Resumos que serão apresentados no 14o Congresso da Sociedade Americana de Terapia Gênica em Seatte no período de 18 a 21 de maio de 2011
1) RNAi Therapy for Dominant Limb Girdle Muscular
Dystrophy Type 1A
Jian Liu, Lindsay M. Wallace, Sara E. Garwick-Coppens, Michael A.
Hauser, Jerry R. Mendell, Scott Q. Harper. Center for Gene Therapy, The Research
Institute at Nationwide Children's Hospital, Columbus, OH; Department of
Pediatrics, The Ohio State University College of Medicine, Columbus, OH;
Molecular, Cellular, and Developmental Biology Graduate Program, The Ohio State
University, Columbus, OH; Department of Neurology, The Ohio State University
College of Medicine, Columbus, OH; Departments of Medicine and Ophthalmology,
Duke University Medical Center, Durham, NC
Limb Girdle Muscular Dystrophy (LGMD) refers to a group of 19 disorders
characterized by progressive wasting and weakness of pelvic and shoulder girdle
muscles. Commonly, patients require wheelchair assistance, and individuals with
some forms of LGMD may also have cardiac and respiratory muscle involvement.
Modest improvements in a relatively limited set of muscles may dramatically
improve patients' quality of life, but there is currently no effective treatment
for LGMD. Gene therapy approaches may hold promise as future LGMD treatments.
Over the last two decades, the feasibility of using gene therapy to treat
muscular dystrophy in general, and LGMD in particular, has improved, but
previous strategies have almost exclusively focused on replacing defective or
missing genes underlying recessive disorders. These gene replacement strategies
are not feasible for treating dominant muscular dystrophies, including the 7
dominant forms of LGMD (called LGMD1). Instead, patients with dominant LGMD1
would likely benefit from reduction of their pathogenic alleles. The recent
emergence of RNA interference (RNAi) as a powerful tool to suppress dominant
disease genes provided a mechanism to accomplish this. In this study, we
developed the first RNAi-based pre-clinical treatment for LGMD1A, which is
caused by dominant mutations in one allele of the myotilin (MYOT) gene. We
generated several MYOT-targeted artificial microRNAs (miMYOT) that significantly
reduced mutant MYOT mRNA and protein in vitro. We then delivered our lead miMYOT
sequence to the T57I transgenic mouse model of LGMD1A using AAV6 vectors, and
data from treated mice were just obtained at the time of abstract submission.
Three months post-injection, we found that miMYOT vectors, but not controls,
significantly reduced soluble mutant MYOT protein to levels undetectable by
immunohistochemical staining in transduced areas and the protein aggregates that
characterize LGMD1A were either absent or very small in treated muscles. We are
currently quantifying these obvious histolopathological improvements, and are
also determining whether our miMYOT vectors will similarly improve overall
muscle weakness in T57I mice using whole muscle specific force assays. These
studies represent important first steps toward translating targeted RNAi gene
therapy approaches for LGMD1A, and may have broader implications. Importantly,
since dominant myopathies arise from mutations in at least 37 different genes
that collectively affect ∼1 in 2,400 to 3,200 individuals, our RNAi strategies
could be adapted to impact a large class of dominant muscle disorders.
2) RNA Interference Improves Myopathic Phenotypes
in Mice Over-Expressing Facioscapulohumeral Muscular Dystrophy Region Gene 1 (FRG1)
Lindsay M. Wallace, Sara E. Garwick-Coppens, Scott Q. Harper. Center
for Gene Therapy, The Research Institute at Nationwide Children's Hospital,
Columbus, OH; Molecular, Cellular and Developmental Graduate Program, The Ohio
State University, Columbus, OH; Pediatrics, The Ohio State University, Columbus,
OH
Muscular dystrophies, and other diseases of muscle, arise from recessive and
dominant gene mutations. Gene replacement strategies may be beneficial for the
former, while gene silencing approaches may provide treatment for the latter. In
the last two decades, muscle-directed gene therapies were primarily focused on
treating recessive disorders. This disparity at least partly arose because
feasible mechanisms to silence dominant disease genes lagged behind gene
replacement strategies. With the discovery of RNA interference (RNAi) and its
subsequent development as a promising new gene silencing tool, the landscape has
changed. In this study, our objective was to demonstrate proof-of-principle for
RNAi therapy of a dominant myopathy in vivo. We tested the potential of
AAV-delivered therapeutic microRNAs, targeting the human Facioscapulohumeral
muscular dystrophy (FSHD) Region Gene 1 (FRG1), to correct myopathic features in
mice expressing toxic levels of human FRG1 (FRG1-high mice). We found
that FRG1 gene silencing improved muscle mass, strength, and histopathological
abnormalities associated with muscular dystrophy in FRG1-high mice.
Specifically, muscles transduced with FRG1-targeted microRNAs (miFRG1) were
normal in size, showed no fibrosis or fat deposition, and had no evidence of
myofiber degeneration or regeneration. Moreover, grip strength testing showed
that miFRG1-treated animals were as strong as wild-type mice, while
control-treated or untreated animals remained significantly weaker. Our results
support the feasibility of using RNAi to target other FSHD candidate genes,
including DUX4, which has recently emerged as the leading pathogenic insult
underlying FSHD. Moreover, this approach potentially applies to as many as 37
different gene mutations responsible for myopathies inherited as autosomal
dominant disorders.
3) Systemic Delivery of RNase H-Active Antisense
Oligos in a Transgenic Mouse Model of Myotonic Dystrophy Type 1
Thurman M. Wheeler, Andrew J. Leger, Sanjay K. Pandey, A. Robert
MacLeod, Masayuki Nakamori, Seng H. Cheng, C. Frank Bennett, Bruce M. Wentworth,
Charles A. Thornton. Department of Neurology, University of Rochester, Rochester,
NY; Genzyme Corporation, Framingham, MA; Isis Pharmaceuticals, Carlsbad, CA
Objective: To test whether systemic delivery of RNase H-active antisense oligos
(ASOs) can reduce or eliminate RNA toxicity in a transgenic mouse model of
myotonic dystrophy type 1 (DM1). Background: DM1 is a dominantly inherited
degenerative disease caused by expression of an expanded CUG repeat (CUGexp) in
the 3′ UTR of the DMPK transcript. CUGexp RNA accumulates in the nucleus,
sequesters poly(CUG) binding proteins, and forms nuclear inclusions.
Trans-dominant effects of the mutant transcript include aberrant pre-mRNA
splicing, dysregulated gene expression, myotonia, and muscular dystrophy. Human
skeletal actin-long repeat (HSA-LR) transgenic mice express CUGexp RNA in the 3′
UTR of an hACTA1 transgene. In this model, the toxic RNA is retained in the
nucleus and induces splicing changes similar to DM1. Antisense knockdown of
pathogenic RNA would be expected to mitigate clinical features of DM1. However,
skeletal muscle is less sensitive to ASO effects because distribution of ASOs to
muscle tissue is low. In previous studies in rodents (n = 10), systemic ASOs
failed to produce target knockdown in muscle, despite strong effects in liver.
Design/methods: ASOs were 2′ methoxyethyl gapmers that were designed to work by
recruiting the nuclear enzyme RNase H to the ASO-RNA heteroduplex. ASOs
targeting the hACTA1 mRNA were screened in cell culture. Active ASOs then were
tested by subcutaneous injection in mice (25 mg/kg biweekly for 4 weeks).
Control mice were treated with saline. Treatment assignments were randomized and
analysis was blinded. Control ASOs targeted Malat1, Pten, and Srb1. Results: Two
hACTA1-targeting ASOs reduced transgene levels by up to 80%. Control ASOs had no
effect. Serum chemistries and histopathology showed no evidence of toxicity.
Knockdown of toxic RNA was associated with elimination of myotonia and
correction of RNA mis-splicing in all muscles examined. These effects persisted
at least 15 weeks after the final dose. By microarray analysis, > 85% of changes
in gene expression were normalized or improved. Another nuclear-retained
non-coding RNA, Malat1, was also sensitive to knockdown in muscle (up to 80%)
using similar RNase H ASO designs. However, ASOs targeting endogenous mRNAs (Pten,
Srb1) did not produce knockdown in muscle, and the distribution of ASOs to
skeletal muscle in the DM1 model was not higher than in WT mice. Conclusions:
Systemic delivery of RNase H-active ASOs was surprisingly effective at reducing
RNA toxicity in a transgenic mouse model of DM1. Nuclear-retained transcripts
may display increased sensitivity to RNase H-active ASOs and enable RNA
knockdown in muscle tissue where biodistribution is low. The mechanism may
relate to residence time of transcripts in the nucleus, the cellular compartment
in which RNase H1 is also located. ASOs that act through the RNase H pathway may
exploit the nuclear retention phenomenon to gain a therapeutic advantage in DM1.
4) Addition of Peptide Therapy To Inhibit NF-κB
Activation to AAV Serotype 9 Mini-Dystrophin Gene Transfer To Treat Muscular
Dystrophy in mdx Mice
Daniel P. Reay, Gabriela A. Niizawa, Jon F. Watchko, Molly Daood,
Eugene Raggi, Paula R. Clemens. Neurology, University of Pittsburgh, Pittsburgh,
PA; Pediatrics, Magee-Women's Research Institute, Pittsburgh, PA; Neurology
Service, Department of Veteran's Affairs Medical Center, Pittsburgh, PA
Systemic gene transfer using serotype 9 adeno-associated vectors (AAV9) is
promising for treatment of preclinical models of Duchenne muscular dystrophy (DMD).
The ability to achieve systemic vector delivery circumvents a significant hurdle
presented by the widespread distribution of skeletal muscle that is best
accessed through the circulation. However, a limitation of systemic gene vector
delivery is that gene transduction levels vary among muscle groups. The addition
of complementary therapy could provide 1) a treatment effect independent of gene
transfer and 2) facilitation of successful gene transfer. Pathological
activation of the nuclear factor κB (NF-κB) signaling pathway has emerged as an
important cause of dystrophic muscle changes in muscular dystrophy. Furthermore,
activation of NF-κB may also inhibit gene transfer by promoting inflammation in
response to the transgene or vector. Therefore, we hypothesized that the
addition of NF-κB inhibition in muscle would complement the therapeutic benefits
of dystrophin gene transfer in the mdx mouse model of DMD. The NF-κB
Essential Modulator (NEMO) binding domain (NBD) peptide, which is utilized in
this study to inhibit NF-κB, is synthesized as a fusion peptide with a protein
transduction domain of 8 lysines (8K) to facilitate intra-cellular delivery. An
AAV9 vector carrying a human codon-optimized mini-dystrophin gene under control
of the cytomegalovirus promoter was given as a single intraperitoneal injection
(1x1011 gc/pup) at age 2-3 days to 16 mdx mice (experimental
mice). Starting at age 4 weeks, 8 of the experimental mice were given
intraperitoneal injections of 8K-NBD peptide dosed at 10mg/kg, 3 times per week
for 5 weeks. The second group of 8 experimental mice received sham
intraperitoneal saline injections. Control groups (n=8/group) included normal
C57BL10 and disease control untreated mdx mice. At sacrifice, ex vivo
physiological studies of diaphragm force production were performed prior to
histological and biochemical assays of diaphragm and limb muscle tissue. In
mdx mice treated with the combination of 8K-NBD peptide and AAV9
mini-dystrophin gene delivery, the quadriceps muscle demonstrated increased
levels of recombinant dystrophin expression (84% of fibers expressing, as
compared to 56% with AAV9 mini-dystrophin gene delivery alone) suggesting that
8K-NBD treatment promoted an environment in muscle tissue conducive to higher
levels of recombinant dystrophin expression. Indices of necrosis and
regeneration were diminished with either AAV9 gene delivery alone or in
combination with 8K-NBD treatment. In diaphragm muscle, transgene expression was
sufficiently high (>95% of muscle fibers expressed dystrophin) that marked
improvements in histological and physiological indices were comparable in the 2
treatment groups. The data support benefit from 8K-NBD treatment to complement
gene transfer therapy for DMD in muscle tissue that receives incomplete levels
of transduction by gene transfer.
5) A Phase I Dose-Escalating Study of AAV1 – γ-Sarcoglycan
Gene Therapy for Limb Girdle Muscular Dystrophy Type 2C
Serge Herson, Fayçal Hentati, Aude Rigolet, Norma B. Romero, Anthony
Behin, France Leturcq, Pascal Laforet, Thierry Maisonobe, Rim Amouri, Hafedh
Haddad, Muriel Audit, Marie-Françoise Rosier-Montus, Bernard Gjata, Carole
Masurier, François M. Lemoine, Pierre Carlier, Jean-Yves Hogrel, Bruno Eymard,
Lee Sweeney, Richard Mulligan, David Klatzmann, Mustapha Cheraï, Didier
Caizergues, Thomas Voït, Olivier Benveniste. Internal Medicine, Université
Pierre et Marie Curie, Assistance Publique-Hôpitaux de Paris, Hôpital
Pitié-Salpêtrière, Paris, France; Department of Molecular Neurobiology and
Neuropathology, National Institute of Neurology, Tunis, Tunisia; Institute of
Myology, Université Pierre et Marie Curie, Assistance Publique-Hôpitaux de
Paris, Hôpital Pitié-Salpêtrière, Paris, France; Laboratoire de Biochimie
Génétique, Assistance Publique-Hôpitaux de Paris, Hôpital Cochin, Paris, France;
Généthon, Evry, France; GenoSafe, Evry, France; UMR 7211, CNRS, Université
Pierre et Marie Curie, Assistance Publique-Hôpitaux de Paris, Hôpital
Pitié-Salpêtrière, Paris, France; Department of Physiology, School of Medicine,
University of Pennsylvania, Philadelphia, PA; Harvard Gene Therapy Initiative,
Department of Genetics, Harvard Medical School, Boston, MA
OBJECTIVE: Safety of dose-escalating AAV1 – γ-sarcoglycan gene therapy (Phase I)
for Limb Girdle Muscular Dystrophy type 2C. BACKGROUND: Gamma-sarcoglycanopathy
or limb girdle muscular dystrophy type 2C (LGMD 2C) is an as yet untreatable
disease caused by autosomal recessively inherited mutations of the γ-sarcoglycan
gene (γSGC). DESIGN/METHODS: Nine non-ambulant LGMD2C patients (2 M, 7 F, age 27
y [16 to 38]), with a del525T homozygous mutation on the γSGC gene and absence
of γSGC immunostaining in muscle biopsy were enrolled. They were divided into 3
groups to receive 3 escalating doses of an AAV1 vector expressing human γSGC
gene under the control of the desmin promoter, by local intramuscular injection
into the extensor carpi radialis muscle. The first group received a single
injection of 3e+9 viral genome (vg) in 100 µl, the second a single injection of
1.5e+10 vg in 100 ul and the third group received 3 concomitant 100 µl
injections at the same site (a total of 4.5e+10 vg). RESULTS: No serious side
effects were observed. All patients became seropositive for AAV1 and one
developed a cytotoxic response again AAV1 capsid. In biopsy specimens taken 30
days after the injections, immunohistochemistry showed γSGC expression in 5/9
patients, with 4.7 to 10.5% of positively stained fibers and detection of γSGC
mRNA by RT-PCR in the 3 patients who received the highest dose. The γSGC protein
became also detectable by Western blot in one patient. CONCLUSIONS: Exogenous γSGC
expression can be obtained in LGMD2C patients following AAV1 gene transfer,
without adverse effects, paving the way for future developments in gene therapy
of hereditary muscle diseases.
6) Immune Tolerance Induction in Canine X-Linked
Muscular Dystrophy with rAAV9-Microdystrophin Transduction
Hiromi Hayashita-Kinoh, Naoko Yugeta, Hironori Okada, Yuko
Nitahara-Kasahara, Tomoko Chiyo, Takashi Okada, Shin'ichi Takeda. Dept. of
Molecular Therapy, National Institute of Neuroscience, NCNP, Tokyo, Japan
Background: Duchenne muscular dystrophy (DMD) is a congenital disease causing
progressive deterioration of skeletal and cardiac muscles because of mutations
in the dystrophin gene. We have previously reported that local injection of
rAAV2 or rAAV8 to canine skeletal muscles without immunosuppression resulted in
insufficient transgene expression with potent immune responses. Also we found
that DCs would trigger an immune response against the rAAV-mediated transduction.
Here we used fetuses of the canine X-linked muscular dystrophy in Japan (CXMDJ)
to investigate the strategy of inducing immunotolerance to the rAAV as well as
the muscle transduction profiles of rAAV9-microdystrophin. Methods: For fetal
transduction, pregnant CXMDJ heterozygote was anesthetized and
fetuses at post-coital day 35 were injected with 1x1012 of
rAAV-CMV-microdystrophin along with 1x1011 of rAAV-CAG-Luciferase
into amniotic fluid with ultrasound guidance. The yolk sacs and umbilical cords
were sampled at delivery, and then transduced rAAV copy numbers were estimated
by qPCR. To expect systemic microdystrophin expression, we additionally injected
rAAV9-CMV-microdystrophin into the jugular vein of 6 weeks old dystrophic dog.
To examine the immunotolerance to the rAAV, purified canine peripheral
leukocytes were exposed to rAAV9-microdystrophin for 4 hours, and then IFN-γ
expression was analyzed using qRT-PCR. Temporal muscles of the rAAV-injected
animals were collected by punch-biopsy at various time points for expression
analysis. In addition, rAAV-injected CXMDJ and non-injected CXMDJ
were compared each other to assess gait function and lameness in the hind limb.
Moreover, using whole body plethysmography, echocardiography and
electrocardiography, we analyzed the respiratory and cardiac functions of the
rAAV-injected dogs. Results: To rAAV transduction to fetus in utero, higher
amount of AAV genome in the yolk sack of the rAAV-injected dogs was detected
compared to that of the rAAV-uninjected control. Expression of IFN-γ in the
purified peripheral blood leukocytes after the rAAV exposure were not induced in
one of the rAAV-injected dogs, suggesting the successful induction of immune
tolerance against rAAV. rAAV-derived microdystrophin expression was confirmed by
qRT-PCR and immunohistochemistry in the transduced dog. Monthly analysis of the
transduced dystrophic dogs demonstrated superior gait function to non-injected
littermate CXMDJ. At 1 year old, respiratory function and cardiac
function of the transduced affected dog was milder form than untransduced
affected dog. Conclusion: Our results demonstrate that induction of oral
immunotolerance against rAAV with long-term transgene expression can be achieved
by direct delivery of rAAV into amniotic fluid. This strategy would be effective
approach to analyze the expression and function of transgene in vivo. These
findings also support the future feasibilities of rAAV-mediated fetal gene
delivery strategies. Furthermore, we plan oral immune tolerance induction after
the delivery to meet clinical settings.
7) Site Directed Gene Repair of the Dystrophin
Gene in Satellite Cells Mediated by PNA-ssODNs Restores Dystrophin Expression
into Skeletal Muscles of a Mouse Model for Duchenne Muscular Dystrophy
Farnoosh Nik-Ahd, Refik Kayali, Carmen Bertoni. Department of
Neurology, University of California Los Angeles, Los Angeles, CA
Gene editing mediated by single stranded oligodeoxynucleoitdes (ssODNs) has
shown to be able to correct single point mutations and to restore dystrophin
expression in several models for Duchenne muscular dystrophy (DMD). This
technology takes advantages of specific repair mechanisms present in the cells
that are able to recognize the presence of mismatches in genomic DNA.
Oligonucleotides complementary to the target sequence but containing a mismatch
at the base targeted for modification are used as templates for the correction
process. Once introduced into the cell, they have been shown to anneal to the
genomic DNA sequence targeted for repair and initiate the repair process leading
to a single nucleotide exchange that is stably inherited throughout cell
division. We have recently demonstrated that ssODNs made of peptide nucleic acid
(PNA-ssODNs) are more effective in inducing single base alterations than
oligonucleotides made of unmodified bases in muscle cells in culture as well as
into skeletal muscle of dystrophin deficient mice. For therapeutic applications,
gene correction strategies mediated by oligonucleotides are likely to require
targeting not only mature myofibers, but also muscle progenitor cells also known
as satellite cells (SC) that actively participate to muscle regeneration and
repair. We have tested the ability of PNA-ssODNs to correct the dystrophin gene
defect in SC of the mdx5cv mouse. This strain has a point
mutation in exon 10 of the dystrophin gene that creates a cryptic splice site
for the dystrophin mRNA and is particularly useful to perform quantitative
analysis of gene correction frequencies at both the mRNA and the genomic DNA
levels. SC isolated from mdx5cv muscles were transfected with
correcting PNA-ssODNs targeting the non-coding strand of the dystrophin gene
defect and transplanted into skeletal muscles of immunosuppressed dystrophin
deficient mice. As control, we used PNA-ssODNs lacking the mismatch. Restoration
of dystrophin expression was assessed in vitro at the mRNA and protein levels.
Gene correction was demonstrated at the genomic level by direct sequencing of
PCR products derived from cells transfected with targeting oligonucleotides and
maintained in culture. Dystrophin was clearly detected in muscles that received
mdx5cv SC transfected with the correcting oligonucleotide but
not in muscles transplanted with SC treated with control PNA-ssODNs. Expression
of dystrophin was stable for up to six months after transplantation as
demonstrated by immunostaining and western blot analyses. These results support
the use of PNA-ssODN as a viable gene editing tool for the treatment of muscle
diseases due to point mutations using cell-mediated regenerative approaches.
Supported by a grant from the Muscular Dystrophy Association of the United
States (MDA)
8) Long-Term Engraftment of Mesenchymal Stromal
Cells That Can Differentiate To Form Myogenic Cells in Dog with Duchenne
Muscular Dystrophy
Yuko Nitahara Kasahara, Hiromi Hayashita Kinoh, Hironori Okada,
Jin-Hong Shin, Akiyo Nishiyama, Sachiko Ohshima Hosoyama, Michiko Wada Maeda,
Akinori Nakamura, Takashi Okada, Shin'ichi Takeda. Department of Molecular
Therapy, National Institute of Neuroscience, National Center of Neurology and
Psychiatry, Kodaira, Tokyo, Japan
Background: Duchenne muscular dystrophy (DMD) is an incurable genetic disease
with early mortality. Multipotent mesenchymal stromal cells (MSCs) could be
potential therapeutics, because they can be transplanted to muscle tissue and
differentiated to form myogenic cells in situ. We developed the
strategies for effective cell transplantation in dystrophic dog. Methods: Canine
CD271-positive (+) MSCs obtained from donor bone marrow cells were enriched by
immunomagnetic isolation. The dog leukocyte antigens (DLA) were analyzed to
determine DLA- identical mating pairs to achieve donor or recipient of
allogeneic transplantation. Cardiotoxin (CTX) was injected into tibialis
anterior (TA) muscles of normal Beagle dog 5 days before MSCs injection. CD271+
MSCs (2x106 cells) transduced with an adenoviral vector expressing
MyoD (Ad-MyoD) were injected into the TA muscles of recipient canine X-linked
muscular dystrophy in Japan (CXMDJ) without immunosuppression. For
intra-arterial injection, MyoD-transduced CD271+ MSCs (5x106
cells) were administered into the femoral artery with transient avascularization
using a tourniquet followed by the injection of papaverin hydrochloride. The
treated muscles were biopsied and analyzed histologically. Autologous CD271+
MSCs (2x106 cells) were transduced with AAV-microdystrophin and
administrated into the TA muscles of DMD dog. Results: CD271+ MSCs
obtained from the normal Beagle dog showed greater growth expansion compared
with CD271-depleted MSCs. MyoD transduction of CD271+ MSCs caused
myogenic differentiation in vitro and myotube formation with late
myogenic markers expression. Without immunosuppression, CD271+ MSCs
in the myogenic cell lineage were transplanted into DLA-identical CXMDJ
and formed clusters of muscle-like tissue at 8 and 12 weeks after the
intra-muscular injection. The engraftment of CD271+ MSCs was also
found at the site of CTX-injured muscle after the intra-arterial injection.
Interestingly, CD271+ MSCs engraftment was improved with the
pre-treatment of papaverin hydrochloride. Immunohistological analysis suggested
that CD271+ MSCs formed muscle-like tissues and exhibited
upregulation of developmental myosin heavy chain as well as dystrophin without
inflammatory cell infiltration. Furthermore, CD271+ MSCs derived from
CXMDJ also showed myotube formation in vitro by the expression
of microdystrophin. We are currently conducting the transplantation of
AAV-microdystrophin-transduced autologous MSCs into the CXMDJ and
will discuss the effects. Conclusion: MyoD-transduced CD271+ MSCs
enabled more efficient realization of MSCs transplantation by the intra-muscular
as well as intra-arterial injection. This strategy of MSCs propagation and
treatment would be promising for the future DMD cell therapy.
9) Systemic Reversal of Dominant Muscle Disease in
a Mouse Model of Muscular Dystrophy
Sergia Bortolanza, Davide Gabellini, Joel R. Chamberlain. Dulbecco
Telethon Institute, Seattle, WA; Division of Regenerative Medicine, San Raffaele
Scientific Institute, Seattle, WA; Medicine, University of Washington, Seattle,
WA
Dominantly inherited genetic disease associated with a gain-of-function
mechanism poses a particular challenge for gene therapy development. A
straightforward therapeutic scheme would be to eliminate the genetic product
that triggers pathogenic processes. We have chosen to pursue an RNAi-based
approached aimed at targeted knockdown of FRG1 mRNA in the FRG1 mouse
model of facioscapulohumeral muscular dystrophy. The FRG1 mouse displays a
variety of typical physical and molecular features of FSHD patients. Using the
FRG1 mouse model we can harness the endogenous cellular RNAi pathway to degrade
mRNA from genes expressing a toxic product in a targeted fashion. We have
packaged extended short hairpin RNA (shRNA) expression cassettes targeting
FRG1 mRNA into adeno-associated virus serotype 6 (AAV6) shuttles for
systemic delivery in vivo. AAV6 was selected as our gene transfer vector
because of its predominant muscle tropism, high efficiency transduction of
muscle cells, and long-term expression of transgenes. Intravenous tail vein
injection of 5 x 1012 vector genomes of the AAV6 FRG1 shRNAs
into dystrophic mice resulted in a significant improvement in muscle function as
measured by treadmill running with a concomitant reversal of phenotypic changes
making the mice indistinguishable from wild-type mice. We also observed
improvements in histological features, including a reduced fiber size, central
nucleation, adipose accumulation, and fibrosis. Accompanying molecular changes
included reduction of FRG1 mRNA by 40-60% relative to saline-injected
controls. Our data indicate that RNAi-mediated mRNA knockdown is a feasible
approach to dominant genetic disease therapy and can be applied after onset of
symptoms of a dominant muscular dystrophy in mice to reverse the course of the
disorder. Application of this method to target relevant components involved in
muscle disease offers a route to clinical application of RNAi for treatment of
FSHD and other dominant muscle disorders.
10) Exploring the Role of Deubiquitinating Enzyme
A20 for Treatment of Muscular Dystrophy in mdx Mice
Rakshita Charan, Gabriela Niizawa, Hiroyuki Nakai, Paula R. Clemens.
Department of Neurology, University of Pittsburgh, Pittsburgh, PA; Neurology
Service, Department of Veteran Affairs Medical Center, Pittsburgh, PA;
Department of Microbiology and Molecular Genetics, University of Pittsburgh,
Pittsburgh, PA
Duchenne muscular dystrophy (DMD) is one of the most common muscle disorders
affecting about 1 in 3500 male births worldwide. It is caused by mutations in
the gene dystrophin, the protein product of which is required for muscle
structure and stability. Studies suggest that the lack of dystrophin protein
leads to dystrophic pathology in skeletal muscle; an important pathologic
mediator is nuclear factor kappa B (NF-κB), a transcription factor that
regulates several genes responsible for stress responses, cell survival and
various inflammatory conditions. NF-κB is pathologically activated in dystrophic
muscle in DMD patients and in the mdx mouse model for DMD. NF-κB activation in
mdx mice is thought to activate protein degradation and cause chronic
inflammation in skeletal muscle. Furthermore, pathological activation of NF-κB
downregulates myogenic regulatory factors and interferes with muscle
regeneration. Attenuation of NF-κB activation in mdx mice has been shown to
improve muscle stability and strength. Thus, strategies to inhibit NF-κB
activation are being actively pursued as a therapeutic option for DMD. A20, a
deubiquitinating enzyme, is known to attenuate NF-κB activation by
deubiquitinating RIP1. We sought to characterize A20 in skeletal muscle and
examine its role as a potential therapeutic target to attenuate NF-κB activation
in DMD. Our studies show that siRNA blockade of A20 significantly increases NF-κB
activation in cultured mdx and control C57BL/10 myotubes in vitro. Since NF-κB
is known to play a role in muscle regeneration and differentiation, we studied
the effect of blocking A20 on muscle differentiation in myoblasts obtained from
these mice. We observe a delay in myoblast differentiation in the absence of A20
indicating a role for A20 in promotion of muscle differentiation. Further
characterization of A20 localization in vivo in mdx muscle demonstrated
expression predominantly in regenerating fibers; interestingly, most are
fast-twitch muscle fibers. A time-profile assessment of A20 expression in mdx
mice compared with control mice showed an increase in A20 protein expression
during the 7-12 week time period in mice that corresponds with the ages when
cycles of severe muscle degeneration and regeneration occur in mdx mice. This
correlation between expression of an NF-κB inhibitor attenuating NF-κB
activation and pathology of DMD is a promising observation to further explore
the efficacy of A20 as a therapeutic target. We are developing adeno-associated
viral vectors carrying A20 expression cassettes in order to test the therapeutic
benefit of A20 over-expression in the mdx model of DMD.
11) Restoration of Dystrophin Expression in the
mdx Mouse Model for Duchenne Muscular Dystrophy (DMD) Induced by RTC13
Refik Kayali, Liutao Du, Jin-Mo Ku, Gladys Completo, Olga Prikhodko,
Yosuf Subat, Hailiang Hu, Michael Jung, Richard A. Gatti, Carmen Bertoni.
Department of Neurology, David Geffen School of Medicine University of
California Los Angeles, Los Angeles, CA; Department of Pathology and Laboratory
Medicine, David Geffen School of Medicine University of California Los Angeles,
Los Angeles, CA; Department of Chemistry and Biochemistry, University of
California Los Angeles, Los Angeles, CA
Molecules that induce ribosomal read-through of nonsense mutations in mRNA and
allow production of a full-length functional protein hold great therapeutic
potential for the treatment of many genetic disorders. Two such read-through
compounds, RTC13 and RTC14, were recently identified by a luciferase-independent
high-throughput screening assay and were shown to have potential therapeutic
functions in the treatment of nonsense mutations. We have tested the ability of
RTC13 and RTC14 to restore dystrophin expression into skeletal muscles of the
mdx mouse model for Duchenne muscular dystrophy (DMD). Intramuscular
injections of RTC13, promoted read-through of the mdx UAA stop codon more
efficiently than gentamicin, PTC124 or RTC14 making it our lead drug candidate.
When administered systemically, RTC13 was shown to restore dystrophin protein in
different muscle groups, including diaphragm and heart. Improved muscle strength
was detected in all treated animals and was accompanied by a significant
decrease in creatine kinase (CK) levels demonstrating that the compound was able
to slow down muscle degeneration and turnover. No signs of toxicity were
detected in mdx after prolonged administration of RTC13 demonstrating that the
compound was well tolerated in mice. The levels of direct bilirubin (DBIL),
blood urea nitrogen (BUN), creatinine, alkaline phosphatase (ALP) and alanine
aminotransferase (ALT) were significantly decreased in RTC13-treated mice as
compared to untreated mdx or mdx mice that received vehicle
alone confirming that restoration of dystrophin expression in muscles was able
to ameliorate some of the secondary pathology associated with the disease in
mdx. Structure activity relationship (SAR) studies were used to optimize the
molecular structure of RTC13 and to identify a derivative that meets optimal
safety profiles while still maintaining maximal read-through activity. These
results advance the development of RTC13 as an effective drug candidate for DMD.
They also offer hope for the treatment of numerous other genetic disorders due
to nonsense mutations and premature termination of protein synthesis.
12) Novel Dual AAV Vectors To Express an
nNOS-Recruiting Mini-Dystrophin Gene for Duchenne Muscular Dystrophy Gene
Therapy
Yadong Zhang, Dongsheng Duan. Department of Molecular Microbiology
and Immunology, School of Medicine, University of Missouri, Columbia, MO
Duchenne muscular dystrophy (DMD) is a lethal childhood muscle disease. We
recently demonstrated that a 7kb synthetic mini-dystrophin gene that can restore
neuronal nitric oxide synthase (nNOS) may yield superior protection in the
murine models of DMD (J Clin Invest. 2009;119: 624-35). Adeno-associated virus (AAV)
holds great promise in treating all affected muscles in the body. However, AAV
application in DMD gene therapy is limited by the 5 kb maximal virion packaging
capacity. We have previously developed a series of dual AAV vector strategies
for doubling AAV packaging capacity. These include overlapping, trans-splicing
and hybrid vector approaches. In dual AAV vectors, a fully functional expression
cassette is split into two parts and each part is individually packaged in an
AAV virion. Reconstitution is achieved through homologous recombination and/or
AAV inverted terminal repeats-mediated viral genome concatamerization. In this
study, we first developed two GFP-dystrophin fusion constructs. In one construct,
GFP is fused in-frame to the 5'-end of the 7 kb mini-dystrophin gene. In another
construct, GFP is fused in-frame to the 3'-end of the 7 kb mini-dystrophin gene.
A series of overlapping and hybrid dual AAV vectors were engineered to express
the GFP-mini-dystrophin fusion gene. Dual AAV vectors were packaged in Y445F
tyrosine mutant AAV serotype-6 vectors. Initial studies in dystrophin-deficient
mdx4cv mice revealed robust mini-dystrophin expression and successful
recruitment of nNOS to the sarcolemma. Additional studies are currently underway
to test functional rescue with these new dual AAV mini-dystrophin vectors (Supported
by NIH and MDA).
13) Fukutin-Related Protein Knock-Down by Systemic
AAV9-shRNA Delivery or by Transgenic Knock-In Mutation Resulted in Similar
Muscle and Heart Pathology of LGMD 2I
Chi-Hsien Wang, Yiumo M. Chan, Bin Xiao, Chunping Qiao, Ru-Hang Tang,
Hui Zheng, Elizabeth Keramaris-Vrantsis, Juan Li, Peijuan Lu, Qilong Lu, Xiao
Xiao. Department of Molecular Pharmaceutics, Eshelman School of Pharmacy,
University of North Carolina-Chapel Hill, Chapel Hill, NC; McColl-Lockwood
Laboratory for Muscular Dystrophy Research, Neuromuscular/ALS Center, Carolinas
Medical Center, Charlotte, NC
Fukutin-related protein (FKRP) has been speculated as a putative
glycosyltransferase. The mutations of the FKRP not only cause late onset and
milder limb-girdle muscular dystrophy 2I (LGMD2I) without central nervous system
defect, but also cause severe allelic diseases such as congenital muscular
dystrophy 1C, Walker-Warburg Syndrome and muscle-eye-brain disease. Since FKRP
knockout mice are embryonic lethal, alternative methods are explored to generate
viable animal models that closely recapitulate LGMD2I clinical phenotypes.
Previously, we successfully knocked down the FKRP expression in the hind limb
muscles for 10 months post i.m. injection (mpj) and recapitulated pathological
and histological signs of LGMD 2I. Here, we investigated two different methods,
AAV9-mediated systemic delivery of shRNA to knock-down FKRP (FKRP KD) in normal
mice and genitic knock-in of the FKRP L276I missense mutation (L276I KI), to see
if we can establish representative LGMD 2I animal models. To evaluate both mice
models, the dystrophic pathologies were monitored. Firstly, central nucleation
was observed in quadriceps of the 4 months old homo FKRP L276I KI mice. For the
FKRP KD mice, it would only be found at 9 mpj. Glycosylation of the
α-dystroglycan was reduced in the quadriceps of both groups at 9 months. We also
found abnormal electrocardiogram (ECG) in the FKRP KD group, i.e., cardio
conduction abnormality (left bundle branch block, LBBB) but there was no
significant change in heart functions, e.g., percent of ejection fraction (EF%):
54.2±5.9 vs control: 54.0±1.6. Differently, the homo FKRP L276I KI mice showed
both LBBB and right bundle branch block (RBBB). Furthermore, heart function
impairment was revealed by echocardiography (Echo), i.e. significant decreases
in EF% and percentage fractional shortening (FS%) in homo FKRP L276I KI mice (EF%:
73.7±10.5 vs. control 82.3±3.6; FS%: 42.5±8.4 vs. control 49.6±3.8). The echo
readings of left ventricular internal dimensions in diastole (LVIDd) and systole
(LVIDs) were also significantly increased in homo FKRP L276I KI mice (LVIDd:
3.3±0.4 vs. control: 2.8±0.3; LVIDs: 1.9±0.5 vs. control: 1.5±0.2), indicating
dilated left ventricle. Moreover, the LV volumes in end-systole (LV-Vol; d),
end-diastole (LV-Vol; s), and mass were significantly increased in homo FKRP
L276I KI mice (LV-Vol; d: 44.0±12.9 vs 30.0±6.4; LV-Vol; s: 12.6±8.0 vs 5.9±1.8;
LV Mass: 91.6±14.9 vs 75.2±14.4), suggesting enlarged left ventricle. In summary,
using two different methods to reduce the FKRP expression in mice, we have
obtained similar LGMD2I phenotypes. But an earlier onset of myopathies and
severe heart failure were found in homo FKRP L276I KI mice. These animal models
should be useful in studying LGMD 2I pathology as well as development of
therapeutic approaches, such as gene therapy.
14) Inhibition of CD26 Activity Enhances
Engraftment of Donor Cells to Regenerating and Dystrophic Skeletal Muscle
Maura H. Parker, Carol Loretz, Rainer Storb, Stephen J. Tapscott.
Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA;
Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA;
Department of Medicine, University of Washington, Seattle, WA; Department of
Neurology, University of Washington, Seattle, WA
Muscle-derived cell transplantation has the potential to effectively treat many
human diseases, including muscular dystrophy. A variety of cell populations
engraft into skeletal muscle of mdx mice, effectively restore dystrophin
expression and reconstitute the satellite cell pool. Yet, a direct and
quantitative comparison of engraftment to determine the most effective cell
population is lacking. We have developed a canine-to-mouse xenotransplantation
model to rapidly and quantitatively compare canine muscle cell engraftment.
Specifically, we demonstrate that canine muscle derived cells engraft into
regenerating mouse muscle, and engraftment is quantifiable and consistent. The
canine-to-mouse model allows us to quantitatively compare cell populations and
modulating factors, and establish priority for transplantation experiments using
a clinically relevant immune tolerant cxmd canine model of muscular
dystrophy. We used the xenotransplant model to show that canine muscle derived
cells sorted for expression of CXCR4 do not display a greater level of
engraftment when compared to a mixed cell population. However, pre-treating a
mixed cell population with diprotin A, a positive modulator of CXCR4-SDF-1
binding, significantly enhances engraftment of donor cells to the mouse
satellite cell niche. Translating these results to the immune tolerant canine,
we demonstrate that injection of diprotin treated donor cells results in a
significantly increased number of muscle fibers expressing dystrophin as
comapred to untreated cells. Temporal regulation of CXCR4/SDF-1 binding may be
an important means of expanding the effective range of engraftment after
transplantation.
15) Antisense Drug Development for Skipping Human
Dystrophin Exons In Vitro and In Vivo Systematically
Bo Wu, Ehsan Benrashid, Peijuan Lu, Caryn Cloer, Allen Zillmer, Mona
Shaban, QiLong Lu. McColl-Lockwood Laboratory for Muscular Dystrophy Research,
Carolinas Medical Center, Charlotte, NC
Antisense therapy has recently been demonstrated with great potential for
frame-restoring of dystrophin mRNA in human muscle cells and in local muscles of
Duchenne muscular dystrophy (DMD) patients. Therapeutic values of exon skipping
critically depend on efficacy of the drugs, antisense oligomers (AOs), for
targeted skipping of human dystrophin exons. Identification of AOs with highest
efficiency and specificity is therefore crucial for AO drug development. In this
study, we applied three cell culture systems, C2C12 myoblasts containing a
GFP-reporter gene expressing human dystrophin exon 50, normal human myoblasts
and a culture of DMD patient-derived skin fibroblasts, to screen AOs targeting
human dystrophin exon 50. The GFP reporter system was most effective and
sensitive for quantitative measurement of exon skipping efficiency. The efficacy
of selected AOs was further investigated with Vivo-Morpholino chemistry in the
hDMD transgenic mouse carrying the full-length human dystrophin gene. Our
results suggest that a combination of in vitro cell culture based selection
systems and a Vivo-Morpholino based evaluation in vivo systemically, provides
stringent screening to identify effective AOs targeting human dystrophin exon
for drug development to treat DMD patients.
16) AAV-Based shRNA Silencing of NF-κB Ameliorates
Muscle Pathologies in mdx Mice
Qing Yang, Ying Tang, Kara Imbrogno, Aiping Lu, Anmin Chen, Fengjin
Guo, Freddie H. Fu, Johnny Huard, Bing Wang. Orthopaedic Surgery, University of
Pittsburgh, Pittsburgh, PA; Orthopaedic Surgery, Tongji Hospital, Huazhong
University of Science and Technology, Wuhan, Hubei, China
Duchenne muscular dystrophy (DMD) is an X-linked genetic muscle disease
affecting 1 of every 3500 male births. Chronic inflammation, promoted by an
up-regulated NF-kappaB (NF-κB) pathway, plays a key role in Duchenne
muscular dystrophy (DMD) patients' pathogenesis. Higher immune alertness, due to
chronic inflammation and immune effector cell infiltration, challenges
recombination adeno-associated viral (AAV) vector-mediated gene replacement
treatment for DMD, especially in large animal models and clinical trials. We and
others found that blocking the NF-κB pathway could be a viable solution to
promote muscle regeneration and to decrease necrosis in mdx mice (a
murine DMD model), by using transgenic and AAV-mediated transfer (dominant-negative
forms of IKKα & β) approaches, without any side effect. Based on the small
interference RNA (siRNA) technology used in this study, we examined whether an
AAV vector carrying small hairpin RNA (shRNA) targeting the NF-κB/p65
subunit, a major subunit of NF-κB associated with inflammation in mdx
mice, inhibits NF-κB activation as well as ameliorates pathological necrosis and
regeneration in the locally-treated gastrocnemius (GAS) muscle of 1 and 4 month
old mdx mice. At 1 month post-treatment, NF-κB/p65 levels in
locally-treated muscles were significantly decreased by the efficient transfer
of specific shRNA in both of the different treated age groups. This led to
remarkable decreases in necrosis and myofiber damage compared to mdx mice
treated by a phosphate-buffered saline (PBS) or a control AAV vector containing
a scrambled shRNA. The shRNA silencing of the NF-κB pathway diminished the
pathological myofiber regeneration in young mdx mice treated at 1 month
of age, but it enhanced the capacity of myofiber regeneration in old mdx
mice treated at 4 months of age. Moreover, quantitative analysis revealed that
central nucleation of the myofibers of treated young mdx muscles was
reduced from 67% to 34%, but centrally-located nuclei were not significantly
reversed to the peripheral position in treated old mdx mice. Our findings
indicate that AAV-mediated NF-κB/p65-shRNA has the capacity to ameliorate muscle
pathologies in mdx mice by degrading mRNA of the NF-κB/p65 subunit.
17) Implication of nNOSμ Delocalization on the
Muscle Force in Dystrophin Deficient and δ-Sarcoglycan Knockout Mice
Dejia Li, Yongping Yue, Yi Lai, Chady H. Hakin, Dongsheng Duan.
Department of Molecular Microbiology and Immunology, University of
Missouri-Columbia, Columbia, MO
The mechanism of force reduction is not completely understood in Duchenne
muscular dystrophy (DMD), a dystrophin-deficient lethal disease. Nitric oxide
regulates muscle force. Interestingly, neuronal nitric oxide synthase μ (nNOSμ),
a major source of muscle nitric oxide, is lost from the sarcolemma in DMD muscle.
We hypothesize that nNOSμ delocalization contributes to force reduction in DMD.
To test this hypothesis, we generated dystrophin/nNOSμ double knockout mice.
Genetic elimination of nNOSμ significantly enhanced force in dystrophin-null
mice. Pharmacological inhibition of nNOS yielded similar results. To further
test our hypothesis, we studied δ-sarcoglycan-null mice, a model of limb-girdle
muscular dystrophy. These mice had minimal sarcolemmal nNOSμ delocalization and
muscle force was less compromised. Annihilation of nNOSμ did not improve their
force either. To determine whether nNOSμ delocalization itself inhibited force,
we corrected muscle disease in dystrophin-null mice with micro-dystrophins that
either restored or did not restore sarcolemmal nNOSμ. Similar muscle force was
obtained irrespective of nNOSμ localization. Additional studies suggest that
nNOSμ delocalization selectively inhibits muscle force in dystrophin-null mice
via nitrosative stress. In summary, we have demonstrated for the first time that
nitrosative stress elicited by nNOSμ delocalization is an important mechanism
underlying force loss in DMD.
18) Evaluation of rAAV6-Microutrophin Expression
Driven by a Highly Active Muscle Specific Regulatory Cassette in Dystrophic Mice
and the Non-Human Primate
Guy L. Odom, Glen B. Banks, Quynh Nguyen, Eric Finn, James M. Allen,
Jacqueline Wicki, Stephen D. Hauschka, Jeffrey S. Chamberlain. Neurology,
University of Washington, Seattle, WA; Biochemistry, University of Washington,
Seattle, WA
Duchenne muscular dystrophy (DMD), the most common inherited muscle disorder of
children, is caused by mutations within the dystrophin gene and results in
progressive muscle wasting. Several genetic intervention strategies are under
investigation, and progress towards clinical gene therapy with adeno-associated
viral (AAV) vector delivered transgenes remains promising. Highly truncated
dystrophin coding sequences have previously been shown to be therapeutic in
small and large animal models of DMD following rAAV gene transfer. However,
given the potential for deleterious cellular immune responses to dystrophin in
human patients, particularly those with large deletions in the dystrophin gene,
the capacity of utrophin (a dystrophin paralog) to act as a therapeutic
transgene is of high interest. To this end, we provide a comparative regulatory
element study evaluating microutrophin expression and functionality in the
highly dystrophic mouse model (mdx/utrn-/-). We also tested microutrophin
expression in a large non-human primate, macaca nemistrina. mdx/utrn-/- mice
were injected intravenously with rAAV6/ microutrophin vectors, carrying either
the ubiquitously active CMV or under control of a novel muscle specific promoter
(MSP) containing modified versions of the M-creatine kinase enhancer and
promoter. Treated mice, displayed alleviation of most of the pathophysiological
abnormalities associated with muscular dystrophy and no significant differences
were observed between animals treated with the two types of vectors. Importantly,
the MSP vector demonstrated a clear lack of expression in non-muscle cell types
both in vivo and in vitro. We have also generated rAAV6 vectors that express a
macaca nemistrina derived microutrophin. Intramuscular injection of this
non-human primate utrophin vector demonstrated widespread expression along the
sarcolemma of monkey myofibers, where it co-localized with endogenous dystrophin.
Consistent with studies in mice, no evidence of a dominant negative effect of
the utrophin expression in wild type macaca nemistrina muscles was observed.
These studies reveal that microutrophin can function not only as a therapeutic
trangene in dystrophic models, but it can also serve as a sensitive reporter
gene to develop systemic gene delivery protocols in large animal models. These
rAAV/microutrophin vectors can also serve as a test system for the potential
immunogenicity of microutrophin. Preliminary studies on the potential
immunogenicity of this delivery system will be presented.
19) Efficient Long-Term Bodywide Expression of an
AAV9-Minidystrophin in the Muscle and Heart of Young Adult GRMD Dogs after
Intravascular Injection without Immune Suppression
Juan Li, Chunping Qiao, Janet Bogan, Dan Bogan, Ruhang Tang, JianBin
Li, Chunlian Chen, Hui Zheng, Jennifer Dow, Joe N. Kornegay, Xiao Xiao. Division
of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North
Carolina, Chapel Hill, NC; Department of Pathology, School of Medicine,
University of North Carolina at Chapel Hill, Chapel Hill, NC
Previously we reported that AAV9 rendered long-term bodywide expression of a
human minidystrophin after intravenous injection in neonatal golden retriever
muscular dystrophy (GRMD) dogs; and that AAV8 achieved long-term expression of a
canine minidys in the limb muscles after isolated limb vein perfusion in young
adult GRMD. Here we report that long-term (≥ 2 years) and bodywide AAV9-minidys
expression was also achieved without any immune intervention in young adult GRMD
dogs, either by isolated limb vein perfusion or by simple intravenous (i.v.)
injection. Methods: 1) Isolated limb perfusion. Three GRMD dogs received 1 x
1013 v.g./kg vector. Dog Jelly (2 mon old; 6.3 kg) was given
AAV9-CMV-opti-hMinidys (codon-optimized human minidys); while Jasper and Peridot
(4 mon old; 12.2 kg and 12.5 kg) were given AAV9-CMV-cMinidys (canine minidys).
The vectors were injected via leg vein with a tourniquet placed at hindlimb
proximal extremity for 10 minutes. Muscle biopsies were taken at 2, 7, 12 & 24
months and analyzed. 2) Intra-venous injection. Dog Duncan (2.5 mon old; 6.5 kg)
was given a simple i.v injection of AAV9-MS-opti-hMinidys (muscle-specific
promoter) at the dose of 4 x 1013 v.g./ kg. Biopsy and necropsy were done at 4
and 12 months. Results: 1) In the limb perfusion study, all 3 dogs had long-term,
widespread minidys expression in all muscle biopsies with improved histology.
The most important finding was that both injected and non-injected limb had
minidys expression. Up to 70% of myofibers were minidys positive in certain
muscle groups without discernable T-cell infiltration. Both legs showed similar
degree of expression. The human minidys persisted for ≥ 2 years (last biopsy) in
Jelly, now 4 years of age. But in Jasper and Peridot, injected with a canine
minidys gene without codon-optimization, stronger expression was always seen in
the vector-injected legs in the 22 months time course. No minidys was detected
in the heart. 2) In the intravenous injection study, dog Duncan showed robust
and uniform minidys expression in the majority of the skeletal muscles for 1
year (duration of the study). The most important finding is the efficient, MS
promoter-mediated cardiac expression, which was previously found very weak or
undetectable in the heart with the CMV promoter. Conclusions: 1) AAV9 achieved
systemic minidys gene delivery after isolated limb perfusion, possibly due to
incomplete tourniquet blockade; 2) A simple i.v. injection of
AAV9-MS-opti-hMinidys achieved robust and long-term bodywide expression; 3)
Efficient cardiacexpression after i.v. injection is most likely due to the use
of muscle-specific promoter; 4) Long-term expression of both human or canine
minidys under the control of different promoters suggest the lack of or minimal
cellular immunity. The mechanisms remain elusive and warrant further studies.
20) Effective Limb Transduction and Phenotypic
Correction after Injection of rAAV8-U7 snRNA in GRMD Dogs
Caroline Le Guiner, Marie Montus, Laurent Servais, Luis Garcia, Yves
Fromes, Jean-Yves Hogrel, Pierre Carlier, Yan Cherel, Philippe Moullier, Thomas
Voit, The AFM-Sponsored Duchenne Consortium. Genethon, Evry, France; INSERM UMR
649, Nantes, France; Institut de Myologie, Paris, France; INRA UMR 703, Nantes,
France
In Duchenne Muscular Dystrophy (DMD) the selective removal by exon skipping of
exons flanking an out-of frame mutation in the dystrophin messenger can result
in in-frame mRNA transcripts that are translated into shorter but functionally
active dystrophin. The goal of our project was to determine in GRMD, the
effective dose of our therapeutic product defined as a recombinant
Adeno-Associated Virus serotype 8 (rAAV8) expressing a modified U7 snRNA
specific for the skipping of exons 5 to 10 of the GRMD dystrophin transcript.
The mode of delivery was the locoregional high-pressure intravenous (IV)
injection of a forelimb. Several groups of GRMD dogs were exposed to different
rAAV8-U7snRNA doses. Each dog was followed ∼3 months after injection. The
primary outcomes were the restoration of dystrophin expression and the
improvement of the tissue pathology in the injected limb compared to the
controlateral limb. The secondary outcomes were the muscle strength correction,
the biodistribution and shedding patterns as well as the immune response against
rAAV8 capsid and dystrophin. Our preliminary results suggest a dose effect of
our therapeutic rAAV. Injection of 2,5E13vg/kg and of 5E12vg/kg of our vector
was able to restore 50 to 80% of Dystrophin expression in the injected limb.
This expression of a semi-functional dystrophin resulted in improvement of
tissue morphology as well as of several functional and MRI parameters. No tissue
inflammation occurred following the procedure. We built a unique network of
laboratories with complementary skills to deliver a GLP-compliant set of
preclinical data to further define the regulatory toxicology studies. The
organization of our network and the results obtained in our GRMD dogs study will
be presented. This project is supported by AFM (Association Française contre les
Myopathies) and by ADNA (Advanced Diagnostics for New Therapeutic Approaches), a
program dedicated to personalized medicine, coordinated by Institut Mérieux and
supported by research and innovation aid from the French public agency, OSEO.
21) Stem Cells Isolated from p65 Deficient
Mice Improved the Histopathology of Skeletal Muscle in Dystrophic Mice
Aiping Lu, Ying Tang, Minakshi Poddar, Bing Wang, Denis C. Guttridge,
Paul D. Robbins, Johnny Huard. Orthopaedic Surgery, University of Pittsburgh,
Pittsburgh, PA; Molecular Virology, Immunology and Medical Genetics, Ohio State
University, Columbus, OH; Microbiogy and Molecular Genetics, University of
Pittsburgh, Pittsburgh, PA
Duchenne muscular dystrophy (DMD) is a deadly genetic disease mainly
characterized by progressive weakening of the skeletal, cardiac and
diaphragmatic muscles. It is critical to find a successful therapy that will
improve the histopathology of the muscles of DMD patients and restore their
normal function. Recent studies have demonstrated that blocking p65, a subunit
of NF-κB, enhances muscle regeneration in injured and diseased skeletal muscle
which suggests that the NF-kB signaling pathway is a contributing factor to the
dystrophic pathology in DMD patients. Previously we demonstrated that muscle
derived stem cells (MDSCs) isolated from the skeletal muscles of heterozygote
P65 knock-out (P65+/-) mice showed better muscle regeneration in vitro and in
vivo compared to the MDSCs isolated from wild-type (WT) mice. We also
demonstrated that the transplantation of P65+/- MDSCs could reduce inflammation.
Based on these results we performed a set of experiments to determine if these
P65+/- MDSCs could alleviate the pathology associated with DMD more efficiently
than wild-type MDSCs. When the p65+/- MDSCs were injected intraperitoneally (IP)
into dystrophin/utrophin deficient (dys-/-:utro-/-, dKO) mice, a reliable mouse
model of DMD, we found that the histopathology improvement in various skeletal
muscles including the gastrocnemius, diaphragm, gluteus maximus and tibialis
anterior muscles by H&E staining. Many centrally nucleated muscle fibers (new
regenerated fibers) were found in the p65+/- MDSC injection group. Mouse IgG
staining showed that there were less necrotic muscle fibers in the mice injected
with p65+/- MDSCs compared to the PBS treated and un-treated muscle. An antibody
against embryonic muscle heavy chain (E-MyHC) was used to evaluate newly
regenerated and pathological muscle fibers and another antibody against F4/80 (a
macrophage marker) was used to analyze the extent of inflammation in the muscle
tissues. Those results showed less inflammation and pathological muscle fibers
within the muscle injected with p65+/- MDSCs compared to untreated and PBS
treated muscles. Trichrome staining showed there were less fibrosis and fewer
necrotic muscle fibers in the mice injected with p65+/- MDSCs compared to PBS
treated and the untreated muscles. Our findings indicated that IP
transplantation of p65+/- MDSCs can decrease macrophage infiltration, fibrosis
formation, and necrosis, while improving muscle regeneration in 4 to 6 week old
dKO mice. We also attempted to inject WT MDSCs isolated from normal animals;
however, the results did not show a similar benefit in the histopathology of the
dKO mice. These results suggest that combining stem cell therapy with anti-NF-κB
therapy is a potential therapeutic approach for the treatment of DMD. Additional
experiments are required to determine whether the benefit observed with NF-κB
inhibition via stem cell therapy is superior than anti-NF-κB gene therapy.
22) Muscle Fiber Type-Predominant Promoter Activity in
Lentiviral-Mediated Transgenic Mouse
Tomohiro Suga, En Kimura, Yuka Morioka, Masahito Ikawa, Sheng Li,
Katsuhisa Uchino, Yuji Uchida, Satoshi Yamashita, Yasushi Maeda, Jeffrey S.
Chamberlain, Makoto Uchino. Neurology, Kumamoto University Graduate School of
Medical Sciences, Kumamoto, Japan; Research Institute for Microbial Diseases,
Osaka University, Suita, Japan; Department of Neurology, University of
Washington School of Medicine, Seattle; Pharmacology, Division of Life Science,
Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto, Japan
Variations in gene promoter/enhancer activity in different muscle fiber types
after gene transduction was noticed, but poorly analyzed. The murine stem cell
virus (MSCV) promoter drives strong, stable gene expression in hematopoietic
stem cells and several other cells, including cerebellar Purkinje cells, however
it has not been studied in striate muscle. We injected a lentiviral vector
carrying an MSCV-EGFP cassette (LvMSCV-EGFP) into tibialis anterior muscles and
observed strong EGFP expression in muscle fibers, primary cultured myoblasts,
and myotubes isolated from injected muscles. We also generated
lentiviral-mediated transgenic mice carrying the MSCV-EGFP cassette and detected
transgene expression in striated muscles. LvMSCV-EGFP transgenic mice showed
fiber type-dependent variations in expression: highest in type I and IIA,
intermediate in type IID/X, and lowest in type IIB fibers. The soleus and
diaphragm muscles, consisting mainly of type I and IIA, are most severely
affected in the mdx mouse model of muscular dystrophy. Further analysis of this
promoter may have the potential to achieve certain gene expression in severely
affected muscles of mdx mice. The Lv-mediated transgenic mouse may prove a
useful tool for assessing the enhancer/promoter activities of a variety of
different regulatory cassettes.
23) Losartan Enhances the Success of Myoblast Transplantation
Raouia Fakhfakh, Yann Lamarre, Daniel Skuk, Jacques P. Tremblay.
Human Genetics Unit, Chul-Chuq, Quebec, QC, Canada
Duchenne muscular dystrophy is a recessive X-linked genetic disease caused by
dystrophin gene mutations. Cell therapy can be a potential approach aiming to
introduce a functional dystrophin in the dystrophic patient myofibers. However,
this strategy produced so far limited results. Transforming growth factor β (TGF-β)
is a negative regulator of skeletal-muscle development and is responsible for
limiting myogenic regeneration. The combination of TGF-β signaling inhibition
with myoblast transplantation can be an effective therapeutic approach in
dystrophin deficient patients. Our aim was to verify whether the success of
human myoblast transplantation in immunodeficient dystrophic mice is enhanced
with losartan, a molecule that down-regulates TGF-β expression. In vitro,
blocking TGF-β activity with losartan increased proliferation and fusion and
decreased apoptosis in human myoblasts. In vivo, human myoblasts
were transplanted in mice treated with oral losartan. Immunodetection of human
dystrophin in Tibialis anterior cross-sections 1 month
post-transplantation revealed more human dystrophin-positive myofibers in these
mice than in non-treated dystrophic mice. Thus blocking the TGF-β signal with
losartan treatment improved the success of myoblast transplantation probably by
increasing myoblast proliferation and fusion, decreasing macrophage activation
and changing the expression of myogenic regulator factors.
24) Interference of Myostatin and TGF-beta Signaling by
Antisense-Mediated Exon Skipping in ALK4⁄5 Receptors
Dwi U. Kemaladewi, Sandra H. van Heiningen, Annemieke Aartsma-Rus,
Gert-Jan B. van Ommen, Peter ten Dijke, Peter A. C. 't Hoen, Willem M. H.
Hoogaars. Human Genetics, Leiden University Medical Center, Leiden, Netherlands;
Molecular and Cell Biology, Leiden University Medical Center, Leiden,
Netherlands
Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder, which is
characterized by the loss of muscle fibers and replacement by fibrotic tissue
due to the lack of the dystrophin protein. Myostatin and Transforming Growth
Factor (TGF)-beta play important roles in regulation of muscle differentiation
and fibrosis, and are emerging as attractive therapeutic targets for DMD
treatment. Both cytokines signal primarily via the overlapping Smad2⁄3-dependent
signaling pathways. In this study we targeted myostatin⁄TGF-beta type 1
receptors Acvr1b (ALK4) and Tgfbr1 (ALK5) using antisense oligoribonucleotides (AON)
targeting regions in the pre-mRNA encoding ligand binding and⁄or kinase domains
of the receptors. Transfection of ALK4 or ALK5 AONs resulted in skipping of the
targeted exon, ∼50% downregulation of the full length transcript and enhanced
myoblast differentiation. In addition, local administration into mdx mice
showed considerable downregulation of full length ALK4 (∼80%) or ALK5 (∼50%),
leading to ∼50% increase of myogenic genes expression and ∼40 % decrease of
fibrotic markers expression. Furthermore, we used these AONs to study how
TGF-beta and myostatin signalings are regulated in different cells origins. We
showed an exclusive requirement of ALK4 for myostatin signaling in myoblasts,
but preference for ALK5 in the other cell types, which suggested that at least
two co-receptors are involved in this cell-type specific regulation of myostatin
signaling. In summary, we were able to use this specific genetic approach to
selectively disrupt myostatin or TGF-beta signaling, dissect the overlapping
pathways and revealed a novel muscle specific mechanism of myostatin signaling.
Systemic administrations are currently ongoing to further assess the therapeutic
benefits of these AONs and to determine the differential effect of ALK4 and ALK5
knockdown on the dystrophic pathology of the mdx mice.
25) PMO Delivery Systems with Bubble Liposomes and Ultrasound
Exposure into Skeletal Muscles of the mdx Mice
Yoichi Negishi, Yuko Ishii, Shoko Sekine, Yoko Endo-Takahashi, Ryo
Suzuki, Kazuo Maruyama, Yukihiko Aramaki. Department of Drug and Gene Delivery
Systems, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences,
Hachioji, Tokyo, Japan; Department of Biopharmaceutics, School of Pharmaceutical
Sciences, Teikyo University, Sagamihara, Kanagawa, Japan
Background: Duchenne muscular dystrophy (DMD) is genetic disorders
causing by mutations in the DMD gene that lead to an absence of functional
protein. Previous reports have shown that a phosphorodiamidate morpholino
oligomer (PMO) designed to skip the mutated exon 23 from the mRNA of murine
dystrophin induce dystrophin expression in dystrophin-deficient mdx mice.
However, efficient delivery method of PMO into the mdx mice still needs to be
developed. Ultrasound (US) in combination with microbubbles has recently been
acquired much attention in the safe method of gene or antisense oligonucleotides
(AOs) delivery. However, microbubbles have problems with size, stability, and
targeting function. Liposomes have been known as drug, antigen, and gene
delivery carriers. To solve the above-mentioned issues of microbubbles, we have
previously developed the polyethyleneglycol (PEG)-modified liposomes entrapping
echo-contrast gas, “Bubble liposomes” (BLs), which can function as a novel gene
delivery tool by applying them with US exposure. In this study, to assess the
feasibility and the effectiveness of BLs for a PMO delivery system into the
mdx mice, we tried to deliver a PMO, which is designed to skip the mutated
exon 23 from the mRNA of murine dystrophin, into skeletal muscles of the mdx
mice by the combination of BLs and US exposure. Methods: A solution of
BLs and PMO for mouse exon 23 was injected into tibialis (TA) muscle of mdx
mice and US exposure (1 MHz, 2 W/cm2, 60 sec., duty cycle 50 %) was
immediately applied at the injection site. Two weeks after the treatment, exon
23 skipping levels were analysed by RT-PCR. The number of dystrophin-positive
fibers after the treatment was also analysed by immunohistochemical analysis.
Results: Mice were euthanized 2 weeks after the injection, the injected
muscles were isolated and analyzed by RT-PCR and by immunohistochemistry. In the
combination of BLs and ultrasound exposure, we found that the PMO significantly
restored the dystrophin mRNA expression and dystrophin-positive fibers by
skipping the mutated exon 23 in the injection site of mdx mice, compared
to the PMO injection alone. Conclusions: These results suggest that PMO
delivery into the muscle of mdx mice can be improved by the combination
of BLs and US exposure. This US-mediated BLs technique may provide an effective
noninvasive method for DMD therapy in the clinical field. Acknowledgments:
This study was supported by Industrial Technology Research Grant Program
(04A05010) in '04 from New Energy and Industrial Technology Development
Organization (NEDO) of Japan.
26) Exploring the Potential of Plasma Pheresis To Circumvent
Pre-Existing Antibodies to AAV8 on Transgene Expression Following Targeted
Vascular Delivery
Louis G. Chicoine, Louise R. Rodino-Klapac, K. Reed Clark, Chrystal
L. Montgomery, Thomas J. Preston, William G. Bremer, Katherine J. Campbell,
Zarife Sahenk, Paul T. Martin, Christopher M. Walker, Jerry R. Mendell.
Pediatrics, The Ohio State University/Nationwide Children's Hospital, Columbus,
OH; Center for Gene Therapy, Research Institute at Nationwide Children's
Hospital, Columbus, OH; Center for Vaccines and Immunity, Research Institute at
Nationwide Children's Hospital, Columbus, OH; Neurology, The Ohio State
University/Nationwide Children's Hospital, Columbus, OH
Gene therapy for muscle disease has taken several positive steps forward. We
recently reported sustained AAV mediated alpha-sarcoglycan (SGCA) gene
expression for as long as 6 months in 5 of 6 limb girdle muscular dystrophy (LGMD2D)
treated patients (Mendell et al. 2010). The one patient that did not express
SGCA was found to have high AAV neutralizing antibodies prior to gene transfer.
With vascular gene therapy to treat multiple muscles as the next logical step
clinically, impediments may lie with the immune response and the presence of
pre-existing antibodies to various AAV serotypes. We tested the hypothesis that
pre-existing antibodies to AAV8 would attenuate transgene expression following
vascular delivery of two potentially therapeutic transgenes
AAV8.MCK.micro-dystrophin.Flag and AAV8.MCK.Galgt2 in a large study of 72
non-human primates. Prior to gene transfer, all animals were pre-screened for
binding antibodies to AAV8. Those animals with a titer of <1:100 were considered
negative, while others (>1:100) were considered positive. Animals were
stratified into positive and negative groups with scheduled sacrifice times at 3
and 6 months (n=9 per group). Vascular delivery to the gastrocnemius muscle of
an isolated limb (Rodino-Klapac et al. 2010) was performed on each animal where
2 x 1012 vg/kg of the appropriate vector was given. None of the
animals suffered noticeable edema or adverse effects from the procedure. Three
or six months after transfer, the macaques were euthanized, and the
gastrocnemius muscle was harvested. Samples of the proximal, central and distal
muscle were stained with anti-FLAG or CT2 antibody. The contralateral
gastrocnemius served as a negative control. Gene expression was visualized in
all subjects and subjects without pre-existing AAV8 antibodies demonstrated
significantly higher transgene expression than subjects with pre-existing
antibodies (62% vs 33% muscle fibers transduced, P ≤ 0.001). Regression analysis
confirmed a direct inverse correlation between the AAV8 antibody titer and the
percent gene expression. From these data we conclude that the presence of
pre-existing antibodies to the vector will attenuate transgene expression.
Plasma pheresis studies appeared to optimize transgene expression and this
procedure would potentially be used to increase the number of patients amenable
to gene therapy and make some patients candidates for retreatment when necessary.
27) Low-Level Expression of a Near-Full Length Dystrophin Improves
Muscle Force and Lifespan in a Strain of Severely Affected Dystrophin/Utrophin
Double Knockout Mice
Dejia Li, Yongping Yue, Dongsheng Duan. Department of Molecular
Microbiology and Immunology, University of Missouri-Columbia, Columbia, MO
Utrophin/dystrophin double knockout mdx (u-dko) mice display severe clinical
symptoms and die prematurely as in Duchenne muscular dystrophy (DMD) patients.
Here we tested the hypothesis that minimal level dystrophin expression can
improve the clinical outcome of u-dko mice. It has been shown that mdx3cv (3cv)
mice express a near-full length dystrophin protein at ∼ 5% of the normal level.
We crossed utrophin-null mutation (all utrophin isoforms are inactivated) to the
3cv background. The resulting uko/3cv mice expressed the same level of
dystrophin as 3cv mice but utrophin expression was completely eliminated.
Surprisingly, uko/3cv mice showed a much milder phenotype. Compared to uko/mdx
mice, uko/3cv mice had significantly higher body weight and stronger specific
muscle force. Most importantly, uko/3cv outlived uko/mdx mice by several folds.
Our results suggest that a threshold level dystrophin expression may provide
vital clinical support in a severely affected DMD mouse model. This finding may
hold clinical implications in developing novel DMD therapies.
28) AAV9-Mediated FKRP Gene Therapy Restores the Expression of
Functional Glycosylation of α-DG
Lei Xu, Pei Lu, Chi-Hsien Wang, Jianbin Li, Elizabeth Keramaris,
Xiao Xiao, Qi Long Lu. McColl-Lockwood Laboratory for Muscular Dystrophy
Research, Department of Neurology, Cannon Research Center, Carolinas Medical
Center, Carolinas Healthcare System, Charlotte, NC; Division of Molecular
Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at
Chapel Hill, Chapel Hill, NC
Mutations in FKRP gene are one of the most common causes of muscular dystrophies,
accounting for more than 19% of all the reported Limb Girdle muscular
dystrophies (LGMD). Currently there is no effective treatment available. Gene
replacement therapy is one of the most promising therapeutic strategy. In this
study we examined the effects of AAV-FKRP in the P448LKI mice via
local and systemic delivery. Mutant mice (12 months) were administrated with
5x109 v.g of AAV9-CB-FKRP to the tibialis anterior (TA) muscle and
1x1012 v.g via introperitoneal injection (i.p.). Muscles were
examined 4 weeks after injections by immunohistochemistry and western blot with
antibodies to FKRP and glycosylated epitope of functional α-dystroglycan. Our
results showed FKRP expression in all muscles including heart, diaphragm and
other skeletal muscles in the mice treated by systemic delivery of AAV9-CB-FKRP.
Consistent with our previous observation, the induced FKRP was mainly localized
to Golgi apparatus within muscle fibers. Expression of FKRP consequently
restored functional glycosylation of α-DG as judged by immunohistochemistry with
the IIH6 antibody in all muscles examined including the cardiac muscle.
Expression of the FKRP transgene and induction of glycosylation of α-DG and its
binding to laminin were confirmed in all the skeletal and cardiac muscles on
Western blots. Our results thus further confirmed that loss of FKRP function is
the cause of hypoglycosylation of α-DG and LGMD2I.
29) Mechanism of Uptaking Morpholino into Dystrophin-Deficient Muscle
Fibers
Yoshitsugu Aoki, Tetsuya Nagata, Toshifumi Yokota, Shinichi Takeda.
Department of Molecular Therapy, National Institute of Neuroscience, National
Center for Neurology and Psychiatry (NCNP), Kodaira, Tokyo, Japan; Research
Center for Genetic Medicine, Children's National Medical Center, Washington, DC
Introduction: A promising therapeutic approach for Duchenne muscular dystrophy (DMD)
is exon skipping using antisense oligonucleotides (AO) such as
phosphorodiamidate morpholino oligomers (PMO). As we already demonstrated using
exon 52 deficient mdx (mdx52) mice, short term systemic treatment of PMO
targeting exon 51 induced highly efficient truncated but functional dystrophin
in body-wide skeletal muscles (Mol Ther.2010;18:1995-2005). This result is
encouraging for the ongoing multinational systemic exon-51 skipping clinical
trial. However, the optimal therapeutic dose of PMO in DMD patients have not
been established. Surprisingly, the mechanism of PMO entry into
dystrophin-deficient fibers remain to be completely elucidated. Purpose: To
verify the mechanism of PMO entry into dystrophin-deficient fibers using mdx52
mice. Methods: The combination of two AOs targeting the 5' and the 3' splice
site was used for skipping exon 51. To research the relation between PMO entry
and dystrophin deficient membrane, a total of 80-640 mg/kg of PMO was injected
into the tail vein of mdx52 and C57BL/6J mice aged 5 wks singly. Muscles were
isolated 2 weeks after the injection and analyzed by RT-PCR. Then, to determine
the relationship between PMO entry and muscle regeneration, PMO (10 μg) was
injected into each tibialis anterior (TA) muscle of mdx52 mice aged 3 to 32 wks
with oral administration of BrdU (0.8 mg/ml for 7 days). Muscles were obtained 2
weeks after the injection and analyzed by RT-PCR, immunohistochemistry and
western blotting. Results: Specific exon 51-skipping and dystrophin expression
were induced in a dose-related manner in mdx52 mice, while on the other hand, no
exon 51-skipping was detected in C57BL/6J mice even at 640 mg/kg. Dystrophin
positive fibers were detected with the greatest numbers when PMO was injected
into TA muscle of mdx52 mice aged 5 wks when the ratio of centrally nucleated
fibers were highly increased. Dystrophin positive fibers were classified into
the two groups: BrdU-labeled regenerated and -unlabeled degenerated fibers in
mdx52 mice.
Conclusion: We suppose that there are two types of PMO entry into
dystrophin-deficient fibers. Interpretation: BrdU-labeled regenerated fibers
could uptake PMO regardless of dystrophin-deficiency and BrdU-unlabeled
degenerated fibers could be 'fibers with leaky membrane'. Our study has a great
potential to exploit an opportunity to optimize antisense therapy using PMO and
to apply the treatment to other neuromuscular diseases.
30) Blocking the NF-κB Pathway Enhances Mini-Dystrophin Efficiency in
Dystrophin/Utrophin Deficient Mice
Qing Yang, Ying Tang, Anmin Chen, Fengjin Guo, Freddie H. Fu, Johnny
Huard, Bing Wang. Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA;
Orthopaedic Surgery, Tongji Hospital, Huazhong University of Science and
Technology, Wuhan, Hubei, China
Chronic inflammation, promoted by an up-regulation of the NF-kappaB (NF-kB)
pathway plays a key role in the progressive muscle wasting and degeneration of
Duchenne muscular dystrophy (DMD) patients. Higher immune alertness in
dystrophic muscle, due to chronic inflammation and immune effector cell
infiltration, challenges recombinant adeno-associated viral (rAAV)
vector-directed DMD gene therapy in dystrophin/utrophin deficient (dys-/-:utro-/-,
dKO) mice, a reliable mouse model of DMD. In our previous study, AAV-mediated
systemic delivery of mini-dystrophin for the treatment of the dKO mice was
effective at partially ameliorating the muscle pathology and increasing function
in these mice, yet it was ineffective at reversing the course of muscle wasting
due to the dystrophic muscle fibrosis, necrosis, and wasting which occurred in
this chronically inflamed environment; therefore, in this study we combined
mini-dystrophin replacement and anti-inflammation. We accomplished this by using
two AAV vectors; one carried a mini-dystrophin gene and the other carried a
short hairpin RNA (shRNA) to block the NF-κB pathway. The goal was to
investigate if blocking inflammation could enhance mini-dystrophin efficiency. A
single intraperitoneal (i.p.) injection was performed in the dKO neonates using
a combination of the two AAV9 vectors which included AAV9-NF-κB/p65-shRNA_ZsGreen
(7 x 1011 v.g. particles/injection) and a low dose of the
AAV9-CMV-hDysΔ3849 vector (5 x 1010 v.g. particles/injection). An
injection of the low dose AAV9-mini-dystrophin vector (5 x 1010 v.g.
particles/injection) served as the control. Mice were sacrificed 10 weeks after
AAV vector injection for muscle histology analysis. The ZsGreen monitored the
shRNA efficiency of AAV delivery in skeletal and cardiac muscles (Green). By
monitoring the ZsGreen expression, we found that the shRNA was delivered to the
skeletal, cardiac and diaphragmatic muscles very efficiently. The efficient
transduction of the diaphragm and cardiac muscles was of particular interest
because these are the muscles whose failure causes the pre-mature death of DMD
patients. A higher level of mini-dystrophin gene expression was observed in the
skeletal and cardiac muscles of the dKO mice treated with both the
mini-dystrophin and NF-κB/p65-shRNA compared to the mice treated with
mini-dystrophin alone. In this study, we found that enhanced mini-dystrophin
expression could be achieved by combining the dystrophin gene replacement with
shRNA silencing to block the NF-κB pathway using two AAV vectors. However, the
simultaneous injection of two rAAV vectors was complex and would be inefficient
for clinical application. This prompts us to develop a single AAV vector which
will carry both the mini-dystrophin and NF-κB/p65-shRNA cassettes. This approach
may offer a more synergistic effect via the combination of gene replacement and
anti-inflammation in a single-step procedure.
31) Noninvasive Monitoring of Whole Body Muscle Function in
Dystrophin-Deficient Dogs
Jin-Hong Shin, Brian B. Greer, Chady H. Hakim, Zhihai He, Dongsheng
Duan. Molecular Microbiology & Immunology, University of Missouri, Columbia, MO;
Electrical and Computer Engineering, University of Missouri, Columbia, MO
Dystrophin-deficient dogs are highly valuable tools for evaluating gene/cell
therapies to treat Duchenne muscular dystrophy (DMD). Unfortunately, robust
muscle functional end points are poorly defined in the canine DMD models. Here,
we described a set of non-invasive methods for whole body muscle function
analysis in normal and affected dogs. Briefly, the gait and voluntary activity
were recorded using high-resolution digital video recorders. Gait recoding was
repeated three times on three different days for each dog. Voluntary activity
monitoring was performed under low-lux red lighting between 7pm and 5am for each
dog. The stride length, stride speed, range of the motion and overall activity
were analyzed using a customer-developed program. Three normal and four affected
dogs of a litter were examined. Affected dogs showed significantly reduced
stride length (affected, 35.7 ± 8.5 cm; normal, 80.2 ± 6.1 cm; p <0.02) and
stride speed (affected, 61.7 ± 16.4 cm/sec; normal, 156.5 ± 23.8 cm/sec; p
<0.03). The range of the motion in forelimb (palm, forearm and wrist) and hind
limb (sole, low leg and ankle) was also significantly decreased in affected dogs.
The affected dogs also showed significantly less movement during overnight
monitoring. In summary, our results suggest that whole body muscle function was
significantly compromised in affected dogs. Non-invasive video recording may
represent a convenient and reliable method to monitor disease progression and
gene/cell therapy effect in canine models of DMD.
32) Recombinase Strategies To Make and Modify iPS Cells
Marisa Karow, Christopher L. Chavez, Alfonso P. Farruggio, Jonathan
M. Gesinger, Joseph C. Wu, Yanru Chen-Tsai, Michele P. Calos. Stanford
University School of Medicine, Stanford, CA
Induced pluripotent stem cells (iPSC) have revolutionized the stem cell field.
iPSC are most often produced by using retroviruses, but the resulting cells are
ill-suited for clinical application. Several alternative strategies to make iPSC
have been developed, but none encompass within them a simple methodology to
facilitate the precise insertion of a therapeutic gene for gene therapy
approaches. Here we report practical strategies to create and genetically modify
murine iPSC that rely on plasmid DNA and the sequential application of three
site-specific recombinases. PhiC31 integrase was used to insert the
reprogramming cassette at preferred locations in the genome, producing iPSC
demonstrated to be pluripotent by a full range of in vitro and in vivo criteria,
including teratoma formation and generation of chimeric mice. The reprogramming
plasmid includes the target attP site for a second site-specific integrase,
which can be utilized for precise insertion of a therapeutic gene into the
target site. Cre recombinase can be employed for clean excision of the entire
reprogramming cassette and most plasmid sequences. We demonstrated the efficient
operation of this type of strategy in accessible cell types, including
fibroblasts and adipose stem cells. Furthermore, to illustrate a clinically
relevant example, we generated iPSC from the A/J limb girdle muscular dystrophy
mouse model, introduced the therapeutic dysferlin gene, and deleted the
reprogramming genes. This simple strategy produces pluripotent cells that are
corrected for a genetic defect and have the potential to be used in a clinical
setting.
33) Sustained Expression of Canine Micro-Dystrophin and Amelioration
of Muscle Function in Dystrophic Dogs Following Large Scale AAV-Mediated
Treatment
Zejing Wang, Rainer Storb, Donghoon Lee, Christine Halbert, Martine
K. Childers, Glen Banks, James Allen, Eric Finn, Martin Kushmeric, Dusty Miller,
Jeffery S. Chamberlain, Stephen J. Tapscott. Transplantation Biology, Fred
Hutchinson Cancer Research Center, Seattle, WA; Medicine, University of
Washington, Seattle, WA; Radiology, University of Washington, Seattle, WA; Human
Biology, Fred Hutchinson Cancer Research Center, Seattle, WA; Neurology and
Regenerative Medicine, Wake Forest University, Charlotte, NC; Neurology,
University of Washington, Seattle, WA
Adeno-associated viral (AAV) vectors as gene delivery vehicles have shown
promise both in preclinical studies and clinical trials for a number of acquired
and inherited diseases, including Duchenne Muscular Dystrophy (DMD). We and
others have shown that dogs and humans mount immune responses against all tested
AAV serotypes, which compromised vector delivery and prevented sustained
therapeutical gene expression. We have developed a transient immunosuppressive
regiment consisting of anti-thymocyte globulin (ATG), cyclosporine (CSP), and
mycophenolate mofetil (MMF), which was effective in preventing immune responses
to AAV and enhanced AAV6-mediated gene delivery to canine skeletal muscle. Here,
we demonstrate that long term expression for 2 years of a species – specific,
functional canine-micro-dys can be achieved in large scale in skeletal muscles
of DMD dogs with ATG/CSP/MMF. Moreover, for the first time we achieved
amelioration of muscle histology and function as a result of sustained
expression of the therapeutic gene as demonstrated both by non invasive magnetic
resonance imaging (MRI) and kinametic gait analysis. In order to identify an
even more effective and less toxic immunosuppressive regimen, we have begun
studies using molecules that block T-cell costimulation. Further, we are also
examining AAV vectors made by a modified production method that minimizes
aberrant packaging of the capsid gene to further reduce immunogenicity in dogs.
In conclusion, the finding of long term dystrophin expression in DMD dogs given
a short course of standard immunosuppression opens the possibility of
translating these strategies to a human DMD trial.
34) Inactivation of Dystrophin Expression Via Cre-loxP Mediated Gene
Excision
Julian N. Ramos, Andrea Arnett, Brian Schultz, Jeffrey S.
Chamberlain. Molecular & Cellular Biology, University of Washington, Seattle, WA;
Neurology, University of Washington, Seattle, WA
Duchenne muscular dystrophy (DMD) is a recessive X-linked disease in which the
dystrophin gene is aberrantly expressed or not at all. Several potential
therapies have demonstrated efficacy in animal models and have proceeded to
human clinical trails. Among these strategies is to express truncated
dystrophins, or micro-dystrophins, in the targeted striated muscle. Although
micro-dystrophin (µDys) constructs have demonstrated a high degree of
functionality in dystrophic models, it is uncertain how stable these truncated
proteins are in adult muscles. To address this question, we generated a double
transgenic floxed-µDys/Cre-ERTM/mdx4cv line expressing micro-dystrophin in
skeletal muscle. Primary myoblast cultures administered 4-hydroxytamoxifen had
<25% µDys-expressing cells by 7 days after the end of the regimen. To ascertain
the persistence of the truncated protein in vivo, Cre-mediated recombination was
induced with tamoxifen delivered intraperitoneally. At 3 weeks post-treatment,
multiple hind limb muscles exhibited less than <50% expression. In order to
accurately gauge the protein's stability, the EDL muscles of floxed-µDys/ mdx4cv
mice were injected with rAAV-CMV-Cre-NLS. This produced a transient knockdown of
dystrophin expression where significant loss was determined by physiological and
morphological aspects, in addition to dystrophin labeling. These results suggest
that a micro-dystrophin with a modular design similar to one described herein
has a maximal half-life of 7 days in vivo.
35) The Polyproline Site in Hinge 2 of Microutrophin Does Not Lead to
Structural Abnormalities like That for Microdystrophin
Glen B. Banks, Guy L. Odom, Eric E. Finn, Jeffrey S. Chamberlain.
Department of Neurology, University of Washington, Seattle, WA
Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease caused
by mutations in dystrophin. A potential therapeutic option for DMD is delivery
of rationally designed truncated (micro) dystrophins using recombinant
adeno-associated virus serotype 6 (rAAV6). However, there remains a possibility
that immune responses against dystrophin could develop in some patients that
would lead to elimination of transduced myofibers. Gene delivery of utrophin, a
paralogue of dystrophin, may be able to circumvent this T-cell mediated immune
response. The truncated utrophin (microutrophinΔR4-R21/ΔCT) that we
previously designed was based on that of microdystrophinΔR4-R23/ΔCT.
We had previously demonstrated in mice that microdystrophins carrying a
polyproline site in hinge 2 could lead to the development of structural
abnormalities in limb muscles, including small myofibers, myotendinous strain
injury, ringed fibers and abnormal neuromuscular synapses. We improved the
microdystrophin design by substituting hinge 2 for hinge 3 (PLoS Genetics,
2010). Here we examined whether the hinge structure of microutrophinΔR4-R21/ΔCT
had a similar impact on transduced myofibers of dystrophic mice. We found that
the polyproline site was conserved in hinge 2 of microutrophinΔR4-R21/ΔCT.
However, microutrophinΔR4-R21/ΔCT could mitigate muscle degeneration
in mdx mice without leading to myotendinous strain injury or ringed
fibers. MicroutrophinΔR4-R21/ΔCT was able to prevent synapse
fragmentation, but was unable to completely restore the synaptic folds.
MicroutrophinΔR4-R21/ΔCT also led to muscle fiber hypertrophy and
pronounced sarcolemmal festoons. Thus, the polyproline site did not adversely
influence the function of microutrophinΔR4-R21/ΔCT like that for
microdystrophinΔR4-R23/ΔCT.