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Genetic engineering strategies to prevent the effects of antibody and complement on xenogeneic chondrocytes

机译：防止抗体和补体对异种软骨细胞影响的基因工程策略

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Advances in animal transgenesis may allow using xenogeneic chondrocytes in tissue-engineering applications for clinical cartilage repair. Porcine cartilage is rejected by humoral and cellular mechanisms that could be overcome by identifying key molecules triggering rejection and developing effective genetic-engineering strategies. Accordingly, high expression of α1,2-fucosyltransferase (HT) in xenogeneic cartilage protects from galactose α1,3-galactose (Gal)-mediated antibody responses. Now, we studied whether expression of a complement inhibitor provides further protection. First, porcine articular chondrocytes (PAC) were isolated from non-transgenic, single and double transgenic pigs expressing HT and moderate levels of human CD59 (hCD59) and their response to human serum was assessed. High recombinant expression of human complement regulatory molecules hCD59 and hDAF was also attained by retroviral transduction of PAC for further analyses. Complement activation on PAC after exposure to 20 % human serum for 24 hours mainly triggered the release of pro-inflammatory cytokines IL-6 and IL-8. Transgenic expression of HT and hCD59 did not suffice to fully counteract this effect. Nevertheless, the combination of blocking anti-Gal antibodies (or C5a) and high hCD59 levels conferred very high protection. On the contrary, high hDAF expression attained the most dramatic reduction in IL-6/IL-8 secretion by a single strategy, but the additional inhibition of anti-Gal antibodies or C5a did not provide further improvement. Notably, we demonstrate that both hCD59 and hDAF inhibit anaphylatoxin release in this setting. In conclusion, our study identifies genetic-engineering approaches to prevent humoral rejection of xenogeneic chondrocytes for use in cartilage repair.
机译：动物转基因的进展可能允许在组织工程应用中使用异种软骨细胞进行临床软骨修复。猪软骨被体液和细胞机制所排斥，可以通过识别触发排斥的关键分子并开发有效的基因工程策略来克服。因此，异种软骨中α1,2-岩藻糖基转移酶（HT）的高表达可防止半乳糖α1,3-半乳糖（Gal）介导的抗体反应。现在，我们研究了补体抑制剂的表达是否提供进一步的保护。首先，从表达HT和中等水平的人CD59（hCD59）的非转基因，单和双转基因猪中分离出猪关节软骨细胞（PAC），并评估它们对人血清的反应。人补体调节分子hCD59和hDAF的高重组表达也可以通过逆转录病毒转导PAC来进行进一步分析。暴露于20％人体血清24小时后，PAC上的补体激活主要触发促炎性细胞因子IL-6和IL-8的释放。 HT和hCD59的转基因表达不足以完全抵消这种作用。但是，阻断性抗Gal抗体（或C5a）和高hCD59水平的组合可提供非常高的保护。相反，高hDAF表达通过单一策略获得了最显着的IL-6 / IL-8分泌降低，但抗Gal抗体或C5a的额外抑制作用却无法提供进一步的改善。值得注意的是，我们证明在这种情况下，hCD59和hDAF均抑制过敏毒素的释放。总之，我们的研究确定了防止软骨修复中异种软骨细胞发生体液排斥的基因工程方法。

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