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    Scientists advance a two-gene approach to curing beta-thalassemia

    Derek Persons

    *A St. Jude team successfully introduced two new genes into blood-producing bone marrow stem cells in an effort to ease beta-thalassemia in mice and reduce treatment side effects.

    The research, published recently in the journal Blood, raises new hope that an individual’s own bone marrow cells can be used with gene therapy to treat and possibly cure beta-thalassemia and other inherited blood disorders, including sickle cell anemia.

    “Now we are talking about new gene combinations and new molecules to expand the hematopoietic stem cells (HSCs),” said Derek Persons, MD, PhD, Hematology, the paper’s senior author. HSCs are the parent cells in bone marrow that give rise to blood cells.

    In this paper, researchers reported the amount of the target protein, fetal-hemoglobin, in red blood cells jumped dramatically and eased disease symptoms in mice transplanted with HSCs carrying one gene to correct beta-thalassemia and another to give those newly corrected cells a survival advantage. The researchers used a lentivirus to ferry the two genes and additional genetic regulatory elements into the defective HSCs in a process known as transduction. The new genes included one to protect against the toxic effects of drugs known as alkylating agents.

    Before some of the transduced HSCs were returned to the mice, they were treated in the laboratory with two such drugs in an effort to kill stem cells that had not incorporated the new genetic material into their own DNA. The goal was to give a survival advantage to the genetically corrected cells. Investigators knew those transduced cells could not prevail if too many uncorrected HSCs remained.

    The approach relies on a gene-therapy system developed at St. Jude. The goal is to develop a strategy for increasing the number of corrected HSCs a patient receives without the need for additional chemotherapy. “This approach could reduce the toxicity of the selection process by reducing or eliminating the need for post-transplantation drug selection,” the researchers noted.

    To work as a beta-thalassemia treatment, Persons said gene therapy must correct the inherited genetic mistake in 15 or 20 percent of a patient’s HSCs.

    Currently, individuals with the most serious form of beta-thalassemia require regular blood transfusions to prevent anemia. Gene therapy offers hope of someday freeing thousands worldwide from the discomfort, risk and cost of life-long transfusion therapy.

    Beta-thalassemia patients are severely anemic because they cannot produce an adequate amount of hemoglobin (Hb), the molecule red blood cells rely on to transport oxygen throughout the body. Normally, hemoglobin is assembled from two different proteins known as alpha-globin and beta-globin. In beta-thalassemia, the genetic instructions for assembling beta-globin are either missing or incorrect. As a result, alpha-globin builds up in the bone marrow and eventually destroys the red blood cells. Untreated, the disease leads to widespread organ damage and premature death.

    In this study another protein, gamma-globin, was a stand-in for beta-globin. Normally, gamma-globin is produced only during fetal development when it combines with alpha-globin to make fetal Hb (HbF), which ferries oxygen throughout the body. 

    Earlier St. Jude research showed that an adequate supply of HbF reverses beta-thalassemia in this same system. Relying on gamma-globin and HbF also protects patients from a potential immune reaction to a new protein—in this case, beta-globin.

    HSC transplants cure the disease in more than 90 percent of beta-thalassemia patients fortunate enough to have genetically matched donors. But such donors are rare.

    For more than two decades, researchers have pursued gene therapy as a way to fix the patient’s own HSCs. The idea is simple. In a process known as transduction, a harmless virus, dubbed a vector, is used as a biological ferry to slip the corrected or missing DNA into the patient’s own HSCs. Ideally, the mature blood cells derived from the HSCs go on to produce enough of the new protein to fix the problem.

    Gene therapy has worked in mice for nearly 10 years. But it has proven tougher to successfully integrate new genes into human HSCs. Currently, clinical trials for other blood disorders have shown low efficiency of gene transfer into human HSCs. Persons said it remains below the 15 to 20 percent needed to correct severe beta-thalassemia and sickle cell disease. The selection scheme used in these experiments provides proof of principle that this method might be the solution.

    Ten years ago, Persons, along with colleagues Brian Sorrentino, MD, and Arthur Nienhuis, MD, both of Hematology, began pursuing a two-gene approach. Along with correcting the underlying gene defect, they proposed adding a second gene to give a survival advantage to transduced HSCs. Persons said this paper demonstrates the strategy works, at least in mice.

    Researchers used a lentivirus to carry genes for human gamma-globin and methylguanine methyltransferase (MGMT) into HSCs. The genes were packaged with additional pieces of DNA, known as regulatory elements. Earlier St. Jude studies demonstrated such regulatory elements improved gene therapy results.

    The MGMT protects cells against a class of drugs known as alkylating agents. In this study, the drugs BCNU and O6-benzylguanine were used to kill HSCs that were not carrying the new genetic information.

    The strategy worked whether chemotherapy to knock out the defective HSCs was given directly to the mice after transplantation or to the treated HSCs in the laboratory before they were transfused into mice.

    Huifen Zhao, PhD, Hematology, showed mice transplanted with pre-treated cells had higher levels of corrected cells and higher levels of engraftment with gamma-globin expressing cells. Zhao is the study’s first author. “The drug-resistant cells engrafted preferentially,” Persons explained.

    Roughly four months after the transplant, 10 of the 17 mice transplanted with pre-treated HSCs were producing therapeutic levels of HbF. That 59 percent compares with just 22 percent of the 18 mice whose transplants did not include chemotherapy to kill uncorrected HSCs. Mice receiving the pre-treated cells also showed the greatest jump in hemoglobin levels.

    Other authors are Tamara Pestina, PhD, Md Nasimuzzaman, PhD, and Phillip Hargrove, all of Hematology; and Perdeep Mehta, of Bioinformatics.

    This research was supported in part by National Heart, Lung, and Blood Institute grants and ALSAC.

    September 2009