Renaissance Orlando, Florida. December 4, 2015
Opening remarks — Barbara Van Husen, president, MPN Research Foundation
“I want to welcome all the CRISPR people, all the MPN people and all the other kinds of people who might be here. For those of you who are investigators it’s always good to be aware there are patients in the room and patient advocates. One of our beliefs is the more the patient community understands about the science the better we can support it.
Last year at ASH in wonderful San Francisco we could not have anticipated havimg this meeting today. While we were all at ASH, Zhenya Senyak took some time out to interview Jennifer Doudna in Berkeley. And it was from Zhenya that we at the Foundation first heard the acronym, CRISPR. What Zhenya told us was this technology was going to change cancer research and medical research in general and he also told us no one would ever apply it to the MPNS unless we did something. We would be the last ones in line regardless of what we know about mutations.
So we funded some additional exploration…and the Foundation’s grant program for 2015 included as one of its focus areas the application of CRISPR technology to MPNs. We got some pretty amazing proposals and actually awarded three two year CRISPR grants to CRISPR experts who are going to be applying this technology to what we know about MPNS.
George Church’s lab [Harvard], Zhaohui Ye at Hopkins and two investigators from Chicago, the University of Illinois. So we feel pretty confident that over the next year or two, along with work being done by other investigators…we’re pretty sure the MPNS won’t be quite at the end of the line when it comes to CRISPR
Another consequence of Zhenya’s work is this meeting because it wasn’t enough to bring CRISPR people and MPN people together, Zhenya was insistent we start thinking of therapeutic uses for the MPN patient. I think he was motivated by the fact that he has watched– and we have all watched– MPN patients who have had to resort to stem cell transplants suffer from Graft Versus Host Disease or die waiting for a match.
So the idea that CRISPR could be applied to stem cell transplant and doing an autologous SCT seemed pretty compelling to us and that’s why we’re sitting around here today.
We’re going to be talking about CRISPR and about MPNs and I’m counting on the Panel discussion to push this topic forward so that MPN patients who do need a stem cell transplant might have better chances. This is the beginning of the dialog, not the end. The best outcome of this meeting is that you all walk out of here thinking about ways to bring this forward.
We at the Foundation want to fund research that makes a difference. And if there are good ideas that make a difference, we’re committed to help them move forward.”
“I’m really happy to be here this morning to talk a little bit about the technology, what it’s capable of, what it actually has done so far and where we think this technology can go.
I’ll start off with a brief summary of the CRISPR/Cas9 system itself and how genome editing works. I’d then like to do a brief tour of proof of concept experiments that have been published in the last year or year and half to give you a flavor for the type of work that’s being done but also the challenges that remain to transform this into a direct therapeutic application.
Finally I’d like to walk you through a little bit of data from our work at Caribou to show how thinking about and very carefully measuring the outcome of genome editing informs the way we think about therapeutic development.”
And for the next fifteen minutes that’s exactly what Rachel did, serving up a short, no holds barred, CRISPR course, clear and precise. She described the CRISPR Cas9 system and its guide RNA that binds to Cas9 and searches through the genome for an identical target DNA sequence, locks on to it and permits Cas9 to make a double stranded break at that site. She talked about critical elements necessary for Cas9 to bind DNA to specific sequence.
But mostly, the mixed audience of physicians, scientists, patients and advocates got to know about how that break was repaired. The cut causing the DNA break or deletion can be repaired by the cell itself from available materials, a Non-Homologous End Joining (NHEJ), likely compromising the protein it coded for. In contrast, the insertion of DNA by CRISPR/Cas9 when successful creates an Homologous Repair (HR). Rachel returned to the issue of NHEJ and HR often enough that even those of us who don’t know the difference between a column and a pipette had a good sense of what might go on within the edited nucleus.
After reviewing several recent CRISPR experiments, all of which underscored the near miraculous progress of the technique of DNA insertion and deletion in the petri dish and mouse models, it became increasingly clear that the rate of success has been low.– generally under 20% efficiency — even though most of the work was being done in cells that are dividing and not terminally differentiated. “Most somatic cells, differentiated cells like muscle cells, do not have high levels of protein machinery to mediate HR.” Traditionally delivery of DNA insertions are accompanied by high levels of mutagenesis
Rachel reviewed the Caribou Biosciences process, the electrophoration delivery, the wait of one or two days, the isolation of the DNA using next generation deep sequencing to understand the outcome of the experiments.
She drove home the point: In general the efficiency of HR in inserting new DNA “is incredibly low.” In insertion experiments, typically in some fraction of cells the double strand break happens but some small fraction of cells have the new DNA inserted, some larger fraction of cells experiences some mutagenesis and a remaining fraction are Wild Type. A new Caribou proprietary system has vastly increased successful edit rates, but a sobering fact remained. The only way to determine if a cell has been successfully edited is to “isolate the DNA.” Destroy the cell. It was an issue the Panel would shortly take up.
For this audience, Ann Mullally was on more familiar ground. “I’m going to run through mutations quickly, highlighting some key points to keep in mind when developing a CRISPR therapeutic strategy. One thing in our favor, we know the genetics that drive MPNs are pretty much defined by two essential mutations, JAK2 and CAL-R plus MPL. So far as cancers are concerned, MPNs are simple genetically. Three genes as a place to start to target…this is probably as good as it gets.
These MPN diseases that arise can be cured by cell transplantation. If we could, in theory, correct the disease in stem cells and return then to the patient we can cure the disease without all the problems of allogenic transplantation.”
Ann then demonstrated how these three mutations are disease initiating. People with detectable mutations in peripheral blood point to early disease indications. And CALR and MPL alone are enough to induce MPNs in mice. There are many secondary or epigenetic mutations as patients progress to MF and in AML patients typically have more leukemic mutations. Of the true drivers, MPL mutations are rare, and occur upstream of JAK2. She also believes repair of calreticulin, a chaperone protein, might be more complex. But the central target, the JAK2 mutation and the JAK STAT pathway enhance the prospects for eventual CRISPR intervention.
Ann talked about the need to move ahead. JAK inhibitors are not enough. There are risks in SCT and our only other current alternative, interferon, works best in early stags of ET and PV, less so in intermediate and higher MF.
The main use of CRISPR in Ann’s lab now is not therapeutic. It is used more as a tool to generate the MPN mutations by designing special guide RNAs. For now, Ann believes that’s the primary practical application of CRISPR/Cas9 in the MPN labs today: Engineer the most common mutations in cells; Study co-mutations and combinatory drugs; Understand the resistance to JAK inhibitors. And, finally, repair MPN mutations in mice. For now.
” If you have a population of cells in a tube or a dish the only information you have about which is right and which is wrong is the genetic information. We have no way in the lab to take that information and sort it to take the good ones in one direction, the bad in the other. The way we figure out whether it’s good or bad is we kill cells and then sequence the DNA.” — Rachel Haurwitz
Tsiporah Shore: There has not been in over 30 years a way to ablate a marrow. Now there is maybe some way to do it and it may take a few years and that would have to be tested in a whole new way…. I’m saying 10 years for not having an ablative regimen. I’m very realistic here.
Technology right now, we don’t have a way to sort. That has to be part of the process. If there’s no way to sorting I’m not able to give a promise to a patient.
Ann Mullally: Can I just say as someone who spends a lot of time writing grants, I think it’s important that we generate a positive energy around some of these things and we try to bring along people who are interested in funding [this research] and help us to grow. That’s a very important part of moving anything forward. If we’re defeated before we begin we’ll never go anywhere. And I understand all the issues of tractability but its important that we generate positive energy to feed the general interest that appears to be there for this type of technology.
Tsiporah Shore: I agree that these are great ideas and we should work toward them but I don’t want patients to feel that I have something that I don’t have. I have patients come to me, referred for transplants. And it’s a lot to take in, what they have to go through. And if you have something that s good and it may still be tough but if you don’t have it, it’s very important to be honest in how you report things to the patient population.
Rachel Haurwitz: I strongly agree with both of you. There’s tremendous potential. There’s a huge amount of work that has to be done, there’s a lot of money to be extracted from Federal agencies, from investors to do that work but at the same time I think you’re exactly right. We have to appropriately communicate to patient populations today, what’s possible today, what may be possible over some reasonable timeline I’m sure many of you have received many of the type of emails that I have received from patients who read an article in the New York Times about any disease that may or may not have a genetic underpinning , send me a personal email saying that’s great I’d love to enroll in your trial. And then having to walk them through where we are as a community, and describe what’s coming next. We must continue to communicate in a fair and reasonable way what the promise is for the future
Jeanne Palmer: I also think an important thing that has to be done is communication with clinicians who are treating this, getting this information out. Sometimes patients come to centers where people know about this but sometimes they go to their local hematologist to get some Jakafi or something from someone who doesn’t know…If we’re talking about an appropriate education and knowledge base it’s also important that we educate the people who are treating them Patients drive a lot of what’s funded. You certainly don’t want to take away their hope but you have to see what’s out there so they can have a trusting relationship. We are able to say OK, yes this is an interesting technology, this is going to greatly advance our knowledge base of the disease and pathology of the disease. But we don’t want them to say “OK I’m going to wait until I can get my Cas9 cells and not get a transplant” and then three years down the road they still don’t have their Cas9 cells but they aren’t going to be able to get a transplant… It’s a question of balance. Like making sure the information gets through like through the MPN Research Foundation that has enormous influence in the community, to MPN patients and clinicians so they can counsel their patients.
Robert Rosen:… CRISPR is very much in the press now, in this morning’s Wall Street Journal, science report. I get we have to manage expectations. There are two tracks here. One is just the CRISPR track by itself, how impressive it is, what you put up there. It’s just amazing. But the second track is the MPN CRISPR track. I’m not sure if you had infinite amounts of money what you’d fund to move it along.
Finally, a note on the issue of selection from an earlier interview with Harvard’s George Church, an MPN Research Foundation grantee who wasn’t at CREATE and a practical approach selection from another MPN Research Foundation grantee, Zhaohui Ye of Johns Hopkins School of Medicine who was unable to attend the Seminar.
The nice thing about the ex vivo route is that whatever your efficiency is you can bump it up by some sort of selective process. You can say I’m only going to reintroduce the cells that meet certain criteria. It’s easiest if the change you’re trying to make results in some change in fluorescence of cell surface antigens. You can sort, selecting for the surface markers. Point is you have that opportunity to screen the population of cells before introducing them. And you don’t have that opportunity if you do it in vivo. And the fact that you can do a whole body ablation gives you a really good way of dealing with the ex vivo part…if that’s where it’s heading. —
It is a promising way to go. There two things you have to be very thorough about are thorough ablation and thorough selection.
- – George Church
” If you could expand HSCs efficiently (like in the case of iPSC). You could expand the edited HSC clonally (starting from single cells) until a larger number of cells are obtained from each clone. You would then take a small portion of them (say 5-10%) from each clone for DNA analysis to know whether the gene correction is successful. The confirmed clones can then be used for transplantation.”
- – Zhaohui Ye
Primary funding for the CREATE Seminar provided by the MPN Research Foundation with the MPN Genetics Network and supporting sponsorship from Dr. Ruben Mesa (Mayo Clinic/Scottsdale), Dr. Richard Silver (Weill-Cornell) and Dr. Srdan Verstovsek (MD Anderson).
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