Rapid, Single-Cell Analysis and Discovery of Vectored mRNA Transfection In Vivo with a loxP-Flanked tdTomato Reporter Mouse.
Rapid, Single-Cell Analysis and Discovery of Vectored mRNA Transfection In Vivo with a loxP-Flanked tdTomato Reporter Mouse.
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DOI：
10.1016/j.omtn.2017.11.005
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发表时间：
2018-03-02
期刊：
影响因子：
--
通讯作者：
Anderson DG
中科院分区：
文献类型：
--
作者：
Kauffman KJ;Oberli MA;Dorkin JR;Hurtado JE;Kaczmarek JC;Bhadani S;Wyckoff J;Langer R;Jaklenec A;Anderson DG
信使核糖核酸疗法有望治疗需要细胞内蛋白表达的疾病，并用于基因组编辑系统，但信使核糖核酸必须转入所需的组织和细胞类型才能有效。通常使用编码报告基因(如萤火虫荧光素酶(FLOC))的mRNA来评估递送mRNAs的纳米颗粒载体；然而，通常不能用FLOC实现单细胞分辨mRNAs表达，因此，如果没有额外的步骤或实验，就不能确定转基因细胞群。为了更快地确定mRNA制剂在体内转染哪些类型的细胞，我们描述了一种基于Cre重组酶(Cre)的系统，该系统在转基因小鼠的转基因细胞中永久表达荧光tdTomato蛋白。在体内应用载体Cre mRNA后，通过Cre介导的tdTomato在块状组织中的表达并以单细胞分辨率显示成功的转基因细胞是可能的。利用这个系统，我们在体内鉴定了现有的mRNA递送载体中以前未知的转基因细胞类型，并开发了一种能够转染肺内皮细胞的新的mRNA配方。重要的是，将编码荧光蛋白的mRNA传递给野生型小鼠的相同配方没有为体内的任何可视化产生足够的信号，这表明我们基于CRE的系统的灵敏度显著提高。我们相信，这里描述的系统可能有助于鉴定和鉴定新组织和细胞类型的信使核糖核酸传递载体。
mRNA therapeutics hold promise for the treatment of diseases requiring intracellular protein expression and for use in genome editing systems, but mRNA must transfect the desired tissue and cell type to be efficacious. Nanoparticle vectors that deliver the mRNA are often evaluated using mRNA encoding for reporter genes such as firefly luciferase (FLuc); however, single-cell resolution of mRNA expression cannot generally be achieved with FLuc, and, thus, the transfected cell populations cannot be determined without additional steps or experiments. To more rapidly identify which types of cells an mRNA formulation transfects in vivo, we describe a Cre recombinase (Cre)-based system that permanently expresses fluorescent tdTomato protein in transfected cells of genetically modified mice. Following in vivo application of vectored Cre mRNA, it is possible to visualize successfully transfected cells via Cre-mediated tdTomato expression in bulk tissues and with single-cell resolution. Using this system, we identify previously unknown transfected cell types of an existing mRNA delivery vehicle in vivo and also develop a new mRNA formulation capable of transfecting lung endothelial cells. Importantly, the same formulations with mRNA encoding for fluorescent protein delivered to wild-type mice did not produce sufficient signal for any visualization in vivo, demonstrating the significantly improved sensitivity of our Cre-based system. We believe that the system described here may facilitate the identification and characterization of mRNA delivery vectors to new tissues and cell types.
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影响因子：
12.4
作者：
Kariko, Katalin;Muramatsu, Hiromi;Welsh, Frank A.;Ludwig, Janos;Kato, Hiroki;Akira, Shizuo;Weissman, Drew
通讯作者：
Weissman, Drew
在体内用脂质纳米颗粒系统地传递了未修饰和假氨酸修饰的mRNA的功效和免疫原性。
影响因子：
14
作者：
Kauffman KJ;Mir FF;Jhunjhunwala S;Kaczmarek JC;Hurtado JE;Yang JH;Webber MJ;Kowalski PS;Heartlein MW;DeRosa F;Anderson DG
通讯作者：
Anderson DG
DOI：
10.1016/j.jconrel.2016.05.031
发表时间：
2016-10-28
期刊：
Journal of controlled release : official journal of the Controlled Release Society
影响因子：
--
作者：
Kim N;Duncan GA;Hanes J;Suk JS
通讯作者：
Suk JS
影响因子：
10.8
作者：
Kauffman, Kevin J.;Dorkin, J. Robert;Anderson, Daniel G.
通讯作者：
Anderson, Daniel G.
影响因子：
15
作者：
Chen, Delai;Love, Kevin T.;Anderson, Daniel G.
通讯作者：
Anderson, Daniel G.