128 Screening of Antisense Oligonucleotides for Human Dystrophin Exon Skipping in a GFP Reporter Culture System with High Sensitivity and Specificity 125 Measurement of Flow Mediated and Nitric Oxide[.]
Trang 1125 Measurement of Flow-Mediated and Nitric
Oxide Synthase-Dependent Vasodilation in Living
Rats Using High Resolution Ultrasound
Christian Heiss,RichardE Sievers, Nicolas Amabile, Shobha
Natarajan, Yerem Yeghiazarians, MatthewL.Springer
1 Department ofMedicine, Division ofCardiology, University of
California, San Francisco, San Francisco, CA
In humans, endothelial function serves as a surrogate marker for
cardiovascular health and is measured as changes in arterial diameter
after temporary ischemia (flow-mediated dilation;FMD) However,
in rodent models of vascular disease,direct measurements of
vas-cular function have been limited to studying isolated blood vessel
fragments ex vivo Here, we present anFMD-related approach to
study conduit arteryvasodilation in anesthetizedrats Diameter and
Doppler-flow measurements were obtained from the femoral artery
using high-resolutionultrasound and automated analysis software
We observed dose-dependent vasodilation using both
endothelium-dependent and endothelium-inendothelium-dependent pharmacologic
vasodila-tors Transient surgically-induced hindlimb ischemia led to reactive
hyperemia with sequential flow velocity increase and femoral artery
dilation, the latter of which was completely abolished by
NO-syn-thase inhibition Reactive hyperemia was mimicked by
adenosine-induced flow increase Repeated measurements were reproducible
on the same day and at one month.To demonstrate the applicability
ofthis approach in a model ofendothelial dysfunction,we show that
FMD is significantly reduced in older animals To our knowledge,
this is the first integrative physiologic model to reproducibly study
FMD in living rodents Notably,this approach demonstrates that
the physiology ofrodent vascular reactivity is concordantwith that
of humans,validating its use for the study of gene-based and other
therapeutic modulators of vascular function
GENE THERAPY FOR MUSCULAR DYSTROPHIES
126 Dp116 Expression in Dystrophin/Utrophin
Double Knockout {mdxiutm -) Mice Restores
Muscle Mass and Viability but Does Not Correct
Dystrophic Pathology
AndreaL.H.Arnett,' Luke M.Judge,' Jeffrey S Chamberlain.'
'Molecular and Cellular Biology, Medical Scientist Training
Pro-gram , University ofWashington , Seattle; 2Neurology, University
ofWashington, Seattle
Mice deficient in both dystrophin and utrophin(mdx.utrnr)
ex-hibit a phenotype similar to that seen in DMD patients,including
severe muscle wasting,skeletal deformities,joint contractures,and
premature death In these mice, the absenceof both dystrophin and
utrophin prevents assembly ofthe dystrophin-glycoprotein complex
(DGC) We previously generated transgenic mice with skeletal
muscle expression of Dpl16 and have introduced this transgene
onto themd:r.utrn : background Dp116 is a non-muscle isoform
of dystrophin that lacks the actin-bindingdomain and the
major-ity of the rod domain found in the full length dystrophin isoform
Thus,it is postulated that Dp 116 has no mechanical link to the actin
cytoskeleton,but can still assemble and stabilize the DGC at the
sarcolemma We now report that Dp 116 expression can dramatically
increase both muscle mass and lifespan inmdxtutrn "and can delay
both formation and progression of kyphosis and joint contractures
However, histological examination ofmuscle from these transgenic
mice reveals signs of muscular dystrophy similar to that seen in
muscles of dystrophin-deficient(mdx) mice, Our results provide
compelling evidence that dystrophin and the DGC participate in
signaling mechanisms that are criticalfor maintenance of muscle
S50
mass Alternatively, Dp 116 may contribute to the structural integ-rity of myofibers via non-canonical binding of Dp 116 to the actin cytoskeleton
127 Functional Capacity of Dystrophins N-Terminal Actin Binding Sequences
Glen B Banks,IPaul Gregorevic,IJames M AlIen,1.2 Eric E Finn,' Jeffrey S.Charnberlain.l-P
1 Department ofNeurology, University ofWashington , Seattle,
WA; 'Department ofBiochemistry: University ofWashington Seattle, Wit.
Duchcnne and Becker muscular dystrophies are caused by mutations in the dystrophin gene Mutations in the N-terminal actin-binding domain (ABO I) of dystrophin typically decrease dystrophin expression and cause clinically more severe phenotypes than do similar mutations elsewhere in the protein Because of the large reduction in protein expression,thc functional capacity
of actin binding sequences in the N-terminal domain is not clear ABO I contains three actin-binding sequences (ABS 1-3) In thc present study we test the pathophysiological effects of in-frame actin-binding sequence deletions from microdystrophin (L'1R4-R23/L'1CT) when expressed in muscles ofdystrophin-deficient mdx mice We delivered microdystrophin into the tibialis anterior muscles
of 2-day-old mdx mice using recombinant adcno-associatcd viral vectors pscudotypcd with the serotype-6 capsids, Mierodystrophin prevented muscle degeneration and loss ofspecific force generating capacity, and partially protected muscles from contraction-induced injury when evaluated at 4 months of age Expression ofmicrodys-trophin lacking ABS3 (ABS I,2~Dys) or ABS2 and 3 (ABS I~Dys)
in the N-terminal domain did not prevent loss of specific force nor protect muscles from contraction-induced injury Furthermore, expression of dystrophins with these N-terminal deletions reduced the overall pereentagc of muscle fibers that were protected from degeneration, compared with the non-deleted microdystrophin Therefore,dystrophin-based constructsappear to require an intact ABO I to restore the contractile properties of skeletalmuscle and prevent ongoing muscle degeneration
128 Screening of Antisense Oligonucleotides for Human Dystrophin Exon Skipping in a GFP Reporter Culture System with High Sensitivity and Specificity
Ehsan Benrashid,' Saafan Malik,'Allen Zillmer,'Randy J Thresherf Jeffrey Rosenfeld,' Qi Long Lu.1
IMcCol/-Locf wood Laboratoryfor Muscular Dystrophy Re-search, Carolinas Medical Center; Charlotte NC; 2Animal Mod-els Core Facility, University ofNorth Carolina Chapel Hill NG.
Frameshift mutations in the dystrophin gene, located on the X chromosome (Xp21),can result in the progressive muscle wasting disease Duchenne'sMuscular Dystrophy (DMD),which affects-a
in 3500 live male births Antisense oligonucleotide (AON) therapy has previously been shown to selectively induce the skipping of particular exons both in vitro and in vivo, by targeting exon
splic-ing enhancer (ESE) regions, 3'-, and 5'-splice sites The DMD phenotype can thus be alleviated to the milder Becker'sMuscular Dystrophy (BMD) form Due to limitations in the quantification of AONefficacyvia RT-PCR and nested PCR assay systems,as well
as practical limitationsofprimary cultures for AON screening, this study employed a GFP reporter culture system to provide for ef-ficient and high throughput screening ofnumerousAON compounds for human dystrophin exon 50 skipping,which could rescue ap-proximately 8% ofDMD mutations AON screening was performed using both 2' -O-mcthyl phosphorothioate (20Mc) and morpholino
Molecul ar Therapy Volume 15 S upplement t••\by 2007
C op~Ti~ht © The Ame ri can $< )(;(1,:1)" o f G ene Therapy
Trang 2(PMO) chemistryvia fluorescencemicroscopy,RT-PCRlnestcd-PCR
the effect ofoverall AON length was also determined on the culture
system The efficient AONs selected were further tested with
deletion; further skipping of E50 can restore the reading frame)
The results showed thatAONs selected with the reporter system are
demonstrate that the in vitro cell culture-based system is a valuable
tool for the screening of effective AONs with high specificity and
antisense therapy in DMD
129 A Myoblast Expansion System for
Improving the Efficiency of Autologous Stem Cell
Therapy To Treat Muscular Dystrophy
'Senator Paul D Wellstone Muscular Dystrophy Co-operative
Research Center, Department ofNeurology; The University0/
Washington School ofMedicine, Seattle, IE,/.
Autologous stem cell-based transplantation is a promising
ap-proach to treating inherited muscle disorders A successful strategy
would be facilitated by regimes that allow tor expansion both in
vitro and in vivo ofcells with myogenic potential A previous study
demonstrated that a chemical inducible dimerizer (CID), AP20 187,
could maintain in vitro proliferation of myoblasts expressing
P36VPGFR-I, a chimeric protein composed of the cytoplasmic
phosphorylation domain of fibroblast growth factor receptor 1
(FGFR-I) and a mutated dimerization domain of FK506 binding
bicistronic expression cassette composed ofa microdystrophin/GPP
fusion gene and the F36VFGFR-1 gene under the control of a
intramuscularly transplanted into mdx tibialis anterior muscles,
expressing microdystrophin/GFP and the developmental isoform of
that in culture, residual AP20187 retained in those CID-expanded
imply that exogenous control of FGFR-I activation in myoblasts
can be used to prevent terminal differentiation and enable
large-scale expansion of myogenic precursors, potentially increasing the
efficiency of myoblast engraftment in muscle disorders
130 Independent Canine Models of Duchenne
Muscular Dystrophy Due to Intronic Insertions of
Repetitive DNA
'Scott-Ritchey Research Center; Auburn University, Auburn, AL;
lSchool 0/Medicine, University ofNorth Carolina - Chapel Hill,
Chapel Hill, NC; 3School ofMedicine, University ofMissouri,
Columbia , Ala.
devcl-opment ofeffective gene therapy for DMD has been and continues
to be a high priority A number of animal models have allowed
most closely recapitulates the clinical presentation, immune system
Molecular Therapy Volume1 5 SupplementI ~ br 2007
C opyright © T he American Soci etyo r Gen eTherapy
best characterized and therefore most commonly employed model Additional canine models would further improve the utility of the dog as a model system as these models would mimic the variety of challenges seen with human patients, including variable transcrip-tion, the presence or absence ofepitopes and the effect of residual
molecular basis These models were identified in the Labrador Re-triever and WelshCorgi breeds In both cases, affected animals can
be identifiedat birth by elevated creatine kinase levels Both Welsh
deficient except for rare revertant fibers They display prominent skeletal muscle pathology identicalto these found in human patients
have identified the molecular basis of each model as the inclusion
of repetitive sequence elements creating novel exons in the cDNA
In both cases these elements have been inserted into introns (intron
13 for the Welsh Corgi and intron 19 for the Labrador Retriever), activating splice acceptor sites already present in the normal intron
characteriza-tion ofthe mutacharacteriza-tions and morphologic features of each ofthese new models provides the informationrequired to employ these models in gene therapy studies These models provide important alternatives
to the available models and they will broaden our understanding of how relevant approaches will function in the face ofdifferent
Virus Vector-Encoding Human Mini-Dystrophin Leads to Functional Correction in Dystrophin Deficient Mice
'Surgery; Children's Hospital ofPhiladelphia, Philadelphia, PA; 'Physiology; University0/Pennsylvania, Philadelphia, PA.
Introduction: Duchenne muscular dystrophy (DMD) is the most common disabling and lethal congenital muscle disorder Patients with DMD experience progressive muscle degeneration and weak-ness until they succumb to respiratory or cardiac failure Currently
virus serotype 9 (AAV2/9)has also been shown to have high muscle
long-term functional correction of'dystrophin deficient mdx mice Methods: Fetuses from pregnant mdx mice underwent vitelline
vein injectionat E14.5 withAAV2/9.CMV.mini-dystrophin Specific skeletal muscles, cardiac muscle, and brain and liver tissue from
treated and age-matched mdx control mice were harvested on week
immunohistochemistry (IHC) of frozen sections and quantified by real-time PCR (Q-PCR) Extensor digitorum longus (EDL) muscles
of the treated and control mice were used in physiological studies
to measure force of maximal twitch, tetanus and eccentric force drop between first and fifth contractions (ECC) Results: Expres-sion of human mini-dystrophin was detected in week 5, 10, and 20 samples using IHC with human specific antibody with no signifi-cant difference in the proportion of transduction among the 3 time
using the human dystrophin probe (hDys) with the ribosomal ISS
S51