By Dean A. Haycock

Special to BioWorld International

Are antisense therapeutics ever going to make financial sense? After years of research and hope, only one antisense agent has been approved. The reason for this disappointing progress does not appear to lie in the rationale behind the technology.

The instructions for synthesizing proteins - both healthy and disease - reside, of course, in DNA. The code is transcribed into RNA before being translated into proteins. Antisense therapeutics are designed to prevent the message from passing from DNA to RNA, before faulty proteins are synthesized. They consist of sections of nucleic acids, called oligonucleotides, that bind complementary sequences of DNA or RNA and prevent them from carrying their encoded messages to the cell's protein synthetic machinery. As a result, disease proteins never get made.

The problems with this clever approach, it turns out, are practical, not conceptual. Antisense agents have not been very successful in the “real world,“ where they have proven to be unstable.

In an attempt to make antisense drugs more stable, chemists gave them a new backbone in the early 1990s. They replaced the sugar phosphate backbone present on all nucleic acids with one made of peptides. These peptide nucleic acids (PNAs) were, as hoped, more stable.

Michael Egholm, a co-inventor of PNA at the University of Copenhagen, in Denmark, and now a group leader at PE Biosystems Inc. (formerly PerSeptive Biosystems), of Framingham, Mass., remembers the response that met the announcement of this type of improved antisense reagent.

“Basically, we were completely overrun by pharmaceutical companies,“ Egholm said. PNAs seemed to be able to solve a lot of the problems that antisense therapeutic developers faced then.

“It was the first analogue that was shown to bind [nucleic acid] with high affinity. It also increased specificity and was not broken down,“ Egholm recalled.

Unfortunately, PNAs were reluctant to cross cell membranes. Their improved stability in the bloodstream offered little therapeutic advantage. They could not make their way into cells to access the RNA which they were designed to bind and inhibit. Interest in PNAs as antisense agents fizzled out, although work continued on their use as tools in molecular biology.

'Sort Of An Obvious Experiment'

Interest in the potential therapeutic applications of PNAs has now been rekindled with the publication of a paper, “Cell penetrating PNA constructs regulate galanin receptor levels and modify pain transmission in vivo,“ in the September issue of Nature Biotechnology. Co-author Ulo Langel, an associate professor at Stockholm University, in Sweden, and his colleagues linked PNAs to naturally occurring “transporter“ peptides, called transportan and pAntennapedia (also known as penetratin-1), that carry molecules through cell membranes.

“It is sort of an obvious experiment to do but it has never been done before. Once you do that, you actually get a very effective antisense probe,“ Egholm said.

The authors demonstrate in both cultured cells and whole animals the advantage of linking peptide transporter proteins to PNAs. They used an antisense PNA directed against RNA that carries the code for the galanin receptor, a molecule that plays a part in the perception of pain. When linked to transportan or penetratin-1, the antisense PNA more effectively enters cells and reduces expression of galanin receptors in cultured cells, compared to PNAs not linked to transporter peptides.

The researchers also succeeded in showing that antisense PNA-penetratin constructs injected directly into the spinal cords of rats effectively decreased the number of galanin receptors in this part of the nervous system. They further demonstrated, using electrophysiological methods, that rats with fewer galanin receptors showed a corresponding decrease in pain responses.

Previously, PNA antisense reagents have been shown to work following injection into cells. This paper is the first to demonstrate that they work following injection into animals.

If, as it appears, the PNA-transporter peptide constructs work by blocking cellular messenger RNA, the approach could have widespread application in the lab and eventually in the clinic, Langel said.

“The first goal of this paper was to show that it was possible to use PNA as antisense, and this, I think, we have shown now,“ Langel told BioWorld International.

Egholm believes the paper has provided “strong indirect proof that this is an antisense mechanism.“ A planned collaboration between the two research groups may prove the mechanism within the next year.

Targets To Include Cancer, Brain Cells

Immune responses to PNA may not present a problem, according to Langel, because low concentrations are effective. Another potential problem with the use of PNA-transporter protein constructs is their lack of specificity. They are not choosy about which cells they enter.

“We will start in October a collaboration in the European community on this penetration problem. One of the ideas is to try to make them cell-selective,“ Langel said. Targets will likely include cancer cells and brain cells.

There are other important applications of the constructs. PNAs designed to specifically block RNAs encoding different proteins could be used to study various biochemical pathways in cells and provide basic knowledge about cell biology and potential new drug targets. And transporter proteins may provide viable means for delivering genes and other therapeutic molecules into cells.

The researchers now are attempting to extend their research to include other antisense targets. They have begun collaborating with PE Biosystems, a division of Perkin-Elmer Corp., of Norwalk, Conn.

Perkin-Elmer owns the patent concerning cell-penetrating PNAs invented by Langel and his co-workers. “Where we at Perkin-Elmer see a use for PNAs in the future is as a means of target validation,“ Egholm said.

In this application, PNAs could serve as less complex but specific alternatives to knockout mice. Lowering the level of a target protein more closely simulates the action of a drug than completely eliminating the entire gene that encodes it.

“[These data show] it is a good technique for introducing PNA, but we also know we can introduce into cells not only PNA but other peptides and, we hope, even small proteins,“ Langel said. *

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