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 .
Claims 17, 21-22, and 24-41 are pending and examined herein.
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 17 October 2025 has been entered.
Claim interpretation
The claim interpretation is repeated for reasons of record as set forth in the previous Office Action.
Claim 17 recites “aada1a” on lines 6 and 9. This is interpreted to be equivalent to the gene “aada”. Absence of evidence to the contrary, such as an outside teaching or additional sequences, it appears the two genes are equivalent. Querying UniProt with a translation of SEQ ID NO: 12, which is the soybean optimized nucleic sequence encoding aada1a according to the sequence listing, returns an alignment with 100% identity to an aada gene (UniProt accession P0AG05, 2021, uniprot.org/uniprotkb/P0AG05/entry):
SEQ ID NO:12 MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPHSDIDLLVTVTVRLDE 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
sp|P0AG05|S3AD_ECOLX MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPHSDIDLLVTVTVRLDE 60
SEQ ID NO:12 TTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAKRELQFGEWQRNDILAG 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
sp|P0AG05|S3AD_ECOLX TTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAKRELQFGEWQRNDILAG 120
SEQ ID NO:12 IFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLFEALNETLTLWNSPPDWA 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
sp|P0AG05|S3AD_ECOLX IFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLFEALNETLTLWNSPPDWA 180
SEQ ID NO:12 GDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQPVILEARQAYLGQEEDRLA 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
sp|P0AG05|S3AD_ECOLX GDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQPVILEARQAYLGQEEDRLA 240
SEQ ID NO:12 SRADQLEEFVHYVKGEITKVVGK* 263
|||||||||||||||||||||||
sp|P0AG05|S3AD_ECOLX SRADQLEEFVHYVKGEITKVVGK- 263
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 17, 21-22, and 26-31 remain rejected under 35 U.S.C. 103 as being unpatentable over Ketkar et al (WO 2008063755 A2) in view of UniProt accession F4IFC5 (uniprot.org/uniprotkb/F4IFC5/entry, integrated into UniProt on July 22, 2015), Berglund et al (2009, Molecular Plant 6: 1298-1309) and Aponte et al (US 20190169626 A1). In light of Applicant’s amendment to the claims the rejection is modified from the set forth in the Office action mailed on 20 June 2025. Applicant' s arguments filed on 17 October 2025 have been fully considered but they are not persuasive.
The claims are broadly drawn to a method for producing a transgenic plant, comprising transforming an explant with a DNA construct comprising a heterologous promoter, a transit peptide with 98% or more identity to SEQ ID NO:3 and a target protein, wherein the target protein is a selectable marker, aadA1a. The method further comprises treating said explant with a selective agent and regenerating a plant from transformed cells, wherein the plant is a soybean, cowpea, hemp, chick pea, pinto bean, or pea plant.
Ketkar et al teaches nucleic acid constructs encoding proteins (see Tables 2 and 10 for exemplary proteins and traits) that comprise one or more expression units, wherein each expression unit comprise a promoter, a nucleic acid encoding a trait of interest and a 3’ untranslated region, and, optionally, an organelle transit peptide sequence (page 15, lines 1-7; instant claims 17 and 21). Ketkar et al further teaches that the promoter can be a promoter from a variety of organisms, for example the viral CaMV 35S promoter (page 16, line 12), which is heterologous to many of the genes described in Table 2 (page 27, Table 2A; instant claims 17 and 21).
Ketkar et al further teaches that the constructs they describe can be targeted to the mitochondria and chloroplast by using a dual targeting peptide as the organelle transit peptide sequence, wherein a ‘pre-sequence’ is added to a nucleic acid of interest (page 18, lines 18-22; instant claim 17 ). Ketkar et al teaches that targeting both organelles makes use of organellar biochemistry more effectively (page 18, line 20-24), they also teach that nucleic acid sequences that encode dual targeting peptides can be identified from nucleic acids coding for proteins that are known to be targeted to both chloroplasts and mitochondria (page 18, lines 24-30).
Ketkar et al teaches a transformation vector for Agrobacterium transformation comprising said nucleic acid constructs, wherein the constructs additionally contain a selectable marker comprising a coding region for aminoglycoside adenyl transferase (aada) which confers resistance to the antibiotic spectinomycin or streptomycin (page 19, lines 28-34; page 20, lines 1-3; instant claims 17 and 26-27).
Ketkar et al teaches transforming plants with the nucleic acids of their invention by microprojectile bombardment (i.e. gene gun; page 20, lines 8-13) or Agrobacterium-mediated transformation (instant claims 27-28).
Ketkar et al teaches a method of generating transgenic plants, comprising: introducing into a cell the nucleic acid constructs taught above, which comprise a dual targeting peptide, a target gene (gene/trait of interest) and a selectable marker; treatment of the cell with a selective agent; and culturing cells that survive the selective agent or score positive in a screening assay to regenerate a plant (page 20, lines 20-34; page 21, lines 1-7; instant claims 17 and 21). Ketkar et al teaches transformed plant cells comprising the nucleic acid of their invention (page 27, lines 1-3; instant claim 17).
Ketkar et al teaches the constructs and methods above in the context of making transgenic corn (a monocot) plants for the breeding of hybrid seed (page 2, lines 25-30).
Ketkar et al does not teach a dual transit peptide with at least 98% sequence identity to SEQ ID NO: 3. Ketkar et al does not teach applying the constructs and methods taught above to a soybean, cowpea, hemp, chick pea, pinto bean, or pea plant. Ketkar et al does not teach an explant with an apical meristem and radicle, wherein the explant is oriented with the apical meristem upwards in a media.
UniProt accession F4IFC5 teaches a sequence wherein the first 65 amino acids share 100% identity with SEQ ID NO: 3 (instant claims 17 and 22). It further teaches that the sequence is a Threonyl-tRNA synthetase and corresponds to the Arabidopsis thaliana gene AT2G04842.
Alignment of SEQ ID NO:3 and UniProt F4IFC5
Query Match 100.0%; Score 331; DB 2; Length 650;
Best Local Similarity 100.0%;
Matches 65; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MASSHSLLFSSSFLSKPSSFTSSLRRFVYLPTRQFWPRQRHGFSTVFAVATEPAISSSGP 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MASSHSLLFSSSFLSKPSSFTSSLRRFVYLPTRQFWPRQRHGFSTVFAVATEPAISSSGP 60
Qy 61 KKAEP 65
|||||
Db 61 KKAEP 65
Berglund et al teaches that AT2G04842 is a protein targeted to the chloroplast and mitochondria and that a construct comprising a nucleic acid encoding the first 65 amino acids of said gene fused to a reporter gene, GFP, was capable of importing the reporter into both mitochondria and chloroplasts (page 1301, col 1-2, bridging paragraph).
Aponte et al teaches a DNA construct comprising an expression cassette, comprising a heterologous promoter, a sequence encoding a dual-transit peptide linked to target gene and selectable resistance gene Acetohydroxyacid synthase (AHAS, which is also known in the art as acetolactate synthase or ALS), and that the dual transit peptide can be linked to ALS mutants which confer herbicide resistance, just as hra (paragraphs 0004-0013; 0146; 0209; 0423). Aponte et al teaches a soybean plant cell (instant claim 17) containing the construct and a method of producing a transgenic plant using the construct, wherein the construct additionally contains a selection marker AHAS, and wherein the steps include: transforming the cells with the construct, treating the cells with the selective agent imazapyr, and culturing the cells that survive (Page 38-40; paragraphs 423-429). Aponte teaches that the dual targeting peptide of their invention, when linked to polypeptides which are heterologous to the transit peptide improved translocation of said polypeptide of interest into the chloroplast and/or mitochondria (0131).
At the time of filing, it would have been prima facie obvious to one having ordinary skill in the art to modify the constructs and methods of Ketkar et al to use the N-terminal portion of AT2G04842, as represented by SEQ ID NO: 3, as a dual transit peptide in a nucleic acid or transformation vector to target a protein to the chloroplast and mitochondria or to produce a transgenic plant. One would have been motivated to do so do given the express suggestion of Ketkar et al that using a dual transit peptide makes use of organellar biochemistry more effectively and given that Aponte et al teach that use of dual peptides improved translocation. One would be further motivated to do so because Ketkar et al teaches that dual transit peptides can be identified from proteins that are known to be targeted to both chloroplasts and mitochondria. One would have a reasonable expectation of success given the express teachings of Berglund et al that shows a dual transit peptide derived from the first 65 amino acid residues of AT2G04842 is capable of transporting a reporter protein to the chloroplast and mitochondria.
At the time of filing, it would have been further prima facie obvious to one having ordinary skill in the art to further modify the methods taught by the combined prior art teachings with the teachings of Aponte et al and apply them to dicotyledonous plants such as soybean. One would have been motivated to do so by the express suggestion of Ketkar et al which suggests applying their methods to “crop breeding decisions” (page 7, lines 18-20). A person skilled in the arts would understand that soybean is an agronomically important crop. One would have had a reasonable expectation of success given the teachings of Aponte that show successful dual targeting of target proteins in soybean plants.
Regarding claim 29, explants derived from the embryos present in soybean seeds, such as a mature embryo axis, are well known in the art and a commonly used explant type. These explants inherently comprise an apical meristem and radicle. Thus, selecting such an explant is an obvious design choice. Further, such an explant could be oriented in a media in one of three ways: apical meristem pointed upwards in a media, apical meristem orientated downwards in a media, or apical meristem orientated parallel to the surface of the media (i.e. surface plated). Again, selecting any one of these finite options would be an obvious design choice.
Regarding claims 30 and 31, the limitations recited therein are merely the intended results of practicing the process steps of claim 17. As set forth above, the combined teachings of the prior art references make obvious all the steps of the method of claim 17. Applicant’s disclosures in the specification teach that practicing the process steps of claim 17 results in transformation frequency greater than 1% and have increased transformation frequency relative to constructs which do not comprise a targeting peptide (see, for example, Tables 1 and 2). Thus, as the combined art references teach all of the limitations of claim 17, practicing an identical method would necessarily yield the same intended results.
Response to Applicant’s remarks filed 17 October 2025 in response to the previous 35 U.S.C 103 rejection:
Applicant argues that none of the cited reference, alone or in combination, provides a reasonable likelihood of success in using the dual transit peptide of SEQ ID NO: 3 in combination with the claimed selectable marker aad1a. Applicant argues that it is important that the specific dual transit peptides disclosed in the application be used in combination with aadA1a or hra to generate transgenic soybean. Applicant argues that Aponte et al teaches unpredictability in the art regarding the use of dual transiting peptides and there is no guarantee of success when employing different dual transit peptides or different combinations of dual transit peptides, selectable markers and plant types. Applicant further argues, in support of the above, that the present application shows that not all dual transit peptides work in combination with the aadA1a or hra selectable marker to generate transgenic, thus there would not be a reasonable expectation of success and successful performance of the claimed methods would not be possible without the inventors' empirical data. In support of this Applicant cites a statement in their own specification: “Consistent with the aadA1a/spectinomycin selection experiments described in Example 1, only the At_dTP targeting peptide enhances hra/imazapyr selection. (Remarks, page 7-8, bridging paragraph; Specification, Example 2, page 32, lines 12-13).
This is not found persuasive.
As a first matter, Examiner notes that combined prior art references do make obvious the use of a dual transit peptide with either aada1a (as required by the rejected claims) or with hra (as taught by Aponte and set forth above) to transform soybean plants Thus it appears that Applicant’s support for their argument that the combined prior art references do not provide a reasonable expectation of success is drawn from the cited teachings of Aponte. Aponte et al’s statement that “although several transit peptides have been described, only a few have been utilized successfully in attempts to target chimeric molecules to chloroplasts and/or mitochondria in higher plants” (Applicant’s Remarks, page 6, last paragraph; Aponte et al, page 1, paragraph 0003) is not sufficient to show that one of skill in the art would not have had a reasonable expectation of success. Aponte et al merely states that few dual transiting peptides have been used, but does not, for example, provide any teaching or suggestions that any dual transiting peptides have been shown to be not effective in plants, or only effective with a certain selectable marker or in certain plants. Aponte et al does not provide any teachings or suggestions that SEQ ID NO: 3 would not be effective when combined with aad1a or hra and used to generate transgenic soybean, cowpea or hemp plants. In fact, Aponte makes no statements regarding the relationship between the efficacy of a dual transit peptide and the choice of selectable marker.
Examiner reminds Applicant, that a guarantee of success is not required for a finding of prima facie obviousness. Rather, it requires that of skill in the art would have had a reasonable expectation of success at the time of filing.
Regarding Applicant’s assertion that the present application shows that not all dual transit peptides work in combination with the aadA1a or hra selectable marker to generate transgenic, thus there would not be a reasonable expectation of success, it does not appear that these arguments are supported by the disclosure of the specification and the data presented therein. The statement cited by Applicant in their remarks references that data provided in Tables 14 and 15. Only a single dual transit peptide is included in this comparison and based on the data presented it appears that transformation frequency is higher when the dual transit peptide is used as compared to no transit peptide or a chloroplast peptide. It is not clear that there is any synergy between the choice of dual transit peptide and selectable marker nor has Applicant provide any evidence that using a different selectable marker (i.e. not aada1a or hra) would affect transformation frequency of the claimed dual transit peptide. Moreover, applicant has taught that both Gm_dTP (a dual targeting peptide from soybean) and At_dTP (i.e. SEQ ID NO:3) both have higher transformation frequencies than other single targeting peptides or no target peptide (See Table 1). Applicant provides no data to gauge how these dual targeting peptides compare to other dual targeting peptides in terms of transformation frequency as no other dual targeting peptides are included in their examples. Nor does applicant include any other selectable markers in their comparisons. Finally, Applicant’s statement is drawn to “enhancing hra/imazapyr selection” but it is not clear how this “enhanced selection” is different from the data regarding transformation frequency presented in Tables 14 and 15 or how “enhanced selection” is inferred from transformation frequency.
Claim 24 remains rejected under 35 U.S.C. 103 as being unpatentable over Ketkar et al (WO 2008063755 A2), UniProt accession F4IFC5 (uniprot.org/uniprotkb/F4IFC5/entry, integrated into UniProt on July 22, 2015), Berglund et al (2009, Molecular Plant 6: 1298-1309), Aponte et al (US 20190169626 A1) as applied to claims 17 above, and further in view of UniProt accession P0AG05 (uniprot.org/uniprotkb/P0AG05/entry, integrated into UniProt on December 20, 2005 ) and Campbell et al (1990, Plant Physiology 92: 1-11). The rejection is repeated for the reasons of record as set forth in the Office action mailed 20 June 2025. Applicant' s arguments filed 17 October 2025 have been fully considered but they are not persuasive.
Claim 24 is drawn to the method of claim 17, wherein the selectable marker is encoded by SEQ ID NO: 12. The specification and sequence listing disclose that SEQ ID NO: 12 is a codon optimized nucleic acid encoding the gene aada1a (page 10, lines 7-11; see Claim Interpretation above).
The teachings of Ketkar et al, Berglund et al, UniProt accession F4IFC5 and Aponte et al are set forth above. The references do not teach a selectable marker encoded by SEQ ID NO: 12.
UniProt accession P0AG05 teaches a sequence encoding an aada gene with 100% identity to an amino acid translation of SEQ ID NO: 12:
SEQ ID NO:12 MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPHSDIDLLVTVTVRLDE 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
sp|P0AG05|S3AD_ECOLX MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPHSDIDLLVTVTVRLDE 60
SEQ ID NO:12 TTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAKRELQFGEWQRNDILAG 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
sp|P0AG05|S3AD_ECOLX TTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAKRELQFGEWQRNDILAG 120
SEQ ID NO:12 IFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLFEALNETLTLWNSPPDWA 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
sp|P0AG05|S3AD_ECOLX IFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLFEALNETLTLWNSPPDWA 180
SEQ ID NO:12 GDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQPVILEARQAYLGQEEDRLA 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
sp|P0AG05|S3AD_ECOLX GDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQPVILEARQAYLGQEEDRLA 240
SEQ ID NO:12 SRADQLEEFVHYVKGEITKVVGK* 263
|||||||||||||||||||||||
sp|P0AG05|S3AD_ECOLX SRADQLEEFVHYVKGEITKVVGK- 263
Campbell et al teaches that codon usage differs between plant species, with different species having a unique codon bias (page 7, column 1, last paragraph; Tables III and IV). Campbell et al further teach that expressing heterologous proteins in host organisms can be improved by altering codons in the target gene to more closely reflect codon usage of the host while maintaining the original amino acid sequence (page 10, col 1, last paragraph).
At the time the invention was made , it would have been prima facie obvious to one having ordinary skill in the art to further modify the method of claim 17 with the teachings of UniProt accession P0AG05 and Campbell et al to include a selectable marker encoded by SEQ ID NO: 12. One would have been motivated to do so because of the express teachings of Ketkar et al to use an aada gene and because of the express teachings of Campbell et al that using codon optimization improves the expression of heterologous proteins in hosts. One would have had a reasonable expectation of success due to the teachings of Campbell et al and because of the routine nature of codon optimization.
Response to Applicant’s remarks filed 17 October 2025 in response to the previous 35 U.S.C 103 rejection:
Applicant argues that as neither POAGO5 nor Campbell teach the use of the dual transit
peptide of SEQ ID NO: 3 in combination with a selectable marker encoded by the aadA1a gene, they fail to overcome the deficiencies of the prior art references as applied to claim 17, and thus claim 24 is not obvious.
This is not found persuasive.
The prior art rejection of claim 24 set forth above does not rely on POAGOS or Campbell teach the use of the dual transit peptide of SEQ ID NO: 3 in combination with a selectable marker encoded by the aadA1a gene or the hra gene. Rather, these prior art references teach SEQ ID NO: 12 (as the sequence of the aad1a gene) and codon optimization. As claim 17 remains rejected—see the Examiner’s response above to Applicant’s arguments directed to the rejection of claim 17—Applicant’s arguments are not persuasive in overcoming the rejection of claim 24.
Claim 25 remains rejected under 35 U.S.C. 103 as being unpatentable over Ketkar et al (WO 2008063755 A2), UniProt accession F4IFC5 (uniprot.org/uniprotkb/F4IFC5/entry, integrated into UniProt on July 22, 2015), Berglund et al (2009, Molecular Plant 6: 1298-1309), Aponte et al (US 20190169626 A1) as applied to claim 17 above, and further in view of Campbell et al (1990, Plant Physiology 92: 1-11). The rejection is repeated for the reasons of record as set forth in the Office action mailed 20 June 2025. Applicant' s arguments filed 17 October 2025 have been fully considered but they are not persuasive.
Claim 25 is drawn to the method of claim 17, wherein the DNA construct comprises SEQ ID NO: 5. The specification and sequence listing disclose that SEQ ID NO: 5 is a codon optimized nucleic acid encoding the transit peptide of SEQ ID NO: 3 (page 11, lines 13-15).
The teachings of Ketkar et al, Berglund et al, UniProt accession F4IFC5 and Aponte et al are set forth above. Additionally, UniProt accession F4IFC5 teaches a sequence with 100% identity to an amino acid translation of SEQ ID NO: 5.
Alignment of SEQ ID NO:5 AA translation and UniProt F4IFC5:
SEQ ID NO: 5 MASSHSLLFSSSFLSKPSSFTSSLRRFVYLPTRQFWPRQRHGFSTVFAVATEPAISSSGP 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
F4IFC5 MASSHSLLFSSSFLSKPSSFTSSLRRFVYLPTRQFWPRQRHGFSTVFAVATEPAISSSGP 60
SEQ ID NO: 5 KKAEP 65
|||||
F4IFC5 KKAEP 65
The references, however, do not expressly teach the codon optimized sequence of SEQ ID NO: 5.
Campbell et al teach that codon usage differs between plant species, with different species having a unique codon bias (page 7, column 1, last paragraph; Tables III and IV). Campbell et al further teach that expressing heterologous proteins in host organisms can be improved by altering codons in the target gene to more closely reflect codon usage of the host while maintaining the original amino acid sequence (page 10, col 1, last paragraph).
At the time the invention was made, it would have been prima facie obvious to one having ordinary skill in the art to further modify the method of claim 17 with the teachings of Campbell et al to include the nucleic acid of SEQ ID NO: 5. One would have been motivated by the express teachings of Campbell et al that using codon optimization improves the expression of heterologous proteins in hosts. One would have had a reasonable expectation of success due to the teachings of Campbell and because of the routine nature of codon optimization.
Response to Applicant’s remarks filed 17 October 2025 in response to the previous 35 U.S.C 103 rejection:
Applicant argues that as Campbell does not teach the use of the dual transit peptide of SEQ ID NO: 3 in combination with a selectable marker encoded by the aadA1a gene, it fails to overcome the deficiencies of the prior art references as applied to claim 17, and thus claim 25 is not obvious.
This is not found persuasive.
The prior art rejection of claim 25 set forth above does not rely on Campbell teach the use of the dual transit peptide of SEQ ID NO: 3 in combination with a selectable marker encoded by the aadA1a gene or the hra gene. Rather, the prior art references teaches codon optimization. As claim 17 remains rejected—see the Examiner’s response above to Applicant’s arguments directed to the rejection of claim 17—Applicant’s arguments are not persuasive in overcoming the rejection of claim 25.
Claims 32-40 are rejected under 35 U.S.C. 103 as being unpatentable over Ketkar et al (WO 2008063755 A2) in view of UniProt accession F4IFC5 (uniprot.org/uniprotkb/F4IFC5/entry, integrated into UniProt on July 22, 2015), Berglund et al (2009, Molecular Plant 6: 1298-1309) and Aponte et al (US 20190169626 A1).
The claims are broadly drawn to a method for producing a transgenic plant, comprising transforming an explant with a DNA construct comprising a heterologous promoter, a transit peptide with 98% or more identity to SEQ ID NO:3 and a target protein, wherein the target protein is a selectable marker, hra. The method further comprises treating said explant with a selective agent and regenerating a plant from transformed cells, wherein the plant is a soybean, cowpea, hemp, chick pea, pinto bean, or pea plant.
Ketkar et al teaches nucleic acid constructs encoding proteins (see Tables 2 and 10 for exemplary proteins and traits) that comprise one or more expression units, wherein each expression unit comprise a promoter, a nucleic acid encoding a trait of interest and a 3’ untranslated region, and, optionally, an organelle transit peptide sequence (page 15, lines 1-7; instant claims 17 and 21). Ketkar et al further teaches that the promoter can be a promoter from a variety of organisms, for example the viral CaMV 35S promoter (page 16, line 12), which is heterologous to many of the genes described in Table 2 (page 27, Table 2A; instant claims 17 and 21).
Ketkar et al further teaches that the constructs they describe can be targeted to the mitochondria and chloroplast by using a dual targeting peptide as the organelle transit peptide sequence, wherein a ‘pre-sequence’ is added to a nucleic acid of interest (page 18, lines 18-22; instant claim 17 ). Ketkar et al teaches that targeting both organelles makes use of organellar biochemistry more effectively (page 18, line 20-24), they also teach that nucleic acid sequences that encode dual targeting peptides can be identified from nucleic acids coding for proteins that are known to be targeted to both chloroplasts and mitochondria (page 18, lines 24-30).
Ketkar et al teaches a transformation vector for Agrobacterium transformation comprising said nucleic acid constructs, wherein the constructs additionally contain a selectable maker comprising a coding region for aminoglycoside adenyl transferase (aada) which confers resistance to the antibiotic spectinomycin or streptomycin (page 19, lines 28-34; page 20, lines 1-3; instant claims 17 and 26-27).
Ketkar et al teaches transforming plants with the nucleic acids of their invention by microprojectile bombardment (i.e. gene gun; page 20, lines 8-13) or Agrobacterium-mediated transformation (instant claims 27-28).
Ketkar et al teaches a method of generating transgenic plants, comprising: introducing into a cell the nucleic acid constructs taught above, which comprise a dual targeting peptide, a target gene (gene/trait of interest) and a selectable marker; treatment of the cell with a selective agent; and culturing cells that survive the selective agent or score positive in a screening assay to regenerate a plant (page 20, lines 20-34; page 21, lines 1-7; instant claims 17 and 21). Ketkar et al teaches transformed plant cells comprising the nucleic acid of their invention (page 27, lines 1-3; instant claim 17).
Ketkar et al teaches the constructs and methods above in the context of making transgenic corn (a monocot) plants for the breeding of hybrid seed (page 2, lines 25-30).
Ketkar et al does not teach a dual transit peptide with at least 98% sequence identity to SEQ ID NO: 3. Ketkar does not teach the selectable marker hra or wherein the selective agent is imazapyr. Ketkar et al does not teach applying the constructs and methods taught above to a soybean, cowpea, hemp, chick pea, pinto bean, or pea plant. Ketkar et al does not teach an explant with an apical meristem and radicle, wherein the explant is oriented with the apical meristem upwards in a media.
UniProt accession F4IFC5 teaches a sequence wherein the first 65 amino acids share 100% identity with SEQ ID NO: 3 (instant claims 17 and 22). It further teaches that the sequence is a Threonyl-tRNA synthetase and corresponds to the Arabidopsis thaliana gene AT2G04842.
Alignment of SEQ ID NO:3 and UniProt F4IFC5
Query Match 100.0%; Score 331; DB 2; Length 650;
Best Local Similarity 100.0%;
Matches 65; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MASSHSLLFSSSFLSKPSSFTSSLRRFVYLPTRQFWPRQRHGFSTVFAVATEPAISSSGP 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MASSHSLLFSSSFLSKPSSFTSSLRRFVYLPTRQFWPRQRHGFSTVFAVATEPAISSSGP 60
Qy 61 KKAEP 65
|||||
Db 61 KKAEP 65
Berglund et al teaches that AT2G04842 is a protein targeted to the chloroplast and mitochondria and that a construct comprising a nucleic acid encoding the first 65 amino acids of said gene fused to a reporter gene, GFP, was capable of importing the reporter into both mitochondria and chloroplasts (page 1301, col 1-2, bridging paragraph).
Aponte et al teaches a DNA construct comprising an expression cassette, comprising a heterologous promoter, a sequence encoding a dual-transit peptide linked to target gene and selectable resistance gene Acetohydroxyacid synthase (AHAS, which is also known in the art as acetolactate synthase or ALS), and that the dual transit peptide can be linked to ALS mutants which confer herbicide resistance, such as hra (paragraphs 0004-0013; 0146; 0209; 0423). Aponte et al teaches a soybean plant cell (instant claim 17) containing the construct and a method of producing a transgenic plant using the construct, wherein the construct additionally contains a selection marker AHAS, and wherein the steps include: transforming the cells with the construct, treating the cells with the selective agent imazapyr, and culturing the cells that survive (Page 38-40; paragraphs 423-429). Aponte teaches that the dual targeting peptide of their invention, when linked to polypeptides which are heterologous to the transit peptide improved translocation of said polypeptide of interest into the chloroplast and/or mitochondria (0131).
At the time of filing, it would have been prima facie obvious to one having ordinary skill in the art to modify the constructs and methods of Ketkar et al to use the N-terminal portion of AT2G04842, as represented by SEQ ID NO: 3, as a dual transit peptide in a nucleic acid or transformation vector to target a protein to the chloroplast and mitochondria or to produce a transgenic plant and to substitute the aada selectable marker with the hra selectable and to use imazapyr as the selective agent. One would have been motivated to do so do given the express suggestion of Ketkar et al that using a dual transit peptide makes use of organellar biochemistry more effectively and given that Aponte et al teach that use of dual peptides improved translocation. One would be further motivated to do so because Ketkar et al teaches that dual transit peptides can be identified from proteins that are known to be targeted to both chloroplasts and mitochondria. One would have been motivated to substitute hra and imazapyr for aada and spectomycin given that Aponte teaches using hra and imazapyr to select transformed soybean and corn plants (in the context of a dual transit peptide) and given that using hra and imazapyr to select transformed plants is common and widespread in the art. One would have a reasonable expectation of success given the express teachings of Berglund et al that shows a dual transit peptide derived from the first 65 amino acid residues of AT2G04842 is capable of transporting a reporter protein to the chloroplast and mitochondria.
At the time of filing, it would have been further prima facie obvious to one having ordinary skill in the art to further modify the methods taught by the combined prior art teachings with the teachings of Aponte et al and apply them to dicotyledonous plants such as soybean. One would have been motivated to do so by the express suggestion of Ketkar et al which suggests applying their methods to “crop breeding decisions” (page 7, lines 18-20). A person skilled in the arts would understand that soybean is an agronomically important crop. One would have had a reasonable expectation of success given the teachings of Aponte that show successful dual targeting of target proteins in soybean plants.
Regarding claim 38, explants derived from the embryos present in soybean seeds, such as a mature embryo axis, are well known in the art and a commonly used explant type. These explants inherently comprise an apical meristem and radicle. Thus, selecting such an explant is an obvious design choice. Further, such an explant could be oriented in a media in one of three ways: apical meristem pointed upwards in a media, apical meristem orientated downwards in a media, or apical meristem orientated parallel to the surface of the media (i.e. surface plated). Again, selecting any one of these finite options would be an obvious design choice.
Regarding claims 39 and 40, the limitations recited therein are merely the intended results of practicing the process steps of claim 32. As set forth above, the combined teachings of the prior art references make obvious all the steps of the method of claim 32. Applicant’s disclosures in the specification teach that practicing the process steps of claim 17 results in transformation frequency greater than 1% and have increased transformation frequency relative to constructs which do not comprise a targeting peptide (see, for example, Tables 1 and 2). Thus, as the combined art references teach all of the limitations of claim 32, practicing an identical method would necessarily yield the same intended results.
Claim 41 is rejected under 35 U.S.C. 103 as being unpatentable over Ketkar et al (WO 2008063755 A2), UniProt accession F4IFC5 (uniprot.org/uniprotkb/F4IFC5/entry, integrated into UniProt on July 22, 2015), Berglund et al (2009, Molecular Plant 6: 1298-1309), Aponte et al (US 20190169626 A1) as applied to claim 32 above, and further in view of Campbell et al (1990, Plant Physiology 92: 1-11).
Claim 41 is drawn to the method of claim 32, wherein the DNA construct comprises SEQ ID NO: 5. The specification and sequence listing disclose that SEQ ID NO: 5 is a codon optimized nucleic acid encoding the transit peptide of SEQ ID NO: 3 (page 11, lines 13-15).
The teachings of Ketkar et al, Berglund et al, UniProt accession F4IFC5 and Aponte et al are set forth above. Additionally, UniProt accession F4IFC5 teaches a sequence with 100% identity to an amino acid translation of SEQ ID NO: 5.
Alignment of SEQ ID NO:5 AA translation and UniProt F4IFC5:
SEQ ID NO: 5 MASSHSLLFSSSFLSKPSSFTSSLRRFVYLPTRQFWPRQRHGFSTVFAVATEPAISSSGP 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
F4IFC5 MASSHSLLFSSSFLSKPSSFTSSLRRFVYLPTRQFWPRQRHGFSTVFAVATEPAISSSGP 60
SEQ ID NO: 5 KKAEP 65
|||||
F4IFC5 KKAEP 65
The references, however, do not expressly teach the codon optimized sequence of SEQ ID NO: 5.
Campbell et al teach that codon usage differs between plant species, with different species having a unique codon bias (page 7, column 1, last paragraph; Tables III and IV). Campbell et al further teach that expressing heterologous proteins in host organisms can be improved by altering codons in the target gene to more closely reflect codon usage of the host while maintaining the original amino acid sequence (page 10, col 1, last paragraph).
At the time the invention was made, it would have been prima facie obvious to one having ordinary skill in the art to further modify the method of claim 32 with the teachings of Campbell et al to include the nucleic acid of SEQ ID NO: 5. One would have been motivated by the express teachings of Campbell et al that using codon optimization improves the expression of heterologous proteins in hosts. One would have had a reasonable expectation of success due to the teachings of Campbell and because of the routine nature of codon optimization.
Response to Applicant’s remarks filed 17 October 2025 regarding claims 32-41 relative to the teachings of Ketkar et al, Aponte et al, and Berglund et al:
Applicant argues, on page 7 of the remarks (paragraph 1) that the new claims are not obvious in view of the cited prior art references because Ketkar et al uses a “distinct transit peptide” and that Ketkar et al fails to teach transforming the claimed plant types, Berglund et al’s teachings are targeted to isolated organelles in vitro, Aponte’s teachings use a dual transiting peptide that differs from the instantly claimed SEQ ID NO: 3, and none of the references teaches hra. Applicant further argues that in light of these alleged deficiencies, none of the cited references provide evidence that claimed construct would function in methods for generating transgenic soybean, cowpea or hemp plants.
This is not found persuasive.
As set forth above and explained in detail the prior art references make obvious the claimed invention and Aponte et al teaches the use of hra as a selectable marker.
In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). No one reference is required to provide all the teachings when a rejection is based on multiple references.
Applicant’s argument that argues that the combination of references does not teach or suggest the method of using the instantly claimed construct is a conclusory statement. Applicant does not provide any arguments directed to the substance or basis of the obviousness rejection based on the combined prior art references.
Finally, regarding applicant’s argument that the cited reference provide no evidence that the claimed construct would function in methods for generating transgenic soybean, cowpea or hemp plants, it appears that Applicant is arguing that the prior art references are not enabled. However, Applicant has not provided any evidence that the combined teachings of the prior art are not enabled. As set forth above in the 35 U.S.C. 103 rejection, it would have been prima facie obvious to one having ordinary skill in the art to modify the constructs and methods of Ketkar et al to use SEQ ID NO: 3 (as taught by Berglund et al), as a dual transit peptide in a nucleic acid or transformation vector to target a protein to the chloroplast and mitochondria or to produce a transgenic plant. One would have been motivated to do so do given the express suggestion of Ketkar et al that using a dual transit peptide makes use of organellar biochemistry more effectively. One would be further motivated to do so because Ketkar et al teaches that dual transit peptides can be identified from proteins that are known to be targeted to both chloroplasts and mitochondria.
Examiner reminds Applicant, that a guarantee of success if not required for a finding of prima facie obviousness. Rather, it requires that of skill in the art would have had a reasonable expectation of success at the time of filing.
Conclusion
No claims are allowed.
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/ALEKSANDAR RADOSAVLJEVIC/ Examiner, Art Unit 1662
/SHUBO ZHOU/Supervisory Patent Examiner, Art Units 1661 and 1662