Abstracts presented on Annual Meeting of
The American Society of Human Genetics (Toronto, Canada - October 26-30/2004)
1) Body-wide delivery of a
microdystrophin gene via intravascular administration of rAAV6 vectors for
treatment of muscular dystrophy.
P. Gregorevic, M. Blankinship, J. Allen, L. Meuse, S. Abmayr, J. Han, J.
Chamberlain. Senator Paul D. Wellstone Muscular Dystrophy Cooperative
Research Center, Dept. of Neurology, University of Washington, Seattle, WA.
Severe neuromuscular disorders, such as Duchenne muscular
dystrophy (DMD), lead to reduced quality of life and premature mortality.
Historically, genetic interventions for these diseases have been limited by an
inability to achieve widespread gene transfer to the affected tissues. Here we
demonstrate a method that, for the first time, enables transduction of the vast
majority of both the cardiac and skeletal musculature of adult mammals via a
single intravenous administration of pseudotype 6 recombinant adeno-associated
virus (rAAV6) vectors. As a means to enhance gene transfer, we have determined
that IV coadministration of vascular endothelium growth factor (VEGF) with
rAAV6 vectors can significantly increase vector accumulation and transgene
expression in striated muscles. Having established in reporter gene studies
that this technique enables tolerable, high-level transgene expression, we
sought to evaluate the potential of this technique for delivering a therapeutic
transgene to the musculature of the mdx dystrophic mouse, a model of DMD. Using
this technique, we have observed that treated mdx mice exhibit expression of
microdystrophin throughout the striated musculature in a manner that is
sufficient to reduce pathological features of the dystrophic phenotype.
Compared with untreated mice, the muscles of treated animals exhibit reduced
susceptibility to contraction-induced injury, and reduced serum creatine kinase
levels, reflecting a global reduction in muscle degeneration. These data are
the first to demonstrate body-wide amelioration of symptoms associated with
dystrophin deficiency in an adult animal following a genetic intervention. In
subsequent studies we have established that these techniques can be adapted to
express alternate transgenes for the treatment of other muscle diseases. We are
currently undertaking studies to determine whether the existing techniques are
effective in animals more similar in size and immunology to humans, and present
relevant data summarizing our progress
2) Therapeutic
antisense-induced exon skipping for Duchenne muscular dystrophy. A. Aartsma-Rus1, A. Janson1,
G. Platenburg2, M. Bremmer-Bout1, J.T. Den Dunnen1,
J.C.T. Van Deutekom1, G-J.B. Van Ommen1. 1) LUMC,
Leiden, the Netherlands; 2) Prosensa, Leiden, the Netherlands.
The severe Duchenne muscular dystrophy (DMD) is mostly caused
by frame disrupting mutations in the dystrophin gene, which result in
non-functional proteins. Mutations that keep the reading frame intact give rise
to internally deleted, semi-functional dystrophins and result in the milder
Becker muscular dystrophy (BMD). Antisense oligonucleotides (AONs) have the
potential to modulate the pre-mRNA splicing such that a specific exon is
skipped. As a result, the reading frame can be restored, which allows the
synthesis of a BMD-like dystrophin.
We have recently demonstrated the skipping of 20 different
human DMD exons using 2'-O-methyl RNA AONs with a full-length phosphorothioate
backbone (2OMePS), and confirmed the therapeutic applicability of this strategy
in cultures from 10 different DMD patients. We now compare the efficacy and
efficiency of 2OMePS AONs to morpholino, locked nucleic acid (LNA) and peptide
nucleic acid (PNA) AONs. While LNA was most efficient in inducing exon
skipping, we find that it acts in a less sequence-specific manner, probably due
to its extremely high RNA affinity. Awaiting further improvements in
oligochemistry, we therefore consider 2OMePS AONs currently the most favourable
compounds.
To facilitate clinical application, multiple in vivo
studies in animal models are ongoing to develop safe and efficient AON-delivery
methods. However, as exon skipping is a sequence-specific therapy, it is
desirable to directly target the human DMD gene. We have therefore set up
sequence-specific human exon skipping in vivo in transgenic mice
carrying the an intact copy of the full-length 2.5 Mb human gene (hDMD).
We injected 2OMePS AONs targeting human exons 44, 46 and 49 into the m.
gastrocnemicus of hDMD mice, and showed skipping of the human (but
not the murine) exons, which persisted for at least 28 days. Based on
pre-clinical data obtained by our group and others, we are currently setting up
a clinical trial aiming at local dystrophin restoration following intramuscular
injections of exon 46 and 51 specific AONs.