The Generation And Gene Correction Of The IPSC Derived From A Deaf Patient With OTOF Gene Mutations

Deafness is a worldwide public health problem.Sensorineural hearing loss?SNHL?caused by damage or dysfunction of cochlear hair cells and auditory nerves is the main reason of irreversible hearing loss.To some extent,hearing aid and cochlear implant techniques can compensate patients' hearing,but ideal etiological therapies for the SNHL itself is expected.The induced pluripotent stem cells?iPSCs?technology can reprogram multiple somatic cells to have similar differentiation potential as embryonic stem cells.Given certain conditions,iPSC can differentiate into otic progenitor cells,hair cell-like cells and spiral ganglion neurons?SGN?-like cells in vitro,and be transplanted back into the body to replace the original damaged or dysfunctional cells.This technology provides new ideas for the treatment of SNHL.Gene defects are the main pathogenic factors causing severe and profound congenital SNHL.Using gene editing technology,mutated genes can be corrected in the iPSCs that were derived from SNHL patients with identified genetic defects.Therefore,the combination of iPSCs and gene editing technology is likely to make the biotherapy of this type of SNHL possible.Auditory neuropathy spectrum disorder?ANSD or auditory neuropathy?is a special type of SNHL manifested with present otoacoustic emission and/or cochlear microphonics,but absent or abnormal auditory brainstem response.OTOF gene mutations are common causes of congenital ANSD.The correction of OTOF gene mutations using gene editing technology can be an essential approach for stem cell transplantation therapy and gene therapy of ANSD.In addition,the iPSCs derived from patients with ANSD caused by OTOF gene mutations can also be used as an ideal cell model to mimic the processes of ANSD in vitro and to study the pathogenesis mechanism of OTOF gene mutations.Therefore,this article will report a case of ANDS caused by OTOF gene mutations,reprogram the patient-derived fibroblasts into iPSC,and try to correct the OTOF gene in iPSC using CRISPR/Cas9 technology.This thesis is divided into the following three parts.In the first part,one case of ANSD was reported.The molecular diagnosis confirmed that the ANSD was caused by OTOF gene compound heterozygous mutations?c.2122C>T/c.5197G>A,NM₁94248?.The patient underwent cochlear implantation with Nucleus CI24 Contour Advance.The results of neural response telemetry?NRT?during the operation and at the time of initial activation showed that all 22 intracochlear electrode contacts can elicit valid evoked compound action potential?ECAP?waveforms.Compared with the results of 7 typical SNHL patients with similar age and hearing loss severity,the t-NRT values of the patient with ANSD caused by OTOF mutations were similar to those of the typical SNHL but were relatively low at the time of initial activation.It suggests that the auditory nerve of the ANSD cases with OTOF gene mutations is integrity in function,and may be more sensitive at the time of initial activation.In the second part,an iPSC line with OTOF gene mutations was generated from the patient with ANSD.By introducing five key transcription factors:OCT3/4,SOX2,KLF4,L-MYC and LIN28,into the fibroblasts derived from the patient,the fibroblasts were induced to reprogram.The positive alkaline phosphatase staining,expression of pluripotent stem cell-specific marker genes and proteins and the potentials of tridermogenesis in vitro showed that the obtained iPSCs matched the internationally accepted iPSC standards.The iPSC genotype validated by Sanger sequencing,which was OTOF gene compound heterozygous mutated?c.2122C>T/c.5197G>A?.The iPSCs then were induced to differentiate into SGN-like cells in vitro using monolayer culture method.The obtained cells expressed SGN-specific marker genes and proteins,suggesting that the obtained iPSCs have the potential to differentiate into inner ear cells.It provides an ideal cell model to mimic the processes of ANSD,and study the pathogenesis mechanism of OTOF gene mutations in vitro.In the third part,an OTOF gene mutation in the iPSCs was corrected by CRISPR/Cas9-mediated homologous recombination repair.By co-transfecting the CRISPR/Cas9 targeting vector with the single-stranded donor oligonucleotide?ssODN?,drug selecting,single clone picking,Sanger sequencing,and restriction enzyme digestion verification,the OTOF 2\22C>T mutation was homozygously corrected in two strains of iPSC.Eight sites with high off-target probability were detected,and no off-target was detected.The CRISPR/Cas9 technology is feasible for accurately repairing specific OTOF gene mutation in patient-derived iPSCs,which provides a new approach to ANSD gene therapy.In conclusion,the auditory nerve function was intact in the ANSD case caused by OTOF gene mutations.ANSD with OTOF gene mutations is an ideal surgical indication for cochlear implantation.The iPSCs derived from ANSD patients can differentiate into inner ear cells,which could mimic the processes of ANSD in vitro and help to understand the pathogenesis of OTOF gene mutations.The CRISPR/Cas9 technology can accurately correct the OTOF gene mutation in iPSCs,making the etiological therapy of this type of ANSD possible.