Targeted Gene Therapy for Haemophilia

We've heard from
Adam Jones, Lecturer at the University of Sunderland about living with haemophilia.  Kathy High, from the Children's Hospital of Philadelphia, offers a gene therapy perspective on treating the disease...

Kathy -   Well, a long term goal of research in the field has been simply to get long term expression of a donated factor 9 gene in a human subject.  So the disease was first corrected in mice about 15 years ago and the effort has been to move successfully from mice to haemophilic dogs, and on into people with haemophilia.  The work that we've been doing that I talked about today though uses a different strategy, not just giving a new normal copy of a factor 9 gene to somebody with haemophilia or to an organism with haemophilia, but rather to go in and seek to actually correct the mutant sequence in an animal with severe haemophilia and restore a normal sequence so that that corrected gene could now be under the control of all the normal regulatory signals about when to go up and when to go down and so forth.  And so, that's the work that I was talking about at the meeting today.  It was accomplished using a synthetic molecule called a zinc finger nuclease that was delivered along with a donor sequence.  The zinc finger nuclease cleaves the DNA at the site that you want to correct and then the cell's own mechanisms use the normal donor to make a repair to the cleavage sight that now installs the normal sequence instead of the mutant sequence.

Chris -   And in that way, the protein, the haemophilia protein missing before is now being made correctly, and so as a result, the person should have a restoration of the blood level so their clotting should go back to normal.

Kathy -   Correct and so, what we've done is show that that can actually be accomplished in a haemophilic mouse.

Chris -   But mouse don't get haemophilia, so this is a mouse you've made?

Kathy -   Right, this is a mouse.  Well actually, mice probably do get haemophilia but they don't survive in the wild with haemophilia.  So yes, this was a mouse that we made in the laboratory and installed the haemophilic mutation and then showed that we can correct it using this approach.

Chris -   So if it works in the mouse, will it work in a person?

Kathy -   Well most therapeutics in haemophilia have actually been tested first in a naturally occurring model of haemophilia - the haemophilia dog - and colonies of haemophilic dogs are maintained at a few universities worldwide and so, our next step will actually be to attempt to carry out the same correction in the haemophilia B dog model.

Chris -   So talk us through the method that you'll use in the dogs then?  What will you do?

Kathy -   Well, we do essentially something very similar to what we've done in the mice.  We have to design a different set of zinc finger nucleases because the dog factor 9 sequence is not identical to the sequence in the mouse that we corrected.  But it will be located in approximately the same place so we'll give the dog an intravenous injection with an AAV [Adeno-Associated Virus] vector that expresses the zinc finger nuclease.  And that will induce this double strand breaks in the dog liver and then at the same time, we will have given this donor with the corrected dog sequence and the cellular mechanisms in the dog liver cells will be triggered and will repair the site where the cleavage is and it should allow expression of normal canine factor 9 in the animal.

Chris -   Why are you using that particular virus, this AAV9.  What's special about it?

Kathy -   So that's a good question and I have to admit that we chose it out of convenience and efficiency, but were you to consider expression in humans or to consider moving this forward into human subjects, it's going to be very important, I think, to identify a vector that will express only short term.  Otherwise, I think you may have safety issues that arise from having the zinc finger nucleases expressed continuously.

Chris -   Sure and the fact that it just goes into the liver, or does it go elsewhere?  Do you know that for a fact?

Kathy -   Well actually, we know that it only expresses in the liver because it has a liver-specific promoter, so it may go to other places.  In fact it does at little levels go to other places, but the promoter does not allow it to express the zinc finger nuclease.

Chris -   So I think this is the system that controls what genes get turned on and they'll only turn on in the liver.

Kathy -   Correct, they'll only turn on in the liver.

Chris -   And so, they make that change in the liver and then hopefully, life longer thereafter, you should preserve the expression of these new proteins.

Kathy -   Well actually, one of the great features of this strategy is that it's a correction in the genome itself and therefore, it will be passed to every daughter cell.  And so, even if the cell gets old and wears out, as long as there are corrections in the stem cells of the liver that give rise to the new cells then you'll propagate the change and we did actually show in the mouse model that we could remove 2/3 of the liver and then as the liver regenerates, you maintain the correction because the residual cells have the correction installed and as they divide and give to new cells, the correction is maintained.

Chris -   So what proportion of the clotting factors in the blood are now the correct one when you do this?

Kathy -   We were only able to correct something like 3 to 7% of the target alleles so that won't give you 100% level of factor 9.  It'll only give you a modest level in the range of 3 to 7%, but haemophilia is one of those wonderful diseases where restoring even a very modest level of normal clotting factor, in the range of 5%, converts the disease from a severe one to a mild one.

Chris -   And if this works the way it does in all these other mammals, it looks encouraging then that you should be able to translate this to the human.

Kathy -   There will be additional issues that I think need to be addressed very carefully as this moves into human subjects for testing and we talked about those in the conference today.  We need to make sure in the human genome that these particular zinc finger nucleases don't cleave other target sites - those are called off-target effects.  So we've analysed those pretty thoroughly in the mouse, but the real tissue of interest of course is human cells, so we'll need to do further analysis in human cells.  So those are the kinds of issues that will need to be addressed before this kind of strategy moves forward for in vivo gene correction, but this type of strategy is already in place for cells that can be manipulated in the laboratory.  So, it's in place for example in T-cells in a trial that is underway for HIV.  Of course, moving gene transfer from ex vivo to in vivo involves another series of considerations that will have to be addressed.

Chris -   One other question that people often raise is, what about the question of spread of the virus outside of the person you're trying to treat?  Is that a risk here or is it constrained and confined just to the person you are administering it to?

Kathy -   Well you know, that's an interesting question and when clinical gene therapy studies  first started, people were very concerned about the risk of what we call horizontal transmission, will the household contacts be at risk for being infected with the virus.  So in the initial studies of AAV gene transfer, that was very extensively looked at.  People were kept in the hospital for 24 hours and we had to collect all their body fluids and try to make sure that the body fluids were not infectious, that they couldn't transmit that to their household contacts.  And so, fortunately that question has been resolved and that doesn't seem to be a big risk.