74 Addition of Peptide Therapy To Inhibit NF κB Activation to AAV Serotype 9 Mini Dystrophin Gene Transfer To Treat Muscular Dystrophy in mdx Mice Molecular Therapy Volume 19, Supplement 1, May 2011[.]
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MUSCULO-SKELETAL GENE & CELL THERAPY
and RTC14, were recently identifi ed by a luciferase-independent
high-throughput screening assay and were shown to have potential
therapeutic functions in the treatment of nonsense mutations We
have tested the ability of RTC13 and RTC14 to restore dystrophin
expression into skeletal muscles of the mdx mouse model for
Duchenne muscular dystrophy (DMD) Intramuscular injections of
RTC13, promoted read-through of the mdx UAA stop codon more
effi ciently than gentamicin, PTC124 or RTC14 making it our lead
drug candidate When administered systemically, RTC13 was shown
to restore dystrophin protein in different muscle groups, including
diaphragm and heart Improved muscle strength was detected in all
treated animals and was accompanied by a signifi cant decrease in
creatine kinase (CK) levels demonstrating that the compound was
able to slow down muscle degeneration and turnover No signs of
toxicity were detected in mdx after prolonged administration of
RTC13 demonstrating that the compound was well tolerated in mice
The levels of direct bilirubin (DBIL), blood urea nitrogen (BUN),
creatinine, alkaline phosphatase (ALP) and alanine aminotransferase
(ALT) were significantly decreased in RTC13-treated mice as
compared to untreated mdx or mdx mice that received vehicle alone
confi rming that restoration of dystrophin expression in muscles was
able to ameliorate some of the secondary pathology associated with
the disease in mdx Structure activity relationship (SAR) studies
were used to optimize the molecular structure of RTC13 and to
identify a derivative that meets optimal safety profi les while still
maintaining maximal read-through activity These results advance
the development of RTC13 as an effective drug candidate for DMD
They also offer hope for the treatment of numerous other genetic
disorders due to nonsense mutations and premature termination of
protein synthesis
Phenotypic Correction after Injection of rAAV8-U7
snRNA in GRMD Dogs
Caroline Le Guiner,1,2 Marie Montus,1 Laurent Servais,3 Luis
Garcia,3 Yves Fromes,1,3 Jean-Yves Hogrel,3 Pierre Carlier,3 Yan
Cherel,4 Philippe Moullier,1,2 Thomas Voit,3 The AFM-Sponsored
Duchenne Consortium.1,2,3,4
1 Genethon, Evry, France; 2 INSERM UMR 649, Nantes, France;
3 Institut de Myologie, Paris, France; 4 INRA UMR 703, Nantes,
France.
In Duchenne Muscular Dystrophy (DMD) the selective removal
by exon skipping of exons fl anking an out-of frame mutation in the
dystrophin messenger can result in in-frame mRNA transcripts that
are translated into shorter but functionally active dystrophin The
goal of our project was to determine in GRMD, the effective dose of
our therapeutic product defi ned as a recombinant Adeno-Associated
Virus serotype 8 (rAAV8) expressing a modified U7 snRNA
specifi c for the skipping of exons 5 to 10 of the GRMD dystrophin
transcript The mode of delivery was the locoregional high-pressure
intravenous (IV) injection of a forelimb Several groups of GRMD
dogs were exposed to different rAAV8-U7snRNA doses Each dog
the restoration of dystrophin expression and the improvement of the
tissue pathology in the injected limb compared to the controlateral
limb The secondary outcomes were the muscle strength correction,
the biodistribution and shedding patterns as well as the immune
response against rAAV8 capsid and dystrophin Our preliminary
results suggest a dose effect of our therapeutic rAAV Injection of
2,5E13vg/kg and of 5E12vg/kg of our vector was able to restore 50
to 80% of Dystrophin expression in the injected limb This expression
of a semi-functional dystrophin resulted in improvement of tissue
morphology as well as of several functional and MRI parameters
No tissue infl ammation occurred following the procedure We built a
unique network of laboratories with complementary skills to deliver a
GLP-compliant set of preclinical data to further defi ne the regulatory toxicology studies The organization of our network and the results obtained in our GRMD dogs study will be presented This project is supported by AFM (Association Française contre les Myopathies) and
by ADNA (Advanced Diagnostics for New Therapeutic Approaches),
a program dedicated to personalized medicine, coordinated by Institut Mérieux and supported by research and innovation aid from the French public agency, OSEO
Engraftment of Donor Cells to Regenerating and Dystrophic Skeletal Muscle
Maura H Parker,1 Carol Loretz,1 Rainer Storb,1,3 Stephen J Tapscott.2,4
1 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA; 2 Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA; 3 Department of Medicine, University of Washington, Seattle, WA; 4 Department of Neurology, University of Washington, Seattle, WA.
Muscle-derived cell transplantation has the potential to effectively treat many human diseases, including muscular dystrophy A
variety of cell populations engraft into skeletal muscle of mdx mice,
effectively restore dystrophin expression and reconstitute the satellite cell pool Yet, a direct and quantitative comparison of engraftment
to determine the most effective cell population is lacking We have developed a canine-to-mouse xenotransplantation model to rapidly and quantitatively compare canine muscle cell engraftment Specifi cally, we demonstrate that canine muscle derived cells engraft into regenerating mouse muscle, and engraftment is quantifi able and consistent The canine-to-mouse model allows us to quantitatively compare cell populations and modulating factors, and establish priority for transplantation experiments using a clinically relevant
immune tolerant cxmd canine model of muscular dystrophy We used
the xenotransplant model to show that canine muscle derived cells sorted for expression of CXCR4 do not display a greater level of engraftment when compared to a mixed cell population However, pre-treating a mixed cell population with diprotin A, a positive modulator
of CXCR4-SDF-1 binding, signifi cantly enhances engraftment of donor cells to the mouse satellite cell niche Translating these results
to the immune tolerant canine, we demonstrate that injection of diprotin treated donor cells results in a signifi cantly increased number
of muscle fi bers expressing dystrophin as comapred to untreated cells Temporal regulation of CXCR4/SDF-1 binding may be an important means of expanding the effective range of engraftment after transplantation
NF-κB Activation to AAV Serotype 9 Mini-Dystrophin
Gene Transfer To Treat Muscular Dystrophy in mdx
Mice
Daniel P Reay,1 Gabriela A Niizawa,1 Jon F Watchko,2 Molly Daood,2 Eugene Raggi,1 Paula R Clemens.1,3
1 Neurology, University of Pittsburgh, Pittsburgh, PA; 2 Pediatrics, Magee-Women’s Research Institute, Pittsburgh, PA; 3 Neurology Service, Department of Veteran’s Affairs Medical Center, Pittsburgh, PA.
Systemic gene transfer using serotype 9 adeno-associated vectors (AAV9) is promising for treatment of preclinical models of Duchenne muscular dystrophy (DMD) The ability to achieve systemic vector delivery circumvents a signifi cant hurdle presented by the widespread distribution of skeletal muscle that is best accessed through the circulation However, a limitation of systemic gene vector delivery
is that gene transduction levels vary among muscle groups The addition of complementary therapy could provide 1) a treatment effect
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MUSCULO-SKELETAL GENE & CELL THERAPY
independent of gene transfer and 2) facilitation of successful gene
transfer Pathological activation of the nuclear factor κB (NF-κB)
signaling pathway has emerged as an important cause of dystrophic
muscle changes in muscular dystrophy Furthermore, activation of
NF-κB may also inhibit gene transfer by promoting infl ammation in
response to the transgene or vector Therefore, we hypothesized that
therapeutic benefi ts of dystrophin gene transfer in the mdx mouse
domain (NBD) peptide, which is utilized in this study to inhibit
NF-κB, is synthesized as a fusion peptide with a protein transduction
domain of 8 lysines (8K) to facilitate intra-cellular delivery An
AAV9 vector carrying a human codon-optimized mini-dystrophin
gene under control of the cytomegalovirus promoter was given as
a single intraperitoneal injection (1x1011 gc/pup) at age 2-3 days to
16 mdx mice (experimental mice) Starting at age 4 weeks, 8 of the
experimental mice were given intraperitoneal injections of 8K-NBD
peptide dosed at 10mg/kg, 3 times per week for 5 weeks The second
group of 8 experimental mice received sham intraperitoneal saline
injections Control groups (n=8/group) included normal C57BL10 and
disease control untreated mdx mice At sacrifi ce, ex vivo physiological
studies of diaphragm force production were performed prior to
histological and biochemical assays of diaphragm and limb muscle
tissue In mdx mice treated with the combination of 8K-NBD peptide
and AAV9 mini-dystrophin gene delivery, the quadriceps muscle
demonstrated increased levels of recombinant dystrophin expression
(84% of fi bers expressing, as compared to 56% with AAV9
mini-dystrophin gene delivery alone) suggesting that 8K-NBD treatment
promoted an environment in muscle tissue conducive to higher
levels of recombinant dystrophin expression Indices of necrosis and
regeneration were diminished with either AAV9 gene delivery alone
or in combination with 8K-NBD treatment In diaphragm muscle,
transgene expression was suffi ciently high (>95% of muscle fi bers
expressed dystrophin) that marked improvements in histological and
physiological indices were comparable in the 2 treatment groups The
data support benefi t from 8K-NBD treatment to complement gene
transfer therapy for DMD in muscle tissue that receives incomplete
levels of transduction by gene transfer
Phenotypes in Mice Over-Expressing
Facioscapulohumeral Muscular Dystrophy Region
Gene 1 (FRG1)
Lindsay M Wallace,1,2 Sara E Garwick-Coppens,1 Scott Q
Harper.1,2,3
1 Center for Gene Therapy, The Research Institute at Nationwide
Children’s Hospital, Columbus, OH; 2 Molecular, Cellular and
Developmental Graduate Program, The Ohio State University,
Columbus, OH; 3 Pediatrics, The Ohio State University, Columbus,
OH.
Muscular dystrophies, and other diseases of muscle, arise
from recessive and dominant gene mutations Gene replacement
strategies may be benefi cial for the former, while gene silencing
approaches may provide treatment for the latter In the last two
decades, muscle-directed gene therapies were primarily focused
on treating recessive disorders This disparity at least partly arose
because feasible mechanisms to silence dominant disease genes
lagged behind gene replacement strategies With the discovery of
RNA interference (RNAi) and its subsequent development as a
promising new gene silencing tool, the landscape has changed In
this study, our objective was to demonstrate proof-of-principle for
RNAi therapy of a dominant myopathy in vivo We tested the potential
of AAV-delivered therapeutic microRNAs, targeting the human
Facioscapulohumeral muscular dystrophy (FSHD) Region Gene 1
(FRG1), to correct myopathic features in mice expressing toxic levels
of human FRG1 (FRG1-high mice) We found that FRG1 gene silencing improved muscle mass, strength, and histopathological abnormalities associated with muscular dystrophy in FRG1-high mice Specifi cally, muscles transduced with FRG1-targeted microRNAs (miFRG1) were normal in size, showed no fi brosis or fat deposition, and had
no evidence of myofi ber degeneration or regeneration Moreover, grip strength testing showed that miFRG1-treated animals were as strong as wild-type mice, while control-treated or untreated animals remained signifi cantly weaker Our results support the feasibility
of using RNAi to target other FSHD candidate genes, including DUX4, which has recently emerged as the leading pathogenic insult underlying FSHD Moreover, this approach potentially applies to
as many as 37 different gene mutations responsible for myopathies inherited as autosomal dominant disorders
Micro-Dystrophin and Amelioration of Muscle Function
in Dystrophic Dogs Following Large Scale AAV-Mediated Treatment
Zejing Wang,1,2 Rainer Storb,1,2 Donghoon Lee,3 Christine Halbert,4
Martine K Childers,5 Glen Banks,6 James Allen,6 Eric Finn,6
Martin Kushmeric,3 Dusty Miller,4 Jeffery S Chamberlain,6
Stephen J Tapscott.1,4,6
1 Transplantation Biology, Fred Hutchinson Cancer Research Center, Seattle, WA; 2 Medicine, University of Washington, Seattle, WA; 3 Radiology, University of Washington, Seattle, WA; 4 Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA;
5 Neurology and Regenerative Medicine, Wake Forest University, Charlotte, NC; 6 Neurology, University of Washington, Seattle, WA.
Adeno-associated viral (AAV) vectors as gene delivery vehicles have shown promise both in preclinical studies and clinical trials for
a number of acquired and inherited diseases, including Duchenne Muscular Dystrophy (DMD) We and others have shown that dogs and humans mount immune responses against all tested AAV serotypes, which compromised vector delivery and prevented sustained therapeutical gene expression We have developed a transient immunosuppressive regiment consisting of anti-thymocyte globulin (ATG), cyclosporine (CSP), and mycophenolate mofetil (MMF), which was effective in preventing immune responses to AAV and enhanced AAV6-mediated gene delivery to canine skeletal muscle Here, we demonstrate that long term expression for 2 years of a species – specifi c, functional canine-micro-dys can be achieved in large scale in skeletal muscles of DMD dogs with ATG/CSP/MMF Moreover, for the fi rst time we achieved amelioration of muscle histology and function as a result of sustained expression of the therapeutic gene as demonstrated both by non invasive magnetic resonance imaging (MRI) and kinametic gait analysis In order to identify an even more effective and less toxic immunosuppressive regimen, we have begun studies using molecules that block T-cell costimulation Further, we are also examining AAV vectors made by a modifi ed production method that minimizes aberrant packaging of the capsid gene to further reduce immunogenicity in dogs In conclusion, the fi nding of long term dystrophin expression in DMD dogs given
a short course of standard immunosuppression opens the possibility
of translating these strategies to a human DMD trial