<i>In vitro</i> evaluation of novel antisense oligonucleotides is predictive of <i>in vivo</i> exon skipping activity for Duchenne muscular dystrophy

Wang, Qingsong; Yin, HaiFang; Camelliti, Patrizia; Betts, Corinne; Moulton, Hong M.; Lee, Hyun Il; Saleh, Amer F.; Gait, Michael J.; Wood, Matthew

doi:10.1002/jgm.1446

Cited by 17 publications

(11 citation statements)

References 43 publications

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“…Thus, in vitro efficacy does not reflect in vivo efficacy. This notion is supported by previous studies, in which 2′Ome PS showed the greatest exon skipping activity in vitro compared to PMO and PNA but the least effective one in vivo locally and systemically [24], [27], [38]. In the current study, we reaffirmed findings from previous studies in mdx mice that demonstrated better in vivo exon skipping efficacy of PMO and PNA compared to negatively charged RNA analog -2′Ome PS, which can be attributed to their higher serum stability and sequence-specificity [27], [31].…”

Section: Discussionsupporting

confidence: 79%

“…In addition, although in vitro models substantially facilitated the optimization of AO sequences for DMD, in vitro models are not completely predictive of in vivo efficacy for some chemistry e.g. PMO (phosphorodiamidate morpholino oligomer) and PNA (peptide nucleic acid), which are notoriously difficult to transfect in vitro , but showing high activity in vivo [24]–[28]. Thus, a readily available and more cost-effective alternative in vivo model which allows researchers to identify uptake in different genetic background before optimization will further speed up the development of DMD AOs.…”

Section: Introductionmentioning

confidence: 99%

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

Cao

Hou

et al. 2014

PLoS ONE

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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.

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Section: Discussionsupporting

confidence: 79%

Section: Introductionmentioning

confidence: 99%

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

Cao

Hou

et al. 2014

PLoS ONE

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“…To date, specific AO sequences have been assessed for their efficiency of exon skipping using cell-based experimental systems, with the optimal sequences then used for in vivo experiments. 11,41,42 However, the in vivo efficacy of phosphorodiamidate morpholino oligomers (PMOs), which are rather difficult to deliver into mammalian cells in culture because of their neutral chemistry, 11,19 could differ from that in vitro. 14 Therefore, we used the mdx52 mouse to screen AO sequences for exon 51-skipping by intramuscular injection in vivo.…”

Section: Discussionmentioning

confidence: 99%

In-frame Dystrophin Following Exon 51-Skipping Improves Muscle Pathology and Function in the Exon 52–Deficient mdx Mouse

et al. 2010

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A promising therapeutic approach for Duchenne muscular dystrophy (DMD) is exon skipping using antisense oligonucleotides (AOs). In-frame deletions of the hinge 3 region of the dystrophin protein, which is encoded by exons 50 and 51, are predicted to cause a variety of phenotypes. Here, we performed functional analyses of muscle in the exon 52-deleted mdx (mdx52) mouse, to predict the function of in-frame dystrophin following exon 51-skipping, which leads to a protein lacking most of hinge 3. A series of AOs based on phosphorodiamidate morpholino oligomers was screened by intramuscular injection into mdx52 mice. The highest splicing efficiency was generated by a two-oligonucleotide cocktail targeting both the 5' and 3' splice sites of exon 51. After a dose-escalation study, we systemically delivered this cocktail into mdx52 mice seven times at weekly intervals. This induced 20-30% of wild-type (WT) dystrophin expression levels in all muscles, and was accompanied by amelioration of the dystrophic pathology and improvement of skeletal muscle function. Because the structure of the restored in-frame dystrophin resembles human dystrophin following exon 51-skipping, our results are encouraging for the ongoing clinical trials for DMD. Moreover, the therapeutic dose required can provide a suggestion of the theoretical equivalent dose for humans.

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“…Antisense oligonucleotide (AO)-mediated exon skipping can restore the open reading frame of mutant DMD pre-mRNA transcripts [3-15] and produce truncated but partially functional dystrophin protein [16-17]. The therapeutic potential of this method has been shown in human subjects following local intramuscular AO injection in two independent clinical trials [10,18].…”

Section: Introductionmentioning

confidence: 99%

Pip5 Transduction Peptides Direct High Efficiency Oligonucleotide-mediated Dystrophin Exon Skipping in Heart and Phenotypic Correction in mdx Mice

et al. 2011

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Induced splice modulation of pre-mRNAs shows promise to correct aberrant disease transcripts and restore functional protein and thus has therapeutic potential. Duchenne muscular dystrophy (DMD) results from mutations that disrupt the DMD gene open reading frame causing an absence of dystrophin protein. Antisense oligonucleotide (AO)-mediated exon skipping has been shown to restore functional dystrophin in mdx mice and DMD patients treated intramuscularly in two recent Phase I clinical trials. Critical to the therapeutic success of AO-based treatment will be the ability to deliver AOs systemically to all affected tissues including the heart. Here we report identification of a series of transduction peptides (Pip5) as AO conjugates for enhanced systemic and particularly cardiac delivery. One of the lead peptide-AO conjugates, Pip5e-AO, showed highly efficient exon skipping and dystrophin production in mdx mice with complete correction of the aberrant DMD transcript in heart, leading to greater than 50% of the normal level of dystrophin in heart. Mechanistic studies indicated that the enhanced activity of Pip5e-PMO is partly explained by more efficient nuclear delivery. Pip5 series derivatives therefore have significant potential for advancing the development of exon skipping therapies for DMD and may have application for enhanced cardiac delivery of other biotherapeutics.

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In vitro evaluation of novel antisense oligonucleotides is predictive of in vivo exon skipping activity for Duchenne muscular dystrophy

Cited by 17 publications

References 43 publications

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

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

In-frame Dystrophin Following Exon 51-Skipping Improves Muscle Pathology and Function in the Exon 52–Deficient mdx Mouse

Pip5 Transduction Peptides Direct High Efficiency Oligonucleotide-mediated Dystrophin Exon Skipping in Heart and Phenotypic Correction in mdx Mice

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