Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Target audience is humans and animal regulatory agencies and practitioners interested in gene therapy. Changes/Problems: When the proposal was initalliy written, all technology was licensed by a biotech company that filed bankruptcy in 2013. The technology has since been purchased by another company, with which I am not working. The result has been for me to develop a new line of research that will be the subject of the next HATCH proposal. What opportunities for training and professional development has the project provided? This project will be used to train an undergraduate intern from Zamorano University, Honduras, in spring 2015. If his performance is satisfactory, he will be able to return to the U.S. to pursue a Master's degree after he completes his undergraduate program. He will be trained in various aspects of molecular biology and cell culture. This work can serve as a basis for his Master's research project. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? Early in the second quarter of 2015, the E. coli cloning strain and the accompanying vector will be completed. Once th new host strain is developed, it will be tested to determine if the system is working properly and growth curves will be conducted to determine the optimal time for inducing the enzyme to remove the "extra" DNA from the plasmid. Conventional plasmid harvest protocols will be used; total yield efficiency of DNA removal will be calculated. During the 3rd and 4th quarters of 2015, a DNA vaccine will be produced to determine system efficacy.
Impacts
What was accomplished under these goals?
As genetic tools for gene therapy and DNA vaccines make each of these technologies more useful in human and animal medicine, there is a growing requirement by the FDA that the components needed for replication of the DNA in a bacterial host not be part of the final product. Numerous attempts by otherstried to minimize the amount of remaining DNA in the final product and have met mixed results. The end product either has unwanted DNA remaining or the procedures to remove the extra DNA result in too much product loss, or the purification costs make the final product cost prohibitive for DNA vaccines, especially for veterinary purposes. For the past year, we have been developing an E. coli strain to grow plasmid DNA for use in gene therapy and DNA vaccines. Our system is unique in that the E. coli strain, when induced, will produce enzymes to remove all extraneous DNA prior to harvest. Once the extra DNA is removed, the vector DNA containing the desired products can be harvest using conventional methods which will increase yield, lower costs, and most importantly, meet FDA requirements for a safe product. To date, the system has been designed, additional seed funding obtained, and the pieces constructed to begin assembling the DNA to be used in making a host strain. It is anticiapted that the host strain will be completed early in 2015.
Publications
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: The market segment served by this project is the pharmaceutical industry, with the individual consumer as the ulitmate target once a therapeutic is approved. Changes/Problems: The Major change in the next reporting period will be the funding source.A private biotech company funded a majority of this work, butit has been disolved therefore other sources of funding through other companies are being pursued to supplement this work. In addition, it is anticipated that additional funds will be pursued in collaboration with UAB and investigators from the LSU School of Veterinary Medicine. What opportunities for training and professional development has the project provided? OUTPUTS: Data from this project has been provided to numerous groups throughout Louisiana in an effort to inform citizens and local and state leaders of the work being conducted as part of the LSU Agricultural Center. Our project aims to establish Baton Rouge in the biotech arena and impact the pharmaceutical industry by lowering drug prices through innovative approaches. In addition, research progress was shared with business collaborators Esperion Therapeutics, Swiftwater Consulting Group, and Cleveland Clinic's Cardiology Department. One journal article and one patent resulted. The initial phase of collaboration has been established with the University of Alabama at Birmingham to investigate using the transposon vectors for treating cystic fibrosis. A material transfer agreement is now in place with the LSU Ag Center and Dr. Cooper's laboratory for receiving human primary epithelial cells. How have the results been disseminated to communities of interest? Results have been disseminated through invited presentations. What do you plan to do during the next reporting period to accomplish the goals? Funding is being pursued to evaluate the new transposon vectors for use in gene therapy to treat cystic fibrosis. The first goal is to establish the best method of DNA delivery to primary airway epithelial cells. Different transfection reagents and DNA concentrations will be tested and the leading candidate optimized for the most number of cells transfected and expressing the dsRed gene. Identification of one of these vectors and delivery methods as superior will result in further testing in collaboration with UAB.
Impacts
What was accomplished under these goals?
The first goal in Objective 1 was met. Using the transposon technology in combination with avian cells, it was demonstrated that a fully formed human monoclonal antibody could be made and secreted from the cell. This was demonstrated with two different monoclonal antibodies. Human FGF2 was also produced and shown to be biolgically active in vitro and promoted wound healing in mice and rats when tested in a burn wound model in vivo. Objective 2 - a significant amount of progress was made on media optimization, however the final anaylsis in the hollow fiber system could not be completed.Objective 3: the new tranposon vectors were completed in August 2013 and a dsRed reporter gene cloned into each one for testing in cell culture. The inital work will be conducted in cell culture to compare protein expression, copy number and site of insertion into the chromosomes. Previous data has demonstrated insertion in up to five dfiiferent avian chromosomes; this data will be used as a baseline for comparison with the new vectors. It is anticipated there will be additional insertion sites and increased protein expression due to a decrease in gene silencing.
Publications
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Data from this project has been provided to numerous groups throughout Louisiana in an effort to inform citizens, and local and state leaders of the work being conducted as part of the LSU Agricultural Center. Our project aims to establish Baton Rouge in the biotech arena and impact the pharmaceutical industry by lowering drug prices through innovative approaches. In addition, research progress was shared with TGRx business collaborators Esperion Therapeutics, Swiftwater Consulting Group, and Cleveland Clinic's Cardiology Department. One journal article and one patent resulted. PARTICIPANTS: Cooper, R (PI), LSU AgCenter; TGRx, Baton Rouge, LA. TARGET AUDIENCES: Biotechnology researchers and economic development investors. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The goal to develop methods for increasing expression of pharmaceutical proteins and delivering DNA for expression of therapeutic proteins is underway. We have begun to investigate DNA delivery platforms that greatly expedite the clone production process for cells producing therapeutic proteins. By coupling our technology with modern bioreactors, significant cost savings can be obtained over traditional manufacturing methods. Our main objectives have been: to use genetic constructs to encode pharmacological proteins; optimization of cell culture media and growth conditions in hollow fiber reactor systems; and design and optimization of genetic constructs for use as transgenes for protein production using transient gene therapy. The FDA had begun to establish a mechanism for regulatory approval of biosimilars. Collaborations between TransGenRx and our lab will result in TransGenRx's ability to meet with FDA representatives and begin establishing approval pathways for our biosimilars in the U.S. market. Our objectives have resulted in a series of experimental goals. Our first goal was to determine if the transposon vector coupled with an avian cell line is capable of producing fully functional monoclonal antibodies and other proteins. This goal was satisfied by TransGenRx using our technology and entering into a commercial contract with Esperion to produce a biopharmaceutical protein to treat atherosclerosis. TransGenRx has completed the first contract and is awaiting the second contract with Esperion. TGRx has developed a growth factor that is demonstrating significant potential in wound healing. This protein can be formulated in a gel to treat pressure sores associated with bed-ridden patients or decubitus ulcers often seen in diabetic patients, or it can be combined with a burn victim's own skin stem cells to treat second and third degree burns with little or no scarring. TGRx has developed a disposable device to spray the growth factor/stem cell solution onto the burn wound which can result in healing in as little as six days. Our team has excellent preliminary data on protein characterization, third party validation, and preliminary animal data. The technology is easily transportable and can be conducted at any burn center with a low speed centrifuge. Once a burn center is properly equipped, the time the skin stem cells are harvested to the time the patient is treated is about 1.5 h. The data generated to date has set the stage for preclinical research and product development leading to clinical trials. This work will consist of two components: 1) animal models for testing skin stem cell isolation and wound treatment, and 2) in vivo testing of the skin stem cell delivery system. It is anticipated the device will fall under the FDA "Pilot Program for Early Feasibility Study Investigational Device Exemption Applications" (FDA-2011-N-0788). This project can be funded based on milestones, i.e. data generation on preclinical work, and each phase of clinical trials based on performance.
Publications
- Q.G. Xue, J. Gauthier, K. Schey, Y. Li, R. Cooper, R. Anderson, J. La Peyre. 2012. Identification of a novel metal binding protein, in plasma of the eastern oysters. Comparative Biochemistry and Physiology Part B 163:74-85.
- Administration of Transposon-based vectors to reproductive organs. Richard K. Cooper and W. C. Fioretti. 2012 US CON 8,283,518
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Results from this project have been provided to numerous groups throughout Louisiana to inform citizens, local, and state leaders. Our project promotes Baton Rouge in the biotech arena and impacts the pharmaceutical industry by lowering drug prices through innovative approaches. In addition, research progress was shared with TransGenRX business collaborators including Esperion Therapeutics, Swiftwater Consulting Group, and Cleveland Clinic's Cardiology Department. PARTICIPANTS: R. Cooper (PI), and F.O. McManus, LSU AgCenter. TARGET AUDIENCES: International and National Scientific communities. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
DNA delivery platforms that greatly expedite the clone production process for cells producing therapeutic proteins is under investigation. By coupling technology with modern bioreactors, significant cost savings can be obtained over traditional manufacturing methods. Collaborations between TransGenRx and our lab has resulted in TransGenRx's ability to meet with FDA representatives and begin establishing pathways for approval of our biosimilars in the U.S. market. Our objectives have resulted in a series of experimental goals. Our first goal was to determine if the transposon vector coupled with an avian cell line is capable of producing fully functional monoclonal antibodies and other proteins. TransGenRx used our technology and entered into a commercial contract with Esperion to produce a biopharmaceutical protein to treat atherosclerosis. TransGenRx has completed the first contract and is pursuing another contract with Esperion within the first quarter of 2012. The second goal of analyzing spent media from avian cell culture to develop a media that will allow for protein expression in hollow fiber systems is still underway. This research has also opened the door for media optimization of another production platform, Hyperstacks from Corning, which could potentially reduce production cost and enhance protein expression. The third goal compares our current transposon-based system with two new transposon vectors to increase insertion efficiency and the ability to overcome silencing. Success with this endeavor has led to evaluating other chromosome insertion systems to further increase our number of incorporation sites.
Publications
- Gene Therapy Using Transposon-based Vectors. R.K. Cooper. Issued 10/11/ 2011. US Patent # 10/ 582,812 Canadian Patent Application No. 2,490,693 entitled: Gene Regulation in Transgenic Animals Using a Transposon-Based Vector. Our File: 56187-309181 (0101CA) Canadian Patent No. 2,490,693
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