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 .
Status of Claims
The amendments submitted on July 2nd, 2026, have been entered.
Claim 32 has been canceled.
Claims 7-8, 10-13, 16, 18-19, and 21-22 are pending in the application and examined in this Office action.
The text of those sections of Title 35 U.S. Code, not included in this action, can be found in a prior Office action.
Objections/Rejections
The objections of claims 7-8, 10-13, 16, 18, 19, and 21-22 are withdrawn in light of the amendments to the claims.
The objection to claim 32 is moot in light of the cancellation of the claim.
Claim Rejections - 35 USC § 101
The rejections of claims 7-8 and 10-13 under 35 USC § 101 are withdrawn in light of amendments to the claims.
Claim rejections under 35 USC § 112 (a) – Written Description
Claims 7-8, 10-13, 16, 18-19, and 21-22 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. This rejection is modified from the rejection presented in the Office Action mailed 04/02/2026.
The claims broadly encompass a tobacco plant or any part thereof and any modification to alter the activity or expression of a CYP94 cytochrome P450 comprising decreased alkaloid and/or TSNA precursor content as compared to an unmodified plant, wherein the CYP94 cytochrome P450 comprising an ortholog of SEQ ID NO: 1-3 or a sequence with at least 80% identity to SEQ ID NO: 1-3.
The instant disclosure describes the knockout of Nitab4.5_0007257g0030.2 encoding a CYP94 cytochrome P450 decreasing alkaloid content in hairy roots of a tobacco plant. Nitab4.5_0007257g0030.2 has a genomic sequence of SEQ ID NO. 1, CDS of SEQ ID NO. 2, and amino acid sequence of SEQ ID NO. 3.
The instant disclosure does not describe any modification that can modulate or decrease the activity or expression of any CYP cytochrome P450 resulting in decreased alkaloid content and/or TSNA content in any part of a tobacco plant or a whole tobacco plant with the modifications, leaf thereof, or tobacco product/blend comprising the modified leaves. The instant disclosure does not describe that an orthologue or sequence with 80% identity to SEQ ID NOs. 1-3 may confer the same function.
Modifications, including mutations, insertions, deletions, and/or substitutions, can have varying
effects on gene expression. Ge, F. et al. (2024, “Review of Computational Methods and Database Sources
for Predicting the Effects of Coding Frameshift Small Insertion and Deletion Variations,” ACS
Publications, 9:2032-2047) teaches, for example, silent mutations may not change the protein structure and thus do not result in increased gene expression, while others, such as frameshift mutations, can cause
a premature stop codon resulting in a truncated protein that is not functional [pg. 2032, col. 1, ¶1]. As the
Applicant only provides the example of the knockout mutation; any type of modification is not reduced to practice. Further, as the claims are broad, modification may encompass natural modification (see claim interpretation). Undue experimentation would be required to be required to confirm that the structure of the mutation or modification performs the function of modulating or decreasing expression and/or activity of a CYP94 cytochrome P450 resulting in decreased alkaloid and/or TSNA content. The instant disclosure lacks sufficient variety of species to reflect the variance within the genus of modification.
Modulating or decreasing any CYP94 cytochrome P450 would not necessarily comprise decreased alkaloid and/or TSNA precursor content. Applicants concede that CYP94C1 in Arabidopsis is thought to be responsible for the inactivation of JA, but that the results of the instant disclosure have unexpectedly demonstrated the opposite effect for the closest homologue in tobacco i.e. that decreasing CYP94 activity and/or expression decreases alkaloid content and thus likely jasmonate signaling [pg. 9, lns. 3-5]. Yang, D. et al. ("Tobacco cytochrome C gene NtCYP94B3s and application thereof in improving content of tobacco jasmonic acid," Publication No. CN 112094855 A, published 12/18/2020, translation included in file wrapper) teaches the tobacco cytochrome gene NtCYP94B3s and the application thereof in improving the content of tobacco jasmonic acid [Abstract]. Yang teaches modulated expression of the tobacco endogenous gene NtCYP94B3 by silencing the gene in the tobacco plant, so that the JA and JA-IIe contents of the tobacco leaf are obviously improved. The content of 12OH-JA-Ile is significantly reduced compared to the WT plant [pg. 3, ¶6]. Thus, jasmonate signaling was not decreased with CYP94B3 silencing, as is speculated in the instant disclosure [pg. 9, lns. 3-5].
Futher, Chen teaches that jasmonic acid mediates many aspects of plant defense responses including nicotine biosynthesis [Abstract]. Chen teaches that jasmonic acid regulates nicotine biosynthetic gene expression through the MYC2 and the jasmonate ZIM‐domain (JAZ) repressors system [pg. 2, col. 1, ¶3]. In the absence of JA, the JAZs bind to MYC2 and form a repression complex, blocking MYC2 from activating nicotine biosynthetic genes. Chen teaches that JA signaling could be attenuated by hydrolysis of JA‐Ile by p450 enzymes CYP94B3 and CYP94C1 [pg. 2, col. 2, ¶3].
Thus, modulating (which encapsulates increasing or altering according to the claim interpretation above) or decreasing a CYP94 cytochrome P450 in any way may not result in a decrease in alkaloid content. In fact, decreasing CYP94 cytochrome P450 through RNAi or T-DNA mutation may increase jasmonic acid, defense responses, and thus increase alkaloid production, such as nicotine. A representative number of species was not described to represent the entire genus of modulating or decreasing the activity or expression of a CYP cytochrome P450. Example 2 in the instant disclosure simply states that homologues of SEQ ID NO. 3 were tested but does not provide additional data to support the claimed invention.
Example 1 of the instant disclosure states that the alkaloid content in the hairy roots was decreased with the knockout mutation, but does not reduce to practice decreased alkaloid content in the tobacco plant, plant part, or harvested leaf thereof. The art teaches that hairy root culture generation to transformed plant has several challenges. Lui, C. et al. (2025, “Reprogramming Hairy Root Cultures: A Synthetic Biology Framework for Precision Metabolite Biosynthesis,” Plants (Basel), 14(13):1928) teaches that scalability, specificity of metabolite biosynthesis, long-term genetic and metabolic stability, and reproducibility of culture performance under varied conditions may prove difficult for hairy root culture [pg. 16, ¶1].
The claims and the instant disclosure provide no guidance with respect to the regeneration of tobacco plants or leaves from hairy roots, merely stating that knockout of the gene leads to a decrease in alkaloid content in leaves. The statistical analysis and provided figure showing the analyzed data is for hairy roots alone [Fig. 1]. The specification lacks sufficient variety of species to reflect the potential variance between hairy roots and any other part of the tobacco plant. It is not clear that the structure in the regenerated plants would have the same function of decreased alkaloid content.
As is known in the art, biological sequences with sequence similarity can sometimes retain functional properties, but they do not always perform the exact same role as the whole sequence. Dib, L. et al. (2012, “Protein Fragments: Functional and Structural Roles of Their Coevolution Networks,” PLoS ONE 7(11): e48124) teaches that fragments along a protein sequence may form functional motifs necessary to the function claimed, without which, the truncated portion may not function [Abstract; pg. 2, col. 1, ¶2]. As 80% sequence identity still allows for great breadth within the claimed sequences, the sequences may not retain the same functionality. The Applicant does not provide working examples of a sequence with 80% identity to any of the sequences in the method as claimed.
Due to the functional unpredictability of sequences with 80% identity and orthologs of the sequences thereof, and breadth of the potential sequences, one of ordinary skill in the art would not be able to make and/or use the full scope of the claimed invention with only reasonable experimentation. Undue experimentation would be required to ensure that any sequence with between 80-100% of any of the three claimed sequences still maintained the functionality of the sequences.
Response to Applicant’s Arguments:
Applicant's arguments filed 07/02/2026 have been fully considered but they are not persuasive. Applicant contends that one of ordinary skill in the art would understand that the gene of an orthologue and its main function are conserved. Applicant argues that the specification provides homologues of CYP94 and that the results demonstrated in the examples would apply to orthologues of the claimed sequences. Applicant asserts that one would have reasonable expectation of success in using sequences with at least 80% sequence identity to the claimed sequences. Applicant contends that hairy root experiments are well known in the art and routinely used to assess the impact of modulating the expression or activity of genes upon alkaloid biosynthesis and that the Examiner has provided no evidence that one skilled in the art would find the statistically significant data implausible.
Regarding the claim of orthologues of SEQ ID NOs: 1-3, although one of ordinary skill would understand that the gene of an orthologue and its main function are generally conserved, the Applicant has not described any orthologues used in the tobacco plant of the invention. The Applicant further contends that “the specification expressly provides that "homologues of CYP94 cytochrome P450 SEQ ID No. 3 are provided in Table 1" and that "[t]he homologous amino acid sequence and/or nucleotide sequence and/or fragments should provide and/or encode a polypeptide which retains the functional activity and/or enhances the activity of the CYP94 cytochrome P450." (Specification, p. 62, 11. 4-6, 15-17.) Thus, it would be expected that the results demonstrated in the examples of the application would apply to orthologues of the claimed sequences.” [Applicant Arguments/Remarks Made in an Amendment filed 07/02/2026].
It appears that the Table 1 homologues are all in Nicotiana tabacum. As stated in MPEP § 2163, to satisfy the written description requirement, a patent specification must describe the claimed invention in sufficient detail that one skilled in the art can reasonably conclude that the inventor had possession of the claimed invention. As orthologues are genes in different species and the Applicant has not provided specific sequences or structural properties, much less a working example showing that an orthologue structure in the tobacco plant would retain the same function as the claims, it is not clear that the Applicant possesses the invention to the broad scope claimed. To the contrary of the Applicant’s arguments, the instant specification recites that “in Arabidopsis thaliana, CYP94C1 is responsible for the final carboxylation of JA-Ile, responsible for inactivation of JA. CYP94C1 in Arabidopsis is thought to be responsible for the inactivation of JA. As described below, the present inventors have unexpectedly demonstrated the opposite effect for the closest homologue in tobacco i.e. that decreasing CYP94 activity and or expression decreases alkaloid content and thus likely also decreases jasmonate signaling.” [pg. 9, lns. 1-6]. This demonstrates the implausibility of orthologues functioning in the same claimed tobacco plant.
The unpredictability of the invention was emphasized in the Office action filed 04/02/2026, providing examples of modulating or decreasing CYP94 cytochrome resulting in increased JA production, defense responses, and thus the increase of certain alkaloids [pg. 9, ¶1-3]. Additionally, the Applicant merely states that the homologues should retain the functional activity and/or enhance the activity of the CYP92 cytochrome P450, suggesting that none of the homologues have been reduced to practice.
Regarding Applicant’s assertion that one would have reasonable expectation of success in using sequences with at least 80% sequence identity to the claimed sequences, as stated in the Office Action filed 04/02/2026, no species were provided to support the broad genus claim of 80% identity to the claimed sequences. For example, SEQ ID NO: 1 is 2147 nucleotides long. 80% identity allows for a total of 429 nucleotide differences, any of which may alter the structure to function relationship. Additionally, as detailed above, a CYP94C1 of Arabidopsis is responsible for the final carboxylation of JA-Ile, responsible for inactivation of JA while the present inventors have unexpectedly demonstrated the opposite effect for the closest homologue in tobacco [pg. 9, lns. 1-6]. [pg. 9, lns. 1-6]. The unexpected results demonstrate the lack of predictability in the sequence structure leading to the claimed function. One of skill in the art would not expect a highly variable sequence provide sufficient function in an unpredictable art.
A "representative number of species" means that the species which are adequately described are representative of the entire genus. Thus, when there is substantial variation within the genus, one must describe a sufficient variety of species to reflect the variation within the genus. See AbbVie Deutschland GmbH & Co., KG v. Janssen Biotech, Inc., 759 F.3d 1285, 1300, 111 USPQ2d 1780, 1790 (Fed. Cir. 2014). MPEP § 2163. The Applicant has provided a single example with 100% identity to SEQ ID NO: 3, thus, a representative number of species are not adequately described.
Lastly, Applicant contends that hairy root experiments are well known in the art and routinely used to assess the impact of modulating the expression or activity of genes upon alkaloid biosynthesis and that the Examiner has provided no evidence that one skilled in the art would find the statistically significant data implausible. Examiner notes that the Office action did not question whether or not hairy root experiments are well known in the art or the plausibility of the statistically significant data. However, the statistically significant data indicates decreased alkaloid content in hairy roots, not in the leaves (see instant specification pg. 69). The Applicant contends on page 7 of Applicant Arguments/Remarks Made in an Amendment that, “the Specification demonstrates that "[k]nock out of Nitab4.5_0007257g0030.2 leads to a decrease in alkaloid content in leaves, in particular a decrease in nicotine, nornicotine, anabasine, PON and anatabine content" and that the data is "representative of 4-8 biological replicates analysed by t-test" with "statistical significance of P value ≤ 0.001." (Specification, p. 69.) On this basis, the specification concludes that "Nitab4.5_0007257g0030.2 is a positive regulator of alkaloid content and is a regulator of pyridine alkaloids in tobacco." (Specification, p. 70, 11. 2-3.).”
Examiner respectfully disagrees with Applicant’s characterization of the results as recited in the specification. The results instead describe that the statistical analysis referred to in the Applicant Arguments/Remarks Made in an Amendment was performed for the alkaloid content of 5-week-old hairy roots with homozygous knock-out mutations. The data that is representative of 4-8 biological replicates shown in Fig. 1 refers to the hairy roots, not the alkaloid content in leaves, as explicitly recited in the specification (see results below, pg. 69 of instant specification).
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The Examiner was not questioning the statistical significance of the results, but instead demonstrating the unpredictability of any modification resulting in the claimed modified tobacco plant or part thereof. Thus, Examiner maintains that while the Applicant has reduced to practice tobacco leaves having been modified by knockout mutation of Nitab4.5_0007257g0030.2 in hairy roots to decrease activity or expression of a CYP94 cytochrome P450 and comprising decreased nicotine, nornicotine, anabasine, PON, and anatabine in hairy roots, the Applicant has not reduced to practice any modification to increase (modulation) or decrease to any alkaloid in the whole tobacco plant or part thereof, as claimed.
Examiner notes that Applicant does not explicitly acknowledge that modulating (or increasing) any CYP94 cytochrome P450 was not reduced to practice as no species were provided to support the claimed genus (see Office action 04/02/2026, pg. 9, ¶3).
Thus, this rejection is maintained.
Claim rejections under 35 USC § 112 (a) – Scope of Enablement
Claims 7, 8, 10-13, 16, 18-19, and 21-22 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 reducing alkaloid content of tobacco hairy roots with homozygous knock-out mutations in Nitab4.5_0007257g0030.2 encoding a CYP94 cytochrome P450, does not reasonably provide enablement for a tobacco plant or any part thereof having been modified in any way to modulate or decrease the activity or expression of any CYP94 cytochrome P450 resulting in decreased alkaloid and/or TSNA precursor content in comparison to a control plant. 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 the invention commensurate in scope with these claims. This rejection is modified from the rejection set forth on pages 10-13 in the Office action dated 04/02/2026.
The claims broadly encompass a tobacco plant or any part thereof and any modification to alter the activity or expression of a CYP94 cytochrome P450 comprising decreased alkaloid and/or TSNA precursor content as compared to an unmodified plant, wherein the CYP94 cytochrome P450 comprising an ortholog of SEQ ID NO: 1-3 or a sequence with at least 80% identity to SEQ ID NO: 1-3.
The specification provides little guidance over the breadth of the pending claims, providing only two examples to support the claims. Example 1 discloses gene editing of Nitab4.5_0007257g0030.2 which encodes a CYP94 cytochrome P450. Gene editing was used to knock out Nitab4.5_0007257g0030.2 in hairy roots and reversed phase high performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used to determine relative content of pyridine alkaloids in hairy roots with the knockout mutations. One figure was provided (Fig. 1) to support the alkaloid content decrease with the mutation, however, this appears to be only data shown in hairy roots, not in any other part of a tobacco plant. Example 2 simply states that “the effects of the homologues of SEQ ID NO. 3, namely those listed in table 1, are tested in assays as described in the above example.” There are no working examples for any orthologs of Nitab4.5_0007257g0030.2 that would indicate to the skilled artisan that a mutation of an ortholog would result in reduced alkaloid content. No data or statistical analysis is provided to support reduced alkaloid content in any tobacco plant organ or cell with the exception of hairy roots. The singular modification that was enabled is the knockout mutation of Nitab4.5_0007257g0030.2. There is no teaching as to other modifications that may modulate or decrease the activity of a CYP94 cytochrome P450 so as to decrease alkaloid or TSNA precursor content.
Biswas, D. et al. (2023, “Hairy root culture: a potent method for improved secondary metabolite production of Solanaceous plants,” Front. Plant Sci. 14:1197555) teaches that hairy roots are primarily generated by Agrobacterium-mediated transformation infection at a wound site, resulting in rapid growth [Abstract]. Hairy roots often have high transformation efficiency and mirror the chemical profile of the plant’s own roots. Zhu, Y. et al. (2024, “Plant hairy roots: Induction, applications, limitations and prospects,” Industrial Crops & Products, 219) teaches that hairy roots as compared to leaves may not regenerate to whole transgenic plants and may require a species-specific method for generating a transgenic plant. Additionally, some regenerated transgenic plants obtained by A. rhizogenes-mediated genetic transformation will show Ri phenotypes (phenotypes from co-cultivation and natural transformation using rhizogenic agrobacteria) or hairy root phenotypes such as loss of apical dominance, altered leaf morphology, shortening of internodes, early flowering, premature leaf senescence, and changes in flower shape [pg. 12, col. 2, ¶2].
Zhu teaches that content of secondary metabolites in plants is usually low and tissue-specific (e.g., present in tissues such as stems, leaves, flowers, fruits, and seeds), which makes the hairy roots not the best platform for the biosynthesis of some secondary metabolites [pg. 12, col. 1, ¶4]. While Tong, A. et al. (2025, “DNA methylation valley as a distinguishing feature occurs in root-specific expressed nicotine-related genes in Nicotiana attenuate,” Front. Plant Sci., 16:1647622) teaches that most nicotine-related genes are synthesized exclusively in the roots [Abstract]. The content of nicotine or any alkaloid and/or TSNA precursor would be unpredictable in the whole plant or part thereof aside from the hairy roots enabled in the instant specification. Though the Applicant concluded in the results of the specification that knockout of the gene leads to a decrease in alkaloid content in leaves, the actual provided data and statistical analysis does not support anything other than a reduction in five specific TSNAs. No other alkaloids or plant parts are shown.
As shown above, the alkaloid content and/or TSNA precursor content in the transgenic tobacco plant, plant or crop bred or frown from the tobacco plant, harvested leaf, or processed leaf, may not predictably have the same result as the hairy roots with the Nitab4.5_0007257g0030.2 knockout shown in Example 1 of the instant specification. Undue levels of experimentation would be required in order to practice the claimed invention. The instant specification does not provide adequate guidance on what alternative modifications may be made to the CYP94 cytochrome P450 other than the mutation. The instant specification does not disclose any other part of the tobacco plant with the exception of hairy roots with the mutation. The skilled artisan would be required to identify alternative mutations and generate transgenic plants therefrom to determine if the tobacco plant comprises decreased alkaloid/TSNA content. In light of the unpredictability and lack of direction provided, one of ordinary skill in the art would not have had reasonable expectation of success in making the claimed invention.
Examiner notes that claim 7 has been amended to recite SEQ ID NOs. for the proposed CYP94 cytochrome P450. However, this does not overcome the scope of enablement rejection for the scope of the modifications possible and the regenerated tobacco plant from the transformed hairy roots due to the maintained recitation of orthologs of the SEQ ID NOs., for which no working examples were provided, or the broad scope of 80% sequence identity as claimed.
Response to Applicant’s Arguments:
Applicant's arguments filed 07/02/2026 have been fully considered but they are not persuasive. Applicant contends that the specification teaches multiple methods by which a skilled person may reduce or eliminate expression.
Examiner respectfully disagrees. Applicant asserts that the specification provides extensive guidance enabling the skilled person to practice the invention. Examiner points out that the claims recite that the plant or part thereof is “modified to modulate or decrease the activity or expression of a CYP94 cytochrome P450.” Based on the definition in the instant specification, modulating may be increasing. Thus, although the specification broadly states methods that could be used for decreasing expression, modulating expression is still not reduced to practice. Further, the Applicant states "[g]ene editing was used to knock out Nitab4.5_0007257g0030.2 in hairy roots" (Specification, p. 69.), showing the results of decreased alkaloid content in hairy roots in Fig. 1. This emphasizes that the Applicant has reduced to practice a knockout modification (i.e., decrease) to reduce the specific alkaloids of nicotine, nornicotine, anabasine, PON, and anatabine, not the genus of any modification to increase or decrease any alkaloid.
Additionally, as detailed above, the instant specification states that a CYP94C1 of Arabidopsis is responsible for the final carboxylation of JA-Ile, responsible for inactivation of JA, while the present inventors have unexpectedly demonstrated the opposite effect for the closest homologue in tobacco [pg. 9, lns. 1-6]. [pg. 9, lns. 1-6]. The unexpected results of the instant invention demonstrate the unpredictability in the art. Thus, although alternative methods were suggested, no working examples were provided. One of skill in the art would not expect any method of modification to increase or decrease the activity of expression of a CYP94 cytochrome P450 to make the invention of a plant with decreased content of any alkaloid and/or TSNA precursor given the unpredictability indicated by the Applicant.
Examiner respectfully notes that Applicant has not acknowledged that transformation of hairy roots may not result in a transgenic plant with the same properties and that nicotine related genes are synthesized exclusively in the roots (see Office action 04/02/2026, pg. 12)
This rejection is modified but maintained.
Claim Rejections under 35 USC § 103
Claims 7-8, 10-13, 16, 18-19, and 21-22 are rejected under 35 U.S.C. 103 as being unpatentable over Luo, J. et al. (2016), “COI1-Regulated Hydroxylation of Jasmonoyl-L-isoleucine Impairs Nicotiana attenuata’s Resistance to the Generalist Herbivore Spodoptera litura,” J. Agric. Food Chem. 64: 2822−2831, in view of Paschold, A. et al. (2007), “Co(i)-ordinating defenses: NaCOI1 mediates herbivore-induced resistance in Nicotiana attenuata and reveals the role of herbivore movement in avoiding defenses,” The Plant Journal, 51(1):79-91, Hart, R. et al. US. Patent Application No. US 20210360960 A1, published 11/25/2021, NCBI Reference Sequence: XM_016659890.1 (PREDICTED: cytochrome P450 94C1-like mRNA [Nicotiana tabacum], originally available 5/3/2016), Heitz, T. et al. (2012), “Cytochromes P450 CYP94C1 and CYP94B3 Catalyze Two Successive Oxidation Steps of Plant Hormone Jasmonoyl-isoleucine for Catabolic Turnover,” The Journal of Biological Chemistry, 287(9):6296-6306) (as cited in IDS filed 1/30/2025). This rejection is modified from the rejection set forth on pages 13-22 in the Office action dated 04/02/2026.
Regarding claims 7, 10, and 11, Luo discloses that phytohormone JA-Ile is well-known as the key signaling molecule that elicits plant defense responses after insect herbivory [Abstract]. Oxidation of JA-Ile is catalyzed by the cytochrome P450s of the CYP94 family and results in the synthesis of 12-hydroxy-jasmonoyl-isoleucine (12OH-JA-Ile). COI1, the receptor of JA-Ile, appeared to transcriptionally control NaCYP94B3 like-1 and-2 and thus regulates the catabolism of its own ligand molecule, JA-Ile. JA-Ile functions as a signaling molecule and induces the production of many defenses including secondary metabolites and proteins, including nicotine (i.e. alkaloid) [pg. 2822, col. 2, ¶1]. Luo teaches that constructed vectors were transformed into Agrobacterium to specifically silence these NaCYP94B3s using the VIGS technology to generate VIGS-EV and VIGS-NaCYP94B3 like 1,-2,-3, and-4 plants [pg. 2826, col. 1, ¶3]. Luo teaches that, consistent with the decreased 12OH-JA-Ile levels in plants silenced in NaCYP94B3 like-1 or-2, the JA-Ile levels increased in these plant [pg. 2826, col. 2, ¶1]. Luo teaches that direct defense traits, namely, TPI activity, nicotine contents, and HGL-DTGs levels, were all highly induced in VIGS NaCYP94B3s due to JA-Ile overproduction caused by blocking JA-Ile hydroxylation [pg. 2827, col. 2, ¶2].
Luo teaches JA, JA-Ile, and 12OH-JA-Ile accumulation patterns in WT and plants transformed with inverted repeat constructs, iraoc, irjar4/6, and ircoi1, which have impaired JA biosynthesis, JA-Ile biosynthesis, and JA perception, respectively. At 1 hour after a wounding treatment, JA levels in iraoc plants were strongly reduced to <3% of the WT levels and JA in ircoi1 plants was ~30% of the WT plants [pg. 2828, col. 1, ¶1]. Luo teaches that ircoi1 plants had decreased transcript levels of NaCYP94B3 like-1 and NaCYP94B3 like-2 (i.e. a tobacco plant having been modified to modulate or decrease the activity or expression of a CYP94 cytochrome P450).
Though the property of decreased nicotine (i.e. alkaloid) content would be inherent to the composition (ircoi1 mutant plant with impaired JA perception) as COI1 is a primary receptor for JA, which triggers the production of such metabolites, Luo does not explicitly teach that the ircoi1 plants had decreased alkaloid/nicotine content.
However, Paschold, teaches COI1 silenced by transformation with an inverted repeat construct (ir-coi1) in Nicotiana attenuata [Abstract]. The induction of TPI activity and the accumulation of nicotine in N. attenuata plants in response to herbivory is mediated by JA signaling [pg. 83, col. 1, ¶2]. Paschold teaches that ir-coi1 plants are male sterile and impaired in JA-elicited direct defense responses, such as nicotine, as compared to a WT control [Abstract; Fig. 3A, as shown below].
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Luo and Paschold do not explicitly teach a motivation to decreased alkaloid content and/or TSNA, however, Hart teaches novel methods of curing tobacco to produce desirable smoke and flavor characteristics while reducing the undesirable decreasing the level of TSNAs and Harmful and Potentially Harmful Constituents (HPHCs) imparted during a conventional fire-curing process and tobacco products thereof [Abstract]. Hart teaches that HPHCs refers to a list established by the U.S. Federal Food and Drug Administration (FDA) by the Federal Food, Drug, and Cosmetic Act of chemicals and chemical compounds identified by the FDA as harmful and potentially harmful to humans [¶26]. This list comprises nicotine, nornicotine, and anabasine, among others.
Luo, Paschold, and Hart do not explicitly teach the tobacco plant or part thereof or the tobacco cell or cell culture of claim 7 wherein the CYP94 cytochrome P450 is encoded by a nucleotide sequence as set out in SEQ ID NO. 1. However, an NCBI BLAST search revealed a predicted cytochrome P450 94C1-like isoform X1 in Nicotiana tabacum (NCBI Reference Sequence: XM_016659890.1) with 100% alignment to SEQ ID NO. 1. Luo does not teach that a P450 94C1 cytochrome in tobacco would be modulated with a coi1 mutant, however, Heitz teaches that along with CYP94B3, CYP94C1 catalyzes oxidation of JA-Ile, and that in coi1 mutants in Arabidopsis, CYP94B3 and CYP94C1 expression was nearly abolished.
Luo teaches that the N. attenuata CYP94 cytochrome P450s of were homologues of Arabodopsis AtCYP94B3 and AtCYP94B1, two enzymes hydroxylating JA-Ile [pg. 2825, col. 1, ¶1]. The four candidates, named NaCYP94B3 like-1,-2,-3, and-4, respectively, were obtained from a search of the N. attenuata genome using the protein sequences of the Arabidopsis CYP94 enzymes. The NaCYP94B3 like-1,-2,-3, and-4 protein sequences, together with other AtCYP94B3- and AtCYP94B1-like proteins from some Solanaceous species and all members of the Arabidopsis CYP94 subfamily were phylogenetically analyzed to show similarities and indicate that they have similar functionality [pg. 2825, col. 2, ¶1]. Thus, although the coi1 mutants with decreased expression of CYP94C1 were in Arabidopsis, as taught by Heitz, there would have been similar functionality to tobacco based on the analysis of Luo, and thus the predicted tobacco CYP94C1 (with 100% identity to SEQ ID NO: 1 of the instant application) would also decrease with the modification of coi1.
Given that Luo teaches an ircoi1 plant that has been modified and modulates or decreases transcript levels of NaCYP94B3 like-1 and-2; given that Paschold teaches that ircoi1 plants comprise decreased nicotine content; given that Hart teaches that lower alkaloid, TNSA and/or nicotine content in tobacco is beneficial to human health; given that NCBI Reference Sequence: XM_016659890.1 is a predicted CYP94C1-like nucleotide sequence; and given that Heitz teaches decreased CYP94C1 expression in coi1 mutants in Arabidopsis, it would have been obvious to one of ordinary skill in the art at the time of filing to decrease or modulate expression of a CYP94 cytochrome P450 in tobacco using the ircoi1 mutant of Luo and Paschold, and thus reduce alkaloid content by blocking JA-elicited direct defense responses, such as nicotine, as taught by Paschold. One would reasonably expect the predicted CYP94C1-like to act similarly given that the tobacco CYP94 cytochrome P450s were previously demonstrated to be homologues to Arabidopsis, as taught by Luo. Thus, the ir-coi1 lines with lower nicotine content and decreased transcript levels of NaCYP94B3 like-1 and NaCYP94B3 like-2 would also have lower CYP94C1-like activity or expression due to the homology to the Arabidopsis genes effected by the coi mutants. This rejection is made to the extent that the claims recite that the tobacco plant has been modified (ir-coi) to modulate or decrease activity or expression of a CYP94 cytochrome P450, and additionally comprise decreased alkaloid and/or TSNA precursor content (nicotine). The claims do not require direct modification of the claimed CYP94 cytochrome P450.
One would have been motivated to lower the alkaloid and/or TSNA content as Hart teaches that these compounds are harmful or potentially harmful to humans. One having ordinary skill in the art would have reasonable expectation of success because modification to decrease activity or expression of a tobacco CYP94 cytochrome P450 was already known to the prior art as taught by Luo.
Regarding claims 8, 12-13, and 16, Paschold teaches generation of transgenic plants with ir-coi1 lines 1 and 2 [pg. 88, col. 1, ¶2]. Paschold teaches that the anther-carrying filaments of the male sterile lines were unable to dehisce and that after drying in a desiccator, they were able to open and release fertile pollen grains [pg. 88, col. 1, ¶2]. The fertile pollen grains were used to pollinate stigmas of open flowers resulting in viable seeds (i.e. a plant propagation material obtained from the tobacco plant). This method was used to generate homozygous progeny of both ir-coi1 lines (i.e. a plant grown from the tobacco plant or part thereof). For analysis of herbivory, detached leaves of ir-coi1 plants were used (i.e. a leaf produced from the tobacco plant; a harvested leaf or cut leaf of the tobacco plant) [pg. 88, col. 2, ¶6]. For reasons detailed above, one would reasonably expect the plant propagation material obtained from the tobacco plant according to claim 7 to have modulated or decreased activity or expression of a CYP94C1-like gene or protein, such as predicted NCBI Reference Sequence: XM_016659890.1.
Regarding claims 18-19 and 21-22, Hart teaches a curing combination process to reduce harmful constituents in cured tobacco and includes cured tobacco having reduced polyaromatic hydrocarbons and TSNAs as compared to conventionally fire-cured tobacco (i.e. processing the tobacco plant or leaf harvested therefrom, wherein the leaf is processed by curing) [Abstract]. Also provided are tobacco products made with tobacco leaf cured using the disclosed processes (i.e. a tobacco blend comprising said cured tobacco material). Hart teaches that any modified or unmodified tobacco plant may be cured [¶64].
It would have been obvious to one of ordinary skill in the art at the time of filing to use the tobacco plant as taught by Luo, Paschold, NCBI Reference Sequence: XM_016659890.1, and Heitz in the process of Hart to further reduce TSNAs in the tobacco product, as Hart teaches that the method may be conducted with any tobacco plant. One of ordinary skill in the art at the time of filing would have been motivated to do so to further reduce harmful constituents as taught by Hart for use in tobacco products. One would have reasonable expectation of success as the physical tobacco leaf as claimed would be able to be processed according to known methods, such as the ones taught by Hart.
Response to Applicant’s Arguments:
Applicant's arguments filed 07/02/2026 have been fully considered but they are not persuasive. Applicants contend that in the rejection of claims 7-8, 10-13, 16, 18-19, and 21-22, none of the cited documents disclose or suggest the presently claimed sequences, that the specification describes the role of these sequences as an unexpected discovery, and that there is no motivation in the cited documents to consider the claimed sequences or to expect that modulating expression of these sequences would result in decreased alkaloid and/or TSNA precursor content.
In the rejection of claim 32, Applicant contends that the sequence XM_016659890.1 is a predicted sequence, there is no suggestion that it has a role in alkaloid biosynthesis, and one would have no reasonable expectation of success in decreasing the expression or activity to decrease alkaloid and/or TSNA precursor content. Further, Applicant argues that the Heitz reference is directed towards Arabidopsis, not tobacco, and that Heitz provides no motivation to modify the activity or expression of a CYP94C1-like isoform in tobacco for the purpose of decreasing alkaloid content. The Applicant contends that the combination of documents is only possible through impermissible hindsight.
In response to applicant's argument that the references fail to show certain features of the invention regarding the rejection of claims 7-8, 10-13, 16, 18-19, and 21-22, it is noted that the features upon which applicant relies (i.e., the sequences previously presented in claim 32) were not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993). In light of the claim amendments, these claims remain rejected under the modified rejection detailed above.
Regarding the rejection of claim 32 (the features of which are now incorporated into claims 7 and 8), in response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, the Examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007).
In this case, Luo teaches an ircoi1 plant that has been modified and modulates or decreases transcript levels of NaCYP94B3 like-1 and-2. Paschold teaches that ircoi1 plants comprise decreased nicotine content. Hart teaches that lower alkaloid, TNSA and/or nicotine content in tobacco is beneficial to human health. NCBI Reference Sequence: XM_016659890.1 is a predicted CYP94C1-like nucleotide sequence. Heitz teaches decreased CYP94C1 expression in coi1 mutants in Arabidopsis. It would have been obvious to one of ordinary skill in the art at the time of filing to decrease or modulate expression of a CYP94 cytochrome P450 in tobacco using the ircoi1 mutant of Luo and Paschold, and thus reduce alkaloid content by blocking JA-elicited direct defense responses, such as nicotine, as taught by Paschold. One would reasonably expect the predicted CYP94C1-like to act similarly given that the tobacco CYP94 cytochrome P450s were previously demonstrated to be homologues to Arabidopsis, as taught by Luo. Thus, the ir-coi1 lines with lower nicotine content and decreased transcript levels of NaCYP94B3 like-1 and NaCYP94B3 like-2 would have predictably lower CYP94C1-like activity or expression due to the homology to the Arabidopsis genes effected by the coi mutants.
The Examiner respectfully disagrees with the Applicant’s characterization of the relied upon sequence, XM_016659890.1. Although XM_016659890.1 is annotated as a predicted sequence, a fairer interpretation of the referenced sequence is that it is annotated as derived from cytochrome P450, for which the Examiner provided adequate rationale for utilization in alkaloid biosynthesis (see, Luo, Paschold, and Hart, pg. 19-21 of Office Action mailed 04/02/2026). There is motivation within Luo and Heitz to suggest that a modification, such as the ircoi1 mutant, which decreases NaCYP94B3 like-1 and-2 in Luo as well as AtCYP94C1 in Heitz, in addition to decreasing nicotine content, as taught by Paschold, would alter CYP92C1-like expression or activity in tobacco as well, given Luo’s teachings that tobacco CYP94 cytochrome P450s were previously demonstrated to be homologues to Arabidopsis.
Additionally, at present, the claims do not require that the modulated or decreased activity or expression of a CYP94 cytochrome P450 must necessarily have a role in alkaloid biosynthesis, rather that the plant additionally comprises decreased alkaloid and/or TSNA precursor content with the modification that modulates or decreases. As the ircoi1 mutant resulted in decreased nicotine content in tobacco, as well as decreased CYP94C1 expression in Arabidopsis, one of skill in the art would reasonably expect that the ircoi1 mutant in tobacco would result in decreased expression of a CYP94C1-like gene in tobacco, given the homology demonstrated by Luo.
In response to applicant's argument that the examiner's conclusion of obviousness is based upon improper hindsight reasoning, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971).
Thus, this rejection is modified due to the claim amendments but is maintained.
Summary
Claims 7-8, 10-13, 16, 18-19, and 22 are rejected.
THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). 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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/EMILY K JOHNSON/Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/Supervisory Patent Examiner, Art Units 1661 & 1662
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