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bioRxiv · 10.1101/2021.06.04.447039

Self-transfecting GMO-PMO and PMO-GMO chimeras enable gene silencing in vitro and in vivo zebrafish model and NANOG Inhibition Induce the Apoptosis in Breast and Prostate Cancer Cells

Kundu, J.·Das, U.·Bose, C.·Bhadra, J.·Sinha, S.
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Abstract

Phosphorodiamidate Morpholino Oligonucleotides (PMOs)-based antisense reagents cannot enter inside cells by itself without the help of any delivery technique which is the last hurdle for their clinical applications. To overcome this limitation, a self-transfecting GMO-PMO or PMO-GMO chimeras has been explored as a gene silencing reagent where GMO stands for guanidinium morpholino oligonucleotides which linked either at the OH- or NH-end of PMOs. GMO not only facilitates cellular internalization of such chimeras but also participates in Watson-Crick base pairing during gene silencing in ShhL2 cells when designed against mGli1 and compared with scrambled GMO-PMO where mutations were made only to the GMO part. GMO-PMO-mediated knockdown of no tail gene resulted no tail-dependent phenotypes in zebrafish and worked even after the delivery at 16-, 32- and 64-cell stages which were previously unachievable by regular PMO. Furthermore, GMO-PMO chimeras has shown the inhibition of NANOG, a key regulator of self-renewal and pluripotency of both embryonic and cancer stem cells. Its inhibition influences on the expression of other cancer related proteins and the respective phenotypes in breast cancer cells and increases the therapeutic potential of taxol. To the best of our knowledge, this is the first report on the self-transfecting antisense reagents since the discovery of guanidinium linked DNA (DNG) and most effective among the all cell-penetrating PMOs reported till date expected to solve the longstanding problem of PMO delivery. In principle, this technology could be useful for the inhibition of any target gene without using any delivery vehicle and should have applications in the fields of antisense therapy, diagnostic and nanotechnology area. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/447039v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1374f3forg.highwire.dtl.DTLVardef@33ca57org.highwire.dtl.DTLVardef@292959org.highwire.dtl.DTLVardef@599b5f_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Kundu, J., Das, U., Bose, C., Bhadra, J., Sinha, S.. 2021-06-04. Self-transfecting GMO-PMO and PMO-GMO chimeras enable gene silencing in vitro and in vivo zebrafish model and NANOG Inhibition Induce the Apoptosis in Breast and Prostate Cancer Cells. https://doi.org/10.1101/2021.06.04.447039

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