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- Title
696. Development of Lipid/Peptide (Lip/Tide) Vectors for Respiratory Gene Transfer.
- Authors
Hart, Stephen L.; Tagalakis, Aris; Writer, Michele J.; Devaney, James; Hailes, Helen C.; Tabor, Alethea B.; Wong, John B.; Bottoms, Steve; McAnulty, Robin J.; Scheule, Ron; Cheng, Seng; Jaffe, Adam
- Abstract
We are developing synthetic,targeted vector systems for the respiratory system to treat diseases such as cystic fibrosis (CF). Lip/Tide-I vectors comprised an integrin-targeting peptide and DOTMA/DOPE which displayed transfected human epithelial cells with low receptor specificity due to a lack of integrin receptors. The integrin-targeting motif was replaced with an epithelial targeting peptide selected by phage display panning on human airway epithelial cells. The cationic lipid was optimised by substituting DOTMA, which has an eighteen-carbon alkyl tail (C18), with a C16 cationic lipid.The epithelial-optimised Lip/Tide vector (Lip/Tide-E) was compared with Lip/Tide-I in transfections of cultured human airway epithelial cells and in vivo by tracheal instillation into murine lungs of fifty microlitres of vector containing 8–16 micrograms of DNA. Lip/Tide-E transfection in human epithelial cells was eight-fold higher than Lip/Tide-I with high specificity of receptor-mediated transfection but did not enhance transfection in vivo owing to species specificity. The lipid enhanced luciferase transfection in human epithelial cells about ten-fold and in murine lung in vivo by two- fold.Luciferase expression in mouse whole lung extracts was compared after one and three doses of Lip/Tide-E vector. Single dose expression levels of Lip/Tide (2,400 RLU/mg) were similar to levels with GL67 (Genzyme), a vector used in CF gene therapy trials, and about twice the level achieved with polyethylenimine (PEI 25 kDa). After three weekly doses of Lip/Tide vector, luciferase expression was equivalent to single dose levels. Immunohistochemical staining of lung sections revealed that the Lip/Tide-transfected luciferase activity was located mainly in airway epithelium, with some further transfection of macrophages, while GL67 activity was largely in macrophages. A moderate Lip/Tide-mediated inflammatory cell infiltration was apparent and levels of inflammatory cytokines from bronchoalveolar lavage fluids, particularly IL-6 and IL-12, were elevated 24 h after transfection. The vector retained its transfection efficiency after nebulisation as evaluated in luciferase assays after in vitro and in vivo administration.In summary, the Lip/Tide-E vector transfection efficiency in human epithelial cells in vitro and in the airway epithelium of mouse lung is superior to Lip/Tide-I and compares well with established vectors such as GL67 and PEI. The modest inflammatory response and the ability to re-administer the Lip/Tide vector to restore gene expression are important requirements of a respiratory gene transfer system although further improvements to the inflammatory profile may be achieved by using CpG-depleted plasmids. The vector is compatible with delivery by aerosol inhalation which requires further evaluation in large animal models.Molecular Therapy (2006) 13, S269–S270; doi: 10.1016/j.ymthe.2006.08.774
- Subjects
GENETIC transformation; EPITHELIAL cells; LUNG disease diagnosis; IMMUNOREGULATION; GENE expression; GENE therapy
- Publication
Molecular Therapy, 2006, Vol 13, pS269
- ISSN
1525-0016
- Publication type
Article
- DOI
10.1016/j.ymthe.2006.08.774