TGFBI R124H突变等位基因沉默在颗粒状角膜缩症2型使用局部siRNA输送
Andrew McLain1, Amanda Kowalczyk1, Paulina Baran-Rachwalska2
1Integrated Diagnostics Laboratory, Northland House, CDHT, Frederick Street, Ulster University, Belfast, UK.
概括
研究人员开发了一种新的siRNA治疗方法,以准TGFBI基因中的R124H突变,这是粒状角膜缩2型的原因. 这种疗法在细胞模型和小鼠模型中都显示出显著的基因沉默,为未来的治疗铺平了道路.
科学领域:
- 眼科医生 眼科 眼科
- 遗传学 遗传学 是一个
- 分子生物学分子生物学
背景情况:
- 基于Oligonucleotide的疗法已经显示出对各种眼睛疾病的前景.
- 颗粒状角膜缩症2型 (GCD2),也称为阿维利诺角膜缩症,是由TGFBI基因的突变引起的,特别是R124H突变.
研究的目的:
- 为TGFBI基因的R124H突变选择和开发一种主要的治疗小干扰RNA (siRNA).
- 在体外和体内GCD2.2模型中评估siRNA的疗效和输送.
主要方法:
- 使用基因步行方法来设计和测试针对R124H TGFBI突变部位的多个siRNA.
- 使用稳定混合脂质纳米颗粒 (sshLNP) 技术传递了SiRNA.
- 在实验室中,在不朽化的人类角膜上皮细胞和原发性人类角膜上皮细胞 (PCEC) 中评估了疗效.
- 在活体中,在表达人类R124H TGFBI转基因的小鼠模型中评估了疗效.
主要成果:
- 一个向R124H TGFBI突变的领先siRNA被确定.
- 在小鼠模型中,局部应用与sshLNP复合的siRNA可使R124H突变TGFBI转基因表达减少22.4%.
- 在初级人类角膜上皮细胞中,单次siRNA治疗导致TGFBI mRNA减少26.6%,TGFBI蛋白减少15.4%.
结论:
- 开发的siRNA证明了降低TGFBI表达的目标有效性,这是GCD2.2的关键因素.
- 由于突变性转基因表达率较低,当前小鼠模型的局限性要求在未来的临床前研究中使用患者衍生细胞的ex vivo模型.
- 这项研究支持基因沉默疗法的潜力,用于治疗诸如GCD2.2之类的角膜 Dystrophies.
相关概念视频
01:22MicroRNAs
MicroRNA (miRNA) are short, regulatory RNA transcribed from introns—non-coding regions of a gene—or intergenic regions—stretches of DNA present between genes. Several processing steps are required to form biologically active, mature miRNA. The initial transcript, called primary miRNA (pri-mRNA), base-pairs with itself forming a stem-loop structure. Within the nucleus, an endonuclease enzyme, called Drosha, shortens the stem-loop structure into hairpin-shaped pre-miRNA. After the pre-miRNA ends...
01:23RNA Interference
RNA interference (RNAi) is a process in which a small non-coding RNA molecule blocks the post-transcriptional expression of a gene by binding to its messenger RNA (mRNA) and preventing the protein from being translated.
This process occurs naturally in cells, often through the activity of genomically-encoded microRNAs. Researchers can take advantage of this mechanism by introducing synthetic RNAs to deactivate specific genes for research or therapeutic purposes. For example, RNAi could be used...
This process occurs naturally in cells, often through the activity of genomically-encoded microRNAs. Researchers can take advantage of this mechanism by introducing synthetic RNAs to deactivate specific genes for research or therapeutic purposes. For example, RNAi could be used...
02:30siRNA - Small Interfering RNAs
Small interfering RNAs, or siRNAs, are short regulatory RNA molecules that can silence genes post-transcriptionally, as well as the transcriptional level in some cases. siRNAs are important for protecting cells against viral infections and silencing transposable genetic elements.
In the cytoplasm, siRNA is processed from a double-stranded RNA, which comes from either endogenous DNA transcription or exogenous sources like a virus. This double-stranded RNA is then cleaved by the ATP-dependent...
In the cytoplasm, siRNA is processed from a double-stranded RNA, which comes from either endogenous DNA transcription or exogenous sources like a virus. This double-stranded RNA is then cleaved by the ATP-dependent...
02:15Experimental RNAi
RNA interference (RNAi) is a cellular mechanism that inhibits gene expression by suppressing its transcription or activating the RNA degradation process. The mechanism was discovered by Andrew Fire and Craig Mello in 1998 in plants. Today, it is observed in almost all eukaryotes, including protozoa, flies, nematodes, insects, parasites, and mammals. This precise cellular mechanism of gene silencing has been developed into a technique that provides an efficient way to identify and determine the...
02:30Small interfering RNAs (siRNA)
Small interfering RNAs, or siRNAs, are short regulatory RNA molecules that can silence genes post-transcriptionally, as well as the transcriptional level in some cases. siRNAs are important for protecting cells against viral infections and silencing transposable genetic elements.
In the cytoplasm, siRNA is processed from a double-stranded RNA, which comes from either endogenous DNA transcription or exogenous sources like a virus. This double-stranded RNA is then cleaved by the ATP-dependent...
In the cytoplasm, siRNA is processed from a double-stranded RNA, which comes from either endogenous DNA transcription or exogenous sources like a virus. This double-stranded RNA is then cleaved by the ATP-dependent...
01:16TGF - β Signaling Pathway
The TGF-β signaling pathway regulates cell growth, differentiation, adhesion, motility, and development. TGF-β ligands that induce TGF-β signaling are synthesized in their latent form. Several proteases or cell surface receptors such as integrins act upon the latent form, releasing the active ligand. There are three types of mammalian TGF-βs: (TGF-β1, TGF-β2, and TGF-β3) that bind as homodimers or heterodimers to TGF-β receptors. The TGF-β receptors are of three kinds RI, RII, and RIII. The RI...