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

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_1/2 of 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. Typically, such efforts rely on a locally injectable delivery systems comprising of either viral or non-viral vectors admixed with fibrin, collagen or hyaluronic acid hydrogel matrices^6-10 that are capable of transiently sustaining gene vectors at the injection site by physically entrapping them in a polymeric network.

Another generally accepted paradigm for localized gene therapy utilizes immobilization of gene vectors on the surface of implanted prosthetic devices^11,12. Permanent medical implants (endovascular, bronchial, urological and gastrointestinal stents, pacemakers, artificial joints, surgical and gynecological meshes, etc.) are used yearly in tens of millions of patients¹³. While generally effective, these devices are prone to complications that are inadequately controlled for by current medical practices^14-17. Implantable prosthetic devices present a unique opportunity to serve as proxy platforms for localized gene therapy treatment. From the pharmacokinetic standpoint, surface derivatization of medical implants with relatively low input doses of gene vectors results in achieving both high local concentrations of gene vectors on the implant/tissue interface and slowing the kinetics of their elimination from this location. As a consequence of protracted residence and enhanced uptake by the targeted cell population, vector immobilization minimizes spread of the gene vector. Thus the inadvertent inoculation of non-target tissues is reduced.

Surface tethering of gene vectors on implantable biomaterials (also termed as substrate-mediated gene delivery or solid phase gene delivery) has been implemented in cell culture and animal experiments using both specific (antigen-antibody^18-20, avidin-biotin^21,22) and non-specific^23-26 (charge, van der Waals) interactions. The covalent attachment of vectors to the surface of the implanted device has been previously considered as non-functional since excessively strong bonds with the surface preclude vector internalization by target cells. Recently it was demonstrated that this limitation can be overcome through the use of spontaneously hydrolysable cross-linker used as the tethers between the modified metallic surface of the stent and capsid proteins of the adenoviral vector^27,28. Moreover, the vector release rate and time course of transgene expression in vitro and in vivo can be modulated with the use of hydrolysable cross-linkers exhibiting different kinetics of hydrolysis²⁸.

The present paper provides a detailed protocol for the reversible covalent attachment of adenoviral vectors to activated metal surface and introduces a useful experimental setup for studying ensuing transduction events in vitro in cultured smooth muscle and endothelial cells and in vivo in the rat carotid model of stent angioplasty.

Protocol

1. Preparation of Cy3-labeled Adenovirus for the Release Experiments Suspend 2 x 1012 particles of Adempty (approximately 2 x 1011 infective units) in 650 µl of carbonate/bicarbonate buffer (CBB; pH 9.3). Dissolve the content of 1 vial (0.2 mg) of amine-reactive fluorescent dye (Cy3(NHS)2) in 1 ml CBB to a final concentration of 0.2 mg/ml. Add 100 µl of the dye solution to virus suspension, vortex for 5 sec and incubate for 1 hr at…

Representative Results

Vector Release Experiments Tethering of adenoviral vectors to the surface of implants, including interventional devices such as endovascular stents, approximates the vector to the disease site, partially obviating the lack of vectors’ physical targeting. However, to be able to achieve therapeutic effects via the transduction of target tissue, the vector must be released from the surface (Figure 2). The use of hydrolysable cross-linkers was hypothesi…

Discussion

The presented protocol describes an operational method for substrate mediated gene delivery achieved through reversible attachment of adenoviral vectors to coatless stainless steel surfaces. While developed for the specific purpose of stent-based gene therapy of vascular restenosis, this technique has much broader applications in the areas of biomaterials, biomedical implants and gene therapy.

Although presented studies have solely utilized stainless steel as a prototypical metal substrate, PA…

Materials

316 stainless steel mesh disks	Electon Microscopy Sciences	E200-SS
Generic 304-grade stainless steel stents	Laserage	custom order
AdeGFP	University of Pennsylvania Vector Core	AD-5-PV0504
AdLuc	University of Pennsylvania Vector Core	AD-5-PV1028
AdEMPTY	University of Pennsylvania Vector Core	A858
Cy3(NHS)2	GE Healthcare	PA23000
Sepharose 6B	Sigma-Aldrich	6B100-500ML
UV 96-well plates	Costar	3635
Fluorometry 96-well plates	Costar	3915
Cell culture 96-well plates	Falcon	353072
Tris(2-carboxyethyl)phosphine hydrochloride (TCEP )	Pierce Thermo Scientific	20490
dithiothreitol (DTT)	Pierce Thermo Scientific	20290
sulfo-LC-SPDP	Pierce Thermo Scientific	21650
Spectrophotometer	Molecular Devices	SpectraMax 190
Spectrofluorometer	Molecular Devices	SpectraMax Gemini EM
Orbital shaker incubator	VWR	1575R
Horizontal airflow oven	Shel Lab	1350 FM
Centra-CL2 centrifuge	International Equipment Company	426
Digital vortex mixerer	Fisher Thermo Scientific	02-215-370
Eclipse TE300 fluorescence microscope	Nikon	TE300
DC 500 CCD camera	Leica	DC-500
7500 Real-Time PCR system	Applied Biosystems	not available
IVIS Spectrum bioluminescence station	Perkins-Elmer	not available
EDTA dipotassium salt	Sigma-Aldrich	ED2P
Bovine serum albumin fraction V (BSA)	Fisher Thermo Scientific	BP1600-100
Tween-20	Sigma-Aldrich	P1379
Dumont forceps	Fine Science Tools	11255-20
A10 cell line	ATCC	CRL-1476
Bovine aortic endothelial cells	Lonza	BW-6002
Luciferin, potassium salt	Gold Biotechnology	LUCK-1Ge
Pluronic F-127	Sigma-Aldrich	P2443-250G
PBS  without calcium and magnesium	Gibco	14190-136
Fetal bovine serum	Gemini Bio-Products	100-106
Penicillin/Streptomycin solution	Gibco	11540-122
DMEM, high glucose	Corning cellgro	10-013-CV
0.25% Trypsin/EDTA	Gibco	25200-056
QIAamp DNA micro kit	Qiagen	56304
Power Sybr Green PCR Master Mix	Applied Biosystems	4367659
MicroAmp Optical 96-well Reaction Plate	Applied Biosystems	N8010560
MicroAmp Optical Adhesive Film	Applied Biosystems	4360954
Cephazolin	Apotex	not available
Loxicom (Meloxicam)	Norbrook	not available
Heparin sodium	APP Pharmaceuticals	not available
Ketavet (Ketamine)	VEDCO	not available
Anased (Xylazine)	Lloid	not available
Forane (Isoflurane)	Baxter	not available
Curved Moria iris forceps	Fine Science tools	11370-31
Curved extra-fine Graefe forceps	Fine Science Tools	11152-10
Dumont #5 forceps	Fine Science Tools	11252-20
Vannas spring scissors	Fine Science Tools	15018-10
Fine scissors – ToughCut	Fine Science Tools	14058-09
Surgical scissors	Fine Science Tools	14101-14
Vicryl suture (5-0)	Ethicon	J385
Suture thread (4/0 silk)	Fine Science Tools	18020-40
Michel suture clips	Fine Science Tools	12040-02
Wound dilator (Lancaster eye specula)	KLS Martin	34-149-07
Hot bead sterilizer	Fine Science Tools	18000-45
Michel suture clip applicator	Fine Science Tools	112028-12
Insyte Autoguard 24G IV catheter	Beckton-Dickinson	381412
2F Fogarty catheter	Edwards Lifesciences	120602F
Teflon tubing	Vention	041100BST
PTA catheter	NuMed	custom order
Gauze pads	Kendall Healthcare	9024
Cotton applicators	Solon Manufacturing	WOD1003
Saline	Baxter	281321
10 ml syringe (Luer-Lok)	Beckton-Dickinson	309604
1 ml syringe (Luer-Lok)	Beckton-Dickinson	309628
Clippers with #40 blade	Oster	78005-314
Transpore surgical tape	3M	MM 15271
Puralube vet ointment	Pharmaderm	not available