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 .
Election/Restrictions
Claims 1, 3, 5, 13, 18-19, 21, 23, 27-29 and 31-32 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to nonelected inventions, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 05/27/2026.
The requirement is deemed proper and is therefore made FINAL.
Claim Status
Claims 1, 3, 5, 7-8, 10-13, 16, 18-19, 21, 23, 27-29 and 31-32 are pending.
Claims 1, 3, 5, 13, 18-19, 21, 23, 27-29 and 31 are withdrawn from examination as being part of non-elected groups.
Claims 7-8, 10-12, 16 and the newly added claim 32 are being examined.
Claim Objections
Claim 10 is objected to because of the following informalities:
Abbreviations should be accompanied by the full name in parentheses, in the first instance in the claims. The full name for “PON” should be recited in claim 10, in parentheses.
Appropriate correction is required.
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, 16 and 32 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 claims contain 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.
It is known in the art that the Nucleoside diphosphate kinase 1 (NDPK1) housekeeping activity provides UTP required as co-substrate for the cytosolic synthesis of UDP-glucose (UDPG) by UDP-glucose pyrophosphorylase (UGPase) (Dorion et al., Plant nucleoside diphosphate kinase 1: A housekeeping enzyme with moonlighting activity, 2018, Plant Signaling & Behavior, 13:e1475804; p.1, right column, para 1, line 9-13). The NDPK family in plants is divided into four types (I-IV) based on phylogenetic analyses and patterns of subcellular localization (Dorion et al.; p.1, left column, para 2, line 1-3). Type I isoforms are represented by one or two genes, depending on the species and are predominantly cytosolic. In various species, expression of NDPK1 is spatio-temporally regulated during growth and development. Down regulation of NDPK1 reduced UTP synthesis but enhanced ATP availability, which, in turn, favors metabolic pathways dependent on adenylates (e.g. starch synthesis) (Dorion et al., p.1, right column, last para, line 4-7). Rajewski et al. (Datura genome reveals duplications of psychoactive alkaloid biosynthetic genes and high mutation rate following tissue culture, 2021, BMC Genomics, 22:201) implicates a NDPK protein, UniProt Accession No. A0ABS8RGT6, having 99% sequence identity to instant SEQ ID NO: 3 (data not shown), in alkaloid biosynthesis in Jimsonweed (Datura stramonium) and tomato (S. lycopersicum), possibly via an undefined/unidentified ATP metabolic process (p.6, Fig. 1E-F).
It is known in the art that UDPG, which is produced by NDPK (Dorion et al.; p.1, right column, para 1, line 9-13), is a major precursor for the synthesis of cellulose, other cell wall carbohydrates (Dorion et al., p.1, right column, para 1, line 9-13) and alkaloids like nicotine (Schwabe et al., Nicotine biosynthesis is completed by cryptic activating glucosylation, 2026, Nature Communications, 17:4221; p.2, right column, last para, line 8-9; Fig. 3, Fig. S4) as well. It is also known in the art that at least one or more members of the NDPK family undergoes glutathionylation, opening a new paradigm of NDPK regulation in plants (Luzarowski et al., Affinity purification with metabolomic and proteomic analysis unravels diverse roles of nucleoside diphosphate kinases, 2017, Journal of Experimental Botany, 68:3487–3499; abstract, line 11-12) as glutathionylation inhibits NDPK activity (Luzarowski et al.; p.3496, right column, para 1, line 4-5).
Claim 32 recites, “…. a sequence which has at least 80% identity to SEQ ID No. 3; or a homologue of SEQ ID No. 3; or b) encoded by a nucleotide sequence as set out in SEQ ID No. 1 or 2; or a nucleic acid sequence which has at least 80% identity to SEQ ID No. 1 or 2; or a homologue of SEQ ID No. 1 or 2…”.
The Applicant describes reducing the expression and/or activity of a single member of the NDPK family in tobacco (Nitab4.5_0000563g0250.2) (spec, p.2, line 21-26; p.88, Example 1, line 3-6) encoding the polypeptide sequence set forth by SEQ ID NO: 3 (spec, p.8, para 1; Fig. 4) leading to decreased alkaloid content (spec, p.88, Example 1, line 3-6). The Applicant describes a Nucleoside Diphosphate Kinase encoded by polynucleotide sequences comprising 100% sequence identity to 2974 (genomic DNA) nucleotide long SEQ ID No: 1 (spec, p.13, line 4-5) and 447 nucleotide long (cDNA) SEQ ID No. 2 (spec, p.13, line 20-21) which encodes a 148 amino acid long specific tobacco NDPK1 (Nitab4.5_0000563g0250.2) polypeptide sequence comprising SEQ ID No. 3 (data not shown). The Applicant does not provide representative examples of other members of the Nucleotide Diphosphate Kinase (NDPK) family in tobacco or from any other plant and reducing the expression and/or activity of the one of more members of the NDPK family in tobacco except Nitab4.5_0000563g0250.2 leading to decreased alkaloid and/or tobacco specific nitrosamine (TSNA) content. The Applicant also does not provide examples of a NDPK having less than 100% sequence identity to SEQ ID No.3 encoded by a gene having less than 100% sequence identity to SEQ ID NO: 1 and/or a cDNA sequence comprising less than 100% sequence identity to instant SEQ ID NO: 2.
The Applicant defines the term “homologue” as “an entity having a certain homology with the subject amino acid sequences and the subject nucleotide sequences. Here, the term "homology" can be equated with "identity"” (spec, p.80, line 22-24). Thus, the Applicant does not define what exact percentage of identity/homology a sequence needs to have to be a “homologue”. Thus, the Applicant does not describe any “homologue” of Nitab4.5_0000563 g0250.2 having less than 100% sequence identity to SEQ ID NOs: 1-3.
Mutating 20% of 148 amino acid long SEQ ID NO: 3 would allow mutating up to 29 amino acid residues along the entire length of the protein. Similarly, mutating up to 20% in 2974 nucleotide long SEQ ID NO: 1 or 447 nucleotide long SEQ ID NO: 2 would allow mutating up to 594 and 89 nucleotides along the entire length of the polynucleotide sequences, respectively, which would allow mutating up to 594 and 89 amino acids in the encoded polypeptide depending on the specific codons of the mutated amino acids. The Applicant does not describe the structure function relationship for the Nucleoside Diphosphate Kinase gene and/or the protein in terms of its function related to alkaloid and/or TSNA precursor content in the plant. The Applicant also does not describe if the activity of Nitab4.5_0000563g0250.2 protein is affected by any post-translational modification(s) including glutathionylation. Moreover, it is known in the art that even a single amino acid change anywhere in the entire length of the protein especially in the active site(s)/domain(s) in a polypeptide can drastically alter its function(s), and it is not possible for any skilled artisan to predict such change without having any structure function relationship.
Considering the breadth of the claims, (lack of representative species of the broad genus claimed), (lack of structure function relationship of the broad genus claimed), (and unpredictability of the art), the 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, 16 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 the gene Nitab4.5_0000563g0250.2 encoding the protein set forth by SEQ ID NO: 3, does not reasonably provide enablement for other genes encoding other Nucleoside Diphosphate Kinase (NDPK) proteins in tobacco. 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 use the invention commensurate in scope with these claims.
It is known in the art that the NDPK family in plants is divided into four types (I-IV) based on phylogenetic analyses and patterns of subcellular localization (Dorion et al.; p.1, left column, para 2, line 1-3). Type I isoforms are represented by one or two genes, depending on the species and are predominantly cytosolic. In various species, expression of NDPK1 is spatio-temporally regulated during growth and development.
The NDPK1 housekeeping activity provides UTP required as co-substrate for the cytosolic synthesis of UDP-glucose (UDPG) by UDP-glucose pyrophosphorylase (UGPase) (Dorion et al.; p.1, right column, para 1, line 9-13). Overexpression increasing NDPK1 activity enhances the capacity for UTP generation and promotes the activity of metabolic pathways reliant on uridinylates (e.g. UDP–glucose and cellulose synthesis) (Dorion et al., bridging paragraph between p. 1-2). On the other hand, down regulation of NDPK1 reduced UTP synthesis but enhanced ATP availability, which, in turn, favors metabolic pathways dependent on adenylates (e.g. starch synthesis) (Dorion et al., p.1, right column, last para, line 4-7). UDPG is the major precursor for the synthesis of cellulose, other cell wall carbohydrates (Dorion et al., p.1, right column, para 1, line 9-13) and alkaloids like nicotine from nicotinic acid (Schwabe et al.; p.2, right column, last para, line 7-12; p.3, left column, para 1, line 1-6; Fig. 3, Fig. S4). Rajewski et al. implicates a NDPK protein, UniProt Accession No. A0ABS8RGT6, having 99% sequence identity to instant SEQ ID NO: 3 (data not shown), in alkaloid biosynthesis in Jimsonweed (Datura stramonium) and tomato (S. lycopersicum) possibly via ATP metabolic process (p.6, Fig. 1E-F).
It is known in the art that UDPG, which is produced by NDPK (Dorion et al.; p.1, right column, para 1, line 9-13), is a major precursor for the synthesis of cellulose, other cell wall carbohydrates (Dorion et al., p.1, right column, para 1, line 9-13) and alkaloids like nicotine (Schwabe et al.; p.2, right column, last para, line 8-9; Fig. 3, Fig. S4) as well.
It is also known in the art that at least one or more members of the NDPK family undergoes glutathionylation, opening a new paradigm of NDPK regulation in plants (Luzarowski et al.; abstract, line 11-12) as glutathionylation inhibits NDPK activity (Luzarowski et al.; p.3496, right column, para 1, line 4-5).
Claim 32 recites, “…. a sequence which has at least 80% identity to SEQ ID No. 3; or a homologue of SEQ ID No. 3; or b) encoded by a nucleotide sequence as set out in SEQ ID No. 1 or 2; or a nucleic acid sequence which has at least 80% identity to SEQ ID No. 1 or 2; or a homologue of SEQ ID No. 1 or 2…”.
The Applicant describes reducing the expression and/or activity of a single member of the NDPK family in tobacco (Nitab4.5_0000563g0250.2) (spec, p.2, line 21-26; p.88, Example 1, line 3-6) encoding the polypeptide sequence set forth by SEQ ID NO: 3 (spec, p.8, para 1; Fig. 4) leading to decreased alkaloid content (spec, p.88, Example 1, line 3-6). The Applicant describes a Nucleoside Diphosphate Kinase encoded by polynucleotide sequences comprising 100% sequence identity to 2974 (genomic DNA) nucleotide long SEQ ID No: 1 (spec, p.13, line 4-5) and 447 nucleotide long (cDNA) SEQ ID No. 2 (spec, p.13, line 20-21) which encodes a 148 amino acid long specific tobacco NDPK1 (Nitab4.5_0000563g0250.2) polypeptide sequence comprising SEQ ID No. 3 (data not shown).
The Applicant does not provide any example or guidance for a skilled artisan how to modify other members of the NDPK family in tobacco or from any other plant and reducing the expression and/or activity of the one of more members of the NDPK family in tobacco except Nitab4.5_0000563g0250.2 leading to decreased alkaloid and/or tobacco specific nitrosamine (TSNA) content. The Applicant also does not provide examples of a NDPK having less than 100% sequence identity to SEQ ID No.3 encoded by a gene having less than 100% sequence identity to SEQ ID NO: 1 and/or a cDNA sequence comprising less than 100% sequence identity to instant SEQ ID NO: 2.
The Applicant defines the term “homologue” as “an entity having a certain homology with the subject amino acid sequences and the subject nucleotide sequences. Here, the term "homology" can be equated with "identity"” (spec, p.80, line 22-24). Thus, the Applicant does not define what exact percentage of identity/homology a sequence needs to have to be a “homologue”.
The Applicant does not describe any “homologue” of Nitab4.5_0000563 g0250.2 having less than 100% sequence identity to SEQ ID NOs: 1-3.
Mutating 20% of 148 amino acid long SEQ ID NO: 3 would allow mutating up to 29 amino acid residues along the entire length of the protein. Similarly, mutating up to 20% in 2974 nucleotide long SEQ ID NO: 1 or 447 nucleotide long SEQ ID NO: 2 would allow mutating up to 594 and 89 nucleotides along the entire length of the polynucleotide sequences, respectively, which would allow mutating up to 594 and 89 amino acids in the encoded polypeptide depending on the specific codons of the mutated amino acids. The Applicant does not describe the structure function relationship for the Nucleoside Diphosphate Kinase gene and/or the protein in terms of its function related to alkaloid and/or TSNA precursor content in the plant. The Applicant also does not describe if the activity of Nitab4.5_0000563g0250.2 protein is affected by any post-translational modification(s) including glutathionylation. Moreover, it is known in the art that even a single amino acid change anywhere in the entire length of the protein especially in the active site(s)/domain(s) in a polypeptide can drastically alter its function(s), and it is not possible for any skilled artisan to predict such change without having any structure function relationship.
Undue trial and error experimentations would be needed to modify one or more members of the NDPK family in tobacco, except Nitab4.5_0000563g0250.2, and/or a NDPK having sequence identity less than 100% (or at least 80%) to instant SEQ ID NOs: 1-3 or a homologue of SEQ ID NOs: 1-3, to reduce the alkaloid and/or TSNA precursor content in the tobacco plant.
Based on breadth of the claims, lack of any working example, lack of guidance in the instant description or in prior art, the specification at the time of the application filed would not have taught one skilled in the art how to make and use the full scope of the claimed invention without performing undue experiments.
Claim Rejections - 35 USC § 102(a)(1)
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 7-8, 10-12 and 32 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Valenti et al. (In the early phase of programmed cell death in Tobacco Bright Yellow 2 cells the mitochondrial adenine nucleotide translocator, adenylate kinase and nucleoside diphosphate kinase are impaired in a reactive oxygen species-dependent manner, 2007, Biochimica et Biophysica Acta, 1767:66–78) in evidence of Dorion et al. (Plant nucleoside diphosphate kinase 1: A housekeeping enzyme with moonlighting activity, 2018, Plant Signaling & Behavior, 13:e1475804) and Schwabe et al. (Nicotine biosynthesis is completed by cryptic activating glucosylation, 2026, Nature Communications, 17:4221).
Claim 7 is drawn to a tobacco cell or cell culture having been modified to decrease alkaloid and/or TSNA precursor content by decreasing the activity or expression of a Nucleoside Diphosphate Kinase.
Valenti et al. describes that the rate of the reactions catalyzed by nucleoside diphosphate kinase (NDPK) is inhibited in a non-competitive-like manner after 2 h of heat shock treatment without affecting cell viability in tobacco (BY2) cells (abstract). The treated cell culture of Valenti et al.is considered “altered or changed”, and therefore “modified”, consistent with Applicant’s definition for “modified” (instant specification, p. 10, last paragraph). Valenti et al. teaches that in spite of no changes in the protein levels, the activity of the NDPK is impaired (reads on to “to decrease the activity”, as recited in claim 1) and reduced to about 50% (p.73, left column, para 3, last 3 lines; p.73, right column, last para, line 5-6) in the early stages of heat-shock due to reactive oxygen species (ROS) (p.67, para 4, line 1-6). The nucleoside diphosphate kinase (NDPK), as taught by Valenti et al., reads on to “a homologue” of SEQ ID NOs: 1-3, as recited in claim 32. Moreover, a tobacco NDPK protein (GenBank accession No. AAX63738, published in 2005) having 100% sequence identity to SEQ ID NO: 3 (data not shown) is known before the effective filing date of the invention.
The Applicant describes that the closest homologue (of tobacco NDPK) in Arabidopsis thaliana is known to play a role in response to reactive oxygen species stress (spec, p.8, line 33-34). Impaired or decreased activity of at least one type of NDPK protein is inherently correlated to decreased UDP Glucose (UDPG) level, which is produced by NDPK (Dorion et al.; p.1, right column, para 1, line 9-13). UDPG is a major precursor for alkaloids including nicotine, as evidenced by Schwabe et al. (p.2, right column, last para, line 8-9; Fig. 3, Fig. S4). Thus, it is inherent that alkaloid content including nicotine (as recited in claims 10-11) in a plant like tobacco (and Arabidopsis) and/or tobacco BY2 cells (and BY2 cell culture) (as recited in claims 7 and 12) would decrease when the UDPG level is decreased compared to an unmodified plant or unmodified (i.e., without the heat shock) cell or cell culture.
The tobacco BY2 cells are used for propagation (of the BY2 cells) and reads on to “plant propagation material comprising decreased activity or expression of a Nucleoside Diphosphate Kinase”, as recited in claim 8.
Conclusion
No claim is allowed.
Communication
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JAY CHATTERJEE whose telephone number is (703)756-1329. The examiner can normally be reached (Mon - Fri) 8.30 am to 5.30 pm..
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J.C.
/Jay Chatterjee/Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/Supervisory Patent Examiner, Art Units 1661 & 1662
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