Our project actually starts with Safety and Security.
Over the last few decades, researchers have focused on RNAi technology for basic research, synthetic biology, and biomedicine. Unfortunately, like what has been discussed in other parts of our wiki, the constitutive knockdown of genes by RNAi may give undesired results, especially in biomedical applications such as gene therapy. It is also problematic for conditional RNAi strategies involving a number of TFs and engineered promoters, which significantly depend on inducer factors and may be toxic to mammalian cells[1].
Out of the many possible effectors of RNAi technology, it has been argued by several scholars that pri-miRNA mimics are the safest option where long-term DNA-induced expression is desirable[2][3]. Pri-miRNA mimics especially avoid the risk of saturation of the endogenous miRNA pathway. Reduced in vivo toxicity was reported following miRNA re-expression via pri-miRNA mimics, when compared to that observed following miRNA re-expression via shRNAs[4].
Our project design specifically focuses on the feasibility of taking advantage of the structural property of pri-miRNA itself and engineering the endogenous silencing pathway. It is consistent with the above-mentioned opinions and efforts for the achievement of a more controllable and safer RNAi application in gene therapy.
General laboratory safety
Figure 1. Waste disposal guidelines
Our lab is under strict safety regulations concerning sterilization and waste disposal. The team members all have lab experience and have already received safety training in their many undergraduate courses. In order to better carry out the project and avoid any possible safety issues, we did further training and paid extra cautions during the experiment. Our PI, Professor Tian helped us standardize the experimental protocols and avoid the possible safety problems in the experiments. She reviewed our experiment plans and gave us specific safety advices on several procedures.
Safe experiments
Our basic method is to build the plasmids in DH5-α E.coli and then co-transform them into HepG2 cell line. The two organisms that we use in our project, DH5-α E.coli and HepG2 cells are of low bio-hazardous risk.
Despite the precautions, there are possible safety issues. On the one hand the cell culture may be polluted by microorganisms, on the other hand the cancer cells may be dangerous to the operator. To avoid such accidents, our team strictly obeys lab safety regulations. During the experiment, everyone wears lab coats, gloves and lab caps to keep a sterile environment for cell culture. After the experiment, we disinfect our hands. If the cells are unfortunately indeed polluted, we immediately sample the cells, identify the microorganisms and add the corresponding antibiotics.
Figure 2: The open bench and clean bench
As for the gene modified E.coli cells, although carefully operated, they are still infectious and pose certain risks. Our team adopted corresponding sterilization methods. The experiments are carried out in clean bench and all of the equipment are sterilized through high pressure steam method. The clean bench and experimental materials should be disinfected by UV light before any operation. The operator must use alcohol tampons to disinfect his/her hands. During the operation, light the alcohol burner.
Safe application
Our gene therapy strategy, if ever to become transgenic drugs, involves multiple genes transferring with two plasmids. The problem is that some cells of the patient is only transfected by one plasmid. Although in principle each of the device has no effect on its own, they can independently encode several proteins or RNA products. The mutagenesis, carcinogenesis, dysmorphogenesis and perhaps other undesirable effects of these products alone are indeed problems to consider and to be researched into. We believe these problems will be solved eventually by further research and clinical trials, if possible. Recent reserach shows that a highly targeting drug delivery system by using Enhanced Permeability and Retention (ERP) effect or Ligand-targeted liposome can reduce the possibility for patients to suffer from such problems[5][6]. Above all, gene therapy remains a promising strategy for future cancer treatment.
Safe Shipment
Our Part has been documented on the Registry as required. We followed the shipment guidelines when submitting our sample. We dried the sample as mini-prepped plasmid DNA and used the Submission Kit to pack it. The package is declared and labeled as DNA (non-hazardous, non-regulated, non-infectious, for research purposes only).
[1] Patel, S., Panchasara, H., Braddick, D., Gohil, N., & Singh, V. (2018). Synthetic small RNAs: Current status, challenges, and opportunities. J Cell Biochem. doi: 10.1002/jcb.27252
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[2] McBride, J. L., Boudreau, R. L., Harper, S. Q., Staber, P. D., Monteys, A. M., Martins, I., . . . Davidson, B. L. (2008). Artificial miRNAs mitigate shRNA-mediated toxicity in the brain: implications for the therapeutic development of RNAi. Proc Natl Acad Sci U S A, 105(15), 5868-5873. doi: 10.1073/pnas.0801775105
[3] Sibley, C. R., Seow, Y., & Wood, M. J. (2010). Novel RNA-based strategies for therapeutic gene silencing. Mol Ther, 18(3), 466-476. doi: 10.1038/mt.2009.306
[4] Costa, P. M., & Pedroso de Lima, M. C. (2013). MicroRNAs as Molecular Targets for Cancer Therapy: On the Modulation of MicroRNA Expression. Pharmaceuticals (Basel), 6(10), 1195-1220. doi: 10.3390/ph6101195
[5] Maeda, H. (2015). Toward a full understanding of the epr effect in primary and metastatic tumors as well as issues related to its heterogeneity. Advanced Drug Delivery Reviews, 91, 3-6.
[6] Noble, G. T., Stefanick, J. F., Ashley, J. D., Kiziltepe, T., & Bilgicer, B. (2014). Ligand-targeted liposome design: challenges and fundamental considerations. Trends in Biotechnology, 32(1), 32-45.
