Stem Cell and Gene Therapy for Sickle Cell and other Genetic Diseases

By Jeffrey Norris

A cure for sickle cell anemia and other life-threatening genetic disorders that arise in the blood is the goal of a new $6.7-million, five-year research project headed by UCSF scientist Y. W. Kan, a pioneer of modern genetics and the diagnosis of genetic diseases before birth.

The pre-clinical research is funded through a competitively awarded grant from the National Institutes of Health (NIH).

The new approach to be explored by Kan and UCSF collaborators, including Dieter Gruenert, PhD, and Marcus Muench, PhD, is based on stem cells and gene therapy. The UCSF researchers will primarily work with stem cells derived by manipulating and reprogramming cells from adults — not with embryonic stem cells. In clinical practice these stem cells would be made from a patient’s own tissue, and the inborn genetic error then would be corrected through genetic engineering.

The procedure that the UCSF scientists are developing is expected to offer a way to circumvent the need to find immunologically compatible bone marrow donors, and eliminate the threat of graft versus host disease — because use of stem cells derived from the patient should not cause unwanted immune responses when put back into the patient.

“This project offers a possibility of curing both newborn and adult patients with their own cells that have been reprogrammed, corrected and converted to cells that will regenerate all of their blood cells, including the immune system,” Kan said.

Kan and colleagues aim to convert a patient’s blood cells into powerful “pluripotent” stem cells. The researchers then will replace a defective portion of a gene in the stem cells using experimental techniques. Finally, they will coax the cells to become the kind of more specialized stem cells — called hematopoietic stem cells — that can specifically regenerate the entire range of blood cells.

Sickle Cell Disease, Beta Thalassemia Among Most Common Genetic Diseases

Initial targets for new treatment are sickle-cell anemia and beta thalassemia. These are the most common and severe genetic diseases caused by defects in a single gene — in this case the gene for beta globin. Beta globin is part of hemoglobin, the molecule in red blood cells that incorporates iron to pick up oxygen in the lungs and deliver it to tissues throughout the body.

These diseases currently can be cured only through transplantation of bone marrow or cord blood. Compatible donors are needed, but not often readily available.

In the United States, sickle cell anemia — now known as sickle cell disease — primarily affects African Americans. In California, about one in 6,600 newborns is born with sickle cell disease.

Anemia, while serious, is only one common manifestation of sickle cell disease. Red blood cells assume a “sickle” shape and can clump together and block small blood vessels during painful episodes that last hours or days and can recur repeatedly.

A patient with sickle cell disease may require frequent blood transfusions, and a patient with beta thalassemia typically requires such transfusions. In patients who cannot receive bone marrow transplants to cure the disease, iron can eventually build up in the body and damage organs as a result of continual transfusions. Organ failure is a common cause of death in these patients. Iron overload often is treated with chelation to remove excess iron. With these treatments many patients now survive to middle age.

Studies indicate that direct medical costs for these diseases with treatment that includes chelation average more than $60,000 dollars per year, per patient.

While the mutation that causes sickle cell disease is most common among people of African origin, beta thalassemia is most common in Mediterranean and Asian populations. Both of these genetic diseases are “recessive,” meaning a child must inherit a disease-causing mutation from both parents to develop major disease.

Innovative Stem Cell and Gene Therapy Approaches

Kan’s early studies to identify DNA mutations responsible for these diseases four decades ago led to some of the first prenatal, DNA-based, diagnostics tests for human disease. Gruenert, the co-director of the new project, is an innovator in the development of gene therapy strategies. The two previously collaborated on an NIH-funded Gene Therapy Core Center at UCSF in the 1990s.

Gruenert has developed a patented method that will be used in the current project to replace the defective beta globin genes in induced pluripotent stem cells. The researchers will develop the induced pluripotent stem cells using blood cells from people with the diseases.

Kan and Gruenert are members of the UCSF Institute for Human Genetics and the Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research. Through their research projects and collaborations they have become familiar with a broad range of stem-cell and gene-therapy techniques.

Since 2007, when Shinya Yamanaka, MD, a UCSF faculty member and Gladstone Institutes investigator, first succeeded in creating immortal pluripotent stem cell populations from skin cells, many of the techniques researchers have used to generate stem cells caused foreign reprogramming DNA to be randomly incorporated into the cellular genome. Such misplaced DNA has raised concerns about the possibility of generating cancers or other physiological disruptions to normal cellular functioning.

The UCSF researchers are exploring ways to safely reprogram cells and generate pluripotent stem cells without introducing unnecessary DNA.

Induced pluripotent stem cells can be grown indefinitely and manipulated to spin off specialized cells of almost any kind. Once the UCSF researchers have created the pluripotent stem cell lines, they will replace the defective stretch of the beta globin gene using a technique called small fragment homologous replacement, which Gruenert first developed in the 1990s and used in gene therapy studies. He previously used the technique to correct physiological defects caused by mutations in the cystic fibrosis gene.

Small fragment homologous replacement enables researchers to perform genetic surgery — swapping out mutation-containing DNA regions that are hundreds of nucleic acid base pairs long. Gruenert will combine small fragment homologous replacement with new techniques used to enhance the efficiency of target-gene modification.

While the research holds great promise, much more work needs to be done before the approach can be applied to patients, Kan said.

Other UCSF scientists who will play a leading role in the newly funded research program include Muench, a senior scientist at Blood System Research Institute, whose research for two decades has focused on understanding the development of hematopoietic cells and on the development of new stem cell transplantation strategies; R. Geoffrey Sargent, PhD, who studies new ways to genetically modify stem cells; and Long-Cheng Li, MD, a pioneer in the use of another tool, called small activating RNA, used to develop induced pluripotent stem cells.

Kan Photo by Cindy Chew