RNA interference (RNAi) is a biological process involving double-stranded RNA (dsRNA) that consists of complementary positive and negative RNA strands corresponding to messenger RNAs (mRNAs) introduced into cells. RNAi serves as a valuable tool for studying gene function, exploring signaling pathways, and developing innovative strategies for gene therapy. Common interference tools mainly include chemically synthesized double-stranded small interfering RNAs (siRNAs), vector-based short hairpin RNAs (shRNAs) and microRNA (miRNA).

How RNA Interference (RNAi) Works?

RNA interference (RNAi) technology harnesses the cell's natural molecular machinery to effectively reduce the expression levels of target genes by short interfering with the action of RNA molecules. Several pathways can induce the production of RNAi, including synthetic molecules, RNAi vectors, and in vitro cleavage. In mammalian cells, short interfering RNA (siRNA), a brief segment of double-stranded RNA, initiates the specific degradation of intracellular target mRNA.

In this process, the antisense strand of the siRNA duplex becomes part of a multiprotein complex known as the RNA-induced silencing complex (RISC). This complex recognizes the corresponding mRNA and cleaves it at specific sites for degradation. The resulting degradation products of the messenger RNA then serve as target molecules for further degradation, ultimately leading to the absence of protein expression.

Fig.1 Cellular mechanism of RNA interference. (Bumcrot D, et al., 2006)

How to Improve siRNA Drugs?

Small interfering RNA (siRNA) drugs represent a novel therapeutic approach based on RNA interference (RNAi) technology, with the potential to specifically silence nearly any therapeutic target.

Fig.2 Small interfering RNA (siRNA) structure and mechanism. (Carugo S, et al., 2023)

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Drug Delivery System Development

To address the challenges associated with siRNA delivery, researchers have developed a variety of delivery methods, which can be primarily categorized into viral vectors and non-viral vectors.

How to Develop shRNA?

Short hairpin RNAs (shRNAs) are molecules with a loop-like structure designed to synthesize sequences that can transcribe and generate small interfering RNAs (siRNAs) complementary to specific regions of target mRNA within cells. These siRNAs bind to the target mRNAs and facilitate their degradation through the mechanism of RNA interference (RNAi), thereby regulating gene expression.

Fig.3 shRNA and siRNA: From Delivery to Processing. (Goel K, et al., 2022)

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How to Construct a miRNA Expression Vector?

miRNA-induced RNA interference mechanism involves cells transcribing primary miRNA molecules. These molecules undergo a series of processing steps in the nucleus, are transported out of the nucleus, and are cleaved by the Dicer enzyme to form mature single-stranded miRNAs. These miRNAs then participate in the initiation and effector phases of RNA interference (RNAi) to achieve post-transcriptional gene silencing.

Fig.4 MicroRNA synthesis and function. (Hayes C N, et al., 2016)

The construction of miRNA expression vectors typically involves designing and synthesizing specific miRNA sequences, which are then cloned into an appropriate vector backbone, such as a plasmid vector. Following this, the vectors are introduced into target cells using transfection techniques for miRNA expression and functional studies.

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RNA Interference Technology in Drug Discovery and Development

Targeted Gene Therapy

Targeting specific genes using siRNA can inhibit or promote gene expression. This technology can be applied to the treatment of various diseases, including cancer, genetic disorders, and viral infections.

Virus Therapy

The use of RNA interference technology to inhibit viral gene expression aims to treat viral infections. This technique has been widely utilized in clinical trials.

Immunotherapy

To treat immune diseases, such as rheumatoid arthritis and inflammatory bowel disease, by modulating the immune response of cells using siRNA.

Gene Editing

RNA interference technology can be utilized to modulate the effects of the CRISPR-Cas9 gene editing system, thereby enabling more precise gene editing.

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