DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Status of the Claims
Claims 1, 5, 8-9, and 12 are canceled.
Claims 2-4, 6-7, 10-11, and 13-19 are pending.
Claims 2-4, 6-7, 10-11, and 13-19 are examined herein.
Claims 2-4, 6-7, 10-11, and 13-19 are rejected.
Priority
Application No. 18/283,884 filed on 09/25/2023 is a 371 of PCT Application No. PCT/CN2022/082830 filed on 03/24/2022, and also claims foreign priority to Chinese Application No. CN202110321591.2 filed on 03/25/2021.
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
The certified copy of the foreign priority document and the English translation of the foreign priority document have been received.
Claim Interpretation
Claim 10 recites “wherein the NiR contains the amino acid sequence shown in SEQ ID NO:1, 3 or 5” which is interpreted as wherein the NiR comprises the amino acid sequence shown in SEQ ID NO:1, 3 or 5.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 2, 4, 11, and 17 are rejected under 35 U.S.C. 103 as being unpatentable over Nishimura (Nishimura, A., Ashikari, M., Lin, S., Takashi, T., Angeles, E. R., Yamamoto, T., & Matsuoka, M. (2005). Isolation of a rice regeneration quantitative trait loci gene and its application to transformation systems. Proceedings of the National Academy of Sciences, 102(33), 11940-11944) and Yin (YIN, G. X., WANG, Y. L., SHE, M. Y., DU, L. P., XU, H. J., MA, J. X., & YE, X. G. (2011). Establishment of a highly efficient regeneration system for the mature embryo culture of wheat. Agricultural Sciences in China, 10(1), 9-17).
Claim 2 is drawn to a method of improving efficiency of transforming exogenous nucleic acid sequences of interest in a plant, comprising: (a) overexpressing Nitrite reductase (NiR) in a cell of the plant; (b) introducing at least one expression construct containing at least one exogenous nucleic acid sequence of interest into the plant cell, wherein the at least one exogenous nucleic acid sequence of interest does not encode NiR; and (c) regenerating into an intact plant from the plant cell, wherein the plant is selected from wheat, strawberry, and tomato.
Claim 4 is drawn to the method of claim 2, wherein the coding nucleic acid sequence of NiR and the at least one exogenous nucleic acid sequence of interest are present in the same expression construct.
Claim 11 is drawn to the method of claim 2, wherein the cell is selected from a protoplast cell, a callus cell, an immature embryo cell, and an explant cell.
Claim 17 is drawn to the method of claim 2, wherein step (a) comprises introducing an expression construct containing a coding nucleic acid sequence of NiR into the cell of the plant.
Regarding claim 2, Nishimura teaches the overexpression of NiR from the high-regeneration rice strain Kasalath in rice cells of the low regeneration rice strain Koshihikari conferred enhanced regeneration ability of the Koshihikari cells (p. 11943, ¶2, Fig. 6). Nishimura also teaches the expression construct includes a GUS reporter gene (i.e. introducing at least one expression construct containing at least one exogenous nucleic acid sequence of interest into the plant cell, wherein the at least one exogenous nucleic acid sequence of interest does not encode NiR).
Regarding claim 4, Nishimura teaches the NiR coding sequence and the GUS reporter gene are present in the same expression construct (Fig. 6a).
Regarding claim 11, Nishimura teaches the cells transformed with the NiR construct are callus (Fig. 6).
Regarding claim 17, Nishimura teaches the coding nucleic acid sequence of NiR is comprised in an expression construct that is introduced into the cell of the plant (Fig. 6a, p. 11943, ¶3).
However, Nishimura teaches the plant is rice, and does not explicitly teach wherein the plant is selected from wheat, strawberry, and tomato.
In analogous art, Yin teaches a method of a highly efficient regeneration system for the mature embryo culture of wheat (title). Yin references the Nishimura paper and describes how the NiR gene was isolated from Kasalath rice line with good mature embryo regeneration ability and was transferred into the calli of low-regeneration rice strain Koshihikari and the transformed calli obtained high regeneration ability (p. 15-16, discussion section, ¶2). Yin teaches that from the regeneration system established in Yin’s study, a good wheat regeneration line was identified and the regeneration related genes TaNiR and TaReg2 were isolated. Yin further teaches of plans to transfer TaNiR, specifically the gene of FJ527909 which encodes an amino acid sequence comprising a sequence that has over 99% sequence identity to instant SEQ ID NO: 3, and TaReg2 into Chinese Spring to test for improvement of this wheat cultivars’ regeneration (p. 16, ¶1).
It would therefore have been obvious to a person of ordinary skill in the art to modify the invention taught by Nishimura to include the limitations of Yin to arrive at the instantly claimed method with a reasonable expectation of success because implementing the method of Nishimura into wheat using a wheat TaNiR gene could be achieved by one having ordinary skill in the art without encountering any special technical difficulties. One having ordinary skill in the art would have been motivated to do so because Yin references the Nishimura paper and methods, then explicitly suggests transferring an identified TaNiR gene into wheat to test for improved regeneration (p. 16, ¶1).
Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over Nishimura and Yin,as applied to claim 2, and further in view of Uniprot Accession No. A0A3B6QJ10 (Uniprot Accession No. A0A3B6QJ10, version 12, published online 12/02/2020).
Claim 10 is drawn to the method of claim 2, wherein the NiR contains the amino acid sequence shown in SEQ ID NO:1, 3 or 5.
Regarding claim 10, Nishimura and Yin teach the limitations of claim 2 as set forth in the previous obviousness rejection. The teachings of Nishimura and Yin as they are applied to claim 2 are set forth previously herein and are incorporated by reference. As previously described, Yin further teaches of plans to transfer TaNiR, specifically FJ527909 which has 99% sequence identity to instant SEQ ID NO: 3, into Chinese Spring to test for improvement of this wheat cultivars’ regeneration (p. 16, ¶1).
However, Nishimura and Yin do not explicitly teach wherein the NiR contains the amino acid sequence shown in SEQ ID NO:1, 3 or 5.
In analogous art, Uniprot Accession No. A0A3B6QJ10, published online 12/02/2020, teaches a wheat-derived protein identified as being involved in nitrite reductase that comprises an amino acid sequence with 100% sequence identity to instant SEQ ID NO: 3.
It would therefore have been obvious to a person of ordinary skill in the art to modify the invention of as taught by Nishimura and Yin to include the amino acid sequence taught by Uniprot Accession No. A0A3B6QJ10 to arrive at the instantly claimed method with a reasonable expectation of success because the amino acid sequence taught by Uniprot Accession No. A0A3B6QJ10 has over 99% sequence identity to the sequence encoded by FJ527909, taught by Yin, and was a known and readily obtainable sequence. One having ordinary skill in the art would have been motivated to combine the teachings because it would have been prima facie obvious to search for known TaNiR sequences, especially those with high sequence identity to the TaNiR sequence taught by Yin, and substitute the protein encoded by the gene taught by Yin with another protein known in the art, the wheat-derived protein involved in nitrite reductase as taught by Uniprot Accession No. A0A3B6QJ10, for the same purpose.
Claims 3, 6-7, 13, 15-16, and 18-19 are rejected under 35 U.S.C. 103 as being unpatentable over Nishimura (Nishimura, A., Ashikari, M., Lin, S., Takashi, T., Angeles, E. R., Yamamoto, T., & Matsuoka, M. (2005). Isolation of a rice regeneration quantitative trait loci gene and its application to transformation systems. Proceedings of the National Academy of Sciences, 102(33), 11940-11944), Kobayashi (Kobayashi, A., Hori, K., Yamamoto, T., & Yano, M. (2018). Koshihikari: a premium short-grain rice cultivar–its expansion and breeding in Japan. Rice, 11(1), 15), and Zafar (Zafar, K., Sedeek, K. E., Rao, G. S., Khan, M. Z., Amin, I., Kamel, R., ... & Mahfouz, M. M. (2020). Genome editing technologies for rice improvement: progress, prospects, and safety concerns. Frontiers in Genome Editing, 2, 5).
Claim 3 is drawn to the method of gene editing in a plant, comprising: (a) overexpressing Nitrite reductase (NiR) in a cell of the plant; (b) introducing at least one expression construct containing at least one exogenous nucleic acid sequence of interest into the plant cell, wherein the at least one exogenous nucleic acid sequence of interest encodes a component of a gene editing system and does not encode NiR; (c) regenerating into an intact plant from the plant cell.
Claim 6 is drawn to the method of claim 3, wherein the gene editing system is selected from a CRISPR system, TALEN, meganuclease and zinc-finger nuclease.
Claim 7 is drawn to the method of claim 6, wherein the gene editing system is a CRISPR system.
Claim 13 is drawn to the method of claim 3, wherein the coding nucleic acid sequence of NiR and the at least one exogenous nucleic acid sequence of interest are present in the same expression construct.
Claim 15 is drawn to the method of claim 3, wherein the cell is selected from a protoplast cell, a callus cell, an immature embryo cell, and an explant cell.
Claim 16 is drawn to the method of claim 3, wherein the plant is selected from wheat, strawberry, rice, corn, soybean, sunflower, sorghum, rape, alfalfa, cotton, barley, millet, sugarcane, tomato, tobacco, cassava and potato.
Claim 18 is drawn to the method of claim 3, wherein step (a) comprises introducing an expression construct containing a coding nucleic acid sequence of NiR into the cell of the plant.
Claim 19 is drawn to the method of claim 7, wherein the gene editing system is a base editing system.
Regarding claim 3, Nishimura teaches the overexpression of NiR from the high-regeneration rice strain Kasalath in rice cells of the low regeneration rice strain Koshihikari conferred enhanced regeneration ability of the Koshihikari cells (p. 11943, ¶2, Fig. 6). Nishimura further teaches the Kasalath NiR gene could also be used as a selection marker for gene transformation into recalcitrant strains of rice without any additional selective agents (p. 11943, ¶2). Nishimura also teaches the expression construct includes a GUS reporter gene (i.e. introducing at least one expression construct containing at least one exogenous nucleic acid sequence of interest into the plant cell, wherein the at least one exogenous nucleic acid sequence of interest does not encode NiR).
Regarding claim 15, Nishimura teaches the constructs comprising the NiR coding sequence were transformed into calli of the low-regeneration rice strain Koshihikari (p. 11943, ¶3).
Regarding claim 16, Nishimura teaches the plant is rice (entire document).
Regarding claim 18, Nishimura teaches the coding nucleic acid sequence of NiR is comprised in an expression construct that is introduced into the cell of the plant (Fig. 6a, p. 11943, ¶3).
However, Nishimura does not explicitly teach:
wherein the at least one exogenous nucleic acid sequence of interest encodes a component of a gene editing system (remaining limitation of claim 3).
The method of claim 3, wherein the gene editing system is selected from a CRISPR system, TALEN, meganuclease and zinc-finger nuclease (claim 6).
the method of claim 6, wherein the gene editing system is a CRISPR system (claim 7).
the method of claim 3, wherein the coding nucleic acid sequence of NiR and the at least one exogenous nucleic acid sequence of interest are present in the same expression construct (claim 13).
the method of claim 7, wherein the gene editing system is a base editing system (claim 19).
Regarding the remaining limitation of claim 3 and claims 6, 7, 13, and 19, in analogous art, Kobayashi teaches Koshihikari (i.e. the low-regenerative rice cultivar described by Nishimura) is the preferred cultivar of Japanese consumers (p. 1, ¶1). However, Kobayashi teaches Koshihikari also has a serious defect of low resistance to rice blast (p. 1, ¶2- p. 2, ¶1). In other analogous art, Zafar teaches CRISPR-Cas9 was used to target OsERF922, a gene responsible for rice blast resistance in rice, and as a result the number of blast lesions was considerably reduced in the edited lines compared to the control plants, highlighting the value of CRISPR-Cas9 for improving rice blast resistance (p. 10, section titled Rice Blast Resistance). Zafar also teaches in contrast to CRISPR/Cas editing, base editing allows mutations to be introduced without producing a double stranded break using cytidine deaminase fused with dCas9 to target the desired location and produce a C to T substitution (Fig. 2).
It would therefore have been obvious to a person of ordinary skill in the art to modify the invention taught by Nishimura to include the limitations of Zafar to arrive at the instantly claimed method with a reasonable expectation of success because Zafar teaches CRISPR/Cas9 can improve resistance to rice blast, and incorporation of the CRISPR system, into the expression construct of Nishimura could be achieved by one of ordinary skill in the art without encountering any special technical difficulties One having ordinary skill in the art would have been motivated to combine the teachings for the purpose of improving regenerative ability and rice blast resistance in rice cultivars including Koshihikari because Nishimura teaches NiR overexpression improved regeneration from callus of the low-regenerative cv. Koshihikari (p. 11943, ¶2, Fig. 6), Kobayashi teaches Koshihikari is a preferred rice cultivar by consumers but has the drawback of low resistance to rice blast(p. 1, ¶1- p. 2, ¶1), and Zafar teaches targeting and mutating the ERF922 gene in rice using CRISPR/Cas9 gene editing system improves rice blast resistance (p. 10, section titled Rice Blast Resistance). Furthermore, it would be prima facie obvious to combine the NiR coding sequence and the gene editing system into a single construct to be introduced into a cell for the purpose of transforming a rice cell a single time and achieve the beneficial outcomes of improved regeneration ability and improved resistance to rice blast. Additionally, it would also be obvious to combine the teachings for the purpose of using the NiR expression as the selection system in the construct also comprising the gene editing system because Nishimura teaches NiR gene could be used as a selection marker for gene transformation into recalcitrant strains of rice without any additional selective agents (p. 11943, ¶2).
Claim 14 is rejected under 35 U.S.C. 103 as being unpatentable over Nishimura Kobayashi, and Zafar as applied to claim 3, and further in view of Yin (YIN, G. X., WANG, Y. L., SHE, M. Y., DU, L. P., XU, H. J., MA, J. X., & YE, X. G. (2011). Establishment of a highly efficient regeneration system for the mature embryo culture of wheat. Agricultural Sciences in China, 10(1), 9-17), Uniprot Accession No. A0A3B6QJ10 (Uniprot Accession No. A0A3B6QJ10, published online 12/02/2020), and Islam (Islam, M. T., Gupta, D. R., Hossain, A., Roy, K. K., He, X., Kabir, M. R., ... & Wang, G. L. (2020). Wheat blast: a new threat to food security. Phytopathology Research, 2(1), 28).
Claim 14 is drawn to the method of claim 3, wherein the NiR contains the amino acid sequence shown in SEQ ID NO:1, 3 or 5.
Regarding claim 14, Nishimura Kobayashi, and Zafar teaches the limitations of claim 3 as set forth in the previous obviousness rejection. The teachings of Nishimura Kobayashi, and Zafar as they are applied to claim 1 are set forth previously herein and are incorporated by reference.
However, Nishimura Kobayashi, and Zafar do not explicitly teach wherein the NiR contains the amino acid sequence shown in SEQ ID NO:1, 3 or 5 (claim 14).
In analogous art, Yin teaches a method of a highly efficient regeneration system for the mature embryo culture of wheat (title). Yin references the Nishimura paper and describes how the NiR gene was isolated from Kasalath rice line with good mature embryo regeneration ability and was transferred into the calli of low-regeneration rice strain Koshihikari and the transformed calli obtained high regeneration ability (p. 15-16, discussion section, ¶2). Yin teaches that from the regeneration system established in Yin’s study, a good wheat regeneration line was identified and the regeneration related genes TaNiR and TaReg2 were isolated. Yin further teaches of plans to transfer TaNiR, specifically the gene of FJ527909 which encodes an amino acid sequence comprising a sequence that has over 99% sequence identity to instant SEQ ID NO: 3, and TaReg2 into Chinese Spring to test for improvement of this wheat cultivars’ regeneration (p. 16, ¶1). In other analogous art, Uniprot Accession No. A0A3B6QJ10, published online 12/02/2020, teaches a wheat-derived protein identified as being involved in nitrite reductase that comprises an amino acid sequence with 100% sequence identity to instant SEQ ID NO: 3. Additionally, Islam teaches there are genes in rice that, when these genes are mutated, they lead to increased resistance to the rice blast disease (p. 2, ¶5). Islam proceeds to explicitly teach identifying these orthologous genes in wheat and then generating their mutations by CRISPR-Cas technology may confer enhanced resistance to wheat blast (p. 2, ¶5).
It would therefore have been obvious to a person of ordinary skill in the art to modify the invention taught by Nishimura Kobayashi, and Zafar to include the limitations of Yin and Uniprot Accession No. A0A3B6QJ10 to arrive at the instantly claimed method with a reasonable expectation of success because implementing the method of Nishimura into wheat using a wheat TaNiR gene could be achieved by one having ordinary skill in the art without encountering any special technical difficulties. One having ordinary skill in the art would have been motivated to do so because Yin references the Nishimura paper and methods, then explicitly suggests transferring an identified TaNiR gene into wheat to test for improved regeneration (p. 16, ¶1). Furthermore, it would therefore have been obvious to a person of ordinary skill in the art to modify the invention of as taught by Nishimura and Yin to include the amino acid sequence taught by Uniprot Accession No. A0A3B6QJ10 to arrive at the instantly claimed method with a reasonable expectation of success because the amino acid sequence taught by Uniprot Accession No. A0A3B6QJ10 has over 99% sequence identity to the sequence encoded by FJ527909, taught by Yin, and was a known and readily obtainable sequence. One having ordinary skill in the art would have been motivated to combine the teachings because it would have been prima facie obvious to search for known TaNiR sequences, especially those with high sequence identity to the TaNiR sequence taught by Yin, and substitute the protein encoded by the gene taught by Yin with another protein known in the art, the wheat-derived protein involved in nitrite reductase as taught by Uniprot Accession No. A0A3B6QJ10, for the same purpose. On having ordinary skill in the art would have been motivated to use the CRISPR/Cas gene editing system in wheat, in combination with the wheat NiR gene, for the purpose of improving wheat regeneration as taught by Yin and conferring resistance to wheat blast as taught by Islam.
Response to Arguments
Applicant argues beginning on p. 5 of remarks dated 09/11/2025 the
following arguments:
(i) The cited references fail to disclose or suggest the method of claim 3
Without conceding to the merits of the rejection and solely in the interest of obtaining issuance of a patent, independent claim 3 has been amended to recite "wherein the at least one exogenous nucleic acid sequence of interest encodes a component of a gene editing system and does not encode NiR." Smedley fails to disclose or suggest a method of gene editing in a plant comprising (a) overexpressing Nitrite reductase (NiR) in a cell of the plant and (b) introducing at least one expression construct containing at least one exogenous nucleic acid sequence of interest into the plant cell, wherein the at least one exogenous nucleic acid sequence of interest encodes a component of a gene editing system and does not encode NiR_as recited in amended claim 3. Indeed, the Office Action acknowledges that "Smedley does not teach:
i. (a) overexpressing [Nitrite] reductase (NiR) in a cell of the plant (remaining
limitation of claim 3)." See Office Action, page 7.
Accordingly, claim 3 is patentable over Smedley.
Applicant further argues that one of skill in the art would not have a motivation nor reasonable expectation of success to arrive at the method of claim 3 in view of Hatzfeld. Smedley, Zhao, and/or Ozawa. Consequently, amended claim 3 is patentable over Hatzfeld, Smedley, Zhao, and/or Ozawa.
This argument has been fully considered and is found not persuasive for
the following reason(s):
In view of the currently amended claims, Applicant’s arguments have been considered but are moot because the new ground of rejection does not rely on any reference applied in the prior rejection of record for any teaching or matter specifically challenged in the argument.
Applicant argues beginning on p. 7 of remarks dated 09/11/2025 the
following arguments:
(ii) The method of claim 3 exhibits unexpected results
The patentability of the method of claim 3 is underscored by the fact that it exhibits unexpected results. As submitted in the previously filed response dated January 23, 2025, the inventors are the first to demonstrate that overexpression of NiR in plants significantly increases the efficiency of gene editing. Indeed, the application states:
The inventor[s] surprisingly found that overexpression of NiR in plant cells can significantly improve the efficiency of regenerating into intact plants from the plant cells as well as the efficiency of transforming exogenous nucleic acid sequences of interest into plants. More surprisingly, the efficiency of gene editing can be improved when the exogenous nucleic acid sequences of interest encode the gene editing system.
See Specification, paragraph [0058].
Moreover, as noted in the previously filed response dated July 21, 2025, Example 2 described that:
UBI-NiR-A3A with [integration] of wheat NiR could improve the regeneration efficiency of wheat transformants by 2.08 times compared to the control UBI-GFP-A3A.
Surprisingly, the number of obtained mutants increased significantly, up to 3 times as that of the control, which was much higher than the improvement of regeneration efficiency. This indicates that nitrite reductase NiR, a plant nitrogen metabolism regulator, could be used as an important regulatory protein to promote the efficiency of plant genetic transformation and, more importantly, to improve the efficiency of genome
editing.
See Specification, paragraph [0097].
In addition to Example 2, Example 3 also illustrates a significant improvement in gene editing efficiency for endogenous genes. Based on the table in Example 3 of the application, one of skill in the art would be able to calculate the regeneration rates and editing rates with the calculation results as shown in the Table provided on p. 7 of remarks.
It follows that based on the results shown in Example 3, the mutation rate upon co- expression of NiR was 4.1 times that of the control group. Collectively, the results of Examples 2 and 3 indicate that, after accounting for the direct effect of regenerant number, the mutation rate still shows an additional 1- to 4-fold increase. Importantly, these results demonstrate that the claimed method achieves a substantial increase in gene editing efficiency.
To further highlight the unexpected results of the claimed method, Applicant herein submits a copy of Yu, Yang, et al. "Enhancing wheat regeneration and genetic transformation through overexpression of TaLAX1." Plant Communications, vol. 5, no. 5, 2024, p. 100738, doi:10.1016/j.xplc.2023.100738 ("Yu") as discussed in the previously filed response dated July 21, 2025. The results described in Yu illustrate that a transformation method to overexpress TaLAX PANICLEl (TaLAX1) does not necessarily lead to an improvement in gene editing efficiency. Yu describes that:
The effect of TaLAXl-A-improved transformation on CRISPR/Cas9-mediated genome editing frequency was also investigated and compared with conventional transformation....
A binary vector containing a cassette with TaLAXl-A-OE, Cas9, and a guide RNA targeting the Q gene... However, the proportion of Q-gene edited TO shoots relative to the total number of TO plants did not differ significantly from that of the control (Figure 4E and Supplemental Data 11). This suggests that the enhanced editing frequencies resulting
from TaLAXl-A can be attributed to improved rates of regeneration.
See Yu, pages 5-6.
Figure 4E and Supplemental Data 11 of Yu clearly demonstrate that there is no significant increase of the gene-edited T0 plants, as compared to the significant increase of the regeneration rate. Thus, a solution capable of increasing "regeneration efficiency" does not necessarily lead to an improvement in "gene editing efficiency." In contrast to Yu, the method of claim 3 achieves a substantial increase in gene editing efficiency. For the sake of the record, this further supports that based on the teachings of Hatzfeld, i.e., enhancing yield-related traits in plants by overexpressing NiR polypeptide, one of skill in the art would not have any motivation or expectation of success that the efficiency of gene editing in a plant could be improved by overexpressing NiR. It follows that one of skill in the art would recognize that the method of claim 3 exhibits unexpected results.
In view of the foregoing arguments, amended claim 3 is patentable over Smedley, Zhao, and/or Ozawa. The other rejected claims depend from claim 3 either directly or indirectly and are also patentable over the cited references for at least the same reasons.
The method of claim 2 is also patentable over the cited references because it exhibits unexpected results. As discussed above in Section I, Examples 2 and 3 of the present application demonstrate that the claimed method achieves a substantial increase in gene editing efficiency. Additionally, Section I also describes how the inventors are the first to demonstrate that overexpression of NiR in plants significantly increases the efficiency of gene editing. Moreover, the results in Yu illustrate that the method of claim 2 is unexpected because it achieves a more substantial increase in gene editing efficiency. Thus, the method of claim 2 exhibits unexpected results.
In view of the foregoing arguments, amended claim 2 is patentable over Zhao and/or Hatzfeld. Claim 10 depends directly from claim 2 and is also patentable over the cited references either alone or in combination for at least the same reasons. Consequently, Applicant requests withdrawal of the rejection.
This argument has been fully considered and is found not persuasive for
the following reason(s):
This is not found persuasive. With regard to Applicant’s argument that Applicant has offered evidence of unexpected and unobvious results, pursuant to MPEP 716.02(b), the evidence relied upon should establish "that the differences in results are in fact unexpected and unobvious and of both statistical and practical significance." Ex parte Gelles, 22 USPQ2d 1318, 1319 (Bd. Pat. App. & Inter. 1992) (Mere conclusions in appellants’ brief that the claimed polymer had an unexpectedly increased impact strength "are not entitled to the weight of conclusions accompanying the evidence, either in the specification or in a declaration."); Ex parte C, 27 USPQ2d 1492 (Bd. Pat. App. & Inter. 1992) (Applicant alleged unexpected results with regard to the claimed soybean plant, however there was no basis for judging the practical significance of data with regard to maturity date, flowering date, flower color, or height of the plant.). In the instant case, Applicant alleges overexpression of NiR in plants significantly increases shoot regeneration and the efficiency of gene editing. Applicant describes NiR-overexpressing explants regenerated 108 shoots from 83 explants (130% regeneration rate) compared to control explants that regenerated 73 shoots from 84 explants (specification, p. 2-3). Applicant also describes the mutation rate of the regenerated shoots overexpressing NiR as 5.56% as compared to controls that had a mutation rate of 1.37% (approximately 4X greater). With regard to the regeneration rate, the basis for regeneration rate appears dependent on total number of shoots produced from each treatment group and not the number of explants that were able to regenerate shoot(s). It is unclear if all explants were able to regenerate shoots, or if a similar number of explants were able to regenerate shoots compared to controls and the explants that regenerated shoots were capable of regenerating more shoots per explant. There is also no description of whether the shoots are the same or different transformation events. Because transformed callus can regenerate multiple shoots from a single transformation event, it is unclear if the mutation rate described is of different mutation events or clones of the same regenerated mutant from a single explant. Although, there does appear to be statistical significance, however it is unclear if the results represent increased regeneration from individual transformation events (and further as a result increased mutants that are clones), or whether NiR overexpression increases the number of explants capable of regenerating shoots and also the number of independent mutation events. With respect to Applicant’s argument regarding overexpression of TaLAX (Yu reference), this argument is moot because the argued gene is a different gene than NiR with a different structure and function.
Further, even if Applicant can make such a showing, MPEP 716.02(c) provides that the evidence of unexpected results must be weighed against evidence supporting prima facie obviousness in making a final determination of the obviousness of the claimed invention. MPEP 716.02(c) directs the examiner to MPEP 716.01(d), which establishes that although the record may establish evidence of secondary considerations which are indicia of nonobviousness, the record may also establish such a strong case of obviousness that the objective evidence of nonobviousness is not sufficient to outweigh the evidence of obviousness. Newell Cos. v. Kenney Mfg. Co., 864 F.2d 757, 769, 9 USPQ2d 1417, 1427 (Fed. Cir. 1988), cert. denied, 493 U.S. 814 (1989); Richardson-Vicks, Inc., v. The Upjohn Co., 122 F.3d 1476, 1484, 44 USPQ2d 1181, 1187 (Fed. Cir. 1997) (showing of unexpected results and commercial success of claimed ibuprofen and pseudoephedrine combination in single tablet form, while supported by substantial evidence, held not to overcome strong prima facie case of obviousness). The showing, when made, must outweigh the rationale in support of a finding of prima facie obviousness provided in the 103 rejection(s). Here, in the prior art Nishimura teaches the overexpression of NiR from the high-regeneration rice strain Kasalath in rice cells of the low regeneration rice strain Koshihikari conferred enhanced regeneration ability of the Koshihikari cells (p. 11943, ¶2, Fig. 6). Nishimura shows transformed calli with the NiR overexpression constructs resulted in significantly increased regeneration rates from what appears to be nearly 0% regeneration in controls to a plate full of many shoots in NiR overexpression transformants (Fig. 6 b-f). In analogous art, Kobayashi teaches Koshihikari (i.e. the low-regenerative rice cultivar described by Nishimura) is the preferred cultivar of Japanese consumers (p. 1, ¶1). However, Kobayashi teaches Koshihikari also has a serious defect of low resistance to rice blast (p. 1, ¶2- p. 2, ¶1). In other analogous art, Zafar teaches CRISPR-Cas9 was used to target OsERF922, a gene responsible for rice blast resistance in rice, and as a result the number of blast lesions was considerably reduced in the edited lines compared to the control plants, highlighting the value of CRISPR-Cas9 for improving rice blast resistance (p. 10, section titled Rice Blast Resistance). In other analogous art, Yin teaches a method of a highly efficient regeneration system for the mature embryo culture of wheat (title). Yin references the Nishimura paper and describes how the NiR gene was isolated from Kasalath rice line with good mature embryo regeneration ability and was transferred into the calli of low-regeneration rice strain Koshihikari and the transformed calli obtained high regeneration ability (p. 15-16, discussion section, ¶2). Yin teaches that from the regeneration system established in Yin’s study, a good wheat regeneration line was identified and the regeneration related genes TaNiR and TaReg2 were isolated. Yin further teaches of plans to transfer TaNiR, specifically the gene of FJ527909 which encodes an amino acid sequence comprising a sequence that has over 99% sequence identity to instant SEQ ID NO: 3, and TaReg2 into Chinese Spring to test for improvement of this wheat cultivars’ regeneration (p. 16, ¶1). From these teachings, it would be obvious to combine the teachings of the prior art to arrive at the instantly claimed invention of a method of improving efficiency of transforming exogenous nucleic acid sequences of interest in a plant, comprising: (a) overexpressing Nitrite reductase (NiR) in a cell of the plant; (b) introducing at least one expression construct containing at least one exogenous nucleic acid sequence of interest into the plant cell, wherein the at least one exogenous nucleic acid sequence of interest does not encode NiR; and (c) regenerating into an intact plant from the plant cell, wherein the plant is selected from wheat, strawberry, and tomato (i.e. claim 2). From these teachings, it would also be obvious to combine the teachings of the prior art to arrive at the instantly claimed invention of the method of gene editing in a plant, comprising: (a) overexpressing Nitrite reductase (NiR) in a cell of the plant; (b) introducing at least one expression construct containing at least one exogenous nucleic acid sequence of interest into the plant cell, wherein the at least one exogenous nucleic acid sequence of interest encodes a component of a gene editing system and does not encode NiR; (c) regenerating into an intact plant from the plant cell (i.e. claim 3) (see full 103 analysis above). Because Nishimura teaches a significant increase in regeneration rate, the evidence of increased regeneration does not appear unexpected or unobvious. Additionally, the evidence of increased efficiency of gene editing is also obvious as a function of inherency because if one of ordinary skill in the art were to conduct the obvious method, this person would also observe the inherent result of increased efficiency of gene editing. In view of the foregoing, Applicant’s evidence is not deemed to outweigh the basis for the rejection
Finally, MPEP 716.02(d) provides that whether the unexpected results are the result of unexpectedly improved results or a property not taught by the prior art, the "objective evidence of nonobviousness must be commensurate in scope with the claims which the evidence is offered to support." In other words, the showing of unexpected results must be reviewed to see if the results occur over the entire claimed range. In re Clemens, 622 F.2d 1029, 1036, 206 USPQ 289, 296 (CCPA 1980). In this case, the scope of the claims appears commensurate with the evidence, however the claims do not require increased regeneration or gene editing efficiency, therefore there is no nexus between the evidence and the claims.
Applicant argues beginning on p. 9 of remarks dated 09/11/2025 the
following arguments:
(i) The cited references fail to disclose or suggest the method of claim 2
Without conceding to the merits of the rejection and solely in the interest of obtaining issuance of a patent, independent claim 2 has been amended to recite "wherein the at least one exogenous nucleic acid sequence of interest does not encode NiR." Zhao is entirely silent with respect to a method of improving efficiency of transforming exogenous nucleic acid sequences of interest in a plant comprising overexpressing nitrite reductase in a plant in cell of the plant, let alone wherein the method comprises introducing at least one expression construct containing at least one exogenous nucleic acid sequence of interest into the plant cell, wherein the at least one exogenous nucleic acid sequence of interest does not encode NiR as recited in amended claim 2. As discussed in the previously filed response dated July 21, 2025, given the differences in the amino acid sequence and function of nitrite reductase compared to nitrate reductase, one of skill in the art would not have a motivation nor a reasonable expectation of success to arrive at the method of amended claim 2 in view of Zhao. Thus, claim 2 is patentable over Zhao.
Hatzfeld does not cure the deficiencies of Zhao. Hatzfeld is entirely silent with respect to a method of improving efficiency of transforming exogenous nucleic acid sequences of interest in a plant comprising (a) overexpressing nitrite reductase in a plant in cell of the plant and (b) introducing at least one expression construct containing at least one exogenous nucleic acid sequence of interest into the plant cell, wherein the at least one exogenous nucleic acid sequence of interest does not encode NiR as recited in amended claim 2. The method described in Hatzfeld has nothing to do with the introduction of an exogenous nucleic acid sequence, which is unrelated to the expression of NiR. It follows that one of skill in the art would not have a motivation nor reasonable expectation of success to arrive at the method of amended claim 2 in view of Zhao and/or Hatzfeld. Thus, amended claim 2 is patentable over Zhao and/or Hatzfeld.
This argument has been fully considered and is found not persuasive for
the following reason(s):
In view of the currently amended claims, Applicant’s arguments have been considered but are moot because the new ground of rejection does not rely on any reference applied in the prior rejection of record for any teaching or matter specifically challenged in the argument.
Conclusion and Inquiries
No claims are allowed.
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JESSICA N. STOCKDALE
Examiner
Art Unit 1663
/JESSICA NICOLE STOCKDALE/Examiner, Art Unit 1663
/CHARLES LOGSDON/Primary Examiner, Art Unit 1662