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 .
Claim Status
Claims 1, 36, 40, 42, 43, 54-56, 59, 61, 67, 68, 71-73, 75 and 93-98 are pending and examined. Claims 2-35, 37-39, 41, 44-53, 57, 58, 60, 62-66, 69, 70, 74 and 76-92 have been cancelled.
The rejection of claim(s) 36, 40, 54, 55, 56 and 59 under 35 U.S.C. 103 as being unpatentable over Akio et al (JP 2013031429 A) and Arimura et al (Patent No. US 10,822,611 B2) and Li et al (2018, Plant Communications, 2:1-13) in view of Cambell (2021, "Using CRISPR-Cas9 to Construct Knockout Mutants in DNA-Repair Genes in Arabidopsis thaliana" , Honors Theses, University of Nebraska-Lincoln. 321. https://digitalcommons.unl.edu/honorstheses/321) is withdrawn as Campbell does not teach cleaving a target sequence in the mitochondria.
Election/Restrictions
Applicant elected the invention of Group I in the reply filed on 08 December 2025. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
Claim 80 was withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Claim Objections
Applicant is advised that should claim 55 be found allowable, claim 56 will be objected to under 37 CFR 1.75 as being a substantial duplicate thereof. When two claims in an application are duplicates or else are so close in content that they both cover the same thing, despite a slight difference in wording, it is proper after allowing one claim to object to the other as being a substantial duplicate of the allowed claim. See MPEP § 608.01(m).
Appropriate action is advised.
Response to Arguments
Applicant traverses the objection because claim 55 introduces a first and second cassette in to the nucleus of the cell whereas claim 56 expresses a first expression cassette in the nucleus and a second in the mitochondria (Applicant reply dated 17 June 2026, p. 7).
This argument is not persuasive because the claims are so close in content that they both cover the same thing, despite a slight difference in wording. Namely, the expression cassettes of claim 55 comprise mitochondrial targeting peptides, the same which is required of the first expression cassette of claim 56 and presumably the second expression cassette of claim 56 (i.e., a mitochondrial targeting peptide is required to cleave the target sequence in the mitochondrial genome).
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 54-56 and 59 REMAIN rejected, and claims 94-96 are NOW 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 54 is drawn to a method comprising introducing into the mitochondria of a cell a polynucleotide encoding a ptxD and further comprises introducing a donor DNA comprising a second polynucleotide encoding a second polypeptide and a third and fourth polynucleotide for recombining the second polynucleotide in the mitochondria and wherein the method further comprises introducing into the mitochondria a recombinant DNA construct comprising a first and second polynucleotide encoding a guide polynucleotide directing a polypeptide to leave a target sequence in an organelle.
The metes and bounds of the claim are indefinite for omitting essential steps, such omission amounting to a gap between the steps. See MPEP § 2172.01. The omitted steps are: a nexus between the introduction of the ptxD gene of claim 36, the donor DNA of claim 40 and the recombinant DNA construct of claim 54.
Namely, it is not clear what the intended purpose of the recombinant DNA construct is as all this construct does is cleave a target sequence without inserting any polynucleotide into the genome of the mitochondria.
Claims 55, 56 and 59 present the same issue and are therefore rejected for the same reason as provided for claim 54.
Claims 94-96 are rejected for depending upon a rejected base claim and for failing to remedy the issues of indefiniteness.
Response to Arguments
Applicant traverses the rejection of the claims in light of the amendments (Applicant reply dated 17 June 2026, p. 9).
However, this argument is not persuasive for the reasons as set forth above. Namely, claim 54 remains directed to a method where (1) ptxD is introduced into mitochondria and (2) a “second” polynucleotide is introduced into the mitochondria and (3) a polynucleotide encoding a polynucleotide guided polypeptide and a guided polypeptide that cleaves at least one “target” sequence.
However, the specification provides no working examples of simultaneously introducing all of (1)-(3) such that the method of (3) is unclear as it appears to be missing a further step. Namely, if (3) occurs without an additional step it is not clear if the target sequence remains cleaved, or, if a sequence of interest is inserted into said cleaved target sequence, or, if the cleaved target sequence is repaired but mutated.
The only example of (3) is Example 12, where gRNA is used to cleave a target sequence to insert ptxD. However, this example does not use both homologous recombination/(2) as encompassed by claim 40 and gRNA/(3) as encompassed by claim 54.
As such, the claims remain rejected for omitting essential steps, such omission amounting to a gap between the steps. See MPEP § 2172.01.
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1, 36, 40, 42, 43, 54-56, 59, 61, 67, 68, 71-73, 75 and 93 REMAIN rejected, and claims 94-98 are NOW rejected, under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for transforming rice callus with plasmid pNAP256, does not reasonably provide enablement for making and/or using the cells and methods as broadly claimed. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make/use the invention commensurate in scope with these claims.
In In re Wands (8 USPQ2d 1400 (CAFC 1988)), the CAFC considered the issue of enablement in molecular biology. The CAFC summarized eight factors to be considered in a determination of "undue experimentation". These factors include: (a) the quantity of experimentation; (b) the amount of guidance presented; (c) the presence or absence of working examples; (d) the nature of the invention; (e) the state of the prior art; (f) the predictability of the prior art; (g) the breadth of the claims; and (h) the relative skill in the art. The factors are analyzed in turn for the instant case as follows:
Here, the claims broadly encompass editing the mitochondrial genome by replacing the start codon of ptxD with any RNA editing site, and doing so while also introducing any cytoplasmic male sterility (CMS) coding region, and further encompass using any guide polynucleotide and polynucleotide guided polypeptide.
Meanwhile, the specification teaches that ptxD having the coding sequence of SEQ ID NO: 66 was codon optimized for expression in the nucleus of yeast and fused with SEQ ID NO: 67 corresponding to the COX4 gene for mitochondrial targeting to yield SEQ ID NO: 68. This was expressed with the TEF1 promoter corresponding to SEQ ID NO: 69. Expression conferred upon yeast transformants the ability to grow on medium with phosphite as the sole phosphorous source (Example 6).
The specification teaches that ptxD having the coding sequence of SEQ ID NO: 70 was codon optimized for expression in the nucleus of rice and fused with SEQ ID NO: 71 corresponding to the RPS10 gene for mitochondrial targeting to yield SEQ ID NO: 68. This was expressed with the UBI promoter corresponding to SEQ ID NO: 39. Expression conferred upon rice callus transformants the ability to grow on medium with phosphite as the sole phosphorous source (Example 7).
The specification teaches that ptxD having the coding sequence of SEQ ID NO: 70 with codons optimized for rice and targeted to the mitochondria enables rice callus to grow on phosphite medium (Example 7).
The specification also teaches that not all guide polynucleotide function as intended to insert a desired gene into the mitochondria of the cell (e.g., see p. 147, ¶ 0422). Thus, the skilled practitioner would not be unable to predictably practice the methods as broadly encompassed by claims 54-56.
Here, the specification fails to provide working examples or supply the appropriate amount of guidance to practice the methods as broadly claimed. Teachings, guidance and working examples are paramount in light of the fact that the skilled artisan appreciates that mitochondrial transformation is a nascent field that has not been applied to the genus of plant cells types as encompassed by the claims.
For example, Kim teaches that there are very few examples of edited mitochondria and that delivering gene editing tools in to the mitochondria remains a challenge (2023, “From ‘science fiction’ to ‘just hard’: Mitochondrial DNA editing inches closer to reality”, Chemical & Engineering News, https://cen.acs.org/biological-chemistry/gene-editing/science-fiction-just-hard-Mitochondrial-DNA-editing-inches-closer-to-reality/101/i17).
With respect to the CMS coding region as encompassed by claim 43, the skilled artisan would be unable to practice the method as broadly claimed in light of the state of the art which teaches that mitochondrial expression of orf79 is toxic to plant regeneration (Kojima et al, 2010, Plant Biotechnology, 27:111-114; see for example the Abstract).
With regards to claims 54-56, the skilled practitioner would be unable to predictably practice the methods as broadly claimed as the specification teaches that not all guide polynucleotide function as intended to insert a desired gene into the mitochondria of the cell (e.g., see p. 137, ¶ 0422).
Regarding RNA editing sites as encompassed by claims 67 and 68, Kobayashi et al teaches that although a few prediction procedures have been proposed they are not easily accessed and the reliability of the prediction result is ambiguous due to the absence of a quantitative index for reliability evaluation, and that the current PPR code may not be sufficient to predict the base preference of all PPR motifs found in natural PPR proteins due to the high degeneracy of PPR motif sequences including the PPR code generating residues (2019, Plant Cell Physiol., 60:862-874; see p. 863, col. 1, ¶ 1).
Moreover, Kobayashi et al teach that to date only 80 or so mitochondrial RNA editing sites and their corresponding site specific recognition PPR proteins have been characterized and that PPR proteins responsible for a large number of editing sites remains unknown, (p. 871, col. 1, ¶ 1).
Kobayashi et al teach that their results suggest a limitation of target RNA editing site prediction as being solely dependent on the PPR code (p. 817, col. 2, ¶ 1 and last ¶). As such, one would be unable to predictably make and/or use the RNA editing sites as encompassed by claim 67 or the RNA editing sites as encompassed by claim 68 which fail to recite any structure associated with said RNA editing sites.
Therefore, in light of the inadequate teachings and guidance in the specification, the lack of working examples, the breadth of the claims which encompass edited mitochondria in cells from any plant and the unpredictable state of the art, the skilled practitioner would be subjected to impermissible undue trial and error experimentation to make and/or use the claimed invention.
Response to Arguments
Applicant traverses the rejection of the claims because the cells and methods are directed to yeast and plants which are exemplified (Applicant reply dated 17 June 2026, p. 12, ¶ 1 and 2).
This argument is not persuasive because Applicant’s own arguments support the proposition that the claims are not enabled for their full scope even after the amendments: “In fact, it is understood in the art that Agrobacterium cannot be directly used to transform mitochondria” (Applicant reply dated 17 June 2026, p. 19, ¶ 1), yet the claims precisely encompass this method.
Applicant traverses the rejection of claim 43 because Kojima demonstrates nuclear expression of orf79 and because the orf79 gene is inserted into the mitochondria at its natural location behind the apt6 gene (Applicant reply dated 17 June 2026, p. 12, last ¶).
This argument is not persuasive because Kojima reports submitochondrial localization and toxicity of orf79 during plant regeneration (p. 111, col. 2, penultimate ¶), and is also not persuasive because the argument is not commensurate in scope with what is claimed: the claims do not require the insertion of orf79 into any particular location of the mitochondrial genome or its natural location.
In fact, Kojiima specifically teaches the precise localization of orf79 within mitochondria might be an important factor causing pollen sterility (p. 113, col. 2, last ¶).
Applicant’s arguments with respect to Kazama are unpersuasive and misleading. Namely, Applicant refers to Figure 1d as evidence that plants may be obtained with the orf79 gene and the CMS phenotype. However, with respect to Figure 1d and Supplementary Table 1 Kazama teaches “To determine whether male fertility was restored, we self-pollinated T0 plants by bagging their panicles to prevent out-crossing and counted the seed set. Recovery of fertility was observed only in orf79-depleted plants”.
With regard to the guide polynucleotide and claims 54-56 Applicant asserts that methods of identifying functional guide polynucleotides are known by one of skill in the art and do not constitute undue experimentation (Applicant reply dated 17 June 2026, p. 13, ¶ 2).
This is not persuasive because the arguments of counsel cannot take the place of evidence in the record. In re Schulze, 346 F.2d 600, 602, 145 USPQ 716, 718 (CCPA 1965). See MPEP 716.01(c)(II). Here, Applicant merely asserts that one knows how to use guide polynucleotides but fails to address that the instant specification teaches that not all guide polynucleotide function as intended to insert a desired gene into the mitochondria of the cell (e.g., see p. 137, ¶ 0422).
Applicant traverses the rejection of claims 67 and 68 because they have been amended to recite an "endogenous mitochondrial RNA editing site." and thus relates to a known RNA editing site from a given organism, and not a theoretical RNA editing site that might be predicted from a given PPR protein sequence (Applicant reply dated 17 June 2026, p. 9, ¶ 2).
This argument is unpersuasive because the claim is not limited to any particular “known” RNA editing site from a given organism.
Claims 1, 36, 40, 42, 43, 54-56, 59, 61, 67, 68, 71-73, 75 and 93 REMAIN rejected, and claims 94-98 are NOW 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.
Instant claims 1, 36, 40, 42, 43, 54-56, 59, 61, 67, 68, 71-73, 75 and 93-98 are drawn broadly to editing the mitochondrial genome of any plant cell type and replacing the start codon of ptxD with any RNA editing site, and doing so while also introducing any CMS coding region, and further encompass using any guide polynucleotide and polynucleotide guided polypeptide.
Meanwhile, the specification describes that ptxD having the coding sequence of SEQ ID NO: 66 was codon optimized for expression in the nucleus of yeast and fused with SEQ ID NO: 67 corresponding to the COX4 gene for mitochondrial targeting to yield SEQ ID NO: 68. This was expressed with the TEF1 promoter corresponding to SEQ ID NO: 69. Expression conferred upon yeast transformants the ability to grow on medium with phosphite as the sole phosphorous source (Example 6).
The specification describes that ptxD having the coding sequence of SEQ ID NO: 70 was codon optimized for expression in the nucleus of rice and fused with SEQ ID NO: 71 corresponding to the RPS10 gene for mitochondrial targeting to yield SEQ ID NO: 68. This was expressed with the UBI promoter corresponding to SEQ ID NO: 39. Expression conferred upon rice callus transformants the ability to grow on medium with phosphite as the sole phosphorous source (Example 7).
The specification describes that ptxD having the coding sequence of SEQ ID NO: 70 with codons optimized for rice and targeted to the mitochondria enables rice callus to grow on phosphite medium (Example 7).
The specification also describes that not all guide polynucleotide function as intended to insert a desired gene into the mitochondria of the cell (e.g., see p. 147, ¶ 0422). Thus, the skilled practitioner would not be unable to predictably practice the methods as broadly encompassed by claims 54-56.
The written description requirement may be satisfied through sufficient description of a representative number of species by disclosing relevant and identifying characteristics such as structural or other physical and/or chemical properties, by disclosing functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show the applicant was in possession of the invention as claimed. See Eli Lilly,119 F.3d at 1568, 43 USPQ2d at 1406.
Here, the specification fails to describe (1) a representative number of species from the broad genus of plant cells comprising an edited mitochondrial genome; (2) a representative number of plant cells comprising a CMS coding region; (3) a representative number of guide polynucleotides with functional activity; and (4) a representative number of species from the broad genus of RNA editing sites that retain functional activity.
Regarding (1), a description of a representative number of plant cells as claimed is critical in light of the fact that the skilled artisan appreciates that mitochondrial transformation is a nascent field that has not been applied to the genus of cell types as encompassed by the claims. For example, Kim describes that there are very few examples of edited mitochondria and that delivering gene editing tools in to the mitochondria remains a challenge (see entire document).
With respect to (2) and the CMS coding region as encompassed by claim 43, the skilled artisan would not be of the opinion Applicant possess the method as broadly claimed in light of the state of the art which describes that mitochondrial expression of orf79 is toxic to plant regeneration (Kojima et al; see Abstract). Moreover, the specification has failed to describe the applicability of introducing a CMS coding region into cells other than those from plants.
With regard to (3) and claims 54-56, the skilled practitioner not be of the opinion that Applicant was in possession of the methods as broadly claimed as the specification describes that not all guide polynucleotide function as intended to insert a desired gene into the mitochondria of the cell (e.g., see p. 147, ¶ 0422).
Regarding (4) and claims 67 and 68, the specification has defined RNA editing sites by function rather than structure. Namely, rather than identify shared characteristics of RNA editing sites, the specification has merely presented a limited number of RNA editing sites via sequence identifiers such that the skilled artisan would be unable to distinguish between those RNA editing sites that have function and those that do not.
This description is critical in light of the state of the art which describes that although a few prediction procedures have been proposed they are not easily accessed and the reliability of the prediction result is ambiguous due to the absence of a quantitative index for reliability evaluation, and that the current PPR code may not be sufficient to predict the base preference of all PPR motifs found in natural PPR proteins due to the high degeneracy of PPR motif sequences including the PPR code generating residues (Kobayashi et al, p. 863, col. 1, ¶ 1).
Moreover, Kobayashi et al describe that to date only 80 or so mitochondrial RNA editing sites and their corresponding site specific recognition PPR proteins have been characterized and that PPR proteins responsible for a large number of editing sites remains unknown, (p. 871, col. 1, ¶ 1).
Kobayashi et al describe that their results suggest a limitation of target RNA editing site prediction as being solely dependent on the PPR code (p. 817, col. 2, ¶ 1 and last ¶). As such, one would be of the opinion that Applicant was in possession of the RNA editing sites as encompassed by claim 67 or the RNA editing sites as encompassed by claim 68 and which fail to recite any structure associated with said RNA editing sites.
Therefore, in light of the failure of the specification to describe a representative number of plant cells, CMS coding regions, guide polynucleotides and RNA editing sites from the genera as claimed, and the state of the art as noted supra, the skilled practitioner would not be led to believe that Applicant possessed the cells and methods as broadly claimed.
Response to Arguments
Applicant traverses the rejection of the claims because the specification describes a donor DNA comprising a second polynucleotide, including a CMS coding region, and integration of such second polynucleotide into the endogenous mitochondrial DNA sequence, because the alleged toxicity of CMS-associated sequences do not demonstrate lack of written description, and because the specification describes the relevant structural features and functional relationship of the donor DNA, homologous recombination sequences, mitochondrial integration, and selection of cells comprising the edited mitochondrial genome (Applicant reply dated 17 June 2026, p. 14)
This argument is not persuasive because the specification fails to describe, in fact, a representative number of species from the broad genus of plant cells comprising an edited mitochondrial genome a representative number of plant cells comprising a CMS coding region or a representative number of guide polynucleotides with functional activity.
Applicant argues with respect to claims 54-56 that guide polynucleotides and polynucleotide-guided polypeptides that cleave target sequences in the mitochondrial genome and can facilitate recovery of cells comprising an edited mitochondrial genome, that the claims do not require possession of every possible guide polynucleotide. Rather, the claims recite guide polynucleotides having the claimed functional relationship with the polynucleotide-guided polypeptide and mitochondrial target sequence (Applicant reply dated 17 June 2026, p. 14, last ¶)
However, this argument is not persuasive because the written description requirement may be satisfied by describing (1) a representative number of species by disclosing relevant and identifying characteristics such as structural or other physical and/or chemical properties or (2) by disclosing functional characteristics coupled with a known or disclosed correlation between function and structure.
Here, the specification fails to do either, and specifically with respect to (2), the specification fails to describe the actual structures that confer the claimed functional activity of guiding and cleaving.
Applicant argues with respect to claims 67 and 68, Applicant has amended the claims to recite an "endogenous mitochondrial RNA editing site." such that the claims are not directed to an open-ended genus of theoretical or predicted RNA editing sites. Applicant argues claim 68 further narrows the endogenous mitochondrial RNA editing site to one from a mitochondrial nad4L gene or a mitochondrial cox2 gene for which the specification provides a description (Applicant reply dated 17 June 2026, p. 15, ¶ 1).
This argument is unpersuasive because the claim is not limited to any particular “known” RNA editing site from a given organism.
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claim(s) 1, 36 and 93 REMAIN rejected, and claims 97 and 98 are NOW rejected, under 35 U.S.C. 102(a)(1) as being anticipated by Akio et al (JP 2013031429 A).
Instant claims 1, 36, 93, 97 and 98 are drawn to a rice cell comprising mitochondria comprising an exogenous pxtD and a method comprising introducing into the mitochondria of a cell a polynucleotide encoding a ptxD, and wherein the cell is a wheat, maize or rice cell.
Akio et al disclose that phosphorous is an important element as a nutrient source for plants and is applied in the form of phosphoric acid but is wasted because it is insolubilized. To remedy this problem in the prior art Akio et al disclose expressing ptxD in a plant conferring the ability to metabolize phosphorous (see Abstract; see ¶ 0030). This gene may be incorporated into the chromosome of mitochondria (¶ 0057).
Akio et al disclose, in fact, that transformed Arabidopsis comprising ptxD was capable of metabolizing phosphoric acid as a nutrient source (¶ 0154). Akio et al claims methods for doing so, and that said method may be practiced in any commercially important plant, including rice cells (see claims 1-4; see ¶ 0013; see also ¶ 0064).
Akio et al disclose this method reduces the cost of producing the plant in addition to reducing soil contaminants, and that applying the method can lead to the selective growth of a desired plant by preventing growth of unwanted plants that are incapable of metabolizing phosphorous acid (¶ 0021 and 0023).
Therefore, a cell comprising mitochondria comprising an exogenous pxtD and a method comprising introducing into the mitochondria of a cell a polynucleotide encoding a ptxD, and wherein the cell is a wheat, maize or rice cell is anticipated by Akio et al.
Response to Arguments
Applicant traverses the rejection of the claims because Kuroda does not describe a cell comprising an edited mitochondrial genome comprising an exogenous polynucleotide encoding phosphite dehydrogenase and does not describe growing the cell under conditions in which the phosphite dehydrogenase is produced, growing the cell in a medium wherein phosphite is present as a primary phosphorus source, and selecting a cell comprising an edited mitochondrial genome comprising the first polynucleotide (Applicant reply dated 17 June 2026, p. 16, ¶2).
This argument is unpersuasive because Akio et al disclose expressing ptxD in a plant conferring the ability to metabolize phosphorous and that it may be incorporated into mitochondria (see Abstract; see ¶ 0030; see also ¶ 0057).
Applicant asserts Kuroda in paragraph [0057] relates to a generic statement as shown in a machine translation, which states: "The chromosome of the host plant into which the gene is incorporated by Agrobacterium infection may be a nuclear chromosome, may be a mitochondrial chromosome, and is not particularly limited." which is a single speculative or unsupported sentence mentioning possible mitochondrial chromosome insertion that is not an enabling anticipatory disclosure (Applicant reply dated 16 June 2026, p. 16, last ¶ and p. 17, ¶ 1).
This argument is not persuasive because on the one hand Applicant argues Kuroda does not disclose mitochondrial transformation but on other hand asserts the art mentions possible mitochondrial chromosome insertion. Thus, Kuroda is deemed to disclose plant mitochondrial transformation based on Applicant’s own statement.
Applicant argues the Office acknowledges that "mitochondrial transformation is a nascent field." And that Kuroda is not accompanied by any working example, protocol, construct, targeting sequence, selection method, confirmation of mitochondrial insertion, or data showing that Agrobacterium-mediated transformation introduced ptxD into a mitochondrial genome. At most, the recited description is a broad, unsupported statement that a chromosome into which the gene is incorporated may be a mitochondrial chromosome (Applicant reply dated 16 June 2026, p. 17, ¶ 1).
This argument is not persuasive because the standard for anticipatory art is not whether any working example, protocol, construct, targeting sequence, selection method, confirmation or data is shown.
Rather, when the reference relied on expressly anticipates or makes obvious all of the elements of the claimed invention, the reference is presumed to be operable. Once such a reference is found, the burden is on applicant to rebut the presumption of operability. In re Sasse, 629 F.2d 675, 207 USPQ 107 (CCPA 1980). see MPEP 2121(I). Here, Applicant has failed to provide any evidence that the prior art is not enabling on its face.
Applicant asserts Kuroda expressly refers to Agrobacterium infection as the method by which the gene is incorporated which is understood to introduce DNA to the nucleus through the action of VirD2 and VirE2 proteins, which possess nuclear localization signals (see Li et al. 2020, Proc. Natl. Acad. Sci. USA 117:26389-26397) and that Kuroda provides no mechanism or basis for a gene to be integrated into the mitochondrion by infection with Agrobacterium. In fact, it is understood in the art that Agrobacterium cannot be directly used to transform mitochondria (Applicant reply dated 17 June 2026, ¶ 2).
This argument is not persuasive because it is not commensurate in scope with what is claimed. Namely, Claims 1 and 36 do not preclude the use of Agrobacterium. The Office’s position is supported by newly added claims 94, 95 and 96 which are expressly limited to introducing DNA via particle bombardment.
Claim Rejections - 35 USC § 103
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.
Claim(s) 36, 40, 42, 43, 61, 71, 72, 73 and 75 REMAIN rejected, and claims 98 is NOW rejected, under 35 U.S.C. 103 as being unpatentable over Akio et al (JP2013031429) in view of Arimura et al (Patent No. US 10,822,611 B2) and in further view of Li et al (2018, Plant Communications, 2:1-13).
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.
Instant claims 36, 40, 42, 43, 61, 71, 72, 73, 75 and 98 are drawn to introducing into a mitochondrion a first polynucleotide encoding pxtD and further comprises introducing a donor DNA comprising a second polynucleotide encoding a second polypeptide and a third and fourth polynucleotide for recombining the second polynucleotide in the mitochondria wherein the second polynucleotide comprises a CMS coding region or a selectable marker providing tolerance to a selective agent, wherein the cell is grown simultaneously or sequentially in the presence of the selectable marker and phosphite, wherein the selectable marker is HPT, and wherein at least 50% of the mitochondrial genomes comprise the edited mitochondrial genome.
Akio et al teach that phosphorous is an important element as a nutrient source for plants and is applied in the form of phosphoric acid but is wasted because it is insolubilized. To remedy this problem in the prior art Akio et al teach expressing ptxD in a plant conferring the ability to metabolize phosphorous (see Abstract; see ¶ 0030). This gene may be incorporated into the chromosome of mitochondria (¶ 0057).
Akio et al teach, in fact, that transformed Arabidopsis comprising ptxD was capable of metabolizing phosphoric acid as a nutrient source (¶ 0154). Akio et al claims methods for doing so, and that said method may be practiced in any commercially important plant such as rice (see claims 1-4; see also ¶ 0064). Homologous recombination may be used to introduce the desired gene and selectable markers such as kanamycin may be included in the plasmid used for transformation (¶ 0055 and 0056; see ¶ 0094).
Akio et al teach this method reduces the cost of producing the plant in addition to reducing soil contaminants, and that applying the method can lead to the selective growth of a desired plant by preventing growth of unwanted plants that are incapable of metabolizing phosphorous acid (¶ 0021 and 0023). Thus, Akio et al teaches selecting for plant cells with edited mitochondria.
Thus while Akio et al clearly teaches, suggests and provides motivation for introducing pxtD into the mitochondria of plants, the issue is whether one would have also introduced an additional polynucleotide for CMS into the mitochondria of the plant.
To this point, Arimura et al teaches it would be desirable to establish a novel method for creating CMS and that if the mitochondrial genome of crops could be directly modified and CMS could be imparted thereto, a variety of CMS would be imparted to a variety of crops and varieties such that CMS could be promptly introduced therein without disturbing the nucleus or the chloroplast genomic sequence, thereby greatly contributing to increased production of crops, stable production, etc. (col. 2, ¶ 1).
Arimura et al teach mito-TALENs to express in rice plants while targeting orf79, and that a plant-derived mitochondrial localization peptide as a signal peptide for transferring TALENs into plant mitochondria should be used to transfer mito-TALENs into plant mitochondria (col. 4, ¶ 1). HPT may be used as a selection marker (e.g., see col. 19, ¶ 1).
Arimura et al teach that methods for introducing a double-strand break into mitochondrial genomic DNA are known in the art and include a method of using ZFN, a method of using CRISPR-Cas9, a method of using TALEN and a method of using various types of restriction enzymes with the method of using TALEN being the most preferred (col. 8, ¶ 1).
Arimura et al teach that when mitochondrial genomic DNA is cleaved using TALEN it is necessary to introduce a gene encoding the TALEN into a nuclear genome, and then to transfer the TALEN expressed in the cytoplasm into the mitochondria and can be obtained by fusing TALEN with a mitochondrial localization signal peptide (col. 8, ¶ 3).
Arimura et al teach that in order to reliably introduce a double-strand break into mitochondrial genomic DNA, a tandem expression Ti plasmid, in which two TALENs (TALEN left and TALEN right; see FIG. 1A) are simultaneously expressed in a single Ti plasmid, and to which a mitochondrial localization signal is added in order to localize it in mitochondria (col. 9, ¶ 1). Thus Arimura et al teach a second, third and fourth polynucleotide as encompassed by the method of claim 40.
Li et al teaches that global demand for crops is increasing and that to feed the rapidly rising population in the face of decreased arable land food production must grow in parallel with reduced inputs. Li et al teaches conventional plant breeding is relatively labor intensive and time consuming whereas genetic engineering is believed to boost crop productivity (p. 1, col. 1).
Plastid transformation to make transgenic plants has been offered as an alternative to nuclear transformation, and the similarity between plastids and mitochondria raise the possibility that the latter could also be transformed (p. 1, col. 1; see also p. 2, col. 2, ¶ 1). However, Li et al teach that the lack of selectable markers for mitochondrial transformation adds to the challenge of mitochondrial DNA editing (p. 9, last ¶). Thus Li et al provides the implicit suggestion that there is a need in the art for selection markers for plant mitochondrial transformation.
Therefore, prior to the effective filing date of the instant invention it would have been prima facie obvious to one of ordinary skill in the art to modify by the method as taught by Akio et al for mitochondrial expression of ptxD to also express a CMS coding region because Akio et al specifically teaches and suggests that additional selection markers may be used.
Thus, by expressing both ptxD and a CMS gene one would impart a feature into a crop plant that reduces cost for production and at the same time provide the additional benefit of another means to select successful transformants while further assisting in breeding new varieties of crops.
One would be motivated to do this additional selection step as Li et al teach that the lack of selectable markers for mitochondrial transformation adds to the challenge of mitochondrial DNA editing. Thus, by applying the teachings of Arimura et al and Li et al to the method of Aiko et al one would be solving the issue of needing markers to select for mitochondrial transformation.
One would have a reasonable expectation of success in doing so because Arimura et al teaches, in fact, the successful knocking out of a CMS gene using mito-TALENs (e.g., see col. 19 and 20).
Response to Arguments
Applicant traverses the rejection of the claims for the same reasons as stated above: Kuroda does not teach or disclose the elements of the instant claims (Applicant reply dated 17 June 2026, p. 18, last ¶).
This argument is not persuasive because Kuroda unequivocally suggests mitochondrial transformation, and provides motivation for expressing ptxD in a rice plant cell.
Applicant’s argument is also not persuasive because Kuroda need not teach every element of the claims as they are not rejected for being anticipated but are rejected for being obvious in light of the art.
Applicant returns the argument that Kuroda is directed to Agrobacterium mediated transformation (Applicant reply dated 17 June 2026, p. 18, last ¶). This argument is not persuasive and has been addressed above: Claims 1 and 36 do not preclude the use of Agrobacterium. The Office’s position is supported by newly added claims 94, 95 and 96 which are expressly limited to introducing DNA via particle bombardment.
In any event, Kuroda teaches the use of particle bombardment for the introduction of exogenous DNA and provides motivation for doing so (e.g., see ¶ 0061 and 0062).
Applicant traverses the rejection of the claims by presenting what Arimura and Li do or do not teach (Applicant reply dated 17 June 2026, p. 19). This argument is not persuasive because one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Rather than explain why it would not have been obvious to modify the method of Kuroda in view of Arimura and Li, Applicant instead merely argues that the teachings of Arimura and Li fail to remedy the purported deficiencies of Kuroda.
Applicant argues that Li teaches away from the instant claims and the instant invention solves long-felt need in the art (Applicant reply dated 17 June 2026, p. 20, ¶ 1), but this argument is not persuasive because as Applicant is aware "the prior art’s mere disclosure of more than one alternative does not constitute a teaching away from any of these alternatives because such disclosure does not criticize, discredit, or otherwise discourage the solution claimed…." In re Fulton, 391 F.3d 1195, 1201, 73 USPQ2d 1141, 1146 (Fed. Cir. 2004). see MPEP 2141.02(VI).
Here, Li does not criticize, discredit, or otherwise discourage arriving at the instantly claimed subject matter and thus cannot be considered to teach away from modifying Kuroda to arrive at the instant invention.
Applicant’s arguments with respect to there being a long-felt need is unpersuasive as Applicant has not demonstrated the specific need for mitochondrial expression of ptxD. Thus, it is immaterial that Applicant is unaware of any report of the use of ptxD for plant mitochondrial transformation.
Claim(s) 36, 67 and 68 REMAIN rejected under 35 U.S.C. 103 as being unpatentable over Akio et al (JP2013031429) in view of Yoo et al (2020, PeerJ, 1-29, 8:e8362 DOI 10.7717/peerj.8362) as evidenced by Kobayashi et al (2019, Plant Cell Physiology, 60(4):862-874) and Zheng et al (2020, Plants, 9:1-14; doi:10.3390/plants9101277).
Instant claims 36, 67 and 68 are drawn broadly to a method comprising introducing into the mitochondria of a cell a polynucleotide encoding a ptxD wherein a sequence encoding a start codon of ptxD is replaced with a RNA editing site from a mitochondrial cox2 gene.
Akio et al teach that phosphorous is an important element as a nutrient source for plants and is applied in the form of phosphoric acid but is wasted because it is insolubilized. To remedy this problem in the prior art Akio et al teach expressing ptxD in a plant conferring the ability to metabolize phosphorous (see Abstract; see ¶ 0030). This gene may be incorporated into the chromosome of mitochondria (¶ 0057).
Akio et al teach, in fact, that transformed Arabidopsis comprising ptxD was capable of metabolizing phosphoric acid as a nutrient source (¶ 0154). Akio et al claims methods for doing so, and that said method may be practiced in any commercially important plant (see claims 1-4). Selectable markers may be included in the plasmid used for transformation (¶ 0056).
Akio et al teach this method reduces the cost of producing the plant in addition to reducing soil contaminants, and that applying the method can lead to the selective growth of a desired plant by preventing growth of unwanted plants that are incapable of metabolizing phosphorous acid (¶ 0021 and 0023).
Thus, while Akio et al reasonably teaches, suggests and provides motivation for introducing ptxD into the mitochondria of plants, the issue is whether one would have done so by replacing the start codon of said gene with an RNA editing site of the cox2 gene.
To this point, Yoo et al teach the use of the cox2 promoter for expression of Cas9 in mitochondria (see Table 1; see also Table 3). Yoo et al specifically teach using the 71 bp-long minimal promoter of said gene (p. 7, last ¶ bridging p. 8).
Meanwhile, Kobayashi et al teaches that cox2 is known to have an RNA editing site (p. 863, col. 2, last ¶), while Zheng et al teach that RNA editing is known to modulate transcript stability and translation efficiency and have proved to improve the stability of functionally relevant secondary structure motifs (see p. 2, ¶ 1; see also p. 6, ¶ 1).
Therefore, prior to the effective filing date of the instant invention it would have been prima facie obvious to one of ordinary skill in the art to modify the method of introducing a ptxD gene into mitochondria as taught by Akio et al by replacing the start codon with an RNA editing site, for example, as taught by Yoo et al because the cox2 RNA editing site has been successfully used to express transgenes of interest.
One would be motivated to do so because it was known that RNA editing is known (p. 863, col. 2, last ¶), while Zheng et al teach that RNA editing is known to modulate transcript stability and translation efficiency and have proved to improve the stability of functionally relevant secondary structure motifs.
Response to Arguments
Applicant traverses the rejection of the claims because the teachings of Kobayashi and Zheng do not cure the purported deficiencies of Kuroda or Yu (Applicant reply dated 17 June 2026, p. 22, ¶ 1).
In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Conclusion
No claim is 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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/JASON DEVEAU ROSEN/Primary Examiner, Art Unit 1662
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