Molecules And Mechanics

Molecules and Mechanics

Tumor Targeting Strategies
Mechanical Stimulation
(Lead Scientists Paul Grayburn MD & Ralph Shohet MD)

One of the most important obstacles to successful gene therapy is effective delivery. In order to capitalize on the discoveries of the Genetic Signatures (Area 2) it will be critical to implement and develop new approaches. One such method uses focussed ultrasound cavitation. The first approved clinical trial of gene therapy began in 1990. Over 300 trials have been initiated in humans and more than 3,000 patients have received gene therapy in some fashion, but the evidence of clinical benefit is scant. During this time, our understanding of the molecular mechanisms of disease has rapidly advanced, providing many new targets for specific genetic therapy.

A major obstacle to successful gene therapy is the lack of an effective delivery system that can be targeted to specific organs or tissues. In tissues other than the liver, the endothelial barrier effectively prevents adenoviral-mediated gene delivery, a fact that severely limits the use of these viral vectors for gene therapy. Dr. Grayburn's laboratory has recently developed a method that overcomes the endothelial barrier. Microbubbles of perfluoropropane gas (mean diameter 3.0 µm) are engineered with an adenovirus vector attached to their albumin shell. The microbubbles are injected IV and ultrasonic stimulation is applied using a standard clinical instrument to the organ of interest using maximal acoustic power and a frequency that is higher than the resonant frequency of the microbubbles. This causes the microbubbles to cavitate, propelling the genetic material into, and through, the local endothelium. Although the microbubbles circulate throughout the body, rupture of the bubbles and concomitant delivery of the genes they contain, occurs specifically at the site where ultrasound is administered. Preliminary data have been obtained demonstrating delivery of b-galactosidase reporter gene expression to the heart or pancreas of rats (Figure). Specific targeting of therapy to selected organs/tissues by ultrasound is ideally suited to treatment of tumors or metastases delivering chemotherapeutic drugs or "suicide" genes to the tumor, while avoiding the adverse side effects of systemic therapy.

Ultrasound-mediated microbubble destruction has been proposed as a method for delivering drugs or genes to specific tissues, including the heart. The technique is noninvasive and provides efficient, tissue-specific targeting. Further work is needed to optimize the echocardiographic parameters for microbubble destruction, to maximize the amount of adenovirus that can be attached to the microbubbles, and to determine the range of reagents amenable to ultrasound-mediated microbubble delivery. We foresee application of this technique to stimulate delivery of various genes, drugs and small molecules (e.g., PNAs),.

Tumor Targeting Strategies
Ligand Discovery
(Lead Scientist Thomas Kodadek, Ph.D.)

Dr. Kodadek's laboratory works in the general field of chemical biology. Research encompasses two broad areas: 1) understanding the mechanisms by which eukaryotic gene expression is regulated, and 2) deciphering the chemical recognition phenomena that underlie specific protein-protein and protein-small molecule interactions. In addition to employing the standard techniques of modern molecular biology and biochemistry, more traditional chemical, spectroscopic, and biophysical methods, are applied including organic synthesis, NMR, CD, and fluorescence spectroscopies. In the gene regulation field focus is on detailed analyses of the mechanism of action of the yeast Gal4 protein, a prototypical transcriptional activator. These studies are done in collaboration with Prof. Stephen Johnston, and employ a wide variety of approaches, from molecular genetics to organic chemistry. Arising from this work are efforts to synthesize organic molecules that are able to mimic certain aspects of activator function and to use these molecules to control gene expression with cell-permeable small molecules, which will be of great value to investigators in the Pre-ICMIC. The ability to design ligands that bind specifically to macromolecules, in particular proteins, is one of the premier goals of chemical biology. To approach this very difficult problem, new combinatorial methods are being developed to isolate peptides that bind polypeptide and small molecule targets with extremely high affinities and specificities. These model complexes should be much more tractable for biophysical analysis than naturally occurring protein-peptide and protein-small molecule complexes. Through these efforts, it is hoped to begin to uncover paradigms that can be used in the de novo design of small molecules with highly specific binding properties. Ligand discovery will be particularly important to capitalize on chromosomal aberrations identified in Area 2 (Genetic Signatures). Molecules may be labeled and their biodistribution and pharmacokinetics assessed.

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Thursday, September 20, 2001

Southwestern In Vivo Cellular and Molecular Imaging Program