SBIR-STTR Award

A novel and versatile protocol, ?ids.PCR? (patent pending), has been developed to uniquely modify gene sequence, and can thus be used to evolve new and desirable properties into gene products. Ids.PCR goes beyond existing "directed evolution" technologies, affording greater diversity of molecules, and yet can be ?tuned? to effect low levels of randomization where desired. Ids.PCR is an acronym for a PCR-based technology that facilitates the random insertion, deletion and/or substitution of mono-or polynucleotides within a DNA sequence. This multifaceted capability facilitates the creation of random libraries of unique genes or sections of genes from which encoded proteins with desired characteristics can be selected and isolated. In conjunction with suitable screening, the net effect is a powerful accelerated evolutionary process that modifies the existing gene sequence to engender desirable/optimal protein characteristics, such as stability and specificity. Applications of ids.PCR include the development and optimization of novel products addressing critical biomedical needs, including protein pharmaceuticals such as protein growth factors, antibodies, and vaccines, as well as optimized enzymes for small molecule drug production. The specific research objective of this proposal for Phase I is to use beta-lactamase as a model system for the production of modified enzymes exhibiting enhanced function and stability. PROPOSED COMMERCIAL APPLICATIONS: The proposed ids. PCR methodology could be utilized to develop unique proteins with desired characteristics for biomedical applications including (as examples): a)Improved protein pharmaceuticals with enhanced stability, improved half-life, and reduced harmful side effects and toxicities. b) Safe and effective vaccines with enhanced immune system stimulating characteristics. Commercially produced protein pharmaceuticals (e.g. G-CSF) could be improved upon

The long range goal of this research is to develop the directed evolution platform technology termed Insertion, Deletion, Substitution random mutagenesis (ids.PCR) for commercial applications in diagnostics. We propose to develop diagnostic reporter enzymes for diagnosis and therapeutic monitoring that require sensitive, specific, quantitative and multiplexed detection of target proteins present in complex mixtures. We will generate beta-lactamase variants that contain inserted amino acid sequences with binding affinity for specific disease-associated target proteins. These functionally enhanced "Diagnostic Reporter Enzymes" will combine the highly efficient catalytic activity of beta-lactamase with specific antibody-like binding affinities, resulting in rapid, specific, selective, and sensitive detection of biological markers associated with disease. For this Phase II research, two commercially relevant protein targets will be used to screen and characterize the evolved enzymes for response to the target alone and in complex patient samples. PROPOSED COMMERCIAL APPLICATIONS: Ids.PCR is a platform evolution technology that has far-reaching potential commercial applications. The demonstration of diagnostic reporter enzymes evolved using this technology would have widespread application in diagnostics and therapeutic monitoring. The goal is to ultimately use these low-cost single molecule reagents to screen large numbers of biological markers for analysis of human diseases in complex patient samples