Genetic Rescue of a Subset of Thyroid Follicular Cells Restores Thyroid Function in Dyshormonogenic <i> Duoxa ^−/− </i> Mice

<b><i>Background:</i></b> Under sufficient iodine supply, dual oxidase (DUOX)-dependent H₂O₂ production constitutes the limiting factor for thyroid hormone (TH) synthesis. Inherited loss-of-function mutations in related genes can trigger congenital hypothyroidism (CH). TH supplementation is not always well-tolerated and requires dose adjustments throughout life. Regenerative medicine directed at thyroid follicular cells (TFCs) could offer an alternative therapy; however, the minimal number of TFCs to be corrected remains unknown. <b><i>Methods:</i></b> Thyroid dyshormonogenesis in <i>Duoxa^-/-</i> deficient mice was rescued by conditional thyroid-specific expression of DUOXA2/DUOX2 subunits. In order to restrict reactivation in a subset of TFCs, low doses of tamoxifen (0.1-2 mg) were injected. Thyroid function was assessed by immunostaining of iodinated-thyroglobulin (iTG). Circulating serum thyrotropin (TSH) and total thyroxin (T4) were quantified, and thyroidal expression of TSH-responsive genes (<i>Nis</i>, <i>Tpo</i>, <i>Tshr</i>) and hepatic deiodinase type-1 (<i>Dio1</i>) was measured. Last, combining iTG immunostaining with Duox2 <i>in situ</i> hybridization, we estimated the fraction of rescued TFCs required to restore follicular TG iodination. <b><i>Results:</i></b> Colloidal iTG⁺ staining in more than 90% of follicles demonstrated the successful rescue of TH biogenesis in doxycycline-induced Tet:<i>Da2D2^+/-</i>;<i>mTg</i>:CreER^T2+/<i>^-</i>;<i>Duoxa^-/-</i> animals (3TA). In contrast, reducing tamoxifen doses to 0.1 mg resulted in unresolved primary CH with thyroid enlargement, induction of <i>Nis</i>, <i>Tpo</i>, and <i>Tshr</i>, decrease of <i>Dio1</i>, and growth delay. Corresponding thyroid sections revealed scattered iTG⁺ colloidal lumens dispersed in histologically altered parenchyma. Nevertheless, we determined that only 11-15% of TFCs need to be reactivated within the follicle to functionally restore iodide organification. Goiter involution was also studied in 3TA following functional oxidase recovery or levothyroxine supplementation. Although thyroid enlargement was similarly reduced in both groups, expression of <i>Nis</i>, <i>Tpo</i>, <i>Tshr</i>, and <i>Dio1</i> more rapidly normalized in genetically rescued 3TA. In these pathological conditions, around 50-70% of iTG⁺ follicles would seem sufficient to recover a healthy thyroid function until two weeks. <b><i>Conclusions:</i></b> Our data in <i>Duoxa^-/-</i> mice suggest that the percentage of TFCs to be corrected is limited to 10-15% per follicle, which could be compatible with future somatic gene therapies. Furthermore, the reconstitution of hormonogenic-competent TFCs successfully results in goiter resolution, to an extent comparable to that of gold-standard TH replacement therapy.