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.
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.
Agency: Department of Health and Human Services | Branch: | Program: STTR | Phase: Phase I | Award Amount: 100.00K | Year: 2008
DESCRIPTION (provided by applicant): The hemophilias (A and B) are rare bleeding disorders toward which much scientific and medical effort has been devoted. In 1840, blood transfusion was used for the first time to stop post-operative bleeding in a hemophi lia patient and in 1968 the first commercial coagulation factor concentrate became available. The cloning of the fVIII gene in 1984 facilitated the development of recombinant fVIII protein products that became commercially available in 1992. This was viewe d as a dramatic therapeutic improvement due to the perceived safety advantage recombinant products have over plasma-derived products, which proved responsible for the infection of thousands of patients with hemophilia with human immunodeficiency virus and/ or hepatitis C virus during the 1980's. Since the development of recombinant fVIII, progress in the treatment of hemophilia A has slowed. The limitations of current treatment are 1) access to fVIII-replacement products, 2) the cost of fVIII- replacement pr oducts, 3) the development of humoral anti-fVIII immune responses that block treatment efficacy and 4) morbidity due to joint disease. State of the art treatment multiple infusions per week of fVIII product. However, many patients are treated only after th e initiation of a bleeding episode and these patients typically develop joint arthropathy due to repeated bleeding into a target joint, e.g. knee. Unless the worldwide fVIII supply increases and prices drop significantly, hemophilia A will remain an import ant heath burden to human society. Therefore, the search for improved therapeutics is warranted. One strategy for improving hemophilia A care is to develop improved recombinant-protein products, e.g. manufactured more efficiently or have increased hemostat ic efficacy. The mission of Expression Therapeutics is to develop products that will improve the treatment of individuals with hemophilia A. Our technology is based on the identification of sequence elements within fVIII that can be modified to increase it s biosynthesis. The goal of the current study is to provide feasibility data supporting the concept that a high-expression fVIII-replacement product can be manufactured more efficiently than traditional human recombinant fVIII products. These data will sup port the development of a novel fVIII-replacement product that will improve the treatment of hemophilia A. PUBLIC HEALTH RELEVANCE: Hemophilia A is a bleeding disorder caused by the insufficiency of a blood clotting factor, designated factor VIII (fVIII). Current treatment for hemophilia relies on infusion of plasma-derived or recombinant fVIII products to restore circulating fVIII activity. Currently, treatment is offered to less than one-third of all patients with hemophilia A patients due to product cost . Unless the worldwide fVIII supply increases and prices drop significantly, hemophilia A will remain an important heath burden to human society and therefore the search for improved therapeutics is warranted.
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.
Agency: Department of Health and Human Services | Branch: | Program: STTR | Phase: Phase I | Award Amount: 100.00K | Year: 2006
DESCRIPTION (provided by applicant): Hemophilia A is a bleeding disorder caused by mutations within the factor VIII (fVIII) gene that result in a deficiency of circulating fVIII activity. Current treatment for hemophilia relies on infusion of plasma-derived or recombinant fVIII to restore circulating fVIII activity. This form of therapy is 1) expensive, 2) hard to maintain due to the frequency and intravenous route of treatment, and 3) only offered to 30% of hemophilia A patients worldwide. The mission of Expression Therapeutics is to develop products that will improve the standard treatment of individuals with hemophilia A. Our technology is based on the identification of sequence elements within fVIII that can be modified to increase its biosynthesis. Gene therapy has the potential to cure hemophilia A. However in three previous clinical trials, the levels of circulating fVIII activity achieved were below that required for therapeutic efficacy and all trials ended following phase I. The goal of the studies proposed in this application is to demonstrate the effectiveness of fVIII high expression elements in a gene transfer-based treatment of hemophilia A. We propose to study the expression of several fVIII transgene constructs in vivo using the murine model of hemophilia A. Additionally, we will study fVIII biosynthesis from human stem/progenitor cells that are genetically modified using recombinant lentiviral vectors containing high expression fVIII transgenes. Demonstrating proof-of-concept that fVIII high expression elements are enabling to gene therapy of hemophilia A will represent a major milestone for Expression Therapeutics in the development of improved therapeutic treatments for hemophilia A. PROJECT NARATIVE Hemophilia A is a bleeding disorder caused by genetic mutation of a blood clotting factor, designated factor VIII (fVIII). Current treatment for hemophilia relies on infusion of plasma-derived or recombinant fVIII to restore circulating fVIII activity. This form of therapy is 1) expensive, 2) hard to maintain due to the frequency and intravenous route of treatment, and 3) only offered to 30% of hemophilia A patients worldwide. Therefore, the development of improved therapeutic treatments for hemophilia A, e.g. gene therapy, is warranted.