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 .
Priority
This application draws priority from an application with the number GB 2201414.6 with a priority date of 02/03/2022. A Certificate of Availability was filed on 04/14/2025.
Restriction/Election
In response to the communication received on March 30th, 2026, from Seiko Okada, the election of Group II, claims 7-8, 10-12, 29, without traversal, is acknowledged. Applicants have further added a new claim, claim 32, which is directed to Group II.
Status of Claims
Claims 1, 3, 5, 13, 18-19, 21-24, 27-28, and 31 are withdrawn under the restriction requirement.
Claim 16 is Canceled.
Claim 32 was Added.
Claims 1, 3, 5, 7-8, 10-13, 18-19, 21-24, 27-29, and 32 are pending.
Claims 7-8, 10-12, 29, and 32 are examined herein.
Claim Interpretation
The phrase “modified” in Claims 7-8, is interpreted by the examiner to mean any possible genetic or non-genetic modification, alteration, or change that may alter flavin adenine dinucleotide (FAD) synthetase activity in a tobacco plant (see the paragraph bridging page 10 and 11 in the specification).
“Modulate” is interpreted to mean increase or decrease as described on page 53 line 19 of the specification.
Claims 7-8, 10-12, and 32 that recite “tobacco plant” are interpreted to mean “a plant in the genus Nicotiana that is used in the production of tobacco industry products” as was stated on page 75 line 34 of the specification.
The “Plant” in Claim 29 is broad and is not explicitly defined in the specification. The “Plant” in Claim 29 is interpreted by the examiner to be synonymous to “Tobacco plant” as this disclosure is not directed to any other species and the abstract recites “plant (i.e. a tobacco plant)”.
FAD synthetase is given the broadest interpretation of:
An enzyme which catalysis the adenylation of flavin mononucleotide (FMN) to form flavin adenine dinucleotide (FAD) coenzyme (See page 11 line 21) or with a FAD synthase binding domain (Page 11 line 34).
In one embodiment, a FAD synthetase comprises an amino acid or nucleotide Page 13 line sequence shown as SEQ ID NOs.1-3, or a sequence which has at least 80% identity thereto, or a homologue thereof (See page 12 line 11, Page 12 line 31, and Page 13 line 15 )
Any sequence with at least 80% identify to sequences found in table 1 of the specification. (See page 12 line 14, Page 12 line 33, and Page 13 line 18 )
Functional variant or Functional Fragment is interpreted to mean a sequence that contains a FAD synthase binding domain which catalysis the adenylation of flavin mononucleotide (FMN) to form flavin adenine dinucleotide (FAD) coenzyme.
Claim Objections
In Claims 7, and 8, 10-12, and 32 depending therefrom, “TSNA” is used as abbreviation. It is suggested to insert a definition for TSNA without bringing in new matter, immediately before the first appearance of “TSNA” in Claim 7; and to enclose the appearance of “TSNA” in parentheses (in Claim 7 only).
In Claims 7 and 29 as well as 8, 10-12, and 32 depending therefrom, “FAD” is used as abbreviation. It is suggested to insert a definition for FAD without bringing in new matter, immediately before the first appearance of “FAD” in Claim 7; and to enclose the appearance of “FAD” in parentheses (in Claim 7 only).
In Claim 10, and Claim 11 depending therefrom, “PON” is used as abbreviation. It is suggested to insert a definition for PON without bringing in new matter, immediately before the first appearance of “PON” in Claim 10; and to enclose the appearance of “PON” in parentheses (in Claim 10 only).
Claim 29 is objected to for referencing the withdrawn Claim 28.
Appropriate correction is required.
Information Disclosure Statement
References listed in IDS documents have been considered by the examiner except where crossed through. References 42 and 58 in IDS filed on 01/22/2025 have not been considered because they were not provided. Instead of providing the document, Applicant uploaded screenshots of where to purchase the book. References 24 and 39 listed in IDS filed on 01/22/2025 also appear to have different titles than the documents uploaded with the same author while reference 25 appears to use the journal as the title in the IDS. The Sawada reference in IDS filed 4/30/2026 does not appear to be uploaded and another document in a language other than English was uploaded.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 10 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 10 draws dependence from Claim 7, which recites decreasing alkaloid and/or TSNA precursor content. However, Claim 10 fails to limit the scope of the claims as it states specific alkaloid’s content can be modulated, which is also interpreted to mean increased. This failure to limit the claims leads to indefiniteness, as it is unclear if alkaloid content should be increased, decreased, or if some alkaloids can be increased while others can be decreased.
Claim Rejections - 35 USC § 112 (a)
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.
Written Description
Claims 7-8, 10-12, 29, and 32 are rejected under 35 USC § 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 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".
The claims are broadly drawn to a tobacco plant or any part thereof having been modified in any way to modulate or decrease the activity or expression of a FAD synthetase and comprising decreased alkaloid and/or TSNA precursor content as compared to an unmodified plant.
Applicant describes:
Virus-induced gene silencing (VIGS) of a FAD synthetase gene (i.e. Nitab4.5_0005997g0050.2) with genomic sequence of SEQ ID NO: 1, the coding sequence of SEQ ID NO: 2, and the amino acid sequence of SEQ ID NO: 3 using the 300-nucleotide cDNA fragment with SEQ ID NO: 43. The TRV vector comprising both (TRV RNA 1 SEQ ID No. 34) and (TRV RNA2) comprising the targeted nucleotide sequence separately propagated in A. tumefaciens.
VIGS silencing Nitab4.5_0005997g0050.2 leading to a significant reduction of nicotine, anabasine, PON, and anatabine.
Applicant does not describe:
Modification of the activity or expression of a FAD synthetase peptide or nucleotide or amino acid sequence with at least 80% identity to SEQ ID NO:1-3, or a functional variants, functional fragments, orthologues, or homologue thereof, other than SEQ ID NOs: 1-3.
If alkaloid content can be increased and TSNA precursors maintained or reduced (i.e., the plant comprising decreased alkaloid and/or TSNA precursor content).
Modulation of a FAD synthetase’s activity in a plant.
Modification of any kind to modulate or decrease the activity or expression of a FAD synthetase, other than VIGS.
Any plant propagation material obtained from the tobacco plant comprising modulated or decreased activity or expression of a FAD synthetase from the heritable modification.
Any plant part with modulated FAD synthetase activity or expression and decreased alkaloid and/or TSNA precursor content, other than tobacco leaves.
The instant claims recite decreased alkaloid and/or TSNA precursor content in comparison to an unmodified plant. With the VIGS modification of Example 1, the instant disclosure describes silencing of FAD synthetase (i.e. Nitab4.5_0005997g0050.2). The results of the relative content of pyridine alkaloids were determined by LC-MS/MS, for analytes nicotine, anabasine, anatabine, nornicotine, PON (see page 103 of the specification). Alkaloid content of 5-week-old tobacco leaves silenced for Nitab4.5_0005997g0050.2 is shown in Fig. 1. Although the Applicant claims that VIGS of Nitab4.5_0005997g0050.2 leads to a decrease in alkaloid content in leaves, Nornicotine was not statistically reduced. Additionally, tobacco plants synthesize other alkaloids not listed in the specification or addressed in this disclosure. Cotinine and β-nicotyrine, are identified as “tobacco minor alkaloids” and are described on page 2 paragraph 2 in Chen, Peter X., et al. "Analysis of minor alkaloids in tobacco: a collaborative study." BEITRAGE ZUR TABAKFORSCHUNG INTERNATIONAL 21.7 (2005): 369.
Given the limited description in the instant specification of a few alkaloids in tobacco plants, a skilled artisan would not have reasonably recognized Applicant to be in possession of the full metes and bounds of the claims at the time the application was filed.
Modifications, including mutations, insertions, deletions, and/or substitutions, can have varying
effects on gene expression. Ge, Fang, et al. "Review of computational methods and database sources for predicting the effects of coding frameshift small insertion and deletion variations." ACS omega 9.2 (2024): 2032-2047 teaches, for example, silent (i.e. synonymous) 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 (see page 1 paragraph 1). Page 2 paragraphs 1-2 in Al Aboud, Nora M., Connor Tupper, and Ishwarlal Jialal. "Genetics, epigenetic mechanism." (2018) teaches that modifications to alter gene expression or activity includes epigenetic changes through alterations in the chromosome, rather than the DNA sequence, and regulating gene expression through chemical modifications. Modification may refer to the alteration of the peptide or gene encoding the peptide by any means, including merely natural modifications. Such modifications may or may not result in an increase or decrease in activity or expression of a FAD synthetase peptide, however, there is not sufficient structure to indicate that any modification would result in the function of increasing or decreasing activity or expression of a FAD synthetase in a reliable and predictable manner.
As the Applicant only provides the one example of the VIGS technique, which is a small species in the large genus of “modification”, all possible modifications are not reduced to practice. Further, it appears that Applicant is reducing to practice decreasing activity FAD synthetase through silencing (i.e. reduction in expression) and thus the plant comprises decreased nicotine content (see Example 1). Modulating, which may be defined as increasing (see, claim interpretation) activity or expression of a FAD synthetase would logically not have the same effect on the alkaloid and/or TSNA precursor content based on the data of Example 1. Therefore, the genus clause of modulating or decreasing the FAD synthetase activity or expression does not provide adequate structure to claim the function and a sufficient number of species were not described to reduce this large genus to practice. Undue experimentation would 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 FAD synthetase 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.
VIGS can serve as an alternative to mutant collections or stable transgenic plants to allow the characterization of gene functions in a wide range of angiosperm species, but is a transient method of genetic transformation. This was described in the abstract of Lange, Matthias, et al. "Virus-induced gene silencing (VIGS) in plants: an overview of target species and the virus-derived vector systems." Virus-Induced gene silencing: Methods and protocols (2013): 1-14. With only a few exceptions, the targeted gene silencing will not be transferred to subsequent generations (see page 10 paragraph 4). These limitations require experiments for each new species or specific tissue targeted, and the optimal time point and location of infection have to be determined experimentally (see page 10 paragraph 4). The Applicant has not reduced to practice VIGS in any tissue other than leaves. The Applicant has not provided any examples of a plant, plant propagation material, or any sort of processed leaf or product produced from the tobacco leaves with the VIGS modification. As VIGS are known to be transient and no examples of a plant grown from the tobacco plant with the modification with modulated or decreased activity or expression of FAD synthetase or decreased alkaloid content are reduced to practice, the Applicant has not sufficiently linked the structure to the function.
Use of FAD synthetase is not prevalent in relation to altering alkaloid and/or TSNA content. FAD synthetase is used to synthesize FAD. FAD and FMN are vital to all living organisms because of their function as irreplaceable cofactors for enzymes participating in a wide variety of metabolic processes. These processes include, but are not limited to, mitochondrial electron transport, photosynthesis, fatty acid oxidation, protein folding, chromatin remodeling, and metabolism of several other biologically relevant compounds, such as nucleotides, amino acids, antioxidants, and other cofactors. In plants, they also play a significant role in signaling as the chromophores in blue-light receptors1. Given the limited research around reduction of expression or activity of plant FAD synthetase, or with regard to decreasing alkaloid and/or TSNA content in tobacco plants, the level of predictability in the art is low.
As in the claim interpretation, FAD synthetase covers a large range of evolutionarily related sequences of amino acids and nucleotides sharing a common ancestor and exhibiting similarity in sequence, structure, or function. The instant disclosure provides a singular example of VIGS of Nitab4.5_0005997g0050.2 purportedly reducing alkaloid content in leaves (Example 1). The VIGS construct uses a 300-nucleotide cDNA fragment of Nitab4.5_0005997g0050.2 (SEQ ID NO: 43). The results show a significant decrease in some alkaloids, but not all possible alkaloids in tobacco (Figure 1). Homologue testing in Example 3 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. Table 1 does not provides all nucleotide and amino acid sequences with 80% similarity to SEQ ID NOs: 1-3 and the specification does not display any data describing the influence of VIGS on these genes or on alkaloid and/or TSNA precursor content.
A related homolog, orthologue or sequence with 80% similarity to FAD synthetase is a broad term for any character, gene, or structure that shares a common ancestral origin. Though homologous/orthologous genes among different plants may perform similar or equivalent functions2. A representative number of species was not described to represent the entire genus of modulating or decreasing the activity or expression of the vastness of the FAD synthetase genus. Sufficient structure was not provided of the homolog-related peptides that share a common ancestor and still maintain the same function in the claims. This was supported by the prior art where the supplementary information from Lynch et al 2022 also provides a FAD synthetase protein with a sequence (see below) that has very low sequence homology to SEQ ID NO:3 but is still classified as a FAD synthetase.
>N.tabacum XP_016463627.1
MEIDKAIRECDDGRLKTKYNNAIYVIKRALALYSVQEVALSFNGGKDSTVLLHLLRAGCFLHEAEENNLR
GDAADGGKTFPIRTIYFESPSAFPEINSFTYEAAATYNIQMDIIRLDFKSGLEALLKANPIRAIFLGVRI
GDPTAVGQEQFSPSSPGWPPFMRVNPILDWSYRDVWAFLLVCKVQYCSLYDQGYTSIGSIHDTVRNALLC
IRNSDNSEEKFKPAYLLADGRLERAGRVKKNPSSVCGKLSSISNGGKMENLNSGSMLTASIISVGDEILF
GTVEDKLGSMLCKKLHSIGWAVSRVAVTRNDIDSVAEEVERRKSTDDMVLIFGGIGPLHSDVTVAGVAKA
FGVRMAPDEEFEEHLRHLIGEKCSGDKNEMALLPEGITELLHHEQLPVPLIKCHNVIILTATNVVELDRQ
WDCLIELAKSNGILVLMDPFVSKCFATTLSDVEVAQPLSKLCAQFPDLYIGGYRRSREGPVVITFEGKDL
SRIEAASQSLCQKFHAGAFSEIE*
Björklund, Åsa K., et al. "Domain rearrangements in protein evolution." Journal of molecular biology 353.4 (2005): 911-923 teaches “proteins are composed of domains, recurrent protein fragments with distinct structure, function and/or evolutionary history” and “The addition of a domain (i.e. fragments) to a protein is likely to alter its function, for example, it has been estimated that single-domain proteins from the same domain family have a 67% chance of having similar functions, whereas the corresponding number for two-domain proteins with just one of the domains in common is 35%” on page 1 paragraphs 1-2. The Applicant does not provide working examples of a functional fragment or functional variant as recited in Claim 32. Due to the functional unpredictability of all orthologs sequences, homologous sequences, and functional fragments and variants thereof, undue experimentation would be required to ensure that all variants and fragments of the claimed sequences still maintained the functionality of reducing the alkaloid and/or TSNA precursor content.
Therefore, given the lack of written description in the instant disclosure with regard to the structural and functional characteristics of the claimed compositions, Applicant does not appear to have been in possession of the claimed genus at the time this application was filed.
Scope of Enablement
Claims 7-8, 10-12, 29, and 32 are 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 Nicotiana plant, or leaf, which has been modified to achieve a reduction in nicotine in comparison to an unmodified Nicotiana plant, wherein the modification is VIGS of Nitab4.5_0005997g0050.2, does not reasonably provide enablement for any modification to modulate or decrease activity or expression of a FAD synthetase, wherein the plant has decreased content of any alkaloid and/or TSNA precursor in comparison to an unmodified plant or propagation material thereof. 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 or use the invention commensurate in scope with these claims.
In re Wands lists a number of factors for determining whether or not undue experimentation would be required by one skilled in the art to make and/or use the invention. These factors are: (1) the quantity of experimentation necessary; (2) the amount of direction or guidance presented; (3) the presence or absence of working examples of the invention; (4) the nature of the invention; (5) the state of the prior art; (6) the relative skill of those in the art; (7) the predictability or unpredictability of the art; (8) the breadth of the claim. In re Wands, 858 F.2d 731, 8 USPQ2d 1400 (Fed. Cir. 1988).
Claims 7-8, 10-12, 29, and 32 are broadly directed to a tobacco plant or part thereof which has been modified by any means to achieve any modulation of FAD synthetase and comprises decreased content of any alkaloid and/or TSNA precursor in comparison to a unmodified plant, as well as any propagate with decreased FAD synthetase activity or expression or decreased alkaloid and/or TSNA precursor content.
Applicant teaches:
Virus-induced gene silencing (VIGS) of a FAD synthetase gene (i.e. Nitab4.5_0005997g0050.2) with genomic sequence of SEQ ID NO: 1, the coding sequence of SEQ ID NO: 2, and the amino acid sequence of SEQ ID NO: 3 using the 300-nucleotide cDNA fragment with SEQ ID NO: 43. The TRV vector comprising both (TRV RNA 1 SEQ ID No. 34) and (TRV RNA2) comprising the targeted nucleotide sequence separately propagated in A. tumefaciens.
VIGS silencing Nitab4.5_0005997g0050.2 leading to a reduction of nicotine, anabasine, PON, and anatabine.
Applicant does not teach:
Modification of the activity or expression of a FAD synthetase peptide or nucleotide sequence with at least 80% identity to SEQ ID NO:1-3, or a functional variants, fragments, orthologues, or homologue thereof, other than SEQ ID NOs: 1-3.
If alkaloid content can be increased and TSNA precursors maintained or reduced (i.e., the plant comprising decreased alkaloid and/or TSNA precursor content).
Modulation of activity of a FAD in a plant.
Modification of any kind to modulate or decrease the activity or expression of a FAD synthetase, other than VIGS.
Any plant propagation material obtained from the tobacco plant comprising modulated or decreased activity or expression of a FAD synthetase from the heritable modification.
Any plant part with modulated FAD synthetase activity or expression and decreased alkaloid and/or TSNA precursor content, other than tobacco leaves.
The instant disclosure and the instant claims do not set forth structural characteristics essential to the FAD synthetase such that one would envision which FAD synthetase would or would not modulate the content of any alkaloid or any TSNA precursor. It is additionally unclear if having sequence identity of at least 80% to SEQ ID NOs: 1-3 would provide sufficient structure to result in the same effect of modulating alkaloid content in a tobacco plant, as posited by the Applicant.
The instant claims are directed to a decrease in activity or expression of a FAD synthetase, meaning that any methodology may be used for any decreased in protein activity or gene expression. This may include anything from a natural modification (i.e., herbivory) to a single point mutation in any part of the gene encoding the peptide. The instant specification is only adequately enabled for the use of one technique, VIGS, for decreasing nicotine content. A VIGS modification would inherently decrease activity or expression of a FAD synthetase (not broadly modulate, which can include increasing activity or expression) based on the nature of the modification, gene silencing.
The instant disclosure only teaches VIGS of Nitab4.5_0005997g0050.2, but does not indicate any other variants or fragments of SEQ ID NOs: 1-3 with a similar functionality of decreased alkaloid content. The instant specification merely states that homologues were tested but does not provide any data to support the conclusion that the fragments, variants, orthologues, or sequences with at least 80% identity to SEQ ID NOs: 1-3 could be used in the invention as claimed. One of ordinary skill in the art would not reasonably be able to make or use the invention to the broad scope it is currently claimed.
For example, SEQ ID NO: 3, the amino acid sequence of the FAD synthetase is 364 amino acids long. Peptides with at least 80% identity to SEQ ID NO: 3 encompass peptides with up to 72 amino acid substitutions relative to SEQ ID NO: 3. There are a large number of possible substitutions with anywhere from 1 to 72 possible amino acids substituted in any position of the 364-length sequence and any other of the 19 possible amino acids substituted. For the potential amino acid substitutions alone at each of the 72 positions, there would be 1972 possibilities, of which, the Applicant has not provided sufficient working examples or direction. While a FAD synthetase domain is described in the specification on page 11 line 35, the instant disclosure fails to provide guidance for how to make polypeptides with 80% identity to the listed sequences of Claim 32 and referenced in Claim 29 which have the claimed function.
Additionally, the exemplified species of the genus claim of 80% identity to SEQ ID NOs: 1-3, are unpredictable. One would not be able to substitute, add or delete portions of the sequence while asserting with certainty that the sequence maintains the function. For example, sites or regions with binding and/or folding activity are critical to the protein’s structure/function relationship and necessitate correct three-dimensional spatial orientation of binding and active sites. Keskin, Ozlem, and Ruth Nussinov. "Favorable scaffolds: proteins with different sequence, structure and function may associate in similar ways." Protein Engineering Design and Selection 18.1 (2005): 11-24 further teaches that even proteins with similar structure may have different functions (see Abstract). The instant specification does not teach any variants, fragments or orthologs to the FAD synthetase as it only provides a single example of Nitab4.5_0005997g0050.2 VIGS. Nitab4.5_0005997g0050.2 has the amino acid sequence of SEQ ID NO: 3, coding sequence of SEQ ID NO: 2, and genomic sequence of SEQ ID NO: 1. The specification fails to provide guidance for how to make or use nucleotides with 80% identity to the SEQ ID NOs: 1-3 with the same function. The Applicants have not provided working examples or prophetic examples of a representative number of plants carrying the modification with such great variety while maintaining the function.
As use of FAD synthetase is not prevalent in relation to altering alkaloid and/or TSNA content, the art does not remedy the deficiencies of the enablement. FAD synthetase is responsible for catalyzing biosynthesis of FAD. FAD and FMN are vital to all living organisms because of their function as irreplaceable cofactors for enzymes participating in a wide variety of metabolic processes. These processes include, but are not limited to, mitochondrial electron transport, photosynthesis, fatty acid oxidation, protein folding, chromatin remodeling, and metabolism of several other biologically relevant compounds, such as nucleotides, amino acids, antioxidants, and other cofactors. In plants, they also play a significant role in signaling as the chromophores in blue-light receptors3. While FAD is a known important cofactor for the afore mentioned cellular processes, “ the responsible catalysts and regulatory mechanisms (i.e. FAD synthetase) remain poorly understood”4. Knockout FAD synthetases could also be lethal as taught by Abbas, Charles A., and Andriy A. Sibirny. "Genetic control of biosynthesis and transport of riboflavin and flavin nucleotides and construction of robust biotechnological producers." Microbiology and molecular biology reviews 75.2 (2011): 321-360.on page 12 paragraph 7, which further emphasizes the necessity of enablement. One of ordinary skill in the art would likely predict that some modifications of a FAD synthetase gene or protein may impair growth of a plant. One would not reasonably predict that a homolog-related peptide or 80% identity, a fragment variant or orthologue thereof, would confer the function of decreased alkaloid and/or TSNA precursor content while also not proving lethal to the plant.
Lastly, VIGS can serve as an alternative to mutant collections or stable transgenic plants to allow the characterization of gene functions in a wide range of angiosperm species, but is a transient method of genetic transformation5. With only a few exceptions, the targeted gene silencing will not be transferred to subsequent generations6. These limitations require experiments for each new species or specific tissue targeted, and the optimal time point and location of infection have to be determined experimentally.
The Applicant has provided only the working example for VIGS in tobacco leaves, not in any other tissue. The Applicant has not provided any working examples of a plant or plant propagation material. As VIGS is known to be transient and no guidance is provided for a plant grown from the tobacco plant with the modification with modulated or decreased activity or expression of a FAD synthetase or decreased alkaloid content, one would not be reasonably enabled to make or use the invention. It would not be predictable to achieve a plant grown from the tobacco plant, or plant propagation material obtained from the tobacco plant that would maintain the same function as the original tobacco plant because the VIGS modification was likely transient. Although there appear to be a few exceptions to this, significant experimentation would be required to ensure the modification could be passed down or would be effective with any FAD synthetase or sequence with 80% similarity to SEQ ID NOs: 1-3.
Thus, the examples provided by the Applicant do not provide adequate working examples to enable the scope of the invention without undue experimentation. Given the breadth of the claims, the lack of guidance and working examples, the unpredictability in the art, and the state of the art, undue experimentation would be required to make and use the claimed invention, and therefore, the invention is not enabled throughout the broad scope of the claims.
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 7-8, 10-12, 29, and 32 are directed to a modification tobacco plant, part, cell, cell culture, propagation material, or tobacco crop from a plant with reduced alkaloid and/or TSNA precursor (i.e. nicotine) content upon modulation or decreased activity or expression of a FAD synthetase. The FAD synthetase can be mutated and would have a protein sequence of SEQ ID NO:3 and is encoded by nucleotide sequences of SEQ ID NO:1-2 or have 80% similarity to SEQ ID NOs:1-3 or be a functional variant or fragment, ortholog, or homolog.
Claims 7-8, 10-12, 29, and 32 are rejected under 35 U.S.C. 103 as being unpatentable over WO 2020069142 A1 (see IDS filed 1/22/2025) in view of Kajikawa, Masataka, et al. "Vacuole-localized berberine bridge enzyme-like proteins are required for a late step of nicotine biosynthesis in tobacco." Plant physiology 155.4 (2011): 2010-2022.
WO 2020069142 A1 is directed to modified host cells comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a Berberine Bridge Enzyme (BBE) or BBE-like polypeptide7. Paragraph 28 teaches “In some embodiments described herein, the BBE or BBE-like polypeptide is a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum”. Paragraph 86 teaches “BBE and BBE-like polypeptides often contain disulfide bonds, glycosylations, and are the only polypeptides known to contain a bi-covalently attached FAD cofactor-thought to increase the enzyme's catalytic potential over the more common mono-covalent FAD”. Paragraph 154 teaches “In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more FAD synthetase polypeptides, reducing or eliminating the expression of the one or more FAD synthetase polypeptides.” This is reiterated in Claims 1-3, 25, 34, and 45 of WO 2020069142 A1. The same limitation of decreasing the expression of a FAD synthetase is present in Claims 7-8, 10-11, 29, and 32 of the Applicant’s present claims. Claim 29 is also directed to a plant with a “mutation” in the FAD synthetase gene which modulates or decreases the activity or expression of FAD synthetase. Paragraph 453 of WO 2020069142 A1 teaches “downregulation of a gene can be accomplished in several ways ….the introduction of mutations that destabilize the protein or reduce catalytic activity achieve a similar goals”.
While reduction of FAD synthetase expression and its relationship to BBE and BBE-like polypeptides is taught in WO 2020069142 A1, it is not explicitly directed to tobacco plant, part, cell, cell culture, propagation material, or tobacco crop from a plant with reduced alkaloid and/or TSNA precursors (limitations recited in Claims 7-8, 10-11, 29, and 32) or a FAD synthetase with at least 80% sequence similarity to the peptide sequence in SEQ ID NO: 3 encoded by the nucleotide sequence with at least 80% sequence similarity to SEQ ID NOs:1-2 (i.e. Claims 29 and 32). It also does not teach plant propagation material obtained from a modified plant (Claim 8) or crop bred or grown from such a plant (Claim 12) .
Kajikawa et al 2010 is directed to berberine bridge enzyme family and their role in nicotine biosynthesis in tobacco plants. It is clearly stated in the passage “In this study, we identified vacuole-localized tobacco flavoproteins, berberine bridge enzyme-like proteins (BBLs), that were required for the synthesis of nicotine, anatabine, anabasine, and anatalline” on page 2 paragraph 3. It also teaches “These tobacco BBLs constitute a distinct clade in the FAD-containing oxidoreductase family that includes berberine bridge enzymes (BBEs)” on page 2 paragraph 4. This reference also teaches mutant tobacco plant lines where BBL expression was decreased (i.e. modulated) using RNAi on page 5 paragraph 2. In the leaves, the amounts of nicotine and nornicotine were much lower in the mutant plants when compared to unmodified plants. This is depicted in Figure 6 B and C. Interestingly enough, while the alkaloids nicotine and nornicotine were reduced (i.e. modulated), another alkaloid’s content, called dihydrometanicotine (DMN) was increased (i.e. modulated). Applicant’s Claims 7-8, 10-11, 29, and 32 were also directed to modified tobacco plants with decreased alkaloid content while Claim 10 is directed to modulated nicotine, nornicotine, PON, anabasine, myosmine, and anatabine content. Additionally, the “Plant and Genetic Transformation” section in the materials and methods section on page 10 describe how mutant plants were generated. The transgenic T1 plants used for the experiment were generated from a transgenic T0 plant. Because plants with the heritable mutation were propagated from an already transgenic plant, these plants read upon Applicant’s Claim 8 (i.e. a plant propagation material obtained from an already modified plant) and Claim 12 (a plant or crop bred from or grown from the modified plant). Because the transgenes (i.e. mutations) were heritable, the plants in the T1 generation contained a mutated nucleic acid molecule, which reads upon the limitation of Claim 29, which recites a mutation comprising a heritable mutation. Other disclosed modified mutant systems with modulated alkaloid and/or TSNA precursor content were hairy roots and BY-2 cells which were described on page 4 paragraph 3 and page 5 paragraph 3. Because they are a plant part, propagation material, and cell culture derived from Nicotiana tabacum, they read upon limitations recited in Claims 7-8, 10-11, and 32. Kajikawa et al 2010 uses the same species of tobacco used in the specification said to comprise the SEQ ID NOs: 1-3 (see Example 2 in Applicant’s specification). Therefore, the plants taught by Kajikawa et al 2010 would also comprise a protein and nucleotide sequences with the same SEQ ID NOs, absent evidence to the contrary, which was a limitation taught in Claims 29 and 32.
It would have been prima facie obvious to combine the teachings of WO 2020069142 A1 and Kajikawa et al 2010 to generate tobacco plants with reduced or modulated alkaloid and/or TSNA content by modulating or decreasing the expression of a FAD synthetase. Kajikawa et al 2010 teaches that BBL enzymes are required for synthesizing alkaloids in tobacco. Both references teach that this class of enzymes require the cofactor FAD. WO 2020069142 A1 teaches that FAD is synthesized by FAD synthetase and teaches engineering an organism with reduced activity and expression FAD synthetase along with a BBL gene. Kajikawa et al 2010 teaches methods on reducing gene expression in tobacco plants resulting in reduced alkaloid and/or TSNA content. It would have been obvious to one of ordinary skill in the art to take the concept of reducing the expression of FAD synthetase from WO 2020069142 A1 and using the methods of Kajikawa et al 2010 to reduce its expression in tobacco. They would also have a reasonably high expectancy of success as FAD is a known cofactor used in the biosynthesis of alkaloids in tobacco and because genes silencing to reduce alkaloid and TSNA precursors is already known in the art regarding this pathway. One having ordinary skill in the art would also have been motivated to reduce the expression or activity of FAD synthetase to generate tobacco plants with reduced alkaloids, as these plants would have increased value as a biomass resource and for the generation of tobacco plants and products with reduced nicotine in view of potential regulation of nicotine ceilings.8
The use of VIGS, quantifying metabolites, and performing in vitro enzyme assays were known and were routinely used in the art at the time the instant application was filed. Similarly, modifications to the polynucleotide, plant, or bacterial compositions, or methods of practice using these compositions, would be mere design choice and routine optimizations of the methods taught by the cited references and the general state of the art, absent evidence to the contrary.
Conclusion
No claims allowed.
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/GEORGE W MEYER/ Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/ Supervisory Patent Examiner, Art Units 1661 & 1662
1 See Page 1 paragraph 1 of Lynch, Joseph H., and Sanja Roje. "A higher plant FAD synthetase is fused to an inactivated FAD pyrophosphatase." Journal of Biological Chemistry 298.12 (2022).
2 Xu, Zilong, et al. "Phylogenetic Inference of Homologous/Orthologous Genes Among Distantly Related Plants." Bio-protocol 13.23 (2023) teaches that homologous/orthologous genes among different plants typically perform similar or equivalent functions, which is theoretically plausible and empirically supported see page 2 paragraph 1.
3 See Page 1 paragraph 1 of Lynch et al 2022.
4 Lynch et al 2022 abstract
5 See the abstract of Lang et al 2013
6 Lang et al 2013 page 10 paragraph 4
7 Paragraph 111 teaches A "modified host cell" (also may be referred to as a "recombinant host cell") may refer to a host cell into which has been introduced a nucleic acid (e.g., a heterologous nucleic acid), e.g., an expression vector or construct. For example, a modified eukaryotic host cell may be produced through introduction into a suitable eukaryotic host cell of a nucleic acid (e.g., a heterologous nucleic acid).
8 See page 4 lines 23-31 of WO 2018/237107 A1 of IDS filed 01/22/2025
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