Antisense PMO Found in Dystrophic Dog Model Was Effective in Cells from Exon 7-Deleted DMD Patient

Background

Antisense oligonucleotide-induced exon skipping is a promising approach for treatment of Duchenne muscular dystrophy (DMD). We have systemically administered an antisense phosphorodiamidate morpholino oligomer (PMO) targeting dystrophin exons 6 and 8 to a dog with canine X-linked muscular dystrophy in Japan (CXMD_J) lacking exon 7 and achieved recovery of dystrophin in skeletal muscle. To date, however, antisense chemical compounds used in DMD animal models have not been directly applied to a DMD patient having the same type of exon deletion. We recently identified a DMD patient with an exon 7 deletion and tried direct translation of the antisense PMO used in dog models to the DMD patient''s cells.

Methodology/Principal Findings

We converted fibroblasts of CXMD_J and the DMD patient to myotubes by FACS-aided MyoD transduction. Antisense PMOs targeting identical regions of dog and human dystrophin exons 6 and 8 were designed. These antisense PMOs were mixed and administered as a cocktail to either dog or human cells in vitro. In the CXMD_J and human DMD cells, we observed a similar efficacy of skipping of exons 6 and 8 and a similar extent of dystrophin protein recovery. The accompanying skipping of exon 9, which did not alter the reading frame, was different between cells of these two species.

Conclusion/Significance

Antisense PMOs, the effectiveness of which has been demonstrated in a dog model, achieved multi-exon skipping of dystrophin gene on the FACS-aided MyoD-transduced fibroblasts from an exon 7-deleted DMD patient, suggesting the feasibility of systemic multi-exon skipping in humans.     

Identification of Small Molecule and Genetic Modulators of AON-Induced Dystrophin Exon Skipping by High-Throughput Screening

One therapeutic approach to Duchenne Muscular Dystrophy (DMD) recently entering clinical trials aims to convert DMD phenotypes to that of a milder disease variant, Becker Muscular Dystrophy (BMD), by employing antisense oligonucleotides (AONs) targeting splice sites, to induce exon skipping and restore partial dystrophin function. In order to search for small molecule and genetic modulators of AON-dependent and independent exon skipping, we screened ∼10,000 known small molecule drugs, >17,000 cDNA clones, and >2,000 kinase- targeted siRNAs against a 5.6 kb luciferase minigene construct, encompassing exon 71 to exon 73 of human dystrophin. As a result, we identified several enhancers of exon skipping, acting on both the reporter construct as well as endogenous dystrophin in mdx cells. Multiple mechanisms of action were identified, including histone deacetylase inhibition, tubulin modulation and pre-mRNA processing. Among others, the nucleolar protein NOL8 and staufen RNA binding protein homolog 2 (Stau2) were found to induce endogenous exon skipping in mdx cells in an AON-dependent fashion. An unexpected but recurrent theme observed in our screening efforts was the apparent link between the inhibition of cell cycle progression and the induction of exon skipping.     

Determination of optimal sites of antisense oligonucleotide cleavage within TNFalpha mRNA

Antisense oligonucleotides provide a powerful tool in order to determine the consequences of the reduced expression of a selected target gene and may include target validation and therapeutic applications. Methods of predicting optimum antisense sites are not always effective. We have compared the efficacy of antisense oligonucleotides, which were selected in vitro using random combinatorial oligonucleotide libraries of differing length and complexity, upon putative target sites within TNFα mRNA. The relationship of specific target site accessibility and oligonucleotide efficacy with respect to these parameters proved to be complex. Modification of the length of the recognition sequence of the oligonucleotide library illustrated that independent target sites demonstrated a preference for antisense oligonucleotides of a defined and independent optimal length. The efficacy of antisense oligonucleotide sequences selected in vitro paralleled that observed in phorbol 12-myristate 13-acetate (PMA)-activated U937 cells. The application of methylphosphonate:phosphodiester chimaeric oligonucleotides to U937 cells reduced mRNA levels to up to 19.8% that of the untreated cell population. This approach provides a predictive means to profile any mRNA of known sequence with respect to the identification and optimisation of sites accessible to antisense oligonucleotide activity.     

Effective Exon Skipping and Dystrophin Restoration by 2′-O-Methoxyethyl Antisense Oligonucleotide in Dystrophin-Deficient Mice

Antisense oligonucleotide (AO)–mediated exon-skipping therapy is one of the most promising therapeutic strategies for Duchenne Muscular Dystrophy (DMD) and several AO chemistries have been rigorously investigated. In this report, we focused on the effect of 2′-O-methoxyethyl oligonucleotides (MOE) on exon skipping in cultured mdx myoblasts and mice. Efficient dose-dependent skipping of targeted exon 23 was achieved in myoblasts with MOE AOs of different lengths and backbone chemistries. Furthermore, we established that 25-mer MOE phosphorothioate (PS) AOs provided the greatest exon-skipping efficacy. When compared with 2′O methyl phosphorothioate (2′OmePS) AOs, 25-mer MOE (PS) AOs also showed higher exon-skipping activity in vitro and in mdx mice after intramuscular injections. Characterization of uptake in vitro corroborated with exon-skipping results, suggesting that increased uptake of 25-mer MOE PS AOs might partly contribute to the difference in exon-skipping activity observed in vitro and in mdx mice. Our findings demonstrate the substantial potential for MOE PS AOs as an alternative option for the treatment of DMD.     

Wild-Type Mouse Models to Screen Antisense Oligonucleotides for Exon-Skipping Efficacy in Duchenne Muscular Dystrophy

A readily available animal model is essential for rapidly identifying effective treatments for Duchenne muscular dystrophy (DMD), a devastating neuromuscular disorder caused by the lack of dystrophin protein, which results from frame-disrupting mutations in the DMD gene. Currently, the mdx mouse is the most commonly used model for antisense oligonucleotide (AO)-mediated exon skipping pre-clinical studies, with a mild phenotype. However, the accessibility of mdx mouse colonies particularly in developing countries can constrain research. Therefore in this study we explore the feasibility of using wild-type mice as models to establish exon-skipping efficiency of various DMD AO chemistries and their conjugates. Four different strains of wild-type mice and six different AO chemistries were investigated intramuscularly and the results indicated that the same exon-skipping efficiency was achieved for all tested AOs as that from mdx mice. Notably, levels of exon-skipping obtained in C57BL6 and C3H and mdx mice were most closely matched, followed by ICR and BALB/C mice. Systemic validation revealed that wild-type mice are less responsive to AO-mediated exon skipping than mdx mice. Our study provides evidence for the first time that wild-type mice can be appropriate models for assessing DMD AO exon-skipping efficiency with similar sensitivity to that of mdx mice and this finding can further accelerate the development of effective DMD AOs.     

In vivo comparison of 2′‐O‐methyl phosphorothioate and morpholino antisense oligonucleotides for Duchenne muscular dystrophy exon skipping

Background

Antisense‐mediated exon skipping is a putative treatment for Duchenne muscular dystrophy (DMD). Using antisense oligonucleotides (AONs), the disrupted DMD reading frame is restored, allowing generation of partially functional dystrophin and conversion of a severe Duchenne into a milder Becker muscular dystrophy phenotype. In vivo studies are mainly performed using 2′‐O‐methyl phosphorothioate (2OMePS) or morpholino (PMO) AONs. These compounds were never directly compared.

Methods

mdx and humanized (h)DMD mice were injected intramuscularly and intravenously with short versus long 2OMePS and PMO for mouse exon 23 and human exons 44, 45, 46 and 51.

Results

Intramuscular injection showed that increasing the length of 2OMePS AONs enhanced skipping efficiencies of human exon 45, but decreased efficiency for mouse exon 23. Although PMO induced more mouse exon 23 skipping, PMO and 2OMePS were more comparable for human exons. After intravenous administration, exon skipping and novel protein was shown in the heart with both chemistries. Furthermore, PMO showed lower intramuscular concentrations with higher exon 23 skipping levels compared to 2OMePS, which may be due to sequestration in the extracellular matrix. Finally, two mismatches rendered 2OMePS but not PMO AONs nearly ineffective.

Conclusions

The results obtained in the present study indicate that increasing AON length improves skipping efficiency in some but not all cases. It is feasible to induce exon skipping and dystrophin restoration in the heart after injection of 2OMePS and unconjugated PMO. Furthermore, differences in efficiency between PMO and 2OMePS appear to be sequence and not chemistry dependent. Finally, the results indicate that PMOs may be less sequence specific than 2OMePS. Copyright © 2009 John Wiley & Sons, Ltd.     

Extensive and prolonged restoration of dystrophin expression with vivo-morpholino-mediated multiple exon skipping in dystrophic dogs

Duchenne muscular dystrophy (DMD) is a severe and the most prevalent form of muscular dystrophy, characterized by rapid progression of muscle degeneration. Antisense-mediated exon skipping is currently one of the most promising therapeutic options for DMD. However, unmodified antisense oligos such as morpholinos require frequent (weekly or bi-weekly) injections. Recently, new generation morpholinos such as vivo-morpholinos are reported to lead to extensive and prolonged dystrophin expression in the dystrophic mdx mouse, an animal model of DMD. The vivo-morpholino contains a cell-penetrating moiety, octa-guanidine dendrimer. Here, we sought to test the efficacy of multiple exon skipping of exons 6-8 with vivo-morpholinos in the canine X-linked muscular dystrophy, which harbors a splice site mutation at the boundary of intron 6 and exon 7. We designed and optimized novel antisense cocktail sequences and combinations for exon 8 skipping and demonstrated effective exon skipping in dystrophic dogs in vivo. Intramuscular injections with newly designed cocktail oligos led to high levels of dystrophin expression, with some samples similar to wild-type levels. This is the first report of successful rescue of dystrophin expression with morpholino conjugates in dystrophic dogs. Our results show the potential of phosphorodiamidate morpholino oligomer conjugates as therapeutic agents for DMD.     

Renadirsen,a Novel 2′OMeRNA/ENA® Chimera Antisense Oligonucleotide,Induces Robust Exon 45 Skipping for Dystrophin In Vivo

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease caused by out-of-frame or nonsense mutation in the dystrophin gene. It begins with a loss of ambulation between 9 and 14 years of age, followed by various other symptoms including cardiac dysfunction. Exon skipping of patients’ DMD pre-mRNA induced by antisense oligonucleotides (AOs) is expected to produce shorter but partly functional dystrophin proteins, such as those possessed by patients with the less severe Becker muscular dystrophy. We are working on developing modified nucleotides, such as 2′-O,4′-C-ethylene-bridged nucleic acids (ENAs), possessing high nuclease resistance and high affinity for complementary RNA strands. Here, we demonstrate the preclinical characteristics (exon-skipping activity in vivo, stability in blood, pharmacokinetics, and tissue distribution) of renadirsen, a novel AO modified with 2′-O-methyl RNA/ENA chimera phosphorothioate designed for dystrophin exon 45 skipping and currently under clinical trials. Notably, systemic delivery of renadirsen sodium promoted dystrophin exon skipping in cardiac muscle, skeletal muscle, and diaphragm, compared with AOs with the same sequence as renadirsen but conventionally modified by PMO and 2′OMePS. These findings suggest the promise of renadirsen sodium as a therapeutic agent that improves not only skeletal muscle symptoms but also other symptoms in DMD patients, such as cardiac dysfunction.     

Antisense-mediated exon skipping: a versatile tool with therapeutic and research applications

Antisense-mediated modulation of splicing is one of the few fields where antisense oligonucleotides (AONs) have been able to live up to their expectations. In this approach, AONs are implemented to restore cryptic splicing, to change levels of alternatively spliced genes, or, in case of Duchenne muscular dystrophy (DMD), to skip an exon in order to restore a disrupted reading frame. The latter allows the generation of internally deleted, but largely functional, dystrophin proteins and would convert a severe DMD into a milder Becker muscular dystrophy phenotype. In fact, exon skipping is currently one of the most promising therapeutic tools for DMD, and a successful first-in-man trial has recently been completed. In this review the applicability of exon skipping for DMD and other diseases is described. For DMD AONs have been designed for numerous exons, which has given us insight into their mode of action, splicing in general, and splicing of the DMD gene in particular. In addition, retrospective analysis resulted in guidelines for AON design for DMD and most likely other genes as well. This knowledge allows us to optimize therapeutic exon skipping, but also opens up a range of other applications for the exon skipping approach.     

Carbohydrate Modifications in Antisense Oligonucleotide Therapy: New Kids on the Block

Abstract

Chemical modifications to improve the efficacy of an antisense oligonucleotide are designed to increase the binding affinity to target RNA, to enhance the nuclease resistance, and to improve cellular delivery. Among the different sites available for chemical modification in a nucleoside building block, the 2′-position of the carbohydrate moiety¹ has proven to be the most valuable for various reasons: (1) 2′-modification can confer an RNA-like 3′-endo conformation to the antisense oligonucleotide. Such a preorganization for an RNA like conformation^2,3,4,5 greatly improves the binding affinity to the target RNA; (2) 2′-modification provides nuclease resistance to oligonucleotides; (3) 2′-modification provides chemical stability against potential depurination conditions pharmacology evaluations and correlation with pharmacokinetic changes are emerging from these novel chemical modifications. Analytical chemistry of modified oligonucleotides before and after biological administration of antisense oligonucleotides with techniques such as capillary gel electrophoresis (CGE) and mass spectrometry help to determine the purity as well as the in vivo fate of these complex molecules. Large-scale synthesis is becoming a tangible reality for antisense oligonucleotides. Nucleic acid chemists and biologists alike are beginning to understand the structure-biological activity in terms of basic physical-organic parameters such as the gauche effect, the charge effect and conformational constraints. Synthesis of chimeric designer oligonucleotides bringing the attractive features of different modifications to a given antisense oligonucleotide sequence to generate synergistic interactions is forthcoming³⁰. These advances along with the potential availability of complete human genome sequence information promise a bright future for the widespread use of nucleic acid based therapeutics.     

Effects of RNA secondary structure on cellular antisense activity

The secondary and tertiary structures of a mRNA are known to effect hybridization efficiency and potency of antisense oligonucleotides in vitro. Additional factors including oligonucleotide stability and cellular uptake are also thought to contribute to antisense potency in vivo. Each of these factors can be affected by the sequence of the oligonucleotide. Although mRNA structure is presumed to be a critical determinant of antisense activity in cells, to date little direct experimental evidence has addressed the significance of structure. In order to determine the importance of mRNA structure on antisense activity, oligonucleotide target sites were cloned into a luciferase reporter gene along with adjoining sequence to form known structures. This allowed us to study the effect of target secondary structure on oligonucleotide binding in the cellular environment without changing the sequence of the oligonucleotide. Our results show that structure does play a significant role in determining oligonucleotide efficacy in vivo. We also show that potency of oligonucleotides can be improved by altering chemistry to increase affinity for the mRNA target even in a region that is highly structured.     

Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates

The potency and specificity of locked nucleic acid (LNA) antisense oligonucleotides was investigated as a function of length and affinity. The oligonucleotides were designed to target apolipoprotein B (apoB) and were investigated both in vitro and in vivo. The high affinity of LNA enabled the design of short antisense oligonucleotides (12- to 13-mers) that possessed high affinity and increased potency both in vitro and in vivo compared to longer oligonucleotides. The short LNA oligonucleotides were more target specific, and they exhibited the same biodistribution and tissue half-life as longer oligonucleotides. Pharmacology studies in both mice and non-human primates were conducted with a 13-mer LNA oligonucleotide against apoB, and the data showed that repeated dosing of the 13-mer at 1–2 mg/kg/week was sufficient to provide a significant and long lasting lowering of non-high-density lipoprotein (non-HDL) cholesterol without increasing serum liver toxicity markers. The data presented here show that oligonucleotide length as a parameter needs to be considered in the design of antisense oligonucleotide and that potent short oligonucleotides with sufficient target affinity can be generated using the LNA chemistry. Conclusively, we present a 13-mer LNA oligonucleotide with therapeutic potential that produce beneficial cholesterol lowering effect in non-human primates.