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 .
Priority
The instant application 17/924,249 filed on 11/09/2022 claims the benefit of priority to PCT/EP2021/062116 filed on 05/07/2021 and EP20174401.8 filed on 05/13/2020. Acknowledgment is made of Applicant's claim for foreign priority under 35 U.S.C. 119 (a)-(d). Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55. As such, the effective filing date of Claims 1-13 is 05/13/2020.
Status of the Claims
Amendments dated 09/23/2025 have been entered.
Claims 1-13 are pending.
Claims 1-13 are examined herein.
Drawings and Nucleotide and/or Amino Acid Sequence Disclosures
Specific deficiency – Nucleotide and/or amino acid sequences appearing in the drawings (FIGS. 3-5 dated 09/23/2025) are not identified by sequence identifiers in accordance with 37 CFR 1.821(d). Sequence identifiers for nucleotide and/or amino acid sequences must appear either in the drawings or in the Brief Description of the Drawings.
Required response – Applicant must provide:
Replacement and annotated drawings in accordance with 37 CFR 1.121(d) inserting the required sequence identifiers;
AND/OR
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required sequence identifiers into the Brief Description of the Drawings, 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.
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.
---The following are modified rejections from those set forth in the Office Action dated 06/23/2025 in view of Applicant’s amendments to the claims.---
Claims 2-3 remain 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.
Claim 2 recites a method step that is interpreted as occurring prior to the crossing step of Claim 1, part (c). Claim 2 recites, in part, “one or more T0 plants having an active construct are crossed with a wild type plant to provide the plurality of progeny plants” which renders the claim indefinite. It remains unclear how the plants produced in the crossing step of Claim 2 could be integrated into the method step of Claim 1 part (c) as a T0 plant that would subsequently be subject to the further crossing step with an isogenic plant as recited in Claim 1 part (c) because the plurality of progeny plants produced in Claim 2 would no longer be considered T0 plants, but rather, and F1 generation of progeny plants. It is unclear how one of ordinary skill in the art could add a precursor step before Claim 1, part (c) and still have all the limitations of part (c) which requires crossing a T0 plant— meaning that one of ordinary skill in the art cannot have an intermediate progeny plant population between Claim 1, part (b) and Claim 1, part (c). It is unclear if the methods steps of Claim 1 part (c) and Claim 2 are meant of occur concurrently, resulting in two populations of progeny plants— one population produced from the isogenic cross and the other produced from the wild-type cross, wherein the sperate populations both undergo the selecting step of Claim 1 part (d). Additionally, Claim 2 could be interpreted as meaning that the step wherein T0 plants expressing the RNA-guided DNA endonuclease are selected from the plurality of T0 plants from Claim 1 to provide one or more T0 plants having an active construct happens before Claim 1 part (c), wherein when the method step of Claim 1 part (c) is executed, the crossing is between a T0 plant having the active construct and a wild-type isogenic plant, which is merely a narrower limitation of the genus of isogenic plants recited in Claim 1 part (c). For purposes of examination, the wild-type plant recited in Claim 2 will be interpreted as a wild-type plant that doesn’t necessarily need to be isogenic relative to the T0 plant. This determination does not relieve the Applicant of their duty to amend the claims in further correspondence.
Claim 3 depends from Claim 2 and is therefore rejected for the same reasons as given above.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 1 and 4-11 are rejected under 35 U.S.C. 103 as being unpatentable over Li et al. (Plant Biotechnology Journal 15.12 (2017): 1566-1576) in view of Fabijanski et al. (DE 69034190 T2, issued 02/23/2006) and Rafalski et al. (Nonmammalian genomic analysis. A practical guide. Academic Press, San Diego (1996): 90-91).
Regarding Claim 1, Li et al. (herein referred to as Li) teaches a RNA-guided endonuclease (RGEN) system as an in vivo desired-target mutator (DMT) in maize, wherein analysis of F1 and F2 plants indicated that the mutations induced by the DMT effect were largely heritable.
Regarding Claim 1 part (a), Li teaches the construction of an RNA-guided Cas9 vector comprising a modified coding sequence of SpCas9 was cloned into a CPB vector behind a maize ubiquitin promoter. The SpCas9 region was amplified and cloned into the CPB vector and the nuclear location signal (NLS) sequence of SV40 and nucleoplasmin were embedded at either end of the Cas9 protein. The guiding RNA sequence 5′-GCGGAGACTAAGTGGctgtagGG-3′, which harbors an SfcI region (lowercase region), was selected to target the maize LG1 locus within the coding region of exon 1 at chromosome 2 from 4265163 to 4268840 (AGP v3.0). The underlined ‘gGG’ represents the proto-adjacent-motif (Figure 2). The maize U6-6 promoter was used to drive the sgRNA gene, and the promoters and sgRNA genes were cloned into the CPB vector (pg. 1573, Materials and Methods, Construction of the RNA-guided Cas9 vector). Approximately 5000 immature wild-type ZC01 embryos from healthy maize were selected and underwent Agrobacterium-mediated transformation under highly stringent bialaphos (Bar) selection (pg. 1567, Results, Regeneration of transgenic maize lines harbouring LG1-targeting RGEN).
Regarding Claim 1 part (b), Li teaches that, in total, 113 independent transformation-positive plants were screened and identified from around 1250 regenerated plants based on PCR with oligonucleotide primers to amplify both the Bar and SpCas genes. The transformation rate was approximately 2.26% under bialaphos selection. The relative exogenous gene copy numbers in the transformants were also evaluated; a total of 97 of 113 (85.8%) transformants harboured one to three copies of the gene (Table S1) (pg. 1567, Results, Regeneration of transgenic maize lines harbouring LG1-targeting RGEN).
Regarding Claim 1 part (c), in Figure S3, Li teaches that the DMT positive T1 plants (derived from two T0 events) comprising the transgene were crossed with an isogenic plant (transgene-free) to produce F1 progeny plants, as shown in the following figure.
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Regarding Claim 1, part (d), Li teaches that when DMT T1 plants that stably expressed the RGEN and harboring mutations to the LG1 locus were crossed with recipient lines, which include the both wild type and isogenic ZC01 line, each line generated the consistent mutant phenotypes in T1 plants, yielding mutation frequencies that ranged from 4.35% to 43.75% (Table 1), wherein the T1 plants carrying the mutation were selected for further crossing (pgs. 1567-1568, Results, The mutation efficiency of LG1-targeting RGEN as a desired-target mutator).
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Li also teaches that the mutation frequencies of the F1 lines that stably expressing RGEN [which were the result of a cross between the DMT T1 line (derived from T0 plants) and the wild-type and isogenic line ZC01] were calculated; each line generated the consistent mutant phenotypes in F1 plants, yielding mutation frequencies that ranged from 21.69 to 34.29% (See ZC01 × CF13-1 (CK*) and ZC01 × CF31-8 (CK*) in Table 1). Sequencing of the expected target regions in wild-type recipient lines showed that they has exactly same sequence at the proto-spacer adjacent motif (PAM) and single guide RNA (sgRNA) targeting region with ZC01 (Figure S2). Thus, the mutation frequency associated with the DTM effect was as fairly high as over 20% (See ZC01 × CF13-1 (CK*) and ZC01 × CF31-8 (CK*) in Table 1) (pgs. 1567-1568, The mutation efficiency of LG1-targeting RGEN as a desired-target mutator).
While Li teaches T1 plants derived from two T0 events that are crossed with isogenic plants not comprising said exogenous DNA to provide a plurality of progeny plants, Li does not explicitly teach the limitation of Claim 1, wherein T0 plants are crossed with isogenic plants not comprising said exogenous DNA to provide a plurality of progeny plants.
Regarding Claim 1, Fabijanski et al. (herein referred to as Fabijanski) teaches that crossing genetically transformed plants (T0 plants) with plants that are isogenic relative to the transformed plant to produce progeny plants that are the result of said crossing, is routine in the art (pg. 20, paragraph 122, right column, lines 20-22).
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention to modify the maize plants that are crossed in the first generation of the DMT method taught by Li to cross the T0 plants taught by Li with the isogenic line taught by Li, rather than the T1 plants that are derived from the T0 plant, to have a first generation of progeny plants that are the result of crossing the transformed plant with an isogenic line, as taught by Fabijanski. Based on the teachings of Rafalski et al., one of ordinary skill in the art would have been motivated to cross the T0 (transformed) plants taught by Li with the isogenic plants taught by Li, to have progeny plants that are the result of crossing the transformed plant with an isogenic line, as taught by Fabijanski because the introgression of a desirable character or trait (gene) from a donor parent plant into an otherwise agronomically acceptable cultivar (recurrent parent) is routine and well known in the art and would create progeny plants that are homozygous at the target locus and that are essentially identical to the recurrent parent (isogenic parent) with the exception of the region surrounding the gene under selection, after a single crossing event (paragraph bridging pgs. 90-91). 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 4, Li teaches that T1 lines harbouring one homologous copy of the LG1-targeted knockout editing machinery transformant were selected (Table 1) as pollen donors to be selfed for seed increase (pg. 1574, right column, DMT crossing and DMT mutation rate).
Regarding Claim 5, Li teaches that T1 transgenic plants carrying the RGEN gene-editing machinery were crossed with its wild-type line, ZC01 (Tables 1, S2), creating F1 plants comprising (CK*; T0 plant crossed with ZC01) DTM effects (See ZC01 × CF13-1 (CK*) and ZC01 × CF31-8 (CK*) in Table 1). Li also discloses that T1 lines harbouring one homologous copy of the LG1-targeted knockout editing machinery transformant were selected (Table 1) as pollen donors to be selfed for seed increase (pg. 1574, right column, DMT crossing and DMT mutation rate), wherein all progeny plants (crosses between the recipient lines and DTM, crosses between the wild-type and isogenic ZC01 and DMT, and selfed plants) were grown in field conditions (pg.1574, left column, DTM crossing and DTM mutation rate).
Regarding Claim 6, Li teaches that the relative exogenous gene copy numbers in the transformants were also evaluated; a total of 97 of 113 (85.8%) transformants harboured one to three copies of the gene (Table S1) (pg. 1567, Results, Regeneration of transgenic maize lines harbouring LG1-targeting RGEN). As such, the specific heritable mutation resulted in a duplication event in the transformants.
Regarding Claim 7, Li teaches the construction of an RNA-guided Cas9 vector comprising a modified coding sequence of SpCas9 was cloned into a CPB vector behind a maize ubiquitin promoter. The SpCas9 region was amplified and cloned into the CPB vector and the nuclear location signal (NLS) sequence of SV40 and nucleoplasmin were embedded at either end of the Cas9 protein (pg. 1573, Materials and Methods, Construction of the RNA-guided Cas9 vector).
Regarding Claim 8, Li teaches the construction of an RNA-guided Cas9 vector comprising guiding RNA sequence 5′-GCGGAGACTAAGTGGctgtagGG-3′, which harbors an SfcI region (lowercase region), was selected to target the maize LG1 locus within the coding region of exon 1 at chromosome 2 from 4265163 to 4268840 (AGP v3.0). The underlined ‘gGG’ represents the proto-adjacent-motif (Figure 2) (pg. 1573, Materials and Methods, Construction of the RNA-guided Cas9 vector). As shown in the following figure, the guide RNA used in the method of the invention is a sgRNA comprising a crRNA fused to a tracrRNA that forms a stem-loop (Table S4).
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Regarding Claim 9, Li teaches in the following figure that in the methods of the present invention (DMT breeding) several generation of progeny plants are backcrossed (plants labeled BC) to create transgene free offspring that have the desired phenotype associated with the heritable mutation (Figure S3). Li discloses that after selection of the expected mutation and a non-Cas9 mutator transformation event from the backcross 1 (BC1) generation and the expected mutation in subsequent BC generations, the targeted gene mutation with 100% of the original receptor background was obtained. In contrast to conventional backcross breeding, the Cas9 mutator breeding scheme avoids the problem of linkage drag encountered during backcross breeding (pg. 1570-1572, paragraph spanning these three pages).
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Regarding Claim 10, Li teaches that approximately 5000 immature ZC01 embryos from healthy maize were selected and underwent Agrobacterium-mediated transformation under highly stringent bialaphos selection. In total, 113 independent transformation-positive plants were screened and identified from around 1250 regenerated plants based on PCR with oligonucleotide primers to amplify both the Bar and SpCas genes. The transformation rate was approximately 2.26% under bialaphos selection. The relative exogenous gene copy numbers in the transformants were also evaluated; a total of 97 of 113 (85.8%) transformants harboured one to three copies of the gene (Table S1) (pg. 1567, Results, Regeneration of transgenic maize lines harbouring LG1-targeting RGEN).
Regarding Claim 11, Li teaches that genomic DNA was extracted from maize T0, T1 and F1 (mutator crossed with recipient lines) lg1 mutants, and the on-target sites were amplified from genomic DNA. The amplicons were designed to surround the intended target site, and PCR was conducted using high-fidelity KOD-plus polymerase and primers spanning the target sites. The amplicons were then purified and digested with SfcI. The restriction site was located at the cleavage site of RNA-guided Cas9, and a mutation changes the RE restriction pattern, which allowed mutants to be quickly distinguished from wild-type individuals (pg. 1573, right column, DNA extraction and PCR-RE assay). Sanger sequencing was used to identify the mutation in the T0 generation. The PCR amplicons obtained from the samples were cloned into the pCR TA clone vector and the M13 R primer was used for sequencing to identify the mutations. The sequencing quality and results were viewed using Sequence Scanner Software ver2.0 (ABI Applied Biosystems) by importing the raw sequencing trace files. The homologous mutant genotypes of T0 individuals were cross-validated across the DNA samples extracted from seedling leaves, young male inflorescences, emerging silk and young ear husks. Mutations were identified in each sample of T0 transformation events based on at least 20 independent high-quality positive sequences from TA clones carrying the PCR amplicons (pg. 1573, left column, Sanger sequencing to identify the target mutation).
Therefore, for all the reasons above, the claimed invention is prima facie obvious.
Response to Arguments
Applicants Remarks on pgs. 5-7 in the reply filed on 09/23/2025 are acknowledged but do not overcome these new rejections. In particular, Applicant’s Remarks rely on several premises, the first of which is that previously cited Fabijanski and Rafalski are not related to a method for the introduction and selection of a specific heritable mutation using RNA-guided DNA endonuclease and guide RNA (Remarks, pg. 5), while also contesting the age of the two references (Remarks, pg. 6).
This is not found persuasive. 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). It is the combination of Li, Fabijanski, and Rafalski that teach methods for introducing and selecting mutations that are largely heritable, not simply the individual disclosures of Fabijanski and Rafalski. Applicant is reminded that the teachings of Li encompass all the “raw materials” that are required by Claim 1, including the RNA-guided DNA endonuclease, guide RNA, T0 generations of plants, and the crossing of plants comprising exogenous DNA with isogenic wild-type lines not comprising the exogenous DNA. The teachings of Fabijanski were cited to submit a disclosure to show that using regenerated T0 plants (like those already taught in Li), rather than T1 plants taught by Li, as the first generation of plants in a cross with another plant is routine and obvious prior to the effective filing date of the claimed invention. It should also be noted that the inclusion of Rafalski is merely meant to provide evidence that one of ordinary skill in the art would have found it routine and well known in the art to introgress a desirable character or trait (gene) from a donor parent plant (such as the T0 generation of plants taught by Li) into an otherwise agronomically acceptable cultivar (such as the wild-type isogenic line taught by Li). Additionally, "The mere age of the references is not persuasive of the unobviousness of the combination of their teachings, absent evidence that, notwithstanding knowledge of the references, the art tried and failed to solve the problem." In re Wright, 569 F.2d 1124, 1127, 193 USPQ 332, 335 (CCPA 1977).
Applicant also argues that “mutagenesis methods not based on RNA-guided DNA endonuclease and guide RNA simply do not allow that the desired mutation is induced in the F1 generation” and as such one of ordinary skill in the art would not have considered Fabijanski and Rafalski in an attempt to modify Li’s methods as proposed (Remarks, pg. 6).
This is not found persuasive. 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). It is the combination of Li, Fabijanski, and Rafalski that teach methods for introducing and selecting mutations that are largely heritable, not simply the individual disclosures of Fabijanski and Rafalski. Applicant is reminded that the teachings of Li encompass all the “raw materials” that are required by Claim 1, including the RNA-guided DNA endonuclease, guide RNA, T0 generations of plants, and the crossing of plants comprising exogenous DNA with isogenic wild-type lines not comprising the exogenous DNA. As such, the disclosures of Fabijanski and Rafalski were not cited to provide a rationale of modifying Li to include the plants or methods of Fabijanski or Rafalski, because Li already discloses all of the elements necessary to accomplish the method in the instant claims. The teachings of Fabijanski were cited to submit a disclosure to show that using regenerated T0 plants (like those already taught in Li), rather than T1 plants taught by Li, as the first generation of plants in a cross with another plant is routine and obvious prior to the effective filing date of the claimed invention. It should also be noted that the inclusion of Rafalski is merely meant to provide evidence that one of ordinary skill in the art would have found it routine and well known in the art to introgress a desirable character or trait (gene) from a donor parent plant (such as the T0 generation of plants taught by Li) into an otherwise agronomically acceptable cultivar (such as the wild-type isogenic line taught by Li).
Applicant also argues that the crossed plants disclosed in Fabijanski are not genetically identical except for the mutant allele of interest because one part is male sterile and the other parent is male fertile (Remarks, pg. 6).
This is not found persuasive. Again, 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). It is the combination of Li, Fabijanski, and Rafalski that teach methods for introducing and selecting mutations that are largely heritable, not simply the individual disclosures of Fabijanski and Rafalski. Applicant is reminded that the teachings of Li encompass all the “raw materials” that are required by Claim 1, including the RNA-guided DNA endonuclease, guide RNA, T0 generations of plants, and the crossing of plants comprising exogenous DNA with isogenic wild-type lines not comprising the exogenous DNA. The teachings of Fabijanski were cited to submit a disclosure to show that using regenerated T0 plants (like those already taught in Li), rather than T1 plants taught by Li, as the first generation of plants in a cross with another plant is routine and obvious prior to the effective filing date of the claimed invention. The fact that the crossed plants disclosed in Fabijanski are not genetically identical except for the mutant allele of interest, is not material to the obviousness rationale set forth in the above rejection because Examiner is not relying on the disclosure of Fabijanski to teach the element of Claim 1, wherein an isogenic line is crossed with the T0 plant. Li already teaches these elements (T0 plants and crossing other generations of plants with isogenic wild-type lines not comprising the exogenous DNA) and Fabijanski is cited to support the fact that using the earliest generation of plants comprising the exogenous DNA (like the regenerated T0 plants already taught in Li), rather than T1 plants taught by Li, as the first generation of plants in a cross with a plant (such as the wild type isogenic lines taught by Li) is routine and obvious prior to the effective filing date of the claimed invention. As further evidence for how routine crossing a regenerated T0 plant is to a isogenic wild-type line, Examiner would like to draw Applicant’s attention to the disclosure of Cigan et al. (WO 2017070032 A1, published 04/27/2017) which teaches regenerated T0 plants derived from Hi-ll immature maize embryos that were crossed with wild type Hi-ll plants and the progeny plants were used for segregation analysis. Sexual transmission of mutated alleles at the expected Mendelian segregation (1:1) was demonstrated in all progeny plants analyzed (pg. 127, lines 1-5). In other words, Cigan teaches that crossing T0 maize plants with isogenic wild type maize plants is routine in the art, for the purpose of producing T1 seeds, wherein the T0 plants and the T1 progeny plants from the seeds can be analyzed for Mendelian segregation frequencies of the mutation, and confirmed for proper inheritance of the mutation and expected (1:1) segregation of the mutated alleles. As such, Applicant is not even the first in the art to use T0 plants in a cross with a isogenic wild-type line in maize, let alone any plant known in the art.
Regarding the Applicant’s disclosure of unexpected results as they relate to crossing the T0 plants with isogenic plants not comprising the exogenous DNA can finding a much higher number of produced mutant plants, Applicants Remarks on pgs. 6-7 do not overcome these rejections. In particular, Applicant’s Remarks rely on the premise that the “much higher number of mutant plants can be obtained by crossing the T0 plants with isogenic plants not comprising the exogenous DNA and a much higher number of seedlings derived from seeds of these crosses can be easily screen” could not be made obvious by the combination of teachings.
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 does not provide any quantitative data to support their claim of unexpected results. Applicant does not provide any quantitative data in the instant specification or drawings that Examiner can access to analyze the practical and statistical significance of the results they are claiming. Applicant has only provided statements in the specification (paragraphs 79-80) and Remarks (pgs. 6-7) that claim unexpected results which are not substantiated with any numerical evidence that can be found in the instant application. Applicant does not provide or define a control that the unexpected results are compared to, to determine the presence of unexpected quantitative data (Essentially, a much higher number of mutant plants compared to what? How many mutant plants would be considered a “higher” number?). It should also be noted that the unexpected results are not reflected in the claim language. Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993). Without any evidence to the contrary, the combination of Li, Fabijanski, and Rafalski would have a reasonable expectation of success in creating some population of progeny plants with the heritable mutation.
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, the evidence does not outweigh the obviousness rational recited in the 35 U.S.C. 103 rejection. The 35 U.S.C. 103 rejection above provides teachings that crossing T0 plants to isogenic wild-type plants is known in the art (based on the combination of Li and Fabijanski and Rafalski) , and that it routine and well known in the art to introgress a desirable character or trait (gene) from a donor parent (such as the regenerated T0 plants taught in Li) into an otherwise agronomically acceptable cultivar (such as the isogenic wild-type lines taught in Li) to create a population of progeny plants that are genetically identical to the recurrent parent except for the fact that the progeny would also have inherited the desirable trait or gene. As such, without any quantitative data to analyze for practical and statistical significance, the claim of unexpected results made by Applicant are not deemed to outweigh the evidence of obviousness in the modified rejections above.
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 the instant case, Applicant’s evidence [statements in the specification (paragraphs 79-80) and Remarks (pgs. 6-7) that claim unexpected results] is presumably based on results obtained using their method for the introduction and selection of a specific heritable mutation in specific plant species and specific plant genotypes relative to the claimed T0 plants. In the singular working example taught by Applicant, the species of plant cell is drawn to a single plant species (tomato) and a specific cultivar (Moneymaker), but none of the claims are limited to this plant. Additionally, the instant claims require that the T0 plant is crossed with an isogenic plant not comprising said exogenous DNA, however the single working example does not provide evidence that an isogenic plant was used in the single embodiment of the invention which presumably caused the unexpected results. Applicant only teaches self-fertilizing the T0 plants (paragraph 123) and crossing the T0 plants to a “wild type plant” (paragraph 124). Self-pollinating the T0 plant would necessarily introduce the exogenous DNA into the progeny plants because the pollen of the regenerated T0 plant would comprise the exogenous DNA that was introduced to the T0 plant cell because the T0 pollen would have the same genotype as the rest of the T0 plant, making a cross that doesn’t include an isogenic line as defined by Applicant (See Remarks, pg. 6). As such, it not clear that the instant application is enabled for crossing the T0 plant to an isogenic line not comprising the exogenous DNA unless the wild-type plant in the instant specification (paragraph 124) is actually a considered both a wild-type and isogenic line. As such, the claims are not deemed to be sufficiently tailored to the evidence.
Claims 2-3 are rejected under 35 U.S.C. 103 as being unpatentable over Li et al. (Plant Biotechnology Journal 15.12 (2017): 1566-1576) in view of Fabijanski et al. (DE 69034190 T2, issued 02/23/2006) and Rafalski et al. (Nonmammalian genomic analysis. A practical guide. Academic Press, San Diego (1996): 90-91) as applied to Claim 1 above, and further in view of Cigan et al. (US Patent Application No. 2015/0082478 A1, published 03/19/2015).
The teachings of Li, Fabijanski, and Rafalski as they are applied to Claim 1 are set forth previously herein and are incorporated by reference.
Regarding Claims 2 and 3, Li teaches that, in total, 113 independent transformation-positive plants (T0 plants) were screened and identified from around 1250 regenerated plants based on PCR with oligonucleotide primers to amplify both the Bar and SpCas genes. The transformation rate was approximately 2.26% under bialaphos selection. The relative exogenous gene copy numbers in the transformants were also evaluated; a total of 97 of 113 (85.8%) transformants harboured one to three copies of the gene (Table S1) (pg. 1567, Results, Regeneration of transgenic maize lines harbouring LG1-targeting RGEN). To prove that stable expression of RGEN could be implemented as an in vivo desired-target mutator (DTM), Li teaches that T1 transgenic plants, derived from two T0 events, carrying the RGEN gene-editing machinery were crossed with its wild-type line, ZC01 (Tables 1, S2), creating F1 plants comprising (CK*; T0 plant crossed with ZC01) DTM effects that were further crossed with its genetic background line, the wild-type and isogenic ZC01 (See ZC01 × CF13-1 (CK*) and ZC01 × CF31-8 (CK*) in Table 1). Li discloses that the F1 maize plants (resulting from a cross between a DMT T1 plant stably expressing the RGEN and the both wild-type and isogenic ZC01 maize line) were subsequently crossed again with the both isogenic and wild-type ZC01 line to quantify the frequency of the mutant phenotypes induced by the in vivo DMT effect among the progeny of crosses with its ‘wild-type’ background, as shown in the following table (See all lines in Table S2).
While Li teaches T1 plants derived from two T0 events that are crossed with wild-type plants to provide a plurality of progeny plants, Li does not explicitly teach the limitation of Claims 2-3, wherein T0 plants are crossed with wild-type plants to provide a plurality of progeny plants.
However, Cigan et al. teaches that crossing T0 maize plants with wild type maize plants is routine in the art, for the purpose of producing T1 seeds, wherein the T0 plants and the T1 progeny plants from the seeds can be analyzed for Mendelian segregation frequencies of the mutation, and confirmed for proper inheritance of the mutation and expected (1:1) segregation of the mutated alleles (paragraph 0826).
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention to modify the maize plants that are crossed in the first generation of the DMT method taught by Li to cross the T0 plants taught by Li with the wild-type plants taught by Li, rather than the T1 plants that are derived from the T0 plant, to have a first generation of T1 progeny seeds and plants that are the result of crossing the transformed plant (T0) with an wild-type line as taught by Cigan. Based on the teachings of Cigan et al., one of ordinary skill in the art would have been motivated to cross the T0 (transformed) plants taught by Li with the wild-type plants taught by Li, to have progeny plants that are the result of crossing the transformed plant with an wild-type line to have a step that allows for confirmation of proper inheritance of the heritable mutation and confirmation of expected Mendelian segregation (paragraph 0826). 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.
Therefore, for all the reasons above, the claimed invention is prima facie obvious.
Response to Arguments
Applicants Remarks on pgs. 7-8 in the reply filed on 09/23/2025 are acknowledged but do not overcome these rejections for the reasons given above in regards to the 35 U.S.C. 103 rejection applied to Claims 1 and 4-11.
Claim 12 is rejected under 35 U.S.C. 103 as being unpatentable over Li et al. (Plant Biotechnology Journal 15.12 (2017): 1566-1576) ) in view of Fabijanski et al. (DE 69034190 T2, issued 02/23/2006) and Rafalski et al. (Nonmammalian genomic analysis. A practical guide. Academic Press, San Diego (1996): 90-91) as applied to Claim 10 above, and further in view of Breyer et al. (Critical reviews in plant sciences 33.4 (2014): 286-330).
Claim 12 is drawn to the method of claim 10, wherein the selection marker is kanamycin resistance or ampicillin resistance.
The teachings of Li, Fabijanski, and Rafalski as they are applied to Claim 10 are set forth previously herein and are incorporated by reference.
However, Li, Fabijanski, and Rafalski do not teach the feature of Claim 12, wherein the selection marker is kanamycin resistance or ampicillin resistance.
Regarding Claim 12, Breyer et al. (herein referred to as Breyer) teaches alternative selection markers for efficient selection of transformation events (plant cells having stably integrated the exogenous DNA) in the production of genetically modified plants. Breyer teaches that antibiotic resistance marker genes (ARMGs) has been demonstrated to be very effective for selection, cost-efficient and applicable to a large number of plant species, wherein nptII that encodes the neomycin phosphotransferase enzyme conferring resistance to the aminoglycoside antibiotics neomycin and kanamycin is one of the most commonly used ARMGs (pg. 287, Introduction, right column). Breyer teaches that Genes conferring herbicide resistance inserted into plants may also be used as selective marker genes (SMGs) and provide therefore for a potential alternative to the use of ARMGs, including the Bar gene from Streptomyces hygroscopicus which confers resistance to phosphinothricin (PPT; glufosinate ammonium) (pg. 289, left column, first full paragraph).
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention to substitute the nptII selective marker taught by Breyer in the place of the bar selective marker gene taught by Li. Based on the teachings of Breyer, one of ordinary skill in the art would have been motivated to make this substitution because Breyer explicitly teaches that nptII and bar selective marker genes are functional equivalents of each other and that using both in a method for modifying a plant cell would result in the ability to determine if a plant cell has been effectively transformed. 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 substituted 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. Therefore, for all the reasons above, the claimed invention is prima facie obvious.
Response to Arguments
Applicants Remarks on pg. 8 in the reply filed on 09/23/2025 are acknowledged but do not overcome these rejections for the reasons given above in regards to the 35 U.S.C. 103 rejection applied to Claims 1 and 4-11.
Claim 13 is rejected under 35 U.S.C. 103 as being unpatentable over Li et al. (Plant Biotechnology Journal 15.12 (2017): 1566-1576) ) in view of Fabijanski et al. (DE 69034190 T2, issued 02/23/2006) and Rafalski et al. (Nonmammalian genomic analysis. A practical guide. Academic Press, San Diego (1996): 90-91) as applied to Claim 11 above, and further in view of Hunter et al. (US 20150285713, published 10/08/2015) .
Claim 13 is drawn to the method of claim 11, wherein the part of the progeny plants is a cotyledon.
The teachings of Li, Fabijanski, and Rafalski as they are applied to Claim 11 are set forth previously herein and are incorporated by reference.
However, Li, Fabijanski, and Rafalski does not teach the feature of Claim 13, wherein the part of the progeny plants is a cotyledon.
Regarding Claim 13, Hunter et al. (herein referred to as Hunter) teaches scutellum sampling to obtain DNA for molecular analysis, wherein scutellum tissue from maize was excised from immature embryos of about 2-4 mm in size and then placed on media for DNA extraction (Example 1). Hunter also teaches that scutellum has a high cell density and therefore, more nuclei per unit of tissue as compared to leaf tissue, resulting in a higher DNA concentration. The high density of DNA makes scutellum tissue an excellent source for genomic DNA extraction (paragraph 0047).
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention to include the scutellum tissue taught by Hunter as one of the plant parts that genomic DNA was extracted from in the methods of Li. Based on the teachings of Hunter, one of ordinary skill in the art would have been motivated to target this tissue type in maize plants because it comprises more nuclei per unit of tissue as compared to leaf tissue, resulting in a higher DNA concentration when extracted. 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 substituted 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. Therefore, for all the reasons above, the claimed invention is prima facie obvious.
Response to Arguments
Applicants Remarks on pg. 8 in the reply filed on 09/23/2025 are acknowledged but do not overcome these rejections for the reasons given above in regards to the 35 U.S.C. 103 rejection applied to Claims 1 and 4-11.
Conclusion
No claims are allowed.
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
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/KELSEY L MCWILLIAMS/Examiner, Art Unit 1663
/Amjad Abraham/SPE, Art Unit 1663