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Stent-based delivery of adeno-associated viral vectors with sustained vascular transduction and iNOS-mediated inhibition of in-stent restenosis

In-stent restenosis remains an important clinical problem in the era of drug eluting stents. Development of clinical gene therapy protocols for the prevention and treatment of in-stent restenosis is hampered by the lack of adequate local delivery systems. Herein we describe a novel stent-based gene delivery platform capable of providing local arterial gene transfer with adeno-associated viral (AAV) vectors. This system exploits the natural affinity of protein G (PrG) to bind to the Fc region of mammalian IgG, making PrG a universal adaptor for surface immobilization of vector-capturing antibodies (Ab). Our results: 1) demonstrate the feasibility of reversible immobilization of AAV2 vectors using vector tethering by AAV2-specific Ab appended to the stent surface through covalently attached PrG, 2) show sustained release kinetics of PrG/Ab-immobilized AAV2 vector particles into simulated physiological medium in vitro and site-specific transduction of cultured cells, 3) provide evidence of long-term (12 weeks) arterial expression of luciferase with PrG/Ab-tethered AAV2Luc, and 4) show anti-proliferative activity and anti-restenotic efficacy of stent-immobilized AAV2iNOS in the rat carotid artery model of stent angioplasty.

The release medium was then replenished and the release was resumed. The experiment was terminated at the 31-day time point.
The amount of AAV2iNOS vector genome associated with the foil samples at each time point was determined by Pico Green assay, as described above. The relative fluorescence intensity values were converted to the vector genome numbers using a calibration curve constructed with the serial dilutions of free AAV2iNOS of a known titer. The surface areas of individual samples were derived from their weights presuming a 50-µm thickness and a 7.95 g/cm 3 specific density of the foil material. Finally, the VG numbers were normalized by the surface area of individual foil coupons and plotted as a function of time.

Immunohistochemistry and dual immunofluorescence
Formalin-fixed, paraffin-embedded destented arterial sections were rehydrated and boiled in epitope retrieval buffer (pH 6) in a pressure cooker for 10 min, followed by permeabilization in 0.5% Tween-20/PBS for 15 min and blocking with 10% horse serum for 15 min. Primary antibodies (anti-DYKDDDDK Tag, Cell Signaling Technology #14793; anti-CD68, BioRad #MCA341) were applied in a 1:100 dilution at 4ºC overnight. Secondary biotinylated anti-mouse (BA-2001) and anti-rabbit (BA-1100) antibodies from Vector Labs were used in a 1:100 dilution at room temperature for 1 hour, followed by the application of the ABC Vectastain system and ImmPACT DAB peroxidase substrate. The slides were further counterstained with Gill 3 hematoxylin.
For dual immunofluorescence staining, rabbit-made anti-FLAG antibody was applied to the slides concurrently with mouse-made anti-cell type marker antibodies (either anti-CD68, or anti-alpha smooth muscle actin), followed by co-treatment with secondary DyLight488-labeled anti-rabbit antibody and biotinylated anti-mouse antibody. Finally, the slides were incubated with DyLight549-labeled avidin and mounted.

PrG/Ab-mediated Ad vector immobilization and transduction
Stainless steel mesh disks were modified with PABT, PEI-PDT, PrG followed by anti-Ad hexon antibody as delineated in the Vector Immobilization Protocol section, prior to the exposure of the samples to 5x10 10 plaque forming units of AdeGFP. The resulting vector loading on the meshes amounted to 1.2x10 8 VG of AdeGFP, as determined by qPCR. Control meshes were formulated omitting anti-Ad hexon antibody attachment step and were exposed to the same AdeGFP suspension as the properly formulated samples. Following PBS washing, the meshes were individually placed in the wells of 96-well plates with subconfluent cultures rat aortic smooth muscle cells. Transduction was assessed 3 days after mesh placement by fluorescence microscopy and fluorimetry. Functionalization of the metal surface with protein G is essential for the subsequent attachment of AAV2-binding Ab as determined by A) fluorescence microscopy (100x original magnification; FITC filter set) of the stainless steel mesh disk treated (right) or untreated (left) with thiolated protein G prior to the exposure to FITC-labeled mouse anti-rabbit antibody ((#31584 Thermo Fisher Scientific; 10 µg/ml in BSA) and B) fluorimetry (485/538 nm) of an undepleted solution of FITC-labeled mouse anti-rabbit antibody 100 ng/ml in 1% BSA) or the same antibody solution depleted with protein G-functionalized and bare metal foil samples. C) The effectiveness of AAV2 immobilization as determined by Pico Green assay was shown to be