Lentiviral vectors mediate nonimmunosuppressive rapamycin analog-induced production of secreted therapeutic factors in the brain: regulation at the level of transcription and exocytosis.

Gene transfer may become a powerful clinical tool for the delivery of secreted therapeutic polypeptides, provided that the in situ production of these peptides can be tightly regulated by the administration of a small inducer molecule. Particularly efficient control may be achieved by simultaneously using two regulation systems that interfere with the biosynthesis of the therapeutic factor at two different levels. Therefore, we have developed a set of two lentiviral vectors containing two regulation systems. These systems are induced by nonimmunosuppressive derivatives of rapamycin ("rapalogs") and allow simultaneous control of expression and of exocytosis of secreted therapeutic polypeptides. The set of vectors was used to produce green fluorescent protein (GFP) and glial cell line-derived neurotrophic factor (GDNF); GFP served as a model factor to demonstrate expression and entry into the exocytotic pathway in transduced cells. The constructs allowed robust in vitro expression and secretion of the polypeptides in the presence of rapalog AP21967. Withdrawal of the inducer resulted in efficient downregulation. In vivo, tightly regulated production of GFP and GDNF was observed after injection of the constructs into the striata of mice. The vectors thus fulfill key requirements for application in gene therapy.

Lentiviral vectors with a defective integrase allow efficient and sustained transgene expression in vitro and in vivo.

Lentivirus-derived vectors are among the most promising viral vectors for gene therapy currently available, but their use in clinical practice is limited by the associated risk of insertional mutagenesis. We have overcome this problem by developing a nonintegrative lentiviral vector derived from HIV type 1 with a class 1 integrase (IN) mutation (replacement of the 262RRK motif by AAH). We generated and characterized HIV type 1 vectors carrying this deficient enzyme and expressing the GFP or neomycin phosphotransferase transgene (NEO) under control of the immediate early promoter of human CMV. These mutant vectors efficiently transduced dividing cell lines and nondividing neural primary cultures in vitro. After transduction, transient GFP fluorescence was observed in dividing cells, whereas long-term GFP fluorescence was observed in nondividing cells, consistent with the viral genome remaining episomal. Moreover, G418 selection of cells transduced with vectors expressing the NEO gene showed that residual integration activity was lower than that of the intact IN by a factor of 500-1,250. These nonintegrative vectors were also efficient in vivo, allowing GFP expression in mouse brain cells after the stereotactic injection of IN-deficient vector particles. Thus, we have developed a generation of lentiviral vectors with a nonintegrative phenotype of great potential value for secure viral gene transfer in clinical applications.