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Bisphosphonate treatment for osteopenia associated with genetic disease in children. S. Carter1, R. Mendoza-Londono1, V.R. Sutton1, C.A. Bacino1, K. Ellis2, R. Shypailo2, B. Lee1. 1) Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; 2) Dept. Pediatrics, CNRC, Baylor College of Medicine.
Osteoporosis is defined as low bone mass
and bone fragility with increased susceptibility to fractures. It is also
defined as bone mineral density (BMD) that is 2.5 SD below the mean peak value
for age-matched controls. Until recently, treatment options for pediatric
osteoporosis have been limited. Bisphosphonates inhibit osteoclast activity and
have been shown to be safe in pediatric applications. Common genetic conditions
associated with low BMD and susceptibility to fractures include osteogenesis
imperfecta (OI), and secondary osteopenia due to to lack of physical activity,
as is seen in patients with neuromuscular disease. We present our experience
treating 14 pediatric patients followed at the Texas Childrens Hospital Skeletal
Dysplasia Clinic between 1999 and 2002. The diagnosis was OI type 3 or 4 in 11
patients and other neuromuscular disease in 3 patients. Ages ranged between
newborn and 15 yrs. (Average 4.5 yrs). The diagnosis of OI was made in the first
two months of life in 9 of the 11 patients. Bone mineral density of the lumbar
spine, femoral head and total body was measured using dual-energy x-ray
absorptiometry (DXA) before treatment and at regular intervals during treatment.
The patients received Pamidronate at a dose of 13.5 mg/kg/year for children
<2-3 years and 9 mg/kg/year for children >3 years. Bone metabolic
parameters (alkaline phosphatase, urine collagen N-telopeptides) showed decrease
in bone turnover. Average L-spine BMD values were Z= -3.86 (Range 6.78 to 2.92)
before treatment and Z= -2.56 (range 3.72 to 1.4) after treatment. Physiologic
response appeared more significant in younger children. Overall there was a
subjective clinical improvement, with decrease in bone pain and frequency of
fractures, resulting in better tolerance of daily activities and well being.
Side effects were minor including transient febrile episodes and mild
hypocalcemia which corrected with supplementation. This experience has
demonstrated that bisphosphonates constitute an important adjuvant treatment for
osteoporosis associated with genetic disease.
Targeted exon skipping as a promising therapeutic tool for Duchenne muscular dystrophy. J.C.T. Van Deutekom1, A. Aartsma-Rus1, M. Bremmer-Bout1, J.A.M. Janson1, W.E. Kaman1, E.J De Meijer1, F. Baas2, J.T. Den Dunnen1, G.J.B. Van Ommen1. 1) Dept Human Genetics, Leiden Univ Medical Ctr, Leiden, Netherlands; 2) Dept Neurology, Academic Medical Ctr, Amsterdam, Netherlands.
Duchenne muscular dystrophy (DMD) is a
lethal muscle disease typically caused by frame-shifting mutations in the DMD
gene that abort the synthesis of the dystrophin protein. In contrast to the gene
therapy studies that are based on gene replacement, we are focusing on gene
correction through the targeted modulation of the splicing of the
patient-specific gene. Using antisense oligoribonucleotides (AONs) directed to
exon-internal sequences, we aim to induce the specific skipping of a target exon
in order to generate a shorter, but in-frame transcript similar to those found
in the corresponding Becker muscular dystrophy (BMD) patients, having milder
phenotypes and longer life expectancies. We have recently demonstrated the
feasibility of AON-induced skipping of 15 different exons within the deletion
hot spot regions. In this study we show the broad therapeutic applicability of
skipping these exons in cultured muscle cells from DMD-patients affected by
different mutations. In fact, through the AON-mediated production of novel,
in-frame transcripts, we detected dystrophin synthesis in at least 70% of
treated muscle cells. We here also show the in vivo feasibility of targeted exon
skipping in mouse muscle tissue. Following intramuscular injections of
mouse-specific exon 46 AONs, we were able to specifically induce exon 46
skipping in a dose-dependent manner. In a subsequent time-series experiment, the
skipping effect was highest at 12 days post-injection with estimated
efficiencies of 20% of total RT-PCR products, and, although gradually
diminishing, continued for at least 29 days. Moreover, using DMD humanized
transgenic mice carrying an integrated 2.7 Mb YAC-derived copy of the human DMD
gene, we were able to test human sequence-specific AONs, and induce the skipping
of human DMD exons, in an experimental mouse background. Our results confirm the
therapeutic potential of AON-induced exon skipping, and contribute to its
further development for DMD gene therapy.
a- and b-sarcoglycan delivery by recombinant adeno-associated virus: efficient rescue of muscle, but differential toxicity. D. Dressman1,2, K. Araishi3, M. Imamura3, T. Sasaoka3, L.A. Liu4, E. Engvall4, E.P. Hoffman1,2. 1) Center for Genetic Medicine, CNMC, Washington, DC; 2) University of Pittsburgh, Department of Molecular Genetics & Biochemistry, Pittsburgh, PA; 3) National Institute of Neuroscience, NCNP, Tokyo, Japan; 4) Burnham Institute, La Jolla, CA.
The sarcoglycanopathies are a group of
four autosomal recessive muscular dystrophies (LGMD 2D, 2E, 2C, and 2F), caused
by mutations of the a, b, g, or d sarcoglycan genes. The d-sarcoglycan deficient
hamster has been the most utilized model for gene delivery to muscle by
recombinant adeno-associated virus (AAV) vectors, however human patients with
d-sarcoglycan deficiency are exceedingly rare with only two patients described
in the United States. Here, we report construction and use of AAV vectors
expressing either a- or b- sarcoglycan, the genes responsible for the most
common forms of the human sarcoglycanopathies. Both vectors showed successful
short-term genetic, biochemical and histological rescue of both a- and
b-sarcoglycan deficient mouse muscle. However, comparison of persistence of
expression in 51 injected mice showed substantial differences between AAV
a-sarcoglycan (a-SG) and b-sarcoglycan (b-SG) vectors. AAV b-SG showed long-term
expression with no decrease in expression for over 21 months after injection,
while AAV a-SG showed a dramatic loss of positive fibers between 28 days and 41
days post-injection (p=0.006). Loss of immuno-positive myofibers was correlated
with significant inflammatory cell infiltrate, primarily macrophages. To
determine if the loss of a-sarcoglycan positive fibers was due to an immune
response or cytotoxic effect of a-sarcoglycan over-expression, SCID mouse muscle
was assayed for cytotoxicity after injection with AAV a- SG, AAV b- SG, or PBS.
The results were consistent with over-expression of a-sarcoglycan causing
significant cytotoxicity. The cytotoxicity of a-sarcoglycan, and not b- or
d-sarcoglycan over-expression, was consistent with biochemical studies of the
hierarchical order of assembly of the sarcoglycan complex. Our data suggests
that even closely related proteins might require different levels of expression
to avoid toxicity and achieve long-term tissue rescue.
Immunostimulatory Properties of Dystrophic Muscle Alter Persistence of Transgenes. E.P. Hoffman1,2, H. Gordish3, K. Araishi4, M. Imamura4, T. Sasaoka4, D. Dressman1,2. 1) Research Center for Genetic Medicine, Children's National Medical Center, Washington, DC; 2) University of Pittsburgh, School of Medicine, Department of Molecular Genetics & Biochemistry, Pittsburgh, PA; 3) University of Pittsburgh, Graduate School of Public Health, Department of Environmental and Occupational Health, Pittsburgh, PA; 4) National Institute of Neuroscience, NCNP, Tokyo, Japan.
Inherited biochemical deficits in
patients with muscular dystrophy result in cycles of degeneration and
regeneration of myofibers. Complementation of the biochemical deficiency in
myofibers has been the major focus of experimental gene delivery in animal
models of dystrophin and sarcoglycan deficiency. Recombinant adeno-associated
virus (AAV) has been shown to deliver genes to mature normal muscle with
long-term persistence, although persistence in dystrophic muscle has been more
variable. We hypothesized that the degeneration/regeneration cycles and immune
cell infiltrate in dystrophic muscle could be immunostimulatory, leading to
increased immune response to a transgene relative to normal muscle. To test this
hypothesis, we delivered both marker (b-galactosidase) and therapeutic genes (b-sarcoglycan)
to dystrophic muscle and to normal muscle using identical AAV vectors. We show
that dystrophic muscle does indeed elicit a stronger immune response to
b-galactosidase, than normal muscle, showing both greater circulating antibodies
(p< 0.05) and greater loss of transduced fibers (p< 0.05). Co-delivery of
marker and therapeutic AAV attenuated the immune response (p<0.05), despite
the use of twice as much AAV. Our results show that pathological muscle is
immunostimulatory towards transgene-delivered proteins, leading to decreased
persistence after AAV delivery. However, biochemical rescue of myofibers by a
therapeutic transgene can attenuate this immune response, and in most cases
leads to immunotolerance and long-term survival of transduced fibers.