Application of siRNA in antiviral therapy

Posted by beauty33 on January 4th, 2022

Induced by siRNA or its biosynthetic precursor RNAi

Gene knockdown by transfection of exogenous siRNA is usually unsatisfactory because the effect is only short-lived, especially in rapidly dividing cells. This can be overcome by generating siRNA expression vectors. The siRNA sequence is modified to introduce a short loop between the two strands. The resulting transcript is short hairpin RNA (shRNA), which can be processed into functional siRNA by Dicer in its usual way. A typical transcription cassette uses an RNA polymerase III promoter (for example, U6 or H1) to direct the transcription of small nuclear RNA (snRNA) (U6 is involved in gene splicing; H1 is RNase) as a component of human RNase P. Theoretically, the resulting siRNA transcript is then processed by Dicer.

The efficiency of gene knockdown can also be improved by using cell extrusion.

The activity of siRNA in RNAi largely depends on its ability to bind to RNA-induced silencing complex (RISC). The binding of duplex siRNA to RISC is followed by the use of endonuclease to untie and cleave the sense strand. The remaining antisense strand-RISC complex can then bind to the target mRNA to initiate transcriptional silencing.

RNA activation

It has been found that dsRNA can also activate gene expression. This mechanism is called \"small RNA-induced gene activation\" or RNAa. It has been shown that dsRNA targeting gene promoters induces effective transcriptional activation of related genes. The use of synthetic dsRNA to prove RNAa in human cells is called \"small activated RNA\" (saRNA). It is not clear whether RNAa is conserved in other organisms.

Post-transcriptional gene silencing

siRNA-induced post-transcriptional gene silencing begins with the assembly of RNA-induced silencing complex (RISC). The complex silences the expression of certain genes by cutting mRNA molecules encoding target genes. To start the process, one of the two siRNA strands (the guide strand) will be loaded into the RISC, while the other strand, the passenger strand, is degraded. Certain Dicer enzymes may be responsible for loading the guide chain into RISC. Then, the siRNA scans and directs the RISC to the completely complementary sequence on the mRNA molecule. It is believed that the cleavage of mRNA molecules is catalyzed by the Piwi domain of the Argonaute protein of RISC. Then the mRNA molecule is accurately cut by cutting the phosphodiester bond between the target nucleotides paired with siRNA residues 10 and 11, counting from the 5\'end. This cleavage causes the mRNA fragments to be further degraded by cellular exonuclease. The 5\'fragment is degraded from its 3\'end by exosomes, and the 3\'fragment is degraded from its 5\'end by 5\'-3\' exonuclease 1 (XRN1). Dissociation of the target mRNA chain from RISC after cleavage allows more mRNA to be silenced. This dissociation process is probably promoted by external factors driven by ATP hydrolysis.

Sometimes cleavage of target mRNA molecules does not occur. In some cases, the endonucleic cleavage of the phosphodiester backbone can be inhibited by the mismatch between the siRNA and the target mRNA near the cleavage site. At other times, even if the target mRNA and siRNA are perfectly matched, the Argonaute protein of RISC lacks endonuclease activity. In this case, gene expression will be silenced by the miRNA induction mechanism.

A simplified version of the Ping-Pong method, which involves the proteins Aubergine (Aub) and Argonaute-3 (Ago3) cleaving the 3\'and 5\'ends of the piRNA.

Piwi-interacting RNA is responsible for transposon silencing, not siRNAs.

Application of siRNA in antiviral therapy

In recent years, the application of RNAi technology in the treatment of viral infectious diseases has received great attention, especially in the treatment of AIDS, hepatitis B, and hepatitis C. The application research is the most active. At present, in the research of siRNA against AIDS, siRNA targeting HIV structural protein genes and long terminal repeat (LTR) can control virus replication; siRNA targeting host cell HIV receptor CD4 gene can effectively control virus entry into host cells , Inhibit the virus infection process. However, CD4 is an indispensable molecule for the normal immune function of the human body, and its expression inhibition will inevitably affect the normal immune function. Therefore, it is envisaged to design siRNA against CCR5, CCR4 and other co-receptors, and it is being tested. In addition, in antiviral therapy, siRNAs designed to target the coding or non-coding regions of the genomes of hepatitis C virus, respiratory syncytial virus, influenza virus, polio virus, etc., have achieved gratifying in vitro inhibition. However, the use of animal experimental models to verify the effect of siRNA in eliminating viruses in vivo needs further research.

Although RNAi is expected to become an effective tool for antiviral therapy, high mutations or base loss of target genes of viral strains, such as HIV and influenza viruses, have become a problem that must be considered when designing siRNA. The discovery of another kind of viral suppressor protein has given researchers a deeper understanding of the antiviral effects of RNAi. Research has found that this viral suppressor is encoded by the viral genome. It was originally discovered in plants, but there are other reports. It also exists in eukaryotes, which competitively bind with RNA interference devices to inhibit RNA interference by blocking the processing of siRNA or the transmission of interference signals, thereby reducing its antiviral effect.

In view of the above influencing factors, in the application of RNAi antiviral therapy, (1) The selection of target sequence is best for the conservative sequence of the virus to reduce the influence of virus mutation. (2) Design a variety of SiRNAs for different target sequences and work together to reduce virus escape. (3) Design siRNA against host genes related to virus entry into cells or virus replication, such as HIV receptor CD4, CCR5, etc., so that even if the virus is highly mutated, the probability of escaping RNA interference will be greatly reduced. Designing siRNA for virus suppressors can reduce or avoid the production of virus suppressors to a certain extent, and with in-depth research on RNA interference, there will be more and more related reports. In summary, although RNAi has made great breakthroughs in the application of antiviral therapy, its application in clinical practice still needs further research.

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