Agency: Department of Health and Human Services | Branch: | Program: STTR | Phase: Phase I | Award Amount: 195.51K | Year: 2009
DESCRIPTION (provided by applicant): Worldwide diabetes is projected to reach epidemic proportions in the next 25 years, however, the prevalence of diabetes in the U.S., India, China, Russia, and Japan is already spiraling out of control. One promising therapy for diabetic conditions linked to the functional or outright loss of insulin-producing cells (i.e. pancreatic beta cells) is simply replenishing them. Two main strategies are being developed: 1) Transplantation of beta cells (cell replacement), and 2) Stimulation of endogenous progenitor/adult stem cells (cellular regeneration). Combined with suppression of an autoimmune response which targets beta cells, such therapies could represent lasting cures. The ultimate goal of this project is to create a diabetes model in which genetic networks that regulate the regeneration of insulin-producing pancreatic beta cells in vertebrates can be elucidated. Because zebrafish have a remarkable capacity for cellular regeneration - and are amenable to forward genetics - mutations can be identified which disrupt the regenerative process. Accordingly, we have adapted an inducible cellular ablation system, termed ZAP, toward the goal of creating new tools for the study of cellular regeneration in zebrafish. The ZAP system can be targeted to any genetically definable cellular subtype and thereby model diseases linked to the loss of a particular cell type (e.g., Type I diabetes). The ZAP system is based on transgenic expression of a fusion protein between a pro-drug converting enzyme and a fluorescent reporter. The enzyme converts otherwise innocuous pro-drugs into cytotoxins, thus adding pro-drugs to the water induces ablation in multiple fish simultaneously. The reporter allows automated quantitative detection of the presence or absence of the targeted cell type. We have recently demonstrated two key findings using transgenic zebrafish expressing ZAPs specifically in beta cells: 1) ZAP-expressing beta cells can be specifically eliminated upon treatment with pro-drug, 2) Upon removal of pro-drug, beta cells are rapidly regenerated over the course of the next few days. Here we propose to create transgenic zebrafish expressing the ZAP system in beta cells. In Phase II efforts, these transgenic animals will be mutagenized and screened for those individuals in which this innate capacity for beta cell regeneration has been disrupted. These mutant lines will provide unique insights into the genetic circuitry underlying the regulation of beta cell regeneration and may be utilized in downstream commercialization efforts for the efficient screening and identification of compounds which stimulate progenitor/stem cells to replace lost beta cells. The specific aims of this proposal are: 1) The creation of transgenic lines that will facilitate ratiometric automated detection of targeted (beta) and control cells (alpha), and 2) Optimization and scaling of quantitative automated detection methods. PUBLIC HEALTH RELEVANCE: The number of people diagnosed with diabetes will reach epidemic proportions worldwide within the first quarter of this century. Therapies aimed at replacing or amplifying the number of insulin-producing cells in the body (the beta cells of the pancreas) show great promise for diabetic conditions which require daily insulin injections. This study proposes to create a series of diabetic disease model organisms. Future studies will utilize these models to identify the genetic networks that regulate the process of beta cell regeneration. Insights gained from generating a diabetes model will help indentify drugs capable of safely stimulating the production of new insulin-producing beta cells - a potential cure for this debilitating disease.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 107.00K | Year: 2004
DESCRIPTION (provided by applicant): Degenerative diseases have become major health issues as the median age of humans has increased. The long-term goal of this project is to identify drugs, which promote the regeneration of specific cell types, thus
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 177.07K | Year: 2011
DESCRIPTION (provided by applicant): Zebrafish are an important animal model system for both basic science research and preclinical disease modeling. Complementary to mammalian models, the zebrafish system facilitates methods that are not practical (e.g.,large-scale forward genetic screens), not possible (in vivo imaging of embryonic development), or not cost-effective (high-throughput chemical screens for drug discovery) in mice or rats. Until recently, however, methods for targeted genetic manipulations(e.g., knockout) have eluded the zebrafish field. Targeted genetic modifications stand as the single most desired methodology of the rapidly growing zebrafish market. The advent of zinc finger nuclease (ZFN)-based genome modification has brought gene knockout, and potentially knock-in, strategies to zebrafish researchers. Yet, the process of identifying appropriate ZFN pairs for a given gene target is not trivial, resulting in only a small number of labs that have successfully applied this approach, to date. Here we propose to test whether customized ZFN pairs from Sigma's Advanced Genetic Engineering (SAGE) group improve the efficiency of creating targeted genetic modifications in zebrafish. If proven effective, we will partner with SAGE - which holds anexclusive license from Sangamo Biosciences for creating ZFN-based animal models - to create a catalog of knockout and knock-in zebrafish. Proprietary techniques that SAGE has applied to other species (e.g. rodents) will be employed both in terms of identifying optimal ZFN targets/pairs and in terms of facilitating ZFN-based modifications. Aim 1: Create three knockout disease models in zebrafish using customized ZFN pairs from SAGE targeting 1three genetic loci: 1) sapje, 2) pink1, 3) kif1b, corresponding to knockout models for Muscular Dystrophy, Parkinson's Disease, and Multiple Sclerosis, respectively. Aim 2: Test knock-in efficiency using customized ZFN pairs from SAGE. Although ZFN-based knockout methods have been validated in zebrafish, ZFN-basedknock-in success has not been demonstrated. For this pilot study, proprietary information from SAGE, our own insights regarding transgenesis, and data from groups that have created ZFN-based knock-ins in other systems, we be employed to introduce a fluorescent reporter into the krox20 locus. Successful demonstration of ZFN-induced knock-in would pave the way for the creation of a catalog highly versatile research models. Success of this Phase I proposal will be followed by scale-up initiatives in Phase II and partnering with SAGE in Phase III (see letter of support) to bring 2 both Knockout zebrafish (KOZTM) and Knock-in zebrafish (KIZTM) models to market. We anticipate that development of a strong disease model catalog, coupled with an extremely strongIP position, will promote lucrative relationships with pharmaceutical partners in our efforts to bring insights afforded by the zebrafish system to bear on human disease. PUBLIC HEALTH RELEVANCE: The ability to manipulate genes in a targeted manner revolutionized the fields of molecular genetics and disease modeling but, until recently, was only applicable in the mouse (e.g., gene knockouts). The advent of zinc finger nuclease (ZFN) technology facilitates targeted gene manipulation in any species in which the genome has been sequenced. Accordingly, Luminomics proposes to create ZFN-induced gene knockout and knock-in models in zebrafish, the fastest growing vertebrate model species, as an off-the-shelf product line of broad appeal to both academic and commercial sectors of the zebrafish research community.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 877.90K | Year: 2007
DESCRIPTION (provided by applicant): Zebrafish have a remarkable capacity for cellular regeneration and are amenable to mutational genetic screens. Accordingly, a transgenic zebrafish system that facilitates the removal of specific cell types and the detection of regenerative "replacement" cells has been developed. This inducible ablation platform, termed ZAP (zebrafish ablation-reporter protein), is based on the principle of cytotoxic pro-drug conversion and can be targeted to any genetically definable cell type. Transgenic expression of ZAPs can therefore be used to model any degenerative condition linked to the loss of specific cells or tissues. ZAPs consist of a pro-drug converting enzyme fused to a fluorescent reporter. The pro-drug converting enzyme converts otherwise innocuous substances into cellular toxins, facilitating inducible elimination of ZAP-expressing cells. The fluorescent reporter allows the presence or absence of targeted cell types to be tracked in living fish. Thus, the ZAP platform provides a means to discovering genetic pathways that regulate the regeneration of specific cell types from discrete adult stem cell populations. Proposed is the creation of a set of transgenic zebrafish lines designed to promote maximum flexibility regarding where, when, how much, and what color ZAPs are expressed. The Gal4-UAS system is employed as a modular component that separates transgene expression into elements conferring timing and location (Gal4-expressing "activator" lines) from elements specifying amount and type of transgene product produced (UAS:reporter "effector" lines). By mating different Gal4 and UAS lines, any expression pattern can be conferred to any reporter/effector gene. An enhancer trap approach, based on Tol2-mediated transposition, will be used to create a series of ~500 Gal4-expressing transgenic lines that allow specific cell types to be targeted. A complimentary series of UAS lines will facilitate control over ZAP expression levels and choice of fluorescent reporter color. Collectively then, these models will promote insight into the development, function, and regeneration of specific cell types. Accordingly, the transgenic lines generated will be characterized in detail and distributed to the larger research community. The short-term goal is to foster elucidation of the molecular mechanisms regulating adult stem cell niches within the regenerative biology research community. The long-term goal of this project is to promote the development of regenerative therapies capable of reversing the debilitating effects of degenerative conditions that plague humankind.
Mathias J.R.,Luminomics, Inc. |
Saxena M.T.,Luminomics, Inc. |
Mumm J.S.,University of Georgia
Future Medicinal Chemistry | Year: 2012
Due to several inherent advantages, zebrafish are being utilized in increasingly sophisticated screens to assess the physiological effects of chemical compounds directly in living vertebrate organisms. Diverse screening platforms showcase these advantages. Morphological assays encompassing basic qualitative observations to automated imaging, manipulation, and data-processing systems provide whole organism to subcellular levels of detail. Behavioral screens extend chemical screening to the level of complex systems. In addition, zebrafish-based disease models provide a means of identifying new potential therapeutic strategies. Automated systems for handling/sorting, high-resolution imaging and quantitative data collection have significantly increased throughput in recent years. These advances will make it easier to capture multiple streams of information from a given sample and facilitate integration of zebrafish at the earliest stages of the drug-discovery process, providing potential solutions to current drug-development bottlenecks. Here we outline advances that have been made within the growing field of zebrafish chemical screening. © 2012 Future Science Ltd. Source