Stent-based delivery of AAV2 vectors encoding oxidation-resistant apoA1

Figure 1. ApoA1 production following AAV2-apoA1(WT) and AAV2-apoA1(4WF) transduction assayed by ELISA and immunofluorescence. (A) Human embryonic kidney cells (HEK-293), rat smooth muscle cells (SMC), rat blood outgrowth endothelial cells (BOEC), and murine macrophages (Raw 264.7) were transduced with AAV2-apoA1(WT) and AAV2-apoA1(4WF). ApoA1 production was determined by ELISA of the media conditioned by transduced cells 3 days post-transduction. (B) Rat endothelial cells (a-c) and rat smooth muscle cells (d-f) were transduced with either AAV2-apoA1(WT) (a and d) or AAV2-apoA1(4WF) (b, e and f) for 3 days, methanol-fixed and reacted with anti-apoA1 antibody except (c), followed by either Cy 3.5- (a, b and c) or AlexaFluor488-labeled (d, e and f) secondary antibodies. Original magnification is 100×.

Figure 2. ApoA1(4WF)-, but not apoA1(WT)-mediated efflux following transduction with the respective AAV2 vectors demonstrates oxidative resistance. Cell culture media conditioned by HEK 293 cells transduced with AAV2-apoA1(WT), AAV2-apoA1(4WF), both containing 10 µg/ml of AAV2-encoded apoA1 variants, and the conditioned media from the non-transduced HEK-293 cells supplemented with 10 µg/ml of recombinant human apoA1 (rhapoA1) were incubated with increasing concentrations of HOCl. Cholesterol efflux assays were then performed on 3H-cholesterol-loaded Raw 264.7 cells incubated with differently oxidized conditioned media from HEK-293 cells for 4 h. *P < 0.05 4WF vs WT. ✞P < 0.05 4WF vs rhapoA1.

Figure 3. Impact of AAV2-apoA1(WT) and AAV2-apoA1(4WF) transduction on the proliferation and migration of SMC and BOEC, reactive oxygen species (ROS) production, and anti-inflammatory responses in endothelial cells. WST-8 assay was used to evaluate the effect of apoA1(WT) and apoA1(4WF) overexpression on proliferation of (A) TNFα (20 ng/ml)-stimulated rat aortic SMC and (B) non-stimulated rat BOEC. The results (both A and B) were normalized by WST-8 assay values produced in confluent cultures of rat SMC (A) and rat BOEC (B) and expressed as the percent of a monolayer confluency. A monolayer scratch injury assay was used to assess migratory capacity of apoA1(WT)- and apoA1(4WF)-transduced rat SMC (C) and rat BOEC (D) compared with non-transduced counterparts. A closure of the gap inflicted by pipette tip was quantified 24 h after the scratch injury. (E) CM-H2DCFDA assay in non-transduced, apoA1(WT)- and apoA1(4WF)-transduced RAEC with/without TNFα stimulation. (F) Attachment of PKH26-labeled rat monocytes to TNFα-stimulated RAEC.

Figure 4. Polybisphosphonate/protein G/antibody-based affinity immobilization of AAV2 particles on stainless steel meshes and cobalt/chromium stents with subsequent vector release and cell transduction. (A) Scanning electron microscopy images of AAV2 particles immobilized on the surface of a stainless steel mesh disk (left panel) vs control bare metal disk (right panel). (B) Underlying HEK-293 cells were transduced by placement of the mesh disk formulated with AAV2-eGFP (left panel) compared to non-transduced cells treated with the control bare metal mesh disk (right panel). (C) Immobilized AAV2 particles were disengaged from the not deployed stents, from the stents deployed in the pig coronary arteries and extracted from the arterial tissue underlying deployed stents. The viral DNA was isolated using a QIAamp DNA minikit and amplified in RT-PCR reaction with AAV2-specific primers. The number of AAV2 genomic copies was quantified using a calibration curve constructed with a known amount of AAV2 genomes.

Figure 5. eGFP expression in non-diseased pig arteries treated with AAV2-eGFP stents. Western blot of protein extracts from coronary arteries receiving AAV2-eGFP stents (4 animals, 8 stents). The animals were euthanized 7 days after stent deployment. The extracted proteins from the stented segments were resolved by PAGE, transferred to a nitrocellulose membrane, and probed with anti-eGFP and anti-β-tubulin (loading control) antibodies.

Figure 6. Long-term expression of the therapeutic transgene, the anti-AAV2 neutralizing antibodies dynamics, and the morphometric results in hypercholesterolemic diabetic pigs. (A) The percent of retained AAV2eGFP transduction capacity after incubation with 1:20 diluted serum from experimental pigs at different time points throughout the study. NAB positivity was denoted at 50% reduction of transducibility of sera-incubated compared to the not incubated AAV2 vectors. (B) Representative images of Verhoeff-van Gieson – stained sections of a normal pig coronary artery and pig coronary arteries treated with BMS, AAV2-eGFP- and AAV2-apoA1(4WF)-eluting stents. (C) Morphometric measurements of the stented segments using 3 different calculation methods (percent of luminal stenosis, neointimal thickness, and the ratio of the neointimal area to the medial area).