By Dean Haycock

Special to BioWorld International

The approach seems almost too simple but it works. Purified or “naked“ DNA, free from protein, can evoke a response from the immune system.

Although no one can explain exactly how naked DNA does it, it has been known for nearly a decade that when injected into an animal's muscle, the DNA can induce an immune response. The type of immune response is determined by the kind of cell the DNA enters. For this reason, there are perhaps more effective ways to deliver DNA vaccines than injecting them into muscle.

One suggestion involves inserting selected DNA into bacteria that are capable of getting into mammalian cells. Once inside, the bacteria would drop off the instructions for making foreign proteins. In theory, these cells will churn out proteins that will induce the immune system to produce long-lasting immunity.

Many disease-causing bacteria, of course, are capable of entering cells. Vaccine developers have asked, why not use pathogenic bacteria in attenuated forms to deliver selected DNA to humans? Several research groups are working on such delivery systems. They have used attenuated Salmonella, a common cause of food poisoning; Shigella, a cause of severe intestinal disorders; and Listeria, a cause of blood poisoning and meningoenchephalitis, as vectors for delivering naked DNA to animal cells or mice. The results suggest that the approach could be effective for delivering potential DNA vaccines.

But not everyone is comfortable with this strategy. Bacteria have a way of reinventing themselves. There is concern that a bug that has been weakened may change back into a killer. It is easy to appreciate this concern. The infectious dose for Shigella is so low that ingestion of as few as 10 to 100 wild-type pathogenic bacteria has produced disease in volunteers.

It would make a lot of people less nervous to use a non-pathogenic bacterial species to deliver DNA. Catherine Grillot-Courvalin, a researcher at the Institut Pasteur, in Paris, and her colleagues describe doing just that in their September Nature Biotechnology paper, titled “Functional gene transfer from intracellular bacterial to mammalian cells.“

E. Coli Entered Mammalian Cells

To engineer a suitable non-pathogenic, “safe“ bacteria, the authors used Escherichia coli bacteria that could not synthesize all the components of its cell wall. These hobbled mutants are more likely to die than to turn virulent. The French scientists prepared the E. coli by inserting a gene for a protein called invasin into them. Normally, invasin enables another bacterium, Yersinia pseudotuberculosis, to invade cells. Expression of the invasin gene in E. coli, which normally cannot enter cells, gave them invasive ability. This ability was, of course, tempered by the inability of the E. coli to synthesize essential components of its cell wall.

Grillot-Courvalin and her co-authors successfully demonstrated that the genetically altered E. coli can enter three types of cultured mammalian cell. After entry, the bacteria conveniently die and release their cellular contents. Among these contents are circular, extrachromosomal bits of DNA called plasmids.

Plasmid DNA, which occurs in many strains of bacteria, replicates separately from bacterial chromosomes. While not essential for bacterial survival, plasmids often provide bacteria with an advantage such as resistance to antibiotics. These small, independent DNA “donuts“ have become important tools for molecular geneticists. Genetic engineers insert genes into them, load them into bacteria and harvest the protein products produced.

In this instance, researchers from the Institut Pasteur transformed E. coli with plasmids containing genes for marker proteins that would indicate the successful delivery of DNA. The markers were a green fluorescent protein (GFP) or beta-galactosidase, an enzyme. Mammalian cultured cells, including human HeLa cells, produced one of these two foreign proteins after being co-cultured with the genetically engineered bacteria. The tell-tale green glow of GFP or the evidence of beta-galactosidase activity indicated that the bacteria delivered the foreign DNA and that the mammalian cells expressed it. This was demonstrated in both dividing cells and quiescent ones. The implication is that DNA that encodes proteins capable of eliciting an immune response in mammals - a DNA vaccine - could be delivered in the same manner.

Low-Motility Plasmid Employed

“This system,“ wrote David Bermudes, associate director of biology at Vion Pharmaceuticals Inc., in New Haven, Conn., “constitutes a new cloak for introducing DNA with vaccine potential.“ Bermudes' comment appears in the same issue of Nature Biotechnology.

To decrease the chances of the genetically modified E. coli passing invasin plasmids onto naturally occurring E. coli, and so making them capable of entering cells, the authors used a plasmid that does not travel very well among bacteria.

The next step is to demonstrate that the modified E. coli can induce an immune response in living animals. “Other people have used Listeria or Salmonella to do that, [and] there is no reason why we should not be able to do that with our E. coli,“ Grillot-Courvalin told BioWorld International.

The technique, of course, has potential for delivering therapeutic genes as well as DNA vaccines. One set of experiments, to be done in collaboration with researchers from Transgene, a private company in Strasbourg, France, will study the effect of intranasal administration of DNA bearing bacteria. Transgene's expertise at detecting gene delivery in the lung should help determine if the new approach might be useful for delivering therapeutic genes to epithelial cells in the lung. In their paper, the authors suggest that the technique might be applied to Burkholderia cepacia, a bacteria that attacks patients with cystic fibrosis (CF).

Since these bacteria can enter epithelial cells, it might be possible to use genetically modified versions to deliver copies of the normal cystic fibrosis transmembrane conductance regulator gene to CF patients in whom the gene is mutated.

The Institut Pasteur has applied for patent protection to cover both DNA vaccine delivery and gene therapy applications of the work described in the report by Grillot-Courvalin and her co-authors. *