After some tragic early setbacks techniques that allow precise genetic manipulation have created a surge of research.
Tiny changes in DNA can have huge consequences. For years, scientists
have been trying to 'fix' these mutations in the hope of treating and
potentially curing some of humanity's most devastating genetic diseases.
After some tragic early setbacks , techniques that allow precise genetic manipulation have created a surge of research.
DNA sequences showing the sickle-cell disease mutation (marked with an
asterisk, top) and the sequence corrected (below) using gene-editing
technology.
Although
most existing treatments for genetic diseases typically only target
symptoms, genetic manipulation or 'gene therapy' goes after the cause
itself. The approach involves either inserting a functional gene into
DNA or editing a faulty one that is already there, so the conditions
most likely to prove curable are those caused by a single mutation.
Sickle-cell disease is a perfect candidate: it is caused by a change in
just one amino acid at a specific site in the β-globin gene. This
results in the production of abnormal haemoglobin proteins that cause
the red blood cells that house them to twist and become sickle shaped.
The distorted cells get sticky, adhere to each other and block blood
vessels, preventing oxygenated blood from flowing through.
Gene
therapy has been used successfully in a handful of patients with immune
disorders, and sickle-cell disease is among researchers' next targets.
The most advanced of these projects is slated to begin clinical trials
by the end of the year, and other trials are set to follow. The
approaches being developed to treat sickle-cell disease take one of two
forms. Conventional gene therapy, also known as gene addition, typically
involves inserting new genes. Usually, a harmless virus is modified
with the gene to be inserted, and this 'viral vector' is mixed with
cells from the patient
in vitro. The virus searches out the cells
and inserts the gene into the cells' DNA, after which the cells are
transplanted into the patient. Conversely, gene editing is more nuanced:
in a molecular cut-and-paste, researchers cut out the faulty DNA
sequence and then insert a piece of laboratory-created DNA. In both
approaches, the modified DNA dictates the formation of a normal, working
protein.
In sickle-cell disease, the only cells that need their
DNA edited are blood stem cells — also known as haematopoietic stem
cells — which are found in bone marrow. These cells continually form new
red blood cells to replace those that are lost, and reprogramming just a
small fraction of them will create enough perfectly formed red blood
cells to eliminate disease symptoms. “Achieving genome editing via
direct repair of blood stem cells represents a high hurdle,” says George
Daley, director of the Stem Cell Transplantation Program at Boston
Children's Hospital in Massachusetts, “but perhaps not an impossible
one.”
Although these approaches are promising, several important
issues must be addressed before they can be used to treat patients, such
as ensuring that the therapies accurately hit their targets and do not
cause irreparable harm to the cells or introduce additional genetic
information that could cause problems such as cancer.
Injecting genes
Gene
addition is poised to become the first sickle-cell gene therapy to be
tested in humans. At the regenerative medicine and stem cell research
centre of the University of California, Los Angeles, molecular
geneticist and physician Donald Kohn is developing protocols for a
clinical trial of this technique that is due to start enrolling patients
by the end of 2014. Doctors will first harvest bone marrow from the hip
bones of patients with sickle-cell disease and then extract
haematopoietic stem cells from the marrow. Using a viral vector, they
will insert a new, working haemoglobin gene into the cells' DNA; the
old, faulty haemoglobin gene will still be present, but it will go
silent as the new gene takes over. The modified cells will then be
infused back into the patient's bloodstream and will migrate to the bone
marrow, where they can provide a continual source of healthy red blood
cells.
Kohn says that this approach has the potential to cure
sickle-cell disease, and with significantly fewer side effects than a
bone marrow transplant — currently the only cure (see
page S14).
He has tested the technique by injecting modified human haematopoietic
stem cells into mice, and found that they were free of sickle cells 2 to
3 months later
1.
The limiting factor in mice, Kohn says, is that they can only sustain
human grafts for that long. In humans, he thinks the correction should
last a lifetime — as long as 50 to 70 years.
One of the challenges
in treating sickle-cell disease with gene therapy is that it is
necessary to extract bone marrow to retrieve haematopoietic stem cells.
With most other diseases, patients can be given drugs that entice these
cells to leave the marrow and enter the bloodstream, where they can be
easily harvested. But in patients with sickle-cell disease, these drugs
can trigger sickle-cell crisis, an acutely painful episode during which
the damaged cells stick together and block blood vessels; the crisis can
be accompanied by anaemia, chest pain, difficulty breathing, blood
trapped in the spleen and liver, even stroke. So researchers must
harvest the bone marrow itself, which can be difficult and slow, and
limits the number of cells that can be collected at one time. Kohn says
that they still do not know whether this approach will yield enough
haematopoietic stem cells for reprogramming. And, like other bone marrow
transplant procedures, the patient still needs to undergo chemotherapy
to kill off the remaining bone-marrow cells to help the genetically
altered ones survive once they are reintroduced into the body.
Talented fingers
Further
away from clinical trials, but potentially a lot more exciting, is gene
editing. The concept was introduced in the 1990s, when artificial
DNA-cutting enzymes known as zinc finger nucleases (ZFNs) were first
engineered. ZFNs bind to a specific section of DNA and create a break at
both ends (see 'Molecular cut-and-paste'). Cells will start to repair
the break, at which point a specific sequence of laboratory-made DNA can
be slotted into the gap. After the DNA is repaired, the cells start to
create healthy copies of the gene.
In parallel to his work on gene addition, Kohn is exploring the
use of ZFNs to edit sickle-cell genes. In collaboration with the firm
Sangamo BioSciences in Richmond, California, he has shown that around 7
%
of haematopoietic cells can be repaired in culture using this
technique, using a viral vector to get the ZFNs into the cells. Because
the repaired cells continue to replicate, this small proportion could be
enough to eventually produce a sufficient amount of working red blood
cells. Kohn says that patients have shown major improvements when just
10–20
% of their donor cells successfully engrafted and started to make new, healthy cells.
The
advantage of gene editing over gene addition (a less complex approach)
is that it provides an actual fix rather than a work around. But ZFNs
are expensive and difficult to program. In 2010, a gene-editing protein
called TALEN (transcription activator-like effector nuclease) was
developed, which uses a similar mechanism as ZFNs but is cheaper and
easier to work with. It was quickly adopted for use in sickle-cell
disease.
At the Salk Institute for Biological Studies, in La
Jolla, California, stem-cell biologist Juan Carlos Izpisua Belmonte uses
TALENs in concert with viral vectors called HDAdVs (helper-dependent
adenoviral vectors) to correct the sickle-cell mutation. Instead of
harvesting haematopoietic stem cells from bone marrow, Izpisua
Belmonte's team takes easily harvestable cells, such as blood, skin or
fat cells, and then turns them into induced pluripotent stem (iPS)
cells, which can be converted into any cell type. The researchers
correct the haemoglobin gene defect
in vitro using gene editing,
then differentiate the repaired iPS cells into blood stem cells. From
there, the researchers have a couple of choices. The repaired cells
could simply be infused into a patient's bloodstream, where they would
make their way into the bone marrow and start to make healthy
haematopoietic cells.
But Izpisua Belmonte is also working on a
cure that could work inside the bone marrow itself. His team is
combining TALENs with a different viral vector, HDAdVs, to boost the
success rate of gene editing, and the researchers are working on a plan
to administer their hybrid vector directly into the bone marrow, so the
genetic fix would take place inside the patient's body. Although each
infusion into the marrow might correct only 1
%
of the cells, ten such procedures over the course of several months —
something Izpisua Belmonte and his research associate Mo Li think is
feasible in terms of time and cost — could alleviate the symptoms of
sickle-cell disease. “Little by little, you are correcting the disease
in vivo,”
says Izpisua Belmonte. So far, this 'hybrid vector' technique has shown
promising efficacy in umbilical-cord blood stem cells.
Sickle-cell
disease results when both copies of the haemoglobin gene are faulty,
and fixing just one of the genes is sufficient to make a big health
improvement. As Li points out, people who carry one copy of the mutated
gene, a genetic condition referred to as 'sickle-cell trait', do not
show symptoms. “In fact, many of the world's best sprinters have the
sickle-cell trait, he says. “Our approaches will most likely restore one
mutated copy to its wild-type sequence, leaving the other copy
untouched.”
CRISPRs (clustered regularly interspaced short
palindromic repeats) are the most recent addition to the gene-editing
toolbox. Whereas ZFNs and TALENs use a protein to lock on to a specific
section of DNA, CRISPRs use a 'guide RNA'. These guide RNAs are much
easier to program than the proteins in TALENs and ZFNs, as well as being
cheaper and more efficient. CRISPRs also make it possible to perform
multiple genetic manipulations in one go. CRISPRs work in combination
with the Cas9 (CRISPR-associated 9) nuclease: after the CRISPR locks on
to the target gene, Cas9 snips both strands of the DNA, disabling the
gene. The approach is less than two years old, yet many researchers are
now working with CRISPRs in parallel with other
in vitro techniques.
Chris Calleri/Georgia Institute of Technology
A researcher corrects a mutation in the β-globin gene that causes sickle-cell disease.
There are safety hurdles to be overcome before gene editing is
used in humans, especially because it involves a permanent change in
the genome. The thorniest issue is 'off-target activity' — unintended
changes to the genome away from the target gene.
Gang Bao, a
biomedical engineer at the Georgia Institute of Technology in Atlanta,
is developing gene-editing strategies for sickle-cell disease and is
paying particular attention to the challenge of limiting off-target
effects. He notes that if erroneous cuts happen in a cancer-causing
gene, they could potentially trigger tumour growth. Even a rate of
off-target activity lower than 1
% could still
pose serious health risks. So that the technology can move forward,
researchers need to have a better understanding of off-target effects.
There are two main issues: determining exactly where the off-target cuts
occur and at what rate.
Bao's group has created software to
predict where the off-target effects might occur for the different
gene-editing techniques. In a paper published in May, his team reported
that their software predicted 114 potential off-target sites across the
whole genome for the CRISPR/Cas9 system, and experiments confirmed 15 of
them by sequencing the cleaved DNA
2.
Izpisua
Belmonte's team is also looking at the rate of unwanted mutations
caused by gene-editing techniques. The group created iPS cell lines and
then edited half of the cells using HDAdVs and TALENs
3,
but left the other half unedited. The edited cells had no more
mutations than the unedited ones, indicating that — in contrast to Bao's
findings for CRISPRs — the use of TALENs does not seem to make cells
any less safe. Although human testing is still a few years off, they say
that these results give them optimism about the potential for gene
editing to work.
The other major challenge for gene-therapy
researchers is ensuring that the edited stem cells survive and generate
healthy red blood cells after they are reinserted into the bone marrow.
Edited cells often die because of the amount of stress they undergo
during therapy. Researchers might be able to improve the cell-survival
rate by delivering other types of cells at the same time, and the speed
of gene editing also seems to be important: the longer the cells are
cultured
in vitro, the less likely they are to survive. “Let's
see if we can perform the whole procedure in four hours instead of four
days,” says Bao.
Future repair toolboxes
Based on his
research so far, Bao thinks that CRISPRs are the best method for
generating DNA breaks, but they are also more likely to cause off-target
activity. TALENs are less efficient than CRISPRs, but they seem to have
fewer off-target effects. The rate of on-target activity for CRISPRs is
between 40
% and 80
%, whereas the on-target rate for TALENs is between 20
% and 50
%,
Bao says. The rate of off-target activity varies depending on the type
of cells and the nuclease used. “If we have a way to overcome the
off-target and [cell-survival] problems, CRISPR is a very promising
technology,” he says.
Kohn has compared ZFNs, TALENs and CRISPRs,
and thinks all three have therapeutic potential for patients with
sickle-cell disease. The techniques are all good at slicing DNA; now the
remaining challenges are delivering them to the target cell and
accurately repairing the gene after the break.
Ultimately, for
sickle-cell gene therapy to become reality, the details must be sorted
out on a large scale. Tinkering with human genes can yield both
devastating and remarkable results, and the difference between the two
often lies in a single nucleic acid of a single gene. This places a
heavy responsibility on the shoulders of every researcher in the field,
but the vast potential of gene therapy makes that burden worthwhile.
