Patent Docs

Special Edition of Scientific American Offers Global Biotechnology Perspective

By Donald Zuhn --

Scientific American has released its second annual examination of the ways in which governments, scientists, industry players, and life science stakeholders are advancing biotechnology innovation around the globe.  The special edition of the consumer science magazine, entitled "Worldview: A Global Biotechnology Perspective," explores biotech's most compelling trends and developments worldwide.

The issue, which was prepared in collaboration with the Biotechnology Industry Organization (BIO), includes the Worldview Scorecard, a list of innovation-capacity scores for 39 countries.  Also included in the issue are a number of articles, such as "Back From The Brink," which looks at economic signs around the world that indicate an improving environment for biotechnology, and a series of articles examining biotechnology in China.

The Worldview Scorecard ranks countries according to their capacities to develop biotechnology, and presents data on a wide range of other topics, including the impact of public policies on biotechnology.  Overall innovation-capacity scores were computed by assessing each country's performance in several individual metrics (e.g., intellectual property protection) on a scale from 0 to 10, with the lowest-ranked country scored as 0 and the highest-ranked country scored as 10 (an explanation regarding the methodology employed by the editors can be found here).  The top dozen countries on this year's scorecard (with final scores) were:

1. United States - 37
2. Singapore - 31
3. Canada - 29
4. Sweden - 28
5. Denmark - 27
5. Finland - 27
5. Israel -27
8. France - 26
8. Iceland - 26
8. Japan - 26
8. Netherlands - 26
8. Switzerland - 26

With regard to IP scores, the United States received the top score (10.00) and Belgium, Canada, Denmark, Finland, France, Ireland, Italy, Japan, and the Netherlands all received scores of 9.05.  The lowest IP scores were given to Iceland (3.82), Brazil (4.18), Russia (4.59), India (4.95), and Mexico (5.49).  A full list of scores can be viewed here.

Rep. Kucinich Introduces Legislation Related to Genetically Engineered Organisms

    By Donald Zuhn --

Last week, Representative Dennis Kucinich (D-OH) (at right) introduced three bills in the House related to the labeling of food containing genetically engineered material, the cultivation and handling of genetically engineered crops, and the establishment of a set of farmer rights regarding genetically engineered animals, plants, and seeds:

• The "Genetically Engineered Food Right to Know Act" (H.R. 5577) would amend the Federal Food, Drug, and Cosmetic Act, the Federal Meat Inspection Act, and the Poultry Products Inspection Act to require that food that contains a genetically engineered material, or that is produced with a genetically engineered material, be labeled accordingly.  The bill states that "[t]he process of genetically engineering foods results in the material change of such foods," and asserts that "Federal agencies have failed to uphold Congressional intent by allowing genetically engineered foods to be marketed, sold and otherwise used without labeling that reveals material facts to the public."  The bill, which has been referred to the House Committees on Agriculture and Energy and Commerce, was co-sponsored by Rep. Peter DeFazio (D-OR), Barney Frank (D-MA), Raul Grijalva (D-AZ), Barbara Lee (D-CA), Jim McDermott (D-WA), Pete Stark (D-CA), and Lynn Woolsey (D-CA).

• The "Genetically Engineered Safety Act" (H.R. 5578) would "prohibit the open-air cultivation of genetically engineered pharmaceutical and industrial crops, . . . prohibit the use of common human food or animal feed as the host plant for a genetically engineered pharmaceutical or industrial chemical, . . . establish a tracking system to regulate the growing, handling, transportation, and disposal of pharmaceutical and industrial crops and their byproducts to prevent human, animal, and general environmental exposure to genetically engineered pharmaceutical and industrial crops and their byproducts, [and] amend the Federal Food, Drug, and Cosmetic Act with respect to the safety of genetically engineered foods."  Noting that "[m]any of the novel substances produced in pharmaceutical crops and industrial crops exhibit high levels of biological activity and are intended to be used for particular medical or industrial purposes, under very controlled circumstances," and that "[n]one of these substances is intended to be incorporated in food or to be spread into the environment," the bill declares that the risks of contamination "necessitate a zero tolerance standard for the presence of pharmaceutical crops and industrial crops and their byproducts in crops used to produce human food or animal feed."  The bill, which has been referred to the House Committees on Agriculture and Energy and Commerce, was co-sponsored by Rep. Peter DeFazio (D-OR), Barney Frank (D-MA), Raul Grijalva (D-AZ), Barbara Lee (D-CA), Pete Stark (D-CA), and Lynn Woolsey (D-CA).

• The "Genetically Engineered Technology Farmer Protection Act" (H.R. 5579) would "provide additional protections for farmers and ranchers that may be harmed economically by genetically engineered seeds, plants, or animals, . . . ensure fairness for farmers and ranchers in their dealings with biotech companies that sell genetically engineered seeds, plants, or animals, [and] assign liability for injury caused by genetically engineered organisms."  The bill states that "[p]olicies promoted by biotech corporations, such as patenting of seeds, depriving farmers the right to save seed, unreasonable seed contracts, and intrusion into everyday farm operations, have systematically acted to remove basic farmer rights enjoyed since the beginning of agriculture and essential for agricultural sustainability and the survival of family farms," and asserts that "[t]he introduction of genetically engineered crops has also created obstacles for farmers, including the loss of markets and increased liability concerns."  The purpose of H.R. 5579 is "[t]o mitigate the abuses upon farmers, a clear set of farmer rights must be established."  Among the rights that would be created by the bill are (1) a requirement for biotech companies to fully disclose the risks of using genetically engineered animals, plants, or seeds, and (2) the prohibition of certain terms and limitations in contracts for the sale of genetically engineered animals, plants, or seeds, including a provision that would "prohibit[] the purchaser from retaining a portion of the harvested crop for future crop planting by the purchaser or that charge[] a fee to retain a portion of the harvested crop for future crop planting."  H.R. 5579 would allow for a maximum $100,000 penalty to be assessed for violations of the Act.  The bill, which has been referred to the House Agriculture, Energy and Commerce, and Judiciary Committees, was co-sponsored by Rep. Peter DeFazio (D-OR), Barney Frank (D-MA), Raul Grijalva (D-AZ), Barbara Lee (D-CA), Pete Stark (D-CA), and Lynn Woolsey (D-CA).

In a press release posted on Rep. Kucinich's website, the Congressman stated that:

To ensure we can maximize benefits and minimize hazards, Congress must provide a comprehensive regulatory framework for all Genetically Engineered products.  Structured as a common-sense precaution to ensure [Genetically Engineered] foods do no harm, these bills will ensure that consumers are protected, food safety measures are strengthened, farmers’ rights are better protected and biotech companies are responsible for their products.

The release noted that Rep. Kucinich had introduced similar bills in previous sessions of Congress.

Not Quite Artificial Life, But We're Getting Closer: Reactions to Venter's Synthetic Cell

    By James DeGiulio --

Recently, we reported that a research team led by Dr. Craig Venter had developed a "synthetic cell" controlled by purely synthetic DNA (see "Dr. Craig Venter Creates First Cell Controlled Entirely by Synthetic DNA").  To evaluate the significance of the discovery, the journal Nature asked eight synthetic-biology experts about the implications for science and society of the "synthetic cell" made by the Dr. Venter's research team ("Life after the synthetic cell").  Generally, most commentators downplayed the gravity of Dr. Venter's discovery, most finding the synthetic cell as falling short of "artificial life."  The following is a summary of each expert's commentary, to illustrate the variety of reactions to Dr. Venter's synthetic cell:

• Dr. Mark Bedau, professor of philosophy and humanities at Reed College in Oregon, noted four issues to keep in mind when forming opinions on Venter's discovery.  First, we now have an unprecedented opportunity to learn about life.  Second, even the simplest forms of life have unpredictable, emergent properties.  Third, these new powers create new responsibilities, since we cannot be sure about the consequences of making new forms of life, and we must "expect the unexpected and the unintended."  Finally, Dr. Bedau notes that we are moving ever-closer to creating life.  Dr. Bedau also notes "a prosthetic genome hastens the day when life forms can be made entirely from nonliving materials."

• Dr. George Church, a geneticist at Harvard Medical School, is of the view that no artificial life was created.  Dr. Church notes that the new bacterium is not changed from the wild-type state in any fundamental sense.  Dr. Church relies on the metaphor that "[p]rinting out a copy of an ancient text isn't the same as understanding the language."  He notes that geneticists already had the ability to make synthetic DNA and get it to function in cells, and that Venter's discovery was exercising that ability on a grander scale.  Dr. Church was interested, however, in the research potential of trimming down genomes, as Dr. Venter did, in order to understand what elements of the genome are essential for speed, efficiency, and robustness.

• Dr. Steen Rasmussen, a professor of physics at the University of Southern Denmark, disputes even calling the discovery a "synthetic cell" because Dr. Venter has only rewritten the genetics "program" running on the "hardware" of the modern cell.  Dr. Rasmussen contrasts the "top-down" approach, which Dr. Venter's discovery represents, and "bottom-up" approaches, which Dr. Rasmussen practices.  According to Dr. Rasmussen, a true synthetic cell can only be created using the "bottom-up" approach, which aims to "assemble life -- including the hardware and the program -- as simply as possible, even if the result is different from what we think of as life."

• Dr. Arthur Caplan, a professor of bioethics at the University of Pennsylvania, stands out from the other commentators by ruling the magnitude of Dr. Venter's discovery as immense, declaring it as "one of the most important scientific achievements in the history of mankind."  Dr. Caplan's commentary is more philosophical than scientific, describing the achievement as undermining a fundamental belief about the nature of life -- that no manipulations of the inorganic would permit the creation of any living thing.  Dr. Caplan fears Dr. Venter's achievement would seem to "extinguish the argument that life requires a special force or power to exist."

• Dr. Steven Benner, of the Foundation for Applied Molecular Evolution, viewed the magnitude of the discovery as mere "synthesis" -- a research strategy that can be applied to any field in which technology allows scientists to design new subject matter.  Dr. Benner finds the discovery analogous to chemical synthesis.  Nonetheless, Dr. Benner was impressed with the discovery, rejecting it as a trivial enhancement of previous gene synthesis.  Dr. Benner also noted the potential to resurrect species of ancient bacteria, so that evolutionary sciences, among others, may benefit from the work.

• Dr. Martin Fussenegger, a professor of biotechnology and bioengineering at ETH Zurich, finds that Dr. Venter's discovery is a "technical tour-de-force," but not much of a conceptual advance.  Indeed, chimera organisms have long been created through breeding and through the transfer of native genomes into denucleated target cells.  Dr. Fussenegger does note, however, that this latest technology will increase the speed with which new organisms can be generated, a thought that he acknowledges as "discomforting."

• Dr. Jim Collins, professor of biomedical engineering at Boston University, echoes the commentary downplaying the advancement as revolutionary.  His view is that Dr. Venter has made an important advance in our ability to re-engineer organisms, yet it does not represent the making of new life from scratch.  Dr. Collins finds Dr. Venter's microorganism to be analogous to a patient who has received an artificial, synthetic heart.  Dr. Collins admits that scientists simply do not know enough about biology to create life.  He states:  "It is like trying to assemble an operational jumbo jet from its parts list -- impossible."

• Dr. David Deamer, professor of biomolecular engineering at University of California, Santa Cruz, also states that Dr. Venter has not created artificial life, noting that the cytoplasm of the recipient cell, among other things, is not synthetic.  However, Dr. Deamer is encouraged that the discovery has the potential for understanding the "origin of life" as we know it.  Dr. Deamer notes that currently "all life arises from existing life.  But perhaps not for much longer."

As can be seen, it appears generally that Dr. Venter's discovery is not going to incite the bioethical pushback it potentially could have, for most scientists surveyed in the Nature article have recognized that DR. Venter has not actually "created" life.  Fortunately, the popular media has also noted this distinction.  In a May 30th New York Times editorial entitled "One Cell Forward," Dr. Venter's discovery of a "synthetic cell" was lauded as "overstated," because it "makes it sound as though [Dr.] Venter had constructed the entire cell, molecule by molecule.  What he has done is create a synthetic genome -- the longest string of DNA to be assembled in a laboratory -- and place it in a bacterium."  Nonetheless, the editorial recognizes that Dr. Venter's discovery is another step closer to artificial life, albeit not as large of a step as claimed, and recognizes that "[t]his newest step in [D]r. Venter's research brings a fresh urgency to the debate -- and the need for some profound decisions."

Patent Docs will continue to update our readers on the debate and decisions regarding Dr. Venter's "synthetic cell" technology.

For additional information regarding this and other related topics, please see:

• "Venter Denies Synthetic Cell Discovery Is Artificial Life, Vatican Agrees," June 6, 2010
• "Dr. Craig Venter Creates First Cell Controlled Entirely by Synthetic DNA," June 1, 2010
• "Playing the Bioterror Card in the Synthetic Biology Debate," December 19, 2007
• "The Synthetic Biology Sky Is Not Falling," December 16, 2007
• "Patenting Life (Really)," June 11, 2007

James DeGiulio has a doctorate in molecular biology and genetics from Northwestern University and is a third-year law student at the Northwestern University School of Law.  Dr. DeGiulio was a member of MBHB's 2009 class of summer associates, and he can be contacted at degiulio@mbhb.com.

Venter Denies Synthetic Cell Discovery Is Artificial Life, Vatican Agrees

    By James DeGiulio --

Last week, we reported that a research team led by Dr. Craig Venter had developed a "synthetic cell" controlled by purely synthetic DNA (see "Dr. Craig Venter Creates First Cell Controlled Entirely by Synthetic DNA")  Dr. Venter and co-author Dr. Daniel Gibson have written an op-ed piece that was recently published in The Wall Street Journal, entitled "How We Created the First Synthetic Cell," which describes their scientific advance and addresses some of the ethical issues involved.  Perhaps fearing a bioethical backlash, the two researchers vehemently deny that they have created life, instead suggesting that they "transformed existing life into new life."  Drs. Venter and Gibson describe their research process as using digitized information to synthesize a modified version of the "naturally occurring" Mycoplasma mycoides genome.

As a historical example, Drs. Venter and Gibson reference the 1967 discovery by Nobel prize winner Dr. Arthur Kornberg (at left), who successfully copied the phiX174 virus DNA.  The authors note that President Lyndon Johnson, at that time, hailed Dr. Kornberg's work as "a very spectacular breakthrough," yet accurately predicted "[i]f you think about some of these decisions the present president is making -- it is going to be a kindergarten class compared to the decisions some future president is going to have to make."  Indeed, President Obama, in a letter last week to the chair of his new bioethics commission, wrote:  "This development raises the prospect of important benefits, such as the ability to accelerate vaccine development.  At the same time, it raises genuine concerns, and so we must consider carefully the implications of this research."  Drs. Venter and Gibson close the piece by welcoming and encouraging such review and dialogue.

The Vatican, after evaluating the methodology of Dr. Venter's creation of the "synthetic cell," has reached the same conclusion put forth by the researchers:  that the discovery is not "creating life" and thus does not move beyond the church's bioethical boundaries.  Furthermore, perhaps surprising to some, the Vatican has publicly praised the discovery.  The Vatican newspaper L'Osservatore Romano published an article calling it an "interesting result" and was optimistic that the development could help cure disease.  Importantly, rather than considering the development as creating life, the article considered that the researchers instead had merely "replaced one of its motors," and that "the DNA, even though it is an excellent engine, is not life."

Though the Vatican's response may appear surprising, the church actually does not officially oppose genetic engineering, so long as the science avoids embryonic stem cells, cloning, or anything else that dabbles in the re-creation of human life.  Nonetheless, the Vatican recognizes that the Venter discovery has taken a substantial step closer to creating life.  Despite the praise in the article, the Venter research team was encouraged to "join courage with caution," when moving forward into such delicate territory.

Patent Docs will report on the scientific community's response to Dr. Venter's "synthetic cell" discovery in a later post.

James DeGiulio has a doctorate in molecular biology and genetics from Northwestern University and is a third-year law student at the Northwestern University School of Law.  Dr. DeGiulio was a member of MBHB's 2009 class of summer associates, and he can be contacted at degiulio@mbhb.com.

Dr. Craig Venter Creates First Cell Controlled Entirely by Synthetic DNA

    By James DeGiulio --

After a 15-year effort, the team of Daniel Gibson (at right), Hamilton Smith, and Craig Venter (below left) have created the first cell controlled by a purely synthetic genome.  In a press release and accompanying press conference, Dr. Venter described the converted cell as "the first self-replicating species we've had on the planet whose parent is a computer."

As reported in the May 20 issue of Science, in an article entitled "Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome," the research team at the J. Craig Venter Institute in Rockville, Maryland designed 1,078 specific cassettes of DNA that were 1,080 base pairs long and ordered them from DNA synthesis company Blue Heron Biotechnology.  To ensure that the assembled genome would be recognizable as synthetic, four of the ordered DNA sequences contained strings of bases that, in code, spell out an e-mail address, the names of the people involved in the project, and a few famous quotations from James Joyce and other authors.  The team then assembled the shorter lengths into a complete Mycoplasma mycoides genome.  The researchers transferred the synthetic genome into a recipient cell from a different bacterial species, replacing the endogenous DNA, and the cell became entirely controlled by the new synthetic genome.  The cell was essentially converted into a different species, for the finished product was capable of replication and had all the expected properties of a Mycoplasma mycoides bacterium.

The cost of the project was $40 million, most of it paid for by Synthetic Genomics, a company Dr. Venter founded.  Not surprisingly, Venter has applied for several patents covering the work, assigning them to his company.  Synthetic Genomics has a contract from Exxon to generate biofuels from algae.  However, the bacterium used by the Venter group is unsuitable for biofuel production, and Dr. Venter said the team would move on to more suitable organisms.  Dr. Venter and his colleagues hope to design new bacteria that will perform useful functions, such as production of new fuel molecules, new food oils, and new biologically derived sources of plastic and chemicals.  Dr. Venter predicted that the first application the world may see could be as soon as next year.

Dr. Venter's "synthetic cell" has the potential to rejuvenate nagging questions of ethics, law, and public safety about artificial life that bio-ethicists have been debating for more than a decade.  However, some scientists downplayed the development, finding it as merely a technical accomplishment, since the new cell can be viewed as an organism with a synthetic genome, not as a synthetic organism.

President Obama has asked the White House bioethics commission to report back to him on the significance of this development, and Nature has published a series of opinions on Dr. Venter's article from synthetic biology experts.  Patent Docs will address the commentary and additional reactions to Dr. Venter's discovery in future posts.

For additional information regarding this and other related topics, please see:

• "Playing the Bioterror Card in the Synthetic Biology Debate," December 19, 2007
• "The Synthetic Biology Sky Is Not Falling," December 16, 2007
• "Patenting Life (Really)," June 11, 2007

James DeGiulio has a doctorate in molecular biology and genetics from Northwestern University and is a third-year law student at the Northwestern University School of Law.  Dr. DeGiulio was a member of MBHB's 2009 class of summer associates, and he can be contacted at degiulio@mbhb.com.

GSK Promotes Open Innovation by Sharing Malaria Inhibitor Compounds

    By James DeGiulio --

Earlier this year, GlaxoSmithKline announced an "open innovation" strategy to help deliver new and better medicines for diseases disproportionately affecting people living in the world's poorest countries.  Making good on that promise, GSK has teamed up with the European Bioinformatics Institute (EMBL-EBI), the U.S. National Library of Medicine (NLM), and Collaborative Drug Discovery (CDD) to make freely available more than 13,500 compounds that could ultimately lead to new treatments for malaria.

The data was generated by screening GSK's library of 2 million compounds for those that demonstrate potent inhibition of the most deadly malaria parasite, Plasmodium falciparum, found primarily in sub-Saharan Africa.  Kinase inhibitors constituted a large proportion of the most potent compounds.  The full results of the study are part of the cover story published in the May 20 issue of Nature ("Thousands of chemical starting points for antimalarial lead identification")

According to the GSK press release, the release of this data marks the first time that a pharmaceutical company has made the structures of so many compounds available at no cost to researchers.  With the structure of the compounds and information about how they affect the malaria parasite, scientists can utilize these compounds for drug discovery or to study their mechanism of parasite inhibition.

The sharing of this data is an example of recent "open source" tactics being applied to drug discovery.  EMBL-EBI will act as the primary repository for the data on this compound set, and will index further information that is deposited.  GSK will add more data as it is generated and external scientists researching these compounds will be asked do the same.  The data on the compounds can be found at the following links:

• European Bioinformatics Institute (EMBL-EBI)
• Pubchem
• Collaborative Drug Discovery

James DeGiulio has a doctorate in molecular biology and genetics from Northwestern University and is a third-year law student at the Northwestern University School of Law.  Dr. DeGiulio was a member of MBHB's 2009 class of summer associates, and he can be contacted at degiulio@mbhb.com.

Gene Therapy Experiencing a Revival

    By James DeGiulio --

After over a decade of suspicion following the death of an 18-year-old patient, gene therapy is experiencing a revival among scientists and drugmakers, according to a recent Bloomberg report ("'Dead as Doornail' Gene Therapy Revival Spurs Genzyme").  Since the patient's death in 1999, scientists have linked many of the safety problems with gene therapy to the modified adenovirus vectors used to deliver the gene.  Current trials utilize a new generation of gene delivery vectors, such as adeno-associated virus (AAV), which trigger a milder immune response than the adenovirus used in previous studies and are consequently much safer.

Recent successes have rejuvenated the gene therapy field.  The Bloomberg report highlights Genzyme, which has invested more than $200 million over the past 10 years on gene therapy technology.  Genzyme's recent Parkinson's disease trial showed a dopamine gene injected into patients' brains effectively reached the targeted nerve cells in the region of the brain controlling movement.  This is an important discovery because efficient gene delivery is a major limitation of gene therapy.  Genzyme is preparing to test the therapy in a larger trial using AAV that could begin next year.

Other recent successes have been reported in high impact factor journals.  The November 6 issue of Science contained a report that showed brain damage in two 7-year-olds with an inherited condition called adrenoleukodystrophy was halted or reversed using gene therapy ("Hematopoietic Stem Cell Gene Therapy with a Lentiviral Vector in X-Linked Adrenoleukodystrophy").  In this study, doctors removed the patients' blood-forming stem cells, inserted gene-bearing viruses into them, and reinfused the patients with the modified cells.

The November 7 issue of The Lancet reported that delivery of a therapeutic gene to patients with Leber's congenital amaurosis resulted in significant vision recovery, with the most improvement noted in children ("Age-dependent effects of RPE65 gene therapy for Leber's congenital amaurosis: a phase 1 dose-escalation trial").  In this study, genes were delivered directly to target cells using AAV virus injected into patients.

Large drug companies are forming partnerships to further gene therapy development.  In January, Novartis licensed gene treatments for hearing loss and balance disorders from GenVec in a deal worth up to $213 million.  In February, startup Tacere Therapeutics announced that Pfizer would license its hepatitis C treatment in an agreement valued at as much as $145 million.

Other trends show the industry's renewed interest in gene therapy.  According to the FDA, there are currently 354 U.S. gene therapy studies under way, up from only 116 worldwide in 2008.  A Jain Pharmabiotech report released this month indicates that a total of 189 companies are currently developing gene therapies, up from only 44 in 1995.

However, despite the recent scientific successes, gene therapy is unlikely to produce revenue for any U.S. company for several years, as no gene therapies have been approved for actual medical use.  Only China has approved gene therapies for two types of cancers.  Nonetheless, there is no shortage of economic optimism.  An October 2008 report from Global Industry Analysts predicts gene therapy products will have revenue of $465 million in 2015.

James DeGiulio has a doctorate in molecular biology and genetics from Northwestern University and is a third-year law student at the Northwestern University School of Law.  Dr. DeGiulio was a member of MBHB's 2009 class of summer associates, and he can be contacted at degiulio@mbhb.com.

GSK Senior Vice President to Start IP Consulting Firm

    By Donald Zuhn --

Sherry Knowles, Senior Vice President, Global Patents for GlaxoSmithKline has announced that she will be leaving GSK in June to start a consulting firm that will focus on IP management, business development, and monetizing assets in the area of pharmaceuticals, biopharmaceuticals, and medical devices.  Ms. Knowles (at right), who has led Global Patents at GSK since 2006, played a significant role in the successful challenge to the claims and continuation rules published by the U.S. Patent and Trademark Office in August of 2007, helping to secure a preliminary injunction enjoining the rules on Halloween in 2007, which injunction was made permanent by the U.S. District Court for the Eastern District of Virginia on April 1, 2008.  Last fall, the USPTO finally rescinded the controversial rules package before the Federal Circuit was to rehear GSK's and Dr. Tafas' appeal en banc.  Ms. Knowles has also been centrally involved in developing GSK's Knowledge Pool for the treatment of neglected tropical diseases in least developed countries.

Managing IP Magazine named Ms. Knowles one of the ten most influential people in Intellectual Property in 2008, and earlier this year, the New Jersey Intellectual Property Lawyers Association awarded Ms. Knowles, as the representative of GSK, the Jefferson Medal for exceptional contribution to Intellectual Property.  In February, Ms. Knowles published an article in the journal Science on "Fixing the Legal Framework for Pharmaceutical Research" (see "Takings' by Congress and the Courts").

Commenting on her upcoming move, Ms. Knowles told Patent Docs that she has thoroughly enjoyed her tenure at GSK and has been "very impressed with the caliber and devotion of the GSK staff in creating and protecting the market for new medicines."  She said she plans to continue her "strong support for the innovator community to help create an environment that fosters and protects investment."  Ms. Knowles noted that she will continue to lead Global Patents until June, and will continue to consult with GSK, subject to availability and the company's needs, after she departs.

For additional information regarding this and other related topics, please see:

• "'Takings' by Congress and the Courts," April 1, 2010
• "GSK Announces 'Open Innovation' Strategy to Combat Neglected Tropical Diseases," February 3, 2010
• "Alnylam to Donate RNAi Patents to Patent Pool," August 10, 2009
• "'New Rules' Officially Rescinded," October 8, 2009
• "Managing Intellectual Property Releases MIP 50," July 28, 2008
• "No April Fool's Joke -- Tafas and GSK Win on Summary Judgment," April 1, 2008
• "GSK Secures Injunction," October 31, 2007

This Just In -- The Anti-commons Aren't So Tragic

    By Kevin E. Noonan --

The problem, according to the academics and the pundits, is that patent exclusivity in certain areas (such as gene patents) could result in a "tragedy of the anti-commons," inhibiting innovation.  One of the many manifestations of this tragedy, according to such august bodies as the National Academy of Science and the Federal Trade Commission, was expected to be an increase in litigation as patentees protected their exclusive rights.  A related issue is the exposure to patent infringement liability for university researchers in the aftermath of the Madey v. Duke case, where the Federal Circuit ruled that university research did not fall under the "common law" experimental use exemption enunciated by Judge Story in Whittemore v. Cutter, 29 F. Cas. 1120, 1121 (C.C.D. 1813), because university research was not performed for "for amusement, to satisfy idle curiosity or for strictly philosophical inquiry," but as part of the university's "business objectives" (teaching and research).  The fear of this liability has led to proposals for basic research to be entitled to a broad exemption.

The underlying grounds for both types of concerns have once again been found to be non-existent, in a study published in the April 4th edition of Nature Biotechnology.  The study, by Ann E. Mills and Patti M. Tereskerz from the Center for Biomedical Ethics and Humanities, program in Ethics and Policy in Heath Systems at the University of Virginia, and entitled "Empirical analysis of major stem cell patent cases: the role of universities" is limited to stem cell patents and litigation involving such patents.  The authors identified these patents as being relevant to questions about the existence of an anti-commons tragedy because such patents were expected to become more important with the policy change on embryonic stem cell research by the Obama administration, and because "there are few or no alternatives to patented technologies that enable embryonic or adult stem cells to be directed into specific cell lineages" and "[t]he interdependence of the technologies involved with these stem cell patents and the fragmentation of their ownership across many organizations could make the task of coordinating access to key technologies an intensive as well as costly process, and so create an anti-commons in the field."

The study builds on earlier work by Bergman and Graf (2007, Nat. Biotechnol. 25:419–24), who indentified three International Patent Classes where most stem cell patents can be identified.  Mills and Tereskerz reviewed these IPCs from U.S. Patent and Trademark Office records for the number of patents in the PTO database in each class claiming stem cells and the number of these patents involved in litigation.  The authors report identifying 67 cases involving 23 patents, where one patent (U.S. Patent No. 5,352,605, owned by Monsanto) was involved in 32 lawsuits.  Of the three IPCs, one (C07K 14/peptides having more than 20 amino acids; 605 relevant patents) showed no patents involved in litigation, while the other two (C12N 5/undifferentiated human, animal or plant cells, e.g., cell lines; tissues; cultivation or maintenance thereof; culture media, 1,163 relevant patents, and C12 15/mutation or genetic engineering; DNA or RNA concerning genetic engineering vectors, their isolation, preparation or purification, 2094 relevant patents) showed no statistically-significant difference in litigation when the fact that some patents are involved in multiple litigations was taken into account.

Turning to the specifics of the parties, 7/67 cases involved university plaintiffs, and 5/28 plaintiffs were universities (18%).  In contrast, only 1/89 defendants were universities.  One plaintiff, Monsanto, showed a decided preference for suing on "older" patents, with U.S. Patent No. 5,352,605 being litigated 32 times, and U.S. Patent 4,940,835 being litigated three times.  Excluding cases involving Monsanto as a plaintiff, there were between one and five litigated cases per year between 1986 and 2007 involving patents from these IPCs, with the trend being markedly downward since 1998.

The authors conclude from their data that for these (stem cell) patents, "the rate of litigation to issued patents in the categories studied is extremely small, which calls into question the claim that a large amount of litigation is causing an anti-commons" (while noting that a patent from these IPCs that was litigated tended to be litigated several times).  The authors also note that, contrary to the popular belief that universities would be defendants, in a significant number of cases universities were plaintiffs, defending their own intellectual property.  (Not noted in the main body of the article was whether industry defendants were involved in suits where universities were plaintiffs, a result expected if university protection of intellectual property was being used to prevent commercial expropriation of technology developed by universities with public monies.)  In this regard, seven of twelve cases involving patents that had been litigated multiple times had university plaintiffs.

Regarding the effects of the Madey decision, the authors state that "it appears that the post-Madey fears of opening a floodgate of litigation against universities is not being realized at the moment."  The authors also compare their results with the results of two earlier studies by Walsh and colleagues (National Research Council, Committee on Intellectual Property Rights in Genomic and Protein Research and Innovation, Reaping the Benefits of Genomic and Protemic Research: Intellectual Property Rights, Innovation, and Public Health, National Academies Press, 2006; and Walsh et al., in Patents in the Knowledge-Based Economy (eds. Cohen W. & Merrill, S.) 285–340 (National Academies Press, Washington, DC, 2003).  These studies showed that university researchers were not sued by patentees for performing basic research (a result supported by several other studies, including Walsh et al., 2003, "Science and the Law: Working Through the Patent Problem," Science 299: 1020; Walsh et al., 2005, "Science and Law: View from the Bench: Patents and Material Transfers," Science 309: 2002-03; Straus, 2002, Genetic Inventions, Intellectual Property Rights and Licensing Practices; Nicol et al., 2003, Patents and Medical Biotechnology: An Empirical Analysis of Issues Facing the Australian Industry, Centre for Law & Genetics, Occasional Paper 6; Nagaoka, 2006, "An Empirical Analysis of Patenting and Licensing Practice of Research Tools from Three Perspectives," presented in OECD Conference in Research Use of Patented Inventions, Madrid).

The authors cite the Walsh studies for the proposition that patentees in industry "welcomed" academic research, because it advanced knowledge about the patented technology.  Indeed, traditional concerns were cited by scientists as being greater impediments than patents:  "'unreasonable terms for obtaining research inputs' was cited by 10% of survey respondents and 'too many patents covering needed research inputs' was cited by only 3% of survey respondents as a reason for project abandonment."

The authors conclude as follows:

We find no evidence that Madey is unleashing a floodgate of litigation in the areas we studied, despite the worry voiced by the three reports cited above.  We also find that for the stem cell patents studied, universities are proactive (and have been before Madey) in protecting their intellectual property, supporting the Madey contention that the boundary between industry and academia is blurring.

And yet, like an urban legend, the myth that university researchers are at risk for patent infringement liability, or that patents cause university researchers to abandon research areas for fear of litigation, or that there is a tragedy of the anti-commons, persist, despite all evidence to the contrary.  The only rational conclusion is that these fantasies support a political agenda that is antithetical to patenting.  Recognizing that these problems do not exist may be the first step in rejecting the several solutions proffered for these purely political reasons.

For additional information regarding this and other related topics, please see:

• "Science Progress Article Examines Impact of Gene Patents on Research," December 21, 2009
• "The Tragedy of a Bad Idea," August 25, 2009
• "Author of Nature Article Provides Rebuttal; Patent Docs Provides Surrebuttal," March 26, 2009
• "Genetic Diagnostic Testing: The 'Anti-Commons' Revisited?" March 25, 2009
• "The Effects of Intellectual Property Protection on Agricultural Research: Patents Are Not the Problem," February 3, 2009
• "Newsweek Joins the Anti-patent Bandwagon," January 26, 2009
• "Science Article Should Help Allay Gene Patenting Fears," December 17, 2008
• "Once Again, The New Yorker Gets It Wrong on Patents," August 10, 2008
• "Once Again, The New Yorker Gets It Wrong on Patents," August 7, 2008
• "BIO Report Indicts 'Patent Reform' Proponents," February 13, 2008
• "The True Tragedy of the Anti-Commons," October 8, 2007
• "The Future of DNA Patenting," February 20, 2007
• "The 'Anti-Commons' Aren't So Tragic, After All," October 27, 2006

"Takings" by Congress and the Courts

    By Kevin E. Noonan --

In a recent article in the journal Science, Sherry Knowles, the Chief Patent Counsel at GlaxoSmithKline, raises several interesting questions regarding the concept of "takings" of property in the patent context.  In a "Policy Forum" article entitled "Fixing the Legal Framework for Pharmaceutical Research," Ms. Knowles (at right) tackles two aspects of such takings regarding branded drugs.  First, she reviews the provisions of the Hatch-Waxman Act that permit a generic drug maker to use an innovator company's safety and efficacy data to support an Abbreviated New Drug Application (ANDA) (calling these provisions of Hatch-Waxman the "forced data-sharing requirement").  Second, she reviews the effects of several Supreme Court and Federal Circuit cases (including KSR International Co. v. Teleflex, Inc., In re Bilksi, and Ariad Pharmaceuticals, Inc. v. Eli Lilly and Co.) on the validity of patents and the consequences of the retroactive application of these rulings on issued patents.  In each case, Ms. Knowles suggests, companies that have invested substantial sums in developing innovative drugs should be entitled to recompense from the government when their data or their patent protection is taken.

With regard to Hatch-Waxman, Ms. Knowles cites the increased costs of bringing a new drug to market today (approximately $1.2 billion) and in the early 1980's (approximately $230 million), the time when Hatch-Waxman was enacted.  She also sets forth the differences in timeframe that innovators and generic drug manufacturers are burdened with.  For the innovator, this involves "10-15 years of pre-approval R&D after a patent application is filed."  In return, an innovator can recoup up to five years of additional patent protection, capped at 14 years after drug approval.  The generic drug maker is constrained by a much shorter period:  an ANDA filer is prohibited from using an innovator's safety and efficacy data for 5 years after drug approval.  Ms. Knowles characterizes this period as a "political compromise" arrived at during negotiations over the Hatch-Waxman Act, but one that was "without substantial supporting economic analysis."  "This period is vastly inadequate today," she says, "and rarely covers the breakeven point for reimbursement of R&D costs.  Because of this, new drugs are almost never developed without patent protection, which limits the number of new drugs created (as not all useful drugs are patentable)."

In those instances where the Hatch-Waxman regime forces an innovator drug company to share its "expensive clinical research data" with a generic drug company, under circumstances where patent protection does not keep the generic drug off the market, Ms. Knowles asks whether the consequent loss of revenue is a "taking" under the Fifth Amendment?  (While the takings clause of the Fifth Amendment is restricted by its terms to takings by the government, the lines between public and private takings have been blurred by the Supreme Court's decision in Kelo v.  City of New London, where the Court held valid the taking of private property by a public entity for the benefit of another private entity.)  The extent of the taking of private data under Hatch-Waxman having "increased sixfold" over the past twenty years, Ms. Knowles asks whether an increase in the data exclusivity period (which would mitigate the deleterious effects of Hatch-Waxman on return of investment) would not be fair.

Ms. Knowles also criticizes the 14-year cap limitation of Hatch-Waxman on patent term extension.  She notes that patents on inventions such as the "Beerella" (U.S. Patent No. 6,637,447) enjoy a longer term than a patent on a metastatic breast cancer therapy (U.S. Patent No. 6,713,485).  This is hardly a societally-useful outcome.  Increasing the data exclusivity period would mitigate these deficiencies of how Hatch-Waxman has been put into practice (in view of our 26 years of experience with the Act and its consequences).

Turning to court decisions that may constitute a taking, Ms. Knowles notes that "[a] patent application filed today on a pharmaceutical innovation will not be used to defend a marker for about 15 years."  Under these circumstances, the state of the law over that time will have significant effects on the capacity of the patent to protect the innovator drug franchise.  While Congress could enact legislation changing the law, this is generally done prospectively, while court decisions apply retrospectively.  While both actions "change the rules of the game," court decisions have a much more drastic effect, since they may "disrupt well-settled expectations, which can affect the outcome of long-term business decisions."

Ms. Knowles then discusses the changes in the law occasioned by the KSR (obviousness), Bilski (patentable subject matter), and Ariad (written description) cases.  Each one of these decisions (or the prospect of these decisions, since the piece was written before the Federal Circuit decided Ariad en banc, and the Supreme Court has yet to decide Bilski) has had the effect of reducing the certainty or expectation of patent protection ex post facto.  She then summarizes the effects of retroactive application of these (and other) judicial decisions:  "[t]hey can facilitate the increase of challenges by generic producers under Hatch-Waxman, . . . stop R&D investment decisions that have already been made and could prevent a product from reaching the market because of patentability issues."  Ms. Knowles also mentions the potential to prevent R&D to take place at all.

These effects can constitute a taking, and the effects of such decisions should be analyzed according to takings law:

An issued patent is property just like a piece of land or a house (citing Consolidated Fruit-Jar Co. v. Wright, 84 U.S. 92, 96 (1872) and Patlex Corp. v. Mossinghoff, 758 F.2d 594, 599 (Fed. Cir. 1985)).  When a federal judicial decision dramatically changes the law, such that a valid patent becomes invalid, has the federal government taken private property in violation of the federal Constitution?  Application of the Fifth Amendment is not clearly limited to legislative and executive action; nothing in its text bars extension of the takings clause to judicial action.

"The time has come," Ms. Knowles contends, "to rethink the right legal framework to promote and protect investment in pharmaceutical research and development."  This rethinking involves increasing the term of data exclusivity under Hatch-Waxman from 5 to 14 years, and for Congress to give appellate courts discretion to have a decision have prospective effect only, should it overrule prior interpretation of patent law.  The benefit of these changes would be to "substantially increase the number of drugs in the R&D pipeline, which would greatly benefit patients and ultimately benefit generic drug companies."

Before wags jump to mention that these changes would also benefit pharmaceutical companies like GSK, it would be well to consider where novel therapies will come from without companies with experience in obtaining (and financing) regulatory approval of such drugs, protecting them with patents, and having the business resources to bring such drugs to market.  It is certainly the case that these companies are for-profit (it's capitalism, after all), but the costs of R&D and regulatory approval, as well as scale-up, production, and distribution are realities that cannot be ignored.  It would be well to remember that, whatever other criticisms may be raised against the pharmaceutical industry, there is no other way for consumers to get new drugs.  It is also good to remember that innovation is not what generic drug companies do (generally), and that without innovator biotechnology and pharmaceutical companies, generic drug makers would have nothing to copy or sell.

House Bill Seeks to Codify President's Stem Cell Order

    By Donald Zuhn --

On Tuesday, Representative Diana DeGette (D-CO) introduced legislation (H.R. 4808) that would amend the Public Health Service Act to provide for human embryonic stem cell research.  H.R. 4808 would codify President Obama's Executive Order 13505, which permitted federal funding of research conducted with human embryonic stem cell lines.  It was therefore fitting that the bill was introduced on the one-year anniversary of the President's reversal of limits imposed by the Bush Administration on embryonic stem cell research (see "President Obama to Lift Stem Cell Limits on Monday" and "President Obama Reaffirms Faith in Science").

Rep. DeGette and bill co-sponsor Rep. Michael Castle (R-DE), who joined the President for last year's announcement concerning the stem cell Executive Order, introduced two stem cell bills (H.R. 872 and H.R. 873) in the House last year.  While the text of the new bill has not yet been made available, both Rep. DeGette and Rep. Castle issued press releases regarding the legislation.

In Rep. DeGette's press release, the Congresswoman said the Stem Cell Research Advancement Act would "ensure a lasting ethical framework overseeing stem cell research at the National Institutes of Health."  Rep. DeGette (at right) noted that since President Obama issued his Executive Order, the NIH had approved 43 stem cell lines.  According to the Congresswoman's press release, H.R. 4808 would provide strong ethical requirements for embryonic stem cell research, require the NIH to maintain guidelines on all human stem cell research and review those guidelines at least every three years, ban the use of federal funding for human cloning under the NIH guidelines, and require a biennial report to Congress on stem cell research.

In his own press release, Rep. Castle (at left) stated that he "continue[s] to share in the view of so many scientists that stem cell research holds great promise for alleviating the suffering of the 100 million American patients living with devastating diseases for which there are no good treatments or cures."  While noting that "[t]he President's Executive Order lifted restrictions that allowed important research to move forward," Rep. Castle contended that "Congress must act to ensure that an over-arching ethical framework is signed into law."

Other co-sponsors of the bill include Rep. Tammy Baldwin (D-WI), Lois Capps (D-CA), Russ Carnahan (D-MO), Charles Dent (R-PA), Gene Green (D-TX), Mark Kirk (R-IL), James Langevin (D-RI), and Ed Perlmutter (D-CO).  On Wednesday, H.R. 4808 was referred to the House Committee on Energy and Commerce, where Rep. DeGette is the Vice Chair and Rep. Baldwin, Capps, and Green are members.

Gates Foundation Seeks to Make This "The Decade of Vaccines"

    By Donald Zuhn --

Last month, Bill and Melinda Gates kicked off the new decade by announcing that their Foundation will be pledging $10 billion over the next ten years to help research, develop, and deliver vaccines to the world's poorest countries.  The announcement came at the World Economic Forum's annual meeting in January.

Noting that "[v]accines already save and improve millions of lives in developing countries," Mr. Gates (at right) declared that "[w]e must make this the decade of vaccines."  He also noted that "[i]nnovation will make it possible to save more children than ever before."  In addition, Mr. and Mrs. Gates urged governments and corporations to increase vaccine investment.  To date, the Bill & Melinda Gates Foundation has provided $4.5 billion for vaccine research, development, and delivery.

Using a model developed by a consortium led by the Institute of International Programs at the Johns Hopkins Bloomberg School of Public Health, the Foundation was able to project the potential impact of vaccines on childhood deaths over the next ten years.  The model predicts that approximately 7.6 million children under 5 years of age can be saved this decade by developing new vaccines to prevent severe diarrhea and pneumonia (both of which are close to becoming available) and extending vaccine coverage in developing countries to 90%.  The model also predicts that an additional 1.1 million children can be saved by introducing a malaria vaccine in the developing world by 2014 (one malaria vaccine candidate is currently in late-stage trials).  In order to reach the Foundation's goal of 90% coverage, however, Mr. Gates stated that billions in additional funding will be required.

The Gateses noted that their $10 billion pledge was inspired by recent progress concerning vaccine research, development, and delivery in the developing world.  For example, the couple pointed to World Health Organization (WHO) data showing that between 2000 and 2009, the percentage of children receiving the basic diphtheria, tetanus, and pertussis (DTP3) vaccine in the poorest countries of the world jumped from 66% to 79%, and that between 2000 and 2008, measles deaths in Africa dropped by 92%.  In addition, a recent New England Journal of Medicine article shows that the introduction of a rotavirus vaccine in South Africa and Malawi reduced cases of severe diarrhea by more than 60%.

Appearing with Mr. and Mrs. Gates was Julian Lob-Levyt, the CEO of the GAVI Alliance, a global health partnership representing stakeholders in immunization from both private and public sectors that was launched at the World Economic Forum ten years ago.  Mr. Gates noted that by coordinating the resources and expertise of vaccine companies, donors, UNICEF, WHO, the World Bank, and developing countries, the GAVI Alliance was "transforming the business of vaccines," and had permitted the vaccination of 257 million additional children, preventing some 5 million deaths.  Looking at the organization's past accomplishments, Mr. Lob-Levyt observed that "[t]he potential to make bigger strides in the coming decade is even more exciting."

Myriad Sticks to Its Business Model

    By Kevin E. Noonan --

Myriad Genetics, one of the preeminent gene-based diagnostics companies, has been targeted by the American Civil Liberties Union, Dan Ravicher’s PUBPAT Organization, a raft of medical researchers and their associations, and individual patients in a lawsuit filed in the Southern District of New York; the avowed aim of the plaintiffs is to get the issue of "gene patenting" before the U.S. Supreme Court (see "Association for Molecular Pathology v. U.S. Patent and Trademark Office").  While the case wends its way through the Federal judicial system, Myriad is continuing its pursuit of diagnostically-relevant cancer markers, the most recent of which is a gene related to susceptibility to pancreatic cancer licensed from the Johns Hopkins University.

The gene encodes PALB2, a tumor suppressor gene reported to be involved in susceptibility to pancreatic cancer in the April 10, 2009 issue of Science (Jones et al., 2009, "Exomic Sequencing Identifies PALB2 as a Pancreatic Cancer Susceptibility Gene," Science 324: 217).  The product of the PALB2 gene is a binding partner of BRCA2, originally identified by its association with breast cancer susceptibility.  The two gene products bind to DNA to repair damage, wherein PALB2 localizes and anchors BRCA2 to damaged DNA in the cell nucleus.  More than 3% of familial pancreatic cancer patients had mutations in PALB2 resulting in the production of a truncated version of the encoded protein.  In contrast, no mutations in the PALB2 gene were found in more than 1,000 normal DNA samples.

Myriad contends that the predictive value of detecting such mutations are due to a 10- to 20-fold increase in the likelihood of developing pancreatic cancer by age 70 for individuals bearing the PALB2 mutation (or mutations in BRCA2 or another tumor suppressor gene, p16).  Early detection is particularly important for pancreatic cancer, which is typically detected only after it has progressed past the stage where intervention has any chance of significantly prolonging life.  Pancreatic cancer is diagnosed in about 200,000 patients worldwide each year, and is fatal almost without exception.

The scope of the difficulty in identifying reliable diagnostic genes is reflected in the following synopsis of the extent of mutations found by the Johns Hopkins researchers in tumor DNA from one pancreatic cancer patient.

Among the 20,661 coding genes analyzed, we identified 15,461 germline variants in Pa10 not found in the reference human genome.  Of these, 7318 were synonymous, 7721 were missense, 64 were nonsense, 108 were at splice sites, and 250 were small deletions or insertions (54% in-frame).

The PALB2 mutation found in tumor DNA from this patient exhibited a germline deletion of four basepairs (TTGT) that produced a frameshift mutation at codon 58, resulting in production of a truncated gene product from this gene.  Additional mutations found associated with pancreatic cancer in the PALB2 gene are a G > T transversion at the 5' splice site of exon 6, a deletion of an A residue in exon 11 (at nucleotide 3116) and a C > T transition mutation in exon 12 (at nucleotide 3256).  Fine structure mapping of mutations found in the PALB2 gene in pancreatic cancer, breast cancer, and Fanconi anemia patients was also reported.

The company estimates that a diagnostic genetic test might be on the market by 2010.

Biogen Idec Receives Method-of-Use Patent on Human ß-Interferon

Implications for Patent Protection for Avonex® Anti-Multiple Sclerosis Drug

    By Kevin E. Noonan --

There is a tendency to blame the process when an undesired outcome occurs.  That is happening in the wake of regulatory filings with the Securities and Exchange Commission regarding U.S. Patent 7,588,755 (the '755 patent), issued on September 15, 2009, and owned by Biogen Idec.  The undesired outcome (by some) is the possibility that this patent will protect Biogen Idec's blockbuster multiple sclerosis drug, Avonex® (human interferon beta 1a), from generic competition for an additional 13 years (until September 15, 2026) after expiration of current patents in May 2013.  That may happen, but a review of the history of the application shows that the patenting process worked as it is supposed to, with no evidence that Biogen Idec was not diligent in prosecuting this application within Patent Office rules.  If anything, it is the diligence with which the Office examined the application that resulted in the delay that may extend Biogen Idec's exclusivity period.

The '755 patent claims priority to a first application filed in 1981 and a divisional application filed in 1989, both abandoned.  The application that resulted in the '755 patent was filed on May 25, 1995, and thus has a patent term of 17 years from issue, determined by U.S. law prior to the adoption of the GATT provision limiting U.S. patent term to 20 years from the earliest priority date.  A history of patent prosecution reveals "why it took so long" for this patent to issue:

The longest period of delay extended from March 12, 1998 to January 15, 2003 -- almost five years -- while prosecution was suspended because of a potential interference.  (For those uninitiated in the arcane world of interferences, the most likely reason for this delay was to give a potential interfering application time to be prosecuted until interfering claims were deemed allowable.)  As it turns out, no interference was declared, and the Examiner renewed prosecution by asserting anticipation rejections based on two patents to Sugano (U.S. Patent Nos. 5,514,567 or 5,326,859).  (Ironically, '755 patent applicant Fiers was involved in an earlier interference against Sugano, and a second party, Revel, almost 20 years ago, in which Sugano prevailed in claims for human ß-interferon.)  The second extensive period of delay involved the appeal of the asserted anticipation rejection, which was complicated by the new formalities rules for appeal briefs promulgated (and strictly enforced) by the Office.  This delayed prosecution of the application to allowance by another 3 years, from June 24, 2005 to May 29, 2008.  Ultimately, the Board of Patent Appeals and Interferences reversed the rejections based on the Sugano patents, paving the way for allowance and issue of the '755 patent (a process that took another 16 months).

In addition to these Patent Office delays, the extended term of this patent is, of course, an artifact of the change in patent term in the U.S. -- the reason the term seems so long today is that we have become accustomed to a 20-year maximum from the earliest priority date, and the prior norm of 17 years from grant date seems anomalous, especially when the term is as extended as it is in this case (31 years from the application filing date, and 45 years from the earliest priority date).  Something like this could not occur today.

Besides these procedural aspects, whether the '755 patent will be useful for preventing generic competition for Avonex® will depend on the scope of the granted claims.  The '755 patent has but three claims, set forth below:

1.  A method for immunomodulation or treating a viral conditions, a viral disease, cancers or tumors comprising the step of administering to a patient in need of such treatment a therapeutically effective amount of a composition comprising:
    a recombinant polypeptide produced by a non-human host transformed by a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of:
    (a) DNA sequences which are capable of hybridizing to any of the DNA inserts of G-pBR322(Pst)/HFIF1, G-pBR322(Pst)/HFIF3 (DSM 1791), G-pBR322(Pst)/HFIF6 (DSM 1792), and G-pBR322(Pst)/HFIF7 (DSM 1793) under hybridizing conditions of 0.75 M NaCl at 68.degree. C. and washing conditions of 0.3 M NaCl at 68.degree. C., and which code for a polypeptide displaying antiviral activity, and (b) DNA sequences which are degenerate as a result of the genetic code to the DNA sequences defined in (a);
    said DNA sequence being operatively linked to an expression control sequence in the recombinant DNA molecule.

2.  The method according to claim 1, wherein said DNA sequence is selected from DNA sequences of the formulae:

ATGACCAACAAGTGTCTCCTCCAAATTGCTCTCCTGTTGTGCTTCTCCACTA
CAGCTCTTTCCATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAGCAA
TTTTCAGTGTCAGAAGCTCCTGTGGCAATTGAATGGGAGGCTTGAATACTGC
CTCAAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCAGC
AGTTCCAGAAGGAGGACGCCGCATTGACCATCTATGAGATGCTCCAGAACAT
CTTTGCTATTTTCAGACAAGATTCATCTAGCACTGGCTGGAATGAGACTATT
GTTGAGAACCTCCTGGCTAATGTCTATCATCAGATAAACCATCTGAAGACAG
TCCTGGAAGAAAAACTGGAGAAAGAAGATTTCACCAGGGGAAAACTCATGAG
CAGTCTGCACCTGAAAAGATATTATGGGAGGATTCTGCATTACCTGAAGGCC
AAGGAGTACAGTCACTGTGCCTGGACCATAGTCAGAGTGGAAATCCTAAGGA
ACTTTTACTTCATTAACAGACTTACAGGTTACCTCCGAAAC,

and

ATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAGCAATTTTCAGTGTC
AGAAGCTCCTGTGGCAATTGAATGGGAGGCTTGAATACTGCCTCAAGGACAG
GATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCAGCAGTTCCAGAAG
GAGGACGCCGCATTGACCATCTATGAGATGCTCCAGAACATCTTTGCTATTT
TCAGACAAGATTCATCTAGCACTGGCTGGAATGAGACTATTGTTGAGAACCT
CCTGGCTAATGTCTATCATCAGATAAACCATCTGAAGACAGTCCTGGAAGAA
AAACTGGAGAAAGAAGATTTCACCAGGGGAAAACTCATGAGCAGTCTGCACC
TGAAAAGATATTATGGGAGGATTCTGCATTACCTGAAGGCCAAGGAGTACAG
TCACTGTGCCTGGACCATAGTCAGAGTGGAAATCCTAAGGAACTTTTACTTC
ATTAACAGACTTACAGGTTACCTCCGAAAC.

3.  The method according to claim 1 wherein the polypeptide is selected from polypeptides of the formulae:

Met-Thr-Asn-Lys-Cys-Leu-Leu-Gln-Ile-Ala-Leu-Leu-
Leu-Cys-Phe-Ser-Thr-Thr-Ala-Leu-Ser-Met-Ser-Tyr-
Asn-Leu-Leu-Gly-Phe-Leu-Gln-Arg-Ser-Ser-Asn-Phe-
Gln-Cys-Gln-Lys-Leu-Leu-Trp-Gln-Leu-Asn-Gly-Arg-
Leu-Glu-Tyr-Cys-Leu-Lys-Asp-Arg-Met-Asn-Phe-Asp-
Ile-Pro-Glu-Glu-Ile-Lys-Gln-Leu-Gln-Gln-Phe-Gln-
Lys-Glu-Asp-Ala-Ala-Leu-Thr-Ile-Tyr-Glu-Met-Leu-
Gln-Asn-Ile-Phe-Ala-Ile-Phe-Arg-Gln-Asp-Ser-Ser-
Ser-Thr-Gly-Trp-Asn-Glu-Thr-Ile-Val-Glu-Asn-Leu-
Leu-Ala-Asn-Val-Tyr-His-Gln-Ile-Asn-His-Leu-Lys-
Thr-Val-Leu-Glu-Glu-Lys-Leu-Glu-Lys-Glu-Asp-Phe-
Thr-Arg-Gly-Lys-Leu-Met-Ser-Ser-Leu-His-Leu-Lys-
Arg-Tyr-Tyr-Gly-Arg-Ile-Leu-His-Tyr-Leu-Lys-Ala-
Lys-Glu-Tyr-Ser-His-Cys-Ala-Trp-Thr-Ile-Val-Arg-
Val-Glu-Ile-Leu-Arg-Asn-Phe-Tyr-Phe-Ile-Asn-Arg-
Leu-Thr-Gly-Tyr-Leu-Arg-Asn,

and

Met-Ser-Tyr-Asn-Leu-Leu-Gly-Phe-Leu-Gln-Arg-Ser-
Ser-Asn-Phe-Gln-Cys-Gln-Lys-Leu-Leu-Trp-Gln-Leu-
Asn-Gly-Arg-Leu-Glu-Tyr-Cys-Leu-Lys-Asp-Arg-Met-
Asn-Phe-Asp-Ile-Pro-Glu-Glu-Ile-Lys-Gln-Leu-Gln-
Gln-Phe-Gln-Lys-Glu-Asp-Ala-Ala-Leu-Thr-Ile-Tyr-
Glu-Met-Leu-Gln-Asn-Ile-Phe-Ala-Ile-Phe-Arg-Gln-
Asp-Ser-Ser-Ser-Thr-Gly-Trp-Asn-Glu-Thr-Ile-Val-
Glu-Asn-Leu-Leu-Ala-Asn-Val-Tyr-His-Gln-Ile-Asn-
His-Leu-Lys-Thr-Val-Leu-Glu-Glu-Lys-Leu-Glu-Lys-
Glu-Asp-Phe-Thr-Arg-Gly-Lys-Leu-Met-Ser-Ser-Leu-
His-Leu-Lys-Arg-Tyr-Tyr-Gly-Arg-Ile-Leu-His-Tyr-
Leu-Lys-Ala-Lys-Glu-Tyr-Ser-His-Cys-Ala-Trp-Thr-
Ile-Val-Arg-Val-Glu-Ile-Leu-Arg-Asn-Phe-Tyr-Phe-
Ile-Asn-Arg-Leu-Thr-Gly-Tyr-Leu-Arg-Asn.

The relevance of these claims to Avonex® will depend on whether Avonex® administration falls within the ambit of the method claim, which requires:

a)  immunomodulation or
b)  treating a viral condition, a viral disease, cancers or tumors
c)  administering to a patient in need of such treatment
d)  a therapeutically effective amount of a composition comprising:
e)  a recombinant polypeptide
f)  produced by a non-human host transformed by a recombinant DNA molecule comprising a DNA sequence
g)  encoding human ß-interferon or sequence homolog thereof
h)  which code for a polypeptide displaying antiviral activity

While elements e) through h) are apparently encompassed by Avonex®, whether these claims will be useful for precluding generic entry will depend on whether treatment of MS involves immunomodulation or viral disease (since it is self-evidently neither cancer or a tumor).  In this regard the scientific literature is littered with reports linking MS with a variety of viral infections (including Epstein-Barr virus and measles virus), but no definitive linkage has been established.  While MS treatment is likely to involve some type of immunomodulation, Biogen Idec would have the burden of establishing such a mechanism should they sue an ANDA filer for infringement.

The importance of the question about the scope of this patent, and its effects on generic competition over Avonex® can be appreciated from the sales figures for the drug in the second quarter of 2009, which amount to almost $600 million (or $2.4 billion per year), according to an Associated Press article cited in Forbes magazine.

As the Forbes article suggests, the outcome of the Avonex® patent regime has relevance to the current follow-on biologics legislation, at least because of the emotional appeal regarding the length of patent protection.  Understanding the history of this patent defuses any apprehension about the likelihood of similar outcomes going forward, and the Avonex® patent estate can be seen to be nothing more than a historical accident that cannot be replicated.  However, the tendency for dissatisfaction with outcomes like this one to guide political activity, and legislation, should not be underestimated.

ChemBridge Makes Chemical Libraries Available Via Public Access Database

    By Donald Zuhn --

Last month, ChemBridge Corp. announced that it would make ten of its most popular chemical screening libraries available to researchers via the Collaborative Drug Discovery (CDD) Public Access database.  The CDD Public Access database, a collection of public data from leading vendors, researchers, scientific literature, and patent resources, is hosted by Collaborative Drug Discovery, Inc. as a service to the research community.  Collaborative Drug Discovery provides web-based software for organizing preclinical research data to help scientists more effectively advance new drug candidates.  Researchers can register for free read-only access to the CDD public data sets here; read-and-write access, including the ability to archive, mine, and collaborate around private data and to import and export both private and public data is also available.  Compounds from the ten ChemBridge libraries being added to the Public Access database can be obtained from the San Diego-based discovery chemistry company.

The libraries being offered by ChemBridge include its DIVERSet, a 50,000 diverse small molecule dataset covering a broad range of pharmacophore space; and CNS-Set, which contains 56,000 molecules with an increased probability of blood brain barrier penetration.  ChemBridge noted that more than 600,000 molecules would be available through the CDD database.  CDD CEO Barry Bunin stated that both the CDD community for private access and the entire researcher community for public access would benefit from the addition of the ten ChemBridge libraries.  Prior to the addition of the ChemBridge molecules, the CDD database contained over a million compounds.

Cancer Drug Development in The New York Times

    By Kevin E. Noonan --

The fastest-growing part of the biotech/pharma industry is anticancer drugs, according to an article in The New York Times today ("For Profit, Industry Seeks Cancer Drugs").  Written by Andrew Pollack, one of the paper's senior business and technology writers, the article highlights the interplay between scientific interest and business opportunity driving this phenomenon.

The article supports its thesis on the recent rise in anticancer drugs with some telling statistics:  there are "more than twice the number of experimental drugs for heart disease and stroke combined, nearly twice as many as for AIDS and all other infectious diseases combined, and nearly twice as many as for Alzheimer's and all other neurological diseases combined."  This corresponds to 860 anticancer drugs currently in clinical trials.

The scientific reasons for these efforts stem from the nature of cancer as a disease.  Robert Weinberg (at left) from the Whitehead Institute at MIT is quoted in the article as saying "Cancer is not a single disease.  It's really dozens, arguable hundreds of diseases."  Bert Vogelstein (below right) from Johns Hopkins is quoted as saying that a typical tumor may have 50-100 genetic mutations, with any two patients having the same type of cancer having as few as five mutations in common.  Accordingly, there are many approaches and molecular targets for small molecule, immunological, and other avenues for attacking cancer cells in an effort to find a cure.  In addition, the situation is ripe for "personalized medicine," or efforts to tailor the treatment to the tumor based on the unique signature of genetic mutations and the corresponding phenotype of an individual's cancer and the targets for therapeutic intervention expressed by each individual's tumor.

But this very complexity is the main reason why cancer remains so daunting after 40 years of concerted efforts to find a cure.  On the one hand, individual cancers even of the same type or in the same tissue vary dramatically in changes in gene expression, chromosomal abnormalities, and cancer-associated phenotypes, so that treatments that are effective for some cancer patients are completely ineffective for others.  This has caused the "war on cancer" to resemble a "grinding war of the trenches," according to an uncredited quote by a cancer researcher.  And intractable clinical situations provide a correspondingly broader scope of opportunities for new drugs effective against cancer in some portion of the patient population.

However, on the other hand, the efficacy of many anticancer drugs is limited at best.  The article cites the instance of drugs like Erbitux® used as a "last ditch treatment for colorectal cancer" providing on average about 90 days of additional life to patients.  And Taceva®, used for treating pancreatic cancer (a cancer having a 3% 5-year survival rate that hasn't changed in the past 100 years), extends life for pancreatic cancer patients on average only 12 days (the basis for its approval by the FDA for this indication).

Since 1998, when there were twelve anticancer drugs in the world's 200 medicines having the greatest sales, the number of anticancer drugs has almost doubled (to 23), and 20 of the 126 drugs having revenues of $1 billion are anticancer drugs.  Despite these efforts, however, only a handful of new anticancer drugs are approved each year (two in 2008, one so far in 2009).  Nevertheless, the article cites studies by IMS Health, a pharmaceutical market research company, for evidence that "[c]ancer drugs have been the biggest category of drugs in terms of sales worldwide since 2006 and in the U.S. since 2008."

One factor driving anticancer drug development is the revenues that can be generated from their sales, which carry a hefty price tag.  Taceva® treatments cost about $3,500 a month, according to the article, and Erbitux® costs $10,000 per month (with an "extra" cost per patient averaging $50,000, based on the average length of treatment course).  These costs have resulted in ImClone having revenues of $1.6 billion in 2008 for Erbitux®, revenues that the article asserts motivated "Eli Lilly & Co. to outbid Bristol-Myers Squibb to acquire ImClone for $6.5 billion."

While these costs are exorbitantly high, the article notes that "[p]atients are often desperate, and insurers risk outrage by denying payments for a[n anti]cancer drug," even when there is little chance of benefit from the treatment.  "Cancer is such an emotional issue that the free market doesn't work like it does for bicycle wheels and umbrellas," according to Robert Erwin, head of the Marti Nelson Cancer Foundation.  Money will flow to anticancer drugs "[a]s long as the health care system will pay the price."  This situation raises special concerns for the healthcare system as a whole, particularly under the current economic climate, the pressures to reduce drug costs, and the amount of investment required to determine whether an anticancer drug lead compound will come to market.  These circumstances are characterized as "the biggest bubble you've ever seen," according to Dr. Mark Ratain (at right), a University of Chicago oncologist.

The confluence of the exponential increase in genetic information over the past decade and the technical ability to identify genetic polymorphism and outright mutation with different cancer phenotypes presents an unprecedented opportunity for developing new anticancer therapeutics.  Mr. Pollack's article is a reminder that the science is only part of the equation, and that the economic realities of drug development and the healthcare marketplace will also have a role in determining how many, and which, of these drugs will be available for the next generation of cancer patients.

Leg Length Variation in Dogs and its Relevance to Human Mutations

    By Kevin E. Noonan --

One of the benefits of the exponential increase in genetic information consequent to the determination of the genomic DNA sequence of numerous species (the various "genome projects" that include the Human Genome Project) is that it permits sequence comparisons between related species.  One result of this new knowledge has been to verify the similarity of developmental control genes throughout the animal kingdom (where genes like the Hox genes in nematode worms and fruit flies have been identified in humans, for example).  Within species, decoded genetic information, and the ability to associate genetic variation between individuals in a species with specific phenotypes has elucidated the genes that control such phenotypes.  Several examples have been reported for domesticated dogs, where size is related to inheritance of different alleles of the insulin-like growth factor-1 gene (IGF-1), and coat length, curliness, and other features are related to inheritance of the FGR-5, KRT-71, and RPSO-2 genes.  Artificial human selection for breed characteristics has contributed to the ability to productively perform these types of association studies.

Leg length in dogs is another phenotypic feature subject to the whims of human breed selection and characteristic of different breeds.  In this week's edition of the journal Science, a group from the National Institutes of Health reports on the genetic basis of this variation (termed chondrodysplasia), and associates its results with a form of human dwarfish with similarities to canine chondrodysplasia (wherein the growth plates in the long bones of the leg calcify prematurely, producing shortened bones with a curved appearance) (Parker et al., 2009, "An Expressed Fgf4 Retrogene Is Associated with Breed-Defining Chondrodysplasia in Domestic Dogs," Science 325: 995-98).

The report is from Elaine Ostrander's lab at the NIH, joined by researchers at the Department of Ecology and Evolutionary Biology at UCLA, the WALTHAM Centre for Pet Nutrition in the UK, the Division of Cardiovascular Medicine at the Oregon Health and Human Science University, the Baxter Institute for Animal Health and the College of Veterinary Medicine and the Department of Biological Statistics and Computational Biology, Cornell University, and the Comparative Orthopedic Laboratory at the University of Missouri.  The study is particularly noteworthy as it identifies an expressed retrogene for fibroblast growth factor 4 (FGF4); retrogenes, more commonly termed pseudogenes, represent reverse-transcribed messenger RNA (thus lacking introns) that have been re-introduced into the genome.  Until recently, these genes were believed to be transcriptionally-silent relics of ancient retroviral infection and were not expected to be transcribed.  It is now recognized that such retrotransposition can, and has occurred at sites containing transcriptional regulatory sequences, as it appears to have in these dogs, leading to a novel form of gene duplication.

Genome-wide association studies (GWAS) using Affymetrix single nucleotide polymorphism (SNP) microarrays were performed on DNA from 835 dogs comprising 76 distinct breeds.  Using American Kennel Club (AKC) breed standards, 95 dogs from the chondrodysplastic breeds (including Pekinese, Welsh corgi, and dachshund) were compared to a control group of 64 non-chondrodysplastic dog breeds comprising 702 animals.  The strongest association in these studies specific for the chondrodysplastic breeds was found on canine chromosome 18 (CFA18).  This association was confirmed by studies on heterozygosity in this region of canine chromosome 18, which was expected and was found to be suppressed, consistent with human selection for chondrodysplasia as a part of the breed standard for these breeds of dogs.  Upon further sequencing analysis, the region was found to contain a processed retrogene of canine FGF4, comprising all the coding exon sequences as well as the 3' untranslated region (UTR) and a polyadenine tract (as expected from an mRNA retrotransposon), but lacking the 5' regulatory sequences of the native canine FGF4 gene (which also resides on canine chromosome 18, about 30 Mb from the FGF4 retrotransposon insertion site).  This gene was found in 175 dogs from 19 breeds exhibiting the chondrodysplasia phenotype, and the gene was found to be homozygous in all but seven of these dogs.  Fortuitously, the group found a single transition mutation difference (a G --> A change in the retrogene) that permitted expression to be differentiated between the retrogene and the native FGF4 gene.  Gene expression studies showed that the retrogene was expressed in the articulate cartilage of the long bones from chondrodysplastic dogs, "whereas only the G allele was detected in cDNA and genomic DNA samples from non-chondrodysplastic dogs."

Finally, these authors found that the FGF4 retrogene was expressed using gene regulatory sequences in a copy of a long interspersed nuclear element (LINE) repeat residing at the retrogene insertion site.  However, analysis of the expression pattern of neighboring genes in adult dog tissues showed that neither the native FGF4 nor the FGF4 retrogene were expressed in adult tissues.  Developmental regulation of FGF4 is believed to be mediated by sequences in the 3' UTR element of the native gene, and the presence of these sequences in the FGF4 retrogene suggests that it is also properly regulated during development.

All modern domestic dog breeds are believed to have descended from the gray wolf, and the fact that chondrodysplastic breeds have been developed several times independently (i.e., without direct ancestral relationships) suggests that the retrotransposition event, and the presence of the FGF4 retrogene, should be present in populations of the ancestral gray wolf.  Using hapolotype analysis over the 24 kb region of canine chromosome 18 comprising the FGF4 retrogene and its insertion site, "this combination was not found in any of the non-chondrodysplastic dogs we tested but was identified in wolves from Europe and the Middle East, supporting fossil evidence that these populations contributed to the early development of the dog."

The authors put forth their hypothesis of how the FGF4 retrogene is involved in chondrodysplasia in dogs, and its relevance to humans:

We hypothesize that atypical expression of the FGF4 transcript in the chondrocytes causes inappropriate activation of one or more of the fibroblast growth factor receptors such as FGFR3.  An activating mutation in FGFR3 is responsible for >95% of achondrodysplasia cases, the most common form of dwarfism in humans, and 60 to 65% of hypochondrodysplasia cases, a human syndrome that is more similar in appearance to breed-defining chondrodysplasia.  . . .  FGF4 induces the expression of sprouty genes, which interfere with the ubiquitin-mediated degradation of the FGF receptors including FGFR3, and overexpression of the sprouty genes can cause chondrodysplastic phenotypes in both mice and humans.

The greater significance of the study can be summarized in no better words than the authors' own:

We have found a single retrotransposition event producing a conserved, expressed retrogene that has strongly focused the evolutionary direction of morphological change in the dog because at least 12% of American breeds share a common phenotype and the retrogene.  This retrogene is actively segregating within the species, has a coding sequence that is identical to that of the source gene, and to the best of our knowledge is the only example of a functional retrogene found in morphologically distinct populations of a single species that is actively maintained by selection.  If such rare mutational events or "sports," as Charles Darwin referred to them in "The Origin of Species," happen only in the evolution of domestic animals, then these systems may be less informative for understanding the origin of evolutionary novelty in wild species.  However, if the molecular phenomenon we have observed represents a class of genomic change associated with dramatic phenotypic evolution, then such genetic changes might be keystone molecular innovations.

The Genetic Basis of Coat Variation in Dogs

    By Kevin E. Noonan --

The genetic age, characterized by numerous genome projects (not the least of which is the Human Genome Project) has focused the public's attention on the influence of our genes on our characteristics.  This has led to questions about whether there is a "gene" for risk-taking, or homosexuality, or aggressiveness.  Indeed, the pervasiveness of the "gene" question for complex traits has produced something of a backlash, illustrated by Richard Lewontin's books, Not in Our Genes and It Ain't Necessarily So, making the reasonable point that genetic reductionism has its limitations.

But genes are certainly implicated in a number of characteristics and traits, and one of the consequences of studying genomes has been the elucidation of these relationships.  In 2007, a report in Science disclosed that body size in domesticated dogs is associated with a particular single nucleotide polymorphism (SNP) in the insulin-like growth factor I gene (IGF-1) (see "From Toy Poodle to Rottweiler:  Why Is Fido So Small (or Large)?").  Last week, in Science, a group from the National Institutes of Health report on another canine phenotype, coat variation, and the genes that are associated with variations in several coat related traits (Cadieu et al., 2009, "Coat Variation in the Domestic Dog Is Governed by Variants in Three Genes," Science DOI: 10.1126/science.1177808).

The report, from Elaine Ostrander's lab at the NIH and researchers at the Veterinary Genetics Laboratory at the University of California, Davis, the Biology Department at the University of Utah, the Department of Ecology and Evolutionary Biology at UCLA, Cornell University's Department of Biological Statistics and Computational Biology, the Faculte de Medicine in Rennes, France, and Affymetrix, involves genome-wide association studies (GWAS) for three coat variations:  the presence or absence of "furnishings" (eyebrows and mustaches as arise in wire-haired dogs), coat length, and coat curliness.  The study involved more than 1,000 dogs from 80 breeds of domestic dogs, and resulted in the identification of three genes associated with variations in coat.  These genes are RSPO2, encoding R-spondin-2; FGF5, encoding fibroblast growth factor 5; and KRT71, encoding keratin-71.  These genes, and specifically SNPs associated with each of these genes, were found to be associated with the presence or absence of furnishings (RSPO2), length (FGF5), and curliness (KRT71).

Unlike the situation with body size, these three genes form a combinatorial group of genes that can be independently segregated (i.e., they all reside on different chromosomes) and can thus create a number of different coat variations depending on the different variant alleles inherited by particular breeds of dog (since coat characteristics are frequently important components of the breed standard for different dog breeds).  Three datasets were used in these experiments:  the first comprised DNA from 96 dachshunds varying as wire-haired (with furnishings), smooth and long-haired coats; 76 Portuguese waterdogs, varying in curl type; and 903 dogs form 80 breeds representing "a wide variety of [coat] phenotypes."  The initial results of polymorphisms segregating with the different coat traits were validated using a larger panel of at least 661 dogs from 106 breeds that included the appropriate controls.

From the dachshund (at right) studies was identified the association of RSPO2 with furnishings as a locus residing on canine chromosome 13 (CFA13).  As reported in the paper, "[a] 718 Kb homozygous haplotype in all dogs fixed with furnishings was located within both the original 3.4Mb haplotype observed in the dachshund-only GWAS, and a 2.8 Mb haplotype identified in crossover analysis within the dachshund pedigree."  From this chromosomal location the RSPO2 gene was identified:

Fine-mapping allowed us to reduce the homozygous region to 238 Kb spanning only the R-spondin-2 (RSPO2) gene, excluding the 5'UTR and the first exon (Fig. 1D; fig. S1 and table S3).  RSPO2 is an excellent candidate for a hair growth phenotype as it is known to synergize with Wnt to activate β-catenin . . . , and Wnt signaling is required for the establishment of the hair follicles . . . .  Moreover, the Wnt/β-catenin pathway is involved in the development of hair follicle tumors or pilomatricomas . . . , which occur most frequently in breeds that have furnishings . . . .  Recent studies have shown that a mutation in the EDAR gene, also involved in the Wnt pathway, is responsible for a course East-Asian hair type found in humans . . ., with some similarity to canine wirehair.

The polymorphism associated with the furnishings trait in dachshunds and confirmed in 704 dogs of varying phenotype is a 167 bp insertion within the 3' untranslated region (UTR) of the RSPO2 gene; "297/298 dogs with furnishings were either homozygous (268) or heterozygous (29) for the insertion, while all 406 dogs lacking the trait were homozygous [for 3' UTR lacking the insertion]."  The authors also report a functional consequence of the insertion, wherein there is a 3-fold increase in RSPO2 expression in muzzle skin from dogs with furnishings, suggesting that the insertion in the 3' UTR influences mRNA stability.

For the association of FGF5 with hair length, the same type of GWAS were performed, locating a polymorphism on canine chromosome 32 (CFA32) at the FGF5 locus.  Here, the polymorphism affected the coding sequence of the protein, resulting in a Cys --> Phe change at a conserved residue (95) in exon 1 of the FGF5 gene (corresponding to a G --> T transversion mutation).  This association was not unexpected, as prior work had shown that the FGF5 gene was associated with a long-haired or "fluffy" phenotype in Welsh corgis.  Long hair was associated with the TT genotype, consistent with a genetically recessive mode of inheritance.  The T allele was found in 91% of all long-haired dogs tested and in only 3.9% of short-haired dogs (with medium-haired dogs having an intermediate 30% frequency level for this allele).  Curiously, Afghan hounds that have particularly long hair do not have the Cys95Phe mutation.

Finally, for the curly coat variation GWAS were performed with Portuguese water dogs (PWD) (at left, First Dog Bo).  These studies revealed a SNP on canine chromosome 27 (CFA27) located in the keratin 71 (KRT71) genetic locus.  As reported in the paper, "Non-curly haired dogs carried the CC genotype [and] curly coated dogs had the TT phenotype," and all three genotypes (CC, CT and TT) were found in PWDs, a breed that expressed curl hair to varying extents.  This SNP is also located in the coding sequence (in the second exon) of the gene, causing an Arg --> Trp change at residue 151.  These findings were also rationalized by the authors:

Keratins are obvious candidates for hair growth . . . and mutations in KRT71 have been described in curly coated mice . . . .  The mutation described in our study is within the second exon of the gene and may affect either or both of two protein domains; a coiled-coil and a prefoldin domain.  Conceivably, sequence alterations in these domains could affect cellular targeting, receptor binding or proper folding of the protein after translation.

These three mutations were sufficient to account for the coat phenotype in 95% of the dogs tested, a total of 622 dogs representing 108 of the 160 dog breeds recognized by the American Kennel Club.  The Supplemental Materials contain a combinatorial diagram of the different variations at the three loci that give rise to at least 7 different coat types (representing the majority of coat types in modern breeds):

* The TT genotype was never found in this combination, even though it would likely also display a short phenotype.

The authors set forth the following conclusions.  First, the "ancestral state" of all three genes (Cys95 in FGF5, Arg151 in KRT71, and absence of an insertion in the 3' UTR in RSPO2) is found in short-haired dog breeds (as well as grey wolves, the ancestors of all modern dog breeds).  Wire-haired breeds (which all have furnishings) have the insertion in the RSPO2 gene.  Curly wire hair is found in dogs having both the RSPO2 insertion and the KRT71 mutation, while longer-haired dog breeds have the FGF5 mutation.  Dogs carrying both the FGF5 mutation and the RSPO2 insertion have long, soft coats and furnishings, whereas dogs with the FGF5 and KRT71 mutations have long, curly coats.  Finally, dogs bearing all three of the variants have long, curly coats, and furnishings.

Elucidation of this genetic architecture of observable phenotypic traits is possible because of the large number of different dog breeds and the great variability, on one hand, and the consistent stability, on the other hand, of genetic transmission in these breeds.  This study suggests that these analytical methods can (and will) be applied to assess the genetic bases of other traits in dogs, and the extension of these results to homologous phenotypes in other mammals including humans.

Photograph of mixed breed terrier (above) by Chris Barber, from the Wikipedia Commons under the Creative Commons license.

Efficient Delivery of siRNA

    By Kevin E. Noonan --

A research paper in the June 2009 edition of Nature Biotechnology (Eguchi et al., 2009, "Efficient siRNA delivery into primary cells by a peptide transduction domain–dsRNA binding domain fusion protein," Nature Biotechnology 27: 567-71) describes a new system for introducing short interfering RNAs (siRNAs) into mammalian cells that may help this technology become practicable for therapeutic uses.  The promise of RNA interference, mediated by siRNAs, has revolutionized the prospects for modulating gene expression as a way to achieve therapeutic aims in disease treatment.  First identified in worms (Fire et al., 1998, "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans," Nature 391: 806–11), plants (Hamilton et al., 1999, "A species of small antisense RNA in posttranscriptional gene silencing in plants," Science 286: 950–52) and then in mammalian cells (Elbashir et al., 2001, "Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells," Nature 411: 494–98), siRNAs form the basis for 81 U.S. patents and 2,125 published patent applications.  Their benefits reside principally in their ability to specifically reduce or even enhance (Li, 2008, "Small RNA-Mediated Gene Activation," In: RNA and the Regulation of Gene Expression: A Hidden Layer of Complexity, Caister Academic Press) gene expression in mammalian cells.

However, a serious impediment to further development of siRNA, particularly for therapeutic uses, is that it is difficult to effectively introduce these molecules into mammalian cells.  siRNAs are double-stranded RNA molecules, typically 21 nucleotides in length and having a 2 nucleotide overhang at the 3' end of each strand.  These molecules are thus large (> 14,000 kDa) and highly negatively charged, and cannot cross the cellular plasma membrane.  Methods for introducing siRNAs have included encapsulation in liposome, complexing with cationic lipids, cholesterol, and condensing polymers, or conjugation with antibodies.  While these methods have proven effective with adherent tumor cells in vitro, they have been less effective in the types of cells that are attractive cellular targets in vivo (such as lymphocytes).  One approach, conjugation with cell-penetrating peptides (PTD) like TAT protein from human immunodeficiency virus or poly(Arg), resulted in cytotoxicity associated with limited siRNA delivery into target cells (Meade et al., 2008, "Enhancing the cellular uptake of siRNA duplexes following noncovalent packaging with protein transduction domain peptides," Adv. Drug Deliv. Rev. 60: 530-36), despite the effectiveness of such PTD carriers in other contexts (Gump et al., 2007, TAT transduction: The molecular mechanism and therapeutic prospects," Trends Molec. Med. 13: 443-48).

The Nature Biotechnology paper is from a group at the Howard Hughes Institute at the University of California, San Diego School of Medicine headed by Steven F. Dowdy.  The paper describes complexes of siRNAs with a fusion protein comprising a TAT-derived PTD conjugated to a double-stranded (ds) RNA-binding domain (DRBD) that binds to siRNA with an avidity of KD ~ 10-9.  A canonical example of such a sequence is the following (provided on-line in supplementary materials):

MGRKKRRQRRRGHSGRKKRRQRRRGHIYPYDVPVPDYAGDPGRKKRRQRRRGDP
AGDLSAGFFMEELNTYRQKQGVVLKYQELPNSGPPHDRRFTFQVIIDGREFPEG
EGRSKKEAKNAAAKLAVEILNEKKAAALEHHHHHH

(where single-underlined sequences are PTD sequences from TAT, the underlined and italicized sequence is an HA-tag, and the boldface sequence is the DRBD from RNA-dependent eIF-2 alpha protein kinase (PKR) protein).  The DRBD sequence non-covalently complexes with siRNA in a way that is distinct from the PTD sequences, which remain competent to mediate transit of the complex through the plasma membrane.

Image obtained from Traversa Therapeutics website.

These complexes were tested in several systems, including human H1299 lung adenocarcinoma cells expressing green fluorescent protein (GFP) and red fluorescent protein, which were used to show specific RNA interference of GFP that was obtained at greater levels than with conventional lipofection techniques.  These results were confirmed by single cell flow cytometry that showed that the entirety of the cell population contained siRNA with no alteration of cellular viability (in contrast with significant cytotoxicity associated with siRNA lipofection).  These results were also confirmed with other cell types, including "primary human fibroblasts, keratinocytes, macrophages and melanoma and glioma cells containing integrated GFP reporter genes."  Substantially similar results were obtained with these cells using siRNAs for glyceraldehyde phosphate dehydrogenase (GAPDH), a housekeeping enzyme.  In these experiments, the effects of the GAPDH siRNAs on the cellular transcriptome were assessed, and were shown to be significantly smaller than the effects of these same siRNAs introduced using lipofection.  In particular, the complexed siRNAs did not activate cellular genes associated with immunogenicity, including interferon-regulated genes, in contrast to lipofected siRNAs.  (siRNA introduction using these complexes did not induce interferon or tumor necrosis factor expression in peripheral blood mononuclear cells, an effect observed with siRNA lipofection.)

In another set of experiments, GFP expression was suppressed in Jurkett T cells, primary murine T cells, and human umbilical vein endothelial cells (HUVEC).  The primary T-cell experiments used siRNAs for CD4 and CD8, and showed specific suppression of each gene by its cognate siRNA and not by the non-cognate siRNA.  (The experiments also showed that expression of an unrelated cell surface marker, CD90, was not affected by siRNA introduction.)  HUVEC cells were treated with GAPDH siRNA and showed significantly lower (essentially undetectable) cytotoxicity and greater delivery than when siRNA were introduced into these cells by lipofection.  Finally, human embryonic stem cells were treated with a number of siRNAs that affected proliferation and differentiation.  Specifically, introduction of complexes of siRNAs for OCT4 caused cell cycle exit and differentiation.

In the most dramatic series of experiments, transgenic mice expressing luciferase in nasal and tracheal passages were treated with luciferase siRNAs complexed with PTD-DRBD.  These experiments showed that luciferase expression could be drastically reduced by these siRNA/PTD-DRBD complexes in vivo.

These results also provide the basis for patent applications in the U.S. and abroad.  The U.S. application is Serial No. 12/278,739, filed August 7, 2008.  Entitled "Transducible Delivery of siRNA by dsRNA Binding Domain Fusions to PTD/CPPS," the application published on April 9, 2009 as U.S. Patent Application Publication No. 2009/0093026, naming Steven F. Dowdy, Jehangir S. Wadia, Bryan Meade, and Akiko Eguchi as inventors.  This application is a U.S. national phase application of International Application No. PCT/US07/03641, filed February 9, 2007 and in turn claiming priority to two U.S. provisional applications (USSN 60/772,787, filed February 10, 2006, and USSN 60/775,638, filed February 21, 2006).  In addition to the experiments disclosed in the research paper, the application also shows EGFR-specific siRNA introduction using PTD-DRBD complexes into EGFR-expressing glioblastoma in mice.  The application also discloses alternative embodiments of PTD and DRBD sequences that can be used in the complexes.

The significance of these results is expressed by the authors as follows:

siRNA delivery has become the rate-limiting barrier to efficient cell culture and preclinical and clinical usage of siRNA therapeutics . . . .  Although current siRNA delivery approaches have merit, they generally do not target the entire population or even a high percentage of cells, especially primary cells, and often result in some degree of cytotoxicity and alternations in cell biology.  In contrast, the PTD-DRBD siRNA delivery approach described here fulfills many of the criteria for and efficient siRNA delivery system for primary cells.  . . .  Because DRBDs bind to dsRNAs (siRNAs) independent of sequence composition, PTD-DRBD could in theory deliver any siRNA into cells.  Lastly, the intranasal knockdown of luciferase demonstrates the in vivo potential of PTD-DRBD-mediated siRNA delivery.

The application is assigned to the Regents of the University of California, but Professor Dowdy is reported to have a start-up biotechnology company, Traversa Therapeutics of La Jolla, CA, and a researcher from Ambion is named on the research paper (but not the patent application).  A serious commitment to commercial development is also evidenced by applicants' recently changing the application status to large entity.

NIH Releases New Stem Cell Rules

    By Kevin E. Noonan --

Yesterday, the National Institutes of Health released guidelines governing requirements (and limitations) for federal funding of research on human embryonic stem cells.  These rules supersede rules promulgated during the Bush administration that so severely constrained the number of funding-eligible human stem cell lines as to effectively prohibit use of federal funds for this research.  These Guidelines were promulgated by Acting Director Raynard S. Kington, and become effective today, July 7, 2009.  The new Guidelines are expected to make up to 700 existing human embryonic stem cell lines eligible for federal funding, a ten-fold increase over the 69 hESC lines approved under the Bush administration (many of which have been acknowledged to be inappropriate for embryonic stem cell research).

The Guidelines were developed by the Institutes in response to Executive Order 13505, entitled "Removing Barriers to Responsible Scientific Research Involving Human Stem Cells" issued March 9, 2009.  Fulfilling a promise President Obama made during his campaign, the Order was intended to permit the Institutes to "support and conduct responsible, scientifically worthy human stem cell research, including human embryonic stem cell (hESC) research, to the extent permitted by law."  The new Guidelines, relating to both intramural and extramural research, "establish policy and procedures under which the NIH will fund such research, and helps ensure that NIH-funded research in this area is ethically responsible, scientifically worthy, and conducted in accordance with applicable law."

The Guidelines are expressly based on the principles that:

1.  Responsible research with hESCs has the potential to improve our understanding of human health and illness and discover new ways to prevent and/or treat illness.

2.  Individuals donating embryos for research purposes should do so freely, with voluntary and informed consent.

Human embryonic stem cells are defined as "cells that are derived from the inner cell mass of blastocyst stage human embryos, are capable of dividing without differentiating for a prolonged period in culture, and are known to develop into cells and tissues of the three primary germ layers," making the distinction that "[a]lthough hESCs are derived from embryos, such stem cells are not themselves human embryos."  The Guidelines establish a new NIH stem cell registry, and stem cells will be eligible for funding if they are listed on the registry or if they comply with Section IIA of the Guidelines.  Cells will be eligible if they are derived from human embryos:

1.  that were created using in vitro fertilization for reproductive purposes and were no longer needed for this purpose;

2.  that were donated by individuals who sought reproductive treatment (hereafter referred to as "donor(s)") and who gave voluntary written consent for the human embryos to be used for research purposes; and

3.  for which all of the following can be assured and documentation provided, such as consent forms, written policies, or other documentation, provided:

a.  All options available in the health care facility where treatment was sought pertaining to the embryos no longer needed for reproductive purposes were explained to the individual(s) who sought reproductive treatment.

b.  No payments, cash or in kind, were offered for the donated embryos.

c.  Policies and/or procedures were in place at the health care facility where the embryos were donated that neither consenting nor refusing to donate embryos for research would affect the quality of care provided to potential donor(s).

d.  There was a clear separation between the prospective donor(s)'s decision to create human embryos for reproductive purposes and the prospective donor(s)'s decision to donate human embryos for research purposes.  Specifically:

i.  Decisions related to the creation of human embryos for reproductive purposes should have been made free from the influence of researchers proposing to derive or utilize hESCs in research.  The attending physician responsible for reproductive clinical care and the researcher deriving and/or proposing to utilize hESCs should not have been the same person unless separation was not practicable.

ii.  At the time of donation, consent for that donation should have been obtained from the individual(s) who had sought reproductive treatment.  That is, even if potential donor(s) had given prior indication of their intent to donate to research any embryos that remained after reproductive treatment, consent for the donation for research purposes should have been given at the time of the donation.

iii.  Donor(s) should have been informed that they retained the right to withdraw consent for the donation of the embryo until the embryos were actually used to derive embryonic stem cells or until information which could link the identity of the donor(s) with the embryo was no longer retained, if applicable.

e.  During the consent process, the donor(s) were informed of the following:

i.  that the embryos would be used to derive hESCs for research;

ii.  what would happen to the embryos in the derivation of hESCs for research;

iii.  that hESCs derived from the embryos might be kept for many years;

iv.  that the donation was made without any restriction or direction regarding the individual(s) who may receive medical benefit from the use of the hESCs, such as who may be the recipients of cell transplants;

v.  that the research was not intended to provide direct medical benefit to the donor(s);

vi.  that the results of research using the hESCs may have commercial potential, and that the donor(s) would not receive financial or any other benefits from any such commercial development;

vii.  whether information that could identify the donor(s) would be available to researchers.

Alternatively (Section IIB), for stem cell lines established prior to promulgation of these Guidelines, eligibility can be established by submitting evidence to a newly-constituted Working Group of the Advisory Committee to the Director showing that "the hESCs were derived from human embryos:  1) that were created using in vitro fertilization for reproductive purposes and were no longer needed for this purpose; and 2) that were donated by donor(s) who gave voluntary written consent for the human embryos to be used for research purposes."

The Guidelines expressly prohibit funding of research on human embryonic stem cells or pluripotent cells introduced into a non-human primate blastocyst or animal breeding experiments involving introduction of human stem cells into non-human primate blastocysts.

The Notice reports that the Institutes received around 49,000 comments in response to the request for public comment published on April 23, 2009 in the Federal Register (74 Fed. Reg. 18578).  These comments came from "patient advocacy groups, scientists and scientific societies, academic institutions, medical organizations, religious organizations, and private citizens," as well as members of Congress.  The Notice contains a synopsis of these comments, which are included in groups identified as title, terminology and background, financial gain, IRB review, donation and informed consent, and monitoring and enforcement actions, among others.  The Notice describes how the draft guidelines were adapted or modified in response to these comments, or the Institutes distinguished or disputed the assertions in the comments.  Of note is the Institutes' assertions that it expects "that stem cell research materials developed with NIH funds, as well as associated intellectual property and data, will be distributed in accordance with the NIH's existing policies and guidance, including ''Sharing Biomedical Research Resources, Principles and Guidelines for Recipients of NIH Grants and Contracts' and 'Best Practices for the Licensing of Genomic Inventions'" (see NIH Policies & Reports page).  In addition, the Institutes will review de novo the circumstances surrounding development of existing stem cell lines rather than grandfathering these cells, applying ethical principles embodied inter alia in the Guidelines and 45 C.F.R. Part 46 Subpart A (Protection of Human Subjects).

The comments section also addresses the potential limitations on federal funding imposed by the Dickey amendment, wherein "an embryo is defined by Section 509, Omnibus Appropriations Act, 2009, Pub. L. 111-8, 3/11/09 . . . as any organism not protected as a human subject under 45 C.F.R. Part 46 that is derived by fertilization, parthenogenesis, cloning or any other means from one or more human gametes or human diploid cells."  The Guidelines state that:

[s]ince 1999, the Department of Health and Human Services (HHS) has consistently interpreted this provision as not applicable to research using hESCs, because hESCs are not embryos as defined by Section 509.  This long-standing interpretation has been left unchanged by Congress, which has annually reenacted the Dickey Amendment with full knowledge that HHS has been funding hESC research since 2001.  These guidelines therefore recognize the distinction, accepted by Congress, between the derivation of stem cells from an embryo that results in the embryo's destruction, for which federal funding is prohibited, and research involving hESCs that does not involve an embryo nor result in an embryo's destruction, for which federal funding is permitted.

While nicely expressing the distinction, it remains to be seen whether Congress acts to refine the Institutes' understanding of these restrictions, in light of the expansion in the scope of human embryonic stem cells intended by these Guidelines to become eligible for federal funding.