By Jeanne Fitzgerald

Gene therapy is a medical breakthrough whose time has come - almost. Headlines for the last three years have proclaimed the amazing discoveries of molecular geneticists as they continue to identify the loci and sites of genes that play a role in genetic diseases. Cystic fibrosis, breast cancer- one by one the place of these dreaded diseases on the map of the human genome is being exposed. In the case of adenosine deaminase deficiency, a rare but deadly disease that causes a nearly complete immunologic deficit, a limited measure of success has been achieved through gene transfer technology.

It is on this last difficult area of gene transfer technology that Dr. Stephen Krawetz, PhD, associate professor of obstetrics and gynecology, Center for Molecular Medicine and Genetics at Wayne State University, is focusing his scientific energy. Using sperm cells and spermatogenesis as a model, Dr. Krawetz has made significant progress in ensuring the successful regulartion and expression of inserted genes.

Dr. Krawetz has been fascinated by the concept of gene regulation since the beginning of his career in science. At an early stage he was influenced by a paper published in Science in 1969 written by Britten and Davidson. In describing a simple yet complex model of gene regulation, they asked the question, How is the specific fate of a cell determined by its genetic makeup? Krawetz and numerous other genetics researchers have been investigating this fundamental mystery in all its permutations and combinations ever since.

Explains Krawetz, the genetic makeup of all cells is identical, but out of that 3 x 10 ⁹ base pairs of DNA, somehow they sort out to determine what specific parts are going to be used in the fate of the cell. How are the parts that will make up a specific cell type - the skin, the hair, the heart, the brain - selected?

In a related issue, he notes, it is believed that if we can understand how cell fate is determined, i.e., how genes are selected for expression, then we may be able to understand the various units or controlling factors that will allow us to change the phenotype of the cell. The next question is, could a mutated skin cell or liver cell or a tumorigenic cell be brought back to the normal phenotype? What is this master switch - if it exists?

The human phenotype (the complete physical, biochemical, and physiologic makeup of the individual), has approximately 210 types of cells. He postulates that perhaps one specific factor is needed to control the phenotype of each cell type, and thus there must be at least 210 different factors to make each different cell. If you add to that one extra factor for a constitueutive factor (meaning that which is expressed in all cells, for example, the glycolytic pathway, an energy procedure, in each cell) then there are 211 types of factors. What actually controls this mechanism is a combinatoric effect, that is, a multitude of factors that control gene expression. The combinatoric effect serves to reduce the number of factors necessary to determine phenotype. For example, if two factors can interact as one factor or the position varies, then that reduces at least by half the total number of factors needed to define the phenotype.

Dr. Krawetz and his associates believe that the problem of continuing gene expression or gene potentiation, lies in an area of the gene called the locus control region or LCR. To understand the importance of the locus control region, it helps to focus first on the three major issues in gene therapy:

1. The copy of a healthy gene that is transferred must be successfully integrated into the genome.

2. Once it's integrated, the next challenge for the gene is to maintain its activity and to assure that it's not silenced, nor activates an otherwise silent gene.

   Medical scientists have come close to overcoming these first two obstacles, but even though the marvel of recombination and stable integration have been accomplished, there is one remaining huge issue, which is:

3. Assuring that the desired gene is going to be appropriately expressed. This is the goal that Dr. Krawetz's lab and a few others have been shooting for. They are searching for a way to insure that a gene is going to be expressed and expressed only in those tissues where expression is appropriate. In other words, if a gene to suppress colon cancer is inserted, the gene must find its way to the specific site where it can do its work. It cannot be activated in the brain, the liver, the heart, or the kidneys. It must end up being expressed in the colon.

To further explore the interesting question of gene expression, Dr. Krawetz and his co-workers have been using human sperm cells as a scientific model. Why sperm? Sperm are an excellent system because it is possible to isolate these cells at various stages of differentiation all the way from the stem cells (spermatogonia) to mature sperm cells. The process of spermatogenesis is like a mini-system of development in terms of differentiation that goes on in the life of the cell. It is one of the few systems that allows us to examine human differentiation. Cell differentiation begins with totipotent stem cells, in a cycle of continual self-renewal, that eventually become committed to a very spcific pathway which leads to mature spermatozoa.

Krawetz has worked with a very specific set of genes in a central locus that package the chromosomes in the head of the sperm. They are called the PRM1, PRM2, and TNP2. His lab was able to show that these three genes are actually linked on a single DNA chromosome, chromosome 16.

Dr. Krawetz was able to clone out that region of the human chromosome and determine the entire nucleotide sequence, the exact string of ACTGs, that make up this region, which revealed a very interesting story. Having learned how the genes evolved and how this particular gene family grew, he discovered that there was a progenitor gene, the PRM1, which duplicated and eventually gave rise to a new gene cluster, the PRM1, PRM2, and TNP2 genes. The PRM1, PRM2, TNP2 gene cluster exists as one large scale genic domain of about 27.5 kilobases in size.

The next step was to define a genic domain by asking what part of that chromosome is actually required for expression. Dr. Krawetz's lab found that genes that are destined to be expressed in sperm had a different chromatin conformation, a different structure in the nucleus than those that are not. They were DNAse I-sensitive. DNAse I is a nonspecific enzyme that randomly chops the DNA and has a marked preference for the DNA that has assumed the open conformation or potentiative state.

In order to show that the genes contained all the information to really adopt a potentiative conformation, Krawetz created a transgenic animal model. He and his coworkers took the entire human protamine locus and inserted that into the mouse genome. Once integrated into the mouse genome and directly incorporated as part of the normal chromosomal makeup, the gene could be transmitted along the line to various offspring and its destiny followed from only where expected, the team knew they had achieved proper regulation. They were also able to show that the gene was expressed and properly regulated regardless of where it had been integrated onto the mouse chromosome which was a very significant finding.

In addition, Krawetz and his team were able to show that there was a direct correlation in terms of expression of the gene to the number of copies that were integrated. In other words, if four copies were integrated four copies of the gene were expressed. This position-independent, copy number- dependent expression of the integrated trans genes allowed them to conclude that, within this DNAse I-sensitive segment, all the information necessary to insure correct expression of each gene in this entire locus, independent of the site of integration had been identified.

In 1992, Krawetz moved from a basic science setting to the strong clinical orientation of the Wayne State department of obstetrics and gynecology. This offered him an opportunity to bring a molecular genetics presence to a clinical setting and become more closely involved with translational research, so he could bring his ideas as quickly as possible from the bench to the bedside. He feels that the resource base provided in terms of access to the clinical setting has opened up a major insight, Whereby eventual patient care needs can be met at the same time interesting problems in basic science are solved.

If grants are any indication of success, then Krawetz is doing very well. His gene system project basesd on protamines is funded by the National Institutes of Health. He also works on another system called Iysyl oxidase, and this work is funded by the American Heart Association.

Krawetz believes his lab is the only lab in the United States that is grappling with potentiation of human spermspecific genes. He notes that there is a lab in Germany that is working with the human sperm model also, using spermatogenesis to answer the questions about protamine.

Which comes back to gene therapy, the ultimate goal of genetic research. At present, emphasizes Krawetz, it is still extremely experimental. When gene therapy was launched at the National Institutes of Health on Sept. 14, 1990, looking at the adenosine deaminase enzyme deficiency, NIH researchers used a retrovirus to transform some cells with the normal copy of adenosine deaminase and reinsert that back into patients, some of whom were able to then produce adenosine deaminase. Those trials are ongoing, and certain patients seem able to retain some of the copies of adenosine deaminase. Adenosine deaminase gene therapy was recently reviewed in Science in two case reports of gene therapy in children. The problem now lies in putting the correct gene in a population of stem cells that can be continually renewed throughout life, thus curing the deficiency.

Medicine is changing at a rapid rate. What was not possible five years ago is now possible. Dr. Stephen Krawetz is convinced that true self-help, that is, deriving therapy from within our own genome, will become more of a reality in the next 10-15 years, and medicine will change even more rapidly than it has in the past. Right now effective therapy for adenosine deaminase deficiency is similar to autologous bone marrow transplantation - a very cumbersome, complex, and somewhat experimental process. Within the next 15 years or so, Krawetz believes that will evolve to something much simpler. Gene therapy will be perhaps in the form of an intermuscular injection given in an outpatient setting. It may not be much more difficult than going in for a flu shot is today.