Ulrike Blömer

A retroviral vector system based on the human immunodeficiency virus (HIV) was developed that, in contrast to a murine leukemia virus-based counterpart, transduced heterologous sequences into HeLa cells and rat fibroblasts blocked in the cell cycle, as well as into human primary macrophages. Additionally, the HIV vector could mediate stable in vivo gene transfer into terminally differentiated neurons. The ability of HIV-based viral vectors to deliver genes in vivo into nondividing cells could increase the applicability of retroviral vectors in human gene therapy.

We describe the construction of a safe, replication-defective and efficient lentiviral vector suitable for in vivo gene delivery. The reverse transcription of the vector was found to be a rate-limiting step; therefore, promoting the reaction inside the vector particles before delivery significantly enhanced the efficiency of gene transfer. After injection into the brain of adult rats, sustained long-term expression of the transgene was obtained in the absence of detectable pathology. A high proportion of the neurons in the areas surrounding the injection sites of the vector expressed the transduced beta-galactosidase gene. This pattern was invariant in animals sacrificed several months after a single administration of the vector. Transduction occurs by integration of the vector genome, as it was abolished by a single amino acid substitution in the catalytic site of the integrase protein incorporated in the vector. Development of clinically acceptable derivatives of the lentiviral vector may thus enable the sustained delivery of significant amounts of a therapeutic gene product in a wide variety of somatic tissues.

We have constructed a new series of lentivirus vectors based on human immunodeficiency virus type 1 (HIV-1) that can transduce nondividing cells. The U3 region of the 5' long terminal repeat (LTR) in vector constructs was replaced with the cytomegalovirus (CMV) promoter, resulting in Tat-independent transcription but still maintaining high levels of expression. A self-inactivating (SIN) vector was constructed by deleting 133 bp in the U3 region of the 3' LTR, including the TATA box and binding sites for transcription factors Sp1 and NF-kappaB. The deletion is transferred to the 5' LTR after reverse transcription and integration in infected cells, resulting in the transcriptional inactivation of the LTR in the proviruses. SIN viruses can be generated with no significant 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 without an internal promoter also successfully transduced neurons in the brain, indicating that the HIV-1 LTR promoter is transcriptionally active in neurons even in the absence of Tat. Furthermore, injection of viruses into the subretinal space of the rat eye showed that wild-type vector transduced predominantly retinal pigment epithelium and photoreceptor cells, while SIN vector was able to transduce other types of retinal cells, including bipolar, Müller, horizontal, and amacrine cells. This finding suggests that the HIV-1 LTR can negatively influence the internal CMV promoter in some cell types. SIN HIV vectors should be safer for gene therapy, and they also have broader applicability as a means of high-level gene transfer and expression in nondividing cells.

The identification of monogenic and complex genes responsible for neurological disorders requires new approaches for delivering therapeutic protein genes to significant numbers of cells in the central nervous system. A lentivirus-based vector capable of infecting dividing and quiescent cells was investigated in vivo by injecting highly concentrated viral vector stock into the striatum and hippocampus of adult rats. Control brains were injected with a Moloney murine leukemia virus, adenovirus, or adeno-associated virus vector. The volumes of the areas containing transduced cells and the transduced-cell densities were stereologically determined to provide a basis for comparison among different viral vectors and variants of the viral vector stocks. The efficiency of infection by the lentivirus vector was improved by deoxynucleoside triphosphate pretreatment of the vector and was reduced following mutation of integrase and the Vpr-matrix protein complex involved in the nuclear translocation of the preintegration complex. The lentivirus vector system was able to efficiently and stably infect quiescent cells in the primary injection site with transgene expression for over 6 months. Triple labeling showed that 88.7% of striatal cells transduced by the lentivirus vector were terminally differentiated neurons.