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

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.

miRNA: A Promising Therapeutic Target in Cancer

“…Recently, two PMO-based drugs, Eteplisren (Exondys 51) and Golodirsen (Vyondis 53 TM ), were approved by FDS for the treatment of Duchenne muscular dystrophy (DMD), owing to their safety and efficacy [ 142 , 143 ]. Later in 2021, Jayanta et al reported that cell-penetrating PMO induced translational repression of genes involved in prostate and breast cancer [ 144 ].…”

Emerging Approaches for Enabling RNAi Therapeutics

“…Researchers also need to explore other potential gene silencing agents apart from non-coding RNAs to achieve targeted gene silencing like GMO-PMO or PMO-GMO chimeras as designed by Professor Sinha and co-workers. [14] Toxicity profile and efficacy of RNAi therapeutics in humans cannot be predicted from in-vitro systems or even from in-vivo murine model due to yet to be determined reasons. [23] It is also not feasible to test the large number of delivery vehicles, which are being developed in non-human primate models.…”

“…[12][13] Recently, Professor Surajit Sinha and his group have developed a GMO-PMO or PMO-GMO chimeras (International Publication Number -WO 2011/018798 A2) (Figure 2c) which are soluble in culture media and can penetrate cells without the need of injection. [14] GMO is guanidinium morpholino oligonucleotides which are linked either at the OH-or NHend of the PMOs.…”

Synthesis and Biophysical Properties of Phosphorodiamidate Piperidino Oligomers

“…PMOs must be chemically modified to improve cellular uptake and pharmacokinetics. , Among the reported modifications, incorporation of a guanidinium linkage or guanidinium-functionalized nucleobase is notable . Sinha’s group demonstrated the cell-penetrating and gene-silencing properties of self-transfecting guanidinium morpholino–PMO chimeras in cell culture and zebrafish .…”