Pharmacokinetics and biodistribution of phosphorodiamidate morpholino antisense oligomers

Amantana, Adams; Iversen, Patrick L.

doi:10.1016/j.coph.2005.07.001

Cited by 140 publications

(94 citation statements)

References 43 publications

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“…In the current study we chose AMOs to correct aberrant diseasecausing ATM splicing because of their stability, their high efficiency for binding target RNA, and their long-term activity (at least 84 h in our experiments) (39)(40)(41). We have demonstrated splicing correction for three distinct prototypes of splicing mutations.…”

Section: Discussionmentioning

confidence: 99%

Correction of prototypic ATM splicing mutations and aberrant ATM function with antisense morpholino oligonucleotides

Du¹,

Pollard²,

Gatti

2007

Proc. Natl. Acad. Sci. U.S.A.

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We used antisense morpholino oligonucleotides (AMOs) to redirect and restore normal splicing of three prototypic splicing mutations in the ataxia-telangiectasia mutated (ATM) gene. Two of the mutations activated cryptic 5 or 3 splice sites within exonic regions; the third mutation activated a downstream 5 splice site leading to pseudoexon inclusion of a portion of intron 28. AMOs were targeted to aberrant splice sites created by the mutations; this effectively restored normal ATM splicing at the mRNA level and led to the translation of full-length, functional ATM protein for at least 84 h in the three cell lines examined, as demonstrated by immunoblotting, ionizing irradiation-induced autophosphorylation of ATM, and transactivation of ATM substrates. Ionizing irradiation-induced cytotoxicity was markedly abrogated after AMO exposure. The ex vivo data strongly suggest that the disease-causing molecular pathogenesis of such prototypic mutations is not the amino acid change of the protein but the mutated DNA code itself, which alters splicing. Such prototypic splicing mutations may be correctable in vivo by systemic administration of AMOs and may provide an approach to customized, mutation-based treatment for ataxia-telangiectasia and other genetic disorders.ataxia-telangiectasia mutated ͉ mutation-based treatment T his study addresses the restoration of normal splicing in three ataxia-telangiectasia mutated (ATM) splicing mutations by exposure of ataxia-telangiectasia (A-T) cells to antisense morpholino oligonucleotides (AMOs). Antisense oligonucleotides have been used to restore pre-mRNA splicing in other disease models (1-8); however, the therapeutic rationale for each varies considerably and, to our knowledge, none has addressed an intranuclear or DNA repair disorder. Furthermore, we believe that A-T offers several advantages for exploring the therapeutic potential of AMOs, such as (i) the availability of many surrogate markers for evaluating ATM function, ex vivo and in vivo, and (ii) a well characterized spectrum of ATM mutations supported by an extensive cell repository of lymphoblastoid cell lines (LCLs) derived from patients with those mutations (9-12). The LCLs allow pre-mRNA mechanisms to be dissected in great detail. The principles gleaned can then be extended to other tissue targets, such as neuronal cells.A-T is a progressive autosomal recessive neurodegenerative disorder resulting from mutations in ATM (13). This gene includes 66 exons and encodes a 13-kb mature transcript with an ORF of 9,168 nt. The ATM protein is a serine/threonine kinase with Ͼ30 phosphorylation targets (14, 15). It affects control of cell cycle checkpoints, repair of dsDNA breaks, responses to oxidative stress, and apoptosis and is a potent tumor suppressor (16)(17)(18)(19). ATM is constitutively expressed in all tissues, primarily in the nucleus. So far no therapy exists for this disorder.ATM protein is not detectable in most A-T patients. A subset of patients with notable protein levels (5-20%) has milder phenotypes (20). Pati...

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Section: Discussionmentioning

confidence: 99%

Correction of prototypic ATM splicing mutations and aberrant ATM function with antisense morpholino oligonucleotides

Du¹,

Pollard²,

Gatti

2007

Proc. Natl. Acad. Sci. U.S.A.

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“…Unmodified PMOs were not assessed in the current study due to their difficulty of uptake in cultured cells (48). Instead, only vivo-PMOs were tested to enhance cell delivery.…”

Section: Discussionmentioning

confidence: 99%

Efficient exon skipping of SGCG mutations mediated by phosphorodiamidate morpholino oligomers

Wyatt¹,

Demonbreun²,

Kim³

et al. 2018

JCI Insight

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“…Morpholinos contain a six-membered morpholine moiety instead of the sugar ribose and phosphorodiamidate linkages (Summerton and Weller 1997). They have a nonionic backbone at physiological pH, making them notoriously hard to transfect in tissue culture experiments (Amantana and Iversen 2005). However, in vivo their nonionic nature results in higher tissue concentrations, due to the lack of nonspecific interactions with cellular components (Amantana and Iversen 2005).…”

Section: Aon Chemistrymentioning

confidence: 99%

“…They have a nonionic backbone at physiological pH, making them notoriously hard to transfect in tissue culture experiments (Amantana and Iversen 2005). However, in vivo their nonionic nature results in higher tissue concentrations, due to the lack of nonspecific interactions with cellular components (Amantana and Iversen 2005). Morpholinos are nontoxic and very stable and have been shown efficient modulators of pre-mRNA splicing (Gebski et al 2003).…”

Section: Aon Chemistrymentioning

confidence: 99%

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

Aartsma‐Rus¹,

Ommen²

2007

RNA

236

177

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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.

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Pharmacokinetics and biodistribution of phosphorodiamidate morpholino antisense oligomers

Cited by 140 publications

References 43 publications

Correction of prototypic ATM splicing mutations and aberrant ATM function with antisense morpholino oligonucleotides

Correction of prototypic ATM splicing mutations and aberrant ATM function with antisense morpholino oligonucleotides

Efficient exon skipping of SGCG mutations mediated by phosphorodiamidate morpholino oligomers

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

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