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[.]
Trang 1912 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,1Alice Hsu,' KerriBostrom,'
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
demon-strated to mediate non-viral integration and long-term expression of
several reporter genes as well as therapeutic genes in mice.Our goal
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-linkedimmunosorbant
as-say(ELISA) for analysisofblood 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 vivostudies,a CMV-regu!ated cEPO
construct was tested by transfection into human HEK 293 cells and
by hydrodynamic injectionintoC57BL/6mice 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,detectable 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
gen-erated by site-directed mutagenesis, we constructed several canine
EPO variants bearing amino acid substitutionspredicted to confer
reduced biological activity while maintaining immunoreactivity in
the human EPO ELISA CMV-regulatcd plasm ids encoding these
cEPO variantswere hydrodynamically injected into C57BLl6 mice,
assaying for immunoreactive material byELISA 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 ThesecEPO 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,1Carsten Rudolph.'
I Pediatrics , Ludwig Maximilians University, Munich , Germany.
Magnetic drug targeting has been previously investigated for
site-specific drug delivery to improve therapeutic efficiency In our
study, weapplied magnetic drug targeting to aerosol gene delivery
to the lungs ofmice by application ofan external magnetic field
dur-S348
ing aerosol application of polyethylen 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 PEI-pDNA 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 expres-sion 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,'JensEriksen.i Julie GehJ.2
I Department 0/ Derm atology, Copenhagen University Ho spital
Gentofle, Hellerup, Denmark; lLaboratOly ofthe Department ofOncology, Copenhagen University Hospital Herlev, Herlev, Denmark
Background :Gene transfer by e1ectroporation (DNA elcctrotrans-fer) 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 expres-sion 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 4 hours, 48 hours or 3 weeks after treatment.Results:
Differentially expressed genes were investigated by microarray analysis, and descriptivestatistics 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 caused
down-regulation of structural proteins e.g sareospans and cata-lytic 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
Molecul ar Therapy Volume15 Supplement I, \by 2007
Co pyright © '111C Am erican Soc iety o f G ene Tl lcr.lpr
Trang 2DNA and eleetroporation was clustering together with injection of
DNA alone and e1ectroporation alone and was furthermore
cluster-ing 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)
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
opti-mised to ensure highly efficient and safe DNAelectrotransfer;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.
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
ex-pression might be required In this original and unpublished work,
we describe a way of reaching a stable level of hematocritin 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 nor
-mal hematocrit in sick mice,and avoiding a deleterious hematocrit
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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,IRuxandra Draghia-Akli.'
'VGX Pharmaceuticals, Immune Therapeutics Division, The Woodlands, TX
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 inthe 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 indifferentmuscles,including tibialis anterior (predomi-nantly 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 ad-ministered 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
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