T H E   N I H    C A T A L Y S T      J A N U A R Y  –   F E B R U A R Y   2000


My purpose here," says Robert Donahue, in his headquarters at 5 Research Court in Rockville, Md., "is to demonstrate that novel and potentially therapeutic vectors are both safe and efficacious"—after they have been evaluated in small animals and before they are tested in human gene therapy trials. "It’s important to make our program visible," Donahue said in an interview. "Preclinical testing enables us to predict whether a vector has the possibility of being pathogenic in humans."

Donahue directs the NHLBI program on gene transfer and bone marrow transplantation in nonhuman primates and, with NHGRI collaborators Rick Morgan and Jay Lozier, has been exploring the potential of an adenoviral vector for human coagulation factor IX as a therapeutic agent in hemophilia B. He is more enthusiastic about other viral candidates than adenovirus as a vector for gene transfer in the treatment of chronic deficiency disorders.Retroviral vectors of murine origin and weakened or "gutless" human lentiviral vectors appear more promising as vectors that can safely target multiple cell types, he said. He and his team currently have six gene therapy projects underway, with a variety of collaborators, pursuing these vectors as conduits for gene expression in multiple lineages of hematopoietic cells. They’re currently looking at green fluorescent marker genes, provided by jellyfish, that enable them to track both the gene and the protein product. They anticipate moving on to genes for drug resistance, factor IX, chronic granulomatous disease, the gamma chain for the T- cell receptor in severe combined immunodeficiency disease, and proteins that could inhibit replication of such viruses as HIV.

The work with factor IX packaged in an adenoviral vector was originally a project in Morgan’s lab, where Lozier developed the vector; it was brought to 5 Research Court for evaluation in nonhuman primates, Donahue recounted.

Delivered alone, the factor IX protein was not immunogenic; delivered in the adenoviral package, however, both the protein and the vector were
immunogenic. This suggests, he said, that an adenoviral vector could be quite useful in a vaccine construct, enhancing the immunogenicity of an antigen, but less as a therapeutic agent for delivery of a protein on a chronic basis. The team determined—and Lozier reported more than a year ago at a meeting of the American Society
of Hematology and again in June 1999 at an American Society of Gene Therapy meeting—that expression of the gene for human coagulation factor IX could be achieved in rhesus monkeys using an E1/E3-deleted adenoviral vector. But the price was acute liver toxicity and coagulopathy.

The study dose ranged from 1 times 1010 to 1 times 1011 plaque-forming units (pfu)/kg. At the low doses, there was no gene expression; at the high doses, gene expression was accompanied by severe liver pathology in response to the vector. Among the findings were increased interleukin-6, decreased serum iron, and significant derangments in liver enzyme, albumin, and bilirubin concentrations and clotting time. These findings were formally published in the December 15, 1999, issue of the journal of the American Society of Hematology (Jay Lozier, Mark Metzger, Robert Donahue, and Richard Morgan. "Adenovirus-mediated expression of human coagulation factor IX in the rhesus macaque is associated with dose-limiting toxicity." Blood 94:3968–3975, 1999). They were also cited during the meeting of the NIH Recombinant DNA Advisory Committee to review adenoviral vectors and the death of a patient in a University of Pennsylvania gene therapy trial (see Gene Therapy Trial and Errors).

"In our study," Donahue said, "administration was intravenous; in the Pennsylvania study, a higher dose was administered directly to the liver, which led to even more damage." He speculated that vector distribution beyond the liver in that study could be attributed to the high dose and noted, too, that the liver’s Kupffer cells are a type of macrophage that processes antigen and releases hematopoietic growth factors that activate an immune response. "There’s a very narrow window between efficacy and toxicity," he observed, noting that converting animal to human dosages in this context demands precision and must take surface area into account. Formulas for computing comparable doses in rodents, dogs, monkeys, and humans can be found in an article by former NCI director Vincent DeVita in the text Cancer: Principles and Practice of Oncology, he said.

—Fran Pollner

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