T 0632/08 (Expression antimicrobial peptides in plastids/AUBURN) 18-01-2011

Summary of Facts and Submissions

I. European patent application no. 01 913 116.8, published as WO 01/64927 (hereinafter "the application as filed"), was refused by the examining division on the grounds that the requirements of Article 56 EPC were not fulfilled. The examining division considered that the set of claims filed with letter dated 30 September 2005 and received by fax on 3 October 2005 did not fulfil the requirements of Article 56 EPC.

II. The applicants (appellants) lodged an appeal against the decision of the examining division. Together with the statement setting out their grounds of appeal, the appellants filed two sets of claims as, respectively, main request and auxiliary request, the former being identical to the set of claims underlying the decision under appeal. As a subsidiary request, oral proceedings were requested.

III. The appellants were summoned to oral proceedings. In a communication pursuant to Article 15(1) of the Rules of Procedure of the Boards of Appeal (RPBA) attached to the summons, the appellants were informed on the board's preliminary, non-binding opinion on some of the issues to be discussed at the upcoming oral proceedings, in particular regarding Articles 84, 83 and 56 EPC for both the main request and the auxiliary request, and Article 123(2) EPC for the auxiliary request. In view of this opinion, the board was inclined to dismiss the appeal.

IV. No substantive reply was received from the appellants which, however, informed the board of their intention not to attend the oral proceedings.

V. Oral proceedings were held on 18 January 2011 in the absence of the appellants. At the end of the proceedings, the board announced the decision that the appeal was dismissed.

VI. The following documents are referred to in the present decision:

D1: WO 99/10513, published on 4 March 1999;

D3: WO 99/06564, published on 11 February 1999;

D4: N.P. Everett, "Design of Antifungal Peptides for Agricultural Applications", Chapter 20 in "ACS Symposium Series. Natural and Engineered Pest Management Agents." Ed. P.A. Hedin et al., 1994, pages 278 to 291.

VII. As stated in point II supra, the appellants' main request was identical to the set of 18 claims underlying the decision under appeal. Claim 1 read as follows:

"1. A stable plastid transformation and expression vector which comprises an expression cassette comprising, as operably linked components in the 5' to the 3' direction of translation, a promoter operative in said plastid, a selectable marker sequence, a heterologous DNA sequence coding for one or more cytotoxic antimicrobial peptides selected from the groups of defensins, PGLA (frog skin), cecropins, apidaecins, melittin, bombinin and magainin, transcription termination functional in said plastid, and flanking each side of the expression cassette, flanking DNA sequences which are homologous to a DNA sequence of the target plastid genome, whereby stable integration of the heterologous coding sequence into the plastid genome of the target plant is facilitated through homologous recombination of the flanking sequence with the homologous sequences in the target plastid genome."

Claims 2 to 8 were embodiments of claim 1. In particular, claims 4 and 5 defined the antimicrobial peptide (AMP) as being, respectively, magainin I or II or an analogue thereof. Claim 6 defined the analogue as MSI-99. Claim 8 further characterized the vector of any of claims 1 to 7 as a universal vector, wherein the flanking DNA sequences were homologous to a spacer sequence of the target plastid genome and the sequence was conserved in the plastid genome of different plant species. Claims 9 to 16 were directed to stably transformed plants which comprised a plastid stably transformed with the vector of any of claims 1 to 8 and the progeny or subsequent generations thereof, including seeds. Claims 17 and 18 related to a method for stably transforming a target plant to control a phytopathogenic bacteria which comprised transforming plastids of said target plant with a vector according to any of claims 1 to 8, selecting transformed plant cells and allowing the transformed plant cells to grow.

VIII. Claim 1 of appellants' auxiliary request read as claim 1 of the main request (supra) except for the addition at the end of the claim of the following feature:

"1. ... and whereby expression for said cytotoxic antimicrobial peptides is at least 21% of the total soluble proteins."

IX. In the decision under appeal, the examining division considered the claimed subject-matter not to be inventive over a combination of documents D3 and D1 with further reference to inter alia document D4.

The examining division stated that document D3, the closest prior art, disclosed the expression of genes encoding antimicrobial peptides (AMPs) in the nucleus of plants and also suggested their expression in plastids of plants. The technical problem to be solved was the provision of an alternative system for the production of AMPs in plants. The solution was the production of a selection of AMPs, singly or in combination, in plastids of plants. This solution was obvious and straightforward in the light of the prior art. Document D1 disclosed vectors for the integration of foreign genes into the genome of plastids, and Example 1 suggested the integration of AMP genes into the genome of plastids. Although the application was the first to teach the stable transformation of plant plastids with AMP genes, the skilled person wishing to act on the obvious suggestion in the prior art to produce AMPs in plastids merely had to clone the AMP genes into the (conventional) universal integration vector of document D1 and would arrive at the vectors of claim 1. The more so since the claims did not require any particular level of expression for the AMP genes or AMPs production and, therefore, the alleged surprising achievement of a high expression of AMP genes in plastids could not be regarded as a special technical feature making a contribution over the prior art. There was no evidence in the prior art, including document D4, to support the allegation of an existing prejudice against the expression of AMP genes into plant plastids based on the lytic properties of the AMPs and their toxicity to plant plastids.

X. The submissions in writing by the appellants, as far as they are relevant to this decision, may be summarised as follows:

Document D3, the closest prior art, was concerned with the expression of AMP genes in plants. The application differed from that prior art in that a high production of AMPs was obtained in plant plastids. The objective technical problem to be solved was the provision of an alternative expression system for AMP genes in plants. The solution was a direct plastid transformation which then needed the exportation of the produced AMPs from said cellular compartment to the cytosol where phytopathogens colonized, to confer disease resistance.

Although document D3 mentioned that two or more expressed AMPs were preferably compartmentalized, it did not suggest direct plastid transformation or exportation of the produced foreign proteins from one cellular compartment to another. Document D3 only exemplified AMP gene constructions for cytosolic and for extracellular localization (Examples 2 and 3). There was no disclosure nor an indication of the means required to achieve a plastid (such as chloroplast) transformation vector to confer disease resistance.

Document D1 disclosed a stable plastid transformation and expression vector competent for the transformation and stable integration into the plastid genome as well as the use of that vector for producing protein-based polymers. However, this vector did not comprise a DNA heterologous sequence encoding one or more AMPs selected from the group of AMPs cited in the claims. Although there was a reference to the production of a factor conferring pathogen resistance such as an AMPs (lytic peptides or chitinase), this reference was found among a long, non-exhaustive list of unrelated proteins, it was not exemplified and its feasibility was not demonstrated. Moreover, since the prior art considered the production of AMPs to be highly toxic for plastids of plants, the skilled person had no reasonable expectation of success.

Thus, there was no motivation to combine the teachings of document D3 with those of document D1. AMPs having a high antibacterial activity were considered in the prior art to have a large potential for toxic activity against plant chloroplasts because of the charge on the chloroplast membranes. The antimicrobial activity of these lytic peptides was known to be associated with the disruption of the membrane of the microorganism. Moreover, no signal protein sequences to export proteins from chloroplasts were known in the prior art, which was why there were no reports of directly engineering the chloroplast genome for disease resistance. Still further, short peptides were known to be highly susceptible to proteolytic degradation in chloroplasts.

Although several foreign genes had been expressed within plastids, such as for introducing herbicide or insecticide resistance, all these genes encoded proteins that functioned within plastids so that when the engineered plastids were consumed by target insects, insecticidal proteins were released inside the insect gut. The use of the plastid compartment to engineer disease resistance required exportation of the foreign proteins from the plastids to the cytosol where phytopathogens colonize. However, neither document D3 nor document D1 taught or suggested engineering of the plastid genome to export the foreign protein to the cytosol to confer disease resistance. Rather, the plastids of the present invention lysed at the site of infection and released AMPs. These AMPs were protected from proteases in the chloroplast and contained so that they were not toxic to chloroplast membranes until lyse at the site of infection. The lysis of the plastids was previously unknown.

XI. The appellants requested that the decision under appeal be set aside and that a patent be granted on the basis of the main request or the auxiliary request, both filed with the grounds of appeal.