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 .
Status of Claims
The amendments submitted on August 25th, 2026, have been entered.
Claim 2 has been canceled.
New claim 31 has been added.
Claims 1, 4-8, 10-14, 16-18, 23, 25-28, and 31 are pending in the application.
Claims 7, 8, 10-14, 16-18, 23, and 25-28 are withdrawn from consideration for being directed to non-elected invention(s).
Claims 1, 4-6, and 31 are examined in this Office action.
The text of those sections of Title 35 U.S. Code, not included in this action, can be found in a prior Office action.
Objections to the Specification
The objections to the specification are withdrawn in light of amendments to the specification.
Objections to the Claims
The objections to claim 2 are moot in light of the claim cancellation.
The objections to claims 1 and 4-6 are withdrawn in light of amendments to the claims.
Rejection under 35 USC § 101
The rejection of claim 2 under 35 USC § 101 is moot in light on the claim cancellation.
The rejection of claims 1 and 4-6 under 35 USC § 101 is withdrawn in light of amendments to the claims.
Rejection under 35 USC § 112(a)
Scope of Enablement
Claims 1, 4-6, and 31 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 production of QA or glycosylated QA in Nicotiana benthamiana, does not reasonably provide enablement for QA or glycosylated QA in any recombinant host cell. 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 or use the invention commensurate in scope with these claims. This is a new rejection necessitated by amendments to the claims.
In re Wands lists a number of factors for determining whether or not undue experimentation
would be required by one skilled in the art to make and/or use the invention. These factors are: (1) the quantity of experimentation necessary; (2) the amount of direction or guidance presented; (3) the
presence or absence of working examples of the invention; (4) the nature of the invention; (5) the state
of the prior art; (6) the relative skill of those in the art; (7) the predictability or unpredictability of the
art; (8) the breadth of the claim. In re Wands, 858 F.2d 731, 8 USPQ2d 1400 (Fed. Cir. 1988).
The Applicant teaches:
Characterization of the saponarioside B biosynthesis pathway in Saponaria officinalis using the S. officinalis transcriptome by production in Nicotiana benthamiana.
The Applicant does not teach:
That QA or glycosylated QA can be produced in any recombinant host cell by the methodology of claim 1 (and depending claims).
The claims are broadly drawn to a method for the production of QA or glycosylated QA in any recombinant host cell. Claim 1 is drawn to a method for the production of QA or glycosylated QA by contacting β-amyrin with an SoC28 oxidase or SoC28C16 oxidase, wherein the recombinant host cell comprises one or more heterologous nucleic acids encoding the SoC28 oxidase or SoC28C16. Claims 4-6 require further enzymes along the biosynthesis pathway. Claim 31 requires that the β-amyrin is produced by contacting OS with a Saponaria officinalis β-amyrin (SobAS), wherein the recombinant host cell comprises one or more heterologous nucleic acids encoding SobAS. Thus, the breadth of the claims is such that any host cell could be used in the method.
The Applicant teaches that the first committed step of triterpene biosynthesis is predicted to be the production of β-amyrin catalyzed by an oxidosqualene cyclase, β-amyrin synthase [pg. 45, lns. 35-37]. The Applicant then follows predicted steps in the saponarioside biosynthetic pathway [pg. 46, lns. 15-40] and using the genes the Applicant had characterized to characterize candidate genes for the next steps in the pathway in Nicotiana benthamiana [pg. 48, lns. 4-42]. The Applicant does not provide sufficient working examples to show QA or glycosylated QA in any recombinant host.
The state of the art suggests that the first step in triterpene biosynthesis involves the cyclization of the linear precursor 2,3-oxidosqualene to a range of diverse scaffolds by a family of enzymes known as oxidosqualene cyclases (OSCs) (Jo, S., et al. 2025. “Unlocking saponin biosynthesis in soapwort.” Nat Chem Biol 21, 215–226. https://doi.org/10.1038/s41589-024-01681-7) [pg. 217, col. 1, ¶2]. Further, the aglycone core of SpA and SpB is QA, which is derived from one of the most common plant triterpenoid scaffolds, β-amyrin.
The method as claimed requires the presence of β-amyrin to contact with a C28 or C28C16 oxidase in the host cell to produce the desired triterpenoid. However, not all host cells have β-amyrin within the cell. β-amyrin is a pentacyclic triterpenoid found primarily in plants (Han, G. et al. 2022. “Antibacterial Mode of Action of β-Amyrin Promotes Apoptosis-Like Death in Escherichia coli by Producing Reactive Oxygen Species.” J. Microbiol. Biotechnol. 32(12): 1547-1552) [pg. 1547, ¶1]. Likely only organisms with the genetic machinery and precursor enzymes would be expected to produce QA or glycosylated QA as claimed. Further, β-Amyrin has been reported to produce mitochondrial ROS that destroy organelles and prevent cell proliferation in microbes [Abstract], demonstrating that the method may not function in a microbial cell, a yeast cell, a HeLa cell, or an insect cell in the same way as the N. benthamiana transformation.
The Applicants have not provided working examples or guidance for any other host cell other than the model species N. benthamiana. It is not clear that the breadth of recombinant host cell, which includes mammalian and microorganism cells, in addition to any plant cell, would be able to produce QA or glycosylated QA. Even among plants, most of which produce β-Amyrin, the Applicant only enables transient expression in a model species, tobacco. Tzfira et al. (2006. “Agrobacterium-mediated genetic transformation of plants: biology and biotechnology.” Current Opinion in Biotechnology. 17:147-154) teaches that not all plants are readily transformable and may depend on the method of transformation, the Agrobacterium strain used, the regeneration capacity, and the plant’s own genetic makeup and defense mechanisms for efficacy of transformation [pg. 150, col. 2, ¶2]. Because of the variability within the art at the time of filing and the lack of predictability within the genus, one of skill in the art would not reliably know how to make or use the invention as claimed.
Although claim 31 further defines that the β-Amyrin is produced by contacting 2,3-oxidosqualene (OS) with a SobAS, this requires that the host cell contains OS. Not all cells have OS and thus would not be able to produce β-Amyrin by the claimed method. For example, E. coli naturally lack 2,3-oxidosqualene (Sun, W. et al. 2023. “Integration of (S)‑2,3‑oxidosqualene enables E. coli to become Iron Man E. coli with improved overall tolerance.” Biotechnology for Biofuels and Bioproducts. 16:191) [pg. 2, col. 2, ¶3].
Thus, given the quantity of experimentation necessary; the amount of direction or guidance presented; the presence or absence of working examples of the invention; the nature of the invention; the state of the prior art; the predictability or unpredictability of the art; and the breadth of the claim, the instant disclosure does not sufficiently enable one of ordinary skill in the art to make or use the invention as claimed.
Response to Applicant’s Arguments:
The rejection of claim 2 under 35 USC § 112(a) is moot in light of the cancellation of the claim.
Applicant’s arguments with respect to claims 1 and 4-6 filed 08/25/2026 have been considered but are moot because the new ground of rejection does not rely on any reference applied in the prior rejection of record for any teaching or matter specifically challenged in the argument.
Examiner notes that the previous rejection is withdrawn and the above rejection is a new rejection necessitated by the claim amendments.
Written Description
Claims 1, 4-6, and 31 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. This is a new rejection necessitated by amendments to the claims.
The instant disclosure describes:
Identification of saponarioside pathway genes based on phylogeny and by co-expression analysis.
Characterization of candidate genes by transient expression in N. benthamiana with the genes along the pathway with full identity to the SEQ ID NOs as claimed.
The instant disclosure does not describe:
That QA or glycosylated QA can be produced in any recombinant host cell by the methodology of claim 1 (and depending claims).
The claims are broadly drawn to a method for the production of QA or glycosylated QA in any recombinant host cell. Claim 1 is broadly drawn to a method for the production of QA or glycosylated QA by contacting β-amyrin with an SoC28 oxidase or SoC28C16 oxidase, wherein the recombinant host cell comprises one or more heterologous nucleic acids encoding the SoC28 oxidase or SoC28C16. Claims 4-6 require further enzymes along the biosynthesis pathway. Claim 31 requires that the β-amyrin is produced by contacting OS with a Saponaria officinalis β-amyrin (SobAS), wherein the recombinant host cell comprises one or more heterologous nucleic acids encoding SobAS.
The instant disclosure teaches characterization of the genes along the pathway to the triterpenoid saponarioside B without reducing to practice any species other than N. benthamiana. Thus, the Applicant has not reduced to practice the variability possible with the host cells other than N. benthamiana.
The Applicant teaches that the first committed step of triterpene biosynthesis is predicted to be the production of β-amyrin catalyzed by an oxidosqualene cyclase, β-amyrin synthase [pg. 45, lns. 35-37]. The Applicant then follows predicted steps in the saponarioside biosynthetic pathway [pg. 46, lns. 15-40] and using the genes the Applicant had characterized to characterize candidate genes for the next steps in the pathway in Nicotiana benthamiana [pg. 48, lns. 4-42]. The Applicant does not provide sufficient working examples to show QA or glycosylated QA in any recombinant host.
The art suggests that the first step in triterpene biosynthesis involves the cyclization of the linear precursor 2,3-oxidosqualene to a range of diverse scaffolds by a family of enzymes known as oxidosqualene cyclases (OSCs) (Jo, S., et al. 2025. “Unlocking saponin biosynthesis in soapwort.” Nat Chem Biol 21, 215–226. https://doi.org/10.1038/s41589-024-01681-7) [pg. 217, col. 1, ¶2]. Further, the aglycone core of SpA and SpB is QA, which is derived from one of the most common plant triterpenoid scaffolds, β-amyrin.
The method as claimed requires the presence of β-amyrin to contact with a C28 or C28C16 oxidase in the host cell to produce the desired triterpenoid. However, not all host cells have β-amyrin within the cell. β-amyrin is a pentacyclic triterpenoid found primarily in plants (Han, G. et al. 2022. “Antibacterial Mode of Action of β-Amyrin Promotes Apoptosis-Like Death in Escherichia coli by Producing Reactive Oxygen Species.” J. Microbiol. Biotechnol. 32(12): 1547-1552) [pg. 1547, ¶1]. One skilled in the art would expect that singularly organisms with the genetic machinery and precursor enzymes to be able to produce QA or glycosylated QA as claimed. Further, β-Amyrin has been reported to produce mitochondrial ROS that destroy organelles and prevent cell proliferation in microbes [Abstract], demonstrating that the structure of the method may not function in a microbial cell as it does in the N. benthamiana transformation.
The Applicants have not reduced to practice any other host cell other than the model species N. benthamiana. It is not clear that the broad genus of recombinant host cell, which includes mammalian and microorganism cells, in addition to any plant cell, would provide the structure to produce QA or glycosylated QA. Even among plants, most of which produce β-Amyrin, the Applicant only reduces to practice transient expression in a model species, tobacco. Tzfira et al. (2006. “Agrobacterium-mediated genetic transformation of plants: biology and biotechnology.” Current Opinion in Biotechnology. 17:147-154) teaches that not all plants are readily transformable and may depend on the method of transformation, the Agrobacterium strain used, the regeneration capacity, and the plant’s own genetic makeup and defense mechanisms for efficacy of transformation [pg. 150, col. 2, ¶2]. Because of the variability within the art at the time of filing, the lack of sufficient variety of species to reflect the variance within the genus, and the lack of correlation between structure and function, the claims lack written description.
Although claim 31 further defines that the β-Amyrin is produced by contacting 2,3-oxidosqualene (OS) with a SobAS, this requires that the host cell contains OS. Not all cells have OS and thus would not be able to produce β-Amyrin by the claimed method. For example, E. coli naturally lack 2,3-oxidosqualene (Sun, W. et al. “Integration of (S)‑2,3‑oxidosqualene enables E. coli to become Iron Man E. coli with improved overall tolerance.” Biotechnology for Biofuels and Bioproducts. 16:191) [pg. 2, col. 2, ¶3]. Since the genus has not been adequately described by structural features or had sufficient species reduced to practice, claims 1, 4-6, and 31 do not meet the written description requirement.
Response to Applicant’s Arguments:
The rejection to claim 2 under 35 USC § 112(a) is moot in light of the cancellation of the claim. Applicant’s arguments with respect to claims 1 and 4-6 have been considered but are moot because the new ground of rejection does not rely on any reference applied in the prior rejection of record for any teaching or matter specifically challenged in the argument.
Examiner notes that the previous rejection is withdrawn and the above rejection is a new rejection necessitated by the claim amendments.
Rejections under 35 USC § 103
Claims 1, 4, and 31 are rejected under 35 U.S.C. 103 as being unpatentable over Osbourn, A. et al. International Publication No. WO 2020/260475 A1. “Transferase Enzymes.” International Publication Date 12/30/2020 (see IDS filed 03/07/2025), in view of Jia, Z. et al. (1998). “Major Triterpenoid Saponins from Saponaria officinalis.” J. Nat. Prod. 61:1368-1373 (see IDS filed 03/07/2025), and Meesapyodsuk, D. et al. (2007). “Saponin Biosynthesis in Saponaria vaccaria. cDNAs Encoding b-Amyrin Synthase and a Triterpene Carboxylic Acid Glucosyltransferase.” Plant Physiology. 143:959-969 (see IDS filed 03/07/2025).
Regarding claims 1 and 31, Osbourn teaches genes and polypeptides for glycosylating quillaic acid in host cells, including the triterpenoid biosynthesis pathway (i.e., a method for the production of a triterpenoid in a recombinant host cell).
The triterpenoid QS-21 consists of a C-30 triterpenoid backbone known as quillaic acid [pg. 1, ln. 28]. Biosynthesis of quillaic acid proceeds from β-Amyrin which is synthesized through cyclisation of the universal linear precursor 2,3-oxidosqualene (OS) by oxidosqualene cyclases (OSCs) (β-amyrin synthase) (i.e., contacting OS with a β-amyrin synthase) [Fig. 2; pg. 1, lns. 34-46]. The β-amyrin scaffold is further oxidized with a carboxylic acid, alcohol and aldehyde at the C-28, C-16, and C-23 positions, respectively, to form oleanolic acid, echinocystic acid, and lastly quillaic acid (QA) (i.e., contacting β-amyrin with a C28 oxidase to oxidize the C28 position of the β-amyrin to a carboxylic acid to form oleanolic acid; contacting oleanolic acid with an oxidase to oxidize the C16 position of the oleanolic acid to an alcohol to form echinocystic acid; contacting echinocystic acid with a C23 oxidase to oxidize the C-23 position of echinocystic acid to an aldehyde to form quillaic acid) (see Fig. 2 below).
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Osbourn teaches that other patent applications, such as WO 2019/122259 describe identification of enzymes participating in QA production. Candidate enzymes were cloned from leaf cDNA. Enzymes were tested by transient co-expression in Nicotiana benthamiana, allowing for the identification of one OSC and three cytochrome P450s (P450s) required for synthesis of β-amyrin and oxidation to quillaic acid.
Osbourn does not explicitly teach wherein the enzymes involved in the biosynthetic pathway are those of Saponaria officinalis, or a C28C16 oxidase, however, quillaic acid is documented in Saponaria officinalis as the sapogenin for the major saponins. Jia teaches that S. officinalis is well known for its detersive property and has been used for soap in the ancient times [pg. 1368, col. 1, ¶1]. Medicinally, it is still used as an expectorant in bronchitis, skin irritation, and rheumatic disorders. Two main triterpenoid saponins were previously established in the 1970s, saponasids A and D, and Jia teaches the isolation of saponariosides A and B from S. officinalis. Two major triterpenoid saponins, named saponariosides A and B, were isolated from the whole plants of Saponaria officinalis and were respectively defined to be 3-O-β-D-galactopyranosyl-(1→2)-[β-d-xylopyranosyl-(1→3)]-β-d-glucuronopyranosyl quillaic acid 28-O-β-d-xylopyranosyl-(1→3)-β-d-xylopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)-β-d-4-O-acetylquinovopyranosyl-(1→4)]-β-d-fucopyranoside (1); 3-O-β-D-galactopyranosyl-(1→2)-[β-d-xylopyranosyl-(1→3)]-β-d-glucuronopyranosyl quillaic acid 28-O-β-D-xylopyranosyl-(1→3)-β-d-xylopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→2)-[β-D-4-O-acetylquinovopyranosyl-(1→4)]-β-D-fucopyranoside (2) [Abstract].
Jia teaches quillaic acid with monosaccharide components identified as fucose, galactose, xylose, quinovose, rhamnose, and glucuronic acid (i.e., quillaic acid or glycosylated quillaic acid) [Chart 1].
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Jia teaches that chemical investigations of S. officinalis have been carried out due its medicinal and commercial importance and that triterpenoid saponins with similar oligosaccharide structures have been isolated in other plants, such as the Gypsophyla paniculata, for the commercial Merck saponin extract [pg. 1372, col. 1, ¶3].
Further, biosynthesis pathways for triterpenoid production are well established in the prior art, as is methodology for determining genes involved in biosynthesis. For example, Meesapyodsuk teaches saponin biosynthesis in Saponaria vaccaria, with the main saponin being vaccaroside [Abstract]. Saponaria vaccaria, another soapwort like Saponaria officinalis, contains bioactive oleanane-type saponins similar to those found in soapbark tree (Quillaja saponaria; Rosaceae). Saponins such as vaccaroside have potentially useful pharmacological activities, including immunogenic, anticholesterolemic, and anticancer activities [pg. 959, col. 1, ¶2]. Thus, Meesapyodsuk teaches a combined polymerase chain reaction and expressed sequence tag approach to identify genes involved in saponin biosynthesis [Abstract].
Meesapyodsuk discloses identification and isolation of β-amyrin synthase, teaching that β-amyrin synthase (SvBS) provides the first step in triterpenoid biosynthesis by converting 2,3-oxidosqualene to β-amyrin [pg. 961, col. 2, ¶2]. The identity of the enzyme was confirmed by expression in yeast, wherein gas chromatography-mass spectrometry analysis of the yeast strain showed a single additional compound as compared to a control. The SvBS amino acid sequence revealed 81%, 80% and 72% identity with the BAS of Glycyrrhiza glabra (GenBank accession no. AB037203), Medicago truncatula (GenBank accession no. AJ430607), and Arabidopsis (Arabidopsis thaliana; GenBank accession no. NM106544), respectively [pg. 961, col. 2, ¶1], while also having 94% identity to SEQ ID NO: 8 of the instant invention (i.e., β-amyrin synthase comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8; see alignment below).
Meesapyodsuk teaches that the next steps in the pathway presumably include: (1) oxidation of β-amyrin at positions 16, 23, and/or 28 [pg. 960, col. 1, ¶1]. Meesapyodsuk teaches that the EST collection, in combination with heterologous expression and other experiments, provides an effective basis with which to uncover the enzymes that catalyze the oxidation, glycosylation, and acyl transfer steps involved [pg. 965, col. 2, ¶1]. Meesapyodsuk teaches that the oxidation of β-amyrin to various sapogenins is of particular interest and relevance to a number of saponin-producing taxa.
Given that Osbourn teaches production of quillaic acid in a recombinant host cell using heterologous nucleic acids expressed in N. benthamiana, as well as methods for identifying the enzymes in other plant species such as Saponaria; given that Jia teaches identification of saponins in S. officinalis; and given that Meesapyodsuk teaches the genes involved the production of a triterpenoid, including contacting OS with a β-amyrin synthase (comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8), such that said OS is converted into β-amyrin and the steps for characterizing the oxidation steps in the pathway in other species; it would have been prima facie obvious to one of ordinary skill in the art at the time of filing to isolate the genes specific to the production of quillaic acid in S. officinalis because of its medicinal and commercial importance as taught by Jia.
One would have reasonable expectation of success as the methodology of isolation from S. officinalis and confirmation of identity of the genes required for quillaic acid gene by expression in yeast and co-expression in N. benthamiana, well as comparable β-amyrin synthase amino acid sequences of other plant species, such as Glycyrrhiza glabra, Medicago truncatula, and Arabidopsis, was already established by Meesapyodsuk and Osbourn. One would further expect success as the methods as taught by Meesapyodsuk were successful in another plant from the Saponaria genus. One would be motivated to use such a method to characterize saponin biosynthesis in S. officinalis, and particularly β-amyrin synthase, given the medicinal importance of saponins and given that Meesapyodsuk teaches that the saponins of the Caryophyllaceae family, such as S. vaccaria and S. officinalis, are almost completely based on β-amyrin. Further, one would be motivated to isolate the enzymes responsible for quilliac acid given the medical significance demonstrated in saponin production in S. officinalis as taught by Jia.
Although Osbourn, Jia, and Meesapyodsuk do not explicitly teach a C28C16 oxidase to oxidize the C28 position and the C16 position, Osbourn teaches C18 and C16 oxidases that accomplish the same function. Given that the pathway was generally known, methods for finding candidate genes for the major biosynthetic steps were known, and methods of expressing heterologous genes in a recombinant N. benthamiana host cell were known, it would have been prima facie obvious to one of ordinary skill in the art at the time of filing to use known methods with a species with a documented saponin biosynthetic pathway.
Regarding claim 4, Osbourn teaches that, as depicted in Figure 3, the branched trisaccharide chain at the C-3 position in QS-21 is initiated with a β-0-glucuronic acid (GlcpA) residue attached at the 3-0 position of QA [pg. 2, lns. 29-32]. The GlcpA residue is then linked to a o-Galactose (Galp) via a 13-1->2 linkage and to a o-Xylose (Xylp) via a 13-1,3 linkage. Osbourn teaches a QA 3-0 glucuronosyl transferase ("QA-GlcA T") capable of transferring o-glucuronic acid ("GlcpA") at the 3-0 position of quillaic acid to form 3[3-{[13-D-glucopyranosiduronic acid]oxy}-quillaic acid ("QA-GlcpA") (i.e., contacting QA with a QA 3-O glucuronosyl transferase to covalently attach D-glucuronic acid to the 3-O position of quillaic acid to form QA-GlcA); QA-GlcpA galactosyl transferase ("QA-GalT") capable of transferring o-Galactose ("Galp") via a 13-1->2 linkage to QA-GlcpA to form 3β-{[β-D-galactopyranosyl-(1->2)-β-Doglucopyranosiduronic acid]oxy}-quillaic acid ("QA-GlcpA-Galp'') (i.e., contacting QA-GlcA with QA-GlcA galactosyl transferase to covalently attach Gal via a β-1->2 linkage to QA-GlcA to form QA-GlcA-Gal); and ("Xylp'') via a 1->3 linkage to QA-GlcpA-Galp to form 3β-{[β - D-xylopyranosyl-( 1->3)-[[3-o-galactopyranosyl-( 1->2) H3-oglucopyranosiduronic acid]oxy}-quillaic acid ("QA-GlcpA-[Galp]-Xylp") (i.e., contacting QA-GlcA-Gal with a QA-GlcA-Gal xylosyl transferase to covalently attach d-Xylose (“Xyl”) via a 1 ,3 linkage to QA-GlcA-Gal to form QA-GlcA-[Gal]-Xyl or QA-Tri) [pg. 4, lns. 5-18].
Osbourn teaches characterization of two glucuronosyl transferases, a galactosyl transferase, and Rhamnosyl, Xylosyl, and dual Rhamnosyl/Xylosyl transferases which permit glycosylation of the 3-0 position of QA with the respective saccharide within the 3-0 branched trisaccharide from Quillaja Saponaria [pg. 2, lns. 40-43], and additionally teaches that the polypeptides or nucleotide sequences may be derived from other plant species [pg. 4, lns. 28-29].
Although the references do not explicitly teach that the CSL, C3Gal, and C3Xyl are from Saponaria officinalis, or have the claimed sequence identity as claimed in the instant application, as detailed above, the references explicitly teach glucuronosyl transferase, galactosyl transferase, and xylosyl transferase within other plant species. The instant claims are generally directed to the identification of enzymes forming the biosynthetic pathway producing saponarioside in S. officinalis, particularly transferases that covalently attach sugars to quillaic acid. Existence of this major saponin in Saponaria was generally known in the art, as taught by Jia. The steps of the covalent attachments are similar to other saponin triterpenoids and taught by Osbourn. Meesapyodsuk teaches that sugars are transferred by the characterized enzymes, forming bonds to the saponin carbon skeleton such as an ester linkage (i.e., covalently attached).
Further, the methods for identifying the genes involved in the pathway were known at the time of filing, as detailed above. The lack of sequences with high similarity or from S. officinalis does not render the claims novel because identification of the genes involved in the saponarioside biosynthetic pathway would have been obvious in view of Osbourn, which disclose the general principle for identifying the genes, as well as the rationale to do so, such as the medical and industrial importance of saponins from S. officinalis. Thus, the teachings of Meesapyodsuk, Osbourn, and Jia combined render obvious claim 4.
Claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over Osbourn in view of Jia and Meesapyodsuk, as applied to claims 4, and further in view of Aharoni, A. et al. International Publication No. WO 2020/049572 A1. “Cellulose-Synthase-Like Enzymes and Uses Thereof.” International Publication Date: 03/12/2020 (see IDS filed 03/07/2025).
Regarding claim 5, as detailed in the rejection of claim 4 above, Osbourn teaches production of quillaic acid in a recombinant host cell using heterologous nucleic acids expressed in N. benthamiana, as well as methods for identifying the enzymes in other plant species such as Saponaria; Jia teaches identification of saponins in S. officinalis; and Meesapyodsuk teaches the genes involved the production of a triterpenoid, including contacting OS with a β-amyrin synthase, such that said OS is converted into β-amyrin and the steps for characterizing the oxidation steps in the pathway in other species.
Osbourn teaches identification of additional enzymes along the pathway by co-expression screening [Example 4], including rhamnosyltransferase and xylosyl transferase [claim 2] heterologously expressed in a host cell. Osbourn, Jia, and Meesapyodsuk do not explicitly teach expression of a fucosyl transferase, however, Aharoni teaches a method of producing a triterpenoid saponin and teaches the key genes and enzymes in the biosynthesis pathway of triterpenoid saponins [Abstract]. Aharoni teaches that there are plentiful reports underlying saponins’ benefits to human health and medical appplications [¶06]. Fig. 20A shows the biosynthetic pathway of spinach saponins comprising co-expressed saponin β-amyrin synthase genes (see Fig. 20A below). Aharoni further teaches identification of key biosynthesis genes in a number of other plant species, including Glycyrrhiza uralensis, Beta vulgaris, Chenopodium quinoa, Medicago sativa, and Glycine max [Table 11] and teaches that the method of producing a triterpenoid saponin includes when the plant cell comprises a cell from a plant in the Caryophyllales order, the plant selected from the Saponaria genus [¶112].
Aharoni teaches the nucleotide and amino acid sequences for triterpenoid biosynthetic pathway in spinach including saponin β-amyrin synthase (SEQ ID NO: 48), cytochrome P450s (SEQ ID NOs: 49 and 52), cytochrome P450 (C-23 oxidase) (SEQ ID NO: 54), glycosyl transferases (UDP-glycosyltransferase; Fucosyl transferase) (SEQ ID NO: 56), glycosyl transferases (UDP-glycosyltransferase) (SEQ ID NO: 58 and 60), glycosyl transferases (UDP-glycosyltransferase; Xylosyl transferase) (SEQ ID NO: 62), acyl transferase (BAHD acyl transferase) (SEQ ID NO: 64), and glucuronic acid transferase (SEQ ID NO: 66) [Table 16]. Aharoni teaches that the biosynthetic intermediate of a triterpenoid saponin comprises oleanolic acid [claim 31]. Fig. 42C shows that β-amyrin is oxidized by cytochromes P450 giving rise to multiple aglycons (bayogenin; serjanic acid; oleanolic acid; medicagenic acid; glycyrrhetinic acid; soyasapogenol A; soyasapogenol B) that are decorated by CSLGs and other glycosyltransferases giving rise to triterpenoid saponins (8 - bayogenin -hexA-hex-hex (M. sativa); 9 - serjanic acid -hexA-hex (Chenopodium quinoa (C. quinoa)); 10 - betavulgaroside IV (B. vulgaris); 11 - yossoside V (S. oleracea); 12 - glycyrrhizin (G. uralensis); 13 - soyasapogenol A-hexA-hex-pent (L. japonicus);14 - soyasaponin VI (G. max)) [¶71].
Aharoni teaches that the most abundant spinach saponin, Yossoside V, has acetyl-fucose, rhamnose and glucose linked to the carboxyl at C-28, as revealed by mass spectrometry [¶948]. Aharoni teaches that a spinach fucosyltransferase (SEQ ID NO: 56) works directly on MA-GlcA to link fucose at position C-28 [¶976]. Next, a glycosyltransferase (SEQ ID NO: 58) transfers a rhamnose onto the fucose [¶977]. Aharoni teaches that Yossoside II is further converted into Yossoside III by SEQ ID NO: 60, which accepts as a substrate only an aglycone having a fucose and rhamnose attached to the C-28 position [¶977].
The references explicitly teach triterpenoid biosynthesis including frucosyl transferase, rhamnosyl transferase, and xylosyl transferase within other plant species and with different sequence identity. The instant claims are generally directed to the identification of enzymes forming the biosynthetic pathway producing saponarioside in S. officinalis, particularly frucosyl transferase, rhamnosyl transferase, and xylosyl transferases resulting in the further enzymatic development of quillaic acid. Existence of this major saponin in Saponaria was generally known in the art, as taught by Jia, and the soapwort saponins found to be similar to that of Q. saponaria. The steps of the covalent attachments are similar to other saponin triterpenoids and taught in both Aharoni and Osbourn and the covalent attachment suggested by Meesapyodsuk, as detailed above. The references teach that the saponin structure can vary based on the plant species and saponin pathway at hand [Meesapydosk, pg. 965, col. 2, ¶2], thus identification of triterpenoid biosynthesis enzymes for saponarioside B would result in the specific linkage as claimed. Further, the methods for identifying the genes involved in the pathway were known at the time of filing, as detailed above.
The lack of sequences with high similarity or from S. officinalis does not render the claims novel because identification of the genes involved in the saponarioside B biosynthetic pathway would have been obvious in view of Aharoni, Osbourn, which disclose the general principle for identifying the genes, as well as the rationale to do so, such as the medical and industrial importance of saponins from S. officinalis, as taught by Meesapydosuk. Thus, the teachings of Meesapyodsuk, Aharoni, Osbourn, and Jia combined make obvious claim 5.
Response to Applicant’s Arguments:
Applicant's arguments filed 08/25/2026 have been fully considered but they are not persuasive. The Applicant contends that in order to meet its burden in establishing a rejection under 35 USC § 103, the Office must demonstrate that the references teach or suggest all claims, a reason for combining them, and provide evidence that the combination would be a predicted success (See Remarks page 21, first paragraph). The Applicant argues that none of the cited references disclose any of the oxidases of claim 1 (SoC28, SoC28C16, or SoC23), or expression of these oxidases in a recombinant host cell (id., page 21, paragraph 4). Further, the Applicant contends that the references do not disclose the series of reactions needed to convert β-amyrin into quillaic acid or the transferase enzymes of claims 4 to 6 (id., page 21, paragraphs 4-5). The Applicant asserts that the isolation of saponins does not make the claimed biosynthetic route obvious and that a skilled person could not reasonably predict the specific S. officinalis oxidases from the biosynthetic pathway characterized in other plant species without the present specification (id., page 21, paragraph 6). Because triterpenoid pathways vary between plants, the Applicant argues that the teachings would not transfer to another species.
Examiner respectfully disagrees.
In response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In this case, Jia teaches the existence of the major saponin in S. officinalis and its uses, providing motivation for isolation of saponins from S. officinalis.
Osbourn teaches genes and polypeptides for glycosylating quillaic acid in host cells, including the triterpenoid biosynthesis pathway. Osbourn further teaches that other patents applications, such as WO 2019/122259, describe identification of enzymes participating in QA production (See Office Action dated 7/8/2026, page 22, paragraph 1). Candidate enzymes were cloned from leaf cDNA. Enzymes were tested by transient co-expression in Nicotiana benthamiana, allowing for the identification of one OSC and three cytochrome P450s (P450s) required for synthesis of β-amyrin and oxidation to quillaic acid. Similarly, Meesapyodsuk teaches a combined polymerase chain reaction and expressed sequence tag approach to identify genes involved in saponin biosynthesis (id., page 12, last paragraph to page 13, first three paragraphs).
The inventive concept of the instant invention appears to be production of a triterpenoid in a recombinant host cell by isolating, characterizing, and expressing S. officinalis enzymes along the QA triterpenoid biosynthetic pathway. Although Osbourn and Meesapyodsuk teach identification and utilization of enzymes within another plant species, the methods for identifying and expressing the enzymes are not novel and could have been readily used by one of ordinary skill in the art at the time of filing. Saponins and saponin biosynthesis were well documented in Saponaria officinalis and the methods as taught by Meesapyodsuk were successful in another plant from the Saponaria genus (id., page 15, paragraph 1). As this was done in related plant species and saponins in S. officinalis are of medical relevance (id., page 15, paragraph 2), one would have reason to combine the methods to isolate such enzymes with reasonable expectation of success. One would have identified the claimed sequences without inventive effort and used them in the method as claimed as the methodology for identifying the sequences was present in the art at the time of filing as was heterologous expression of enzymes along the QA pathway.
With regard to Applicant’s argument that none of the cited references disclose any of the oxidases of claim 1 (SoC28, SoC28C16, or SoC23), as disclosed in the Office Action (page 21, paragraph 2), Osbourn teaches the β-amyrin scaffold is further oxidized with a carboxylic acid, alcohol and aldehyde at the C-28, C-16a and C-23 positions, respectively, to form oleanolic acid, echinocystic acid, and lastly quillaic acid (QA). As taught in Aharoni and Osbourn, methods for identifying genes involved in the pathway were known at the time of filing and rely on next-gen sequencing, transcriptome analysis and exploitation of knowledge from similar pathways, such as Saponaria vaccaria and Quillaja saponaria (id., page 23, paragraph 1). Insofar as the claim limitations rely on known principles of gene identification in the art, identification of the genes involved in the saponarioside biosynthetic pathway would have been obvious in view of Aharoni, Osbourn, which disclose the general principle for identifying the genes, as well as the rationale to do so, such as the medical and industrial importance of saponins from S. officinalis. It would not have been inventive to identify the oxidases of claim 1 and express them in a recombinant host cell. As detailed above, Osbourn does explicitly teach the sequence of oxidation reactions needed to convert β-amyrin into quillaic acid [Osbourn, Fig. 2].
In response to Applicant’s assertation that the isolation of saponins does not make the claimed biosynthetic route obvious and that a skilled person could not reasonably predict the specific S. officinalis oxidases from the biosynthetic pathway characterized in other plant species without the present specification, Meesapyodsuk, Aharoni and Osbourn teach characterization, identification, and confirmation of key genes in saponin triterpenoid biosynthesis pathways, including those of QA (See Office Action, page 25, paragraph 1). In particular, Aharoni, Osbourn, and Meesapyodsuk teach β-amyrin synthase for β-amyrin synthesis from OS and Aharoni and Osbourn further teach oxidation at positions C-28, C-16, and C-23 to form quillaic acid.
The references teach key genes in saponin triterpenoid biosynthesis, but within other plant species. The instant claims are generally directed to the identification of enzymes forming the biosynthetic pathway producing QA in S. officinalis. Existence of quillaic acid in Saponaria was known in the art, as taught by Jia. The steps of the pathway are similar to other saponin triterpenoids, as taught in both Aharoni and Osbourn. Meesapydsuk additionally teaches that soapwort contains saponins, such as QA, similar to those found in Quillaja Saponaria [Abstract]. Further, the methods for identifying the genes involved in the pathway were known at the time of filing. As taught in Aharoni and Osbourn, the methods rely on next-gen sequencing, transcriptome analysis and exploitation of knowledge from similar pathways, such as Saponaria vaccaria and Quillaja saponaria. Insofar as the claim limitations rely on known principles of gene identification in the art, identification of the genes involved in the QA biosynthetic pathway would have been obvious in view of Aharoni, Osbourn, which disclose the general principle for identifying the genes, as well as the rationale to do so, such as the medical and industrial importance of saponins from S. officinalis.
One skilled in the art would have been able to use known methodology to identify, characterize, and express in a recombinant host cell genes from S. officinalis along the QA biosynthetic pathway. Although triterpenoid pathways vary between plants, one would not need the instant specification in order to use known methodology for a known triterpenoid in S. officinalis. One would have identified the transferases according to the combined teachings of Jia, Osbourn, Aharoni, and Meesapyodsuk as they were suggested to produced glycosylated QA in the combined references.
Summary
Claim 6 is deemed free of the prior art given the failure of the prior art to teach or reasonably suggest the application of quinovosyl transferase from Saponaria officinalis with 50% identity to SEQ ID NO: 36. Aharoni teaches that takes part in the last step of saponin biosynthesis in spinach by transferring the acetyl group by a member of the benzylalcohol acetyl-, anthocyanin-O-hydroxy-cinnamoyl-, anthranilate-N-hydroxy-cinnamoyl/benzoyl-, deacetylvindoline acetyltransferase (BAHD) superfamily of acyltransferases reported to be involved in triterpenoid saponin biosynthesis. However, the references do not teach or reasonably suggest the application of quinovosyl transferase from Saponaria officinalis with 50% identity to SEQ ID NO: 36, thus the subject matter of claim 6 appears to be free of the prior art due to the recitation of quinovosyl transferase from Saponaria officinalis with 50% identity to SEQ ID NO: 36.
Claims 1, 4-6, and 31 are rejected.
Applicants’ amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR § 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/EMILY K JOHNSON/Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/Supervisory Patent Examiner, Art Units 1661 & 1662
