Date: 15th June 2020
Hearing loss is the most common neurological disorder, and around 466 million people worldwide have disabling hearing loss. Around 50% of inner ear disorders are caused by genetic mutations, and those in genes such as the transmembrane channel-like 1 (TMC1) gene can cause hereditary hearing loss. Now scientists have used gene editing to repair a single nucleotide mutation in TMC1 mice in vivo to partially restore hearing in mice.
TMC1 encodes a protein that forms mechanosensitive ion channels in sensory hair cells of the inner ear and is required for normal auditory function. Previous work, from one of the senior authors Jeffrey Holt, had shown that replacing the TMC1 gene using a synthetic adenoviral vector (AAV) could restore deafness in the mice. In fact, Holt and co-senior author, David Liu, have a long history working together on TCM1, and together showed that lipid-mediated in vivo delivery of Cas9–guide RNA complexes could ameliorate hearing loss in a TCM1 mutated mouse model for a dominantly inherited form of genetic deafness.
However, now the team wanted to see whether it was possible to correct a single recessive mutation in TMC1 responsible for hearing loss – rather than replacing the entire gene. The mouse model Baringo was used, caused by a recessive mutation in TMC1, which leads to rapid deterioration of inner ear hair cells causing profound deafness in mice at just 4 weeks of age.
The team from Boston Children’s Hospital and the Broad Institute of MIT and Harvard, US, used base editing – pioneered in the Liu lab- to precisely repair the mutation. DNA base editors comprise of a catalytically disabled nuclease fused to a nucleobase deaminase enzyme, converting one base or base pair into another, without creating a double-stranded DNA break.
To begin, the team tested several optimised cytosine base editors (CBE) and guide RNAs in embryonic fibroblasts derived from Baringo mice, settling on a CBE derived from an activation-induced cytidine deaminase (AID).
However, whilst in vitro delivery of base editors is relatively easy, they are too large to be delivered by a single AAV in vivo. To circumvent this problem the team used dual AAVs and a so-called split-intein delivery system. Here the base editor was split into two components, and conjugated to one half of a split intein. Inteins are proteins than can excise themselves from host proteins via protein splicing, and once split (either naturally or artificially) they can carry out a trans-splicing reaction leading to the formation of a new peptide bond between the two polypeptides originally flanking the intein. In this context here, it led to the reconstitution of the base editor. The two fragmented parts of the split-intein CBE were each delivered by a separate AAV (AID-CBE AAVs).
To test the dual system in vivo the dual AID-CBE AAVs were injected into the inner ears of Baringo mice at postnatal day 1. Cochlea dissection from the injected Baringo mice showed that there was up to 51% reversion of the c.A545G mutation back to the wild type sequence c.A545A in the Tmc1 transcripts, as a result of the gene editing. On further analysis the team saw the restoration of inner hair cell sensory transduction and hair cell morphology in the treated mice compared to the control mice, and transiently rescued low-frequency hearing 4 weeks after injection. Although an estimated 25% of the inner ear cells would have received both AAVs – both are required for gene editing- this was enough to partially restore hearing in the treated mice.
Conclusions and future applications:
The team here have used base editing for the first time to restore the hearing of mice with a known recessive genetic mutation. It is also the first time base editing has been used for a genetic sensory disorder. These findings provide a groundwork for potential treatment of recessive hearing loss and support further development of base editing to correct pathogenic point mutations.
Currently there are several clinical trials ongoing to assess the safety of CRISPR-Cas gene editing, and early patient data is showing promising but mixed results. In the main body of this work, the editing of the cells has been performed in vitro and the edited-cells have been transplanted back in to the patient. That said, we reported back in March this year that Allergan and Editas Medicine had dosed a patient with world’s first ever in vivo CRISPR medicine into the eye. Whilst base editing is still a relatively ‘new’ tech, it is thought that off-target effects are low, therefore this may provide a highly valuable translatable in vivo editing tool. As with the eye it is tempting to speculate that due to the fact the organ is duplicated (one can be treated at a time), and relatively peripheral, the ear is a good ‘first’ testing grounds for such therapies. The question arises as to whether this might be the first-ever base editor to reach clinical trials in the not so distant future? Certainly with Lui as a scientific founder or co-founder of six biotechnology and therapeutics companies, including Editas Medicine and Beam Therapeutics this tech has a good pedigree.
However, with this in mind there are several optimisations and improvements that must be made. Whilst the edited-mice registered sounds as quiet as 60 decibel (untreated mice couldn’t detect 110 decibels), they couldn’t detected softer 30 decibel sounds. Furthermore, it was noted that the treated mice also started to show a decline in hearing after 6 weeks. As only 25% of cells in the inner ear were subjected to gene editing it is hoped by increasing the efficiency of the delivery system that a more pronounced, and sustained hearing improvement could be achieved. In support of this the team did find that those 25% of cells receiving both AAvs recovered 100% of their function.
With the recent demonstration that multiplexed precise base editing could edit up to three target sites simultaneously in primates, together with the work here, it places base editing as a strong emerging contender in the gene editing market and extends its range of applicable diseases.
For more information please see the press releases from the Broad Institute or Harvard University
Yeh, W.-H., O. Shubina-Oleinik, J. M. Levy, B. Pan, G. A. Newby, M. Wornow, R. Burt, J. C. Chen, J. R. Holt and D. R. Liu (2020). “In vivo base editing restores sensory transduction and transiently improves auditory function in a mouse model of recessive deafness.” Science Translational Medicine 12(546): eaay9101.