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431. Targeted Restoration of Dystrophin Expression in DMD by Peptide-Conjugated Antisense Oligonucleotides
Oligonucleotide Therapies for Diseases of Muscle and Nerve administration. We used an RNAse protection assay to show that, in response to Ad injection, transcription of pro-inflammatory genes, including IL-1α, IL-1β, KC, MIP-2, and TNF-α, was activated in the spleen to a much higher degree than in the liver. Activation of transcription for these genes was observed using a wide range of Ad doses and as early as 10 min after intravenous Ad injection. To better understand the molecular mediators involved in the induction of antiAd inflammatory responses, we administered Ad in type I IFN-R KO and IL-1RI KO mice. Analysis of pro-inflammatory gene activation in these mice revealed that the induction of most of the analyzed genes was completely dependent on IL-1RI signaling and was similar to wild type mice in type I IFN-R KO mice. Because IL-1RI can be activated by both IL1α and IL-1β, we further analyzed which of these two cytokines was involved in the initiation of Ad-induced inflammation. Administration of Ad in Casp-1-KO mice only partially suppressed KC and MIP-2 activation, suggesting that IL-1β is not the key cytokine responsible for activation of the anti-Ad inflammatory cascade. Next, we injected wild type mice with either IL-1α- or IL-1βblocking antibodies prior to Adv administration. These experiments showed that although both of the cytokines contribute to the anti-Ad response, IL-1α plays the main role in the induction of a cascade of inflammatory chemokines. Because association between endosomal rupture and mature IL-1α production is known, and because Ad actively ruptures the endosomes upon entry into the cell, we next evaluated the levels of cytokine induction after injection of mice with the Ad mutant ts1, which is unable to escape from the endosomal compartment. Analysis of pro-inflammatory gene activation revealed that ts1 virus also strongly induced IL-1RI-dependent activation of KC and MIP-2 and other analyzed cytokines and chemokines. However, ts1 failed to induce an inflammatory cytokine cascade in Caspase-1 KO mice or wild type mice pre-injected with anti-IL-1β antibodies. Collectively, our study demonstrates that Ad entry into cells engages distinct, but synergistic, molecular pathways to activate a cascade of inflammatory cytokines and chemokines downstream of IL-1R signaling. Our data further suggests that Ad recognition by cells does not require Ad entry into the cytoplasm and can occur within cellular endosomal compartments. This study improves our understanding of the molecular mechanisms responsible for the induction of acute anti-Ad inflammatory responses in vivo and may provide the basis for designing safer Ads for gene therapy applications.
Oligonucleotide Therapies for Diseases of Muscle and Nerve 430. A Morpholino-Cell-Penetrating Peptide Conjugate Caused Effective Exon-Skipping in Heart and Skeletal Muscles of MDX Mice
Natee Jearawiriyapaisarn,1,2 Hong M. Moulton,3 Brian Buckley,4 Jennifer Roberts,2 Peter Sazani,4 Suthat Fucharoen,1 Patrick L. Iversen,3 Ryszard Kole.2 1 Thalassemia Research Center and Institute of Molecular Biology and Genetics, Mahidol University, Nakhonpathom, Thailand; 2 Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC; 3AVI BioPharma, Inc., Corvallis, OR; 4 Ercole Biotech, Research Triangle Park, Durham, NC.
switching oligonucleotides. After four once daily intraperitoneal injections at 12.5 mg/kg/day, all conjugates exhibited broad tissue distribution with varied degree of splicing correction in different tissues. The B conjugate showed high splicing correction in the heart (60%), diaphragm (100%) and quadriceps (100%) of the mice. The muscles of heart, diaphragm and quadriceps are important targets for the treatment of Duchenne muscular dystrophy (DMD) with splice switching oligonucleotides. To determine whether the B peptide-conjugated PMO would be applicable for DMD treatment, the M23D-B PPMO, targeted to a donor splice site of dystrophin intron 23, was assessed for exon 23 skipping efficiency in mdx mice, a model for DMD. In these mice, a nonsense mutation in exon 23 of the DMD gene prevents dystrophin production. Four once daily intravenous injections of M23D-B at 12.5 mg/kg/day resulted in persistent exon skipping in dystrophin mRNA and produced high and sustained dystrophin protein expression in the heart, diaphragm and multiple skeletal muscles of treated mice without causing any detectable toxicity. The results suggest that the X/B containing PPMO can be applicable to therapeutic modulation of gene expression.
431. Targeted Restoration of Dystrophin Expression in DMD by Peptide-Conjugated Antisense Oligonucleotides
HaiFang Yin,1 YiQi Seow,1 Hong M. Moulton,2 Patrick L. Iversen,2 Jordan K. Boutilier,2 Matthew Wood.1 1 Physiology, Anatomy and Genetics, University of Oxford, Oxford, Oxfordshire, United Kingdom; 2AVI BioPharma Inc., Corvallis, OR. Duchene Muscular Dystrophy (DMD) is a severe muscle disorder caused by mutations in the dystrophin gene. The efficacy of antisense oligonucleotide (AO)-mediated exon skipping for the restoration of dystrophin has been established in animal models and in DMD patients. However there remain significant limitations to this therapeutic approach due to the lack of effective systemic AO delivery to muscle. Here we investigate systemic muscle-specific AO delivery by testing AOs directly conjugated to cell penetrating peptides (CPPs) alone or in combination with tissue-specific homing peptides (e.g. muscle-specific peptide, MSP). Morpholino chemistry AOs were directly conjugated to CPPs alone or in combination with homing peptides and evaluated in mdx mice following weekly systemic delivery. Effective exon skipping and dystrophin expression were induced in body-wide skeletal muscles at extremely low AO doses of 3mg/kg. This is the first time that targeted AO delivery to muscle and successful body-wide restoration of dystrophin expression have been achieved at such low AO doses. In parallel we also report the discovery and characterization of a novel delivery formulation which facilitates AO uptake in muscle. A series of studies have shown that this delivery formulation enhances the delivery of AOs of different chemistry (e.g. 2-OMeRNA, PNA and morphlino), depends on the activity of specific muscle membrane transporters, and that it induces significant restoration of dystrophin expression in muscle compared with commonly used delivery formulations. In summary, we report data demonstrating the potential of muscle-specific homing peptides, CPPs and novel delivery formulations for the targeted restoration of dystrophin in DMD.
Conjugation of arginine-rich cell-penetrating peptides (CPPs) to phosphorodiamidate morpholino oligomers (PMO) has been shown to enhance the intracellular delivery of PMO to modulate gene expression, in particular to alter pre-mRNA splicing. In searching for more active, stable and less toxic peptide-PMO conjugates (PPMOs), we designed a series of PPMOs (termed A through I) carrying CPPs that consisted of eight arginine (R) and a variable number of 6-aminohexanoic acid and/or β-alanine residues. Their functional biodistribution and toxicity were initially evaluated in EGFP-654 transgenic mice, a positive readout mouse model for splice Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
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Oligonucleotide Therapies for Diseases of Muscle and Nerve 430. A Morpholino-Cell-Penetrating Peptide Conjugate Caused Effective Exon-Skipping in Heart and Skeletal Muscles of MDX Mice
Natee Jearawiriyapaisarn,1,2 Hong M. Moulton,3 Brian Buckley,4 Jennifer Roberts,2 Peter Sazani,4 Suthat Fucharoen,1 Patrick L. Iversen,3 Ryszard Kole.2 1 Thalassemia Research Center and Institute of Molecular Biology and Genetics, Mahidol University, Nakhonpathom, Thailand; 2 Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC; 3AVI BioPharma, Inc., Corvallis, OR; 4 Ercole Biotech, Research Triangle Park, Durham, NC.
switching oligonucleotides. After four once daily intraperitoneal injections at 12.5 mg/kg/day, all conjugates exhibited broad tissue distribution with varied degree of splicing correction in different tissues. The B conjugate showed high splicing correction in the heart (60%), diaphragm (100%) and quadriceps (100%) of the mice. The muscles of heart, diaphragm and quadriceps are important targets for the treatment of Duchenne muscular dystrophy (DMD) with splice switching oligonucleotides. To determine whether the B peptide-conjugated PMO would be applicable for DMD treatment, the M23D-B PPMO, targeted to a donor splice site of dystrophin intron 23, was assessed for exon 23 skipping efficiency in mdx mice, a model for DMD. In these mice, a nonsense mutation in exon 23 of the DMD gene prevents dystrophin production. Four once daily intravenous injections of M23D-B at 12.5 mg/kg/day resulted in persistent exon skipping in dystrophin mRNA and produced high and sustained dystrophin protein expression in the heart, diaphragm and multiple skeletal muscles of treated mice without causing any detectable toxicity. The results suggest that the X/B containing PPMO can be applicable to therapeutic modulation of gene expression.
431. Targeted Restoration of Dystrophin Expression in DMD by Peptide-Conjugated Antisense Oligonucleotides
HaiFang Yin,1 YiQi Seow,1 Hong M. Moulton,2 Patrick L. Iversen,2 Jordan K. Boutilier,2 Matthew Wood.1 1 Physiology, Anatomy and Genetics, University of Oxford, Oxford, Oxfordshire, United Kingdom; 2AVI BioPharma Inc., Corvallis, OR. Duchene Muscular Dystrophy (DMD) is a severe muscle disorder caused by mutations in the dystrophin gene. The efficacy of antisense oligonucleotide (AO)-mediated exon skipping for the restoration of dystrophin has been established in animal models and in DMD patients. However there remain significant limitations to this therapeutic approach due to the lack of effective systemic AO delivery to muscle. Here we investigate systemic muscle-specific AO delivery by testing AOs directly conjugated to cell penetrating peptides (CPPs) alone or in combination with tissue-specific homing peptides (e.g. muscle-specific peptide, MSP). Morpholino chemistry AOs were directly conjugated to CPPs alone or in combination with homing peptides and evaluated in mdx mice following weekly systemic delivery. Effective exon skipping and dystrophin expression were induced in body-wide skeletal muscles at extremely low AO doses of 3mg/kg. This is the first time that targeted AO delivery to muscle and successful body-wide restoration of dystrophin expression have been achieved at such low AO doses. In parallel we also report the discovery and characterization of a novel delivery formulation which facilitates AO uptake in muscle. A series of studies have shown that this delivery formulation enhances the delivery of AOs of different chemistry (e.g. 2-OMeRNA, PNA and morphlino), depends on the activity of specific muscle membrane transporters, and that it induces significant restoration of dystrophin expression in muscle compared with commonly used delivery formulations. In summary, we report data demonstrating the potential of muscle-specific homing peptides, CPPs and novel delivery formulations for the targeted restoration of dystrophin in DMD.
Conjugation of arginine-rich cell-penetrating peptides (CPPs) to phosphorodiamidate morpholino oligomers (PMO) has been shown to enhance the intracellular delivery of PMO to modulate gene expression, in particular to alter pre-mRNA splicing. In searching for more active, stable and less toxic peptide-PMO conjugates (PPMOs), we designed a series of PPMOs (termed A through I) carrying CPPs that consisted of eight arginine (R) and a variable number of 6-aminohexanoic acid and/or β-alanine residues. Their functional biodistribution and toxicity were initially evaluated in EGFP-654 transgenic mice, a positive readout mouse model for splice Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
S163