ISIS Press Release 29/07/05
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
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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