The lncRNA 44s2 Study Applicability to the Design of 45-55 Exon Skipping Therapeutic Strategy for DMD

Abstract

In skeletal muscle, long noncoding RNAs (lncRNAs) are involved in dystrophin protein stabilization but also in the regulation of myocytes proliferation and differentiation. Hence, they could represent promising therapeutic targets and/or biomarkers for Duchenne and Becker muscular dystrophy (DMD/BMD). DMD and BMD are X-linked myopathies characterized by a progressive muscular dystrophy with or without dilatative cardiomyopathy. Two-thirds of DMD gene mutations are represented by deletions, and 63% of patients carrying DMD deletions are eligible for 45 to 55 multi-exons skipping (MES), becoming BMD patients (BMDΔ45-55). We analyzed the genomic lncRNA presence in 38 BMDΔ45-55 patients and characterized the lncRNA localized in introns 44 and 55 of the DMD gene. We highlighted that all four lncRNA are differentially expressed during myogenesis in immortalized and primary human myoblasts. In addition, the lncRNA44s2 was pointed out as a possible accelerator of differentiation. Interestingly, lncRNA44s expression was associated with a favorable clinical phenotype. These findings suggest that lncRNA44s2 could be involved in muscle differentiation process and become a potential disease progression biomarker. Based on these results, we support MES45-55 therapy and propose that the design of the CRISPR/Cas9 MES45-55 assay consider the lncRNA sequences bordering the exonic 45 to 55 deletion.

Keywords: Becker muscular dystrophy (BMD); Duchenne muscular dystrophy (DMD); lncRNA; long noncoding RNA; ncRNA.

Figure 1

Neo-intron of deleted 45-55 exons in Duchenne muscular dystrophy (DMD) gene. (A) Schematic representation of the whole genome sequencing (WGS) detection of the deletion breakpoint (black boxes) in the DMD gene of 18 patients BMDΔ45-55. (B) Schematic representation of the canonic exon 45-55 deletion neo-intron with the lncRNA (colored boxes for lncRNA and arrows to indicate the transcription sense. The exons 1 to 44 and 56 to 79 are illustrated in clear red boxes).

Figure 2

lncRNA profile in BMDΔ45-55 patients (n = 38). (A) lncRNA detection by PCR from diagnostic DNA, the genomic presence of each lncRNA was annotated by P and the absence by N. (B) Scheme presentation of the most frequent lncRNA genomic presence profiles. (C) Phenotype correlation with lncRNA genomic clusters. Abbreviations: P = present lncRNA sequence, N = absent lncRNA sequence, MBMD = Mild Becker muscular dystrophy (BMD), IBMD = Intermediate BMD, SBMD = Severe BMD, ex = exon).

Figure 3

lncRNA profile in control and DMD/BMD patients. (A) Genomic profile of control, DMD/BMD patient selected for the expression analysis in muscular biopsy. The genomic presence of each lncRNA was annotated by P. (B) Assessment of relative lncRNA expression in the muscular biopsies of a healthy subject and DMD and BMD patients by RT-qPCR (Mean ± SD); lncRNA from the healthy subject was the control and set to 1 (respective p-values are indicated * p < 0.05, 44s2: ** p = 0.001, *** p < 0.0005) by two-way ANOVA test. Abbreviations: DMD = Duchenne muscular dystrophy, BMD = Becker muscular dystrophy.

Figure 4

lncRNA genomic profile and expression assessment in human myoblasts. (A) DNA and RNA profiling in muscular biopsy (MB) from healthy subject and immortalized human myoblasts from healthy subject (Hthy) and DMDΔ45-52 deletion patient (DMD). (B) Proliferation assessment at different timelines (day 0 to day 3/P0-P3) in human immortalized (healthy subject = Hthy-I, DMDΔ45-52 patient = 45-52-I) and primary human myoblasts from healthy subject (Hthy), BMDΔ45-55 (45-55), and DMDΔ45-52(45-52) patients. (C) lncRNA expression profile at day 1 (P1) and 3 of proliferation(P3) (left) in human primary and (right) in immortalized myoblasts. (D) lncRNA expression was analyzed by RT-qPCR at 3 and 5 days (D3, D5) of differentiation (left) in human primary and (right) in immortalized myoblasts (black columns illustrate nc44s, red columns = nc44s2, green columns = nc55s, violet columns = nc55as). Data represent the average from three independent experiments (Mean ± SD); * p < 0.05, ** p < 0.01, and *** p < 0.001 by two-way ANOVA test).

Figure 5

nc44s2 overexpression study during myogenesis in human primary myoblasts. (A) Growth charts depicting the proliferation rate in 3 cell lines; red color illustrates the control condition, green the nc44s2 in human primary myoblasts issued from healthy subjects (Hthy), BMDΔ45-55 patients (45-55), and DMDΔ45-52 patient (45-52). (B) MyoD and Myf5 expression assessment by RT-qPCR at 3 days of proliferation from three independent experiments performed in duplicates (significant increase in MyoD expression in 45-55 * p = 0.02 by two-way ANOVA). (C) MyHCIIb and MyoD expression at 3 days of differentiation from three independent experiments performed in duplicates (significant increase in 45-55 of MyHCIIb, ** p = 0.03 by two-way ANOVA) (Means ± SD). (D) Immunofluorescence experiment illustrating the myotubes at 3 days of differentiation in three primary human myoblasts visualized at X10 magnification. The fusion index was evaluated by myotubes nuclei count/total nuclei number and compared between OE and control conditions in at least three representative images at three days of differentiation (** p < 0.004 by two-way ANOVA) (Means ± SD).

Figure 6

Dystrophin mRNA expression assessment. (A) mRNA dystrophin expression assessed by RT-qPCR at 3 days of proliferation (left) and at 3 days of differentiation (right) in human primary myoblasts issued from healthy subjects (Hthy), BMDΔ45-55 patients (45-55), and DMDΔ45-52 patient (45-52) from three independent experiments, performed in duplicates; * p = 0.01 by one-way ANOVA (Means ± SD). (B) mRNA dystrophin expression after lnc44s2 OE in the same cell lines at 3 days of proliferation and differentiation from three independent experiments, performed in duplicates; p = 0.01 by two-way ANOVA (Means ± SD). Abbreviations: ctr = AAV9-control condition, nc44s2 = AAV9-44s2-lncRNA OE.

Figure 7

Schematic representation of lncRNA 44s2 role during regeneration in dystrophic skeletal muscle.