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Lentiviral vector mobilization following HIV-1 infection of vector-transduced cells poses biosafety risks to vector-treated patients and their communities. The self-inactivating (SIN) vector design has reduced, however, not abolished mobilization of integrated vector genomes. Furthermore, an earlier study demonstrated the ability of the major product of reverse transcription, a circular SIN HIV-1 vector comprising a single- long terminal repeat (LTR) to support production of high vector titers. Here, we demonstrate that configuring the internal vector expression cassette in opposite orientation to the LTRs abolishes mobilization of SIN vectors. This additional SIN mechanism is in part premised on induction of host PKR response to double-stranded RNAs comprised of mRNAs transcribed from cryptic transcription initiation sites around 3'SIN-LTR's and the vector internal promoter. As anticipated, PKR response following transfection of opposite orientation vectors, negatively affects their titers. Importantly, shRNA-mediated knockdown of PKR rendered titers of SIN HIV-1 vectors comprising opposite orientation expression cassettes comparable to titers of conventional SIN vectors. High-titer vectors carrying an expression cassette in opposite orientation to the LTRs efficiently delivered and maintained high levels of transgene expression in mouse livers. This study establishes opposite orientation expression cassettes as an additional PKR-dependent SIN mechanism that abolishes vector mobilization from integrated and episomal SIN lentiviral vectors.

Integration-deficient lentiviral vectors (IDLVs) have been shown to transduce a wide spectrum of target cells and organs in vitro and in vivo and to maintain long-term transgene expression in nondividing cells. However, epigenetic silencing of episomal vector genomes reduces IDLV transgene expression levels and renders these safe vectors less efficient. In this article, we describe for the first time a complete correction of factor IX (FIX) deficiency in hemophilia B mice by IDLVs carrying a novel, highly potent human FIX cDNA. A 50-fold increase in human FIX cDNA potency was achieved by combining two mechanistically independent yet synergistic strategies: (i) optimization of the human FIX cDNA codon usage to increase human FIX protein production per vector genome and (ii) generation of a highly catalytic mutant human FIX protein in which the arginine residue at position 338 was substituted with leucine. The enhanced human FIX activity was not associated with liver damage or with the formation of human FIX-directed inhibitory antibodies and rendered IDLV-treated FIX-knockout mice resistant to a challenging tail-clipping assay. A novel S1 nuclease-based B1-quantitative polymerase chain reaction assay showed low levels of IDLV integration in mouse liver. Overall, this study demonstrates that IDLVs carrying an improved human FIX cDNA safely and efficiently cure hemophilia B in a mouse model.

Nonintegrating lentiviral vectors present a means of reducing the risk of insertional mutagenesis in nondividing cells and enabling short-term expression of potentially hazardous gene products. However, residual, integrase-independent integration raises a concern that may limit the usefulness of this system. Here we present a novel 3' polypurine tract (PPT)-deleted lentiviral vector that demonstrates impaired integration efficiency and, when packaged into integrase-deficient particles, significantly reduced illegitimate integration. Cells transduced with PPT-deleted vectors exhibited predominantly 1-long terminal repeat (LTR) circles and a low level of linear genomes after reverse transcription (RT). Importantly, the PPT-deleted vector exhibited titers and in vitro and in vivo expression levels matching those of conventional nonintegrating lentiviral vectors. This safer nonintegrating lentiviral vector system will support emerging technologies, such as those based on transient expression of zinc-finger nucleases (ZFNs) for gene editing, as well as reprogramming factors for inducing pluripotency.

The feasibility of using nonintegrating lentiviral vectors has been demonstrated by recent studies showing their ability to maintain transgene expression both in vitro and in vivo. Furthermore, human immunodeficiency virus-1 (HIV-1) vectors packaged with a mutated integrase were able to correct retinal disease in a mouse model. Interestingly, these results differ from earlier studies in which first-generation nonintegrating lentiviral vectors yielded insignificant levels of transduction. However, to date, a rigorous characterization of transgene expression from the currently used self-inactivating (SIN) nonintegrating lentiviral vectors has not been published. In this study, we characterize transgene expression from SIN nonintegrating lentiviral vectors. Overall, we found that nonintegrating vectors express transgenes at a significantly lower level than their integrating counterparts. Expression from nonintegrating vectors was improved upon introducing a longer deletion in the vector's U3 region. A unique shuttle-vector assay indicated that the relative abundance of the different episomal forms was not altered by the longer U3 deletion. Interestingly, the longer U3 deletion did not enhance expression in the corpus callosum of the rat brain, suggesting that the extent of silencing of episomal transcription is influenced by tissue-specific factors. Finally, and for the first time, episomal expression in the mouse liver was potent and sustained.

The capacity to efficiently transduce nondividing cells, shuttle large genetic payloads, and maintain stable long-term transgene expression are attributes that have brought lentiviral vectors to the forefront of gene delivery vehicles for research and therapeutic applications in a clinical setting. Our discussion initiates with advances in lentiviral vector development and how these sophisticated lentiviral vectors reflect improvements in safety, regarding the prevention of replication competent lentiviruses (RCLs), vector mobilization, and insertional mutagenesis. Additionally, we describe conventional molecular regulatory systems to manage gene expression levels in a spatial and temporal fashion in the context of a lentiviral vector. State of the art technology for lentiviral vector production by transient transfection and packaging cell lines are explicitly presented with current practices used for concentration, purification, titering, and determining the safety of a vector stock. We summarize lentiviral vector applications that have received a great deal of attention in recent years including the generation of transgenic animals and the stable delivery of RNA interference molecules. Concluding remarks address some of the successes in preclinical animals, and the recent transition of lentiviral vectors to human clinical trials as therapy for a variety of infectious and genetic diseases.

Lentiviral vectors can confer high levels of gene transfer and transgene expression in a variety of cell types. However, the biodistribution and toxicity after intravenous administration have not been reported. To address these issues of biodistribution and toxicity, an HIV-1-based vector, HR'cmvGFP, was administered to normal BALB/c mice by tail-vein injection. Nine different organs and bone marrow were evaluated by real-time quantitative PCR (QPCR) assay capable of a broad range of quantitation (5-log fold) to detect as few as one copy of the green fluorescent protein gene (GFP) per 10(5) cells. Four days after vector administration, high levels of transgene and gene expression were observed in liver, spleen, and bone marrow in all animals. By 40 days after injection, GFP levels had decreased in liver and spleen, but bone marrow exhibited a consistently high level of transgene. This finding was consistent with the increase in both GFP frequency and expression levels observed in peripheral blood by fluorescence-activated cell-sorting (FACS) analysis. Between 0 and 1% transgene was detected in all other organs. No significant pathologic lesions were found attributable to vector in any of the tissues examined. The observation of bone marrow transduction after intravenous vector administration suggests the possibility of an in vivo approach to stem cell gene therapy.

The ability to transduce non-dividing cells is a unique feature of lentiviruses which distinguishes them from simple retroviruses. This feature was the major incentive for the development of the lentivirus vector system. Lentivirus vectors can deliver and integrate > 8 kb of transgenic DNA into target cell genomes without inducing a host immune response against the transduced cells. Thus lentivirus vector-based gene delivery can be considered the most efficient method by which transgenes can be incorporated into the host cell genome and maintain long-term expression. This review describes the major developments in the lentivirus vector system, which significantly improve vector biosafety, vector production and transgene expression. The success and difficulties in reverting disease phenotypes by lentivirus vectors carrying therapeutic genes in various animal models including beta-thalassemia and Parkinson's disease and the implications of these studies for future gene therapy clinical trials are also discussed.

To facilitate the generation of SIN lentivirus vector-producer cell lines, we have developed a novel conditional SIN (cSIN) lentivirus vector, which retains its SIN properties in normal target cells yet can be produced at high titers from tetracycline-regulated packaging cell lines. The design of the cSIN vector is based on replacing the vector U3 transcription regulatory elements with the Tet-responsive element, which allows vector production exclusively in cells expressing the synthetic Tet-regulated transactivator (tTA). In contrast minimal vector production ( approximately 200 IU/ml) is obtained in target cells that do not express the tTA, even in the presence of all HIV-1 proteins. Following transduction of the Tet-regulated SODk1 lentivirus vector-packaging cell line with the cSIN vector, high titers of cSIN recombinant vector (>10(6) IU/ml) could be generated, which efficiently transduced terminally differentiated neurons in normal rat brain.

Lentiviral vectors can deliver and express genes in a wide variety of dividing and nondividing cells. These include terminally differentiated neurons, myotubes, hepatocytes, and hematopoietic stem cells. We now describe the generation of lentiviral vectors in which the expression of the transgene can be regulated. We have developed an inducible lentiviral vector system that contains the entire tetracycline (Tet)-regulated system developed by H. Bujard and colleagues. The novel vector expresses the GFP reporter gene and the tetracycline transactivator under the control of the tetracycline-inducible promoter and the human CMV promoter, respectively. In vitro transduction of human 293 cells resulted in a very low basal expression of GFP in the presence of the effector substance doxycyline. Withdrawal of doxycyline induced a more than 500-fold increase in transgene expression. Switching transgene expression "off and on" did not change either the kinetics or the magnitude of induction. Maximal suppression of GFP mRNA transcription was achieved within 24 h of addition of the drug; however, due to the slow turnover rate of GFP, green fluorescent cells could be detected up to 10 days following doxycyline treatment. Following transduction of rat brain with recombinant lentiviruses, doxycyline-regulated GFP expression could be observed in terminally differentiated neurons. Specifically, by adding or withdrawing doxycyline from the rats' drinking water, induction and suppression of GFP expression could be regulated in vivo. These studies show that an inducible lentiviral vector can deliver and regulate transgene expression in vivo. We believe that regulated gene expression is an essential tool for successful gene therapy approaches.

Lentivirus vectors can transduce dividing and nondividing cells. Using three-plasmid transient transfections, high-titer (>10(9) IU/ml) recombinant lentivirus vectors pseudotyped with vesicular stomatitis virus G (VSV-G) protein can be generated (T. Kafri et al., Nat. Genet. 17:314-317, 1997; H. Miyoshi et al., Proc. Natl. Acad. Sci. USA 94:10319-10323, 1997; L. Naldini et al., Science 272:263-267, 1996). The recombinant lentiviruses can efficiently infect brain, liver, muscle, and retinal tissue in vivo. Furthermore, the transduced tissues demonstrated long-term expression of reporter genes in immunocompetent rodents. We now report the generation of a tetracycline-inducible VSV-G pseudotyped lentivirus packaging cell line which can generate virus particles at titers greater than 10(6) IU/ml for at least 3 to 4 days. The vector produced by the inducible cell line can be concentrated to titers of 10(9) IU/ml and can efficiently transduce nondividing cells in vitro and in vivo. The availability of a lentivirus packaging cell line will significantly facilitate the production of high-titer lentivirus vectors for gene therapy and study of human immunodeficiency virus biology.

Autoimmune destruction of islets in the pancreas leads to the development of insulin-dependent diabetes mellitus (IDDM). Replacement of insulin-producing tissue by transplantation of islets provides a cure to disease but requires immunosuppression or a means of controlling anti-graft immune responses. To promote islet survival we have utilized a local approach by expressing immunoregulatory molecules in islet grafts. The results presented here show that the human immunodeficiency virus (HIV)-based lentiviral vector is capable of stably transducing whole islets. Foreign reporter gene expression was observed both in vitro and in vivo 30 days after transplantation. Grafts containing insulin-positive beta-islet cells expressing foreign protein indicate that transduction does not interfere with glucose regulation. The absence of inflammatory infiltrates in grafts suggests that transduction does not activate the immune system. When islets transduced with an HIV vector expressing IL-4 were transplanted into diabetes-prone mice, animals were protected from autoimmune insulitis and islet destruction. As demonstrated by proliferative and cytokine analysis, protection was consistent with a switching of islet-antigen-specific T cell responses toward a Th2 phenotype. These results suggest that HIV-based lentivirus vectors can efficiently transduce islet cells with genes encoding potentially therapeutic molecules, for possibly managing diabetes.

Successful gene therapy approaches will require efficient gene delivery and sustained expression of the transgene in recipients. A variety of methods, ranging from direct DNA delivery to infection with recombinant viruses containing foreign genes, have been developed, but they all have some major limitations that restrict their utility. We have described a human lentiviral (HIV)-based vector that can transduce non-dividing cells in vitro and deliver genes in vivo. With this vector, expression of transgenes in the brain has been detected for more than six months--the longest period tested so far. Because lentiviral vectors are pseudotyped with vesicular stomatitis virus G glycoprotein (VSVG; ref. 8), they can transduce a broad range of tissues and cell types. We now describe the ability of lentiviral vectors to introduce genes directly into liver and muscle. Sustained expression of green fluorescent protein (GFP), used as a surrogate for therapeutic protein, can be observed for more than 22 weeks in the liver. Similar long-term expression (more than eight weeks) was observed in transduced muscle. In contrast, little or no GFP could be detected in liver or muscle transduced with the Moloney murine leukaemia virus (M-MLV), a prototypic retroviral based vector. At a minimum, 3-4% of the total liver tissue was transduced by a single injection of 1-3 x 10(7) infectious units (I.U.) of recombinant HIV vector. Furthermore, no inflammation of recruitment of lymphocytes could be detected at the site of injection. Animals previously transduced with a lentiviral vector can be efficiently re-infected with lentiviral vectors. Additionally, we show that the requirement for lentiviral accessory proteins to establish efficient transduction in vivo is tissue dependent.

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.