Science in Society Archive

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.

  1. Genetics & Bio-Defence Research Rescue Biotech Slump
  2. Gene Therapy Risks Exposed
  3. Death Sentence on Cloning
  4. Pig Organ Transplants Dangerous & Costly
  5. 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.

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?

Article first published 30/09/02



Sources

  1. Nakai H, Montini E, Fuess S, Storm TA, Grompe M and Kay MA. AAV serotype 2 vectors preferentially integrate into active genes in mice. Nature Genet. 2003, 34, 297-302
  2. "Harmful potential of viral vectors fuels doubts over gene therapy", Ericka Check Nature 2003, 423, 573-4.
  3. Schroder ARW, Shinn P, Chen H, Berry C, Ecker JR and Bushman F. HIV-1 integration in the human genome favors active genes and local hotspots. Cell 2002, 110, 521-9.
  4. Yamada T, Iwasaki Y, Tada H, et al., Nanoparticles for the delivery of genes and drugs to human hepatocytes," Nature Biotechnology, DOI:10.1038/nbt843, June 29, 2003.<http://www.nature.com/nbt/>

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