Biotech century ending?
This miniseries charts the further collapse of the biotech empire, particular in the supposedly ‘highly lucrative’ biomedical sector since the latter part of 2000. It is now desperately grasping for support from the taxpayer by hyping genetics and bio-defence. Don’t be fooled.
- Genetics & Bio-Defence Research Rescue Biotech Slump
- Gene Therapy Risks Exposed
- Death Sentence on Cloning
- Pig Organ Transplants Dangerous & Costly
- Animal Pharm Folds
Gene Therapy Risks Exposed
First test it on patients then study the risks.
Dr. Mae-Wan Ho and
Prof. Joe Cummins report on the
damning revelations from studies that should have been carried out before
patients were treated.
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Geneticist Mark Kay and his team at Stanford University examined a viral
vector that has been used in gene therapy trials of haemophilia and cystic
fibrosis. It turns out that the virus has the potential to cause the same
problems that led to leukemia in the severe combined immune deficiency
SCID trial in Paris last year. The study involves a different vector, made
from the adeno-associated virus (AAV), not known to cause disease in
humans. But they now show that the vector integrates itself more often
into genes than other regions of DNA.
Unlike the retrovirus used as a vector in the SCID trials, which tend to
integrate into the cell’s genome, AAV integrates much less often.
Nevertheless, geneticists can’t be sure that it does not cause cancer
when it does integrate.
By extracting DNA from liver cells from mice injected with the AAV
vectors, the team found 72% of the integrations were into a region
containing a gene. If it were random, the vector would have interrupted a
gene no more than 40% of the time.
In addition, chromosomal deletions of up to 2kb were detected at all 14
integrations sites examined. Most of the deletions were less than 0.3kb.
All the genes targeted by the AAV vectors were expressed. There was a
preference for introns (non-coding regions of genes) over exons (coding
regions).
The same tendency to integrate into genes has been discovered earlier
for retroviruses such as HIV-1. Studies on integration in vitro
have found that DNA binding proteins, bound to target DNA, can block
integration by obstructing access of integration complexes. In contrast,
DNA bending proteins such as nucleosomes (protein complexes that wind and
package DNA strands into chromatin) can actually promote integration. On
the nucleosome, the positions of maximal DNA distortion were particularly
favoured for integration. The researchers infected a human lymphoid cell
line with HIV or an HIV-based vector, and cloned 524 junctions between
viral and cellular DNA. The sequences were then determined and mapped on
the human genome sequence. As a control, 111 sites were generated by
integration in vitro into naked human DNA and their genomic
distribution compared with the in vivo integration sites.
Genes were found to be clearly preferred integration targets in vivo
but not in control naked DNA. There was a strong correlation between gene
activity and integration targets, particularly for genes that were active
in cells after infection with HIV vector. Hotspots for integration were
also detected, including a 2.5kb region that contained 1% of the
integration events. Some 69% integration sites were in gene regions, a
highly significant departure from random. For the in vitro naked
DNA control, 35% were in transcription units (gene regions). The human
genome is about 33% transcription units, so the frequency of integration
in genes in vitro was not significantly different from random.
The integration sites tend to cluster. No clustering was found in the
control. There were seven regional hotspots of 100kb, four of which
contained a gene. High local gene density correlated with all regional
hotspots. The targeted genes in all four cases were active, and all
increased in activity after infection by 2 to 3 fold.
HIV integration was favored in Alu elements (short mobile
genetic elements that are now increasingly recognized to be playing a key
role in ‘natural genetic engineering’ the genome). That may be
because Alu elements are enriched in gene-rich regions.Within
genes, integration favored in introns over exons.
Thus, sites of HIV integration in the human genome are not random, but
enriched in active genes and regional hotspots, probably due to increased
accessibility to chromosomal DNA in transcribed regions, or it may be
promoted at active genes by favourable interactions between the ‘pre-integration
complex’ of the HIV and locally bound transcription factors.
Such results are making researchers seek better ways to target vectors
to specific regions of DNA, and to develop vectors that don’t
integrate into DNA at all. Kay says that he has taken numerous precautions
to protect the 14 haemophilias he has treated. Why didn’t he do the
experiments before treating the patients?
Meanwhile, a team led by Shun’ichi Kuroda of Osaka University,
reported in the June 29 advance online issue of Nature Biotechnology
that they used yeast to produce nanometer-scale (average 80nm) hollow
vesicles bound by lipid membranes studded with hepatitis B virus (HBV)
envelope L protein to act as gene delivery vehicles. These particles are
readily purified by ultra-centrifugation, and are free of viral genomes.
Their hollow interiors can be stuffed with transgenic DNA or drugs by
electroporation (creating temporary holes in the membrane using an
electric field). These were tested both in vivo and ex vivo.
A plasmid expressing green fluorescent protein (GFP) was introduced into
the L particles by electroporation, and used to transfect various human
cancer cells. Only human liver carcinoma cell lines HepG2 and NuE took up
the vesicles with an efficiency of nearly 100%. However, another human
carcinoma cell line, PLC/PRF/5, which releases HBV surface antigen
particles containing the L protein, could not be transfected with the
L/GFP particles. When injected into mice carrying graft of tumours, the
L/GFP particles were only taken up by tumours derived from NuE cells, but
not in tumours derived from non-liver cell lines.
When the human F9 gene encoding clotting factor IX was
incorporated into the L particles and injected intravenously into mice
carrying a tumour derived from different cells lines, plasma collected
showed only mice carrying the liver carcinoma NuE tumour synthesize the
blood clotting protein, and continued for at least a month. By changing
the protein on the surface of the particles, it was possible to target
delivery of drugs or DNA to other cell types. One potential obstacle to
using the method, Kuroda admits, is that although the particles appear to
be nontoxic, they are "somewhat immunogenic". That’s
probably an understatement, given that similar vesicles were used in an
HBV vaccine by the same team.
Opinion is divided as to whether this gene therapy delivery system is
as safe as it seems. It could reduce many of the well-known risks of viral
vectors; above all, the generation of infectious viruses and insertion
into genes that could trigger cancer. Furthermore, viral vectors cannot be
targeted to specific cells lines, and can also induce potent inflammatory
immune responses.
Kuroda is reported to be developing a "stealth version" of
the L particles to minimize the immune response. However, there is still a
lot we don’t know about the immune response, and previous attempts to
manipulate it has all too often ended in making things worse.
For example, a specifically designed myelin basic protein peptide was
used in attempted immunotherapy of multiple sclerosis in a phase II
clinical trial in 2000. The peptide was poorly tolerated, and the trial
had to be halted. Three patients developed exacerbations of multiple
sclerosis.
One nagging question remains in the present study. In the in vivo
experiment in mice successfully transfected with L particles
expressing human blood clotting factor, the protein started disappearing
on day 30, and by day 42, had vanished completely. What happened to the
cells that had taken up the L particles? What happened to the mice? The
toxicity test carried out consisted of five four-week old mice injected
with 500ug of the L particles expressing the human clotting factor 9,
which were reported to have "survived for more than 2 weeks,
indicating that the median lethal dose (LD50) is >20mg/kg." That
meant the mice did die. Is that reassuring? What did the mice die of?
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