RESUMOS QUE SERÃO APRESENTADOS NO CONGRESSO ANUAL DA SOCIEDADE AMERICANA DE TERAPIA GÊNICA, BOSTON, MAIO DE 2008
[531] Gait Disturbances in Animals with Muscular
Dystrophy
Thomas G.
Hampton, Ajit Kale, Ivo Amende, Hemmi N. Bhagavan, Case G. VanDongen R&D, The
CuraVita Corporation, Boston, MA; Medicine, Saint Elizabeths
Medical Center at Tufts, Boston, MA; R&D, BioBreeders, Inc., Watertown, MA
Gene therapy holds great promise as a treatment for muscular dystrophy. Gait
analysis is increasingly becoming of interest to researchers in muscular
dystrophy. Clinically, muscular dystrophy results in gait disturbances, and gait
analysis is routinely used to aid in diagnosis and treatment. The
dystrophin-deficient [mdx] mouse, and the delta-sarcoglycan-deficient [BIO TO2]
hamster are excellent models to study muscular dystrophy and the efficacy of
gene therapy. Yet, little is known about gait in these animal models. Here, we
examine gait in mdx mice and BIO TO2 hamsters to identify physio-markers of
muscular weakness in animals as they walk on a motorized treadmill belt. We
demonstrate, for the first time, significant functional differences in forelimb
gait vs. hind limb gait in dystrophin-deficient mice and BIO TO2 hamsters. The
gait of 1 month old hamsters is similar to that of 12 week old mice, with
comparable stride lengths [7
cm], stepping frequencies[4
Hz], and relative contributions of stance [65%]
and swing [35%]
to stride duration. Decreases in hind limb propulsion and increases in hind paw
eversion are characteristic of muscle weakness in mice and dystrophic hamsters.
We further demonstrate that hind limb propulsion decreases and hind paw eversion
are characteristic of neonatal mouse pups that are just able to support their
weight and walk on a treadmill [
16 days of age]. Taken together, two distinct gait metrics are common to two
different animal models of muscle weakness, indicating that gait analysis in
rodent models may provide phenotypic information regarding muscle strength.
Identification of muscular dysfunction in the mdx mouse and BIO TO2 hamster will
be important in examining the effectiveness of gene therapy for muscular
dystrophy.
[537] Effect of Gender on Phenotype in Golden
Retriever Muscular Dystrophy
Joe N. Kornegay,
Daniel J. Bogan, Janet R. Bogan Department of Pathology and Laboratory Medicine
and the Gene Therapy Center, School of Medicine, University of North
Carolina-Chapel Hill, Chapel Hill, NC
Background. Duchenne muscular dystrophy (DMD) is an X-linked recessive
disorder affecting approximately 1 of 3,500 newborn human males in whom absence
of the protein dystrophin causes progressive degeneration of skeletal and
cardiac muscle. Spontaneous forms of X-linked muscular dystrophy due to
dystrophin deficiency have been identified in mice, multiple dog breeds, and
cats. Unlike the dystrophin-deficient mdx mouse, which remains relatively normal
clinically, affected dogs develop progressive, fatal disease similar to DMD. The
most studied canine condition is golden retriever muscular dystrophy (GRMD). So
as to better utilize the GRMD model in therapeutic trials, we have evaluated the
diseases
natural history using phenotypic tests. Affected dogs have joint contractures
and weakness of individual and grouped muscles. Importantly, by comparing serial
measurements of these tests, one can document improvement or delayed progression
of disease. Previously published studies have suggested that gender affects
phenotype in both the mdx mouse and GRMD dog. Homozygous females reportedly have
less severe clinical signs, due presumably to various factors, including effects
of estrogen on muscle regeneration and inflammation. Such a gender effect could
influence interpretation of preclinical studies in which both male and female
animals are used. Materials and Methods. To further clarify the effect of
gender on the GRMD phenotype, we evaluated several phenotypic tests in untreated
homozygous females (F; n = 24) and heterozygous males (M; n = 27) at 6 months of
age. Newborn GRMD dogs were identified based on elevation of serum creatine
kinase and subsequently developed characteristic clinical signs. Genotype was
confirmed by PCR in most dogs. Methods for measuring tibiotarsal joint angle and
torque force have been published. Cranial sartorius circumference measurements
were made by encircling the muscle with suture at the time of biopsy. Paired
t-tests (parametric) or Mann-Whitney rank sum test (non-parametric) were used.
Results. Male and female values (mean
SD) for tibiotarsal joint angles (M = 150.59
10.86o, F = 145.25
14.47o; p = 0.14); torque force generated by either isometric
tibiotarsal tetanic flexion (M = 0.438
0.114 N/kg, F = 0.448
0.152 N/kg; p = 0.79) or extension (M = 2.109
1.071 N/kg, F = 2.171
0.722 N/kg; p = 0.604); tetanic extension/flexion ratio (M = 5.71
4.24, F = 5.64
3.14; p = 0.95); and cranial sartorius circumference (M = 3.03
0.69 mm/kg, F = 3.40
0.96 mm/kg; p = 0.134) did not differ. Conclusions. Results from these
tests do not confirm an effect of gender on phenotype in GRMD dogs at 6 months
of age. However, given that gender-based differences in the degree of muscle
regeneration and inflammation have been documented in mdx mice and other murine
models of muscle injury, care should be taken in designing and evaluating
preclinical studies in which both male and female GRMD dogs are used.
[536] Functional Correlation in Golden Retriever Muscular Dystrophy
Joe N. Kornegay,
Daniel J. Bogan, Janet R. Bogan Department of Pathology and Laboratory Medicine
and the Gene Therapy Center, School of Medicine, University of North
Carolina-Chapel Hill, Chapel Hill, NC
Background. Most preclinical studies using the golden retriever muscular
dystrophy (GRMD) model of Duchenne muscular dystrophy (DMD) have documented
benefit based on pathologic or genetic features rather than functional tests. We
have shown that GRMD dogs treated with prednisone have increased isometric
tibiotarsal joint tetanic extensor force and a paradoxical decrease in flexor
force. The decrease in flexor force may have occurred because prednisone reduced
early flexor muscle necrosis and subsequent functional hypertrophy. In contrast
to our finding of a paradoxical decrease in tibiotarsal joint flexion force in
prednisone-treated dogs, another group showed that GRMD dogs given
mesoangioblasts had increased values, perhaps reflecting differences in the
timing or mechanism of the two treatments. These results emphasize the need to
carefully consider functional endpoints used in the GRMD model. Materials and
Methods. Newborn GRMD dogs were identified based on elevation of serum
creatine kinase. Genotype was confirmed by PCR in most dogs. Functional tests
were evaluated at 6 months of age. Tibiotarsal joint angle and torque force were
measured as previously described and correlated in 51 dogs. Cranial sartorius
circumference was measured by encircling the muscle with suture at the time of
biopsy and correlated with tibiotarsal joint angle in 49 dogs. Correlation
coefficients were done using a simple coefficient equation. Results.
There was a high correlation between tibiotarsal joint isometric tetanic force
and angle. Values (mean
SD) for extension (2.138
0.915 N/kg) correlated directly (r = 0.54; p < 0.0001; power = 0.987), while
those for flexion (0.443
0.132 N/kg) correlated inversely (r = - 0.70; p < 0.0001; power = 1.00) with
joint angle (148.08
12.84o). Tibiotarsal joint angle (148.00
12.97o) and cranial sartorius circumference (3.204
0.839 mm/kg) correlated inversely (r = -0.70; p < 0.0001; power = 1.00). Dogs
with weak extension and strong flexion force values and larger cranial sartorius
muscles tended to have tibiotarsal joint flexor contractures. Conclusions.
Contracture and muscle strength scores in DMD patients generally correlate and
deteriorate synchronously. Joint contractures occur due to an imbalance in the
strength of agonist and antagonist muscles. Weakness of the antagonist extensor
muscle correlates highly with flexor contracture severity. Thus, as opposing
extensor muscles weaken, flexor contractures worsen. A similar high correlation
was seen between tibiotarsal joint angle and extensor force in GRMD dogs of this
study. In addition, tibiotarsal joint flexor force and joint angle correlated
inversely, suggesting that flexor muscle functional hypertrophy could contribute
to contractures. The inverse correlation between cranial sartorius circumference
and tibiotarsal joint angle is consistent with our previously published findings
and suggests that hypertrophy of this muscle may play a role analogous to
iliotibial band tightening in DMD.
[540] RNAi Targeting of FRG1: A Potential Therapy
for Facioscapulohumeral Muscular Dystrophy (FSHD)
Sara E. Garwick,
Jennifer L. Allen, Lindsay M. Wallace, Jorge A. Torres, Rossella Tupler, Scott
Q. Harper Center for Gene Therapy, The Research Institute at Nationwide Childrens
Hospital, Columbus, OH; Pediatrics, The Ohio State University, Columbus, OH;
Molecular, Cellular, and Developmental Biology Program, The Ohio State
University, Columbus, OH; Biomedical Sciences, Universita d
Modena e Reggio Emilia, Modena, Italy; Program in Gene Function and Expression,
University of Massachusetts Medical School, Worcester, MA
Facioscapulohumeral muscular dystrophy (FSHD) is the third most common muscular
dystrophy, affecting 1 in 20,000 people. It is a dominantly inherited disorder
characterized by progressive and asymmetric wasting of facial, shoulder, and
limb muscles. In
20%
of patients, pelvic and abdominal muscles are also affected, resulting in
wheelchair dependence. Symptoms typically arise in late adolescence to adulthood,
though more severe juvenile onset cases occur in 5-10% of cases. FSHD is caused
by contraction of a specific haplotype of subtelomeric repetitive elements (D4Z4
repeats) on human chromosome 4q. Though this mutation was identified
15
years ago, efforts to develop FSHD therapies have been hindered by the inability
to establish a clear underlying pathogenic mechanism for the disease. Currently,
no treatment exists. One well-supported model suggests that D4Z4 contractions
alter normal chromatin structure leading to aberrant up-regulation of chromosome
4 genes. To date, the best FSHD candidate gene is FRG1, which is localized to
human 4q, elevated in some FSHD patient biopsies, and recent transgenic mouse
studies support that FRG1 over-expression in muscle leads to FSHD-associated
phenotypes. We hypothesize that reducing expression of FRG1 levels may offer a
potential treatment for the FSHD. RNA interference (RNAi) has emerged as a
powerful tool to reduce expression of any gene of interest in a
sequence-specific manner. As such, RNAi is a leading candidate strategy for FSHD
therapy. Here we use vector- and transgenic-based approaches to test treatment
of dominant FSHD through FRG1 inhibition. First, we developed 14 different
microRNA shuttle vectors targeting human FRG1 (miFRG1). Using adeno-associated
viral vectors, we are testing the ability of our lead miFRG1 sequences to reduce
over-expressed FRG1 and improve FSHD-associated symptoms in FRG1-high
mice. Second, we created a method to investigate the potential reversibility of
FSHD, by generating 4 lines of doxycycline (dox)-responsive mice to inducibly
over-express, or normalize, FRG1 in muscle (TRE.FRG1 x MCK.rTTA). These mice
represent a genetic method to reduce FRG1 to normal levels after onset of
dystrophy, thereby serving as a gold standard model for RNAi therapy. This work
is an important first step toward establishing a potential treatment for FSHD
targeting increased FRG1 expression. Moreover, we are establishing
proof-of-principle for a method that can be broadly applied if additional
FSHD-related genes are identified in the future.
[528] Development of Strong Muscle-Specific
Promoters for Gene Therapy of Duchenne Muscular Dystrophy
Yue Zeng,
Marilyne Blain, Mehdi Bendjelloul, Claire Guilbault, Kenneth E. Hastings, George
Karpati, Bernard Massie, Renald Gilbert Genomics & Gene Therapy Vectors,
Biotechnology Research Institut, NRC, Montreal, QC, Canada; Neuromuscular
Research Group, Montreal Neurological Institute, Montreal, QC, Canada; Molecular
Biology, University of Montreal, Montreal, QC, Canada
Duchenne muscular dystrophy (DMD) is fatal muscle disease caused by mutations in
the dystrophin gene. Delivery of a functional dystrophin cDNA to muscle fibers
is a promising approach for the treatment of DMD. The ideal vector that carries
the dystrophin expression cassette should not only allow high short term
dystrophin expression level in muscle, but it should also provide stable and
long-term expression. To reach this goal, we have developed powerful muscle
specific promoters derived from the genetic elements of the human slow isoform
of troponin I gene (TnIS). We generated several constructs containing one to
four copies of the TnIS upstream enhancer (USE) or truncated USE (USE)
fused to the minimal promoter of the TnIS gene. Transient transfection studies
in myotube cultures showed that constructs containing three or more USE or
USE
were stronger than CMV and comparable to the powerful hybrid CMV enhancer/-actin
promoter (CB). Moreover, these constructs had only a very weak activity in
non-muscle cells. The strength of the constructs containing three copies of USE
or
USE
(USEx3 and
USEx3)
was similar to CMV and CB after in vivo electroporation of plasmid DNA into
mouse muscle. We have also prepared gutless adenovirus expressing
-galactosidase
regulated by USEx3 andUSEx3
and we are currently testing their efficacy. In summary, multimerization of USE
and
USE
generated powerful muscle-specific regulatory elements that could be useful for
dystrophin gene replacement therapy of DMD.
[347] The Use of CpG Modifications Enhances Gene
Correction Levels Mediated by Oligonucleotides in the Mdx Mouse Model for
Duchenne Muscular Dystrophy
Carmen Bertoni,
Arjun Rustagi, Thomas A. Rando Neurology, UCLA, Los Angeles, CA; Neurology and
Neurological Sciences, Stanford, Palo Alto, CA
Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder
characterized by complete absence of dystrophin expression in skeletal muscles.
Gene editing mediated by single stranded oligodeoxynucleotides (ssODNs) has the
potential to treat both single point mutations as well as deletions that cause
frame shift of the dystrophin mRNA. We have focused on the development of new
vectors capable of activating specific repair mechanisms to direct the repair
process specifically on the sequence of the genomic DNA targeted for correction.
The Methyl Binding Protein 4 (MBD4) is a glycosylase 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 and activate MBD4. The ability of modified ssODN to increase gene
correction levels were assessed in muscle precursor cells in vitro using
a reporter construct carrying a single point mutation in the gene encoding Green
Fluorescent Protein (GFP). Muscle precursor cells were transfected with the
reporter gene and stable clones were selected. CpG-mediated ssODNs showed a two
to four fold increase in gene correction frequencies compared with unmodified
ssODNs. Similarly, targeting oligonucleotides containing CpG modifications but
unable to act through the base excision repair pathway failed to show
significant increases in gene repair. The feasibility of using modified ssODNs
for the treatment of DMD was tested in the mdx mouse. We have designed ssODNs
complimentary to the coding or the non-coding strand of the donor site of exon
23 to induce skipping of the exon responsible for the lack of dystrophin in mdx
and restore its expression. The ability of ssODNs containing CpG modifications
to increase gene repair was studied in vitro and in vivo. The
amount of dystrophin protein restored was significantly increased by the use of
ssODNs designed to activate MBD4. Studies conducted on muscle cells in culture
demonstrated up-regulation of MBD4 mRNA and the activation of the base excision
repair mechanism through which MBD4 acts. 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 immunohistochemistry and at
the mRNA level by RT-PCR. Immunostaining analysis of mdx-injected muscles
demonstrated the efficacy of ssODN containing CpG modifications of increasing
the expression of functional dystrophin in vivo. The single base pair
alteration was confirmed at the genomic level using restriction endonuclease
analysis of total DNA isolated from muscles injected with targeting ssODN.
Dystrophin expression was stable for at least four months after injection (the
latest time point analyzed). Control oligonucleotides homologous to the region
of the genomic DNA targeted for repair but unable to induce the single base pair
alteration had no effects.
[427] AAV-Mediated Gene Therapy Strategies in a
Canine Model of Duchenne Muscular Dystrophy
Zejing Wang,
Stanly Riddell, Donghoon Lee, James Allen, Martin Kushmeric, Jeffrey Chamberlain,
Stephen Tapscott, Rainer Storb Transplantation Biology, Fred Hutchinson Cancer
Research Center, Seattle; Immunology, Fred Hutchinson Cancer Research Center,
Seattle; Radiology, University of Washington, Seattle; Neurology, University of
Washington, Seattle; Human Biology, Fred Hutchinson Cancer Research Center,
Seattle
Duchenne Muscular Dystrophy (DMD) in both humans and dogs (cxmd) is a
lethal, X-linked muscle disease due to lack of an anchor protein, dystrophin,
cause by deletions or mutations in the dystrophin gene. Adeno-associated virus (AAV)-mediated
micro-dystrophin (m-dys) delivery to skeletal muscle has been successful in
restoring muscle function in mdx mice. Our previous studies in wild-type
and cxmd dogs have demonstrated that direct intramuscular injection of
AAV2 or AAV6 carrying different promoter-transgene cassettes resulted in robust
cellular immune responses to AAV capsid proteins. Recent evidence of
immune-mediated loss of AAV vector persistence in human trials also suggested
that immune modulation might be necessary to achieve long-term transgene
expression. We now demonstrated that the induced immune response to AAV vectors
could be averted by a brief course of immunosuppression with a combination of
anti-thymocyte globulin, cyclosporine, and mycophenolate mofetil, which
permitted long-term and robust expression of a canine m-dys transgene in the
skeletal muscle of cxmd dogs, restored localization of components of the
dystrophin-associated protein complex at the muscle membrane, and decreased need
for muscle regeneration. To further characterize the specificity of the observed
T cell responses, we generated an overlapping peptide panel spanning the full-
length capsid protein VP1 of AAV6 and used an ELISpot assay to measure antigen
specific T cell secretion of interferon-g. Our preliminary results identified 7
peptides containing potential immunogenic epitopes from two different dogs,
including one common sequence recognized by both dogs, and suggested that AAV
capsid proteins played at least partial roles in induction of T cell responses
following intra-muscular injection in dogs. We have initiated studies to
determine the usefulness of non-invasive magnetic resonance imaging (MRI)
compared to muscle biopsies, in assessing immune responses to AAV vector and the
effect of restoring expression of dystrophin in dystrophic muscle. Our
preliminary data suggested a trend for water relaxation properties (transverse
relaxation time) T2 values to be longer in cxmd dogs than in normal
controls. The same trend was also found in AAV-treated limbs with T2 tends to
return towards control values. In conclusion, our studies suggested that AAV
capsid proteins can induce immune responses in dog muscles, and transient
immunosuppressive modulation can diminish this response for sustained transgene
expression. Furthermore, MRI technology has the potential for non-invasive
monitoring efficiency of AAV-mediated gene therapy. These methodologies can be
directly translated to treat DMD patients.
[784] Systemic Delivery of AAV8 In Utero Results in High Level
Gene Expression in Diaphragm: Treatment Implications for Duchenne Muscular
Dystrophy
Bhanu M.
Koppanati, Juan Li, Bing Wang, Molly Daood, Xiao Xiao, Jon F. Watchko, Paula R.
Clemens Department of Neurology, University of Pittsburgh, Pittsburgh, PA; Gene
Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC;
Department of Orthopedic Surgery, University of Pittsburgh, Pittsburgh, PA;
Department of Pediatrics, Magee-Womens
Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA;
Neurology Service, Department of Veterans Affairs Medical Center, Pittsburgh, PA
One of the major challenges in the treatment of muscle disorders such as
Duchenne muscular dystrophy (DMD) which affects many muscle groups is achieving
efficient, widespread transgene expression in muscle. In utero gene
transfer can potentially address this problem by accomplishing gene delivery
when the tissue mass is small and the immune system is still immature. In our
previous study we tested in utero AAV1 gene delivery using
intraperitoneal administration to embryonic day 16 (E-16) pups. We observed high
levels of transduction in diaphragm and intercostal muscle, but no detectable
levels in limb muscle. Recently newer AAV serotypes such as AAV8 have
demonstrated widespread and high transgene expression in skeletal muscles and
diaphragm by systemic injections in adults and neonates. We have, therefore,
tested AAV8 gene delivery by intraperitoneal administration in E-16 mice in
utero. Using a AAV8 carrying a lacZ transgene, we observed high
levels of transduction of diaphragm and more moderate levels of transduction of
multiple limb muscles and heart. Furthermore we are extending these studies to
the DMD disease model, the mdx mouse, using an AAV8 vector carrying an
internally-deleted dystrophin cDNA. Preliminary data show high levels of
recombinant dystrophin expression in the diaphragm with functional benefit
measured by in vitro force studies. Our current studies demonstrate the
potential of AAV8 to achieve widespread muscle transduction in utero and
suggest the possibility of therapeutic potential for DMD.
[378] Body-Wide Restoration of Dystrophin
Expression and Amelioration of Pathology in Dystrophic Dogs Using a Morpholino
Cocktail
Toshifumi Yokota,
Qi-long Lu, Terence A. Partridge, Masanori Kobayashi, Akinori Nakamura,
Shin'ichi Takeda, Eric P. Hoffman Center for Genetic Medicine, Childrens
National Medical Center, Washington, DC; Neuromuscular/ALS Center, Carolinas
Medical Center, Charlotte, NC; Dpt of Molecular Therapy, National Center of
Neurology and Psychiatry, Kodaira, Tokyo, Japan
Duchenne muscular dystrophy (DMD) is one of the most prevalent types of muscular
dystrophy and is characterized by rapid progression of muscle degeneration that
occurs early in life. Mutations in the dystrophin gene cause DMD and Becker
muscular dystrophy (BMD), a milder allelic form of DMD. In general, DMD patients
carry mutations which cause premature translation termination (nonsense or frame
shift mutations), while in BMD patients dystrophin is reduced either in
molecular weight (derived from in-frame deletions) or in expression level. It is
noteworthy that some BMD patients with quite large deletions, nonetheless show
only very mild or asymptomatic clinical or laboratory evidence of muscle disease.
This raises the possibility of using anti-sense-mediated removal of one or more
exons around the site of the original mutation so as to induce loss of
additional exons from DMD mRNA and thus restore the translational reading frame
to convert DMD to a milder BMD phenotype. Restoration of reading frame of
dystrophin by antisense-mediated exon skipping of mRNA has been demonstrated in
the mdx mouse model of DMD. Here, we describe the development, testing and
systemic application of a cocktail of antisense phosphorodiamidate morpholino
oligomers (PMOs; morpholinos) designed to promote the skipping of exons 6 and 8
to restore dystrophin expression in canine X-linked muscular dystrophy (CXMD), a
clinically severe dog (beagle) model of DMD. In muscle cultures, each anti exon-6
antisense oligo (AO) alone induced efficient 6-9 skipping whereas, for
intramuscular injection a three morpholino cocktail was required to restore
dystrophin expression. Systemic infusions of 120-200 mg/Kg of this cocktail,
weekly or bi-weekly for 5-22 weeks into three 2-7 months old dystrophic dogs
induced recovery of dystrophin expression in skeletal muscle throughout the body
accompanied by decreased muscle inflammation and improved exercise ability with
no evidence of toxicity. Such multi-exon skipping could be potentially
applicable for more than 90% of DMD patients with dystrophin deletion mutations
and offers the prospect of selecting deletions that optimize the functionality
of the dystrophin protein.
[535] Electrocardiographic Improvement of Mdx
Heart by Transduction with rAAV9-Microdystrophin
Jin-Hong Shin,
Sachiko Ohshima, Kasahara Yuko, Takashi Okada, Shin'ichi Takeda Molecular
Therapy, National Institute of Neuroscience, NCNP, Kodaira, Tokyo, Japan;
Neurology, Graduate School of Medicine, Pusan National University, Busan,
Republic of Korea; Neurology, Graduate School of Medical Sciences, Kyushu
University, Fukuoka, Japan
Background: Duchenne muscular dystrophy is a lethal X-linked disorder due
to deficiency of the dystrophin and characterized by progressive deterioration
of skeletal and cardiac muscles. Cardiomyopathy, although not evident in early
cases, comprises major cause of mortality in advanced patients. Common
electrocardiographic abnormalities in human patients include tachycardia,
shortened PR interval, and reversed R/S ratio. We have previously reported
improvement of skeletal muscle pathology and specific force by the transduction
of microdystrophin into mdx mice, a most well-known model of
Duchenne muscular dystrophy. To investigate the therapeutic effect of the
microdystrophin on the mdx mice heart, we systemically transduced
mdx mice by the AAV9 vector expressing the microdystrophin (rAAV9-microdystrophin).
Methods: Four weeks old mdx mice (n=5) were transduced with the
rAAV9-microdystrophin driven by the CMV promoter (3.0 x 1012
v.g./body) via the tail vein. Four weeks as well as 20 weeks after transduction,
the electrocardiogram was taken under anesthesia, and compared with that of
age-matched C57BL10 mice (n=5) and untransduced mdx mice (n=5). Autonomic
blockade was performed with intraperitoneal injection of either atropine (0.5
mg/kg body weight) or propranolol (1.0 mg/kg), or both. Baroreflex sensitivity
was tested by intraperitoneal injection of phenylephrine (3.0 mg/kg) after
adrenergic blockade with propranolol. Results: Immunofluorescence
staining revealed that more than 90% of the cardiac myofibers were transduced
with the microdystrophin. HR was elevated (p<0.01) in the mdx mice
group compared to C57BL10 mice, while there was not significant difference
between transduced and untransduced group. PR interval was significantly shorter
(p<0.05) in the untransduced mdx mice than C57BL10 mice at the age of 8
weeks, while the microdystrophin-transduced mdx mice showed
prolonged PR interval toward that of the C57BL10. R/S ratio from right
precordial lead markedly increased in the untransduced mdx mice compared
to C57BL10 at the age of 6 months, which became significantly decreased in the
transduced mdx mice (p<0.05). The differences persisted more than 20
weeks after transduction. PR interval, heart rate, and heart rate variability
were all responsive to autonomic challenges in any group, although sympathetic
blockade could not completely revert the PR shortening of mdx mice.
Conclusion: Although cardiac pathology in mdx mice is not evident
before 3 months of age, we could detect several abnormalities in the
electrocardiographic profiles, as early as 8 weeks old. The changes were
improved by systemic transduction of the microdystrophin, supporting
therapeutic effect on cardiac dysfunction. The response to autonomic challenge
suggests that these electrocardiographic abnormalities are partly due to
upregulated sympathetic tone to compensate the cardiac function in the
presymptomatic mdx heart, leaving the possibility of abnormalies in the
conduction system.
[539] Long Term Benefit of AAV/Antisense-Mediated
Exon Skipping in Dystrophic Mice
Fernanda G. De
Angelis, Michela A. Denti, Tania Incitti, Valentina Cazzella, Irene Bozzoni
Genetics and Molecular Biology, University of Rome
La
Sapienza,
Rome, Italy
Deletions and point mutations in the dystrophin gene cause either the severe
progressive myopathy Duchenne Muscular Dystrophy (DMD) or the milder Becker
Muscular Dystrophy, depending on whether the translational reading frame is lost
or maintained. Since internal in-frame deletions in the protein produce only
mild myopathic symptoms it is possible, by skipping specific mutated exons, to
restore a partially corrected phenotype. Exon skipping in the dystrophin mRNA
can be achieved in the mdx mouse model by the use of chimeric antisense U1snRNA.
We designed and produced Adeno-Associated Viral (AAV) vectors carrying antisense
sequences against the splice junctions of dystrophin exon 23 of the mdx mouse
and we tested their activity in vivo. Local intramuscular delivery of
AAV-antisense RNAs restores dystrophin and muscle strength, while systemic
delivery results in body-wide rescue of dystrophin synthesis and functional
recovery. Finally we have analyzed the long-term antisense efficacy 18 months
after a single systemic injection. We showed that the U1-antisense construct is
still present and able to rescue dystrophin synthesis at levels sufficient to
mantain an effective regeneration capacity and an almost normal muscle phenotype.
The absence of immune response against the transgene, together with data coming
from non human primates showing the persistence of expression from AAV vectors
in muscle for more than 6 years, suggest that a long-term treatment for human
DMD patients might be feasible.
[529] Preservation of Muscle Force in Mdx3cv Mice
Correlates with the Low-Level Expression of a near Full-Length Dystrophin
Protein
Dejia Li,
Yongping Yue, Dongsheng Duan Department of Molecular Microbiology and Immunology,
University of Missouri, Columbia, MO
Complete absence of dystrophin causes Duchenne muscular dystrophy (DMD).
Dystrophin restoration at
20% level reduces muscle pathology and improves muscle force. Levels lower than
this are considered therapeutically irrelevant. Interestingly, less than 20%
dystrophin expression is seen in some Becker muscular dystrophy (BMD) patients.
To understand the role of low-level dystrophin expression, we compared muscle
force and pathology in mdx3cv and mdx4cv mice. Dystrophin was eliminated in
mdx4cv mice. But mdx3cv mice expressed a near full-length dystrophin protein at
5%
of the normal level. Consistent with previous reports, we found dystrophic
skeletal muscle pathology in both strains. Surprisingly, mdx3cv extensor
digitorium longus (EDL) muscle showed significantly higher tetanic force and it
was also more resistant to eccentric contraction-induced injury. Furthermore,
mdx3cv forelimb grip force was stronger. Immunostaining revealed utrophin
up-regulation and detectable dystrophin-associated glycoprotein complex assembly
on the sarcolemma in both strains. Our results suggest that a sub-therapeutic
level expression of a near full-length membrane-bound dystrophin may have
contributed to muscle force preservation in mdx3cv mice. This finding may help
to explain the benign clinical phenotype in some BMD patients.
[432] Full Rescue of Dystrophin Expression in
Cardiac, Smooth and Skeletal Muscles by Antisense Oligonucelotide-Induced Exon
Skipping
Qi Long Lu,
Peijuan Lu, Bo Wu McColl-Lockwood Laboratory for Muscular Dystrophy Laboratory,
Neuromuscular/ALS Center, Carolinas Medical Center, Charlotte, NC
Frameshift and nonsense mutations in the dystrophin gene cause Duchenne muscular
dystrophy (DMD). Skipping the mutated exon(s) with antisense oligonucleotides (AON)
can restore reading-frame and production of dystrophin proteins. We demonstrated
early that functional levels of dystrophin can be induced by intramuscular
delivery of 2-O-methyl
phosphorothioate AONs (2O
MePS) in the mdx mouse, a model of DMD. Effective exon skipping and dystrophin
induction can also be achieved by systemic delivery of the AONs. To improve the
efficiency of exon skipping, we examined AONs with various chemistries and found
that phosphorodiamidate morpholino oligomer (PMO) offers significantly higher
efficiency than 2O
MePS in exon skipping and dystrophin induction. PMO delivered systemically is
able to restore functional levels of dystrophin in skeletal muscles. However,
considerable variation exists within and between skeletal muscles. More
disappointing, AON of all chemistries fail to induce meaningful levels of exon
skipping and dystrophy production in the cardiac muscle. However, restoration of
functional levels of dystrophin in heart and respiratory muscles is critical for
treating DMD. To overcome the delivery difficiency with bare AONs, we examined
several delivery-enabling modifications to PMO. We are now able to induce
effective exon skipping and restore near normal levels of dystrophin expression
in all body muscles, including smooth muscles and cardiac muscle by systemic
delivery of delivery-enabled PMOs. This is associated with significantly
improved muscle functions. Antisense therapy offers realistic hope for the
treatment of majority of DMD patients.
[1000] Methodologies To Enhance Systemic Gene
Transfer to Musculature in Large Animal Models Using AAV6 Vectors
Paul Gregorevic,
Brian R. Schultz, James M. Allen, Eric Finn, Caitlin Doremus, Jeffrey B.
Halldorson, Jeffrey S. Chamberlain Paul D. Wellstone Muscular Dystrophy
Cooperative Research Center, Department of Neurology, University of Washington,
Seattle, WA; Molecular and Cellular Biology, Medical Scientist Training Program,
University of Washington, Seattle, WA
Severe neuromuscular disorders such as Duchenne muscular dystrophy are
associated with profound structural and functional disruption of muscles
throughout the body. Therefore, it is necessary to develop genetic interventions
of therapeutic potential that can access affected muscle fibers body-wide. We
have previously reported therapeutically successful delivery of rAAV6 vectors
throughout the musculature of dystrophic mice. Towards a systemic intervention
with clinical potential, we are currently seeking to enhance the efficiency of
rAAV6 transduction in small and larger mammals. In mice, we have compared the
tissue-specific expression levels of reporter genes driven by either the CMV or
CAG promoter, finding that CAG produces stronger transgene expression levels
overall, but also increases expression in non-muscle tissue, especially liver.
Simultaneous injection of proteasome inhibitors along with rAAV6 has little
effect in striated muscle; however, transgene expression in other tissues is
dramatically increased. This suggests that different mechanisms of transduction
may occur in different tissues. For larger animal studies, we are utilizing a
wild-type dog model for systemic rAAV6 dissemination experiments. Although a
single rAAV6 injection in a mouse can lead to whole-body transduction, including
heart, diaphragm, and limb muscles, a larger animal may benefit from multiple
routes of vector administration. In initial experiments, we performed either a
jugular vein infusion or a localized systemic injection into the femoral artery.
Jugular vein delivery of 10^13 vector genomes produces substantial reporter gene
expression in both the heart and the diaphragm. Localized infusion of the same
vector genome quantity into the femoral artery produces robust expression in
muscles immediately distal to the infusion site. In these studies, using human
reporter genes, animals receiving immune suppression exhibit greater transgene
expression than animals not immune suppressed. Additional studies in progress
are comparing different infusion protocols for the relative ability to transduce
limb muscles versus internal muscles, such as heart and diaphragm, and the use
of single versus multiple injections for systemic gene transfer.
[999] Effective Transduction of Dystrophic Dogs
with rAAV Serotype 8
Sachiko Ohshima,
Jin-Hong Shin, Akiyo Nishiyama, Katsutoshi Yuasa, Yuko Kasahara, Takashi Okada,
Shin'ichi Takeda Molecular Therapy, National Institute of Neuroscience, NCNP,
Kodaira, Tokyo, Japan
Background: Duchenne muscular dystrophy (DMD) is an X-linked, lethal
disorder of the striated muscle caused by mutations in the dystrophin
gene, which encodes a large sub-sarcolemmal cytoskeletal protein dystrophin. The
absence of dystrophin associated with the loss of dystrophin-glycoprotein
complex from the sarcolemma results in progressive muscle weakness,
cardiomyopathy, and early mortality. Several treatment modalities have been
attempted to correct the dystrophic phenotypes, but more effective therapy still
needs to be developed. A recombinant adeno-associated virus (rAAV) has been
utilized in the various preclinical and clinical studies. However, many
questions associated with the host immune reaction have been raised and innate
immune response against the rAAV has not been studied. Here we investigated the
transduction efficiency and immune response by using the rAAV8 and rAAV2 in the
muscles of normal Beages and canine X-linked muscular dystrophy in Japan (CXMDJ).
Methods: The rAAV8 or rAAV2 encoding the lacZ gene driven by the
CMV promoter in the range of 1 x 1011 to 1 x 1013 g.c./muscle
was directly injected into the anterior tibial muscle. The rAAV8 at a dose of 1
x 1014 g.c./kg was also injected into the unilateral hind limb via
the lateral saphenous vein of the normal Beagles at 5-12 weeks old by using the
limb-perfusion method. The CXMDJ at 5-12 weeks old were also
transduced with the rAAV8 encoding the microdystrophin gene by the same
method. The transduced muscles were sampled 4 weeks after the injection to
analyze histological findings. To investigate innate immunity against the rAAV,
bone marrow-derived dendritic cells were differentiated using GM-CSF as well as
IL-4 and infected by the rAAV2 or 8. Levels of the cytokine and costimulating
factor mRNA of the transduced dendritic cells of Beages were evaluated by
qRT-PCR. Results: Efficient
-galactosidase
transduction was confirmed in the canine skeletal muscles with either
intramuscular or intravenous injection of the rAAV8. Microdystrophin expression
in the CXMDJ muscle with limb-perfusion method was more extensive
than that with intramuscular injection. rAAV-mediated transduction with either
injection protocol was associated with the lymphocyte infiltration. qRT-PCR
analysis of the rAAV-transduced dendritic cells suggested that mRNA levels of
the costimulating factors as well as interferon
were higher in the cells transduced with the rAAV2 than that with the rAAV8.
Discussion: The rAAV8 is the efficient tool for the therapeutic gene
delivery into the dystrophic canine skeletal muscle. rAAV8-mediated gene
transfer showed effective transgene expression, but roles of dendritic cells in
the innate immune response must be further investigated to improve transduction
protocol. We are currently conducting MR imaging and torque measurement of the
microdystrophin-transduced CXMDJ to investigate the therapeutic
efficiency.
[349] The Functional Capacity of
R4-R23
Microdystrophin Is Improved by Switching Hinge 2 with Hinge 3
Glen B. Banks,
Ariana C. Combs, Caitlin Doremus, Leonard Meuse, Eric E. Finn, James M. Allen,
Jeffrey S. Chamberlain Senator Paul D Wellstone Muscular Dystrophy Cooperative
Research Center, Department of Neurology, University of Washington, Seattle, WA
In skeletal muscle, dystrophin provides a flexible connection between actin and
the dystrophin glycoprotein complex at the myotendinous junction (MTJ),
sarcolemma and neuromuscular synapse. Dystrophin-deficient muscles are highly
susceptible to contraction-induced injury and they undergo repeated cycles of
necrosis and regeneration. Dystrophin contains an N-terminal actin binding
domain, a large central rod domain, a cysteine rich region and a C-terminal
domain (Fig. 1A). The central rod domain contains 24 spectrin repeats, 4 hinge
regions and a second actin-binding domain (Fig. 1A). Large deletions in the rod
domain can minimally affect the functional capacity of dystrophin and usually
lead to a more mild form of DMD, called Becker muscular dystrophy. Our
laboratory previously developed a highly functional truncated dystrophin called
R4-R23/CT
microdystrophin that prevents muscle degeneration in dystrophin-deficient mdx
mice when intravenously delivered using recombinant adeno-associated virus
pseudotyped with serotype 6 capsids (rAAV6). We found here that expression of
R4-R23/CT
microdystrophin led to chronic myotendinous strain injury. This injury led to an
increase in utrophin and
7-integrin
expression in addition to ringed fibers, where the peripheral myofibrils form
rings around the central myofibrils. Interestingly, the sarcolemma of these
muscles was protected from contraction-induced injury in an isometric stretch
assay, better than wild-type mice. We could circumvent these abnormalities by
replacing hinge 2 from the central rod domain of
R4-R23/CT
microdystrophin with hinge 3 (H2-R24/CT+H3
microdystrophin). To compare the efficacy of these two microdystrophins we
delivered a non-saturating dose (2e12 vg) of rAAV6-microdystrophin intravenously
into 3 week old mdx mice. We examined the gastrocnemius and tibialis anterior
muscles 5 months after injection. Greater than 60% of the mdx muscle fibers
expressed both of the microdystrophins. We found that
R4-R23/CT
microdystrophin significantly reduced the central nuclei from 80% in mdx mice to
16% in dystrophin positive fibers (P < 0.001). However,
H2-R24/CT+H3
microdystrophin was more effective, reducing the central nuclei to 2% in
dystrophin positive fibers (P < 0.001). The muscle fiber area was significantly
increased in
H2-R24/CT+H3
microdystrophin positive fibers compared to
R4-R23/CT
microdystrophin positive fibers (P < 0.001). Both of these microdystrophins were
equally effective at restoring peak force production and protecting the muscles
from contraction-induced injury. Thus, we have developed a highly functional
microdystrophin with greater potential for gene therapy of DMD.
[233] Pre-Clinical Biodistribution and Toxicity
Analysis Following Intravenous Delivery of AAV6 Vectors
Daniel Stone,
Ying Liu, Zong-Yi Li, Robert Strauss, Eric E. Finn, James M. Allen, Jeff S.
Chamberlain, Andre Lieber Division of Medical Genetics, Department of Medicine,
University of Washington, Seattle, WA; Department of Pathology, University of
Washington, Seattle, WA; Department of Neurology, University of Washington,
Seattle, WA; California Institute for Quantitative Biosciences, University of
California Berkeley, Berkeley, CA
In recent years a common strategy to increase the gene delivery efficiency of a
parental viral vector has been to use different serotypes of the same virus.
Specifically, vectors based entirely on novel serotypes, or containing regions
of other serotypes, have been utilized. Using this approach adeno-associated
virus (AAV) vectors based on serotype 6 (AAV6) were found to efficiently
transduce muscle in vivo after intravenous delivery, when compared to parental
AAV2 vectors. A number of studies have demonstrated widespread transduction of
skeletal muscle after AAV6 delivery, and due to these encouraging pre-clinical
results it is being considered for use in the treatment of muscular dystrophy.
As a prelude to future clinical studies, we investigated the biodistribution and
safety profile of AAV6 following intravenous delivery to mice. Mice were
injected with a dose of 2
1012 viral genomes and analyzed for CBC, serum markers, vector
biodistribution, cytokine/chemokine induction and tissue inflammation at 30
minutes, 6 or 72 hours post injection. AAV6 had minimal effect on circulating
blood cell levels, with only a moderate increase in leukocyte levels at 72 hours,
while serum levels of ALT and sCD62p were unaffected, indicating no
hepatotoxicity or coagulation activation. Vector biodistribution showed more
vector in serum than blood cells after 30 minutes and 6 hours, while the major
sites of tissue sequestration were the liver and spleen. Vector was found in all
tissues tested and for all tissues the level of vector genomes dropped
significantly between 6 and 72 hours. Analysis of gene transcription for IL-1,
IL-6, TNF-,
MCP-1 and IP-10 revealed that AAV6 induced significant transcription in liver,
while in spleen only transcription of MCP-1 was seen. Analysis of serum showed
significant rises in levels of TNF-
at 30 minutes post injection, MCP-1 at 6 hours post injection, and IL-6 at 30
minutes and 6 hours post injection. Immunohistochemistry using liver sections
and antibodies against markers for leukocytes, monocytes/macrophages,
granulocytes/neutrophils, lymphocytes or NK cells demonstrated a lack of
inflammatory infiltrate at any time point tested. When taken together our data
suggest that AAV6 vectors show non-specific biodistribution, and are relatively
non-toxic following intravenous delivery. Although there is some indication of
AAV6 induced toxicity, it is mostly transient, and has almost completely
subsided by 72 hours post injection. AAV6 remains a promising tool for future
use in the treatment of muscular dystrophy.
[1001] Experimental Targeted Gene Therapy for
Quadriceps Muscle Weakness for Sporadic Inclusion Body Myositis (sIBM) with
Implications for Other Neuromuscular Disorders
Janaiah Kota,
Chrystal L. Montgomery, Amy Eagle, Danielle Tucker, Chalonda Handy, Amanda M.
Haidet, Louise R. Rodino-Klapac, Kim M. Shontz, Christopher J. Shilling, K. Reed
Clark, Zarife Sahenk, Jerry R. Mendell, Brian K. Kaspar The Research Institue at
Nationwide Childrens
Hospital, Columbus, OH
Sporadic inclusion body myositis (sIBM) is the most common acquired muscle
disease occurring over age 50 with a prevalence of 5 per 100,000. Clinical
features include: male preponderance (males 2:1 females), selective muscle
atrophy (particularly quadriceps muscle), and weakness in knee extension with
frequent falls and loss of ambulation. The cause remains enigmatic. Muscle
pathology demonstrates combined muscle degeneration and inflammation.
Immunosuppressive (IS) therapy can reduce mononuclear cell infiltration in
muscle without sustained clinical benefit. A translational strategy targeting
sIBM would improve quadriceps muscle strength and diminish inflammation.
Therapeutic strategies for neuromuscular disorders have focused on enhancement
of muscle mass and strength. Follistatin has been demonstrated to bind to
myostatin, a negative regulator of muscle mass, and functions as a potent
myostatin antagonist. Several studies, including work from our laboratory have
demonstrated the potential of follistatin based on rodent models of muscular
dystrophy. In our studies, delivery of follistatin led to increased muscle mass
and size along with decreased pathological markers of the disease, demonstrating
significant therapeutic promise for advancing to clinical studies. To this end
we combined gene transfer of rAAV1.follistatin to the quadriceps of
Cynamologous macaque with immunosuppressive therapy employing
tacrolimus and myocophenylate mofetil (MMF). This IS regimen serves two roles in
sIBM: promoting safe passage for gene transfer and reduction of the inflammatory
milieu. Four months post injection, the effect of rAAV.follistatin was assessed
on the injected muscle in comparison to nave
control. Gross observation of the muscle demonstrated unequivocal increase in
muscle size. By histological exam, fiber size was significantly increased
predominantly affecting type 2 muscle fibers (nave,
68.4 um10.0,
follistatin, 87.015
um). The effects were greatest at the injection site but spread throughout the
muscle. This may be related to the secretion of the peptide reaching sites
beyond the immediate injection area in light of detection of follistatin in the
serum of treated animals exceeding the baseline levels. In support of secreted
follistatin reaching sites beyond the transduced muscle, we found a shift in
fiber size in remote muscles such as tibialis anterior and gastrocnemius muscles.
The immunosuppressive drugs had no adverse effects on the general health of
animals, assessed by observation and blood chemistries and there were no organ
abnormalities at necropsy. In summary, the ability of follistatin to cause
hypertrophy in higher animals warrants its consideration for clinical
development to treat human muscle disorders. sIBM is one example but other
conditions would also benefit, including several forms of muscular dystrophy.
The necessity for IS therapy in non-inflammatory neuromuscular disorders will
require further study.
[266] Transplantation of Muscle-Derived Stem Cells
Genetically Engineered To Express Vascular Endothelial Growth Factor (VEGF)
Decreases Fibrosis in Dystrophic Muscle
Bridget M. Deasy,
Joseph M. Feduska, Thomas R. Payne, Johnny Huard Stem Cell Research Center,
Childrens
Hospital of Pittsburgh, Pittsburgh, PA; Department of Bioengineering, University
of Pittsburgh, Pittsburgh, PA; Department of Orthopaedic Surgery, University of
Pittsburgh, Pittsburgh, PA; Department of Molecular Genetics & Biochemistry,
University of Pittsburgh, Pittsburgh, PA; Cook Myosite Inc., Pittsburgh, PA
Transplantation of allogeneic or genetically-engineered autologous
muscle-derived stem cells (MDSCs) into the skeletal muscle of
dystrophin-deficient mdx mice, a model for Duchenne Muscular Dystrophy,
is able to regenerate dystrophin-positive skeletal muscle fibers. Here, we
examined the role of VEGF signaling in MDSC-mediated cell therapy for muscular
dystrophy. We used MDSCs which were genetically engineered to express human
vascular endothelial growth factor (VEGF165, MDSC-VEGF) or the
VEGF-specific antagonist, soluble Flt1 (sFlt1, MDSC-Flt). After transducing the
cells, there was no change in the marker profile of the cells (CD34, Sca-1, or
desmin) or in the ability of the cells to differentiate into myotubes in
vitro. In vivo, our studies show a significant decrease in fibrosis
at the site of transplantation of cells engineered with VEGF165 (VEGF
secretion levels 105
106 ng/mL/5E5 cells) when compared to non-engineered cells.
In contrast, we observe a significant decrease in vascularization and an
increase in fibrosis in the injected muscle with cells engineered to express
sFlt1 as compared to the transplantation of control MDSCs. We detected a
significant positive correlation between vascularization and skeletal muscle
regeneration. At the same time, we did not observe any significant increase of
skeletal muscle regeneration (as measured by the number of new
dystrophin-positive fibers) in the engraftments using MDSC-VEGF or MDSC-Flt
cells as compared to control MDSCs. These findings suggest that an increase in
vascularization and a decrease in skeletal muscle fibrosis in dystrophic tissue
are induced by the secretion of VEGF by donor MDSCs.
[998] Long-Term Mini-Dystrophin Expression without
Immunosuppression in GRMD Dogs after AAV8-Mediated Gene Delivery by Hydrodynamic
Limb Vein Injection
Joseph Kornegay,
Chunlian Chen, Juan Li, Janet Bogan, Dan Bogan, Jiwei Chen, Bing Wang, Tong Zhou,
Richard J. Samulski, Xiao Xiao Department of Pathology School of Medicine,
School of Medicine, Univerity of North Carolina at Chapel Hill, Chapel Hill, NC;
Division of Molecular Pharmaceutics, School of Pharmacy, Univerity of North
Carolina at Chapel Hill, Chape Hill, NC; Gene Therapy Center,, School of
Medicine, Univerity of North Carolina at Chapel Hill, Chapel Hill, NC; Dept. of
Orthopedic Surgery, University of Pittsburgh, Pittsburgh, PA
Golden retriever muscular dystrophy (GRMD) dogs are employed as a large animal
model of Duchenne muscular dystrophy (DMD) for AAV vector mediated gene therapy
studies. Specifically, AAV serotype 8 (AAV8) is selected as the vector of choice
for the delivery of a canine mini-dystrophin gene, which is under the
transcriptional control of CMV promoter. The AAV8-CMV-cMinidys vector was
injected into the hind legs of multiple young adult GRMD dogs (5 kg to 10 kg in
weight) by the hydrodynamic limb vein injection method (isolated retrograde limb
vein perfusion). The vector dose used in each dog was at 1 x 10e13 v.g/kg body
weight. The injection volumes ranged from 10 ml/kg to 50ml/kg with a consistent
injection rate of 1 ml/second. During and immediate after injection, blood
circulation in the hindlimbs was block by a tourniquet for a total of 10
minutes. No overt vector-related adverse effect was observed during and after
the procedure. At various time points, muscle biopsy samples were taken and
analyzed for mini-dystrophin expression and immune responses. Canine
minidystrophin gene expression was detected at both short term and long term (6
months) time points post vector injection. There was no discernable CTL
responses against the canine minidystrophin gene. Immunofluorescent staining of
CD4+ and CD8+ cells on muscles from vector-injected legs and the contralateral
saline-injected control legs revealed no statistic differences. Furthermore, a
normal dog similarly perfused with an AAV8-CMV-GFP vector also showed strong GFP
expression at 2 week and 10 week time points without CTL immune responses. By
contrast, a normal dog similarly perfused with an AAV2-CMV-GFP vector triggered
robust CTL responses. These results suggest that the AAV8 vectors triggers
minimal or no CTL responses against the canine minidystrophin in GRMD dogs and
GFP in normal dogs when delivered by the limb perfusion method.
[527] Gene Delivery to Dystrophic Diaphragm by the
Helper-Dependent Adenovirus Vector (HDAdv)
Mediated Full-Length Dystrophin Expression
Masatoshi
Ishizaki, Ryoko Kawano, Tomohiro Suga, Yuji Uchida, En KImura, Yasushi Maeda,
Makoto Uchino Neurology, Kumamoto University, Kumamoto City, Japan; Sojo
Univetsity, Kumamoto City, Japan
Backgrouud Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting
disease that causes respiratory or cardiac failure and results in death at about
20 years of age. Ventilatory insufficiency is a central problem in the
management of DMD patients. One of the most important problem against the DMD
gene therapy is the huge size of dystrophin cDNA. We have generated a
helper-dependent adenovirus vector, which has a cloning capacity of up to 37kb,
that carried myc-tagged murine full-length dystrophin cDNA. In this study we
evaluated the therapeutic effect of the HDAd-mediated full-length dystrophin
gene transfer into severe dystrophic diaphragm of utrophin/dystrophin double
knockout mice (dko mice) by the intraperitoneal injection. Method and Results ?We
have constructed HDAd vector contained the murine full-length dystrophin
expression cassette and myc-tag,integral protein (HDAdv-mFLmyc-dys). Each 7-day-old
dko mice were injected with the HDAdv-mFLmyc-dys (1.41012
particles/ml, 100?l) by the intraperitoneal injection. In the diaphragm, the
transgene was widely expressed (mean 38%, max 61%) and prevented the dystrophic
changes pathologically in injected dko mice. The contractile property of the dko
diaphragm was measured to assess the correction of muscle function as well as
pathological improvements. Furthermore, we showed that respiratory function of
injected dko mice recovered by using whole-body plethysmography and their
lifespan became longer. Conclusion These results offer a hopeful prospect for
DMD therapy. Therapeutic gene transfer with HDAd may ameliorate DMD patients.
[538] Transgenic Mini-Dystrophin Expression in
Skeletal Muscles of Mdx:utrn-/- Double KO Mice Ameliorates Dystrophic Phenotypes
and Remarkably Extends Life Span
Peiqi Hu, Bing
Wang, Juan Li, Jiwei Chen, Chunping Qiao, Chunlian Chen, Xiao Xiao Division of
Molecular Pharmaceutics, School of Pharmacy, University of North Carolina,
Chapel Hill, NC
Mice deficient in both dystrophin and utrophin (mdx:utrn-/-) manifest phenotypes
similar to that seen in Duchenne muscular dystrophy (DMD) patients, including
severe muscle wasting, skeletal deformities, joint contractures and premature
death, and have been considered as a better animal model than the mdx mice. We
previously created mini-dystrophin genes and demonstrated that AAV-mediated
local intramuscular delivery of the minigenes into the muscle of mdx mice
ameliorated mdx dystrophic histopathology and improved contractile functions. In
this study, we have generated transgenic mdx:utrn-/- mice expressing
mini-dystrophin 3849 (containing 5 central rods) under the control of a
shortened muscle-specific creatine kinase promoter (dMCK). Immunofluorescent
staining revealed widespread expression in multiple skeletal muscles, mostly in
the fast twitch myofibers, but no detectable mini-dystrophin in the cardiac and
diaphragm muscles. Compared with phenotypic mdx:utrn-/- double knockout mice,
the expression of mini-dystrophin 3849 transgene in the transgenic mdx:utrn-/-
mice resulted in 1) dramatically restoring life span. All observed mice (n=9)
lived longer than 40 weeks and 70% of them (6 in 9 mice) lived longer than 80
weeks, whereas 50% survival rate for dKO mice (n=17) were 9 weeks. 2) restoring
fertility of both males and females. 3) preventing growth retardation, kyphosis
formation and joint contractures.
4) significantly improving muscle strength and contractile function. 5)
ameliorating dystrophic pathology. We conclude that mini-dystrophin 3849 gene is
effective to genetically rescue muscle deficiencies, improve the overall health
and prolong the life span of the severe DMD mice, and interestingly, the these
data indicate that restoration of dystrophin in heart and diaphragm is not
critically required for the prolongation of life span.
[532] Promoter, Vector Serotype and Route of
Delivery Influence
-Sarcoglycan
Persitent Expression after Gene Transfer in Mouse
Marc Bartoli,
Jérôme Poupiot, Isabelle Richard LGMD Department, Genethon, Evry, France
We previously demonstrated a wide and sustained transgene expression in gene
transfer experiments using recombinant pseudotype-1 adeno-associated virus (rAAV1)
vectors expressing the human
-sarcoglycan
cDNA, constituting a promising approach for the treatment of limb girdle
muscular dystrophy type 2D (LGMD2D). However, we observed that injected mice
developed serologic-mediated immune response towards the transgene. We evaluated
this immune response against
-sarcoglycan
by testing different promoters (CMV, C5-12, desmin), AAV serotypes (1, 6 and 9)
and administration-routes (intramuscular, intraarterial and intravenous). We
observed that i) use of a weak and muscle-specific promoter (desmin) induced the
lowest cellular infiltration, ii) AAV6 serotype is more immunogenic than AAV1
and 9 and iii) systemic administration by intravenous injection of the vector
prevented all cellular infiltrations and allowed
-sarcoglycan
expression persistence. In parallel, to further examine the observed immune
response, we carried-out detection of serotype specific antibodies and
demonstrated that conditions leading to the loss of expression of
-sarcolgycan
correlated with the presence of IgG2a isotypes. These findings showed that the
gene transfer design influences
-sarcolgycan
persitent expression and that IgG2a anti--sarcoglycan
antibodies detection could be useful to monitor deleterious immune response.
[534] A Gene Therapy Strategy Utilizing
Follistatin Combined with Micro-Dystrophin Gene Replacement Dramatically
Improves Force Generation in Mdx Mice
Louise R.
Rodino-Klapac, Paul M. L. Janssen, Anil Birdi, Amy Eagle, Kim M. Shontz,
Chrystal L. Montgomery, K. Reed Clark, Jerry R. Mendell, Brian K. Kaspar Center
for Gene Therapy, Research Institute at Nationwide Childrens
Hospital, Columbus, OH; Pediatrics, The Ohio State University, Columbus, OH;
Physiology and Cell Biology, The Ohio State University, Columbus, OH
Duchenne muscular dystrophy (DMD) is a devastating disease of childhood
characterized by severe muscle weakness leading to loss of ambulation and
premature death. Gene replacement strategies using mini- or micro-dystrophin
cassettes small enough for AAV packaging incompletely restore muscle function.
Clinically this mandates a complementary approach for functional restoration.
Prior studies demonstrated a role for muscle specific expression of IGF-1 (Barton
et al 2002; Abmayr et al 2005) to enhance muscle function. Our goal is to expand
the repertoire of potential agents augmenting function for translational
treatment for DMD patients. Follistatin 344 is an alternatively spliced
circulating myostatin inhibitor with predominate effects limited to skeletal
muscle, resulting in increased muscle mass. To test its capacity in a
pre-clinical setting, we treated aged 6 month old mdx mice with a combinatorial
therapy of rAAV.follistatin 344 and rAAV.micro-dystrophin by direct muscle
injection into the tibialis anterior/extensor digitorum (EDL) complex. The
physiologic affects of combinatorial therapy were established six months after
gene transfer including studies addressing the order of transgene delivery with
comparisons to the effects of either transgene alone. We found significant
improvements in both maximum force generation and resistance to damage by
repeated eccentric contractions in all treatment groups with the most
significant improvement in muscles treated with both follistatin and
micro-dystrophin (p<0.05). These findings correlated with improved hindlimb grip
strength and reversal of dystrophic features by histopathology. Increased force
by follistatin expression exceeded mere increase in muscle size in mdx muscle,
addressing concerns that size alone might account for improved muscle function.
Complete necropsies and reproductive capacity of mice undergoing gene therapy
demonstrated no evidence of off target affects. In summary, although AAV gene
therapy treatment for DMD is limited by the inability to deliver full-length
dystrophin, combinational therapy with follistatin provides a means to
compensate for the limitations of small dystrophin gene replacement expanding
our translational options.
[533] MMP-1 Gene Therapy Enhances Myoblast
Migration after Implanting into MDX Mice
Yong Li, William
Wang, Haiying Pan, Mia Jefferson Dept. of Orthopaedic Surgery, University of
Pittsburgh, School of Medicine, Pittsburgh, PA; Stem Cell Research Center,
Childrens
Hospital of Pittsburgh, Pittsburgh, PA; Dept. of Pathology, University of
Pittsburgh, School of Medicine, Pittsburgh, PA
The challenge in myogenic cell transplantation for Duchenne Muscular Dystrophy
is quick death and poor migration of donor cells that significantly limits this
technique application. Current treatment involves numerous injections which
result in not only pain and discomfort, but also the development of additional
scar tissue after the injections. Therefore, the development of a novel
therapeutic approach by which to enhance the migratory properties of the
transplanted cells that will reduce the number of injection sites, promote
widespread cell fusion, and improve muscle healing would be very significant.
Matrix metalloproteinase type 1 (MMP1), a naturally occurring collagen-digesting
enzyme, can eliminate the existing fibrous scar in different tissues including
skeletal muscle tissues. Additionally, MMP1 also is able to enhance cell
migration. Therefore, the current experiment use of retrovirus vector transfer
MMP1 gene into C2C12 myoblasts prior transplanted into skeletal muscle of MDX/SCID,
a dystrophic/immunodeficient mouse model. Results show that MMP1 gene transfer
increases C2C12 myoblast migration, differentiation and significantly enlarges
the dystrophin-positive muscle graft within the dystrophic skeletal muscle after
implanting into MDX/SCID mice. We also detected some LacZ, a transplanted
myoblasts tracker marker co-localized with dystrophin positive myofibers within
diaphragm muscles at two weeks following systemic injection of the MMP1
gene-transferred C2C12 myoblast. These results support the notion that MMP1 is
able to increase myoblast migration and may help spread donor cells to all types
of muscles after systemic delivery. Our overall goal is to identify a novel
technique via local and/or systemic delivery that upon transplantation will
stimulate cell migration in vivo, improving dystrophin gene transfer into
the dystrophic muscle fibers.
[997] Systemic AAV-9 Delivery in Normal Dog Leads
to High-Level Persistent Transduction in Whole Body Skeletal Muscle
Yongping Yue,
Arkasubhra Ghosh, Chun Long, Brian Bostick, Bruce F. Smith, Joe N. Kornegay,
Dongsheng Duan Dept of Molecular Microbiology and Immunology, University of
Missouri, Columbia, MO; Dalton Cardiovascular Research Center, University of
Missouri, Columbia, MO; Scott-Ritchey Research Center, Auburn University, Auburn,
AL; Dept of Pathology and Laboratory Medicine, University of North Carolina -
Chapel Hill, Chapel Hill, NC
Adeno-associated virus (AAV) is a promising vector for muscle disease gene
therapy. The dog model represents an ideal intermediate system prior to human
trials. Previous attempts to deliver AAV directly to canine muscle have largely
failed to achieve efficient transduction because of a strong immune response. In
this study, we evaluated systemic AAV-9 gene delivery in newborn dogs by local
and systemic delivery. Transgene expression was examined at different time
points after AAV infection by biopsy or whole body necropsy. In contrast to the
previous reports of low expression and strong immune reaction in dog muscle, we
observed efficient transduction at 4 weeks following intramuscular gene delivery.
Importantly, systemic gene delivery resulted in impressive whole body skeletal
muscles transduction for up to 6 months. In more than 20 different muscle groups
(including head, neck, chest, abdominal, thoracic and pelvic limbs), we observed
80% transduction in the majority of muscles throughout the entire muscle length.
Taken together, our results provide the first evidence that systemic AAV
delivery can reach multiple muscles in a large animal and that body size is not
a barrier to intravascular AAV gene transfer. Our results raise the hope of
whole body correction for many muscle diseases such as Duchenne muscular
dystrophy. (Supported by NIH and MDA).
[1004] Oligodeoxynucleotide-Mediated Gene
Correction of the Dystrophin Gene In Utero
Lingzhi Cai,
Carmen Bertoni, Thomas A. Rando, Paula R. Clemens Neurology, University of
Pittsburgh, Pittsburgh, PA; Neurology, UCLA, Los Angeles, CA; Neurology,
Stanford University, Stanford, CA; Neurology Service and GRECC, VAPAHCS,
Stanford, CA; Neurology Service, Veterans Affairs Medical Center, Pittsburgh, PA
Duchenne muscular dystrophy (DMD) is a progressive muscle disease that is caused
by mutations in the dystrophin gene. There is pathologic evidence that the
disease process in DMD is ongoing at birth, supporting efforts to develop
treatments that could be instituted at the earliest point during fetal life.
Gene correction strategies are attractive because of their ongoing benefit to
all progeny of the corrected cell. Previous studies demonstrated the feasibility
of making single base changes in the dystrophin gene using single-stranded
oligodeoxynucleotides (ssODNs) in skeletal muscle. In this study, ssODN gene
correction technology was applied to fetal skeletal muscle in utero, with
a goal of gene correction in muscle progenitor cells. MDX92b and MDX93b are
ssODNs that are complementary to the transcribed and nontranscribed strands,
respectively, of the intron 22/exon 23 splice site of the mdx mouse
dystrophin gene. Specific gene correction was expected to result in skipping of
exon 23, creating an in-frame deletion of the exon that contains the nonsense
mutation that results in dystrophin deficiency in the mdx mouse. An ssODN,
homologous to the coding strand of the targeted sequence but lacking the
mismatch and therefore unable to induce the specific single base alteration, was
used for negative control experiments. Doses of 25g
or 50g
of ssODN were injected intramuscularly into the hind limb of embryonic day 16
(E-16) mdx mice in utero. Muscles from mice treated with control
or targeting ssODNs were harvested and tested for dystrophin protein expression
by immunostaining at 4 weeks, 9 weeks, 4 months and 6 months of age. There was
an average increase from 8 (4 weeks) to 53 (6 months) dystrophin-positive fibers
with the 25g
dose of ssODN and an average increase from 47 (9 weeks) to 130 (6 months)
dystrophin-positive fibers with the 50g
dose. No dystrophin-positive fibers were observed in untreated mdx mice
nor those treated with the control ssODN at any time-point. In ssODN treated
muscles, dystrophin-positive fibers harbored fewer centrally-placed nuclei, as
compared to uncorrected muscle fibers, indicating that gene correction protected
against muscle fiber degeneration. In summary, these data demonstrate that gene
correction of muscle cells in utero is feasible. The temporal increase in
the number of dystrophin-positive fibers following a single injection of a
targeting ssODN suggests that muscle precursor cells underwent gene correction
in utero.
[433] Restoration of Dystrophin Expression in
Skeletal and Cardiac Muscle by Systemic Delivery of MorpholinoE23-Vivo Porter
Oligonucleotide
Bo Wu, Yongfu
Li, Paul A. Morcos, Timothy J. Doran, Peijuan Lu, Qi Long Lu McColl-Lockwood
Laboratory for Muscular Dystrophy Laboratory, Neuromuscular/ALS Center,
Carolinas Medical Center, Charlotte, NC; Gene Tools, LLC, Philomath, OR
Antisense oligonucleotide (AONs) is able to restore dystrophin expression by
targeted skipping of exon(s) disrupting reading frame of the gene in the
dystrophic mdx mouse. However, low delivery efficiency leads to high
variability and failure of dystrophin induction in skeletal and cardiac muscle
respectively. In this study, we examined a Morpholino oligomer conjugated with
an guanidine significantly improved dystrophin production in both skeletal and
cardiac muscles in the mdx mice in vivo. Intramuscular injection of
morpholinoE23-vivoporter induced dystrophin expression in almost 100% fibers of
TA muscle. Single intravenous injection of morpholinoE23-vivoporter restored
dystrophin expression in skeletal muscles at the levels equivalent to the
injection of 50 times unmodified morpholinoE23. Repeated injection at biweekly
interval achieved almost normal levels of dystrophin expression in all skeletal
muscles body-wide and partially in cardiac muscle without detectable immune
response. Intraperitoneal injections of the morpholinoE23-vivoporter achieved
normalization of dystrophin expression only in the diaphragm and abdomen muscles,
indicating a local rather than systemic delivery effect. Morpholino with
delivery-enabling modifications offers realistic prospects for the treatment of
a majority of DMD patients.
[431] Targeted Restoration of Dystrophin
Expression in DMD by Peptide-Conjugated Antisense Oligonucleotides
HaiFang Yin,
YiQi Seow, Hong M. Moulton, Patrick L. Iversen, Jordan K. Boutilier, Matthew
Wood Physiology, Anatomy and Genetics, University of Oxford, Oxford, Oxfordshire,
United Kingdom; AVI BioPharma Inc., Corvallis, OR
Duchene Muscular Dystrophy (DMD) is a severe muscle disorder caused by mutations
in the dystrophin gene. The efficacy of antisense oligonucleotide (AO)-mediated
exon skipping for the restoration of dystrophin has been established in animal
models and in DMD patients. However there remain significant limitations to this
therapeutic approach due to the lack of effective systemic AO delivery to muscle.
Here we investigate systemic muscle-specific AO delivery by testing AOs directly
conjugated to cell penetrating peptides (CPPs) alone or in combination with
tissue-specific homing peptides (e.g. muscle-specific peptide, MSP). Morpholino
chemistry AOs were directly conjugated to CPPs alone or in combination with
homing peptides and evaluated in mdx mice following weekly systemic delivery.
Effective exon skipping and dystrophin expression were induced in body-wide
skeletal muscles at extremely low AO doses of 3mg/kg. This is the first time
that targeted AO delivery to muscle and successful body-wide restoration of
dystrophin expression have been achieved at such low AO doses. In parallel we
also report the discovery and characterization of a novel delivery formulation
which facilitates AO uptake in muscle. A series of studies have shown that this
delivery formulation enhances the delivery of AOs of different chemistry (e.g.
2-OMeRNA, PNA and morphlino), depends on the activity of specific muscle
membrane transporters, and that it induces significant restoration of dystrophin
expression in muscle compared with commonly used delivery formulations. In
summary, we report data demonstrating the potential of muscle-specific homing
peptides, CPPs and novel delivery formulations for the targeted restoration of
dystrophin in DMD.
[920] Over-Expression of Follistatin in Myoblasts
Increases Their Proliferation and Differentiation, and Improves the Graft
Success
Basma F.
Benabdallah, Manaf Bouchentouf, Joel Rousseau, Jacques P. Tremblay Pharmacologie,
CRH Sainte-Justine, Montreal, QC, Canada; Medecine Experimentale, Hopital
General Juif - Universite Mc Gill, Montreal, QC, Canada; Genetique Humaine, CHUL,
Quebec, QC, Canada; Genetique Humaine, CHUL, Quebec, QC, Canada
Duchenne muscular dystrophy is caused by the absence of functional dystrophin
protein, leading to the myofiber membrane instability and progressive muscle
atrophy. Myoblast transplantation in dystrophic muscles is a potential therapy
for the disease, as it permits the long term restoration of dystrophin
expression in transplanted muscles. However, the success of this approach is
limited by the short period of muscle repair which follows myoblast
transplantation. Myostatin, known as a powerful inhibitor of muscle growth, is
involved in terminating the period of muscle repair following injury by blocking
myoblast proliferation and delaying myoblast differentiation. Follistatin forms
a complex with myostatin preventing its interaction with its receptor and thus
blocking the myostatin signal. Here, we used a lentivirus to over-express the
follistatin protein in normal myoblasts to block the myostatin signaling. Our
results first confirmed the over-expression of the human follistatin into
lentivirus transduced myoblasts, and second showed that the over-expression of
the follistatin protein in normal human myoblasts improved in vitro their
proliferation rate by about 1,5 folds after 96 h and also their differentiation
rate by about 1,6 and 1,8 folds respectively in the absence and in the presence
of recombinant myostatin. Finally, our data demonstrated that the engrafment of
those transduced human normal myoblasts with the follistatin lentivirus into
SCID mouse muscles was enhanced by 2 folds.
[1003] Myostatin Propeptide Gene Delivery in
Normal Dogs Via AAV Vector Increased Muscle Fiber Sizes
Chunping Qiao,
Janet Bogan, Juan Li, Hui Zheng, Zhenhua Yuan, Jianbin Li, Dan Bogan, Joe
Kornegay, Xiao Xiao Molecular Pharmaceutics, UNC School of Pharmacy, Chapel
Hill; Department of Pathology & Laboratory Medicine, UNC, Chapel Hill
The ultimate purpose of this study is to evaluate the therapeutic efficacy of
myostatin inhibition by means of AAV-mediated gene transfer in the DMD large
animal model golden retriever muscular dystrophy (GRMD) dogs, hopefully paving
the way for future clinical studies. The immediate purpose of this study is to
see whether delivery AAV vector encoding myostatin propeptide (AAV-MPRO) gene in
normal dogs can increase their muscle fiber sizes, as it does in mouse. We have
delivered AAV8 vector containing the MRPO gene by the hydrodynamic limb vein
injection technique in the hind leg of 3-month-old normal dogs. The body weight
ranged from 6.3 kg to 9.7 kg. For each dog, the vector was delivered into
one-leg, and the contralateral leg served as controls. A tourniquet was used to
block the blood circulation of the entire injected hind leg (lower and upper
legs and buttocks) for 10 minutes during vector injection. The vector dose was
1013 v.g/kg body weight. During and after vector injection in 3
months, no vector-related adverse event was observed. ELISA assay of sera has
detected the presence of MRPO protein in the vector-injected dogs over the
background levels of uninjected dogs. MRI imaging of the hind legs revealed
increased sizes in some dogs but not in other. Muscle biopsy on a number of
muscles of both legs was performed. Samples were examined for vector
distribution by Real-time PCR and muscle histology by HE staining. Real-time PCR
detected AAV-MPRO-Fc vector DNA in a majority of biopsied muscles from the
vector injected legs, The copy numbers ranged between 0.01 v.g. to 1.5 v.g./nucleus.
In two of the vector-perfused dogs, we observed muscle myofiber size increases
on the injected legs when compared to the same muscle of the uninjected control
legs. Particularly for dog Ramone, the gastrocnemius muscle from the injected
leg displayed at least 50% myofiber size increase comparing with the muscle from
control leg. Our preliminary studies demonstrated that delivering AAV vector
encoding myostatin propeptide gene into normal dog could induce muscle
hypertrophy and increase their myofiber sizes. Currently we are still analyzing
the data and plan to do more experiments.
[351] A Novel Mini-Dystrophin Gene Restores
Neuronal Nitric Oxide Synthase (nNOS) to the Sarcolemma
Yi Lai, Yongping
Yue, Dejia Li, Chun Long, Luke Judge, Brian Bostick, Jeffrey S. Chamberlain,
Dongsheng Duan Dept of Mole Microbio and Immun, Univ of Missouri-Columbia,
Columbia, MO; Dept of Neurology, Univ of Washington, Seattle, WA
Duchenne Muscular Dystrophy (DMD) is the inherent disease due to the loss of
dystrophin protein. Gene therapy is one of the most promising means to cure this
disease. Dystrophin is composed of the N-terminal, rod, cysteine-rich and
C-terminal domains. The rod domain consists of 24 spectrin-like repeats.
Dystrophin is located on the sarcolemma of myofibers and it connects the
extracelluar matrix with cytoskeletal F-actin filaments. Dystrophin assembles
dystroglycans, sarcoglycans, dystrobrevins, syntrophins and nNOS into the
dystrophin-associated protein complex (DGC) on the sarcolemma. The loss of
dystrophin leads to the secondary loss of DGC members, including nNOS from the
sarcolemma. The absence of sarcolemmal nNOS is a critical factor in DMD
pathogenesis. Restoring sarcolemmal nNOS is thus considered an important goal in
DMD therapy. The prevailing hypothesis is that dystrophin C-terminal domain
recruits syntrophin to DGC and syntrophin then recruits nNOS to DGC through a
PDZ-PDZ domain interaction. However, recent studies suggest that syntrophin can
also be restored to the sarcolemma through a C-terminal domain independent
pathway. The presence of syntrophin is insufficient by itself to restore nNOS.
We hypothesized that a region in dystrophin rod domain is also required to
recruit nNOS to DGC. To test this hypothesis, we generated a series of
mini-dystorphin genes carrying different lengths of rod domain. We used the
H2-R19
minigene as starting template and added back the missing repeats one by one. The
H2-R19
minigene is the best characterized mini-dystrophin gene and it is also the most
potent candidate gene besides full-length gene. To identify regions responsible
for nNOS restoration, we transfected our newly synthesized minigenes into mdx
muscles and performed single and double immunostaining as well as in situ nNOS
activity assays. We also included a full-length human dystrophin plasmid as
positive control. The transfected plasmids were distinguished from revertant
fibers by a human dystrophin specific antibody and a sereis of epitope-specific
antibodies. Minigene/nNOS double positive, minigene positive/nNOS negative and
minigene negative/nNOS positive fibers were quantified from a total of 58
transfected muscles. Our results demonstrated that adding an additional 1, 2, or
3 repeats was not sufficient to restore nNOS. However, in the presence of 4
additional repeats, 97.8 % of transfected myofibers restored nNOS. This is very
close to what we observed with full-length gene plasmid (98.9 %). Taken together,
we have shown that dystrophin rod domain is critical for restoring sarcolemmal
nNOS. This finding challenges the prevailying hypothesis and sheds new light on
the mechanism of nNOS restoration in muscle. Importantly, the new minigene we
described here represents an ideal candidate gene for adeno-associated virus (AAV)
mediated gene therapy and lentiviral mediated stem cell therapy. Therapies based
on this novel minigene are predicted to lead to a better recovery of muscle
function than current mini-/micro-genes. (Supported by NIH
[1002] Delivery of NEMO Binding Domain-Protien
Transduction Domain Fusion Peptide to Young Mdx Mice Yields Increased
Regeneration and Decreased Necrosis in Hindlimb and Diaphragm Muscles
Daniel P. Reay,
Michele Yang, Khaleel K. Rheman, Denis C. Guttridge, Paul D. Robbins, Paula R.
Clemens Department of Neurology, University of Pittsburgh, Pittsburgh, PA;
Childrens
Hospital of Philadelphia, Philadelphia, PA; Department of Molecular Genetics and
Biochemistry, University of Pittsburgh, Pittsburgh, PA; Human Cancer Genetics
Program, The Ohio State University, Columbus, OH; Neurology Service, Veterans
Affairs Medical Center, Pittsburgh, PA
Peptide-mediated therapies are a potential addition to the treatment modalities
for Duchenne muscular dystrophy (DMD), providing the opportunity to complement
viral and non-viral gene transfer and cellular transfer approaches. The
activation of NF-B
is a critical factor that results in muscle degeneration and wasting and is
observed in muscle of human DMD and in the mdx mouse, a genetic and
biochemical model for DMD. The NF-B
intracellular pathway marks a critical point at which muscle degeneration may be
averted, as NF-B
plays a key role in increasing transcription of pro-inflammatory cytokines and
decreasing transcription of muscle regulatory factors. In the absence of
dystrophin, signaling pathways activate NF-B,
leading to nuclear translocation and increased transcription of many genes that
are ultimately detrimental to muscle. In order to circumvent this process,
specific peptides containing the NEMO binding domain (NBD), have been generated
that interrupt the IKK
subunit (NEMO) of the IB
kinase complex, such that IKK can no longer phosphorylate IB,
effectively blocking activation of NF-B.
To increase the delivery of the peptide to widespread muscle cells throughout
the body, a fusion peptide was prepared by linking the NBD peptide to different
protein transduction domains (PTDs). In a prior study, treatment of mdx
mice with an NBD peptide fused to the antennapedia gene homeobox domain (ANTP)
resulted in decreased muscle inflammation and increased muscle fiber
regeneration. In this study, we treated mdx mice at 4-5 weeks of age with
an intraperitoneal injection of different PTD-NBD peptides (HIV Tat homeodomain
(TAT), poly-lysine (8K), and ANTP fused to either wild type or mutant NBD) on a
time course of three injections per week for a total of 4 and 7 weeks of
treatment. Upon completion of the study, the tibialis anterior (TA) muscle and
diaphragm were excised and analyzed for morphologic features of muscle
regeneration and necrosis and for NF-B
activity. Increased regeneration and decreased necrosis was observed after 4
weeks of treatment in the TA muscle and after both 4 and 7 weeks of treatment in
the diaphragm muscles. Additionally, decreased NF-B
activity was revealed by electrophoretic mobility shift assay (EMSA). There was
some variation in both morphologic results and in the degree of effect on NF-B
activity depending on the specific PTD. These studies provide evidence that
PTD-NBD peptide therapy has the potential for therapeutic effect in DMD by
modulating downstream pathways of dystrophin-deficiency.
[930] Gastrocnemius Targeted Vascular Delivery of
AAV8 in Non-Human Primates
Louis G.
Chicoine, Louise R. Rodino-Klapac, Chrystal L. Montgomery, Nathaniel A. Walton,
Janaiah Kota, Brian D. Coley, Reed K. Clark, Jerry R. Mendell Center for Gene
Therapy, The Research Institute at Nationwide Childrens
Hospital, Columbus, OH; Radiology, Nationwide Childrens
Hospital, Columbus, OH
DMD is the most common severe form of childhood muscular dystrophy. Ambulation
is lost in early teenage years and premature death is encountered in the early
twenties from loss of pulmonary function and cardiomyopathy. Effective gene
replacement therapy using adeno-associated virus (AAV) requires vascular
delivery to reach multiple muscle groups. In the mdx mouse we demonstrated that
rAAV8 can successfully cross the vascular barrier and deliver the
micro-dystrophin gene when perfused through the femoral artery. Before such a
major undertaking can be proposed clinically, it is critical to optimize
delivery conditions and volumes in the non-human primate, a model more closely
simulating the clinical paradigm. The premise for our current study is that
efficiency of AAV gene delivery through a vascular barrier is enhanced in a
larger fluid volume by exposing vector to a greater endothelial surface area and
or greater number of capillary junctions, permitting greater muscle transduction.
We tested this hypothesis by targeting the left gastrocnemius muscle of 3 adult
rhesus macaques with AAV8.CMF.eGFP (1 x 1012 vg/kg) using a
fluoroscopically guided catheter, which was positioned beyond the popliteal
artery entering the sural artery of the left gastrocnemius. Two tourniquets were
then placed on the leg, one just proximal to the catheter tip and one below the
gastrocnemius to create a tight compartment for gene delivery. The region was
pre-flushed in each of the macaques with one of the following volumes 0.5, 1.0,
or 2.5 ml/kg of PBS with tourniquets snug but not tight enough to completely
occlude the vessels. After this pre-flush, tourniquets were tightened and the
eGFP containing vector was delivered in volumes equal to 0.5, 1.0, and 2.5 ml/kg
over 1 minute. The region remained isolated for 10 minutes (dwell time) and was
then flushed with an equal volume of PBS. The tourniquets were released and the
arterial catheter removed. The gastrocnemius muscles and other organs were
harvested 3 weeks later. qPCR analysis showed that the 2.5 ml/kg treated
gastrocnemius contained the greatest abundance of eGFP transduced gene as well
as the widest distribution of eGFP expression within the muscle. The levels of
transduction were confirmed by western blot analysis. Biodistribution analysis
by qPCR also showed limited delivery to systemic organs and contralateral muscle.
Only the spleen and liver contained measurable amounts of eGFP by qPCR. We
conclude that AAV8.CMV.eGFP delivered to the non-human primate through a
fluoroscopically-guided catheter efficiently transduces the gastrocnemius muscle,
increasing in proportion to volume of fluid used for vector delivery. We
anticipate using this approach for clinical delivery of rAAV carrying
micro-dystrophin or in other appropriate gene restoration strategies.
[430] A Morpholino-Cell-Penetrating Peptide
Conjugate Caused Effective Exon-Skipping in Heart and Skeletal Muscles of MDX
Mice
Natee
Jearawiriyapaisarn, Hong M. Moulton, Brian Buckley, Jennifer Roberts, Peter
Sazani, Suthat Fucharoen, Patrick L. Iversen, Ryszard Kole Thalassemia Research
Center and Institute of Molecular Biology and Genetics, Mahidol University,
Nakhonpathom, Thailand; Lineberger Comprehensive Cancer Center, University of
North Carolina, Chapel Hill, NC; AVI BioPharma, Inc., Corvallis, OR; Ercole
Biotech, Research Triangle Park, Durham, NC
Conjugation of arginine-rich cell-penetrating peptides (CPPs) to
phosphorodiamidate morpholino oligomers (PMO) has been shown to enhance the
intracellular delivery of PMO to modulate gene expression, in particular to
alter pre-mRNA splicing. In searching for more active, stable and less toxic
peptide-PMO conjugates (PPMOs), we designed a series of PPMOs (termed A through
I) carrying CPPs that consisted of eight arginine (R) and a variable number of
6-aminohexanoic acid and/or
-alanine
residues. Their functional biodistribution and toxicity were initially evaluated
in EGFP-654 transgenic mice, a positive readout mouse model for splice switching
oligonucleotides. After four once daily intraperitoneal injections at 12.5
mg/kg/day, all conjugates exhibited broad tissue distribution with varied degree
of splicing correction in different tissues. The B conjugate showed high
splicing correction in the heart (60%), diaphragm (100%) and quadriceps (100%)
of the mice. The muscles of heart, diaphragm and quadriceps are important
targets for the treatment of Duchenne muscular dystrophy (DMD) with splice
switching oligonucleotides. To determine whether the B peptide-conjugated PMO
would be applicable for DMD treatment, the M23D-B PPMO, targeted to a donor
splice site of dystrophin intron 23, was assessed for exon 23 skipping
efficiency in mdx mice, a model for DMD. In these mice, a nonsense
mutation in exon 23 of the DMD gene prevents dystrophin production. Four once
daily intravenous injections of M23D-B at 12.5 mg/kg/day resulted in persistent
exon skipping in dystrophin mRNA and produced high and sustained dystrophin
protein expression in the heart, diaphragm and multiple skeletal muscles of
treated mice without causing any detectable toxicity. The results suggest that
the X/B containing PPMO can be applicable to therapeutic modulation of gene
expression.
[844] An Improved Protocol for Intra-Arterial
Injection in Mouse
Qiang Liu, Zhong
Yang, Yaming Wang Anesthesia, Brigham & Womens
Hospital, Harvard Medical School, Boston, MA
An improved protocol for intra-arterial injection in mice Qiang Liu, Zhong Yang,
Yaming Wang Department of Anesthesia, Brigham
Womens
Hospital, Harvard Medical School, Boston MA 02115 Duchenne muscular dystrophy (DMD)
is an X-linked lethal degenerative disease characterized by widespread muscle
damage throughout the body. It is caused by a mutation in the coding region of a
large membrane protein, dystrophin. Dystrophin deficiency reduces the stability
of skeletal muscle cell membranes. Given the syncitial nature of skeletal
myofibers, fusion of diseased muscle cells with therapeutic cells expressing
wild type dystrophin may lead to a cell based therapy for this devastating
disease. However, systemic delivery of therapeutic cells to muscles remains a
major challenge. Recent studies shown that some cells with stem cell like
properties can transmigrate from blood into damaged muscles and consequentially
contribute to muscle regeneration upon intra-arterial injection. Thus, the use
of intra-arterial injection techniques has increased rapidly. The small size of
the mouse femoral artery coupled with its high blood pressure presents technical
challenges for such delivery methods. Here we describe a new protocol that has
improved our success rate substantially while avoiding permanent occlusion of
the femoral artery and the usage of heparin. In Brief: 1. Separation of the
femoral artery: In order to expose the artery and avoid collateral damage to
adjacent structures, the artery will be separated from associated nerves and
veins carefully with fine-tip forceps. 2. Creation of a small incision on the
wall of the femoral artery: After occlusion of the proximal end of the femoral
artery and vein with a microvessel clip, a small incision will be made on the
wall of the artery using a 30G1/2 needle tip. 3. Insertion of a blunt-tipped
MicroFilTm 36 gauge needle through the incision into the lumen of the artery:
The titanium alloy MicroFilTm 36 gauge needle, is 10cm long, 20
m
ID and 90
m
OD and is equipped with a syringe adapter at one end (World Precision
Instruments, USA). It is flexible, yet rigid enough for this use. 4. Cell
injection: Cells diluted in 100l
HBSS will be injected through the MicroFilTm needle slowly into the artery. The
MicroFilTm 36 gauge needle will be carefully removed 5 minutes after injection.
Pressure will be applied to the incision site by a wet cotton tip for 2 minutes
to minimize bleeding before releasing the microvessel clip. This protocol has
allowed us to achieve a greater than 90% success rate. We encountered no
post-operative complications related to the procedure. This is a simple,
reliable and practical approach that resolves a technical difficulty faced by
many labs.
[93] High-Level Muscle Specific Transgene
Expression in Cultures of Myotubes Using Lentiviral Vectors
Marc-Andre
Robert, Marilyne Blain, Sofien Dessolin, Antoine Caron, Sophie Broussau, Lucie
Bourget, Bernard Massie, Renald Gilbert Genomics and Gene Therapy Vectors,
Biotechnology Reasearch Institute, Montreal, QC, Canada; Molecular Biology,
University of Montreal, Montreal, QC, Canada; Neuromuscular Research Group,
Montreal Neurological Institute (McGill University), Montreal, QC, Canada
Lentiviral vectors (LV) are promising vehicles for gene transfer applications,
because they can integrate their genome into the cells chromosomes and thus
provide stable transgene expression. LV can accommodate relatively large
transgene (at least 6 kb) and they can transduce dividing and non-dividing cells.
LV carrying minidystrophin could thus be used to treat patients suffering from
Duchenne muscular dystrophy by restoring the dystrophin function with a stem
cells or a gene therapy approach. Because unrestricted and widespread dystrophin
expression could be immunogenic or even toxic to dividing cells (myoblasts or
stem cells), we have constructed a small and efficient muscle specific promoter
(USEx3,
600pb)
derived from the slow troponin I gene. In the present study, we compared the
strength, the stability and the muscle specificity of LV carrying GFP regulated
by
USEx3
(LV-USEx3)
or by the cytomegalovirus (CMV) promoter (LV-CMV). Mouse C2C12 myoblasts were
transduced with both LVs and GFP expression was monitored by flow cytometry in
actively dividing myoblasts, or by fluorescent microscopy and by western blot in
differentiated myotube cultures. In the myoblasts, the fluorescence index of the
cell population ([% of fluorescent cells] X [mean fluorescence intensity]) was
stable for at least three weeks and was 5 to 20-folds higher in the cells
transduced with LV-CMV. However, after differentiation into myotubes, the GFP
expression level of the cells transduced with LV-USEx3
was greater than for LV-CMV. These data show that
USEx3,
in the context of LV, can provide stable, specific and high-level transgene
expression in differentiated muscle cultures.
[346] Heart Specific Min-Dystrophin Expression
Rescues Systolic but Not Diastolic Function in Mdx Mice
Brian P. Bostick,
Yongping Yue, Chun Long, Nathaniel Marschalk, Jing Chen, Dongsheng Duan
Molecular Microbiology and Immunology, University of Missouri School of Medicine,
Columbia, MO
Heart failure is a leading cause of death in Duchenne muscular dystrophy (DMD).
Significant progress has been made in developing gene and cell therapies for
treating DMD skeletal muscle disease. Unfortunately, there is little knowledge
of the suitability of these therapies for the heart. Notable among the skeletal
muscle therapies is the 6 kb
H2-R19
mini-dystrophin gene. The
H2-R19
minigene has been shown to completely rescue skeletal muscle pathology and
restore muscle force. In this study, we set out to test the suitability of the
H2-R19
minigene for the treatment of DMD cardiomyopathy. We developed a series of
transgenic mice with heart specific expression of the
H2-R19
dystrophin. After backcrossing these mice to the congenic mdx background, we
performed a comprehensive evaluation of heart structure and function.
Morphological examination revealed a rescue from dystrophic pathology. Most
notable was the strengthened cardiomyocyte sarcolemmal integrity in the Evans
blue uptake assay. Additionally, Masson trichrome staining demonstrated a
complete absence of fibrosis. Heart restricted expression of the
H2-R19
minigene improved uphill treadmill endurance compared to mdx mice. ECG analysis
showed normalization of the PR interval and cardiomyopathy index. However, the
heart rate, QRS duration and QT interval did not reach normal levels. Left
ventricular catheterization proved the most informative. The
H2-R19
transgenic mdx mice had normalization of baseline systolic parameters. Notably,
end-systolic pressure, end-systolic volume and maximal rate of pressure
development (dP/dt max) returned to normal levels. Analysis of baseline
diastolic parameters showed an incomplete rescue. The end-diastolic volume, time
constant of isovolumetric relaxation (tau) and maximal rate of left ventricular
relaxation (dP/dt min) remained uncorrected. This systolic-diastolic discrepancy
resulted in a normal ejection fraction, while stroke volume and cardiac output
were only partially improved. Challenging the heart with dobutamine revealed
that the
H2-R19
minigene was capable of restoring normal dobutamine response. Further,
H2-R19
transgenic mdx mice undergoing dobutamine stress had a normal survival. To
explore the mechanisms underlying the incomplete rescue, we expressed the
H2-R19
minigene on the normal C57BL/10 (BL10) background. The
H2-R19
dystrophin profoundly displaced wild-type dystrophin. However, the
cardiovascular profile of these mice was not affected. Taken together, our
results provide the first comprehensive analysis of a therapeutic
mini-dystrophin gene in the heart. The incomplete rescue of DMD heart disease by
the
H2-R19
minigene suggests that the heart may have different requirements for gene
therapy than skeletal muscle. Additionally, these findings highlight the
potential importance of the skeletal muscle in modulating heart function. (Supported
by grants from the NIH and the MDA).
[348] A Human Artificial Chromosome (HAC) Vector
with about 2.4 Mb-Human Dystrophin Genome Including Native Expression Control
Elements
Hidetoshi
Hoshiya, Yasuhiro Kazuki, Satoshi Abe, Masato Takiguchi, Yuichi Iida, Yoshinori
Watanabe, Mitsuhiko Osaki, Naoyo Kajitani, Toko Yoshino, Yasuaki Shirayoshi,
Mitsuo Oshimura Department of Biomedical Science, Regenerative Medicine and
Biofunction,, Graduate School of Medical Science,Tottori University, Yonago,
Tottori, Japan; Research Center for Bioscience and Technology, Tottori
University, Yonago, Tottori, Japan; Division of Regenerative Medicine and
Therapeutics, Tottori University, Yonago, Tottori, Japan
Duchenne muscular dystrophy (DMD) is caused by mutation or deletion in the 2.4
Mb dystrophin gene. This gene has highly complex expression patterns, and
various isoforms are expressed by at least seven promoters and alternative
splicing. For gene therapy of DMD, there are a number of points that should be
considered, i.e., the tissue-specific expression, period, localization,
expression level, and isoforms. Moreover, it is necessary to avoid damaging hosts
chromosome, and to be expressed semipermanently. We have previously produced a
Human Artificial Chromosome (HAC) vector by deleting all genes on human
chromosome 21. The purpose of this study is to develop a dystrophin expression
vector that has the potential for physiological expression of dystrophin.
Because the dystrophin is the biggest gene, no vector with whole dystrophin
genomic region has been developed. Thus, we constructed a HAC vector with only
2.4Mb human dystrophin genomic region including its own transcriptional
regulatory elements. A loxP site was inserted to a proximal locus of the
dystrophin genome of human chromosome X (h.Chr.X) in homologous recombination
proficient chicken DT40 cells. Genes on telomere-side from the dystrophin genome
were deleted by the telomere truncation in the DT40 cells. This modified h.Chr.X
fragment including the dystrophin genome was transferred to CHO cells containing
the HAC vector by microcell-mediated chromosome transfer (MMCT). The dystrophin
genome was cloned into the HAC vector by the Cre-mediated site-specific
translocation. This vector was designated as Dys-HAC. The Dys-HAC was
transferred to mouse embryonic stem (ES) cells from the CHO cells. To test the
human dystrophin expression in vivo, the chimeric mice were produced from the ES
cells with the Dys-HAC. We confirmed the human dystrophin expression of various
tissue-specific isoforms in the chimeric mice with the Dys-HAC by RT-PCR
analysis. In addition, the GFP on the Dys-HAC was expressed in all chimeric
tissues, suggesting that the Dys-HAC was stably maintained in vivo. Mesenchymal
stem cells (MSCs) have great potential to differentiate into functional skeletal
muscle and are considered as candidates for transplantation therapy. Thus, the
Dys-HAC was transferred to human MSCs and to test the mitotic stability of the
Dys-HAC. The Dys-HAC was stably maintained up to 75 PDLs without a selection in
human MSCs. In conclusion, the Dys-HAC may be a useful tool for an ex vivo
therapy for DMD. Furthermore, using stem cells derived from multiple potential
sources combined with the HAC-mediated gene delivery may comprise this useful
treatment for genetic defects such as DMD in a near future.
[864] Polyplex Nanomicelle Promotes Intravenous
Gene Transfer to Skeletal Muscle
Keiji Itaka,
Katsue Morii, Kensuke Osada, Kazunori Kataoka Division of Clinical Biotechnology,Center
for Disease Biology and Integrative Medicine, Graduate School of Medicine, The
University of Tokyo, Tokyo, Japan; Department of Materials Science and
Engineering, Graduate School of Engineering, The University of Tokyo, Tokyo,
Japan
Skeletal muscle is an attractive target for clinical gene therapy, especially
for muscular dystrophy and peripheral ischemic diseases. Long-term secretion of
functional protein such as erythropoietin from the transgene into the muscle is
also beneficial for the treatment of various chronic diseases. For this purpose,
a variety of delivery methods using naked DNA has been investigated. In this
study, a novel gene carrier for intravenous transfer to muscle tissue using
increased pressure was proposed. This carrier is a polyplex nanomicelle composed
of plasmid DNA and poly(ethylene glycol)-poly(lysine) (PEG-PLys) block copolymer.
The nanomicelle has the core-shell structure with dense PEG palisades
surrounding the core to compartmentalize the condensed pDNA. The nanomicelles
containing luciferase-encoding pDNA were injected from great saphenous vein with
a tourniquet placed on the upper hind limb to restrict blood flow into and out
of the hind limb. A substantial transgene expression was observed on the
skeletal muscles of the injected limb by IVIS observation and the histological
analysis. Compared to naked DNA, the nanomicelle achieved more than one-order
higher luciferase expression as well as the more sustained expression profile up
to two months. Interestingly, the transgene expressions by nanomicelles on the
skeletal muscles were strongly correlated to the expressions using a cell-free
transcription and translation assay system, where the ideal composition of
nanomicelles showed much higher expressions than naked DNA. These results
suggest that, by the intravenous injection with isolation of the limb by
tourniquet, the nanomicelles transferred the pDNA under the optimal condition
for the gene expression into the muscle fibers. Thus, the intravenous injection
of nanomicelles is a promising technique to achieve sufficient and long-term
transgene expressions on the skeletal muscles.