Reducing immunoreactivity of porcine bioprosthetic heart valves by genetically-deleting three major glycan antigens, GGTA1/β4GalNT2/CMAH
Reducing immunoreactivity of porcine bioprosthetic heart valves by genetically-deleting three major glycan antigens, GGTA1/β4GalNT2/CMAH
复制标题
通过基因删除三种主要聚糖抗原 GGTA1/β 4GalNT2/CMAH 来降低猪生物假体心脏瓣膜的免疫反应性
DOI：
10.1016/j.actbio.2018.03.055
复制
发表时间：
2018-05-01
影响因子：
9.7
通讯作者：
Dai, Yifan
中科院分区：
文献类型：
--
作者：
Zhang, Runjie;Wang, Ying;Dai, Yifan
源于猪的生物心脏瓣膜（BHVs）在临床上广泛用于心脏瓣膜置换。然而，与慢性钙化相关的受体免疫反应会导致生物心脏瓣膜的结构性瓣膜退变（SVD）。猪生物心脏瓣膜上两个特征明确的表位与SVD有关，包括半乳糖 -α1,3 - 半乳糖（αGal）和N - 羟乙酰神经氨酸（Neu5Gc），它们的合成分别由α(1,3)半乳糖基转移酶（由GGTA1基因编码）和CMP - Neu5Ac羟化酶（由CMAH基因编码）催化。据报道，来自αGal基因敲除猪的生物心脏瓣膜与人血清的免疫反应显著降低。此外，与来自αGal基因敲除猪的生物心脏瓣膜相比，来自αGal/Neu5Gc双缺失猪的瓣膜可进一步减少人IgM/IgG结合。最近，另一种猪异种抗原Sd(a)被鉴定出来，它由β - 1,4 - N - 乙酰 - 半乳糖胺基转移酶2（β4GaINT2）产生。为了探究来自GGTA1、CMAH和β4GaINT2三基因敲除（TKO）猪的组织是否会进一步减少人抗体与猪心包的结合，通过CRISPR/Cas9介导的基因打靶成功培育出TKO猪。我们的结果显示，TKO猪的αGal、Neu5G和Sd(a)表达为阴性，人IgG/IgM与心包的结合极少。此外，对TKO猪心包的胶原蛋白组成和物理特性分析表明，消除这三种异种抗原对猪心包的物理特性没有显著影响。我们的结果证明TKO猪将是生物心脏瓣膜的理想来源。
重要性声明
心脏瓣膜置换手术是治疗病变心脏瓣膜的一种成熟的救命疗法。由戊二醛固定的猪或牛组织制成的生物心脏瓣膜在临床上广泛使用，但表现出与年龄相关的结构性瓣膜退变，这与针对生物心脏瓣膜的免疫反应有关。本研究通过CRISPR/Cas9介导的基因打靶消除了商业生物心脏瓣膜上存在的三种主要异种抗原，即半乳糖 -α1,3 - 半乳糖（αGal）、N - 羟乙酰神经氨酸（Neu5Gc）和β - 1,4 - N - 乙酰 - 半乳糖胺基转移酶2（β4GaINT2）的聚糖产物。转基因猪心包显示免疫原性降低，但与野生型猪心包的胶原蛋白组成和物理特性相当。我们的数据表明，来自TKO猪的生物心脏瓣膜是目前来自野生型猪的生物心脏瓣膜的一种有前景的替代品。（C）2018 Acta Materialia Inc. 由Elsevier Ltd.出版。保留所有权利。
Bioprosthetic heart valves (BHVs) originating from pigs are extensively used for heart valve replacement in clinics. However, recipient immune responses associated with chronic calcification lead to structural valve deterioration (SVD) of BHVs. Two well-characterized epitopes on porcine BHVs have been implicated in SVD, including galactose-alpha 1,3-galactose (alpha Gal) and N-glycolylneuraminic acid (Neu5Gc) whose synthesis are catalyzed by alpha(1,3) galactosyltransferase (encoded by the GGTA1 gene) and CMP-Neu5Ac hydroxylase (encoded by the CMAH gene), respectively. It has been reported that BHV from alpha Gal-knockout pigs are associated with a significantly reduced immune response by human serum. Moreover, valves from alpha Gal/Neu5Gc-deficient pigs could further reduce human IgM/IgG binding when compared to BHV from alpha Gal-knockout pigs. Recently, another swine xenoantigen, Sd(a), produced by beta-1,4-N-acetyl-galactosaminyl transferase 2 (beta 4GaINT2), has been identified. To explore whether tissue from GGTA1, CMAH, and beta 4GaINT2 triple gene-knockout (TKO) pigs would further minimize human antibody binding to porcine pericardium, TKO pigs were successfully produced by CRISPR/Cas9 mediated gene targeting. Our results showed that the expression of alpha Gal, Neu5G and Sd(a) on TKO pigs was negative, and that human IgG/IgM binding to pericardium was minimal. Moreover, the analysis of collagen composition and physical characteristics of porcine pericardium from the TKO pigs indicated that elimination of the three xenoantigens had no significant impact on the physical proprieties of porcine pericardium. Our results demonstrated that TKO pigs would be an ideal source of BHVs.Statement of significanceSurgical heart valve replacement is an established lifesaving treatment for diseased heart valve. Bioprosthetic heart valves (BHVs) made from glutaraldehyde-fixed porcine or bovine tissues are widely used in clinics but exhibit age-dependent structural valve degeneration (SVD) which is associated with the immune response against BHVs. Three major xenoantigens present on commercial BHVs, Galactosea alpha 1,3 galactose (alpha Gal), N-glycolylneuraminic acid (Neu5Gc) and glycan products of beta-1,4-N-acetyl-galactosaminyl transferase 2 (beta 4GaINT2) are eliminated through CRISPR/Cas9 mediated gene targeting in the present study. The genetically modified porcine pericardium showed reduced immunogenicity but comparable collagen composition and physical characteristics of the pericardium from wild-type pigs. Our data suggested that BHVs from TKO pigs is a promising alternative for currently available BHVs from wild-type pigs. (C) 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.