HAMBURG, Germany — Larova Biochemie GmbH, of Teltow, Germany, focuses on the enzymatic production of nucleosides and nucleotides, the building blocks of DNA, and has just completed a DM11 million (US$6.7 million) financing round.

With new enzymatic technologies, Larova plans to synthesize chirally pure antisense oligonucleotides by the ton. Moving in a rapidly expanding market, the company is looking for industry partners and is already starting preparations for a second financing round.

The company, founded in 1992, is based on a surprising discovery. Biochemist Roxana Havlina found that several important steps of DNA metabolism are not catalyzed by different enzymes, but are carried out by a single multifunctional enzyme instead.

"I was working with Lactobacillus leichmannii," Havlina, founder of Larova, told BioWorld International. "And when I was studying the enzymes of the DNA metabolism in this organism, I noticed that several of the enzymes described in the literature were extremely similar in terms of physico-chemical properties such as molecular weight, iso-electrical point, etc. In addition, their kinetics and the way their activity was regulated had much in common."

After an additional year in the laboratory, Havlina proved that, in Lactobacillus, enzymatic reactions such as kinase, reductase, transferase, deaminase and polymerase activities were not performed by different proteins but by different activity centers of a single multifunctional enzyme.

Havlina then examined several commercially available, analogous enzymes from other organisms and found the same properties. "E. g., a highly purified DNA polymerase I from E. coli distributed by a big lab supplier has nucleoside kinase and adenosin desaminase activities as well," she said. "And there are a lot of other examples. So it is very clear that multifunctionality of this enzyme group is an universal phenomenon."

These findings not only have consequences for textbooks and enzyme nomenclature. "First, it is an important step towards a better understanding of DNA metabolism, especially DNA replication," Havlina said. "This could help to explain a lot of defects and disease mechanisms. Second, now, as we understand kinetics, activation and inhibition of this multifunctional enzyme, we can exactly mimic the sequence of activities that actually takes place in the cell. Hence, procedures become faster and more simple. Moreover, if you look at these multifunctional enzymes as most versatile tools it is easy to recognize that, by adding new substrates or co-enzymes, a lot of unimagined reactions become possible."

Havlina decided to set up her own enterprise and file for a patent on the new enzymatic tools. The European patent covering 12 countries was granted in December of last year. Patents in Japan and the U.S. are pending.

Currently, Larova uses the enzyme technology to produce nucleosides, nucleotides and oligonucleotides for customers in academia and commercial research. Oligonucleotides today are usually produced by chemical synthesis. This process leads not only to considerable amounts of solvent residues, but to unwanted byproducts and impurities. Using its enzyme technology, Larova produces chirally pure isomeric fractions with high purity. "The knowledge provides us with a considerable advantage over our competitors," Havlina said.

"Today, we produce on the order of several hundred grams, but we are negotiating with a possible partner to help us scale up to on the order of several tons," she said.

"Market demand for oligonucleotides is already big," Havlina added. "And, as antisense oligonucleotides, ribozymes, and aptamers are very valuable therapeutics, we expect the market to grow rapidly in the next couple of years." Natural nucleosides from fish sperm are source materials for antisense-oligonucleotide synthesis today, but supply is limited and not sufficient for ton-scale production.

Havlina said she is confident that the chemically modified nucleotides that form the basis of the second-generation antisense oligonucleotides can be produced enzymatically by Larova's technology in high quality and quantity as well. Currently, they can be synthesized in very poor yield only.

In June, Larova closed a DM11 million private financing round with KBB-Kapital-Beteiligungsgesellschaft, of Potsdam, Germany, and Technologie-Beteiligungs-Gesellschaft, of Bonn, Germany. Additional funds from the federal Bundeministerium fur Bildung und Forschung (BMBF) and a U.S. venture capital company to support expansion to the continental U.S. In order to develop industrial scale production technologies, Larova will enter a second financing round soon.

Together with companies like Bayer AG, of Leverkusen, Germany, and Lion Biosciences AG, of Heidelberg, Germany, Larova participates in the BMBF-sponsored "protein structure production site," a project to build an automated protein fermentation and purification factory.

Larova employs a staff of 13 and expects to grow to 100 employees by 2005. "Our aim is to become one of the world's leading research and production sites in the area of nucleic acid enzyme technology," Havlina said. *

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