415. Conditional Expression of a Suicide Gene by the Telomere Reverse Transcriptase Promoter for Post-Therapeutic Deletion of Insertional Mutagenesis

Abstract

The success of gene therapy for human X-linked severe combined immunodeficiency (X-SCID) has been recently clouded by the occurrence of post-therapeutic cancers caused by vector-associated insertional mutagenesis. Improving the safety feature of integrating vectors becomes imperative and challenging. We demonstrated a gene expression cassette that can be possibly exploited in integrating vector systems to eliminate the rare but existing adverse events. The Herpes simplex virus thymidine kinase (HSV-TK) gene under the transcriptional control of the human telomerase reverse transcriptase (hTERT) promoter was incorporated into a self-inactivating HIV-based lentiviral vector. Theoretically, the hTERT promoter is silent in normal somatic cells and re-activated in tumor cells. Therefore, normal gene-corrected cells should not express HSV-TK. However, if some gene-corrected cells subsequently become tumorigenic and the hTERT promoter is re-activated, application of ganciclovir (GCV), a clinically used anti-virus drug, will achieve selective deletion of the cancerous cells. To prove the principle, we transduced tumor cells, including HT-1080 cells (fibrosarcoma), 293T (transformed human embryonic kidney cells), SCCLSU-1 (primary head and neck squamous cell carcinoma cells), and Jurkat cells (acute T cell leukemia cells), and normal cells, including primary human airway fibroblast cells and human marrow mesenchymal stem cells, with 10 MOIs of the HIV-CMV-EGFP-hTERT-TK vector and the HIV-CMV-EGFP control vector, respectively. GCV application only eradicated the tumor cells transduced with vector containing the hTERT-TK element, but had no effects on the normal cells received an identical treatment, suggesting the hTERT-TK cassette in the lentiviral vector was adequate to differentiate tumor cells from normal, thus destroying tumor cells selectively. HT1080 cells with single HIV-CMV-EGFP-hTERT-TK integration was implanted into left flanks of nu/nu mice and the cells with HIV-CMV-EGFP integration to the right flanks of the same animals. After establishment of tumors, GCV administration ablated the left-side tumors but not the right-side tumors. These results suggest the potential of hTERT-TK cassette in integrating vectors as a safety valve for human gene therapy to control post-therapeutic insertional mutagenesis.