Increased erythropoiesis in mice injected with submicrogram quantities of pseudouridine-containing mRNA encoding erythropoietin.
Increased erythropoiesis in mice injected with submicrogram quantities of pseudouridine-containing mRNA encoding erythropoietin.
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DOI：
10.1038/mt.2012.7
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发表时间：
2012-05
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体外转录mRNA的优化进展使mRNA介导的治疗更接近现实。在培养的细胞中，我们最近实现了高水平的翻译与高效液相色谱（HPLC）纯化，在体外转录的mRNA含有修饰的核苷假尿苷。重要的是，假尿苷使mRNA无免疫原性。在这里，使用促红细胞生成素（EPO）编码的mRNA与transIT-mRNA复合，我们在体内评估这一新一代的mRNA。单次注射100 ng（0.005 mg/kg）mRNA显著升高小鼠血清EPO水平6小时，并维持4天。相比之下，含有尿苷的mRNA产生的EPO水平低10-100倍，仅持续1天。从假尿苷mRNA翻译的EPO是功能性的，并引起网织红细胞计数和血细胞比容的显着增加。10 ng mRNA使网织红细胞数量加倍。每周注射100 ng EPO mRNA足以将红细胞压积从43%增加到57%，并通过继续治疗维持。即使注射大量的假尿苷-mRNA，血浆中也检测不到炎性细胞因子。使用猕猴，我们也可以检测到显着增加血清EPO水平后，腹腔注射恒河猴EPO mRNA。这些结果表明，HPLC纯化的，含有假尿苷的mRNA编码的治疗性蛋白质具有很大的临床应用潜力。
Advances in the optimization of in vitro-transcribed mRNA are bringing mRNA-mediated therapy closer to reality. In cultured cells, we recently achieved high levels of translation with high-performance liquid chromatography (HPLC)-purified, in vitro-transcribed mRNAs containing the modified nucleoside pseudouridine. Importantly, pseudouridine rendered the mRNA non-immunogenic. Here, using erythropoietin (EPO)-encoding mRNA complexed with TransIT-mRNA, we evaluated this new generation of mRNA in vivo. A single injection of 100 ng (0.005 mg/kg) mRNA elevated serum EPO levels in mice significantly by 6 hours and levels were maintained for 4 days. In comparison, mRNA containing uridine produced 10–100-fold lower levels of EPO lasting only 1 day. EPO translated from pseudouridine-mRNA was functional and caused a significant increase of both reticulocyte counts and hematocrits. As little as 10 ng mRNA doubled reticulocyte numbers. Weekly injection of 100 ng of EPO mRNA was sufficient to increase the hematocrit from 43 to 57%, which was maintained with continued treatment. Even when a large amount of pseudouridine-mRNA was injected, no inflammatory cytokines were detectable in plasma. Using macaques, we could also detect significantly-increased serum EPO levels following intraperitoneal injection of rhesus EPO mRNA. These results demonstrate that HPLC-purified, pseudouridine-containing mRNAs encoding therapeutic proteins have great potential for clinical applications.
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