Delivery of CRISPR/Cas9 using AAV-PHP.B in the inner ear leads to allele-specific inactivation of the mutated Tmc1 allele and protects auditory function in Beethoven mice

Hearing Loss (HL) is the most prevalent sensorineural disorder, affecting 360 million people worldwide. As genetic causes lead to 50% of pre-lingual deafness, gene therapy is considered as a potential therapeutic strategy. Mutations in the TMC1 gene are linked to both autosomal dominant (DFNA36) and recessive (DFNB7/11) forms of non-syndromic HL. DFNA36 is caused by a TMC1 point mutation (p.M418K) and characterized by a postlingual form of HL, with an onset in the mid-teen years, which leaves a window for therapeutic intervention. The natural disease progression is replicated in the heterozygous Tmc1Bth/+ Beethoven mice, a useful model for the testing of gene therapies. Here, we develop an AAV-based allele-specific gene targeting approach using the CRISPR/Cas9 technology to selectively disrupt and knock-out the Bth-Tmc1 allele. To deliver this system to the neonatal inner ear, we used the AAV-PHP.B vector, which showed effective targeting of both inner and outer hair cells throughout the mouse cochlea (>90% efficacy). This transduction efficacy allowed us to consider a double vector approach, with one vector used to express the spCas9 nuclease whereas a second vector was co-injected to express the gRNA selectively targeting the Bth-Tmc1 allele. The results obtained in the Tmc1Bth/+ mice showed long-lasting improvement of the auditory function, with a significant protection of the auditory brainstem response and the distortion product otoacoustic emissions. Furthermore, the treated mice showed a significantly increased startle reflex response. Altogether, these results support the use of AAV-based gene therapy to target genetic defects in hair cells leading to HL.