California Pacific Currents 2004
Spell Check for the Cure: Dieter Gruenert Pursues a New Form of Gene Therapy for Two Inherited Diseases
One in every 500 African-American babies and one in every 1,000–1,400 Hispanic-American babies is born with sickle cell anemia. All told in the United States, about 72,000 people are affected by this inherited blood disorder, which causes fatigue, pain, infections, and other serious symptoms. New treatments and bone marrow transplantation have helped those with sickle cell anemia to lead productive lives, but complications and hospitalizations are still all too common.
About one in 2,500 Caucasian babies in the US has cystic fibrosis (CF), making it the most prevalent serious genetic disorder in this population. Most of these babies develop lung infections or digestive symptoms in their first year. Even though early diagnosis and modern drugs have helped the 30,000 or so Americans now living with CF, the average lifespan is still only 30 to 35 years.
Sickle cell anemia and CF are the twin targets of pioneering research in gene therapy being pursued by Dieter Gruenert, PhD, Senior Scientist at the California Pacific Medical Center Research Institute. “We’re focusing on two of the most prevalent genetic disorders found in the country,” says Dr. Gruenert.
Genetic Roots: Black and White
Both diseases are caused by errors in the genes that tell the body how to make a certain protein. In sickle cell disease, the oxygen-carrying hemoglobin protein in red blood cells is defective. This is because ß-globin—one of the proteins that is a component of hemoglobin—carries a specific mutation (an error in the sequence of DNA letters that spell out the code for a particular protein). The abnormal hemoglobin molecule alters the shape of the blood cells. Normally smooth and donut-shaped, red blood cells in patients with the disease become hard and pointed (the so-called “sickle cells”). Instead of gliding smoothly through the smaller blood vessels, sickle-shaped cells often get stuck, blocking blood flow, causing pain, and damaging organs like the liver, spleen, lungs, and eyes. Because sickle cells are fragile, they also die sooner, leading to a chronic shortage of red blood cells and extreme fatigue.
The underlying problem in CF is also a faulty protein—called the cystic fibrosis transmembrane conductance regulator (CFTR)—primarily made by epithelial cells that line the inside of organs and passageways that need to be moist. The CFTR protein transports salt and water in and out of the epithelial cells that line the narrow passageways of the lung and pancreas. When these cells don’t make a CFTR that works correctly, the mucus gets very thick and plugs up these passageways. Mucus accumulates in the lungs, pancreas, and intestines and leads to breathing difficulties, lung infections, and malnutrition.
A Refined Approach for Gene Therapy
Researchers are attempting to restore fully functional ß-globin and CFTR genes into a patient’s own cells to stimulate production of healthy proteins. In most of these “gene therapy” efforts, scientists have inserted copies of the healthy gene into the patient’s cells. This approach can produce the whole healthy protein within cells in the laboratory and in animals. However, progress in treating humans with serious genetic diseases has been slower than expected. Dieter Gruenert thinks he knows why. “The classic approach to gene therapy, fixing a genetic defect with a copy of the healthy gene, is an easy way to do it. We’ve been using this approach for years. The problem with it is that we still don’t know what happens when the gene is inserted and expressed in a cell that doesn’t normally express it. The normal cell controls on these genes are disrupted.”
Dr. Gruenert warns that the random insertion and activation of genes, independent of the cellular regulatory machinery, may lead to problems. “This may be fine in treating diseases like cancer where your goal is to kill cells,” he explains, “but if you want to fix the cell, you may run into problems in the long term.” He describes a widely reported gene therapy trial where two of 14 patients developed leukemia because the healthy gene was inserted in a region that ultimately altered the cell’s ability
The Advantages of Genome Editing
To circumvent these problems, Dr. Gruenert worked with Karl Kunzelmann, MD, who was a fellow in Dr. Gruenert’s laboratory and is now at Regensburg University in Germany, to develop and patent a technique called small fragment homologous replacement (SFHR). This work pioneered a novel class of gene therapy that has been dubbed variously as “genome editing,” “genetic targeting,” or “genetic surgery.”
In the genome editing strategy, small lengths of specially synthesized DNA with the “correct spelling” are packaged and inserted into cells carrying the mutation. At this point, the cell’s own spell-check machinery takes over, removing the mutated portion of the gene and replacing it with the correct genetic information supplied by the therapeutic DNA fragment. While the molecular mechanism behind this genetic find-and-replace function remains a mystery, it does work.
“The whole idea,” Dr. Gruenert explains, “is to go in and modify the patient’s DNA while maintaining the integrity of the gene. If we fix a gene and its protein is supposed to be made in a cell, it will be. But if it’s not supposed to be, the gene will remain silent. You have a cell-appropriate process that is less likely to be detrimental.”
Paving His Own Way, Building His Own Tools
Because CF and sickle cell are considered relatively rare disorders, most research funding comes from private individuals, foundations, and the government. Thus, Dr. Gruenert, like many academic researchers, must pave his own way with grants to pursue his novel line of genetic research. This interest began for him in the late 1980s with his work at the University of California, San Francisco, continued with his founding of the Human Molecular Genetics Unit at the University of Vermont, and remains the focus of his current research at California Pacific.
The self-sufficiency shown by Dr. Gruenert in securing research grants is also reflected in his history of creating the sophisticated tools needed to advance his research. In the late 1980s, for example, he created the first lab-grown human lung cells that carry the normal CF gene, as well as others that have the mutant CF gene. His cell lines remain the gold standard for gaining a better understanding of CF gene function and for early testing of new CF treatments.
The Challenges Ahead
Now that the principles of genome editing have been demonstrated, Dr. Gruenert’s research is aimed at boosting the target cell’s uptake of the gene fragments and maximizing the number of cells expressing the normal hemoglobin or CFTR proteins. This involves changing the length of the DNA fragments and experimenting with the microscopic lipid sacs (fat bubbles) that deliver the fragments. As part of this optimization process, he also continues to investigate the mysterious enzymes within cells that guide the DNA fragments to their proper location and then help cut and paste the targeted DNA sections.
Dr. Gruenert is also looking ahead to other likely hurdles for testing in patients. “CF is a hard nut to crack,” he says. “You not only have the genetic defect but you also have a lot of tissue damage in the airways because of the constant infections and the scarring of the tissues.”
This damage means that CF patients may lack the living lung cells needed to receive the DNA fragments. To get around this problem, Dr. Gruenert plans to take stem cells derived from the patient’s own bone marrow and make them both delivery vehicles for the spell-checked genes as well as repair systems to regenerate the damaged tissues. “This way,” he says, “we could avoid immune reactions to foreign tissue. The cells would migrate to the damaged organ, become epithelial lung cells, and express the normal CF gene.”
“In sickle cell disease,” he says, “using the patient’s own bone marrow cells also makes perfect sense since this is where the red blood cells originate. It basically becomes a slightly modified version of the bone marrow transplant that is already the most effective treatment of sickle cell disease.”
Stressing that his work is in the early stages, Dr. Gruenert says there is growing interest in gene targeting approaches. So what are the chances that those thousands of kids born this year with sickle cell disease or CF will eventually benefit from some type of gene therapy?
“Pretty reasonable,” according to Dr. Gruenert. “Currently in CF, we have reached somewhat of a plateau in survival at around age 35 because many of the conventional treatment options have been exhausted. Many patients with sickle cell disease also have limited treatment options. To get beyond this, we absolutely need to come up with a gene- and cell-based therapy that will correct the genetic defect and revitalize the red blood cells and lungs.”
Finding cures for sickle cell anemia and CF is obviously a life-long quest for Dr. Gruenert. But for him it’s about more than the science. He often speaks directly with hematologists, pulmonologists, and patients and their families to learn about their struggles and needs, and to update them on research progress. And through his volunteer role as genetics advisor with many national organizations, he has heard the hopes and fears of the parents of children diagnosed with sickle cell anemia and CF. In response, he has dedicated himself to exploring a gene therapy cure.