Abstract
BackgroundAging, noise, infection, and ototoxic drugs are the major causes of human acquired sensorineural hearing loss, but treatment options are limited. CRISPR/Cas9 technology has tremendous potential to become a new therapeutic modality for acquired non-inherited sensorineural hearing loss. Here, we develop CRISPR/Cas9 strategies to prevent aminoglycoside-induced deafness, a common type of acquired non-inherited sensorineural hearing loss, via disrupting the Htra2 gene in the inner ear which is involved in apoptosis but has not been investigated in cochlear hair cell protection.ResultsThe results indicate that adeno-associated virus (AAV)-mediated delivery of CRISPR/SpCas9 system ameliorates neomycin-induced apoptosis, promotes hair cell survival, and significantly improves hearing function in neomycin-treated mice. The protective effect of the AAV–CRISPR/Cas9 system in vivo is sustained up to 8 weeks after neomycin exposure. For more efficient delivery of the whole CRISPR/Cas9 system, we also explore the AAV–CRISPR/SaCas9 system to prevent neomycin-induced deafness. The in vivo editing efficiency of the SaCas9 system is 1.73% on average. We observed significant improvement in auditory brainstem response thresholds in the injected ears compared with the non-injected ears. At 4 weeks after neomycin exposure, the protective effect of the AAV–CRISPR/SaCas9 system is still obvious, with the improvement in auditory brainstem response threshold up to 50 dB at 8 kHz.ConclusionsThese findings demonstrate the safe and effective prevention of aminoglycoside-induced deafness via Htra2 gene editing and support further development of the CRISPR/Cas9 technology in the treatment of non-inherited hearing loss as well as other non-inherited diseases.
Highlights
Aging, noise, infection, and ototoxic drugs are the major causes of human acquired sensorineural hearing loss, but treatment options are limited
We found that the expression levels of the apoptotic factors Casp3, Casp9, Diablo, and Htra2 were significantly higher in neomycin-treated cochleae compared to saline controls
GFP-positive cells were sorted by fluorescence-activated cell sorting (FACS) at 48 h after transfection with SpCas9–guide RNAs (gRNAs), and the cells were cultured for another 7 days
Summary
Noise, infection, and ototoxic drugs are the major causes of human acquired sensorineural hearing loss, but treatment options are limited. We develop CRISPR/Cas strategies to prevent aminoglycoside-induced deafness, a common type of acquired non-inherited sensorineural hearing loss, via disrupting the Htra gene in the inner ear which is involved in apoptosis but has not been investigated in cochlear hair cell protection. The treatment options are limited and there have been no proven preventative or regenerative interventions for high-risk individuals so far Aminoglycosides, such as gentamicin, sisomicin, streptomycin, kanamycin, and neomycin, are broad-spectrum antibiotics that are effective against aerobic gram-negative bacteria. There have been some attempts to protect auditory and vestibular function from aminoglycosideinduced ototoxicity, including reducing the uptake of aminoglycosides in cochlear hair cells and protecting hair cells by ameliorating intracellular cytotoxicity [18] None of these strategies has been approved for the treatment of drug-induced deafness, and there is still tremendous demand to explore novel protective compounds and therapeutic strategies. To develop new therapeutic strategies for acquired non-inherited SNHL, we selected the mouse model of ototoxic deafness caused by aminoglycoside antibiotics
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