DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Status
Claims 1-18 are pending.
Claims 1-18 are examined on the merits.
Claim Objections
Claim 2 is objected to because it recites “AIPQF[M/N][I/V]G (SEQ ID NO: 356)”. SEQ ID NO: 356 is a nucleotide sequence. The appropriate sequence identifier is SEQ ID NO: 355.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Scope of Enablement
Claims 1-4 and 6-18 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specifications, while being enabling for particular Arabidopsis and Steria PPO sequence background and selected substitutions, including mutations at positions 362, 365, and 479 of SEQ ID NO: 1 and positions 360, 363, and 477 of SEQ ID NO: 2; does not reasonably provide enablement for the full scope of the claimed PPO genus encompassing PPO enzymes and functional fragments having mutations at those positions or residues corresponding thereto across substantially divergent PPO sequence backgrounds, including sequences having as little as 30% sequence identity to the disclosed PPO sequence. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention commensurate in scope with these claims.
An “analysis of whether a particular claim is supported by the disclosure in an application requires a determination of whether that disclosure, when filed, contained sufficient information regarding the subject matter of the claims as to enable one skilled in the pertinent art to make and use the claimed invention.” MPEP 2164.01. “A conclusion of lack of enablement means that. . . the specification, at the time the application was filed, would not have taught one skilled in the art how to make and/or use the full scope of the claimed invention [i.e. commensurate scope] without undue experimentation.” In re Wright, 999 F.2d 1557,1562, 27 USPQ2d 1510, 1513 (Fed. Cir. 1993); MPEP 2164.01.
In In re Wands, 858 F.2d 731,8 USPQ2d 1400 (Fed. Cir. 1988), several factors implicated in determination of whether a disclosure satisfies the enablement requirement and whether any necessary experimentation is “undue” are identified. These factors include, but are not limited to:
(A) The breadth of the claims;
(B) The nature of the invention;
(C) The state of the prior art;
(D) The level of one of ordinary skill;
(E) The level of predictability in the art;
(F) The amount of direction provided by the inventor;
(G) The existence of working examples; and
(H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure. In re Wands, 858 F.2d 731,737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988). No single factor is independently determinative of enablement; rather “[i]t is improper to conclude that a disclosure is not enabling based on an analysis of only one of the above factors while ignoring one or more of the others.” MPEP 2164.01. Likewise, all factors may not be relevant to the enablement analysis of any individual claim.
The enablement issue concerns the breadth of the claimed modified PPO enzyme genus, including the breadth of PPO sequence variation, functional fragments, mutations at the recited or “corresponding” amino acid positions, mutation identities and combinations, and the resulting PPO-inhibitor resistance across that scope.
Claim 1 encompasses a modified PPO enzyme or functional fragment thereof having at least one mutation at residue 362, 365, and/or 479 of SEQ ID NO: 1, or a residue corresponding thereto. The claim does not limit the PPO enzyme to a particular sequence-identity threshold, nor does it limit the mutation at these positions to the particular amino-acid substitutions experimentally demonstrated in the specification. Claims 3 and 8-10 further encompass various combinations of mutations at additional positions or residues corresponding thereto.
The breadth of the claimed genus is further illustrated by claim 7, which encompasses PPO sequence having as little as 30% sequence identity to SEQ ID NO: 1, 2, 153, or 154 while retaining mutations at specified or corresponding positions. Consistent with this breadth, the specification states that modified PPO enzymes may have as little as 30% sequence identity to numerous disclosed PPO sequences or functional fragments thereof (p5-8). The specification further expressly describes sequences having at least 30% identity to SEQ ID NO: 1 having mutations at positions 362, 365, and/or 479, or corresponding positions, and sequences having at least 30% identity to SEQ IG NO: 2 having mutations at positions 360, 363, and/or 477, or corresponding positions (p9-10). Claim 12 likewise extends to PPO sequences having as little as 30% sequence identity to a large collection of disclosed SEQ ID NOs. The specification itself additionally defines PPO homologues as functionally equivalent proteins capable of carrying out the PPO enzymatic reaction and expressly contemplates homologous having sequence identity as low as 20-40%. Thus, the enablement concern is not based merely on the proposition that a protein having approximately 30% identity could not retain PPO enzymatic activity. Rather, the issue is whether the disclosure enables the claimed herbicide-resistance mutations and their functional effects across such divergent PPO sequence backgrounds.
The specification provides substantial experimental disclosure for particular PPO sequence backgrounds and particular variants. Specifically, Example 1 (p76) describes a site-saturation variant library based on Arabidopsis thaliana PPO1 in which 352 amino-acid positions were individually substituted with codons encoding each of the other 19 amino acids or were deleted, followed by screening for herbicide tolerance (Example 1, p76; Table 1). Example 2 (p82-83), then selected a subset of herbicide-tolerance mutations identified form the AtPPO1 screen for inclusion in a combinatorial variant library (Example 2, p82-83; Table 3). The specification further identifies particular substitutions at the principal positions, including cysteine or phenylalanine at position 362, methionine or leucine at position 365, and methionine or asparagine at position 479 of SEQ ID NO: 1, together with corresponding substitutions in SEQ ID NO: 2 (p9-10). Thus, the disclosure provides concrete teaching for selected substitutions in identified PPO backgrounds rather than a generalized demonstration that arbitrary mutations at the recited positions operate across the entire claimed sequence genus.
With respect to claims 2 and 3, the specification provides concrete disclosure of the L479M/N substitutions and particular multi-mutation combinations within the Arabidopsis PPO1 background. Thus, the enablement concern is not whether those specifically disclosed embodiments can be made and used. Rather, because claims 2 and 3 depend from claim 1 and retain scope encompassing PPO homologues and functional fragment having mutations at “corresponding” residues, the issue is whether the disclosure enables those limitations across the broader claimed PPO sequence genus without undue experimentation.
The specification also provides particular examples of determining corresponding positions. It explains that positions 362, 365, and 479 of SEQ ID NO: 1, which includes the naturally occurring transit peptide, correspond respectively to positions 328, 331, and 445 of transit-peptide-free SEQ ID NO: 153, and that positions 360, 363, and 477 or SEQ ID NO: 2 correspond respectively to positions 325, 328, and 442 of SEQ ID NO: 154 (p28, line 15-35). The specification also teaches that corresponding positions may be identified by sequence alignment and conserved regions. Accordingly, the issue is not simply whether a skilled artisan could computationally identify a nominally corresponding residue. Rather, identification of a corresponding residue does not establish that mutation of that residue will produce the dame effect on PPO catalytic activity or PPO-inhibitor resistance in a substantially different sequence background. The specification does not provide a general structure-function relationship demonstrating that the local structural environment governing the effect of the mutation is sufficiently conserved throughout the claimed genus.
The experimental plant disclosure is likewise concentrated on selected PPO sequence backgrounds. Example 4 (p89-90) describes expression of Arabidopsis PPO1 and variants thereof and Setaria PPO1 and variants thereof in transgenic tobacco. Example 5 (p90-91) testes such transgenic tobacco plants for resistance to PPO-inhibiting compounds and scores herbicidal damage at 7 and 14 days (Example 5, p90-91; Tables 8-24). Thus, although the specification provides working plant examples, the examples remain focused on identified Arabidopsis and Setaria PPO sequences and selected variants thereof. Moreover, the specification demonstrates that the identity and combination of mutations materially affect herbicide resistance. For example, the specification reports that AtPPO1 V002 had higher IC50 values than AtPPO1 V030 for all tested herbicides except fomesafen, and identifies the additional L479M substitution in V002 as the distinguishing mutation (Example 3; Table 6). This experimental result indicates that the functional effect is dependent on the particular mutation or combination rather than being attributable merely to the presence of any mutation at a recited position.
Dayan (Franck E. Dayan et. al., Biochimica et Biophysica Acta 1804 (2010) 1548–1556) further demonstrates that the effect of a PPO mutation on catalytic function and inhibitor resistance is dependent on the structural context of the mutation and the particular inhibitor (Title and Abstract). Dayan studies a Gly210 deletion in PPO2. Although Gly210 is adjacent rather than directly within the PPO active site, the deletion decreased catalytic efficiency, significantly altered inhibitor interactions, destabilized the α-8 helix-capping region, and enlarged the active-site cavity by approximately 50% (Abstract). Dayan therefore demonstrates that alteration of even a residue outside the immediate active site can substantially affect PPO architecture, catalytic properties, and inhibitor binding.
Bi (Bo Bi et. al., Pest Management Science (2020) 76:pp1786-1794) likewise demonstrates that the functional consequence of a PPO mutation may be inhibitor-specific. The A212T mutation altered the PPO1 active-site environment and conferred resistance to oxidization, while the resistant plants were not resistant to the structurally unrelated PPO inhibitors lactofen, flumioxazin, and sulfentrazone (Abstract). Structural modeling attributed the oxadiazon resistance to mutation-induced changes in the active site that altered inhibitor binding thus, the art demonstrates that PPO-inhibitor resistance cannot necessarily be extrapolated merely from the presence of a mutation at a PPO residue; the effect depends upon the particular substitution, its surrounding structural environment, and the inhibitor being tested.
The low sequence-identity boundary therefore does not establish lack of PPO function, A divergent protein may remain a functional PPO, as contemplated by the specification. However, retention of the general PPO enzymatic reactions distinct from preservation of the local structural environment required for a particular mutation to confer PPO-inhibitor resistance. Neither the specification nor the working examples establish that the resistance effect of mutations identified principally in the Arabidopsis and Seteria PPO backgrounds can be predictably extrapolated to PPO homologues and functional fragments throughout the full claimed sequence space.
Accordingly, to practice the full claimed scope, a person of ordinary skill would need to identify PPO sequences throughout the claimed sequence space; identify a residue corresponding to the recited residue in each divergent PPO sequence or functional fragment; determine whether modification of that corresponding residue has the same functional consequence in the particular sequence background; select among possible substitutions and combinations of substitutions; determine the corresponding residue in each divergent PPO sequence or functional fragment; introduce one or more possible mutations at the recited positions; determine whether the resulting protein retains PPO enzymatic activity; and, where required, experimentally determine whether the enzyme or plant exhibits resistance or decreased inhibition in response to PPO-inhibiting compounds. Although the specification provides assays and screening techniques for identifying successful variants, particularly in Examples 1-5 (p76-91), the disclosure does not provide a predictive structure-function relationship that permits successful members throughout the full claimed genus to be identified without extensive empirical screening.
The breadth of the claims is large because the genus varies along multiple dimensions, including PPO sequence background, sequence identity, functional-fragment boundaries, corresponding residue, mutation identity, mutation combination, and response to PPO inhibitors. The specification provides considerable direction and numerous working examples, particularly in Examples 1-5 (p76-91), but those examples are concentrated in particular PPO sequence backgrounds and selected variants. The specification does not provide sufficient guidance or a general structure-function correlation demonstrating that those results can reasonably be extrapolated to PPO sequences throughout the claimed sequence space, particularly sequences approaching 30% identity.
Therefore, the amount of experimentation necessary to identify operative embodiments across the full claimed scope would be undue, and the specification does not enable the full scope of claims 1-4 and 6-18 as presently claimed.
Written Descriptions
Claim 1-4, and 6-18 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
The Federal Circuit has clarified the application of the written description requirement. The court stated that a written description of an invention "requires a precise definition, such as by structure, formula, [or] chemical name, of the claimed subject matter sufficient to distinguish it from other materials". University of California v. Eli Lilly and Co., 119 F.3d 1559, 1568; 43 USPQ2d 1398, 1406 (Fed. Cir. 1997). The court also concluded that "naming a type of material generally known to exist, in the absence of knowledge as to what that material consists of, is not description of that material". Id. Further, the court held that to adequately describe a claimed genus, Patent Owner must describe a representative number of the species of the claimed genus, and that one of skill in the art should be able to "visualize or recognize the identity of the members of the genus". Id.
The claims are rejected for lacking adequate written description support regarding the broad scope of the following.
The written description issue concerns the breadth of the claimed modified PPO genus, particularly PPO homologs and functional fragments having mutations at the recited or “corresponding” amino-acid positions across widely varying sequence backgrounds, rather than the specifically disclosed Arabidopsis and Setaria embodiments.
Claim 1 broadly encompasses a modified PPO enzyme or functional fragment thereof having at least one mutation at residues 362, 365 and/or 479 of SEQ ID NO: 1, or a residue corresponding thereto, without limiting the PPO to a particular sequence-identity threshold or limiting the mutation to the particular substitutions identified experimentally. Claim 7 expressly encompasses sequence having as little as 30% identity to SEQ ID NO: 1, 2, 153 or 154, together with mutations at the specified or corresponding positions. Claim 12 similarly encompass sequences having as little as 30% identity to a large number of disclosed PPO sequences.
The specification expressly states that modified PPO enzymes may have as little as 30% sequence identity to numerous reference PPO sequences may encompass functional fragments thereof (p5-8). The specification more specifically describes PPO sequences having at least 30% identity to SEQ ID NO: 1 with mutations at positions 362, 365, and/or 479, or corresponding positions, and PPO sequences having at least 30% identity to SEQ ID NO : 2 with mutations at positions 360, 363, and/or 477, or corresponding positions (p9-10).
The disclosure also identifies numerous specific combinations involving additional amino-acid positions, including residues 305, 361, 404, 462, 431, and 461 in combination with the principal positions (p10-13). These disclosures demonstrate possession of the expressly identified substitutions and combinations, but do not by themselves demonstrate possession of every mutation at the recited or corresponding positions throughout the full breadth of the claimed PPO sequence genus.
With respect to claims 2 and 3, the specification provides express support for particular embodiments within the claimed scope. The specification expressly identifies L479M and L479N substitutions in Arabidopsis PPO and discloses specific multi-mutation combinations corresponding to those recited in claim 3. Thus, the written description rejection is not based on an absence of disclosure of those particular Arabidopsis embodiments. Rather, because claims 2 and 3 depend from claim 1 and retain the broader scope encompassing PPO homologs and functional fragments having mutations at “corresponding” residues, the issue is whether the disclosed Arabidopsis and Setaria species reasonably demonstrate possession of that broader genus across substantially divergent PPO sequence backgrounds.
The working examples are likewise focused on specifically selected PPO backgrounds, Example 1 (p76) generates and screens a site-saturation library based on Arabidopsis thaliana PPO (p76, Example 1; Table 1). Example 2 (p82-83) uses selected herbicide-tolerance mutations identified form that AtPPO1 screen to form a combinational AtPPO1 library (p82-83, Example 2; Table 3). Examples 4-5 (p89-91) principally examine Arabidopsis PPO1 and Setaria PPO1 sequences and variants expressed in transgenic tobacco and tested against PPO-inhibiting herbicides.
The specification additionally provides particular examples of residue correspondence. It states the positions 362, 365, and 479 of SEQ ID NO: 1 correspond respectively to positions 328, 331, and 445 of SEQ ID NO: 153, and that positions 360, 363, and 477 of SEQ ID NO: 2 correspond respectively to positions 325, 328, an d442 of SEQ ID NO: 154 (p28, line 15-35). These disclosures demonstrate possession of those expressly identified correspondence relationships. However, the specification does not disclose structural features common to PPO sequences through the entire claimed sequence range that would allow a skilled artisan to recognize, form the disclosure itself, every claimed “corresponding” residue in PPO sequence extending toward 30% identity and in functional fragment thereof.
Moreover, the disclosure of the principal mutation sites is not accompanied by a generalized structure-function rule showing the arbitrary substitutions at those sites define a commonly possessed genus. Instead, the specification particularly identities only selected substitutions e.g., 362C/F, 365M/L, and 478M/N (p9-10), and experimentally evaluated selected sequence combinations. The experimental results further show that particular mutation combinations produce different effects; for example, the additional L479M substitution in AtPPO1 V002 was associated with increased IC50 relative to AtPPO1 V030 for most tested herbicides (Example 3; Table 6).
Thus, although the specification provides numerous actual species, sequence variants, and experimental examples, those species are concentrated around particular Arabidopsis and Setaria PPO sequence backgrounds and selected mutation. The disclosure does not provide species representative of the full breadth created by PPO sequences approaching the claimed low-identity boundaries, functional fragments, and the full range of residues encompassed by the term “corresponding” residues, nor does it identify structural features common to that full genus that would permit a skilled artisan to recognize its members.
Accordingly, the original disclosure does not reasonably convey to a person of ordinary skill in the art the inventors had possessed, as of the filing date, of the full genus of modified PPO enzymes and functional fragments encompassed by claims 1-4 and 6-18.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1, 4, 6, 7, 9, and 11-18 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Boynton (John E. Boynton et. al., US7586023B1, Application 1996-12-27, Publication 2009-09-08).
Claim 1 recites a modified Protoporphyrinogen Oxidase (PPO) enzyme or functional fragment thereof, wherein the modified Protoporphyrinogen Oxidase or functional fragment thereof comprises at least one mutation at one or more amino acid residues selected from residues 479, 365 and/or 362 of SEQ ID NO:1, or residues corresponding thereto.
Boynton discloses a modified protoporphyrinogen oxidase (PPO) enzyme having a mutation corresponding to Val365 of the Arabidopsis PPO. Specifically, Boynton identifies SEQ ID NO: 11 as the Arabidopsis thaliana PROTOX/PPO coding sequence and expressly states that the resistance mutation identified in Val365 in the Arabidopsis PROTOX gene of SEQ ID NO: 11, with the mutation resulting in a Val to Met substitution (p32, line 13-17). Boynton further discloses creation of herbicide-resistant plant PPO genes by site-directed mutagenesis such that the corresponding valine is substituted with methionine and the resulting modified PPO exhibits resistance to PPO-inhibiting herbicides (p31-32, Example 11).
Sequence analysis further establishes that nucleotides from 16 to1629 of Boynton SEQ ID NO: 11 encode an amino-acid sequence having 100% identity to SEQ ID NO: 1 of the instant application (p43-44; alignment below).
Accordingly, the V365M modification discloses by Boynton is a mutation at residue 365 of SEQ ID NO: 1, as presently claimed. Thus, Boynton discloses a modified PPO having at least one mutation at a residue selected from 479, 365, and/or 362 of SEQ ID NO: 1.
Therefore, claim 1 is anticipated by Boynton.
Claim 4 recites modified PPO enzyme or functional fragment thereof of claim1, wherein the modified PPO enzyme or functional fragment thereof comprises an amino acid sequence having at least 30% to at least 99% sequence identity to: any one of SEQ ID NOs including 58.
For the same reasons set forth above with respect to claim 1, Boynton discloses a modified Arabidopsis PPO comprising the V365M mutation, wherein sequence analysis establishes that the underlying Boynton Arabidopsis PPO sequence is 100% identical to SEQ ID NO: 1 of the instant application (see alignment below). Applicant’s specification identifies SEQ ID NO: 58 as an Arabidopsis PPO sequence comprising L479M relative to SEQ ID NO: 1 (see below alignment).
Boynton’s V365M PPO differs from SEQ ID NO: 58 at residues 365 and 479 and therefore has approximately 99.6% amino-acid sequence identity to SEQ ID NO: 58, thereby satisfying the sequence-identity limitation of claim 4.
Therefore, claim 4 is anticipated by Boynton.
Claim 6 recites the modified PPO enzyme or functional fragment thereof of claim 1, wherein the modified PPO enzyme or functional fragment thereof is a PPO1 enzyme; optionally wherein the modified PPO enzyme or functional fragment thereof, comprises an increased resistance to a compound that inhibits a PPO enzyme relative to a control PPO enzyme and/or has decreased percentage inhibition in response to a compound that inhibits a PPO enzyme relative to a control PPO enzyme.
Boynton discloses that the modified PPO is a plant PPO enzyme and that introduction of the Val to Met substitution produces an herbicide-resistant PPO enzyme (p31-32, Example 11). Example 13 expressly compares a cDNA encoding the herbicide-sensitive PPO with a cDNA encoding the corresponding Val to Met herbicide-resistant PPO and reports that cells expressing the sensitive PPO are strongly inhibited by the PPO inhibitor whereas cells expressing the resistant PPO continue to grow (p33, line 42-58, Example 13). Boynton concludes that this difference results from the differing inhibitory effect of the compound on the PPO enzymes (p3 line56-67; p4 line 1-12) .
Because the protein encoded by SEQ ID NO: 11 is 100% identical to instant SEQ ID NO: 1 before introduction of the V365M mutation, the disclosed modified Arabidopsis PPO corresponds to the PPO1 enzyme recited in claim 6 (see alignment below). Boynton therefore discloses a PPO1 enzyme according to claim 1 having increased resistance and/or decreased inhibition in response to a PPO-inhibiting compound relative to the corresponding unmodified PPO.
Therefore, claim 6 is anticipated by Boynton.
Claim 7 recites the modified PPO enzyme or functional fragment thereof of claim 1, wherein the modified PPO enzyme or functional fragment thereof comprises:(a) an amino acid sequence having at least 30% - 99% sequence identity to SEQ ID NO: 1 and at least one mutation at one or more amino acid residues selected from residues 362, 365 and/or 479 of SEQ ID NO: 1.
Boynton’s Arabidopsis PPO sequence encoded by SEQ ID NO: 11 is, as established by sequence analysis, 100% identical to instant SEQ ID NO: 1 before introduction of the V365M mutation. The resulting Boynton V365M protein therefore differs from instant SEQ ID NO: 1 by only the expressly introduced mutation at residue 365 and satisfies the sequence-identity requirement of claim 7. Boynton expressly discloses the V365M substitution at this position.
Thus, Boynton discloses an amino-acid sequence satisfying the recited sequence-identity threshold to SEQ ID NO: 1 and having at least one mutation at residue 365 or SEQ ID NO: 1. Therefore, claim 7 is antedated by Boynton.
Claim 9 recites the modified PPO enzyme or functional fragment thereof: (i) of claim 1, wherein;(a) the mutation at residue 362 of SEQ ID NO: 1 or residues corresponding thereto comprises a substitution to a cysteine or phenylalanine;(b) the mutation at residue 365 of SEQ ID NO: 1 or residues corresponding thereto comprises a substitution to a methionine or a leucine; and/or (c) the mutation at residue 479 of SEQ ID NO: 1 or residues corresponding thereto comprises a substitution to a methionine or asparagine.
Boynton expressly discloses substitution of the valine corresponding to Val365 of the Arabidopsis PPO with methionine, i. e., V365M.
Accordingly, Boynton expressly discloses the 365M alternative encompassed by claim 9.
Therefore, claim 9 is antedated by Boynton.
Claim 11 recites the modified PPO enzyme or functional fragment thereof of claim 6: wherein the modified PPO enzyme or functional fragment thereof comprises or consists of a sequence according to SEQ ID NOs: 188 - 190, 209, 210, 248 - 250, 269, 270, 276- 288 or 290– 302.
For the same reasons set forth above with respect to claims 1 and 6, Boynton discloses a modified Arabidopsis PPO comprising the V365M mutation. Sequence alignment of Boynton SEQ ID NO: 11 with SEQ ID NO: 190 of the instant application shows that SEQ ID NO: 190 corresponds to the Arabidopsis PPO sequence of Boynton lacking the N-terminal transit peptide, and that Boynton residue 365 aligns with residue 331 of SEQ ID NO: 190 (see below). The corresponding aligned amino-acid sequence is otherwise identical. Thus, Boynton discloses the modified PPO enzyme comprising the sequence of SEQ ID NO: 190 as recited in claim 11.
Therefore, claim 11 is antedated by Boynton.
Claim 12 recites the modified PPO enzyme or functional fragment thereof of claim 1, wherein the modified PPO enzyme further comprises a transit peptide; optionally wherein the transit peptide comprises a mitochondrial transit peptide and/or a chloroplastic transit peptide.
Boynton discloses that the Arabidopsis PPO protein is a chloroplast-localized PPO having a chloroplast-targeting signal peptide (p15 line18-21). In particular, Boynton identifies the known Arabidopsis PPO cDNA as encoding a protein having PPO activity and a chloroplast-targeting signal peptide. Boynton further discloses that sequences encoding a signal or transit peptide, including chloroplast transit peptides, may be associated with the herbicide-resistant PPO-coding sequence to direct the PPO enzyme to its desired site of action (p16 line 29-60).
Boynton therefore discloses the modified PPO of claim 1 further comprising a transit peptide as required by claim 12.
Therefore, claim 12 is antedated by Boynton.
Claim 13 recites a nucleic acid comprising a polynucleotide encoding a modified PPO enzyme or functional fragment thereof according to claim 1; or a recombinant vector comprising the nucleic acid.
For the same reason set forth above with respect to claim 1, Boynton expressly discloses a nucleic acid encoding the modified herbicide-resistant PPO and a recombinant vector comprising that nucleic acid. Example 12 discloses preparation of a cDNA encoding the herbicide-resistant PPO by introducing the Val to Met resistance mutation (p32). Example 13 states that the resulting cDNA encoding herbicide-resistant PPO is cloned into plasmid vector pUC118. Boynton also generally discloses introducing the herbicide-resistant PPO coding sequence into plasmids or other vectors for expression (p33).
Thus, Boynton expressly discloses both a polynucleotide encoding the modified PPO of claim 1 and a recombinant vector comprising such nucleic acid.
Therefore, claim 13 is antedated by Boynton.
Claim 14 recites a plant or part thereof comprising a modified PPO enzyme or functional fragment thereof according to claim 1 wherein the plant or part thereof comprises an increased resistance to a compound that inhibits a PPO enzyme relative a control plant and/or has decreased percentage inhibition in response to a compound that inhibits a PPO enzyme relative to a control plant; further optionally wherein the control plant is a wild type plant, or a plant comprising a modified PPO enzyme according to SEQ ID NO: 124.
For the same reasons set forth above with respect to claim 1, Boynton discloses the modified Arabidopsis thaliana PPO having a V365M mutation corresponding to residue 365 of SEQ ID NO: 1 of the instant application (p32, line 13-17). Boynton expressly discloses introducing a genetically engineered PPO gene having the resistance-conferring amino-acid substitution into plants or plant cells, wherein expression of the DNA confers PPO-inhibiting herbicide resistance. Boynton further expressly provides plants and plant cells upon which such resistance is conferred by the disclosed method (p2, line 46-67).
Accordingly, Claim 14 is anticipated by Boynton.
Claim 15 recites a method of producing a plant or part thereof, the method comprising: modifying the plant or part thereof to comprise a PPO enzyme according to claim 1; optionally wherein modifying the plant or part thereof comprises transforming the plant or part thereof with a nucleic acid or a recombinant vector.
For the same reasons set forth above with respect to claim 1, Boynton discloses the modified Arabidopsis thaliana PPO having a V365M mutation (p32, line 13-17). Boynton discloses introducing the herbicide-resistant PPO coding sequence into plant cells, including in a chimeric gene construct or plasmid/vector, selecting transformed PPO-inhibitor-resistant plant cells, culturing the transformed cells, and regenerating PPO-inhibitor-resistant plant therefrom (p12, line 9-23; p12 line 24-49).
Accordingly, Claim 15 is anticipated by Boynton.
Claim 16 recites a method of controlling undesired vegetation in the vicinity of a plant or part thereof or at a locus for growth of the plant or part thereof, the method comprising applying an effective amount of at least one PPO-inhibiting herbicide to the vicinity of the plant, the locus for growth of the plant or to the plant or part thereof, wherein said plant or part thereof comprises the plant or part thereof of claim 14, wherein the effective amount of said PPO-inhibiting herbicide controls undesired vegetation in said vicinity or at said locus.
For the same reasons set forth above with respect to claim 1, Boynton discloses the modified Arabidopsis thaliana PPO having a V365M mutation (p32, line 13-17). Boynton expressly discloses controlling PPO-inhibitor-sensitive plants in a field of PPO-inhibitor-resistant crop plants by applying an effective amount of PPO-inhibiting herbicide to inhibit growth of the sensitive plants (p18, line 21-28).
Accordingly, Claim 15 is anticipated by Boynton.
Claim 17 recites using of a compound which inhibits a PPO enzyme in combination with a plant or part thereof according to claim 14.
For the same reasons set forth above with respect to claim 1, Boynton discloses the modified Arabidopsis thaliana PPO having a V365M mutation (p32, line 13-17). Boynton discloses cultivation of PPO-inhibitor-resistant crop plants in the presence of PPO-inhibiting herbicides for weed control and expressly discloses application of PPO-inhibiting herbicides to fields containing such resistant plants (p2, line 13-23).
Claim 18 recites the modified PPO enzyme of claim 6 , wherein the compound is an herbicide selected from the group of compounds, which include formesafen and oxyfluorfen.
Boynton expressly identifies PPO-inhibiting herbicides as including the diphenyl ether herbicides formesafen and oxyfluorfen (p5, line 13-18). Boynton further discloses that the modified PPO DNA has the ability to confer resistance to PPO-inhibiting herbicides when expressed in plants and cells (p4, line 44-59).
Because disclosure of either formesafen or oxyfluorfen satisfies one alternative of the Markush group of claim 18, claim 18 is anticipated by Boynton.
Claim 5 is rejected under 35 U.S.C. 102(a)(1) as being anticipated by Volrath (Sandra L. Volrath et. al., US6084155A, Application 1998-06-22, Publication 2000-07-04).
Claim 5 recites a modified Protoporphyrinogen Oxidase (PPO) enzyme or functional fragment thereof, wherein the modified Protoporphyrinogen Oxidase or functional fragment thereof comprises mutations at amino acid residues 305 and 426 of SEQ ID NO: 1, or residues corresponding thereto, wherein the mutations are substitutions of 305L and 426V.
Volrath discloses an Arabidopsis thaliana protox-1 protein according to SEQ ID NO: 2 (p6, line 57-58). Sequence comparison establishes that Volrath SEQ ID NO: 2 is 100% identical to SEQ ID NO: 1 of the instant application (see below). Volrath identifies the AraC-2 resistance site as Tyr426 of Arabidopsis PPO1 SEQ ID NO: 2, corresponding to the tyrosine codon at nucleotides 1306-1308 of SEQ ID NO:1 (p56, line 28-35; p55, line 55-59). Volrath further discloses substitution of this same tyrosine residue with valine, designated pAraC-2Val, to yield a functional herbicide-resistant Protox enzyme (p56, line 28-35). further discloses substitution of serine at amino acid residue 305 with leucine (AraC305Leu) (p56, line 53-59). Example 13 expressly discloses combining the AraC305 Leu mutation with the AraC-2Val mutation and reports increased tolerance to protox-inhibiting herbicide (p57, Example 13). Accordingly, Volrath discloses a modified PPO enzyme comprising the 305L and 426V substitutions of SEQ ID NO: 1, as required by claim 5.
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Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 8 and 10 are rejected under 35 U.S.C. §103 as being unpatentable over Boynton (US7586023B1) as apply to claim 1, in view of Volrath (US6084155A).
Claim 1 as the teachings of Boynton are discussed above.
Claims 8 and 10 interpreted as dependent of claim 1.
Claim 8 recites the modified PPO enzyme or functional fragment thereof of: claim 1, wherein modified PPO enzyme or functional fragment thereof further comprises one or more further mutations at one or more amino acid residues selected from residues 305, 361, 404, 426, 431, 461, and/or 479 of SEQ ID NO: 1 or residues corresponding thereto; wherein:(a) mutation at residue 305 of SEQ ID NO: 1 or residues corresponding thereto comprises a substitution to a leucine;(b) mutation at residue 361 of SEQ ID NO: 1 or residues corresponding thereto comprises a substitution to cysteine, aspartic acid, glutamine or threonine;(c)mutation at residue 404 of SEQ ID NO: 1 or residues corresponding thereto comprises a substitution to alanine;(d) mutation at residue 426 of SEQ ID NO: 1 or residues corresponding thereto comprises a substitution to leucine, cysteine, methionine, threonine or valine;(e) mutation at residue 431 of SEQ ID NO: 1 or residues corresponding thereto comprises a substitution to alanine, phenylalanine or arginine; and/or (f) mutation at residue 461 of SEQ ID NO: 1 or residues corresponding thereto comprises a substitution to glutamine.
For the same reasons set forth above with respect to claim 1, Boynton teaches the modified Arabidopsis thaliana PPO having a V365M mutation corresponding to residue 365 of SEQ ID NO: 1 of the instant application.
Boynton does not teach the further S305L mutation recited in claim 8. Claim 8 requires a further mutation at residue 305 wherein residue 305 is substituted with leucine.
Volrath teaches that serine at amino acid residue 305 of Arabidopsis PPO SEQ ID NO: 2 is substituted with leucine (AraC305Leu) and that this second-site mutation increases tolerance to PPO-inhibiting herbicides (p56, line 53-59). Volrath further teaches combining AraC305Leu with previously identified herbicide-resistant PPO mutants and reports that the additional S305L mutation significantly increased growth in the presence of PPO-inhibiting herbicide ( p57, Example 13).
It would have been obvious to one of ordinary skill in the art to further modify Boynton’s V365M herbicide-resistant PPO to include Volrath’s S305L mutation because Volrath expressly teaches S305L as a second-site mutation for enhancing the herbicide resistance of previously resistant PPO enzymes, with a reasonable expectation of obtaining increased PPO-inhibitor tolerance.
Accordingly, Claim 8 is prima facie obvious over Boynton and Volrath.
Claim 10 recites the modified PPO enzyme or functional fragment thereof of claim 1, comprising a combination of mutations at residues:(a) 365, 426, and 479 of SEQ ID NO: 1, or residues corresponding thereto;(b) 305, 404, 426, 431, and 479 of SEQ ID NO: 1, or residues corresponding thereto;(c) 305 and 365 of SEQ ID NO: 1, or residues corresponding thereto;(d) 305, 362 and 404 of SEQ ID NO: 1, or residues corresponding thereto;(e) 305, 361, 365, 431 and 479 of SEQ ID NO: 1, or residues corresponding thereto;(f) 305, 426 and 479 of SEQ ID NO: 1, or residues corresponding thereto;(g) 426, 461 and 479 of SEQ ID NO: 1, or residues corresponding thereto;(h) 305, 361 and 365 of SEQ ID NO: 1, or residues corresponding thereto;(i) 305, 361, 362, 426 and 479 of SEQ ID NO: 1, or residues corresponding thereto;(j) 361 and 365 of SEQ ID NO: 1, or residues corresponding thereto;(k) 365, 404, and 479 of SEQ ID NO: 1, or residues corresponding thereto; or (1) 305, 404, and 479 of SEQ ID NO: 1, or residues corresponding thereto.
For the same reasons set forth above with respect to claim 1, Boynton teaches the modified Arabidopsis thaliana PPO having a V365M mutation corresponding to residue 365 of SEQ ID NO: 1 of the instant application.
Boynton does not teach the claimed combination of mutations at residues 305 and 365. Claim 10 expressly recites, as alternative (c), mutations at residues 305 and 365 of SEQ ID NO: 1, or corresponding residues.
Vorath teaches substitution of S305 with leucine (S305/AraC305 Leu) in Arabidopsis PPO and teaches that S305L enhances the herbicide resistance of previously resistant PPO mutants (p56, line 53-59). Volrath further expressly teaches combining S305L with undependably identified PPO-resistance mutations to obtain increased PPO-inhibitor tolerance ( p57, Example 13).
It would have been obvious to one of ordinary skill in the art to add Volrath’s S305L mutation to Boynton’s V365M PPO, because Volrath expressly teaches combining S305L with an already herbicide-resistant PPO to further increase herbicide tolerance. The resulting PPO would comprise mutations at residues 305 and 365, as recited in alternative (c) of claim 10.
Accordingly, claim 10 is prima facie obvious over Boynton and Volrath.
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to YANXIN SHEN whose telephone number is (571)272-7538. The examiner can normally be reached Monday-Friday.
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/YANXIN SHEN/ Examiner, Art Unit 1663
/WEIHUA FAN/ Primary Examiner, Art Unit 1663
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