Melanoma Genomics—Techniques and Implications for Therapy
Sanjiv S. Agarwala, Vernon K. Sondak in Melanoma, 2008
A relatively recent advent in molecular biology has been the discovery of cellular mechanisms of gene regulation in response to the introduction of double-stranded RNA into eukaryotic organisms such as nematodes. On introduction of double-stranded RNA into a cell, an adenosine triphosphate (ATP)-dependent cleavage of the dsRNA into smaller 21 to 25 nucleotide fragments occurs. This is mediated by the dsRNA-specific endonuclease, Dicer, producing siRNAs with a phosphate group on their 5’ end, and a hydroxyl group on their 3’ end. These fragments are then incorporated into a protein complex where they are unwound into single-stranded RNAs that then target their corresponding mRNAs for degradation by recruiting them to a protein complex known as the RNA-induced silencing complex (RISC) (73). Because of the transient nature of siRNA transfection, researchers have attempted to make more stable transfectants, known as short hairpin RNA (shRNA). This again harnesses the endogenous machinery of the cell, as shRNAs are designed so that they are transcribed off a sequence that contains a stem or hairpin loop using an RNA polymerase promoter (74). However, the mechanisms of shRNA processing are not yet clear, and the efficiency of this technique is not yet on a par with the transient technique of siRNA delivery. For both techniques, validation of protein down-regulation following message reduction is key.
Gene Delivery for Intervertebral Disc
Raquel M. Gonçalves, Mário Adolfo Barbosa in Gene and Cell Delivery for Intervertebral Disc Degeneration, 2018
An additional apoptotic marker that has been recently designated as a target for IDD gene therapy is C/EBP homologous protein (CHOP), whose expression is triggered by endoplasmic reticulum stress provoked by biomechanical stress exerted on AF cells, thus leading to apoptosis. Zhang et al. (2014) combined the use of a lentiviral vector and RNAi by the introduction in the viral genome of a short hairpin RNA (shRNA) designed to bind and inhibit CHOP expression. Rat AF cells were cultured and transduced in vitro and then underwent cyclic tension loading; the experimental group showed decreased apoptosis and prolonged CHOP inhibition. Subsequently, the authors tested the effectiveness of this vector in vivo by an intradiscal injection in a rat IDD model. At 7 weeks after injection, MRI scans showed a higher intradiscal intensity in the experimental group than in the control group; corresponding findings were detected at the histological assay, where the injected group demonstrated no or mild signs of IDD. In addition, apoptosis was significantly decreased in the experimental group (Zhang et al. 2011).
The Cell and Cell Division
Anthony R. Mundy, John M. Fitzpatrick, David E. Neal, Nicholas J. R. George in The Scientific Basis of Urology, 2010
Some years ago it was found in plants and some primitive organisms that short sequences of RNA forming a hairpin shape could regulate the half-life of mRNA. Such short regulatory sequences are in fact used in cell biology experiments to “knock down’’ the function of certain genes. It was then found that the production of such short hairpin RNA (shRNA) sequences is an important part of the regulation of mammalian gene function. The expression of such shRNA sequences is now being actively studied in human health and disease.
Follicle-stimulating hormone peptide-conjugated nanoparticles for targeted shRNA delivery lead to effective gro-α silencing and antitumor activity against ovarian cancer
Published in Drug Delivery, 2018
Shan-Shan Hong, Ming-Xing Zhang, Meng Zhang, Yi Yu, Jun Chen, Xiao-Yan Zhang, Cong-Jian Xu
We previously developed a short interfering RNA (siRNA) delivery system consisting of a polyethylene glycol (PEG)-polyethylenimine (PEI) copolymer modified with FSH β 33–53 peptides to improve the stability, circulation time, and delivery efficiency of the system. This system could mediate the highly selective delivery of siRNA into ovarian clear cell carcinoma cells ES-2 (Hong et al., 2013). However, the acute toxicity observed in vivo limited our work. The reason for the high toxicity might be that a low amount of PEG grafting leads to polyplex aggregation. The molecular weight and conjugation ratio of PEG on PEI are related to DNA association and polyplex aggregation, considering that PEG conjugation offers colloid stability and biocompatibility for the PEI-DNA complex (Smith et al., 2015). Moreover, our previous study used siRNA for target gene knockdown. However, the silencing effect of siRNA is transient. Short hairpin RNA (shRNA) is a stem-loop RNA that also silences a specific gene via RNAi (Lam et al., 2015). Compared with siRNA, it provides long-lasting gene silencing and comparable efficiency (Gvozdeva et al., 2016).
Choice of nanocarrier for pulmonary delivery of cancer therapeutics
Published in Expert Opinion on Drug Delivery, 2020
Patients with lung cancer are treated with several therapeutic procedures such as surgery, radiotherapy, chemotherapy, and molecular-targeted therapies. Chemotherapy is usually administered to the patients as neo-adjuvant or adjuvant therapy. It is an essential treatment mode especially in advanced lung cancer where metastasis prevails. Gene therapy has been the recent focus of research [2,3]. Small interfering ribonucleic acid (siRNA), short hairpin RNA (shRNA) and microRNA (miRNA) are examples of RNAi-based therapeutics that can either be delivered through systemic administration or local administration to negate the expression of intended gene and cancer. The delivery of RNAi-based therapeutics through systemic administration may bring about adverse effects such as liver toxicity, therapeutic instability, and stimulation of immune response. Pulmonary administration of RNAi-based therapeutics is deemed to be a better delivery approach in targeting the cancer tissue [4].
Lessons learned from lung and liver in-vivo gene therapy: implications for the future
Published in Expert Opinion on Biological Therapy, 2018
Joost van Haasteren, Stephen C. Hyde, Deborah R. Gill
Some of the diseases targeted by ex-vivo gene therapy harbor a mutation that causes a proliferative advantage to cells that have been corrected (such as SCID). This will, in time, cause an amplification of cells that are corrected since they will out-proliferate non-corrected cells. Ultimately, as long as this amplification is not unregulated, this advantage is likely to benefit the treated patient. Such an effect is not readily observed in in-vivo lung or liver gene therapy, but examples do exist, and a proliferative advantage can be established artificially. For example, correcting the common PI*Z mutation of AAT deficiency or inhibiting its expression with a microRNA prevents the accumulation of PI*Z polymers in the hepatocytes and give those cells a survival advantage over non-corrected cells that have a tendency to undergo stress-induced apoptosis [87]. A more synthetic approach was taken by Nygaard and colleagues, who developed a vector that, in addition to expressing a therapeutic transgene, also expressed a short hairpin RNA (shRNA) that protects against a toxic drug [88]. Cells that do not have this vector integrated succumb to the drug and allow cells that do harbor the shRNA to outgrow them and at the same time express the therapeutic transgene.
Related Knowledge Centers
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