Li Huawei/Shu Yilai team developed CRISPR/Cas9 gene editing to prevent sensorineural hearing loss

More than 1.
3 billion people worldwide suffer from acquired hearing loss, and sensorineural hearing loss (SNHL) is the most common.
The causes of deafness mainly include noise, ototoxic drugs, aging, genetics, infection and other factors [1].
There are no treatable drugs (except for sudden deafness).

Ototoxic deafness caused by aminoglycosides (AGs) is one of the main types of acquired SNHL.

For patients who require multiple courses of intravenous medication (for example, tuberculosis and cystic fibrosis patients), the pathogenicity rate of AGs ototoxicity even exceeds 50%, but clinically effective prevention and treatment strategies are lacking.

The mitochondrial serine protease Omi/HtrA2 encoded by Htra2 gene can combine with X-linked inhibitor of apoptosis protein (XIAP) to promote cell apoptosis.

In the previous study, the research group found that the expression level of Htra2 gene in the cochlea after neomycin injury increased significantly, which may be involved in the pathogenesis of ototoxic deafness [2].

CRISPR/Cas9 gene editing has made some progress in the field of gene therapy for hereditary deafness [3-6], but there is no research on the prevention and treatment of acquired non-hereditary SNHL.

On March 22, 2021, Professor Li Huawei from the Eye, Ear, Nose and Throat Hospital Affiliated to Fudan University and a research team from Shu Yilai published a research paper Prevention of acquired sensorineural hearing loss in mice by in vivo Htra2 gene editing in Genome Biology.

The study designed two CRISPR/Cas9 systems (SpCas9 and SaCas9 systems) targeting the Htra2 gene, which were carried into the inner ear of mice through the new adeno-associated virus (AAV) Anc80L65 to achieve effective editing of the Htra2 gene in vivo and promote the survival of cochlear hair cells.
Significantly improve the hearing function of mice after exposure to aminoglycoside antibiotics.

This is the first internationally successful study based on CRISPR/Cas9 gene editing to prevent and treat acquired non-hereditary sensorineural hearing loss.

The research first constructed the SpCas9 system for the Htra2 gene, designed three specific guide RNAs (gRNAs), and tested the cleavage activity of the three gRNAs in the HEI-OC1 cell line in vitro, and found that Sp-g1, Sp-g2 The in vitro editing efficiency of Sp-g3 and Sp-g3 were 87.
27 ± 0.
02%, 71.
72 ± 1.
27% and 74.
88 ± 0.
77%, respectively.

Afterwards, the protective effect of the Anc80L65–SpCas9 system on cochlear hair cells was verified in the organ of Corti of the cochlea cultured in vitro.

AAV-Anc80L65 can efficiently transduce the inner and outer hair cells of the cochlea, but the packaging capacity of AAV is limited, and it is difficult to package the entire SpCas9 system with a single vector.

The study adopted the split-SpCas9 strategy, split SpCas9, and packaged with double AAV; multi-site cutting to improve knockout efficiency.

In the neomycin injury model of Corti's organ in vitro, the constructed Anc80L65–SpCas9–Htra2-gRNA therapeutic system can knock out the Htra2 gene, promote the survival of cochlear hair cells, and effectively prevent the ototoxicity of aminoglycoside antibiotics.

After the effectiveness of the gene editing system was confirmed in an in vitro neomycin injury model, the efficacy of the system was further explored in vivo.

Since the Anc80L65–SpCas9 system needs to be divided into double AAVs, this may affect the transduction efficiency of the entire Cas9 system in hair cells.

Therefore, this study also constructed the Anc80L65–SaCas9 single AAV vector system to test its preventive effect on neomycin ototoxic deafness.

Studies have found that in vivo neomycin injury models, both SpCas9 and SaCas9 systems can effectively prevent ototoxic deafness caused by aminoglycoside antibiotics.

Mice pretreated with the AAV-SpCas9 system showed more hair cells to survive 2 weeks after neomycin injury, especially in the middle and bottom circle areas of the cochlea.

The gene therapy system also has a significant protective effect on the hearing function of mice.

The mice with the most significant hearing improvement had an ABR threshold of 8 kHz that was 40 dB lower than that of the contralateral ear.

The experiment observed that 8 weeks after neomycin injury, the hearing protection effect of the AAV-SpCas9 system still lasted for a long time.

The AAV–SaCas9 system can also promote the survival of the middle and bottom loop hair cells of the cochlea and improve hearing.

The ABR threshold of the injected ear of 4-week-old mice was significantly lower than that of the uninjected ear at all frequencies tested (4, 8, 16, 24, and 32 kHz), with an average protection threshold of 10-20 dB.

In mice at 6 weeks of age, the protective effect is still very obvious, and the best threshold improvement reaches 50 dB.

The research team carried out the safety assessment of the AAV-CRISPR/SpCas9 and SaCas9 systems at the same time.
During the observation period, no obvious off-target situation was found, and there was no effect on the hearing of wild-type mice, indicating that the treatment system is safe.

In summary, this study proves that the Htra2 gene is a potential therapeutic target for preventing ototoxicity of aminoglycoside antibiotics; it also proves that Htra2 editing with a gene editing system can safely and effectively prevent the ear caused by aminoglycoside antibiotics.
Toxic deafness provides a scientific basis for the intervention of acquired deafness in clinical transformation.

Professor Li Huawei and Researcher Shu Yilai from the Eye, Ear, Nose and Throat Hospital Affiliated to Fudan University are the co-corresponding authors, and postdoctoral fellow Gu Xi, postdoctoral fellow Wang Daqi, and assistant experimentalist Xu Jijiao are co-first authors.

The first unit and communication unit of the research are both the Eye, Ear, Nose and Throat Hospital of Fudan University.

The team of Professor Li Huawei and the research team of Shu Yilai from the Eye, Ear, Nose and Throat Hospital Affiliated to Fudan University have long been committed to gene therapy for genetic deafness based on CRISPR/Cas9 gene editing, clinical transformation, hair cell regeneration, and hearing protection research, and long-term full-time postdoctoral recruitment.

Resume delivery: yilai_shu@fudan.
edu.
cn or hwli@shmu.
edu.
cn for reprinting.
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Link to the original text: https://genomebiology.
biomedcentral.
com/articles/10.
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