CRISPR—no, it’s not a new cutting-edge kitchen gadget for low fat diets, rather it was first recognized by Osaka University researcher Yushizumi Ishino in 1987, who found sequences of interrupted repeats in microbes was unusual because repeated sequences are typically arranged consecutively along DNA. Evidence then came that CRISPR is an adaptive immune system. When a microbe is invaded by a virus, the first stage of the immune response is to capture viral DNA and insert it into a CRISPR locus in the form of a spacer. These spacers can be cut out, but damages the DNA.
Mazhar Adli, PhD, and his team have developed a technique to prevent genes from carrying out their function without causing the extensive DNA damage the current approach requires. This is important because silencing genes allows scientists to understand what individual genes do and identify the ones that cause disease. Gene silencing also may one day let doctors better treat and even cure genetic diseases with abnormal gene activity.
Our genes – the blueprints for life – are segments of the long double strands of DNA in our cells. To silence genes using the current approach, the CRISPR system cuts both strands. Doing this too often causes the cells to die –– a major limitation for CRISPR. Even cuts that are not fatal to cells can have unintended effects that result from the body’s efforts to repair the broken DNA.
Adli’s approach, on the other hand, avoids cutting the DNA altogether. Instead, it takes advantage of the fact that DNA is made of four main building blocks: cytosine, adenine, guanine and thymine. Adli’s method lets scientists use CRISPR to convert one building block into another to artificially create what are called stop codons – the “off” switches that naturally occur at the end of genes. Turn cytosine into thymine, for example, and the whole gene is silenced, meaning there is no protein production from that gene. ABILITY caught up with Dr. Adli to dig deeper into this innovative system that is revolutionizing the new era of science.
Carol Brown: Can you tell us the quick background on CRIPSR [pronounced “krisper”]?
Mazhar Adli: About 30 years ago, Japanese scientists discovered this weird sequence in bacteria, and they called them “Clustered Regularly Interspaced Short Palindromic Repeats”. So CRIPSR.
Brown: Makes me think of food.
Adli: (laughs) Thirty years ago we didn’t know anything about it, and in 2005 people started to realize that some of these genetic sequences in between those palindromic sequences actually doesn’t belong to bacteria, it belongs to some viruses. That of course immediately sparked an idea that somehow the bacteria is able to store a portion of virus genetic information into its own genome. And if the bacteria is doing this, evolutionarily it has to provide some sort of advantage to bacteria. One of the ideas is that somehow, maybe this is an immune system. In a way, you are carrying a photograph of your enemy in your own pocket. If you encounter that enemy again, you can protect yourself better. People started to work on it. How does bacteria use the CRIPSR system as an immunity against viruses?
In 2012 the first major paper was published describing that bacteria actually use this—in this CRIPSR, there’s a protein called a Cas9 protein, and it uses short RNA. Part of its short RNA belongs to the virus, so it’s kind of a complementary sequence to the virus DNA. And this is how bacteria recognizes a virus. As soon as it recognizes this short RNA, it can cleave the DNA from the virus. This gives greater protection to the bacteria, so they can survive better with the viruses.
This sparked the idea that, what if you put this protein and this short RNA in mammalian cells? Can this be used so we can go and cleave specific regions in the mammalian genome? And they showed it also works in the mammalian system. Since you have an enzyme now and RNA, a very short, RNA molecule, you can custom-design it. This gives you the ability to send this protein to anywhere you want in the genome to create these breaks or double-stem breaks in the DNA.
Brown: You’re talking about cleaving. That’s not your approach?
Adli: Right, this is not what we are doing. This is how traditional CRIPSR works. Basically, it goes and cleaves the DNA. After it cleaves the DNA, the cell has to repair this DNA, and when it makes the repair, it makes some random mutations. In a way, it’s kind of genetic scars that are left behind. Some of these scars cause mutations and cause what we call knock-out. Basically, the gene cannot produce a protein because of these random mutations. This is how the traditional CRIPSR works. When people say “CRIPSR-mediated gene silencing,” this is what they mean, the silencing as a result of these DNA breaks and due to these random mutations left behind from the breaks.
What we are doing is completely different. First of all, the traditional CRIPSR works very efficiently with the DNA double-strand breaks and with the scars, but the problem is two-fold. One, we cannot predict what’s going to happen to that DNA. It’s a completely random insertion and deletion at those sites. Second, in our genome, we usually have more than one copy of a gene. In normal cells we have at least two copies. But in the research environment, in cancers and other situations, we have multiple copies of a gene, and when there is a DNA the cell thinks something catastrophic is happening, that the cell is dying and is undergoing what we call apoptosis. It’s a programmed cell death.
So it’s not because we silence the gene or because the gene is no longer present, it just due to excessive DNA damage due to the activity of CRIPSR. So we wanted to overcome this challenge. We are not making any double-stem breaks, we are changing the genetic code. Do you have any biology background?
Brown: You said the word “biology”? What does that mean? (laughter) My undergrad is in biology, but that doesn’t mean I know much today.
Adli: I don’t know how simple I should go?
Brown: Good point. I’d guess the simpler the better.
Adli: We found around 17,000 genes we can target this way, and, as you know, we have roughly 20,000 genes. The way the genetic information is being tracked or translated, we have this strip of codes. This is how the genes code for proteins, with this strip of codes. Some of this strip of codes code for nothing, basically, we call them “stop.” When the gene comes toward the end of a gene, we have these stop codons. We are changing one base, one nucleotide, into another one directly. This allows us to create these stop codons early on in the genes, so there are no strand breaks. By creating these stop codons, when the gene is being translated, the gene is being cut right at the beginning of it. There’s no protein production from there on.
This way the cell does not realize there is a strand break because there is no strand break, and since there is no DNA damage, there’s cell viability, and the cell’s survival returns to normal. In the other case, if you create a stem break, the cell undergoes stress, and in certain cases it doesn’t survive; it’s undergoing cell death, or it reduces its overall viability. You’re kind of making the cell sick when you make a double-strand break, when you cleave the DNA. But in our case, we are converting one genetic code directly into another one, and by editing these genetic codons, we are stopping the protein expression.
Brown: Can you give us an example of what that allows potentially to happen?
Adli: For a lot of diseases, including cancer, sometimes we have an apparently active gene. We just want to silence that gene: to inactivate that gene. Our way allows us to directly do this. The second thing is, research-wise, we can now apply this method and understand the function of genes. We can do this one gene at a time. We can also screen many, many genes, thousands and thousands of genes. In the screening scenarios, for example, we can delete, in genetic terms, we can say “gene deletion” or “knock-out.” We can silence: we can inactivate genes. Instead of inactivating one gene at a time, we can inactivate thousands of genes in a population of cells, and then we can let these cells, for example, form a tumor and see which gene is the most important in terms of tumor formation, or which gene is most important in terms of responding to a drug.
So the method we are using is called “base editor,” rather than just CRIPSR, because the conventional CRIPSR, as I just said, goes through the DNA double-strand breaks. But this base editor is converting one base into another one, and it’s much more precise genetic editing. The major advantage of this approach is that it’s much more precise. Let’s say one cell out of a thousand undergoes the mutation or the genetic editing you desire. With our approach, it’s 60% of the cells, or in certain cases an even higher percentage of the cells have the desired genetic editing.
Let’s say you want to edit the word “c-o-n-t-r-o-l.” With CRIPSR, it will randomly shuffle some letters in that region. We are saying that, if the word is “c-o-n-t-r-o-l,” we just want to replace “n” and “t,” we can do this with this new method. It’s a much more precise way of genetic editing. And more importantly, it is safer, because the cell is not sensing DNA damage because we don’t make any strand breaks, any DNA breaks.
Brown: Are there plans for clinical trials?
Adli: At the moment it’s not at clinical trials, but we are hoping that we can apply that base editing technology to certain diseases, cystic fibrosis is one of them, and potentially, hemophilia is another one. Hopefully we can make these precise genetic changes in these diseases. Of course, we have to first show in the resource environment, in the research lab, that this is working in the human cell lines, and then show in the animal models that it is working. Then it will come to the clinical trials. Right now it’s not at the clinical trial level yet.
Brown: Have you tried animals yet?
Adli: We can make these editings in animals, yes. We’ve tried them in animals, and it works in vivo as well. Right now we’re trying to see what is the best disease model to apply this to, because we need to pick a disease model where we can deliver this systematically to the patient. For example, a muscle disease, let’s say muscular dystrophy, we probably will not be able to use this method because of the delivery issue. We can correct genetic mutations in human cell lines, but the bottleneck is the delivery of this system to the whole body. Locally, for example, you can inject it, and locally you may do some corrections. If you inject it into the muscles of the arm, you correct those muscles, but the other muscles in the body will not be corrected.
However, certain genetic diseases, let’s say blood malignancies, when there’s something wrong with the blood cells, you can correct the blood stem cell. Once you do that, you can introduce that stem cell to the patient, and it will repopulate the entire blood system. Every cell in the blood system can be corrected in this way. That’s why blood malignancies are probably the early diseases where this will be applicable.
The second disease setting is cystic fibrosis-type diseases, where the lungs are the area affected. You can have the patient inhale the virus carrying this agent, and this genetic mutation will be corrected in some of the cells. The good thing is that you don’t need to correct 100% of the cells in the lungs to get rid of the disease symptoms. If you correct only 10% of the cells, you eliminate 90% of the disease symptoms. This is another disease where this will be applicable early on. We are excited and trying to work in these directions to identify a disease where we can apply this more efficiently.
Brown: Are you the only ones working in this? Are other labs around the world working this way?
Adli: While CRIPSR is relatively new, a lot of people are working on this technology and improving it further, refining and perfecting it. Ours is one of the labs developing CRIPSR-based tools. Companies also are investing heavily in this research. Everyone is picking his/her own niche. Some are using the traditional CRIPSR. Usually the research labs are invested in perfecting the technology and some big companies are using the existing technology and trying to go to the clinic as soon as possible.
Brown: The way you’re approaching it, is that unique?
Adli: Yes. What we call the CRIPSR stop, changing these genetic codons to silence genes, is a technique we came up with and first published on. Other labs have worked on the CRIPSR base-editing technology, but we invented the CRIPSR stop: stopping the protein expression by using base editing.
Brown: Now that you’ve invented this, will that be knowledge other universities and labs around the world can use?
Adli: We are making all the reagents, everything, open-source. I am a big fan of open-source. We’ve already published all our findings. We’ve put all our plasmids and all our reagents out. Everything we’ve found is out there right now. We want other people to try it and further improve the technology. We are also improving it. We patented it, but for research purposes, it’s freely available to everyone who wants to use it in the research environment. We are hoping other people will use it and show that it’s efficient and safe, most importantly.
Brown: Are you looking for the best disease to work on?
Adli: Yes, I am reading a lot about genetic diseases. I am talking to the clinical people about these genetic diseases. As a research lab in academia, I’m trying to pick the best disease model to apply this technology to for a couple of reasons. One is that there are certain diseases a lot of companies are invested in it because there is a large volume of patients, and they can make money off of it. But for me, there are some rare diseases, and this could be directly applicable, and a company may not invest in it because there is not a large number of patients. You know how the pharmaceutical industry works. If they see there are enough patients, then they invest in these kinds of diseases.
I as a scientist whose research is funded by taxpayers’ money, I am also interested in the diseases where a company may not wish to invest in it, but I can make some contribution to that area.
Brown: When you say “rare,” you couldn’t work with undiagnosed genetic diseases?
Adli: If it’s undiagnosed, of course we don’t know what it is. But the rare diseases, let’s say you have one in 5,000 people, one in 10,000, pharmaceutical companies are most likely not going to invest anything in it. Sometimes we call them an “orphan disease,” very rare genetic diseases. Usually in these situations, foundations or research labs are trying to find a cure for those kinds of diseases.
Brown: If legs are atrophying, you’d be able to inject modified cells in the areas where the muscles are having issues?
Adli: That is the current way some labs are invested in using CRIPSR for muscular dystrophies. There is good success in local areas. In cell lines, we can definitely cure many genetic diseases. The bottleneck is how to deliver this systematically to patients. You can inject in local areas, but in the long term, this is not seen as a cure, because you have to inject many, many times, and if that muscle cell dies, your cure is gone. But if you could find a stem cell where you can make this correction and give that adult stem cell to the patient, in theory you would eliminate the disease for the rest of the patient’s life.
Brown: Even if you didn’t, wouldn’t you be at least slowing down progression?
Adli: Yes. With those local injections it should slow down the progression. But for some muscles, for example, the diaphragm or internal muscles, cardio muscles, there the problem is how to deliver to those kinds of muscles.
If the diagnosis is done, injections will definitely be one way to slow it down. Researchers are exploring different avenues in this approach. One school of thought says we should find the genetic reason, try to correct the genetic code, make this correction, and this will be a cure. Other people say that for a lot of these diseases, we may not be able to identify the genetic cause or make the correction, but we can identify the major driver of this genetic abnormality. For example, in some muscle dystrophies, there is not a genetic mutation, but there is an independent accumulation of certain metabolites in the cells. They say that if we can neutralize the metabolites, we would get rid of the symptoms they cause. Those approaches say you can give certain drugs that will hopefully systematically reduce the overall accumulation of those metabolites.
All these approaches are equally viable. Hopefully in the near future we’ll have drug-based things that will eliminate some of the metabolites and help stop this progression.
Brown: What do you think your timeline is for human trials?
Adli: That honestly depends on the funding situation. When the diseases are rare and the risk is higher, getting funding gets tighter. The early things for which we can easily get funding is to say we’re going to apply this to cancer or other diseases that affect many, many people. For rare diseases, I am currently preparing an application for private foundations. Those are the ones where we can hopefully get some funding to start to do this kind of research. Unfortunately, to be able to do any kind of research, I need to hire a post-doc-level person, pay his/her salary and pay for the reagents. For this one, I am currently writing funding grants hoping to get some pilot projects going.