Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells
Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells
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DOI：
10.1182/blood-2006-04-015024
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发表时间：
2006-12-15
期刊：
影响因子：
20.3
通讯作者：
Sahin, Ugur
中科院分区：
文献类型：
--
作者：
Holtkamp, Silke;Kreiter, Sebastian;Sahin, Ugur
树突状细胞（DC）转染体外转录，RNA编码，肿瘤相关抗原的连续转移最近进入临床试验作为一种有前途的方法，用于癌症免疫治疗。然而，RNA作为一种潜在的药物化合物和临床开发的一个关键方面的药物动力学探索仍然悬而未决。在研究RNA分子的不同结构修饰对DC中编码蛋白的动力学的影响时，我们鉴定了位于编码区3'的组分，其有助于更高的转录稳定性和翻译效率。通过使用定量逆转录-聚合酶链反应（RT-PCR）和eGFP变体来测量转录物量和蛋白质产量，我们表明与较短的多聚（A）高相比，测量120个核苷酸的多聚（A）高，具有游离3'端的未掩蔽的多聚（A）高而不是用不相关的核苷酸延伸的多聚（A）高，以及在编码区和poly（A）尾之间从头到脚克隆的2个连续的β-珠蛋白3'非翻译区，每个独立地增强RNA稳定性和翻译效率。转染细胞表面抗原特异性肽/MHC复合物的密度及其刺激和扩增抗原特异性CD 4（+）和CD 8（+）T细胞的能力也增加。总之，我们的数据提供了优化RNA转染的DC疫苗的策略和定义此类疫苗制剂的放行标准的基础。
Adoptive transfer of dendritic cells (DCs) transfected with in vitro-transcribed, RNA-encoding, tumor-associated antigens has recently entered clinical testing as a promising approach for cancer immunotherapy. However, pharmacolkinetic exploration of RNA as a potential drug compound and a key aspect of clinical development is still pending. While investigating the impact of different structural modifications of RNA molecules on the kinetics of the encoded protein in DCs, we identified components located 3' of the coding region that contributed to a higher transcript stability and translational efficiency. With the use of quantitative reverse transcription-polymerase chain reaction (RT-PCR) and eGFP variants to measure transcript amounts and protein yield, we showed that a poly(A) tall measuring 120 nucleotides compared with a shorter one, an unmasked poly(A) tall with a free 3' end rather than one extended with unrelated nucleotides, and 2 sequential beta-globin 3' untranslated regions cloned head to tall between the coding region and the poly(A) tail each independently enhanced RNA stability and translational efficiency. Consecutively, the density of antigen-specific peptide/MHC complexes on the transfected cells and their potency to stimulate and expand antigen-specific CD4(+) and CD8(+) T cells were also increased. In summary, our data provide a strategy for optimizing RNA-transfected DC vaccines and a basis for defining release criteria for such vaccine preparations.