Abstract
Usher syndrome type III (USH3) characterized by progressive loss of vision and hearing is caused by mutations in the clarin-1 gene (CLRN1). Clrn1 knockout (KO) mice develop hair cell defects by postnatal day 2 (P2) and are deaf by P21-P25. Early onset profound hearing loss in KO mice and lack of information about the cochlear cell type that requires Clrn1 expression pose challenges to therapeutic investigation. We generated KO mice harboring a transgene, TgAC1, consisting of Clrn1-UTR (Clrn1 cDNA including its 5′ and 3′ UTR) under the control of regulatory elements (Atoh1 3′ enhancer/β-globin basal promoter) to direct expression of Clrn1 in hair cells during development and down regulate it postnatally. The KO-TgAC1 mice displayed delayed onset progressive hearing loss associated with deterioration of the hair bundle structure, leading to the hypothesis that hair cell expression of Clrn1 is essential for postnatal preservation of hair cell structure and hearing. Consistent with that hypothesis, perinatal transfection of hair cells in KO-TgAC1 mice with a single injection of AAV-Clrn1-UTR vector showed correlative preservation of the hair bundle structure and hearing through adult life. Further, the efficacy of AAV-Clrn1 vector was significantly attenuated, revealing the potential importance of UTR in gene therapy.
Highlights
USH3 is caused by mutations in the clarin-1 (CLRN1) gene, which encodes a four-transmembrane protein (CLRN1) closely related to tetraspanins[6,7]
The associated virus (AAV) based preservation of hearing in the new USH3 model (KO-Transgene Atoh1-enhacer-Clrn-1 (TgAC1) mice) provide proof of concept that gene therapy is effective in mitigating sensory deficit associated with clarin-1 protein (CLRN1) mutation
From P90 to P150, the mean difference in auditory evoked brainstem response (ABR) threshold between KO-TgAC1 mice treated with AAV-Clrn1-without untranslated region (UTR) (n = 10) and untreated KO-TgAC1 mice (n = 6) (Fig. 6D) is ≈5 dB sound pressure level (SPL), and the difference is not statistically significant. These results demonstrate that AAV-mediated gene therapy can prevent deafness in the USH3 background, and AAV-Clrn1-UTR is the causal variant associated with the potent/ stable therapeutic effect
Summary
USH3 is caused by mutations in the clarin-1 (CLRN1) gene, which encodes a four-transmembrane protein (CLRN1) closely related to tetraspanins[6,7]. Answers to these questions are critical to understanding the role of Clrn[1] in the ear and in guiding future efforts to develop viable therapies for USH3 patients To address these questions, we generated KO mice carrying a transgene, TgAC1, that consisted of Clrn1-UTR (Clrn[1] cDNA flanked by 5′ and 3′ UTR) under the control of regulatory elements (Atoh1 3′ enhancer/β-globin basal) known to direct expression of the target gene in hair cells during development and significantly down regulate it after birth (
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