Abstract
BackgroundFor improved uptake of oligonucleotide-based therapy, the oligonucleotides often are coupled to peptides that facilitate entry into cells. To this end, novel cell-penetrating peptides (CPPs) were designed for mediating intracellular uptake of oligonucleotide-based therapeutics. The novel peptides were based on taking advantage of the nuclear localization properties of transcription factors in combination with a peptide that would bind putatively to cell surfaces. It was observed that adding a glutamate peptide to the N-terminus of the nuclear localization signal (NLS) of the Oct6 transcription factor resulted in a novel CPP with better uptake and better nuclear colocalization than any other peptide tested.ResultsUptake of the novel peptide Glu-Oct6 by cancer cell lines was rapid (in less than 1 hr, more than 60% of DU-145 cells were positive for FITC), complete (by 4 hr, 99% of cells were positive for FITC), concentration-dependent, temperature-dependent, and inhibited by sodium azide (NaN3). Substitution of Phe, Tyr, or Asn moieties for the glutamate portion of the novel peptide resulted in abrogation of novel CPP uptake; however none of the substituted peptides inhibited uptake of the novel CPP when coincubated with cells. Live-cell imaging and analysis by imaging flow cytometry revealed that the novel CPP accumulated in nuclei. Finally, the novel CPP was coupled to a carboxyfluorescein-labeled synthetic oligonucleotide, to see if the peptide could ferry a therapeutic payload into cells.ConclusionsThese studies document the creation of a novel CPP consisting of a glutamate peptide coupled to the N-terminus of the Oct6 NLS; the novel CPP exhibited nuclear colocalization as well as uptake by prostate and pancreatic cancer cell lines.
Highlights
For improved uptake of oligonucleotide-based therapy, the oligonucleotides often are coupled to peptides that facilitate entry into cells
Neither a PMSA-targeted peptide nor TAT resulted in efficient peptide nucleic acid (PNA) uptake in DU-145 cells Because PMSA is overexpressed on most prostate cancer cells, it is an attractive target for therapeutic delivery
TAT peptide, a 13-amino acid peptide comprised of residues 48 to 60 of the human immunodeficiency virus-1 TAT protein, has been shown to be an efficient cell-penetrating peptides (CPPs) for delivery of anti-viral oligonucleotides and PNAs [3]; as is the case for either a carboxy-terminal peptide (EEE), it had not been fully evaluated in most cancer lines for delivery of PNAs
Summary
For improved uptake of oligonucleotide-based therapy, the oligonucleotides often are coupled to peptides that facilitate entry into cells. To this end, novel cell-penetrating peptides (CPPs) were designed for mediating intracellular uptake of oligonucleotide-based therapeutics. Experimental therapeutic approaches using oligonucleotides for prostate and pancreatic cancer are actively investigated in many laboratories, including ours [1,2]. Such inhibitors are attractive in theory but lack a practical method for delivery in the clinical setting. As for pancreatic cancer, the antennepedia protein Antp when coupled to the tumor suppressor p16 successfully inhibited cell growth [9], and the insulin-like growth factor loop 1 peptide IGF1 is being tried for imaging of early pancreatic tumors [10]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
