Annual Meeting of American Society of Gene & Cell Therapy - May 17-20, Washington, USA
1) Laminin-111: A Highly Potent
Therapeutic Agent To Treat Duchenne Muscular Dystrophy
Sebastien Goudenege, Yann Lamarre, Nicolas Dumont, Joel Rousseau,
Skuk Daniel, Jacques P. Tremblay. Unité de recherche de recherche en Génétique
Humaine, Université Laval, Quebec City, QC, Canada; Unité CRML, Université Laval,
Quebec City, QC, Canada.
Muscle disorders such as Duchenne muscular dystrophy (DMD) still need effective
treatments and myoblast transplantation is considered as a potential approach to
repair damaged skeletal muscles. Duchenne muscular dystrophy is a genetic
disease that usually begins in childhood and results in the complete loss of
dystrophin and the dystrophin glycoprotein complex (DGC) from muscles. The loss
of dystrophin and the consequent absence of the DGC in the sarcolemma appear to
be key players in the development of muscular dystrophy. Indeed the lack of link
between the contracting apparatus and the extracellular matrix leads to frequent
damage to the sarcolemma triggering muscle fiber necrosis. Laminins are major
proteins in the extracellular matrix. The most studied isoform, laminin-111 is
normally present in skeletal and cardiac muscles in mice and humans but only
during embryonic development. As tissues mature, it disappears and is replaced
by other forms of laminin. In this study, we show that intramuscular injection
of laminin-111 reduced the damage to sarcolemma, as indicated by a reduction
Evans blue dye positive fibers. As a consequence of better muscle state, the
inflammatory reaction characterized by macrophage infiltration was drastically
reduced. These changes induced by laminin-111 not only increased resistance in
dystrophin-deficient muscle but also increased the muscle strength (44 %).
Moreover, we also demonstrated that the co-injection of laminin-111 with
myoblasts significantly improved this cell transplantation in RAG-mdx mice:
i.e., the total number of human dystrophin fibers resulting from the fusion of
the transplanted myoblasts with the dystrophin negative fibers was significantly
increased (33 %). To explain the improvement of success in transplantation, we
showed that laminin-111 increase proliferation (28 %) and drastically migration
(743 %) in vitro. These results strongly suggest that the simple
injection of laminin-111 protein may a simple and safe therapy to prevent
repetitive cycles of injury, fibrosis and loss of muscle function in DMD and to
improve the success in myoblast transplantation.
2) Safety and Feasibility
of Transvenous Limb Perfusion with Saline in Human Muscular Dystrophy
William Powers, Zheng Fan, Keith Kocis, Robert Valley, Manisha
Chopra, James Howard, Jr, Joseph Muenzer. Department of Neurology, University
of North Carolina School of Medicine, Chapel Hill, NC; Department of
Anesthesiology, University of North Carolina School of Medicine, Chapel Hill, NC;
Department of Pediatrics, University of North Carolina School of Medicine,
Chapel Hill, NC; Wellstone Muscular Dystrophy Cooperative Research Center,
University of North Carolina, Chapel Hill, NC; Gene Therapy Center, University
of North Carolina, Chapel Hill, NC.
High-pressure retrograde transvenous limb perfusion has been successfully used
to deliver plasmid DNA and AAV-minidystrophin transgenes into skeletal muscle in
experimental animals. Translating this promising technique to humans with
muscular dystrophy requires addressing multiple safety and logistical aspects
including analgesia, vascular access and substantially larger infusion volumes.
We are conducting an IRB-approved dose escalation study of transvenous single
limb perfusion with 0.9% saline in adults with Becker and limb-girdle muscular
dystrophies starting with infusions of 5% of limb volume. We are monitoring the
following parameters to determine safety: (1) systemic cardiovascular function
(2) limb tissue compartmental pressures (3) limb tissue oximetry (4) Doppler
ultrasonography for local venous and arterial damage (5) electrodiagnostic
studies for local nerve damage (6) local muscle function (7) serum and urine
muscle enzymes and (8) renal function. To date, four subjects have been studied
with written informed consent (Table). Limb volume was determined by water
immersion. An 18 or 20 g intravenous catheter was inserted into the distal
lesser saphenous vein. A Zimmer ATS 2000 single cuff tourniquet was placed just
above the knee. Infusion of normal saline was carried out with a Belmont FMS
2000 Rapid Infuser. Cardiac function was continously monitored by
echocardiography while observing for any intracardiac microcavitations
indicative of saline leakage during the infusion. In subject 1, analgesia and
anxiolysis was provided with fentanyl and midazolam. In subjects 2-4, a
combination of fentanyl, midazolam and propofol was administered. No subject
complained of any post procedure pain other than due to needle punctures. No
adverse safety events occurred in any of the monitored parameters. Conclusion:
We have demonstrated that high-pressure retrograde transvenous limb perfusion
with saline up to 15% of limb volume at these infusion parameters is safe and
feasible. We will escalate the volume to a maximum of 40% of limb volume to
determine the maximum safe perfusion parameters and then document the
effectiveness of different infusion parameters to produce entry of fluid into
muscle by T2 MRI. These studies will serve as a basis for future gene therapy
clinical trials.
Infusion Parameters | ||||||
Age | Diagnosis | Tourniquet Pressure (mm Hg) | Max Infusion Line Pressure (mm Hg) | Volume (% limb) | Volume (mL) | Flow Rate (mL/min) |
31 | LGMD2A | 310 | 290 | 5 | 245 | 60 |
35 | LGMD2A | 310 | 300 | 5 | 200 | 67 |
38 | LGMD | 310 | 300 | 10 | 700 | 70 |
27 | Becker MD | 310 | 295 | 15 | 975 | 80 |
3) Induction of Oral
Immunotolerance to rAAV9-Microdystrophin in Canine X-Linked Muscular Dystrophy
Hiromi H. Kinoh, Naoko Yugeta, Hironori Okada, Yuko N. Kasahara,
Takashi Okada, Shin'ichi Takeda. Department of Molecular Therapy, National
Institute of Neuroscience, NCNP, Kodaira, 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 neonates and 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: Normal canine (1 day after birth) dogs were injected with the 1x1013
of rAAV9-CAG-Luciferase into the jugular vein. Two or four weeks after the rAAV
injection, expression of rAAV-derived luciferase in the skeletal muscle and
cardiac muscle was analyzed. 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 examine the immunotolerance to the rAAV,
purified canine peripheral leukocytes were exposed to rAAV9-microdystrophin for
4 hours, and then IFN-β expression was analyzed using RT-qPCR. To expect
systemic microdystrophin expression, we additionally injected
rAAV9-CMV-microdystrophin into the jugular vein of 6 weeks old dystrophic dog.
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.
Results: In the case of transduction of neonate, transgene expression was not
significant by immunohistochemistry. In transduction of fetus, 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-β 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 RT-qPCR
and immunohistochemistry in the transduced dog. Monthly analysis of the
transduced dystrophic dogs demonstrated superior gait function to non-injected
littermate CXMDJ.
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.
4) A New Therapeutic
Approach for Duchenne Muscular Dystrophy: Restoration of the Dystrophin Reading
Frame with a Meganuclease
Jacques P. Tremblay, Pierre Chapdelaine, Christophe Pichavant, Joel
Rousseau, Frédéric Pâques. Unité de Génétique Humaine, CRCHUQ, Québec, QC,
Canada; Cellectis Genome Surgery, Cellectis S.A., Romainville, France.
Mutations in Duchenne muscular dystrophy are either inducing a nonsense codon or
a frame shift. Meganucleases can be engineered to induce double strand break at
specific DNA sequences. These breaks are repaired by Non Homologous End Joining
(NHEJ), which results in insertions or deletions (Indels) of a few base pairs.
To verify whether meganucleases could be used to restore the normal reading
frame of a dystrophin gene with a frame shift mutation, we have inserted a 29 bp
sequence containing a target sequence for a meganuclease. The co-transfection in
293FT cells of the target micro-dystrophin plasmid and of the appropriate
meganuclease restored the micro-dystrophin expression. Meganucleases also
restored micro-dystrophin expression in myoblasts and in muscle fibers in vivo.
The mutation of the targeted micro-dystrophin sequence was confirmed by PCR
amplification followed by digestion with the Surveyor enzyme and by cloning and
sequencing of the amplicons. These experiments are thus a proof of principle
that meganucleases engineered to target appropriate sequences in dystrophin gene
would be able to restore the normal reading frame of that gene in DMD patients
with out of frame deletion. Meganucleases targeting a sequence including or near
nonsense mutation could also be used to delete it.
5) Cell Therapeutic
Approach to Duchenne Muscular Dystrophy Using Myogenic Differentiation of
Multipotent Mesenchymal Stromal Cells
Yuko N. Kasahara, Hiromi H. Kinoh, Hironori Okada, Jin-Hong Shin,
Akiyo Nishiyama, Sachiko O. Hosoyama, Michiko W. Maeda, Akinori Nakamura,
Takashi Okada, Shin'ichi Takeda. Department of Molecular Therapy, National
Institute of Neuroscience, NCNP, Kodaira, Tokyo, Japan.
Background: Duchenne muscular dystrophy (DMD) is an incurable genetic disease
with early mortality. We studied gene and cell therapeutic approach to DMD in
dogs using myogenic differentiation of bone marrow-derived multipotent
mesenchymal stromal cells (MSCs) to establish an efficient protocol. Because of
safety and utility, MSCs would make a larger contribution to clinical benefit
than iPS cells. Since current methods of myogenic differentiation of MSCs are
generally inefficient, we employed MyoD, the master switch protein for myogenic
differentiation, to convert MSCs into myogenic cells. We also investigated the
strategies for cell expansion, and delivery route 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- matched mating pairs to
achieve donor or recipient of allogeneic transplantation. Cardiotoxin (CTX) was
injected into tibialis anterior (TA) and extensor carpi ulnaris (ECU) muscles 5
days before MSCs injection. MSCs transduced with adenoviral vector expressing
MyoD (Ad-MyoD) were injected into the CTX- injured TA (2x106 cells)
and ECU muscles (1x106 cells) of recipient normal dog with oral
administration of cyclosporine and mycophenolate mofetil. We also injected 1 or
2x106 of MSCs into DMD dog. For intra-arterial administration,
MyoD-transduced MSCs (5x106 cells) were administered into femoral
artery with transient avascularization using a tourniquet. The treated muscles
were biopsied and analyzed histologically. Results: CD271-enriched MSCs obtained
from normal dog showed 20-fold higher growth expansion compared with
CD271-depleted MSCs. MyoD transduction of the CD271-enriched MSCs revealed in
vitro myogenic differentiation and myotube formation. MSCs transplanted to
the DLA- matched recipient dog were observed as several clusters in the injected
muscles 4 weeks after the intramuscular injection. The engraftment of MSCs was
also successful with the immunosuppressant-free transplantation into DMD dog by
the intramuscular injection. Furthermore, extensive engraftment of MSCs was
detected at the site of the CTX-injured muscle 2 weeks after the intra-arterial
injection. Immunohistological analysis suggested that most of MSCs formed
muscle-like tissues with the upregulation of developmental myosin heavy chain.
Conclusion: MyoD-transduced MSCs enabled more efficient realization of MSCs
transplantation by the intramuscular as well as intra-arterial injection.
Although further study is required for effective differentiation in vivo,
this strategy of MSCs propagation and treatment would be promising for the
future DMD cell therapy.
6) Gentamicin Treatment of
Duchenne Muscular Dystrophy Reinforces the Potential for Mutation Suppression
Therapy
Vinod Malik, Louise R. Rodino-Klapac, Laurence Viollet, Cheryl Wall,
Wendy King, Roula Al-Dahhak, Sarah Lewis, Christopher J. Shilling, Janaiah Kota,
John Hayes, John D. Mahan, Katherine J. Campbell, Brenda Banwell, Majed Dasouki,
Victoria Watts, Kumaraswamy Sivakumar, Ricardo Bien-Willner, Kevin M. Flanigan,
Zarife Sahenk, Richard J. Barohn, Christopher M. Walker, Jerry R. Mendell. Gene
Therapy, The Research Institute at Nationwide Childrens Hospital, Columbus, OH;
Pacific University, Forest Grove, OR; The Ohio State University, Columbus, OH;
vaccines & Immunity, The Research Institute at Nationwide Childrens Hospital,
Columbus; University of Toronto, Toronto, ON, Canada; University of Kansas,
Kansas City, KS; Neuromuscular Research Center, Scottsdale, AZ.
Duchenne Muscular Dystrophy (DMD) is a severe X-linked genetic muscle disease
caused by nonsense mutations of the dystrophin gene in about 15% of cases.
Pre-clinical studies in mdx mice demonstrate that gentamicin suppression of
premature termination codons allows readthrough, thereby translating the full
length protein. Clinical attempts to reproduce these findings in DMD patients
have been inconsistent. In the current clinical study we have attempted to
address several issues: 1) the biopotency of gentamicin comparing stop codon DMD
subjects with patients harboring frameshift mutations; 2) the feasibility of
long-term gentamicin administration considering that readthrough would be an
ongoing requirement; 3) the percent dystrophin expression required to provide
clinically meaningful outcomes; 4) the potential immunogenicity of newly
expressed dystrophin epitopes induced by stop codon readthrough. Four DMD
cohorts received gentamicin. In a short-term 14-day study, Cohort 1 (n = 10)
stop codon DMD patients and Cohort 2 (n = 8) frameshift DMD subjects were
treated with daily gentamicin 7.5mg/kg/day. The serum creatine kinase (CK)
dropped by 50% in the stop codon patients (11,320 ± 2575 U/L to 5429 ± 1317 U/L,
p < 0.007) but not in the frameshift subjects. Based on these findings an
unprecedented six month delivery study was implemented with weekly or biweekly
gentamicin (7.5mg/kg) potentially permitting an accumulating dystrophin pool to
reach therapeutic levels. After 6-months of gentamicin, dystrophin levels
significantly increased (p = 0.027) reaching levels 13% to 15% of normal
accompanied by a drop in serum CK, stabilization of muscle strength indicating a
shift in natural history rate of decline, and a slight increase in forced vital
capacity. It appeared that stable transcripts escaping nonsense mediated decay
or resulting from alternative splicing predicted the greatest increase of
dystrophin following gentamicin. Novel immunogenic epitopes were found in
post-treatment biopsies by antigen specific IFN-g ELISpots. Taken together our
results confirm that aminoglycoside antibiotics suppresss nonsense mutations and
support on-going efforts to achieve drug-induced mutation suppression of stop
codons. Immunogenic epitopes resulting from readthrough emphasize the importance
of monitoring T cell immunity during clinical gene manipulation trials.
7) Long Term Antisense
Treatment in Dystrophic Mouse Models for Duchenne Muscular Dystrophy
Annemieke Aartsma-Rus, Christa L. de Winter, Hans A. Heemskerk,
Judith C. van Deutekom, Gert-Jan B. van Ommen. Human Genetics, Leiden
University Medical Center, Leiden, Netherlands; Proesensa Therapeutics, Leiden,
Netherlands.
Antisense-mediated reading frame restoration is presently one of the most
promising therapeutic approaches for Duchenne muscular dystrophy (DMD). In this
approach, antisense oligoribonucleotides (AONs) induce specific exon skipping
during pre-mRNA splicing of mutated dystrophin transcripts. This is aimed to
restore the disrupted open reading frame and allow synthesis of internally
deleted, partly functional Becker-like dystrophin proteins. The approach is
theoretically applicable to over 70% of all patients, with exon 51 skipping
being applicable to the largest group of patients (13% of all mutations). Proof
of concept has been achieved in cultured muscle cells from patients carrying
different mutation types, in the mdx mouse model, and recently in DMD patients
in clinical trials after local or subcutaneous (systemic) treatment of PRO051, a
2'-O-methyl phosphorothioate modified AON. In each case AON treatment resulted
in skipping of the targeted exon and dystrophin restoration in the absence of
adverse effects. A subsequent trial where patients are treated systemically has
recently been completed successfully and a 6-months followup trial using the
most effective dosage is underway by Prosensa.
Due to AON turnover, repeated treatment is necessary. Therefore, long term
safety and efficacy of subcutaneous 2'O-methyl phosphorothioate AON treatment
was tested in mouse models with varying levels of severity: mdx mice (mild
phenotype) and mdx mice with one utrophin allele (mdx +/-; intermediate
phenotype). Mice were treated with weekly SC injections of 200 mg/kg for up to 6
months. This was well tolerated during treatment and liver and kidney weights
and serum parameters were similar for 4, 8, 12 and 24 week treated mice compared
to saline treated controls at the end of treatment. In both models treatment
resulted in significantly improved serum creatine kinase (marker for muscle
quality) and rotarod running time (marker for muscle function) compared to the
controls. In the more severely affected mdx +/- mice the therapeutic effect was
larger. Time course experiments revealed that exon skipping levels increased for
the first 12 weeks, but remained constant after that. Protein analysis revealed
a similar pattern.
These results indicated that long term subcutaneous treatment with 2'-O-methyl
phosphorothioate AONs is safe and efficient in dystrophic mouse models, which is
encouraging for future long term trials in patients, which are currently planned
by Prosensa Therapeutics.
8) Lentiviral Vector
Mediated Delivery of Full-Length Dystrophin for Gene Therapy of Muscular
Dystrophy
En Kimura, Katsuhisa Uchio, Tomohiro Suga, tatsuya Koide, Yuji
Uchida, Yasushi Maeda, Satoshi Yamoshita, Jeffrey S. Chamberlain, Makoto Uchino.
Neurology, Kumamoto University Graduate School of Medical Sciences, Kumamoto,
Japan; Pharmacology, Sojo University, Kumamoto, Japan; Neurology, University of
Washington School of Medicine, Seattle, WA.
Duchenne muscular dystrophy (DMD) is an inherited severe muscle wasting disorder,
and there is currently no effective treatment. DMD causes respiratory and/or
cardiac failure and results in death at about 20 years of age. Lentiviral
vectors are an efficient gene delivery tool for skeletal muscle fibers as well
as myogenic progenitor cells in vivo and ex vivo. The integration ability of
lentiviral vectors is a huge advantage for targeting DMD myogenic cells, which
require the missing gene product, dystrophin, to be expressed permanently, which
can not be achieved with non-integrating vectors. We have shown that stable
transduction of myogenic stem cells in vivo using lentiviral vectors
could be of benefit for treating dystrophic muscles in mdx mice.
Injection of micro-dystrophin expressing lentiviral vectors into neonatal mdx
muscles resulted in widespread and stable expression of micro-dystrophin for at
least two years, and led to a marked amelioration of dystrophic pathology.
As the carrying capacity of lentiviral vector has been considered to be limited
to ∼9 kb, it can package truncated versions of dystrophin gene, such as
the micro- or mini-dystrophins. Although some groups have shown
that larger constructs can be packaged, little data is available on the titers
that can be obtained for fully packed particles. Recently, several modifications
of lentiviral vector production methods have been successfully employed to
generate relatively higher titers of vector stocks. Consequently, we have
explored the ability to package the full-length dystrophin cDNA in a
VSVG-pseudotyped lentiviral vector. Although the functional titer was still
relatively low compared with smaller dystrophin cDNAs, it was able to deliver
the full-length transgene into mdx myoblasts. These genetically corrected
mdx myoblasts successfully expressed the full-length 427 kDa of
dystrophin protein, which could also be provided to dystrophin deficient
myotubes in vitro by cell fusion in co-cultures. After intramuscular
transplantation, these genetically modified myogenic cells supplied full-length
dystrophin protein to mdx muscle fibers. Our candidate strategy with
lentiviral vectors carrying full-length dystrophin cDNAs could be useful for
ex vivo cell therapies.
9) Transient Expression of
a Therapeutic Dystrophin Transgene in Duchenne Muscular Dystrophy Revealed by T
Cell Mediated Immunity
Jerry R. Mendell, Katherine Campbell, Louise R. Rodino-Klapac,
Zarife Sahenk, Christopher Shilling, Sarah Lewis, Dawn Bowles, Steven Gray,
Chengwin Li, Gloria Galloway, Vinod Malik, Brian Coley, K. Reed Clark, Juan Li,
Xiao Xiao, Jade Samulski, Scott McPhee, R. Jude Samulski, Christopher M.
Walker. Research Institute at Nationwide Childrens Hospital, Columbus, OH;
University of North Carolina, Chapel Hill, NC.
Duchenne muscular dystrophy (DMD) is an X-linked genetically inherited disease
of childhood characterized by progressive muscle weakness resulting from
mutations in the dystrophin gene. Recombinant adeno-associated virus (rAAV)
vectors can package miniaturized versions of the dystrophin gene less than half
of the 11 kb coding sequence. Sustained expression of these smaller dystrophin
proteins in rAAV transduces dystrophic muscle of mdx mice providing
partial restoration of muscle force generation paving the way for a clinical
trial. The minidystrophin transgene used in this study encoded the actin binding
domain, 5 rod spectrin repeats (R1, 2, 22, 23, and 24), 3 hinge domains (H1, 3
and 4), and the cysteine-rich domain driven by the human cytomegalovirus early
promoter. Vector genomes were packaged in a hybrid AAV2 capsid with 5 amino acid
substitutions designed to minimize recognition by serum neutralizing antibodies.
Six subjects with frame-shifting deletions in the dystrophin gene were enrolled
in a phase I pilot clinical gene therapy trial. Vector was injected into one
biceps muscle. Four hours prior to gene transfer, subjects received intravenous
methylprednisolone (2.0 mg/kg) to reduce potential inflammation caused by needle
manipulation. Subjects 001, 002, and 003 received low dose vector (6x1011
vg) while subjects 004, 005, and 006 received a dose 5-fold higher (3x1012
vg). No adverse events were observed. Treated and control contralateral biceps
muscles were sampled at day 42 (subjects 001, 003, 004, 006) or day 90 (subjects
002, 005) to assess for vector genomes and minidystrophin protein. Vector DNA
ranged from 0.01 to 2.56 genome copies per diploid genome in the treated but not
the control side in study subjects. Expression of dystrophin protein was not
sustained after gene transfer. T cell immunity against non-self and self
dystrophin epitopes was detected in a subset of participants. In one subject,
CD4+ and CD8+ T cells were targeted to non-self dystrophin epitopes encoded by
the transgene in a domain of a large frame-shifting endogenous dystrophin gene
deletion. In two subjects, dystrophin specific T cells were detected in a
pre-treatment ELISpot IFN-γ assay that targeted dystrophin self-epitopes
expressed on revertant muscle fibers. In one of these individuals the frequency
of self-reactive T cells increased rapidly after expression of the dystrophin
transgene in skeletal muscle. This study demonstrates for the first time the
potential for cellular immune response to therapeutic transgene products in
humans treated with rAAV vectors. Recall of auto-reactive T cells also has
important clinical significance for strategies to increase expression of
functional dystrophin in DMD and should be considered in design and monitoring
of experimental therapies for this disease. (ClinicalTrials.gov ref. no.
NCT00428935).
10) Optimization of AAV
Vectors for Improved Therapeutic Protein Expression in the Canine Models for
Duchenne Muscular Dystrophy
Alock Malik, Marilyn Mitchell, Andrew Mead, Mike Petrov, Hansell
Stedman. University of Pennsylvania, Philadelphia, PA.
A major bottleneck in the development of effective gene therapy for Duchenne
Muscular Dystrophy is the design of vectors that yield the highest possible
expression of dystrophin or utrophin in the target tissues. The magnitude of
this challenge has come into clearest focus during the transition from small to
large animal models in the preclinical setting. We recently initiated studies of
limb-wide, vascular gene transfer in the dystrophic dog using AAV vectors based
on internally deleted versions of the wild type canine dystrophin and utrophin
cDNA sequences. Early in the course of these studies, the need for higher levels
of protein expression normalized to the administered vector dose prompted a
parallel effort to further optimize the entire vector expression cassette.
Iterative improvements were made through the analysis and serial modification of
plasmids encoding identical proteins using distinct coding sequence and
transcriptional cassettes. Systematic screening, first in HEK 293 cells and then
in mdx mouse muscle allowed us to determine the single best expression cassette
from a pool of plasmids with different promoters (ubiquitous, muscle specific
and hybrid-synthetic promoters), introns, poladenylational signals,
transcriptional enhancer elements, and peptide coding sequences. The best
combination of these improvements thus far has provided at least a 10-fold
higher level of recombinant protein expression than our first generation
cassette, with further improvements under additional analysis. Selected versions
of these transcriptional cassettes are being further characterized in the
context of myotrophic AAV vectors following administration to mdx mice and GRMD
dogs. 10-fold or greater improvements due to the optimization process should
greatly accelerate the pace of therapeutic trials in the dystrophic dogs.
11)
Rescue from Respiratory Dysfunction by Transduction of Full-Length
Dystrophin to Diaphragm Via the Peritoneal Cavity in Muscular Dystrophy Mouse
Ishizaki Masatoshi, Yasushi Maeda, En Kimura, Makoto Uchino.
Neurology, Kumamoto University, Kumamoto, Japan.
Duchenne muscular dystrophy (DMD) is an inherited severe muscle wasting disorder
with no effective therapy so far. DMD causes respiratory or cardiac failure as
well as the muscle waste. Among the various symptoms the respiratory
insufficiency is a major mortal cause of DMD patients at about 20 years of age.
So, the improvement of respiratory function will make the patient's life longer.
Here we first report a sensitive procedure using wholebody plethysmography to
monitor respiratory parameters detected in the utrophin/dystrophin double
knockout mouse (dko mouse), showing quite similar systemic symptoms to human DMD
including restrictive ventilatory impairment.
Furthermore, we show that high efficient dystrophin-transduction to dko's
diaphragm achieved by simple intraperitoneal injection of a helper-dependent
adenovirus vector (HDAdv) containing the full-length dystrophin expression
cassette (HDAdv-mFLmyc-dys). In spite of dystrophin expression only in the
diaphragm could result in the rescue from the ventilatory impairment (increased
tidal volume and improvement of compensatory hyperpnea). Our result suggests
that DMD patient's mortal ventilatory impairment may be improved by technically
easy means of the intraperitoneal injection of HDAdv.
Method and Results We have constructed helper-dependent adenovirus vector (HDAdv)
contained the murine full-length dystrophin expression cassette (HDAdv-mFLmyc-dys).
Each 7-day-old dko mice were injected with the HDAdv-mFLmyc-dys by the
intraperitoneal injection. In the diaphragm, the transgene was widely expressed
and prevented the dystrophic changes pathologically in injected dko mice.
Furthermore, We showed that respiratory function of injected dko mice recovered
by using wholebody plethysmography. Conclusion These total assessment system,
including wholebody plethysmography, may be useful to evaluate the therapeutic
approaches for the neuro-muscular disease models. Moreover, therapeutic gene
transfer with HDAdv may ameliorate respiratory insufficiency of DMD patients in
future.
12) First Generation
AAVmicroutrophin Vector Infused into the Isolated Pelvic Limb of a Canine Model
for Duchenne Muscular Dystrophy
Mihail Petrov, Marilyn Mitchell, Alock Malik, Andy Mead, Frederick
Balzer, Leonard Su, Jacqueline Farag, Benjamin Kozyak, Kapil Gopal, Charles
Bridges, Janet Bogan, Martin Childers, Joe Kornegay, Hansell Stedman. Surgery,
University of Pennsylvania, Philadelphia, PA; University of North Carolina,
Chapel Hill, NC; Wake Forest Univ., Winston Salem, NC.
Germline and somatic gene transfer of internally deleted dystrophin and utrophin
coding sequences into dystrophic mice has provided evidence for phenotypic
amelioration. Myotrophic AAV vectors have been shown to be amenable to a
vascular route of administration, suggesting that partial phenotypic correction
should be feasible in a large animal disease model. To this end, we initially
injected the isolated pelvic limbs of dogs hemi- or homozygous for the GRMD
mutation (dystrophin intron 6 splice acceptor site) with a “first generation”
AAV6 vector containing a constitutive promoter/enhancer driving transcription of
a “microutrophin” cassette based on the wild type cDNA sequence. The afferent,
transvenular extravasation route of administration used recapitulates that
previously shown to transduce essentially 100% of the skeletal muscle fibers in
the mature canine leg (Su, Gopal, et al, 2005). A proportion of the dogs
underwent transient single agent immunosuppression using a protocol previously
shown to prevent inhibitory antibody formation in a canine model for hemophilia
B (Arruda, Stedman, et al, 2005). Dogs were injected with vector at one of three
doses. Follow up studies for force transduction were performed by a group of
investigators blinded as to the AAV dose, identity of the injected limb, and the
presence or absence of prior immunosuppression. In a group of dogs receiving the
highest dose of AAV.utrophin-1 (10E13.5vg/kg), the ratio of torque developed by
the treated vs. untreated limb is 1.07+/-.2. Five of six dogs in this group
showed flexion strength improvement on the treated side. If one disallows the
data from the one confounding dog on the grounds that a minor technical problem
occurred (electrode migration during force transduction, as suspected from the
discrepancy between serial measurements in this dog) the average ratio among
remaining dogs is 1.15+/-.08. Importantly, with this combination of treatment
and immunosuppressive regimen the muscles did not become weaker, as one might
have expected with an AAV-induced myositis. Interestingly, data on other
non-immunosuppressed groups of dogs suggest that the vector might have caused a
subclinical myositis. Among dystrophic dogs receiving even lower doses of
AAV.utrophin-1 without cyclophosphamide, the ratio of treated to untreated limb
torque was 0.94+/-0.05. Moreover, among non-dystrophic dogs receiving the
unrelated AAV.F.IX (containing a “self” transgene) without immunosuppression,
the ratio of treated to untreated limb strength was 0.85+/-0.06. In conjunction
with emerging trends in AAV-based clinical investigation, these observations
heighten the impetus to formally address the prevention of cellular immune
response directed against input AAV capsid antigens.
13) Cell Therapy of
Muscular Dystrophy with Engineered Cd133+ Cells
Andrea Farini, Mirella Meregalli, Daniele Parolini, Marzia Belicchi,
Simona Maciotta, Paola Razini, Joao da Silva Bizario, Luis Garcia, Nereo
Bresolin, Yvan Torrente. Dept of Neurological Sciences, Fondazione IRCCS
Policlinico of Milan, Università degli Studi, Dino Ferrari Center, Milan, Italy;
AADM/UNAERP, San Paolo, Brazil; Facultè de Medecine Pierre et Marie Curie, UMR S
787, INSERM/UPMC, Institut de Myologie, Paris, France.
DMD is a genetic disease caused by mutations in dystrophin gene. Forced
exclusion (skipping) of a single or multiple exons can restore the reading
frame, giving rise to a shorter, but functional dystrophin protein. We selected
the GRMD dog, that shares with DMD patient progressive clinical signs and severe
myopathy with contractures and premature death. We isolated CD133+ cells from
skeletal muscle biopsies of GRMD dogs and we transduced them with lentiviral
vectors constructed to convey antisense oligonucleotides able to eliminate the
mRNA segment from exon 6 to 8. Under appropriate sedation, the dogs received
arterial systemic injections through a catheter introduced in the left femoralis
artery and reached the aortic arch at the level of the left subclavia: cells
were released mainly in the two large arteries under fluoroscopic guidance in
order to provide the whole body musculature. Serial injections of the engineered
CD133+ cells do not stimulate an immunoreaction in the treated dogs. The
delivery of these cells results in a partial recovery of stiffness and
ambulation disability of the treated dogs. The muscle biopsies of the
transplanted dogs showed clusters of dystrophin positives fibers. This approach
should offer a preclinical evidence for future therapies based on autologous
transplantation.
14) Systemic Delivery of
Antisense Morpholino Corrects RNA Mis-Splicing and Reduces Myotonia in a
Transgenic Mouse Model of Myotonic Dystrophy Type 1
Thurman M. Wheeler, Yong-Fu Li, Krzysztof Sobczak, Paul A. Morcos,
Charles A. Thornton. Department of Neurology, University of Rochester,
Rochester, NY; Gene Tools, LLC, Philomath, OR.
Objective: To test whether morpholinos modified for systemic delivery can
reverse symptoms in a transgenic mouse model of myotonic dystrophy type 1 (dystrophia
myotonica type 1; DM1). Background: DM1, the most common muscular dystrophy in
adults, is a dominantly inherited disorder characterized by expression of a
toxic RNA that leads directly to symptoms. DM1 is caused by expression of an
expanded CUG (CUGexp) repeat in the DM protein kinase mRNA. Nuclear foci of
CUGexp RNA sequester RNA binding proteins in the muscleblind-like (MBNL) family,
leading to abnormal alternative splicing of select genes. Mis-splicing of the
muscle chloride channel, ClC-1, leads to myotonia in DM1. Intramuscular
injection of antisense morpholino targeting the CUGexp RNA can displace Mbnl1
protein and eliminate myotonia in a transgenic mouse model of DM1 (Wheeler,
2009). However, muscle uptake of unmodified morpholino after systemic delivery
is low. Morpholinos coupled to an octa-guanidine dendrimer (Vivo Morpholinos)
are designed for enhanced tissue uptake after systemic delivery. Design/methods:
200 μg of Vivo Morpholino (Gene Tools) was injected into the tail vein of HSALR
transgenic mice twice weekly for two months. Two dendrimer-modified morpholinos
were tested. One morpholino was a CAG repeat, previously shown to displace Mbnl1
protein from CUGexp RNA in mice. The sequence of the second morpholino
previously demonstrated selective suppression of exon 7a of ClC-1 (ClC-1
antisense) in mice (Wheeler, 2007). Control mice were injected with vehicle
alone (saline). Treatment assignments were randomized. Injections and
electromyography were blinded. Results: In mice treated with the CAG repeat
oligo, RNA mis-splicing of several transcripts was improved and myotonia was
reduced in all 3 hindlimb muscles tested. Treatment with ClC-1 antisense
completely reversed ClC-1 mis-splicing and eliminated myotonia in all 3 hindlimb
muscles tested. ClC-1 protein was restored to the muscle surface membrane in
both groups of morpholino-treated mice. Identical dosing for one month produced
a partial effect. Intravenous injection of saline had no effect. Conclusions: By
using a dendrimer-modified morpholino, whole-body correction of RNA mis-splicing
and reduction of myotonia can be achieved in a transgenic mouse model of DM1.
These results support the feasibility and effectiveness of systemic delivery of
antisense morpholino as treatment for DM1.
15) Gene Transfer-Driven
Inhibition of Cardiac Ankyrin Repeat Protein as a Potential Therapy for the
Correction of Muscular Dystrophies
Nathalie F. Danièle, Laurence Suel-Petat, Isabelle Richard.
Research, Généthon, Evry, France.
Muscular dystrophies (MD) are a group of genetic disorders characterised by
progressive muscle degeneration and weakness, with a broad range of genetic and
phenotypic variations. Up until now, the treatments, which are only palliative,
aim at alleviating the symptoms. The research of new treatments, mainly
orientated towards gene therapy, is largely impeded by the fact that each
therapy has to be tailored for the targeted disease. In order to find a
therapeutic target eligible for the correction of the largest possible number of
MD, we previously screened the expression of pivotal proteins in several major
MD and demonstrated that Cardiac Ankyrin Repeat Protein
(CARP), a transcription regulator, is constitutively up-regulated in every
muscular dystrophy considered, including the most common form Duchenne Muscular
Dystrophy. Whether this up-regulation is beneficial or detrimental for the
muscle tissue remains unknown, although the anti-proliferative function of CARP
is consistent with an aggravation of the phenotype. In the present work, we
assess the down-regulation of CARP as a potential therapy in the
alpha-sarcoglycan deficient mouse model. We demonstrate that CARP
over-expression occurs in both muscle fibres and mononucleated cells in these
animals. In order to target each of these tissues, two different CARP-specific
Sh-RNA sequences, previously validated in cellular models, were vectorized into
rAAV-[2/1] or SIN-lentiviral vectors. The vectors are injected directly in the
TA muscle of young animals (3-weeks or 3-days old, depending on the nature of
the vector) and the treatments efficiencies are assessed after 1 and 3 months of
expression. Altogether, the analyses of these protocols will be useful to
ascertain whether CARP is a suitable therapeutic target for the treatment of MD.
16) Multiple Exon-Skipping
Using Cell-Penetrating Morpholinos for Dystrophic Dogs
Toshifumi Yokota, Takashi Saito, Nobuyuki Urasawa, Tetsuya Nagata,
Akinori Nakamura, Ryszard Kole, Peter Sazani, Terence Partridge, Shin'ichi
Takeda, Eric Hoffman. Research Center for Genetic Medicine, Children's National
Medical Center, Washington, DC; Department of Molecular Therapy, National
Institute of Neuroscience, Kodaira, Tokyo, Japan; AVI BioPharma, Inc., Bothell,
WA.
Duchenne muscular dystrophy (DMD), the most common and fatal X-linked myopathy,
and its milder form, Becker muscular dystrophy (BMD), are caused by mutations in
the DMD gene. Exon skipping using antisense oligonucleotides is currently one of
the most promising molecular therapies for DMD. The exon skipping approach leads
to the production of internally deleted, in-frame mRNA transcripts that are
translated into truncated dystrophin protein that has missing amino acids, but
retains some functions like Becker dystrophy. Previously we reported first
successful exon-skipping treatment in body-wide skeletal muscles in Canine
X-linked muscular dystrophy (CXMD) using a cocktail of phosphorodiamidate
morpholino oligomers (PMOs, morpholinos) targeting exon 6 and exon 8 of
dystrophin mRNA in vivo (multiple exon-skipping) (Ann Neurol 2009;65:667–676;
Efficacy of systemic morpholino exon-skipping in Duchenne dystrophy dogs).
However, unmodified morpholinos exhibit inefficient delivery to the heart
leading to dystrophin induction with barely detectable in the cardiac muscle.
Here, we sought to recover the expression of dystrophin in cardiac muscles in
dystrophic dogs using morpholinos conjugated to short arginine-rich
cell-penetrating peptides (PPMOs) and morpholinos conjugated with octa-guanidine
dendrimer (vivo-morpholinos), and demonstrated that the delivery moieties
significantly improved dystrophin production in both skeletal and cardiac
muscles. The efficacies of vivo-Morpholinos and PPMOs are similar after local
injections in dystrophic dogs. Single intravenous or cardiac catheter injection
with PPMOs at 12 mg/kg restored dystrophin expression in cardiac muscles as well
as body-wide skeletal muscles. No obvious toxicity was detected by blood tests
and histology. Our results show the potential of PMO conjugates as therapeutic
agents for DMD and many other diseases by targeting mRNAs.
17) Validation of
Intravascular Administration of Adeno-Associated Vvirus for Muscular Dystrophies
in a Non-Human Primate Model
Aravind Asokan, Julia Hegge, Joshua C. Grieger, Chengwen Li, Swati
Yadav, Jade J. Samulski , Xiao Xiao, Juan Li, Jon Wolff, R. Jude Samulski, Scott
W. J. McPhee. University of North Carolina, Chapel Hill, NC; Mirus
BioCorporation, Madison, WI; Asklepios BioPharmaceutical Inc, Chapel Hill, NC.
Duchenne muscular dystrophy (DMD) results from mutations in the dystrophin gene,
and is characterized by severe skeletal muscle deterioration in the first
decades of life. Effective genetic interventions will likely require systemic
intravascular delivery of the therapeutic to target this widespread and large
volume of target tissue. We are developing Adeno Associated Virus (AAV) mediated
minidystrophin gene delivery and have previously conducted a Phase I trial of
intramuscular administration in patients with DMD. The next step in clinical
development is the progression to regional limb delivery via an intravascular
route of administration. The Hydrodynamic Intravascular Infusion technique was
initially developed to mediate efficient naked DNA transfer to an individual
limb. This is achieved by transient restriction from systemic circulation using
a tourniquet secured around the upper limb to isolate the vasculature. A large
volume is then rapidly delivered via a line into a distal superficial vein. We
have adapted this technique to evaluate regional delivery of multiple AAV
serotypes including 2.5, 8, 9 and the novel capsid 2i8 to the lower limb. We
tested reporter gene delivery via the saphenous vein in Rhesus macaques and
quantified luciferase expression levels in a large number of lower hind limb
muscle groups. We studied infusion volumes ranging up to 35% of limb volume, and
the procedure was well tolerated by all animals. With AAV 2i8 we observed the
highest luciferase expression levels after low volume (<0.1mL/mL limb volume)
delivery. AAV2i8 vector is a novel capsid with a reengineered receptor footprint
to mediate systemic muscle transduction in conjunction with detargeting of the
liver. These results provide preclinical feasibility to support advancing
regional AAV minidystrophin gene delivery to the limbs of patients with DMD.
18) AAV9-Mediated Catalase
Expression in Mitochondria Improves Exercise Performance in mdx Mice
Dejia Li, Dongsheng Duan. Department of Molecular Microbiology and
Immunology, University of Missouri, Columbia, MO.
Duchenne muscular dystrophy (DMD) is a lethal degenerative muscle disease caused
by dystrophin deficiency. The precise mechanisms by which absence of dystrophin
results in the muscular dystrophy remain not fully understood. It has been
suggested that increased oxidative stress may contribute to myofiber necrosis
and degeneration. Here we tested whether reducing mitochondrial free radical
could ameliorate muscle disease in the mdx model of DMD. The human catalase gene
was engineered with the mitochondria-leading sequence for targeted expression in
the mitochondria (Schriner et al Science 308:1909, 2005). The mitochondrial
catalase gene was packaged in adeno-associated virus serotype-9 (AAV9) and
delivered to neonatal mdx mice. Histopathology and muscle force were examined
three months later. We did not detect morphology improvement in muscle section,
neither was serum creatine kinase level reduced. However, the eccentric
contraction-induced damaged was mitigated in the extensor digitorium longus
muscle. When challenged with downhill treadmill running, AAV-infected mice
significant outperformed untreated mice. Taken together, our results suggest
that the mitochondria-derived reactive oxygen species may contribute to muscle
weakness in mdx mice. Strategies to reduce oxidative stress in
mitochondria may represent a viable approach to treat muscular dystrophy.
19) Genetic Evidence for a
Structural Role of α–dystrobrevin-3 within the Dystrophin-Glycoprotein Complex
Guy L. Odom, Glen B. Banks, Dewayne Townsend, Marvin E. Adams,
Joseph M. Metzger, Stanely C. Froehner, Jeffrey S. Chamberlain. Neurology,
University of Washington, Seattle, WA; Integrative Biology & Physiology,
University of Minnesota, Minneapolis, MN; Physiology and Biophysics, University
of Washington, Seattle, WA.
α-Dystrobrevin (αDB) is an integral component of the dystrophin-glycoprotein
complex (DGC) at the sarcolemma of myofibers. Mice lacking αDB1-3 (adbn-/-) in
striated muscle present with several phenotypic changes consistent with
dystrophy including myopathy, prominent neuromuscular and myotendinous junction
defects, and a mild cardiomyopathy. Decreasing amounts of αDB from the
sarcolemma contributes to the severity of disease in several muscular
dystrophies including Duchenne muscular dystrophy (DMD). Both αDB1 and αDB2
transgenes restore the skeletal muscle abnormalities in the adbn-/- to varying
degrees because of subtle differences in their location within the sarcolemma
and varying functional (phosphorylation/protein-interaction) domains contained
in each protein(Grady et al., 2003; Grady et al., 1999; Grady et al., 2000).
Because αDB3 is the shorter isoform which lacks these functional domains,
including being unable to bind directly to dystrophin, it has previously been
assumed to play only a negligible role in the DGC. Here, overall cardiac
functional workload was assessed in wild type and mutant mice using a dobutamine
stress test protocol followed by measurement of hemodynamic properties. We
measured 18 ECG parameters at rest, and at 5 time points. None of these
parameters showed significant differences, suggesting a relatively normal
electrical activity. We next analyzed the role of αDB3 in skeletal muscle by
systemically delivering αDB3 to adbn-/- mice with recombinant adeno-associated
virus serotype 6. The results demonstrated αDB3 to be an important DGC component
within skeletal muscle, showing localization to the sarcolemma despite being
unable to bind dystrophin, and a robust interaction with ankyrin as demonstrated
by in vitro co-immunoprecipitation, suggesting a cooperative role toward the
stabilization of myofibers. Overall, expression in vivo of αDB3 resulted in the
mitigation of prominent dystrophic abnormalities including the prevention of
muscle degeneration, synapse fragmentation, and shallow folds within the
myotendinous junction. These results suggest an important structural role of
αDB3 whose absence can contribute to sarcolemma fragility in DMD and other
muscular dystrophies.
20) Characterization of A20
as an Inhibitor of NF-κB Activation in Dystrophic Mice
Rakshita Charan, Paula R. Clemens. Department of Neurology,
University of Pittsburgh, Pittsburgh, PA; Department of Veterans Affairs Medical
Center, Veterans Affairs, Pittsburgh, PA.
Of all muscular dystrophies, Duchenne muscular dystrophy (DMD) is the most
common affecting about 1 in 3500 male births worldwide. The disease 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 structural
support in dystrophin-deficient muscle fibers may be responsible for muscle
pathology in progressive muscular dystrophy. It is also known that nuclear
factor-kappa B (NF-κB), which is a nuclear transcription factor, is up regulated
in dystrophic muscle in DMD patients as well as in the mouse model for DMD (mdx).
NF-κB regulates several genes responsible for stress responses, cell survival
and various inflammatory conditions. Thus, the up regulation of this
transcription factor is thought to activate protein degradation and cause
chronic inflammation in skeletal muscle. Furthermore, NF-κB downregulates
myogenic regulatory factors and this process likely interferes with muscle
regeneration. Attenuating NF-κB activation in these dystrophic mice has been
shown to improve muscle stability and strength. Strategies of inhibition of NF-κB
activation are being actively pursued as a therapeutic option for DMD. One of
the NF-κB pathway attenuators, A20 is a deubiquitinating enzyme, known to
inhibit NF-κB activation by deubiquitinating RIP1; ubiquitination of RIP1 is
essential for NF-κB activation.
Our aim is to characterize A20 in skeletal muscle and establish its role as a
potential therapeutic target as attenuator of the NF-κB pathway activation in
DMD. We show that blocking of A20 using A20siRNA increases NF-κB activation in
mdx as well as control C57BL/10 mice myotubes. We further characterized
localization of A20 in muscle and established that A20 is expressed
predominantly in fast-twitch muscle fibers. Interestingly, we also observed that
in mdx muscle, A20 is over-expressed in regenerating fibers. To study the
localization of A20 through the life of the mdx mouse, we did a time-profile
assessment of A20 expression and compared it with control mice. We see an
increase in A20 protein expression during the 7-10 week time period in mice,
which is correlative of the ages when severe degeneration and regeneration
cycles take place in mdx mice. This is the first observation of a correlation
between expression of an NF-κB inhibitor and pathology of DMD.
Our studies support the utility of therapeutic manipulation of A20 to promote NF-κB
inactivation in dystrophic muscle fibers and provide a potential therapy for DMD.
21) Impact of the Host
Environment on the Control of Gene Transfer-Induced Immune Responses with
miRNA142.3p-Regulated Vectors
Florence Boisgérault, David-Alexandre Gross, Maxime Ferrand, Sylvie
Darocha, Jérôme Poupiot, Marc Bartoli, Isabelle Richard, Anne Galy. UMR951,
Genethon, Evry, France; CNRS UMR8587, Genethon, Evry, France.
Recombinant AAV2/1 vectors have emerged as strong candidates for gene transfer
into skeletal muscle. However their efficacy is partially limited by the
development of anti- transgene immune responses which affect long-term muscular
reconstruction in the context of muscular dystrophies. In a murine model of
limb-girdle muscular dystrophy type 2D (Sgca-null), administration of rAAV2/1
expressing the alpha-sarcoglycan (sgca) transgene under control of the
ubiquitous CMV promoter results in a transient expression of the protein in
skeletal muscle whereas an efficient and more stable expression was obtained
using a muscle-specific promoter. To understand how transgene expression
regulates the development of immune responses, a reporter (Sgca-HY) transgene
was modified by the introduction of two epitopes of the murine male HY antigen,
and used to follow transgene-specific CD4+ and CD8+ T cell responses in mice of
the C57BL/6 background. With this system, a single i.m. injection of the
rAAV-CMV-sgca-HY vector in C57BL/6 or in Sgca-null mice induced a strong
cellular CD4 and CD8-mediated immune response resulting in an extensive
mononuclear cell infiltrate in the injected muscle associated with muscular
destruction and loss of gene-modified cells. To determine the impact of direct
antigenic presentation on immune responses, the transgene was de-targeted from
hematopoietic cells by inserting miRNA142.3 target sequences into the expression
cassette. This strategy abrogated the development of effector anti-transgene CD4+
and CD8+ cellular immune responses in C57Bl/6 mice, enabling long-term transgene
persistence. This approach was also tested in the pathological sgca-null model
but was not as effective as in normal mice. Following rAAV-mediated gene
transfer in sgca-null mice, transgene expression was improved but not maintained
over time. The mir142.3p-regulated vector prevented the development of
transgene-specific CD8+ T cell responses but failed to control CD4+ T cell
reactivity. These data suggest that miRNA142.3p-regulated expression systems are
useful to reduce the initial immunogenicity of the transgene after rAAV-mediated
gene delivery into normal skeletal muscle but may not be entirely efficient to
control antigen presentation to CD4+ T cells in an inflammatory context. This
suggests that controlling anti-transgene CD4+ T cell responses following rAAV2/1-mediated
gene delivery is essential to establish long-term transgene persistence.
22)
Feasibility and Effectiveness of Exon 51 skipping in Human-Like mdx
Mutation
Yoshitsugu Aoki, Toshifumi Yokota, Takashi Saito, Akinori Nakamura,
Tetsuya Nagata, Hitoshi Okazawa, Shin'ichi Takeda. Department of Molecular
Therapy, National Institute of Neuroscience, National Center of Neurology and
Psychiatry (NCNP), Kodaira, Tokyo, Japan; Medical Research Institute, Tokyo
Medical and Dental School University Graduate School, Bunkyo-ku, Tokyo, Japan;
Research Center for Genetic Medicine, Children's National Medical Center,
Washington, DC.
Background: Duchenne muscular dystrophy (DMD) is caused by the lack of
dystrophin at the sarcolemma. Systemic delivery of anti-sense oligonucleotide
(AO) is a promising approach to therapy for Duchenne muscular dystrophy (DMD).
Current clinical trials for DMD involve exon 51-skipping but are unaccompanied
by animal model studies of recovery of skeletal muscle function with this skip.
Here, we use the exon 52-deletion mdx mouse to screen AO sequences for skipping
exon 51, making assessment of molecular and functional efficacy. Methods: We
tested a series of phosphorodiamidate morpholino oligomers (PMOs) singly and in
combination against exon 51 of the mouse DMD gene, and screened for molecular
efficacy by intramuscular injection and then intravenously injected an optimized
PMO combination (160 mg/kg/each, once weekly for 7 weeks) into mdx52 mice. The
efficiency and efficacy of exon 51 skipping were tested at the mRNA, protein,
histological, and functional levels. Results: The highest splicing efficiency of
an individual sequence (50-55%) was that targeting the 3' splice site
(+10-15bp). But, yet more effective (75%), was a two-AO combination targeting
both 5' and 3' splice sites. This, on systemic delivery, induced 10-40% of
wild-type dystrophin expression in all muscles, accompanied by amelioration of
dystrophic pathology and improvement of skeletal muscle function. Blood tests
and histological examination of liver and kidney indicated no evidence of
toxicity. Interpretation: This demonstration of efficient exon 51-skipping
leading to clear improvement in skeletal muscle function, supports the
pre-clinical rationale for current human trials. However, since the greatest
efficacy was associated with targeting of dual sequences unrelated to those in
the current human trials, further testing and selection of target sequences is
indicated.
23)
Sustained Alpha-Sarcoglycan Gene Expression in LGMD2D Following Gene
Transfer
Jerry R. Mendell, Louise R. Rodino-Klapac, Xiomara Rosales-Quintero,
Brian D. Coley, Gloria Galloway, Sarah Lewis, Vinod Malik, Christopher J.
Shilling, Barry J. Byrne, Thomas Conlon, Katherine J. Campbell, William G.
Bremer, Christopher M. Walker, Zarife Sahenk, K. Reed Clark. Center for Gene
Therapy, Research Institute at Nationwide Children's Hospital, Columbus, OH;
Pediatrics, University of Florida College of Medicine, Gainesville, FL; Center
for Vaccines and Immunology, The Research Institute at Nationwide Children's
Hospital, Columbus, OH.
Limb-girdle muscular dystrophy (LGMD) type 2D is defined as a deficiency of
a-sarcoglycan (α-SG), a sarcolemmal transmembrane protein contributing to
membrane stability. There are few if any treatment options for this disease. We
previously reported a successful trial of SGCA gene transfer in three patients
with persistence of gene expression for at least three months. The number of α -SG
positive fibers reached 57%, 69%, and 62% and western blot showed a 4 to 5 fold
increase in each subject with the maximum restoration of α-SG in any block 76%
of normal control muscle. Persistent gene expression was complemented with
restoration of the full sarcoglycan complex and an increase in mean fiber
diameter in one subject analyzed at 3 months. To evaluate whether long term gene
expression was achievable, a second cohort of 3 patients was evaluated at 6
months. As in the first cohort, a double-blind, randomized controlled trial was
initiated in LGMD2D subjects using rAAV1 with full length human SGCA under
control of a truncated muscle creatine kinase (tMCK) promoter. The extensor
digitorum brevis (EDB) muscle received 3.25 X 1011 vector genomes on
one side, while the control side was given saline. All analyses were done prior
to breaking the blind. The entire EDB muscle was removed from both sides at 6
months in all three subjects. Transgene specific PCR analysis demonstrated
expression on only one side in each subject which corresponded with gene
expression. Subject 1 increased α -SG by 2 fold on the side of gene transfer
reaching wild-type levels. This was accompanied by an increase in mean muscle
fiber diameter, 28.2 ± 11.1 to 52.2 ± 13.1. Subject 2 demonstrated robust gene
expression, again equivalent to wild-type levels on the side of gene transfer.
MHCI and CD4, CD8 mononuclear cells were upregulated on the side of gene
transfer in subjects 1 and 2. ELISpots for antigen specific production of IFN-γ
secretion were monitored beginning pre-gene transfer. Subject 1 had a transient
minimal response to AAV1 capsid pools at days 14 and 28. Patient 2 showed no
response to α-SG or AAV1 capsid peptide pools. In contrast to the first two
patients, Subject 3 had pre-existing immunity to AAV1 (neutralizing antibodies)
and an ELISpot IFN-γ response within the first week to AAV following gene
transfer suggesting a memory response. Levels of gene expression were also
reduced in this subject compared to others. Overall the promising results from
this study with gene expression persisting for 6 months lays the foundation for
potentially achieving long-term sustained correction of the underlying gene
defect in muscular dystrophy. However, the influence of pre-existing immunity to
AAV may affect initial muscle fiber transduction or sustained gene expression.
24)
ncRNAs Originating from the Dystrophin Gene as Biomarker for Assessing
Antisense Therapy
Matteo Bovolenta, Annarita Armaroli, Marcella Neri, Simona Brioschi,
Marina Fabris, Chiara Scotton, Sofia Falzarano, Paola Rimessi, Emanuele Valli,
Giovanni Perini, Luciano Merlini, Francesca Gualandi, Alessandra Ferlini.
Department of Diagnostic Medicine, Section of Medical Genetics, University of
Ferrara, Ferrara, Italy; Department of Biology and Evolution, University of
Bologna, Bologna, Italy.
The DMD gene is the largest in the human genome, it spans 2.2 Mb and is made of
79 exons and 7 isoforms, all finely regulated and expressed in specific tissues.
Mutations in this gene lead to three distinct phenotypes: Duchenne Muscular
Dystrophy (DMD), Becker Muscular Dystrophy and X-linked dilated cardiomyopathy.
Therapeutic approaches are now becoming reality in DMD, nevertheless, clinical
outcome measures may not always be sensitive enough to detect small changes in
disease progression/regression and after short treatment periods. It is
therefore imperative to identify enrichment endpoints, as biomarkers, able to
document benefits of the treatment at early stages and at the individual level.
Recently, genome-wide approaches to monitor transcription have revealed a
notable number of non coding RNAs (ncRNAs) with many regulatory functions. To
address this issue we designed a novel gene-specific Gene Expression tiling
array covering the full DMD gene and used it to search for non-coding
transcripts in polyA+ RNAs from human brain, heart, skeletal muscle and skin.
The search identified 13 sense and 2 antisense oriented transcripts, originating
from dystrophin introns/UTR regions. Six of these ncRNAs (four sense polyA+
ncRNAs, located adjacent to dystrophin known promoter regions, and the 2
antisense) were validated through Northern blotting and fully characterized by
RACE PCR and sequencing. Their length ranges from 1800 to 2800 bp, 5 were
unspliced, but one was spliced in several isoforms. Compartmentalisation studies
demonstrated that all the six ncRNAs are located in the nucleus. None of these
has an open reading frame, suggesting that they belong to the long non-coding
RNA category. These ncRNAs are highly represented in human heart, skeletal
muscle and brain, known tissues of elective dystrophin expression. CGH-DMD
analysis in 5 DMD patients with dystrophin exons 45-50 deletion (thus eligible
for exon 51 skipping antisense therapy) allowed us to define the deletion
breakpoints therefore predicting the loss/maintenance at the genomic level of
the ncRNAs region. One sense oriented ncRNA, which localises within intron 44
and the corresponding genomic region was deleted in only one DMD boy, was
consistently missing in his myogenic cells. Further studies are in progress in
order to profile the expression of these ncRNAs in patients' myogenic cells
before and after AONs treatment for exploring if these transcripts may be used
as muscle transcriptomic biomarkers for monitoring the impact of novel treatment
in dystrophinopathies.
25)
Expression of Micro-Dystrophin and Full-Length Dystrophin in Mouse and
Dog Muscles
Christophe Pichavant, Pierre Chapdelaine, Joe N. Kornegay, João C.
S. Bizario, Xiao Xiao, Jacques P. Tremblay. CHUL, Quebec, QC, Canada;
University of North Carolina School of Medicine, Chapel Hill; Muscular Dystrophy
Research Center, Ribeirão Preto-SP, Brazil.
Duchenne muscular dystrophy (DMD) is an X-linked genetic disease characterized
by the absence of dystrophin in the muscle. Since the phenotype of the
dystrophic dog is closer to the human phenotype than the mdx mouse, we
have studied the introduction of the dog dystrophin in dystrophic dog muscles.
To achieve that, two different isoforms of the dystrophin were used in our
experiments: the dog micro-dystrophin (µdys) and the full-length dog dystrophin
(FLDYS). Muscle precursor cells (MPCs) were transduced with a lentivirus coding
for the µDys in fusion with a V5 tag (µDys.V5) and transplanted in
immunodeficient mouse and dog muscles. A non viral gene therapy, the
electrotransfer, was also used to introduce the plasmids coding for the µDys.V5
cDNA and for the FLDYS cDNA. These plasmids were electrotransferred in
immunodeficient mouse, dog and dystrophic dog muscles.
Concerning the use of the µDys.V5 in mouse muscles, the percentage of V5
positive fibres was more important in the muscles treated by ex vivo gene
therapy than those electrotransferred with the plasmid. Dog muscles were also
transplanted with dog MPCs transduced with the µDys.V5 lentivirus. No V5
expression was detected in the treated muscles one month following the graft.
However, accumulations of CD8+ cells were observed in these muscles. A biopsy of
another muscle transplanted with the same transduced cells has been also studied
at two months and as expected, no transgene was found and also no CD8+ cell. Dog
muscles were also electrotransferred with the µDys.V5 plasmid and two weeks
later, fibres expressing this dystrophin were co-localised or not with CD8+
cells. To introduce the FLDYS into muscles, only the electrotransfer technique
was used since the plasmid containing this transgene was large (∼17 kb). Our
results showed that the electrotransfer of this large plasmid into mouse muscles
allowed the FLDYS expression in the treated muscles. The electrotransfer of
FLDYS in a dystrophic dog muscle led also to the expression of dystrophin.
However, accumulations of mononuclear cells were observed in this muscle, these
lymphocyte infiltrations were co-localised or not with the myofibers expressing
the FLDYS.
In conclusion, the introductions of the µDys or of the FLDYS by ex vivo
gene therapy or by electrotransfer are potential approaches to restore the
dystrophin in DMD patients. The next step will be to improve the efficiency of
the delivery methods and also to verify whether the specific immune response
producing the rejection of the myofibers is due to our transgene, to our
bacterial backbone sequence or to other components.
26)
Screening of Nature Compounds for Enhancing Antisense
Oligonucleotide-Induced Exon Skipping of Dystrophin Gene In Vitro and
In Vivo
Bo Wu, Caryn Cloer, Peijuan Lu, Mingxing Wang, Wei Li, Kazuo Koike,
Qi Long Lu. McColl-Lockwood Laboratory for Muscular Dystrophy Research,
Carolinas Medical Center, Charlotte, NC; Department of Pharmaceutical Sciences,
Toho University, Narashino, Chiba, Japan.
Antisense oligonucleotide-mediated exon skipping therapy has demonstrated great
potential for treating Duchenne muscular dystrophy (DMD). Phosphorodiamidate
morpholino oligomer (PMO) is the most widely used and being applied in clinical
trials. However unmodified PMO has limited efficacy due to low potency and
ineffciency in systemic delivery, especially failure to restore dystrophin in
heart. Recently our and others' groups have found that modifications of PMO with
cell-penetrating peptides and non-peptide dendrimeric octa-guanidine
significantly improve the delivery of PMO and achieved short-term rescue of
dystrophin expression in body-wide muscles including the cardiac muscle with
improved pathology and partial restoration of muscle functions. However, the
safety of the long-term use of positively charged polymers remains to be
investigated. Here we developed a drug screening system using C2C12 myoblasts
and myotubes expressing a reporter GFP with its reading frame disrupted by the
insertion of a targeted dystrophin exon to screen and identify nature compounds
capable of enhancing the exon skipping effect by specific antisense oligomers.
We identified a series of nature compounds (5) capable of increasing exon
skipping efficiency when used in combination with PMO in vitro. Three compounds
were able to enhance PMO-induced exon skipping two fold in TA muscles of
dystrophic mdx mouse by local injection when compared with unmodified PMO.
Onecompound significantly increased systemic effect of PMO in the cardiac and
skeletal muscles with reduction of serum creatine kinase levels. Nature
compounds could be explored for enhancing specific exon skipping with antisense
oligomers for DMD therapy.
27)
External, Non-Invasive Monitoring of Progressive Cardiorespiratory
Dysfunction in a Canine Model of DMD
Andrew Mead, Alock Malik, Mihail Petrov, Martin Childers, Janet
Bogan, Joseph Kornegay, Hansell Stedman. University of Pennsylvania,
Philadelphia, PA; Wake Forest University, Winston-Salem, NC; University of North
Carolina, Chapel Hill, NC.
The GRMD (Golden Retriever Muscular Dystrophy) model for Duchenne Muscular
Dystrophy (DMD) mimics human disease progression with regard to both
histopathology and locomotive function. However, unlike DMD, the GRMD model
exhibits a high degree of variability in both the severity and speed of
progression, even among littermates. This aspect of the model poses challenges
for assessing efficacy in pre-clinical therapeutic trials of gene therapy, and
requires further improvement in quantitative measures of disease progression.
Electrical stimulation of distal limb muscles has provided the most reliable
quantitative data, however this requires serial episodes of general anesthesia,
with attendant risks of cardiopulmonary complication in the dystrophic dogs.
Moreover, the data are limited to locomotive muscle function. Most non-invasive
means of assessment, such as running times, are unreliable due to their
volitional nature, and fail to dissect the cardiac, respiratory and locomotive
components of disease progression. Here we investigate the use of a non-invasive
device that records EKG, 3D accelerometry, and 2-band plethysmography in a
wireless package that has negligible effect on animal behavior, the “Lifeshirt”
(Vivometrics). In particular we simultaneously examine cardiac and respiratory
function, two physiological parameters relevant to longevity in DMD. 1. We
hypothesize that the progressive loss of ventilatory reserve caused by
degeneration of the diaphragm will manifest, in the GRMD model, as a progressive
respiratory paradox, (i.e. asynchrony in the chest wall and abdominal wall
excursion) during and immediately after mild exertion. The degree of paradox
will correlate with trans-diaphragmatic pressure development during stimulated
spontaneous breathing and phrenic nerve stimulation at the time of necropsy. 2.
Progressive heart failure, also a hallmark of DMD in humans, will manifest first
as parasympathetic withdrawal measured by depressed respiratory sinus arrhythmia
in affected dogs as compared to unaffected littermates. Decreased RSA would be
expected to correlate with impaired cardiac contractility as measured by more
direct means, e.g. Langendorff isolated perfused heart studies performed post
mortem.
Data obtained using the "Lifeshirt"system on GRMD pups reveals that by 6 months
of age there are early indicators of respiratory and cardiac dysfunction. In
preliminary studies of 11 dystrophic and 7 normal dogs under 8 months of age RSA
was significantly reduced in the affected group (p<.006), and respiratory phase
angle was higher (p<.05). Affected pups were also marked by the complete absence
of diaphragm-intensive panting behavior. These differences between normal and
affected animals, and among affected animals at different stages of disease
progression increased markedly after moderate voluntary exercise.
Standardization of these approaches will enhance the GRMD model's role in
translational studies of gene therapy for cardiac and respiratory muscle disease
in DMD.
28)
Splice Site Strength and Nonsense-Associated Exon Skipping in the DMD
Gene
Kevin M. Flanigan, Diane M. Dunn, Jerry R. Mendell, Alan Pestronk,
Julaine M. Florence, United Dystrophinopathy Project Consortium, Robert B.
Weiss. Center for Gene Therapy, Nationwide Children's Research Institute,
Columbus, OH; Department of Neurology, Washington University, St. Louis, MO;
Department of Human Genetics, University of Utah, Salt Lake City, UT.
Understanding the molecular pathogenesis of DMD gene mutations is more
compelling than ever considering evolving treatment strategies that include gene
replacement therapy, exon skipping, and nonsense mutation readthrough. Nonsense
mutations can be associated with a spectrum of phenotypes: Duchenne Muscular
Dystrophy (DMD) with loss of walking by age 12, Becker Muscular Dystrophy (BMD)
with continued walking after age 15, and an intermediate (IMD) phenotype with
loss of walking between ages 12 and 15. In some cases, a nonsense mutation has
been shown to ablate an exon splice enhancer (ESE) or create an exon splice
suppressor (ESS) motif, resulting in altered mRNA splicing with a resultant
milder phenotype. To assess the generality of this mechanism, we evaluated 166
unique nonsense mutations found in 210 patients with well-established phenotypes.
Among mutation sites in which mutation-induced exon skipping would be predicted
to result in an out-of-frame transcript, only 5% resulted in BMD or IMD (n=4)
and 95% resulted in DMD (n=77). In contrast, among mutations in an in-frame
flanking exon context, 18% (n=15) resulted in BMD or IMD, and 82% (n=70)
resulted in DMD. Thus, in BMD or IMD the exonic sequence context is typically
in-frame (15/19) (P=.014 by Fisher's exact test), although the presence of an
in-frame exon context is not by itself predictive of BMD. We calculated the
splice site strengths of in- versus out-of-frame exons using maximum entropy (MaxEnt),
multiple dependence decomposition (MDD), first order Markov model (MM), and a
weight matrix model (WMM). On average, in-frame exons have weaker splice site
(ss) signals than out-of-frame exons, most notably in the 5'ss strength (MaxEnt
p=0.006; MDD p=0.065; MM p=0.029; WMM p=0.049). The subset of in-frame exons
that are enriched for BMD nonsense mutations have on average weaker 3'ss
strength compared to the other in-frame exons (MaxEnt p=0.015; MDD p=0.015; MM
p=0.053; WMM p=0.015). Thus, exons with in-frame BMD nonsense mutations appear
to have the weakest splice site signals of all the DMD exons, as measured by the
mean difference between consensus values for 3'ss and 5'ss strengths (MaxEnt:
13.21 vs. 15.96 for in-frame BMD vs. in-frame DMD, and 13.21 vs. 16.93 for
in-frame BMD vs. out-of-frame exons). In contrast, BMD mutations are not more
frequently associated with either the ablation of ESE motifs or the creation of
an ESS site than are DMD mutations (Mann-Whitney-Wilcoxon rank sum test, p=0.7).
Our data suggest that in BMD, ESE ablation or ESS creation signals are not the
sole determinant of nonsense mutation-associated exon skipping, but rather that
the effects of these exonic signal alterations are manifest on a background of
weak intrinsic splice site signals within a restricted set of exons. An improved
understanding of DMD splice site metrics may shed light on the design of
antisense oligonucleotides, and the interpretation of results from upcoming
clinical trials of exon skipping.
29)
The Polyproline Site in Hinge 2 Influences the Functional Capacity of
Truncated Dystrophins
Glen B. Banks, Luke M. Judge, James M. Allen, Jeffrey S. Chamberlain.
Department of Neurology, University of Washington, Seattle, WA.
Mutations in dystrophin can lead to Duchenne muscular dystrophy or the more mild
form of the disease, Becker muscular dystrophy (BMD). The hinge 3 region in the
rod domain of dystrophin is particularly prone to deletion mutations. In-frame
deletions of hinge 3 are predicted to lead to BMD, however the severity of
disease can vary considerably. Here we performed extensive structure-function
analyses of truncated dystrophins with modified hinges and spectrin-like repeats
in mdx mice. We found that the polyproline site in hinge 2 profoundly
influences the functional capacity of a microdystrophinΔR4-R23/ΔCT
with a large deletion in the hinge 3 region. Inclusion of polyproline in
microdystrophinΔR4-R23/ΔCT led to small myofibers (12% smaller than
wild-type), Achilles myotendinous disruption, ringed fibers and aberrant
neuromuscular junctions in the mdx gastrocnemius muscles. Replacing hinge 2 of
microdystrophinΔR4-R23/ΔCT with hinge 3 significantly improved the
functional capacity to prevent muscle degeneration, increase muscle fiber area
and maintain the junctions. We conclude that the rigid α-helical structure of
the polyproline site significantly impairs the functional capacity of truncated
dystrophins to maintain appropriate connections between the cytoskeleton and
extracellular matrix.
30)
Restoration of Wild-Type DMD mRNA in an Immortalized Human
Fibroblast Line from an Exon 8-9 Duplicated DMD Patient
Ling Zhao, Gillian Butler-Browne, Vincent Mouly, Soraya Chaouch,
Kamel Mamchaoui, Steve D. Wilton, Kevin M. Flanigan. Human Genetics, University
of Utah, Salt Lake City, UT; UPMC Université Paris and Institut de Myologie,
Paris, France; University of Western Australia, Perth, Australia; Center for
Gene Therapy, Nationwide Children's Research Institute, Columbus, OH.
Both 2'O-methyl and morpholino antisense oligonucleotides are currently in
trials as a method of altering pre-mRNA splicing in patients with Duchenne
Muscular Dystrophy (DMD). The goal is to convert out-of-frame DMD
mutations into in-frame transcripts, resulting in a milder Becker Muscular
Dystrophy phenotype. DMD duplications account for around 5% of all
patient mutations. Skipping of only the duplicate exons would restore a
wild-type DMD transcript, and be predicted to have an even greater
clinical response than that expected from restoring the reading frame around a
genomic deletion to make a BMD-like dystrophin isoform. To study skipping of
duplicated exons, we developed immortalized fibroblast clonal cell lines derived
from a patient with a tandem duplication of exons 8 and 9 in the DMD gene
(F43401-ht-MyoD) and from a normal control (WT-ht-MyoD). Cells were immortalized
by stable lentiviral transfection with the human telomerase gene, followed by a
tet-inducible MyoD construct; similar cells have been demonstrated to express
dystrophin protein following transplantation into immunodeficient mice (Chaouch
S, Hum Gene Ther. 2009 Jul;20(7):784-90.). The patient cells demonstrated growth
curves similar to wild type. Induction of MyoD expression with doxycycline (2 ug/ml)
resulted in the transdifferentiation of F43401-ht-MyoD fibroblasts into a
myogenic lineage, with 95% of cells expressing desmin at day 4. Five days after
doxycycline treatment, cells were treated with 2'O-methyl modified AONs directed
toward either exon 8, exon 9, or both exons 8 and 9 at four concentrations (25,
50, 100, and 300 nM), and harvested 5 days later for RNA extraction and rtPCR
analysis using primers spanning exons 7 through 10. Treatment with exon 8 AONs
resulted in significant skipping of both exons 8 and 9, with a maximum effect at
100 nM resulting in 86% wild type transcript. AONs directed to either exon 9 or
exons 8 and 9 in combination showed no significant wild type mRNA. However,
significant dystrophin protein expression was not detected in either wild-type
or AON-treated cells maintained up to 12 days post-transdifferentiation.
Diminished DMD mRNA levels in these lines in comparison to primary
myoblasts may explain this paucity of dystrophin expression. Nevertheless, such
clones have several potential benefits as tools for exploring exon skipping or
other therapies. In particular, the robust inducible transdifferentiation avoids
variability between experiments in viral myoD transfection, and thus may
facilitate comparative studies of AON chemistries and sequences. Our data
confirm that skipping of both exons 8 and 9 occurs with the use of only a single
exon 8 AON, as previously found in normal and DMD primary cell lines, and
suggest that skipping of selected DMD exonic duplications may be
titratable in a fashion that allows restoration of wild type DMD mRNA.
31)
Pre-Injection of Polymers Can Increase the In Vivo Transduction
Efficiency of Adeno-Associated Virus Type 2
Gilles Moulay, Sylvie Boutin, Carole Masurier, Daniel Scherman,
Antoine Kichler. Genethon, BP60, Evry Cedex, France; CNRS UMR 8151 – U1022
Inserm, Unité de Pharmacologie Chimique et Génétique et d'Imagerie, Université
Paris Descartes, Chimie Paristech, Paris Cedex, France.
Background The success of muscular dystrophy gene therapy requires
widespread and stable gene delivery. In this context, adeno-associated virus (AAV)
has attracted great attention as an optimal vehicle for body-wide gene delivery.
However, for the successful treatment of a disease such as Duchenne muscular
dystrophy large amounts of recombinant vector are essential. Injection of very
high doses of viral vectors not only raises questions about the technical
feasibility of the large scale production but also about the overall safety of
the approach. One way to overcome both problems would be to find strategies able
to increase the transduction efficiency in vivo. In the present work, we
investigated whether polymers can act as adjuvants to increase the efficiency of
AAV-2 vector after systemic injection. Methods Our strategy consists in
the injection of cationic or anionic polymers before intravenous administration
of Balb/c mice with AAV-2 encoding the reporter gene mSeAP (a murine secreted
alkaline phosphatase). The transduction efficacy was followed by quantifying the
mSeAP in serum at different time points. Vector biodistribution was evaluated in
different tissues by quantification of the mSeAP protein as well as by real time
PCR of the recombinant viral genomes. Results The pre-injection of a
cationic polymer resulted in a 4 to 12-fold increase of seric mSeAP levels.
Histochemical analysis showed the appearance of mSeAP positive muscle fibers
with this strategy when no staining was visible in control AAV-2 injected mice.
The PCR data confirmed an overall increased tissue transduction by AAV-2.
Pre-injection of an anionic polymer resulted in a 2-fold increase of mSeAP
expression in serum. Interestingly, while this latter increase is moderate this
strategy permitted to significantly reduce the neutralizing antibody titer
raised against the AAV-2 capsid. Conclusion Our results show that
strategies of pre-injection of polymers can be used either to improve the
overall transduction of systemically administered AAV-2 or to reduce the humoral
response against the capsid proteins.
32)
Successful Expression of a R16/17 Micro-Dystrophin Gene in Dystrophic
Dogs with a Tyrosine-Mutant AAV Vector and a Brief Course of Immune Suppression
Jin-Hong Shin, Yi Lai, Yongping Yue, Arun Srivastava, Bruce F.
Smith, Dongsheng Duan. Molecular Microbiology and Immunology, University of
Missouri, Columbia, MO; Division of Cellular Biology and Molecular Therapy,
University of Florida, Gainesville, FL; Department of Pathobiology, Auburn
University, Auburn, AL.
Adeno-associated virus (AAV)-mediated micro-dystrophin expression is a promising
strategy to treat Duchenne muscular dystrophy (DMD). Two recent reports
demonstrated micro-dystrophin expression in dog models of DMD with traditional
AAV serotypes. While their results are encouraging, there are several
limitations. First, the micro-dystrophin used in these studies cannot restore
neuronal nitric oxide synthase (nNOS) to the sarcolemma. Second, the
transduction efficiency of traditional AAV serotypes may not meet the clinical
need. Third, a complicated immune suppression scheme was used to prevent immune
rejection. To further improve AAV micro-dystrophin gene therapy, we tested a
novel R16/17 microgene, a tyrosine-mutant AAV-6 vector and an abbreviated immune
suppression protocol in the golden retriever muscular dystrophy (GRMD) model. In
contrast to the previously published microgenes, the R16/17 microgene recovers
membrane-associated nNOS expression and enhances exercise performance. Recent
studies also suggest that mutating surface tyrosine residues can significantly
boost AAV transduction efficiency. We delivered 2.85 x 1012 v.g.
particles of the tyrosine-mutant AAV-6 canine R16/17 microgene vectors to the
forelimb muscles of three GRMD dogs. Immune suppression with oral cyclosporin
and mycophenolate mofetil started at one week before injection and continued for
five weeks. Injection sites were marked with a tattoo dye. Muscle samples were
obtained at 3, 7 and 11 weeks after injection. Robust micro-dystrophin
expression was observed in all the time points. No scene of extensive
inflammatory cell infiltration was observed at any time point. Taken together,
our results suggest that tyrosine-mutant AAV-6-mediated R16/17 microgene
expression may represent a viable approach for DMD gene therapy. Further, a
relatively brief course of immune suppression may suffice the need of
pre-clinical studies in the dog model.
33)
Reverse Perfusion Affords Efficient Whole-Body Gene Transfer to Muscle
in the Large Animal
Frederick Balzer, Jacqueline Farag, Alock Malik, Mihail Petrov,
Andrew Mead, Benjamin Kozyak, Kapil Gopal, Marilyn Mitchell, Connie Choi, Jessie
Chen, David Low, Charles Yarnall, Charles Bridges, Hansell Stedman. Surgery,
University of Pennsylvania School of Medicine, Philadelphia, PA.
* contributed equally to this work Proof of concept studies in small animal
models of severe muscular dystrophy have shown survival benefit following
intraperitoneal or intravenous injection of myotrophic adeno-associated virus (AAV)
vectors of serotypes 1, 6 and 8. Before translating these discoveries into
clinical therapy, a major limitation must be addressed: the vascular systems of
larger terrestrial vertebrates are substantially less permeable than their
homologues in mice, in order to manage the hydrostatic pressure gradients
associated with gravitation, acceleration and changes in posture. This presents
a problem of access, since only after binding to serotype-specific receptors on
the cell surface are AAV particles internalized and transported to the nucleus.
This is especially relevant to the muscular dystrophies, in which diagnosis is
usually made after the young patient has achieved an age of three years and a
standing height of three feet. To approximate this scenario, limb-wide,
vector-mediated gene transfer to striated myocytes in the mature large animal
model has relied upon transient alterations in endothelial permeability
utilizing approaches that require isolation from the systemic circulation.
Rapidly applied post-capillary venular pressures of 50 to 100 torr are needed to
achieve extravasation of vector in the normothermic limb, supporting the
hypothesis that vector transport occurs primarily by way of reversibly induced,
pressure-dependent gaps in this anatomically unique portion of the endothelial
sheet. We further hypothesized that, by carefully manipulating the circulation
and body temperature to protect critical organ systems from acute injury, one
could safely elevate peripheral venous pressure to the range of 80 torr during
vector infusion and achieve efficient, body-wide transport of vector from
vascular space to muscle interstitium in the maturing large animal.
This required development of a novel technology for atraumatic balloon occlusion
of the full length of the aorta and vena cavae, in order to isolate the viscera
and restrict pressure and vector to the more peripheral muscle compartments
during a period of protective deep hypothermia and cardiac arrest. We now report
the evolution of this approach to optimize safety while realizing an average
300-fold enhancement in the efficiency of body-wide AAV1-mediated gene transfer
to striated muscle in the adolescent dog.
34)
Combining Gene and Stem Cell Therapy in the Treatment of
Dysferlinophaties
Daniele Parolini, Claire Navarro, Andrea Farini, Mirella Meregalli,
Marzia Belicchi, Paola Razini, M. Krahn, Louis Garcia, Nicolas Lévis, Yvan
Torrente. Dept. of Neurological Sciences, Università degli Studi di Milano,
Milan, Italy; Laboratoire de Genetique Moleculaire, Hopital d'Enfants de la
Timone, INSERM U910 Genetique Medicale et Genomique Fonctionnelle, Marseille,
France; UMRS787, INSERM/UPMC, Institut de Myologie, Faculté de Médecine Pierre
et Marie Curie, Paris, France.
Mutations in gene encoding dysferlin are involved in two main muscular
dystrophies: Miyoshi myopathy and Limb-Girdle Muscular Dystrophy 2B. Both
diseases are characterized by progressive weakness and skeletal muscle wasting.
Dysferlin is expressed in skeletal and cardiac muscles, where its main function
is membrane repair. So far, no treatment is available and development of
effective therapies remains a big challenge. New hopes are coming from stem cell
and gene therapies. Based on work developed in DMD, we investigated the
feasibility of stem cell engineering by exon-skipping in dysferlinopathies. We
focused our efforts on a patient carrying a deletion in exon 22 found at
heterozygous composite state with a large deletion (Δ25-29) predicted to be
in-frame. According to the absence of protein observed, we supposed a
destabilization and/or degradation of this predicted in-frame truncated mRNA and/or
protein. In this study both exons 22 and 23 needed to be removed to restore the
ORF and allow production of a truncated functional protein. As proof of
principle of the exon-skipping feasibility, we firstly tested if the skipped
dysferlin product can be correctly expressed. To achieve this aim, constructs
encoding deleted forms of dysferlin were transfected into HEK and 3T3 cells. The
truncated dysferlin Δ22-23, mimicking the skipped allele of our patient, was
detected in WB, correctly addressed to the membrane and functional.
Interestingly, also the protein Δ25-29 was produced, suggesting that the absence
of dysferlin in our patient is most probably due to mRNA instability. Finally, a
third deleted form was produced carrying the deletion Δ22-29, suggesting that
all these exons can be removed without affect dysferlin expression. We then
designed antisense oligonucleotides (AONs) able to target acceptor or donor
splice sites as well as ESE sequences of exons 22 and 23. Their ability to skip
exons of interest was tested on human normal myoblasts and circulating CD133
stem cells isolated from the patient. Unfortunately, very low skipping
efficiency was observed and results were not reproducible. To overcome exon
skipping difficulties, we developed a strategy based on complete dysferlin
delivery by lentivirus vector in blood-derived CD133 stem cells isolated from
the same and unskippable patients. The produced vector allowed dysferlin
expression both in vitro, in human fibroblasts and mononucleated cells
from our patients, and in vivo after intramuscular injection of
transduced CD133 stem cells in the scid/blAJ mouse model. We strongly
believe that the combination of gene and stem cell therapy represent a useful
tool for new therapeutic approaches in dysferlinopathies. Moreover, lentivirus
carrying complete dysferlin can be useful to bypass all unskippable
mutations, as those located in the transmembrane domain or other essential parts
of the gene.
35)
Late Gestation Intrauterine Gene Transfer of AAV9 in Non-Human Primates
Preferentially Transduces the Central Nervous System, Liver, Skeletal and
Cardiac Musculature
C. N. Mattar, N. Johana, A. Fisk, A. Biswas, J. Lu, M. H. Tan, L. G.
Tan, A. Rahim, S. Waddington, M. Choolani, J. Chan. Experimental Fetal Medicine
Group, National University of Singapore, Singapore, Singapore; University of
Queensland, Brisbane, Australia; DSO National Laboratories, Singapore, Singapore;
University College, London, United Kingdom; Reproductive Medicine, KK Women's
and Children's Hospital, Singapore, Singapore.
Introduction
Monogeneic diseases resulting in irreversible brain damage are potential
candidates for intrauterine gene transfer. IUGT mediates access to specific
organ compartments not easily targeted in adult recipients and may circumvent a
nullifying immune response to the transgene and vector. AAV9 targets neonatal
neuronal tissues and adult glia when administered systemically, suggesting
different environmental influences affecting vector tropism during development
and post-natal life. Here we examined vector biodistribution and transduction
efficacy, transgene expression and immune response to AAV2/9 following late
gestation IUGT in non-human primates. Materials and methods
M fasicularis females were time-mated and 1x1013 vector genomes of
scAAV2/9-CMV-eGFP were injected via the fetal intrahepatic vein under ultrasound
guidance. Maternal viraemia was determined with qPCR following serial blood
sampling. Selective maternal tissue sampling was performed after delivery of
infants. At predetermined time-points comprehensive necropsies were performed on
injected offspring and transgene expression assayed through stereoscopic
bioimaging, immunohistochemistry and molecular analyses. Results
Two fetuses received 3.3 to 4.0x1013 vg/kg (birth weight) of
scAAV9-GFP at 0.9G. Low-level maternal viraemia was detected between 5 minutes
and 48 hours following IUGT with peak levels at 1.8 and 2.6 vectors/µl plasma.
Offspring were delivered one week later. Despite an increased anti-AAV9 antibody
response in mothers and infants persisting up to 3 months following IUGT, GFP
expression was prominent in the maternal ovarian cortex, mononuclear cells and
skin of both mother and infant sampled at delivery. Varied levels of maternal
tissue transduction were demonstrated at delivery with levels ranging from >300
vg/dg in skeletal muscle to >1800 vg/dg in mononuclear cells. Stereoscopic
fluorescence microscopy at necropsy demonstrated prominent GFP expression in
skeletal muscles, diaphragm, dorsal root ganglia, myocardium and renal cortex.
Multiorgan vector distribution was noted with a wide range of transduction
levels, from <2 copies per cell in the intestines to several-fold higher in the
liver. Immunohistochemistry confirmed the expression of GFP in the central
nervous system, renal cortex, liver, seminiferous tubules and myocardium.
Conclusions
A single dose of AAV9 delivered systemically in late gestation resulted in
widespread vector biodistribution with robust transgene expression in skeletal
muscle, diaphragm, myocardium, kidney, central nervous system and dorsal root
ganglia. Pre-existing maternal immunity to AAV9 did not neutralize transgene
expression in injected offspring. Thus AAV9 may be a useful vector with which to
target such genetic diseases as spinal muscular atrophy, cystic fibrosis and
Duchenne muscular dystrophy.
36)
Monitoring and Suppression of Immune Responses to AAV-Mediated Gene
Therapy in Canine Muscle
Zejing Wang, Rainer Storb, Donghoon Lee, Martin Kushmeric, Baocheng
Chu, Jeffery Chamberlain, Stainly Riddell, Stephen Tapscott. Transplantation
Biology, Fred Hutchinson Cancer Research Center, Seattle, WA; Radiology,
University of Washington, Seattle, WA; Neurology, University of Washington,
Seattle, WA; Immunology, Fred Hutchinson Cancer Research Center, Seattle, WA;
Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA.
The use of Adeno-associated viral (AAV) vectors as a gene delivery vehicle has
shown promise both in preclinical studies and clinical trials for a number of
acquired and inherited diseases, including Duchenne Muscular Dystrophy (DMD). We
previously demonstrated that direct intramuscular injections of rAAV2 or rAAV6
in wild type and DMD dogs resulted in robust T-cell responses to viral capsid
proteins. Others have shown that cellular immunity to AAV capsid proteins
coincided with liver toxicity and elimination of transgene expression in a trial
of human hemophilia B. It has been suggested that activation of T cells after
AAV injection in mice was due to binding of the virion to heparan sulfate
proteoglycans (HSPG), which facilitates its uptake by dendritic cells. Both AAV2
and AAV6 contain HSPG binding motifs. Hence, we compared the immunogenicity of
capsids from AAV6 to those from AAV1, which is a serotype without a
heparin-binding domain, and AAV1-E531K which has an introduced heparin-binding
site. We found that immune responses were elicited independent of HSPG binding.
We also confirmed the utility of ELISpot assays for detecting such responses
with cells derived from peripheral blood. AAV vectors are currently being used
in human trials for intramuscular delivery of transgenes. An important component
of human clinical trials will be non-invasive monitoring of inflammation at
sites of gene delivery. We tested whether magnetic resonance imaging (MRI) could
be used for non-invasive detection of inflammatory reactions to rAAV vectors in
muscle. Studies on dogs injected with different vectors (rAAV6, rAAV1 and
rAAV1-E531K) showed that MRI accurately detected local inflammatory responses
following intramuscular rAAV injection. We previously demonstrated that immune
response to AAV vectors in DMD dogs could be averted by a brief course of
immunosuppression with anti-thymocyte globulin, cyclosporine, and mycophenolate
mofetil, which resulted in long-term and robust expression of transgenes in the
skeletal muscle. In order to reduce toxicities associated with these
immunosuppressants, we have initiated studies of alternative, efficient but less
toxic regimens through T-cell co-stimulation blockade with or without rapamycin.
In conclusion, our studies suggested that heparin-binding ability of a given AAV
serotype did not alter induction of T-cell responses after intramuscular
injection in dogs. Further, the studies validated the utility of ELISpot assays
and MRI for monitoring immune and inflammatory responses to rAAV vectors in dogs.
Therefore, these assays should be part of future human clinical trials of AAV
gene therapy to monitor immune responses. Finally, immunosuppression is probably
required when applying AAV-mediated gene therapies to treat human patients.
37)
Irradiation of Adult mdx Mice Prior to Full-Length Murine
Dystrophin cDNA Transfer Results in a Delayed and Diminished Anti-Dystrophin
Host Immune Response
Saman Eghtesad, Heng Zheng, Hiroyuki Nakai, Michael W. Epperly,
Paula R. Clemens. Neurology, University of Pittsburgh, Pittsburgh, PA;
Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA;
Radiation Oncology, University of Pittsburgh Cancer Institute, Pittsburgh, PA;
Neurology Service, Veterans Affairs Medical Center, Pittsburgh, PA.
Duchenne muscular dystrophy (DMD) is a fatal, genetic disorder in which
dystrophin-deficient muscle progressively degenerates, and for which dystrophin
gene transfer could be an effective treatment. The host immune response to
dystrophin protein, however, is an obstacle to successful therapeutic gene
expression. Understanding the host immune response induced by dystrophin will
facilitate discovery of strategies to prolong expression of recombinant
dystrophin protein in dystrophic muscle. Using whole-body irradiation of the
dystrophic mdx mouse prior to intramuscular high-capacity
adenoviral-mediated gene transfer of a full-length, murine, dystrophin cDNA, we
temporally removed the host immune system. A 600rad dose removed peripheral
immune cells before gene transfer and was followed by self-reconstitution of
immunity. A 900rad dose removed central and peripheral immune cells and was
followed by adoptive transfer of wild-type syngeneic whole bone marrow after
gene transfer. Control mice were either left untreated or received gene transfer
without irradiation. We observed a delayed anti-dystrophin humoral response that
correlated with a decreased, but not complete loss of dystrophin expression in
both 600rad and 900rad irradiated, vector-treated mice. Non-irradiated,
vector-treated mice showed a complete loss of muscle dystrophin expression that
correlated with an earlier anti-dystrophin humoral response and necrotic muscle
fibers surrounded with infiltrating CD4+ and CD8+ T cells.
Although the levels of infiltrating T cells in muscle of vector-treated mdx
mice were similar whether or not the mice were irradiated, the pattern of T cell
infiltration was more dispersed in those mice that were irradiated and did not
appear to be focally attacking individual muscle fibers. With 900rad, but not
600rad, irradiation, we observed a significantly increased number of
infiltrating regulatory T cells in vector-treated muscles. In conclusion, by
studying antigen-specific humoral responses to dystrophin and cellular
infiltration in muscle in the setting of dystrophin gene transfer and temporal
removal of the host immune system, our data support that dystrophin gene
transfer induces an anti-dystrophin immune reaction that is comprised of both
humoral and cell-mediated responses. Immunity induced in the dystrophic host, in
response to dystrophin gene transfer, is associated with loss of
dystrophin-transduced fibers. This immunity is delayed, but not eliminated by
temporal removal of the host central or peripheral immune response. Therefore,
immunity induced by recombinant dystrophin expression will have significant
implications for dystrophin gene therapy for DMD.
38)
Identifying Genes and Signaling Pathways Regulating the Quiescent State
in Muscle Stem (Satellite) Cells
Kristy Boyle, Jeffrey S. Chamberlain. Department of Neurology, The
University of Washington School of Medicine, Seattle, WA.
Duchenne muscular dystrophy (DMD) is an X-linked degenerative disease affecting
approximately 1/3,500 newborn males and is caused by mutations in the dystrophin
(dmd) gene. Loss of dystrophin causes membrane instability of skeletal muscle
fibers resulting in degeneration and subsequent cycles of regeneration within
the musculature. Regeneration is mediated by the resident stem cells, termed
satellite cells, which are located between the external lamina and the
sarcolemma of adult skeletal muscle fibers. Satellite cells are generally
quiescent however, in response to muscle disease, injury, exercise and growth
they become activated, proliferate, and differentiate into post-mitotic
myofibers. As endogenous muscle stem cells, satellite cells are an attractive
population for cell-mediated therapy of DMD however the mechanisms governing the
quiescence, activation and self-renewal of these cells remain to be fully
elucidated. Quiescence is a poorly understood cell state that appears to be
under active control and is not simply a state of inactive transcription.
Understanding the mechanisms that control satellite cell quiescence may provide
key insights into muscle regeneration and enhance the ability to manipulate
these processes in order to further understand the potential for satellite cells
to be used as a cell therapy. To investigate this, we isolated satellite cells
from the hind limb muscles of WT mice by collagenase/dispase digestion and
contaminating CD45+/Mac-1+/Sca-1+ cells were subsequently removed by MACS cell
separation. The resulting population was approximately 80% pure as shown by
immunostaining, and cell cycle analysis revealed that over 80% of the resulting
myogenic cell population was in G0, indicating a high proportion of quiescent
cells in the isolated population. A previously reported microarray study
identified a collection of approximately 500 genes expressed more highly in
quiescent compared to activated satellite cells, which included three
Kruppel-like factors (Klf4, 9, and 15), and the Oncostatin M receptor (OSMR)
(1). By RT-PCR we confirmed that the expression of all three Klf genes was
increased in quiescent satellite cells and that expression subsequently
decreased during a time-course of activation in culture. Furthermore, this
decrease in expression correlated with increasing expression of MyoD, which is
upregulated in activated satellite cells, therefore indicating a potential role
for the Klf genes in the regulation of satellite cell quiescence. Lastly,
exogenous administration of OSM to satellite cells in vitro resulted in retained
expression of the Klf genes over a three day activation period and a concomitant
decrease in MyoD expression, indicating a potential link between OSM signaling
and Klf gene expression. (1) Fukada et al. (2007) Stem cells. 25(10):2448-59
39)
Gene and Cell Therapy Approaches Tto Treating the Dysferlinopathies
Bradley A. Williams, Nilah Monnier, Douglas E. Albrecht, Nupur Garg,
Laura E. Rufibach, Esther Hwang, Angela Salerno, Plavi Mittal. Jain Foundation
Inc., Bellevue, WA.
The dysferlinopathies, including Limb Girdle Muscular Dystrophy 2B (LGMD2B) and
Miyoshi myopathy, are a group of inherited diseases caused by mutations in the
gene encoding dysferlin. These diseases are characterized by progressive
skeletal muscle wasting, typically beginning in the late teenage years. Our
foundation supports targeted research projects that address fundamental
roadblocks and challenges in the use of gene and cell therapy to treat the
dysferlinopathies. Gene therapy is complicated by the fact that the dysferlin
gene is large (roughly 6 kb, above the packaging capacity of AAV vectors) and
that the structural subdomains important for its function have not been
characterized. We are supporting the development of a dual-vector AAV strategy
in which the two halves of the dysferlin gene are delivered independently to
muscle fibers, where they are reassembled by trans-splicing into the full-length
gene. We are also supporting the development of non-viral methods to deliver and
integrate the full-length dysferlin gene into cells, including transfection of
full-length dysferlin and phiC31 integrase into autologous patient-derived
muscle stem cells. In addition, we are supporting structural studies of the
dysferlin protein, which may suggest combinations of subdomains with the
potential to form a functional mini-dysferlin small enough to permit packaging
into a single AAV vector. In the muscle stem cell therapy field, we have found
that a major challenge is the lack of standardized methods for comparing and
contrasting the stem cell populations being studied in different laboratories,
and we are actively promoting efforts to develop these standards. In addition,
we are supporting the development of methods to increase the regeneration
capacity of existing muscle progenitor cells in dysferlinopathy patients, as
well as supporting basic studies on the role of dysferlin in myogenic cells. We
believe that pursuing all of these avenues of research in parallel has the
greatest potential to realize our goal of an approved therapy for
dysferlinopathy patients in the foreseeable future.
40)
In Vivo Reprogramming into the Myogenic Lineage and Improving
Engraftment in the mdx Mouse Model of DMD
Lindsey A. Muir, Jeffrey S. Chamberlain. Neurology, University of
Washington, Seattle, WA.
Duchenne muscular dystrophy (DMD) is characterized in skeletal muscle by cycles
of myofiber necrosis and regeneration leading to loss of muscle fibers and
replacement with fibrotic connective and adipose tissue. Direct lineage specific
reprogramming of patient-derived adult stem cells is a possible approach for
cell therapy aimed at regeneration of muscle. Use of primary fibroblasts for
dystrophin delivery in the mdx4cv mouse model of DMD offers potential advantages
over conventional myoblasts in relative ease of retrieval, expansion, and
culturing, and may be a promising alternative for use in autologous cell
transplantation. We have shown that primary murine fibroblasts transduced by
lentivirus with a tamoxifen-inducible form of the myogenic regulator MyoD [MyoD-ER(T)]
and transplanted into dystrophic muscles enables tamoxifen-dependent
regeneration of myofibers that express micro- and mini-dystrophin. The success
of future translational studies will depend on reproducible methods for cell
delivery and achieving maximal engraftment, which eventually must be explicitly
delineated for a clinical setting. In order to explore whether delivery of
reprogrammed fibroblasts improves function in dystrophic muscle, we are
investigating methods for maximizing engraftment, in terms of both cell survival
and contribution to muscle fibers. We are currently using reprogrammed murine
dermal fibroblasts and a whole muscle mononuclear population to test delivery
methods (volume and number), use of chemical and toxin-based injury protocols,
and myoblast and fibroblast mitogens. We are additionally investigating the role
of the host immune system in engraftment by evaluating differences between
immune-competent and immune-deficient mdx hosts.
41)
AAV Microgene Transfer Reveals the Cellular Motif for
Dystrophin-Mediated Sarcolemmal Neuronal Nitric Oxide Synthase (nNOS)
Localization
Yi Lai, Yongping Yue, Dongsheng Duan. Molecular Microbiology and
Immunology, School of Medicine, University of Missouri, Columbia, MO.
Duchenne muscular dystrophy (DMD) is a severe inherited muscle disease caused by
dystrophin deficiency. Over the last few years, several highly abbreviated
micro-dystrophin genes have been developed for DMD gene therapy. Though
promising, these microgenes remain suboptimal. An important function of
dystrophin is to anchor neuronal nitric oxide synthase (nNOS) to the sarcolemma.
We recently found that dystrophin spectrin-like repeats 16 and 17 (R16/17) are
crucial for membrane-associated nNOS localization. However, the underlying
mechanism is not clear. A better understanding of the nNOS recruiting motif in
dystrophin may guide us develop more effective microgene therapy. Each
dystrophin spectrin-like repeat contains three α-helices. Here we tested the
hypothesis that R16/17-mediated nNOS anchoring depends on the correct α-helix
phasing and composition. To determine the impact of the α-helix phasing, we
sequentially deleted one of the six α-helices of R16/17 in the ΔR2-15/ΔR18-23/ΔC
microgene. Adeno-associated virus (AAV) was used to introduce these microgenes
to dystrophin-null mdx mice. The original ΔR2-15/ΔR18-23/ΔC microgene
efficiently recruited nNOS to the membrane. However, none of the modified
microgenes restored sarcolemmal nNOS. To determine the contribution of α-helix
composition, we swapped each α-helix of R16/17 with the corresponding α-helix of
repeat 18 and performed a yeast-two-hybrid assay. Except for the first α-helix
of repeat 17, replacing other α-helices of R16/17 did not compromise nNOS
interaction. To confirm the in vitro finding, we engineered these chimeric
repeats into the ΔR2-15/ΔR18-23/ΔC microgene and performed AAV gene transfer
study. In contrast to the yeast-two-hybrid result, in vivo screening suggest
that the second, third α-helix of repeat 16 and the first α-helix of repeat 17
are all required for anchoring nNOS. In summary, our results suggest that the
correct phasing and composition are critical for dystrophin-mediated sarcolemmal
nNOS localization. An intact R16/17 is essential for micro-dystrophin based DMD
gene threrapy.
42)
High Force Myosin Correlated with the Early Degeneration of Jaw Closing
Muscle in the GRMD Model of DMD
Andrew F. Mead, Alock Malik, Mihail Petrov, Martin Childers, Marilyn
Mitchell, Janet Bogan, Joe Kornegay, Brittani Nickens, Hansell Stedman.
University of Pennsylvania, Philadelphia, PA; Wake Forest University,
Winston-Salem, NC; University of North Carolina, Chapel Hill, NC.
Duchenne Muscular Dystrophy (DMD) is a disease caused by the deficiency of the
cytoskeletal protein dystrophin, and characterized by the progressive
degeneration of muscle. Dystrophin's normal physiological roles contribute to
membrane stability during forceful contraction and to sarcoplasmic calcium
regulation. In the maintenance of a colony of dystrophin null dogs it was noted
that essentially all animals exhibited progressive trismus within the first year.
This symptom complicates the clinical management of many dogs, often requiring
hand feeding of pureed solids to support skeletal growth and to avoid
accelerated general clinical deterioration. Trismus has not been reported in the
constellation of disease-related problems affecting patients with DMD. We
hypothesized that the discrepancy relates to another genetic difference between
the species: among carnivores and primates, only humans lack the powerful
masticatory muscle (MYH16) myosin because of a frameshifting deletion unique to
the hominid lineage. Recent studies have shown that the MYH16+ muscle fibers of
carnivores develop significantly higher specific force than all other muscle
fibers from the same animals. We further hypothesized that the combined presence
of MYH16 and absence of dystrophin in a single muscle fiber would further
increase the susceptibility to sarcolemmal damage during forceful contracture.
Histological examination of the masticatory and locomotive muscles in the GRMD
model using a novel, epitope-directed antibody against a unique surface loop of
the MYH16 myosin shows that fibers in the temporalis that express MYH16 are
preferentially susceptible to necrosis. Further studies are underway to
precisely correlate the relationships between histological indices of
myonecrosis/apoptosis and the expression of isoforms of myosin and several
proteins involved in calcium signaling and homeostasis
43)
AAV-Mediated SERCA2a Expression Improves the Electrocardiographic
Profile of Aged mdx Mice
Jin-Hong Shin, Brian P. Bostick, Yongping Yue, Roger J. Hajjar,
Dongsheng Duan. Molecular Microbiology and Immunology, University of Missouri,
Columbia, MO; Cardiovascular Research Institute, Mount Sinai School of Medicine,
New York, NY.
Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by
dystrophin deficiency. Dilated cardiomyopathy and subsequent heart failure is a
major cause of death in DMD. Recent studies suggest that AAV-mediated
sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) expression holds great
promise for the treatment of heart failure. Here we hypothesize that
AAV-mediated SERCA2a expression may ameliorate cardiomyopathy in DMD. To test
this hypothesis, we injected AAV9-SERCA2a into 12-month-old female mdx
mice (n=5) via the tail vein. Mdx mice are the most commonly used animal
model for DMD. These mice develop cardiomyopathy characteristic of DMD when they
reach 20 months of age. Eight months after AAV9-SERCA2a administration, we
compared electrocardiographic changes of SERCA2a-treated mice with age- and
sex-matched untreated mdx (n=9) and normal C57BL/10 (n=7) mice. We
observed significant improvement in several ECG parameters (P<0.01,
compared to those of untreated mdx mice). The tachycardia seen in mdx
mice (655 ± 11 bpm) was reversed after SERCA2a treatment (541 ± 18 bpm).
Untreated mdx mice showed characteristic PR interval reduction (normal,
40.7 ± 1.4 msec; mdx, 35.1 ± 0.8 msec) and QT interval prolongation
(normal, 20.8 ± 0.4 msec; mdx, 27.8 ± 0.8 msec). These indices were fully
normalized in SERCA2a-treated mice (PR interval, 41.4 ± 1.5 msec; QT interval,
20.6 ± 1.2 msec). The cardiomyopathy index was also completely normalized
(normal, 0.78 ± 0.07; untreated mdx, 1.11 ± 0.05; SERCA2a-treated mdx,
0.69 ± 0.06). Interestingly, QRS duration and Q wave amplitude were not altered
by SERCA2a therapy. In summary, our results suggest that AAV-mediated SERCA2a
expression improves the electrocardiographic profile in aged mdx mice.
Future hemodynamic studies are warranted to further corroborate the therapeutic
benefit of SERCA2a in end-stage dystrophic heart disease.
44)
Increased Muscle Regeneration in Hindlimb and Diaphragm of mdx
Mice Treated with AAV9 Mini-Dystrophin and Octalysine-NEMO Binding Domain
Peptide
Daniel P. Reay, Geno Raggi, Bing Wang, Xiao Xiao, Paul D. Robbins,
Paula R. Clemens. Neurology, University of Pittsburgh, Pittsburgh, PA;
Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA; Microbiology and
Molecular Genetics, University of Pittsburgh, Pittsburgh, PA; Department of
Veteran's Affairs, Pittsburgh, PA; School of Pharmacy, University of North
Carolina, Chapel Hill, NC.
The loss of a functional, membrane-localized dystrophin protein is the primary
cause of Duchenne muscular dystrophy (DMD). The muscle inflammatory infiltrates
and failure of muscle regeneration that are secondary to dystrophin loss have
recently been shown to be due, in part, to activation of the nuclear factor κB (NF-κB).
Dystrophic muscle shows substantially increased nuclear accumulation of NF-κB,
which is well known to regulate cytokines, other inflammatory molecules and
myogenic proteins. Toward the development of therapy for DMD, we designed an
experiment to test the combination of dystrophin gene replacement and inhibition
of NF-κB activation. For dystrophin gene replacement therapy, we utilized an
AAV9 vector to systemically deliver a mini-dystrophin transgene to 3 day old
neonatal dystrophin-deficient mdx mice. Beginning at 4 weeks of age, mice
were then treated with tri-weekly injections of octalysine-NEMO binding domain
peptide (8K-NBD), to inhibit NF-κB activation. Mice treated with either AAV9
mini-dystrophin vector alone or AAV9 mini-dystrophin vector plus 8K-NBD peptide
exhibited mini-dystrophin expression and reduced necrosis in the quadriceps and
diaphragm, but mice treated with the AAV9 mini-dystrophin vector plus 8K-NBD
peptide additionally demonstrated increased levels of muscle regeneration in
quadriceps and diaphragm. EMSA analysis for NF-κB confirmed decreased levels of
nuclear NF-κB in 8K-NBD-treated mdx mice. Our data suggest that high
levels of mini-dystrophin gene transfer are required to demonstrate a reduction
in the nuclear accumulation of NF-κB in muscle of mdx mice treated with
vector alone. Overall, treatment of mdx mice with AAV9 mini-dystrophin
gene replacement therapy in combination with NBD peptide/NF-κB inhibitory
therapy may provide critical insight into potential treatments for DMD.
45)
A Comparison of Adeno-Associated Virus Mediated Gene Transfer into
Healthy and Dystrophin-Deficient Skeletal Muscles
Gilles Moulay, Carole Masurier, Pascal Bigey, Daniel Scherman,
Antoine Kichler. Genethon, BP60, Evry Cedex, France; Unité de Pharmacologie
Chimique et Génétique et d'Imagerie, CNRS UMR 8151 – U1022 Inserm, Université
Paris Descartes, Chimie Paristech, Paris Cedex, France.
Background
Skeletal muscle is an attractive gene therapy target for several reasons: it is
easily accessible and constitutes about 30% of the normal adult body mass; it
has an abundant blood vascular supply, thus providing an efficient transport
system for the secretion of proteins into the circulation; and it provides a
very permissive physiological environment for adeno-associated virus (AAV)
mediated gene transfer. However, gene therapy in pathologic, in particular
dystrophic muscle may be more difficult than in healthy tissue due to
degenerative-regenerative processes as well as to the inflammatory context.
In the present work, we followed the expression levels of a secreted inhibitor
of the pro-inflammatory TNF cytokine in healthy and dystrophic muscles after AAV-2/6
mediated transduction. Methods
AAV-2/6 vectors encoding chimeric proteins inhibiting TNF-a were injected into
either mdx mice (a mouse model of Duchenne muscular dystrophy in a C57Bl/10
background) or in healthy C57Bl/10 mice. The chimeric proteins that were used
are the human and murine tumor necrosis factor soluble receptor I fused with the
murine heavy immunoglobulin chain (h and mTNFR-Is/mIgG1, respectively). We
conducted an AAV-2/6 dose-response study and we determined the expression
kinetic of the transgenic proteins by ELISA. Further, we followed the antibody
response against the transgene and studied the expression pattern in the
injected muscles by immunostaining. Results
Our two main findings are that: 1- when injecting an equivalent dose, the
transduction efficiency by AAV-2/6 is reduced in dystrophic muscles as compared
to the healthy ones; 2- the humoral response against the transgenic protein is
stronger in the mdx mouse.
Taken together, our results underscore that the inflammatory context of
dystrophic muscles has to be taken into consideration when designing gene
therapy approaches. Not only the levels of expression are lower in the
dystrophic muscle but the inflammatory context favours immune response against
the transgene.
46)
Sarcolemmal nNOS Anchoring Reveals a Functional Difference between
Dystrophin and Utrophin
Yongping Yue, Dejia Li, Luke Judge, Akshay Bareja, Yi Lai, Kay E.
Davies, Jeffrey S. Chamberlain, Dongsheng Duan. Department of Molecular
Microbiology and Immunology, The University of Missouri, Columbia, MO;
Department of Neurology, The University of Washington, Seattle, WA; Department
of Physiology Anatomy and Genetics, University of Oxford, Oxford, United Kingdom.
Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by
dystrophin deficiency. In normal muscle, dystrophin help maintain sarcolemmal
stability by linking the extracellular matrix and the cytoskeleton. Further,
dystrophin recruits neuronal nitric oxide synthase (nNOS) to the sarcolemma.
Reduced sarcolemmal integrity has been considered as a major pathogenic
mechanism in DMD. Interestingly, recent studies suggest that failure to anchor
nNOS to the membrane also contributes to muscle fatigue. Over the last two
decades, a great variety of therapeutic modalities have been explored to treat
DMD. A particularly attractive approach is to increase utrophin expression.
Utrophin shares considerable sequence homology, structural similarity and
functional properties with dystrophin. Here, we test the hypothesis that
utrophin also brings nNOS to the sarcolemma. The full-length utrophin cDNA was
expressed in dystrophin-deficient mdx mice by gutted adenovirus or via
transgenic over-expression. Subcellular nNOS localization was determined by
immunofluorescence staining, in situ nNOS activity staining and microsomal
preparation western blot. Despite supra-physiological utrophin expression, we
did not detect nNOS at the sarcolemma. Our results suggest that full-length
utrophin does not anchor nNOS to the sarcolemma. This finding may have important
implications in developing utrophin-based DMD therapies.
47)
Detailed Physical Mapping of Deletion Breakpoints in the GSHP Dog Model
for DMD
Alock Malik, Acong Xu, Jesse Chen, Andrew Mead, Martin Childers,
Janet Bogan, Scott Schatzberg, Joe Kornegay, Hansell Stedman. University of
Pennsylvania, Philadelphia, PA; Wake Forest University, Winston-Salem, NC;
University of North Carolina, Chapel Hill; University of Georgia, Athens.
The German Short Haired Pointer model for Duchenne muscular dystrophy (GSHPMD)
may provide a unique opportunity to study the immunologically relevant aspects
of gene therapy for DMD as it might provide an unmitigated dystrophin null
environment unlike the other known animal models for DMD (e.g. mdx, GRMD, in
which point mutations result in dystrophin deficiency with the potential for
reversion or read-through by alternative splicing). Although earlier studies
revealed the GSHPMD mutation to be a visible deletion within the p21 region of
the canine X chromosome, the precise boundaries of this deletion and their
relationship to the flanking genes are not yet known. In order to localize the
deletion breakpoints a PCR based strategy was used. A 9Mbp region flanking the
canine dystrophin gene was targeted to strategically design various primers to
amplify the region syntenic to the human Xp21 region. Based on the PCR
amplification profile the deletion boundaries were quickly resolved to within a
few thousand base pairs. Our data suggest that the deletion covers approximately
3Mbp upstream of the 5'UTR and ends very close to the 3'UTR knocking out the
entire dystrophin gene and most likely affecting at least one flanking gene,
BCMP1, as well. We anticipate sequencing across the deletion breakpoint in the
coming weeks, with the opportunity to define the most probable genomic mechanism
resulting in the deletion. Accurate description of the breakpoints of the
deletion will help in early screening and carrier detection in GSHPMD colony,
and will allow a precise depiction of the exons removed. The GSHPMD model may
play a key translational role in the future development of therapies for DMD
because of the anticipated complete absence of any dystrophin open reading frame
and hence utility as a surrogate, from the standpoint of potential immunological
response to recombinant dystrophin, for all DMD gene deletions found in the
human gene pool. This model may also represent a unique opportunity to define
the physiological role of the highly conserved “brain cell membrane protein 1” (BCMP1),
a highly expressed, brain-specific, putative four-transmembrane protein
previously suggested to be a candidate gene for X-linked mental retardation.
48)
Pre-Existing Antibodies to AAV8 Attenuates Micro-Dystrophin Expression
Following Targeted Vascular Delivery
Louise R. Rodino-Klapac, Chrystal Montgomery, William Bremer, Nancy
Davis, Kimberly Shontz, Vinod Malik, Katherine Campbell, Thomas J. Preston,
Zarife Sahenk, K. Reed Clark, Brian D. Coley, Christopher M. Walker, Jerry R.
Mendell, Louis G. Chicoine. Center For Gene Therapy, The Research Institute at
Nationwide Children's Hospital, Columbus, OH; Pediatrics, The Ohio State
University, Columbus, OH; Center for Vaccines and Immunity, The Research
Institute at Nationwide Children's Hospital, Columbus, OH; Heart Center,
Nationwide Children's Hospital, Columbus, OH.
Duchenne muscular dystrophy (DMD) is the most common muscle disease of childhood
and is caused by mutations within the dystrophin gene. Thus it is potentially
amenable to gene therapy. Several gene replacement strategies are under
investigation and progress towards clinical gene therapy with adeno-associated
virus (AAV) delivered transgenes remains promising. An impediment to successful
vascular gene therapy is the presence of pre-existing antibodies that
cross-react with various AAV serotypes. We tested the hypothesis that
pre-existing antibodies to AAV8 would attenuate transgene expression following
vascular delivery of a rAAV8.micro-dystrophin vector to the gastrocnemius of
non-human primates. Rhesus macaques (n=6) were stratified into two groups based
on AAV8 binding antibody sero-status (plus or minus). Animals were intubated and
placed under isoflurane anesthesia and the femoral artery was isolated and
cannulated with the goal of targeting the gastrocnemius muscle for vascular
delivery of vector. Prior to vector administration, a prevector flush of saline
(2.5 ml/kg) was given over 1 minute. This was immediately followed by occluding
blood flow to the extremity using proximal and distal tourniquets to
compartmentalize the gastrocnemius. rAAV8.MCK.micro-dys.FLAG, was infused over 1
minute at a dose of 2 × 1012 vg/kg in 2.5 ml/kg of Tris buffered
saline. The extremity remained isolated from the circulation for 10 minutes. A
postvector flush (2.5 ml/kg) was infused over 1 minute and then the tourniquets
were released. Bleeding was controlled by direct pressure and the wound was
surgically closed. None of the animals suffered noticeable edema or adverse
effects from the procedure. Three months after transfer, the macaques were
euthanized, and the gastrocnemius muscle harvested. Samples of proximal, central
and distal muscle were stained with anti-FLAG antibody and the percentages of
muscle fibers expressing the transgene were measured. The contralateral
gastrocnemius served as a negative control. Efficient micro-dystrophin
expression was visualized in all six subjects. However, subjects without
pre-existing AAV8 antibodies demonstrated more transgene expression compared to
subjects with pre-existing antibodies (% positive muscle fibers: Proximal 71.3 ±
3.5; Central 82.0 ± 4.0; Distal 71.0 ± 6.5 vs. Proximal 26.0 ± 3.2; Central 35.7
± 1.8; Distal 29.3 ± 0.3, respectively; P ≤ 0.003). From these data we conclude
that the presence of pre-existing binding antibodies attenuate transgene
expression. We speculate that immune modulation of patients with plasmapheresis
may enhance transgene expression in cases where pre-existing vector antibodies
are present.
49)
Regulatory Cassettes Derived from the Slow Troponin I Gene Confer
High-Level Expression in Skeletal Muscle after Gene Transfer Mediated by
Helper-Dependent Adenovirus and Recombinant AAV
Rénald Gilbert, Mehdi Bendjelloul, Yué Zeng, Claude Guérin, Nancy
Larochelle, George Karpati, Bernard Massie, Josephine Nalbantoglu. Genomics &
Gene Therapy Vectors, Biotechnology Research Institute, Montreal, QC, Canada;
Neuromuscular Research Group, Montreal Neurological Institute, Montreal, QC,
Canada; Département de Microbiologie et Immunologie, Université de Montréal,
Montreal, QC, Canada.
Viral vectors used for gene replacement therapy of muscle diseases, such as
Duchenne muscular dystrophy, require regulatory elements that can confer strong
and muscle-specific expression of the therapeutic gene product. To achieve this
goal, we generated regulatory cassettes by linking three (ΔUSEx3) or four (ΔUSEx4)
copies of the truncated 60-pb upstream enhancer (ΔUSE) of human slow troponin I
gene. ΔUSE has been reported to drive pan-muscle specific expression in
transgenic mice. In addition, we have previously shown, using naked DNA, that ΔUSEx3
confers strong and skeletal muscle-specific expression in cell culture and in
vivo. To evaluate the activity of ΔUSEx3 and ΔUSEx4 in the context of viral
vectors, we constructed helper-dependent adenovirus (HD) expressing
β-galactosidase (β-gal) regulated by ΔUSEx3, ΔUSEx4, or by the hybrid CMV
enhancer/β-actin (CB) promoter. We also constructed recombinant AAV vectors
expressing the green fluorescent protein (GFP) regulated by ΔUSEx4 or CMV. The
β-gal activity of HD-ΔUSEx3(β) and HD-ΔUSEx4(β) was 1% of HD-CB(β) in cultured
non-muscle cells and 20 to 90% in differentiated myotubes. After intramuscular
injection of normal and mdx mouse muscle, the number of transduced fibers
and the β-gal activity of HD-ΔUSEx3(β) and HD-ΔUSEx4(β) corresponded to 20 to
80% of the value obtained with HD-CB(β). Notably, the number of transduced
fibres after intramuscular injection of AAV-ΔUSEx4(GFP) in adult mdx
muscle was similar to the value obtained under the same conditions using AAV-CMV(GFP).
In summary, the strength, muscle specificity and small size of ΔUSEx3 and ΔUSEx4
render them very attractive for gene transfer applications in skeletal muscle.
50)
Blocking the Myostatin Signal Improves the Success of Human Myoblast
Transplantation in Dystrophic Mice
Raouia Fakhfakh, Annick Michaud, Jacques P. Tremblay. Human
Genetics Unit, Chul-Chuq, Quebec, QC, Canada.
Duchenne muscular dystrophy (DMD) is a recessive disease caused by a dystrophin
gene mutation. Myoblast transplantation permits to introduce the dystrophin gene
in dystrophic muscle fibers. However, the success of this approach is reduced by
the short duration of the regeneration following the transplantation, which
reduces the number of hybrid fibers. Myostatin is a negative regulator of
skeletal-muscle development and responsible for limiting regeneration. It binds
with high affinity to the ActRIIB, which initiates signaling through a smad2/3-dependent
pathway. Our aim was to verify whether the success of the myoblast
transplantation is enhanced by blocking the myostatin signal with expression of
dominant negatif mutants of activin type IIB receptor, (dnActRIIB). In vitro,
human myoblasts is infected with a lentivirus carrying or not dnActRIIB.
Increase proliferation and fusion of dnActRIIB myoblastes is the result of
blocking myostatin activity. This inhibition regulates the expression of
myogenic regulatory factors (MRLFs). In vivo, dnActRIIB myoblastes was
transplanted in immunodifiscient dystrophic mice (Rag/mdx). Dystrophin
immunostaining in the tissue cross-sections of tibialis anterior muscles of RAG/mdx
mice 3 weeks post-transplantation revealed more human dystrophin-positive
myofibers with dnActRIIB myoblasts than controls. Blocking the myostatine signal
allowed the success of myoblast transplantation to improve, the fusion and the
proliferation of myoblastes to increase and regulat expression of myogenic
regulator factors.