Introduction to gene-directed therapies (a LinkAGE webinar)

[00:00:00] Professor Kate Tatton-Brown: Good afternoon and welcome to LinkAGE, our new webinar series from the Genomics Education Program. LinkAGE stands for linking research with genomic education, so academia with genomic education. And the purpose of the series is to really horizon scan what is going on in that research setting and try to show how it can be applied to the clinical setting. So really seeing those green shoots of research. The point about this webinar series is it has a clear educational narrative, so we really hope that you’ll enjoy many of the talks that we have in the series.

[00:00:36] A few housekeeping points: you’ll be automatically muted and your camera will be off. When it comes to the Q&A at the end. Please use the Q&A tab and please save your questions for the Q&A. At the end, you’ll be asked to evaluate the talk and then you can request a CPD certificate. You only get that CPD certificate if you have completed the evaluation form. And to let you know that we will be recording this webinar, and the series, and that the recording will be sent to you later.

[00:01:08] Our next webinar is on the 21st of February, which James will also be presenting, and that will be on CRISPR therapeutics. So a big thank you to Dr. James Patterson to come and talk today. So, James studied medicine before undertaking a PhD with Sir Paul Nurse at the Crick Institute on fission yeast cell-size control.

[00:01:30] James is now a founder of Xap Therapeutics, which is a biotech company developing CRISPR engineered iPS cell-derived delivery vehicles for complex therapeutics. So James is very much going to set the scene for us, today, on gene-directed therapies, thinking about ex vivo and in vivo approaches and new advances in this landscape, and thinking about the challenges that are associated with gene-directed therapies.

[00:02:00] So, a big thank you, James, for coming to speak to us today and for sharing your expertise over the next 45 minutes. And I’m very much looking forward to your talk. Over to you.

[00:02:11] Dr James Patterson: Thank you very much for that introduction, and thank you for giving me the opportunity to speak. So I’m now gonna try and share my screen.

[00:02:19] Hopefully there’s no hiccups here.

[00:02:21] Okay, thank you everyone for joining. Today I’m going to be giving an introduction to gene-directed therapies. Just briefly, I do work at companies so a quick disclosure.

[00:02:31] And so an overview of what we’re going to be, or what I’m going to be, discussing today are: an introduction to gene-directed therapies; an overview of gene expression and how we can target it therapeutically; discussion of ex vivo gene-directed therapeutic approaches; and also then insight into in vivo gene-directed therapies; followed up by some insight into the next generation approaches and also some considerations on the economics and ethics of these novel therapies, and really gene-directed medicines is a huge space.

[00:03:04] So I just wanted to lay out some scope here so that no one is disappointed. So, I wanna give a broad overview of the different chemical and biological entities and delivery vehicles you’ll likely start to encounter within the clinic over the next few years. And I’m gonna focus this mostly on the treatment of diseases with a genetic basis – not really looking at oncology treatment. So yeah, I’m talking about CAR T-cell which are also, in a way gene-directed therapies. However I think that space is a, it’s a talk in its own right. And I should, I hope you should be with an understanding of the key risks and issues with these modalities and where they’re going in the future, and, for an in-depth focus on CRISPR, you’ll have to attend my next talk.

[00:03:44] So to start off with, I just wanna say gene therapies are coming, whether you like it or not. Cell and gene therapies are more likely to be approved in small molecules. So this is something that pharma is very interested in because they spend a lot of money developing a drug and they’re obviously very interested in seeing it get approved.

[00:04:01] Drugs targeting a genetically implicated target are four times more likely to be approved as well. And the FDA estimates there will be 10 to 27 gene therapy approvals a year, by 2025. And you can see here in this plot, on the right, that the percent of the new drug applications that feature cell- and gene-therapy technologies is rapidly rising.

[00:04:23] So why are we seeing this increased interest in gene therapies now? What’s been the catalyst here? And really I think it’s a combination of different factors. So it’s the availability of genetic information, which has expanded massively since the early 2000s. Obviously, with the sequencing of the human genome, but not only that, also the sequencing of lots of human genomes, which has enabled novel targets to be discovered.

[00:04:48] I think there’s been a huge increase in the availability of different gene targeting modalities. So, all these interesting technologies like CRISPR, like small interfering RNA, which started off really being used a lot by researchers in the lab, which have now transitioned into the clinic.

[00:05:05] The platform nature of gene-directed medicines. This idea that all gene medicines or many of them are based on different combinations and nucleotides, means that once you build up manufacturing technology around joining these nucleotides together in a certain order, and once you show that it works for one clinical indication – so POC ( proof of concept) – it’s very rapid to expand into novel targets.

[00:05:28] So once you start to see, once you start to get some clinical signal and have some success, it’s very easy to expand that success into novel diseases, which is really the appeal of these platform companies and platform technologies. And also really something, at least something small, but good, that comes out of Covid is the success of the mRNA vaccine. And this has really increased confidence in gene medicine generally as a modality, and also enabled the delivery technologies so that nanoparticle – that delivers their mRNA vaccine – it’s also now been shown to work in patients.

[00:05:59] Changing gears a bit and going back to the beginning, what is gene expression? And I think we need to understand what gene expression is, in order to understand how to target therapies towards it. And what I mean by gene expression is really the central dogma, which was first proposed by Francis Crick, and this is the idea that of information flow from DNA to RNA to protein. So DNA is this heritable substance which encodes for all of the proteins that are present in your body, and your proteins are all the things that are functional and do all the jobs that cells need to do to survive and to specialise into different cell types. And RNA is the intermediary between these two.

[00:06:42] So if we take a closer look at the central dogma and consider it in the context of a eukaryotic or a human cell. The cell is set up into multiple, different, broad zones.

[00:06:55] So you can imagine there’s the nucleus, which is the location of the DNA; you have the cytoplasm, which is the location of the mature mRNA; and also within the cytoplasm you have the ribosome, which is the convertor (or the factory), which converts mRNA into protein. And what to really take away from this picture is this idea that your DNA has all the instructions for every mRNA that you’re going to make, and only certain regions of that instruction manual are read at any one time in any cell in your body.

[00:07:33] And different cells express different mRNAs depending on the job that they’re meant to do. So the mRNA compliment of a cell is a direct reflection of its function of the proteins that it will go on to make, and you’ll see that a lot of the gene therapeutics target different points in this pathway.

[00:07:54] We’re diving in a bit more into what we mean by the code and how this information is stored. If you take a look at this DNA, obviously, if you could dive into a cell and look at the chemistry, it doesn’t exactly look like a squiggle. In reality these little bars and this double stranded nature of the DNA is driven by complimentary base pairing between nucleotides.

[00:08:15] DNA and RNA are both polymers of a specific chemical compound and nucleotide. And these nucleotides can be joined together into long strings and different nucleotides and the different sequence of these nucleotides encode information. See, I think everyone in this call will know that these nucleotides are ‘A’, ‘T’, ‘G’ and ‘C’, and that A pairs with T and that G pairs with C, and this is how you are able to perform a double strand in DNA.

[00:08:47] And when you perform transcription and read DNA and produce RNA, that is complimentary to the DNA strand that encodes it. So you can see here, for instance, the example RNA sequence below is U-A-C. This sequence is complimentary to the A-T -G present in the DNA. In RNA instead of T it’s using U, but for all intents and purposes, it’s practically the same.

[00:09:13] So the important thing about the RNA code is that once you’re encoding a protein, it’s a triplet code. So a combination of three nucleotides of RNA encode for a specific amino acid. And this is important, but it’s important about how you consider mutations – we’ll get that point – but it also just serves to show how information has flowed from the DNA to RNA, and then subsequently is converted into protein by the ribosome. And this is really the basis of the central dogma. So I think at this point I can give a definition for today’s talk on gene-directed therapies. And that’s really any medicine that harnesses a specific combination nucleotides to drive therapeutic function.

[00:09:57] So you’ll see there’s loads of different ways that we can use nucleotides, or use nucleotides targeting a specific nucleotide sequences, to drive therapeutic function with a range of different therapeutic modalities. So before we move on to that, I think we need to dig a bit more into how a cell actually decides what it means to express.

[00:10:19] And this is important because it impacts how we can therapeutically impact gene expression in cells.

[00:10:24] So, if you look at the cell, instead of now seeing a simple linear DNA region what I’ve highlighted here is a sort of a rough design of a gene. And a gene in the cell is the independent unit that’s able to express an mRNA, and that mRNA go on to make a protein.

[00:10:43] And the genes are composed of promoters, which tell the cell when and where to express a certain mRNA. And there’s a terminator region, which tells the cell or tells whatever is reading this region of DNA code, when to stop expressing mRNA. So it’s a discreet unit. Something that’s important to consider is that distant regions of DNA within the cell, within the nucleus, can also impact the functionality and the expression of individual genes.

[00:11:12] So it’s both very close, or ‘in cis’ as the geneticists would say, a regulation of gene expression. And there’s also trans regulation, so long distance modulation of which gene is expressed in cells. And this gives us tools or ideas for how we can modulate the expression in a cell with therapeutic tools.

[00:11:33] And really this is driven in the cell by transcription factors. And transcription factors are proteins which bind to specific sequences within promoters and enhancers and tell the cell whether or not they should express a certain protein-coding gene. So transcription factors are effectively the instruction manual for how to read the full instruction manual, that is the DNA. So each specific cell type will have a certain set of transcription factors that dictate that cell’s function based on what mRNA is expressed.

[00:12:06] So after you start expressing your mRNA, it’s a little bit more complicated than just a single gene that’s produced. You must first produce a pre-mRNA, so this is present in the nucleus. And the reason this is a pre-mRNA is because genes aren’t so simple that they express a fully linear sequence that can be translated into a protein. They often, especially in humans, contain a huge range of introns. And introns are non-coding sequences that are present on mRNA molecules. They can have functions in regulating expression of the gene, and they also give the cell optionality in how, or what the protein may code for in this gene. If you take a look at this pre-mRNA here, it’s relatively simple. It has three exons and two introns. And what will happen in intron splicing is that the green regions or the introns are literally chopped out, and the red regions are glued back together, and this produces a mature mRNA.

[00:13:07] The cell can choose to skip different exons, and so you can also imagine a mature mRNA here, which skips the middle exons (the middle red block) and that might produce, from the same gene, a protein of different functionality from the three-exon protein, because now your coding sequence has changed.

[00:13:28] So once you produce your mature mRNA you are also subject to potential regulation within the cytoplasm of the cell. And there are small regulatory RNAs, so small regions of RNA that bind complimentarily to the mRNA molecule and can regulate its translation or drive that mRNA to be destroyed. And these are really, really potent therapeutic modalities as well as being things with the cell uses itself to further tune gene expression.

[00:14:00] So where can this all go wrong? Because DNA mutations can impact every step of this. Of course ones that we think about commonly are loss-of-function mutations, or recessive gene mutations present in the protein coding sequence of gene.

[00:14:16] You can also have gain-of-function mutations which result in dominant genetic diseases. So, dominant genetic diseases, for instance Huntington’s disease, various hypercholesterolemias are also autosomal dominant; loss-of-function mutations, cystic fibrosis or sickle cell.

[00:14:32] Missense and nonsense mutations are just descriptions of alterations of the genetic code level. So nonsense mutation is the introduction of an early stop codon so that your ribosome stops reading your gene halfway through. And it stopped, obviously detrimental if all the interesting bits are after that stop codon. You can also get large insertion-deletions which can clearly mess up the protein-coding capacity of the gene. And you can also often get these repeat expansions, so cells really struggle with copying repetitive regions in a gene. And again, this is the basis for Huntington’s disease.

[00:15:06] You can also get some mutations that alter transcription factor binding sites, and this can also result in disease. One classical example is a type of alpha thalassaemia. So this is a disease where your red blood cells are unable to produce certain haemoglobins, and in a certain alpha thalassaemia you can it can be caused by introduction of a transcription factor binding sites motif in the wrong place, you end up with aberrant transcriptions. So aberrant mRNA production, which disrupts the actual mRNA production you want to get. And again, you can think about how we might fix that with certain gene-editing approaches. You can also have mutations that alter your exon choice and can result in, for instance, exon skipping, the production of alternative spice products or even the inclusion of introns – which again disrupt your protein coding sequence.

[00:15:57] If you look at ALS and SMA, these are two severe neurological diseases. They’re caused by mutations in genes that alter splicing. So this happens in trans. So you have a gene that’s important for splicing. For instance, SMN in SMA and mutation of this prevents you from splicing genes properly in the nervous system resulting in a range of different severe neurological phenotypes.

[00:16:20] There’s a particular dilated cardiomyopathy, which results in extension of the three prime splice site in lamin A, and various muscular dystrophies can also be heavily impacted by splicing-driven mutations, so Duchenne and Becker as muscular dystrophies.

[00:16:36] Interestingly, you can also find diseases caused by mutations in microRNA binding sites or in microRNAs themselves. For instance, loss of microRNA-361 causes a choroideraemia. And there’s a particular, hereditary spastic paraplegia, which results from mutation of microRNA binding sites in gene called RE-1.

[00:17:00] And these, I’m just highlighting here that all these regulatory mechanisms have really strong phenotypes and have strong impacts when their functionality is lost. How can we learn from the importance of these mechanisms in order to design therapeutics that will change the biology of the disease and end up treating patients?

[00:17:20] And gene-directed therapeutic modalities really target different steps of these pathways.

[00:17:26] So yeah, starting off the top, you can see I’ve listed some gene editing enzymes. So CRISPR, zinc-finger nucleases and TALENs. And these enzymes act at the level of the DNA, so the heritable substance of the cell. And they can either disrupt genes, they can switch genes on and off if you modify them to express certain genetic fusion proteins, and they can also switch genes off less permanently. Instead of disrupting the gene, they can inhibit the transcription of the gene. So they’re almost, sort of, designer transcription factors.

[00:18:00] Again, I think that would be more the subject of the next talk.

[00:18:02] ASOs are small DNA fragments that bind to RNAs and they can block splice sites. So they can alter the exon choice in certain genes depending on where you target. We can also use a range of small RNA species to alter mRNA expression. So small interfering RNAs, microRNAs and short hairpin RNAs, are commonly used in research labs and commonly studied to modulate gene expression in cell models. But they also make for brilliant therapeutics as I’ll go on to show.

[00:18:32] And then there’s also the very obvious approach of re-expressing genes. So if you have a disrupted protein-coding gene, for instance, because it has a recessive mutation and you want to re-express the functional version, you can use viral vectors, for instance, to reintroduce the DNA. For this we’ll reintroduce the gene for that protein back into the cell. A key difference between the adeno-associated virus and lentiviruses is that (A) these deliver their DNA gene into the nucleoplasm of the cell, so it’s not integrated into the host genome. So it’s not heritable if that cell goes on to divide. However, lentivirus integrates this gene directly into the host genome, so it’s heritable. Once that cell goes onto divide.

[00:19:20] These viral vectors can then express mRNAs, which can make proteins. But importantly they can also express shRNAs, they can produce short hairpin RNAs, which then can then regulate endogenous genes.

[00:19:35] And they can also express the mRNA for these various gene editing tools. So they’re quite modular in terms of the different therapeutics and actions they could be used to employ.

[00:19:46] Looking again on the right here of different delivery vehicles. Exosomes and LNPs can be a small, almost fat bubbles that can hold RNA species and deliver them into a cell.

[00:19:56] So LNPs have featured heavily recently in news because they were used to deliver the mRNA for the Covid vaccine. Exosomes, more in the research stage, and I’ll have a slide on them later. And if you’re talking about editing cells outside of the body and putting them back into the patient, you can also use a strategy called the electroporation. And this is literally zapping a cell and then forcing in these nucleic acids into a cell and then altering their phenotype in the desired manner. And really the reason I’m adding these delivery vehicles on the slide is because, I hope you can see that we have a huge range of talk to modulate how genes expressed, and which genes they expressed where. And really I think the key takeaway from this talk is that we’ve got a lot of tools now. We’ve got a lot of tools in our arsenal for how to modify genetic disease. And really it’s the delivery vehicles, I think, that need a little to be desired. And there’s active work, including our company, but also many others trying to improve delivery.

[00:20:59] So yes, it’s the problem of delivery.

[00:21:00] There’s two broad roots to get to get these gene medicines into cells and they can be, sort of subdivided into in vivo gene-directed therapies. So these are therapies that you put into the patient and then they go and do their job somewhere inside the patient.

[00:21:15] And then there’s ex vivo therapies where you take out a particular cell type that is diseased in the patient, or could impact the patient therapeutically, and then you do all your engineering outside of the patient in some lab somewhere. And then ship those cells, once you’ve engineered, them back to the hospital, the doctor, and they get administered to the patient. And ex vivo gene therapies, as you can imagine, because you’re playing with isolated cells or engineering isolated cells, they’re slightly less technically complex.

[00:21:47] However, they’re much more logistically complex because you have to organise isolation for cells, shipping out, and you have to make effectively a personalised product for every patient. So, I’m gonna move into ex vivo therapies, but before we do that, I want to talk about some issues that are universal to all these therapies.

[00:22:03] So one issue that’s universal is something called cell autonomous immunity, or effectively innate immune system. And really this is an issue for any kind of gene-directed medicine. And the reason for this is that cells don’t like having novel nucleic acids or novel genes introduced into them because usually when that’s happening it’s because they’re infected with the virus.

[00:22:25] So they have a huge range of strategies to try and detect any nucleic assets that have been introduced into them. And they can do this by having particular receptors in particular locations in the cell. For instance, in a cell’s normal lifespan, it would never really expect to see DNA floating around in the cytoplasm – except during mitosis but has ways of switching that off.

[00:22:46] Similarly within the endosomes, so endosomes are intracellular. Imagine once a cell samples some media or takes something up, it can be stored in the endosome. If it detects RNA or DNA within this compartment, it should never really be seeing RNA there, so it assumes it’s a virus.

[00:23:05] And the end result of this really is that cells trigger an inflammatory response. They express interferons, they can get sick and die. So it’s quite a difficult, difficult thing to overcome. And different chemical modifications on chemically synthesised RNAs and nucleic acids have allowed us to evade some of these strategies. It’s a key reason that the mRNA therapies, mRNA vaccine that recently came out, was able to be functional. Because it uses a particular nucleic acid chemistry that allows it to evade some of these immune responses. And there’s active research and ongoing into designing better gene-associated virus vectors that, for instance, don’t trigger these response pathways.

[00:23:47] So just something to be aware of.

[00:23:49] Another universal potential issue is payload immunity. So if you are thinking about a patient who has a recessive genetic disease resulting in the absence of a protein, they likely never actually developed tolerance to that gene. Intolerance is our body’s ability to recognise self, due to training of the immune system in early development. And the cause of many autoimmune conditions is this loss of tolerance.

[00:24:11] So you can imagine the situation now where you have a patient, for instance, let’s say a haemophilia-A patient who is bleeding consistently because they don’t express factor VIII, and you introduce factor VIII into them. While it’s not technically a foreign protein, it is foreign to that patient because that patient all intents and purposes, has never seen factor VIII.

[00:24:33] So you can get massive immune responses to these otherwise therapeutic proteins. And this is a really hard thing to overcome. And it’s also potentially large issue if you imagine engineering a patient’s haematopoietic stem cells to express one of these proteins and then do a bone marrow transplant with a cell expressing that protein back into the patient. If it then becomes target for an immune response, you can imagine it could wipe out the patient’s entire bone marrow system, which would be quite catastrophic.

[00:25:02] So these are just two things to consider for, broadly, across ex vivo and in vivo approaches.

[00:25:07] Starting off with ex vivo approaches. One of the key modalities that has really come to fore is autologous haematopoietic stem cell therapies.

[00:25:19] And the reason for this is that haematopoietic stem cell transplants are routinely performed. We’re very good at harvesting haematopoietic stem cells from patients and from the healthy donors.

[00:25:29] We know that there are quite a few diseases now that have been treated with allogeneic or donor derived – so allogeneic and donor derived can be used effectively interchangeably, spanning beyond even the sort of classical haematopoietic space. For instance, haematopoietic stem cell transplants have been used to treat lysosomal storage disorders, metabolic diseases, and clearly being used for haemoglobinopathies, and they’re also using oncology.

[00:25:55] The problem with these allogeneic treatments is that you never really have a 100% match between the patient and the donor. So there’s always this risk of rejection or even a graft-versus-host disease where the inputs HSCs form cells are gonna go on to attack the patients.

[00:26:12] If they’re in a perfect world, you would take out a patient’s haematopoietic stem cells, engineer them somewhere, and then put them back in. And in assuming you hadn’t introduced a protein that was immunogenic to the patient, the patient should be able to live a happy and normal life and never have to think about their disease again.

[00:26:30] And one space where I think there’s been quite a lot of really exciting work increasingly in the haemoglobinopathies, and these are thalassemias and sickle cell disease. Both diseases that have really major unmet need. So, thalassemia’s in severe, or up to very severe, anaemias where patients need to be on lifelong blood transfusions.

[00:26:51] And this causes a whole host of issues beyond the original anaemia because you’re constantly being given blood transfusions, you can end up iron overloaded. There’s risk associated with constantly having transfusions. And if you had a sort of once-and-done treatment it would be really transformative for these patients.

[00:27:09] Sickle cell disease is caused by particular mutations in haemoglobin. And this disease is also truly awful. Patients end up with severely painful attacks where particular organs can become ischemic. And there’s really no good sort of medical treatment available for these yet. So really a disease with high unmet need.

[00:27:32] And there’s been now multiple approaches using gene-directed medicine to treat haemoglobinopathies. So, these haemoglobinopathies include both sickle cell disease, and beta thalassemia. They’re both caused by mutations in the beta globin gene, and there are both CRISPR-based approaches. So version one of this is driven by knock-out of a gene, which results in upregulation of fetal haemoglobin expression. And there are future variants coming back based on the correction of beta-globin mutations. And these treatments are delivered to HSCs, that are being taken out of the patients, through electroporation.

[00:28:11] There are also the lentiviral approaches where you have a virus, an lentivirus, which is effectively derived from the HIV virus-backbone. And this introduces another copy of beta globin into the cell on top of its sort of normal globin state so that this gene is now re-expressed and potentially rescues what’s going on in the cells.

[00:28:35] And, I just want to give you a bit more detail now on the lentivirus itself.

[00:28:41] The lentivirus is an integrated viral delivery vehicle, and it’s really proven useful in ex vivo engineered therapies, both in the CAR T-cell space and also in HSCs, which we are discussing here. And there’s been a lot of work over the last 20-or-so years really producing a good integrative virus, which evades some of the safety.

[00:29:03] Obviously, if you’re integrating a gene somewhere randomly into the genome of a target cell, there’s a risk that your integration event it could happen into an essential gene, could knock out a tumour suppressor, and this could result in clonal expansion of that cell, especially the formation of a leukaemia. Luckily these lentiviruses seem very good at not jumping into essential genes, and so there hasn’t, hadn’t been any recent cases of clonal expansions, or any sort of leukaemic events.

[00:29:29] This is in contrast to the early days in the first generation versions of these, which are based on gamma retroviruses, which did cause some severe diseases in patients.

[00:29:38] And, so some good news, basically, is that a treatment has already been approved based on this. So this is really seeing gene-directed therapeutics coming into clinic.

[00:29:47] Zynteg lo has been produced by Bluebird Bio, and it’s also based on lentivirus-based approach, where the patient’s own HSC is extracted, modified and reinfused.

[00:29:59] Unfortunately, these therapies are very expensive. And this therapy has not been approved by NICE. It has been approved in Europe for the treatment of beta thalassaemia.

[00:30:08] You can see here an example. I think every, every dot here is a transfusion event. And after administration of zynteglo, you can see that patients remain transfusion-free for extended periods of time, with very few patients having to receive transfusions. So really transformative to these patients. And sickle cell trials are underway.

[00:30:28] Changing gears a bit now to think about how CRISPR therapeutics are administered. I’d tell you a little bit more about electroporation, but really is as simple as zapping cells in the presence of RNA and protein.

[00:30:40] So CRISPR engineering, what you do is you get a purified cas 9 – which is the sort of cutting element of the CRISPR complex – and you mix it with the guide RNA – which targets this cutting enzyme to particular locations on the genome. Again, based on complimentary base pairing. Once you pre-mix these, you zap them into cells and they go off and edit their target locus. And the approach that CRISPR therapeutics is using to treat beta thalassaemia and sickle cell disease is based on reactivation of haemoglobin F.

[00:31:11] And it’s actually quite an interesting mechanism where a certain population of patients was identified to have high levels of fetal haemoglobin expression. And was identified that these patients had an absence of expression of BCL11a. So BCL11a’s job is to switch off expression of fetal haemoglobin and switch on expression of adult haemoglobin, including the beta-globin which features the sickle mutation.

[00:31:38] So in, in sickle cell disease patients, for instance, they’ll start off at a very young age as being practically healthy. And then as they switch off the fetal haemoglobin expression, they become sick. So we have activated fetal haemoglobin. It’s one strategy to prevent this. So by knocking down expression of BCL11a using CRISPR, the idea is to generate healthy erythrocytes.

[00:32:02] And this is also in clinical development at the moment by CRISPR therapeutics partnered with Vertex. And their regulatory filings are underway. They’re likely to be the first ever gene-directed therapy that is approved. The costs are still unknown. And again, here you can see in beta thalassaemia the region up until – this is sort bars indicating the length of time the patient has been transfusion free, with dark being better. And then here in sickle cell disease, from the start of treatment, you can see that none of these patients effectively had any vaso-occlusive events. So they haven’t experienced any of the pain that they used to be subject to.

[00:32:38] Extending out beyond haemoglobin, including the haemoglobinopathy, sorry, I’m struggling with that word.

[00:32:43] Haematopoietic stem cell treatments have also been used to treat neurometabolic disorders, and these are really generally caused by buildup of toxic metabolites in patients that have recessive mutations in enzymes that usually clear these metabolites. And one that’s closer to home, that’s just recently been approved is libmeldy from Orchard Therapeutics. The company that came out of work at Great Ormond Street Hospital.

[00:33:06] A really great success story. Yeah. With a really terrible genetic disease called metachromatic leukodystrophy, now being treated by this autologous gene-engineered and cell therapy.

[00:33:15] So, ex vivo section summarised: ex vivo HSC based gene-directed therapeutics, they’re making their way into clinical practice as we speak, and there’s a whole raft that the new one’s coming through present in clinical trials. They’re really life-changing for patients. They’re once-and-done therapeutics. They work extremely well in the cases where they’re currently being demonstrated. There’s this complexity of the manufacturing process, which is something to overcome. They have a high cost, which impacts patient access. And there are still some safety issues, but they’re not generally related to the actual gene-directed part of the therapy, it’s just that haematopoietic stem cell transplants generally employ a really toxic cocktail of drugs, for instance busulfan, because you have to clear out your old bone marrow and then replace it with young, newly engineered bone marrow.

[00:34:02] But there’s active development of really much more non-toxic conditioning regimes, which should hopefully take away for these to be much more routinely used. And just there are some off-topic issues that have mostly remained hypothetical, and this is insertional mutagenesis. And also potential issues of guide specificity with the CRISPR/Cas.

[00:34:21] Thinking beyond HCS. CAR-T cell therapies are in development. So these are genes being introduced to T-cells, which then go off and treat cancers. People are working on novel and immune evasion strategies to develop universal cell therapies.

[00:34:36] And, regardless of cell type, though, what I told you today about lentiviruses and electroporation are generally the methods of use that people use to develop, to engineer cells ex vivo.

[00:34:47] Okay, so now to move on to in vivo therapeutics.

[00:34:51] And really with in vivo therapies, the problem is hitting enough of the right cells.

[00:34:55] So in vivo gene-directed therapies, they’re usually logistically simpler to manufacture than autologous cell therapies because, you can imagine, you produce your delivery vehicle and you administer it to a patient in the safety of the doctor’s office, and it does its job in the patient. You don’t have to ship away their tissue somewhere else to be engineered.

[00:35:12] There’s a range of different delivery approaches available. However, the major reason that these drugs fail, they struggle in the clinic, is that they’re not present at the right concentration at the right place, or they don’t deliver this gene to the right place. Despite the fact that they would work, because they’ve been shown to work really in grey models, in grey mouse models and also grey cell models.

[00:35:31] So really this idea of a therapeutic window, of getting the right dose in the right place, but not getting that dose everywhere else in the patient, is really important.

[00:35:40] And. Just to give you an idea the scale of this problem, if you imagine injecting a patient with one of these delivery vehicles, it’s usually gonna be intravenous and it has to travel up through the circulation, through the lungs – which are massive surface area, massive space, for these delivery vehicles to escape into the parenchyma – g et tracked somewhere, get taken up by the immune cells. Then they have to filter through kidney, spleen, and most importantly liver, which are then involved here. Then evade the immune health in the circulation, which are trying to get rid of them. They have to evade degradation enzymes, with full of RNase, which is specifically designed to break down any free-RNA – which is a compound present in most of these gene-directed therapies.

[00:36:22] Then have to somehow escape the circulation into a tissue of interest.

[00:36:26] And if you’re in the bloodstream, this is especially a problem in the brain but it’s also a problem in a lot of other tissues where endothelial cells blanket the hole, obviously to hold blood inside your circulatory system. It’s quite hard to get out in the right place. They then need to evade immune cells again with a resident in the tissue. They then need to get into their target cell. They then need to traffic this medicine into the nucleus of the cell, evading all of those cell-intrinsic immunity pathways that are designed to recognise any of these gene therapies. And then finally, they need to express enough cargo in that cell to have a therapeutic effect.

[00:36:59] So you can see how the odds are really stacked against in vivo therapeutics. But I’m gonna share some stories, which shows us a lot of hope. A lot of things are working, especially in the liver.

[00:37:09] So, in vivo gene medicines, these have really emerged as the major vehicles of choice. These are adeno-associated viruses. These, unlike lentiviruses, are non-integrated but it can randomly happen. They are good for targeting non-dividing cells. There is some potential for immunogenicity and inflammatory risks because the capsid is a virus.

[00:37:30] There’s a range of different AV subtypes, which target different organs, so choice of subtype is vital to match with your actual clinical indication. Another issue with these is that their genome size is quite small, so it’s quite hard to fit some therapeutic genes in within them. Especially an issue in Duchenne muscular dystrophy where some developed a mini gene or a mini dystrophin gene, which is able to overcome this problem.

[00:37:55] They’re generally used in the liver, brain or in muscle indications. And one other thing to be aware of is pre-existing humoral immunity. So AEs don’t just exist in the context of therapeutics, but we’ve become exposed to them throughout life and we can develop immune responses to them. And this severely hampers, if you do already have preexisting antibodies to an AAV, really hampers its ability to get to the right tissue sort of stacks the odds even more against them.

[00:38:21] And so often in trials, people would be screened for preexisting antibodies and then excluded. So there’s a whole population of people that might never be able to access AAV therapeutics.

[00:38:31] So moving on though, let’s talk about some more successful stories here. So actually the first gene medicine that’s ever approved was Luxturna and Spark. And this was a topically administered AAV delivered behind the retina. And its treatment for disease caused mutations in RPE65. And so this AAV reintroduces the RPE65 gene. And what you can see on this plot here is the level of light patients needed to navigate a dark room, or a sort of maze.

[00:39:04] And, I think this is quite clever from a clinical perspective, about how they design clinical trials for eye diseases. Where you can dose one eye initially and look to see therapeutic benefit in that eye and then move on and use the other eye as a control. So the untreated eye. And so you can see here that the green eye was dosed first. And it allowed patients to walk through the room at much lower light conditions than the control eye when they blanketed one eye, and that when they finally dosed the second eye, it also brought up brought down light level that patients can use to navigate this room. It’s a very nice clinical design for a trial.

[00:39:36] I am aware that we’ve got some time constraints. It was 45 minutes or an hour, but happy to continue, or I can start to wrap up a bit. Someone?

[00:39:49] Professor Kate Tatton-Brown: I think let’s, thank you very much James. Let’s start to slowly wrap up with the summary because we have got another session in a month.

[00:39:55] Dr James Patterson: Okay. I do want to talk about one other therapeutic: siRNA therapies. So as mentioned, small interfering RNAs are able to modulate, are able to alter gene expression by binding to target mRNAs and down regulating the expression. And because of this modular platform nature they’re really able to potentially target any gene.

[00:40:16] Delivery beyond the liver is a major hurdle. And the first approved therapy is a drug called patisarin, which has been used to treat hereditary TTR amyloidosis. And some of you may already have come across another drug called inclisiran, which is targets PCSK9 of dyslipidaemia.

[00:40:32] All approved sRNA therapeutics target the liver.

[00:40:35] There have been four approved in the last four years. All from Alnylam which is really a stunning example of how the single platform approach can work incredibly well.

[00:40:44] And just want to say the last thing here on PCSK9 as a gene target. Makes a really a textbook genomically identified target, so PCSK9 is a gene involved in regulation of cholesterol.

[00:40:56] And back in 2003, it was identified as a gene association with autosomal dominant hypercholesterolaemia. In 2005 mutations were identified in PCSK9 that actually protected patients and resulted in hypocholesterolaemia.

[00:41:10] So you have a gene now that’s implicated in high and low levels of cholesterol.

[00:41:15] So based on this, monoclonal antibodies inhibiting PCSK9 were developed, and they are now pretty much revolutionised hypercholesterolaemia treatment coming in to replace statins, or be used in conjunction with statins. And now in inclisiran has been approved in siRNA targeting, targeting this pathway. And a beautiful thing about inclisiran is that it has to be dosed twice a year, while the antibodies are dosed twice a month.

[00:41:42] Now CRISPR-knockout-based approaches to targeting PCSK9 are in development, which would effectively require dosing once in a lifetime to fully protect against cardiovascular incidence.

[00:41:54] So. On that note I will jump to a conclusion slide. And this is just to say always remember that patients test these drugs and we should never move off the bench too fast.

[00:42:05] Just want to highlight that there was this, in the early days of gene therapy a sadly a patient who died. There were a few patients who died, but one that really became quite public was Jesse Gelsinger. And because of his death in 1999, there were a range of safety issues and potential, sort of, safety issues that were identified in gene therapies.

[00:42:23] And it resulted in the gene therapy dark age as funding evaporated in public perception turned sour. Now 24 years later, I have to say that’s no longer the case, and gene therapy is now safer. They’re highly effective and they’re really moving from bench to bedside and saving patients’ lives with much less risk.

[00:42:42] So silver lining here on where things can go if you give it time to progress and do things safely. So thank you for your time. And please let me know if you have any questions.

[00:42:51] Dr Hassan Shakeel: Hi James. Thanks for that. Really informative talk, spanning a bunch of different gene-directed therapies. And thanks again for agreeing to do this and then the session next month.

[00:43:05] If it’s okay with you, I’m going to pose you a couple of questions that the audience have asked. So we’ll get right into it and if anyone does have any further questions, please just post them in the chat and we’ll gradually work through them.

[00:43:17] And please don’t be offended if I don’t pick your questions. There will, I imagine be quite a few, so we’ll try and work through as many as possible.

[00:43:24] So the first one, James, if it’s okay, is do you think gene-directed therapies are likely to overtake conventional pharmacological agents in treatments in rare diseases, specifically?

[00:43:37] Dr James Patterson: Rare diseases specifically? I think they will. I’m trying to think if they already have, that’s what I’m wondering.

[00:43:45] I think in terms of the, in terms of the rare diseases that have very well described monogenic causes and in terms of the efficacy, I think there’s, it’s very hard to make an argument to the fact that they haven’t overtaken small molecule conventional therapies in terms of efficacy.

[00:44:00] I think really the key to this is expanding access and expanding these sort of manufacturing of some of these delivery vehicles and making them safer. Because the problem is that you think about where these patients are that have a lot of rare diseases, they’re not always at Great Ormond Street Hospital able to have an ex vivo gene therapy that should.

[00:44:18] And really to truly treat these diseases and you need to have something that you can shift somewhere, have it stored easily and then administer to a patient in a non, very, not the most up-to-date setting. And I think that’s really how we’re gonna treat rare diseases. I think it will come.

[00:44:33] Dr Hassan Shakeel: Yeah. Yeah. I think I’m most same opinion, but I guess we’ll watch this space.

[00:44:39] Okay, so next question. You’ve given us a really nice overview of a bunch of different gene-directed therapeutic technologies, actually from ASOs to CRISPR and everything else in between siRNAs and what-have-you. In your view, and I think I might know the answer to this already based on your company, but in your view which technology is likely to have the most clinical impact in the coming future and why?

[00:45:08] Dr James Patterson: It’s also a difficult question cause it’s almost what tool are you gonna use to skin your cat?

[00:45:13] You if you have if you have a monogenic disease and you know that if you correct that mutation and you can access it and you want to only do it once, and then clearly something like CRISPR is the tool you’re gonna reach for.

[00:45:27] And it’s a once-and-done therapy. Patients should be able to live the rest of their life without any worry. However, if you have something that’s more sporadic then maybe you don’t want a permanent modification. Maybe you do want something that’s more, more temporary. And there, I think siRNA therapeutics, mRNA therapies those still have, those will have a bigger potential impact.

[00:45:45] Course delivering CRISPR and allowing to control the epigenome, which would be something to talk about in the next session. I think there’s the scope there to do transient modifications too.

[00:45:55] Dr Hassan Shakeel: Yep. Okay. Next question. And you have touched on this already actually, so there are, I think a big challenge is the earlier safety concerns when one thinks about gene targeted therapies and their mechanisms of delivery. But are there any other challenges specifically when you’re trying to convert these into common clinical practice of gene-directed therapies, in your mind?

[00:46:22] Dr James Patterson: So again, I think it’s almost like a matrix of safety issues, one associated with your delivery vehicle. And then the next column being, what are you delivering. Something that’s a permanent modification like, let’s say a nuclease-based CRISPR is gonna be chopping your genome somewhere. So you’re probably quite worried about off targets.

[00:46:40] Then there are novel CRISPR approaches that don’t make cuts and they’re potentially safer, just from moving up on the safety space.

[00:46:47] Something like an mRNA is reasonably safe, but again, if you chemically modify it to escape some of these inflammatory process and then thinking about viruses again, I think AAVs that well, they’re very successful there is quite a lot of information about inflammation, especially in the muscular dystrophy space. They’ve been some worrying signs of quite severe inflammatory responses, which have put patients at risk.

[00:47:10] So again, safety issues are really, really tough to predict. Preclinical models are vital before you head into a patient. Just be very clever about what you do so you don’t put patients at risk.

[00:47:21] Dr Hassan Shakeel: Okay. As we are running short on time, I’m going to pick one more question, if that is okay. And I think this touches on quite an important issue that you’ve touched on actually. Do you think nanotechnology is the future of in vivo delivery of these gene-directed therapies? And if so, why?

[00:47:41] Dr James Patterson: Nanotechnology, it’s a very a very broad if some of these things fit into nanotechnology, in LNP is almost a nanoparticle. If maybe if you’re thinking of these nanorobots that can actually make decisions. And detect things. I think it’s probably still a way off from little nanorobots delivery therapies and recognizing specific molecules themselves to deliver therapeutics. But I’d class LNP in some of these nanoparticle things as nanotechnology.

[00:48:08] Dr Hassan Shakeel: Perfect. Thank you, very much, Dr Patterson, James. And I’m going to now hand over to Professor Tatton-Brown to wrap up what has been our first LinkAGE seminar. Thanks again, James.

[00:48:21] Dr James Patterson: Thank you very much.

[00:48:22] Professor Kate Tatton-Brown: Brilliant. And big thank you from me to James, for a fabulous overview of gene-directed therapies.

[00:48:30] You covered so much information there, but in very clear way. So thank you very much for that. And thank you very much Hassan for fielding the Q&A. So that brings us to the close of this first LinkAGE webinar. I hope you’ve all enjoyed it. As I mentioned at the beginning, there is an evaluation form that will be popping up soon. And if you follow the QR code that’ll take you to it.

[00:48:52] If you would like a CPD certificate for your attendance today, then we would just ask that you fill out that evaluation questionnaire first of all.

[00:48:59] So our next webinar is on the 21st of February where James will be talking about CRISPR. And then after James’s webinar we’ll be handing over to other speakers who’ll be fulfilling the rest of our gene-directed therapies chapter.

[00:49:13] So please have a look on our website for the forthcoming webinars. And please make sure that you sign up for them in advance. This will be recorded and you’ll get access to the recording afterwards, so you’ll have a chance to really think about everything that James has shared with us and really assimilate that learning.

[00:49:30] So that brings us to the close with one final, big thank you to James and to you all for attending today. Good evening and have a lovely evening. Bye.