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 .
Election/Restrictions
Applicant’s election of Group I in the reply filed May 4, 2026 is acknowledged. Group I includes claims 1, 4, 7-12, 16, 17(a), 19-20, 22-23, 26, and 29-31, directed to a Catharanthus roseus plant and methods of preparing/using the plant by introducing a gain-of -function mutation in CrDELLA1 and/or CrDELLA2. Claims 13-14 and 17(b) are withdrawn from further consideration as being directed to a nonelected invention.
Response to Remarks
Applicant’s statement that it does not agree with the characterization of the cited prior art has been considered. However, the statement appears to related to the restriction requirement and does not present a substantive traversal of any rejection on the merits. Applicant’s election of Group I is acknowledged, and the elected claims are examined on the merits below.
Claim Status
Claims 1, 4, 7-12, 16, 17(a), 19-20, 22-23, 26, and 29-31 are pending.
Claims 1, 4, 7-12, 16, 17(a), 19-20, 22-23, 26, and 29-31 are examined on the merits.
Sequence Rules
This application contains sequence disclosures that are encompassed by the definitions for nucleotide and/or amino acid sequences set forth in 37 CFR 1.821(a)(1) and (a)(2). However, this application fails to comply with the requirements of 37 CFR 1.821 through 1.825.
Nucleotide sequences disclosed in Table 1 are not accompanied by sequence identifiers (SEQ ID NOs).
Full compliance with the sequence rules is required in response to this Office action. A complete response to this Office action must include both compliance with the sequence rules and a response to the issues set forth herein. Failure to fully comply with both of these requirements in the time period set forth in this Office action will be held to be non-responsive.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1, 4, 7-12, 16, 17, 19-20, 22-23, 26, and 29-31 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.
Claim 1, 7-9 recites “one or more DELLA transcription factors”, and claims 4, 10-12, 17, 19, and 20 further recites “CrDELLA1 and/or CrDELLA2”. However, the claims do not identify the recited DELLA transcription factors, CrDELLA1, or CrDELLA2 by SEQ ID NO, accession number, amino acid sequence, nucleic acid sequence, or other objective structural boundaries. Because “CrDELLA1” and “CrDELLA2” appear to be applicant-assigned nomenclature, the claims do not clearly define which C. roseus DELLA genes/proteins are encompassed or which sequences are being modified, deleted, or mutated. Accordingly, the metes and bounds of the claimed plant, methods, and cell are unclear.
Dependent claims 16, 22-23, 26, and 29-31 are included in this rejection because they do not include additional limitations to resolve the ambiguity.
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
Claims 1, 4, 7-12, 16, 17, 19-20, 22-23, 26, and 29-31 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.
Claims 1 is directed to a broad functional genus of Catharanthus roseus plants comprising “one or more DELLA transcription factors having enhanced activity” and being “capable of enhanced production of vindoline”. The claim encompasses transgenic plants, selectively bred plants, endogenous or heterologous DELLA proteins, and any mechanism resulting in enhanced DELLA activity and enhanced vindoline production. The claim is not limited to a specific CrDELLA activity and enhanced vindoline production. The claim is no limited to a specific CrDELLA sequence, specific mutation, specific expression level, specific regulatory mechanism, or specific transformation or breeding strategy. Thus, claim 1 encompasses a broad genus of DELLA-enhanced plants defined primarily by desired functional results.
In contrast to the breadth of claim 1, the specification discloses only two identified C. roseus DELLA proteins, CrDELLA1 (CRO_T106013; SEQ ID NO:18) and CrDELLA2 (CRO_T106004; SEQ ID NO:19), identified through BLASTP homology and conserved-domain analysis. The experimental disclosure is limited primarily to: (i) simultaneous silencing of CrDELLA1/CrDELLA2, (ii) silencing of CrGID1a/CrGIC1b, (iii) PAC treatment experiments, and (iv) selected truncation constructs including CrDELLA1Δ1-209 and CrDELLA1Δ1-112. The specification does not disclose representative DELLA variants spanning the full scope of the claimed genus, including the broad range of DELLA proteins, mutations, allelic variants, transgenic plants, and selectively bred plants encompassed by claim 1.
The specification additionally does not identify structural features common to the claimed DELLA genus that correlated with the claimed phenotype of enhanced vindoline production. The disclosure generally states that DELLA proteins interact with JAZ, PIF, FID1, and COP1 proteins and participate in light and jasmonate signaling crosstalk. However, the specification does not establish which sequence variations, mutation sites, deletion boundaries, allelic modifications, or expression changes retain DELLA functionality while also producing enhanced vindoline biosynthesis. DELLA proteins are described as interacting with “over 300 different transcription factors” (instant application, pa0008), indicating broad pleotropic regulatory activity and substantial functional complexity. The disclosure therefore does not provide sufficient structure-function correlation to permit extrapolation from the limited disclosed CrDELLA embodiments to the full claimed functional genus.
The disclosed experimental data further demonstrate variability and context dependence rather than possession of a predictable genus. The specification reports that silencing CrDELLA1/ CrDELLA2 produced moderate decrease in selected vindoline pathway genes in certain developmental contexts (pa0087-0088; Fig 4A-4D), but repeated experiments did not consistently reproduce broad vindoline pathway suppression (pa0089). The specification further reports that CrGID1a/1b silencing resulted primarily in non-significant increases in vindoline pathway gene expression (pa0090, fig 5A-5B). In addition, PAC treatment increased certain vindoline pathway genes while significantly decreasing D4H expression and showing no effect on T16H2 (pa0091-0096, fig 6A-6B). The specification additionally states that vindoline pathway induction is highly dependent on tissue specificity, developmental stage, jasmonate signaling, and light conditions (pa0005-0007). Collectively, these disclosures demonstrate context-dependent and variable biological responses rather than possession of a predictable genus of CrDELLA/CrGID1 modifications that reliably confer the full scope of the claimed enhanced vindoline, vinblastine, and/or vincristine production phenotypes.
The unpredictability of the claimed vindoline-production phenotype is further supported by Magnotta (Mary Magnotta et. al., Phytochemistry (2006) 67: 1758–1764), which reported that vindoline biosynthesis in Catharanthus roseus is complex and controlled by developmental, environmental, tissue-specific, and cell-type-specific factors (p1758, Introduction). Magnotta further taught that conversion of tabersonine to vindoline requires multiple strictly ordered enzymatic reactions, and identified a low-vindoline cultivar having approximately 10-fold lower tabersonine-16-hydroxylase activity that a normal vindoline-producing cultivar (p1758, Abstract). Additionally, Edge (Alison Edge et. al., Planta (2018) 247(1):155-169) reported that a single amino acid substitution in tabersonine 3-reductase (T3R H189Y) dramatically altered vindoline pathway metabolite accumulation and reduced T3R biochemical activity by approximately 95%, resulting in reduced vindoline accumulation and increased tabersonine epoxide intermediates (p155, right column, pa1). These studies demonstrate that vindoline biosynthesis is highly sensitive to precise pathway perturbations and biological context.
This conclusion is further supported by Applicants’ own later publication, Cole-Osborn (Lauren F. Cole-Osborn et. al., Plant Molecular Biology (2025) 115:72, pp1-26), which states that “Our results do not unequivocally prove that DELLAs directly regulate vindoline biosynthesis in C. roseus”, but instead only “provide some evidence suggesting” that increasing DELLA protein levels “could lead to increased vindoline biosynthesis”. The same publication characterizes the work as encouraging “further inquiries” into the role of DELLA proteins in TIA regulation (p3, right column, pa4). This statement is consistent with the specification’s limited and variable experimental disclosure and further supports that the application does not reasonably convey possession of the full genus of DELLA-enhanced D. roseus plants recited in claim 1.
Accordingly, the specification’s limited DELLA-related disclosure does not provide sufficient representative species or structural guidance to support the broad genus of DELLA-enhanced vindoline-producing plant encompassed by claim 1.
Accordingly, the specification does not provide a representative number of species or common structure features sufficient to demonstrate possession of the full genus of DELLA-enhanced C. roseus plants encompassed by claim 1 at the time of filing.
Claims 10-12, 17, 19 and 20 further broaden the unsupported genus by reciting broad classes of gain-of-function mutations in CrDELLA1 and/or CrDELLA2. Claim 11 recites mutations that inhibit GID1 binding, inhibit DELLA degradation in the presence of gibberellic acid, or disrupt DELLA-COP1 binding. These limitations are defined primarily by desired functional outcome rather than by specific structural feature. Although the specification generally describes DELLA N-terminal DELLA and TVHYNP motifs as being involved in GID1-mediated degradation and describes DELLA-COP1 interaction, the specification does not identify which mutations across CrDELLA1 and CrDELLA2 maintain proper DELLA structure, interaction capability, regulatory function, and vindoline-enhancing activity.
Claims 12 and 19 are particularly broad because they recite deletion of “up to 112 amino acids” at the N-terminus of CrDELLA1 and/or CrDELLA2. The specification broadly lists numerous possible deletion ranges and endpoints, including deletions of the first 50-220 amino acids and specific deletions at 50, 60, 70, 80, 90, 100, 110, 112, 115, 120, 150, 175, 200, 209, or 220 amino acids (pa0074). However, the working examples appear limited to selected constructs including CrDELLA1Δ1-209 and CrDELLA1Δ1-112 (pa0061, pa0102, fig 2A-2B). The specification does not disclose representative species across the full claimed deletion gens or establish that the full deletion range retains DELLA functionality and enhanced vindoline production capability.
Claim 8 additionally recites activation of both the upstream terpenoid indole alkaloid pathway from geraniol and tryptophan to tabersonine and the downstream vindoline pathway from tabersonine to vindoline. However, the specification reports that upstream TIA pathway genes TDC and G10H were not affected by DELLA silencing experiments (pa0087, pa0071, and fig 12). The specification does not reasonably convey possession of DELLA-enhanced plant capable of activating the full pathway breadth recited in claim 8.
Claims 16, 22, 23, 26, 29, and 30 further extend the claims to enhanced vinblastine and/or vincristine production. Vinblastine and vincristine biosynthesis involves additional downstream metabolic coupling, precursor availability, compartmentalization, developmental regulation, and pos-harvest processing. The speciation does not disclose representative plants across the claimed DELLA mutation genus that demonstrably produce enhanced vinblastine and/or vincristine throughout the full scope of the claims. Rather, the claims extrapolate broad downstream alkaloid production from limited and variable vindoline-pathway observations.
Claim 31 is deficient for the same reasons because it recites a cell obtained from the insufficiently described plant claim 1, or a C. roseus cell bearing identical genetic modifications compared to said plant.
Dependent claims 4, 7, and 9 are included in this rejection because the additional limitation doe not cure the lack of written description support for the full scope.
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 1, 4, 7, 9-10, 16, 17, 19, 20, 22-23, 29, and 31 are rejected under 35 U.S.C. §103 as being unpatentable over Liu (Yongliang Liu et. al., Plant Physiology (2019) Vol. 180, pp1336–1350) in view Ito (Takeshi Ito et. al., PLANT SIGNALING & BEHAVIOR (2018)VOL.13, NO. 3, pp1-3), and further in view of Willige (Björn C Willige et. al., Plant Cell (2007);19(4):1209-20).
Liu teaches regulation of light-induced vindoline biosynthesis in Catharanthus roseus (p1336, Abstract). Liu identifies CrGATA1 as an activator of light-induced vindoline biosynthesis and teaches that CrGATA1 induces vindoline pathway genes, including T16H2, T3O, T3R, D4H, and DAT (p1336, Abstract). Liu further teaches CrPIF1 represses CrGATA1 and DAT promoter activities through binding to the G-box or PBE-box motifs (p1344, left column, pa2) and functions as a repressor of the vindoline pathway (p1336, Abstract). Accordingly, Liu teaches that CrPIF1 negatively regulates CrGATA1 and vindoline biosynthesis in C. roseus (p1336, Abstract).
Ito teaches that DELLA proteins are central regulators of gibberellin signaling and function as transcriptional as transcriptional regulators. Ito further teaches that DELLA proteins interact with PHYTOCHROME INTERACTING FACTORs (PIFs) to inhibit their DNA-binding activity (p1, left column, pa 2).
Willige teaches that DELLA proteins contain an N-terminal DELLA domain essential for GA-dependent proteasomal degradation (p1209, Abstract). Willige further teaches that mutations in or deletion of the DELLA domain, including the Arabidopsis gai-1, 17 amino-acid deletion, stabilize DELLA proteins by preventing interaction with the GID1A GA receptor, thereby conferring GA-insensitive, gain-of-function DELLA activity (p1209, left column, pa1; p1210, left column, pa3).
Liu taches that CrPIF1 represses CrGATA1 and vindoline-pathway genes and functions as a repressor of vindoline biosynthesis in C. roseus. Ito teaches that DELLA proteins interact with PIFs to inhibit their DNA-binding activity. Willige teaches that DELLA-domain gain-of-function mutations stabilize DELLA proteins by preventing GA/DID1-mediated degradation. Therefore, a person of ordinary skill in the art would have been motivated to increase DELLA activity in C. roseus to inhibit CrPIF1-mediated repression of CrGATA1 and vindoline-pathway genes, thereby enhancing vindoline biosynthesis.
Claim 1 recites a Catharanthus roseus (C. roseus) plant comprising one or more DELLA transcription factors having enhanced activity compared to a naturally occurring C. roseus plant, wherein the plant is capable of enhanced production of vindoline compared to said naturally occurring C. roseus plant; wherein the plant is a transgenic plant or a plant obtained by selective breeding.
Under BRI, “DELLA transcription factors having enhanced activity” includes increased DELLA protein level, increased DELLA stability, reduced DELLA degradation, or increased DELLA-mediated inhibition of downstream repressors such as PIF.
Liu teaches a C. roseus plant system in which vindoline biosynthesis is enhanced by de-repression/activation of the CrPIF-CrGATA1 regulatory module (Fig 7, also see below). Ito teaches the DELLA proteins inhibit PIF DBA-binding activity (p1, left column, pa2). Willige further teaches that DELLA-domain mutations stabilize DELLA proteins by preventing GA/GID1-mediated degradation, thereby conferring gain-of-function DELLA activity. Therefore, it would have been obvious to increase DELLA activity in C. roseus to inhibit CrPIF-mediated repression and enhance vindoline production.
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Claim 4 recites the plant of claim 1, wherein the plant has a higher level of CrDELLA1 and/or CrDELLA2 proteins than said naturally occurring C. roseus plant.
For the same reasons set forth for claim 1. Ito teaches that increased/stabilized DELLA protein activity results from reduce GA/GID1-mediated DELLA degradation (p1, left column, pa1). Thus, a plant having a higher level of DELLA protein would have been obvious as a way to increase DELLA activity and inhibit PIF repression.
Claim 7 recites the plant of claim 1, wherein one or more DELLA transcription factors activate a biosynthetic pathway leading to vindoline synthesis.
Under BRI, “activate a biosynthetic pathway leading to vindoline synthesis” includes derepressing or increasing expression of vindoline-pathway genes.
For the same reasons set forth for claim 1. Liu teaches that CrGATA1 activates vindoline pathway genes and that CrPIF1 represses CrGATA1 and vindoline pathway genes (Fig 7). Ito teaches that DELLA inhibits PIF activity (p1, left column, pa2). Therefore, enhanced DELLA activity would have been expected to activate/derepress the biosynthetic pathway leading to vindoline synthesis.
Claim 9 recites the plant of claim 7, wherein said one or more DELLA transcription factors bind and inhibit a jasmonate zim domain protein (JAZ) and/or a phytochrome interacting factor protein (PIF).
Under BRI, “bind and inhibit PIF” includes DELLA interaction with PIF that inhibits PIF DNA-binding activity, as taught by Ito.
For the same reasons set forth for claim 7. Ito expressly teaches that DELLA proteins interact with PIFs and inhibit their DNA-binding activity (p1, left column, pa2). Liu teaches CrPIF1 is a repressor of Cr-GATA1 and vindoline biosynthesis (see fig 7). Thus, DELLA inhibition of PIF corresponds to the claimed DALLA inhibition of PIF.
Claim 10 recites to the plant of any of the preceding claims, wherein the plant comprises a CrDELLA1 and/or CrDELLA2 gene harboring a gain of function mutation.
Under BRI, a “gain-of-function mutation” in CrDELLA1/ CrDELLA2 includes a mutation that increases DELLA activity by increasing DELLA protein stability, reducing GA/GID-mediated degradation, or increasing DELLA-mediated repression of downstream transcription factors such as PIFs.
Claim 10 is obvious for the same reasons set forth for claim 1. Ito teaches that DELLA proteins are degraded through GA/GID1-mediated recruitment to the ubiquitin E3 ligase complex and subsequent degradation by the 26S proteasome (p1, left column, pa1). Ito further teaches that DELLA proteins interact with PIFs and inhibit their DNA-binding activity (p1, left column, pa2). Liu teaches that CrPIF represses CrGATA1 and vindoline-pathway genes and functions as a repressor of vindoline biosynthesis in C. roseus. Willige taches that DELLA-domain mutations, including the 17-amino-acid deletion in gai-1, stabilize DELLA proteins by preventing GA/GID1-mediated interaction and degradation (p1209, left column, pa1; p1210, left column, pa3).
Therefore, it would have been obvious to introduce a gain-of-function mutation in CrDELLA1 and/or CrDELLA2 to stabilize DELLA activity, inhibit CrPIF-mediated repression, and enhance vindoline biosynthesis.
Claim 16 recites the plant of claim 1, wherein the plant is also capable of enhanced production of vinblastine and/or vincristine upon processing leaves of the plant to promote reaction of vindoline and catharanthine, compared to said naturally occurring C. roseus plant.
Under BRI, “capable of enhanced production of vinblastine and/or vincristine upon processing leaves” encompasses plants having increased vindoline available as precursor substrate for downstream vinblastine and/or vincristine production after leaf processing.
Claim 16 is obvious for the same reasons set forth for claim 1. Liu further teaches that vinblastine and vincristine are derived from coupling vindoline and catharanthine (p1336, Abstract). Thus, enhancing vindoline production in the plant would have predictably increased the available vindoline precursor for vinblastine and/or vincristine production upon leaf processing.
Claim 17 recites a method of preparing a C. roseus plant capable of enhanced vindoline production. The method comprising the steps of:(a) introducing a gain of function mutation in a CrDELLA1 gene and/or a CrDELLA2 gene into a C. roseus plant.
Claim 17 is obvious for the same reasons set forth for claim 1 and 10. Liu teaches that inhibition of CrPIF1 repression increases CrGATA1/vindoline-pathway gene expression and vindoline accumulation in C. roseus (fig 7). Ito teaches the stabilized DELLA proteins inhibit PIF activity. Therefore, it would have been obvious to prepare a C. roseus plant capable of enhanced vindoline production by introducing a gain-of-function mutation in CrDELLA1 and/or CrDELLA2 to increase/stabilize DELLA activity and inhibit CrPIF1-mediated repression of vindoline biosynthesis. Willige teaches introducing DELLA-domain gain-of-functions that stabilize DELLA proteins, and Liu/Ito provide the reason to apply such stabilized DELLA activity in C. roseus to enhance vindoline production.
Claim 17 is obvious.
Claim 19 recites the method of claim 17, wherein the gain of function mutation comprises deleting up to 112 amino acids at the N-terminus of CrDELLA1 and/or CrDELLA2.
For the same reasons set forth for claim 17, Willige taches that DELLA-domain mutations, including the 17-amino-acid deletion in gai-1, stabilize DELLA proteins by preventing GA/GID1-mediated interaction and degradation (p1209, left column, pa1; p1210, left column, pa3). Accordingly, claim 19 is obvious over Liu, Ito and Willige.
Claim 20 recites the method of claim 17, wherein point mutations in a CrDELLA1, CrDELLA2, CrGIDla, and/or CrGID1b gene are introduced by radiation or chemical agent mutagenesis or by selective breeding combined with screening for increased levels of CrDELLA1 protein and/or CrDELLA2 protein.
For the same reasons set forth for claim 17, Willige teaches DELLA-domain mutations that stabilize DELLA proteins, impair GA-induced DELLA degradation, impair GID1 interaction, and confer GA-insensitive gain-of-function DELLA phenotypes. Willige further teaches DELLA-domain deletions and mutations that increases DELLA activity through reduced degradation/stabilization.
Because Ito teaches DELLA proteins inhibit PIF activity, and Liu teaches CrPIF repressed vindoline biosynthesis in C. roseus, a person of ordinary skill in the art would have been motivated to introduce known grain-of-function DELLA mutations, such as the stabilized DELLA mutants taught by Willige, into C. roseus to increase DELLA activity and suppress CrPIF1-mediated repression of vindoline biosynthesis.
Accordingly, claim 20 is prima facie obvious over Liu in view of Ito and Willige because the claimed DELLA gain-of-function mutant plant would have been obtain by applying known DELLA-stabilizing mutations to the C. roseus DELLA/PIF/vindoline regulatory system using routine plant molecular biology and transformation techniques, with a reasonable expectation of enhancing vindoline biosynthesis.
Claim 22 recites a method of producing vinblastine and/or vincristine, the method comprising the steps of: (a) providing the plant of claim 1; and (b) growing the plant under conditions suitable for the production of precursors of vinblastine and/or vincristine in the plant.
For the same reasons set forth for claim 1. Liu teaches that vinblastine and vincristine are derived from coupling of vindoline and catharanthine (p1336, Abstract). Therefore, providing the DELLA-enhanced plant of Claim 1 and growing the plant under conditions suitable for production of vinblastine/vincristine precursors would have been obvious, because enhanced vindoline production would increase the available vindoline precursor.
Claim 22 is obvious over Liu, Ito and Willige.
Claim 23 recites the method of claim 22, further comprising contacting the transgenic plant with a compound that reduces the level of gibberellic acid in the plant.
For the same reasons set forth for claim 22. Willige teaches treatment with the GA biosynthesis inhibitor paclobutrazol (PAC), which reduce GA levels and increase DALLA protein accumulation/stability (p1210, right column, pa4). Accordingly, claim 23 is obvious.
Claim 29 recites the method of claim 22, further comprising the steps of: (c) subjecting leaves of the plant to a treatment that enhances alkaloid biosynthesis in leaves of the plant; and (d) waiting for a period of time, during which vindoline and catharanthine accumulate in said leaves.
Under BRI, “a treatment that enhances alkaloid biosynthesis” includes light treatment. Liu teaches that light treatment induces vindoline-pathway gene expression and increases vindoline accumulation in C. roseus leaves/seedling (p1336, Abstract). Thus, subjecting leaves to a treatment that enhances alkaloid biosynthesis and waiting for vindoline and catharanthine accumulation would have been obvious.
Claim 31 recites a cell obtained from the plant of claim 1, or a C. roseus cell bearing identical genetic modifications compared to said plant.
For the same reasons set forth for claim 1, because the plant of claim 1 would have been obvious over Liu, Ito, and Willige, a cell obtained from that plant, or a C. roseus cell bearing the same genetic modification, would also have been obvious.
Claims 11-12 are rejected under 35 U.S.C. §103 as being unpatentable over Liu (2019) in view Ito (2018), Willige (2007) as applied claim 10 and further in view of Blanco-Touriñán (Noel Blanco-Touriñán et. al., PNAS (2020) vol.117, no. 24, pp13792–13799).
Claim 10 as the teaching of Liu, Ito and Willige is discussed above.
Claim 11 is interpreted as depend of claim 10.
Claim 11 recites the plant of claim 10, wherein the gain of function mutation is selected from mutations that inhibit gibberellic acid insensitive dwarf la (GIDla) and/or gibberellic acid insensitive dwarf lb (GIDlb) binding to DELLA, mutations that inhibit degradation of DELLA in the presence of gibberellic acid, and mutations that disrupt DELLA-COP 1 binding.
Under BRI “the gain of function mutation” includes any mutation in CrDELLA1 and/or CrDELLA2 that increase DELLA activity by preventing or reducing normal negative regulation of DELLA, including mutations that: (i) inhibit GIDla/GIDlb binding to DELLA; (ii) inhibit gibberellic-acid-mediated DELLA degradation; and (ii) disrupt DELLA-COP1 binding.
For the same reasons set forth for claims 1 and 10, Willige teaches that DELLA-domain mutations inhibit GID1-mediated DELLA interaction and degradation, thereby stabilizing DELLA protein and conferring gain-of-function DELLA activity (p1213, both left and right column). Blanco-Touriñán further teaches that COP1 physically interacts with DELLA proteins and promotes DELLA destabilization/degradation (p13792, Abstract). Therefore, mutations that inhibit GID1 binding, inhibit DELLA degradation in the presence of GA, and/or disrupt DELLA-COP1 binding would have been obvious ways to stabilize DELLA proteins and increase DELLA activity. Accordingly, claim 11 is obvious over Liu, Ito, Zhang and Blanco-Touriñán.
Claim 12 recites the plant of claim 11, wherein the gain of function mutation inhibits GID1a and/or GIDlb from binding to DELLA, and wherein the mutation comprises an N-terminal deletion of up to 112 amino acids of CrDELLA1 and/or CrDELLA2.
For the same reasons set forth for claim 11, Willige teaches that the Arabidopsis gai-1 gain-of-function mutation contains a 17-amino-acid deletion in the conserved N-terminal DELLA domain and that partial or full deletions of the DELLA domain stabilize DELLA proteins and confer GA insensitivity (p1209, Abstract; left column, pa1; p1210, left column, pa3). Therefore, deleting up to 112 amino acids at the N-terminus of CrDELLA1 and/or CrDELLA2 would have been an obvious way to inhibit GID1-mediated DELLA degradation and increase DELLA activity.
Claim 8 is rejected under 35 U.S.C. §103 as being unpatentable over Liu (2019) in view Ito (2018) and Willige (2007) as applied claim 7, and further in view of Liu (Yongliang Liu et. al., Biotechnology Letters (2021) 43:2085–2103)
Claim 7 as the teaching of Liu, Ito and Willige is discussed above.
Claim 8 is interpreted as depend of claim 7.
Claim 8 recites the plant of claim 7, wherein the one or more DELLA transcription factors activate a biosynthetic pathway from geraniol and tryptophan to tabersonine (i.e., terpenoid indole alkaloid (TIA) pathway) as well as a biosynthetic pathway from tabersonine to vindoline (vindoline pathway).
Under BRI, “activate a biosynthetic pathway from geraniol and tryptophan to tabersonine … as well as … from tabersonine to vindoline” requires activation of both the upstream TIA pathway and downstream vindoline pathway.
For the same reasons set forth for claim 7, Liu 2021 teaches that C. roseus TIA biosynthesis includes upstream and midstream pathway genes including TDC, STR, SGD, and related TIA biosynthetic genes, and downstream vindoline pathway genes including T16H2, T3O, T3R, D4H, and DAT (fig. 2, also see below). Liu 2021 further teaches that upstream and midstream TIA biosynthetic genes are coordinatively regulated with downstream vindoline-pathway genes through interconnected transcriptional regulatory networks involving CrMYC2m ORCA factors, CrGATA1, and CrPIF1 (p2096, left column, pa2, and Fig. 2). Liu 2019 teaches that CrPIF represses CrGATA1 and vindoline-pathway genes, while Ito and Willige teach that DELLA proteins inhibit PIF activity and that stabilized DELLA proteins enhance DELLA signaling through impaired GID1-mediated degradation. Therefore, modulation of DELLA-PIF singling would reasonably have been expected to influence both upstream TIA biosynthesis and downstream vindoline biosynthesis.
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Claim 26 is rejected under 35 U.S.C. §103 as being unpatentable over Liu (2019) in view Ito (2018) and Willige (2007) as applied claim 22, and further in view of Liu (2021) and Zhang (Hongtao Zhang, et. al., The Plant Journal (2011) 67, 61–71).
Claim 22 as the teaching of Liu, Ito and Willige is discussed above.
Claim 26 is interpreted as depend of claim 22.
Claim 26 recites the method of claim 22, further comprising contacting the plant with a plant defense hormone.
For the same reasons set forth for claim 22 and 8, Liu 2021 teaches that jasmonate-responsive regulatory networks, including CrMYC2 and ORCA factors, regulate upstream/midstream TIA biosynthetic genes in C. roseus (fig 2). Liu 2021 further teaches that upstream/midstream TIA biosynthesis and downstream vindoline biosynthesis are coordinately regulated through interconnected transcriptional regulatory networks in C. roseus (p2096, left column, pa2, and Fig. 2). Zhang teaches that C. roseus alkaloid biosynthesis is regulated by jasmonate-responsive transcriptional control (p61, Abstract). Specifically, Zhang identify CrMYC2 as a major activator of methyl jasmonate-responsive ORCA gene expression and teaches that MeJA-responsive expression of alkaloid biosynthesis genes in C. roseus is controlled by a transcription factor cascade (p61, Abstract). Because jasmonate/methyl jasmonate is a plant defense hormone, it would have been obvious to contact the DELLA-enhanced C. roseus plant with a plant defense hormone to further induce TIA/alkaloid biosynthesis and increase precursor production.
Accordingly, claim 26 is obvious of Liu 2019, Ito, Willige, Liu 2021 and Zhang.
Claim 30 is rejected under 35 U.S.C. §103 as being unpatentable over Liu (2019) in view Ito (2018), and Willige (2007) as applied claim 29, further in view of Magnotta (2006).
Claim 29 as the teaching of Liu, Ito and Willige is discussed above.
Claim 30 is interpreted as depend of claim 29.
Claim 30 recites the method of claim 29, further comprising the steps of:(e) harvesting said leaves; (f) homogenizing the harvested leaves in a buffer solution, whereby said vindoline and catharanthine are released from cells of the harvested leaves and one or more enzymes involved in biosynthesis of vincristine and/or vinblastine are also released from cells of the harvested leaves; and (g) incubating the homogenized leaves, whereby vincristine and/or vinblastine are produced from reaction of said vindoline and catharanthine.
For the same reasons set forth for claim 29, Magnotta teaches that vinblastine and vincristine are formed from oxidative coupling of catharanthine and vindoline, and that vindoline biosynthesis proceeds through multiple enzymatic reactions in C. roseus tissues (p1758, Introduction). Magnotta further teaches harvesting young leaves, homogenizing the harvested tissue in extraction buffer, preparing crude protein extracts (p1763, left column, pa1), and performing incubation-based enzyme assays using the extract (p1763, right column, pa1).
Therefore, one leaves containing increased vindoline and catharanthine were obtained, it would have been obvious to harvest and homogenize the leaves in buffer and incubate the homogenate to release alkaloids and endogenous enzymes and promote production of vinblastine and/or vincristine from vindoline and catharanthine.
Conclusion
No claims are allowed.
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/YANXIN SHEN/ Examiner, Art Unit 1663
/WEIHUA FAN/ Primary Examiner, Art Unit 1663
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