Frederick Enright, Cooper, Richard K.
Frederick M. Enright and Richard K. Cooper
Genetic therapy is the introduction of a gene or group of genes into an animal to either correct the result of an abnormal gene or to form a new product that has a beneficial effect for the animal or for those using products derived from the animal.
Called a “DNA construct,” the newly introduced material has a specific genetic code to allow the desired gene to be inserted into the animal’s own genes. The construct also has a promoter responsible for forcing the gene for the new product to be read, or expressed, and a specific genetic code to stop the expression of the gene.
The DNA construct offers potential in cancer research. Physicians have long sought treatments for cancer that destroy cancer cells only. Most existing cancer treatments destroy not only cancer cells but also normal cells. The dreaded side effects of cancer chemotherapeutic drugs—anemia, fever, weakness and digestive upsets—are because of the drugs’ destruction of normal cells.
Likewise, the DNA construct is of interest to veterinarians and animal scientists who have searched for a way to sterilize pets, such as dogs and cats, without surgery. Surgical procedures, either castration in males or removal of the ovaries and uterus in females, are expensive, time-consuming and involve risk to the animal.
In the past 10 years, scientists at the LSU AgCenter have studied the effectiveness of membrane-disrupting peptides linked to ligands (small molecules that bind other molecules, frequently a hormone to its receptor) to destroy cancer cells selectively and to sterilize animals without surgery. The membrane-disrupting, peptide-ligand compounds are small proteins. They have the unique characteristic of having one side of the peptide molecule able to interact and bind to fat (lipid in the cell’s membrane) with the other side of the molecule preferring to interact with water (a major component of the fluid around the outside of cells). Because of this “split personality,” these molecules bind to lipids in the cell membrane and result in membrane disruption. While some cells are more susceptible to disruption than others (depends on the types of lipids composing their membranes), in general, these membrane-disrupting peptides can destroy all types of cells. By themselves, they do not demonstrate much selectivity in killing cells.
Many cells in the body have unique proteins embedded in their cell membranes that recognize ligands. The ligands react with or join to membrane proteins called receptors much like a key (the ligand) fits into a lock (the receptor). Thus, if scientists know about the “lock,” the “key” can be found to fit it. The key may be used to direct drugs to specific cells with the appropriate lock.
Prostatic, breast, testicular and ovarian cancer cells (called carcinomas) frequently have receptors (locks) on their surfaces that bind to reproductive hormones (keys). Gonadotropin releasing hormone (GnRH) is a small peptide in the brain that binds to special cells in the pituitary gland called gonadotropic cells. When GnRH binds to them, these pituitary cells manufacture and secrete two other larger protein molecules called luteinizing hormone (LH) and follicle stimulating hormone (FSH). The two hormones travel in the bloodstream from the pituitary and bind with cells in the testes of males and ovaries of females and result in sperm production and maturation in the male and in egg production and maturation in the female. Not only do these ligands play an important role in reproduction, they are important in the life cycle of selected cancer cells.
Because of their limited selectivity toward the cells they destroy, LSU AgCenter scientists have combined membrane-disrupting peptides with either the GnRH ligand or another ligand, which is the 15 amino acid segment of the beta chain of chorionic gonadotropin, referred to as beta CG. CG is an LH-like hormone made by the placenta during pregnancy. Normal cells and cancer cells with receptors for LH will bind to CG because of this 15 amino acid binding site.
These membrane-disrupting peptides linked to either GnRH or to beta CG have been studied for their ability to selectively destroy human prostatic, breast and ovarian carcinomas transplanted into “nude” mice. These are mice with a genetic defect in the immune system that prevents them from rejecting human tumors. If left untreated, these transplanted cells will grow and spread (metastasize) and eventually kill these mice.
This research has been conducted at the Pennington Biomedical Research Center in Baton Rouge, which is also part of the Louisiana State University System. To date, these hormone-linked peptides have been successful in selectively destroying the primary (original tumor mass) and metastatic tumor cells. More than 700 mice have been treated with these compounds. Unlike other cancer chemotherapeutic drugs, these membrane-disrupting peptides linked to ligands selectively destroy only the cancer cells and the gonadotropic cells in the pituitary or testicular and ovarian cells having either GnRH or LH receptors. The only observed side effects of these compounds were a loss of fertility in males and females. Other vital organs in the treated mice remain normal.
In parallel studies conducted by LSU AgCenter scientists, the effects of GnRH or beta CG-linked peptides on reproduction in normal female and male mice, rats, rabbits, fish, pigs and dogs have been studied. These studies have demonstrated specificity in the rapid destruction of gonadotropic cells in the pituitary gland by the GnRH-linked peptide or destruction of testicular cells or ovarian cells by the beta CG-linked peptide. That was the successful outcome of the research. However, a single treatment of animals with these peptides resulted in only a temporary loss of reproductive function.
For example, studies of mice receiving a single treatment showed that for the first two weeks following treatment, the number of gonadotropic cells in the pituitary gland was reduced by 80 percent. By three to four weeks after treatment, gonadotropic cells in the pituitary gland were returning. By five to six weeks post-treatment, the number of gonadotropic cells was equal to the number of these cells in untreated mice. Thus, our hope of long-term sterilization following one injection of these peptides was not realized.
Though repeated injections of the peptides prolong the sterilization effect, this would be impractical for long-term sterilization of pets or livestock. There are several potential solutions to this problem. One is to package the peptides for slow release in the animal. We plan to conduct studies using small osmotic pumps filled with the peptide. These pumps are implanted under the skin and will “trickle” out small amounts of peptides for extended periods. A second solution lay in introducing genes that cause the animal itself to produce the peptides. This second approach is appealing because of the low cost of genetic constructs as compared to the cost of synthesizing peptides.
Data collection began on the effects of these membrane-disrupting, peptide-ligand compounds on naturally occurring mammary gland adenocarcinomas in dogs and cats. Richard Cooper, professor in the Department of Veterinary Science, had previously invented a unique vector with the ability to efficiently insert stable, foreign DNA into catfish. Using Cooper’s vector, DNA constructs of two different peptides (Phor11 and Phor14) linked to either GnRH or to beta CG were made. By using the genes for the linked peptides, the animal itself could produce the drugs.
Initial studies to determine the safety of the genetic constructs were carried out in mice and dogs. Preliminary studies using DNA constructs for membrane-disrupting peptides and ligands have demonstrated that the DNA constructs are safe and that there is the same degree of specificity in the destruction of cells with either LH or GnRH surface receptors as observed with the peptide-ligand compounds. Initial results in female dogs suggest that the effects on the reproductive organs following exposure to DNA constructs persist for at least 116 days. Additional studies are needed to determine the length of this extended sterilization.
Peptide therapy studies in dogs and cats with advanced mammary gland adenocarcinomas have begun. Both dogs and cats have demonstrated excellent responses to the treatments. Following introduction of the genetic constructs, their tumors have undergone cellular death and have decreased dramatically in size. We are not claiming a cancer cure but are optimistic about the ability to effect these cancers. Additional work is necessary to learn to manipulate the constructs and treatments depending upon the situation. We need to determine how long dog and cat cells will express the constructs. Prolonged expression may be desirable in some cases while in others transient expression would be preferable. The basic construct and the proteins being expressed may require modifications. Fine tuning the genetic constructs will require several additional years of research.
(This article appeared in the fall 2003 issue of Louisiana Agriculture.)