Technique for genetically modifying blood stem cells brings cure for blood diseases closer

The condition of mice with a genetic blood disease called beta-thalassemia improved significantly following treatment of their blood forming cells with a gene that enabled them to produce the type of hemoglobin normally found only in the fetus.

These findings, by investigators at St. Jude Children’s Research Hospital, are published in the October issue of Blood.

Beta-thalassemia is a genetic blood disease in which the red blood cells have an abnormal form of the oxygen-carrying molecule called hemoglobin (Hb). Normal Hb is made up of two alpha-globin proteins and two beta-globin proteins. The defective red blood cells in beta-thalassemia arise from hematopoietic stem cells (HSCs) in which both genes for beta-globin are either missing or mutated. In the absence of beta-globin, the other building block of Hb, alpha-globin, accumulates and eventually destroys the red blood cell. HSCs are parent cells in the bone marrow that give rise to blood cells.

The St. Jude team successfully treated beta-thalassemia in mice by using a newly developed vector—or biological ferry composed of DNA housed within a harmless virus—to shuttle a therapeutic gene into the defective HSCs. The gene allowed red cells that were derived from the HSCs to make gamma-globin, a protein that acted as a substitute for beta-globin. The resulting Hb molecule, composed of alpha- and gamma-globin, is called fetal Hb (HbF) because it normally occurs only during the fetal stage of development. The use of HbF prevents the need to introduce normal beta-globin into the body in someone who has never had it before. It is possible that the sudden introduction of beta-globin could trigger an immune response against this protein.

The vector, which used a virus called lentivirus to carry the gamma-globin gene, also carried a special set of additional pieces of DNA called regulatory elements. These elements were part of a section of the normal chromosome containing the globin locus control region (LCR), which has overall operational control over the expression of the gene. The St. Jude team used these regulatory elements that normally control the beta-globin gene and put them into the vector with the gamma-globin gene. The entire vector, named mLar-beta-delta-gamma-V-5, was then used to transduce (genetically modify) HSCs.

Previously, the St. Jude team had used a different vector that required more than one gamma-globin gene to insert itself into the cell; red cells derived from transduced HSCs could then make significant amounts of HbF. This vector did not consistently produce high levels of gamma-globin, according to Derek Persons, M.D., Ph.D., assistant member of St. Jude Hematology-Oncology. Persons is senior author of the Blood report. The ability of this previous vector to trigger production of gamma-globin varied, depending on where in the chromosome it landed.

“Our new vector is more dependable, and we need to get only one copy of it into a stem cell to produce significant amounts of fetal hemoglobin,” Persons said.

The present study showed that the control elements significantly improved the ability of a single gamma-globin gene to produce this protein in HSCs. The HSCs then produced enough HbF to reverse beta-thalassemia. The success of this technique is important because it showed that only one copy of the gene needed to be inserted into an HSC in order to increase the likelihood that sufficient quantities of the protein would be made. By minimizing the number of copies of a therapeutic gene shuttled into an HSC, researchers can reduce the chance that one of them would inadvertently insert itself into the middle of a normal gene on a chromosome and disrupt it.

“When it comes to avoiding disruption of chromosomes while improving the efficacy of a single gene used to modify a cell, less is definitely more,” Persons said.

In the experiment, mice that received transplants of stem cells modified with mLar-beta-delta-gamma-V-5 had total HbF levels of 17 to 33 percent, which led to significant improvement in their disease. This contrasted with the results in the study using the previously constructed vector, d432 beta-delta-gamma, which found that none of five mice treated with stem cells modified by this vector had HbF levels more than 15 percent.

“The enhancement in expression of the gamma-globin gene that was key to the success of this new vector was due to the additional controlling elements we added,” said Hideki Hanawa, M.D., Ph.D. Hanawa is the first author of the Blood article and a former postdoctoral student in Persons’ laboratory.

Despite the promise of the lentivirus in improving the efficiency of human HSC gene transfer, some uncertainty remains as to whether this technique alone will result in enough genetically modified HSCs to produce enough red blood cells containing HbF to successfully treat beta-thalassemia in humans. However, Persons and his colleagues previously demonstrated (Blood. 2003;102:506-513) a potential technique for enriching the relatively small population of HSCs modified by vectors.

“This combination of using a more effective vector and enriching the population of HSCs that are genetically modified by the gamma-globin gene could one day offer patients with hemoglobin diseases the promise of a cure,” Persons said.

Other authors of this paper are Phillip W. Hargrove, Steven Kepes, Deo K. Srivastava, and Arthur W. Nienhuis.

This work was supported in part by the National Heart, Lung and Blood Institute (NIH); a Cancer Center Support (CORE) grant; and ALSAC.