Precise detection of CRISPR-Cas9 editing in hair cells in the treatment of autosomal dominant hearing loss

Cui, Chong; Wang, Daqi; Huang, Bowei; Wang, Fang; Chen, Yuxin; Lv, Jun; Zhang, Luping; Lei, Han; Liu, Dong; Chen, Zhengyi; Li, Geng-Lin; Li, Huawei; Shu, Yilai

doi:10.1016/j.omtn.2022.07.016

Cited by 25 publications

(17 citation statements)

References 60 publications

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“…Bth/+ controls exhibited hearing loss that progressed to profound levels by 20 wk of age, consistent with prior reports (5,8,15). In light of previous successful rescue with an allele-specific strategy using the AAV2/9 serotype (6), rescue of mouse models of DFNB7/11 using gene replacement, and partial preservation of auditory function in Tmc1 Bth/+ mice with Tmc2 gene replacement alone (12,14), we compared treatment efficacy of AAV2/ 9.miSafe.mTmc1 with AAV2/9.miTmc1D11.mTmc1 to test the efficacy of Tmc1 expression augmentation (gene replacement alone) and with RNAi + gene replacement (Fig 3B).…”

Section: Design Of Mutation-agnostic Rnai With Gene Replacement Strategysupporting

confidence: 91%

“…Autosomal dominant non-syndromic hearing loss arises because of pathogenic variants with dominant-negative, gain-of-function, or haploinsufficiency mechanisms of action, and accounts for ∼15–20% of genetic hearing loss diagnoses ( 3 , 4 ). Several cochlear gene therapies have leveraged adeno-associated virus (AAV) to deliver RNAi or CRISPR/Cas9 constructs to reduce or rescue hearing impairment in neonatal mouse models of autosomal dominant non-syndromic hearing loss ( 5 , 6 , 7 ). Although these treatments represent substantial advancements toward the clinical application of gene therapy to address hearing loss, delivery before the onset of murine hearing at ∼P12–P15 limits direct applicability to the human cochlea, which is mature at birth ( 8 , 9 , 10 ).…”

Section: Introductionmentioning

confidence: 99%

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Mutation-agnostic RNA interference with engineered replacement rescuesTmc1-related hearing loss

et al. 2022

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Hearing loss is the most common sensory deficit, of which genetic etiologies are a frequent cause. Dominant and recessive mutations inTMC1, a gene encoding the pore-forming subunit of the hair cell mechanotransduction channel, cause DFNA36 and DFNB7/11, respectively, accounting for ∼2% of genetic hearing loss. Previous work has established the efficacy of mutation-targeted RNAi in treatment of murine models of autosomal dominant non-syndromic deafness. However, application of such approaches is limited by the infeasibility of development and validation of novel constructs for each variant. We developed an allele-non-specific approach consisting of mutation-agnostic RNAi suppression of both mutant and WT alleles, co-delivered with a knockdown-resistant engineered WT allele with or without the use of woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) to augment transgene expression. This therapeutic construct was delivered into the mature murine model of DFNA36 with an AAV vector and achieved robust hair cell and auditory brainstem response preservation. However, WPRE-enhancedTmc1expression resulted in inferior outcomes, suggesting a role for gene dosage optimization in futureTMC1gene therapy development.

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Section: Design Of Mutation-agnostic Rnai With Gene Replacement Strategysupporting

confidence: 91%

Section: Introductionmentioning

confidence: 99%

Mutation-agnostic RNA interference with engineered replacement rescuesTmc1-related hearing loss

et al. 2022

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“…In conclusion, because of its small size, SaCas9 stands out among many Cas9s and has been involved in more developments for therapeutic applications [ 31 ]. Many engineering efforts are being taken to address its functional deficiencies, such as the off-target effects and low efficiency, etc.…”

Section: Discussionmentioning

confidence: 99%

Full-Length Model of SaCas9-sgRNA-DNA Complex in Cleavage State

Zhu

Qian

et al. 2023

IJMS

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Staphylococcus aureus Cas9 (SaCas9) is a widely used genome editing tool. Understanding its molecular mechanisms of DNA cleavage could effectively guide the engineering optimization of this system. Here, we determined the first cryo-electron microscopy structure of the SaCas9-sgRNA-DNA ternary complex. This structure reveals that the HNH nuclease domain is tightly bound to the cleavage site of the target DNA strand, and is in close contact with the WED and REC domains. Moreover, it captures the complete structure of the sgRNA, including the previously unresolved stem-loop 2. Based on this structure, we build a full-length model for the ternary complex in cleavage state. This model enables identification of the residues for the interactions between the HNH domain and the WED and REC domains. Moreover, we found that the stem-loop 2 of the sgRNA tightly binds to the PI and RuvC domains and may also regulate the position shift of the RuvC domain. Further mutagenesis and molecular dynamics simulations supported the idea that the interactions of the HNH domain with the WED and REC domains play an important role in the DNA cleavage. Thus, this study provides new mechanistic insights into the DNA cleavage of SaCas9 and is also useful for guiding the future engineering of SaCas9-mediated gene editing systems.

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“…Dominant-negative variants in KCNQ4 nearly abolish this current, resulting in buildup of intracellular potassium and abolition of membrane repolarization, ultimately resulting in hair cell death manifesting as a predominantly high frequency, progressive hearing loss. Two laboratories recently and contemporaneously leveraged CRISPR/Cas9-based targeted disruption in murine models of DFNA2A [24,25]. In both cases, intracochlear delivery of sgRNA/SpCas9 resulted in partial preservation of hearing.…”

Section: Autosomal Dominant Nonsyndromic Hearing Lossmentioning

confidence: 99%

Advances in cochlear gene therapies

Klimara

Smith

2023

Current Opinion in Pediatrics

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Purpose of review Hearing loss is the most common sensory deficit and in young children sensorineural hearing loss is most frequently genetic in etiology. Hearing aids and cochlear implant do not restore normal hearing. There is significant research and commercial interest in directly addressing the root cause of hearing loss through gene therapies. This article provides an overview of major barriers to cochlear gene therapy and recent advances in preclinical development of precision treatments of genetic deafness. Recent findings Several investigators have recently described successful gene therapies in many common forms of genetic hearing loss in animal models. Elegant strategies that do not target a specific pathogenic variant, such as mini gene replacement and mutation-agnostic RNA interference (RNAi) with engineered replacement, facilitate translation of these findings to development of human therapeutics. Clinical trials for human gene therapies are in active recruitment. Summary Gene therapies for hearing loss are expected to enter clinical trials in the immediate future. To provide referral for appropriate trials and counseling regarding benefits of genetic hearing loss evaluation, specialists serving children with hearing loss such as pediatricians, geneticists, genetic counselors, and otolaryngologists should be acquainted with ongoing developments in precision therapies.

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Precise detection of CRISPR-Cas9 editing in hair cells in the treatment of autosomal dominant hearing loss

Cited by 25 publications

References 60 publications

Mutation-agnostic RNA interference with engineered replacement rescuesTmc1-related hearing loss

Mutation-agnostic RNA interference with engineered replacement rescuesTmc1-related hearing loss

Full-Length Model of SaCas9-sgRNA-DNA Complex in Cleavage State

Advances in cochlear gene therapies

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