Tebas P.,University of Pennsylvania |
Stein D.,Jacobi Medical Center |
Binder-Scholl G.,University of Pennsylvania |
Mukherjee R.,University of Pennsylvania |
And 24 more authors.
Blood | Year: 2013
We report the safety and tolerability of 87 infusions of lentiviral vector-modified autologous CD4 T cells (VRX496-T; trade name, Lexgenleucel-T) in 17 HIV patients with well-controlled viremia. Antiviral effects were studied during analytic treatment interruption in a subset of 13 patients. VRX496-T was associated with a decrease in viral load set points in 6 of 8 subjects (P = .08). In addition, A → G transitions were enriched in HIV sequences after infusion, which is consistent with a model in which transduced CD4 T cells exert antisense-mediated genetic pressure on HIV during infection. Engraftment of vector-modified CD4 T cells was measured in gut-associated lymphoid tissue and was correlated with engraftment in blood. The engraftment half-life in the blood was approximately 5 weeks, with stable persistence in some patients for up to 5 years. Conditional replication of VRX496 was detected periodically through 1 year after infusion. No evidence of clonal selection of lentiviral vector-transduced T cells or integration enrichment near oncogenes was detected. This is the first demonstration that gene-modified cells can exert genetic pressure on HIV. We conclude that gene-modified T cells have the potential to decrease the fitness of HIV-1 and conditionally replicative lentiviral vectors have a promising safety profile in T cells. This study is registered at www.clinicaltrials.gov as number NCT00295477. © 2013 by The American Society of Hematology.
Kimpel J.,Chemotherapeutisches Forschungsinstitut Georg Speyer Haus |
Braun S.E.,Harvard University |
Qiu G.,Harvard University |
Wong F.E.,Harvard University |
And 12 more authors.
PLoS ONE | Year: 2010
Although a variety of genetic strategies have been developed to inhibit HIV replication, few direct comparisons of the efficacy of these inhibitors have been carried out. Moreover, most studies have not examined whether genetic inhibitors are able to induce a survival advantage that results in an expansion of genetically-modified cells following HIV infection. We evaluated the efficacy of three leading genetic strategies to inhibit HIV replication: 1) an HIV-1 tat/rev-specific small hairpin (sh) RNA; 2) an RNA antisense gene specific for the HIV-1 envelope; and 3) a viral entry inhibitor, maC46. In stably transduced cell lines selected such that. 95% of cells expressed the genetic inhibitor, the RNA antisense envelope and viral entry inhibitor maC46 provided the strongest inhibition of HIV-1 replication. However, when mixed populations of transduced and untransduced cells were challenged with HIV-1, the maC46 fusion inhibitor resulted in highly efficient positive selection of transduced cells, an effect that was evident even in mixed populations containing as few as 1% maC46-expressing cells. The selective advantage of the maC46 fusion inhibitor was also observed in HIV-1-infected cultures of primary T lymphocytes as well as in HIV-1-infected humanized mice. These results demonstrate robust inhibition of HIV replication with the fusion inhibitor maC46 and the antisense Env inhibitor, and importantly, a survival advantage of cells expressing the maC46 fusion inhibitor both in vitro and in vivo. Evaluation of the ability of genetic inhibitors of HIV-1 replication to confer a survival advantage on genetically-modified cells provides unique information not provided by standard techniques that may be important in the in vivo efficacy of these genes. © 2010 Kimpel et al.
Baranyi L.,Lentigen Corporation |
Doering C.B.,Emory University |
Denning G.,Expression Therapeutics |
Gautney R.E.,Expression Therapeutics |
And 5 more authors.
Human Gene Therapy Methods | Year: 2013
Lentiviral vectors (LVs) are widely recognized as the most efficient method for the stable delivery of nucleic acid sequences into mammalian cells. Using erythropoietin (EPO), recombinant factor VIII (fVIII), and an anti-CD20 antibody as model proteins, we demonstrate advantages of LV-based gene delivery to achieve high production levels by transduced cells. Highly productive cell clones were able to incorporate up to 100 vector copies per cellular genome, without selection or gene amplification, and were isolated without extensive screening of a large number of clones. The LV transgenes were shown to be distributed throughout the genome, as visualized by fluorescent in situ hybridization. High-expressing clones producing 100-200 pg/cell/day of EPO were isolated and characterized. EPO production was demonstrated for at least 5 months of continuous culture without selection, during which all the clones displayed high levels of glycosylation despite production levels at 10-20 g/liter. To demonstrate the utility of LV technology for multiple classes of proteins, cell lines producing fVIII and an anti-CD20 antibody were also developed. Cell clones demonstrating high levels of fVIII (100 clot units/ml and anti-CD20 antibody as high as 40-100 pg/cell/day) were isolated and characterized. LV-transduced cells and plasmid-transfected cells were compared for protein production per transgene copy. LV-transduced cells produced significantly higher levels of protein per copy of transgene than plasmid-transfected cells did. This study demonstrates the utility of LV technology for rapid generation of highly productive and stable cell lines over conventional plasmid transfection methods, significantly decreasing the time, cost, and risk of the manufacture of proteins and other complex biological molecules. © 2013, Mary Ann Liebert, Inc.
Spencer H.T.,Children's Healthcare Of Atlanta |
Denning G.,Expression Therapeutics |
Gautney R.E.,Expression Therapeutics |
Dropulic B.,Lentigen Corporation |
And 6 more authors.
Molecular Therapy | Year: 2011
Patients with hemophilia A present with spontaneous and sometimes life-threatening bleeding episodes that are treated using blood coagulation factor VIII (fVIII) replacement products. Although effective, these products have limited availability worldwide due to supply limitations and product costs, which stem largely from manufacturing complexity. Current mammalian cell culture manufacturing systems yield around 100νg/l of recombinant fVIII, with a per cell production rate of 0.05pg/cell/day, representing 10,000-fold lesser production than is achieved for other similar-sized recombinant proteins (e.g. monoclonal antibodies). Expression of human fVIII is rate limited by inefficient transport through the cellular secretory pathway. Recently, we discovered that the orthologous porcine fVIII possesses two distinct sequence elements that enhance secretory transport efficiency. Herein, we describe the development of a bioengineered fVIII product using a novel lentiviral-driven recombinant protein manufacturing platform. The combined implementation of these technologies yielded production cell lines that biosynthesize in excess of 2.5mg/l of recombinant fVIII at the rate of 9pg/cell/day, which is the highest level of recombinant fVIII production reported to date, thereby validating the utility of both technologies. © The American Society of Gene & Cell Therapy.
Norell H.,Medical University of South Carolina |
Zhang Y.,Medical University of South Carolina |
McCracken J.,University of Chicago |
Martins Da Palma T.,Medical University of South Carolina |
And 9 more authors.
Cancer Immunology, Immunotherapy | Year: 2010
Objective clinical responses can be achieved in melanoma patients by infusion of T cell receptor (TCR) gene transduced T cells. Although promising, the therapy is still largely ineffective, as most patients did not benefit from treatment. That only a minority of the infused T cells were genetically modified and that these were extensively expanded ex vivo may have prevented their efficacy. We developed novel and generally applicable retroviral vectors that allow rapid and efficient selection of T cells transduced with human TCRs. These vectors encode two TCR chains and a truncated CD34 molecule (CD34t) in a single mRNA transcript. Transduced T cells were characterized and the effects of CD34-based enrichment of redirected T cells were evaluated. Both CD8+ and CD4+ T cells could be transduced and efficiently co-expressed all introduced transgenes on their surface. Importantly, more than fivefold enrichment of both the frequency of transduced cells and the specific anti-tumor reactivity of the effector population could be achieved by magnetic beads-based enrichment procedures readily available for clinical grade hematopoietic stem cell isolation. This CD34-based enrichment technology will improve the feasibility of adoptive transfer of clinically relevant effectors. In addition to their enhanced tumor recognition, the enriched redirected T cells may also show superior reactivity and persistence in vivo due to the high purity of transduced cells and the shortened ex vivo culture. © 2010 Springer-Verlag.
Felizardo T.C.,U.S. National Institutes of Health |
Foley J.,U.S. National Institutes of Health |
Steed K.,U.S. National Institutes of Health |
Dropulic B.,Lentigen Corporation |
And 2 more authors.
Autophagy | Year: 2013
We hypothesized that rapamycin, through induction of autophagy and promotion of an antiapoptotic phenotype, would permit lentiviral (LV)-based transgene delivery to human T-Rapa cells, which are being tested in phase II clinical trials in the setting of allogeneic hematopoietic cell transplantation. Manufactured T-Rapa cells were exposed to supernatant enriched for a LV vector encoding a fusion protein consisting of truncated CD19 (for cell surface marking) and DTYMK/ TMPK?, which provides "cell-fate control" due to its ability to phosphorylate (activate) AZT prodrug. LV-transduction in rapamycin-treated T-Rapa cells: (1) resulted in mitochondrial autophagy and a resultant antiapoptotic phenotype, which was reversed by the autophagy inhibitor 3-MA; (2) yielded changes in MAP1LC3B and SQSTM1 expression, which were reversed by 3-MA; and (3) increased T-Rapa cell expression of the CD19-DTYMKΔ fusion protein, despite their reduced proliferative status. Importantly, although the transgene-expressing T-Rapa cells expressed an antiapoptotic phenotype, they were highly susceptible to cell death via AZT exposure both in vitro and in vivo (in a human-into-mouse xenogeneic transplantation model). Therefore, rapamycin induction of T cell autophagy can be used for gene therapy applications, including the CD19-DTYMKΔ cell-fate control axis to improve the safety of T cell immuno-gene therapy. © 2013 Landes Bioscience.
Lentigen Corporation, Boro Oy Inc. and Chang | Date: 2013-03-27
The present invention relates to lentiviral vectors for gene therapy, cancer treatment, producing recombinant proteins, such as antibodies and vaccines, and other therapeutic purposes. Novel lentiviral vectors are disclosed, e.g., comprising helper sequences in opposite orientations and/or minimally functional LTR sequences, which can be used to prepare high efficiency transduction vectors. Vectors are also designed to express silencing RNA and antisense polynucleotides.
Dropulic B.,Lentigen Corporation
Human Gene Therapy | Year: 2011
Lentiviral vectors have been successfully used in the clinic and they are increasingly being used for nonclinical applications. They are capable of stably transducing a broad range of mammalian cell types, including nondividing cells, with high efficiency. This review summarizes the evolving molecular design of lentiviral vectors, describing how they have improved since their first description. Lentiviral vector safety and issues surrounding genotoxicity are discussed. Examples of successful application of lentiviral vectors in laboratory and preclinical research are described. These include functional genomics, target validation, protein manufacturing, in vivo imaging, transgenic animals, and stem cell research. © 2011 Mary Ann Liebert, Inc.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 5.12M | Year: 2010
DESCRIPTION (provided by applicant): This Phase II proposal is a continuation of our Phase I award, Lentiviral Vectors for TCR Immunotherapy Targeted to Melanoma. We have successfully achieved the milestones laid out in our initial application. We generated a lentiviral gene vector capable of expressing a T cell receptor (TCR) specific for the tyrosinase:368-376 epitope, and demonstrated the activity of this cloned TCR in in vitro and in vivo models. The targeting of the melanoma-associated differentiation antigen by T cells transduced with this specific TCR will create a new therapeutic option for patients with melanoma. According to the American Cancer Society melanoma is currently the sixth most common cancer in men and the seventh most common cancer in American women. In this proposal we will generate clinical grade TCR vector, transduce patient-derived T cells, and then initiate a phase I clinical trial to evaluate the safety of this procedure. Recent findings from the NCI indicate that a transient lymphopenia induced by chemotherapy is essential for therapeutic effect. The trial we propose will be the first to try this procedure outside of the confines of the NCI, and will establish a new paradigm for the treatment of melanoma in the hospital setting. Secondarily this grant will move product development for the lentiviral vector expressing this TCR another step forward in the critical path of product development, and further key corporate goals of Lentigen to become the leader in clinical application of lentiviral vector technology. It is clear that immunotherapy will be a key feature for effective control of melanoma, a type of tumor for which current therapies do not offer satisfactory results. Lentiviral vectors have been evaluated in Phase I trials in HIV/AIDS. This proposal will be the first to use lentiviral technology in the treatment of melanoma. Our milestones will be 1) To generate GMP grade lentiviral vector, establishing release criteria for clinical use, and 2) to transduce patient T cells with this vector and infuse them according to our phase I FDA clinical trial- designed to evaluate the safety of transduced T cell infusion in lymphodepleted patients. In summary, Lentigen Corp. along with Dr. Michael Nishimura and his clinical team at the Medical University of South Carolina are uniquely positioned to provide a comprehensive evaluation of engineered human T cells in a clinical setting that can be generalized to other centers treating this life-threatening malignancy. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to develop a novel and improved immunotherapy for melanoma, a tumor for which current therapies do not offer satisfactory results. This therapy will feature the activation of immune cells that will be manipulated in the laboratory and infused back into the patient in order to eliminate melanoma tumor cells. Because of its great potential to offer a solution for those patients failing other therapies, this therapy will have significant relevance for cancer patients with melanoma and health care providers in the United States and worldwide.
Lentigen Corporation | Entity website
Lentiviral Vectors for Translational and Commercial Cell and Gene TherapyLentigen is a leading provider of custom lentiviral vectors for pre-clinicaland clinical applications. We are passionate about innovative technologies that can transform the practice of medicine ...