RESUMOS QUE SERÃO APRESENTADOS NO 8o ENCONTRO ANUAL DA SOCIEDADE ANUAL DA SOCIEDADE AMERICANA DE TERAPIA GÊNICA - 1 a 5 DE JULHO DE 2005, ST LOUIS, MISSOURI
1) Antisense Therapy for Duchenne Muscular
Dystrophy: A Realistic Possibility
Qi L. Lu, Adam Rabinowitz, HaiFang Yin,
Julia Alter, George Bou-Gharios, Terrence Partridge. Muscular Dystrophy
Laboratory, Neuromuscular/ALS Center, Carolinas Medical Center, Charlotte, NC;
Muscle Cell Biology, MRC Clinical Science Centre, London, United Kingdom;
Department of Renal Medicine, Imperial College, Hammersmith Hospital Campus,
London, United Kingdom
Duchenne Muscular Dystrophy (DMD) is the most common form of muscular dystrophy
affecting 1 in every 3500 live male births. The disease is characterized by
severe muscle wasting and weakness, which becomes clinically evident between the
ages of 3 to 5 years. The milder form of the disease is Becker muscular
dystrophy (BMD) with a spectrum of phenotypes ranging from almost asymptomatic
to mild forms of DMD. Both DMD and BMD are the results of mutations in the
dystrophin gene. The phenotypic distinction between DMD/BMD can be explained by
the reading-frame theory where DMD is caused by frame-shifting or nonsense
mutations leading to premature termination of protein synthesis whereas BMD is
caused by mutations resulting in altered, but in-frame transcripts. One
promising alternative approach for treatment of DMD is antisense oligonucleotide
(AO)-mediated gene correction at RNA level (antisense therapy). This technique
uses small oligonucleotides to correct frame-shifting or nonsense mutations by
skipping a mutated exon or exons which disrupt the reading frame in such a way
that restores reading frame of the dystrophin transcript. This results in the
expression of a truncated, but at least partially functional dystrophin.
Antisense therapy is uniquely applicable to DMD treatment for many reasons.
First, the gene consists of 79 exons and muscle form of dystrophin protein can
be divided into amino terminal, rod, cysteine-rich and carboxy terminal domains.
However, the rod domain of the dystrophin gene appears not to be critical for
its functions. Second, the majority of DMD mutations occur within this
non-critical region of the dystrophin gene. Thus correction of frame-shifting
mutations by skipping the mutated exon or other exons necessary for restoration
of reading frame will retain critical functions of the protein. We have
demonstrated that specifically designed 2-O-methyl
phosphorothioate AO (20MeAO) delivered by intramuscular injections was able to
effectively skip the mutated region (exon) of the dystrophin gene in animal
model of DMD with functional improvement in the muscles. Dystrophin induced by
20MeAO remains at detectable levels even 3 months after intramuscular injections.
This instigated clinical trials in several countries. We now demonstrated that
the AOs delivered by simple intra-venous injections can induce dystrophin
expression in body-wide skeletal muscles up to therapeutic levels. The
simplicity and safety of the antisense therapy provide a realistic possibility
for treatment of the majority of DMD mutations.
2) Ex Vivo Gene Therapy for Duchenne
Muscular Dystrophy: Lentiviral and PhiC31 Integrase Approaches
Simon P. Quenneville, Pierre Chapdelaine,
Joël Rousseau, Michele P. Calos, Jacques P. Tremblay. Genetique Humaine, CRCHUL,
Sainte-Foy, QC, Canada; Department of Genetics, Stanford University School of
Medecine, Stanford, CA
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 and a non viral method
were tested to perform the genetic modification. The co-transfection (nucleofection)
of a PhiC31 integrase and a dystrophin expressing plasmid as already led to a
stable expression of the full length dystrophin1. This was the
biggest expression cassette ever stabilized in primary cultured human myoblasts.
Using this method, we are now capable of transferring transgene expressing cells
into a muscle, leading to expression into the muscle fibers. However, this
method is not very efficient. This problem could be solved using viral vectors.
We have 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. We were able
to detect the expression of both transgenes into these muscle fibers one month
after the engraftment. Evans bleu will be injected in the blood of the mice
before an eccentric exercise. We will verify whether the expression of
dystrophin in the muscle fibers reduces their damage during the exercise and
thus the incorporation of Evans blue. This work indicates that ex vivo
gene therapy is a possible approach to treat Duchenne muscular dystrophy.
1-Quenneville SP et al., Mol Ther. 2004 Oct;10(4):679-87.
3) Immunity to AAV-Mediated Gene Therapy in
a Random-Bred Canine Model of Duchenne Muscular Dystrophy
Zejing Wang, Michael J. Blankinship, Paul
Gregorevic, Marie-Terese Little, Rainer J. Storb, James M. Allen, Stephen J.
Tapscott, Jeffrey S. Chamberlain, Christian S. Kuhr. Transplantation Biology,
Fred Hutchinson Cancer Research Center, Seattle, WA; Human Biology, Fred
Hutchinson Cancer Research Center, Seattle, WA; Neurology, University of
Washington, Seattle, WA
Introduction: Duchenne muscular dystrophy (DMD) is caused by mutations in
the dystrophin gene. Studies in the mdx mouse model of DMD have shown that
muscle membrane integrity and function can be improved by AAV-mediated delivery
of a functional dystrophin protein. To assess the potential clinical utility of
treating human DMD patients with AAV-mediated gene delivery, we performed a
series of direct intra-muscular injections in random-bred normal dogs and in
dogs with muscular dystrophy caused by a dystrophin mutation (xmd dogs).
Methods: AAV serotypes 2 and 6 carrying different promoter-transgene
cassettes were produced as previously described for the murine studies and
purified either on a heparin column or with a combination of heparin column and
cesium chloride gradient. Direct intramuscular injections of virus (1012,
1010, or 108 viral genomes per site) in a total volume of
250 ul were performed. The injection sites were biopsied under anesthesia
between 2 and 12 weeks after injection. Results: Injection of either AAV6
or AAV2 expressing CMV-b-galactosidase (b-gal) induced a strong inflammatory
response containing both CD8+ and CD4+ T-lymphocytes with peak tissue
destruction 4 weeks following viral injection. A similar robust immune response
was seen with injection of AAV6-RSV-alkaline-phosphatase, AAV6-CMV-canine-factor-IX
(cFIX) and with empty AAV6 capsid alone. Additional purification of the
AAV6-CMV-cFIX by centrifugation through a cesium chloride gradient did not
diminish the immune response. Continuous immunosuppression with cyclosporine (CSP)
and mycophenolate mofetil (MMF) largely prevented the immune response to
AAV6-CMV-b-gal in a normal dog for up to four weeks and permitted robust
transgene expression. The same immunosuppressive regimen did not prevent an
immune response to AAV6-CMV-cFIX or AAV6-CMV-human-micro-dystrophin in an xmd
dog, suggesting that a more aggressive immunosuppressive regimen might be
necessary in the xmd dog. Conclusions: Taken together, our results
suggest that AAV capsid proteins, or proteins associated with the capsid, elicit
significant immune responses when directly injected into skeletal muscle of
normal random-bred dogs. The combination of CSP and MMF effectively prevents the
immune responses in a normal dog, but not in an xmd dog, possibly due to the
pre-existing inflammatory nature of the DMD muscle disease or due to genetic
variation in a random-bred animal population. The robust immune responses to
AAV2 and AAV6 in random-bred dogs contrast with the lack of an immune response
in studies by others in inbred mice and dogs. Further studies will be necessary
to determine the nature of the immune responses and to develop appropriate
immunosuppressive regimen for AAV gene delivery to xmd muscle.
4) Stable Genome Alteration of the
Dystrophin Gene for the Treatment of Duchenne Muscular Dystrophy (DMD) Due to
Frame-Shift Mutations Using Oligonucleotide-Mediated Gene Editing
Carmen Bertoni, Thomas A. Rando. Neurology,
Stanford Universdity School of Medicine, Stanford, CA
Duchenne muscular dystrophy is an X linked neuromuscular disorder characterized
by lack of dystrophin gene expression. Mutations in the dystrophin gene include
single point mutations, large deletions and frame shift mutations. Genome
editing of the dystrophin gene represents an attractive approach to restoration
of dystrophin into skeletal muscle of DMD patients. Oligonucleotide-mediated
single base substitution has the potential to completely correct the mutation in
the case of single point mutations, or to restore the reading frame of the
dystrophin gene to correct frame shift mutations. Furthermore
oligonucleotide-mediated gene correction allows the gene to remain under the
control of its own regulatory mechanisms.
We have tested the applicability of genome editing of the dystrophin gene in the
mdx mouse 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 single
stranded oligonucleotides (ssODNs) complimentary to the coding or the non-coding
strand of the donor site of exon 23. Modifications were inserted into the
oligonucleotides in an attempt to recruit specific repair processes present in
eukaryotic cells and increase the efficiency of the repair process. For these
kinds of studies, muscle cells represent an very appealing system since the
repair processes involved can be characterized in dividing cells (proliferating
muscle precursor cells) as well as mature myofibers at the terminally
differentiated stage, thus providing further insight in the process that
regulate gene correction.
CpG modifications were introduced at the 3
end of ssODNs as well as 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. Correction of the dystrophin gene was shown to occur at the
genomic level using all types of oligonucleotides and was shown to be 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 confirm the applicability of genome
editing in cases in which DMD is due to single point and frame-shift mutations.
5) Embryonic Stem Cell-Mediated
Regenerative Therapy for Duchenne Muscular Dystrophy
Shiro Ozasa, Shigemi Kimura, Makoto
Matsukura, Kaori Ito, Makoto Ikezawa, Hiroe Kawano, Teruhisa Miike, Kenichi
Yamamura, Kimi Araki, Kuniya Abe Hitoshi Niwa. Department of Child Development,
Kumamoto University Graduate School, Kumamoto, Japan; Institute of Molecular
Embryology and Genetics, Kumamoto University Graduate School, Kumamoto, Japan;
Research & Development Team for Mammalian Cellular Dynamics, BioResource Center,
RIKEN Tsukuba Institute, Tsukuba, Ibaragi, Japan; Laboratory of Pluripotent Cell
Studies, RIKEN center for developmental Biology, Kobe, Hyogo, Japan
Introduction
Duchenne muscular dystrophy (DMD) is a severe muscle degenerative disorder
caused by mutations in the dystrophin gene. Embryonic stem cell (ES cell)
transplantation is one of the more promising therapies because ES cells are
available in large quantities and can serve to systemically restore affected
muscles of DMD patients. Although ES cells have shown to be capable of
differentiating into various tissues and cell types, it has been difficult to
induce differentiation of ES cells specifically into muscle fibers only. We are
proposing a method to overcome this impediment by establishing genetically
engineered ES cells which harbor a tetracycline-regulated expression vector
encoding the myogenic transcriptional factor MyoD.
Methods and Results
ZHTc6 is a mouse-derived ES cell line in which Tet-Off System is integrated.
ZHTc6 can be induced to express the POU transcriptional factor Oct-3/4 on the
removal of doxycycline, a derivative of tetracycline. We cultured ZHTc6 without
feeders in LIF-supplemented medium. We constructed Supertargeting Vector-MyoD,
which is targeted upon the electroporation to invoke the homologous
recombination and replace the inducible Oct-3/4 gene of ZHTc6 with MyoD gene.
Southern-blot analysis revealed that three out of 6 clones were recombinants.
The recombinants, hereinafter referred to as ZHTc6-MyoDs, were induced to
differentiate nonspecifically in the differentiation medium in the absence of
doxycycline. Western analysis of the differentiated cells revealed the
expression of MyoD, desmin, myogenin and fast myosin heavy chain (fMHC). The
involvement of MyoD in myotube formation was substantiated by colocalization of
MyoD with the muscle-specific protein dystrophin as revealed by
immunocytochemical analysis. Desmin and fMHC were also detected by
immunocytochemical analysis. Serial flow cytometric analysis revealed that the
undifferentiated ZHTc6-MyoDs initially expressed Sca1 and c-kit at a high level
(more than 90%), but the expression gradually decreased during differentiation.
To the contrary, only a small fraction (less than 1%) of ZHTc6-MyoDs expressed
CD34 throughout the differentiation. We intramuscularly injected 2-day-differentiated
ZHTc6-MyoDs into gastrocnemius muscles of adult mdx-nude mice. Seven weeks after
injection, tumors developed in the injected area to the size of 20 mm in
diameter. Examination of the tumor specimen uncovered the presence of the
clusters of dystrophin-positive myofibers surrounded by granular tumor cells.
Conclusion
In this system, we can maintain ES cells undifferentiated in the presence of
doxycycline and induce ES cells to undergo myogenic specification on the removal
of doxycycline in vitro.
6) Development of rAAV-Based Skeletal and
Cardiac Muscle Regulatory Cassettes for Gene Therapy of Duchenne Muscular
Dystrophy
Maja Z. Salva, Charis L. Himeda, Phillip
Tai, Michael J. Blankinship, Paul Gregorevic, James M. Allen, Leonard A. Meuse,
Jeffrey S. Chamberlain, Stephen D. Hauschka. Bioengineering, Univeristy of
Washington, Seattle, WA; Biochemistry, University of Washington, Seattle, WA;
Neurology and Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research
Center, University of Washington, Seattle, WA
A promising gene therapy approach for treatment of DMD involves systemic
delivery of rAAV vectors encoding microdystrophin. This method was shown to
mediate efficient transduction of all striated muscles and high-level transgene
expression driven by the ubiquitous CMV promoter. However, the use of viral
promoters increases the risk of an immune response, largely due to transgene
expression in antigen-presenting cells, and may thus be inadequate for long-term
therapy. An ideal regulatory cassette for gene therapy of DMD thus needs to
direct high-level, tissue-specific expression in skeletal and cardiac muscle and
be short enough to package into the rAAV microdystrophin construct.
Our lab has developed a series of muscle-specific regulatory cassettes based on
the murine muscle creatine kinase (MCK) gene enhancer and promoter regions. The
previous
best
small cassette, CK6 (580bp) drives microdystrophin expression in skeletal muscle
following systemic rAAV delivery, at 10% of the level of the CMV promoter.
However, expression in heart and diaphragm is very low. To improve expression,
we combined the wild type MCK enhancer with a 190bp enhancer region from the
murine alpha myosin heavy chain (MyHC) gene, which is expressed at high levels
in cardiac muscle. Addition of the MyHC enhancer increased activity in MM14
skeletal myocytes by 5-fold as assessed by alkaline phosphatase reporter gene
activity. Furthermore, high-level expression was detected in skeletal muscle, as
well as heart and diaphragm after systemic delivery in mice of rAAV6 vectors
encoding these constructs. The activity of the hybrid cassette was 7-fold higher
in heart and 3-fold higher in diaphragm and soleus (predominantly slow-twitch
muscle fibers) when compared to the wild type MCK cassette, while the activity
in tibialis anterioris (predominantly fast-twitch muscle fibers) was unchanged.
Expression was restricted to muscle tissue, as evidenced by nearly undetectable
levels in the liver, spleen, lung, and aorta. Additional cassettes comprising
the MyHC enhancer and various modifications of the minimal MCK cassette have
also been tested in MM14 skeletal myocytes. The strongest cassettes were shown
to possess 12-fold higher activity than the wild type MCK cassette, and were
about 55% as active as the CMV promoter. These contained a 63bp deletion within
the MCK enhancer, addition of a 50bp region downstream of the transcription
initiation site, and a mutation that creates a consensus initiator-binding site
from the terminal transferase promoter. We are currently testing activity of
these constructs in mouse muscle and non-muscle tissues following systemic
delivery of rAAV6 vectors. The strongest tissue-specific cassette will be
evaluated for long-term expression of therapeutic levels of microdystrophin in
the mdx mouse model for DMD.
7) Enhanced Plasmid-Mediated Dystrophin
Expression in the mdx Mouse Model for Duchenne Muscular Dystrophy by a
PhiC31 Integrase Plasmid System
Carmen Bertoni, Sohail Jarrahian, Thurman
M. Wheeler, Yining Li, Eric C. Olivares, Michele P. Calos, Thomas A. Rando.
Neurology, Stanford University School of Medicine, Stanford, CA; Genetics,
Stanford University School of Medicine, Stanford, CA
Duchenne muscular dystrophy is caused by lack of dystrophin expression in
skeletal muscles and is characterized by progressive degeneration of muscle
fibers. 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. Concerns, however, have been raised about
the ability of extrachromosomal vectors to sustain gene expression for prolonged
periods of time at levels that are therapeutic.
We have tested the ability of the phiC31 integrase system to integrate plasmid
vectors stably into the chromosomes of terminally differentiated myonuclei and
maintain transgene expression over prolonged periods of time. 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 phiC31
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 a bioluminescence live imaging
system (BLIS). Shortly after delivery, the level of gene expression obtained in
muscles that had received pCSI was 10-fold higher than in muscles receiving pCS.
This increase in the gene expression level was maintained for up to 2 years
after injection. Site-specific integration of the luciferase attB plasmid
was confirmed by PCR analysis.
The beneficial effects of phiC31-mediated integration were also tested in the
mdx mouse model for DMD. Muscles were co-injected with an attB
plasmid carrying the luciferase reporter gene driven by the CK6 promoter and an
attB plasmid carrying the full-length dystrophin gene, also driven by the
CK6 promoter, in the presence of either pCS or pCSI. BLIS analysis showed
persistent luciferase expression only in muscles co-injected with the integrase
expression vector. The level of dystrophin expression was higher in muscles
co-injected with pCSI, as demonstrated by RT-PCR analysis. Six months after gene
delivery, the overall increase in dystrophin expression resulted in more than
twice as many dystrophin-positive fibers in pCSI-injected muscles, compared to
muscles injected with pCS. Distribution of dystrophin along the length of the
fibers was also greater in muscle receiving the integrase plasmid. Plasmid
integration was also confirmed in muscles co-injected with pCSI. CD4 and CD8
staining showed no increase in the inflammatory response in muscles that
received integrase. Site-specific integration of plasmid DNA mediated by phiC31
integrase enhance plasmid-mediated gene expression and has a relevant clinical
application for muscle diseases such as DMD.
8) Sustained Whole-Body Functional Rescue
by Systemic Delivery of AAV8 Vectors in Heart Failure and Muscular Dystrophy
Hamsters
Tong Zhu, Liqiao Zhou, Satsuki Mori, Zhong
Wang, Charles Mctiernan, Chunping Qiao, Chunlian Chen, Daowen Wang, Juan Li,
Xiao Xiao. Molecular Genetics and Biochemistry, University of Pittsburgh School
of Medicine, Pittsburgh, PA; Cardiovascular Institute, University of Pittsburgh
School of Medicine, Pittsburgh, PA; Cardiology, Tongji Hospital,Huazhong Science
& Technology University, Wuhan, Hubei, China
The success of muscular dystrophy gene therapy requires widespread and stable
gene delivery with minimal invasiveness. Using the delta-sarcoglycan (SG)-deficient
hamster (TO-2), a congestive heart failure and muscular dystrophy hamster model,
here we show that a single injection of double-stranded adeno-associated virus
serotype 8 vector carrying human delta-sarcoglycan gene (dsAAV8-SG),
without the need of any physical or pharmaceutical interventions, achieved
nearly complete gene transfer and tissue-specific expression in the heart and
skeletal muscles of the diseases hamsters.
Broad and sustained ( >12 months) restoration of the missing delta-sarcoglycan
gene in the TO-2 hamsters corrected muscle cell membrane leakiness throughout
the body, and normalized serum creatine kinase levels (an 50-100 fold drop).
Histology examination revealed minimal or absence of central nucleation,
fibrosis, and calcification in the skeletal muscle as well as the heart. Whole
body functional analysis such as treadmill running showed dramatic improvement,
similar to the wild-type F1B hamsters. Furthermore, cardiac function studies
such as echocardiography revealed significantly increased %FS and decreased
LVEDD and LVESD in the treated TO-2 hamsters. The survival time of treated TO-2
hamsters was dramatically prolonged.
To the summary, our study demonstrates that systemic AAV8 vector-mediated gene
transfer could persistently ameliorate of cardiac and skeletal muscle pathology,
profoundly improve cardiac and whole-body functions, and significantly prolong
the life-span of the treated TO-2 hamsters. Future research is warranted to see
if such profound gene delivery efficiency as well as therapeutic efficacy can be
duplicated in large animals and eventually in human patients.
9) Dystrophin in Vascular Smooth Muscle Is
Important for Duchenne Muscular Dystrophy Therapy
Kaori Ito, Shigemi Kimura, Gail D. Thomas,
Shiro Ozasa, Makoto Matsukura, Makoto Ikezawa, Hiroe Ueno, Kowashi Yoshioka,
Misao Suzuki, Takeshi Miwa Teruhisa Miike. Department of Child Development,
Faculty of Medical and Pharmaceutical Sciences Kumamoto University Graduate
School, Kumamoto, Japan; Department of Internal Medicine, Division of
Hypertension, University of Texas Southwestern Medical Center, Dallas, TX;
Division of Transgenic Technology Center for Animal Resources and Development
(CARD), Kumamoto University, Kumamoto, Japan; Department of Oncogene Research,
Research Institute for Microbial Deseases, Osaka University, Suita, Osaka, Japan
Duchenne muscular dystrophy (DMD) is an X-linked fatal disease caused by
mutations of the gene encoding the cytoskeletal protein dystrophin, which is
associated with a complex of proteins that spans the membrane and effectively
links the cytoskeleton to the extracellular matrix. Despite the abundance of
information about the molecular basis of this disease, there is currently no
effective treatment for DMD because the mechanism by which dystrophin deficiency
produces the clinical phenotype is poorly understood. Our previous data showed
that dystrophin expression is normally detectable in smooth muscle cells (SMCs)
as well as in skeletal muscle cells. Likewise, it is defective in SMCs of DMD
patients and mdx mice, an animal model of DMD. Recent studies have revealed that
focal multicellular myocytolytic lesions in muscular dystrophies may represent
functional vascular abnormalities, regardless of whether or not they are primary.
Although vascular dysfunction in DMD patients and mdx mice has been also
reported, it is considered that the dystrophin deficiency of skeletal muscle and/or
vascular endothelium, not vascular smooth muscle cells (VSMCs), is responsible
for the vascular abnormalities.
We have hypothesized that dystrophin deficiency of VSMCs leads to vascular
dysfunction and exacerbates muscle pathology. Here we generated transgenic mice
expressing14Kb full-length human dystrophin cDNA under the transcriptional
control of the smooth muscle alpha-actin promoter. These mice were then crossed
with mdx mice, resulting in three independent SMTg/mdx lines, that express
dystrophin only in SMCs. The expression pattern was detectable by
semi-quantitative RT-PCR analysis and immunohistochemical staining, which showed
the specific expression of transgene in SMCs.
We assessed the vasoconstrictor response to sympathetic stimuli to determine if
dystrophin expression of VSMCs affects the blood flow regulation during exercise.
In contrast to mdx mice, alpha-adrenergic vasoconstriction in SMTg/mdx mice is
significantly attenuated during muscle contraction, resulting in amelioration of
the regulation of blood flow. Furthermore serum CK levels of SMTg/mdx mice were
significantly reduced in comparison with those of mdx mice. This is the first
report of a functional role for dystrophin in VSMCs and the resulting partial
restoration of phenotype. These results suggest that VSMCs can be an effective
target for treatment of DMD. Further, we believe that our SMTg/mdx mouse model
is worth exploring to gain a better understanding of the functions of dystrophin
in VSMCs and the pathophysiology of DMD patients.
10) An Ideal Therapeutic Agent for Duchenne
Muscular Dystrophy Involving a Gutted Adenovirus Expressing Full-Length Utrophin
Jatinderpal R. Deol, Renald Gilbert, Mylene
Bourget, Joon-Shik Moon, 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
Duchenne muscular dystrophy (DMD) is characterized by necrosis and progressive
loss of muscle fibers. DMD patients have a mutation in the gene encoding
dystrophin (dys), a large sarcolemmal membrane-associated cytoskeletal protein.
Gene replacement therapy using fully deleted adenoviral vectors shows great
potential for the eventual treatment of DMD.These vectors are less immunogenic
than their predecessors and have the capacity to carry the full-length dys cDNA
(13 kb). The introduction of fully deleted adenoviral (AdV) vectors encoding
full-length dys into muscles leads to significant improvement of the dystrophic
phenotype of the mdx mouse, an animal model of DMD. However, the
introduction of dys, a neoantigen, into mdx muscle causes immune
responses resulting in increased appearance of inflammatory infiltrates and
damage to the muscle. An alternative approach is to use utrophin (utr), a
functional homologue of dys, normally present only at the neuromuscular junction.
This approach of simply increasing the expression of a protein that is already
present in dystrophic muscle would prevent any immune response to the transgene
product. Thus, we have created a fully deleted AdV encoding full-length murine
utr cDNA regulated by a powerful hybrid promoter consisting of the chicken
b-actin promoter and CMV enhancer. Cells infected with the purified recombinant
AdV showed strong utr expression levels as determined by Western blot analysis.
Furthermore, during in vivo studies, high transduction levels were
obtained in the tibialis anterior (TA) muscles of mdx mice. The vector
was administered to cohorts of 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. In the first group of neonates (n=5) at 10 days
post-injection the mean number of sarcolemmal utr-positive fibers in injected vs
control-injected TA was 1596 +/- 297 vs 114 +/- 76, which corresponds to
58
% transduction level. The mean number of utr-positive fibers in the adults (n=7)
was 685 +/- 505 and 112 +/- 80 for viral and control injected TAs respectively,
which corresponds to
23
% transduction level. The numbers obtained correspond to an approximate 14 and 6
fold increase in utr expression over control levels in neonates and adults.
These results were further validated by consistent utr levels observed by
Western blots using muscle sections. Subsequent in vivo evaluations will
be performed at 30, 60, 90, 180, and 360 days post-injection. The injected
muscles will be examined for utr expression, the restoration of the
dys-associated protein complex, and the reversal of the physiological dystrophic
phenotype. Considering our preliminary data thus far, helper-dependent, fully
deleted (gutted) AdV expressing full length utr promises to be the ideal agent
for gene replacement therapy in dys-deficiency states.
11) A Novel Approach of Gentamicin Therapy
for Duchenne Muscular Dystrophy Using Hybrid Liposome and Establishment of a
System To Identify the Patients Eligible for the Treatment
Shigemi Kimura, Kaori Ito, Shro Ozasa,
Hiroe Kawano, Makoto Ikezawa, Makoto Matsukura, Ryuichi Ueoka, Yoko Matsumoto,
Msafumi Matsuo, Yasuhiro Takeshima Teruhisa Miike. Department of Child
Development, Kumamoto University Graduate School, Kumamoto, Japan; Department of
Applied Life Science, Sojo University, Kumamoto, Japan; Department of Pediatrics,
Kobe University Graduate School of Medicine, Kobe, Japan
Aminoglycoside antibiotics have been found to suppress nonsense mutations
located in the defective dystrophin gene in mdx mice, suggesting a possible
treatment for Duchenne muscular dystrophy (DMD). However, the gentamicin therapy
is hindered by several problems. First, a high dose of gentamicin, which is
needed to induce a read-through of the nonsense mutations in DMD patients, can
not be given because of oto- and nephrotoxicity. Even if this dose could be
tolerated, this high concentration cannot be sustained due to the short
half-life of gentamicin. Lastly, identifying patients that are applicable for
this therapy is difficult because: 1) only 5 to 13% of DMD patients have
nonsense mutations in the dystrophin gene, 2) it is challenging to find nonsense
mutations in the gene because dystrophin cDNA is very long (14kb), and 3) the
efficiency of aminoglycoside-induced read-through is dependent on the kind of
nonsense mutation.
In order to overcome the dose and concentration issues, we have investigated the
use of hybrid liposomes. Mdx mice, given hybrid liposome encapsulated gentamicin
(340mg/kg) intraperitoneally for 2 weeks, did not show oto- and nephrotoxicity.
In contrast, only mdx mice injected with the same dose of gentamicin only
exhibited ototoxicity. In addition, the efficiency of dystrophin positive fibers
in the mdx mice injected with hybrid liposome encapsulated gentamicin was higher
than in the gentamicin only injected mice; serum CK levels remained lower in the
hybrid liposome group than in the gentamicin only group. Therefore, the results
suggest that the hybrid liposome encapsulated gentamicin is more effective for
DMD patients than genamicin alone. In order to resolve the patient
identification problem, we have previously developed a system for identifying
candidates that qualify for aminoglycoside therapy. Fibroblasts from 9 DMD
patients with nonsense mutations of the dystrophin gene were isolated, induced
to differentiate to myogenic lineage by AdMyoD, and exposed to gentamicin. The
dystrophin expression in gentamicin exposed myotubes was monitored by in vitro
dystrophin staining and western blotting analysis. The results showed that the
gentamicin therapy is far more effective for DMD patients that have nonsense
mutation TGA than for patients that have nonsense mutation TAA and TAG.
12) Efficient Expression of the 6 kb
Mini-Dystrophin Gene by Trans-Splicing Adeno-Associated Viral (AAV) Vector
Restores the Entire Dystrophin-Associated Glycoprotein Complex and Reduces
Contraction-Induced Damage in the Mdx Mouse Model of Duchenne Muscular
Dystrophy
Yi Lai, Yongping Yue, Mingju Liu, Arka
Ghosh, John F. Engelhardt, Jeffrey S. Chamberlain, Dongsheng Duan. Department of
Molecular Microbiology and Immunology, University of Missouri, Columbia, MO;
Department of Anatomay and Cell Biology, University of Iowa; Department of
Neurology, University of Washington
Duchenne muscular dystrophy (DMD) is the most common lethal muscle disease
caused by mutations in the dystrophin gene. Adeno-associated virus (AAV) has
been used to express the 3.8 kb C-terminal deleted micro-dystrophin gene for DMD
gene therapy. Despite amelioration of some disease-associated changes, the
microgene is not fully functional. Trans-splicing AAV vectors have been
developed recently to express the 6 kb
H2-R19
mini-dystrophin gene, the most competent gene besides the full-length gene. Our
recent studies suggest that the RNA processing represents a critical barrier in
trans-splicing AAV vector. In this study, we first screened a series of
endogenous exon/intron/exon junctions by the RNase protection assay (RPA). The
56/56/57, 60/60/61 and 63/63/64 junctions hold the highest theoretic splicing
values and may represent the most favorable sites to split the dystrophin gene.
We generated synthetic mini-constructs carrying the splicing signals from each
of above junctions. We also inserted the inverted terminal repeat junction in
the middle of the intron. As a control, we included the 53/53/54 junction that
has a low splicing value. The most efficient splicing was achieved in the
63/63/64 junction where the ratio of the spliced to unspliced RNA (S/U ratio)
reached 7.1
0.9. The 60/60/61 and the 53/53/54 junctions yielded medium S/U ratios of 3.0
0.4 and 2.5
0.3, respectively. The 56/56/57 junction resulted in the lowest S/U ratio of 1.0
0.6. We also quantified the level of accumulated mRNA. Surprisingly, the highest
mRNA was obtained from the 60/60/61 junction (255.1
20.6, relative unit), followed by the 63/63/64 (139.5
6.4), the 53/53/54 (48.8
20.6) and the 56/56/57 (0.8
0.2) junctions. We next generated two sets of AAV-6 trans-splicing vectors based
on the 60/60/61 and the 63/63/64 junctions, respectively. 2 x 1010
particles of each set of vectors were delivered to the anterior tibialis (TA)
muscle of 2-m-old mdx mice. Mini-dystrophin expression was quantified at 1 m
post-infection by imunofluorescence staining. Consistent with the RPA results,
trans-splicing vectors based on the 60/60/61 junction yielded 34
fold higher expression. To further evaluate the therapeutic potential, we
delivered the 60/60/61 trans-splicing vectors (donor/acceptor co-infection, or
donor or acceptor single infection) to both the TA and the extensor digitorium
longus (EDL) muscles of the 2-m-old mdx mice. At 3 m post-infection,
transduction efficiency in co-infected TA muscle reached 60-80%. Importantly,
the entire dystrophin-associated glycoprotein complex (including dystroglycan,
sarcoglycan, dystrobrevin and syntrophin) was restored in co-infected muscle.
Furthermore, trans-splicing vector mediated mini-dystrophin expression provided
significant protection against eccentric contraction induced injury in the EDL
muscle.
13) Recombinant Adeno-Associated Viral (rAAV)
Microdystrophin Vectors as Therapeutic Tools for Duchenne Muscular Dystrophy (DMD)
Takis Athanasopoulos, Ian Graham, Helen
Foster, Norma Perez, Adelin Vulin, Vanessa Hill, Stewart Fabb, Luis Garcia,
Olivier Danos, George Dickson. 1Centre for Biomedical Sciences, School of
Biological Sciences, Royal Holloway University of London, Egham, Surrey, United
Kingdom; Gnthon
III, Evry, Paris, Cedex, 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 (that could incorporate the full dystrophin cDNA cassette) are
formidable obstacles to the development of gene therapy. AAV vectors overcome
many of the problems associated with other vector systems but accommodate
limited transgene capacity (<5kb). More than 8 AAV vector serotypes have been
identified to date with certain serotypes (1, 5, 6, 7) displaying more
favourable tropism in transducing muscle fibers compared to the traditionally
used AAV2.
A human microdystrophin cDNA (<3.8 kb) rationally designed in our lab based on
patient and preclinical data incorporated and successfully packaged in an AAV-2
based vector cassette, under the control of a CMV promoter, effectively restored
DAP complex and inhibited myofibre degeneration after local intramuscular
administrations in nude/mdx mice, as reported previously. Other human
microdystrophin versions have been developed in various labs worldwide
displaying different degrees of functionality.
However certain issues including optimization of transduction, safety and
immunogenicity vector/transgene profiling and species and tissue (muscle)
specificity still remain to be fully addressed. Here we report data on
production of human, canine and murine microdystrophin cDNA constructs via
PCR-mediated overlap extension synthesis, incorporating either constitutive or
muscle-specific promoter elements and optimal Kozak sequences for
transcriptional improvement and packaging of the cassettes into recombinant AAV
vectors of alternative serotypes (1, 5, 6) by pseudotyping. These vectors are
currently fully screened and their functionality determined/compared by
extensive in vitro and in vivo studies utilising local and
systemic routes of administration (i.m., i.p., i.v. and intra-arterial) in
dystrophic and non-dystrophic preclinical models.
14) Evaluation of Immune Responses to
Dystrophin in Humanized mdx HLA-A*0201 Dystrophic Mice: Application in Duchenne
Muscular Dystrophy Gene Therapy after U7 snRNA-Mediated Exon Skipping
Florent Ginhoux, Sabrina Turbant, Marylène
Leboeuf, David A. Gross, François Lemonnier, Luis Garcia, Olivier Danos, Jean
Davoust. Immunology, Genethon, Evry, France; Immunology, Institut Pasteur,
Paris, France
Cellular immune responses may compromise long-term expression in
Dystrophin-based gene therapy treatments. To predict cytotoxic T-cell responses
mediated by dystrophin (DYST), we designed a new H-2-negative HLA-A*0201
transgenic mouse model breaded on mdx background bearing a stop mutation in DYST
exon 23. After DYST plasmid injection we identified a shared mouse/human
specific DYST epitope located on exon 24, which elicits HLA-A*0201-restricted
cytotoxic T cell activities in all mice. We found however, that rescue of
dystrophic muscle by a single administration of an AAV vector expressing
antisense sequences linked to a modified U7 small nuclear RNA (Goyenvalle A,
Vulin A, Fougerousse F, Leturcq F, Kaplan JC, Garcia L, Danos O., Science. 2004,
306 : 1796-9) restores long term expression of DYST in muscle fibres and does
not induce CTL activity against DYST in mdx HLA-A*0201 mice. This persistent
exon skipping removes exon 23 and restores DYST exon 24 on the dystrophin
messenger mRNA of the mdx HLA-A*0201 mouse. Several procedures were then
attempted to induce muscle inflammation secondary to exon skipping. They failed
to trigger effector CTL responses and failed to trigger immune rejection of
rescued muscles fibres. We conclude that in contrast to delivery of DYST plasmid,
rescuing dystrophic muscle through U7 snRNA-mediated exon skipping does not
induce a primary immune response directed by exon 24 DYST sequence. The
knowledge of HLA-A*0201-restricted human DYST peptides will be of importance to
assay the occurrence of immune responses in HLA-A*0201 positive humans enrolled
in future clinical studies.
15) Successful Myoblast Transplantation to
Duchenne Muscular Dystrophy Patients
Jacques P. Tremblay, Daniel Skuk, Sylvain
Michel, Raynald Roy, Jean-Guy Lachance, Hélène Senay, Deschênes Louise, Goulet
Marlyne, Brigitte Roy, Jean-Pierre Bouchard. Gntique
Humaine, Centre de Recherche du CHUL, Qubec,
QC, Canada; Nphrologie,
CHUQ Pavillon Hotel Dieu, Qubec,
QC, Canada; Neurology, Centre Hospitalier Affili,
Qubec,
QC, Canada
Nine Duchenne muscular dystrophy (DMD) patients received injections of myoblasts
obtained from skeletal muscle biopsies of normal donors. Cells were injected in
1 cm3 of the Tibialis anterior by 25 or 100 parallel injections. We
performed similar patterns of saline injections in the contralateral muscles as
controls. The patients received tacrolimus for immunosuppression. Muscle
biopsies were performed at the injected sites 4 weeks later. We observed
dystrophin-positive myofibers in the cell-grafted sites: in 8 of of 9 patients.
In these 8 patients, the percentages of dystrophin positive fibers ranged from
3.5 to 26%. Eight patients had identified dystrophin-gene deletions and thus for
four of these patients these results were obtained using mAbs specifically to
epitopes coded by the deleted exons. Donor-dystrophin was absent in the control
sites. For the other patients there were several folds more dystrophin-positive
myofibers in the cell-grafted muscle than in the control muscle.
Donor-dystrophin transcripts were detected by RT-PCR (using one primer reacting
with a sequence in the deleted exons) in 8 patients. No antibodies were detected
in the host serum against the donor myoblasts. Therefore, significant dystrophin
expression can be obtained in the skeletal muscles of DMD patients following
specific conditions of cell delivery and immunosuppression.
16) Highly Efficient Exon-Skipping and
Sustained Correction of Muscular Dystrophy Using an Adeno-Associated Viral
Vector
Aurelie Goyenvalle, Adeline Vulin, Jean
Claude Kaplan, France Leturcq, Olivier Danos, Luis Garcia. DMD Laboratory,
Genethon & CNRS UMR8115, Evry, France; Laboratoire de Biochimie et de Gntique
Molculaire,
Hopital Cochin, Paris, France
In Duchenne Muscular Dystrophy (DMD), the lack of Dystrophin results in a series
of catastrophic events that lead to muscle fiber death and muscle wasting.
Dystrophin is a modular protein with a central rod domain composed of 24
spectrin-like repeats and truncated proteins missing some of these repeats can
be fully functional or at least partly deficient as seen in patients with mild
(Becker) forms of DMD. In a majority of severe DMD patients that presents out of
frame alterations of the dystrophin transcript, a translational open reading
frame would be restored by eliminating selected exon(s) from the pre mRNA. Exon
skipping naturally occurs during dystrophin mRNA processing giving rise to rare
revertant fibers that contain shortened proteins. It can be forced using
antisense sequences delivered as synthetic oligonucleotides, or attached to a
small nuclear (sn) RNA, notably U7. A
double-target
U7SmOPT containing antisense sequences from introns 22 and 23 (U7-SD23/BP22 )
was introduced in an AAV vector and packaged as an AAV2/1 pseudotype. Adult mdx
mice were injected into the Tibialis Anterior, or submitted to whole limb
arterial perfusion, and analysed between 2 and 13 weeks. Dystrophin transcripts
missing exon 23 were detected by RT-PCR, representing 15% of the amplified
material at two weeks and becoming the major species (60 to 70%) at one month.
The Dystrophin protein was readily detected both by Western blot on muscle
extracts and by immunofluorescence on tissue sections were restoration of the
Dystrophin Associated Complex was also documented. The levels of rescued
dystrophin was 3 % of normal at 2 weeks and 50 to 80% thereafter. The treated
muscles were shown to recover normal contractile and mechanical properties by
measuring resistance to eccentric contractions. Fiber resistance to
exercise-induced damage was also restored, as seen using Evans Blue exclusion.
The level and stability of U7-induced exon skipping that we observe in vivo in
the context of an AAV vector is unparalleled, and provides a strong rational for
the development of this approach in the clinic.
17) Lentivirus Mediated Dystrophin
Expression in mdx Muscles
En Kimura, Sheng Li, Brent Fall, Miki
Haraguchi, Leonard Meuse, Jeffery S. Chamberlain. Department of Neurology,
University of Washington School of Medicine, Seattle, WA; Medicine and
Biochemistry, University of Washington School of Medicine, Seattle, WA; Senator
Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, University of
Washington School of Medicine, Seattle, WA
In dystrophic muscles of the mdx mouse, a model for Duchenne muscular
dystrophy, activated-satellite cells (myoblasts) proliferate in the muscle
micro- environment during regeneration and degeneration processes. Most of these
cells terminally differentiate and form myofibers, while small numbers are
stored adjacent to myofibers as mitotically quiescent satellite cells for future
muscle regeneration.
Satellite cells and other
stem
cells are an attractive target for genetic modification by lentiviral vectors,
since these vectors enable stable gene expression of a transgene following
integration into the host cells
genomic DNA. We have generated a series of lentiviral vectors that express
various reporter genes and truncated mini- and micro- version of the dystrophin
gene, and have tested their transduction ability in various myogenic precursors
both in vitro and in vivo. Skeletal muscles of newborn mice may be
considered a good target for lentiviral vectors, because of the relatively high
numbers of activated muscle progenitor cells contributing to muscle growth and
satellite cell pools. Therefore, we tried targeting satellite cells or muscle
progenitor cells using lentiviral vectors expressing dystrophin micro-genes
using several different approaches.
Much higher levels of transduction were obtained following intramuscular
injection into neonatal muscles, which are rich with active myoblasts, compared
with muscles from young and adult mice; both muscle fibers and primary cultured
satellite cells stably expressed a GFP marker gene delivered via
lentiviral vectors. Injection of lentiviral vectors expressing a functional
micro-dystrophin/eGFP fusion protein into neonatal mdx muscles resulted
in stable expression of dystrophin for at least one year, the longest time point
current analyzed. Functional studies of the effects of this transduction are in
progress. These data show that myogenic stem cells can be stably transduced with
lentiviral vectors and can contribute to stable muscle regeneration in
dystrophic muscle by enabling continuous expression of micro-dystrophin, which
may have implications for gene therapy of Duchenne muscular dystrophy.
18) Plasmid-Mediated Gene Transfer in
mdx Mice Using Mini- and Micro-Dystrophin Constructs
Leland E. Lim, Carmen Bertoni, Thomas A.
Rando. Neurology and Neurological Sciences, Stanford University School of
Medicine, Stanford, CA; Geriatrics Research, Education, and Clinical Center, VA
Palo Alto Health Care System, Palo Alto, CA
The mdx mouse is a homologue of Duchenne muscular dystrophy (DMD), which
is due to mutations in dystrophin, a subsarcolemmal protein associated with the
actin cytoskeleton. Though the pathological changes observed in the mdx
mouse are different from those of human DMD, the mdx mouse is an
excellent model in which to study potential gene therapy approaches. One
technique is the use of
naked
plasmid DNA as a method of gene transfer. Previous work has shown that
dystrophin can be restored to the sarcolemma by intramuscular injection of a
full-length dystrophin cDNA construct. However, expression has been limited to a
small percentage of fibers close to the injection site. Furthermore, the large
size of the dystrophin cDNA presents obstacles to the design of effective
vectors and efficient uptake into muscle fibers. Here, we report the use of two
truncated dystrophin plasmid constructs introduced using electroporation into
the tibialis anterior muscles of young mdx mice. These constructs have
large deletions of the central rod domain while preserving the critical N- and
C-terminal regions. We tested whether these truncated constructs would be more
efficacious than the full-length dystrophin cDNA by virtue of improved delivery
and uptake. In order to follow the expression of dystrophin over time in living
mice, we used a luciferase reporter construct introduced into muscle along with
the dystrophin constructs. Luciferase activity can be followed non-invasively
using a bioluminescent imaging system, and the persistence of luciferase
activity in mdx mouse muscle is dependent upon the expression of
dystrophin to prevent muscle fiber breakdown and concomitant loss of luciferase
plasmids. Our results show that the truncated dystrophin constructs are at least
as effective as full-length dystrophin in preserving luciferase activity. Also,
sarcoglycan and dystroglycan components are present at the sarcolemma of the
dystrophin-expressing fibers, indicating that the truncated dystrophin
constructs are capable of restoring a functional dystrophin-glycoprotein complex.
Current analyses are focusing on the relative distribution of plasmids in muscle
to compare delivery of constructs of different sizes. We are also analyzing the
effectiveness of dystrophin expression by assessing central nucleation. Finally,
our current studies suggest that the longitudinal distribution along the length
of individual fibers is critical to its ability to protect that fiber over the
long term from the degenerative process. At this stage, we conclude that the
smaller dystrophin proteins are effective in ameliorating the dystrophic
features of mdx muscle fibers, and may be superior to full length
dystrophin constructs for the purpose of plasmid-mediated gene transfer for the
treatment of Duchenne muscular dystrophy.
19) Canine Mini-Dystrophin Gene Transfer by
AAV1 in mdx Mice Ameliorates Dystrophic Pathology and Protects Membrane
Integrity
Bing Wang, Juan Li, Liqiao Zhou, Chunping
Qiao, Mengnan Tian, Tong Zhu, Janet Bogan, Joseph Kornegay, Xiao Xiao. Dept. of
Molecular Genetics and Biochemistry and Dept. of Orthopedic Surgery, University
of Pittsburgh, Pittsburgh, PA; Department of Veterinary Medicine and Surgery,
University of Missouri, Columbia, MO
Duchenne muscular dystrophy (DMD) is the most common and lethal genetic muscle
disorders, affecting one in 3,500 males. No efficacious treatment is currently
available for DMD. While the mdx mouse is the most widely used small
animal model for DMD, the large animal model, golden retriever muscular
dystrophy (GRMD) dog, displays more similarities to human DMD patients in
clinical and pathological characteristics. Therefore, the GRMD dog is considered
a clinically more relevant and therapeutically more challenging DMD model.
To investigate AAV-mediated gene therapy for DMD in the canine model, we have
cloned and constructed a canine version of mini-dystrophin gene. The dystrophin
cDNA was partially cloned via RT-PCR from normal dog muscle. The N-terminus, 5
central rod domains and the cysteine-rich domain were spliced together,
generating the canine mini-dystrophin gene of 3.8 kb, which was subsequently
cloned into the AAV vector driven by the CMV promoter. Before testing the
mini-dystrophin in dogs that have limited availability, we first tested the
construct in the mdx mice to see if the primary biological functions
could be observed.
We show that the canine mini-dystrophin gene is efficiently expressed in both
neonatal and adult mdx mice at one to three months after AAV1 vector
injection. Immunostaining of serial muscle thin-sections, using antibodies
against rods 1
2 region of the dystrophin and against a, b, and g sarcoglycans separately,
revealed the expression of canine minidystrophin gene and the restoration of the
missing sarcoglycans at the plasma membrane. Importantly, the missing nNOS
associated with myofiber membrane was also partially restored and coincided
mini-dystrophin. H
E staining of the vector-treated muscle displayed normal histology and the lack
of fibrosis and infiltration, when compared to the contralateral untreated
gastrocnemius. We found that the central nuclei have been reduced from 70% to
25% in adult mdx group, and over 98% of nuclei were peripherally located
in areas of young mdx muscle expressing canine mini-dystrophin. Finally,
in vivo muscle cell membrane integrity test after Evans blue injection also
showed improvement and the exclusion of EBD dye leakage in
mini-dystrophin-positive areas. Thus, the results obtained in mdx mice
pave the way for further testing the canine mini-dystrophin in the GRMD dog
model.
20) An AAV Vector-Mediated Gene Transfer
into Canine Skeletal Muscle
Madoka Ikemoto, Katsutoshi Yuasa, Madoka
Yoshimura, Akiyo Nishiyama, Yuko Miyagoe-Suzuki, John McC Howell, Shin'ichi
Takeda. Department of Molecular Therapy, National Institute of Neuroscience,
NCNP, Kodaira, Tokyo, Japan; Faculty of Pharmacy, Musashino University,
Nishi-tokyo, Tokyo, Japan; Division of Veterinary and Biomedical Sciences,
Murdoch University, Perth, Western Australia, Australia
Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscle disorder caused
by mutations in the dystrophin gene (14 kb cDNA). An adeno-associated virus (AAV)
vector-mediated gene transfer is one of attractive approaches to the treatment
of DMD, but it has a limitation in insertion size up to 4.9 kb. We recently
demonstrated that the AAV vector-mediated micro-dystrophin cDNA transfer could
ameliorate dystrophic phenotypes in skeletal muscles of dystrophin-deficient
mdx mice (Mol Ther 10: 821-828, 2004). For clinical application, it is
important to examine therapeutic effects and the safety issue in larger animal
models, such as dystrophic dogs. We established a colony of beagle-based canine
X-linked muscular dystrophy in Japan (Exp Anim. 52: 93-97, 2003). To investigate
transduction efficiency in canine skeletal muscle using an AAV vector, we
injected the AAV vector encoding the LacZ gene driven by a CMV promoter (1.0-2.0
x 1013 vg/ml, 100-500
l/muscle)
into skeletal muscles of normal dogs.
-galactosidase
(-gal)
was expressed only in few fibers at 2 weeks after the injection, and not
detected at 4 or 8 weeks after the injection. Instead, large numbers of
mononuclear cells appeared around
-gal-expressing
fibers. To clarify mechanisms of low transduction and cellular infiltration in
canine muscle after transfer of the AAV vector, we examined viral infectivity
in vitro, cytotoxicity and immune responses of AAV vector transduction in
vivo. First, we infected the AAV vector into canine primary myotubes. This
in vitro study showed that the AAV vector could allow higher transgene
expression in canine myotubes than in murine ones. Second, we tested whether
injection of AAV particles elicit cytotoxicity or not. When a promoter-less AAV
vector expressing no transgene (5 x 1012 vg/muscle) was injected into
canine muscle, almost no infiltrating cells was observed in injected muscle.
Third, we investigated immune responses. A lot of CD4- or CD8-positive cells
were detected in clusters of infiltrating cells, together with elevated serum
level of anti--gal
IgG. To confirm low transduction depending on immune response, dogs received
daily oral administration of cyclosporine (20 mg/kg/day) from
5
day of the introduction of the AAV vector. Immunosuppression largely but not
completely improves transduction efficiency of the AAV vector. These results
suggest that AAV vector-mediated gene transfer elicits stronger immune responses
in canine muscle, but immune responses against transgene products can not
thoroughly explain the phenomenon. Cellular toxicity of transgene products might
also participate in these infiltrations, while cytotoxicity and immunity of the
AAV particles themselves can be negligible based on the result of a
promoter-less AAV vectors. It is indispensable to know the molecular background
of excess immune responses and cellular toxicity in canine models to establish
AAV vector-mediated gene transfer in dystrophic patients.
21) Overlapping Adeno-Associated Viral (AAV)
Vector Mediated Gene Transfer Is Dependent on Viral Serotype and the Transgene
Sequence in Skeletal Muscle
Arkasubhra Ghosh, Yongping Yue, Dongsheng
Duan. Department of Molecular Microbiology and Immunology, University of
Missouri, Columbia, MO
The small packaging capacity is one of the major hurdles for adeno-associated
virus (AAV)-mediated gene therapy. The overlapping approach has been developed
recently to expand the AAV packaging capacity (Duan et al, Mol. Ther. 4:383,
2001; Halbert et al, Nat. Biotechnol.20:697, 2002). In this approach, a large
gene is split into two partially overlapping fragments and separately packaged
into two AAV vectors, including the upstream (carrying the 5-end
of the gene) and the downstream (carrying the 3-end
of the gene) vectors. Transgene expression is achieved after homologous
recombination of the overlapping region between the upstream and the downstream
vectors in co-infected cells. Despite the promising proof-of-principle results
in the lung with AAV-6 alkaline phosphatase (AP) overlapping vectors, the
transduction efficiency in skeletal muscle has been very disappointing. With AAV-2
LacZ overlapping vectors, the efficiency is only 0.37% of that from an intact
AAV-2 LacZ vector. This level of expression is far from sufficient to treat
muscular dystrophy. In this study, we examined two potential rate-limiting
factors in the overlapping approach, including AAV serotype and the transgene
sequence. Previous studies suggest that AAV transduction in muscle is influenced
by viral serotype. AAV-6 mediates much higher gene expression than AAV-2 in
muscle. To test whether AAV-6 can improve overlapping vector-mediated gene
transfer in muscle, we delivered a total of 1 x 10e9 viral genome particles of
LacZ overlapping vectors (AAV-2 or AAV-6; 5 x 10e8 particles of each vector
including the upstream and the downstream vectors) to the anterior tibialis (TA)
muscle of 6-week-old BL10 mice. As a control, we also delivered 5 x 10e8
particles of the intact AAV LacZ vector to the contra-lateral TA muscle.
Transduction efficiency was quantified at 6 weeks later by scoring the
percentage of LacZ positive myofibers. Consistent with previous reports, less
than 0.03% of myofibers were transduced by AAV-2 overlapping vectors. However,
AAV-6 overlapping vectors yielded 35.2
5.7% transduction. This equals 42.25% of that of the transduction efficiency
from an intact AAV-6 LacZ vector. To determine whether the transgene sequence
effects the transduction efficiency, we compared AAV-6 LacZ and AAV-6 AP
overlapping vectors. Surprisingly, the transduction efficiency of AP overlapping
vectors (80.4
2.8%) reached that of the intact AP vector (84.1
1.9%). In summary, our findings suggest that AAV-6 overlapping vectors represent
a promising approach to deliver certain larger therapeutic genes for muscular
dystrophy gene therapy.
22) Evaluation of Gene Transfer Efficacy
Mediated by AAV1 and AAV6 Vectors in Skeletal Muscle of Adult Mice Following
Different Routes of Administration
Christel Rivière, Karine Poulard, Gabor
Veres, Olivier Danos, Anne M. Douar. Gene Transfert Department - CNRS UMR 8115,
Genethon, Evry, France
Skeletal muscle represents an important target tissue for gene therapy
application due to the large number of genetic muscular disorders including
Duchene Muscular Dystrophy. In addition the muscle may be also used as a
delivery platform to express therapeutic proteins into the bloodstream for
non-muscular metabolic disorders. Adeno-Associated Virus 1-based vector (rAAV1)
is often considered as the most efficient AAV serotype to deliver genes into the
muscles. In this study, we compared the performance of AAV pseudotyped (based on
AAV2 genome) vectors expressing a secreted form of the murine alkaline
phosphatase, (mSEAP) with capsid from serotype 1 and 6, in mouse skeletal muscle
using different delivery methods: direct injection into individual muscle
(intramuscular, IM), local limb distribution of vectors via the circulation
(intra-arterial delivery, IA) or systemic delivery (intravenous administration,
IV). We injected the maximal volume that each of these route allowed. We
analyzed two parameters: the levels of a reporter circulating protein (), a
relevant parameter for a depot organ strategy, and the extent of fiber
transduction in the muscles, a relevant parameter in view of a muscular disorder
therapeutic approach.
We compared rAAV1 and rAAV6 encoding mSEAP in a dose response study following IM
administration in adult C57Bl/6 mice. Both vectors showed strong and equivalent
levels of transduction at the highest doses (6x108 vg and 3x109
vg), while at the lowest dose injected (1.5x1010 vg), rAAV6 was 3-fold
more efficient than rAAV1 in transducing muscle, suggesting a differential
threshold of efficiency at sub-optimal doses.
In addition, the potential for systemic gene transfer after IV of rAAV1 and
rAAV6 at the whole body level was investigated. For the two doses tested (1x1011
vg and 3x1011 vg). AAV6 vector led to 3-fold higher levels of
circulating mSEAP levels than rAAV1. In addition, a widespread transduction of
both skeletal and cardiac tissues was observed with rAAV6 after histochemical
detection of mSEAP. However, in term of circulating protein level, IV
administration was less efficient than IM injection since the same level of
circulating mSEAP was achieved with 3x1011 vg using IV delivery
compared to3x109 vg for IM delivery.
We have also evaluated intra-arterial delivery via the femoral artery of rAAV1
and rAAV6 at two doses (1x1011 vg and 3x1011 vg). This
route yielded a 10 fold higher mSEAP levels in the serum than IV and led to
robust transgene expression pattern in the hind limb muscles.
Finally, in an attempt to increase gene transfer efficacy in muscle after IV
delivery, we co-injected the VEGF, with either rAAV1 or rAAV6. For both vectors,
the presence of VEGF did not show a positive impact on transgene expression
levels.
In conclusion, our results show that despite the high similarity between AAV1
and AAV6 capsid sequences, rAAV6 performance for muscle transduction are
superior to rAAV1 with all IM, IA or IV delivery routes. This finding prompts us
to consider AAV6 vectors as one of the most efficient viral agent to deliver
gene in skeletal muscle and further clinical applications.
23) Identification of Differentially
Expressed Genes in Duchenne Muscular Dystropy Utilising RNAi Technology:
Possible Targets for Gene Therapy
Mohammad Mahdi Ghahramani Seno, Ian R.
Graham, Ken Laing, Marita Pohlschmidt, Takis Athanasopoulos, Mark Crompton,
George J. Dickson. Centre for Biomedical Research, Royal Holloway-University of
London, Egham, Surrey, United Kingdom; Bacterial Microarray Group London, St
Georges
Hospital Medical School
University of London, London, United Kingdom
Duchenne Muscular Dystrophy (DMD) is one of a group of genetically heterogeneous
muscular dystrophies that are characterized by progressive weakness and wasting
of skeletal muscle. Loss of myofibres occurs in response to a deficiency of
dystrophin, a protein which is believed to be responsible for myofibre
maintenance and integrity. Dystrophin forms a link between the cytoskeleton and
the membrane-spanning dystrophin-associated glycoprotein complex (DAPC),
indicative of a structural role for dystrophin.
The application of gene therapy protocols for DMD still presents many daunting
challenges due partly to intrinsic features of the dystrophin gene. Hence,
improvement in the understanding of the underlying primary molecular events
leading to a dystrophic pathology might pave the way for the discovery of new
starting points.
Here we present a strategy to use RNAi technology to study the events occurring
in muscle cell development due to dystrophin deficiency. RNAi has been proven to
be a powerful technology to study molecular effects due to knockdown of single
genes. We have used a series of siRNAs to target and knock down the expression
of dystrophin in primary cultures of mouse muscle, and subsequently used
transcriptomic array analysis to identify genes whose expression was affected in
response to dystrophin deficiency. The data obtained from this experiment, which
include some very interesting potential new targets, are currently being
analysed. We are also developing a recombinant adeno-associated virus (rAAV)
vector expressing an shRNA targeting dystrophin. The use of such rAAV-shDNA
vectors will enable us to target dystrophin in vivo to obtain a better and
potentially curative insight into the pathophysiology of DMD.
24) Perivascular CD45-:Sca-1+:CD34-
Cells Are Derived from Bone Marrow Cells and Participate in Dystrophic Skeletal
Muscle Regeneration
Sheng Li, Morayma Reyes, En Kimura, Jessica
Foraker, Miki Hagakura, Leonard Meuse, Brent Fall, Jeffrey S. Chamberlain.
Department of Neurology, University of Washington School of Medicine, Seattle,
WA; Muscular Dystrophy Cooperative Research Center, University of Washington
School of Medicine, Seattle, WA
Multiple mechanisms may account for bone marrow (BM) cell incorporation into
myofibers following muscle damage. Here, we demonstrated that mouse CD45-:Sca-1+:CD34-
cells may play a role in the maturation of skeletal muscles and regeneration of
mdx4cv dystrophic skeletal muscles, an animal model for
Duchenne muscular dystrophy. To understand the origin of CD45-:Sca-1+:CD34-
cells in mouse skeletal muscle, we reconstituted lethally irradiated wild type
or mdx4cv mice with unfractionated BM cells from transgenic
mice ubiquitously expressing green fluorescence protein (GFP). 1, 2, and 6
months post-transplantation, we analyzed the skeletal muscle mononuclear cells
from the recipients by flow cytometry for GFP, CD45-PerCP, Sca-1-PE, and
CD34-APC. To our surprise, we found BM-derived (GFP+) CD45-:Sca-1+:CD34-
cells in the skeletal muscles of these GFP+ BM transplant recipients.
We also demonstrated that these BM-derived cells were localized in the
perivascular tissue by immuno-staining and that their frequency increased with
time. We were interested in the potential clinical application of these cells
for muscle diseases. Thus, we sorted CD45-:Sca-1+:CD34-
cells by fluorescence activated cell sorting (FACS) skeletal muscle mononuclear
cells and cultured them in several stem cell media (recipes), including a
low-serum medium containing specific cytokines for isolating multipotent adult
progenitor cells (MAPCs). MAPCs can be isolated from skeletal muscle and BM and
differentiate to form myotubes in vitro and in vivo. Strikingly,
we found that MAPCs were enriched up to 40 folds by sorting this population from
skeletal muscle mononuclear cells. Concomitantly with the increase in frequency
of BM-derived muscle CD45-:Sca-1+:CD34- cells,
frequency of BM-derived muscle MAPCs also increased with time
post-transplantation in dystrophic muscles. Furthermore, BM-derived muscle MAPCs
displayed similar phenotypes of endogenous muscle MAPCs, suggesting a potential
mechanism of BM cell migration to dystrophic skeletal muscles. To understand how
these BM-derived cells migrate to the muscle and once in the muscle how they
mobilize, we investigated the in vitro chemotatic response of GFP+
(BM derived) muscle MAPCs and CD45-:Sca-1+ cells isolated
from muscles of GFP+ BM transplant recipients. We found that these
cells were highly chemoattrated to stroma derived factor, SDF-1, a
chemo-attractant for cells expressing CXCR4. We also observed higher frequency
of BM-derived CD45-:Sca-1+:CD34- cells in
mdx dystrophic muscle than wild type muscle, which may be explained by
higher expression levels of SDF-1 in mdx dystrophic muscles. Taken
together, our results suggest that dystrophic muscles recruit BM cells that
localize in perivascular tissues and can be defined as CD45-:Sca-1+:CD34-.
This population when cultured enriches for MAPCs and can participate in muscle
regeneration in dystrophic muscles.
25) AAV-Mediated Myostatin Propeptide Gene
Delivery Results in Growth and Hypertrophy of Skeletal but Not Cardiac Muscles
Chunping Qiao, Jianbin Li, Tong Zhu,
Chunlian Chen, Terry O'Day, Jon Watchko, Juan Li, Xiao Xiao. Department of MGB,
University of Pittsburgh, Pittsburgh, PA; Dept. of Pediatrics, University of
Pittsburgh, Pittsburgh, PA; Dept. of Orthopaedic Surgery, University of
Pittsburgh, Pittsburgh, PA
Muscular dystrophies are inherited myogenic disorders characterized by
progressive muscle wasting and weakness. Because of the lack of effective
treatment with traditional pharmaceutical agents, gene therapy and stem-cell
therapy have been vigorously explored. Besides gene replacement therapy, a
different form of gene therapy, i.e., expression of
booster
genes,
has been investigated, aiming at alleviating the secondary deficiencies in
muscular dystrophies rather than the primary ones. Blockade of myostatin is one
of such strategies. Myostatin is a member of the TGF-beta family and a negative
regulator of skeletal muscle growth. Studies using transgenic mice or antagonist
antibody against myostatin showed promotion of muscle growth and amelioration of
muscular dystrophy. However, gene delivery of myostatin inhibitors has not been
reported.
In this study we examined whether myostatin propeptide gene transfer into normal
mice by AAV vectors could increase muscle mass and strength. AAV vector carrying
myostatin propeptide (MPRO) gene was delivered into neonate and adult BL10 mice
respectively. For neonates study, AAV serotype 8 carrying MPRO gene was
delivered into 3 to 5 days old mice by intraperitoneal injection. Two months
after vector injection we observed very significant hypertrophy of the skeletal
muscles. The treated mice gained body weight and had less fat when compared to
the controls. The skeletal muscle mass including TA, GAS, Quadriceps, and
Diaphragm from the treated mice were significantly larger (p<0.01). Myofiber
diameters of the treated mice were larger than the untreated ones. The
hypertrophy was not observed in the hearts of the treated mice despite the gene
expression there. However, the larger muscle did not yield stronger force. The
peak twitch and peak tetanic force of the treated muscle did not increase
significantly, and the treated mice also did not increase performance on the
treadmill. For the adult study, AAV-MPRO gene was delivered by local and
systemically. Local intramuscular injection study indicated that the treated
muscle mass also increased significantly. However, the treated muscle showed
increased central nucleation, suggesting muscle regeneration. The study of
systemically delivery of AAV-MPRO gene to the adult mice is under way. In
conclusion, these data have shown for the first time that muscle mass can be
increased by gene delivery of myostatin propeptide in normal mice. Further
studies on muscular dystrophy animal models are warranted.
26) Real Time Imaging of Myoblast
Transplantation Using the Human Sodium Iodide Symporter (hNIS) as Reporter Gene
Manaf Bouchentouf, Basma F. Benabdallah,
Joel Rousseau, Marcel Dumont, Jacques P. Tremblay. Human Genetic unit, CHUL,
Ste-Foy, QC, Canada; Radiology Unit, HSFA, Quebec, QC, Canada
BACKGROUND: The quantification of the graft success is a key element to
evaluate the efficiency of cellular therapies for several pathologies such as
Duchenne muscular dystrophy. This study describes a novel approach to evaluate
the success of myoblast transplantation (i.e., the survival or the transplanted
cells and of the muscle fibers that they have formed) by real-time imaging.
METHODS: C2C12 myoblasts were first transfected with a
plasmid containing the human sodium iodide symporter (hNIS) gene. The specific
uptake of 99mTCO4 by the hNIS positive myoblasts was
demonstrated in vitro, no accumulation of 99mTCO4
was observed within the control cells. The cells were then transplanted into the
Tibialis anterior (TA) muscle of mdx mice. Following
intra-peritoneal administration of Na99mTCO4,
scintigraphies (g-radiographs) were performed depicting hNIS-dependent 99mTCO4
uptake within the TA. RESULTS: Preliminary experiments have
demonstrated that the resolution of the camera used is 2 mm. In vitro the
cells expressing the hNIS were able to incorporate the Na99mTCO4
and were also able to form myotubes. In vivo image
acquisition of transplanted mice TA revealed that hNIS positive cells
were able to incorporate the Na99mTCO4. CONCLUSIONS:
This approach permitted to evaluate the progression of the transplantation
and the graft success without having to biopsy the animals during the follow-up
period.
27) Overexpression of the Myostatin
Antagonist Follistatin in Normal Myoblasts Genetically Modified with a
Lentivirus
Basma F. Benabdallah, Joel Rousseau, Manaf
Bouchentouf, Jacques P. Tremblay. Human Genetic, CHUL-CHUQ-Laval University,
Sainte Foy, QC, Canada
Background: Duchenne muscular dystrophy is a severe myopathy caused by
the absence of a functional dystrophin in muscles. Transplantation of normal
myoblasts is a potential therapy that permits to restore the expression of the
dystrophin in transplanted muscles by the formation of hybrid dystrophin
positive fibers. However, the success of this approach is compromised by the
limited regeneration of damaged muscle. Myostatin, the most powerful inhibitor
of muscle growth identified to date, is regulated by different antagonist
proteins such as follistatin. Our purpose is to block the myostatin signal in
mdx and SCID host mice by the respective transplantation of normal murine
and human myoblasts genetically modified with a follistatin lentivirus.
Methods: 293T packaging cell line was used to produce a lentivirus coding
for the short form of follistatin protein under the control of a cytomegalovirus
promoter. Normal murine and human myoblasts were infected with the lentivirus to
overproduce the follistatin protein. The surexpression of the follistatin in
both transfected 293T cells and infected murine and human normal myoblasts was
verified by immuno-cytochemistry assay and Western blot. A lentivirus containing
the eGFP gene was used as a control. The differentiation of control and
lentivirus-follistatin infected myoblasts was also evaluated by the
determination of the fusion index.
Results: The immunocytochemistry assay showed that the follistatin was
clearly overexpressed in both pCMV-Fst transfected 293T cells and infected
murine and human normal myoblasts compared with the control cells transfected or
infected with the eGFP lentivirus. The Western blot also demonstrated that the
follistatin was effectively overexpressed in pCMV-Fst transfected 293T cells and
infected murine and human normal myoblasts compared with the control cells
transfected or infected with the eGFP lentivirus. The fusion index test also
showed that myoblasts infected with the follistatin lentivirus formed more
myotubes than myoblasts infected with the eGFP-lentivirus when cultured in
differentiation conditions.
Conclusions: The transplantation of normal murine or human myoblasts
overexpressing follistatin, in mdx or SCID mouse respectively in order to
antagonize the myostatin signaling, could be a good approach to improve the
success of the potential cellular therapy of Duchenne myopathy.
28) Development of AAV-Mediated Gene
Therapy for Murine Models of Genetic Diseases Affecting the Heart
Christina A. Pacak, Cathryn Mah, Gabriel
Gaidosh, Melissa Lewis, Raquel Torres, Kevin Campbell, Glenn A. Walter, Barry J.
Byrne. Molecular Genetics and Microbiology, University of Florida, Gainesville,
FL; Powell Gene Therapy Center, University of Florida, Gainesville, FL;
Physiology, University of Florida, Gainesville, FL; Cellular and Molecular
Therapy, University of Florida, Gainesville, FL; HHMI, University of Iowa, Iowa
City, IA
The long term goal of this project is to develop a clinically relevant gene
therapy approach for the treatment of genetic diseases affecting the heart. Due
to its small size, safety and proven ability to persist for long periods of time
in muscle, adeno-associated virus (AAV) has emerged as a promising cardiac gene
delivery vehicle. We have sought to determine the most advantageous combination
AAV serotype and vector delivery route for the transduction of cardiomyocytes
in vivo. Both intra-venous (iv) and intra-cardiac (ic) injection routes were
compared by injecting 1x1011 and 5x1010 (respectively)
vector particles of AAV-CMV-LacZ per mouse neonate of 3 different serotypes AAV1,
AAV8 and AAV9. Tissue analysis included both x-gal staining on tissue sections
to visualize expression and the quantitative
-galactosidase
enzyme detection assay. Our results show that iv administration of AAV9 results
in 30-fold more efficient transduction of cardiac tissue than AAV1. Moreover,
hearts injected with AAV9 displayed a global distribution of transgene
expression suggesting this serotype has no transduction site preference within
cardiac tissue. 2 murine models for cardiac dysfunction; the alpha-sarcoglycan (asg
-/-) knockout model for Limb Girdle Muscular Dystrophy Type 2D (LGMD-2D)
and the acid-alpha glucosidase (gaa-/-) knockout model for
Pompe Disease are currently being characterized functionally and morphologically
using both non-invasive MRI and histological techniques. LGMD-2D is the result
of mutations in the alpha sarcoglycan (ASG) gene. This model displays the
development of dystrophic lesions in cardiac and skeletal muscle. We have
developed a non-invasive assay using MRI that enables us to locate and measure
the random development of lesions within the muscles of asg -/-
mice. We have demonstrated functional correction and prevention of disease
progression in skeletal muscles injected with AAV-ASG. Studies are currently
underway to demonstrate the same effect in cardiac tissue. The cells of the
gaa-/- mouse contain lysosomes enlarged with glycogen due to a
GAA enzyme deficiency. Previously, our lab has demonstrated that delivery of the
human GAA gene to the skeletal muscle of this model is therapeutically
beneficial. To evaluate gene delivery to the cardiac tissue of this model we are
currently characterizing the cardiac phenotype of disease and assessing our
ability to prevent disease presentation following optimized delivery of the
human GAA gene. In conclusion, our maker-gene study has established that iv
delivery using AAV9 is the most clinically advantageous combination of AAV
serotype and delivery route for the transduction of cardiac tissue in vivo.
We are now in the process of using this delivery method to demonstrate disease
correction in two mouse models of cardiomyopathy.
29) Decorin Promotes Differentiation of
Myoblasts into Myotubes That Express Slow MyHC In Vitro
Yong Li, Juan Li, Ying Tang, Xiao Xiao,
Johnny Huard. Dept. of Orthopaedic Surgery, Childrens
Hospital and University of Pittsburgh, Pittsburgh, PA; Dept. of Molecular
Genetic and Biochemistry, University of Pittsburgh, Pittsburgh, PA
Decorin, a small leucine-rich proteoglycan, is a key regulator in extracellular
matrix assembly and cell proliferation. It also has a specific effect on the
migration and differentiation of embryonic skeletal muscle cells (1). We have
demonstrated that decorin can prevent muscle fibrosis, enhance muscle
regeneration, and improve the functional recovery of injured skeletal muscle
(2). However, the mechanism behind decorins
effect on muscle regeneration is unclear. In the current study, we used an
AAV-mDecorin plasmid to transfect a mouse decorin gene into myoblasts (C2C12
cells) and evaluated the phenotypic changes during the resultant myoblast
differentiation. Our results revealed that myoblasts engineered to express
decorin undergo differentiation more readily than do normal myoblasts in vitro.
This enhanced differentiation may be due to the stimulation of P21, an important
cyclin-dependent kinase inhibitor that is up-regulated during muscle cell
differentiation (3). We also observed up-regulated expression of myogenic
proteins (MyoD, Myogenin, Myf5, and Myf6) by these decorin-transfected (CD)
cells. This study demonstrates that decorin treatment resulted in the
overexpression of slow myosin heavy chain (MyHC) and the production of slow
myotubes by CD cells in vitro. These results suggest that decorin may promote
muscle regeneration through the activity of slow MyHCexpressing
muscle fibers. These results indicate that decorin improves skeletal muscle
healing not only by preventing fibrosis, but also by promoting muscle
regeneration. These findings could explain why decorin treatment can lead to
nearly complete functional recovery of injured muscle (2).
References:
1. Villena J, et al. J Cell Physiol 2004;198:16978.
2. Fukushima K, et al. Am J Sports Med 2001;29:394402.
3. Halevy O, et al. Science. 1995;267:1018-21.