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 .
Restriction/Election
In response to the communication received on March 30th, 2026, from Seiko Okada, the election of Group II, claims 7-8, 10-13, 16, 18-19, and 21-22, without traversal, is acknowledged. Applicants have further added a new claim, claim 32, which is directed to Group II.
Priority
Applicant’s claim for the benefit of a prior-filed application NO: GB2201415.3 filed February 3rd, 2022, and PCT/GB2023/050204 filed January 30th, 2023, under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged.
Thus, the earliest possible priority for the instant application is February 3rd, 2022.
Information Disclosure Statement
The information disclosure statements (IDSs) submitted on January 22, 2025 and April 30th, 2026 were considered, initialed, and attached hereto. A signed copy of the list of references cited is included with this Office Action.
Status of Claims
Claims 1, 3, 5, 7-8, 10-13, 16, 18-19, 21-23, 27-29, and 31-32 filed March 30th, 2026 are pending.
Claims 1, 3, 5, 23, 27-29, and 32 are withdrawn under the restriction requirement.
Claims 7-8, 10-13, 16, 18-19, 21-22, and 32 are examined herein.
Specification
Applicant is reminded of the proper language and format for an abstract of the disclosure.
The abstract should be in narrative form and generally limited to a single paragraph on a separate sheet within the range of 50 to 150 words in length. The abstract should describe the disclosure sufficiently to assist readers in deciding whether there is a need for consulting the full patent text for details.
The language should be clear and concise and should not repeat information given in the title. It should avoid using phrases which can be implied, such as, “The disclosure concerns,” “The disclosure defined by this invention,” “The disclosure describes,” etc. In addition, the form and legal phraseology often used in patent claims, such as “means” and “said,” should be avoided.
Claim Objections
In claims 7, and 8, 10-13, 16, 18-19, 21-22, 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 8, 10-13, 16, 18-19, 21-22, and 32 depending therefrom, “DPH3” is used as abbreviation. It is suggested to insert a definition for DPH3 without bringing in new matter, immediately before the first appearance of “DPH3” in claim 7; and to enclose the appearance of “DPH3” 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 19 is objected to because of the additional period at the end of the claim.
Claim 32 is objected to because of the comma instead of a period at the end of the claim and because claim 32 recites in part “wherein the DPH3 homolog-related peptide comprises an amino acid sequence as set out in SEQ ID No. 3… or is encoded by a nucleotide sequence as set out in SEQ ID No. 1 or 2.” The claim should be amended to clarify that the DPH3 homolog-related peptide comprises the amino acid sequence of SEQ ID No. 3 or is encoded by the nucleotide sequences SEQ ID No. 1 or 2. The claim should be amended to recite, for example, “wherein the DPH3 homolog-related peptide comprises the amino acid sequence of SEQ ID No. 3… or is encoded by the nucleotide sequence of SEQ ID No. 1 or 2.”
Appropriate correction is required.
Claim Interpretation
The term “DPH3 Homolog-related peptide” is recited in claim 7 (and claims 8, 10-13, 16, 18-19, 21-22, and 32 depending therefrom). The instant specification states that the terms "DPH type domain containing protein" and "DPH3 Homolog-related peptide" or "DPH3 Homolog-related protein" are used interchangeably herein and that an illustrative sequence of a DPH type domain containing protein or DPH3 Homolog-related peptide from tobacco is shown in SEQ ID NO: 3 [pg. 10, lns. 30-35]. Thus, a sequence with high identity to SEQ ID NO: 3 is taken to be a DPH3 homolog-related peptide. The recitation of “homolog-related peptide” under the broadest reasonable interpretation is taken to mean a large range of evolutionarily related short sequences of amino acids sharing a common ancestor and exhibiting similarity in sequence, structure, or function1. The art suggests that this is a dipthamide biosynthesis peptide2, but there is no defined source or definition for the peptide in the instant disclosure. Thus, this is taken to mean a sequence sharing similar structure and/or function to the disclosed sequences.
The term "modifying" or "modified" recited in claim 7 (and claims 8, 10-13, 16, 18-19, 21-22, and 32 depending therefrom) as used herein means a plant (e.g. a tobacco plant) or nucleic acid sequence that has been altered or changed by any means, including naturally, as defined in the instant disclosure [pg. 10, lns. 8-10].
The term “modulated” as recited in claim 7 (and claims 8, 10-13, 16, 18-19, 21-22, and 32 depending therefrom) is defined as either increasing or decreasing in the instant disclosure [pg. 31, ln. 16]. Thus, modulated may refer to decreasing, increasing, or any change.
The term “unmodified plant” as recited in claim 7 (and claims 8, 10-13, 16, 18-19, 21-22, and 32 depending therefrom) is defined as a plant which has not been modified according to the present invention in which all relevant features are the same, as defined in the instant disclosure [pg. 11, lns. 10-14].
The term “plant propagation material” as recited in claim 8 is taken to mean any plant matter taken from a plant from which further plants may be produced. This may be a seed, plant calli or plant clumps, as defined in the specification [pg. 44, lns. 25-29].
Claim Rejections – 35 USC § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. 112(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, 10-13, 16, 18-19, 21-22, 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 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 DPH3 Homolog-related peptide and comprising decreased alkaloid and/or TSNA precursor content as compared to an unmodified plant.
Applicant describes:
Virus-induced gene silencing (VIGS) of Nitab4.5_0002165g0050.2 (which has the 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 a 300-nucleotide cDNA fragment of Nitab4.5_0005997g0050.2 (SEQ ID NO: 21). The TRV VIGS vector comprised both TRV RNA1 (SEQ ID NO: 22) and TRV RNA2 propagated into A. tumefaciens [Example 1].
VIGS of Nitab4.5_0002165g0050.2 causing a significant reduction in nicotine within a Nicotiana plant as compared to an unmodified control plant [Example 1].
Applicant does not describe:
Modulation of any alkaloids or TSNA precursors other than nicotine.
Modification of the activity or expression of a DPH3 homolog-related peptide with at least 80% identity to SEQ ID NO: 1-3, or a functional variants, fragments or orthologues 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 DPH3 homolog-related peptide.
Modification of any kind to modulate or decrease the activity or expression of a DPH3 homolog-related peptide, other than VIGS.
Any plant propagation material obtained from the tobacco plant comprising modulated or decreased activity or expression of a DPH3 homolog-related peptide from the heritable modification.
Any plant part with modulated DPH3 homolog-related peptide activity or expression and decreased alkaloid and/or TSNA precursor content, other than tobacco leaves.
Alkaloids are a vast genus of molecules found primarily as secondary metabolites in plants. However, several other types of alkaloids are known to be produced by other organisms, such as animals, fungi and bacteria, and some alkaloids are specific to certain plants. For example, Papaver somniferum (opium poppy) is known for producing unique benzylisoquinoline alkaloids, mainly morphine, codeine, and thebaine, which are not produced by tobacco plants (Beaudoin, G. et al., 2014, “Benzylisoquinoline alkaloid biosynthesis in opium poppy”, Planta, 240(1):19-32, doi: 10.1007/s00425-014-2056-8).
The instant claims recite decreased alkaloid and/or TSNA precursor content in comparison to an unmodified plant (which is taken to mean a control tobacco plant under the same conditions as the modified plant, see claim interpretation). With the VIGS modification of Example 1, the instant disclosure describes silencing of Nitab4.5_002165g0050.2. The results of the relative content of pyridine alkaloids were determined by LC-MS/MS, for analytes nicotine, nicotine d4, anabasine, anatabine, nornicotine, nornicotine d4, PON, and PON d4 [pg. 70]. Alkaloid content of 5-week-old tobacco leaves silenced for Nitab4.5_002165g0050.2 is shown in Fig. 1. Although the Applicant claims that VIGS of Nitab4.5_002165g0050.2 leads to a decrease in alkaloid content in leaves, in particular a decrease in nicotine, anabasine, and PON, Fig.1 shows that only the reduction in nicotine content was statistically significant.
Given the limited description in the instant specification of only Nicotiana plants and the alkaloids present in tobacco, 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, 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]. Aboud, M. et al. (Genetics, Epigenetic Mechanism. [Updated 2023 Aug 14]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2026 Jan-. Available from: https://www.ncbi.nlm.nih.gov/ books/NBK532999/) teaches that modifications to alter gene expression or activity includes epigenetic changes through alterations in the chromosome, rather than the DNA sequence, regulating gene expression through chemical modifications [pg. 1, ¶1-2]. Modification may refer to the alteration of the peptide or gene encoding the peptide by any means, including merely natural modifications, such as a reaction to abiotic or biotic stress. Such modifications may or may not result in an increase or decrease in activity or expression of a DPH3 homolog-related 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 DPH3 homolog-related peptide in a reliable and predictable manner.
As the Applicant only provides the one, specific example of the VIGS technique, which is a small species in the large genus of “modification”, any methodology of modification is not reduced to practice. Further, it appears that Applicant is reducing to practice decreasing activity or expression of a DPH3 homolog-related peptide through silencing and thus the plant comprises decreased nicotine content [Example 1]. Modulating, which may be defined as increasing (see, claim interpretation) activity or expression of a DPH3 homolog-related peptide 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 peptide 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 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 DPH3 homolog-related peptide 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 (Lange, M, et al. 2013, “Virus-Induced Gene Silencing (VIGS) in Plants: An Overview of Target Species and the Virus-Derived Vector Systems”, In: Becker, A. (eds) Virus-Induced Gene Silencing. Methods in Molecular Biology, vol 975. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-278-0_1) [Abstract]. With only a few exceptions, the targeted gene silencing will not be transferred to subsequent generations [pg. 10, ¶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. Gene silencing may only be successful when the consequences of the target gene VIGS (i.e., decreased activity or expression of a DPH3 homolog-related peptide comprising decreased alkaloid and/or TSNA precursor content) do not exceed the stability of the VIGS vector. 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 is 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 a DPH3 homolog-related peptide or decreased alkaloid content are reduced to practice, the Applicant has not sufficiently linked the structure to the function.
Use of DPH3 homolog-related peptides is not prevalent in relation to altering alkaloid and/or TSNA content. Zhang et al. (2022, “Translational fidelity and growth of Arabidopsis require stress-sensitive diphthamide biosynthesis”, Nature Communications, 13:4009) teaches DPH1-DPH7 are responsible for catalyzing biosynthesis steps of diphthamide, a post-translationally modified histidine residue of eukaryotic TRANSLATION ELONGATION FACTOR2 (eEf2) [Abstract]. Zhang teaches that diphthamide contributes to the functionality of the translational machinery monitored by plants to regulate growth. Diphthamide remains only partly understood across cellular physiology. Lack of diphthamide modification on eEF2 is reported to cause severe defects in humans and mutant mice dying during embryonic development, while single-celled organisms and mammalian cell cultures lacking diphthamide remain viable with elevated rates of -1 ribosomal frameshifting in protein biosynthesis of yeast and mouse mutant cells lacking diphthamide [pg. 2, col. 1, ¶2]. Zhang teaches knockout dph1 mutants in Arabidopsis, wherein the mutants exhibit TARGET OF RAPAMYCEIN (TOR) kinase activity attenuation, autophagy activation, and dwarfing in plants as a result of a reduction in cell proliferation in leaves and roots [pg. 2, col. 1, ¶3]. Zhang concludes that Arabidopsis requires DPH1 function to maintain cell proliferation at levels sustaining normal growth rates, as well as the full capacity for TOR activation [pg. 9, col. 2, ¶5].
Given the limited research around DPH3, and therefore DPH3 homolog-related peptides, with regard to decreasing alkaloid and/or TSNA content in tobacco plants, the level of predictability in the art is low. One of ordinary skill in the art would likely predict that some modifications of a DPH3 homolog-related peptide may impair growth of a plant if it retained a similar biological function to DPH3 in other species given that Zhang teaches that DPH1-DPH7 are important for catalyzing diphthamide biosynthesis.
As in the claim interpretation, “DPH3 homolog-related peptide” covers a large range of evolutionarily related short sequences of amino acids sharing a common ancestor and exhibiting similarity in sequence, structure, or function. The instant disclosure provides a singular example of VIGS of Nitab4.5_002165g0050.2 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: 21). The results show a significant decrease in nicotine only [Fig. 1]. Homologue testing in 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. Table 1 provides only three homologues and does not display any data describing the influence of VIGS on the genes or further on alkaloid and/or TSNA precursor content.
The recitation of DPH3 homolog-related peptide is defined only in the specification using exemplary language. A related homolog of a DPH3 peptide 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 functions3, the broader recitation of “homolog-related” does not guarantee the same functional connotation as just “homolog”. A representative number of species was not described to represent the entire genus of modulating or decreasing the activity or expression of a DPH3 homolog-related peptide given the vastness of the 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.
As is known in the art, fragments of biological sequences 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]. The Applicant does not provide working examples of a fragment of a sequence or a variant functioning in the method as claimed in claim 32. Due to the functional unpredictability of paralogs, and fragments and variants thereof, and breadth of the potential fragments and variants, undue experimentation would be required to ensure that all variants and fragments of the claimed sequences still maintained the functionality of the sequences.
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, 10-13, 16, 18-19, 21-22, and 32 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_0002165g0050.2, does not reasonably provide enablement for any modification to modulate or decrease activity or expression of a DPH3 homolog-related peptide, wherein the plant has decreased content of any alkaloid and/or TSNA precursor in comparison to an unmodified plant, or plant products 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, 10-13, 16, 18-19, 21-22, and 32 are broadly directed to a tobacco plant or part thereof which has been modified by any means to achieve any modulation of a DPH3 homolog-related peptide and comprises decreased content of any alkaloid and/or TSNA precursor in comparison to a modified plant, as well as any propagate with decreased DPH3 homolog-related peptide activity or expression or decreased alkaloid and/or TSNA precursor content.
Applicant teaches:
Virus-induced gene silencing (VIGS) of Nitab4.5_0002165g0050.2 (which has the 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 a 300-nucleotide cDNA fragment of Nitab4.5_0005997g0050.2 (SEQ ID NO: 21). The TRV VIGS vector comprised both TRV RNA1 (SEQ ID NO: 22) and TRV RNA2 propagated into A. tumefaciens [Example 1].
VIGS of Nitab4.5_0002165g0050.2 causing a significant reduction in nicotine within a Nicotiana plant as compared to an unmodified control plant [Example 1].
Applicant does not teach:
Modulation of any alkaloids or TSNA precursors other than nicotine.
Modification of the activity or expression of a DPH3 homolog-related peptide with at least 80% identity to SEQ ID NO: 1-3, or a functional variants, fragments or orthologues 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 DPH3 homolog-related peptide.
Modification of any kind to modulate or decrease the activity or expression of a DPH3 homolog-related peptide, other than VIGS.
Any plant propagation material obtained from the tobacco plant comprising modulated or decreased activity or expression of a DPH3 homolog-related peptide from the heritable modification.
Ant plant part with modulated DPH3 homolog-related peptide activity or expression and decreased alkaloid and/or TSNA precursor content, other than tobacco leaves.
While alkaloids are a vast genus of molecules found primarily as secondary metabolites in plants, several other types of alkaloids are known to be produced by other organisms, such as animals, fungi and bacteria, and some alkaloids are specific to certain plants. For example, Papaver somniferum (opium poppy) is known for producing unique benzylisoquinoline alkaloids, mainly morphine, codeine, and thebaine, which are not produced by tobacco plants (Beaudoin, G. et al., 2014, Benzylisoquinoline alkaloid biosynthesis in opium poppy”, Planta, 240(1):19-32, doi: 10.1007/s00425-014-2056-8). The instant disclosure and the instant claims do not set forth structural characteristics essential to the DPH3 homolog-related peptide such that one would envision which DPH3 homolog-related peptides 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 DPH3 homolog-related peptide, 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 DPH3 homolog-related peptide (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_0002165g0050.2, 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 would be able to be used in the invention as claimed. One of ordinary skill in the art would not reasonable 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 DPH3 homolog-related peptide is 242 amino acids long. Peptides with at least 80% identity to SEQ ID NO: 3 encompass peptides with up to 48 amino acid substitutions relative to SEQ ID NO: 3. There are a large number of possible substitutions with anywhere from 1 to 48 possible amino acids substituted in any position of the 242-length sequence and any other the other 19 possible amino acids substituted. For the potential amino acid substitutions alone at each of the 48 positions, there would be 1948 possibilities, of which, the Applicant has not provided sufficient working examples or direction. The instant disclosure fails to provide guidance for how to make polypeptides with 80% identity to the listed sequences of claim 3 which have the claimed function.
Additionally, the exemplified species of the genus claim of 80% identity to SEQ ID NOs: 103, 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, O. et al. (2005, “Favorable scaffolds: proteins with different sequence, structure and function may associate in similar ways”, Protein Engin. Des. & Selec., 18(1):11-24) further teaches that even proteins with similar structure may have different functions [Abstract]. The instant specification does not teach any variants, fragments or orthologs to the DPH3 homolog-related peptide as it only provides a single example of Nitab4.5_002165g0050.2 VIGS. Nitab4.5_002165g0050.2 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 DPH3 homolog-related peptides is not prevalent in relation to altering alkaloid and/or TSNA content, the art does not remedy the deficiencies of the enablement. DPH1-DPH7 are responsible for catalyzing biosynthesis steps of diphthamide, a post-translationally modified histidine residue of eukaryotic TRANSLATION ELONGATION FACTOR2 (eEf2) (Zhang et al. (2022, “Translational fidelity and growth of Arabidopsis require stress-sensitive diphthamide biosynthesis”, Nature Communications, 13:4009) [Abstract]. Diphthamide is known to contribute to the functionality of the translational machinery monitored by plants to regulate growth, but remains only partly understood across cellular physiology. Knockout dph1 mutants in Arabidopsis exhibited TARGET OF RAPAMYCEIN (TOR) kinase activity attenuation, autophagy activation, and dwarfing in plants as a result of a reduction in cell proliferation in leaves and roots [pg. 2, col. 1, ¶3]. Thus, Arabidopsis requires DPH1 function to maintain cell proliferation at levels sustaining normal growth rates, as well as the full capacity for TOR activation [pg. 9, col. 2, ¶5].
As DPH1-7 are responsible for diphthamide biosynthesis, it is likely that DPH3 has some similar functionality, but given the limited research around DPH3, and therefore DPH3 homolog-related peptides, with regard to decreasing alkaloid and/or TSNA content in tobacco plants, the level of predictability in the art is low. One of ordinary skill in the art would likely predict that some modifications of a DPH3 homolog-related peptide may impair growth of a plant if it retained a similar biological function to DPH3 in other species given that Zhang teaches that DPH1-DPH7 are important for catalyzing diphthamide biosynthesis. 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.
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 transformation (Lange, M, et al. 2013, “Virus-Induced Gene Silencing (VIGS) in Plants: An Overview of Target Species and the Virus-Derived Vector Systems”, In: Becker, A. (eds) Virus-Induced Gene Silencing. Methods in Molecular Biology, vol 975. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-278-0_1) [Abstract]. With only a few exceptions, the targeted gene silencing will not be transferred to subsequent generations [pg. 10, ¶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. Gene silencing may only be successful when the consequences of the target gene VIGS (i.e., decreased activity or expression of a DPH3 homolog-related peptide comprising decreased alkaloid and/or TSNA precursor content) do not exceed the stability of the VIGS vector.
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, plant propagation material, or any sort of processed leaf or product produced from the tobacco leaves with the VIGS modification. 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 DPH3 homolog-related peptide 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 DPH3 homolog-related peptide.
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 § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(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, 10-13, 16, 18, and 32, are rejected under 35 U.S.C. 102(a)(2) as being anticipated by Coffin, M., “Genes and Uses for Plant Enhancement”, International Publication NO: WO 2009134339 A2, published 11/05/2009 (see, IDS filed 01/22/2025).
Claim 7 recites a tobacco plant or part thereof or a tobacco cell or cell culture, having been modified to modulate or decrease the activity or expression of a DPH3 Homolog-related peptide, and comprising decreased alkaloid and/or TSNA precursor content in comparison to an unmodified plant or unmodified cell or cell culture.
Claim 8 recites a plant propagation material obtained from the tobacco plant according to claim 7, the plant propagation material comprising modulated or decreased activity or expression of a DPH3 Homolog-related peptide, and decreased alkaloid and/or TSNA precursor content in comparison to a plant propagation material obtained from an unmodified tobacco plant.
Claim 10 recites the tobacco plant or part thereof or tobacco cell or cell culture according to claim 7, wherein the content of one or more alkaloids selected from nicotine, nornicotine, PON, anabasine, myosmine, and anatabine is modulated or decreased.
Claim 11 recites the tobacco plant or part thereof or tobacco cell or tobacco cell culture according to claim 10, wherein the nicotine content is decreased.
Claim 12 recites a plant or a crop bred or grown from the tobacco plant or part thereof or tobacco cell or cell culture according to claim 7, the plant or the crop comprising modulated or decreased activity or expression of a DPH3 Homolog-related peptide, or decreased alkaloid and/or TSNA precursor content in comparison to a plant or a crop bred or grown from an unmodified tobacco plant or part thereof or an unmodified tobacco cell or cell culture.
Claim 13 recites a product or a leaf produced from the tobacco plant or part thereof or a tobacco cell or cell culture according to claim 7, the product or the leaf comprising modulated or decreased activity or expression of a DPH3 Homolog-related peptide, or decreased alkaloid and/or TSNA precursor content in comparison to a product or a leaf produced from an unmodified tobacco plant or part thereof or an unmodified tobacco cell or cell culture.
Claim 16 recites a harvested leaf or a cut harvested leaf of the tobacco plant according to claim 7, or obtained from a plant propagated from a propagation material obtained from the tobacco plant according to claim 7, the harvested leaf or the cut harvested leaf comprising modulated or decreased activity or expression of a DPH3 Homolog-related peptide, or decreased alkaloid and/or TSNA precursor content in comparison to a harvested leaf or a cut harvested leaf obtained from an unmodified tobacco plant.
Claim 18 recites A processed leaf: obtained by processing the tobacco plant according to claim 7;obtained from a plant or a harvested leaf thereof, wherein the plant was propagated from the plant propagation material obtained from the tobacco plant according to claim 7; or obtained by processing a harvested leaf or a cut harvested leaf of the tobacco plant according to claim 7, the processed leaf comprising modulated or decreased activity or expression of a DPH3 Homolog-related peptide, or decreased alkaloid and/or TSNA precursor content in comparison to a processed leaf obtained by processing an unmodified tobacco plant or a harvested leaf or a cut harvested leaf thereof.
Claim 32 recites a tobacco plant or part thereof or a tobacco cell or cell culture according to claim 7, wherein the DPH3 Homolog-related peptide: a) comprises an amino acid sequence as set out in SEQ ID NO: 3; or a functional variant or functional fragment or orthologue of SEQ ID NO: 3; or a sequence which has at least 80% identity to SEQ ID NO: 3; or a homologue of SEQ ID NO: 3; or b) is encoded by a nucleotide sequence as set out in SEQ ID NO: 1 or 2; or a functional variant or functional fragment or orthologue of 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.
Regarding claim 7, 10-11, and 32, Coffin discloses transgenic seeds for crops with enhanced agronomic traits, providing recombinant DNA molecules for expression or suppression of a protein (i.e., modulate or decrease the activity of a peptide) [Abstract]. Coffin discloses that tobacco is a plant of interest for the production of transgenic plants having enhanced traits (i.e., a tobacco plant or part thereof or a tobacco cell or cell culture having been modified) [pg. 77, ln. 9]. Coffin discloses SEQ ID NO: 1607 through SEQ ID NO: 94613, homologs to the proteins cognate to genes used in trait improving [pg. 36, lns. 14-17].
Coffin teaches that "homolog" means a protein that performs the same biological function as a second protein, including those identified by sequence identity search [pg. 9, lns 8-10]. Homologs of the proteins in the invention are identified by comparison of the amino acid sequence of the protein to amino acid sequences of proteins from the same or different plant sources [pg. 35, lns. 27-30]. Thus, homolog is used herein to describe proteins that are assumed to have functional similarity by inference from sequence base similarity [pg. 36, lns. 13-14].
Coffin discloses SEQ ID NO: 23048 with 93.5% identity to instant SEQ ID NO: 3 (i.e., wherein the DPH3 homolog-related peptide comprises an amino acid sequence as set out in SEQ ID NO: 3; or a functional variant or functional fragment or orthologue of SEQ ID NO: 3; or a sequence which has at least 80% identity to SEQ ID NO: 3; or a homologue of SEQ ID NO: 3) (see, alignment below). With high sequence identity to SEQ ID NO: 3, this is taken to be a DPH3 homolog-related peptide (see, claim interpretation). As Coffin discloses plant sequences and their use for altering plant phenotypes by overexpression or down regulation and teaches a DPH3 homolog-related peptide, a reduced alkaloid and/or TSNA content is inherent to the plants as claimed. Although Coffin does not explicitly teach that the alkaloid and/or TSNA precursor content (nicotine, noricotine, PON, anabasine, myosmine, and anatanine) is decreased, the function would inherently come from the taught structure.
Query Match 93.5%; Score 1180.5; Length 243;
Best Local Similarity 94.2%;
Matches 229; Conservative 5; Mismatches 8; Indels 1; Gaps 1;
Qy 1 MAIQRL-LPLFFLLISSLTFLAQSRSDTNHVYSPCADAKVQKSDGFSFGIAFSSRTSFFL 59
|||||| | | |||||||| |||||||||||||||||||||:||||||||||:||||||:
Db 1 MAIQRLPLLLVFLLISSLTLLAQSRSDTNHVYSPCADAKVQRSDGFSFGIAFASRTSFFV 60
Qy 60 NSSVQLSPCDKRLSLSSANSQIAVFRPKVDEISLLTINTTNFFPDSYGGYMVAFAGRKYA 119
|||||||||||||||||||||||||||||||||||||||::|||||||||||||||||||
Db 61 NSSVQLSPCDKRLSLSSANSQIAVFRPKVDEISLLTINTSSFFPDSYGGYMVAFAGRKYA 120
Qy 120 ARSLPAFVANNTFTVTSFTLVLEFKKGRLENLYWKRDGCSSCSGNSNFVCLNDQDCAIRT 179
|||||||||| |||||||||| |||||||||||||||||||||||||||||| |||||||
Db 121 ARSLPAFVANGTFTVTSFTLVHEFKKGRLENLYWKRDGCSSCSGNSNFVCLNGQDCAIRT 180
Qy 180 NNCKNRGGNVDCSLGIQLTFSGTDKHESVFNSWFEVKNLRQYSLYGLYSNLRSSLTDQYN 239
|||||||||||||||||||||||||| ||||||||||||||||||||||||| |||||||
Db 181 NNCKNRGGNVDCSLGIQLTFSGTDKHASVFNSWFEVKNLRQYSLYGLYSNLRGSLTDQYN 240
Qy 240 KFF 242
|||
Db 241 KFF 243
Regarding claim 8, Coffin teaches that seeds of transgenic plants are provided by this invention can be used to propagate more plants containing the trait-improving recombinant DNA constructs of this invention (i.e., plant propagation material obtained from the plant).
Regarding claim 12, Coffin teaches methods for manufacturing non-natural, transgenic seed that can be used to produce a crop of transgenic plants with an enhanced trait resulting from expression of stably-integrated, recombinant DNA in the nucleus of the plant cells (i.e., a plant or crop bred or grown from the tobacco plant or part thereof or tobacco cell or cell culture) [pg. 3, lns. 4-6].
Regarding claims 13 and 16, in the provided examples, Coffin teaches that the plant parts were harvested at day 49 for dry weight measurements (i.e., a product or a leaf produced from the tobacco plant or part thereof or a tobacco cell or cell culture; a harvested leaf of the tobacco plant) [Example 1].
Regarding claim 18, Coffin teaches that transgenic plants can be used to provide plant parts according to the invention for regeneration or tissue culture of cells or tissues containing the described constructs. Plant parts for these purposes can include leaves or any other portion of the plant which can be used to regenerate additional transgenic plants, cells, protoplasts or tissue culture (i.e., a processed leaf: obtained from a plant or a harvested leaf thereof, wherein the plant was propagated from the plant propagation material obtained from the tobacco plant according to claim 7) [pg. 80, lns. 3-10].
Thus, Coffin anticipates claims 7-8, 10-13, 16, 18, and 32.
The art does not explicitly teach nor suggest a leaf processed by curing, fermenting, pasteurizing or a combination thereof, cured tobacco material, or a tobacco blend from tobacco comprising a modification to modulate or decrease activity of a DPH3 homolog-related peptide and decreased alkaloid and/or TSNA precursor content in comparison to an unmodified plant.
Claims 12-13, 16, 18-19, and 21-22, are rejected under 35 U.S.C. 102(a)(2) as being anticipated by Pramod, S. et al. International Publication No. WO 2021247740 A1, “Compositions and Methods for Producing Tobacco Plants and Products Having Altered Alkaloid Levels,” published 12/09/2021. This rejection is made to the extent that the claims are interpreted to require merely decreased alkaloid and/or TSNA precursor content in comparison to a product or a leaf produced from an unmodified tobacco plant. Thus, the claimed material would be indistinguishable from other tobacco material having been modified to have lower alkaloid and/or TSNA precursor content as it does not require modulated activity or expression of a DPH3 homolog-related peptide.
Claim 12 recites a plant or a crop bred or grown from the tobacco plant or part thereof or tobacco cell or cell culture according to claim 7, the plant or the crop comprising decreased alkaloid and/or TSNA precursor content in comparison to a plant or a crop bred or grown from an unmodified tobacco plant or part thereof or an unmodified tobacco cell or cell culture.
Claim 13 recites a product or a leaf produced from the tobacco plant or part thereof or a tobacco cell or cell culture according to claim 7, the product or the leaf comprising decreased alkaloid and/or TSNA precursor content in comparison to a product or a leaf produced from an unmodified tobacco plant or part thereof or an unmodified tobacco cell or cell culture.
Claim 16 recites a harvested leaf or a cut harvested leaf of the tobacco plant according to claim 7, or obtained from a plant propagated from a propagation material obtained from the tobacco plant according to claim 7, the harvested leaf or the cut harvested leaf decreased alkaloid and/or TSNA precursor content in comparison to a harvested leaf or a cut harvested leaf obtained from an unmodified tobacco plant.
Claim 18 recites a processed leaf: obtained by processing the tobacco plant according to claim 7; obtained from a plant or a harvested leaf thereof, wherein the plant was propagated from the plant propagation material obtained from the tobacco plant according to claim 7; or obtained by processing a harvested leaf or a cut harvested leaf of the tobacco plant according to claim 7, the processed leaf comprising decreased alkaloid and/or TSNA precursor content in comparison to a processed leaf obtained by processing an unmodified tobacco plant or a harvested leaf or a cut harvested leaf thereof.
Claim 19 recites the processed leaf according to claim 18, wherein the leaf is processed by curing, fermenting, pasteurizing or a combination thereof, or wherein the leaf is a cut processed leaf
Claim 21 recites cured tobacco material made from the tobacco plant or a part thereof according to claim 7, or a harvested leaf of the tobacco plant according to claim 7, or a processed leaf obtained by processing the tobacco plant or part thereof according to claim 7, the cured tobacco material comprising decreased alkaloid and/or TSNA precursor content in comparison to a cut tobacco material made from an unmodified tobacco plant, a part thereof, a harvested leaf thereof, or a processed leaf thereof.
Claim 22 recites a tobacco blend comprising said cured tobacco material of claim 21.
Pramod teaches compositions and methods for producing tobacco plants and products having altered alkaloid levels [Abstract]. Pramod teaches that a variety of factors affect tobacco alkaloid levels including genotype, environment, fertilization, and agronomic practices [¶5]. Genetic studies using the low alkaloid Burley 21 (LA BU21) lines indicated that two unlinked loci contribute to nicotine levels in the tobacco leaf. Pramod teaches a modified tobacco plant, or part thereof, comprising a genetic modification in a gene and downregulating the expression or activity of the gene [¶9]. Pramod additionally teaches harvesting from low nicotine varieties [¶260], cured tobacco material from the tobacco [claim 12], leaf processing by processes such as fermenting [¶222], and a tobacco blend made from the cured tobacco [¶17] (i.e., a product produced, a harvested leaf, a processed leaf, wherein the leaf is processed by fermenting, cured tobacco material, a tobacco blend).
The products of the instant claims simply require that the plant or the crop comprises modulated or decreased activity or expression of a DPH3 homolog-related peptide or decreased alkaloid and/or TSNA precursor content in comparison to a plant or a crop bred or grown from an unmodified tobacco plant or part thereof. Therefore, a plant or crop bred or grown from the plant of claim 7, or products thereof, need not have modulated or decreased activity or expression of a DPH3 homolog-related peptide. Thus, there is nothing of record to distinguish the plants and products of claims 12-13, 16, 18-19, 21-22 from those of the prior art, as Pramod teaches decreased alkaloid content in comparison to an unmodified plant.
Conclusion
No claims allowed.
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/EMILY K JOHNSON/Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/Supervisory Patent Examiner, Art Units 1661 & 1662
1 Incardona, F. et al. (1995), “How much are homologous peptides homologous?” J. Theor. Biol. 175:437-455.
2 UniProt (https://www.uniprot.org/uniprotkb/Q21102/entry) teaches that diphthamide biosynthesis protein 3 (DPH3) is required for the first step of diphthamide biosynthesis.
3 Wu, Z. et al. (2023, “Phylogenetic Inference of Homologous/Orthologous Genes Among Distantly Related Plants,” Commun. Biol. DOI: 10.1038/s42003-023-04849-4) teaches that homologous/orthologous genes among different plants typically perform similar or equivalent functions, which is theoretically plausible and empirically supported [pg. 2, ¶1].
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