Progress 10/01/05 to 09/30/10
Outputs
OUTPUTS: The objectives were to test vectors designed to incorporate into the chicken genome, specifically germ cells, and use these vectors to deliver genes expressing pharmaceutical proteins with human and veterinary potential. Numerous methods for gene delivery were optimized, including testicular injection, cardiac injection, and direct oviduct injection. Research using liver cells led to a protein manufacturing platform currently being commercialized by TransGenRx, Inc. (TGRx). To date, twelve different proteins have been produced in the cell culture system; and six have been tested for biological activity and shown to be as active, if not superior, to products currently marketed. Research on transgene integration into avian chromosomes resulted in one doctoral thesis and three peer reviewed publications during the course of the project. PARTICIPANTS: Dr. Richard Cooper, Professor, Principal Investigator, Jackie McManus, Developed cell culture techniques for cell lines and helped developed cloning protocols. Ellen Case, Evaluated different methods for cell transfection and helped develop clone selection protocols. Laynette Spring, assisted in clone selection and determining growth parameters for cell lines. Fuad Odeh and Amanda Broussard developed Western blot assays and ELISA's for protein detection and characterization. Laura Peak, Ashley Borrel and Donndi Harper developed vector construction strategies, built vectors to be tested and sequenced all DNA Brandye Smith conducted all PCR on genomic and mRNA samples from transgenic birds and and cell lines. TARGET AUDIENCES: The technology developed in this project was partially funded by TransGenRx, Inc. under the Research Agreement and the License and Royalty agreement with the LSU Agricultural Center. The technology is being commercialized to develop human phamaceutical proteins at a reduced cost PROJECT MODIFICATIONS: Due to the issues observed with gene silencing in transgenic quail and chickens and a discovery made using cell culture, a major shift was made in 2007 to focus efforts on developing and commercializing protein production in cells. Research led to control elements that resulted in proteins being expressed at high levels in cell culture and have indicated a cost effective, alternative approach to pharmaceutical protein production is very feasible. The research conducted in cell culture has also resulted in technology that may yield an entirely new cell line as well as DNA vectors that will make it possible to overcome gene silencing, have use in gene therapy, and increase protein output in cell culture.
Impacts
The goal of this project was to develop a platform technology for producing transgenic chickens capable of expressing human pharmaceutical proteins in their egg white and/or yolk. The tubular gland cells lining the oviduct that make egg white proteins are among the most prolific protein producing cells known, and the egg white itself is a rather simple biological fluid from which to purify pharmaceutical proteins expressed from transgenes. This project resulted in an extensive knowledge base on gene expression and delivery in transgenic chickens and quail. Heritability was demonstrated through G2 in chickens and G3 in quail. Vectors containing proteins controlled by regulatory elements specific for expression in the oviduct or liver were built and tested. This research resulted in the development of promoters capable of expressing proteins at a rate higher than previously observed. Methods developed with testicular injected males resulted in >50% transgenic offspring, while cardiac injected females resulted in >90% transgenic offspring from the eggs laid up to 5 d post-injection (this procedure was designed to target developing oocytes). Mating of testicular injected males with cardiac injected females yielded greater than 95% transgenic offspring. Gene silencing was determined to be an issue in transgenic offspring despite testing a variety of insulator elements designed to prevent it. Silencing was determined to be based on the insertion site; expression was determined to occur in the brain of the transgenic birds. Chromosome analysis on G3 animals demonstrated the transgene on 1 to 5 chromosomes per genome, but copy number was not determined. These data resulted in the design of two vectors to target different insertion sites to overcome gene silencing; these will be built and tested in the new project. A result of intensive research to overcome gene silencing led to development of regulatory elements that express transgenes at a very high rate in avian liver cells. Cell culture methods were developed for tubular gland (oviduct) and liver cells to serve as a testing platform for expression before putting a gene in a bird. This finding has resulted in a protein production platform being commercialized by TGRx. These cells have been shown to secrete 90% of the protein made making them well suited for a perfusion culture system. These cells also glycosylate proteins very similar to human cells and have resulted in the production of new molecular entities that are being investigated for potential use in humans. This production platform has proven to be a low cost approach to producing biosimilar (generic) proteins coming off patent through 2015 and is anticipated to allow true generic drug pricing on these drugs. Most of this research resulted in 41 patent applications or issued patents jointly owned by the LSU Ag Center and TGRx.
Publications
- Lacey R. McNally, William G. Henk, and Richard K. Cooper. 2005. Laser Pressure Catapulting From Teflon-Coated Coverslip Slides Followed by β-Actin Gene Identification in Japanese Quail Macrochromosomes and Microchromosomes. Journal of Microscopy 218: 219-224.
- Boudreaux, C. M., R. K. Cooper, F. M. Enright, and R. E. Corstvet. 2005. Cecropin B transgene expression and protection against Mannheimia haemolytica 1:A infection of the bovine nasal mucosa. Am J Vet Res. 66: 1922-1930.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: The goal to develop methods for producing low cost pharmaceutical proteins is being realized. Proof-of-concept was obtained on six different pharmaceutical proteins in 2009, and discoveries were made that demonstrate the ability to make glycosylated proteins very similar to the protein glycosylation patterns in humans. This resulted in at least two new innovator drugs that can be termed "biobetters (better than the original drug)." The results obtained in 2009 demonstrate the feasibility of producing generic or biobetter drugs at a cost that allows generic pricing. There were three patents issued in 2009 and 11 patent applications filed. These findings have allowed TransGenRx, Inc. to begin commercializing the technology with plans to enter the market place on a limited basis in 2010. PARTICIPANTS: This project provided training for one research assistant professor, two post-docs, and four undergraduate students in 2009. Experience was gained in cell culture, molecular biology, protein detection and purification, and large scale production in cGMP manufacturing. Eleven personnel received GMP training in SOP preparation, clean room validation and operation, and large scale production in hollow fiber bioreactors. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Currently, no regulatory pathway exists to approve a generic (biosimilar) protein-based pharmaceutical drug in the U.S. The Federal Government is working with the FDA to establish a mechanism for regulatory approval, but the pathway is not anticipated to be in place for three to five years. Once a clear pathway has been defined, production of a generic protein pharmaceutical will be difficult if there is not an advance in the current production technology. Using current methods does not offer a significant enough price reduction to decrease costs by the needed 40% for a generic drug to be competitive. The goal of this project from the beginning has been to develop a system that allows a high rate of pharmaceutical protein production at a low cost. Using our DNA delivery technology to force gene incorporation, the oviduct of chickens was targeted to produce pharmaceutical proteins in egg white. In 2009, while developing a protein expression system for chickens, a discovery was made that allows cell culture to be coupled with new bioreactor technology resulting in low cost production of proteins. This research has also resulted in the production of innovator drugs that may be considered as "biobetter." The impact on consumers is lower medication costs; and in at least two instances with our biobetter compounds, the potential for little or no toxicity when compared to the innovator drugs currently being marketed.
Publications
- No publications reported this period
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Activities: The goal of this project is to develop cheaper methods of protein pharmaceutical production in order to lower the end cost of the drug and make the drugs more widely available. To this end, research using cell culture to increase protein expression in chickens has resulted in expression rates in cell culture that have not been previously possible. As a result, TransGenRx, Inc. (TGRx) is applying this technology in an effort to commercialize the production of human growth hormone, monoclonal antibodies, and interferon. Another unexpected research result was the discovery a new drug entity - naturally glycosylated human interferon. Interferon currently on the market is produced in E. coli and has to be pegylated. The process we have developed produces naturally glycosylated interferon that is as biologically active as the current product and is glycosylated in the same position as native human interferon. Three new patent applications have been submitted on the production method and products being developed. Events: The recent advances in this technology was presented at the CPHI conference in Frankfurt, Germany (Oct 1-4, 2008). Products: In addition to the products mentioned above, I am currently training 4 students. Two of the students are learning to apply our technology to gene therapy applications using cell culture as a model and I have two students learning to work in a clean room environment. These students are gaining experience that will make them very marketable in this field, and we are hoping to one day expand the curriculum into a degree program or a portion of a program that allows an emphasis on working with cell culture in a cleanroom environment. Currently, this segment of the workforce is missing in Louisiana. Another anticipated product is the creation of biotech jobs with TGRx. Application of our technology to the biologics industry will allow the growth of a biotech business in Louisiana with a potential for significant growth within the next 18 months. PARTICIPANTS: Jackie McManus, M.S. Instructor. Cell Culture Production Manager. Oversees clone production and Bioreactor operation. Ellen Case, B.S., Research Associate in cell culture. Day to day care for cell culture experiments, cell transfection and clone selection. Laynette Spring, B.S., Research Associate in cell culture. Day to day care for cell culture experiments, cell transfection and clone selection. Amanda Broussard, B.S. Research Associate. Responsible for developing and running ELISA-based assays for determining protein quantity and clone suitability. Fuad Odeh, Ph.D. Post-Doctoral Researcher. In charge of target protein characterization using ELISAs, SDS PAGE and Western Blots. Jay Marcin, B.S., Research Associate. Assists in compliance with Quality Control/Quality Assurance, S.O.P. preparation and training. Adriane Bercegeay, B.S. Research Associate. Construction of DNA vectors and Bioinformatics for optimizing gene expression. Billy Dudley, Ph.D. Post-Doctoral Researcher. Responsible for cleanroom operations in compliance with GMP/FDA guidelines, Bioreactor scheduling for protein production, and inventory control. Holly Bofinger, B.S. Research Associate. Responsible for PCR analysis on transgenic birds and their offspring and transfected cell lines. Brandye Gaudin, B.S. Research Coordinator. Designs and oversees the PCR and DNA sequencing laboratories. Laura Peak, M.S., Research Associate. Vector production and Quality Control. Responsible for constructing DNA vectors for use in chickens and cell culture (first 9 months of 2008) and developing SOP's and running Nova Bioflex Analyzer for Quality control on incoming media and Bioreactor samples (duties shifted in last quarter of 2008). Ashley Borrel, B.S. Research Associate. Production of vectors for use in chickens and in cell culture, DNA sequencing and vector design. Donndi Harper, B.S. Research Associate. Production of vectors for use in chickens and in cell culture, DNA sequencing and vector design. Shane Castille, B.S. Research Associate. Poultry manager in charge of maintaining normal and transgenic chickens associated with the project. TARGET AUDIENCES: The immediate efforts stemming from this project have been two fold: 1.) the transfer of knowledge to TGRx in order to establish a biotech industry in the Baton Rouge area; and 2.) the training of students in the areas of molecular biology, protein chemistry, cell culture, animal husbandry (poultry), and clean room operations in compliance with the FDA. The target audiences first are the students and the knowledge and training they receive. The second is more long term and will be all peoples that may be affected by the production of lower cost protein-based drugs, such as insulin, monoclonal antibodies, etc. Our goal is to ultimately have an impact on healthcare by providing true generic biologic drugs. PROJECT MODIFICATIONS: This project was developed in order to produce transgenic chickens capable of expressing pharmaceutical proteins in the whites and yolks of their eggs. This portion of the project is still under development. However, in the course of developing cell culture techniques to optimize gene expression in chickens, a discovery was made that allows more rapid commercialization using cell culture production methods. The adaption of our technology to traditional cell culture techniques and new cell culture bioreactor systems will allow higher protein production in a fraction of the space as current cell culture methods allow. The transfer of this technology to TGRx allows quicker entry into the commercial arena.
Impacts
There are several outcomes of the research described above. First, the increased protein production in cell culture is allowing TGRx to enter commercial production in early 2009. Once commercial, this will represent a significant transfer of LSU Ag Center technology from the lab to the commercial arena. This has the potential for impacting the local and state economy through the creation of jobs and tax revenue, creating a strong biotech company in the Baton Rouge area with the potential for attracting additional businesses, and providing an opportunity to train students with the skills needed to work in the biotech industry. Another significant outcome is the discovery of a new drug entity. Early indications are that this drug may be much less toxic than what is currently available, be more efficacious due to a longer half life, and be more readily available to a wider range of neurological disorders due to the decreased expense of the final product.
Publications
- Cooper, R.K. and W. C. Fioretti. 2008 Provisional Patent Application No. 61/100,075 entitled NOVEL VECTORS FOR PRODUCTION OF ANTIBODIES IN VITRO.
- Cooper, R. K. and W. C. Fioretti. 2008. Provisional Patent Application No. 61/100,116 entitled NOVEL VECTORS FOR PRODUCTION OF INTERFERON IN VITRO.
- Cooper, R. K. and W. C. Fioretti. 2008. Provisional Patent Application No. 61/100,157 entitled NOVEL VECTORS FOR PRODUCTION OF GROWTH HORMONE IN VITRO.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Several items of note were achieved on this project in 2007. First, office actions were granted on several patent applications which should result in patents being awarded in 2008. Second, significant progress was made in developing transfecting reagents and protocols from bulk reagents that greatly reduces the cost when compared to commercially prepared transfecting reagents. Techniques were developed using linear and branched PEI's that when complexed with DNA can target specific tissue and cell types. Third, DNA vectors were developed and tested that have alleviated some of the problems associated with gene silencing. These vectors were tested extensively in cell culture in order to determine the appropriate combination of elements to prevent gene silencing. The result was significant expression in cell culture beyond anything previously seen. In fact, the expression was so good that efforts are underway to commercialize the technology which will result in TGRx having two
commercial products during the third quarter of 2008.
PARTICIPANTS: Collaborators through TGRx, Inc. William Fioretti, President of TGRx and biochemist Hansen T. Bordelon, Ph.D., protein isolation Scot Floyd, M.S., Poultry Manager LAES employees: Dr. Billy Dudley, post-doc and coordinator of protein isolation and characterization team. Jackie McManus, MS, Coordinator of cell culture production facility Dr. Fuad Odeh, post-doc, protein characterization Dr. Maria Diaz, post-doc, genomics and transgene applications Laura Peak, MS, vector design and construction Brandye Gaudin, PCR analysis of transgenic animals Ashley Fruge, vector development Adriane Bercegeay, vector development and bioinformatics Donndi Harper, vector development Ellen Case, cell culture Amanda Broussard, protein characterization Laynette Spring, Immunohistochemistry Jay Marcin, egg processing, separation Holly Bofinger, poultry production.
TARGET AUDIENCES: Pharmaceutical companies with protein products; scientists with an interest in transgenic animals or in gene therapy.
PROJECT MODIFICATIONS: The major project modification was the development of cell culture as a tool for commercial production. The techniques developed to reduce or eliminate gene silencing when combined with our vector technology have provided an avenue for commercial production that had not been previously considered. The result will be an ability to enter commercial production in 2008.
Impacts
In the near term, the studies to alleviate gene silencing have resulted in technology that could make TGRx profitable during 2008. Two companies have already expressed interest in purchasing any products produced using our cell culture system. This would fulfill a goal of having an LAES technology licensed and developed by a Louisiana-based start up company begin generating income and in collaboration with the efforts of the LETC, keep Louisiana technology in Louisiana. The result will be additional job opportunities and another step towards establishing a biotech industry in our state. Long term, the results obtained in 2007 will result in cheaper ways to make transgenic animals and increase expression of transgenes in chickens. The net result will mean more jobs in Louisiana, especially through support businesses that arise and the promise of cheaper protein-based pharmaceuticals due to cheaper production methods.
Publications
- Chandler, J. E., T. Taylor, A. Canal, R. Cooper, E. Moser, M. McCormick, S. Willard, H. Rycroft, and G. Gilbert. 2007. Calving sex ratio as related to the predicted Y-chromosome-bearing spermatozoa ratio in bull ejaculates. Theriogenology 67: 563-571.
- Xue Q.-G., Itoh N., Schey K.L., Li Y., Cooper R. and La Peyre J.F. 2007. A new lysozyme from the eastern oyster (Crassostrea virginica) indicates adaptive evolution of i-type lysozymes in bivalve mollusks. Cellular Molecular Life Sciences 64:82-95.
- Itoh N., Xue Q.-G., Li Y., Cooper R. and La Peyre J.F. 2007. cDNA cloning and tissue expression of plasma lysozyme in the eastern oyster, Crassostrea virginica. Fish and Shellfish Immunol. 23:957-968.
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Progress 01/01/06 to 12/31/06
Outputs
A series of DNA vectors designed to optimize protein expression in the egg white of laying hens was constructed during 2006. These vectors contain various regulatory elements designed for oviduct specific expression and increased secretion of the target protein. As each vector was completed, it was tested in cell culture to determine if the components were working correctly and that the target protein was expressed in proper form. Vectors that function correctly will be tested in birds during 2007. Germ line male and female birds will be developed in January and evaluated for transgene presence and protein expression when sexual maturity is reached.
Impacts
This project is designed to lower the cost and increase production capacity for protein-based pharmaceutical drugs. It will have local, national, and international impact by providing jobs for Louisiana and lowering drug costs. Another benefit is the increased production capacity to produce drugs that would not ordinarily be produced due to the expense associated with the capital investment required for the production facility. FDA approved chicken houses can be built for 1/100th the cost of a traditional cell culture facility.
Publications
- Lacey R. McNally, William G. Henk, and Richard K. Cooper. 2006. Chromosomal Localization of a Proinsulin Transgene in Japanese Quail by Laser Pressure Catapulting. Transgenic Research 15: 427-433.
- Transposon-based vectors having elements for regulation of gene silencing. R. K. Cooper. Patent Application Submitted May 2006.
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