Driving Drug Development with CRISPR/Cas9 | Vital Science: S2, E06
- Hello, and welcome to episode six of "Vital Science." Since the discovery of DNA's helical structure and subsequent cracking of the human genome, we've seen a rise in molecular technologies that are driving laboratory practice and clinical translation. In the past 20 years, these advances have resulted in an ability to alter genetic pathways on our quest to understand and cure disease.
As you'll hear in a bit, molecular manipulation has been around for decades with scientists first modifying the genes of bacteria. You may have heard of one such technology, Clustered Regularly Interspaced Short Palindromic Repeats by its more common name CRISPR, but what is CRISPR exactly? That's what we'll dive into today. To provide some background, CRISPR uses a synthetic guide RNA molecule and an enzyme typically Cas9 from the bacterial immune system to edit messenger RNA. Now a common practice in labs around the world, CRISPR is laying the foundation for breakthroughs in agriculture, veterinary medicine, and drug development.
So how are people using this exciting in vitro technology? Our host Gina Mullane sat down with our resident experts, executive director of Discovery Sciences, Dr. David Fischer, and manager of our CNS portfolio, Dr. Carina Peritore, to talk about the history, applications, and potential of CRISPR.
(upbeat music) - Welcome to the "Vital Science" podcast to my guests, David Fischer and Carina Peritore. Carina, can you please introduce yourself? - Hi, thank you, Gina. I'm a product manager and neuroscience discovery at Charles River Labs. I've been here for a little over two years now. - Wonderful, tell us about your background.
How did you find yourself as a product manager at Charles River? - Sure, so I studied neurochemistry for my PhD and then moved on to an academic post-doc in neuroscience. From there in industry post-doc in neuroscience where I worked in IPS derived differentiation protocols for neurons and did a little bit of gene editing using CRISPR. And from there moved into the commercial side of science where I'm now helping to facilitate crosstalk between the different business units at Charles River Labs and helping enhance visibility of our neuroscience portfolio. - Wonderful, sounds like a fun job you have there. - It is. - And David, welcome. Can you please share your background with us as well? - Thank you, Gina, yes of course.
So I've got a PhD in molecular genetics and similar to Carina, I went and did a post-doc in neuroscience before moving into industry, and I've been with Charles River Labs for 15 years now, where I really work in the early stages of drug discovery, trying to help design programs and move them forward towards the clinic. - And is that work rewarding, David? - It is, and especially nowadays. Some of the drug discovery programs can happen so fast that you can actually see drugs move into the clinic in just a couple of years. - Wow, who would have thought that? Well, it's wonderful to have you both here today for a unique format of our podcast.
We're gonna have a conversation on the exciting topic of gene therapy. So our series on in vitro technologies that have changed the world led us to both of you to share your knowledge and perspective on this exciting technology that's rapidly advancing disease treatment. So I'll start with you, David.
Can you explain what this technology allows scientists to do and why it is adventitious to other gene editing tools? - Yeah, so we were gonna discuss CRISPR Cas9 technology as a gene editing platform, as sorta the next generation of gene therapy. And the real advantage over other technologies is the versatility that you can really design to home it in on particular genes, particular mutations, and do all kinds of tricks in cells, both in the lab, in cell culture, but also in vivo, and of course, a lot of our clients and collaborators are now progressing into clinical trials as well. - It does seem like a pretty common practice nowadays. It seems that genetic engineers have designed systems to send DNA at any desired location for a while, but it required scientists to assemble protein to hone in, like you mentioned, on every new target sequence, which could be a very tedious process and then comes along CRISPR.
And this technology was interesting mostly to microbiologists, but it's now become ubiquitous in biology labs over the past few years. I'm wondering, David, if you might be able to elaborate a little bit on what the hype is for CRISPR specifically and maybe how it works. - Yeah, absolutely. Yes, I should said coming out, before CRISPR Cas9, there were some other technologies that allowed you to in principle, make these edits in cells, take out particular sequences, insert sequences, that it would be a laborious process because you've had to design for instance zinc finger proteins, or TALENs to snip particular sequences. And you may need to screen different variants to find the ones that work best.
And then you could perform your gene editing experiments. With CRISPR, you basically look up the sequence that you want to edit. You order a guide RNA to match that sequence and you co-transfect with Cas9.
So the enzyme is universal and it's programmed with an RNA molecule. So by change the sequence of the RNA molecule you change it to whatever sequence you want to address. And that is the basic difference. You don't need to change the enzyme. The enzyme is the same, it's a constant. You just change the little tool that hones it into the right sequence.
- And that's basically why the two Nobel prize winners won last year for that exact technology, right? - Yeah, so Jennifer Doudna and Emmanuelle Charpentier won the Nobel Prize for Chemistry in 2020. And what of course typically happens with Nobel Prizes is that these are awarded decades after these discoveries were made. In this case it's within a decade. So I think the researchers did most of the research that this prize was awarded for in 2010, 2011, '12. So we're less than a decade away from that.
And I think the reason that no prize was awarded so quickly is that not only is this is a truly innovative platform, but it's also a very robust technology. Tens of thousands of researchers are using this day in day out in their lab and coming up with all kinds of different variations on the same theme, but the basic principle, this RNA guided genome editing technology, that was the discovery that is a Nobel Prize was awarded for. (soothing music) - Dr. Fischer notes that Nobel Prizes in scientific disciplines, typically like chemistry, come years after ground has been broken. Presumably this is because the number of researchers doing great work has grown exponentially and it could take years to validate some of these discoveries. What's truly novel and revolutionary, the awarding of the 2020 Nobel Prize that Doudna and Charpentier draws attention to the fact that CRISPR is a game-changing technology, one that lays the foundation for a whole new approach to therapeutic discovery and development.
As you'll hear in a bit, Dr. Fischer describes the value of CRISPR beyond gene therapy to improve the way we screen drug candidates. So that makes sense that thousands and thousands of researchers are looking at CRISPR now. And do you think it's because of that transition from the discovery in bacteria to mammalian cells? - Yeah, so obviously there are lots of researchers that do incredible work in microbiology.
That's the number of researchers studying human cells or animal cells is significantly larger. And it was always relatively straightforward to make edits in bacteria, but it was always extremely difficult to make genetic edits in mammalian cells like human cells. So clearly this has really changed the game significantly. And that's why it's such a popular technology. In addition to just making single gene edits, this technology also allows you to do large library screens looking for novel function associated with genes, very similar actually to the RNI screens that were are such a boom in the early parts of the century, because it's the same thing. You just generate libraries of these guide RNAs and together with Cas9, screen them for phenotypes.
And for instance, cell survival, you can do this in cancer cells to identify genes that stop cancer cells in their tracks. - David, you mentioned so many different types of cell types that scientists have been successful with. Where are we with neurons or are there any other situations where the process has been really successful? - So in cell culture, neurons are not really a problem.
You can make edits, you can knock out genes in neurons using CRISPR Cas9. Of course, anything that's takes weeks to different shades in cell culture is more difficult. So also the genome editing in neurons is a little bit more difficult than in a rapidly proliferating cell line. I think where the difficulty really comes is if you want to then apply this in vivo in an animal or in the clinic because neurons, of course, there are major cell type in the brain. The brain is a relatively large organ and it's difficult to just get the delivery worked out for the combination of Cas9 and the guide RNAs.
It's a relatively large enzyme, so it just barely fits in everyone's most popular gene therapy factor, AAV. So I think we're still trying to optimize this, perhaps trying to identify a little bit shorter versions of Cas9 so that they fit more comfortably in AAV. And once we get the delivery sorted out, then I think that there's going to be a lot of potential for editing neurons in vivo as well. - Yeah, and I can just add to that. A lot of researchers are trying to edit genes in human IPS-derived lineages and it's still on the very infancy and earliest stages and it's not trivial.
But would you agree with that, David? - Yeah, totally. STEM cells are more difficult as well. And that's because STEM cells have this natural barrier against genome edits. They want to remain picture perfect, no mutations.
So they try to resist genome engineering as much as possible. So it is a little bit more difficult as well in STEM cells because of that increased DNA repair capacity that they have. - Yeah, so even with trials and tribulations of using human IPS derived cells to edit, a lot of researchers anticipate being able to use those cells to then infuse into patients to correct whatever mutations need to be corrected for a specific genetic disorder. But recently it seems that for a rare genetic retinal disease, so infants who are born with this disease are essentially blind, doctors have used CRISPR to try to treat patients by editing their genes while they're still inside their body. And so usually, like I mentioned before, scientists would take the cells out of the patient's body, edit their cells, and then put them back into their body. Since doctors have figured out a way to get CRISPR inside the body to do essentially a genetic surgery within the patient.
Do you know how successful that's been or if there's many more examples of this? - You know, I think that there's a very good point. What we're seeing if you look at at the early stage clinical trials and there is no phase three trial for any CRISPR drug ongoing at the moment. So these are all early clinical trials where the primary endpoint is most likely safety and hints of efficacy, but certainly not proof of efficacy. The vast majority are ex-vivo edits, where our patient cells are taken out. For instance, their T-cells are taken out, gene is changed, knocked out, inserted.
And then after the cells are carefully checked, they're put back into the patient and allowed to do their job. The eye is indeed a unique organ in that researchers have already figured out to deliver drugs locally to the eye and there're number of different drugs that are given by injections to the eye. So because of this very localized delivery that doesn't spread to the body, researchers have used the same approach with CRISPR Cas9 just do editing in the retina of these patients. So it's unique setting, and it allows to also very quickly understand if there is any improvement because of the the rapid changes you can measure with physio acuity. So I think that that is a clear example of an in vivo edit, and researchers are, of course, looking for other organs to treat, but we need to figure out delivery.
So an organ like the liver is so much larger than the eye that a localized injection probably it doesn't work. So you need to give an intravenous injection so that you hit the whole liver. So you need much more of the vectors and the reagents to perform the edits. - Oh, it's so interesting.
It makes sense that if there's a direct local injection to the area of interest, like the eye, it's easier to use CRISPR by itself as a therapy. I mean, it seems like there are some associated risks with gene editing. Anytime DNA is broken when it's in the process of being repaired, there's the potential for genes or chromosomes to rearrange and potentially cause unwanted mutations that could lead to cancer.
So if the wrong piece of DNA is cut, it would seem essential that the DNA to be cut outside of the patient and then infused back in. So if there are errors, it's not devastating to the patient. I dunno, what are your thoughts on that? - Yeah, I think that that is indeed probably the most pressing points with genetic engineering, genome engineering in clinical studies to understand the potential for off-target edits and the impact that those habits may have later on because researchers are trying to optimize the enzymes, the Cas9 enzymes and select guide RNAs that have a much higher fidelity, so much lower off-target sequences.
But if you edit a million cells, there may be one cell where one gene is mutated that you didn't intend to. So with next gen sequencing and other technologies, researchers are carefully looking to understand what kind of frequency of targets edits we see, and also if there are particular hotspots in a genome. Of course, genes that you'd want to avoid are like ONCOR genes, tumor suppressor genes. And at the moment, it doesn't seem that there is a preference for such genes, what people have reported is relatively random.
So that is good. And clearly, the ex vivo edits allow you to carefully test the cells or samples of cells to understand what your targets look like. (soothing music) - After Doctors Fischer and Paradore talked about how CRISPR works, Gina asked our guests to talk about some of the therapeutic areas where we might see CRISPR-based therapies hit the market in our lifetime and what the challenges to developing such therapies might be. (soothing music) - So what other diseases do you see CRISPR being used to treat? Is it used presently for any? I've seen stories about treating sickle cell disease and other neurodegenerative diseases? - Yeah, so in early clinical studies, there are are different platforms, not only CRISPR, but also some of the other, the zinc fingers, the TALENs, MegaTALENs. In the early clinical studies for sickle cell disease, where you haven't gotten an ex vivo treatment of progenitors or types of cancer where it's something similar like CAR Ts, where T-cells are programmed to attack the cancer cells.
So clearly these two areas, not blood-born diseases, either genetic defects like sickle cell disease, beta thalassemia, or cancer cells of blood cells are being addressed in the clinic at moment. Again, phase one, phase two trials, nothing in phase three. - So, David, I wonder if you could elaborate a little bit more on some of the challenges in using CRISPR as a genetic therapy? - Yeah, so there are, of course, always challenges with any new technology, but I think we are in a very fortunate time that gene therapy has also come of age.
We also now have a number of oligonucleotide based drugs, both siRNAs and antisense oligonucleotides approved as drugs. And it's really this combination of understanding how to generate the gene therapy vector. For instance, AAV, how to package that, how to deliver that, and understanding how we can also deliver these oligonucleotide drugs that basically allows you as sort of Lego bricks put this new platform together. So we're using a bit of AAV gene therapy.
We're using some RNA based oligonucleotides, understanding how we've addressed delivery, immunogenicity, of course. So some people will have pre-existing antibodies against AAVs because this is a virus that although it is not pathogenic, it does occur in the population. There could also be antibodies against Cas9, but we have ways to address this to understand if there are any consequences. So really learning from the other technologies really helps speed up the development of genetic engineering in the clinic. - And when you mentioned ASOs, I was just curious, why would someone develop an ASO versus a CRISPR for a genetic therapy? - So ASOs are wonderful tools, and nowadays also wonderful drugs, but not every mutation can be addressed with an antisense oligonucleotides There're basically two main mechanisms. One is excellent skipping.
And the other one is knock down of the messenger RNAs through RNSH, but there are of course mutations where you don't want to reduce the expression of the messenger RNA or there is no accent to skip, but you want to fix a point mutation and an ASO basically doesn't allow you to do that. So here a CRISPR Cas9 would allow you to, for instance, replace that part of of the DNA with the mutation or with the new base editors, just flip one nucleotide back to what it's supposed to be. So clearly there are mutations that you cannot address with an ASO, at least not with the current form. - And what about diseases that don't have a genetic defect? Can CRISPR help with something like HIV, where could the viral genes that have taken up residence in the genome of infected people be specifically cut out in that instance? - There are studies ongoing in HIV, and they're not trying to cut out the HIV genome from the cells in the patient, but rather by taking T-cells from the patient and making them resistant to HIV by taking out one of the receptors for cell entry. You create cells that you can put back into patients, and they will over time compete out the effected cells because they cannot be infected with HIV.
And this is a way that researchers hope to potentially build a cure for HIV by finally taking out all the cells that are effected, replacing them with cells that are inert and resistant. (soothing music) - I never realized how far we've come in the field of genetic engineering. And yet it seems as though we're only scratching the surface. In his long career as a molecular geneticist and in vitro discovery scientist, Dr. Fischer certainly has seen a lot. How will CRISPR impact his future projects in drug development in general? Carina wrapped up their conversation with an eye toward the future, getting Dr. Fischer's thoughts on what comes next. (soothing music) - So, wow it sounds like we might not be there yet for HIV.
Still in the earliest stages. Do you have any ideas of what the future holds for this editing? I know we talked about perhaps neurodegenerative diseases or cancer treatments. What do you see in the near future? - So what I think we'll see is more tools becoming identified. So Cas9 was identified from one species of bacteria. As we all know, there are thousands, if not millions of different species of bacteria and a large number of these will have similar enzymes.
So we may find an enzyme that is just a bit smaller, fits more comfortably in a gene therapy vector. Or we may find the enzymes that have slightly different activity. We've already seen base editors being engineered.
They're important at the moment the rather large so they don't fit very well into AAV. So we also need to improve on that to make them smaller and also understand what their off-target activity is. But I think with all these steps being taken, we are gonna build a platform that hopefully at a certain point, we can just plug and play different guides RNAs for different mutations and create a whole host of different drugs, all using the same principle with just that switch changed to guide RNA. - Yeah, it's nice to hear that researchers are looking also outside of the Cas9 system for genetic engineering possibilities.
Very exciting. - Well, that was a really interesting conversation today. Thank you both for sharing your unique and really valuable perspectives. - Thank you, Gina. You're welcome.
(upbeat music) - It sounds as though CRISPR is worth the hype, and I'm excited to see where this new technology will take us. What excites you about what you heard today from our guests? What are your questions? Let us know by sending an email to firstname.lastname@example.org. For our listeners who are looking for more information about what we covered with David and Carina today, please review our episode notes where you'll find links and resources to explore this topic further. Thank you all for joining us for another episode of "Vital Science."