Gene Therapy

Lipofection of nondividing cells is inefficient because much of the transfected DNA is retained in endosomes, and that which escapes to the cytoplasm enters the nucleus at low rates. To improve the final ratelimiting step of nuclear import, we have used the nonclassical nuclear localization signal (NLS) containing the M9 sequence of heterogeneous nuclear ribonucleoprotein (hnRNP) A1. This approach has been validated in endothelial cells, neurons, and mouse embryonic stem cells.

During the delivery of plasmid DNA for gene therapy often provokes an inflammatory response that reduces transgene expression. Cationic lipids for lipofection lack pharmacological activity despite the hydrophobicity of many drug candidates that could be exploited. We have developed a one-step synthesis of a water-soluble, dexamethasone–spermine (DS) cationic lipid that has potent gene transfer capability in confluent endothelial cells when used with the neutral lipid, dioleoylphosphatidylethanolamine (DOPE). DS retains partial corticosteroid character as quantified by the rapid translocation of glucocorticoid receptor to the nucleus and by dosedependent transactivation from a glucocorticoid response element. DS has anti-inflammatory activity in vivo in the mouse thioglycollate model of inflammation. In a mouse lung model, DS:DOPE resulted in significantly less interferon-g production at Day 1 and elevated transgene expression at Days 1 and 7 postintranasal instillation compared to DCChol: DOPE.

We formulated adenovirus (AdV) vectors with cationic steroid liposomes containing dexamethasone–spermine (DS)/dioleoylphosphatidylethanolamine (DOPE) in an effort to overcome the lack of apically expressed AdV vector receptors on airway epithelial cells and to reduce the inflammation associated with AdV vector exposure. An AdV vector (1 to 2.5 x10^11 genome copies) expressing human placental alkaline phosphatase or B-galactosidase (LacZ) was delivered alone or complexed with DS/DOPE, DC-Chol/DOPE, or dexamethasone to C57Bl/6 mice via intranasal instillation. Formulation of the AdV vector with DS/DOPE and DC-Chol/DOPE resulted in transgene expression targeted only to the airway epithelial cells with minimal expression in alveolar cells, while AdV alone caused high alveolar transduction. The DS/DOPE and dexamethasone formulations greatly reduced cellular infiltrates compared to AdV vector alone, while formulation with DC-Chol/DOPE did not. Viral vectors can be formulated with DS/DOPE to improve targeting to the airway epithelium in vivo and to attenuate vector-induced inflammation through the pharmacological activity of DS.

Publications