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
1. Claims 47-50, 52-53, 67, and 69-75 are pending and examined to the extent of the elected species.
Claims 42-46 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on December 15, 2025.
Restrictions/Elections
2. The Office acknowledges receipt of Applicant’s restriction election filed December 15, 2025. Applicant elects Group II, claims 47-50, 52-53, 67, and 69-75, drawn to a method for producing a Beta vulgaris plant or plant cell having an engineered protoporphyrinogen oxidase 2 (PPO2) protein comprising an amino acid substitution at position 126 of SEQ ID NO:3 and a plant produced thereby, without traverse. Applicant further elects the species of the in-frame deletion of glycine at a position corresponding to 208 and/or 209 for the additional mutation of claims 48-50, SEQ ID NO:69, SEQ ID NO:93, and SEQ ID NO:15, without traverse.
Priority
3. This application is a 371 of PCT/IB2022/058290 filed on September 02, 2021. The Office acknowledges receipt of Applicant’s domestic priority document Provisional App. No. 63/240,271 filed September 02, 2021.
Information Disclosure Statement
4. The information disclosure statements (IDS) submitted on October 29, 2024, October 20, 2025, and December 16, 2025 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements have been considered. Signed copies are attached.
Specification
5. The disclosure of the specification is objected to because of the following:
The use of the term “BLAST®” (p. 21, ln. 31), which is a trade name or a mark used in commerce, has been noted in this application. The term should be accompanied by the generic terminology; furthermore, the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Appropriate correction is required.
Abstract
6. The Abstract of the specification is objected to because of the following:
The abstract of the disclosure does not commence on a separate sheet in accordance with 37 CFR 1.52(b)(4) and 1.72(b). A new abstract of the disclosure is required and must be presented on a separate sheet, apart from any other text.
Applicant is reminded of the proper language and format for an abstract of the disclosure.
The abstract should be in narrative form and generally limited to a single paragraph on a separate sheet within the range of 50 to 150 words in length. The abstract should describe the disclosure sufficiently to assist readers in deciding whether there is a need for consulting the full patent text for details.
The language should be clear and concise and should not repeat information given in the title. It should avoid using phrases which can be implied, such as, “The disclosure concerns,” “The disclosure defined by this invention,” “The disclosure describes,” etc. In addition, the form and legal phraseology often used in patent claims, such as “means” and “said,” should be avoided (e.g., “producing said plants” in lns. 2-3).
Claim Objections
7. Claims 47-50, 52-53, 67, and 69-75 are objected to because of the following:
If the “PPO2” of claim 47 is “protoporphyrinogen oxidase 2”, it is recommended Applicant amend claim 47 such that the meaning of the PPO2 acronym is spelled out in full with the acronym in parentheses at its first recitation within the claims. Alternatively, if the “PPO2” of claim 47 is not protoporphyrinogen oxidase 2, then the recitation of PPO2 is unclear and claim 47 may be found indefinite under 35 U.S.C. 112(b).
Claim 47 contains a grammatical error in the recitation of “comprising: introducing”. The colon is superfluous and should be removed because there is only a single embodiment described after the colon.
Regarding claim 47, it is suggested Applicant insert the phrase “at the position” between “amino acid substitution” and “corresponding to” in ln. 3 for the purpose of grammatical clarity.
Regarding claim 48, it is suggested Applicant insert the term “position” after each of the recitations of “a position corresponding to” in lns. 4-9. It is suggested Applicant make similar amendments to claims 67, 70, 72, and 75.
Applicant is advised that should claim 52 be found allowable, claim 67 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). Though claim 52 includes an additional limitation requiring the produced plant be resistant or tolerant to one or more herbicides, the Office notes that Applicant’s disclosure indicates increased PPO2 herbicide resistance as the goal and function of the method of claim 47 (i.e., the targeted substitution of position 126 of SEQ ID NO:3)[00005], [00138-00141], [00146], [00148]. Therefore, the plant of claim 67, which comprises a substitution of position 126 of SEQ ID NO:3 must inherently comprise resistance and/or tolerance to one or more herbicides. If the plant of claim 67 does not necessarily comprise increased herbicide resistance and/or tolerance, then the method of claim 47 may be deemed unpredictable and subject to further rejection under 35 U.S.C. 112(a).
Regarding claim 70, there is a grammatical error; it is recommended Applicant amend “genomic region corresponding to between position 3653 and 3698” to “genomic region corresponding to the region between positions 3653 and 3698”.
Regarding claim 74, it is recommended Applicant amend “wherein the PPO2 protein is at least 99% identical to” to “wherein the PPO2 protein shares at least 99% sequence identity with” to clarify that is the sequences of the recited proteins that are being compared.
Dependent claims are included.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
8. 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.
9. Claims 48-50 and 70-75 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
The metes and bounds of claim 48 are indefinite because it is unclear if the recited method comprises a single introduced nucleic acid or multiple introduced nucleic acid. The recitation of a plant or plant cell further comprising a nucleic acid suggests the introduction of an additional nucleic acid. Applicant is required to clarify the intended recitation. Does claim 48 recite the introduction of an additional nucleic acid?
The metes and bounds of claim 70 are undefined because it is unclear if the recitation of “wherein the method comprises” is intended to clarify the “introducing” step recited in ln. 2 of claim 47 or is intended to recite an additional claim embodiment.
Regarding claim 74, the antecedent basis of “the PPO2” protein in ln. 3 is unclear. Does said recitation refer to the “engineered PPO2” protein in claim 74, ln. 2, the wildtype PPO2 protein (i.e., SEQ ID NO:3), or to the “engineered PPO2” protein of claim 47, lns. 1-2?
Dependent claims are included.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(a)
10. The following is a quotation of the first paragraph of 35 U.S.C. 112(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.
11. Claims 47-50, 52-53, 67, and 69-75 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Regarding claims 47 and 67, Applicant has not provided a representative number of PPO2 polypeptides comprising substitution of the amino acid at the position corresponding to position 126 of SEQ ID NO:3 and having increased resistance to PPO inhibitor herbicides to reasonably convey possession of the claimed invention commensurate in scope with the claims.
Claims 47 and 67 encompass transgenic B. vulgaris plants comprising a PPO2 polypeptide comprising an amino acid substitution at the position corresponding to position R126 of SEQ ID NO:3. The claims encompass any substitution at position R126 and therefore encompass 19 amino acids. The recited PPO2 polypeptide is not limited to SEQ ID NO:3 or peptides obtained from any specific plant species and is herein interpreted to encompass any and all PPO2 polypeptides which share the proteins regions identified in the art to be conserved among PPO2 proteins (Rangani et al., Frontiers in plant science. 2019; 10:568 (U); p. 05, Figure 1). Accordingly, the claims encompass PPO2 proteins from at least 12 species and comprising 19 potential amino acid variations in each protein; thus, the claims encompass at least 228 distinct PPO2 proteins. The limitation regarding any PPO2 proteins comprising any substitution at the position corresponding to R126 of SEQ ID NO:3 is broad and lacks adequate written description.
The Office notes that the claims are silent as to whether the recited method of claim 47 results in the production of a plant that is resistant to PPO inhibitor herbicides and as to whether the plant of claim 67 is resistant to PPO inhibitor herbicides. However, if the method of claim 47 does not result in the production of a PPO inhibitor herbicide resistant plant as indicated in the specification, then claim 47 and its dependents will be assessed for utility under 35 U.S.C. 101. Likewise, if the plant of claim 67 is not resistant to PPO inhibitor herbicides as indicated in the specification, then claim 67 will be assessed for utility under 35 U.S.C. 101.
Applicant describes the generation of transgenic Beta vulgaris protoplasts, calli, and plants comprising a Cas9- or Cpf1-targeted gene mutation to introduce an arginine-to-leucine substitution of the amino acid at the position corresponding to position 126 of SEQ ID NO:3 of a PPO2 protein (see: Figure 4)[00136-00137], [00142]. Applicant also indicates the presence of at least one PPO2 gene allele comprising the R126L mutation confers resistance to PPO inhibitor herbicides[00138-00141], [00146], [00148]. Applicant discloses the tolerance of 22 genotypes of B. vulgaris plants to PPO inhibitor herbicides (pp. 46-47, Table 6), wherein 8 of those genotypes (HTC003, HTC007, HTC013, HTC015, HTC018, HTC024, HTC046, and HTC056) comprise the recited R126L mutation. Applicant also discloses B. vulgaris protoplasts, calli, and/or plants comprising additional mutations besdies the R126L mutation (p. 42, Table 4; p. 48, Table 7)[00143].
Applicant does not describe examples of B. vulgaris plants demonstrating increased resistance to PPO inhibitor herbicides and comprising non-leucine substitutions at position 126 of SEQ ID NO:3. Applicant provides no examples wherein R126 has been substituted for the other 18 available non-leucine amino acids. Applicant provides no indication or guidance as to which of the possible substitutions results in the desired phenotype. The state of the art (Rangani et al., Frontiers in plant science. 2019; 10:568 (U)) teaches that substitution of position R128 of A. palmeri PPO2, which corresponds to position R126 of SEQ ID NO:3, with glycine (p. 02, right column, first full paragraph), methionine (p. 02, right column, first full paragraph), or leucine (p. 07, “G399A Mutant PPO Confers Broad Resistance to PPO-Inhibiting Herbicides”; p. 09, Table 2) interferes with hydrogen-bonding interactions and inhibits a broad spectrum of PPO-inhibitor herbicides (p. 10, left column, third full paragraph). However, Rangani also teaches that the R126L substitution is insufficient to provide resistance to several PPO-inhibitor herbicides that do not rely on hydrogen-bonding to R126 (p. 09, Table 2, p. 10, left column, third full paragraph, lns. 14-17). The state of art also teaches that protein chemistry is highly unpredictable. For example, substitution of Tyr-38 with Phe or Trp did not abolish protein activity while replacement of Tyr-38 with other amino acids severely reduced or abolished protein activity, which indicates that even a single amino acid change can unpredictably alter the function of polypeptide variants (Lazar et al., Molecular and Cellular Biology, 1989; 9(2):860-864 (U); Abstract; p. 860, “Detection and biological activity of yeast-secreted proteins of mutant TGF-a.”). Accordingly, even though the substitution of specific amino acids (i.e., glycine, leucine, or methionine) for the arginine at position 126 of SEQ ID NO:3 results in increased resistance to specific PPO-inhibitor herbicides, other amino acid substitutions are not predictably functional with regard to specific herbicides and one of ordinary skill in the art could not predict which amino acid substitutions at position 126 of SEQ ID NO:3 would result in increased resistance to specific PPO inhibitors besides the three species disclosed by Applicant and the prior art (i.e., methionine, leucine, and glycine). Because the claims encompass polypeptides that are not predictably functional and are not described by either Applicant or the prior art, the specification does not describe species over the full scope of the encompassed sequences/structures.
MPEP § 2163 states that the written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice, or by disclosure of relevant, identifying characteristics, i.e., structure or other physical and/or chemical properties, by 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 claimed genus. A “representative number of species” means that the species which are adequately described are representative of the entire genus. See, e.g., AbbVie Deutschland GMBH v. Janssen Biotech, 759 F.3d 1285, 111 USPQ2d 1780 (Fed. Cir. 2014). When there is substantial variation within a genus, as here in which the genus comprises over 200 distinct PPO2 polypeptides, one must describe a sufficient variety of species to reflect the variation within the genus. A single PPO2 polypeptide sequence comprising a leucine substitution at the position corresponding to R126 of SEQ ID NO:3 does not provide adequate written description for other substitutions at the position corresponding to position R126 of SEQ ID NO:3.
Claims 48-50, 70-73, and 75 do not address the limitation regarding non-leucine substitutions of position 126 and therefore, also lack adequate written description.
Though claims 52-53 provide additional limitations regarding the function of the substitution at position 126, said limitations provide no guidance as to which non-leucine amino acids will result in increased herbicide tolerance and/or resistance. Regarding claim 53, Rangani teaches that it is difficult to predict which and if the substitution of a particular amino acid at position R126 of SEQ ID NO:3 will result in resistance to a specific PPO inhibitor herbicide (p. 09, Table 2). Therefore, claims 52-53 also lack adequate written description.
Claim 69 narrows the scope of the possible substitutions to alanine, glycine, leucine, isoleucine, and methionine. Claim 74 narrows the scope of the possible substitution to that of the elected sequence of SEQ ID NO:15, which comprises an R126L mutation (Figure 2), and SEQ ID NOs:9, 12, 18, and 21, which comprise mutations of R126 to alanine, glycine, isoleucine, and methionine, respectively. As stated above, the function of the non-leucine amino acids in the substitution of position 126 remains unpredictable absent further disclosure from Applicant or the prior art. Therefore, the substitutions of alanine and isoleucine at position R126 of SEQ ID NO:3 are not sufficiently described and claims 69 and 74 also lack adequate written description.
Accordingly, there is a lack of adequate written description to inform a skilled artisan that Applicant was in possession of the claimed invention at the time of filing.
Claim Rejections - 35 USC § 103
12. 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.
13. Claims 47-50, 52-53, 67, and 69-75 are rejected under 35 U.S.C. 103 as being unpatentable over Rangani et al. (Frontiers in plant science. 2019; 10:568 (U)) as evidenced by GenBank Accession No. QBB02367.1 (NCBI. 2019 (W)) in view of McElroy et al. (WO2021/061830, published 04/01/2021 (Applicant’s IDS)) and further in view of NCBI Reference Sequence: XM_010694988.2 (NCBI. 2016 (X)).
Regarding claim 47, Rangani teaches a method for producing a plant or plant cell having an engineered protoporphyrinogen oxidase 2 protein comprising an amino acid sequence substitution corresponding to position 126 of SEQ ID NO:3 (Abstract; p. 02, right column, first and second full paragraphs; p. 05, Figure 1). As evidenced by the attached sequence alignment (GenBank Accession No. QBB02367.1), R128 of the protoporphyrinogen oxidase 2 protein taught by Rangani corresponds to position R126 of the instant SEQ ID NO:3. Furthermore, Rangani teaches that the position corresponding to R126 of SEQ ID NO:3 is highly conserved in the PPO2 amino acid sequences of several crop species (p. 05, Figure 1) and that substitution of the arginine at the position corresponding to R126 of SEQ ID NO:3 reduces binding to PPO inhibitor herbicides (p. 10, left column, third full paragraph).
Rangani does not teach targeted genome editing or the sugar beet B. vulgaris.
However, McElroy teaches introducing a nucleic acid mutation by targeted genome editing which results in a A212T substitution in a sugar beet (and other crop species) protoporphyrinogen oxidase (PPO) to impart resistance to a PPO inhibitor herbicide to the plant (Claims 1-4 and 7-10; p. 01, lns. 25-28; p. 02, lns. 1-34; p. 03, ln. 23-p. 04, ln. 4).
Reference Sequence: XM_010694988.2 teaches a B. vulgaris PPO2 nucleic acid and protein sequence having at least 99% sequence identity to SEQ ID NO:3.
The combination of Rangani, McElroy, and Reference Sequence: XM_010694988.2 teaches a method for producing a sugar beet B. vulgaris plant or plant cell having an engineered protoporphyrinogen oxidase 2 protein comprising introducing a nucleic acid mutation by targeted genome editing which results in an amino acid sequence substitution corresponding to position 126 of SEQ ID NO:3.
The level of ordinary skill in the plant biotechnology art is high as evidenced by both Rangani and McElroy. It would have been prima facie obvious to one of ordinary skill in the art to combine the teachings of Rangani and McElroy to produce a B. vulgaris plant comprising a targeted nucleic acid mutation resulting in a substitution of the arginine at the position of the PPO2 polypeptide taught by Reference Sequence: XM_010694988.2 corresponding to position 126 of SEQ ID NO:3. One would have been reasonably motivated to do so for the following reasons. Rangani teaches that substitution of the arginine at the position corresponding to position 126 of a PPO2 polypeptide inhibits binding of certain PPO inhibitor herbicides (p. 10, left column, third full paragraph) and confers medium to high herbicide resistance to plants comprising said mutation (p. 02, right column, second full paragraph). McElroy suggests a need to develop herbicide-resistant crops to allow the application of broad-spectrum and/or non-selective herbicides to reduce the growth of weeds in farm fields (p. 01, lns. 10-24), discloses that amino acid substitutions within highly conserved regions of PPO proteins can disrupt protoporphyrinogen oxidase activity (p. 34, Example 9), and suggests the use of targeted gene editing tools including CRISPR to introduce said substitutions (p. 02, lns. 18-34) into crop plants including sugar beet (Claims 1-4 and 7-10). The Office notes that position 212 of the PPO protein taught by McElroy (see: McElroy p. 34, lns. 17-25) corresponds to position 210 of the PPO2 protein taught by Rangani (see: Rangani, p. 05, Figure 1); McElroy compares the engineered PPO protein to an A. tuberculatus PPO2 protein sequence, which is also disclosed in Figure 5 of Rangani. Given Rangani’s teachings regarding substitution of the arginine at the position corresponding to position 126 of a PPO2 polypeptide and McElroy’s teachings regarding targeted substitution mutations within highly conserved regions of PPO proteins, one of ordinary skill in the art would have been motivated, with a reasonable expectation of success, to introduce targeted substitutions at position 126 of the sugar beet PPO2 to produce a sugar beet plant with increased resistance to PPO-inhibitor herbicides. One of ordinary skill would have been motivated to modify sugar beet specifically because of its global importance as a crop plant and McElroy’s direct suggestions to modify sugar beet PPO proteins. Accordingly, one of ordinary skill in the art would have been motivated to produce the claimed invention without any surprising or unexpected results.
Regarding claim 48, in addition to the teachings discussed above, Rangani teaches a nucleic acid encoding a PPO2 protein comprising an in-frame deletion of the glycine at the position corresponding to position 208 and/or 209 of SEQ ID NO:3 (p. 02, right column, first and second full paragraphs). As disclosed in the attached sequence alignment, position 210 of the A. palmeri PPO2 sequence disclosed by Rangani corresponds to position 208 of the instant SEQ ID NO:3.
Regarding claims 49-50, Rangani is silent to the allelic location of the deletion at the position corresponding to G208 and/or G209 of SEQ ID NO:3. However, the allelic location of the deletion at the position corresponding to G208 and/or G209 of SEQ ID NO:3 with respect to the location of the substitution at the position corresponding to R126 would not impact the function of said deletion, because the deletion is sufficient to impart herbicide resistance to a plant comprising only said deletion. Furthermore, Applicant has provided no evidence or assertion of surprising or unexpected results for a plant comprising hetero-allelic (claim 50) or homo-allelic (claim 49) mutations. See MPEP 716 and MPEP 2183.
Regarding claims 52 and 67, the combined teachings of Rangani, McElroy, and Reference Sequence: XM_010694988.2 are as discussed above. As stated above, Rangani does not teach a B. vulgaris plant. However, McElroy teaches an herbicide resistant B. vulgaris (i.e., sugar beet) plant (Claims 1-4; p. 01, lns. 25-28; p. 02, lns. 1-34). Therefore, the combined teachings of Rangani, McElroy, and Reference Sequence: XM_010694988.2 teach an herbicide resistant B. vulgaris (i.e., sugar beet) plant comprising an amino acid substitution at the position corresponding to the arginine at position 126.
Regarding claim 53, in addition to the teachings discussed above, Rangani teaches resistance to acifluorfen, fomesafen, lactofen, fluoroglycofen-ethyl, oxyfluorofen, flumioxazin, azadenidin, carfentrazone-ethyl, sulfentrazone, fluthiacet-methyl, oxadiargyl, oxadiazon, pyraflufen-ethyl, saflufenacil, trifludimoxazin, and S-3100 (p. 09, Table 2; p. 09, Table 3).
Regarding claim 69, in addition to the teachings discussed above, Rangani teaches substituting R126 with leucine (p. 09, Table 2).
Regarding claim 70 and 72, in addition to the teachings discussed above, Rangani teaches exposure to at least one herbicide that inhibits PPO2 (p. 03, “Fomesafen Dose-Response Assay”; p. 04, “Cross-Resistance to Foliar-Applied PPOHerbicides”; and p. 04, “Recombinant Expression, Purification, and in vitro Inhibition Studies of PPO2”).
Rangani does not teach protoplasts or gRNA targeting a genomic region corresponding to between positions 3653 and 3698 (claim 70) or positions 3679 and 3698 (claim 72) of SEQ ID NO:1.
However, McElroy teaches transforming/transfecting a protoplast obtained from B. vulgaris cells with a genome editing system to generate a transfected protoplast, wherein the genome editing system comprises: i) a Cas enzyme (p. 04, lns. 3-4), at least one guide RNA (p. 03, ln. 25), and at least one single strand donor template designed to introduce a substitution/modification at a target site (p. 03, ln. 26); exposing the transfected protoplast to a selective pressure of at least one herbicide that inhibits PPO (i.e., oxadiazole; p. 04, lns. 1-2); selecting a protoplast comprising a substitution at the desired site (p.03, ln. 31-p.04, ln. 1); and regenerating a plant from said selected protoplast to produce a B. vulgaris plant having an engineered PPO2 protein (p. 04, lns. 30-34; p. 05, lns. 1-8; p. 21, lns. 17-34).
The combination of Rangani, McElroy, and Reference Sequence: XM_010694988.2 teaches transfecting a protoplast obtained from B. vulgaris cells with a genome editing system to generate a transfected protoplast, wherein the genome editing system comprises: i) a Cas enzyme, at least one guide RNA targeting the genomic sequence encoding the position corresponding to R126 of SEQ ID NO:3, and at least one single strand donor template designed to introduce a substitution/modification at the position corresponding to R126 of SEQ ID NO:3; exposing the transfected protoplast to a selective pressure of at least one herbicide that inhibits PPO; selecting a protoplast comprising a substitution at the position corresponding to R126 of SEQ ID NO:3; and regenerating a plant from said selected protoplast to produce a B. vulgaris plant having an engineered PPO2 protein. Though both Rangani and McElroy are silent to the sequence of the guide RNA targeting the PPO2 gene sequence, one of ordinary skill in the art would be capable of identifying the genomic region encoding the position corresponding to R126 of SEQ ID NO:3 of the B. vulgaris PPO2 gene and the design and use of CRISPR/Cas gene editing constructs, primers, and guide RNAs is routine in the art. Furthermore, the sequence of guide RNAs is inherent to the target site and one of ordinary skill in the art would be sufficiently capable to design donor DNA template comprising the desired amino acid substitutions. Accordingly, it would be both obvious and well within the skill of one of ordinary skill in the art to design guide RNAs to target the genomic region encoding the position corresponding to R126 of SEQ ID NO:3. Applicant’s specific guide RNAs would not provide any surprising or unexpected results in comparison to any other guide RNAs designed to target the desired region.
Regarding claim 71, the combined teachings of Rangani, McElroy, and Reference Sequence: XM_010694988.2 are as discussed above. Rangani does not teach a plasmid-free method. However, McElroy teaches a plasmid-free methods of transformation including particle bombardment, microinjection, and electroporation (p. 21, lns. 17-28).
Regarding claim 73, the combined teachings of Rangani, McElroy, and Reference Sequence: XM_010694988.2 are as discussed above. Rangani does not teach SEQ ID NO:69 or SEQ ID NO:93. However, as stated above in the rejection of claims 70 and 72 under 35 U.S.C. 103, the combination of Rangani, McElroy, and Reference Sequence: XM_010694988.2 teaches the design and use of CRISPR/Cas enzymes, guide RNAs, a donor DNA template to introduce targeted substitutions at the position corresponding to R126 of SEQ ID NO:3, it would be both obvious and well within the skill of one of ordinary skill in the art to design guide RNAs and donor templates to modify the genomic region encoding the position corresponding to R126 of SEQ ID NO:3, and Applicant’s specific guide RNAs and donor template would not provide any surprising or unexpected results in comparison to any other guide RNAs or donor template designed to target the desired region.
Regarding claim 74, in addition to the teachings discussed above, Rangani teaches an R128L mutation. As noted above, position 128 of the PPO2 protein taught by Rangani corresponds to position 126 of SEQ ID NO:3. SEQ ID NO:15 represents a variant of SEQ ID NO:3 comprising an R126L mutation (Applicant’s specification dated 02/29/2024; p. 05, Table 1; Sequence Listing of the instant application). Therefore, SEQ ID NO:15 is obvious in view of the teachings of Rangani.
Regarding claim 75, the combined teachings of Rangani, McElroy, and Reference Sequence: XM_010694988.2 are as discussed above. Rangani is silent to SEQ ID NO:1. However, SEQ ID NO:1 represents the genomic sequence of B. vulgaris PPO2 ((Applicant’s specification dated 02/29/2024; p. 04, Table 1). As further demonstrated by Applicant’s disclosure, B. vulargis PPO2 can also be encoded by cDNA sequence of SEQ ID NO:2, which has less than 90% sequence identity with SEQ ID NO:1 (Applicant’s sequence listing). Thus, SEQ ID NO:3 can be encoded by various nucleic acid sequences. Accordingly, one of ordinary skill in the art could express an engineered PPO2 protein comprising a substitution of the arginine at the position corresponding to position 126 of SEQ ID NO:3, wherein the PPO2 protein is encoded by a nucleic acid sequence having at least 99% sequence identity to the sugar beet PPO2 sequence taught by Reference Sequence: XM_010694988.2. Such a PPO2 protein would have the same function as an identical protein expressed from Applicant’s sequence recited in claim 75. Applicant has made no assertion and provided no evidence that the recited nucleic acid sequence having 90% identity to SEQ ID NO:1 provides any surprising or unexpected results when compared to the cited sequences available to one of ordinary skill.
Accordingly, one of ordinary skill in the art would have been motivated to produce the claimed invention without any surprising or unexpected results.
Conclusion
14. No claim is allowed.
Examiner’s Contact Information
15. Any inquiry concerning this communication or earlier communications from the examiner should be directed to DEQUANTARIUS JAVON SPEED whose telephone number is (703)756-4779. The examiner can normally be reached M-F; 9AM-5PM ET.
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/DEQUANTARIUS JAVON SPEED/Assistant Examiner, Art Unit 1663
/Amjad Abraham/SPE, Art Unit 1663