A sidestep produces
results:http://www.sciencedaily.com/releases/2012/07/120701191613.htm
D.
Scientists Develop Alternative to Gene Therapy
ScienceDaily (July 1, 2012) --- Scientists at The Scripps Research
Institute have discovered a surprisingly simple and safe method to
disrupt specific genes within cells. The scientists highlighted the
medical potential of the new technique by demonstrating its use as a
safer alternative to an experimental gene therapy against HIV infection.
"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.
The new technique, reported in /Nature Methods/ on July 1, 2012, employs
zinc finger nuclease (ZFN) proteins, which can bind and cut DNA at
precisely defined locations in the genome. 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.
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.
"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.
*Questioning Assumptions*
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, Barbas said.
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 damage.
"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.
*Hope for 'Tiny Factories' of Health*
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.
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