Dr. Phillip Sharp Video (Text Version)
Title of Talk: New Science of RNA and New Opportunities for Therapies
Natasha Kyprianou, PhD, PCRP Integration Panel Chair:
Now it is my distinct honor and indeed a privilege to introduce one of the most prominent world leaders in molecular biology and biochemistry as our first keynote speaker this evening. Dr. Phillip Sharp is a known Nobel Laureate who serves as the Institute Professor at the Caulk Institute for Integrative Cancer Research at the Massachusetts Institute of Technology in Cambridge. Dr. Sharp’s scientific career has followed a phenomenal and very impressive part.
Born and raised in Kentucky, he received his doctoral degree in chemistry from the University of Illinois and his postdoctoral Fellowship training at Cold Spring Harbor Laboratory in New York. At a relatively young age he became a Professor and subsequently head of the Department of the Biology at MIT, a position held until 1999. In 2000, Dr. Sharp was the Founding Director of the McGovern Institute for Brain Research. His brilliant work is focused on the molecular biology of gene expression as it relates to cancer and mechanisms of RNA splicing.
His landmark achievement in science was the very discovery of RNA splicing in 1977. This revolutionary discovery fundamentally changed the way scientists think and understand the structure of genes, and then—Dr. Sharp, the 1993 Nobel Prize in Physiology and Medicine. Dr. Sharp is an elected member of the National Academy of Sciences, the Institute of Medicine, and the American Academy of Arts and Sciences. But perhaps the most humbling honor that he has received, and I just found out this evening, and I would like to share it with you is that a school in Kentucky has been named after Dr. Sharp in his honor.
This evening Dr. Sharp will be sharing with us his exceptional vision about a new science of RNA and new opportunities for therapies. Dr. Sharp, on behalf of the Congressionally Directed Medical Research Program for Prostate Cancer, it is my honor to welcome you to talk in the scientific meeting of the IMPaCT Meeting. Thank you for honoring us.
Phillip Sharp, PhD; Institute Professor, Caulk Institute for Integrative Cancer Research, Massachusetts Institute of Technology
Thank you Natasha. It’s an honor to be asked to speak to this IMPaCT Meeting. I actually—when I accepted the invitation knew very little about the Prostate Cancer Research Program, in particular, this meeting and I really look forward to coming and learning more about this program. And what I have heard so far has only encouraged me and excited me about the whole—the whole affair.
Natasha commented that I have a school named after me in Kentucky. The only interesting thing about that dedication and it appearing in the MIT Press is that one of my colleagues at MIT looked me dead in the eyes and said Phil, from now you cannot screw up because you can’t let those kids down. So there is a little bit of—of pressure here.
So I want to begin by commenting that I am associated with a company called Alnylam and I’m going to use some data from Alnylam to illustrate a point in my comments tonight. So I want you to be aware of that.
What I’m going to talk about is new science and new opportunities for therapy. And I am going to speak about scientific discoveries and if there’s a take home message from my comments tonight, the take home messages are we really still know very little about biological systems, particularly the human cell. In fact, we didn’t even know using the analogy to a car that the car had a brake. But we now know cells have a brake, and a brake is a whole new small molecule called RNA. It’s not new as a molecule but its function is new. I’m going to talk about that, and I’m going to talk about the new science of RNA; none of the science I’m going to talk about today—tonight is over 10 years old. It is a whole new opening and it’s an innovative area that I think has great implications for cancer research and prostate cancer as well.
Now at the bottom here you see the Koch Institute for Integrative Cancer Research at MIT. People usually don’t think of MIT as having a Cancer Research Program. We’ve had a Cancer Center for 30 years; I’ve been Director of the Cancer Center and I’ve spent my whole life in that Cancer Center, and we have had a significant amount of impact understanding the basic science of cancer. However, we’ve just undergone a change in objectives and—and motivations with the establishment of the Koch Institute for Integrative Cancer Research. I show you the new building we dedicated this past Thursday. I show you 25 or so photographs circulating that new building. Half of those people are engineers; half of those scientists—half, the other half are scientists and cell biologists working on cancer. And what we’ve done in this new Koch Institute and, I will illustrate how we interact in my comments tonight, is integrate engineering, material science, how to make nanoparticles, how to do other analytical techniques, how to do chemistry on materials, and cancer—trying to translate our understanding of the knowledge of cancer into treatment more effective and more rapidly and as well to advance our understanding of this disease.
So let’s start with some science. About 10 years ago, two scientists, middle-age, Andy Fire and Craig Mello, discovered this process called RNA interference, only 10 years ago. And what they discovered was that you could take double-strand RNA and I’ll illustrate that in a moment, and introduce it into cells and it would effectively silence the expression of the gene. And since that work in just about 2000, we’ve understood now that the—these small RNA processes that I’ll talk about, silence genes at multiple stages, silence genes in every cell in our body, and in fact, silences genes in our germ cells in ways we don’t understand, yet are likely to be very important, and as well, in a new class of RNAs that we’ve just got the first hint of 2 years ago called long non-coding RNAs as a large family, there’s a whole other family of RNAs in our cells that are likely to be fundamentally important that for the first time we’re able to see them only 2 years ago due to the ability to do deep sequencing in RNA.
So what am I talking about? I’m talking about a process that’s illustrated here where we’re looking at the chemistry of the process which we were able to work out with—and Greg Hannon at Cold Spring Harbor where there’s RNA, double-strand RNA, which is very much like DNA, but it’s slightly different in structure. It’s made from DNA, copied from DNA; it’s processed by an enzyme called dicer into small interfering RNAs and I’ll talk more about those in a moment. They then enter a protein complex that’s in every cell in our body, takes one of those strands and looks for complements like fitting to a glove, and destroys the RNA if it has a complement.
Now this system we’ll talk about. Over here, I show you this system in plants and in worms. The system actually amplifies itself and that’s the immune system of plants and worms. So we didn’t even know this system existed in essence until about 10 years ago. So it’s really, really fundamentally new.
So what I showed you and commented on is a gene silencing by these small interfering RNAs which are illustrated here as two strands. If we introduce them in the cells, they will pair with our messenger RNA and silence the gene from that messenger RNA—any gene. I can make these RNAs in the laboratory and I can silence any gene in your body by using these RNAs, and they have been fundamentally changing tool in our research in laboratories. But more importantly in what I’m going to talk about at the moment is that using this tool to silence RNAs is very similar therapeutically as using small molecules to drug a protein in the cytoplasm of the cell because that silences a specific gene product or silencing a protein on the surface of the cell by using an antibody.
So we could possibly have a whole new therapeutic modality where we could silence and treat any gene we want to in terms of the context of the disease. So the challenge then is how to introduce these small RNAs into the cells in your body so that if we wanted to treat a disease in a cancer cell we could get these therapeutic gene silencing RNAs into those cells. In essence, biology has attacked that problem by using viruses to introduce pieces of DNA or RNA into the—the cells in your body and the way viruses do this is that they enter the bloodstream, circulate to the bloodstream, come out of the bloodstream into extravascular space, target cells and their surface, enter the cell and release the nucleic acid into the interior of the cell. So a virus can actually introduce the equivalent of these small RNAs into any cell in essence in the body if they’re designed or selected to do so.
So our challenge in terms of taking this new science that’s only about 10 years old and trying to translate it into a therapeutic modality that might be useful is this issue of how to deliver these small RNAs to cells in the body of a human.
So you try to design a nanoparticle that looks like a virus. So what do we do? You take materials and you design with novel lipids and that’s these green components; insert and mix with cholesterol and those are the purple components. Add components on the surface that actually protects the nanoparticle from—from attaching to surfaces that it shouldn’t and putting these small RNAs into the nanoparticles to actually be targeted to specific cells in the body or to be taken up by specific cells in the body. And this is the creation of a nanoparticle. These nanoparticles can be anywhere from 30 nanometers, size of the little virus, to 100 nanometers the size of a big virus, and you can make them in a laboratory using very new technology and new ways of doing things. That’s our engineering friends and the materials that go into this is also part of the manipulation of engineering.
So where are we at in this process of taking this science and possibly using it in a therapeutic modality? I want to make one point about this approach. This approach is compart—modularization of a pharmaceutical because what you’re doing is creating a vehicle that actually is usable for any different—any small RNA to any gene in the body. So if you can work this chemistry out to make this deliverable then these small RNAs can be targeted to any gene and I’ve taken a—a pharmaceutical problem of how to make a specific drug from years to months and how to deliver it—I can use a standard vehicle, and deliver my therapeutic specifically with a standard vehicle.
So if this technology works and I believe it is working now and will work better, this technology has the possibility of being a transformative technology for pharmaceuticals in a significant way.
So where are we at? This is our technology that started 2 years ago. Let me describe this experiment. This is taking these nanoparticles with novel lipids that have been made or selected to have these pharmacological properties, introducing a small RNA into them that silences the gene expressed in the liver called factor seven, and asking by different dosage, here’s a decreasing dosage of these small RNAs with nanoparticle formulations—what the level of dose is required to silence this gene to 50% in the liver of a mouse. And you see that it’s at .3 milligrams per kg with this material, and then the last 2 years that has moved two orders of magnitude down to .3 mg per kg.
So we’ve made very significant advances in our technology by making different materials in those nanoparticles to deliver these small RNAs into the liver cells and silenced genes in those liver cells. How does that work? Well it actually works very similar to how a virus works in terms of interacting with cells. We have been able by different—by chemical and cell biological methods to identify the steps that occur in the uptake of these nanoparticles and the release of the RNA into the cell to silence genes. In this case, factor seven as a gene to be silenced. So here’s a nanoparticle that’s delivered into the bloodstream. It interacts with a protein in the bloodstream called Apo-E. It binds to the surface, moves out of the bloodstream, and attaches itself to a receptor on the surface of the cell and that receptor internalizes that particle and in that vesicle the cell makes the vesicle acidic. These lipids need to then unfold under those acidic conditions and release the contents of the RNA into the cell.
And that process occurs now with significant efficiency so that every cell in the liver can be silenced. The gene within it can be silenced by delivery of these small RNAs.
Now I’m going to speak later in a—in an example about how many genes are mutated in most human cancers. As you well know it’s multiples, and I can also tell you that we can introduce multiple small RNAs into these particles and silence combinations of genes as well as we can silence one gene. So not only can we hit one gene but we can actually hit pathways with this technology. How efficient is that process? Once we actually introduce the RNA within a liver and this is again delivery to a mouse, we—and this is the small interfering RNA to a gene that’s expressed only in the liver of the mouse, what you see is the liver RNA content decreasing as we increase dose and at one nanogram of this RNA per gram of liver, we’re getting 50% silencing of that gene, meaning that only a few hundred to a thousand of these small RNAs per cell in this tissue is sufficient to almost completely silence this gene.
So this technology is a technology in which we are able to take a chemically synthesized small RNA targeted to a gene of choice in the body, introduce it with a capsule of nanoparticle that has a particular chemical property and then use it possibly as a therapeutic. This technology has just been tested as taken into human clinical trials as of about a year ago—they began. They’re escalating doses in liver cancer; it’s a technology that I think has promise for many different settings and many different types of malignant activity.
There’s other technologies that are also delivering these small RNAs to cells and I suspect that what we will see over time and time is probably within the next decade is a number of therapeutic modalities that can vastly expand the fraction of drugs—genes, sorry, the fraction of genes that is targetable within a cell and new modalities to make those diseases more controllable and treatable and curable than they are now.
So new science within a decade is left—is leading us to new therapeutic approaches to treat disease. But new science is also leading us to understand diseases in new ways and I want to use the rest of my comments this evening to talk about new insights into how our cells function and how our cells change during the development of cancer.
We discovered—not we, the field discovered, but some of my colleagues at MIT were very important in it—that in 2001 that your genome, the genome of man, encodes about 1,000 new genes that code for small RNAs just as I’ve been describing called micro-RNAs. These are encoded in our genome; they are expressed as hair-pinned RNAs in the nucleus of a cell. That’s what this shows. They’re processed through Drosha to make a hair pin that is processed through dicer. This is a—a gene I will comment about in a moment and make something that looks like small interfering RNAs that I’ve mentioned before and that then enters a complex with Argonaut, and if these RNA sequences is perfectly complementary to a message, it will cleave the message and if it’s partially complementary to a message, it will suppress the activity of the message.
I will talk about how many genes these types of RNAs interact with and what the code is that we’ve been able to figure out as a field over the last several years, but when I mentioned that until about a decade ago we didn’t know how cells functioned in terms of having a brake, we didn’t know this system existed until about 2001 in human cells. And I’m hoping I will be able to convince you that it’s profound importance in how cancer occurs.
So the code for interacting with these small RNAs is illustrated here. This shows you the RNA sequences of a small interfering RNA shown here. There’s 21 nucleotides in these small RNAs, much like the small interfering RNAs I mentioned before, and they pair to the messenger RNA expressed from a gene and silence that message and that’s their function. The silencing is normally only partial, down-tuning, so the rules for this have been worked out over the last several years that the micro-RNAs interact with messenger RNAs by targeting seeds in the 3' UTR, in the open but some sites are in the open reading frame; the evidence for micro-RNA targeting is—there’s some evidence there’s another modality but it’s very rare. Most of the modality is through the seed interactions. Most of the protein silencing in our cells is only a modest change, one and twofold, 1.5 to twofold; it can be up to 10-fold but it’s mostly a modest change and that’s surprising because what I’m going to tell you is that changes in this process can have profound effects on the development of cancer yet as we understand it—it modulates the activities of genes modestly in most of our cells.
The silencing mechanism we know something about; it’s a translation and we actually believe this works in the cytoplasm and not in the nucleus of cells.
Half of our—all our genes, the evidence is pretty strong that half of all the genes that are expressed in our body are regulated by these small RNAs. The number of small RNAs look like they’re 1,000 and therefore we find that half of our messages in our cells that are expressed to make our skin, bones, and blood and change in cancer are—half of them are under the control of these small micro-RNAs and there’s actually four or five micro-RNAs that appear to be interacting with each of those messengers in the cell.
So here’s an example of the nature of those interactions. When we look at specifics and I won’t take you through the science that—that—experiments that demonstrates this but this is a gene that if overexpressed in cells can drive those cells to become malignant. It’s HMGA2 and what you see here is a number of codes of different color, small RNAs and you see about 15 sites in the 3' UTR of that gene that interact with these small RNAs and controls and suppresses its activity and actually suppress the activity of this gene in terms of forming cancer.
So now we turn to the issue of what is the process by which these genes are involved in cancer? What I—we know that most cancer cells have a multitude of mutations that drive those cells to become malignant and have under—uncontrolled growth or changes in their growth properties and the properties of being able to move from different sites in the body to other sites in the body—metastatic growth. And what is shown here is a diagram that Hanahan and Weinberg put together showing all these red names are points in pathways within our cells that are known if mutated can either drive the cell to form cancer or if lost can promote the development of cancer. And you see these organized in pathways and this worked out over about 25 years of cancer biology. Most of those genes now have drugs or many of them—at least most of these pathways—are now drugged, so that these are pathways that can be manipulated to treat cancer in personalized care.
Now we know that mixed within that—those pathways and critical for the control of those pathways is a large number of these small RNAs that suppress the activity of these genes that cause cancer. For example, this small RNA let 7 is suppressing this RAS pathway that can drive the cell to undergo proliferation and to convert to malignancy. This micro-RNA 15 and 16 suppresses BCR2 and suppresses cell—cell death, promotes cell death in terms of promotion to malignancy.
So let’s use a few of those examples and talk about a few of these RNAs and changes in these RNAs that actually control our—our—control the development of cancer. So for example, this cluster of small micro-RNAs, 17 to 92 were identified early on as being amplified in B-cell lymphomas. The genes for these micro-RNAs were amplified in B-cell lymphomas. We know they suppress a—a gene that controls cell death and therefore as they suppress this cell death gene, they’re actually promoting cancer. They control the gene that controls growth signals. As they suppress that, they’re actually controlling promoting cell growth. This suppresses cell growth.
So these are acting as oncogenes amplified in cells or stimulated by the activity of a regulator and they promote cell proliferation. And all of these micro-RNAs are tumor suppressors. Loss of activity of these micro-RNAs from genes actually promotes the growth of cancer and, in fact, this particular gene here let 7 which I mentioned before is a gene that targets KRAS and HMGA2 and we know that loss of this activity promotes the rate and growth and spread of cancer. And in fact, most of the micro-RNAs that are present in our cells controlling half of our genes are involved in this process of suppressing the activity of genes that promote the growth of cancer cells. And I’ll illustrate that with an experiment from a colleague at MIT in a moment.
But let’s turn to the evidence that in humans the level of this micro-RNA has an effect. This is non-small cell lung cancer from 150 patients. In those cells, the level of these small RNAs, let 7 as a family have been determined in all 150. This cluster here has low levels of that regulatory RNA, and this cluster has high levels of that regulatory RNA. And if we look at survival versus the low or high what you clearly see is that these small RNAs are correlating with progression to terminal much more clearly correlating with progression of the disease.
So these small RNAs are in some ways regulating the growth properties and the malignancies of the cells in ways that are critical to the outcome of this disease.
So then you turn to the question and I’ll use this as a—a general question; can a decrease in the regulation by this new family of regulatory factors, micro-RNAs, as frequently observed in cancer—can this promote malignancies. And this experiment has been addressed by Tyler Jacks and Madhu Kumar, colleagues at MIT. Tyler Jacks is a Professor and Director of the Koch Institute and what they did was to create an animal model of the development of sarcoma in a mouse and to do that they take and make conditional expression, meaning you can inactivate two genes or activate this gene KRAS, which is known to be activated in a large fraction if not over half of all human cancers, inactivate p53 which is again inactivate a large fraction of all human cancers, and if you do that by itself in the mouse, you’ll develop a sarcoma in about 100 days.
So the question then—this experiment is if you delete this gene dicer, which I commented on before, is the gene that controls the level of these small RNAs as required for synthesis, then what is the consequence in terms of the rate of development of cancer in this animal model. And probably I will relate to the rate of development of cancer in humans. So what is seen when you do that experiment is if the dicer and the micro-RNA levels are—are normal you get cancer in this animal model in about 200 days; if you delete one copy of this dicer gene, reducing the level of the small RNAs by a factor of two, you get cancer in 100 days, accelerated at a rate twofold. If you delete both copies it suppresses the rate. And what that tells you is that this gene and these small RNAs are contributing to the general progression of the malignancies in these cells and as a haploinsufficient tumor suppressor—means one copy promotes cancer growth; two copies doesn’t but I’ll tell you it doesn’t because the cells die if they have two copies missing.
So if you look—then look at human cancers, breast cancer, kidney, intestine, liver, and lungs, what fraction of human malignancies had mutations in one copy of the two copies in human cells, you see that almost 50% in some cancers have lost one copy of this dicer, activity required for the synthesis of these small RNAs and therefore have lost the brakes that compel that cell to not grow in an uncontrolled fashion.
So these RNAs are actually critical for controlling the rate of development of cancer and the actual progression of the disease.
I won’t take you through the data because time is passing, but what I can tell you is that the actual small RNAs are really unusual in the way they work in biological systems. Though they control disease processes, such as the rate of developmental—the rate of development of cancer and the progression of cancer, though they control the way our cells undergo division from one cell type to another cell type to make the morphological three-dimensional physiological activity of an organism, they’re not essential for the survival of cells.
So we can delete these small RNAs by deleting all the dicer activity; that’s what this slide shows and that’s what shows that the small RNAs are actually deleted but if we take those cells and inject them into a new mouse what we see is that you can develop in the mouse a malignant state with these cells missing all those small RNAs. It just takes a little longer.
Now if we stress these cells by using chemotherapeutic compounds what we know is that in this cell state where there’s no small RNAs these cells die more readily than the cells that contain these small RNAs. So though they’re not essential, they allow the cells to under—undergo or respond appropriately to the cell stress. So this whole system that I’m talking about or this micro-RNA regulation system in cells stabilizes cells against growth, stabilizes developmental transitions so that cells as they undergo developmental transitions from one state to another know their cell state and they control the responses of cells to stress and are absolutely essential for controlling responses to stress.
So I’ll conclude my comments by saying the following: over the last decade we have made several fundamental discoveries related to RNA. One of them is at—in our cells because of this micro-RNA system that I just talked about, we can introduce small RNAs into those cells; it’ll enter this micro-RNA pathway that’s used in every cell in our body to control genes and it can be used to silence the activity of a gene in that cell. And that’s raised the possibility that we could develop a whole new therapeutic approach to treatment of disease by using these small RNAs and nanoparticles and other technology to deliver these RNAs to cells. And it holds promise over time of a whole new approach to treatment.
Then the other thing it tells you is that though we’ve been studying the biological activity of cells and cells undergoing transformation to malignant cells over the last 50 years, it wasn’t until little less than a decade ago we realized that within each of those cells there’s a whole system that stabilizes the gene expression activity within those cells and controls those cells’ growth and malignant transformation and that loss of that activity is a fundamentally important aspect of the progression of cancer.
So we have made a lot of progress; we’ve got to translate that progress into treatment and prevention of disease but we also have a lot of things we don’t know about yet in biological systems that are going to be fundamentally important in terms of future treatment and more effective treatment of diseases, such as cancer and prostate cancer specifically. Thank you.