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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR
1.17(e), was filed in this application after final rejection. Since this application is eligible for continued
examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the
finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's
submission filed on 11/20/2025 has been entered.
Priority
Acknowledgement is made of applicant’s claim for benefit under 35 U.S.C. 119(e). As such the effective filing date of Claims 1-18 is 13 March 2020.
Status of the Claims
Amendments dated 11/07/2025 have been entered.
Claims 1-18 are pending.
Claims 1-18 are examined herein.
The rejection of Claim 18 under 35 U.S.C. 103 as being unpatentable over Streitner et al. (WO 2019/185609 A1, published 10/03/2019; IDS Document) in view of Abbitt et al. (WO 2018/183878 A1; published 10/04/2018), Marand et al. ( Biochimica et Biophysica Acta (BBA)-Gene Regulatory Mechanisms 1860.1 (2017): 131-139), and Kiran et al. (Plant physiology 142.1 (2006): 364-376) is withdrawn in view of Applicant’s Remarks (pgs. 8-9) dated 11/07/2025 which were found persuasive.
Nucleotide and/or Amino Acid Sequence Disclosures
Specific deficiency – Nucleotide and/or amino acid sequences appearing in the specification [See pgs. 5, 25, 44, 50; Table 1, Table 2, Table 3, Table 6, Table 7, Table 9, Table 13, Table 14 for any nucleic acid with at least 10 nucleotides having at least 4 of the nucleotides NOT designated as “n” and any peptide/protein with at least 4 defined amino acids (meaning NOT “X”)] are not identified by sequence identifiers in accordance with 37 CFR 1.821(d).
Required response – Applicant must provide:
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required sequence identifiers, consisting of:
A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
A copy of the amended specification without markings (clean version); and
A statement that the substitute specification contains no new matter.
Specific deficiency - This application contains sequence disclosures in accordance with the definitions for nucleotide and/or amino acid sequences set forth in 37 CFR 1.821(a)(1) and (a)(2). However, this application fails to comply with the requirements of 37 CFR 1.821 - 1.825.
The sequence disclosures are located on pgs. 5, 25, 44, 50; Table 1, Table 2, Table 3, Table 6, Table 7, Table 9, Table 13, Table 14 —any nucleic acid with at least 10 nucleotides having at least 4 of the nucleotides NOT designated as “n” and any peptide/protein with at least 4 defined amino acids (meaning NOT “X”)] .
Required response – Applicant must provide:
A "Sequence Listing" part of the disclosure, as described above in item 1); as well as
An amendment specifically directing entry of the "Sequence Listing" part of the disclosure into the application in accordance with 1.825(b)(2);
A statement that the "Sequence Listing" includes no new matter in accordance with 1.825(b)(5); and
A statement that indicates support for the amendment in the application, as filed, as required by 37 CFR 1.825(b)(4).
If the "Sequence Listing" part of the disclosure is submitted according to item 1) a) or b) above, Applicant must also provide:
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required incorporation-by-reference paragraph, consisting of:
A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
A copy of the amended specification without markings (clean version); and
A statement that the substitute specification contains no new matter;
If the "Sequence Listing" part of the disclosure is submitted according to item 1) b), c), or d) above, Applicant must also provide:
A replacement CRF in accordance with 1.825(b)(6); and
Statement according to item 2) a) or b) above.
Claim Objections
Claim 7 is objected to because of the following informalities:
Claim 7, line 2 should be amended to recite “…comprising the sequence “TATA” from a…”
Appropriate correction is required.
Claim Rejections - 35 USC § 112
New Matter
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.
---This is a new rejection from those set forth in the Office Action dated 09/08/2025 necessitated by Applicant’s amendments to the claims dated 11/07/2025.---
Claims 1-18 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 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. All dependent claims are included in these rejections unless they contain a limitation that overcomes the deficiencies of the parent claim from which they depend.
Claims 1, 8-15, and 17-18 have been amended to require that the modified TATA box and the heterologous expression modulating element are functionally linked to cooperatively modulate expression of an endogenous polynucleotide.
While the function of modulating expression of an endogenous polynucleotide by a modified TATA box and a functionally linked expression modulating element was originally described in the instant specification, this particular type of modulation of expression through the functionally linked compositions of matter (unspecified “cooperatively”) was not envisioned in the application as originally filed. There is not support in the specification for the amended claim limitation. There is not even a single mention in the disclosure of the term “cooperatively”, nor does there appear to be any implicit support that Applicant’s envisioned a method of modulating expression of an endogenous polynucleotide that has this particular and unspecified type of modulation of expression. Therefore, the claims are rejected for introducing new matter in the application.
Claim Rejections - 35 USC § 112
Indefiniteness
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.
---This is a new rejection from those set forth in the Office Action dated 09/08/2025 necessitated by Applicant’s amendments to the claims dated 11/07/2025.---
Claims 1-18 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention. All dependent claims are included in these rejections unless they contain a limitation that overcomes the deficiencies of the parent claim from which they depend.
The recitation of “cooperatively” in Claims 1, 8-15, and 17-18 renders the claims indefinite. As discussed in the 35 U.S.C. 112(a) new matter rejection above, neither the instant specification nor the instant claims provide any disclosure or definition of the term “cooperatively” in such a way that one of ordinary skill in the art would be reasonably apprised of the metes and bounds of the claim limitation. It is unclear what degree of expression modulation is required for the expression to be considered cooperative based on the relationship of the functionally linked modified TATA box and heterologous EME. Additionally, it is unclear how one of ordinary skill in the art would be able to determine if the modulation of expression is not “cooperatively” occurring based on the relationship of the functionally linked modified TATA box and heterologous EME. How is the term “cooperatively” measured to determine if the modulation of expression has occurred cooperatively? For purposes of examination, this claim limitation will be interpreted to encompass any degree of expression modulation (i.e., increase or decrease) in a plant cell comprising a modified TATA box in a genomic locus and a heterologous EME. This determination does not alleviate Applicant’s duty to amend the claim in any further correspondence.
Claim Rejections - 35 USC § 103
---These are modified rejections from those set forth in the Office Action dated 09/08/2025 necessitated by Applicant’s amendments to the claims dated 11/07/2025.---
Claims 1-17 are rejected under 35 U.S.C. 103 as being unpatentable over Streitner et al. (WO 2019/185609 A1, published 10/03/2019; IDS Document) in view of Abbitt et al. (WO 2018/183878 A1; published 10/04/2018) and Marand et al. (Biochimica et Biophysica Acta (BBA)-Gene Regulatory Mechanisms 1860.1 (2017): 131-139).
Regarding Claim 1, Streitner et al. (herein referred to as Streitner) teaches a method for increasing the expression level of a nucleic acid molecule of interest in a cell, preferably a plant cell (Abstract, Claim 1), wherein the expression of an endogenous nucleic acid molecule is increased up to many-fold of what is achieved under the control of a promoter without the introduced activating elements (pg. 3, second paragraph). In the method for increasing the expression level of a nucleic acid molecule of interest in a cell, Streitner teaches a cell (preferably a plant cell) comprising introducing into the cell a promoter activating nucleic acid sequence (Claim 1, part ia), and in one embodiment of the invention, inserting the promoter activating nucleic acid sequence into a recipient promoter controlling the expression of the nucleic acid molecule of interest in the cell at a position upstream of the transcription start site of the recipient promoter controlling the expression of the nucleic acid molecule of interest (Claim 1, part iiia). In one embodiment of the invention, Streitner teaches modifying one or more TATA box motif(s) present in the promoter activating nucleic acid sequence inserted or introduced in step iiia) or present in the recipient promoter by addition and/or substitution and/or deletion of one or more nucleotides for converting the one or more TATA box motif(s) into one or more TATA box motif(s) having increased or higher relative score(s) when matching or aligning the one or more modified TATA box motif(s) to the TATA box consensus (Claim 1, part iiic).
Regarding Claim 8, Streitner teaches a method for increasing the expression level of a nucleic acid molecule of interest in a cell, preferably a plant cell (Abstract, Claim 1), wherein the expression of an endogenous nucleic acid molecule is increased up to many-fold of what is achieved under the control of a promoter without the introduced activating elements (pg. 3, second paragraph). In the method for increasing the expression level of a nucleic acid molecule of interest in a cell, Streitner teaches a cell (preferably a plant cell) comprising introducing into the cell a promoter activating nucleic acid sequence (Claim 1, part ia), and in one embodiment of the invention, inserting the promoter activating nucleic acid sequence into a recipient promoter controlling the expression of the nucleic acid molecule of interest in the cell at a position upstream of the transcription start site of the recipient promoter controlling the expression of the nucleic acid molecule of interest (Claim 1, part iiia). In one embodiment of the invention, Streitner teaches modifying one or more TATA box motif(s) present in the recipient promoter by addition and/or substitution and/or deletion of one or more nucleotides for converting the one or more TATA box motif(s) into one or more TATA box motif(s) having increased or higher relative score(s) when matching or aligning the one or more modified TATA box motif(s) to the TATA box consensus (Claim 1, part iiic).
Regarding Claim 9, Streitner teaches a method for increasing the expression level of a nucleic acid molecule of interest in a cell, preferably a plant cell (Abstract, Claim 1), wherein the expression of endogenous or exogenous nucleic acid molecules is increased up to many-fold of what is achieved under the control of a promoter without the introduced activating elements (pg. 3, second paragraph). In the method for increasing the expression level of a nucleic acid molecule of interest in a cell, Streitner teaches a cell (preferably a plant cell) comprising introducing into the cell a promoter activating nucleic acid sequence, a chimeric promoter, a delivery system, or a nucleic acid construct or expression cassette (Claim 1, part ia), or introducing into the cell means for site-specific modification of the nucleic acid sequence of a recipient promoter controlling the ex pression of the nucleic acid molecule of interest (Claim 1, part ib), and optionally, introducing into the cell a site-specific nuclease or an active fragment thereof, or providing the sequence encoding the same, the site-specific nuclease inducing a double-strand break at a predetermined location, preferably wherein the site-specific nuclease or the active fragment thereof comprises a zinc-finger nuclease, a transcription activator-like effector nuclease, a CRISPR/Cas system, including a CRISPR/Cas9 system, a CRISPR/Cpf1 system, a CRISPR/C2C2 system a CRISPR/CasX system, a CRISPR/CasY system, a CRISPR/Cmr system, an engineered homing endonuclease, a recombinase, a transposase and a meganuclease, and/or any combination, variant, or catalytically active fragment thereof; and optionally when the site-specific nuclease or the active fragment thereof is a CRISPR nuclease: providing at least one guide RNA or at least one guide RNA system, or a nucleic acid encoding the same (Claim 1, part ii), and inserting the promoter activating nucleic acid sequence into a recipient promoter controlling the expression of the nucleic acid molecule of interest in the cell at a position upstream or downstream of the transcription start site of the recipient promoter controlling the expression of the nucleic acid molecule of interest (Claim 1, part iiia). In one embodiment of the invention, Streitner teaches modifying one or more TATA box motif(s) present in the recipient promoter by addition and/or substitution and/or deletion of one or more nucleotides for converting the one or more TATA box motif(s) into one or more TATA box motif(s) having increased or higher relative score(s) when matching or aligning the one or more modified TATA box motif(s) to the TATA box consensus (Claim 1, part iiic).
Regarding Claim 10, Streitner teaches a delivery system that comprises the promoter activating nucleic acid sequence and/or the chimeric promoter, and/or means for site-specific insertion or introduction of the promoter activating nucleic acid sequence into a recipient promoter, wherein the nucleic acid construct or expression cassette comprises the promoter activating nucleic acid sequence and/or the chimeric promoter (Claim 1, part (c)(iii)). In one embodiment of the invention, Streitner teaches modifying one or more TATA box motif(s) present in the recipient promoter by addition and/or substitution and/or deletion of one or more nucleotides for converting the one or more TATA box motif(s) into one or more TATA box motif(s) having increased or higher relative score(s) when matching or aligning the one or more modified TATA box motif(s) to the TATA box consensus (Claim 1, part iiic).
Regarding Claim 11, Streitner teaches method for increasing the expression level of a nucleic acid molecule of interest in a cell, preferably a plant cell (Claim 1), wherein the cell or organism is a plant cell or plant, and/or wherein the recipient promoter and/or the donor promoter is/are a plant promoter, and/or wherein the recipient promoter and the donor promoter are different (Claim 3), wherein the promoter activating nucleic acid sequence comprising one or more contiguous stretch(es) of nucleotides isolated from a donor promoter, wherein the donor promoter is a promoter of a gene having a high expression level, and/or one or more TATA box motif(s) of a donor promoter or one or more TATA box motif(s) having a relative score of greater than 0.8 when matching or aligning the one or more TATA box motif(s) to TATA box consensus, and/or iii. comprises one or more pyrimidine patch (Y patch) promoter element(s) of a donor promoter (Claim 1). In one embodiment of the invention, Streitner teaches modifying one or more TATA box motif(s) present in the recipient promoter by addition and/or substitution and/or deletion of one or more nucleotides for converting the one or more TATA box motif(s) into one or more TATA box motif(s) having increased or higher relative score(s) when matching or aligning the one or more modified TATA box motif(s) to the TATA box consensus (Claim 1, part iiic).
Regarding Claim 12, Streitner teaches a method for increasing the expression level of a nucleic acid molecule of interest in a cell, preferably a plant cell (Abstract, Claim 1), wherein the expression of an endogenous nucleic acid molecule is increased up to many-fold of what is achieved under the control of a promoter without the introduced activating elements (pg. 3, second paragraph). In the method for increasing the expression level of a nucleic acid molecule of interest in a cell, Streitner teaches a cell (preferably a plant cell) comprising introducing into the cell a promoter activating nucleic acid sequence (Claim 1, part ia), and in one embodiment of the invention, inserting the promoter activating nucleic acid sequence into a recipient promoter controlling the expression of the nucleic acid molecule of interest in the cell at a position upstream of the transcription start site of the recipient promoter controlling the expression of the nucleic acid molecule of interest (Claim 1, part iiia). In one embodiment of the invention, Streitner teaches modifying one or more TATA box motif(s) present in the promoter activating nucleic acid sequence inserted or introduced in step iiia) or present in the recipient promoter by addition and/or substitution and/or deletion of one or more nucleotides for converting the one or more TATA box motif(s) into one or more TATA box motif(s) having increased or higher relative score(s) when matching or aligning the one or more modified TATA box motif(s) to the TATA box consensus (Claim 1, part iiic).
Regarding Claim 13, Streitner teaches a method for increasing the expression level of a nucleic acid molecule of interest in a cell, preferably a plant cell (Abstract, Claim 1), wherein the expression of an endogenous nucleic acid molecule is increased up to many-fold of what is achieved under the control of a promoter without the introduced activating elements (pg. 3, second paragraph). In the method for increasing the expression level of a nucleic acid molecule of interest in a cell, Streitner teaches a cell (preferably a plant cell) comprising introducing into the cell a promoter activating nucleic acid sequence (Claim 1, part ia), and in one embodiment of the invention, inserting the promoter activating nucleic acid sequence into a recipient promoter controlling the expression of the nucleic acid molecule of interest in the cell at a position upstream of the transcription start site of the recipient promoter controlling the expression of the nucleic acid molecule of interest (Claim 1, part iiia). In one embodiment of the invention, Streitner teaches modifying one or more TATA box motif(s) present in the promoter activating nucleic acid sequence inserted or introduced in step iiia) or present in the recipient promoter by addition and/or substitution and/or deletion of one or more nucleotides for converting the one or more TATA box motif(s) into one or more TATA box motif(s) having increased or higher relative score(s) when matching or aligning the one or more modified TATA box motif(s) to the TATA box consensus (Claim 1, part iiic).
Regarding Claim 14, Streitner teaches a method for increasing the expression level of a nucleic acid molecule of interest in a cell, preferably a plant cell (Claim 1), comprising introducing into the cell a promoter activating nucleic acid sequence, a chimeric promoter, a delivery system, or a nucleic acid construct or expression cassette (Claim 1, part ia), or introducing into the cell means for site-specific modification of the nucleic acid sequence of a recipient promoter controlling the ex pression of the nucleic acid molecule of interest (Claim 1, part ib), and optionally, introducing into the cell a site-specific nuclease or an active fragment thereof, or providing the sequence encoding the same, the site-specific nuclease inducing a double-strand break at a predetermined location, preferably wherein the site-specific nuclease or the active fragment thereof comprises a zinc-finger nuclease, a transcription activator-like effector nuclease, a CRISPR/Cas system, including a CRISPR/Cas9 system, a CRISPR/Cpf1 system, a CRISPR/C2C2 system a CRISPR/CasX system, a CRISPR/CasY system, a CRISPR/Cmr system, an engineered homing endonuclease, a recombinase, a transposase and a meganuclease, and/or any combination, variant, or catalytically active fragment thereof; and optionally when the site-specific nuclease or the active fragment thereof is a CRISPR nuclease: providing at least one guide RNA or at least one guide RNA system, or a nucleic acid encoding the same (Claim 1, part ii), and inserting the promoter activating nucleic acid sequence into a recipient promoter controlling the expression of the nucleic acid molecule of interest in the cell at a position upstream or downstream of the transcription start site of the recipient promoter controlling the expression of the nucleic acid molecule of interest (Claim 1, part iiia). Streitner also teaches a cell or organism or a progeny thereof or a part of the organism or progeny thereof, a) in which a promoter activating nucleic acid as defined in any one of claims 1 to 4 is inserted or introduced (Claim 9). In one embodiment of the invention, Streitner teaches a conversion of an original TATA box into a TATA box from an activating DNA element, wherein the original TATA-box of a promoter of interest (e.g. Zm-prom1 ) is exchanged for the specific TATA-box being part of a small activating DNA element (e.g. E59) wherein the exchange is positioned 23 bp upstream of the TSS (pg. 75, Example 6, paragraph 3). The modified promoter Zm-prom1v3 was 9.75-fold activated compared to the unmodified Zm-prom1 (pg. 75, Example 6, paragraph 5). Any plants regenerated from the corn transformants used in the method of Example 6 would be rendered obvious by the disclosure of the corn transformants in Example 6.
Regarding Claim 15, Streitner teaches a method for increasing the expression level of a nucleic acid molecule of interest in a cell, preferably a plant cell (Abstract, Claim 1), wherein the expression of endogenous or exogenous nucleic acid molecules is increased up to many-fold of what is achieved under the control of a promoter without the introduced activating elements (pg. 3, second paragraph), wherein the promoter activating nucleic acid sequence causes an increased expression of the nucleic acid molecule of interest upon site-specific insertion (Claim 1). In one embodiment of the invention, Streitner teaches base editing to modify an endogenous TATA box motif to generate sequence of an activating DNA element (Example 7, pgs. 75-76) wherein the base editing of the endogenous TATA box (modifying the endogenous TATA box) increased endogenous promoter transcript levels in the genome edited callus and subsequent regenerated corn shoots (Example 7, pg. 76) In Example 7, Streitner teaches that base editors coupled to a catalytically impaired Cas or Cpf1 effector, or any other CRISPR nuclease can mediate targeted transitions of C-G to T-A and of A-T to G-C by either using a cytosine deaminase or an adenine deaminase evolved to process DNA (Example 7, pg. 75; Figure 1(B1, B2); Figure 10C). In Example 8, Streitner teaches the conversion of original TATA-box into activating DNA elements, wherein the original TATA-box of a promoter of interest (e.g ZmZEPI ) is converted into small activating DNA element (e.g E59, E53f, E55a) by using site specific mutagenesis (base editing). The exchange was positioned ~33 bp upstream of the TSS (given with bases CAA in this case), wherein The effect is measured in a transient assay system based on corn leaf bombard- ment with respective promoter-reporter constructs followed by luciferase measurement (see Figure 10C).The modified promoter ZmZEPI v1 was activated 5,9-fold, the modified promoter ZmZEPI v2 was activated 15,6-fold and the modified promoter ZmZEPI v3 was acti- vated 10,5-fold compared to the unmodified ZmZEPI promoter (Example 8, pg. 77).
Regarding Claim 17, Streitner teaches a teaches a method for increasing the expression level of a nucleic acid molecule of interest in a cell, preferably a plant cell (Abstract, Claim 1), wherein the expression of endogenous or exogenous nucleic acid molecules is increased up to many-fold of what is achieved under the control of a promoter without the introduced activating elements (pg. 3, second paragraph), wherein the promoter activating nucleic acid sequence causes an increased expression of the nucleic acid molecule of interest upon site-specific insertion (Claim 1). In one embodiment of the invention, Streitner teaches modifying one or more TATA box motif(s) present in the promoter activating nucleic acid sequence inserted or introduced in step iiia) or iiib) or present in the recipient promoter by addition and/or substitution and/or deletion of one or more nucleotides for converting the one or more TATA box motif(s) into one or more TATA box motif{s) having increased or higher relative score{s) when matching or aligning the one or more modified TATA box motif{s) to the TATA box consensus (Claim 1, part iiic).
However, Streitner does not teach the feature of Claims 1, 8-15, and 17 wherein the regulatory region further comprises one or more copies of a heterologous expression modulating element, wherein the heterologous expression modulating element is at least 20 nucleotides upstream from the modified TATA box, wherein the modified TATA box and the heterologous expression modulating element are functionally linked to cooperatively modulate expression of the endogenous polynucleotide or the feature of Claim 6, wherein the expression modulating element is within 250 nucleotides from the TATA box.
Regarding Claims 1, 8-15, and 17, Abbitt et al. (herein referred to as Abbitt) teaches a method of modulating expression of an endogenous polynucleotide in a plant cell, the method comprising altering one or more nucleotides in a regulatory region of the endogenous polynucleotide such that the regulatory region of the polynucleotide comprises an expression modulating element having at least one copy of a polynucleotide sequence selected from the group consisting of SEQ ID NOS: 1-68 (SEQ IDs for expression modulating elements) (Claim 1), wherein the alteration of one or more nucleotides is by genome modification (Claim 2), wherein the expression modulation element is present within about 10 to about 5000 bp from a transcriptional start site of the endogenous polynucleotide (Claim 3), wherein the expression modulation element further comprises additional copies of the expression modulating element such that about 2X to 10X copies of the expression modulating elements are present in the regulatory region of the endogenous polynucleotide (Claim 4), wherein the expression modulation element further comprises additional copies of the expression modulating element such that about 2X to 10X copies of the expression modulating elements are present in the regulatory region of the endogenous polynucleotide (Claim 5). Additionally, Abbitt defines expression modulating elements to be “a nucleotide sequence that up or down-regulates the expression of one or more plant genes. EME may have one or more copies of the same sequence arranged head-to-head, tail-to- head, or head-to-tail or a combination thereof configurations. EMEs are derived from plant sequences, or from bacterial or viral enhancer elements” (pg. 11, lines 11-15) , wherein an enhancer element “may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter” (pg. 11, lines 28-29) .
Regarding Claims 1, 6, 8-15, and 17, in one embodiment of the invention, Abbitt teaches that 4 copies of EME1 (expression modulating element) (SEQ ID NO: 1 ) or 1 copy of EME2 (SEQ ID NO: 5) were inserted 20 base pairs upstream of the TATA box (Location 2 in Figure 1) in a maize protoplast cell. Abbitt teaches that when the EME1 sequence is inserted in the promoter region (either 520 or 20 bp upstream of the TATA box within the promoter) (paragraph bridging pgs. 52-53), the expression levels of the reporter gene are significantly increased, while insertion in the other 5 locations within the expression cassette resulted in a significant decrease of the expression of the reporter gene. A significant increase in gene expression was observed when EME2 (SEQ ID NO: 5) was inserted in locations within the promoter (Locations 1 and 2 in Figure 1; both 520 and 20 bp upstream of the TATA box in the promoter) (paragraph bridging pgs. 52-53).
Regarding the limitation of Claims 1, 8-15, and 17, wherein the modified TATA box and the heterologous expression modulating element are require to be “functionally linked”, the instant specification defines “functionally linked” as “…the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other.” (pg. 15, lines 25-27). The broadest reasonable interpretation of the term “ functionally linked” in view of the Applicant’s disclosure, encompasses any nucleotide sequence(s) that have some unspecified degree of an effect on the function of other nucleotide sequence(s). In light of this interpretation of the broad term “functionally linked”, Abbitt teaches in Example 8 (pg. 55; Figure 4) that the insertion of an EME 20 bp upstream of the TATA box of the promoter (ZmGOS2) resulted in the greatest increase in promoter expression compared to all other insertion locations. As such, Abbitt teaches that the EME inserted 20 base pairs upstream of the TATA box of the ZmGOS2 promoter inherently has some degree of functional linkage to the ZmGOS2 TATA box, because the presence of the EME affected the function (cooperatively facilitated expression) of the ZmGOS2 promoter by increasing the expression of ZmGOS2, which means (insofar as the Applicant’s has defined “functionally linked”) the function of the ZmGOS2 promoter’s TATA box was affected to some degree as well.
Marand et al. (2017) teaches that enhancers are important cis-regulatory DNA elements that regulate transcription programs by recruiting transcription factors and directing them to the promoters of target genes in a cell-type/tissue-specific manner. The expression of a gene can be regulated by one or multiple enhancers at different developmental stages and/or in different tissues (Abstract). Marand et al. (2017) also teaches that newer generation enhancer traps contain a minimal promoter including a TATA box and a transcription start site, which is fused to the 5′ end of a reporter gene, typically β-galactosidase (GUS) or green fluorescent protein (GFP) (pg. 135, left column, 2nd full paragraph).
It would have been obvious to one of ordinary skill in the art at the time of the effective filing date of the claimed invention to introduce the expression modulating elements taught by Abbitt exactly 20 nucleotides upstream from the modified TATA-box taught by Streitner. Based on the teachings of Marand, that explicitly teaches it is routine in the art to include enhancer sequences to regulate gene expression in programs designed to modulate transcription of genes of interest, a person of ordinary skill in the art would have been motivated to include the expression modulating elements taught by Abbitt in the regulatory region that comprises the modified TATA-box taught by Streitner because it is routine in the art to include enhancer elements in a regulatory region that comprises a TATA box, to modulate the expression of a gene in a plant cell. Additionally, based on the teachings of Abbitt, one of ordinary skill in the art would have been motivated to include the expression modulating element at least 20 base pairs upstream of the modified TATA box taught by Streitner, because Abbitt explicitly recites that EME placement as a functional embodiment of the invention with a reasonable expectation of success in modulating the expression of an endogenous gene of interest (Abbitt, Figure 1; pg. 53, lines 2-7). Furthermore, one of ordinary skill in the art would have been motivated to introduce the expression modulating element taught by Abbitt exactly 20 base pairs upstream of the modified TATA box taught by Streitner, because Abbitt explicitly teaches that inserting an EME precisely 20 base pairs upstream of the TATA box provides the greatest amount of increased expression (modulation of expression) of a gene of interest— specifically an endogenous gene of interest (ZmGOS2 promoter) relative to the plant cell species (maize protoplast) used in the methods taught by Abbitt (Abbitt; Example 8, Figure 4). At the most basal level, the teachings of Abbitt and Streitner are both directed to methods of modulating expression of modulating expression of an endogenous polynucleotide in a plant cell, and because adding enhancer elements to the regulatory region of a gene in a plant cell is well known in the art as evidenced by Marand, one of ordinary skill in the art would have been further motivated to include the EME taught by Abbitt in the regulatory region comprising the modified TATA box taught by Streitner to modulate gene expression, specifically no less than 20 nucleotides from the modified TATA-box in the case of increasing gene expression. The rationale to support a conclusion that the claims would have been obvious is that all the claimed elements were known in the prior art, and one of ordinary skill could have combined these elements as claimed with no change to their respective functions. Thus the combination yielding predictable results would have been expected by a skilled artisan.
Regarding Claim 2, Streitner teaches that for the step of method comprising inserting the promoter activating nucleic acid sequence into a recipient promoter controlling the expression of the nucleic acid molecule of interest in the cell at a position upstream of the transcription start site of the recipient promoter controlling the expression of the nucleic acid molecule of interest, in one embodiment of the invention the promoter activating nucleic acid sequence inserted or introduced during this step can be “CTATATATA” (E59b) (pg. 32, paragraph 1).
Regarding Claim 3, Streitner teaches that for the step of method comprising inserting the promoter activating nucleic acid sequence into a recipient promoter controlling the expression of the nucleic acid molecule of interest in the cell at a position upstream or downstream of the transcription start site of the recipient promoter controlling the expression of the nucleic acid molecule of interest, in one embodiment of the invention the promoter activating nucleic acid sequence inserted or introduced during this step can be “CTATATATA” (E59b) (pg. 32, paragraph 1).
Regarding Claim 4, Streitner teaches the insertion or modification to introduce the promoter activating nucleic acid sequence into the recipient promoter in step iiia) is at a position 500 nucleotides or less, preferably 150 nucleotides or less upstream of the transcription start site of the nucleic acid molecule of interest (Claim 1, pg. 801, part a).
Regarding Claim 5, in one embodiment of the invention, Streitner teaches a conversion of an original TATA box into a TATA box from an activating DNA element, wherein the original TATA-box of a promoter of interest (e.g. Zm-prom1 ) is exchanged for the specific TATA-box being part of a small activating DNA element (e.g. E59) wherein the exchange is positioned 23 bp upstream of the TSS (pg. 75, Example 6, paragraph 3).
Regarding Claim 7, in one embodiment of the invention, Streitner teaches a conversion of an original TATA box into a TATA box from an activating DNA element, wherein the original TATA-box of a promoter of interest (e.g. Zm-prom1 ) is exchanged for the specific TATA-box being part of a small activating DNA element (e.g. E59) wherein the exchange is positioned 23 bp upstream of the TSS (given with bases TGA in this case) (pg. 75, Example 6, paragraph 3).
Regarding Claim 16, in one embodiment of the invention, Streitner teaches that the promoter activating nucleic acid sequence described in any of the embodiments above comprises one or more TATA box motif(s) of the donor promoter or one or more TATA box motif(s) having a relative score of greater than 0.8, greater than 0.81 , greater than 0.81 , greater than 0.82, greater than 0.83, greater than 0.84, greater than 0.85, greater than 0.86, greater than 0.87, greater than 0.88, greater than 0.89, greater than 0.90, greater than 0.91 , greater than 0.92, greater than 0.93, greater than 0.94, greater than 0.95, greater than 0.96, greater than 0.97, greater than 0.98, or greater than 0.99 when matching or aligning the one or more TATA box motif(s) to TATA box consensus (pg. 31, paragraph 1).
Therefore, for all the reasons above, the claimed invention is prima facie obvious.
Response to Arguments
Applicant’s Remarks on pgs. 6-8 in the reply filed on 11/07/2025 are acknowledged but do not overcome these modified rejections. In particular, Applicant’s rely on the premise that Streitner, Abbitt, or Marand, alone or in any combination do not teach or suggest a functional integration of the modified TATA box and the expression modulating element (EME) within the same regulatory region or that "... the heterologous expression modulating element is at least 20 nucleotides upstream from the modified TATA box....", wherein the claimed invention is not a mere aggregation of known elements, but a configuration requiring functional integration of a modified TATA box and an EME that results in expression outcomes (Remarks pg. 6).
This is not found persuasive as Applicant is reading limitations into the claims that do not exist in their currently amended form. Newly amended Claims 1, 8-15, and 17-18 merely require that the modified TATA box and the heterologous expression modulating element are “functionally linked”. Applicant has defined the term “functionally linked” in the instant specification as “…the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other.” (pg. 15, lines 25-27). Therefore, the broadest reasonable interpretation of the term “functionally linked” in view of the Applicant’s disclosure encompasses any nucleotide sequence(s) that have some unspecified degree of an effect on the function of other nucleotide sequence(s), not “functional integration”. As stated before, Streitner explicitly teaches modifying one or more TATA box motif(s) that are already present in the recipient promoter in a plant cell (endogenous polynucleotide) by addition and/or substitution and/or deletion of one or more nucleotides for converting the one or more TATA box motif(s) into one or more TATA box motif(s) having increased or higher relative score(s) when matching or aligning the one or more modified TATA box motif(s) to the TATA box consensus (Streitner, Claim 1, part iiic). A fuller review of the prior art also teaches functional linkage at least as disclosed in the present application, wherein Abbitt teaches functional linking between a heterologous EME that is 20 base pairs upstream of TATA box, wherein the presence of the EME taught by Abbitt affected the function (cooperatively facilitated expression) of the ZmGOS2 promoter by increasing the expression of ZmGOS2, which means (insofar as the Applicant’s has defined “functionally linked”) the function of the ZmGOS2 promoter’s TATA box was affected to some degree as well (Abbitt, Example 8, pg. 55; Figure 4). The motivation to combine these elements into a single invention is presented in the modified 35 U.S.C. 103 rejection above.
Applicant also argues that Streitner does not teach or suggest altering one or more nucleotides in a regulatory region of the genomic locus comprising the endogenous polynucleotide to create a modified TATA box in the regulatory region, wherein the regulatory region further comprises one or more copies of a heterologous expression modulating element, wherein the modified TATA box and the heterologous expression modulating element are functionally linked to cooperatively modulate expression of the endogenous polynucleotide, and wherein the heterologous expression modulating element is at least 20 nucleotides upstream from the modified TATA box (Remarks, pg. 7)
This is not found persuasive. MPEP 2145 Section IV states that “One cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., Inc., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). First, Streitner explicitly teaches modifying one or more TATA box motif(s) present in a promoter activating nucleic acid sequence inserted or introduced to a plant cell or a TATA box that is already present in the recipient promoter (endogenous genomic locus) by addition and/or substitution and/or deletion of one or more nucleotides for converting the one or more TATA box motif(s) (endogenous TATA box motif located in an endogenous genomic locus) into one or more TATA box motif(s) (modified TATA box motif) having increased or higher relative score(s) when matching or aligning the one or more modified TATA box motif(s) to the TATA box consensus (Streitner, Claim 1, part iiic). As such, even if Streitner does not teach the instantly claimed “heterologous expression modulating element, wherein the modified TATA box and the heterologous expression modulating element are functionally linked to cooperatively modulate expression of the endogenous polynucleotide, wherein the heterologous expression modulating element is at least 20 nucleotides upstream from the modified TATA box” it is the combination of Streitner and Abbitt, with a motivation to combine taught by Marand, that teaches the claimed elements.
Applicant also argues that Abbitt discusses EMEs and their positioning relative to Transcription Start Site (TSS), but not its functional integration with modified TATA box (Remarks, pg. 7).
This is not found persuasive. MPEP 2145 Section IV states that “One cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., Inc., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). As such, it is the combination of Streitner and Abbitt, with a motivation to combine taught by Marand, that teaches the claimed elements. Additionally, Abbitt explicitly teaches expression modulating elements positioning relative to TATA boxes (See Abbitt, paragraph bridging pgs. 52-53). Regarding Applicant’s argument that Abbitt does not teach EMEs functional integration with modified TATA box— Applicant is reading limitations into the claims that do not exist in their currently amended form. Newly amended Claims 1, 8-15, and 17-18 merely require that the modified TATA box and the heterologous expression modulating element are “functionally linked”. Applicant has defined the term “functionally linked” in the instant specification as “…the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other.” (pg. 15, lines 25-27). Therefore, the broadest reasonable interpretation of the term “functionally linked” in view of the Applicant’s disclosure encompasses any nucleotide sequence(s) that have some unspecified degree of an effect on the function of other nucleotide sequence(s), not “functional integration”.
Applicant argues that Marand discusses enhancer function and minimal promoters but lacks specific teachings or enabling disclosure on engineered regulatory constructs (Remarks, pg. 7).
This is not found persuasive. MPEP 2145 Section IV states that “One cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., Inc., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). As such, it is the combination of Streitner and Abbitt, with a motivation to combine taught by Marand, that teaches the claimed elements.
Applicant argues that “there is no clear reasoning for combining Streitner with Abbitt and Marand, and that using an applicant's disclosure as a blueprint to reconstruct the claimed invention from isolated pieces of the prior art contravenes the statutory mandate of § 103 which requires judging obviousness at the point in time when the invention was made. See Grain Processing Corp. v. American Maize-Prods. Co., 840 F.2d 902,907 (Fed. Cir. 1988)” (Remarks, pg. 11, bottom of first full paragraph).
This is not found persuasive. In response to Applicant's argument that the examiner's conclusion of obviousness is based upon improper hindsight reasoning, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971). Applicant fails to acknowledge the rationale to combine Streitner and Abbitt in view of the teachings of Marand. Marand et al. (2017) teaches that enhancers are important cis-regulatory DNA elements that regulate transcription programs by recruiting transcription factors and directing them to the promoters (which comprise TATA boxes) of target genes in a cell-type/tissue-specific manner. The expression of a gene can be regulated by one or multiple enhancers at different developmental stages and/or in different tissues (Abstract). Marand et al. (2017) also teaches that newer generation enhancer traps contain a minimal promoter including a TATA box and a transcription start site (pg. 135, left column, 2nd full paragraph). Based on the teachings of Marand, that explicitly teaches it is routine in the art to include enhancer sequences to regulate gene expression in programs designed to modulate transcription of genes of interest, a person of ordinary skill in the art would have been motivated to include the expression modulating elements taught by Abbitt (which are defined as enhancers; Abbitt, pg. 11, lines 28-29) in the regulatory region that comprises the modified TATA-box taught by Streitner because it is routine in the art to include enhancer elements in a regulatory region that comprises a TATA box (such as the promoter comprising a TATA box taught by Streitner), to modulate the expression of a gene in a plant cell. Additionally, based on the teachings of Abbitt, one of ordinary skill in the art would have been motivated to include the expression modulating element at least 20 base pairs upstream of the modified TATA box taught by Streitner, because Abbitt explicitly recites that EME placement as a functional embodiment of the invention with a reasonable expectation of success in modulating the expression of an endogenous gene of interest (Abbitt, Figure 1; pg. 53, lines 2-7). Based on the teachings of Abbitt, one of ordinary skill in the art would have been motivated to introduce the expression modulating element taught by Abbitt exactly 20 base pairs upstream of the modified TATA box taught by Streitner, because Abbitt explicitly teaches that inserting an EME precisely 20 base pairs upstream of the TATA box provides the greatest amount of increased expression (modulation of expression) of a gene of interest— specifically an endogenous gene of interest (ZmGOS2 promoter) relative to the plant cell species (maize protoplast) used in the methods taught by Abbitt (Abbitt; Example 8, Figure 4).
As such, the claims remain rejected for being prima facie obvious in view of the prior art.
Closest Prior Art
Claim 18 appears to be free of the prior art, given the failure of the prior art to teach or suggest modulation of expression of endogenous polypeptides using modified/non-native TATA boxes and introduced heterologous expression modulating elements in combination to engineer repression/decreased expression of the endogenous polypeptide of interest. The closest prior art in regards to Claim 18 can be found in combination of Streitner et al. (WO 2019/185609 A1, published 10/03/2019; IDS Document) in view of Abbitt et al. (WO 2018/183878 A1; published 10/04/2018), Marand et al. ( Biochimica et Biophysica Acta (BBA)-Gene Regulatory Mechanisms 1860.1 (2017): 131-139).
Streitner teaches method for increasing the expression level of a nucleic acid molecule of interest in a cell, preferably a plant cell (Abstract, Claim 1), wherein the expression of endogenous or exogenous nucleic acid molecules is increased up to many-fold of what is achieved under the control of a promoter without the introduced activating elements (pg. 3, second paragraph). In one embodiment of the invention, Streitner teaches a method further comprising modifying one or more TATA box motif{s) present in the recipient promoter by addition and/or substitution and/or deletion of one or more nucleotides for converting the one or more TATA box motif(s) into one or more TATA box motif{s) having increased or higher relative score{s) when matching or aligning the one or more modified TATA box motif{s) to the TATA box consensus (Claim 1, part iiic).
Abbitt teaches a method of modulating expression of an endogenous polynucleotide in a plant cell, the method comprising altering one or more nucleotides in a regulatory region of the endogenous polynucleotide such that the regulatory region of the polynucleotide comprises an expression modulating element having at least one copy of a polynucleotide sequence selected from the group consisting of SEQ ID NOS: 1-68 (Claim 1), wherein the alteration of one or more nucleotides is by genome modification (Claim 2), wherein the expression modulation element is present within about 10 to about 5000 bp from a transcriptional start site of the endogenous polynucleotide (Claim 3), wherein the expression modulation element further comprises additional copies of the expression modulating element such that about 2X to 10X copies of the expression modulating elements are present in the regulatory region of the endogenous polynucleotide (Claim 4), wherein the expression modulation element further comprises additional copies of the expression modulating element such that about 2X to 10X copies of the expression modulating elements are present in the regulatory region of the endogenous polynucleotide (Claim 5). Additionally, Abbitt defines expression modulating elements to be “a nucleotide sequence that up or down-regulates the expression of one or more plant genes. EME may have one or more copies of the same sequence arranged head-to-head, tail-to- head, or head-to-tail or a combination thereof configurations. EMEs are derived from plant sequences, or from bacterial or viral enhancer elements” (pg. 11, lines 11-15) , wherein an enhancer element “may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter” (pg. 11, lines 28-29) . In one embodiment of the invention, Abbitt also teaches that 4 copies of EME1 (SEQ ID NO: 1 ) or 1 copy of EME2 (SEQ ID NO: 5) were inserted either 520 base pairs upstream of the TATA box (Location 1 in Figure 1) or 20 base pairs upstream of the TATA box (Location 2 in Figure 1). Abbitt teaches that when the EME1 sequence is inserted in the promoter region, the expression levels of the reporter gene are significantly increased while insertion in the other 5 locations within the expression cassette resulted in a significant decrease of the expression of the reporter gene. A significant increase in gene expression was observed when EME2 (SEQ ID NO: 5) was inserted in locations within the promoter (Locations 1 and 2 in Figure 1) (pg. 53, lines 2-7). As such, the disclosure of Abbitt teaches away from the limitation in the present invention, wherein the placement of the EME at least 20 basepairs upstream of the TATA box would need to decrease expression of the endogenous gene of interest. Abbitt teaches the opposite, wherein the location of the EME being 20 base pairs away from the TATA-box would vastly increase expression of the endogenous gene of interest. Essentially, if one of ordinary skill in the art wanted to decrease expression of an endogenous gene of interest, based on the disclosure of Abbitt, one of ordinary skill in the art would not place the EME at least 20 base pairs upstream of the TATA box motif.
While Marand explicitly teaches it is routine in the art to include enhancer sequences to regulate gene expression in programs designed to modulate transcription of genes of interest, the disclosure of Abbitt would not lead one of ordinary skill in the art to add an enhancer element at least 20 base pairs upstream of the modified TATA box taught by Streinter, if the purpose of the invention was to decrease expression of the endogenous gene of interest.
Although the prior art teaches methods of modifying a TATA-box in a regulatory region of a plant cell, to study effect of mutations at each position in a 13-nucleotide-long prototype TATA-box on promoter function in leaf tissue, wherein promoter function was inhibited due to the modifications of the TATA-box (“knock out” of TATA box motif) [See Kiran et al. (Plant physiology 142.1 (2006): 364-376); Abstract]—if the purpose of a skilled artisan’s invention was to modify the endogenous TATA box in such a way that causes decreased expression of the TATA box, leading to decreased expression of an endogenous gene of interest, one of ordinary skill in the art would not have any motivation or rationale to simultaneously introduce an expression modulating element at least 20 base pairs upstream (a position known in the art to increase expression of a gene of interest) of the “knocked out” modified TATA box.
Conclusion
No claims are allowed.
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/KELSEY L MCWILLIAMS/Examiner, Art Unit 1663
/BRATISLAV STANKOVIC/Supervisory Patent Examiner, Art Units 1661 & 1662