Probing Gene Therapy

Researchers meet to share insight on bleeding disorders
Author: Katarina Grande

Editor’s Note: This article was funded through an educational grant from Novo Nordisk.

Abuzz with excitement, nearly 100 researchers from a dozen countries filed into the National Hemophilia Foundation’s 11th Workshop on Novel Technologies and Gene Transfer for Hemophilia at The Children’s Hospital of Philadelphia on March 2 and 3, 2012. From the scientists who focus on a particular section of a single molecule to those who study bleeding disorders in a global context, the world’s foremost researchers in the field met to share breakthroughs and ideas, and to cultivate new collaborations.

FIX Gene Therapy Trial Shows Early Promise

One globe-spanning research team brought exciting announcements on the progress of its clinical trial using gene therapy for hemophilia B, factor IX (FIX) deficiency. Edward Tuddenham, MD, professor at University College London, lights up when asked about the status of the first seven trial participants. “They’ve all reduced treatment or have no treatment,” he says. Patients with a therapeutic response produced enough FIX to suspend prophylactic infusions to prevent bleeding episodes. Two participants still infused, based more on personal preference than necessity. The trial is being conducted in stages, so the first participant received the gene therapy almost two years ago, the seventh recently. The initial participant has maintained a level of about 2% of FIX activity and remains off prophylaxis, says Andrew Davidoff, MD, Chair of Surgery at St. Jude Children’s Research Hospital in Memphis. Tuddenham and his colleague, Amit Nathwani, MD, PhD, faculty member at the University College London Cancer Institute, treat and provide clinical care for the trial participants, and Nathwani, Davidoff, and Art Nienhuis, MD, collaborated to develop the preclinical data on which the trial is based. The clinical-grade vector was manufactured at the GMP facility at St Jude’s.

The development of the perfect vector, the delivery vehicle for the gene, straddles the realms of art and science. The vector must slip under the radar of the immune system, fit the size requirements for carrying the gene, not incorporate itself permanently into the human genome and place the gene in the correct location. Viruses emptied of their disease-causing agents can do this successfully, but they must be selected and retooled carefully. Davidoff and team opted for an adeno-associated virus (AAV) vector that they modified. This FIX gene-containing vector can be given to patients via peripheral vein injection, a safer alternative to more invasive options that directly target the liver—the ultimate destination. The team’s vector has tropism for the liver, which is where the FIX gene begins its work. Despite this progress, the clotting factor levels that were attained in the St Jude-UCL study were insufficient to offer protection in the face of serious trauma or injury, and additional modifications of the vectors are needed to boost factor IX levels. Transient  toxicity, treated with steroids, is likely due to an immune reaction against the gene-modified cells as shown by Kathy High and Frederico Mingozzi of the Center for Cellular and Molecular Therapeutics at the Children’s Hospital of Philadelphia in Pennsylvania.

Paul Monahan, MD, outlined plans for a trial by investigators at UNC-Chapel Hill that will investigate an AAV vector similar to the one reported by Davidoff, but encoding a variant factor IX protein with higher enzyme activity.

Progress in gene therapy for hemophilia A will require more research before progressing to clinical trials, because FVIII is a much larger molecule than FIX and more engineering is required to fit it into an AAV vector. Nevertheless, Thierry VandenDriessche and his colleagues at the Department of Gene Therapy & Regenerative Medicine of the University of Brussels in Belgium have squeezed FVIII into AAV and expressed high levels in mice at low vector doses. But because immune responses and risk of inhibitor are more common for all types of treatment in hemophilia A, these issues will need to be analyzed carefully.

Patient Panel Provides Perspective

The workshop’s Patient Perspective Panel was comprised of four participants who addressed attendees, sharing their reactions to the gene therapy developments and other research. They also shared their stories of either living with or caring for a family member with a bleeding disorder. The currently available therapies are good, panelists said. Though challenges such as frequent prophylactic factor infusions, occasional joint pain and the pervasive fear of a severe bleed still exist, treatment for bleeding disorders has moved forward. To illustrate this point, panelist Phil Kucab, a second-year medical student at Wayne State University School of Medicine in Detroit, Michigan, who has severe hemophilia A, described his family’s history with the bleeding disorder. Two of his uncles had hemophilia in the 1970s; neither made it past age 17, each due to a severe bleed and complications with treatment. “Treatment has come such a long way since I was born. You could make the argument that we already cured hemophilia, if you define a cure as getting rid of joint problems and joint disease, bleeds and risk of viral transmission,” Kucab says.

A Therapeutic Cost

Though treatment improvements are clear in the developed world, ultimately economics may play a role in the equation for treating bleeding disorders for much of the rest of the globe. To treat hemophilia B, the annual cost per patient is around 150,000 British pounds (about $240,000), Tuddenham estimates. Since gene therapy is a one-time or at least very infrequent treatment, Tuddenham predicts that the costs to the patient will be lower. “But actually, where we think this is really going to make a difference is in the developing countries,” he says. Without the infrastructure to develop and manufacture safe products, people with bleeding disorders who live in such places have limited treatment options.

Editing Genes

Parallel to Doering’s research are other projects that take different routes to gene therapy, many still in early stages. Much of the gene therapy research has focused on the premise that if a new functional copy of the gene can be inserted into the genome, the problem of expression will be solved. To understand the concept behind this approach, imagine an alphabet with a strangely shaped letter “F.” Once a regular letter F is inserted successfully, a complete regular-letter alphabet results. The strangely shaped letter now sits next to the newly inserted regular F, but it can essentially be overlooked in favor of the new F. If there were a way to edit the letter instead of placing a whole new copy into the alphabet, perhaps longer term, more sustainable expression could result in terms of gene therapy. This is the approach of Katherine A. High, MD. Instead of inserting a new gene copy, an “edited” gene can pass the corrected version on to new cells. High’s team is using a pair of genetic scissors called  “zinc finger nucleases” to snip out the defective part of the FIX gene in people with severe hemophilia B and replace it with a fully functional version. The researchers have tried this in mice and observed stable FIX expression of 20%. This is an encouraging early stage result, since going from less than 1% to more than 5% expression levels can change a person’s hemophilia diagnosis from severe to mild. Further work is needed to see if it can be extrapolated to large animal models.

Improved Factor Traveling Through Trials

As participants in the workshop’s Patient Perspective Panel noted, research guiding the development of improved factor products is desirable alongside gene therapy studies. “The current treatments are good. They are safe, they are reliable for the most part, and they pretty much take care of our patients. But there’s a lot of room for improvement, and that’s why we’re here,” says Lisa A. Michaels, MD, of Bayer HealthCare Pharmaceuticals, Inc., in Montville, New Jersey. Her team is working on new versions of recombinant FVII (BAY 86-6150) and FVIII (BAY 94-9027) that last longer, ideally necessitating fewer prophylactic infusions. The two products are on their way to a phase 2/3 clinical trial to study the safety and effectiveness in people with hemophilia. Already, BAY 86-6150 has shown a half-life (the amount of time it takes for the infused factor to decrease by half) of five to seven hours, double that of the currently available rFVIIa product.

After years traveling through the many stages of safety testing, three different factor molecules developed by Novo Nordisk are heading toward advanced clinical testing. The molecules will be used in new factor products and include a longer-lasting FVIII recombinant called “turoctocog alfa,” a longer-lasting FIX recombinant named “N9GP,” and a FVIIa recombinant called “vatreptacog alfa” for individuals with inhibitors, meaning people whose bodies have developed antibodies to the very factor replacement products with which they treat themselves. “We have taken on the challenge of improving an already very good bypassing agent, NovoSeven®, understanding that there is need for higher efficacy in the management of bleeding episodes in patients with inhibitors,” says Stephanie Seremetis, MD, vice president for Medical and Science in Haemostasis Global Development at Novo Nordisk.

Scientists at Biogen Idec Hemophilia are in late stage clinical trials with longer-lasting, protein-engineered FIX and FVIII, as reported by Medical Director Snejana Krassova, MD. They have used a natural pathway to recycle the molecules and keep them in the circulation longer. Scientists at CSL Behring are also devising longer lasting FVIIa and FIX products. Together, these modifications to FVIIa, FVIII, and FIX may represent the next step in changing how hemophilia is managed. The clinical trials are evaluating whether patients can manage with less frequent dosing regimens.

Bypassing Inhibitors

When standard treatment regimens no longer work, people with hemophilia face a particularly challenging situation. And, almost one-third of people with hemophilia—up to 25% with hemophilia A and 4% with B—will develop inhibitors. Strategies for treating hemophilia in this population focus on the clotting cascade. Although FVIII and FIX are generally required for clotting—this is why treatments for hemophilia A and B focus on replacing these missing proteins—when inhibitors develop, other routes to forming a blood clot can be targeted using bypassing agents.

Besides the Bayer, CSL Behring and Novo Nordisk teams developing improved FVIIa molecules, another team, led by Joerg Schuettrumpf, MD, a researcher at the Institute of Transfusion Medicine and Immune Hematology of the German Red Cross Blood Donor Service Baden-Wuerttemberg-Hessen, is addressing the challenge of inhibitors by using altered FIX molecules called variants that trigger a pathway that causes clotting without requiring FVIII.  So far, the variants have promoted clotting in mice with inhibitors to FVIII. Schuettrumpf’s team plans to eventually incorporate these molecules into existing treatment strategies for people with inhibitors.

Slow-motion Clotting Cascade

Rodney Camire, PhD, associate professor of pediatrics at the University of Pennsylvania in Philadelphia, and Lacramioara Ivanciu, PhD, a post-doctoral fellow in Camire’s lab and recipient of NHF’s Judith Graham Pool Postdoctoral Research Fellowship, study another detour along the clotting highway, using “zymogen-like” variants. Zymogens are enzymes (molecules that speed up chemical reactions) that need a nudge before rushing into action. Slowing them down means the molecule will last longer. Camire’s molecule of focus, FXa, another protein along the clotting pathway, has a very short half-life. The variant Camire’s team developed lasts longer than the original molecule, using an alternate route on the clotting pathway that avoids the road leading to FVIII.

Local Research, Global Impacts

A thematic pathway woven through the workshop was simple, yet important: local research leads to global impacts. Developing factor that lasts longer, for example, could bolster access for the two-thirds of the patients throughout the world who cannot receive regular prophylaxis, says panelist Perseus Patel, a senior at the University of California–Los Angeles. “You can give those kids a chance to take factor, do physical therapy, get their joints relatively strong enough so they can live average lifestyles and now you’re really changing an entire generation,” he says. Patel moved to the US from Mumbai, India, when he was 10 to get treatment for severe hemophilia A. He started a self-infusion camp in India as a high school student. He represents one member of an increasingly global community of innovators, researchers and providers who work toward improving the lives of people with bleeding disorders worldwide.

Researchers attending this year’s workshop reflected on progress made since 1996, the workshop’s first year. “We’ve covered many different topics over these last 16 years, going from protein therapies to a lot of focus on gene therapy—and a lot of ups and downs in gene therapy over the years. And now we’re pretty well equally distributed with very exciting activities occurring in both protein engineering and gene therapy,” says meeting Co-Chair Glenn Pierce, MD, PhD. Researchers credited the patients for their active participation in clinical trials and ongoing advocacy efforts. After 41 passionately delivered research presentations, reflections from the Patient Perspective Panel “remind us of why we come to work every morning,” says Pierce.

The workshop was organized by Glenn Pierce, MD , PhD (Senior Vice President in Global Medical Affairs at Biogen Idec in Weston, Massachusetts), Kathy High, MD (Director of the Center for Cellular and Molecular Therapeutics at the Children’s Hospital of Philadelphia in Pennsylvania and Investigator at Howard Hughes Medical Institute), David Lillicrap, MD, (Professor of Pathology at  Queens University, Kingston, Canada),  Thierry VandenDriessche, PhD (Professor, Director Department of Gene Therapy & Regenerative Medicine at the University of Brussels, Belgium) and Steven Pipe, MD (Associate Professor of Pediatrics and Pathology, Director Division of Pediatric Hematology and Oncology at the University of Michigan).