The National Hemophilia Foundation's Sixth Annual Workshop on Gene Therapies for Hemophilia
Authors: Gina Kirchweger
Journal: Molecular Therapy (2003)

Each year, approximately 80 scientists gather together to discuss their progress in the struggle to deliver sustainable expression of Factors VIII or IX to hemophilia patients. However, the last ten to twelve years have shown that gene therapy to cure hemophilia is fairly challenging in practical terms, according to Glenn Pierce, Vice President of clinical development at Avigen, Inc., and National Hemophilia Foundation (NHF) board member. High up on the agenda of this year's meeting were strategies to prevent an immune response against the therapeutic proteins (inhibitor formation)—one of the major practical challenges—and the problem of insertion mutagenesis when using retroviral vectors. Although past gene transfer studies have shown that missing clotting factors can be replaced, the host's immune system nearly always produces a response that causes the rejection of the transfected cells, or generates neutralizing antibodies that limit the lifespan of the therapy. The Salk Institute's Inder Verma, who co-chaired the workshop with Pierce is convinced that “the major bane of gene therapy will always be the immunological consequences.” Increasingly, however, researchers are learning how to dodge an attack by the immune system. A crucial prerequisite to obtain therapeutic levels of circulating factor VIII or IX is the development of immunological tolerance to the proteins. Roland W. Herzog (Children's Hospital of Philadelphia) is studying whether transgene expression alone can induce tolerance to human coagulation factor IX (hF.IX). He introduced the gene into an immunocompetent murine model of hemophilia B, using liver-specific regulatory sequences to drive expression of the gene from an AAV 2-based vector. He subsequently immunized the mice with hF.IX protein to test the reaction of the immune system. The rate of tolerance induction in the mice varied between 20-100% depending on transgene expression levels and the genetic background of the recipients. The weaker the extent of cytokine release after immunization in control mice, the easier it was to induce tolerance after expression of the transgene in the same mice, he found. Mice that had been successfully tolerized to the antigen did not produce anti-hF.IX antibodies, and had substantially reduced in vitro T-cell responses after immunization with hF.IX. Injections into skeletal muscle—a mode of delivery that is falling increasingly out of favor with the community—did not induce tolerance and resulted in a strong immune response. “There is a good chance that you get tolerance when you introduce your gene via hepatic transfer, but I don't want to leave you with the impression that you always will,” Herzog explained. Herzog was unable to generate tolerance in Fas-deficient mice, suggesting a role for Fas- and Fas ligand-mediated apoptosis in the elimination of F.IX recognizing lymphocytes. Oral Alpan and Polly Matzinger (National Institutes of Allergy and Infectious Disease) tackled the immunological problem with a different approach. They fed hemophilia B mice the missing clotting factor to induce oral tolerance. Needing a safe and inexpensive source of biologically active F.IX, they teamed up with William Velander (Virginia Tech), who is producing recombinant hF.IX in the milk of transgenic pigs. Previous attempts to produce hF.IX in ruminants by other researchers had failed to generate animals that secreted significant amounts of the factor. Velander thinks that the ability of the pig mammary gland to modify recombinant proteins with y-carboxyglutamic acid—carbohydrate structures having terminal N-acetylneuraminic acid—and phosphorylation on serine residues, was key to their success. Each transgenic sow produces 100-300 liters of harvestable milk containing approximately 100-1000 units (U)/ml of hF.IX activity as measured by partial thromboplastin time. The hemophilia B mice received about 125-250 U/kg of hF.IX every other day for up to two months. “When we looked at the mice, we discovered the very astonishing fact that feeding the milk to the mice not only prevented their immune response to hF.IX but also treated their bleeding disorder,” reported Alpan. The mice no longer bled profusely, but had perfectly normal clotting times. This effect lasted for up to two days after a single post-prandial feeding. “Orally administered factor IX is emerging as a promising option to induce immunological tolerance in patients in preparation for gene therapy,” says Alpan. The researchers saw the same effect when they fed commercially available hF.VIII diluted in milk—although it had to be administered in lower doses because the protein is notoriously difficult to produce and no high producing transgenic animals currently exist. Some workers are trying to overcome this obstacle by tinkering with the amino acid sequence of factor VIII to improve the stability and yield of the secreted protein. Encouraged by their success in mice, Alpan and Velander fed 25 U/kg of hF.IX to Erika, a normal mixed breed dog. Two hours after the feeding, the serum levels of F.IX had risen to about 120 to 130 percent above normal, as measured by both ELISA and activated partial thromboplastin time. Next, they plan to expand their studies to hemophilia B dogs. “Although it is difficult to extrapolate from mice to dogs and to humans, our results are very encouraging. For a certain number of human patients, oral therapy may even be an option,” Velander opines. Washington University's Katherine Parker Ponder's vehicle of choice for gene delivery is based on moloney murine leukemia virus-derived retroviral vectors (RVs), which trans-port genes only into dividing cells. Ponder injected RVs expressing canine factor IX (cF.IX) intravenously into new-born UNC hemophilia B dogs, whose hepatocytes exhibit active replication. In each of three dogs treated in this way, whole blood clotting time was corrected and remained with-in the normal range for the duration of evaluation (1, 4 and 13 months, respectively). More importantly, none of the dogs developed anti-cF.IX antibodies. To exclude the possibility of pre-existing tolerance to cF.IX, she injected five wild type dogs two or three days after birth with an RV expressing human F.IX. Each animal showed stable expression that averaged 10% of normal. “The neonatal approach seems to induce tolerance to the transgene,” Ponder concluded, while cautioning against jumping to the conclusion that the approach might work equally well in human newborns, whose immune systems are much further developed. So far, Ponder has followed eleven dogs up to thirty months and has yet to detect any sign of cancer. Thus, insertional mutagenesis from integration into the chromosome does not appear to be associated with tumorigenesis in this system. Insertional mutagenesis was on the minds of many of the participants, due to the development of leukemia by two out of eleven boys in the French X-SCID gene therapy trial. In both cases, the murine retrovirus-based vector integrated near the LMO-2 oncogene, which was subsequently activated. Scientists have long worried that retroviral vectors might land in the wrong place in the human genome and inadvertently wreak genetic havoc. Calculations presented by Christof von Kalle (University of Cincinnati) suggested that rather than being a coincidence, the insertion of the vector used in the French study near the LMO-2 gene may occur in about one out of 100,000 integrations. Since millions of bone marrow cells had been modified ex vivo and returned to the boys, such insertions might crop up in most, if not all patients, he concluded. A further insertion has now turned up near the LMO-2 locus in a third child in the French trial, but fortunately the boy shows no sign of leukemia at this stage. “Stem cell transduction is so much more efficient than it used to be. In a tragic sense, the French workers became victims of their own success,” said von Kalle. Gene therapists hope that this problem will prove specific to the gene or methods used in the X-SCID study. However, experiments presented by Frederic Bushman (Salk Institute) point to a potential problem when using HIV and related retroviruses as gene transfer agents. He infected human cells with viruses and developed a high throughput method that allows him to pinpoint a large number of different integration sites simultaneously. He reported that the viruses are more likely to land in or near genes—especially in active ones—than in intergenic regions. Even more alarming was the fact that some favored hotspots clustered around highly expressed oncogenes. Bushman suggested that “oncogenes that are most at risk could be monitored after gene therapy.” He and von Kalle plan to join forces to create a combined database of integration events. Retroviruses are not the only vectors that pose a risk for insertional mutagenesis. A certain percentage of injected AAV-based vectors, currently a major workhorses for the delivery of genes encoding factor VIII or Factor IX, also integrate into the host genome. When Hiroyuki Nakai and Mark Kay from the Stanford University School of Medicine forced the proliferation of hepatocytes from mice injected with AAV-based vectors, episomal vector DNA was lost. Nevertheless, about 5% of the actively expressing vector remained, and was integrated in the host genome. When they analyzed the insertion sites, they also found a preference for integration into transcriptionally active regions. The mechanisms of integration by retroviruses and AAV are inherently different, and the relevance of the AAV data to the concerns raised by the recent cases of retrovirus-linked leukemia in X-SCID patients is not known. Michele Calos (Stanford University School of Medicine) thinks she has found a solution to the vexing problem of genomic vector insertion: a site-specific integration system mediated by phage ^C31-derived integrase. The gene of interest is placed on a plasmid along with a recognition site and introduced into the target cells together with a plasmid coding for the integrase. In collaboration with Kay, Calos tested the technique by injecting a hF.IX plasmid into the tail vein of normal mice. Within a week, mice that received the injection produced twelve times more Factor IX than littermates that received the injection without the integrase. Furthermore, the injected mice sustained normal levels of hF.IX for more than a year. Although the mouse genome contains at least 53 potential integration sites, Calos found the Factor IX gene in only two locations, with a location on chromosome 2 by far the more common. She said that for each tissue there may exist particular sites that are more likely to be insertion targets. Her group is now testing the technique in different human tissues to ensure that no integration site is near a potential oncogene. “We need to look in different tissues to see where the hotspot is before we can go ahead,” Calos said. Thus, a multitude of approaches and ideas to overcome the practical challenges of gene transfer to treeat hemophilia are currently being put to the test in different labs around the world. Only time will tell which ones will be abandoned and which ones will prove their worth in clinical trials.