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Lexington, KY, United States

Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 220.20K | Year: 2016

DESCRIPTION provided by applicant ParaTechs Corporation aims to provide researchers with advanced technology and methods to simplify procedures for laboratory animal models While actively supporting efforts to `reduce replace and refineandapos animal use ParaTechs provides safe and efficient alternatives for surgical procedures We have already brought to market a nonsurgical embryo transfer device NSETTM which replaces surgical embryo transfers with a nonsurgical method that takes only seconds and does not require anesthesia We have developed a new protocol for nonsurgical artificial insemination AI of mice which replaces existing protocols for surgical AI and is a technical advancement for existing nonsurgical AI protocols We plan to use this newly developed protocol to revolutionize the process of archiving and global distribution of mouse models through a novel sperm cryopreservation and strain recovery system The use of mouse strains in research is one of the most important tools used to study mammalian gene function While hundreds of thousands of mouse strains have been catalogued and cryopreserved sharing of strains is becoming increasingly difficult as regulatory constraints and the cost of shipping live animals increase We can solve this problem and provide a technically superior alternative with the cryopreservation and insemination Candamp I device This system will integrate storage and shipment of cryopreserved sperm with nonsurgical artificial insemination The device will be suitable for storage of cryopreserved sperm will be shippable will allow viable sperm recovery upon thawing and then will be used directly for nonsurgical artificial insemination This device will eliminate the need to perform in vitro fertilization IVF upon thawing of cryopreserved sperm greatly reducing the number of mice required for the procedure and replacing a surgical method of artificial insemination both of which promote animal health and welfare Therefore the goals of this project are to design manufacture and test a Candamp I device for mice The goals will be accomplished in two aims First a Candamp I device prototype will be designed and produced Second the feasibility of using the Candamp I device prototype for sperm cryopreservation and mouse strain recovery will be tested This new technological breakthrough will provide an alternative to cryorecovery of mouse strains by in vitro fertilization methods the currently preferred but technically challenging and labor intensiv strain recovery method In doing so the Candamp I device will provide a substantial cost savings to researchers and a convenient method for shipping and strain recovery PUBLIC HEALTH RELEVANCE The ability to genetically modify the laboratory mouse is now producing an ever rising number of increasingly valuable and unique strains to investigate human disease While maintaining these mouse strains has become a significant and recurring effort and budgetary commitment cryopreservation of strains in sperm repositories has proven successful as a means to permanently and economically preserve them ParaTechs proposes to design manufacture and test a cryopreservation and insemination Candamp I device for the purpose of integrating cryopreservation storage shipping and strain recovery of mice through a newly developed nonsurgical artificial insemination method

Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase II | Award Amount: 1.04M | Year: 2015

DESCRIPTION provided by applicant The baculovirus expression vector system BEVS has been successfully utilized to produce thousands of proteins for use as vaccines and therapeutics as well as for studies of protein structure and function One limitation of BEVS is the propensity of baculoviruses to accumulate transposon insertions into the fp k gene leading to the andquot few polyhedra FP andquot phenotype This mutation shifts the balance of virus production from occlusion derived viruses which are not infectious in tissue culture to budded viruses BV which are the form of virus that is used in baculovirus expression Higher levels of BV would be advantageous for BEVS users but FP mutants are also deficient in transcription from the polyhedrin promoter that drives expression of target genes Baculoviruses also rapidly accumulate defective interfering particles DIP which are often linked to a sharp decrease in target gene expression due to deletion of the target gene and or the viral genes needed for its expression One factor promoting DIP formation is transposition into fp k The goal of this proposal is to limit deleterious effects of transposition into fp k while taking advantage of the fact that elimination of the FP K activity significantly increases BV production During Phase I two strategies an inducible construct for controlling fp k and an fp k deletion mutant coupled with a cell line constitutively expressing FP K were explored for regulating FP K expression Both strategies sought to produce high titer virus during the amplification stage of baculovirus infection and enable a switch to high level transcription from the polyhedrin promoter during the recombinant protein expression phase Results document achievement of Phase I objectives with deletion of fp k from the virus and complementary expression from an engineered cell line enabling the predicted control of budded virus and recombinant protein production However the inducible construct did not provide sufficient FP K control due to the toxicity of the heavy metal inducer to insect cells Therefore in Phase II we will develop an improved inducible construct with tighter transcriptional regulation using an ecdysone receptor based inducible promoter validate the beneficial effects of FP K regulation using viruses that express intracellular and secreted yellow fluorescent protein YFP for BEVS and BacMam mammalian expression technology develop user friendly fp k mutant backbones for simplified BEVS and BacMam cloning and demonstrate their utility with several medically relevant transgenes and test the fp k expression system in the context of vankyrin enhanced BEVS VE BEVSTM technology ParaTechsandapos VE BEVS products delay death and lysis of baculovirus infected cells thereby boosting target protein expression up to fold Many BEVS users would want to incorporate both vankyrin and FP K technologies therefore it is important to determine whether they are compatible Taken together completion of Phase II objectives will enable users of the BEVS to control and optimize production of BV and recombinant protein expression PUBLIC HEALTH RELEVANCE The baculovirus expression vector system BEVS is commonly used for production of proteins for structure function studies vaccines and therapeutics Unfortunately many researchers are not aware that certain mutations rapidly accumulate leading to lower levels of target gene expression ParaTechs has developed a strategy to prevent these mutations while simultaneously providing higher levels of virus production stable genome maintenance and target gene expression

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 200.98K | Year: 2011

DESCRIPTION (provided by applicant): Most cell culture lines are anchorage-dependent and require surface attachment for proliferation. For industrial production, increased surface area can be provided by microcarrier beads, but the associated increases incost and the resulting complexity of manipulation may preclude their use. Due to these limitations, anchorage-independent cells are preferred for the production of biopharmaceuticals, but appropriate anchorage-independent cells are not available for all applications; e.g., production of vaccines and cell-type specific proteins. This proposal explores the use of a novel insect virus protein to transform adherent cells to cells that can thrive in suspension culture. ParaTechs has identified a cell line fromAgrotis ipsilon (black cutworm), which provides levels of recombinant protein expression that are 3-to-10 times higher than the standard Sf9 cell line. Unfortunately, the cells are strongly adherent, which makes them unsuitable for large-scale protein production. We intend to stably transform the A. ipsilon cells with an insect virus gene that causes a loss of adhesion in hemocytes. We predict that expression of this protein in transformed cells will enable them to grow in suspension culture. In combinationwith our Vankyrin-Enhanced Baculovirus Expression Vector System (VE-BEVS), which has the ability to increase protein production per cell by a factor of 4 to 22, we anticipate achieving a level of protein expression per cell that is at least 12-to-220 times higher than currently possible. This dramatic increase in yield will be significant for all BEVS users, from individual researchers to large biopharmaceutical companies. We also will apply this technology to adherent mammalian tissue cultures. A simple method for converting mammalian cells from anchorage-independent to suspension culture would make a significant contribution to the production of vaccines and bio-therapeutics, and would result in improved human health. ParaTechs personnel are experiencedbaculovirologists and cell biologists. The protocols use standard technologies that are routinely adopted in our company. We do not anticipate difficulty with the experimentation. Current literature strongly supports our hypothesis and we are confident ofour success. PUBLIC HEALTH RELEVANCE: The future of human medicine will involve a dramatic increase in development of protein-based drugs and subunit vaccines, whose production will require large-scale propagation of tissue culture cells in suspension culture. Most continuous cell lines, however, are anchorage-dependent, and no method exists to routinely and easily transform adherent cells to suspension culture. ParaTechs will test an insect virus gene that causes hemocytes to lose adhesion for the ability to adapt anchorage- dependent insect and mammalian cells to suspension culture. Achievement of this goal would make a significant contribution to biopharmacology and human health.

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 1.03M | Year: 2011

DESCRIPTION (provided by applicant): The use of research animals in research has been essential to development of vaccines and study of most human diseases. Modern rodent research is increasingly powerful with the ability to manipulate the genomes of miceand rats such that they closely mimic complex human diseases such as Alzheimer's and artherosclerosis. Generating transgenic mice and rats, requires that embryos that are manipulated by researchers be transferred into recipient female mice where they can complete their development. Up to this point, these transfers have required surgical procedures in which the embryo is implanted into the uterus. Recently, a device has been developed that enables these embryo transfers to be performed without surgery whicheliminates the post-operative recovery period thereby reducing pain of the animals. This proposal investigates and expands upon the utility of this embryo transfer device by establishing the optimal ages and mouse strains for embryo transfer (Aim 1), determining whether embryonic stem cells can be effectively and efficiently transferred with this device (Aim 2), determining whether the device is useful for artificial inseminations (Aim 3), directly visualizing the device in the uterus which may enable design improvements (Aim 4) and quantifying measures of stress relative to surgical procedures to support widespread adoption of this procedure in the research community (Aim 5). The primary project objective is to produce data that will prove the efficacy ofthis embryo transfer device and thereby enable researchers to replace surgical procedures with a non-surgical method that is equally effective. Proving that this non-surgical transfer method is effective under a range of conditions will expand its use overthis range of applications and thereby maximize its impact in enabling researchers to refine and reduce the number of surgical procedures performed in mice. PUBLIC HEALTH RELEVANCE: Animal use in research is essential for biomedical research and with the developing abilities to genetically engineer mice for research; these animals are becoming even more important as they can be modified to mimic most human diseases. Genetic engineering of mice requires, as one of many other things, the ability to transfer embryos into mice where they complete their development. This project supports further development of a device that enables these embryo transfers to be performed without surgery and therefore greatly reduces the pain category that these research animals experience and thereby supports progress in biomedicine.

Agency: National Science Foundation | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 150.00K | Year: 2014

The broader impact/commercial potential of this Small Business Innovation Research (SBIR) Phase I project is the development of a technology to sustain the efficacy of transgenic plants expressing the Bacillus thuringiensis (Bt) toxin. Transgenic expression of Bt toxins is a major tool for the control of insect pests in agriculturally important crops. Bt transgenic technology has been deployed on over one billion acres because it improves crop yields and reduces the environmental problem of using toxic chemical pesticides. Insects, however, are becoming increasingly resistant to the Bt toxin, which is threatening its sustainability. This project proposes to develop alternative means to suppress populations of important lepidopteran pest(s) targeted by Bt. There is no greater threat to sustainability of the Bt technology than the development of resistance, with current evidence indicating that resistance is increasingly impacting producers globally. Novel strategies to suppress or eliminate insect populations resistant to Bt represent a significant commercial opportunity. This SBIR Phase I project proposes to develop a novel biological control agent for use in specifically suppressing insect populations that are developing resistance to the Bt toxin. This project will produce a biological control agent, and test it for the ability to replicate in the target insect and determine if it has the predicted pathology. Once the agent is developed and its properties evaluated in the laboratory, experiments will be performed to establish methods for efficient infection of target pests. Phase II will assess impacts on pest populations in microcosm experiments with data used to develop population models of effects in the field. Successful experimental results will develop the technology to the point of field testing and commercialization planned for Phase III.

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