Table 3 In vivo studies utilizing CRISPR/Cas-based therapeutic strategies for genetic HL

KCNQ4

Encoding Kv7.4 that affects the ionic homeostasis of inner ear

DFNA2

SpCas9

Using a dual adeno-associated virus (AAV) package targeting outer hair cells, in vivo gene editing was Iapplied to disrupt the dominant-negative allele in Kcnq4

The strategy enhanced the functional Kv7.4 channel activity, and partially restored hearing function in a murine model, even at low gene editing efficacies

[184]

SaCas9-KKH

AAV-SaCas9-KKH-g3 system was injected into the inner ears of Kcnq4^+/G229D mice

The AAV-SaCas9-KKH-g3 agents could effectively and specifically edit the Kcnq4^G229D alleles in Kcnq4^+/G229D mice

[185]

MYO6

Affecting hair bundle via modulating differentiation and development of stereocilia bundles

DFNA22

SaCas9-KKH

AAV-PHP.eB vector-mediated in vivo delivery of SaCas9-KKH-sgRNA complexes was used to specifically knock out the Myo6^C442Y allele in Myo6^WT/C442Y mice

Rescue of auditory function was observed up to 5 months in the AAV-SaCas9-KKH-Myo6-g2-treated Myo6^WT/C442Y mice

[186]

TMC1

Encoding a pore-forming subunit that is crucial for mechanosensory transduction channels

DFNA36

SpCas9

Injection of Cas9-gRNA-lipid complexes into the cochlea of neonatal Tmc1^Bth/+ mice aimed to selectively disrupt dominant Tmc1^Bth/WT alleles associated with HL

The strategy substantially reduced progressive HL in neonatal Tmc1^Bth/WT mice, with higher hair cell survival rates and lower ABR thresholds

[187]

SaCas9-KKH

Fourteen Cas9/gRNA combinations were screened for specific and efficient disruption of the Tmc1^Bth/WT allele

The strategy selectively and efficiently disrupted the Tmc1^Bth/WT mutant allele, and AAV-mediated delivery prevented HL in Beethoven mice up to one year post transduction

[189]

SpCas9

Dual delivery of SpCas9 and gRNA in separate AAV9-PHP.B vectors selectively disrupts a dominant Tmc1 allele and preserves hearing in Beethoven mice

The results show that dual vector delivery of SpCas9/gRNA with AAV9-PHP.B can effectively and selectively target the Tmc1^Bth/WT allele and preserve hearing function of Beethoven mice

[190]

DFNB7/11

SpCas9

The dual AID-CBEmax AAVs were injected into the inner ears of Baringo mice to genetically correct the Tmc1 c.A545G point mutation

The strategy mediated in vivo base editing of Tmc1^Y182C/Y182C to improve auditory function in Baringo mice with recessive HL

[114]

DFNA36

RfxCas13d (CasRx)

The AAV-PHP.eB-CasRx-sgRNA3 was delivered into the inner ears of Beethoven mice to deregulate the expression of the Tmc1^Bth transcript

The strategy mediated the efficient and selective in vivo RNA knockdown of the Tmc1^Bth mutation

[200]

PCDH15

Encoding a subunit of the tip link that is crucial for mechanosensory transduction channels

DFNB23

SpCas9

NHEJ-mediated nonrandom editing was used to o correct a frameshift mutation in the postmitotic hair cells in vivo by injectoporating m-3j-gRNA1 and Cas9 into Pcdh15^av3j/av3j cochleae

Half of the animals gained improvements in auditory responses, and balance function is restored in the majority of injected Pcdh15^av3j/av3j mutant mice

[192]

CDH23

Affecting intercellular adhesion via causing structural abnormalities in the stereocilia

DFNB12

Cas9 mutant D10A

Targeted CRISPR/Cas9-mediated HDR was used to correct the Cdh23^ahl allele directly in C57BL/6NTac zygotes

The strategy efficiently corrected the Cdh23^ahl allele in C57BL/6NTac mice, with complete abrogation of both the progressive HL and sensory cell degeneration phenotypes

[194]

SLC26A4

Encoding the anion exchanger pendrin that affects the ionic homeostasis of inner ear

DFNB4

SaCas9

A plasmid co-expressing SaCas9 and engineered sgRNAs delivered both into Neuro2a cells and primary mouse embryonic fibroblasts

The programmed sgRNAs and donor template packed into rAAVs induced HDR-mediated genome modification of the c.919-2A splicing site in the murine Slc26a4 locus

[13]

MITF

Encoding a transcription factor that affects the proliferation and differentiation of neural crest-derived melanocytes

Waardenburg syndrome 2A

SpCas9

The CRISPR-Cas9-mediated HDR using ssODN and donor DNAs performed to correct the MITF c.740T > C mutation of the WS2A pig model

The strategy achieved precise correction of this point mutation and successfully rescued the anophthalmia and HL phenotype

[15]

Klhl18

May affect the actin core of stereocilia

Progressive, predominantly low-frequency recessive HL

SaCas9-KKH

The SaCas9-KKH-sgRNA2 were delivered using AAV9 and AAV-PHP.eB into the inner hair cells of homozygous Klhl18lowf mice to correct the point mutation (Chr9:110455454 C > A)

The strategy repaired the mutation in Klhl18, leading to significant restoration of hearing function in treated mice

[14]

MYO6

Affecting hair bundle via modulating differentiation and development of stereocilia bundles

DFNA22

Cas13X

The AAV-PHP.eB–mxABE was injected into the inner ears of Myo6^WT/C442Y mice for in vivo RNA correction of Myo6^C442Y

The strategy resulted in 4.22 ± 0.68% base editing efficiency for correction of mutant allele (Myo6C442Y) to WT (Myo6) and ameliorated the auditory function in Myo6^WT/C442Y mice

[113]