Sickle cell anemia is a paradox. The molecular basis underlying thisgenetic disease is well-understood. A single amino acid change in thea-globin gene results in formation of a fibrous variant of the oxygen-carrying protein, hemoglobin. This, in turn, causes sickling orcrinkling of red blood cells, making them fragile and rupture-prone.

Yet, despite this detailed mechanistic knowledge, effective treatmentof sickle cell disease remains elusive. As a result, the afflicted, whoare almost exclusively black, must live with painful acute episodesthat cause physical impairment and, often, eventually death. Atpresent, the only effective therapy is replacement of the offending redblood cell precursors by bone marrow transplantation, but matcheddonors are too rare to make this treatment common.

However, a potential cure for sickle cell disease may ultimatelyemerge as a result of work reported in the current issue of theProceedings of the National Academy of Sciences (PNAS), datedMarch 28. In their article, Ken Takekoshi and colleagues from theHarvard-MIT Division of Health Sciences and Technology describethe successful retroviral vector-based transfer of an engineeredhybrid a-globin gene into mouse cells that serve as a model systemfor differentiating red blood cells. They engineered this modifiedgene to prevent formation of the sickle cell variant of hemoglobinwhile causing expression of high levels of the modified a-globin.

Hybrid Globin Gene Prevents Sickling

Hematologist Philippe Leboulch, senior author of the PNAS paper,told BioWorld Today he created a hybrid a-globin gene thatexpresses a protein "retaining the overall structure of human a-globin." He added, "We incorporated specific coding sequences intothe hybrid from the delta-globin gene, a gene normally expressed inthe fetus. The amino acid residues encoded by these fetal genesequences are the ones responsible for the known anti-sickling effectof delta-globin."

Leboulch continued: "We then solved the problem of thecharacteristic low expression of transfected a-globin genes. Weplaced our hybrid gene under the control of specific sequences fromthe so-called a locus control region located far upstream of the a-globin coding sequences."

But the retroviral vectors constructed with this hybrid gene wereunstable. As Leboulch said, "By eliminating some sequences fromintron 2 and some splicing signals in the hybrid gene, we were finallyable to get stable retroviral vector transmission." The resultingexpression level of this hybrid gene was almost 85 percent that of thenaturally occurring a-globin gene in their in vitro model system ofred blood cell differentiation.

Pointing The Hybrid Gene At The Marketplace

Leboulch reported that he and the PNAS paper's other senior author,Irving London, a physician and molecular biologist, in 1994 formed acompany to develop gene therapy options for hematopoietic stemcells called Innogene Pharmaceuticals Inc., in Cambridge, Mass. "Wehave obtained exclusive licenses from MIT to the enablingtechnology for our work," Leboulch said.

Innogene's founders said they have identified the reason whyretroviral vectors, in general, do not efficiently infect non-dividingcells. Leboulch said that "Retroviral vector complexes are nottransported to the nucleus. We are now adding viral nuclearlocalization signals to our vectors to see if we can transport thesecomplexes to the nucleus of non-dividing cells." Achieving this, he concluded, would make almost anycell in the body accessible to gene therapists.

In an effort to further strengthen its position, Innogene is in theprocess of merging with Genetix Inc., also of Cambridge, which has aproprietary position in retroviral packaging cell lines. Leboulch saidthat the merged company is in the final stage of obtaining equityinvestment and research and development support from two majorpharmaceutical firms. "With all of this in place," Leboulch observed,"we hope to be well-positioned to move into animal trials so that wecan assess the efficacy of gene therapy in human patients."

Conventional Therapy Also Advances

While current drug therapy for sickle cell disease is disappointing,new clinical trial results have renewed interest in less futuristictherapy. As hematologist Ronald Nagel from the Albert EinsteinMedical School in New York told BioWorld Today, "NIH [NationalInstitutes of Health] has cut short its clinical trial using hydroxyureaas a treatment for sickle cell disease because of its dramatic successin reducing painful crises in sufferers. While the idea of usinghydroxyurea is not new, this is the first time it has been evaluated inan effectively controlled double-blind study."

"Hydroxyurea is a cytotoxic agent, as is vincristine," observed Nagel."While the side effects of these drugs can be serious, they also clearlyaffect delta-globin expression which, in turn, reduces sickling. Delta-globin levels increased as much as eight percent in the clinical trial.While not a major increase, it has a major effect. Apparently, theeffect of hydroxyurea is not solely linked to delta-globin expression."

Nagel pointed out that the idea behind Leboulch's work had been theaccepted gene therapy strategy for about eight years. "Many labshave been working with a variety of vectors to try and express usefula-globin variants," said Nagel. "More recently, other gene therapyapproaches have been added, such as expression of ribozymes thatspecifically cleave a-globin mRNA."

Gene therapy for sickle cell disease has opened a new door on thetreatment of an old and refractory disease. But, more traditionalroutes of drug therapy also continue to advance. Researchers wonderwhether these two approaches will be competitive or complementary.n

-- Chester Bisbee Special To BioWorld Today

(c) 1997 American Health Consultants. All rights reserved.

No Comments