GENE TRANSFER FOR B-THALASSEMIA

July 26, 2010 — Preclinical trials of a genetic treatment for β-thalassemia have demonstrated the potential to successfully correct the genetic error underlying this disease. The normal β-globin gene was inserted into patients' hemopoietic cells from bone marrow aspirates, increasing β-globin production to levels typically found in carriers of the mutation.

The study, led by an Italian group based at the San Raffaele Scientific Institute in Milan, was reported online July 21 in EMBO Molecular Medicine.

β-thalassemia is a genetic disease caused by the absent or reduced ability to produce β-globin — a protein that constitutes 2 of the 4 protein chains in normal hemoglobin. If 1 of a person's 2 genes is mutated, the individual is a carrier, with only mild anemia. However, patients with 2 mutant genes show symptoms of severe anemia including pallor; dark urine; slow growth; enlargement of the liver, heart, and spleen; and bone problems. The disease occurs primarily among individuals of Mediterranean, African, or Asian heritage.

Standard treatment involves transfusions as often as every 2 to 4 weeks, introducing additional problems such as infection and iron overload, with complications involving heart and kidney damage.

"Treatments are limited to lifelong regular blood transfusions, and iron chelation to prevent fatal iron overload," said senior author Giuliana Ferrari, PhD, from the San Raffaele Telethon Institute for Gene Therapy, in a press release. "The alternative is bone marrow transplantation, an option open to less than 25% of patients."

"Iron overload is due to [the] hemolytic process, higher absorption, and to transfusions," added Dr. Ferrari in an email to Medscape Medical News.

Participants in the study were 44 patients with β-thalassemia major or intermedia, from 2 to 15 years old, of varied Mediterranean origins (eg, Palestinian, Iraqi Kurd, Lebanese, Syrian, and others), who represented a variety of mutations in the β-globin gene. All were enrolled in bone marrow transplant programs.

Investigators obtained bone marrow samples from the patients as a source of CD34+ cells (hemopoietic cells). As the report explains: "Correction of β-thalassemia by gene therapy requires gene transfer in stem/progenitor cells and high levels of β-globin gene expression in the differentiated erythroid progeny." The CD34+cells were transduced with a lentiviral vector, LV GLOBE, engineered to contain the normal β-globin gene, and were then cultured for 14 days.

As part of this procedure, the CD34+ progenitor cells are also treated with cytokines "to maximize the efficiency of gene transfer in CD34 cells, since the cells are committed to cell cycle," Dr. Ferrari told Medscape Medical News. "In this condition the vector works best, rather than in really quiescent cells."

Gene transfer efficiency ranged from 37% to 95%, with an average of 65%. Analysis of transduction efficiency in cultured cells found that the proportion of cells from patients with β-thalassemia producing normal hemoglobin was comparable with that of normal individuals (44.6% ± 6.7% compared with 53.8% ± 7.2%). Transduced cells from patients with β-thalassemia who had produced low levels of β-globin also significantly increased their production of normal hemoglobin (P < .05).

In cells from 2 control patients, the ratio of β-globin/α-globin synthesis was .97 and .79. In transduced cells that had completely lacked β-globin production, the ratio improved to .30, and cells that had previously exhibited low β-globin production achieved a ratio of .35.

The study also investigated the tendency for the viral vectors to integrate the normal β-globin gene at a disruptive location, especially at loci involving protooncogenes. In the 4 patients with β-thalassemia who were analyzed, the vectors integrated at 403 unique sites. There was a low tendency (6.4% of the time) to "integrate at recurrent sites (hot spots)" in the genome. Although 3 hot spots were in cancer-related genes, in general the vector exhibited "no tendency...to integrate into protooncogenes."

A "Close Up" article in the same issue of Molecular Medicine drew 3 messages from the study. First, the manipulations involved in transduction with the viral vector did not impair hemopoietic cells' function or general gene expression. Second, at low copy number (about 84% of the hemopoietic cell colonies in culture contained 1 to 4 copies of the vector), the vector could correct β-globin expression, leading to red blood cell formation in offspring of the transduced cells. Finally, the LV GLOBE vector behaves like other lentiviral vectors, with no bias toward integrating in oncogenes.

"This work represents the kind of translational studies that are required to move human investigations forward but are often very difficult to fund and publish," wrote coauthor David A Williams, MD, chief of the Division of Hematology/Oncology, and director of translational research, Children's Hospital Boston, Massachusetts.

Asked about his expectations for use of genetic therapies, Dr. Williams told Medscape Medical News via email: "There will be applications more widely in monogenic diseases in hematology [and] immune diseases, but also in other organ systems (eg, the eye studies). There will be applications in nonmonogenic diseases such as autoimmune or rheumatologic disease and cancer," said Dr. Williams, and "potentially in [central nervous system] degenerative processes."

For β-thalassemia, Dr. Williams was cautiously optimistic about gene therapy replacing bone marrow transplants: "Difficult to say at this point, but if gene therapy were to uniformly work and not have excessive toxicities, it might well be the preferred therapy. Its advantage is that it eliminates risk of [graft-versus-host disease] and eliminates need to find a donor. However," he concluded, "we are still away from this being reality."

"Allogeneic transplantation is the cure of choice in case of [a] compatible donor," agreed Dr. Ferrari. "Gene therapy is still a trial whose benefit needs to be proven."

The authors of the study and of the Close Up article have disclosed no relevant financial relationships.

EMBO Mol Med. Published online July 21, 2010.