Success of gene therapy in hemophilia B serves as ‘seminal event’ for researchers, patients

A study published in The New England Journal of Medicine in December 2011 highlighted a potential breakthrough in gene therapy, documenting how British researchers used the technique to successfully treat six patients with hemophilia B.

Katherine A. High, MD, the William H. Bennett Professor of Pediatrics at the University of Pennsylvania School of Medicine, as well as an attending hematologist and director of the Center for Cellular and Molecular Therapeutics at The Children’s Hospital of Philadelphia, led a round table discussion during the HemOnc Today section editors’ retreat April 27-28 to further explore the study results, their potential implications and the future of gene therapy in hemophilia B.

A transcript of that round table discussion follows.

Dr. High: Hemophilia B has long been a goal for people interested in gene transfer. The reason for that is, most people with the disease have circulating levels of Factor IX of less than 1%, but if you could just raise the levels to the range of 5%, you would convert the person’s disease from severe to mild. This has been easy to do in mice and hemophilic dogs but surprisingly difficult to do in humans.

The first trial, in which this kind of vector was infused into the liver, was reported in 2006. The patients at the highest dose got therapeutic levels of Factor IX in the range of 10%, but they only lasted for about 6 weeks and they disappeared at the same time that the patients developed an elevation in liver enzymes.

Results of a more recent trial, jointly sponsored by St. Jude Children’s Research Hospital in Memphis and University College London and the Royal Free Hospital in London, were published in December in The New England Journal of Medicine.

One of the important changes to the protocol in this trial was that they stipulated that if they saw a rise in liver enzymes or a drop in the Factor IX level, they would start the patient on high-dose steroids and give a course of steroids for 4 to 8 weeks to try to protect the patient from what appeared to be an immune response to the viral vector capsid and rescue the transduced cells and keep them from being destroyed by the patient’s own immune system.

Trial results

Dr. High: Just to backtrack, what they observed in the first trial was something that looked a little bit like an autoimmune hepatitis but, in fact, it was alloimmune hepatitis. It was a patient mounting an immune response to the vector capsid.

In the trial in London, in the first two dose cohorts, there was long-term expression at low levels around 2% of normal, but for people with hemophilia, moving from less than 1% to 2% is clinically meaningful in terms of how much they need to rely on clotting factor concentrates. But in the high-dose group, the patients initially had levels of around 8%, and that would really make them mild hemophiliacs, and that would be a very important change.

One of those people did develop this transaminase elevation. It was unclear at first what was going on because it happened much later than had been observed in the previous trial, so there was some confusion about whether this was something that should trigger the steroids, but he was eventually put on that. He leveled out at around 2%.

The other patient in the high-dose cohort ended up with a level of about 6%, so he is a very happy individual now. He apparently plays rugby on the weekends, which is an unusual leisure activity for someone with severe hemophilia, but I guess he used to dose himself with factor before he played. Now he doesn’t have to.

This is a very important landmark for people with hemophilia.

One of the ironies of this is that it is coming to fruition at the same time that the major manufacturers of clotting factor concentrates are in the late stages of investigation with new, longer-acting clotting factor concentrates. So it is expected that these will go to licensing within the next year or two.

The half-life of these new, longer-acting Factor IX products is on the order of 90 hours, instead of the 12 to 24 hours of currently available clotting factors, and it is expected that patients will only need to dose themselves about twice a month.

The question is: Where will these different types of therapies fit in, and will they have any ramifications for the 80% of the world’s hemophilia population who currently do not have access to optimal treatment? The treatment we use today, infusion of recombinant protein, costs about $300,000 a year if you do prophylactic infusion in an adult male. Consequently, people in North America and Western Europe are managed that way but, in most other countries, the consumption of factor is a good deal less.

How will these new therapies go forward? Will these results be replicated, and can they be extended, for example, to Factor VIII? Those are some of the questions that people in the field are thinking about right now.

Costs are unclear

Dr. Green: Is there an estimate about whether the new, longer-lived forms will be more expensive?

Dr. High: I would expect, based on previous experience, that the manufacturers will advance the opinion that they’ll be making a better product available for a similar cost so the total yearly cost to the patient would not rise, but it would be easier and more convenient for the patient.

Dr. Green: What is the cost of the adenovirus treatment?

Dr. High: Because there are no adeno-associated virus (AAV) products on the market, it is very difficult to predict. The cost of goods for the manufacturer of recombinant AAV goes down every year. At Children’s Hospital, we have a trial running for an eye product and, of course, the doses of that are logs lower than for a hemophilia product, but I know in that case the cost of goods is less than the cost of a single dose of clotting factor in an adult male. I believe the cost of goods will not be the prohibitive expense, but, of course, there’s a lot that needs to be done in terms of development of these promising results.

Dr. Bertino: Have they really looked at the integration sites and it is also in bone marrow stem cells or just liver cells?

Dr. High: This vector is infused intravenously, so the infusion procedure is easy and convenient for the patient. AAV is stabilized predominantly in a non-integrating form, so in terms of what the FDA and other regulatory agencies expect about analysis of integration sites, it’s less challenging than it is for retroviral and lentiviral vectors, which rely on integration in order to work. However, as you have mentioned, whenever you put foreign DNA into a cell, you run some risk that there will be integration events, and at least in animal livers, this has been observed with the types of doses that are being used in the hemophilia trials.

For the subjects who have been enrolled on these trials, liver biopsy is not part of the trial protocol, so there has been no analysis of the target organ in human subjects. In terms of vector shedding in the body fluids, that’s been followed and all of the body fluids have been positive for some short period of time after the vector infusion, but nothing is persistently positive.

AAV immunity

Dr. Boxer: One-third of the adult population has immunity to adeno-associated virus, so what’s going to be the outcome of these patients, and are other vectors being developed?

Dr. High: Most of us have been exposed to the wild-type virus from which this vector is engineered, usually during childhood. It doesn’t cause any known illness, but something like 30% of the population has circulating antibodies to AAV, and if you try to infuse the vector through the bloodstream, those antibodies are powerfully neutralizing and those people won’t get a therapeutic effect. Right now, those people are excluded from trials that rely on intravenous infusion. Studies have looked at plasmapheresis as a way to get rid of the antibody transiently, and there are drugs, such as Rituxan (rituximab; Genentech and Idec Pharmaceuticals), that can get rid of B cells. Those issues will need to be addressed in further studies, but right now those people are excluded from the trials. That’s a big proportion of the population, so people will be motivated to figure out a way to include them.

Dr. Jacob: In the presentation of your group at the American Society of Hematology Annual Meeting in December, I got the impression you are using an adeno-associated virus 8 now. My impression was that very few people actually have been exposed to that, whereas your previous studies were with adeno-associated virus 2, which was a common problem of immunity. Have I got that wrong?

Dr. High: Between the first trial and the second trial, a different AAV vector was used, a switch as you said from serotype 2 to serotype 8. The prevalence of neutralizing antibodies in the population is lower for 8, but it is still fairly substantial. These capsid structures are pretty highly conserved from one serotype to another, so the antibodies cross-react fairly extensively. Even though it’s a lower number than for AAV 2, it’s still fairly substantial. The other realization has been that, we thought at first that only fairly high titers would prevent transduction and it’s become clear that even modest titers will prevent transduction. That’s part of the reason for that change in numbers of how many people are prevented from getting the therapy by the antibodies.

Impact of hepatitis

Dr. Rao: This presentation at ASH was a seminal event from the perspective of patients who have hemophilia and for the entire field of gene therapy. It made the point that we can achieve gene therapy, and it can be effective.

One specific question: Many of these patients are positive for hepatitis C virus (HCV) or hepatitis B virus. The older patients probably have a higher incidence, and this has been a limitation in current trials in selecting individuals for gene therapy. Is that going to be an issue and, if it is, how would that get handled?

Dr. High: That’s a very good question, and it’s a difficult issue for the trials. In the first trial, using AAV2, because we had not expected this alloimmune hepatitis, we enrolled subjects who were HCV-positive so they could be HCV RNA viral load-positive and still enter the trial, and no adverse events were seen, including in these people. Now, because we know that a certain percentage of people who get that high dose are likely to need a course of prednisone for 4 to 8 weeks, HCV RNA viral load-positive patients are excluded because people are worried about putting them on steroids for 4 to 8 weeks. The question is whether it will be possible to fashion a regimen that will be effective against this transient immune response against the capsid but that will be safe for people who are HCV RNA viral load-positive. What we think, after conferring with hepatologists, is that for individuals who clear on the new anti-HCV regimens, it will be possible to allow these people to enter the trials.

Dr. Rao: There will eventually be a place for both the newer products that are coming down the pike, as well as gene therapy, simply because there will be a huge population of hemophiliacs who will not be candidates for gene therapy. There will be an indication for both.

Dr. High: Yes, and your question points to something important. There is success in a well-defined patient group, but it enables us to both extend that result and begin to work on some of these other problems. What do you do about people who have antibodies to AAV? What do you do about people who are HCV RNA viral load-positive and can’t go on steroids? Is there another regimen that you can give them? A lot of these people go to transplant. They get immunosuppression. There should be a regimen that is usable in that situation. We just have to figure those things out.

Factor IX vs. Factor VIII

Dr. Coleman: Why did you choose Factor IX instead of Factor VIII?

Dr. High: The AAV vector can accommodate a transgene of about 5 kilobases (kb). For Factor IX, the coding region alone is about 1.5 kb and the coding region of even B-domain deleted Factor VIII is 4.4 kb, so you can put it in there, but you have to engineer a very small promoter and a very small intron and so forth to get that Factor VIII in.

Dr. Rao: Most hemophiliacs have Factor VIII deficiency, not Factor IX deficiency. Because there are limitations based on the size of Factor VIII molecule, there are some alternative methods that have been proposed. One is using megakaryocytes and platelets to deliver Factor VIII, driven by a platelet-specific promoter, in which case they are actually delivering Factor VIII to the site of the bleed, since platelets accumulate there and release their contents. Moreover, should gene therapy use Factor VII-a expression to correct the hemostatic abnormality given that expressing Factor VIII has had some limitations?

Dr. High: It has been possible to make an AAV vector that expresses Factor VIII, and I suspect you will see a clinical trial of that begin within the next 2 years. In terms of the other strategies that you discussed, I’m a hematologist, so I was always attracted to the idea of trying to put one of these genes into a hematopoietic cell, including a megakaryocyte. What prevents people from moving to that right away is that, for all of the strategies so far that rely on genetic modification of autologous hematopoietic cells, you have had to do some sort of myeloablative conditioning to make a niche for the transduced cells. That has been unattractive to hemophilia patients. Everybody is still looking for a solution, medicine in a bottle that you can take down off a shelf and inject and you’re finished, and bone marrow transplantation is a little more involved than that. However, a good niche for that strategy may be, as you mentioned, inhibitor patients who have failed on immune tolerance induction because, as you say, if you can package the Factor VIII into granules within a platelet and they release the granule contents at the site of a bleed, you may have a way to get around an inhibitor. I wonder if that’s the place to begin with that strategy.

Dr. Rao: How about the approach expressing Factor VII-a?

Dr. High: We have done a lot of work with VII-a. The challenge for moving that into the clinical arena will be that it is likely that you are going to need a regulatable promoter, so it would be a promoter that can raise the levels of VII-a up or down based on an oral drug. The reason for that is, although it’s all right to have constitutive expression of Factor VIII or Factor IX, people may not feel the same way about constitutive expression of Factor VII-a.

Future of gene therapy

Dr. Gordon Smith: Where are we going to be 2 years down the line in terms of gene therapy?

Dr. High: It’s difficult to answer that question. There are no licensed gene therapy products at this point in ICH countries, so the regulatory pathway is not entirely clear. There are products that have been through phase 3 or that are approaching phase 3. It may be that, within a couple of years, we will at least have a road map of what is required for licensing. The product we use to treat Factor IX deficiency was licensed on a trial of around 50 patients. It’s possible that large trials won’t be required, but until there are licensed gene therapy products, it will be difficult to comment on that.

Dr. Jacob: You talk about the immune suppression of your vector, which you evidently can handle fairly readily. Are there other suspicions that, without an immunologic attack, your gene is just going to peter out anyway? Is there any evidence that occurs in animal trials?

Dr. High: The data in dogs have yielded a surprising finding. Ten years after an injection, these hemophilic dogs are still expressing Factor IX at the levels that you observe at about 6 weeks when they hit a plateau. I find that very surprising. I would have expected the levels to slowly decay. It will be important to see what happens in these first subjects, whether they maintain the levels for 2 years — which they seem to have done already — or longer than that, and whether re-administration is going to become an issue. For that reason, what people would like to do is be able to give enough vector to get an initial level in the range of 20% to 40%, so you’d have a comfortable margin even if there were a slow decline.

Dr. Coleman: One of the reasons you’re so successful is the liver cells just don’t turn over as rapidly as hematopoietic cells because it’s been tried. They tried to put in the NDR1 gene so they could deliver high doses of chemotherapy, but they were never very successful. The plating efficiency was poor. Liver cells, if you have damage, can divide, but I gather they don’t turn over so quickly. Do you know the half life or mitotic rate?

Dr. High: I have tried to find out from hepatologists what they expect the life span of a hepatocyte to be and I have not been able to find that out. You are right, it could be addressed experimentally.

Dr. Coleman: It would be very interesting to see if the daughter cells also carry the gene.

Dr. High: Certainly based on animal studies, we believe the daughter cell will not carry the donated gene, but then I cannot explain why dogs would still have the same level 10 years later.

Dr. Coleman: You might want to consider taking out a large portion of the liver from the dog and let him regenerate and see what happens. I don’t know whether you’ve done those experiments.

Dr. High: We’ve done those experiments in mice. What happens then is, if you’re sitting at about 100%, you remove 80% of the mouse’s liver. As the liver regenerates, you do not get the gene passed to the daughter cells and the levels go very low.

A more affordable alternative

Dr. Jacob: Assuming that one ultimately could get this therapy approved, if this is one intravenous infusion of a product, would this not be an incredible situation for poor countries — one injection compared with no possibility of the $300,000-a-year kind of recombinant type of therapy?

Dr. High: If the data continue the way they are going now, this could be a situation where you could make a single intervention and then return the patient to his home setting. He would not need a great deal of follow-up and he would be much safer than he is now if he is a person who bleeds and has to solve difficult transportation issues to get to a place where he can get the factor. It seems to me that it would certainly have a great deal to offer for that setting. The questions are: What will be the availability, who will control the distribution and what will the cost be?

Dr. Rao: My view is that, in most developing countries, it will be difficult to afford the annual costs of the replacement with recombinant products. If gene therapy is established, and I think it will be, that will be the way for most of those countries to go rather than depend on recurrent use of products.

Dr. High: I don’t disagree with you. This has been a complex product to manufacture. It has a protein coat with a DNA sequence that is the active agent, so entities that want to develop the product are going to have to become expert at a fairly complex manufacturing process, but obviously there are individuals everywhere who can do that.

Katherine A. High, MD, the William H. Bennett Professor of Pediatrics at the University of Pennsylvania School of Medicine, as well as an attending hematologist and director of the Center for Cellular and Molecular Therapeutics at The Children’s Hospital of Philadelphia, led the round table discussion.

Other participants included Ralph Green, MD, a professor in the department of medical pathology and laboratory medicine at UC Davis Medical Center; Joseph R. Bertino, MD, associate medical editor of HemOnc Today; Laurence Boxer, MD, professor and director of pediatric hematology/oncology at the University of Michigan Medical Center; Harry S. Jacob, MD, FRCPath(Hon), chief medical editor of HemOnc Today; A. Koneti Rao, MD, Sol Sherry Professor of Medicine and section chief of hematology at Temple University School of Medicine; Morton Coleman, MD, director of the Center for Lymphoma and Myeloma at Weill Cornell Medical College; and Edward Gordon-Smith, MD, MSc, FRCPath, a professor at St. George’s Hospital Medical School in London.

References:

• Manno CS. Nat Med. 2006;12:342-347.
• Nathwani AC. N Engl J Med. 2011;365:2357-2365.