DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Priority
Applicant’s claim for the benefit of a prior-filed application no. 63/367,397 filed June 30, 2022 under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Acknowledgment is made of applicant’s claim for foreign priority of prior-filed application no. EP23305155.6 filed February 6, 2023 and application no. PCTEP2023068108 filed June 30, 2023 under 35 U.S.C. 119 (a)-(d). The certified copies have been filed in instant application.
Thus, the earliest possible priority for the instant application is June 30, 2022.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on April 2, 2025 was considered, initialed, and attached hereto. A signed copy of the list of references cited is included with this Office Action.
The listing of references on pages 25-26 of the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered. Examiner notes that some references have been submitted in the IDS, however, “Finney, D.J. (1971) Probit Analysis. 3rd Edition, Cambridge University Press, Cambridge,” has not.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code (for example, pg. 17, ln. 25; pg. 21, ln. 20; pg. 25, ln. 11). 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.
Status of Claims
Claims 1-5 filed 12/13/2024 are pending and examined herein.
Claim Interpretation
The Sequence Listing (XML file) dated 12/13/2024 denotes the organism of SEQ ID NO. 1 as Bacillus thuringiensis despite the specification disclosing that SEQ ID NO. 1 was isolated from Aneurinibacillus migulanus. Claims 1-5 referencing SEQ ID NO. 1 are interpreted to be in relation to the organism Aneurinibacillus migulanus.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-5 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
All claims are drawn to pesticidal activity based on a polypeptide having at least 70% sequence identity to SEQ ID NO. 1. In the specification, Example 1 shows that SEQ ID NO. 1 was generated by isolating DNA samples from the bacteria Aneurinibacillus migulanus from a public sequence collection. These were aligned using BLASTP against a Gpp34 protein sequence with demonstrated insecticidal activity. SEQ ID NO. 1, GUN0040A, had 42% identity to the Gpp34 protein sequence selected (SEQ ID NO. 2) and is designated as a Gpp (Cry) 34-like insecticidal protein in the sequence listing of the instant application. GUN0040A was cloned and harvested for recombinant protein purification for insect larval activity assays [Example 2]. Transformed bacterial whole cells expressing GUN0040A protein were used to evaluate efficacy against the coleoptera species Western corn rootworm (WCR), displaying greater WCR larval mortality and growth inhibition compared to negative controls [Example 3]. The GUN0040A protein alone showed that it acts as a single protein toxin in an artificial diet-overlay bioassay, not requiring another toxin to display control coleopteran [Example 4]. GUN0040A was transiently expressed in N. benthamiana and evaluated for phytotoxicity with Southern Corn Rootworm (SCR) [Example 6]. Western Corn Rootworm efficacy was tested with the transgenic plants and their progenies [Example 7].
The Applicant does not reduce to practice a polypeptide with less than the full-length sequence of SEQ ID NO. 1. In the specification, there are no provided variants or fragments retaining the claimed biological function. No description is given as to the relative function of the different regions or domains of the claimed sequence and it is only described and tested as a complete sequence. A representative number of species were not adequately described to reflect the variation that could arise with a sequence with 70% identity to SEQ ID NO. 1 with confirmed pesticidal activity against WCR and SCR. Gpp34/35 (previously Cry34/35) is related to Cry pesticidal proteins, which have variable specificity to target pests and are hierarchically organized by sequence similarity, as described in Crickmore (2021, “A structure-based nomenclature for Bacillus thuringiensis and other bacteria-derived pesticidal proteins,” 186:107438, as cited in the IDS filed 01/07/2026). Although GUN0040A is not a Cry-related protein, it was considered as a gene candidate due to its similarity in sequence and insecticidal activity to Gpp34Aa1 in the instant application.
Crickmore teaches that Cry-related pesticidal proteins are categorized by a group (Cry1, Cry 2…) where toxins share less than 45% amino acid identity with proteins from another group, a capital letter (Cry1A, Cry1B etc) is given when they share less than 78% identity, a small letter (Cry1Aa, Cry1Ab etc) when toxins share more than 70% but less than 95% identity [pg. 1, col. 2 ¶1]. Crickmore teaches that this hierarchy is preferred over previously used nomenclature, where proteins were classified according to their insecticidal activity, as a limitation of this system was that proteins that shared sequence homology often had different insecticidal specificities, requiring them to be put in different primary classification group [pg. 1, col. 1 ¶1].
Further, Bravo notes that the Cry proteins have evolved similar modes of action but different insect specificity [pg. 19, col. ¶2] (2012, Microbial Biotechnology, 6(1):17-26). Additionally, receptor recognition by Cry toxins has been recognized as a key step of Cry toxicity that is fundamental for insect specificity [pg. 19, col. 2, ¶1] This has been shown in the case of Cry3Aa that has insecticidal activity against coleopteran larvae like Colorado potato beetle (Leptinotarsa decemlineata), but shows very low toxicity against Western corn rootworm (Diabrotica virgifera virgifera) [pg. 20, col. 1, ¶2].
In a study to identify Cry proteins with unique modes of action for insecticidal activity against Spodoptera frugiperda (fall armyworm), Chae showed that even with 97.4% amino acid sequence identity to a potent toxin, a polypeptide showed negligible activity against fall armyworm (2022, Toxins, 14, 852). Thus, a high level of sequence specificity relative to the pest at hand would likely be required in proteins related to Cry to determine that the polypeptide will have pesticidal activity. No other sequences or variants thereof were reduced to practice in the instant application.
“Pesticidal activity” is defined within the instant specification as “the proteins or polypeptides or toxin of the present disclosure is insecticidal and is able to induce the stunting (sub-lethal effect) and/or killing (lethal effect of insect pests)” [¶30]. The specification further states that the plant of the claims “is protected from infection by plant pathogens such as Western Corn Rootworm (Diabrotica virgifera virgifera LeConte), Northern Corn Rootworm (Diabrotica barbarberi Smith & Lawrence)), and Southern Corn Rootworm (Diabrotica undecimpunctata)” [¶10]. The Applicant specifies that whole cell bacterial cultures were tested against a variety of lepidopteran crop pests to explore the specificity of the insecticidal activity of GUN0040A protein [Example 3]. Spodoptera frugiperda (J. E. Smith) (Fall armyworm), Helicoverpa zea (Corn earworm), and Ostrinia nubilalis (European corn borer) were tested using a lepidopteran specific diet. GUN0040A protein did not exhibit elevated mortality or growth inhibition against any of the lepidopteran larvae (Spodoptera frugiperda, Helicoverpa zea and Ostrinia nubilalis). Thus, only the efficacy of GUN0040 protein against the Coleoptera species WCR and SCR were reduced to practice, not the genus claim of “pesticidal activity”, which seems to indicate a larger variety of pathogens can be targeted by the claimed polypeptide. As shown above, the effectivity of pesticidal activity on different pests varies greatly with the Cry-related protein based on the sequence, mode of action, and insect specificity.
Lastly, the Applicant provides only the example of transformation in N. benthamiana, yet claimed the broad genus of all plants for effective transformation and protection from a plant pathogen. Not all plants are readily transformable and may depend on the method of transformation, the regeneration capacity, and the plant’s own genetic makeup and defense mechanisms for efficacy of transformation [National Academies Press (US); 2004. Ch. 2, Methods and Mechanisms for Genetic Manipulation of Plants, Animals, and Microorganisms.].
With the exception of a transformed N. benthamiana plant or host cell comprising a recombinant nucleic acid molecule encoding a polypeptide having full length sequence identity to SEQ ID NO. 1, wherein the polypeptide has pesticidal activity against Western and Southern corn rootworm, the Applicant has not reduced to practice any sequence having at least 70% identity to SEQ ID NO. 1 in the method of protecting a plant (i.e., all possible plants) from a plant pathogen (i.e., all possible plant pathogens).
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 3-5 are rejected under 35 U.S.C. 103 as being unpatentable over Deter et al. (2002). “Pesticidal Genes and Methods of Use.” (WO 2022236060 A1, cited in IDS). (App. No. US 2022028078 W, filed 2022-05-06, published 2022-11-10), in view of NCBI hypothetical protein [Aneurinibacillus migulanus], first deposited 08/19/2015, (Acc. No. WP_052520572.1) (sequence cited in IDS filed 04/02/2025).
Claim 3 recites a recombinant nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having at least 70% sequence identity to SEQ ID NO: 1, wherein the polypeptide has pesticidal activity.
Claim 4 recites a vector comprising a recombinant nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having at least 70% sequence identity to SEQ ID NO:1, wherein the polypeptide has pesticidal activity.
Claim 5 recites a transformed host cell comprising a nucleic acid encoding a recombinant nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having at least 70% sequence identity to SEQ ID NO: 1, wherein the polypeptide has pesticidal activity.
Regarding claim 3, Deter teaches a polypeptide having pesticidal activity [claim 1], a nucleic acid molecule that encodes said polypeptide [claim 7], and the nucleic acid molecule of claim 7, wherein the nucleic acid molecule is a recombinant nucleic acid molecule [claim 9]. Deter discloses that the polypeptide comprising the amino acid sequence of the recombinant pesticidal proteins is encoded by a nucleotide sequence, or fragments and variants thereof that retain pesticidal activity, set forth in SEQ ID NO: 2 [¶12 & 112]. Deter teaches that the pesticidal proteins provided are toxin proteins from bacteria and exhibit activity against certain pests [¶13]. The pesticidal proteins are biologically active (e.g., pesticidal) against pests including insects, fungi, nematodes, and the like [¶12]. Deter provides examples of pesticidal activity against numerous pests, including rootworm (Western corn rootworm, e.g., Diabrotica virgifera, Northern corn rootworm,e.g., Diabrotica longicornis barberi; and Southern corn rootworm, e.g., Diabrotica undecimpunctata) [Example 3, ¶34 and 24]. Deter also specifies that said lepidopteran pest, hemipteran pest, or said coleopteran pest may be resistant to one or more strains of Bacillus thuringiensis or one or more toxin proteins produced by one or more strains of Bacillus thuringiensis [claim 32].
Regarding claim 4, Deter teaches a DNA construct comprising a heterologous promoter operably linked to a nucleotide sequence that encodes a polypeptide, wherein the polypeptide has pesticidal activity [claim 10], wherein the promoter drives expression in a plant cell [claim 11], and a vector comprising the DNA construct [claim 14].
Regarding claim 5, Deter discloses a host cell comprising the DNA construct comprising a heterologous promoter operably linked to a nucleotide sequence that encodes a polypeptide, wherein the polypeptide has pesticidal activity [claim 15].
Regarding the limitation of 70% identity to SEQ ID NO. 1 set forth in claims 3-5, Deter discloses that the pesticidal proteins are biologically active (e.g., pesticidal) against pests including insects, fungi, nematodes, and the like [¶12]. Deter teaches that “fragments” or “biologically active portions” include polypeptides comprising a sufficient number of contiguous amino acid residues to retain the biological activity, i.e., have pesticidal activity [¶43]. Such biologically active portions can be prepared by recombinant techniques and evaluated for pesticidal activity. A “fragment” comprises at least 8 contiguous amino acids [¶43] and a “variant” is intended polypeptides having an amino acid sequence that is “at least about 70% identical” to any of the recombinant pesticidal proteins [¶48]. Deter additionally provides examples of variants and fragments of the proteins [Table 1].
SEQ ID NO. 2 of Deter is a Gpp (Cry34/35) gene class DNA encoded protein (APG06384.0) with 69.2% identity to SEQ ID NO. 1 of the instant application (see alignment below). SEQ ID NO. 2 of Deter is a Lysinibacillus sp. and is noted as an aergerolysin protein. The proteinaceous inclusions of Bacillusthuringiensis (Bt) are called crystal proteins or δ-endotoxins (or Cry proteins), which are toxic to members of the class Insecta and other invertebrates [¶14] The Cry34/Cry35 binary toxin of Bt is known to kill insects, including Western corn rootworm (the pathogen of used in the examples of the instant application), a significant pest of corn [¶34]. Deter discloses that the classification of Cry34 has been revised to be in the Gpp class of aegerolysin like pesticidal proteins, such as Gpp34a [¶35]. Deter specifies that a variant of SEQ ID NO. 2 could be about 70% identical and comprise a sufficient number of contiguous amino acid residues to retain pesticidal activity, and further states that biologically active portions can be prepared by recombinant techniques and evaluated for pesticidal activity. Given the technical problem at hand (obtaining a polypeptide for a transgenic plant showing pest resistance), and the methods and high sequence similarity presented by Deter, one of ordinary skill in the art would have arrived at the claimed subject matter.
Query Match 69.2%; Score 408; Length 114;
Best Local Similarity 68.4%;
Matches 78; Conservative 13; Mismatches 23; Indels 0; Gaps 0;
Qy 1 MSAREVHITVINVSSFELQLESKTYLNHGEWILTPTNVPEGGNLNFRADSDGFATGAEGS 60
|||||||| ||||| |||||| | | |||| ||||| ||: |||||||| || ||:
Db 1 MSAREVHIEVINVSKNELQLESNTNLKHGEWKSTPTNVSAGGDEKFRADSDGFMTGVEGT 60
Qy 61 IFYTVPDGEIKLYFDDPYVGSNAFAATTSSPSVSVQAVGSSGNVCKVMYVITNK 114
||| |||||| ||||:|:|||| |:| :|||:: :|:| ||: |:| |:||||
Db 61 IFYKVPDGEIALYFDNPFVGSNDFSAHSSSPTIQAKAIGGSGDSCEVTYLITNK 114
Deter does not explicitly teach 70% identity to SEQ ID NO. 1 and does not explicitly state that a sequence matching SEQ ID NO. 1 of the instant application would retain the function of pesticidal activity. However, a hypothetical protein (Aneurinibacillus migulanus) (Acc. No. WP_052520572.1) with 100% sequence identity to that of SEQ ID NO. 1 was first deposited on 08/19/2015 in the GenBank (see alignment below).
Score
Expect
Method
Identities
Positives
Gaps
235 bits(600)
4e-78
Compositional matrix adjust.
114/114(100%)
114/114(100%)
0/114(0%)
Query 1 MSAREVHITVINVSSFELQLESKTYLNHGEWILTPTNVPEGGNLNFRADSDGFATGAEGS 60
MSAREVHITVINVSSFELQLESKTYLNHGEWILTPTNVPEGGNLNFRADSDGFATGAEGS
Sbjct 1 MSAREVHITVINVSSFELQLESKTYLNHGEWILTPTNVPEGGNLNFRADSDGFATGAEGS 60
Query 61 IFYTVPDGEIKLYFDDPYVGSNAFAATTSSPSVSVQAVGSSGNVCKVMYVITNK 114
IFYTVPDGEIKLYFDDPYVGSNAFAATTSSPSVSVQAVGSSGNVCKVMYVITNK
Sbjct 61 IFYTVPDGEIKLYFDDPYVGSNAFAATTSSPSVSVQAVGSSGNVCKVMYVITNK 114
Even though Acc. No. WP_052520572.1 was annotated as hypothetical protein at the time of the first deposit, it would inherently have the function of pesticidal activity presented in the instant application as the function of a sequence follows its structure. Acc. No. WP_052520772.1 is noted as aegerolysin and has 100% sequence identity to SEQ ID NO. 1 of the instant application, which claims the function of pesticidal activity, thus the function of pesticidal activity would inherently belong to the hypothetical protein, as well. This sequence is inherently recombinant, and would include a recombinant nucleic acid molecule comprising a nucleotide sequence encoding said polypeptide (i.e. having at least 70% identity to SEQ ID NO. 1 of the instant application). A vector and a transformed host cell comprising a recombinant nucleic acid molecule comprising a nucleotide sequence encoding said polypeptide could be routinely accomplished by methods known to one of ordinary skill in the art with a known sequence.
Subject Matter Free of Art
Claims 1 and 2 contain subject matter free of prior art. Even though a protein with 100% sequence identity to SEQ ID NO. 1 was disclosed in the art prior to the time of filing of the instant application, the prior art does not provide significant teachings or motivation to suggest a method of protecting a plant from infection by a plant pathogen and a transformed plant comprising a recombinant nucleic acid molecule encoding a polypeptide with SEQ ID NO. 1, in view of the fact that the disclosed protein was hypothetical. The prior art fails to suggest such a method and product thereof, thus claims 1 and 2 have subject matter free of prior art.
Conclusion
No claims allowed.
Contact Information
Any inquiry concerning this communication or earlier communications from the examiner should be directed to EMILY K. JOHNSON whose telephone number is (571)272-5761. The examiner can normally be reached Monday - Friday 7:30 am - 5:00 pm.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Bratislav Stankovic can be reached at 571-270-0305. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/EMILY K JOHNSON/Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/Supervisory Patent Examiner, Art Units 1661 & 1662