Could CRISPR-CAS9 be a new strategy for the treatment of deafness?
Tmc1 is a component of transmembrane channels in mammalian hair cells. The mutation of the gene Tmc1 causes autosomal dominant hearing loss. The researchers developed a genome-editing approach to modify the mutation.
The perception of sound in the human inner ear begins in the sensory hair cells (HCs) of the cochlea. HCs convert sound vibrations into electrical signals, which are transmitted via spiral ganglion neurons to the brain through the eighth cranial nerve. The transformation of sound in neural signal is due to the presence of mechanotranduction channels. Deafness is the most common sensory deficit in humans, due to environmental or genetic factors, causing above all the death of HCs. Among the most common treatments, we remember genetic approaches with viral vectors, the cochlear implant and generation of HCs from stem cells [1]. In 2017 a group of researchers guided by David R. Liu, conducted a study about the treatment of autosomal dominant deafness by in vivo delivery of genome editing agents [2]. They chose as animal model the so-called Beethoven (Bth) mice, characterized by dominant mutation of the gene Tmc1 (c.T1235A, which causes a substitution of Met to Lys) [3]. Tmc1 is an essential component of transmembrane channels: it’s believed that it is the pore-forming subunit (with Tmc2) of the hair cell transduction channel [4]. The mutation T1235A causes altered Ca2+ permeability, reduces single-channel current levels, HCs death and progressive hearing loss.
The researchers developed a genome-editing approach to target the mutation, designing and validating, both in vitro and in primary fibroblasts, genome editing agents. The method chosen was CRISPR-Cas9, a complex formed by an endonuclease (Cas9) and a single-guide RNA (sg-RNA), which leads the nuclease to a complementary DNA sequence. Cas9 cleaves the target sequence, that can be repaired by the cell creating mutations (and silencing the gene) or giving an exogenous DNA sequence that can replace the mutated one. The first step was to identify a sgRNA that preferentially disrupts the dominant deafness-associated allele in the Tmc1 gene. They tested in vitro the ability of four candidates sgRNAs to specifically target the Tmc1Bth allele rather than the wild-type Tmc1. The most efficient sgRNA showed the highest rate (10%) of targeted indels in mutant Tmc1Bth fibroblast and its ability to cleave Tmc1Bth allele 23-fold more efficiently than the wild-type allele. Second, the researchers used sequencing and in silico methods to analyze the potential presence of off-target sites. They identified ten off-target sites and measured the indel frequency at each of them, following DNA nucleofection or ribonucleotide protein (RNP) lipid-delivery. The latter showed modification of only one off-target site (1.2% indel), compared with DNA nucleofection (with 8% indel at nine off-target sites), consistent with literature [5]. Therefore, the best way to deliver the Cas9-Tmc1-mut3 complex was lipid-mediated. Seventeen cationic lipids were tested, but the highest indel rate of the target locus was observed with Lipofectamine2000 (12% indel).
Then, the researchers analyzed the ability of Cas9-Tmc1-mut3 complex to target the Tmc1Bth allele in vivo in Tmc1Bth/+Tmc2Δ/Δ mice, to isolate the effect of editing the mutant allele. They observed a decline in transduction currents from inner HCs, proving the disruption of the Tmc1Bth allele. Next, they focused on hair cell survival: injection of Cas9-Tmc1-mut3 complex in Tmc1Bth/+Tmc2+/+ (Tmc1Bth/+) mice showed an enhanced survival of HCs compared to uninjected ears. They also analysed the auditory brainstem responses (ABRs), the sound-evoked neural outputs of the cochlea, due to inner HCs. The treated Tmc1Bth/+ mice showed lower ABR thresholds in relation with untreated Tmc1Bth/+ ones, consistent with an enhanced cochlear function. Then, they measured the distortion product otoacustic emissions (DPOAEs), a way to quantify the amplification provided by outer HCs. DPOAE thresholds were lightly elevated in treated Tmc1Bth/+ mice, probably due to damage of outer HCs. Furthermore, the researchers evaluated acoustic startle response, as behavioral measure of hearing rescue. Injected Tmc1Bth/+ mice had significant startle response following acoustic stimulus, especially after 110 dB and 120 dB, differently to the uninjected Tmc1Bth/+ mice, in which no response was detected. The Cas9-Tmc1-mut3 complex delivery causes the disruption of Tmc1Bth allele; thanks to this effect, it restores the hearing function in Tmc1Bth/+ mice. Nowadays, the treatment is not applicable to homozygous mutant mice because the hearing rescue requires the presence of at least one wild-type allele.
Considering the unpredictability of the technique, it could cause mutations, as oncogene’s activation as oncosuppressor’s deactivation. It would be interesting to perform sequencing after the injection, to know the precise DNA sequence modification. Furthermore, during the transduction current measurement of Tmc1Bth/+Tmc2Δ/Δ mice, the untreated mutant mice showed higher current levels than the wild-type mice, inconsistent with previous statements [6]. The results obtained could deserve further searches to better understand the role of Tmc1Bth mutation in the channel’s mechanism. Assessing the DPOAEs measures, the elevations in thresholds (in untreated mutant mice) resulted smaller than those of ABR and according with reports that Bth mutation affects the most the inner HCs than outer HCs. The researchers observed that DPOAE thresholds were slightly elevated in injected ears: this is consistent with outer HCs damage, perhaps because of the injection procedure, as suggested in the paper. The possibility to cause damage is a limitation of the technique and it should be considered in view of the development of human treatment.
In conclusion, the technique is effective in the treatment of autosomal dominant deafness in heterozygous Bth mice, ensuring high specificity and low off-target sites. Nevertheless, more studies are necessary to make human treatment possible.
References
- Geleoc GSG, Holt JR. 2014. Sound Strategies for Hearing Restoration. Science. May; 344: 596-+.
- Gao X, Tao Y, Lamas V, Huang MQ, Yeh WH, Pan BF, Hu YJ, Hu JH, Thompson DB, Shu YL, et al. 2018. Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents. Nature. Jan; 553: 217-+.
- Pan BF, Geleoc GS, Asai Y, Horwitz GC, Kurima K, Ishikawa K, Kawashima Y, Griffith AJ, Holt JR. 2013. TMC1 and TMC2 Are Components of the Mechanotransduction Channel in Hair Cells of the Mammalian Inner Ear. Neuron. Aug; 79: 504-515.
- Pan BF, Akyuz N, Liu XP, Asai Y, Nist-Lund C, Kurima K, Derfler BH, Gyorgy B, Limapichat W, Walujkar S, et al. 2018. TMC1 Forms the Pore of Mechanosensory Transduction Channels in Vertebrate Inner Ear Hair Cells. Neuron. Aug; 99: 736-+.
- Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH, Maeder ML, Joung JK, Chen ZY, Liu DR. 2015. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nature Biotechnology. Jan; 33: 73-80.
- Corns LF, Johnson SL, Kros CJ, Marcotti W. 2016. Tmc1 Point Mutation Affects Ca2+ Sensitivity and Block by Dihydrostreptomycin of the Mechanoelectrical Transducer Current of Mouse Outer Hair Cells. Journal of Neuroscience. Jan; 36: 336-349.