Gene therapy

Hemophilia Gene Therapy Breakthroughs

Gene therapy continues to emerge as hemophilia treatment
Author: Katarina Maria Grande

Hemophilia researchers from across the globe gathered at the National Hemophilia Foundation’s (NHF’s) workshop on Novel Technologies and Gene Transfer for Hemophilia February 22–23, 2008, at The Children’s Hospital of Philadelphia to discuss research, ask questions and learn about the field’s latest findings. Biochemists had the chance to see progress on a clinical level, while physicians received a glimpse of the basic science discoveries.

As gene therapy continues to emerge as a potentially viable treatment for hemophilia, researchers presented a range of findings on the subject. These included mechanisms using viruses as transporters, or “vectors,” for therapeutic gene delivery; viral vector composition; immune responses to vectors; vector delivery systems; bioengineered stem cells and nonviral methods for gene delivery.

“There’s even more distinction between presentations at this meeting compared to the last two or three. Rather than focus on a number of types of methodologies, we’ve actually increased the diversity of approaches. That tells us that this is more difficult than we anticipated,” says Glenn Pierce, MD, PhD, co-organizer of this and the previous workshops, of Bayer HealthCare, Berkeley, California. Pierce and the other organizers presented questions to the research community. Some were answered in the presentations; others will drive the research of the scientists returning to their laboratories.

The Goal

In theory, it’s a simple idea: Overcome a defective gene by plugging in a healthy one. The cells with the new gene then make enough error-free copies of the gene to produce a desired function, such as production of clotting factors. Gene transfer therapy seems to offer the perfect solution for diseases like hemophilia that are caused by a mutation on a single gene—the gene that codes for factor VIII in hemophilia A and factor IX in hemophilia B.

The severity of hemophilia is measured on a gradient scale; the less clotting factor present, the more serious the disease. If the amount of intact factor VIII-producing cells increases by even a small percentage, the effect on daily life could be substantial—less bleeding into joints, less frequent prophylactic injections and less worry. “We know from many years of experience that a small amount of clotting factor—factor VIII or factor IX—will prevent the acute and chronic consequences of factor deficiency,” says David Lillicrap, MD, of Queen’s University in Kingston, Canada, a workshop organizer and one of many hemophilia experts who attended the meeting.

Rather than subject people with severe hemophilia to regular injections of bioengineered clotting factor, it would be advantageous to push—and sustain—factor levels above the threshold. Although this has been achieved through gene therapy in hemophilic dogs, people with hemophilia have yet to experience equal success.

The Research

Viral Vectors

Though hemophilia is caused by a defect in a single gene, its simplicity stops there. The gene that codes for factor VIII is large and cumbersome. It requires a cleverly developed vehicle to transport it into cells. By engineering a virus to “infect” cells with a healthy factor VIII gene, researchers have been relatively successful. Dirk Grimm, PhD, of the University of Heidelberg in Germany, is working with an adeno-associated viral (AAV) vector as a carrier for the factor VIII gene. As a small, nondisease-causing virus capable of infecting many cell types, AAV is an attractive vector candidate. “We’re basically trying to take natural evolution one step further, modifying the natural virus for therapeutic purposes,” Grimm says. He is altering an AAV gene by borrowing genetic material from its relatives, attempting to create one that infects liver cells that produce factor VIII.

The use of viral vectors, however, has potential problems. If the virus can integrate itself into the genome, becoming a permanent presence in the body, it could be dangerous. “The biggest safety problem in the past has been that these vectors can cause cancer,” says Basil Golding, MD, of the US Food and Drug Administration. Though rarely reported in studies using integrating viral vectors, it can happen. The virus’ hitchhiking DNA inserts itself into the target cell genome, potentially causing a mutation if inserted where it doesn’t belong. However, most of the DNA that the AAV delivers does not integrate into the recipient’s genome. On the other hand, Thierry VandenDriessche, PhD, University of Leuven in Belgium and workshop co-organizer, has worked with both integrating and nonintegrating vectors to deliver coagulation factor genes. He has found benefits to both DNA delivery systems, but indicated that it is too early to identify one approach as the best. 

The other barrier facing viral vectors is the wrath of the immune system. Even if an adeno-associated virus does not cause disease, the body still considers it foreign and the immune system mounts an attack. “The delivery of DNA itself does not seem to be the problem anymore. Safety seems to have become a bigger issue than efficacy, which used to be a big issue in the past couple of years,” says ­Katherine A. High, MD, The Children’s Hospital of Philadelphia, a workshop organizer and pioneer in understanding this immune response.

Nonviral Vectors

Instead of using viruses to insert the factor VIII gene into cells, Michele Calos, PhD, of Stanford University in Palo Alto, California, injects DNA directly into a vein near the liver of hemophilic mice. An enzyme helps sneak the DNA into the body’s genome—not randomly, but in consistent locations. This induces the activity of the gene while reducing the carcinogenic risk. “Simplicity is an advantage when you’re doing something like this. We have a good chance to not encounter some really difficult biological roadblocks,” says Calos. In an experiment on mice with hemophilia B that were injected with factor IX, 20% of their liver cells made the missing factor protein. “These are definitely therapeutic levels,” Calos reports.

Since Calos has effectively demonstrated that her nonviral methodology works in small animals, her lab is running experiments on pigs to see if results are similar. Soon, the experiments will be conducted on monkeys, the final model before trying the process on humans. Although the DNA embeds itself safely into the genome, the physical act of injecting DNA into the liver vein is being assessed for safety. There is concern that the method could potentially encourage cancerous growth, since the cells around the injection site become agitated and respond by dividing rapidly. Although tumor growth as a result of hydrodynamic delivery has never been observed in the mice models, Calos’ team will closely monitor the process in larger animals. 

Stem Cells

Another version of gene therapy now in development uses stem cells. Rather than injecting viral vectors into the body, this method first plugs the gene into hematopoietic stem cells (those that have differentiated enough to become bone marrow cells), which can then morph into various blood cell types. Next, researchers insert the cells into the body, like a miniature version of organ transplantation.

“We’ve got a two-pronged approach: modifying the factor VIII to optimize its expression properties and, at the same time, combining it with a technology that has been proven to work clinically to cure a genetic disease,” says Christopher Doering, PhD, of Emory University School of Medicine in Atlanta. Doering’s lab found that a genetically modified version of a pig factor VIII gene is much more effective at secreting the protein than its human counterpart. His research team plugged this gene into mouse stem cells, transplanted them into mice and found consistently high levels of factor VIII in the blood. A year later, the mice were still producing their own factor VIII at normal levels.

With this type of transplantation, the immune system must first be weakened. Otherwise, it will do its job of recognizing the new cells as foreign invaders that need to be destroyed. Of course, altering the immune system, even temporarily, has its risks. Doering’s lab continues working to find ways to transplant the modified stem cells with the least amount of immunosuppression.

Bioengineered Clotting Factors

As many scientists focus on future therapies for hemophilia, others are perfecting what already works—therapeutic injections of the missing clotting factor. The aim of this research is to make clotting factors with longer half-lives, decreasing the frequency of injections. 

Therapeutic factor VIII is no longer derived mainly from transfused blood. Instead, scientists use cultured cells to make copies of the protein. This is achieved by introducing the gene that codes for factor VIII into well-characterized­ animal cells cultured in the laboratory. Within one day, a cell will pick up the DNA and integrate it into its own genome. After a few billion cells are made, they are transferred to fermentation vats where functional factor VIII or factor IX is produced, ready for clinical use after protein purification. As a natural process of the body’s regulation of the clotting cycle, enzymes known as ­proteases constantly gobble up factor VIII, making its lifespan in the bloodstream short. That’s where the University of Michigan’s Randal Kaufman, PhD, and colleagues come in. They’re altering factor VIII’s genetic sequence to make it last longer in the bloodstream. To do this, Kaufman’s lab is examining the pathway by which factor VIII is regulated and then destroyed.

Kaufman’s lab found that stressed-out cells don’t fold the large and clunky factor VIII protein very well. Misfolded factor VIII causes a mess in the endoplasmic reticulum (ER), the cell organelle that handles protein folding. Kaufman and his colleagues have found a way to reduce intracellular stress by feeding the cells antioxidants. “Antioxidants help protein folding in the ER,” Kaufman says of his results. As the amount of properly folded factor VIII increases, more of it is able to exit the cell, where it can be used for protein replacement or, potentially, for gene therapy.

Numerous other presentations focused on altering factors VII, VIII and IX to improve upon the normally existing proteins. These modified proteins have features that can be used for improved protein therapy and, in some cases, gene transfer. The excitement generated by the molecular biologists and protein biochemists could be felt at the meeting, as some of the most promising protein versions are on the pathway toward clinical testing in patients. 

Clinical Trials

Clinical trials are pushing forward, slowly and deliberately. They begin with a few human subjects receiving a low dose of gene vector. The subjects are monitored closely, often one at a time. Amit Nathwani, MD, PhD, of University College London, is preparing for a clinical trial for hemophilia B that will assess the safety of a vector designed by colleagues at St. Jude Children’s Research Hospital in Memphis, Tennessee. In his large animal study done in macaques, Nathwani says, “Expression in these three animals is between 8% and 13% at 18 months after delivery.” These results are similar to those obtained a few years ago by Pierce, High and colleagues at Avigen and The Children’s Hospital of Philadelphia. The prospect of duplicating the results in patients who will undergo the therapy is promising. Maintaining 18 months of factor VIII expression in a human patient would signify a major breakthrough in clinical hemophilia research. High’s group has initiated another clinical trial using a slightly different AAV vector than Nathwani’s. It will assess the use of transient immunosuppression to obtain multiyear cures, as shown in mice, dogs and macaques.

Pathways to Improved Treatment

As clinical scientists get closer to improved treatment with each experiment, teams of researchers are working behind the scenes on a smaller scale. The immunologists are figuring out how a single component of the immune system works to reject cells that contain viral vectors, the biochemists are elucidating the pathway by which factor is expressed and the geneticists are studying how rebuilt genes work. While this research is less showy and more technical, it is crucial to the ultimate understanding of hemophilia as a disease.

“Although the concept of gene transfer and some of these other modifications are relatively straightforward, the pragmatics and the biology are anything other than straightforward,” Lillicrap says. “We need to focus our understanding on the basic mechanisms. Unless we can do that, we’re not going to be able to move ahead with confidence and success.”

The pairing of a comprehensive understanding of each component of hemophilia with macro-level research, such as clinical trials, could ultimately be the key to a cure. Although there are no clear-cut answers yet, the field is expanding as different methodologies for gene therapy are tried and tested.

“There is now increasing diversity,” adds Pierce. “For the individual with hemophilia, it’s a very exciting time. The current treatments, which are very safe and effective, will get even better through research on modified clotting factor proteins and improved transfer of clotting factor genes.”