The power and promise of zinc finger nuclease mediated genome editing
Fyodor Urnov, PhD, is Project Leader and Senior Scientist at Sangamo
BioSciences, Inc. where he co-developed human genome editing with engineered
zinc finger nucleases (ZFNs). Dr Urnov previously led the company’s
research and development efforts in deploying genome editing for crop trait
engineering (in partnership with Dow Agrosciences) and in generation of engineered
cell lines for manufacturing, improved generation of transgenic animals
and as research reagents (in partnership with Sigma-Aldrich). In his current
role as Project Leader for the Hemoglobinopathies, Dr Urnov heads Sangamo’s
collaboration with Biogen to develop genome editing as a one time, lasting
treatment for beta-thalassemia and sickle cell disease. Dr Urnov is also an associate
adjunct professor in the department of Molecular and Cell Biology at
the University of California, Berkeley. Dr Urnov received his PhD from Brown
University and holds a BSc in Biology from Moscow State University. He is an
author on more than 60 scientific publications and an inventor on more than
90 issued and pending US patents related to ZFN technology.
QQ You have been working in the field of gene editing for
a number of years – can you briefly explain how you
got into this area and your particular expertise?
I received my PhD from Brown University where I studied how
proteins bind to DNA and turn genes on and off; following this I
conducted my postdoctoral research at the NIH doing more of the
same. Then 15 years ago I was brought into Sangamo with the goal of
trying to build next-generation approaches to dealing with the challenge
of genetic disease. As I’m a basic scientist and not a physician, at that time
my background was not in any particular genetic disease; however, I found
it thrilling that a general solution to the problem of how to change the sequence
of DNA with high efficiency and precision inside the nucleus of
living human cells in fact emerged out of the basic investigations of protein–
DNA interactions, mechanisms of DNA repair, processes that most people
think are rather fundamental and haven’t an immediate applied relevance.
This is probably the second time
in the history of biomedicine where
something this basic has had such a
translational impact; the first instance
was the discovery of recombinant
DNA which emerged from studies of
bacterial defence and resulted in the
development of recombinant insulin
and monoclonal antibodies. Genome
editing arose from the studies of protein–DNA interactions, mechanisms
of double strand break repair, understanding how to deliver nucleic acids
to cells, and yet here we are with the conceptual equivalent of Microsoft
Word for the human genome.
QQ And why is it that we are now starting to see so much
excitement and interest in gene editing?
In many ways we’ve always wanted to do this haven’t we? The
notion of improving the human predicament by changing DNA
is an old one and probably on some level predates the discovery
of DNA in the late nineteenth century. We first showed that human
genes can be rewritten with high precision and efficiency nearly a decade
ago and at the time it wasn’t clear to us whether we would be able
to make it sufficiently broad and useful, in other words could we make
various types of edits, not just correct genes as we did initially for the
genetic mutation that gives rise to bubble boy disease – or severe combined
immune deficiency (SCID)? Would we be able to knock genes
out? What about more than one gene at a time? Would we be able to
integrate genes into specific locations to allow their sustained function?
Or edit the genes of model organisms important in biomedicine such
as the rat or pig? Over the 6 years that followed our initial discovery of
human genome editing, we and our collaborators in academia as well
as other academic groups who have been using zinc finger nucleases
(ZFNs) demonstrated conclusively that the answer to those questions
is an affirmative yes.
Yet whilst it was evident that genome editing will change the way people
approach experimentation both in basic research and translational settings,
the main question that remained was: how do you make that initial double
strand break in the DNA?
Then two discoveries were made essentially back to back that expanded
the access of the average researcher to the tools required to make that initiating
break. The first was our discovery of a second nuclease class called
TAL effector nucleases (TALENs) that are assembled using more Lego-like
principles than ZFNs, which are more sophisticated set of molecular scissors.
Then of course most recently Emmanuelle Charpentier and Jennifer
Doudna made their landmark discovery that Cas9 is an RNA-guided
nuclease. The field immediately realized that the previous 7 or 8 years
of toolbox building that we’d generated with ZFNs and then with
TALENS, could be taken and deployed wholesale to the cause of
genome editing with CRISPR/Cas9. All the tools existed but the
path to initiating the break was the question and that’s what Cas9
has made so easy for everyone.
An additional factor that has also impacted the advancement of
gene editing is that gene sequencing has become cheap and very efficient.
We have the means of editing genes but of course we need to
know what to edit and to what form. That part of the puzzle is what
facile sequencing has really enabled.
You can sequence different organisms to understand the basis of
trait differences or sequence the DNA of a patient who is presenting
with a particular condition. Before the emergence of gene editing,
you would just stare at that sequence and feel helpless; but now with
not one but three different gene editing platforms available it is easy
to understand why people empowered with the ability to not just
read DNA but change it, are doing so.
Q:At Sangamo you work with Zinc Fingers – what
benefits are unique to using this specific type of
nuclease?
As you can imagine this is a topic I could talk about at considerable
length. The challenge in using a nuclease for the treatment
or prevention of disease is twofold: potency and specificity.
Zinc fingers are the best studied and most sophisticated nuclease
platform for which both the potency and specificity metrics meet
the demands of deployment at clinical scale. As an example, in collaboration
with Biogen we are advancing genome editing of human
hematopoietic stem and progenitor cells (HSPCs) as a potential
treatment for the b-hemoglobinopathies – sickle cell disease and
b-thalassemia. We discovered that the human genome contains a
specific region which if disabled by genome editing creates a disease-
protective phenotype in the erythrocyte progeny of the edited
HSPCs. Remarkably, as we’ve shown in this work recently published
in Nature Methods, it is a highly specific process – you have to cut
to within one base pair of a specific position in the human genome
to create that desired protective effect. Now this highlights a unique
benefit of the ZFN platform in that it allows the placement of that
double-strand break to that level of precision.
The other challenge is of molecular specificity and we’ve focused
on ensuring that the nucleases we build attain clinical-grade specificity
with respect to genome-wide action. ZFNs are of course
Mother Nature’s own invention for engaging specific loci in the human
genome – they co-evolved with the human genome to allow
the potent and specific regulation of specific gene loci. In many ways
we are borrowing her invention and developing ways to engineer
the zinc fingers to attain maximum potency and specificity of action
It is this ability to cut very precisely where we need to cut and to
do so while maintaining clinical-grade specificity of action within
the nucleus that drives our reliance specifically on the ZFNs rather
than the other nuclease platforms.
Q Sangamo is one of the leaders in moving gene
editing from the preclinical into the clinical
setting – with a couple of INDs accepted by the
FDA for HIV and b-hemoglobinopathies – can
you tell us about some of the key challenges in
making this translation step?
12 years ago, following the first demonstration that
we could engineer ZFNs to create a double-strand break,
we very quickly realized that there are numerous downstream
considerations that you must address before this
can become clinically actionable. The first consideration is
that of deploying this approach in the cell type or setting that is
clinically relevant. With specific focus on our programs in HIV
and b-hemoglobinopathies, the challenge was how to genome
edit at clinical-scale potency and specificity in a whole patient
dose of cells – millions and potentially billions of human T cells
or HSPCs. The challenge of cell husbandry and safe and effective
delivery of the nuclease was a critical issue for us to resolve, but
I’m delighted to report that after a great deal of work we have
charted a path to ex vivo genome editing of T cells and human
HSPCs.
The second consideration is building a panel of assays to evaluate
the preclinical safety of genome editing prior to the cells
being transplanted into patients in our trial. Here we have benefited
greatly from an essentially collaborative effort with the
regulatory authorities – FDA and NIH – in building a comprehensive
panel of assays. These enable us to assess the safety of our
genome editing approach in a way that is appropriately balanced
relative to the risk–benefit profile in the context of a particular
disease indication.
In summary, deploying your nuclease to make the DNA break
in the right cell type and then assessing the specificity and safety
of that editing in a manner that is commensurate with what you
are trying to achieve clinically has been a formidable challenge
that I’m delighted to report we have overcome. We are advancing
to the clinic an approach for in vivo genome editing and have
just received unanimous approval for our clinical study protocol
for hemophilia B and MPS I (Hurler Syndrome) from the NIH
Recombinant DNA Advisory Committee (RAC). Once reviewed
by the FDA, this trial will be the first in vivo genome editing for
any nuclease platform.
Q As a trailblazer in clinical gene editing, Sangamo
has paved an unchartered path through the
regulatory landscape – what are the key learnings
from this experience with regulatory bodies?
\
Dialogue is essential. The specific branch of the FDA that we
work with is CBER – the Center for Biologics Evaluation and Research
– they fully understand that cell and gene therapies are experimental,
that we’re not developing a small molecule for a particular
disease indication with an existing history of preclinical and clinical
development. The FDA has an established path for the progressive
discussion of both assaying the safety as well as the clinical issues for
proposed clinical deployment.
Over the past decade we have
benefited tremendously from
being able to engage the FDA
in reviewing our proposals,
receiving very constructive
feedback and addressing that
feedback. I‘m very proud to
report that not only are we in
clinical trials with autologous
edited T cells but that we have
an open IND for the editing of HSPCs in HIV. We are also on track
for filing an IND for in vivo genome editing before the end of 2015
as well as an IND for b-thalassemia in the first half of next year. The
reason I mention these timelines is that when I talk about a dialogue
with the agency this isn’t hypothetical – we are in discussions and
regulatory dialogue with them all the time and they have been a
great partner to work with. And the other agency is of course the
NIH RAC which is staffed by people who have been working in the
field of cell and gene therapy their entire lives and so they have been
really constructive with their feedback as well.
Q When moving to the clinical setting, what are the
potential safety risks and how do you mitigate these?
The first thing to understand is that this is very much disease
indication specific and I can give you some insight into
how we approach safety in our ex vivo therapy for beta-hemoglobinopathies.
We have built a comprehensive panel of assays
that assess the safety of genome editing to both the genome and
the “stemness” phenotype of human HSPCs. What’s interesting is
that having watched this field over the last decade, the technology
just does not stand still. The world around us continues to develop
new approaches to evaluate biological systems that really didn’t exist
even 10 years ago. When I was a graduate student in the early 1990s
at Brown I used to perform a procedure called Sanger sequencing
and it took me a month to determine the DNA sequence of 1000
base pairs; yet today we have next generation sequencing where for
a fraction of the cost and within 48 hours you can determine the
DNA sequence of thousands of loci or sequence the entire genome
in a population of cells.
Assays evolve and our ways of looking at the safety of gene editing
has also evolved as the technology becomes more sophisticated. An
important thing to understand is that when we assess the safety of
what we are doing, we don’t really stand still with respect to what our
clinical-grade reagents are. The research-stage reagents that we utilize
to obtain initial read outs of efficacy with ZFNs both in vitro and potentially
in animal models, we can assess their safety rather rapidly and
then, if necessary, essentially optimize the reagents further to attain
maximum on-target and genome-wide specificity read outs.
Q Sangamo’s lead product for HIV targets the
CCR5-encoding gene – can you briefly explain
the scientific rationale behind the selection of this
target and your approach to disrupt this gene?
The age of genomics has brought us this remarkable discovery
that natural selection has non-uniformly distributed
disease-relevant alleles in humans. We are all familiar with lactase-
persistent alleles that are more prevalent in parts of the world
where people drink milk. And in the mid-90s here in the San Francisco
Bay area, the remarkable observation was made that some people
who have been exposed to HIV appear to remain overt disease
symptom free. It was very rapidly determined by DNA sequencing
that these individuals are ‘natural mutants’ – namely they are homozygous
for the loss-of-function allele of a gene called CCR5 which
encodes the co-receptor for HIV entry into the cell.
Timothy Ray Brown at this point is probably one of the best
known names in biomedicine – but he’s not a scientist, he is in fact
the famous Berlin patient who has been effectively cured of his HIV
infection by allogeneic bone marrow transplant of HSPCs that are
homozygous for this disease-protective allele of CCR5. Whilst this
is fantastic for Timothy, this approach is just not scalable worldwide
to HIV patients. We reasoned therefore, that based on this
very strong epidemiological and public health data indicating that
people homozygous for the knock out allele of CCR5 are protected
from infection by R5 tropic HIV. Furthermore, looking at Timothy
Ray Browns’ experience we posited that you could attempt to
recreate this HIV-protective genotype in the cells of HIV-positive
individuals in the hopes of essentially creating a compartment of the
immune system that is protected from HIV infection.
Q In collaboration with the University of
Pennsylvania, you have treated over 70 HIV
patients with this ZFN-mediated gene editing
approach – can you share with us some of your
clinical experiences and outcomes to date?
We are excited to report that the treatment is well tolerated
to date and that we have evidence of an antiviral effect including
subjects that have demonstrated control of viral load for
an extended period while remaining off anti-retroviral therapy
(ART). For example in our most recent cohort, we have shown that
two out of our three subjects have sustained functional control of viral
load in the absence of ART. We also have a cohort of immunologic
non-responders that we were
happy to see have demonstrated
a decrease in the size of their
HIV reservoir at 36 months.
We were greatly encouraged by
how well this has gone and now
have a Phase 1 study, for which
we have an open IND, for the
same approach but this time in
HSPCs and this study is being
conducted at the City of Hope in Southern California. And you may
we have an open IND, for the
same approach but this time in
HSPCs and this study is being
conducted at the City of Hope in Southern California. And you may
ask: “why go from T-cells to stem cells?” The logic here is that we are
attempting to protect additional compartments of the hematopoietic
tree from HIV infection, for example macrophages and dendritic cells.
Q How durable does this response appear to be?
Will there be a need or option to re-dose patients?
Re-dosing is an option and we are evaluating the delivery
of ZFNs to the T cells in the form of in vitro transcribed messenger
RNA (mRNA) which absolutely gives us the option
to re-dose. With respect to the durability of response, we’ve seen
modified cells persisting in our subjects out over 4 years post-transplant
– the longest time point studied to date. Granted, 4 years is
not a lifetime, but we are greatly encouraged by the durability we’ve
observed so far.
Q HIV is renowned for its ability to evade immune
detection through its presence in latent
reservoirs in the body – can gene editing impact
these reservoirs and cure patients vs functionally
cure?
Looking at the cohorts 1 through 3 in one of our earlier
studies, for whom we have up to 36 months of follow-up
data, one could argue that the answer to this question is yes.
We’ve observed a mean 0.9 log decrease in HIV reservoir in nine
out of nine subjects and in some individuals this decrease is actually
substantially greater. We are excited to observe an increase in genome
editing in the CD4+ central stem cell memory compartment
and whilst I’m not an immunologist, my qualified immunologist
colleagues assure me that this compartment of the immune system
lasts the lifetime of a human. Therefore, being able to modify those
cells and potentially protect them from HIV infection gives us great
hope that we are in fact creating a lifetime effect.
Q Whilst HIV is a global healthcare issue, large
patient populations are found within developing
nations – do you think therefore that it’s possible
that gene editing technologies will one day
replace existing ART which is currently a cheaper
and comparatively easy to deliver to patients?
This as you can imagine is a topic that we think about a lot at
Sangamo and the issue of cost is nuanced. ART really changed
the prognosis of patients with HIV but it is a lifetime treatment and
patients must take multiple pills daily. When I was in high school
I was a huge fan of Queen and it was such a tragedy when Freddie
Mercury succumbed to the disease; and it’s fantastic – as a basketball
fan – to see Magic Johnson is alive and well. But we must contend
with the fact that the life-time cost of ART is not unsubstantial. The
big hope of course with genome editing is that one creates a functional
cure with a one-time treatment or a potentially short regimen
of re-dosing.
The other issue to consider regarding ART is that it’s evident
that there are certain patients who are immunologic non-responders,
whose immune system never completely recovered
from the initial assault from the virus. Helping these people is
a real challenge and that’s why we enrolled them in some of our
cohorts. Ultimately ours is an experimental therapy still in clinical
development, but as I mentioned, to date the treatment has
been well tolerated and we have some people with viral control
in the absence of ART.
In addition to cost, a lifetime regimen of ART is clearly associated
with side effects that in many patients, create serious non-compliance
issues, namely they have to make a choice of whether to take
the medication or not. Our hope is that this will be a non-issue with
a genome editing approach because a human being once edited will
not need to comply with a therapy anymore as the therapy will have
been complete.
Q Sangamo is also applying ZFN gene editing to the
hemoglobinopathies – can you tell us about the
selected target and how it was identified?
As I sometimes point out to my colleagues – I love being
second. My colleague Michael Holmes, PhD, at Sangamo was the
scientific leader responsible for taking our first genome editing approach
to the clinic. As mentioned, the approach to editing CCR5
in HIV patients was based upon epidemiologic and public health
data that showed that there is a naturally occurring disease-protective
genetic variation – namely the knockout mutation of CCR5.
In reference to my point of going second, I am the scientific leader
in our collaboration with Biogen where we are looking to deploy
the same fundamental principle – which is to rely on naturally occurring
disease-protective genetic variation – to the hemoglobinopathies,
the most prevalent genetic diseases globally. In Thailand alone
there are 300,000 people with b-thalassemia; 100,000 people with
sickle cell disease in the USA and that many neonates born annually
with sickle cell disease in Nigeria alone. Therefore they truly represent
a substantial public health burden.
One of the remarkable things about these diseases is the large
variability in clinical presentation of the disease: some individuals
are relatively disease free whilst others are severely ill despite having
the same underlying genetic mutation. This led people to study this
disparity and 4 years ago studies started to emerge that this is in
fact due to the protective effects of mutations at other loci in the
genome. These individuals are not single but double mutants; they
have the disease-causing mutation and then they have a disease-protective
mutation in addition. Researchers started to look at where
that disease-protective variation lies and were greatly surprised to
find it in the gene bcl11a. Now the name of this gene is actually a
find it in the gene bcl11a. Now the name of this gene is actually a
misnomer – it should really be called multi-functionally important
human gene that happens to be the key regulator of human fetal
globin! In utero or immediately post birth we produce a different
b-hemoglobin to that which we make as adults. This hemoglobin
is call fetal hemoglobin, which is quickly shut off and its synthesis
in our erythrocytes is replaced by adult hemoglobin. In individuals
with sickle cell disease or b-thalassemia, they are in this unfortunate
position whereby Mother Nature doesn’t realize that their adult
hemoglobin is the mutant form and thus when they switch from a
perfectly functioning fetal hemoglobin to mutant adult hemoglobin
they develop the disease. That is unless they have this second mutation
in bcl11a in which case the switch in hemoglobin production is
incomplete and they synthesize sufficient levels of fetal hemoglobin
throughout life which protects them from the fact that their adult
hemoglobin is mutant.
Greatly encouraged by the fact that there are people with much
milder or essentially no disease symptoms if they have this protective
mutation, our strategy is to recreate this disease-protective genetic
variation in HSPCs of people with b-thalassemia and subsequently
with sickle cell disease. I hope that that we will create a one-time
approach that will be broadly applicable against both of these hemoglobinopathies
where we take HSPCs from a person with either disease,
genetically engineer them in a way that selectively eliminates
expression of the bcl11a from the erythropoietic tree, transplant the
cells back into the subject and cross all fingers other than zinc in
the hope that we get a sustained elevation of fetal hemoglobin. If
we achieve that elevation then we know that will confer protection
against the disease.
Q You are also working on in vivo applications
for diseases such as hemophilia and lysosomal
storage disorders – what are the main differences
and challenges in applying gene editing in vivo
versus ex vivo?
Deployment in vitro is carried out in a more controlled environment:
you harvest the T or stem cells from a subject and
they are either in a bag or a cuvette in front of the operator at
all times. Therefore you build non-clinical safety assays and efficacy
assays that are focused on the fact that you are working with cells
that are in front of you. With in vivo genome editing you develop
your genome editing tool, deliver it to the subject but then Mother
Nature takes her course so the challenge there, that we believe we
have successfully met, is to build a comprehensive panel of preclinical
safety and efficacy assays that adequately assess the potency and
specificity of that approach.
One of the things that I find remarkable and most translationally
exciting about the in vivo genome editing approach – and I must
credit my colleagues Edward Rebar, PhD, and Michael Holmes,
PhD, who are leading the development of our in vivo editing – is
that we are potentially able to treat a range of monogenic disorders
by the targeted editing of just one locus. In essence, we believe we
are building an in vivo protein replacement platform and the strategy
here was to identify a human gene that is expressed to a very
high level in the human liver but is
also non-essential so that its loss of
expression causes no ill effects. And
one such gene is albumin. The data
we have generated thus far support
the hypothesis that for monogenic
diseases such as lysosomal storage
disorders or hemophilia B, we will
be able to replace the human albumin
gene in the liver of an affected
individual with the open reading frame of the gene which is disabled
by mutation. Following genome editing with ZFNs, the hepatocyte,
which is our natural ‘engine’ for the secretion of protein into the
bloodstream, no longer secretes albumin but now faithfully secretes
the protein we have just integrated into it. I find this truly thrilling
– this notion that we have for lack of a more elegant term, a ‘plug
and play’ locus on the human genome that we are hopeful we can
develop as a broad approach for the treatment of monogenic diseases
that are addressable in this way.
Q Sangamo is collaborating with Biogen and Shire
on the development of couple of clinical products
– what are the key considerations when looking
to collaborate with industry partners?
In many ways the proverbial saying: “it takes a village to
raise a child” is highly relevant here. We’ve discussed the tremendous
efforts in academia upon which we have relied in building
this gene editing platform. As we move toward the clinic, for the
specific indications that we are targeting which affect a large number
of people worldwide, being able to collaborate with a company like
Biogen or Shire is wonderful because they bring the extraordinary
might that big biotech and big pharma possess. Obviously it’s good
to be able to work with someone who is also focused on the same
therapeutic areas that we are targeting, but more than that - they
need to be excited about the fact that this is not a small molecule or
a biologic. We are building genome editing as a therapeutic modality
and we are hopeful to look for synergy – which is certainly the
case with Biogen and Shire. We are the genome editing people, we
live and breathe zinc finger nucleases, so it’s incredibly important to
be able to partner with organizations that have their own expertise
that is relevant to the disease indication we are pursuing.
Q How do you envisage the field evolving over the
next 5 years – what progress do you hope to see?
I am very much a glass half full kind of person – I am thrilled
about the progress that has been made so far. Over the next 5
years we will have data, not only from the more advanced stages of
clinical development of our genome editing in T cells in HIV but
also of this approach in our trial of CCR5 editing in HSPCs. Following
on from this will be the application of genome editing of HSPCs
for b-thalassemia or sickle cell diseases. Data from deploying editing
in HSPCs for these three indications will really teach the field more
broadly about what genome editing ex vivo can do to address the
challenge of infectious and monogenic diseases of the blood. Should
the data look good, which I’m incredibly optimistic that they will,
then this will fundamentally change the way we think about dealing
with those diseases clinically.
Following closely on from this is our approach of genome editing
in vivo and our initial approach to rewriting the gene expression
programme of the hepatocyte to become an in vivo protein synthesis
machine. Once again, if we are able to demonstrate that the liver
genome was rewritable safely and effectively allows an improvement
in the predicament of patients with hemophilia or lysosomal storage
diseases, again I think the field as a whole will start to look very
differently at how we approach the clinical management of these
conditions.
Last but not least, if you had told me when I was a graduate student
that 20 years from now you will be able to engineer molecular
scissors that will be able to within base pair precision disable a locus
in the human genome in a clinical-scale dose of HSPCs that would
then retain every metric of viability and functionality to allow an
autologous transplant for the treatment of HIV or beta thalassemia,
I don’t know how I would have reacted, but that would have sounded
incredibly futuristic and yet here we are. The lesson from this
and from other developments of technologies, such as deep sequencing,
is that we should not underestimate the progress of technology.
Engineering of nucleases, of cell husbandry, cell processing and of
course delivery modalities is advancing at a pace that is just breath
taking. Therefore good and accurate prognoses for the next 5 years
are hard to provide because who knows what is currently being invented.
My take on this is really formulated by Alan Kay who was
one of the pioneers of computer science: “the best way to predict the
future is to invent it”. I am very much a believer in that paradigm
– we are currently inventing what the next 5 years are going to look
like. Exciting they will be, that’s for sure.
http://insights.bio/cell-and-gene-therapy-insights/2015/12/11/dr-fyodor-urnov-the-power-and-promise-of-zinc-finger-nuclease-mediated-genome-editing/
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