Application of siRNAs in gene therapy
Application of siRNAs in gene therapy Introduction: Small interfering RNAs (siRNAs) are effector molecules in the process of RNA interference (RNAi). Fire et al [1] injected double-stranded RNA into C. elegans in 1998 RNA, dsRNA) molecules degrade the target mRNA containing homologous sequences in the cytoplasm. Subsequent studies have shown that RNAi phenomenon is widespread in most eukaryotes such as fungi, Arabidopsis, zebrafish, fruit flies, mice and rats In biology. Early application of long dsRNA in mammalian cells can cause non-specific interferon response and lead to cell death. Later genetics and biochemical studies proved that cutting dsRNA into 21-28 nucleotide siRNAs does not cause interferon Response, and can effectively degrade target mRNA containing homologous sequences. Therefore, siRNAs are rapidly developing into new tools for studying gene function and as a new method of treatment. 1. Silencing mechanism of siRNAs siRNAs are produced by the RNase-III family of endonucleases known as Dicer in the process of cutting naturally occurring long dsRNA in the cytoplasm. Dicer cuts long dsRNA into 21-28 nucleotide siRNA pairs Strand. In addition to the phosphate at the 5 'end and the hydroxyl group at the 3' end, this siRNA double strand also has an overhang composed of 2 nucleotides at the 3 'end. SiRNA double strand and RNA-induced silencing complex (RNA-induced The silencing complex (RISC) is cleaved into single strands of siRNA after binding, and the homologous target mRNA that is completely complementary to the single strand of siRNA is cleaved and degraded by RISC to achieve gene silencing. Now siRNA can be chemically synthesized like a ribozyme Methods or through the vector to express double-stranded short hairpin-like RNA (shRNA) into the cell, the latter can be converted into siRNA in the cell to exert gene silencing effect. Some studies have proved that siRNAs have other silencing mechanisms For example, in several organisms, the ability to modify cellular chromatin through the RNAi pathway leads to gene silencing at the transcription level [2-3]. 2. Gene silencing efficiency and safety of siRNAs Whether the various gene silencing methods can efficiently target the target is still a key issue for its application in treatment. Harborth et al [4] studies have shown that RNA binding proteins, secondary structure and tertiary structure of mRNA can affect the silencing of siRNA Efficiency. Most studies have confirmed that siRNAs are more effective than oligodeoxyribonucleic acids (ODNs) and have a longer duration of action. The half-maximum inhibitory concentration (IC50) of siRNAs acting on the same target is more than that of phosphorous Acyl-modified ODNs are 100-1 000 times lower. Although the efficiency of siRNAs with ribozymes and / or DNases has not been systematically compared extensively, studies by Drew et al [5] have shown that siRNAs are more efficient than ribozymes and / or DNases. More efficient, and RNA with a long hairpin structure can more strongly inhibit the expression of target genes than the ribozyme of the hammerhead structure. 3. Expression vector of siRNAs Because siRNAs can be obtained either by chemical synthesis or by vector expression, this makes it possible for targeted drugs to be used in gene therapy. Vectors expressing siRNAs usually contain RNA polymerase III promoter (pol III) ) Or RNA polymerase II promoter (pol II) sequence, first transcribed to generate shRNA similar to the miRNA precursor, and then turned into siRNA in the cell to exert gene silencing effect. In vivo and culture tissue cells using vectors expressing siRNA It can integrate into the genome for a long time and "knock out" endogenous genes. Many studies have proved that adenovirus vectors, adeno-associated viral (AAV) vectors, retrovirus vectors and lentiviral vectors can be effectively transfected. In vivo and in cultured cells. Shinagawa et al [10] studies have shown that expression vectors containing pol II can produce shRNA composed of hundreds of base pairs in vivo without inducing non-specific interferon responses, which is used for siRNAs. It provides a safe new method for mammals. 4. Application of siRNAs in living body Electroporation, local injection or intravenous injection have been able to successfully introduce chemically synthesized siRNAs, plasmids expressing siRNAs, and viruses expressing siRNAs into mammalian cells. However, it is difficult to evaluate which method can cause genes more effectively. Silence. The siRNA dissolved in normal saline was injected into the rat tissues by high pressure through the tail vein of the mouse, and the siRNA has been successfully introduced into the rat tissues. Among them, the silencing efficiency of the target gene in the liver is more than 90%, while in the lung, kidney, spleen, and pancreas The gene silencing efficiency is slightly lower [11-12]. The gene silencing effect caused by this method generally lasts for several days, and in some cases can exceed 1 wk, and the efficiency of gene silencing varies depending on the species. 5. Gene therapy based on siRNAs Although the application of siRNA in mammalian cells is only 4 years, he is rapidly developing into a new method of gene therapy. If siRNAs can effectively reach the liver through tail vein injection, he will be widely used in the treatment Various liver diseases. By silencing the expression of endogenous apoptotic genes in the liver, mice pretreated with anti-apoptotic enzyme Caspase-8 or anti-Fas cell death receptor siRNAs can effectively prevent acute liver function induced by various agents Failure; using the same siRNA can also treat liver damage that has already occurred [11-12]. SiRNAs achieve antiviral purposes by inhibiting the virus itself or the cofactors necessary for viral transcription. The hepatitis B virus (HBV) genome and Co-transfection of siRNAs can effectively reduce the replication level and protein synthesis of HBV [17]. Wohlbold et al [18] proved that siRNAs can effectively silence the expression of abnormal BCR-ABL fusion genes without affecting the normal c-BCR and c-ABL. Transcription, which provides a new method for the treatment of chronic myeloid leukemia with Ph chromosome positive.
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miRNAs (microRNAs) are a class of non-coding small-molecule RNAs that have similar functions to siRNAs and regulate intracellular gene expression. Mature miRNAs are hairpin-like structure precursors composed of 70 nucleotides in the cytoplasm that are sheared into A single strand composed of 21-22 nucleotide molecules. After they are assembled into a protein complex (miRNP), they are complementary to the 3 'untranslated region of the mRNA in the ribosome to prevent translation of the mRNA. If the homologous target mRNA is completely Complementary binding, then miRNAs, like siRNAs, can cyclically degrade target mRNA through a positive feedback pathway.
Low concentration of siRNAs can start the process of gene silencing, because they can quickly and specifically bind to RISC, thereby reducing the possibility of binding to non-specific proteins, which is conducive to reducing the non-specific response of siRNAs in treatment. Studies have shown that transfection of moderate concentrations of siRNAs does not cause systemic non-specific reactions [6]. There are also three studies [7-9] that non-specific reactions caused by siRNAs are related to the concentration of siRNAs, cell type, transfection reagents and siRNAs. The way of transfection is related. These non-specific reactions include the interferon response caused by stimulating gene subtypes, but the research is not as people think, the resulting interferon response does not affect cell growth.
Viral vectors expressing siRNAs provide new methods for studying the gene function of mammals, and bring new hope for the treatment of major human diseases. Several viruses have been designed to express siRNAs. Recombinant AAV can be used in mammalian dividing cells And long-term expression of siRNA in quiescent cells. They are usually integrated into the host genome in the form of episomes at random and low frequency. Studies [13] have shown that injecting AAV vectors expressing siRNA into the rat brain can cause up to 7 weeks. Gene silencing effect. Viral vectors expressing siRNA in tissue models of HIV infection have also been successfully used in therapy [14].
There are now several new ways to introduce siRNAs into the body. Recent studies [15] have shown that large molecules can promote the transdermal absorption of several small molecules, including siRNAs. This will facilitate the transdermal absorption of siRNAs into the systemic blood circulation Play the role of therapeutic drugs. Introducing aerosols containing siRNAs into the lungs can also play a role in gene therapy [16].
The double-stranded siRNAs in plasma can resist the degradation of endonuclease, but this does not mean that they can stably exist in the body. Because unmodified siRNAs cannot quickly enter the cell or have low affinity for plasma proteins. It is cleared by the body earlier. If it is not a method of expressing siRNAs, it must be chemically modified siRNAs to be more effective as a method of gene therapy. There are studies [19-20] that are using thiophosphoryl modified siRNA to improve the cell Uptake ability, thereby enhancing the effect of gene silencing. Last year, the US FDA approved modified siRNAs for clinical new drug trials for the treatment of patients with age-related macular degeneration (age-related macular degeneration) [21]. Recently Soutschek et al [22] Injecting cholesterol-modified anti-apoB siRNA through the tail vein of mice can effectively silence the overexpression of homologous target genes, and confirmed that the modified anti-apoB siRNA caused a lower level of cholesterol in mice than apoB gene knockout. The levels of mice are similar, which realizes the possibility of siRNA as an intravenous therapeutic drug.
As a new gene therapy method, siRNAs have aroused the interest of many researchers, largely because of its low toxicity and specificity as a regulator of endogenous gene expression in cells. On the other hand, it is more specific than ODNs, Ribozymes have stronger gene silencing efficiency. However, there are still some challenges in applying siRNAs to clinical treatment, such as the best way to introduce siRNAs into cells and how to obtain higher efficiency, how to avoid off-target phenomena and non-specific reactions. Therefore, Further research on the mechanism of RNAi will enrich our understanding of gene expression regulation and will also benefit the practical application of gene therapy.