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
Examiner notes that Applicant does not agree with the characterization of the special technical feature [pg. 8], but Applicant does not explicitly elect to traverse the restriction requirement. Because the Applicant did not distinctly and specifically point out supposed errors in the restriction requirement, the election has been treated as an election without traverse. See MPEP § 818.03(a).
Applicant’s election of Group I (claims 1-7, 9, 10, 13, and 21) and “NUDX13” (taken to mean NUDIX 13) in the reply filed on 1/14/2026 is acknowledged.
Priority
Application No. 18/730,449 filed 7/19/2024 is a 371 of PCT Application No. PCT/US23/60894 filed on 01/19/2023, which claims benefit to Provisional Application No. 63/301,026 filed 1/19/2022.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 7/19/2024 and 3/20/2025 were considered, initialed, and attached hereto. A signed copy of the list of references cited is included with this Office Action.
The listing of references on several pages of the Specification is not a proper information disclosure statement (i.e. pg. 85, ¶184). For example, 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.
Status of Claims
Claims 1-7, 9-10, 13, 16-18, 21-24, and 27-29 are pending.
Claims 16-18, 22-24, and 27-29 are withdrawn from consideration as being drawn to non-elected inventions.
Claims 1-7, 9-10, 13, and 21 filed 1/14/2026 examined herein.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code (for example on pg. 65, ¶141 and 142]. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
Claim Objections
In claim 1, and those depending therefrom, “NUDIX” is used as abbreviation. It is suggested to insert a definition for NUDIX without bringing in new matter, immediately before the first appearance of “NUDIX” in claim 1; and to enclose the appearance of “NUDIX” in parentheses (in claim 1 only).
Claim 21 is objected to for the recitation of “an” engineered plant of claim 1. It is suggested to rewrite to recite “the” engineered plant of claim 1. Appropriate correction is required.
Claim Interpretation
The claims are drawn to increased gene expression in a transgenic plant. Although no specific utility is referenced in the claims, the plants and methods of growing/propagating/harvesting are for use in phytoremediation based on Applicant’s disclosure.
The term NUDIX is used without a definition in claim 1. This gene is taken to encompass the Nudix (nu cleoside di phosphate linked to some moiety, X) hydrolases, including names NUDX and NUDT, as well as additional alternative names in some plant species such as Arabidopsis: ATDCP2 (AtNUDT27) and ATASPP (AtNUDT14) (Krasezewka, E., et al. 2008, “The plant Nudix hydrolase family,” Acta Biochimica Polonica, 55(4): 663-671, as cited in IDS filed 3/20/2025). The Applicant cites the Krasezewka publication when referencing the 27 NUDIX enzymes found in Arabidopsis (NUDIX1-27) [pg. 31, ¶103]. Additionally, in the Applicant Remarks Made in an Amendment filed 1/14/2026, the Applicant elects “NUDX13” from the group of NUDIX genes (NUDIX 1-27) [pg. 9].
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.
Claims 1, 10 and 21 are rejected under 35 U.S.C. 101 because the claimed invention is directed to non-statutory subject matter. The claims do not fall within at least one of the four categories of patent eligible subject matter because the claimed invention is directed to a judicial exception (i.e., a law of nature, a natural phenomenon, or an abstract idea) without significantly more. Claims 1 and 21 are determined to be directed to subject matter that is naturally occurring, or to a law of nature/natural principle or natural phenomenon. The rationale for this determination is explained below.
The claims recite an engineered plant comprising increased NUDIX gene expression or increased amount of NUDIX gene products and a method comprising growing the plant, however the limitation of “engineered plant” is interpreted as being structurally equivalent to a natural plant as it is not linked to any recited structural features that would distinguish the engineered plant from a naturally occurring plant. The instant specification discloses that "engineered" is used to include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of a modified nucleic acid including those engineered plants initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic [¶64]. Further, the Applicant discloses that the term "engineered" does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation. However, transgenic plant techniques used to make such an engineered plant can be used, for example, to repair a mutation in a plant and thereby revert the plant to one that is genetically identical to a wild-type plant (Kmiec et al., U.S. Patent Application Publication 2003/0236208 A1, paragraphs 0096-0107). Because the term “engineered” is not linked to any recited structural features that would distinguish the plant from one that is naturally occurring, the claims are drawn to any plant.
The limitation of increase NUDIX gene expression, increased amount of one or more NUDIX gene products, or increased activity of one or more NUDIX enzymes, as compared to a suitable control does not amount to significantly more than the judicial exception because NUDIX genes are naturally ubiquitous and highly conserved across plant species. Liu, Y. et al. (2022, “Nudix hydrolase 14 influences plant development and grain chalkiness in rice,” Front. Plant Science, 13) teaches that AtNUDT7 is induced by naturally occurring biotic stress, including drought, salinity, and plant immunity [pg. 2, col. 1, ¶2]. This indicates that under natural conditions, such as a drought, AtNUDT7, a NUDIX gene product, would be induced or increase in amount as compared to a suitable control.
Additionally, the increase in NUDIX gene expression would include NUDIX 13 listed in claim 10, as this protein has been characterized as a novel member of the Arabidopsis thaliana family of Nudix enzymes (Olejnik, K. et al. (2007), Cloning and characterization of AtNUDT13, a novel mitochondrial Arabidopsis thaliana Nudix hydrolase specific for long-chain diadenosine polyphosphates,” FEBS Journal, 274:4877-4885 (as cited in IDS filed 3/20/2025)). Olejnik teaches that mitochondrial Nudix hexaphosphate hydrolase AtNUDT13 could be involved in the turnover of ATP and ADP during plant stress [pg. 4882, col. 1, ¶1]. Claim 21, a method comprising growing the plant of claim 1, would be a natural process as the plant may be identical to one that would appear in nature and fluctuate in NUDIX gene expression or gene products.
This judicial exception is not integrated into a practical application because the claimed invention is directed to a naturally occurring process. The claimed plants have the inherent properties that are recited in the claims. The claim does not include additional elements that are sufficient to amount to significantly more than the judicial exception because the claim is directed to a naturally occurring composition and process.
Claim Rejections - 35 USC § 112(a)
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 2, 4-7, and 13 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
The claims are broadly drawn to an engineered plant comprising overexpression of NUDIX genes, gene products, or enzymes. Claim 2 is drawn to modified NUDIX genes and/or gene products producing an engineered plant that overexpresses NUDIX polypeptides. Claims 4-7 and 13 are additionally drawn to modification including mutations, insertions, deletions, substitutions, or any combination thereof, or other modifications, such as post-translational modification and epigenome modification. Claims 6, 7, and 13 are further drawn to modification of the mRNA to increase protein translation or mRNA stability.
The instant specification reduces to practice Arabidopsis gain-of-function plants overexpressing a subset of NUDIX enzymes, including NUDIX13 [¶262]. The specification reduces to practice transgenic lines that contain the CaMV 35S promoter driving expression of NUDIX13 fused to a C-terminal GFP tag ¶263].
The instant specification does not reduce to practice that any modification, including any mutation, insertion, deletion, substitution or any combination thereof, of NUDIX genes would produce an engineered plant that increased expression of NUDIX genes as compared to a control. The instant specification does not reduce to practice a modified epigenome that increases NUDIX gene expression, or what modifications of the epigenome would provide the desired function and how to implement such modifications. The instant specification does not reduce to practice a modified NUDIX mRNA, wherein the mRNA has been modified as compared to an unmodified control to increase protein translation, mRNA stability, or both, or post-translational modifications such that activity of the one or more modified polypeptides is increased as compared to the control.
Modifications, including mutations, insertions, deletions, and/or substitutions can have varying effects on gene expression. Ge, F. et al. (2024, Review of Computational Methods and Database Sources for Predicting the Effects of Coding Frameshift Small Insertion and Deletion Variations,” ACS Publications, 9:2032-2047) teaches, for example, silent mutations may not change the protein structure and thus do not result in increased gene expression, while others, such as frameshift mutations, can cause a premature stop codon resulting in a truncated protein that is not functional [pg. 2032, col. 1, ¶1]. As the Applicant only provides the example of the use of transgenic lines that contain the CaMV 35S promoter driving expression of NUDX13, the modification of the endogenous NUDIX gene comprising any mutation, insertion, deletion, substitution or combination thereof is not reduced to practice. Undue experimentation would be required to be required to confirm that the structure of the mutation performs the function of increasing NUDIX gene expression and the specification lacks sufficient variety of species to reflect the variance within the genus of modification.
The variability of engineered epigenome modification in plants is influenced by several factors, including the specific tools used for editing, the targeted genes, and the environmental conditions under which the modifications are applied. Qi, Q. et al. (2023, “Advances in Plant Epigenome Editing Research and Its Application in Plants,” Molecular Sciences, 24, 3442) teaches that epigenetic changes, such as DNA methylation, histone modifications, and non-coding RNA changes, can affect genome stability and gene expression [pg. 2, ¶1]. Different editing platforms, such as dCas9, TALEs and ZFPs, can have variable results, with CRISPR able to precisely target and edit specific epigenetic features. By fusing the dCas9 protein with methylation- and histone-modifying enzymes, the expression of the target gene can be directly regulated by epigenetic modification of the gene. These epigenomic alterations can effectively design target traits and also activate silenced genes to perform new functions in plant development. However, not all epigenetic modifications have the same function or would have the same effect on gene expression, for example DNA methylation typically silences genes, while histone modifications can either promote or suppress gene expression. RNA-associated silencing can also influence gene expression by interacting with mRNA or affecting histone modifications [pg. 6, ¶4]. Thus, any modification epigenome of claim 4 would not necessarily increase NUDIX gene expression.
The instant specification indicates that epigenome editing can be accomplished utilizing a programmable nuclease system or other approach to provide targeted epigenomic modification to the endogenous genomic site of interest, and that methods and techniques are generally well known in the art [¶180]. The only example in the instant specification does not reduce to practice any modification or methodology of modification of the epigenome, including DNA methylation and/or histone methylation of a histone associated with the one or more NUDIX genes, as claimed in claim 5. The instant specification provides no guidance or way of determining how to arrive at the implementation of such modifications other than stating that the methods and techniques are well known in the art and could be performed with a NUDIX gene.
Similarly, the instant specification states that modifications of mRNA may be genetically encoded in the DNA or that the mRNA may be directly modified, yet does not provide an example of mRNA modification related to the NUDIX genes. Chmielowska-Bąk, J. et al. (2019, “In search of the mRNA modification landscape in plants,” BMC Plant Biology, 19:421) teaches that mRNA modification may occur through natural chemical modification of mRNA. This includes adenine methylated at the N6 position (m6A), cytosine methylated at the 5′ position (m5C), 8-hydroxyguanosine (8-OHG) and 8-nitroguanosine (8-NO2G) [pg. 2, col. 1, ¶1]. Chmielowska-Bąk teaches that methylation of adenosine at the N6 position (m6A) is the most widely studied internal mRNA modification [pg. 2, col. 1, ¶2]. Chmielowska-Bąk teaches that m6A might affect mRNA metabolism in many distinct ways– by increasing its translocation from nucleus to cytoplasm, modulating stability, enhancing translation or affecting splicing [pg. 3, col. 1, ¶2]. However, Chmielowska-Bąk teaches that alterations in methylation/demethylation machinery may also lead to severe developmental defects, disturbed development of vascular tissues, hampered inflorescence internodes and root growth [pg. 2, col. 2, ¶2]. Chmielowska-Bąk teaches that oxidative mRNA modifications, such as 8-hydroxyguanosine (8-OHG), can hamper translation, resulting in a decreased level of encoded proteins [pg. 4, col. 2, ¶2]. Thus, any modification of mRNA as compared to an unmodified control will not necessarily result in increased protein translation, mRNA stability, or both. The specification, although it stated that mRNA modifications are well known in the art, does not reduce to practice that any mRNA modification would perform the given function or that a modified NUDIX mRNA or nucleic acid modifications thereof that increase translation or mRNA stability. The different modifications of mRNA would not be expected to act similarly. It is unreasonable to conclude that any modification would result in the claimed function. Undue experimentation would be necessary to confirm that a modification of NUDIX mRNA results in the engineered plant.
The specification fails to provide adequate written description to support claims 2, 4-7, and 13. The limited example provided of transgenic lines containing the promoter driving expression of NUDIX 13 do not describe the claimed genus. Therefore, one of ordinary skill in the art at the time of filing would not have recognize the Applicant to be in possession of the claimed invention. Thus, claims 2, 4-7, and 13 do not meet the written description requirement.
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.
Claims 1-5 and 21 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Ogawa, T. et al. (2009), Overexpression of an ADP-ribose pyrophosphatase, AtNUDX2, confers enhanced tolerance to oxidative stress in Arabidopsis plants,” The Plant Journal, 57:289–301 (as cited in IDS filed 7/19/2024).
Claim 1 recites an engineered plant comprising: a. increased NUDIX gene expression as compared to a suitable control; b. increased amount of one or more NUDIX gene products as compared to a suitable control; c. increased activity of one or more NUDIX enzymes as compared to a suitable control; or d. any combination of (a) to (c).
Claim 2 recites the engineered plant of claim 1, wherein the engineered plant comprises one or more modified NUDIX genes and/or gene products thereby producing an engineered plant that overexpresses one or more NUDIX polypeptides as compared to a suitable control.
Claim 3 recites the engineered plant of claim 1, wherein the one or more NUDIX gene products are NUDIX mRNA, NUDIX polypeptide(s), or both.
Claim 4 recites the engineered plant of claim 1, wherein the engineered plant comprising increased NUDIX gene expression a. comprises one or more exogenous NUDIX genes or a portion thereof that encodes a NUDIX polypeptide; b. comprises one or more modified endogenous NUDIX genes; c. comprises a modified epigenome thereby increasing NUDIX gene expression; or d. any combination of (a) to (c).
Claim 5 recites the engineered plant of claim 4, wherein a. the one or more modified endogenous NUDIX genes comprise one or more mutations, insertions, deletions, substitutions, or any combination thereof in the coding or non- coding region of the one or more modified endogenous NUDIX genes such that expression of the one or more modified endogenous NUDIX genes are increased as compared to an unmodified control; b. the modified epigenome comprises decreased DNA methylation of one or more NUDIX genes and/or histone methylation of a histone associated with the one or more NUDIX genes thereby increasing gene expression of the one or more NUDIX genes; c. the modified epigenome comprises increased histone acetylation of a histone associated with the one or more NUDIX genes thereby increasing gene expression of the one or more NUDIX genes; or d. any combination of (a) to (c).
Claim 21 recites a method comprising: growing, propagating, harvesting, and/or cultivating an engineered plant of claim 1.
Regarding claim 1, Ogawa explicitly discloses the effect of overexpression of AtNUDX2 on levels of ADP-ribose, NAD(H) and ATP, and PAR activity in Arabidopsis [pg. 290, col. 2, para 2]. Ogawa teaches that Nudix hydrolases, characterized by a conserved Nudix motif, GX5EX7REVXEEXGU, where U is usually Ile, Leu or Val [pg. 290, col. 1, ¶3]. Ogawa teaches that overexpression of AtNUDX2, encoding ADP-ribose pyrophosphatase, confers enhanced tolerance of oxidative stress on Arabidopsis plants, resulting from maintenance of NAD+ and ATP levels by nucleotide recycling from free ADP-ribose molecules under stress conditions [Abstract]. Ogawa teaches the generation of transgenic Arabidopsis plants overexpressing AtNUDX2 under the control of the CaMV 35S promoter (Pro35S:AtNUDX2) (i.e. engineered plant) [pg. 291, col. 1, ¶1]. A. tumefaciens transformed by electroporation was used to infect Arabidopsis via the vacuum infiltration method and the seedlings grown on basic MS medium in petri dishes before being transferred to soil [pg. 298, col. 1, para 2]. A Western blot analysis showed an increase of AtNUDX2 protein in Pro35S:AtNUDX2 plants compared with control plants (i.e. increased amount of one or more NUDIX gene products as compared to a suitable control) [Figure 2a].
Regarding claims 2 and 3, Ogawa teaches the screening of a large number of activation-tagged mutants in Arabidopsis [pg. 290, col. 2, ¶3]. An activation T-DNA-tagging screen can identify mutants that are caused by gain-of-function [pg. 290, col. 1, ¶2]. Ogawa teaches that the large number of mutants were screened using paraquat (PQ), an agent which causes oxidative stress. A single mutant line (pqr-216) with enhanced tolerance to oxidative stress was isolated, with the resistance phenotype likely due to increased gene expression caused by insertion of T-DNA at a single site [pg. 290, col. 2, ¶3]. Thermal asymmetric interlaced PCR analysis showed that the T-DNA was inserted 2.1 kbp upstream from the predicted translation start point of a gene, At5g47650, encoding AtNUDX2 (Figure 1c) (i.e. engineered plant comprising a modified NUDIX gene). Ogawa explicitly discloses that the AtNUDX2 transcript was found to be 10.6 +/- 2.7-fold higher in pqr-216 than in the wild-type plants [Figure 1d; pg. 291, col. 1, ¶1]. Ogawa teaches a Western blot analysis using a polyclonal antibody raised against the recombinant AtNUDX2 protein showed accumulation of a polypeptide of 32 kDa in pqr-216 plants, which corresponds to the deduced molecular mass of the AtNUDX2 protein (31.6 kDa) (i.e. wherein the one or more NUDIX gene products are NUDIX polypeptides) [Figure 1d].
Regarding claim 4, Ogawa teaches the generation of AtNUDX2-overexpressing plants comprising increased NUDIX gene expression [Abstract]. Ogawa teaches isolation of RNA from Arabidopsis plants and the cDNA encoding AtNUDX2 protein amplified and cloned into pST-Blue T-vector [Pg. 298, col. 1, ¶2]. The cDNA encoding the open reading frame of AtNUDX2 was then cloned into an XbaI/SacI site downstream of the CaMV 35S promoter of binary vector pDH123. Agrobacterium tumefaciens was transformed with the construct and used to infect Arabidopsis [i.e. wherein the engineered plant comprises one or more exogenous NUDIX genes].
Regarding claim 5, Ogawa teaches a PQ resistant line pqr-216 with T-DNA inserted upstream from the predicted translation start point of a gene encoding AtNUDX2 (i.e. engineered plant comprising increased NUDIX gene expression comprising one or more modified endogenous NUDIX genes) [pg. 290, col. 2, ¶3]. Ogawa teaches that the insertion of T-DNA at a single site was recognized as the catalyst for increased gene expression and ultimately the phenotype of PQ resistance (i.e. wherein the one or more modified endogenous NUDIX genes comprise one or more insertions in the coding or non-coding region of the genes). Ogawa teaches that the pqr-216 mutant had higher AtNUDX2 gene expression as compared to the wild-type plants (i.e. such that the expression of the one or more endogenous NUDIX genes are increased as compared to an unmodified control) [pg. 291, col. 1, ¶1].
Regarding claim 21, Ogawa explicitly discloses infecting Arabidopsis with the AtNUDX2 vector. T1 seedlings were selected on basic MS medium in Petri dishes before transfer to soil. T3 seeds were harvest from the plants and used to the experiments (i.e. growing, propagating, harvesting, and/or cultivating the plant of claim 1) [pg. 298, col. 1, ¶2].
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.
Claims 6 and 7 are rejected under 35 U.S.C. 103 as being unpatentable over Ogawa (as cited in IDS filed 7/19/2024) as applied to claims 1-5 above, and further in view of Ugar, S. et al., US Application Publication No. US 2017/0009244 A1, published 2017 (as cited in IDS filed 7/19/2024).
Claim 6 recites the engineered plant of claim 1, wherein the engineered plant comprising increased amount of one or more NUDIX gene products as compared to a suitable control comprises (a) a modified NUDIX mRNA, wherein the mRNA has been modified as compared to an unmodified control to increase protein translation, mRNA stability, or both; (b) a modified NUDIX polypeptide, wherein the polypeptide has been modified as compared to an unmodified control to increase protein stability; or both (a) and (b); (c) one or more modified NUDIX polypeptides comprises one or more amino acid mutations, insertions, deletions, substitutions, or any combination thereof such that activity of the one or more modified NUDIX polypeptides is increased as compared to a control; (d) comprises one or more post-translational modifications such that activity of the one or more modified NUDIX polypeptides is increased as compared to a control; or (e) any combination of (a)-(d).
Claim 7 recites the engineered plant of claim 6, wherein the modified NUDIX mRNA comprises (a) one or more mutations, insertions, deletions, substitutions, or any combination thereof such that translation and/or stability of the NUDIX mRNA is increased; (b) one or more nucleic acid modifications that increases translation and/or mRNA stability; or both (a) and (b).
Regarding claims 6 and 7, Ogawa teaches the engineered plant of claim 1, yet does not explicitly teach the engineered plant comprising increased amount of one or more NUDIX gene products as compared to a suitable control comprising a modified NUDIX mRNA, wherein the mRNA has been modified as compared to an unmodified control to increase protein translation, mRNA stability, or both; (b) a modified NUDIX polypeptide, wherein the polypeptide has been modified as compared to an unmodified control to increase protein stability; or both (a) and (b); (c) one or more modified NUDIX polypeptides comprises one or more amino acid mutations, insertions, deletions, substitutions, or any combination thereof such that activity of the one or more modified NUDIX polypeptides is increased as compared to a control; (d) comprises one or more post-translational modifications such that activity of the one or more modified NUDIX polypeptides is increased as compared to a control; or (e) any combination of (a)-(d).
However, Ugar teaches modification of RNA for increased transcript stability and translation efficiency, in particular mRNA and an increase in mRNA translation (modified mRNA to increase protein translation as compared to an unmodified control) [¶011]. Ugar teaches that RNA having an open-ended poly(A) sequence was found to be translated more efficiently than RNA having a poly(A) sequence with a masked terminus and that a double 3′-untranslated region (UTR), in particular of the human beta-globin gene, in an RNA molecule improves translation efficiency in a way which clearly exceeds the total effect to be expected using two individual UTRs [¶012]. A combination of the above-described modifications was found according to the invention to have a synergistic influence on the stabilization of RNA and the increase in translation. Ugar teaches that any nucleic acid (DNA/RNA)-based vector systems (for example plasmids, adenoviruses, poxvirus vectors, influenza virus vectors, alphavirus vectors, and the like) may be used to introduce the vector into a host cell [¶065]. Ugar teaches that the modifications may be introduced into expression vectors and utilize them for the purpose of increasing transcription of recombinant nucleic acids and expression of recombinant proteins in cell-based systems. This includes, for example, enzymes. Ugar teaches that the host cells are any which can be transformed or transfected with an exogenous nucleic acid, comprising prokaryotic (e.g. E. coli) or eukaryotic cells (e.g. yeast cells and insect cells) [¶126].
Given that Ogawa teaches the engineered plant of claim 1, wherein AtNUDX2 gene expression is increased in Arabidopsis and given that Ugar teaches modified mRNA that increases protein translation and stability, it would have been prima facie obvious to one of ordinary skill in the art at the time of filing to combine the method overexpression of Ogawa with the method of modification of Ugar. One would have been motivated to combine the overexpression with the modified mRNA to increase protein translation and stability. One would have reasonable expectation of success as the overexpression of the AtNUDX gene would upregulate endogenous genes which could then be modified by the mRNA modifications proposed by Ugar. Ugar specifies that these modifications may be used with an enzyme (such as NUDIX) and that any host cell which may be transformed would be applicable for use.
Claim 9 is rejected under 35 U.S.C. 103 as being unpatentable over Ogawa (as cited in IDS filed 7/19/2024) as applied to claims 1-5 above, and further in view of Lee, K. et al. (2019), “CRISPR/Cas9-mediated targeted T-DNA integration in rice,” Plant Molecular Biology, 99:317-328.
Claim 9 recites the engineered plant of claim 1, wherein the engineered plant comprises a CRISPR-Cas system or component(s) thereof.
As above, Ogawa teaches a PQ resistant line pqr-216 with T-DNA inserted upstream from the predicted translation start point of a gene encoding AtNUDX2 (i.e. engineered plant comprising increased NUDIX gene expression comprising one or more modified endogenous NUDIX genes) [pg. 290, col. 2, ¶3]. Ogawa teaches that the insertion of T-DNA at a single site was recognized as the catalyst for increased gene expression and ultimately the phenotype of PQ resistance (i.e. wherein the one or more modified endogenous NUDIX genes comprise one or more insertions in the coding or non-coding region of the genes). Ogawa teaches that the pqr-216 mutant had higher AtNUDX2 gene expression as compared to the wild-type plants (i.e. such that the expression of the one or more endogenous NUDIX genes are increased as compared to an unmodified control) [pg. 291, col. 1, ¶1]. Thus, Ogawa teaches the engineered plant of claim 1, but does not explicitly teach the plant of claim 1 wherein the engineered plant comprises a CRISPR-Cas system of component thereof.
However, Lee teaches that T-DNA integration into the rice genome may be precisely targeted with a combined CRISPR/Cas9 system and Agrobacterium-mediated transformation. Lee teaches the use of a standard Agrobacterium binary vector to construct a T-DNA that contains a CRISPR/Cas9 system using SpCas9 and a gRNA targeting the exon of the rice AP2 domain-containing protein gene Os01g04020 to allow for efficiently generated targeted T-DNA insertions [Abstract]. Lee teaches that this system can be easily applied to other plants, ensuring optimal expression of transgenes without unwanted insertional mutagenesis [pg. 326, col. 1, ¶2; col. 2, ¶2].
It would have been prima facie obvious to one of ordinary skill in the art at the time of filing that the T-DNA insertion upstream from the translation start point of a gene encoding AtNUDX2 could be used in tandem with CRISPR/Cas9. One would have been motivated to do so based on the higher accuracy and lack of unwanted insertional mutagenesis.
Claims 10 and 13 are rejected under 35 U.S.C. 103 as being unpatentable over Ogawa (as cited in IDS filed 7/19/2024) as applied to claims 1-5 above, and further in view of Olejnik, K. et al. (2007), Cloning and characterization of AtNUDT13, a novel mitochondrial Arabidopsis thaliana Nudix hydrolase specific for long-chain diadenosine polyphosphates,” FEBS Journal, 274:4877-4885 (as cited in IDS filed 3/20/2025).
Claim 10 recites the engineered plant of claim 1, wherein the NUDIX gene and/or gene products are NUDIX 13.
Claim 13 recites the engineered plant of claim 4, wherein the exogenous or endogenous NUDIX gene and/or gene products are NUDIX 13.
Regarding claims 10 and 13, Ogawa teaches the engineered plant of claim 1 and 4, but does not explicitly teach the plant wherein the NUDIX genes/gene products are NUDIX 13. However, Olejnik teaches a cDNA corresponding to the At3g26690 gene, which encodes a Nudix protein (AtNUDT13) with predicted mitochondrial localization, was isolated from an Arabidopsis thaliana library [Abstract]. Olejnik teaches that AtNUDT13 protein, a novel member of the A. thaliana Nudix family of enzymes, specifically catalyzes the hydrolysis of long-chain diadenosine polyphosphates and is located in mitochondria [pg. 4881, col. 2, ¶4]. Olejnik teaches that the mitochondrial Nudix hexaphosphate hydrolase AtNUDT13 could be involved in the turnover of ATP and ADP (e.g. during stress) [pg. 4882, col. 1, ¶1] and that the study of plants in which the activities of the AtNUDT13 hydrolase have been altered by gene disruption or overexpression are in progress [pg. 4882, col. 2, ¶1].
Given that Ogawa teaches the engineered plant of claim 1 and 4, but teaches overexpression or increased NUDIX gene expression of AtNUDX2, and given that Olejnik teaches the At3g26690 gene, which encodes a Nudix protein (AtNUDT13), it would have been prima facie obvious to one of ordinary skill in the art at the time of filing to modify the engineered plant of Ogawa to overexpress or increase the AtNUDT13 hydrolase instead of AtNUDX2. One would be motivated to modify the engineered plant as Olejnik teaches AtNUDT13 may be involved in the turnover of ATP and ADP during times of stress. It would be an obvious gene variant of the Nudix hydrolases to try, as Ogawa teaches that overexpression of AtNUDX2 helps in the maintenance of NAD+ and ATP levels by nucleotide recycling from free ADP-ribose molecules under stress conditions, thus conferring enhanced tolerance of oxidative stress on Arabidopsis plants [Abstract]. Olejnik suggests that studies in overexpression of NUDT13 are already underway. Additionally, the gene reported in Olejnik for NUDT13 is the same the enzyme for NUDIX13 of the instant application (At3g26690).
Conclusion
No claims allowed.
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/EMILY K JOHNSON/Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/Supervisory Patent Examiner, Art Units 1661 & 1662