DEVELOPMENT OF A SELF-INACTIVATING LENTIVIRUS VECTOR

We have constructed a new series of lentivirus vectors based on human immunodeficiency virus type 1 (HIV-1) that can transduce nondividing c ells. The U3 region of the 5' long terminal repeat (LTR) in vector con structs was replaced with the cytomegalovirus (CM) promoter, resulting in Tat-independent transcription but still maintaining high levels of expression. A self-inactivating (SIN) vector was constructed by delet ing 133 bp in the U3 region of the 3' LTR, including the TATA box and binding sites for transcription factors Spl and NF-kappa B. The deleti on is transferred to the 5' LTR after reverse transcription and integr ation in infected cells, resulting in the transcriptional inactivation of the LTR in the proviruses. SIN viruses can be generated with no si gnificant decreases in titer. Injection of viruses into the rat brain showed that a SIN vector containing the green fluorescent protein gene under the control of the internal CMV promoter transduced neurons as efficiently as a wild-type vector. Interestingly, a wild-type vector w ithout an internal promoter also successfully transduced neurons in th e brain, indicating that the HIV-1 LTR promoter is transcriptionally a ctive in neurons even in the absence of Tat. Furthermore, injection of viruses into the subretinal space of the rat eye showed that wild-typ e vector transduced predominantly retinal pigment epithelium and photo receptor cells, while SIN vector was able to transduce other types of retinal cells, including bipolar, Muller, horizontal, and amacrine cel ls. This finding suggests that the HIV-1 LTR can negatively influence the internal CMV promoter in some cell types. SIN HIV vectors should b e safer for gene therapy, and they also have broader applicability as a means of high-level gene transfer and expression in nondividing cell s.