Intratracheally administered LNA gapmer antisense oligonucleotides induce robust gene silencing in mouse lung fibroblasts.
Intratracheally administered LNA gapmer antisense oligonucleotides induce robust gene silencing in mouse lung fibroblasts.
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DOI：
10.1093/nar/gkac630
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发表时间：
2022-08-26
影响因子：
14.9
通讯作者：
Watts, Jonathan K.
中科院分区：
文献类型：
--
作者：
Shin, Minwook;Chan, Io Long;Cao, Yuming;Gruntman, Alisha M.;Lee, Jonathan;Sousa, Jacquelyn;Rodriguez, Tomas C.;Echeverria, Dimas;Devi, Gitali;Debacker, Alexandre J.;Moazami, Michael P.;Krishnamurthy, Pranathi Meda;Rembetsy-Brown, Julia M.;Kelly, Karen;Yukselen, Onur;Donnard, Elisa;Parsons, Teagan J.;Khvorova, Anastasia;Sontheimer, Erik J.;Maehr, Rene;Garber, Manuel;Watts, Jonathan K.
肺是一个复杂的器官，各种细胞类型具有不同的作用。已经在肺中研究了反义寡核苷酸（ASO），但是由于缺乏适当的分析方法，确定它们在每种细胞类型中的有效性一直是具有挑战性的。我们采用了三种不同的方法来研究不同细胞类型内的沉默功效。首先，我们使用谱系标记物在流式细胞术中鉴定细胞类型，并同时测量ASO诱导的细胞表面蛋白CD 47或CD 98沉默。其次，我们应用单细胞RNA测序（scRNA-seq）来测量不同细胞类型中的沉默功效;据我们所知，这是scRNA-seq首次应用于测量寡核苷酸治疗的功效。在这两种方法中，成纤维细胞对局部递送的ASO最敏感，在内皮细胞中也具有显著的沉默。第三，我们通过沉默主要在成纤维细胞中表达的两个靶标Mfap 4和Adam 33，证实了成纤维细胞中的稳健沉默是广泛适用的。在独立的方法中，我们证明了血管内施用LNA缺口体ASO在肺成纤维细胞中强烈诱导基因沉默。成纤维细胞中ASO诱导的基因沉默是持久的，单次给药后持续4-8周。因此，肺成纤维细胞与作为治疗剂的ASO很好地一致。
The lung is a complex organ with various cell types having distinct roles. Antisense oligonucleotides (ASOs) have been studied in the lung, but it has been challenging to determine their effectiveness in each cell type due to the lack of appropriate analytical methods. We employed three distinct approaches to study silencing efficacy within different cell types. First, we used lineage markers to identify cell types in flow cytometry, and simultaneously measured ASO-induced silencing of cell-surface proteins CD47 or CD98. Second, we applied single-cell RNA sequencing (scRNA-seq) to measure silencing efficacy in distinct cell types; to the best of our knowledge, this is the first time scRNA-seq has been applied to measure the efficacy of oligonucleotide therapeutics. In both approaches, fibroblasts were the most susceptible to locally delivered ASOs, with significant silencing also in endothelial cells. Third, we confirmed that the robust silencing in fibroblasts is broadly applicable by silencing two targets expressed mainly in fibroblasts, Mfap4 and Adam33. Across independent approaches, we demonstrate that intratracheally administered LNA gapmer ASOs robustly induce gene silencing in lung fibroblasts. ASO-induced gene silencing in fibroblasts was durable, lasting 4–8 weeks after a single dose. Thus, lung fibroblasts are well aligned with ASOs as therapeutics.
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影响因子：
14.9
作者：
Jafar-Nejad P;Powers B;Soriano A;Zhao H;Norris DA;Matson J;DeBrosse-Serra B;Watson J;Narayanan P;Chun SJ;Mazur C;Kordasiewicz H;Swayze EE;Rigo F
通讯作者：
Rigo F
影响因子：
4
作者：
Crooke ST
通讯作者：
Crooke ST
影响因子：
14.9
作者：
Frankish A;Diekhans M;Ferreira AM;Johnson R;Jungreis I;Loveland J;Mudge JM;Sisu C;Wright J;Armstrong J;Barnes I;Berry A;Bignell A;Carbonell Sala S;Chrast J;Cunningham F;Di Domenico T;Donaldson S;Fiddes IT;García Girón C;Gonzalez JM;Grego T;Hardy M;Hourlier T;Hunt T;Izuogu OG;Lagarde J;Martin FJ;Martínez L;Mohanan S;Muir P;Navarro FCP;Parker A;Pei B;Pozo F;Ruffier M;Schmitt BM;Stapleton E;Suner MM;Sycheva I;Uszczynska-Ratajczak B;Xu J;Yates A;Zerbino D;Zhang Y;Aken B;Choudhary JS;Gerstein M;Guigó R;Hubbard TJP;Kellis M;Paten B;Reymond A;Tress ML;Flicek P
通讯作者：
Flicek P
影响因子：
7
作者：
Derr A;Yang C;Zilionis R;Sergushichev A;Blodgett DM;Redick S;Bortell R;Luban J;Harlan DM;Kadener S;Greiner DL;Klein A;Artyomov MN;Garber M
通讯作者：
Garber M
DOI：
10.1513/pats.200803-025hr
发表时间：
2008-09-15
期刊：
Proceedings of the American Thoracic Society
影响因子：
--
作者：
Franks, Teri J;Colby, Thomas V;Williams, Mary C
通讯作者：
Williams, Mary C