Quantitative Measurement of Cytosolic and Nuclear Penetration of Oligonucleotide Therapeutics.
Quantitative Measurement of Cytosolic and Nuclear Penetration of Oligonucleotide Therapeutics.
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DOI：
10.1021/acschembio.1c00830
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发表时间：
2022-02-18
影响因子：
4
通讯作者：
Kritzer, Joshua A.
中科院分区：
文献类型：
--
作者：
Deprey, Kirsten;Batistatou, Nefeli;Debets, Marjoke F.;Godfrey, Jack;VanderWall, Kirstin B.;Miles, Rebecca R.;Shehaj, Livia;Guo, Jiaxing;Andreucci, Amy;Kandasamy, Pachamuthu;Lu, Genliang;Shimizu, Mamoru;Vargeese, Chandra;Kritzer, Joshua A.
开发有效寡核苷酸疗法的一个主要障碍是缺乏对其细胞质和细胞核渗透的了解。为了解决这个问题，我们将氯烷渗透测定（CAPA）应用于寡核苷酸治疗。 CAPA 用于定量反义寡核苷酸 (ASO) 和 siRNA 的胞质递送，并探索各种常用化学修饰及其模式的影响。我们通过探索血清的影响、比较活性数据和 CAPA 数据以及评估氯烷标签及其连接子化学的影响来评估潜在的伪影。我们还使用病毒转导将 CAPA 扩展到上皮细胞和神经元细胞系的核区室。使用这种增强的方法，我们测量了一组化学多样化的修饰 RNA 的 48 小时核穿透时间过程。展望未来，CAPA 将成为一种有用的工具，用于解构治疗相关细胞类型中寡核苷酸治疗剂的内体摄取、逃逸到细胞质中以及亚细胞运输的复杂过程。人们对寡核苷酸治疗的细胞内化知之甚少。通过应用新颖的检测方法，我们提供了寡核苷酸治疗剂向不同区室和细胞类型运输的定量数据。
A major obstacle to the development of effective oligonucleotide therapeutics is a lack of understanding about their cytosolic and nuclear penetration. To address this problem, we have applied the chloroalkane penetration assay (CAPA) to oligonucleotide therapeutics. CAPA was used to quantitate cytosolic delivery of antisense oligonucleotides (ASOs) and siRNAs and to explore the effects of a wide variety of commonly used chemical modifications and their patterning. We evaluated potential artifacts by exploring the effects of serum, comparing activity data and CAPA data, and assessing the impact of the chloroalkane tag and its linker chemistry. We also use viral transduction to expand CAPA to the nuclear compartment in epithelial and neuronal cell lines. Using this enhanced method, we measured a 48-hour time course of nuclear penetration for a panel of chemically diverse modified RNAs. Moving forward, CAPA will be a useful tool for deconvoluting the complex processes of endosomal uptake, escape into the cytosol, and subcellular trafficking of oligonucleotide therapeutics in therapeutically relevant cell types. The cellular internalization of oligonucleotide therapeutics is poorly understood. By applying novel assays, we provide quantitative data on the trafficking of oligonucleotide therapeutics to different compartments and cell types.
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影响因子：
14.9
作者：
Deprey, Kirsten;Batistatou, Nefeli;Kritzer, Joshua A.
通讯作者：
Kritzer, Joshua A.
影响因子：
16.6
作者：
Liu Y;Dodart JC;Tran H;Berkovitch S;Braun M;Byrne M;Durbin AF;Hu XS;Iwamoto N;Jang HG;Kandasamy P;Liu F;Longo K;Ruschel J;Shelke J;Yang H;Yin Y;Donner A;Zhong Z;Vargeese C;Brown RH Jr
通讯作者：
Brown RH Jr
影响因子：
2.9
作者：
Kang, SH;Cho, MJ;Kole, R
通讯作者：
Kole, R
影响因子：
4
作者：
Coles, Andrew H.;Osborn, Maire F.;Khvorova, Anastasia
通讯作者：
Khvorova, Anastasia
影响因子：
18.2
作者：
Lacroix, Aurelie;Vengut-Climent, Empar;Sleiman, Hanadi F.
通讯作者：
Sleiman, Hanadi F.