SNF-6 is an
acetylcholine transporter interacting with the dystrophin complex in
Caenorhabditis elegans
Nature,
2004
Hongkyun
Kim, Matthew J. Rogers, Janet E. Richmond & Steven L. McIntire - USA
Muscular dystrophies are among the most
common human genetic diseases and are characterized by progressive muscle
degeneration. Muscular dystrophies result from genetic defects in components of
the dystrophin–glycoprotein complex (DGC), a multimeric complex found in the
muscle cell plasma membrane. The DGC links the intracellular cytoskeleton to
the extracellular matrix and is thought to be important for maintaining the
mechanical integrity of muscles and organizing signalling molecules. The exact
role of the DGC in the pathogenesis of disease has, however, remained
uncertain. Mutations in Caenorhabditis elegans DGC genes lead to specific
defects in coordinated movement and can also cause muscle degeneration. Here we
show that mutations in the gene snf-6 result in phenotypes indistinguishable
from those of the DGC mutants, and that snf-6 encodes a novel
acetylcholine/choline transporter. SNF-6 mediates the uptake of acetylcholine
at neuromuscular junctions during periods of increased synaptic activity. SNF-6
also interacts with the DGC, and mutations in DGC genes cause a loss of SNF-6
at neuromuscular junctions. Improper clearing of acetylcholine and prolonged
excitation of muscles might contribute to the pathogenesis of muscular
dystrophies.
Nucleofection of
Muscle-Derived Stem Cells and Myoblasts with C31 Integrase: Stable Expression
of a Full-Length-Dystrophin Fusion Gene by Human Myoblasts
Molecular
Therapy, 2004
Simon
P. Quenneville,Pierre Chapdelaine,JoJl Rousseau, Jean Beaulieu, Nicolas J.
Caron, Daniel Skuk, Philippe Mills, Eric C. Olivares, Michele P. Calos, and
Jacques P. Tremblay - Canada
Ex vivo gene therapy offers a potential
treatment for Duchenne muscular dystrophy by transfection of the dystrophin
gene into the patient’s own myogenic precursor cells, followed by
transplantation. We used nucleofection to introduce DNA plasmids coding for
enhanced green fluorescent protein (eGFP) or eGFP-dystrophin fusion protein and
the phage C31 integrase into myogenic cells and to integrate these genes into a
limited number of sites in the genome. Using a plasmid expressing eGFP, we
transfected 50% of a mouse muscle-derived stem cell line and 60% of normal
human myoblasts. Co-nucleofection of a plasmid expressing the C31 integrase and
an eGFP expression plasmid containing an attB sequence produced 15 times more
frequent stable expression, because of site-specific integration of the transgene.
Co-nucleofection of the C31 integrase plasmid and a large plasmid containing
the attB sequence and the gene for an eGFP–full-length dystrophin fusion
protein produced fluorescent human myoblasts that were able to form more
intensely fluorescent myotubes after 1 month of culture. A nonviral approach
combining nucleofection and the C31 integrase may eventually permit safe
autotransplantation of genetically modified cells to patients.
A Facile
Nonviral Method for Delivering Genes and siRNAs to Skeletal Muscle of
Mammalian Limbs
Molecular
Therapy. 2004,10(2):386-398
James E. Hagstrom, Julia Hegge, Guofeng Zhang, Mark Noble,
Vladimir Budker, David L. Lewis, Hans Herweijer and Jon A. Wolff - USA
Abstract
Delivery is increasingly being recognized as
the critical hurdle holding back the tremendous promise of nucleic acid-based
therapies that include gene therapy and more recently siRNA-based therapeutics.
While numerous candidate genes (and siRNA sequences) with therapeutic potential
have been identified, their utility has not yet been realized because of
inefficient and/or unsafe delivery technologies. We now describe an
intravascular, nonviral methodology that enables efficient and repeatable
delivery of nucleic acids to muscle cells (myofibers) throughout the limb
muscles of mammals. The procedure involves the injection of naked plasmid DNA
or siRNA into a distal vein of a limb that is transiently isolated by a
tourniquet or blood pressure cuff. Nucleic acid delivery to myofibers is
facilitated by its rapid injection in sufficient volume to enable extravasation
of the nucleic acid solution into muscle tissue. High levels of transgene
expression in skeletal muscle were achieved in both small and large animals
with minimal toxicity. Evidence of siRNA delivery to limb muscle was also
obtained. The simplicity, effectiveness, and safety of the procedure make this
methodology well suited to limb muscle gene therapy applications.
Mattie Bremmer-Bout, Annemieke Aartsma-Rus, Emile J. de
Meijer, Wendy E. Kaman, Anneke A. M. Janson, Rolf H. A. M. Vossen, Gert-Jan B.
van Ommen, Johan T. den Dunnen and Judith C. T. van Deutekom - The Netherlands
The therapeutic potential of frame-restoring
exon skipping by antisense oligonucleotides (AONs) has recently been
demonstrated in cultured muscle cells from a series of Duchenne muscular
dystrophy (DMD) patients. To facilitate clinical application, in vivo
studies in animal models are required to develop safe and efficient AON-delivery
methods. However, since exon skipping is a sequence-specific therapy, it is
desirable to target the human DMD gene directly. We therefore set up human
sequence-specific exon skipping in transgenic mice carrying the full-size human
gene (hDMD). We initially compared the efficiency and toxicity of
intramuscular AON injections using different delivery reagents in wild-type
mice. At a dose of 3.6 nmol AON and using polyethylenimine, the skipping levels
accumulated up to 3% in the second week postinjection and lasted for 4 weeks.
We observed a correlation of this long-term effect with the intramuscular
persistence of the AON. In regenerating myofibers higher efficiencies (up to
9%) could be obtained. Finally, using the optimized protocols in hDMD
mice, we were able to induce the specific skipping of human DMD exons without
affecting the endogenous mouse gene. These data highlight the high sequence
specificity of this therapy and present the hDMD mouse as a unique model
to optimize human-specific exon skipping in vivo.