Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers

Ilia Fishbein; Scott P. Forbes; Richard F. Adamo; Michael Chorny; Robert J. Levy; Ivan S. Alferiev

doi:10.3791/51653

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Method Article

Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers

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DOI:

10.3791/51653

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August 12th, 2014

Ilia Fishbein¹ , Scott P. Forbes¹ , Richard F. Adamo¹ , Michael Chorny¹ , Robert J. Levy¹ , Ivan S. Alferiev¹

¹Department of Pediatrics, Division of Cardiology, The Children's Hospital of Philadelphia, University of Pennsylvania

Summary

These studies report on reversible attachment of adenoviral gene vectors to coatless metal surfaces of stents and model mesh disks. Sustained release of transduction-competent viral particles contingent upon hydrolysis of cross-linkers used for vector immobilization results in a durable site-specific transgene expression in vascular cells and in stented arteries.

Abstract

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically narrowed segments of coronary and peripheral arteries. Endovascular stents capable of tunable release of genes with anti-restenotic activity may present an alternative strategy to presently used drug-eluting stents. In order to attain clinical translation, gene-eluting stents must exhibit predictable kinetics of stent-immobilized gene vector release and site-specific transduction of vasculature, while avoiding an excessive inflammatory response typically associated with the polymer coatings used for physical entrapment of the vector. This paper describes a detailed methodology for coatless tethering of adenoviral gene vectors to stents based on a reversible binding of the adenoviral particles to polyallylamine bisphosphonate (PABT)-modified stainless steel surface via hydrolysable cross-linkers (HC). A family of bifunctional (amine- and thiol-reactive) HC with an average t_1/2of the in-chain ester hydrolysis ranging between 5 and 50 days were used to link the vector with the stent. The vector immobilization procedure is typically carried out within 9 hr and consists of several steps: 1) incubation of the metal samples in an aqueous solution of PABT (4 hr); 2) deprotection of thiol groups installed in PABT with tris(2-carboxyethyl) phosphine (20 min); 3) expansion of thiol reactive capacity of the metal surface by reacting the samples with polyethyleneimine derivatized with pyridyldithio (PDT) groups (2 hr); 4) conversion of PDT groups to thiols with dithiothreitol (10 min); 5) modification of adenoviruses with HC (1 hr); 6) purification of modified adenoviral particles by size-exclusion column chromatography (15 min) and 7) immobilization of thiol-reactive adenoviral particles on the thiolated steel surface (1 hr). This technique has wide potential applicability beyond stents, by facilitating surface engineering of bioprosthetic devices to enhance their biocompatibility through the substrate-mediated gene delivery to the cells interfacing the implanted foreign material.

Introduction

The effectiveness of gene therapy as a therapeutic modality is hampered by the poor targeting capacity of gene therapy vectors^1,2. The lack of proper targeting results in sub-therapeutic levels of transgene expression at the target location and leads to a wide dissemination of vectors to non-target organs³, including those responsible for mounting immune responses against both the vector and encoded therapeutic product^4,5. One potential means to offset the promiscuity of transduction and to promote targeting is to introduce gene vectors at the desired location in a form that precludes their free dissemination via blood and lymph. Typic....