Towards a cure for deafness in DFNA9: loading lipid nanoparticles with CRISPR-Cas9 to safely target dominant-negative mutations in the COCH gene in vitro and in vivo.

Hearing impairment is the most frequent sensory deficit in the human population, affecting 440 million people worldwide, whereby loss of hearing and balance has a significant impact on quality of life and society. Hearing loss is also listed by the World Health Organization as a priority disease for research into therapeutic interventions to address public health needs. DFNA9 (DeaFNess Autosomal 9) is an autosomal dominant hearing disorder caused by different heterozygous gain-of-function mutations in the COCH gene (Coagulation Factor C Homology) and is characterized by vestibular dysfunction and progressive late-onset (3rd-5th decade) sensorineural hearing loss (SNHL) leading to deafness. Within Belgium and the Netherlands, there are >1000 patients affected by the p.P51S COCH mutation, one of the heterozygous mutations causing DFNA9, who – in the current absence of a disease modifying therapy – will develop deafness. A promising approach to tackle the pathophysiological mechanism of DFNA9 and stop or delay the onset of SNHL is to introduce a CRISPR-Cas9 mRNA based therapy into the inner ear, delivered by a Lipid nanoparticle (LNP) to disrupt or downregulate the expression of mutant COCH protein. Until today, most researchers focus on the use of viral vectors, mostly AAV, to apply gene therapy in vivo in the inner ear. However, many people have already been exposed to AAV earlier in life and generated specific antibodies. Therefore, using LNP could provide a suitable alternative as they are already been proven to be safe when injecting a large population (COVID-19 mRNA vaccines). Moreover, using Cas9 mRNA instead of Cas9 DNA will only lead to a temporary expression of the Cas9 protein which will largely decrease the change on an evoked immune response against Cas9. To develop a cochlear gene therapy than can be translated to a human setting, a highly innovative humanized DFNA9 mouse model was generated. These mice bear humanized COCH harbouring the p.P51S mutation and thus mimic the genotype of DFNA9 patients. The goal of this project is: (1) assessing the safety and targeted delivery of LNP injected in vivo in the inner ear of our humanized DFNA9 mouse model (2) assess the use of LNP for in vitro delivery of Cas9/gRNA complexes to specifically downregulate the production of mutant cochlin in embryonic fibroblasts derived from our humanized DFNA9 mouse model.

Keywords:DFNA9, HEARING & DEAFNESS, COCHLEA, GENE THERAPY

Disciplines:Otology