Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 1.77M | Year: 2011
DESCRIPTION (provided by applicant): Sleeping Beauty (SB) is a transposon system that has been extensively shown to be capable of mediating integration of new gene sequences into the chromosomes of target cells and tissues. At Discovery Genomics, Inc. (DGI), we are working to develop the SB transposon system for human gene therapy, with hemophilia as our lead project. During initial Phase II SBIR studies, we demonstrated effective delivery of SB transposon DNA to the liver of dogs. Single or double ballooncatheters were used to achieve whole or partial occlusion of the liver followed by rapid, high volume retrograde infusion of DNA containing solution into the hepatic venous circulation. Results from these experiments (reporter gene expression was observedfor six weeks following infusion) place DGI at the forefront of non-viral gene therapy efforts targeting the liver in large animals. Based on these results and recent feedback from the Food and Drug Administration, in this Phase II competing renewal application we propose further preclinical studies to address issues of safety, scale-up to the size of humans, and efficacy in a large animal model of hemophilia. The Specific Aims are; (i) To develop double balloon catheters capable of delivering SB transposonDNA to the liver in humans and to test the safety and effectiveness of these catheters for DNA delivery in pigs as a large animal model of comparable size to human beings; (ii) To extend the duration of transgene expression after delivery of SB transposons to the liver of normal dogs, using DGI's unique canine secreted alkaline phosphatase (cSEAP) reporter system; (iii) To deliver SB transposons encoding canine clotting factor IX (cFIX) to the liver of cFIX deficient dogs, testing for long-term expressionof cFIX and improved clotting function as a large animal model for SB mediated gene therapy of hemophilia B. Results from these studies will provide necessary preclinical data for subsequent submission of an Investigational New Drug application with the FDA for treatment of hemophilia B using the SB transposon system, with subsequent growth and commercial development of Discovery Genomics, Inc. PUBLIC HEALTH RELEVANCE: In this grant application, studies are proposed to develop a new approach for treating hemophilia by non-viral gene therapy using an integrating DNA element (a transposon) called Sleeping Beauty. The experiments described in the proposal will establish conditions for delivery of Sleeping Beauty DNA in pigs as an animal model similar insize to humans, and in hemophilic dogs as a model for treatment of human hemophilia.
Agency: Department of Health and Human Services | Branch: | Program: STTR | Phase: Phase I | Award Amount: 174.01K | Year: 2007
DESCRIPTION (provided by applicant): Discovery Genomics, Inc. is focused on development of the Sleeping Beauty (SB) transposon system as a non-viral means of integrating new gene sequences in cells and tissues for therapeutic purposes. Here we propose to develop a method for improved targeting of the SB system to specific cell types following systemic injection by coupling a targeting tether to the plasmid carrier of the transposon. This approach will allow 'universal' vectors that could carry any gene and be directed to any cell type without modification of the transposase or transposon for each cellular target. This project will dovetail with DGI's ongoing SBIR project to deliver Factor VIII and IX genes in appropriate animal model systems. Thus, this project has high-impact potential because the techniques we will develop can be used for treatment of multiple diseases using a 'universal' non-viral vector system whose properties will be applicable to many therapeutic transgenes. The Specific Aims of the project are to: 1. Construct a Liver- directed Targeting Tether (LTT) vector system to increase uptake of SB transposons into liver cells. This aim will be accomplished by construction of a LTT targeting tether comprised of a LexA DNA-binding domain fused to a ligand specific for hepatocytes and construction of a plasmid, pKLAT2, that contains an SB transposon plus multimeric LexA operator sites to which the targeting tether can bind. 2: Evaluate the efficiency in cultured liver cells (HuH7 and HepG2) of a LTT targeting tether to enhance uptake and transposition of a pKLAT2 transposon vector. HeLa cells will serve as a control for cells that lack appropriate liver-specific receptors. 3: Evaluate the efficiency of a LTT targeting tether to enhance uptake and transposition of a pKLAT2 transposon vector into liver cells in mice.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 837.42K | Year: 2007
DESCRIPTION (provided by applicant): Fanconi anemia (FA) is a rare, autosomal recessive inherited disease caused by the absence of any of eight different proteins that regulate DNA repair. Affected individuals suffer from bone marrow failure early in life. FA can be effectively treated by allogeneic hematopoietic stem cell transplant. However, most patients lack a matched related donor, and there is a marked increase in morbidity and mortality following transplant using stem cells from an unrelated source. Discovery Genomics, Inc. (DGI) is developing its Sleeping Beauty (SB) transposon system as a non- viral vector for gene therapy of FA. The two-component SB system consists of a transposon (inverted repeats flanking a therapeutic gene of interest) and a transposase that catalyzes excision of the transposon at the ends of the IR's and then integration into host cell chromosomal sequence. In Phase I studies, DGI demonstrated the successful loading of transposon DNA and RNA into cultured hematopoietic cells, and then correction of human lymphoblastoid cells deficient in Fanconi anemia complementation group C (FANC-C) by SB-mediated transposition. In this Phase II application, the overall goal is to provide key preclinical data that will be necessary to support the anticipated efficacy of SB-mediated gene therapy for FA. In this regard, there are two key questions that will need to be addressed; (i) What is the effectiveness of SB-mediated FANC gene insertion in the treatment of an animal model of FA? (ii) For the human trial, what is the clinically applicable methodology that will be used for introduction of FANC-encoding transposon DNA into human hematopoietic stem cells (HSC)? There are two Specific Aims: Aim 1. Evaluate Sleeping Beauty transposon-mediated integration and long-term expression of the FANC-C gene in hematopoietic stem cells of FANC-C deficient mice as a model for SB-mediated gene therapy for Fanconi anemia. Aim 2. Evaluate Sleeping Beauty transposon-mediated integration and long-term expression in human CD34+ hematopoietic stem cells. For both of these Aims, SB transposon DNA and transposase-encoding sequence will be introduced into HSC using Cyto Pulse electroporation technology. Results from these experiments will provide the technical basis for achieving transposon-mediated integration and long-term expression in hematopoietic stem cells, the cell population that will be targeted for FA gene therapy, and will also provide the commercial basis for growth of DGI in the development of FA gene therapy using the SB transposon system. This project describes the development of a non-viral gene transfer method for treatment of Fanconi anemia, a rare disorder of the blood, by Discovery Genomics, Inc. (DGI), a small biotech startup company. Successful treatment of Fanconi anemia using DGI's transposon system is intended to provide proof of principle for the potential application of this technology to the treatment of other disorders of the blood as well.
Agency: Department of Health and Human Services | Branch: | Program: STTR | Phase: Phase I | Award Amount: 290.92K | Year: 2008
DESCRIPTION (provided by applicant): The overall goal of Discovery Genomics, Inc. is to develop the Sleeping Beauty transposon system for human gene therapy. To achieve this to treat hemophilia, we must be able to direct uptake and long-term expression of therapeutic genes in targeted tissues such as the liver. Only one method of plasmid delivery has been effective for high-level gene expression in the liver in mice - the rapid, high-pressure delivery known as hydrodynamic injection. In the mouse, hydrodyna mic delivery of DNA requires injection of a large volume (10% vol/wt) of a DNA solution through the tail vein in less than 10 seconds. For larger animals, this procedure is conjectured to be unacceptable on a whole animal basis. In this highly focused proj ect we propose to develop catheter-based, local hydrodynamic delivery to the liver of dogs that will require minimal surgery and amounts of therapeutic DNA. These features are especially important for the delivery of Factor IX and Factor VIII-expressing tr ansposons for treatment of hemophilia. Recognizing that delivery to only one cell type in the liver (and other organs) is not possible, we will to couple physical and biological controls over gene expression to our transgenic constructs that will reduce th eir expression in hematopoietic cells to reduce undesirable immunological responses. This project will dovetail with our ongoing SBIR project to deliver Factor VIII and IX genes in dogs via open-chest surgery for treatment of hemophilia. The Specific Aims of the project are to 1) develop appropriate transposons with reporter genes and recovery cassettes that can be used to evaluate the efficacy of hydrodynamic delivery in dogs, 2) determine an optimal, catheter-based delivery procedure that can be used to i nject DNA with minimal surgical intervention into canine livers, and 3) demonstrate that long-term gene expression will result from transposition of Sleeping Beauty transposons into canine hepatocytes using the procedure(s) developed in Aim 2. Publi c Health Relevance: Delivery of Sleeping Beauty Transposons to Dog Liver for Gene Therapy. There currently is no efficient and effective method for delivery of Sleeping Beauty transposons to liver in humans for gene therapy. The goal of this project is to develop a transposon delivery system that will allow hydrodynamic delivery of SB transposons in dogs as a model large animal for humans.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 270.67K | Year: 2010
DESCRIPTION (provided by applicant): Primary immune deficiencies comprise a group of inherited genetic disorders caused by interruption of normal lymphoid development. These diseases are considered prime candidates for gene therapy by introduction of the missing gene into hematopoietic stem cells that can then differentiate into lymphoid cells, and thus restore immunity. X-linked severe combined immunodeficiency (X-SCID), caused by a mutation in the gene encoding the common ( chain gene ((c) of the receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-213, is one of the more common of the primary immunodeficiencies. Clinical gene therapy trials for X-SCID have focused on the use of retroviruses as an integrating viral vector for introduction of the (c gene, resulting in restoration of immunity in patients that have engrafted with transduced, autologous hematopoietic stem cells. However, serious adverse events have emerged in these clinical trials, namely a T-cell leukemia-like syndrome developing in 5 of 21 treated patients. Therefore, safer, alterative therapies are needed. At Discovery Genomics, Inc. (DGI), we are working on developing the Sleeping Beauty (SB) transposon system for gene therapy, including the targeting of hematopoietic stem cells for treatment of primary immunodeficiencies. The two-component SB system consists of a transposon (inverted repeats (IR's) flanking a therapeutic gene of interest) and a transposase that catalyzes excision of the transposon at the ends of the IRs and then integration into host cell chromosomal sequence. As a lead immunodeficiency, here we propose that X-SCID may be treatable without the use of a virus by combining the power of electroporation for introduction of DNA into cells, along with use of DGI's Sleeping Beauty transposon system to achieve integration and long-term (c gene expression. In this Phase I application, the overall goal is to establish conditions for SB-mediated gene therapy for X-SCID. In this regard, there are two key questions that will need to be addressed: (i) Will SB-mediated transposition of the (c gene allow functional correction of lymphoid cells? (ii) What is the effectiveness of SB-mediated (c gene insertion in the treatment of an animal model of X-SCID? There are two Specific Aims: Aim 1. Evaluate the effectiveness of Sleeping Beauty transposons for correction of (c chain deficiency in a lymphoblastoid cell line derived from an X-linked SCID patient. Aim 2. Evaluate Sleeping Beauty-mediated transposition and long term expression of (c gene in hematopoietic stem cells derived of X-SCID mice, as a model for SB-mediated gene therapy of X-SCID. Successful accomplishment of these goals will provide evidence for the effectiveness of the SB system to mediate non-viral gene transfer in murine hematopoietic stem cells (previously undemonstrated), as well as provide key preclinical data for the development of SB transposons for X-SCID as a lead primary immunodeficiency. PUBLIC HEALTH RELEVANCE: Results from these experiments will provide the technical basis for achieving transposon-mediated integration and long-term expression in hematopoietic stem cells, the cellular target inr gene therapy of primary immunodeficiencies. In addition, it will provide the basis for the development of the SB transposon system for treatment of other hematologic diseases.