Centre for Comparative Genomics, Discovery Way, Murdoch University, Perth, WA, 6150, Australia.
Perron Institute for Neurological and Translational Science, Nedlands, WA, 6009, Australia.
Sci Rep. 2017 May 9;7(1):1613. doi: 10.1038/s41598-017-01559-w.
Integrin alpha-4 (ITGA4) is a validated therapeutic target for multiple sclerosis (MS) and Natalizumab, an antibody targeting ITGA4 is currently approved for treating MS. However, there are severe side effects related to this therapy. In this study, we report the development of a novel DNAzyme that can efficiently cleave the ITGA4 transcript. We designed a range of DNAzyme candidates across various exons of ITGA4. RNV143, a 30mer arm-loop-arm type DNAzyme efficiently cleaved 84% of the ITGA4 mRNA in human primary fibroblasts. RNV143 was then systematically modified by increasing the arm lengths on both sides of the DNAzymes by one, two and three nucleotides each, and incorporating chemical modifications such as inverted-dT, phosphorothioate backbone and LNA-nucleotides. Increasing the arm length of DNAzyme RNV143 did not improve the efficiency however, an inverted-dT modification provided the most resistance to 3' → 5' exonuclease compared to other modifications tested. Our results show that RNV143A could be a potential therapeutic nucleic acid drug molecule towards the treatment for MS.
整合素 alpha-4（ITGA4）是多发性硬化症（MS）的一个经过验证的治疗靶点，针对 ITGA4 的那他珠单抗（Natalizumab）目前已被批准用于治疗 MS。然而，这种治疗方法存在严重的副作用。在这项研究中，我们报告了一种新型 DNA 酶的开发，该酶可以有效地切割 ITGA4 转录本。我们设计了一系列跨越 ITGA4 不同外显子的 DNA 酶候选物。RNV143 是一种 30 个碱基的臂环臂型 DNA 酶，能有效地切割人原代成纤维细胞中 84%的 ITGA4 mRNA。然后，我们通过在 DNA 酶的两侧各增加一个、两个和三个核苷酸来系统地修饰 RNV143，并引入了一些化学修饰，如反向-dT、硫代磷酸酯骨架和 LNA-核苷酸。然而，增加 DNA 酶 RNV143 的臂长并没有提高效率，与其他测试的修饰相比，反向-dT 修饰提供了对 3'→5' 外切核酸酶的最大抗性。我们的结果表明，RNV143A 可能是一种有潜力的治疗性核酸药物分子，可用于治疗 MS。
