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A dual-AAV approach restores fast exocytosis and partially rescues auditory function in deaf otoferlin knock-out mice.

ABSTRACT: Normal hearing and synaptic transmission at afferent auditory inner hair cell (IHC) synapses require otoferlin. Deafness DFNB9, caused by mutations in the OTOF gene encoding otoferlin, might be treated by transferring wild-type otoferlin cDNA into IHCs, which is difficult due to the large size of this transgene. In this study, we generated two adeno-associated viruses (AAVs), each containing half of the otoferlin cDNA Co-injecting these dual-AAV2/6 half-vectors into the cochleae of 6- to 7-day-old otoferlin knock-out (Otof -/-) mice led to the expression of full-length otoferlin in up to 50% of IHCs. In the cochlea, otoferlin was selectively expressed in auditory hair cells. Dual-AAV transduction of Otof -/- IHCs fully restored fast exocytosis, while otoferlin-dependent vesicle replenishment reached 35-50% of wild-type levels. The loss of 40% of synaptic ribbons in these IHCs could not be prevented, indicating a role of otoferlin in early synapse maturation. Acoustic clicks evoked auditory brainstem responses with thresholds of 40-60 dB. Therefore, we propose that gene delivery mediated by dual-AAV vectors might be suitable to treat deafness forms caused by mutations in large genes such as OTOF.

SUBMITTER: Al-Moyed H

PROVIDER: S-EPMC6328916 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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A dual-AAV approach restores fast exocytosis and partially rescues auditory function in deaf otoferlin knock-out mice.

Al-Moyed Hanan H Cepeda Andreia P AP Jung SangYong S Moser Tobias T Kügler Sebastian S Reisinger Ellen E

EMBO molecular medicine 20190101 1

Normal hearing and synaptic transmission at afferent auditory inner hair cell (IHC) synapses require otoferlin. Deafness DFNB9, caused by mutations in the <i>OTOF</i> gene encoding otoferlin, might be treated by transferring wild-type otoferlin cDNA into IHCs, which is difficult due to the large size of this transgene. In this study, we generated two adeno-associated viruses (AAVs), each containing half of the otoferlin cDNA Co-injecting these dual-AAV2/6 half-vectors into the cochleae of 6- to ...[more]

PMID: 30509897

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Project description:Otoferlin is involved in neurotransmitter release at the synapse between inner hair cells (IHCs) and auditory nerve fibres, and mutations in the OTOF gene result in severe to profound hearing loss. Abnormal sound-evoked cochlear potentials were recorded with transtympanic electrocochleography from four children with otoferlin (OTOF) mutations to evaluate physiological effects in humans of abnormal neurotransmitter release from IHCs. The subjects were profoundly deaf with absent auditory brainstem responses and preserved otoacoustic emissions consistent with auditory neuropathy. Two children were compound heterozygotes for mutations c.2732_2735dupAGCT and p.Ala964Glu; one subject was homozygous for mutation p.Phe1795Cys, and one was compound heterozygote for two novel mutations c.1609delG in exon 16 and c.1966delC in exon 18. Cochlear potentials evoked by clicks from 60 to 120 dB peak equivalent sound pressure level were compared to recordings obtained from 16 normally hearing children. Cochlear microphonic (CM) was recorded with normal amplitudes from all but one ear. After cancelling CM, cochlear potentials were of negative polarity with reduced amplitude and prolonged duration compared to controls. These cochlear potentials were recorded as low as 50-90 dB below behavioural thresholds in contrast to the close correlation in controls between cochlear potentials and behavioural threshold. Summating potential was identified in five out of eight ears with normal latency whilst auditory nerve compound action potentials were either absent or of low amplitude. Stimulation at high rates reduced amplitude and duration of the prolonged potentials, consistent with neural generation. This study suggests that mechano-electrical transduction and cochlear amplification are normal in patients with OTOF mutations. The low-amplitude prolonged negative potentials are consistent with decreased neurotransmitter release resulting in abnormal dendritic activation and impairment of auditory nerve firing.

Project description:The synergistic activation of transcription factors can lead to thyroid progenitor cell speciation. We have previously shown in vitro that mouse or human stem cells, expressing the transcription factors NKx2-1 and Pax8, can differentiate into thyroid neo-follicular structures (TFS). We now show that syngeneic mouse TFS when implanted into hypothyroid TSH receptor knockout (TSHR-KO) mice can ameliorate the hypothyroid state for an extended period. ES cells derived from heterozygous TSHR-KO blastocysts were stably transfected with Nkx2-1-GFP and Pax8-mcherry constructs and purified into 91.8% double positive cells by flow cytometry. After 5 days of activin A treatment these double positive cells were then induced to differentiate into neo-follicles in Matrigel for 21 days in the presence of 500μU/mL of TSH. Differentiated TFS expressing thyroglobulin mRNA were implanted under the kidney capsule of 4-6 weeks old TSHR-KO mice (n=5) as well as hind limb muscle (n=2) and anterior chamber of one eye (n=2). Five of the mice tested after 4 weeks were all rendered euthyroid and all mice remained euthyroid at 20 weeks post implantation. The serum T4 fully recovered (pre-bleed 0.62 ± 0.03 to 8.40 ± 0.57 µg/dL) and the previously elevated TSH became normal or suppressed (pre-bleed 391 ± 7.6 to 4.34 ± 1.25 ng/dL) at the end of the 20 week observation period. The final histology obtained from the implanted kidney tissues showed only rudimentary thyroid follicular structures but which stained positive for thyroglobulin expression. The presence of only rudimentary structures at the site of implant on these extended animals suggested possible migration of cells from the site of implant or an inability of TFCs to maintain proper follicular morphology in these external sites for extended periods. However, there were no signs of tumor formation and no immune infiltration. These preliminary studies show that TSHR-KO mice are a useful model for orthotropic implantation of functional thyroid cells without the need for thyroidectomy, radioiodine ablation or anti thyroid drug control of thyroid function. This approach is also proof of principle that thyroid cells derived from mouse ES cells are capable of surviving as functional neo-follicles in vivo for an extended period of 20 weeks.

Dataset Information

A dual-AAV approach restores fast exocytosis and partially rescues auditory function in deaf otoferlin knock-out mice.

Publications

A dual-AAV approach restores fast exocytosis and partially rescues auditory function in deaf otoferlin knock-out mice.

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