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 1-20 are pending. Claims 1-20 are examined in the instant application. All previous rejections not set forth below have been withdrawn.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Response to Amendments
Status of Rejections from action:
In regard to Claims 1-20 under 112(a) is written description is modified in view of amendments.
In regard to Claims 1-20 under 112(a) enablement is modified in view of amendments.
In regard to Claims 1-20 under 103 is modified in view of amendment.
Priority
Acknowledgment is made of Applicant' s claim for foreign priority under 35 U.S.C. 119 (a)-(d). Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55. This application is claiming the benefit of Application No. JP2022-026111 filed February 22, 2022.
Claim Rejections - 35 USC § 112(a)(Written Description)
Claims 1-19 REMAIN rejected and Claim 20 is NOW 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.
Applicant’s disclosure is as follows.
The Applicant describes a tobacco plant comprising both mutants alleles in the endogenous Lycopene 2-Cyclase (LCY-e) genes. These genes are mutated, Nicotiana sylvestris (SEQ ID NO: 1) and Nicotiana tomentosiformis (SEQ ID NO: 2), to ensure the enzyme’s function is suppressed across the entire genome (see page 9 paragraphs [0028] and [0030]) resulting in “β-damascenone, β-damascone, and β-cyclocitral that were increased only in the double mutant” (p. 63-64, table 4, and fig.1).
The claimed invention lacks adequate written description for the following reasons. Claims 1-20 are directed to a tobacco plant that comprises mutant SEQ ID NO: 1 (LCY-e-S protein) and SEQ ID NO: 2 (LCY-e-T protein) resulting in the tobacco leaves having at least a 10% increase of apocarotenoid compared to the wild-type plant.
The specification does not describe a representative number of amino acid sequences from the genus of sequences with at least 95% identity to SEQ ID NO: 1 and SEQ ID NO: 2 resulting in the tobacco leaves having at least a 10% increase of apocarotenoid.
A polypeptide with at least 95% identity to SEQ ID NOs:1 and 2 would have 26 amino acid substitutions relative to SEQ ID NOs: 1 and 2, respectively.
While one skilled in the art can generate a population of sequences having 95% sequence identity to SEQ ID NO:1 and SEQ ID NO: 2, it is unpredictable which species within the population would also have at least a 10% increase of apocarotenoid. The disclosure of SEQ ID NO:1 and SEQ ID NO: 2 is not representative of sequences having 95% sequence identity to SEQ ID NO:1 and SEQ ID NO: 2 and have at least a 10% increase of apocarotenoid. No common structure or motif is disclosed.
Applicant has described one structure/sequence which is not deemed to be a representative number of structures/sequences from the genus of sequences having 95% sequence identity to SEQ ID NOs: 1 and 2 that results in at least a 10% increase of apocarotenoid.
Because of the lack of a description of a representative number of structures/sequences the impact of 5% variation of SEQ ID NOs:1 and 2, one skilled in the art would not know the structures that confer the desired trait. Therefore, one skilled in the art would find it hard to believe that the Applicant is in possession of such a large genus of structures that will increase apocarotenoid content in tobacco leaves and increases apocarotenoid by 10% compared to the wild-type.
Accordingly, there is lack of adequate description to inform a skilled artisan that Applicant was in possession of the claimed invention at the time of filing. See Written Description guidelines published in Federal Register/ Vol.66, No. 4/ Friday, January 5, 2001/ Notices; p. 1099-1111.
Response to Arguments
Applicant argues both rejections be reconsidered and withdrawn in view of the narrowing amendment of the claims to recite that the leaves have an apocarotenoid content of 10% or more than an apocarotenoid content of leaves of a wild-type plant. Additionally, Applicant notes the robust disclosure in the specification "mutation" processes and "suppressed functions" ([0039] - [0087]), which is commensurate in scope with the claims as amended (Remarks, June 18, 2026, pgs 8-9).
This argument has been fully considered but not found persuasive.
MPEP says," An adequate written description of the invention may be shown by any description of sufficient, relevant, identifying characteristics so long as a person skilled in the art would recognize that the inventor had possession of the claimed invention. See, e.g., Purdue Pharma L.P. v. Faulding Inc., 230 F.3d 1320, 1323, 56 USPQ2d 1481, 1483 (Fed. Cir. 2000)." (MPEP, 2163/II,A,3, (a)).
In the instant case, The Office agrees there is adequate information for one skilled in the art to reproduce SEQ ID NO:1 and 2, however one skilled in the art cannot predictably reproduce mutated sequences having at least 95% sequence identity to SEQ ID NO: 1 and SEQ ID NO:2 with reduction of activity and increase of apocarotenoid by 10%. The specification fails to provide a representative number of sequences with such mutations and tobacco plants comprising said sequences with the resulting claimed phenotype.
Additionally, the claims are not commensurate in scope with what is claimed. Namely, the amendments apply to particular nucleic and amino acid structures and not the variants of sequences as claimed.
Also, the issue is not whether one can make and use the claimed plants. The issue is whether Applicant has possession of the claimed plants, and the fact that the specification discloses how to do something does not address whether Applicant actually possess the plants as claimed comprising the variant polynucleotide and polypeptide sequences that give rise to the apocarotenoid content as claimed.
At least for these reasons, the rejection is maintained.
Claim Rejections - 35 USC § 112(a)(Enablement)
Claims 1-19 REMAIN rejected and Claim 20 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a double mutant tobacco plant comprising both mutations in SEQ ID NO: 1 SEQ ID NO: 2 to increase levels of β-damascenone, β-damascone, and β-cyclocitral as exemplified on page 53 of the instant specification, does not reasonably provide enablement for a polypeptide having as little as 95% sequence identity to SEQ ID NOs:1 and 2 having suppressive activity and resulting in an increase of apocarotenoid of 10% or more than the wild-type. 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.
(A) The breadth of the claims
The breadth of the claims encompasses any tobacco plant comprising polypeptides having any structure within 95% sequence identity to SEQ ID NOs:1 and 2, provided that the function of said polypeptides has been suppressed and increases apocarotenoid by 10% compared to the wild-type.
(B) The nature of the invention.
The nature of the claimed invention is directed to any tobacco plant comprising polypeptides having SEQ ID NOs: 1 and 2 which reduces LCY-e activity and increases apocarotenoid by 10% compared to the wild-type.
(C) The state of the prior art
The state of the prior art does not teach polypeptides having 95% sequence identity to SEQ ID NOs: 1 and 2 or the structures that confer function such that when these polypeptides are mutated functional activity is suppressed and result in increasing apocarotenoid by 10% compared to the wild-type.
(D) The level of one of ordinary skill
The level of one of ordinary skill in the art is high.
(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.
The claimed invention lacks adequate enabling guidance for the following reasons. Claims 1-20 are directed to a tobacco plant that comprises mutations in sequences having at least 95% sequence identity to SEQ ID NO: 1 (LCY-e-S protein) and SEQ ID NO: 2 (LCY-e-T protein) resulting in a decrease of expression of LCY-e activity and increases apocarotenoid by 10% compared to the wild-type.
The claims encompass an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 1 and 2.
The only LCY-e-S and LCY-e-T taught is from Nicotiana being SEQ ID NOs: 1 and 2 and that when mutated increase β-damascenone, β-damascone, and β-cyclocitral levels. The specification does not teach the adequate amount of direction or guidance to arrive or identify the amino acid sequences from the genus of sequences with at least 95% identity to SEQ ID NO: 1 and 2. From the teachings of instant specification one skilled in the art cannot predict which mutations in the structures having 95% sequence identity to SEQ ID NO:1 and SEQ ID NO: 2 as broadly claimed will suppress LCY-e activity and increase apocarotenoid by 10%.
The specification teaches 3 tobacco lines having single point mutations that suppress gene activity: 2 lines having 78Th glutamine and 145th tryptophan of the LCY-e-S protein and 1 line at the 7th arginine of the LCY-e-T (spec. p. 53). While Applicant teaches that SEQ ID NOs:1 and 2 demonstrate specific instances where targeted mutations result in the suppression of gene activity and increased apocarotenoid, there is not enough clear guidance on the mutations to a sequence with having 95% sequence identity to SEQ ID NO: 1 and 2 will suppress LCY-e activity and increase apocarotenoid by 10%.
Applicant does not teach enough examples, besides 3 examples of single point mutations, for LCY-e-S and LCY-e-T shared by tobacco plants that would allow one skilled in the art to predict their structures which lead to a functional LCY-e-S or LCY-e-T polypeptide such that when they are mutated functional activity will be suppressed and increase apocarotenoid by 10%.
The specification fails to TEACH or fails to provide GUIDANCE for making variants with at least 95% sequence identity to SEQ ID NOs: 1 and 2 retain functional activity across different tobacco plants. Consequently, it remains unknown whether mutation in a polypeptide having at least 95% sequence identity to SEQ ID NOs: 1 and 2 will reduce LCY-e activity and increase apocarotenoid by 10%. The lack or guidance and the lack of working examples means one skilled in the cannot make and use a polypeptide having as little as 95% sequence identity to SEQ ID NOs: 1 and 2.
A polypeptide with at least 95% identity to SEQ ID NOs: 1 and 2 would have 26 amino acid mutations each, which encompasses additions, deletions, substitutions and any combination thereof within the relative to SEQ ID NOs: 1 and 2. These polypeptides would encompass 1926 distinct protein variants, respectively. In the absence of guidance indicating where in the sequence of SEQ ID NOs: 1 and 2 such variations can be sustained, undue trial and error experimentation would be required to make the claimed polypeptide which would retain the activity of SEQ ID NO: 1 and 2 and can be subsequently mutated to suppress LCY-e activity and increase apocarotenoid by 10%.
Therefore, while the examples teach that mutating the specific sequences of SEQ ID NOs: 1 and 2 in a tobacco plant result in increased β-damascenone, β-damascone, and β-cyclocitral levels, the specification fails to teach enough mutations besides 3 examples that suppress activity/expression of SEQ ID NOs: 1 and 2. Applicant has not taught enough mutations in enough sequences found with those having 95% or more identity to SEQ ID NO: 1 or 2 that can be mutated to suppress LCY-e activity and increase apocarotenoid by 10%..
Because of the lack of working examples of sequences with at least 95% sequence identity having suppressed LCY-e activity and increase apocarotenoid by 10%, the lack of information on conserved regions required for desired increase apocarotenoid activity, and the impact of 5% variation of SEQ ID NOs: 1-2, there is not enough guidance to predictably make and/or use the claimed sequences to predictably produce mutated sequences having suppressed LCY-e activity and increase apocarotenoid by 10%.
Given the breadth of the claims, the lack of sufficient guidance, the absence of working examples regarding the structure of a mutated polypeptide having at least 95% sequence identity to SEQ ID NOs: 1 and 2 which confer functional activity, the state of the prior art, and unpredictability in the art, one skilled in the art cannot make and use the claimed invention as commensurate in scope with the claims without excessive burden and undue experimentation.
For at least this reason, the Specification does not teach a person with skill in the art how to make and/or use the subject matter within the full scope of these Claims.
Response to Arguments
Applicant traverses the rejection for the same reasons provided to overcome the written description rejection (Remarks, June 18, 2026, pgs 8-9).
These arguments are not persuasive for the reasons as set forth above. While the specification teaches plants with particular sequences can be made and used, the specification does not teach that the genus of sequences as claimed predictably produce a plant with an apocarotenoid content that can be far greater than 10%.
Therefore, the rejection is maintained.
Claim Rejections - 35 USC § 103
Claims 1-19 REMAIN rejected and Claim 20 is NOW rejected under 35 U.S.C. 103 as being unpatentable over Shi et al. (“Molecular cloning and functional characterization of the lycopene ε-cyclase gene via virus-induced gene silencing and its expression pattern in Nicotiana tabacum.” International journal of molecular sciences vol. 15,8 14766-85. 22 Aug. 2014, doi:10.3390/ijms150814766 (Previously cited)) and in view of Senger et al., (WO 2020049155 A1 (Previously cited)).
a. In regard to claims 1, 3, 6-9, 11 and 14-17, Shi et al. teach “the sequence identity between Ntε-LCY1 and Ntε-LCY2 was up to 97%, so it was difficult to specifically silence one copy” suggesting that the sequence identity between ε-LCY genes are high (see page 14776 Discussion middle section). Additionally, Shi et al. teach the amino acid sequence of Ntε-LCY1 and Ntε-LCY2 having 99.6% and 99.2% sequence identity to Applicant SEQ ID NOs: 1 and 2, respectively (see alignment previous Office action). Top of Form
Shi et al. teach suppressing Ntε-LCY transcript levels (i.e. promotion of mRNA degradation, such as in claim 3 and 11) (p.14774 fig. 6). Shi et al. teach that “[a] conserved fragment of two Ntε-LCY cDNAs was selected as an RNAi effective element, and the silencing efficiency achieved up to 90%–95% reduction in transcript levels. As expected, β-branch carotenoid concentrations all increased in the leaves of TRV-ε-lcy plants and the lutein content decreased”, suggesting that that both Ntε-LCY1 and Ntε-LCY2 were silenced (i.e. suppression of a function of a first endogenous gene and a function of a second endogenous gene) (p.14777). Moreover, Shi et al. teach that “lower ε-LCY expression was beneficial for photosynthesis” and to “manipulate tobacco carotenoids” (p. 14778).
In regard to claims 2 and 10, Shi et al. teach “[v]irus-induced gene silencing (VIGS) has proven to be a powerful tool for the rapid analysis of gene function. It is possible to target most genes and downregulate the mRNA levels in a sequence-specific manner [27]” (p. 14768). This suggest that mRNA transcription levels are decreased (p.14777).
In regard to claims 4 and 12, since Shi et al. teach reducing transcription of said genes, the production of said polypeptides are also reduced.
In regard to claims 5 and 13, since Shi et al. teach reducing transcription of said gene the translation of the polypeptides encoded by said gene are also reduced.
In regard to claims 18 and 19, Shi et al. teach a freeze-dried tobacco leaf (i.e. cured leaf) comprising inhibited SEQ ID NOs: 1 and 2 (p. 14781 sec. 4.9). Additionally, Shi et al. teach flue-cured tobacco leaves and improving carotenoid content in mature tobacco leaves for the tobacco industry (i.e. a tobacco product) (p. 14767 and see image below). Since, Shi et al. provides motivation and suggested to improve carotenoid content in tobacco products, one skilled in the art would be motivated to inhibit SEQ ID NOs:1 and 2 to modulate carotenoid content.
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In regard to claim 20, Since Shi et al. teach the same method on reducing the activity of SEQ ID NO: 1 and SEQ ID NO: 2 in tobacco to increase carotenoid, then the tobacco plant of Shi et al. also has increased β-damascenone, β-damascone, and β-ionone. The specification states that “Examples of the apocarotenoids, which refer to the degraded products of carotenoids, include aroma constituents (for example, β-damascenone, β-ionone, and megastigmatrienone)” (pg.2 para. 0004). In other words, since Shi et al. increases carotenoids then in turn also increases apocarotenoids such as β-damascenone, β-damascone, and β-ionone.
Shi et al. teach silencing gene LCY-e activity but does not specifically teach mutations in SEQ ID NOs: 1 and 2. Additionally, Shi et al. do not teach producing progeny seed via crossing.
In regard to claims 1, 3, 6-9, 11 and 14-17, Senger et al. teaches that “reducing the copy number of functional (e.g. expressed) genes, can for example be mediated e.g. by adding or expressing an antisense molecule, cosuppression molecule, an antibody, ribozyme, siRNA, microRNA, ta-siRNA, a cosuppression molecule, or RNAi, by mutation or deletion of a gene sequence, expressing or improving the activity of a negative expression element or by other methods known to the person skilled in the art or mentioned herein” (p.57-58). Suggesting that one skilled in the art would know whether to mutate or silence because to overall outcome is to suppress activity regardless of the technique used.
Lastly, Senger et al., teach to produce “[s]eedling progeny of transgenic plants” (p. 65 line 6) by crossing plants with said mutation with another plant not comprising a mutation (p. 34 line 3).
Therefore, prior to the effective filing date, it would have been prima facie
obvious to one of ordinary skill in the art to modify the teachings of Shi et al. by instead substituting the mutagenesis method as taught by Senger et al. because RNAi and mutagenesis are functionally equivalent in the sense that they both inhibit the gene of interest.
One skilled in the art would have a reasonable expectation of success in this
approach because (1) Shi et al. actually shows inhibiting expression/activity of the SEQ ID NO: 1 and 2 using RNAi, and (2) mutagenesis techniques are well known in the art to suppress gene function as taught by Senger et al. so one would expect the same results that if one skilled in the art were to mutate SEQ ID NOs: 1 and 2 rather than use RNAi.
Furthermore, it would be obvious to one of ordinary skill in the art to make offspring or progeny comprising said mutations because doing so would save time, money, and resources by not having to mutate the plant again and would lead to plants with the desired phenotype (i.e. inhibition of LCY-e activity).
Response to Arguments
Applicant's arguments filed 06/18/2026 have been fully considered but they are not persuasive. Applicant argues 1) Senger is a very general reference that would not motivate one skilled in the art to motivate the disclosure of Shi in a manner that meet the limitations of Applicant's claims; 2) the primary Shi reference does not disclose of SEQ ID NOs. 1 and 2 (Office Action page 17); and 3) the narrowing amendment of the claims to recite that the leaves have "an apocarotenoid content of 10% or more than an apocarotenoid content of leaves of a wild-type plant.
In regard to Applicants 1st argument that there is no motivation to combine Senger and Shi, Senger was used to show that one skilled in the art would know whether to mutate or silence a gene because the overall outcome is to suppress activity regardless of the technique used.
Thus, the Office has provided motivation to modify the teachings of Shi because using RNAi as opposed to mutagenesis is substituting one known method for another that leads to the predictable inhibition of a gene of interest. In fact, Senger even suggests that mutation and RNAi are functionally interchangeable methods.
Moreover, one is motivated to do so because β-branch carotenoid concentrations all increased in the leaves of TRV-ε-lcy plants and the lutein content decreased” and because lower ε-LCY expression was beneficial for photosynthesis” and to “manipulate tobacco carotenoids.
In regard to the Applicants 2nd argument, it is noted that this is a 103 rejection and not a 102 rejection. Thus, Shi does not need to specifically teach a sequence having 100% sequence identity to SEQ ID NO: 1 or 2. It is also noted that this argument is not commensurate in scope with what is claimed because the instant claims do not require a sequence having 100% sequence identity to SEQ ID NO: 1 or 2.
Here, the Office has not argued that Shi needs to be routinely optimized to arrive at the instant claims. Rather, the Office has argued that RNAi and mutagenesis are functionally equivalent methods because they both inhibit expression of a gene of interest. Thus, there is no need to select any particular variable from any list of possibilities to arrive at the instant claims.
Applicant’s other arguments with respect to Senger are unpersuasive because one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Here, the teachings Applicant refers to are not relevant as the Office has explained that Shi is cited to demonstrate that the one of ordinary skill in the art understands that RNAi and mutagenesis are functionally interchangeable methods.
Applicant’s 3rd argument that the claim was narrowed to include 10% apocarotenoid is unpersuasive because it is not commensurate in scope with what is claimed. This amendment only applies to inhibiting expression of SEQ ID NO:1 and SEQ ID NO:2, and not to sequence variants having as little as 95% sequence identity to SEQ ID NO:1 and SEQ ID NO:2. Also, it is noted that there is no upper limit to the amount of apocarotenoid content so that this additional limitation is not commensurate in scope with the variants of sequences that are encompassed by the claims.
Therefore, the rejection is maintained.
Conclusion
No claims are allowed.
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/C.J.O./Examiner, Art Unit 1663 /JASON DEVEAU ROSEN/Primary Examiner, Art Unit 1662
