Package Deal

Hemophilia gene therapy trials start delivering the goods
Author: Sarah Aldridge

Update, November 2014: The St. Jude/UCL gene therapy trial now has data from all 10 participants, to be published in the New England Journal of Medicine. The six people treated with the highest dose of factor IX (FIX) have experience a more than 90% reduction in bleeds and in their use of factor product. Their median annual number of bleeds dropped dramatically from 15.5 to 1. Of the seven subjects who were on prophylaxis before the trial, four have discontinued it. Researchers are still experimenting with a faster process to manufacture the genetically engineered virus that carries the FIX gene into the body. For the study it took six months to make enough for the 10 participants.

Original article: When it comes to mailing packages using ground transportation, the US Postal Service has stiff competition. Other companies also promise to deliver parcels quickly, safely and in one piece. When it comes to gene therapy trials for people with hemophilia, different vehicles are competing to carry the packages. Currently, viruses lead the fleet, delivering healthy genes to cells that can begin producing normal levels of factor protein.

After a nearly 15-year hiatus, many believe gene therapy is about to deliver the goods. Preliminary results from a clinical trial in the UK show that several patients with severe hemophilia B, or factor IX (FIX) deficiency, no longer need factor product. A few infuse less often. Following on its heels are three clinical trials in the US using gene therapy for patients with FIX deficiency.

But gene therapy is not quite ready for routine administration. Clinical trials for hemophilia A, or factor VIII (FVIII) deficiency, are a few years down the road. Those for FIX are testing ways to manage immune responses. And none of the gene therapy clinical trials has yet to include children. Yet all are vying to create the perfect package deal.

Creating the perfect package

Gene therapy entails correcting a genetic defect by inserting healthy genes into living cells, which then produce functional levels of the missing or deficient protein. Hemophilia is a good candidate for gene therapy because it is caused by a genetic mutation in either the FVIII or FIX gene.

The FIX gene is easier to work with than the FVIII gene for several reasons. “It’s not an enormous gene,” says Paul E. Monahan, MD, attending physician, Comprehensive Hemophilia Diagnostic and Treatment Center at the University of North Carolina at Chapel Hill. That’s in contrast to the FVIII protein, which is large, is coded for by a sizable gene and travels a complicated path in cells before it can be secreted into the bloodstream, he says. Although FIX is normally made in the liver and modified by liver cells, other tissues can make it following gene therapy, Monahan says.

Another advantage of FIX is related to inhibitor, or antibody, formation. “It doesn’t seem to set off alarms with the immune system quite as easily as FVIII does,” says Monahan. Lastly, FVIII is typically bound to von Willebrand factor. “So all the things that make it a little bit more complex to make as recombinant protein also make it more complex to express in gene therapy,” he says.

Viruses provide speedy delivery

Viruses are some of the most reliable vehicles, called vectors, to carry healthy genes into the body. Like express mail, they are sent to a specific site. They then latch onto receptors on cells, taking over the cells’ machinery and instructing them to produce factor protein. By genetically altering these viruses, investigators have been able to insert the desired gene, which is then carried to the site where it is produced.


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Currently, the adeno-associated virus (AAV) is out-competing­ other courier viruses to carry the FIX gene. Of its 11 serotypes, or subtypes, AAV serotype 8 has an affinity for the liver—that’s where FVIII and FIX proteins are normally produced.

AAV-8 has several other advantages. “By itself it does not cause disease in humans,” says Ulrike M. Reiss, MD, director of clinical hematology in the Department of Hematology at St. Jude Children’s Research Hospital in Memphis. It needs a helper virus for that. “The vector made from AAV-8 usually does not integrate in the chromosomes, so you have a lesser chance of it entering human DNA.”

The procedure is simple: The patient is given a single infusion of the vector-containing gene through a peripheral vein in the arm.

FIX gene therapy clinical trials

2013 could be called the year of FIX gene therapy trials. Three clinical trials are being conducted on adult patients with severe hemophilia B. Each is slightly different, and the combined data will help researchers fine-tune the process of gene therapy for hemophilia. All of the studies are using AAV-8 as the gene delivery vector. All are currently enrolling patients.

St. Jude/UCL assembles the package prototype

St. Jude Children’s Research Hospital and University College London (UCL) collaborated on a gene therapy trial for FIX patients that paved the way for the current trials in the US. Results from the first six patients will be published in The New England Journal of Medicine sometime this fall; results from the other four patients, treated with the highest dose on this trial, will be published in the next few months.

Prior to the trial, the first six men in the study were on various prophylaxis regimens, says Reiss. Now, four of them have stopped. “The two patients on the highest dose now express factor levels between 3% and 6%,” she says. That means they moved from a diagnosis of severe hemophilia (<1% of normal clotting factor) to moderate hemophilia, which is 1% to 5%, according to the National Hemophilia Foundation. “Hardly any of them have had a bleeding event,” Reiss says. Some have returned to favorite outdoor activities, including cricket, hiking and soccer. One ran a marathon.

The patients in the low- and intermediate-dose groups have expressed factor levels of 1% to 2% and 2% to 3%, respectively. Now, three years after the trial ended, two of them remain off prophylaxis; the other two use it, but on extended intervals, says Reiss.

Two of the high-dose subjects developed transaminitis, an inflammation of the liver, about eight weeks after the infusion. The patients were not sick, but before they could develop symptoms, they were treated with a tapering dose of the oral steroid prednisolone for a few weeks. “They had an immediate response to that,” Reiss says. “The liver function tests normalized within a few days and the FIX levels were preserved.”

This immune response was likely caused by antigens, or proteins, on the viral capsids. Capsids are the vessels that contain the AAV-8/FIX genetic material, and carry and deposit it into the host cell. “Most viruses make an overabundance of the capsid shell,” Monahan says. Of the vector particles that were infused, nearly 90% were empty capsids, which triggered the immune system to label them as foreign and mount a defense.

For the new study in the US, enrolling patients this fall, the capsid antigen issue will be corrected. “We will add an additional purification step at the end of the production line to remove the empty capsids,” Reiss says.

Another concern is the risk that the AAV-8/gene package could wind up in reproductive cells. That’s why sperm cells are assessed. “In the patients we have infused so far, the vector cleared from the semen within about three weeks,” says Reiss. Study participants are asked to use contraception or a barrier method until at least three negative tests are confirmed, she says.

UNC Chapel Hill modifies the contents

Patients are being recruited for a collaborative study by the Gene Therapy Center at UNC–Chapel Hill and its strategic biotechnology partner, Asklepios Biopharmaceutical Inc., with support from the National Institutes of Health. The vector will be infused at the Mount Sinai Medical Center in New York and at the University of California-Davis in Sacramento. Subjects will be followed by their hematologist.

The study builds on the findings of the St. Jude/UCL team. Instead of having 80% to 90% empty capsids, the vector preparation will have only 5% to 10%. “Our hope is to avoid the immune response observed previously,” says Monahan. His other hope is that by using the “FIX Padua” variant of the protein, which has increased potency, levels of protein production similar to those already demonstrated in previous trials will result in five to six times enhanced blood clotting activity.

“That might be associated not just with having a low risk of spontaneous bleeding, but hopefully no joint bleeding at all,” he says. Ultimately, Monahan believes the FIX Padua variant will sustain even higher factor levels. “We hope to be able to escalate to a dose that gives us between 10% to 40% activity,” he says.

Children’s Hospital of Philadelphia rewraps the parcel

Katherine A. High, MD, is the director of the Center for Cellular and Molecular Therapeutics at the Children’s Hospital of Philadelphia (CHOP), where another gene therapy trial is being conducted. High expects to enroll nine to 15 subjects with baseline FIX levels of 2% or less.

Like the other trials, patients diagnosed with hepatitis C virus (HCV) and/or human immunodeficiency virus (HIV) are not automatically excluded. “We’re looking for individuals who are HCV RNA viral load negative,” High says. Those subjects’ bodies are not actively producing HCV particles. If they’ve been on HCV therapy and cleared the virus, they are eligible to participate.

“It’s all right if they’re HIV positive, as long as they’re stable on a HAART (highly active antiretroviral therapy) regimen or have an adequate CD4 cell count,” says High. However, as with the other trials, patients who test positive for AAV antibodies will be excluded.

The CHOP investigation is a dose escalation study. “If we do find a dose that gives levels of at least 3% in each of the first two subjects, then we’ll enroll a total of five subjects at that dose before going up to the next dose,” says High. Patients will be monitored closely. “They come back once a week for blood drawing for the first 12 weeks,” she says. That’s followed by monthly blood tests for the next year.

The study at CHOP is unique in the type of DNA used. “The other trials are using a piece of double-stranded DNA—it’s called a self-complementary vector design,” says High. In contrast, she and her colleagues are using a single-stranded vector. “The advantage is that it’s easier to manufacture, with better yields. It also results in a more homogeneous [pure] product.”

Detours and obstacles

AAV is a common virus that infects humans, so the older you are, the more likely you have been exposed to it. Once identified as “nonself,” the immune system produces antibodies to fight AAV if it is encountered again. “Those antibodies can circulate in high enough titers that if they tried to do a gene therapy application using AAV-based serotype, it’s possible that our immune system would wipe out the delivery system,” says Steven Pipe, MD, pediatric medical director, Hemophilia and Coagulation Disorders Program at the University of Michigan in Ann Arbor. “We wouldn’t get the same efficacy.”

Reiss estimates that because up to one-third of the US population has antibodies to AAV-8, there is a need for other serotypes to serve as viable vectors. “In nonhuman primates, the AAV5 vector containing the FIX gene has been used and has worked well,” she says.

Although the UK patients from the St. Jude/UCL trial have maintained stable FIX levels for three years, long-term stability is an unknown. The reality of re-dosing is on Pipe’s mind. “If they were re-dosed, would they have the same efficacy with another exposure, or would the immune response be primed?” he asks.

So far, no children have been enrolled in the gene therapy studies for several reasons. “We will observe the adults for a couple more years to ensure that the vector does not integrate into the DNA, posing a risk for cancer development, which has been reported with other types of vectors,” says Reiss. Also, it is uncertain if children will display long-term FIX expression. “Because their livers are still growing, there may be a small chance that the FIX expression decreases over time,” she says.

Because these phase I trials are evaluating safety and efficacy, inhibitor patients must be excluded, says Pipe. But subsequent phases may be more open. “I believe it would be entirely plausible to pursue a protocol that included inhibitor patients, even with the existing applications, and see if it did have any tolerizing effect,” he says.

In hot pursuit

Gene therapy trials for other factor deficiencies are down the road. Researchers are using an AAV vector for FVII and for FVIII. “The first FVIII clinical trials using an AAV vector could be tried in humans within a couple of years, so it’s coming close,” Reiss says.

The financial support for research and development, along with patients’ commitment to clinical trials, has been the impetus to create the perfect package to deliver healthy genes. “We’re going to have some truly transformative ­therapies that are going to come out of all this activity,” Pipe says.