RESEARCH THAT WILL BE PRESENTED IN ANNUAL MEETING OF AMERICAN SOCIETY OF GENE THERAPY 2012, MAY 2012
Demonstration of Systemic Exon 45-55 Multiple
Skipping in Dystrophic mdx52 Mice
Yoshitsugu Aoki, Tetsuya Nagata, Akinori Nakamura, Takashi Saito,
Jun Tanihata, Stephanie Duguez, Kanneboyina Nagaraju, Eric Hoffman, Terence
Partridge, Toshifumi Yokota, Shin'ichi Takeda. Department of Molecular Therapy,
National Institute of Neuroscience, National Center of Neurology and Psychiatry
(NCNP), Kodaira, Tokyo, Japan; Department of Medicine (Neurology and
Rheumatology), Shinshu University School of Medicine, Matsumoto, Nagano, Japan;
Research Center for Genetic Medicine, Children's National Medical Center,
Washington, DC; Department of Medical Genetics, School of Human Development,
Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Canada
Background: Duchenne muscular dystrophy (DMD), the most common form of muscular
dystrophy, is caused by a lack of dystrophin. A most promising therapeutic
approach is antisense oligo-mediated elimination of frame disrupting mutations
by exon skipping. Two major hurdles are, limited applicability, and uncertain
function of the resulting quasi-dystrophin. Skipping of exon 45-55 at the
mutation hot-spot would address both issues. Here, we tested the feasibility of
antisense oligo-mediated exon 45-55 multiple skipping in mdx52 mice which harbor
a deletion mutation in exon 52 of the Dmd gene. Methods: We newly designed 87
antisense oligo sequences against the ten exons between exons 45 and 55 except
exon 52. We screened a series of phosphorodiamidate morpholino oligomers with
cell-penetrating moiety (vPMOs) singly and in ten-oligo cocktail using H-2Kb-tsA58
(H-2K)-mdx52 myogenic cells. Then we tested the molecular efficacy by
intramuscular injection and intravenously injected an optimized vPMOs cocktail
(0.6 mg/kg each of the ten-oligo, biweekly for 18 weeks) into 5-week-old mdx52
mice. The efficiency and efficacy of exon 45-55 multiple skipping were tested at
the mRNA, protein, histological and functional levels. Results: We demonstrated
skipping of all 10 exons in H-2K-mdx52 myotubes. Better still, in dystrophic
mdx52 mice, systemic injections of such ten-oligo cocktail induced extensive
dystrophin expression at the subsarcolemma in skeletal muscles throughout the
body with 15% dystrophin protein levels on average. The expression of
β-dystroglycan and α1-syntrophin were fully restored, but neuronal nitric oxide
synthase (nNOS) was not detected by western blotting. Dystrophic pathology and
skeletal muscle function were improved without any detectable toxicity after the
systemic vPMO cocktail treatment. Conclusion: We demonstrated an effective
rescue by systemic exon 45-55 multiple skipping in a DMD animal model.
Interpretation: This represents a major advance on the simple use of exon
skipping to change the phenotype from DMD to its milder form Becker muscular
dystrophy (BMD), because, on the basis of clinical reports, it could rescue more
than 63% of patients with deletion mutations, while spontaneous deletions of
this region are associated with asymptomatic or exceptionally mild phenotypes
with almost normal life expectancy.
RNAi Therapy for Dominant Limb Girdle Muscular
Dystrophy Type 1A
Jian Liu, Lindsay Wallace, Sara E. Garwick-Coppens, Darcy Nelson,
Carol Davis, Susan V. Brooks, Michael E. Hauser, Jerry R. Mendell, Scott Q.
Harper. The Research Institute at Nationwide Children's Hospital, Columbus, OH;
Duke University Medical Center, Durham, NC; University of Michigan School of
Medicine, Ann Arbor, MI
Limb Girdle Muscular Dystrophy (LGMD) refers to a group of 22 disorders
characterized by progressive wasting and weakness of pelvic and shoulder girdle
muscles. Commonly, patients require wheelchair assistance, and individuals with
some forms of LGMD may have cardiac and respiratory muscle involvement. Modest
improvements in a limited set of muscles may dramatically improve patients'
quality of life, but there is currently no effective treatment. Over the last
two decades, the feasibility of using gene therapy to treat muscular dystrophy
has improved, but previous strategies have almost exclusively focused on
replacing defective or missing genes underlying recessive disorders. These
strategies are not feasible for treating dominant muscular dystrophies,
including the 7 dominant forms of LGMD (LGMD1). Instead, patients with dominant
LGMD1 would benefit from reduction of their pathogenic alleles. In this study,
we developed the first RNAi-based pre-clinical treatment for LGMD1A, which is
caused by dominant mutations in one allele of the myotilin (MYOT) gene. Our
strategy involved delivering MYOT targeted artificial microRNAs (miMYOT) to
muscles of the T57I transgenic mouse model of LGMD1A using AAV6 vectors. We
found that miMYOT vectors, but not controls, significantly reduced soluble
mutant MYOT protein and MYOT aggregates that characterize LGMD1A were either
absent or very small in treated muscles. This reduction was accompanied by
significantly recovery in function, as indicated by improvements in muscle mass,
treadmill running performance, and whole muscle force in treated T57I mice. We
are currently quantifying these obvious histopathological improvements, and are
determining whether our miMYOT vectors will improve overall muscle weakness in
T57I mice. These studies represent important first steps toward translating
targeted RNAi gene therapy approaches for LGMD1A.
Genetic Correction of Dystrophin by Engineered
Nucleases
David G. Ousterout, Pablo Perez-Pinera, Matthew T. Brown, Charles A.
Gersbach. Biomedical Engineering, Duke University, Durham, NC
Duchenne Muscular Dystrophy (DMD) is the most common hereditary monogenic
disease, occurring in about 1 in 3500 male births. DMD is caused by a genetic
defect in dystrophin, an essential musculoskeletal protein. The absence of
dystrophin leads to muscle weakness and wasting, resulting in fatal respiratory
and cardiac disease. At present, there are no treatments that can effectively
address the poor life expectancy and quality of life of these patients. Recent
studies have demonstrated the potential of a new class therapeutics based on
targeted gene editing by designer nucleases. These nucleases take advantage of
natural DNA repair mechanisms to create desired changes to a genetic sequence.
Importantly, zinc-finger nucleases (ZFNs) against the HIV-1 co-receptor CCR5 are
in an ongoing Phase 1/2 and two Phase 1 clinical trials, demonstrating the
feasibility of this type of therapeutic approach. Other studies are utilizing
ZFNs to correct genetic mutations associated with sickle cell anemia, X-linked
severe combined immunodeficiency, hemophilia B, and alpha-1 anti-trypsin
deficiency. This project utilizes synthetic nucleases to edit and correct the
dystrophin gene as a novel potential therapy for DMD. The advantage of this
method is that the native dystrophin gene is restored, presumably including all
of the major isoforms and functions of dystrophin. We designed ZFNs and TALE
nucleases (TALENs) targeted to exon 51 of the human dystrophin gene that
potentially address greater than 13% of all DMD mutations. ZFNs were generated
using modular assembly (MA) and Context-Dependent Assembly (CoDA). TALENs were
generated using the Golden Gate Assembly method. Five MA-ZFNs, seven CoDA ZFNs,
and twelve TALENs were tested for gene editing activity at the endogenous
dystrophin gene in human K562 cells. Two MA-ZFNs, three CoDA ZFNs, and four TALE
nucleases were highly active, modifiying 6-13% of alleles as measured by the
Surveyor nuclease assay. One ZFN of interest binds directly in exon 51 and
mediated homology-directed repair in 6% of alleles using plasmid- and
single-strand oligonucleotide-based donor templates. One TALEN mediated
high-efficiency gene editing in 13% of alleles. Assuming that one third of these
NHEJ events results in reading frame restoration, this approach has a potential
gene correction rate of up to 4.3%. These nucleases were also active in
immortalized myoblasts from DMD patients with an NHEJ rate of 1-12% as measured
by Surveyor. We also detected successful homology-directed repair in these DMD
cells after co-delivering the designer nucleases with a donor template designed
to correct the dystrophin gene. We predict that this will lead to restored
dystrophin function and expression in the corrected cells. Ongoing studies are
assessing dystrophin gene correction and protein restoration in DMD
patient-derived myoblasts in vitro and by in vivo muscle transplantation into
immunodeficient mice. The development of these nucleases therefore presents a
transformative approach to treating DMD.
Muscle Force Improvement by Long-Term Systemic
Expression of an AAV9 Minidystrophin after Delivery in Young Adult GRMD Dogs
without Immune Suppression
Juan Li, Chunping Qiao, Dan Bogan, Ruhan Tang, Janet Bogan, Tao Bian,
Jianbin Li, Jennifer Dow, Zhenhua Yuan, Joshua C. Grieger, Richard Jude Samulski,
Joe Kornegay, Xiao Xiao. Division of Molecular Pharmaceutics, Eshelman School of
Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC; Gene
Therapy Center, School of Medicine, University of North Carolina at Chapel Hill,
Chapel Hill, NC
Recently we reported efficient and long-term transgene expression without immune
suppression in a young adult golden retriever muscular dystrophy (GRMD) dog, by
a single intravenous (i.v.) injection of an AAV9 human minidystrophin vector
containing a muscle-specific (MS) promoter (ASGCT 2011 abstract). However, we
did not examine muscle functions in that dog. To repeat the gene expression
results and also to examine immunology and muscle force improvement, we carried
out an experiment using 8 young GRMD dogs from the same litter randomly divided
into 2 groups, 2 males and 2 females per group for the gene therapy study. In
this study,we first pre-screened anti-AAV9 antibodies by an in vitro infection
assay and found no detectable neutralizing antibodies in all the dogs. At 12
weeks of age, four dogs (weight 5.8kg, 7.1kg, 7.5kg, 7.9 kg respectively) were
injected with the AAV9-MS-human Mini-dystrophin vector by a leg vein at the dose
of 3.0x1013/kg. The other 4 dogs were used as untreated controls.
Initial muscle biopsies at 4 weeks post injection showed detectable but low
levels of minidys expression by immunofluorescent (IF) staining and western blot.
The second muscle biopsies at four month post injection, however, showed
significantly increased gene expression levels. The results of quantitative PCR
of vector DNA were also consistent with the IF staining and western blot data.
ELISPOT assays against minidys peptide library at 7 weeks post AAV injection
revealed no differences in all 8 GRMD dogs and a normal control dog. None of
them had readings above background level (50 spots/106 PBM cells). We
next investigated if muscle forces could be improved after AAV9-MS-human
Mini-dystrophin vector gene therapy. Several phenotypic markers were evaluated
in dogs at both 2 (pretreatment) and 6 months. The principal outcome parameter
was tetanic isometric tibiotarsal joint extension force, which showed consistent
decline in GRMD dogs in our natural history studies. Values for the gene therapy
treated dogs increased from 1.75 N/kg ± 0.345 at 2 months to 2.05 N/kg ± 0.356
at 6 months, while those of untreated dogs decreased from 1.32 N/kg ± 0.11 to
1.09 ± 0.270 (p < 0.01). The muscle force data are in general agreement with
long-term expression of minidystrophin gene. In addition, other therapeutic
parameters such as muscle MRI and joint angle measurement are being investigated.
In conclusion, the above results further demonstrated that long-term
minidystrophin gene expression could be achieved by a single i.v. injection
without immune suppression. More importantly, muscle functions could be
significantly improved as a result of AAV9-MS-minidystrophin gene therapy.
RNA Interference Inhibits DUX4-Induced Muscle
Toxicity In Vivo: Implications for a Targeted FSHD Therapy
Lindsay M. Wallace, Sara E. Garwick-Coppens, Jian Liu, Jacqueline
Domire, Scott Q. Harper. Molecular, Cellular, and Developmental Biology Graduate
Program, The Ohio State University, Columbus, OH; Center for Gene Therapy, The
Research Institute at Nationwide Children's Hospital, Columbus, OH; Integrated
Biomedical Sciences Graduate Program, The Ohio State University, Columbus, OH;
Department of Pediatrics, The Ohio State University, Columbus, OH
Facioscapulohumeral muscular dystrophy (FSHD), one of the most common inherited
muscle disorders, is characterized by asymmetric wasting and weakness in muscles
of the face, scapula, and limbs. The clinical features of FSHD were described
over a century ago, and a chromosomal deletion that reduces the copy number of
D4Z4 repeats on chromosome 4q35 was linked to the disease in 1992. Unfortunately,
because these D4Z4 contractions do not completely remove any obvious genes, the
pathogenic mechanisms underlying FSHD remained unresolved. As a result, FSHD
research for the last two decades has been primarily focused on understanding
pathogenesis, while translational research for the disease has been largely
lacking. Today no targeted treatment for FSHD exists, but recent findings
suggest the barrier to translation may be lowering. Several studies, including
our work, support an FSHD pathogenesis model involving over-expression of the
pro–myopathic gene, DUX4. Thus, DUX4 inhibition may be a promising therapeutic
strategy for FSHD. In this study, we tested a pre-clinical RNAi-based DUX4 gene
silencing approach as a prospective treatment for FSHD. We used adeno-associated
viral (AAV) vector-delivered therapeutic microRNAs to correct DUX4-associated
myopathy in vivo. We found that our DUX4-targeted microRNAs (miDUX4)
significantly reduced DUX4 RNA and protein and protected mouse muscles from
DUX4-induced damage. Specifically, miDUX4-treated muscles showed minimal to no
myofiber degeneration, regeneration or activation of apoptotic pathways, while
control-treated muscles had widespread damage associated with elevated DUX4
levels. These histological improvements were also associated with a functional
improvement measured by grip strength. Our results provide proof–of–principle
for RNAi therapy of FSHD through DUX4 inhibition, and importantly, this strategy
can be modified to target other genes that may be mis-expressed in FSHD.
A Novel Rationally Designed AAV Micro-Utrophin
Vector Recruits nNOS to the Sarcolemma
Yi Lai, Junling Zhao, Yongping Yue, Dongsheng Duan. Molecular
Microbiology and Immunology, University of Missouri, Columbia, MO
Sarcolemmal neuronal nitric oxide synthase (nNOS) is crucial for normal muscle
function. Absence of sarcolemmal nNOS plays a critical role in the pathogenesis
of muscular dystrophy. Duchenne muscular dystrophy (DMD) is caused by mutations
in the dystrophin gene. Dystrophin anchors nNOS to the sarcolemma. Utrophin is a
homolog of dystrophin and has been extensively investigated as a candidate gene
for DMD gene therapy. Unfortunately, unlike dystrophin, utrophin cannot recruit
nNOS to the sarcolemma. Inability of utrophin to restore sarcolemmal nNOS may
significantly attenuate its therapeutic effect. To overcome this drawback of
utrophin, we set to engineer novel utrophin genes that can fix nNOS to the
muscle cell membrane. We recently found that dystrophin spectrin-like repeats 16
and 17 (R16/17) mediate sarcolemmal nNOS localization. Utrophin R15/16 shares
great homology with utrophin R16/17. In this study, we examined whether
replacing utrophin R15, or R16 or both with respective dystrophin R16 and R17
can result in novel utrophin genes capable of nNOS anchoring. A series of four
repeats micro-utrophin AAV vectors were generated. All micro-utrophin genes
contained a flag tag, utrophin N-terminus, utrophin repeats 1, and 22 and
utrophin cysteine-rich domain. The two repeats between utrophin R1 and R22 were
either original utrophin R15/16 or engineered with dystrophin R16/17
substitution. The micro-utrophin gene was packaged into AAV-9 and delivered AAV
viruses to the tibialis anterior muscle of utrophin and dystrophin double
knockout mice. One month after injection, we examined microgene expression and
sarcolemmal nNOS localization. Robust expression was observed in all AAV
infected muscle. As expected, the microgene construct carrying original utrophin
R15/16 did not recover sarcolemmal nNOS. Constructs with either dystrophin R16
or R17 failed to restore nNOS to the sarcolemma. However when both repeats were
replaced, sarcolemmal nNOS expression was restored. Our results suggest that
both dystrophin R16 and R17 are required for nNOS localization. Further,
dystrophin R16/17 is sufficient for sarcolemmal nNOS expression in the context
of utrophin. The novel micro-utrophin gene described here may hold great promise
for DMD gene therapy.
AAV-Mediated Gene Therapy in Limb-Girdle Muscular
Dystrophy 2I (LGMD2I) Mouse Model
Chi-Hsien Wang, Bin Xiao, Chungping Qiao, Yiumo M. Chan, Ru-Hang
Tang, Juan Li, Elizabeth Keramaris-Vrantsis, Peijuan Lu, Qilong Lu, Xiao Xiao.
Department of Molecular Pharmaceutics, Eshelman School of Pharmacy, University
of North Carolina-Chapel Hill, Chapel Hill, NC; McColl-Lockwood Laboratory for
Muscular Dystrophy Research, Neuromuscular/ALS Center, Carolinas Medical Center,
Charlotte, NC
Fukutin-related protein (FKRP) is a putative glycosyltransferase. The genetic
mutations of the FKRP cause a wide spectrum of disease phenotypes ranging from
late onset and milder limb-girdle muscular dystrophy 2I (LGMD2I) without central
nervous system defect to severe allelic diseases such as congenital muscular
dystrophy 1C (CMD1C), Walker-Warburg Syndrome (WWS) and muscle-eye-brain disease
(MEB), both with defects in the central nervous system. Currently, neither
effective treatment is available for FKRP-related muscular dystrophies. Recently,
we have created a novel and viable FKRP knockin mouse model L276Ineo+ with
disease phenotypes recapitulating the mild LGMD2I. The availability of this
unique animal model enabled us to test postnatal AAV-mediated gene therapy for
LGMD2I in this study. The optimized FKRP cDNA was cloned into AAV vector
containing ubiquitous CB promoter (CMV enhancer and beta-actin promoter), and
different dosages of AAV9-CB-opti-FKRP-myc vectors were delivered into neonatal
homozygous L276Ineo+ mice via intraperitoneal injection. Untreated or
AAV9-CB-GFP-treated homozygotes were used as controls. The mice have been
treated for over a year. Overexpression of FKRP in homozygous L276Ineo+ mice
significantly improved muscle morphology, pathology, and muscle membrane leakage.
Specifically, the presence of pronounced central nucleation is one of the
characteristics of the muscle of homozygous L276Ineo+ mice. However, the central
nucleation was barely found in the AAV FKRP vector-treated muscle. The serum
creatine kinase level, an indication of muscle leakage, was also significantly
decreased after the treatment (816.77±546.80 in homozygous L276Ineo+ mice vs
151±57.11 in treated mice). More importantly, the motor functions of the treated
mice has been greatly improved as evidenced by treadmill running test (homozygous
L276Ineo+ mice: 353.8±172.9 meters vs AAV-treated mice: 697.4±97.9 meters) and
grip force measurement (homozygous L276Ineo+ mice: 0.1713±0.0429 kg vs
AAV-treated mice: 0.2296±0.0127kg). In summary, our preliminary data indicated
that AAV-mediated gene therapy offers great therapeutic effects in the LGMD2I
mouse model. Overexpression of FKRP in treated mice ameliorated muscle pathology,
improved muscle physiology, and normalized serum creatine kinase level.
rAAV9-Mediated Microdystrophin Gene Transfer with
Immune Tolerance Induction Improves Dystrophic Phenotype of Canine X-Linked
Muscular Dystrophy
Hiromi Hayashita-Kinoh, Naoko Yugeta, Hironori Okada, Yuko
Nitahara-Kasahara, Tomoko Chiyo, Takashi Okada, Shin'ichi Takeda. Department of
Molecular Therapy, National Institute of Neuroscience, NCNP, Kodaira, Tokyo,
Japan
Background: Duchenne muscular dystrophy (DMD) is a congenital disease causing
progressive deterioration of skeletal and cardiac muscles because of mutations
in the dystrophin gene. We reported that local injection of rAAV2 or rAAV8 into
canine skeletal muscles without immunosuppression resulted in insufficient
transgene expression with immune responses. Here we transduced fetuses of the
canine X-linked muscular dystrophy in Japan (CXMDJ) to investigate
the strategy of inducing immune tolerance to the rAAV as well as the therapeutic
effects of rAAV9-microdystrophin. Methods: For fetal transduction, pregnant CXMDJ
heterozygote was anesthetized and fetuses at post-coital day 35 were injected
with 1x1012 g. c. of rAAV9-CMV-microdystrophin along with 1x1011
g. c. of rAAV9-CAG-luciferase into amniotic fluid under ultrasonic guidance. To
examine the immune tolerance to the rAAV, purified canine peripheral leukocytes
were exposed to rAAV9-microdystrophin for 4 hours, and then IFN-γ induction was
evaluated using qRT-PCR. For systemic microdystrophin expression, we
additionally injected rAAV9-CMV-microdystrophin into the jugular vein of 6 weeks
old CXMDJ. Transduced littermates were periodically compared each
other to assess gait function. Moreover, we analyzed the respiratory and cardiac
functions of the rAAV-injected dogs by using whole body plethysmography,
echocardiography and electrocardiography. Skeletal muscles of the rAAV-injected
animals were sampled to examine rAAV-derived microdystrophin expression at 28
months of age. Results: IFN-γ expression in the purified peripheral blood
leukocytes after the rAAV exposure was not induced in one of the rAAV-injected
dogs, suggesting successful induction of immune tolerance against rAAV. The
animal was additionally transduced with the rAAV9-CMV-microdystrophin at 6 weeks
old. rAAV-derived microdystrophin expression in the tibialis anterior muscles
and cardiac muscles was confirmed by immunohistochemistry in the transduced dog.
Monthly analysis of the transduced dystrophic dogs demonstrated improved gait
function compared to the non-injected littermate CXMDJ. At 1 year old,
respiratory function and cardiac function of the transduced affected dog was
better than those of the untransduced affected dog. Macroscopic findings of
heart and diaphragm of the transduced CXMDJ dog maintained nearly
normal, suggesting that the combination of the immune tolerance induction and
single additional intravenous injection of the rAAV-microdystrophin achieved
successful long-term transgene expression with improved dystrophic phenotype.
Conclusion: Our results demonstrate that induction of oral immune tolerance
against rAAV with long-term transgene expression can be achieved by direct
delivery of rAAV into amniotic fluid. We also showed that rAAV-mediated
microdystrophin transduction of the dystrophic dog improved their dystrophic
phenotype. These findings support the therapeutic benefits of microdystrophin
and future feasibilities of rAAV-mediated gene therapy strategies.
Increased Overexpression of Human Alpha7 Integrin
as a Potential Therapy for Duchenne Muscular Dystrophy
Kristin N. Heller, Chrystal L. Montgomery, Vinod Malik, Kimberly M.
Shontz, Paul M. L. Janssen, K. Reed Clark, Louise R. Rodino-Klapac, Jerry R.
Mendell. The Research Institute at Nationwide Childrens Hospital, Columbus, OH;
Department of Pediatrics, The Ohio State University, Columbus, OH
Duchenne Muscular Dystrophy (DMD) is a severe muscle disease caused by mutations
in the dystrophin gene. Dystrophin helps link integral membrane proteins to the
actin cytoskeleton and stabilizes the sarcolemma during muscle activity. We have
focused on a unique approach utilizing adeno-associated virus (AAV) to develop a
treatment for DMD using the mdx mouse model. Our strategy involves the
upregulation of α7 integrin, a laminin receptor in skeletal and cardiac muscle
that links the extracellular matrix to the actin skeleton, similar to that of
the dystrophin-glycoprotein complex. There is an increase in α7 in patients with
DMD and the mdx mouse and α7 has been proposed to be an important modifier of
dystrophic symptoms. Burkin et al. showed that transgenic expression of the rat
isoform in the mdx/utr -/- promoted satellite cell proliferation and activation,
maintenance of muscle integrity, promoted hypertrophy and reduced cardiomyopathy.
We have generated rAAV8.MCK.human α7 integrin and delivered it to the lower limb
of mdx mice through isolated limb perfusion. As mdx mice have endogenous α7
expression, we measured overexpression human α7 as a biomarker of efficacy.
Outcome measures include reversal of pathology and improvement in muscle
strength and resistance to eccentric contraction induced injury in the EDL
muscle. We have demonstrated approximately fifty percent of fibers in the TA and
EDL overexpressing human α7 integrin after delivery through the femoral artery.
The increase of human α7 integrin in skeletal muscle significantly protected
against loss of force compared with untreated (contralateral) muscles, although
measurement of normalized specific force in rAAV8.MCK.huα7 treated and untreated
contralateral mdx EDL muscles show no significant change. α7 integrin's
protection against eccentric contraction gives hope that this treatment will
preserve the muscle phenotype over time. In addition, as an endogenous protein
it has the benefit of being a non-immunogenic. We are currently treating mdx/utrn-/-
mice to determine if there is a similar improvement in muscle physiology and
investigating the mechanism that is involved in the protection from eccentric
contraction induced injury.
Systemic Delivery of nNOS-Recruiting
Mini-Dystrophin AAV Vectors Ameliorated Muscular Dystrophy in a Mouse DMD Model
Yadong Zhang, Dongsheng Duan. Molecular Microbiology and Immunology,
University of Missouri School of Medicine, Columbia, MO
The nNOS-recruiting mini-dystrophin gene is a promising candidate gene for
Duchenne muscular dystrophy (DMD) gene therapy. We recently developed a set of
dual AAV vectors (YZ27/YZ22) to express the nNOS-recruiting minigene. Local
injection yielded robust transduction and sarcolemmal nNOS localization (Zhang &
Duan Hum Gene Ther 23:98-103, 2012). Here, we examined the therapeutic efficacy
of this dual vector set following systemic gene transfer. We packaged YZ27/YZ22
in AAV-9 and administered to 3-week-old dystrophin-deficient mdx mice via tail
vein injection. Four months later we examined minigene expression,
histopathology and muscle force. Immunohistochemistry and Western blot revealed
broad mini-dystrophin expression in skeletal muscle. Inflammation and central
nucleation were reduced in treated mice. Physiology assay showed significant
improvement of specific muscle force in both anterior tibialis and extensor
digitorum longus muscles. These data have further demonstrated therapeutic
efficacy of the nNOS recruiting mini-dystrophin dual AAV vectors. Further
evaluation of this vector set in large animal models may set the foundation for
future clinical trial (Supported by NIH and MDA).
Adeno Associated Virus (AAV) Mediated Follistatin
Gene Transfer Toxicology Studies in Preparation of Phase I/II Clinical Trial
Janaiah Kota, Christopher Shilling, Chrystal Montgomery, Sarah
Lewis, Adam Bevan, Kim Shontz, Yuuki Kaminoh, Kristin Heller, Xiomara Rosales,
Laurence Viollet, Kevin Flanigan, K. Reed Clark, Brian Kaspar, Zarife Sahenk,
Jerry Mendell. Center for Gene Therapy, The Research Institute at Nationwide
Childrens Hospital, Columbus, OH
Inhibition of myostatin signaling is a therapeutic strategy to improve muscle
size and strength for muscle disease. Follistatin has been demonstrated as a
potent myostatin antagonist. In our previous pre-clinical work in both rodents
and non-human primates, intramuscular (IM) administration of an alternatively
spliced follistatin isoform, FS344, delivered using AAV serotype1 effectively
improved muscle mass and strength with no off target effects. To establish the
safety of rAAV1.CMV.follistatin344 for clinical trial use, a dose-ranging
toxicology study was carried out in mice including a dose 10-fold greater than
that planned for a phase I/II gene therapy clinical trial. C57BL/6 mice were
randomized to three experimental arms delivering rAAV1.CMV.follistatin344
bilaterally to quadriceps muscle by IM injection. Dose ranging corresponded to
the highest clinical dose (2x1012 vg/kg) or 10-fold higher (2x1013
vg/kg) compared to vector diluent. Animals were necropsied at 6, 12, 24 and 36
weeks (10 animals/timepoint) post injection (PI). Outcomes included: Body weight,
hematology, chemistry panels, reproductive hormones, immune studies, viral
vector biodistribution and histopathology of 24 organs from each animal. No
treatment related adverse clinical signs were observed throughout the study.
None of the animals showed abnormalities in hematology, clinical chemistry
parameters and reproductive hormones and viral DNA dissipated in non-injected
tissues by 24 weeks. At 6 weeks PI, in high dose animals, scattered focal
infiltrates of inflammatory cells were observed in the injected muscle
consistent with T-cell immunity to AAV1 as identified by IFN-g ELISpot, but no
other pathology was observed in any of the organs. Increase in quadriceps muscle
mass was observed at all-time points that correlated with myofiber hypertrophy
and muscle follistatin expression. Mean fiber diameter increased and remained
persistent from 6 weeks (63.3 ±2.1 µm) to 36 weeks (65.9 ± 2.1 µm) PI compared
to controls (46.6 ± 5.1µm). The density of PaX7 positive nuclei increased
rapidly at 6 weeks, prior to the occurrence of hypertrophic-multinucleated
muscle fibers at 12 weeks. No circulating antibody to follistatin was observed.
In conclusion, this study shows rAAV1.CMV.follistatin344 is safe and well
tolerated at the proposed clinical doses and causes satellite cell proliferation
and significant muscle hypertrophy by fusion with differentiated satellite cells.
Early transient inflammation in muscle is consistent with an immune response to
AAV1. These results support this approach for clinical trial use
Abbreviated Dystrophins Restore the Passive
Properties of the Extensor Digitorum Longus Muscle in Dystrophin-Null Mice
Chady H. Hakim, Dongsheng Duan. Molecular Microbiology and
Immunology, University of Missouri, Columbia, MO
Muscle and joint stiffness is a major clinical feature in Duchenne muscular
dystrophy (DMD) patients. DMD is one of the most common lethal inherited muscle
wasting diseases in childhood and it is caused by the loss of the dystrophin
protein. We recently showed that the extensor digitorum longus (EDL) muscle of
mdx mice (a DMD mouse model) exhibits disease-associated muscle stiffness.
Abbreviated forms of the dystrophin gene (micro and mini-dystrophin) are the
leading candidate genes for DMD gene therapy. Unfortunately, it has never been
clear whether these abbreviated genes can mitigate muscle stiffness. To address
this critical question, we examined the passive properties (elastic and viscous
properties) of the EDL muscle in transgenic mdx mice that express either a
representative minigene (ΔH2-R15) or one of the two most promising microgenes (ΔR2-15/ΔR18-23/ΔC
or ΔR4-23/ ΔC). The passive properties were measured in 6, 14, and 20-month-old
transgenic mice and the results were compared to those of age and sex-matched
normal and mdx mice. Surprisingly, despite significant truncation of the gene,
the elastic and viscous properties were completely normalized in every strain of
transgenic mice we examined. Our results demonstrate for the first time that
abbreviated dystrophin genes can effectively treat muscle stiffness in a DMD
model. Our findings provide important support to further develop mini-/micro-dystrophin
gene therapy.
A Novel Cell Therapy for Muscular Dystrophy by
Bone Marrow Stromal Cell: Mesenchymal Stem Cell Derived from Bone Marrow Can
Affect Skeletal Muscle Regeneration
Yasushi Maeda, Asuka Koga, Masatoshi Ishizaki, Tomohiro Suga.
Neurology, Kumamoto University Faculty of Life Sciences, Kumamoto, Japan;
Neurology, Kumamoto Saishunso National Hospital, Koshi, Kumamoto, Japan
Bone marrow stromal cells are stem cells differentiating in various mesenchymal
tissues: bone, fat, skeletal muscle and so on. These cells are called MSC and
widely noticed as an ideal cell for regenerative medicine. Its immunomodulatory
competence becomes evident as well. MSC having these multi biological properties
is very interesting in therapeutic application for various diseases. We
hypothesized that MSC may affect the regeneration of mesenchymal tissue like
skeletal muscle. In order to prove this hypothesis, we repetitively transplanted
MSC (dko-MSC) from a muscular disease model mouse, dystrophin / utrophin double
knockout mouse (D/U dko), into D/U dko mouse's peritoneal cavity. Nine times
transplantations, two millions of dko-MSC in each transplantation, were carried
out by 8-week-old. The transplantation obviously improved the body build,
skeletal structure and locomotor activity.
Histologically, the transplantation made myofiber hypertrophic comparing to the
wild-type mouse's myofiber. Surprisingly, transplantation drastically prolonged
life span.
We can prove that MSC modifes the skeletal muscle regeneration. Although the
mechanism is still unknown, autologous MSC transplantation to patient suffering
from muscular dystrophy will become a novel therapy. More importantly, this cell
therapy may be applied not only to muscular dystrophy but also common myopathy.
An Approach for Systemic Delivery of Embryonic
Stem Cells into Mouse Skeletal Muscle
Shigemi Kimura, Kowashi Yoshioka, Shiro Ozasa. Child Development,
Kumamoto University Graduate School, Kumamoto, Japan
We established genetically engineered ES (ZHTc6-MyoD) cells that harbor a
tetracycline-regulated expression vector encoding myogenic transcriptional
factor MyoD, for the therapy of muscle diseases, especially Duchenne muscular
dystrophy (DMD). Almost all the ZHTc6-MyoD cells were induced into muscle
lineage after removal of tetracycline. The undifferentiated ZHTc6-MyoD cells are
Sca-1+ and c-kit+, but CD34-, all well-known markers for mouse hematopoietic
stem cells. In addition, they are able to maintain themselves in the
undifferentiated state, even after one month of culture. Therefore, it is
possible to obtain a large quantity of ZHTc6-MyoD cells in the undifferentiated
state that maintain the potential to differentiate only into muscle lineage.
Additionally, the cells were infected with lentiviral vector carrying GFP gene
as maker. We injected the infected cells into vein of mdx mice, a model mouse of
DMD, for systemic delivery. A few GFP positive myofibers were detected in the
muscle of the mice. In addition, GFP positive cells were isolated from skeletal
muscle of the injected mice, and differentiated to muscle lineage. Finally the
myotubes beat. Therefore, our ES cells have considerable therapeutic potential
for treating muscle diseases.
Strategy for rAAV-Mediated Transduction of Common
Marmoset Skeletal Muscle To Generate NHP DMD Model
Hironori Okada, Hidetoshi Ishibashi, Hiromi Hayashita-Kinoh, Tomoko
Chiyo, Yuko Nitahara-Kasahara, Takashi Okada, Shin'ichi Takeda. Department of
Molecular Therapy, National Institute of Neuroscience, NCNP, Kodaira, Tokyo,
Japan; Department of Neurophysiology, National Institute of Neuroscience, NCNP,
Kodaira, Tokyo, Japan
[Background] Duchenne muscular dystrophy (DMD) caused by dystrophin gene
mutation is the most frequent and severe form of muscular dystrophy. Therapeutic
methods developed for DMD have been assessed using mouse and dog model (e.g. mdx,
GRMD, and CXMDJ) to reveal the interspecies differences of immune
response. Bisides, motor, emotional, and cognitive functions affected by lack of
dystrophin in central nervous system are left as issues unable to be assessed by
mouse and dog model, even if therapies were achieved to prolong patient's life.
Therefore, animal models with more advanced immune system and higher brain
function are required to comprehensively assess DMD therapeutics. Common
marmoset has various advantages as non-human primate (NHP) model, such as small
body, short gestation, multiple birth, and short sexual maturation. Although
generation of GFP transgenic common marmosets with gene-transfer to
pre-implantation embryo by lentiviral vector (rLV) was reported, this method
needs many donor and recipient animals for pre-implantation embryo. On the other
hand, adeno-associated virus vector (rAAV) is capable of safe gene transfer and
long-term transgene expression with a wide range of tissue specificity along
with transduction of non-dividing cells. In order to realize high-throughput
generation of DMD model marmoset using rAAV, we investigated in vivo
transgene expression patterns with rAAV transduction. [Methods] rAAV9
carrying CAG promoter-driven EGFP (rAAV9-CAG-EW) was injected into adult
marmoset TA and ECR muscles. rAAV1-CAG-EW was i.p. injected into new born
marmosets. Marmoset dystrophin gene was estimated from their genomic sequences
obtained from the UCSC Genome Browser website. For induction of frame-shift
mutation by exon skipping, h51AON1 anti-sense morpholino or U7 promoter-driven
snRNA which carries h51AON1 sequence, optimized Sm binding sequence, and hnRNP
A1 binding site (U7cjE51) was constructed. rAAV9-U7cjE51 was injected into adult
marmoset TA and ECR muscles. [Results] After rAAV9-CAG-EW injection into
adult marmoset muscles, stringent expression of EGFP was observed at 2 weeks.
However, diverse diminution of EGFP expression, fibrosis, and lymphocyte
invasion were observed by 10 weeks. Interestingly, proficient EGFP expression in
TA muscle was successfully observed at 14 months after rAAV1-CAG-EW i.p.
injection into new born marmoset. Both h51AON1 morpholino and h51AON1 snRNA
induced exon 51 skipping in a primary myoblasts of marmoset. Five weeks after
rAAV9-U7cjE51 injection into adult marmoset muscles, exon 51 skipping was
observed at the site of transduction. [Conclusion] Long-term effective
muscle transduction after systemic injection into new born marmoset was
confirmed. Also, induction of frame-shift mutation in marmoset dystrophin was
favorably induced in vivo. This strategy with rAAV transduction would be
useful for generating DMD and various models in marmoset.
Intravenous PMO Delivery System with Bubble
Liposomes and Ultrasound Exposure into Skeletal Muscles of the mdx Mice
Yoichi Negishi, Yuko Ishii, Takuo Kojima, Hitomi Shiono, Shoko
Sekine, Yoko Endo-Takahashi, Ryo Suzuki, Kazuo Maruyama, Yukihiko Aramaki.
Department of Drug Delivery and Molecular Biopharmaceutics, School of Pharmacy,
Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan;
Department of Biopharmaceutics, School of Pharmaceutical Sciences, Teikyo
University, Sagamihara, Kanagawa, Japan
Background: Duchenne Muscular Dystrophy (DMD) is genetic disorders caused by
mutations in the DMD gene that lead to the absence of essential muscle protein
dystrophin. Recently, it is expected that exon skipping, mediated by antisense
oligonucleotides (AOs), is one of the most promising methods for restoration of
dystrophin expression in DMD treatment. However, efficient delivery method of
AOs is still required for the DMD treatment. Ultrasound (US) in combination with
microbubbles has recently been acquired much attention in the safe method of
gene or antisense oligonucleotides (AOs) delivery. However, microbubbles have
problems with size, stability, and targeting function. Liposomes have been known
as drug, antigen, and gene delivery carriers. To solve the above-mentioned
issues of microbubbles, we have previously developed the polyethyleneglycol (PEG)-modified
liposomes entrapping echo-contrast gas, “Bubble liposomes” (BLs), which can
function as a novel gene delivery tool by applying them with US exposure. In
this study, to achieve a regional PMO delivery into the mdx mice, we
tried to deliver a PMO, which is designed to skip the mutated exon 23 from the
mRNA of murine dystrophin, into skeletal muscles of the mdx mice by
intraveneous injection and subsequently the treatment of BLs and US exposure.
Methods: Mdx mice (5 weeks old, male) were anesthetized with
pentobarbital throughout each procedure. Prior to each injection, a tourniquet
was placed around the upper hind limb to restrict blood flow into and out of the
limb. A solution of BLs and PMO for mouse exon 23 was injected into the great
saphenous vein of mdx mice. All intravenous injections were performed
using a syringe pump. Subsequently, US exposure (1 MHz, 2 W/cm2, duty
cycle 50 %) was immediately applied to the hamstring muscle. Two weeks after the
treatment, exon 23 skipping levels were analysed by RT-PCR. The number of
dystrophin-positive fibers after the treatment was also analysed by
immunohistochemical analysis. Results: Mice were euthanized 2 weeks after the
single injection, the injected muscles were isolated and analyzed by RT-PCR and
by immunohistochemistry. In the combination of BLs and ultrasound exposure, we
found that the PMO significantly restored the dystrophin mRNA expression and
dystrophin-positive fibers by skipping the mutated exon 23 in the injection site
of mdx mice, compared to the PMO injection alone. The dystrophin-positive
fibers can be found in the wide area of muscle of mdx mice. Conclusions:
By the intravenous delivery of PMO into muscle using BLs and US, the efficient
PMO delivery into the muscle of mdx mice can be achieved with mild US
application. Taken together, this ultrasound-mediated BL technique using veins
may be an effective method for DMD therapy.
Efficient Hydrodynamic Limb Perfusion of an AAV9
Mini-Dystrophin in Adult GRMD Dogs by a Clinically Used, Pressure- and
Speed-Controlled Pump
Juan Li, Chunping Qiao, Janet Bogan, Jianbin Li, Ruhan Tang, Dan
Bogan, Jennifer Dow, Zheng Jane Fan, William J. Powers, Richard Jude Samulski,
Joe Kornegay, Xiao Xiao. Division of Molecular Pharmaceutics, Eshelman School of
Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC; Gene
Therapy Center, School of Medicine, University of North Carolina at Chapel Hill,
Chapel Hill, NC; Dpatartment of Neurology, School of Medicine, University of
North Carolina at Chapel Hill, Chapel Hill, NC
Hydrodynamic limb perfusion is a simple gene delivery method to the muscle
tissues. Previously we have used a speed-controlled pump (Harvard pump) to
deliver AAV vectors into the hind limb muscles of large animals upon occlusion
of the blood vessels with a rubber tourniquet. However, it is highly desirable
to use clinically proven safe settings with controllable injection pressure and
speed and FDA-approved equipment. Recently, Fan et al published safety data in a
clinical trial in muscular dystrophy patients with hydrodynamic saline injection
in the occluded limbs, using a clinical Belmont high volume pump and a blood
pressure cuff. But it was unclear if the same setting could achieve efficient
gene vector delivery to muscle tissues. Here we used the clinical equipment and
setting to test in a large animal model of muscular dystrophy, the GRMD dogs,
for therapeutic gene delivery in limbs. A custom-designed, cone-shaped blood
pressure cuff was used to fit the upper hind limb for better occlusion. The
initial experiment with an AAV-GFP revealed that the vector was absorbed by the
in-path metal heating coils of the Belmont pump. No gene expression was detected.
To bypass the heating coils, we added a smaller clinical pump to deliver the
undiluted vector in parallel with the Belmont pump that delivers the saline
diluent. The vector and saline were mixed in the tubing immediate before the
catheter. The two pumped were synchronized for injection speed and total volume.
Two adult GRMD dogs (14 months old; weighed 15.5 kg and 15.3 kg) were recruited
in the AAV9 minidys vector perfusion study. First, the lower hind limb volume
was measured by displacement in a water-filled container. The cuff was placed
above the knee at a marked line. A 20-gauge catheter was inserted into the great
saphenous vein. The Belmont pump was set at an injection speed of 80 ml/min and
pressure limit at 300 mmHg, the same as in the human studies. The total
injection volume was set at 40% of the occluded lower limb. After the cuff was
inflated to 310 mmHg, the two pumps, one with the vector and the other with the
saline, were started simultaneously. Out of the three legs injected, two had
good occlusion. Limb muscle biopsies were performed at 3 weeks post injection.
Immunofluorescent staining and Western blot of the muscle revealed robust
expression of the minidystrophin in all samples, both anterior and posterior
compartment of the legs. Additional biopsies will be done at longer time points.
The preliminary study validated the effectiveness of the clinical procedure in
achieving therapeutic gene delivery in the DMD dog model. It also warrants
further investigation and optimization of the hydrodynamic limb perfusion
procedure for future clinical trials.