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
This application 17/911,930 filed on 09/15/2022 is a national phase application under 35 U.S.C. § 371 that claims priority to International Application No. PCT/US21/22389 field on 03/15/2021, which claims priority to U.S. Provisional Patent Application Serial No. 62/989,725, field on 03/15/2020.
A certified copy of priority document for the International Application No. PCT/US21/022389 field on 03/15/2021 has been submitted of the record by Applicants on 09/15/2022.
It is noted that newly added claim 70 filed on 11/20/2025 is not supported by U.S. Provisional Patent Application Serial No. 62/989,725, field on 03/13/2020. Accordingly, the priority of claim 70 is 03/15/2021, the filing date of International Application No. PCT/US21/022389.
Claim status
Applicant’s response filed on 11/20/2025 is acknowledged. Claims 1, 5, 27 and 30 are amended. Claims 6-7, 10-12, 17-19, 25-26, 28-29, 31-55, and 57-69 are cancelled. Claim 70 is newly added. Claims 1-5, 8-9, 13-16, 20-24, 27, 30, 56, and 70 are pending.
Claim 27, 30 and 56 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 07/28/2025.
Claims 1-5, 8-9, 13-16, 20-24, and 70 are currently under examination.
Summary of this Final Office Action
Previous rejection of claims 1-3, 8-9, 13, and 22 under 35 U.S.C. 102(a)(1) as being anticipated by Shinmyo et al. (2004) (Shinmyo et al., piggyBac-mediated somatic transformation of the two-spotted cricket, Gryllus bimaculatus, Dev Growth Differ, 2004 Aug;46(4):343-9. doi: 10.1111/j.1440-169x.2004.0075 1.x.) is withdrawn as claims 1, 5, 27 and 30 are amended.
Previous rejection of under claims 1, 3-4, 14-16, and 20-23 under 35 U.S.C. 103 as being unpatentable over Shinmyo et al. (2004) (Shinmyo et al., piggyBac-mediated somatic transformation of the two-spotted cricket, Gryllus bimaculatus, Dev Growth Differ, 2004 Aug;46(4):343-9. doi: 10.1111/j.1440-169x.2004.0075 1.x.) in view of Uhlírová et al. (2002) (Uhlírová et al., Heat-inducible transgenic expression in the silkmoth Bombyx mori, Dev Genes Evol. 2002 Apr;212(3):145-51. doi: 10.1007/s00427-002-0221-8. Epub 2002 Mar 1.) is withdrawn as claims 1, 5, 27 and 30 are amended.
Previous rejection of under claims 1, 5, 7, and 24 under 35 U.S.C. 103 as being unpatentable over Shinmyo et al. (2004) (Shinmyo et al., piggyBac-mediated somatic transformation of the two-spotted cricket, Gryllus bimaculatus, Dev Growth Differ, 2004 Aug;46(4):343-9. doi: 10.1111/j.1440-169x.2004.0075 1.x.) in view of Lee et al. (2015) (Lee et al., Production of antibacterial Bombyx mori cecropin A in mealworm-pathogenic Beauveria bassiana ERL1170, J Ind Microbiol Biotechnol. 2015 Jan;42(1):151-6. doi: 10.1007/s10295-014-1551-z. Epub 2014 Nov 28.), is withdrawn as claims 1, 5, 27 and 30 are amended.
New ground of rejections of under 35 U.S.C. 112(b) and under 35 U.S.C. 103 necessitated by claim amendment filed on 11/20/2025 are documented in this Final office action.
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.
Claim 70 is 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.
A broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). In the present instance, claim 70 recites the broad recitation “at least 2 mg”, and the claim also recites “at least 45 mg” which is the narrower statement of the range/limitation. The claim(s) are considered indefinite because there is a question or doubt as to whether the feature introduced by such narrower language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims.
It is noted that claim 70 as written encompasses “at least 2 mg” of any recombinant protein expressed from any number of transformed insects recited in claim 1.
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.
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.
Claims 1-3, 5, 8-9, 13, and 22 are rejected under 35 U.S.C. 103 as being unpatentable over Shinmyo et al. (2004) (Shinmyo et al., piggyBac-mediated somatic transformation of the two-spotted cricket, Gryllus bimaculatus, Dev Growth Differ, 2004 Aug;46(4):343-9. doi: 10.1111/j.1440-169x.2004.0075 1.x.) in view of Eckermann et al. (2018) (Eckermann et al., Hyperactive piggyBac transposase improves transformation efficiency in diverse insect species
Insect Biochem Mol Biol., 2018 Jul:98:16-24. doi: 10.1016/j.ibmb.2018.04.001. Epub 2018 Apr 10) and Zhan et al. (2020) (Zhan et al., Genomic landscape and genetic manipulation of the black soldier fly Hermetia illucens, a natural waste recycler, Cell Res. 2020 Jan;30(1):50-60. doi: 10.1038/s41422-019-0252-6. Epub 2019 Nov 25; cited as NPL CC in the IDS filed on 09/15/2022).
Regarding claims 1-2, 8-9, and 22, Shinmyo et al. (2004) teaches that “Transgenic insects have been artificially produced to study functions of interesting developmental genes, using insect transposons such as piggyBac. In the case of the cricket, however, transgenic animals have not yet been successfully artificially produced. In the present study, we examined whether the piggyBac transposon functions as a tool for gene delivery in embryos of Gryllus bimaculatus. We used either a piggyBac helper plasmid or a helper RNA synthesized in vitro as a transposase source. An excision assay revealed that the helper RNA was more effective in early Gryllus eggs (which reads on germline recited in instant claim 3) to transpose a marker gene of eGFP than the helper plasmid containing the piggyBac transposase gene driven by the G. bimaculatus actin3/4 promoter. Further, only when the helper RNA was used, somatic transformation of the embryo with the eGFP gene was observed. These results suggest that the piggyBac system with the helper RNA may be effective for making transgenic crickets.” (See Abstract).
Regarding claim 3, the Gryllus eggs taught by Shinmyo et al. reads on the limitation “germline of the insect”.
Regarding claim 13, the limitation “the recombinant protein is an antigen” reads on eGFP taught by Shinmyo et al. because the limitation “an antigen” encompasses any other foreign substance which induces any immune response in the body of a subject with an active immune system.
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Fig. 1. Polymerase chain reaction (PCR) excision assay in Gryllus embryos. (A) Diagram of the PCR excision assay. When piggyBac functions in Gryllus eggs, excision events should
occur in the donor plasmid. pBacexfp and pBac-exrp primers were used to detect the excision, while act-fp and act-rp primers were used to examine if the donor plasmid was rescued. In the
case of pBGact-eGFP, the size of excision diagnostic band becomes 1060 bp (5.2 kbp without
excision). Arrows indicate the positions and directions of the primers used in PCR analyses. Arrowheads indicate inverted terminal repeats. (B) In the case of the helper plasmid (P), the
diagnostic band was amplified at 72 h, but not 12, 24 and 48 h after egg laying (hAEL). In the case of the helper RNA (R), the diagnostic band was amplified from 12 to 72 hAEL. The diagnostic bands were not detectable in the absence of the helper plasmid or helper RNA (H-). We obtained the same results in every experiment (n = 5).
Regarding claims 8 and 22, Shinmyo et al. (2004) further teaches that “The helper RNA may be efficiently translated in early developing Gryllus eggs. In order to know when and where helper piggyBac RNA is translated in developing Gryllus eggs, we used eGFP mRNA transcribed in vitro, instead of the helper RNA. We injected the eGFP mRNA into the vicinity of the female pronucleus. Fluorescence of eGFP was observed in 174 (68%) out of 256 embryos at 42 hAEL, whereas 45 (18%) embryos did not develop. Some fluorescence spots of eGFP were first observed in the eggs at 8 hAEL. At 10 hAEL, signal spots were observed in the posterior half of the egg (Fig. 2A). At 20 hAEL, the spots were distributed all over the egg and concentrated at the posterior pole in the egg (Fig. 2B). At 30 hAEL, the spots were aggregated in a belt-like shape at approximately 10–30% egg length from the posterior end where early embryo forms (Fig. 2C). (See right column, page 346).
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Fig. 2. Translation patterns of eGFP mRNA injected in the egg of G. bimaculatus eGFP mRNA was injected into the vicinity of the female pronucleus of the egg (A-C), or the vicinity of the posterior pole (D, E). (A-C), eGFP fluorescence at 10 (A), 20 (B) and 30 (C) hAEL. At 30 hAEL, the spots were aggregated in a belt-like shape (boxed) at approximately 10-30% egg length from the posterior end where the early embryo forms as shown in (F). (D, E) eGFP fluorescence at 10 (D) and 40 (E) hAEL. (F) A schematic illustration of the embryo (green) in the egg (ivory) at 40 hAEL. The upper and lower sides are dorsal (D) and ventral (V), respectively. The left and right sides are anterior (A) and posterior (P), respectively. Bars, 0.5 mm (A–E).
Shinmyo et al. (2004) does not explicitly teach genetic manipulation of genus of insect Hermetia recited in claim 1, and species of insect Hermetia illucens recited in claim 5.
Regarding genus of fly Hermetia recited in claim 1, and species of fly Hermetia illucens recited in claim 5 in the context of diverse transgenic insects (more specifically, fly) generated by piggyBac transposase, Eckermann et al. (2018) teaches that “Even in times of advanced site-specific genome editing tools, the improvement of DNA transposases is still on high demand in the field of transgenesis: especially in emerging model systems where evaluated integrase landing sites have not yet been created and more importantly in non-model organisms such as agricultural pests and disease vectors, in which reliable sequence information and genome annotations are still pending. In fact, random insertional mutagenesis is essential to identify new genomic locations that are not influenced by position effects and thus can serve as future stable transgene integration sites. In this respect, a hyperactive version of the most widely used piggyBac transposase (PBase) has been engineered. The hyperactive version (hyPBase) is currently available with the original insect codon-based coding sequence (ihyPBase) as well as in a mammalian codon-optimized (mhyPBase) version. Both facilitate significantly higher rates of transposition when expressed in mammalian in vitro and in vivo systems compared to the classical PBase at similar protein levels. Here we demonstrate that the usage of helper plasmids encoding the hyPBase - irrespective of the codon-usage - also strikingly increases the rate of successful germline transformation in the Mediterranean fruit fly (Medfly) Ceratitis capitata, the red flour beetle Tribolium castaneum, and the vinegar fly Drosophila melanogaster. hyPBase-encoding helpers are therefore highly suitable for the generation of transgenic strains of diverse insect orders. Depending on the species, we achieved up to 15-fold higher germline transformation rates compared to PBase and generated hard to obtain transgenic T. castaneum strains that express constructs affecting fitness and viability. Moreover, previously reported high sterility rates supposedly caused by hyPBase (iPB7), encoded by ihyPBase, could not be confirmed by our study. Therefore, we value hyPBase as an effective genetic engineering tool that we highly recommend for insect transgenesis (See Abstract).
Regarding genus of fly Hermetia recited in claim 1, and species of fly Hermetia illucens recited in claim 5, Zhan et al. (2020) teaches that “The black soldier fly (BSF), Hermetia illucens (Diptera: Stratiomyidae), is renowned for its bioconversion of organic waste into a sustainable source of animal feed. We report a high-quality genome of 1.1 Gb and a consensus set of 16,770 gene models for this beneficial species. Compared to those of other dipteran species, the BSF genome has undergone a substantial expansion in functional modules related to septic adaptation, including immune system factors, olfactory receptors, and cytochrome P450s. We further profiled midgut transcriptomes and associated microbiomes of BSF larvae fed with representative types of organic waste. We find that the pathways related to digestive system and fighting infection are commonly enriched and that Firmicutes bacteria dominate the microbial community in BSF across all diets. To extend its potential practical applications, we further developed an efficient CRISPR/Cas9-based gene editing approach and implemented this to yield flightless and enhanced feeding capacity phenotypes, both of which could expand BSF production capabilities. Our study provides valuable genomic and technical resources for optimizing BSF lines for industrialization” (See Abstract).
Zhan et al. further teaches that “Flies mainly feed during the larval stage. The efficiency of consuming organic waste would be improved if the larval stage of BSF could be stably extended. Metamorphosis in insects is controlled by a cascade of hormones and neuropeptides. By screening genes involved in this peptidergic signaling pathway, we focused on a gene encoding the prothoracicotropic hormone (PTTH), which contributes to molting and metamorphosis by initiating the signaling cascade that results in the biosynthesis and release of ecdysone. Knockout of Ptth in BSF dramatically delayed the pupation process of BSF larvae. The genome of BSF encodes a single copy of Ptth, with marginal conservation with the Drosophila ortholog except for the N-terminal end (Supplementary information, Fig. S9). We designed two sgRNAs, targeted to the second and fourth exons, to disrupt HiPtth substantially in vivo (Fig. 6a, b). Upon CRISPR/Cas9-mediated ablation of HiPtth, the average duration of the last larval instar increased from 4–5 days in controls to > 85 days in mutant larvae of any mosaic forms of disrupted HiPtth. We also found that both the body size (Fig. 6c) and weight (Fig. 6d) of the Ptth mutants were significantly larger than wild type (See right column, page 55).
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Fig. 6 Mutagenesis of Ptth leads to increased feeding capacity in BSF larvae. The CRISPR/Cas9 system was used to induce mutations at the HiPtth locus in H. illucens. a Schematic representation of the exon/intron boundaries of the HiPtth gene. Exons are shown as boxes; thin lines represent introns; numbers are fragment lengths in base pairs (bp). Target site (TS) locations are noted and PAM sequences are shown in red. b Sequences of the targeted region in the HiPtth locus in the mutants. The PAM sequence is in red. The numbers of nucleotides deleted in each line are indicated on the right. c Morphology of HiPtth mutants showing their greater size relative to wild type (WT) controls. d Average body weights of mutants and control (n = 30; mean values ± SEM)
A skilled artisan would have been motivated to incorporate the teachings of Eckermann et al. (2018) and Zhan et al. (2020) into the teachings of Shinmyo et al. (2004) because (i) Shinmyo et al. (2004) teaches “piggyBac-mediated somatic transformation of the two-spotted cricket, Gryllus bimaculatus” (See title); (ii) Eckermann et al. (2018) teaches “Hyperactive piggyBac transposase improves transformation efficiency in diverse insect species” (See Title), including germline transformation in the Mediterranean fruit fly (Medfly) Ceratitis capitata, the red flour beetle Tribolium castaneum, and the vinegar fly Drosophila melanogaster (See Abstract); and (iii) Zhan et al. (2020) teaches “Genomic landscape and genetic manipulation of the black soldier fly Hermetia illucens, a natural waste recycler (See Abstract).
There would have been a reasonable expectation of success to reach claimed transformed insect of instant claims 1-3, 5, 8-9, 13, and 22 because (i) Shinmyo et al. (2004) succeeded in making somatically transformed Gryllus embryos with the helper piggyBac RNA and the donor with eGFP (See left column, page 349); (ii) Eckermann et al. (2018) demonstrate that the usage of helper plasmids encoding the hyPBase - irrespective of the codon-usage – also strikingly increases the rate of successful germline transformation in the Mediterranean fruit fly (Medfly) Ceratitis capitata, the red flour beetle Tribolium castaneum, and the vinegar fly Drosophila melanogaster (See Abstract); and (iii) Zhan et al. (2020) developed a CRISPR/Cas9-based genome editing approach in BSF and implemented this technology to test the function of some modified the black soldier fly (BSF), Hermetia illucens genes in vivo (See right column, page 55).
Applicant’s arguments
Applicant submits that the corresponding present claims do not recite an insect of Gryllus
bimaculatus. Shinmyo does not teach a transformed insect of the genus Hermetia, as recited in the present claims. Accordingly, Applicant respectfully requests reconsideration and withdrawal of the rejection.
Response to Applicant’s arguments
Applicant’s arguments have been addressed in the new grounds of rejection under 35 U.S.C. 103 documented above.
Claims 4, 14-16, 20, 21, 23, and 70 are rejected under 35 U.S.C. 103 as being unpatentable over Shinmyo et al. (2004) (Shinmyo et al., piggyBac-mediated somatic transformation of the two-spotted cricket, Gryllus bimaculatus, Dev Growth Differ, 2004 Aug;46(4):343-9. doi: 10.1111/j.1440-169x.2004.0075 1.x.) in view of Eckermann et al. (2018) (Eckermann et al., Hyperactive piggyBac transposase improves transformation efficiency in diverse insect species Insect Biochem Mol Biol., 2018 Jul:98:16-24. doi: 10.1016/j.ibmb.2018.04. 001. Epub 2018 Apr 10) and Zhan et al. (2020) (Zhan et al., Genomic landscape and genetic manipulation of the black soldier fly Hermetia illucens, a natural waste recycler, Cell Res. 2020 Jan;30(1):50-60. doi: 10.1038/s41422-019-0252-6. Epub 2019 Nov 25; cited as NPL CC in the IDS filed on 09/15/2022) as applied to claims 1-3, 5, 8-9, 13, and 22 above, and further in view of Uhlírová et al. (2002) (Uhlírová et al., Heat-inducible transgenic expression in the silkmoth Bombyx mori, Dev Genes Evol. 2002 Apr;212(3):145-51. doi: 10.1007/s00427-002-0221-8. Epub 2002 Mar 1.).
The combined teachings of Shinmyo et al. (2004), Eckermann et al. (2018), and Zhan et al. (2020) have been documented above in the rejection of claims 1-3, 5, 8-9, 13, and 22 under 35 U.S.C. 103.
The combined teachings of Shinmyo et al. (2004) Shinmyo et al. (2004), Eckermann et al. (2018), and Zhan et al. (2020) do not explicitly teach (i) the exogenous gene is stably heritable recited in claim 4, which depends from claims 1 and 3; and integrated into insect genome recited in claim 14; (ii) Drosophila heat shock protein 70 (hsp70) promoter recited in claim 15; (iii) reared insect, a larva, an egg and an adult insect, recited in claims 16, 21, and 23. (iv) a pupa recited in claim 20, and (v) the insect expresses at least 2 mg of the recombinant protein recited in claim 70.
(i)-(ii) Regarding the limitations recited in instant claim 4, which depends from claims 1 and 3, and the limitations claims 14-15, Uhlírová et al. (2002) teaches that “Germline transformation with new transposon vectors now enables causal tests of gene function via ectopic protein expression or RNA interference in non-drosophilid insects. The problem remains of how to drive the transgene expression in vivo. We employed germline transformation using the piggyBac 3xP3-EGFP vector to test whether the Drosophila heat shock hsp70 promoter will be active in the live silkworm. We modified the original vector by cloning the coding sequence for Bombyx nuclear receptor Ftz-F1 between the hsp70 promoter and the terminator. Three independent transgenic lines expressing the Pax-6-driven EGFP marker in larval and adult photoreceptors were obtained with efficiencies of up to 1.7% of fertile G0 adults that gave GFP-positive progeny. Chromosomal integration of the transposon was confirmed with inverse PCR. Heat induction of the transgenic BmFtz-F1 was proven at both the mRNA and protein levels. RT-PCR data showed that the Drosophila heat shock promoter was functional in all three transgenic lines. Although basal activity was apparent at 25°C, 1 h at 42°C induced BmFtz-F1 mRNA at different stages of development and in diverse tissues. The relative levels of induction differed among the transgenic lines. Northern blot hybridization detected transgenic BmFtz-F1 only after heat shock and low levels of the mRNA were still present 6 h after the heat treatment. Immunostaining of epidermis using anti-BmFtz-F1 antibody showed a clear increase of nuclear signal 90 min after a heat shock. (See Abstract, Uhlírová et al., 2002). The design of a piggyBac vector for inducible transgene expression is shown in Fig. 2.
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Fig. 2 Design of a piggyBac vector for inducible transgene expression. The marker gene EGFP is located behind the Pax-6 artificial promoter driving expression in photoreceptor cells
(Horn and Wimmer 2000. A heat-shock-inducible cassette with BmFtz-F1 transgene was cloned using AscI and FseI restriction enzymes. HSpro and F1rm are primers used for RT-PCR to identify the transgene expression only. For inverse PCR, HaeIII restriction sites and two sets of primers (LF + LR or RF + RR) within the piggyBac arms are shown.
(iii) Regarding the limitations recited in instant claims 16, 21-23, Uhlírová et al. teaches that “From 2,873 eggs coinjected with pBac{hsFtz-F1} and a transposase helper plasmid (see Materials and methods, page 146) in three separate trials, we obtained three independent transgenic lines. Transformation efficiency thus varied between 0 and 1.7% of fertile G0 adults that gave EGFP-positive progeny (Table 1). This is low relative to piggyBac transformation efficiencies achieved previously in Nistari silkmoth: 3.9% by Tamura et al. (2000), 17% in Musca domestica (Hediger et al. 2001), 35% in D. melanogaster and even 60% transformants in the flour beetle Tribolium castaneum (Berghammer et al. 1999). The universal activity of the artificial 3xP3-TATA promoter based on Pax-6/Eyeless binding sites (Berghammer et al. 1999) enabled us to detect green fluorescence from mid-embryogenesis through the adult stage (Fig. 3) Besides the simple larval eyes (stemmata) and adult compound eyes, the EGFP marker was visible in CNS and ventral ganglia (Fig. 3). All of the larval stemmata and adult ommatidia were EGFP-positive, but the signal in each ommatidium was only visible from a particular angle due to blocking by pigment. Described fluorescence patterns were similar to those previously seen in flies and silkmoth carrying the same 3xP3 promoter (Horn et al. 2000; Thomas et al. 2002). (See bridging paragraph, pages 147-148, Uhlírová et al., 2002).
(iv) Regarding limitation “a pupa” recited in claim 20, Uhlírová et al. explicitly teach both a molting larva and adult developmental stages of silkmoth transformed with the pBac{hsFtz-F1} vector in Fig. 3 (page 148). In this regard, it is noted that a larva is the active, feeding stage that emerges from the egg, whereas a pupa is a relatively inactive, transformative stage where the larva changes into an adult insect.
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Fig. 3A-D Expression of enhanced GFP marker detected at different developmental stages of silkmoth transformed with the pBac{hsFtz-F1} vector. A Seven-day-old embryo showing green fluorescence in the developing larval stemmata (arrowhead) and nervous ganglia (arrow). Yellow-green color of the rest of the body is caused by autofluorescence. B Adult compound eyes. EGFP signal in most of the ommatidia is invisible from one angle due to eye pigmentation. C Arrowhead shows new second instar stemmata in a molting larva. D Stemmata of a newly ecdysed second instar larva (arrowhead). Arrow shows EGFP, cotransported with eye pigment along the axon.
(v) Regarding limitation “the insect expresses at least 2 mg of the recombinant protein” recited in claim 70, Uhlírová et al. teach that “To determine whether the transgenic BmFtz-F1 mRNA is also translated into the protein, we stained epidermis of day-1 fifth instar larvae with anti-BmFtz-F1 antibody. Confocal images in Fig. 6 show that nuclear staining in epidermal cells of transgenic animals strongly intensified 90 min after a 90-min heat treatment (See right column, page 149).
It is noted that claim 70 as written encompasses “at least 2 mg” of any recombinant protein expressed from any number of transformed insects recited in claim 1.
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Fig. 6 Immunocytochemistry of epidermis using anti-BmFtz-F1 antibody. Day-1 fifth instar transgenic larvae (line 208) were treated at 42°C for 90 min and dorsal abdominal epidermis was dissected 90 min after the heat shock (right). Epidermis of a transgenic
larva without heat shock was used for a control (left). Samples were stained with anti-BmFtz-F1 antibody (dilution 1:200) and Cy3-conjugated secondary antibody (1:2,000). The images
were captured by confocal microscopy under identical brightness and contrast settings
A skilled artisan would have been motivated to incorporate the teachings of Uhlírová et al. (2002) into the combined teachings of Shinmyo et al. (2004), Eckermann et al. (2018), and Zhan et al. (2020) because Drosophila heat shock hsp70 promoter is a well-established heat inducible promoter utilized for modulating exogenous gene expression taught by Uhlírová et al. (2002).
There would have been a reasonable expectation of success to reach claimed transformed insect of instant claims 4, 14-16, 20, 21, 23, and 70 because Uhlírová et al. (2002) explicitly teaches chromosomal integration of the transposon was confirmed with inverse PCR. Heat induction of the transgenic BmFtz-F1 was proven at both the mRNA and protein levels.
Applicant’s arguments
Applicant submits that the corresponding present claims do not recite an insect of Gryllus
bimaculatus. Shinmyo and Uhlfrova do not teach a transformed insect of the genus Hermetia, as
recited in the present claims. Accordingly, Applicant respectfully requests reconsideration and
withdrawal of the rejection.
Response to Applicant’s arguments
Applicant’s arguments have been addressed in the new grounds of rejection under 35 U.S.C. 103 documented above.
Claims 24 and 70 are rejected under 35 U.S.C. 103 as being unpatentable over Shinmyo et al. (2004) (Shinmyo et al., piggyBac-mediated somatic transformation of the two-spotted cricket, Gryllus bimaculatus, Dev Growth Differ, 2004 Aug;46(4):343-9. doi: 10.1111/j.1440-169x.2004.0075 1.x.) in view of Eckermann et al. (2018) (Eckermann et al., Hyperactive piggyBac transposase improves transformation efficiency in diverse insect species Insect Biochem Mol Biol., 2018 Jul:98:16-24. doi: 10.1016/j.ibmb.2018.04. 001. Epub 2018 Apr 10) and Zhan et al. (2020) (Zhan et al., Genomic landscape and genetic manipulation of the black soldier fly Hermetia illucens, a natural waste recycler, Cell Res. 2020 Jan;30(1):50-60. doi: 10.1038/s41422-019-0252-6. Epub 2019 Nov 25; cited as NPL CC in the IDS filed on 09/15/2022) as applied to claims 1-3, 5, 8-9, 13, and 22 above, and further in view of Lee et al. (2015) (Lee et al., Production of antibacterial Bombyx mori cecropin A in mealworm-pathogenic Beauveria bassiana ERL1170, J Ind Microbiol Biotechnol. 2015 Jan;42(1):151-6. doi: 10.1007/s10295-014-1551-z. Epub 2014 Nov 28.)
The combined teachings of Shinmyo et al. (2004), Eckermann et al. (2018), and Zhan et al. (2020) have been documented above in the rejection of claims 1-3, 5, 8-9, 13, and 22 under 35 U.S.C. 103.
The combined teachings of Shinmyo et al. (2004), Eckermann et al. (2018), and Zhan et al. (2020) do not explicitly teach (i) the limitations “a biomass produced from the transformed” recited in claim 24, and (ii) the limitation “the insect expresses at least 2 mg of the recombinant protein” recited in claim 70.
It is noted that claim 70 as written encompasses “at least 2 mg” of any recombinant protein expressed from any number of transformed insects recited in claim 1.
(i) Regarding the limitations recited in claim 24, Lee et al. (2015) teaches that “Efforts are underway to produce antimicrobial peptides in yellow mealworms (Tenebrio molitor), which can be developed as more effective and safer animal feed additives. In this work, we expressed Bombyx mori (Bm) cecropin-A in mealworms by the infection of transformed entomopathogenic Beauveria bassiana ERL1170. The active domain of Bm cecropin A gene was tagged with a signal sequence of B. bassiana for extracellular secretion, and the fragment was inserted into ERL1170 by the restriction enzyme-mediated integration method. Transformant D-6 showed antibacterial activity against Bacillus subtilis and Listeria monocytogenes. Against T. molitor larvae, D-6 had similar mortality to wild-type, and D6-infected mealworm suspension showed strong antibacterial activity against the two bacteria, but not in the wild-type-infected mealworms, thereby increasing the value of mealworms as animal feed additives.” (See Abstract, and Fig. 1, Lee et al., 2015).
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Fig. 1 Construction of the fungal transformation vector, pBARKS1-Bbs-cecropin A containing B. bassiana signal (Bbs) and B. mori (Bm) cecropin A. a Integration of the egfp expression cassette to pBARKS1; and b exchange of egfp with Bbs-Bm cecropin A fragment.
Lee et al. (2015) further teaches that “The D-6-infected mealworm suspension showed
strong antibacterial activity against the two bacteria on LB medium in 3 days, but not in the wild type-infected mealworms (Fig. 3b). The B. mori cecropin A was possibly accumulated in T. molitor larvae, and the infected mealworms could be used as an antibacterial material in
animal feeds. However, one concern about the utility of the system is the reproducibility of the process (concentration of active cecropin A in the final product in different batches). It can be overcome when the transformant stably keeps the gene over the generations and inoculation factors such as larval stage and quantity of conidia are consistent in batches. (See bridging paragraph, pages 154-155, Lee et al., 2015). Moreover, Lee et al. (2015) concludes that “This work suggests that B. mori cecropin A was successfully expressed in yellow mealworms by the infection of transformed B. bassiana ERL1170 containing the cecropin A-expression cassette. The transformant-infected mealworms showed strong antibacterial activity against B. subtilis
and L. monocytogenes, thereby increasing the value of mealworms as animal feed additives. (See left column, page 155).
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Fig. 3 Antibacterial activity of the transformant D-6-infected mealworm (T. molitor) powder. a Percentage (%) of live T. molitor larvae after the treatment of wild-type and D-6 conidial suspensions (1 × 107 conidia/mL). In 7 days, mycelial outgrowth was observed in
both of wild-type and D-6 treatments; and b Antibacterial activity of the infected mealworm powder against B. subtilis and L. monogenesis.
(ii) Regarding the limitations recited in claim 70, Lee et al. (2015) teaches that “A transformant with strong antibacterial activity was selected, and it was designated as D-6. The transformant had a stable growth on the Czapek’s solution agar containing PPT during sub-cultures, whereas wild type did not grow on the selection medium (Fig. 2a). On the SDA/4 medium, D-6 showed a slightly different morphology (more mycelial mass was observed) compared to the wild-type (Fig. 2a), and “Transcription of Bbs-cecropin A gene was confirmed by RT-PCR analysis (Fig. 2b) and the expression of cecropin A was confirmed by western blot analysis (Fig. 2c) (See left column, page 154).
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Fig. 2 Characterization of B. mori cecropin A-producing transformant D-6. a Mycelial growth of wild-type and transformant D-6 on Czapek’s solution agar containing phosphinothricin (PPT) and SDA/4 (without PPT); b Reverse transcription (RT) PCR of wild type and transformant D-6 (B. bassiana 18S rRNA was used as a control); c SDS-PAGE and western blot of wild-type and D-6 supernatants; and d Antibacterial activity of wild-type and transformant D-6 against B. subtilis and L. monogenesis on LB agar in three days
A skilled artisan would have been motivated to incorporate the teachings of Lee et al. (2015) into the combined teachings of Shinmyo et al. (2004), Eckermann et al. (2018), and Zhan et al. (2020) to reach claimed “biomass produced from the transformed insect of claim 1” as recited in instant claim 24 and claimed “the insect expresses at least 2 mg of the recombinant protein” recited in claim 70 because “Functional proteins can be produced in insects by the infection of genetically engineered-entomopathogenic fungi, and ultimately the mycotized insects can be used as raw materials for feed additives. Yellow mealworm, Tenebrio molitor L. (Coleoptera Tenebrionidae) has been added to animal feeds, because it has large amount of nutrition.” (See right column, page 151, Introduction, Lee et al., 2015).
There would have been a reasonable expectation of success to reach claimed “biomass produced from the transformed insect of claim 1” as recited in claim 24 and claimed “the insect expresses at least 2 mg of the recombinant protein” recited in claim 70 because Lee et al. (2015) explicitly teaches that “B. mori cecropin A was successfully expressed in yellow mealworms by the infection of transformed B. bassiana ERL1170 containing the cecropin A-expression cassette. The transformant-infected mealworms showed strong antibacterial activity against B. subtilis and L. monocytogenes, thereby increasing the value of mealworms as animal feed additives.” (See left column, page 155).
Applicant’s arguments
Applicant submits that the corresponding present claims do not recite an insect of Gryllus
bimaculatus. Shinmyo and Lee do not teach a transformed insect of the genus Hermetia, as recited
in the present claims. Accordingly, Applicant respectfully requests reconsideration and withdrawal
of the rejection.
Response to Applicant’s arguments
Applicant’s arguments have been addressed in the new grounds of rejection under 35 U.S.C. 103 documented above.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682