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Xenotransplantation. 2019 May 22:e12524. doi: 10.1111/xen.12524. [Epub ahead of print]

Generation of cattle knockout for galactose-α1,3-galactose and N-glycolylneuraminic acid antigens.

Author information

1
Avantea, Laboratory of Reproductive Technologies, Cremona, Italy.
2
Fondazione Avantea, Cremona, Italy.
3
Centre de Recherche en Transplantation et Immunologie, UMR 1064, INSERM, Université de Nantes, Nantes, France.
4
Institut de Transplantation Urologie Néphrologie (ITUN), CHU Nantes, Nantes, France.
5
IECM, Immuno-endocrinology, EA4644 Oniris, University of Nantes, USC1383 INRA, Oniris, Nantes, France.
6
Cardiac Surgery Unit - Department of Cardiac, Thoracic and Vascular Sciences and Public Health - Padova University School of Medicine and CORIS, Padova, Italy.
7
Infectious Diseases and Transplantation Division, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), Hospitalet de Llobregat, Barcelona, Spain.
8
Reparative Therapy of the Heart, Vall d'Hebron Research Institute (VHIR) and Department of Cardiac Surgery, University Hospital Vall d'Hebron, Autonomous University of Barcelona (AUB), Barcelona, Spain.
9
Department of Thoracic and CardioVascular Surgery, Nantes Hospital University, Nantes, France.
10
The George S. Wise Faculty of Life Sciences, Department of Cell Research and Immunology, Tel Aviv University, Tel Aviv, Israel.
11
Transplant Immunology Unit, Padua General Hospital, Padua, Italy.

Abstract

Two well-characterized carbohydrate epitopes are absent in humans but present in other mammals. These are galactose-α1,3-galactose (αGal) and N-glycolylneuraminic acid (Neu5Gc) which are introduced by the activities of two enzymes including α(1,3) galactosyltransferase (encoded by the GGTA1 gene) and CMP-Neu5Gc hydroxylase (encoded by the CMAH gene) that are inactive in humans but present in cattle. Hence, bovine-derived products are antigenic in humans who receive bioprosthetic heart valves (BHVs) or those that suffer from red meat syndrome. Using programmable nucleases, we disrupted (knockout, KO) GGTA1 and CMAH genes encoding for the enzymes that catalyse the synthesis of αGal and Neu5Gc, respectively, in both male and female bovine fibroblasts. The KO in clonally selected fibroblasts was detected by polymerase chain reaction (PCR) and confirmed by Sanger sequencing. Selected fibroblasts colonies were used for somatic cell nuclear transfer (SCNT) to produce cloned embryos that were implanted in surrogate recipient heifers. Fifty-three embryos were implanted in 33 recipients heifers; 3 pregnancies were carried to term and delivered 3 live calves. Primary cell cultures were established from the 3 calves and following molecular analyses confirmed the genetic deletions. FACS analysis showed the double-KO phenotype for both antigens confirming the mutated genotypes. Availability of such cattle double-KO model lacking both αGal and Neu5Gc offers a unique opportunity to study the functionality of BHV manufactured with tissues of potentially lower immunogenicity, as well as a possible new clinical approaches to help patients with red meat allergy syndrome due to the presence of these xenoantigens in the diet.

KEYWORDS:

CMAH ; GGTA1 ; Neu5Gc; bioprosthetic Heart Valve (BHV); cattle; knockout; xenotransplantation; αGal

PMID:
31115108
DOI:
10.1111/xen.12524

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