#268 ‒ Genetics: testing, therapy, editing, association with disease risk, autism, and more | Wendy Chung, M.D., Ph.D.

25:03And so conditions like sickle cell disease and other hemoglobinopathies, we knew at the protein level well before we knew at the DNA level. So that was something pretty characteristic. Other cases, we knew based on enzymatic activity. And so we could see biochemically in a test tube, if you will, what the reaction that was run. So we knew about many of those things before we ever knew the exact DNA sequence or exactly what the genetic variants were that caused those conditions. And by the time you were a graduate student, so still pre -Him and Genome sequence, right around the time that Rudy Libel is figuring out what left and is and things like that, how much resolution did you have into what a gene looked like at that time?

25:42So it was pretty painful at the time. We would use these things called linkage maps to try and figure out what chromosome something a condition was on, be able to get closer through linkage analysis to the right neighborhood, the right zip code, eventually the right address. us. When we did this, we didn't have great signposts to be able to even figure out where we were within this. We didn't have things like structures of genes, references. So as you were doing this, you were sequencing not just one person with a disease, but also you had to sequence, quote, unquote, normal people or average people for comparison.

26:13We didn't have that as something you could just look up online. We didn't have the internet as an example at the time to be able to see, you know, have investigators work together from around the world is what's much more sort of old school passing papers back and forth and you know meeting in various locations. So there's a lot slower, a lot more laborious. And with this I have to say, and you had mentioned Rudy Leibold, he did have this big bold idea in terms of cloning a gene for obesity and it's the first time he put in a grant putting out that idea. People thought it was just totally ridiculous.

26:45The idea that you could, we called it positional cloning, but identify a gene solely based on its position within in the genome, understanding and not requiring any understanding of the biology or physiology, but just purely based on genetics and genetic mapping, people thought it was impossible to do. So, and of course, subsequently, whole generation of disease, whole generation of scientists found diseases that way. If you can imagine that process was often a decades -long process or longer. I mean, this was not something you did very quickly. So, I often tell people, you know, the first gene that I cloned took eight years, the last gene I cloned took eight hours.

27:22So, you know, this is just absolutely astronomically different in terms of how we now find disease genes. Yeah, it's very interesting. I looked at a graph, the speed that you're describing even exceeds Moore's law. It's on a Moore's law trajectory with an enormous step function when high throughput sequence came along, which we can probably get to later because I think it's important for folks to understand that. What was the first organism for which we had a whole genome sequence? within that it was certainly a microorganism I don't remember if it's E. coli or something similar in terms of a bacteria but definitely a very small organism yeast or another important part of our library and so being able to understand those small organisms certainly much easier you know even when it comes to the complexity of the human genome some of us would say that well it's been announced repeatedly that the human genome project has been finished but it's only been recently even that as we say Tila me or Tila me we've been able to see the sequence really, the entirety, including some of the cryptic portions that are hard to read through.

28:22So there's still things we have to discover even within the human genome. So before the human genome was sequenced, which I think was around 2000. The first announcement, yes. There was an expectation that humans would have how many genes, based on the understanding of how many genes these far, far simpler organisms had. So there were estimates for some people as many as 100 ,000 genes by comparison, I think current estimates are about 20 ,000 genes in terms of the complexity of people, human sideos. But yet the complexity at the individual gene level is probably more complicated than we appreciated.

28:57Our ability to have different, what we call, isoforms or versions of the way genes are cut and pasted together or how they're utilized in different ways over time and space by different organs or cell types, any one gene could be made into a dozen or more different gene products. And so some of that complexity was not at the level of the individual gene, but how that gene is reused in slightly different ways. And so anyway, it was shocking. The first time I think we appreciated that it was about 20 ,000 genes in the genome, there were definitely people that were surprised by that. Yeah, it seems like such a small number given the variation between individuals.

29:40In fact, outside of identical twins, we have what, 8 billion people on this planet all with distinct genomes. And yet, the homology between us is how strong, like in other words, how similar are we all genetically? So we're all 99 .9 % the same, about 1 in 1 ,000 base pairs is what you and I probably differ by on average. So as humans, we're pretty similar to each other. And most of the genetic variants, differences that we have are not meaningful. You know, they don't cause any differences in terms of the way our body's function. On the other hand, you know, as something as subtle as one in three billion base pairs can be the difference between life and death, can be the difference in terms of the way the body or the brain function.

30:22So small nucleotide differences can be profound, depending on what genes and when those genes work. So basically three billion base pairs, 20 ,000 genes, 46 chromosomes, is sort of the hierarchy of organization. At what point, well maybe just explain to folks the difference between coding and non -coding portions of the gene. So when you think about all of those ASTs, G's, and C's that you talked about, it's a relatively small portion of that information that gets moved from the DNA to the, eventually, the protein. So the portions of that that are made into the proteins are about, I don't know, let's say for round numbers about a percentage and a half of all of that DNA sequence.

31:06That other say 98 and a half percent. There's a lot of it that to be honest we have no idea what it does. There are certain portions we do understand. They're very important for regulation to know where and when and how much that gene is expressed. There may be other things that are subtle in terms of being able to attract binding factors, transcription factors, other things that may modify the DNA, biochemical changes to the DNA itself, which may affect expression. And there are also what people have called junk DNA as well in there. Repetitive sequences that probably don't do anything positive for us, but get carried along in the ride.

31:43But there's a lot that we also don't know. We don't know everything clearly about this, and there may even be disease -causing variations that are in that space that we haven't even recognized yet to your point. It's a small minority that actually encodes ultimately what we think of as most of what forms the body, physically forms the body in terms of proteins. And in 2000, when the Imagenome Project results were announced, what fraction of those 3 billion and base pairs were identified. So within that, I don't know, we'll say round numbers about 70 % or so. Interesting. And did that include all of the coding segments?

32:21Or was it not yet understood at that time? What fraction of those were coding and non -coding? The majority of the coding was identified at that time to answer your question. There were a few portions of the genome that were hard to read out for various reasons, or hard to map, and put the pieces together. One of the things, just for the listener, to realize is that there was a bit of a jigsaw puzzle. When we did, and when we do do the sequencing in many cases, we're not sequencing. I use the term telomere to telomere or end -to -end along the chromosome. So it's not as if we get one continuous strand of the DNA sequence that comes off the sequencers, where we can just read through it and know it comes together.

33:00In many cases, we have pieces of it, and we have to informatically put the pieces back together and put it back into the right order. or in some cases that's because we have overlap between those pieces. And so we can see based on overlap, this is the first piece, that must be the second piece, that the third piece based on the overlaps. And so we make these things called contigs or contiguous sequences of DNA and put the puzzle and the pieces together in that way. There's certain regions of the genome that are complicated. They're what we call repetitive sequences. And so they may not be unique and it may be hard to even sequence through those regions.

33:36And so putting those pieces back together in the right order, in some cases, has been challenging to do. And so sometimes you'll hear some of us as geneticists say, there's a dark matter, or there are some cryptic regions of the genome that we haven't been able to really dig into. And that's because of some of these complexities of the sequences there and our ability to sequence through them. So, even despite what you're saying in terms of knowing the genes or even knowing portions of the sequences, we didn't necessarily know that it was all part of one gene or that we had all the pieces are put it all together yet.

34:08And so some genes and some diseases were easier to crack than others as a result. So today if somebody goes out and gets a commercial genetic test, what's the difference between someone who goes out and gets a whole genome sequence and say someone that goes to one of the over the counter sequencing services, like a 23 in me? What's the difference in the analysis and what's the difference in the information? This is an important question and if people are listening this is time to perk up and listen closely. So there's a big difference in not all genetic testing is the same and I'm not being critical of any of the companies that do this, but just to realize they're trying to serve a different purpose.

34:48So 23 and me as an example or ancestry .com is another example. Those are more things that are not medically sort of targeted. They're not trying to answer a specific medical question of, do you have an increased risk of breast cancer, do you have an increased risk of heart attack? They're really not getting at that level of detail. Just as an example, ancestry .com is very good at being able to understand your heritage, you're literally where your family is from, where your ancestors are from. It's quite detailed at this point in terms of being able to say what part of the world your family comes from, if you might be adopted as an example, not know about your hair, or to your ancestry, be able to give you some of that.

35:27And I'll also say for better or for worse, if you're trying to find this out, you may identify some of your blood relatives, some people who you would know from a family reunion, some people you might not know for some reason. And people sometimes find out about that. Like I said, even people who are adopted, I've known to find some of their, actually, their birth parents that way. So that's one type of thing, but that's not really for the intention of identifying information for a medical purpose. And so I just want to warn the listeners that if you get something back or more importantly if you don't get something back from those tests, it doesn't mean an all clear for your health.

36:03It doesn't mean that you're free of cancer or won't have any increased risk. So on the other hand, there are other tests that are really designed for a medical purpose to answer a question. And you didn't ask about this specifically, but many of the listeners will know that they would have gotten a test, for instance, if they were thinking about having children, planning a family, wanting to know if they are children or if they were at increased risk of having a child with something like TASACs disease or cystic fibrosis, one of those other recessive conditions that we alluded to. And so that's not with the attention for personal health so much, as I said, thinking about future children or families.

36:40And let me be clear, this is not necessarily about abortion, but this is about being able to care for a child long term and think about reproductive options. So that's another use case and a very common use case in terms of what people will do. Another common use case is for thinking about cancer risk. And so some people may have a family history of cancer. Some people may say their particular heritage is such that, for instance, if they happen to be a Jewish ancestry, they may be concerned because they know there's a higher chance of having a BRCA, so we're called breast cancer one or BRCA one or two mutation.

37:13So some people do a very targeted test, and I'm emphasizing targeted, very specific clinical question. And again, it's answering that question. It's not necessarily giving a genetic clean bill of health for everything. It's very focused. On the other hand, you alluded to what I'll call a genomic test, and I'm going to make a distinction between genetic and genomic. And what I mean by genomic when I'm saying this is it's really including as we talked about all genes. So it's not focused on just a handful of genes. It's really focused on the genes and the genome, those 20 ,000 genes. You can look at just the coding regions that we talked about before, those looking at the protein sequence that we call that in the aggregate in XOM, because those little pieces that code the genes are Xons, EXOM, and when you put them together in the aggregate, we call it the XOAM.

38:10Other individuals are interested in knowing all three billion of their base pairs. They're entire genetic sequence, and we call that a genome, and that will include everything, both the coding and the non -coding regions. I think of that in terms of genome sequence as being in some ways the THE genetic test, right? It's all encompassing. One can blind yourself to look at very focused subsets of genes based on a clinical indication, or you can look at everything because you want to look at everything about your health, or because maybe you don't know all of the genes for your particular symptoms, and you have to be all encompassing in terms of that.

38:46And we may get to some of those use cases, but there are many conditions that are genetically heterogeneous, or have many different genes that can cause them. And so we want to be all encompassing in terms of looking at that. Even though we can sequence all that data right now, we can't interpret it at all. So as an example. Out of those 20 ,000 genes, we have now assigned functions with disease for about 7 ,000. But that still means that for over 50 % of those genes, we don't know of a gene disease association. In even, I will say, because this happens to me with fair frequency, even when I think I know about an association of a gene with a disease, if we study it further, we'll realize that they don't map just one to one.

39:31There may be more than one disease associated with a gene. And so there's still things that we're figuring out about what those genes do. So Wendy, let's just talk technically about those different options that you laid out. And let's start with the most comprehensive. So I've had a whole genome sequence done. And I believe it was done off a couple of tubes of blood, maybe even just one tube of blood if I recall, but no more than two. So 10CC of blood at the most. So I sent that over to, it was a university that did it with part of a clinical trial. Well, what did that university do with those two tubes of blood to extract the insight to read the three billion base pairs that make up my whole genome?

40:09So I'm guessing, but I'm guessing that what they did is in the white blood cells, in that tuba blood, they opened those up to extract what we call the genomic DNA. So the DNA included in each of the nuclei of those white blood cells. From that, they, depending on how they did this, they may have captured out specific fragments and then read through all of that using what we call short -read sequencing. Again, my guess in terms of this. That short -read sequencing was probably less than 100 nucleotides for each little fragment. As they did that, they then had to do that, informatic computational step of putting all of those pieces together.

40:49In most cases, they were probably able to do that because they had a reference sequence. They knew what the average person looked like and they put your sequence right on top of that. And to the extent that those mapped uniquely, they could put that all together. There may have been those some of your sequence where they didn't know where it fit. And so it got sort of put aside in an area that they didn't even analyze. But there were some of your sequence that probably got put aside that they didn't know where it fit, how the pieces fit together. And in fact, the reason I say this, and this is very, very highly technical, is that we're not perfect at doing this.

41:22And so there are times when there's information over here on the side where we haven't used. As they did that, they're then able to read out. And depending on the purpose of what your analysis was, they could read out and they could say, well, for instance, if you are interested, I don't know anything about your own medical situation. But if they said, well, we're interested in knowing whether or not you have PKU. They could look at your phenylyl and ehydroxylase gene. they could read out all the sequence. And warn you, as I said, one in the thousand base pairs, there's gonna be a difference in this.

41:50And so they see a difference, then they have to do an interpretation. And the interpretation is actually a lot more sophisticated than one might imagine, because again, there are literally tens of thousands of genetic variants in your genome, and what they mean, and whether or not they do anything whatsoever is hard to know. Each of us has what people think of as mutations, or genetic variants that are associated with disease and cause a problem. Each one of us has those. Some that are very, very powerful. Some that are kind of wimpy, and they infer some very small risk. But in the aggregate you put together a lot of these little wimpy genetic variants, and it may amount to something more substantial.

42:31So depending on when your genome was sequenced, when it was most recently interpreted, you might have gotten really profound, powerful information in terms of taking care of yourself, and you might have gotten the sort of, we don't see much here, you know, sort of you go on your merry way. And for the average person, my guess is if you were middle age when this was done, for the average middle age person, it's mostly, we don't see much here because you've survived, hopefully as a relatively healthy person to this point, that you've essentially made it through some of the most devastating things we can see in the genome.

43:03One more question before we leave the whole genome sequence and talk about the whole exome sequence. One thing that they learned in me that was quite interesting was that I was mosaic for a certain gene. This was only realized because as part of this clinical trial, everyone in my family was sequenced, and one of my children had a full copy of the gene, which they got from me, but I was mosaic for it, so I didn't have very much of it. Can you explain what that means? So this can come up in a couple of different ways and I can talk more or less about this if you're interested, but you would think we're the same every cell in our body exactly the same.

43:40But in fact, that's not true. And your viewers or your listeners rather will realize this when we think of cancer. Our genomes are not stable over our life course from the point of conception to the point of death. And when you think about every cell division, if you have to copy over three billion letters, we're not perfect. And we have spell checkers to try and catch these things. but our body doesn't always catch them. And over time, mutations can accumulate in the body and of course as they accumulate this may lead to a barren cell growth, which is essentially what cancer is. And so cancer is at the heart of genetic disease, but oftentimes not from the genes you're born with, but for the changes that happen over your life course.

44:19Now, in certain individuals, and I'm guessing this was the case when you just described your family, this will be true not just of your skin cells. for instance, if there's too much UV damage to your skin in the summertime, but this can happen in your germ line as well. So it can happen in the egg and the sperm. And when this happens, again, as what we call somatic mutations or mutations over the life course, again, if they're in the germ line, they can be passed down to the next generation. And so you can be what we call a germ line mosaic. You can be a mosaic and mosaic just like a tile pattern that you see in a bathroom or something, right?

44:53where you have some color tiles, one color and some other tiles, another color, there are different patterns, because some cells have the mutation and some don't. And so in the same way, you can have what we call gonadol mosaic, meaning that the germ line is affected. In some cases, you can see those gonadol mosaicism, that mosaicism in the blood as well. So what you were talking about in terms of getting a blood sample, you might see that a certain fraction of the cells have those mutations in the blood. And then if you see them in the next generation as well, you'll know that it was transmitted through the germ line.

45:24I'll share an interesting factoid for individuals. The number of those mutations in the germ line actually increases over the life course. And so in particular, if you think about the biological process for spermatogenesis with men, those sperm continue to divide over the life course and those mutations can continue to to accumulate over the life course. And so in fact, some of the conditions that are associated with denovon mutations or new mutations, we see the frequency of that being greater for parents, for instance, who are older parents at the time of conception than for parents who are younger at the time of conception.

46:02And it's not, you know, that it's like astronomically exponentially higher. It's a linear process for those types of mutations, but we do see those increasing over the life course. Historically, we would assume that women are more susceptible to that via age. I mean, the rate of either aneuploidy or mutation seems to rise more sharply with women earlier, starting probably in mid to late 30s. You are pointing out that the same is true of spermatogenesis. Am I correct in saying that the egg seems more impacted by the sperm? And if so, why is that? Is it a more complex division? So what you're bringing up is that meiosis in the two sexes is different and it is susceptible to different underlying biological processes.

46:46So as you're saying, for women, if you look at the curve in terms of problems and you ploy to your sex or not sex, but rather chromosome differences, namely Down syndrome is what many people think about, increases with advanced maternal age. And that has to do essentially with the stickiness of the chromosomes at meiosis in the ability to separate or not. And so the curve that is associated with that, which many people learned at some point, is that there's an inflection. It's not a linear relationship with maternal age, but as you said in the mid -30s, it starts to increase more significantly.

47:21And as a result of that, there's a whole sort of medical way that we can follow women when they're pregnant to try and pick up if they're interested, those particular chromosome issues. the difference when it comes to what we call DNA sequence differences. So again, not whole chromosomes, but single letters is that that process of being able to have the cell divide and replicate and copy over that information happens at every single cell division and there's a certain probability that that will happen. And so that's a linear relationship in men. And based on the biology men obviously from a reproductive point of view may have children over a larger period of time so we can see greater differences across men as they're reproducing.

48:04So biology is a little bit different between the two sexes. We have a clear sense why you see more of these meiotic differences with age. In other words, what is the fundamental characteristic of aging that is driving that? Is it sort of like evolution says, well, I don't care because I don't want you to reproduce after a certain age, you know, again, not to anthropomorphize evolution, but therefore I'm not going to preserve the integrity of your genome beyond a certain age. I mean, I'm curious as to see, see, me that strikes me as an explanation, but not the reason. The reason must be something more fundamental at the level of an aging hallmark process.

48:46I hadn't thought of the question quite that way, but I do think if you think about throughout out all of human history, the age at which people were having, reproducing and having children has skewed much younger than it is in terms of current society. So I think we have pushed the boundaries to a certain extent in terms of what the biology historically has been. I don't know if we had a, you know, use before date in terms of ovaries and gonads before, but that I think just historically has been what's happened. So within that, the fundamental biology for women has been proteins that are responsible, as I said in terms of meiosis and separating of the chromosomes and being able to have, if you remember when meiosis starts in females, it actually is starting way, way early ingestation.

49:32So even during fetal life and so those proteins have to be working in intact from before a fetus is even born, before a child is born, lasting all the way through whenever that pregnancy is conceived essentially when those gametes are finally dividing. So that could be 30, 40 or more years for that process to have to work. And that's kind of asking a lot when you think about the biology. For men, I'll just throw in another interesting factoid that for some of these mutations that arise during the process of gameted genesis, there are even what we call selfish sperm mutations. That is that certain mutations may even give a selective advantage to those spermatocytes where they may have a reproductive division advantage, for instance.

50:16And so we may see more of those in terms of the biology of what we see in the next generation because of the effect they have even in terms of directly on the sperm. So lots of biology at the root of that. So let's go one step down from the whole genome sequence to the whole exome sequence. So if as part of my blood test, I only wanted the whole exome sequence. Are they actually doing the whole genome sequence and just reporting out the exomes or are they actually doing something technically different? So to answer the question, you have to read the fine print on your genetic test report or your clinical trial consent or whatever it is.

50:51So I don't know the answer, you know, without knowing that. It certainly was the case that a few years ago, not that long ago, it was so expensive to sequence a genome that we rarely did it. There was really just cost prohibitive. The exome was a really good shortcut because we didn't know what a lot of the other information meant anyway. So we were kind of it would have been just throwing it away. Increasingly, we have a better sense of what the non -coding regions do. We have better ability to interpret and recognize genetic variants. And so I would say as the sequencing costs have been coming down, there's more of a shift to going to genomes rather than exomes.

51:29But truly, most of what is medically used at this point is in the coding space. So even if you're sequencing a genome, it's still focused on the coding regions. On a research basis though, very different. And of course we have to do research before we can apply it clinically. A lot of the research now is understanding what all of those regions do and being able to eventually use that information clinically. So I do think within the next decade we're going to see a shift. And we may even shift from this what we call short -read sequencing of these 100 -base pair fragments, even to things that are much longer in terms of accuracy, able to read through some of the greater the areas that are more complex and probably that we've been missing out on before.

52:08And a short read, you said, is about 100 base pairs? What's your minus, yeah. So what's the technical limitation for making that longer? So as you do this, there's just lower and lower fidelity for each base pair that you do. And so at some point, you start getting errors within your readout. Then you don't want to have too many errors, because you can't distinguish between what are true biology versus what are artifacts of what you're doing in the laboratory. How are you correcting the errors? If you're getting even one error every in one short read sequence that would give you, we only differ from each other by one and a half percent of those base pairs, that would be, for example, you wouldn't be able to use this information in court.

52:47How do we preserve the fidelity of this to make such bold claims as, hey, we found this blood at the scene of the crime and we absolutely know dispositively it belongs to this individual? We do it not just once as the answer. So in fact, when we read this out, we have a term called redepth. It's how many times we look at any one nucleotide in the genome, and we don't look at each position just once. Depending on how much we want to spend doing it, we might look at the redepth of 30x. So we might look at every one position on average 30 times. And if you see out of the 30 times 29 of one nucleotide, one of the other, you say, oh, that one, the odd ball there, that's probably just a sequencing error.

53:28that's an artifact of our method in the lab. The other 29 are the ones that we want to pay attention to. In some cases, again, I bring in cancer. It becomes very important to know for those somatic mutations that weren't there from birth, that they may be there in a small number of cells, but cells that can be very, very dangerous for cancer. And so we may increase the redep to be very, very high. We may go up to 1 ,000X in terms of redep to make sure we've got the accuracy to see something reproducibly even in 10 % of cells. but again, we're 10%, you need 10 % of a large number to have the assurance that what you're seeing is in fact representative of the underlying biology, not an artifact.

54:07Let's talk a little bit more about an example there. So you brought up BRCA1 and BRCA2. So typically the genetic tests that would be done off a whole genome sequence or even a targeted sequence because let's say a person says I want specifically to be tested for breast cancer genetics, but you're still testing on the actual DNA taken from white blood cells, correct? So these days we do things lots of different ways. And just again, for some of the listeners, we've tried to make everything we do more accessible. This is a fundamental sort of core value for me. So as an example, we can even now do things from cheek swabs, from saliva samples, from blood samples.

54:44So again, we try and make it less invasive, easier to do, even potentially from home. And so for some people who have done something like 23 and me that you may have even done it that way Let's pick one of those examples a cheek swab or a blood test and you're gonna do this enormous depth of readout to really make sure that there are No copies of the bracket gene or any of the other genes that you're looking at how many genes by the way when we do breast cancer Genetic screen how many known genes are we looking for so interestingly It's a little bit of ala carte. So what I mean by that is it's your choice, it's your choice either as a doctor ordering the test, it's your choice as a patient that's getting the test.

55:24In some cases, you know you have a family history of a BRCA mutation. And so within that case, we may know the exact address to go to and it's a very simple plus minus readout. And we don't have to do a whole genome sequence for that. We just need to look at one gene and say, yeah, your nay, it's there or not there for that particular variant. And beyond that, they're even, I alluded to this before, but there are certain variants that are seen in certain communities. So as an example, if you happen to be Ashkenazi, Jewish, there are three different spots in BRCA one or two that account for the vast majority of all mutations in those two genes.

55:58And if you know that, we can take a shortcut and we can basically say, for literally, a small fraction of the cost of sequencing a genome, boop, boop, boop, we look at those three spots, we get yay or nay, and you've got most of the information that you need. to your point. There are some people who come in with a family history of breast cancer, and they say, but I want to be careful. And so in that circumstance, we may do a panel of 50 different genes, 5O different genes that will cover most of the genes that we see for hereditary, not just breast cancer, but ovarian cancer, colon cancer, the most common cancers that we see that are driven by germline or inherited genetic factors.

56:37So for round numbers, 50 is a good number when you're trying to be really comprehensive. If you said, just give me the focus, breast cancer things. It might be more like 10. You've said this twice now, but I think it's helpful for folks to listen. When last I checked, maybe 5 % of cancer was accounted for by germline mutations. 95 % of cancer is accounted for by somatic mutations. Is that still accurate, would you say? I'd say I'm going to modify that just a little bit, not to be a contrarian, but for the genes that all call monogenic, highly penetrant. Let me unpack that a little bit. Monogenic.

57:12Monogenic single gene, highly penetrant, high probability that over the life course, you'll develop cancer if you have this particular genetic variant. So when you limit yourself to that, yes, about 5 % of cancers are due to those powerful single genes, high probability of cancer. Now on the other hand, over time, we've realized that there are additional genes that all called moderate risk genes. Many of those genes may confer something like a 2 to 3 -fold increased risk as opposed to something like a 10 -fold increased risk. So there's another probably 5 % or so that are due to those. And then there's this other thing that we call polygenic risk.

57:52Poly meaning multiple, genic genes, polygenic multiple genes. And the number of genes we oftentimes look at in those circumstances may be anywhere from 100 to 100s, or even in some cases thousands of genetic variants all mathematically summed together to understand what the risk is associated with that package. All of us have genetic variants that go into that polygenic risk. And part of the question is, along a distribution, are you at the high end of that risk curve or you at the low end or at the average end? And so within that, this is now something that is not clinically being utilized routinely, but we are on a research point of view trying to understand clinical implementation for now those polygenic risks for cancer.

58:39Assuming that that amounts to, I don't know, we'll figure it out, but that might amount to 10 % of cases. You'd say, well, look, 20 % of cancer has a genetic component, as opposed to, and it's broken down into those three categories of monogenic highly penetrant. I think the second category was it monogenic, not highly penetrant or not monogenic. I call it monogenic moderate risk. Moderate penetrant and that polygenic. Right. And those would be the three. And then going back to this case of, say, the breast cancer example, a woman says, I just want to do a deep dive on breast cancer. I don't know which genes it is because all my families deceased, but four women in my family have died of breast cancer.

59:21We're going to do this cheek swab and you're going to look at 10 genes that are associated, plus whatever the polygenic genes are. Why is it that you don't need to look directly at breast cells? Why is it that we can infer that what we see in a cheek cell and an epithelial cell rather in the cheek or in a monocyte in the blood is also captured in mammary tissue? Good question and the answer is it's probably not. So what you're doing when you're doing the cheek sample, the blood sample, is you're really getting at the germ line. So you're mostly getting at what you are born with, what that inherited susceptibility is.

1:00:02As we talked about though, your genes are changing over your life course. Your cells are changing over your life course. The cancer doesn't happen overnight. You don't go from a normal cell to a cancer cell overnight. There's a progression in terms of going through this. And so there are other ways that people have thought about that. I'll call it a liquid biopsy. So this idea that you might be able to, and it's a slightly different test than what I was describing before. But where you would look for these somatic mutations, you describe this before. But when you're looking for that needle in a haystack, if you've got a tumor that's going to slough off some of that DNA into the circulation, you might be able to see some of that fragmented DNA floating around, and you might be able to pick up some of those mutations that might be reflective of that mammary cell that's either gone or I and is a cancer, but maybe not something that you're detecting on mammography or something else.

1:00:56And so this is in some ways been the holy grail of being able to do cancer screening. It's not quite ready for prime time yet. And people think about it more, I would say, right now for thinking about recurrence of cancer. So how do you monitor someone who's had a previous cancer diagnosis? You think they're all clear in seeing whether or not they've had a recurrence. The other use case people have thought about is someone who might be at high risk of cancer, so someone who's identified in whatever reason based on an exposure, based on a genetic profile, but it's not ready yet for population screening in terms of being able to pick up cancers at an earlier stage.

1:01:32We're still relying on other things to do that. So let's now talk about what happens at the level of the 23 and me's and the ancestries and companies that are obviously doing something far less than a whole genome sequence or even a whole exome sequence just on the basis of the cost at which they can offer these things. What are they technically doing with the epithelial cell of your cheek or the saliva or the white blood cells that they get? Again, and I'll say, read the fine print of what you sign on the consent form. Number one, it may change over time and I don't represent any of those companies so I don't want to miss speak in terms of what they're doing.

1:02:10They are in general though, number one, not trying to detect cancer. So any of what I talked about, not the purpose of what they're doing, they're in general not trying to read out the genome, at least not for the purpose of getting you medical information for what I call news you can use to manage your own healthcare. They're largely doing it in a way that I'll call more recreational. And so with doing that for any of you who have done 23 in me, you may find out something about, for instance, If you were to eat asparagus, what your urine might smell like or what your ear wax might be like or if you're lactose intolerant.

1:02:43They are things that are related to how the biology of your body works. They are related to genetic variance. So those two things go together, but they're not telling you based on your ear wax if you're going to have major problems with hearing loss down the road or cancer risk or things like that. So that's why I use the term recreational in that way. But what are they technically doing? Depending on the company and depending on what they're doing, they're oftentimes reading out what we call single nucleotide polymorphisms or so -called SNPs. So they're not reading out the entirety of your genome.

1:03:13They're not reading out all three billion base pairs. They are selectively going in and saying, at this exact address, do you have an A or do you have a G? At this other address here, do you have a C or do you have a G? And based on that, they may selectively look at those particular variants and say, with your profile, I know that your family originally came from Egypt. or wherever it is, you know, in terms of being able to look at Amstressry, where they may say, based on looking at this, I know this particular genetic variant may predispose you to be lactose intolerant. I would expect that you're going to have problems in terms of eating ice cream for dessert tonight.

1:03:49You know, that's generally the type of thing they're reporting out. Depending on, again, the company in the terms of the agreements, there may be differences, but generically, that's what many of them are doing. And so if a gene differs by more than one nucleotide, a snip is not of much use, unless you sample two snips in the same gene. It gets tricky. So technically a single nucleotide polymorphism could be a genetic variant that has a big, big effect on a gene and could, from a medical point of view, be very, very impactful. So it's not just the size that matters. It's, you know, as some people would say, location, location, location, it's like real estate.

1:04:26So it all matters which variant we're talking about. But in general, the ones that they're looking at are not the ones that are medically impactful. They're just normal variants that are innocent by standards, but help us understand where our ancestors came from. For the most part, that's what the companies are doing. Although they do comment on some important ones, so you mentioned isoforms as normal variants. So the ApoE gene has three isoforms, the two, three, the four isoform. all are relatively common. I mean, the three being the most common than the four, the two is not that common, but all would be considered, quote unquote, normal variants.

1:05:03One obviously comes with a much higher risk of neurodegenerative disease. A 23 in me test does read out that prediction. Presumably that tells us that those genes only really differ by one base pair, correct? The two, the three, the four only differ by one base pair. That's therefore the only place they need to sample. But I've seen in 10 years several instances of a miss call, meaning the SNP read ends up not matching with the more rigorous exome sequence. Why do you think that's the case? Does that surprise you? I will say that in doing this, I'm not someone that has been asked to go in and QC or do quality control or anything like that for the laboratories.

1:05:48I have known of things as simple as these are done in plates that oftentimes are 12 by 8 plates and if you flip the plate the other way you can have sample switches and you've got a different person being read out for a different thing It can be something as simple as a logistic like that and I have seen that lab error before There can be sample switches at multiple places and at the end of the day I will say that if you are doing something from the recreational side to something where you are gonna make a major healthcare decision. You are, as a woman, for instance, going to go through and have a mastectomy, get a second opinion.

1:06:23Like, be sure that this is really you, and it's really the result you think it is before you do anything irreversible or, you know, go out and buy that big life insurance policy. And I would say that in general, you know, if you were getting a second opinion, back cancer diagnosis. You mentioned the Guardian study earlier. Can you say a little bit more about what that study is? So the Guardian study, as you can imagine, based on what I started out the conversation with Final Cutineria is I've always been wanting to be able to get information that people could use to be able to maximize health and being able to just be the best person they could be.

1:06:57And I started out in 1996 again had started out in the space of PKU decades ago and had been studying a disease called spinal muscular atrophy for about a decade with colleagues of mine. And this is a neurodegenerative condition and used to be the most common genetic it caused of death for children less than two years of age. And I realized, starting in 2016, that we were just at the cusp of potentially a treatment that might slow down or stop the neurodegeneration. Yet tragically, if we didn't identify babies before they started showing symptoms, it would be too late. That is, we'd have this window of opportunity.

1:07:36So we started out a newborn screening program for SMA and then babies that were identified through that had the option if they wanted to, of going to a clinical trial. That ended up being quite synergistic in the sense that we did identify babies who would have been predicted to have the most severe type of SMA. They did get into early clinical trials right away. They did benefit from those that helped in terms of the ultimate evidence that was necessary to show the efficacy of those treatments. And because of that, and because we were able to show that we could do it technically and that people wanted it, SMA has been added to the recommended universal screening panel for babies across the United States.

1:08:13And so now four million babies born each year in the United States are screened for SMA. And we have three FDA approved treatments, including a one -and -done gene therapy. So babies can now be identified within the first week or two of life, get a one -and -done IV infusion of the gene and go on to have much, much better life if not be quote -unquote normal. We've seen as far as we can see so far. So that got me thinking about doing this on larger scale. We've since done newborn screening study for Dushane muscular dystrophy and just recently actually was FDA approved a treatment for DMD, Dushane's muscular dystrophy.

1:08:47But I'm, I don't know, I'm getting older and I'm getting more impatient and I didn't want to do these one by one. I started thinking about how could we do these at scale for population help, but not just for one condition at a time, but how could we do it for dozens or hundreds or potentially even more conditions. And so So the guardian study actually stands for something. It stands for genomic uniform screening against rare diseases in all newborns. And if you put the letters together from that, it spells out guardian. And the idea behind that is to take that same newborn screening dried blood spot that we use already for PKU that we talked about.

1:09:25Sequence the genome. We don't need to read out everything in the genome. We only read out the genes that we consent people to read so that they consent to in the study. And those genes are genes that have, I call it, news we can use. Information that has treatments immediately available. And in planning this study for almost four years or just over four years with families, we had many, many iterations about what they wanted, what they wanted us to screen for, what should be, what should enable them to be the best parents and give their children the best chance in a healthy life. And we thought about also this dynamic change in what we'd have treatments for.

1:10:03The fact that the world is changing rapidly and we wanted the flexibility that if a new treatment became approved tomorrow, boom, we could instantly change the screen and be able to implement that. We wouldn't have to wait a decade to gather the evidence to do that. We wanted to be nimble and flexible. So the reason for using the genome as the backbone is it gives us that infinite flexibility to be able to adapt and to be able to move the field forward. So we've been doing the Guardian study in New York City since September 2022 and right now have screened just over 3 ,000 babies. And it really has been remarkable to me in terms of being able to see just the broad support from our community in doing this.

1:10:42In New York City, you should realize you prop most of the listeners probably do you realize the wonderful diversity we have in New York City. That is that of the people who participate, it's not just white folks, it's not just people who are from Ireland, we were talking about Irish individuals in P .K .U. but it's people from around the world. We have about a quarter of people of European ancestry, of Latina ancestry, of Black ancestry, of Asian and other ancestry. So it becomes really, I think, representativeness, or representative, basically, of the world. And it also is geared to leave no baby behind, because newborn screening is kind of this one universal thing where everyone goes through the health system in the same way.

1:11:23And by making this free and being able to allow everyone to enter if they so chose, we can really see also what information people want and what they don't want. Within this, I guess one of the things that's been refreshing to me is to see that about 74 % of parents that we approach and offer this to decide they want to do this. And that's an important number to me. When we did this for SMA, the number was 93%. When we did at this for too shane muscular dystrophy, it was 84%. And so within this, it's not 100 % of people who want any of these genetic screens, and that's perfectly fine. I'm not trying to force anyone to do anything, but it's also not 10%.

1:12:02The majority of parents are saying, yes, there's something I can do to ensure that I have a healthier child's, like give it to me, like help me be a better parent. Why would I not wanna do this, is mostly what we hear? Within this, I also appreciate, and we do this with regular newborn screening, just the traditional newborn screening. And we realize that traditional newborn screening isn't perfect. I never thought it was, nothing ever is. But we realize that adding this additional dimension, and we've even done it for PKU within this study, this additional dimension helps us to do a better job.

1:12:33And so just as an example, we've also identified part of newborn screening identifies some children with severe combined immunodeficiency, a problem where you can have an overwhelming infection and die from this, but treatments is available, including a bone marrow transplant. And so because of that, we have as part of newborn screening a way to screen and identify some, but not all children that have that. We've added this now genome sequencing to enrich and improve that. And in fact, identify a baby that was missed by our traditional newborn screening for Skid, yet is at increased risk in terms of this overwhelming infection, but yet with the opportunity to intervene at an early stage which one a bone marrow transplant will be most effective.

1:13:17And so there are numerous examples where we've identified whether it's Wilson's disease, whether it's severe combined immunodeficiency, whether it's a congeroplasia, but other conditions that are treatable that we just needed to identify those babies. And as we've done that, the number of children that, and I just know, because I've been practicing in New York City for 25 years, I know sort of how people navigate the system and how they get through and we've been able to really get to many of the people who are usually unfortunately left behind either because they're immigrants, they don't speak the language, they don't have the same health insurance, but individuals that we're seeing come out positive for this are very, very different in terms of reflecting our community than the people who navigate the health care system and get into CS.

1:14:00And we realize based on other studies that we've done that most of the children that would have been diagnosed, if ever they were diagnosed on average aren't diagnosed until somewhere between 8, 9, 10 years of age. And so we're intervened or were able to identify them literally a decade earlier before a lot of damage has been done to their body. So it's just the beginning, you know, 3000 is great. I think it demonstrates that we can do this. I think it tells us what our community wants out of this. It shows us some pitfalls in terms of how it's hard to do and what we need to do to do it better.

1:14:34But I do fully believe that this both derisks this in terms of being able to also have groups that are working on therapies, be able to realize that this is something. There's an opportunity now for treatment for this and is a powerful way of moving forward health equity, at least for children for the next generation. Is this something that's done only at Columbia or is it a multi -center New York hospital endeavor? So right now this is done through our New York Presbyterian Hospital system, so it's not just Columbia, but it's through this hospital network. It's only so far in those hospitals, but based on our success for this, we are figuring out how we can be able to expand this more broadly and really think about this as I said, as integrating within the public health infrastructure, not trivial to do this on scale.

1:15:19As an example, doing this in New York State, if we were to do this for every baby, we'd need to do it for about 210 ,000 babies a year. So no small feat, but something that we're gaining the experience, you know, what the pain points are and how to solve for them. is this all funded by NIH? So within this, as you can imagine, this is not inexpensive to do. So in fact, none of this is funded by NIH. NIH, I won't go into all the details, but NIH is able to fund programs that are about this big. People may not see this if they're just listening to me, but very small amount. This ends up being about two orders of magnitude, larger in cost than anything that NIH can fund.

1:15:57And so it's a challenge in terms of, as you think about, big bold new transformative ideas. How do we as a scientific community accomplish these? And so we've done this by putting together many different stakeholders. I don't think any one group could be able to take this on. And truth be told, we're not completely there with the funding. I think we needed to demonstrate that we actually could do this in this first feasibility stage before gaining the resources to do this with what I hope will be at least a hundred thousand babies to get to the sample size we need to see some of these rare conditions and to know what the outcomes are and that we really can screen for them.

1:16:32So what's the actual cost of doing the sequence for each baby so to look at those 250 samad conditions? What's the bench cost? So as we started out doing this round number $1 ,000 per baby. So thinking about generating the data and interpreting the data, getting it back to folks cost of about $1 ,000 per baby, the goal is to be able to do this and get it down by the order of magnitude? Can we get it down to $100 per baby as an example? And in doing that, can we think about the economic impact? Most importantly, the health impact for the baby, but as we think about as a society, how to be able to afford doing this, we are doing the economic analysis to understand.

1:17:13But the good thing is sequencing cost or decreasing, analysis and interpretation cost or decreasing, more of this can be done in automated ways, as we understand what normal variation is for people around the world. And that's one of the critical factors is doing that around the world. Now that thousand dollars is a fully loaded cost. That's the interpretation, that's the overhead, that's the PI time and such. The sequencing cost must be significantly less than that, given that aluminum could do a whole genome sequence for a thousand dollars now, right? Even over the course of this study, the sequencing costs have come down.

1:17:45If you can imagine it, and we just started it like I said September 2022, less than a year ago. But already the sequencing costs have come down. I expect they'll continue to come down in terms of this. and so data generation certainly can be done for well less than a thousand dollars now, but as you said part of it is the interpretation and we have study staff that explain the study to everyone, explain results to apparently so, yep, includes multiple pieces. So at the outset of this discussion you mentioned that SMA actually has a successful gene therapy. I was not aware of that. Yes. How many of these single gene highly penetrant conditions that children are born with, be it inborn areas of metabolism or inarity, genetic diseases, etc.

1:18:27How many of them have FDA approved gene therapies already? Not very many of them. So really the shining example is SMA or spinal muscular atrophy. There are very few gene therapies that are now approved. There are now, for instance, I mentioned Dushan muscular dystrophy, hemophilia. There are a few other conditions, but it is still literally a handful for gene therapy, literally gene therapy. Others have treatments available. And I think for many of those, I'll just give one example that we've had through the Guardian study, a condition called Wilson syndrome, for instance, that leads to ultimately liver failure and need for liver transplant.

1:19:05The treatment that we give children for this is zinc. So it's something that it doesn't require gene therapy. It doesn't need anything that fancy. We can simply use zinc to out -compete copper and make sure that we don't end up with a copper over -ofer blood situation, and we have treatments that are very well -tolerated, pennies a day in terms of doing this. We hope extremely effective long -term. So, although gene therapy is wonderful, I want to underscore we don't always need gene therapy, as long as we have that early diagnosis. If there were three diseases today that you see in a pediatric practice that would be most amenable to gene therapy, in terms of some aggregate score of the technical nature of doing the gene therapy and the lack of alternative therapies elsewhere and the number of kids afflicted.

1:19:54If you were sort of to take that as your triple proxy, what would be, if you could wave a magic wand, what would be the three diseases you would want to put high on the list of gene therapy targets? That's a great question. I don't think I've ever thought that through exactly in that way. So there are a lot of neurological conditions. Let me start with that in terms of places that we are woefully behind in terms of treatments. And I'll give you one shining example of TASACs disease as an example. Many individuals, the way they've dealt with this because there's no treatments, is simply not having children or not having children together or going through other reproductive options.

1:20:33But things like TASACs disease is just a terrible condition. And it has relatively high population prevalence to one of your points. and has really nothing available in terms of treatment today. And so those children are born healthy normal children and die within the first few years of life with a degenerative course oftentimes associated with epilepsy. So a condition, tastes, acts, or a condition like that, I think fulfills all the criteria that you're talking about. Other similar conditions like that, although we still need to understand treatability or things like fragile X, another condition that we see in this case x -linked.

1:21:11By the, you can tell from the name fragilex similar in terms of high frequency, really nothing in terms of treatability at this point. And perhaps the ability, it's a, we can get into it or not, but the gene therapy for this is a little bit trickier. Different strategy from a technical point of view, and then one would take for tastesacks, so whether it's gene therapy or gene editing or other types of things, there would be a different technical strategy. And then I would say they're just a large group. I won't pick one, but large groups of inborn errors in terms of liver disease that primarily affect the liver.

1:21:44So whether you're talking about something like maple syrup, urine disease, progaryonic acidemia, but something like that, that we have good programs in place to identify those children's and our treatments. They're just not up to snuff yet. They're not quite as good as they should be. Well, let's talk about how genetic therapy works. So, God, probably 23 years ago, we had a tragedy in one of the most highly publicized examples of early gene therapy. The tragedy, of course, really didn't have to do directly with the gene therapy. It had to do with the vector that was used to deliver it and the young man who received that gene.

1:22:22I can't remember what it was for. It was for an informer of metabolism as well, wasn't it? Eurea cycle defect called Ornithine transcarb amylase deficiency. Yeah, and Jesse, what was his last name? Jesse Gelsinger. Gelsinger, yeah. And this was at Penn at Chop, right? That's right. About the year 2000, if my memory is correct, maybe 99. I think that's about right. Maybe off by eight. Yeah. And so let's talk about that. Let's talk technically about that. So they used an ad no virus, but explain what that means and what was the state of the art 25 -ish years ago? And let's contrast that with what's being done today.

1:22:55For those of you who are listening, ad no virus is obviously causes the common cold. And so whether it's adenor, adenor associated virus, we oftentimes use that as the vector or the delivery vehicle to deliver genes. Within this, the viruses are manipulated. They're engineered, so to speak, so that they're not going to be contagious. So even though you might get a cold or pass it along to someone, you're not going to do that with gene therapy. Yet there are problems with this because the common cold is common. And so people may have been infected with adenoviruses and their body may try and mount an immune response when it's infected, as it would be with a cold.

1:23:32And that's where a lot of the mischief comes in. And unfortunately, it hasn't ended with Jesse and Jesse's death. There have been other deaths in the gene therapy space with others that have had a response to the vectors. Oftentimes an immunological response to that. In Jesse Gelsinger's case, it was the vector in the gene therapy who was targeted at the liver, we've talked about a little bit of that already, but sometimes there can be an overwhelming response from the liver where the liver starts to fail, where there's an immune response that goes on. And so, as I said, this has not been solved completely at this point.

1:24:10There have been other genetic therapies, other diseases, not just liver diseases, but where there have been similar responses. And I think one of the things that we've learned from this is we have to be very careful with people with underlying liver disease when it comes to this because a fragile liver can get tiptofer, especially with adenovirus. We also have to be careful about who's been exposed to those viruses, so sometimes we do screens to be able to see who might have one of these responses. But ultimately, and partly because of that, people have also been trying to figure out other delivery systems, other vehicles, other ways of being able to get those genes into cells that may not be as toxic or problematic.

1:24:47How may understand a little bit, you know, so an adenovirus is very common, Presumably, in the case of Jesse, he'd already been exposed to some antigen that was related to this. And so he already had memory B cells and memory T cells that were ready to mount a healthy immune response should he have been exposed to that very common adenovirus again. The fact that he had such a harsh response to the gene therapy was that because of the dose of adenovirus that he received, or does that imply that he would have had some sort of catastrophic multi -system organ failure? Had he just had another exposure to that exact adenovirus as a cold and that as you said was a function of his underlying liver health.

1:25:29So it's a very tricky situation and I say this because there will be some either patients or doctors advising patients about genetic therapies or genetic therapy trials in the future. It's tricky in the following sense. You're right that there's a dose response in terms of the immunological response, and so you don't want to go too high on the dose because you don't want to have too big a response. On the other hand, with these gene therapies, you oftentimes get one chance at this in terms of doing this because once you've given the therapy, the body is going to amount an immune response to that and would neutralize that same therapy if you were to give that again.

1:26:07And so the tricky thing about this is, you don't wanna go too high and you don't wanna go too low, because if you under -dose it and if you don't get enough in and that's your one shot on goal, you've burned it. And so within this, it's a tricky situation to figure out how to get it just, right? And as it is with many clinical trials when you're first in human, you don't know, right? It is first in human. Oftentimes there are non -human primates in terms of trying to figure out as much as you can, but it's still not a person, and each person is unique. And so as you're doing it, it is a tricky situation in terms of getting it right.

1:26:38When you do the first person, you learn and you figure out from there whether you're going to go higher or lower, but there is someone who's going to be the first person. And there's no way to engineer the adenovirus to make it invisible to the immune system while still able to insert its DNA package into the cell. So there's certainly things that we do to try and do this better. As I alluded to, one of the advantages for someone, for instance, with SMA, where we're dosing them at a week or two of life is they haven't had the common cold. They haven't been exposed to these things. They've got a fresh immune system.

1:27:11So the likelihood they're going to have a response like this is much lower. So we haven't been seeing those types of things with newborns that we've been treating with SMA. There are other vectors, there are not just vectors, other delivery vehicles that we use that are not viruses. And so that's something else that in terms of developing new technologies, things that may not pose the same problems. I don't want to guarantee that that's going to be the case, but certainly with the experience we've had, even from the COVID vaccine, from mRNA vaccines, and having delivery systems that were basically lipid nanoparticles to deliver nucleic acids to cells, We've learned a tremendous amount from millions of people who have been treated with that and some of those technologies may prove to be helpful with other delivery systems.

1:27:56Maybe this is a good time to explain what CRISPR is and how it factors into gene editing. Although I guess before we do that explain what is required to change a gene. So I don't know, pick someone with sickle cell anemia. So, if you wanted to use gene therapy to fix, quote unquote, sickle cell anemia, my vague recollection from medical school biochemistry is that was a one amino acid change, correct? Correct. It was valine one of them. Yep, so the eighth amino acid, exactly in hemoglobin beta, is that there are two different sort of variations or flavors at that amino acid you can change, but that causes sickle cell disease, yes.

1:28:40Okay. So if you want to permanently change that, it's not enough to do it in the red blood cells that are floating around in the bloodstream because they're going to be trashed in this plane a couple of months from now. You must change the DNA of the stem cells in the marrow, correct? It's exactly right. You have to get those progenitor cells from which the future generations of red cells will be derived. And let's assume you had the correct gene sequence. That's not hard to do. we know what that is, and we can put that into a virus, and maybe just explain to people why viruses are great vehicles.

1:29:15What is it about a virus that makes it an ideal candidate here? Well, the nice thing about a virus is it was designed by Mother Nature to infect ourselves, right? So it's pretty good at being able to do that. The viruses that you're talking to in the way that I think about them are often helpful for gene addition. So where you've got a protein product that hasn't shown up for work, it's not working, it's a loss of function, it's not present. You need to be able to deliver or add back that gene. It may not be integrated into your genome, and in fact, there are probably some advantages if it's not, but the virus can bring that in and bring it into the cells.

1:29:51And to your point, and this is important, many times that means bringing it into stem cells so that they can have the longevity of continuing to populate the body over time. And so is it safe to say that the real challenge is that step. It's not just putting the corrected version of hemoglobin, the gene for the corrected version of hemoglobin into the virus of choice. It's figuring out how to get that to selectively, in fact, a progenitor stem cell within the bone marrow. Is that presumably why we don't yet have genetic therapy for sickle cell anemia? Well, sickle cell is interesting in that there are a couple different strategies people think of.

1:30:31So one is gene editing is the term I'm gonna use. So it's fixing the gene in sort of where it is, not adding a gene, but actually fixing the gene that's present. Another strategy that people think about, again, for the Efficient Nautos who are thinking about that, as I mentioned hemoglobin beta in terms of the adult form of hemoglobin, there's also fetal hemoglobin that's made in utero. And so with that, that actually has a very tight binding of oxygen because the fetus needs to be able to get oxygen from the maternal blood, and it can substitute for adult hemoglobin, actually quite efficiently.

1:31:08And so one thing one can do is actually turn up the amount of fetal hemoglobin expression, and essentially be the incite -to version of your gene therapy. It's just manipulating the gene expression, but for a gene that's already present in those individuals. And so there are ways of manipulating gene expression in that case for fetal hemoglobin. So there are multiple ways to scan a cat, so to speak. Very technically different in terms of what we administer to people and what we're changing in terms of the gene therapy. But I want to pick up on what you were talking about with gene editing because that's yet something else.

1:31:43That's really in sight too. Yeah, and I want to come back to that. Let's go back to one more thing on the gene addition. If you were to add a gene for a corrected version of beta hemoglobin, would you actually run into a problem now where you're making too much hemoglobin? And half of it is appropriate and half of it is sickle and therefore not. And you could argue you're creating more problems because you've doubled the hemoglobin, but you still have the issue where this cells sickle and create all of the distal ischemia that the person has. So is that the real reason that nobody's interested in doing an addition therapy for sickle selenemia?

1:32:22So that's exactly right. Just for the listeners, we don't use the gene addition strategy for sickle selenemia because we'd have to dilute out, so to speak, so much of the hemoglobin with the sickling that physiologically rerun into other problems. Okay. So gene editing would hands down be the best solution for certainly a situation like that, and probably many cases could it be done with high fidelity and ease. So I guess let's talk about gene editing in any way you see fit in whatever way you want to tell the story. I mean, gene editing in and of itself is a whole podcast, I suppose, but what's the medium version of that story?

1:33:02So from a simplistic way of thinking about this, it's going in and insight to being able to correct the genetic variant. Now realizing we've talked about single nucleotide variants, those are the easiest ones to edit. There's just one single base pair that needs to be flipped. It actually matters what that base pair is. If you have to change an A to a G or a C to a T, believe it or not, there are different base editors that can do different types of nucleotides, which is. But there are many mutations that are not just single nucleotides. There may be multiple nucleotides. There may be multiple repeats, so these sort of complex things that we talked about with fragile X.

1:33:38There may be entire chunks of chromosomes. They get very complicated. And furthermore, it may be when you think about population genetics, you may have a mutation distribution across a gene that may be quite heterogeneous. Cycle cell is an easy one. In the way that you mentioned, you're talking about the same position for everyone with cyclocell disease. A couple different nucleic acids, but it's the same position essentially. Whereas other genetic conditions, it may be that almost everyone has a different mutation. So you have lots of different things you need to edit, and that may be easier or more difficult to do.

1:34:12So there are some nuances in terms of that. You mentioned CRISPR. So CRISPR -Cas9 is something that many will know because Nobel laureates were awarded for this amazing discovery, which was by the way I will say just really good science with creative women who are thinking about other ways to use it. It wasn't fundamentally the discovery was not made with the intention of doing genetic engineering or you know genetic manipulations, but really smart people thinking about it. The CRISPR -Cast9 system has, I think of it as an Achilles heel of a double stranded DNA break. So it fundamentally in terms of being able to make the correction has to cut the DNA, cutting the two strands of the DNA to make the correction.

1:34:55And that fundamentally leads to some instability as the cell repairs that process, which you use the word fidelity, which I like the use of that term because it really is all about fidelity and potential off -target effects, where you inadvertently introduce other than the intended correction, other genetic changes, and sometimes those other genetic changes can cause mischief. We call them off -target effects. So things where they may inadvertently destroy the gene, cause other changes to the gene, cause other changes to other genes that you weren't even trying to target, but it can lead to problems of, essentially, promiscuity or inaccuracy or low fidelity.

1:35:34This is, to me, in my opinion, the Achilles heel in terms of that particular system. So there are others in terms of thinking about other technologies, other strategies that in terms of doing a double -stranded DNA break will do a single -stranded DNA break. So it'll nick just one of the two copies, which in terms of the process that the cellular machinery has for the repair of that ends up being a much higher fidelity system. So there are lower off target, lower error rates. It tends to be a more robust system. So this is still very, very early. I want to underscore this very early in terms of doing this.

1:36:11There are lots of other complexities besides the machinery of what I just described was prime editing, but other complexities in terms of the machinery to go in and make the changes, what types of mutations can be repaired. Primediting has strategies to be able to do, just not single nucleotides, but much more complex mutations to be able to fix. But ultimately, it's a vehicle for delivery. It's getting in early enough before the damage is done to the body. It's being able to get to the body safely that you need to. It's sort of multiple pieces of the puzzle that have to all be solved simultaneously to get the whole package to work.

1:36:51So we're not quite there yet, but I'm optimistic that we are, as a lot of scientists working together realizing that this may be one sort of solution eventually when all the components are there that may be scalable to deal with many different types of genetic conditions. You were alluding to the differences in strategies between TASAX and Fragile X syndrome. Do we have enough information now in this discussion for you to explain the different strategies there? I think so. So let's start with TASACs. TASACs is due to an enzyme that's missing. We talked about recessives. It's a recessive condition.

1:37:25This is a degenerative condition. And so you want to be able to get in early for all the reasons that we've talked about, and you could do a gene addition. The gene, the enzyme, is missing, so you can just pop it back in. It doesn't have to integrate. You just need to get it early enough to do its job and not to cross any mystery along the way. So that strategy would be a good strategy. The tough part, you need to get it into the brain. You know, as you're thinking about the delivery system, brain is a complex organ. So you want to be able to get it throughout the brain for function. What does that mean, Wendy?

1:37:54That means you'd have to introduce a, like, an intranasal virus. Is there something in glial cells and neurons? Where does this enzyme normally get made? Within this, as you said, there are multiple ways to access the brain. It's obviously a protected space in terms of the blood brain barrier. I doubt we're going to be able to do it with intranasal. Although, you know, there is some that you can get by putting this in that way. Some cases we do intrathecal, so for women who've had an epidural, it's basically the same way that we access the space for an epidural. In some cases, for anyone who's thought about chemotherapy that we give for brain cancer, sometimes we actually have to do it into the ventricle.

1:38:31Sounds a little bit barbaric, but we go through the skull and being able to do the injections there. But my point is it's not as simple as a simple intravenous infusion. It's not like we can just give it purerfully and get it to the brain where we need to. So it's challenging in that way and as we think about it, in some cases we need to get throughout the brain even into deep nuclei or different parts of the brain. So as an example, if you were to inject it and you had a high concentration on the left but it didn't get to the right, that would be a problem. You need to be able to get even distribution as we're doing this.

1:39:03Anyway, we'll take sex though. It is the case that as I was saluting to before, you probably don't need to get to 100 % of the protein or the enzyme that's there, even 50 % I'm sure is enough and it's possible you can get down to 20 % and that would be just fine. And so that's one strategy. Fragilex is a little bit more complicated. The actual mutation itself is what we call a trinucleotide repeat, a repeat that's too big and so we need to be able to make it smaller within doing this. It also has the same problems in terms of being in the brain but it's not just simply adding back some additional fragile X protein.

1:39:40So we can't just make it a gene addition strategy. We've got to really think about the gene editing that I was alluding to where you're fixing, shrinking the size of that repeat back down to the normal size. What's unknown, and I think one of the things we don't know until we do it in people, is what is that window of treatability? And just to be provocative to listeners think about this, is that window, even if guardian work perfectly and we could identify these babies with this within the first week of life, is that early enough? Where's my hope is that for many conditions that will be, it's possible that we'll need to go even earlier.

1:40:15And so there are some people that have thought about even in utero, gene therapy or genetic treatments for some conditions, not all of them, but for some conditions where it might be necessary to get even during development, fetal development to have the maximal effect. So I'm not saying we're going there anytime soon, but just to sort of think through that, there may be imperfect solutions unless one gets to the right time and the right place and to your point, the right cell type even. And fragile X is also a recessive condition so it can only impact women presumably because you would need both copies of the X.

1:40:48So fragile X, we do call it an X -linked recessive, but what that also means is that it's mostly males who are affected because males only have the one X if they have that repeat expansion. the males will be affected. Females can be affected, although it's much more unusual. Because they would need to have got the X from their father as well. That's it. I want to come back and talk more at the end of our discussion about kind of the future of gene editing and the ethics around it and things like that, which I'm sure is something you've thought a lot about. Before we do that, I want to talk about some of the more complex diseases that clearly have a genetic component, but they're probably much more polygenic.

1:41:26So let's start with what your colleagues done the hall are doing with respect to obesity. How much do we understand about the genetics of obesity? And does genetic therapy play any role there? So I'll start out about talking with obesity, but I may switch gears at some point soon after that. So obesity, we have ways of calculating heritability. It's to give us a scientific insight of how genetic is a certain condition. So you can do this by looking at twins, for instance. You can look at identical twins. You can fraternal twins, and you can see how similar they are in terms of body mass index, adiposity, things like that as measures of obesity.

1:42:06And it does end up being highly heritable. It's not the most heritable factor, but it is highly heritable. So that sort of points in one direction that genes are important. What's the heritability of obesity, by the way? So the heritability running somewhere around 50 % for round numbers, or 0 .5. other conditions that are extremely heritable, of course, are closer to one. So as an example, and by comparison, type two diabetes or non -insulin -dependent diabetes, more heritable, more strongly genetic in terms of that. And type one? Type one diabetes less heritable, complex interaction, both of your immune system and the genetics that govern your immune system, but also what you're exposed to early on in sort of the cross -reaction your immune system has between self and non -self.

1:42:50So a little bit different model. On the other hand, clearly there are all called environmental differences. And so you can look at what's happened to the average body mass index of the average American over the last generation. Argenes haven't changed, but on the other hand, by most measures, you can see that we're more prosperous. In general, the average body mass index has increased. And there have been some interesting studies looking at particular groups of Pima Native Americans, for instance, that have genetically the same genes. They come from the same original community, but they live in different environments, one in which it's more sort of a traditional environment in terms of the amount of access to colorically dense foods and the amount of physical energy that's expended on a day -to -day basis.

1:43:37And those same original groups, but in two different environments, you see much more obesity in the one group that has red access to, again, obesity -genic foods versus the other that doesn't. So again, strongly suggesting that it's not just the genes in terms of this. Now on the other hand, and this goes back to you are describing Rudy Leibohl and his original work in terms of identifying leptin and the leptin receptor through positional cloning methods, leptin and the leptin receptor in terms of mutations in those genes do not account for the vast majority of obesity. Very rare. I've certainly had patients with these conditions, but that's just because of the nature of who I am, but very rare.

1:44:17Most people would not have seen this. On the other hand, understanding the fundamental biology of how body rate is regulated and governed critical in terms of understanding those two molecules. And my point in this is I don't know if it's going to be for obesity, but it may be for other conditions like myocardial infarctions or coronary artery disease that knowing about the biology and sort of the final common mechanism or the final common pathway through which the biology is regulated, one may have ways of either pharmacologically or some will say in terms of gene therapy being able to make permanent manipulations.

1:44:55So statins as an example in terms of treatment for hyperclosterolemia, There may be various different genetic mechanisms by which one has an increased risk for a heart attack. Yet statins seem to work for a lot of different people. And some have thought that, for instance, rather than using that as a medication, would there be a way of genetically making a manipulation? So it's kind of a one -time and not having to require continued ongoing therapy. So I'm not saying that this is exactly where obesity treatment is going to be going. And I am in a good way excited that we certainly have better treatments for the first time, I think, for obesity now than we had five or 50 years ago.

1:45:35So we may be going in a better direction. We didn't really talk about epigenetics, but I think obesity might be a good time to do a little bit of backtracking and explain what the epigenome is and how it changes. Not just over a person's life, but perhaps more importantly from one generation to the next. And the reason I'm asking the question, is I wonder if it's playing a role in the propagation of obesity across generations, even though as you pointed out, we're not really experiencing much genetic drift in the period of time that we're seeing an explosion in obesity. And so with all of that said, my question is ultimately, do you think epigenetic changes could be explaining the increase we see in obesity as a susceptibility to a besogenic environmental factors.

1:46:29I think the bottom line is we don't know, but for those who don't know what the term epigenetics is, break it down, epi above, and then genetics the genes. And so there are chemical modifications that happen to the genome which are used to affect gene regulation. Some of those chemical modifications include methylation and those are dynamic. They can change over the life course. they can change by cell type and their ways to be able to coordinate regulation of potentially groups of genes. They're tricky to analyze from a methodological point of view scientifically. They're tricky because they do vary over the life course and they vary by cell type or tissue.

1:47:10And so using, for instance, we've talked about this a lot, but using a blood sample as a matter of trying to get the epigenetic profile for what's going on in your brain or your pancreas doesn't always work. And it's hard to even know whether or not it works, because it's not as if we're going in and doing pancreatic or brain biopsies on most people. So, you know, you can do things in animal models, you can get some indirect evidence, but it's hard to know for sure whether or not this is truly answering the question you're trying to answer. So I'll say there's a lot of conjecture in the area of epigenetics and hard to know for sure exactly what that is.

1:47:46On the other hands, I will say that we've known about something called the agudymouse, which is a mouse model for obesity, and depending on how much folate you give that mouse, for instance, while the dam, the mother mouse is carrying her pregnancy, her little mice, depending on the amount of folate in her diet, does affect the epigenetics. Folate is used in terms of methylation for the DNA, and so you can see a readout of the effect. And depending on that, you can see a change in the coat color, you can see a change in the obesity for these agudy mice and for their progeny. And there are things in terms of, as you said, transgenerational, potentially.

1:48:23We don't entirely know the mechanism of how that might be occurring, whether it's epigenetics or other things, but there are things that we can see. But these are complicated. So I'll just say, I personally don't feel like scientifically. We have all the evidence to make definitive conclusions at this point, but one wonders about what many different contributors could be. Although I have to guess that this is not going to be the major, major driver. Let's pivot to autism now. Autism is in the news all the time it seems and certainly appears that it's increasing in frequency and unclear how much of that is due to an increase in diagnosis and recognition versus how much of that is triggered by other environmental factors.

1:49:04But there doesn't seem to be much confusion around the fact that there's a strong genetic component to it. So let's start with that. Based on all of the twin studies, what is the heritability of autism? I will say to your point, autism is even within the name a spectrum. So it's not just one condition, it's a spectrum and it goes from severe what some people will call profound autism. It can be associated with intellectual disabilities to other individuals at the mild quote unquote milder and who are quite talented in many ways yet have social challenges. So within that entire spectrum, if one includes everything within that, the heritability is estimated to be approximately 0 .8, although some individuals will save in as high as 0 .9.

1:49:49The point within that though is that it's not 100%. And in fact, we do know of times over the life course in particular, prenatal and early child to a better important to the developing brain and where changes in exposure beyond the genes can play a role. So as an example, prematurity is one of the more common, if you will, exposures, but in terms of what happens to the developing brain, and if you are born when you're 26 weeks old, much higher probability of autism than if you're born at, to remember, 40 weeks. And so there are other factors beyond just the genes that are involved. But clearly, the other point that I'll make about heritability is one calculates heritability as a measure of the inherited genetic factors.

1:50:34But you've mentioned it once already. One of the factors in autism is that there are de novo or new genetic variants that occur for the first time in the individual with autism. Those individuals aren't captured in that measure of heritability, because heritability is fundamentally trying to get it transmitted genetic variants that are going from parent child and those do no -bow genetic events are new in the child. And so there are genetic aspects not included in heritability, if that makes sense. Yep. So what are the genes that seem to be responsible for autism? So depending on who you ask and how you want to define this, there I think everyone would agree there are at least 100 genes that have been identified with high confidence as being associated with autism.

1:51:19Depending on how rigorous you want to be about this process, you know, some people would say that we estimate that there are at least a thousand genes and we probably, you know, are about a third of the way there in terms of having some sense of those genes. Not surprisingly, those genes are genes that are in the brain, they're expressed in the brain, they function in the brain, not surprisingly. And many of those genes are especially active during development. And so what I mean is intrauterine fetal development within the brain specifically. And what do they code for? I mean, how many of those genes would be genes in the exome versus the intron?

1:51:56Most of the ones we recognize, underscore the ones we recognize are in the coding sequence, but that's a limitation of what we recognize. We do realize that statistically we see that there is a signal in the non -coding space, but we have less evidence to implicate specific genes or specific genetic variants individually in the non -coding space, because the effect size or how powerful they are is somewhat reduced compared to those coding sequences. The other issue is not just where in the genes, but what genes are involved. And so the genes that are involved fundamentally can be genes that function at the synapse, so the connections between brain cells and communicate between brain cells.

1:52:38That happens to be one thing that's quite important. They can be cells that are rather genes that are important in regulation of genes and gene networks. Many of them are transcription factors, histone modifiers. We talked even about epigenetics, some of those genes that may be responsible for those epigenetic changes, that they often, I think of them as having multiple downstream genes that they affect. So it's not having a very, you know, sort of focus. It's more universal effect that they have. Those genes that have that more global effect, oftentimes have a more global effect on brain function and cognition.

1:53:13So it may not be that it's autism only, but they may also be associated with intellectual disabilities, they may be associated with epilepsy, they may be associated with more sort of global effects in terms of brain function. And to the extent that that term autism is used across the spectrum, there are oftentimes those individuals described as profound autism. So there can be different things, there can also be, I'll just put as an example, we mentioned, I'll go back to PKU, believe it or not. So I happened to run a very large autism study called Spark. And within Spark, we identified a teenage young man who actually has his autism as a responsible of undiagnosed PKU.

1:53:52Even autism can be caused by, you know, full circle, an inborn era of metabolism where there are toxic things that build up in the brain and then cause that dysfunction of the brain. So not everything is a sort of primarily in terms of the brain, but things that can diffuse to and have an effect on the function of the brain. But to be clear, autism is a clinical diagnosis in the same way that familial hypercholestralemia is a phenotypic diagnosis. It's a diagnosis in the case of FH where LDL cholesterol has to be above 190 milligrams per desoleter off treatment. and it's incredibly heterogeneous in terms of the genes that are responsible to my last count.

1:54:33I think there were more than 3 ,500 genes that could produce that phenotype of high LDL cholesterol. So autism is the same, right? The diagnosis is clinical. It's a phenotypic defined disease and maybe up to a thousand genes involved in that or a thousand different ways to get there or more, right? Exactly right. So it is a DSM diagnosis in terms of clinical behavioral criteria. I know this gets confusing for people, but one can have a gene that's identified as causal, but the diagnosis is still a behavioral diagnosis. Simply a gene associated with that. And as you said, not just one single gene, it doesn't map one to one.

1:55:11In fact, no one gene or genetic factor accounts for more than one percent of individuals who have that clinical diagnosis of autism. So approximate prevalence of autism today? Round numbers 2%, you were looting 2 up before, but this number has fluctuated over time, whether it's for all the reasons you said, but about 2 % today. Does it just seem like it's more or is this really a function of greater awareness? So I think it's a function of several things. It doesn't help that the definition has changed over time, so literally the DSM diagnostic criteria have changed over time and so for that, not surprising, the prevalence has changed over time.

1:55:49In a good way, there is greater recognition and diagnosis as you alluded to. We've seen this in particular for underserved individuals that are more frequently diagnosed now. So I think the disparities are decreasing, and I think that's a good thing. But there are also, I'll say, maybe there are things that are changing in terms of society, changing the biology. I don't know. We haven't been able to put our finger on that, but there are possible contributing factors with that. And then there's also a motivation to a certain extent in terms of the way our society works to be able to access resources.

1:56:21And so people that may not have been motivated to get a label per se, they may still have known it, they may have thought it to themselves, but they didn't necessarily seek a diagnosis or label, except that there were resources, educational resources, support resources that were important, and we wanna make sure those individuals get those resources. What are some other, both neurologic and non -neurologic sequelae of autism, or call it comorbid conditions with autism? So I think that's a good way to phrase it. Comorbid conditions is one of the things that I think about. So as an example, some individuals will have epilepsy associated with their autism.

1:56:56For some individuals that epilepsy will be recognized very early. For some individuals, it won't come until the teenagers are adolescents. But that can be incredibly important. Within this, there are behavioral co -occurring diagnoses, for instance, of anxiety is quite frequent, ADHD or attention issues, again, quite frequent. And I think some things were just beginning to understand, although I think it's incredibly important is that most of what we know about autism is based on individuals below the age of 20. Those are the individuals who've been studied most. And I think there's a whole lot we don't know about adults with autism.

1:57:30And I can say I do know some conditions that are associated as degenerative conditions as well. That when people are adults, there may be particular subtypes of autism that are neurodegenerative because the genes that are involved are responsible for neuro maintenance, being able to sustain the brain and continue functioning. And when they're not functioning at some point, start having things associated like Parkinsonism. Some subtypes that may be associated with increased risk of, we mentioned obesity, believe it or not, some of these same genes may also predispose to obesity, especially with some of the medications we use to treat some of these behavioral conditions, even increase the effects, the metabolic effects and weight gain and diabetes associated with that.

1:58:12And there may be other things as well. But to a large extent I would say it's under -recognized and we have a lot of more gaps in our knowledge. But many people who continue to need those that understanding. And I think earlier you used a term of precision medicine. I don't mean it to sound like a cliche, but you can imagine that it's a large percentage, 2 % of the population, rate, heterogeneity, everyone doesn't need to have the same sort of rules that they're following, the same rule book or the same management guidelines and how do we get greater specificity to not overburden people, but yet to be able to also, you know, allow them to achieve their full potential and lead their healthiest lives.

1:58:48You mentioned that most of what we know about autism is based on studying people who are up to but below typically 20 years old. Does that suggest that prior to about the year 2000, there was nobody really studying this, because, presumably, if we were, we would know about what people look like later in life today. So, many of the adults with autism, number one, were not diagnosis having autism. They may have had, you know, some of these challenges, but things have just changed over time. And so, having a label, having a diagnosis has changed with society. Other individuals who were studied 20 years ago have not been followed longitudinally.

1:59:27And that's hard. Although there have been some epidemiological studies like Framingham that have followed individuals over long periods of time, it's hard to be able to do that. People move, people, you know, investigators lose funding, people die. I mean, you know, lots of things that happen. And so just knowing what someone looks like at two and that same person at 22, there are very few studies in terms of in children what that looks like. And so I think that's been a large part of the problem as well. I mean, I'm shocked to hear that the heritability is as high as it is, 0 .8 to 0 .9. What is it for other DSM conditions, such as bipolar disorder and schizophrenia?

2:00:05So bipolar disorder and schizophrenia are certainly much lower, especially in even things like depression, major depression, lower still. So this is actually one of the highest heritable factors in terms of behavioral health or psychiatric conditions. And just approximately what are the heritability factors for those other conditions you just mentioned? So more in the neighborhood of 0 .5 to 0 .6 or for something like major depression even lower, maybe more like 0 .3. Wow. What is the implication, by the way, if the heritability is 0 .8 to 0 .9, that almost implies for certain that a person with autism will have an autistic child, doesn't it?

2:00:45Well, that's only half the equation, right? So it takes two to tango in terms of making a child. So both individuals are contributing genetic factors as we're thinking about this. And so, and it's the combination of those factors together within that combination. So if you're talking about... Even if we're not talking about polygenic combinations. So in other words, of those, I think you said, 100 to 1000 genes that seem to be implicated in autism. Some individuals with autism may only have one of those genes, correct? Oh, absolutely. So some of them may have only one gene that's the predominant sort of contributor in terms of this.

2:01:21Some will be more of that polygenic combination of factors. But a single gene individual has a 50 % chance of passing that gene under their offspring and assuming it's fully penetrant. They would have effectively a 40 to 50 % chance of transmitting autism to their offspring. That's correct. Now for the types of genes that you mentioned, many of those individuals actually won't go on to have their own families. And so one of the factors within this is that those highly penetrant single gene factors, many individuals, for instance, you can imagine if they're not living independently, if they're not verbal, you know, they won't pass those genes down.

2:01:57Let's pivot for a moment to cardiovascular disease. There are a couple of things that stand out from a genetic perspective. One I've already mentioned briefly, which is FH. The other is LPLitLA attached to the LPA gene. I believe that L .P. Little A is the most prevalent arthrogenic condition that is genetically associated. Roughly one in ten people have an elevated that. That would be another great example of how gene addition therapy would be of no use because you'd want to be gene editing that. What else do we know about cardiovascular disease beyond those two special cases of elevated L .P.

2:02:33Little A and familial hyperclestrolemia? How much of the rest of AACVD appears to be heritable? Well, the interesting thing is, we alluded to this a little bit, when we think about my cardinal infarctions, hyperlibidemia, in that sort of cardiovascular health, there may be genetic factors that are numerous, but from a therapeutic point of view, we may not target all of those genetic contributors, there may be final common biology. And so I think that's a theme that I see most in terms of thinking about coronary artery disease, my cartilage infarction, things like that. Within the cardiovascular disease space, though, I do think they're interesting.

2:03:12I'll just give one other example in terms of a relatively common but rare disease that of cardiomyopathies. And so when you think about genetic cardiomyopathies, they affect on average about one in 500 individuals. And so that's not one in a million. It's also not 10 % of the population, right, or somewhere in the And I will say that I was waiting to see the data come out, but we've known about many of those genes for a long time. We've known about the mutations. We've known about frequency. We've known about natural history and waiting to see whether or not genetic therapies would be effective for those conditions.

2:03:48And it's not yet in people, but I will say the early data are looking promising in terms of animal models to be able to reverse this or prevent this. I still think the heart is a tricky organ to be fudzing with. I'll just put it that way. It always makes me a little bit nervous because electrical things can happen very suddenly and it can be quite dangerous. So that always makes me a little bit nervous to think about genetic therapies for the heart. But like I said, some of the early data from the side mints in particular look like there could be promising roads ahead. And like I said, it's a common condition that otherwise, often times, we treat with transplant.

2:04:24when we with the heart finally fails and so it would be lovely if didn't have to wait for hearts for transplant. Yeah, it's interesting. So going back to what you said about the ASEVD side, it almost sounds to me like you're saying that if genetic therapies and treatments are going to be deployed against conditions, especially like FH, you would really do it to more mimic the drug than you would to try to correct the defect. And that makes a lot of sense in the case of FH because of the heterogeneity, right? You know, to have 5 ,000 different gene therapies for the 5 ,000 different genes that can be altered in the result of hyperlipidemia is a bad idea, whereas if you can simply knock out PCSK9 as a gene, you basically take care of everyone.

2:05:15And so let's talk about what that means technically. So So presumably there are genetic therapies that are already in the works at looking at targeting PCSK9. PCSK9 inhibitors as a class of drug have been perhaps the most exciting drug class introduced in the last decade. And the results have lived up to the hype. I mean, when Helen Hobbs first made the discovery of the individuals that were both hyper and hypofunctioning PCSK9, I still remember reading those papers 15 years ago thinking, This is too good to be true. This will not pan out. That was my dumb prediction. This will not pan out. And I was wrong.

2:05:56I'm delighted to be wrong. So what does that look like now? What does the gene therapy look like to silence a gene in this case without creating some unintended consequence? It's going to start out with not just, you know, the average person who might have a higher risk, it's going to start out at the extreme for someone we talked about Jesse Gelsinger, who's going to be willing to be the first in and try this and be that brave first person. And so I won't claim that I've designed the clinical trial that goes with this, but just to say it's going to start at that extreme. And there are going to be a lot of complexities.

2:06:27I will say, and I'm sure many listeners are thinking of this, the cost with gene therapies is prohibitive right now. It's not as if we could, if any single gene therapy were $3 million, dollars, which is not uncommon for current gene therapies. We can't afford to spend $3 million per person with the number of people who are at risk in the US population. That SMA single gene therapy is about a $3 million treatment. I'll say the range of genetic therapies right now runs between about $1 and $3 million. So depending on which ones are out there and it gets complicated. Sorry, just to say one thing about that, it is worth keeping that in perspective, right?

2:07:05I'm not going to sort of advocate one way or the other, but it's not uncommon to spend a million dollars on chemotherapy at the end of life for a year's worth of life extension. And if you contrast that with a million dollar gene therapy in infancy that gives 80 or 90 years of life extension, it at least puts those two treatments in context. I agree with you 100%. Health economists have been trying to get at that in terms of the value, you know, if we're talking about true value in terms of this and I'm not going to pit one against the other in terms of this, but you do have to think about, and I would argue not just the healthcare system cost to the person, but it's the societal cost to the family, to the community.

2:07:50There's a lot of cost that goes into this if you do the economic analysis accurately. On the other hand, you know, to say that that might be worthwhile in one case, you do have to think about scaling, and I'll just throw out a number for you. 10 % of the U .S. population has a rare genetic condition. They may or may not know it, but that's just true in terms of these monogenic factors that we've been talking about. So that's 10 % of the population. If you now think about the percentage of the population that's obese or that has obesity or type 2 diabetes or some of these other common conditions, that someday might be treated by these one and done types of things, then it becomes in terms of a society, what can we afford to do, what are the competing other healthcare costs or other societal costs in general that we have and how are we going to write these?

2:08:39I will say that I'm confident that as we have more ability to understand how to do this, there's a lot of fixed costs but the marginal cost is not nearly as high. I think you know what I mean by that in terms of both what it takes to design the clinical trials to do the manufacturing, to do the monitoring, you know, all of these things, they won't scale linearly. And so I do think we'll have some cost realization that we can recoup. But, you know, I think those are the big questions that we think about is how are we going to afford this? And what are the key things that we need to do to enable doing this on scale?

2:09:13To your point, what are the competing alternatives? If it is, you know, a medication that you're taking every day, if you can't really get, to a good point in terms of the MI risk or anything in terms of heart health or stroke health or other things. So all of those will go into it and eventually a health economist will price this out and figure out what a reason of all fee is to charge for such therapies. You know, again, just going back to what it costs today on the gene therapy side. It still seems like, I don't say this to be disparaging the field at all because the field is remarkable, But I just say it is more of an observation of where we are relative to say gene sequencing.

2:09:48We're still in its infancy, aren't we? Oh, absolutely. I mean, as sad as it is to say, given what you said about Jesse Gelsinger's case was more than 20 years ago, we are still at our infancy in terms of being able to realize all the potential. I don't even think we have all the technologies or the vehicles or delivery systems that we're going to eventually be effective. Again, thinking it through that way, in the year 2000, it cost one billion dollars to sequence a human genome. Today it costs one thousand dollars. So that's a six log reduction in cost. In part through Moore's law, in part through the step function change of high throughput sequencing.

2:10:31We don't need a six log reduction in the cost of gene therapy to make it readily available. quite frankly, a two -log reduction would make this a game changer. Does that strike you as something that's feasible in the next two decades? I would say yes. There are going to be certain catalytic transformative breakthroughs that will make and enable those changes that you're talking about. So again, I'll go back to what we did with the COVID vaccines. It was incumbent upon everyone to be able to come up with solutions and the solutions that allowed for the adaptability and even changing the vaccine on the fly were remarkable to me, at least from a scientific point of view.

2:11:14And I know some people may push back on me, but that is my true belief. If you could think about the same way with delivering an mRNA vaccine and doing the same thing and realize that it's different for genetic gene addition and doing it, but it's not entirely different in terms of how to do this. And so as you're talking about, I call it rinse and repeat. but it's being able to do this and having the infrastructure, the delivery system, the regulatory system, the manufacturing process, all of those things. Once you get this and get this down, there are ways to scale this and to be able to, if the gene fits, if it's a certain size, if there's certain mutations to be able to do this repeatedly.

2:11:50So I do have that hope, but there are going to be the function, I think we're going to see, I call it a step function, right? So you're going to see, it's not going to be linear, it's not going to be, right? So that's We'll see. So last year I read Walter Isaacson's biography of Jennifer Daudna and the story of the discovery of CRISPR. I thought it was a fantastic book. I can't recommend it highly enough to people who are listening or watching. I'm sure you've read it. There was a fantastic discussion of the ethics of this. And once you realize the power of gene editing, you very quickly start to pivot away from the discussion we're having today, which is a child is born with TASACS disease.

2:12:29This child is going to be dead in a couple of years, and it's a very ugly death. There's really nobody in their right mind that wouldn't be in favor of a therapy there. We could go through all the examples we've talked about, and there's nobody I can imagine that's going to say if a woman is born with a brach emutation, and she has the choice between a gene edit to fix it or or a mastectomy to remove her breast, we'd probably prefer the former, if for no other reason, then it ends the gene there, and her daughter won't get it, her son won't get it, et cetera. How can people think about the next layer of complexity, which is, well, should we be able to take a person who has an apoe for isoform and turn that into an apoe III isoform, or an apoe2 isoform, which would actually come with significant protection against neurodegenerative disease.

2:13:25That's a slightly different case because of course the penetrance and the risk profile is different. So, how do you, as a scientist, think about this because I think that both ethicists and scientists need to be a part of this discussion? So I agree completely and I have to admit. So one thing I think we universally agree on, I hope, is that we're not doing gene editing gene manipulation to affect the next generation. So we're not looking for things that are transmissible in the germ line. We're not trying to create superhuman where we're trying to, you know, fiddle with the genes to either correct them or to be able to enhance them as the term that I'll use that's transmissible.

2:14:07I think that's a line scientist in it. This is... Sorry, would that be true even with something like TASACs or cystic fibrosis or things like that? That's what this consensus among the scientific community is, is that again, that you would treat the soma or the body of the person that might be at risk or have those diseases, but you wouldn't try and do manipulations that would be transmissible to the next generation. I see. So my argument for Braka is only partially correct. I argued that gene therapy would be favorable because it would spare the woman a mastectomy and spare the risk of transmission.

2:14:42You're saying, no, you would only do it in the somatic cells, not the germline. She could still transmit that gene to her daughter. That's correct. That's the current consensus, scientifically. The other part of it is this tricky thing that you're talking about, which is enhancement in terms of it's not correcting something that's problematic. It's enhancing the body the way it is or trying to, in some sense, prevent a disease process. But I will say the enhancement. Once you get to the point of enhancement, that's a trigger word for we shouldn't go there. And part of this is that we're not as smart as we think we are.

2:15:17You know, we can think that something's not going to have off -target effects, that it's not going to disrupt a gene inadvertently. But I think in the short term, what saves us is that the risk profile, given the uncertainty in the long term, is so high that there aren't going to be either scientists or people, I think, who are going to, I hope, go for things that are trivial in terms of enhancements. I'd say that's the first part of this. But to your point in the 8 -0 -Eath -234 situation, I think is one that people think about. I also think that although probably the average listener here is more sophisticated about this, I think the average person walking down Broadway here is not going to think about this in quite the same way.

2:15:58And so they're going to think about things that will be enhancements, whether it's being able to increase your earning potential, being able to be taller, more athletic, you know, funnier, you know, there are multiple dimensions in which people would, and people would survey to figure out, you know, how they would value certain attributes, you know, would definitely be thinking about doing that. I think at this point, it has to be just enhancement is a line that people are, I hope, not crossing that it really is about disease and being able to make people healthier as we're doing this. But to the extent that there are certain medical industries that are not covered by insurance, that people that are not regulated, there are certain parts of the world that do things differently, where people can go and seek certain things.

2:16:41I do hope that there is a consensus scientifically about places we don't go, because otherwise there will be ways that people will find to do things. I mean, is it worth just explaining the story of kind of the initial blow up around crisper and what took place in China and how that brought the scientific community in some ways closer around this consensus. So the circumstance in China was something I have to admit was I thought was a little unusual. So it was a circumstance in which the CCR -5 gene was manipulated to try and prevent children from getting infected with HIV. By manipulating that gene, it's not as if that was curing a disease to your point.

2:17:23It's not as if it was preventing a disease that was a foregone conclusion. It was preventing transmission of infectious agent that those children were at increased risk for, but it was not a foregone conclusion that they would have been HIV or HIV positive. And so... Wasn't it also the case that there was very little chance they were going to be because these were children born of IVF and the sperm are washed in that situation. And therefore, because in this case, I believe the father was HIV positive and the mother HIV negative, but it was a situation where it was almost entirely possible to prevent the transmission of HIV to the offspring.

2:18:00Is that correct? Right. That was my point in terms of I don't think heroic measures needed to be made, to be able to go. There were perfectly, I think, reasonable ways that are very effective, not just reasonable, but very effective. So it was an on -sort of selection of a use case. I guess is the point that I'm making scientifically. If I were going to do something, I would have done it for this use case. But regardless of that, the scientific community also just, I think rallied around that align had been crossed, right? That this was essentially a form of enhancement. It wasn't something that was saving a life, saving a high probability of something for which there were no other treatments, anything else.

2:18:36And but I think, you know, the other point that I made is true, which is that this is, the world is global and, you know, scientists are in all sorts of places. And it only takes one person to be able to do something that's crossing a line and there are people that there is tourism, medical tourism in places and people I've seen go to, you know, where they feel that there's something that they think is important that they'll seek. And so I think it is important for hopefully people to uphold certain ethical standards and for us to, you know, have those guiding principles so that people know where those lines are.

2:19:09Do you think it would have been different if the first use of crisper in humans was to do something that we could all agree on would be a great use case? In other words would the field be in a different place today? So what year was this that that I'm blanking on the name of the scientist who did this? But I know he's been basically, if not put in jail, certainly put out of science. I can't recall the year either, but I'm trying to think of what would have been that perfect use case. What were you thinking? Sickle cell. Let's say you took a kid or tastes acts for that matter. You took a kid who had a disease that was going to significantly impair the quality of their life.

2:19:46You used gene editing to fix the gene and produce phenotype that could have a normal life expectancy. So again, same technology, but far better application. I think what most people in the field, and this is usually possible, is that rather than trying to diddle the genes, if you're at the stage of an embryo within vitro fertilization, you can simply do selection of an embryo that doesn't have that genetic risk, and therefore or you're not increasing the risk of something off target, and you're still accomplishing what it is that you were sent out to achieve. I have seen some ethicists make the argument that if there were a very limited number of embryos and that were not possible to do, would that be a circumstance in which, like you said, for the purposes of direct therapy for a very bad disease in which that couple had no other alternative in terms of having a biological child, would that be possible?

2:20:43Knowing that that would affect the germ line. So as opposed to what we were talking about before would be something. And that is what I would say as gets us close to the edge in terms of thinking about that, sort of the use case that you were talking about, but not as a matter of routine and not a routine goal in terms of the intention to do in a universal way. It's incredibly fascinating. If you had a crystal ball, you know, and you could look into the future in 2040, probably you're coming to the end of your career, you're thinking about retiring, but you're looking back at a 40 plus year career in this space that has probably seen more transformation than most fields in all of medicine.

2:21:27What would you not be surprised to see happening in 2040 in this field? I would not be surprised that diagnosis is trivial. I do hope we're getting to that point. I think there are not just with the cost of sequencing decreasing, but with machine learning, artificial intelligence, the ability to ingest huge amounts of information, genomic information, clinical information, other information, the diagnostic ability in medicine in general. And I won't say this is limited just to genetics and genomics, but as especially true in this field, diagnostics will be hopefully trivialized. And I hope, although I can't guarantee it's going to happen, I hope from an equity point of you will be more accessible to more people around the world, just because that cost barrier will decrease.

2:22:18When I'm not sure of is on the therapeutic point of view to what we've been talking about, how much of that we will have realized within 20 years. While I'm optimistic, we will have gotten a lot of that done. I'm not sure how much it will penetrate, either parts of society in the United States are globally in terms of what we'll be able to do. And this is just, I think it's very hard for me to predict timing. And I think Bill Gates has a quote similar to this. You know, I do think within some points in the next hundred years we'll get to this point, whether it's the next 20 years or not, and exactly what percentage of individuals we will have been able to serve.

2:22:54I'm not sure because there are going to be as I alluded to these kind of step functions, these transformative things that it's just very hard for me to predict, given that I feel like we got stuck for the last 20 years, but I do feel like we're gaining momentum. But there's, it doesn't take much in society and in science to get us stuck. So I think that's just a word of caution. I would not have predicted. I hate to make things about COVID, but I would not have predicted in society what's happened in the last 10 years or some of the trajectories we've had. So I will not use my crystal ball, and I'll say we'll be much farther ahead.

2:23:27But I don't know, big confidence interval where we're going to be in 20 years. Well, Wendy, congratulations on your new role at Austin Children's Hospital, obviously for folks not aware that's certainly probably one of the three most preeminent children's hospitals in North America, and maybe you would argue the single most. But anyway, that sounds like a wonderful opportunity. I assume you'll be able to stay involved in Spark and Guardian as a PI, as those tend to expand. So, especially, I want to thank you for making time on the day that you're literally moving to Boston to make time to sit down for so long.

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