Effective siRNA delivery and target mRNA degradation using an amphipathic peptide to facilitate pH-dependent endosomal escape.
Effective delivery of siRNA (small interfering RNA) into the cells requires the translocation of siRNA into the cytosol. One potential delivery strategy uses cell-delivery peptides that facilitate this step. In the present paper, we describe the characterization of an amphipathic peptide that mediates the uptake of non-covalently bound siRNA into cells and its subsequent release into the cytosol. Biophysical characterization of peptide and peptide/siRNA mixtures at neutral and lysosomal (acidic) pH suggested the formation of α-helical structure only in endosomes and lysosomes. Surprisingly, even though the peptide enhanced the uptake of siRNA into cells, no direct interaction between siRNA and peptide was observed at neutral pH by isothermal titration calorimetry. Importantly, we show that peptide-mediated siRNA uptake occurred through endocytosis and, by applying novel endosomal-escape assays and cell-fractionation techniques, we demonstrated a pH-dependent alteration in endosome and lysosome integrity and subsequent release of siRNA and other cargo into the cytosol. These results indicate a peptide-mediated siRNA delivery through a pH-dependent and conformation-specific interaction with cellular membranes and not with the cargo.
Department of RNA Therapeutics, Merck & Co. Inc., 770 Sumneytown Pike, West Point, PA 19486, USA. rene_bartz@merck.com
René
R
Bartz
Haihong
H
Fan
Jingtao
J
Zhang
Nathalie
N
Innocent
Craig
C
Cherrin
Stephen C
SC
Beck
Yi
Y
Pei
Aaron
A
Momose
Vasant
V
Jadhav
David M
DM
Tellers
Fanyu
F
Meng
Louis S
LS
Crocker
Laura
L
Sepp-Lorenzino
Stanley F
SF
Barnett
D001324:Autoantigens / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D016503:Drug Delivery Systems / Q000379:methods*; D004526:Efficiency; D011992:Endosomes / Q000187:drug effects* / Q000378:metabolism; D018390:Gene Targeting / Q000379:methods; D018014:Gene Transfer Techniques; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology*; D034622:RNA Interference / Q000187:drug effects / Q000502:physiology; D020871:RNA Stability / Q000187:drug effects* / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000494:pharmacology; D012261:Ribonucleoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000097585:SS-B Antigen
D001324:Autoantigens; D010446:Peptide Fragments; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012261:Ribonucleoproteins; D000097585:SS-B Antigen