Past Seminar Series

The patterns of gene expression, protein post-translational modifications, covalent and non-covalent associations, and how these may be affected by changes in the environment, cannot be accurately predicted from DNA sequences.

Therefore, proteome characterization is increasingly viewed as a necessary complement to complete sequencing of the genome. Approaches for proteome characterization are presently based upon mass spectrometric analysis of 2-D gel separated proteins. However, this approach remains constrained by the speed of the 2-D gel separations, the sensitivity needed for protein visualization, the speed and sensitivity of subsequent mass spectrometric analyses for identification, and the limitations of spot visualization for quantitation.

Our objective is to circumvent the limitations of this approach by directly characterizing the cell's polypeptide constituents by combining fast separations and the mass accuracy and sensitivity obtainable with Fourier transform ion cyclotron resonance mass spectrometry. Protein identification is based upon global approaches for protein digestion and accurate peptide mass analysis for the generation of "Accurate Mass Tags". In the same analysis precise measurements of relative protein expression can be obtained by the inclusion of a stable-isotope labeled "reference proteome", enabling measurements of protein expression with precisions of better than 10% and sensitivities in the attomole range. We are also exploring the use of several stable isotope labeling technology that facilitate protein identification and quantitation, as well as advance capabilities for the analysis of mammalian proteomes. The status of the high throughput proteomics capability will be presented, along with initial results for application to several microorganisms and preliminary results for the extension to mammalian proteomes.

Oncosis is developing a series of new clinical and research instruments based on its Photosis(tm) technology platform for highly-accurate, high-throughput, laser-based processing of living cells. Laser-based processing of cells can induce many important responses, including cell death, optoporation (selective gene transduction of individual cells), stimulation of a reporter molecule, or even inactivation of a specific molecule or mRNA transcript within the cell. Photosis(tm) incorporates rapid optical scanning of biological samples, image analysis, and high-speed laser beam steering to hit specific cells within the sample. Targeted cells can be identified by parameters such as size, shape, and fluorescence. Once identified, individual cells are targeted with a short laser pulse to induce the desired response. The current prototype Photosis(tm) instrument processes hundreds of millions of cells in an hour under closed sterile conditions, enabling the first application of tumor cell purging from autologous NHL stem cell transplants. Adaptations of this instrument will target other clinical and research markets where Photosis(tm) technology advantages provide clear value. Photosis(tm) is competitive with flow cytometry in terms of speed, yield and purity, and significantly expands the number of cell types (e.g. adherent, fragile, or infected cells, etc.) that can be efficiently processed. These factors are critical in applications such as cDNA library construction, gene expression analysis using DNA microarrays, proteomics, and the establishment of primary cell lines. Beyond cell purification, Photosis(tm) can potentially be useful for optoporation in gene therapy applications, and for rapid interrogation of individual cells in high-through.

Phylos is developing technology that broadly impacts the way cancer is understood, diagnosed, and treated therapeutically. Research activities are outlined as follows:

• Mechanisms that underlie cancer. Phylos has developed libraries of cellular proteins derived from mRNA. These libraries can be used in an in vitro approach to trace pathways of interacting proteins that cause cancer, and offer a new and powerful way to map the underlying mechanisms of the disease.
• Cancer target discovery and diagnosis. Phylos is developing a two-tiered approach towards the discovery and identification of cancer markers. Using its PROfusion(tm) screening technology, Phylos is engaged in identifying high affinity binding agents that recognize secreted cancer markers. These binders are next placed on a microdiagnostic device, the PROfile(tm) protein chip. This chip has the capacity to profile thousands of marker proteins. Detection of rare proteins (down to single molecule) is feasible with the PROfile(tm) chip.
• Cancer Imaging and Therapy. Phylos technology has the capacity to identify the highest affinity and selective antibodies, due to the in vitro selection scheme that the PROfusion(tm) system provides as well as the large libraries of molecules that can be screened. Phylos is also developing an antibody mimic framework protein derived from a small 8.5 kD domain of human fibronectin. The fibronectin molecules will have potential uses therapeutically (avoiding antibody intellectual property), as imaging agents (with improved tumor penetration, due to the size of the molecules), and as immunotoxins (where previously the strategy has failed due to the size and binding affinity of the agents).

Our goal is to create specialized variations of the process of classical breeding for applications to a diversity of target sequences in diverse fields. We have developed a variety of 'molecular breeding' formats for single genes, pathways, episomes, viruses and partial and whole genomes. An important advantage of this approach over alternative approaches is that it does not require much information. Molecular breeding, also called DNA shuffling, is a reliable method for recombination of pools of related sequences. Libraries of chimeras are constructed from homologous DNA sequences obtained from natural diversity. A pool of the best clones obtained after one cycle of screening for improved function is re-shuffled to create the next library of chimeras. The screening consists of a variety of high and low throughput analytical techniques to identify positive combinations of sequences while removing negative combinations of sequences. The application of this process to a broad range of specific examples will be described.

A major goal of The Center for Biomedical Inventions is to develop new technologies for immune manipulation. We have identified 6 areas of invention required to revolutionize vaccinology. One of them was to develop systematic methods of vaccine identification. Expression library immunization was invented to screen pathogen genomes for the best vaccine candidates. The results of such screens have yielded some surprising results. We have also produced new approaches to quickly screen sequenced genomes. One form of this technology, linear expression elements, was used to screen the parapox genome for new immunological reagents with success. A second area is to develop more creative ways to manipulate the immune response. Two new technologies - inducible vaccines and genetic tolerization will be described. A third area is to make vaccines more effective. Progress toward targeting dendritic cells in vivo will be described.