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Baltimore, MD, United States

The present invention relates to a designer or recombinant ubiquitin ligase molecule that includes a toxin binding domain that is specific for a toxin active fragment, wherein the toxin active fragment is an enzymatically active fragment of one or more toxins or toxin serotypes; and an E3-ligase domain that comprises an E3-ligase or polypeptide that facilitates E2-mediated ubiquitination of the toxin active fragment. In an embodiment, the composition further includes a delivery system that allow the designer ubiquitin ligase to enter the cell. The present invention further includes methods for treating an individual intoxicated with a toxin by administering the designer ubiquitin ligase of the present invention.

Synaptic Research, Llc | Date: 2011-06-13

A cell based assay for detection for protease activity is disclosed. In the assay a cell is engineered to express a protease substrate with at least one label, preferably on its C-terminus. Cleavage of the substrate by the protease that recognizes it results in a C-terminal fragment and a N-terminal fragment, where the fragment having the label is subject to ubiquitin proteasome degradation. The assay measures the disappearance of the label due to degradation of the fragment to which it is attached. A cell free assay is also described for detection of protease activity. In the cell free assay, the protease substrate is expressed in a solution that includes the elements of the ubiquitin proteasome pathway for degradation of the fragment. The assay measures the disappearance of the label attached to the fragment that results from cleavage by the protease.

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 324.52K | Year: 2011

DESCRIPTION (provided by applicant): Induced pluripotent stem cells (iPSC) have tremendous potential for regenerative medicine in future. However, there are obstacles to overcome before the iPSC can be used therapeutically. One major hurdle is the tool used to generate iPSC from adult somatic cells. We believe that the delivery of reprogramming proteins directly rather than the genes that encode these proteins is the safest means for generating iPSC. We propose to engineer Clostridium difficile toxin TcdB as a cytosolic delivery vehicle for embryonic transactivators that can reprogram adult somatic cells to induced pluripotent stem cells (iPSC) and/or tissue specific cells. C. difficile toxin TcdB, exhibits multiple modular domains that provide all the mechanisms that allow the toxin efficiently deliver its N-terminal enzymatic domain (GT domain) to cytosol. We will engineer TcdB as a universal protein delivery vehicle by replaceing the enzymatic domain with a protein cargo to provide therapeutic benefits.We will initially append a chimeric transacivator to the N-terminus of the toxin or replace the GT domain with the chimeric transactivator. Using a high-throughput reporter cell line system, we will first examine how effectively the engineered recombinantprotein can deliver a biologically active cargo. We will then test if the engineered TcdB can deliver biologically active reprogramming factors (Oct4 and Sox2) to another reporter cell line and assess their ability to differentiate mouse embryonic stem cells with the help of our collaborators. Finally, we will use the TcdB delivery vehicle to generate of iPSC from mouse embryonic fibroblasts after we demonstrate that we can deliver those reprogramming factors efficiently to the well-characterized reportercells. Other methods of delivering proteins, such as the TAT protein transduction domain, will be tested in parallel to compare the efficiency of our novel system to established techniques. We believe this TcdB-based protein delivery vehicle will be a valuable tool for generating iPSC for research purposes, drug development, and toxicology studies. We also anticipate the development of TcdB-based biologics for regenerative medicine. PUBLIC HEALTH RELEVANCE: This project will investigate the use of anew protein delivery vehicle to introduce reprogramming factors to adult skin cells in order to convert them to induced pluripotent stem cells (iPSC). This approach relies on proteins rather than genes to create iPSC, which avoids the potential dangers ofgenetic modification. These iPSC have enormous potential to replace embryonic stem cells for regenerative medicine.

A novel protein delivery system to generate induced pluripotent stem (iPS) cells is described. The delivery system comprises a construct with a receptor binding domain that recognizes a receptor in a somatic cell, a translocation domain that allows the transfer of an inducer into the cytosolic space, and a cargo bearing domain to which the inducer is attached and facilitates transfer of the inducer into the cell.

Synaptic Research, Llc and The Regents Of The University Of California | Date: 2012-10-26

This invention provides diatom-based vaccines.

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