Aggregation and Disaggregation of Morpholino Oligomers in Solution.

Morpholino oligomers are effective antisense molecules to regulate gene expression and the US FDA has approved a Morpholino drug for the treatment of Duchenne muscular dystrophy. However, it has been observed that the antisense activities of aqueous solutions of some Morpholinos decrease over time. We hypothesize that the decreased activity is caused by the formation of soluble aggregates of the Morpholinos. Here, we analyzed three Morpholino sequences by size exclusion chromatography and found two of them have over time formed soluble aggregates in water. The degree of aggregation is sequence-, temperature-, and time-dependent. We describe a simple procedure for detecting and breaking down the aggregates to return the Morpholinos to their monomeric forms.

Crowding induces live cell extrusion to maintain homeostatic cell numbers in epithelia.

For an epithelium to provide a protective barrier, it must maintain homeostatic cell numbers by matching the number of dividing cells with the number of dying cells. Although compensatory cell division can be triggered by dying cells, it is unknown how cell death might relieve overcrowding due to proliferation. When we trigger apoptosis in epithelia, dying cells are extruded to preserve a functional barrier. Extrusion occurs by cells destined to die signalling to surrounding epithelial cells to contract an actomyosin ring that squeezes the dying cell out. However, it is not clear what drives cell death during normal homeostasis. Here we show in human, canine and zebrafish cells that overcrowding due to proliferation and migration induces extrusion of live cells to control epithelial cell numbers. Extrusion of live cells occurs at sites where the highest crowding occurs in vivo and can be induced by experimentally overcrowding monolayers in vitro. Like apoptotic cell extrusion, live cell extrusion resulting from overcrowding also requires sphingosine 1-phosphate signalling and Rho-kinase-dependent myosin contraction, but is distinguished by signalling through stretch-activated channels. Moreover, disruption of a stretch-activated channel, Piezo1, in zebrafish prevents extrusion and leads to the formation of epithelial cell masses. Our findings reveal that during homeostatic turnover, growth and division of epithelial cells on a confined substratum cause overcrowding that leads to their extrusion and consequent death owing to the loss of survival factors. These results suggest that live cell extrusion could be a tumour-suppressive mechanism that prevents the accumulation of excess epithelial cells.

An octaguanidine-morpholino oligo conjugate improves muscle function of mdx mice.

INTRODUCTION: Skeletal muscles of mdx mice lack functional levels of dystrophin due to a mutation in Dmd exon 23. Morpholino antisense oligomers can induce expression of a truncated dystrophin by redirecting splicing to skip processing of exon 23. METHODS: We tested whether systemic administration of Vivo-Morpholino, an octaguanidine delivery moiety-Morpholino conjugate that targets exon 23 (VMO23), restored function to muscles of mdx mice. RESULTS: Extensor digitorum longus (EDL) muscles of mdx mice were weaker, less powerful, and showed greater functional deficits after eccentric contractions than normal. VMO23 treatment normalized EDL force and power of mdx mice and eliminated their exaggerated sensitivity to eccentric contractions. Diaphragm muscle strips from mdx mice also produced lower-than-normal force and power, and these variables were restored to normal, or near-normal, levels by VMO23 treatment. CONCLUSION: These results provide a functional basis for continuing development of VMO23 as a treatment for Duchenne muscular dystrophy.

Octa-guanidine morpholino restores dystrophin expression in cardiac and skeletal muscles and ameliorates pathology in dystrophic mdx mice.

Steric-block antisense oligonucleotides (AONs) are able to target RNAs for destruction and splicing alteration. Reading frame restoration of the dystrophin transcript can be achieved by AON-mediated exon skipping in the dystrophic mdx mouse model. However, simple, unmodified AONs exhibit inefficient delivery systemically, leading to dystrophin induction with high variability in skeletal muscles and barely detectable in cardiac muscle. Here, we examined a Morpholino oligomer conjugated with a dendrimeric octaguanidine (Vivo-Morpholino) and demonstrated that the delivery moiety significantly improved dystrophin production in both skeletal and cardiac muscles in mdx mice in vivo. Single intravenous (IV) injections of 6 mg/kg Vivo-MorpholinoE23 (Vivo-ME23) generated dystrophin expression in skeletal muscles at the levels higher than the injection of 300 mg/kg unmodified ME23. Repeated injections at biweekly intervals achieved near 100% of fibers expressing dystrophin in skeletal muscles bodywide without eliciting a detectable immune response. Dystrophin protein was restored to approximately 50 and 10% of normal levels in skeletal and cardiac muscles, respectively. Vivo-Morpholinos showed no signs of toxicity with the effective dosages and regime, thus offering realistic prospects for the treatment of a majority of Duchenne muscular dystrophy (DMD) patients and many other diseases by targeting RNAs.

Design and synthesis of dendritic molecular transporter that achieves efficient in vivo delivery of morpholino antisense oligo.

Safe and efficient in vivo delivery of Morpholino antisense oligos was probably the last and most difficult challenge for the broad application of antisense in animal research and therapeutics. Several arginine-rich peptides effective for in vivo delivery of Morpholino antisense oligos require rather complex and expensive procedures for synthesis and conjugation. This work describes the design and synthesis of a dendritic transporter in a most concise manner where the selection of the core scaffold, functional group multiplication, orthogonal protecting group manipulation, solid phase conjugation, and off-resin perguanidinylation of the transporter structure are all orchestrated for efficient assembly. We utilized triazine as a core to provide a site for on-column conjugation to the Morpholino oligo and to anchor functional side arms which, after extension, multiplication, and deprotection, are subsequently converted from primary amines to the eight guanidinium headgroups that serve for transport across cell membranes. Intravenous administration of the delivery-enabled Morpholino into a splice-reporter strain of transgenic living mice results in de novo expression of splice-corrected green fluorescent protein in a broad range of tissues and organs in those treated mice. This rigorously demonstrates that this new dendritic transporter achieves effective delivery of a Morpholino oligo into the cytosol/nuclear compartment of cells systemically in vivo. The practical conjugation process may overcome any availability limitation for routine use by the scientific community, and the efficient delivery ability of this transporter may advance the application of Morpholino antisense technology in animals.

In vivo correction of a Menkes disease model using antisense oligonucleotides.

Although the molecular basis of many inherited metabolic diseases has been defined, the availability of effective therapies in such disorders remains problematic. Menkes disease is a fatal neurodegenerative disorder due to loss-of-function mutations in the ATP7A gene encoding a copper-transporting P-type Atpase. To develop therapeutic approaches in affected patients, we have identified a zebrafish model of Menkes disease termed calamity that results from splicing defects in the zebrafish orthologue of the ATP7A gene. Embryonic-recessive lethal mutants have impaired copper homeostasis that results in absent melanin pigmentation, impaired notochord formation, and hindbrain neurodegeneration. In this current study, we have attempted to rescue these striking phenotypic alterations by using a series of antisense morpholino oligonucleotides directed against the splice-site junctions of two mutant calamity alleles. Our findings reveal a robust and complete correction of the copper-deficient defects of calamity in association with the generation of the WT Menkes protein in all rescued mutants. Interestingly, a quantitative analysis of atp7a-specific transcripts suggests that competitive translational regulation may account for the synthesis of WT protein in these embryos. This in vivo correction of Menkes disease through the rescue of aberrant splicing may provide therapeutic options in this fatal disease and illustrates the potential for zebrafish models of human genetic disease in the development of treatments based on the principles of interactions of synthetic oligonucleotide analogues with mRNA.

Vivo-Morpholinos: a non-peptide transporter delivers Morpholinos into a wide array of mouse tissues.

We have developed a new transporter structure that provides effective delivery of Morpholino antisense oligomers into a wide variety of tissues in living mice. This transporter comprises a dendritic structure assembled around a triazine core which serves to position eight guanidinium head groups in a conformation effective to penetrate cell membranes. This transporter structure is conjugated to a Morpholino oligomer to form a delivery-enabled product referred to as a Vivo-Morpholino. Vivo-Morpholinos are shown to effectively enter and function within cultured cells in the presence of 100% serum using a rigorous positive test system based on correction of a defined splicing error in a pre-messenger RNA. In addition, Vivo-Morpholinos are demonstrated to enter into a wide variety of tissues in a similar positive test system in transgenic mice, as evidenced by correction of the targeted splicing error in all tissues assessed, including near-complete splice correction in the small intestine, colon, stomach, liver kidney, and a number of muscles. Finally, Vivo-Morpholinos, which target the exon-skipping of exon 23 harboring a premature termination codon in the mdx mouse model, effectively restore the reading frame of dystrophin and restore expression of a functional dystrophin protein.

Achieving targeted and quantifiable alteration of mRNA splicing with Morpholino oligos.

This work represents the first guide for using steric-block antisense oligos as tools for effective and targeted modification of RNA splicing. Comparison of several steric-block oligo types shows the properties of Morpholinos provide significant advantages over other potential splice-blocking oligos. The procedures and complications of designing effective splice-blocking Morpholino oligos are described. The design process requires complete pre-mRNA sequence for defining suitable targets, which usually generate specific predictable messengers. To validate the targeting procedure, the level and nature of transcript alteration is characterized by RT-PCR analysis of splice modification in a beta-globin splice model system. An oligo-walking study reveals that while U1 and U2 small nuclear RiboNucleoProtein (snRNP) binding sites are the most effective targets for blocking splicing, inclusion of these sites is not required to achieve effective splice modifications. The most effective targeting strategy employs simultaneously blocking snRNP binding sites and splice-junctions. The work presented here continues to be the basis for most of the successful Morpholino oligos designed for the worldwide research community to block RNA splicing.

Fgf3 and Fgf8 dependent and independent transcription factors are required for otic placode specification.

The vertebrate inner ear develops from the otic placode, an ectodermal thickening that forms adjacent to the presumptive hindbrain. Previous studies have suggested that competent ectodermal cells respond to signals from adjacent tissues to form the placode. Members of the Fgf family of growth factors and the Dlx family of transcription factors have been implicated in this signal-response pathway. We show that compromising Fgf3 and Fgf8 signaling blocks ear development; only a few scattered otic cells form. Removal of dlx3b, dlx4b and sox9a genes together also blocks ear development, although a few residual cells form an otic epithelium. These cells fail to form if sox9b function is also blocked. Combined loss of Fgf signaling and the three transcription factor genes, dlx3b, dlx4b and sox9a, also completely eliminates all indications of otic cells. Expression of sox9a but not dlx3b, dlx4b or sox9b requires Fgf3 and Fgf8. Our results provide evidence for Fgf3- and Fgf8-dependent and -independent genetic pathways for otic specification and support the notion that Fgf3 and Fgf8 function to induce both the otic placode and the epithelial organization of the otic vesicle.

Pho85 phosphorylates the Glc7 protein phosphatase regulator Glc8 in vivo.

The budding yeast Glc7 serine/threonine protein phosphatase-1 is regulated by Glc8, the yeast ortholog of mammalian phosphatase inhibitor-2. In this work, we demonstrated that similarly to inhibitor-2, Glc8 function is regulated by phosphorylation. The cyclin-dependent protein kinase, Pho85, in conjunction with the related cyclins Pcl6 and Pcl7 comprise the major Glc8 kinase in vivo and in vitro. Several glc7 mutations are dependent on the presence of Glc8 for viability. For example, glc7 alleles R121K, R142H, and R198D are lethal in combination with a glc8 deletion. We found that glc7-R121K is lethal in combination with a pho85 deletion. This finding indicates that Pho85 is the sole Glc8 kinase in vivo. Furthermore, glc7-R121K is also lethal when combined with deletions of pcl6, plc7, pcl8, and pcl10, indicating that these related cyclins redundantly activate Pho85 for Glc8 phosphorylation in vivo. In vitro kinase assays and genetic results indicate that Pho85 cyclins Pcl6 and Pcl7 comprise the predominant Glc8 kinase.

A zebrafish sox9 gene required for cartilage morphogenesis.

The molecular genetic mechanisms of cartilage construction are incompletely understood. Zebrafish embryos homozygous for jellyfish (jef) mutations show craniofacial defects and lack cartilage elements of the neurocranium, pharyngeal arches, and pectoral girdle similar to humans with campomelic dysplasia. We show that two alleles of jef contain mutations in sox9a, one of two zebrafish orthologs of the human transcription factor SOX9. A mutation induced by ethyl nitrosourea changed a conserved nucleotide at a splice junction and severely reduced splicing of sox9a transcript. A retrovirus insertion into sox9a disrupted its DNA-binding domain. Inhibiting splicing of the sox9a transcript in wild-type embryos with splice site-directed morpholino antisense oligonucleotides produced a phenotype like jef mutant larvae, and caused sox9a transcript to accumulate in the nucleus; this accumulation can serve as an assay for the efficacy of a morpholino independent of phenotype. RNase-protection assays showed that in morpholino-injected animals, the percent of splicing inhibition decreased from 80% at 28 hours post fertilization to 45% by 4 days. Homozygous mutant embryos had greatly reduced quantities of col2a1 message, the major collagen of cartilage. Analysis of dlx2 expression showed that neural crest specification and migration was normal in jef (sox9a) embryos. Confocal images of living embryos stained with BODIPY-ceramide revealed at single-cell resolution the formation of precartilage condensations in mutant embryos. Besides the lack of overt cartilage differentiation, pharyngeal arch condensations in jef (sox9a) mutants lacked three specific morphogenetic behaviors: the stacking of chondrocytes into orderly arrays, the individuation of pharyngeal cartilage organs and the proper shaping of individual cartilages. Despite the severe reduction of cartilages, analysis of titin expression showed normal muscle patterning in jef (sox9a) mutants. Likewise, calcein labeling revealed that early bone formation was largely unaffected in jef (sox9a) mutants. These studies show that jef (sox9a) is essential for both morphogenesis of condensations and overt cartilage differentiation.