Research Reports: November 1994

Release Date:
Research Reports

Research Reports , a feature service by the Office of Research Reports of the National Institute of General Medical Sciences, is designed to let you know what some of our grantees are doing. A list of some recent NIGMS grant awards is also included.

This issue of Research Reports contains the following stories:

Engineering Bubbles to Carry Drugs
Tracking a New "Drug"--Antisense DNA
Drug Discovery Hits the Fast Lane
Manipulating Genes for Therapeutic Goals

All of these stories report on different attempts to design and deliver new therapeutic substances, especially nucleic acids.

Basic Research and Drug Development

The past decade has seen an enormous growth in the number of companies seeking to develop the techniques of genetic engineering for the treatment of diseases. Since the late 1970's, recombinant DNA technology has enabled researchers to splice new genetic sequences into bacterial DNA and engineer microbes to produce useful substances. More recently, scientists have been exploring other ways to use the rapidly developing technology for therapeutic purposes, including ways to use nucleic acids (DNA and its molecular relative, RNA) themselves like drugs. Many scientists who were conducting basic research in the field of genetics have become involved in clinical applications of their work, including methods of drug design. Because drugs are worthless unless they can be delivered to their "work site" in the body without being destroyed, these scientists are also concerned with drug delivery.

The four stories contained in this issue of Research Reports describe some of the new directions researchers are heading. Although clearly targeted toward therapeutic intervention, most of this research is still at a fairly experimental level. It remains to be seen which, if any, methods will have a significant clinical impact. All of the new methods described here grew out of basic, relatively untargeted, research.

Engineering Bubbles to Carry Drugs

Hundreds of potentially valuable drugs have been devised that, for various reasons, can't be used. Many of these are waiting for scientists to invent a way to "sneak" them past the body's ever-vigilant defenses and into the sites where they are needed. Over the years, many clever drug delivery systems have been proposed. Although no perfect "magic bullets" have been developed so far, much progress is being made.

One drug delivery vehicle that researchers have recently been improving is the liposome. For years, scientists have known that fatty substances called phospholipids will spontaneously form microscopic bubbles in solution and that these bubbles will encapsulate drugs that can be dissolved in the solution. It was also known that such a phospholipid coating could protect the drug from degradation by the body. In many ways, liposomes looked like a good possibility for the delivery of some drugs.

There were many problems, however--finding a way to produce liposomes on an industrial level, getting them to entrap enough of the drug to be therapeutic, directing them to the desired site, producing liposomes with the desired electrical charge (so they will encapsulate electrically charged substances), and producing stable liposomes that don't leak or change shape and size, but that can be degraded by the body after they have delivered their drug.

NIGMS grantee Moo Jung Cho, Ph.D., and his colleagues at the School of Pharmacy, University of North Carolina at Chapel Hill, are testing the feasibility of developing "crosslinked" liposomes, which are devoid of stability-related problems and yet are biodegradable once they have delivered their drug. Stability is important because liposomes that increase in size can cause blockage of fine blood vessels. Increases in size can also cause intravenously administered liposomes to congregate in parts of the body where they are not wanted.

Dr. Cho is synthesizing two phospholipid compounds that form liposomes and that will crosslink as the result of simple chemical manipulations. He describes his efforts at crosslinking as an attempt to create a "net" of chemical bonds that would greatly strengthen the forces that hold the liposome molecules together, thus stabilizing liposome size.

Dr. Cho anticipates that liposomes will be particularly useful in carrying drugs to macrophages, the body's scavenger cells. This is because macrophage cells are among the early defenders of the body against attack by foreign invaders and are therefore quick to seek out and engulf the liposomes. In the process, the liposome would deliver the drug to the macrophage.

Diseases like leishmaniasis and malaria are particularly hard to treat because the disease-causing agents live within the macrophage, which protects them from the immune system. Liposomes could work like a "Trojan horse," sneaking into the macrophage and then destroying the agent as they release their drug. In addition, macrophage-like cells that engulf liposomes might themselves serve as drug delivery systems. Another function of this type of cells is to rush to diseased tissue, so a drug delivered to them would then be delivered to the diseased tissue.

Liposomes could also be used to deliver nucleic acids, which are otherwise destroyed by the body. [Therapeutic nucleic acids, called nucleotides, are discussed in the following stories in this issue of Research Reports .] Researchers are working to develop liposomes that carry a positive electrical charge, a necessary step before the liposomes will form a complex with nucleic acids, which are negatively charged.

Several small biotech companies are seeking to bring liposome-delivered drugs to market. Currently, both an antifungal drug and an anticancer drug designed to be delivered in liposomes have reached clinical trials. The liposomes enable the antifungal agent to circulate in the bloodstream. This prevents the drug, which is very toxic, from damaging the kidneys and other organs. The liposomes also greatly increase the length of time the anticancer drug can circulate. If these drugs are successful and are approved for general use, they may be just the beginning of an important role for liposome-delivered therapeutics.

Tracking a New "Drug"--Antisense DNA

Can DNA--or the related molecule RNA--be used as a drug? In theory, yes. There are several ways researchers imagine using DNA or RNA to treat or prevent diseases. One of these methods is called antisense technology. The goal of antisense technology is to block a DNA message. Usually this means to prevent it from being delivered by messenger RNA (mRNA) into the cytoplasm, where it would be translated into a protein. There are many diseases where specific protein inhibition would be therapeutic.

NIGMS grantee Francis C. Szoka, Ph.D., and his colleagues at the University of California, San Francisco are working to develop techniques to get antisense molecules to disrupt gene function. Their specific interest is in blocking the production of proteins known as inflammatory mediators in order to treat conditions such as septic shock and arthritis. They are also interested in liposomes as possible vehicles for delivery of their antisense "drugs" because nucleotides are quickly destroyed by the body and need to be shielded until they reach their targets.

A DNA molecule is composed of a long double helix of nucleotide bases--adenine (A) bound to thymine (T) and guanine (G) bound to cytosine (C). A block of three bases of one strand (called the sense strand) of the DNA double helix codes for one amino acid building block of proteins. For example, C-A-G codes for the amino acid glutamine.

The other DNA strand (called the antisense strand) is the template for the production of mRNA. It is believed that antisense nucleotides could be developed that would bind to mRNA, blocking it from delivering its message and thus preventing a protein from being made.

During replication, the bound DNA strands separate. At this point, it is also possible that an antisense molecule could successfully target the sense strand and block the message in that way. A third potential use of antisense technology might be to get the antisense molecules to block DNA messages by binding to the double helix, producing a nonfunctional triple sequence.

There are problems with antisense technology, however. Researchers need to be sure that the antisense molecules are actually interfering with the target cellular nucleotides and not with some other aspect of the cell's activity. They need to deliver the strands of antisense nucleotides, which are called oligonucleotides or "oligos," directly to the living cell. There is also often a problem with getting nucleotides to bind tightly enough to the target mRNA.

Recently, Dr. Szoka and his colleagues demonstrated for the first time that oligos do bind to their target nucleotides inside the living cell. They did this by following microinjected oligos, which were fluorescently labeled, with a laser microscope. This work is important because now oligos of different chemistries, primary sequences, and delivery routes can be studied optically.

Dr. Szoka is also experimenting with the use of liposomes to deliver oligonucleotides. Oligonucleotides are expensive and liposomes, by protecting them, might permit far fewer to be a therapeutic dose. An ideal target for liposome-encapsulated antisense oligos would be diseases that involve macrophages or those that involve organ systems, such as the liver or spleen, where macrophages accumulate in large numbers. Macrophages produce the inflammatory agents that are involved in septic shock and arthritis. An antisense strand that would inhibit production of these agents could be therapeutic for these conditions.

With the new techniques pioneered by Dr. Szoka and his colleagues, researchers can now better study how antisense oligos work and make necessary modifications to improve their effectiveness.

Drug Discovery Hits the Fast Lane

Scientists spend much time and effort looking for new therapeutic agents. Many drugs are arrived at by first screening tens of thousands of candidate compounds, most of which come from such natural sources as plants. But finding a drug from a natural source may require searching rain forests, oceans, and even mud puddles for substances with bioactive properties. Then, when a therapeutic substance is found, it must be manufactured in quantity and at reasonable cost. For these reasons, it takes a very long time to get drugs to the stage where they can even begin the long process of clinical screening and eventual approval for use in treating human disorders.

In theory, any compound that can bind to and disrupt a disease-causing substance might be useful in treating the disease. Because it is now known that specific short nucleotide sequences can bind to most proteins, scientists are examining these sequences as possible drugs. Unlike antisense sequences, short nucleotide sequences could be used when neither the target's sequence nor its shape is known. The sequences work by binding directly to proteins rather than by inhibiting the process of protein production, as antisense sequences do.

The process of screening for therapeutic nucleotide sequences would be very similar to screening for any candidate drug compound. In order to locate candidate sequences, many sequences would be tested for binding, with the eventual "winners" then entering into the process of drug development.

Many scientists are interested in developing different methods for greatly speeding up the process of identifying binding oligonucleotides. Termed "combinatorial technology," these processes may save millions of dollars in the research and development costs of drugs.

NIGMS grantee Lawrence Gold, Ph.D., of the University of Colorado at Boulder and his colleagues were studying RNA-protein and DNA-protein interactions when they developed SELEX, a method that greatly simplifies the identification of binding sites. The acronym stands for Systematic Evolution of Ligands by Exponential Enrichment. SELEX enables RNA's and DNA's that bind to specific proteins to be isolated from large populations of random sequences. The scientists speculated that SELEX might be used to discover oligonucleotides that will bind to any target molecule. If so, this would be an extremely important development in the search for candidate molecules for a variety of diagnostic and therapeutic purposes.

Dr. Gold and his colleagues discovered SELEX as a spinoff of his studies of an enzyme that speeds up the addition of molecules to the DNA chain. They wanted to learn how the enzyme bound to a specific region on the mRNA, so they devised an experiment to test all of the very large number of RNA sequences that were candidates for binding.

The experiments were successful and while studying the results, the researchers were convinced that nucleotides had the potential to form many unpredictable but possibly useful shapes. They decided to use their SELEX system to screen huge quantities of random nucleotide sequences for their ability to bind to specific proteins. These experiments were highly successful and many promising sequences have already been identified.

The researchers believe SELEX may be able to shorten the time required to come up with viable drug candidates from years to months. Last year, a company called NeXagen (which licensed SELEX from the University of Colorado) took less than 4 months to generate a library of short, stable chains of nucleotides; screen them for the ability to bind and block basic fibroblast growth factor (a promoter of cell proliferation); and move several of the compounds into animal tests as potential treatments for cancer and other conditions.

NeXagen uses SELEX to make quite quickly trillions of different oligonucleotides to be screened. The huge pool of candidate molecules permits the discovery of compounds that are more specific than those found in nature or than are synthesized by traditional means.

Recently, chemists using SELEX published a paper in Science describing how they managed to identify an oligonucleotide that could discriminate very accurately between theophylline, a common medication for asthma, and caffeine, which differ from each other by only a single chemical group. The pool of molecules screened by the researchers contained 100 trillion different random short-sequence RNA molecules. Because the level of discrimination shown by the "winning" oligonucleotide is 100 times better than the current chemical--a monoclonal antibody--used to monitor blood theophylline levels, this work may lead to a new assay to increase the safety and effectiveness of theophylline.

Dr. Gold is particularly interested in developing and studying oligonucleotides that may serve as novel antiviral, anti-inflammatory, and anticancer compounds. He thinks that single-stranded oligonucleotides will be better than peptides (small proteins) or antibodies with respect to their diagnostic and therapeutic potential, partly because oligonucleotides are able to fold into extremely stable structures. Dr. Gold is also exploring liposome-mediated delivery of both the RNA and DNA molecules. In the future, the dual technologies of SELEX and liposome design may together provide "drugs" that, in all likelihood, would not otherwise have been found.

Manipulating Genes for Therapeutic Goals

An appealing way to treat disease is to use gene therapy to get the cell to make its own medicine. Ever since the discovery many decades ago that simple "errors" in a single gene can cause devastating diseases, scientists have dreamed of manipulating genes to correct these disorders. However, even though researchers can now precisely identify many genetic errors and synthesize new DNA in which these errors are corrected, they have not succeeded in discovering foolproof methods of using the synthesized DNA to correct errors in living human beings. But there are other ways of using genes for therapeutic purposes. For example, NIGMS grantee Stuart L. Schreiber, Ph.D., and his colleagues at Harvard University, together with Gerald R. Crabtree, Ph.D., and his colleagues at Stanford University, have designed a system that may someday become an important new form of human gene therapy.

In an example of a highly productive collaboration between biologists and chemists, the researchers used hybrid genes to force cells to make artificial receptors that can be controlled by specially engineered drugs. The work raises the possibility of a new kind of therapy in which individuals could get their own bodies to manufacture a therapeutic substance when needed and to cease making the substance when it is no longer needed. For example, instead of injecting insulin, someone with diabetes could simply swallow a drug that would turn on modified insulin genes, which could then be turned off by swallowing another drug.

Dr. Schreiber is a chemist whose field of research is cell signaling pathways (how messages from the outside are relayed through the cell to the nucleus, where specific genes respond) in the immune system. His discoveries helped define some of the fundamental features of these signaling pathways.

In recent years, Dr. Schreiber has made dramatic discoveries about an aspect of T cell signaling that may have far-reaching implications for our understanding of these systems. In 1989, he discovered that proteins called immunophilins are intermediaries in the T cell's message-relaying system. One immunophilin, which he and his coworkers first described, is FKBP, which binds to a potent immunosuppressive drug called FK506. FK506, which is derived from a Japanese soil fungus, is administered in tiny doses following organ transplants to inhibit the immune system from attacking the new organ. But until Dr. Schreiber's structural account of the fit between FK506 and FKBP, no one knew exactly how the drug worked.

In the current studies, Drs. Schreiber, Crabtree, and coworkers engineered cultured T cells to take up a "reporter" gene that would send an easily observed signal when turned on and make synthetic receptors for a molecule called FK1012 that they created by combining two modified FK506 molecules. When they added FK1012 to the solution, these molecules bound to the synthetic receptor and turned the gene on. The gene could be turned off by adding a version of FK506 called FK506-M, which knocked the FK1012 from the artificial receptor.

FK1012 appears to be a nontoxic substance, which makes it look like a good candidate for use in humans. The researchers envision a possible form of gene therapy that would involve removing some of a patient's T cells and engineering them to carry a gene for a therapeutic molecule and a receptor for FK1012. They would then reintroduce the T cells and control their production of the therapeutic molecules by doses of FK1012 or FK506-M.

Normally, T cells circulate throughout the body, but if the "drugs" needed to be delivered to a specific site, it is possible the T cells could be equipped with targeting molecules that would cause them to home in to the desired location. It is also possible that engineered T cells could be programmed to kill specific cells. One early use for T cells that are programmed to kill may be to knock out target cells in model systems to study their effects.

Although the elegant experiments of Drs. Schreiber and Crabtree are still a long way from any specific clinical application, a biotechnology company has already expressed interest in their work.

Recent NIGMS Grant Awards

This sampling of titles from the more than 3,000 grant awards NIGMS makes each year is designed to give you an idea of the basic research the Institute supports. Among the awards that were made in 1994 are the following:

American National Red Cross
W. Scott Argraves: "Fibronectin Receptor-Cytoplasmic Interaction"

Baylor College of Medicine
Andrea Ballabio: "XIST Gene and Its Role in X-Inactivation"

Boston College
T. Ross Kelly: "Synthetic Active Sites for Catalysis of Bond Formation"

Cold Spring Harbor Laboratory
David L. Spector: "Spatial Organization of Gene Expression"

Colorado State University
Robert W. Woody: "Optical Properties of Biological Macromolecules"

Cornell University
Charles F. Aquadro: "Comparative Molecular Population Genetics of Drosophila"

Duke University
R. Bruce Nicklas: "Analysis and Control of Chromosome Movement"

Harvard University
Welcome W. Bender: "Molecular Genetics of the Bithorax Complex"

Johns Hopkins University
Jef D. Boeke: "Transposition Mechanisms"

Louisiana State University Medical Center, Shreveport
Edwin A. Deitch: "Burn and Trauma Induced Infections from the Gut"

Massachusetts Institute of Technology
Rick L. Danheiser: "Pericyclic Reactions for Organic Synthesis"

North Carolina State University, Raleigh
Kelly G. Tatchell: "Genetic Analysis of Protein Phosphatase I in Yeast"

Ohio State University
Michael H. Zehfus: "Compact Domains in Proteins"

Pennsylvania State University Hershey Medical Center
Thomas M. Krummel: "Fetal Wound Repair--Cellular and Biochemical Mechanisms"

Princeton University
James R. Broach: "Mating Type Silencing and Switching in Yeast"

Purdue University
William A. Cramer: "Structure-Function of Photosynthetic Cytochrome Complex"

Rutgers, the State University of New Jersey, New Brunswick
Helen M. Berman: "Biomolecular Structures and Interactions"

Scripps Research Institute
Kim D. Janda: "Catalytic Antibodies"

Stanford University
Lubert Stryer: "Optical Studies of Protein Structure and Function"

State University of New York at Buffalo
Jiali Gao: "Biomolecular Interactions and Enzymatic Processes"

University of Arizona
Roy R. Parker: "Regulation of mRNA Turnover in Yeast"

University of California, Berkeley
Thomas W. Cline: "Regulation of Sex-Specific Genes in Drosophila"

University of California, Irvine
William A. Fonzi: "Regulation of Dimorphism in Candida albicans "

University of California, Riverside
Bryan K. Epperson: "Spatial Correlations of Population Genetic Variation"

University of Chicago
Theodore L. Steck: "Acidosomes in Dictyostelium discoideum "

University of Colorado Health Sciences Center
James P. Hoeffler: "Protein/Protein Interactions of CREB/ATF Proteins"

University of Florida
Thomas C. Rowe: "Topoisomerase II-Active Drugs in Mitochondria"

University of Georgia
Richard B. Meagher: "Differential Expression of the Diverse Plant Actin Genes"

University of Illinois at Chicago
Michael L. Sinnott: "Catalytic Consequences of Experimental Evolution"

University of Illinois, Urbana-Champaign
Andrew H. Wang: "X-Ray Diffraction of Nucleic Acid Structures"

University of Kansas, Lawrence
Robert E. Palazzo: "Centrosome Maturation in Vitro "

University of Kentucky
Isabel Mellon: "Mechanisms of DNA Repair in Active Genes"

University of Maryland, College Park
Daniel E. Falvey: "Photochemical Aspects of DNA Photorepair"

University of Missouri, Columbia
Suresh C. Tyagi: "Regulators of Neutrophil Proteases"

University of North Carolina, Chapel Hill
Steven W. Matson: "Enzymatic Mechanisms of E. coli DNA Helicases"

University of Oregon
Stephen J. Remington: "Structure, Mechanism and Regulation of Glycerol Kinase"

University of Pennsylvania
Tracy K. McIntosh: "Endorphins in Shock and Trauma"

University of Pittsburgh
Timothy R. Billiar: "Nitric Oxide and Hepatic Function in Sepsis and Trauma"

University of Rochester
Terry Platt: "RNA Transcription Termination and 3' End Processing"

University of Texas Health Science Center, San Antonio
John C. Lee: "Dynamics and Topography of Yeast Ribosomes"

University of Utah
David J. Stillman: "Molecular Mechanisms in Mother-Specific Transcription"