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 .
Restriction/Election
Applicant's election with traverse of Group I (claims 1-21) in the reply filed on 12/24/2025 is acknowledged. The traversal is on the ground(s) that Group I and Group II are related as product and process of use. This is found persuasive, and as a courtesy to the Applicant, the claims of Group I and Group II will be rejoined. However, the species election of RUBY (SEQ ID NO: 1), which reads on claims 1-7 and 16-21 is still deemed proper and is therefore made FINAL.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code (page 51, Example 3, paragraph 0121). Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
Claim Objections
Claim 3 is objected to because of the following informalities: the claim lacks an article prior to the word “selectable”. It is suggested to write ---wherein the selectable marker--- for proper antecedence.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 17 and 18 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 17 recites “wherein the split between the N-terminal fragment and the C-terminal fragment of NpuDnaE occurs at amino acid position within the first 110 amino acids”. The term NpuDnaE is not accompanied by a SEQ ID NO identifier, so it is unclear what sequence will have a split within the first 110 amino acids, or at position N102:I103 (instant claim 18). Without an accompanying SEQ ID NO identifier for reference, both the term NpuDnaE and the location of the split within are rendered meaningless.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1-4, 16, and 19-22 are rejected under 35 U.S.C. 103 as being unpatentable over CHENG (Cheng et al, Pub. No.: US 2020/0263197 A1; Pub. Date: Aug. 20, 2020; included on IDS dated 01/14/2025) in view of YADAV (Yadav et al., Pub. No.: US 2004/0172688 A1; Pub. Date: Sep. 2, 2004).
Claim 1 recites “[a] split selectable marker system for plant co-transformation, the system comprising:
A) a first vector comprising:
(i) from 5' to 3', and operably linked:
a first promoter,
a nucleotide sequence encoding an N-terminal fragment of a selectable marker protein;
a nucleotide sequence encoding an N-terminal fragment of an intein; and
a first terminator;
wherein the nucleotide sequence encoding the N-terminal fragment of the selectable marker protein is linked, in frame, to the nucleotide sequence encoding the N- terminal fragment of the intein;
and
(ii) a first gene of interest, wherein the first gene of interest comprises a promoter, a coding sequence, and a terminator; and
B) a second vector comprising:
(i) from 5' to 3', and operably linked:
a second promoter,
a nucleotide sequence encoding a C-terminal fragment of the intein;
a nucleotide sequence encoding a C-terminal fragment of the selectable marker protein; and
a second terminator;
wherein the nucleotide sequence encoding the C-terminal fragment of the intein is linked, in frame, to the nucleotide sequence encoding the C-terminal fragment of the selectable marker protein;
and
(ii) a second gene of interest, wherein the second gene of interest comprises a promoter, a coding sequence, and a terminator;
wherein upon expression in a plant cell, the N-terminal fragment and the C-terminal fragment of the intein join the N-terminal fragment and the C-terminal fragment of the selectable marker protein to form a peptide bond”.
In regard to claims 1 and 22, CHENG teaches and claims a split intein selectable marker system for the production and selection of transgenic cells (i.e., a split selectable marker system for plant co-transformation) (Cheng, Abstract).
CHENG teaches and claims a method comprising delivering to eukaryotic cells (i.e., a method of co-transforming plant cells, the method comprising delivering DNA vectors into a plant cell (instant claim 22))
(a) a first vector comprising (i) a nucleotide sequence encoding an N-terminal fragment of a selectable marker protein (i.e., a nucleotide sequence encoding an N-terminal fragment of a selectable marker protein), which is upstream from a nucleotide sequence encoding an N-terminal fragment of an intein (i.e., from 5’ to 3’; a nucleotide sequence encoding an N-terminal fragment of an intein) and (ii) a nucleotide sequence encoding a first molecule of interest (i.e., a first gene of interest); and
(b) a second vector comprising (i) a nucleotide sequence encoding a C-terminal fragment of the intein (i.e., a nucleotide sequence encoding a C-terminal fragment of an intein), which is upstream from a C-terminal fragment of the selectable marker protein (i.e., from 5’ to 3’; a nucleotide sequence encoding an N-terminal fragment of a selectable marker protein) and (ii) a nucleotide sequence encoding a second molecule of interest (i.e., a second gene of interest),
wherein the N-terminal fragment and the C-terminal fragment of the intein catalyze joining of the N-terminal fragment and the C-terminal fragment of the selectable marker protein to produce a full-length selectable marker protein (i.e., wherein the N-terminal fragment and the C-terminal fragment of the intein join the N-terminal fragment and the C-terminal fragment of the selectable marker protein to form a peptide bond) (Cheng, page 1, paragraph 0006; claim 1).
CHENG teaches a vector includes a promoter operably linked to a nucleic acid encoding a fragment of an intein and a fragment of selectable marker protein; a vector also comprises a promoter operably linked to a nucleic acid, such as a transgene, encoding a molecule of interest (i.e., operably linked) (Cheng, page 7, paragraph 0050).
CHENG teaches delivering to a composition comprising eukaryotic cells two or more vectors, wherein each vector comprises (i) a nucleotide sequence encoding a selectable marker protein fragment linked to an N-terminal intein protein fragment and/or a C-terminal intein protein fragment and (ii) a nucleotide sequence encoding a molecule of interest, wherein the intein protein fragments, when joined in frame (i.e., linked, in-frame) to form full-length function proteins, catalyze joining of the selectable marker protein fragments to produce a full-length selectable marker protein (Cheng, page 1, paragraph 0006).
CHENG teaches a first promoter and a second promoter (see Fig. 1A below; Pr = promoter) (i.e., a first promoter and a second promoter) (Cheng, Fig. 1A).
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Cheng does not explicitly teach a first terminator or a second terminator. Cheng does not explicitly teach a first gene of interest, wherein the first gene of interest comprises a promoter, a coding sequence, and a terminator, or a second gene of interest, wherein the second gene of interest comprises a promoter, a coding sequence, and a terminator.
However, Yadav teaches constructs and methods to introduce a protein splicing mechanism into plants by employing inteins and transgenes. Inteins function effectively in plants when they contain plant optimized codons, leading to their self-excision from a protein precursor and ligation of the extein fragments to produce a mature or active protein in the plant. This mechanism can be utilized to assemble exteins into large protein polymers (including structural proteins and bioactive proteins), hybrid protein polymers, and circular protein polymers. Further, by selectively choosing promoters responsive to various inducers, plant tissues, or plant developmental states, it is possible to control the protein splicing mechanism so as to produce complex mature and active protein products under selected environmental conditions, in selected plant tissues, at selected development stages, or in selected plant generations. This permits use of the intein-mediated protein splicing reaction as a means to activate regulatory protein factors and enzymes, and thus control gene expression and metabolism. The present invention and its embodiments therefore can benefit plant-based protein polymer production methods and have use in agronomic practice for various other agricultural and industrial applications (Yadav, page 5, paragraph 0073).
Yadav teaches that a split intein is comprised of two distinct polypeptides or proteins, referred to as the “N-terminal” or N-intein (abbreviated as IntN, Intn or Int-n) and the “C-terminal” or C-intein (abbreviated as IntC, Intc or Int-c) because of their homology to the N-terminal and C-terminal regions of non-split inteins, respectively. Together IntN and IntC polypeptides, when operably linked to foreign polypeptides, possess all necessary functionality to complete a trans-protein splicing reaction, whereby the two foreign “extein” fragments are ligated together by formation of a peptide bond (Yadav, page 5, paragraph 0080).
Yadav further teaches that an intein cassette refers to a synthetic construct that minimally includes an intein or a portion thereof, and an extein. This encompasses split intein constructs that have the structures of ExtN-IntN and IntC-ExtC. An intein cassette may possess intervening sequences between the intein sequence and extein fragment that are destined to produce a mature, active protein. These intervening sequences may include, for example, regulatory sequences (e.g., promoters and 3′ terminators) (i.e., a first terminator and a second terminator) (Yadav, page 6, paragraph 0082).
Additionally, Yadav teaches an expression cassette that contains a DNA sequence for a selected gene product (i.e., a transgene) for expression in the host cell. This expression cassette typically includes a 5′ promoter region, the transgene ORF, and a 3′ terminator region, with all necessary regulatory sequences required for transcription and translation of the ORF (i.e., a first gene of interest, wherein the first gene of interest comprises a promoter, a coding sequence, and a terminator, and a second gene of interest, wherein the second gene of interest comprises a promoter, a coding sequence, and a terminator) (Yadav, page 9, paragraph 0119).
Yadav teaches plasmids pGISN containing the 35S-Pro::GUSn/Intn::NOS-T chimeric gene, and pGISC containing the 35-Pro::Intc/GUSc::NOS-Ter chimeric gene (Yadav, Example 15, page 28, paragraph 0329; Figure 19 below).
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At the time the instant application was filed, it would have been obvious and within the scope of one of ordinary skill in the art to construct a split intein selectable marker system for the production and selection of transgenic cells as taught by Cheng, additionally comprising a first terminator and a second terminator; and a first gene of interest, wherein the first gene of interest comprises a promoter, a coding sequence, and a terminator, and a second gene of interest, wherein the second gene of interest comprises a promoter, a coding sequence, and a terminator, as taught by Yadav. One would have been motivated to combine the teachings of Cheng and Yadav to better regulate the transcription, RNA processing or stability, or translation of the associated coding sequence. Thus, one of ordinary skill in the art would have a high expectation of success by combining the teachings of Cheng and Yadav.
In regard to claim 2, Yadav teaches suitable regulatory sequences will include, but are not limited to: constitutive plant promoters, plant tissue-specific promoters, plant developmental stage-specific promoters, inducible plant promoters and viral promoters (i.e., wherein the first promoter and the second promoter are each an inducible or constitutive promoter) (Yadav, page 6, paragraph 0092).
In regard to claims 3 and 4, Yadav teaches transformation markers include:
non-destructive screenable reporters (e.g., green fluorescent and luciferase genes) (i.e.,
wherein selectable marker protein is a protein that produces a visible signal; wherein the visible signal is
a fluorescent signal) (Yadav, page 12, paragraph 0145).
In regard to claim 16, Cheng teaches and claims that the first intein is a NpuDnaE intein and the second intein is a NpuDnaE intein (i.e., wherein the intein is NpuDnaE) (Cheng, page 6, paragraph 0031; claim 11).
In regard to claims 19-21, Yadav teaches and claims plant hosts for transformation. Specific examples of useful host plant are:
Food plants (annuals): asparagus (Asparagus), banana (Musa), barley (Hordeum), blueberry (Vaccinium), broad bean (Vicia), cacao (Theobroma), capsicum pepper (Capsicum), carrot (Daucus), cassava (Manihot), corn (Zea), cucumber (Cucumis), eggplant (Solanum), Lentil (lens), lettuce (Lactuca), mango (Mangifera), oilseed rape, canola, cabbage, broccoli, cauliflower (Brassica), oat (Avena), onions (Allium), papaya (Carica), peas (Pisum), peanut (Arachis), pineapple (Ananas), pinto bean, mung bean, lima bean (Phaseolus), potato (Solanum), pumpkin, zucchini (Cucurbita), radish (Raphanus), rice (Oryza), rye (Secale), sesame (Sesame), spinach (Spinaceae), sorphum (Sorphum), soybean (Glycine), strawberry (Fragaria), sugarcane (Saccharum), sugar beet (Beta), sunflower (Helianthus), sweet potato (Ipomoea), tomato (Lycopersicom), watermelon (Citrullus), wheat (Triticum), and yam (Dioscorea).
Non-food plants (annuals): alfalfa (Medicago), amaranth (Amaranthus), angelica (Agelica), arabidopsis (Arabidopsis), castorbean (Ricinus), cotton (Gossypium), colewort (Crambe), dandelion (Taraxacum), flax (Linum), hemp (Cannabis), jojoba (Simmondsia), jute (Corchorus), kenaf (Hibiscus), lupine (Lupinus), petunia (Petunia), plantain (Plantago), sisal (Agave), snapdragon (Antirrhinum), switch grass (Panicum), and tobacco (Nicotiana).
Arboreous plants: apple (Malus), acacia (Acacia), chestnut (Castanea), citrus (Citrus), coconut (Cocos), coffee (Coffea), cypress (Cupressus), eucalypti (Eucalyptus), grape (Vitis), hemlock (Tsuga), hickory (Carya), maple (Acer), oak (Quercus), pear (Pyrus), peach, plum, cherry (Prunus), pine (Pinus), poplar (Populus), rose (Rosa), spruce (Picea), and walnut (Juglans) (i.e., wherein the plant is an herbaceous or woody plant; wherein the herbaceous plant is selected from the group comprising Arabidopsis thaliana, Brassica rapa, Glycine max, Nicotiana benthamiana, Oryza sativa, Solanum lycopersicum, Solanum tuberosum, Panicum virgatum, Sorghum bicolor, and Zea mays; wherein the woody plant is selected from the group comprising Citrus sinensis, Eucalyptus grandis, Malus domestica, Populus tremula x P. alba INRA 717-1B4, Prunus persica, and Vitis vinifera) (Yadav, page 12, paragraphs 0148-0151; claim 30).
Claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over CHENG (Cheng et al, Pub. No.: US 2020/0263197 A1; Pub. Date: Aug. 20, 2020; included on IDS dated 01/14/2025) and YADAV (Yadav et al., Pub. No.: US 2004/0172688 A1; Pub. Date: Sep. 2, 2004) as applied to claims 1-4, 16, and 19-21 above, and in further view of HE (He et al., 2020, Horticultural Research, Vol. 7(152), pp. 1-6; included on IDS dated 01/14/2025).
Claim 5 recites “[t]he system of claim 4, wherein the selectable marker protein is RUBY”.
Cheng and Yadav teach the system of claim 4.
Neither Cheng nor Yadav explicitly teaches wherein the selectable marker protein is RUBY.
However, He teaches that reporters have been widely used to visualize gene expression, protein localization, and other cellular activities, but the commonly used reporters require special equipment, expensive chemicals, or invasive treatments. He constructs a new reporter RUBY that converts tyrosine to vividly red betalain, which is clearly visible to naked eyes without the need of using special equipment or chemical treatments. He shows that RUBY can be used to noninvasively monitor gene expression in plants. Furthermore, He shows that RUBY is an effective selection marker for transformation events in both rice and Arabidopsis. The new reporter will be especially useful for monitoring cellular activities in large crop plants such as a fruit tree under field conditions and for observing transformation and gene expression in tissue culture under sterile conditions (He, Abstract, page 1).
He further demonstrates that the synthetic cassette of betalain biosynthetic genes was able to produce betalain in Arabidopsis and in rice (i.e., wherein the selectable marker protein is RUBY), providing a visible color for monitoring gene expression and plant transformation. He believes that RUBY will be very useful in large plants such as fruit trees and in field conditions. Because RUBY does not require either special equipment or expensive substrates, RUBY provides a cost-effective reporter and RUBY is a convenient alternative to the existing reporters. Betalain is a natural product and was shown to have health benefits. Using RUBY as a reporter has less environmental and health concerns compared with antibiotic and/or herbicide resistance markers (He, page 5, left column, second paragraph).
At the time the instant application was filed, it would have been obvious and within the scope of one of ordinary skill in the art to use RUBY as the selectable marker as taught by He, in the split intein selectable marker system for the production and selection of transgenic cells as taught by Cheng, additionally comprising a first terminator and a second terminator; and a first gene of interest, wherein the first gene of interest comprises a promoter, a coding sequence, and a terminator, and a second gene of interest, wherein the second gene of interest comprises a promoter, a coding sequence, and a terminator, as taught by Yadav. One would have been motivated to combine the teachings of Cheng, Yadav, and He knowing that RUBY is useful in large plants such as fruit trees and in field conditions; RUBY does not require either special equipment or expensive substrates; RUBY provides a cost-effective reporter; and using RUBY as a reporter has less environmental and health concerns compared with antibiotic and/or herbicide resistance markers. Thus, one of ordinary skill in the art would have a high expectation of success by combining the teachings of Cheng, Yadav, and He.
Claims 17 and 18 are rejected under 35 U.S.C. 103 as being unpatentable over CHENG (Cheng et al, Pub. No.: US 2020/0263197 A1; Pub. Date: Aug. 20, 2020; included on IDS dated 01/14/2025) and YADAV (Yadav et al., Pub. No.: US 2004/0172688 A1; Pub. Date: Sep. 2, 2004) as applied to claims 1-4, 16, and 19-21 above, and in further view of ZETTLER (Zettler et al., 2009, FEBS Letters, Vol. 583(5), pp. 909-914).
Claim 17 recites “[t]he system of claim 16, wherein the split between the N-terminal fragment and the C-terminal fragment of NpuDnaE occurs at amino acid position within the first 110 amino acids”.
Cheng and Yadav teach the system of claim 16.
Neither Cheng nor Yadav explicitly teaches wherein the split between the N-terminal fragment and the C-terminal fragment of NpuDnaE occurs at amino acid position within the first 110 amino acids.
However, Zettler teaches that in protein splicing, an internal protein element, the intein, excises itself out of a precursor protein with concomitant linkage of the two flanking polypeptide sequences, the exteins, by a native peptide bond. The intein domain can also be split into two pieces; in this case two separate polypeptides first associate to reconstitute the active intein followed by the ligation of the fused N- and C-extein sequences through protein trans-splicing. This reaction is highly valuable for a number of in vitro and in vivo applications in the fields of biotechnology, protein and cellular chemistry, as well as molecular and structural biology. For example, it has been used for protein semi-synthesis and selective chemical modification of proteins, generation of cyclic peptides and proteins, segmental isotopic labelling, conditional control of protein function, and construction of reporters of protein-protein interactions. In this study, we have undertaken a first in vitro characterization of the naturally split Npu DnaE intein using purified constructs (Zettler, Introduction, page ).
Zettler teaches the protein trans-splicing reaction schemes of the Npu DnaE intein constructs used in the study. The amino acids flanking the splice junctions are indicated. The IntN part consists of 102 aa, the IntC part of 36 aa (see Figure below) (i.e., wherein the split between the N-terminal fragment and the C-terminal fragment of NpuDnaE occurs at amino acid position within the first 110 amino acids; wherein the split between the N-terminal fragment and the C-terminal fragment of NpuDnaE occurs at amino acid position N102:I103) (Zettler, Figure 1, page ).
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At the time the instant application was filed, it would have been obvious and within the scope of one of ordinary skill in the art to split the NpuDnaE intein at position 102/103 as taught by Zettler, in the split intein selectable marker system for the production and selection of transgenic cells as taught by Cheng, additionally comprising a first terminator and a second terminator; and a first gene of interest, wherein the first gene of interest comprises a promoter, a coding sequence, and a terminator, and a second gene of interest, wherein the second gene of interest comprises a promoter, a coding sequence, and a terminator, as taught by Yadav. One would have been motivated to combine the teachings of Cheng, Yadav, and Zettler knowing that the NpuDnaE fusion constructs taught by Zettler were converted to the splice products in excellent yields from about 55% to 90%. Thus, one of ordinary skill in the art would have a high expectation of success by combining the teachings of Cheng, Yadav, and Zettler.
Summary
No claim is allowed.
However, claims 6-7 are deemed free of the prior art. A thorough search of the prior art did not disclose the selectable marker RUBY split at an amino acid position within the first 240 amino acids of SEQ ID NO: 1 or at amino acid position L231:C232 of SEQ ID NO: 1. Accordingly, claims 6-7 are objected to as being dependent upon a rejected base claim (instant claim 5), but may be allowable if re-written in independent form including all of the limitations of the base claim and any intervening claims.
Correspondence
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CHRISTINA MEADOWS
Examiner
Art Unit 1663
/CHRISTINA L MEADOWS/Examiner, Art Unit 1663
/Amjad Abraham/SPE, Art Unit 1663