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 .
Claim Status
Claims 1, 4, 6, 8,15,24, 26, 28,30, 32, 34,38, 41, 43, 50, 55, 64, 66, 68, and 69 are pending.
Claims 1, 4, 6,8,15,24, 26, 28,30, 32, 34,38, 41, 43, 50, 55, 64, 66, 68, and 69are examined on the merits.
Claim Objections
Claim 30 is objected to because of the following informality: "the mutated plant part, plant part, or plant cell," Appropriate correction is required.
Claims 8 and 69 are objected to because of the following informality: “oil content is comprises” is grammatically incorrect. Appropriate correction is required.
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.
Claim 1, 4, 6, 8, 15, 26, 28, 30, 32, 34, 38, 41, 43, 55, 66, 68,and 69 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.
Claims 1 is rejected as indefinite for the recitation “homolog” thereof encoding an AIP2 E3 ligase. In specification, “According to some embodiments, the term “homology” or “homologous” refers to identity of two or more nucleic acid sequences; or identity of two or more amino acid sequences; or the identity of an amino acid sequence to one or more nucleic acid sequence. According to some embodiments, the homology is a global homology, e.g., a homology over the entire amino acid or nucleic acid sequences of the invention and not over portions thereof. The degree of homology or identity between two or more sequences can be determined using various known sequence comparison tools which are described in WO2014/102774 (according to the calculation, the homolog could be as low as 80% identity of two nucleotide sequences or amino acid sequences, respectively ) (paragraph 0087).”
The recitation of a “homolog” and sequences having “80%” identity lacks a recognized meaning in the art and the specification, does not provide a clear definition of what is meant by “homolog” or as low as “80%” identity (e. g., degree of sequence identity, length of comparison, or reference sequence). From the definition, “homology”/ 80% identity is a relative term , and can be read many ways (ortholog vs paralog; close vs distant; protein vs DNA, within a species vs across species). Accordingly, one of ordinary skill in the art cannot ascertain the metes and bounds of claims 1 with reasonable certainty.
Dependent claims 4, 6, 8, 26 and 28 are included in this rejection because they do not include additional limitations to resolve the ambiguity.
Claim 30 is rejected as indefinite for the recitation “homolog” for the same reasons set forth with respect to claim 1. Dependent claims 32, 34, 38, 41, 43, 66, 68, and 69 are included in this rejection because they do not include additional limitations to resolve the ambiguity.
Claim 66 is rejected as indefinite for the recitation “population”. In specification, the inventors recite “the term “population” refers to a set comprising any number, including one, of individuals, objects” (paragraph 0089). “one” is not a population. One of ordinary skill in the art cannot ascertain the metes and bounds of “population” with reasonable certainty.
Dependent claims 68 and 69 are included in this rejection because they do not include additional limitations to resolve the ambiguity.
Claim 15 is rejected as being indefinite for dependency on canceled claim.
Claim 55 is rejected as being indefinite for dependency on canceled claim
Claim 66 is rejected as being indefinite for dependency on itself.
Claim 15 is interpreted as depends on claim 1.
Claim 55 is interpreted as depends on claim 30. Claim 66 is interpreted as depends on claim 30. It should be noted that such treatment does not relieve Applicants of the responsibility of responding to this rejection. Moreover, if the intended meaning of the claim is different than that posited by the Examiner, additional 35 U.S.C. § 112 and prior art rejections may be readily applied in a subsequent office action.
Claim Rejections - 35 USC § 112
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.
Written Descriptions
Claim 1, 4, 6, 8, 15, 24, 26, 28, 30, 32, 34, 41, 50, 64, 66, 68, and 69 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.
The Federal Circuit has clarified the application of the written description requirement. The court stated that a written description of an invention "requires a precise definition, such as by structure, formula, [or] chemical name, of the claimed subject matter sufficient to distinguish it from other materials". University of California v. Eli Lilly and Co., 119 F.3d 1559, 1568; 43 USPQ2d 1398, 1406 (Fed. Cir. 1997). The court also concluded that "naming a type of material generally known to exist, in the absence of knowledge as to what that material consists of, is not description of that material". Id. Further, the court held that to adequately describe a claimed genus, Patent Owner must describe a representative number of the species of the claimed genus, and that one of skill in the art should be able to "visualize or recognize the identity of the members of the genus". Id.
The claims are rejected for lacking adequate written description support regarding the broad scope of the following.
Claims 1, 4, 6, 8, 26, 28, 30, 32, 34, 38, 43, 50, 66, 68, and 69 regards the “homologs” of AIP2 genes.
In specification, the homolog could be as low as 80% identity of two nucleotide sequences or amino acid sequences, respectively (paragraph 0087). Under BRI, homolog or as low as 80% identity encompasses a vast genus of sequence that can differ at up to 20% of positions while still meeting the limitation, including variants with extensive substitutions/indels across the entire region. As discussed under 112(b), this identity-only boundary is so broad that it captures innumerable sequences without specifying which differences are permitted or excludes (e. g., which residues/regions must be conserved to maintain the recited gene/ protein and associated function).
While additional claim limitations could narrow the scope (such as restricting the homolog to specified SEQ ID NOs, to orthologs of a particular AIP2 gene, or to sequences meeting a defined % identity across a defined length), the claims as presently drafted do not provide such boundaries, and a POSITA would not be reasonably apprised of what genes are encompassed as “homolog”.
Claims 1 and 30 regards a plant or plant part “comprising a genetic mutation”. The recitation of “genetic mutation” is drafted as an open-ending genus that encompasses an essentially unlimited number of different edits (e. g., substitutions, insertions, deletions, frameshifts, regulatory-region changes, and combinations thereof) across the claimed genes, many of which would have unpredictable or non-functional effects. The specification provides only a limited set of exemplified mutations and does not supply sufficient guidance to identify which mutation types, positions, and allele combinations will reliably produce the recited outcome across the full scope,
Claims 1, 4, 6, 8, 15, 24, 26, 28, 30, 64, 66, 68 and 69 regards the “a plant or plant part”, the term “plant” is drafted as a broad genus encompassing many species with materially different genomes, transformation/editing efficiencies, gene copy members, regulatory architectures, and trait expression response. The specification working examples and guidance, are limited to a narrow subset (e. g., one species and particular genotypes/targets) and do not teach how to reliably introduce the claimed mutation and obtain the recited phenotype across the full scope of plants without extensive species-by-species optimization, regeneration, and phenotypic testing, which would require undue experimentation.
The claims are drawn broadly to plants and plant parts of any species comprising a genetic mutation in at least one API2 gene or homolog thereof, or in a regulatory region thereof, that results increased protein and /oil content over a control, and further encompass both increased and decreased API2 expression or activity and a wide range of trait magnitudes (e. g., 0.5 to 100% increase in protein and /or oil content).
The specification as filed only describes specific Glycine max AIP2 (Glyma.17g220500 and Glyma.14g105900 , SEQ ID NO: 1-7), a limited number of defined deletions (3 alleles with deletions in exon-2 region from Glyma.17g220500 or Glyma.14g105900) in those genes and /or their regulatory regions, and screening of soybean plants carrying those particular mutations for AIP2 expression, AIP2 activity, and seed protein and /or oil content (Examples 1-4, table 1).
The disclosure does not provide sufficient representative examples, structural guidance, or “blaze marks” to demonstrate possession of the full scope of the genus now claimed, including (1) AIP2 “homologs” across different plant species, (2) all types and locations of mutations in coding and regulatory regions that would be expected to increase protein and /or oil content, and (3) the full range of claimed increases in protein and/or oil content across such diverse plants and homologs.
The ABI3-interacting protein 2 (AIP2) gene encodes a RING motif containing E3 ligase that can interact with and polyubiquitinate the B3-domain transcription factor Abscisic Acid-Insensitive 3 (ABI3). AIP2 likely targets ABI3 for post-translational destruction. ABI3 is specifically expressed in seeds and is known to play a role in seed maturation, accumulation of seed storage proteins and controlling expression of many other regulators of metabolic pathways (paragraph 0005 instant application). Plants have evolved complex, integrated, and overlapping signaling pathways to maintain a plastic growth habit in response to stresses such as drought, salt, cold, as well as hormonal cues such as abscisic acid (ABA). The ABA signaling pathway is showing below. AIP2 does not function independently in ABA signaling, but instead operates as one component of a complex regulatory network in which multiple factors jointly influence ABA responses and downstream traits.
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(Parinita Agarwa, Structural Dynamics, Evolutionary Significance, and Functions of Really Interesting New Gene Proteins in Ubiquitination and Plant Stress)
There are big group of AIP2 homologs/orthologs in different species with different functions. The OsDSG1 is the rice ortholog of AIP2 and also possesses E3 ubiquitin ligase activity. OsDSG1 interacts with OsABI3, the rice equivalent of ABI3, and targets it for degradation, negatively regulating the plant’s tolerance to salt and drought stresses within an ABA-dependent pathway (Parinita Agarwa, Structural Dynamics, Evolutionary Significance, and Functions of Really Interesting New Gene Proteins in Ubiquitination and Plant Stress: A Review, DNA and Cell Biology Vol. 44, No. 5, 2025). More example comes from Arabidopsis, SALT-AND DROUGHT-INDUCED RING FINGER1 (SDIR1) encodes a RING-H2-type E3 ubiquitin ligase and SDIR1-overexpressing Arabidopsis lines show ABA hypersensitivity and ABA-related phenotypes during germination, enhanced ABA-induced stomatal closure, and drought tolerance. Han (Guoliang Han et. al., RING Zinc Finger Proteins in Plant Abiotic Stress Tolerance, Front. Plant Sci., 13 April 2022) further points that SDIR1 orthologs in other plant species were found to have similar functions, such as DELAYED SEED GERMINATION 1 (OsDSG1) and SALT-AND DROUGHT-INDUCED RING FINGER 1 (OsSDIR1) in rice, ZmRFP1 in maize and ABI3-INTERACTING PROTEIN 2 (AtAIP2) in Arabidopsis. Agarwa shows a big list of AIP2 homologs in E3 ligase family.
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These homologs come from different plant species, target to different substrates, and have different functions. While these proteins are closely related homologs with similar functions, extending the claims to more remote homologs would encompass proteins with increasingly diverse and potentially unrelated functions. Thus, the term “homolog” is overly broad in this content because homologous genes from different species, or even within the same species, can have substantially different functions; reciting “AIP2 homologs” sweeps in a large and functionally diverse set of sequences that are not shown or described in the specification. Furthermore, not all of the AIP2 genes or homologs are known to be functionally linked to seed protein and/or oil yield, and the specification does not demonstrate that perturbation of any given AIP2 will necessarily increase protein and/or oil content, let alone do so within the broad percentage ranges recited in the claim.
Even in same species, high similarity AIP2 may have different functionality. Gao (Dong-Yao Gao, et. al., Functional analyses of an E3 ligase gene AIP2 from wheat in Arabidopsis revealed its roles in seed germination and pre-harvest sprouting, Volume56, Issue5, Pages 480-491 26 November 2013) discovers, TaAIP2A and TaAIP2B from wheat, are identified to encode E3 ligase transcriptional factors with the typical RING domain, respectively. TaAIP2A and TaAIP2B function through interaction with wheat Viviporous-1 (TaVp1) negatively regulate the ABA signaling pathway and play important roles in seed germination. TaAIP2A and TaAIP2B have only one amino acid difference between these two homologies in wheat. However, compared with the genomic sequence of TaAIP2A, that of TaAIP2B had 31 and 161 bp insertions in the first intron, 89 bp deletions in the second intron, and 201 bp insertions in the third intron, respectively. It remains unclear the effects of these intron differences on the expression levels of these two homologues. However, the expression profiles of these two homologous genes in wheat were quite different throughout seed developmental stages and in mature embryos upon water stratification. Phenotype analyses indicated that whereas TaAIP2A performed almost the same roles as its counterpart in Arabidopsis in seed germination, flowering time, and ABA responsiveness, TaAIP2B played a stronger role in these aspects. As indicated in the result, the cDNA sequence homology between TaAIP2A and TaAIP2B was 98.56%. No differences in promoter regions of both TaAIP2 genes were detected in the tested three wheat varieties. Only big variations existed in the intron region of these two genes. Therefore, the differences in the intron region may have a critical role in determining the functional performance of these two genes. Mutates any endogenous homolog is not reasonably expected to yield the same result, because each homolog can have a different expression profile, tissue specificity and biological function; accordingly, not every mutation in any indigenous homolog will produce the claimed phenotype.
Zhang (Xiuren Zhang et.al., The AIP2 E3 ligase acts as a novel negative regulator of ABA signaling by promoting ABI3 degradation, Genes Dev., 2005 Jul 1;19(13):1532-43) points out, AIP2 has a stronger binding affinity for the B2 + B3 domain of ABI3 than the A1 + B1 domain, but only ubiquitinates the latter, suggesting that its interaction domains and polyubiquitination sites are different from each other. It is possible that there are other substrates to share a common E3 ligase with ABI3. With the fact that AIP2 transcripts were ubiquitously present in all plant tissues, whereas ABI3 was exclusively expressed in developing and mature siliques and seeds (Zhang), AIP2 may have other targets, AIP2 may have functions other than targets ABI3 for post-translational destruction. In other example, Gao shows the similar dual function of TaAIP2 in wheat, on one hand, by negatively regulating the ABA signaling pathway, TaAIP2 played an important role in seed germination and PHS resistance through interaction with TaVp1 in wheat; on the other hand, it would be interesting to explore the potential targets of TaAIP2 and its roles in regulating the proteolysis of other B3 domain factors in wheat.
Considering that the Arabidopsis genome encodes 43 protein members of the B3 domain family, AIP2 is also implicated in the regulated proteolysis of these B3 domain factors in vivo, which means, AIP2 might have additional, pleotropic functions, so randomly alter AIP2 can affect other related phenotypes, and changes made to obtain the claimed trait may also undesirably impact other API2-regulated pathways. Below is the list from Agarwa for the number of E3-ubiquitin ligase genes (AIP2 homologs) from Arabidopsis and Oryza sativa.
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Taken together, the above considerations indicated that the inventors have not demonstrated possession of the full scope of the claimed invention. The claims encompass mutations in any endogenous AIP2 homolog across diverse plant species and tissue, yet homologs can differ markedly in expression profile, tissue specificity, and function, and mutating any indigenous homolog is not reasonably expected to yield the same protein/oil phenotype. In addition, AIP2 does not act independently in ABA signaling but functions within a complex regulatory network and may have other pleiotropic roles, so random changes to AIP2 can impact multiple related phenotypes and not every mutation will work to produce the claimed trait. In view of the limited number of specific soybean AIP2 alleles and mutations actually described, and the lack of guidance enabling the skilled artisan to identify, across the full breadth of claimed homologs and species, which particular modifications will provide the recited increase in protein and/or oil content without undue experimentation, the specification does not reasonably convey that the inventors were in possession of the entire scope of the claimed invention as of the filing date.
Scope of Enablement
Claims 1, 4, 6, 8, 15, 24, 26, 28, 30, 32, 34, 41, 64, 66, 68, and 69 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specifications, while being enabling for editing the specific Glycine max AIP2 genes explicitly identified by SEQIID NO: 1 and 4 (and the corresponding allele sequences); the allele combinations and specific deletions (e. g., deletions of nt 1199-1212 and/or 1108-1114/1109-1118) recited in claim 24 and claim 64; does not reasonably provide enablement for the full scope of the claimed invention, the broader genus of AIP2 gene or homolog. 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 and/or use the invention commensurate in scope with these claims.
An “analysis of whether a particular claim is supported by the disclosure in an application requires a determination of whether that disclosure, when filed, contained sufficient information regarding the subject matter of the claims as to enable one skilled in the pertinent art to make and use the claimed invention.” MPEP 2164.01. “A conclusion of lack of enablement means that. . . the specification, at the time the application was filed, would not have taught one skilled in the art how to make and/or use the full scope of the claimed invention [i.e. commensurate scope] without undue experimentation.” In re Wright, 999 F.2d 1557,1562, 27 USPQ2d 1510, 1513 (Fed. Cir. 1993); MPEP 2164.01.
In In re Wands, 858 F.2d 731,8 USPQ2d 1400 (Fed. Cir. 1988), several factors implicated in determination of whether a disclosure satisfies the enablement requirement and whether any necessary experimentation is “undue” are identified. These factors include, but are not limited to:
(A) The breadth of the claims;
(B) The nature of the invention;
(C) The state of the prior art;
(D) The level of one of ordinary skill;
(E) The level of predictability in the art;
(F) The amount of direction provided by the inventor;
(G) The existence of working examples; and
(H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure. In re Wands, 858 F.2d 731,737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988). No single factor is independently determinative of enablement; rather “[i]t is improper to conclude that a disclosure is not enabling based on an analysis of only one of the above factors while ignoring one or more of the others.” MPEP 2164.01. Likewise, all factors may not be relevant to the enablement analysis of any individual claim.
Claims 1, 4, 6, 8, 26, 28, 30, 32, 34, 38, 43, 50, 66, 68, and 69 regards the “homologs” of AIP2 genes.
Claims 1, 4, 6, 8, 15, 24, 26, 28, 30, 64, 66, 68 and 69 regards the “a plant or plant part”, which comprising a genetic mutation, comprises increased protein, expression level of…, comprises… etc..
Claims 1 and 30 regards a plant or plant part “comprising a genetic mutation”.
The breath of the genera has been discussed above.
In contrast, the specification provides only a limited number of working examples in soybean (Glycine max) and focuses on two specific GmAIP2 genes, Glyma.17g220500 and Glyma.14g105900 (SEQ ID Nos 1-7 and table 1). The disclosure describes editing promoter/5’UTR regions of these two soybean AIP2 genes and assaying expression changes in protoplasts or reporter constructs (example 1-2). The term “regulatory region of an AIP2 gene” includes not only promoter region, it could include in introns and 3’UTRs. For example, Cao mentions, two TaAIP2 genes in wheat has same promoter regions, but intron region has a critical role in determining the functional performance of the two genes. In fact, without any guidance, or thorough understanding of each AIP2 gene, it would be difficult to know which part of regulatory regions are critical for gene expression. Inventors also recognize in soybean AIP2, “most regulatory motifs have not been discovered or are poorly understood”. the specification itself acknowledges that most regulatory motifs are unknown or poorly understood, and that only a minority of gRNAs tested across a promoter region produce and overexpression outcome, with responsive promoter regions lacking shared sequence or positional features across genes (paragraph 0291-0294).
AIP2 homolog represents a large family, and individual AIP2 genes and homologs can have different expression pattens and biological functions, and they cannot be assumed to behave identically with respect to the claimed protein and /oil traits. For example, Cao mentioned two TaAIP2 genes TaAIP2a and TaAIP2b have only one amino acid difference, but they have different roles in seed germination, flowering time, and ABA responsiveness, and are quite different throughout seed developmental stages and in mature embryos upon water stratification. Han point out a homolog of AIP2, SDIR1 encodes a RING-H2-type E3 ubiquitin ligase and SDIR1-overexpressing Arabidopsis lines show ABA hypersensitivity and ABA-related phenotypes during germination, enhanced ABA-induced stomatal closure, and drought tolerance. Han further point out other AIP2 homologs in other plants, such as DELAYED SEED GERMINATION 1 (OsDSG1), and SALT-AND DROUGHT-INDUCED RING FINGER 1 (OsSDIR1) in rice, ZmRFP1 in maize, and ABI3-INTERACTING PROTEIN 2 (AtAIP2) in Arabidopsis. AIP2 represents a large gene family, and individual AIP2 genes and homologs can have different function, so they cannot all be assumed to behave identically with respect to the claimed protein and /or oil traits.
The specification describes generating a small set of particular deletions (14 bp in Glyma.14g105900; 7 bp and 10 bp in Glyma.17g220500 in exon-2 of the gene respectively ) in soybean T0/T1 plants (example3). As we discuss above, B3-domain transcription factor abscisic acid-insensitive 3 (ABI3) is a central regulator in ABA signaling, ABI3-interacting protein (AIP2), which contains a RING motif, can polyubiquitinate ABI3 in vitro. The AIP2 E3 ligase activity is abolished when RING motif is mutated (Zhang). It’s very clear, because the RING motif of AIP2 is critical for its E3 ligase function, functionally meaningful modification must occur in this conserved motif, and changes elsewhere in the protein are not reasonably expressed to produce the same effect. According , the specification would need to provide meaningful guidance as to where within the AIP2 coding or regulatory sequence and how to introduce mutations to obtain the claimed increase in protein and /or oil content.
In summary, given this limited disclosure, one of ordinary skill in the art would not be able to practice the full scope of the claimed invention without undue experimentation. The claims encompass (1) numerous plant species beyond soybean; (2) a broad genus of “AIP2 homologs” that can differ in sequence, expression profile, tissue specification, and biological function; and (3) essentially any type and any location of mutation in both coding and regulatory regions, while still requiring an increased in seed protein and/or oil content across the full breath of the genus and over wide percentage ranges. The specification does not provide predictive guidance or criteria for identifying, in non-soybean species or across diverse AIP2 homologs, which particular mutations in coding regions or regulatory regions will increase seed protein and/or oil content, nor does it teach how to achieve the recited magnitude of trait change across that breath.
Instead, the disclosure relies on iterative design and screening of gRNAs and mutant even within soybean, in a context where regulatory motifs are largely unknown and only a fraction of tested sites yield the desired expression outcome.
Thus, in view of the unpredictability discussed above, the lack of enabling guidance from either the instant disclosure or the art, and breath and diversity of the embodiments encompassed by the claimed genera, the lack of sufficient working examples, and the level of the art at the time of the invention, one of ordinary skill in the art must rely on undue trial and error experimentation to make and test the numerous AIP2s and homologs from different plant species in different coding region and regulatory region of targeted genes, in order to make and/or use the invention within the full scope of these Claims.
For at least this reason, the Specification does not teach a person with skill in the art how to make and/or use the subject matter within the full scope of these Claims.
Claim Rejections - 35 USC § 102
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.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1, 6, 8, 26, 28, 30,34, 38, 66, and 68 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Shen (Bo Shen et. al., RNAi and CRISPR–Cas silencing E3-RING ubiquitin ligase AIP2 enhances soybean seed protein content, Journal of Experimental Botany, Vol. 73, No. 22 pp. 7285–7297, 2022)
Claim 1 recites a plant or plant part comprising a genetic mutation in at least one native abscisic acid-insensitive 3 (abi3)-interacting protein 2 (AIP2) gene or homolog thereof encoding an AIP2 E3 ligase or in a regulatory region of the at least one native AIP2 gene or homolog thereof encoding an AIP2 E3 ligase, wherein the mutated plant or plant part comprises increased protein and/or oil content as compared to a control.
Under BRI, claim 1 encompasses any plant or plant part comprising at least one endogenous AIP2 gene and /or “homolog thereof”, where the “homolog” is read broadly to include any gene related to AIP2 by sequence similarity (including orthologs and paralogs, absent an objective identity threshold or other boundary), and the claim is satisfied by any detectable increase - at any time, in any tissue, and by any mechanism, (1)the expression level of the native AIP2 gene/homology and /or (2) the expression level or activity of an AIP2 E3 ligase encoded thereby, as compared to a control plant or plant part with “and/or” permitting satisfaction by either increased gene expression or increased ligase expression/activity or both.
Shen disclosed soybean plants in which native AIP2 genes Glyma.17g220500 (AIP2a)(SEQ ID NO:4 in instant claims) and Glyma.14g105900 (AIP2b)(SEQ ID NO: 1 in instant claims) are genetically modified (p7287).
Shen discloses, CRISPR-Cas9 is used to introduce small deletions in exon1 of AIP2a and AIP2b in elite soybean(“4-bp deletion” frame-shift mutants and “3-bp deletion” in-frame mutants) which constitute genetic mutations in the native AIP2 genes (p7291).
Shen also discloses, a seed-specific RNAi construct using a 349-bp AIP2 fragment silences both native AIP2 genes, which is likewise a genetic modification of the native AIP2 loci that alters AIP2 expression (p7286).
Shen further discloses that these mutated plants have increased seed protein content relative to non-transgenic controls, with no significant decrease in seed oil content, and a higher protein + oil index (e. g., RNAi and CRISPR lines showing increases of about 1.7-7 percentage points of protein with essentially unchanged oil) (p7291). Thus, Shen discloses plants or plat parts with increased “protein and /or oil content” as broadly recited in claim 1.
Accordingly, Shen discloses each and every element of claim 1, including (1) a plant or plant part having a genetic mutation in at least on native AIP2 gene encoding an AIP2 E3 ligase, and (2) increased protein and /or oil content compared to a control, and therefore anticipates claim 1 under 35 USC § 102(a)(1)
Claim 6 is drawn to the plant of claim 1, wherein the expression level of the native AIP2 gene or homolog, and/or the expression level or activity of the encoded AIP2 E3 ligase, is reduced or abolished.
Shen’s RNAi lines show >90% reduction in AIP2 transcript abundance in seeds relative to wild-type controls and consequently reduced AIP2 protein and activity (p7289).
Shen’s CRISPR mutants introduce frameshift deletions and in-frame deletions in exon 1 that compromise AIP2 function, thereby reducing activity of the AIP2 E3 ligase compared to wild type. Shen explicitly correlates these mutations with increased see protein content while maintaining oil (p7291).
Thus, Shen disclosed plants in which AIP2 gene expression and /or AIP2 E3 ligase activity is reduced or abolished relative to control, and the plants exhibit increased protein content. This meets all limitation of claim 6.
Claim 8 further recites that the increased protein and /or oil content “comprises a 0.5% to 100% or 0.5% to 20% increase as compared to the control”.
Shen reports increases in seed protein content of approximately 1.7-7 percentage points (absolute) in CRISPR mutants and about 7 percentage points in the RNAi line, relative to the control, with no significant reduction in oil (p7291). These increase fall within the claimed ranges of 0.5-100% or 0.5-20% increase relative to a control.
Shen discloses plants satisfying the additional limitations of the specified magnitude of protein increase of claim 8.
Claim 26 recites a population of plants or plant parts comprising the plant or plant part of claim 1.
Instant application recites “the term “population” refers to a set comprising any number, including one, of individuals, objects”(paragraph 0089). The population could be one. Shen discloses multiple independent RNAi events and multiple CRISPR-edited lines. Thus, Shen disclosed a population of plants or plant parts comprising the plant or plant part of claim 1.
Claim 28 recites a seed or protein composition produced from the plant or plant part of claim 1, or a population of plants or plant parts comprising the plant or plant part of claim 1.
Shen provides detailed analysis of seeds harvested from the AIP2-RNAi line and the CRISPR-edited plants, including wet-chemistry determination of protein, oil, and carbohydrate composition and FT-NIR/SS-NIR analysis (p7291, table 1). These seeds are “seed produced from the plant or plant part of claim 1” and the reported protein composition is a “protein composition produced from” such plants. Since the seeds from Shen’s study come from AIP2-mutated plants with increased protein content relative to controls, they meet the limitations of claim 28.
Claim 30 recites a method to increase a plant’s protein and /or oil content by using an RNA-guided nuclease and gRNA to edit an endogenous AIP2 gene (or it’s homolog) or its regulatory region, creating a mutant from which a plant/plant part is generated that has higher protein and /or oil than a control.
Shen discloses each of these steps:
Shen uses a type II CRISPR-Aas9 system (and RNA-guided nuclease) in soybean, with a single guide RNA (GM-AIP2-CR1) designed to target exon 1 of both native AIP2 genes (AIP2a and AIP2b) (p7287).
Shen introduces constructs encoding Cas9 and the guide RNA into soybean embryonic tissues (embryonic axes) by particle bombardment, creating transformed plant cells that express the RNA-guided nuclease and gRNA (p7287).
The Cas9/gRNA complex binds the AIP2 genomic target and cleaves the DNA at the protospacer/PAM site. Sequencing confirms mutations at the expected cleavage site, including 3-bp and 4-bpdeletions in exon1 of AIP2a and AIP2b (p7287).
These edited T0 plants are propagated to T1/T2 generations, and seeds from the edited plants are analyzed for composition. Shen reports that T2 seeds from AIP2 mutants have increased protein content (1.7-2.3 percentage) with no significant reduction in oil relative to wild-type controls(p7289-p7290).
Thus, Shen disclosed a method in which: (1) editing reagents comprising a CRISPR RNA-guided nuclease and gRNA target native AIP2 genes are introduced into plant cells; (2) the nuclease cleaves the AIP2 genomic sequence and mutations are introduced at the cleavage site; and (3) plants regenerated from these cells produce seeds with increased protein content relative to controls. This meets every limitation of claim 30.
Claim 34 limits claim 30 by requiring that the edit reduces or abolishes expression of the native AIP2 gene (homolog) and/or reduces or abolishes expression or activity of the encoded AIP2 E3 ligase compared to a control.
Shen’s CRISPR edits reduce AIP2 function, and Shen expressly correlates these mutation with reduced AIP2 function, and Shen expressly correlates these mutation with reduced AIP2 activity and increased protein content in the resulting seeds. In the RNAi approach, Shen achieve >90% transcript knockdown (p7289), which is a clear “reduced “expression level.
Accordingly, the method of Shen’s CRISPR editing satisfied the additional limitation of claim 34.
Claim 38 recites that the increased protein and /or oil content “comprises a 0.5% to 100% or 0.5% to 20% increase as compared to the control”.
Shen’s methods yield quantified increases in seed protein content within the claimed ranges (1.7-2.3 percentage in CRSIPR-edited lines, and 7% in RNAi lines), while maintaining oil content. These values fall within the 0.5-100% and 0.5-20% ranges recited in claim 38.
Claim 66 is interpreted as dependent of claim 30.
Claim 66 covers a plant/plant part/cell or plant population produced by claim 30, where seed from the resulting plants has increased protein and/or oil content versus a control.
Shen ‘s CRISPR-Cas9 editing method meets all elements of claim 30. Shen shows that seeds from these plants have increased protein content with unchanged oil relative to wild type (p7290). Thus, Shen discloses plants and plant populations as in claim 66
Claim 68 recites “A seed or protein composition produced from the plant, plant part, plant cell, or population of plants of claim 66, wherein the seed comprise increased protein and/or oil content compared to a control”.
Shen provides detailed compositional analysis of seeds harvested from the CRISPR-edited AIP2 mutants produced by the CRISPR method, showing increased protein content relative to wild type. These seeds are “seed produce from” the plants of claim 66 and the reported protein values constitute a “protein composition produced from “ such plants, with increased protein compared to control.
Therefore, Shen discloses the subject matter of claim 68.
Claim 69 further specifies that the increased protein and /or oil content in the plant/plant part/population or the seed composition “comprises a 0.5% to 100% or 0.5% to 20% increase as compared to the control”.
Shen’s reported protein increases (for both RNAi and CRISPR) fall within these claimed ranges. Thus, Shen’s plants and seeds satisfy the quantitative limitation of claim 69.
Claims 1 and 4 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by 20190352658 A1 (Eliot M. Herman, Engineering high-protein-content soybeans, Publication of 2019-11-21).
Claim 4 depends from claim 1, and requires that, relative to a control, the plant/plant part has increased (1) expression of at least one native AIP2 gene (or it’s homolog), and/or (2) expression level or E3 ligase activity of the AIP2 protein encoded by that gene/homolog.
Under BRI, claim 4 covers any plant or plant part comprising at least one native AIP2 gene (or any “homolog” thereof), wherein, relative to a control, the plant/plant part exhibits an increased (1) expression level of the AIP2 gene (or it’s homolog), and/or (2) expression level or E3 ligase activity of the AIP2 protein.
Herman discloses that AIP2 (SEQ ID NO: 2 (XP_003550230.1) / LOC100800189/ GLYMA_17G220500, which is identical to SEQ ID NO:4 in instant claims) over-expression lines of AiP2-7 and AiP2-14 show protein contents of about 42-43 % with ~17% oil, whereas the control shows about 36% protein and ~18.5% oil, yielding a higher protein + oil index(~60% vs. ~54% (Herman example 4 and table 1, paragraph 0033). Claims1 and 4 are anticipated from Herman.
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.
Claims 15, 24, 32, 41, 43, 50, 55, 64, and 69 are rejected under 35 U.S.C. §103 as being unpatentable over Shen (Bo Shen et. al., RNAi and CRISPR–Cas silencing E3-RING ubiquitin ligase AIP2 enhances soybean seed protein content, Journal of Experimental Botany, Vol. 73, No. 22 pp. 7285–7297, 2022) as apply to claim 1, in view of US 20190352658 A1 (Eliot M. Herman, Engineering high-protein-content soybeans, Publication of 2019-11-21), and further in view of Cui (Yingbo Cui et al., Review of CRISPR/Cas9 sgRNA Design Tools, Interdisciplinary Sciences: Computational Life Sciences, volume 10, pages 455-465, 2018)
Claim 1 as the teachings of Shen are discussed above.
Claim 15 is interpreted as dependent of claim 1.
Claim 15 recites genetic mutation is at least partially in exon 2 of a native Glycine max AIP2 gene comprising the nucleic acid sequence set forth as SEQ ID NO: 1 and/or 4.
Shen teaches using RNA-guided nuclease (e. g., CRISPR/Cas) to introduce small indels in endogenous plant genes at exon 1 sites to modulate agronomic traits, and that targeting coding exon1 to disrupt or modulate gene function (p7290).
Shen does not teach choose exon 2 for CRISPR to modulate AIP2 function.
Herman teaches that altering expression of the native soybean AIP2 (E3 ligase) gene (SEQ ID NO:1/2) results in high-protein seeds and identifies the AIP2 coding sequence used to obtain the trait (claim 6).
Cui teaches that selection of specific CRISPR target sites within a known gene is performed using standard sgRNA design tools that scan exons and output multiple candidate target sites.
It would have been obvious to one of ordinary skill in the art, motivated by Herman to modify the AIP2 locus to obtain the high-protein trait and following the genome-editing strategies of Shen and the guide-design practices of Cui, to target exon 2 of the native Glycine mas AIP2 gene (SEQ ID NO:1 and/or 4) with CRISPR editing, such that the resulting genetic mutation is at least partially in exon 2. The limitation “at least partially in exon 2” therefore represents an obvious selection of a specific exonic region within the known AIP2 gene and does not impart patentability. Accordingly, claim 15 is obvious over the combined teaching of the prior arts.
Claim 24 further limits the subject matter of the claim 1 to a soybean plant or plant part who’s native AIP2 gene has one of several specified deletion genotypes/allele combinations, including homozygous or heterozygous deletions at nucleotides 1199-1212 (SEQ ID NO:1) and or/1108-1114 or 1109-1118 (SEQ ID NO:4) , with the permitted outcomes alternatively expresses as particular allele sets defined by SEQ ID Nos 3/4/6/7 (and sometimes SEQ ID NO:1) across the AIP2 loci.
Shen teaches using CRISPR/Cas to introduce small deletions at defined exonic positions of endogenous plant genes, and that plants carrying homozygous and heterozygous deletion alleles at one or more duplicated /allelic loci are routinely generated and selected to fix desirable traits (p7291) .
Shen do not teach the specific deletions at nucleotides 1199-1212 and 1108-1114/1109-1118 of exon 2 of the native Glyecine max AIP2 gene (and the corresponding SEQ IDN: 1, 3, 4, 6, 7 alleles) represent specific small in-frame/frameshift indels at CRISPR cut sites within the claimed exon region, and the various homozygous/heterozygous allele combinations recited in items (i)-(xiv) correspond to predictable zygosity configurations obtained by standard selfing and crossing of edited plants.
Herman teaches that altering AIP2 expression in soybean (using defined AIP2 constructs) yield high-protein seeds and discloses the AIP2 coding sequence, thereby motivating the skilled artisan to obtain AIP2 loss- or gain-of-function alleles in soybean to achieve similar protein and /or oil improvements (claim 6-13).
Cui teaches that guide RNAs are routinely designed along an exon to generate multiple nearby protospacer site capable of producing different but functionally similar indels. Thus, once Herman teaches that AIP2 editing in soybean is desirable and Shen teaches CRISPR editing of plant exons to create small deletions and to stack edited alleles, it would have been obvious to produce soybean plants with small deletions in exon 2 of AIP2 at positions such as 1108-1118 and 1199-1212 and to obtain homozygous and heterozygous combinations thereof through routine breeding. The particular numeric combinations listed in claim 24 therefore represent an obvious selection from a finite set of predicable allelic configurations and do not confer patentable distinction. Accordingly, claim 24 is obvious over the combined teaching of the prior arts. Accordingly, claim 24 is obvious over the combined teaching of the prior arts.
Claim 30 as the teachings of Shen are discussed above.
Claim 41 recites the method of claim 30, wherein the at least one RNA-guided nuclease cleaves a target site in the at least one native AIP2 gene or homolog thereof, or in a regulatory region of the at least one native AIP2 gene or homolog thereof, and wherein the genetic mutation is introduced at the cleaved target site, wherein the target site comprises a nucleic acid sequence set forth as SEQ ID NO: 9.
Shen teaches a method of increasing protein and/or oil content by editing an endogenous AIP2 gene in a plant using an RNA-guided nuclease and a guide RNA that directs cleavage within the AIP2 locus, thereby introducing a mutation and altering seed composition.
Shen does not teach how to modulate regulatory region of AIP2 genes.
Herman teaches “Genetic elements of this invention can include soybean in origin including the primary transgene as well as the controlling upstream and downstream elements” (paragraph 0022).
Cui teaches that selection of specific CRSPR target sites (20-nt protospacers adjacent to a PAM) within a known gene is routine and accomplished using standard sgRNA design tools, and that multiple alternative target sequences within the same gene are typically identified and scored. It would have been obvious to one of ordinary skill in the art, motivated to implement Shen’s AIP2-editing strategy for the high-protein trait taught by Herman, to select and suitable CRISPR target site within AIP2 coding or regulatory region, including a site corresponding to SEQ ID NO:9, as a routine matter of sgRNA design per Cui.
Thus, the additional limitation that “the target site comprises a nucleic acid sequence set forth as SEQ ID NO:9” represents an obvious choice among equivalent target sites in the API2 gene and does not confer patentability. Accordingly, claim 41 is obvious over the combined teaching of the prior arts.
Claim 43 recites the method of claim 41, wherein the at least one RNA-guided nuclease comprises a CRISPR (Clustered Regularly Interspaced Palindromic Repeats) nuclease.
Shen expressly uses a CRISPR-Cas9 nuclease (an RNA-guided CRISPR nuclease) to edit endogenous AIP2 in soybean.
Shen does not teach “ at least one RNA-guided nuclease comprises a CRISPR nuclease”
Cui makes explicit detail method for designing gRNA, and implementing CRISPR-based editing in plant.
Accordingly, claim 43 is obvious over the combined teaching of the prior arts.
Claim 50 recites the method of claim 30, wherein the at least one gRNA comprises:
(a) a nucleic acid sequence that shares at least 80% sequence identity with the nucleic acid sequence set forth as SEQ ID NO: 8; or (b) a nucleic acid sequence set forth as SEQ ID NO: 8.
Claim 30 as the teachings of Shen are discussed above. Shen teaches use of a guide RNA that targets exon 1 of endogenous AIP2 genes in soybean to direct a CRISPR-Cas9 nuclease to cleave AIP2 and create mutations that alter seed traits.
Shen do not teach the specific gRNA sequences.
Herman teaches that altering AIP2 expression both increase and decrease in soybean seeds yields a high-protein trait.
Cui teaches that sgRNA spacer sequence (20-nt protospacers) are routinely designed and optimized within a known gene sequence using standard rules and software, and that closely related for activity and specificity.. It would have been obvious to one of ordinary skill in the art, implementing Shen’s AIP2 editing to achieve the high-protein trait suggested by Herman, to design and use guide RNAs targeting the AIP2 locus having the specific spacer sequence of SEQ ID NO:8 or sequences with at least 80% identity, as routine variants selected by standard sgRNA design and optimization practices per Cui.
Therefore, the recitation that the at least one gRNA comprises SEQ ID NO: 8 or a sequence having at least 80% identity thereto does not render claim 50 non-obvious. Accordingly, claim 50 is obvious over the combined teaching of the prior arts.
Claim 55 is interpreted as dependent of claim 30.
Claim 55 recites genetic mutation is at least partially in exon 2 of a native Glycine max AIP2 gene comprising the nucleic acid sequence set forth as SEQ ID NO: 1 and/or 4.
Shen teaches using RNA-guided nuclease (e. g., CRISPR/Cas) to introduce small indels in endogenous plant genes at exon 1 sites to modulate agronomic traits, and that targeting coding exon1 to disrupt or modulate gene function (p7290).
Shen does not teach choose exon 2 for CRISPR to modulate AIP2 function.
Herman teaches that altering expression of the native soybean AIP2 (E3 ligase) gene (include all of the exons) (SEQ ID NO:1/2) results in high-protein seeds and identifies the AIP2 coding sequence used to obtain the trait, (claim 6).
Cui teaches that selection of specific CRISPR target sites within a known gene is performed using standard sgRNA design tools that scan exons and output multiple candidate target sites which should including exon2, to generate indels that modify gene function. It would have been obvious to one of ordinary skill in the art, implementing Herman’s AIP2-based high-protein strategy with the editing approaches of Shen and the design guidance of Cui, to select and use guides that cut within exon 2 of the native Glycine max AIP2 gene ( SE ID NO: ¼) so that the resulting genetic mutation is at least partially in exon 2. The additional limitation in claim 55 therefore reflects a routine choice of target exon within a known gene and does not render the method non-obvious. Accordingly, claim 55 is obvious over the combined teaching of the prior arts.
Claim 64 further limits the subject matter by reciting a particular configuration, stacking, or section scheme involving a defined AIP2 mutant alleles (e. g., a specific exon-2 deletion) in combination with another allele or trait, such as a specified homozygous/heterozygous state across AIP2a and AIP2b, or combining the AIP2 mutant with ahigh-protein background, while requiring increased seed protein (and optionally maintained oil).
Shen teaches CRISPR-based editing of endogenous plant genes to introduce small deletions (e. g., 1-30bp, up to ~100bp) at defined exonic positions and to generate lines carrying homozygous and heterozygous combinations of edited alleles at duplicated loci, with the expectation that such allelic combinations provide graded modulation of gene function.
Shen teaches that CRISRP-edited AIP2 alleles can be combined and segregated into various genotypic configurations (AIP2a HOM/AIP2b, AIP2a HOM/AIP2b HET, AIP2a HET/AIP2b HOM, and wild type) and that these different zygosity combinations show graded but similar increases in seed protein content and protein + oil index relative to wild type. Shen therefore explicitly teaches that it is routine to fix, combine, and pyramid different AIP2 alleles within a breeding program and that the choice of particular homozygous or heterozygous configuration is a matter of quantitative optimization of the trait, not a qualitatively new invention.
Shen does not teach the specific deletions at nucleotides 1199-1212 and 1108-1114/1109-1118 of exon 2 of the native Glyecine max AIP2 gene (and the corresponding SEQ IDN: 1, 3, 4, 6, 7 alleles) represent specific small in-frame/frameshift indels at CRISPR cut sites within the claimed exon region, and the various homozygous/heterozygous allele combinations recited in items (i)-(xiv) correspond to predictable zygosity configurations obtained by standard selfing and crossing of edited plants.
Herman teaches that altering expression of soybean AIP2 yields a high-protein trait and discloses the relevant AIP2 sequence. Herman similarly teaches that high-protein AIP2 traits can be stacked or combined with other genetic backgrounds, that AIP2 high-protein lines can be crossed with other transgenic lines (e. g., β-carotene lines, Triple Null lines) to create stacked traits, and that multiple independent content, protein + oil index, and seed quality traits. Herman therefore reinforces that a POSITA would reasonably expect to combine specific AIP2 alleles and backgrounds to tune protein and oil traits.
Cui teaches that sgRNA design tools identify multiple nearby protospacer sites within an exon, leading to a spectrum of closely related indels at and around the cut sites, and that such variants ae routinely isolated and characterized. In view of these teaching, once exon 2 of AIP2 is targeted as in Herman /Shen, it would have been obvious to obtain edited soybean plants with small deletions at the exon-2 positions corresponding to nucleotides 1199-1212 of sequence.
In view of Shen’s explicitly teaching that different AIP2 zygosity combinations (single and double mutants) produce predictable increments in protein content, and Herman’s teaching that high-protein AIP2 alleles can be stacked with other traits and backgrounds, a person of ordinary skill in the art would have found it obvious to select and particular AIP2 genotype or stacked combination (including that recited in claim 64) that delivers the desired high-protein phenotype, using routine breeding, crossing, and selection methods. Choosing a specific zygosity pattern or trait stack among the finite set taught by Shen and Herman constitutes nothing more than routine optimization and design choice, with predicable results and a clear motivation to achieve higher protein and improved protein +oil index. Accordingly, claim 64 would have been obvious over Shen in view of Herman.
Claims 32 are rejected under 35 U.S.C. §103 as being unpatentable over of Shen (Bo Shen et. al., RNAi and CRISPR–Cas silencing E3-RING ubiquitin ligase AIP2 enhances soybean seed protein content, Journal of Experimental Botany, Vol. 73, No. 22 pp. 7285–7297, 2022) as apply to claim 30, in view of US 20190352658 A1 (Eliot M. Herman, Engineering high-protein-content soybeans, Publication of 2019-11-21)
Claim 30 as the teachings of Shen are discussed above.
Claim 32 recites the method of claim 30, wherein the edit increase expression of the native AIP2 gene (or homolog) and/or increase expression level or activity of the encoded AIP2 E3 ligase relative to a control.
Shen teaches that Glyma.17g220500 (AIP2a) and Glyma.14g105900 (AIP2b)are the same soybean AIP2 gene and confirms that CRISPR-Cas9 editing of exon 1 of AIP2a and/or AIP2b produces knockout and in -frame deletion/ frameshift deletion alleles that increase seed protein content in elite soybean cultivar 93Y21, again without reducing oil content and with a higher protein + oil index relative to wild-type.
Shen does not teach increasing the expression of AIP2 gene will increase seed protein content without reducing oil content.
Herman teaches that soybean seed protein content is tightly regulated by ABA-controlled “proteome rebalancing” and identifies AIP2, an E3 RING ubiquitin ligase, as a key negative regulator of the ABI3 transcription factor in this pathway (examples 1-3). Herman further teaches that altering expression of the native soybean AIP2 gene (SEQ ID NO: 2 (XP_003550230.1) / LOC100800189/ GLYMA_17G220500) in seeds, either by over-expressing AIP2 under the oleosin promoter or silencing intrinsic AIP2 by RNAi, produces soybean seeds with elevated protein content relative to a control line (example 4, table 1). Herman teaches that AIP2 over-expression lines of AiP2-7 and AiP2-14 show protein contents of about 42-43 % with ~17% oil, whereas the control shows about 36% protein and ~18.5% oil, yielding a higher protein + oil index(~60% vs. ~54% (Herman example 4 and table 1, paragraph 0033). Herman also teaches that AIP2 RNAi lines similarly increase total seed protein, and that both AIP2 over-expression and AIP2 silencing “results in additional protein accumulation” without major changes in storage protein composition, indicating a global increase in seed protein mediated by AIP2-depedent ABA signaling (example 4-6, also metabolomics/proteomics discussion at paragraph 0035-0036). Thus, Herman teaches that modulating AIP2 expression in either direction (up or down ) in soybean seeds is a predictable and effective way to obtain high-protein seeds with elevated protein and increased protein + oil output.
A person of ordinary skill in the art, starting from Herman’s explicit demonstration that AIP2 over-expression in seeds increases total seed protein and the protein +oil index (Herman, table1-2) and knowing that AIP2 and ABI3 are nodes in the ABA regulon controlling seed storage protein accumulation (Herman, example 2-3), would have been motivated to obtain high-protein soybean plants by any routine method that increases AIP2 expression or activity in seeds, including by introducing a mutation in a native AIP2 gene or its regulatory region that elevates AIP2 expression, as an alternative to introducing an exogenous AIP2 expression cassette. Shen supplies the conventional genome-editing tools and teaches that CRISPR editing of the endogenous AIP2 loci in soybean is feasible and effective. Combining Herman’s teaching that AIP2 over-expression yields high-protein seeds with Shen’s teaching that CRISPR editing of endogenous AIP2 genes is an established approach in soybean would have rendered it obvious to a POSITA to produce a “mutated plant” having a genetic change in a native AIP2 gene or its regulatory region such that AIP2 expression or activity is increased relative to a control and seed protein is increased, as recited in claim 32.
Whether the desired high-protein trait is achieved by increasing or decreasing AIP2 expression is merely a matter of routine design choice among art-recognized alternatives that Herman show are both effective in increasing seed protein (paragraph 0033-0036).
Accordingly, claim 32 is unpatentable over Herman in view of Shen.
Conclusion
No claims are allowed.
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Any inquiry concerning this communication or earlier communications from the examiner should be directed to YANXIN SHEN whose telephone number is (571)272-7538. The examiner can normally be reached Monday-Friday.
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/YANXIN SHEN/ Examiner, Art Unit 1663
/WEIHUA FAN/ Primary Examiner, Art Unit 1663
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