Joseph M. Ladowski

Recent advances in genome editing and creation of genetically modified pigs.

The field of xenotransplantation is benefiting greatly from recent advances in genetic engineering. The efficiency and pace with which new model animals are being created has dramatically sped progress towards clinical relevance. Endonuclease-driven genome editing now allows for the efficient generation of targeted genetic alterations. Herein we review the available methods of genetic engineering that have been successfully employed to create genetically modified pigs.

The contemporary management of renal artery aneurysms

BACKGROUND Renal artery aneurysms (RAAs) are rare, with little known about their natural history and growth rate or their optimal management. The specific objectives of this study were to (1) define the clinical features of RAAs, including the precise growth rate and risk of rupture, (2) examine the current management and outcomes of RAA treatment using existing guidelines, and (3) examine the appropriateness of current criteria for repair of asymptomatic RAAs. METHODS A standardized, multi-institutional approach was used to evaluate patients with RAAs at institutions from all regions of the United States. Patient demographics, aneurysm characteristics, aneurysm imaging, conservative and operative management, postoperative complications, and follow-up data were collected. RESULTS A total of 865 RAAs in 760 patients were identified at 16 institutions. Of these, 75% were asymptomatic; symptomatic patients had difficult-to-control hypertension (10%), flank pain (6%), hematuria (4%), and abdominal pain (2%). The RAAs had a mean maximum diameter of 1.5 ± 0.1 cm. Most were unilateral (96%), on the right side (61%), saccular (87%), and calcified (56%). Elective repair was performed in 213 patients with 241 RAAs, usually for symptoms or size >2 cm; the remaining 547 patients with 624 RAAs were observed. Major operative complications occurred in 10%, including multisystem organ failure, myocardial infarction, and renal failure requiring dialysis. RAA repair for difficult-to-control hypertension cured 32% of patients and improved it in 26%. Three patients had ruptured RAA; all were transferred from other hospitals and underwent emergency repair, with no deaths. Conservatively treated patients were monitored for a mean of 49 months, with no acute complications. Aneurysm growth rate was 0.086 cm/y, with no difference between calcified and noncalcified aneurysms. CONCLUSIONS This large, contemporary, multi-institutional study demonstrated that asymptomatic RAAs rarely rupture (even when >2 cm), growth rate is 0.086 ± 0.08 cm/y, and calcification does not protect against enlargement. RAA open repair is associated with significant minor morbidity, but rarely a major morbidity or mortality. Aneurysm repair cured or improved hypertension in >50% of patients whose RAA was identified during the workup for difficult-to-control hypertension.

Humoral Reactivity of Renal Transplant-Waitlisted Patients to Cells From GGTA1/CMAH/B4GalNT2, and SLA Class I Knockout Pigs

Background Antipig antibodies are a barrier to clinical xenotransplantation. We evaluated antibody binding of waitlisted renal transplant patients to 3 glycan knockout (KO) pig cells and class I swine leukocyte antigens (SLA). Methods Peripheral blood mononuclear cells from SLA identical wild type (WT), &agr;1, 3-galactosyltransferase (GGTA1) KO, GGTA1/ cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) KO, and GGTA1/ CMAH /b1,4 N-acetylgalactosaminyl transferase (B4GalNT2) KO pigs were screened for human antibody binding using flow cytometric crossmatch (FCXM). Sera from 820 patients were screened on GGTA1/CMAH/B4GalNT2 KO cells and a subset with elevated binding was evaluated further. FCXM was performed on SLA intact cells and GGTA1/SLA class I KO cells after depletion with WT pig RBCs to remove cell surface reactive antibodies, but leave SLA antibodies. Lastly, human and pig reactive antibodies were eluted and tested for cross-species binding and reactivity to single-antigen HLA beads. Results Sequential glycan KO modifications significantly reduce antibody binding of waitlisted patients. Sera exhibiting elevated binding without reduction after depletion with WT RBCs demonstrate reduced binding to SLA class I KO cells. Human IgG, eluted from human and pig peripheral blood mononuclear cells, interacted across species and bound single-antigen HLA beads in common epitope-restricted patterns. Conclusions Many waitlisted patients have minimal xenoreactive antibody binding to the triple KO pig, but some HLA antibodies in sensitized patients cross-react with class I SLA. SLA class I is a target for genome editing in xenotransplantation.

Silencing Porcine CMAH and GGTA1 Genes Significantly Reduces Xenogeneic Consumption of Human Platelets by Porcine Livers.

Background A profound thrombocytopenia limits hepatic xenotransplantation in the pig-to-primate model. Porcine livers also have shown the ability to phagocytose human platelets in the absence of immune-mediated injury. Recently, inactivation of the porcine ASGR1 gene has been shown to decrease this phenomenon. Inactivating GGTA1 and CMAH genes has reduced the antibody-mediated barrier to xenotransplantation; herein, we describe the effect that these modifications have on xenogeneic consumption of human platelets in the absence of immune-mediated graft injury. Methods Wild type (WT), ASGR1−/−, GGTA1−/−, and GGTA1−/−CMAH−/− knockout pigs were compared for their xenogeneic hepatic consumption of human platelets. An in vitro assay was established to measure the association of human platelets with liver sinusoidal endothelial cells (LSECs) by immunohistochemistry. Perfusion models were used to measure human platelet uptake in livers from WT, ASGR1−/−, GGTA1−/−, and GGTA1−/− CMAH−/− pigs. Results GGTA1−/−, CMAH−/− LSECs exhibited reduced levels of human platelet binding in vitro when compared with GGTA1−/− and WT LSECs. In a continuous perfusion model, GGTA1−/− CMAH−/− livers consumed fewer human platelets than GGTA1−/− and WT livers. GGTA1−/− CMAH−/− livers also consumed fewer human platelets than ASGR1−/− livers in a single-pass model. Conclusions Silencing the porcine carbohydrate genes necessary to avoid antibody-mediated rejection in a pig-to-human model also reduces the xenogeneic consumption of human platelets by the porcine liver. The combination of these genetic modifications may be an effective strategy to limit the thrombocytopenia associated with pig-to-human hepatic xenotransplantation.

Silencing the porcine iGb3s gene does not affect Galα3Gal levels or measures of anticipated pig-to-human and pig-to-primate acute rejection.

The Galα(1,3)Gal epitope (α‐GAL), created by α‐1,3‐glycosyltransferase‐1 (GGTA1), is a major xenoantigen causing hyperacute rejection in pig‐to‐primate and pig‐to‐human xenotransplantation. In response, GGTA1 gene‐deleted pigs have been generated. However, it is unclear whether there is a residual small amount of α‐Gal epitope expressed in GGTA1−/− pigs. Isoglobotrihexosylceramide synthase (iGb3s), another member of the glycosyltransferase family, catalyzes the synthesis of isoglobo‐series glycosphingolipids with an α‐GAL‐terminal disaccharide (iGb3), creating the possibility that iGb3s may be a source of α‐GAL epitopes in GGTA1−/− animals. The objective of this study was to examine the impact of silencing the iGb3s gene (A3GalT2) on pig‐to‐primate and pig‐to‐human immune cross‐reactivity by creating and comparing GGTA1−/− pigs to GGTA1−/−‐ and A3GalT2−/−‐double‐knockout pigs.

Modified glycan models of pig-to-human xenotransplantation do not enhance the human-anti-pig T cell response

UNLABELLED Genetically modified porcine models of pig-to-human xenotransplantation offer the most immediate answer to a growing shortage of available solid organs. Recently a modified porcine glycan model has been discovered that reduces human antibody binding to levels comparable with allograft standards. As this background provides an answer to the problem of acute humoral xenograft rejection (AHXR), it is important to consider the impact these modifications have on measures of cell-mediated rejection. The objective of this study was to examine the impact of currently relevant glycan knockout models of pig-to-human xenotransplantation in a lymphocyte proliferation assay. To accomplish these goals, genetically modified pigs were created through CRISPR/Cas9-directed silencing of the GGTA1, and CMAH genes. Peripheral blood mononuclear cells (PBMCs) and spleen cells were obtained from these animals and used as a source of stimulation for human responders in one-way mixed lymphocyte reactions. The response was tested in the presence and absence of clinically available immunomodifiers. CONCLUSIONS Clinically relevant glycan knockout models of pig-to-human xenotransplantation do not enhance the human-anti-pig cellular response. Currently available and conventional immunosuppression has the capacity to mediate the human xenogeneic T cell response to these knockout cells.

Transcatheter Aortic Valve Replacement Versus Aortic Valve Bypass: A Comparison of Outcomes and Economics.

BACKGROUND Transcatheter aortic valve replacement (TAVR) is currently offered to patients who are high-risk candidates for conventional surgical aortic valve replacement. For the past 37 years, off-pump aortic valve bypass (AVB) has been used in elderly patients at our center for this similarly high-risk group. Although TAVR and AVB were offered to similar patients at our center, comparisons of clinical outcomes and hospital economics for each strategy were not reported. METHODS We reviewed the clinical and financial records of 53 consecutive AVB procedures performed since 2008 with the records of 51 consecutive TAVR procedures performed since 2012. Data included demographics, hemodynamics, The Society of Thoracic Surgeons (STS) risk score, extent of coronary disease, and ventricular function. Follow-up was 100% in both groups. Hospital financial information for both cohorts was obtained. Mean risk score for the TAVR group was 10.1% versus 17.6% for AVB group (p < 0.001). RESULTS Kaplan-Meier hospital rates of 3- and 6-month survival and of 1-year survival were 88%, 86%, 81%, and 61% and 89%, 83%, 83%, and 70% for the TAVR and AVB groups, respectively (p = 0.781). Two patients who had undergone TAVR had a procedure-related stroke. The one stroke in an AVB recipient was late and not procedure related. At discharge, mild and moderate perivalvular and central aortic insufficiency were present in 31% and 16% of TAVR recipients, respectively; no AVB valve leaked. Transvalvular gradients were reduced to less than 10 mm Hg in both groups. The average hospital length of stay for the AVB-treated patients was 13 days, and it was 9 days for the TAVR-treated patients. Median hospital charges were

The fate of human platelets exposed to porcine renal endothelium: a single-pass model of platelet uptake in domestic and genetically modified porcine organs

253,000 for TAVR and

Examining the Biosynthesis and Xenoantigenicity of Class II Swine Leukocyte Antigen Proteins

158,000 for AVB. Mean payment to the hospital was

Transcatheter Aortic Valve Replacement Versus Aortic Valve Bypass

65,000 (TAVR) versus