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 .
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 11/3/25 has been entered.
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 62-69 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.
Claims 62 and 65 depend from a cancelled claim. The remainder of the claims are rejected because they depend from claim 62 or 65. For purposes of the instant examination, claims 62 and 65 are interpreted as they depend from claim 59.
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 59, 62-69, and 71-73 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.
The claims are directed to a composition comprising a dsRNA of any length (i.e. 1000 nucleotides) that comprises a smaller fragment sequence that is complementary or identical to at least 21 contiguous nucleotides of a Varroa Destructor mRNA.
The specification does not adequately describe the structure required for the function. The specification discloses: [00118] In an aspect, a nucleic acid molecule provided herein comprises between 20 and 5000 nucleotides, between 20 and 2500 nucleotides, between 20 and 1000 nucleotides, between 20 and 900 nucleotides, between 20 and 800 nucleotides, between 20 and 700 nucleotides, between 20 and 600 nucleotides, between 20 and 500 nucleotides, between 20 and 400 nucleotides, between 20 and 300 nucleotides, between 20 and 200 nucleotides, between 20 and 150 nucleotides, between 20 and 100 nucleotides, between 20 and 75 nucleotides, between 20 and 50 nucleotides, between 20 and 40 nucleotides, between 20 and 30 nucleotides, between 20 and 25 nucleotides, between 20 and 28 nucleotides, between 50 and 500 nucleotides, or between 100 and 1000 nucleotides in length.
However, the specification does not describe even a single species of dsRNA that is 5000 nucleotides in length, for example, that is complementary or identical to at least 21 contiguous nucleotides of a Varroa Destructor mRNA and has the required function.
The specification discloses three dsRNA trigger nucleic acid molecules (SEQ ID NOs: 1-3, Table 6) that each over 300 nucleotides in length, which is not commensurate in scope or representative of any dsRNA of any length (i.e. 5000 nt as disclosed in the specification) that is complementary or identical to as little as 21 contiguous nucleotides of a Varroa Destructor mRNA and has the required function.
The structure recited encompasses a large genus of dsRNAs that would not likely have the function of inhibiting the expression of any specific target gene. Additionally, without further description of the structure required for the function, one would not be able to recognize that applicant was in possession of the entire genus of dsRNA molecules within the instantly recited composition as claimed.
The MPEP states that for a generic claim, the genus can be adequately described if the disclosure presents a sufficient number of representative species that encompass the genus. See MPEP § 2163. If the genus has a substantial variance, the disclosure must describe a sufficient variety of species to reflect the variation within that genus. See MPEP § 2163. Although the MPEP does not define what constitute a sufficient number of representative species, the courts have indicated what do not constitute a representative number of species to adequately describe a broad genus. In Gostelli, the courts determined that the disclosure of two chemical compounds within a subgenus did not describe that subgenus. In re Gostelli, 872, F.2d at 1012, 10 USPQ2d at 1618. Additionally, in Carnegie Mellon University v. Hoffman-La Roche Inc., Nos. 07-1266, -1267 (Fed. Cir. Sept. 8, 2008), the Federal Circuit affirmed that a claim to a genus described in functional terms was not supported by the specification’s disclosure of species that were not representative of the entire genus. Furthermore, for a broad generic claim, the specification must provide adequate written description to identify the genus of the claim. In Regents of the University of California v. Eli Lilly & Co. the court stated:
"A written description of an invention involving a chemical genus, like a description of a chemical species, 'requires a precise definition, such as by structure, formula, [or] chemical name,' of the claimed subject matter sufficient to distinguish it from other materials." Fiers, 984 F.2d at 1171, 25 USPQ2d 1601; In re Smythe, 480 F.2d 1376, 1383, 178 USPQ 279, 284985 (CCPA 1973) ("In other cases, particularly but not necessarily, chemical cases, where there is unpredictability in performance of certain species or subcombinations other than those specifically enumerated, one skilled in the art may be found not to have been placed in possession of a genus ...") Regents of the University of California v. Eli Lilly & Co., 43 USPQ2d 1398.
The Guidelines for Examination of Patent Applications under the 35 USC § 112, first paragraph, “Written Description” Requirement”, published at Federal Register, Vol. 66, No. 4, pp. 1099-1111 outline the method of analysis of claims to determine whether adequate written description is present. The first step is to determine what the claim as a whole covers, i.e., discussion of the full scope of the claim. Second, the application should be fully reviewed to understand how applicant provides support for the claimed invention including each element and/or step, i.e., compare the scope of the claim with the scope of the description. Third, determine whether the applicant was in possession of the claimed invention as a whole at the time of filing.
To achieve the desired function, it appears that the structure is required to be of a shorter length than the claimed genus which has no length limitation. With respect to siRNAs, as single species of dsRNA that are generally complementary to 21 nucleotides, Elbashir et al. (The EMBO Journal, Vol. 20, No. 23, pages 6877-6888, 2001) teaches that duplexes of 21-23 nt RNAs are the sequence specific mediators of RNAi and that even single mismatches between the siRNA duplex and the target mRNA abolish interference (abstract and page 6888).
The claims encompass very long dsRNA, for example, that can trigger RNAi. Such dsRNA with 21 contiguous nucleotides that are either complementary or identical to the target would not likely result in the required function. For example, Parrish et al. (Molecular Cell, Vol. 6, 1077–1087, November, 2000) teach that sequences of 1000 bp trigger RNAi (page 1078).
Therefore, the specification does not adequately describe the genus of dsRNA molecules that are recited within the instant composition.
Thus, having analyzed the claims with regard to the Written Description guidelines, it is clear that the specification does not disclose a representative number of species for RNAi agents within the instant enormous genus that are inhibitory of the target as claimed. Thus, one skilled in the art would be led to conclude that Applicant was not in possession of the claimed invention at the time the application was filed.
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 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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 59, 62-69, and 71-73 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ben-Chanoch et al. (US 2012/0157512 A1) and Moran et al. (US 2019/0015528 A1), in view of Ward et al. (US 10,190,118 B2), Cohen et al. (US 8,025,552 B2), Ingberg et al. (US 9,540,642 B2), and Stanojevic et al. (Bulgarian Journal of Agricultural Science, 15 (No 4) 2009, 307-311).
Ben-Chanoch et al. teach: [0017] According to an aspect of some embodiments of the present invention there is provided a nucleic acid construct comprising a polynucleotide expressing a nucleic acid agent downregulating expression of a gene product of an insect pathogen, the polynucleotide operably linked to a cis-regulatory element operable in plants.
Ben-Chanoch et al. teach: [0018] According to an aspect of some embodiments of the present invention there is provided a cell comprising a nucleic acid construct comprising a polynucleotide expressing a nucleic acid agent downregulating expression of a gene product of an insect pathogen, the polynucleotide operably linked to a cis-regulatory element operable in plants.
Ben-Chanoch et al. teach: [0019] According to some embodiments of the invention, the cell is a plant cell.
Ben-Chanoch et al. teach: [0020] According to some embodiments of the invention, the cell is an Agrobacterium.
Ben-Chanoch et al. teach: [0021] According to an aspect of some embodiments of the present invention there is provided a plant expressing a nucleic acid construct comprising a polynucleotide expressing a nucleic acid agent downregulating expression of a gene product of an insect pathogen, the polynucleotide operably linked to a cis-regulatory element operable in plants.
Ben-Chanoch et al. teach: [0080] As used herein, the phrase "Varroa destructor mite" refers to the external parasitic mite that attacks honey bees Apis cerana and Apis mellifera. The mite may be at an adult stage, feeding off the bee, or at a larval stage, inside the honey bee brood cell (instant claim 59).
Ben-Chanoch et al. teach: [0113] In specific embodiments, suitable insect pathogen specific siRNA can be an IAPV, Nosema or Varroa specific siRNA, corresponding to IAPV sequences SEQ ID NOs: 256752, 256761, 256762 and 311552, Nosema cerana sequences SEQ ID NOs: 55-59, and Varroa destructor sequences SEQ ID NOS: 253194, 254090 and 259289 (instant claim 59).
Ben-Chanoch et al. teach: [0152] It is recognized that the polynucleotides comprising sequences encoding the nucleic acid agent can be used to transform organisms to provide for host organism production of these components. Such host organisms include baculoviruses, bacteria, and the like. In this manner, the combination of polynucleotides encoding the nucleic acid agent may be introduced via a suitable vector into a microbial host, and said host applied to the environment, or to plants or animals. The term "introduced" in the context of inserting a nucleic acid into a cell, means "transfection" or "transformation" or "transduction" and includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be stably incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
Ben-Chanoch et al. teach: Methods and compositions for transforming plants to express polynucleotides capable of gene silencing gene expression in pathogens of beneficial insects such as IAPV, Nosema species and Varroa mites, and methods for using the transgenic plants for reducing infection and susceptibility of bees to Colony Collapse Disorder are provided (abstract) (instant claim 59).
Ben-Chanoch et al. recite:
1. A nucleic acid construct comprising a polynucleotide expressing a nucleic acid agent downregulating expression of a gene product of an insect pathogen, said polynucleotide operably linked to a cis-regulatory element operable in plants.
2. The nucleic acid construct of claim 1, wherein said nucleic acid agent is selected from the group consisting of a dsRNA, an antisense RNA and a ribozyme.
3. The nucleic acid construct of claim 2, wherein said dsRNA is selected from the group consisting of siRNA, shRNA and miRNA (instant claim 59).
4. The nucleic acid construct of claim 1, said cis regulatory element being active in floral nectary tissues, extrafloral nectarines and/or pollen.
5. The nucleic acid construct of claim 1, wherein said nucleic acid agent is greater than 15 base pairs in length.
6. The nucleic acid construct of claim 5, wherein said nucleic acid agent is 19 to 25 base pairs in length (instant claim 59).
7. The nucleic acid construct of claim 5, wherein said nucleic acid agent is greater than 30 base pairs in length.
8. The nucleic acid construct of claim 1, wherein said insect is a bee (instant claim 59).
9. The nucleic acid construct of claim 1, wherein said insect pathogen is selected from the group consisting of a virus, a bacteria, a parasitic protozoan, a fungus a nematode and a mite.
10. The nucleic acid construct of claim 1, wherein said insect pathogen is a virus.
19. The nucleic acid construct of claim 1, wherein said insect is a bee and said pathogen is a Varroa destructor mite (instant claim 59).
20. The nucleic acid construct of claim 19, wherein said gene product is an Varroa specific mRNA encoding a polypeptide selected from the group consisting of NADH dehydrogenase subunit 2, ATP synthetase subunit 8, ATP synthetase subunit 6, sodium channel and cytochrome oxydase subunit I.
21. The nucleic acid construct of claim 1, wherein said insect is a bee and said nucleic acid construct comprises at least two nucleic acid agents, each of said nucleic acid agents downregulating expression of at least one gene product of at least one bee pathogen (instant claim 59).
31. A method of reducing the susceptibility of an insect to a disease caused by an insect pathogen, the method comprising providing to the insects plants expressing the nucleic acid construct of any of claims 1 to 24, such that said insect feeds from said plants and ingests said nucleic acid agent, thereby reducing the susceptibility of said insect to said pathogen.
32. The method of claim 31, wherein said insect is a pollinating insect or a herbivorous insect.
33. A method for reducing the susceptibility of a bee to a disease caused by an insect pathogen comprising providing the bee with a plant expressing the nucleotide construct of any of claims 11 to 20 such that said bee feeds from said plants and ingests said nucleic acid agent, thereby reducing the susceptibility of said bee to said pathogen.
Therefore, it was known in the art to treat or prevent a Varroa destructor infestation of a beehive or colony via providing to the beehive or bee colony a composition comprising a siRNA targeted to a Varroa destructor mRNA (instant claim 59).
Ben-Chanoch et al. do not teach incorporation of a carbohydrate and two or more food grade preservatives.
Moran et al. teach: Provided herein are genetically engineered bacteria that are native to a host insect microbiome. Further provided are methods of inducing RNA interference in an insect, such as a bee, by administering the genetically engineered bacteria (abstract).
Moran et al. teach: [0005] RNA interference (RNAi) is a powerful technique to specifically downregulate gene expression in many eukaryotes by providing dsRNA with sequence identity to eukaryotic genes. RNAi has been shown to function in honey bees previously, and has been used to knock down bee genes to investigate their function and also improve bee health by lowering pathogen burden (e.g., viral and varroa mite). Injection or feeding of double stranded RNA (dsRNA) has been shown to trigger RNAi in honey bees, leading to molecular cascades that can degrade invading viral RNA and kill pests (e.g., Varroa mites).
Moran et al. teach: [0006] In one embodiment, the invention relates to a microbial composition comprising one or more bacteria genetically engineered to express at least one heterologous nucleic acid, wherein the one or more bacteria are native to the microbiome of a host insect.
Moran et al. teach: [0007] In one embodiment, the host insect is selected from the group consisting of a honey bee and a bumble bee.
Moran et al. teach: [0016] In one embodiment, the composition is a bee-ingestible composition.
Moran et al. teach: [0017] In one embodiment, the bacteria are present as a live suspension. In one embodiment, the bacteria are present as a lyophilized powder. In one embodiment, the composition is in solid form. In one embodiment, the composition is in liquid form. In one embodiment, the composition comprises protein. In one embodiment, the composition comprises pollen. In one embodiment, the composition is a sucrose solution. In one embodiment, the composition is a corn syrup solution. In one embodiment, the composition comprises a carbohydrate or sugar supplement.
Moran et al. teach: [0018] In one embodiment, the invention relates to an insect comprising a microbial composition comprising one or more bacteria genetically engineered to express at least one heterologous nucleic acid, wherein the one or more bacteria are native to the microbiome of a host insect.
Moran et al. teach: [0021] In one embodiment, the invention relates to a method for downregulating expression of a target gene product, comprising administering an effective amount of a microbial composition comprising one or more bacteria genetically engineered to express at least one heterologous nucleic acid, wherein the one or more bacteria are native to the microbiome of a host insect to the host insect, wherein said bacteria express an inhibitor of said target gene product.
Moran et al. teach: [0022] In one embodiment, the target gene product is a gene from an organism selected from the group consisting of a pathogen, a parasite, a virus, a mite, Acute Bee Paralysis Virus (ABPV), Kashmir Bee Virus (KBV), Israeli Acute Paralysis Virus (IAPV), Nosema ceranae, Deformed Wing Virus, and Varroa destructor mite.
Moran et al. teach: [0023] In one embodiment, the insect is selected from the group consisting of a bee, a honey bee, a forager, a hive bee, a pupae, an adult bee, and a bee colony parasite.
Moran et al. teach: [0187] As detailed herein, bee feeding is common practice amongst bee-keepers, for providing both nutritional and other, for example, supplemental needs. Bees typically feed on honey and pollen, but have been known to ingest non-natural feeds as well. Bees can be fed various foodstuffs.
Moran et al. teach: [0070] In some cases, Colony Collapse Disorder (CCD) of honeybees can be due to Varroa mite infections. Varroa mites are suspected of acting as vectors for a number of honey bee pathogens, including Deformed Wing Virus (DWV), Kashmir Bee Virus (KBV), Acute Bee Paralysis Virus (ABPV) and Black Queen Cell Virus (BQCV), and may weaken the immune systems of their hosts, leaving them vulnerable to infections. Accordingly, certain embodiments of the present disclosure provide methods of reducing the susceptibility of honeybees to Colony Collapse Disorder (CCD). The inhibitory nucleic acid molecules (e.g., dsRNA) may be complementary to the mRNA of the target gene product. For example, the inhibitory nucleic acid molecule can be complementary to region of a mRNA comprising at least 10, 15, 20, 21, 22, 23, 24, or 25 contiguous nucleotides. In some aspects, the inhibitory nucleic acid molecule can be complementary to region of a mRNA comprising at least 50, 100, 150, 200, 300 400 or 500 contiguous nucleotides or essentially the entire mRNA. In some aspects a dsRNA comprise at least 10, 15, 20, 21, 22, 23, 24, or 25 complementary nucleotides. For example, the dsRNA produced by the engineered bacteria may comprise a sequence complementary to Varroa destructor mite mRNA of and capable of inducing degradation of the Varroa destructor-specific mRNA.
Therefore, it was known to incorporate a carbohydrate into compositions for delivery of bee-ingestible siRNAs targeting Varroa Destructor mites. It would have been obvious to incorporate the carbohydrate into the composition of Ben-Chanoch et al. as a matter of design choice for delivery. It is noted that the prior art is not required to teach applicants intended use language of improving the stability of the dsRNA, but rather is required to teach each structural requirement of the composition. However, dsRNA would be expected to have improved stability when incorporated into a composition with preservatives as compared to naked dsRNA.
It would have been obvious to deliver the dsRNA of Ben-Chanoch et al. from a composition composing a carbohydrate instead of delivery from a plant as a matter of design choice, because it was known to deliver siRNAs to bees from compositions with carbohydrates, as evidenced by Moran et al. Both Moran et al. and Ben-Chanoch et al. are evidence of methods of delivering dsRNA targeting Varroa destructor mRNA to bees.
Since it was known to incorporate various carbohydrates and foodstuffs, it would have been obvious to incorporate a food grade preservative as a matter of design choice with the expectation of preserving the composition.
Moran et al. teach: [0191] Also provided is a method for reducing the susceptibility of a bee to a disease caused by pathogens, the method effected by feeding the bee on an effective amount of a nucleic acid or nucleic acid construct comprising a nucleic acid agent downregulating expression of a polypeptide of the bee pathogen and/or causing cleavage and/or degradation of a bee pathogen RNA. Methods for reducing the susceptibility of a bee colony or bee-hive to bee pathogens by feeding oligonucleotides and/or polynucleotides are envisaged. Thus, in some embodiments, the present invention can be used to benefit any numbers of bees, from a few in the hive, to the entire bee population within a hive and its surrounding area. It will be appreciated, that in addition to feeding of oligonucleotides and/or polynucleotides for reduction of the bee pathogen infection and infestation, enforcement of proper sanitation (for example, refraining from reuse of infested hives) can augment the effectiveness of treatment and prevention of infections.
Ben-Chanoch et al. and Moran et al. are each evidence of delivery of a composition comprising a dsRNA targeting Varroa destructor mRNA, wherein Moran et al. offer motivation to incorporate a carbohydrate. The references do not teach incorporation of two or more food grade preservatives.
However, it would have been obvious to incorporate sodium benzoate and potassium sorbate because Stanojevic et al. teach that these agents are preservatives and have antimicrobial effects and act synergistically on food-spoiling bacteria and fungi and therefore have potential use in the food industry (abstract).
Therefore, it would have been obvious to incorporate these agents into a solution that is intended to feed an organism, including the bee feeding solution of Ben-Chanoch et al. and Moran et al. with a reasonable expectation of the benefits taught by Stanojevic et al.
Additionally, Ward et al. teach: Provided herein are methods and compositions for modulating gene expression in insects by administering a composition comprising an RNA effector molecule and a delivery agent. Methods are provided for controlling pest populations by inhibiting insect growth, development, survival, reproduction and/or viability. Also provided herein are methods for treating or preventing disease in an insect caused by a pathogen or by external factors (e.g., pollution, environment, stress, weather, etc.) (abstract).
Ward et al. teach: Pests including insects, arachnids, crustaceans, fungi, bacteria, viruses, nematodes, flatworms, roundworms, pinworms, hookworms, tapeworms, trypanosomes, schistosomes, botflies, fleas, ticks, mites, lice and the like are pervasive in the human environment, and a multitude of means have been utilized for attempting to control infestations by these pests. Compositions for controlling infestations by microscopic pests such as bacteria, fungi, and viruses have been provided in the form of antibiotic compositions, antiviral compositions, and antifungal compositions. Compositions for controlling infestations by larger pests such as nematodes, flatworm, roundworms, pinworms, heartworms, tapeworms, trypanosomes, schistosomes, and the like have typically been in the form of chemical compositions which can either be applied to the surfaces of substrates on which pests are known to infest, or to be ingested by an infested animal in the form of pellets, powders, tablets, pastes, or capsules and the like.
Ward et al. teach: The iRNAs included in the compositions featured herein encompass a dsRNA having an RNA strand (the antisense strand) having a region that is typically 9-36 nucleotides in length, e.g., 30 nucleotides or less, generally 19-24 nucleotides in length, that is substantially complementary to at least part of an mRNA transcript of an insect pest or an insect pathogen.
Ward et al. teach: Recently, horizontal transmission of honeybee viruses has been documented in bee colonies, for example, transmission of deformed wing virus (DWV) and Kashmir Bee Virus (KBV) by the parasitic mite Varroa destructor, as well as some evidence of virus in honeybee eggs and young larvae, life stages not parasitized by Varroa mites. Vertical transmission of multiple viruses from mother queens to their offspring in honeybees has also been recently demonstrated, as well as viruses in feces of queens, suggesting a role for feeding in virus transmission. Moreover, honeybee viruses have been detected in tissues of the gut, suggesting that viruses could be ingested by queens from contaminated foods and passed into the digestive tract, which then acts as a major reservoir for viral replication. Indeed, viruses might penetrate the gut wall and move into the insect hemocoel, spreading infections to other tissues.
Ward et al. teach: The oligonucleotide can be linked to a food component of the insect, such as a food component for a mammalian pathogenic insect and/or agricultural pest for ease of delivery and/or in order to increase uptake of the oligonucleotide by the insect. Ingestion by an insect permits delivery of the insect control agents and results in modulation of a target gene in the host. Methods for oral introduction include, for example, directly mixing an oligonucleotide with the insect's food, spraying the oligonucleotide in the insect's habitat or field, as well as engineered approaches in which a species that is used as food is engineered to express an oligonucleotide, then fed to the insect to be affected.
Ward et al. teach: According to an aspect of some embodiments of the present invention there is provided a method for increasing the tolerance of a bee to a disease caused by a pathogen comprising feeding the bee an effective amount of the oligonucleotide comprising a nucleic acid sequence down-regulating expression of a gene product of a bee pathogen or a nucleic acid construct comprising the oligonucleotide, thereby increasing the tolerance of the bee to the pathogen.
Ward et al. teach: According to some embodiments of the invention the feeding comprises providing a liquid bee-ingestible composition or a solid bee-ingestible composition.
Ward et al. teach incorporation of carbohydrates. Ward et al. teach: Carbohydrate based targeting ligands include, but are not limited to, D-galactose, multivalent galactose, N-acetyl-D-galactose (GalNAc), multivalent GalNAc, e.g. GalNAC2 and GalNAc3; D-mannose, multivalent mannose, multivalent lactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent fucose, glycosylated polyaminoacids and lectins.
Ward et al. teach incorporation of corn syrup, sugar syrup, sucrose, as well as food-based ingredients as trimethylamine, putrescine, bacterial or yeast volatiles or metabolites, ammonium acetate, ammonium carbonate or other ammonia-emitting compounds as well. Therefore, it would have been obvious in view of Ward et al. to incorporate more than one carbohydrate (instant claims 59, 62, and 63).
Ward et al. teach incorporation of preservatives. Ward et al. teach: Preservatives include antimicrobial, anti-oxidants, chelating agents and inert gases. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like.
Ward et al. teach: A large variety of non-emulsifying materials is also included in emulsion formulations and contributes to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants.
Therefore, Ward et al. offer motivation for more than one preservative.
Ward et al. teach that antimicrobials, such as potassium sorbate, nitrates, nitrites, and propylene oxide, protect the bioactive agents from microbial destruction can be added in amounts from 0.1% to about 2% by weight (instant claims 59 and 68).
Ward et al. teach incorporation of citric acid or benzoic acid to reduce degradation. Ward et al. teach incorporation of vitamin A, B, B12, C, or E (instant claims 65 and 66).
Ward et al. teach that additives added to the formulation are typically added in amounts from about .001% to about 10% by weight and teaches incorporation of a carrier lipid at higher percentages. Determination of percentage of each component is considered to be a matter of routine optimization of design parameters and is routine to one of ordinary skill in the art (instant claims 59, 67-69, 71, and 72).
Given the teachings of Ward et al., it would have been obvious to provide a composition comprising a dsRNA targeted to Varroa destructor, at least one carbohydrate, and one or more food grade preservatives to honeybees with a reasonable expectation of success. Ward et al. offers clear motivation for targeting Varroa destructor and teaches incorporation of more than one type of carbohydrate and food grade preservative.
Although Ward et al. does not teach sodium benzoate specifically, Ward et al. teaches incorporation of potassium sorbate and other food grade preservatives and sodium benzoate was a known food grade preservative, as taught by Stanojevic et al.
It is considered a matter of routine optimization to determine the weight % for each component and well within the technical grasp of one of ordinary skill in the art.
When formulating a composition for controlling an arthropod parasite in honeybees, it would have been obvious to incorporate vitamin C because Ward et al. teaches incorporation of vitamin C and Cohen et al. teaches that Vitamin C is included as a primary nutrient source in compositions for diet formulations for honeybees. Cohen et al. teach that the formulations of the invention provide a complex mixture of nutrients in amounts and proportions effective to support growth and development of honey bees. Cohen et al. teach incorporation of sucrose as a sugar source and potassium sorbate as an anti-fungal and/or anti-microbial agent. Cohen et al. teaches that other anti-fungal and/or antimicrobial agents are known in the art, which would include sodium benzoate as taught by Stanojevic et al. Cohen et al. teaches incorporation of each ingredient at varying percentages for normal bee feeding (claims) (i.e. claim 1 recites 0-1.5% antifungal agent, claim 5 recites 65-85 % sugar syrup, claim 8 specifies sucrose) (instant claims 59, 67, and 68).
Ingberg et al. teach: This application provides and discloses anti-parasitic, anti-pest or insecticidal nucleic acid molecules and their calmodulin target genes for the control of arthropod parasites and pests. This application further provides methods and compositions for the control and treatment of parasites and pests in Apis mellifera (honey bee) hives (abstract).
Ingberg et al. recites:
A bee-ingestible, bee-absorbable, mite-ingestible, or mite-absorbable composition comprising an excipient and a nucleic acid molecule having a sequence that is at least 96% identical or complementary to at least 21 contiguous nucleotides of SEQ ID NO: 3.
SEQ ID NO: 3 is a Varroa destructor target sequence.
2. The composition of claim 1, wherein said nucleic acid sequence is a dsRNA.
3. The composition of claim 1, wherein said excipient is selected from the group consisting of protein, pollen, carbohydrate, polymer, liquid solvent, sugar syrup, sugar solid, and semi-solid feed.
4. The composition of claim 2, wherein said dsRNA is an siRNA.
5. The composition of claim 1, wherein said nucleic acid sequence comprises at least 23 contiguous nucleotides of SEQ ID NO: 3.
6. The composition of claim 2, wherein said dsRNA sequence comprises at least 23 contiguous nucleotides of SEQ ID NO: 3.
7. A nucleic acid construct comprising a nucleic acid that encodes a sequence that is at least 95% identical or complementary to SEQ ID NO: 3 operably linked to a promoter sequence functional in a host cell and capable of producing a dsRNA when introduced into said host cell.
8. The nucleic acid construct of claim 7, further comprising at least one regulatory element selected from the group consisting of translation leader sequences, introns, enhancers, stem-loop structures, repressor binding sequences, termination sequences, pausing sequences, and polyadenylation recognition sequences.
9. The nucleic acid construct of claim 7, wherein said host cell is selected from the group consisting of a bacteria cell and a yeast cell.
10. A method of providing a composition to a honeybee, comprising providing the bee an effective amount of the composition of claim 1, whereby the nucleic acid is present in honeybee tissue.
11. The method of claim 10, wherein said calmodulin gene sequence is a Varroa destructor calmodulin gene sequence.
12. The method of claim 10, wherein said honeybee is a forager or a hive bee.
13. The method of claim 11, wherein said honeybee is a bee of a colony and said providing reduces the susceptibility of said bee colony to Varroa destructor, reduces the parasitation of said bee colony by Varroa destructor, or reduce the parasite load of said bee colony by Varroa destructor.
14. The method of claim 11, wherein said providing reduces the susceptibility of said honeybee to Varroa destructor, reduces the parasitation of said honeybee by Varroa destructor, or reduce the parasite load of said honeybee by Varroa destructor.
15. The composition of claim 1, further comprising one or more nucleic acid molecules having a second nucleic acid sequence that is essentially identical or essentially complementary to a different region of a calmodulin gene sequence.
Inberg et al. is evidence that siRNAs targeting instantly disclosed SEQ ID NO: 3 is a Varroa destructor target sequence for a bee-ingestible composition comprising a carbohydrate.
Response to Arguments
Applicant argues that each of the references do not teach the complete combination of the dsRNA, a carbohydrate, and a food grade preservative. However, the instant rejection is a rejection under 35 USC 103 rather than 35 USC 102 and therefore each reference is not relied upon for teaching the entire invention. Therefore, the recited improvement of stability of the preamble does not impart a specific structural limitation. The structural limitations of the claims are considered obvious in view of the cited references.
Importantly, the claims are not limited to any specific carbohydrate and any specific combination of preservative that have demonstrated an unexpected result, but are rather directed to any carbohydrate and any of the food grade preservatives of claim 59, which is an enormous genus of possible combinations of agents.
Moran et al. teaches incorporation of a carbohydrate into a composition for delivery of bee-ingestible nucleic acids. Incorporation of a carbohydrate into bee feeding nucleic acid compositions is not novel.
Applicant argues that the Ben-Chanoch and Moran references focus on
genetic engineering of plants or bacteria. Ben-Chanoch et al. and Moran et al. are evidence that it was known to feed dsRNA to bees to target Varroa Destructor mRNA. Each reference utilizes a different composition, but are each evidence that it was obvious to deliver the dsRNA to the honeybee to target Varroa Destructor mRNA.
Feeding dsRNA to bees was known in the prior art. Ben-Chanoch et al. teach that application of RNA interference technology for insects that are plant pests and other plant pests has been suggested. Ben-Chanoch et al. teach that moderate RNAi-type silencing of insect genes by feeding has been demonstrated and that dsRNA absorbance via honey has also been demonstrated [0011]. Ben-Chanoch et al. teach that direct application of dsRNA to the commercially grown honeybees can be facilitated by providing it in bee feed, but there is a need for improved methods of providing the beneficial insects with protection from pathogenic disease agents [0016].
Ben-Chanoch et al. teach a cell comprising a nucleic acid construct comprising a polynucleotide expressing a nucleic acid agent downregulating expression of a gene product of an insect pathogen, the polynucleotide operably linked to a cis-regulatory element operable in plants [0018]. Ben-Chanoch et al. focus on feeding dsRNA to insects via the plant and teaches dsRNA feeding to bees [0026]-[0028].
Ben-Chanoch et al. teach: According to an aspect of some embodiments of the present invention there is provided a method for reducing the susceptibility of an bee to a disease caused by an insect pathogen comprising providing the bee with a plant expressing a nucleic acid construct comprising a polynucleotide expressing a nucleic acid agent down-regulating expression of a gene product of a IAPV, Nosema and/or Varroa bee pathogen, the polynucleotide operably linked to a cis-regulatory element operable in plants, such that the bee feeds from the plants and ingests the nucleic acid agent, thereby reducing the susceptibility of the bee to the pathogen [0028].
Moran et al. teaches incorporation of a carbohydrate into a composition for delivery of bee-ingestible nucleic acids. Incorporation of a carbohydrate into bee feeding nucleic acid compositions is not novel.
Applicant argues that claim 33 of Ben-Chanoch teaches providing bees with plants expressing nucleotide constructs. In either instance, one skilled in the art would understand that any use of potential food compositions would be irrelevant to the disclosure of Ben-Chanoch. Ben-Chanoch is evidence that it was known to deliver dsRNA to the honeybee to target Varroa Destructor mRNA. It certainly is not novel to formulate the dsRNA into a composition. Ben-Chanoch teaches that dsRNA can be incorporated into bee feed and teaches feeding plants to the bee containing the dsRNA.
Applicant argues that Moran teaches the delivery of bacteria engineered to express nucleic acids and discusses combining such bacteria with a carbohydrate. Applicant respectfully disagrees that incorporating a carbohydrate into the composition of Ben-Chanoch is a matter of design choice for delivery. As already noted, the delivery disclosed in Ben- Chanoch is the expression by plants. There actually is no "composition" contemplated by Ben-Chanoch into which it would even be appropriate to add a carbohydrate. Contrary to applicants argument, Ben-Chanoch clearly teaches methods and compositions for transforming plants to express polynucleotides capable of gene silencing gene expression in Varroa mites (see abstract).
Additionally, regardless of incorporation of the carbohydrate into the composition of Ben-Chanoch, it was known in the art to formulate compositions for dsRNA delivery via feeding honeybees to target Varroa mites, as evidenced by Moran. The references are cited to demonstrate that it was known to obvious to deliver the dsRNA to the honeybee to target Varroa Destructor mRNA.
Applicant argues that the Ward reference is primarily directed to treating an insect disease that involves administering a dsRNA that includes specific nucleotide modifications for purposes of increasing molecular stabilization of the dsRNA. Though the general focus of stabilization of RNA is through nucleotide modifications, Ward does contemplate delivery of dsRNA via an insect food source. Ward provides a passing mention of certain additives that one skilled in the art might include in an insect food source, but fails to provide any disclosure of a certain combination of preservatives that possess particular RNA stabilizing effects. Ward is relied upon in its entirety and applicant attempting to dismiss the teachings of Ward is not convincing. Ward is not requir3ed to teach applicants intended use language of improving the stability of dsRNA because the instant claims are composition claims, not method claims. Ward offers clear motivation to incorporate dsRNA into an insect food source and offers motivation to combine preservatives.
Applicant argues that Ward does not teach a composition comprising two or more food grade preservatives that are selected from the group consisting of a sorbate, a sorbate salt, potassium sorbate, sorbic acid, a benzoate, a benzoate salt, sodium benzoate, benzoic acid, citric acid or a tocopherol. Ward is not relied upon for this.
Applicant argues that none of the prior art suggests combining two or more food grade preservatives selected from the group in claim 1 in combination with dsRNA, much less with a carbohydrate source that comprises at least 60% b/w of the composition. It is the combination of references that are relied upon for teaching the entirety of the instant claims (see the rejection, above).
Applicant argues that in addition, the data presented in the present application teaches that the combination of a benzoate salt and a potassium salt with the dsRNA molecule unexpectedly increased the stability and steadfastness of the dsRNA molecule. Contrary to applicant’s argument, the specification does not present data for combining any combination of any sorbate, any sorbate salt, potassium sorbate, sorbic acid, any benzoate, or any benzoate salt; as well as any possible carbohydrate at a concentration of at least 60%, by weight, and an unexpected increase in the stability and steadfastness of any dsRNA molecule of any length that has at least 21 nucleotides identical or complementary to a Varroa destructor mRNA.
Applicant argues that Example 1 discusses the increased stability of dsRNA by using a formulation with two food grade preservatives (specifically sodium benzoate and potassium sorbate). Tables 7 and 8 show that dsRNA combined with a carbohydrate and both of sodium benzoate and potassium sorbate was much more stable and degraded significantly slower than dsRNA combined with carbohydrate alone when exposed to conditions meant to simulate the conditions of a bee hive. This example is not commensurate in scope with any dsRNA molecule of any length that has at least 21 nucleotides identical or complementary to a Varroa destructor mRNA and as any possible carbohydrate at a concentration of at least 60%, by weight.
Even with regards to siRNAs and specifically potassium sorbate and sodium benzoate, which is not identical to the instant claim scope, it was known to deliver dsRNA to honeybees to target Varroa Destructor mRNA; it was known to deliver siRNA to honeybees via feeding; and it would have been obvious to incorporate sodium benzoate and potassium sorbate because Stanojevic et al. teach that these agents are preservatives and have antimicrobial effects and act synergistically on food-spoiling bacteria and fungi and therefore have potential use in the food industry (abstract).
Therefore, it would have been obvious to incorporate these agents into a solution that is intended to feed an organism, including the bee feeding solution of Ben-Chanoch et al. and Moran et al. with a reasonable expectation of the benefits taught by Stanojevic et al.
Applicant argues that Example 2 addresses the use of a single food grade preservative (sodium benzoate) without the addition of a second food grade preservative. As discussed in Example 2 "the addition of a second food-grade preservative ... in Example 1 ... further improved the stability of dsRNAs over time" as compared to the formulations set forth in Example 2, Table 9 which contained only a
single food grade preservative. This dramatic difference in stabilizing dsRNA by using two or more food grade preservatives is nowhere contemplated by the cited art. The ability of a second food grade preservative to boost the stability of dsRNA is not an obvious design choice and not suggested in the prior art. Contrary to applicants argument, it was known in the art that these two agents are preservatives and have antimicrobial effects and act synergistically on food-spoiling bacteria and fungi and therefore have potential use in the food industry, as taught by Stanojevic et al.
Applicant argues that the Cohen reference describes certain compositions for feeding honeybees, but is silent concerning dsRNA, let alone addressing the challenges of exogenous delivery of dsRNA to mites or other arthropod pests and the instability of such compositions in field conditions. There is no suggestion that the Cohen teachings could be modified to include dsRNA, but even if there were, there is no teaching to include a dsRNA as well as a food grade preservative to enhance stability of such hypothetical dsRNA addition. Cohen is not relied upon for teachings regarding dsRNA. It would have been obvious to incorporate Vitamin C because Cohen et al. teaches that Vitamin C is included as a primary nutrient source in compositions for diet formulations for honeybees. In a formulation of dsRNA targeting Varroa destructor mRNA and a carbohydrate to feed to honeybees, which is obvious in view of references other than Cohen, it would have been ob