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Biotechnology and intellectual property As yet, the national and international law on the patentability of replicating organisms is uncertain, though the issuing of the "oncomouse" patent was indisputably a milestone in the patenting of life. The discussion has now progressed beyond merely the question of whether living organisms can be patented, and is now concerned with establishing solid criteria and re-interpreting existing patent law with regard to biological material. In this article (in two parts), R. S. Crespi examines both historical and recent developments, and the criteria courts have applied in granting or rejecting applications. The recently introduced EC directives and recommendations for the patenting of microbial and higher organisms, and their likely effects on court decisions are considered. Part I (see below) deals with the kinds of patent that can be obtained for inventions arising from research in general microbiology, and in the fields of recombinant DNA and hybridoma technology. The examples (presented in this section) of patents that have come before the courts are concerned with the production of substances of interest in human medicine and diagnostics, and the disputed points are rather specific ones which the court was called upon to settle in a particular context. Part 2 (see next month's TIBTECH) discusses some of the more general questions investigated by the international official bodies, including those which are especially relevant to the patenting of inventions in agricultural biotechnology.
Part 1: Patenting in biotechnology R. Stephen Crespi Many research scientists still ::eIntellectual property is a subject which attracts a great deal of interest gard the patent law as a 'jungle' al.~cl. on the part of those engaged in the find many of its concepts strange. development of biotechnology. This For example, the notion of what is is not surprising considering that the 'inventive' or 'obvious', compared to patent system provides an arena in what is already known, is not easy to which disputes over the ingenuity explain to those involved directly in and practical significance of new research, and whose criteria are biotechnology processes and prod- rather different from those of the ucts are settled. As part of this con- lawyers and judges who must deal tentious process, deciding who has with these issues in the legal arena. legal priority between competitive In this article (parts I and 2), it is research groups or industrial firms is proposed to outline how biotechusually a key issue which guarantees nological innovation is accommothe interest of the parties involved dated in the basic framework of patent laws that were created some and of observers alike. Once considered rather unfashion- time ago, and to explore the modern able or ethically dubious by scien- initiatives which have been taken to tists, the patenting of inventions improve the situation. arising from basic research (which may be commercially significant) is The international dimension Patent law has a long tradition of now viewed more favourably by many. With this changing climate, it international co-operation to solve is an easy step to become rather problems which are not confined to critical of the patent law and its one or a few countries. The most shortcomings in relation to any par- celebrated example of this is the Paris Convention of 18831 , of which ticular technology. there are now 100 member states, R. S. Crespi (patent consultant) is at and which establishes the basic prinBlackbird Cottage, Walton Avenue, ciple of equal treatment for domestic and foreign inventors. Where invenBognor Regis, Sussex P021 1NP, UK. (~) 1991, Elsevier Science Publishers Ltd (UK) 0167- 94130/91/$2.00
tars working in different countries seek to patent the same invention, the Convention allows an international priority to be claimed based on the filing of a patent application initially in one member state and ~ubsequently in others. The notion of a 'priority date ~ obtained in this way is very important in patent law because for almost all countries the party with the earliest date wins the contest, subject to certain provisos 1 which will not be detailed here. The main instrument of international collaboration in these matters is the World Intellectual Property Organisation (wIPe), based in Geneva, which administers the Paris Convention and all subsequent international conventions, w I P e often takes the initiative, or is sometimes prompted by a member state, to address a particular problem area but the Conventions and other results of their work must be ratified by member states and introduced into their national laws if they are to be effective. Once a member state has ratified a Convention it can be required to comply fully with its provisions but, in practice, w I P e does not act as an enforcement agency. In more recent times, the next international grouping of major significance to come into existence
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1980. This provides that culture colin the field of patents was the Eurolections can apply to be officially pean patent system. The legal basis recognized under the treaty as 'interof this system is the European Patent national depositary authorities' Convention (EPC)2 of 1973 which (IDAs) in which material may be began operation in 1978, and now deposited for these purposes. Any has 14 member states. All member states of the European Community IDA in any member state can be (EC) (except Portugal and Ireland) selected by the patent applicant for belong to the EPC, and some non-EC the deposit of the relevant biological states (Austria, Sweden and Switzer- material, and this deposit will sufland) belong. The EPC has the dis- fice for all member states in which tinction of being the first patent the applicant files for patent protecstatute to introduce specific pro- tion. The first major critical investivisions for biotechnology inventions. One of these concerns the use gation of the international patent of 'culture collections' as patent protection available for biotechdepositories for the placement of nology was published in 1985 by the microorganisms referred to in patent Organisation for Economic Coapplications. The other provision operation and Development (OECD)3. deals with the exclusion from As part of an extended survey of the patentability of certain plant-related industrial and social impact of bioinventions. These particular pro- technology, OECD examined the visions have been highly contro- patent law and made positive recomversial from the outset and continue mendations for reform. At the same time WIPO began a (geographically) to be hotly debated. even wider study, through its Committee of Experts on BiotechnoThe work of international logical Inventions 4 which has now organizations been working for over five years to Since the early 1970s, there has been general recognition of the fact provide 'suggested solutions' of the that biotechnology is a special case. problems of patent law in this field. First, it is undeniable that living In October 1988, the European material is far too complex to be Commission entered the arena with fully described with the precision a proposal for a directive to EC required by current patent law when member states s to solve these writing a specification for an inven- problems by a uniform approach tion which enables it to be put into throughout the Community. To practical use by workers of ordinary appreciate the efforts of these interskill in the art. In short, this problem national bodies it is necessary first to is one of 'reproducibility' from the outline what is patentable in this written description, and it is this technology. which has led to the practice of depositing the relevant biological Patentability ofmicrobiological material in a culture collection as a inventions After more than a century of exsupplement to the written text. Another problem is one which stems perience ranging over many other from the nature of biological ma- fields of technology, patent law has terial itself, which can be replicated established many important prein vast quantities from minute cedents (especially in the field of amounts of starting material. Because chemical inventions) which have of this, loss of legal control of pro- been taken over into their microprietary biological material can have biological counterparts with satisserious consequences for the pro- factory results. In classical microprietor. A third problem is that of biology (i.e. where the invention is variability on continued replication based on the discovery or developof original biological material and ment of a new microorganism, or the the question of 'sameness' between use of a particular organism, new or ancestral material and its descen- known), the usual types of patent dants after multiple generations. which can be obtained are summarThrough the WIPO channel, the ized in Box 1. The patent types are practice of depositing microorgan- discussed in the light of the main isms for patent purposes became types of 'claim' used in patents to international in the Budapest Treaty define what the patent covers. Patent of 19771 which came into force in claims are not 'advertising slogans'
but are verbal definitions of the rights in process, product, or other terms. For convenience, Box I refers to microorganisms, but it is now recognized that the categories apply equally to other types of biological material including cell lines and plant or animal cells. It should be noted that it is normal to have as many as possible of the claim types shown in Box 1 in any one patent in order to protect all ways of commercial exploitation of the basic invention. Current issues The early difficulties which appeared to be an obstacle to the granting of patents for living organisms have now been overcome and there is a clear trend towards allowing such patents, at least in the major industrialized countries. Undoubtedly, the decision of the US Supreme Court in 1980 in the Chakrabarty case 8 to allow the following claim to a microorgan.ism was of prime influence in this connection: 'A bacterium from the genus Pseudomonas containing therein at least two stable energy-generating plasmids, each of said plasmids providing a
separate hydrocarbon degradative pathway.' The subject of this claim is an organism made more effective in treating oil spills by manipulating a natural Pseudomonas strain (which contains only one such plasmid). Many examples of the categories listed in Box 1 will likewise be products and processes devised by human intervention in the works of Nature. However, the question remains as to whether something that already exists in Nature can be patented, whether it be an inanimate substance, an enzyme, a microorganism (or higher life form) or a gene? This is known as the 'product of Nature' problem in patent law and it has a long history.
Natural product patents Patent law makes a distinction between 'discovery' and 'invention' and excludes mere discovery from patentability. Some might say that the isolation of substances from Nature is mere discovery and not invention. The European Patent Office has rejected this line of reasoning and, in its official
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---Box I
Patent types: microbiological inventions Process inventions
Product inventions
• Producing a new microorganism Methods for producing new microorganisms can be protected in the form of process claims. The normal screening methods used in classical microbiology to isolate naturally occurring organisms would not usually rank as processes of the patentable kind. But methods for improving the properties of such organisms e.g. to reduce pathogenicity or otherwise enhance their biological utility would be patentable. An important proviso here is that the method must be a reproducible one. Novel methods of cultivating microorganisms could also
• End products of biosynthesis The most valuable product patents are those which cover a novel product produced by a known or, more usually, a newly discovered or newly developed organism. The most celebrated examples of these are the antibiotic patents which dominated the pharmaceutical patent scene for some decades after the second world war. The products will be protected in so-called 'product per se' claims which cover the substance itself (i.e. without being restricted to any one particular process of production). The process described in the patent application will be microbiological but the product claim would also cover the same product if produced by chemical synthesis.
be patentable e.g. using special media or other culture conditions. Constructing new organisms by genetic recombination or cell fusion now provide the more common types of method or process patent. • Usesofmicroorganisms Claims to the use of new (or known) organisms in industrial processes are now rather frequent. Use patents are really in the same category as process patents since their legal interpretation is often much the same. In some instances a 'use claim" can be a preferable form of protecting the invention in broad terms (e.g. 'the use of strain X to control agricultural pests'). Use claims are also convenient when the process is a generally known type of process and the novelty is primarily in the kind of microorganism used, for example, in fermentation-type processes to produce a secondary metabolite or alternatively where the biomass itself is useful, e.g. as edible protein for human consumption or animal feed. Other examples would be the use of the organism to extract valuable metals or to clean up an effluent or to perform a biotransformation reaction.
Guidelines 7, analyses the question as follows: 'To find a substance freely occurring in Nature is also mere discovery and therefore unpatentable. However, if a substance found in Nature has first to be isolated from its surroundings and a process for obtaining it is developed, that process is patentable. Moreover, if the substance can he properly characterized either by its structure, by the process by which it is obtained or by other parameters and it is "new" in the absolute sense of having no previously recognized existence, then the substance per se may be patentable. An example of such a case is that of a new substance which is discovered as being produced by a microorganism.' The example given in this Guideline would apply to antibiotics and other microbial metabolites, but another equally acceptable example is that of a newly isolated microorganism. The Guideline speaks of products having 'no previously
• The new microorganism Patents in which new microorganisms are themselves the subject of product claims were at one time highly controversial and still attract some opposition from those who are unhappy with the idea of 'patenting life'. The microorganism must be defined in an acceptable way to form the subject of a product claim and herein lies the opportunity for imaginative drafting, a topic which would require more discussion than space will allow. • Compositions (formulations) Claims to formulations of microorganisms designed to utilize their special properties are now very common. They will often be used in addition to 'use' claims. If the microorganism is 'known' (i.e. already available to the skilled person) the 'use' claim, and this type of product claim, will be the typical choice of claim. In the composition claim it will be important to define the other components of the composition as broadly as possible.
workable method of making pure product in quantity could patent not only the specific method but also could claim the pure product per se. The classical example of this was the production of vitamin B12 by fermentation and its isolation in bulk as the pure substance 8. Before 1951, vitamin B12 had been available only as crude liver extracts which were of no use for therapeutic application. A US District court upheld a 'product per se' claim to the pure vitamin defined by chemical and physical characteristics 9. Purified natural products The basic question of patenting The question of patenting purified products has arisen more than once purified natural products is being in US case law. The early court de- considered once again in current US cisions pointed to the conclusion litigation over patents for purified that a known substance cannot be naturally occurring proteins. These inpatented as a pure product solely clude Factor VIII (the blood clotting on the basis of its purity. However, factor important in the treatment where the substance was known of hemophilia) and erythropoietin previously only in the form of crude (the hormone which stimulates extracts which were of no practical the formation of red blood cells). use, an inventor who hau devised a In both cases the patent discloses
recognized existence' and is therefore rather limited. Much research in the natural-product field is concerned with active substances which are known to exist, the object being to isolate the substance in highly pure form or to devise synthetic methods for its production. The most topical example of this is the preparation of pure proteins by means of sophisticated purification techniques or by recombinant DNA methods.
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a particular method of purifying or isolating the protein. However, the claims are not limited to products produced by these methods and they also cover the proteins defined solely in terms of units of activity, a parameter connected with the degree of purity which these methods have made possible. The following is a short account of the way the US courts have approached these issues.
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In the meantime a patent on a recombinant method for producing VIII:C was issued in July 1988 to Genetics Institute Inc. The Scripps-Genentech litigation has had its ups and downs 11 for both parties, but is now close to a conclusion. • A m g e n Inc. vs. Genetics Institute Inc.
Erythropoietin is the natural protein which stimulates the production of red blood cells. Amgen holds • Scripps Clinic a n d Research US patent 12 for the preparation of Foundation vs. Genentech A method of separating Factor VIII erythropoietin by recombinant methprocoagulant activity protein (VIII:C) ods. However, the Amgen patent from the yon Willebrand factor pro- claims DNA sequences coding for tein (VIII:RP) with which it is com- the protein as well as the corresplexed in plasma was patented by ponding vectors and transformed Scripps m. The method of preparation cells, but the recombinant protein of the purified VIII:C component is not claimed as such. The US described in the patent involves re- Patent Office do not usually allow moval of VIII:RP from the VIII:C/ claims of the latter kind where the VIII:RP complex present in plasma protein is already known. In parallel with Amgen's patent (or a commeccial concentrate) by adsorption on a specific monoclonal application, Genetics Institute Inc. antibody. The patent has claims to (GI) had filed an application on a this particular method of purifi- method of purifying erythropoietin cation and claims to the product of from human urine by reverse-phase HPLC 13. However, unlike the such a method. Scripps realized later that they Scripps case, GI made sure of having wanted broader product claims to broad product claims in this patent, the purified product than.~hose ~hey including the following: had obtained in the patent. They 'Homogeneous erythropoietin charactherefore filed for an additional terized by a molecular weight of about patent in December 1983 and ob34 000 daltons on SDS PAGE, movetained Reissue patent 32,011 in ment as a single peak on reverse phase high performance liquid chromaOctober 1985. The reissue patent tography and a specific activity of at contained the following product least i60 000 IU per absorbance unit claim: at 280 nanometers.' 'A human VIII:Cpreparation having a The dispute in this case 14.15 inpotency in the range of 134 to 1172 volves similar issues to those in the units per ml and being substantially Factor VIII case although some diffree of VIII:RP.' ferences exist. This claim is directed to a product in The two instances of patent disterms of its potency and purity, but pute outlined above are examples of makes no mention of the method by conflict between a natural product which it is obtained. It would there- patent and a recombinant DNA fore appear to cover a purified VIII:C patent in which it has to be decided made by any method whatsoever. It who is free to operate commercially would thus cover Factor VIII:C with the recombinant protein. In produced by recombinant methods these two examples, and especially and having the specified potency. in the second, one question for the Genentech had produced the recom- court is whether a claim based enbinant product and were sued by tirely on the purification of the Scripps for infringement of their natural product can be permitted reissue patent. Genentech had them- also to cover the protein as made by selves applied to patent their tech- recombinant methods. The more nology but even if they were to basic issue, however, is whether succeed it would not affect the claims to purified natural products question as to whether their product are legally valid in such broad terms is dominated by the Scripps patent. as those used in the above examples.
The new biotechnology (recombinant DNA) In a recombinant DNA patent it will be common to have claims of the following types: (1) Recombinant protein products (and alleles, variants, derivatives). (2) DNA sequences coding for the products of (1). (3) Vectors containing the DNA sequences of (2). (4) Microorganisms, cell lines and other organisms transformed with vectors (3). (5) Processes for constructing the microorganisms etc. of (4). (6) Processes of producing products as in (1), by expression of DNA sequences in (2), in a recombinant host organism. • The Genentech vs. Wellcome case on tissue plasminogen activator 16-1e, under Genentech's UK patent 19, is a prime example of relying on a claim of type (1): 'Human tissue plasminogen activator as produced by recombinant DNA technology.' On commercial grounds, this type of claim was exactly what was required, but it failed on legal grounds, being held obvious, first, because a product of this kind was a known desirable objective and, second, because the product was obtained by using known methods of gene cloning. Thus, for the British Courts, being first in the race (to clone and express genes for known proteins) is not enough to support so broad a legal monopoly in such circumstances. The main problem here, for the patentee, was the absence of any commercially valuable fall-back position in the other claims of the patent which the court might have been prepared to sustain. There is no example yet of a non-British court taking the same line as in the t-PA case. The corresponding US patent 2° has no final product claim of the above kind but has claims of types (21, (3) and (4) noted above: 'A DNA isolate consisting essentially of a DNA sequence encoding human tissue plasminogen activator.' • The nerve growth factor case involved a decision by the US Patent Office Board of Appeals on an appli-
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cation dealing with a recombinant nerve growth factor protein 21. The decision was released for publication in March 1989. The Board rejected a claim to the protein defined by amino acid sequence, and freedom from other proteins of human origin.
'Human [5-NGFcomprising the amino acid sequence Ser-Ser-Ser-His... [full sequence given]... Lys-Ala-Val-Arg and which is free of other proteins of human origin.' The applicant's method of preparation was by using recombinant DNA techniques but the words of the claim are not limited to this method and would presumably extend to the natural protein purified by some totally different method. Nevertheless the US Patent Office decided to assume this method as implied in the claim because of the way the purity of the product was expressed. The claim was rejected in the absence of evidence that the recombinant product was significantly different from the natural material or possessed any unexpected properties.
Hybridoma technology
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• In the Wistar Institute case z3 the following claim came under scrutiny in the British Patent O;~ice: 'A process for producing viral antibodies comprising fusing a viral antibody producing cell and a myeloma cell to provide a fused cell hybrid, culturing said hybrid and collecting the viral antibodies.' The main distinction between this and the known basic process appears to rest on the term 'viral'. The UK Patent Office took the view that since the basic monoclonal antibody technique had already been published it was obvious to apply this technique to an area where it had already been considered valuable (i.e. for viruses). The Patent Office also would not allow a specific claim to the particular hybridoma developed for producing monoclonal antibodies against influenza virus. The applicant had not shown that the preparation of hybridomas of this type required anything other than the application of known techniques and moreover none of the hybridomas could be said to have particularly unusual advantages. It must be pointed out however that the US Patent Office did not take the same approach and in fact granted a US patent containing this broad process claim 24.
The patenting of hybridomas and other cell lines is based, in many countries, on the principles established for microorganism patents. Patents can be granted for novel • In the Hybritech case 25 the parent myelomas used to make hy- patentee successfully sued a number bridomas, the family of derived hy- of companies under its US patent 2e. bridomas and specific hybridomas, The invention lay in the use of the production of monoclonal monoclonal antibodies in place of antibodies in process or product prior art polyclonal antibodies in a terms, as well as applications known sandwich assay system. When the US Patent Office originally of these in therapy or diagnostics. Many patents are being granted on examined the application, it argued the basis of the use of monoclonal that it would be obvious to use antibodies and systems containing monoclonal antibodies in place of them in diagnostic assays. In these, polyclonals in conventional imthe claims are directed to the method munoassay protocols. This objection of assay, to particular reagent com- was overcome by including in the positions or to combinations of claims a numerical limitation regarding the affinity (binding power) materials useful in diagnostic kits. After the publication in 197522 of of the antibodies. In the District the basic Milstein-K6hler technique Court the patent was held invalid on and the subsequent appreciation of the grounds of obviousness, but this its more general importance it was was over-ruled by the Court of reasonable to assume that the patent- Appeals for the Federal Circuit ability of any application of this because the prior art was 'devoid of general procedure must rest on some any suggestion that monoclonal antispecial and non-obvious property or bodies can be used in the same advantage of the particular system fashion as polyclonals'. Also it was influenced by the commercial succonstructed.
cess of the patentee's {Hybritech) product and found that a three-year time gap between the first availability of monoclonals and the sale of the patentee's kits was long enough to indicate lack of obviousness. • By contrast, in Unilever PLC vs. Boehringer Mannheim 27 (immuneglobulins) the Technical Board of Appeals of the European Patent Office has held that the replacement of polyclonal antibodies by monoclonal antibodies in an immunepurification process required no inventive step at the priority date of the opposed patent (February 1981). The Unilever claims were to a process for the recovery of immuneglobulins of high purity and potency from milk by selective binding to a low-affinity monoclonal antibody specific to one or more of the immunoglobulins and itself bound to an insoluble carrier material. It was clear that the novelty lay only in the use of monoclonal antibodies in substitution for the prior use of monospecific polyclonal antibodies in the same basic process. This substitution was held to be the 'next logical step' and the 'desired logical and obvious step' to improve the purification process of the nearest prior art.
Concluding remarks The inconsistency of approach by the courts of different countries renders it difficult to make the predictions that patent lawyers are frequently asked to provide. If not a total jungle the situation is at least tangled, but this stems just as much from the complexities of the technology as from the law itself. It is bound to take time for the courts to become so universally familiar with biotechnology inventions that more uniform judgements result.
References NB UK case citations are to 'Reports of Patent Cases' (RPC). US case citations are to United States Patent Quarterly {USPQ). 1 Available from World Intellectual Property Organisation Publication (1989) Geneva 2 European Patent Convention (1987) 4th edn, available from European Patent Office, Munich 3 Bieir, F. K., Crespi, R. S. and Straus, J. (1985) Biotechnology and Patent Pro-
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tection - An International Review, Organisation for Economic Cooperation and Development 4 World Intellectual Property Organisation (1988} Industrial Property Protection of Biotechnological Inventions, Documents Bio T/CE/IV/2 and 3 5 European Commission (1988) Proposal for a Directive on the Legal Protection of Biotechnological Inventions, 496 final SYN 159, Brussels 6 Diamond vs. Chukrabarty 206 USPQ
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7 EPO Guidelines Tart C, Chapter IV,
[li
p. 31 8 (Aug. 1951) US Patent 2, 563, 794 9 Merck & Co. vs. Chase Chemical Co.
10 11 12 13 14 15 16 17 18
155 USPQ 139 (Nov. 1982) US Patent 4, 361, 509 (1989) 11 USPQ 2d 1187 (Oct. 1987) US Patent 4,703,008 (June 1987) US Patent 4,667,195 (1989) lO USPQ 2d 1906, 1909 (1989) 13 USPQ 2d 1737-1798 (1989) RPC 1473 Crespi, R. S. (1989) Nature 337, 26 Crespi, R. S. (1989) Intellectual Property in Business, Vol. 1, Issue 2,
(](IHHHli/iil//
Does co-suppression of sense genes in transgenic plants involve antisense RNA? Experiments with transgenic plants have shown that endogenous genes, or genes of bacterial origin stably integrated into the nuclear genome, can be down-regulated by antisense genes (see Refs 1,2 for reviews). Recently, it has been reported that sense transgenes also effectively down-regulate the expression of chalcone synthase and dihydroflavonol-4-reductase in petunia flowers 3'4 and polygalacturonase in tomato fruit s . These results, together with a number of other genetic phenomena, were reviewed by Jorgensen in his recent article 6, though no clear mechanism for the inhibition of gene expression was suggested. We propose that antisense RNA, generated by transcription of the 'wrong' DNA strand of the inserted sense transgene, is responsible for down-regulation of the endogenous gene in these transgenic plants. This unexpected transcription of antisense RNA could arise from a converging promoter, either within or near the inserted T-DNA. If this proposal is correct, it will be necessary to re-design expression cassettes for Work with transgenic plants. We have noted several striking similarities between downregulation of gene expression by sense and antisense transgenes. (1) Both are remarkably effective and specific for the target gene 4.s.7.e.
(2) They cause a dramatic reduction in the amount of mRNA that accumulates from the endogenous gene 3-5.7-1°. {3) Both lead to the production of similar fragments of endogenous mRNA s. (4) Mutual gene inactivation, termed cosuppression by Jorgensen 6, is a feature of both sense and antisense gene action 3-5'11. Inhibition of gene expression by antisense RNA is presumed to depend on specific base pairing between sense and antisense transcripts ~.2. Since the rate of transcription of the endogenous gene appears not to be affected ~2, it is probable that the antisense transcripts base-pair with pre-mRNA or mRNA and prevent its accumulation post-transcriptionally, because the hybrids formed are unstable and rapidly degraded 9.13. We speculate that inactivation of gene expression by sense transgenes also involves antisense RNA (Fig. 1). A common antisense mechanism would explain the remarkable similarity in the effect of sense and antisense genes in transgenic tomatoes 5 and petunia flowers 6. Sense and antisense genes have been used to down-regulate genes in the anthocyanin biosynthesis pathway in petunia flowers, creating unusual white patterns on a red or blue background 3.4.s.1°. Although there is considerable variation in both sense and antisense trans-
25-27 19 (Feb. 1986) UK Patent 2,119,804 20 (Aug. 1988) US Patent 4,766,075 21 Ex parte Gray, 10 USPQ 2d 1922 22 K~hler, G. and Milstein, C. (1975) Nature 256, 495 23 (1983) Wistar Institute's UK Patent Application 2,000, 186, RPC 255 24 (April 1980) US Patent 4,196,265 25 Hybritech vs. Monoclonal Antibodies Inc. 231 USPQ 81 26 (Mar. 1983) US Patent 4,376,110 27 Unilever vs. Boehringer Mannheim,
EPO Decision T499/88 (in press)
formants, the patterns produced by sense transgenes are different from those produced by antisense transgenes 4. This suggests that the gene responsible for down-regulation in the sense transformants has a different promoter from the one responsible in the antisense transformants. At first sight, such a conclusion seems to contradict the evidence, since sense and antisense transgene constructs frequently contain the same promoter 3-s'9'1°. However, in the published studies reporting gene inactivation by s e n s e transgenes, the T-DNA transferred to plants contain the sense transgene driven by the CaMV 35S promoter located so that it converges with the chimaeric nos NPT II kanamycin resistance gene used for selection of transgenic plants 3-5 (Fig. 1). With such a construct, it is possible that readthrough transcription could occur from the nos NPT II gene, continuing past the normal processing and polyadenylation sites at the 3' end of the mRNA, producing low levels of antisense RNA from the adjacent sense transgene. There are examples where transcription termination by RNA polymerase II occurs as a progressive reduction in transcription, with heterogeneous 3' endpoints, rather than an abrupt termination at a specific site 14. Furthermore, a number of studies have shown that the 3' processing of a nascent transcript does not necessarily disrupt the transcription complex, allowing readthrough of the polymerase into other regions of the DNA 14-17. These antisense transcripts might not be full length, but sequences as short as 41 bp have been shown to be effective TM, and low concentrations of antisense RNA
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Biotechnology and intellectual property As yet, the national and international law on the patentability of replicating organisms is uncertain, though the issuing of the "oncomouse" patent was indisputably a milestone in the patenting of life. The discussion has now progressed beyond merely the question of whether living organisms can be patented, and is now concerned with establishing solid criteria and re-interpreting existing patent law with regard to biological material. In this article (in two parts), R. S. Crespi examines both historical and recent developments, and the criteria courts have applied in granting or rejecting applications. The recently introduced EC directives and recommendations for the patenting of microbial and higher organisms, and their likely effects on court decisions are considered. Part I (see below) deals with the kinds of patent that can be obtained for inventions arising from research in general microbiology, and in the fields of recombinant DNA and hybridoma technology. The examples (presented in this section) of patents that have come before the courts are concerned with the production of substances of interest in human medicine and diagnostics, and the disputed points are rather specific ones which the court was called upon to settle in a particular context. Part 2 (see next month's TIBTECH) discusses some of the more general questions investigated by the international official bodies, including those which are especially relevant to the patenting of inventions in agricultural biotechnology.
Part 1: Patenting in biotechnology R. Stephen Crespi Many research scientists still ::eIntellectual property is a subject which attracts a great deal of interest gard the patent law as a 'jungle' al.~cl. on the part of those engaged in the find many of its concepts strange. development of biotechnology. This For example, the notion of what is is not surprising considering that the 'inventive' or 'obvious', compared to patent system provides an arena in what is already known, is not easy to which disputes over the ingenuity explain to those involved directly in and practical significance of new research, and whose criteria are biotechnology processes and prod- rather different from those of the ucts are settled. As part of this con- lawyers and judges who must deal tentious process, deciding who has with these issues in the legal arena. legal priority between competitive In this article (parts I and 2), it is research groups or industrial firms is proposed to outline how biotechusually a key issue which guarantees nological innovation is accommothe interest of the parties involved dated in the basic framework of patent laws that were created some and of observers alike. Once considered rather unfashion- time ago, and to explore the modern able or ethically dubious by scien- initiatives which have been taken to tists, the patenting of inventions improve the situation. arising from basic research (which may be commercially significant) is The international dimension Patent law has a long tradition of now viewed more favourably by many. With this changing climate, it international co-operation to solve is an easy step to become rather problems which are not confined to critical of the patent law and its one or a few countries. The most shortcomings in relation to any par- celebrated example of this is the Paris Convention of 18831 , of which ticular technology. there are now 100 member states, R. S. Crespi (patent consultant) is at and which establishes the basic prinBlackbird Cottage, Walton Avenue, ciple of equal treatment for domestic and foreign inventors. Where invenBognor Regis, Sussex P021 1NP, UK. (~) 1991, Elsevier Science Publishers Ltd (UK) 0167- 94130/91/$2.00
tars working in different countries seek to patent the same invention, the Convention allows an international priority to be claimed based on the filing of a patent application initially in one member state and ~ubsequently in others. The notion of a 'priority date ~ obtained in this way is very important in patent law because for almost all countries the party with the earliest date wins the contest, subject to certain provisos 1 which will not be detailed here. The main instrument of international collaboration in these matters is the World Intellectual Property Organisation (wIPe), based in Geneva, which administers the Paris Convention and all subsequent international conventions, w I P e often takes the initiative, or is sometimes prompted by a member state, to address a particular problem area but the Conventions and other results of their work must be ratified by member states and introduced into their national laws if they are to be effective. Once a member state has ratified a Convention it can be required to comply fully with its provisions but, in practice, w I P e does not act as an enforcement agency. In more recent times, the next international grouping of major significance to come into existence
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1980. This provides that culture colin the field of patents was the Eurolections can apply to be officially pean patent system. The legal basis recognized under the treaty as 'interof this system is the European Patent national depositary authorities' Convention (EPC)2 of 1973 which (IDAs) in which material may be began operation in 1978, and now deposited for these purposes. Any has 14 member states. All member states of the European Community IDA in any member state can be (EC) (except Portugal and Ireland) selected by the patent applicant for belong to the EPC, and some non-EC the deposit of the relevant biological states (Austria, Sweden and Switzer- material, and this deposit will sufland) belong. The EPC has the dis- fice for all member states in which tinction of being the first patent the applicant files for patent protecstatute to introduce specific pro- tion. The first major critical investivisions for biotechnology inventions. One of these concerns the use gation of the international patent of 'culture collections' as patent protection available for biotechdepositories for the placement of nology was published in 1985 by the microorganisms referred to in patent Organisation for Economic Coapplications. The other provision operation and Development (OECD)3. deals with the exclusion from As part of an extended survey of the patentability of certain plant-related industrial and social impact of bioinventions. These particular pro- technology, OECD examined the visions have been highly contro- patent law and made positive recomversial from the outset and continue mendations for reform. At the same time WIPO began a (geographically) to be hotly debated. even wider study, through its Committee of Experts on BiotechnoThe work of international logical Inventions 4 which has now organizations been working for over five years to Since the early 1970s, there has been general recognition of the fact provide 'suggested solutions' of the that biotechnology is a special case. problems of patent law in this field. First, it is undeniable that living In October 1988, the European material is far too complex to be Commission entered the arena with fully described with the precision a proposal for a directive to EC required by current patent law when member states s to solve these writing a specification for an inven- problems by a uniform approach tion which enables it to be put into throughout the Community. To practical use by workers of ordinary appreciate the efforts of these interskill in the art. In short, this problem national bodies it is necessary first to is one of 'reproducibility' from the outline what is patentable in this written description, and it is this technology. which has led to the practice of depositing the relevant biological Patentability ofmicrobiological material in a culture collection as a inventions After more than a century of exsupplement to the written text. Another problem is one which stems perience ranging over many other from the nature of biological ma- fields of technology, patent law has terial itself, which can be replicated established many important prein vast quantities from minute cedents (especially in the field of amounts of starting material. Because chemical inventions) which have of this, loss of legal control of pro- been taken over into their microprietary biological material can have biological counterparts with satisserious consequences for the pro- factory results. In classical microprietor. A third problem is that of biology (i.e. where the invention is variability on continued replication based on the discovery or developof original biological material and ment of a new microorganism, or the the question of 'sameness' between use of a particular organism, new or ancestral material and its descen- known), the usual types of patent dants after multiple generations. which can be obtained are summarThrough the WIPO channel, the ized in Box 1. The patent types are practice of depositing microorgan- discussed in the light of the main isms for patent purposes became types of 'claim' used in patents to international in the Budapest Treaty define what the patent covers. Patent of 19771 which came into force in claims are not 'advertising slogans'
but are verbal definitions of the rights in process, product, or other terms. For convenience, Box I refers to microorganisms, but it is now recognized that the categories apply equally to other types of biological material including cell lines and plant or animal cells. It should be noted that it is normal to have as many as possible of the claim types shown in Box 1 in any one patent in order to protect all ways of commercial exploitation of the basic invention. Current issues The early difficulties which appeared to be an obstacle to the granting of patents for living organisms have now been overcome and there is a clear trend towards allowing such patents, at least in the major industrialized countries. Undoubtedly, the decision of the US Supreme Court in 1980 in the Chakrabarty case 8 to allow the following claim to a microorgan.ism was of prime influence in this connection: 'A bacterium from the genus Pseudomonas containing therein at least two stable energy-generating plasmids, each of said plasmids providing a
separate hydrocarbon degradative pathway.' The subject of this claim is an organism made more effective in treating oil spills by manipulating a natural Pseudomonas strain (which contains only one such plasmid). Many examples of the categories listed in Box 1 will likewise be products and processes devised by human intervention in the works of Nature. However, the question remains as to whether something that already exists in Nature can be patented, whether it be an inanimate substance, an enzyme, a microorganism (or higher life form) or a gene? This is known as the 'product of Nature' problem in patent law and it has a long history.
Natural product patents Patent law makes a distinction between 'discovery' and 'invention' and excludes mere discovery from patentability. Some might say that the isolation of substances from Nature is mere discovery and not invention. The European Patent Office has rejected this line of reasoning and, in its official
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---Box I
Patent types: microbiological inventions Process inventions
Product inventions
• Producing a new microorganism Methods for producing new microorganisms can be protected in the form of process claims. The normal screening methods used in classical microbiology to isolate naturally occurring organisms would not usually rank as processes of the patentable kind. But methods for improving the properties of such organisms e.g. to reduce pathogenicity or otherwise enhance their biological utility would be patentable. An important proviso here is that the method must be a reproducible one. Novel methods of cultivating microorganisms could also
• End products of biosynthesis The most valuable product patents are those which cover a novel product produced by a known or, more usually, a newly discovered or newly developed organism. The most celebrated examples of these are the antibiotic patents which dominated the pharmaceutical patent scene for some decades after the second world war. The products will be protected in so-called 'product per se' claims which cover the substance itself (i.e. without being restricted to any one particular process of production). The process described in the patent application will be microbiological but the product claim would also cover the same product if produced by chemical synthesis.
be patentable e.g. using special media or other culture conditions. Constructing new organisms by genetic recombination or cell fusion now provide the more common types of method or process patent. • Usesofmicroorganisms Claims to the use of new (or known) organisms in industrial processes are now rather frequent. Use patents are really in the same category as process patents since their legal interpretation is often much the same. In some instances a 'use claim" can be a preferable form of protecting the invention in broad terms (e.g. 'the use of strain X to control agricultural pests'). Use claims are also convenient when the process is a generally known type of process and the novelty is primarily in the kind of microorganism used, for example, in fermentation-type processes to produce a secondary metabolite or alternatively where the biomass itself is useful, e.g. as edible protein for human consumption or animal feed. Other examples would be the use of the organism to extract valuable metals or to clean up an effluent or to perform a biotransformation reaction.
Guidelines 7, analyses the question as follows: 'To find a substance freely occurring in Nature is also mere discovery and therefore unpatentable. However, if a substance found in Nature has first to be isolated from its surroundings and a process for obtaining it is developed, that process is patentable. Moreover, if the substance can he properly characterized either by its structure, by the process by which it is obtained or by other parameters and it is "new" in the absolute sense of having no previously recognized existence, then the substance per se may be patentable. An example of such a case is that of a new substance which is discovered as being produced by a microorganism.' The example given in this Guideline would apply to antibiotics and other microbial metabolites, but another equally acceptable example is that of a newly isolated microorganism. The Guideline speaks of products having 'no previously
• The new microorganism Patents in which new microorganisms are themselves the subject of product claims were at one time highly controversial and still attract some opposition from those who are unhappy with the idea of 'patenting life'. The microorganism must be defined in an acceptable way to form the subject of a product claim and herein lies the opportunity for imaginative drafting, a topic which would require more discussion than space will allow. • Compositions (formulations) Claims to formulations of microorganisms designed to utilize their special properties are now very common. They will often be used in addition to 'use' claims. If the microorganism is 'known' (i.e. already available to the skilled person) the 'use' claim, and this type of product claim, will be the typical choice of claim. In the composition claim it will be important to define the other components of the composition as broadly as possible.
workable method of making pure product in quantity could patent not only the specific method but also could claim the pure product per se. The classical example of this was the production of vitamin B12 by fermentation and its isolation in bulk as the pure substance 8. Before 1951, vitamin B12 had been available only as crude liver extracts which were of no use for therapeutic application. A US District court upheld a 'product per se' claim to the pure vitamin defined by chemical and physical characteristics 9. Purified natural products The basic question of patenting The question of patenting purified products has arisen more than once purified natural products is being in US case law. The early court de- considered once again in current US cisions pointed to the conclusion litigation over patents for purified that a known substance cannot be naturally occurring proteins. These inpatented as a pure product solely clude Factor VIII (the blood clotting on the basis of its purity. However, factor important in the treatment where the substance was known of hemophilia) and erythropoietin previously only in the form of crude (the hormone which stimulates extracts which were of no practical the formation of red blood cells). use, an inventor who hau devised a In both cases the patent discloses
recognized existence' and is therefore rather limited. Much research in the natural-product field is concerned with active substances which are known to exist, the object being to isolate the substance in highly pure form or to devise synthetic methods for its production. The most topical example of this is the preparation of pure proteins by means of sophisticated purification techniques or by recombinant DNA methods.
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a particular method of purifying or isolating the protein. However, the claims are not limited to products produced by these methods and they also cover the proteins defined solely in terms of units of activity, a parameter connected with the degree of purity which these methods have made possible. The following is a short account of the way the US courts have approached these issues.
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In the meantime a patent on a recombinant method for producing VIII:C was issued in July 1988 to Genetics Institute Inc. The Scripps-Genentech litigation has had its ups and downs 11 for both parties, but is now close to a conclusion. • A m g e n Inc. vs. Genetics Institute Inc.
Erythropoietin is the natural protein which stimulates the production of red blood cells. Amgen holds • Scripps Clinic a n d Research US patent 12 for the preparation of Foundation vs. Genentech A method of separating Factor VIII erythropoietin by recombinant methprocoagulant activity protein (VIII:C) ods. However, the Amgen patent from the yon Willebrand factor pro- claims DNA sequences coding for tein (VIII:RP) with which it is com- the protein as well as the corresplexed in plasma was patented by ponding vectors and transformed Scripps m. The method of preparation cells, but the recombinant protein of the purified VIII:C component is not claimed as such. The US described in the patent involves re- Patent Office do not usually allow moval of VIII:RP from the VIII:C/ claims of the latter kind where the VIII:RP complex present in plasma protein is already known. In parallel with Amgen's patent (or a commeccial concentrate) by adsorption on a specific monoclonal application, Genetics Institute Inc. antibody. The patent has claims to (GI) had filed an application on a this particular method of purifi- method of purifying erythropoietin cation and claims to the product of from human urine by reverse-phase HPLC 13. However, unlike the such a method. Scripps realized later that they Scripps case, GI made sure of having wanted broader product claims to broad product claims in this patent, the purified product than.~hose ~hey including the following: had obtained in the patent. They 'Homogeneous erythropoietin charactherefore filed for an additional terized by a molecular weight of about patent in December 1983 and ob34 000 daltons on SDS PAGE, movetained Reissue patent 32,011 in ment as a single peak on reverse phase high performance liquid chromaOctober 1985. The reissue patent tography and a specific activity of at contained the following product least i60 000 IU per absorbance unit claim: at 280 nanometers.' 'A human VIII:Cpreparation having a The dispute in this case 14.15 inpotency in the range of 134 to 1172 volves similar issues to those in the units per ml and being substantially Factor VIII case although some diffree of VIII:RP.' ferences exist. This claim is directed to a product in The two instances of patent disterms of its potency and purity, but pute outlined above are examples of makes no mention of the method by conflict between a natural product which it is obtained. It would there- patent and a recombinant DNA fore appear to cover a purified VIII:C patent in which it has to be decided made by any method whatsoever. It who is free to operate commercially would thus cover Factor VIII:C with the recombinant protein. In produced by recombinant methods these two examples, and especially and having the specified potency. in the second, one question for the Genentech had produced the recom- court is whether a claim based enbinant product and were sued by tirely on the purification of the Scripps for infringement of their natural product can be permitted reissue patent. Genentech had them- also to cover the protein as made by selves applied to patent their tech- recombinant methods. The more nology but even if they were to basic issue, however, is whether succeed it would not affect the claims to purified natural products question as to whether their product are legally valid in such broad terms is dominated by the Scripps patent. as those used in the above examples.
The new biotechnology (recombinant DNA) In a recombinant DNA patent it will be common to have claims of the following types: (1) Recombinant protein products (and alleles, variants, derivatives). (2) DNA sequences coding for the products of (1). (3) Vectors containing the DNA sequences of (2). (4) Microorganisms, cell lines and other organisms transformed with vectors (3). (5) Processes for constructing the microorganisms etc. of (4). (6) Processes of producing products as in (1), by expression of DNA sequences in (2), in a recombinant host organism. • The Genentech vs. Wellcome case on tissue plasminogen activator 16-1e, under Genentech's UK patent 19, is a prime example of relying on a claim of type (1): 'Human tissue plasminogen activator as produced by recombinant DNA technology.' On commercial grounds, this type of claim was exactly what was required, but it failed on legal grounds, being held obvious, first, because a product of this kind was a known desirable objective and, second, because the product was obtained by using known methods of gene cloning. Thus, for the British Courts, being first in the race (to clone and express genes for known proteins) is not enough to support so broad a legal monopoly in such circumstances. The main problem here, for the patentee, was the absence of any commercially valuable fall-back position in the other claims of the patent which the court might have been prepared to sustain. There is no example yet of a non-British court taking the same line as in the t-PA case. The corresponding US patent 2° has no final product claim of the above kind but has claims of types (21, (3) and (4) noted above: 'A DNA isolate consisting essentially of a DNA sequence encoding human tissue plasminogen activator.' • The nerve growth factor case involved a decision by the US Patent Office Board of Appeals on an appli-
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cation dealing with a recombinant nerve growth factor protein 21. The decision was released for publication in March 1989. The Board rejected a claim to the protein defined by amino acid sequence, and freedom from other proteins of human origin.
'Human [5-NGFcomprising the amino acid sequence Ser-Ser-Ser-His... [full sequence given]... Lys-Ala-Val-Arg and which is free of other proteins of human origin.' The applicant's method of preparation was by using recombinant DNA techniques but the words of the claim are not limited to this method and would presumably extend to the natural protein purified by some totally different method. Nevertheless the US Patent Office decided to assume this method as implied in the claim because of the way the purity of the product was expressed. The claim was rejected in the absence of evidence that the recombinant product was significantly different from the natural material or possessed any unexpected properties.
Hybridoma technology
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• In the Wistar Institute case z3 the following claim came under scrutiny in the British Patent O;~ice: 'A process for producing viral antibodies comprising fusing a viral antibody producing cell and a myeloma cell to provide a fused cell hybrid, culturing said hybrid and collecting the viral antibodies.' The main distinction between this and the known basic process appears to rest on the term 'viral'. The UK Patent Office took the view that since the basic monoclonal antibody technique had already been published it was obvious to apply this technique to an area where it had already been considered valuable (i.e. for viruses). The Patent Office also would not allow a specific claim to the particular hybridoma developed for producing monoclonal antibodies against influenza virus. The applicant had not shown that the preparation of hybridomas of this type required anything other than the application of known techniques and moreover none of the hybridomas could be said to have particularly unusual advantages. It must be pointed out however that the US Patent Office did not take the same approach and in fact granted a US patent containing this broad process claim 24.
The patenting of hybridomas and other cell lines is based, in many countries, on the principles established for microorganism patents. Patents can be granted for novel • In the Hybritech case 25 the parent myelomas used to make hy- patentee successfully sued a number bridomas, the family of derived hy- of companies under its US patent 2e. bridomas and specific hybridomas, The invention lay in the use of the production of monoclonal monoclonal antibodies in place of antibodies in process or product prior art polyclonal antibodies in a terms, as well as applications known sandwich assay system. When the US Patent Office originally of these in therapy or diagnostics. Many patents are being granted on examined the application, it argued the basis of the use of monoclonal that it would be obvious to use antibodies and systems containing monoclonal antibodies in place of them in diagnostic assays. In these, polyclonals in conventional imthe claims are directed to the method munoassay protocols. This objection of assay, to particular reagent com- was overcome by including in the positions or to combinations of claims a numerical limitation regarding the affinity (binding power) materials useful in diagnostic kits. After the publication in 197522 of of the antibodies. In the District the basic Milstein-K6hler technique Court the patent was held invalid on and the subsequent appreciation of the grounds of obviousness, but this its more general importance it was was over-ruled by the Court of reasonable to assume that the patent- Appeals for the Federal Circuit ability of any application of this because the prior art was 'devoid of general procedure must rest on some any suggestion that monoclonal antispecial and non-obvious property or bodies can be used in the same advantage of the particular system fashion as polyclonals'. Also it was influenced by the commercial succonstructed.
cess of the patentee's {Hybritech) product and found that a three-year time gap between the first availability of monoclonals and the sale of the patentee's kits was long enough to indicate lack of obviousness. • By contrast, in Unilever PLC vs. Boehringer Mannheim 27 (immuneglobulins) the Technical Board of Appeals of the European Patent Office has held that the replacement of polyclonal antibodies by monoclonal antibodies in an immunepurification process required no inventive step at the priority date of the opposed patent (February 1981). The Unilever claims were to a process for the recovery of immuneglobulins of high purity and potency from milk by selective binding to a low-affinity monoclonal antibody specific to one or more of the immunoglobulins and itself bound to an insoluble carrier material. It was clear that the novelty lay only in the use of monoclonal antibodies in substitution for the prior use of monospecific polyclonal antibodies in the same basic process. This substitution was held to be the 'next logical step' and the 'desired logical and obvious step' to improve the purification process of the nearest prior art.
Concluding remarks The inconsistency of approach by the courts of different countries renders it difficult to make the predictions that patent lawyers are frequently asked to provide. If not a total jungle the situation is at least tangled, but this stems just as much from the complexities of the technology as from the law itself. It is bound to take time for the courts to become so universally familiar with biotechnology inventions that more uniform judgements result.
References NB UK case citations are to 'Reports of Patent Cases' (RPC). US case citations are to United States Patent Quarterly {USPQ). 1 Available from World Intellectual Property Organisation Publication (1989) Geneva 2 European Patent Convention (1987) 4th edn, available from European Patent Office, Munich 3 Bieir, F. K., Crespi, R. S. and Straus, J. (1985) Biotechnology and Patent Pro-
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tection - An International Review, Organisation for Economic Cooperation and Development 4 World Intellectual Property Organisation (1988} Industrial Property Protection of Biotechnological Inventions, Documents Bio T/CE/IV/2 and 3 5 European Commission (1988) Proposal for a Directive on the Legal Protection of Biotechnological Inventions, 496 final SYN 159, Brussels 6 Diamond vs. Chukrabarty 206 USPQ
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7 EPO Guidelines Tart C, Chapter IV,
[li
p. 31 8 (Aug. 1951) US Patent 2, 563, 794 9 Merck & Co. vs. Chase Chemical Co.
10 11 12 13 14 15 16 17 18
155 USPQ 139 (Nov. 1982) US Patent 4, 361, 509 (1989) 11 USPQ 2d 1187 (Oct. 1987) US Patent 4,703,008 (June 1987) US Patent 4,667,195 (1989) lO USPQ 2d 1906, 1909 (1989) 13 USPQ 2d 1737-1798 (1989) RPC 1473 Crespi, R. S. (1989) Nature 337, 26 Crespi, R. S. (1989) Intellectual Property in Business, Vol. 1, Issue 2,
(](IHHHli/iil//
Does co-suppression of sense genes in transgenic plants involve antisense RNA? Experiments with transgenic plants have shown that endogenous genes, or genes of bacterial origin stably integrated into the nuclear genome, can be down-regulated by antisense genes (see Refs 1,2 for reviews). Recently, it has been reported that sense transgenes also effectively down-regulate the expression of chalcone synthase and dihydroflavonol-4-reductase in petunia flowers 3'4 and polygalacturonase in tomato fruit s . These results, together with a number of other genetic phenomena, were reviewed by Jorgensen in his recent article 6, though no clear mechanism for the inhibition of gene expression was suggested. We propose that antisense RNA, generated by transcription of the 'wrong' DNA strand of the inserted sense transgene, is responsible for down-regulation of the endogenous gene in these transgenic plants. This unexpected transcription of antisense RNA could arise from a converging promoter, either within or near the inserted T-DNA. If this proposal is correct, it will be necessary to re-design expression cassettes for Work with transgenic plants. We have noted several striking similarities between downregulation of gene expression by sense and antisense transgenes. (1) Both are remarkably effective and specific for the target gene 4.s.7.e.
(2) They cause a dramatic reduction in the amount of mRNA that accumulates from the endogenous gene 3-5.7-1°. {3) Both lead to the production of similar fragments of endogenous mRNA s. (4) Mutual gene inactivation, termed cosuppression by Jorgensen 6, is a feature of both sense and antisense gene action 3-5'11. Inhibition of gene expression by antisense RNA is presumed to depend on specific base pairing between sense and antisense transcripts ~.2. Since the rate of transcription of the endogenous gene appears not to be affected ~2, it is probable that the antisense transcripts base-pair with pre-mRNA or mRNA and prevent its accumulation post-transcriptionally, because the hybrids formed are unstable and rapidly degraded 9.13. We speculate that inactivation of gene expression by sense transgenes also involves antisense RNA (Fig. 1). A common antisense mechanism would explain the remarkable similarity in the effect of sense and antisense genes in transgenic tomatoes 5 and petunia flowers 6. Sense and antisense genes have been used to down-regulate genes in the anthocyanin biosynthesis pathway in petunia flowers, creating unusual white patterns on a red or blue background 3.4.s.1°. Although there is considerable variation in both sense and antisense trans-
25-27 19 (Feb. 1986) UK Patent 2,119,804 20 (Aug. 1988) US Patent 4,766,075 21 Ex parte Gray, 10 USPQ 2d 1922 22 K~hler, G. and Milstein, C. (1975) Nature 256, 495 23 (1983) Wistar Institute's UK Patent Application 2,000, 186, RPC 255 24 (April 1980) US Patent 4,196,265 25 Hybritech vs. Monoclonal Antibodies Inc. 231 USPQ 81 26 (Mar. 1983) US Patent 4,376,110 27 Unilever vs. Boehringer Mannheim,
EPO Decision T499/88 (in press)
formants, the patterns produced by sense transgenes are different from those produced by antisense transgenes 4. This suggests that the gene responsible for down-regulation in the sense transformants has a different promoter from the one responsible in the antisense transformants. At first sight, such a conclusion seems to contradict the evidence, since sense and antisense transgene constructs frequently contain the same promoter 3-s'9'1°. However, in the published studies reporting gene inactivation by s e n s e transgenes, the T-DNA transferred to plants contain the sense transgene driven by the CaMV 35S promoter located so that it converges with the chimaeric nos NPT II kanamycin resistance gene used for selection of transgenic plants 3-5 (Fig. 1). With such a construct, it is possible that readthrough transcription could occur from the nos NPT II gene, continuing past the normal processing and polyadenylation sites at the 3' end of the mRNA, producing low levels of antisense RNA from the adjacent sense transgene. There are examples where transcription termination by RNA polymerase II occurs as a progressive reduction in transcription, with heterogeneous 3' endpoints, rather than an abrupt termination at a specific site 14. Furthermore, a number of studies have shown that the 3' processing of a nascent transcript does not necessarily disrupt the transcription complex, allowing readthrough of the polymerase into other regions of the DNA 14-17. These antisense transcripts might not be full length, but sequences as short as 41 bp have been shown to be effective TM, and low concentrations of antisense RNA
