156 research outputs found
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In Vivo Delivery of Morpholino Oligos by Cell-Penetrating Peptides
Morpholino oligos (Morpholinos) are widely used tools for knocking down gene expression and are currently in a clinical trial for treatment of Duchene muscular dystrophy. A Morpholino analog has been in a clinical trial as a potential anti-bioterrorism agent for inhibiting replication of deadly Marburg viral infection. The cellular uptake of Morpholinos can been greatly increased by conjugation with cell-penetrating peptides (CPP). The use of the CPP-Morpholino conjugates (PPMOs) in vivo has been broadly demonstrated in viral, bacterial, genetic and other diseases. The following aspects of PPMOs will be discussed in this paper including chemistry, stability, antisense specificity, mechanism of cellular uptake, in vivo efficacy, tissue distribution, pharmacokinetics, toxicity and the human clinical trials. PPMOs are powerful research tools for studying gene function in animals and their properties are being improved as potential human therapeutic agents.Keywords: Antisense, PPMO, Cell-penetrating peptide, Duchenne muscular dystrophy, Morpholino oligo
Cell-penetrating peptides as transporters for morpholino oligomers: effects of amino acid composition on intracellular delivery and cytotoxicity
Arginine-rich cell-penetrating peptides (CPPs) are promising transporters for intracellular delivery of antisense morpholino oligomers (PMO). Here, we determined the effect of L-arginine, D-arginine and non-α amino acids on cellular uptake, splice-correction activity, cellular toxicity and serum binding for 24 CPP−PMOs. Insertion of 6-aminohexanoic acid (X) or β-alanine (B) residues into oligoarginine R8 decreased the cellular uptake but increased the splice-correction activity of the resulting compound, with a greater increase for the sequences containing more X residues. Cellular toxicity was not observed for any of the conjugates up to 10 μM. Up to 60 μM, only the conjugates with ⩾ 5 Xs exhibited time- and concentration-dependent toxicity. Substitution of L-arginine with D-arginine did not increase uptake or splice-correction activity. High concentration of serum significantly decreased the uptake and splice-correction activity of oligoarginine conjugates, but had much less effect on the conjugates containing X or B. In summary, incorporation of X/B into oligoarginine enhanced the antisense activity and serum-binding profile of CPP−PMO. Toxicity of X/B-containing conjugates was affected by the number of Xs, treatment time and concentration. More active, stable and less toxic CPPs can be designed by optimizing the position and number of R, D-R, X and B residues
Delivery of steric block morpholino oligomers by (R-X-R)4 peptides: structure–activity studies
Redirecting the splicing machinery through the hybridization of high affinity, RNase H- incompetent oligonucleotide analogs such as phosphoramidate morpholino oligonucleotides (PMO) might lead to important clinical applications. Chemical conjugation of PMO to arginine-rich cell penetrating peptides (CPP) such as (R-Ahx-R)4 (with Ahx standing for 6-aminohexanoic acid) leads to sequence-specific splicing correction in the absence of endosomolytic agents in cell culture at variance with most conventional CPPs. Importantly, (R-Ahx-R)4–PMO conjugates are effective in mouse models of various viral infections and Duchenne muscular dystrophy. Unfortunately, active doses in some applications might be close to cytotoxic ones thus presenting challenge for systemic administration of the conjugates in those clinical settings. Structure–activity relationship studies have thus been undertaken to unravel CPP structural features important for the efficient nuclear delivery of the conjugated PMO and limiting steps in their internalization pathway. Affinity for heparin (taken as a model heparan sulfate), hydrophobicity, cellular uptake, intracellular distribution and splicing correction have been monitored. Spacing between the charges, hydrophobicity of the linker between the Arg-groups and Arg-stereochemistry influence splicing correction efficiency. A significant correlation between splicing correction efficiency, affinity for heparin and ability to destabilize model synthetic vesicles has been observed but no correlation with cellular uptake has been found. Efforts will have to focus on endosomal escape since it appears to remain the limiting factor for the delivery of these splice-redirecting ON analogs
A Dystrophin Exon-52 Deleted Miniature Pig Model of Duchenne Muscular Dystrophy and Evaluation of Exon Skipping
Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder caused by mutations in the DMD gene and the subsequent lack of dystrophin protein. Recently, phosphorodiamidate morpholino oligomer (PMO)-antisense oligonucleotides (ASOs) targeting exon 51 or 53 to reestablish the DMD reading frame have received regulatory approval as commercially available drugs. However, their applicability and efficacy remain limited to particular patients. Large animal models and exon skipping evaluation are essential to facilitate ASO development together with a deeper understanding of dystrophinopathies. Using recombinant adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer, we generated a Yucatan miniature pig model of DMD with an exon 52 deletion mutation equivalent to one of the most common mutations seen in patients. Exon 52-deleted mRNA expression and dystrophin deficiency were confirmed in the skeletal and cardiac muscles of DMD pigs. Accordingly, dystrophin-associated proteins failed to be recruited to the sarcolemma. The DMD pigs manifested early disease onset with severe bodywide skeletal muscle degeneration and with poor growth accompanied by a physical abnormality, but with no obvious cardiac phenotype. We also demonstrated that in primary DMD pig skeletal muscle cells, the genetically engineered exon-52 deleted pig DMD gene enables the evaluation of exon 51 or 53 skipping with PMO and its advanced technology, peptide-conjugated PMO. The results show that the DMD pigs developed here can be an appropriate large animal model for evaluating in vivo exon skipping efficacy
Simulating chemistry efficiently on fault-tolerant quantum computers
Quantum computers can in principle simulate quantum physics exponentially
faster than their classical counterparts, but some technical hurdles remain.
Here we consider methods to make proposed chemical simulation algorithms
computationally fast on fault-tolerant quantum computers in the circuit model.
Fault tolerance constrains the choice of available gates, so that arbitrary
gates required for a simulation algorithm must be constructed from sequences of
fundamental operations. We examine techniques for constructing arbitrary gates
which perform substantially faster than circuits based on the conventional
Solovay-Kitaev algorithm [C.M. Dawson and M.A. Nielsen, \emph{Quantum Inf.
Comput.}, \textbf{6}:81, 2006]. For a given approximation error ,
arbitrary single-qubit gates can be produced fault-tolerantly and using a
limited set of gates in time which is or ; with sufficient parallel preparation of ancillas, constant average
depth is possible using a method we call programmable ancilla rotations.
Moreover, we construct and analyze efficient implementations of first- and
second-quantized simulation algorithms using the fault-tolerant arbitrary gates
and other techniques, such as implementing various subroutines in constant
time. A specific example we analyze is the ground-state energy calculation for
Lithium hydride.Comment: 33 pages, 18 figure
Sustained Dystrophin Expression Induced by Peptide-conjugated Morpholino Oligomers in the Muscles of mdx Mice
Cell-penetrating peptides (CPPs), containing arginine (R), 6-aminohexanoic acid (X), and/or β-alanine (B) conjugated to phosphorodiamidate morpholino oligomers (PMOs), enhance their delivery in cell culture. In this study, the potency, functional biodistribution, and toxicity of these conjugates were evaluated in vivo, in EGFP-654 transgenic mice that ubiquitously express the aberrantly spliced EGFP-654 pre-mRNA reporter. Correct splicing and enhanced green fluorescence protein (EGFP) upregulation serve as a positive readout for peptide-PMO (PPMO) entry into cells and access to EGFP-654 pre-mRNA in the nucleus. Intraperitoneal injections of a series of PPMOs, A-N (12 mg/kg), administered once a day for four successive days resulted in splicing correction in numerous tissues. PPMO-B was highly potent in the heart, diaphragm, and quadriceps, which are key muscles in the treatment of Duchenne muscular dystrophy. We therefore investigated PPMO M23D-B, designed to force skipping of stop-codon containing dystrophin exon 23, in an mdx mouse model of the disease. Systemic delivery of M23D-B yielded persistent exon 23 skipping, yielding high and sustained dystrophin protein expression in body-wide muscles, including cardiac muscle, without detectable toxicity. The rescued dystrophin reduced serum creatinine kinase to near-wild-type levels, indicating improvement in muscle integrity. This is the first report of oligonucleotide-mediated exon skipping and dystrophin protein induction in the heart of treated animals
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Fructose Promotes Uptake and Activity of Oligonucleotides With Different Chemistries in a Context-dependent Manner in mdx Mice
Antisense oligonucleotide (AO)-mediated exon-skipping therapeutics shows great promise in correcting frame-disrupting mutations in the DMD gene for Duchenne muscular dystrophy. However, insufficient systemic delivery limits clinical adoption. Previously, we showed that a glucose/fructose mixture augmented AO delivery to muscle in mdx mice. Here, we evaluated if fructose alone could enhance the activities of AOs with different chemistries in mdx mice. The results demonstrated that fructose improved the potency of AOs tested with the greatest effect on phosphorodiamidate morpholino oligomer (PMO), resulted in a 4.25-fold increase in the number of dystrophin-positive fibres, compared to PMO in saline in mdx mice. Systemic injection of lissamine-labeled PMO with fructose at 25 mg/kg led to increased uptake and elevated dystrophin expression in peripheral muscles, compared to PMO in saline, suggesting that fructose potentiates PMO by enhancing uptake. Repeated intravenous administration of PMO in fructose at 50 mg/kg/week for 3 weeks and 50 mg/kg/month for 5 months restored up to 20% of wild-type dystrophin levels in skeletal muscles with improved functions without detectable toxicity, compared to untreated mdx controls. Collectively, we show that fructose can potentiate AOs of different chemistries in vivo although the effect diminished over repeated administration.This is the publisher’s final pdf. The article is copyrighted by the author(s) and published by Nature Publishing Group on behalf of the American Society of Gene and Cell Therapy. It can be found at: http://www.nature.com/mtna/journal/v5/n6/full/mtna201646a.htmlKeywords: antisense oligonucleotide, Duchenne muscular dystrophy, exon skipping, fructos
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Long-Term Rescue of Dystrophin Expression and Improvement in Muscle Pathology and Function in Dystrophic Mdx Mice by Peptide-Conjugated Morpholino
Exon skipping is capable of correcting frame-shift and nonsense mutations of
Duchenne Muscular Dystrophy (DMD). Phase II clinical trials in UK and Netherlands
have reported induction of dystrophin expression in muscles of DMD patients by
systemic administrations of both phosphorodiamidate morpholino oligomers (PMO)
and 2’O methyl phosphorothioate. Peptide-conjugated PMO (PPMO) offers
significantly higher efficiency than PMO with the ability to induce near normal levels
of dystrophin and restores functions in both skeletal and cardiac muscles. Here, we
examined one year systemic efficacy of PPMO targeting exon 23 in dystrophic mdx
mice. LD50 of the PPMO was approximately 85mg/kg. Half life of the dystrophin
expression was about 2 months in skeletal muscles but shorter in cardiac muscle.
Biweekly injection of 6 mg/kg PPMO produced higher than 20% dystrophin
expression in all skeletal muscles, and up to 5% in cardiac muscles with
improvement in muscle function and pathology, and reduction in the levels of serum
creatine kinase (CK). Monthly injections of 30mg/kg PPMO restored dystrophin to
more than 50% normal levels in all of skeletal muscles, but 15% in cardiac muscle.
This was associated with greatly reduced serum CK levels, near normal histology,
and functional improvement of skeletal muscles. Our result demonstrated for the
first time that one year regular administration of PPMO could be safely applied to
achieve significant therapeutic effect in animal models
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Context Dependent Effects of Chimeric Peptide Morpholino Conjugates Contribute to Dystrophin Exon-skipping Efficiency
We have recently reported that cell-penetrating peptides (CPPs) and novel chimeric peptides containing CPP (referred as
B peptide) and muscle-targeting peptide (referred as MSP) motifs significantly improve the systemic exon-skipping activity
of morpholino phosphorodiamidate oligomers (PMOs) in dystrophin-deficient mdx mice. In the present study, the general
mechanistic significance of the chimeric peptide configuration on the activity and tissue uptake of peptide conjugated PMOs
in vivo was investigated. Four additional chimeric peptide-PMO conjugates including newly identified peptide 9 (B-9-PMO and
9-B-PMO) and control peptide 3 (B-3-PMO and 3-B-PMO) were tested in mdx mice. Immunohistochemical staining, RT-PCR and
western blot results indicated that B-9-PMO induced significantly higher level of exon skipping and dystrophin restoration than
its counterpart (9-B-PMO), further corroborating the notion that the activity of chimeric peptide-PMO conjugates is dependent on
relative position of the tissue-targeting peptide motif within the chimeric peptide with respect to PMOs. Subsequent mechanistic
studies showed that enhanced cellular uptake of B-MSP-PMO into muscle cells leads to increased exon-skipping activity in
comparison with MSP-B-PMO. Surprisingly, further evidence showed that the uptake of chimeric peptide-PMO conjugates of
both orientations (B-MSP-PMO and MSP-B-PMO) was ATP- and temperature-dependent and also partially mediated by heparan
sulfate proteoglycans (HSPG), indicating that endocytosis is likely the main uptake pathway for both chimeric peptide-PMO
conjugates. Collectively, our data demonstrate that peptide orientation in chimeric peptides is an important parameter that
determines cellular uptake and activity when conjugated directly to oligonucleotides. These observations provide insight into
the design of improved cell targeting compounds for future therapeutics studies.This is the publisher’s final pdf. The article is copyrighted by the American Society of Gene & Cell Therapy and published by the Nature Publishing Group. It can be found at: http://www.nature.com/mtna/index.html.Keywords: Antisense oligonucleotide, Exon skipping, Chimeric peptide conjugate, Duchenne muscular dystroph
Modification of Hydrophilic and Hydrophobic Surfaces Using an Ionic-Complementary Peptide
Ionic-complementary peptides are novel nano-biomaterials with a variety of biomedical applications including potential biosurface engineering. This study presents evidence that a model ionic-complementary peptide EAK16-II is capable of assembling/coating on hydrophilic mica as well as hydrophobic highly ordered pyrolytic graphite (HOPG) surfaces with different nano-patterns. EAK16-II forms randomly oriented nanofibers or nanofiber networks on mica, while ordered nanofibers parallel or oriented 60° or 120° to each other on HOPG, reflecting the crystallographic symmetry of graphite (0001). The density of coated nanofibers on both surfaces can be controlled by adjusting the peptide concentration and the contact time of the peptide solution with the surface. The coated EAK16-II nanofibers alter the wettability of the two surfaces differently: the water contact angle of bare mica surface is measured to be <10°, while it increases to 20.3±2.9° upon 2 h modification of the surface using a 29 µM EAK16-II solution. In contrast, the water contact angle decreases significantly from 71.2±11.1° to 39.4±4.3° after the HOPG surface is coated with a 29 µM peptide solution for 2 h. The stability of the EAK16-II nanofibers on both surfaces is further evaluated by immersing the surface into acidic and basic solutions and analyzing the changes in the nanofiber surface coverage. The EAK16-II nanofibers on mica remain stable in acidic solution but not in alkaline solution, while they are stable on the HOPG surface regardless of the solution pH. This work demonstrates the possibility of using self-assembling peptides for surface modification applications
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