DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Priority
Applicant’s claim for the benefit of a prior-filed U.S. provisional patent application (No. 63/335,292) under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Applicant has not complied with one or more conditions for receiving the benefit of an earlier filing date under 35 U.S.C. 119(e) as follows:
The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later-filed application must be sufficient to comply with the requirements of 35 U.S.C. 112(a) or the first paragraph of pre-AIA 35 U.S.C. 112, except for the best mode requirement. See Transco Products, Inc. v. Performance Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994).
The disclosure of the prior-filed provisional application, Application No. 63/335,292, fails to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph for one or more claims of this application. The provisional application does not provide support for SEQ ID NOs. 2 and 5-14. Accordingly, claims 22 and 23 of the instant application cannot claim the effective filing date of the provisional, but are accorded the benefit of 04/27/2023 as the filing date of a prior-filed PCT application (PCT/US2023/066309) that does provide support for the SEQ ID NOs. above.
Adequate support is provided for the remaining claims (claims 14-21 and 24-25) to claim the benefit of the earliest priority date of 04/27/2022. Examiner notes that SEQ ID NO. 3 of the provisional appears to have been renumbered to SEQ ID NO. 1 in the instant application (as well as SEQ ID NO. 4 to NO. 3, and SEQ ID NO. 5 to NO. 4). They are considered synonymous for examination purposes.
Information Disclosure Statement
Initialed and dated copy of Applicant’s information disclosure statement (IDS) filed on 10/24/2024 is attached to the instant Office Action. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
The listing of references on page 16 of the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Specification
The disclosure is objected to for lacking capitalization of Agrobacterium throughout. Additionally, Glycine max should be italicized and the “m” on max lowercase in the specification.
Appropriate correction is required.
Claims
Claim 14-25 are pending and are examined in this Office Action.
Claim Objections
Claim 14 is objected to because of the following informalities: The dash after “…in a plant cell comprising:” is not grammatically correct and should be removed. Claim 14 is further objected to for reciting in line 1 the transitional term “comprising” followed by method steps. It is suggested to insert the phrase ---, the method--- or ---, said method--- before “comprising” in line 1 of claim 14.
Claims 15-16 are objected to for improperly capitalizing “Max” in line 2, as well as lacking italicization on Glycine max.
Claim 17 is objected to for improperly capitalizing the species name “Max” in line 3.
Claim 19 is objected to because of the following informalities: The claim lacks a period at the end of the sentence.
Claim 20 is objected to for use of the acronym “ALCR/aclA” without spelling out the full term.
Claim 21 is objected to because of the following informalities: Claim 21 recites the method of claim 14, wherein the RNAi molecule comprises a nucleotide sequence according to SEQ ID NO. 1. A nucleotide sequence could be interpreted as a dimer or trimer of SEQ ID NO. 1, or SEQ ID NO. 1 in its entirety, making the claim unclear. Examiner suggest amending the claim to recite “the nucleotide sequence according to SEQ ID NO. 1,” as written in claim 18 for clarity and consistency.
Claims 22-23 are objected to for the recitation of “SEQ ID NO’s” appearing plural in line 2. Examiner suggests amending to “SEQ ID NOs.” in line 2 of claims 22 and 23.
Claim 24 is objected to because of the following informalities: The claim lacks a period at the end of the sentence and requires capitalization of Agrobacterium.
Appropriate correction is required.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claim 25 is rejected under 35 U.S.C. 101 because the claimed invention is directed to a product of nature without significantly more. The claim recites a squalene compound. This judicial exception is not integrated into a practical application because the additional elements do not contribute meaningful limitation to the natural product.
Claim 25 recites a squalene compound produced by the method of claim 14, wherein a method of upregulating squalene production in a plant cell comprises expressing in a plant cell a heterologous nucleotide sequence, operably linked to a promoter, encoding a RNAi molecule directed against one or more of the plant’s endogenous squalene epoxidase enzymes, wherein said RNAi molecule downregulated the downstream conversion of squalene into sterols. Squalene is a natural compound regularly extracted from plants, such as Olea europea, Amaranthus sp., Glycine max, and Zea mays, for various cosmetic, pharmacological, and nutritional uses (Lozano-Grande, 2018, pg. 8, col. 1, ¶ 5). The method of the instant application of manipulating the sterol biosynthesis pathway and silencing the enzyme responsible for conversion of squalene into sterols would not change the inherent properties of the squalene product produced by the method as compared to a squalene compound found in nature.
Thus, the claim does not include additional elements that are sufficient to amount to significantly more than the judicial exception because, when considered in combination, they do not add to the inventive concept.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 22 and 23 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. Claims 22 and 23 recite the limitation "said squalene epoxidase paralogs". There is insufficient antecedent basis for this limitation in the claim as preceding claim 14 does not recite squalene epoxidase paralogs. Examiner notes that 22 and 23 potentially should depend from claim 17 which does recite squalene epoxidase paralogs.
Claim 16 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention. Claim 16 recites the method of claim 15, wherein said Glycine max plant cell comprises a Glycine max plant seed. It is unclear how the plant cell can comprise a plant seed. The method of upregulating squalene production in a plant cell comprises expressing a sequence encoding a RNAi molecule against one or more of the plant’s endogenous squalene epoxidase enzymes within a plant cell. The instant specification discloses that an Agrobacterium intermediate vector can be utilized for ultimate transformation of SQE-RNAi into soybean seed. A method of getting a plant seed into a plant cell is unknown in the art. For the purpose of examination and in the interest of compact prosecution, this is taken to mean that the plant cell is derived from a Glycine max plant seed.
Claim Rejections - 35 USC § 112(a) – Written Description
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 22 and 23 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 claims contain 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.
Claim 22 recites the method of claim 14, wherein said squalene epoxidase paralogs are selected from: SEQ ID NOs. 5-14, or a fragment or variant thereof.
Claim 23 recites the method of claim 14, wherein said squalene epoxidase paralogs are selected from: SEQ ID NOs. 5-7, and 9-12, or a fragment or variant thereof.
The claims are directed to a genus of squalene epoxidase paralogs selected from SEQ ID NOs. 5-14, or a fragment or variant thereof. As the claims do not define a genus or species of the squalene epoxidase paralogs, the broadest reasonable interpretation is that the sequences, and fragments and variants thereof, may come from any genus or species.
The instant specification discloses that, in a preferred embodiment, the RNAi molecule of the invention targets and inhibits one or more squalene epoxidase paralogs in Glycine max, specifically inhibiting 7 squalene epoxidase paralogs and their associated isoforms [pg. 4, lns. 4-12]. SEQ ID NOs. 5-12 are the squalene epoxidase paralogs and SQ ID NOs. 13 and 14 are their associated isoforms [pg. 4, lns. 13-17]. However, the specification additionally discloses that while the methods and compositions for the production of squalene in transgenic plants are namely for soybeans, this method could also be used in seeds harvest from other commonly farmed crops such as corn, rice and rapeseed [pg. 1, lns. 5-10]. The specification does not further define that the sequences, or variants or fragments thereof, must come from Glycine max. “Variant” is defined as distinct enzymes or genes of a second family or species that most often has functional structural or genomic similarities [pg. 8, lns. 15-20]. This definition suggests that less often, the distinct enzymes or genes of a second family or species would not have functional structural or genomic similarities and may not confer the function as claimed. “Fragment” is defined as a truncated portion of the peptide or gene that still retains its function [pg. 8, lns. 22-24]. The specification provides only biosynthesis of squalene in germinating soybeans [Example 1], despite the claims being directed to a broad genus of all possible plant cells.
For example, Laranjeira et al. (2015) discloses the presence of several squalene epoxidase genes in most plants and their phylogenetic diversity. Plant squalene epoxidases form multicopy gene families with high diversity, suggesting differing evolutionary roles involving plant squalene epoxidases and functional specialization of plant squalene epoxidase paralogs [pg. 1091, col. 1, ¶4]. Manzoor et al. (2021) further discloses that in Arabidopsis, for example, six SQE (1 to 6) proteins have been identified and functionally characterized. Out of the six, only three Arabidopsis SQEs (SQE 1 to 3) displayed epoxidase activity in the functional assay, and the rest (SQE4 to 6) had no illustrative activity, suggesting the functional divergence of SQE homologs [pg. 992, col. 2, ¶ 1]. Thus, the squalene epoxidase paralogs known in the prior art are unpredictable and vary across plant species. Additionally, fragments or variants thereof may refer to dimers or trimers of provided sequences which are not reduced to practice.
The claims lack written description because the genus of squalene epoxidase paralogs, and fragments or variants thereof, claimed have substantial variance while the specification lacks sufficient variety of species to reduce such variation to practice. As the claims specify that a fragment or variant of the provided sequences could be used, but does not provide examples or support in the specification past the Glycine max paralogs and their associated isoforms, there is a lack of common structural and functional attributes between the claimed genus and what appears to be possessed at the time of filing.
Examiner notes that amending the claims to recite with more specificity the full length of the SEQ ID NOs. correlated to the 7 squalene epoxidase paralogs and their associated isoforms might help to overcome this rejection.
Claim Rejections - 35 USC § 112(a) – Scope of Enablement
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 22 and 23 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for the SEQ ID NOs. of Glycine max listed in their entirety, does not reasonably provide enablement for a fragment or variant thereof. 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 the invention commensurate in scope with these claims.
Claim 22 recites the method of claim 14, wherein said squalene epoxidase paralogs are selected from: SEQ ID NOs. 5-14, or a fragment or variant thereof.
Claim 23 recites the method of claim 14, wherein said squalene epoxidase paralogs are selected from: SEQ ID NOs. 5-7, and 9-12, or a fragment or variant thereof.
A fragment or variant of SEQ ID NO. 5-14 may include dimers and trimers of any of the listed sequences, indicating great breadth in the claims. Though a “fragment” is defined in the specification as a truncated portion of the peptide or gene that still retains its intended function, and “variant” is defined as most often maintaining functional, structural, or genomic similarities, this is not enough for one of ordinary skill in the prior art to determine which fragments or variants of the listed sequences may confer the same function as the squalene epoxidase paralogs. Moreover, the genus or species of the sequences and fragments and variants thereof is not limited to soy in the claims as it is in the example provided in the specification, adding breadth to the claims [Example 1].
As is known in the art, fragments of biological sequences can sometimes retain functional properties, but they do not always perform the exact same role as the whole sequence. Fragments along a protein sequence may form functional motifs necessary to the function claimed, without which, the truncated portion may not function (Dib et al., 2012). Similarly, variants of genetic sequences do not always have the same function as the original (reference) sequence with as few as four substitutions being capable of converting the function of the sequence (Gerlt et al., 2000). The Applicant does not provide working examples of a fragment of a sequence or a variant functioning in the method as claimed. Additionally, as suggested in Laranjeira (2015), plant squalene epoxidases form multicopy gene families with high diversity and plant squalene epoxidases may have specialized functionality [pg. 1091, col. 1, ¶4].
Due to the functional unpredictability of paralogs, and fragments and variants thereof, and breadth of the potential fragments and variants across species, one of ordinary skill in the art would not be able to make and/or use the full scope of the claimed invention with only reasonable experimentation.
Undue experimentation would be required to ensure that all variants and fragments of the claimed sequences still maintained the functionality of the sequences which would be needed to ensure the method as claimed functions to upregulate squalene production in a plant cell.
Examiner notes that amending the claims to recite with more specificity the full length of the SEQ ID NOs. correlated to the 7 squalene epoxidase paralogs and their associated isoforms might help to overcome this rejection.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(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 14-16, 19, and 24-25 are rejected under 35 U.S.C. 102(a)(2) as being anticipated by Aharoni et al. (US 20220217934 A1 - U.S. patent application of WO 2020/049572 as filed in IDS 10/24/2024).
Claim 14 recites a method of upregulating squalene production in a plant cell comprising: expressing in a plant cell a heterologous nucleotide sequence, operably linked to a promoter, encoding a RNAi molecule directed against one or more of the plant’s endogenous squalene epoxidase (SQE) enzymes, wherein said RNAi molecule downregulates the downstream conversion of squalene into sterols.
Claim 15 recites the method of claim 14, wherein said plant cell comprises a Glycine max plant cell.
Claim 16 recites the method of claim 15, wherein said Glycine max plant cell comprises a Glycine Max plant seed.
Claim 19 recites the method of claim 14, wherein said promoter comprises an inducible promoter.
Claim 24 recites the method of claim 14, wherein said plant cell comprises a soybean seed derived from a soybean plant transformed by Agrobacterium mediated transformation, or a soybean seed transformed by Agrobacterium mediated transformation.
Claim 25 recites a squalene compound produced by the method of claim 14.
Regarding claim 14, Aharoni discloses methods of reducing the content of phytosterol, a derivative thereof, a metabolite thereof, or a biosynthetic intermediate thereof [¶ 159]. The method comprises expressing in a plant cell a heterologous polynucleotide sequence encoding a heterologous gene, which may be operably linked to a promoter [¶174 & 267]. In some embodiments, the heterologous gene may be endogenous and may be squalene epoxidase [¶ 211]. Expression of the heterologous gene or polynucleotide is silenced, repressed, or reduced, e.g., by deletion, insertion or modification, or by introduction of at least one silencing molecule targeted to the heterologous gene [¶107]. The silencing molecule may be selected from the group consisting of RNA interference molecule (i.e. RNAi molecule directed against squalene epoxidase) [¶ 381].
Aharoni further specifies that squalene epoxidase is an enzyme involved in triterpenoid (i.e. sterol) biosynthesis [¶ 1029]. The genetically modified plant comprises reduced content of the phytosterol, or a derivative thereof, a metabolite thereof, or a biosynthetic intermediate thereof; compared to the corresponding unmodified plant [¶ 160]. Thus, as squalene epoxidase is responsible for the conversion of squalene into sterols, RNAi molecule directed against squalene epoxidase would downregulate the conversion of squalene into sterols, therefore upregulating squalene production. Squalene upregulation, the intended goal recited in the preamble, would be accomplished by virtue of the method steps of Aharoni with expression in a plant cell of a heterologous sequence, operably linked to a promoter, encoding a RNAi molecule directed against an endogenous squalene epoxidase, lacking evidence to the contrary.
Regarding claim 15 and 16, Aharoni explicitly discloses that said plant cell from the genetically modified plant above comprises a cell from a plant in the Fabales order [¶ 111]. Ahroni discloses that the plant may be Glycine max [¶ 654]. Aharoni further teaches that the plant cell is comprised in a plant or in its portion, wherein the portion may be a plant seed [¶ 120].
Regarding claim 19, Aharoni explicitly discloses that the promoter is an inducible promoter in some embodiments [¶ 238].
Regarding claim 24, Aharoni explicitly discloses that, in some embodiments, expression vectors used to produce genetically modified plant cells include Agrobacterium (ie. Agrobacterium mediated transformation) [¶ 737]. Some embodiments encompass the genetically modified cell comprised in a plant or portion thereof [claim 80] and further encompass seeds of the genetically modified plant [¶ 258], which can be soy [¶ 129].
Regarding claim 25, Aharoni discloses that, in some embodiments, a phytosterol, or biosynthetic intermediate thereof (i.e., squalene compound), may be extracted from the transformed plant [¶ 125 & ¶ 183].
Accordingly, Aharoni anticipated the claimed invention of claims 14-16, 19, and 24-25.
Claim 25 is further rejected under 35 U.S.C. 102(a)(1) as being anticipated by Lozano-Grande et al. (2018). “Plant Sources, Extraction Methods, and Uses of Squalene.” International Journal of Agronomy. 1829160.
Claim 25 recites a squalene compound produced by the method of claim 14.
The claim is drawn to a squalene compound produced by the method of upregulating squalene production in a plant cell comprising expressing in a plant cell a heterologous nucleotide sequence, operably linked to a promoter encoding a RNAi molecule targeted to one or more endogenous squalene epoxidase enzymes to downregulate conversion of squalene to sterols. This claim is interpreted as a product-by-process claim.
Lozano-Grande teaches plant sources, extraction methods, and uses of squalene, specifying that squalene is used in response to stress conditions in plants and that the concentration of squalene is dependent on the species, harvest conditions, and extraction method [pg. 8, col. 2, ¶ 1]. Lozano-Grande discloses extraction methodology to obtain the squalene compound from various species including Glycine max [pg. 8, col. 2, ¶ 2].
It is noted that, “[e]ven though product-by-process claims are limited by and defined by the process, determination of patentability is based on the product itself. The patentability of a product does not depend on its method of production. If the product in the product-by-process claim is the same as or obvious from a product of the prior art, the claim is unpatentable even though the prior product was made by a different process.” In re Thorpe, 777 F.2d 695, 698, 227 USPQ 964, 966 (Fed. Cir. 1985)”. Furthermore, "[b]ecause validity is determined based on the requirements of patentability, a patent is invalid if a product made by the process recited in a product-by-process claim is anticipated by or obvious from prior art products, even if those prior art products are made by different processes." Amgen Inc. v. F. Hoffman-La Roche Ltd., 580 F.3d 1340, 1370 n 14, 92 USPQ2d 1289, 1312, n 14 (Fed. Cir. 2009). See also Purdue Pharma v. Epic Pharma, 811 F.3d 1345, 117 USPQ2d 1733 (Fed. Cir. 2016). See MPEP § 2113.
In the instant case, the squalene compound is produced through the methodology of claim 14, but the produced squalene compound is indistinguishable from the compound found in Lozano-Grande/one found in nature. The instant specification discloses that the squalene was extracted from soybean tissue via 3:1 Isopropanol:Soybean (V/W) incubated overnight at 37C [Example 1], which was a method previously proposed by Lozano-Grande (i.e. Organic solvents) [pg. 8, col. 2, ¶ 2]. The product, obtained by any method, remains the same. The patentability of a product does not depend on its method of production and the claimed squalene compound does not appear to be different from that which is found in nature and has been previously extracted from plants.
Accordingly, Loranzo-Grande anticipated the claimed invention of claim 25.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claim 17 is rejected under 35 U.S.C. 103 as being unpatentable over Aharoni as applied to claims 14-16, 19, and 24 above, and further in view of Monzoor et al. (2021) “Transcriptome-wide identification of squalene epoxidase genes from Glycyrrhiza glabra L.: expression analysis and heterologous expression of GgSQE1 suggest important role in terpenoid biosynthesis.” Protoplasma. 258(5):991-1007.
Claim 17 recites the method of claim 14, wherein said heterologous nucleotide sequence comprises a hairpin RNAi molecule targeting the expression of squalene epoxidase paralogs in Glycine max.
As applied above, Aharoni teaches genetically modified cells and plants having increased or decreased heterologous gene expression, wherein the gene can be squalene epoxidase and may be silenced through RNAi. The heterologous nucleotide sequence may be operably linked to a promoter, which may be inducible. Aharoni discloses that the RNAi molecule directed against the endogenous gene can be a single-stranded hairpin polynucleotide (ie. Hairpin RNAi molecule targeting the expression of an endogenous gene) [¶ 385].
Although Aharoni teaches that the targeted gene may be squalene epoxidase and may be in endogenous to soy (i.e. squalene epoxidase in Glycine max) [¶ 211, 128 & 145], Aharoni does not explicitly teach that these endogenous genes are squalene epoxidase paralogs. However, Monzoor discloses that studies have demonstrated varied and differential expression of squalene epoxidase gene paralogs from several plant species, indicating their regulatory/diverse function [pg. 1001, col. 2, ¶ 1]. Monzoor further discloses that The NCBI database reports 426 squalene epoxidases from plants with 49 submissions from Fabaceae family covering only nine genera [pg. 992, col. 2, ¶ 1]. Among the Fabaceae family, maximum submissions are reported from Glycine max. Paralogs are well known in the prior art and the Applicants themselves disclose that “paralog” refers to genes that are related by replication within a genome. This suggests that squalene epoxidase genes and paralogs would inherently be within the Glycine max genome and could be targeted by the hairpin RNAi molecule of Aharoni. One of ordinary skill in the art would be motivated to use known Glycine max squalene epoxidases in the method of Aharoni as Manzoor suggests that squalene epoxidase genes are significant as 2,3-oxidosqualene is the upstream precursor metabolite for several industrial and pharmaceutically important molecules and would be a useful pathway for manipulation [pg. 1005, col. 1 ¶ 2]. One would additionally have reasonable expectation of success as Aharoni suggest that the plant utilized can be Glycine max [¶ 654].
Claim 20 is rejected under 35 U.S.C. 103 as being unpatentable over Aharoni as applied to claims 14-16, 19, and 24 above, and further in view of Jepson et al. (WO 20010109357 A2 – as improperly incorporated by reference in the instant specification dated 10/24/2024). Examiner notes that this reference was not listed in the IDS filed 10/24/2024.
Claim 20 recites the method of claim 19, wherein said inducible promoter comprises an ALCR/alcA ethanol switch.
As applied above, Aharoni teaches genetically modified cells and plants having increased or decreased heterologous gene expression, wherein the gene can be squalene epoxidase and may be silenced through RNAi. The heterologous nucleotide sequence may be operably linked to a promoter, which may be inducible. Aharoni does not explicitly teach the inducible promoter comprising an ALCR/alcA ethanol switch.
Jepson discloses a chemically inducible promoter based on the alcA/alcR promoter system, wherein an expression cassette comprises a first promoter operatively linked to the alcR regulator protein sequence obtainable from Aspergillus nidulans which encodes the AlcR regulator protein, and an inducible promotor obtainable from the alcA gene promoter of Aspergillus nidulans operatively linked to a target gene [Abstract & pg. 18 ¶ 2]. Jepson discloses that this system provides higher levels of inducible expression in transgenic plants and higher proportion of high expressing lines in the primary transformant plant population [Abstract]. Jepson further teaches that a range of promoters are known to be operative to plants and it would advantageous to have a choice of a variety of promoters to select the most suitable promoter [pg. 1 ¶ 5]. Utilizing this inducible promoter would be an obvious variant to try over other promoters and one of ordinary skill in the art would be motivated to use such promoter due to the enhanced inducible expression in plant populations disclosed by Jepson. One of ordinary skill in the art would have reasonable expectation of success using this inducible promoter in the method of Aharoni as Jepson suggests soybean as an example of plants that can be genetically modified with this promoter, which was the plant used in the example of the instant application [pg. 24, ¶ 2].
Claims 22 and 23 are rejected under 35 U.S.C. 103 as being unpatentable over Aharoni as applied to claims 14-17, 19, and 24 above and further in view of Famodu et al. (2004). “New isolated polynucleotide encoding a squalene metabolic enzyme, useful as probes in gene mapping, in producing transgenic plants or in plant breeding to develop lines having desired phenotypes.” Acc. ADE28085 (PN US 6630617 B1).
As applied above, Aharoni teaches genetically modified cells and plants having increased or decreased heterologous gene expression, wherein the gene can be squalene epoxidase and may be silenced through RNAi. The heterologous nucleotide sequence may be operably linked to a promoter. Aharoni does not explicitly teach the squalene epoxidase paralog SEQ ID NO. 5, or a fragment or variant thereof.
Famodu teaches nucleic acid fragments encoding enzymes involved in squalene metabolism in plants and seeds. Famodu discloses a soybean squalene monooxidase protein (squalene epoxidase), SEQ ID NO. 14 with 99.7% identity to SEQ ID NO. 5 (alignment shown below)[¶ 9]. Famodu explicitly discloses that the nucleic acid fragments of the instant invention may be used to create transgenic plants in which the disclosed polypeptides are present at higher or lower levels than normal and that this would have the effect of altering the relative sterol composition in those cells [¶ 56]. As this sequence is a known squalene epoxidase protein in the prior art, it would have been prima facie obvious to one of ordinary skill in the art to use this sequence in the method of Aharoni. One would be motivated to use this sequence with reasonable expectation of success as it is in Glycine max, as specified in Aharoni, and because Famodu specifically suggests use of this protein in a transgenic plant to alter relative sterol composition.
Query Match 99.7%; Score 2710; Length 523;
Best Local Similarity 100.0%;
Matches 523; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MVDPYVLGWIICAVLSLVALRNFAFARKNRCHSSETDATRRAENVTTAAGECRSSSRDGD 60
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Db 1 MVDPYVLGWIICAVLSLVALRNFAFARKNRCHSSETDATRRAENVTTAAGECRSSSRDGD 60
Qy 61 VDVIIVGAGVAGSALAHTLGKDGRRVLVIERDLSEQDRIVGELLQPGGYLKLIELGLEDC 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 VDVIIVGAGVAGSALAHTLGKDGRRVLVIERDLSEQDRIVGELLQPGGYLKLIELGLEDC 120
Qy 121 VEKIDAQLVFGYALFKDGKHTRLSYPLEKFHSDVAGRSFHNGRFIQRMREKAASLSNVRL 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 VEKIDAQLVFGYALFKDGKHTRLSYPLEKFHSDVAGRSFHNGRFIQRMREKAASLSNVRL 180
Qy 181 EQGTVTSLLEEKGVIKGVHYKTKDSQELSACAPLTVVCDGCFSNLRRSLCNPKVDVPSHF 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 EQGTVTSLLEEKGVIKGVHYKTKDSQELSACAPLTVVCDGCFSNLRRSLCNPKVDVPSHF 240
Qy 241 VGLILESCELPYANHGHVILGDPSPVLFYRISSSEIRCLVDVPGQKVPSISNGEMTNYLK 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 VGLILESCELPYANHGHVILGDPSPVLFYRISSSEIRCLVDVPGQKVPSISNGEMTNYLK 300
Qy 301 TVVAPQIPPELHDSFVAAVDKGNIRTMPNRSMPAAPYPTPGALLMGDAFNMRHPLTGGGM 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 TVVAPQIPPELHDSFVAAVDKGNIRTMPNRSMPAAPYPTPGALLMGDAFNMRHPLTGGGM 360
Qy 361 TVALSDIVVLRNLLRPLRDLNDAPGLCKYLESFYTLRKPVASTINTLAGALYKVFCASPD 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 TVALSDIVVLRNLLRPLRDLNDAPGLCKYLESFYTLRKPVASTINTLAGALYKVFCASPD 420
Qy 421 PARKEMRQACFDYLSLGGLFSEGPVSLLSGLNPRPLSLVLHFFAVAIYGVGRLLLPFPSP 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 PARKEMRQACFDYLSLGGLFSEGPVSLLSGLNPRPLSLVLHFFAVAIYGVGRLLLPFPSP 480
Qy 481 KRMWIGVRLISSASGIILPIIKAEGVRQMFFPATVPAYYRNPP 523
|||||||||||||||||||||||||||||||||||||||||||
Db 481 KRMWIGVRLISSASGIILPIIKAEGVRQMFFPATVPAYYRNPP 523
Subject Matter Free of Art
The full length SEQ ID NO. 1 appears to be free of the prior art. Claim 18 and 21 are objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims. Claim 21 has additional claim objection informalities that require correction.
Conclusion
No claims are allowed.
Claims 18 and 21 have subject matter free of art.
Contact Information
Any inquiry concerning this communication or earlier communications from the examiner should be directed to EMILY K. JOHNSON whose telephone number is (571)272-5761. The examiner can normally be reached Monday - Friday 7:30 am - 5:00 pm.
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/EMILY K JOHNSON/Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/Supervisory Patent Examiner, Art Units 1661 & 1662