Small hairpin RNA
A small hairpin RNA ( shRNA for short ) is an RNA molecule that forms a hairpin structure and can be used to artificially shut down genes with the help of RNA interference (RNAi). The expression of shRNA is mostly mediated by plasmids or viral vectors. The choice of promoter is crucial for reliable shRNA expression. Initially, polymerase III promoters such as U6 and H1 were used, which, however, did not allow any local or temporal control.
Therefore, polymerase II promoters are now used instead. shRNA is a beneficial mediator of RNAi because it has a relatively low rate of degradation. However, it also has disadvantages because it requires the use of expression vectors, which leads to safety concerns.
The introduction of plasmids into cells with the aid of transfection can be achieved in vitro using commercially available reagents. However, this method cannot be used in vivo and is therefore limited.
The use of bacterial vectors for shRNA expression in cells is a fairly new method. It could be shown that recombinant bacteria of the species Escherichia coli , which contain shRNA plasmids, caused target genes in the intestine to be shut down after being fed to mice.
Constructs based on adeno-associated viruses (AAV), adenoviruses or lentiviruses can be used as viral vectors . Adeno-associated and adenoviruses do not mutate the target cell's genome; however, the transgene is lost after cell division. Lentiviruses integrate into the genome and are passed on after cell division; however, the genetic makeup of the target cell mutates. This problem can be circumvented by using integrase-deficient lentiviruses.
Mechanism of action
After integrating a lentiviral vector into the host genome, the shRNA is transcribed in the cell nucleus by polymerase II or III, depending on the type of promoter. This transcript mimics pri- microRNA (microRNA primary) and the enzyme Drosha processed. The resulting pre-shRNA is exported from the nucleus into the cytoplasm . The RNA is then processed further by the Dicer enzyme and loaded onto the RNA-induced silencing complex ( RISC ). The sense of meaning (passenger) is dismantled; the opposite sense strand (guide) is hybridized with the complementary sequence on a target mRNA . In the case of perfect complementarity, RISC cuts the mRNA; in the case of incomplete compl. RISC prevents the translation of the mRNA. In both cases, the shRNA silences the gene.
Applications in gene therapy
Because of the ability of shRNA to specifically and permanently silence genes, there is great interest in its use for gene therapy applications. Commercial company Gradalis developed the FANG vaccine , which is used to treat advanced tumor diseases. The vaccine consists of a bifunctional shRNA against the immunosuppressive mediators TGFβ1 and β2. Autologous tumor cells have been isolated from patients and transformed with a plasmid containing the bifunctional shRNA and GM-CSF encoded ex vivo by electroporation transfected. These cells were then rendered incapable of dividing with radiation and injected into the patient. The vendor has also developed a bifunctional shRNA that targets Stathmin-1 and is introduced into the tumor using lipoplex technology.
shRNA-based therapies face some difficulties. The greatest challenge is administration. shRNA is usually brought into a cell via a nucleotide vector, which is accompanied by safety concerns in the patient. Viral vector-based gene therapy could trigger a systemic immune response in the organism or destroy important genes such as tumor suppressors in the genome of the target cell . A possible oversaturation of the RISC is also a problem. If the shRNA is expressed in too large an amount, the cell is no longer able to process natural endogenous RNAs such as microRNAs . In addition, the therapeutic shRNA could inadvertently shut down other genes.
Individual evidence
- ↑ P. Svoboda, P. Stein, RM Schultz: RNAi in mouse oocytes and preimplantation embryos: effectiveness of hairpin dsRNA. In: Biochem Biophys Res Commun . 287, 2001, pp. 1099-1104. PMID 11587535 .
- ↑ Zhaohui Wang, Donald D. Rao, Neil Senzer, John Nemunaitis: RNA Interference and Cancer Therapy. In: Pharmaceutical Research 28, 2011, pp. 2983-2995. doi: 10.1007 / s11095-011-0604-5 .
- ↑ Shuanglin Xiang, Johannes Fruehauf, Chiang J Li: Short hairpin RNA - expressing bacteria elicit RNA interference in mammals. In: Nature Biotechnology 24, 2006, pp. 697-702. doi: 10.1038 / nbt1211 .
- ↑ Angelo Lombardo, Pietro Genovese, Christian M Beausejour, Silvia Colleoni, Ya-Li Lee, Kenneth A Kim, Dale Ando, Fyodor D Urnov and others: Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery. In: Nature Biotechnology. 25, 2007, pp. 1298-1306. doi: 10.1038 / nbt1353 . ISSN 1087-0156
- ↑ Neil Senzer, Minal Barve, Joseph Kuhn, Anton Melnyk, Peter Beitsch, Martin Lazar, Samuel Lifshitz, Mitchell Magee and others: Phase I Trial of “bi-shRNAifurin / GMCSF DNA / Autologous Tumor Cell” Vaccine (FANG) in Advanced Cancer. In: Molecular Therapy. 20, 2011, pp. 679-686. doi: 10.1038 / mt.2011.269 . ISSN 1525-0016
- ^ KA Whitehead, JE Dahlman, RS Langer, DG Anderson: Silencing or stimulation? siRNA delivery and the immune system. In: Annu Rev Chem Biomol Eng . 2, 2011, pp. 77-96. doi: 10.1146 / annurev-chembioeng-061010-114133 .