DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Status of claims
The applicant’s amendment filed 5/1/2025 has been entered.
Claims 1-4, 9 had/have been canceled by the applicant.
Claims 5-7 have been amended.
In summary, claims 5-8, 10-11 are pending and examined in this office action.
The application is re-open. Accordingly, the action is non-final.
Priority
Instant application 17627410, filed 01/14/2022, is a National Stage entry of PCT/JP2020/028981, International Filing Date: 07/29/2020, and claims foreign priority to 2019-142038, filed 08/01/2019.
A certified copy of 2019-142038 was filed 1/14/2022. However, the English translation is missing.
It is suggested to file a certified English translation to perfect the priority.
Claim Rejections - 35 USC 112
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.
Scope of enablement
Claims 5-7 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 method of using high concentration of ascorbic acid at 100-300 mM initial concentration and/or 25-80 mM final concentration in Nicotiana plant, but does not reasonably provide enablement for a method of using high concentration of ascorbic acid at 100-300 mM initial concentration and/or 25-80 mM final concentration in a genus of plants except Nicotiana plant. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention commensurate in scope with these claims.
Claims are broadly drawn to methods comprising administering ascorbic acid, a salt thereof, or a solvate thereof to a genus of plants by contacting (particularly spraying) the plants at an initial concentration of 100 to 300 mM (claims 5-6), or by adding to an Agrobacterium suspension for infecting a genus of plants at a final concentration of 25-80 mM (claim 7), for inhibiting cell death induced by transient protein expression in the genus of plants (preambles).
Transient expression of proteins, for example by Agrobacterium infiltration, can induce plant cell death, in the art, and according to the specification ([0001]-[0003]).
The specification provides examples that serve as guidance of spraying different concentrations of ascorbic salt (sodium ascorbate) to Nicotiana benthamiana leaves (Examples 1-2 in [0065]-[0092]), The applicant obtained the results of increased fresh tissue weight and increased GFP expression at high initial concentration, particularly at initial concentration of 200 mM for spraying (amount or final concentration of spraying is not disclosed) (See figures 3B, 4B and 5B).
The specification also provides examples that serve as guidance of adding different concentrations of sodium ascorbate to Agroinfiltration liquid for infiltrating Nicotiana benthamiana leaves (Examples 3-4 in [0093]-[0103]). The applicant obtained the results of increased hCu1 protein expression at high final concentration, particularly at final concentration of 40 mM that is comparable to the effect of spraying at initial concentration of 200 mM (see [0098] and figure 13B).
In the art, Nosaki at al (Prevention of necrosis caused by transient expression in Nicotiana benthamiana by application of ascorbic acid. Plant Physiology. P832-835, 2021) teach that In Nicotiana plants, high concentrations of applied ascorbic acid, like 200 mM or even 300 mM, are generally well-tolerated and primarily function as an antioxidant that suppresses stress symptoms like necrosis, rather than causing toxicity (p833, figure 1).
Thus, the specification, supported by the art, reasonably enables that, at high concentrations, for example initial concentration of 200 mM (amount or final concentration not disclosed) or final concentration of 40 mM, have the effect of inhibiting cell death of Nicotiana plant tissue.
However, the specification fails to provide any example serving as guidance of using high concentrations of ascorbic acid having the effect of inhibiting cell death in the tissues of the genus of all plants except in Nicotiana plant.
Critically, in the art, high concentrations of ascorbic acid cause toxicity to plant tissues. For example, Toth et al (Concentration Does Matter: The Beneficial and Potentially Harmful Effects of Ascorbate in Humans and Plants. ANTIOXIDANTS & REDOX SIGNALING. P1516-1533, 2018) teach that ascorbic acid (Asc) plays an essential role as a scavenging reactive oxygen species (ROS), preventing the overaccumulation of ROS in plant (p1516, Abstract; p1517, right col, last para). However, at high doses, Asc has toxicity and harmful effects in plants and the mechanism thereof (p1523-1527, fig 5 in p1524).
For another example, Castro et al (Ascorbic acid toxicity is related to oxidative stress and enhanced by high light and knockdown of chloroplast ascorbate peroxidases in rice plants. Theor. Exp. Plant Physiol. p41–55, 2018) teach that high concentrations of ascorbic acid (AA) causes toxicity in rice leaves. Castro et al teach that at concentrations of 20-50 mM, AA caused toxicity (p48, fig 2), and that at a concentration of 50 mM, AA caused tissue toxicity in all tests in rice leaves (p49, figs 4-5).
The MW of AsA is 176 g/mol. Thus, 1 mg/L of AsA is about 0.0057 millimolar (mM), or 1000 mg/L is about 5.7 mM.
More convincingly, Akram et al (Ascorbic Acid-A Potential Oxidant Scavenger and Its Role in Plant Development and Abiotic Stress Tolerance. Frontiers in Plant Science, p1-17, 2017) teach and demonstrated that at low doses or concentrations, below 2000 mg/L (11.4 mM), ascorbic acid (AsA) improves plant cell conditions and yields in all examples (Table 1 in pages 9-10). However, in the only example that the ascorbic acid (AsA) concentration is high, at 50, 100 and 150 mM, AsA caused toxicity indicated by decreased flavonoids, anthocyanins and total soluble sugars in a sunflower plant (same Table 1, the 3rd row from the last row).
In summary, in the art, there is no example that ascorbic acid in a concentration higher than 25 mM, being initial or final concentration, caused beneficial effect in plants other than in Nicotiana plant, not to mention have the effect of inhibiting cell death.
In contrary, in higher concentration, ascorbic acid caused toxicity in plants. CHRISTENHUSZ et al (The number of known plants species in the world and its annual increase. Phytotaxa 261: 201–217, 2016) teach that there are about 374,000 species of plants, and 308, 312 vascular plant species (p201, Abstract).
MPEP 2164 provides the Enablement Requirement. MPEP section 2164.01 states “Any analysis of whether a particular claim is supported by the disclosure in an application requires a determination of whether that disclosure, when filed, contained sufficient information regarding the subject matter of the claims as to enable one skilled in the pertinent art to make and use the claimed invention.
The standard for determining whether the specification meets the enablement requirement was cast in the Supreme Court decision of Mineral Separation v. Hyde, 242 U.S. 261, 270 (1916) which postured the question: is the experimentation needed to practice the invention undue or unreasonable? That standard is still the one to be applied. In re Wands, 858 F.2d 731, 737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988). Accordingly, even though the statute does not use the term “undue experimentation,” it has been interpreted to require that the claimed invention be enabled so that any person skilled in the art can make and use the invention without undue experimentation.
In this particular case, Nicotiana is the only guided plant that is demonstrated to tolerant to high dose/concentration of ascorbic acid and to result in inhibiting cell death induced by transient protein expression. At higher concentration, ascorbic acid did not lead to such result, but rather caused demonstrated toxicity in plants in multiple references.
Therefore, given the claim breadth, lack of guidance in the specification, insufficient working examples in the instant specification, unpredictability in the prior art, undue experimentation would have been required by one skilled in the art to make and use the invention as broadly claimed.
Claims 8, 10-11 limit the plant to Nicotiana plant, thus, are excluded.
Lacking written description
Claims 5-7 are rejected under 35 U.S.C. 112(a), 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 pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention.
To claim a genus under the written description requirement, the applicant is required to describe a representative number of species to reflect the variation within the genus or structures sufficient to define the genus. The factors to be considered include disclosure of complete or partial structure, physical and/or chemical properties, functional characteristics, structure/function correlation, methods of making the claimed product, or any combinations thereof.
By court’s statement in Regents of the Univ. of Cal. v. Eli Lilly, 119 F.3d 1559, 1566, 43 USPQ2d 1398, 1404 (Fed. Cir. 1997), a written description of an invention “requires a precise definition, such as a structure, formula, or chemical name, of the claimed subject matter sufficient to distinguish it from other materials”; further, a written description of a claimed genus requires a description of a representative number of species of the claimed genus, and one of skill in the art should be able to “visualize or recognize the identity of the members of the genus”.
Claims are broadly drawn to methods comprising administering ascorbic acid, a salt thereof, or a solvate thereof to a genus of plants by contacting (particularly spraying) the plants at an initial concentration of 100 to 300 mM (claims 5-6), or by adding to an Agrobacterium suspension for infecting a genus of plants at a final concentration of 25-80 mM (claim 7).
The claimed function is for inhibiting cell death induced by transient protein expression in the genus of plants (preambles).
The structure of ascorbic acid, a salt thereof, or a solvate thereof is taught in prior art and well-known. The general function of ascorbic acid as an antioxidant is also taught in prior art and well-known.
Transient expression of proteins, for example by Agrobacterium infiltration, can induce plant cell death, in the art, and according to the specification ([0001]-[0003]).
The specification provides examples of spraying different concentrations of ascorbic salt (sodium ascorbate) to Nicotiana benthamiana leaves (Examples 1-2 in [0065]-[0092]), The applicant obtained the results of increased fresh tissue weight and increased GFP expression at high initial concentration, particularly at initial concentration of 200 mM for spraying (amount or final concentration of spraying is not disclosed) (See figures 3B, 4B and 5B). The specification also provides examples of adding different concentrations of sodium ascorbate to Agroinfiltration liquid for infiltrating Nicotiana benthamiana leaves (Examples 3-4 in [0093]-[0103]). The applicant obtained the results of increased hCu1 protein expression at high final concentration, particularly at final concentration of 40 mM that is comparable to the effect of spraying at initial concentration of 200 mM (see [0098] and figure 13B).
In the art, Nosaki at al teach that In Nicotiana plants, high concentrations of applied ascorbic acid, like 200 mM or even 300 mM, are generally well-tolerated and primarily function as an antioxidant that suppresses stress symptoms like necrosis, rather than causing toxicity (p833, figure 1).
Thus, the specification, supported by the art, reasonably describes that ascorbic acid, at high concentrations, for example initial concentration of 200 mM (amount or final concentration not disclosed) or final concentration of 40 mM, have the effect or function of inhibiting cell death of Nicotiana plant tissue.
However, the specification fails to describe using high concentrations of ascorbic acid having the effect or function of inhibiting cell death in the tissues of the genus of all plants except in Nicotiana plant. In another word, Nicotiana plant is the only plant that is described by the applicant.
In the art, high concentrations of ascorbic acid cause toxicity to plant tissues. For example, Toth et al teach that ascorbic acid (Asc) plays an essential role as a scavenging reactive oxygen species (ROS), preventing the overaccumulation of ROS in plant (p1516, Abstract; p1517, right col, last para). However, at high doses, Asc has toxicity and harmful effects in plants and the mechanism thereof (p1523-1527, fig 5 in p1524).
For another specific example, Castro et al teach that high concentrations of ascorbic acid (AA) causes toxicity in rice leaves. Castro et al teach that at concentrations of 20-50 mM, AA caused toxicity (p48, fig 2), and that at a concentration of 50 mM, AA caused tissue toxicity in all tests in rice leaves (p49, figs 4-5).
The MW of AsA is 176 g/mol. Thus, 1 mg/L of AsA is about 0.0057 millimolar (mM), or 1000 mg/L is about 5.7 mM.
Convincingly, Akram et al teach and demonstrated that at low doses or concentrations, below 2000 mg/L (11.4 mM), ascorbic acid (AsA) improves plant cell conditions and yields in all examples (Table 1 in pages 9-10). However, in the only example that the ascorbic acid (AsA) concentration is high, at 50, 100 and 150 mM, AsA caused toxicity indicated by decreased flavonoids, anthocyanins and total soluble sugars in a plant (sunflower, same Table 1, the 3rd row from the last row).
In summary, in the art, there is no example that ascorbic acid in a concentration higher than 25 mM, being initial or final concentration, caused beneficial effect in plants other than in Nicotiana plant. In contrary, in higher concentration, ascorbic acid caused toxicity in plants.
Regarding the description of a representative number of species, CHRISTENHUSZ et al (The number of known plants species in the world and its annual increase. Phytotaxa 261: 201–217, 2016) teach that there are about 374,000 species of plants, and 308, 312 vascular plant species (p201, Abstract). The genus of plants are heterogenous in structure.
Thus, Nicotiana plant is the only described plant, which does not describe the common structure feature of the genus of 374, 000 plant species, and is not sufficient to represent the genus of plants.
Therefore, the application has not met either of the two elements of the written description requirement as set forth in the court’s decision in Eli Lilly, and has not shown her/his possession of the claimed genus at the time of the application.
Claims 8, 10-11 limit the plant to Nicotiana plant, thus, are excluded.
Remarks
Prior does not teach or suggest using ascorbic acid at a concentration higher than 25 mM, being initial or final concentration, caused beneficial effect in plants other than in Nicotiana plant, not to mention have the effect of inhibiting cell death induced by transient expression process. In contrary, high concentrations of ascorbic acid caused toxicity in plants other than in Nicotiana plant.
In addition, Norkunas et al (Improving agroinfiltration‑based transient gene expression in Nicotiana benthamiana. Plant Methods, p1-14, 2018) teach that in Nicotiana plant, ascorbic acid, at 5 mM has significant effect on increased GUS activity as compared to control 0. However, at 10 mM has no effect on GUS activity as compared to control 0, and at 50 mM and 100 mM has decreased GUS activity as compared to control 0 (p7, fig 3b), although the concentrations did not cause significant toxicity in Nicotiana plant.
Moreover, the applicant provides examples of spraying different concentrations of ascorbic salt (sodium ascorbate) to Nicotiana benthamiana leaves, and demonstrated the results of increased fresh tissue weight and increased GFP expression at high initial concentration. The applicant also provides examples of adding different concentrations of sodium ascorbate to Agroinfiltration liquid for infiltrating Nicotiana benthamiana leaves, and demonstrated the results of increased hCu1 protein expression at high final concentration, particularly at final concentration of 40 mM that is comparable to the effect by spraying at initial concentration of 200 mM. Thus, at the very least, such result is deemed unexpected, for example, as compared to that of Norkunas et al above.
Therefore, no art rejections are made.
Response to Arguments
According to the decision of 11/4/2025, the application is re-open. The arguments of 5/1/2025 and of 8/15/2025 have been fully analyzed and considered. Accordingly, the office action is non-final.
All of the rejections are re-written. The arguments of 5/1/2025 and of 8/15/2025 are regarding the previous rejections, and are not applicable to the instant rejections.
Conclusion
Claims 5-7 are rejected.
Claims 8, 10-11 are objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
Contact information
Any inquiry concerning this communication or earlier communications from the examiner should be directed to WAYNE ZHONG whose telephone number is (571)270-0311. The examiner can normally be reached 8:30am to 5:00pm EST.
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/Wayne Zhong/
Primary Examiner, Art Unit 1662