In the beginning, hemophilia victims _ who lack clotting factor VIII_ used to die young of uncontrollable blood loss.Then, scientists found out how to extract factor VIII from human donorblood. Later, they learned how to screen that gift of life for hepatitisand HIV contamination.Exogenous factor VIII serves as an injectable "tourniquet" to quenchhemorrhagic crises, making up for the missing or mutated gene thatcauses hemophilia.More recently, in the early 1980s, Genentech Inc., of South SanFrancisco, and Genetics Institute, of Cambridge, Mass., independentlycloned and expressed the gene for recombinant human factor VIII,which is now commercially available.Now, the 20,000 hemophilia sufferers in the U.S. may well bewondering whether, and when, gene therapy will replace theiremergency injections of the clotting factor, so they can make their ownfactor VIII as needed.At least a dozen biotechnology companies are working to perfect genetherapy for hemophilia. One apparent road block is the unwieldy sizeof the factor VIII cDNA sequence, which hinders its insertion into thepatient's cells. The gene encodes 2,351 amino acids.Seeking to overcome this obstacle, a group at the Research Institute ofMolecular Pathology in Vienna, a facility of Boehringer-Ingelheim, hasinserted a stubbier factor VIII gene sequence, which lacks its 980-amino-acid B domain.Molecular biologist Max L. Birnstiel, the institute's managing director,reports preclinical gene therapy using the foreshortened gene in thecurrent issue of the Proceedings of the National Academy of Sciences(PNAS). Its title sums up his team's approach:"In vivo production of human factor VIII in mice after intrasplenicimplantation of primary fibroblasts transfected by receptor-mediated,adenovirus-augmented gene delivery."Mice whose spleens received surgical implants of fibroblaststransfected ex vivo with truncated factor VIII genes expressed themissing protein in their blood at levels that reached "production ratesof 1000 milliunits per 106 cells in 24 hours." This yield, Birnstielwrote, "clearly exceeded the limits required for therapy."Prior in vitro studies achieved factor VIII output "sufficient to generatetherapeutic plasma levels in mice," and exceeded "the values obtainedin vitro with other gene transfer techniques by a factor of 10 to 100."Birnstiel told BioWorld Today that these comparisons reflect only invitro, not in vivo, experiments conducted by other gene therapy groups.Birnstiel's gene-therapy recipe started by transfecting mousefibroblasts with a defective human adenovirus modified by polylysine,and complexed to a B-domain-deleted human factor VIII expressionplasmid.Fibroblasts transformed by this ex vivo gene therapy construct werethen surgically injected into the spleens of six four-week-old femalemice. Control mice had the package implanted into a muscle, a route,Birnstiel said, that had never successfully incorporated the factor VIIIgene.He described the steps performed by each component in his genedelivery package:y the factor VIII gene, minus its B domain, is complexed to itsinactivated-adenovirus vector;y polylysine condenses the DNA into a tiny ball of nucleic acid, thebetter to penetrate the target cell;y transferrin, a molecule conjugated to the polylysine, attaches theDNA vector to the target fibroblast cell's surface receptor;y the adenovirus vector penetrates the cell's endosome-dimpledmembrane, allowing the DNA to enter the cytoplasm by endocytosis. Itavoids the protein-destroying lysosomes, which would degrade theDNA, and enters the nucleus, by mechanisms unknown.Birnstiel said that the experiment, designed only to prove the principleof his transfection method, chose to implant the package in the spleen,"for the first time in an animal model." In human subjects, he observed,"one can always introduce the transfected cells into the portal veinleading to the liver, a presumed primary organ for factor VIIIexpression."He pointed out that although the factor VIII gene is stymied in muscletissue, factor IX _ which activates factor VIII in the blood-clottingcascade _ "being a much smaller molecule, works very nicely inmuscle, where it is secreted at high levels."The Vienna institute's overall transfection approach, he said, is patent-pending. It is well known to American researchers laboring in thehemophilia gene therapy vineyard. Among these, one major player isSan Diego-based Vical, Inc., which last December entered into acollaborative arrangement with Baxter Health Care Corp., of Deerfield,Ill. (See BioWorld Today, Dec. 15, 1993, p. 1.)Vical's director of business development, Robert Zaugg, told BioWorldToday, "We and Birnstiel have done some work together."As to the Vienna procedure, Zaugg questioned the practicality of aproduct that would be patient-specific. "Given the presence of foreign,probably immunogenic, components, could you give their productmore than once without a major immune response?"Queried on this caveat by BioWorld Today, Birnstiel replied: "This is apoint that is often raised. But while the components are immunogenic,we have found no immune reaction to cells transformed by our method.So this in the end may not be a problem."
Hemophilia Gene Therapy ProjectsCompany CollaboratorGenetic Therapy, Inc., CytoTherapeutics, Inc., Gaithersburg, Md. Providence, R.I.Genetics Institute, Cambridge, Mass.Transkaryotic Therapies, Inc., Cambridge, Mass.Gene Medicine, Inc., Houston
Somatix Therapy Corp., Baxter Health Care Corp. Alameda, Calif. (joint program, using (Somatix also has a implant system; program of its own) Baxter Health Care Corp.,
Vical, Inc., San Diego Baxter Health Care Corp., (a program looking at direct DNA injection )Viagene, Inc., San Diego Miles, Inc., Kankakee, Ill.TargeTech, Inc., Meriden, Conn.Targeted Genetics, Seattle (details lacking)SyStemix, Inc., Palo Alto, Calif. (details lacking)
Source: William Johnston, vice president and general manager ofBaxter Health Care Corp.'s Gene Therapy Unit
-- David N. Leff Science Editor
(c) 1997 American Health Consultants. All rights reserved.