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 .
Application Status
The action is written in response to applicant’s correspondence received on 4/13/2026. Claims 1-59 are currently pending in the instant application.
Priority
The instant application claims priority to US Provisional Application 63/039,506, filed on 6/19/2020.
Election/Restriction
Applicant’s election of the inventions of Group I, claims 1-36, drawn to a minicell comprising an inhibitory RNA targeting a target sequence of a pathogen is acknowledge in the response filed on 10/29/2025. Applicant’s election of the species 1) dsRNA as the inhibitory RNA molecule recited in claims 6 and 26, 2) chitin synthase as the target sequence recited in claim 9, 3) Chitin Synthase gene as the target sequence recited in claim 10, 4) SEQ ID NO: 18 as the target sequence recited in claims 11 and 12, 5) Botrytis as the pathogen recited in claim 13, and 6) strawberry of the subject plan, in the response filed on 4/13/2026, is acknowledged.
Election was made without traverse in the reply filed on 10/29/2025 and 4/13/2026.
Claims 37-59 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to non-elected inventions.
Applicant is reminded that upon the cancelation of claims to a non-elected invention, the inventorship must be corrected in compliance with 37 CFR 1.48(a) if one or more of the currently named inventors is no longer an inventor of at least one claim remaining in the application. A request to correct inventorship under 37 CFR 1.48(a) must be accompanied by an application data sheet in accordance with 37 CFR 1.76 that identifies each inventor by his or her legal name and by the processing fee required under 37 CFR 1.17(i).
Claims 1-36 are under examination of the merits.
Specification
The abstract of the instant application is objected to because the abstract states “a method for controlling pests and pathogens with novel systems and compositions…”. The term novel is inherently evaluative and subjective, and not objective and descriptive, as required by MPEP § 608.01(b). A patent abstract is a concise statement of the technical disclosure of the patent and should include that which is new in the art to which the invention pertains. The abstract should not refer to purported merits or speculative applications of the invention and should not compare the invention with the prior art. See form paragraph 6.14 regarding the content of the abstract.
A correct abstract of the disclosure is required and must be presented on a separate sheet, apart from any other text. See MPEP § 608.01(b).
REQUIREMENTS FOR PATENT APPLICATIONS CONTAINING NUCLEOTIDE AND/OR AMINO ACID SEQUENCE DISCLOSURES
Items 1) and 2) provide general guidance related to requirements for sequence disclosures.
37 CFR 1.821(c) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.821(a) must contain a "Sequence Listing," as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.821 - 1.825. This "Sequence Listing" part of the disclosure may be submitted:
In accordance with 37 CFR 1.821(c)(1) via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter "Legal Framework") as an ASCII text file, together with an incorporation-by-reference of the material in the ASCII text file in a separate paragraph of the specification as required by 37 CFR 1.823(b)(1) identifying:
the name of the ASCII text file;
ii) the date of creation; and
iii) the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(1) on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation-by-reference of the material in the ASCII text file according to 37 CFR 1.52(e)(8) and 37 CFR 1.823(b)(1) in a separate paragraph of the specification identifying:
the name of the ASCII text file;
the date of creation; and
the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(2) via the USPTO patent electronic filing system as a PDF file (not recommended); or
In accordance with 37 CFR 1.821(c)(3) on physical sheets of paper (not recommended).
When a “Sequence Listing” has been submitted as a PDF file as in 1(c) above (37 CFR 1.821(c)(2)) or on physical sheets of paper as in 1(d) above (37 CFR 1.821(c)(3)), 37 CFR 1.821(e)(1) requires a computer readable form (CRF) of the “Sequence Listing” in accordance with the requirements of 37 CFR 1.824.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed via the USPTO patent electronic filing system as a PDF, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the PDF copy and the CRF copy (the ASCII text file copy) are identical.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed on paper or read-only optical disc, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the paper or read-only optical disc copy and the CRF are identical.
Specific deficiencies and the required response to this Office Action are as follows:
Specific deficiency – Nucleotide and/or amino acid sequences appearing in the specification are not identified by sequence identifiers in accordance with 37 CFR 1.821(d). The instant specification is objected to as the sequences provided in paragraph 0234 for the PCR primers exceed 10 nucleotides and the SEQ ID NOs are not provided.
Required response – Applicant must provide:
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required sequence identifiers, consisting of:
A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
A copy of the amended specification without markings (clean version); and
A statement that the substitute specification contains no new matter.
The use of the term BioFlo® (paragraph 0228), Monarch®, Direct-zol™ (paragraph 0234), Biotek Take3™, Quant-iT™ RiboGreen™ (see paragraph 0235), which is a trade name or a mark used in commerce, has been noted in this application. The term should be accompanied by the generic terminology; furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Written Description
Claims 1-39 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.
Breadth of the Claims
With regards to the breadth of the claims, the instant claim 1 broadly recites a biocontrol composition comprising a minicell comprising at least one polynucleotide encoding an inhibitory RNA molecule that is directed to at least one target sequence in a pathogen. In the instant specification, applicant defines “biocontrol” or “biological control” refers to the control of pests by interference with their ecological status. As the claims are drawn to a composition, any composition that has the structure of the claim (a minicell that encodes at least one polynucleotide encoding an inhibitory RNA molecule directed to a pathogen) has the inherent function of “biocontrol” unless the term “biocontrol” provides structural limitations to the recited claim, please see MPEP § 2112.01. As it stands, claim 1 broadly claims a minicell, encoding any polynucleotide that encodes any inhibitory RNAi molecule (siRNA, RNAi, dsRNA, etc.) that targets any pathogen. Regarding claim 21, though the applicant limits the target pathogen by provided SEQ ID NOs that is directed to B. cinerea, claim 21 still maintains the broadness of any inhibitory RNAi molecule.
Species Described by Complete Structure or Reduction to Practice
With regards to species described by complete structure or reduction to practice, the instant specification provides the SEQ ID NOs in paragraph (0256) which discloses the different sequences of various regions of B. cinerea, targeting the Chs3a and Chs3b gene. Applicant provides working examples of minicell production (see paragraph 0221), construction design and expression of minicells-encapsulated dsRNAs (see paragraph 0226), and the effects of minicells encapsulated dsRNAs as a biofungicide on B. cinerea (see paragraph 0239). Applicant does further disclose the effects of minicells on other fruit-rot fungal pathogens such as Alternaria alternata and Penicillium expansum. However, absent evidence to the contrary, three species reduced to practice is not representative of the immensely broad genus of any and all “pathogens” as interpreted in claim 1. Furthermore, applicant discloses the amino acid sequences SEQ ID NO: 18, 20, 22, and 24 as protein targets for RNAi in claims 11 and 12. Absent evidence to the contrary, the provided SEQ ID NOs are 100% identical to the target proteins. For example, a protein blast search of SEQ ID NO: 18, or XP_001557191.1, reveals 100% identity to BcCHSIIIa (Botrytis cinerea B05.10), as shown below. Therefore, applicant does not disclose a broad range of sequences that could encompass the claim language of claim 11 and 12 which recites a target amino acid sequence with at least 90% identity to the claimed SEQ ID NOs.
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Level of Predictability and State of the Prior Art
With regards to level of predictability in the art and state of the art, the level of predictability is high. It has been well established, that at the time of filing, the use of minicells encoding a RNAi molecule has been successfully shown to inhibit the growth of certain plant-related pathogens as evidenced by Shakeel et al. (WO 2019/060903 A1, published 3/28/2019). Furthermore, the state of the art also shows that RNAi is being explored as a suitable method to treat grey mold, cause by the pathogen B. cinerea as evidenced by of Islam et al. (RNAi-Based Biofungicides as a Promising Next-Generation Strategy for Controlling Devastating Gray Mold Diseases, Int. J. Mol. Sci., Volume 21, Issue 6, all pages, published 2/12/2020).
Taken together, given the broadness of the claim 1 and 21, it is clear the applicant does not have possession of all embodiments of the invention as claimed. These embodiments include any inhibitory RNA molecule as applicant only discloses examples of dsRNA, which is a fraction of the entire genus of RNAi, such as ssRNA, antisense RNA, siRNA, miRNA, etc. Applicant also broadly claims the use of minicells to target any pathogen, which reads on any organism, wherein the examples provided in the instant specification only direct the use of minicells encoding dsRNA to target to Botrytis cinerea, Alternaria alternata, and Penicillium expansum. Furthermore, the provided SEQ ID NOs of 18, 20, 22, or 24, absent evidence to the contrary, shows 100% identity to amino acid sequence targets which does not encompass the range of at least 90% identity as recited in claim 11 and 12.
In view of the foregoing, it is concluded that the instant specification fails to adequately describe the claimed invention in such a way to reasonably convey to one skilled in the relevant art that the instant co-inventors had possession of the claimed invention at the time the application was filed. Claims 1, 11, 12 and 21, and subsequent dependent claims are rejected under U.S.C. 112(a), Written Description.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1-8, and 16-20 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Shakeel et al. (WO 2019/060903 A1, published 3/28/2019).
Though there is a common assignee (AgroSpheres, Inc.) and common inventor (Shakeel, Ameer Hamza), this publication qualifies as 102(a)(1) prior art as the publication date falls outside the 1-year grace period of the earliest effective filing date (5/19/2020).
Regarding claim 1, Shakeel teaches an agriculture formulation which comprises 1) intact minicell comprising at least one biologically active compound, wherein the biologically active compound is selected from the group consisting of a) a nucleic acid…b) a biocontrol compound…c) a biostimulant compound (see paragraph 0009). In some embodiments, said biologically active compound is said nucleic acid that is selected from a group consisting of an antisense nucleic acid, a double-stranded RNA (dsRNA), a short-hairpin RNA (shRNA), a small-interfering RNA (siRNA), a microRNA (miRNA)…etc. (see paragraph 0010).
Shakeel further teaches where the term “biologically active” indicates that a composition or compound itself has a biological effect, or that it modifies, causes, blocks, reduces, limits the production or activity of…A “biological effect” may be but is not limited to one that impacts a biological process in a plant, one that impacts a biological process in a pest, pathogen, or parasite (paragraph 0050).
Furthermore, as Shakeel teaches a biological composition comprising a minicell and a nucleic acid molecule that is inhibitory RNA directed to a pathogen which is identical to the invention of claim 1, the down regulation of the target sequence, or where the disease caused by the pathogen is suppressed upon application of the composition compared to a control pathogen lacking the composition, is an inherent function of the composition. Please see MPEP § 2112.01, where for product and apparatus claims, “When the structure recited in the reference is substantially identical to that of the claims, claimed properties or functions are presumed to be inherent”, absent evidence to the contrary. Therefore, the downregulation of at least one target sequence in a pathogen, and where the disease caused by the pathogen is suppressed compared to a control is an inherent feature of the composition taught by Shakeel.
Regarding claim 2, Shakeel teaches where compositions described herein can comprise an agriculturally acceptable carrier (see paragraph 0232).
Regarding claim 3, Shakeel teaches where in some embodiments, said minicell is derived from a prokaryotic cell, a gram-negative bacterial cell, a gram-positive bacterial cell, or a eukaryotic cell (see paragraph 0009).
Regarding claim 4, Shakeel teaches where E. coli rely on the min system in order to ensure proper replication of parent cells into daughter cells where the genes associated with the system work together to control the placement of the Z-ring (see paragraph 0169). This system can be manipulated in order to shift the Z-ring to the polar end of the cell which excludes the nucleoid DNA upon completion of replication. This will form achromosomal and/or anucleate cells, causing asymmetrical cell division (see paragraph 0170).
Regarding claim 5, Shakeel teaches where the present disclosure provides the production of minicells from HT115 (DE3) using genetically-engineered techniques. HT115 (DE3) is a RNAi Feed strain, which is a RNase III – deficient E. coli strain with IPTG-inducible T7 Polymerase activity (see paragraph 0198).
Regarding claim 6, Shakeel teaches in some embodiments, said biologically active compound is said nucleic acid that is selected from a group consisting of an antisense nucleic acid, a double-stranded RNA (dsRNA), a short-hairpin RNA (shRNA), a small-interfering RNA (siRNA), a microRNA (miRNA)…etc. (see paragraph 0010).
Regarding claim 7, Shakeel teaches where, in some embodiments, the anucleated cell-based platforms can be utilized to encapsulate externally and/or exogenously produced dsRNA that is first produced outside of the anucleate minicell (see paragraph 0104).
Regarding claim 8, Shakeel teaches where in the context of the present disclosure, operably linking a heterologous promoter to an endogenous gene means genetically inserting a heterologous promoter sequence in front of an existing gene, in the location where that gene is naturally present (see paragraph 0073). Shakeel teaches where promoter refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA and the promoter or “enhancer” may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be comprised of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments (see paragraph 0081). Shakeel teaches where “recombinant construct” or “expression construct” may comprise an artificial combination of nucleic acid fragments and where “operably linked means the sequential arrangement of the promoter polynucleotide according to the disclosure with a further oligo- or polynucleotide, resulting in transcription of said further polynucleotide (see paragraph 0083).
Finally, Shakeel teaches where the anucleated cell-based platform uses bacterial cells lacking ribonucleases, has an RNA polymerase promoters to produce dsRNA used for RNA interference of a target. This bacterial cell I then modified to produce minicells where the dsRNA encapsulated within them (see paragraph 0103. Furthermore, the anucleated cell-based platforms can be utilized to internally express dsRNA from a recombinant plasmid capable of producing dsRNA inside of the anucleate minicell or where the anucleated cell-based platforms can be utilized to encapsulate externally and/or exogenously produced dsRNA that is first produced outside of the anucleate minicell then delivered to its target within the anucleate minicell (see paragraph 0104).
Regarding claim 16, Shakeel teaches where an anucleated-cell based platform herein can be mixed with an agriculturally acceptable carrier, where the carrier can be a solid carrier, or a liquid carrier, and in various forms including microspheres, powders, emulsions, and the like (see paragraph 0233).
Regarding claim 17, Shakeel teaches where the carrier may confer a variety of properties, such as increased stability, wettability, or dispersibility (see paragraph 0233).
Regarding claim 18, Shakeel teaches example 15, where the minicells encapsulating biologically active compounds can be formulated as a liquid, dry composition, powder, granule, seed coating, drench, in-furrow composition, or foliar spray. The formulated minicells encapsulated active compounds can be applied to a target cell found in a plant, pest, an insect, a worm, fungi,..etc. (see paragraph 0431). Though Shakeel does not directly quote spraying as a method of application, application of the composition using a foliar spray inherent involves the function of spraying.
Regarding claim 19 and 20, Shakeel teaches the present disclosure also teaches exemplary crops as targets, where such crops can include strawberries (see paragraph 0307).
In view of the foregoing, claims 1-8, and 16-20 are rejected under U.S.C. 102(a)(1) as being anticipated by Shakeel, evidence above.
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 9-11 and 13-15 are rejected under 35 U.S.C. 103 as being unpatentable over Shakeel et al. (WO 2019/060903 A1, published 3/28/2019) in view of Islam et al. (RNAi-Based Biofungicides as a Promising Next-Generation Strategy for Controlling Devastating Gray Mold Diseases, Int. J. Mol. Sci., Volume 21, Issue 6, all pages, published 2/12/2020) and Arbelet et al. (Disruption of the Bcchs3a Chitin Synthase Gene in Botrytis cinerea is Responsible for Altered Adhesion and Overstimulation of Host Plant Immunity, Molecular Plant-Microbe Interactions, Volume 23, Issue 10, pgs. 1324-1334, published 6/22/2010).
Regarding claims 9-11, and 13-15, Shakeel teaches the composition of the independent claim 1, which recites a biocontrol composition comprising at least one polynucleotide encoding an inhibitory RNA molecule that is directed to at least one target sequence in the pathogen.
Regarding claims 9-11, Shakeel does not teach wherein the target sequence is selected from genes encoding Argonaute family proteins, such as chitin synthases (claim 9), where the target sequence is a Chitin Synthase 3a gene or a 3b gene, etc. (claim 10), or where the inhibitory RNA molecule targets a nucleic acid sequence encoding an amino acid sequence at least 90% identical to SEQ ID NO: 18 (claim 11).
Regarding claims 13-15, Shakeel does not teach where the pathogen is Botrytis (claim 13), specifically Botrytis cinerea (claim 14 and 15).
Regarding claims 9, Islam teaches where the virulence genes of the B. cinerea are possible target for the RNAi-based fungicides and where mutations in the B. cinerea chitin synthase genes reduce the virulence of B. cinerea in different plant species (see section 5). Furthermore, Islam teaches where dsRNA can be used to silence the regions of the B. cinerea genes that are essential for fungicide resistance, rendering the existing fungicides more effective (see section 5).
Regarding claim 10, Islam does not specifically teach where the target sequence is a Chitin Synthase 3a or 3b gene. Regarding claim 10, Arbelet teaches where disruption of the Chitin Synthase 3a gene (BcCHS3a) alters B. cinerea virulence (see introduction).
Regarding claim 11, Islam teaches where the virulence genes of the B. cinerea are possible target for the RNAi-based fungicides. Though Islam does not specifically teach where the inhibitory RNA molecule targets an amino acid sequence with 90% identity to SEQ ID NO: 18, the instant specification provides guidance on SEQ ID NO: 18. According to the table in paragraph 0256 of the instant specification, SEQ ID NO: 18 encode for the BcCHS3a protein (XP_001557191), which is the unmodified, naturally occurring BcCHS3a protein in Botrytis cinerea, absent evidence to the contrary. Given that Islam involves targeting Chitin Synthase of B. cinerea with RNAi, and Arbelet teaches specifically BcCHS3a is a target where disruption of said gene affects B. cinerea virulence, it is obvious that the naturally occurring BcCHS3a, which has 100% identity to SEQ ID NO: 18, would be a target for RNAi fungicide disclosed in Islam.
Regarding claim 13-15, Islam teaches where B. cinerea is one of the most critical agro-economic phytopathogens that has been reported to cause gray mold disease in more than 1000 plant species where RNAi-based biofungicides that use dsRNA to target essential fungal genes are considered an emerging approach for controlling devastating gray mold disease (see abstract of Islam).
It would have been obvious to one with ordinary skill in the art, before the effective filing date of the claimed invention, to have combined the teachings of Shakeel, Islam, and Arbelet to arrive at a biocontrol composition comprising a minicell encoding an inhibitory RNA molecule that is directed to target a sequence in a pathogen, wherein the sequence encodes for an amino acid sequence that is at least 90% identical to SEQ ID NO: 18 or BcCHS3a.
One would expect a reasonable chance of success as Shakeel shows minicells can be used to introduced to a plant wherein the composition has a biological effect which blocks, reduces, limits the production or activity of…and where a “biological effect” may be but is not limited to one that impacts a biological process in a plant, one that impacts a biological process in a pest, pathogen, or parasite (paragraph 0050). Furthermore, Islam teaches that RNAi fungicides are an emerging technology to target chitin synthase in B. cinerea.
One would be motivated to combine these arts as Islam teaches where B. cinerea is one of the most critical agro-economic phytopathogens that has been reported to cause gray mold disease in more than 1000 plant species where RNAi-based biofungicides that use dsRNA to target essential fungal genes are considered an emerging approach for controlling devastating gray mold disease (see introduction). Islam also teaches where Chitin synthase can be a potential target for RNAi-based fungicides (see section 5 of Islam) and Arbelet teaches where disruption of BcCHS3a in B. cinera results in altered adhesion and delayed virulence (see introduction of Arbelet). Given the breadth of Shakeel, wherein the minicell composition can be used to target a variety of pathogens and plants, one would be motivated to use a minicell composition that would provide a biologically altering composition that does not have the negative effects of pesticides (Shakeel teaches in paragraph 0005 where there is a continuing concern about the negative effects of pesticides on human health and the surrounding environment), which can target BcCHS3a or SEQ ID NO: 18, and lowers the virulence of B. cirenea, which is known to have a pathogenic effect on thousands of plant species (see Islam, introduction), including strawberries.
In view of the foregoing, claims 9-11 and 13-15 are rejected under 35 U.S.C. 103 as being prima facie obvious, before the effective filing date.
Claims 21-36 are rejected under 35 U.S.C. 103 as being unpatentable over Shakeel et al. (WO 2019/060903 A1, published 3/28/2019) in view of Islam et al. (RNAi-Based Biofungicides as a Promising Next-Generation Strategy for Controlling Devastating Gray Mold Diseases, Int. J. Mol. Sci., Volume 21, Issue 6, all pages, published 2/12/2020) and Arbelet et al. (Disruption of the Bcchs3a Chitin Synthase Gene in Botrytis cinerea is Responsible for Altered Adhesion and Overstimulation of Host Plant Immunity, Molecular Plant-Microbe Interactions, Volume 23, Issue 10, pgs. 1324-1334, published 6/22/2010).
Regarding claim 21-36, the teachings of Shakeel are described above but will be reiterated:
Regarding claim 21, Shakeel teaches a biocontrol composition comprising a minicell comprising at least one polynucleotide encoding an inhibitory RNA molecule that targets a nucleic acid sequence that encodes a polypeptide within a cell of a target (see paragraph 0009).
Regarding claims 22-23, Shakeel teaches where compositions described herein can comprise an agriculturally acceptable carrier (see paragraph 0232) and where said minicell is derived from a prokaryotic cell, a gram-negative bacterial cell, a gram-positive bacterial cell, or a eukaryotic cell (see paragraph 0009).
Regarding claim 24, Shakeel teaches where E. coli rely on the min system in order to ensure proper replication of parent cells into daughter cells where the genes associated with the system work together to control the placement of the Z-ring (see paragraph 0169). This system can be manipulated in order to shift the Z-ring to the polar end of the cell which excludes the nucleoid DNA upon completion of replication. This will form achromosomal and/or anucleate cells, causing asymmetrical cell division (see paragraph 0170).
Regarding claim 25, Shakeel teaches where the present disclosure provides the production of minicells from HT115 (DE3) using genetically-engineered techniques. HT115 (DE3) is a RNAi Feed strain, which is a RNase III – deficient E. coli strain with IPTG-inducible T7 Polymerase activity (see paragraph 0198).
Regarding claim 26, Shakeel teaches in some embodiments, said biologically active compound is said nucleic acid that is selected from a group consisting of an antisense nucleic acid, a double-stranded RNA (dsRNA), a short-hairpin RNA (shRNA), a small-interfering RNA (siRNA), a microRNA (miRNA)…etc. (see paragraph 0010).
Regarding claim 27, Shakeel teaches where, in some embodiments, the anucleated cell-based platforms can be utilized to encapsulate externally and/or exogenously produced dsRNA that is first produced outside of the anucleate minicell (see paragraph 0104).
Regarding claim 28, Shakeel teaches where in the context of the present disclosure, operably linking a heterologous promoter to an endogenous gene means genetically inserting a heterologous promoter sequence in front of an existing gene, in the location where that gene is naturally present (see paragraph 0073). Shakeel teaches where promoter refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA and the promoter or “enhancer” may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be comprised of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments (see paragraph 0081). Shakeel teaches where “recombinant construct” or “expression construct” may comprise an artificial combination of nucleic acid fragments and where “operably linked means the sequential arrangement of the promoter polynucleotide according to the disclosure with a further oligo- or polynucleotide, resulting in transcription of said further polynucleotide (see paragraph 0083).
Finally, Shakeel teaches where the anucleated cell-based platform uses bacterial cells lacking ribonucleases, has an RNA polymerase promoters to produce dsRNA used for RNA interference of a target. This bacterial cell I then modified to produce minicells where the dsRNA encapsulated within them (see paragraph 0103. Furthermore, the anucleated cell-based platforms can be utilized to internally express dsRNA from a recombinant plasmid capable of producing dsRNA inside of the anucleate minicell or where the anucleated cell-based platforms can be utilized to encapsulate externally and/or exogenously produced dsRNA that is first produced outside of the anucleate minicell then delivered to its target within the anucleate minicell (see paragraph 0104).
Regarding claims 29-31, Shakeel teaches the composition of claim 21.
Regarding claim 32, Shakeel teaches where an anucleated-cell based platform herein can be mixed with an agriculturally acceptable carrier, where the carrier can be a solid carrier, or a liquid carrier, and in various forms including microspheres, powders, emulsions, and the like (see paragraph 0233).
Regarding claim 33, Shakeel teaches where the carrier may confer a variety of properties, such as increased stability, wettability, or dispersibility (see paragraph 0233).
Regarding claim 34, Shakeel teaches example 15, where the minicells encapsulating biologically active compounds can be formulated as a liquid, dry composition, powder, granule, seed coating, drench, in-furrow composition, or foliar spray. The formulated minicells encapsulated active compounds can be applied to a target cell found in a plant, pest, an insect, a worm, fungi,..etc. (see paragraph 0431). Though Shakeel does not directly quote spraying as a method of application, application of the composition using a foliar spray inherent involves the function of spraying.
Regarding claim 35 and 36, Shakeel teaches the present disclosure also teaches exemplary crops as targets, where such crops can include strawberries (see paragraph 0307). Regarding claims 21-36, Shakeel does not teach wherein the inhibitory RNA molecule that targets a nucleic acid sequence encoding an amino acid sequence is at least identical to SEQ ID NO: 18 or Chitin Synthase (3a or 3b). Shakeel also does not teach where the pathogen is specifically Botrytis cinera.
Regarding claims 21-36, Islam teaches where the virulence genes of the B. cinerea are possible targets for the RNAi-based fungicides. Though Islam does not specifically teach where the inhibitory RNA molecule targets an amino acid sequence with 90% identity to SEQ ID NO: 18, the instant specification provides guidance on SEQ ID NO: 18. According to the table in paragraph 0256 of the instant specification, SEQ ID NO: 18 encode for the BcCHS3a protein (XP_001557191), which is the naturally occurring BcCHS3a protein in Botrytis cinerea, absent evidence to the contrary. Arbelet teaches where disruption of the Chitin Synthase 3a gene (BcCHS3a) alters B. cinerea virulence (see introduction). Given that Islam involves targeting Chitin Synthase of B. cinerea with RNAi with minicells (see introduction), and Arbelet teaches specifically BcCHS3a as a target where disruption of said gene affects B. cinerea virulence, it is obvious that the naturally occurring BcCHS3a, which is identical to SEQ ID NO: 18, would be a target for RNAi fungicide disclosed in Islam.
It would have been obvious to one with ordinary skill in the art, before the effective filing date of the claimed invention, to have combined the teachings of Shakeel, Islam, and Arbelet to arrive at a biocontrol composition comprising a minicell encoding an inhibitory RNA molecule that is directed to target a sequence in a pathogen, wherein the sequence encodes for an amino acid sequence that is at least 90% identical to SEQ ID NO: 18 or BcCHS3a.
One would expect a reasonable chance of success as Shakeel shows minicells can be used to introduced to a plant wherein the composition has a biological effect which blocks, reduces, limits the production or activity of…and where a “biological effect” may be but is not limited to one that impacts a biological process in a plant, one that impacts a biological process in a pest, pathogen, or parasite (paragraph 0050). Furthermore, Islam teaches that RNAi fungicides are an emerging technology to target chitin synthase in B. cinerea.
One would be motivated to combine these arts as Islam teaches where B. cinerea is one of the most critical agro-economic phytopathogens that has been reported to cause gray mold disease in more than 1000 plant species where RNAi-based biofungicides that use dsRNA to target essential fungal genes are considered an emerging approach for controlling devastating gray mold disease (see introduction). Islam also teaches where Chitin synthase can be a potential target for RNAi-based fungicides (see section 5 of Islam) and Arbelet teaches where disruption of BcCHS3a in B. cinera, which is identical to SEQ ID NO: 18 absent evidence to the contrary, results in altered adhesion and delayed virulence (see introduction of Arbelet). Given the breadth of Shakeel, wherein the minicell composition can be used to target a variety of pathogens and plants, one would be motivated to combine these arts to create and use a minicell composition which would provide a biologically altering composition targeting SEQ ID NO: 18 in Botrytis cinerea that does not have the negative effects of pesticides (Shakeel teaches in paragraph 0005 where there is a continuing concern about the negative effects of pesticides on human health and the surrounding environment) in order to protect strawberry plants from said pathogen.
In view of the foregoing, claims 21-36 are rejected under 35 U.S.C. 103 as being prima facie obvious before the effective filing date.
Conclusion
No claim are allowed.
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/D.T.Y./Examiner, Art Unit 1635
/RAM R SHUKLA/Supervisory Patent Examiner, Art Unit 1635
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