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
This application claims priority to PCT/US2023/071018 which has a priority date of 28 July 2022 and a WIPO certificate was filed 02/25/2025.
Status of the Claims
Claims 1-5,8,12-13,19-20, 23,25,28,32-33,45,47, and 75-77 are pending.
Claims 1-5,8,12-13,19-20, 23,25,28,32-33,45,47, and 75-77 are examined herein.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of
browser-executable code (see paragraph 138). 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.
Information Disclosure Statement
The references indicated in the specification, such as in paragraphs 39 and 46, are not listed in a proper information disclosure statement. 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.
Claim Interpretation
“Genomic modification” of Claim 1 appears to be defined in paragraph 0067. Modifications can include an insertion, nucleic acid substitution, nucleic acid deletion, and any combination thereof.
The phrase “leghemoglobin complex comprising the leghemoglobin protein associated with a heme group” (i.e. Claim 45) is interpreted to mean leghemoglobin protein associated with a heme group (porphyrin bound to iron) which can be observed as a pink or red color and is described in paragraph 14. This phrase is also interpreted by the examiner to describe a “hemelated” leghemoglobin protein as recited in Claim 76.
The examiner interpreted the mutant plants described in the specification transformed with the vectors in example 1 and used to generate Table 11 are co-expressing the glutamyl-tRNA reductase and ferrochelatase gene.
Claim Objections
Claim 19 and 20 are objected to for drawing dependence from canceled Claim 18.
Claim 5, 13, 20, and 25 have two spellings of reticulum.
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.
Claim 76 is 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 joint inventor (or for application subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 76 uses the phrase “at least about” which is an indefinite phrase. As written, the metes and bounds of the claim are unclear. For example, is 48% at least about 50%?
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 1-3,5,8,12-13,19-20,28,32-33,45,47, and 75-77 are rejected under 35 USC § 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 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".
The claims are broadly drawn to a plant leaf comprising leghemoglobin and a genomic modification where leghemoglobin is inserted into a native non-leghemoglobin gene such that the leghemoglobin coding sequence replaces all or part of the native non-leghemoglobin gene coding sequence, insertion of a heterologous regulatory enhancer or promoter to a native leghemoglobin coding sequence, and a substitution or deletion to a native leghemoglobin’s coding sequence which increases leghemoglobin’s expression.
Applicants describe the generation of an alfalfa mutant that synthesizes leghemoglobin, with SEQ ID NO: 2, in its leaves. These expression vectors were introduced into alfalfa by Agrobacterium based transformation. The tested expression vectors contained the leghemoglobin fused to a RUBISCO activase (RCA2) or RUBISCO small subunit (SSU) promoter along with or without a RUBISCO SSUSP or KDEL targeting sequence. Example 2 also describes the vectors contained the two soybean porphyrin pathway genes, glutamyl-tRNA reductase and ferrochelatase, which the examiner interprets to mean was included in the vectors used to transform the alfalfa. The applicant disclosed the alfalfa transformant that produced the most leghemoglobin was transformed with the RCA2 promoter and no signal sequence, and leghemoglobin protein content can be seen in Table 11. With the exception of a random Agrobacterium based transformation into alfalfa, no other genomic modifications to any other plant are provided.
Applicant defines “leghemoglobin protein” or “leghemoglobin” as a globulin protein or polypeptide, whether unfolded or folded into a monomer and which may or may not have associated with it a heme group in paragraph 22. With the exception of protein with a SEQ ID NO:2, applicant does not describe any other leghemoglobin genes.
Applicant further describes and claims other non-leghemoglobin genes (i.e. RUBISCO, vegetative storage protein, or RUBISCO Activase gene) can be fully or partially replaced with leghemoglobin in paragraph 61. These genes were identified because of their preferential expression in leaves, and methods for using CRISPR/cas9 systems, specific gRNAs, and Agrobacterium-mediated transformation to excise all or part of these genes is described in Examples 4-6 in paragraphs 115-126. With the exception of soybean genomic nucleic acid and protein sequences shown in tables 5, 7, and 9, applicant does not describe any other RUBISCO, vegetative storage protein, or RUBISCO Activase genes.
Applicant’s claims encompass modifying a native leghemoglobin gene to where it is operably linked to a target sequence and/or its regulatory sequences can be modified which was described in paragraph 7, 13, and 57 where it identifies the RUBISCO small subunit and RUBISCO activase isoform 2 promoters. Paragraph 62 describes the native leghemoglobin gene can be edited so it is operably linked to a targeting sequence. However, there are no examples or description in the disclosure on how to accomplish this.
Applicants further describe that mutants can be generated by co-expressing porphyrin pathway genes in paragraph 65 and lists glutamyl-tRNA reductase, a ferrochelatase, a glutamate-1-semialdehyde 2, a 1-aminomutase, an aminolevulinate dehydratase, a hydroxymethylbilane synthase, a urophorphyrinogen III synthase, a urophorphyrinogen decarboxylase, a coporphyrinogen III oxidase, and a protoporphyrinogen oxidase. This is reiterated in Example 2. With the exception or porphyrin pathway genes in table 3, Applicant does not describe any other porphyrin pathway genes.
Regarding the broad definition of “leghemoglobin”, there are many enzymes not recognized as leghemoglobin that fit the description provided in paragraph 22 of the disclosure (i.e. globular protein that may or may not have associated with a heme group). For example, cytoglobin is described to have a globular structure and have a heme group in the active site and is described on page 15 paragraph 6 of Vlasova, Irina I. "Peroxidase activity of human hemoproteins: keeping the fire under control." Molecules 23.10 (2018): 2561. This protein matches the description described in the specification; however, it is involved in many processes (i.e. collagen synthesis, antifibrotic activity, and suppression of cell oncogenic transformation) which is different than the role of leghemoglobin (i.e. protect the nitrogenase enzyme from oxygen inactivation and to facilitate oxygen flow to the nitrogen-fixing bacteria) taught by the specification in paragraph 2. Because proteins other than leghemoglobin fit this description, it could be unclear if any globular protein being expressed in a leaf would read upon the limitations of independent Claims 1 and 12.
There is also lack of clear description regarding Claims 4 and 23 which are directed to replacing RUBISCO, vegetative storage protein, or a RUBISCO Activase genes. Nagarajan, Ragupathi, and Kulvinder S. Gill. "Evolution of Rubisco activase gene in plants." Plant molecular biology 96.1 (2018): 69-87 teaches a “[a]lthough almost all the studied photosynthetic organisms contain RCA [i.e. RUBISCO activase gene], a detailed analysis of the gene number, duplications, deletions, exon–intron structure and their evolution in important plant species is still lacking”. This indicates that one of ordinary skill in the art may not know if they were inserting a leghemoglobin gene in a native RUBISCO Activase gene in any plant.
Additionally, Sadravi, Shekoofeh, June Lee, and Jianfeng Xu. "Advances in promoter engineering strategies for enhanced recombinant protein expression in plants." Frontiers in Plant Science 16 (2025): 1747353 teaches “native promoters often contain cis-regulatory elements whose functions are not fully understood. This complexity can complicate efforts to fine-tune expression levels or engineer predictable promoter performance across different systems”. Because native cis-regulatory elements are not well understood, it would be difficult for one of ordinary skill in the art to identify if they were modifying a native leghemoglobin enhancer or promoter sequence in all plant species.
Applicant claims 10 porphyrin pathway genes in Claims 8 and 28 which would encompass many genes and homologs when directed to all plant species as directed to in Claims 1 and 12. Weisman, Caroline M., Andrew W. Murray, and Sean R. Eddy. "Many, but not all, lineage-specific genes can be explained by homology detection failure." PLoS biology 18.11 (2020): e3000862 teaches that homologs can exist but a mere BLAST result would not necessarily detect them as there is no statistical similarities between them and is descried on page 2 in paragraphs 1-3. This implies that there are likely homologous genes to the disclosed soybean glutamyl-tRNA reductase, a ferrochelatase, a glutamate-1-semialdehyde 2, a 1-aminomutase, an aminolevulinate dehydratase, a hydroxymethylbilane synthase, a urophorphyrinogen III synthase, a urophorphyrinogen decarboxylase, a coporphyrinogen III oxidase, and a protoporphyrinogen oxidase genes that would require characterization and significant experimentation and trial and error for one of ordinary skill in the art to determine if they were transforming a plant with one of these genes or modified a plant to induce their expression in leaves.
Therefore, given the lack of written description in the instant disclosure with regard to the structural and functional characteristics of the claimed compositions, Applicant does not appear to have been in possession of the claimed genus at the time this application was filed.
Scope of Enablement
Claims 1-3,5,8,12-13,19-20,28,32-33,45,47, and 75-77 are rejected under 35 U.S.C. § 112(a), first paragraph, because the specification, while being enabling for producing leghemoglobin with SEQ ID NO: 2 in an alfalfa leaf, does not reasonably provide enablement for producing leghemoglobin in all plant species leaves with all possible claimed genomic modifications. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims. All dependent claims are included in these rejections unless they contain a limitation that overcomes the deficiencies of the parent claim from which they depend.
Applicants provides an alfalfa mutant that synthesizes leghemoglobin with SEQ ID NO: 2 in its leaves. These expression vectors were introduced into alfalfa by Agrobacterium based transformation. The tested expression vectors contained the leghemoglobin fused to a RUBISCO activase (RCA2) or RUBISCO small subunit (SSU) promoter along with or without a RUBISCO SSUSP or KDEL targeting sequence. Example 2 also recites the vectors contained the two soybean porphyrin pathway genes, glutamyl-tRNA reductase and ferrochelatase, which the examiner interprets to mean was included in the vectors used to transform the alfalfa. The applicant disclosed the alfalfa transformant that produced the most leghemoglobin was transformed with the RCA2 promoter and no signal sequence, and protein content can be seen in Table 11. With the exception of a random Agrobacterium based transformation into alfalfa, no other genomic modifications in any other plant are provided.
Applicant defines “leghemoglobin protein” or “leghemoglobin” as a globulin protein or polypeptide, whether unfolded or folded into a monomer and which may or may not have associated with it a heme group in paragraph 22. With the exception of protein with a SEQ ID NO:2, applicant does not describe any other leghemoglobin genes.
Applicant further teaches and claims other non-leghemoglobin genes (i.e. RUBISCO, vegetative storage protein, or RUBISCO Activase gene) can be fully or partially replaced with leghemoglobin in paragraph 61. These genes were identified because of their preferential expression in leaves, and methods for using CRISPR/cas9 systems, specific gRNAs, and Agrobacterium-mediated transformation to excise all or part of these genes is described in Examples 4-6 in paragraphs 115-126. With the exception of soybean genomic nucleic acid and protein sequences shown in tables 5, 7, and 9, applicant does not describe any other RUBISCO, vegetative storage protein, or RUBISCO Activase genes.
Applicant’s claims encompass modifying a native leghemoglobin gene to where it is operably linked to a target sequence and/or its regulatory sequences can be modified which was described in paragraph 7, 13, and 57 where it identifies the RUBISCO small subunit promoter or RUBISCO activase isoform 2 promoters. Paragraph 62 teaches the native leghemoglobin gene can be edited to be operably linked to a targeting sequence. However, there are no examples or description in the disclosure on how to accomplish this.
Applicants further teaches that mutants can be generated by co-expressing porphyrin pathway genes in paragraph 65 and lists glutamyl-tRNA reductase, a ferrochelatase, a glutamate-1-semialdehyde 2, a 1-aminomutase, an aminolevulinate dehydratase, a hydroxymethylbilane synthase, a urophorphyrinogen III synthase, a urophorphyrinogen decarboxylase, a coporphyrinogen III oxidase, and a protoporphyrinogen oxidase. This is reiterated in Example 2. With the exception or porphyrin pathway genes in table 3, Applicant does not describe any other porphyrin pathway genes from any other species.
Regarding the definition of “leghemoglobin”, there are many enzymes not recognized as leghemoglobin that fit the definition provided in paragraph 22 of the disclosure (i.e. globular protein that may or may not have associated with a heme group. For example, cytoglobin is described to have a globular structure and have a heme group in the active site and is described on page 15 paragraph 6 of Vlasova et al 2018. This protein matches the description described in the specification; however, it is involved in many processes (i.e. collagen synthesis, antifibrotic activity, and suppression of cell oncogenic transformation) which is different than the role of leghemoglobin (i.e. protect the nitrogenase enzyme from oxygen inactivation and to facilitate oxygen flow to the nitrogen-fixing bacteria) taught by the specification in paragraph 2. Because the disclosure only provides one working example of a leghemoglobin gene and the definition of leghemoglobin is broad, one of ordinary skill in the art would not reasonably be able to make or use the invention to the broad scope it is currently claimed.
Additionally, it is known in the art that RUBISCO small subunit mutants show reduced protein expression according to Atkinson, Nicky, et al. "Rubisco small subunits from the unicellular green alga Chlamydomonas complement Rubisco‐deficient mutants of Arabidopsis." New Phytologist 214.2 (2017): 655-667. The Arabidopsis 1a3b mutant, which show reduced expression of small rubisco subunit genes, shows reduced growth and is described on page 2 paragraph 4. The reduction in protein content is seen in Figure 3b. This implies that inserting a leghemoglobin gene in a RUBISCO gene may actually reduce protein content instead of increasing its production and may result in a stunted plant. The specification lack guidance on how to avoid inhibiting plant growth and preventing reduction of protein yield.
Additionally, Sadravi et al 2025 teaches “native promoters often contain cis-regulatory elements whose functions are not fully understood. This complexity can complicate efforts to fine-tune expression levels or engineer predictable promoter performance across different systems”. This indicates that engineering a native leghemoglobin gene’s expression by modifying its native promoter to express leghemoglobin in leaves would be unpredictable and require undue trial and error experimentation to one of ordinary skill in the art.
Regarding co-expressing porphyrin pathway genes in plants with leghemoglobin production, Phung, Thu-Ha, et al. "Porphyrin biosynthesis control under water stress: sustained porphyrin status correlates with drought tolerance in transgenic rice." Plant physiology 157.4 (2011): 1746-1764 teaches “Each enzymatic step involved in porphyrin synthesis is tightly regulated to avoid the phytotoxic accumulation of intermediates”. This indicates that co-expressing one of the afore listed porphyrin pathway genes in a plant leaf could lead to the accumulation of phytotoxic intermediates which would have unknown effects on the plant and leghemoglobin production.
It is noted that, while Applicants provide data in Table 11 showing expression of leghemoglobin in an alfalfa leaf, the claims are directed to a method of increasing leghemoglobin in all possible plant leaves with many possible genetic modification (see Claim 1); however, Applicants have provided no evidence for increased leghemoglobin levels in any plant other than alfalfa and only provided one genetic modification process using Agrobacteria-mediated transformation, which is a random insertion event in the genome as taught by the abstract of Gelvin, Stanton B. "Integration of Agrobacterium T-DNA into the plant genome." Annual review of genetics 51 (2017): 195-217.
Given the absence of guidance in the specification, and given the relatively high level of unpredictability in the art, one of skill in the art cannot predict the effect of introducing the myriad of claimed genetic modification and their causal relationship to increased leghemoglobin levels in all possible plant leaves. Accordingly, given the lack of guidance in the instant Specification, undue trial and error experimentation would have been required for one skilled in the art to use the claimed invention.
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.
Claims 1-3,5,8,12-13,19-20,28,32-33,45, and 47 are rejected under 35 U.S.C. 103 as being unpatentable over the teachings of (I) Schaeffer, Scott M., and Paul A. Nakata. "CRISPR/Cas9-mediated genome editing and gene replacement in plants: transitioning from lab to field." Plant Science 240 (2015): 130-142 in view of (II) Barata, Reinaldo M., et al. "Targeting of the soybean leghemoglobin to tobacco chloroplasts: effects on aerobic metabolism in transgenic plants." Plant Science 155.2 (2000): 193-202, (III) US 2019/0292217 Al, and (IV) US 20150361441 A1.
Schaeffer et al 2015 teaches technology to genetically modify plant genomes is already prevalent in the field of engineering crops with improved traits. This is described on page 4 paragraph 2 in the “gene knock-in or replacement” section. It teaches a “CRISPR/Cas9 gene knock-in or gene replacement system is designed to facilitate the insertion, removal, or replacement of a specific gene(s) within a given genome. The site-specific insertion of protein tags, modified promoters, and other regulatory sequences are also possible.” This means methods to replace non-leghemoglobin genes, insert heterologous regulatory enhancers or promoters, or introducing substitutions or deletions of native regulatory enhancers or promoters for the production of leghemoglobin in leave are already known in the art and address the limitations (i.e. Claims 1-2, and 19).
While Schaeffer et al 2015 teaches methods of gene knock-in or gene replacement, it does not explicitly teach expressing leghemoglobin in a leaf.
Barata et al 2000 is directed to expressing leghemoglobin under the 35S promoter and is disclosed on page 2 paragraph 2 in the passage “In the present work, targeting of heterologous Lb (i.e. leghemoglobin) to tobacco chloroplasts (i.e. organelles in leaves and plastids) was performed to determine whether Lb can be correctly imported and processed inside the organelle and to examine the effects on photosynthesis, carbohydrates, chlorophyll and the activities of enzymes involved in oxidative stress.” Because the leghemoglobin was operably linked to a heterologous regulatory element, the leaves of this plant read upon the limitations of the plant leaf in Claims 12 and 13. Barata et al 2000 also teaches that the plants they generated contained 0.01-0.1% total soluble proteins and was taught in the abstract and page 4 paragraph 5. The protein was also extracted from leaves (page 4 paragraph 2) which was a limitation in Claims 1-3,5,8,12-13,19-20,28,32-33,45, and 47 which are directed to producing leghemoglobin in leaves. Plants described in Barata et al 2000 were not co-expressing a recombinant construct comprising any of the genes listed in Claim 32 [(i) a recombinant construct comprising a sequence encoding a glutamyl tRNA reductase, or a truncated portion thereof, (ii) a recombinant construct comprising a sequence encoding a ferrochelatase, (iii) a recombinant construct comprising a sequence encoding a glutamyl tRNA reductase binding protein, and (iv) a recombinant construct comprising a sequence encoding an aminolevulinic acid synthase)]. Page 4 paragraph 4 teaches the Rubisco transit peptide was fused to the soybean leghemoglobin sequence to target the leghemoglobin to the chloroplasts (i.e. plastid) as was recited in Claims 5, 13, and 20.
Schaeffer et al 2015 and Barata et al 2000 do no not teach a leghemoglobin polypeptide sequence having at least 90% similarity to SEQ ID NO:2 or a plastid or endoplasmic reticulon targeting sequence with 95% identity with SEQ ID NOs: 53 or 54. It also does not teach co-expression of a porphyrin pathway gene in a leaf. Additionally, Barata et al 2000 does not teach production of leghemoglobin in any plant species other than tobacco or if the protein was associated with a heme group.
US 2019/0292217 Al is directed to methods and materials for upregulating heme biosynthesis in plants, and more particularly, to methods for recombinantly producing heme-loaded heme polypeptides (i.e. leghemoglobin) in transgenic plants, plant cells, or seeds having upregulated heme biosynthesis (see paragraph 2). Paragraph 22 and Figure 2 teaches a polynucleotide sequence for soybean leghemoglobin that encodes the amino acid sequence that is 100% similar to applicant’s SEQ ID NO: 2 (see alignment below) which was a limitation of (Claims 2 and 33). It also teaches the limitations of Claims 5,13, and 20 by teaching “In some embodiments, the nucleic acid construct further includes a targeting sequence that can be used to direct the heme polypeptide (i.e. leghemoglobin) and/or heme biosynthesis polypeptide to one of several different intracellular compartments, including, for example, the endoplasmic reticulum (ER), mitochondria,[and] plastids (such as chloroplasts)”. It also teaches the recombinant constructs can contain a porphyrin pathway gene operably linked to a heterologous regulatory element in example 5 on paragraph 79. The Leghemoglobin gene was transformed into Arabidopsis with the porphyrin pathway gene, ferrochelatase. This is reiterated in Claims 1 and 15 of US 2019/0292217 Al and reads upon limitations in Applicant’s Claims 8 and 28. Paragraph 5 teaches that the transgenic plant can be a soy plant (i.e. soybean which has the native leghemoglobin polypeptide sequence of SEQ ID NO:2 and is a limitation of Claim 2) and alfalfa, while claim 52 teaches a method of producing a heme-loaded heme-polypeptide which is described in paragraph 31 to mean a prosthetic group bound to iron in the center of a porphyrin ring which were limitations of Applicant’s Claim 45. This implies the heme containing protein described in US 2019/0292217 Al would also be a pink or red color, absent evidence to the contrary.
100.0% identity in 145 residues overlap; Score: 721.0; Gap frequency: 0.0%
Sequence1 1 MGAFTEKQEALVSSSFEAFKANIPQYSVVFYTSILEKAPAAKDLFSFLSNGVDPSNPKLT
Sequence2 1 MGAFTEKQEALVSSSFEAFKANIPQYSVVFYTSILEKAPAAKDLFSFLSNGVDPSNPKLT
************************************************************
Sequence1 61 GHAEKLFGLVRDSAGQLKANGTVVADAALGSIHAQKAITDPQFVVVKEALLKTIKEAVGD
Sequence2 61 GHAEKLFGLVRDSAGQLKANGTVVADAALGSIHAQKAITDPQFVVVKEALLKTIKEAVGD
************************************************************
Sequence1 121 KWSDELSSAWEVAYDELAAAIKKAF
Sequence2 121 KWSDELSSAWEVAYDELAAAIKKAF
*************************
Schaeffer et al 2015, US 2019/0292217 Al, and Barata et al 2000 do not teach a plastid or endoplasmic reticulum targeting sequence with SEQ ID NO: 53 or 54 or sequence with 95% identity.
US 20150361441 A1 teaches a sequence (SEQ ID NO:479) with 100% similarity to Applicant’s SEQ ID NO:53 and identifies it as a chloroplast (i.e. plastid) signal peptide. These were limitations expressed in Claims 5, 13, and 20 and an alignment is below.
SEQ ID NO 479
LENGTH: 56
TYPE: PRT
ORGANISM: Artificial sequence
FEATURE:
OTHER INFORMATION: transit peptide from soybean rubisco small subunit precursor
(AAA82069)
Query Match 100.0%; Score 276; Length 56;
Best Local Similarity 100.0%;
Matches 55; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MASSMISSPAVTTVNRAGAGMVAPFTGLKSMAGFPTRKTNNDITSIASNGGRVQC 55
|||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MASSMISSPAVTTVNRAGAGMVAPFTGLKSMAGFPTRKTNNDITSIASNGGRVQC 55
It would have been prima facie obvious to combine the teachings of Schaeffer et al 2015 in view of Barata et al 2000, US 2019/0292217 Al, and US 20150361441 A1 to generate a leaf comprising a genomic modification where leghemoglobin is inserted into a non-leghemoglobin gene, inserting a heterologous regulatory promoter or enhancer, or modifying (i.e. substitution or deletion) a native leghemoglobin promoters or enhancers such that leghemoglobin is expressed in the leaf. Doing so would have inherently generated a plant leaf with 0.05% or 0.1% of the total leaf protein (i.e. Claims 3 and 12). One having ordinary skill in the art would have a reasonable expectation of success as the leghemoglobin gene has been expressed in multiple plant genomes along with plastid targeting sequences and with porphyrin pathway genes. Additionally, overexpression of leghemoglobin in leaves has already been successfully demonstrated in Barata et al 2000. Motivation for why one of ordinary skill in the art would chose to overexpress leghemoglobin in the leaves is taught in paragraph 7 of US 20160340411 A1, which teaches promoters can be used to express heme-containing protein (i.e. leghemoglobin) under control of tissue (i.e. leaves) specific promoters. Paragraph 104 provides further motivation for production in leaves as they are taught to be “bulky and easily harvestable”. The insertion of the leghemoglobin gene in a non-leghemoglobin gene or modification to native leghemoglobin gene regulator or promoter sequences are design choices known in the art. The criticality of any one of these genetic modifications over any other (i.e. random insertion with a constitutive promoter) has not been demonstrated or explained by the Applicant.
Claims 75-77 are rejected under 35 U.S.C. 103 as being unpatentable over the teachings of Schaeffer et al 2015 in view Barata et al 2000 as applied to Claim 1 above, and further in view of Barata et al 2000, Jung, S., et al. "Functionality of soy protein produced by enzyme‐assisted extraction." Journal of the American Oil Chemists' Society 83.1 (2006): 71-78 and US 20160340411 A1.
Claims 75-77 are directed to methods of extracting protein from a plant, an isolated product, and a protein fraction from the leaf of Claim 1.
Barata et al 2000 teaches plants producing leghemoglobin in the leaves in the abstract and teaches the protein was extracted from homogenized leaf tissue after filtering the permeant on page 4 paragraph 2 which was a limitation of Claim 75 while protein from leaves is a limitation in Claims 75-77. Barata et al 2020 also teaches that the leaves contain up to 0.1% leghemoglobin of total soluble proteins. Because they extracted soluble protein, the extracted fraction would also comprise 0.1% leghemoglobin by weight of total protein after filtering (i.e. Claim 77).
Barata et al 2000 does not teach the use of cellulase, hemicellulose, or a pectinase. It also does not teach the limitations of an isolated product comprising at least 0.2% leghemoglobin by weight of total protein wherein at least about 50% of the leghemoglobin is hemelated with an iron group.
Jung et al 2006 is directed to “the potential of enzymes to increase soy protein extractability without causing protein degradation”. It describes protein was extracted from soy flakes in the presence of cellulase, hemicellulose, and/or pectinase for the controlled breakdown of plant tissue structure (i.e. polysaccharides) for increased protein extraction (see page 3 paragraph 3). They found a 3 hour extraction of defatted soy flakes increased the yield of protein by 17% with cellulase (i.e. Claim 75) and was described on page 3 paragraph 4. The following paragraphs in the Jung et al 2006 explain other plant tissue treated with pectinase.
Barata et al 2000 and Jung et al 2006 do not teach the limitations of an isolated product comprising at least 0.2% leghemoglobin by weight of total protein wherein at least about 50% of the leghemoglobin is hemelated with an iron group.
US 20160340411 A1 is directed to methods and material for producing heme-containing polypeptides, and more particularly, to producing heme-containing polypeptides in recombinant bacterial cells such as Bacillus cells or in recombinant plants or plant cells (see paragraph 2). Paragraphs 219 teaches isolated purified protein can account for 0.2% or more of the protein component by weight. Paragraph 178 teaches “Heme content can refer to the percentage of polypeptide molecules that comprise the correct amount of heme moieties”. It also teaches heme content of a polypeptides can be at least 50%.
It would have been prima facie obvious to combine the teachings of Schaeffer et al 2015 in view of Barata et al 2000 as applied to Claim 1, and further in view of Barata et al 2000, Jung et al 2006, and US 20160340411 A1 to extract protein from leaves comprising leghemoglobin using cellulase, hemicellulose, or pectinase, collecting an isolate comprising 0.2% leghemoglobin by weight of total protein where 50% hemelated leghemoglobin, and a fraction comprising 0.1% leghemoglobin by weight of total protein. Limitations recited in the claims (i.e. an isolated product comprising at least 0.2% leghemoglobin by weight of total protein wherein at least about 50% of the leghemoglobin is hemelated with an iron group and a protein fraction comprising at least 0.1% leghemoglobin by weight of total protein) are mere optimization of the methods and a design choice of the inventor and their criticality has not been demonstrated or described. One of ordinary skill in the art would have a reasonable expectation of success that utilizing enzymes to degrade the polysaccharides of the leaf would aid in extracting higher quantities of protein as leghemoglobin has already been extracted from plant leaves and the use of cellulase has been demonstrated to aid in protein extraction from plant tissue (i.e. soy flakes). The motivation for why the inventor would want to extract leghemoglobin from the plants is described in paragraph 3 of US 20160340411 A1 which teaches “there is a continuing need for methods to produce proteins at large scale for industrial and food purposes”.
Subject Matter Free of Art
Claims 4, 23 and 25 appear to recite subject matter free of art.
Claim 4 and 23 are directed to replacing native non-leghemoglobin genes selected from the group consisting of a RUBISCO gene, a vegetative storage protein, a RUBISCO activase gene, or any combination thereof with a leghemoglobin gene. Claim 25 draws dependence from Claim 23 and is directed to introducing an insertion comprising a plastid or endoplasmic reticulum targeting sequence (with SEQ ID NO:53 or 54) operably linked to the leghemoglobin coding sequence.
The closest prior art appears to be Su, Lingtao, et al. "Large-scale integrative analysis of soybean transcriptome using an unsupervised autoencoder model." Frontiers in Plant Science 13 (2022): 831204 which demonstrates the Rubisco gene identified as “glyma.13g046200 “ in paragraph 117 of the specification are highly expressed in the leaves and can be seen on page 6 paragraph 4 of Su et al 2022. The use of plastid and endoplasmic reticulon targeting sequence were previously rejected (see rejections for Claim 5, 13 and 20).
Conclusion
No claims are allowed.
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/GEORGE W MEYER/Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/Supervisory Patent Examiner, Art Units 1661 & 1662
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