An alternative strategy to increasing influenza virus replication for vaccine production in chicken embryo fibroblast (DF-1) cells by inhibiting interferon alpha and beta using peptide-conjugated phosphorodiamidate morpholino oligomers.

Introduction. Influenza is a global health issue causing substantial health and economic burdens on affected populations. Routine, annual vaccination for influenza virus is recommended for all persons older than 6 months of age. The propagation of the influenza virus for vaccine production is predominantly through embryonated chicken eggs.Hypothesis/Gap Statement. Many challenges face the propagation of the virus, including but not limited to low yields and lengthy production times. The development of a method to increase vaccine production in eggs or cell lines by suppressing cellular gene expression would be helpful to overcome some of the challenges facing influenza vaccine production.Aims. This study aimed to increase influenza virus titres by using a peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO), an antisense molecule, to suppress protein expression of the host genes interferon alpha (IFN-α) and interferon beta (IFN-ß) in chicken embryo fibroblast (DF-1) cells.Methods. The toxicity of PPMOs was evaluated by cytotoxicity assays, and their specificity to inhibit IFN-α and IFN-ß proteins was measured by ELISA. We evaluated the potential of anti-IFN-α and anti-IFN-ß PPMOs to reduce the antiviral proteins in influenza virus-infected DF-1 cells and compared the virus titres to untreated controls, nonsense-PPMO and JAK/STAT inhibitors. The effects of complementation and reconstitution of IFN-α and IFN-ß proteins in PPMO-treated-infected cells were evaluated, and the virus titres were compared between treatment groups.Results. Suppression of IFN-α by PPMO resulted in significantly reduced levels of IFN-α protein in treated wells, as measured by ELISA and was shown to not have any cytotoxicity to DF-1 cells at the effective concentrations tested. Treatment of the self-directing PPMOs increased the ability of the influenza virus to replicate in DF-1 cells. Over a 2-log10 increase in viral production was observed in anti-IFN-α and IFN-ß PPMO-treated wells compared to those of untreated controls at the initial viral input of 0.1 multiplicity of infection. The data from complementation and reconstitution of IFN-α and IFN-ß proteins in PPMO-treated-infected cells was about 82 and 97% compared to the combined PPMO-treated but uncomplemented group and untreated group, respectively. There was a 0.5-log10 increase in virus titre when treated with anti-IFN-α and IFN-ß PPMO compared to virus titre when treated with JAK/STAT inhibitors.Conclusions. This study emphasizes the utility of PPMO in allowing cell cultures to produce increased levels of influenza for vaccine production or alternatively, as a screening tool to cheaply test targets prior to the development of permanent knockouts of host gene expression.

The RNA helicase DHX16 recognizes specific viral RNA to trigger RIG-I-dependent innate antiviral immunity.

Type I interferons (IFN-I) are essential to establish antiviral innate immunity. Unanchored (or free) polyubiquitin (poly-Ub) has been shown to regulate IFN-I responses. However, few unanchored poly-Ub interactors are known. To identify factors regulated by unanchored poly-Ub in a physiological setting, we developed an approach to isolate unanchored poly-Ub from lung tissue. We identified the RNA helicase DHX16 as a potential pattern recognition receptor (PRR). Silencing of DHX16 in cells and in vivo diminished IFN-I responses against influenza virus. These effects extended to members of other virus families, including Zika and SARS-CoV-2. DHX16-dependent IFN-I production requires RIG-I and unanchored K48-poly-Ub synthesized by the E3-Ub ligase TRIM6. DHX16 recognizes a signal in influenza RNA segments that undergo splicing and requires its RNA helicase motif for direct, high-affinity interactions with specific viral RNAs. Our study establishes DHX16 as a PRR that partners with RIG-I for optimal activation of antiviral immunity requiring unanchored poly-Ub.

Development of DG9 peptide-conjugated single- and multi-exon skipping therapies for the treatment of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is primarily caused by out-of-frame deletions in the dystrophin gene. Exon skipping using phosphorodiamidate morpholino oligomers (PMOs) converts out-of-frame to in-frame mutations, producing partially functional dystrophin. Four single-exon skipping PMOs are approved for DMD but treat only 8 to 14% of patients each, and some exhibit poor efficacy. Alternatively, exons 45 to 55 skipping could treat 40 to 47% of all patients and is associated with improved clinical outcomes. Here, we report the development of peptide-conjugated PMOs for exons 45 to 55 skipping. Experiments with immortalized patient myotubes revealed that exons 45 to 55 could be skipped by targeting as few as five exons. We also found that conjugating DG9, a cell-penetrating peptide, to PMOs improved single-exon 51 skipping, dystrophin restoration, and muscle function in hDMDdel52;mdx mice. Local administration of a minimized exons 45 to 55-skipping DG9-PMO mixture restored dystrophin production. This study provides proof of concept toward the development of a more economical and effective exons 45 to 55-skipping DMD therapy.

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.

Enhanced delivery of peptide-morpholino oligonucleotides with a small molecule to correct splicing defects in the lung.

Pulmonary diseases offer many targets for oligonucleotide therapeutics. However, effective delivery of oligonucleotides to the lung is challenging. For example, splicing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) affect a significant cohort of Cystic Fibrosis (CF) patients. These individuals could potentially benefit from treatment with splice switching oligonucleotides (SSOs) that can modulate splicing of CFTR and restore its activity. However, previous studies in cell culture used oligonucleotide transfection methods that cannot be safely translated in vivo. In this report, we demonstrate effective correction of a splicing mutation in the lung of a mouse model using SSOs. Moreover, we also demonstrate effective correction of a CFTR splicing mutation in a pre-clinical CF patient-derived cell model. We utilized a highly effective delivery strategy for oligonucleotides by combining peptide-morpholino (PPMO) SSOs with small molecules termed OECs. PPMOs distribute broadly into the lung and other tissues while OECs potentiate the effects of oligonucleotides by releasing them from endosomal entrapment. The combined PPMO plus OEC approach proved to be effective both in CF patient cells and in vivo in the mouse lung and thus may offer a path to the development of novel therapeutics for splicing mutations in CF and other lung diseases.

CX3CR1 Is a Receptor for Human Respiratory Syncytial Virus in Cotton Rats.

Respiratory syncytial virus (RSV) has been reported to use CX3CR1 in vitro as a receptor on cultured primary human airway epithelial cultures. To evaluate CX3CR1 as the receptor for RSV in vivo, we used the cotton rat animal model because of its high permissiveness for RSV infection. Sequencing the cotton rat CX3CR1 gene revealed 91% amino acid similarity to human CX3CR1. Previous work found that RSV binds to CX3CR1 via its attachment glycoprotein (G protein) to infect primary human airway cultures. To determine whether CX3CR1-G protein interaction is necessary for RSV infection, recombinant RSVs containing mutations in the CX3CR1 binding site of the G protein were tested in cotton rats. In contrast to wild-type virus, viral mutants did not grow in the lungs of cotton rats. When RSV was incubated with an antibody blocking the CX3CR1 binding site of G protein and subsequently inoculated intranasally into cotton rats, no virus was found in the lungs 4 days postinfection. In contrast, growth of RSV was not affected after preincubation with heparan sulfate (the receptor for RSV on immortalized cell lines). A reduction in CX3CR1 expression in the cotton rat lung through the use of peptide-conjugated morpholino oligomers led to a 10-fold reduction in RSV titers at day 4 postinfection. In summary, these results indicate that CX3CR1 functions as a receptor for RSV in cotton rats and, in combination with data from human airway epithelial cell cultures, strongly suggest that CX3CR1 is a primary receptor for naturally acquired RSV infection. IMPORTANCE The knowledge about a virus receptor is useful to better understand the uptake of a virus into a cell and potentially develop antivirals directed against either the receptor molecule on the cell or the receptor-binding protein of the virus. Among a number of potential receptor proteins, human CX3CR1 has been demonstrated to act as a receptor for respiratory syncytial virus (RSV) on human epithelial cells in tissue culture. Here, we report that the cotton rat CX3CR1, which is similar to the human molecule, acts as a receptor in vivo. This study strengthens the argument that CX3CR1 is a receptor molecule for RSV.

TMPRSS2 Is the Major Activating Protease of Influenza A Virus in Primary Human Airway Cells and Influenza B Virus in Human Type II Pneumocytes.

Cleavage of influenza virus hemagglutinin (HA) by host cell proteases is essential for virus infectivity and spread. We previously demonstrated in vitro that the transmembrane protease TMPRSS2 cleaves influenza A virus (IAV) and influenza B virus (IBV) HA possessing a monobasic cleavage site. Subsequent studies revealed that TMPRSS2 is crucial for the activation and pathogenesis of H1N1pdm and H7N9 IAV in mice. In contrast, activation of H3N2 IAV and IBV was found to be independent of TMPRSS2 expression and supported by an as-yet-undetermined protease(s). Here, we investigated the role of TMPRSS2 in proteolytic activation of IAV and IBV in three human airway cell culture systems: primary human bronchial epithelial cells (HBEC), primary type II alveolar epithelial cells (AECII), and Calu-3 cells. Knockdown of TMPRSS2 expression was performed using a previously described antisense peptide-conjugated phosphorodiamidate morpholino oligomer, T-ex5, that interferes with splicing of TMPRSS2 pre-mRNA, resulting in the expression of enzymatically inactive TMPRSS2. T-ex5 treatment produced efficient knockdown of active TMPRSS2 in all three airway cell culture models and prevented proteolytic activation and multiplication of H7N9 IAV in Calu-3 cells and H1N1pdm, H7N9, and H3N2 IAV in HBEC and AECII. T-ex5 treatment also inhibited the activation and spread of IBV in AECII but did not affect IBV activation in HBEC and Calu-3 cells. This study identifies TMPRSS2 as the major HA-activating protease of IAV in human airway cells and IBV in type II pneumocytes and as a potential target for the development of novel drugs to treat influenza infections.IMPORTANCE Influenza A viruses (IAV) and influenza B viruses (IBV) cause significant morbidity and mortality during seasonal outbreaks. Cleavage of the viral surface glycoprotein hemagglutinin (HA) by host proteases is a prerequisite for membrane fusion and essential for virus infectivity. Inhibition of relevant proteases provides a promising therapeutic approach that may avoid the development of drug resistance. HA of most influenza viruses is cleaved at a monobasic cleavage site, and a number of proteases have been shown to cleave HA in vitro This study demonstrates that the transmembrane protease TMPRSS2 is the major HA-activating protease of IAV in primary human bronchial cells and of both IAV and IBV in primary human type II pneumocytes. It further reveals that human and murine airway cells can differ in their HA-cleaving protease repertoires. Our data will help drive the development of potent and selective protease inhibitors as novel drugs for influenza treatment.

Anchor peptide captures, targets, and loads exosomes of diverse origins for diagnostics and therapy.

Exosomes are circulating nanovesicular carriers of macromolecules, increasingly used for diagnostics and therapeutics. The ability to load and target patient-derived exosomes without altering exosomal surfaces is key to unlocking their therapeutic potential. We demonstrate that a peptide (CP05) identified by phage display enables targeting, cargo loading, and capture of exosomes from diverse origins, including patient-derived exosomes, through binding to CD63-an exosomal surface protein. Systemic administration of exosomes loaded with CP05-modified, dystrophin splice-correcting phosphorodiamidate morpholino oligomer (EXOPMO) increased dystrophin protein 18-fold in quadriceps of dystrophin-deficient mdx mice compared to CP05-PMO. Loading CP05-muscle-targeting peptide on EXOPMO further increased dystrophin expression in muscle with functional improvement without any detectable toxicity. Our study demonstrates that an exosomal anchor peptide enables direct, effective functionalization and capture of exosomes, thus providing a tool for exosome engineering, probing gene function in vivo, and targeted therapeutic drug delivery.

Systems-based analysis of RIG-I-dependent signalling identifies KHSRP as an inhibitor of RIG-I receptor activation.

Retinoic acid-inducible gene I (RIG-I) receptor recognizes 5'-triphosphorylated RNA and triggers a signalling cascade that results in the induction of type-I interferon (IFN)-dependent responses. Its precise regulation represents a pivotal balance between antiviral defences and autoimmunity. To elucidate the cellular cofactors that regulate RIG-I signalling, we performed two global RNA interference analyses to identify both positive and negative regulatory nodes operating on the signalling pathway during virus infection. These factors were integrated with experimentally and computationally derived interactome data to build a RIG-I protein interaction network. Our analysis revealed diverse cellular processes, including the unfolded protein response, Wnt signalling and RNA metabolism, as critical cellular components governing innate responses to non-self RNA species. Importantly, we identified K-Homology Splicing Regulatory Protein (KHSRP) as a negative regulator of this pathway. We find that KHSRP associates with the regulatory domain of RIG-I to maintain the receptor in an inactive state and attenuate its sensing of viral RNA (vRNA). Consistent with increased RIG-I antiviral signalling in the absence of KHSRP, viral replication is reduced when KHSRP expression is knocked down both in vitro and in vivo. Taken together, these data indicate that KHSRP functions as a checkpoint regulator of the innate immune response to pathogen challenge.

New paradigms for understanding and step changes in treating active and chronic, persistent apicomplexan infections.

Toxoplasma gondii, the most common parasitic infection of human brain and eye, persists across lifetimes, can progressively damage sight, and is currently incurable. New, curative medicines are needed urgently. Herein, we develop novel models to facilitate drug development: EGS strain T. gondii forms cysts in vitro that induce oocysts in cats, the gold standard criterion for cysts. These cysts highly express cytochrome b. Using these models, we envisioned, and then created, novel 4-(1H)-quinolone scaffolds that target the cytochrome bc1 complex Qi site, of which, a substituted 5,6,7,8-tetrahydroquinolin-4-one inhibits active infection (IC50, 30 nM) and cysts (IC50, 4 µM) in vitro, and in vivo (25 mg/kg), and drug resistant Plasmodium falciparum (IC50, <30 nM), with clinically relevant synergy. Mutant yeast and co-crystallographic studies demonstrate binding to the bc1 complex Qi site. Our results have direct impact on improving outcomes for those with toxoplasmosis, malaria, and ~2 billion persons chronically infected with encysted bradyzoites.

Effective dystrophin restoration by a novel muscle-homing peptide-morpholino conjugate in dystrophin-deficient mdx mice.

Antisense oligonucleotide (AO)-mediated splice correction therapy for Duchenne muscular dystrophy has shown huge promise from recent phase 2b clinical trials, however high doses and costs are required and targeted delivery can lower both of these. We have previously demonstrated the feasibility of targeted delivery of AOs by conjugating a chimeric peptide, consisting of a muscle-specific peptide and a cell-penetrating peptide, to AOs in mdx mice. Although increased uptake in muscle was observed, the majority of peptide-AO conjugate was found in the liver. To search for more effective muscle-homing peptides, we carried out in vitro biopanning in myoblasts and identified a novel 12-mer peptide (M12) showing preferential binding to skeletal muscle compared to the liver. When conjugated to phosphorodiamidate morpholino oligomers, ~25% of normal level of dystrophin expression was achieved in body-wide skeletal muscles in mdx mice with significant recovery in grip strength, whereas <2% in corresponding tissues treated with either muscle-specific peptide-phosphorodiamidate morpholino oligomer or unmodified phosphorodiamidate morpholino oligomer under identical conditions. Our data provide evidences for the first time that a muscle-homing peptide alone can enhance AO delivery to muscle without appreciable toxicity at 75 mg/kg, suggesting M12-phosphorodiamidate morpholino oligomer can be an alternative option to current AOs.

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 frameshift and nonsense mutations in Duchenne muscular dystrophy. Phase 2 clinical trials in the United Kingdom and the Netherlands have reported induction of dystrophin expression in muscle of Duchenne muscular dystrophy patients by systemic administration of both phosphorodiamidate morpholino oligomers (PMO) and 2'-O-methyl phosphorothioate. Peptide-conjugated phosphorodiamidate morpholino offers significantly higher efficiency than phosphorodiamidate morpholino, with the ability to induce near-normal levels of dystrophin, and restores function in both skeletal and cardiac muscle. We examined 1-year systemic efficacy of peptide-conjugated phosphorodiamidate morpholino targeting exon 23 in dystrophic mdx mice. The LD(50) of peptide-conjugated phosphorodiamidate morpholino was determined to be approximately 85 mg/kg. The half-life of dystrophin expression was approximately 2 months in skeletal muscle, but shorter in cardiac muscle. Biweekly injection of 6 mg/kg peptide-conjugated phosphorodiamidate morpholino produced >20% dystrophin expression in all skeletal muscles and ≤5% in cardiac muscle, with improvement in muscle function and pathology and reduction in levels of serum creatine kinase. Monthly injections of 30 mg/kg peptide-conjugated phosphorodiamidate morpholino restored dystrophin to >50% normal levels in skeletal muscle, and 15% in cardiac muscle. This was associated with greatly reduced serum creatine kinase levels, near-normal histology, and functional improvement of skeletal muscle. Our results demonstrate for the first time that regular 1-year administration of peptide-conjugated phosphorodiamidate morpholino can be safely applied to achieve significant therapeutic effects in an animal model.

Development of peptide-conjugated morpholino oligomers as pan-arenavirus inhibitors.

Members of the Arenaviridae family are a threat to public health and can cause meningitis and hemorrhagic fever, and yet treatment options remain limited by a lack of effective antivirals. In this study, we found that peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) complementary to viral genomic RNA were effective in reducing arenavirus replication in cell cultures and in vivo. PPMO complementary to the Junín virus genome were designed to interfere with viral RNA synthesis or translation or both. However, only PPMO designed to potentially interfere with translation were effective in reducing virus replication. PPMO complementary to sequences that are highly conserved across the arenaviruses and located at the 5' termini of both genomic segments were effective against Junín virus, Tacaribe virus, Pichinde virus, and lymphocytic choriomeningitis virus (LCMV)-infected cell cultures and suppressed viral titers in the livers of LCMV-infected mice. These results suggest that arenavirus 5' genomic termini represent promising targets for pan-arenavirus antiviral therapeutic development.

Functional rescue of dystrophin-deficient mdx mice by a chimeric peptide-PMO.

Splice modulation using antisense oligonucleotides (AOs) has been shown to yield targeted exon exclusion to restore the open reading frame and generate truncated but partially functional dystrophin protein. This has been successfully demonstrated in dystrophin-deficient mdx mice and in Duchenne muscular dystrophy (DMD) patients. However, DMD is a systemic disease; successful therapeutic exploitation of this approach will therefore depend on effective systemic delivery of AOs to all affected tissues. We have previously shown the potential of a muscle-specific/arginine-rich chimeric peptide-phosphorodiamidate morpholino (PMO) conjugate, but its long-term activity, optimized dosing regimen, capacity for functional correction and safety profile remain to be established. Here, we report the results of this chimeric peptide-PMO conjugate in the mdx mouse using low doses (3 and 6 mg/kg) administered via a 6 biweekly systemic intravenous injection protocol. We show 100% dystrophin-positive fibers and near complete correction of the dystrophin transcript defect in all peripheral muscle groups, with restoration of 50% dystrophin protein over 12 weeks, leading to correction of the DMD pathological phenotype and restoration of muscle function in the absence of detectable toxicity or immune response. Chimeric muscle-specific/cell-penetrating peptides therefore represent highly promising agents for systemic delivery of splice-correcting PMO oligomers for DMD therapy.

Peptide-morpholino conjugate: a promising therapeutic for Duchenne muscular dystrophy.

Steric-blocking oligos can correct reading frame errors or skip premature termination codons. For Duchenne muscular dystrophy (DMD), systemic administration of oligos produces limited delivery into muscle cells. Conjugation to a cell-penetrating peptide greatly enhances muscle uptake of morpholino oligos. A peptide-morpholino conjugate (PPMO) restored dystrophin in mdx mice to > 80% and 50% of normal levels in skeletal and cardiac muscles, respectively, after a single intravenous 30-mg/kg injection. Six injections over 3 months restored dystrophin to nearly normal levels in all muscles. One PPMO injection daily at 12 mg/kg each for 4 days caused exon skipping clearly detectable in the muscles of the mdx mice 9 weeks later, showing prolonged activity. PPMO significantly improved muscle pathology, strength and function, and the survival rate of mice whose hearts were challenged by chemical-induced heart failure. No toxicity or immunogenicity was detected. Our studies demonstrated that muscle functions can be restored with a low dose of PPMO, making it a promising therapeutic for DMD.

Cell-penetrating peptide-conjugated antisense oligonucleotides restore systemic muscle and cardiac dystrophin expression and function.

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.

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.

Effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modified morpholino oligomer.

Antisense oligonucleotide-mediated exon skipping is able to correct out-of-frame mutations in Duchenne muscular dystrophy and restore truncated yet functional dystrophins. However, its application is limited by low potency and inefficiency in systemic delivery, especially failure to restore dystrophin in heart. Here, we conjugate a phosphorodiamidate morpholino oligomer with a designed cell-penetrating peptide (PPMO) targeting a mutated dystrophin exon. Systemic delivery of the novel PPMO restores dystrophin to almost normal levels in the cardiac and skeletal muscles in dystrophic mdx mouse. This leads to increase in muscle strength and prevents cardiac pump failure induced by dobutamine stress in vivo. Muscle pathology and function continue to improve during the 12-week course of biweekly treatment, with significant reduction in levels of serum creatine kinase. The high degree of potency of the oligomer in targeting all muscles and the lack of detectable toxicity and immune response support the feasibility of testing the novel oligomer in treating Duchenne muscular dystrophy patients.

Cell penetrating peptide conjugates of steric block oligonucleotides.

Charge neutral steric block oligonucleotide analogues, such as peptide nucleic acids (PNA) or phosphorodiamidate morpholino oligomers (PMO), have promising biological and pharmacological properties for antisense applications, such as for example in mRNA splicing redirection. However, cellular uptake of free oligomers is poor and the utility of conjugates of PNA or PMO to cell penetrating peptides (CPP), such as Tat or Penetratin, is limited by endosomal sequestration. Two new families of arginine-rich CPPs named (R-Ahx-R)(4) AhxB and R(6)Pen allow efficient nuclear delivery of splice correcting PNA and PMO at micromolar concentrations in the absence of endosomolytic agents. The in vivo efficacy of (R-Ahx-R)(4) AhxB PMO conjugates has been demonstrated in mouse models of Duchenne muscular dystrophy and in various viral infections.

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-alpha amino acids on cellular uptake, splice-correction activity, cellular toxicity and serum binding for 24 CPP-PMOs. Insertion of 6-aminohexanoic acid (X) or beta-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 microM. Up to 60 microM, only the conjugates with > or = 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.