Development Of Antisense Drugs To Treat Duchenne Muscular Dystrophy

ObjectiveDuchenne muscular dystrophy(DMD)is a devastating neuromuscular disease which is caused by nonsense or frameshift mutations in the DMD gene,resulting in the lack of dystrophin.DMD is a X-linked disease and its incidence in male infants is about 1/3500.Clinically,DMD patients suffer progressive and irreversible muscle damage and atrophy,eventually dying at the age of 20-30.Therefore,searching for a medicine that can promote the DMD exon 51 skipping is urgent for the treatment of DMD.RNA splicing is a critical part of post-transcriptional processing of gene expression in eukaryotes.Abnormalities in this process can cause many diseases.In recent years,antisense nucleic acid technology has been proven to correct gene splicing errors to achieve the purpose of treating human diseases.For example,nusinersen can effectively correct SMN2 gene splicing errors and increase the inclusion of exon 7 to treat spinal muscular atrophy(SMA).Antisense nucleic acid technology can also be used to promote exon skipping to correct reading frame shifts caused by gene mutations.The DMD gene is one of the largest genes in human and contains 79 exons.The large region of exons 43-53 is a hot spot where mutations occur,usually causing the original open reading frame to be destroyed and protein translation termination.One of the drug development strategies for such mutations is to use antisense oligonucleotides(ASO)to promote the skipping of exon 51 to restore the reading frame.Although the newly produced dystrophin protein has lost several exons,the final product retains most of the amino acid sequence.Therefore,the protein retains basic biological functions,and the patient's symptoms will be greatly improved.According to statistics,this method can be applied to about 13%of DMD patients.There is an ASO drug(eteplirsen)that can treat DMD patients by promoting the skipping of exon 51,but its effect is very limited,especially since there is no protein evidence to prove its therapeutic effect.This project aims to design and screen a new generation of ASO drugs with extremely high efficacy as the target of exon 51 skipping.MethodsUsing SLIC method to construct DMD minigene;using site-directed mutagenesis to delete multiple consecutive fragments on minigene;using site-directed mutagenesis to replace the sequence on SMN1-pCI minigene to explore motifs that can inhibit splicing;designing complementary ASO sequences targeting on exon 51 and its neighboring introns then performing initial screening and further screening(micro walk);screening for second-generation(SG)ASO;exploring the dose effect of ASO;second-generation ASOs that targeting exon 51 were designed to induce exon 51 skipping.Some of morpholino modified second-generation ASOs were transfected into human skeletal muscle cells,and the effect of second-generation ASOs in muscle cells was analyzed.All plasmids and ASOs were transferred into HEK293 cells or human skeletal muscle cells with Lipo2000,and the exon splicing changes of the minigene were detected by fluorescent labeling semi-quantitative RT-PCR.ResultsAfter the DMD minigene was successfully constructed,a series of deletion mutations were first made in exon 51.No mutation was observed in the splicing of exon 51.It is inferred that there is no strong splicing regulatory element on this exon.The results confirmed that DMD exon 51 is not a cassette alternative splicing exon.Then we designed and screened a large number of ASOs targeting exon 51 and the intron sequences on both sides.It was found that several ASOs have obvious inhibitory effect on the splicing of exon 51,and the effect is better than eteplirsen.The mechanism is worthy of further study.We optimized the further screening of three primary screening ASOs near their targets and found that;one named as 022012 was found to be most inhibitory during an optimization screen.To further improve the efficacy of ASOs,we tried to attach a general inhibitory motif to one side or both side of an ASO.By testing several known splicing silencer motifs,we found that the TAGGGTTAGGG sequence is the strongest in inhibiting splicing,so it can be used as a potential tag for designing second-generation ASO,When exploring second-generation ASOs,an ASO named SG0236 emerged as the strongest splicing inhibitor;it can promote DMD exon 51 skipping at very low concentrations.Dose response analysis showed a significant effect at 1 nM,which reduced the inclusion rate of exon 51 to less than 50%,while 10 nM reduced the inclusion rate to 1%;after transfecting some morpholino second-generation ASOs into human skeletal muscle cells,it was found that the effect of SG0236 was still better than other second-generation ASOs.The above results laid the foundation for future in vivo experiments.ConclusionAntisense therapeutics that induces DMD exon 51 skipping can be applied to about 13%of DMD patients.We designed and screened numerous ASOs in HEK293 cells and found several ASOs that potently promote exon 51 skipping.We further designed second-generation ASOs,and identified SG0236 that induces exon 51 skipping at very low concentrations.Compared to eteplirsen,SG0236 exhibited a superior effect on promoting DMD exon 51 skipping in both HEK293 and human skeletal muscle cells.This ASO drug candidate has the potential to treat DMD,and further studies in a mouse model are in progress.