Dr. Arthur Burghes discusses antisense oligomer treatment in an SMA mouse model
Guest: Arthur Burghes, PhD, professor of Molecular and Cellular Biochemistry, Molecular Genetics and Neurology, The Ohio State University
Access an abstract of this month’s featured research article: A single administration of morpholino antisense oligomer rescues spinal muscular atrophy in mouse. Hum Mol Genet. 2011 Dec 30. [Epub ahead of print]
Kevin Flanigan: Welcome to this month in muscular dystrophy. I'm Kevin Flanigan from the Center for Gene Therapy at Nationwide Children's Hospital in Columbus, Ohio.
Each month on this podcast, we invite authors of recent publications to discuss how their work improves our understanding of inherited neuromuscular diseases and what they work might mean for the treatment of these diseases.
It's my great pleasure to have today as our guest, Dr. Arthur Burghes, Professor of Molecular and Cellular Biochemistry, Molecular Genetics and Neurology at the Ohio State University.
Arthur Burghes: Thank you, Kevin.
Kevin Flanigan: It's a great pleasure to have you here to talk about your recent paper in fact so recent that is just available online, I think, at present.
Arthur Burghes: Correct.
Kevin Flanigan: Antisense oligomer treatment as a therapy release and rescue in the mouse model of spinal muscular atrophy and I'll remind our readers there's a link to this paper on our website where you can see the abstract yourself.
Arthur, why don't we begin with spinal muscular atrophy. Tell us what is SMA
Arthur Burghes: Well, spinal muscular atrophy is a disease of the motor neurons which is in the spinal cord. So those neurons don't function correctly and the reason they don't function correctly it's because they don't have sufficient SMN. They do have some SMN but just not enough of it.
Kevin Flanigan: So SMN is this protein from the gene called survival of motor neuron.
Arthur Burghes: Right.
That's correct. It's a protein and it's made by two genes in the body. One is called SMN1 and lo and behold, the other one is called SMN2. They lie adjacent to each other but then not equivalent to each other.
Kevin Flanigan: So not enough of SMN causes an absence of this protein in quite severe clinical syndrome...
Arthur Burghes: Not an absence of the protein, just what genetically called a hypomorph or too little of the protein. So, the SMN2 does produce some protein. Just not enough of it and that basis of some therapies for this disorder is to treat that gene into making sufficient SMN and make it look like SMN1. And that's the basis of this current paper were talking about, yes.
Kevin Flanigan: So before we get to the paper, let's go back just a minute for some of our listeners who are unfamiliar with the clinical syndrome. So, there's actually spinal muscular atrophy (SMA). There's several types of it, actually.
Arthur Burghes: That is correct. I mean three or four types depending on how detail you want to get into the clinical classifications. But this type 1 which is the most severe form. It also interestingly has the least copies of SMN2, type 2 is intermediate, and type 3 is mild and some people use the type 4 classification as well.
Kevin Flanigan: And this type 1 is, my listenters may know, very severe disease. Children never walk in fact and some don't go home from the hospital even.
Arthur Burghes: Correct.
Type 1 or Werdnig-Hoffman's, it's infantile so usually presentation is before the age of six months, depending on the level of respiratory intervention, death can occur before the age of two although nowadays with some of the respiratory interventions considerably longer lives.
Kevin Flanigan: So, we have these two genes, SMN1 and SMN2, and the severity of disease goes along with how much, how many copies of SMN2 one has.
Arthur Burghes: Yes. Basically, how many copies you have and how well they work. So some SMN2 has worked a little better. But basically that's correct. More SMN2 copies, better off you are.
Kevin Flanigan: All right. And tell us about the differing in the splicing in exon 7, the difference that happens with the disease itself.
Arthur Burghes: OK.
So the major difference between SMN1 and SMN2 is in the degree with which incorporates the exon 7. So, there's actually a single nucleotide change that is critical, a C to T change between the two genes. So, in the case of SMN1, it has a C in exon 7 and in SMN2, it has a T six base pairs in exon 7. And that single nucleotide change basically alters the ability to incorporate that exon into the final transcripts.
So how much of that exon 7 gets into the messenger RNA which is the piece that is going to go on to make the SMN protein. So, in essence, if you have an SMN2 gene, it's not going to be very good in incorporating that exon into the transcripts, it does produce a protein but that protein that is producing is of two forms.
Most of the form lacks exon 7. It doesn't oligomerize and it doesn't stick to itself and therefore gets rapidly degraded. There's a little bit of full-length SMN and it's that little bit of full-length SMN that is made that allows the cell to live, but it's not enough for motor neurons.
Kevin Flanigan: Not enough to rescue them. They die without it. So actually, this is a great introduction. We're talking this paper as about splicing and some of our listeners know, some of our other podcasts have been about alteration of splicing. Just a reminder, when we talk about splicing, splicing is this fairly complex process where the pieces of the gene that are not all assembled into the final blueprint if you will. They get spliced together, stitched together to make a final blueprint for the protein.
So, in this case, this is an interesting paper for our listeners because it's different from some of the other ones we've discussed. In this case that SMN2 inefficiently incorporates, insufficiently stitches in exon 2 and your therapy is directed toward increasing that.
Arthur Burghes: Yes, exon 7.
Kevin Flanigan: So, how do you increase the incorporation of exon 7? How does one go about doing this:?
Arthur Burghes: Well, there's a number of ways that you could conceive of this, and I'm not going to go through all of those. I'm just going to go to how this one works.
Kevin Flanigan: Terrific. OK.
Arthur Burghes: And that is, there is a splice inhibitor in what's called the intron which is the sequence right adjacent to the exon. So if you can block that splice inhibitor from binding certain proteins, notably in this case, it's protein called hnRNP A1, doesn't really matter. But if you can block it, then you'll increase the amount of exon 7 in the final transcript.
So, the therapy works by binding an antisense oligonucleotide to that negative element in the RNA which now results in more of the exon 7 being spliced in because this negative regular is not working anymore.
Kevin Flanigan: So to restate it, one little way of, you know, where all of us think of exon as pretty discreet elements but really they have this defining in control elements. You mentioned inhibitors, there's splice enhancers as well and so forth, so here your antisense oligomer really masks that splicing silence or element, is that right?
Arthur Burghes: That is absolutely correct. I just want to go back one step. Often you draw our gene which is a genomic DNA level and it's got exons but then it actually transcribes the whole component into what's called an hnRNA or pre-mRNA.
And so you'll going from the DNA to the pre-mRNA which contains everything, exons, introns and every bit, and that is where the antisense oligo is going to bind. It's going to bind to that pre-mRNA which is before it goes into splicing and therefore, then all the signals come into play, into splicing way, where you stitched together the final messenger rRNA which then gets exported to...
Kevin Flanigan: To make...
Arthur Burghes: To make the protein in the sarcoplasm.
Kevin Flanigan: So this is interesting. So, your model here is to mask the silencer and if anybody's listening to this, who's listened to our other podcasts for example about Deuchenne muscular dystrophy, in those examples, the idea was to actually mask an enhancer element, to splice out another exon to take another piece that make a deletion bigger but to restore what we call an open reading frame, a translatable fraction.
Arthur Burghes: Yes.
Kevin Flanigan: So here, it's different. We want to mask the silencer, to put in this exon, it's pretty insufficiently done.
Arthur Burghes: It's exactly the reciprocal of the Deuchenne case where you're trying to skip an exon and therefore you're going to block what enhances incorporation of that exon. Here you're doing the opposite. You're blocking a silencer to increase the incorporation.
Kevin Flanigan: Well, this is quite interesting that brings us right up to your paper which again I remind our listeners that they can find the link to the paper on our website itself. In our paper, you used morpholino oligomer and used it in animal called SMN delta 7. So, could you explain for a little bit what this animal model is and what it looks like.
Arthur Burghes:Yes. OK, for sure. So, a number of years ago, I don't care to say how many, we created an animal model of SMA.
So, the first thing we did, together with Michael Sendtner in Germany, is to actually breed the deletion with a copy of SMN2. So we put the SMN2 copy into a null background.
Kevin Flanigan: Because mice don't have their own SMN2.
Arthur Burghes: Yes. Mice do not have two copies of SMN. They don't have SMN2. So you have to first put the SMN2 in and then you have to create the knockout of the gene. So the two components were put together to mimic SMA in the mouse or the genetics of it.
Kevin Flanigan: This is now a standard, a quite standard model.
Arthur Burghes: Right. And then we also put in a second expression of the delta 7 transgene and the reason for that was it made it slightly milder. It actually extended the life to 14 days.
Kevin Flanigan: So, 14 days is the average lifespan.
Arthur Burghes: Average lifespan of a knockout. So a minus minus mask.
Kevin Flanigan: Right. So they're missing the SMN1 copy and they live for 14 days. And if you treat them with your antisense morpholino oligomer, what happens?
Arthur Burghes: Well, we treated it at different concentrations and so one thing I should say here is that there are different chemistries of these antisense oligomers. And we're not the only people who have treated SMA mice with antisense oligomers, there's a different chemistry called an MOE chemistry developed by Isis and Adrian Krainer's Group and Marco Passini have treated with these antisense oligo's. We've used morpholino's. We like morpholino's because they appear to have very low toxicity in the CMS in our hands. And so we've treated at different concentrations going up to extremely high concentrations and seen no toxicity first of all.
Kevin Flanigan: When you say treated, we're still talking here like in your paper about injecting it intrathecally we call it, is it?
Arthur Burghes: OK. So that's a very good point and I should have mentioned this right off the bat. So actually this work in my lab is done by Paul Porensky who's actually a resident neurosurgeon. So it's a collaboration between a molecular genetics lab which is mine and actually a neurosurgery person because they have skills that I do not posses. And so, it's actually injected into the ventricle of the brain.
Kevin Flanigan: So directly into the spinal fluid that bathe the brain.
Arthur Burghes: Yes. The CSF that bathes the brain and just as an aside people would think that's an exceptionally complicated procedure but at least according to the neurosurgeons, you know, taking or delivering for instance in cancers, drugs in children to the brain is not on standard that you can do indwelling catheters and various things into the ventricles of the brain.
So this is not...
Kevin Flanigan: Not impossible to imagine as a therapy.
Arthur Burghes: This is not far off and the difference or one thing that comes up when you switch to humans is like intrathecal delivery, so you got two options. Do I give it in an indwelling catheter into the ventricles which breath the spinal cord or do I give it intrathecally into the fluid is more directly or closer to the spinal cord?
Kevin Flanigan: But in this model you went intrathecally into the...
Arthur Burghes: We went intra ICV, so intracerebroventricularly. And the reason you do that in the mouse because the mouse is...
Kevin Flanigan: It's a bigger target...
Arthur Burghes: Yes. It's small.
Kevin Flanigan: So what happened? You used different dosages here and what did you see in the life expectancy and so on?
Arthur Burghes: So in the life expectancy we immediately extended the life expectancy if you average overall the concentrations we used to over a hundred days. So you go from 14 days to over a hundred days.
Kevin Flanigan: So this is quite extraordinary and quite clear and statistically significant...
Arthur Burghes: Yes. It's one single dose over a hundred days.
Kevin Flanigan: And this one along with incorporations, of showing the incorporation of axons.
Arthur Burghes: Yes. So, when you go and analyze the tissue by either a technique called the first transcripts phase PCR which simply analyzes the message or using a digital PCR and what you find is a large increase incorporation of exon 7. It's a full-length SMN and a large increase in the protein.
Kevin Flanigan: So one of the things I found very interesting about your paper was giving this as a single dose at a very early age had this prolonged effect. Is that realistic or comparable do you think to what we understand about the human disease?
Arthur Burghes: I think the full answer to that question, we don't really fully know. OK.
So what I would say is it goes along with certain other studies we done with inducible transgenes. It seems the critical period is over the neonatal period. What I can say is when we induce SMN there, we get a major effect in the mice.
Do we fully model the plateau of the so called plateau phase in the severe mice that we're looking at? I think that's questionable. I can't really answer whether the mouse has fully models that phase. So the question of whether introducing an ASO or any other therapy in an SMA patient that's had it for a long period of time a one benefit that's going to have is really a question that needs to be addressed in clinical trial in human patients. It cannot be fully addressed in the mouse model.
Kevin Flanigan: Right. But it's truly supports the idea of really quite early clinical... if these animal models suggest that we should be thinking of trials in the youngest patients as soon as we can identify.
Arthur Burghes: I think that's the most likely to be have the biggest efficacy.
Kevin Flanigan: Well, what are the next steps then in this project for you and your laboratory? Where is this heading?
Arthur Burghes: The first thing we're doing is to go and look in the pig. But the reason is that another group actually at the University of Missouri has produced a pig that has SMN2 in it. So the first thing is in a large animal that is close in size to a human. So, for instance if you get a five-day-old piglet that is approximately 2 kilograms, what we going to do is put the antisense oligo, the ASO into the pig at five days of age through the intracisternal and ask the question, how is the biodistribution of the antisense oligo to alter the splicing of SMN2?
So, this is like a prelude to the human trials.
Kevin Flanigan: To human trials.
Arthur Burghes: Because if you going to inject it to humans, I would like to know what the biodistribution is, how far am I affecting the splicing, am I affecting it at the lumbar cord and the cervical cord and everywhere else I need to get it.
Kevin Flanigan: Right. Clearly an important issue to think about giving this to humans.
Arthur Burghes: And that's the first thing. And the second thing is safety. So we would like to start to do studies in larger organisms as well as setup or the controls and everything else you need to do to ensure this very high safety before you venturing to a clinical trial with this antisense oligo.
Kevin Flanigan: Right.
Well, it's certainly an exciting work and I appreciate you taking the time to come and explain it to our listeners today.
So thank you very much for joining us.
Arthur Burghes: Thank you.
Kevin Flanigan: This podcast is brought to you by Nationwide Children's Hospital. You can find out more about the muscular dystrophy program and ongoing clinical trials at Nationwide Children's at our website, nationwidechildrens.org/muscular-dystrophy-podcast. You'll also find the link to the abstract of Dr. Burghes work that be discussed today.
Thank you for joining us.