16 Modification of Ad5 Hexon Hypervariable Regions Circumvents Pre Existing Ad5 Neutralizing Antibodies vector having all viralgenes deleted resulted in improved long term expression Therefore, it is[.]
vector having all viral genes deleted resulted in improved long-term expression Therefore, it is still possible that, besides E2a and E4 genes, there may exist another viral gene, which is expressed under the lack ofEIA gene expression, causes cellular immune response and hampers long-term expression of transgene So, we searched another viral gene which is expressed without E IA gene expression, by usingFGAdV harboringa transgeneundercontrolof the modified chickenp-actinpromoter(CAG promoter).Wefoundthat the protein IX (piX) gene, encoding a minor component of viral capsid of 14 kD, was co-expressed together with the transgene The induction ofpiX gene expression was dependent on the sorts ofthe promoter used: the CMV promoter also induced the pIX expression but the EFla promoter did not In the wild-type adenovirus genome, pIX gene is located within the EIB region, sharing poly (A) signal with the other EI B genes whereas having its own promoter In all of the FG AdV to date, pIX gene retains its own promoter and located immediately right to the inserted expression unit of the transgene So we suppose that inserted heterologous promoters may activate the pIX native promoter by their enhancer effect.Next we examined the in vivo effect of heterologous promoters in C57BLl6 mice by i.v injection AdV bearing CAG promoter caused ALT elevation significantly as expected, but AdV bearing EFla promoter or no promoter/transgene gave no detectable elevation Furthermore a high-level transgene expression of AdV expressing human growth hormone under the EFIa promoter was observed for at least six months These results suggest that pIX co-expression induced by heterologous promoters may be at least one of thc cause of AdVinduced host immune response and that EFla promoter is valuable for long-term expression of first-generationAdV 15 Engineering a Recombinant Adenovirus for Targeted Transduction of Neutrophils for Use as Anti-Inflammatory Cell Vehicles Justin C Roth,1 Michael O Alberti,' Larisa Pereboeva,' Svetlana Kornarova, I Stanton L Gerson.' David T Curiel.I I Division ofHuman Gene Therapy Departments a/Medicine Pathology, and Surgery; and Gene Therapy Center: University of Alabama, Birmingham, Al ; lDivision 0/ Hematology/Oncology and Comprehensive Cancer Center; Case Western Reserve University and University Hospitals a/Cleveland, Cleveland, Oil Inflammation is a hallmark of many diseases, including arthritis, asthma, atheroselerosis, and cancer Neutrophils home to and infiltrate tissues in response to inflammatory signals Thus, we sought to utilize ncutrophils as vehicles for delivering therapeutic payloads to sites of inflammation.Adenovirus vectors have bccn shown to be efficient ex vivo gene transfer agents to a variety of cell types However, neutrophils not express the coxsackievirus adenovirus receptor (CAR) and arc therefore resistant to transduction with Adenovirus (Ad) vectors We have previously demonstrated the ability to retarget Ad vectors to specific cell types using genetic methods Therefore, we set out to genetically modify the adenovirus fiber to express a neutrophil-specific binding peptide in place of the fiber knob We hypothesized that the resulting Ad vectors would have enhanced neutrophil transduction efficiency, Further, ablation of the fiber knob would reduce CAR-mediated transduction of non-target cells A heptapeptide phage library was panned against murine bone marrow leukocytes in vitro and in vivo, and phage clones were eluted from distinct FACS-isolatedpopulations Several consensus sequences were identified after repeated rounds of panning The phage clones were further characterized by flow cytornctry; biotinylatcd clone preps were added to whole bone marrow labeled with lineage-specificantibodies, and bound phage was detected with an avidin-APC conjugate A neutrophil binding consensus sequence was identified using both panning strategies This peptide sequence was then synthesized and biotinylated for Molecular Therapy Volume 15.Supplement I ~I.t Coprriglll © The American Soci ety of Gene Therapy r 2007 further characterizationusing flowcytometry,The synthetic peptide bound to Gr-I +/CD 11 b+/B220'/CD4'/TerlI9' leukocytes, further demonstrating specificity for neutrophils This peptide sequence was then genetically incorporated into a recombinant Ad5 fiber that lacks knob, and contains a fibritin-foldon element to maintain fiber trimerization Neutrophil binding specificity was maintained in the context of the recombinant fiber, Viral genomcs containing the recombinant fiberwere then rescued in the fiber-complementing cell line, 293F28 The resulting virions were mosaic and contained equivalent levelsofboth wild type and recombinant fiber, indicating that the recombinant fiber is stable and incorporated into virions Our results demonstrate that a neutrophil-specific peptide can be functionally incorporated into an Ad fiber protein and displayed on virions These studies will determine the enhanced transduction efficiency ofneutrophils ex vivo and will establish the feasibility of an exciting new approach to utilize these cells as vehicles for the treatment of inflammatorydisease 16 Modification of Ad5 Hexon Hypervariable Regions Circumvents Pre-Existing Ad5 Neutralizing Antibodies Joseph T Bruder,' Elena Semenova,' Charlie Thomas,' Noelle B Patrcrson.! Maureen E Stefaniak! Keith Limbach.i C R King, I Denise L Doolan.' 'Research, Genl'ec, Inc., Gaithersburg, MD; 11'vIalaria Program, Naval Medical Research Center; Silver Spring, I'.-ID Adenovirus type (Ad5) based vectors are capable of generating robust and protective T cell and antibody responses in animal models and arc currently being evaluated in clinical trials for HIV and malaria However, the high prevalence ofncutralizing antibodies to AdS in human populationshas the potential to limit the effectiveness of AdS-based vaccines The majority of serotype-specific neutralizing antibodies recognize determinants on the hexon and fiber capsid proteins, and hexon-specific neutralizing antibodies appear to be the most prevalent and potent in vivo Epitopes targeted by these hexon-specific neutralizing antibodies have been mapped to the hypervariablc domains of the hexon protein, contained within exposed loops at the surface of the capsid Our objective was to develop a modified adenovector vaccine platform designed to circumvent the perceived problem of pre-existing anti-adenovirus immunity prevalent in human populations Accordingly, we have deletedthe hypervariableloopsfromthe hexonproteinwithinanAd5 adenovector vaccine and replaced them with the loops derived from selected serotypes with low prevalence in humans Wesuccessfully substituted the Ad5 FGI hypcrvariablc loop with loops from Ad2 (group C), Ad34 (group B) and Ad43 (group D) adenoviruses, We also attempted to substitute the large DEI loop in its entirety with loops fromAd2, Ad34 andAd43 but, ofthese substitutions,only the Ad2 hypervariable loop swap was capable of beingconverted into a virus vector.The Ad5 vector carrying the Ad2 hypcrvariablc loops (Ad5,l-12) grew to high titers, expressed antigen at levels similar to Ad5 vectors, and was not neutralizedbyAdS hexon-specific neutralizing antibodies Immunogenicity evaluation of these vectors for T cell and antibody responses to PyCSP in the presence or absence of preexisting AdS and Ad2 neutralizing antibodies is ongoing and results of this analysis will be presented Since our firstattempts to produce adenovectors carrying the DEI loop derived fromAd43 or Ad34 did not result in viable virus, we focused on precisely swapping the small hypervariable loops within the DEI loop of Ad5 hexon with sequences from these non-prevalent serotypes Using this strategy, we were successful at generatingAd5 vectors carrying hypervariable loops from rare serotypes We will present data on the evaluation of these new vectors for avoidance of pre-existing antibodies in vitro and in the mouse model for malaria In addition, by makingchimerasbetweendifferentversionsof AdS vectorscarryS7 ing the complete or smaller substitutionsin the hypervariableloops we intend to map the residues that are required for the structural integrityof the capsid as well as define the epitopes for adenovirus neutralizingantibodies PHYSICAL METHODS OF DELIVERY 17 GnRH-Expressing Plasmid Delivered by Constant-Current Electroporation Can Regulate the Reproductive Axis in Horses William A Storer,I Donald F Thompson, Jr,2 Kenneth Bondioli.l Amir S Khan,' Patricia A Brown.' Ruxandra Draghia-Akli.' 'Agricultural Sciences, McNeese State University, Lake Charles , LA; 2SclI001 0/Animal Sciences, LAES, LSU Agricultural Center, Baton Rouge, LA; JVGX Pharmaceuticals Immune Therapeutics Division, The Woodlands TX Gonadotropin releasing hormone (GnRH) therapy has shown promiseas a treatmentfor reproductive dysfunction Chronic, pulsatile administration ofGnRH increases LHand testosterone secretion Pulsatile therapy using the peptide hormone is impractical due to the long-term and labor intensive nature of the treatment A novel plasmid-mediated delivery system for GnRH (pGnRH)expression was evaluated using constant-current electroporation (CCE) Ten reproductively sound stallions ranging in age from to 25 yr were used in this study On d 0, stallions in the firstgroup received mg of a pGnRH (n = 3); the second group received mg of pGnRH (n = 3); and the third group received mg of a control plasmid expressing secreted embryonic alkaline phosphatase, pSEAP (n = 4) All plasmids were delivered by i.m injection and CCE Blood samples were collected from all stallions twice weekly beginning I wk before treatment through wk after treatment On d 21, all stallions were fitted with an indwellingjugular catheter and I hr later received a challenge injection of GnRH (0.1 ug/kg of BW, i.v.), Blood samples were collected to assess the pituitary-gonadal response to GnRH Samples were analyzed for LH, FSH, and testosterone Semen evaluationwas conductedon the last ejaculates from each stallion Treatment with pGnRH resulted in a 16% increase (P < 0.05) in plasma concentrationsof testosteroneabove controls in samples collected twice weekly by d 35 post treatment (Figure I, *p < 0.05); the increase was maintained for the duration of sample collection (d 58) PlasmaconcentrationsofLH and FSH were not different betweengroups GnRH challenge revealed a 71 % increase (P < 0.0 I) in plasma LH response and a non-significant increasedresponsein testosteroneconcentrations Conversely, concentrations of FSH were similar between groups in response to the GnRH challenge.Testosteroneproduction was accompanied by an increase in LH secretionafter GnRHchallengewithoutany adverse physiological effects, such as down-regulation of gonadotropinsor semenmorphology In conclusion, we haveshown ina relevantlarge animal model that GnRH plasmiddelivery by CCE has potential~s a safe and effectivehormonedeliverysystem.These resultswarrant future researchinto the potentialapplicationsof this technologyfor both veterinary usc and usc in humans S8 0.75 J - - pGnRH - - SEAP 0.5 E Cl 0.25 e Q) e -~ Q) III III Q) I- -0.25 -0.5 -0.75 -1 -10 10 20 30 40 50 60 Day 18 Non-Viral Cutaneous Gene Transfer DNA Using In Vivo Electroporation Richard Heller,' ? Mark J Jaroszeski.P Yolmari Cruz, I Amy Donate ,' Bernadette Ferraro,' Richard Gilbcrt.i-' Kenneth Ugcn.l-' Loree C Hellcr.'-' 'Molecular Medicine , University ofSouth Florida, Tampa, FL; ' Chemtcal Engineering, University ofSouth Florida, Tampa, FL; 'Cemer for Molecular Delivery, University ofSouth Florida, Tampa, FL The easy accessibility of skin makes it an excellent target for gene transfer protocols.Cutaneousdiseases can be treated directly In addition, the skin is a suitable target for delivering expressed proteinssystemically To fully take advantageof skin as a target for gene transfer, it is importantto establish an efficientand reproducible delivery system EIectroporation as a tool for the delivery of plasmid DNA is a strong candidate to meet these delivery criteria Electroporation of skin is a simple, direct, in vivo methodto deliver genes for therapy Previously, we demonstratedthat eleetroporation could be used to deliver plasmid DNA to the skin This work was performed ina mousemodeland utilizednon-penetrating, rigidplate electrodes To fully evaluate the potential of this approach it was critical to evaluate delivery in an animal model with skin thickness and structuresimilar to human Forthe experimentsreportedhere,a guinea pig modelwas used to evaluatedelivery ln addition,an electrode system that would allow for largerareas to be treated without increasing the applied voltagewasneeded This newelcetrodedesign consistsof an arrayof electrodesembeddedin a flexible matrix.The size of the array can be increaseddependenton the applicationand the cutaneousarea to be treated Various e1eetroporation parameters were evaluatedand comparedto standardplate electrodes for levels of expression Resultsclearly showed that the array inducedsimilar reportergene expressionas obtained withstandard plate electrodes Highest expression was obtained with pulse conditions of 225 V/ em and 150 ms Notably, muscle stimulation was greatly reduced when pulses were applied with the electrode array as opposed to the standard plate electrodes.This approach is being evaluated for the potential for delivering DNA vaccines to the skin Preliminary experiments utilizing a plasmid encoding for Hepatitis B surface antigen have demonstratedthat high antibody titers can be induced after two applications(prime/boost).Resultspresented here further demonstrate that electroporation can be used to augment the efficiency of direct injectionof plasmid DNAto skin in largeranimals and may have utility in several applications including delivery of DNA vaccines (Supported by a researchgrant from the NIH (RO I EB00544I) and alsosupported bythe Centerfor MolecularDelivery, Univ of South Florida) Molecular Therapy Volume 15, Supplcrncn I, \by 2007 C ()ppi ~ht © · 11 11; American Society of Gene Th erapy ... levels of expression Resultsclearly showed that the array inducedsimilar reportergene expressionas obtained withstandard plate electrodes Highest expression was obtained with pulse conditions of. .. stallions in the firstgroup received mg of a pGnRH (n = 3); the second group received mg of pGnRH (n = 3); and the third group received mg of a control plasmid expressing secreted embryonic alkaline... University ofSouth Florida, Tampa, FL; '' Chemtcal Engineering, University ofSouth Florida, Tampa, FL; ''Cemer for Molecular Delivery, University ofSouth Florida, Tampa, FL The easy accessibility of skin