient generation of genetically distinct transplantation 2006;13:547–559 pigs in a single pregnancy using multiplexed single-guide RNA and carbohydrate 3. Hering BJ, Wijkstrom M, Graham ML, et al. Prolonged diabetes reversal selection. Xenotransplantation 2015;22:20–31 after intraportal xenotransplantation of wild-type porcine islets in immunosup- 23. Casu A, Echeverri GJ, Bottino R, et al. Insulin secretion and glucose me- pressed nonhuman primates. Nat Med 2006;12:301–303 tabolism in alpha 1,3-galactosyltransferase knock-out pigs compared to wild- 4. Cardona K, Korbutt GS, Milas Z, et al. Long-term survival of neonatal type pigs. Xenotransplantation 2010;17:131–139 porcine islets in nonhuman primates by targeting costimulation pathways. Nat 24. Manell E, Hedenqvist P, Svensson A, Jensen-Waern M. Establishment of a Med 2006;12:304–306 refined oral glucose tolerance test in pigs, and assessment of insulin, glucagon 5. van der Windt DJ, Bottino R, Casu A, et al. Long-term controlled normo- and glucagon-like peptide-1 responses. PLoS One 2016;11:e0148896 glycemia in diabetic non-human primates after transplantation with hCD46 25. Burlak C, Paris LL, Lutz AJ, et al. Reduced binding of human antibodies to transgenic porcine islets. Am J Transplant 2009;9:2716–2726 cells from GGTA1/CMAH KO pigs. Am J Transplant 2014;14:1895–1900