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Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has ushered in a new era of gene therapy. In this study, we aimed to demonstrate precise CRISPR/Cas9-mediated genome editing of the splicing mutation c.919-2A > G in intron 7 of the SLC26A4 gene, which is the second most common causative gene of congenital hearing loss. We designed candidate single-guide RNAs (sgRNAs) aimed to direct the targeting of Staphylococcus aureus Cas9 to either exon 7 or exon 8 of SLC26A4. Several of the designed sgRNAs showed targeting activity, with average indel efficiencies ranging from approximately 14% to 25%. The usage of dual sgRNAs delivered both into Neuro2a cells and primary mouse embryonic fibroblasts resulted in the successful removal of large genomic fragments within the target locus. We subsequently evaluated genome editing in the presence of artificial donor templates to induce precise target modification via homology-directed repair. Using this approach, two different donor plasmids successfully introduced silent mutations within the c.919-2A region of Slc26a4 without evident off-target activities. Overall, these results indicate that CRISPR/Cas9-mediated correction of mutations in the Slc26a4 gene is a feasible therapeutic option for restoration of hearing loss.

The-state-of-art CRISPR/Cas9 is one of the most powerful among the approaches being developed to rescue fundamental causes of gene-based inheritable diseases. Several strategies for delivering such genome editing materials have been developed, but the safety, efficacy over time, cost of production, and gene size limitations are still under debate and must be addressed to further improve applications. In this study, we evaluated branched forms of the polyethylenimine (PEI) - branched PEI 25 kDa (BPEI-25K) - and found that it could efficiently deliver CRISPR/Cas9 plasmids. Plasmid DNA expressing both guide RNA and Cas9 to target the Slc26a4 locus was successfully delivered into Neuro2a cells and meditated genome editing within the targeted locus. Our results demonstrated that BPEI-25K is a promising non-viral vector to deliver the CRISPR/Cas9 system in vitro to mediate targeted gene therapy, and these findings contribute to an understanding of CRISPR/Cas9 delivery that may enable development of successful in vivo techniques.

For successful wound treatment, therapeutics must be delivered directly to the wound. Various issues restrict the delivery of antibiotics to wounds, including the barrier mannered by necrotic tissue and biofilms, which create an extracellular polymeric layer that impedes the efficient administration of therapeutics. For achieving break of the necrotic tissue barrier and biofilm, in addition, improving antibiotics penetration through a painless administration, we fabricated porous polymer coatings on microneedles (MNs) which had the ability of automatic “release” therapeutics in response to wound pH conditions. In the pores of the porous polymer film, the model drug was packed using aqueous gelatin porogen, and the porous layer was coated with a Eudragit S100 film to cap the pores to prevent drug leakage and provide a wound pH-responsive drug release. By combining the advantages of porous and pH-responsive polymer coatings, the coated MNs exhibited remarkably enhanced therapeutic results. This formulation showed both in vivo (in rats) and in vitro (in phosphate-buffered saline and in porcine skin) wound pH-sensitive drug release with rapid responsiveness. At healthy skin pH (pH 4.5), an insignificant release was noticed for MNs in the test media. However, drug release considerably increased when MNs were exposed to wound pH conditions (pH 7.5). The present study provides proof-of-concept evidence that developed MNs have the potential of enhanced treatment protocol for wound infections with the flexibility of coating materials and antimicrobials and offers significant scope for further variations and advancement.

Hereditary hearing loss (HHL) is a common genetically heterogeneous disorder, which follows Mendelian inheritance in humans. Because of this heterogeneity, the identification of the causative gene of HHL by linkage analysis or Sanger sequencing have shown economic and temporal limitations. With recent advances in next-generation sequencing (NGS) techniques, rapid identification of a causative gene via massively parallel sequencing is now possible. We recruited a Korean family with three generations exhibiting autosomal dominant inheritance of hearing loss (HL), and the clinical information about this family revealed that there are no other symptoms accompanied with HL. To identify a causative mutation of HL in this family, we performed whole-exome sequencing of 4 family members, 3 affected and an unaffected. As the result, A novel splicing mutation, c.763+1G>T, in the solute carrier family 17, member 8 (SLC17A8) gene was identified in the patients, and the genotypes of the mutation were co-segregated with the phenotype of HL. Additionally, this mutation was not detected in 100 Koreans with normal hearing. Via NGS, we detected a novel splicing mutation that might influence the hearing ability within the patients with autosomal dominant non-syndromic HL. Our data suggests that this technique is a powerful tool to discover causative genetic factors of HL and facilitate diagnoses of the primary cause of HHL.

Cisplatin (cis-diaminedichloridoplatinum(II), cis-[PtCl2(NH3)2]) is an effective chemotherapeutic agent in the treatment of several types of malignant solid tumors but its clinical use is associated with ototoxicity. Several studies have investigated the effect of antioxidants on cisplatin-induced ototoxicity in mice. The triazole KPR-A020 has been shown to play a protective role against mitochondrial dysfunction by reducing the production of mitochondrial reactive oxygen species (ROS). The effect of KPR-A020 on cisplatin-induced ototoxicity was examined using cultures of cochlear explants. Healthy mice were randomly divided into 4 groups: control, treated with cisplatin alone (CP), treated with cisplatin and KPR-A020 (CP + KPR-A020), and treated with KPR-A020 alone (KPR-A020). The cochlear explants were harvested for histological and immunohistochemical examinations. Biochemical analyses of the explants revealed that pre-treatment with KPR-A020 prevented an increase in mitochondrial ROS levels. Moreover, the CP + KPR-A020 group showed better hair cell survival than the CP group. Immunohistochemical examinations of cochlear explants stained with anti-caspase-3 revealed greater immunopositivity in the CP group. The CP + KPR-A020 group showed significantly less immunopositivity than the CP group (P < 0.05). Thus, it appears that KPR-A020 protects hair cells in the organ of Corti from cisplatin-induced toxicity by decreasing the production of mitochondrial ROS. The results of this study suggest that KPR-A020 can be used as an antioxidant and antiapoptotic agent to prevent hearing loss caused by cisplatin induced-oxidative stress.

Hereditary spastic paraplegia (HSP) is a heterogeneous group of genetic disorders characterized by lower-limb spastic paralysis. We report on a family with three generations of autosomal dominant inheritance of HSP caused by a novel heterozygous splice-site mutation (c.303 + 2 T > C) in REEP1 that was confirmed by RFLP analysis. Carriers of the mutation, including one asymptomatic individual, showed a mild HSP phenotype with a wide range of intrafamilial variation. All symptomatic carriers had ankle contractures in addition to other classical clinical symptoms of HSP. Clinicians should suspect REEP1-related HSP in patients who show ankle contractures with other symptoms of HSP and should consider that these patients have asymptomatic carriers within their family.

Background Myosin is a key protein involved in regulating the shape and motility of cells. The MYH9 and MYH14 genes, which encode non-muscle myosin heavy chain IIA (NMMHC II-A) and IIC (NMMHC II-C), respectively, are expressed in the inner ear. These myosin genes are known to be associated with autosomal dominant non-syndromic hearing loss (ADNSHL); however, genetic studies in patients with ADNSHL in Korea have rarely been reported. Methods We analyzed the MYH9 and MYH14 genes in 75 Korean patients with ADNSHL. Results We identified 4 possible pathogenic variants: a novel variant p.F1303L and 2 previously reported variants (p.R1730C and p.R1785C) in the MYH9 gene, and a novel variant p.A1868T in the MYH14 gene. All the variants were located in the myosin tail domain, which is essential for the interaction of myosin with actin. These variants were predicted to be possibly pathogenic by functional prediction tools and were absent in 100 unrelated normal controls. Conclusion These results suggest that all the variants identified in this study have a strong potential to affect the structural stability and/or function of non-muscle myosin in the inner ear, which might lead to ADNSHL. This study establishes the link between the genotype and development of ADNSHL and contributes to the establishment of Korean database for hereditary hearing loss.

Amikacin is a semi-synthetic aminoglycoside widely used to treat infections caused by gentamicin-resistant gram-negative organisms and nontuberculous mycobacteria. However, the use of this agent often results in ototoxicity due to the overproduction of reactive oxygen species (ROS). Galangin, a natural flavonoid, has been shown to play a protective role against mitochondrial dysfunction by reducing mitochondrial ROS production. In this study, the effect of galangin on amikacin-induced ototoxicity was examined using cultures of cochlear explants. Immunofluorescent staining showed that treatment of inner hair cells (IHCs) and outer hair cells (OHCs) with galangin significantly decreased damage induced by amikacin. Moreover, pretreatment with galangin resulted in decreased amikacin-provoked increase in ROS production in both types of hair cells by MitoSOX-red staining. Attenuation of apoptotic cell death was assessed immunohistochemically using active caspase-3 antibody and with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, compared to explants exposed to amikacin alone (P

Pannexins (Panx) are a family of proteins that share sequences with the invertebrate gap junction proteins, innexins, and have a similar structure to that of the vertebrate gap junction proteins, connexins. To date, the Panx family consists of 3 members, but their genetic sequences have only been completely determined in a few vertebrate species. Moreover, expression of the Panx family has been reported in several rodent tissues: Panx1 is ubiquitously expressed in mammals, whereas Panx2 and Panx3 expressions are more restricted. Although members of the Panx family have been detected in mammals, their genetic sequences in avian species have not yet been fully elucidated. Here, we obtained the full-length mRNA sequences of chicken PANX genes and evaluated the homology of the amino acids from these sequences with those of other species. Furthermore, PANX gene expression in several chicken tissues was investigated based on mRNA levels. PANX1 was detected in the brain, cochlea, chondrocytes, eye, lung, skin, and intestine, and PANX2 was expressed in the brain, eye, and intestine. PANX3 was observed in the cochlea, chondrocytes, and bone. In addition, expression of PANX3 was higher than PANX1 in the cochlea. Immunofluorescent staining revealed PANX1 in hair cells, as well as the supporting cells, ganglion neurons, and the tegmentum vasculosum in chickens, whereas PANX3 was only detected in the bone surrounding the cochlea. Overall, the results of this study provide the first identification and characterization of the sequence and expression of the PANX family in an avian species, and fundamental data for confirmation of Panx function.

Cisplatin is an effective antineoplastic drug that is widely used to treat various cancers; however, it causes side effects such as ototoxicity via the induction of apoptosis of hair cells in the cochlea. Alpha-lipoic acid (ALA) has been reported to exert a protective effect against both antibiotic-induced and cisplatin-induced hearing loss. Therefore, this study was conducted to (1) elucidate the mechanism of the protective effects of ALA against cisplatin-induced ototoxicity using in vitro and ex vivo culture systems of HEI-OC1 auditory cells and rat cochlear explants and (2) to gain additional insight into the apoptotic mechanism of cisplatin-induced ototoxicity. ALA pretreatment significantly reduced apoptotic cell death of the inner and outer hair cells in cisplatin-treated organ of Corti explants and attenuated ototoxicity via marked inhibition of the increase in the expression of IL-1β and IL-6, the phosphorylation of ERK and p38, the degradation of IκBα, the increase in intracellular levels of ROS, and the activation of caspase-3 in cisplatin-treated HEI-OC1 cells. This study represents the first histological evaluation of the organ of Corti following treatment with ALA, and these results indicate that the protective effects of ALA against cisplatin-induced ototoxicity are mediated via the regulation of MAPKs and proinflammatory cytokines.

The TMPRSS3 gene (DFNB8/10), which encodes a transmembrane serine protease, is a common hearing loss gene in several populations. Accurate functions of TMPRSS3 in the hearing pathway are still unknown, but TMPRSS3 has been reported to play a crucial role in inner ear development or maintenance. To date, 16 pathogenic mutations have been identified in many countries, but no mutational studies of the TMPRSS3 gene have been conducted in the Korean hearing loss population. In this study, we performed genetic analysis of TMPRSS3 in 40 unrelated Korean patients with autosomal recessive hearing loss to identify the aspect and frequency of TMPRSS3 gene mutations in the Korean population. A total of 22 variations were detected, including a novel variant (p.V291L) and a previously reported pathogenic mutation (p.A306T). The p.A306T mutation which has been detected in only compound heterozygous state in previous studies was identified in homozygous state for the first time in this study. Moreover, the clinical evaluation identified bilateral dilated vestibules in the patient with p.A306T mutation, and it suggested that p.A306T mutation of the TMPRSS3 gene might be associated with vestibular anomalies. In conclusion, this study investigated that only 2.5% of patients with autosomal recessive hearing loss were related to TMPRSS3 mutations suggesting low prevalence of TMPRSS3 gene in Korean hearing loss population. Also, it will provide the information of genotype-phenotype correlation to understand definite role of TMPRSS3 in the auditory system.

Auditory neuropathy spectrum disorder (ANSD) is caused by dys-synchronous auditory neural response as a result of impairment of the functions of the auditory nerve or inner hair cells, or synapses between inner hair cells and the auditory nerve. To identify a causative gene causing ANSD in the Korean population, we conducted gene screening of the OTOF, DIAPH3, and PJVK genes in 19 unrelated Korean patients with ANSD. A novel nonsense mutation (p.Y1064X) and a known pathogenic mutation (p.R1939Q) of the OTOF gene were identified in a patient as compound heterozygote. Pedigree analysis for these mutations showed co-segregation of mutation genotype and the disease in the family, and it supported that the p.Y1064X might be a novel genetic cause of autosomal recessive ANSD. A novel missense variant p.K1017R (c.3050A>G) in the DIAPH3 gene was also identified in the heterozygous state. In contrast, no mutation was detected in the PJVK gene. These results indicate that no major causative gene has been reported to date in the Korean population and that pathogenic mutations in undiscovered candidate genes may have an effect on ANSD.

Objectives SLC26A4 (PDS) mutations are common cause of congenital hearing loss in East Asia. Hearing loss caused by PDS mutations tends to have delayed presentation; thus universal newborn hearing screening (UNHS) can be less effective in these patients. We examined the efficiency of newborn hearing screening test in patients with bi-allelic PDS mutations. Methods Forty-three patients with sensorineural hearing loss were recruited. Patients had an enlarged vestibular aqueduct and biallelic PDS mutations. Among them, newborn hearing screening test had been performed on 14. The remaining 29 patients did not undergo newborn hearing screening test. Another 15 patients without a PDS mutation but who had sensorineural hearing loss were also recruited as a comparison group. We reviewed the hearing loss history of the children using medical records and parent interviews. Results Among 14 patients with PDS mutation, four (28.6%) passed newborn hearing screening test in both ears and six (42.9%) passed in one ear. In contrast, only 2 of 15 (13.3%) children without a PDS mutation passed newborn hearing screening test bilaterally. The age at confirmation of bilateral hearing loss in bilateral “pass” patients with PDS mutation was 31.5 ± 17.9 months, which was significantly delayed compared to the age for bilateral “refer” children (1.75 ± 0.96 months) (p Conclusion The UNHS is not an accurate tool for predicting long-term hearing loss in patients with PDS mutations. We recommend that genetic screening be combined with UNHS, particularly in communities with a high prevalence of PDS mutations, to better identify children in need of early habilitation.

CHARGE syndrome is an autosomal dominant congenital disorder known to be caused by the haploinsufficiency of the CHD7 gene. Heterozygous mutations in the CHD7 gene have been identified in approximately 60–70% of patients clinically diagnosed with CHARGE syndrome. Although there have been many reports on the mutational spectrum of the CHD7 gene in patients with CHARGE syndrome worldwide, little is known about this syndrome in the Korean population. In this study, three Korean patients with CHARGE syndrome including one patient with Patau syndrome were evaluated for genetic analysis of the CHD7 gene using direct sequencing of all 38 exons and the flanking intronic regions. One nonsense and two novel missense mutations were identified in the CHD7 gene. Clinical symptoms caused by the missense mutations were much milder compared to the nonsense mutation, confirming the previously determined genotype–phenotype correlation in CHARGE syndrome. Our study demonstrates the importance of mutational screening of CHD7 in patients who have been diagnosed with other syndromes but display clinical features of CHARGE syndrome.

A number of genes responsible for hearing loss are related to ion recycling and homeostasis in the inner ear. Connexins (Cx26 encoded by GJB2, Cx31 encoded by GJB3 and Cx30 encoded by GJB6) are core components of gap junctions in the inner ear. Gap junctions are intercellular communication channels and important factors that are associated with hearing loss. To date, a molecular genetics study of GJB3 and GJB6 as a causative gene for hearing loss has not been performed in Korea. This study was therefore performed to elucidate the genetic characteristics of Korean patients with nonsyndromic sensorineural hearing loss and to determine the pathological mechanism of hearing loss by analyzing the intercellular communication function of Cx30 and Cx31 variants. Sequencing analysis of the GJB3 and GJB6 genes in our population revealed a total of nine variants, including four novel variants in the two genes. Three of the novel variants (Cx31-p.V27M, Cx31-p.V43M and Cx-30-p.I248V) and two previously reported variants (Cx31-p.V84I and Cx30-p.A40V) were selected for functional studies using a pathogenicity prediction program and assessed for whether the mutations were located in a conserved region of the protein. The results of biochemical and ionic coupling tests showed that both the Cx31-p.V27M and Cx31-p.V84I variants did not function normally when each was expressed as a heterozygote with the wild-type Cx31. This study demonstrated that two variants of Cx31 were pathogenic mutations with deleterious effect. This information will be valuable in understanding the pathogenic role of GJB3 and GJB6 mutations associated with hearing loss.

Mutations of the TECTA gene, which encodes alpha-tectorin, are associated with both dominant (DFNA8/A12) and recessive (DFNB 21) modes of inherited nonsyndromic sensorineural hearing loss, respectively. Although clinical data and genetic analysis for TECTA gene have been reported from different groups, there is no report that compound heterozygous mutations in the TECTA gene result in nonsyndromic sensorineural hearing loss. Here, we identified a missense mutation (p.C1691F) and a splicing mutation (c.6162 + 3insT), one in each TECTA allele, in the patient with hearing loss. Also, we demonstrated that the splicing mutation results in the abnormal skipping of an exon, which leads to a truncated protein as determined by exon-trapping analysis. To the best of our knowledge, this is the first report of an in vitro functional study of splice site mutations in the TECTA gene.

Hearing loss is a common communication disorder caused by various environmental and genetic factors. Hereditary hearing loss is very heterogeneous, and most of such cases involve sensorineural defects in the auditory pathway. There are currently 57 known autosomal dominant non-syndromic hearing loss (DFNA) loci, and the causative genes have been identified at 22 of these loci. In the present study, we performed a genome-wide linkage analysis in a Korean family segregating autosomal dominant hearing loss. We observed linkage on chromosome 1p34, and at this locus, we detected a novel mutation consisting of an 18 nucleotide deletion in exon 4 of the KCNQ4 gene, which encodes a voltage-gated potassium channel. We carried out a functional in vitro study to analyze the effects of this mutation (c.664_681del) along with two previously described KCNQ4 mutations, p.W276S and p.G285C. Although the c.664_681del mutation is located in the intercellular loop and the two previously described mutations, p.W276S and p.G285C, are located in the pore region, all mutants inhibit normal channel function by a dominant negative effect. Our analysis indicates that the intercellular loop is as significant as the pore region as a potential site of pathogenic effects on KCNQ4 channel function.

Autosomal dominant mutations in the transcription factor POU4F3 gene are associated with non-syndromic hearing loss in humans; however, there have been few reports of mutations in this gene worldwide. We performed a mutation analysis of the POU4F3 gene in 42 unrelated Koreans with autosomal dominant non-syndromic hearing loss, identifying a novel 14-bp deletion mutation in exon 2 (c.662del14) in one patient. Audiometric examination revealed severe bilateral sensorineural hearing loss in this patient. The novel mutation led to a truncated protein that lacked both functional POU domains. We further investigated the functional distinction between wild-type and mutant POU4F3 proteins using in vitro assays. The wild-type protein was completely localized in the nucleus, while the truncation of protein seriously affected its nuclear localization. In addition, the mutant failed to activate reporter gene expression. This is the first report of a POU4F3 mutation in Asia, and moreover our data suggest that further investigation will need to delineate ethnicity-specific genetic background for autosomal dominant non-syndromic hearing loss within Asian populations.

Summary Objectives Mutations in the GJB2, GJB6 and SLC26A4 genes are a frequent cause of hearing loss in a number of populations. However, little is known about the genetic causes of hearing loss in the Korean population. Methods We sequenced the GJB2 and GJB6 genes to examine the role of mutations in these genes in 22 hearing loss patients. We also sequenced the SLC26A4 gene in seven patients with inner ear malformations, including enlarged vestibular aqueduct (EVA) revealed by computer tomography. Results Coding sequence mutations in GJB2 were identified in 13.6% of the patients screened. Two different mutations, 235delC and T86R were found in three unrelated patients. The 235delC was the most prevalent mutation with an allele frequency of 6.9% in our patient group. No mutations, including 342-kb deletion, were found in GJB6 gene. Three different variants of SLC26A4 were identified in the EVA patients, including one novel mutation. Four EVA patients carried two mutant alleles of SLC26A4, and at least one allele in all patients was the H723R mutation, which accounted for 75% of all mutant alleles. Conclusions Our results suggest that GJB2 and SLC26A4 mutations together make up a major cause of congenital hearing loss in the Korean population. Further studies may be able to identify other common variants that account for a significant fraction of hearing loss in the Korean population.

Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder that is characterized by branchial cysts or fistulae, external ear malformations and/or preauricular pits, hearing loss and renal anomalies. Recent advances in molecular genetics have shown a human homologue of the Drosophila 'eyes absent' gene (EYA1) on chromosome band 8q13.3 to be the most common cause of BOR syndrome. Several mutations have been identified in the EYA1 gene in patients with BOR syndrome worldwide. Here, we report a second Korean family with BOR syndrome with a novel nonsense EYA1 mutation.

Individual differences in perception are ubiquitous within the chemical senses: taste, smell, and chemical somesthesis [1–4]. A hypothesis of this fact states that polymorphisms in human sensory receptor genes could alter perception by coding for functionally distinct receptor types [1, 5–8]. We have previously reported evidence that sequence variants in a presumptive bitter receptor gene (hTAS2R38) correlate with differences in bitterness recognition of phenylthiocarbamide (PTC) [9–11]. Here, we map individual psychogenomic pathways for bitter taste by testing people with a variety of psychophysical tasks and linking their individual perceptions of the compounds PTC and propylthiouracil (PROP) to the in vitro responses of their TAS2R38 receptor variants. Functional expression studies demonstrate that five different haplotypes from the hTAS2R38 gene code for operatively distinct receptors. The responses of the three haplotypes we also tested in vivo correlate strongly with individuals' psychophysical bitter sensitivities to a family of compounds. These data provide a direct molecular link between heritable variability in bitter taste perception to functional variations of a single G protein coupled receptor that responds to compounds such as PTC and PROP that contain the N-C=S moiety. The molecular mechanisms of perceived bitterness variability have therapeutic implications, such as helping patients to consume beneficial bitter-tasting compounds—for example, pharmaceuticals and selected phytochemicals.

The ability to taste phenylthiocarbamide (PTC) is a classic phenotype that has long been known to vary in human populations. This phenotype is of genetic, epidemiologic, and evolutionary interest because the ability to taste PTC is correlated with the ability to taste other bitter substances, many of which are toxic. Thus, variation in PTC perception may reflect variation in dietary preferences throughout human history and could correlate with susceptibility to diet-related diseases in modern populations. To test R. A. Fisher’s long-standing hypothesis that variability in PTC perception has been maintained by balancing natural selection, we examined patterns of DNA sequence variation in the recently identified PTC gene, which accounts for up to 85% of phenotypic variance in the trait. We analyzed the entire coding region of PTC (1,002 bp) in a sample of 330 chromosomes collected from African (n=62), Asian (n=138), European (n=110), and North American (n=20) populations by use of new statistical tests for natural selection that take into account the potentially confounding effects of human population growth. Two intermediate-frequency haplotypes corresponding to “taster” and “nontaster” phenotypes were found. These haplotypes had similar frequencies across Africa, Asia, and Europe. Genetic differentiation between the continental population samples was low (FST=0.056) in comparison with estimates based on other genes. In addition, Tajima’s D and Fu and Li’s D and F statistics demonstrated a significant deviation from neutrality because of an excess of intermediate-frequency variants when human population growth was taken into account (P

Gene therapy for treating genetic hearing loss has made astonishing progress with biotechnical advance, but it still remains the difficulty to treat the disorder ultimately. A diversity of viral vectors are used in gene therapy as vehicle for delivery of therapeutic gene. Moreover, the successful outcome of the gene therapy depends on timing and delivery route as well as titer of virus. Here, we demonstrate that gene therapy by in utero is a promising tool for treating genetic hearing loss. We used adeno-associated virus serotype 1 (AAV1) to express therapeutic gene in mice. To evaluate the safety and efficiency of virus, we transferred an AAV1-GFP into the otocyst at embryonic day 12.5, and the contralateral ear was used as a control. After the gene transfer, a strong expression of transgene was observed without ototoxicity within cochlea and the hearing ability was unaffected by AAV. These results were identified by several histological interpretations and auditory brainstem responses, respectively. We also examined a successful expression of an AAV1-MsrB3-GFP within hair cells in the same way. Together, these results indicate that gene therapy by in utero is potential strategy to treat genetic hearing loss by monogenic mutations. It may apply to treat the deficiency of the MsrB3 gene which results in autosomal recessive non-syndromic hearing loss, DFNB74, in humans.