120 Proper Timing of VEGF Expression Is Required for the Formation of Stable and Mature Blood Vessels disease However, SVGs have limited durability Furthermore, there are no effective treatments to pr[.]
disease However, SVGs have limited durability Furthermore, there are no effective treatments to prevent SVG failure Gene transfer to SVGs at the time of implantation has been introduced as a method to modify the degenerative process and prevent vein graft failure The most common viral vector employed has been recombinant adenovirus and therefore, the duration of experimental transgene expression has been relatively short compared to the anticipated clinical life of the grafts It is unelear whether early and transient transgene expression will correlate with late freedom from graft failure In contrast to adenoviral vectors, adeno-associated viral (AAV) vectors have been shown to provide stable long-term transgene expression in vivo and are more clinically applicable since they induce less inflammatory response and are not associated with human pathogenic conditions Very little is known regarding transduction of AAV vectors in veins of higher mammals, ineluding humans Successful treatment ofSVGs in patients with AAV vectors requires a more comprehensive understanding ofthe transduction process I-Iere we define the optimal conditions for AAV mediated transduction of canine and human saphenous vein utilizing an in vitro model Human saphenous vein segments were acquired after coronary bypass surgery Canine saphenous vein specimens were obtained from Class A and Class B mongrels after euthanasia These specimens were treated intraluminally with a suspension of either Adenoviral or AAV luciferase vectors Parameters which were tested included: physical (dose, duration of exposure, temperature, distension pressure, oncotic pressure); chemical (proteosome inhibitors, heparin , pluorinic gel); and genetic (capsid optimization) Veinsegments were maintained in culture for days, after which they were assayed for viability and luciferase activity Interestingly, of the AAV caps ids examined, a genetically modified AAV3b variant (termed SASTG) yielded substantially greater luciferase activity than eitherAAV I or AAV2; expression levels approached, in some p instances, that ofthe adenoviral luciferase vector Luciferase activity was dose dependent with Ix I0 12 tvp demonstrating highest transduction efficiency The transduction efficiency ofAAV-SASTG luciferase was not affected by oncotic pressure, however, it was increased at higher luminal distention pressures This study demonstrates that AAV is an effective vector for transgene delivery to canine and human saphenous vein grafts Moreover, the AAV-SASTG capsid modification provides superior transduction efficiency in this model as compared to AAVI and AAV2 SASTG has transduction efficiency that in some instances approaches adenovirus This combined with preliminary data that show SASTG 's improved function in the setting ofheparin, makes SASTG the ideal capsid type for gene therapy for coronary artery bypass grafting Preliminary studies arc currently underway examining this improved transduction efficiency in a canine vein graft model These data will also be reported 119 NOS2 Gene Transfer with a Synthetic Vector Inhibits Neointimal Hyperplasia in a Rabbit Vein Graft Model Qing-Hai Mcng,' Scott A Irvine; Jean R Mcliwan ,' Stephen L Hart.' I Molecular Immunology Unit, UCL Institute ofChild Health, London, United Kingdom; 'Cenne for Cardiovascular Medicine and Biology, University College London, London, United Kingdom Vein graft failure is a main subject of vascular investigations Efforts have been focused on preventing neointimal hyperplasia which is the major cause of long term failure Proliferation and migration of vascular smooth muscle cells and adventitial fibroblasts contribute significantly to neointima formation and have become the target of gene therapy However, the lack of safe and efficient vectors has significantly hindered the progress in this field We have developed a synthetic vector system termed a Receptor-Targeted Molecular Therapy Volume15 Supplement I ~ br 2007 Copyright © T he American Soci ety ot Gen e Therapy Nanocomplex (RTN) which comprises a cationic lipid, a cationic targeting peptide and plasmid DNA, to elicit synergistic effects on gene delivery The RTN system has demonstrated efficient and consistent gene transfer in many cell types and animal models In this study, we aim to transfer therapeutic genes, TIMP-I (tissue inhibitor of metalloprotcinasc-l ) and iNOS (inducible nitric oxide synthase), to the vessel wall in a rabbit vein graft model to evaluate the efficacy ofthe RTN vector in vascular gene therapy Rabbit vein graft surgery with cuffed anastomosis was performed to position a fragment of jugular vein to carotid artery Ex vivo gene delivery was carried out, adventitially with ends of vein graft clipped, by immersing vein graft for 20 minutes in a saline solution of the RTN vector Efficiency of gene delivery was firstly evaluated with a reporter gene, GFP, in cultured rabbit aorta rings Incubation of the rings, for periods as short as 15 minutes , with RTN formulations could elicit efficient transfection with GFP, with peak expression observed on day-5 Delivery of~-Galactosidase in vivo in the vein graft model led to efficient transfection throughout the vein wall on day Another 29 New Zealand white rabbits (3.0-3.5kg) were divided into groups of vein graft surgery and gene transfer with RTNs carrying different genes: I) TIMP-I group (n=7); 2) iNOS group (n=7); 3) pCI control group (n=8): vehicle plasmid containing no functional gene; 4) surgery control group (n=7): no RTN vector added in the saline solution Vein graft samples were collected on day-28 and morphometry analysis was performed on paraffin sections with Elastin-VG staining Significant inhibition ofneointima formation was observed in iNOS group compared to control groups in terms of both neointimal area (60% reduction compared to surgery group and 77% reduction to pCI group) and thickness (41% reduction compared to surgery group and 46% reduction to pCI group , standardized by the ratio of neointima to medial layer) However, TIMP-I gene transfer showed no Inhibitory effect on neointimal hyperplasia In addition, plasmid DNA was detected by peR in vein graft samples but not in organ samples (lung, liver, kidney, and testis) from iNOS transfected rabbits The results of this study demonstrate the efficacy of RTN vectors in vascular gene therapy, which suggests a novel selection of a safe and efficient vector to deliver genes to vascular system In addition , we have confirmed the therapeutic effects of iNOS, but not TIMP-I , gene transfer on neointimal hyperplasia in vein graft disease 120 Proper Timing of VEGF Expression Is Required for the Formation of Stable and Mature Blood Vessels Sabrina Tafuro, I Eduard Ayuso,' Serena Zacchigna, I Lorena Zentilin,' Franca Dore,' Mauro Giacca.' 'Molecular Medicine Laboratory; International Centre for Genetic Engineering and Biotechnolgoy (ICGEB), Trieste, Italy; /Centre ofAnimal Biotechnology and Gene Therapy (CBATEG), Universitat Autonoma de Barcelona, Barce/ona, Spain; 'Struuura Complessa di Medicina Nucleare, Az Ospedaliero-Universitaria Ospedali Riuniti, Trieste, Italy AAV vectors represent an outstanding tool for pro-angiogenic gene transfer, given the specific tropism ofthese vectors for skeletal and cardiac muscle cells We have previously observed that the injcction ofAAV-VEGF in the mouse skeletal musele induced marked nco-vascularization, persisting for several months To establish the kinetics of the angiogenic response and the duration of the VEGF stimulus required to promote functional vessel formation, we now constructed novel AAV vectors in which the expression ofVEGF165 was regulated by an inducible Tet-On system When the vectors were injected in the mice tibialis anterior muscle , the expression of the VEGF mRNA was found to be strictly dependent upon the administration ofdoxyclycline in the drinking water To our surprise, we discovered that VEGP expression for a period of 15 days led to S47 the formation ofan unstable vasculature, which regressed when gene expression was turned off In contrast, the continuous expression ofthe factor for at least 30 days was required for the formation for stable vessels, that persisted upon withdrawal of the stimulus To determine the functional capability of the newly formed vessels, we performed static scintigraphy using a gamma camera equipped with pinhole collimator (Siemens Ecam), after 30 days ofVEGF expression and after withdrawal ofdoxycycline for 15 days At both time points functional images of the mice legs were acquired by the injection of 3.7 mBq of99mTe Tetrofosmin in basal condition and of37 MBq after IO of hind-limb muscle contraction, to mimic physiological exercise We discovered that the vasculature formed by continuous VEGF expression not only did not increase basal blood flow, but it even decreased perfusion after muscle exercise This remarkable finding was probably related to a pathological increase in the leakiness of the newly formed vasculature and to the formation of artero -venous shunts that exclude perfusion of the microvasculature In contrast, the vessels persisting after cessation ofthe VEGF stimulus were found to maintain functional perfusion after increased physiological blood flow demand When analyzed histologically by the injection of'fluorescent lectins, the vasculature formed after continuous VEGF expression consisted ofcapillaries or increased diameter and abnormal shape, which showed remarkably altered permeability, Together, these results clearly indicate that functional blood vessel formation requires an appropriate duration ofthe VEGF stimulus, casting doubts on the potential ofVEGF gene therapy trials that exploit systems with short-term or unregulated expression of this therapeutic gene 121 A Pressured-Freeze Method To Make Multi-Functional Echogenic Liposomes for Image-Guided and Ultrasound-Controlled Oligonucleotide Delivery feet ofNF-kappa decoy on the inhibition ofVSMC proliferation by 30% This drug delivery system possesses unique properties for cell targeting, ultrasound detection, drug encapsulation, triggered drug release and ultrasound-enhanced uptake.The method present here also applicable for siRNA delivery UlJlnound enhllrtced the InhIlltlon eITeet01NF kapp B decoy on lhe proflteraUon of vascular smoot h muscle ceUs Mean ~ SO.n=3 1m , - - - - - - - - - - - - - - - - - 'ii ~ • E • II) Iu i'E + • g :> E" i 2~ ~~ ",.c: "'".c: :"b c~ u tEa ++ O~ C IIJ "~p; o~ +++ Shaoling Huang,' Robert C Macbonald ,' David D Mcl'herson.' 122 AAV2 Pseudotyped with AAV9 Capsid Is an Efficient Vector for Vascular Gene Delivery Ilia Fishbein,' Meizan Lai.' Robert J Levy.' NF-kappaB decoy is a novel drug for atheroma treatment, and mechanism to enhance local delivery may have important clinical implications Echogcnie liposomes that co-encapsulate gas and NF-kappaB decoy will allow directed ultrasound image-guided delivery and controlled release Liposomes containing air were prepared by conventional procedures of hydrating the lipid film, sonication, freezing and thawing A single, but critical, modification of this method involved pressurization of lipid dispersion with air after sonication to encapsulate air into Iiposomes After equilibration, the sample was frozen The pressure is reduced to atmospheric and the suspension thawed This method allows the echogenic liposome to be made anionic or cationic For anionic liposomes, NF-kappaB decoy was encapsulated in hydration step For cationic liposomes, NF-kappaB decoy was added after freezethawing The echogenicity ofdrug-loaded Iiposomes was measured by a 20 MHz intravascular ultrasound catheter Ultrasound-triggered release was achieved by applying I MHz ultrasound at W/cm2 for lOs This Iiposomal preparation results excellent air content (30 III per mg Iiposomes), high ultrasound refclectivity and sufficient NF-kappaB decoy loading efficiency When added into anionic liposomes, 15+1-3% of NF-kappaB decoy was encapsulated in lipsomes A single ultrasound application triggered 40% of release, When added into cationic echogenic liposomes, 90% of NF-kappaB decoy can be associated with liposomes Cationic eehogenic liposomes containing air and NF-kappaB decoy were incubated with cultured vascular smooth muscle eells(VSMC) to study their effects on cell proliferation Ultrasound enhanced the uptake of NF-kappaB decoy into VSMC and thus enhanced the biologic cf- Background and hypothesis Effective gene therapy of vascular disorders remains an elusive target The main reasons contributing to the relative inefficacy of vascular gene therapy are poor transfectivity/transducibility of cell types in the arterial wall, and the short duration oftransgene expression Adena-associated virus ofserotype pscudotyped with serotype capsid (AAV2/9) has been previously demonstrated to achieve higher transduction of heart and skeletal muscle than the parent AAV2 vector However, no study to date has investigated the capability ofAAV2/9 to transduce vasculature Thus, we hypothesized that the hybrid AAV2/9 serotype may possess significant transduction potency toward arterial tissue in vivo Herein we report on an extremely robust reporter gene transduction in rat carotid arteries achieved by local delivery of AAV2/9 Methods Subconfluent monolaycrs of rat aortic smooth muscle (AW) and bovine aortic endothelial (BAEC) cells were transduced with AAV2(cmv)GFP, AAV2/9( cmv )GFP and Ad5( emv)GFP at GCIccI! and particle/cell ratio of Ix I O~ The resulting transgene expression was determined by live cell fluorometry To investigate efficacy of respective viral vectors in vivo, 50 III volumes or the suspensions comprising 5xl0 1U AAV2/9 GC or 5xl0 1U physical particles of AdS (both encoding firefly luciferase under control ofthe CMV promoter) were intravascularly delivered for to isolated endothelium-denuded segments ofrat common carotid arteries The ensuing arterial reporter expression was studied by serial intravital bioluminescence imaging Results In A I0 cells experiments, the peak GFP expression achieved with both AAV serotypes at days post-transduction was 18-fold (AAV2/9) and IS-fold (AAV2) lower, than the exp ression at 48 hours following Add-mediated transduction In BAEC cultures AAV2 transduction resulted in 56-fold lower OFP expression levels , 'Internal Medicine, Division ofCardiology, UniversityofTexas Health Science Center: Houston, TX; ]BiochemistIJ\ Molecular Biology and Cell Biology Northwestern University, Evanston, IL S48 'Pediatric Cardiology, The Childrens Hospital ofPhiladelphia Philadelphia, PA Molecular Therapy Volume 15 Supp lement Iã \ br 2007 Copyright â "111(: Amcricm Society o f Gen e Therap y .. .the formation ofan unstable vasculature, which regressed when gene expression was turned off In contrast, the continuous expression ofthe factor for at least 30 days was required for the formation. .. required for the formation for stable vessels, that persisted upon withdrawal of the stimulus To determine the functional capability of the newly formed vessels, we performed static scintigraphy... 30 days ofVEGF expression and after withdrawal ofdoxycycline for 15 days At both time points functional images of the mice legs were acquired by the injection of 3.7 mBq of9 9mTe Tetrofosmin