TERAPIA GÊNICA NA DISTROFIA MUSCULAR - RESUMOS QUE SERÃO APRESENTADOS NO 10o ENCONTRO ANUAL DA SOCIEDADE AMERICANA DE TERAPIA GÊNICA - DE 30 DE MAIO A 3 DE JUNHO EM SEATTLE, USA
1)
Antisense Oligonucleotide Induced Exon Skipping as a Therapy for Duchenne
Muscular Dystrophy
Steve D. Wilton, Abbie A. Fall, Penny M.
Harding, Patrick Iversen, Susan Fletcher Centre for Neuromuscular and
Neurological Disorders, University of Western Australia, Perth, Western
Australia, Australia; Research and Development, AVI Biopharma, Corvallis, OR
Antisense oligonucleotides (AOs) can be used to re-direct normal splicing
patterns of a pre-mRNA in order to excise selected exons from the mature mRNA.
We have been developing AO induced targeted exon skipping as a potential therapy
for Duchenne muscular dystrophy (DMD), the most common severe form of childhood
muscle wasting. The huge dystrophin gene (79 exons spanning 2.4 Mb) encodes a
protein that links the actin cytoskeleton and a complex of proteins embedded in
the sarcolemma. Protein-truncating mutations in the dystrophin gene result in a
non-functional protein, with subsequent loss of structural integrity of the
muscle fibres. An X-linked disease, affected males show signs of muscle weakness
from 3-5 years of age and are restricted to a wheelchair by age 12 years. Becker
muscular dystrophy (BMD) also arises from dystrophin mutations, but these are
typically in-frame, and allow synthesis of an internally deleted protein, which
can be of near normal function. Some BMD patients are asymptomatic and diagnosis
may only be made later in life, despite missing substantial regions of the
dystrophin gene. We have developed a panel of AOs that can induce skipping of
each exon of the predominant muscle specific isoform. Forty one of the 77 exons
targeted for excision could be removed with high efficiency with single AOs,
although no obvious pre-mRNA motif was consistently targeted. The remaining
exons were more stubborn and were excised at lower efficiencies, despite many
AOs being designed and evaluated. One approach found to induce very efficient
skipping of these recalcitrant exons was the use of select AO combinations.
Consisting of AOs that were often inactive when used individually, some AO
cocktails could induce efficient exon skipping after in vitro transfection in
the nanomolar range. AO design and delivery are both crucial aspects of
developing a viable therapy. We have observed that trends in exon skipping
efficiency induced by AOs composed of 2'-O-methyl
modified bases on phosphorothioate backbone (2OMeAOs) were also seen when the
corresponding sequences were applied as phosphorodiamidate morpholino oligomers
(PMOs). That is, the design of 2OMeAOs can be extrapolated to the morpholino
chemistry. However, the more robust PMOs seem much better suited to in vivo
applications as shown by substantial systemic dystrophin expression being
induced in a mouse model of muscular dystrophy after repeated weekly
intraperitoneal administration of a PMO coupled to a cell penetrating peptide
tag at a dose of 5 mg/kg. PMOs have already entered clinical trials for other
conditions. A clinical trial is scheduled to commence in the UK in 2007 using a
PMO directed at dystrophin exon 51. This compound should restore the reading
frame in DMD cases arising from responsive dystrophin mutations occurring in the
major deletion hotspot. The PMO will be delivered by an intramuscular injection,
and if safety concerns are met and proof-of-principle demonstrated in human
dystrophic muscle, systemic administration will follow as soon as possible.
2)
Prolonged AAV-Mediated Dystrophin Expression in a Canine Model of Duchenne
Muscular Dystrophy with a Brief Course of Immunosuppression
Zejing Wang, Christian S. Kuhr, James M.
Allen, Eric Finn, Paul Gregorevic, Jeffrey S. Chamberlain, Stephen J. Tapscott,
Rainer F. Storb Transplantation Biology, Fred Hutchinson Cancer Research Center,
Seattle, WA; Neurology, University of Washington, Seattle, WA; Human Biology,
Fred Hutchinson Cancer Research Center, Seattle, WA
Introduction: Duchenne muscular dystrophy (DMD) is caused by mutations in
the dystrophin gene. Studies in the mdx mouse model of DMD have shown that
muscle membrane integrity and function can be improved by AAV-mediated delivery
of a functional dystrophin protein. Our previous studies have demonstrated that
direct intramuscular injection of AAV2 or AAV6 in wild-type random bred dogs
resulted in a robust immune response to capsid, or capsid-associated proteins.
Recent evidence of immune-mediated loss of AAV vector persistence in human
trials also suggests that immune modulation might be necessary to achieve
successful long-term transgene expression. In this study, we assessed the
potential of various immunosuppression regimens in prolonging AAV-mediated
transgene expression in both random-bred normal dogs and in dogs with muscular
dystrophy caused by a dystrophin mutation (cxmd dogs). Methods:
AAV serotype 6 carrying different promoter-transgene cassettes were produced as
previously described for murine and canine studies. Direct intramuscular
injections of vectors at 1x1011 vector genomes per site in a total
volume of 250 l
were performed. Different immunosuppression regimens were utilized between 4 to
18 weeks. The injection sites were biopsied under anesthesia between 4 and 30
weeks after injection for histology, immune responses and transgene expression
analysis. Results: Daily immunosuppression with cyclosporine (CSP) and
mycophenolate mofetil (MMF) largely prevented the immune response to
AAV6-CMV-LacZ in a normal dog for up to four weeks and permitted robust
transgene expression. The same immunosuppressive regimen did not as completely
prevent an immune response to AAV6-CMV-cFIX or AAV6-CMV-human-micro-dystrophin
(h--dys)
in a cxmd dog, suggesting that a more aggressive immunosuppressive
regimen might be necessary. A brief course of five days anti-thymocyte globulin
(ATG) treatment was given to cxmd dogs injected with either AAV6-CMV- h--dys
or AAV6-CMV-canine micro-dystrophin (c--dys)
to deplete T lymphocyte, and followed by daily use of both CSP and MMF. This
regimen successfully prevented the immune responses and allowed long-term
expression of transgenes in dystrophic muscles. Conclusions: Taken
together, our results suggest that the combination of CSP and MMF effectively
prevents the immune responses in a normal dog, while a more potent regimen with
the combination of ATG, CSP and MMF is required in cxmd dogs, possibly
due to the pre-existing inflammatory nature of the DMD muscle disease. The
latter immunosuppression regimen is sufficient to permit long-term and robust
expression of a c--dys
transgene in the skeletal muscles of a cxmd dog, and its expression
restored localization of components of the dystrophin-glycoprotein complex at
the muscle membrane. This protocol has potential applications to human clinical
trials to enhance AAV-mediated gene therapies for various human diseases.
3) A
Clinically Relevant Gene Therapy Approach for Duchenne Muscular Dystrophy by
Vascular Delivery of Micro-Dystrophin
Louise R. Rodino-Klapac, Paul M. Janssen,
Chyrstal L. Montgomery, Ryan Jensen, Louis G. Chicoine, K. Reed Clark, Jerry R.
Mendell Center for Gene Therapy, Columbus Childrens
Research Institute, Columbus, OH; Physiology and Cell Biology, The Ohio State
University, Columbus, OH
Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder with
monogenic mutations setting the stage for successful gene therapy treatment.
Long-term therapeutic goals include systemic delivery of a small dystrophin
transgene, micro-dystrophin, delivered by adeno-associated virus (AAV) via the
vasculature. Current and past clinical trials have limited gene delivery to
direct intramuscular injection. As we proceed toward our goal of widespread
muscle transduction, we anticipate progress in a stepwise fashion, where the
transgene is delivered by selective catheterization to branches of the femoral
artery. This approach of isolated lower limb perfusion (ILP) permits a clinical
trial for muscular dystrophy assessing both safety and efficacy as for the
following reasons: 1) selective delivery of vector to lower limb muscles can
produce clinically meaningful results; 2) the lower limb can be
compartmentalized to prevent spread of virus to other organ systems providing an
important measure of safety; 3) delivery of virus in a compartmentalized system
provides safe passage for the virus since pre-existing immunity to AAV may
preclude muscle transduction. We first tested ILP of micro-dystrophin in the mdx
mouse with the goal of comparing the efficiency of AAV serotypes [AAV1, 6, or
AAV8] in crossing the vascular barrier leading to widespread gene expression in
muscle in a manner different from previous studies, not relying on excessive
pressure, volume, or pharmacologic agents to combat vascular resistance. A
micro-dystrophin construct was used with features previously described by Harper
et al (2002), deleting the untranslated regions and C-terminus, retaining
spectrin repeats 1-3 and 24, and hinges 1, 2, and 4 of full-length dystrophin,
under control of a muscle specific promoter, and the addition of an intron to
enhance gene expression. Comparative studies conclude that AAV6 and AAV8 deliver
and transduce micro-dystrophin by ILP more efficiently than AAV1, with micro-dystrophin
levels > 85% for both serotypes. Functional significance was established in
extensor digitorum longus (EDL) by demonstrating increased maximum force
generation and protection against eccentric contractions (P< 0.05).
Extending these studies to non-human primate; we have successfully translated
ILP vascular delivery using AAV8 to cross the endothelial barrier of muscle
vasculature by specifically targeting the lower limb muscles with a fluoroscopy
guided catheter to deliver AAV8.GFP (green fluorescent protein). These findings
and ongoing studies set the stage for a future clinical trial in DMD patients
with vascular delivery of the micro-dystrophin transgene.
4)
Independent Canine Models of Duchenne Muscular Dystrophy Due to Intronic
Insertions of Repetitive DNA
Bruce F. Smith, Joe N. Kornegay, Dongsheng
Duan Scott-Ritchey Research Center, Auburn University, Auburn, AL; School of
Medicine, University of North Carolina
-Chapel
Hill, Chapel Hill, NC; School of Medicine, University of Missouri, Columbia, MO
Duchenne muscular dystrophy (DMD) is the most common X-linked disease and
inherited myopathy of humans. As such, development of effective gene therapy for
DMD has been and continues to be a high priority. A number of animal models have
allowed the testing of novel approaches in order to determine their validity
prior to application to human patients. Of these, the canine model most closely
recapitulates the clinical presentation, immune system responses and body mass
of human patients. Several canine mutations have been identified, although the
Golden Retriever remains the best characterized and therefore most commonly
employed model. Additional canine models would further improve the utility of
the dog as a model system as these models would mimic the variety of challenges
seen with human patients, including variable transcription, the presence or
absence of epitopes and the effect of residual mutant or
"revertant"
protein. We have characterized two additional canine models of DMD at the level
of histology, morphology and molecula"
basis".
These models were identified in the Labrador Retriever and Welsh Corgi breeds.
In both cases, affected animals can be identified at birth by elevated creatine
kinase levels. Both Welsh Corgis and Labrador Retrievers have a relentlessly
progressive and ultimately fatal course of disease . Both models are dystrophin
deficient except for rare revertant fibers. They display prominent skeletal
muscle pathology identical to these found in human patients such as variable
fiber size, central nucleation, fiber splitting, fatty infiltration, macrophage
infiltration, fibrosis and calcification. We have identified the molecular basis
of each model as the inclusion of repetitive sequence elements creating novel
exons in the cDNA. In both cases these elements have been inserted into introns
(intron 13 for the Welsh Corgi and intron 19 for the Labrador Retriever),
activating splice acceptor sites already present in the normal intron sequence.
In both cases, the inserted sequences contain in-frame stop codons leading to
early termination of translation. The characterization of the mutations and
morphologic features of each of these new models provides the information
required to employ these models in gene therapy studies. These models provide
important alternatives to the available models and they will broaden our
understanding of how relevant approaches will function in the face of different
mutations and clinical manifestations in the human population.
5) A
Myoblast Expansion System for Improving the Efficiency of Autologous Stem Cell
Therapy To Treat Muscular Dystrophy
Sheng Li, Brent Fall, Miki Haraguchi,
Jeffrey S. Chamberlain Senator Paul D Wellstone Muscular Dystrophy Co-operative
Research Center, Department of Neurology, The University of Washington School of
Medicine, Seattle, WA
Autologous stem cell-based transplantation is a promising approach to treating
inherited muscle disorders. A successful strategy would be facilitated by
regimes that allow for expansion both in vitro and in vivo of
cells with myogenic potential. A previous study demonstrated that a chemical
inducible dimerizer (CID), AP20187, could maintain in vitro proliferation
of myoblasts expressing F36VFGFR-1, a chimeric protein composed of the
cytoplasmic phosphorylation domain of fibroblast growth factor receptor 1
(FGFR-1) and a mutated dimerization domain of FK506 binding protein (F36V).
Here, we generated a lentiviral vector carrying a bicistronic expression
cassette composed of a microdystrophin/GFP fusion gene and the F36VFGFR-1 gene
under the control of a synthetic muscle-specific promoter. The mdx
myoblasts transduced with this vector were prevented from differentiating and
were expanded in culture medium containing AP20187, but lacking FGF-2. When
intramuscularly transplanted into mdx tibialis anterior muscles, these
AP20187-expanded cells formed large clusters of myofibers expressing
microdystrophin/GFP and the developmental isoform of myosin heavy chain.
Although CID-expanded myoblasts differentiated to form myotubes and myofibers in
skeletal muscle, we found that in culture, residual AP20187 retained in those
CID-expanded cells dramatically delayed myotube formation. These observations
imply that exogenous control of FGFR-1 activation in myoblasts can be used to
prevent terminal differentiation and enable large-scale expansion of myogenic
precursors, potentially increasing the efficiency of myoblast engraftment in
muscle disorders.
6)
Gene Therapy for Duchenne Muscular Dystrophy by the Helper-Dependent Adenovirus
Vector (HDAdv) -Mediated
Full-Length Dystrophin Expression
Masatoshi Ishizaki, Ryoko Kawano, Yuji
Uchida, En Kimura, Makoto Uchino, Yasushi Maeda Neurology, Graduate School of
Medical Sciences, Kumamoto University, Kumamoto, Japan
Backgrouud Duchenne muscular dystrophy (DMD) is a progressive muscle wasting
disorder caused by the absense of dystrophin. Among various obstacles against
the DMD gene therapy, the huge cDNA size, 14kb, limits the use of many kinds of
virus-based vectors. We have generated a helper-dependent adenovirus vector,
which has a cloning capacity of up to 37kb, that carried myc-tagged murine
full-length dystrophin cDNA. In this study we evaluated the therapeutic effect
of the HDAd-mediated full-length dystrophin gene transfer into mdx mice and
utrophin/dystrophin double knockout mice (dko mice), which are severely
dystrophic mice. Method and Results [We have constructed HDAd vector contained
the murine full-length dystrophin expression cassette and myc-tag,integral
protein (HDAdv-mFLmyc-dys). (1) Gene delivery by multiple intramuscular
injection into neonatal dko mice Each 7-day-old dko mice were injected with the
HDAdv-mFLmyc-dys into the following muscle groups: lower limbs, upper limbs and
lattissimus dorsi. The transgene was widely expressed and prevented the
dystrophic changes pathologically and physiologically in injected dko mice. We
observed the restoration of dystrophin and dystrophin associated proteins, and
nNOS. Furthermore, motor performance in injected dko mice could be improved and
their lifespan became longer. (2) Systemic gene delivery intravenously in young
adult mdx mice Young adult mdx mice were injected with the HDAdv-mFLmyc-dys via
the tail vein. Body-wide expression of full-length dystrophin expression was
detected in the skeletal muscle and diaphragm. Conclusion These results offer a
hopeful prospect for DMD therapy. Therapeutic gene transfer with HDAd may
ameliorate DMD patients.
7)
Inhibition of Myostatin by Gene Therapy Increases Muscle Mass and Strength in a
Mouse Model of Muscular Dystrophy
Amanda M. Haidet, Liza Rizo, Chalonda R.
Handy, Chris J. Shilling, Zarife Sahenk, Jerry R. Mendell, Brian K. Kaspar
Integrated Biomedical Science Graduate Program, The Ohio State University,
Columbus, OH; Center for Gene Therapy, Columbus Childrens
Research Institute, Columbus, OH; Department of Pediatrics, The Ohio State
University, Columbus, OH
Increasing the size and strength of muscles represents a promising therapeutic
strategy for musculoskeletal disorders. Significant interest has focused on
myostatin, a negative regulatory factor of muscle growth. Inhibition of
myostatin significantly increases muscle mass. Several proteins including
follistatin, follistatin-related gene (FLRG) and growth and differentiation
factor-associated serum protein 1 (GASP-1) inhibit myostatin. We have cloned the
genes expressing these proteins into Adeno-associated viral (AAV) vectors.
Administering AAV1 at a single time point by intramuscular injection provides a
long-lasting therapeutic benefit. Wild-type mice injected in the hindlimbs with
1 x 1011 viral particles of AAV1-Follistatin, FLRG, or GASP-1 have
shown increased overall body mass, with a subsequent 30% increase in hindlimb
and forelimb grip strength compared to AAV1-GFP treated controls. We have
evaluated these animals over 1.5 years following gene injection and have found
no untoward effects due to post-natal gene delivery of these muscle-enhancing
factors. The overall strength increase was greatest for animals receiving
AAV1-Follistatin followed by AAV1-FLRG and then AAV1-GASP-1. We next tested the
potential of AAV1-Follistatin to increase muscle mass and strength and delay
muscle deterioration in the mdx mouse model of Duchenne muscular dystrophy (DMD).
DMD is an X-linked recessive disease resulting in the wasting of skeletal
muscles and cardiac function, ultimately resulting in death. Mdx animals were
injected bilaterally in the hindlimbs with AAV1-Follistatin at 3 weeks of age
and followed for 6 months before sacrifice. These animals showed a 30-50%
increase in skeletal muscle mass, enhanced muscle grip strength, and decreased
serum creatine kinase levels compared to AAV1-GFP controls. These findings
indicate that follistatin slows or delays muscle damage. Histological analysis
of AAV1-Follistatin treated muscles demonstrated myofiber hypertrophy at the
local site of injection and at remote sites, including paraspinal and upper limb
muscles reinforcing conclusions that circulating follistatin had biological
efficacy. No adverse organ pathology was detected and animals were capable of
normal reproduction in preliminary studies. Furthermore, mdx animals treated
with AAV1-Follistatin at 6.5 months of age also showed increased muscle
strength, demonstrating the ability of follistatin to improve strength in aged
animals. These results suggest that inhibition of myostatin by myostatin
inhibitory proteins, in particular follistatin, delivered by viral mediated gene
therapy represents a promising therapeutic strategy that warrants consideration
for clinical trials in human muscle diseases.
8)
In Vitro Identification
of Optimal Exon-Skip Targets for Treatment of Duchenne Muscular Dystrophy Using
a Human Muscle Cell Line
Nathaniel A. Walton, Clifford J. Beall,
Jerry R. Mendell, K. Reed Clark Center for Gene Therapy, Columbus Childrens
Research Institute, Columbus, OH; Department of Pediatrics, The Ohio State
University, Columbus, OH
Introduction: DMD is a chronic, debilitating disease of childhood caused
by the absence of dystrophin. Muscle biopsies from at least half of dystrophin
deficient patients show revertant myofibers expressing the mutant protein. This
is likely related to second-site mutations that restore the reading frame or
possibly create alternative or cryptic splice-sites. Clinical trials are
underway to exploit similar mechanisms to skip exons and restore the dystrophin
reading frame in DMD patients using anti-sense oligonucleotides (AONs) and
morpholinos. While this method holds promise, the potential need for
re-administration and excessive production costs keeps enthusiasm in check. A
potential option to achieve the same goal is through the use of modified snRNA
genes with anti-sense exon/intron sequences delivered by adeno-associated virus
(AAV) to disrupt spliceosomal recognition of the targeted exon during pre-mRNA
processing. In making such a system clinically applicable to as many patients as
possible, we have developed a system to identify potential anti-sense targets
through recognition of exon splice enhancer (ESE) motifs. To achieve this goal,
we have established a myocyte cell line derived from a patient with an exon 33
stop codon mutation. We describe a matrix assay to screen potential anti-sense
targets in tandem that results in enhanced dual-target snRNA vectors.
Methods: AONs (2-O-methyl
phosphothioate) were designed to target multiple ESE motifs using the ESE finder
program. Myotube cultures were differentiated using serum starvation and
incubation at 37o. AONs (10 nM - 200 nM) were introduced by
transfection using Lipofectin and total RNA isolated 24 hr post-transfection.
RT-PCR (100 ng of template) was performed using dystrophin specific primers
located in exons 31 and 35. The full-length amplicon was 626 bp, while mRNA with
exon 33 skipped yielded a 470 bp amplicon. Results: Six AONs were
synthesized that were predicted to block recognition of 2 or more contiguous ESE
binding motifs. AONs were transfected alone or in tandem at several final
concentrations (12.5, 25 and 50 nM) into mature (6 days post-differentiation)
myotube cultures. AON matrix analysis using RT-PCR revealed that individual AONs
were not as efficient (based on minimal titration end-point) in producing
skipped transcripts as several dual AON combinations. Currently, we are
evaluating the optimal AONs combinations in dual-target snRNA AAV vectors in
this culture system. Discussion: Specific targeting with AONs to regions
within exons should increase specificity by avoiding canonical splice donor and
acceptor sites. We identified several active anti-sense target regions that when
combined in tandem allowed activity down in the low nanomolar range (12.5 nM).
Our data suggest that targeting two regions within an exon facilitates
disruption of spliceosomal recognition that can be incorporated into AAV vectors
for sustained delivery of anti-sense sequences at doses within the expectation
of a clinical trial needed to achieve exon exclusion and possible therapeutic
efficacy.
9)
Gait in BioTO-2 and Bio14.6 Dystrophic Hamsters
Thomas G. Hampton, Ivo Amende, Ajit Kale,
Scott Mccue, Hemmi N. Bhagavan, Anton H. M. Terpstra, Case VanDongen R
D,
Biobreeders Inc., WaterTown, MA; R
&D,
MouseSpecifics Inc., Boston, MA
The delta-sarcoglycan-deficient hamster strains BIO 14.6 and BIO TO2 are
excellent models to study muscular dystrophy and the efficacy of gene therapy.
Gait disturbances, important clinically, have not yet been described in these
hamster models. Accordingly, we compared the gait of BIO 14.6 (n=12) and BIO TO2
(n=12) dystrophic hamsters to healthy BIO F1B (n=12) control hamsters. We used
ventral plane videography to determine gait indices in 3-mo and 9-mo old male
BIO 14.6, BIO TO2, and BIO F1B hamsters walking on a transparent treadmill belt
at 16 cm/s. Gait indices were based on ~10
consecutive strides for each of the 4 limbs. We also studied 1-mo old BIO TO2
(n=4) and F1B (n=4) hamsters and found kinematic and postural changes in both
BIO 14.6 and BIO TO2 hamsters, including significantly shorter swing, stride,
and stance durations. Stride length was
~13% shorter (P<0.05) in
BIO 14.6 and BIO TO2 dystrophic hamsters at 3-mo and 9-mo of age compared to BIO
F1B hamsters. Propulsion duration of the hind limbs, an indicator of muscle
strength, was shorter in 9-months BIO 14.6 (236
+14
ms) and BIO TO2 hamsters (244
+8
ms) compared to BIO F1B (303
+11
ms; P<0.05). Braking duration, reflecting generation of ground reaction
forces, was delayed in 9-mo old BIO14.6 (142
+7
ms) and BIO TO2 hamsters (185
+9
ms) compared to BIO F1B (112
+14
ms; P<0.05). Hind paw eversion, evidence of muscle weakness, was greater
in 9-mo old BIO TO2 than in BIO F1B hamsters (20.1
+1.1o
vs. 6.7 +1.7o;
P<0.05). The propulsive deficit was apparent in BIO 14.6 and BIO TO2 at
3-mo and in BIO TO2 animals at 1-mo of age. Our quantitative analysis of gait
demonstrates gait disturbances in hamsters with muscular dystrophy that commence
as early as 1-mo of age. Our findings show that these dystrophic BIO hamsters
recapitulate the functional features of human muscular dystrophy. Early
detection and quantitative measures of these gait abnormalities will accelerate
the development of gene therapy for treating muscular dystrophy.
10)
Retrograde Axonal Transport of AAV Vectors Allows Extended Restoration of
Dystrophin Expression by Exon Skipping in mdx Mice Brain
Elise Peltekian, Ros Carine, Carole
Gruszczynski, Cyrille Vaillend, Serge Laroche, Luis Garcia, Olivier Danos
Duchenne Muscular Dystrophy, Genethon/CNRS UMR 8115, Evry, France; Duchenne
Muscular Dystrophy, Genethon/CNRS UMR 8115, Evry, France; NAMC, CNRS UMR 8620,
Orsay, France; NAMC, CNRS UMR 8620, Orsay, France; Duchenne Muscular Dystrophy,
Genethon/CNRS UMR 8115, Evry, France; Hopital Necker, Inserm U781, Paris,
France; Duchenne Muscular Dystrophy, Genethon/CNRS UMR 8115, Evry, France
Dystrophin is selectively localized in cortical, hippocampal and cerebellar
neurons. In addition to the progressive muscle wasting, one third of Duchenne
patients suffer a moderate to severe form of mental retardation. Behavioural
studies also suggest that the loss of dystrophin expression in mdx mice
brain results in a mild cognitive deficit and memory impairment. We have
recently developed an original approach to obtain a complete, functional and
enduring restoration of dystrophin in the skeletal muscle of mdx mice [Goyenvalle
et al., 2004]. The muscle rescue was achieved by means of vectorized antisense
sequences that are potentially able to eliminate the mutated exon 23 of the
dystrophin gene in this mutant, thereby allowing a local restoration of
dystrophin expression. AAV vector considerably enhanced the proper sub cellular
localization of antisense sequences and their inclusion into the spliceosome.
This vector has been shown to be particularly efficient for gene transfer into
pyramidal neurons of the hippocampus. We show here that dystrophin expression in
the mdx mice dorsal hippocampus was achieved by a bilateral intracerebral
stereotaxic administration of an AAV-vector expressing antisense sequences
linked to a modified U7 small nuclear RNA (snRNA). mRNA analyses and protein
detection by immunohistochemistry and Western blotting were carried out on
serial brain tissue sections. The DNA sequence confirmed that the 688 bp band
resulting from RT-PCR corresponds to the exon 23-skipped mRNA. Moreover, this
exon 23-skipped mRNA and the corresponding protein were selectively detected in
a remote cerebral strucuture, the entorhinal cortex, directly connected to the
hippocampus. These findings suggest a retrograde axonal transport of AAV
particles to this afferent neuroanatomic area, such allowing the rescue of
dystrophin expression in this neuronal netwotk. These results already suggest
that mRNA manipulation may allow the reversion of genetic alterations in the
mature brain. One main interest of this approach is the restoration of the lost
protein not only in the target structure of the brain but also in the target
cells, within the complex cell population of the brain tissue. Potentially, exon
skipping may be used to selectively inactivate the expression of genes involved
in cognitive processes and synaptic plasticity. It may open new opportunities
for therapeutic strategies targeted to a functional neuroanatomical structure.
11)
Restoration of Dystrophin Expression in the Diaphragm of the Dystrophic mdx Mice
by Polymer-Tagged Morpholino Antisense Oligonucleotides
Bo Wu, Pei Juan Lu, Jignya Ashar, Ehsan
Benrashid, Elizabeth Keramaris, Yong Fu Li, Paul A. Morcos, Qi Long Lu McColl-Lockwood
Laboratory for Muscular Dystrophy Research, Cannon Research Center, Carolinas
Healthcare System, Charlotte, NC; Gene Tools, LLC, Philomath, OR
Duchenne and Becker muscular dystrophies are allelic disorders arising from
mutations in the dystrophin gene. Duchenne muscular dystrophy (DMD) is
characterised by nonsense or frame-shifting mutations in the dystrophin gene
that result in an absence of functional protein, dystrophin, while Becker
muscular dystrophy is usually caused by in-frame deletions resulting in
shortened yet partially functional dystrophin. The application of antisense
oligonucleotides (AOs) is a promising strategy for the treatment of DMD. This
treatment removes specific exons from the dystrophin mRNA transcript with AOs,
thus altering pre-mRNA processing to restore the mRNA reading frame and to
express shortened but functional Becker-like protein. Local and systemic
deliveries of phosphodiamidate morpholino oligonucleotides (PMOs) have been
demonstrated to effectively induce exon skipping and dystrophin expression in
the Golden retrieval muscular dystrophic (GRMD) dog and the dystrophic mdx mouse
. However, systemic delivery of PMOs in both models also demonstrated that
expression of dystrophin can not be achieved homogeneously in all muscle of the
body with especially poor induction in the cardiac muscle and diaphragm. Efforts
have been made to search for and achieve improved delivery and improved
antisense oligonucleotide efficiency. In this study, we examine the effect of
PMOs tagged at the 3 prime with an arginine-based polymer (Vivoporter, supplied
by GeneTools) on skipping of E23 of the dystrophin gene in the mdx mice in vivo.
We found that just 10g
morpholino- vivoporter improved local efficiency of PMO induced exon skipping
with almost 100% TA muscle fibres expressing dystrophin. Intraperitoneal
injection of the morpholino- vivoporter achieved full induction of dystrophin in
the diaphragm and abdomen muscles, but limited dystrophin expression in other
skeletal and cardiac muscles. Since DMD severely affects respiratory muscles
specifically the diaphragm, resulting in progressive deteriorations and failure
of respiratory system, full induction of dystrophin in these muscles by i.p.
injection may provide a realistic alternative for the rescue of the respiratory
functions in DMD.
12)
Screening of Antisense Oligonucleotides for Human Dystrophin Exon Skipping in a
GFP Reporter Culture System with High Sensitivity and Specificity
Ehsan Benrashid, Saafan Malik, Allen
Zillmer, Randy J. Thresher, Jeffrey Rosenfeld, Qi Long Lu McColl-Lockwood
Laboratory for Muscular Dystrophy Research, Carolinas Medical Center, Charlotte,
NC; Animal Models Core Facility, University of North Carolina, Chapel Hill, NC
Frameshift mutations in the dystrophin gene, located on the X chromosome (Xp21),
can result in the progressive muscle wasting disease Duchennes
Muscular Dystrophy (DMD), which affects
~1 in 3500 live male
births. Antisense oligonucleotide (AON) therapy has previously been shown to
selectively induce the skipping of particular exons both in vitro and
in vivo, by targeting exon splicing enhancer (ESE) regions, 3'-,
and 5'-splice
sites. The DMD phenotype can thus be alleviated to the milder Beckers
Muscular Dystrophy (BMD) form. Due to limitations in the quantification of AON
efficacy via RT-PCR and nested PCR assay systems, as well as practical
limitations of primary cultures for AON screening, this study employed a GFP
reporter culture system to provide for efficient and high throughput screening
of numerous AON compounds for human dystrophin exon 50 skipping, which could
rescue approximately 8% of DMD mutations. AON screening was performed using both
2-O-methyl
phosphorothioate (2OMe) and morpholino (PMO) chemistry via fluorescence
microscopy, RT-PCR/nested-PCR analysis, as well as flow cytommetry. After
initial selection of AONs, the effect of overall AON length was also determined
on the culture system. The efficient AONs selected were further tested with
normal human myoblasts and relevant patient fibroblasts (with hEx51 deletion;
further skipping of E50 can restore the reading frame). The results showed that
AONs selected with the reporter system are highly effective for specific exon
skipping in all cells. Our results demonstrate that the in vitro cell
culture-based system is a valuable tool for the screening of effective AONs with
high specificity and reliability, both of which are essential for clinical
trials involving antisense therapy in DMD
13)
rAAV Type 8-Mediated Extensive Therapeutic Gene Delivery into Skeltal Muscle of
alpha-Sarcoglycan
Deficient Mice
Akiyo Nishiyama, Beryl N. Ampong, Jin-Hong
Shin, Hiroyuki Nakai, Takashi Okada, Shin'ichi Takeda Molecular Therapy,
National Institute of Neuroscience, NCNP, Kodaira, Tokyo, Japan; Molecular
Genetics and Biochemistry, University of Pittsburgh School of Medicine,
Pittsburgh, PA
Backgrounds: Autosomal recessive limb-girdle muscular dystrophy type 2D
(LGMD2D) is caused by the mutations in the
alpha-sarcoglycan
gene (alpha-SG)
and the absence of alpha-SG
results in a reduction of whole SG complex, composed of
alpha-,
beta ,gama,
alpha-SG,
from the sarcolemma. The recombinant adeno-associated virus (rAAV) is expected
to be useful to transduce alpha-SG
cDNA. However, the transduction by the rAAV type 2 (rAAV2) encoding
alpha-SG
cDNA showed cytotoxicity with immune response in the
alpha-SG-deficient
mice. The rAAV2 only allows transduction of the gene through local injection,
but the rAAV type 8 (rAAV8) enables systemic deliverly of the gene through
intravenous injection. We examined the therapeutic effects of the rAAV8 encoding
the alpha-SG
cDNA on the alpha-SG-deficient
mice. Methods: We generated the rAAV2 or rAAV8 encoding the
alpha-SG
cDNA driven by a CMV promoter (rAAV2-alpha-SG
or rAAV8-alpha-SG).
We injected the rAAVs into the anterior tibial (TA) muscle of the
alpha-SG-deficient
mice. The neonatal or adult alpha-SG-deficient
mice were injected with the 1 x 1011 vg or 5 x 1011 vg of
rAAVs, respectively. Moreover, we injected the rAAV8-alpha-SG
(5 x 1012 vg) into the tail vein of the 5-week-old
alpha-SG-deficient
mice for systemic delivery. Results and Discussion: At 4 weeks after the
injection of the rAAV8-alpha-SG
into alpha-SG-deficient
TA muscle, expression of alpha-SG
was widely distributed in the posterior leg muscles, including the TA, extensor
digitorum longus, gastrocnemius, soleus, and tibialis posterior muscles. In
particular, the rAAV8-alpha-SG
effectively transduced the posterior leg muscles as well as the heart of the
adult mice. In contrast, the rAAV2-alpha-SG
merely achieved local transduction of muscle fibers in the TA muscle. Together
with wide expression of alpha-SG,
expression of other SGs was also detected in the posterior leg after the local
injection of the rAAV8-alpha-SG.
The rAAV8-alpha-SG-transduced
muscle did not induce the severe immune response or cytotoxicity after the low
dose injection of the rAAV8-alpha-SG.
In association with the rAAV8-alpha-SG
transduction, the alpha-SG-deficient
muscle significantly decreased the degeneration-regeneration cycle. Furthermore,
the contractile force of the rAAV8-alpha-SG-injected
muscle has been largely improved compared to the non-injected muscles. By
systemic delivery of the rAAV8-alpha-SG
into the adult alpha-SG-deficient
mice, we observed the alpha-SG
expression in the various tissues including the skeletal muscle, cardiac muscle,
and liver at 4 weeks. Conclusion: The rAAV8 is an effective tool to
deliver therapeutic genes into the dystrophic skeletal muscle and enables wide
distribution of the gene. This extensive rAAV8-mediated
alpha-SG
transduction in the limb-girdle muscular dystrophy type 2D model mice would pave
the way for the future clinical application.
14)
Induction of Dystrophin Expression by Exon Skipping in mdx Mice by PNA
and PNA-Peptide Conjugates
HaiFang Yin, QiLong Lu, Matthew J. A. Wood
Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford,
Oxfordshire, United Kingdom; McColl-Lockwood Laboratory,
Neuromuscular/Amyotrophic Lateral Sclerosis Center, Carolinas Medical Center,
North Carolina
Duchenne muscular dystrophy (DMD) is the most common, serious form of muscular
dystrophy, arising from mutations in the dystrophin gene that preclude the
synthesis of functional protein. Antisense oligonucleotides (AOs) have been used
to manipulate splicing of the pre-mRNA to induce specific exon skipping and
thereby restore the reading frame and expression of functional dystrophin. The
efficacy of this approach has now been established in animal models and in DMD
patients. In this report, we describe in vitro and in vivo studies examining the
effects of peptide nucleic acid oligonucleotides (PNA) and PNA with peptide
conjugates including TAT, muscle-specific peptide (MSP), AAV6 functional domain
(AAV6), AAV8 functional domain (AAV8) on exon skipping efficiency. In cultured
mdx mouse cells treated with unmodified neutral PNA or PNA-peptide
conjugates, RT-PCR showed the efficient skipping of exon 23. In vivo, single
intramuscular injections of PNA or PNA-peptide conjugates into TA muscles of
mdx mice resulted in substantial numbers of dystrophin-positive fibres at 2
weeks after injection, with no apparent cytotoxicity. Mice of different ages
treated with PNA and PNA-peptide conjugates all showed significant improvement
in dystrophin expression compared with untreated mdx mice. Western
analysis confirmed dystrophin expression in these muscles. Dose-response
experiments demonstrated that PNA and PNA-AAV6, PNA-AAV8 conjugates showed
dose-dependent characteristics. Surprisingly, there was no significant
difference between the PNA and PNA-peptides conjugates in both delivery and exon
skipping efficiency. Together, our results to date suggest that PNA and PNA-peptide
conjugates have potential for the treatment of DMD.
15)
Complementary Dystrophin and Utrophin Expression Prevents Severe Dilated
Cardiomyopathy in 2-Year-Old Mdx Mice
Brian P. Bostick, Yongping Yue, Chun Long,
Dongsheng Duan Molecular Microbiology and Immunology, School of Medicine,
University of Missouri, Columbia, MO
Background. A cure for dystrophin-deficient muscular dystrophy
requires concomitant correction in both skeletal muscle and the heart. Recent
advance in gene transfer technique has made it possible to deliver a therapeutic
dystrophin gene to more than half cardiomyocytes. We have previously shown that
dystrophin expression in 50% of cardiomyocytes ameliorated stress-induced
cardiomyopathy in young dystrophin-null mdx mice. However, it is not clear
whether this level of expression can halt end-stage heart disease. Here we
evaluated the protective effect of partial dystrophin expression in aged mdx
mice that displayed severe dilated cardiomyopathy. Methods and Results.
To mimic 50% transduction of the myocardium, heterozygous female mice were
generated by crossing normal and affected animals. We confirmed that half of the
heart cells in these mice expressed dystrophin. Heart function and pathology
were evaluated using a comprehensive panel of anatomical, histopathological and
in vivo physiological assays in 2-year-old mice. Heart weight/body weight
ratio was completely normalized. Morphology study revealed an absence of cardiac
fibrosis and calcification. Importantly, all the ECG and hemodynamic parameters
were corrected. Further studies suggest that the protection was not due to
expansion of dystrophin positive cells in the aged heart. Interestingly, a
double immunostaining for dystrophin and utrophin revealed complementary
expression in the heart of heterozygous mice. Conclusions. We
demonstrated that dystrophin expression in half cardiomyocytes is sufficient to
eliminate severe heart disease in aged mdx mice. We propose a model where the
interspersing dystrophin-positive cells form a latticework that functions as a
reinforcing steel grid to strengthen the mechanic stability of the whole heart.
Since utrophin is usually up-regulated in DMD patients, our results raise the
hope of ameliorating dystrophic cardiomyopathy by partial gene transfer.
(Supported by grants from the National Institutes of Health and the Muscular
Dystrophy Association).
16) In Utero Delivery
of Adeno-Associated Virus Vector-Encoding Human Mini-Dystrophin Leads to
Functional Correction in Dystrophin Deficient Mice
Anthony Y. Tsai, Sasha Bogdanovich,
Christina F. Hughes, Masayuki Endo, Jesse Vrecenak, Jeremy Traas, Philip Zoltick,
Tejvir S. Khurana, Tim Brazelton, Alan W. Flake Surgery, Childrens
Hospital of Philadelphia, Philadelphia, PA; Physiology, University of
Pennsylvania, Philadelphia, PA
Introduction: Duchenne muscular dystrophy (DMD) is the most common disabling and
lethal congenital muscle disorder. Patients with DMD experience progressive
muscle degeneration and weakness until they succumb to respiratory or cardiac
failure. Currently there is no cure for DMD. Human mini-dystrophin with
truncation of the repeating rod domain has been shown to ameliorate dystrophic
histopathology and restores membrane integrity. Adeno-associated virus serotype
9 (AAV2/9) has also been shown to have high muscle specificity. Here, we
investigated the possibility of using in utero delivery of AAV2/9
vector-mediated mini-dystophin to achieve long-term functional correction of
dystrophin deficient mdx mice. Methods: Fetuses from pregnant mdx
mice underwent vitelline vein injection at E14.5 with
AAV2/9.CMV.mini-dystrophin. Specific skeletal muscles, cardiac muscle, and brain
and liver tissue from treated and age-matched mdx control mice were
harvested on week 5, 10 and 20. Human mini-dystrophin expression was detected by
immunohistochemistry (IHC) of frozen sections and quantified by real-time PCR
(Q-PCR). Extensor digitorum longus (EDL) muscles of the treated and control mice
were used in physiological studies to measure force of maximal twitch, tetanus
and eccentric force drop between first and fifth contractions (ECC). Results:
Expression of human mini-dystrophin was detected in week 5, 10, and 20 samples
using IHC with human specific antibody with no significant difference in the
proportion of transduction among the 3 time points. Q-PCR of heart, TA and
soleus at the 20-week time point using the human dystrophin probe (hDys) with
the ribosomal 18S probe as the endogenous control detected a normalized hDys/18S
RNA ratio of 4.46+3.04,
2.29+1.12,
and 2.76+3.86,
respectively. Functional studies showed that ECCs demonstrated significantly
(P<0.01) less reduction in treated vs. control animals (4.30+2.02%
vs.14.01+6.54%)
respectively. There was no difference in force observed during twitch (93.00+26.94mN
vs. 103.15+31.15mN)
or tetanus (459.17+98.81mN
vs. 528.24141.77mN)
between treated and untreated animals respectively. Conclusion: These data
demonstrate that systemic in utero administration of AAV 2/9 vector
encoding for human mini-dystrophin can 1) attain significant transgene
expression in skeletal muscles and the heart corroborated by Q-PCR results 2)
achieve sustained expression of the transgene without evidence of immune
response and 3) result in functional improvement in the mdx model of
muscular dystrophy. In utero gene transfer remains an attractive strategy
for the treatment of the muscular dystrophies.
17)
Inhibiting Myostatin in Dystrophic Mice with Follistatin Improves the Success of
Myoblast Transplantation
Basma F. Benabdallah, Manaf Bouchentouf,
Joel Rousseau, Pascal Bigey, Annick Michaud, Pierre Chapdelaine, Daniel Scherman,
Jacques P. Tremblay Genetique Humaine, Centre de Recherche du CHUL, Quebec, QC,
Canada; Genetique Humaine, Centre de Recherche du CHUL, Quebec, QC, Canada;
Genetique Humaine, Centre de Recherche du CHUL, Quebec, QC, Canada; U640, Inserm,
Paris, France; Genetique Humaine, Centre de Recherche du CHUL, Quebec, Canada;
Ontogenie et Reproduction, Centre de Recherche du CHUL, Quebec, QC, Canada;
U640, Inserm, Parid, France; Genetique Humaine, Centre de Recherche du CHUL,
Quebec, France
Duchenne muscular dystrophy is a recessive disease due to a mutation in the
dystrophin gene. Myoblasts transfer permits to introduce dystrophin gene in
dystrophic muscle fibers. However, this approach success is reduced by the short
duration regeneration post-transplantation; wich reduces the number of hybrid
fibers. In this manuscript, the aim was to verify whether the success of the
myoblast transplantation is enhanced by the blockade of the myostatin signal
with an antagonist, follistatin. Three different approaches were studied to
over-express the follistatin in mdx transplanted muscles. First,
transgenic follistatin/mdx mice were generated; second, a follistatin
plasmid was electroporated in mdx muscles, and finally, follistatin was
induced in mdx mice muscles by a histone deacetylase inhibitor treatment.
All the three approaches induced an improvement of the success of the myoblast
transplantation. Moreover, a fiber hypertrophy was also observed in all muscles,
proving that the inhibition of the myostatin by the follistatin may represent a
good method to improve myoblast transplantation and increase muscle function.
The inhibition of the myostatin by follistatin could be a promising novel
therapeutic approach toward the treatment of muscle wasting in muscle diseases
such as Duchenne muscular dystrophy in combination with myoblast
transplantation.
18)
Distinct Transduction Profiles in the Dystrophic Dogs with rAAV Serotype 8
Sachiko Ohshima, Jin-Hong Shin, Akiyo
Nishiyama, Katsutoshi Yuasa, Hiroyuki Nakai, Takashi Okada, Shin'ichi Takeda
Molecular Therapy, National Institute of Neuroscience, NCNP, Kodaira, Tokyo,
Japan; Molecular Genetics and Biochemistry, University of Pittsburgh School of
Medicine, Pittsburgh, PA
Background: Duchenne muscular dystrophy (DMD) is an X-linked, lethal
disorder of the striated muscle caused by mutations in the dystrophin
gene, which encodes a large sub-sarcolemmal cytoskeletal protein. The absence of
dystrophin associated with the loss of dystrophin-glycoprotein complex (DGC)
from the sarcolemma results in progressive muscle weakness, cardiomyopathy, and
early death. Several treatment modalities have been attempted to correct the
dystrophic phenotypes, but effective therapy still awaits to be developed. A
recombinant adeno-associated virus (rAAV) serotype 2 (rAAV2) have been utilized
in the various preclinical and clinical studies, but the rAAV serotype 8 (rAAV8)
demonstrated the better gene transfer efficiency in the skeletal and cardiac
muscle. Here we investigated the transduction profiles with the rAAV2 and rAAV8
in the muscles of canine X-linked muscular dystrophy in Japan (cxmdJ).
Methods: The rAAV2 or rAAV8 encoding the lacZ gene driven by a CMV
promoter was injected into the tibialis anterior and extensor carpi ulnaris of
the normal beagles at 6-10 weeks old (1x1011 - 2x1013 vg).
We did biopsy of the transduced muscle 2 weeks after the injection and analysed
histologically to examine beta-gal
expression and immune response. We also injected the rAAV8 encoding the
microdystrophin (M3, cCS2)
gene driven by the CMV promoter into the tibialis anterior and extensor carpi
ulnaris of cxmdJ at 6-8 weeks old (1x1012 - 2x1013
vg) and examined the efficiency of rAAV8. The transduced muscle has been sampled
4 weeks after the injection to perform histochemical and immunohistochemical
analysis, and Western blotting. Results: We detected the greater number
of the beta-gal-positive
fibers in the rAAV8-transduced canine skeletal muscles than those in the
rAAV2-transduced muscles 2 weeks after the injection. Moreover, we can detect
much less inflammatory cell infiltration in the rAAV8-transduced muscle than the
rAAV2-transduced muscles. Immunohistochemistory of CD4, CD8, CD11b, and MHC
class I, revealed less CTL-mediated immune response with the rAAV8 than those
with the rAAV2. However, we can detect a certain degree of immune response with
a high dose of rAAV8. Consequently, extensive microdystrophin expression in the
rAAV8-injected skeletal muscle of cxmdJ was achieved.
Discussion: rAAV8-mediated gene transfer showed effective transgene
expression with less immune responses than those in the rAAV2-mediated
transduction. Even though rAAV8-mediated gene transfer is less immunogenic, it
is indispensable to clarify the appropriate condition of virus titer and dose
according to the encoding gene. We are currently attempting either the limb
perfusion or systemic delivery through saphenous vein in cxmdJ
to examine the rAAV8-mediated transduction efficiency of microdystrophin.
Conclusion: The rAAV8 is the efficient tool for the therapeutic gene
delivery into the dystrophic canine skeletal muscle. The systemic
microdystrophin transduction with the rAAV8 would be promising for the future
DMD gene therapy.
19)
Intravascular Delivery of AAV-9 Leads to Efficient Transduction of Multiple
Muscles in Dog
Yongping Yue, Janet Bogan, Arka Ghosh, Dan
Bogan, Chun Long, Bruce F. Smith, Joe N. Kornegay, Dongsheng Duan Molecular
Microbiology and Immunology, University of Missouri, Columbia, MO; Dalton
Cardiovascular Research Center, University of Missouri, Columbia, MO;
Scott-Ritchey Research Center, Auburn University, Auburn, AL; Pathology and
Laboratory Medicine, University of North Carolina, Chapel Hill, NC
Duchenne muscular dystrophy (DMD) is the most common childhood fatal muscle
disease. Since nearly every muscle in the body is affected, an effective DMD
gene therapy will require efficient transduction of all body muscles.
Proof-of-concept body-wide transduction has been demonstrated in mice with adeno-associated
viruses (AAV). However, the size of the human body is several hundreds fold
larger than that of a mouse. Translating results in the mouse to human patients
remains a titanic challenge. The size of a dog is only several fold different
from that of a human patient. The dog model therefore represents an ideal system
to test whether intravascular delivery can target multiple muscles prior to
human clinical trials. Previous attempts to deliver AAV directly to canine
muscle have largely failed to achieve efficient transduction. This is mainly due
to a strong immune response. In this study, we evaluated whether systemic gene
delivery in newborn dogs could avoid inducing immune responses and lead to high
level expression. An AAV-9 vector carrying the human placental alkaline
phosphatase reporter gene was injected into neonatal dogs via the jugular vein
in the absence of immuno-suppressive drugs. Transgene expression was examined by
histochemical staining at 2, 3, 4, and 6 weeks after AAV infection in the
cranial sartorius, vastus lateralis, long digital extensor and the lateral head
of the gastrocnemius muscles. In contrast to the previous reports of low
expression and strong immune reaction in dog muscle, we observed high-level
persistent expression in multiple muscles. In some sections, up to 95% of
myofibers were transduced. Taken together, our results provide the first
evidence that systemic AAV delivery can reach multiple muscles in a large animal
and that body size is not a barrier to intravascular AAV gene transfer. Our
results raise the hope of whole body correction for many systemic diseases (such
as DMD) in human patients. (Supported by grants from the National Institutes of
Health and the Muscular Dystrophy Association).
20)
Ameliorating Dystrophic Pathology Via AAV-Mediated Gene Delivery of Myostatin
Propeptide
Chunping Qiao, Jianbin Li, Bing Wang, Juan
Li, Xiao Xiao Molecuar Pharmaceutics, UNC Shool of Pharmacy, Chapel Hill, NC;
Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA
Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene,
is the most common, disabling and lethal muscle disease. Myostatin has been
extensively documented as a negative regulator of muscle growth. Myostatin
blockades therefore offers an effective strategy for treating a number of muscle
degenerative diseases, including sarcopenia and muscular dystrophy. In this
study, we investigated whether gene delivery of myostatin inhibitors,
specifically, the propeptide, could improve muscle growth and ameliorate the
pathologies of DMD in mouse model. The serotype 8 AAV-MPRO76AFc vector was
delivered into 3-month-old mdx mice by simple tail vein injection. The treated
mdx mice started to gain weight two weeks after vector injection (p<0.01), and
the difference became highly significant after one and half months of treatment
(p<0.001). The average serum CK level of treated mice (610
+261
IU/L) was slightly lower than mdx mice (906
+289)
(n=5 for each group, p=0.06), although it was still far above the wild type
mouse (6.7 +2.3
IU/L). We then did cryo-thin-sectioning on TA muscle, and performed histology
examination. The treated muscle showed improved muscle pathology with more
uniformed muscle fiber sizes and less mononuclear cells. The treated muscle also
displayed less fibrosis. Because of the limited number of studied animals, at
present time, we are still in the progress to evaluate whether AAV-MPRO76AFc
gene delivery into mdx mice can increase their muscle strength. In conclusion,
our study indicated that delivering myostatin propeptide gene offered
therapeutic benefit in Duchenne Muscular Dystrophic mice.
21)
Antisense Oligonucleotide Selection for Exon Skipping by a Readout Reporter
System with High Capacity and Specificity
Allen E. Zillmer, Qi L. Lu, Jeffrey
Rosenfeld McColl-Lockwood Laboratory for Muscular Dystrophy Research, Carolinas
Medical Center, Charlotte, NC
Duchenne muscular dystrophy (DMD) presents a unique opportunity to apply
antisense technology (AON). Since the majority of DMD mutations occur in a
region non-critical to the gene function, exons or groups of exons can be
skipped by AONs resulting in truncated but functional protein. The critical
factor determining the efficiency of exon skipping is the AON selection. RT-PCR
detection of mRNA with target exon skipped in human myoblast culture is highly
variable. In this study, we explore the use of an expression vector-based
readout reporter system for the selection of AONs. This system utilizes EGFP
reporter to demonstrate the efficiency of specific AON on exon skipping. The
sequence of a human dystrophin intron was inserted into a specific site within
the EGFP coding region. The insertion however did not disrupt the reading frame
of the gene as the intron sequence was recognized by the transcription machinery
and reliably spliced out of the transcripts. The insertion of E23 of the mouse
dystrophin together with 900bp of its two flanking introns produced a chimeric
transcript with the insertion of E23 in the EGFP transcript, leading to the
disruption of EGFP reading frame. Targeting the transcripts with specific AONs
to E23 removed the exon and restored the expression of EGFP. The levels of EGFP
expression reliably indicated the efficiency of AON both in vitro and in vivo.
The high levels of EGFP expression and consistency in culturing the transfectant
C2C12 myoblast provide highly sensitive and reliable measurement for the
selection of AON with highest potency in skipping specific human dystrophin exon.
We conclude that this system will be complementary to the human myoblast cell
culture systems, providing high capacity to distinguish the differential
antisense effect and to select most potent AON for skipping many dystrophin
exons required for treating the majority of DMD.
22)
The Evaluation of Novel Photoreceptor Specific Promoters in an EIAV Lentiviral
Vector for Targeted Gene Expression in the Photoreceptors of the Eye
On Kan, Marjorie Nicoud, Sharifah Iqball,
Stuart Sims, Jian Kong, Peter Gouras, Rando Allikmets, Stuart Naylor, Katie
Binley Biological Systems, Oxford BioMedica (UK) Ltd, Oxford, United Kingdom;
Ophthalmology and Pathology
&Cell
Biology, Columbia University, New York
Many human diseases resulting in blindness such as Stargardt disease (STGD)
primarily affect the photoreceptors. STGD is a macular dystrophy caused by a
mutation in the photoreceptor ATP binding transporter gene (ABCR) resulting in
the accumulation of the toxic lipofuscin pigment A2E. This leads to degeneration
of RPE and photoreceptor cells. We wanted to develop an EIAV based lentiviral
vector that displays photoreceptor specific gene expression and evaluate these
vectors in the murine ABCR-/- k/o model of Stargardt disease. We
investigated the ability of several truncated human photoreceptor specific
promoters: rhodopsin (Rho), beta-phosphodiesterase
(PDE) and retinitis pigmentosa 1 (RP1) to drive reporter gene expression in
vitro, by means of transient plasmid transfections in various cell lines,
including the human retinoblastoma Y-79. Subsequently, the promoters were
transferred to the EIAV lentivector platform for further evaluation in vitro
and in vivo. These promoters were either evaluated alone or in
conjunction with an enhancer element derived from the human interphotoreceptor
retinoid binding protein gene (IRBP). We discovered that the PDE promoter
coupled with three copies of the IRBP enhancer element showed significant
expression in Y-79 cells, comparable to the CMV promoter, but showed little or
no activity in non-photoreceptor cell types in vitro. Subretinal delivery
of these EIAV vectors into mice demonstrated that reporter gene expression was
restricted to the photoreceptor cell layer. The PDE promoter coupled with a
single IRBP enhancer element displayed the strongest photoreceptor specific
expression in vivo. We will present data to show that these novel
photoreceptor promoters can be used to drive ABCR expression in the
photoreceptors of ABCR-/- k/o mice resulting in a significant
reduction in A2E accumulation and thus show promise for addressing this disease
in the clinical setting.
23)
Viral Mediated Transfer of the ELOVL4 Gene Can Result in High Level ELOVL4
Protein Expression in Human RPE Cells, Increasing the Major Anti-Inflammatory
Retinal Fatty Acid DHA
Sergey S. Seregin, Maria O. Tikhonenko,
Julia V. Busik, Andrea Amalfitano Departments of Microbiology and Molecular
Genetics, Pediatrics, Physiology, Michigan State University, East Lansing, MI
The highly conserved Elongation of very long chain fatty acids-like 4 protein
(ELOVL4) belongs to a family of proteins functioning in elongation of long chain
fatty acids. ELOVL4 is expressed in retina, skin, brain and testis suggesting
important roles for ELOVL4 in these tissues. Inherited mutations in the ELOVL4
gene are known to account for autosomal dominant Stargardt-like macular
degeneration, and autosomal dominant macular dystrophy (ADMD), possibly due to
significant decreases in retinal production of very long chain fatty acids (VLCFA).
All of these abnormalities are associated with the loss of functional ELOVL4
protein, thus gene therapy approaches based upon transduction of the ELOVL4
gene, and expression of ELOVL4 protein in retinal tissues, may be a viable
strategy for several retinal diseases. To establish the proof of concept for
this goal, we investigated ELOVL4 gene delivery via the use of an Adenovirus
(Ad) based gene transfer vector. Ad based vectors are known to transduce a wide
variety of cell types, are the most widely utilized gene transfer vector
currently utilized in human clinical gene therapy trials, and most importantly,
are currently being safely utilized in advanced human clinical trials for
treatment of other retinal diseases. A recombinant rat or human ELOVL4 cDNA was
generated, and each was placed under transcriptional control of a CMV
enhancer/promoter element, within an Ad shuttling plasmid. Recombination with an
E1-,E3- Ad plasmid, resulted in the successful generation of an [E1-,E3-]Ad+(rat
or human)ELOVL4 vector. These recombinant Ads were successfully amplified on 293
cells, and utilized to infect human Retinal Pigmented Epithelial (hRPE) cells.
The hRPE cells infected with either human, or rat ELOVL4 recombinant Ads
expressed up to 25 fold higher protein levels of ELOVL4 relative to hRPE cells
infected with a control (CMV-LacZ) Ad vector, as determined by Western blotting
of proteins derived from the infected cells. More importantly, the increased
levels of ELOVL4 expression were also associated with significantly elevated
levels of docosahexaenoic acid (DHA) in the hRPE cells, as determined by RP-HPLC
followed by UV and evaporative light scatter detection methods. These data
provide proof of concept that efficient transfer of the ELOVL4 gene can be
accomplished in hRPE cells, and that over-expression of ELOVL4 protein can
increase the levels of the major retinal anti-inflammatory fatty acid DHA.
Future studies will determine how useful this approach will be in relevant
animal models of retinal disease.
24)
Effect of Insulin-Like Growth Factor-1 (IGF-1) on Global Gene Expression
Profiles in Murine Skeletal Muscle
C. Ramana Bhasker, Shaochun Song, Theodore
Friedmann Center for Molecular Genetics, Dept. of Pediatrics, University of
California San Diego, La Jolla, CA
The peptide hormone IGF1 is a powerful mitogen that plays vital roles in the
action of growth hormone and is a central participant in growth and function of
skeletal muscle. It has been shown to bring about important therapeutic effects
in the mdx mouse model of muscular dystrophy and other forms of muscular and
neuromuscular disease such as a mouse model of ALS. There is little detailed
understanding of the global effects of IGF-1 on global patterns of gene
expression. To develop a more thorough understanding of the mechanisms of action
of IGF-1, we have recently undertaken microarray-based and proteomic studies of
IGF-1 on murine myoblasts in culture and in skeletal muscle in vivo. We report
here a description of the acute in vivo effects of IGF-1 on global patterns of
gene expression in skeletal muscle of the mouse, using an Affymetrix (mouse
genome 430 2.0 array) platform and data analysis with the Vampire Software (SDSC
and UCSD) and the Genespring Software (Agilent/Silicon Genetics). Our results
indicate that more than 200 genes representing many functional families are
down-regulated while far fewer genes are up-regulated by IGF1 at 1, 2, 4 and 6
hours after subcutaneous administration of IGF-1. In contrast, IGF-1 produces a
far greater degree up-regulation of many gene families in cultured myoblasts.
Interestingly, after the in vivo exposure to IGF-1, some important
muscle-specific genes are strongly down-regulated while a number of small and
large ribosomal subunit proteins are up-regulated, consistent with stimulated
protein synthesis These kinds of detailed global gene expression and proteomic
studies represent powerful approaches to understanding the mechanisms of action
of IGF-1 and related growth factors, both in the development of disease
phenotypes and in their treatment.
25)
Spliceosome-Mediated RNA Trans-Splicing for Muscular Dystrophies
Chunping Qiao, Jianbin Li, Bing Wang, Juan
Li, Xiao Xiao Department of Molecular Pharmaceutics, UNC School of Pharmacy,
Chapel Hill, NC; Department of Orthopaedic Surgery, University of Pittsburgh,
Pittsburgh, PA
Spliceosome-mediated RNA trans-splicing (SMaRT) is a novel RNA-reprogramming
strategy. It offers several potential advantages over traditional gene
replacement therapy, such as reduced transgene size and elimination of ectopic
expression of the repaired product by retaining endogenous regulation. In this
study, we explored the potential application of SMaRT technology for muscular
dystrophies. We use the Duchenne muscular dystrophy mouse, the mdx, as a model
system, in which the mutated gene dystrophin was shown to carry a premature stop
codon in exon 23. We intended to use pre-trans-splicing molecules (PTM)
containing mouse dystrophin intron 22 sequence linked the C-terminal coding
region of dystrophin to intercept the RNA splicing before exon 23. The resultant
is a trans-spliced mRNA encoding a functional dystrophin. To examine the
trans-splicing efficiency in cultured cells, we first created a target plasmid
by inserting the dystrophin intron 22 into mini-dystrophin gene 3858, between
Rod 2 (R2) and Rod 22 (R22) regions, generating plasmid
pAAV-cmv-3858-spolyA-in22. We then constructed three PTMs containg different
binding domains of intron 22 and the C-half of the mini-dystrophin 3990 with a
myc tag. The PTM plasmids were respectively co-transfected with the target
plasmid for trans-splicing into 293 cells. Trans-splicing events were monitored
by both RT-PCR and western blot. Sequencing results of RT-PCR fragment confirmed
the expected trans-splicing products, while western blot showed expected myc-tagged
mini-dystrophin. We then packaged those three PTMs into adeno-associated viral
vector and injected them into the leg muscle of three-month-old mdx mice.
Two months after vector injection, the mice were sacrificed and the injected
muscles were carefully dissected. The trans-splicing events were analyzed by
both immunofluorescent (IF) staining and RT-PCR. IF staining indicated low
percentage of mini-dystrophin positive cells, while RT-PCR failed to detect
trans-splicing product. Our results indicated that SMaRT technology offers some
potential for the treatment of muscular dystrophies; however, its efficiency has
to be improved to render therapeutic effect.
26)
Isolation, Characterization, and Myogenesis of Satellite Cells Derived from
Skeletal Muscle
Nicholas Ieronimakis, Gayathri Balasundaram,
Jeffrey S. Chamberlain, Morayma Reyes Pathology, University of Washington,
Seattle, WA; Neurology, University of Washington, Seattle, WA
Satellite cells constitute the natural stem cell reservoir for regeneration in
adult skeletal muscle. The regenerative role and capability of satellite cells
(SC) in skeletal muscle makes them a primed candidate for treatment of
degenerative diseases such as muscular dystrophy. However, the challenge of
isolation and expansion of pure satellite cell populations has encumbered their
use in clinical applications. We report here the direct isolation of SC from
skeletal muscles by fluorescence-activated cell sorting based on the expression
of Sca-1, CD34, CD31 and CD45 cell surface antigens. Sca-1 is predominantly a
marker of hematopoietic stem cells and as we have reported a marker of
endothelial cells in the skeletal muscle. Conveniently SC are Sca-1 negative.
CD34 is also a marker of hematopoietic stem cells and some endothelial cells,
but in the skeletal muscles CD34 is highly expressed by SC. By utilizing a host
of antibodies we have isolated a very distinct and homogenous population
recognized as Sca-1-, CD31-, CD34+
and CD45- from both wt and mdx (dystrophyn -/-) mice, ages
ranging from newborn to 25 months. Because some hematopoietic and endothelial
cells also express these markers we excluded all CD45+ cells (hematopoietic)
and CD31+ (endothelial) cells. Furthermore, the forward and size
scatter pattern of this population corresponds with the size morphology of SC as
a homogenously small mononuclear population. Depending on age, this population
represents 0.5-2% of all mononuclear cells derived from skeletal muscles. The
abundance of SC in the skeletal muscle declines with age though more severely in
mdx mice to almost undetectable levels by 25 month. RT-PCR analysis and
immunohistochemistry of freshly sorted cells confirms this population expresses
many satellite cell markers such as Pax7, NCAM, MyoD, Myf5, Syndecan 3, CD34,
and c-met while lacking expression of endothelial markers TEK, vWF, and Flt.
Nearly 100% of these cells express Pax7 and Myf5 signifying this population to
be very pure and homogenous. In addition, staining of freshly FACS-sorted SC for
NCAM and c-met are polarized which correlates with polarization of these markers
in muscle tissue sections towards the basal lamina. Interestingly, SC derived
from mdx mice express lower levels of NCAM, MyoD, Myf5 and syndecan 3, perhaps
due to impaired myogenesis. We have culture-expanded these cells in vitro in
F10C with 15% horse serum and 10 ng/ml bFGF, obtaining clones of more than 1000
SC (>10 cell doublings) for seven days. These cells can differentiate into
robust myotubes when bFGF is withdrawn and horse serum is reduced to 1.5%. To
demonstrate the myogenic potential of these cells, Sca-1-,
CD31-, CD45-, CD34+ cells
were FACS-sorted from GFP mice and directly injected intramuscularly in the
tibialis anterior muscles of mdx mice. Two weeks after transplantation multiple
green myofibers were seen throughout the TA muscle, demonstrating the
regenerative capability of these cells. This new approach using flow cytometry
to directly isolate SC will be extremely useful in studying their biology for
the development of regenerative treatments for muscular diseases.
27)
Global Force Improvement by Systemic AAV Delivery of
-Sarcoglycan
Transgene in Deficient Mice
Marc Bartoli, Francoise Fougerousse, Jérôme
Poupiot, Olivier Danos, Isabelle Richard LGMD, Genethon, Evry, France
-sarcoglycanopathy
(Limb Girdle Muscular Dystrophy type 2D, LGMD2D) is a recessive muscular
disorder caused by deficiency in -sarcoglycan,
a transmembrane protein part of the dystrophin-associated complex. We
constructed recombinant adeno-associated virus (rAAV) vectors expressing the
human -sarcoglycan
cDNA under the control of a muscle specific promoter. Efficient and sustained
transgene expression with correct sarcolemmal localization and without evident
toxicity was obtained after intra-arterial injection into the both hindlimbs of
a LGMD2D murine model. Transgene expression resulted in restoration of the
sarcoglycan complex, histological improvement, membrane stabilization with full
rescue of the contractile force deficits and stretch sensibility that led to an
increase of the global activity of the animals. We will also present the
analyses carried-out to monitor the immune response against the transgene. This
poster establishes the feasibility for whole body AAV-mediated
-sarcoglycan
gene transfer as a therapeutic approach
28)
Viral Based Therapeutic for LGMD Type 2D Deficiencies Demonstrates Long-Term
Results in Alpha-Sarcoglycan Deficient Animals
Louise R. Rodino-Klapac, Christopher J.
Shilling, Zarife Sahenk, K. Reed Clark, Jerry R. Mendell Center for Gene
Therapy, Columbus Childrens
Research Institute, Columbus, OH
Limb-girdle muscular dystrophy (LGMD) type 2D is characterized by skeletal
muscle weakness and results from mutations occurring in the
alpha-sarcoglycan
gene. Localized in the sarcolemma, the sarcoglycans (alpha,
beta, gamma, delta) are a
subcomplex of the dystrophin-associated proteins (DAP). Alpha-sarcoglycan (alpha-SG)
deficiency is the most common form of sarcoglycan-LGMD and no therapeutic
treatments are currently available. The
alpha-SG mouse model
provides an opportunity to test translational treatment approaches. Prior
studies have suggested that AAV-mediated
alpha-SG
gene transfer could not sustain expression because of transgene toxicity,
potentially precluding clinical gene transfer for LGMD2D. Herein, we describe
our in vivo work comparing alpha-SG
gene expression from either the ubiquitously expressed cytomegalovirus (CMV)
promoter, or muscle specific promoters, muscle creatine kinase (MCK) and desmin
(DES) in the alpha-SG
KO mouse in the context of rAAV gene delivery. We injected the tibialis anterior
(TA) muscle of 4-6 week old alpha-SG
KO mice with rAAV1.alpha-SG
(CMV, MCK, DES promoter) at low (3 x 109 vg) and high (3 x 1010
vg) doses. Sustained gene expression was observed irrespective of promoters at
six and twelve weeks post gene transfer. Quantitation of
alpha-SG
gene expression by fiber counts yielded similar levels of myofiber transduction
for CMV and MCK at the 6 week high dose, 61.4% versus 64.4% respectively.
However, alpha-SG
expression using the DES promoter was significantly lower at the high dose with
34% of the myofibers transduced. Similar levels of expression were seen at the
12 week time point for MCK and DES, while CMV exhibited a 25% reduction in
expression. Our studies show sustained and robust gene expression well beyond
the time at which transgene cytotoxic effects were previously reported using
rAAV2. CMV.alpha-SG.
Mononuclear cell analysis in our studies showed no evidence of infiltrating B or
T cell subsets or macrophages. Differences in our findings compared to previous
work could relate to AAV serotype. In summary, our data demonstrate robust
alpha-SG
gene expression as long as 3 months using AAV1, with no reduction in expression
using the muscle specific MCK promoter. These findings enhance the possibility
for gene therapy as a potential treatment option for LGMD2D and support our
current efforts for launching a gene therapy study for this disease.
29)
Functional Analysis of alpha-Dystrobrevin-3 in Striated Muscle with rAAV6 Gene
Transfer
Guy Odom, Glen Banks, James Allen, Marv
Adams, Leonard Meuse, Miki Haraguchi, Stan Froehner, Jeffrey Chamberlain
Neurology, University of Washington, Seattle, WA; Physiology and Biophysics,
University of Washington, Seattle, WA
Alpha Dystrobrevin (alphaDb)
is a member of the dystrophin subfamily of proteins, and is an integral
component of the dystrophin-glycoprotein complex (DGC) at the sarcolemma of
striated muscles. Decreasing amounts of dystrobrevin from the sarcolemma
contributes to the severity of disease in several muscular dystrophies including
DMD and LGMD. Mice lacking the three isoforms of
alphaDb
(alphaDb-/-)
found in skeletal muscle present with several phenotypic changes consistent with
dystrophy including a mild cardiomyopathy, myopathy, fragmented neuromuscular
synapses and shallow folds within the muscle-tendon junction. Both
alphaDB1
and alphaDB2
transgenes restore the skeletal muscle abnormalities in the DB knockout to
varying degrees because of subtle differences in their location within the
muscles membrane and varying functional domains contained in each protein.
Because alphaDB3
lacks many of these functional domains it has previously not been considered a
relevant isoform of alphaDb.
In the present study we tested whether
alphaDb-3 has any
functional significance in skeletal muscle integrity, myotendonous junction
folding, and maintenance of the neuromuscular synapses. We intravenously
administered recombinant adeno-associated viral (rAAV6) vectors containing GFP-tagged
alphaDb-2
or alphaDb-3
transgenes in order to transduce the striated musculature of
alpha-Db
knockout mice. The mice were injected at 4 weeks of age and analyzed at 8 and 12
weeks after administration. alphaDb
expression was viewed using immunohistochemistry of frozen muscle sections.
Muscle integrity was analyzed in frozen sections stained with hematoxylin and
eosin. Neuromuscular synapses were analyzed in wholemount teased muscle fibers
stained with rhodamine conjugated alpha-bungarotoxin. The lengths of myotendonus
junctions of gastrocnemius muscle were viewed using transmission electron
microscopy. We also tested how alphaDB3
might interact with the DGC in vitro and in vivo utilizing coimmunoprecipitation
assays. Muscles throughout alphaDB-/-
mice were transduced with rAAV6-DB2 and rAAV6-DB3.
alphaDB2
and alphaDB3
both localized to the sarcolemma and were concentrated within the folds of the
myotendinous and neuromuscular junctions.
alphaDb3
and alphaDb-2
were equally effective at preventing central nucleation in most skeletal muscle
fibers, preventing fragmentation of neuromuscular synapses and restoring folds
at the myotendinous junction. alphaDB2
was more effective than alphaDB3
in preventing fingerlike projections from protruding out of the synaptic
borders. In coimmunoprecipitation studies,
alphaDb-3
was demonstrated to interact with the sarcoglycan subcomplex through an
individual sarcoglycan interaction in vitro. In vivo coimmunoprecipitation of
alphaDB3
from whole muscle lysates resulted in the majority of the DGC components being
detected. alphaDb-3
is a significant component of the DGC that functions to prevent muscle
degeneration, synapse destabilization and myotenindous junction defects within
skeletal muscle. These results suggest the reduction of
alphaDB3
from the sarcolemma in various muscular dystrophies likely contributes to their
pathogenesis in a similar manner to
alphaDB1 and
alphaDB2.
30)
Dp116 Expression in Dystrophin/Utrophin Double Knockout (mdx:utrn-/-)
Mice Restores Muscle Mass and Viability but Does Not Correct Dystrophic
Pathology
Andrea L. H. Arnett, Luke M. Judge, Jeffrey
S. Chamberlain Molecular and Cellular Biology, Medical Scientist Training
Program, University of Washington, Seattle; Neurology, University of Washington,
Seattle
Mice deficient in both dystrophin and utrophin (mdx:utrn-/-)
exhibit a phenotype similar to that seen in DMD patients, including severe
muscle wasting, skeletal deformities, joint contractures, and premature death.
In these mice, the absence of both dystrophin and utrophin prevents assembly of
the dystrophin-glycoprotein complex (DGC). We previously generated transgenic
mice with skeletal muscle expression of Dp116 and have introduced this transgene
onto the mdx:utrn-/- background. Dp116 is a non-muscle isoform
of dystrophin that lacks the actin-binding domain and the majority of the rod
domain found in the full length dystrophin isoform. Thus, it is postulated that
Dp116 has no mechanical link to the actin cytoskeleton, but can still assemble
and stabilize the DGC at the sarcolemma. We now report that Dp116 expression can
dramatically increase both muscle mass and lifespan in mdx:utrn-/-
and can delay both formation and progression of kyphosis and joint contractures.
However, histological examination of muscle from these transgenic mice reveals
signs of muscular dystrophy similar to that seen in muscles of dystrophin-deficient
(mdx) mice. Our results provide compelling evidence that dystrophin and
the DGC participate in signaling mechanisms that are critical for maintenance of
muscle mass. Alternatively, Dp116 may contribute to the structural integrity of
myofibers via non-canonical binding of Dp116 to the actin cytoskeleton.
31)
Targeting Adenovirus Vector to Neural Cell Adhesion Molecule (NCAM) for Gene
Delivery to Developing Muscle
Bhanu Munil Koppanati, Christoph Volpers,
Joel Kaar, Florian Kreppel, Alan J. Russell, Stefan Kochanek, Carl Lagenaur,
Paula R. Clemens Department of Neurology, University of Pittsburgh School of
Medicine, Pittsburgh, PA; Center for Molecular Medicine (ZMMK) and Institute for
Genetics, University of Cologne, Cologne, Germany; McGowan Institute for
Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA; Division of
Gene Therapy, University of Ulm, Helmholtzstrasse, Ulm, Germany; Department of
Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA;
Neurology Service, Department of Veterans Affairs Medical Center, Pittsburgh, PA
Duchenne muscular dystrophy (DMD) is a debilitating disorder affecting
approximately 1 in 3500 males. Most primary muscle disorders, such as DMD,
affect multiple widespread muscle groups. One significant concern hindering
successful gene transfer to muscle is inefficient gene delivery to muscle cells.
Gene delivery in utero and vector targeting to a receptor highly
expressed and relatively specific for developing muscles are strategies with the
potential to overcome this difficulty. Several strategies have been attempted to
achieve vector targeting of various cells. In this study genetic modification of
adenoviral fiber protein to contain the Immunoglobulin G-binding domain of
Staphylococcus aureus protein A combined with post-assembly vector
modification with a targeting antibody was pursued. We tested whether a
genetically modified adenoviral vector containing a synthetic Immunoglobulin
G-binding domain in the capsid conjugated to anti-NCAM antibody can target NCAM,
a cell surface receptor on embryonic muscle, to increase muscle gene delivery
in vitro. In order to ablate native tropism and thus detarget the modified
vector, the vector was conjugated with increasing concentrations of polyethylene
glycol (PEG). We demonstrated that PEG ablated native tropism in a
dose-dependent fashion. Furthermore, in order to retarget the vector to NCAM-expressing
myoblasts, the vector was incubated with increasing concentrations of anti-NCAM
antibody, prior to conjugation with PEG. We observed an antibody-dependent
increase in gene transfer in primary myoblasts, but not in control HepG2 cells.
Further increases in antibody concentration resulted in a reduction of gene
transfer. In addition, when primary myoblasts preincubated with an RGD peptide
were infected with the PEG-treated vector-targeting ligand complex, we observed
a reduction in gene delivery suggesting that the modified vector could be using
the same pathway for internalization as the unmodified vector. Further studies
are underway to study intracellular trafficking of the vector in muscle cells.
Our results encourage us to pursue studies of the modified vector in utero,
with a hope for synergistic benefits of combined fetal gene delivery and
cell-specific targeting.
32)
Effect of Animal Age and the Route of Administration on Systemic AAV-9
Transduction
Brian P. Bostick, Arkasubhra Ghosh,
Yongping Yue, Chun Long, Donsheng Duan Molecular Microbiology and Immunology,
School of Medicine, University of Missouri, Columbia, MO
Recombinant adeno-associated virus serotype-9 (rAAV-9) has recently been
identified as an exceptional vector for systemic gene transfer. Most studies
have focused on the transduction profile following intravenous delivery. It is
not well appreciated how the route of administration (intra-venous vs.
intra-arterial) and the age of the animal at the time of gene transfer (neonatal
vs. adult) influence the transduction profile. To examine the effect of the
administration route, we injected newborn C57Bl/10 mice with AV.RSV.AP (an
rAAV-9 vector carrying the alkaline phosphatase (AP) reporter gene; 12 x 1010
vg/g body weight) either intravenously via the facial vein, or intra-arterially
via the left ventricle. To determine the effect of age, we compared 40-hr-old
neonatal mice (12 x 1010 vg/g body weight, 3.5 x 1011
vg/mouse) and 7-wk-old adult mice (7 x 1010 vg/g body weight, 1 x 1012
vg/mouse) injected intravenously. Transgene expression was evaluated at
~12
weeks post-injection. In general, regardless of the age or the route of
administration, we observed broad transduction in all body skeletal muscle with
the highest expression in the diaphragm and tongue muscles. Interestingly,
muscles composed predominantly of slow twitch fibers (such as the soleus muscle)
showed less expression. Alkaline phosphatase activity in the soleus muscle was
significantly lower than that in the tibialis anterior muscle (27.97
+7.97
units/mcg
protein vs. 144.57
+35.81
units/mcg
protein, respectively; p < 0.05). We also observed high transduction in cardiac
muscle in all groups. In addition to the strong striated muscle expression, we
found consistent lung transduction (including small conducting airways and
alveolar cells) in all groups. The route of administration seemed to have
minimal effect on the overall transduction profile. However, the age at the time
of injection seemed to play a role in several tissues/organs. Both the liver and
the kidney showed higher expression when AAV was delivered to adult mice. Adult
mice also showed relatively high transduction in vascular endothelial cells and
vascular smooth muscle. The most striking difference was seen in the retina. The
ganglion cell layer was strongly transduced following neonatal administration
but not after adult delivery. Taking together, our results provide additional
support for the use of rAAV-9 in systemic gene therapy. Efficient transduction
via both venous and arterial routes allows maximal flexibility in a clinical
setting. However, the age-associated differences should be considered when
designing gene therapy protocols with rAAV-9. (Supported by grants from the
National Institutes of Health, the Cystic Fibrosis Foundation and the Muscular
Dystrophy Association).
33)
The AAV9 Capsid Preferentially Transduces Cardiac Tissue and Demonstrates Unique
Behavior In Vivo
Christina A. Pacak, Cathryn S. Mah, Bijoy
Thattaliyath, Melissa A. Lewis, Sean Germain, Glenn A. Walter, Barry J. Byrne
Department of Molecular Genetics and Microbiology, University of Florida,
Gainesville, FL; Powell Gene Therapy Center, University of Florida, Gainesville,
FL; Department of Pediatric Cardiology, University of Florida, Gainesville, FL;
Department of Cellular and Molecular Therapy, University of Florida,
Gainesville, FL; Department of Physiology and Functional Genomics, University of
Florida, Gainesville, FL
Pompe disease is a form of muscular dystrophy and metabolic myopathy caused by a
deficiency in the enzyme acid alpha glucosidase (GAA). A lack of GAA results in
the accumulation of glycogen in lysosomes and consequent dysfunction in cells
throughout the body that can result in cardiorespiratory failure. Previously, we
have demonstrated that intravenous administration of AAV2/9 to adult and newborn
mice and non-human primates can result in efficient transduction of cardiac
tissue. Here we demonstrate through MRI, ECG, biochemical, and histological
analysis that intravenous administration of AAV2/9 carrying a therapeutic
transgene in newborn (prior to disease presentation) or adult (where cardiac
abnormalities were present) mice can ameliorate the cardiac phenotype in a mouse
model of Pompe disease (Gaa-/-). AAV2/9 treatment normalizes
the shortened PR interval in Gaa-/- mice by an average of 5 ms
(from 22% shorter than wild-type to only 11% shorter). Cardiac MR demonstrated a
return of the EF% to wild-type control levels following therapeutic gene
delivery at both ages. Furthermore, NMR, EM and PAS staining revealed clearance
of glycogen and healthy sub-cellular morphology in the hearts of treated mice.
Ex vivo force mechanics on excised diaphragm sections demonstrated an
improvement in the contractile force of treated mice and CT scans show a
distinct decrease in spinal kyphosis. In conclusion, these data show that IV
administration of rAAV2/9-CMV-Gaa can both prevent and correct the
cardiac phenotype and provide global improvement in a mouse model of Pompe
disease warranting application of this approach to the clinic setting to assess
its efficacy in humans.
34)
Scalable Method for Purification of Recombinant Adeno-Associated Viral Vector (rAAV)
Type 6
Jingmin Zhou, Bern Hauck, J. Fraser Wright,
Katherine A. High, Guang Qu Center for Cellular and Molecular Therapeutics, The
Childrens
Hospital of Philadelphia, Philadelphia, PA
Recombinant adeno-associated viral vector serotype 6 (rAAV-6) has been shown to
be highly efficient for muscle gene delivery, including delivery to skeletal and
cardiac muscle. This unique property makes rAAV-6 an especially attractive
vector to deliver therapeutic genes for diseases such as Duchenne muscular
dystrophy. However, systemic gene delivery will likely require production and
purification of large quantities of this vector, and therefore readily scalable
purification processes are critical for clinical development. We previously
reported a scalable purification process for rAAV type 2 and successfully
implemented the process into cGMP manufacturing to purify clinical vectors (Qu
et al, J. Virol. Meth. 2007 Mar.140 183-92). Here we described a fully scalable,
column chromatography-based method for rAAV-6 purification. The method developed
included cation exchange chromatography followed by an anion exchange column
chromatography to remove cell culture process impurities (HEK293 cell and cell
culture medium -derived),
and a final single CsCl gradient centrifugation step to remove AAV-6 empty
capsids, a product related impurity. In contrast to results obtained during
process development for AAV-2 vector purification by ion exchange chromatography
and AAV-6 vector purification by heparin resin chromatography, the rAAV-6
particles were found to be very sensitive (ie. easily detached from the ion
exchange resins) to the washes using buffers containing ionic detergents such as
N-lauroyl sarcosine or sodium cholate. These detergent washes could not be used
in rAAV-6 cation exchange column chromatography to remove cellular proteins.
Highly efficient vector purification was achieved by optimizing washing and
elution conditions, including pH, salt concentration and volume of the washing
and elution buffers. Typically 80% of the starting / feed-stream vector genomes
were recovered through the two-column process, and highly purified, empty capsid-free
rAAV-6 vector was obtained in the final preps. The vector preps have been shown
to be effective by transduction in vitro.
35)
Phase I-IIa Gene Therapy Protocol for LGMD 2C: Clinical Strategy and
Implications
Hafedh Haddad, Didier Caizergues, Aude
Rigolet, Carole Masurier, Anne-Marie Douar, Bernard Gjata, Patricia
Noguiez-Hellin, Muriel Audit, Loic Millot, Otto Merten, Pascal Laforet, France
Leturcq, Thomas Voit, Bruno Eymard, Pierre Carlier, Olivier Benvéniste, François
Lemoine, Norma Romero, David Klatzmann, Jean-Yves Hogrel, Marie Rosier-Montus,
Patrice Denèfle, Serge Herson, Anne-Marie Masquelier Labs, Genethon, Evry,
France; Hopital,
La Pitie-Salpetriere,
Paris, France; Institut de Myologie, (IDM), Paris, France; Hopital,
Cochin, Paris, France
Limb girdle muscular dystrophy type 2C (LGMD 2C) is a rare autosomal recessive
muscular disorder caused by mutations in the gamma sarcoglycan (gama-SG)
gene, the most frequently occurring one being the 525delT mutation. Patients
with LGMD 2C commonly present with proximal and progressive muscular weakness
before the age of 10 and become wheelchair bound by age 12 on average.
Cardiomyopathy and respiratory insufficiency may develop during the course of
the disease and lead to poor prognosis and premature death. After completion of
the pre-clinical work in collaboration with Harvard Gene Therapy Initiative and
University of Pennsylvania School of Medicine, GENETHON has obtained an Orphan
Drug Designation by EMEA in October 2004 and approval from the French Agencies
in November2006 to initiate a phase I/IIa clinical gene therapy trial using a
serotype 1 adeno-associated virus (AAV1) vector harboring the human
gama-sarcoglycan
gene under the control of a desmin promoter. The current investigation center is
the Department of Internal Medicine and the Institute of Myology at La Pitie-Salpetriere
Hospital in Paris. The investigational product is administered by a single
intramuscular injection into the radial muscle.The primary objective of this
phase I-IIa is to evaluate the clinical safety of local intramuscular injection
of the gene therapy product and tolerance with a 6 months follow-up. Secondary
objectives are to monitor local and systemic immune responses, assess
histological modifications and gene transfer into injected muscles, including
sarcoglycan protein complex expression and therapeutic gene distribution. A
total of 9 patients, aged above 15 and harboring the 525delT mutation, will be
enrolled sequentially in the study and assigned to 3 cohorts with a single
dose-escalation. A 6 month follow-up is planned for each patient. Evaluation
will address clinical histological, morphological, biological, immunological and
functional parameters as well as MRI. First patient has been treated in Paris in
December 2006. The phaseI-IIa is planned to last 18 months. In view of the phase
IIb of this clinical trial in which systemic administration will be performed,
important preclinical and clinical points to consider are being tackled and will
be presented.
36)Transplantation of Autologous
Genetically Modified Myoblasts: A Possible Treatment for Recessive Muscular
Dystrophies
Jacques P. Tremblay, Simon Quenneville,
Pierre Chapdelaine, Zoé Coulombe, Christophe Pichavant Human Genetic, CRCHUL,
Quebec, Canada
Autologous myoblasts are possible vectors to deliver genes into muscle fibers of
patients affected by various recessive muscular dystrophies. These genes may be
incorporated in the myoblasts in culture by transfection or nucleofection with a
plasmid using the Phi C31 integrase system. The gene of interest may also be
transduced with a retrovirus, a lentivirus or a dual high-capacity (hc)
adenovirus (Ad)-adeno-associated virus (AAV) hybrid vector (HV) that can deliver
two full-length dystrophin-encoding modules into target cells. These various
methods have been used successfully by our research team to introduce
successfully the micro-dystrophin gene and the full length dystrophin gene in
myoblasts of animal models or of patients affected by Duchenne Muscular
Dystrophy (DMD). These genetically modified cells were than transplanted with
success in muscles leading to the expression of the transgene in thousands of
muscle fibers, even in non-human primates. We have also used lentiviral vector
to introduce a normal UDP-GlcNAc 2-epimerase / N-acetylmannosamine kinase (GNE)
gene into myoblasts of patients affected by Hereditary Inclusion Body Myopathy (HIBM).
This genetic correction permitted to increase the synthesis of sialic acid in
the genetically corrected cells and thus increase the sialylation of the surface
glycoproteins of these cells. To avoid the potential toxicity of the transgene
expression in myoblasts the transgene may be place under the control of a muscle
specific promoter so that it is expressed only following fusion with the host
muscle fibers. The genetic modification of myoblasts is also an excellent way to
introduce U7snRNA to induce exon skipping. This approach has permitted the
expression of a quasi-dystrophin both in vitro and in vivo. These
results demonstrate the feasibility of ex vivo gene for many recessive
muscular dystrophies.
37)
Vascular Endothelial Growth Factor Overexpression Improved Survival and
Engraftment of Human Myoblasts into SCID Mouse Muscles
Manaf Bouchentouf, Basma F. Benabdallah,
Pascal Bigey, Jacques P. Tremblay Human Genetics, CHUL, Quebec, Canada;
Laboratoire de Pharmacologie Chimique et Genetique,
Inserm U640 -
CNRS, Paris, France
Background: Transplantation of muscle precursor cells (MPC) or
myoblasts is considered as a potential approach to repair damaged skeletal
muscles in a number of recessive muscular dystrophies, in particular Duchenne
muscular dystrophy (DMD). This approach is however limited by the death of the
majority of the cells within four days following their injection. The purposes
of this study were to evaluate whether ischemia was implicated in the early
death of myoblasts transplanted into SCID mouse TA muscles and to evaluate the
effect of VEGF overexpression on their survival, proliferation and graft
success. Methods: In vitro apoptosis was induced by culturing
myoblast in 6 well plates at 75% of confluence with serum free medium 24 hours
in hypoxic environment. To evaluate effect of VEGF165 on transplanted myoblasts,
intramuscular injections were performed into TA muscles of 18 SCID mice using a
30-gauge needle insulin syringe. Nine mice were injected with 40
mcg
of pCEP4-VEGF plasmid ressuspended in 40
mcl
of 0,9% sterile NaCl and nine other mice were injected with 40
mcg
of pCEP4 empty plasmid ressuspended in 40
mcl
of 0,9% sterile NaCl. Electroporation was performed using two stainless steel
plate electrodes placed on the injected muscles and eight square electric pulses
of 20 ms length with 200 V/cm voltage. Hypoxia within transplanted human
myogenic cells was detected using the Hypoxyprobe-1 kit. To quantify the in
vivo cell mortality, myoblasts were radio-labeled with [methyl-14C]
thymidine (50 mCi/mmol). Radio-labeled cells (1x106) were injected
into 8 sites of the TA muscle using a glass micro-pipette.The amount of
radio-label within each TA muscle was measured on DNA extracts using liquid
scintillation counter Results: VEGF165 overexpression on human
myoblast enhanced their survival in vitro but had no effect on their
proliferation. Electroporation of SCID mouse TA muscles with pBabe-VEGF-Puro
vector promoted VEGF165 production. Electroporation of SCID mouse TA muscles
with vector coding for the VEGF165 reduced transplanted myoblast death but did
not improve their proliferation. VEGF165 overexpression enhanced myoblast graft
success. Conclusion: Our results suggest a role of hypoxia in the
death of transplanted myoblast and that VEGF overproduction could be an
alternative to improve their survival.
38)
AAV Vector-Mediated Expression of Follistatin Stimulates Skeletal Muscle
Hypertrophy and Can Enhance Functional Performance in Models of Muscle Disease
Paul Gregorevic, Norman A. Meznarich, James
M. Allen, Miki Haraguchi, Leonard Meuse, Eric Finn, Jeffrey S. Chamberlain Paul
D Wellstone Muscular Dystrophy Cooperative Research Center, Department of
Neurology, The University of Washington, Seattle, WA
Life threatening loss of skeletal muscle strength is associated with a host of
conditions including muscular dystrophies, aging, cancer, and chronic
obstructive pulmonary disease. Interventions that enhance muscle function in
patients offer the potential for considerably improving quality of life and
extending lifespan. We demonstrate that an intervention utilizing rAAV6 vectors
carrying an expression cassette encoding the short form of human follistatin can
stimulate muscle fiber hypertrophy to increase murine skeletal muscle mass by
100% and force producing capacity by >50% within 6 weeks of intramuscular
injection. The hypertrophic effects mediated by this intervention are
constrained to the region of transduction, but intravascular administration of
this vector to mice facilitates body-wide skeletal muscle hypertrophy and
increases contractile capacity on a similar time scale without loss of effect
for over 16 months. This potent and rapidly acting intervention represents a
powerful model system to study the biology of skeletal muscle adaptation, but
may also prove valuable as a timely intervention where rapid loss of muscle
strength is a serious health concern. We have ascertained that interventions
incorporating rAAV6-Fst administration can restore muscle mass and function in
murine models of muscle disease including aging-related muscle wasting, where
the muscles of treated animals approach the functional capability of muscles
from young adult animals. Ongoing work is aimed at utilizing rAAV6-mediated
expression of follistatin to study the mechanisms governing muscle adaptation,
and to establish the therapeutic potential for treatment of conditions caused or
complicated by loss of skeletal muscle strength.
39)
Intravenous Injection Cofactors of Adeno-Associated Virus Produce Varying
Tissue-Specific Transduction Levels in Adult Mice
Brian R. Schultz, Paul Gregorevic, Eric
Finn, Caitlin Doremus, Leonard Meuse, Miki Haraguchi, James M. Allen, Jeffrey S.
Chamberlain Molecular and Cellular Biology, University of Washington, Seattle,
WA; Medical Scientist Training Program, University of Washington School of
Medicine, Seattle, WA; Department of Neurology, University of Washington School
of Medicine, Seattle, WA
Adeno-associated virus (AAV) is a promising vector for gene replacement therapy
in many genetic diseases. In particular, recombinant AAV serotype 6 (rAAV6) has
been utilized to administer a therapeutic gene with beneficial results in mouse
models of Duchenne muscular dystrophy (DMD). In genetic diseases affecting
tissues throughout the body, such as DMD, effective methods of systemic
transduction are essential. Manipulating intervention conditions, such as vector
dose and co-delivery of transduction-facilitating cofactors, may be important
for optimizing the efficacy of viral-vector gene therapy. At increasing doses of
intravenously delivered rAAV6-CMV-hPLAP to adult C57BL/6 mice, cardiac muscle
and diaphragm display a progressive dose-response; however, soleus and tibialis
anterior muscles exhibit measurable transduction only after receiving a
threshold level of vector genomes above that at which heart and diaphragm begin
to show transduction. Administering vector in conjunction with VEGF, heparin
sodium, VEGF + heparin sodium, or mouse serum albumin, or under conditions of
anesthesia or exercise, results in varying degrees of transduction increase or
decrease depending on the muscle group. Another factor to consider is the viral
capsid itself. In the preparation of rAAV6, intact virions lacking DNA ("empty
capsids")
are produced in excess of those virions that contain vector genomes ("full
capsids").
During vector preparation, full capsids are routinely separated from empty
capsids by density gradient ultracentrifugation. Adding an excess of empty
capsids into the intravenous injection solution increases transduction to
varying degrees, again depending on muscle group. These results suggest that the
vector genome dose and/or cofactors involved with intravenous injection of rAAV6
can be tailored to target tissue groups and/or levels of transduction.