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Johnston J.M.,Emory University | Denning G.,Expression Therapeutics | Moot R.,Emory University | Whitehead D.,Emory University | And 4 more authors.
Gene Therapy | Year: 2014

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. © 2014 Macmillan Publishers Limited.


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.


Patent
Expression Therapeutics | Date: 2013-07-01

A system and method of adapting host cells to suspension cell culture and suspension cell lines ATCC PTA-12593 and ATCC PTA-12461 produced thereby are disclosed. The method includes the serial replating of substantially undiluted culture cells onto a surface area until cell clumps are visualized and then, upon cell clumping, moving the cells into a suspension culture system.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 296.32K | Year: 2012

DESCRIPTION (provided by applicant): The manufacturing of recombinant protein-based biopharmaceuticals is a complex, labor and capital intensive endeavor. It currently is accepted that the use of mammalian cells is a requirement for the production of mosthuman proteins, which typically contain extensive post-translational modifications that are not performed by prokaryotes and single-celled eukaryotes. Although mammalian cells such as Chinese hamster ovary cells and baby hamster kidney cells can faithfullybiosynthesize most human proteins, the efficiency is dramatically lower than is achieved by bacterial or yeast cells. Of the recombinant proteins currently marketed, coagulation factor VIII is manufactured with the lowest efficiency and is by far the mostexpensive on a per unit mass basis. Other expensive and difficult to manufacture biopharmaceuticals include the other coagulation factors (e.g. factors IX and VIIa), interferon, and hormone-based cytokines. The goal of the current application is to develop a technology that can improve the production volume and pharmacy price of these difficult to manufacture recombinant biologics. Expression Therapeutics has developed a new baby hamster kidney-derived (BHK) cell line, designated BHK-MS, which can be cultured and expanded indefinitely in serum and blood product-free medium under suspension bioreactor conditions. In the current studies, we are 1) developing and validating the suspension adapted BHK-MS cell line for superior coagulation factor VIII and factorIX production, and 2) performing a pilot-scale manufacturing run and analyzing the biochemical and pharmaceutical characteristics of BHK-MS biosynthesized recombinant factor VIII and factor IX, which will serve as a model, difficult to manufacture biopharmaceuticals. PUBLIC HEALTH RELEVANCE: The long-term commercial objective of the current project is to commercialize a suspension cell-based biomanufacturing platform designed for improved volumetric productivity of complex, hard to manufacture biologics. Low-yield manufacturing processes currently in place often result in limited product availability and high-product costs. Expression Therapeutics has developed a patent-pending cell line designed for efficient manufacturing of recombinant biologics.The studies proposed herein are designed to provide proof-of-concept of the efficiency and scalability, as well as, rigorous preclinical validation and characterization of this novel manufacturing platform.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 378.46K | Year: 2012

DESCRIPTION (provided by applicant): The overall goal of this proposal is to conduct late-stage preclinical studies to support a pilot clinical trial of hematopoietic stem cell transplantaton gene therapy for hemophilia A. In a series of recent studies, wehave shown that the transplantation of genetically-engineered hematopoietic stem cells can restore factor VIII (fVIII) activity to curative levels in hemophilia A mice and that human hematopoietic stem cells are readily transduced with recombinant lentivirus encoding a genetically-engineered fVIII transgene. To date, we are the only group that has obtained sustained therapeutic fVIII expression levels in hemophilia A mice using non- myeloablative transplantation regimens that are in routine clinical practice. Our gene therapy approach uses a bioengineered fVIII transgene (termed ET-3) that achieves normal fVIII activity levels (1 unit/ml) at hematopoietic stem cell transduction efficiencies (1-5%), which are achieved currently in human gene therapy clinicaltrials. We have generated extensive preclinical data using ET-3 demonstrating proof-of-concept that hematopoietic stem cells genetically engineered with a lentivirus vector encoding ET-3, coupled with a non-myeloablative transplant regimen, can be used totreat hemophilia A. We now propose to first conduct pre-IND meetings with the FDA to direct final preclinical testing of ET-3. Therefore, our late stage testing will be based on FDA guidance. Second, we will generate our final preclinical data set using clinical- grade (GMP) lentiviral vector encoding ET-3, which will specifically test the safety and effectiveness of the clinical product. It is anticipted that a follow up phase II project will be submitted in support of the actual clinical trial. Four organizations have partnered to accomplish these goals, including: i) Expression Therapeutics, LLC, a biotechnology company founded on the high expression fVIII technology, ii) Emory University, where the conception and proof of concept of high expression fVIII technology occurred, iii) Children's Healthcare of Atlanta, financial supporter of the Gene Therapy Program at Emory University and investor in Expression Therapeutics, LLC, and iv) Lentigen Corporation, a company dedicated to the successful clinical application of lentiviral vectors and holder of the largest lentiviral vector intellectual property portfolio. Lentigen will generate the clinical-grade recombinant lentivector that will be used in the proposed studies including the clinical trial.PUBLIC HEALTH RELEVANCE: Insufficient expression of the blood clotting factor VIII results in the bleeding disorder hemophilia A. Current treatment for this disease consists of difficult, lie-long, intravenous infusion of plasma-derived or recombinant factor VIII to restore circulating factor VIII activity levels and is currently offered to less than one-third of all hemophilia A patients due to high product cost and limited availability. Gene therapy offers a potential cure fo this debilitating and, in many parts of the world, lethal disease. We have shown that transplantation of bone marrow cells genetically-modified to express an engineered factor VIII protein is a feasible treatment for hemophilia A. In the current application, we propose to conduct late-stage pre-clinical testing to support approval of a first in man clinical gene therapy trial.


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 224.22K | Year: 2016

DESCRIPTION provided by applicant The development of transformative therapeutics including the possibility of a cure though gene therapy continues to be the major research and development activity in the commercial hemophilia space For example there are several company sponsored ongoing clinical trials of recombinant adeno associated viral rAAV encoding coagulation factor IX FIX for the treatment of hemophilia A Furthermore as a result of these trials there now exists extensive safety and initial efficacy data for multiple rAAV FIX products However progress in the translation of this approach to hemophilia A has been hampered by significant hurdles including the size complexity immunogenicity instability and biosynthetic inefficiency of coagulation factor VIII FVIII in comparison to FIX Now through the identification of liver specific transcriptional regulatory modules LTRMs that are smaller tha existing enhancer promoters combinations and a liver enhanced codon optimization algorithm used to engineer the coagulation factor VIII FVIII transgene we have been able to design rAAV vectors encoding FVIII with genome sizes under nucleotides that have unprecedented potency The proposed milestones of the current project are to characterize and select a lead candidate LTRM optimized for FVIII mRNA and transgene product output rAAV vector manufacture yield and rAAV product consistency and to identify a lead candidate AAV FVIII transgene These proof of concept studies will be performed using the preclinical murine model of hemophilia A The data obtained should facilitate the selection and preclinical validation of a lead candidate liver directed AAV FVIII expression cassette combining these novel technologies exclusively available to Expression Therapeutics ET Therefore ET is in an opportunistic position to capitalize on the ongoing rAAV FIX hemophilia B clinical findings and incorporate preclinical Randamp D and clinical protocol development strategies into this high priority rAAV FVIII project This existing clinical gene therapy knowledge combined with our hemophilia A experience and innovative technologies should enable ET to overcome the major hurdles facing commercialization of liver directed rAAV FVIII therapy for hemophilia A PUBLIC HEALTH RELEVANCE The goal of this project is test new technologies developed initially for liver directed recombinant adeno associated viral vector gene therapy of hemophilia A Our group has developed both liver expression and size optimized transcription regulatory modules and a liver optimized coagulation factor VIII transgene that should overcome the existing barriers to rAAV based gene therapy of hemophilia A The preclinical data obtained from the proposed studies should facilitate the selection of a lead gene therapy vector candidate


Johnston J.M.,Emory University | Denning G.,Expression Therapeutics | Doering C.B.,Emory University | Spencer H.T.,Emory University
Gene Therapy | Year: 2013

We previously compared the expression of several human factor VIII (fVIII) transgene variants and demonstrated the superior expression properties of B domain-deleted porcine fVIII. Subsequently, a hybrid human/porcine fVIII molecule (HP-fVIII) comprising 91% human amino-acid sequence was engineered to maintain the high-expression characteristics of porcine fVIII. The bioengineered construct then was used effectively to treat knockout mice with hemophilia A. In the current study, we focused on optimizing self-inactivating (SIN) lentiviral vector systems by analyzing the efficacy of various lentiviral components in terms of virus production, transduction efficiency and transgene expression. Specifically, three parameters were evaluated: (1) the woodchuck hepatitis post-transcriptional regulatory element (WPRE), (2) HIV versus SIV viral vector systems and (3) various internal promoters. The inclusion of a WPRE sequence had negligible effects on viral production and HP-fVIII expression. HIV and SIV vectors were compared and found to be similar with respect to transduction efficiency in both K562s and HEK-293T cells. However, there was an enhanced expression of HP-fVIII by the SIV system, which was evident in both K562 and BHK-M cell lines. To further compare expression of HP-fVIII from an SIV-based lentiviral system, we constructed expression vectors containing the high expression transgene and a human elongation factor-1 alpha, cytomegalovirus (CMV) or phosphoglycerate kinase promoter. Expression was significantly greater from the CMV promoter, which also yielded therapeutic levels of HP-fVIII in hemophilia A mice. Based on these studies, an optimized vector contains the HP-fVIII transgene driven by a CMV internal promoter within a SIV-based lentiviral backbone lacking a WPRE. © 2013 Macmillan Publishers Limited All rights reserved.


PubMed | Georgia Institute of Technology, Expression Therapeutics and Emory University
Type: Journal Article | Journal: Gene therapy | Year: 2014

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 1nM 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 Therapeutics | Entity website

Agarose gel electrophoresis of a DNA restriction digest used to ensure quality Currently, hemophilia A is treated by prophylactic or on-demand intravenous administration of either plasma-derived or recombinant factor VIII products. Current limitations of treatment include the development of humoral anti-factor VIII immune responses and the cost of treatment ...

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