Small hairpin RNA
A Small hairpin RNA ( shRNA short ) is an RNA molecule that forms a hairpin structure and can be used to shut down genes artificially by means of RNA interference (RNAi ). The expression of shRNA is usually mediated by plasmids or viral vectors. The choice of promoter is crucial for a reliable shRNA expression. First, we used the polymerase III promoters such as U6 and H1, however, did not allow for spatial or temporal control.
Therefore, instead promoters of pol II are now used. shRNA is an advantageous mediators of RNAi, since it has a relatively low degradation rate. However, it also has drawbacks, since it requires the use of expression vectors, which has safety concerns result.
The introduction of plasmids into cells by means of transfection can be achieved by commercially available reagents in vitro. But this method is not available in vivo and thus limited.
The use of bacterial vectors for shRNA expression in cells is a relatively new method. It has been shown that recombinant bacteria of the species Escherichia coli, which contain shRNA plasmids, after feeding to mice caused a cessation of target genes in the intestine.
As viral vectors constructs based on adeno-associated viruses ( AAV), adenovirus or lentivirus can be used. Adeno-associated adenoviruses and not mutate the genome of the target cell; however, is lost after cell division, the transgene. Lentiviruses integrate into the genome and are passed after cell division; mutate so but the genome of the target cell. This problem can be circumvented by use of integrase -deficient lentivirus.
Mechanism of Action
After integration of the lentiviral vector into the host genome, the shRNA in the cell nucleus by a polymerase II or III is transcribed depending on the nature of the promoter. This transcript mimicking pri - miRNA (primary miRNA ) and is processed by the enzyme Drosha. The resulting pre- shRNA is exported from the nucleus to the cytoplasm. The RNA is then further processed by the enzyme Dicer, and downloaded to the RNA-induced silencing complex (RISC). The sense strand ( passenger) is degraded; the antisense strand ( guide) is hybridized to the complementary sequence of a target mRNA. In the case of perfect complementarity RISC cuts the mRNA; in case of incomplete Exhaust RISC prevents translation of the mRNA. In both cases, the shRNA shuts down the gene.
Applications in Gene Therapy
Because of the ability of the shRNA to specific and permanent gene silencing, there is great interest in the use for gene therapy applications. The commercial providers gradalis developed the CATCH vaccine, which is used for the treatment of advanced cancer. The vaccine consists of a bifunctional shRNA against the immunosuppressive mediators TGFß1 and ß2. Autologous tumor cells have been isolated from the patient and transfected with a plasmid encoding the bifunctional shRNA and GM-CSF ex vivo by electroporation. These cells were then made unable to divide radiation and injected into the patient. The provider has also developed a bifunctional shRNA directed against stathmin -1 and is introduced into the tumor with lipoplex technology.
ShRNA -based therapies are faced with some difficulties. The biggest challenge is the administration. shRNA is usually brought about a Nukleotidvektor in a cell, which is accompanied in the patient safety concerns. Viral vector-based gene therapy could trigger a systemic immune response in the body or destroy important genes such as tumor suppressors in the genome of the target cell. A possible saturation is also a problem of the RISC dar. If the shRNA is expressed in too large amount, the cell is not able to process natural edogene RNA as microRNAs. In addition, the therapeutic shRNA could inadvertently shut down other genes.