Multiply attenuated, self-inactivating lentiviral vectors efficiently transduce human coronary artery cells in vitro and rat arteries in vivo

Endothelial cells (ECs) in normal vessels are poorly transducible by retroviral vectors, which require cell division for gene transduction. Among retroviruses, lentiviruses have the unique ability to integrate their genome into the chromatin of nondividing cells. Here we show that multiply attenuated, self-inactivating, lentiviral vectors transduce both proliferating and growth-arrested human umbilical vein ECs (HUVECs), human coronary artery ECs (HCAECs), and human coronary artery smooth muscle cells (HCASMCs), with high efficacy. Lentiviral vectors containing the enhanced green fluorescence protein (EGFP) transgene driven by either the cytomegalovirus or the elongation factor-1alpha promoter, but not the phosphoglycerate kinase promoter, directed high-level EGFP expression in endothelial and smooth muscle cells. The endothelium-specific Tie2 promoter also directed transgene expression in ECs. Re-insertion of cis-acting sequences from pol of human immunodeficiency virus type 1 (HIV-1) into the vectors improved transgene expression. A lentiviral vector containing the vascular endothelial growth factor transgene promoted EC proliferation and sprouting in vitro. In vivo gene transfer was studied by lumenal infusion of vector containing solutions into rat carotid arteries. Lentivirus-mediated EGFP gene transfer was observed in approximately 5% of ECs. Lentiviral vectors containing the LacZ transgene achieved detectable beta-galactosidase activity in rat arteries, albeit at a lower level compared with adenoviral vectors. This difference was mainly due to the lower concentration of lentiviral vector preparations. Lentivirus-mediated gene transfer was associated with minimal neointimal hyperplasia and scant inflammatory cell infiltrates in the media and adventitia. These observations indicate that lentiviral vectors may be useful for genetic modifications of vascular cells in vitro and in vivo.     

Onco-retroviral and lentiviral vector-based gene therapy for hemophilia: preclinical studies

Hemophilia A and B gene therapy requires long-term and stable expression of coagulation factor VIII (FVIII) or factor IX (FIX), respectively, and would need to compare favorably with protein replacement therapy. Onco-retroviral and lentiviral vectors are attractive vectors for gene therapy of hemophilia. These vectors have the potential for long-term expression because they integrate stably in the target cell genome. Whereas onco-retroviral vectors can only transduce dividing cells, lentiviral vectors can transduce a broad variety of cell types irrespective of cell division. Several preclinical and clinical studies have explored the use of onco-retroviral and, more recently, lentiviral vectors for gene therapy of hemophilia A or B. Both ex vivo and in vivo gene therapy approaches have been evaluated, resulting in therapeutic FVIII or FIX levels in preclinical animal models. Whereas in vivo gene therapy using onco-retroviral or lentiviral vectors often led to long-term FVIII or FIX expression from transduced hepatocytes, ex vivo approaches were generally hampered by either low or transient expression of FVIII or FIX levels in vivo and/or inefficient engraftment. Furthermore, immune responses against the transgene product remain a major issue that must be resolved before the full potential of these vectors eventually can be exploited clinically. Nevertheless, the continued progress in vector design combined with a better understanding of vector biology may ultimately yield more effective gene therapy approaches using these integrating vectors.     

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.     

Design of an HIV-1 lentiviral-based gene-trap vector to detect developmentally regulated genes in mammalian cells

The recent development of HIV-1 lentiviral vectors is especially useful for gene transfer because they achieve efficient integration into nondividing cell genomes and successful long-term expression of the transgene. These attributes make the vector useful for gene delivery, mutagenesis, and other applications in mammalian systems. Here we describe two HIV-1-based lentiviral vector derivatives, pZR-1 and pZR-2, that can be used in gene-trap experiments in mammalian cells in vitro and in vivo. Each lentiviral gene-trap vector contains a reporter gene, either beta-lactamase or enhanced green fluorescent protein (EGFP), that is inserted into the U3 region of the 3' long terminal repeat. Both of the trap vectors readily integrate into the host genome by using a convenient infection technique. Appropriate insertion of the vector into genes causes EGFP or beta-lactamase expression. This technique should facilitate the rapid enrichment and cloning of the trapped cells and provides an opportunity to select subpopulations of trapped cells based on the subcellular localization of reporter genes. Our findings suggest that the reporter gene is driven by an upstream, cell-specific promoter during cell culture and cell differentiation, which further supports the usefulness of lentivirus-based gene-trap vectors. Lentiviral gene-trap vectors appear to offer a wealth of possibilities for the study of cell differentiation and lineage commitment, as well as for the discovery of new genes.     

Intercellular delivery of a herpes simplex virus VP22 fusion protein from cells infected with lentiviral vectors

Effective gene therapy depends on the efficient transfer of therapeutic genes and their protein products to target cells. Lentiviral vectors appear promising for virus-mediated gene delivery and long-term expression in nondividing cells. The herpes simplex virus type 1 tegument protein VP22 has recently been shown to mediate intercellular transport of proteins, raising the possibility that it may be helpful in a setting where the global delivery of therapeutic proteins is desired. To investigate the effectiveness of lentiviral vectors to deliver genes encoding proteins fused to VP22, and to test whether the system is sufficiently potent to allow protein delivery from transduced cells in vitro and in vivo, fusion constructs of VP22 and the enhanced green fluorescent protein (EGFP) were prepared and delivered into target cells by using HIV-1-based lentiviral vectors. To follow the spread of VP22-EGFP to other cells, transduced COS-7 cells were coplated with a number of different cell types, including brain choroid plexus cells, human endothelial cells, H9 cells, and HeLa cells. We found that VP22-EGFP fusion proteins were transported from transduced cells to recipient cells and that such fusion proteins accumulated in the nucleus and in the cytoplasm of such cells. To determine the ability to deliver fusion proteins in vivo, we injected transduced H9 cells as well as the viral vector directly into the brain of mice. We present evidence that VP22-EGFP fusion proteins were transported effectively from lentivirus transduced cells in vivo. We also show that the VP22-EGFP fusion protein encoded by the lentivirus is transported between cells. Our data indicate that such fusion proteins are present in the nucleus and in the cytoplasm of neighboring cells. Therefore, lentiviral vectors may provide a potent biological system for delivering genes encoding therapeutic proteins fused to VP22.     

Efficient lentiviral transduction of human cord blood CD34(+) cells followed by their expansion and differentiation into dendritic cells

OBJECTIVE: To support immune reconstitution after cord blood transplantation, immunotherapy using gene-modified dendritic cells (DCs), the most potent antigen-presenting cells, can be a powerful strategy for preventing infection and recurrence. To investigate the applicability of lentiviral vector-transduced DCs compared to retroviral vectors, we transduced umbilical cord blood (CB) CD34(+) cells, then expanded and differentiated them into DCs. MATERIALS AND METHODS: We transduced CB CD34(+) cells by vesicular stomatitis virus G-protein pseudotyped self-inactivating lentiviral vector or retroviral vectors carrying the enhanced green fluorescent protein gene. The cells were expanded in the stroma-dependent culture system and transferred to the culture condition for developing DCs. The efficiency of transduction and expression of the transgene in severe combined immunodeficiency (SCID) mice-repopulating cells (SRCs) and DCs were compared between lentiviral vector and retroviral vectors. Induced DCs were cocultured with allogeneic or autologous T cells to test the ability to present antigens. RESULTS: CB CD34(+) cells transduced by lentiviral vector and expanded ex vivo sustained stable transgene expression and multipotentiality by assessing SRCs assay and clonogenic assay of bone marrow cells from the transplanted mice. DCs derived from these cells expressed green fluorescent protein and surface markers CD1a, CD80, and HLA-DR and showed potent allo-stimulatory activity as well as nontransduced DCs did. On the other hand, we did not detect transgene expression in SRCs and DCs transduced by retroviral vectors. CONCLUSION: Gene-modified DCs derived from ex vivo expanded CB CD34(+) cells transduced by lentiviral vector will be useful in future immunotherapy protocols.     

HIV-1 vectors: fulfillment of expectations, further advancements, and still a way to go

The ability of lentiviral vectors to transduce and stably integrate their genomes into non-dividing cells was the major reason for the development of the HIV-1 based vector gene delivery system. The first VSV-G pseudotyped lentiviral vectors fulfilled these expectations by ferrying large genetic payloads to non-dividing cells in vitro and in vivo. Here we discuss advances in HIV-1 vector systems which lead to improvement in biosafety, transduction efficiency, longevity and regulation of transgene expression, and vector production. The successful use of the advanced HIV-1 based vector system opened new avenues in establishing transgenic animal models for basic research. Additionally, we describe accomplishments using HIV-1 based vectors to correct pathological courses of incurable diseases in preclinical animal models including Parkinson's disease and beta-thalassemia.     

Herpes simplex virus thymidine kinase-mediated suicide gene therapy for hepatocellular carcinoma using HIV-1-derived lentiviral vectors

BACKGROUND/AIMS: Gene therapy is a promising approach for treatment of hepatocellular carcinoma (HCC). However, transduction of non-tumoral hepatocytes may lead to severe hepatitis when using suicide gene therapy approaches. The aim of our study was to evaluate the gene transfer efficiency into HCC cells and normal hepatocytes using human immunodeficiency virus (HIV)-derived lentiviral vectors in vitro and in vivo. METHODS: Lentiviral vectors encoding for the LacZ gene or the fusion gene HSV-Tk/GFP were tested in vitro in human HCC cells and human hepatocytes in primary culture and in vivo in a chemically induced rat model of HCC. RESULTS: We show that HIV-1-derived lentiviral vectors are efficient in transducing HCC cells in vitro and in vivo. No significant transduction of non-tumorous hepatocytes was observed in vivo whatever the route of administration used. Measurement of tumor growth following direct intratumoral injection of a lentiviral vector containing the HSV-Tk gene and GCV treatment showed a strong antitumoral efficacy in the absence of normal liver toxicity. CONCLUSIONS: These observations suggest that lentiviral vectors allow an antitumoral effect with low liver toxicity when using suicide gene therapy approach and could be efficient tools for HCC gene therapy.     

Cre-lox-regulated conditional RNA interference from transgenes

We have generated two lentiviral vectors for conditional, Cre-lox-regulated, RNA interference. One vector allows for conditional activation, whereas the other permits conditional inactivation of short hairpin RNA (shRNA) expression. The former is based on a strategy in which the mouse U6 promoter has been modified by including a hybrid between a LoxP site and a TATA box. The ability to efficiently control shRNA expression by using these vectors was shown in cell-based experiments by knocking down p53, nucleophosmin and DNA methyltransferase 1. We also demonstrate the usefulness of this approach to achieve conditional, tissue-specific RNA interference in Cre-expressing transgenic mice. Combined with the growing array of Cre expression strategies, these vectors allow spatial and temporal control of shRNA expression in vivo and should facilitate functional genetic analysis in mammals.     

Specific transgene expression in human and mouse CD4+ cells using lentiviral vectors with regulatory sequences from the CD4 gene

Achieving cell-specific expression of a therapeutic transgene by gene transfer vectors represents a major goal for gene therapy. To achieve specific expression of a transgene in CD4(+) cells, we have generated lentiviral vectors expressing the enhanced green fluorescent protein (eGFP) reporter gene under the control of regulatory sequences derived from the CD4 gene--a minimal promoter and the proximal enhancer, with or without the silencer. Both lentiviral vectors could be produced at high titers (more than 10(7) infectious particles per milliliter) and were used to transduce healthy murine hematopoietic stem cells (HSCs). On reconstitution of RAG-2-deficient mice with transduced HSCs, the specific vectors were efficiently expressed in T cells, minimally expressed in B cells, and not expressed in immature cells of the bone marrow. Addition of the CD4 gene-silencing element in the vector regulatory sequences led to further restriction of eGFP expression into CD4(+) T cells in reconstituted mice and in ex vivo-transduced human T cells. Non-T CD4(+) dendritic and macrophage cells derived from human CD34(+) cells in vitro expressed the transgene of the specific vectors, albeit at lower levels than CD4(+) T cells. Altogether, we have generated lentiviral vectors that allow specific targeting of transgene expression to CD4(+) cells after differentiation of transduced mice HSCs and human mature T cells. Ultimately, these vectors may prove useful for in situ injections for in vivo gene therapy of HIV infection or genetic immunodeficiencies.     

A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA

We describe the use of lentiviral vectors expressing small interfering RNAs (siRNAs) to knock down the expression of specific genes in vitro and in vivo. A lentiviral vector capable of generating siRNA specific for GFP after transduction of 293T-GFP cell lines showed no GFP fluorescence. Furthermore, no GFP-specific RNA could be detected. When eggs from GFP-positive transgenic mice were transduced with lentivirus-expressing siGFP virus, reduced fluorescence could be seen in blastocysts. More interestingly, pups from F(1) progeny, which expressed siGFP, showed considerably diminished fluorescence and decreased GFP. We propose that an approach of combining transgenesis by lentiviral vectors expressing siRNAs can be used successfully to generate a large number of mice in which the expression of a specific gene(s) can be down-regulated substantially. We believe that this approach of generating "knockdown" mice will aid in functional genomics.     

Targeting transgene expression to antigen-presenting cells derived from lentivirus-transduced engrafting human hematopoietic stem/progenitor cells.

Hematopoietic stem cells (HSCs) represent an important target for the treatment of various blood disorders. As the source of critical cells within the immune system, genetic modification of HSCs can also be used to modulate immune responses. The effectiveness of HSC-mediated gene therapy largely depends on efficient gene delivery into long-term repopulating progenitors and targeted transgene expression in an appropriate progeny of the transduced pluripotent HSCs. Self-inactivating (SIN) lentiviral vectors have been demonstrated to be capable of transducing mitotically inactive cells, including HSCs, and accommodating a nonviral promoter to control the transgene expression in transduced cells. In this study, we constructed 2 SIN lentiviral vectors, EF.GFP and DR.GFP, to express the green fluorescent protein (GFP) gene controlled solely by the promoter of either a housekeeping gene EF-1alpha or the human HLA-DRalpha gene, which is selectively expressed in antigen-presenting cells (APCs). We demonstrated that both vectors efficiently transduced human pluripotent CD34+ cells capable of engrafting nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. When the EF.GFP vector was used, constitutive high-level GFP expression was obtained in all the human HSC progeny detectable in NOD/SCID mice and in subsequent in vitro differentiation assays, indicating that engrafting human HSCs have been transduced. In contrast, the DR.GFP vector mediated transgene expression specifically in human HLA-DR+ cells and highly in differentiated dendritic cells (DCs), which are critical in regulating immunity. Furthermore, human DCs derived from transduced and engrafted human cells potently stimulated allogeneic T-cell proliferation. This study demonstrated successful targeting of transgene expression to APCs/DCs after stable gene transduction of pluripotent HSCs.     

A helper-dependent adenovirus vector system: Removal of helper virus by Cre-mediated excision of the viral packaging signal

Adenoviruses are attractive vectors for the delivery of foreign genes into mammalian cells for gene therapy. However, current vectors retain many viral genes that, when expressed at low levels, contribute to the induction of a host immune response against transduced cells. We have developed a helper-dependent packaging system for production of vectors that have large regions of the genome deleted. Helper viruses were constructed with packaging signals flanked by loxP sites so that in 293 cells that stably express the Cre recombinase (293Cre), the packaging signal was efficiently excised, rendering the helper virus genome unpackageable. However, the helper virus DNA was replicated at normal levels and could thus express all of the functions necessary in trans for replication and packaging of a vector genome containing the appropriate cis-acting elements. Serial passage of the vector in helper virus-infected 293Cre cells resulted in an ≈10-fold increase in vector titer per passage. The vector could be partially separated from residual helper virus by cesium chloride buoyant density centrifugation. Large scale preparations of vector yielded semi-purified stocks of ≈10¹⁰ transducing virions/ml, with <0.01% contamination by the E1-deleted helper virus. This system should have great utility for the generation of adenovirus-based vectors with increased cloning capacity, increased safety and reduced immunogenicity.     

Improved adenovirus packaging cell lines to support the growth of replication-defective gene-delivery vectors.

Adenovirus (Ad) vectors have been extensively used to deliver recombinant genes to a great variety of cell types in vitro and in vivo. Ad-based vectors are available that replace the Ad early region 1 (E1) with recombinant foreign genes. The resultant E1-deleted vectors can then be propagated on 293 cells, a human embryonal kidney cell line that constitutively expresses the E1 genes. Unfortunately, infection of cells and tissues in vivo results in low-level expression of Ad early and late proteins (despite the absence of E1 activity) resulting in immune recognition of virally infected cells. The infected cells are subsequently eliminated, resulting in only a transient expression of foreign genes in vivo. We hypothesize that a second-generation Ad vector with a deletion of viral genes necessary for Ad genome replication should block viral DNA replication and decrease viral protein production, resulting in a diminished immune response and extended duration of foreign gene expression in vivo. As a first step toward the generation of such a modified vector, we report the construction of cell lines that not only express the E1 genes but also constitutively express the Ad serotype 2 140-kDa DNA polymerase protein, one of three virally encoded proteins essential for Ad genome replication. The Ad polymerase-expressing cell lines support the replication and growth of H5ts36, an Ad with a temperature-sensitive mutation of the Ad polymerase protein. These packaging cell lines can be used to prepare Ad vectors deleted for the E1 and polymerase functions, which should facilitate development of viral vectors for gene therapy of human diseases.