Development of a receptor-targeted gene delivery system using CXCR4 ligand-conjugated cross-linking peptides.
BACKGROUND
Success in gene therapy greatly depends on the efficiency of nucleic acid delivery. Important features of the carriers for gene delivery should include an enhanced transfection ability, targeting of specific receptors and low toxicity. In the present study, we characterized CXCR4-targeted cross-linking peptides modified with an N-terminal fragment of chemokine stromal cell-derived factor-1α as carriers for gene delivery.


METHODS
We studied three variants of DNA/carrier complexes with different targeting ligand content. The physicochemical characteristics of the complexes, including their DNA-binding and protective ability, interaction with glycosaminoglycans and size, were determined. Transfection efficacy was studied in cell lines with different levels of CXCR4 expression (HeLa, A172, CHO, Е.А.hy926) and also in human mesenchymal stem cells (hMSCs). The influence of the ligand content on the efficacy of transfection was studied by means of chlorpromazine blockage of clathrin-mediated endocytosis, competition with CXCR4-antagonist AMD3100, and valproic acid treatment of hMSCs.


RESULTS
CXCR4-targeted peptides were evaluated for their physicochemical properties and in vitro transfection capacities. Ligand-modified carriers were found to be 10- to 50-fold more effective than unmodified carriers in CXCR4-positive cells. By contrast, their transfection efficacy in CXCR4-negative cells was similar to unmodified carriers. Experiments with chlorpromazine demonstrated receptor-specific transfection in A172 cells. The transfection efficacy of CXCR4-targeted carriers in AMD3100-treated HeLa cells was reduced by two-fold compared to the untreated control. Valproic acid treatment resulted in a four- to 15-fold increase of transfection efficacy for ligand-modified carriers in hMSCs.


CONCLUSIONS
CXCR4-targeted cross-linking peptides should be considered as useful tools for nonviral gene delivery into tumor and mesenchymal stem cells.
Laboratory for Prenatal Diagnostics of Inherited Diseases, D. O. Ott Research Institute of Obstetrics and Gynecology RAMS, Saint-Petersburg, Russia.
Anna
A
Egorova
Maria
M
Bogacheva
Anastasia
A
Shubina
Vladislav
V
Baranov
Anton
A
Kiselev
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D003432:Cross-Linking Reagents / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D046911:Macromolecular Substances / Q000737:chemistry / Q000494:pharmacology; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D019718:Receptors, CXCR4 / Q000378:metabolism*; D014162:Transfection
D003432:Cross-Linking Reagents; D008024:Ligands; D046911:Macromolecular Substances; D010455:Peptides; D019718:Receptors, CXCR4; D004247:DNA
CXCR4; cancer cells; cross-linking peptides; human mesenchymal stem cells; targeted gene delivery