The broad-leafed tobacco plant has for centuries been smoked, chewed and sniffed, all leisure activities linked to serious health issues.

But work being done by St. Louis-based Chlorogen Inc. might change the reputation of tobacco for the better, introducing it as an important crop for efficiently making therapeutic proteins to combat gynecological cancers and other diseases.

"What we think is our sort of sweet spot is we’re focusing on some hard-to-make molecules, proteins that are difficult if not impossible to make to a commercial scale in other traditional systems," said David Duncan, Chlorogen’s president and CEO.

With $6 million in venture funding secured last month, the company is transitioning from two years of technology development to now applying its chloroplast transformation technology (CTT) to produce proteins for human therapy. Initially, it will focus on TGF-beta molecules, at least 30 of which are known to have therapeutic value. One candidate in particular appears to have an effect on ovarian cancer in animal models. But producing those proteins in mammalian cells, in yeast or in bacteria, is either not possible or not economical.

"These particular molecules tend to cannibalize, tend to kill the very cells that you try to grow them in, so the production is dramatically reduced," Duncan told BioWorld Today. "It’s very inefficient and very costly, but they don’t act that same way in the cellular component of the tobacco plant."

Chlorogen expresses its proteins through chloroplasts, unlike the more traditional method of creating them in the nucleus of the cell. While there is only one nucleus for every cell, there are about 100 chloroplasts in each cell and between 10 and 100 copies of DNA for every chloroplast. It is a high-expressing system compared to nuclear transformation and one that provides up to 10,000 opportunities to express a copy of a protein.

The company’s method of genetically transforming chloroplasts is significant because it addresses a major environmental concern about gene transfer.

"Tobacco is not a food or feed crop, and the chloroplasts are maternally inherited only," Duncan said, "so there’s no risk of the DNA getting into the pollen."

The seed is first produced in a greenhouse, and then taken out to a field. Chlorogen has experiment stations in Kentucky, South Carolina and Missouri and uses a few private farmers to do the work. A 10- to 15-acre tobacco crop could produce a year’s supply of drug, and only a few acres could produce as much protein as a $100 million factory, the company said.

Chlorogen holds a patent on a genetic sequence that instructs proteins, introduced with a gene gun, to function only within the chloroplasts. Genes that enter other regions of the cell, such as the nucleus, are not functional. The company also secured the only other competing property estate from Rutgers University, and now has all patents and pending patents covering the area of research.

The U.S. Department of Agriculture has endorsed Chlorogen’s CTT method of producing proteins, Duncan said.

"They’ve taken a hard look at the chloroplast transformation system," he said, "and believe it to be a preferred way to be in the field of producing proteins because it’s so environmentally friendly."

Tobacco is an ideal plant to use for expressing proteins because it is simple to transform, has a large biomass, can be harvested several times during a season and enables rapid scale-up since each plant can produce about 1 million seeds.

So far, Chlorogen’s method has expressed proteins such as human serum albumin for therapeutic and non-therapeutic applications, interferon for liver diseases, and insulin-like growth factor for diabetes and other diseases. Other potential products include vaccines against cholera, anthrax or plaque, as well as proteins for crop improvements, such as insect, herbicide, disease and drought resistance.

The platform "is an enabling technology," Duncan said. "As such, it really fits across a fairly broad application, such as vaccines for biodefense, proteins for agriculture, proteins for food."

Chlorogen said that protein-based drugs derived from plants have an advantage over chemical-based treatments in that they are produced more efficiently, are safer and cheaper. While mammalian cells are being isolated and replicated in fermenters, factories that do the work are quite expensive and produce only limited quantities of proteins. The inefficiencies may not only drive up the costs of drugs, but it may deter manufacturers from developing cures for rare diseases.

Since receiving $5.8 million through its first venture funding in May 2003, Chlorogen has built an operation of 11 employees and six consultants. Its investors include Burrill & Co. Inc., of San Francisco; Harris & Harris Group, of New York; Prolog Ventures, of St. Louis; and Redmont Venture Partners, of Birmingham, Ala. Joining those investors in the latest $6 million Series B round was Finistere Partners LLC, of San Diego.

While Chlorogen builds a therapeutic pipeline, it also is pursuing collaborations in the food, biopolymers and biodefense markets, and it has an out-licensing strategy for CTT.

Duncan hopes to have its first product ready to enter the clinic within two years, at which time it will seek a partner.

"Our forte will be in the early stages of development, expressing the protein, manufacturing the protein," he said, "but we will then look for a partner to take it forward through clinical testing."

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