Transduction Efficiency Of Lentiviral Vectors Research Articles

Enhancing therapeutic efficacy of in vivo platelet-targeted gene therapy in hemophilia A mice

AbstractOur previous studies demonstrated that intraosseous (IO) infusion of lentiviral vectors (LVs) carrying a modified B domain–deleted factor VIII (FVIII) transgene driven by a megakaryocyte-specific promoter (GP1Bα promoter; G-F8/N6-LV) successfully transduced hematopoietic stem cells (HSCs) to produce FVIII stored in the platelet α-granules. Platelet FVIII corrected the bleeding phenotype with limited efficacy in hemophilia A (HemA) mice with and without preexisting anti-FVIII inhibitors. The present study sought to further enhance the therapeutic efficacy of this treatment protocol by increasing both the efficiency of LV transduction and the functional activity of platelet FVIII. A combined drug regimen of dexamethasone and anti-CD8α monoclonal antibody enhanced the percentage of transduced bone marrow and HSCs over time. In G-F8/N6-LV–treated HemA mice, significant improvement in phenotypic correction was observed on day 84. To improve platelet FVIII functionality, genes encoding FVIII variant F8X10K12 with increased expression or F8N6K12RH with increased functional activity compared with F8/N6 were incorporated into LVs. Treatment with G-F8X10K12-LV in HemA mice produced a higher level of platelet FVIII but induced anti-FVIII inhibitors. After treatment with combined drugs and IO infusion of G-F8/N6K12RH-LV, HemA mice showed significant phenotypic correction without anti-FVIII inhibitor formation. These results indicate that new human FVIII variant F8/N6K12RH combined with immune suppression could significantly enhance the therapeutic efficacy of in vivo platelet-targeted gene therapy for murine HemA via IO delivery. This protocol provides a safe and effective treatment for hemophilia that may be translatable to and particularly beneficial for patients with preexisting inhibitory antibodies to FVIII.

Construction of a novel lentiviral vector knocking down PD-1 via microRNA and its application in CAR-T cells

By inserting microRNAs into the intron of EF1α promoter, we constructed a novel lentiviral vector knocking down PD-1 gene via microRNA and applied it to CAR-T cells. Lentiviral transduction efficiency and PD-1-silencing efficiency were detected by flow cytometry. PD-1 expression was detected by Western blotting. Relative expression of microRNA was measured by Q-PCR. Cytotoxicity of CAR-T cells based on this vector was tested by luciferase bioluminescence and flow cytometry. Compared with lentiviral vector with microRNA transcribed by U6 promotor, the transduction efficiency of lentiviral vector with microRNA which was inserted into the intron of EF1α promoter was more significant, and the knockdown rate of PD-1 was more than 90%, which was validated by flow cytometry and Western blotting. And the relative expression level of microRNA in Jurkat cells transduced with this novel lentiviral vector was shown by Q-PCR. Compared with normal CAR-T cells, CAR-T cells based on this vector showed stronger cytotoxicity against PD-L1 positive Raji cells. We successfully constructed a novel lentiviral vector that knocked down PD-1 via microRNA and verified the superiority of its transduction efficiency and knockdown efficiency of PD-1. CAR-T cells based on this vector can exert a more powerful cytotoxicity, thus providing theoretical support for the subsequent treatment of PD-L1 positive tumors.

The Relationships between Target Gene Transduction, Engraftment of HSCs and RBC Physiology in Sickle Cell Disease Gene Therapy

The Relationships between Target Gene Transduction, Engraftment of HSCs and RBC Physiology in Sickle Cell Disease Gene Therapy

Staurosporine Increases Lentiviral Vector Transduction Efficiency of Human Hematopoietic Stem and Progenitor Cells

Lentiviral vector (LVV)-mediated transduction of human CD34+ hematopoietic stem and progenitor cells (HSPCs) holds tremendous promise for the treatment of monogenic hematological diseases. This approach requires the generation of a sufficient proportion of gene-modified cells. We identified staurosporine, a serine/threonine kinase inhibitor, as a small molecule that could be added to the transduction process to increase the proportion of genetically modified HSPCs by overcoming a LVV entry barrier. Staurosporine increased vector copy number (VCN) approximately 2-fold when added to mobilized peripheral blood (mPB) CD34+ cells prior to transduction. Limited staurosporine treatment did not affect viability of cells post-transduction, and there was no difference in in vitro colony formation compared to vehicle-treated cells. Xenotransplantation studies identified a statistically significant increase in VCN in engrafted human cells in mouse bone marrow at 4 months post-transplantation compared to vehicle-treated cells. Prostaglandin E2 (PGE2) is known to increase transduction efficiency of HSPCs through a different mechanism. Combining staurosporine and PGE2 resulted in further enhancement of transduction efficiency, particularly in short-term HSPCs. The combinatorial use of small molecules, such as staurosporine and PGE2, to enhance LVV transduction of human CD34+ cells is a promising method to improve transduction efficiency and subsequent potential therapeutic benefit of gene therapy drug products.

Differential Effects of Strategies to Improve the Transduction Efficiency of Lentiviral Vector that Conveys an Anti-HIV Protein, Nullbasic, in Human T Cells.

Nullbasic is a mutant form of HIV-1 Tat that has strong ability to protect cells from HIV-1 replication by inhibiting three different steps of viral replication: reverse transcription, Rev export of viral mRNA from the nucleus to the cytoplasm and transcription of viral mRNA by RNA polymerase II. We previously showed that Nullbasic inhibits transduction of human cells including T cells by HIV-1-based lentiviral vectors. Here we investigated whether the Nullbasic antagonists huTat2 (a Tat targeting intrabody), HIV-1 Tat or Rev proteins or cellular DDX1 protein could improve transduction by a HIV-1 lentiviral vector conveying Nullbasic-ZsGreen1 to human T cells. We show that overexpression of huTat2, Tat-FLAG and DDX1-HA in virus-like particle (VLP) producer cells significantly improved transduction efficiency of VLPs that convey Nullbasic in Jurkat cells. Specifically, co-expression of Tat-FLAG and DDX1-HA in the VLP producer cell improved transduction efficiency better than if used individually. Transduction efficiencies could be further improved by including a spinoculation step. However, the same optimised protocol and using the same VLPs failed to transduce primary human CD4+ T cells. The results imply that the effects of Nullbasic on VLPs on early HIV-1 replication are robust in human CD4+ T cells. Given this significant block to lentiviral vector transduction by Nullbasic in primary CD4+ T cells, our data indicate that gammaretroviral, but not lentiviral, vectors are suitable for delivering Nullbasic to primary human T cells.

Prostaglandin E2 Increases Lentiviral Vector Transduction Efficiency of Adult Human Hematopoietic Stem and Progenitor Cells.

Gene therapy currently in development for hemoglobinopathies utilizes ex vivo lentiviral transduction of CD34+ hematopoietic stem and progenitor cells (HSPCs). A small-molecule screen identified prostaglandin E2 (PGE2) as a positive mediator of lentiviral transduction of CD34+ cells. Supplementation with PGE2 increased lentiviral vector (LVV) transduction of CD34+ cells approximately 2-fold compared to control transduction methods with no effect on cell viability. Transduction efficiency was consistently increased in primary CD34+ cells from multiple normal human donors and from patients with β-thalassemia or sickle cell disease. Notably, PGE2 increased transduction of repopulating human HSPCs in an immune-deficient (nonobese diabetic/severe combined immunodeficiency/interleukin-2 gamma receptor null [NSG]) xenotransplantation mouse model without evidence of in vivo toxicity, lineage bias, or a de novo bias of lentiviral integration sites. These data suggest that PGE2 improves lentiviral transduction and increases vector copy number, therefore resulting in increased transgene expression. As a result, PGE2 may be useful in clinical gene therapy applications using lentivirally modified HSPCs.

Lentiviral Vector-based Insertional Mutagenesis Identifies Genes Involved in the Resistance to Targeted Anticancer Therapies

The high transduction efficiency of lentiviral vectors in a wide variety of cells makes them an ideal tool for forward genetics screenings addressing issues of cancer research. Although molecular targeted therapies have provided significant advances in tumor treatment, relapses often occur by the expansion of tumor cell clones carrying mutations that confer resistance. Identification of the culprits of anticancer drug resistance is fundamental for the achievement of long-term response. Here, we developed a new lentiviral vector-based insertional mutagenesis screening to identify genes that confer resistance to clinically relevant targeted anticancer therapies. By applying this genome-wide approach to cell lines representing two subtypes of HER2(+) breast cancer, we identified 62 candidate lapatinib resistance genes. We validated the top ranking genes, i.e., PIK3CA and PIK3CB, by showing that their forced expression confers resistance to lapatinib in vitro and found that their mutation/overexpression is associated to poor prognosis in human breast tumors. Then, we successfully applied this approach to the identification of erlotinib resistance genes in pancreatic cancer, thus showing the intrinsic versatility of the approach. The acquired knowledge can help identifying combinations of targeted drugs to overcome the occurrence of resistance, thus opening new horizons for more effective treatment of tumors.

High-throughput screening identifies compounds that enhance lentiviral transduction.

A difficulty in the field of gene therapy is the need to increase the susceptibility of hematopoietic stem cells (HSCs) to ex vivo genetic manipulation. To overcome this obstacle a high-throughput screen was performed to identify compounds that could enhance the transduction of target cells by lentiviral vectors. Of the 1280 compounds initially screened using the myeloid-erythroid-leukemic K562 cell line, 30 were identified as possible enhancers of viral transduction. Among the positive hits were known enhancers of transduction (camptothecin, etoposide and taxol), as well as the previously unidentified phorbol 12-myristate 13-acetate (PMA). The percentage of green fluorescent protein (GFP)-positive-expressing K562 cells was increased more than fourfold in the presence of PMA. In addition, the transduction of K562 cells with a lentiviral vector encoding fVIII was four times greater in the presence of PMA as determined by an increase in the levels of provirus in genetically modified cells. PMA did not enhance viral transduction of all cell types (for example, sca-1(+) mouse hematopoietic cells) but did enhance viral transduction of human bone marrow-derived CD34(+) cells. Notably, the percentage of GFP-positive CD34(+) cells was increased from 7% in the absence of PMA to greater than 22% in the presence of 1 nM PMA. PMA did not affect colony formation of CD34(+) cells or the expression of the hematopoietic markers CD34 and CD45. These data demonstrate that high-throughput screening can be used to identify compounds that increase the transduction efficiency of lentiviral vectors, identifying PMA as a potential enhancer of lentiviral HSC transduction.

Expression of Mutated Huntingtin Fragment in the Putamen Is Sufficient to Produce Abnormal Movement in Non-human Primates

Huntington's disease (HD) is a neurological disorder characterized by striatal degeneration, motor symptoms and complex neuropsychiatric alterations. There is currently no genetic model of HD in non-human primates (NHPs). In this study we investigated neuropathological and behavioral changes following injections of lentiviral vectors encoding a fragment of mutated huntingtin (Htt171-82Q) into the dorsolateral sensorimotor putamen of macaques. In the first study, we injected Htt171-82Q into one hemisphere and a lentiviral vector encoding Htt171-19Q or saline into the other, and studied the animals for 9 weeks. During this period, when apomorphine was administered into Htt171-19Q/82Q animals, it induced progressive chorea, dystonia and ipsilateral turning behavior, whereas animals infected with Htt171-19Q/19Q showed no abnormal behavior. After 9 weeks, the putamen of animals infected with Htt171-82Q presented neuritic and nuclear Htt aggregates, reactive astrocytes and loss of the neuronal marker NeuN. In a second study, we injected Htt171-82Q bilaterally into the dorsolateral putamen. From week 15 after infection, these animals progressively developed spontaneous dyskinesia of the legs, arms, and trunk and, in one case, tics that persisted for up to 30 weeks. The present study constitutes a proof-of-principle for the development of a genetic model of HD in NHP.

Factors that influence VSV‐G pseudotyping and transduction efficiency of lentiviral vectors—in vitro and in vivo implications

Pseudotyping viral vectors with vesicular stomatitis virus glycoprotein (VSV-G) enables the transduction of an extensive range of cell types from different species. We have discovered two important parameters of the VSV-G-pseudotyping phenomenon that relate directly to the transduction potential of lentiviral vectors: (1) the glycosylation status of VSV-G, and (2) the quantity of glycoprotein associated with virions. We measured production-cell and virion-associated quantities of two isoform variants of VSV-G, which differ in their glycosylation status, VSV-G1 and VSV-G2, and assessed the impact of this difference on the efficiency of mammalian cell transduction by lentiviral vectors. The glycosylation of VSV-G at N336 allowed greater maximal expression of VSV-G in HEK293T cells, thus facilitating vector pseudotyping. The transduction of primate cell lines was substantially affected (up to 50-fold) by the degree of VSV-G1 or VSV-G2 incorporation, whereas other cell lines, such as D17 (canine), were less sensitive to virion-associated VSV-G1/2 quantities. These data indicate that the minimum required concentration of virion-associated VSV-G differs substantially between cell species/types. The implications of these data with regard to VSV-G-pseudotyped vector production, titration, and use in host-cell restriction studies, are discussed.

Efficient Viral Transduction of Murine Hematopoietic Stem Cells Using HIV-1 Gag-Pol Cross Packaged Simian Immunodeficiency Viral Backbone.

Lentiviral vectors have been shown to infect non-dividing cells, including hematopoietic stem cell [HSC], and HIV lentiviral vector has been studied extensively in preclinical models. However low HIV lentiviral vector transduction efficiency compared to retroviral vectors, is seen in murine HSC, hampering transplantation and long-term expression of transgene in the recipients. Furthermore, concerns remain regarding the safety of HIV based vectors. Simian Immunodeficiency Viral [SIV] vectors could be safer since the parent virus does not cause disease in humans. However, to model this approach has been difficult because native SIV vectors do not transduce murine cells. We have generated a bicistronic SIV lentiviral SIN vector, containing MGMT and firefly luciferase genes linked by a self-cleavage FMDV 2A sequence. The SIV backbone was kindly provided by Dr. Donald Kohn (University of Southern California). The transgenes are controlled by the MND promoter, which has been shown to express well in murine hematopoietic stem cells. The vector was generated by cross-packaging SIV RNA with HIV-1 ΔR8.91 packaging plasmid and VSVG pseudotyped envelope (Ref. Retrovirology 2005, 2:55). Unconcentrated viruses had an average titer of 1E+06 iu/ml, which was similar to HIV-1 lentiviral vectors. In vitro, HIV-1 cross-packaged SIV-mnd-MGMT-2A-Luc vector was able to transduce both human and murine cell lines with no reduction of expression for 10 weeks. In addition, this cross-packaged SIV vector was also able to transduce primary murine bone marrow cells from Balb/C mice with low MOI of 0.5 to 1. Transduced primary murine bone marrow cells maintained transgene expression during a 4 week culture. To analyze in vivo expression, Balb/C bone marrow cells were transduced for 48 hrs in cytokines with the HIV-1 packaged SIV vector and transplanted into irradiated recipients. We used bioluminescent imaging (BLI) to monitor the transgene expression and the dynamic engraftment of transduced murine bone marrow cells. At MOI of 0.5 or 5, transduction efficiencies in murine progenitor cells were 24.4% and 46.7% respectively by PCR of transgene from CFU colonies. Bioluminescent imaging indicated similar engraftment patterns of transduced bone marrow cells by HIV-1 lentiviral vector or cross-packaged SIV lentiviral vector, as early as day 5. Consistent BLI signals indicated sustained expression of transgene in SIV vector transduced bone marrow cells beyond 30 days. With this study, cross-packaged SIV SIN vector could be used as a potential gene transfer vector in both preclinical murine studies and perhaps in clinical trials.

Efficient transduction of neurons using Ross River glycoprotein-pseudotyped lentiviral vectors

Lentiviral vectors are promising tools for CNS gene transfer since they efficiently transduce the cells of the nervous system in vivo. In this study, we have investigated the transduction efficiency of lentiviral vectors pseudotyped with Ross River virus glycoprotein (RRV-G) (RRV-G-pseudotyped lentiviral vectors (RRV-LV)). The RRV is an alphavirus with an extremely broad host range, including the cells of the central nervous system. Previous studies have shown that lentiviral vectors can be efficiently pseudotyped with this envelope protein and have demonstrated promising features of such vectors, including the possibility to establish stable producer cell lines. After injection of RRV-LV expressing green fluorescent protein into different structures in the rat brain we found efficient transduction of both neurons and glial cells. By using two cell-type-specific promoters, neuron-specific enolase and human glial fibrillary acidic protein, we demonstrated cell-specific transgene expression in the desired cell type. Ross River virus glycoprotein-pseudotyped lentiviral vectors also transduced human neural progenitor cells in vitro, showing that receptors for the RRV-G are present on human neural cells.

361. Lentiviral Vector Transduction Efficiency and Biosafety Profile of Adult Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) offer tremendous potential in regenerating diseased or injured tissues due to their ability to differentiate into various different cell types including cartilage, bone, fat and other connective tissue. Lentiviral vector transduced MSCs are of interest to gene therapy as they can be used to deliver genes to sites of injury after systemic delivery, serve as a platform for controlled therapeutic gene expression and have the potential for modification by over-expression of differentiation factors. Therapeutic use of transduced MSCs must also satisfy questions regarding biosafety such as the effect of transduction on cellular phenotype and differentiation capability and the risk of insertional mutagenesis caused by integrating viral vectors.

738. Optimizing a Lentiviral Vector System for Efficient Transduction of Primary Rat Cardiovascular Cells In Vitro

Cardiovascular disease (CVD) is the number one cause of mortality in the United States. However, little progress has been made in the development of effective gene therapy treatments for cardiovascular pathologies. Recently, lentiviral vectors based on HIV-1 have been developed and optimized, and have ideal characteristics for gene therapy applications to treat CVD, particularly in myocardium and vascular smooth muscle. To achieve our goal of efficient gene transfer in vivo, in vitro systems needed to be developed to assay modified lentiviral vector transduction into isolated primary rat cardiomyocytes and vascular smooth muscle cells (VSMCs). In our initial studies, lentiviral vector transduction efficiency was examined using vectors pseudotyped with the vesicular stomatitis virus G protein (VSV-G) and containing the bacterial lacZ gene driven by a number of different viral and cellular promoters, including phosphoglycerokinase (PGK), cytomegalovirus (CMV), ubiquitin (Ub) and elongation factor 1a (EF1a), CMV-EF1a hybrid (CEF) and cardiac-specific promoter α-myosin heavy chain (αMHC). All of the promoters were found to function in the cardiovascular cells, even at MOI's below 1. The promoter with the highest transduction efficiency was CEF (3.37 × 105 T.U./ng p24; n=5 preparations in triplicate) as determined by end-point dilution and p24 Gag ELISA, whereas the PGK promoter resulted in the lowest lentiviral vector titer (7.14 × 102 T.U./ng p24; n=5 preparations in triplicate). The CEF promoter was then used to determine lentiviral vector transduction efficiency in isolated rat primary VSMCs from the jugular vein and thoracic aorta. The VSMCs were difficult to infect, with multiple infections (MOI=10/infection) being necessary to achieve high infectivity (i.e., greater than 85% of the VSMCs). Interestingly, the cells from the aorta were slightly more transducible than those from the jugular vein. The relatively low infectivity in the VSMCs may be one reason that VSV-G pseudotyped lentivectors have been unable to effectively transduce these cells in vivo. We have analyzed each of the promoters to assess their ability to drive gene expression in primary rat cardiomyocytes. Following the infection of the cardiomyocytes at a MOI of 1, we determined the β-gal transgene expression for each of the promoters, and found that the PGK promoter produced the highest level of β-gal protein (1497 ± 129 pg β-gal/mg total protein; n=5 preparations in triplicate) and no β-gal expression was observed in the mock treated cells. No significant differences were observed in lentiviral vector transduction and/or gene expression using lentiviral vectors that either contained or were devoid of the accessory proteins, specifically vif, vpr, vpu and nef. Current studies are being performed with these lentivectors to determine if the in vitro experiments in the primary cardiomyocytes correlate with in vivo transduction using different routes of administration into the heart. These studies should help to better illuminate lentiviral vectors as a gene therapy tool for use in cardiovascular disease in basic and clinical research. Funded by NHLBI 18766–01.

The effect of age on hepatic gene transfer with self-inactivating lentiviral vectors in vivo

It is known that cellular proliferation, by either compensatory regeneration or direct hyperplasia, can augment lentiviral vector transduction into hepatocytes in vivo. For this reason, the present study was designed to determine if adolescent mice (3½ weeks of age), which still have relatively proliferating livers, would have differential transduction compared to older (7 weeks of age) mice. Self-inactivating lentiviral vectors containing the human α1-antitrypsin (hAAT) promoter driving the expression of either the bacterial lacZ gene or the hAAT cDNA were generated for these studies. We found that adolescent mice given lentiviral vectors expressing lacZ (50 μg p24/mouse) via intravenous administration had a significantly higher level of hepatocyte transduction as measured by X-gal staining of liver sections compared to the 7-week-old mice. In addition, serum hAAT levels were nearly 40-fold higher in 3½-week-old mice administered lentiviral vectors expressing hAAT (50 μg p24/mouse) compared to the 7-week-old mice. Moreover, the incorporation of a matrix attachment region from immunoglobulin κ significantly increased transduction of hepatocytes in vivo. Although there was a small reduction in the circulating levels of hAAT, likely due to an immune response against the transgene product, gene expression was sustained for the duration of the study (30 weeks in total). In conclusion, the present study strongly demonstrates that lentiviral vector transduction efficiency and transgene expression were significantly enhanced in adolescent compared to older mice.

Endostatin gene transfer inhibits joint angiogenesis and pannus formation in inflammatory arthritis.

Rheumatoid arthritis is a prevalent example of an inflammatory angiogenic disease, which is mediated by pro-inflammatory and pro-angiogenic cytokines such as tumor necrosis factor (TNF). To evaluate the effect of the potent anti-angiogenic factor endostatin on TNF-induced inflammatory arthritis, we injected an endostatin-expressing lentiviral vector directly into the joints of human TNF-transgenic mice before the onset of disease. Histological analysis of the injected joints 8 weeks later revealed that endostatin reduced blood vessel density within the synovial tissues and an overall mean arthritis index. In vitro and in vivo examination of the potential mechanism by which endostatin inhibited the arthritis revealed that endostatin blocks TNF-induced activation of JNK and JNK-dependent pro-angiogenic gene expression. These data suggest a novel mechanism by which endostatin inhibits angiogenesis, and demonstrates the potential utility of anti-angiogenic gene therapy for treatment of inflammatory arthritis.