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Successful Gene-Editing to Reverse Deafness Scores Another Win for Gene Therapy
By Sahana Shankar, Ph.D. Candidate
In a collaborative study between Boston’s Children Hospital, and the Broad Institute, researchers successfully use base editing to restore hearing in deaf mice.
Hereditary hearing loss can be caused by mutations in many genes and is a matter of concern in the US, where up to 10% of people have hearing loss. It is a chronic condition with no current therapy. Management relies on sound amplification by hearing aids. A possible line of treatment for genetic diseases is gene therapy. This could be gene replacement or gene silencing. However, in the case of recessive loss-of-function mutations, base-editing could be a one-time strategy to correct the mutation and restore gene function permanently.
Base-editing in vivo is akin to running a spell-check on the mutated gene and changing the point mutation to the correct sequence. Conceptually simple, the modalities of introducing a base-editor enzyme and evaluating its efficiency and sensitivity are quite challenging. The Holt lab has worked out the genetic causes of hearing loss and develops gene therapy approaches. In 2011, they identified TMC1 (Trans-membrane channel-like 1) protein required for hearing and balance. It is involved in mechanosensitive ion channels in the sensory hair cells of the inner ear. Mutations in the Tmc1 gene have been implicated in hereditary hearing loss. The Liu lab has worked on CRISPR-Cas9 gene-editing for a dominant Tmc1 mutation in a mouse model. This collaboration from the Holt and Liu labs, published in Science Translational Medicine, demonstrated that base-editing is a potential tool for gene therapy and focused on the p.Y182C or c.A545G mutation of Tmc1.
The first author, Wei Hsi Yeh, designed multiple base editors that could correct point mutations. However, base editors are too large to fit into AAVs (adeno-associated virus), which are typically used as vehicles to deliver cargo into cells. By splitting the editor into 2 AAVs, the team was able to co-infect the cells with two halves of the base editor, which would rejoin and target the Tmc1 gene for editing. They evaluated four base editors for targeting and editing Tmc1 in mouse embryonic fibroblasts (MEFS) and chose the AID-BE4max editor which had 21-fold more efficient editing and low indel frequency (1.9%) at the target locus.
Analysis of the genomic DNA of Baringo MEFs with CIRCLE-seq suggested insubstantial off-target editing. Cochlea dissection of Baringo mice (a mouse model for Tmc1-mediated hearing loss) helped elucidate the effect of the c.A545G mutation on the inner and outer hair cells (IHCs and OHCs) to abolish hearing. Ear cells have to convert sound waves to electric currents to convey them to the brain to process. The IHCs and OHCs in Baringo mice lacked sensory transduction currents.
Injecting the base editor into inner ear hair cells of the Baringo mouse at P1 (postnatal day 1) resulted in Tmc1 editing in hair cells at an average frequency of 10-51% and restoration of IHC and OHC morphology and sensory transduction current.
To investigate auditory function, the team checked for auditory brainstem responses (ABR- the lowest sound pressure needed to generate identifiable brainstem waveforms) at P30 (postnatal day 30). For WT control, the ABR was 30dB. Baringo mice had no ABRs at maximum sound at 110dB, indicating deafness. In Tmc1-edited mice, the ABR was 60dB, suggesting a 50dB improvement. There was a loss of unedited cells and a decline in hearing in 4-6 weeks. While this is a significant improvement from profound deafness in untreated mice, editing frequency needs to be higher to sustain the restoration of IHC and OHC function.
Jeffrey Holt, Director of Otolaryngology research at the F.M. Kirby Neurobiology Center at Boston Children’s, and co-senior author of the study said, “This research is very important for the pediatric community here at Boston Children’s Hospital and elsewhere because about 4,000 babies are born each year with genetic hearing loss. And, we feel this is a big step beyond the field of hearing restoration and for the broader field focused on the treatment of genetic disorders”. David Liu, a member at Broad Institute and director of Broad’s Merkin Institute for Transformative Technologies in Healthcare, is the other co-senior author.
The authors acknowledge that base-editing in humans may require more off-target analysis. A 5 to 50dB improvement in low-frequency hearing may still not be enough to understand spoken language, and further improvements in efficiency will be necessary to advance this method into clinical practice. However, AAV-mediated Tmc1 base-editing in vivo can restore auditory sensory defects caused by recessive mutations. There are over 70 mutations in the Tmc1, and further work would involve editing each mutation, one at a time. This proof-of-concept study can support further research into gene therapy-based interventions of genetic diseases.
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