Gene Therapy a Step Closer to Mass Production

EUREKA project E! 3371 Gene Transfer Agents has made great advances in the development of novel non-viral carriers able to introduce genetic material into the target cells. These new agents, derivatives of cationic amphiphilic 1,4-dihydropyridine (1,4-DHP), avoid the problems of the recipient's immune system reacting against a viral carrier.

The project partners have developed methods to produce them in large amounts, which solves another of the problems with viral delivery. But the greatest advantage is that the new compounds are significantly more effective at delivering DNA into cell nuclei than other standard synthetic carriers; increasing the chance of the DNA successfully controlling the defective genes, and the disease.
Gene therapy involves the insertion of DNA into human cells within the body to treat disease. The technique is still in its early days, and has been demonstrated successfully only in the last decade. Most investigation has been into the possibilities for treating hereditary diseases related to a genetic defect, and the technique also has potential uses in treating the early stages of cancer, and in cardiovascular and neurodegenerative diseases.
Gene therapy faces many difficulties as a practical method; not the least of which is that DNA is a large and complicated structure which needs to be delivered and attached to the correct section of the recipient's set of DNA. A number of methods are in use or under investigation for introducing DNA into cells (a process known as transfection) -- using viruses, chemical agents or physical injection.
Viruses or chemical carriers
With viral carriers, the DNA to be introduced is injected into the virus, which carries it into the cell by way of a vesicle formed around the virus particle by the cell wall. Once inside the cell, the vesicle breaks down and the virus injects the DNA into the cell's nucleus. The viral route does, however, have major disadvantages. The immune system of the person receiving treatment often interferes with viral activity; and viruses can have unpredictable mutagenic side-effects. Also large-scale production of viral vectors is problematic.
A wide range of chemical agents are already known to be able to form a complex of 1,4-DHP with DNA and deliver it into the recipient's cells. These agents are much easier to produce on a large scale than viruses and do not usually cause an immune response. However they are not so effective at introducing the DNA as the viral carriers.
Seeking the best of both worlds
The challenge facing the partners in the EUREKA project was to combine the effectiveness of the viral vectors with the production advantages and lack of immune response shown by chemical agents. Scientists at the Latvian Institute of Organic Synthesis and the University of Kuopio in Finland had discovered new groups of possible DNA transfer agents: 1,4-DHP derivatives. These compounds were found to be more effective in gene transfer than two widely-used standard gene delivery agents (known as DOTAP and PEI 25) and the discovery was covered by a patent. This finding offered the exciting prospect of better efficiency from a non-viral carrier.
Professor Arto Urtti of Helsinki University (formerly from Kuopio) explains: "When these compounds are in solution and DNA is added, they bind together. The large, loose DNA molecule collapses and tiny particles of about 10-50nm in diameter are formed, composed of both DNA and carrier. When you present this to the cells, the nanoparticles bind to the cell surface, which folds inwards to form a vesicle within the cell. The particles then escape from the vesicle, releasing the DNA.
Researchers at Helsinki University found that out of all the compounds tested, the most effective were those which succeeded in transferring DNA into the nucleus. The mechanism by which the DNA enters the nucleus is not yet clearly understood, but it is known that gene transfer is more effective in cells which are actively dividing, e.g. cancer cells.
Dr Aiva Plotniece, Dr Arkadijs Sobolevs and their colleagues at the Latvian Institute then set out to synthesise dozens of different DHP derivative compounds. Dr Plotniece comments: "The great advantage of these compounds is the biologically active 1,4-DHP fragment, which with proper substitution, can show certain biological and physico-chemical properties. During the project we have designed different 1,4-DHPs, which allowed us to establish structure-activity relationships."
The third project partner, the independent Latvian chemical producer Bapeks, contributed its experience of larger-scale synthesis and advised the Latvian Institute researchers on how best to scale up the synthesis methodology. The compounds were then distributed to a number of other research colleagues in Latvia, Finland and Lithuania for further study. At present, project partners feel that the main uses will be in laboratory experiments, and much further research is needed before they can be used for gene transfer in the human body.
Partners in the EUREKA project believe that although more research is needed, the project has been very successful. "It was the first big, important project for us" says Dr Sobolevs. "We have significantly widened the potential uses of self-assembling 1,4-dihydropyridine derivatives into nanomedicine, gene delivery and even into drug delivery systems." The project team found that EUREKA support helped greatly in preparing, managing and reporting the project. It was also through EUREKA that the other partners were introduced to Bapeks.

Scientists 'Boot Up' a Bacterial Cell With a Synthetic Genome

Scientists have developed the first cell controlled by a synthetic genome. They now hope to use this method to probe the basic machinery of life and to engineer bacteria specially designed to solve environmental or energy problems.

Scanning electron micrographs of M. mycoides JCVI-syn1. Samples were post-fixed in osmium tetroxide, dehydrated and critical point dried with CO2 , then visualized using a Hitachi SU6600 scanning electron microscope at 2.0 keV.

The study will be published online by the journal Science, at the Science Express website, on May 20.
The research team, led by Craig Venter of the J. Craig Venter Institute, has already chemically synthesized a bacterial genome, and it has transplanted the genome of one bacterium to another. Now, the scientists have put both methods together, to create what they call a "synthetic cell," although only its genome is synthetic.
"This is the first synthetic cell that's been made, and we call it synthetic because the cell is totally derived from a synthetic chromosome, made with four bottles of chemicals on a chemical synthesizer, starting with information in a computer," said Venter.
"This becomes a very powerful tool for trying to design what we want biology to do. We have a wide range of applications [in mind]," he said.
For example, the researchers are planning to design algae that can capture carbon dioxide and make new hydrocarbons that could go into refineries. They are also working on ways to speed up vaccine production. Making new chemicals or food ingredients and cleaning up water are other possible benefits, according to Venter.
In the Science study, the researchers synthesized the genome of the bacterium M. mycoides and added DNA sequences that "watermark" the genome to distinguish it from a natural one.
Because current machines can only assemble relatively short strings of DNA letters at a time, the researchers inserted the shorter sequences into yeast, whose DNA-repair enzymes linked the strings together. They then transferred the medium-sized strings into E. coli and back into yeast. After three rounds of assembly, the researchers had produced a genome over a million base pairs long.
The scientists then transplanted the synthetic M. mycoides genome into another type of bacteria, Mycoplasm capricolum. The new genome "booted up" the recipient cells. Although fourteen genes were deleted or disrupted in the transplant bacteria, they still looked like normal M. mycoides bacteria and produced only M. mycoides proteins, the authors report.
"This is an important step we think, both scientifically and philosophically. It's certainly changed my views of the definitions of life and how life works," Venter said.
Acknowledging the ethical discussion about synthetic biology research, Venter explained that his team asked for a bioethical review in the late 1990s and has participated in variety of discussions on the topic.
"I think this is the first incidence in science where the extensive bioethical review took place before the experiments were done. It's part of an ongoing process that we've been driving, trying to make sure that the science proceeds in an ethical fashion, that we're being thoughtful about what we do and looking forward to the implications to the future," he said.
This research was funded by Synthetic Genomics, Inc. Three of the authors and the J. Craig Venter Institute hold Synthetic Genomics, Inc. stock. The J. Craig Venter Institute has filed patent applications on some of the techniques described in this paper.

How Ducks Host Influenza Unharmed: Could Findings Shield Humans from Bird Flu Viruses?

A University of Alberta-led research team has discovered an influenza detector gene that could potentially prevent the transmission of the virus to humans.

Mallard duck with fifteen ducklings. Researchers have identified the genetic detector that allows ducks to live, unharmed, as the host of influenza. 

Katharine Magor, a U of A associate professor of biology, has identified the genetic detector that allows ducks to live, unharmed, as the host of influenza. The duck's virus detector gene, called retinoic acid inducible gene -- I, or RIG-I, enables a duck's immune system to contain the virus, which typically spreads from ducks to chickens, where it mutates and can evolve to be a human threat like the H5N1 influenza virus. The first human H5N1 cases were in Hong Kong in 1997. Eighteen people with close contact to chickens became infected and six died.
Magor's research shows chickens do not have a RIG-I gene. A healthy chicken can die within 18 hours after infection, but researchers have successfully transferred the RIG-I gene from ducks to chicken cells. The chicken's defenses against influenza were augmented and RIG-I reduced viral replication by half.
One potential application of this research could affect the worldwide poultry industry by production of an influenza-resistant chicken created by transgenesis.
The work of Katharine Magor, her U of A PhD candidate Megan Barber, and researchers from the United States (Jerry Aldridge and Robert Webster) was published March 22, in the online, early edition of Proceedings from the National Academy of Sciences.