ABSTRACTS THAT WILL BE PRESENTED IN THE 9TH ANNUAL MEETING OF THE AMERICAN SOCIETY OF GENE THERAPY, 2006, MAY 31- JUNE 4, BALTIMORE
1) Systemic Administration of
rAAV6-Microdystrophin Preserves Muscle Function and Extends Lifespan in the
Dystrophin-/Utrophin- Mouse Model of Severe Muscular Dystrophy
Paul Gregorevic, Michael J. Blankinship, Elina Minami, James M.
Allen, Charles E. Murry, Jeffrey S. Chamberlain Department of Neurology,
University of Washington, Seattle, WA; Department of Pathology, University of
Washington, Seattle, WA
Gene replacement is a promising strategy for treating severe neuromuscular
disorders such as Duchenne muscular dystrophy (DMD). However, the therapeutic
potential of miniaturized dystrophin expression cassettes has not been
rigorously assessed because traditional gene transfer systems cannot
simultaneously transduce the important skeletal muscles, and the heart of
animals that model muscular dystrophies. Furthermore, because of limited
prospects for efficient gene transfer, previous studies have typically assessed
the effects of localized interventions in the comparatively robust
dystrophin-deficient mdx mouse, which poorly models the progressive, severe
dystrophy that shortens the lives of DMD patients. We recently established a
method that facilitates the delivery of genes to striated muscles throughout
adult animals via intravenous administration of recombinant adeno-associated
viral vectors (rAAV vectors). This advance has enabled us to rigorously evaluate
systemic administration of our most promising microdystrophin construct as a
prospective treatment for DMD in the dystrophin-/utrophin- double-knockout
mouse. This model experiences severe and progressive muscular dystrophy that
shortens lifespan by 75% compared with wild-type and mdx mice. We observed that
systemic administration of rAAV6-microdystrophin vectors restores dystrophin
expression in important skeletal muscles body-wide and throughout the mouse
heart. Restoration of dystrophin expression considerably reduced key indices of
muscle disease (such as muscle fiber degeneration) in hindlimb and diaphragm
muscles. Treatment also enhanced the force-producing capacity, and resistance to
contraction-induced injury of limb and respiratory muscles, and dramatically
reduced serum-based indicators of body-wide muscle fiber degeneration.
Importantly, we have established that these effects of intravenous
rAAV6-microdystrophin delivery preserve the muscle mass and physical mobility of
dystrophic mice, and considerably extend their lifespan. These data provide
evidence that systemic administration of AAV vectors is a promising strategy to
achieve whole body treatment of pathology associated with muscular dystrophy,
and as such may lead to a treatment for DMD and other severe neuromuscular
disorders.
2) Ex Vivo Gene Therapy for Duchenne
Muscular Dystrophy: Lentiviral Vectors, Exon Skipping and PhiC31 Integrase
Approaches
Simon P. Quenneville, Pierre Chapdelaine, Joel Rousseau, Jacques P.
Tremblay Human Genetic, CRCHUL, Quebec, QC, Canada
Duchenne muscular dystrophy is the most severe muscular dystrophy. It is caused
by the absence of dystrophin in muscle fibers. This absence lead to increased
muscle damage, loss of muscle mass, loss of strength, respiratory and heart
failure. This disease as currently no treatment. Myogenic cells transplantation
is a possible cure for Duchenne muscular dystrophy. However, allogeneic graft
success relies on the use of efficient but toxic immunosuppressive drugs. The
use of these drugs is a major problem that could be solved by the use of ex vivo
gene therapy. This method consists in genetically modifying patient myoblasts
before their auto-transplantation. In this study, a viral (lentiviral) and a non
viral (integrase PhiC31) method were tested to perform the genetic modification.
Therapeutic sequences included microdystrophin, dystrophin and exon skipping
cassettes.
Co-transfection (nucleofection) of a PhiC31 integrase and a dystrophin
expressing plasmid was used to perform the genetic modification. We have also
generated eGFP, eGFP-microdystrophin and exon skipping lentiviral vectors with
CMV and MCK promoters. Following in vitro modification of dystrophic cells (mdx
mice derived) and normal human myoblasts, the cells were engrafted into mdx and
SCID mice muscles.
The nucleofection led to a stable expression of the full length dystrophin. This
was the biggest expression cassette ever stabilized in primary cultured human
myoblasts. Following transplantation, the dystrophin expression was observed
into a muscle, leading to the apparition of the dystrophin associated proteins
a-sarcoglycan and b-dystroglycan. However, this method is not very efficient. We
have also generated eGFP and eGFP-micro-dystrophin expressing lentiviral vectors
under the control of the CMV and MCK promoters. Following in vitro infection of
human myoblasts, the cells were engrafted into SCID mice muscles. Both
transgenes were expressed into the muscles one month after the engraftment. The
expression of microdystrophin also led to the apparition of a-sarcoglycans in
the dystrophic model mice. We have also used a lentiviral vectors coding for an
exon skipping cassette allowing us to skip the exon 51. In vivo experiments in
SCID mice as shown that it is possible to express a quasi dystrophin protein
with cells originating from a DMD patient. This work indicates that ex vivo
gene therapy is a possible approach to treat Duchenne muscular dystrophy.
3) Recombinant AAV Gene Delivery of Follistatin
for Muscle Enhancement in Models of Muscular Dystrophy
Liza Rizo, Chris Shilling, Amanda Haidet, Priya Umapathi Umapathi,
Zarife Sahenk, Jerry R. Mendell, Brian K. Kaspar Gene Therapy and Neuromuscular
Research, Columbus Children's Research Institute/The Ohio State University,
Columbus, OH
Objective: To determine the efficacy of recombinant adeno-associated
virus (rAAV) delivering follistatin (FS), a potent inhibitor of myostatin, to a
mouse model for limb-girdle muscular dystrophy and wild-type animals.
Background: LGMD2D is a debilitating muscle disease of children and young
adults. There is no proven treatment to delay the disease progression.
Inhibition of myostatin, a negative growth modulator for muscle, can
functionally improve normal or dystrophic muscle. This has been observed across
species lines, through genetic manipulation, naturally occurring mutations or
protein-specific antibodies. We have employed a novel gene therapy approach
using a rAAV vector carrying a FS transgene.
Design/Methods: Four-week-old wild-type, mdx, or alpha-sarcoglycan
deficinet mice were injected bilaterally into the quadriceps and tibialis
anterior muscles with 1x10e10 viral genomes of rAAV1-FS and evaluated for muscle
strength and endurance using hindlimb and forelimb grip strength as well as
Rotarod. At age 120 days, gross muscle evaluation and analysis of muscle weight,
fiber number, and cross-sectional area (CSA) on H&E sections was performed with
the addition of a FS ELISA assay.
Results: We found increased serum FS levels accompanied by a local and
remote increase in muscle mass in treated animals (p<0.05). Muscle hypertrophy
was observed in rAAV-FS mice versus controls. Motor function tests showed
statistically significant improvements in muscle strength in FS-treated mice.
Conclusions/Relevance: This data supports a therapeutic effect in
muscular dystrophy by myostatin inhibition. This approach represents a
clinically applicable gene therapy method to enhance muscle mass and function in
muscular dystrophy with potential for treatment of other muscle diseases. No
toxicity was encountered. The FS transgene could also be used to complement gene
replacement therapy. For example, adding muscle mass in patients with more
advanced dystrophies could improve their candidacy for gene replacement therapy.
4) C-Terminal Truncated Microdystrophin Recruits
Dystrobrevin and Syntrophin to the Dystrophin-Associated Glycoprotein Complex
and Reduces Muscular Dystrophy in Symptomatic Utrophin/Dystrophin Double
Knock-Out Mice
Yongping Yue, Mingju Liu, Dongsheng Duan Molecular Microbiology and
Immunology, University of Missouri, Columbia, MO
C-terminal truncated (C)
microdystrophin is being developed for Duchenne muscular dystrophy (DMD) gene
therapy. Encouraging results have been achieved in the mdx mouse model.
Unfortunately, mdx mice do not display the same phenotype as human patients.
Evaluating
C
microdystrophin in a symptomatic model will be of significant relevance to human
trial. Utrophin/dystrophin double knock-out (u-dko) mice were developed to mold
severe dystrophic changes in human patients. In this study we evaluated
therapeutic effect of the
R4-R23/C
microdystrophin gene (R4/C)
after serotype-6 adeno-associated virus (AAV-6)-mediated gene transfer in
neonatal u-dko muscle. At two months after gene transfer, percentage of
centrally nucleated myofiber was reduced from 89.2% to 3.4% and muscle weight
was normalized. Furthermore, we have demonstrated for the first time that
C
microdystrophin can eliminate interstitial fibrosis, macrophage infiltration and
restore dystrobrevin and syntrophin to the dystrophin-associated glycoprotein
complex. Interestingly neuronal nitric oxide synthase was not restored. The most
impressive results were achieved in muscle force measurement. Neonatal gene
therapy increased twitch and tetanic specific force. It also brought the
response to eccentric contraction-induced injury to the normal range. In summary,
our results suggest that the
R4/C
microgene holds great promise in preventing muscular dystrophy.
5) A Pseudo-Exon Derived from an Intronic
Insertion Is Responsible for Duchenne-Like Muscular Dystrophy in the Welsh Corgi
Dog
Bruce F. Smith, Joseph N. Kornegay Scott-Ritchey Research Center,
Auburn University, Auburn, AL; College of Veterinary Medicine, University of
Missouri, Columbia, MO
Duchenne Muscular Dystrophy (DMD) is the most common X-linked inherited disease,
with an incidence of 1 in 3500 male births. This disease is relentlessly
progressive and usually fatal by early adulthood. A wide range of mutations in
the dystrophin gene have been characterized for DMD, as well as the less severe
form, Becker's Muscular Dystrophy. DMD presents gene therapy challenges due to
the size of the gene and resulting cDNA and the wide variety and complexity of
the mutations involved. Animal models for DMD have been described in the mouse,
cat and dog. The disease in mice and cats is significantly less severe than that
seen in humans, while the canine disease, characterized by muscle hypertrophy,
followed by muscle loss, fibrosis and death, closely mimics disease progression
in affected humans. As is expected from the frequency of mutation in the human
population, DMD has been recognized in several breeds of dogs including the
Golden Retriever, German Short-Haired Pointer, Rottweiler, Labrador Retriever,
West Highland White and Welsh Corgi. Where the mutation has been determined,
each breed has demonstrated a new mutation. Here we report the identification of
the mutation responsible for DMD in the Welsh Corgi. Affected dogs can be
recognized at, or shortly after, birth by increased serum creatine kinase levels.
These dogs show progressive muscle atrophy and fibrosis, stunted growth,
contractures and consequent debilitation. However, several of the affected males
have lived to sexual maturity. Muscle biopsies from affected Corgis show that
most fibers remain unstained for dystrophin, while rare fibers show dystrophin
staining using a variety of antibodies. Sequence analysis of the cDNA indicates
a 166 base insertion between exons 13 and 14, based on the human exon structure.
The inserted sequence shows a high degree of sequence similarity to a canine
LINE-1 element. Sequence analysis of intron 13, which is approximately 25kb,
indicates that there is an insertion in the intron. The insertion is immediately
downstream of an AG dinucleotide pair and contains the 166 bases seen in the
cDNA followed by a GT dinucleotide pair and additional inserted sequence. Thus
the insertion utilizes a putative intronic 3'splice acceptor site and a 5'
splice donor to form a novel exon, which is spliced into the mature mRNA. An
in-frame stop codon in this novel exon results in a truncated dystrophin protein,
which is non-functional. Staining of occasional fibers with antibodies to
epitopes located downstream of this insertion indicates that this exon may be
skipped or that alternative splicing and/or promoter use may produce a product.
This large animal model, with its defined mutation, will be useful in a variety
of ways, including the use of gene repair approaches to skip the inserted exon,
studies of spicing mechanisms in dystrophin, and studies of other gene therapy
approaches.
6) Antisense Oligonucleotide Therapy for Duchenne
Muscular Dystrophy: From Cell Culture to Clinical Trial
Jignya Ashar, Terence Partridge, Qi L. Lu MocColl-Lockwood
Laboratory for Muscular Dystrophy Research, Carolinas Medical Center, Charlotte,
NC; Research Center for Genetic Medicine, Children's National Medical Center,
Washington, DC
Duchenne muscular dystrophy (DMD) results from non-sense or frame-shifting
mutations in the dystrophin gene. The majority of these mutations can be
corrected by removal of one extra exon to create shortened, but in-frame
transcripts and produce at least partially functional proteins. Earlier studies
showed that antisense oligonucleotide (AON)-mediated splicing alteration can
achieve specific skipping of targeted exon, providing the potential for the
treatment of DMD. This promise was firmly established when we demonstrated that
AON can specifically remove the mouse dystrophin exon 23 containing a nonsense
point mutation and restore the functional levels of dystrophin protein in muscle
via intramuscular administration. More recently, we further demonstrated that
systemic delivery of AON can effectively restore dystrophin expression in
body-wide muscles. These data have prompted much of the current enthusiasm for
clinical trials with AONs. We also demonstrated that modifications of AON
chemistry are critical for achieving therapeutical effect by antisense therapy.
Morpholinos is one of the most effective chemistry for inducing functional
levels of dystrophin in bodywide skeletal muscles with improved force generation.
We have been examining several chemistries with various delivery systems using
mdx mouse as an in vivo model system to search for more effective chemistry and
clinically applicable delivery approaches to achieve long-term restoration of
dystrophin expression. Individual human dystrophin exon has been selected for
systematic screening of AON sequences to maximize the potential of antisense
effect as a preparation for clinical trials. We are also developing animal
models to select AON sequences for targeting individual human dystrophin exon
and to examine the therapeutic capacity of exon skipping in vivo. In
conclusion, our results have provides realistic hope for the treatment of a
majority of DMD patients.
7) Development of Recombinant Novel
Adeno-Associated Viral (rAAV) Vectors Encoding Optimised Microdystrophin cDNAs
for Duchenne Muscular Dystrophy (DMD)
Takis Athanasopoulos, Ian Graham, Capucine Trollet, Helen Foster,
Norma Perez, Vanessa Hill, Phillippe Moullier, George Dickson Biochemistry,
Centre for Biomedical Sciences, Royal Holloway University of London, Egham,
Surrey, United Kingdom; Gene Therapy Lab, Genethon, Evry, Paris, France;
Laboratoire de Therapie Genique, Inserm, Nantes, France
Duchenne muscular dystrophy (DMD) is a lethal genetic muscle disorder affecting
1:3500 male individuals, caused by recessive mutations in the dystrophin gene.
The size of the gene (2.4Mb) and mRNA (14kb) in addition to immunogenicity
problems and inefficient transduction of mature myofibres by currently available
vector systems are formidable obstacles to the development of gene therapy. AAV
vectors overcome many of the problems associated with other vectors but
accommodate limited transgene capacity (<5kb), allowing only truncated versions
of dystrophin (microdystrophin) to be packaged. More than 9 AAV vector serotypes
have been identified to date with certain serotypes(1, 5-9) displaying more
favourable tropism in transducing muscle compared to the traditionally used AAV2.
Human microdystrophin (mdys) cDNAs (<3.8 kb) rationally designed in our lab
based on patient and preclinical data were packaged in AAV-based vector
cassettes, under the control of CMV or Spc512 muscle synthetic promoter,
effectively expressed mdys (CMV>Spc512), restored DAP complex and inhibited
myofibre degeneration after local intramuscular administrations in nude/mdx
and mdx mice. Expression of microdystrophins using e.g. an AAV1CMVhmdys
vector was observed at high levels via i.m (>90%), i.p. (20-25%), but not via
systemic i.v. route of administration. However, certain issues including
systemic delivery, optimization of mdys cDNA constructs, AAV transduction
efficiency, safety and immunogenicity while retaining species and tissue (muscle)
specificity, still remain to be fully elucidated.
Here we report on production of codon/sequence optimised, transcriptionally
enhanced, species-specific (human, canine and murine) microdystrophin cDNA
cassettes (based on
3788 and
R4-23 published sequences) incorporating either constitutive or muscle-specific
promoter elements. These cassettes offered 3-5 fold increase in expression in
comparison to conventional configurations. Incorporation of Kozak/WPRE elements
for transcriptional enhancement, packaging of the cassettes into recombinant AAV
vectors of novel alternative serotypes (6, 7, 8
9) by pseudotyping and
AAV-stealth
approaches have been envisaged. Resulting rAAV6 and rAAV8 pseudotyped vectors
encoding species-specific comdys have been produced at titers>10E12 vg/mL and
are currently screened utilising local and systemic routes of administration in
preclinical models of the disease.
8) Enhanced Level of Gene Correction Mediated by
Oligonucleotides Containing CpG Modification in the mdx Mouse Model for Duchenne
Muscular Dystrophy
Carmen Bertoni, Arjun Rustagi, Thomas A. Rando Neurology, Stanford
University, Stanford, CA; Neurology Service and GRECC, VAPAHCS, Palo Alto, CA
Single point mutations and large deletions of the dystrophin gene are
responsible for Duchenne muscular dystrophy (DMD) a severe neuromuscular
disorders characterized by complete absence of dystrophin expression in skeletal
muscles. Gene editing mediated by single stranded oligonucleotides (ssODNs)
represents an appealing option to treat the disease since it has the potential
to treat both single point mutations as well as deletions that cause frame shift
of the dystrophin mRNA. The major limitation of the technology has been so far
the low level of genomic correction detected in muscle cells.
We have focused on the development of new vectors capable to activate specific
repair mechanisms and capable of directing the repair process specifically on
the sequence of the genomic DNA targeted for correction. The methyl binding
protein 4 (MBD4) takes an active role in the base excision repair mechanism and
is highly expressed in muscles. In addition to containing a binding site for the
methylcytosine, MBD4 contains also a specific glycosilase capable of recognizing
a T to G transversion at CpG sites and direct the conversion of the thymine into
methylcytosine. CpG modifications were introduced on the mutating base of the
targeting oligonucleotide in the attempt to mimic a deamination of
methylcytosine which results in the activation of MBD4.
All the studies were conducted using the mdx mouse as a model for DMD. This
strain contains a stop codon in exon 23 of the dystrophin gene that is
responsible for the absence of dystrophin protein in skeletal muscles. As a
target for the single base substitution we have chosen the splice boundary of
exon 23 of the mouse dystrophin gene in order to induced exon skipping to bypass
the nonsense mutation and induce expression of internally deleted but functional
dystrophin proteins. We have designed ssODNs complimentary to the coding or the
non-coding strand of the donor site of exon 23. CpG modifications were
introduced at the targeted base. The ability of these modified ssODNs to
increase gene repair was studied in muscle cells both in vitro and in
vivo. The level of dystrophin protein expression was significantly increased
by the use of ssODNs containing CpG modifications on the targeting base. Studies
conducted on muscle cells in culture demonstrate up-regulation of MBD4 mRNA and
the activation of the base excision repair mechanism through which MBD4 acts,
demonstrating the specificity of the repair process recruited by the ssODNs.
Correction of the dystrophin gene was shown to occur at the genomic level and
was stable over prolonged periods of time. In muscle cells in culture,
restoration of dystrophin expression was analyzed at the protein level by
western blot and immunostaining analysis and at the mRNA level by RT-PCR. In
vivo analysis also showed restoration of dystrophin in skeletal muscles of
injected mice by immunostaining. These studies demonstrate that the efficacy of
oligonucleotide-mediated gene correction can be increased by improving
oligonucleotide design.
9) Phi C31 Integrase System Enhances Dystrophin
Gene Expression in Skeletal Muscle of Mouse Models for Duchenne Muscular
Dystrophy
Carmen Bertoni, Sohail Jarrahian, Thurman M. Wheeler, Yining Li,
Eric C. Olivares, Michele P. Calos, Thomas A. Rando Neurology, Stanford
University, Stanford, CA; Genetics, Stanford University, Stanford, CA
Duchenne muscular dystrophy (DMD) is caused by lack of dystrophin expression in
skeletal muscles. To be effective, gene therapy approaches to DMD need to target
a large number of fibers in the muscle, and the distribution of dystrophin
through the fiber length needs to be sufficient to prevent fiber degeneration.
Plasmid-based gene therapies have been shown to be a valid approach to the
treatment of a variety of disorders including DMD. Among the limitations of this
technology are the restricted distribution of plasmid following direct
intramuscular injection, the challenge of achieving widespread plasmid delivery
by systemic injection, and the loss of extrachromosomal plasmid over time. In
order to address the latter issue, which could prove to be a major limitation of
therapeutic efficacy in DMD, we have studied the effects of targeted plasmid
integration using a phage integrase (Phi C31) that has been shown to mediate the
integration of suitably modified plasmids into the mammalian genome. Using a
luciferase expression plasmid, we were able to monitor plasmid gene expression
non-invasively in living mice by bioluminescence imaging (BLI). Tibialis
anterior muscles of wild-type animals were injected with a plasmid carrying a
luciferase reporter gene under the control of the CK6 muscle specific promoter
and a fC31 integrase attB site. One group of muscles was co-injected with
an equal amount of a CMV-driven integrase-expressing plasmid (pCSI) to direct
site-specific integration, while contralateral muscles received empty vector (pCS).
All muscles were subjected to electroporation to achieve a high level of plasmid
delivery, and expression was followed using BLIS. Shortly after delivery, the
level of gene expression obtained in muscles that had received pCSI was 5- to
10-fold higher than in muscles receiving pCS. This increase in the gene
expression level was not due to differences in delivery efficiency and was
maintained for up to 2 years after injection.
Co-injection of an integrase plasmid resulted in up to 10-fold higher levels of
sustained luciferase expression, and we demonstrated a direct correlation
between levels of expression and plasmid integration. Likewise, dystrophin
expression in mdx muscle was enhanced by co-injection with an integrase plasmid.
Using a combination of dystrophin and luciferase plasmids, we were able to
follow the functional benefit of dystrophin expression in the dystrophic muscle.
The expression of dystrophin slowed the loss of luciferase expression associated
with muscle degeneration, and that protection was even greater when the
dystrophin plasmid was co-injected with the integrase plasmid. In the presence
of integrase, dystrophin expression was enhanced in individual fibers and was
detectable along the entire length of a subset of fibers. Those fibers were
protected indefinitely. These data demonstrate the importance of both the level
and the distribution of dystrophin expression to achieve therapeutic efficacy
and that the efficacy can be enhanced by targeted plasmid integration.
10) Gene Therapy for Duchenne Muscular Dystrophy
Using a Gutted Adenovirus Expressing Utrophin
Jatinderpal R. Deol, Renald Gilbert, Gawiyou Danialou, Mylene
Bourget, Joon-Shik Moon, Basil J. Petrof, Josephine Nalbantoglu, George Karpati
Neuromuscular Research, Montreal Neurological Institute, Montreal, QC, Canada;
Genomics and Gene Therapy Vectors, Biotechnology Research Institute, Montreal,
QC, Canada; Department of Neurology, UMDNJ-New Jersey Medical School, Newark, NJ;
Respiratory Division, Meakins Christie Laboratories, Montreal, QC, Canada
Duchenne muscular dystrophy (DMD) is characterized by a mutation in the gene
encoding a sarcolemmal protein, dystrophin (dys) and the deficiency of dys leads
to progressive loss of muscle fibers. Gutted adenovirus (AdV) vectors are less
immunogenic than their predecessors and have the capacity of carrying large DNA
inserts such as the full-length dys cDNA (13 kb). The use of gutted AdV vectors
encoding full-length dys leads to significant improvement of the dystrophic
phenotype of the mdx mouse, an animal model of DMD. However, dys behaves
as a neoantigen in genetic dys deficiency states such as the mdx and it
causes deleterious immune responses in the treated muscles. An alternative
approach would be to use utrophin (utr), a functional homologue of dys, which is
normally present only at the sarcolemma of the neuromuscular and myotendinous
junctions. Therefore, utr is not expected to behave as a neoantigen in
dys-deficient states. In fact, transgenic mdx mice expressing
extrasynaptic utr in muscle fibers have shown functional and histological
improvement. Previous findings have also shown utrophin, when expressed via
a first generation adenovirus to be sufficient and adequate in replacing
dystrophin at the sarcolemma and preventing the symptoms of DMD. Thus, in an
attempt to minimize the vector immunogenicity and to harness the therapeutic
potential of utr, we created a fully deleted AdV encoding full-length utrophin.
The vector was administered to cohorts of mdx neonates (2-4 day old) and
adults (5 to 7 week old) at doses of 1.45 x 1010 and 4.35 x 1010
virus particles respectively into the tibialis anterior (TA) muscles. In
neonates at 10 days post-injection (pi), the mean number of extrasynaptic utr+
fibers in vector vs control-injected TA was 1596 vs 114, corresponding to 58 %
transduction level. At 60 days pi, the level of transduction was found to be
near 52% corresponding to approximately 794 utr+ fibers. The levels in adults at
10 days pi were 685 and 112 for vector and control injected TAs respectively,
corresponding to a 23% transduction level. At the 10 day timepoint, there was a
14- and 6- fold increase in utr expression over control levels in neonates and
adults. In adults at 30 days pi, the mean number of utr + fibers was 770
corresponding to 35% transduction level. However, that number dropped
substantially to an average of 80 fibers at 60 days pi and remained between 80
and 120 up to the 180 days pi. At no time point were anti-utrophin antibodies
detected. The decline of the initial high utr transduction of the TA muscles
associated with time may be due to the fact that the initial extrasynaptic utr
level is not sufficiently high to prevent the natural progression of the
dystrophic phenotype. Further augmentation of the initial utr transduction
should be helpful in order to completely and functionally compensate for dys.
11) Dystrophin Expression in Muscles of Duchenne
Muscular Dystrophy Patients Following High-Density Injections of Normal Myogenic
Cells
Jacques P. Tremblay, Daniel Skuk, Marlyne Goulet, Brigitte Roy,
Pierre Chapdelaine, Raynald Roy, Francine Dugré, Jean-Pierre Bouchard, Jean-Guy
Lachance, Michel Sylvain, Louise Deschênes, Hélène Senay Anatomy/Physiology,
Laval Universite, Quebec, QC, Canada; Immunologie, CRCHUL, Quebec, QC, Canada;
Departement de Microbiologie, CHUL, Quebec, QC, Canada; Departement
Neuropediatrie, CHUL, Quebec, QC, Canada; Neurologie, Hopital de l'Enfant-Jesus,
Quebec, QC, Canada; Service de Nephrologie, Hotel-Dieu de Quebec, Quebec, QC,
Canada; Service de Microbiologie, Hotel-Dieu de Quebec, Quebec, QC, Canada
A clinical trial was conducted to test a new protocol of normal muscle-precursor
cell (MPC) allotransplantation in skeletal muscles of Duchenne muscular
dystrophy (DMD) patients. The protocol consisted on using high-density
cell-injections as a method for cell delivery and a tacrolimus-based
immunosuppression as a method to control acute rejection. Cultured MPCs obtained
from one of the patient's parents were implanted in 0.25 or 1 cm3 of a Tibialis
anterior in 9 DMD patients. MPC injections were placed 1 to 2 mm from each other,
and a similar pattern of saline injections was done in the contralateral muscle.
The patients were immuno-suppressed with tacrolimus. Muscle biopsies were
performed at the injected sites 4 weeks later. In the biopsies of the
cell-grafted sites, there were myofibers expressing donor's dystrophin in 8
patients. The percentage of myofibers expressing donor's dystrophin varied from
3.5% to 26%. Evidence of small-myofiber neoformation was observed in some
patients. Donor-derived dystrophin transcripts were detected by RT-PCR in the
cell-grafted sites in all patients. The protocol of immunosuppression was
sufficient to obtain these results, although it is not certain whether acute
rejection was efficiently controlled in all the cases. In conclusion,
intramuscular allotransplantation of normal MPCs can induce the expression of
donor-derived dystrophin in skeletal muscles of DMD patients, although this
expression is restricted to the sites of MPC injection.
12) Muscle Specific and Efficient Transgene
Expression Using the Fast and Slow Troponin I Promoters in Cell Culture
Marilyne Blain, Angela Kumar, Kenneth E. Hastings, George Karpati,
Bernard Massie, Renald Gilbert Genomics & Gene Therapy Vectors, Biotechnology
Research Institute, NRC, Montreal, QC, Canada; Neuromuscular Research Group,
Montreal Neurological Institute, McGill University, Montreal, QC, Canada
Gene replacement therapy for muscular diseases, such as Duchenne muscular
dystrophy, using viral vectors requires strong promoters with the ability to
confer muscle specific expression of the therapeutic gene product. Because
skeletal muscle consists of a mixture of slow and fast muscle fibers, an ideal
promoter for such application should direct efficient expression in both muscle
fiber types. The genetic elements regulating expression of the fast and slow
isoforms of troponin I (TnI), an abundant muscle protein, are relatively well
characterized. Theoretically, a combination of vectors expressing transgenes
regulated by TnIfast and TnIslow enhancers/promoters could be used to deliver
strong and muscle specific transgene expression in every skeletal muscle fiber
of the body.
To evaluate the usefulness of the TnIfast and TnIslow enhancers/promoters for
gene therapy, we have compared their strengths with the cytomegalovirus (CMV)
and with the short (1.3-kb) murine muscle creatine kinase (MCK) promoters in
cell culture. Plasmids expressing
-galactosidase
(-gal)
regulated by MCK, CMV or TnIfast / TnIslow enhancers/promoters were constructed.
The TnIfast construct includes the minimal promoter (200-bp) of the quail
TnIfast gene linked to three copies (150-bp each) of that gene's first-intron
enhancer element (IRE). The TnIslow construct includes the minimal promoter
(100-bp) of the human TnIslow gene linked to that gene's upstream enhancer
element of 160-bp (USE). Except for MCK, a small intron (100-bp) derived from
SV40 was inserted between the promoter and
-bal.
293A cells and C2C12 myoblasts were transfected with the different constructs
and
-gal
expression was compared 48 hrs later. Analyses were also performed in culture of
C2C12 myotubes 10 days after serum withdrawal.
In 293 cells, MCK-, TnIfast-, and TnIslow-driven expression was at least 1300
folds weaker than CMV. In non-differentiated myoblasts, TnIslow-driven
expression was 3.5 and 15 times better than TnIfast- and MCK-driven expression,
respectively. Both TnIfast and TnIslow enhancers/promoters provided high-level
-gal
expression in myotubes (35-40 % of CMV, and at least 10 times stronger than
MCK).
Our data confirmed the excellent muscle specificity and strength of TnIfast and
TnIslow enhancer/promoter elements. Their relative small size makes them very
attractive for usage in vectors with limited insert capacity such as AAV. These
excellent properties warrant further in vivo gene transfer studies using
viral vectors.
13) Efficient and Selective Gene Transfer Directed
to Muscle by Tropism-Modified Adeno-Associated Virus Vector
Chi-Yi Yu, Bing Wang, Zhong Wang, Zhongren Cao, Juan Li, Xiao Xiao
Department of Orthopaedic Surgery, School of Medicine, University of Pittsburgh,
Pittsburgh, PA
Duchenne muscular dystrophy (DMD) is a lethal genetic muscle disorder that
affects one in 3500 males. Transfer of the therapeutic gene into the diseased
muscles has great potential for the treatment of DMD patients. Gene delivery
vehicles based on recombinant adeno-associated virus (rAAV) have been generated
as promising vectors for the correction of genetic diseases. However, rAAV
primarily transduces the liver after systemic administration, reducing its
efficiency for gene transfer at skeletal muscle. The question of how to deliver
the therapeutic genes into most of the diseased myofibers specifically becomes a
challenge. Our goal is to develop an efficient and muscle-specific AAV vector
for systemic gene delivery. In this study, we genetically incorporated the
muscle-targeting peptide into AAV2 capsid and evaluated its efficiency and
selectivity for muscle targeting.
In order to validate the targeting effect of the muscle-targeting peptide for
AAV-mediated gene transfer, differentiated C2C12 myotubes were infected with
modified and wild-type vectors harboring a luciferase reporter gene. The
modified AAV2 viruses were able to transduce the differentiated C2C12 myotubes
2- to 3-fold better than wild-type AAV2. We further determined cell type
specificity of the modified AAV2 targeting. Transductions of undifferentiated
C2C12 myoblasts, HeLa cells, HepG2 cells, and 293 cells resulted in 97% ~ 99%
decreases in reporter gene activities compared to the vector carrying a
wild-type capsid. This indicated the modified AAV2 viruses significantly
increased their selectivity for targeting. We next examined if heparin could act
as a competitor to impact the transduction efficiency of the modified AAV2 in
C2C12 myotubes since a primary receptor for AAV2 has been identified to be
heparan sulfate proteoglycan. The result showed that the modified AAV2-mediated
transduction of C2C12 myotubes was not affected by heparin. Significant gene
transduction was maintained even in the presence of heparin when C2C12 myotubes
were infected with the modified AAV2 vectors. Heparin dependence was also
determined by the heparin-affinity column analysis. In contrast to wild-type
AAV2 vector, peptide-modified AAV vector was substantially detected in the
column wash, consistent with the cell-transduction studies.
Our work is the first time muscle tropism was increased through
peptide-engineered AAVs. First, we demonstrated that enhanced transduction of
myotubes is mediated by this muscle-targeting peptide. Second,
peptide-engineering AAVs decreased their transduction in non-muscular control
cell lines, such as HepG2 and 293 cells. Third, we found the modified AAV2 did
not require the heparin-dependent mechanism for muscular targeting. In summary,
this study demonstrates the ability of genetically engineered gene therapy
vectors for selective delivery of genes to target muscle cells. These results
make us believe the use of this muscle-targeting peptide might facilitate the
generation of efficient and muscle-specific AAV vectors for systemic gene
delivery in the future.
14) Histone Deacetylase Inhibitors Improve Gene
Transfer To Mature Skeletal Muscle
Nancy Larochelle, Jatinderpal R. Deol, Paul Holland, George Karpati,
Josephine Nalbantoglu Neuroimmunology, Montreal Neurological Institute,
Montreal, QC, Canada
AdV-mediated gene transfer occurs inefficiently in adult skeletal muscle due to
downregulation of the primary adenovirus receptor CAR during muscle maturation.
We have shown previously that AdV-mediated gene transfer is enhanced in CAR
transgenics, indicating that prior modulation of CAR expression results in very
efficient AdV transducibility of mature skeletal muscle. We hypothesized that
transient CAR expression could be achieved through activation of the
transcription of the endogenous CAR gene using histone deacetylase (HDAC)
inhibitors. HDACs are known to regulate gene expression via the modulation of
chromatin structure by histone acetylation and deacetylation.
To assess the effect of histone deacetylase inhibitors on CAR expression and
adenovirus-mediated gene transfer, mouse myoblasts (C2C12) were pre-treated with
the HDAC inhibitor valproic acid (VPA) and infected with AdVCMVlacZ.
Quantification of
-galactosidase
activity 24 hours post-infection revealed a 5-100 - fold increase in enzyme
activity which depended on the multiplicity of infection (MOI). CAR expression,
in cells treated with VPA, increased by a factor 3 as determined by Western
blotting. VPA treatment had minimal effect on C2C12 myoblast transducibility by
an Ad35 expressing lacZ, indicating that the effect is specific for the Ad5
receptor CAR. Similar treatment of primary myoblast cultures derived from a
patient afflicted with Duchenne muscular dystrophy (DMD) also produced a
comparable 5-300 - fold increase in
-galactosidase
activity when the VPA-treated cells were transduced with AdCMVlacZ at various
MOI.
To assess in vivo effects, adult normal and dystrophin-deficient (mdx)
mice were treated with VPA by intraperitoneal injection (ip). Gene transfer was
evaluated 7 days after intramuscular injection of AdCMVlacZ. In normal mice, no
difference was seen in the number of transduced fibers and in the
-galactosidase
activity between VPA-treated animals and controls. On the other hand, a 2-fold
increase was detected in mdx mice pretreated with VPA compared to
non-pretreated mice. This augmentation in transduced fibers in mdx
muscles correlated with an increase in CAR transcript (up to 7-fold) as
determined by qRT-PCR.
The HDAC inhibitor valproic acid renders muscle cells more susceptible to
adenovirus-mediated gene transfer. This may have future implications for
muscle-directed gene therapy in muscular dystrophies.
15) rAAV2/9 Mediated Gene Delivery of Acid
-Glucosidase
Corrects the Cardiac Phenotype in a Mouse Model of Pompe Disease
Christina A. Pacak, Cathryn Mah, Kerry O. Cresawn, Melissa A. Lewis,
Sean Germain, Barry J. Byrne Molecular Genetics & Microbiology, University of
Florida, Gainesville, FL; Cell & Molecular Therapy, UF, Gianesville, FL;
Pediatric Cardiology, UF, Gianesville, FL
The long term goal of this project is to develop a clinically relevant gene
therapy approach for the treatment of Pompe Disease. Pompe Disease is a form of
muscular dystrophy and metabolic myopathy caused by mutations in the acid alpha
glucosidase (GAA) gene. An insufficient amount of GAA leads to the accumulation
of glycogen in lysosomes and consequent cellular dysfunction. Cardio-respiratory
failure typically occurs in the early onset patients within the first year of
life. We have characterized the cardiac phenotype in our mouse model (gaa-/-)
at various ages. Through ECG analysis we observe a shortened PR interval by 3
months of age (gaa-/-33.41+1.35ms, cont 44.95+1.58ms)
mimicking the conduction phenotype in the human Pompe population. Abnormal
amounts of glycogen are observed in lysosomes as demonstrated by the periodic
acid shift (PAS) stain. MRI analysis shows a decrease in stroke volume (SV)(gaa-/-36.13+1.19ul,
cont 51.84+3.59ul) and a decrease in cardiac output (CO)(gaa-/-7.95+0.26ml/min,
cont 11.40+0.79ml/min) at 3 months and an increase in mass (gaa-/-181.99+10.7mg,
cont 140.79+5.12mg) by 12 months. This model of cardiac dysfunction is
being used to develop a cardiac gene delivery technique which can be applied to
many genetically inherited cardiomyopathies. Previously, we have shown that
intra-venous (IV) delivery of recombinant adeno-associated virus type 1 (rAAV2/1)
pseudotype capsid carrying the CMV-hgaa construct to 1 day old gaa-/-
neonates restores GAA activity in mice. Also, LacZ transgene delivery using the
IV administration route and rAAV2/9 pseudotype capsid resulted in 200 fold
higher levels of expression in cardiac tissue than rAAV2/1. Additional
experiments showed transduction following delivery to adults. We have now
combined rAAV2/9 with the clinically relevant IV administration route in order
to deliver the human GAA (hgaa) gene to gaa-/- mice. Neonates
treated with rAAV2/9-CMV-hgaa at a range of doses (4x105vg
4x108vg 4x1010vg) have demonstrated sustained PR interval
correction (39.38+2.42 ms). PAS stains as well as NMR analysis have shown
less glycogen accumulation in cardiac tissue of treated gaa-/- mice as
compared to untreated mice. MRI analysis shows an increase in SV and CO. Adult
gaa-/- mice have also been treated using this strategy and are being
assessed in order to determine if we have the ability to reverse the effects of
Pompe Disease in mice already presenting the cardiac phenotype. While the focus
of this project is on correction of the cardiac phenotype of Pompe Disease, our
systemic delivery route, use of the CMV promoter and the fact that GAA is a
secreted enzyme all promote correction throughout the body. GAA activity is
observed in other tissues of treated mice including skeletal muscles and liver.
These studies have demonstrated the ability of rAAV2/9 to be administered
systemically using the IV delivery route, transcend the vasculature, transduce
tissues throughout the body and ultimately prevent presentation of the cardiac
phenotypes of Pompe Disease.
16) Correction of Ventilation in Glycogen Storage
Disease Type II Mice after Gel-Mediated Delivery of Adeno-Associated Virus
Serotype 1 Vectors
Cathryn Mah, Lara R. DeRuisseau, Christina A. Pacak, Melissa A.
Lewis, David D. Fuller, Barry J. Byrne Pediatrics, Div of Cell and Molecular
Therapy, and Powell Gene Therapy Center, University of Florida, Gainesville, FL;
Physical Therapy, University of Florida, Gainesville, FL
Glycogen storage disease type II (GSDII) is an autosomal recessive disorder
caused by a lack of functional acid
-glucosidase
(GAA) and results in massive storage of glycogen in lysosomal compartments. It
is characterized by progressive skeletal muscle weakness and affected patients
suffer severe respiratory insufficiency, oftentimes requiring mechanical
ventilation. Previously, we demonstrated that a novel gel-mediated method of
delivery of recombinant adeno-associated virus serotype 1 (rAAV2/1) vector to
diaphragm in a mouse model of GSDII (Gaa-/-) could restore
therapeutic levels of diaphragmatic GAA enzymatic activity with concomitant
clearance of glycogen in vivo. We have further characterized the effects
of this treatment method on ventilatory function. Similar to the GSDII patient
population, we demonstrate in Gaa-/- mice that there is an
age-related progressive weakening of diaphragm contractile strength that is
accompanied by ventilation impairments. We administered 1x1011
particles rAAV2/1-CMV-GAA to diaphragms of Gaa-/- mice at 3,
9, and 21-mos of age via the gel method. Diaphragm contractile strength and
respiratory function were assessed by measuring force-frequency relationships
and by barometric whole-body plethysmography, respectively. For plethysmography,
ventilation was measured in conscious, unrestrained mice under conditions of
normoxia (FIO2:0.21, FICO2: 0.00) and hypercapnia (FIO2:
0.21, FICO2: 0.07) to assess the extended range of ventilatory
capacity. In mice treated at 3 mos of age, we show a significant improvement in
contractile force at 6 mos (peak force of 24.833.31 vs 16.53+0.74 N/cm2)
that is sustained through 1 yr (21.59+1.59 vs 13.94+1.15 N/cm2)
of age as compared to age-matched untreated controls. Under conditions of
normoxia, the ratio of minute ventilation (VE; mL/min) to expired CO2
(VE/VCO2) (18.65+0.73 vs 13.3+0.74), and
peak inspiratory flow (mL/sec) (4.11+0.17 vs 3.21+0.29) were
significantly improved (p<0.05) in mice treated at 3 mos of age and
tested at 6 mos as compared to untreated controls. However, these improvements
did not persist to 1 yr of age. In mice treated at 9 and 21 mos of age, there
was significant improvement in contractile function in treated diaphragms 3 mos
post-treatment (peak force of 21.28+1.49 at 1 yr and 17.21+0.29 N/cm2
vs 12.71+0.94 N/cm2 at 2 yrs of age, respectively) as compared
to age-matched untreated controls. In all rAAV2/1-treated mice, minute
ventilation and peak inspiratory flows were significantly improved during
hypercapnia. These results demonstrate that gel-mediated delivery of rAAV2/1
vectors improve ventilatory function in a mouse model of muscular dystrophy.
17) Novel Tissue-Specific Regulatory Cassettes
Direct High-Level Transgene Expression in Skeletal and Cardiac Muscle
Maja Z. Salva, Charis L. Himeda, Phillip Tai, Eiko Nishiuchi, Eric
E. Finn, James M. Allen, Paul Gregorevic, Michael J. Blankinship, Leonard A.
Meuse, Jeffrey S. Chamberlain, Stephen D. Hauschka Bioengineering, University of
Washington, Seattle, WA; Biochemistry, University of Washington, Seattle, WA;
Neurology, University of Washington, Seattle, WA
Systemic delivery of rAAV6 vectors can achieve efficient transduction of the
entire striated musculature, making this an attractive strategy for gene therapy
of Duchenne Muscular Dystrophy. However, this delivery method also transduces
cells in many non-muscle tissues, which may cause therapeutic problems including
toxicity and a transgene-directed immune response. These problems could be
avoided by limiting transgene expression through the use of muscle-specific
promoters. Unfortunately, the expression levels of such promoters following
systemic delivery of rAAV vectors are lower than viral promoters in most
skeletal muscles and negligible in cardiac muscle. The goal of this project is
to design regulatory cassettes that drive high levels of skeletal and cardiac
muscle-specific transgene expression while also being sufficiently short
(<800bp) to fit into rAAV vectors containing microdystrophin cDNA (3.8-kb). Our
lab has developed a series of muscle-specific regulatory cassettes based on the
enhancer and promoter of the murine muscle creatine kinase (MCK) gene. The CK6
cassette, previously thought to be the strongest modification, has higher
activity than the wild type cassette in skeletal muscle, but very low activity
in cardiac and diaphragm muscle. We have designed new cassettes to increase
expression, and their activities have been evaluated in various muscles and
non-muscle tissues following systemic delivery of rAAV6 vectors expressing a
human placental alkaline phosphatase reporter gene. First, we designed a 570-bp
MCK-based cassette, CK7, which exhibited activity levels similar to CK6 in
quadriceps, a predominantly fast-twitch muscle, but had significantly higher
activity in cardiac muscle, diaphragm, and soleus, a predominantly slow-twitch
muscle. To further improve expression, especially in cardiac muscle, we added a
190-bp enhancer from the murine alpha-myosin heavy chain gene (MHC)
to the CK7 cassette to produce MHCK7, a 770-bp cassette that has the highest
overall activity. When compared to CK6, the activity of MHCK7 was about 400-fold
higher in cardiac muscle and 10-fold higher in soleus, which was equivalent to
the activity of the CMV promoter. Staining for AP activity in cross-sections of
both muscles revealed strong expression in virtually all fibers. The activity in
diaphragm was about 40-fold higher, but was still considerably lower than the
CMV promoter, with only about 20% of the fibers staining positive. Finally, the
activity in quadriceps muscle was equivalent to CK6. The activity of MHCK7 in
kidney, aorta, brain, testes, and intestine was at background levels, while a
very slight increase in activity, about 50-200-fold lower than in muscle, was
detected in liver, spleen, and lung. Based on these results, the MHCK7 cassette
is currently being tested with respect to its capacity for driving
microdystrophin expression in mdx mice following intramuscular and systemic
injections of rAAV6 vectors.
18) Non-Invasive Measurement of Maximal Strength
in the Normal and Dystrophic Dog Quadriceps Using Magnetic Stimulation of the
Femoral Nerve
Frederick J. Balzer, Martin K. Childers, Janet R. Bogan, Daniel J.
Bogan, Joe N. Kornegay, Hansell H. Stedman Surgery, University of Pennsylvania
School of Medicine, Philadelphia, PA; Physical Medicine and Rehabilitation,
School of Medicine University of Missouri-Columbia, Columbia, MO; Medicine and
Surgery, College of Veterinary Medicine University of Missouri-Columbia,
Columbia, MO
FJB and MKC contributed equally to this study. We have previously reported in
detail the first gene transfer method to achieve virtually 100% myofiber
transduction in proximal limb muscles of the dog - thus establishing the
feasibility of high efficiency somatic gene transfer to striated muscle in a
large mammal. Our group is currently undertaking to study the efficacy of
therapeutic gene delivery in a canine model for Duchenne Muscular Dystrophy.
Towards this end, our group has developed a novel, completely non-invasive,
non-volitional assessment of proximal (quadriceps) muscle strength in the
dystrophic dog using magnetic stimulation of the femoral nerve. Using a computer
controlled 1.4 Tesla electro-magnetic coil capable of inductively triggering
isolated single twitches up to complete fusion and sustained supramax titanic
contraction, we initially studied quadriceps force produced by inductive
magnetic stimulation of the femoral nerve in normal dogs with the result that
supramaximal stimulation is rapidly achieved and well tolerated in lightly
sedated dogs. Following this, we repeated the same experiment on a
representative set of dystrophic dogs from the GRMD colony. Again the magnetic
stimulation was well tolerated in this context, importantly in the oldest dog
studied, a lightly sedated 18 month old GRMD male. Statistical analysis of the
quadriceps tetanic force data for normal and GRMD dogs reveals that the general
approach is highly reproducible. The average 8 month old GRMD dog tested had a
maximal quadriceps force of 1.79 N/kg body weight, only 46% that of even the
youngest normal dogs tested (3.91 N/kg at 7 weeks). In contrast, the previous
tests of relative force showed that GRMD dogs doubled their strength in the TA/EDL
muscle group from 3 to 6 months and thereafter maintained approximately 100% of
the value found in the youngest normal dogs tested. Importantly, for the
quadriceps force as measured by the present systems, the left versus right limb
correlation is even better than that previously observed for tibiotarsal
extension, and can be assessed in younger dogs. Based on our current experience
we anticipate straightforward extrapolation of this approach to the measurement
of quadriceps force in pups at 4 weeks, providing baseline information before
the onset of clinically overt muscle weakness. We anticipate that the use of
this test sequence in the DMD large animal model will allow us to define the
response to regional gene therapy in terms of clinically relevant, statistically
robust efficacy endpoints that are internally consistent and replicative.
19) Increased Transgene Expression of Dystrophin
in mdx Muscle by RNAi-Mediated Silencing of Calpain Expression
Makiya Nishikawa, Kazuhiro Hirata, Kazuya Machida, Yuki Takahashi,
Katsutoshi Yuasa, Shin'ichi Takeda, Yoshinobu Takakura Graduate School of
Pharmaceutical Sciences, Kyoto University, Kyoto, Japan; Faculty of Pharmacy,
Musashino University, Nishitokyo, Japan; Department of Molecular Therapy,
National Institute of Neuroscience, NCNP, Kodaira, Japan
Plasmid DNA-based dystrophin gene transfer can be a way to cure Duchenne
muscular dystrophy (DMD) at the molecular level. However, there are several
technical difficulties in DMD gene therapy. Although intravascular delivery of
naked plasmid DNA solves some of the problems associated with the delivery of
dystrophin gene to widespread muscle cells, a marked increase in the expression
level of dystrophin may be required for effective DMD gene therapy. Several
studies have reported that the cellular free calcium is increased in dystrophic
muscle and this increase activates calcium-dependent proteases, calpains.
Because calpains degrade dystrophin, the silencing of calpain expression in
dystrophic muscle cells may improve transgene expression of dystrophin. In this
study, we used RNA interference (RNAi) to silence calpain expression in muscle
cells. To this end, we constructed plasmid DNA expressing small interfering RNA
(siRNA) targeting one of three isoforms of calpain, under the control of U6
promoter (psiCalp). Cationic liposome-mediated transfection of psiCalp targeting
-calpain,
m-calpain or p94 (calpain 3) resulted in significant reduction in the
corresponding mRNA level in C2C12 myoblasts. The reduction in the mRNA level was
associated with the reduced calpain activity in the cells. Total calpain
activity in the gastrocnemius muscle of mdx mice (3- to 8-weeks-old) was
found to be significantly greater than that of control (C57BL/10) mice.
Intramuscular injection of 10100
g
psiCalp into mdx muscle reduced the calpain activity. Then, plasmid DNA
encoding a human micro-dystrophin cDNA (CS1, 4.9 kb), pCAG-CS1, was injected
into the gastrocnemius muscle of 3-weeks-old mdx mice. Intramuscular
injection of naked pCAG-CS1 resulted in significant, but low expression of the
micro-dystrophin. On the other hand, co-injection of psiCalp greatly increased
the expression of the micro-dystrophin, which was confirmed by immunostaining
and immunoblot analysis. These results indicate that RNAi-mediated silencing of
calpain expression is effective in increasing transgene expression of
micro-dystrophin in mdx muscle. Although the silencing might be transient,
the restoration of (micro-)dystrophin expression could inhibit aberrant
activation of calpains in muscle cells.
20) Transplantation of SM/C-2.6+ Satellite Cells
Transduced with Micro-Dystrophin CS1 cDNA by Lentiviral Vector into mdx Mice
Madoka Ikemoto, So-ichiro Fukada, Akiyoshi Uezumi, Satoru Masuda,
Ampong Beryl Nyamekye, Hiroyuki Miyoshi, Hiroshi Yamamoto, Yuko Miyagoe-Suzuki,
Shin'ichi Takeda Department of Molecular Therapy, National Institute of
Neuroscience, NCNP, Kodaira, Tokyo, Japan; Subteam for Manipulation of Cell Fate,
RIKEN Tsukuba Institute, Tsukuba, Ibaraki, Japan; Department of Immunology,
Osaka University, Suita, Osaka, Japan
Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscle disorder caused
by mutations in the dystrophin gene (14 kb cDNA). Cell therapy is one of
attractive approaches to the treatment of DMD. Satellite cells reside beneath
the basal lamina and play major roles as stem cells in growth and repair of
adult skeletal muscle. Satellite cells are expected to be an ideal cell source
for cell therapy of DMD, but identification and accumulation of a pure satellite
cell population in situ was very difficult. We developed a cell sorting system
to purify quiescent satellite cells from intact mouse skeletal muscle using a
recently developed novel monoclonal antibody SM/C-2.6 (Exp. Cell Res. 296:
245-255, 2004). First, to determine the regenerative capacity of SM/C-2.6+ cells,
we injected three kinds of cells, freshly isolated quiescent cells, passaged
cells and primary myoblasts derived from GFP-Tg mice into skeletal muscles of
dystrophin-deficient mdx mice. Grafting of freshly isolated SM/C-2.6+ cells
resulted in a large number of GFP-positive fibers at 4 weeks after the injection,
whereas grafting of the same number of passaged SM/C-2.6+ cells and primary
myoblasts lead to significantly fewer GFP-positive fibers. Surprisingly,
culturing of SM/C-2.6+ cells only once after isolation hardly reduced their
regenerative capacity. It is, therefore, possible to expand SM/C-2.6+ cells to
some extent in vitro without reduction of their regenerative capacity. Next, to
test whether autologous, genetically corrected satellite cells may represent a
possible tool for the therapy of DMD, we tried to isolate SM/C-2.6+ cells from
skeletal muscles of mdx mice and transduced them with lentiviral vector. In
dystrophic muscle, the cells other than satellite cells exceedingly infiltrate
and proliferate. Therefore, we purified satellite cells from mdx muscle using
antibodies to additional surface markers (CD45, CD31 and Sca-1). When SM/C-2.6+,
CD45-, CD31-, Sca-1- cells (mdx SM/C-2.6+ cells) were sorted and cultured in
vitro for 4 days, more than 95% of them expressed MyoD. To genetically correct
these cells, we produced a third-generation lentiviral vector expressing
micro-dystrophin CS1 (BBRC. 293: 1265-1272, 2002; Mol Ther 10: 821-828, 2004)
and then infected freshly isolated mdx SM/C-2.6+ cells with the lentiviral
vector at MOI of 200 for 16 hours. Under these conditions, more than 90% of the
cells were efficiently transduced. We, then, transplanted SM/C-2.6+ cells
transduced with CS1 into mdx muscles. Four weeks after the injection, many
fibers expressed micro-dystrophin on the sarcolemma. These data indicate that
mdx SM/C-2.6+ cells transduced with micro-dystrophin by lentiviral vector can
efficiently contribute to muscle regeneration. Thus, the combination of
SM/C-2.6+ cells and lentiviral vector represents a promising approach to cell
therapy of DMD.
21) Morpholino Antisense Oligonucleotide Induced
Exon Skipping Efficiently Restores Dystrophin Expression in the mdx Mouse
Dominic J. Wells, Caroline McCulley, Anna Graham, Ke Liu, Kim E.
Wells Cellular and Molecular Neuroscience, Imperial College London, London,
United Kingdom
Although antisense oligonucleotides (AO) have been used most commonly to inhibit
gene expression, they can also be used to modify splicing of the primary
transcript by targeting the exon splicing enhancers or the 3'/5' splice sites.
Recent studies have shown that the morpholino chemistry is particularly useful
as such AOs are not readily degraded. The mdx mouse is dystrophin deficient due
to a premature stop codon in exon 23. Using an AO that targets exon 23, we have
examined the longevity of exon skipping by RT-PCR and dystrophin
immunocytochemistry and western blots. Skipped dystrophin transcript can still
be detected 14 weeks after a single intramuscular injection of morpholino AO and
dystrophin is clearly present at least 4 months post treatment. This is
substantially longer than the expression seen in similar studies using a 2-O-methyl
phosphorothioate AO. We have used similar techniques to examine the dose/volume
relationship in preparation for clinical trials of this approach in Duchenne
muscular dystrophy (DMD). As the diaphragm of the mdx mouse is the muscle that
most closely resembles the pathology in DMD, exhibiting substantial fibrosis and
fibre loss, we have used the diaphragm to test the effect of increased fibrosis
in reducing AO uptake by the muscle fibres by treating mice at different ages.
Delivery of AO in mice younger than 2 weeks can largely prevent the development
of dystrophic pathology and treatment at later ages reduces the membrane
fragility as determined by uptake of Evans Blue dye. These pre-clinical studies
demonstrate the effectiveness of the morpholino based AO in inducing long-lived
expression of dystrophin and this approach is now being taken forward to a UK
clinical trial in DMD by the MDEX Consortium.
22) Long-Term Expression of Mini-Human Dystrophin
in Transgenic mdx Mice Improves Dystrophic Muscle Functions
Bing Wang, Juan Li, Chunlian Chen, Xiancheng Jiang, Terry O' Day,
Jon Watchko, Xiao Xiao Orthopedic Surgery, University of Pittsburgh, Pittsburgh,
PA; Pediatrics, Magee-Womens
Research Institute, University of Pittsburgh, Pittsburgh, PA; Anatomy and
Molecular Cell Biology, The State University of New York, New York, NY
BACKGROUND: Duchenne muscular dystrophy (DMD) is a lethal genetic muscle
disorder caused by recessive mutations in the dystrophin gene and affects one in
every 3500 males. The clinical and pathological characteristics of DMD patients
show progressive myopathy of skeletal and cardiac muscles and premature death of
patients. The human "mini- or micro -dystrophines" with different truncated
central rod domains have been proven to ameliorate dystrophic pathology. In this
study, our purpose is to investigate the long-term expression of human
mini-dystrophin and therapeutic benefits in transgenic mdx mice.
METHODS: Four lines of transgenic mdx mouse (Tg-Dys3849-mdx) were generated. The
3.8 kb human mini-dystrophin containing five rods (R1-2, R22-24) of the central
region and two hinges (H1 and H4) was driven by a truncated creatine-kinase
promoter with two modified enhancers (dMCK). The gene expression cassette was
cloned in an AAV vector plasmid for creation of transgenic mice. After five
generations of back-crossing with the mdx mice, one of the lines containing a
single copy of the minigene was used to test the gene expressions and muscle
functions.
RESULTS: 1) the human mini-dystrophin expression was found a majority of the of
skeletal muscles (Quad, GAS, TA, forelimb, abdominal, intercostals, thoracic and
lumbar spinal muscles), but only very limited gene expressions in DIA and no
expression in cardiac muscle; 2) Preference of gene expression was found in fast
twitch myofibers over the slow fibers; 3) the DAGs such as sarcoglycans and nNOS
were restored at sarcolemma and coincided with human mini dystrophin expression;
4) the morphology of dystrophic muscle expressing the human mini-dystorphin was
improved and central nuclei were reduced to < 2%; 5) myofiber membrane integrity
was improved by EBD test; 6) Improvement in treadmill running and grip force was
observed in transgenic mice at 6 and 10 months when compared with the
littermates (p<0.05); Also, tetanic force and specific force of TA muscle were
significantly increased at 6, 10 and 20 months (p<0.05); 7) pseudohypertrophy
was not found in GAS and TA mass (p<0.05).
Taken together, this study demonstrates that the human mini-dystrophin with 5
central rods can effectively ameliorate the pathology and improve the functions
of the dystrophic muscles in mdx mice.
23) Rescue of Dystrophin in the GRMD Dog by
Multi-Exon Skipping Using Engineered U7 snRNAs
Adeline Vulin, Aurelie Goyenvalle, Ines Barthelemy, France Leturcq,
Jean-Claude Kaplan, Olivier Danos, Stephane Blot, Luis Garcia Maladie de
Duchenne, Genethon - CNRS UMR 8115, Evry, France, Metropolitan; Laboratoire de
Neurobiologie, ENVA, Maisons Alfort, France, Metropolitan; Laboratoire de
Biochimie et de Genetique Mol, Institut Cochin, Paris, France, Metropolitan
Mutations in the dystrophin gene that create frame-shifts or premature stop
codons on the mRNA are responsible of the severe Duchenne Muscular Dystrophy (DMD),
while mutations that preserve the reading frame result in either milder or
asymptomatic phenotypes. The exon skipping stratagem makes use of compounds
designed to target key motifs on pre-mRNA to re-direct splicing in order to
eliminate detrimental exons and rescue the production of shorter-proteins still
functional. We have achieved persistent "on demand alternative splicing" that
removes several consecutive exons on the dystrophin mRNA of the GRMD dog, by
single administration of AAV vectors expressing antisense sequences coupled to a
modified U7 short nuclear (sn) RNA. We report the sustained production of
dystrophin at physiological levels in entire groups of muscles at the scale of a
large size animal model of DMD.
24) Design and Optimization of U7snRNAs for
Skipping of Exon 51 in DMD: Promising Tools for Future Clinical Trials
Aurelie Goyenvalle, Adeline Vulin, Stephanie Lorain, Annemieke
Aartsma-Rus, Judith C. T. van Deutekom, Olivier Danos, Luis Garcia DMD
Laboratory, Genethon, Evry, France, Metropolitan; Department of Human Genetics,
Leiden University Medical Center, Leiden, Netherlands
Most cases of Duchenne muscular dystrophy (DMD) are caused by dystrophin gene
mutations that disrupt the mRNA reading frame. In some cases, forced exclusion
of a single exon can restore the reading frame, given rise to a shorter, but
still functional dystrophin protein (so called quasi-dystrophin). One potential
treatment of the disorder has utilized antisense oligoribonucleotide (AO) to
induce removal of disease-associated exons during pre-mRNA processing. Indeed,
this approach has been successfully used in DMD cells in vitro with antisense
sequences against splice junctions of exon 51. Skipping of this exon would
theoretically restore a functional quasi-dystrophin in a significant subset of
DMD patients with
45-50,47-50,48-50,
49-50,
50
and
52
genotypes. However, since the AO are not self-renewed, they can not achieve long
term correction. To overcome this limitation, we have introduced antisense
sequences into small nuclear RNAs (snRNA) and vectorized them in AAV and
lentiviral vectors.
We have designed AAV and lentiviral vectors harboring chimeric U7 snRNA carrying
antisense sequences against exon 51 of the human dystrophin gene (U7-ex51).
Lentiviral vectors expressing this U7-ex51 were tested on human myoblasts,
whereas AAV vectors were injected in the transgenic hDMD mice (carrying the
human dystrophin gene).
We confirmed the skipping of the exon 51 in vitro in human myoblasts after
transduction with the lentiviral vector encoding U7-ex51 by RT-PCR. We also
detected the skipping of the exon 51 after intramuscular injection of an
AAV-U7ex51 vector in the transgenic hDMD mouse.
We have also tested the efficacy of these vectors to restore dystrophin
expression in myoblasts from patients with
49-50
and
52
deletions.
In this study, we provide evidence that efficient skipping of exon 51 can be
achieved in human cells and also in vivo after intramuscular injection in a
transgenic hDMD mice through U7snRNA shuttle. These results offer very promising
tools for clinical treatment of DMD.
25) Improvement of Muscle Mass Using shRNA
Targeting Myostatin or Activin Receptor IIb
Julie Dumonceaux, Solenne Marie, Luis Garcia Maladie de Duchenne,
Genethon, Evry, France
Duchenne Muscular Dystrophy (DMD) is the most severe degenerative disorder of
skeletal and cardiac muscle. DMD patients show a progressive muscle weakness
which begins in early childhood. Our goal is to stop this continuous muscle
wasting.
Myostatin is a negative regulator of skeletal muscle mass. This protein binds to
its cell-surface receptor (Activin receptor IIb, AcvRIIb) to inhibit both
proliferation and differentiation of myoblasts. In vivo, it was shown that
myostatin deficient mice show a double mass phenotype compared to wild type
animals. Our hypothesis is that inactivation of myostatin function may
consequently results in an increase of muscle mass in DMD patients as well as in
its murine model, the mdx mouse.
We have investigated RNA interference technique to inhibit functions of
myostatin. We decided to target AcvRIIb as well as the myostatin. Mice were
injected intra muscularly using an AAV carrying the AcvRIIb shRNA or myostatin
shRNA. One of the myostatin shRNA inhibits 95% of the mRNA expression.
Preliminary results also show that a 50 % down regulation of AcvRIIb in mdx mice
results in an 15% increase in muscle mass. The next step is now to combine on a
same AAV vector the exon skipping strategy (ie AAV-U7-SD23/BP22) and the
inactivation of myostatin function (ie AAV-AcvRIIb shRNA). This vector may serve
to both rescue dystrophin and improve muscle mass.
26) Haute Couture Vectors: Solving Clinical
Problems with Custom Designed AAV Vectors
Dawn E. Bowles, Chengwen Li, Paul E. Monahan, Joseph E. Rabinowitz,
Mavis McKenna, Jude Samulski Gene Therapy Center, University of North Carolina
at Chapel Hill, Chapel Hill, NC; Department of Biochemistry and Molecular
Biology, University of Florida, Gainesville, FL
Compelling evidence that therapeutic transgene levels can be achieved from
adeno-associated virus (AAV) vectors has not been observed in any human gene
therapy application to date. We show that AAV capsids can be tailored to solve
such clinical problems and describe here a new generation of AAV vectors
approved for use in clinical studies.
The availability of capsid DNA sequences from AAV serotypes exhibiting elevated
tissue transduction profiles, the absence of cross-neutralizing antibody between
certain serotypes (e.g. AAV2 and AAV1), and the three-dimensional structure of
the AAV2 capsid served as a roadmap for AAV2 capsid surface positions to change
as well as replacement amino acid candidates in the generation of these new
vectors.
A subset of these rationally designed AAV2 capsid variants exhibited enhanced
skeletal muscle transduction, diminished recognition by human sera, and the
ability to overcome the presence of AAV2 neutralizing antibodies all without
disrupting current AAV2 purification parameters. These chimeric AAV vector
displayed identical viral yield as parent virus AAV2, vector stability, and
long-term gene expression in vivo. Further engineering determined that
the insertion of only one amino acid in the AAV2 capsid was responsible for both
the phenotypes of skeletal muscle enhanced transduction as well as resistance to
anti-AAV2 neutralizing antibodies. Validation of these chimeric AAV vectors in
clinical trials is on-going and outcome should provide essential data required
for testing in human disorders such as muscular dystrophy and hemophilia B.
Notes: DEB and CL contributed equally to this work. Present addresses of DEB and
JER: Department of Surgery, Duke University, DUMC, Durham, NC (DEB). Center for
Translational Medicine, Thomas Jefferson University, Philadelphia, PA (JER).
27) Physiological Correction of Glycogen Storage
Disease Type II Using Adeno-Associated Virus Serotype 1 Vectors
Cathryn Mah, Christina A. Pacak, Kerry O. Cresawn, Lara R.
DeRuisseau, Sean Germain, Melissa A. Lewis, David D. Fuller, Barry J. Byrne
Pediatrics, Div. Cell and Molecular Therapy and Pediatric Cardiology, University
of Florida, Gainesville, FL; Powell Gene Therapy Center, University of Florida,
Gainesville, FL; Interdisciplinary Program in Biomedical Sciences, Genetics
Advanced Concentration, University of Florida, Gainesville, FL; Graduate Program
in the Dept of Physiological Sciences, College of Veterinary Medicine,
University of Florida, Gainesville, FL; Physical Therapy, College of Public
Health and Health Professions, University of Florida, Gainesville, FL
Glycogen storage disease type II (GSDII) is caused by a lack of functional
lysosomal acid
-glucosidase
(GAA) and results in massive storage of glycogen in lysosomal compartments,
ultimately leading to fatal severe hypertrophic cardiomyopathy and respiratory
insufficiency. Previously, we demonstrated the ability of a single intravenous
administration of recombinant adeno-associated virus serotype 1 (rAAV2/1) vector
to restore therapeutic levels of cardiac and diaphragmatic GAA enzymatic
activity with concomitant clearance of glycogen in vivo in a mouse model
of GSDII (Gaa-/-). We have further characterized both cardiac
and respiratory function in rAAV2/1-treated animals one year post-treatment.
Similar to the GSDII patient population, electrocardiogram (ECG) measurements
(P-R interval) are significantly shortened in the mouse model. In rAAV2/1-treated
mice, we show a significant improvement in cardiac conductance with prolonged
P-R intervals of 39.34+1.6 ms, as compared to untreated controls (35.58+0.57
ms) (p<0.05). In addition, using cardiac magnetic resonance imaging (MRI)
we note a marked decrease in cardiac left ventricular mass from 181.99+10.70
mg in untreated age-matched controls to 141.97+19.15 mg in the rAAV2/1-treated
mice. Furthermore the mice displayed increased diaphragmatic contractile force
to approximately 90% of wild-type peak forces with corresponding significantly
improved ventilation (particularly in frequency, minute ventilation, and peak
inspiratory flow), as measured using barometric whole body plethysmography.
These results demonstrate that in addition to biochemical and histological
correction, rAAV2/1 vectors can mediate sustained physiological correction of
both cardiac and respiratory function in a model of fatal cardiomyopathy and
muscular dystrophy.
28) Optimizing Intron Splicing Signal Overcomes
the mRNA Accumulation Barrier in Trans-Splicing AAV Vectors
Yi Lai, Dongsheng Duan Department of Molecular Microbiology and
Immunology, School of Medicine, University of Missouri-Columbia, Columbia, MO
Trans-splicing adeno-associated viral (AAV) vectors hold great promise in many
gene therapy applications. We have recently shown that rational selection of the
gene splitting site and the endogenous intron sequence can lead to extremely
efficient trans-splicing vectors compatible to that of a single intact AAV
vector (Lai et al Nature Biotechnology 23:1435, 2005). A key factor in
constructing effective trans-splicing vectors is the mRNA production from the
reconstituted viral genome. In the dystrophin gene (a therapeutic gene for
Duchenne muscular dystrophy), the 60/60/61 (exon/intron/exon) junction yields
the highest mRNA level while the 63/63/64 junction is the most efficiently
spliced junction when tested in the reconstituted trans-splicing viral genome.
However, only vectors based on the 60/60/61 junction can produce therapeutic
level protein. Many endogenous introns carry sub-optimal splicing signals. It is
not clear whether optimizing intron sequence can further improve trans-splicing
AAV vectors. In this study, we evaluated the effect of replacing the endogenous
intron with a synthetic intron that matches perfectly with the conserved motif.
We first generated two additional constructs p60/synthetic/61 and p63/synthetic/64.
In these constructs, the endogenous introns were replaced by the synthetic
intron in p60/60/61 and p63/63/64, respectively. Similar to the parent
constructs, the newly synthesized constructs also carried the double-D inverted
terminal repeat junction in the middle of the intron. RNase protection assay (RPA)
was used to determine the level of unspliced and spliced RNA, and the relative
splicing efficiency. At 48 hrs after transfection, RNA was extracted for RPA.
The splicing indices in intron 60 are quite compatible to those of the conserved
motif. Substitution of the endogenous intron with the synthetic intron resulted
in marginal but statistically not significant improvement. The mRNA level in p60/60/61
was 236.5 + 132.5 (relative unit) and it was 300.2 + 86.6 in p60/synthetic/61
(p > 0.05). However, the same replacement in the 63/64 exon junction led to a
three-fold increase in accumulated mRNA from 124.3 + 5.8 to 362.4 +
32.3 (p < 0.05). We next examined the relative splicing efficiency.
Interestingly, the synthetic intron did not augment the already efficient
splicing in the 63/63/64 junction. However, the relatively poor splicing in p60/60/61
was improved. The ratio of spliced to unspliced RNA increased from 2.8 +
0.3 in p60/60/61 to 7.7 + 1.9 in p60/synthetic/61. The percentage of
splicing also increased from 72.6 % to 85.2 %.To further explore the molecular
mechanisms, we quantified the pre-mRNA stability. At 48 hrs after transfection,
actinomycin D was added to cell culture to stop de novo RNA synthesis. RNA was
extracted at 0, 0.5, 1 and 3 hrs after actinomycin D treatment. In these studies,
we observed increased pre-mRNA stability in both 60/synthetic/61 and 63/synthetic/64
transcripts. Taken together, our results suggest that optimizing intron sequence
may boost the transduction efficiency of trans-splicing AAV vectors.
29) Efficient Dystrophin Production in Response to
Local and Systemic Administration of Morpholino Antisense Oligonucleotides in
the Mdx Mouse
Ian R. Graham, Francesca C. Thorogood, Helen Foster, George Dickson
Centre for Biomedical Sciences, Royal Holloway - University of London, Egham,
Surrey, United Kingdom
Duchenne muscular dystrophy (DMD) is an X-linked recessive inherited disease
that affects 1 in 3,500 male births. It is characterised by severe muscle
wasting and muscle degeneration. Affected patients are frequently
wheelchair-bound prior to 12 years and usually die in their late teens or early
twenties as a result of respiratory or cardiac failure. DMD arises as a result
of frameshift mutations (often gross deletions) in the gene encoding dystrophin,
a 427kDa protein of the muscle sarcolemma that functions as a molecular scaffold
connecting the intracellular actin cytoskeleton with the extracellular matrix.
Such mutations result in a complete absence of dystrophin protein and lead to
the DMD phenotype, for which there is currently no cure.
Gene therapy approaches have traditionally been based on gene augmentation
strategies, in which a functional copy of the dystrophin cDNA, or truncations
thereof, has been introduced on viral or plasmid vectors. In an alternative
strategy, we and others have been developing the use of antisense
oligonucleotides (AO) to induce the exclusion of specific exons during the
splicing of dystrophin pre-mRNA. We have used the mdx mouse, a
naturally-occurring model of DMD carrying a stop codon in exon 23 of the
dystrophin gene, to demonstrate that exclusion (skipping) of exon 23 in response
to AO does indeed result in de novo production of dystrophin by modulation of
the splicing pathway.
Recent advances in oligonucleotide chemistry and design have allowed us to
compare the efficiency of delivery and bioactivity of AOs based on morpholino
phosphorodiamidate backbones with those of the previous 2'-O-methyl chemistry.
Again using the mdx mouse, we have performed dose-response and time-course
studies of morpholino AOs targeted to exon 23, by both intramuscular and
intravenous delivery. In this way, we have demonstrated widespread and efficient
AO-induced dystrophin production by both delivery routes, in a manner which
shows AO therapy to be a potentially viable treatment for DMD in a clinical
setting.
30) Effective Repetitive Dystrophin Gene Transfer
into Skeletal Muscle of Adult mdx Mice Using a Helper-Dependent Adenovirus
Vector Expressing the Coxsackievirus and Adenovirus Receptor (CAR) and
Dystrophin
Yuji Uchida, Yasushi Maeda, En Kimura, Makoto Uchino Department of
Neurology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto,
Japan
The helper-dependent adenovirus (HDAd) vector is less immunogenic and has a
larger cloning capacity of up to 37 kb enough to carry the full-length
dystrophin cDNA. However, high and long-term expression of dystrophin transduced
to mature muscle still remains difficult. One of the main reasons for this is
that the expression of the coxsakievirus and adenovirus receptor (CAR) is very
low in mature muscle. We have constructed two different HDAd vectors. One
contains the LacZ and the murine full-length dystrophin expression cassette (HDAdLacZ-dys),
and the other is a new, improved vector containing the CAR and the dystrophin
expression cassette (HDAdCAR-dys). We initially demonstrated high dystrophin
expression and prevention of the dystrophic pathology in mdx muscle injected
during the neonatal phase with HDAdLacZ-dys. Furthermore, we demonstrated that
repeated injections of HDAdCAR-dys into mature muscle led to approximately nine
times greater dystrophin-positive fibers in number than a single injection,
thereby recovering the expression of dystrophin-associated proteins. This data
has also shown that HDAdCAR-dys enabled administration of adenovirus (Ad) vector
to the host with pre-existing immunity to the same serotype of Ad. Repetitive
injections of the HDAd vector containing the CAR and the dystrophin expression
cassette could improve the efficiency of subsequent dystrophin gene transfer to
mature mdx muscle. This result suggests that our new HDAd vector will provide a
novel gene therapy strategy for Duchenne muscular dystrophy. In addition, we
injected mdx mice intraperitoneally with HDAdLacZ-dys to transduce into
diaphragm.