Clinical Research

A NOVEL GENE THERAPY APPROACH TO TISSUE SPECIFIC CYTOTOXICITY IN THYROID CELLS

LoGerfo P et al
Columbia University College of Physicians and Surgeons, New York, NY

ABSTRACT.
Total thyroidectomy is a common procedure for patients suffering from thyroid cancer. Thyroglobulin is known to be completely endemic to thyroid tissure, and is an indicator of factors which turn on human thyroglobulin (hTg) promoters. Herpes simplex virus type 1 thymidine kinase (TK) has been used as a suicide mechanism in gene therapy for cancer. We have devised a novel gene therapy approach using a thyroglobulin promoter to drive thymidine kinase, which would result in tissue specific cell death in thyroid tissue.

We received a 200 bp human thyroglobulin proximal promoter with a 1.4 kb enhancer region housed in a pBLCAT3 plasmid. The CAT region was cut out of the plasmid using XhoI and EcoRI restriction enzymes. Herpes simplex virus type 1 thymidine kinase (HSV-1 TK) phosphorylates some nucleoside analogs, with high affinity for the guanosine nucleoside analogs of ganciclovir (GCV). Once phosphorylated, the analogs inhibit DNA replication by chain termination. A mutant TK known to have a 43 fold increase in susceptibility to ganciclovir administration was amplified by PCR with primers which added XhoI at the 5-prime and EcoRI at the 3-prime ends. The PCR product was also placed in a TA vector for easier future amplication and restriction enzyme digest. The mTK gene and the hTg plasmid with enhancer were then ligated, and amplified by maxiprep. The resulting hTg-mTK gene was then sequenced using primers to check for the mTK insert. The resulting plasmid was digested with XhoI and EcoRI, and the appropriate bands appearing after running a 1% agarose gel.

The positive control was created using the pALTER-MAX plasmid (Promega) containing a cytomegalovirus promoter (CMV) positioned before a multiple cloning region. The CMV plasmid was opened using XhoI and EcoRI restriction enzymes, and the mTK was inserted and verified using the same techniques associated with the creation of the hTg-mTK plasmid.