Zinc finger nuclease proteins provide simple, safe alternative to
11 July 2012
Zinc finger nuclease (ZFN) proteins have a surprising ability
to easily enter cells and disrupt specific genes within cells, according
to research The Scripps Research Institute in the US.
ZFN proteins can bind and cut DNA at precisely defined locations
in the genome. The Scripps Research scientists simply added ZFN
proteins directly to cells in a lab dish and found that the proteins
crossed into the cells and performed their gene-cutting functions
with high efficiency and minimal collateral damage. The new
technique was reported in Nature Methods on July 1, 2012.
ZFNs are coming into widespread use in scientific experiments and
potential disease treatments, but typically are delivered into cells
using potentially risky gene therapy methods.
“We showed that we can modify the genomes of cells without the
troubles that have long been linked to traditional gene therapy
techniques,” said the study’s senior author Carlos F Barbas III, who
is the Janet and Keith Kellogg II Professor of Molecular Biology and
Chemistry at The Scripps Research Institute.
“This work removes a major bottleneck in the efficient use of ZFN
proteins as a gene therapy tool in humans,” said Michael K Reddy,
who oversees transcription mechanism grants at the National
Institutes of Health’s (NIH) National Institute of General Medical
Sciences, which helped fund the work, along with an NIH Director’s
Pioneer Award. "The directness of Dr Barbas's approach of ‘simply’
testing the notion that ZFNs could possess an intrinsic
cell-penetrating ability is a testament to his highly creative
nature and further validates his selection as a 2010 recipient of an
NIH Director’s Pioneer Award.”
ZFNs, invented in the mid-1990s, are artificial constructs made
of two types of protein: a “zinc-finger” structure that can be
designed to bind to a specific short DNA sequence, and a nuclease
enzyme that will cut DNA at that binding site in a way that cells
can’t repair easily. The original technology to make designer zinc
finger proteins that are used to direct nucleases to their target
genes was first invented by Barbas in the early 1990s.
Scientists had assumed that ZFN proteins cannot cross cell
membranes, so the standard ZFN delivery method has been a
gene-therapy technique employing a relatively harmless virus to
carry a designer ZFN gene into cells. Once inside, the ZFN gene
starts producing ZFN proteins, which seek and destroy their target
gene within the cellular DNA.
One risk of the gene-therapy approach is that viral DNA—even if
the virus is not a retrovirus—may end up being incorporated randomly
into cellular DNA, disrupting a valuable gene such as a tumor-suppressor
gene. Another risk with this delivery method is that ZFN genes will
end up producing too many ZFN proteins, resulting in a high number
of “off-target” DNA cuts. “The viral delivery approach involves a
lot of off-target damage,” said Barbas.
In the new study, Barbas and his colleagues set out to find a
safer ZFN delivery method that didn’t involve the introduction of
viruses or other genetic material into cells. They experimented
initially with ZFN proteins that carry extra protein segments to
help them penetrate cell membranes, but found these modified ZFNs
hard to produce in useful quantities. Eventually, the scientists
recognized that the zinc-finger segments of ordinary ZFNs have
properties that might enable the proteins to get through cell
membranes on their own.
“We tried working with unmodified ZFNs,
and lo and behold, they were easy to produce and entered cells quite
efficiently,” Barbas said.
New strategy against HIV
Next, the team showed how the new technique could be used in a
ZFN-based strategy against HIV infection.
The AIDS-causing retrovirus normally infects T cells via a T cell
surface receptor called CCR5, and removing this receptor makes T
cells highly resistant to HIV infection. In 2006, an HIV patient in
Berlin lost all signs of infection soon after receiving a bone
marrow transplant to treat his leukemia from a donor with a CCR5
gene variant that results in low expression of the receptor.
Disrupting the CCR5 gene in T cells with a ZFN-based therapy might
be able to reproduce this dramatic effect.
“The idea is to protect some of the patient’s T cells from HIV,
so that the immune system remains strong enough ultimately to wipe
out the infection,” said Barbas.
A gene therapy that uses ZFNs to disrupt CCR5 genes in T cells
and reinfuses the modified T cells into patients is currently in
clinical trials. Barbas and his team showed that they could achieve
the same effect with their simpler ZFN-delivery method. They added
ZFN proteins directly to human T cells in a culture dish and found
that within hours, a significant fraction of the ZFN-treated cells
showed sharp reductions in CCR5 gene activity.
After several applications of ZFNs, aided by a special cooling
method that improves the ability of the proteins to get across cell
membranes, the scientists were able to inactivate CCR5 genes with an
efficiency approximating that of the gene therapy-based approach,
The new approach also appeared to be safer. A
DNA-based method the team used for comparison or the viral-based
methods reported in the literature by others ended up producing ZFNs
for up to several days, causing a significant amount of off-target
DNA damage. But the directly delivered ZFN proteins remained intact
within cells for only a few hours, causing minimal off-target
“At some off-target locations where the gene therapy approach
frequently causes damage, we saw no damage at all from this new
technique,” said Barbas.
Turning skin cells into therapeutic stem cells
The team tested its direct ZFN-delivery technique with a variety
of other cell types and found that it works with particularly high
efficiency in human skin “fibroblast” cells. Researchers now are
working on advanced therapies in which they harvest such fibroblasts
from patients and reprogram the cells’ gene-expression patterns so
that they effectively become stem cells. These induced stem cells
can then be modified using ZFNs and other genome-editing techniques.
When reinfused into a patient, they can produce millions of
therapeutic progeny cells over long periods.
Such techniques may one day be used to treat a vast array of
diseases. Barbas, who has been developing anti-CCR5 strategies for
more than a decade, wants to start with a ZFN-based therapy that
disrupts the CCR5 gene in hematopoietic stem cells. These
blood-cell-making stem cells, reinfused into an HIV patient, would
become tiny factories for producing HIV-resistant T cells.
“Even a small number of stem cells that carry this HIV-resistance
feature could end up completely replacing a patient’s original and
vulnerable T cell population,” he said.
The other authors of the paper, “Targeted gene knockout by direct
delivery of ZFN proteins,” are first author Thomas Gaj, and Jing Guo,
Yoshio Kato, and Shannon J. Sirk, all members of the Barbas
laboratory during the study.
Thomas Gaj, Jing Guo, Yoshio Kato, Shannon J. Sirk, and Carlos
Barbas. Targeted gene knockout by direct delivery of ZFN proteins.
Nature Methods, July 1, 2012;