Design and Analysis of Effects of Triplet Repeat Oligonucleotides in Cell Models for Myotonic Dystrophy

Abstract

Myotonic dystrophy type 1 (DM1) is caused by DM protein kinase (DMPK) transcripts containing an expanded (CUG)n repeat. Antisense oligonucleotide (AON)-mediated suppression of these mutant RNAs is considered a promising therapeutic strategy for this severe disorder. Earlier, we identified a 2′-O-methyl (2′-OMe) phosphorothioate (PT)–modified (CAG)7 oligo (PS58), which selectively silences mutant DMPK transcripts through recognition of the abnormally long (CUG)n tract. We present here a comprehensive collection of triplet repeat AONs and found that oligo length and nucleotide chemistry are important determinants for activity. For significant reduction of expanded DMPK mRNAs, a minimal length of five triplets was required. 2′-O,4′-C-ethylene-bridged nucleic acid (ENA)–modified AONs appeared not effective, probably due to lack of nuclear internalization. Selectivity for products from the expanded DMPK allele in patient myoblasts, an important requirement to minimize unwanted side effects, appeared also dependent on AON chemistry. In particular, RNase-H–dependent (CAG)n AONs did not show (CUG)n length specificity. We provide evidence that degradation of long DMPK transcripts induced by PS58-type AONs is an RNase-H independent process, does not involve oligo-intrinsic RNase activity nor does it interfere with splicing of DMPK transcripts. Our collection of triplet repeat AONs forms an important resource for further development of a safe therapy for DM1 and other unstable microsatellite diseases. Myotonic dystrophy type 1 (DM1) is caused by DM protein kinase (DMPK) transcripts containing an expanded (CUG)n repeat. Antisense oligonucleotide (AON)-mediated suppression of these mutant RNAs is considered a promising therapeutic strategy for this severe disorder. Earlier, we identified a 2′-O-methyl (2′-OMe) phosphorothioate (PT)–modified (CAG)7 oligo (PS58), which selectively silences mutant DMPK transcripts through recognition of the abnormally long (CUG)n tract. We present here a comprehensive collection of triplet repeat AONs and found that oligo length and nucleotide chemistry are important determinants for activity. For significant reduction of expanded DMPK mRNAs, a minimal length of five triplets was required. 2′-O,4′-C-ethylene-bridged nucleic acid (ENA)–modified AONs appeared not effective, probably due to lack of nuclear internalization. Selectivity for products from the expanded DMPK allele in patient myoblasts, an important requirement to minimize unwanted side effects, appeared also dependent on AON chemistry. In particular, RNase-H–dependent (CAG)n AONs did not show (CUG)n length specificity. We provide evidence that degradation of long DMPK transcripts induced by PS58-type AONs is an RNase-H independent process, does not involve oligo-intrinsic RNase activity nor does it interfere with splicing of DMPK transcripts. Our collection of triplet repeat AONs forms an important resource for further development of a safe therapy for DM1 and other unstable microsatellite diseases. IntroductionMyotonic dystrophy type 1 (DM1) is an autosomal dominant, trinucleotide repeat expansion disease and the most common form of adult onset muscular dystrophy.1Groenen P Wieringa B Expanding complexity in myotonic dystrophy.Bioessays. 1998; 20: 901-912Crossref PubMed Scopus (70) Google Scholar,2Wheeler TM Thornton CA Myotonic dystrophy: RNA-mediated muscle disease.Curr Opin Neurol. 2007; 20: 572-576Crossref PubMed Scopus (132) Google Scholar,3Harper PS Myotonic Dystrophy. WB Saunders, London, UK2001Google Scholar The multisystemic symptoms are highly variable and involve different tissues and organs, including skeletal muscle, heart, brain, eyes, reproductive systems, and the gastrointestinal tract. No cure to slow or stop the disease process is currently available.The genetic basis of DM1 is an unstable (CTG)n expansion in the 3′-untranslated region of the DM protein kinase (DMPK) gene. Length of the (CTG)n segment is correlated with age of onset and severity of disease.1Groenen P Wieringa B Expanding complexity in myotonic dystrophy.Bioessays. 1998; 20: 901-912Crossref PubMed Scopus (70) Google Scholar In the healthy population, the CTG repeat is polymorphic in length, with a triplet number that ranges from 5 to 37 (mostly below 15),4Zerylnick C Torroni A Sherman SL Warren ST Normal variation at the myotonic dystrophy locus in global human populations.Am J Hum Genet. 1995; 56: 123-130Crossref PubMed Scopus (1) Google Scholar whereas disease alleles may contain between 50 and >2,000 triplets.1Groenen P Wieringa B Expanding complexity in myotonic dystrophy.Bioessays. 1998; 20: 901-912Crossref PubMed Scopus (70) Google ScholarThe DM1 pathogenic mechanism involves an RNA gain-of-function. Repeat-containing transcripts from the expanded allele accumulate in cell nuclei and alter the of with disease most for and protein 1 TM Thornton CA Myotonic dystrophy: RNA-mediated muscle disease.Curr Opin Neurol. 2007; 20: 572-576Crossref PubMed Scopus (132) Google Scholar In adult DM1 the in of these splicing of in which of splicing For and of may caused by splicing of muscle and TM Thornton CA Myotonic dystrophy: RNA-mediated muscle disease.Curr Opin Neurol. 2007; 20: 572-576Crossref PubMed Scopus (132) Google Scholar on and C B of is with in myotonic PubMed Scopus Google with DM1 that (CUG)n RNA show that of RNA are of RNA and in myotonic Genet. PubMed Scopus Google Scholar that the of expanded DMPK transcripts to In the different with the to expanded DMPK transcripts RNA RNase-H activity or RNA Wieringa B therapy in myotonic dystrophy: on RNA Genet. 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PubMed Scopus Google Scholar and and to TM P of RNA in myotonic PubMed Scopus Google Scholar DM1 and J of myotonic dystrophy protein kinase (DMPK) and in human myotonic dystrophy PubMed Scopus Google AONs and to the (CUG)n repeat. and chemistry are in and for and AONs and by The which was to an from and from AONs to the 5 AON was at a AON of in to on 5 of or in to For oligo between and was to a of was RNA was of was the with the that was and 5 of a to the at a of 50 of the and of the was RNA was the AONs by kinase in the of and on by cell was by in containing 1 5 cell was and in cell of 1 and The on for and a cell was with 5 cell at 37 for or was by and was was by the of of The oligo was with and in on a in at for and to RNA was the RNA to the was TM P of RNA in myotonic PubMed Scopus Google Scholar human DMPK and by or and with or for RNA of at for J a to for PubMed Scopus Google Scholar in the for and in a for and of nucleic acid PubMed Scopus Google Scholar to of and products on and to and triplet repeat are in RNA was to the with in a of For in of AONs to the RNA to the and for on the 1 of was in a to In was The for was for number was for and for other products on by of was for at the for of J J for of PubMed Scopus Google Scholar of was to in and to by of this was in a of containing 5 The and of the The No and in to the or A was for to the of and the PubMed Scopus Google Scholar and for We that these not by the and an J B A of by of Google Scholar of at by a by B A of an for the of phosphorothioate in PubMed Scopus Google Scholar with a and with the at 37 for 1 and to and the was and for at was an The with a The The was acid was at a The and to of the in was a of in A containing was for cell on The of the containing or in and including and A was with a and For of oligo we a DM1 patient or with or or and in 1 and on on an and to with Wieringa B and of splicing myotonic dystrophy protein kinase (DMPK) with Genet. PubMed Scopus Google Scholar or and of by of was for at in triplet repeat (CUG)n and (CAG)n RNA was by in of a and The was by a a expansion of the (CTG)n repeat in myotonic dystrophy is by and Genet. PubMed Scopus Google Scholar and and to triplet repeat and RNAs in of the in to the AON to expanded triplet repeat RNA was with a and of in 50 and for at 37 was to a of and and at on a in and to and RNAs with AONs in the of 50 50 and 1 for at 37 by RNase-H–dependent (CUG)n RNA was in 50 50 5 and 1 and with AON in the of RNase-H for 1 at 37 by with the that the was to of to between AONs and (CUG)n oligo (CUG)n RNA was in and 1 and with AON for at 37 in the of or by The of AONs to the was from by and from healthy human The AONs to in and in a of and the at 37 for The was by the on and to the and by oligonucleotide was a this oligo to the J P of an oligonucleotide of by to with Google and an or a of in are the was to a between and number of CTG triplets in alleles in human between considered significant P and with with AON in cell with of including with and of AONs in with of triplet repeat transcripts and by with of a (CUG)n with a of with to of AONs with (CAG)n splicing of the in the with in RNA a or tract. with with in this with in this IntroductionMyotonic dystrophy type 1 (DM1) is an autosomal dominant, trinucleotide repeat expansion disease and the most common form of adult onset muscular dystrophy.1Groenen P Wieringa B Expanding complexity in myotonic dystrophy.Bioessays. 1998; 20: 901-912Crossref PubMed Scopus (70) Google Scholar,2Wheeler TM Thornton CA Myotonic dystrophy: RNA-mediated muscle disease.Curr Opin Neurol. 2007; 20: 572-576Crossref PubMed Scopus (132) Google Scholar,3Harper PS Myotonic Dystrophy. WB Saunders, London, UK2001Google Scholar The multisystemic symptoms are highly variable and involve different tissues and organs, including skeletal muscle, heart, brain, eyes, reproductive systems, and the gastrointestinal tract. No cure to slow or stop the disease process is currently available.The genetic basis of DM1 is an unstable (CTG)n expansion in the 3′-untranslated region of the DM protein kinase (DMPK) gene. Length of the (CTG)n segment is correlated with age of onset and severity of disease.1Groenen P Wieringa B Expanding complexity in myotonic dystrophy.Bioessays. 1998; 20: 901-912Crossref PubMed Scopus (70) Google Scholar In the healthy population, the CTG repeat is polymorphic in length, with a triplet number that ranges from 5 to 37 (mostly below 15),4Zerylnick C Torroni A Sherman SL Warren ST Normal variation at the myotonic dystrophy locus in global human populations.Am J Hum Genet. 1995; 56: 123-130Crossref PubMed Scopus (1) Google Scholar whereas disease alleles may contain between 50 and >2,000 triplets.1Groenen P Wieringa B Expanding complexity in myotonic dystrophy.Bioessays. 1998; 20: 901-912Crossref PubMed Scopus (70) Google ScholarThe DM1 pathogenic mechanism involves an RNA gain-of-function. Repeat-containing transcripts from the expanded allele accumulate in cell nuclei and alter the of with disease most for and protein 1 TM Thornton CA Myotonic dystrophy: RNA-mediated muscle disease.Curr Opin Neurol. 2007; 20: 572-576Crossref PubMed Scopus (132) Google Scholar In adult DM1 the in of these splicing of in which of splicing For and of may caused by splicing of muscle and TM Thornton CA Myotonic dystrophy: RNA-mediated muscle disease.Curr Opin Neurol. 2007; 20: 572-576Crossref PubMed Scopus (132) Google Scholar on and C B of is with in myotonic PubMed Scopus Google with DM1 that (CUG)n RNA show that of RNA are of RNA and in myotonic Genet. PubMed Scopus Google Scholar that the of expanded DMPK transcripts to In the different with the to expanded DMPK transcripts RNA RNase-H activity or RNA Wieringa B therapy in myotonic dystrophy: on RNA Genet. PubMed Scopus Google Scholar of DMPK was by of these between and transcripts was and safe for therapeutic in DM1 an transcripts from the expanded allele and DMPK transcripts or transcripts containing (CUG)n length of the (CUG)n repeat is the polymorphic between mutant and DMPK the repeat forms an therapeutic the that and mutant DMPK transcripts are in the (CUG)n length may by to this mutant transcripts accumulate we on the and of an oligonucleotide to the (CUG)n TM P of RNA in myotonic PubMed Scopus Google Scholar is a (CAG)7 2′-O-methyl (2′-OMe) phosphorothioate which silences expanded (CUG)n transcripts in DM1 cell and in AON at the expansion of a (CAG)n to interfere with the (CUG)n TM of RNA by of protein on triplet repeat PubMed Scopus Google Scholar or nucleic (CAG)n to RNase-H activity for degradation of RNase degradation of RNA in myotonic dystrophy type PubMed Scopus Google Scholar a nuclear RNA which pathogenic DMPK in DM1 a C A C of in DM1 by PubMed Scopus Google we on that AONs for of expanded DMPK repeat AONs with different and in an for in a DM1 cell human DMPK transcripts. AON length and nucleotide chemistry appeared to for therapeutic A of AONs was in DM1 patient of this are important for in with AONs and for development of a therapy for a in of this we on on in by of these