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Podcast » CRISPR Based T Cell Editing

First Author Conversations Podcast

Episode 5: CRISPR Based T Cell Editing, Theodore Roth, Ph.D., UCSF

TIL Frequency & Tumor Reactivity

Posted: Sep 24th, 2019

Genome editing technologies have proliferated in recent years as have different types of immunotherapies. What possibilities exist at the intersection of these advances? In this episode Tracy interviews Theo Roth, Marson Lab at UCSF.

Theo is the first author on a recent paper in Nature entitled Reprogramming Human T-Cell Function and Specificity With Non Viral Genome Targeting.

In recent episodes of this podcast we have discussed manipulating the stemness of T-cells as well as identifying populations of T-cells that are reactive to tumor neo-antigens. Here, we are talking about rapidly engineering immune cell genomes. How quickly can in be done (in terms of helping a patient) and what are the limitations on the scale of changes that could be made? Can it be done without the use of viral vectors?

Theo describes the work to "knock-in" new sequences and ensure they integrate in the correct locations, under the correct regulation.

Full Transcript »

Speaker 1:
Welcome to first author conversations, a podcast presented by GenScript. We cover the latest high impact peer review publications in life science. We invite the authors to talk about their studies and the behind the scenes stories. Here's your host, Tracy Yin.
Tracy:
Hi everyone. This is Tracy Yin. Thanks for tuning in. Our guest today is Theodore Roth. Theo graduated from Stanford University with a Bachelor's degree in Biology and a Master's degree in Biomedical Informatics. Currently he's under MD/PHD training at University of California San Francisco. And he recently successfully defended his PHD thesis. Congrats Theo!
Theo Roth:
Uh-Huh (affirmative), Thank you Tracy.
Tracy:
Last July, Theo, and your colleagues published in Nature your study of Reprogramming Human T-Cell Function and Specificity With Non Viral Genome Targeting. Before we dive in, will you please let us know your research focus and interest and just briefly introduce yourself?
Theo Roth:
Yeah, so my name's Theo Roth. I uh, the last couple of years have focused on studying the intersection between a genome editing technologies, which have obviously rapidly proliferated the last few years and primary human immune cells or, and their application to cell based therapies. So we set kind of at the intersection of gene editing, immunology and cell therapy, hopefully to develop new therapeutics for cancer and autoimmune disease.
Tracy:
Thank you Theo. So, what made you look into non-viral method despite the fact that viral vectors are commonly used in therapeutic engineering?
Theo Roth:
Yeah. When we started these projects we were trying to consider what the best methodologies would be in order to enable rapid engineering of immune cell genomes. And this is kind of what we're really getting at is putting new DNA sequences, integrating new instructions into the genomes of human immune cells in order that we can make those cells into a therapeutic drug. The challenge there and the key technical limitation is how quickly you can make those modifications and any limitations that you have on the types of modifications that you can make.
Theo Roth:
So with traditional viral vectors, which have both packaging capacities randomly integrate their sequences and also are relatively time consuming and can be expensive to produce, we decided to take a chance and see if there, you know, taking advantage of these new CRISPR/Cas9 approaches, could we actually develop a technology that had some advantages over current viral vector based approaches, and that's what we ended up devoting a lot of our time to developing a non-viral approach that allowed us to do not only similar things to what we're done over randomly integrating vectors, but to have a very simple technology, it's cheaper, faster, simpler to use, but also allows you to target the integration of your DNA. So you both have a simpler technology, but also you can choose where in the genome it goes by combining your DNA templates with the CRISPR/Cas9 components to target the genomic site.
Tracy:
Yes, so you just touched the point, that's the limitations of using viral vectors. And what's special about the primary T-cells? Is there any special difficulties?
Theo Roth:
Yeah, so we've applied our non-viral knock-in methodology to many different cell types, but one of the most exciting ones therapeutically is primary human immune cells and especially primary human T-cells. And now the difficulty with primary T-cells has been that they can be, rather than a cell lines that will grow forever in a dish, primary T-cells only can only expand them for a limited time in vitro. And that made it, that made the complexity and the cost of using viral vectors even more apparent because you can't grow yourselves up forever, you can't select a single clone that has just so happens to have the modification you want. But the importance of using these primary T-cells is really that, these are the final cell type that will be the basis for a cell based therapies, especially for cancer and auto immune disease. So if we can make a methodology that worked really well in that final cell type, then we're devoting our efforts to what's going to be the most therapeutically relevant approaches and we're not going to develop any technologies that may be helpful for cell line work, but which wouldn't be able to be used in the actual therapeutic cell type.
Tracy:
So in the past, many scientists tried to knock in long DNA sequences into primary T-cells, and in most of the cases, the T-cells died. In your methodology and in your publication, you described your group was able to repair a disease causing genetic mutation in T-cells from children with a rare genetic form of auto immune and can you elaborate this work for us?
Theo Roth:
Yeah. So like I said, one of the most exciting things about, these gene editing technologies in the way that we've applied it into human T-cells is that, like I mentioned, we get to choose the exact site in the genome where we integrate new DNA sequences. And this is really useful when in the case of genetic diseases where we know exactly what the cause of the diseases, it's a certain mutation that we've characterized and we know exactly where in the genome it is. We've sequenced the patients, whole-exome or whole-genome sequencing is now pulling out, you know, rare disease causing mutations for many undiagnosed diseases.
Theo Roth:
The question is there's never been a drug for you know, there's never going to be drugs for each individual one if we're still developing individual small molecules or antibody based therapies. And so with this gene editing approach though, because it's so easy to target new sequences, we can go in exactly into the site where the genetic mutation is in the patient's own cells and correct just that specific mutation. And so, there's a lot more clinical concerns to think about it for each individual type of therapy. But in one case that we've identified already there a family that has a rare monogenic autoimmune disease in the protein called CD 25, which is also known as the IL-2 receptor which helps to allow T-cells to proliferate.
Theo Roth:
And this mutation is causing a lot of problems in the patient cells. But what we've been able to do is to show that we could use our technology to isolate patient's own immune cells from the affected children in this family and actually correct that causative mutation. And now we're pursuing, you know, more clinical studies, based on to see whether that would be a viable therapeutic option for this family.
Tracy:
So in this work, how did you choose what type of DNA templates to use? I guess we're getting into more technical details right now, and there are many different types, like linear double stranded DNA shot, single stranded DNA in long single stranded DNA, as well as viral genome, and I guess, maybe you can tell us how you chose, why you chose single stranded DNA to be used as DNA template.
Theo Roth:
Yeah. So there's a lot of different types of formats of DNA template that can be used, but it all gets down to a very straight forward question of you just, you need a DNA template that can be used for the process of homology directed repair, which is how the cell is going to fix the double stranded DNA break or the break that you introduce using a gene editing components like CRISPR/Cas9. And you want them to repair that break with a DNA sequence that you provide. And in doing so also integrate a little bit of new DNA as well. So what you need to do is you have to get that DNA template though, into the site in the cell where that repair's going to happen, which means into the nucleus. And so the initially, the easiest thing for us to use was very short sequences of single stranded DNA, chemically synthesize the same way that DNA Oligos or primers can be chemically synthesized at large quantities. And we would electroporate that piece of DNA in at the same time as our gene editing components.
Theo Roth:
Now the long-term concern is that when we can only use chemically synthesize DNA, we were limited in the total size of DNA that we can use. And that's just because chemical synthesis can only get up to a couple hundreds, about 200 total base pairs of DNA. So that meant that we couldn't integrate very large sequences. We could do some work on correcting individual mutations, which is very exciting. But as we wanted to do larger, larger knock-ins to change T-cell function in larger, bigger and better ways, we moved to other couple other types. The most commonly used type in the field is to actually use a non-integrating virus, into put your new DNA sequence inside the viruses' genome.
Theo Roth:
And so you use the virus as a way to get that DNA sequence into the nucleus because that's where the virus is going to put it anyway. And then because it's integrates deficient, it doesn't for a large part, but not 100%. You want to integrate into the target genome, and have potentially some safety concerns there. However, that's still necessitated making a virus. And so we wanted to look and see whether we could do it without a viral template at all. And that's where we started using longer sequences of non viral DNA, such as double stranded DNA sequences that were either cut out of a plasmid or made by PCR or long single stranded DNA sequences, which also, which can work as well. And so we found, but kind of surprisingly as we've developed the methodology, we found that we could use these purely non-viral sequences to integrate very large pieces, large DNA elements into the genome. And that really was kind of our breakthrough that allowed us to start exploring a lot of exciting applications.
Tracy:
So using CRISPR/Cas9 system, we also look very carefully into the specificity. So how can we ensure that we target the specific loci and not others?
Theo Roth:
Yeah, there's a lot of questions around this and it's something that, you know, the field needs to be very aware of. There's two broad overall concerns and went over doing these type of gene editing experiments. One is that the editing components that CRISPR/Cas9 in our case but could be TALENs or zinc fingers, could be cutting at sites, others sites in the genome besides the on-target site that you want it to be cutting at, and that could potentially pose safety concerns. There's been lots of exciting, technical development around this area producing higher fidelity versions of CRISPR/Cas9, new rules for how you design guide RNA's to target this on target sequences, very selectively and specifically. And there's new stuff coming out all the time around this. And so it's, you know, the field is rapidly developing.
Theo Roth:
The second main concern is that if you're integrating new DNA sequences, would that new DNA sequence go into the genome at a site that you didn't want it to? And here is where we have to be very rational about what we're talking about. And so what we found is that with our non-viral templates, when we use double stranded DNA, we do observe some integration of that double stranded DNA sequence at other sites in the genome besides the on-target locus. Now it's a very, very small amount, but we do, we can observe it when we use single stranded DNA sequences, long single stranded DNA, we actually observe less off target integrations, at least relative to what we can observe in our assays. Now the bigger question though is that, so far and including an FDA approved T-cell therapies, the way that new DNA sequence has been edited has been with randomly integrating viral vectors.
Theo Roth:
And so in the case here, there isn't even really a concept of on-target versus off-target integration or an edit because there is no target site. It's essentially 100% of the integrations are all off-target. And so even with the simple to use double stranded DNA, we already find that probably, well 90% or more at least of the observed a knock-ins that we see are all at the on target locus. And that number is even higher for the single stranded DNA. So, we think that already we've been able to drastically improve the safety profile by moving from the viral randomly integrating viral vectors, which have no target site, which in our 100% off-target integrations to our non-viral system that is as a very high degree of on target integration.
Tracy:
That's very impressive. So, what's the maximum length that you have succeeded in insertion?
Theo Roth:
So we're continuing to drive efficiency improvements and stuff to our methods all the time. You know, we've only been working on optimizing these approaches for a year or two. The thing to realize is that what we find is kind of inverse relationship between the size of the construct that we knock in and the efficiency of that knock-in. And so as we go to larger and larger constructs, the efficiency goes down a little bit and that just continues as you get higher. So we don't think there's necessarily a theoretical, a fundamental limit to the size, but rather that it would eventually be too inefficient that you wouldn't see knock-ins, the largest that we've done our ourselves so far and achieved pretty good knock-in rates has been well over 3kb so far. But really we just haven't tested larger things than that.
Tracy:
And so you mentioned that you've been continuing working to further improve the efficiency. Can you tell us more about that?
Theo Roth:
Yes, absolutely. I did my undergraduate degree at Colgate University in upstate New York. I majored in biochemistry there, and that's where I got my first longer term lab work experience. I worked in Roger Rowlett's lab, where we studied carbonic anhydrase and that's where I got an appreciation for understanding substrates of enzymes, how those enzymes work, some protein chemistry. And I found myself really interested in lab work and the only thing that I wanted more out of that was some sort of applicability to human health.
Theo Roth:
Yeah, since our initial study, we've continued to drive new approaches and new ways that we can further improve the knock-in efficiencies and the size of our constructs, how many constructs we can knock in at a single time. And so we've done this in a couple of exciting ways so far that we have a recent bioRxiv paper out about this, but we've improved the way that we can complex the CRISPR/Cas9 RNP with our DNA template. And we've also improved some ways that allow for more efficient electroporation by adding in some chemical components that improve the delivery of our DNA and RNP components into the cell. And this has helped improve both the efficiency as well as the viability of our cells and really enabled us to increase the size and the scale of our knock-ins.
Tracy:
And for the homology arms, do you have any suggestions about the lengths?
Theo Roth:
Yeah, since we target in many different sites in the genome, and well at any individual side you can always be useful to optimize. As a general rule, we found that going with about 300 base pair homology arms around a large insertion usually yields about the most optimal knock-in efficiencies.
Tracy:
For the new DNA that you are trying to integrate with the endogenous elements, you have any suggestions on the reagents that are involved, what kind of ratio or any pattern we need to follow?
Theo Roth:
Yeah, there are just a lot of small technical details just to make sure that, you know, you're using the right just to the exact right amount of DNA and things like that. You know, same thing with the DNA template and the RNP if you use too much then you get low cell viability, and you know, and that doesn't help for your editing efficiencies and if you use too little then your efficiencies are going to be lower than what they could be.
Theo Roth:
Now one of the most exciting things about these targeted gene integrations is that now you can start to take advantage of choosing a specific target site that allows you to integrate your new DNA sequence with certain endogenous DNA elements in order to make new constructs that maybe you couldn't do purely synthetically. And some of the exciting things that we've done has been like to replace at the T-cell receptor locus to replace just the specificity of the receptor while keeping the endogenous regulatory elements and endogenous signaling components. And so that allows us to use a smaller insertion as well as taking advantage of an endogenous regulatory circuit that we would never be able to fully recapitulate, with a purely a synthetic or a purely viral construct that we were driving our new gene also say a high expression viral promoter.
Tracy:
So, and then talking about the safety, any concerns I believe there are many concerns surrounding CRISPR mediated indel formation and any thoughts about that?
Theo Roth:
Yeah, I think we have to be, you know, we always, we have to be very cautious when we're using any new types of methodologies. And we also have to be very, rational about what's been done already and what has been proved to be safe. And so some of this can vary depending on the type of the cell type that's being used. So what's been found from the gene therapy field is that, when doing, you know, randomly integrating viral vector based therapies in hematopoietic stem cells, that has led to a variety of off target integrations, and you know, deleterious effects that ended up causing a couple patient deaths in various clinical trials, in the early 2000s especially. But you know, I'm young as well. But that's in contrast to what we see in primary T-cell based therapies.
Theo Roth:
Mostly because the proliferative or oncogenic capacity of primary T-cells is quite a bit less than the earlier stem cells. But what that means is that we've already integrated or disrupted DNA in your cells that went into patients, in probably just about every potential target site in the genome that there is. And so, and there's shown to be very very little risk of oncogenic transformation in the human T-cells. So, as we're thinking about what therapies are heading into the clinic, we know we have to be very cautious, but also thinking about there's probably going to be a level of acceptable off target editing and mutations that make sense in primary T-cells, which has been shown by the thousands of patients that have already been dosed with Chimeric Antigen receptor T-cell therapies where again, there's every integration is off target and that's going to be very different compared to stem cell based therapies or [inaudible] stem cell based therapies or there has been shown to be a very real and very concerning risk of oncogenic transformation in gene edited product.
Tracy:
And so can we rapidly screen many potential therapeutic sequences? I know like what Judd Hultquist group in Northwestern did, they created a high-throughput CRISPR/Cas9 platform to analyze the role of host factors in HIV infection and pathogenesis.
Theo Roth:
Yeah. So, I've been with these, one of the exciting things about these gene editing tools, is that they are so simple that allows us to start doing much larger scale applications into a screening approaches in order to try to rapidly identify new target sites or sequences that can we can be used both for research applications in therapies. Judd, who is an old collaborator of ours, along with the others in the Marson lab in the Krogan lab at UCSF helped develop a high throughput approach to do genome knockout greens where they would array individual knockouts into 96 well plates and be able to test many different knockouts in one by one in each well, but one of the things that, you know, and we are developing similar approaches that allow us to do the same thing but with large knock-ins as well, both in race and also in, for large pool applications as well. And so that is really, I hope it's going to be driving the pace of development in this field and further driving the efficacy and the efficiency of gene editing applications, especially based on integration of new DNA and new cassettes.
Tracy:
Yeah. So we just touch base two application fields of this immersion technology, including treatment of cancer and auto immune as well as the infectious disease. So to further apply this into clinical fields, are there any bottlenecks that we definitely need to overcome and what are we doing now to tackle the challenge?
Theo Roth:
Yeah. Yes, a big thing is continuing to drive the simplicity, the efficiency of our methodologies. A big thing is to continue to think about smart ways that we can integrate new DNA and to overcome the specific challenges of different therapeutic settings, whether that's auto immune or cancer or infectious disease. And I think that maybe the biggest thing is kind of being ready to make a big shift in the field when it comes to our conception of what types of modifications and the types of gene modified cell therapies that we can make are in realizing that making these targeted integrations is now not only possible, but almost as efficient as doing the traditional randomly integrating variance. And that this opens up a lot of new applications that we've never really thought about before and I hope are really, are going to drive the pace of therapeutic development more quickly than it has been traditionally in the cell therapy field.
Tracy:
That's great. Thank you Theo and thank you all for listening. We look forward to future progresses of this emerging technology. Thanks Theo.
Theo Roth:
Thank you Tracy.