Peptide-based intracellular shuttle able to facilitate gene transfer in mammalian cells.
Loligomers are peptide-based intracellular vehicles able to penetrate cells and self-localize into distinct cellular compartments. Loligomers can be rapidly assembled by automated solid-phase approaches and were designed to act as nonviral, nonlipophilic intracellular shuttles. One nucleus-directed loligomer, termed loligomer 4, was evaluated for its ability to function as a transfection agent. Loligomer 4 readily associates with plasmids to form noncovalent complexes. The migration of loligomer 4-plasmid complexes into cells was monitored by flow cytometry and fluorescence microscopy. Populations of plasmids labeled with 7-AAD exist either free or in association with loligomer 4 inside cells and are visible throughout the cytosol and nucleus of chinese hamster ovary (CHO) cells. Loligomer 4-plasmid complexes were not cytotoxic to cells and were readily imported by most cells (>70%). CHO cells were transfected with complexes of loligomer 4 and plasmids harboring luciferase, green fluorescent protein or beta-galactosidase reporter genes. The transfection efficiency of loligomer 4-plasmid DNA complexes was greater when cells were maintained as suspensions instead of monolayers. Transfections could be performed with cells suspended in serum-containing medium. The observed levels of transfection, however, were modest with 5-10% of CHO cells expressing either a green fluorescent protein or the enzyme beta-galactosidase. Loligomers have recently been observed in vesicular compartments [Singh, D., Kiarash, R., Kawamura, K, LaCasse, E. C., and Gariépy, J. (1998) Biochemistry 37, 5798-5809] and differences between levels of cellular import and transfection efficiency may well reflect the need to optimize the release of loligomers and their complexes from these compartments in future designs. In summary, loligomer 4 behaves as a stable, soluble and effective transfection agent. These results demonstrate the feasibility of designing loligomers able to act as intracellular guided agents aimed at gene transfer applications.
Singh
D
D
Division of Molecular and Structural Biology, Department of Medical Biophysics, University of Toronto and the Ontario Cancer Institute, Princess Margaret Hospital, 610 University Avenue, Toronto, Canada M5G 2M9.
Bisland
S K
SK
Kawamura
K
K
Gariépy
J
J
D000818:Animals; D001943:Breast Neoplasms / Q000378:metabolism; D016466:CHO Cells; D019556:COS Cells; D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D004247:DNA / Q000008:administration & dosage* / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D019793:Fluorescein / Q000378:metabolism; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D009842:Oligopeptides / Q000008:administration & dosage / Q000138:chemical synthesis / Q000378:metabolism*; D014677:Pharmaceutical Vehicles / Q000008:administration & dosage / Q000138:chemical synthesis / Q000378:metabolism; D010957:Plasmids / Q000008:administration & dosage / Q000378:metabolism; D014162:Transfection / Q000379:methods; D014407:Tumor Cells, Cultured
D009842:Oligopeptides; D014677:Pharmaceutical Vehicles; D004247:DNA; D019793:Fluorescein