Edith Pfister, Ph.D., Assistant Professor, University of Massachusetts Medical
This talk will cover the basics of therapies designed to lower huntingtin: what do we know and what don’t we know. It will introduce the basic mechanisms of huntingtin lowering and gene therapy. The goal is to introduce the language of huntingtin lowering and of gene therapy and to come away with a basic understanding of how they work.
John Fremer
We would like to welcome you to the Sanguine Speaker Series webinar, Gene Therapy for HD: Sorting Truth from Hype. Today’s webinar is hosted by Edith Pfister, PhD, who is an assistant professor at the University of Massachusetts Medical School. Dr. Pfister, over to you.
Edith Pfister
Thank you very much. And thanks for having me do this presentation. I’m very excited to do it. So, just by conflict of interest statement, I am a co-inventor on a patent for gene therapy for HD, which I do occasionally make money from. All right. So, an overview of my talk. What I’m going to do is I’m going to give a brief introduction to genetic diseases, I’m going to talk a little bit about what gene therapy is, I’m going to give you a few approved gene therapies and then I’m going to talk about gene therapy specifically for Huntington’s disease. And I’m going to basically compare that to some of the approved therapies so we can look at what the challenges are.
Edith Pfister
Okay. So, what I’m showing you here is a piece of DNA. And so this is the root cause of genetic diseases, is you have a change in DNA. The DNA is then transcribed into another nucleic acid molecule called RNA. And you can have a change that causes the RNA… After the RNA is translated into protein, you’re going to have a change that causes the RNA not to be made and then the protein not to be made. You can also have a change that just causes a different RNA to be made and then you get a protein that potentially doesn’t have function or it has a different function.
Edith Pfister
And so, genetic diseases are caused by mutations in DNA. They can be inherited or they can occur spontaneously. So, you can have a parent who doesn’t have a disease and then the mutation can occur in their child. They can be caused by a loss of gene function, an increase in gene product or a change in function. And your symptoms can be serious or they can be mild. So it’s not necessarily a death sentence, just any genetic disease and they can appear at or before birth or they can appear later in life. So, what is gene therapy? Gene therapy is an introduction of genetic material, DNA or RNA into cells to replace augment or reduce the product of a mutated gene. So, how does this work? So, if you want to replace a gene, say you have a loss of function. So, you have a protein that’s not being made, you’re going to introduce a normal copy of the gene and that’s going to be made into a normal at mRNA and then a normal protein.
Edith Pfister
If you have abnormal protein or you want to get rid of the gene product, then you’re you’re going to introduce this other type of nucleic acid that’s going to inhibit the mRNA. So, basically what happens is the mRNA gets cut and then you don’t get a protein. And how do we introduce the genetic material? Now, the way that I’m going to tell you about, because this is the way that the products that I work on work, is I’m going to tell you about this viral vector called adeno-associated viral vector. And so, adeno- associated virus is a naturally occurring virus. It’s a small virus, it has a genome of its own and this genome contains the genes that help it to replicate, make more of itself. And so, when it enters into cells, those genes are going to be turned on, it’s going to make more of itself and then be released from the cells. What we do in the lab, is we take out that AAV gene and we replace it with what’s called a transgene. And this is the genetic material that you want to introduce into the animal or the patient.
Edith Pfister
And we do this and we make these in a culture dish. And so what we do is we introduce this vector with the transgene in it and we also introduce the other gene products that can help it replicates separately. And so what happens is that this transgene, AAV gets packaged and that’s what gets [inaudible 00:05:16]. So, it doesn’t have the genes that can replicate itself, it just has the genetic material you want to introduce. And so that then is made into DNA. And the important thing to remember here is AAV gene therapy adds a new piece of genetic material. So, if you want to do gene replacement with an AAV, what you’re going to put in is that normal gene is going to go between those loops, which are called ITRs. And that’s going to make normal RNA, going to make normal protein.
Edith Pfister
If you want to do gene silencing, what we introduce is this other type of RNA. It’s going to be made into an RNA called an artificial microRNA. And what this does, is this causes the mRNA of the gene that you want to reduce to be cut or cleaved and then you don’t get the abnormal gene product. So, now I’m going to talk just a little bit, because I know this is always a big, big question about these other systems, CRISPR/Cas9. So this is not a way of introducing DNA into the cell. The difference with CRISPR is that you actually are modifying the patient or the animal or the cell genomic DNA. And so this is derived from a bacterial protein, you have a protein and you have a guide RNA and they go in and they can then cut the genomic DNA. And then you get a change in the DNA.
Edith Pfister
So this is as opposed to if you were introducing something new like a new gene product or a microRNA, this actually edits the patient’s own DNA. And so this is actually not something that is in the clinic now, but it always comes up as a question. And you might introduce this… I should just also say you might introduce this using an AAV. So, just to compare the types of gene therapy to drive this home. So expression of a transgene or microRNA using a viral vector like AAV does not produce a change in the patient’s DNA. It is permanent, or at least very long lasting. Whereas CRISPR/Cas9 is going to produce a change in DNA and it’s also permanent and long lasting.
Edith Pfister
All right. So now I’m going to talk a little bit about approved gene therapies. So, first I’m going to talk a little bit about SMA, which I think is a really good example that can teach us some lessons about Huntington’s disease. So what is SMA? It’s a degenerative muscle disorder, class of five by age of onset. So, type one SMA is onset very, very early. Babies before six months and they don’t achieve any developmental milestones. They don’t sit up, nothing. Type two is they do achieve sitting, but not walking and then there’s a progressive decline. Type three is childhood. So, they achieve walking and then there’s a type four, which is on a later onset. So the first therapy of this type of therapy approved for SMA was spinraza, which is a gene modification. We don’t really call it gene therapy because it’s not permanent. It’s something called an antisense oligonucleotide, which is not permanent. It doesn’t stay inside the cell forever. It degrades.
Edith Pfister
But what it did is in the case of SMA, the problem is this deleted or mutated SMN1 gene, and then there’s also an SMN2 gene, which is very, very similar to SMN1. So, everybody has both of these. Some people have more copies of SMN2 than others. So it’s highly similar, but it is normally produced at low levels. And so what the ASO does is it changes the SMN2 so that it’s more like SMN1 by changing the way that mRNA is made. And then you get more SMN protein and that makes patients better. This was initially approved for the most severe form of SMN, but it does appear that some older patients have seen some benefit. And there’s a partial response rate. The second therapy I’m going to talk about is a classic gene therapy, it’s called Zolgensma, and this is an AAV like I talked about. And what it does, is it introduces a copy of SMN1 into the patient and it’s delivered to the CSF or blood depending on the age of the patient. And it’s most effective in the youngest patients.
Edith Pfister
And that’s partially because younger patients are smaller and it distributes throughout the body or throughout the CNS better in younger patients because they’re smaller. All right. Another approved gene therapy is that Luxtruna, which is a gene therapy for the eye. So this is a therapy for a congenital form of blindness that’s caused by a loss of the RPE65 gene and it’s delivered by injection into the back of the eye. And this is a little bit… There are some similarities here to Huntington’s disease in that the injection is very local and you’re really just trying to get the eye. So, gene therapy for HD. Where are we right now? So, Huntington’s disease is caused by a repeat expansion, a CAG repeat expansion in the Huntington’s gene. And this repeat expansion is down here in exon one. So normally it’s less than 36 repeats of CAG. CAG, CAG, CAG 36 times. In patients is greater than 36 CAGs, and so this is a really large gene, you have a small change down here and that causes disease.
Edith Pfister
So to compare this to SMA, SMA is caused by a loss of function or deficiency. And natural duplications of SMN2 provide evidence that there is a therapeutic benefit to introducing SMN2. HD is caused by a toxic mutant protein. And there’s very little known about the effect of reducing normal Huntington and we really don’t even know at what stage we can reduce the toxic Hunnington and have a therapeutic effect. So SMA affects motor neurons in the spinal cord and this is accessible. So it’s accessible by the CSF in the blood, whereas HD affects the striatum and deep brain structures and they’re inaccessible or they’re accessible primarily through direct injection into the brain. SMA1 has an infantile onset at least in type one and a rapid progression and developmental milestones are really well known. So it’s pretty easy to determine whether kids are breaching those developmental milestones, losing those developmental milestones.
Edith Pfister
It’s relatively easy to track. And because they’re children and they’re smaller, the dosing is easier in young patients, whereas HD has an adult onset in a relatively slow progression, which is difficult to track because it’s very heterogeneous and we have a lack of established biomarkers. So what does gene therapy for HD look like? So what we do and what other groups have done, is we’re introducing an artificial microRNA that targets the Huntington gene. And we are targeting both normal and the mutant Huntington. So, not just the mutant Huntington. And this is because it’s actually fairly difficult to specifically target the mutant Huntington. And what happens when you introduce this microRNA, is that a protein that’s already in the cell called Argonaut, takes up this microRNA and it cleaves both the mutant and the normal Huntington mRNA. Now, I told you that Huntington affects the deep brain structures. And so, one of the things that we have to think about is how to get into those deep brain structures. And what we’ve done is we’ve looked in sheep and we’ve injected the AAV into the sheep, into the striatum in sheep.
Edith Pfister
And you can see this bright part is where the AAV is in the sheep. And so you can see that it’s a very limited distribution. So we have to think about how much therapeutic benefit that limited distribution is going to have. And the same thing is true of an ASL. So as I said, HD has an adult onset, slow progression. The progression is difficult to track and there’s a lack of established biomarkers. The toxic mutant protein. There’s very little known about the effect of reducing Huntington, so we know that we can probably reduce Huntington up to about 50% because there are people who have only one copy, but we don’t know what happens if you reduce Huntington more than that. And we don’t even really know how much therapeutic benefit you’re going to get from reducing Huntington. Oh, so here’s some frequently asked questions about gene therapy.
Edith Pfister
So, is gene therapy a cure? And so my answer is not really. So, we know that gene therapy is… That can have curative potential, but it’s not necessarily a complete cure. And one of the problems is of course, what I just mentioned, that you have a limited spread that you may have lost some function before you actually put the gene therapy in. So there are effects that may have already occurred that you can’t cure. So, not really. Now, will insurance pay for gene therapy? I don’t really know the answer to this. My limited knowledge of this is probably, it seems like the companies are working really hard with insurance to make sure that there are payment options that there’s some reimbursement for people, for hospitals, et cetera. So, the answer is probably. And can gene therapy reduce the chance of having a child with a genetic disease? And in this case the answer is no. So, gene therapy does not change the cells in your body that are responsible for reproduction. They don’t change the germs cells. And like I said actually, most gene therapies right now have a very limited distribution.
Edith Pfister
So you’re really only affecting the structures that cause disease, but there are options. So for HD, pre-implantation diagnosis has been available for a while. And so that is an option for HD. That is not a gene therapy. And I actually think I went through this very fast. Oops. So, these are the people who work with me. I want to thank all of these people for their work. And I will take any questions now.
Lisa Scimemi
Edith, some of the questions have come in. Besides Huntington’s and SMA, is gene therapy being used in research for other rare diseases?
Edith Pfister
A lot of rare diseases are very… There’s a lot of work on very rare diseases. There’s work in cancer, there’s a lot of work in cancer, muscular dystrophy and several others. They’re just not approved therapies yet, but the rate of approvals is accelerating. So, we do expect to see quite a few gene therapies in the future.
Lisa Scimemi
The next question is, is the MRI you showed demonstrating where the contrast agent localized and not the vector itself?
Edith Pfister
Yes, it is. That is the contrast agent. And you’re right, it may not be the vector. When we’ve done GFP staining, it’s a very similar area. I’m not going to claim it’s exactly the same, but it’s very similar.
Lisa Scimemi
You mentioned that the CRISPR/Cas9 is not in the clinic, is that something that will be changing in the near future?
Edith Pfister
I don’t know how close that is. There’re a lot of questions about that. I think that first cell therapies where you take the cells out and make a change to them and then reintroduce them, that may happen. I don’t think CRISPR… For example, CRISPR in an AAV is going to come anytime soon because there’s just no way to turn it off.
Lisa Scimemi
What trends junction efficiency are you getting with the system in your in vivo models?
Edith Pfister
So, it looks like it’s about 50% of neurons based on GFP screening.
Lisa Scimemi
How does this help HD patients and their symptoms?
Edith Pfister
Right. So, that’s a complicated question. So, the idea is that you would put an AAV in before either right when the patient became symptomatic or before they’re symptomatic, that would be ideal because what you’re going to do then is you’re going to reduce the mutant Huntington and you’re going to spare those neurons. You won’t get neuronal death. And so the idea is then you don’t get symptom progression. It seems to me and really, we don’t know the answer to this unlikely that you would get a reversal in symptoms. It’s most likely that you would just halt the disease where it was. And particularly if we’re targeting the striatum, that’s thought to be the place where the motor symptoms are centered. So, you would get a halting of the motor symptoms. Now, if we can treat the cortex, we might get some of the cognitive, emotional symptoms as well. So, it’s work that’s being done to figure that out, but it’s really hard to do that kind of work in a mouse to figure out exactly what the symptom is going to be. So that’s something that is likely going to be answered in clinical trials.
Lisa Scimemi
When are your clinical trials starting?
Edith Pfister
So, there are… Our personal clinical trials, we’re not at clinical trials yet. It’s out of my hands now, it’s been licensed to a company. So, I don’t do that part. There are clinical trials, the ASO, there’s antisense oligonucleotide in clinical trials right now. There’s also the one allele-specific antisense oligonucleotide is also in clinical trials. That’s a little more complicated. I can talk about that ad nauseam, because that was original project. And then there is this year, I believe it’s been a little bit delayed by COVID as has everything. But this year there’s a clinical child starting with an AAV, that’s very similar to the one I described and I think they started enrolling.
Lisa Scimemi
What is the average age that a patient would be going under gene therapy for Huntington’s?
Edith Pfister
That’s an interesting question. And I think it’s going to depend a little bit on what… For example, what the people running the clinical trials decide, when they decide they want to treat. Now, okay. I said my personal preference is to actually have something be effective, you need to treat fairly early. So my guess is if you have onset at the age of 40, you would want to treat at about the age of 40, at the age of onset. Ideally, you might even want to treat before onset because by the time you see symptoms in patients they’ve lost maybe 50% of cells in the striatum. So in theory, you want to start before that, but there’s always the question of whether there’s going to be detrimental effects as well. So, I think they’re initially probably going to try to start right after onset. So, that would be average age of onset is maybe 40.
Lisa Scimemi
Do you know how big the clinical trials will be?
Edith Pfister
I don’t know that. I’m not a clinical person.
Lisa Scimemi
That is all the questions we have. I’ll turn it back over to you for some closing remarks.
Edith Pfister
Okay. Well, thank you very much for listening to this talk. I am happy I am at UMass Medical School. I’m happy to answer questions. You can find my email through the medical school, if you have questions, I’m always happy to talk. I do hope that these therapies are in the clinic soon and I hope that we can help a lot of patients. Thank you.
John Fremer
Thank you, Dr. Pfister, and thanks everyone for joining this Sanguine, S3 webinar, Gene Therapy for HD: Sorting Truth from Hype. For a list of upcoming webinars or to request patient samples, please visit researcher.sanguinebio.com. Thank you all again and enjoy the rest of your day.