Decreased levels of myotonic dystrophy protein kinase (DMPK) and delayed differentiation in human myotonic dystrophy myoblasts

Furling, Denis; D, Lemieux; Taneja, Krishan; Puymirat, Jack

doi:10.1016/s0960-8966(01)00226-7

Cited by 87 publications

(78 citation statements)

References 37 publications

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“…As shown in Figure 3b, the growth of normal and DM1 myoblasts was very similar, with a doubling time of about 36 h, as previously reported. 25 The growth of DM1 myoblasts expressing either 5 0 UTR antisense RNAs or (CUG)13 antisense RNAs or the GFP gene was also similar with that of noninfected DM1 myoblasts. This indicates that the antisense vector has no toxic effect on DM1 cells.…”

Section: Resultsmentioning

confidence: 75%

“…25,32 Here we showed that retrovirus producing (CUG) 13 antisense RNA has no effect on the proliferative rate of DM1 myoblasts; however, it restores the differentiation of infected DM1 myoblasts as indicated by the increase in the fusion index, in the mean number of nuclei per myotube as well as in the uptake of glucose. The specificity of these effects is supported by the fact that two other retrovirus producing the GFP protein or an antisense RNA against the 5 0 UTR have no effect on the levels of DMPK mRNAs nor on the growth and differentiation of DM1 myoblasts.…”

Section: Targeting Of Mutant Dmpk Transcripts D Furling Et Almentioning

confidence: 88%

“…The percentage of myogenic cells in the culture was determined by immunocytochemistry analysis using anti-desmin antibody as described previously. [24][25][26] Southern blot analysis showed a length of CTG repeats of about 750 in DM1 myoblasts. 25 For myoblast differentiation, cells were subsequently cultured in a-MEM supplemented with 10 mg/ml insulin, 10 ng/ml epidermal growth factor and 0.5 mg/ml BSA.…”

Section: Muscle Cellsmentioning

confidence: 99%

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Viral vector producing antisense RNA restores myotonic dystrophy myoblast functions

Furling

Doucet²,

Ma³

et al. 2003

Gene Ther

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Myotonic dystrophy (DM1) is caused by the expansion of a trinucleotide repeat (CTG) located in the 3 0 untranslated region of the myotonic dystrophy protein kinase gene, for which currently there is no effective treatment. The data available suggest that misregulation of RNA homeostasis may play a major role in DM1 muscle pathogenesis. This indicates that the specific targeting of the mutant DMPK transcripts is essential to raise the rationale basis for the development of a specific gene therapy for DM1. We have produced a retrovirus which expresses a 149-bp antisense RNA complementary to the (CUG)13 repeats and to the 110-bp region following the repeats sequence to increase the specificity. This construct was introduced into human DM1 myoblasts, resulting in a preferential decrease in mutant DMPK transcripts, and effective restoration of human DM1 myoblast functions such as myoblast fusion and the uptake of glucose. It was previously shown that delay of muscle differentiation and insulin resistance in DM1 are associated with misregulation of CUGBP1 protein levels. The analysis of CUGBP1 levels and activity in DM1 cells expressing the antisense RNA indicated a correction of CUGBP1 expression in infected DM1 cells. We therefore show that current antisense RNA delivered in vitro using a retrovirus is not only capable of inhibiting mutant DMPK transcripts, but also can ameliorate dystrophic muscle pathology at the cellular levels. Gene Therapy (2003) 10, 795-802.

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Section: Resultsmentioning

confidence: 75%

Section: Targeting Of Mutant Dmpk Transcripts D Furling Et Almentioning

confidence: 88%

Section: Muscle Cellsmentioning

confidence: 99%

See 1 more Smart Citation

Viral vector producing antisense RNA restores myotonic dystrophy myoblast functions

Furling

Doucet²,

Ma³

et al. 2003

Gene Ther

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“…2 ). Myoblast cell culture models 5,6 and subsequently a transgenic mouse model 7 have provided strong evidence for the involvement of RNA containing expanded CUG repeat tracts in aspects of DM1 skeletal muscle pathology. However, there is no clear model of RNA toxicity in the heart, and instead it has been suggested that DM1 cardiac pathology may be due to misexpression of DMPK 8,9 .…”

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confidence: 99%

Reversible model of RNA toxicity and cardiac conduction defects in myotonic dystrophy

et al. 2006

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Myotonic dystrophy (DM1), the most common muscular dystrophy in adults, is caused by an expanded (CTG) n tract in the 3′ UTR of the gene encoding myotonic dystrophy protein kinase (DMPK) 1 , which results in nuclear entrapment of the 'toxic' mutant RNA and interacting RNAbinding proteins (such as MBNL1) in ribonuclear inclusions 2 . It is unclear if therapy aimed at eliminating the toxin would be beneficial. To address this, we generated transgenic mice expressing the DMPK 3′ UTR as part of an inducible RNA transcript encoding green fluorescent protein (GFP). We were surprised to find that mice overexpressing a normal DMPK 3′ UTR mRNA reproduced cardinal features of myotonic dystrophy, including myotonia, cardiac conduction abnormalities, histopathology and RNA splicing defects in the absence of detectable nuclear inclusions. However, we observed increased levels of CUG-binding protein (CUG-BP1) in skeletal muscle, as seen in individuals with DM1. Notably, these effects were reversible in both mature skeletal and cardiac muscles by silencing transgene expression. These results represent the first in vivo proof of principle for a therapeutic strategy for treatment of myotonic dystrophy by ablating or silencing expression of the toxic RNA molecules.Common features of adult-onset DM1 include myotonia, progressive skeletal muscle loss, cardiac conduction defects, smooth muscle dysfunction, cataracts and insulin resistance 2 . The normal number of CTG repeats (n = 5 to ~30) is higher (n = 50 to >3,000) in individuals with DM1 (ref. 1 ). Unlike the wild-type transcript, mutant DMPK mRNA forms nuclear aggregates 3,4 and is thought to trigger dominant effects by aberrant interactions with or altered activity of RNA splicing factors, principally members of the muscleblind-like (MBNL) family (such as MBNL1) and the CUG-BP and ETR3-like factor (CELF) family (such as CUG-BP1), leading to abnormal splicing of specific RNAs such as chloride channel (Clcn1), insulin Correspondence should be addressed to M.S.M. (mahadevan@virginia.edu). 4 These authors contributed equally to this work. AUTHOR CONTRIBUTIONSM.S.M., R.S.Y., Q.Y., C.D.F.-M., T.D.B. and L.H.P. performed experimental work and data analysis. S.B. generated the transgene constructs. M.S.M. was responsible for conceptual design and execution. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. One potential therapeutic approach in DM1 is to get rid of the toxic RNA from cells. However, it is unclear if this will alleviate the effects of the disease. We used the tetracycline (Tet) inducible system with the reverse tetracycline transactivator (rtTA) to generate double transgenic mice harboring (i) a Tet-responsive, DMPK promoter 10,11 -driven transgene (named GFP-DMPK 3′ UTR) expressing the DMPK 3′ UTR mRNA as part of a GFP transcript, and (ii) a constitutively expressed rtTA transgene (Fig. 1a) Fig. 1). Notably, RNA blots of skeletal muscle RNA showed two major species due to alternative use of polyadenylation signal...

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“…21 In DM1, a strategy to remove or correct mutant mRNA is appropriate in view of evidence that the accumulation of mutant mRNA in the nucleus is the primary basis of the disease. 3,22 Approaches targeted at the mRNA level to remove mutant mRNA, namely the use of antisense, 23 nuclear-retained hammerhead ribozyme 4 and siRNA, 5 have been shown to be able to remove mutant mRNA in in vitro DM1 cells. However, the removal of wild-type mRNA together with mutant mRNA that occurs with all these techniques may have potential untoward consequences.…”

Section: Discussionmentioning

confidence: 99%

Correction of dystrophia myotonica type 1 pre-mRNA transcripts by artificial trans-splicing

et al. 2008

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Dystrophia myotonica type 1 (DM1), the most common muscular dystrophy in adults, results from expansion of a CTG repeat in the 3 0 -untranslated region of the dystrophia myotonica protein kinase gene (DMPK). Correction of the mutant DMPK transcript is a potential therapeutic strategy in DM1. We investigated the efficacy of artificial trans-splicing molecules (ATMs) to target and correct DMPK transcripts. ATMs designed to target intron 14 of DMPK pre-mRNA transcripts were tested for their ability to trans-splice the transcripts of a DMPK mini-gene construct and the endogenous DMPK transcripts of human myosarcoma cells (CCL-136). On agarose gel electrophoresis analysis, six of eight ATMs showed trans-splicing efficacy when applied to DMPK mini-gene construct transcripts, of which three were able to trans-splice endogenous DMPK pre-mRNA transcripts in myosarcoma cells, with trans-splicing efficiency ranging from 1.81 to 7.41%. These findings confirm that artificial trans-splicing can repair DMPK pre-mRNA and provide proof-of-principle evidence for this approach as potential therapeutic strategy for DM1.

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Decreased levels of myotonic dystrophy protein kinase (DMPK) and delayed differentiation in human myotonic dystrophy myoblasts

Cited by 87 publications

References 37 publications

Viral vector producing antisense RNA restores myotonic dystrophy myoblast functions

Viral vector producing antisense RNA restores myotonic dystrophy myoblast functions

Reversible model of RNA toxicity and cardiac conduction defects in myotonic dystrophy

Correction of dystrophia myotonica type 1 pre-mRNA transcripts by artificial trans-splicing

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