A Concentrated and Stable Aerosol Formulation of Cationic Lipid:DNA Complexes Giving High-Level Gene Expression in Mouse Lung

Abstract

Advances in gene therapy vectors and techniques hold promise for treatment of many inherited and acquired diseases. For lung indications, especially those involving the epithelium, delivery of the gene therapy vehicle ideally will involve the use of an aerosol. Aerosol delivery of transgenes using cationic lipids is currently limited by the ability to generate highly concentrated formulations of lipid:DNA complexes that are stable and retain their activity following aerosolization. We have examined many of the variables inherent in aerosolizing cationic lipid gene delivery vehicles and have devised a new formulation that incorporates small amounts of a polyethylene glycol-containing lipid. This formulation has allowed the preparation of concentrated dispersions of cationic lipid:plasmid DNA (pDNA) complexes (> 20 mM pDNA) at approximately 10-fold higher concentrations than previously reported. Most of the pDNA in these formulations was bound to the lipid component and thereby protected from nebulizer-induced shearing; the pDNA also maintained full biological activity both in vitro and in vivo. This new formulation thus represents a significant improvement over current methods to prepare concentrated, active cationic lipid gene delivery vectors, and provides a new tool with which to test gene transfer to the lung.

Pegylated GL67 lipoplexes retain their gene transfection activity after exposure to components of CF mucus

“…We studied cationic, non-pegylated (DOTAP:DOPE) and pegylated (GL67) lipoplexes, respectively ( Figure 1). As GL67-based lipoplexes lacking DMPE-pEG 5000 , were not available, 14 we investigated cationic DOTAP:DOPE lipoplexes as a model for lipoplexes without pEG. Cationic lipoplexes based on DOTAP have also been used in a clinical trial for CF gene therapy.…”

“…This fulfills the initial purpose for inclusion of DMPEpEG 5000 , which was to minimize the precipitation of the lipoplexes when they were prepared at high concentrations. 14 Nonetheless, 4 h after preparation a small amount of the GL67 lipoplexes did precipitate.…”

“…The GL67:pDNA molar ratio in these lipoplexes was 0.75:1 (molar concentration of pDNA based on an average nucleotide molecular mass of 330 g/mol) and the final pDNA concentration in the lipoplexes was 0.126 g/ l. The calculation of the +/-charge ratio of the GL67 lipoplexes was difficult as the pKas of the nitrogen atoms (which are necessary to calculate the amounts of positive charges) are unknown. Compared with other types of lipoplexes, the GL67-based lipoplexes described above were most effective after aerosol delivery into the lungs of mice, 14 and consequently, were used in a clinical trial by Alton et al 4 …”

Biodistribution and transgene expression with nonviral cationic vector/DNA complexes in the lungs

“…27 Confocal microscopy studies revealed that lipoplexes, but not polyplexes, aggregated in the lumen of bronchi and co-localized either totally (DOTAP) or partially (GL-67A) with the surfactant protein A. GL-67A was chosen since it was shown to be a more efficient lipid formulation than Gene Therapy other commercial lipids, such as DOTAP, for mouse and human lung transfection. 9,28 It includes a neutral lipid, DOPE, and a polyethylene glycol-containing lipid, DMPE-PEG5000, which stabilizes the bilayer configuration. It could be possible that these adjunctive components might have influenced the interaction with surfactant.…”

“…[1][2][3][4] Considering only nonviral DNA delivering agents, several previous works have achieved gene transfer with cationic lipids (lipofection) and polymers (polyfection) to the lungs of laboratory animals by either direct instillation in the nose and trachea [5][6][7] or via the aerosol administration. 8,9 Systemic intravenous infusion of cationic vector/DNA complexes has been attempted and shown to transfect preferentially the lung tissue. 7,[10][11][12][13] The most important determinants for cationic lipid-mediated gene transfer through intravenous administration are the cationic lipid structure, the charge ratio (+/−) between the cationic vector and DNA, [14][15][16] slightly positive PEI complexes as compared with DNA alone, whereas cationic lipid-based vectors, DOTAP and GL-67A, gave not significant luciferase activity.…”