Inducible expression of herpes simplex virus thymidine kinase from a bicistronic HIV1 vector

The possibility of protecting human CD4+ lymphocytes from human immunodeficiency virus type 1 (HIV1) infection, through a suicide mechanism elicited by the HIV1 transcription apparatus itself, offers a potentially useful approach for gene therapy of the acquired immunodeficiency syndrome. A replication-defective lentiviral HIV1 vector (HYIRES-TK) was designed to carry both the hygromycin (Hy) phosphotransferase gene for positive selection and the thymidine kinase (TK) gene of herpes simplex virus driven by the viral long terminal repeat (LTR). The internal ribosome entry site (IRES) from encephalomyocarditis virus was placed between the two genes for their efficient simultaneous translation. Transient expression of active TK into transfected COS-1 cells was shown to be induced by Tat and Rev over a detectable basal level. By providing the missing viral proteins in trans, recombinant viruses were generated and used to transduce Jurkat cells. The Hy-resistant population of cells was sensitive to ganciclovir (GCV) and acyclovir (ACV), a result consistent with a basal level of TK expression. Cocultivation of transduced cells with cells chronically infected with HIV in the presence of 10 microM ACV, a concentration non-toxic for the uninfected cells, resulted in increased killing of cells transduced with the HY-IRES-TK vector. These data indicate that two genes can be expressed from the viral LTR in the context of an HIV1 vector, with the aid of an IRES sequence. The expression is inducible by the HIV proteins Tat and Rev and it is possible to specifically kill infected cells with subtoxic concentrations of drug. To decrease the sensitivity of the transduced cells towards GCV, a variant vector expressing a truncated TK was constructed. The truncated version was expressed at levels similar to those of wild-type TK but induced sensitivity towards GCV in transduced cells that was intermediate between that of untransduced cells and of cells expressing wild-type TK.