Retrovirus-Mediated Gene Transfer into Canine Thyroid Using an Ex Vivo Strategy

Abstract

We describe studies in a canine model aimed at establishing methods for ex vivo gene delivery to thyroid follicular cells. Canine follicular cells were harvested from tissue obtained by unilateral lobectomy, grown in thyrotropin-containing media, and transduced with amphotropic retroviral vectors carrying Escherichia coli β-galactosidase or Tn7 neomycin-resistance genes. Up to 30% of cells were transduced with retroviral vectors containing the neomycin resistance gene, and transduced cells could be selected with G418. Significantly, transduced and selected cells exhibited the morphology of thyroid follicular cells and continued to express thyroglobulin. To assess the viability of cultivated and transduced cells for transplantation, cells were stained with the vital fluorescent dye Dil, recovered by trypsinization, and transplanted into the contralateral thyroid lobe of autologous animals. Engraftment was demonstrated by fluorescence microscopy and identification of proviral sequences 7–10 days after transplantation. Proviral transcripts were evident using coupled reverse transcription and the polymerase chain reaction using total RNA from transplanted glands. Thyroid follicular cells may represent an attractive target for gene therapy due to their proliferative potential, their large protein synthetic and secretory capacity, and their susceptibility to regulation. The thyroid might be a target for therapy of congenital or acquired thyroid diseases as well as disorders requiring regulated expression of proteins in the circulation. This work demonstrates the feasibility of ex vivo gene delivery to thyroid follicular cells that may be used in future investigations.

Overview summary

Many different organs have been considered as targets for somatic gene therapy. O'Malley et al. describe a novel target for gene therapy, the thyroid, and demonstrate the feasibility of a series of molecular, cellular, and surgical manipulations required to deliver genetic material to this organ in a dog model. This work establishes a methodological basis for considering the therapeutic potential of gene delivery to the thyroid for congenital or acquired thyroid disease or disorders requiring systemic circulation of therapeutic gene products.