Gene Therapy Frees β-Thalassemia Patient From Transfusions for More Than 2 Years

Jacquelyn K. Beals, PhD

September 16, 2010

September 16, 2010 — Treating β-thalassemia with gene therapy has enabled a young adult patient who received his first transfusion at age 3 years to live without transfusions for more than 2 years. The report, published September 16 in Nature, also describes the partial dominance of a cell clone overexpressing a truncated HMGA2 mRNA, which has remained stable for 15 months.

β-thalassemia is one of a group of β-hemoglobinopathies, the most common heritable diseases around the world. The disorder is caused by a recessive genetic mutation leading to nonproduction or reduced production of β-globin, which makes up 2 of the 4 globin chains in human hemoglobin. The deficit of normally functioning hemoglobin results in fewer mature red blood cells and anemia.

Most β-thalassemia patients originate from India, central or southeast Asia, the Mediterranean region, the Middle East, or northern Africa. This study focused on compound βE0-thalassemia, more common in southeast Asia, in which 1 allele (β0) is nonfunctioning and the other (βE) is a mutant allele whose mRNA may either be spliced correctly (producing a mutated βE-globin) or incorrectly (producing no β-globin). This genotype causes a severe thalassemia, with half of the affected patients requiring transfusions.

Gene therapy for β-thalassemia is being pursued by several groups around the world. The use of lentiviral vectors reduces or avoids the problems encountered in 2002/2003 when gene therapy trials for X-SCID (X-linked severe combined immunodeficiency), using retroviral vectors, led to several cases of leukemia and cessation of the trials.

Lentiviral Vectors Offer Advantages

"There are 3 reasons why [lentiviral vectors] are more advantageous than retroviral vectors.... Lentiviral vectors are very effective at modifying or transducing quiescent cells — cells that do not divide — and bone marrow stem cells, at any given time, most of them are quiescent," said senior author Philippe Leboulch, MD, professor, Department of Medicine, Harvard Medical School and Brigham & Women's Hospital, Boston, Massachusetts; director of the Commissariat à l'Energie Atomique's Institute of Emerging Diseases and Innovative Therapies, Fontenay-aux-Roses, France, and professor of medicine and cell biology, University of Paris, in a telephone interview with Medscape Medical News.

The second reason is that "retroviral vectors are pretty simple beasts, if you will, and the size of what they contain in terms of gene length is short, and they don't have the machinery to prevent the viral RNA from being spliced before packaging, so it gets rearranged very commonly....

"Lentiviral vectors have the...[machinery to] prevent splicing of the viral RNA before packaging, even if it is very long and complex," explained Dr. Leboulch. "For globin gene disorders, we needed to use the full-length gene with the introns, we needed to add the promoter of the gene, we needed to add enhancer elements, so it was very long and complex, and only lentiviral vectors were able to carry that well.

"The third reason is that lentiviral vectors have the tendency to alter gene expression upon integration less frequently than retroviral vectors. So it's safer," Dr. Leboulch noted.

Transfusion-Free Since June 2008

In the present study, a lentiviral vector was used to transduce the human β-globin gene into purified blood and marrow cells obtained from the patient. The transduced gene coded for a mutated β-globin with "anti-sickling properties," allowing it to be distinguished from normal adult β-globin.

"Our vector does not contain the completely wild-type β-globin gene," said Dr. Leboulch. "We introduced a point mutation in the coding region.... That mutation allows us to measure the output of expression from the integrated gene and differentiate that from transfusions," and also from the small amount of β-globin that patients with β+-thalassemia express.

The 18-year-old patient received the transplant of gene-modified cells in June 2007, and his last transfusion in June 2008. His hemoglobin levels are presently stable at 9 to 10 g/dL (normal values in men: 13 - 18 g/dL), and about a third of the hemoglobin contains the form introduced by the viral vector.

Between 21 and 33 months after the transplant, the percentage of vector-bearing cells in his blood gradually increased:

  • whole blood: from 6.3% to 10.8%,

  • T lymphocytes: from 0.9% to 1.7%,

  • B lymphocytes: from 5.3% to 9.3%,

  • granulocytes-monocytes: from 12.3% to 18.7%, and

  • erythroblasts: from 1.9% to 2.9%.

Normal β-globin received previously in transfusions has been undetectable for more than 17 months.

Keeping an Eye on HMGA2

The authors emphasized that a prominent integration site of the vector was in the HMGA2 gene, causing "transcriptional activation" of this gene and giving rise to a clone of cells. A plateau was reached at which 45% of nucleated blood cells that contained the vector had a HMGA2 integration site. However, vector-bearing cells were still in the minority, and 28 months posttransplant, the percentages of blood cells with a HMGA2 integration site were relatively low: 3% of nucleated blood cells, 8% of granulocytes-monocytes, and 2% of erythroblasts.

"So it's not as if it's taking over," clarified Dr. Leboulch. "It's just that it is more dominant than other [clones], so it's more obvious that it is there."

In addition, although HMGA2 expression increased many-fold 16 months after the transplant (compared with before the transplant), HMGA2 was expressed only in erythroblasts — no HMGA2 mRNA was detected in granulocytes or monocytes, in spite of their higher prevalence of HMGA2 integration sites.

HMGA2 plays a role in embryonic development, is considered an oncogene, and is expressed in some benign tumors, so its activation elicited some concern. "The clone may remain homeostatic or be a prelude to multistep leukaemogenesis," the report states. However, at present, expression of HMGA2 mRNA has been stable for 15 months, and the clone of cells has also stabilized.

"Whenever a hematologist or oncologist hears, in terms of your blood system, that one clone is that dominant, that frequent, that's usually associated with a bad thing. One [clone] shouldn't take over that much," said Derek A. Persons, MD, PhD, from the Department of Hematology and Oncology, St. Jude Children's Research Hospital, Memphis, Tennessee, in a telephone interview with Medscape Medical News. Dr. Persons also commented on the study in a News & Views article in Nature.

"But you've got to remember, it's still only 7% of all the [blood] cells in the body. And the key thing is that it stopped growing, it plateaued, and it stayed stable," said Dr. Persons. "Cancer doesn't stay stable, so this guy doesn't have cancer. He's got his clone, and we don't know what's going to happen to it. Like I said at the ending of the [News & Views article], the only way we're going to really ever know is, we watch him."

The study was supported by grants from the National Institutes of Health and L’Association française contre les myopathies. Dr. Leboulch is the cochair of bluebird bio (formerly Genetix Pharmaceuticals) scientific advisory board. Dr. Persons does research in this field and is putting forth a gene therapy trial for β-thalassemia but reported no conflicts of interest.

Nature 2010;467:318-323, 277-278.

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