Methods in molecular biology (Clifton, N.J.), 2011
Antisense oligonucleotides (AOs) are effective splice switching agents and have potential as ther... more Antisense oligonucleotides (AOs) are effective splice switching agents and have potential as therapeutics via the exclusion or inclusion of specific target gene exons to ameliorate and modify disease progression. The leading example is Duchenne muscular dystrophy (DMD), a fatal muscle degenerative disease, where AO-mediated skipping of specific DMD gene exons can restore the disrupted DMD open reading frame, leading to the production of functional dystrophin protein and ameliorate the DMD phenotype in animal models. Clinical proof-of-concept has recently been shown in two successful, independent Phase I clinical trials. These trials both followed local intramuscular treatments, and the challenge now is to develop and test systemic protocols, which will be required for treatment-aimed disease modification. Recently, a number of groups have demonstrated the promise of AOs directly conjugated to cell-penetrating peptides (CPPs) as having significant potential for systemic delivery and ...
Molecular therapy : the journal of the American Society of Gene Therapy, 2008
Duchenne muscular dystrophy (DMD) is the most common and severe form of muscular dystrophy, arisi... more Duchenne muscular dystrophy (DMD) is the most common and severe form of muscular dystrophy, arising from mutations in the dystrophin gene that preclude the synthesis of functional protein. Antisense oligonucleotides (AOs) have been shown to induce specific exon skipping and thereby restore the reading frame and expression of functional dystrophin. In this report, we examine the effects of peptide nucleic acid (PNA) oligonucleotides and PNAs conjugated with peptides including TAT, muscle-specific peptide (MSP), adeno-associated virus 6 (AAV6) functional domain (AAV6), and AAV8 functional domain (AAV8), on exon skipping in vitro and in vivo. Efficient skipping of targeted exon 23 was achieved in cultured mdx myoblasts with PNA and PNA-peptide conjugates. Furthermore, single intramuscular injections of PNA and all PNA-peptide conjugates resulted in significant numbers of dystrophin-positive fibers in the injected tibialis anterior (TA) muscles of mdx mice, with no apparent local toxici...
This study examined the feasibility of using the promoter of the pig parotid secretory protein (P... more This study examined the feasibility of using the promoter of the pig parotid secretory protein (PSP) gene for expression of the phytase transgene in mouse models. The pig parotid secretory protein gene is specifically expressed at high levels in the salivary glands. The 10-kb upstream promoter region of the gene necessary for tissue-specific expression has been identified. We have constructed phytase transgenes composed of the appA phytase gene from Escherichia coli driven by the upstream promoter region of the pig PSP gene with a 3' tail of either bovine growth hormone or the pig PSP gene polyadenylation signal. Transgenic mouse models with the construct showed that the upstream region of the pig PSP gene is sufficient for directing the expression of phytase transgenes in the saliva. Expression of salivary phytase reduced fecal phytate by 8.5 and 12.5% in 2 transgenic mouse lines, respectively. These results suggest that the expression of phytase in salivary glands of monogastr...
The key regulatory elements for PKD2 transcription remain unclear. To identify these core element... more The key regulatory elements for PKD2 transcription remain unclear. To identify these core elements, we characterized porcine PKD2 promoter with bioinformatics and molecular tools and found porcine PKD2 promoter bearing typical features of enriched CpG and less TATA. Further studies demonstrated that the core region was located in fragment -483 to +100. Subsequent biophysical binding assays and mutation experiments revealed that G4 motif and Sp1 are critical regulators for mediating the transcription of porcine PKD2. Moreover, the same regulatory pattern was reproduced in human PKD2 promoter region, indicating the critical role of G4 and Sp1 in regulating PKD2.
Exon-skipping oligonucleotides are a well-researched therapeutic strategy for Duchenne&am... more Exon-skipping oligonucleotides are a well-researched therapeutic strategy for Duchenne's muscular dystrophy (DMD). Despite remarkable successes in animal models with intramuscular and intravenous delivery of unmodified oligonucleotides, the ability to specifically target both normal and dystrophic muscle with a simple peptide ligand could decrease the therapeutic dose required and reduce the potential for toxicity. Thus, 3 rounds of in vivo phage display utilizing a 12-mer peptide library were performed with mdx mice and a peptide motif with potential for targeting to muscle but not liver was identified. This motif was shown to have enhanced binding affinity to C2C12 myoblasts over a scrambled control peptide and in vivo application of a fluorescein-labeled peptide containing the identified motif resulted in increased specificity for the heart and quadriceps muscle after tail-vein administration in C57BL/6 mice. This work has many potential applications for oligonucleotide or drug delivery to muscle for myopathies.
Duchenne muscular dystrophy (DMD) is the most common and severe form of muscular dystrophy, arisi... more Duchenne muscular dystrophy (DMD) is the most common and severe form of muscular dystrophy, arising from mutations in the dystrophin gene that preclude the synthesis of functional protein. Antisense oligonucleotides (AOs) have been shown to induce specific exon skipping and thereby restore the reading frame and expression of functional dystrophin. In this report, we examine the effects of peptide nucleic acid (PNA) oligonucleotides and PNAs conjugated with peptides including TAT, muscle-specific peptide (MSP), adeno-associated virus 6 (AAV6) functional domain (AAV6), and AAV8 functional domain (AAV8), on exon skipping in vitro and in vivo. Efficient skipping of targeted exon 23 was achieved in cultured mdx myoblasts with PNA and PNA-peptide conjugates. Furthermore, single intramuscular injections of PNA and all PNA-peptide conjugates resulted in significant numbers of dystrophin-positive fibers in the injected tibialis anterior (TA) muscles of mdx mice, with no apparent local toxicity. Similar effects of exon skipping and dystrophin expression were obtained in mice of all ages. PNA and PNA-AAV6, PNA-AAV8 conjugates induced dystrophin expression in a dose-dependent manner. Our results demonstrate that PNAs have a higher efficiency of exon skipping than 2'O methyl phosphorothioate AOs do, and have a potential use in AO chemistry for antisense therapy of DMD.
Induced splice modulation of pre-mRNAs shows promise to correct aberrant disease transcripts and ... more Induced splice modulation of pre-mRNAs shows promise to correct aberrant disease transcripts and restore functional protein and thus has therapeutic potential. Duchenne muscular dystrophy (DMD) results from mutations that disrupt the DMD gene open reading frame causing an absence of dystrophin protein. Antisense oligonucleotide (AO)-mediated exon skipping has been shown to restore functional dystrophin in mdx mice and DMD patients treated intramuscularly in two recent phase 1 clinical trials. Critical to the therapeutic success of AO-based treatment will be the ability to deliver AOs systemically to all affected tissues including the heart. Here, we report identification of a series of transduction peptides (Pip5) as AO conjugates for enhanced systemic and particularly cardiac delivery. One of the lead peptide-AO conjugates, Pip5e-AO, showed highly efficient exon skipping and dystrophin production in mdx mice with complete correction of the aberrant DMD transcript in heart, leading to >50% of the normal level of dystrophin in heart. Mechanistic studies indicated that the enhanced activity of Pip5e-phosphorodiamidate morpholino (PMO) is partly explained by more efficient nuclear delivery. Pip5 series derivatives therefore have significant potential for advancing the development of exon skipping therapies for DMD and may have application for enhanced cardiac delivery of other biotherapeutics.
Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that abolish the synthes... more Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that abolish the synthesis of dystrophin protein. Antisense oligonucleotides (AOs) targeted to trigger excision of an exon bearing a mutant premature stop codon in the DMD transcript have been shown to skip the mutated exon and partially restore functional dystrophin protein in dystrophin-deficient mdx mice. To fully exploit the therapeutic potential of this method requires highly efficient systemic AO delivery to multiple muscle groups, to modify the disease process and restore muscle function. While systemic delivery of naked AOs in DMD animal models requires high doses and is of relatively poor efficiency, we and others have recently shown that short arginine-rich peptide-AO conjugates can dramatically improve in vivo DMD splice correction. Here we report for the first time that a chimeric fusion peptide (B-MSP-PMO) consisting of a muscle-targeting heptapeptide (MSP) fused to an arginine-rich cell-penetrating peptide (B-peptide) and conjugated to a morpholino oligomer (PMO) AO directs highly efficient systemic dystrophin splice correction in mdx mice. With very low systemic doses, we demonstrate that B-MSP-PMO restores high-level, uniform dystrophin protein expression in multiple peripheral muscle groups, yielding functional correction and improvement of the mdx dystrophic phenotype. Our data demonstrate proof-of-concept for this chimeric peptide approach in DMD splice correction therapy and is likely to have broad application.
Antisense oligonucleotides (AOs) have the potential to induce functional dystrophin protein expre... more Antisense oligonucleotides (AOs) have the potential to induce functional dystrophin protein expression via exon skipping by restoring in-frame transcripts in the majority of patients suffering from Duchenne muscular dystrophy (DMD). AOs of morpholino phosphoroamidate (PMO) and 2'-O-methyl phosphorothioate RNA (2'Ome RNA) chemistry have been shown to restore dystrophin expression in skeletal muscle but not in heart, following high-dose systemic delivery in murine models of muscular dystrophy (mdx). Exploiting the cell transduction properties of two basic arginine-rich cell penetrating peptides, we demonstrate widespread systemic correction of dystrophin expression in body-wide muscles and cardiac tissue in adult dystrophic mdx mice, with a single low-dose injection of peptide-conjugated PMO AO. This approach was sufficient to restore uniform, high-level dystrophin protein expression in peripheral muscle and cardiac tissue, with robust sarcolemmal relocalization of the dystrophin-associated protein complex and functional improvement in muscle. Peptide-conjugated AOs therefore have significant potential for systemic correction of the DMD phenotype.
Methods in molecular biology (Clifton, N.J.), 2011
Antisense oligonucleotides (AOs) are effective splice switching agents and have potential as ther... more Antisense oligonucleotides (AOs) are effective splice switching agents and have potential as therapeutics via the exclusion or inclusion of specific target gene exons to ameliorate and modify disease progression. The leading example is Duchenne muscular dystrophy (DMD), a fatal muscle degenerative disease, where AO-mediated skipping of specific DMD gene exons can restore the disrupted DMD open reading frame, leading to the production of functional dystrophin protein and ameliorate the DMD phenotype in animal models. Clinical proof-of-concept has recently been shown in two successful, independent Phase I clinical trials. These trials both followed local intramuscular treatments, and the challenge now is to develop and test systemic protocols, which will be required for treatment-aimed disease modification. Recently, a number of groups have demonstrated the promise of AOs directly conjugated to cell-penetrating peptides (CPPs) as having significant potential for systemic delivery and ...
Molecular therapy : the journal of the American Society of Gene Therapy, 2008
Duchenne muscular dystrophy (DMD) is the most common and severe form of muscular dystrophy, arisi... more Duchenne muscular dystrophy (DMD) is the most common and severe form of muscular dystrophy, arising from mutations in the dystrophin gene that preclude the synthesis of functional protein. Antisense oligonucleotides (AOs) have been shown to induce specific exon skipping and thereby restore the reading frame and expression of functional dystrophin. In this report, we examine the effects of peptide nucleic acid (PNA) oligonucleotides and PNAs conjugated with peptides including TAT, muscle-specific peptide (MSP), adeno-associated virus 6 (AAV6) functional domain (AAV6), and AAV8 functional domain (AAV8), on exon skipping in vitro and in vivo. Efficient skipping of targeted exon 23 was achieved in cultured mdx myoblasts with PNA and PNA-peptide conjugates. Furthermore, single intramuscular injections of PNA and all PNA-peptide conjugates resulted in significant numbers of dystrophin-positive fibers in the injected tibialis anterior (TA) muscles of mdx mice, with no apparent local toxici...
This study examined the feasibility of using the promoter of the pig parotid secretory protein (P... more This study examined the feasibility of using the promoter of the pig parotid secretory protein (PSP) gene for expression of the phytase transgene in mouse models. The pig parotid secretory protein gene is specifically expressed at high levels in the salivary glands. The 10-kb upstream promoter region of the gene necessary for tissue-specific expression has been identified. We have constructed phytase transgenes composed of the appA phytase gene from Escherichia coli driven by the upstream promoter region of the pig PSP gene with a 3' tail of either bovine growth hormone or the pig PSP gene polyadenylation signal. Transgenic mouse models with the construct showed that the upstream region of the pig PSP gene is sufficient for directing the expression of phytase transgenes in the saliva. Expression of salivary phytase reduced fecal phytate by 8.5 and 12.5% in 2 transgenic mouse lines, respectively. These results suggest that the expression of phytase in salivary glands of monogastr...
The key regulatory elements for PKD2 transcription remain unclear. To identify these core element... more The key regulatory elements for PKD2 transcription remain unclear. To identify these core elements, we characterized porcine PKD2 promoter with bioinformatics and molecular tools and found porcine PKD2 promoter bearing typical features of enriched CpG and less TATA. Further studies demonstrated that the core region was located in fragment -483 to +100. Subsequent biophysical binding assays and mutation experiments revealed that G4 motif and Sp1 are critical regulators for mediating the transcription of porcine PKD2. Moreover, the same regulatory pattern was reproduced in human PKD2 promoter region, indicating the critical role of G4 and Sp1 in regulating PKD2.
Exon-skipping oligonucleotides are a well-researched therapeutic strategy for Duchenne&am... more Exon-skipping oligonucleotides are a well-researched therapeutic strategy for Duchenne's muscular dystrophy (DMD). Despite remarkable successes in animal models with intramuscular and intravenous delivery of unmodified oligonucleotides, the ability to specifically target both normal and dystrophic muscle with a simple peptide ligand could decrease the therapeutic dose required and reduce the potential for toxicity. Thus, 3 rounds of in vivo phage display utilizing a 12-mer peptide library were performed with mdx mice and a peptide motif with potential for targeting to muscle but not liver was identified. This motif was shown to have enhanced binding affinity to C2C12 myoblasts over a scrambled control peptide and in vivo application of a fluorescein-labeled peptide containing the identified motif resulted in increased specificity for the heart and quadriceps muscle after tail-vein administration in C57BL/6 mice. This work has many potential applications for oligonucleotide or drug delivery to muscle for myopathies.
Duchenne muscular dystrophy (DMD) is the most common and severe form of muscular dystrophy, arisi... more Duchenne muscular dystrophy (DMD) is the most common and severe form of muscular dystrophy, arising from mutations in the dystrophin gene that preclude the synthesis of functional protein. Antisense oligonucleotides (AOs) have been shown to induce specific exon skipping and thereby restore the reading frame and expression of functional dystrophin. In this report, we examine the effects of peptide nucleic acid (PNA) oligonucleotides and PNAs conjugated with peptides including TAT, muscle-specific peptide (MSP), adeno-associated virus 6 (AAV6) functional domain (AAV6), and AAV8 functional domain (AAV8), on exon skipping in vitro and in vivo. Efficient skipping of targeted exon 23 was achieved in cultured mdx myoblasts with PNA and PNA-peptide conjugates. Furthermore, single intramuscular injections of PNA and all PNA-peptide conjugates resulted in significant numbers of dystrophin-positive fibers in the injected tibialis anterior (TA) muscles of mdx mice, with no apparent local toxicity. Similar effects of exon skipping and dystrophin expression were obtained in mice of all ages. PNA and PNA-AAV6, PNA-AAV8 conjugates induced dystrophin expression in a dose-dependent manner. Our results demonstrate that PNAs have a higher efficiency of exon skipping than 2'O methyl phosphorothioate AOs do, and have a potential use in AO chemistry for antisense therapy of DMD.
Induced splice modulation of pre-mRNAs shows promise to correct aberrant disease transcripts and ... more Induced splice modulation of pre-mRNAs shows promise to correct aberrant disease transcripts and restore functional protein and thus has therapeutic potential. Duchenne muscular dystrophy (DMD) results from mutations that disrupt the DMD gene open reading frame causing an absence of dystrophin protein. Antisense oligonucleotide (AO)-mediated exon skipping has been shown to restore functional dystrophin in mdx mice and DMD patients treated intramuscularly in two recent phase 1 clinical trials. Critical to the therapeutic success of AO-based treatment will be the ability to deliver AOs systemically to all affected tissues including the heart. Here, we report identification of a series of transduction peptides (Pip5) as AO conjugates for enhanced systemic and particularly cardiac delivery. One of the lead peptide-AO conjugates, Pip5e-AO, showed highly efficient exon skipping and dystrophin production in mdx mice with complete correction of the aberrant DMD transcript in heart, leading to >50% of the normal level of dystrophin in heart. Mechanistic studies indicated that the enhanced activity of Pip5e-phosphorodiamidate morpholino (PMO) is partly explained by more efficient nuclear delivery. Pip5 series derivatives therefore have significant potential for advancing the development of exon skipping therapies for DMD and may have application for enhanced cardiac delivery of other biotherapeutics.
Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that abolish the synthes... more Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that abolish the synthesis of dystrophin protein. Antisense oligonucleotides (AOs) targeted to trigger excision of an exon bearing a mutant premature stop codon in the DMD transcript have been shown to skip the mutated exon and partially restore functional dystrophin protein in dystrophin-deficient mdx mice. To fully exploit the therapeutic potential of this method requires highly efficient systemic AO delivery to multiple muscle groups, to modify the disease process and restore muscle function. While systemic delivery of naked AOs in DMD animal models requires high doses and is of relatively poor efficiency, we and others have recently shown that short arginine-rich peptide-AO conjugates can dramatically improve in vivo DMD splice correction. Here we report for the first time that a chimeric fusion peptide (B-MSP-PMO) consisting of a muscle-targeting heptapeptide (MSP) fused to an arginine-rich cell-penetrating peptide (B-peptide) and conjugated to a morpholino oligomer (PMO) AO directs highly efficient systemic dystrophin splice correction in mdx mice. With very low systemic doses, we demonstrate that B-MSP-PMO restores high-level, uniform dystrophin protein expression in multiple peripheral muscle groups, yielding functional correction and improvement of the mdx dystrophic phenotype. Our data demonstrate proof-of-concept for this chimeric peptide approach in DMD splice correction therapy and is likely to have broad application.
Antisense oligonucleotides (AOs) have the potential to induce functional dystrophin protein expre... more Antisense oligonucleotides (AOs) have the potential to induce functional dystrophin protein expression via exon skipping by restoring in-frame transcripts in the majority of patients suffering from Duchenne muscular dystrophy (DMD). AOs of morpholino phosphoroamidate (PMO) and 2'-O-methyl phosphorothioate RNA (2'Ome RNA) chemistry have been shown to restore dystrophin expression in skeletal muscle but not in heart, following high-dose systemic delivery in murine models of muscular dystrophy (mdx). Exploiting the cell transduction properties of two basic arginine-rich cell penetrating peptides, we demonstrate widespread systemic correction of dystrophin expression in body-wide muscles and cardiac tissue in adult dystrophic mdx mice, with a single low-dose injection of peptide-conjugated PMO AO. This approach was sufficient to restore uniform, high-level dystrophin protein expression in peripheral muscle and cardiac tissue, with robust sarcolemmal relocalization of the dystrophin-associated protein complex and functional improvement in muscle. Peptide-conjugated AOs therefore have significant potential for systemic correction of the DMD phenotype.
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