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 .
Claim Status
Claims 1-13, 15-16, 18-19, 22-23, 25, and 28 are pending.
Claims 1-13, 15-16, 18-19, 22-23, 25, and 28 are examined on the merits.
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 1-13, 15-16, 18-19, 22-23, 25, and 28 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 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.
The claims are rejected for lacking adequate written description support regarding the broad scope of the following.
Independent claim 1 broadly encompasses a plant whose heritable genetic material comprises a gene encoding a transportable carbonic anhydrase protein under the control of a promoter active in root cells, such that the carbonic anhydrase is transported from the roots into the extracellular environment. Independent claim 4 broadly encompasses a polynucleotide comprising a promoter active in plant roots, a carbonic anhydrase sequence, and a signal peptide sequence capable of directing transport into the extracellular environment. Independent claim 25 broadly encompasses a method for increasing the capacity of a plant to sequester carbon in soil by altering the heritable genetic material of the plant such that a carbonic anhydrase protein is transported form the roots into the extracellular environment.
The specification provides working examples only for limited constructs. Specifically, the working examples use soybean carbonic anhydrase Glyma.19G135900 (SEQ ID NO: 1 and 8) and carbonic anhydrase from “Conticribra weissflogii” (SEQ ID NO: 2 and 9) (Examples 1, 5, 8, and 11). The working examples use limited transport sequences, including a carrot extension signal peptide (SEQ ID NO:3) and a pea BP80 signal peptide/transmembrane domain (SEQ ID NO: 4 and 5) (Examples 2, 9 and 11). The working examples use limited root-active promoters, including soybean expansin promoter pGmEXPA7 (SEQ ID NO: 6) and soybean pGmPIN2b promoter (SEQ ID NO:7) (Examples 2, 6, and 12). The working examples demonstrate the constructs in “Arabidopsis thaliana and Glycine max” plants (Examples 2-15).
The claims, however, are much broader than the working examples. The claims encompass all carbonic anhydrase having subtypes, including α, β, ϒ, δ, ζ, η, θ, and ι carbonic anhydrase; carbonic anhydrase sequences having as little as 65% identity to SEQ ID NO: 1 or SEQ ID NO: 2; promoter sequences having as little as 65% identity to SEQ ID NO: 6 or 7; signal peptide sequences having as little 65% identity to SEQ ID NO: 3 or 4; any promoter active in root cells; and a broad genus of plants, including numerous crop plants and trees.
The specification does not provide representative species across the full scope of the claimed genera, including representative carbonic anhydrases from each claimed subtype, representative 65% identity variants that retain the required activity and transport properties, representative promoters across the full genus of root-active promoters, or representative examples across the broad claimed plant and tree species. The specification also does not identify common structural features that would allow one of ordinary skill in the art to recognize that applicant possessed the full scope of the claimed transportable carbonic anhydrases, root-active promoters, signal peptides, and plant genera.
According, the specification does not reasonably convey to one of ordinary skill in the art that applicant was in possession of the full scope of independent claims 1, 4, and 25, and dependent claims 2-3, 5-13, 15-16, 18-19, 2-23, and 28.
Scope of Enablement
Claims 1-13, 15-16, 18-19, 22-23, 25, and 28 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specifications, while being enabling for limited working embodiments using soybean carbonic anhydrase SEQ ID NO: 1 and Conticribra weissflogii carbonic anhydrase SEQ ID NO: 2, the carrot extensin signal peptide SEQ ID NO: 3, the pea BR80 signal peptide SEQ ID NO: 4, the soybean expansin promoter pGmEXPA7 SEQ ID NO: 6, and the soybean pGmPIN2b promoter SEQ ID NO: 7, primarily in Arabidopsis thaliana and soybean; does not reasonably provide enablement for the full scope of the claimed invention, including carbonic anhydrases from essentially any species, carbonic anhydrase sequence have as little as 65% identity to SEQ ID NO: 1 or 2, promoter sequences having as little as 65% identity to SEQ ID NO: 6 or 7, signal peptide sequence having as little as 65% identity to SEQ ID NO: 3 or 4, any promoter active in root cells, broad signal peptides derived from secreted proteins generally, and numerous crop plants and trees. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention commensurate in scope with these claims.
An “analysis of whether a particular claim is supported by the disclosure in an application requires a determination of whether that disclosure, when filed, contained sufficient information regarding the subject matter of the claims as to enable one skilled in the pertinent art to make and use the claimed invention.” MPEP 2164.01. “A conclusion of lack of enablement means that. . . the specification, at the time the application was filed, would not have taught one skilled in the art how to make and/or use the full scope of the claimed invention [i.e. commensurate scope] without undue experimentation.” In re Wright, 999 F.2d 1557,1562, 27 USPQ2d 1510, 1513 (Fed. Cir. 1993); MPEP 2164.01.
In In re Wands, 858 F.2d 731,8 USPQ2d 1400 (Fed. Cir. 1988), several factors implicated in determination of whether a disclosure satisfies the enablement requirement and whether any necessary experimentation is “undue” are identified. These factors include, but are not limited to:
(A) The breadth of the claims;
(B) The nature of the invention;
(C) The state of the prior art;
(D) The level of one of ordinary skill;
(E) The level of predictability in the art;
(F) The amount of direction provided by the inventor;
(G) The existence of working examples; and
(H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure. In re Wands, 858 F.2d 731,737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988). No single factor is independently determinative of enablement; rather “[i]t is improper to conclude that a disclosure is not enabling based on an analysis of only one of the above factors while ignoring one or more of the others.” MPEP 2164.01. Likewise, all factors may not be relevant to the enablement analysis of any individual claim.
Independent claim 1 broadly encompasses a plant comprising a gene encoding a transportable carbonic anhydrase protein under the control of a promoter active in root cells, such that the carbonic anhydrase is transported from the roots into the extracellular environment. Independent claim 4 broadly encompasses a polynucleotide comprising a promoter active in plant roots, a carbonic anhydrase sequence, and a signal peptide sequence capable of directing transport into the extracellular environment of a plant root. Independent claim 25 broadly encompasses a method for increasing the capacity of a plant to sequester carbon in soil by altering the heritable genetic material of the plant such that a carbonic anhydrase protein is transported from the roots into the extracellular environment.
The specification provides working examples only for limited combinations of carbonic anhydrases, promoters, signal peptides, and plant species. For example, the specification demonstrates soybean carbonic anhydrase SEQ ID NO: 1 and Conticribra weissflogii carbonic anhydrase SEQ ID NO: 2, carrot extensin signal peptide SEQ ID NO: 3, pea BP80 signal peptide SEQ ID NO: 4, soybean expansin promoter pGmEXPA7 SEQ ID NO:6, and soybean pGmPIN2b promoter SEQ ID NO: 7, primarily in Arabidopsis thaliana and soybean plants.
The claims, however, encompass substantially broader subject matter, including all carbonic anhydrase subtypes, carbonic anhydrases from essentially any species, carbonic anhydrase sequence having as little as 65% identity to SEQ ID NO:1 or 2, promoter sequence having as little as 65% identity to SEQ ID NO: 6 or 7, signal peptide sequences having as little as 65% identity to SEQ ID NO: 3 or 4, any promoter active in root cells, broad signal peptides derived from secreted proteins generally, and numerous crop plants and trees.
The specification does not provide sufficient guidance to determine which carbonic anhydrase variants across the full claimed scope retain enzymatic activity, are properly expressed in root cells, are transported into the extracellular environment, remain active extracellularly, and increase carbon sequestration. Although, the specification states that any carbonic anhydrase enzyme would be expected to work equivalently, the working examples test only soybean and Conticribra weissflogii carbonic anhydrases.
The specification also does not provide sufficient guidance to determine which promoters across the full scope of “promoter active in root cells” provide sufficient root expression for extracellular carbonic anhydrase activity and carbon sequestration. The specification uses soybean expansin promoter pGmEXOA7 and soybean pGmPIN2b promoter, but states generally that any root promoter selected from any species would be expected to function equivalently. Such general statements do not enable the full scope without screening and optimization.
The specification further does not provide sufficient guidance to determine which signal peptides or signal peptide variants across the full claimed scope successfully direct different carbonic anhydrase proteins into the extracellular environment of plant roots. The working examples use carrot extensin signal peptide SEQ ID NO: 3 and pea BP80 signal peptide SEQ ID NO: 4, but the claims encompass broad signal peptides and variants having as little as 65% identity.
Undue experimentation would also be required to practice the claimed invention across the broad plant genus recited in the claims, including numerous crop plants and trees. The working examples are primarily limited to Arabidopsis thaliana and soybean. The specification does not provide sufficient guidance showing that the claimed combinations of carbonic anhydrases, promoters, signal peptides, and sequence variants will function across the full range of claimed plants and trees to produce extracellular carbonic anhydrase activity and increased carbon sequestration.
Accordingly, one of ordinary skill in the art would have to engage in substantial screening and optimization to identify which carbonic anhydrases, 65% identity variants, root-active promoters, signal peptides, plant species, and combinations thereof successfully provide extracellular carbonic anhydrase activity and increased carbon sequestration.
Dependent claims 2-3, 5-13, 15-16, 18-19, 2-23, and 28 are deficient for the same reasons because they depend or otherwise rely on the same non-enabled subject matter.
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 1-6, 9, 11, 15-16, 19, 22, 23, and 28 are rejected under 35 U.S.C. §103 as being unpatentable over Djokovic (Slavica Djonovic et. al., WO2016200987A1, Application 2016-06-08, Publication 2016-12-15) in view of Borisjuk (Nikolai V. Borisjuk et. al., Nature biotechnology (1999) VOL 17, pp466-469).
Claim 1 recites a plant whose heritable genetic material comprises a gene encoding a transportable carbonic anhydrase protein under the control of a promoter active in root cells, such that the carbonic anhydrase is transported from the roots of the plant into the extracellular environment.
Djonovic teaches a soybean gene Glyma.19G135900 (SEQ ID NO: 4050) encoding an alpha carbonic anhydrase 4. Djonovic further teaches SEQ ID NO: 4050 is 100% identical to applicant’s SEQ ID NO: 1 (see alignment below), and Glyma. 19G135900 expresses in root tissues (Table 8D, p268, and see below).
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Djonovic does not expressly teach modifying the carbonic anhydrase so that it is transported form plant roots into the extracellular environment.
Borisjuk teaches engineering plant to continuously secrete recombinant proteins from root s into hydroponic medium, i. e., “rhizosecretion”, and teaches that secretion of recombinant proteins is depended on the presence of an endoplasmic reticulum signal peptide fused to the recombinant protein sequence. Borisjuk further teaches that GFP targeted to the root secretory pathway localized to the cell walls and intercellular space/apoplast and accumulated in root exudates, and an active bacterial xylanase is released from roots of transgenic plants into the surrounding medium (p466 Abstract; p466 left column, pa3; Fig 1; and Fig 2).
It would have been obvious to one of ordinary skill in the art to modify the root-expressed soybean carbonic anhydrase gene taught by Djonovic by adding a known signal peptide as taught Borisjuk, so that the carbonic anhydrase would enter the plant secretory pathway and be transported from the roots into the extracellular environment. A person of ordinary skill would have had a reasonable expectation of success because Borisjuk demonstrates root section of multiple unrelated heterologous proteins, including GFP, SEAP, and bacterial xylanase, and reports that the secreted proteins retained biological activity (Abstract; Fig 1-3).
Accordingly, claim 1 would have been obvious over Djonovic in view of Borisjuk.
Claim 2 recites the plant of claim 1, wherein the carbonic anhydrase is over-expressed in the extracellular environment of the root cells in comparison to a plant whose heritable genetic material does not comprise a gene encoding a transportable carbonic anhydrase protein under the control of a promoter active in root cells. Claim 3 recites the plant of claim 1, wherein the gene encoding the transportable carbonic anhydrase protein further comprises a signal peptide sequence for directing transport of the carbonic anhydrase into the extracellular environment of the plant root.
Regarding claim 2, Borisjuk teaches that recombinant proteins were secreted into root exudates/hydroponic medium, and that SEAP accumulated in much higher amounts in the medium than in the root tissues, with 79% of total SEAP activity associated with the culture medium and 21% associated with root tissue and intercellular fluid after 32 days (Abstract; Fig 3; p468, left column and right column). Therefore, Borisjuk teaches overexpression/ accumulation of recombinant protein in the extracellular root environment relative to control plants lacking the secreted recombinant construct.
Regarding claim 3, Borisjuk teaches that protein secretion was dependent on the presence of an ER signal peptide fused to the recombinant protein sequence and teaches use of ER signal peptides to direct recombinant proteins into the secretory pathway and root exudates (Abstract; Fig 2A; Fig 3A; p466, right column, pa3; p468, both left and right column).
According, claims 2 and 3 would have been obvious over Djonovic in view of Borisjuk.
Claim 4 recites a polynucleotide sequence comprising a promoter, a carbonic anhydrase sequence, and a signal peptide sequence capable of directing transport of the carbonic anhydrase into the extracellular environment of a plant root.
For the same reason set forth above with respect to claim 1, Djonovic teach the carbonic anhydrase sequence, Borisjuk teaches root secretion construct comprising a promoter, a recombinant protein coding sequence, and an ER signal peptide. Including GFP fused to a calreticulin ER signal peptide under the mas2’ promoter and SEAP comprising a signal peptide under the mas2’ promoter. Borisjuk further teaches that the mas2’ promoter is preferentially active in plant roots and was used for root secretion of recombinant proteins (p466 left and right column; Fig 2A; Fig 3A).
According, claim 4 would have been obvious over Djonovic in view of Borisjuk.
Claim 5 recites the plant according to claim 3, wherein the signal peptide sequence is either natively encoded or translationally fused to the carbonic anhydrase sequence to ensure transport of the carbonic anhydrase to the extracellular environment of the plant root. Claim 6 recites the plant according to claims 3, wherein the signal peptide sequence for directing transport is derived from a secreted protein that is transported to the extracellular environment by a protein secretion pathway.
For the same reason set forth above with respect to claim 3, Borisjuk teaches that the signal peptide is fused to the recombinant protein seduce and that protein secretion depends on the ER signal peptide fused to the recombinant protein (Abstract; Fig 2A; Fig 3A). Borisjuk teaches ER signal peptide from the abundant ER protein, calreticulin of Nicotiana plumbaginifolia. This signal peptide originated from a tobacco species closely related to the transgene recipient, thus increasing the chances for effective ER targeting, which means the calreticulin is a secreted protein that is transported to the extracellular environment by a protein secretion pathway (p466 right column, pa3). Borisjuk further teaches that the signal peptide directs recombinant proteins through the secretory pathway into the apoplast/root exudate (Fig 2C-2G).
According, claims 5 and 6 would have been obvious over Djonovic in view of Borisjuk.
Claim 9 recites the plant according to claim 1, wherein the promoter is root-specific.
Borisjuk teaches promoters used for recombinant protein expression in plants, including mas2’ promoter, and teaches that the mas2’ promoter is preferentially active in plant root (p466, right column, pa1).
According, claim 9 would have been obvious over Djonovic in view of Borisjuk.
Claim 11 recites the plant according to claim 1, wherein the carbonic anhydrase is an α, β, ϒ, δ, ζ, η, θ, or ι subtype carbonic anhydrase.
Djonovic teaches that gene Glyma.19G135900 encoding alpha carbonic anhydrase 4 (Table 8D, p268).
Accordingly, claim 11 would have been obvious over Djonovic in view of Borisjuk.
Claim 15 recites a plant cell, cell line or progeny thereof comprising the polynucleotide sequence of claim 4. Claim 19 recites a plant part, plant tissue, plant organ, plant cell, plant protoplast, embryo, callus, cell culture, pollen grain or seed, derived or obtained from the plant cell line or progeny according to claim 15. Claim 22 recites a bacterium comprising the polynucleotide of claim 4. Claim 23 recites plant comprising the polynucleotide of claim 4 stably integrated into the genome thereof. Claim 28 recites a processed plant product obtained from the plant of claim 1, wherein the processed product comprises a detectable nucleic acid sequence of the gene.
Borisjuk teaches plant transformation vectors and transgenic plants comprising recombinant protein expression cassettes for root secretion, including constructs for GFP, SEAP, and xylanase, and teaches transformation of tobacco plants with these constructs (Abstract; “Rhizosecretion of GFP”; “Rhizosecretion of human alkaline phosphatase”; “Experimental protocol”). Borisjuk further teaches plant cells and transgenic plants comprising SEAP constructs and Agrobacterium-mediated transformation of tobacco (p469, “Plant transformation and cultivation”). Borisjuk teaches bacteria, plants, plant cells, tissues, and seeds comprising plant expression cassettes (p469, “Plant transformation and cultivation). Borisjuk teaches that plant seeds expressing recombinant proteins may be processed into plant-derived products, including protein-containing seed material, flour , meal, or other processed seed products (p468, pa3-4). Thus, Borisjuk shows that it is known to obtain processed plant products from recombinant/protein-expressing plants or seeds.
Accordingly, claim 15, 19, 22, 23 and 28 would have been obvious over Djonovic in view of Borisjuk.
Claim 16 recites the plant, according to claim 1 wherein the plant is a crop plant, selected from the group consisting of: corn, soybean, pea, cotton, canola, camelina, potato, tomato, sugar beet, cassava, sweet potato, alfalfa, wheat, barley, sorghum, oat, sorghum, millet, rye, teff, rice plant, or clover, cress, brassicas, vetch and prairie grasses, or a tree selected from the group consisting of poplar, spruce, pine, eucalyptus, oil palm and rubber.
Djonovic teaches to engineer soybean and maize to improve agronomic traits in plants (claim 2), and Borisjuk teaches tobacco plants engineered for root secretion of recombinant proteins. Accordingly, claim 16 would have been obvious over Djonovic in view of Borisjuk.
Claim 18 recites the plant, according to claim1,wherein more carbon is sequestered into the extracellular environment of the plant in comparison to a plant whose heritable genetic material does not comprise a gene encoding a transportable carbonic anhydrase protein under the control of a promoter active in root cells.
For the same reason set forth above with respect to claim 1, Borisjuk teaches that secretion of recombinant protein such as GFP targeted to the root secretory pathway localized to the cell walls and intercellular space/apoplast and accumulated in root exudates, and that active bacterial xylanase was released from roots of transgenic plants into the surrounding medium (Fig 2). Roots of transgenic plants were clearly distinguished from untransformed controls by the bright green fluorescence of GFP visible over the slight background fluorescence of non-transformed roots (Fig. 2B); and the proportion of GFP in the cellular protein fraction isolated from roots of the carGFP transformants was much lower than that in their apoplast or root exudates (Fig. 2F).
It would have been obvious to one of ordinary skill in the art to modify the root-expressed soybean carbonic anhydrase gene taught by Djonovic by adding a known signal peptide as taught Borisjuk, so that the carbonic anhydrase would enter the plant secretory pathway and be transported from the roots into the extracellular environment, and thereby resulting in secretion of carbonic anhydrase into the extracellular environment, where the enzyme would be expected to catalyze the hydration of carbon dioxide outside the root cells, thereby increasing carbon sequestration in the extracellular environment relative to a plant lacking the transgene.
Claim 18 is obvious over Djonovic in view of Borisjuk.
Claim 7 is rejected under 35 U.S.C. §103 as being unpatentable over Djonovic (WO2016200987A1) in view of Borisjuk (1999) as apply to claim 3, and further in view of Haseloff (Jim Haseloff et. al., US20050132432A1, Application 2002-09-20, Publication 2005-06-16).
Claim 3 as the teachings of Djonovic and Borisjuk are discussed above.
Claim 7 is interpreted as dependent of claim 3.
Claim 7 recites the plant according to claim 3, wherein the signal peptide sequence is derived from the extensin protein.
For the same reason set forth above with respect to claim 3, Haseloff teaches a carrot extensin-GFP fusion, and provide the extensin sequence SEQ ID NO: 13 (p13), wherein the alignment shows that Haseloff SEQ ID NO:13 comprises a portion that is 100% identical to SEQ ID NO: 3 of the instant application (see below).
Accordingly, claim 7 would have been obvious over Djonovic in view of Borisjuk and Haseloff.
Claims 8 and 10 is rejected under 35 U.S.C. §103 as being unpatentable over Djonovic (WO2016200987A1) in view of Borisjuk (1999) as apply to claim 1, and further in view of Moore (Desmond A Moore et. al., CN114555806A, Application 2020-10-15, Publication 2022-05-27).
Claim 1 as the teachings of Djonovic and Borisjuk are discussed above.
Claims 8 and 10 are interpreted as dependent of claim 1.
Claim 8 recites the plant according to claim 1, wherein the promoter is derived from a gene from the same plant species or variety as the plant.
For the same reason set forth above with respect to claim 1, Moore teaches that the promoter used in a plant expression cassette may be native or homologous to the plant host, and explains that a native or homologous promoter is a promoter found in the native plant into which the promoter is introduce (pa0016). Thus, Moore teaches using a promoter derived from the same plant species as the transformed plant.
Claim 10 recites the plant according to claim 1, wherein the carbonic anhydrase further comprises a transmembrane domain or a membrane anchor sequence.
For the same reason set forth above with respect to claim 1, Moore teaches plant expression cassettes in which an N-transgene is operably linked to a membrane-specific hydrophobic MeSH targeting sequence that targets the expressed protein to an anchor membrane, and teaches that the expressed protein may be immobilized or anchored in the membrane (Abstract; pa0005-0006; “Disclosure of Invention”).
It would have been obvious to include a transmembrane domain or membrane anchor sequence with the carbonic anhydrase construct where membrane localization or anchoring was desired, because Moore teaches that such membrane-anchoring sequences improve transgene expression and/or performance in plants.
Accordingly, claim 10 would have been obvious over Djonovic in view of Borisjuk and Moore.
Claims 12 and 13 are rejected under 35 U.S.C. §103 as being unpatentable over Djonovic (WO2016200987A1) in view of Borisjuk (1999) as apply to claim 1 and 3, and further in view of Moore (CN114555806A), and in view of Orozco (Emil Orozco et. al., US20050138691A1, Application 2004-11-12, Publication 2005-06-23).
Claims 1, and 4 as the teachings of Djonovic and Borisjuk are discussed above.
Claim 12 is interpreted as dependent of claim 1.
Claim 13 is interpreted as dependent of claim 3.
Claim 12 recites the plant according to claim 1, wherein the gene encoding a transportable carbonic anhydrase protein comprises SEQ ID NO: 1 or 3EQ ID NO: 2 or a sequence having at least 65% identity to SEQ ID NO: 1 or SEQ ID NO: 2, optionally wherein the promotor comprises SEQ ID NO: 6 or SEQ ID NO: 7 or a sequence having at least 65% identity to SEQ ID NO: 6 or SEQ ID NO: 7.
Claim 13 recites the plant according to claim 3, wherein the gene encoding a transportable carbonic anhydrase protein comprises SEQ ID NO: 1 or SEQ ID NO: 2 or a sequence having at least 65% identity to SEQ ID NO: 1 or SEQ ID NO: 2, optionally wherein the promotor comprises SEQ ID NO: 6 or SEQ ID NO: 7 or a sequence having it least 65% identity to SEQ ID NO: 6 or SEQ ID NO: 7; and wherein the signal peptide sequence comprises SEQ ID NO: 3 or SEQ ID NO: 4 or a sequence having at least 65% identity to SEQ ID NO: 3 or SEQ ID NO: 4.
For the same reason set forth above with respect to claim 1 and 3, Djonovic SEQ ID NO: 4050 is 100% identical to current application SEQ ID NO: 1 (see below); regarding the signal peptide sequence, Haseloff teaches a carrot extensin-GFP fusion, wherein the alignment shows that Haseloff SEQ ID NO:13 comprises a portion that is 100% identical to SEQ ID NO: 3 of the instant application (see below); regarding alternative signal peptide/membrane targeting sequence, Moore teaches SEQ ID NO: 218 is 98.5% identical to current application SEQ ID NO: 4 (see below alignment); regarding promoter sequence, Orozco SEQ ID NO: 31 is 100% identical to the overlapping portion of applicant’s SEQ ID NO: 7 (see alignment below), and Orozco identifies SEQ ID NO: 31 as a soybean auxin transport protein EST (Table 1; pa0023 and pa0054). While an EST is not a promoter, the 100% identity between Orozco SEQ ID NO:31 and the overlapping portion of applicant’s promoter SEQ ID NO: 7, therefore, Orozco teaches the claimed sequence identity requirement for the optional SEQ ID NO: 7 promoter limitation.
Because the cited art teaches the claimed sequences and the sequence alignment results, the claimed “at least 65% identity” limitations are met.
Accordingly, claim 12 and 13 would have been obvious over Djonovic in view of Borisjuk, Moore, and Orozco.
Claims 18 and 25 are rejected under 35 U.S.C. §103 as being unpatentable over Djonovic (WO2016200987A1) in view of Borisjuk (1999) as apply to claim 1, and further in view of Sauze (Joana Sauze et. al., Biogeosciences (2018) 15, 597–612).
Claim 1 as the teachings of Djonovic and Borisjuk are discussed above.
Claim 18 is interpreted as dependent of claim 1.
Claim 18 recites the plant, according to claim1,wherein more carbon is sequestered into the extracellular environment of the plant in comparison to a plant whose heritable genetic material does not comprise a gene encoding a transportable carbonic anhydrase protein under the control of a promoter active in root cells.
For the same reason set forth above with respect to claim 1, Borisjuk teaches that secretion of recombinant protein such as GFP targeted to the root secretory pathway localized to the cell walls and intercellular space/apoplast and accumulated in root exudates, and that an active bacterial xylanase was released from roots of transgenic plants into the surrounding medium (Fig 2). Roots of transgenic plants were clearly distinguished from untransformed controls by the bright green fluorescence of GFP visible over the slight background fluorescence of nontransformed roots (Fig. 2B); and The proportion of GFP in the cellular protein fraction isolated from roots of the carGFP transformants was much lower than that in their apoplast or root exudates (Fig. 2F). Sauze further teaches that carbonic anhydrase activity accelerates CO2 hydration and can enhance conversion of CO2 into bicarbonate/carbonate species involved in carbon sequestration (Abstract).
It would have been obvious to one of ordinary skill in the art to modify the root-expressed soybean carbonic anhydrase gene taught by Djonovic by adding a known signal peptide as taught Borisjuk, so that the carbonic anhydrase would enter the plant secretory pathway and be transported from the roots into the extracellular environment, and thereby resulting in secretion of carbonic anhydrase into the extracellular environment, where the enzyme would be expected to catalyze the hydration of carbon dioxide outside the root cells, thereby increasing carbon sequestration in the extracellular environment relative to a plant lacking the transgene.
Claim 18 is obvious over Djonovic in view of Borisjuk and Sauze.
Claim 25 recites a method for increasing the capacity of a plant to sequester carbon in the soil, the method comprising altering the heritable genetic material of the plant such that a carbonic anhydrase protein is transported from the roots of the plant into the extracellular environment.
For the same reason set forth above with respect to claim 18, Djonovic and Borisjuk teach the modified plant and root secretion of recombinant protein as set forth above. Sauze further teaches that carbonic anhydrase activity accelerates CO2 hydration and can enhance conversion of CO2 into bicarbonate/carbonate species involved in carbon sequestration (Abstract).
It would have been obvious to combine the root-secreted carbonic anhydrase system taught by Djonovic and Borisjuk with the carbon-sequestration teaching of Sauze, because secreting carbonic anhydrase from roots into the surrounding extracellular/root environment would have been expected to increase local CO2 hydration and thereby incase the plant’s capacity to sequester carbon in the soil.
Claim 18 is obvious over Djonovic in view of Borisjuk and Sauze.
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Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to YANXIN SHEN whose telephone number is (571)272-7538. The examiner can normally be reached Monday-Friday.
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/YANXIN SHEN/ Examiner, Art Unit 1663
/WEIHUA FAN/ Primary Examiner, Art Unit 1663
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