Efficient transduction of primary human B lymphocytes and nondividing myeloma B cells with HIV-1-derived lentiviral vectors

We studied the transduction of primary human B lymphocytes and myeloma cells with lentiviral vectors. In peripheral blood B cells that had been activated with helper T cells (murine thymoma EL-4 B5) and cytokines, multiply attenuated HIV-1-derived vectors pseudotyped with vesicular stomatitis virus (VSV) G-envelope protein achieved the expression of green fluorescence protein (GFP) in 27% +/- 12% (mean +/- 1 SD; median, 27%) of B cells in different experiments. When compared in parallel cultures, the transducibility of B cells from different donors exhibited little variation. The human cytomegalovirus (CMV) promoter gave 4- to 6-fold higher GFP expression than did the human elongation factor-1alpha promoter. A murine retroviral vector pseudotyped with VSV G protein proved inefficient even in mitotically active primary B cells. B cells freshly stimulated with Epstein-Barr virus were also transducible by HIV vectors (24% +/- 9%), but B cells activated with CD40 ligand and cytokines resisted transduction. Thus, different culture systems gave different results. Freshly isolated, nondividing myeloma cells were efficiently transduced by HIV vectors; for 6 myelomas the range was 14% to 77% (median, 28%) GFP(+) cells. HIV vectors with a mutant integrase led to no significant GFP signal in primary B or myeloma cells, suggesting that vector integration was required for high transduction. In conclusion, HIV vectors are promising tools for studies of gene functions in primary human B cells and myeloma cells for the purposes of research and the development of gene therapies.     

Lentiviral-mediated gene transfer into haematopoietic stem cells

OBJECTIVES: Lentiviral vectors can transduce nondividing cells. As most haematopoietic stem cells (HSCs) are nondividing in vivo, lentiviral vectors are promising viral vectors to transfer genes into HSCs. DESIGN AND SETTING: We have used HIV-1 based lentiviral vectors containing the green fluorescent protein (GFP) gene to transduce umbilical cord blood CD34+ and CD34+/CD38- cells prior to transplantation into NOD/SCID mice. RESULTS: High level engraftment of human cells was obtained and transgene expression was seen in both myeloid and lymphoid lineages. Bone marrow from the primary transplant recipients mice was transplanted into secondary recipients. GFP expression was seen in both lymphoid and myeloid cells in the secondary recipients 6 weeks posttransplantation. Human haematopoietic progenitor colonies were grown from both primary and secondary recipients. Over 50% of the haematopoietic colonies in these recipients were positive for the GFP transgene by PCR. Following inverse PCR, amplified fragments were sequenced and integration of the vector into human genomic DNA was demonstrated. Several vectors containing different internal promoters were tested in NOD/SCID mice that had been transplanted with transduced CD34+ and CD34+/CD38- cells. The elongation factor-1alpha (EF-1alpha) promoter gave the highest level of expression, both in the myeloid and lymphoid progeny of the engrafting cells. CONCLUSIONS: These data collectively indicate that candidate human HSCs can be efficiently transduced with lentiviral vectors and that the transgene is highly expressed in their progeny cells.     

Anti-HIV-1 gene expressing lentiviral vectors as an adjunctive therapy for HIV-1 infection

Lentiviral based gene therapy may provide a valuable addition to the current anti-HIV arsenal. Many lentiviral vector systems have been described including those based on feline immunodeficiency virus (FIV), human immunodeficiency virus 1 (HIV) and 2 (HIV-2/SIV) as well as replication incompetent, self-inactivating (sin) vs. conditionally replicating (mobilizable) vectors. Lentiviral vectors offer promise in treating HIV-1 infection as they are capable of stably transducing both dividing and nondividing cells, specifically those cells involved in HIV-1 replication and immune restoration: T-cells, hematopoietic stem cells, and dendritic cells. Moreover, some of the HIV-1 and 2 based vectors can be mobilized by wildtype HIV-1 in vivo and spread to those cells targeted by the virus as well as can compete with viral RNA for packaging and access to viral proteins such as Tat and Rev required for viral replication. Finally, lentiviral vectors can be designed to express therapeutic anti-HIV-1 genes, which specifically target various stages of viral replication. Many candidate RNA based anti-HIV-1 genes have been expressed from lentiviral vectors including ribozymes and anti-sense RNA [1]. Recently, small interfering RNAs (siRNAs) have been shown to potently suppress HIV replication [2-6]. This review will focus on the current status of lentiviral vector development and the feasibility of using lentiviral vectors in delivering anti-HIV genes, specifically ribozymes, and siRNAs as a therapeutic approach to employ in conjunction with current anti-retroviral therapies.     

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.     

HIV, but not murine leukemia virus, vectors mediate high efficiency gene transfer into freshly isolated G0/G1 human hematopoietic stem cells

Recent studies have opened the possibility that quiescent, G₀/G₁ hematopoietic stem cells (HSC) can be gene transduced; lentiviruses (such as HIV type 1, HIV) encode proteins that permit transport of the viral genome into the nucleus of nondividing cells. We and others have recently demonstrated efficient transduction by using an HIV-1-based vector gene delivery system into various human cell types including human CD34⁺ cells or terminally differentiated neurons. Here we compare the transduction efficiency of two vectors, HIV-based and murine leukemia virus (MuLV)-based vectors, on untreated and highly purified human HSC subsets that are virtually all in G₀/G₁. The HIV vector, but not MuLV vector supernatants, transduced freshly isolated G₀/G₁ HSC from mobilized peripheral blood. Single-step transduction using replication-defective HIV resulted in HSC that expressed the green fluorescent protein (GFP) transgene while retaining their stem cell phenotype; clonal outgrowths of these GFP⁺ HSC on bone marrow stromal cells fully retained GFP expression for at least 5 weeks. MuLV-based vectors did not transduce resting HSC, as measured by transgene expression, but did so readily when the HSC were actively cycling after culture in vitro for 3 days in a cytokine cocktail. These results suggest that resting HSC may be transduced by lentiviral-based, but not MuLV, vectors and maintain their primitive phenotype, pluripotentiality, and at least in vitro, transgene expression.     

Lentiviral vector transduction of NOD/SCID repopulating cells results in multiple vector integrations per transduced cell: risk of insertional mutagenesis

Efficient vector transduction of hematopoietic stem cells is a requirement for successful gene therapy of hematologic disorders. We asked whether human umbilical cord blood CD34(+)CD38(lo) nonobese diabetic/severe combined immunodeficiency (NOD/SCID) repopulating cells (SRCs) could be efficiently transduced using lentiviral vectors, with a particular focus on the average number of vector copies integrating into these primitive progenitor cells. Mouse bone marrow was analyzed by fluorescence-activated cell-sorter scanner and by semiquantitative polymerase chain reaction (PCR) to determine the transduction efficiency into SRCs. Lentiviral vector transduction resulted in an average of 22% (range, 3%-90%) of the human cells expressing green fluorescent protein (GFP), however, multiple vector copies were present in human hematopoietic cells, with an average of 5.6 +/- 3.3 (n = 12) copies per transduced cell. To confirm the ability of lentiviral vectors to integrate multiple vector copies into SRCs, linear amplification mediated (LAM)-PCR was used to analyze the integration site profile of a selected mouse showing low-level engraftment and virtually all human cells expressing GFP. Individually picked granulocyte macrophage colony-forming unit colonies derived from the bone marrow of this mouse were analyzed and shown to have the same 5 vector integrants within each colony. Interestingly, one integration site of the 5 that were sequenced in this mouse was located in a known tumor-suppressor gene, BRCA1. Therefore, these findings demonstrate the ability of lentiviral vectors to transduce multiple copies into a subset of NOD/SCID repopulating cells. While this is efficient in terms of transduction and transgene expression, it may increase the risk of insertional mutagenesis.     

Stable and efficient gene transfer into the retina using an HIV-based lentiviral vector

The development of methods for efficient gene transfer to terminally differentiated retinal cells is important to study the function of the retina as well as for gene therapy of retinal diseases. We have developed a lentiviral vector system based on the HIV that can transduce terminally differentiated neurons of the brain in vivo. In this study, we have evaluated the ability of HIV vectors to transfer genes into retinal cells. An HIV vector containing a gene encoding the green fluorescent protein (GFP) was injected into the subretinal space of rat eyes. The GFP gene under the control of the cytomegalovirus promoter was efficiently expressed in both photoreceptor cells and retinal pigment epithelium. However, the use of the rhodopsin promoter resulted in expression predominantly in photoreceptor cells. Most successfully transduced eyes showed that photoreceptor cells in >80% of the area of whole retina expressed the GFP. The GFP expression persisted for at least 12 weeks with no apparent decrease. The efficient gene transfer into photoreceptor cells by HIV vectors will be useful for gene therapy of retinal diseases such as retinitis pigmentosa.     

Stable transduction of quiescent CD34(+)CD38(-) human hematopoietic cells by HIV-1-based lentiviral vectors

We compared the efficiency of transduction by an HIV-1-based lentiviral vector to that by a Moloney murine leukemia virus (MLV) retroviral vector, using stringent in vitro assays of primitive, quiescent human hematopoietic progenitor cells. Each construct contained the enhanced green fluorescent protein (GFP) as a reporter gene. The lentiviral vector, but not the MLV vector, expressed GFP in nondivided CD34(+) cells (45.5% GFP+) and in CD34(+)CD38(-) cells in G0 (12.4% GFP+), 48 hr after transduction. However, GFP could also be detected short-term in CD34(+) cells transduced with a lentiviral vector that contained a mutated integrase gene. The level of stable transduction from integrated vector was determined after extended long-term bone marrow culture. Both MLV vectors and lentiviral vectors efficiently transduced cytokine-stimulated CD34(+) cells. The MLV vector did not transduce more primitive, quiescent CD34(+)CD38(-) cells (n = 8). In contrast, stable transduction of CD34(+)CD38(-) cells by the lentiviral vector was seen for over 15 weeks of extended long-term culture (9.2 +/- 5.2%, n = 7). GFP expression in clones from single CD34(+)CD38(-) cells confirmed efficient, stable lentiviral transduction in 29% of early and late-proliferating cells. In the absence of growth factors during transduction, only the lentiviral vector was able to transduce CD34(+) and CD34(+)CD38(-) cells (13.5 +/- 2.5%, n = 11 and 12.2 +/- 9.7%, n = 4, respectively). The lentiviral vector is clearly superior to the MLV vector for transduction of quiescent, primitive human hematopoietic progenitor cells and may provide therapeutically useful levels of gene transfer into human hematopoietic stem cells.     

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.     

Development of gene therapy for blood disorders by gene transfer into haematopoietic stem cells

Haematopoietic stem cells (HSCs) are important target cells for gene therapy of blood disorders due to their pluripotency and ability to reconstitute haematopoiesis following myeloablation and transplantation. HSCs can 'self-renew' and generate new stem cells. Genetically modified stem cells are therefore expected to last a lifetime in the recipient following blood and marrow transplantation, and can potentially cure haematological disorders. Oncoretroviral vectors have been the main vectors used for HSCs because of their ability to integrate into the chromosomes of their target cells. Because oncoretroviral vectors require dividing target cells for successful localization of the preintegration complex and subsequent chromosomal integration of the provirus, only the dividing fraction of the target cells can be transduced. As only a small fraction of haematopoietic stem cells is dividing at any one time, oncoretroviral vector transduction of human HSCs has been low in clinical trials. However, patients with severe combined immune deficiency-X1 (SCID-X1) have recently been treated successfully by gene therapy of autologous bone marrow cells using oncoretroviral vectors containing the common gamma chain gene. While several additional disorders may potentially be treated successfully using oncoretroviral gene transfer to HSCs, many disorders may require much higher gene transfer efficiency than was achieved in the SCID-X1 study. Therefore, lentiviral vectors have recently emerged as promising vectors for human HSCs because they can transduce dividing and nondividing HSCs efficiently, and may become the vectors of choice in the future for treatment of blood disorders where a large fraction of HSCs has to be corrected.     

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.     

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.     

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.     

High-efficient lentiviral vector-mediated gene transfer into primary human NK cells

OBJECTIVE: The long-term transfection of genes into primary natural killer (NK) cells without disrupting normal cellular functions has been proven to be difficult with currently available gene-transfer methods. In this study, we establish a lentiviral vector-based technique for improved gene transfer into human NK cells in vitro and we report on high-efficient transduction of freshly isolated as well as cultured primary NK cells. METHODS: Freshly isolated or primary cultured human NK cells, as well as the human NK cell line YTS, were transduced with replication-incompetent human immunodeficiency virus (HIV)-based lentiviral vector bearing a GFP reporter gene or a gene of interest under the control of the elongation factor 1alpha (EF1alpha) promoter. Transduction efficiencies were monitored by flow cytometry. RESULTS: A long-term transgene expression was detected in up to 98% of YTS NK cells, whereas in freshly isolated or primary cultured NK cells exposed to interleukin (IL)-2 plus IL-12 upon infection, efficiency was in the range of 50% to 90%. Moreover, in freshly isolated quiescent NK cells a transfection efficiency of 18% to 20% was achieved without stimulation. Notably, no major phenotypic and functional modifications were observed in transduced cells with respect to control cells: the expression levels of activating receptors, CD69-antigen induction as well as cytotoxic function were unaffected. CONCLUSION: Results of our study demonstrate that NK cells can be efficiently transduced by lentiviral vectors.     

Gene transfer in humans using a conditionally replicating lentiviral vector

We report findings from a clinical evaluation of lentiviral vectors in a phase I open-label nonrandomized clinical trial for HIV. This trial evaluated the safety of a conditionally replicating HIV-1-derived vector expressing an antisense gene against the HIV envelope. Five subjects with chronic HIV infection who had failed to respond to at least two antiviral regimens were enrolled. A single i.v. infusion of gene-modified autologous CD4 T cells was well tolerated in all patients. Viral loads were stable, and one subject exhibited a sustained decrease in viral load. CD4 counts remained steady or increased in four subjects, and sustained gene transfer was observed. Self-limiting mobilization of the vector was observed in four of five patients. There is no evidence for insertional mutagenesis after 21-36 months of observation. Immune function improved in four subjects. Lentiviral vectors appear promising for gene transfer to humans.     

Efficient ex vivo gene transfer into non-human primate hepatocytes using HIV-1 derived lentiviral vectors

BACKGROUND/AIMS: Lentivirus-mediated ex vivo gene therapy is becoming a promising approach for the treatment of liver metabolic disorders. However, the feasibility of this approach needs to be studied in large animal models. The purpose of this study was to evaluate the efficacy of ex vivo gene transfer into Macaca hepatocytes with two different HIV-1 derived lentiviral vectors. METHODS: A self-inactivating lentivector was constructed to express GFP under the control of the hepatic apolipoprotein A-II promoter. Freshly isolated and thawed hepatocytes were transduced in suspension with lentiviral vectors expressing the GFP gene under the control of a ubiquitous promoter (EF1-alpha) and the apolipoprotein A-II promoter. Transduced thawed hepatocytes were transplanted into the spleen of newborn mice, and livers analyzed 4 and 12 weeks after transplantation. RESULTS: We show that lentivectors are efficient in transducing hepatocytes in suspension either freshly isolated or cryopreserved. We also show that thawed and transduced hepatocytes engrafted and participated in liver growth after transplantation into newborn mice and that the apolipoprotein A-II promoter is functional. CONCLUSIONS: Our data show that transplantation of transduced hepatocytes into monkeys should allow to evaluate the fate of transplanted cells and transgene expression in a pre-clinical model of ex vivo gene therapy.