Zinc is “the Barry Manilow of heavy metals—really dull and really boring. It’s not fancy, like iron or copper.” But as Adrian Woolfson, BM BCh PhD, reaches his first-year anniversary as Sangamo’s executive vice president of Research and Development, he is pinning big hopes on zinc fingers, which make up the first proven genome editing technology pioneered by the Bay-Area-based biotech.
Woolfson recently spoke exclusively to GEN EDGE correspondent Julianna LeMieux, PhD, about Sangamo’s progress and aspirations in gene therapy, gene editing, as well as the promise of new technologies for genome editing and delivery. (This interview has been lightly edited for length and clarity.)
JULIANNA LEMIEUX: Hemophilia studies are going to be watched very closely this year. What are you doing at Sangamo that is different from your competitors?
ADRIAN WOOLFSON: Hemophilia is probably the commonest severe bleeding disorder. About 1 in 5,000 babies are born with severe hemophilia. From an early age, they get recurrent bleeds into their joints and muscle… It is very disruptive and requires a huge multidisciplinary team to look after them. You need to think of this as a kind of lifelong burden of therapy, and that even with the therapy, you get suboptimal protection. So there is a huge unmet medical need.
What we’re looking for is a safe gene therapy, with few or no side effects, where you have a predictable therapy that provides a good chance that it would get you into the therapeutic range in a predictable timescale, and a reliable therapy that, once it gets there, stays there.
The data we presented at ASH [American Society of Hematology] 2019 demonstrate the potential for this medicine to be very differentiated from our competitors. First, it was very well tolerated. Second, at the high dose, all five patients got into the normal reference range within 5–7 weeks of therapy. All five patients did not get any bleeds beyond the three weeks of the protocol where factor use is still allowed.
It is safe, and as far as we can see. Furthermore, patients 7 and 8, who respectively went out to 44 and 37 weeks respectively, showed durability. The two patients most recently treated, at 22 and 12 weeks of follow up, also demonstrated similar patterns of factor VIII expression…
So we’re happy for those reasons — the rapid kinetics, the fact that they all got to the levels quickly that, when they got there, they stayed there, and up to a maximum duration of follow up, which is 44 weeks. Even at the lower-dose cohorts, the patient factor VIII levels stayed rock solid.
Another thing is that the patients treated in the high-dose cohort were ethnically diverse, and all of them were quite heavily factor therapy replacement dependent when they came into the study. We also saw a really nice dose response in the study…
There was a lot of buzz about the data at ASH. I spoke to a number of international thought leaders, both from the scientific and investment community, and they all were really happy with what they were seeing.
LEMIEUX: How many patients are enrolled in the trial?
WOOLFSON: There are 11 patients in total, five in the expansion, and then two in each of the low-dose cohorts. We have no more plans to enroll further patients, we are on the process of transferring the IND to Pfizer, and we have completed the manufacturing transfer, as well. Pfizer have announced publicly that they have begun to [enroll] patients into the lead-in cohort, which is the ‘run in’ precursor to the phase III study, which will be moving forward next year.
LEMIEUX: Tell us a bit about the nuts and bolts of the therapy.
WOOLFSON: We are using an AAV6 to deliver the FVIII construct. We’re the only people doing that. We believe that this is likely to contribute to the success of our therapy. At Sangamo, we have been engineering cassettes for gene editing and other purposes for many years. We believe that our know-how and skills in engineering cassettes has played a big part of the success of this program in terms of the behavior of the factor VIII cassette once it is inside the cell, the kinetics, reliability and so on. There are a considerable number of cassette modifications, and we have optimized codon optimization, regulatory elements and so on.
We have handed over the manufacturing to Pfizer, and per our agreement, Pfizer will discuss the details of the manufacturing process for phase III. Needless to say, they are planning to start the study in 2020. Adequate provision has been made to enable that.
LEMIEUX: Sangamo has zinc finger genome editing, zinc finger gene expression, and traditional gene therapy. Is the balance tilting in favor of one or the other?
WOOLFSON: We see ourselves as a genomic medicine company. That means that we have four key platforms which address genetic diseases using different modalities. These four platforms are discrete but also interlocked, because the skill sets that you need to operate each of them are interchangeable.
We have a gene therapy platform—hemophilia A, which we’re handing over to Pfizer. We have also got a wholly owned gene therapy program, which is Fabry disease, and will be treating the first patients in 2020. We also have a PKU gene therapy program which is heading towards an IND. Behind that, we have two other gene therapy programs in the early preclinical stages.
Our second platform is the ex vivo gene editing platform – and our beta thalassemia and sickle-cell disease programs are already in the clinic. Then we have our ongoing work with Kite-Gilead on the CD19 allogeneic CAR-T cell.
We also have our TX200 autologous Treg program, a fascinating first-in-human and a first-in-class. This breaks into a new frontier of cellular therapy using a similar approach to CAR-T cells, but using a different type of cell, the T-regulatory cell, to transduce the CAR construct into.
There, we are trying to suppress the immune system. We are taking end-stage renal failure patients who need a kidney transplant, but who are mismatched in HLA-A2 with their donor, and trying to see whether the Tregs localize into the donor kidney, check that it is safe, and then try to reduce the immune suppression that these patients have to address the fact that they have had a transplantation from an HLA-mismatched donor. We’re very excited by the CAR-Treg program.
Third is our in vivo genome editing program, that is, in a sense, the most challenging and also the most exciting. We’ve presented some data where we treated patients with MPS II, and we believe that at the highest dose we had clear evidence that we had edited in vivo for the first time in history a living patient, and we had managed to insert the gene for IDS, which encodes the missing protein in MPS II, into the safe harbor region of the albumin locus in the liver of MPS II patients. We observed a substantial degree of synthesis of IDS in one patient, and also saw evidence in two other patients that we had integrated the transgene successfully.
The patient who had the substantial synthesis was actually overweight and received roughly double the dose of the other patients. We realized that we had actually—based upon the hemophilia A threshold effect— probably underdosed in the MPS II study data presented at WORLD (We’re Organizing Research on Lysosomal Diseases) 2019.
Indeed it made us realize that we had probably dosed three to five times lower than we needed to. But we have a bunch of different levers that we’re now examining, ways that we can get the MPS II in vivo editing program back into the clinic, including increasing the dose, and making a second-generation AAV.
We’ve also made second-generation zinc fingers that are more potent and are trying to put two zinc fingers into a single AAV. And we’ve got a second-generation donor. Using one or more of those methods, we believe we’re going to get the in vivo genome editing working.
Our fourth platform is a transcriptional regulation platform, using zinc finger protein transcription factors, where we are trying to repair the expression of key genes involved in pathology. For example, in tauopathies like Alzheimer’s, we’re able to suppress the production of tau. That is pretty easy… but in diseases like Huntington’s disease, you have to discriminate between the healthy allele (~20 CAG repeats) and the diseased allele (~40 repeats or more). This is a very difficult discrimination. But we have found actually that we could differentially take out the diseased allele whilst preserving the wild-type allele. This is something you could never have done with CRISPR, because there are no PAM sequences for CRISPR to bind anywhere near.
The reason we can do this is because zinc finger proteins exhibit a higher-order behavior whereby they exhibit an apparent cooperativity effect—where individual ZFPs appear to link together and enable this difficult discrimination.
We are also exploring another approach, which is to epigenetically edit in diseases like Fragile X and Rett syndrome.
We also have a couple of other approaches in the CNS. One is where we can do mitochondrial editing by adding on mitochondrial transport sequences to zinc finger proteins, and where we can actually get them into the mitochondria. Again, that is something you cannot most likely do with CRISPR, because RNA cannot get into mitochondria. Also, we’re using our zinc finger nucleases to engineer T-regulatory cells to suppress inflammation in the brain in the case of multiple sclerosis, for example.
We imagine Sangamo as evolving in three successive but inter-linked and overlapping waves of technical evolution. In wave one, the here and now, we’re focusing principally on gene therapy. It is something that we know is low-risk. We’ve shown we can do it. We have de-risked it with hemophilia A…
But we’re also doing some of these other things, like the ex vivo editing, and the CAR-T cell stuff. And we’re beginning to gear up to do the regulatory work. As we move into wave two, we’ll still be doing the gene therapy, and the ex vivo work. But we’ll make the regulatory work, mainly on CNS, a more pronounced feature of what we are doing.
As we move into wave three, genome engineering will become a core feature, as will next-generation cell therapy. We’re exploring a number of different areas including induced pluripotent stem cells, with a view to bringing down the cost of goods of cellular therapy and democratizing it, and also increasing the volume of patients that we can treat.
With in vivo editing, we’re also exploring new functionalities. We have to date focused principally on zinc finger nucleases to cut DNA and using transcriptional regulation by adding on functional domains that can repress, and also epigenetic domains that can change methylation states. But at our R&D day, we announced something really important —we’ve been working on a very new way to functionalize zinc finger proteins with a functionality we call an integrase.
The integrase is able to specifically target any position in the nuclear or mitochondrial genome, and without cutting the DNA at all, to introduce a DNA cargo of almost an unlimited size. It is able to integrate this with great specificity. This could be a real game-changer and transformative to the whole field.
The integrase we’ve developed is far more efficient than many other integrases that have been described in the literature. So this could be a very important breakthrough which could transcend anything else that anyone can do, including prime editors, for example! We have not described it publicly—you’re going to have to wait until we publish the paper, which will be sometime toward the end of the year.
LEMIEUX: Adrian, which technique is personally the most exciting to you?
WOOLFSON: It is difficult to just talk about one of them, because I think if T-regulatory cells work, and we de-risk them in this current study, they could be as important for autoimmune diseases and inflammatory diseases as CAR-T cells have proved to be for cancer. That in itself would open up a whole new frontier.
I think that in vivo genome editing with an integrase is the other key area, and I think that reengineering cell function in iPSC cells using editors and integrases, as well, is going to be the other key frontier.
But using a whole toolkit of different functionalities, from transcriptional regulators to activators, repressors, nucleases, epigenetic editors, and also these integrases is going to be really revolutionary, because, for example, you could imagine, making CAR-T cells in vivo. You could get rid of the middleman, get rid of manufacturing altogether and just introduce a big construct into the hematopoietic stem cells in a living person.
With an integrase, you can do that. Because we can put in pretty much a cassette of any size into any position in the genome, and if successful that would be transformative. We could even imagine putting whole metabolisms into living patients, and really big genes, like the gene for muscular dystrophy.
I think that this has the potential to open up a whole new future, and I think ultimately gene editing will be disruptive to gene therapy, because it is permanent. You can do it early on in life, and you can also multiplex with the integrase. You can introduce many genes, and that may end up being relevant as we come to understand the biology of polygenic diseases. I think it would become increasingly important to be able to edit multiple genes at once.
The tools we have right now will be critically important in going beyond the engineering capacity of just doing CARs, for example—I’m talking about cellular therapy. We have the comprehensive toolkit that would be required to engineer these cells in a manner which goes beyond what anybody is doing now.
One of the problems with multiplex editing now with a nuclease is you introduce the risk of recombination. You’re limited in how many genes you can knock out in one go. But if you have methods that can destroy genes without cutting, then you can knock out or knock in as many as you like. Your ability to engineer cells and reimagine their genomes is really significantly greater… I’m hoping that the Treg platform will be really, really disruptive and have a huge therapeutic impact in autoimmune and inflammatory diseases.
LEMIEUX: For these integrases, you need to be able to deliver them. How important is the delivery aspect?
WOOLFSON: When I joined Sangamo in January 2019, I made delivery one of my key priorities. We now have around 14 different programs at Sangamo focused on different aspects of delivery, including a microevolutionary approach where we used direct evolution of AAVs to generate a new portfolio of serotypes, which we select in nonhuman primates. This work is proving successful, we have already generated a series of candidates which appear to be significantly outperforming the wild-type candidates. We’re focused on making candidates that have an improved ability to selectively target the brain.
But we’re also exploring other technology platforms, like liquid nanoparticles, and they may be very well suited to delivering integrases, because the integrase itself is not particularly big. But if you want to deliver a really big DNA template, 20–50 kilobases, then you could stick that onto a lipid nanoparticle with no problem.
We’re also exploring other orthogonal methodologies which are very differentiated, in addition to AAVs and lipid nanoparticles. There are a number of different modalities that we are looking at, and some of those are being done in collaboration with some world-class academic institutions, and we are in-licensing, and others that we are doing in-house. I’m very confident that we have made some significant inroads, and that we will systematically crack delivery across all the indications where we are required to do that.
LEMIEUX: Why are zinc finger nucleases so important at Sangamo?
WOOLFSON: What we are most excited about is the zinc finger protein platform. But we still believe, as do many others, that it is the best-in-class gene editing platform. And there is a reason why companies like Pfizer, Sanofi, Takeda and Kite-Gilead have come to work with us… We’re still leading the world in pioneering this technology and in publishing in cutting-edge journals. Our publication record is extraordinary: we have published one Nature Medicine papers, two Nature Communications papers and a Nature Biotechnology paper within the last 12 months, all with zinc finger proteins.
Let me tell you why we believe that zinc finger proteins are the preferred editors and platform of choice. First, because they are proteins. Unlike a CRISPR guide sequence made from RNA, the fact that zinc finger proteins are proteins means that they are definable and engineerable, and also tunable. They’re like mini-machines that can be tweaked and redesigned, and that gives us incredible capacity to optimize their behavior.
For example, one of the papers that was published by my colleague Ed Rebar showed that by slowing down the rate of cutting in a Fok1 nuclease, we could paradoxically make the ZFN more efficient, because it had less off-target, because when it went to the wrong sequence, it fell off before it cut. We were also able to remove some of the amino acid that nonspecifically touched DNA, and thereby increased the selectivity and the potency.
Another feature of zinc finger proteins is that when you look at their structure, they are much smaller than CRISPR. That means two things. They can be delivered more effectively but also they can get into chromatin much more easily. They can navigate their way in, whereas CRISPR/Cas9 constructs are really big and bulky…
We can find a zinc finger protein that can target any position, more or less, within the nuclear genome, but also within the mitochondrial genome. Unlike CRISPR, which is constrained by the presence of an effective PAM motif, zinc finger proteins do not have that restriction. There are PAM deserts in the genome where you can never land a CRISPR. We don’t have that problem. Not only that, we can generate multiple zinc fingers to target each position, which recognize each position in a very different way. Furthermore, we find it very easy to generate these solutions. It’s not like we have to search for hundreds of thousands of zinc fingers to find ones that work…
Another important difference is that zinc finger proteins are natural, human proteins which are naturally functioning as transcription factors. As I’m speaking to you, 3% of my genome is engaged in making zinc finger proteins.
We are using a library of synthetic versions of the natural zinc finger proteins. That is very different to CRISPR constructs derived from bacteria, which have a high immunogenicity. That is why humans have so much preexisting immunity to Cas9, whereas there is far less immunogenic potential if you use a zinc finger protein…
For all of those reasons, we think that zinc finger proteins win hands down as the preferred genome editing technology. And we have a very deep knowledge of working with zinc finger proteins, which is hard for anybody else to emulate.