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914 Gene Expression Profiles in Skeletal Muscle after DNA Electrotransfer Indicate a Highly Efficient and Safe Gene Therapy Platform 912 Canine Erythropoietin as a Secreted Reporter System for the Stu[.]
912 Canine Erythropoietin as a Secreted Reporter System for the Study of Sleeping Beauty-Mediated Long-Term Expression in the Dog R Scott Mcivor,' Erik R Olson , Alice Hsu,' Kerri Bostrom,' Susan Sonntag,' Kurt Lalsresh ,' Jason Bell,' Perry B Hackett.' I Discovery Genomics, lnc., Minneapolis, MN; lR&D Systems, Minneapolis, MN; JGenetics, Cell Biology and Development, University ofMinnesota, Minneapolis, MN The Sleeping Beauty transposon system (SB) has been demonstrated to mediate non-viral integration and long-term expression of several reporter genes as well as therapeutic genes in mice Our goa l is to test the effectiveness of SB in achieving long-term expression of therapeutic genes in the dog as a large animal model for gene therapy in humans We previously reported murine erythropoietin (EPO) as a convenient reporter for which long-term expression can be monitored using a sensitive enzyme-linked immunosorbant assay (ELISA) for analys is of blood samples regularly collected from mice treated with murine EPO-encoding transposons (Molecular Therapy 13: 617, 2006) To adapt this system for monitoring ofgene expression in the dog, the canine EPO coding sequence was isolated from a canine cDNA library To test the effectiveness of the eEPO sequence as a reporter for in vivo studies, a CMV-regu!ated cEPO construct was tested by transfection into human HEK 293 cells and by hydrodynamic injection into C57BL/6 mice Supernatants from the transfected 293 cells contained substantial levels ofmaterial that was immunoreactive in the human EPO ELISA, demonstrating the effectiveness ofthis assay for monitoring expression ofcanine EPO Plasma prepared from blood collected 24 hours after injection ofthc animals also contained substantiallcvels ofimmunorcactive canine EPO, detec table by ELISA Blood samples collected one week post-injection showed a substantial increase in hematocrit (from 50 up to a range of 64 to 70), demonstrating the biological activity of cEPO when expressed in mice after hydrodynamic injection of the CMV-regulated construct These results verify that canine EPO can be used as a sensitive secretable reporter that is detectable by ELISA of plasma samples, and that the mouse can be used as a test system for cEPO expression and biological activity One of the problems encountered in long-term expression of biologically active EPO is a severe erythrocytosis that can be lethal for the test animal Based on structure-function studies reported for human EPO variants generated by site-directed mutagenesis, we constructed several canine EPO variants bearing amino acid substitutions predicted to confer reduced biological activity while maintaining immunoreactivity in the human EPO ELISA CMV-regulatcd plasm ids encoding these cEPO variants were hydrodynamically injected into C57BLl6 mice, assaying for immunoreactive material by ELISA and for biological activity by increased hematocrit We found that the S lODE form of cEPO was undetectable by ELISA, but was also biologically inactive However, the G 145A and K45D variants of cEPO both exhibited reduced biological activity while immunoreactivity was largely retained The se cEPO variants will provide useful reporters for studies in the dog should long-term expression ofwild-type cEPO prove to cause health problems for the testing of SB transposons in this large animal system 913 Aerosol Gene Delivery to the Lungs of Mice Guided by Magnetic Forces Petra Dames ,' Eugenia Lesina, Carsten Rudolph.' Pediatrics, Ludwig Maximilians University, Munich, Germany I Magnetic drug targeting has been previously investigated for site-specific drug delivery to improve therapeutic efficiency In our study, we applied magnetic drug targeting to aerosol gene delivery to the lungs ofmice by application ofan external magnetic field durS348 ing aerosol application of poly ethylen imine (PEI)-pDNA particles in combination with superparamagnetic iron oxide nanoparticles (SPION) In previous inhalation studies in mice, we found that inhaled aerosol droplets containing SPIONs could be enriched in lungs when an electromagnet was positioned above the lung during inhalation Deposition rates ofco-delivered plasmid DNA correlated with SPION deposition, In this study, we examined gene expression in the lungs after aerosol delivery guided by magnetic forces using pDNA coding for firefly luciferase Additionally, pDNA deposition in mice lungs was quantified by real time PCR.Branched PEl (25kDa)pDNA complexes together with SPIONs were applied to mice placed in a whole-body nebulization device As an external magnetic field a small cubic (10 mm3) permanent magnet was fixed on the fur of mice above the lungs with tissue glue After aerosol application of SPIONs coated with PEl (12.5 mgml-I) and co-delivery of PEIpDNA complexes (0.1 mgml-I, 125:1), we detected 4-fold higher luciferase expression in mice lungs with than without magnetic field application But expression was 7.5-fold lower compared to PEI-pDNA aerosol application Therefore, we performed in vitro experiments co-transfecting BEAS-2B cells with PEI-pDNA and SPIONs at different ratios As observed in vivo, luciferase expression was deereascd compared to PEI-pDNA transfection at high SPION/pDNA ratios But expression was increased about 2-fold by magnetic forces when lower ratios ofSPIONs to pDN A (15: I) were used Further in vivo experiments were performed using PEI-pONA (0.2 mgml-l ) and lower concentration ofSPIONs (3.0 mgml-l ), We detected a 1.5-fold increase ofpDNA deposition whcn an external magnetic field was applied, but we could not measure any luciferase expression in mice lungs with IVIS 100 imaging system (Xenogen, Alameda, CA) In conclusion, we suggest that it is in principal possible to enhance pDNA deposition and gene expression in mice lungs after aerosol delivery by magnetic forces, but further efforts are necessary to increase gene expression in future 914 Gene Expression Profiles in Skeletal Muscle after DNA Electrotransfer Indicate a Highly Efficient and Safe Gene Therapy Platform John R Zibert,' Pernille H Moller,' Jens Eriksen.i Julie GehJ.2 I Department 0/ Dermatology, Copenhagen University Hospital Gentofle, Hellerup, Denmark; lLaboratOly ofthe Department ofOncology, Copenhagen University Hospital Herlev, Herlev, Denmark Background: Gene transfer by e1ectroporation (DNA elcctrotransfer) to muscles results in high lcvcllong term transgenic expression, showing great promises for systemic treatments of e.g cancers and protein deficiency syndromes However, little is known about the adverse effects of DNA electrotransfer on muscle fibres We have therefore investigated transcriptional changes through gene expression profile analyses, as well as morphological changes evaluated by histological analysis DNA eleetrotransfer was obtained using a combination of a short high voltage pulse (HV, 1000 V/cm, 100 us) followed by a long low voltage pulse (LV, 100 V/cm, 400 ms); a pulse combination optimised for safe and efficient gene transfer Muscles were transfeeted with green fluorescent protein (GFP) and excised at hours, 48 hours or weeks after treatment Results: Differentially expressed genes were investigated by microarray analysis, and descripti ve statistics were performed to evaluate the effects of I) electroporation, 2) DNA injection, and 3) time after treatment The biological significance of the results was assessed by gene annotation and supervised cluster analysis and validated by quantitative RT-PCR Generally, electroporation alone cau sed down-regulation of structural proteins e.g sareospans and catalytic enzymes such as phosphoenolpuryvate carboxykinases, DNA injection alone induced down-regulation of intracellular transport proteins e.g sentrins Three weeks after treatment, the injection of Molecular Therapy Volume15 Supplement I, \by 2007 Copyright © '111C American Societyo f Gene Tllcr.lpr DNA and eleetroporation was clustering together with injection of DNA alone and e1ectroporation alone and was furthermore clustering very close to the untreated controls This indicates a safe gene therapy platform with transient effects on the muscle fibres after three weeks (see the figure) peak after treatment More generally, this strategy could be applied for functional studies of any given circulating protein instead of generating transgenic animals 916 Muscle Characteristics Affect Plasmid Expression Following Electroporation in Both Large and Small Animals Patricia A Brown,' Amir S Khan; Melissa A Pope, I Ruxandra Draghia-Akli.' 'VGX Pharmaceuticals, Immune Therapeutics Division, The Woodlands, TX EP DNA Time Most interestingly, no changes in the expression of proteins involved in inflammatory responses or muscle regeneration was detected, indicating limited muscle damage and regeneration The morphological changes indicated structural changes with loss ofcell integrity and striation pattern in some fibres after DNA injection and electroporation treatment, while electroporation alone caused minor loss of striation pattern but preservation of cell integrity Conclusion: The small and transient changes found in the gene expression profiles arc of great importance, as this indicates that DNA electrotransfer is safe with minor effects on the muscle host cells Certainly the I-1V+LV pulse combination used have been optimised to ensure highly efficient and safe DNA electrotransfer; These findings are essential for introducing this gene therapy platform in the clinic with focus on systemic treatment regimes for a variety of diseases like cancers 915 Controlled Long-Lasting Hematocrit Increase by EPO Plasmid Electrotransfer Emmanuelle Fabre ,' Daniel Scherman; Pascal Bigey.' 'INSERM U640; CNRS UMR8151; Chemical and Genetic Pharmacology Laboratory University Paris Descartes, Ecole Nationale Superieure de Chimie de Paris; INSERM; CNRS, Paris, France In vivo electroporation (EP) has been established as a simple, efficient and reproducible method for the intramuscular delivery of therapeutic plasm ids, DNA vaccines, oligonucleotides, and siRNA Expression levels are increased by several orders of magnitude with EP over 1M injection alone Obtaining the optimal expression in animal models involves taking into account several different variables, including: animal species and age , plasmid size , and components of the expression cassette and backbone, plasmid formulation and concentration, and electroporation conditions However, pre-clinical studies outlining the effect ofmuscle type (i.e fiber type composition) and its characteristics (fat and fiber content versus muscle fiber content) have not been well described in the literature While muscles in smaller animals such as the rodent can have predominantly one type of fiber types (type I, type II and its subtypes), larger animals have mostly mixed fiber types muscles; nevertheless fiber type percentage may change during development and/or activity and training Our recent studies have indicated that the choice ofmuscle lor injection and EP is vital to obtain the highest possible plasmid expression levels A reporter plasmid expressing secreted embryonic alkaline phosphatase (SEAP) was administered to mice in different muscles, including tibialis anterior (predominantly type II fibers) or quadriceps muscle (mixed muscle) Serum SEAP levels were over 50% higher (p == 0.002) when the plasmid was administered in the tibialis muscle When the footpad of mice was injected and electroporated, the flexor digitorum brevis muscle fibers expressed well the plasmid, while only low expression was detected in the interstitial fat and conjunctive tissue Splenius, pectoralis, and semitendinosus muscles in young horses were administered the SEAP plasmid loll owed by identical conditions of electroporation Pectoralis muscle obtained the highest expression at Day 21, and correlated with a predominance of type ([ fibers in these well trained horse (more than twice as high as the splenius and ten times the levels achieved in the semitendinosus) In a previous study in pigs, a series of muscles (semitendinosus, sternocranialis, longissimus dorsi, brachialis, masseter, and footpad) were injected with SEAP-expressing plasmid at identical conditions Longissimus dorsi yielded higher expression as compared to semitendinosus, sternocranialis and brachialis Transfection of masseter (15-30% type I fibers) and footpad muscles yielded the lowest expression The EP used in these studies was preformed using a constant current electroporation (CCE) device The results ofstudies suggest that the careful choice oftarget muscle for transfection with vaccine and/or therapeutic plasm ids in clinical studies is critically important and that CCE can be used to deliver these vectors successfully The potential applications of gene transfer include gene therapy, DNA vaccination or gene functional study According to the desired application, different level, kinetics and localization of gene expression might be required In this original and unpublished work , we describe a way of reaching a stable level of hematocrit in beta thalassemic mice after several electrotransfer treatments with low doses of EPO encoding DNA plasmid Our results suggest that a therapeutic level of EPO can be reached , inducing an almost normal hematocrit in sick mice , and avoiding a deleterious hematocrit Molecular Therapy Volume IS Supplement ~by Cop yright © The Americ m Society o r Gene Therapy 2007 S349 ... of any given circulating protein instead of generating transgenic animals 916 Muscle Characteristics Affect Plasmid Expression Following Electroporation in Both Large and Small Animals Patricia... of proteins involved in inflammatory responses or muscle regeneration was detected, indicating limited muscle damage and regeneration The morphological changes indicated structural changes with... small and transient changes found in the gene expression profiles arc of great importance, as this indicates that DNA electrotransfer is safe with minor effects on the muscle host cells Certainly