Safe Gene Therapy At Last?

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29 Jul 2005 15:59:25 -0000

Safe Gene Therapy At Last?

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ISIS Press Release 29/07/05

 

Safe Gene Therapy At Last?

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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. http://www.i-sis.org.uk/full/SGTALFull.php

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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

http://www.i-sis.org.uk/isisnews.php).

 

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 http://www.i-sis.org.uk/isisnews.php);

although microarray analyses themselves are of questionable

reliability ( " Biotech wonder tool in disarray " , SiS 26

http://www.i-sis.org.uk/isisnews.php).

 

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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