Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 211166, China.
Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 211166, China; Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing 211166, China.
Acta Biomater. 2018 May;72:196-205. doi: 10.1016/j.actbio.2018.03.055. Epub 2018 Apr 7.
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-α1,3-galactose (αGal) and N-glycolylneuraminic acid (Neu5Gc) whose synthesis are catalyzed by α(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 αGal-knockout pigs are associated with a significantly reduced immune response by human serum. Moreover, valves from αGal/Neu5Gc-deficient pigs could further reduce human IgM/IgG binding when compared to BHV from αGal-knockout pigs. Recently, another swine xenoantigen, Sd(a), produced by β-1,4-N-acetyl-galactosaminyl transferase 2 (β4GalNT2), has been identified. To explore whether tissue from GGTA1, CMAH, and β4GalNT2 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 αGal, 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.
Surgical 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 α1,3 galactose (αGal), N-glycolylneuraminic acid (Neu5Gc) and glycan products of β-1,4-N-acetyl-galactosaminyl transferase 2 (β4GalNT2) 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.
生物瓣（BHV）来源于猪，广泛应用于临床心脏瓣膜置换。然而，受者的免疫反应与慢性钙化有关，导致 BHV 的结构瓣膜退化（SVD）。两种在猪 BHV 中特征明确的表位与 SVD 有关，包括半乳糖-α1,3-半乳糖（αGal）和 N- 糖基神经氨酸（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 敲除猪的 BHV 与人血清的免疫反应显著降低。此外，与来自 αGal 敲除猪的 BHV 相比，来自 αGal/Neu5Gc 缺陷猪的瓣膜可进一步降低人 IgM/IgG 结合。最近，另一种猪异种抗原 Sd(a)由β-1,4-N-乙酰半乳糖胺基转移酶 2（β4GalNT2）产生。为了探讨 GGTA1、CMAH 和β4GalNT2 三基因敲除（TKO）猪的组织是否会进一步降低人抗体与猪心包的结合，我们通过 CRISPR/Cas9 介导的基因靶向成功地产生了 TKO 猪。我们的结果表明，TKO 猪的 αGal、Neu5G 和 Sd(a)表达为阴性，人 IgG/IgM 与心包的结合最小。此外，对 TKO 猪的心包胶原组成和物理特性的分析表明，三种异种抗原的消除对猪心包的物理特性没有显著影响。我们的结果表明，TKO 猪将是生物瓣的理想来源。
心脏瓣膜置换术是一种成熟的挽救生命的治疗方法，用于患有心脏瓣膜疾病的患者。戊二醛固定的猪或牛组织制成的生物瓣（BHV）在临床上广泛应用，但存在与 BHVs 免疫反应相关的年龄依赖性结构瓣膜退化（SVD）。本研究通过 CRISPR/Cas9 介导的基因靶向消除了商业 BHV 上存在的三种主要的异种抗原，即半乳糖-α1,3 半乳糖（αGal）、N- 糖基神经氨酸（Neu5Gc）和 β-1,4-N-乙酰半乳糖胺基转移酶 2（β4GalNT2）的糖基产物。通过基因靶向敲除了这三种主要的异种抗原后，经过基因修饰的猪心包显示出免疫原性降低，但与野生型猪的心包胶原组成和物理特性相当。我们的数据表明，来自 TKO 猪的 BHV 是目前可用的来自野生型猪的 BHV 的一种有前途的替代物。
