Nanotech 2010

Engineering Acid- Responsive Lipid Envelopes around Adenovirus for Efficient Gene Delivery

W.T. Al-Jamal, J. Van den Bossche, A. Yilmazer, B. Tian, K. Kostarelos
The School of Pharmacy, UK

Keywords: adenovirus, liposomes, gene delivery, pH-sensitive

Abstract:

Gene therapy involves the delivery of a functional gene by a vector into target cells, resulting in a desired therapeutic effect. Adenovirus (Ad) has shown a great promise in gene therapy (1,2). However, in vivo studies have reported an immunogenic response and a overwhelming accumulation and gene expression in the liver resulting in significant hepatoxicity. These issues currently inhibit the use of this vector for clinical genetic therapies. Such limitations have been overcome by engineering an artificially enveloped Ad using zwitterionic and cationic lipid formulations (3,4). However, this resulted in a significant reduction of gene expression in vitro due to the poor release of the lipid bilayer enveloped virus from the endosomal compartment that hinders their gene expression capacity. In the present work, we have explored the use of pH-sensitive DOPE:CHEMS (6:4) lipid-envelope to enhance the virus release from the endosome following endocytosis. Artificially enveloped Ad were prepared by lipid film hydration followed by sonication. The surface engineered Ad were characterised by transmission electron microscopy (TEM), atomic force microscopy (AFM), dot blot, dynamic light scattering and zeta potential measurements. DOPE:CHEMS envelopment exhibited good stability at physiological pH (7.4) but immediately collapsed and released the naked virions at pH 5.5. Furthermore, the gene expression of recombinant Ad encoding for beta-galactosidase (β-gal) reporter gene enveloped in DOPE:CHEMS showed high levels of gene expression when tested in different cell lines, comparable to naked Ad. These transfection results were further confirmed by studying the intracellular trafficking of fluorescently-labelled, Cy3-Ad using confocal laser scanning microscopy (CLSM). Interestingly, Cy-3 Ad enveloped in DOPE:CHEMS showed a uniform fluorescence distribution within the cytoplasm indicating Ad endosomal release. In addition, pH- sensitive enveloped Ad injected directly into human cervical adenocarinoma (C33a) xenografts grown on the flank of nude mice showed same level of gene expression to naked Ad. In conclusion, this type of artificially-enveloped Ad offers a promising tool in gene delivery since high level of Ad gene expression can be maintained and expected to dramatically improve the innate Ad immunogenicity and hepatotoxicity in vivo.
 
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