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8-Humans: Safe gene therapy at last?

-------------------------------- GENET-news -------------------------------

TITLE:  Safe Gene Therapy At Last?
SOURCE: The Institute of Science in Society, UK, by Mae-Wan Ho
DATE:   29 Jul 2005

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Safe Gene Therapy At Last?

Gene defect corrected without inserting foreign DNA.
Dr. Mae-Wan Ho investigates

A fully referenced version of this article is posted on ISIS members'
website. Details here http://

A research team in a company in Richmond, California, claims to have
corrected the gene mutation associated with the fatal X-linked severe
combined immune deficiency (X-SCID) in human cells without insertion
foreign DNA into their genomes, and published their results online in the
journal Nature 2 June. This raises hope of a safer form of gene therapy
after three infants in Paris with X-SCID, who received gene therapy
through their own bone marrow cells - isolated, genetically modified in
the laboratory and injected back into the patient - came down with
leukaemia ("Gene therapy woes", SiS 26

In the latest experiments, the human cells were treated with the
company's patented "zinc-finger nucleases" (ZFNs). ZFNs are proteins made
up of "fingers" of about 30 amino acids, stabilized by a zinc atom. Each
finger binds to a specific combination of DNA bases and is attached to
nuclease, a DNA cutting enzyme. By using different combinations of amino
acids, they can be designed to bind to DNA at the exact site where the
gene is mutated to cut it out. This triggers the cell's repair mechanism,
which corrects the gene using a copy of the correct gene sequence
provided in a plasmid, in a process of homologous recombination, in which
the replacement depends on similarity in DNA sequence between the
replacement and the resident copy of the gene.

Infants with X-SCID have a mutated gene on their X- chromosome that makes
their immune system unable to function. More than 10 infants in the
Necker Hospital in Paris, France had been treated with conventional gene
replacement therapy since 2000 using a retrovirus as the vector (gene
carrier) to insert the correct gene sequence into their bone-marrow
cells. But the retroviral vector carrying the correct gene sequence
cannot be targeted, so it ends up inserting in wrong places in the
genome. To-date, three infants have developed leukaemia because the
retroviral vector inserted near an oncogene (cancer-related gene),
causing it to over-express, and the cell to multiple out of control. One
of the infants has died earlier this year.

The ZFNs are highly specific. Each finger recognizes 3-4 base pairs of
DNA via a single alpha-helix formed by the finger, and several fingers
can be linked in tandem to recognize a broad spectrum of DNA sequences
with high specificity. Earlier work from another laboratory has shown
that a zinc finger can be linked to a non-specific DNA- cutting domain of
a DNA-cutting enzyme to produce the ZFN, which then cuts specifically at
the zinc finger recognition site. An important feature is that two ZFNs
bind to the same gene, in a precise orientation and spacing relative to
each other, to create a double-strand break in the DNA, which then
triggers the repair mechanism.

Mathew Proteus at the University of Texas Southwestern Medical Center,
Dallas, Texas, a co-author of the Nature paper, had earlier used the
technique to correct a marker gene in human cells. But he only managed to
correct a few percent of the cells.

In the latest paper, they succeeded in modifying 18 percent of the cells
without the need to select for them with selectable markers such as
antibiotic resistance or fluorescent proteins. The advance was due to a
more elaborate combination of zinc fingers than used previously, which
are optimised for binding and cutting. A pair of four- fingered ZFNs,
each binding to 12 base pairs (24 in all), home in precisely on the
target between the pair of ZFNs, a mutation hotspot in the X-SCID gene,
and replacing it with the correct copy.

In one experiment, they isolated single clones of cells after giving them
the ZFNs and the correct copy of the gene, and found that 13.2% of the
clones had converted one of the two X-chromosomes, while 6.6% had both X
chromosomes corrected.

The researchers did other experiments confirming the findings, and
demonstrated that corresponding changes occurred in levels of mRNA and
protein expressed from the corrected gene.

The corrected gene sequence appeared to be stable for at least one month
afterwards, and analysis showed there was no gross mis-integration of
extra DNA or rearrangement or scrambling at the site of correction.

The company's aim is to take blood from patients, correct the genetic
defect in the blood cells and then infuse the cells back into the
patients. Besides X-SCID, other 'single gene' diseases such as sickle
cell anaemia or beta- thalassemia can also be treated, and perhaps immune
cells could also be altered to prevent infection with HIV.

Dana Carroll, a biochemist at University of Utah, Salt Lake City, who has
used ZFN to correct genes in fruit flies, said, "FN-induced gene
targeting places the normal gene at its normal chromosomal location,
where it should have no untoward genetic consequences." But he warned
that side- effects cannot be excluded.

Is it safer?

The results look quite impressive, and as pointed out in the Nature
article, "the 'hit and run' mechanism of ZFN action uncouples the
therapeutically beneficial changes made to the genome from any need to
integrate exogenous DNA, while still generating a permanently modified cell."

This new technique thus avoids all the hazards associated with the viral
vector and foreign gene constructs with aggressive promoter to force the
cells to express the foreign gene, and also appears to be specific: the
PCRs and Southern blots (which probe for the corrected gene sequences)
all look quite clean. Further tests that could have been performed are
genomic and expressed sequence microarrays, and protein gels, to see if
other genes have also been corrected and/or changes in the pattern of RNA
and protein expressed have occurred. It was microarray analysis that
first alerted the gene therapy community to the problems of the
'precision' gene therapy of RNA interference hailed as 2002's
"breakthrough of the year" ("Controversy over gene therapy
'breakthrough'", SiS 26; although
microarray analyses themselves are of questionable reliability ("Biotech
wonder tool in disarray", SiS 26

It would also be important to show that the corrected protein does not
cause side effects, such as immune rejection in patients whose bodies may
treat the protein as 'foreign'.


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