Parrett Brian J, Yamaoka Satoko, Barry Michael A
Virology and Gene Therapy (VGT) Graduate Program, Mayo Clinic, Rochester, MN 55902, USA.
Department of Medicine, Division of Infectious Diseases, Mayo Clinic, Rochester, MN 55902, USA.
Mol Ther Methods Clin Dev. 2024 Dec 19;33(1):101402. doi: 10.1016/j.omtm.2024.101402. eCollection 2025 Mar 13.
Lipid nanoparticles (LNPs) are often liver tropic, presenting challenges for LNP-delivered mRNA therapeutics intended for other tissues, as off-target expression in the liver may increase side effects and modulate immune responses. To avoid off-target expression in the liver, miR-122 binding sites have been used by others in viral and non-viral therapeutics. Here, we use a luciferase reporter system to compare different copy numbers and insertion locations of miR-122 binding sequences to restrict liver expression. We inserted one to five miR-122 binding sites into the 5' or 3' untranslated regions (UTRs) of luciferase mRNAs and tested them in LNPs and via systemic intravenous and local intramuscular injections in mice. Our results showed no significant differences in de-targeting efficacy between mRNAs harboring one or multiple miR-122 binding sites or between those with 5' or 3' UTR placements. To test the impact of miR-122 binding sites on antibody response to a mRNA vaccine, Ebola virus matrix protein VP40 mRNAs were modified with or without miR-122 binding sites and injected in mice intramuscularly. This work reinforces the utility of miR-122 binding sites while providing a comparison of these sites to aid the future development of LNP-mRNA therapies for non-hepatic tissues.
脂质纳米颗粒（LNPs）通常具有肝脏靶向性，这给旨在用于其他组织的LNP递送的mRNA疗法带来了挑战，因为肝脏中的脱靶表达可能会增加副作用并调节免疫反应。为了避免在肝脏中出现脱靶表达，其他人已在病毒和非病毒疗法中使用了miR-122结合位点。在这里，我们使用荧光素酶报告系统来比较miR-122结合序列的不同拷贝数和插入位置，以限制肝脏表达。我们将一到五个miR-122结合位点插入荧光素酶mRNA的5'或3'非翻译区（UTRs），并在LNPs中以及通过在小鼠体内进行全身静脉注射和局部肌肉注射来对它们进行测试。我们的结果表明，含有一个或多个miR-122结合位点的mRNA之间，或5'或3'UTR位置的mRNA之间，在去靶向功效上没有显著差异。为了测试miR-122结合位点对mRNA疫苗抗体反应的影响，对带有或不带有miR-122结合位点的埃博拉病毒基质蛋白VP40 mRNA进行修饰，并在小鼠体内进行肌肉注射。这项工作强化了miR-122结合位点的实用性，同时对这些位点进行了比较，以帮助未来开发用于非肝组织的LNP-mRNA疗法。
