Gene Editing: The Future of Care for Inborn Errors of Immunity

Gene editing has moved from the laboratory to the clinic and is likely to transform treatment for our patients with inborn errors of immunity. In this episode, Caroline Kuo, MD, explains genome editing technologies and how these tools may enable precise, potentially curative therapies. 

Transcript

Rebecca Saff, MD, PhD, FAAAAI 0:13

Hello, and welcome to Conversations in the World of Allergy, the podcast of the American Academy of Allergy, Asthma & Immunology. I'm your host, Dr. Rebecca Saff. Allergy and immunology is a field that's evolving at an incredible pace, and staying current isn't just important, it's essential. This podcast brings you conversations with leading experts to explore the latest advances, challenge how we think about core topics, and ultimately help us deliver the best care to our patients. Gene editing has moved from the laboratory to the clinic with the first CRISPR-based therapies now approved for sickle cell disease. In our patients, it is likely to transform the treatment for patients with inborn errors of immunity where genetic mutations have been identified. In this episode, we explore the evolution of genome editing technologies and discuss how these tools may enable precise, potentially curative therapies for patients with inborn errors of immunity. We also examine the challenges that must be overcome before these approaches become routine clinical practice. Today we welcome Dr. Carolyn Kuo. Dr. Quo is an associate professor of pediatrics in the Division of Allergy, Immunology, and Neumatology at UCLA, whose clinical and research interests are in immune deficiencies and recurrent infections. She is an investigator on gene therapy trials for primary immune deficiencies at UCLA. She is one of the authors of What a Clinician Needs to Know About Genome Editing in the Journal of Allergy and Clinical Immunology and Practice. And I'm looking forward to her helping us understand gene editing. So we're ready to explain it to our patients. Dr. Kuo, thank you so much for taking the time to join us today and welcome to the podcast. Yeah, of course. Thank you so much for having me. I'd just like to start by getting to know you a little bit better. Besides the introduction, can you tell us a little bit about yourself?

Caroline Kuo, MD 1:50

I'm currently at the University of California, Los Angeles, and I'm a native to Southern California. So I grew up here. I went to college here, I went to medical school at USC, did my my residency and fellowship also in in LA. So, you know, I'm used to this warm sunny weather, and um I have a husband and and two young daughters, a two-year-old and five-year-old. So outside of work, I don't get to do a lot of the things that I used to do. But it is filled up with a lot of you know activities for the kids, a lot of play dates, a lot of birthday parties, you know, a phase of life that I'm I'm enjoying, and they take me for a wild ride. Definitely a two and a five-year-old are their own uh hobby. You don't have time for other.

Rebecca Saff, MD, PhD, FAAAAI 2:38

Yes. What made you choose allergy immunology as a field? And particularly what got you interested in inborn areas of immunity?

Caroline Kuo, MD 2:45

Yeah, actually, you know, when I I when I was a pediatric resident, I thought I was gonna go into neonatology. I was very close to um had actually received an offer to to join the fellowship program after my training and was very close to accepting that. And then shortly thereafter, had a patient that was admitted that ultimately had autosomal dominant hyper-IgE syndrome. And and I didn't really know that I was interested in immunology, but I did find myself not being able to stop reading about him. Um, it was one of the, he was one of the few patients that made me, you know, sit by myself in the dark at home, you know, when I should have been resting, and just wanting to read everything I could about that disease. And I think it was through him that I realized, oh, I I like this. I I I like this topic. It fascinates me. And it's something that I I want to pursue further. Um, so I would say that you know, he was my initial drive to look into allergy and immunology. And when I was applying for fellowship programs, I also realized that a lot of the immunologists that I was meeting did a lot of research, not all basic science, but some basic science, a lot of clinical research. And you know, there was a lot of curiosity in people that I met in the field. Um, and I I was really attracted to that and and really wanted that for my own career.

Rebecca Saff, MD, PhD, FAAAAI 4:16

Curiosity will really take you far because it gets you to learn and read about all these things.

Caroline Kuo, MD 4:21

Yeah, definitely.

Rebecca Saff, MD, PhD, FAAAAI 4:23

So gene therapy has been used and studied now for actually decades, I think. Um, what are the successes that we've seen in gene therapy and how many people have really undergone gene therapy outside of just the trials that we read about?

Caroline Kuo, MD 4:36

Yeah, great question. So when I think about gene therapy, um I sort of break it down into three big buckets. So I think of gene therapy of hematopoietic stem cells, so bone marrow stem cells, which I'm more familiar with. Um, but you know, that could include, you know, um treatments for severe combined immune deficiency, for beta-thalassemia, for sickle cell disease, adrenal leukodystrophy, those kinds of diseases. And then the second bucket are immunotherapy, so modification of T cells of a patient's own T cells so that they're able to target cancer cells. And you know, there's there's several T cell car T cells or T cell receptor immunotherapies that have been approved. And then the third bucket I think of are sort of in vivo AAV therapy. So um AAV stands for adeno-associated virus, so using an adeno-associated virus to deliver the normal gene to various organs. And so that's been used in the eye for congenital forms of blindness, for neurologic diseases like spinal muscular dystrophy, um, and for coagulation disorders like factor deficiencies. Um, and so there have certainly been FDA-approved treatments in each of those buckets. In terms of the numbers of patients that have been treated, you know, there unfortunately isn't a central kind of registry of who's been treated. I would say the fewest probably have been in the hematopotic stem cell realm. And then more patients have been treated with immunotherapy as well as the AAV therapy. So, you know, I think thousands of patients worldwide have been treated, for example, for the spinal muscular atrophy, um, for spinal muscular atrophy using AAV.

Rebecca Saff, MD, PhD, FAAAAI 6:35

And why do you think there hasn't been more gene therapy in inborn errors of immunity? Um, what are the challenges that that group of patients might have in particular?

Caroline Kuo, MD 6:45

Yeah, so I think some of that is historical. So in the early clinical trials for inborn errors of immunity, um, there were complications. So there was what we call insertional oncogenesis when we use the older forms of viral vectors to deliver a normal copy of the gene. Um, so when when those cases occurred, we were using um retroviral vectors and they like to integrate in parts of the DNA or in genes that are active, that are actively transcribed. And so that also meant that they were more likely to integrate near proto-oncogenes. Um, and so you know, if we integrated a gene cassette near proto-oncogene, that could lead to turning on the gene that we inserted in an uncontrolled fashion. Um, the good news is that's been, you know, addressed largely by the most recent generation of viral vectors. So these are HIV-derived viral vectors, which we call lentiviral vectors, that have been stripped of its um virulence factors, so they're not harmful to us, but they still retain the ability to hold a DNA cargo, so you know, our therapeutic transgene, and they still retain the ability to deliver that to cells. Um, you know, so so I think the insertional oncogenesis remains a concern, but really has been addressed by some of the more um you know recent versions of viral vectors. Um, I think the other thing too is that you know there's a lot of inborn errors of immunity, and and as you know, that list grows every year. You know, we now have over 500 genes that we know can cause immune deficiency, and and in each of those diseases many times are really rare. And you know, I think there are inherent problems with developing gene therapies for rare diseases. You know, it's many times few patients are available for treatment. The development of gene therapy is takes many years and it's very costly. And by the time it does get to FDA approval and marketing, it means that you know these treatments are very, very, very expensive. And so, you know, I think it's just inherent to inborn areas of immunity that you know we're treating you know many of these rare diseases. And and I think until recently, we were using viral vectors, which randomly add DNA to cells. And some immune deficiencies are not amenable to that kind of gene correction where you have a gene that's randomly added and is on all the time. There's a there's many immune deficiencies in genes that are tightly regulated. And so when you do gene therapy by viral vector gene addition, you can't necessarily regulate when that gene turns on or turns off.

Rebecca Saff, MD, PhD, FAAAAI 9:44

And now the shift, there's been a shift kind of from this gene therapy to more of this genome editing idea. And is that the kind of movement of the viral vector instead of using that, actually using these techniques to really edit the gene, is my understanding.

Caroline Kuo, MD 9:59

Yeah, you know, I think there's still very much a role for viral vector gene therapy because it's tried and true and it works, and it can be life-saving and curative, because they, you know, there are many diseases where having a gene on all the time is okay. So, so for example, adenosine, DMA, severe combined immune deficiency, so ABA skid, um, it's okay if if that gene is turned on all the time. And so viral vector gene therapy works really well for that disease. You know, I think because there's been so much work done with viral vectors, we also know how they behave and we know that they are relatively gentle on these delicate hematopoietic stem cells and and that cells that receive that viral vector generally do pretty well. So I do think that there still is a role for it, and it's certainly you know still always something we consider whenever we tackle a new disease. You know, is this you know, is this genetic defect amenable to gene addition, just random gene addition? But as you mentioned, there's been you know a large shift to gene editing, and and that's where we go in and um and edit essentially. So we go to exactly where we want to go in the genome. So usually we go to the site of where that gene is, and we either site specifically insert the entire coding C DNA to that where we want it so that it remains under control of endogenous control elements, or we go in and we make little changes, like if we know you know certain base pairs are incorrect or certain little segments are wrong, we can go in and fix that. And so, you know, I think that's very attractive because that can address all of the immune deficiencies that are due to genes that are tightly regulated, and you know, we don't run the risk of insertional oncogenesis, which we can have with random viral vector integration, because now we're going specifically to where we want it and we're making the changes that we want.

Rebecca Saff, MD, PhD, FAAAAI 12:05

There's a lot of different techniques that are now used in gene gene editing, um, and they all have these different acronyms. Could you walk us through maybe some of the primary forms of gene editing that editing that have been used?

Caroline Kuo, MD 12:18

Yeah, sure. So, you know, I think many people now have heard of CRISPRCAS9, you know, not just in scientific literature, but in the Lay News. And, you know, it's really, you know, revolutionized the field of gene editing. But you know, we can't forget about the the other gene editing tools that came out right before it. So, you know, one one of which is ZFNs or zinc finger nucleases, um, and that was uh followed thereafter with talons or transcription activator-like effector nucleases. And then, you know, then that was followed with CRISPR-Cas9, which has really kind of changed everything. But I think of all three of them very similarly. So they all have a DNA binding domain, so something that will recognize that you can design to recognize a very specific sequence of DNA. So that's very important because that's that's what allows the targeting. And they also all have a DNA cleavage domain because you know the goal is to make a cut in a specific sequence of DNA that then allows you to insert whatever therapeutic sequence you want to put there. ZFNs and talons are a little bit more similar in that they use these protein modules that are sort of linked together, these repetitive protein modules that are that are pretty similar but differ slightly so that they can recognize each of the different, you know, DNA-based pairs. And so you can imagine if you stitch together a series of the these protein modules, that then you can make them specific for your DNA sequence sequence of interest. The other thing is talons and ZFNs, they work in pairs. So you have a left arm that binds one strand of DNA upstream, and then you have a right arm that binds the other strand of DNA downstream, and then they cut somewhere in between that. They are very different from CRISPR-Cas9. So CRISPRs use a guide, a short guide RNA. So it's now it's RNA, it's not a protein. The RNA is what gives it its specificity, and it and you can you know design this guide RNA to be complementary to whatever you're trying to target, and it sort of zips along your DNA and looks for its complementary sequence, and when it finds it, it stops and then it recruits Cas9, which is what does the cutting at a very specific part of this guide. So, you know, they're they're both all of these technologies are similar because you have to target DNA and then you have to cut it. But the reason, you know, CRISPR-Cas9 really revolutionized the field is because they're so easy to make. You know, I will say when I when I started in the field, I you know, I think I was very fortunate when I started. I started right when talons came out and right before CRISPR-Cas9 came out. So when I started the work, zinc finger nucleases had to be made by a company, and it was difficult to make a good pair of ZFNs. Then there was a paper that came out describing how you can make talons on your own in the lab. And so that's actually what I started working off with. But I struggled for a year just to get a couple of good pairs of talons just to cut DNA. I wasn't even correcting anything yet. And now we can have undergrads make really efficient guides in a week. So you can see how that really, you know, changes the field. When we started with CRISPR, we also felt like, oh, but you know, maybe it's gonna be very nonspecific. Maybe there's gonna be a lot of off-target cutting because now the specificity of a CRISPR is this 20-base pair guide RNA. So, how is that going to be better compared to the ZFNs and talons that are these two arms of proteins that have to come together? They these arms, both of the arms recognize different DNA sequences, they have to both bind, they have to come together, and then a cut can happen. You know, and yes, off-target activity can occur, but it turns out that CRISPRs can be quite specific as well. And so, you know, we still definitely use EFNs and talons, but really in in sort of the day-to-day and thinking of new therapeutics, you know, I think many people in the field kind of go straight to CRISPR-Cas9.

Rebecca Saff, MD, PhD, FAAAAI 16:51

Are there disadvantages of CRISPR? I mean, it's such an amazing technology. Um, it's so specific, it's short, you know, kind of all of these things. What are the disadvantages of it?

Caroline Kuo, MD 17:03

Um, so I think, you know, one is there still can be off-target effects. Um, so you know, there are certainly, you know, many instances where we've identified a good guide RNA that is, you know, very specific to our target, that has very high activity where we want it to cut. But then when we do genome-wide assessment for cutting of that of DNA by that same guide, we do pick up there. Are cases where you have off-target activity that's higher than the on-target activity. And, you know, so of course, in those cases, it's very easy to say, okay, we're not going to use this guide. But what about kind of more rare off-target activities? You know, we we can we certainly pick those up. And and sometimes it's hard to know what the effects are when it's in intragenic regions or in introns. We sort of assume it's probably okay, but you know, we don't we don't know for sure. We're still, you know, faced with the difficult task of delivering CRISPR-Cas9 reagents to the right cells. They still, you know, delivery is always a big problem with anything related to gene editing or gene therapy. And so we we we still you know need to get them to the right place. And finally, the most common form of CRISPR-Cas9 that we use of the most common form of Cas9 is actually from a bacteria that many of us have encountered. And so there's always this theoretical risk of you know, could we be giving this to an individual that already has pre-formed antibodies to Cas9? And could that decrease the efficiency of gene editing?

Rebecca Saff, MD, PhD, FAAAAI 18:44

There is a CRISPR-approved therapy for sickle cell disease. It's available. Um, but my understanding is that actually it's been there's been some a fewer number of people that have received that therapy than was expected. Um, why do you think that is?

Caroline Kuo, MD 18:59

The therapy you're referring to is called Kaskevi, and you did very well in clinical trials. And and the way that this works is it's it's a CRISPR, and instead of fixing a gene, so it actually knocks out an enhancer of another gene called BCL11A. And what BCL1A typically does is it is a repressor of fetal hemoglobin production. So, you know, during development, we switch from fetal to to adult hemoglobin. But fetal hemoglobin can't sickle because it has the gamma subunits, it doesn't have the beta subunits. And so, you know, it has a benefit in that if you can make more fetal hemoglobin, that those, you know, that can't sickle, but also it has higher affinity for oxygen. And so what's what's really nice about this approach is it's a knockout approach. So all you need to do is take design a guide and um to target that locus, and you you knock out that gene and you've achieved therapeutic benefit. Um, it's different from many, you know, the approach for many other diseases where we need to design a CRISPR to cut DNA, open up the DNA, and then we also have to deliver the therapeutic gene, the corrective gene, and have that integrate properly at the right location. So Caskevi is really, you know, I think done really well because you know, they don't need to fix anything, they just knock out a gene. And those patients in the clinical trial did really well. There really was a very significant decrease in hospitalizations and sickle crises.

Rebecca Saff, MD, PhD, FAAAAI 20:34

I remember reading the New England Journal of Paper and just being amazed at the you know how beneficial it was, how effective, like very few downsides.

Caroline Kuo, MD 20:43

Yes, yeah, exactly. And you know, technically there's tens of thousands of patients who could be eligible for the treatment just in the US. But to my knowledge, and you know, I I I only know what I you know can read about, but to my knowledge, there's only probably been a couple hundred um patients that have received this this therapy. And I think it's a reflection of you know the challenges in the field that we're still facing. So one is, you know, this still is a transplant. It's an autologous transplant, so you're receiving your own cells back. So it's great that you don't have the challenges with graph versus host disease or or graph rejection, but it's still a transplant, meaning that you still have to make room in your bone marrow to allow those gene-modified cells to find residence. And the way that we do that is through bu sulfan. And that's a you know, a chemotherapeutic agent. You know, it's still required, it still, you know, will result in infections and you know, could result in infertility and and bad mucositis, and you know, still requires hospitalization. So, so yes, it's better than an allogenaic transplant, but you know, it's Not, it's still not a walk in the park. It's still a big commitment. And at this point in time, you know, because we have to collect stem cells from a patient, usually through phoresis, and then we have to take those cells and they have to be gene modified. This can only occur at specific treatment centers. You can't just walk into any hospital and expect them to be able to carry this out. You know, they have to be able to collect the cells, they have to be able to process cells. They get actually shipped to a central location for them to be gene modified, but then you have to be able to take those cells back, you have to be at a place where there's transplant medicine, and then you'd be able to reinfuse them. So that you know, a lot has to go on. And then the manufacturing and quality checks that have to occur are very extensive. Um, and so and it could take many months. So, you know, I would say it's it's very involved still. And um, patients have to be healthy enough without severe organ damage in order to qualify them for studies, and many times that's because of um them having to receive B-sulfan conditioning. You know, they have to be healthy enough to receive that without having too many side effects. And then finally, the cost of the product is $2.2 million. And so, you know, I think that's certainly justified because, you know, I think receiving the therapy is is essentially curative and it prevents so many hospitalizations and downstream issues, but you know, that makes insurance authorization very complex and very long-winded.

Rebecca Saff, MD, PhD, FAAAAI 23:45

And so base editing is another form of gene editing that's been described. It's a way I think that you can edit the DNA without having a double strand break. How does that work? And is it being used?

Caroline Kuo, MD 23:58

Yeah, so so yeah, great, great question because um there are now newer CRISPR Cas9 technologies that are coming out that are very exciting for everyone working working on this. So base editing is exactly what you said. It doesn't cause a double strand of break in the DNA, it creates a nick in the DNA. So just cutting one strand. And I think of it like a like a pencil and eraser. So you can go in and take out the you know, erase that one base pair that's mutated and write in the correct base pair. And so what is this composed of? It's still at you know, at its heart, it still started off with CRISPR Cas9. So you still have a guide that directs the editing to the right place, so you still need that, and and you also still need a Cas9 because that's kind of where the enzymatic activity is. But the Cas9 has been modified, so it only creates a NIC, so it only cuts one strand of DNA, and that Cas9 is now fused to another protein, which is a deaminase enzyme. And what that deaminase enzyme does is it can chemically change one DNA base into another. Um, so currently there's two forms of base editors. We have cytosine base editors that can change a C to a T, or if you think of the opposite strand, a G to an A. Um, and then we also have adenine base editors that can convert an A to a G, or on the other strand, a T to a C. So, you know, it it does mean that we are a little bit limited because these two base editors can only make transition changes, meaning a peering to a peering, or pyrimidine to a perimidine.

Rebecca Saff, MD, PhD, FAAAAI 25:59

You have to have a very specific type of mutation for that to be applicable.

Caroline Kuo, MD 26:03

Yeah, exactly. I think you know, the the big advantages over sort of traditional CRISPR-Cas9 gene editing is that, you know, as we've mentioned several times, there's no double-stranded break in the DNA. And that's beneficial for many reasons because you know, our cells don't like DNA double-stranded breaks. If they occur, you know, they occur when we're exposed to UV radiation or, you know, just in general, you know, stress and to the cells, but they want to repair that. When there's enough of those breaks, they sort of recognize this as a as a danger signal they may want to get rid of the cell or cut out a bunch of that DNA. So it's good to not cut both strands of the DNA. And you know, then we also prevent translocation. So you can imagine if you cut one portion of your DNA really well, and let's say there's an off-target effect somewhere else that also cuts really well, those two chromosomes could come together. Um, and so we don't we don't run that risk. Thus far, base editors have been quite efficient. I think to date, I think the adenine base editors are more efficient than the cytosine base editors in sort of generally speaking, but you know, they are very efficient and they are they can be efficient to levels that are therapeutic. It's it's permanent, you know. So we you know we we make that one change and it's permanent. And we can use it for many diseases. I think, you know, I've heard in in many talks and read in papers that maybe one-third of diseases can be corrected by just base editors. Um, but the disadvantages are also related, right? So we're limited to those changes, so we can't change all the other um base pairs that need to be changed. We can't fix deletions or insertions, or and we can't fix things that are multiple base pairs in a row. And you know, by we call it bystander editing. So bystander editing can still occur. So if you think about like, oh, I there's an A, there's a mutation that's an A that I want to change into a G. Well, what if there's A's that are next to it? You know, we only have four DNA base pairs, so it's it's highly likely that you might have other A's in in the vicinity. And so, you know, it does take some designing of these guides and finesse to try to only change the A that you want and minimize these bystander effects.

Rebecca Saff, MD, PhD, FAAAAI 28:28

So, which of the inborn errors of immunity do you think we're going to see first off that this these therapies coming to fruition?

Caroline Kuo, MD 28:37

You know, I think one is the diseases where gene regulation is very important. So, for example, um X-Lean typer IgM due to defects in C D40 ligand, the C D40 ligand gene is very tightly regulated. You know, it's expressed very lowly on T cells in all of us when we're not sick. And and during times of infection, it's very quickly upregulated. And even before we get better, you know, that CD ligand expression goes down. And so we know that if we keep CD ligand expression on constitutively, so on all the time, that in mouse models that can result in abnormal lympho proliferation. So I think um gene editing, base editing is going to be really helpful for those kinds of diseases. It's already been put in through early, you know, very small and early phase clinical trials for X-linked CGD, for CD40 ligand, and you know, they have had really excellent results based on preliminary data. Um, so I think we're just going to see more and more of this.

Rebecca Saff, MD, PhD, FAAAAI 29:42

Absolutely. Um and where are we in these treatments? Do you think you're going to see something approved within the next couple of years? Do you think we're we're further than that?

Caroline Kuo, MD 29:53

Um no, I I think I think we will. I think we will. I think we're very close. And you know, I think something that really changed um the way that we think about base editors was baby KJ. So he's the little baby that was treated at UPenn, who was born with a metabolic disorder that is very serious. And his clinicians and his the scientific team there, along with other collaborators, were able to put together a in vivo base editor to help ameliorate his disease, I think within nine months, a crazy short period of time. And I think that really has changed the way that many of us think about base editing. Because, you know, at least personally, I for the most part was working with sort of traditional CRISPR-Cas9 and trying to integrate the entire therapeutic gene in the right location. And the reason that I really focused on that was because I thought this is a one-size fits-all. So if I can integrate a corrective gene where it needs to go, then I can treat anyone with that particular disease regardless of which mutation they have. They can even have a deletion, they can have an insertion, they can have anything. And I because what I'm inserting is going to override anything downstream. But that has a lot of its own challenges because you know I mentioned earlier that hematopoietic stem cells are delicate and they don't like to be manipulated and they don't like people sticking in DNA where it doesn't belong. And um, and so it's, you know, we can we can gene edit really efficiently in a dish, but when we put those into in vivo models, those gene editing rates go down. And I think it's a reflection of the of the toxicity to matter what extent cells. The great thing about base editing is that the edit is contained within the editing tool, the base editor, the guide and the cast. We don't have to deliver a corrective piece of DNA, which is toxic to cells. I think that lack of a DNA double-stranded break, cells like that. They don't, they it doesn't turn on these alarms, you know, that kind of uh result in an immune response. And so we're able to efficiently edit these primitive uh stem cells, and we're able to put them into in vivo models or even humans and have them remain. And so I think given how scientifically advanced we are with base editors and given the excellent results with baby KJ, the FDA has you know now become more open to this idea of platform technologies. This they call it a plausible mechanism pathway, where you can go to them to get an IND for a let's say a base editor for a single mutation in a gene, and that will require the full package. You know, it's you're gonna have to do everything that is typically required of an IND package currently. But you can go back to them. Let's say you you you get it approved and you treat a patient and it's it works well, you can go back to them and you can add on additional mutations without that whole package. There's gonna be certain elements that are still going to be required, like on-target editing and off-target analysis, but they're not gonna require the costly um work that's required to get a you know a new IND for every mutation. And so I used to be kind of down on base editors because I thought, how am I gonna get funding to address every mutation and get a new IMD for every mutation? But ever since this new pathway came out, I think you know, this is definitely you know much more feasible. My lab and many others are very actively working on this to try to get at least one mutation in you know for IMD approval and just continue to continue to add more to that.

Rebecca Saff, MD, PhD, FAAAAI 33:58

And do you still need that conditioning regimen that we were discussing for these base editing?

Caroline Kuo, MD 34:04

We do. Right now we do, because we just we need room, we need to make room in the bone marrow. You know, what once we collect those stem cells, we need room to put them back. Um, and so they do receive, they generally will need um conditioning, and the conditioning regimens will differ depending on the type of immune deficiency. Some immune deficiencies like skid, where you know their immune system is sort of an empty bag, will will need can can can get by with reduced conditioning. Other immune deficiencies, particularly ones where there's immune dysregulation, that's part of the clinical picture, they're probably going to need more because you not only need to make space, but you have to get rid of you know the autoreactive cells.

Rebecca Saff, MD, PhD, FAAAAI 34:48

Do you see these conditioning regimens getting better over time? You know, are there medications that are on the horizon that may not be as toxic as B cell band, for example?

Caroline Kuo, MD 34:57

Yeah, definitely. I think it's something we're all waiting for. You know, I think many groups are working on these less toxic conditioning regimens. Some of them are antibody-based, where you know you can introduce antibodies that are specific to certain markers of stem cells, and it'll selectively get rid of those and so that there's not as much toxicity to other cells. And I think the other thing that you know we all are looking forward to is sort of this increase in attention to in vivo geneticity. So rather than having to take cells out of a person and fixing them, what if we just deliver what we need to those cells in a human, which maybe wouldn't require conditioning because we're, you know, we're we're targeting the stem cells that are already in their natural niche.

Rebecca Saff, MD, PhD, FAAAAI 35:49

And do you think that there'll be room for gene editing outside of the hematopoietic compartment?

Caroline Kuo, MD 35:55

Yeah, definitely. You know, I think there's definitely going to be a need for gene editing of some differentiated immune cells, um, like for example, T cells or B cells. So, you know, one example I can think of, and I I brought up CD40 ligand deficiency already, but you know, that that immune deficiency is due to a defect of CD40 ligand that's on T cells. And, you know, because many patients are transplanted when they're already quite ill or they've already had pre-existing infections, the risk of giving them conditioning for either allogenaic or autologous hematopoietic stem cell transplant is reactivation of those previous infections or ongoing infections. And so in those situations, when we when I think about gene therapy, perhaps the safest approach would be that we collect stem cells from them for sort of a permanent or a curative gene therapy, but simultaneously collect T cells from them so that we can correct their T cells and infuse them into them sort of as a bridge so that they have some protection while we're waiting for them to engraft.

Rebecca Saff, MD, PhD, FAAAAI 37:12

You don't have that period of time where they're so immunosuppressed while you're waiting for that engraftment.

Caroline Kuo, MD 37:17

Yeah, exactly.

Rebecca Saff, MD, PhD, FAAAAI 37:19

Yeah. And then what are some of the specific immune or inborn errors of immunity that you see kind of being early earlier targets because of the therapies that we currently have? You mentioned CD40 ligand. Are there other ones kind of in that category where you can really see it being an earlier early adopter of gene editing?

Caroline Kuo, MD 37:37

Yeah, I think um, you know, I think any of the immune deficiencies that have already been treated with viral vector gene therapy could be early adopters, because if there's anything that you know potentially went wrong with with lentiviral vector gene therapy or maybe not went wrong, maybe just because of the disease process is not as amenable to gene addition. I think you know, because there's already this precedence for gene therapy that they could easily transition over to gene editing. And then, of course, I think just you know, in addition to C before you lie again, any disease where there remains the need for tight regulation of the gene.

Rebecca Saff, MD, PhD, FAAAAI 38:18

And what do you think is the next development of technology that we're waiting for? What do you think is going to be the next CRISPR?

Caroline Kuo, MD 38:27

Well, you know, I think um one thing I didn't talk about that's sort of related to base editing that came came out kind of around or shortly thereafter was is something called prime editing. And I had mentioned to you that, you know, base editing is is limited in terms of the the base pairs that it can change. And so prime editing in instead of being like a pencil, like an eraser and pencil, is more like a search and replace. It's kind of been described, where the very similar components are still there, but that guide now has been extended. So we still use that guide to go to the right place in the genome, but that guide is now longer. We call it now a peg RNA, so prime editing guide RNA. So that it's it's been extended so that it includes the correction that we want to achieve. So we don't need to introduce any other elements, but it it has a template for the correction we want. And it could be any base pair change, it could be writing, inserting in a few deletions that had been lost, it could be replacing several different base pairs at once. And so now we have this peg RNA that's longer, and the CAS9 is still a nic A, so it only nicks one strand of DNA, but it's now fused to a reverse transcriptase. And so when the CAS9 comes in, that reverse transcriptase will recognize the little template that we have attached to the peg and write that in and correct the mutation that we want to correct. And so it's a little bit more complex. There's there's more elements involved in prime editing, but it it does enable many, many more different types of corrections. And so, you know, when I think about improved editing technologies, I think of it kind of like that. Like we're just going to have, you know, we're just going, and it's not me, but other groups are going to keep, you know, working off what we already have, making these technologies more specific, more efficient, and maybe not require so many elements. You know, when we talk about clinical trial, the fewer things you need to bring into a clinical protocol, the easier it is to get it approved and the easier it is to get it to the population. You know, I don't know what's coming up, but that's sort of what I think of is we're gonna continue to improve upon what we have just to make things more efficient and safer.

Rebecca Saff, MD, PhD, FAAAAI 41:01

And do you think cost-wise, do you think this is going to be something that insurance will eventually cover? Um, because you are doing it young, you're saving all these hospitalizations, or you think that's still gonna be a significant barrier for a lot of patients?

Caroline Kuo, MD 41:14

Yeah, I think that the the cost will be justified. I think that if you know these therapies are um curative, you know, when calculate out how much money you're saving in terms of that individual's lifetime hospitalizations and medications and treatments, it will be worthwhile, but it'll still be very expensive for all the reasons I mentioned before, in terms of meeting specialized centers and and you know, have being able to process cells and do the gene, the the manufacturing of the product. But I think that it will improve because I think that there's been a lot more focus on in vivo gene therapy. So that takes away the need for cell processing. You know, now we just have to make the gene editors and get them into the to a patient. It's complicated, but people are doing it, it's being achieved. And many times this is being done through lipid nanoparticles. So, you know, I said delivery is a is a big issue. So, you know, right now people are focusing on on LNPs to carry the cargo and get them to the right place. So I think as delivery methods improve, and as we start to be able to deliver gene therapy reagents in a patient, that's really going to decrease the cost, and that's really going to make this a therapy that's more available. Because now you may receive a product in a vial rather than having to be a center that collects cells, sends it somewhere, have to condition them, receive the product and give it back and monitor them. And so so I think it's on the horizon.

Rebecca Saff, MD, PhD, FAAAAI 42:52

And that's already in clinical trials, these kind of treatments where you don't need the conditioning regimen where you can just introduce it.

Caroline Kuo, MD 42:59

Yes, yes. And so, for example, you know, relevant to the field of allergy immunology, there's an active clinical trial for hereditary angioedema. Um and that's CRISPR-based, and that's in vivo. And my understanding is that the outcomes have been quite good. And again, it's an it's another um, it's a very smart approach. So, you know, similar to the sickle cell CRISPR gene editing that I described earlier, it's not correcting the gene that's responsible for HAE. It's not correcting SERP SERPING G1. Instead, it's knocking out another gene that produces precalocrine. And by having less precalicrine, then you have less bradykinin production. So you're you're not as likely to have these swelling episodes. It's a it's a very creative way of addressing the disease sort of semi indirectly. And you know, it's it's being delivered by lipid nanoparticles. And what's also a You know, really great for this particular disease is that lipid nanoparticles like to go to the liver. They naturally travel to the liver. And that's where, you know, a lot of the pathology stems from in HAE. And so, you know, they were able to design, you know, highly specific and efficient, you know, CRISPR guides. And they were able to deliver it by LMPs in vivo. The reason it hasn't quite happened yet for diseases that are in the hematopoietic stem cell compartment is because LMPs don't naturally go to the bone marrow. They're all going to go to the liver first. And so that requires additional engineering of lip and nanoparticles in order to get them to the right site. So that's definitely being done by many groups and it will happen. But it does make sense that this HAE and Vogene editing occurred first because it kind of all came together in a way where we were able to deliver reagents to the right organ in a very efficient manner.

Rebecca Saff, MD, PhD, FAAAAI 45:04

How do you target? What are some techniques people are using to target the hematoproduc stem cells with these lipid particles?

Caroline Kuo, MD 45:11

So you can design lipid nanoparticles to target specific antibodies or receptors that stem cells express. Of course, you know, anything you add on to a lipid nanoparticle is going to change its property. So change its ability to travel to the right place, change its ability to carry cargo. And so I think there's a lot of very, there's a lot of finesse that needs to go into to making a good targeting lipid nanoparticle. But you know, it does require addition of other elements onto the surface of the lipid nanoparticle to get it to the right place.

Rebecca Saff, MD, PhD, FAAAAI 45:46

Does it make it more immunogenic as well, or not really?

Caroline Kuo, MD 45:50

I think it could be.

Rebecca Saff, MD, PhD, FAAAAI 46:08

Yes.

Caroline Kuo, MD 46:09

Which is amazing. Definitely.

Rebecca Saff, MD, PhD, FAAAAI 46:11

And tells us how important it is to continue to discover the genes that are causing this inborn narrative immunity because we can't target them unless we know the mutations that need to be targeted.

Caroline Kuo, MD 46:20

So yeah, yes, definitely. You know, I, you know, I do get emails from patients who, you know, are asking if gene therapies can be developed for their particular diseases. And, you know, I would say a good number of them come to me without a gene, right? They have a true clinical presentation of of immune of immune deficiency or immune dysregulation, but there has been no gene identified. And in those situations, it's really, you know, it's really disheartening to say, no, I can't because I don't know what to do. I don't know what to target. Um, and so certainly this continued, you know, this curiosity that kind of is in our field is so important because, you know, identifying what we can target, what is causing the disease is is going to be important for developing new therapeutics.

Rebecca Saff, MD, PhD, FAAAAI 47:06

Well, I think that's a great place to end. The curiosity is in our DNA as allergy immunologists, and we have to continue to uh to brainstorm and develop these ideas.

Caroline Kuo, MD 47:15

Yes.

Rebecca Saff, MD, PhD, FAAAAI 47:16

Yeah. Well, thank you so much.

Caroline Kuo, MD 47:18

Yeah, of course. Thank you for having me today.

Rebecca Saff, MD, PhD, FAAAAI 47:21

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