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 .
Amendments
In the reply 7/27/2026, Applicant has amended Claims 1, 3, 6-8, 10-11, 18, 20-22, 24-25, and 27-28, cancelled claims 9, 34, and 35 and added new claims, Claims 36-38.
Applicant’s election of the following invention without traverse in the reply filed on 2/02/2026 is acknowledged.
Group I, claims 1-8, 10-18, 20-33, and 36-38, drawn to methods of modulating gene expression.
Claim 19 is pending but withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a non-elected invention, there being no allowable generic or linking claim.
Rejoinder
The species election requirement for Group I, as set forth in the Office action mailed on 10/02/2025, has been reconsidered. The species election requirement for Group I is hereby withdrawn. Claims 12, 14, and 32, directed to previously non-elected species are no longer withdrawn from consideration.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 7/27/2026 was filed after the mailing date of the non-final Office action on 3/24/2026. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Withdrawn Objection to Specification
The objection to the specification for referring to “SEQ ID NO:874”, has been withdrawn in light of Applicant’s amendments to the specification.
Withdrawn Claim Objections
The objection to Claim 6 has been withdrawn due to Applicant’s amendment to spell out the abbreviation “sgRNA”.
Withdrawn 35 USC § 112(b)
The prior rejection of Claims 1-11, 13, and 15-18 under 35 U.S.C. § 112(b) pre-AIA 2nd paragraph as being indefinite is withdrawn in light of Applicant’s amendments of Claim 1 to describe the transcription modulating protein, and Claims 3 and 11 to remove the phrases “such as”.
Withdrawn 35 USC § 102
The prior rejection of Claims 1, 4-8, 10, 13, 15, 18, 20-28, and 33 under 35 U.S.C. 102(a)(1)/(a)(2) as being anticipated by Klavins et al., (US2017/0369892, filed 6/22/2017, published 12/28/2917) is withdrawn in light of Applicant’s amendment of Claim 1 to limit the cell to a mammalian cell and to include a nuclear translocation step, which are limitations Klavins does not anticipate.
Withdrawn 35 USC § 103
The prior rejection of Claims 2-3 under 35 U.S.C. 103 as being unpatentable over Klavins et al., (US2017/0369892, filed 6/22/2017, published 12/28/2917) is withdrawn in light of Applicant’s amendment of Claim 1.
The prior rejection of Claims 11, 16-17, 21, 29-31 under 35 U.S.C. 103 as being unpatentable over Klavins et al., (US2017/0369892, filed 6/22/2017, published 12/28/2917) is withdrawn in light of Applicant’s amendment of Claim 1 to include a nuclear translocation step for claim 11, 16-17, and to include an in vivo mammalian embodiment for claims 21, 29-31, which is a limitation Klavins does not make obvious.
Furthermore, the Examiner finds Applicant’s arguments persuasive that “NPR1 cannot function as a degron”, and the “exact molecular mechanism and structural outcome of SA binding to plant NPR1 was highly controversial and poorly understood”. Applicant convincingly argues that those skilled in the art “would not expect the use of NPR1 or its transactivation domain (TAD) in mammalian cells would result in an intact, functional, SA-inducible nuclear translocator.”
New Claim Rejections - 35 USC § 112(a)
(Written Description)
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.
Claims 1-8, 10-18, 20-33, and 36-38 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention.
Independent claims 1 and 20 encompass a genus of methods for providing modulated gene expression comprising (a) a mammalian cell comprising (i) a target gene under the control of a transcriptional element modulated by a genus of transcription modulating proteins or functional fragments thereof, and (ii) a chimeric molecule comprising the genus of transcription modulating proteins or functional domains thereof, a genus of salicylic acid (SA) binding domains from a NPR gene and optionally a NLS; and (b) contacting the mammalian cell with salicylic acid to modulate gene expression, while the specification discloses no methods for modulating gene expression.
In regard to the scope of the chimeric molecules comprising the genus of transcription modulating proteins or functional domains thereof, Applicant’s specification describes the genus of transcription modulating proteins to include CRISPR gene such as Casp9/Cpf1, TALENS, and other factors such as VP16, FoxA and MyoD (p. 3, Summary, p. 13-15, Section B). Applicant specification describes the scope of transcription modulating proteins include both negative modulators of transcription and inducers of transcription (p. 3, Summary). However, Applicant’s specification describes no methods of modulating gene expression with any type of transcription modulating protein or functional domains thereof.
In regard to the scope of the chimeric molecules comprising the genus of salicylic acid (SA) binding domains from a NPR gene, Applicant’s specification describes the genus of SA binding domains from NPR genes as including the NPR1, NPR3, and NPR4 genes (p. 10, 3rd para.). Applicant specification further indicates the scope of the NPR1-like proteins suitable for the invention include those from Malus pumila, Malus hupehensis, Oryza sativa, Populus trichocarpa, Nicotiana tabacum, Nicotiana glutinosa, Vitis vin[fera, Vitis aestivalis c.v. Norton, Gossypium hirsutum, Pyrus pyrifolia, Ipomoea batatas, Carica papaya, Musa acummata, Musa spp. ABB, Solanum lycopersicum, Brassica juncea, Glycine mas, Jheobroma cacao, Saccharwn spp., Coffea arabica, Phalaenopsis aphrodite, Trilicum aestivum L., Beta vulgaris, Persea americana, Cocos nucifera L., Gladiolus hybridus, Brassica napus, Arachis hypogaea, Lilium 'Sorbonne' and Eucalyptus grandis. However, as stated above Applicant’s specification describes no methods of modulating gene expression with any type of NPR gene, and only describes a method of inducing nuclear translocation with a limited genus of chimeric molecules comprising the Arabidopsis thaliana NPR1 gene or a specific TAD fragment thereof linked to a mCherry fluorescent protein.
Dependent claims 2-4, and 36 encompass a genus of methods comprising a subgenus of method for expressing the chimeric molecule.
Dependent claims 5-6, 14-15, 23, 25, 32-33 encompass a genus of methods comprising a subgenus of transcription modulating proteins or functional domains thereof.
Dependent claims 7-8, 10, 18, 22-24 encompass a genus of methods comprising a subgenus of chimeric proteins with various NLSs and solubility domains.
Dependent claims 11, and 38 encompass a genus of methods comprising a subgenus of chimeric proteins with a NPR TAD.
Dependent claims 12-13, and 16-17, 26-30 encompass a genus of methods comprising a subgenus of target genes.
Dependent claims 21, 31 and 37 encompass a genus of methods comprising a subgenus of mammalian cells in vivo.
Under the written description guidelines (see MPEP 2163) the Examiner is directed to determine whether one skilled in the art would recognize that the Applicant was in possession of the claimed invention as a whole at the time of filing. The following considerations are critical to this determination.
To satisfy the written description requirement, a patent specification must describe the claimed invention in sufficient detail that one skilled in the art can reasonably conclude that the inventor had possession of the claimed invention. See, e.g., Moba, B.V. v. Diamond Automation, Inc., 325 F.3d 1306, 1319, 66 USPQ2d 1429, 1438 (Fed. Cir. 2003); Vas-Cath, Inc. v. Mahurkar, 935 F.2d at 1563, 19 USPQ2d at 1116. An original claim may lack written description support when (1) the claim defines the invention in functional language specifying a desired result but the disclosure fails to sufficiently identify how the function is performed or the result is achieved or (2) a broad genus claim is presented but the disclosure only describes a narrow species with no evidence that the genus is contemplated. See Ariad Pharms., Inc. v. Eli Lilly & Co., 598 F.3d 1336, 1349-50 (Fed. Cir. 2010) (en banc). The written description requirement is not necessarily met when the claim language appears in ipsis verbis in the specification. "Even if a claim is supported by the specification, the language of the specification, to the extent possible, must describe the claimed invention so that one skilled in the art can recognize what is claimed. The appearance of mere indistinct words in a specification or a claim, even an original claim, does not necessarily satisfy that requirement." Enzo Biochem, Inc. v. Gen-Probe, Inc., 323 F.3d 956, 968, 63 USPQ2d 1609, 1616 (Fed. Cir. 2002).
Accordingly, to satisfy the written description requirement, the specification must describe the claimed invention in sufficient detail that one skilled in the art can reasonably conclude that the inventor had possession of the claimed invention. Vas-Cath, Inc. v. Mahurkar, 935 F.2d 1555, 1562-63, 19 USPQ2d 1111 (Fed. Cir. 1991). See also MPEP 2163.
ACTUAL REDUCTION TO PRACTICE
In regard to claims 1 and 20 encompassing a genus of methods for providing modulated gene expression comprising (a) a mammalian cell comprising (i) a target gene under the control of a transcriptional element modulated by a genus of transcription modulating proteins or functional fragments thereof, and (ii) a chimeric molecule comprising the genus of transcription modulating proteins or functional domains thereof, a genus of salicylic acid (SA) binding domains from a NPR gene and optionally a NLS; and (b) contacting the mammalian cell with salicylic acid to modulate gene expression, as stated supra, the specification discloses no methods for modulating gene expression. Applicant’s specification describes no methods of comprising (a) a target gene and Applicant’s specification describes no method for making and using (b) a chimeric protein comprising a transcription modulating protein or functional domain thereof with a salicylic acid binding domain from a NPR protein.
However, Applicant’s specification does describe a method of contacting a mammalian cell with specific concentrations of SA (about 2.5 mM) to induce nuclear translocation of a limited genus of mCherry-NPR1 fusion proteins. Specifically, Applicant’s specification only describes a method using the NPR1 gene from Arabidopsis thaliana. Furthermore, as far as functional domains of the NPR1 protein, Applicant’s specification only disclose the TAD domain from amino acids 513-593 from Arabidopsis thaliana NPR1.
Importantly, Applicant’s specification indicates that the N-terminal or C-terminal position of the NRP1 protein or its TAD domain in the chimeric molecules can significantly influence nuclear translocation. Specifically, Table 1 of the disclosure (see below) demonstrates that the mCherry-NPR1 chimeric molecule exhibited “No translocation”, while the mCherry-NPR1-TAD, NPR1-TAD-mCherry and NPR1-mCherry did to varying degrees depending on the concentration of SA used (see Fig. S4). Thereby indicating that position of the transcription modulating protein relative to the NPR1 protein is critical to nuclear translocation.
PNG
media_image1.png
745
1333
media_image1.png
Greyscale
Furthermore, Applicant’s arguments filed 7/27/2026 indicate that the “exact molecular mechanism and structural outcome of SA binding to plant NPR1 was highly controversial and poorly understood”. Applicant argues that those skilled in the art “would not expect the use of NPR1 or its transactivation domain (TAD) in mammalian cells would result in an intact, functional, SA-inducible nuclear translocator.”
Accordingly, Applicant did not demonstrate a reduction to practice of methods for providing modulated gene expression comprising (a) a mammalian cell comprising (i) a target gene under the control of a transcriptional element modulated by a genus of transcription modulating proteins or functional fragments thereof, and (ii) a chimeric molecule comprising the genus of transcription modulating proteins or functional domains thereof, a genus of salicylic acid (SA) binding domains from a NPR gene and optionally a NLS; and (b) contacting the mammalian cell with salicylic acid to modulate gene expression, nor did Applicant adequately set forth in terms of distinguishing identifying characteristics as evidenced by other descriptions of the invention that are sufficiently detailed to show that Applicant was in possession of the claimed genus of methods.
DISCLOSURE OF STRUCTURE
The Applicant has provided no examples of a methods for providing modulated gene expression comprising (a) a mammalian cell comprising (i) a target gene under the control of a transcriptional element modulated by a genus of transcription modulating proteins or functional fragments thereof, and (ii) a chimeric molecule comprising the genus of transcription modulating proteins or functional domains thereof, a genus of salicylic acid (SA) binding domains from a NPR gene and optionally a NLS; and (b) contacting the mammalian cell with salicylic acid to modulate gene expression. Certainly, with the help of a cell culture facility, a skilled artisan could make modified mammalian cell comprising a target gene under the control of a transcriptional element modulated by a genus of transcription modulating proteins or functional fragments thereof. Furthermore, a skilled artisan could make a chimeric molecule comprising the genus of transcription modulating proteins or functional domains thereof and a genus of salicylic acid (SA) binding domains from a NPR gene and optionally a NLS. These were known proteins and genes.
However, neither the specification nor the art indicate a relationship between the structure of the claimed genus of modified mammalian cells and chimeric proteins and the ability to successfully modulate gene expression in a cell, much less a cell in vivo, by administering salicylic acid.
SUFFICIENT RELEVANT IDENTIFYING CHARACTERISTICS
[AltContent: textbox ([img-media_image2.png])]Applicant’s specification describes that the Arabidopsis thaliana gene NPR1 has been shown to directly bind SA, which causes a conformational change in NPR1 that is accompanied by the release of the C-terminal transactivation domain (TAD) for the N-terminal autoinhibitory BTB/POZ domain (see Fig. 4E, adjacent)
However, the prior art is silent with respect to methods of modulating gene expression in a cell comprising any chimeric protein that comprises any NRP protein or fragment thereof by administering salicylic acid. In fact, as stated supra, Applicant’s specification admits that “exact nature and outcome of the molecular interaction between NPR1 and SA remains controversial”, as is the proposed role for this interaction on influencing nuclear translocation (p. 9., 3rd para.).
Moreover, the breadth of the claims encompass a genus of modified mammalian cells, in vitro or in vivo, that can undergo modulated gene expression (in any direction by any amount) by expressing a genus of chimeric molecules that comprise a genus of transcription modulating proteins fused in any configuration with any NPR protein, yet the present specification provides limited guidance and no description of methods for modulating gene expression by administering salicylic acid to said genus of engineered cells and chimeric proteins, therefore the skilled artisan would not know what rational approach to take to make and use the genus of modified chimeric proteins with any predictable outcome on modulating gene expression. Therefore, it is incumbent on the applicant to provide this nexus between structure and function, in order to be given credit for possession of the claimed genus of methods.
STATE OF THE ART & QUANTITY OF EXPERIMENTATION
The method of practicing the claimed invention is not well established. Although the plant NPR genes, transcription modulating proteins (e.g, Cas9 and TALENs) and the transactivation domains (e.g., VP16) were known in the state of the art, one of skill in the art would neither expect nor predict a successful outcome to modulate gene expression in a mammalian cell in vitro or in vivo by administering salicylic acid to the claimed genus of chimeric molecules.
The following describes the state of the art with respect to salicylic acid regulation of NPR1. Specifically, the prior art of Fu et al. (Nature, 2012, 486:228-231, see IDS filed 7/27/2026) examines the nuclear translocation of Arabidopsis NRP1 in plant cells, and teaches that SA controls the nuclear translocation of NPR1 through cellular redox changes, wherein NPR1 is retained in the cytoplasm as an oligomer through redox-sensitive intermolecular disulphide bonds. After induction, these disulphide bonds are reduced, releasing NPR1 monomers into the nucleus, where NPR1 acts as a cofactor for transcription factors (p. 228, 3rd para.). Importantly, NPR1 does not bind DNA on its own. Thus, one of ordinary skill would not have predicted that a chimeric protein comprising NRP1 alone would have been able to modulate gene expression. Furthermore, as noted by Applicant’s remarks, one of ordinary skill would not have expected that the same redox conditions would have existed in mammalian cells so as to allow NPR1 to translocate to the nucleus.
The following describes the state of the art with respect to transcription modulating proteins or functional domains thereof. Specifically, the prior art of Konermann et al., (Nature, 2015, 517:583-588) indicates that conventional transcription modulating proteins such as dCas9 directed by a sgRNA typically achieve little to no transcriptional regulation (p. 583, 2nd para.). Konermann teaches the dCas9 complex must be optimized to include other adaptor proteins to recruit a transcriptional modulating complex (p. 583, last para., see Fig. 2B). Furthermore, despite engineering the sgRNA to incorporate protein-interacting aptamers to predictably allow modulated gene expression, Konermann acknowledges that future studies must address “additional selection criteria that are critical for guide efficacy” (p. 587, Discussion, last para.). For example, Konermann teaches that sgRNA must be screened across at least 1000 bp of a target gene’s transcriptional start site (TSS) to find optimal sgRNA that can modulate gene expression (p. 584, last two para.). Furthermore, Konermann discusses other programable DNA-binding proteins such as TALENS (p. 583, 2nd para.), but dismisses these as difficult to be scaled to facilitate genomic perturbation because each and every TALEN must be individually designed and empirically tested for binding a specific regulatory region in a target gene. Thus, one of ordinary skill would not have predicted that a chimeric protein comprising NRP1 with any transcription modulating protein or functional fragment thereof would have been able to modulate gene expression.
Finally, with regard to using salicylic acid to modulate gene expression in a mammalian cell in vivo, it is well known that a serum level of 100 mg/dL (which is about 7 mM) is generally classified as a critical threshold associated with severe toxicity in acute ingestions of salicylates (e.g., aspirin). Thus, since Applicant’s method requires 2.5 mM for at least 30 minutes to see a translocation of a chimeric mCherry-NPR1 fusion protein to the nucleus of a mammalian cell, an effective dose of salicylic acid to achieve the claimed method in vivo has not been described.
Applicant has claimed a genus of methods to modulate gene express in a mammalian cell in vitro and in vivo, yet the specification has not disclosed such methods in any cell, has not set forth in terms of distinguishing identifying characteristics as evidenced by other descriptions of the invention (e.g., in vitro target gene binding and gene modulation assays) that are sufficiently detailed to show that Applicant was in possession of the claimed genus of methods. Furthermore, the state of the art indicated that practicing the claimed method is not well established with the claimed function in successfully modulating gene expression in a mammalian cell would require undue experimentation, and one of skill in the art would neither expect nor predict the claimed method of gene modulation would be successful according to the claimed genus of chimeric molecules.
CONCLUSION
Therefore, the Examiner concludes that there is insufficient written description of the instantly claimed genus methods of modulating gene expression in a mammalian cell comprising administering an effective amount salicylic acid, wherein the genus of chimeric molecules are translocated to the nucleus and/or modulate expression of a target gene.
New Claim Rejections - 35 USC § 112(a)
(Scope of Enablement)
Claims 1-8, 10-18, 20-33, and 36-38 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification does not reasonably provide enablement for the genus of methods for providing modulated gene expression comprising (a) a mammalian cell comprising (i) a target gene under the control of a transcriptional element modulated by a genus of transcription modulating proteins or functional fragments thereof, and (ii) a chimeric molecule comprising the genus of transcription modulating proteins or functional domains thereof, a salicylic acid (SA) binding domain from a NPR gene and optionally a NLS; and (b) contacting the mammalian cell with salicylic acid to induce nuclear translocation of the chimeric molecule, thereby modulating gene expression. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to practice the invention according to these claims.
SCOPE OF THE INVENTION
Independent claims 1 and 20 encompass a genus of methods for providing modulated gene expression comprising (a) a mammalian cell comprising (i) a target gene under the control of a transcriptional element modulated by a genus of transcription modulating proteins or functional fragments thereof, and (ii) a chimeric molecule comprising the genus of transcription modulating proteins or functional domains thereof, a genus of salicylic acid (SA) binding domains from a NPR gene and optionally a NLS; and (b) contacting the mammalian cell with salicylic acid to modulate gene expression, while the specification discloses no methods for modulating gene expression.
In regard to the scope of the chimeric molecules comprising the genus of transcription modulating proteins or functional domains thereof, Applicant’s specification describes the genus of transcription modulating proteins to include CRISPR gene such as Casp9/Cpf1, TALENS, and other factors such as VP16, FoxA and MyoD (p. 3, Summary, p. 13-15, Section B). Applicant specification describes the scope of transcription modulating proteins include both negative modulators of transcription and inducers of transcription (p. 3, Summary). However, Applicant’s specification describes no methods of modulating gene expression with any type of transcription modulating protein or functional domains thereof.
In regard to the scope of the chimeric molecules comprising the genus of salicylic acid (SA) binding domains from a NPR gene, Applicant’s specification describes the genus of SA binding domains from NPR genes as including the NPR1, NPR3, and NPR4 genes (p. 10, 3rd para.). Applicant specification further indicates the scope of the NPR1-like proteins suitable for the invention include those from Malus pumila, Malus hupehensis, Oryza sativa, Populus trichocarpa, Nicotiana tabacum, Nicotiana glutinosa, Vitis vin[fera, Vitis aestivalis c.v. Norton, Gossypium hirsutum, Pyrus pyrifolia, Ipomoea batatas, Carica papaya, Musa acummata, Musa spp. ABB, Solanum lycopersicum, Brassica juncea, Glycine mas, Jheobroma cacao, Saccharwn spp., Coffea arabica, Phalaenopsis aphrodite, Trilicum aestivum L., Beta vulgaris, Persea americana, Cocos nucifera L., Gladiolus hybridus, Brassica napus, Arachis hypogaea, Lilium 'Sorbonne' and Eucalyptus grandis. However, as stated above Applicant’s specification describes no methods of modulating gene expression with any type of NPR gene, and only describes a method of inducing nuclear translocation with a limited genus of chimeric molecules comprising the Arabidopsis thaliana NPR1 gene or a specific TAD fragment thereof linked to a mCherry fluorescent protein.
Dependent claims 2-4, and 36 encompass a genus of methods comprising a subgenus of method for expressing the chimeric molecule.
Dependent claims 5-6, 14-15, 23, 25, 32-33 encompass a genus of methods comprising a subgenus of transcription modulating proteins or functional domains thereof.
Dependent claims 7-8, 10, 18, 22-24 encompass a genus of methods comprising a subgenus of chimeric proteins with various NLSs and solubility domains.
Dependent claims 11, and 38 encompass a genus of methods comprising a subgenus of chimeric proteins with a NPR TAD.
Dependent claims 12-13, and 16-17, 26-30 encompass a genus of methods comprising a subgenus of target genes.
Dependent claims 21, 31 and 37 encompass a genus of methods comprising a subgenus of mammalian cells in vivo.
The factors to be considered in determining whether undue experimentation is required are summarized In re Wands 858 F.2d 731, 8 USPQ2nd 1400 (Fed. Cir, 1988). The Court in Wands states: “Enablement is not precluded by the necessity for some 'experimentation.'” Clearly, enablement of a claimed invention cannot be predicated on the basis of quantity of experimentation required to make or use the invention. “Whether undue experimentation is needed is not a single simple factual determination, but rather is a conclusion reached by weighing many factual considerations.” (Wands, 8 USPQ2d 1404). The factors to be considered in determining whether undue experimentation is required include: (1) the quantity of experimentation necessary, (2) the amount or direction or guidance presented, (3) the presence or absence of working examples, (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, and (8) the breadth of the claims. While all of these factors are considered, a sufficient amount for a prima facie case is discussed below.
The office has analyzed the specification in direct accordance to the factors outlined in In re Wands. MPEP 2164.04 states: "[W]hile the analysis and conclusion of a lack of enablement are based on factors discussed in MPEP 2164.01(a) and the evidence as whole, it is not necessary to discuss each factor in written enablement rejection." These factors will be analyzed, in turn, to demonstrate that one of ordinary skill in the art would have had to perform "undue experimentation" to make and/or use the invention.
ACTUAL REDUCTION TO PRACTICE
In regard to claims 1 and 20 encompassing a genus of methods for providing modulated gene expression comprising (a) a mammalian cell comprising (i) a target gene under the control of a transcriptional element modulated by a genus of transcription modulating proteins or functional fragments thereof, and (ii) a chimeric molecule comprising the genus of transcription modulating proteins or functional domains thereof, a genus of salicylic acid (SA) binding domains from a NPR gene and optionally a NLS; and (b) contacting the mammalian cell with salicylic acid to modulate gene expression, as stated supra, the specification discloses no methods for modulating gene expression. Applicant’s specification describes no methods of comprising (a) a target gene and Applicant’s specification describes no method for making and using (b) a chimeric protein comprising a transcription modulating protein or functional domain thereof with a salicylic acid binding domain from a NPR protein.
However, Applicant’s specification does describe a method of contacting a mammalian cell with specific concentrations of SA (about 2.5 mM) to induce nuclear translocation of a limited genus of mCherry-NPR1 fusion proteins. Specifically, Applicant’s specification only describes a method using the NPR1 gene from Arabidopsis thaliana. Furthermore, as far as functional domains of the NPR1 protein, Applicant’s specification only disclose the TAD domain from amino acids 513-593 from Arabidopsis thaliana NPR1.
Importantly, Applicant’s specification indicates that the N-terminal or C-terminal position of the NRP1 protein or its TAD domain in the chimeric molecules can significantly influence nuclear translocation. Specifically, Table 1 of the disclosure (see below) demonstrates that the mCherry-NPR1 chimeric molecule exhibited “No translocation”, while the mCherry-NPR1-TAD, NPR1-TAD-mCherry and NPR1-mCherry did to varying degrees depending on the concentration of SA used (see Fig. S4). Thereby indicating that position of the transcription modulating protein relative to the NPR1 protein is critical to nuclear translocation.
PNG
media_image1.png
745
1333
media_image1.png
Greyscale
Furthermore, Applicant’s arguments filed 7/27/2026 indicate that the “exact molecular mechanism and structural outcome of SA binding to plant NPR1 was highly controversial and poorly understood”. Applicant argues that those skilled in the art “would not expect the use of NPR1 or its transactivation domain (TAD) in mammalian cells would result in an intact, functional, SA-inducible nuclear translocator.” Accordingly, Applicant did not demonstrate a reduction to practice of a method of modulating gene expression in a mammalian cell in vitro or in vivo, nor did Applicant demonstrate using any engineered mammalian cell with any chimeric molecule how to make and use the claimed invention.
STATE OF THE ART & QUANTITY OF EXPERIMENTATION
The method of practicing the claimed invention is not well established. Although the plant NPR genes, transcription modulating proteins (e.g, Cas9 and TALENs) and the transactivation domains (e.g., VP16) were known in the state of the art, one of skill in the art would neither expect nor predict a successful outcome to modulate gene expression in a mammalian cell in vitro or in vivo by administering salicylic acid to the claimed genus of chimeric molecules.
The following describes the state of the art with respect to salicylic acid regulation of NPR1. Specifically, the prior art of Fu et al. (Nature, 2012, 486:228-231, see IDS filed 7/27/2026) examines the nuclear translocation of Arabidopsis NRP1 in plant cells, and teaches that SA controls the nuclear translocation of NPR1 through cellular redox changes, wherein NPR1 is retained in the cytoplasm as an oligomer through redox-sensitive intermolecular disulphide bonds. After induction, these disulphide bonds are reduced, releasing NPR1 monomers into the nucleus, where NPR1 acts as a cofactor for transcription factors (p. 228, 3rd para.). Importantly, NPR1 does not bind DNA on its own. Thus, one of ordinary skill would not have predicted that a chimeric protein comprising NRP1 alone would have been able to modulate gene expression. Furthermore, as noted by Applicant’s remarks, one of ordinary skill would not have expected that the same redox conditions would have existed in mammalian cells so as to allow NPR1 to translocate to the nucleus.
The following describes the state of the art with respect to transcription modulating proteins or functional domains thereof. Specifically, the prior art of Konermann et al., (Nature, 2015, 517:583-588) indicates that conventional transcription modulating proteins such as dCas9 directed by a sgRNA typically achieve little to no transcriptional regulation (p. 583, 2nd para.). Konermann teaches the dCas9 complex must be optimized to include other adaptor proteins to recruit a transcriptional modulating complex (p. 583, last para., see Fig. 2B). Furthermore, despite engineering the sgRNA to incorporate protein-interacting aptamers to predictably allow modulated gene expression, Konermann acknowledges that future studies must address “additional selection criteria that are critical for guide efficacy” (p. 587, Discussion, last para.). For example, Konermann teaches that sgRNA must be screened across at least 1000 bp of a target gene’s transcriptional start site (TSS) to find optimal sgRNA that can modulate gene expression (p. 584, last two para.). Furthermore, Konermann discusses other programable DNA-binding proteins such as TALENS (p. 583, 2nd para.), but dismisses these as difficult to be scaled to facilitate genomic perturbation because each and every TALEN must be individually designed and empirically tested for binding a specific regulatory region in a target gene. Thus, one of ordinary skill would not have predicted that a chimeric protein comprising NRP1 with any transcription modulating protein or functional fragment thereof would have been able to modulate gene expression.
Finally, with regard to using salicylic acid to modulate gene expression in a mammalian cell in vivo, it is well known that a serum level of 100 mg/dL (which is about 7 mM) is generally classified as a critical threshold associated with severe toxicity in acute ingestions of salicylates (e.g., aspirin). Thus, since Applicant’s method requires 2.5 mM for at least 30 minutes to see a translocation of a chimeric mCherry-NPR1 fusion protein to the nucleus of a mammalian cell, an effective dose of salicylic acid to achieve the claimed method in vivo has not been described.
Since the prior art at the effective filing date of the present application did not provide guidance for modulating gene express according to the claimed method, it is incumbent upon the instant specification to do so. The physiological art is recognized as unpredictable (MPEP 2164.03). As set forth in In re Fisher, 166 USPQ 18 (CCPA 1970), compliance with 35 USC 112, first paragraph requires: “That scope of claims must bear a reasonable correlation to scope of enablement provided by specification to persons of ordinary skill in the art; in cases involving predictable factors, such as mechanical or electrical elements, a single embodiment provides broad enablement in the sense that, once imagined, other embodiments can be made without difficulty and their performance characteristics predicted by resort to known scientific laws; in cases involving unpredictable factors, such as most chemical reactions and physiological activity, scope of enablement varies inversely with degree of unpredictability of factors involved.” Moreover, the courts have also stated that reasonable correlation must exist between scope of exclusive right to patent application and scope of enablement set forth in the patent application (27 USPQ2d 1662 Ex parte Maize!.). In view of the foregoing, due to the lack of sufficient guidance provided by the specification regarding the issues set forth above, the state of the relevant art, and the breadth of the claims, it would have required undue experimentation for one skilled in the art to make and use the instant broadly claimed invention.
CONCLUSION
In conclusion, since the art teaches that success of said method is prone to influence by multiple factors, and is highly unpredictable with respect to modulating gene expression in mammalian cells in vitro and in vivo with chimeric molecules by administering salicylic acid, and the specification does not provide ample guidance with respect to achieving the unexpected results, one would be burdened with undue experimentation to use the claimed invention.
New Claim Rejections - 35 USC § 112(b)
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.
Claims 20-33 and 38 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 pre-AIA the applicant regards as the invention.
Claim 20 recites the limitation "the" salicylic acid responsive transcription modulating protein. There is insufficient antecedent basis for this limitation in the claim. Dependent claims are included in the basis of this rejection because they do not clarify the salicylic acid responsive transcription modulating protein.
Claim 20 recites the broad recitation for the chimeric molecule comprising “a functional domain of (a) salicylic acid responsive transcription modulating protein” and the claim also recites the chimeric molecule comprises “a salicylic acid binding domain from a plant non-expressor of pathogenesis-related gene (NPR)”, which is the narrower statement of the limitation. A broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) is considered indefinite, since the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). Dependent claims are included in the basis of this rejection because they do not clarify the chimeric molecule.
Claim 23 recites the limitation "the" transactivation domain. There is insufficient antecedent basis for this limitation in the claim.
New Claim Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 31 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Specifically, claim 31 draws to a method modulating gene expression in a eukaryotic cell, which does NOT narrow the scope of claims 20 and 21where the cell is a mammalian cell. Applicant may cancel the claim, amend the claim to place the claim in proper dependent form, rewrite the claim in independent form, or present a sufficient showing that the dependent claim complies with the statutory requirements.
New Claim Objections
Claims 1 and 20 are objected to because of the following informalities: instant claims use the phrase “functional fragment” and “functional domain” interchangeably. Applicant should be consistent with their terminology. As the phrase “functional fragment” is not found in the specification, the Examiner recommends using “functional domain” throughout.
New Objection to Specification
Applicant uses the alpha/numeric identifiers of S1-S4 for Figures 9-12. All sheets of drawings must be numbered in the center of either the top or the bottom of each sheet but not in the margin in numbers larger than those used as reference signs in order to avoid confusion with the latter. For drawings, a separate series of page numbers is to be used. The number of each sheet of the drawings must consist of two Arabic numerals separated by an oblique stroke, the first being the sheet number and the second being the total number of sheets of drawings. For example, “2/5” would be used for the second sheet of drawings where there are five in all.
Different figures on the sheets of drawings must be numbered in Arabic numerals consecutively and independently of the numbering of the sheets and, if possible, in the order in which they appear. This numbering should be preceded by the expression “Fig.”
Conclusion
No claims are allowed.
Examiner Contact Information
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ARTHUR S LEONARD whose telephone number is (571)270-3073. The examiner can normally be reached on Mon-Fri 9am-5pm.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, James Doug Schultz can be reached on 571-272-0763. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of an application may be obtained from the Patent Application Information Retrieval (PAIR) system. Status information for published applications may be obtained from either Private PAIR or Public PAIR. Status information for unpublished applications is available through Private PAIR only. For more information about the PAIR system, see http://pair-direct.uspto.gov. Should you have questions on access to the Private PAIR system, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative or access to the automated information system, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/ARTHUR S LEONARD/Examiner, Art Unit 1631
