AL AI N F I SCHE R

F
or more than 40 years
1
, gene therapy has
been predicted to be a way to cure inher-
ited diseases that are caused by defec-
tive copies of a gene. The typical approach to
gene therapy is to add a functional copy of the
mutated gene to the genome, using a geneti-
cally engineered retroviral vector to transport
the copy into diseased cells (Fig.1a). Although
gene addition has been used to treat certain
inherited diseases, such as severe combined
immunodeficiencies
2,3
, its success has been
mitigated by side effects
4
. A different strategy,
based on repair of the defective gene, is there-
fore an attractive alternative. In a paper pub-
lished on Natures website today, Genovese
etal.
5
report progress in the optimization of
such a strategy.
Gene-repair techniques rely on artificial
nuclease enzymes that specifically target the
mutated genetic sequence and create a DNA
break. If a DNA template for the desired
replacement sequence is provided, the mutated
stretch can be repaired by an exchange of
genetic information known as homologous
recombination (Fig.1b)
6
. One benefit of this
strategy is that it takes advantage of an innate
method of recombination used by the cell to
repair harmful DNA breaks, and so does not
adversely affect other genomic regions, such
as regulatory sequences. Gene addition, on the
other hand, relies on a semi-random integra-
tion method that may disrupt genetic regula-
tory sequences, because the extra sequence
integrates imprecisely within the genome. This
has been shown
4
to cause toxic side effects, for
example vector-mediated activation of cancer-
causing oncogenes.
Zinc-finger nucleases (a zinc-finger protein
that binds to the desired sequence of DNA,
combined with a nuclease, which cleaves DNA
wherever the molecule binds) were the first
enzymes to be designed for gene repair and
have been shown to work in cell lines
6
. Other
nucleases for example, those modified
from artificial restriction enzymes known as
TALENs, or from RNA-guided enzymes based
on the bacterial CRISPR-associated system
have since been used
6
to correct genetic muta-
tions in human cell lines, including induced
pluripotent stem cells (iPSCs), which have
been genetically reset and can give rise to most
cell types in the body. Genovese and colleagues
have used this approach to correct muta-
tions in a fraction of human haemato poietic
stem cells (HSCs), which are a key target for
treating inherited disorders associated with
blood cells.
How did the authors achieve this? The hurdle
to overcome lay in the fact that homologous
recombination occurs only in cycling cells (in
the S/G2 phase of the cell cycle), and so the
procedure is ineffective in adult stem cells such
as HSCs, which are mostly quiescent. Using
several tricks, Genovese et al. achieved effi-
cient integration of a green fluorescent protein
sequence into the genome of human HSCs at
chosen sites, including a protein-coding region
(exon 5) of the IL2RG gene that is mutated
in patients with X-linked severe combined
immunodeficiency syndrome (SCID-X1).
To prepare HSCs for gene repair, Genovese
and colleagues stimulated the cells with signal-
ling molecules called cytokines for two days,
and on the second day used a viral vector to
introduce a recombination template. They
then used electric currents to permeabilize the
cells, and added a Zn-finger nuclease designed
to target the desired DNA sequence. It is likely
that pretreatment with cytokines made the
HSCs less sensitive to the toxic effects of intro-
ducing the nucleases
4
and, more importantly,
that the treatment prompted some of the cells
to enter the cell cycle and become capable of
mediating homologous recombination. In
addition, the authors treated the cells with
two compounds dimethyl prostaglandinE
2

GENE THERAPY
Repair and replace
One approach to treating inherited diseases is repairing the defective genes, but
this has proved challenging in stem cells. An optimized protocol has now been
developed that allows gene repair in blood-cell precursors.
Figure 1 | Strategies for treating inherited diseases with gene therapy. a, In gene addition, stem cells
harbouring a genetic mutation are pre-stimulated to prepare them for genetic manipulation, and then a
functional copy of the mutated gene is transported into the cells in a retroviral vector. The functional gene
integrates into the genome at a semi-random site, restoring normal gene expression. b, An alternative
approach is gene repair, whereby a nuclease enzyme engineered to create DNA breaks around the mutated
site and a DNA template for the functional sequence are added to pre-stimulated cells using a viral vector.
The mutated sequence is then replaced with the functional copy by homologous recombination (white
dotted lines). Genovese et al.
5
have optimized pre-stimulation during gene repair in haematopoietic stem
cells, by treating the cells both with factors that prevent early differentiation (dmPGE
2
and SR1) and with
signalling molecules called cytokines that decrease the cells sensitivity to the toxic effects of the nuclease.
a Gene addition b Gene repair
Stem cell
Mutated
gene
Cell
pre-stimulation
Cell
pre-stimulation
with dmPGE
2
, SR1
and cytokines
Semi-random
integration
Normal gene
product
Normal gene
product
Retroviral
vector
Viral
vector
Functional
gene
Engineered
nuclease
DNA
template
| N A T U R E | 1
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doi:10.1038/nature13344
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(dmPGE
2
)

and SR1, an inhibitor of the aryl-
hydrocarbon receptor protein that prevent
premature differentiation, keeping the HSCs
in a stem-cell state.
Genovese and co-workers protocol enabled
site-specific genome editing in some of the
HSCs. When the treated cells were transferred
into immunodeficient mice, edited cells were
detected for up to 18 weeks, and could even be
transferred to a second recipient. When their
technique was applied to HSCs taken from
a patient with SCID-X1, the authors found
that gene correction had occurred in 311%
of progenitor cells and the myeloid cells that
differentiated from them.
This work is undoubtedly a step towards
using gene repair for gene therapy. Correc-
tion of SCID-X1 is known
2
to require only a
few IL2RG-expressing HSCs, and so it may be
that this technology can be used as it stands to
treat patients with SCID-X1. But before that
can be tested, it will be necessary to evaluate
the frequency at which the protocol causes
harmful DNA mutations, brought about by the
cells attempts to repair off-target DNA breaks
through an alternative, error-prone mode of
recombination. It is reassuring, however, that
Genovese and co-workers observed few such
off-target effects when they tested their proto-
col in a precancerous cell line.
If this method of gene repair in HSCs is to
be applied to diseases that demand a higher
level of gene correction than SCID-X1
(for example, WiskottAldrich syndrome,
adreno leukodystrophy, metachromatic leuko-
dystrophy or -thalassaemia
710
), further opti-
mization is required. For example, the design
of nucleases that increase the fidelity of DNA
cuts, combined with the in vitro expansion of
treated HSCs before transfusion might increase
the efficiency of the protocol. It is interesting
to note that an improved gene-addition proto-
col, which uses self-inactivating viral vectors
designed to reduce activation of host genes,
including oncogenes, has led to the successful
and apparently safe treatment of the inherited
disorders mentioned above
710
. Competition
between the two strategies is likely to continue
for some time.
Ultimately, gene repair seems to be an attrac-
tive strategy for targeting stem cells, such as
HSCs, that have been taken from tissues affected
by inherited disorders. As technology that
allows reprogramming of differentiated cells
to stem-cell states improves, gene correction
in reprogrammed cells such as iPCSs or
induced HSCs
11
engineered from the patients
blood cells will probably become a preferable
option. Until this goal is reached, however, it is
good to see that Genovese and colleagues have
made another advance in extending the useful-
ness of gene therapy.
Alain Fischer is at the Imagine Institute,
Hpital Necker-Enfants Malades, Paris 75015,
France, and at the Collge de France, Paris.
e-mail: alain.fischer@inserm.fr
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