The St. Jude team overcame the obstacle posed by the large number of defective hematopoietic stems cells (HSCs) producing faulty red blood cells in beta-thalassemia or sickle cell disease. The large numbers of defective HSCs thwart attempts by gene therapy to reverse the disease. HSCs are parent cells in the bone marrow that give rise to blood cells.
The researchers also performed the difficult task of integrating genes into an HSC's own DNA so the HSCs function normally.
St. Jude investigators said their results offer promise for developing gene therapy to treat blood diseases in humans caused by defective hemoglobin, i.e., hemoglobin that either lacks a critical protein called beta-globin or that contains a mutated form of the protein. Hemoglobin is the oxygen-carrying protein in red blood cells. Replacing red blood cells that carry defective hemoglobin with red cells that have normal hemoglobin is a potential strategy for curing these disorders.
Beta thalassemia (Cooley's anemia) occurs when the hemoglobin molecule lacks the beta-globin molecule that is part of the hemoglobin molecule. Children with untreated thalassemia have reduced production and survival of their faulty red blood cells. Left untreated, children with this disease die in the first decade of life. When treated by transfusions that supply normal blood cells, these children can survive into their late teens. But unless they are also treated for iron overload from blood transfusions they will eventually succumb to heart failure.
In sickle cell disease, an abnormal gene for beta-globin causes hemoglobin molecules in the red blood cell to clump together and distort the cell into the shape of a sickle. Instead of flowing freely, sickle-shaped red cells sludge and block blood vessels. This cutoff of blood flow can cause pain, stroke, leg ulcers, bone damage and other medical problems.
The St. Jude researchers chose beta-thalassemia and sickle cell disease as targets for their gene therapy study because both diseases could potentially be treated by modifying HSCs with normal genes for gamma-globin, which is usually produced only during fetal life.
The St. Jude strategy could let physicians treat beta-thalassemia and sickle-cell disease with a minimal number of genetically modified cells, eliminating the need to use radiation and intensive chemotherapy to rid the body of defective HSCs. Radiation and intensive chemotherapy cause severe side effects (e.g., nausea, hair loss) that significantly degrade patient quality of life.
"The success of this procedure in mice encourages us to continue developing gene therapy for hemoglobin disorders in children," said Arthur W. Nienhuis, M.D., St. Jude director and an author of a report on this work that will appear in an upcoming issue of the journal Blood. "Such treatment could eliminate the serious complications these diseases can cause, such as anemia, slow growth and stroke, without the need for irradiation," Nienhuis said.
Overcoming obstacles of insertion and enrichment
Genetically modified HSCs cannot reverse a hemoglobin disease if there are too many uncorrected red blood cells being made by defective HSCs, said Brian P. Sorrentino, M.D., director of St. Jude Experimental Hematology. "But the current technology didn't allow genetically modified HSCs to quickly take over red blood cell production in the bone marrow. So we developed a way to give normal HSCs a survival advantage over abnormal HSCs in the body. This way the population of genetically modified cells became naturally enriched--or enlarged--in the mice, Sorrentino said."
The St. Jude team enriched the population of HSCs using a virus (oncovirus) to carry a gene called MGMT into the cells. MGMT protects against the toxic effect of a drug called TMZ. The investigators infused HSCs with the MGMT gene into mice, and then treated the mice with TMZ. The toxic drug eliminated the defective HSCs while the donor cells with the MGMT gene survived and multiplied.
This technique, called in vivo selection, allowed the genetically modified HSCs to assume production of red blood cells inside the mice after TMZ treatment eliminated defective HSCs. In the absence of competition from defective HSCs, the genetically modified cells significantly increased their numbers and replaced the beta-thalassemic red cells with normal red cells. Using this technique, the St. Jude researchers achieved in treated mice an average 67-fold enrichment of normal red blood cells--even though they infused into the mice low, non-therapeutic levels of the genetically modified HSCs that produce these red cells.
The investigators also overcame the obstacle of getting both the gamma-globin gene and the MGMT genes to integrate into the cell's own DNA. They adapted a previously developed technique that used a virus to ferry genes into cells. The virus--called lentivirus--is a family of viruses that includes HIV, the cause of AIDS. Certain non-disease-causing genes in the virus ensure that the therapeutic gene becomes integrated into the DNA of HSCs.
Once integrated into the DNA, the genes were duplicated each time the cells divided, and were passed on to future generations of HSCs, which produced red blood cells.
Successfully inserting both genes at one time into a patient's HSCs would permit physicians to enrich the genetically modified cells that produce normal gamma-globin, according to Sorrentino.
"Our finding gives us hope that we might one day be able to help patients with hemoglobin diseases generate healthy blood cells in their own bodies," said Derek Persons, assistant member in the St. Jude Department of Hematology/Oncology. Persons is the lead author of the report. "The technique we've pioneered will allow us to enrich the population of cells carrying the normal gene by eliminating competing, defective cells, without using radiation or intensive chemotherapy."
The use of radiation or high doses of chemotherapy to destroy abnormal HSCs--a process called myeloablation--is already commonly used in the treatment of blood cancers, and is very toxic. The use of chemical treatment instead of radiation--a process called non-myeloablation, or conditioning--is less toxic, but still reduces the burden of abnormal cells. Non-myeloablation is especially attractive to researchers who want to develop relatively non-toxic treatments for blood diseases that require eliminating large quantities of abnormal stem cells.
Other authors of this study include Esther R. Allay, Nobukuni Sawai, Phillip W. Hargrove, Thomas P. Brent and Hideki Hanawa.
This work was supported by NHLBI, a Cancer Center Support (CORE) Grant and ALSAC.
St. Jude Children's Research Hospital
St. Jude Children's Research Hospital, in Memphis, Tennessee, was founded by the late entertainer Danny Thomas. The hospital is an internationally recognized biomedical research center dedicated to finding cures for catastrophic diseases of childhood. The hospital's work is supported through funds raised by ALSAC. ALSAC covers all costs not covered by insurance for medical treatment rendered at St. Jude Children's Research Hospital. Families without insurance are never asked to pay. For more information, please visit www.stjude.org.