Development Of Therapeutic Anusense Oligonucleotides To Treat Hearing Loss Caused By SLC26A4 C.919-2A>G Mutation

Background and Objective:Hearing loss is the most common sensory disorder,which occurs in approximately 1/1000 newborns,with～50%cases attributed to genetic defects.Hearing loss has a profound negative impact on daily life of the patients and increases social costs significantly.To date,hundreds of causative genes for hereditary hearing loss(HHL)have been identified.Among them,recessive mutations of SLC26A4,the second leading cause of HHL worldwide,are the most common cause for both Pendred syndrome(PS,MIM 274600)and enlarged vestibular aqueduct(EVA,OMIM 600791).SLC26A4 is highly expressed in the thyroid,inner ear and kidney,and encodes a 780 aa transmembrane protein called pendrin,functioning as an anion exchanger that transports negatively charged ions such as chloride,iodide,bicarbonate,formate and thiocyanate across the cell membrane.In the inner ear,the main role of pendrin may maintain a stable pH environment of endolymph.A wide spectrum of mutations occurs in the SLC26A4,including 163 pathogenic mutations.Mutation of SCL26A4 may result in reduction or loss of its anion transport activity in the inner ear,leading to failure to acquire normal hearing.The c.919-2A>G mutation is the most prevalent pathogenic mutation in Chinese populations,accounting for～60%of all SLC26A4 disease mutant forms.This mutation destroys the 3’ splice site of intron 7,as a result,the downstream exon 8 is predominantly skipped,causing a frame shift which generates a truncated dysfunctional protein.A variety of strategies can be used to correct the aberrant splicing of pre-mRNA.In recent years,a small molecule drug and an antisense oligonucleotide(ASO)drug,which can correct the aberrant splicing of SMN2,have been approved for spinal muscular atrophy(SMA)treatment.ASO has gradually become a new platform for drug discovery.Therefore,we try to design ASOs that can correct exon 8 skipping of SLC26A4 c.919-2A>G mutant in this study.Experimental Design and Methods:SLC26A4 c.919-2A>G(A-2G)mutant minigene was established.Then,we systematically analyzed the effects of splicing factors,including multiple SR proteins and hnRNP proteins,on exon 8 splicing of the mutant via overexpression in HEK293 cells.Deletion and mutation were used to identify key splicing regulatory elements(SREs).And the splicing factor which binds to a specific SRE was verified by RNA pull down,overexpression,knockdown,and the MS2-CP system.Meanwhile,ASO walk was used to screen the most effective ASO that corrects exon 8 skipping in the A-2G minigene system.Then,the lead ASO was verified in the peripheral blood mononuclear cells(PBMCs)of homozygous c.919-2A>G patient and several tissues of a humanized Slc26a4 c.919-2A>G mouse model.Results:We constructed an A-2G mutant minigene.Then we uncovered multiple proteins that modulate exon 8 inclusion of the mutant.Among them,hnRNP A1 inhibited exon 8 splicing through binding to an intron splicing silencer(ISS)in intron 8.The lead ASO was obtained through ASO walk in HEK293 cells.Cholesterol-conjugated ASO significantly promoted exon 8 inclusion in PBMCs derived from a homozygous patient.In humanized mouse model,the lead ASO significantly improved exon 8 inclusion in the liver.Conclusion:We found that multiple splicing factors modulate exon 8 splicing and one of them is hnRNP A1 that represses exon 8 inclusion through binding to an ISS.We also identified an ASO candidate that has therapeutic potential to treat hearing loss caused by the c.919-2A>G mutation in SLC26A4.