Lung delivery studies using siRNA conjugated to TAT(48-60) and penetratin reveal peptide induced reduction in gene expression and induction of innate immunity.
The therapeutic application of siRNA shows promise as an alternative approach to small-molecule inhibitors for the treatment of human disease. However, the major obstacle to its use has been the difficulty in delivering these large anionic molecules in vivo. In this study, we have investigated whether siRNA-mediated knockdown of p38 MAP kinase mRNA in mouse lung is influenced by conjugation to the nonviral delivery vector cholesterol and the cell penetrating peptides (CPP) TAT(48-60) and penetratin. Initial studies in the mouse fibroblast L929 cell line showed that siRNA conjugated to cholesterol, TAT(48-60), and penetratin, but not siRNA alone, achieved a limited reduction of p38 MAP kinase mRNA expression. Intratracheal administration of siRNA resulted in localization within macrophages and scattered epithelial cells and produced a 30-45% knockdown of p38 MAP kinase mRNA at 6 h. As with increasing doses of siRNA, conjugation to cholesterol improved upon the duration but not the magnitude of mRNA knockdown, while penetratin and TAT(48-60) had no effect. Importantly, administration of the penetratin or TAT(48-60) peptides alone caused significant reduction in p38 MAP kinase mRNA expression, while the penetratin-siRNA conjugate activated the innate immune response. Overall, these studies suggest that conjugation to cholesterol may extend but not increase siRNA-mediated p38 MAP kinase mRNA knockdown in the lung. Furthermore, the use of CPP may be limited due to as yet uncharacterized effects upon gene expression and a potential for immune activation.
Moschos
Sterghios Athanasios
SA
Biopharmaceutics Research Group, Airways Disease, National Heart and Lung Institute, Imperial College, London, UK. s.moschos@imperial.ac.uk
Jones
Simon Wyn
SW
Perry
Mark Michael
MM
Williams
Andrew Evan
AE
Erjefalt
Jonas Sten
JS
Turner
John James
JJ
Barnes
Peter John
PJ
Sproat
Brian Stephen
BS
Gait
Michael John
MJ
Lindsay
Mark Andrew
MA
D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000494:pharmacology*; D002460:Cell Line; D057846:Cell-Penetrating Peptides; D002784:Cholesterol / Q000378:metabolism; D016503:Drug Delivery Systems; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects* / Q000502:physiology; D015696:Gene Products, tat / Q000737:chemistry*; D007113:Immunity, Innate / Q000187:drug effects* / Q000502:physiology; D008168:Lung / Q000502:physiology*; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000737:chemistry / Q000494:pharmacology*; D013997:Time Factors; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D002352:Carrier Proteins; D057846:Cell-Penetrating Peptides; D015696:Gene Products, tat; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D002784:Cholesterol; C414409:penetratin; D048051:p38 Mitogen-Activated Protein Kinases
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