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Michael J. Gait - One of the best experts on this subject based on the ideXlab platform.
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identification of a peptide for systemic brain delivery of a Morpholino Oligonucleotide in mouse models of spinal muscular atrophy
Nucleic Acid Therapeutics, 2017Co-Authors: Fazel Shabanpoor, Suzan M Hammond, Frank Abendroth, G Hazell, Wood Mja, Michael J. GaitAbstract:Splice-switching antisense Oligonucleotides are emerging treatments for neuromuscular diseases, with several splice-switching Oligonucleotides (SSOs) currently undergoing clinical trials such as for Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA). However, the development of systemically delivered antisense therapeutics has been hampered by poor tissue penetration and cellular uptake, including crossing of the blood-brain barrier (BBB) to reach targets in the central nervous system (CNS). For SMA application, we have investigated the ability of various BBB-crossing peptides for CNS delivery of a splice-switching phosphorodiamidate Morpholino Oligonucleotide (PMO) targeting survival motor neuron 2 (SMN2) exon 7 inclusion. We identified a branched derivative of the well-known ApoE (141-150) peptide, which as a PMO conjugate was capable of exon inclusion in the CNS following systemic administration, leading to an increase in the level of full-length SMN2 transcript. Treatment of newborn SMA mice with this peptide-PMO (P-PMO) conjugate resulted in a significant increase in the average lifespan and gains in weight, muscle strength, and righting reflexes. Systemic treatment of adult SMA mice with this newly identified P-PMO also resulted in small but significant increases in the levels of SMN2 pre-messenger RNA (mRNA) exon inclusion in the CNS and peripheral tissues. This work provides proof of principle for the ability to select new peptide paradigms to enhance CNS delivery and activity of a PMO SSO through use of a peptide-based delivery platform for the treatment of SMA potentially extending to other neuromuscular and neurodegenerative diseases.
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bi specific splice switching pmo Oligonucleotides conjugated via a single peptide active in a mouse model of duchenne muscular dystrophy
Nucleic Acids Research, 2015Co-Authors: Fazel Shabanpoor, Amer F. Saleh, Matthew Ja Wood, Graham Mcclorey, Peter Jarver, Michael J. GaitAbstract:The potential for therapeutic application of splice-switching Oligonucleotides (SSOs) to modulate pre-mRNA splicing is increasingly evident in a number of diseases. However, the primary drawback of this approach is poor cell and in vivo Oligonucleotide uptake efficacy. Biological activities can be significantly enhanced through the use of synthetically conjugated cationic cell penetrating peptides (CPPs). Studies to date have focused on the delivery of a single SSO conjugated to a CPP, but here we describe the conjugation of two phosphorodiamidate Morpholino Oligonucleotide (PMO) SSOs to a single CPP for simultaneous delivery and pre-mRNA targeting of two separate genes, exon 23 of the Dmd gene and exon 5 of the Acvr2b gene, in a mouse model of Duchenne muscular dystrophy. Conjugations of PMOs to a single CPP were carried out through an amide bond in one case and through a triazole linkage (‘click chemistry’) in the other. The most active bi-specific CPP–PMOs demonstrated comparable exon skipping levels for both pre-mRNA targets when compared to individual CPP–PMO conjugates both in cell culture and in vivo in the mdx mouse model. Thus, two SSOs with different target sequences conjugated to a single CPP are biologically effective and potentially suitable for future therapeutic exploitation.
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Parallel Synthesis of Cell-Penetrating Peptide Conjugates of PMO Toward Exon Skipping Enhancement in Duchenne Muscular Dystrophy
Nucleic Acid Therapeutics, 2014Co-Authors: Liz O'donovan, Itaru Okamoto, Andrey A. Arzumanov, Donna Williams, Peter J Deuss, Michael J. GaitAbstract:We describe two new methods of parallel chemical synthesis of libraries of peptide conjugates of phosphorodiamidate Morpholino Oligonucleotide (PMO) cargoes on a scale suitable for cell screening prior to in vivo analysis for therapeutic development. The methods represent an extension of the SELection of PEPtide CONjugates (SELPEPCON) approach previously developed for parallel peptide-peptide nucleic acid (PNA) synthesis. However, these new methods allow for the utilization of commercial PMO as cargo with both C- and N-termini unfunctionalized. The synthetic methods involve conjugation in solution phase, followed by rapid purification via biotin-streptavidin immobilization and subsequent reductive release into solution, avoiding the need for painstaking high-performance liquid chromatography purifications. The synthesis methods were applied for screening of PMO conjugates of a 16-member library of variants of a 10-residue ApoE peptide, which was suggested for blood-brain barrier crossing. In this work the conjugate library was tested in an exon skipping assay using skeletal mouse mdx cells, a model of Duchene's muscular dystrophy where higher activity peptide-PMO conjugates were identified compared with the starting peptide-PMO. The results demonstrate the power of the parallel synthesis methods for increasing the speed of optimization of peptide sequences in conjugates of PMO for therapeutic screening.
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development of a general methodology for labelling peptide Morpholino Oligonucleotide conjugates using alkyne azide click chemistry
Chemical Communications, 2013Co-Authors: Fazel Shabanpoor, Michael J. GaitAbstract:We describe a general methodology for fluorescent labelling of peptide conjugates of phosphorodiamidate Morpholino Oligonucleotides (PMOs) by alkyne functionalization of peptides, subsequent conjugation to PMOs and labelling with a fluorescent compound (Cy5-azide). Two peptide–PMO (PPMO) examples are shown. No detrimental effect of such labelled PMOs was seen in a biological assay.
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Pip6-PMO, a new generation of peptide-Oligonucleotide conjugates with improved cardiac exon skipping activity for DMD treatment
Molecular Therapy - Nucleic Acids, 2012Co-Authors: Corinne Betts, Amer F. Saleh, Caroline Godfrey, Thibault Coursindel, Suzan M Hammond, Andrey A. Arzumanov, Michael J. Gait, Matthew Ja WoodAbstract:Antisense Oligonucleotides (AOs) are currently the most promising therapeutic intervention for Duchenne muscular dystrophy (DMD). AOs modulate dystrophin pre-mRNA splicing, thereby specifically restoring the dystrophin reading frame and generating a truncated but semifunctional dystrophin protein. Challenges in the development of this approach are the relatively poor systemic AO delivery and inefficient dystrophin correction in affected non-skeletal muscle tissues, including the heart. We have previously reported impressive heart activity including high-splicing efficiency and dystrophin restoration following a single administration of an arginine-rich cell-penetrating peptide (CPPs) conjugated to a phosphorodiamidate Morpholino Oligonucleotide (PMO): Pip5e-PMO. However, the mechanisms underlying this activity are poorly understood. Here, we report studies involving single dose administration (12.5 mg/kg) of derivatives of Pip5e-PMO, consecutively assigned as Pip6-PMOs. These peptide-PMOs comprise alterations to the central hydrophobic core of the Pip5e peptide and illustrate that certain changes to the peptide sequence improves its activity; however, partial deletions within the hydrophobic core abolish its efficiency. Our data indicate that the hydrophobic core of the Pip sequences is critical for PMO delivery to the heart and that specific modifications to this region can enhance activity further. The results have implications for therapeutic PMO development for DMD.
Fazel Shabanpoor - One of the best experts on this subject based on the ideXlab platform.
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amphiphilic lipopeptide significantly enhances uptake of charge neutral splice switching Morpholino Oligonucleotide in spinal muscular atrophy patient derived fibroblasts
International Journal of Pharmaceutics, 2017Co-Authors: Behnoosh Tajikahmadabad, Anastasios Polyzos, Frances Separovic, Fazel ShabanpoorAbstract:Splice-switching antisense Oligonucleotides (SSOs) are emerging therapeutics with two SSOs recently approved by the FDA for Duchenne muscular dystrophy and spinal muscular atrophy. SSOs are administered without any delivery vector and require large doses to achieve the therapeutic benefit, primarily due to their poor cellular uptake. Although cell-penetrating peptides (CPP) have shown great potential in delivering SSOs into cells, their capacity as delivery vector is limited. Here we have studied the effect of lipid conjugation on the cell permeability of a known CPP (ApoE). Myristic acid was coupled at the N-terminus of ApoE to a C-terminal cysteine residue. The myristoylated ApoE (Myr-ApoE) was conjugated to a maleimide functionalised phosphorodiamidate Morpholino Oligonucleotide (PMO). The Myr-ApoE-PMO conjugate showed no cytoxicity and had significantly higher efficiency in cell permeability with 30% higher splice-switching activity compared to ApoE-PMO. The self-assembly properties of this amphiphilic lipopeptide-PMO conjugate was assessed. Transmission electron microscopy showed formation of nanoparticles with amphiphile behaviour and spherical structure. The self-assembly of Myr-ApoE-PMO into nanoparticles enabled it to better bind to cell membranes and to be more efficiently taken up by fibroblast cells. These results showed that modification of physico-chemical properties of peptides to produce peptide amphiphiles enhances cellular uptake and can be used as an efficient delivery vector for therapeutic SSOs.
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identification of a peptide for systemic brain delivery of a Morpholino Oligonucleotide in mouse models of spinal muscular atrophy
Nucleic Acid Therapeutics, 2017Co-Authors: Fazel Shabanpoor, Suzan M Hammond, Frank Abendroth, G Hazell, Wood Mja, Michael J. GaitAbstract:Splice-switching antisense Oligonucleotides are emerging treatments for neuromuscular diseases, with several splice-switching Oligonucleotides (SSOs) currently undergoing clinical trials such as for Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA). However, the development of systemically delivered antisense therapeutics has been hampered by poor tissue penetration and cellular uptake, including crossing of the blood-brain barrier (BBB) to reach targets in the central nervous system (CNS). For SMA application, we have investigated the ability of various BBB-crossing peptides for CNS delivery of a splice-switching phosphorodiamidate Morpholino Oligonucleotide (PMO) targeting survival motor neuron 2 (SMN2) exon 7 inclusion. We identified a branched derivative of the well-known ApoE (141-150) peptide, which as a PMO conjugate was capable of exon inclusion in the CNS following systemic administration, leading to an increase in the level of full-length SMN2 transcript. Treatment of newborn SMA mice with this peptide-PMO (P-PMO) conjugate resulted in a significant increase in the average lifespan and gains in weight, muscle strength, and righting reflexes. Systemic treatment of adult SMA mice with this newly identified P-PMO also resulted in small but significant increases in the levels of SMN2 pre-messenger RNA (mRNA) exon inclusion in the CNS and peripheral tissues. This work provides proof of principle for the ability to select new peptide paradigms to enhance CNS delivery and activity of a PMO SSO through use of a peptide-based delivery platform for the treatment of SMA potentially extending to other neuromuscular and neurodegenerative diseases.
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bi specific splice switching pmo Oligonucleotides conjugated via a single peptide active in a mouse model of duchenne muscular dystrophy
Nucleic Acids Research, 2015Co-Authors: Fazel Shabanpoor, Amer F. Saleh, Matthew Ja Wood, Graham Mcclorey, Peter Jarver, Michael J. GaitAbstract:The potential for therapeutic application of splice-switching Oligonucleotides (SSOs) to modulate pre-mRNA splicing is increasingly evident in a number of diseases. However, the primary drawback of this approach is poor cell and in vivo Oligonucleotide uptake efficacy. Biological activities can be significantly enhanced through the use of synthetically conjugated cationic cell penetrating peptides (CPPs). Studies to date have focused on the delivery of a single SSO conjugated to a CPP, but here we describe the conjugation of two phosphorodiamidate Morpholino Oligonucleotide (PMO) SSOs to a single CPP for simultaneous delivery and pre-mRNA targeting of two separate genes, exon 23 of the Dmd gene and exon 5 of the Acvr2b gene, in a mouse model of Duchenne muscular dystrophy. Conjugations of PMOs to a single CPP were carried out through an amide bond in one case and through a triazole linkage (‘click chemistry’) in the other. The most active bi-specific CPP–PMOs demonstrated comparable exon skipping levels for both pre-mRNA targets when compared to individual CPP–PMO conjugates both in cell culture and in vivo in the mdx mouse model. Thus, two SSOs with different target sequences conjugated to a single CPP are biologically effective and potentially suitable for future therapeutic exploitation.
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development of a general methodology for labelling peptide Morpholino Oligonucleotide conjugates using alkyne azide click chemistry
Chemical Communications, 2013Co-Authors: Fazel Shabanpoor, Michael J. GaitAbstract:We describe a general methodology for fluorescent labelling of peptide conjugates of phosphorodiamidate Morpholino Oligonucleotides (PMOs) by alkyne functionalization of peptides, subsequent conjugation to PMOs and labelling with a fluorescent compound (Cy5-azide). Two peptide–PMO (PPMO) examples are shown. No detrimental effect of such labelled PMOs was seen in a biological assay.
Matthew Ja Wood - One of the best experts on this subject based on the ideXlab platform.
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bi specific splice switching pmo Oligonucleotides conjugated via a single peptide active in a mouse model of duchenne muscular dystrophy
Nucleic Acids Research, 2015Co-Authors: Fazel Shabanpoor, Amer F. Saleh, Matthew Ja Wood, Graham Mcclorey, Peter Jarver, Michael J. GaitAbstract:The potential for therapeutic application of splice-switching Oligonucleotides (SSOs) to modulate pre-mRNA splicing is increasingly evident in a number of diseases. However, the primary drawback of this approach is poor cell and in vivo Oligonucleotide uptake efficacy. Biological activities can be significantly enhanced through the use of synthetically conjugated cationic cell penetrating peptides (CPPs). Studies to date have focused on the delivery of a single SSO conjugated to a CPP, but here we describe the conjugation of two phosphorodiamidate Morpholino Oligonucleotide (PMO) SSOs to a single CPP for simultaneous delivery and pre-mRNA targeting of two separate genes, exon 23 of the Dmd gene and exon 5 of the Acvr2b gene, in a mouse model of Duchenne muscular dystrophy. Conjugations of PMOs to a single CPP were carried out through an amide bond in one case and through a triazole linkage (‘click chemistry’) in the other. The most active bi-specific CPP–PMOs demonstrated comparable exon skipping levels for both pre-mRNA targets when compared to individual CPP–PMO conjugates both in cell culture and in vivo in the mdx mouse model. Thus, two SSOs with different target sequences conjugated to a single CPP are biologically effective and potentially suitable for future therapeutic exploitation.
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Pip6-PMO, a new generation of peptide-Oligonucleotide conjugates with improved cardiac exon skipping activity for DMD treatment
Molecular Therapy - Nucleic Acids, 2012Co-Authors: Corinne Betts, Amer F. Saleh, Caroline Godfrey, Thibault Coursindel, Suzan M Hammond, Andrey A. Arzumanov, Michael J. Gait, Matthew Ja WoodAbstract:Antisense Oligonucleotides (AOs) are currently the most promising therapeutic intervention for Duchenne muscular dystrophy (DMD). AOs modulate dystrophin pre-mRNA splicing, thereby specifically restoring the dystrophin reading frame and generating a truncated but semifunctional dystrophin protein. Challenges in the development of this approach are the relatively poor systemic AO delivery and inefficient dystrophin correction in affected non-skeletal muscle tissues, including the heart. We have previously reported impressive heart activity including high-splicing efficiency and dystrophin restoration following a single administration of an arginine-rich cell-penetrating peptide (CPPs) conjugated to a phosphorodiamidate Morpholino Oligonucleotide (PMO): Pip5e-PMO. However, the mechanisms underlying this activity are poorly understood. Here, we report studies involving single dose administration (12.5 mg/kg) of derivatives of Pip5e-PMO, consecutively assigned as Pip6-PMOs. These peptide-PMOs comprise alterations to the central hydrophobic core of the Pip5e peptide and illustrate that certain changes to the peptide sequence improves its activity; however, partial deletions within the hydrophobic core abolish its efficiency. Our data indicate that the hydrophobic core of the Pip sequences is critical for PMO delivery to the heart and that specific modifications to this region can enhance activity further. The results have implications for therapeutic PMO development for DMD.
Sidney Altman - One of the best experts on this subject based on the ideXlab platform.
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combined effect of a peptide Morpholino Oligonucleotide conjugate and a cell penetrating peptide as an antibiotic
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Donna Wesolowski, Dulce Alonso, Sidney AltmanAbstract:A cell-penetrating peptide (CPP)–Morpholino Oligonucleotide (MO) conjugate (PMO) that has an antibiotic effect in culture had some contaminating CPPs in earlier preparations. The mixed conjugate had gene-specific and gene-nonspecific effects. An improved purification procedure separates the PMO from the free CPP and MO. The gene-specific effects are a result of the PMO, and the nonspecific effects are a result of the unlinked, unreacted CPP. The PMO and the CPP can be mixed together, as has been shown previously in earlier experiments, and have a combined effect as an antibiotic. Kinetic analysis of these effects confirm this observation. The effect of the CPP is bacteriostatic. The effect of the PMO appears to be bacteriocidal. An assay for mutations that would alter the ability of these agents to affect bacterial viability is negative.
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Basic peptide-Morpholino oligomer conjugate that is very effective in killing bacteria by gene-specific and nonspecific modes
Proceedings of the National Academy of Sciences, 2011Co-Authors: Donna Wesolowski, Hyun Seop Tae, Neeru Gandotra, Paula Montero Llopis, Ning Shen, Sidney AltmanAbstract:Basic peptides covalently linked to nucleic acids, or chemically modified nucleic acids, enable the insertion of such a conjugate into bacteria grown in liquid medium and mammalian cells in tissue culture. A unique peptide, derived from human T cells, has been employed in a chemical synthesis to make a conjugate with a Morpholino Oligonucleotide. This new conjugate is at least 10- to 100-fold more effective than previous peptides used in altering the phenotype of host bacteria if the external guide sequence methodology is employed in these experiments. Bacteria with target genes expressing chloramphenicol resistance, penicillin resistance, or gyrase A function can effectively be reduced in their expression and the host cells killed. Several bacteria are susceptible to this treatment, which has a broad range of potency. The loss in viability of bacteria is not due only to complementarity with a target RNA and the action of RNase P, but also to a non-gene-specific tight binding of the complexed nontargeted RNA to the basic polypeptide-Morpholino Oligonucleotide.
J C Smith - One of the best experts on this subject based on the ideXlab platform.
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defining synphenotype groups in xenopus tropicalis by use of antisense Morpholino Oligonucleotides
PLOS Genetics, 2005Co-Authors: Amer A Rana, Clara Collart, Michael J Gilchrist, J C SmithAbstract:To identify novel genes involved in early development, and as proof-of-principle of a large-scale reverse genetics approach in a vertebrate embryo, we have carried out an antisense Morpholino Oligonucleotide (MO) screen in Xenopus tropicalis, in the course of which we have targeted 202 genes expressed during gastrula stages. MOs were designed to complement sequence between −80 and +25 bases of the initiating AUG codons of the target mRNAs, and the specificities of many were tested by (i) designing different non-overlapping MOs directed against the same mRNA, (ii) injecting MOs differing in five bases, and (iii) performing “rescue” experiments. About 65% of the MOs caused X. tropicalis embryos to develop abnormally (59% of those targeted against novel genes), and we have divided the genes into “synphenotype groups,” members of which cause similar loss-of-function phenotypes and that may function in the same developmental pathways. Analysis of the expression patterns of the 202 genes indicates that members of a synphenotype group are not necessarily members of the same synexpression group. This screen provides new insights into early vertebrate development and paves the way for a more comprehensive MO-based analysis of gene function in X. tropicalis.