Derick G. Wansink

CRISPR/Cas9-Induced (CTG⋅CAG)n Repeat Instability in the Myotonic Dystrophy Type 1 Locus: Implications for Therapeutic Genome Editing

Myotonic dystrophy type 1 (DM1) is caused by (CTG⋅CAG)n-repeat expansion within the DMPK gene and thought to be mediated by a toxic RNA gain of function. Current attempts to develop therapy for this disease mainly aim at destroying or blocking abnormal properties of mutant DMPK (CUG)n RNA. Here, we explored a DNA-directed strategy and demonstrate that single clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-cleavage in either its 5′ or 3′ unique flank promotes uncontrollable deletion of large segments from the expanded trinucleotide repeat, rather than formation of short indels usually seen after double-strand break repair. Complete and precise excision of the repeat tract from normal and large expanded DMPK alleles in myoblasts from unaffected individuals, DM1 patients, and a DM1 mouse model could be achieved at high frequency by dual CRISPR/Cas9-cleavage at either side of the (CTG⋅CAG)n sequence. Importantly, removal of the repeat appeared to have no detrimental effects on the expression of genes in the DM1 locus. Moreover, myogenic capacity, nucleocytoplasmic distribution, and abnormal RNP-binding behavior of transcripts from the edited DMPK gene were normalized. Dual sgRNA-guided excision of the (CTG⋅CAG)n tract by CRISPR/Cas9 technology is applicable for developing isogenic cell lines for research and may provide new therapeutic opportunities for patients with DM1.

Antisense transcription of the myotonic dystrophy locus yields low-abundant RNAs with and without (CAG)n repeat

ABSTRACT The unstable (CTG·CAG)n trinucleotide repeat in the myotonic dystrophy type 1 (DM1) locus is bidirectionally transcribed from genes with terminal overlap. By transcription in the sense direction, the DMPK gene produces various alternatively spliced mRNAs with a (CUG)n repeat in their 3′ UTR. Expression in opposite orientation reportedly yields (CAG)n-repeat containing RNA, but both structure and biologic significance of this antisense gene (DM1-AS) are largely unknown. Via a combinatorial approach of computational and experimental analyses of RNA from unaffected individuals and DM1 patients we discovered that DM1-AS spans >6 kb, contains alternative transcription start sites and uses alternative polyadenylation sites up- and downstream of the (CAG)n repeat. Moreover, its primary transcripts undergo alternative splicing, whereby the (CAG)n segment is removed as part of an intron. Thus, in patients a mixture of DM1-AS RNAs with and without expanded (CAG)n repeat are produced. DM1-AS expression appears upregulated in patients, but transcript abundance remains very low in all tissues analyzed. Our data suggest that DM1-AS transcripts belong to the class of long non-coding RNAs. These and other biologically relevant implications for how (CAG)n-expanded transcripts may contribute to DM1 pathology can now be explored experimentally.

A low absolute number of expanded transcripts is involved in myotonic dystrophy type 1 manifestation in muscle

Muscular manifestation of myotonic dystrophy type 1 (DM1), a common inheritable degenerative multisystem disorder, is mainly caused by expression of RNA from a (CTG·CAG)n-expanded DM1 locus. Here, we report on comparative profiling of expression of normal and expanded endogenous or transgenic transcripts in skeletal muscle cells and biopsies from DM1 mouse models and patients in order to help us in understanding the role of this RNA-mediated toxicity. In tissue of HSALR mice, the most intensely used ‘muscle-only’ model in the DM1 field, RNA from the α-actin (CTG)250 transgene was at least 1000-fold more abundant than that from the Dmpk gene, or the DMPK gene in humans. Conversely, the DMPK transgene in another line, DM500/DMSXL mice, was expressed ∼10-fold lower than the endogenous gene. Temporal regulation of expanded RNA expression differed between models. Onset of expression occurred remarkably late in HSALR myoblasts during in vitro myogenesis whereas Dmpk or DMPK (trans)genes were expressed throughout proliferation and differentiation phases. Importantly, quantification of absolute transcript numbers revealed that normal and expanded Dmpk/DMPK transcripts in mouse models and DM1 patients are low-abundance RNA species. Northern blotting, reverse transcriptase–quantitative polymerase chain reaction, RNA-sequencing and fluorescent in situ hybridization analyses showed that they occur at an absolute number between one and a few dozen molecules per cell. Our findings refine the current RNA dominance theory for DM1 pathophysiology, as anomalous factor binding to expanded transcripts and formation of soluble or insoluble ribonucleoprotein aggregates must be nucleated by only few expanded DMPK transcripts and therefore be a small numbers game.

Trinucleotide-repeat expanded and normal DMPK transcripts contain unusually long poly(A) tails despite differential nuclear residence

In yeast and higher eukaryotes nuclear retention of transcripts may serve in control over RNA decay, nucleocytoplasmic transport and premature cytoplasmic appearance of mRNAs. Hyperadenylation of RNA is known to be associated with nuclear retention, but the cause-consequence relationship between hyperadenylation and regulation of RNA nuclear export is still unclear. We compared polyadenylation status between normal and expanded DMPK transcripts in muscle cells and tissues derived from unaffected individuals and patients with myotonic dystrophy type 1 (DM1). DM1 is an autosomal dominant disorder caused by (CTG)n repeat expansion in the DMPK gene. DM1 etiology is characterized by an almost complete block of nuclear export of DMPK transcripts carrying a long (CUG)n repeat, including aberrant sequestration of RNA-binding proteins. We show here by use of cell fractionation, RNA size separation and analysis of poly(A) tail length that a considerable fraction of transcripts from the normal DMPK allele is also retained in the nucleus (~30%). They carry poly(A) tails with an unusually broad length distribution, ranging between a few dozen to >500 adenosine residues. Remarkably, expanded DMPK (CUG)n transcripts from the mutant allele, almost exclusively nuclear, carry equally long poly(A) tails. Our findings thus suggest that nuclear retention may be a common feature of regulation of DMPK RNA expression. The typical forced nuclear residence of expanded DMPK transcripts affects this regulation in tissues of DM1 patients, but not through hyperadenylation.

Assisted delivery of antisense therapeutics in animal models of heritable neurodegenerative and neuromuscular disorders: a systematic review and meta-analysis

Antisense oligonucleotide (AON)-based therapies hold promise for a range of neurodegenerative and neuromuscular diseases and have shown benefit in animal models and patients. Success in the clinic is nevertheless still limited, due to unfavourable biodistribution and poor cellular uptake of AONs. Extensive research is currently being conducted into the formulation of AONs to improve delivery, but thus far there is no consensus on which of those strategies will be the most effective. This systematic review was designed to answer in an unbiased manner which delivery strategies most strongly enhance the efficacy of AONs in animal models of heritable neurodegenerative and neuromuscular diseases. In total, 95 primary studies met the predefined inclusion criteria. Study characteristics and data on biodistribution and toxicity were extracted and reporting quality and risk of bias were assessed. Twenty studies were eligible for meta-analysis. We found that even though the use of delivery systems provides an advantage over naked AONs, it is not yet possible to select the most promising strategies. Importantly, standardisation of experimental procedures is warranted in order to reach conclusions about the most efficient delivery strategies. Our best practice guidelines for future experiments serve as a step in that direction.

Abnormalities in Skeletal Muscle Myogenesis, Growth, and Regeneration in Myotonic Dystrophy

Myotonic dystrophy type 1 (DM1) and 2 (DM2) are autosomal dominant degenerative neuromuscular disorders characterized by progressive skeletal muscle weakness, atrophy, and myotonia with progeroid features. Although both DM1 and DM2 are characterized by skeletal muscle dysfunction and also share other clinical features, the diseases differ in the muscle groups that are affected. In DM1, distal muscles are mainly affected, whereas in DM2 problems are mostly found in proximal muscles. In addition, manifestation in DM1 is generally more severe, with possible congenital or childhood-onset of disease and prominent CNS involvement. DM1 and DM2 are caused by expansion of (CTG•CAG)n and (CCTG•CAGG)n repeats in the 3′ non-coding region of DMPK and in intron 1 of CNBP, respectively, and in overlapping antisense genes. This critical review will focus on the pleiotropic problems that occur during development, growth, regeneration, and aging of skeletal muscle in patients who inherited these expansions. The current best-accepted idea is that most muscle symptoms can be explained by pathomechanistic effects of repeat expansion on RNA-mediated pathways. However, aberrations in DNA replication and transcription of the DM loci or in protein translation and proteome homeostasis could also affect the control of proliferation and differentiation of muscle progenitor cells or the maintenance and physiological integrity of muscle fibers during a patient’s lifetime. Here, we will discuss these molecular and cellular processes and summarize current knowledge about the role of embryonic and adult muscle-resident stem cells in growth, homeostasis, regeneration, and premature aging of healthy and diseased muscle tissue. Of particular interest is that also progenitor cells from extramuscular sources, such as pericytes and mesoangioblasts, can participate in myogenic differentiation. We will examine the potential of all these types of cells in the application of regenerative medicine for muscular dystrophies and evaluate new possibilities for their use in future therapy of DM.

G.P.132

Myotonic dystrophy type 1 (DM1) is a multisystemic neuromuscular disorder caused by an expanded CTG repeat in the 3′ UTR of the DM protein kinase (DMPK) gene, resulting in formation of toxic RNA aggregates that interfere with RNA splicing and dysregulate normal cell function. RNA-modulation by antisense oligonucleotides (AONs) represents an interesting therapeutic approach for DM1 and is currently in pre-clinical development. We have previously obtained molecular proof-of-concept for a panel of AONs that target the CUG expansion and result in efficient knockdown of toxic RNA in DM1 patient myoblasts with different repeat expansion lengths and in cultured muscle cells derived from DM500 mice. To facilitate systemic AON delivery and uptake, we used a 7-amino acid linear muscle targeting peptide (PP08) and conjugated this to different CUG-targeting AONs. We have shown that these peptide-conjugated AONs are well tolerated and do not result in complement activation in human and monkey plasma or induce cytokine release in human whole blood in vitro safety assays. Subcutaneous administration of peptide-conjugated AONs in different DM1 mouse models (DM500, HSALR) resulted in enhanced tissue PK levels in several tissues of relevance for DM1 such as skeletal muscle and brain, indicating that the peptide-AON displayed efficient tissue uptake and was even able to pass the blood–brain-barrier. The higher tissue levels were accompanied by an increased knockdown of mutant RNA compared to non-conjugated AONs. Besides inducing a significant RNA knockdown, peptide-conjugated AONs resulted in a mild but statistically significant reduction of myotonia in gastrocnemius and tibialis muscles of HSALR mice, as determined by EMG analysis. These data demonstrate that peptide-AONs targeting the CUG-repeat have potential in a multisystemic therapeutic approach for DM1.

Cell Membrane Integrity in Myotonic Dystrophy Type 1: Implications for Therapy

Myotonic Dystrophy type 1 (DM1) is a multisystemic disease caused by toxic RNA from a DMPK gene carrying an expanded (CTG∗CAG) n repeat. Promising strategies for treatment of DM1 patients are currently being tested. These include antisense oligonucleotides and drugs for elimination of expanded RNA or prevention of aberrant binding of this RNA to RNP proteins. A significant hurdle for preclinical development along these lines is efficient systemic delivery of compounds across endothelial and muscle and brain cell membranes. It has been reported that DM1 patients show elevated levels of muscular markers in their serum and that splicing of dystrophin, an essential protein for muscle membrane structure, is abnormal. Therefore, we studied cell membrane integrity in DM1 mouse models commonly used for preclinical testing. We found that membranes in skeletal muscle, heart and brain were impermeable to Evans Blue Dye. Creatine kinase levels in serum were similar to those in wild type mice and expression of dystrophin protein was unaffected. Also in patient muscle biopsies cell surface distribution of dystrophin was normal. Combined, our findings show that cells in DM1 tissues have a normal functional membrane, which forms a barrier that must be overcome in future work towards effective drug delivery.