Our group has previously demonstrated stent-based gene delivery with either viral or plasmid vectors. However, these previous studies utilized bulky PLGA or collagen stent coatings, known to cause inflammatory reactions in stented arteries. In the present experiments we successfully attached adenoviruses either directly, or via anti-adenovirus antibodies to the steel surface of stents using chemical coordination with biphosphonates

Alferiev, Ivan S

Connolly, Jeanne M

Fishbein, Ilia

Levy, Robert J

Vohs, John M

Wilensky, Robert L

Wong, Gordon Sek-Yin

English

ScholarlyCommons@Penn

University of PennsylvaniaScholarlyCommonsDepartmental Papers (CBE) Department of Chemical & BiomolecularEngineering5-2003An Ultra-Thin Polymer Coating for the Tetheringof Adenoviral Vector to the Surface of CoronaryStentsIlia FishbeinThe Children's Hospital of PhiladelphiaIvan S. AlferievThe Children's Hospital of PhiladelphiaGordon Sek-Yin WongUniversity of PennsylvaniaJohn M. VohsUniversity of Pennsylvania, vohs@seas.upenn.eduJeanne M. ConnollyThe Children's Hospital of PhiladelphiaSee next page for additional authorsFollow this and additional works at: http://repository.upenn.edu/cbe_papersPart of the Biochemical and Biomolecular Engineering Commons, Materials Science andEngineering Commons, and the Polymer Science CommonsThis paper is posted at ScholarlyCommons. http://repository.upenn.edu/cbe_papers/173For more information, please contact repository@pobox.upenn.edu.Recommended CitationFishbein, I., Alferiev, I. S., Wong, G., Vohs, J. M., Connolly, J. M., Wilensky, R. L., & Levy, R. J. (2003). An Ultra-Thin Polymer Coatingfor the Tethering of Adenoviral Vector to the Surface of Coronary Stents. Molecular Therapy, 7 (5, Supplement), S329-S330.http://dx.doi.org/10.1016/S1525-0016(16)41295-5An Ultra-Thin Polymer Coating for the Tethering of Adenoviral Vector tothe Surface of Coronary StentsAbstractOur group has previously demonstrated stent-based gene delivery with either viral or plasmid vectors.However, these previous studies utilized bulky PLGA or collagen stent coatings, known to cause inflammatoryreactions in stented arteries. In the present experiments we successfully attached adenoviruses either directly,or via anti-adenovirus antibodies to the steel surface of stents using chemical coordination withbiphosphonates.DisciplinesBiochemical and Biomolecular Engineering | Chemical Engineering | Engineering | Materials Science andEngineering | Polymer ScienceAuthor(s)Ilia Fishbein, Ivan S. Alferiev, Gordon Sek-Yin Wong, John M. Vohs, Jeanne M. Connolly, Robert L. Wilensky,and Robert J. LevyThis technical report is available at ScholarlyCommons: http://repository.upenn.edu/cbe_papers/173Molecular Therapy 	 ® !" S329	851. Suppression of Chemical Burn-InducedNeovascularization by Subconjunctival Injectionof Adeno-Associated Virus Vector ExpressingAngiostatinLai Li-Ju,1,2 Li Juan,2 Tsao Yeou-Ping,1 Xiao Xiao.21Department of Ophthalmology, Chang Gang Memorial Hospital,Taipei, Taiwan; 2Department of Molecular Genetics andBiochemistry, University of Pittsburgh, Pittsburgh, PA, UnitedStates.Ocular neovascularization (NV) is one of the leading courses ofblindness. Many ocular surface diseases such as dry eye syndrome,ocular cicatritial phemphigoid (OCP), limbal insufficiency and sequelof chemical burn frequently lead to cornea NV. The underlingmechanisms of these diseases include chronic inflammation,autoimmune response and stem cell loss. This major ocularcomplication can led to corneal scarring, edema, lipid deposition,and inflammation that may not only significantly alter visual acuity,but also worsen the prognosis of the subsequent penetratingkeratoplasty (PKP). Since ocular surface diseases usually haveprotracted courses, the control of their respective detrimentalmechanisms usually depend on long term application of steroid orcytotoxic or immunosuppressive medications such ascyclophosphamide and cyclosporine A. Local and systemic sideeffects create dilemma for clinician. On the other hand, therecombinant AAV vector is currently considered as one of the mostpromising viral vectors for gene therapy. In this study, we investigatethe efficacy of AAV vectors in a rat model of experimental cornealNV generated by silver nitrate (AgNO3) cauterization. The vectorscontained either the mouse angiostatin gene (AAV-angiostatin) orthe control green fluorescent protein gene (AAV-GFP). Two weeksafter subconjunctival injection the vectors, gene transfer efficiencyand therapeutic efficacy were evaluated under slit-lampbiomicroscopy every other day. Vessel length was measured byfluorescent isothiocyanate (FITC)-coupled concanavalin A (ConA)lectin angiography. AAV-angiostatin successfully suppresses thechemical-burn induced corneal NV as shown by Angiographyanalysis. The areas free from blood vessel invasion in the corneacorrelated with the sites of virus injection. The therapeutic effectlasted for the entire 12-week duration of our experiments, whileAAV-GFP expression persisted more than 6 months. This is thefirst report that subconjunctival injection of AAV-angiostatin cansignificantly inhibit the formation of corneal NV lesion. Importantly,subconjunctival injection can be used as a unique reversible genetherapy method. Therefore, the AAV vector represents a promisinggene delivery system for efficient and long-term transduction inocular tissue. Our study demonstrated the feasibility of gene therapyapproaches for the treatment of corneal NV diseases. It may alsobecome clinically useful for a variety of ocular diseases involvingNV, such as wound- and inflammation-related corneal angiogenesiswith limbal insufficiency, which is still pharmacologically untreatable.852. Development of a Retroviral VectorContaining the Erythroid Band 3 PromoterFlanked by the Chicken β-Globin 5’HS4 InsulatorRobert I. Liem,1 Tiffany F. Frazar,1 Nancy E. Seidel,1 Lisa J.Garrett,2 Patrick G. Gallagher,3 David M. Bodine.11Genetics and Molecular Biology Branch, National HumanGenome Research Institute, Bethesda, MD, United States;2Genetic Disease Research Branch, National Human GenomeResearch Institute, Bethesda, MD, United States; 3Department ofPediatrics, Yale University School of Medicine, New Haven, CT,United States.Expression of genes in retroviral vectors is subject to the epigeneticeffects of surrounding heterochromatin after integration, resulting intransgene silencing or position effect variegation (PEV).  It has beenshown that an insulator element from the chicken β-globin locus(5’HS4) serves as a barrier against such position effects and therebypromotes long-term, uniform expression of a transgene but themechanism by which an insulator suppresses PEV has not beenelucidated. The erythroid anion exchanger 1 (AE1 or band 3)promoter is active only in erythroid cells.  Using a transgenic mouseassay, we have analyzed human γ-globin gene expression usingpromoter elements from the erythroid form of the AE1 gene.Transgenic mice containing 2 different AE1 promoter sequences, -1738 to +22 (9 lines) or -357 to +22 (9 lines), fused to the γ-globingene coding region 5 bases upstream of the initiation codon showederythroid specific expression of the transgene with an average AE1/γ-globin mRNA level of 13.5±0.07% and 7.3±0.04% of total mouseα-globin mRNA per transgene copy, respectively.  With bothpromoters, γ-globin expression was position independent and copynumber dependent.  However, γ-globin distribution in circulatingerythrocytes was variegated in 14/18 lines.  We hypothesized thatflanking the 5’ and 3’ ends of the -1738 to +22 AE1/γ-globin geneconstruct with the chicken 5’HS4 would suppress PEV, resulting inmore uniform expression of the transgene.  In transgenic micecontaining the insulated, long AE1/γ-globin construct, expression ofthe transgene was erythrocyte specific, position independent (6/6lines express) and copy number dependent (p = 0.0001).  Transgenicmice had an average AE1/γ-globin mRNA level of 19.8±0.03% oftotal mouse α-globin mRNA per transgene copy.  AE1/γ-globinmRNA from the non-insulated construct initiated at cryptic sitesupstream of the start site used by the endogenous gene.  In contrast,the insulated AE1/γ-globin construct initiated transcription at thestart site used by the endogenous gene.  Distribution of γ-globin inall 6 lines was also uniform, with 100% of erythrocytes containinghuman γ-globin.  We conclude that the erythroid AE1 promotercontains an insulator element upstream of position -1738.Furthermore, the chicken 5’HS4 insulator suppresses PEV bydirecting transcription initiation to the correct start site.  We haveconstructed an oncoretroviral vector containing the -357 to +22AE1/γ-globin gene with the chicken 5’HS4 insulator in the 3’LTR.Stable transduction of the AE1/γ-globin gene and transfer of 5’HS4to the 5’LTR was demonstrated by Southern blot analysis oftransduced K562 cell DNA.  We hypothesize that an insulatedAE1/γ-globin vector will express γ-globin at relatively high levels inprimary erythroid cells.	853. An Ultra-Thin Polymer Coating for theTethering of Adenoviral Vector to the Surface ofCoronary StentsIlia Fishbein,1 Ivan S. Alferiev,1 Gordon S. Wong,2 John M. Vohs,2Jeanne M. Connolly,1 Robert L. Wilensky,3 Robert J. Levy.11Cardiology, The Children’s Hospital of Philadelphia,Philadelphia, PA; 2Chemical Engineering, University ofPennsylvania, Philadelphia, PA; 3Cardiology, University ofPennsylvania Hospital, Philadelphia, PA.Our group has previously demonstrated stent-based gene deliverywith either viral or plasmid vectors. However, these previous studiesutilized bulky PLGA or collagen stent coatings, known to causeinflammatory reactions in stented arteries. In the present experimentswe successfully attached adenoviruses either directly, or via anti-adenovirus antibodies to the steel surface of stents using chemicalcoordination with bisphosphonates. Bisphosphonate derivativesof polyallylamine possessing primary amines (PAA-BP), or bothprimary amines and latent thiols (PrSSPAA-BP) were customsynthesized. The dynamics of PrSSPAA-BP adsorption to the Molecular Therapy 	 ® !" S330	surface of 316L stainless steel was studied using a compoundcomprised of a fluorescent marker, dansyl, attached to the polymerbackbone through a cleavable bond. Fluorometric studies showedfast (less than 1 hour) saturation of the steel surface with PrSSPAA-BP. Estimation of the bound polymer amount (10-12 M/cm²)corresponds to monolayer deposition of the polymer. Additionally,XPS analysis confirmed the presence of the polymer and showedthat the thickness of polymer coating does not exceed 5 nm. Reactionbetween primary amines in the polymer coating and the bifunctionalcross-linker, SPDP, was employed to introduce thiol-reactive,pyridyldithio (PDT) groups on the stent surface. A panel of reducedanti-adenoviral antibodies (anti-hexon, anti-fiber and anti-knob) wasused for the virus-tethering purposes. Monoclonal anti-knobantibody (AkAb) was found to be the most effective in terms of theamount of tethered AdV (108-109/cm²). The attachment of AdV toAkAb-primed surface was profoundly inhibited (87%) by a 1000-fold excess of knob protein. Alternatively, vector immobilizationwas achieved by SPDP modification of AdV with subsequentcovalent tethering to deprotected thiols in the PrSSPAA-BP coating.Disulfide bridges emerging during the latter immobilization schememay be cleaved by endogenous reducing compounds, regeneratingviable AdV particles. However, the direct tethering procedure is farless effective than Ab-mediated immobilization both in terms ofbound vector amount, and overall transduction success. Stainlesssteel meshes with tethered AdV-GFP placed in confluent culturesof A10 cells have driven 90% transduction of cells within 50 μm ofthe mesh borders, while the same amount of free virus (MOI=1)does not result in measurable cell transduction. Moreover, meshesrecovered from the A10 culture and transferred to new culturesretained their transducing potential up to a fourth round of relocation.Stents bearing AkAb-tethered AdV-GFP were deployed in rat carotidarteries (n=8). The extent of transgene expression in the stentedarteries was determined by immunohistochemistry 1, 3 and 7 daysafter deployment. GFP-positive cells were abundant in arterial mediaand adventitia at 7 days. In conclusion, we studied new strategiesfor AdV immobilization on the stent surface, which allow site-specific tethering of a clinically significant amount of the recombinantAdV. In vitro and in vivo studies demonstrated high transductionefficacy and superb localization of the reporter transgene expression.854. A Novel Adenovirus Vector ProducingMultiple Isoforms of VEGF Reverses Hind LimbIschemia in the Mouse at Lower Doses ThanEquivalent Vectors Expressing Only a SingleVEGF IsoformPaul R. Whitlock,1 Neil R. Hackett,1 Philip L. Leopold,1 Todd K.Rosengart,2 Ronald G. Crystal.11Weill Medical College of Cornell University, New York, NY;2Evanston Northwestern Healthcare, Evanston, IL.Alternate splicing is a process that permits the expression ofmultiple protein isoforms with overlapping biological propertiesfrom a single gene. As an example of an alternately spliced gene wehave used human vascular endothelial growth factor (VEGF) whosepre-mRNA is spliced to give seven isoforms of which three,VEGF121, VEGF165 and VEGF189, represent >99% of the VEGFexpressed. Physiological angiogenesis occurs in the context of multipleisoforms determined by alternate splicing, resulting in VEGF proteinswith overlapping properties. By contrast, as presently predictedtherapeutic angiogenesis uses only one of these isoforms. Thehypothesis of this work is that angiogenic gene therapy with thegenomic form of VEGF expressing the three major isoforms, couldbe more potent than a vector expressing a single isoform. Anadenovirus vector (AdVEGF-All) expressing a cDNA/genomichybrid gene capable of expressing multiple human VEGF isoformswas constructed and shown to express VEGF121, VEGF165 andVEGF189 in a 2:2:1 ratio. The AdVEGF-All vector was given byintramuscular injection in a model of mouse hind limb ischemia,involving excision of a 0.6 cm portion of the iliac artery. Assessmentof blood flow recovery was then monitored up to 21 days using alaser Doppler scanner. At 3 wk, tissues were harvested and stainedfor vonWillebrand factor. Initial post-operative scans showed bloodflow in the ischemic limb to be 9 ± 1% of that in the non ischemiclimb. At a dose of 108 particle units, flow recovered to 92 ± 9% inAdVEGF-All treated animals by day 21, compared with recoveryto 95 ±8% in AdVEGF121 treated animals and 50 ± 4% in AdNullcontrols (p>0.5 comparing AdVEGF-All to AdVEGF121; p < 0.05comparing AdVEGF-All and AdVEGF121 to AdNull). For groupstreated with 108 particle units the ratio of vessel numbers assessedby histology were 1.42 ± 0.21 for VEGF-All, 1.39 ±0.22 forVEGF121, and 0.67 ±0.17 for AdNull. Strikingly, however, at adose of 105 particle units, the recovery was 79 ± 8% in AdVEGF-All treated animals by day 21, compared to only 56 ± 3% inAdVEGF121 treated animals and 47 ± 3% in AdNull controls(p<0.05 comparing AdVEGF-All to AdVEGF121 and AdNull).Interestingly, a mixture of vectors coding individually for VEGF121, 165, and 189 made up to a total dose of 105 particle units in aratio of 2:2:1, mediated recovery to 87 ±7% (p>0.5 comparingAdVEGF-All to mixture). Histological analysis showed that theratio of vessel numbers in the ischemic compared to the control limbin animals treated at a dose of 105 particle units was 0.61 ± 0.01 forAdNull, 0.63 ±0.21 for AdVEGF121, and 1.43 ± 0.39 for AdVEGF-All. We conclude that a mixture of VEGF isoforms provides a morepotent angiogenic response than a single isoform, suggesting thatthe individual isoforms function synergistically, an observation withimportant implications for gene and recombinant protein therapy.Dr. Crystal has equity in, is a consultant to, and receives sponsoredresearch funds from, GenVec, Inc., Gaithersburg, Maryland, apublicly-traded biotechnology company.855. A Genetically Engineered PlasmidEncoding a Zinc Finger VEGF-ActivatingTranscription Factor Induces Angiogenesis in theRabbits with Hind-Limb IschemiaQunsheng Dai,1 Jianhua Huang,1 Bruce Klitzman,2 Joe Rokovich,3Ed Rebar,4 Brian Johnstone,4 Christopher Kontos,1 Brian Annex.11Department of Medicine, Division of Cardiology, DukeUniversity and Durham Veterans Affairs Medical Center,Durham, NC, United States; 2Division of Plastic-Surgery, DukeUniversity Medical Center, Durham, NC, United States; 3EdwardsLifesciences LLC, Irvine, CA, United States; 4SangamoBioSciences, Inc., Richmond, CA, United States.BACKGROUND: Peripheral arterial disease (PAD) afflictshundreds of thousands of patients and is major cause of morbidityand mortality. Therapeutic angiogenesis seeks to treat PAD via thegrowth and proliferation of new blood vessels. Unfortunately thisapproach has met little success when single agents have beenemployed. The purpose of this study was to investigate the role ofintra-muscular injection of plasmid-DNA encoding zinc finger-basedvascular endothelial growth factor (VEGF)-activating transcriptionfactors (ZFP-VEGF) that will enhance multiple VEGF isoforms inTA muscle following surgically induced hind-limb ischemia.METHODS: In total, 23 New Zealand White rabbits underwentfemoral artery ligation and excision and were divided into two groupsat 10 days post-operatively. One group received a single unilateral(ischemic limb TA muscle) intra-muscular injection of ZFP-VEGF(500mg) (n=13) and the other (control) group (n=10), included b-galactosidase injection (500mg) (n=7) and sham treatment (n=3).The ischemic and contra-lateral TA muscles of all animals wereharvested 21 days post-operatively. In a subgroup of these rabbits,blood flow in ischemic and non-ischemic limbs (contra-lateral limb)

An Ultra-Thin Polymer Coating for the Tethering of Adenoviral Vector to the Surface of Coronary Stents

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An Ultra-Thin Polymer Coating for the Tethering of Adenoviral Vector to the Surface of Coronary Stents

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