Two genes with but a single promoter are out to improve the successrate of gene therapy.The dual construct's first target is Gaucher's disease, a hereditarylysosomal storage disorder, in which lack of the gene forglucocerebrosidase causes cells to build up huge backlogs ofglucosylceramide. Grotesquely enlarged liver and spleen, plusbreakdown of bone structure, mark Gaucher's, and condemn itssufferers to endless agony, ruined health and, often, early death.Genzyme Corp., of Cambridge, Mass., supplies placenta-extractedhuman glucocerebrosidase as maintenance therapy, at costs to patientsaveraging $140,000 a year. The company is developing a recombinantversion of the enzyme at its pilot plant in Framingham, Mass. It expectsto reach full-scale production early next year. (See BioWorld Today,Dec. 6, 1993, p. 1.)NIH's Recombinant-DNA Advisory Committee, RAC, last yearauthorized two companies to treat Gaucher's by gene therapy. Theirapproved protocols use a retroviral vector to insert the gene forglucocerebrosidase ex vivo in progenitor CD34 blood cells. (SeeBioWorld Today, June 6, 1993, p. 5).Current Gaucher's Gene Therapy TrialsGenetic Therapy, Inc. of Gaithersburg, Md., recently began treatingpatients with this protocol at the NIH and the University of SouthernCalifornia. The other RAC-approved clinical trial, by Theragen Inc., ofAnn Arbor, Mich., will begin in October at the University ofPittsburgh, the firm's manager of regulatory affairs, KatherineKaufmann, told BioWorld Today. It will enroll five patients."Often, when you put a gene into a cell," says Ira Pastan of theNational Cancer Institute, "for some reason its expression diminisheswith time, or the gene stops functioning."Pastan, who heads NCI's Laboratory of Molecular Biology, puttogether the two-gene-one-promoter vector package, and tried itsuccessfully on a continuous line of mouse fibroblast cells in vitro. TheMDR gene's P-glycoprotein targeted the cells' plasma membrane; thepassenger gene's glucocerebrosidase homed in on the lysosomes. Thebone marrow of these mice proved resistant to doxorubicin, taxol, VP-16 topoisomerase, and other cytotoxins that trigger MDR expression.Pastan's paper in the current (April 12) Proceedings of the NationalAcademy of Sciences is titled: "Drug-selected coexpression of humanglucocerebrosidase and P-glycoprotein using a bicistronic vector."Besides the missing Gaucher's gene, this construct expresses themultidrug resistance gene, (MDR), as selectable marker. It encodeshuman P-glycoprotein. The double-acting promoter, which drives bothgenes, comes from the genome of the Harvey murine sarcoma virus."This promoter for some reason is very active," Pastan told BioWorldToday, "so we use it in our retroviral vector."In explaining his strategy, Pastan notes that "If you had a vector inwhich you wanted to express two unrelated genes to make twoproteins, one way in the past was to use separate promoters." But oneof the promoters could shut down.To prevent that, he placed his single promoter before the MDR gene,which activated its expression. Then he hooked up the second gene bya virus-derived internal "ribosome landing pad. Because viruses, likebacteria, can connect their genes, the ribosome, which falls off at theend of the first gene, can start up again at the beginning of the secondgene."Why a multidrug resistance gene? In Pastan's game plan, its purpose isto protect most of the patient's bone marrow from cytotoxiccompounds administered to make room for the therapeutic Gaucher'sgene.Human, Mouse Tests PendingTo test this concept, two human trials are planned, at the NIH and theSloan-Kettering Cancer Center in New York. Their purpose is limitedto showing that "you can get expression of the MDR gene in bonemarrow in people," Pastan said.The people will be women with breast cancer, from whom bonemarrow is removed and stored while they undergo high-dose, cell-killing chemotherapy or radiation of their tumors. When the stockpiledbone marrow is reinfused, it will have the MDR gene added so that ifand when the bone marrow repopulates, it will be resistant to anti-cancer drugs.If the patients should then be re-treated with chemotherapy, their bonemarrow would survive these cytotoxic agents."So when you treat a patient with a drug that will kill normal bonemarrow cells," Pastan said, "those carrying the MDR gene will expandand express it. And the passenger gene associated with it would alsotherefore expand and be expressed."Multidrug resistance is nature's way of protecting delicate tissues, suchas colon and liver, from poisonous chemicals. The 170-kiloDaltonMDR gene product, P-glycoprotein, pumps such naturally occurringcytotoxic substances _ notably, cancer chemotherapy compounds _out of cells, to the frustration of oncologists.Also on Pastan's drawing board, a transgenic Gaucher's-disease mousemodel, which lacks glucocerebrosidase expression, will be tested invivo with the two-gene therapy vector.The NCI scientist trusts that his construct will provide one-stoprestoration of a Gaucher's patient's glucosylceramide metabolism. "Wewould hope," Pastan said, "that you could take the cells from a patient,insert the gene, put them back in _ and only have to do it once."Goals, Yes _ Predictions, No"Our goal," he said fervently, "is to provide an alternative to expensiveinjections of glucocerebrosidase. That's the goal of gene therapygenerally. We're just trying to improve what others are doing, a way toexpress genes more efficiently."He added, "If current gene therapy protocols work, and don'textinguish the gene, that's fine. My guess is that in time they will fail,and this would be a way to assure continued expression. But we'll see."He makes no prediction as to when or if the bicistronic construct willreach human trials. "That brings up an ethical problem, which needs tobe resolved. The drugs currently used to ensure expression of the MDRgene are mostly anti-cancer drugs, which are cytotoxic to normal bonemarrow cells. Should you destroy some normal marrow to ensureexpression of a passenger gene? I think that's an ethical question thatneeds to be dealt with."

-- David N. Leff Science Editor

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