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Prenatal electroporation-mediated gene transfer restores Slc26a4 knock-out mouse hearing and vestibular function.

ABSTRACT: The otocyst, an anlage of the inner ear, presents an attractive target to study treatment strategies for genetic hearing loss and inner ear development. We have previously reported that electroporation-mediated transuterine gene transfer of Connexin30, utilizing a monophasic pulse into Connexin30^-/- mouse otocysts at embryonic day 11.5, is able to prevent putative hearing deterioration. However, it is not clear whether supplementary gene transfer can rescue significant morphological changes, caused by genetic deficits. In addition, with the transuterine gene transfer technique utilized in our previous report, the survival rate of embryos and their mothers after treatment was low, which became a serious obstacle for effective in vivo experiments. Here, we set out to elucidate the feasibility of supplementation therapy in Slc26a4 deficient mice, utilizing biphasic pulses, optimized by modifying pulse conditions. Modification of the biphasic pulse conditions during electroporation increased the survival rate. In addition, supplementation of the target gene cDNA into the otocysts of homozygous Slc24a4 knockout mice significantly prevented enlargement of the endolymphatic space in the inner ear areas; moreover, it rescued hearing and vestibular function of mice in vivo.

SUBMITTER: Takeda H

PROVIDER: S-EPMC6884448 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Prenatal electroporation-mediated gene transfer restores Slc26a4 knock-out mouse hearing and vestibular function.

Takeda Hiroki H Miwa Toru T Kim Min Young MY Choi Byung Yoon BY Orita Yorihisa Y Minoda Ryosei R

Scientific reports 20191129 1

The otocyst, an anlage of the inner ear, presents an attractive target to study treatment strategies for genetic hearing loss and inner ear development. We have previously reported that electroporation-mediated transuterine gene transfer of Connexin30, utilizing a monophasic pulse into Connexin30<sup>-/-</sup> mouse otocysts at embryonic day 11.5, is able to prevent putative hearing deterioration. However, it is not clear whether supplementary gene transfer can rescue significant morphological c ...[more]

PMID: 31784581

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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

Prenatal electroporation-mediated gene transfer restores Slc26a4 knock-out mouse hearing and vestibular function.

Publications

Prenatal electroporation-mediated gene transfer restores Slc26a4 knock-out mouse hearing and vestibular function.

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