Department of Surgery, University of Minnesota Twin Cities, Minneapolis, Minn; Institute of Cardiovascular Sciences, University College London, London, United Kingdom.
Department of Surgery, University of Minnesota Twin Cities, Minneapolis, Minn; Institute of Cardiovascular Sciences, University College London, London, United Kingdom.
J Thorac Cardiovasc Surg. 2023 Oct;166(4):e142-e152. doi: 10.1016/j.jtcvs.2023.02.007. Epub 2023 Feb 15.
Heart valve implantation in juvenile sheep to demonstrate biocompatibility and physiologic performance is the accepted model for regulatory approval of new biological heart valves (BHVs). However, this standard model does not detect the immunologic incompatibility between the major xenogeneic antigen, galactose-α-1,3-galactose (Gal), which is present in all current commercial BHVs, and patients who universally produce anti-Gal antibody. This clinical discordance leads to induced anti-Gal antibody in BHV recipients, promoting tissue calcification and premature structural valve degeneration, especially in young patients. The objective of the present study was to develop genetically engineered sheep that, like humans, produce anti-Gal antibody and mirror current clinical immune discordance.
Guide RNA for CRISPR Cas9 nuclease was transfected into sheep fetal fibroblasts, creating a biallelic frame shift mutation in exon 4 of the ovine α-galactosyltransferase gene (GGTA1). Somatic cell nuclear transfer was performed, and cloned embryos were transferred to synchronized recipients. Cloned offspring were analyzed for expression of Gal antigen and spontaneous production of anti-Gal antibody.
Two of 4 surviving sheep survived long-term. One of the 2 was devoid of the Gal antigen (GalKO) and expressed cytotoxic anti-Gal antibody by age 2 to 3 months, which increased to clinically relevant levels by 6 months.
GalKO sheep represent a new, clinically relevant advanced standard for preclinical testing of BHVs (surgical or transcatheter) by accounting for the first time for human immune responses to residual Gal antigen that persists after current BHV tissue processing. This will identify the consequences of immune disparity preclinically and avoid unexpected past clinical sequelae.
在幼年绵羊中植入心脏瓣膜以证明生物相容性和生理性能是新的生物心脏瓣膜（BHV）获得监管批准的公认模型。然而，这种标准模型无法检测到所有当前商业 BHV 中存在的主要异种抗原半乳糖-α-1,3-半乳糖（Gal）与普遍产生抗 Gal 抗体的患者之间的免疫不相容性。这种临床差异导致 BHV 受者产生诱导性抗 Gal 抗体，促进组织钙化和早期结构瓣膜退化，尤其是在年轻患者中。本研究的目的是开发类似于人类的基因工程绵羊，产生抗 Gal 抗体并反映当前的临床免疫差异。
将 CRISPR Cas9 核酸酶的向导 RNA 转染到绵羊胎儿成纤维细胞中，在羊α-半乳糖基转移酶基因（GGTA1）的外显子 4 中产生双等位基因移码突变。进行体细胞核移植，将克隆胚胎转移至同步受体。对克隆后代进行 Gal 抗原表达和自发产生抗 Gal 抗体的分析。
4 只存活的绵羊中有 2 只长期存活。其中 1 只（GalKO）缺乏 Gal 抗原，并在 2 至 3 个月大时表达细胞毒性抗 Gal 抗体，到 6 个月时增加到具有临床相关性的水平。
GalKO 绵羊通过首次考虑到当前 BHV 组织处理后仍然存在的残留 Gal 抗原引起的人类免疫反应，代表了 BHVs（外科或经导管）的新的、具有临床相关性的高级标准。这将在临床前识别免疫差异的后果，并避免意外的临床后遗症。
