Prevention of acquired sensorineural hearing loss in mice by in vivo Htra2 gene editing

Abstract

Background Aging, 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. Results The 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. Conclusions These 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.

HtrA2/Omi mitigates NAFLD in high-fat-fed mice by ameliorating mitochondrial dysfunction and restoring autophagic flux

“…HtrA2/Omi has been reported to be involved in CCl 4 -induced liver fibrotic and age-related autophagic deficiency [ 3 , 23 ]. In order to investigate whether alteration of HtrA2/Omi existed in hepatocyte in the progression of NAFLD, we validated the expression of HtrA2/Omi.…”

Progress in Gene Editing Tools and Their Potential for Correcting Mutations Underlying Hearing and Vision Loss

“…The CRISPR/Cas9-mediated inhibition of Htra2 significantly decreased neomycin-induced apoptosis, promoted hair cell survival, and improved hearing function in drug-treated mice. The best results were obtained using the Anc80L65-SaCas9-Htra2 gRNA system, probably thanks to a more efficient transduction rate of the hair cells using single AAVs (Gu et al, 2021). The injected ears showed sustained (up to 8 weeks) and significant improvement in auditory brainstem response threshold, up to 50 dB at 8 kHz for the SaCas9 system (Gu et al, 2021).…”

“…A recent study harnessed CRISPR/Cas9 editing agents to tackle this apoptotic pathway by targeting the Htra2 gene, which encodes a proapoptotic mitochondrial serine protease. Because Htra2 was identified among a set of highly overexpressed genes after drug-induced inner ear damage, two CRISPR/Cas9 systems, SpCas9 and SaCas9, were used to disrupt its expression in the inner ear (Gu et al, 2021). The CRISPR/Cas9-mediated inhibition of Htra2 significantly decreased neomycin-induced apoptosis, promoted hair cell survival, and improved hearing function in drug-treated mice.…”

“…The best results were obtained using the Anc80L65-SaCas9-Htra2 gRNA system, probably thanks to a more efficient transduction rate of the hair cells using single AAVs (Gu et al, 2021). The injected ears showed sustained (up to 8 weeks) and significant improvement in auditory brainstem response threshold, up to 50 dB at 8 kHz for the SaCas9 system (Gu et al, 2021). The protective effect did not cover all sound frequencies, as no beneficial outcomes were observed in the basal turn of the cochlea.…”

Strategies for High-Efficiency Mutation Using the CRISPR/Cas System

“…Compared to fluorescent proteins, the antibiotic-based method offers an alternative strategy that does not require expensive equipment but needs more time (Moriarity et al, 2014;Liesche et al, 2016). Although numerous antibiotic resistance genes have been applied in various fields, such as hygromycin (Moriarity et al, 2014), neomycin (Gu et al, 2021), zeocin (Kobayashi et al, 2019), gentamicin (Mulsant et al, 1988) and puromycin (Pandey et al, 2021), marker-free strategies are the preferred method, ameliorating public concerns for the biosafety of antibiotic resistance genes. Another non-fluorescence activated cell sorting-based enrichment method is antigen gene H-2K k , which has a high enrichment efficiency with magnetic bead separation (Wei et al, 2001).…”