Genzyme Lipid 67 is a cholesterol derivatized with spermine to create a cationic lipid. GL67 has been proven to efficiently mediate gene transfer into mammalian cells and was a key formulation component that successfully completed clinical studies for the delivery of pDNA via inhalation.
Reference Reading
1. Comparison of the gene transfer efficiency of mRNA/GL67 and pDNA/GL67 complexes in respiratory cells
Oliwia Andries, Marina De Filette, Joanna Rejman, Stefaan C De Smedt, Jo Demeester, Mario Van Poucke, Luc Peelman, Cindy Peleman, Tony Lahoutte, Niek N Sanders. Mol Pharm. 2012 Aug 6;9(8):2136-45. doi: 10.1021/mp200604h.
Complexes between mRNA and GL67: DOPE: DMPE-PEG5000 (GL67) liposomes were formulated and characterized. Subsequently, the in vitro and in vivo expression characteristics of mRNA/GL67 complexes and pDNA/GL67 complexes, each produced at their optimal ratio, were compared in respiratory cells. Transfection of A549 cells with mRNA/GL67 complexes resulted in a much faster expression than after transfection with pDNA/GL67 complexes. The percentage of GFP-positive cells after mRNA and pDNA transfection peaked after 8 and 24 h, respectively. At these time points the percentage of GFP-positive cells was two times higher after mRNA transfection than after pDNA transfection. Furthermore, the efficacy of mRNA/GL67 complexes was independent of the cell cycle. This was in sharp contrast with pDNA/GL67 complexes that caused only a weak expression in nondividing cells. This confirms that the nuclear barrier is a crucial obstacle for pDNA but not for mRNA. Finally, mRNA/GL67 and pDNA/GL67 complexes encoding luciferase were administered intranasally to the lungs of mice. The mRNA/GL67 complexes did not give rise to a measurable luciferase expression in the murine lungs. In contrast, a detectable bioluminescent signal was present in the lungs of mice that received the pDNA/GL67 complexes. We showed that mRNA/GL67 complexes have a lower stability in biological fluids. Consequently, this may be an explanation for their lower performance in vivo.
2. Pegylated GL67 lipoplexes retain their gene transfection activity after exposure to components of CF mucus
N N Sanders, S C De Smedt, S H Cheng, J Demeester. Gene Ther. 2002 Mar;9(6):363-71. doi: 10.1038/sj.gt.3301663.
The highly viscous secretions lining the upper airways and bronchi of cystic fibrosis (CF) patients may pose a significant barrier to successful gene therapy of the lung. In this report we examined the influence of CF mucus components (albumin, DNA, mucin and phospholipids) on the gene transfection activity of cationic DOTAP-based lipoplexes and pegylated GL67-based lipoplexes which previously have been used in CF clinical studies. Upon exposure of the cationic DOTAP: DOPE lipoplexes to either albumin, linear DNA or mucin (at concentration ratios expected to occur in vivo) a significant decrease in gene transfection activity was observed. This was primarily due to aggregation of the lipoplexes. However, exposure of pegylated GL67 lipoplexes to the same components did not affect their gene transfection activity. Indeed, it was determined that CF mucus components did not interact significantly with these pegylated GL67 lipoplexes. These results suggest that charge shielding of cationic gene carriers with pEG may favor their physicochemical stability in CF mucus and thereby aid in preserving their transfection activity.
3. Transfection efficiency and toxicity following delivery of naked plasmid DNA and cationic lipid-DNA complexes to ovine lung segments
Michael Emerson, Louise Renwick, Stephen Tate, Susan Rhind, Elspeth Milne, Hazel Alsop Painter, A Christopher Boyd, Gerry McLachlan, Uta Griesenbach, Seng H Cheng, Deborah R Gill, Stephen C Hyde, Alison Baker, Eric W Alton, David J Porteous, D David S Collie. Mol Ther. 2003 Oct;8(4):646-53. doi: 10.1016/s1525-0016(03)00233-8.
We defined, using a novel large animal model system, the acute pathologic response to localized pulmonary administration of either naked plasmid DNA (pDNA) or cationic lipid-pDNA complexes (pDNA: GL67) and related such responses to concomitant indicators of transfection efficiency, namely levels of chloramphenicol acetyl transferase (CAT) protein and mRNA in specific lung tissue compartments. We instilled doses of 0.2, 1, and 5 mg pDNA to spatially distinct lung segments in six anesthetized sheep and doses of 0.2, 1, and 5 mg pDNA: GL67 to a further six sheep. Twenty-four hours after gene delivery the sheep were euthanized and necropsy examination with sampling of relevant tissues was carried out. Levels of plasmid-derived CAT-specific mRNA and CAT protein in samples derived from segments treated with either pDNA or pDNA: GL67 increased in relation to the administered dose. Levels of mRNA and protein expression were greater for pDNA: GL67 than for pDNA alone. A significant correlation was observed between mRNA and protein expression in samples derived from airways treated with pDNA: GL67. Histopathological changes following administration of both pDNA and pDNA: GL67 were characterized by a neutrophilic inflammation predominantly oriented on airways. The severity of the inflammatory response appeared to correlate with the administered dose of DNA and was generally more severe for pDNA: GL67.
