879. Transduction Enhancement by the Adenovirus/Polymer Complexes Derivatized with Protein Transduction Domain Peptides
Authors: Ilia Fishbein, Ivan S. Alferiev, Richard Gaster, Michael Chorny, Origene Nyanguile, Robert J. Levy
Journal: Molecular Therapy (2005)

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Background: Cellular uptake of group C adenoviruses critically depends on the availability of the cognate Coxsackie Adenovirus Receptors (CARs) on the cell surface. In cardiovascular tissues paucity of CARs is a limiting factor for the successful application of adenoviral vectors. Additionally, immune responses elicited by Ad infection diminish transgene expression due to the elimination of free Ad and transduced cells. Polymer modification of adenoviral surface potentially addresses these problems by masking Ad particles from neutralizing antibodies and providing a convenient platform for the conjugation of ligands possessing transduction-facilitating properties. We report here that a covalent modification of Ad surface with a photoactivatable polymer derivatized with protein transduction domains (PTD), TAT and Antp, increases transduction efficiency.

Methods: A positively charged, thiol-reactive, photoactivatable polyallylamine (PAA) derivative was coupled with TAT-Cys and Antp-Cys. Ad samples suspended in water or PBS of different ionic strength (x1 and x5) were admixed with PTD-derivatized and unmodified polymers to obtain final polymer/Ad ratios ranging from 1:100 to 1:5 (w/w). Initial charge-based polymer/virus attachment was rendered covalent by 15 sec UV light (350 nm) illumination. Surface charge and size of the Ad/polymer complexes were measured by zeta-potentiometry and dynamic light scattering, respectively. Ad surface modification was examined by transmission electron microscopy (TEM) employing colloidal anionic gold. Transduction of a rat aortic smooth muscle cell line (A10) and primary bovine aortic endothelial cells (BAEC) was assessed by the fluorescence microscopy and the fluorimetry of cell lysates. In some experiments the cells were pretreated with the recombinant soluble knob protein (5 |[mu]|g/ml) prior to the Ad infection.

Results: Association of Ad with unmodified polymer increased surface charge of the vector from |[minus]|31.67 mv to |[minus]|11.89 mv, while the interaction with the Antp- and TAT-modified polymers inverted the surface charge to +3.75 mv and +12.09 mv, respectively. Recharging of the Ad surface was confirmed by the TEM demonstrating the attraction of 5 nm anionic gold particles to the surface of the Ad modified with TAT-derivatized polymer, but not to the surface of naive Ad. The association of Ad-GFP with PTD-derivatized polymers resulted in 3-7-fold increase of GFPexpression in cells, whereas unmodified polymer-derivatized virus demonstrated the same efficiency as the naive Ad. Pretreatment of A10 cells with knob protein caused 86% transduction inhibition with free and unmodified polymer-derivatized Ad, while the transgene expression of the PTD/polymer-modified Ad was not affected. Optimization of the transduction protocol revealed maximal enhancement of transgene expression at a polymer/Ad ratio of 1:10. Also, complexes prepared in x5 PBS achieved higher GFP expression than those prepared in water or x1 PBS, which correlated with smaller size of complexes (330 nm vs 1360 and 1060 nm diameter, respectively).

Conclusion: Covalent attachment of PDT-decorated polymers to the Ad surface increases transduction in vitro.