DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Priority
Acknowledgment is made of applicant’s claim for foreign priority Application No. CN202210276159.0 and PCT/CN2022/083735 under 35 U.S.C. 119 (a)-(d). The certified copy has been filed in Application No. 18/849,919, filed on 9/23/2024 with the abstract of Application No. PCT/CN2022/083735 translated to English.
It is noted, however, that applicant has not filed a complete English translated copy as required by 37 CFR 1.55. Thus, the earliest possible priority date for the instant application is 9/23/2024. Submission of an English translation is required to perfect foreign priority.
Status of Claims
Claims 1-9 filed 9/23/2024 are pending and examined herein.
Information Disclosure Statement
Initialed and dated copy of Applicant’s information disclosure statement (IDS) filed on 9/23/2024 is attached to the instant Office Action. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
The listing of references on page 16 [¶102] of the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code (for example, pg. 8, ¶20 & 25; pg. 9, ¶20; pg. 10, ¶63). 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.
The disclosure is objected to for lacking italics in writing Agrobacterium and Arabidopsis thaliana throughout. Appropriate correction is required.
Claim Interpretation
Claim 1, and claims 2-9 depending therefrom, recite a “functional gene” connected to a signal peptide coding gene and a 3’UTR within each of the four expression cassettes. Claim 9 further limits the functional gene to being a damage-induced gene. The specification does not provide a definition for functional gene. It is therefore taken to mean any gene with functional activity or any gene that is biologically active.
Claim 1, and claims 2-9 depending therefrom, recite that the promoter, 5’UTR, 3’UTR, and the terminator of the expression cassettes are all “derived from jasmonic acid-induced expression genes.” The specification does not define the meaning of “derived” or how the expression genes are derived from jasmonic acid induction. Based on the art-accepted meaning, “derived” from jasmonic acid-induced expression genes is taken to mean that the sequences of the expression cassette components come from any gene that is directly or indirectly induced by jasmonic acid.
Claim Objection
In claims 1 and 2, “UTR” is used as abbreviation. It is suggested to insert a definition for UTR without bringing in new matter, immediately before the first appearance of “UTR” in claim 1; and to enclose the appearance of “UTR” in parentheses (in claim 1 only).
In claim 2, “LOX2”, “VSP2”, “LOX3”, and “VSP1” are used as abbreviation(s). It is suggested to insert a definition for LOX2, VSP2, LOX3, and VSP1 without bringing in new matter, immediately before the first appearance of “LOX2”, “VSP2”, “LOX3”, and “VSP1” in claim 2; and to enclose the appearance of “LOX2”, “VSP2”, “LOX3”, and “VSP1” in parentheses.
In claims 3 and 4, “PR1”, “PR3”, “PR5”, and “PDF1.2” are used as abbreviation(s). It is suggested to insert a definition for PR1, PR3, PR5, and PDF1.2 without bringing in new matter, immediately before the first appearance of “PR1”, “PR3”, “PR5”, and “PDF1.2” in claim 3; and to enclose the appearance of “PR1”, “PR3”, “PR5”, and “PDF1.2” in parentheses (in claim 3 only).
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 6 and 7 are rejected under 35 U.S.C. 101 because the claimed invention is directed to non-statutory subject matter. The claim(s) does/do not fall within at least one of the four categories of patent eligible subject matter because "use" of the DNA molecule of claim 1 (referenced in claim 6) or the biomaterial of claim 5 (referenced in claim 7) does not purport to claim a process, machine, manufacture, or composition, and is thus not among the categories of patentable inventions
Claims 6 and 7 are additionally rejected under 35 U.S.C. 101 because the claimed invention is not supported by either a specific and substantial asserted utility or a well-established utility. The claims fail to point out any active, positive steps for the delineating how this use of the DNA molecule or the biomaterial is actually practiced in damage-induced expression of plant functional genes, improving the expression of plant functional genes, or in plant stress resistance. A claim must clearly recite the utilizing step to have a specific and substantial asserted utility.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 6 and 7 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claims 6 and 7 are “use” claims that attempt to claim a process without setting forth any steps involved in the process, the process being damage-induced expression of plant functional genes, improvement of expression of plant functional genes, and plant stress resistance using a DNA molecule or a biomaterial containing the DNA molecule. It is unclear how you would use the DNA molecule or the biomolecule: would you spray it over plants, inject it into the plants, soak the plants?
Examiner notes that, in contrast, claims 8 and 9 are examples of proper method claims with steps set forth to articulate the 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.
Written Description
Claims 1-9 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 directed to a DNA molecule comprising four expression cassettes, cassettes A-D, each comprising a unique promoter, 5’UTR, 3’UTR, and terminator, all derived from “jasmonic acid-induced expression genes.” The claims do not limit the range of jasmonic-acid induced expression genes allowing a broad genus of genes. The claims also state that there is a functional gene connected with the signal peptide coding gene and a 3’UTR, but do not further define the structure of the functional gene, and do not require that the functional gene is derived from jasmonic acid-induced expression genes like the expression cassette components.
The instant specification describes four expression genes of Arabidopsis thaliana induced by jasmonic acid, AtLOX2, AtVSP2, AtLOX3, and AtVSP1, randomly selected with their respective promoter, terminator, and 3’ and 5’UTR regions obtained from TAIR. The claims simply state that the expression cassette elements must be derived from jasmonic acid-induced expression genes, claiming a much broader genus than the cassette elements derived from only LOX2, VSP2, LOX3, and VSP1 in Arabidopsis reduced to practice, and not defining the meaning of jasmonic acid-induced.
Hickman et al. (2017, “Architecture and Dynamics of the Jasmonic Acid Gene Regulatory Network,” The Plant Cell, 29:2086-2105) teaches that the jasmonic acid (JA) pathway has been shown to be complex, with a broad range of defense-related proteins and secondary metabolites, including, but not limited to the herbivory markers VSP1 (VEGETATIVE STORAGE PROTEIN 1) and VSP2 [pg. 2090, col. 1, ¶4], the regulators JAZ1 (JASMONATE ZIM-DOMAIN 1), 2, 5, 7, 8, 9, 10, and 13, MYC2, ANAC019, ANAC055, RGL3, and JAM1, and the biosynthesis genes biosynthesis genes LOX2 (LIPOXYGENASE 2), LOX3, AOS, AOC1, AOC2, AOC3, ACX, and OPR3 [pg. 2095, col. 1, ¶1]. Additionally, JA production triggers antagonistic or synergistic action of other hormones produced during parasitic interactions, such as salicylic acid, ethylene, and abscisic acid [ pg. 1, col. 2, ¶2]. With the broad genus of jasmonic acid-induced expression genes, the promoter, the 5’UTR, the 3’UTR, and the terminator are not limited to the large group of genes related to the JA pathway above. They may instead be genes along various pathogen defense hormonal pathways induced by jasmonic acid that have not been reduced to practice. For example, repression of different defense related genes, such as NPR4 and MYB51, which encode regulators that promote salicylic acid responses and indolic glucosinolates, as well as key immune regulators EDS1 and PAD4, are antagonistically influenced by JA, yet JA induction causes an overrepresentation of genes related to growth and development, including primary metabolism and auxin signaling [pg. 2094, col. 1, ¶2; pg. 2094, col. 2, ¶1].
Thus, claim 1, and claims 3-9 depending therefrom, lack written description because the genus of jasmonic acid-induced expression genes has substantial variance while the specification lacks sufficient variety of species to reflect such variance and there is a lack of common structural attributes of the claimed genus. Examiner notes that this rejection may be overcome by further defining the claimed derived jasmonic acid-induced expression genes. Examiner also notes that claim 2 is not rejected by the same rationale, as it further limits the jasmonic acid-induced expression genes to AtLOX2, AtVSP2, AtLOX3, and AtVSP1, as described in the instant specification.
Further regarding claims 1, 2, and 5-9, the four expression cassettes (A-D) each comprise a signal peptide derived from “secretory protein.” The claims do not limit the range of secretory proteins, allowing a broad genus of proteins. The instant application reduced to practice the signal peptides of secretory proteins in Arabidopsis thaliana searched through literature and TAIR [¶61]. The signal peptide screening resulted in four protein genes, AtPR1, AtPR3, AtPR5, and AtPDF1.2. According to claim 1, the signal peptide may be any derived from a secretory protein.
Hu et al. (2021, “Advances and perspectives in discovery and functional analysis of small secreted proteins in plants,” Horticulture Research,8(130)) teaches that secretory proteins have been found to play diverse roles in various processes, including plant growth and development, plant response to abiotic and biotic stresses, and beneficial plant–microbe interactions [Abstract]. Hu also discloses that there are potentially a lot of secretory proteins that have not been discovered in plant gnomes, thus indicating a large genus for signal peptides derived from secretory proteins, some of which are yet to be documented. Additionally, the genus claim of signal peptides from secretory proteins is not limited to singularly plant secretory proteins, broadening the genus. The Applicant has only reduced to practice four signal peptides from four secretory proteins (AtPR1, AtPR3, AtPR5, and AtPDF1.2). There is significant variability within the genus that is not captured in the four described species, thus claim 1, and claims 3-9 depending therefrom, do not comply with the written description requirement.
More specifically defining the secretory proteins and/or signal peptides might help overcome the stated rejections. Examiner also notes that claims 3 and 4 overcome this rejection because they further limit claim 1 by providing specific signal peptides.
Further regarding claims 1-9, the claims also state that there is a functional gene connected with the signal peptide coding gene and a 3’UTR, but do not further define the structure of the unspecified functional gene, and do not require that the functional gene is derived from jasmonic acid-induced expression genes like the expression cassette components. As “functional gene”, as recited in claim 1, and claims 2-9 depending therefrom, was not defined in the specification and was defined in the claim interpretation above as any gene that plays an active role in biological processes, the claims are interpreted to mean that ANY gene that plays an active role in biological processes would be applicable in the recombinant vector.
The specification reduces to practice only four expression cassettes using cotton GhJAZ8 as the functional gene in the constructed plant recombinant expression vector [¶63]. The GhJAZ8 gene sequence was optimized by the codon preference of maize, wheat, soybean, and rice to facilitate the integration of the gene into the expression cassettes.
Coates et al. teaches that it is unlikely that one optimal expression chassis exists for all recombinant proteins, as they each have different capabilities, and different recombinant proteins have various levels of complexity (2022, “Optimizing expression and extraction of recombinant proteins in plants”, Frontiers in Plant Science, 13). Coates further teaches that some proteins require specific post-translational modifications or are more membranous, meaning gene optimization would need to occur or they would necessitate a different vector [pg. 2, col. 1, ¶1]. Further, it is not specified that the functional gene is a plant functional gene. The different genes found in the genus of functional genes would not be expected to act similarly in the DNA molecule as claimed.
Thus, claim 1, and claims 2-9 depending therefrom, lack adequate written description because the genus of functional genes has substantial variance while the specification lacks sufficient variety of species to reflect such variance and there is a lack of common structural attributes of the claimed genus. Amending the claims to further define the claimed functional gene(s) might help overcome the stated rejections.
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 1, 5, and 8-9 are rejected under 35 U.S.C. 103 as being unpatentable over Raja, V. et al. (2021), “Pyramiding ascorbate–glutathione pathway in Lycopersicum esculentum confers tolerance to drought and salinity stress,” Plant Cell Reports, 41:619-637, in view of Mason, H. et al. (1993), “ldentification of a Methyl Jasmonate-Responsive Domain in the Soybean vspB Promoter,” The Plant Cell. 5:241-251, and Wantanabe, S. et al. (2013), “Pathogen Infection Trial Increases the Secretion of Proteins Localized in the Endoplasmic Reticulum Body of Arabidopsis,” Plant Physiology, 163:659-664.
Claim 1 recites a DNA molecule, wherein the DNA molecule comprises four expression cassettes, and each expression cassette respectively comprises a promoter, a 5'UTR connected with the promoter, a signal peptide coding gene connected with the 5'UTR, a functional gene connected with the signal peptide coding gene, a 3'UTR connected with the functional gene and a terminator connected with the 3'UTR; the promoter, the 5'UTR, the 3'UTR and the terminator are all derived from jasmonic acid-induced expression genes; the signal peptide is derived from secretory protein; the nucleotide sequences of the functional genes in respective expression cassettes are different, and the nucleotide sequences of the promoter, the 5'UTR, the 3'UTR, the signal peptide coding gene and the terminator in each expression cassette are different or the same.
Claim 5 recites a biomaterial, wherein the biomaterial is a recombinant vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ containing the DNA molecule of claim 1.
Claim 8 recites a method for improving plant stress resistance, wherein the method comprises expressing a functional gene in a recipient plant with the DNA molecule according to claim 1.
Claim 9 recites the method according to claim 8, wherein the functional gene is a damage-induced gene.
Regarding claim 1, Raja teaches Glutathione Ascorbate (AsA-GSH) overexpression lines by stacking genes of the AsA-GSH pathway isolated from Penniserum glaucoma. Raja teaches expression of AsA-GSH pathway genes, PgSOD, PgAPX, PgGR, PgDHAR and PgMDHAR, under a T7 promoter in tomato plant system to improve plant defense [Abstract]. The individual cassettes were prepared for each gene having a T7 promoter and a 5’ ribulose bisphosphate carboxylase small chain (RbcS) UTR at the upstream of the gene and a 3’ RbcS UTR followed by T7 terminator at the downstream of the gene and T7 RNA polymerase gene cloned under Rd29A promoter (i.e. at least four individual expression cassettes, each comprising a promoter, a 5’UTR, a functional gene, a 3’UTR, and a terminator) [pg. 620, col. 2, ¶4]. The in vitro pyramiding of the genes and Rd29A-T7 RNA polymerase in pMDC100 (binary vector) was achieved by multi-round gateway technology using LR clonase enzyme II [pg. 620, col. 2, ¶3]. The recombinant pMDC100 vector was introduced into LBA4404 strain of Agrobacterium and the selection raised transgenic tomato plants were confirmed by amplifying NPTII gene used as section marker (Table S1). After this, the transgenic lines (T0) were self-crossed to obtain T1 seeds [¶2].
Raja teaches that many endeavors to develop oxidative stress resistance in plants by manipulating
antioxidant genes have been performed such as in Medicago sativa, cotton, Nicotiana tabacum, Solanum malongena, Oryza sativa, Synechococcus elongatus, and Solanum tuberosum [pg. 620, col. 1, ¶2]. Thus, manipulation of multiple genes through “gene stacking” for improving the stress tolerance
is important for engineering complex traits in plants [pg. 620, col. 1, ¶2]. Consequently, pyramiding multiple genes from different sources into a single plant using multiple expression cassettes is now emphasized for the development of genotypes with resistance against several stresses [pg. 620, col. 2, ¶1].
Raja does not explicitly teach that the promoter, the 5’UTR, the 3’UTR and the terminator are all derived from jasmonic acid-induced expression genes, or that the cassettes include a signal peptide coding gene.
However, Mason teaches that JA or methyl jasmonate (MeJA) is a primary regulator of VSP expression [pg. 241, col. 1, ¶1]. Vsp mRNA accumulation in wounded soybean tissue is preceded by an increase in JA/MeJA in the wounded tissue. lnhibitors of LOX that have the potential to inhibit JA/MeJA biosynthesis also block vsp mRNA accumulation in wounded tissue. Furthermore, the vsp genes are inducible in leaves and cell cultures by JA/MeJA. This suggests that these proteins may have some function in plant defense, in addition to their role in substrate storage [pg. 242, col. 1, ¶1]. Mason teaches that the vspB (gene encoding VSP in soybean) promoter is responsive to sugars, MeJA, and wounding in transgenic tobacco plants [pg. 242, col. 1, ¶2].
Mason teaches gus fusions containing the vspB 5' region and either the vspB 3' region or substitution of a nopaline synthase (nos) terminator in place of the vspB 3'end, showing that the vspB 5' domain alone conferred responsiveness to MeJA [pg. 242, col. 1, para 4; Fig. 1]. Wounding of leaves still attached to transgenic plants by crushing the dista1 one-third of the leaf also caused an increase in GUS activity for plants with the vspB 5' and 3' regions [pg. 242, col. 2, ¶2; Fig. 2]. Mason teaches that a fragment from the 3' end of vspB was obtained by polymerase chain reaction using the parent clone as a template, primers incorporated a Sacl site at the 5’ end and a EcoRl site at the 3’ end, and the PCR product was digested and ligated into pBllOl-X in place of the nopaline synthase (nos) terminator to yield pBIlO1-XV (i.e. jasmonic acid-induced terminator in place of nos).
Mason does not explicitly teach that the cassettes include a signal peptide coding gene, however, Wantanabe teaches the secretion of proteins localized in the endoplasmic reticulum body of Arabidopsis [Abstract]. Wantanabe teaches synthesis of secretory pathogenesis-related (PR) proteins and protein genes (PDF1.2a, PDF1.2b, PDF1.2c, and PDF1.3) encoding plant defensin (PDF), which belong to a class of PR proteins [pg. 659, col. 2, ¶2]. Wantanabe teaches that in response to a fungal pathogen, enhanced secretion of proteins localized in ER bodies occurs [pg. 661, col. 1, ¶1]. Wantanabe teaches that PDF1.2a and several PR proteins have a signal peptide (SP) and teaches transgenic Arabidopsis plants expressing GFP fused to a SP of PDF1.2a [pg. 661, col. 1, ¶2-3]. Wantanabe teaches that SP likely requires additional sequences to achieve enhanced secretion [pg. 661, col. 2, ¶1].
Given that Raja teaches expression of over four expression cassettes in a single line for a pathway related to plant stress defense, comprising a promoter, 5’UTR, 3’UTR, terminator, and functional gene for each cassette; given that Mason teaches expression of VSP genes, including a vsp specific promoter, 5’ and 3’ UTR, and terminator; and given that Wantanabe teaches that PR and PDF genes encode SPs and teaches expression of secretory SPs for response to a fungal pathogen, it would be prima facie obvious to one of ordinary skill in the art at the time of filing to modify a multiple cassette vector with the Gateway method as shown by Raja, using genes in the jasmonic acid pathway for defense against wound-induced pathogenesis with JA induced expression genes such as VSP and LOX suggested by Mason, with a signal peptide related to plant defense such as PR and PDF genes as shown by Wantanabe. One of ordinary skill in the art would have been motivated to create such a construct for jasmonic acid-induced expression genes as Wantanabe suggests that JA signaling pathways play important roles in plant immunity against microbes, including fungal pathogens, and as the construct taught by Raja was previously demonstrated as an effective strategy for developing stress resistant crops. One of ordinary skill in the art would have been motivated to add a signal peptide to the amino acid sequence of the recombinant target protein sequence to enhance secretion, specifically using SPs from PR and PDF proteins, which are known SPs involved in plant pathogen defense.
Regarding claim 5, Raja teaches overexpression of genes along a pathway activated to alleviate ROS production and mitigate its deleterious effects in response to different environmental stressors [Abstract]. Using the stacked expression cassettes, Raja teaches transgenic seedlings infiltrated with Agrobacterium containing the constructed recombinant vector (i.e. a biomaterial, wherein the biomaterial is a recombinant vector, recombinant microorganism, transgenic plant tissue, or a transgenic plant organ). With the obvious rationale above, it would have been prima facie obvious to one of ordinary skill in the art to combine the stacking of expression cassette with Gateway taught by Raja, with the jasmonic acid-induced expression genes taught by Mason, including the signal peptide taught by Wantanabe.
Regarding claims 8 and 9, Raja teaches genetic editing for improving resistance to stress in plants by overexpressing AsA-GSH lines in recipient plants for enhancing resistance of ROS production and damage [Abstract], while Mason teaches the use of VSP in conferring resistance to MeJA and wounding, specifying that wounding caused increased expression due to JA accumulation (i.e. functional gene is damage-induced) [pg. 242, col. 2, ¶3]. According to the obvious rationale above, it would have been prima facie obvious to one of ordinary skill in the art to combine the stacking of expression cassette with Gateway taught by Raja, with the jasmonic acid-induced expression genes taught by Mason, including the signal peptide taught by Wantanabe to use in a method for improving plant stress resistance, wherein the functional gene is expressed in a recipient plant and is damage-induced.
Claim 2 is rejected under 35 U.S.C. 103 as being unpatentable over Raja, in view of Mason and Wantanabe, as applied to claims 1, 5, and 8-9 above, and further in view Wasternack, C. et al. (2013), “Jasmonates: biosynthesis, perception, signal transduction and action in plant stress response, growth and development. An update to the 2007 review in Annals of Botany,” Annals of Botany, 111:1021-1058.
Claim 2 recites the DNA molecule according to claim 1, wherein the four expression cassettes are expression cassette A, expression cassette B, expression cassette C and expression cassette D; the promoter of the expression cassette A is p-AtLOX2, and the nucleotide sequence of the p-AtLOX2 is the 1st-896th positions of SEQ ID No. 1; the 5'UTR of the expression cassette A is AtLOX2-5'UTR, and the nucleotide sequence of the AtLOX2-5'UTR is the 897th-1088th positions of SEQ ID No. 1; the 3'UTR of the expression cassette A is AtLOX2-3'UTR, and the nucleotide sequence of the AtLOX2-3'UTR is the 1548th-1672nd positions of SEQ ID No. 1; the terminator of the expression cassette a is t-AtLOX2, and the nucleotide sequence of the t-AtLOX2 is 1673rd-1872nd positions of SEQ ID No. 1; the promoter of the expression cassette B is p-AtVSP2, and the nucleotide sequence of the p-AtVSP2 is the 1873rd-2964th positions of SEQ ID No. 1; the5'UTR of the expression cassette B is AtVSP2-5'UTR, and the nucleotide sequence of the AtVSP2-5'UTR is the 2965th-3344th positions of SEQ ID No. 1; the 3'UTR of the expression cassette B is AtVSP2-3'UTR, and the nucleotide sequence of the AtVSP2-3'UTR is the 3829th-4078th positions of SEQ ID No. 1; the terminator of the expression cassette B is t-AtVSP2, and the nucleotide sequence of the t-AtVSP2 is the 4079th-4278th positions of SEQ ID No. 1;the promoter of the expression cassette C is p-AtLOX3, and the nucleotide sequence of the p-AtLOX3 is 4279th-5278th positions of SEQ ID No. 1; the 5'UTR of the expression cassette C is AtLOX3-5'UTR, and the nucleotide sequence of the AtLOX3-5'UTR is the 5279th-5467th positions of SEQ ID No. 1; the 3'UTR of the expression cassette C is AtLOX3-3'UTR, and the nucleotide sequence of the AtLOX3-3'UTR is the 5892nd-6747th positions of SEQ ID No. 1; the terminator of the expression cassette C is t-AtLOX3, and the nucleotide sequence of the t-AtLOX3 is 6748th-6947th positions of SEQ ID No. 1; the promoter of the expression cassette D is p-AtVSPI, and the nucleotide sequence of the p-AtVSPI is the 6948th-7947th positions of SEQ ID No. 1; the 5'UTR of the expression cassette D is AtVSP1-5'UTR, and the nucleotide sequence of the AtVSPI-5'UTR is the 7948th-8259th positions of SEQ ID No. 1; the 3'UTR of the expression cassette D is AtVSP1-3'UTR, and the nucleotide sequence of the AtVSP1-3'UTR is the 9409th-9607th positions of SEQ ID No. 1; the terminator of the expression cassette D is t-AtVSPI, and the nucleotide sequence of the t-AtVSPI is the 9608th-9807th positions of SEQ ID No. 1.
The DNA molecule of claim 1 is rejected over Raja, in view of Mason and Wantanabe, as disclosed above. Although Raja, Mason, and Wantanabe combined make obvious the DNA molecule of claim 1 and teach signal peptides related to PR and PDF, they do not explicitly disclose the JA gene expression cassette components recited in claim 2.
However, Wasternack discloses the role of jasmonate signaling pathways in stress responses and development, including identification of proteins important to biosynthesis and signaling. Wasternack teaches multiple LOXs of Arabidopsis, including proteins LOX2 and LOX3, demonstrating the importance of LOX2 in the bulk of JA formation upon wounding [pg. 1022, col. 2, ¶2], and LOX3 in the synthesis of jasmonate [pg. 1023, col. 2, ¶1]. Wasternack teaches that along the JA signaling cascade, defense response it elicited through the expression of VSP2, the most prominent marker gene of JA signaling [pg. 1032, col. 1, ¶1], and VSP1 is expressed downstream of a master regulator, transcription factor MYC2 [pg. 1032, col. 1, ¶3]. Wasternack additionally teaches a LOX3 promoter involved in JA biosynthesis [pg. 1048, col. 1, ¶3], as well as transgenic lines of Arabidopsis, tomato, and Medicago truncatula over-expressing genes along the JA pathway [pg. 1027, col. 1, ¶2; pg. 1028, col. 1, ¶4; pg. 1042, col. 1, ¶2].
It would have been prima facie obvious to one of ordinary skill in the art to use genes such as LOX2, LOX3, VSP1, and VSP2 from the model organism Arabidopsis thaliana, as Wasternack teaches that these are known genes are important to the JA signaling cascade. One would be motivated to utilize such genes, or promoters, 3’ and 5’UTRs, and terminators, from these genes because of their influence on wound induced JA promotion disclosed by Wasternack. Based on the use of vsp genes and expression cassette components in transgenic plants and the suggestion that VSP and LOX proteins function in plant defense disclosed by Mason, and the expression of multiple genes from different sources in a single plant disclosed by Raja, one of ordinary skill in the art at the time of filing would have been able to create multiple expression cassettes with the genes at hand as their functions and components were known in the art. The positions of the individual gene elements within SEQ ID NO. 1 would be simply design choices by one of ordinary skill in the art when constructing the expression vectors, lacking evidence to the contrary.
Claim 3 and 4 are rejected under 35 U.S.C. 103 as being unpatentable over Raja, in view of Mason and Wantanabe, as applied to claims 1, 5, and 8-9 above, and further in view of Zhu, T. et al., (2002), “Novel polynucleic acid segment useful for modulating gene expression within a cell by posttranscriptional gene silencing, and for augmenting a plant cell genome,” WO200281695-A2, Parrow, M. et al., (2014), “Composition useful to make ethanol from plant or partially metabolized plant, comprises transgenic soy plant that has been transformed with gene expressing enzyme i.e. capable of partially metabolizing lignin, hemicellulose or cellulose,” US20170233753-A1, and Broekaert, W. et al., (1998), “Protecting plants against pathogens by inducing defensin genes - by stimulating ethylene or jasmonate pathways, also new promoter of defensin gene from Arabidopsis,” WO9800023-A2.
Claim 3 recites the DNA molecule according to claim 1, wherein the signal peptide is selected from sp-AtPRI, sp-AtPR3, sp-AtPR5 and sp-AtPDF 2; the amino acid sequence of sp-AtPRl is SEQ ID No.2, the amino acid sequence of sp-AtPR3 is SEQ ID No.3, the amino acid sequence of sp-AtPR5 is SEQ ID No.4, and the amino acid sequence of sp-AtPDF1.2 is SEQ ID No. 5.
Claim 4 recites the DNA molecule according to claim 3, wherein the signal peptide coding gene is the coding gene of sp-AtPR1, sp-AtPR3, sp-AtPR5 or sp-AtPDF1.2, and the coding gene of sp-AtPR1 is a DNA molecule with a nucleotide sequence of 201089th-1163rd positions of SEQ ID No. 1; the coding gene of sp-AtPR3 is a DNA molecule with a nucleotide sequence of 3345th-3440th positions of SEQ ID No. 1; the coding gene of sp-AtPR5 is a DNA molecule with a nucleotide sequence of 5468th-5533rd position of SEQ ID No. 1; the encoding gene of sp-AtPDFI.2 is a DNA molecule with the nucleotide sequence of 8260th-8343rd positions of SEQ ID No. 1.
The DNA molecule of claim 1 is rejected over Raja, in view of Mason and Wantanabe, as disclosed above. Although Raja, Mason, and Wantanabe combined make obvious the DNA molecule of claim 1 and teach signal peptides related to PR and PDF, they do not explicitly disclose the sequences of the signal peptides recited in claim 3.
Zhu, however, teaches inducible expression of genes in some transgenic plants, wherein vectors may be constructed and employed in the intracellular targeting of a specific gene product within the cells of a transgenic plant or in directing a protein to the extracellular environment [pg. 83, ¶4]. Zhu teaches that targeting can be achieved by providing the polypeptide with an appropriate targeting peptide sequence, such as a secretory signal peptide (for secretion or cell wall or membrane targeting, a plastid transit peptide, a chloroplast transit peptide, a mitochondrial target peptide, a vacuole targeting peptide, or a nuclear targeting peptide, and the like) [pg. 88, ¶2]. This is generally achieved by joining a DNA sequence encoding a transit or signal peptide sequence to the coding sequence of a particular gene. The resultant transit, or signal, peptide transports the protein to a particular intracellular, or extracellular destination, respectively [pg. 83, ¶4].
Zhu teaches that the choice of DNA segments to be delivered to the recipient cells depends on the desired trait, including disease resistance or tolerance, or stress tolerance and/or resistance [pg. 88, ¶4; pg. 89, ¶1]. Zhu teaches that based on blast searches of DNA databases, genes encoding proteins or protein domains fall into several classes such as silencing-related RNA and DNA metabolism, signal transduction (protein kinases, receptors and calmodulin), and stress-related and pathogenesis-related (PR) proteins [pg. 109, ¶6]. Zhu provides sequences for Arabidopsis thaliana proteins modulated by posttranslational gene silencing and discloses Acc. No. ABP81196, designated as pathogenesis-related protein 1 (PR-1) with 100% identity to SEQ ID NO. 2 of the instant application, as well as GenBank Acc. No. ABP81190, designated as a pathogenesis-related protein 5 (PR-5) with 100% identity to SEQ ID NO. 4 of the instant application (see alignments below).
SEQ ID NO. 2 aligned with Acc. No. ABP81196:
Query Match 100.0%; Score 122; Length 161;
Best Local Similarity 100.0%;
Matches 25; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MNFTGYSRFLIVFVALVGALVLPSK 25
|||||||||||||||||||||||||
Db 1 MNFTGYSRFLIVFVALVGALVLPSK 25
SEQ ID NO. 4 aligned with Acc. No. ABP81190:
Query Match 100.0%; Score 103; Length 239;
Best Local Similarity 100.0%;
Matches 22; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MANISSIHILFLVFITSGIAVM 22
||||||||||||||||||||||
Db 1 MANISSIHILFLVFITSGIAVM 22
Zhu does not teach a signal peptide of AtPR3 with SEQ ID NO. 3 or of AtPDF1.2 with SEQ ID NO. 5, however, Parrow teaches a genetically modified soy plant that has been transformed with genes expressing enzymes capable of partially metabolizing lignin, hemicellulose, or cellulose.
Parrow teaches that a typical synthetic gene for expression in soybean seeds contains a seed-specific promoter, followed by an enhancer element sequence, followed by a sequence encoding a signal peptide (e.g. a native signal peptide, the Arabidopsis thaliana plant signal peptide or the gene of interest signal peptide) followed by the open reading frame of the desired protein product, followed by a terminator sequence (e.g. 35S, Nos, Bar, vsp, etc.) [¶127]. Together, these above elements constitute a gene cassette. Parrow teaches that the signal peptide sequence from Arabidopsis thaliana chitinase protein was included because it has been used by other inventors to successfully target heterologous proteins to the secretory pathway [¶158]. Parrow teaches Arabidopsis thaliana chitinase proteins with 100% identity to SEQ ID NO. 3 of the instant application (see alignment to SEQ ID NO. 54 below) [Table 6 and 7A, SEQ ID NO. 54 and 59]. Although this is designated as a chitinase protein, it is known in the art that among PR protein groups, PR-3 is categorized as chitinase1 with activity in transgenic plants for alleviation of pathogenesis2.
SEQ ID NO. 3 alignment with SEQ ID NO. 54 of Parrow:
Query Match 100.0%; Score 160; Length 33;
Best Local Similarity 100.0%;
Matches 32; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MPPQKENHRTLNKMKTNLFLFLIFSLLLSLSS 32
||||||||||||||||||||||||||||||||
Db 1 MPPQKENHRTLNKMKTNLFLFLIFSLLLSLSS 32
Zhu and Parrow do not teach an AtPDF1.2 signal peptide with the amino acid sequence of SEQ ID NO. 5, however, Broekaert teaches protection against plant pathogens by inducing defensin genes (PDF) through stimulation of ethylene or jasmonate pathways.
Broekaert teaches a method of protection against necrotrophic pathogens, specifically fungi [Abstract]. Broekaert teaches that plants attacked by microbial pathogens are able to induce expression of defense-related genes encoding PR proteins [pg. 1, lns. 8-10]. Broekaert teaches that PR-1, PR-2 and PR-5 are systematically induced upon pathogen infection and that the invention is a composition that is capable of inducing expression of a PDF gene in order to protect a plant against attack by pathogens [pg. 4, lns. 29-31]. When Arabidopsis species are used, the defensin may be the product of the plant defensin gene PDF1.2 [pg. 4, lns. 4-5]. Broekaert explicitly teaches an Arabidopsis thaliana plant defensin (PDF1.2) with 100% sequence identity to SEQ ID NO. 5 that is induced by stimulating the jasmonate and ethylene pathways for plant defense (see alignment below) [pg. 3, lns. 3-4]. In the development of a marker gene for the induction of jasmonate defense pathways in Arabidopsis and tobacco, Broekart teaches that the Arabidopsis PDF1.2 gene promoter was cloned based on the PDF1.2 cDNA sequence, the genomic sequence of which includes a coding sequence for the PDF1.2 signal peptide [Example 8].
Query Match 100.0%; Score 130; Length 80;
Best Local Similarity 100.0%;
Matches 28; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MAKFASIITLIFAALVLFAAFDAPAMVE 28
||||||||||||||||||||||||||||
Db 1 MAKFASIITLIFAALVLFAAFDAPAMVE 28
Given that Raja, Mason, and Wantanabe in combination teach the DNA molecule of claim 1 and given that Wantanabe teaches that PR and PDF genes encode SPs and teaches expression of secretory SPs for response to a fungal pathogen, it would be prima facie obvious to one of ordinary skill in the art at the time of filing to modify a multiple cassette vector, with particular signal peptides related to plant defense such as PR and PDF as shown by Wantanabe. Given that Broekart teaches that PR-1 and PR-5 from Arabidopsis are induced upon pathogen infection and PDF1.2 is induced by stimulating jasmonate, and a 100% match to AtPDF1.2 with SEQ ID NO. 5 of the instant application; given that Zhu teaches that inducible expression of genes in transgenic plants can be accomplished by joining a secretory signal peptide sequence to the coding sequence of a particular gene for a desired trait, including disease resistance, with a 100% match to the AtPR-1 with SEQ ID NO. 2 and AtPR-5 with SEQ ID NO. 4 of the instant application; and given that Parrow teaches Arabidopsis thaliana chitinase signal peptide used to target heterologous proteins to the secretory pathway and using expression cassette elements related to the jasmonic acid pathway, such as a vsp terminator sequence, as well as a 100% match to the AtPR3 with SEQ ID NO. 3, one of ordinary skill in the art would be motivated to use these signal peptides in pathogen defense for their demonstrated success in plant genetic transformation and for the fact that they are in the model species Arabidopsis thaliana.
It would have been prima facie obvious to one of ordinary skill to use any of these sequences in the constructed cassettes as signal peptides because they are found in the model species Arabidopsis thaliana, and are known secretory proteins with high expression and signal peptides. As the sequences above were known to the prior art, and their function as secretory signal peptides and relation to the jasmonic pathway/plant defense responses clearly defined, they would have been obvious to try in the instant invention to achieve the result of pathogen defense.
Regarding claim 4, the recited positions within SEQ ID NO. 1 would be simply design choices by one of ordinary skill in the art when constructing the expression vectors, lacking evidence to the contrary.
Conclusion
No claims allowed.
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/EMILY K JOHNSON/Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/Supervisory Patent Examiner, Art Units 1661 & 1662
1 Edreva et al. (2005, Pathogenesis-related proteins: Research progress in the last 15 years, Gen. Appl. Plant Physiology, 31(1-2):105-124) teaches that among PR protein groups, PR-3 is designated as chitinase. Plant-resistance to several fungal infections has been greatly enhanced by the overexpression of PR-chitinases genes in heterologous and homologous systems.
2 Datta et al. (2001, Enhanced resistance to sheath blight by constitutive expression of infection-related rice chitinase in transgenic elite indica rice cultivars, Plant Science, 160(3):405-414) teaches the introduction of PR-3 chitinase gene from Rhizoctonia solani into infected rice plants into various susceptible indica rice cultivars. The transformants showed higher resistance to the sheath blight disease caused by R. solani.