Abstract
Although rapidly becoming a valuable tool for gene silencing, regulation or editing in vitro, the direct transfer of small interfering ribonucleic acids (siRNAs) into cells is still an unsolved problem for in vivo applications. For the first time, we show that specific modifications of antisense oligomers allow autonomous passage into cell lines and primary cells without further adjuvant or coupling to a cell-penetrating peptide. For this reason, we termed the specifically modified oligonucleotides "cell membrane-crossing oligomers" (CMCOs). CMCOs targeted to various conserved regions of human immunodeficiency virus (HIV)-1 were tested and compared with nontargeting CMCOs. Analyses of uninfected and infected cells incubated with labeled CMCOs revealed that the compounds were enriched in infected cells and some of the tested CMCOs exhibited a potent antiviral effect. Finally, the CMCOs did not exert any cytotoxicity and did not inhibit proliferation of the cells. In vitro, our CMCOs are promising candidates as biologically active anti-HIV reagents for future in vivo applications.
Highlights
In in vitro experiments, antisense technology represents a simple method for the treatment of diseases as diverse as viral infections, cancer and inflammatory, metabolic or neurodegenerative diseases
“Conventional” peptide nucleic acids (PNAs) molecules are poorly water soluble compared with DNA, and permeation of the cell membrane is a general problem for PNAs
PNA monomer building blocks are commercially available, and optically pure cell membrane–crossing oligomers” (CMCOs) monomer building blocks (R-configuration according to Cahn-Ingold-Prelog priority rules) were synthesized according to previous reports [18,23,24]
Summary
Antisense technology represents a simple method for the treatment of diseases as diverse as viral infections, cancer and inflammatory, metabolic or neurodegenerative diseases. There are efforts toward antisense drug discovery using cellular ribonucleic acids (RNAs) as molecular targets, but the effective delivery of these oligos in vivo is problematic. Among existing antisense strategies are peptide nucleic acids (PNAs), which were discovered in the context of gene targeting and gene therapeutic drugs. PNAs contain a chargeneutral pseudo-peptide backbone [1,2] that confers high chemical stability and resistance against degradation by diverse nucleases. PNAs were not degraded by endogenous proteases and peptidases during 2-h incubations in human serum, bacterial cell extracts or mouse ascites [3,4]. The effective delivery of inhibitory antisense reagents by complex formation of the small interfering RNAs (siRNAs)/
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