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 $\epsilon$, arbitrary single-qubit gates can be produced fault-tolerantly and using a limited set of gates in time which is $O(\log \epsilon)$ or $O(\log \log \epsilon)$; 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

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

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

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

Studies on metal-organic frameworks of Cu(II) with isophthalate linkers for hydrogen storage

Hydrogen (H2) is a promising alternative energy carrier due to its environmental benefits, high energy density and its abundance. However, development of a practical storage system to enable the “Hydrogen Economy” remains a huge challenge. Metal-organic frameworks (MOFs) are an important class of crystalline coordination polymers constructed by bridging metal centers with organic linkers, and show promise for H2 storage due to their high surface area and tuneable properties. We summarize our research on novel porous materials with enhanced H2 storage properties, and describe frameworks derived from 3,5-substituted dicarboxylates (isophthalates) that serve as versatile molecular building blocks for the construction of a range of interesting coordination polymers with Cu(II) ions. A series of materials has been synthesised by connecting linear tetracarboxylate linkers to {Cu(II)2} paddlewheel moieties. These (4,4)-connected frameworks adopt the fof-topology in which the Kagomé lattice layers formed by {Cu(II)2} paddlewheels and isophthalates are pillared by the bridging ligands. These materials exhibit high structural stability and permanent porosity, and the pore size, geometry and functionality can be modulated by variation of the organic linker to control the overall H2 adsorption properties. NOTT-103 shows the highest H2 storage capacity of 77.8 mg g−1 at 77 K, 60 bar among the fof-type frameworks. H2 adsorption at low, medium and high pressures correlates with the isosteric heat of adsorption, surface area and pore volume, respectively. Tri-branched C3-symmetric hexacarboxylate ligands with Cu(II) give highly porous (3,24)-connected frameworks incorporating {Cu(II)2} paddlewheels. These ubt-type frameworks comprise three types of polyhedral cage: a cuboctahedron, truncated tetrahedron and a truncated octahedron which are fused in the solid state in the ratio 1:2:1, respectively. Increasing the length of the hexacarboxylate struts directly tunes the porosity of the resultant material from micro- to mesoporosity. These materials show exceptionally high H2 uptakes owing to their high surface area and pore volume. NOTT-112, the first reported member of this family reported, adsorbs 111 mg g−1 of H2 at 77 K , 77 bar. More recently, enhanced H2 adsorption in these ubt-type frameworks has been achieved using combinations of polyphenyl groups linked by alkynes to give an overall gravimetric gas capacity for NU-100 of 164 mg g−1 at 77 K, 70 bar. However, due to its very low density NU-100 shows a lower volumetric capacity of 45.7 g L-1 compared with 55.9 g L-1 for NOTT-112, which adsorbs 2.3 wt% H2 at 1 bar, 77K. This significant adsorption of H2 at low pressures is attributed to the arrangement of the {Cu24(isophthalate)24} cuboctahedral cages within the polyhedral structure. Free metal coordination positions are the first binding sites for D2, and in these ubt-type frameworks there are two types of Cu(II) centres, one with its vacant site pointing into the cuboctahedral cage and another pointing externally. D2 molecules bind first at the former position, and then at the external open metal sites. However, other adsorption sites between the cusp of three phenyl groups and a Type I pore window in the framework are also occupied. Ligand and complex design feature strongly in enhancing and maximising H2 storage, and, although current materials operate at 77 K, research continues to explore routes to high capacity H2 storage materials that can function at higher temperatures