iPSCs
The risks of mutagenesis and oncogenesis are recognized as major safety concerns for this method if labs were to use an inappropriate concentration of vectors. 21 The reprogramming efficiency of episomal vectors is generally lower than that of integrating vectors, such as retroviral or lentiviral vectors. They require optimization of vector concentration and cell density to be viable as an efficient reprogramming and iPSC generation method. 22 This means that it may need experts with more experience to ensure that the right levels of concentration for safety, efficiency, and effectiveness are achieved, limiting its accessibility. 23
The polycistronic minicircle DNA nonviral vector method of iPSC generation
The minicircle DNA vector reprogramming method involves the delivery of supercoiled DNA that has had its normal bacterial DNA removed. This supercoiled DNA is called a ‘ minicircle ’ . A single minicircle vector can be used to generate multiple transgene-free iPSCs from adult human cells. 24 It does this by using electroporation to deliver the minicircles, within a buffer solution, into the cell. Then the factors Oct4, Sox2, Klf4 and L-Myc act to reprogram the cell into an iPSC. Multiple recombinase systems have been used to generate minicircles, including phage λ integrase, phiC31 recombinase, Flp recombinase, ParA resolvase, and Cr. 25
Benefits and drawbacks
Polycistronic minicircle DNA nonviral vectors have several benefits when generating iPSCs. This method is typically cheaper than other methods of iPSC generation. Compared to some other methods, minicircle DNA has higher transfection efficiencies and longer ectopic expression due to its lower activation of DNA-silencing mechanisms. 26 The electroporation of a single polycistronic vector to generate iPSCs can reduce transgene insertion into the genome and the plasmids are unable to self- replicate within an individual which reduces the risk to patients. Furthermore, since multiple
21 Trevisan, M., Desole, G., Costanzi, G., Lavezzo, E., Palù, G., & Barzon, L. (2017). Reprogramming Methods Do Not Affect Gene Expression Profile of Human Induced Pluripotent Stem Cells. International Journal of Molecular Sciences, 18 . 22 Bang, J.S., Choi, N.Y., Lee, M., Ko, K., Lee, H.J., Park, Y.S., Jeong, D., Chung, H., & Ko, K. (2018). Optimization of episomal reprogramming for generation of human induced pluripotent stem cells from fibroblasts. Animal Cells and Systems, 22 , 132 - 139. 23 Bang, J.S., Choi, N.Y., Lee, M., Ko, K., Lee, H.J., Park, Y.S., Jeong, D., Chung, H., & Ko, K. (2018). Optimization of episomal reprogramming for generation of human induced pluripotent stem cells from fibroblasts. Animal Cells and Systems, 22 , 132 - 139. 24 Jia, F., Wilson, K.D., Sun, N., Gupta, D.M., Huang, M., Li, Z., Panetta, N.J., Chen, Z., Robbins, R.C., Kay, M.A., Longaker, M.T., & Wu, J.C. (2010). A nonviral minicircle vector for deriving human iPS cells. Nature Methods, 7 , 197-199. 25 ‘ Minicircle DNA. ’ Minicircle DNA - an overview | ScienceDirect Topics. Accessed March 1, 2023. https://www.sciencedirect.com/topics/medicine-and-dentistry/minicircle-dna. 26 Jia, F., Wilson, K.D., Sun, N., Gupta, D.M., Huang, M., Li, Z., Panetta, N.J., Chen, Z., Robbins, R.C., Kay, M.A., Longaker, M.T., & Wu, J.C. (2010). A nonviral minicircle vector for deriving human iPS cells. Nature Methods, 7 , 197-199.
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