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

• DOI: 10.1111/xen.12524
• Publication Date: 2019-05-22T09:32:12Z
• Authors: Andrea Perota; Irina Lagutina; Roberto Duchi; Elisa Zanfrini; Giovanna Lazzari; Jean Paul Judor; Sophie Conchon; Jean Marie Bach; Tomaso Bottio; Gino Gerosa; Cristina Costa; Manuel Galiñanes; Jean Christian Ro...; Vered Padler‐Kara...; Emanuele Cozzi; Jean Paul Soulillou; Cesare Galli
• Keywords: Male; 0301 basic medicine; 570; Knockout; Transplantation, Heterologous; knockout; Neu5Gc; 630; Mixed Function Oxygenases; Animals, Genetically Modified; Gene Knockout Techniques; 03 medical and health sciences; Carn de boví; xenotransplantation; Antigens, Heterophile; Cytidine Monophosphate; Animals; Humans; Heart valves; Bioprosthesis; 2. Zero hunger; [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology; GGTA1; Galactose; αGal; Original Articles; CMAH; Fibroblasts; Vàlvules cardíaques; Bioprosthetic Heart Valve (BHV); Galactosyltransferases; cattle; Heart Valve Prosthesis; bioprosthetic Heart Valve (BHV); Xenotransplantation; Cattle; Female; Neuraminic Acids; Beef; bioprosthetic Heart Valve (BHV); cattle; CMAH; GGTA1; knockout; Neu5Gc; xenotransplantation; αGal; [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology; Food Hypersensitivity

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