Novel Exon-Skipping Therapeutic Approach for the DMD Gene Based on Asymptomatic Deletions of Exon 49

Abstract

Exon skipping is a promising therapeutic approach. One important condition for this approach is that the exon-skipped form of the gene can at least partially perform the required function and lead to improvement of the phenotype. It is therefore critical to identify the exons that can be skipped without a significant deleterious effect on the protein function. Pathogenic variants in the DMD gene are responsible for Duchenne muscular dystrophy (DMD). We report for the first time a deletion of the in-frame exon 49 associated with a strikingly normal muscular phenotype. Based on this observation, and on previously known therapeutic approaches using exon skipping in DMD for other single exons, we aimed to extend the clinical use of exon skipping for patients carrying truncating mutations in exon 49. We first determined the precise genomic position of the exon 49 deletion in our patients. We then demonstrated the feasibility of skipping exon 49 using an in vitro AON (antisense oligonucleotide) approach in human myotubes carrying a truncating pathogenic variant as well as in healthy ones. This work is a proof of concept aiming to expand exon-skipping approaches for DMD exon 49.

Keywords: AON; DMD; deletion; exon skipping; genetics; muscular dystrophy; myopathy; pathological mechanisms; therapy.

Figure 1

(a) Schematic representation of dystrophin (violet), a rod-shaped protein containing four main functional domains: an actin-binding amino-terminal domain (ABD1); a central rod domain composed of 24 spectrin-like repeats (R1–R24, represented by circles) interrupted by four proline-rich parts (H1–H4) which gives it more flexibility (represented by diamonds); a cysteine-rich domain; a carboxy-terminal domain. A second actin-binding domain (ABD2) extends from R11 to R17. Dystrophin has two membrane lipid-binding domains (LBD), the first one comprises the repeats R1 to R3 whereas the second one (LBD2) comprises repeats R4 to R19. This places dystrophin very near the sarcolemma with a large part of its central rod domain lying along the phospholipid membrane. R19 (LBD2), which is coded partially by exon 49, is represented by a dashed white circle. In the cellular context, dystrophin forms a complex with other proteins (DG: dystroglycans, SGC/SPN: sarcoglycan–sarcospan complex, DB: dystrobrevin, SYN: syntrophins). This complex plays an important role in signal transduction in addition to its mechano-protective role, which is indispensable for contractions and proper muscle function [3]. (DGC: dystrophin glycoproteins complex, ECM: extracellular matrix); (b) Schematic representation of exons 50, 49 and 48 in DMD (rectangles). The identified genomic deletion of exon 49 is represented by the dashed horizontal line. Genomic positions are mentioned using (hg19/GRCH37).

Figure 2

Long-range PCR results showing the amplified fragment containing the breakpoint junction of the exon 49 deletion, using Q5 High-Fidelity DNA Polymerase. We note that we have the same 750 bp fragments for the 5 individuals. All patients are identified by the same letter as in the text. (-) signs mark the negative control lanes (PCR amplification without DNA). For patient D (daughter of patient E), PCR had to be redone using a new source of DNA because of the degradation of the first source.

Figure 3

(a) Schematic representation of exon 49 DNA sequence in a DMD patient carrying a frameshift variant (asterisk). AON A and B, transfected in WT and mutated myotubes, are represented in grey and black respectively and hybridized to their complementary sequence. AON A masks the canonical (ag) splice site (red), AON B masks a region rich in ESE (exonic splicing enhancer), represented by horizontal lines (color reflects density of ESE presence); (b) Results of AON transfection into WT and mutated myotubes are presented. In the dashed rectangle: RT-PCR results after RNA extraction from transfected WT myotubes are shown on electrophoresis migration gel; primers located in exons 47 and 51 were used. In WT, myotubes bands without exon 49 represent 11.68% with AON A alone, 16.87% with AON B alone and 24.42% using AON A plus B. The second migration gel represents the results of transfected myotubes carrying the c.7186_7187insT pathogenic variant (RT-PCR). Exon skipping was done using AON B alone (first lane) and compared to the control (second lane). The band without exon 49 represents 63.28% of all amplified bands. The sequence of represented bands is shown with their respective chromatograms. (ES: Exon skipping, WT: Wild type).