Ironically, two inherited maladies that evolved to ward off malariacause widespread suffering in their own right, far from malarialareas.Sickle-cell anemia inflicts painful, intermittent "crises" on African-Americans, and on blacks elsewhere, whose ancestors inhabited themosquito-infested lands of Africa. There, to this day, malaria is agreater scourge than sickle-cell anemia. In the U.S., one in 573newborns, regardless of race, inherit it.Beta-thalassemia affects large populations in Italy, Greece, Israeland other Mediterranean regions, where malaria once reigned, aswell as their emigrant compatriots in the U.S.Both hereditary anemias result from a mutated globin gene. Nowgene therapy is taking the latest in vitro steps to some day treat, andcure, sickling and thalassemia by supplying their patients'impoverished red blood cells with a replacement fetal gene.Globin is the stuff that erythrocytes _ red blood cells _ are madeof. It represents 94 percent of those cells' substance. The other sixpercent is heme, the iron-complex that completes the recipe foroxygen-carrying hemoglobin (Hg).A newborn infant's red blood cells still contain smatterings of theprenatal globin that saw it through the fetal stages of gestation. Aspecific mutation in the beta-locus of the adult globin gene causessickle cell anemia A different mutation produces abnormally lowamounts of beta-globin in thalassemia.A Cloning Construct to Combat SicklingThese are the targets on which gene therapists at the NationalHeart, Lung and Blood Institutite (NHLBI), and collaboratingcenters, are levelling their sights, with sickling for starters.Their paper in the current Proceedings of the National Academy ofSciences (PNAS), dated Oct. 11, bears the title: "Recombinantadeno-associated virus (rAAV)-mediated expression of a human g-globin gene in human progenitor-derived erythroid cells."What they have done, said Jeffrey Miller, the article's first author,and a clinical fellow at NHLBI's Hematology Branch, is take cellsfrom the bone marrow of a patient with sickle cell anemia andinfect them with a recombinant AAV vector. "This 4,725-nucleotidecloning genome contains the normal gamma-globin gene," he toldBioWorld Today, "plus the necessary regulatory sequences." Withthis construct, Miller added, "we transduced a group of CD34+cells, which are a population of bone-marrow progenitor cells thatincludes stem cells."The team grew these cells in the presence of growth factors,primarily erythropoietin, which is necessary to make Hg. "Many ofthe colonies that formed," Miller said, "were positive for thepresence of the viral genome, and showed evidence that it wasbeing expressed at the level of messenger RNA."Fetal Cells Without Any FetusMoreover, pools of transduced colonies had more of the formedhemoglobin coming from the gamma-globin genes than from thenon-transduced cells. And the ultimate sign of success: Theproportion of F (for "fetal") hemoglobin -- on which therapeutichopes are based -- was 40 percent in those transduced cells, ascompared with 26 percent in controls..How the researchers contrived this fetal-globin enrichment, Millerexplained, was by fractionating the Hg variants, "A, A2, F, or inthis case, S (for "sickle") as they came off the high-pressure liquidchromatography column."He added, "It's known that increasing fetal content in sickled cellsreduces the sickling phenomenon. So our purpose was to see ifvirus infecting the cell would result in increasing their F Hgcontent. Many of the cells did indeed express the correct gene forfetal Hg production."For treating beta-thalassemia," he confirmed, "the approach wouldbe the same, with only the target gene varying."That approach, as reported in PNAS, Miller observed, embodiesthree innovations: "First, it's an adeno-associated virus vector.Second, the vector itself contains no neomycin marker gene, so thatdispels any question as to whether such a gene might interfere withthe expression of other genes contained in these vectors. Thirdly,the most novel thing about this is a real hint that we may indeed beable to get globin gene expression in primary cells from patientswith sickle cell anemia."Looking to the future, Miller theorized what the somatic genetherapy clinical application would be: "Once we were able to makethese virions in sufficient quantity, we would use them to infectpatients with sickle cell anemia ex vivo, and so essentially havethem make the anti-sickling globin chain, that is, the F Hg."He foresees two next steps: "One, develop in vivo models towardwhich we have work in progress. Two, enlist biotechnology tocome up with better production methods in the vector itself."Once we have an adequate viral production that shows a lack oftoxicity in an in vivo model," Miller continued, "then the futurewould obviously be to think along the lines of therapy in humans.But we're not there, of course."Thalassemia Therapy Then, Now and FutureSpeaking as a clinician who sees patients as well as their cells,hematologist Miller concluded with feeling, "I'd like to make thepoint that the potential is very nice. It will be nice to see if this workpays off in the clinical arena, eventually."BioWorld was unable to reach for comment hematologist MartinCline of the University of California at Los Angeles. In July 1980,Cline attempted gene therapy for two young women with beta-thalassemia, one in Italy, the other in Israel. He administeredrecombinant globin genes mixed with their bone marrow. The effortwas not clinically successful, but may go down in biotechnologyhistory as the first documented gene therapy trial.There are roughly 8,000 to 10,000 beta-thalassemia patients in theU.S., estimates Ralph Cazzetta, director of patient services at theCooley's Anemia Foundation in New York. (Thalassemia is alsoknown as Cooley's Anemia.)For now, Cazzetta told BioWorld Today, the only therapy for thedisorder is Desferal, an iron-chelating agent. "It's infusedsubcutaneously for eight to 10 hours a night," he explained, "torelieve iron overload brought about by the multiple bloodtransfusions that thalassemia patients must get, to replace their losthemoglobin."Cazzetta added that, contrary to popular impression, thalassemia isvery prevalent in China and India as well as in the Mediterraneanregion.

-- David N. Leff Science Editor

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