BUSINESS
Companies Designing Genetic Code Blocking Drugs To Treat Disease Small young research firms have set their sights on drugs that inactivate the genetic process to halt production of disease-causing proteins Ann M. Thayer, C&EN Northeast News Bureau
Advances in synthetic chemistry and molecular genetics in the past several years have led to new approaches to the d e v e l o p m e n t of therapeutic drugs. Whereas traditional drugs work to inhibit an enzyme or protein, these new approaches involve blocking genetic messages to turn off production of disease-causing proteins at the source. And, unlike gene therapy, which aims to insert needed genetic information, code blocking looks to inhibit malfunctioning or deleterious genes as a means to control disease. In the past few years, several small companies have started up around what is often described as the ultimate in rational drug design. Rational drug design looks for a clear and predictable relationship between a disease-causing target and a drug. Here the target is a nucleotide sequence on a single-stranded messenger RNA (mRNA), which encodes for disease-causing proteins, or doublestranded DNA from which mRNA is transcribed. Once a target sequence is determined, a complementary or an antisense DNA sequence that will bind and inactivate the genetic message or process can be synthesized. Genetic code blocking compounds are attractive drug candidates for several reasons. At 15- to 25-nucleotides long, an antisense oligonucleotide is expected to be highly selective in its ability to recognize and bind to its
target sequence. In fact, a single mismatch in complementary nucleic acid bases can reduce the affinity for hybridization by several orders of magnitude. This high specificity is expected to result in few side effects. And, as a significant number of diseases have genetic origins, there is the potential for a wide range of applications and sizable markets. (The current pharmaceutical market is estimated at $20 billion.) This fact has not been missed by venture capitalists who have recently been investing in young, researchoriented companies. Typical of these is Gilead Sciences of Foster City, Calif., formed in 1987 with $12 million in financing. Michael Riordan, founder, president, and chief executive of-
ficer, brought together the research expertise of Peter B. Dervan of California Institute of Technology, Douglas Melton of Harvard University, a n d H a r o l d W e i n t r a u b of Fred Hutchinson Cancer Research Center in Seattle. This s u m m e r Gilead signed a five-year, $20 million deal with the British drug firm, Glaxo. Glaxo will take a minority interest in the company and retain rights for any therapeutic agents developed for the treatment of cancer. Private companies like Gilead are working in the areas of viral and inflammatory diseases and oncogenebased cancers—diseases for which traditional therapeutics have not been overly successful. With relatively well-characterized genomes, virus-
How antisense oligonucleotide inhibits protein production - Protein
DNA produces messenger RNA (mRNA)
DNA-
Normal cell activity • mRNA produces protein
mRNA
v
y
Antisense oligonucleotide
Cell with antisense treatment • Antisense molecule binds to mRNA • Protein production is blocked
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Business es are considered prime candidates. Antisense drugs aim to inhibit the growth or replication of viruses, ei ther destroying them entirely or re ducing them to levels at which a body's defenses might be able to take over. Human immunodeficiency vi rus and herpes simplex and hepatitis Β viruses are frequent targets. In-vitro studies have shown that micromolar levels of code-blocking com pounds can inhibit such viruses and some oncogene expression. Although pursuing similar diseas es, companies are moving in differ ent directions with regard to cellular targets and synthetic oligonucleotide chemistry. Gilead's work is directed at both RNA- and DNA-targeting agents. Whereas single-stranded mRNA in a cell's cytoplasm is some times considered more accessible, DNA or gene targets in the nucleus offer the advantage of only one or two copies per cell rather than the thousands of copies of mRNAs. "Essentially, what we are trying to do is mimic the activity of proteins that already exist in the nucleus," says Riordan. "These transcription factors bind to specific gene promotor sites and very selectively regulate the activity of individual genes." However, the chemistry of forming a "triplex," that is, an oligonucleotide sequence that binds to double-stran ded DNA, generally is considered to be somewhat more challenging. Another firm, Triplex Pharmaceu tical, focuses on the triple-helix tech nology developed by Michael E. Hogan at Baylor College of Medi cine. Formed in 1989 and located in The Woodlands, Tex., the company has received about $2.5 million in venture capital financing. Research focuses on understanding the bind ing of triplex-forming oligonucle otides and their construction to opti mize stability and uptake into cells. Natural oligonucleotides are not expected to work well as therapeutic drugs because they are readily de graded by enzymes. Thus, research ers are investigating a variety of oli gonucleotide analogs. Modifications in chemical structure are being eval uated for their stability, ability to permeate cells, toxicity, and affinity to hybridize with targets. Companies are reluctant to discuss specific chemical modifications and 18
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Modified oligonucleotides block genetic code Modified oligonucleotides that bind to DNA or messenger RNA (mRNA) and prevent gene expression are used in genetic code blocking. These mole cules must be sequence-specific, sta ble, and compatible with cellular media. Inherently, an antisense sequence of nucleotide bases is complementary to and has high affinity for the targeted DNA or mRNA sequence. Changes in the phosphodiester sugar backbone of synthetic antisense DNA strands help to increase resistance to nucleases. First-generation compounds include the replacement of oxygen atoms in one or more phosphate groups. Such modifi cations also can alter an oligonucle otide's solubility in water, but may in crease lipophilicity and allow easier penetration of cell membranes. Other modifications being investigated are the replacement of one or more phosphate group linkages. Formacetal, carba mate, or sulfamate linkages have an added advantage in not creating chiral centers that make purification diffi cult. The addition of pendant groups
designs for oligonucleotides. Howev er, first-generation molecules that consisted of simple phosphate group derivatives are leading to more so phisticated oligonucleotide chemis tries. These include oligonucleotides, with enzymatic activity or ligands that can crosslink, that disable or de grade the hybrids they form with RNA or DNA. In analogy to tradi tional pharmaceutical products, and in contrast to drugs developed by biotechnology, the new classes of compounds being developed are very well defined chemically and structurally, explains Riordan, and therefore are expected to sustain strong patent protection. Isis Pharmaceuticals, Carlsbad, Cal if., is looking at one class of oligonu cleotide called phosphorothioates for use in antisense RNA applica tions. With the substitution of sulfur for a phosphate group oxygen, the phosphorothioate oligonucleotide hybridizes to mRNA and is resistant to nucleases. Phosphorothioate olig onucleotides can be synthesized by automated techniques. However, be
X 0 S CH3 OR
Phosphate group Phosphodiester Phosphorothioate Methylphosphonate Alkylphosphotriester [R = CH3, CH2CH3, CH(CH3)2]
Base = Adenine, guanine, cytosine, or
or other substitutions in the sugarphosphate or base structure are now becoming part of synthetic oligonucle otide chemistry.
ing anionic, like natural DNA, these compounds are water soluble but may not pass easily through lipoidal cell membranes. Isis has a partner ship with Applied Biosystems, a leading manufacturer of equipment for DNA synthesis. Isis was set up in early 1989 with $5.2 million in funding and an ini tial focus on acute diseases and those amenable to topical application. Its founders include Stanley Crooke, David Ecker, and Christopher Mirabelli from SmithKline & French Laboratories, the pharmaceutical re search arm of SmithKline Beecham. Crooke, now chief executive officer of Isis, had been president of R&D for SmithKline Beckman for six years, before the company was merged into SmithKline Beecham. In late October, Isis entered into two major collaborative agreements. A five-year, $30 million cooperative research agreement with Ciba-Geigy will focus on four molecular targets possibly involved in causing cancer, inflammation, dermatological dis eases, and viral infections. Ciba-Gei-
gy will receive exclusive worldwide marketing rights for all products except pharmaceuticals in the U.S. The second joint agreement is with Rhône-Poulenc and calls for joint pursuit of a "mutually selected molecular disease target" in the area of inhibiting the expression of oncogenes. Financial terms of the cooperative research program have not been made public, but Isis is to receive research funding, milestone payments, and domestic codevelopment rights. Rhône-Poulenc will hold an exclusive license for therapeutic antisense products discovered under the agreement, and Isis will receive royalties. Synthecell, Rockville, Md., is also investigating and developing phosphorothioate, as well as other, antisense oligonucleotides. Unlike the young venture-capital-based companies in this field, Synthecell began in August 1987 to develop and manufacture customized biomolecules including synthetic DNA, peptides, and small proteins. In working to supply its customers with synthetic biomolecules and oligonucleotides for research, explains James Hawkins, Synthecell president and chief executive officer, the company developed a profitable product base from which it now hopes to sustain long-term development in the area of antisense therapeutics. With a background that includes Baylor College of Medicine and the National Institutes of Health, Hawkins is directing the company toward near-term development of diagnostic and therapeutic oligonucleotides for RNA targets. Experienced in synthetic methods, the firm also is developing large-scale production and advanced synthetic techniques. Hawkins is also editor-in-chief of a new peer-reviewed journal, Antisense Research & Development. Focusing on another form of oligonucleotide chemistry is Genta, an early 1989 spin-off of the biotechnology DNA probe company, GenProbe. In two rounds of venture capital financing, the San Diego-based Genta raised $8.6 million. Founder Thomas Adams, now chairman and chief executive officer, was a founder and chairman of Gen-Probe. GenProbe, acquired by Chugai Pharmaceutical in early 1989 for $110 mil-
lion, retains an interest in Genta. Genta says that it is seeking corporate partnerships and multiyear, multimillion-dollar research agreements in exchange for marketing rights. Having licensed the methyl phosphonate oligonucletide chemistry developed by Paul Ts'o and Paul S. Miller at Johns Hopkins University, Genta is working to develop therapeutic agents for viral diseases and cancers. Methyl phosphonate oligo-
nucleotides have been shown to be effective antisense RNA agents against the herpes simplex virus in animal cells. Genta says it is conducting preclinical development of oligonucleotides for treatment of one viral and one cancer disease. It expects to be filing investigational new drug applications in the next 12 months, which will allow it to begin human clinical trials. Although animal studies are be-
To Reach The Top Of The Fine Chemicals Industry, We Started At The Base. How did Nobel Chemicals get to be a world leader in Nitration and Nitric Acid Oxidation? By continually expanding our base technologies. By providing complete programs from synthetic design to custom manufacture. And by perfecting "forward integration"— environmental, quality and cost control from raw material to intermediate to end product. In short, we became a world leading chemical supplier by doing what we do best. Creating pure chemistry. Call or write Nobel Chemicals for more information on these base technologies:
Condensation
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R3 Nobel Chemicals Nobel Industries Sweden Please contact: U.S. Outside of U.S. Nobel Chemicals, Inc. Fine Chemicals Division 1530 Valley Center Parkway S-691 85 Karlskoga Suite 110 Sweden Bethlehem, PA 18017, USA Phone: 46 586 83000 Phone: (215) 866-4100 FAX: 46 586 32147 FAX: (215) 867-4577 {CLE 17 ON READER SERVICE CARD December 3, 1990 C&EN
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Business coming more common, the success of genetic code blocking therapeutics has not yet been proven in humans. Most companies hesitate to estimate a time frame for drug development, although some observers suggest that it will be at least five to 10 years to complete the early stages, reach clinical trials, and enter the regulatory approval process. Multimillion-dollar investments and partnerships with large pharmaceutical firms will still be required, Hawkins suggests, as the small, young firms in the field have just started the development process. With only a handful of companies now, he anticipates as many as 15 to 20 players in the next two years. In addition, there are remaining scientific and technological hurdles, and possibly regulatory questions, that will have to be addressed for what is viewed as a potentially revolutionary new class of drugs that interact with the human genome. Genetic code blocking agents have yet to face the usual Food & Drug Ad-
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ministration tests. Some existing antiviral agents are based on nucleoside chemistry, but questions as to the impact of synthetic DNA oligonucleotides if incorporated into human genetic material are expected to arise. Assuming that gene sequences will eventually be determined and the mechanisms of complex genetic diseases discerned, the remaining scientific questions primarily center on effective delivery of code-blocking drugs to cellular targets in the human body. Effective delivery involves getting the necessary concentration of drug to the target site, having it permeate the cell, persist, and disable its target. One delivery route may be through the attachment of a cell-specific protein or peptide. Other routes include increasing membrane permeation and uptake—one commonly mentioned is encapsulation in liposomes—or choosing gene targets that have the most profound effect on disease and exist in the fewest copies.
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A related issue will be the ability to scale up oligonucleotide synthesis economically for large-scale production. By most estimates, dose requirements without cellular targeting, higher potency, or low-copy targets would have to be high to achieve effective drug concentrations. Extending the currently available research-scale synthetic technologies to produce amounts needed for animal or human studies, not to mention commercial production, is expected to be extremely timeconsuming and expensive. "If you look at the commercial challenges for this field/' says Riordan, "it all boils down to decreasing dose requirements or increasing potency." However, optimization of large-scale chemical synthesis techniques for DNA production is expected to be within the realm of drug-development expertise, says Hawkins, especially among the large pharmaceutical firms who are becoming partners with the small firms. D
