RESUMEN
Recessive PJVK mutations that cause a deficiency of pejvakin, a protein expressed in both sensory hair cells and first-order neurons of the inner ear, are an important cause of hereditary hearing impairment. Patients with PJVK mutations garner limited benefits from cochlear implantation; thus, alternative biological therapies may be required to address this clinical difficulty. The synthetic adeno-associated viral vector Anc80L65, with its wide tropism and high transduction efficiency in various inner ear cells, may provide a solution. We delivered the PJVK transgene to the inner ear of Pjvk mutant mice using the synthetic Anc80L65 vector. We observed robust exogenous pejvakin expression in the hair cells and neurons of the cochlea and vestibular organs. Subsequent morphologic and audiologic studies demonstrated significant restoration of spiral ganglion neuron density and hair cells in the cochlea, along with partial recovery of sensorineural hearing impairment. In addition, we observed a recovery of vestibular ganglion neurons and balance function to WT levels. Our study demonstrates the utility of Anc80L65-mediated gene delivery in Pjvk mutant mice and provides insights into the potential of gene therapy for PJVK-related inner ear deficits.
Asunto(s)
Terapia Genética , Pérdida Auditiva Sensorineural , Ratones , Animales , Células Ciliadas Auditivas/metabolismo , Pérdida Auditiva Sensorineural/genética , Pérdida Auditiva Sensorineural/terapia , Pérdida Auditiva Sensorineural/metabolismo , Cóclea/metabolismo , Fenotipo , Proteínas/genéticaRESUMEN
Recessive variants of the SLC26A4 gene are an important cause of hereditary hearing impairment. Several transgenic mice with different Slc26a4 variants have been generated. However, none have recapitulated the auditory phenotypes in humans. Of the SLC26A4 variants identified thus far, the p.T721M variant is of interest, as it appears to confer a more severe pathogenicity than most of the other missense variants, but milder pathogenicity than non-sense and frameshift variants. Using a genotype-driven approach, we established a knock-in mouse model homozygous for p.T721M. To verify the pathogenicity of p.T721M, we generated mice with compound heterozygous variants by intercrossing Slc26a4+/T721M mice with Slc26a4919-2A>G/919-2A>G mice, which segregated the c.919-2A > G variant with abolished Slc26a4 function. We then performed serial audiological assessments, vestibular evaluations, and inner ear morphological studies. Surprisingly, both Slc26a4T721M/T721M and Slc26a4919-2A>G/T721M showed normal audiovestibular functions and inner ear morphology, indicating that p.T721M is non-pathogenic in mice and a single p.T721M allele is sufficient to maintain normal inner ear physiology. The evidence together with previous reports on mouse models with Slc26a4 p.C565Y and p.H723R variants, support our speculation that the absence of audiovestibular phenotypes in these mouse models could be attributed to different protein structures at the C-terminus of human and mouse pendrin.
Asunto(s)
Pérdida Auditiva/genética , Transportadores de Sulfato/química , Transportadores de Sulfato/genética , Animales , Modelos Animales de Enfermedad , Técnicas de Sustitución del Gen , Pérdida Auditiva/metabolismo , Pérdida Auditiva/patología , Homocigoto , Humanos , Masculino , Ratones , Mutación Missense , Fenotipo , Dominios Proteicos , Especificidad de la Especie , Transportadores de Sulfato/metabolismoRESUMEN
Recessive variants of the SLC26A4 gene are globally a common cause of hearing impairment. In the past, cell lines and transgenic mice were widely used to investigate the pathogenicity associated with SLC26A4 variants. However, discrepancies in pathogenicity between humans and cell lines or transgenic mice were documented for some SLC26A4 variants. For instance, the p.C565Y variant, which was reported to be pathogenic in humans, did not exhibit functional pathogenic consequences in cell lines. To address the pathogenicity of p.C565Y, we used a genotype-based approach in which we generated knock-in mice that were heterozygous (Slc26a4+/C565Y), homozygous (Slc26a4C565Y/C565Y), and compound heterozygous (Slc26a4919-2A>G/C565Y) for this variant. Subsequent phenotypic characterization revealed that mice with these genotypes demonstrated normal auditory and vestibular functions, and normal inner-ear morphology and pendrin expression. These findings indicate that the p.C565Y variant is nonpathogenic for mice, and that a single p.C565Y allele is sufficient to maintain normal inner-ear physiology in mice. Our results highlight the differences in pathogenicity associated with certain SLC26A4 variants between transgenic mice and humans, which should be considered when interpreting the results of animal studies for SLC26A4-related deafness.
Asunto(s)
Modelos Animales de Enfermedad , Estudios de Asociación Genética/métodos , Predisposición Genética a la Enfermedad/genética , Pérdida Auditiva Sensorineural/genética , Mutación , Transportadores de Sulfato/genética , Animales , Genotipo , Pérdida Auditiva Sensorineural/metabolismo , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Fenotipo , Transportadores de Sulfato/fisiología , Acueducto Vestibular/metabolismo , Acueducto Vestibular/patologíaRESUMEN
Sensorineural hearing loss is one of the most common sensory disorders worldwide. Recent advances in vector design have paved the way for investigations into the use of adeno-associated vectors (AAVs) for hearing disorder gene therapy. Numerous AAV serotypes have been discovered to be applicable to inner ears, constituting a key advance for gene therapy for sensorineural hearing loss, where transduction efficiency of AAV in inner ear cells is critical for success. One such viral vector, AAV2/Anc80L65, has been shown to yield high expression in the inner ears of mice treated as neonates or adults. Here, to evaluate the feasibility of prenatal gene therapy for deafness, we assessed the transduction efficiency of AAV2/Anc80L65-eGFP (enhanced green fluorescent protein) after microinjection into otocysts in utero. This embryonic delivery method achieved high transduction efficiency in both inner and outer hair cells of the cochlea. Additionally, the transduction efficiency was high in the hair cells of the vestibules and semicircular canals and in spiral ganglion neurons. Our results support the potential of Anc80L65 as a gene therapy vehicle for prenatal inner ear disorders.
RESUMEN
Betel quid is one of the most widely used psychoactive substances, and is consumed by approximately 10% of the world's population. In addition to its carcinogenicity, betel quid has also been reported to affect many organs, including the brain, heart, lungs, gastrointestinal tract, and reproductive organs. As betel quid contains several neurotoxic ingredients, we hypothesize that it also possesses ototoxicity and may lead to sensorineural hearing impairment (SNHI). In this study, we investigated the contribution of betel quid consumption to SNHI in a large clinical cohort, and validated the pathogenetic mechanisms in ex vivo tissue explants. We enrolled a total of 2364 volunteers, and determined their audiologic results based on Z-scores converted from their original frequency-specific hearing thresholds. Using generalized linear regression, we identified a positive correlation between betel quid consumption and the Z-scores across different frequencies. Subsequently, we explored the toxicity of arecoline, the main neuroactive component of betel quid, on tissue explants from murine cochleae. Arecoline reduced cell activity in the explant cultures and induced apoptosis in the hair cells, probably through the effects of oxidative stress. These findings have expanded the potential hazards of betel quid to common neurological disorders, and provide insights into preventive strategies against SNHI caused by neurotoxic substances.
Asunto(s)
Areca/efectos adversos , Arecolina/toxicidad , Pérdida Auditiva Sensorineural/inducido químicamente , Estrés Oxidativo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Apoptosis , Audiología , Índice de Masa Corporal , Femenino , Humanos , Modelos Lineales , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Neurotoxinas/toxicidad , Estudios Prospectivos , Factores de RiesgoRESUMEN
SLC26A4 is the second most frequent gene implicated in congenital hearing loss after GJB2 mutations. Here, we report the generation of induced pluripotent stem cells (iPSCs), from a patient who was carrying a homozygous c.919-2A>G variant in the SLC26A4 gene. This is the most common variant of SLC26A4 gene in the Chinese population and the second most prevalent one in other Asian countries. The established patient-derived iPSC displayed all the features of pluripotent stem cell markers and had the ability to differentiate into all of the three germ layers and possessed a normal karyotype.
Asunto(s)
Bocio Nodular/patología , Pérdida Auditiva Sensorineural/patología , Células Madre Pluripotentes Inducidas/citología , Sitios de Empalme de ARN/genética , Transportadores de Sulfato/genética , Diferenciación Celular , Línea Celular , Reprogramación Celular , Femenino , Bocio Nodular/genética , Pérdida Auditiva Sensorineural/genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Cariotipo , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Polimorfismo de Nucleótido SimpleRESUMEN
Recessive mutations in the GJB2 gene are the most common genetic cause of hearing loss in humans. By using the Sendai-virus delivery system, we generated induced pluripotent stem cells (iPSCs) from the peripheral blood mononuclear cells of a female patient with the p.V37I (c.109Gâ¯>â¯A) mutation, a GJB2 mutation highly prevalent in the Asian population. The resulting iPSCs had a normal karyotype. The iPSCs also showed pluripotency, as confirmed by immunofluorescence staining, and differentiated into the three germ layers in vivo. This cellular model will provide a useful platform for investigating the pathogenic mechanisms of deafness related to GJB2 mutations.