SBIR-STTR Award

There is a growing list of organisms that have been completely sequenced and the entire human genome will be sequenced in the foreseeable future. For example, Borrelia burgdorferi, chlamydia, helicobacter pylori and tuberculosis encode 850, 900, 1,600 and 4,000 genes, respectively. Straightforward, rapid and economical methods are available to obtain the necessary primers and generate pure preparations of each of these genes by PCR, but conventional PCR products are not transcriptionally active so they can not be used directly in functional assays. The typical way to create transcriptionally active genes is to clone the PCR fragments into an expression vector, transform and grow bacteria, and purify the plasmid. Although this process can lead to large quantities of plasmid useful for a variety of different experiments, it would greatly facilitate research and functional screening if the initial PCR product were transcriptionally active. The purpose of this proposal is to develop a practical method for generating transciptionally active PCR fragments, which can be used directly in in vitro transfection assays and in vivo. The method will enable high throughput functional screening of a very large number of genes, on a scale that is not possible today. A kit designed to accomplish this will be developed and made available to scientists. PROPOSED COMMERCIAL APPLICATIONS: The resources from this grant will be used to develop a practical method for generating transciptionally active PCR fragments, which can be used directly in in vitro transfection assays and in vivo. This method will give investigators the ability to create very large libraries of 1000s of transcriptionally active genes from the knowledge of their sequence

DNA vaccines offer an attractive alternative to create effective vaccines that are inexpensive to manufacture, and can be widely distributed. One of the most difficult tasks in developing a DNA vaccine is the identification of the antigen that will stimulate the most effective immune response against the pathogen, particularly when the genome of the infectious organism is large. A technology developed by Gene Therapy Systems Inc. under a previously funded SBIR grant (R43 AI47641-01) will be applied to the general problem of how to identify potent DNA vaccine antigens from complex organisms. The technology called Transciptionally Active PCR (commercial name "TAP ExpressTM") is a method for generating functional PCR fragments that can be used directly in in vitro transfection assays and in vivo. TAP fragments are as active as supercoiled plasmids in all assays examined including DNA vaccine immunizations, enabling high throughput functional screening of a very large number of genes, on a scale that has not been previously possible. In this proposal several enhancements will be added to the technology and it will be used to screen 424 malaria antigens with the goal of identifying new DNA vaccine antigens that have improved protective immunological activity. PROPOSED COMMERCIAL APPLICATION: The resources from this grant will be used to continue the development of a practical method for generating transcriptionally active PCR (TAP) fragments, which can be used directly in in vitro transfection assays and in vivo. A high throughput vaccine antigen screening system will be developed to enable the identification of potent vaccine antigens from complex organisms such as malaria.

Thesaurus Terms:
genetic transcription, method development, nucleic acid sequence, polymerase chain reaction, transfection, vector vaccine genetic enhancer element, genetic promoter element, genetic screening, genetic terminator element, plasmid, transfection /expression vector biotechnology, laboratory mouse